future perspectives in clinical research

Clinical Research Trends & Insights for 2023

In our 2023 preview, 14 experts from WCG share the important shifts, trends, regulations, and priorities that will inform clinical trial development this year and beyond.

After the disruptions to the clinical research field in 2020 and 2021, many looked forward to 2022 as a year of “getting back to normal.” Instead, we found that many pandemic-associated changes have forced us to look at a lot of issues through a new lens.

Certainly, “decentralized trials” dominated our conversations, and considerations of the importance of proactive efforts to facilitate the inclusion of historically underrepresented populations in research is finally being recognized. Both biopharma sponsors and clinical sites are finding their footing again, allowing them to renegotiate some old practices from new perspectives (e.g., investigator oversight responsibilities in decentralized trials, how much of site monitoring really needs to be on-site).

Our clinical trials continued to evolve in response to new therapeutic advances, with more protocols incorporating complex design elements, earlier evaluations of efficacy, and an increasing number of platform studies to move potential drug candidates to decisions more quickly.

2023 looks like it will be just as exciting. In this report, WCG experts from a variety of areas talk about what they’re watching, expecting, and looking forward to in 2023. We’re exploring study design, study conduct, new proposed regulations and guidance, and even at the types of therapeutic agents moving into clinical investigation. 

Thank you for joining us as we look ahead to the next exciting year!

Explore Insights:

Perspectives on clinical research sites in 2023, redesigning the clinical research landscape, diversity strategies and empowering sites to support them, the fda’s recent notice of proposed rulemaking (nprm) and guidance, diversifying clinical research by providing research participants payments, clinical trial technology integration now and into the future, interoperability required for the future of clinical trials, quality by design and slowing down to speed up, the emerging use of adjudication in oncology for determining patient eligibility, mrna: not just for vaccines.

Current Trends in Neurodevelopmental and Rare Pediatric Disease

Neuroscience Research Trends to Pay Attention to in 2023

Epilepsy treatments and research: where we started and where we are heading, watch our 2023 trends & insights webinar.

Hear our online panel of clinical trial experts discuss the future of clinical trials, and answer questions from your peers.

This engaging discussion is facilitated by Jill Johnston, Chief Innovation Officer of WCG.

Sandra Smith, RN, MSN, AOCN

Senior Vice President, Clinical Solutions & Strategic Partnerships, WCG

To combat the tumultuous conditions of the industry, research organizations have been forced to look internally to identify operational efficiencies over the last two years, and institutional reconfiguration will continue to evolve in 2023. Centralization of research services, embedding enabling technologies, and establishing research partnerships – both with service providers and with sponsors – will remain key themes in addressing site capacity challenges. Many non-institutional research sites will aggregate into networks to achieve higher operational performance.

Human capital remains critical, and the skillset for research expertise will continue to be in high demand in 2023. Sites are reporting fewer vacant positions, but turnover will persist, and training the newly hired, less experienced research personnel will remain critical. Sponsor-supported staffing augmentation solutions will be key to trial conduct for many organizations.

Oncology will endure as a dominant therapeutic area in clinical research in 2023. Approximately 50 percent of all clinical trials started in 2022 1 were in oncology, with more than 1M patients (about the population of Delaware) needed to complete those open trials 2 . Increasing trial complexity will challenge sites with narrow inclusion/exclusion criteria, multiple study arms (i.e., 19-20 percent increase in four to five arm trials in oncology) 3 , and an increasing number of amendments per clinical trial (i.e., 57 percent increase since 2017) 4 .

Declining average rates of enrollment per site 5 and the need for increased enrollment diversity will accelerate sponsor interest in opening trials at community-based sites and in supporting new investigators/sites. Rapid trial activation, bringing trial access to new participant populations, and utilization of decentralized trial components, will be critical components for trial success.

References:

1, 2, 3, 4, 5 WCG Knowledge Base TM , 2022

Jamie Harper, MHA

Director of Site Engagement and Relations, WCG

In 2023, clinical trial access and improving participant diversity will remain key concerns within the clinical research and healthcare community. Compounded by the number of physician investigators continuing to decrease, the industry will be tasked with developing solutions to ensure scientific medical advancements continue while supporting these diverse populations. Identifying and supporting new physician investigators at research sites, especially those ingrained into the community setting, is critical to offsetting these concerns and accelerating advancements.

Redesigning the clinical trial landscape to mitigate these challenges and create an environment that allows engaged community physicians to provide clinical trial access to the communities they serve is a must. By engaging community sites and physicians who may not have previously considered clinical research as an option, it presents a path toward addressing the concerns of clinical trial access and participant diversity. However, this pathway will require the clinical research industry to provide support for the tools and mentorship needed to successfully conduct compliant clinical trials.

The new environment will require evolution of the current collaborations between sponsors, clinical research organizations (CROs), third-party clinical research vendors, and experienced physician investigators. This collaboration’s success will depend on a shared focus to cultivating a redesigned clinical research landscape through inclusion.

Katherine Cornish, PhD, PMP

Former Associate Director, Patient Advocacy & Clinical Trial Diversity, WCG

As the FDA (U.S. Food and Drug Administration) moves closer to finalizing the current draft guidance on establishing diversity plans 1 to improve clinical trial participation of underrepresented racial and ethnic populations, it becomes imperative to ensure that the strategies outlined in these plans are translated to the site level in intentional and actionable ways.

Sponsors must empower their sites by providing pragmatic strategies that site staff can use to effectively identify, engage, enroll, and retain underrepresented populations. This should be done through the creation of site-focused diversity plans that tie into the overarching diversity plan. Key partnerships with advocacy groups, community-based organizations, and other similar entities outlined in diversity plans at a national level should be translated to individual sites locally. Sponsors must also provide the necessary support to ensure these strategies can be enacted by their sites.

Additionally, these strategies must be identified, documented, and mobilized early in the trial lifecycle; they must be thought about proactively rather than as rescue methods. Sponsors should reach out to the FDA to discuss their diversity plans as early as possible – certainly prior to study start. Thinking about these approaches early on can also ensure that the necessary support is preemptively budgeted at the site level, allowing sites to initiate local advocacy and community engagement efforts from the start. This ensures that sites are prepared and ready when recruitment begins and can enact their action plans from first patient to last visit.

• https://www.fda.gov/media/157635/download

Dave Borasky, MPH, CIP

Vice President, IRB Compliance WCG IRB

The year 2022 ended with two interesting developments for Institutional Review Boards (IRBs) and industry on the FDA front. First, the FDA published two Notices of Proposed Rulemaking (NPRMs) 1,2 that  are intended to bring greater harmony between the FDA regulations for human subjects research, and the Common Rule regulations that apply to U.S. federally funded research that is not subject to FDA regulation. While this harmonization is expected – it was mandated by the 21st Century Cures Act – it was not clear when the NPRMs would be issued. Harmonization on issues of informed consent requirements and the use of a single IRB for multisite trials will improve the review and implementation of clinical research in 2023 and beyond.

The second late-year offering from the FDA was the draft guidance Ethical Considerations for Clinical Investigations of Medical Products Involving Children . Through this guidance, the FDA has finally provided clear direction on the expectation that IRBs use component analysis when reviewing research with children that includes multiple research-related interventions or procedures. Component analysis, which has the IRB assess each study procedure (and each study arm), considering the risks and benefits, is often confusing to sponsors. The guidance also provides direction on the FDA’s thinking on what constitutes a “minor increase over minimal risk” (21 CFR 56.102(i)), which is not defined in the regulation, but represents an important threshold for reviewing research involving children, particularly in the context of component analysis.

• https://www.federalregister.gov/documents/2022/09/28/2022-21088/protection-of-human-subjects-and-institutional-review-boards
• https://www.federalregister.gov/documents/2022/09/28/2022-21089/institutional-review-boards-cooperative-research

Kelly Fitzgerald, PhD

IRB Executive Chair and Vice President, IBC Affairs WCG IRB

Payment to research participants is a critical aspect of diversifying clinical research participation. There will be a continued increase in awareness of this issue in 2023. IRBs are often seen as a barrier to paying participants, and that may have been true in the past, but the thinking on this topic has evolved significantly in the last few years, informed in part by the recognition of the importance of research participants as partners rather than as research “subjects. ”1

IRBs are tasked with ensuring that research-recruiting and consenting processes do not exert undue influence on participants, and high payments may be seen by IRB members as unduly influential 2 . Some people believe that payments could incentivize someone to participate in activities they would otherwise choose not to do, particularly people with low incomes. However, when an IRB insists on lower payments, participants who have less free time, less available income, or more burdensome lives are less likely to participate, and the study ends up with a participant population that does not match society because those people who can bear the financial burdens of research participation will participate.

The clinical research world now recognizes that payments can incentivize without being unduly influential. While there is a perception that IRBs will not approve high payments for participants, this is changing. During the last 24 months, out of approximately 10,000 IRB reviews, only 13 reviews resulted in the IRB specifically requiring modifications to the payment plan (see table), and no records included a request to decrease the proposed payments to participants.

IRB members reflect the values and norms of their communities, and the events of the last few years have led to the awareness of the importance, both scientifically and ethically, of diverse representation in clinical trials. To improve in this area, there must be support for higher payments for research participation and a reliance on mechanisms other than limiting payment to ensure participants are recruited ethically. In 2023, there needs to be more discussions around accurately assessing participant costs for research participation and providing just compensation for their service 3 . 

• National Academies of Sciences, Engineering, and Medicine 2022. Improving Representation in Clinical Trials and Research: Building Research Equity for Women and Underrepresented Groups. Washington, DC: The National Academies Press. https://doi.org/10.17226/26479.
• Payment and Reimbursement to Research Subjects, FDA, Jan 2018.
• Largent EA, Lynch HF. Paying Research Participants: The Outsized Influence of “Undue Influence”. IRB. 2017 Jul-Aug;39(4):1-9. PMID: 29038611; PMCID: PMC5640154.

Sonia Abrol

Senior Vice President, Product and Strategy, WCG Velos

The COVID-19 pandemic made many stakeholders in clinical research reassess their technology infrastructure. To sustain and accelerate research, they prioritized taking various research management workflows online to vendor managed systems, ensuring centralized, easy, and secure access to data. As sites and sponsors expanded their technology portfolio, sites are now carrying a bigger technology burden. They are supporting the costs of multiple site technologies and then putting additional time and effort into entering data in both site and sponsor systems. Approximately 90 percent of data needed by sponsors now lives in one or more site systems 1 . 

Additionally, the uptick in adoption of innovative technologies for helping with decentralized trials, patient recruitment, patient retention, and engagement has produced new data silos for sites and sponsors. As a result, clinical research stakeholders are recognizing the need for more interoperability and workflow-driven integrations.

In 2023, sites and sponsors will continue to make technology integration one of their top priorities as they select the right solutions, enhance their technology infrastructure, and budget for such investments. Sites starting their journeys toward integrated ecosystems may begin with key multi-level integrations between their e-regulatory/e-Binder, EHR/EMR, CTMS, IRB and Financial systems. Those that are already ahead of the curve will continue to expand and develop new integration workflows by connecting one silo at a time.

In parallel, technology vendors will continue to work toward offering more open systems and actively defining their own integration roadmaps to help sites and sponsors gain operational efficiencies and reduce the technology burden on users.

Also, it will be interesting to see what the gained momentum in collaboration among sites, sponsors, and vendors will bring in 2023. As these consortia and partnerships come together to help define data exchange standards, map best workflows among site systems and site-sponsor systems and prioritize implementation of cost-effective and standard technology integrations, we will see remarkable growth and acceleration in conducting clinical research and drug development.

• WCG Knowledge Base TM , 2022

Rahul Bafna

Former Chief Product Officer, WCG

The use of healthcare technology at the consumer level has exploded over the past decade. This started with the broad availability and adoption of wearable devices, taking advantage of smaller sensors, better batteries, and always-on connectivity. Over this same time period, there has been a ton of investment in healthcare technology for clinicians – the HITECH Act of 2009 was designed to accelerate medical product development. Many of these advancements in technology have the potential to be transformative in the realm of clinical trials, which for decades have suffered from low patient participation, unrepresentative study populations, heavy operational burden, and long timelines. The key to making these advancements work in real-world settings is the seamless interoperability of patient data. 

As an example, patient-trial matching is one of the most time-consuming operations of running a trial. Multiple software platforms exist that can match eligible patients – based on the clinical record stored in one or more EHRs and lab systems – to the various inclusion and exclusion criteria of trials. But for this matching to happen, these solutions need access to those clinical records in a standardized format. Once found, matches need to be pushed back into the physician’s daily workflow – often the EHR – to drive patient conversations, consent and enrollment. Once enrolled, technology can simplify or even automate the data collection necessary for some types of studies. This will be critical for enabling decentralized trials (DCT).

DCTs will allow more patients to participate, potentially from underserved communities who would benefit from trial designs that require fewer or no site visits. The use of technology can also enable more frequent collection of study data, and any data collected in novel systems will need to be pushed back to a unified source, such as an EHR or EDC. 

Making sense of clinical data from multiple sources – whether for clinical trials or routine care – requires that data to be collected, retained, transformed, transmitted, standardized, and made available to researchers and clinicians at the right time and in the correct format. Interoperability between all these systems is critical to driving innovation and bringing new treatments to patients. For several years now, the Office of the National Coordinator for Health Information Technology (ONC) has been leading the charge to improve the interoperability of patient clinical data through regulation – mandating specific data standards that enable data to be exported in standardized formats. The 21st Century Cures Act, which includes interoperability standards not only for accessing clinical data but also for enabling new workflows and within platforms like EHRs, will continue to drive advancement in 2023. 

Cristin MacDonald, PhD

VP Client Delivery, WCG Avoca

The year 2023 should be seen as an opportunity for everyone in the clinical research industry to embrace the concept of “slowing down to speed up.” While the concept of Quality by Design is not new, the newly finalized ICH E8 guidance, EU CTR, along with the upcoming ICH E6 R3 and FDA’s Diversity guidance are all examples of the regulators prioritizing efficiency in clinical trial execution by forcing quality into the forefront of study design.

Whether it is by requiring that sponsors show their diversity strategies in advance of launching a pivotal trial, or by subjecting sponsors to regulatory holds for failed submissions, it is time to spend more resources early in study planning. It is time to slow down, think ahead, and ensure quality is built in study design.

Gone are the days of rushing to a first subject in timeframe and reactive protocol amendments that have negative time and budget impacts. Entirely new benchmarks need to be established that encourage study teams to invest the appropriate time and effort in study planning and reward those who demonstrate rapid study completion with minimal amendments by leveraging the proper resourcing up front.

Shaena Kauffman

Executive Director, Operations EAC, WCG

One of the most interesting trends over the past year has been the increased use of Endpoint Adjudication Committees (EACs) to determine patient eligibility, especially in oncology clinical trials. This trend will likely continue in 2023. Adjudication of inclusion criteria, or eligibility adjudication, has been used in clinical trials where inclusion criteria are subjective. The independent assessment by an EAC gives regulators and scientists confidence in the comparability of clinical trial subjects. Previously, this type of adjudication has been applied to areas such as progressive neurological disease or heart failure, where medical expertise is required, or eligibility is subjective. However, there is a rising trend in using EACs to confirm oncology disease progression to demonstrate a consistent evaluation for patient eligibility and provide real-time feedback to the Investigator study sites.

In the area of disease progression, adjudication has the potential to mitigate variability and increase the strength of the clinical trial results.  In addition to bolstering the clinical trial data against the regulatory and scientific rigors, EAC members can serve as a resource to investigator study sites that may need access to experts to assess some of the more complex patient eligibility criteria. Of course, the most significant benefits are that adjudication of inclusion criteria reduces protocol deviations and enforces scientific rigor through a pre-randomization, standardized, objective assessment. The use of eligibility EACs will continue to expand in 2023 as sponsors and sites report the benefits.

Daniel Kavanagh , PhD

Senior Scientific Advisor, Gene Therapy, WCG

Reflecting a new emphasis on cell and gene therapy (CGT) for 2023, the FDA recently announced a new “Super Office,” the Office of Therapeutic Products (OTP), to replace the former Office of Tissues and Advanced Therapies. Included in the announcement are plans to increase hiring and issue more CGT guidance documents in the coming year.

The year 2022 saw many new advances in cell and gene therapy, including FDA approvals for new products and supplemental indications for genetically modified cellular therapies and vectored gene therapies. In 2023, one area to watch closely is that of “vectorless” mRNA therapeutics. Synthetic mRNA is a powerful tool that can program cells for new therapeutic functions. When combined with rapidly developing nanoparticle technologies, mRNA therapeutics may be targeted to a wide array of cell types and tissues. One potential application of gene transfer technology is to bypass the complex in-vitro manufacturing process currently required for biologics and turn the body’s own cells into temporary factories to produce antibodies, enzymes, or vaccine antigens. Many such approaches in development rely on mRNA.

Currently approved gene therapies tend to produce long-lasting or permanent changes in cellular programming and may induce immune responses that prevent re-dosing. For these reasons, many gene therapies are designed for “one-and-done” curative administration. This offers new hope to patients and their families, but also presents challenges for safety, commercialization, and reimbursement. In contrast, mRNA molecules are normally short-lived and exert transient effects on cell biology. Thus, for some applications, mRNA is an important alternative to DNA-based therapies, with potential advantages for safety, re-dosing, and commercialization.

Current Trends in Neuro-developmental and Rare Pediatric Disease

Scott Hunter, PhD

Senior Scientific Expert, Neurodevelopment Disorders, WCG

In 2023, there will continue to be an emphasis on developing new outcome measures that can be used across rare pediatric and Central Nervous System (CNS) diseases. This has been an area of shared concern among caregivers regarding challenges observed with their impacted children that affect both individual and family engagement and quality of life. Moving from broader cognitive measures that are often experienced as less sensitive, focus has moved more to communication (specifically receptive and expressive language skills in areas including Fragile-X, Angelman, Rett, Autism Spectrum Disorders) and motor control (e.g., Rett, Angelman, Fragile-X).

Seeking greater sensitivity with scales developed, foundation and patient advocacy partners are developing mechanisms to build disorder-specific scales to address key areas of caregiver and patient concern, while also collaborating across indications to support better measurement design. The goal of this is to amplify acceptance by the FDA of caregiver and clinician report measures (ClinROs and patient reported outcomes, or PROs) in the rare disease space.

We will move toward using wearables and engaging more directly with samples collected from these technologies that can be expert reviewed and rated. Collecting simultaneous biological and behavioral outcomes through direct movement assessment, recording of communication efforts, and video monitoring of responses, provides significant multipoint data that can be aggregated in tandem with use of performance measurements in rare and neurodevelopmental disease.

Mark G. A. Opler, PhD, MPH

Chief Research Officer, WCG Endpoint Solutions

The last five years have brought a remarkable burst of innovation in neuroscience drug development. From the approval of the first, rapid-acting agent to treat depression, to the past year’s massive surge of activity in the study of psychedelic compounds, to the ongoing, exploratory use of wearables and other technology-oriented methods for data collection, there has been a tremendous degree of change. In the coming year, we’ll see some of these exciting yet unproven innovations begin to demonstrate their promise in a more substantive way with enduring, real-world impacts. 

A few of the most important trends to watch include: 

• Late-Stage Studies of Psychedelics and Cannabinoids:  The first large-scale, Phase III industry-sponsored studies of psilocybin by Compass Pathways should help prove the viability of the psychedelics movement in neuroscience. The published results of the Compass Phase II program, if confirmed in Phase III, would solidify this while also clarifying the direction that this exciting new class of treatments is likely to take in years to come. 
• Development of Wearables and “Digital Biomarkers ”: Although regulatory agencies have held off on explicitly approving the use of novel endpoints related to wearable devices or similar device-rated digital biomarkers, the proliferation of these tools as exploratory endpoints is creating a growing wealth of data. As the evidence base for them continues to grow, standardization and adoption will almost certainly follow.

Mike Cioffi

Senior Vice President, Clinical Solutions and Strategic Partnerships, WCG

Currently, there are more than 30 antiseizure medications (ASMs) available to clinicians to treat patients. But even with such a significant amount of treatment options, there are still over 30 percent of individuals who do not respond to common ASMs and are addressed as “drug-resistant.” 1 This will drive innovation in both pharmacological and non-pharmacological interventions aimed at improving symptoms and quality of life for patients along with their caregivers.

Research is ongoing in many areas, and we will see important advances in the field in the coming years. An increased application of technologies to aid in epilepsy management is likely. Smartwatches that can detect imminent seizures and alert caregivers to the location of the patient, and apps that can inform clinicians with real-time information on their patients’ status and seizure counts are all on the horizon.

Precision medicine has begun to take a more prominent role as we understand more about the pathogenesis of epilepsies. Significant advancement has been made in identifying the genetics of epilepsies, and the discovery of gene mutations responsible for a large portion of patients with developmental and epileptic encephalopathies (DEEs) 2 . 

Additionally, the search for biomarkers to guide drug development is moving at a fast pace and will potentially allow clinicians to improve diagnostic accuracy, predict response to ASMs, and improve outcomes for patients.

While there are certainly limitations to current technologies and biomarker identification, and despite precision medicine being further researched, this multidisciplinary approach will redefine our ability to improve the treatment and management of epilepsy in the future.

• Kalilani L, Sun X, Pelgrims B, Noack-Rink M, Villanueva V. The epidemiology of drug-resistant epilepsy: a systematic review and meta-analysis.  Epilepsia.  (2018) 59:2179–93. doi: 10.1111/epi.14596
• Perucca P, Bahlo M, Berkovic SF. The genetics of epilepsy. Annu Rev Genomics Hum Genet. 2020;21(1):205–30

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• Review Article
• Published: 27 January 2023

Pharmacogenomics: current status and future perspectives

• Munir Pirmohamed   ORCID: orcid.org/0000-0002-7534-7266 1  

Nature Reviews Genetics volume  24 ,  pages 350–362 ( 2023 ) Cite this article

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• Pharmacogenomics
• Therapeutics

Inter-individual variability in drug response, be it efficacy or safety, is common and likely to become an increasing problem globally given the growing elderly population requiring treatment. Reasons for this inter-individual variability include genomic factors, an area of study called pharmacogenomics. With genotyping technologies now widely available and decreasing in cost, implementing pharmacogenomics into clinical practice — widely regarded as one of the initial steps in mainstreaming genomic medicine — is currently a focus in many countries worldwide. However, major challenges of implementation lie at the point of delivery into health-care systems, including the modification of current clinical pathways coupled with a massive knowledge gap in pharmacogenomics in the health-care workforce. Pharmacogenomics can also be used in a broader sense for drug discovery and development, with increasing evidence suggesting that genomically defined targets have an increased success rate during clinical development.

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M.P. has received partnership funding for the following: the Medical Research Council (MRC) Clinical Pharmacology Training Scheme (co-funded by MRC and Roche, UCB, Eli Lilly and Novartis) and a PhD studentship jointly funded by the Engineering and Physical Sciences Research Council (EPSRC) and Astra Zeneca. He also has unrestricted educational grant support for the UK Pharmacogenetics and Stratified Medicine Network from Bristol-Myers Squibb. He has developed an HLA genotyping panel with MC Diagnostics but does not benefit financially from this. He is part of the Innovative Medicines Initiative (IMI) consortium ARDAT ( www.ardat.org ). None of the funding received is related to the current paper.

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Pirmohamed, M. Pharmacogenomics: current status and future perspectives. Nat Rev Genet 24 , 350–362 (2023). https://doi.org/10.1038/s41576-022-00572-8

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Cancer is a major public health problem worldwide with more than an estimated 19.3 million new cases in 2020. The occurrence rises dramatically with age, and the overall risk accumulation is combined with the tendency for cellular repair mechanisms to be less effective in older individuals. Conventional cancer treatments, such as radiotherapy, surgery, and chemotherapy, have been used for decades to combat cancer. However, the emergence of novel fields of cancer research has led to the exploration of innovative treatment approaches focused on immunotherapy, epigenetic therapy, targeted therapy, multi-omics, and also multi-target therapy. The hypothesis was based on that drugs designed to act against individual targets cannot usually battle multigenic diseases like cancer. Multi-target therapies, either in combination or sequential order, have been recommended to combat acquired and intrinsic resistance to anti-cancer treatments. Several studies focused on multi-targeting treatments due to their advantages include; overcoming clonal heterogeneity, lower risk of multi-drug resistance (MDR), decreased drug toxicity, and thereby lower side effects. In this study, we'll discuss about multi-target drugs, their benefits in improving cancer treatments, and recent advances in the field of multi-targeted drugs. Also, we will study the research that performed clinical trials using multi-target therapeutic agents for cancer treatment.

Introduction

Cancer is a leading cause of worldwide death and the most prevalent disease, with an estimated 19.3 million new cancer cases around the world in 2020 [ 1 ]. Therefore, its early detection and effective treatment development are crucial for managing this life-threatening disease.

Limitations of conventional chemotherapeutic agents, lack of specificity in existing epigenetic targeting drugs, and drug resistance are among the main challenges in cancer therapy [ 2 , 3 ]. Through decades, different strategies have been developed for cancer treatment such as immunotherapy, gene therapy, epigenetic therapies, etc. [ 4 , 5 , 6 ]. While numerous cancer types may initially respond to chemotherapy, they can eventually develop resistance to it [ 7 ]. The ability of cancer cells to develop resistance against traditional treatments, and the growing number of drug-resistant cancers highlights the need for more research and the development of new treatments [ 8 ].

Targeted therapy, also known as precision medicine, blocks cancer cell growth by interfering with specific molecules needed for cancer development and growth, instead of simply interfering with all rapidly dividing cells like traditional chemotherapy [ 9 ]. Tamoxifen was the first targeted cancer therapy approved in the 1970s. It blocks the growth of estrogen receptor (ER)-positive breast cancer cells by binding to the estrogen receptor and preventing estrogen from binding [ 10 ]. The targeting therapy can be classified into single- and multi-targeting agents.

Single-target therapy has been a major advance in cancer treatment, but it has limitations. When single-target therapy fails, the alternative strategy is multi-target therapy, which includes polypharmacological drugs or drug combinations [ 11 ].

Polypharmacology involves targeting multiple tumor growth and progression-related pathways, making it more effective in treating complex diseases and drug-resistant cancers. Studies in polypharmacology could reveal new off-targets for current drugs, offering insight into drug side effects and toxicities. Furthermore, it can aid drug repurposing by identifying new indications or therapeutic targets for existing drugs [ 12 ]. Despite the optimistic outlook on multi-target therapy, overcoming challenges such as appropriate target selection is crucial for enhancing treatment efficacy [ 13 ].

Herein, we'll dive into the concept of polypharmacology, its potential, challenges, and future perspectives. Also, we'll argue the recent multi-target drug studies and potential therapeutic targets for developing anticancer agents in few prevalence malignancies.

Single-, combination-, and multi target directed ligands-therapies; what's the difference?

Cancer treatments can be categorized based on the way therapeutic agents are employed into single agents, combination, and multi-target directed ligands (MTDLs) which are described as follows.

Monotherapy, also known as single-target therapy, aims to combat cancer by selectively attacking certain genes and proteins responsible for the survival and proliferation of malignant cells [ 14 ]. Unlike conventional chemotherapy drugs that exhibit a lack of selectivity towards cancer cells versus normal cells, this method ensures reduced harm to healthy cells, consequently minimizing the occurrence of substantial toxicity and side effects [ 15 ]. While monotherapy has shown some efficacy in certain cases, it may not be effective for all patients because the tumor cells can become resistant to monotherapies [ 16 ].

Combination therapy is a therapeutic modality that employs combining two or more agents with different mechanisms of action to achieve synergistic effects against cancer [ 17 ]. Since the discovery of new pharmacological anti-cancer agents is arduous and costly, it is essential to identify more effective methods that are economically viable [ 18 ]. While monotherapy is still applicable in some cases, combination therapy is increasingly recognized for its effectiveness and broad treatment coverage in managing complex diseases like cancer [ 19 ]. However combination therapy is a feasible option, there are remain challenges such as cost-effectiveness [ 20 ], and identifying the best drug combinations [ 21 ] which will be discussed in the next section.

Drug resistance is an important issue with current treatments which can be overcome using MTDLs [ 22 ]. MTDLs are a new class of drugs that target multiple receptors/enzymes simultaneously leading to better efficacy, preventing drug resistance development, and also combating it [ 23 ]. This strategy also has the potential to lower the required dosage of individual drugs, reducing the risk of adverse effects and enhancing treatment outcomes [ 7 ]. On the other hand, designing selective MTDLs with high affinity to their targets while avoiding off-target effects is a significant challenge in MTDLs treatments [ 24 ]. Understanding the pharmacokinetics (PK) and pharmacodynamics (PD) of designed ligands is another challenge, however, computer-aided drug designing tools provided applications for describing PK (i.e. absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties) of designed drugs more predictably [ 25 ]. MTDLs reveal great superiority in comparison to mono- & combination-therapies which will further be discussed.

Cancer and the necessity of using multi-targeted drugs

Cancer resistance is affected by Darwinian law, intra-tumor cell heterogeneity, and compensatory pathways often result in the tumor cells' survival [ 26 ]. It is the main challenge with monotherapies [ 8 ], which is attributed to up to 90% of cancer-associated deaths [ 27 ] and can be caused by various factors. Under the treatment pressure, cancer cells can adapt molecular and cellular mechanisms to evade the effects of the drug, often evolve into more aggressive or metastasis phenotypes, and limit the success of monotherapies [ 28 ].

Combination therapies have shown great potential for cancer treatment reducing monotherapy's defects [ 29 , 30 , 31 ]. They improve treatment outcomes, lead to synergistic anticancer effects, overcome clonal heterogeneity, and reduce drug resistance probability [ 32 , 33 , 34 ]. However, it's challenging to identify an effective combination [ 21 , 35 ]. Combination therapy can also lead to side effect accumulation. They may include the sum of each drug's known side effects or completely unexpected side effects caused by drug-drug interactions [ 17 , 36 ]. The treatment-related adverse events resulting from combination therapy had led to dose reduction or discontinuation reported in several studies [ 37 , 38 , 39 , 40 , 41 ]. Therefore, computational methods are employed to predict the right combinations for cancer treatment [ 42 , 43 ]. In comparison, the MTDLs are constructed of a single compound designed for multifunctional properties with fewer side effects and more predictable toxicity. Also, combination therapies indicated higher utility values than monotherapies but they were generally more expensive [ 38 ]. While the MTDLs can modulate multiple targets simultaneously making it cost-effective as monotherapies and high efficacy as combination therapies in administration for patients [ 44 ]. Additionally, the potential for useful drug combinations is restricted by the risk of side effects, drug interactions, and technological challenges in obtaining stable pharmaceuticals. However, in theory, the number of useful combinations is unlimited if the molecular structure is properly selected and optimized. Also, in practice, it is most feasible to obtain ligands based on two to five pharmacophores [ 45 ]. Moreover, the drugs regimen of a combination therapy can indicate different absorption and distribution profiles which can affect the treatment outcomes. Also, administering doses or timings for agents of a combination treatment regimen with different half-lives is also challenging [ 46 ]. In contrast, using the computational approaches in designing MTDLs provides more predictable PK & physicochemical features resulting in more desirable ADMET profile of designed drugs [ 47 ]. In addition, it's generally easier to optimize the dose for a multi-targeted ligand than to do so separately for the components of the combination therapy regimen. Lastly, the clinical trial approval in a combination therapy requires each drug to be investigated separately, and then in combination with each other which is cost- and time-consuming while, MTDLs are time- & cost-efficient for clinical trials since a single compound is involved in the study [ 45 ].

Overall, multi-targeted treatments, specifically MTDLs, can provide several benefits in cancer treatment leading to improved overall survival with decreased side effects for cancerous patients.

Strategies for developing MTDLs

The methods commonly used to develop MTDLs can be classified into two categories. The first category involves a random screening approach, while the second category utilizes a knowledge-based approach to combine scaffolds from different active molecules with known activity against a particular target. This latter approach is referred to as the framework combination approach [ 48 ].

Random screening involves using quantitive structure–activity relationship (QSAR) and/or virtual screening to discover an anti-cancer agent. QSAR serves as a valuable tool for uncovering the interplay between structure and activity within smaller congeneric compound series and enables the understanding of physicochemical and biological properties of the molecules for further targeting in cancer treatment [ 49 , 50 ]. On the other hand, virtual screening allows for the docking of thousands or even millions of compounds to bind to proteins associated with cancer in a relatively cost-effective way. By doing so, it can help in the discovery of potential inhibitors for specific proteins or entire signaling pathways involved in the development of cancer [ 51 ].

The framework combination approach is a knowledge-based method to discover multi-target drugs by combining drugs/pharmacophores for developing a new hybrid molecule with the desired activity toward multiple targets [ 52 ]. The molecular components or individual partners can come together covalently to form a molecular matrix, by fusing, merging, or linking [ 53 ].

The fused strategy combines two or more distinct biologically active pharmacophoric moieties, usually via a zero-length linker or a spacer, to form a new molecular hybrid [ 54 ]. While merged strategy involves merging pharmacophores into one molecule, resulting in the development of a unique, smaller chemical compound with retained pharmacological properties but notably different chemical traits [ 55 ]. The combined agents might hold onto the functional properties of one or both of the overlapping drugs [ 54 ]. Also, this strategy can lead to a resulting compound with reduced molecular weight compared to fusing/linking strategies employment [ 56 ]. Furthermore, the merging requires in-depth knowledge about the side chains interaction and the conformation that affects the compound function while the linking strategy is simpler [ 57 ]. The linking is the binding of two compounds that bind within their pharmacophores together through a linker (cleavable or not) to obtain a new compound capable of aiming multiple targets at the same time [ 58 , 59 ]. For example, trastuzumab emtansine, an FDA-approved drug [ 60 ], is an MTDL that linked an anti-HER2/neu antibody with emtansine (a microtubule inhibitor) through MCC (4-[N-maleimidomethyl]cyclohexane-1-carboxylate) linker [ 45 ]. Furthermore, the linking strategy also makes designing a variety range of hybridizations possible in comparison to the merge & fused strategies [ 61 ]. Although these strategies are utilized prevalently in neurological disorders, their principles are also applied in developing anticancer agents to achieve more efficient treatments [ 45 , 54 ]. Last, a schematic overview of MTDLs deigning strategies is depicted in Fig.  1 .

The schematic view of strategies that have been utilized to develop multi-target anti-cancer agent to combat cancer

Current cancer multi-target therapeutics

The current cancer treatments mostly target receptor tyrosine kinases (RTKs). They're transmembrane receptors that play a role in many cellular processes, including growth, differentiation, and metabolism [ 62 ]. RTKs are key regulators of cancer cell growth and metastasis. Dysregulation of RTK signaling can lead to a variety of human diseases, including cancer [ 15 , 63 ]. Its alterations are common in a wide variety of cancers, highlighting their importance in cancer progression and making them promising therapeutic targets [ 64 ].

The JAK/STAT signaling pathway is a critical player in cancer treatment and multi-target therapy, with abnormal activation observed in various solid malignancies such as breast, lung, liver, head and neck, and stomach cancers [ 65 ]. This heightened JAK/STAT signaling has been associated with poorer prognoses, including increased recurrence rates and reduced overall survival [ 66 ]. Consequently, targeting this pathway holds promise for therapeutic interventions in cancer, showing efficacy in modulating the progression of solid tumors [ 67 ]. In summary, the JAK/STAT signaling pathway presents substantial therapeutic opportunities and is a key focus for multi-target therapy in solid malignancies.

The NF-κB pathway is another crucial regulator facilitating communication between inflammation and cancer at various levels [ 68 ]. Activation of NF-κB leads to the induction of several target genes, including those that promote cell proliferation and inhibit apoptosis [ 69 ]. Additionally, NF-κB signaling interacts with multiple other pathways, such as STAT3, AP1, interferon regulatory factors, NRF2, Notch, WNT–β-catenin, and p53. Notably, all recognized hallmarks of cancer involve NF-κB activation [ 70 , 71 ]. Alterations in the NF-κB pathway are frequently observed in both solid and hematopoietic malignancies, promoting tumor cell proliferation and survival [ 72 ]. Excessive activation of the NF-κB-signaling pathway has been documented in various tumor tissues, making research on this pathway for targeted cancer therapy a significant area of interest [ 73 ]. Studies have shown that inhibition of NF-κB, either by knocking out RelA or IKK2 or by overexpressing a dominant negative form of IκBα, significantly reduces tumor volume, lowers tumor grade, and prolongs survival in mouse models [ 71 , 74 , 75 ].

An overview of these three pathways and their involvement in cancer development, progression, and overall survival is depicted in Fig.  2 . Next, we will further review multi-target drugs in cancer treatment.

Overview of activation/inhibition of receptor tyrosine kinase (RTK)/Phosphoinositide 3-kinases (PI3K)/Akt, JAK/STAT, and nuclear factor κB (NF-κB) pathways. These pathways involve tumor cell survival, proliferation, differentiation, metabolism, apoptosis, and protein synthesis. Due to their function, these receptors and downstream molecules have been targeted over decades to increase overall survival and tumor progression inhibition. The receptors are named as an example because each signaling pathway's initiating receptors contain a variety of receptors. The black boxes describe the anticancer small molecules with their respective targets. They act through inhibiting the activity of proteins/molecules which are involved in cancer development and progression. Vascular endothelial growth factor receptor (VEGFR), G-protein coupled receptors (GPCR), epidermal growth factor receptor (EGFR), and tumor necrosis factor receptor (TNFR)

Imatinib (Glivec) is a first-generation multi-targeted tyrosine kinase inhibitor (TKI) that received Food and Drug Administration (FDA) approval in 2001 for malignant metastatic or unresectable gastrointestinal stromal tumors (GISTs) [ 76 ]. It's a 2-phenyl amino pyrimidine derivative that has been used to treat chronic myeloid leukemia (CML), and advanced anaplastic thyroid cancer [ 77 , 78 ]. Imatinib acts by inhibiting Bcr-Abl, c-KIT, and platelet-derived growth factor (PDGF) tyrosine kinase activity through binding to their ATP-binding site [ 78 , 79 , 80 ]. According to the Fu et al. study [ 81 ], it indicated that imatinib's most adverse events include skin color change (55.6%) and edema (38.9%). The drug resistance related to imatinib was lysosomal sequestration that affects its target site concentration [ 82 ]. Furthermore, it has been suggested that glucose transporter (GLUT)-1 is involved in the acquisition of imatinib resistance by GIST cells, which can be overcome by combining WZB117 & imatinib [ 83 ].

Lapatinib is a first-generation quinazoline based TKI that inhibits epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) reversibly [ 15 , 84 , 85 , 86 ]. HER2 overexpression has been observed in 20 to 30% of breast cancers which is related to more aggressive disease and higher mortality [ 87 ]. Also, the overexpression of EGFR was observed in 16–36% of breast cancer cases [ 88 ]. The FDA approved lapatinib in March 2007 for treating advanced or metastatic breast cancer patients with overexpression of HER2 [ 89 ].

The lapatinib clinical trials were conducted on hormonally untreated prostate and metastatic urothelial bladder cancer, but there was no reported significant antitumor activity [ 90 , 91 ]. In a phase II trial, the treatment with lapatinib did not show significant efficacy in inducing tumor regression for non-small cell lung cancer (NSCLC) in 75 patients studied [ 92 ]. On the other, lapatinib therapy is associated with a significant reduction in various forms of pain, including musculoskeletal pain, headache, bone pain, and pain in extremities, in cancer patients [ 93 ]. Its resistance is associated with a widespread reprogramming of glycolysis, which is mediated by phosphorylation and is accompanied by changes in metabolites and increased sensitivity to glycolysis inhibition [ 94 ]. The most common toxicities are diarrhea and rash, which are mostly mild to moderate in severity [ 95 ]. In most cases, symptoms are mild and do not lead to drug discontinuation [ 96 ].

Sorafenib as the first oral multi-kinase inhibitor was approved by the FDA for the treatment of patients with advanced renal cell carcinoma (RCC) in 2005, advanced unresectable hepatocellular carcinoma (HCC) in 2007, and advanced radioiodine-refractory differentiated thyroid carcinoma in 2013 [ 97 , 98 , 99 ]. It inhibits the activity of the serine-threonine kinases Raf-1 and B-Raf, the receptor tyrosine kinase activity of vascular endothelial growth factor receptor (VEGFR)-1/2/3, platelet-derived growth factor receptor β (PDGFR-β), c-Kit, RET, and FLT3 [ 15 , 100 , 101 ]. Several studies have reported diarrhea, hand-foot syndrome, rash, and fatigue as the most common adverse events related to sorafenib treatment [ 102 , 103 , 104 ]. Also, drug discontinuation due to intolerance or toxicities was responsible for 16% of cancerous patients [ 104 ]. Tumor cells could exhibit primary resistance or acquired resistance. In primary resistance, patients have low response rates at the initial treatment with sorafenib and gene polymorphism may play a crucial part in regulating the function of sorafenib. Many factors such as intratumor genetic heterogeneity may induce acquired resistance following sorafenib treatment, thus other treatment options should be provided [ 105 ]. HCC cell's metabolic characterization changes are also associated with their resistance to sorafenib and can be overcome by combination with aspirin [ 106 ].

Pazopanib, an oral second-generation TKI, has been approved by the FDA (2009) for RCC and soft tissue sarcoma treatment [ 107 , 108 ]. Preclinical studies have suggested that pazopanib inhibits both angiogenic and oncogenic signaling pathways by VEGFR, PDGFR, fibroblast growth factor receptor (FGFR), and c-Kit inhibition [ 109 ]. It downregulates the mitogen-activated protein kinase (MAPK) signaling pathway through the inhibition of pan-RAF [ 110 ]. Interestingly, in a phase I study, 58% of patients demonstrated > 50% reduction in tumor blood flow at Day 8 of treatment, which increased to 91% at Day 22 [ 111 ]. Pazopanib is associated with several adverse effects, with hypertension as the most common one, followed by cytopenia, proteinuria, prolonged QT interval, elevated liver enzymes, diarrhea, nausea, and fatigue [ 103 , 111 , 112 ]. Pazopanib has indicated significant potential as a treatment option for NSCLC [ 113 ], breast cancer, urothelial carcinoma [ 114 ], thyroid cancer [ 115 ], and GIST [ 116 ].

Sunitinib, an oral multikinase inhibitor, received first approval from the FDA in January 2006 for treating advanced RCC. Subsequently, it has gained global approval for this use as well as for treating GISTs and advanced pancreatic neuroendocrine tumors in patients who are resistant or intolerant to imatinib [ 117 ]. It also has shown potential antitumor activity in various other malignancies, such as thyroid, lung, bladder, pancreatic, and esophageal carcinomas, gliomas, and sarcomas [ 118 ]. Sunitinib exerts its anti-angiogenesis effect by inhibiting RTKs including EGFR, FGFR-1, PDGFR-β, VEGFR-1/2/3, RET, FLT3, KIT, and CSF1R through competitive binding to their adenosine triphosphate (ATP) pocket [ 15 , 119 , 120 ]. In 2011, it was approved by the FDA for the second time to treat progressive, well-differentiated pancreatic neuroendocrine tumors [ 121 ]. There are a few ways to the drug resistance of sunitinib. One of them is autophagy-flux-associated sunitinib lysosomal sequestration which leads to the isolation of the drug from the cytoplasm in endoplasmic cells [ 122 ]. It also can promote epithelial-mesenchymal transition (EMT) in metastatic RCC cells, leading to resistance to sunitinib treatment [ 123 ]. Moreover, the most common side effects include diarrhea, nausea, asthenia, and fatigue which many studies focused on managing the drug's related drug resistance [ 121 , 124 , 125 ].

This second-generation TKI is another quinazoline-based orally active small molecule that exhibits potent inhibitory activity against multiple targets, including VEGFR-2 and -3, EGFR, and the rearranged during transfection (RET) receptors [ 126 , 127 ]. Vandetanib significantly disrupts the EGFR-induced production of angiogenic growth factors, leading to an "indirect" impact on angiogenesis in vivo [ 128 ]. The FDA approved vandetanib in April 2011 for symptomatic or progressive medullary thyroid cancer (MTC) in patients with unresectable locally advanced or metastatic disease treatment [ 129 ]. The common adverse events are reported diarrhea, hypertension, QTc prolongation, and fatigue [ 130 ]. Among these, QTc prolongation which significantly increased during treatment with vandetanib should be well-considered due to its life-threatening effect [ 131 ]. Genetic alterations, including DNA mutations and epigenetic modifications, contribute to the resistance of medullary thyroid carcinoma to tyrosine kinase inhibition. To overcome this resistance, a potential strategy involves targeting these genetic alterations by adding further therapeutic agents [ 132 ].

Axitinib is a second-generation targeted drug that selectively inhibits VEGFR 1, 2, and 3 tyrosine kinase activity [ 133 ]. It was first recommended for FDA approval by the Oncology Drug Advisory Committee (ODAC), and full approval was granted in January 2012 for the treatment of patients with advanced RCC [ 134 , 135 ]. This antiangiogenic drug improved the overall survival of patients with head and neck squamous cell carcinoma [ 136 ]. The common expected side effects of this indazole-based agent are hypertension (16%), fatigue (11%), and diarrhea (11%) [ 137 , 138 ]. Another point to be considered is to monitor proteinuria before initiation and periodically during treatment. So if moderate to severe proteinuria develops, the dose is reduced or even temporarily the treatment stops [ 134 ]. Generally, the majority of side effects are manageable with supportive care and dose modification [ 139 ]. So far, there has been no report of drug resistance to this drug.

Cabozantinib

It's a second-generation multi-targeted TKI with inhibitory effects against C-mesenchymal-epithelial transition factor (C-MET), VEGFR2, RET, KIT, AXL, and FLT3, all of which play a role in the pathogenesis of liver cancer [ 140 ]. Cabozantinib was approved by the FDA for advanced RCC (2016) [ 141 ], HCC (2019) [ 142 ], and differentiated thyroid cancer (2021) [ 143 ]. It provides a substantial clinical advantage over sunitinib when used as the first-line therapy for patients with metastatic RCC [ 144 ]. Additionally, sunitinib-induced resistance can be overcome using cabozantinib in the treatment of RCC [ 145 ]. Furthermore, HCC cells overexpressed C-MET up to 40% [ 146 ], and tumor cells with low C-MET levels exhibited primary resistance to C-MET inhibitors such as cabozantinib. However, rational combinations show the potential to overcome this resistance [ 147 ]. The most reported side effects associated with the treatment were hypertension, fatigue, and diarrhea [ 148 ].

Regorafenib

Regorafenib is a sorafenib-derived, multitargeted kinase inhibitor approved by the FDA in 2017. This second-generation TKI has demonstrated beneficial effects in the treatment of advanced HCC, metastatic colorectal cancer, and GISTs [ 149 ]. The drug targets RAS/RAF/MEK/ERK pathway by inhibiting the VEGFR, PDGFR, FGFR, KIT, and RET [ 150 , 151 ]. It also can suppress AXL signaling, inhibit STAT3, and promote cell death in triple negative breast cancer [ 152 ]. Moreover, the colon cancer cell's growth and survival can be affected by regorafenib-induced generation of reactive oxygen species and synergistically enhanced oxaliplatin-induced cell growth inhibition [ 153 ]. Regorafenib's effectiveness and safety have been demonstrated in several studies. It increases patient survival and disease progression prevention which is more appealing than sorafenib due to its greater potential for RTK inhibition [ 154 ]. Also, a clinical study by Pavlakis et al. indicated regorafenib potential in the treatment of refractory advanced gastro-oesophageal cancer [ 155 ]. The side effects consist of hand-foot skin reaction, hypertension, and fatigue [ 156 ]. It has shown that HCC patients with higher topoisomerase IIα expression had shorter overall survival, but its inhibition reverses drug resistance to regorafenib [ 151 ].

Lorlatinib is a multi-target drug and a third-generation tyrosine kinase inhibitor that can target anaplastic lymphoma kinase (ALK) and ROS1 [ 157 ]. Besides common side effects of lorlatinib including hypercholesterolemia, hypertriglyceridemia, edema, weight gain, and peripheral neuropathy [ 158 ], it has been approved twice by the FDA. The first one was in November 2018 for previously treated ALK-Positive metastatic NSCLC [ 159 ]. In March 2021, loratinib (brand name Lorbrena) was approved for the second time by the FDA for first-line treatment of patients with metastatic ALK-positive NSCLC [ 160 ]. The results of a clinical trial involving 296 patients compared the effectiveness of lorlatinib versus crizotinib. The findings indicated that lorlatinib offers advantages over crizotinib and supports its use for patients with or without baseline brain metastases [ 161 ]. Lorlatinib resistance can be caused by various mechanisms, such as ALK rearrangement in NSCLC. To overcome the resistance, some combinations such as combination with gilteritinib has been shown promising effects in silico in ALK-positive lung cancer cells [ 162 ].

Lenvatinib is an FDA-approved (2018) TKI drug for the treatment of RCC, unresectable or advanced HCC, and radioactive iodine-refractory differentiated thyroid cancer [ 163 ]. It has been investigated due to its therapeutic effects in advanced endometrial cancer [ 164 ], adenoid cystic [ 165 ], medullary thyroid [ 166 ], and anaplastic thyroid carcinomas [ 167 ]. In a comparative clinical study, lenvatinib demonstrated similar overall survival to sorafenib in untreated advanced HCC as a first-line treatment [ 168 ]. Lenvatinib prevents tumor angiogenesis through inhibition of VEGFR-1, -2, and -3, and also blocks the proliferation of tumor cells through inhibition of FGFR-1, FGFR-2, FGFR-3, & FGFR-4, PDGFRα, RET, and c-KIT [ 169 ]. Hypertension, fatigue, weight loss, diarrhea, and nausea are the most reported adverse effects of this medication [ 170 ]. The acquired resistance with administration of lenvatinib in advanced HCC may be caused by increased activation of EGFR and insulin-like growth factor 1 receptor (IGF1R)/insulin receptor (INSR) [ 171 ]. To potentially overcome or delay resistance to the anti-tumor effects of lenvatinib, combining multiple drugs to simultaneously inhibit different angiogenic pathways could be a promising future strategy [ 172 ].

Entrectinib

It's an orally active, small-molecule TKI for tropomyosin receptor kinases (TRK)-A/B/C, ROS1, and ALK that can cross the blood–brain barrier (BBB) [ 173 , 174 ]. Entrectinib received breakthrough and priority designations from the FDA (in August 2019) and European Medicines Agency (EMA) for the treatment of neurotrophic tyrosine receptor kinase (NTRK)-positive solid tumors in adults and children with no standard options as well as adults with ROS1 + NSCLC [ 173 ]. This second-generation agent has a significant potential for treating primary and metastatic central nervous system (CNS) tumors with no adverse off-target activity [ 175 ]. The most studied adverse effects include fatigue, paresthesia, dysgeusia, myalgia, and nausea [ 176 ]. A study described a rare entrectinib resistance mechanisms in ROS1-rearranged NSCLC [ 177 , 178 ]. Another study by Russo et al. [ 179 ] analyzed NTRK1 mutations that drive resistance to TRK Inhibitors. However, further assessments are also required for the occurrence percentage of the mutations.

Last, Table  1 summed up the drugs that have been described above with their respective details. Also, the drug's FDA approval timeline has been depicted in Fig.  3 . It shows that FDA-approved multi-target drugs had an upward trend which indicates their effectiveness and as a result, scientist's interests. In addition, the approval of these multi-target drugs by the FDA further underscores the potential of multi-target therapies in enhancing the outcomes of cancer treatment. Noteworthy, as it obvious most of multi-target drugs that developed in recent years are multi TKI, however, targeting novel biomarkers and different pathways at the same time using MTDLs approach would be a great opportunity to overcome RTK-induced resistance in cancerous cell [ 7 , 180 , 181 ].

The timeline of multi-targeted drugs with their FDA approval history. Chronic myeloid leukemia (CML), renal cell carcinoma (RCC), gastrointestinal tumors (GISTs), hepatocellular carcinoma (HCC), medullary thyroid cancer (MTC), non-small cell lung cancer (NSCLC).

Potential targets for development of a novel multi-target cancer treatment

Colorectal, lung, and prostate cancers are among the leading causes of cancer-related deaths in the United States [ 182 ]. Therefore, targeting the potential biomarkers in these three prevalent malignancies can result in more effective multi-target agents leading to a reduction in the cancer population worldwide. Below, the potent targets (i.e. highly expressed markers or markers with the expression limited to tumor cells) have been introduced with their respective role in cancer development, progression, and survival.

The NSCLC is a heterogeneous malignancy that accounts for ∼ 85%–87% of all lung cancers [ 183 ], which is the leading cause of cancer-related deaths worldwide [ 184 ]. The statistics recorded 1.28 million new NSCLC cases from 2010 to 2017 in United States [ 185 ].

Several proteins have been found to play crucial roles in NSCLC's development, progression, and survival. One of these markers is EGFR with an overexpression between 40–80% in advanced NSCLC patients [ 186 ]. This receptor is a member of the ErbB family that can initiate and progress the NSCLC by regulating both apoptosis and cell proliferation [ 184 , 187 ]. The HER2, another ErbB family member, indicates RTK activity with an overexpression range of 2.4% to 38% [ 188 ]. Moreover, the overexpression of RTK's downstream signaling pathway molecules including phosphorylated-Akt (p-Akt) and -mTOR (p-mTOR) was observed in 78% & 46.7% of NSCLC patients, respectively [ 189 ]. The RTK/Ras/PI3K/Akt pathway promotes oncogenesis by affecting cell proliferation & growth, apoptosis, and angiogenesis, so its inhibition could be beneficial for patients [ 190 ]. In addition, the eukaryotic translation initiation factor 4E (eIF4E) is a protein with a crucial role in the initiation of protein synthesis [ 191 ]. The phospho-eIF4E expression has been found to correlate with p-Akt indicating that eIF4E activation plays a crucial role in the NSCLC progression and its upregulation has been found in 39.9% of NSCLC-diagnosed patients [ 189 ].

The AIB1 is a known potent transcriptional coactivator of estrogen receptor α that functions through direct contact with the nuclear receptor, and the overexpression (in 48.3% cases) is associated with shortened patient survival and acts as a biomarker for NSCLC patients with poor prognosis [ 183 , 192 ]. The C-MET alterations are also associated with NSCLC's poor prognosis and its expression upregulates in 25–75% of diagnosed cases [ 193 ]. It is responsible for the drug resistance in most of lung cancerous cells [ 194 ]. Another tumor marker that mediates critical processes for cancer progression, such as migration, cell adhesion, and tumorigenesis is osteopontin. Its expression rate in tumor cells is 67.8%, while only 20.2% of normal lung tissues express this oncogenic protein [ 195 , 196 ].

A study by Maeda et al. [ 197 ] found that carcinoembryonic antigen has the potential for targeting NSCLCs with a high level of expression (in ~ 35–60% cases) and is involved in tumor cell proliferation, adhesion, and migration [ 198 ]. The junction adhesion molecule (JAM)-A is a protein expressed on endothelial-, epithelial-, and immune cells as well as platelets [ 199 ]. The high expression of JAM-A occurred in 37% of NSCLC in comparison to the normal tissues which significantly correlates with TNM stage, lymph node metastasis, and a decrease in overall survival [ 200 ].

Prostate cancer

Prostate cancer (PC) is the second cause of death and the first place of new cases of cancer in the United States among males [ 201 ]. The growth of prostate tumors is dependent on androgens [ 202 ] and about 80–90% of cases rely on androgens at the initial diagnosis [ 203 ]. Furthermore, the CUB domain-containing protein 1 (CDCP1) is a transmembrane protein that serves as a substrate for SRC family kinases and can cause tumor progression [ 204 ]. It's found to be overexpressed in approximately 50% of metastatic biopsies and around 30% of primary tumors [ 205 ].

The remodeling and spacing factor 1 (RSF1) protein has also been suggested to contribute to cancer progression, as its expression levels have been found to increase in more advanced pathological stages, lymph node metastasis, higher Gleason scores, and increased tumor cell proliferation [ 206 , 207 ]. Detectable levels of RSF1 expression were observed in 79.2% of the 16,456 interpretable PC studied [ 206 ]. The prostate tumor overexpressed-1 (PTOV1) is a protein with 80% overexpression in patients with prostate intraepithelial neoplasia, and it's linked to prostate cancer progression. It also accumulates and alters the cancer cell's biological behavior [ 208 ]. A protein from the G-protein coupled receptors (GPCRs) family called prostate-specific GPCR 2 (PSGR2) is a receptor whose expression is restricted to human prostate tissue and exhibits distinct expressions in normal and tumor tissues [ 209 ]. The overexpression of this protein in normal and tumor tissues has a significant difference (62% of examined patients) [ 210 ].

The receptor-interacting protein kinase 2 (RIPK2) is also a predictive marker and can influence disease progression [ 211 ]. It's gained or amplified in approximately 65% of lethal metastatic castration-resistant PC and can stabilize the c-Myc transcription factor [ 212 ]. Caveolin-1 is a membrane protein highly expressed in PC and it's associated with disease progression, castration resistance, and biochemical recurrence [ 213 ]. Out of 197 cases of prostate cancer in the Chen et al. study, 111 cases were reported caveolin-1 positive (56.35%) [ 214 ]. Furthermore, elevated levels of fibroblast growth factor 8 (FGF8) in PC have been linked to reduced patient survival rates, and this association remains present even in cases of androgen-independent disease. Around 50% of clinically localized human PC express increased FGF8, while 80% or more of advanced cancers express increased FGF8 [ 215 ]. Trefoil factor 3 (TFF3) has the ability to activate ERK1/2, a crucial element of the MAPK signaling pathway. This activation ultimately leads to the promotion of tumor cell proliferation [ 216 ]. The studies have reported that TFF3 overexpression is observed in over 40% of PC cells [ 217 , 218 ].

Colorectal cancer

Colorectal cancer (CRC) is the third most frequently diagnosed and the second most fatal cancer for both males and females [ 219 ]. Globally, there is a rise in the occurrence of CRC among young adults [ 220 ]. These facts highlight the importance of new potential and novel targets for the development of anti-CRC therapeutic agents. The coiled-coil domain containing 34 (CCDC34) is a protein whose overexpression is related to CRC apoptosis reduction and metastasis enhancement and is thought to be affected via survivin, Bcl-2, N-cadherin, and E-cadherin regulation. The protein-positive rate is reported in 74.12% of patients' tissues [ 221 ]. The G-protein-coupled prostaglandin E receptor 2 (PTGER2) is a receptor that plays a crucial role in the CpG island methylator phenotype (CIMP), tumoral microsatellite instability (MSI), and survival. Out of the 516 colorectal cancers that were studied, PTGER2 overexpression was found in 169 tumors, which accounts for 33% of the total [ 222 ]. Furthermore, numerous studies have demonstrated the involvement of cyclin B1 in cancer cell differentiation, growth, apoptosis, and resistance to chemotherapy [ 223 , 224 , 225 , 226 ]. In 88% of the patients with CRC, cyclin B1 was found to be overexpressed compared to the non-neoplastic colorectal mucosa cells [ 225 ].

Mutations that deactivate the adenomatous polyposis coli (APC) gene and result in the increased activity of the Wnt signaling pathway play a crucial role in initiating the development of CRC and its progression [ 227 , 228 ]. The APC-related mutations account for approximately 80% of CRC cases [ 229 ]. This evidence indicates the potential of targeting the Wnt signaling pathway. The overexpression of TP53 protein (TP53 +), which is involved in lymphatic and vascular invasion, is detected in 53% of stage III CRC patients [ 230 , 231 ]. It also has been investigated that adjuvant chemotherapy benefit in stage III CRC is restricted to cases with low-level TP53 protein expression [ 231 ]. Moreover, the serine/arginine-rich splicing factor 3 (SRSF3) is another potential target that its high expression is associated with cell proliferation, migration, invasion, and metastasis [ 232 ]. The SRSF3 has been reported to be negative or weakly positive in 80% of patients with metastatic stage IV colorectal cancer, which was markedly related to poor survival, so it's not a good aim for advanced CRC patients [ 233 ]. But overall, the percentage of SRSF3 overexpression in CRC has been reported to be approximately 70.6% which makes it favorable especially in earlier stages [ 234 ].

The introduced potential biomarkers for developing new anti-cancer MTDLs can be targeted whether in inhibition of the exact protein or gene downregulation. Lastly, the above mentioned malignancies with their respective biomarkers & overexpression percentages are depicted in Fig.  4 .

The NSCLC, colorectal, and prostate introduced selected proteins with their respective overexpression percentage as potential targets for drug development. These targets introduced due to their high overexpression which make them more desirable agents for cancer treatment

Future perspective

According to the 2020 statistics, there were approximately 19.3 million new cases of cancer and 10 million cancer-related deaths worldwide [ 1 ]. This indicates the emergence of exploration of the complexities and drug resistance associated with this disease. In recent decades, treatments have focused on targeting therapy which started with monotherapy and continued with combination therapies, and in recent years multi-targeted therapy has been introduced to find novel and more effective cancer treatments.

Polypharmacology involves the design and utilization of pharmaceutical agents that can act on multiple targets or disease pathways. This approach offers the potential to develop more effective drugs by specifically modulating multiple targets. Recent advancements in the computational biology approach lead to AI-based tools development for generating small molecules in silico more precisely with the employment of deep learning/reinforcement learning methods [ 235 , 236 , 237 ]. These web servers paved the way for the de novo design of molecules by providing knowledge-based machine learning algorithms, so drugs with more efficacy and lower toxicity become more achievable for both experts and non-experts. Then, designed small molecules can be further optimized by their affinity to selected targets using molecular docking web servers such as NeuralDock [ 238 ]. Also, for similairization of real-world interaction between ligands and their respective targets, molecular dynamics can be performed. Apart from these applications, AI-based methods provide opportunities for drug repurposing which are helpful in designing a multi-target drug [ 239 , 240 ]. In addition, the identification of protein's structure and function which is necessary in the process of in silico drug designing has been facilitated by single crystal X-ray diffraction (SC-XRD), nuclear magnetic resonance (NMR), and cryo-electron microscopy (Cryo-EM) methods. On the other hand, the current multi-targeted agents are focused on small molecule deployment while the potency of peptide-based multi-targeted drugs has not been well-considered. Peptides offer significant therapeutic potential due to their high binding affinities, selectivity, specificity, and efficacy. They can also bind to surfaces, making them useful for targeting "undruggable" targets. Additionally, the diverse side chains in peptides provide a wide range of potential therapeutic targets.

All in all, MTDLs offer promising opportunities for targeting complex diseases such as cancer, either in small molecules or peptide conformations, and should be considered in hard-to-treat malignancies.

Cancer treatment has become more necessary in recent years due to high rate of cancer cases worldwide. Also, treatment-induced drug resistance is another challenge that had an upward trend in recent years. These highlight the emergence of developing novel treatment strategies for combating cancer more effectively to overcome drug resistance. In the last decades, scientists moved on from monotherapy to combination therapy and recently multi-targeted agents due to the promised application provided by multi-target drugs. Moreover, the traffic of FDA-approved multi-targeted therapeutics after 2010 indicates the interest of researchers in this field. However, there are challenges in multi-target drug development such as PK/PD predictability. Recent advancements in computational biology unlocked new tools for designing hybrid compounds capable of targeting different biomarkers synergistically with desired PK features. Despite the progress in computational biology, the knowledge of drug designers is really important because the employment of these tools is not solely sufficient for achieving more effective drugs with favorable outcomes. Overall, polypharmacology, especially MTDLs, indicates reliable potential for overcoming cancer resistance.

Availability of data and materials

No datasets were generated or analysed during the current study.

Abbreviations

Deoxyribonucleic acid

Estrogen receptor

Multi-target directed ligand

Absorption, distribution, metabolism, excretion, and toxicity

Pharmacokinetic

Pharmacodynamics

Receptor tyrosine kinase

Tyrosine kinase inhibitor

Food and Drug Administration

Gastrointestinal stromal tumor

Chronic myeloid leukemia

Platelet-derived growth factor

Epidermal growth factor receptor

Human epidermal growth factor receptor 2

Hepatocellular carcinoma

Renal cell carcinoma

Vascular endothelial growth factor receptor

Platelet-derived growth factor receptor

Fibroblast growth factor receptor

Mitogen-activated protein kinase

Non-small cell lung cancer

Epithelial-mesenchymal transition

Medullary thyroid cancer

Rearranged during transfection

Oncology Drug Advisory Committee

C-mesenchymal-epithelial transition factor

Anaplastic lymphoma kinase

Insulin-like growth factor 1 receptor

Insulin receptor

Tropomyosin receptor kinases

Blood-brain barrier

European Medicines Agency

Neurotrophic tyrosine receptor kinase

Phosphorylated-Akt

Phosphorylated-mTOR

CUB domain-containing protein 1

Remodeling and spacing factor 1

G-protein coupled receptors

Prostate-specific G-protein coupled receptor 2

Prostate tumor overexpressed-1

Receptor-interacting protein kinase 2

Fibroblast growth factor 8

Trefoil factor 3

Coiled-coil domain containing 34

G-protein-coupled prostaglandin E receptor 2

CpG island methylator phenotype

Microsatellite instability

Adenomatous polyposis coli

Serine/arginine-rich splicing factor 3

Single crystal X-ray diffraction

Nuclear magnetic resonance

Cryo-electron microscopy

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The authors would like to express their gratitude to the Cancer Research Center, Semnan University of Medical Sciences, Semnan, Iran.

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Doostmohammadi, A., Jooya, H., Ghorbanian, K. et al. Potentials and future perspectives of multi-target drugs in cancer treatment: the next generation anti-cancer agents. Cell Commun Signal 22 , 228 (2024). https://doi.org/10.1186/s12964-024-01607-9

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Nonalcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis: Current Issues and Future Perspectives in Preclinical and Clinical Research

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• DOI: 10.3390/ijms21249646

Nonalcoholic fatty liver disease (NAFLD) is a continuum of liver abnormalities often starting as simple steatosis and to potentially progress into nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma. Because of its increasing prevalence, NAFLD is becoming a major public health concern, in parallel with a worldwide increase in the recurrence rate of diabetes and metabolic syndrome. It has been estimated that NASH cirrhosis may surpass viral hepatitis C and become the leading indication for liver transplantation in the next decades. The broadening of the knowledge about NASH pathogenesis and progression is of pivotal importance for the discovery of new targeted and more effective therapies; aim of this review is to offer a comprehensive and updated overview on NAFLD and NASH pathogenesis, the most recommended treatments, drugs under development and new drug targets. The most relevant in vitro and in vivo models of NAFLD and NASH will be also reviewed, as well as the main molecular pathways involved in NAFLD and NASH development.

Keywords: hepatocellular carcinoma; metabolic syndrome; non-alcoholic fatty liver disease; steatohepatitis; steatosis.

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Alternative routes into clinical research: a guide for early career doctors

• Related content
• Peer review
• Phillip LR Nicolson , consultant haematologist and associate professor of cardiovascular science 1 2 3 ,
• Martha Belete , registrar in anaesthetics 4 5 ,
• Rebecca Hawes , clinical fellow in anaesthetics 5 6 ,
• Nicole Fowler , haematology clinical research fellow 7 ,
• Cheng Hock Toh , professor of haematology and consultant haematologist 8 9
• 1 Institute of Cardiovascular Sciences, University of Birmingham, UK
• 2 Department of Haemostasis, Liaison Haematology and Transfusion, University Hospitals Birmingham NHS Foundation Trust, Birmingham
• 3 HaemSTAR, UK
• 4 Department of Anaesthesia, Plymouth Hospitals NHS Trust, Plymouth, UK
• 5 Research and Audit Federation of Trainees, UK
• 6 Department of Anaesthesia, The Rotherham NHS Foundation Trust, Rotherham Hospital, Rotherham
• 7 Department of Haematology, Royal Cornwall Hospitals NHS Trust, Treliske, Truro
• 8 Liverpool University Hospitals NHS Foundation Trust, Prescott Street, Liverpool
• 9 Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool
• Correspondence to P Nicolson, C H Toh p.nicolson{at}bham.ac.uk ; c.h.toh{at}liverpool.ac.uk

Working in clinical research alongside clinical practice can make for a rewarding and worthwhile career. 1 2 3 Building research into a clinical career starts with research training for early and mid-career doctors. Traditional research training typically involves a dedicated period within an integrated clinical academic training programme or as part of an externally funded MD or PhD degree. Informal training opportunities, such as journal clubs and principal investigator (PI)-mentorship are available ( box 1 ), but in recent years several other initiatives have launched in the UK, meaning there are more ways to obtain research experience and embark on a career in clinical research.

Examples of in-person and online research training opportunities

These are available either informally or formally, free of charge or paid, and via local employing hospital trusts, allied health organisations, royal colleges, or universities

Research training opportunities

Mentorship by PIs at local hospital

Taking on formal role as sub-investigator

Journal clubs

Trainee representation on regional/national NIHR specialty group

API Scheme: https://www.nihr.ac.uk/health-and-care-professionals/training/associate-principal-investigator-scheme.htm .

eLearning courses available at https://learn.nihr.ac.uk (free): Good clinical practice, fundamentals of clinical research delivery, informed consent, leadership, future of health, central portfolio management system.

eLearning courses available from the Royal College of Physicians. Research in Practice programme (free). www.rcplondon.ac.uk

eLearning courses available from the Medical Research Council (free). https://bygsystems.net/mrcrsc-lms/

eLearning courses available from Nature (both free and for variable cost via employing institution): many and varied including research integrity and publication ethics, persuasive grant writing, publishing a research paper. https://masterclasses.nature.com

University courses. Examples include novel clinical trial design in translational medicine from the University of Cambridge ( https://advanceonline.cam.ac.uk/courses/ ) or introduction to randomised controlled trials in healthcare from the University of Birmingham ( https://www.birmingham.ac.uk/university/colleges/mds/cpd/ )

This article outlines these formal but “non-traditional” routes available to early and mid-career doctors that can successfully increase research involvement and enable research-active careers.

Trainee research networks

Trainee research networks are a recent phenomenon within most medical specialties. They are formalised regional or national groups led by early and mid-career doctors who work together to perform clinical research and create research training opportunities. The first of these groups started in the early 2010s within anaesthetics but now represent nearly every specialty ( box 2 ). 4 Trainee research networks provide research training with the aim of increasing doctors’ future research involvement. 5

A non-exhaustive list of UK national trainee led research networks*

Acute medicine.

No national trainee research network

Anaesthesia

Research and Audit Federation of Trainees (RAFT). www.raftrainees.org

Cardiothoracic surgery

No national trainee-specific research network. National research network does exist: Cardiothoracic Interdisciplinary Research Network (CIRN). www.scts.org/professionals/research/cirn.aspx

Emergency medicine

Trainee Emergency Medicine Research Network (TERN). www.ternresearch.co.uk

Ear, nose, and throat

UK ENT Trainee Research Network (INTEGRATE). www.entintegrate.co.uk

Gastroenterology

No national trainee research network. Many regional trainee research networks

General practice

No national trainee-specific research network, although national research networks exist: Society for Academic Primary Care (SAPC) and Primary Care Academic Collaborative (PACT). www.sapc.ac.uk ; www.gppact.org

General surgery

Student Audit and Research in Surgery (STARSurg). www.starsurg.org . Many regional trainee research networks

Geriatric Medicine Research Collaborative (GeMRC). www.gemresearchuk.com

Haematology (non-malignant)

Haematology Specialty Training Audit and Research (HaemSTAR). www.haemstar.org

Haematology (malignant)

Trainee Collaborative for Research and Audit in Hepatology UK (ToRcH-UK). www.twitter.com/uk_torch

Histopathology

Pathsoc Research Trainee Initiative (PARTI). www.pathsoc.org/parti.aspx

Intensive care medicine

Trainee Research in Intensive Care Network (TRIC). www.tricnetwork.co.uk

Internal medicine

No national trainee-led research network. www.rcp.ac.uk/trainee-research-collaboratives

Interventional radiology

UK National Interventional Radiology Trainee Research (UNITE) Collaborative. https://www.unitecollaborative.com

Maxillofacial surgery

Maxillofacial Trainee Research Collaborative (MTReC). www.maxfaxtrainee.co.uk/

UK & Ireland Renal Trainee Network (NEPHwork). www.ukkidney.org/audit-research/projects/nephwork

No national trainee-led research network

Neurosurgery

British Neurosurgical Trainee Research Collaborative (BNTRC). www.bntrc.org.uk

Obstetrics and gynaecology

UK Audit and Research Collaborative in Obstetrics and Gynaecology (UKAROG). www.ukarcog.org

The National Oncology Trainee Collaborative for Healthcare Research (NOTCH). www.uknotch.com

Breast Cancer Trainee Research Collaborative Group (BCTRCG). https://bctrcguk.wixsite.com/bctrcg

Ophthalmology

The Ophthalmology Clinical Trials Network (OCTN). www.ophthalmologytrials.net

Paediatrics

RCPCH Trainee Research Network. www.rcpch.ac.uk/resources/rcpch-trainee-research-network

Paediatric anaesthesia

Paediatric Anaesthesia Trainee Research Network (PATRN). www.apagbi.org.uk/education-and-training/trainee-information/research-network-patrn

Paediatric haematology

Paediatric Haematology Trainee Research Network (PHTN). https://b-s-h.org.uk/about-us/special-interest-groups/paediatric-sig/phtn

Paediatric surgery

Paediatric Surgical Trainees Research Network (PSTRN). www.pstrnuk.org

Pain medicine

Network of Pain Trainees Interested in Research & Audit (PAIN-TRAIN). www.paintrainuk.com

Palliative care

UK Palliative Care Trainee Research Collaborative (UKPRC). www.twitter.com/uk_prc

Plastic surgery

Reconstructive Surgery Trials Network (RSTN). www.reconstructivesurgerytrials.net/trainees/

Pre-hospital medicine

Pre-Hospital Trainee Operated Research Network (PHOTON). www.facebook.com/PHOTONPHEM

Information from Royal College of Psychiatrists. www.rcpsych.ac.uk/members/your-faculties/academic-psychiatry/research

Radiology Academic Network for Trainees (RADIANT). www.radiantuk.com

Respiratory

Integrated Respiratory Research collaborative (INSPIRE). www.inspirerespiratory.co.uk

British Urology Researchers in Surgical Training (BURST). www.bursturology.com

Vascular surgery

Vascular & Endovascular Research Network (VERN). www.vascular-research.net

*limited to those with formal websites and/or active twitter accounts. Correct as of 5 January 2024. For regional trainee-led specialty research networks, see www.rcp.ac.uk/trainee-research-collaboratives for medical specialties, www.asit.org/resources/trainee-research-collaboratives/national-trainee-research-collaboratives/res1137 for surgical specialties, and www.rcoa.ac.uk/research/research-bodies/trainee-research-networks for anaesthetics.

Networks vary widely in structure and function. Most have senior mentorship to guide personal development and career trajectory. Projects are usually highly collaborative and include doctors and allied healthcare professionals working together.

Observational studies and large scale audits are common projects as their feasibility makes them deliverable rapidly with minimal funding. Some networks do, however, carry out interventional research. The benefits of increasing interventional research studies are self-evident, but observational projects are also important as they provide data useful for hypothesis generation and defining clinical equipoise and incidence/event rates, all of which are necessary steps in the development of randomised controlled studies.

These networks offer a supportive learning environment and research experience, and can match experience with expectations and responsibilities. Early and mid-career doctors are given opportunities to be involved and receive training in research at every phase from inception to publication. This develops experience in research methodology such as statistics, scientific writing, and peer review. As well as research skills training, an important reward for involvement in a study is manuscript authorship. Many groups give “citable collaborator” status to all project contributors, whatever their input. 6 7 This recognises the essential role everyone plays in the delivery of whole projects, counts towards publication metrics, and is important for future job applications.

Case study—Pip Nicolson (HaemSTAR)

Haematology Specialty Training, Audit and Research (HaemSTAR) is a trainee research network founded because of a lack of principal investigator training and clinical trial activity in non-malignant haematology. It has led and supported national audits and research projects in various subspecialty areas such as immune thrombocytopenia, thrombotic thrombocytopenic purpura, venous thrombosis, and transfusion. 8 9 10 Through involvement in this network as a registrar, I have acted as a sub-investigator and supported the principal investigator on observational and interventional portfolio-adopted studies by the National Institute for Health and Care Research (NIHR). These experiences gave me valuable insight into the national and local processes involved in research delivery. I was introduced to national leaders in non-malignant haematology who not only provided mentorship and advice on career development, but also gave me opportunities to lead national audits and become involved in HaemSTAR’s committee. 10 11 These experiences in leadership have increased my confidence in management situations as I have transitioned to being a consultant, and have given me skills in balancing clinical and academic roles. Importantly, I have also developed long term friendships with peers across the country as a result of my involvement in HaemSTAR.

Associate Principal Investigator scheme

The Associate Principal Investigator (API) scheme is a training programme run by NIHR to develop research skills and contribute to clinical study delivery at a local level. It is available throughout England, Scotland, Wales, and Northern Ireland for NIHR portfolio-adopted studies. The programme runs for six months and, upon completion, APIs receive formal recognition endorsed by the NIHR and a large number of royal colleges. The scheme is free and open to medical and allied healthcare professionals at all career grades. It is designed to allow those who would not normally take part in clinical research to do so under the mentorship of a local PI. Currently there are more than 1500 accredited APIs and over 600 affiliated studies across 28 specialties. 12 It is a good way to show evidence of training and involvement in research and get more involved in research conduct. APIs have been shown to increase patient recruitment and most people completing the scheme continue to be involved in research. 12 13

Case study—Rebecca Hawes

I completed the API scheme as a senior house officer in 2021. A local PI introduced me to the Quality of Recovery after Obstetric Anaesthesia NIHR portfolio study, 14 which I saw as a training opportunity and useful experience ahead of specialist training applications. It was easy to apply for and straightforward to navigate. I was guided through the six month process in a step-by-step manner and completed eLearning modules and video based training on fundamental aspects of running research projects. All this training was evidenced on the online API platform and I had monthly supervision meetings with the PI and wider research team. As well as the experience of patient recruitment and data collection, other important aspects of training were study set-up and sponsor communications. Key to my successful API scheme was having a supportive and enthusiastic PI and developing good organisational skills. I really enjoyed the experience, and I have since done more research and have become a committee member on a national trainee research network in anaesthesia called RAFT (Research and Audit Federation of Trainees). I’ve seen great enthusiasm among anaesthetists to take part in the API scheme, with over 150 signing up to the most recent RAFT national research project.

Clinical research posts

Dedicated clinical research posts (sometimes termed “clinical research fellow” posts) allow clinicians to explore and develop research skills without committing to a formal academic pathway. They can be undertaken at any stage during a medical career but are generally performed between training posts, or during them by receiving permission from local training committees to temporarily go “out of programme.” These positions are extremely varied in how they are advertised, funded, and the balance between research and clinical time. Look out for opportunities with royal colleges, local and national research networks, and on the NHS Jobs website. Research fellowships are a good way to broaden skills that will have long term impact across one’s clinical career.

Case study—Nicole Fowler

After completing the Foundation Programme, I took up a 12 month clinical trials fellow position. This gave me early career exposure to clinical research and allowed me to act as a sub-investigator in a range of clinical trials. I received practical experience in all stages of clinical research while retaining a patient facing role, which included obtaining consent and reviewing patients at all subsequent visits until study completion. Many of the skills I developed in this post, such as good organisation and effective teamwork, are transferable to all areas of medicine. I have thoroughly enjoyed the experience and it is something I hope to talk about at interview as it is an effective way of showing commitment to a specialty. Furthermore, having a dedicated research doctor has been beneficial to my department in increasing patient involvement in research.

Acknowledgments

We would like to thank Holly Speight and Clare Shaw from the NIHR for information on the API scheme.

*These authors contributed equally to this work

Patient and public involvement: No patients were directly involved in the creation of this article.

PLRN, MB, and CHT conceived the article and are guarantors. All authors wrote and edited the manuscript.

Competing interests: PLRN was the chair of HaemSTAR from 2017 to 2023. MB is the current chair of the Research and Audit Federation of Trainees (RAFT). RH is the current secretary of RAFT. CHT conceived HaemSTAR.

Provenance and peer review: Commissioned; externally peer reviewed.

• Downing A ,
• Morris EJ ,
• Corrigan N ,
• Bracewell M ,
• Medical Academic Staff Committee of the British Medical Association
• ↵ RAFT. The start of RAFT. https://www.raftrainees.org/about
• Jamjoom AAB ,
• Hutchinson PJ ,
• Bradbury CA ,
• HaemSTAR Collaborators
• ↵ National Institute for Health and Care Research. Associate Principal Investigator (PI) Scheme. 2023. https://www.nihr.ac.uk/health-and-care-professionals/career-development/associate-principal-investigator-scheme.htm
• Fairhurst C ,
• Torgerson D
• O’Carroll JE ,
• Warwick E ,
• ObsQoR Collaborators

The current status and future perspectives of clinical boron neutron capture therapy trials

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• Published: 20 April 2024

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Several hundreds of patients have been so far treated in clinical trials with boron neutron capture therapy (BNCT).

This is a non-systematic review of clinical trials with BNCT, with special emphasis on the more recent trials.

The conducted trials have been relatively small single-arm studies and included mostly the patients with head and neck carcinomas resistant to traditional treatment modalities and glioblastomas. In general, the efficacy results have been promising and BNCT has been relatively well tolerated, even in the patients who have already been treated with conventional radiotherapy or chemoradiation. The most frequent adverse events have been similar to those associated with the conventional radiotherapy. At present, there is no evidence how the efficacy of BNCT would compare to the standard treatment modalities in earlier treatment lines.

Conclusions

Most of the existing studies have been performed with reactor-based facilities, but there is now a rapidly increasing number of linear accelerator-based BNCT sites, and the clinical research is apparently activating again. This, combined with the increased knowledge on cancer biology and novel types of oncological therapies, opens possibilities to study innovative boron carriers and to combine BNCT with modern oncological therapies in the future clinical trials. To conduct larger phase III trials, multicenter approaches are encouraged to be applied, keeping in mind the importance of joint instructions and quality control measurements.

Avoid common mistakes on your manuscript.

1 From reactors to linear accelerators

The principle of BNCT was first presented by astrophysicist Gordon Locher in 1936, but the first clinical BNCT trials enrolling glioblastoma patients after debulking craniotomy were published not until in mid-50’s [ 1 , 2 , 3 , 4 ]. In these studies, no patients survived beyond one year and substantial toxicities, such oedema, necrosis, and refractory shock were recorded in all patients. Clinical BNCT trials were mostly halted until late 1960’s when a new generation of especially Japanese groups using sodium borocaptate (BSH) as a boron carrier reported trial results, again using mostly patients with glioblastoma [ 5 , 6 , 7 , 8 ]. The field continued as active until the millennium, after which several BNCT nuclear reactor facilities were closed. Now, there is a rapidly increasing number of linear accelerator-based BNCT sites and the clinical research is apparently activating again.

2 Overview of the efficacy and toxicity in clinical BNCT trials

So far, several hundreds of patients have been treated with BNCT using neutrons obtained from a nuclear facility and with boronophenylalanine (L-BPA) and/or BSH as the boron carrier, but the trials have been almost solely single-site studies without a comparison arm. By far most of the current evidence comes from head and neck carcinomas and glioblastomas and due to the radiobiological principles of BNCT, also other BNCT-treated tumors have been mostly superficially located. As usually in oncological trials, the development of a new method has started from the tumor types mostly refractory to standard treatments. In all conducted studies, the efficacy results have been compared to the historical controls if anything, which may potentially cause misleading interpretations of toxicity in tumor types such as metastases and melanomas, where the evidence of re-irradiation efficacy is scarce.

Although the multidisciplinary approach is especially emphasized in the treatment of head and neck cancers, external radiotherapy is one of the mainstays in the treatment of early, locally advanced and metastatic head and neck cancers. Of all tumor types, BNCT has been under the most active clinical research in head and neck carcinomas, with some exceptions specifically in tumors with squamous cell carcinoma histology. The number of patients in each trial has been low, up to 30 patients. In the reported modern phase I/II trials the efficacy has still been encouraging with objective response rates more than 70% in heavily pre-treated patients and even complete responses in more than half of the patients have been reported in several trials [ 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 ]. In a trial with locally recurred head and cancer and previous photon irradiation to a cumulative dose of 50 to 98 Gy, the 2-year locoregional recurrence-free survival was 27%, 2-year progression-free survival 20%, and 2-year overall survival 30% [ 9 ]. The most frequently reported adverse events across the different head and neck cancer trials have included mucositis, oral pain and fatigue, but the rate of grade 3 adverse events have been relatively low. In sum, the available data suggest BNCT as an effective and moderately well tolerated treatment in locally recurrent, inoperable head and neck cancer with relatively high response rates.

Glioblastoma is a malignancy with an aggressive behavior and with extremely rare long-term survivors. External radiotherapy with temozolomide after debulking surgery has been the major first-line treatment modality for over decade [ 19 ]. In the BNCT context, trials enrolling glioblastoma patients have recruited a larger number of patients per trial compared with e.g. the trials with head and neck cancer patients, and notably also patients without previous radiotherapy have been included in the glioblastoma trials. Although the latest published BNCT clinical trial including specifically glioblastoma patients dates back to 2011, the results have been promising and without no alarming rates of adverse events in the trials conducted in the modern BNCT era. For example, Joensuu et al. and reported 1-year overall survival rate of 61% and Hideghety et al. 50%, with study sizes of 72 and 36 patients, respectively [ 14 , 20 ]. The results are consistent with more recent studies, also [ 21 , 22 ]. There is also preliminary data regarding BNCT efficacy in patients with high-grade meningiomas. In a retrospective series of 44 recurrent and refractory high-grade meningiomas treated with a reactor-based BNCT, median overall survival of 29.6 months was observed [ 23 ]. Promisingly, also grade 3 recurrent and refractory meningiomas had an overall survival of 21.6 months, while in grade 2 recurrent and refractory meningiomas median overall survival was 44.0 months. These results are far better than reported from e.g. octreotide analogue and tyrosine kinase inhibitor studies in this patient population [ 24 , 25 , 26 ]. One patient in the meningioma BNCT series died of disseminated intravascular coagulation syndrome, otherwise no unexpected toxicity signals were reported either in the meningioma trials.

The treatment landscape of cutaneous melanomas has been reformed during the last decade after the successful introduction of immuno-oncological compounds, mainly checkpoint inhibitors, and BRAF/MEK inhibitors [ 27 ]. Cutaneous melanomas are considered as radioresistant tumors, but in early phase clinical trials BNCT has demonstrated substantial efficacy. The published response rates have been even tremendous with complete responses observed in 70–75% of the patients [ 28 , 29 ]. Some preliminary results suggest that BNCT could be effective especially in melanoma subtypes of non-nodular histology [ 30 ]. Although skin ulcers and necrosis have been reported, long-term safety overall seems very reasonable [ 31 ].

In addition to glioblastomas, head and neck cancers and cutaneous melanomas, there have been single clinical BNCT trials e.g. in the patients with liver metastases, hepatocellular carcinomas, sarcomas and extramammary Paget’s disease in the modern BNCT era [ 11 , 32 , 33 , 34 , 35 , 36 , 37 ]. Still, these studies have included only very few patients, which makes the interpretation of efficacy and toxicity of BNCT in these tumor types currently impossible.

3 Special issues when conducting clinical BNCT trials

As discussed above, BNCT has been demonstrated as a safe method to treat several tumor types with promising efficacy during the last 25 years in investigator-initiated trials. As new linear accelerator-based BNCT facilities are continuously built and several commercial companies are also involved in the research, larger phase II, even phase III trials are expected to be launched in the near future, which will provide BNCT a possibility to become as one of the standard treatment options in several clinical situations.

Still, there are several unsolved issues in the clinical development of BNCT. As there are still few BNCT sites globally and the slow patient recruitment has been a bottleneck in many BNCT trials, all efforts should be undertaken to develop multicenter trial approaches. This would also enhance the generalizability of the obtained results, not mentioning the unification and dissemination of good BNCT practices created when designing the trials. To conduct such multicenter studies, it is obvious that both the unified quality control and operating instructions are in the key role for their success. These include, but are not limited to boron dosing and boron measurements, joint response evaluation and the standardization of the neutron beam characteristics. The radiobiological perspectives of BNCT are not in the scope of this review, but it is obvious that complex BNCT dose distribution with low and high LET components requires special knowledge, and normalization. Similarly, outside of the primary focus of this review, the studies on the toxicity and efficacy of novel boron carriers have special considerations. The binary nature of BNCT means that novel boron carriers, without their own therapeutic effect, have to be tested at first in cancer patients without irradiation, yielding to a rare occasion in the development of oncological treatments with no potential benefit for a patient.

4 Potential combinational targets in BNCT clinical trials

In the future, in phase II-III trials BNCT could be studied to combine it with several intriguing targets. Optimally, these strategies could help to overcome the resistance mechanisms of both BNCT and the combined treatment and increase synergy without an increased toxicity. Boronated epidermal growth factor receptor (EGFR) binding antibodies have shown promising results in preclinical studies, but also very preliminary, successful clinical experiences for combining BNCT with chemoradiotherapy including EGFR antibody cetuximab have been reported [ 38 , 39 , 40 , 41 ]. As BNCT is effective only locally, the combination with e.g. checkpoint inhibitors may complement BNCT by eliminating the micrometastatic disease. There is no any literature available on combining BNCT with checkpoint inhibitors, although these drugs have revolutionized the treatment of BNCT-sensitive melanomas and are approved also for the treatment of locally advanced and metastatic head and neck cancers [ 27 , 33 ]. Despite the lack of any clinical evidence, it is not foreseen that checkpoint inhibitors would affect to resistance mechanisms for BNCT. Instead, destroying tumor cells with BNCT could in theory lead to the increased efficacy of checkpoint inhibitors by the increased release of neoantigens [ 42 ].

In contrast to BNCT, chemotherapy is effective only in cancer cells in the proliferative phases of the cell cycle, but could also complement the efficacy of BNCT by destroying subclinical metastases. Adding BNCT to the standard of care, external radiotherapy and temozolomide, in patients with glioblastoma yielded encouraging median survival time of 21.1 months and 2-year overall survival of 45.5%, although the final results are still unpublished (NCT00974987) [ 21 ]. Several tumor-targeted boron delivery systems with chemotherapeutic agents such as doxorubicin and paclitaxel are in the early phases of the development [ 43 , 44 ].

Conventional photon radiotherapy is dependent from oxygen, while BNCT needs especially sufficient and even tumor to normal tissue boron intake ratio to be effective and predictable. Hypothetically, photon radiotherapy could provide means to ameliorate dose shortages in poorly boron-intaking tumors. Still, poorly vascularized tumors are likely to have both deoxygenation and reduced boron intake. These two radiotherapy modalities have little expected cross-resistance and have been successfully combined in preclinical models [ 45 ]. In a non-randomized controlled clinical trial of 21 patients with newly diagnosed glioblastoma, BNCT followed by external radiotherapy led to a substantial improvement of median survival time compared with BNCT alone, without no worrying toxicity signals [ 46 ]. If BNCT could be beneficial to combine e.g. with hadrontherapies or FLASH radiotherapy, will likely remain as an unsolved issue for long.

5 Conclusions

The binary nature of BNCT requires special solutions for clinical BNCT research. So far, data from hundreds of patients, mainly with head and neck carcinomas, is available from single-site, controlled phase I-II clinical trials with promising results and acceptable toxicity. There is no at present evidence if BNCT would be most effective in earlier treatment lines. Currently there are only two registered BNCT trials in ClinicalTrials.gov enrolling patients (NCT05737212, NCT05601232), but the number of clinical BNCT studies is expected to be a rapidly growing field later in this decade [ 47 ]. In the nearest future, it is likely that BNCT will be tested in basket trials with several tumor types. To conduct still lacking comparative phase III trials, multicenter approaches are encouraged to be applied, keeping in mind the importance of joint instructions and quality control measurements.

Data availability

As a review article, there is no original data in this manuscript.

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Karihtala, P. The current status and future perspectives of clinical boron neutron capture therapy trials. Health Technol. (2024). https://doi.org/10.1007/s12553-024-00862-7

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Clinical research study designs: The essentials

Ambika g. chidambaram.

1 Children's Hospital of Philadelphia, Philadelphia Pennsylvania, USA

Maureen Josephson

In clinical research, our aim is to design a study which would be able to derive a valid and meaningful scientific conclusion using appropriate statistical methods. The conclusions derived from a research study can either improve health care or result in inadvertent harm to patients. Hence, this requires a well‐designed clinical research study that rests on a strong foundation of a detailed methodology and governed by ethical clinical principles. The purpose of this review is to provide the readers an overview of the basic study designs and its applicability in clinical research.

Introduction

In clinical research, our aim is to design a study, which would be able to derive a valid and meaningful scientific conclusion using appropriate statistical methods that can be translated to the “real world” setting. 1 Before choosing a study design, one must establish aims and objectives of the study, and choose an appropriate target population that is most representative of the population being studied. The conclusions derived from a research study can either improve health care or result in inadvertent harm to patients. Hence, this requires a well‐designed clinical research study that rests on a strong foundation of a detailed methodology and is governed by ethical principles. 2

From an epidemiological standpoint, there are two major types of clinical study designs, observational and experimental. 3 Observational studies are hypothesis‐generating studies, and they can be further divided into descriptive and analytic. Descriptive observational studies provide a description of the exposure and/or the outcome, and analytic observational studies provide a measurement of the association between the exposure and the outcome. Experimental studies, on the other hand, are hypothesis testing studies. It involves an intervention that tests the association between the exposure and outcome. Each study design is different, and so it would be important to choose a design that would most appropriately answer the question in mind and provide the most valuable information. We will be reviewing each study design in detail (Figure  1 ).

Overview of clinical research study designs

Observational study designs

Observational studies ask the following questions: what, who, where and when. There are many study designs that fall under the umbrella of descriptive study designs, and they include, case reports, case series, ecologic study, cross‐sectional study, cohort study and case‐control study (Figure  2 ).

Classification of observational study designs

Case reports and case series

Every now and then during clinical practice, we come across a case that is atypical or ‘out of the norm’ type of clinical presentation. This atypical presentation is usually described as case reports which provides a detailed and comprehensive description of the case. 4 It is one of the earliest forms of research and provides an opportunity for the investigator to describe the observations that make a case unique. There are no inferences obtained and therefore cannot be generalized to the population which is a limitation. Most often than not, a series of case reports make a case series which is an atypical presentation found in a group of patients. This in turn poses the question for a new disease entity and further queries the investigator to look into mechanistic investigative opportunities to further explore. However, in a case series, the cases are not compared to subjects without the manifestations and therefore it cannot determine which factors in the description are unique to the new disease entity.

Ecologic study

Ecological studies are observational studies that provide a description of population group characteristics. That is, it describes characteristics to all individuals within a group. For example, Prentice et al 5 measured incidence of breast cancer and per capita intake of dietary fat, and found a correlation that higher per capita intake of dietary fat was associated with an increased incidence of breast cancer. But the study does not conclude specifically which subjects with breast cancer had a higher dietary intake of fat. Thus, one of the limitations with ecologic study designs is that the characteristics are attributed to the whole group and so the individual characteristics are unknown.

Cross‐sectional study

Cross‐sectional studies are study designs used to evaluate an association between an exposure and outcome at the same time. It can be classified under either descriptive or analytic, and therefore depends on the question being answered by the investigator. Since, cross‐sectional studies are designed to collect information at the same point of time, this provides an opportunity to measure prevalence of the exposure or the outcome. For example, a cross‐sectional study design was adopted to estimate the global need for palliative care for children based on representative sample of countries from all regions of the world and all World Bank income groups. 6 The limitation of cross‐sectional study design is that temporal association cannot be established as the information is collected at the same point of time. If a study involves a questionnaire, then the investigator can ask questions to onset of symptoms or risk factors in relation to onset of disease. This would help in obtaining a temporal sequence between the exposure and outcome. 7

Case‐control study

Case‐control studies are study designs that compare two groups, such as the subjects with disease (cases) to the subjects without disease (controls), and to look for differences in risk factors. 8 This study is used to study risk factors or etiologies for a disease, especially if the disease is rare. Thus, case‐control studies can also be hypothesis testing studies and therefore can suggest a causal relationship but cannot prove. It is less expensive and less time‐consuming than cohort studies (described in section “Cohort study”). An example of a case‐control study was performed in Pakistan evaluating the risk factors for neonatal tetanus. They retrospectively reviewed a defined cohort for cases with and without neonatal tetanus. 9 They found a strong association of the application of ghee (clarified butter) as a risk factor for neonatal tetanus. Although this suggests a causal relationship, cause cannot be proven by this methodology (Figure  3 ).

Case‐control study design

One of the limitations of case‐control studies is that they cannot estimate prevalence of a disease accurately as a proportion of cases and controls are studied at a time. Case‐control studies are also prone to biases such as recall bias, as the subjects are providing information based on their memory. Hence, the subjects with disease are likely to remember the presence of risk factors compared to the subjects without disease.

One of the aspects that is often overlooked is the selection of cases and controls. It is important to select the cases and controls appropriately to obtain a meaningful and scientifically sound conclusion and this can be achieved by implementing matching. Matching is defined by Gordis et al as ‘the process of selecting the controls so that they are similar to the cases in certain characteristics such as age, race, sex, socioeconomic status and occupation’ 7 This would help identify risk factors or probable etiologies that are not due to differences between the cases and controls.

Cohort study

Cohort studies are study designs that compare two groups, such as the subjects with exposure/risk factor to the subjects without exposure/risk factor, for differences in incidence of outcome/disease. Most often, cohort study designs are used to study outcome(s) from a single exposure/risk factor. Thus, cohort studies can also be hypothesis testing studies and can infer and interpret a causal relationship between an exposure and a proposed outcome, but cannot establish it (Figure  4 ).

Cohort study design

Cohort studies can be classified as prospective and retrospective. 7 Prospective cohort studies follow subjects from presence of risk factors/exposure to development of disease/outcome. This could take up to years before development of disease/outcome, and therefore is time consuming and expensive. On the other hand, retrospective cohort studies identify a population with and without the risk factor/exposure based on past records and then assess if they had developed the disease/outcome at the time of study. Thus, the study design for prospective and retrospective cohort studies are similar as we are comparing populations with and without exposure/risk factor to development of outcome/disease.

Cohort studies are typically chosen as a study design when the suspected exposure is known and rare, and the incidence of disease/outcome in the exposure group is suspected to be high. The choice between prospective and retrospective cohort study design would depend on the accuracy and reliability of the past records regarding the exposure/risk factor.

Some of the biases observed with cohort studies include selection bias and information bias. Some individuals who have the exposure may refuse to participate in the study or would be lost to follow‐up, and in those instances, it becomes difficult to interpret the association between an exposure and outcome. Also, if the information is inaccurate when past records are used to evaluate for exposure status, then again, the association between the exposure and outcome becomes difficult to interpret.

Case‐control studies based within a defined cohort

Case‐control studies based within a defined cohort is a form of study design that combines some of the features of a cohort study design and a case‐control study design. When a defined cohort is embedded in a case‐control study design, all the baseline information collected before the onset of disease like interviews, surveys, blood or urine specimens, then the cohort is followed onset of disease. One of the advantages of following the above design is that it eliminates recall bias as the information regarding risk factors is collected before onset of disease. Case‐control studies based within a defined cohort can be further classified into two types: Nested case‐control study and Case‐cohort study.

Nested case‐control study

A nested case‐control study consists of defining a cohort with suspected risk factors and assigning a control within a cohort to the subject who develops the disease. 10 Over a period, cases and controls are identified and followed as per the investigator's protocol. Hence, the case and control are matched on calendar time and length of follow‐up. When this study design is implemented, it is possible for the control that was selected early in the study to develop the disease and become a case in the latter part of the study.

Case‐cohort Study

A case‐cohort study is similar to a nested case‐control study except that there is a defined sub‐cohort which forms the groups of individuals without the disease (control), and the cases are not matched on calendar time or length of follow‐up with the control. 11 With these modifications, it is possible to compare different disease groups with the same sub‐cohort group of controls and eliminates matching between the case and control. However, these differences will need to be accounted during analysis of results.

Experimental study design

The basic concept of experimental study design is to study the effect of an intervention. In this study design, the risk factor/exposure of interest/treatment is controlled by the investigator. Therefore, these are hypothesis testing studies and can provide the most convincing demonstration of evidence for causality. As a result, the design of the study requires meticulous planning and resources to provide an accurate result.

The experimental study design can be classified into 2 groups, that is, controlled (with comparison) and uncontrolled (without comparison). 1 In the group without controls, the outcome is directly attributed to the treatment received in one group. This fails to prove if the outcome was truly due to the intervention implemented or due to chance. This can be avoided if a controlled study design is chosen which includes a group that does not receive the intervention (control group) and a group that receives the intervention (intervention/experiment group), and therefore provide a more accurate and valid conclusion.

Experimental study designs can be divided into 3 broad categories: clinical trial, community trial, field trial. The specifics of each study design are explained below (Figure  5 ).

Experimental study designs

Clinical trial

Clinical trials are also known as therapeutic trials, which involve subjects with disease and are placed in different treatment groups. It is considered a gold standard approach for epidemiological research. One of the earliest clinical trial studies was performed by James Lind et al in 1747 on sailors with scurvy. 12 Lind divided twelve scorbutic sailors into six groups of two. Each group received the same diet, in addition to a quart of cider (group 1), twenty‐five drops of elixir of vitriol which is sulfuric acid (group 2), two spoonfuls of vinegar (group 3), half a pint of seawater (group 4), two oranges and one lemon (group 5), and a spicy paste plus a drink of barley water (group 6). The group who ate two oranges and one lemon had shown the most sudden and visible clinical effects and were taken back at the end of 6 days as being fit for duty. During Lind's time, this was not accepted but was shown to have similar results when repeated 47 years later in an entire fleet of ships. Based on the above results, in 1795 lemon juice was made a required part of the diet of sailors. Thus, clinical trials can be used to evaluate new therapies, such as new drug or new indication, new drug combination, new surgical procedure or device, new dosing schedule or mode of administration, or a new prevention therapy.

While designing a clinical trial, it is important to select the population that is best representative of the general population. Therefore, the results obtained from the study can be generalized to the population from which the sample population was selected. It is also as important to select appropriate endpoints while designing a trial. Endpoints need to be well‐defined, reproducible, clinically relevant and achievable. The types of endpoints include continuous, ordinal, rates and time‐to‐event, and it is typically classified as primary, secondary or tertiary. 2 An ideal endpoint is a purely clinical outcome, for example, cure/survival, and thus, the clinical trials will become very long and expensive trials. Therefore, surrogate endpoints are used that are biologically related to the ideal endpoint. Surrogate endpoints need to be reproducible, easily measured, related to the clinical outcome, affected by treatment and occurring earlier than clinical outcome. 2

Clinical trials are further divided into randomized clinical trial, non‐randomized clinical trial, cross‐over clinical trial and factorial clinical trial.

Randomized clinical trial

A randomized clinical trial is also known as parallel group randomized trials or randomized controlled trials. Randomized clinical trials involve randomizing subjects with similar characteristics to two groups (or multiple groups): the group that receives the intervention/experimental therapy and the other group that received the placebo (or standard of care). 13 This is typically performed by using a computer software, manually or by other methods. Hence, we can measure the outcomes and efficacy of the intervention/experimental therapy being studied without bias as subjects have been randomized to their respective groups with similar baseline characteristics. This type of study design is considered gold standard for epidemiological research. However, this study design is generally not applicable to rare and serious disease process as it would unethical to treat that group with a placebo. Please see section “Randomization” for detailed explanation regarding randomization and placebo.

Non‐randomized clinical trial

A non‐randomized clinical trial involves an approach to selecting controls without randomization. With this type of study design a pattern is usually adopted, such as, selection of subjects and controls on certain days of the week. Depending on the approach adopted, the selection of subjects becomes predictable and therefore, there is bias with regards to selection of subjects and controls that would question the validity of the results obtained.

Historically controlled studies can be considered as a subtype of non‐randomized clinical trial. In this study design subtype, the source of controls is usually adopted from the past, such as from medical records and published literature. 1 The advantages of this study design include being cost‐effective, time saving and easily accessible. However, since this design depends on already collected data from different sources, the information obtained may not be accurate, reliable, lack uniformity and/or completeness as well. Though historically controlled studies maybe easier to conduct, the disadvantages will need to be taken into account while designing a study.

Cross‐over clinical trial

In cross‐over clinical trial study design, there are two groups who undergoes the same intervention/experiment at different time periods of the study. That is, each group serves as a control while the other group is undergoing the intervention/experiment. 14 Depending on the intervention/experiment, a ‘washout’ period is recommended. This would help eliminate residuals effects of the intervention/experiment when the experiment group transitions to be the control group. Hence, the outcomes of the intervention/experiment will need to be reversible as this type of study design would not be possible if the subject is undergoing a surgical procedure.

Factorial trial

A factorial trial study design is adopted when the researcher wishes to test two different drugs with independent effects on the same population. Typically, the population is divided into 4 groups, the first with drug A, the second with drug B, the third with drug A and B, and the fourth with neither drug A nor drug B. The outcomes for drug A are compared to those on drug A, drug A and B and to those who were on drug B and neither drug A nor drug B. 15 The advantages of this study design that it saves time and helps to study two different drugs on the same study population at the same time. However, this study design would not be applicable if either of the drugs or interventions overlaps with each other on modes of action or effects, as the results obtained would not attribute to a particular drug or intervention.

Community trial

Community trials are also known as cluster‐randomized trials, involve groups of individuals with and without disease who are assigned to different intervention/experiment groups. Hence, groups of individuals from a certain area, such as a town or city, or a certain group such as school or college, will undergo the same intervention/experiment. 16 Hence, the results will be obtained at a larger scale; however, will not be able to account for inter‐individual and intra‐individual variability.

Field trial

Field trials are also known as preventive or prophylactic trials, and the subjects without the disease are placed in different preventive intervention groups. 16 One of the hypothetical examples for a field trial would be to randomly assign to groups of a healthy population and to provide an intervention to a group such as a vitamin and following through to measure certain outcomes. Hence, the subjects are monitored over a period of time for occurrence of a particular disease process.

Overview of methodologies used within a study design

Randomization.

Randomization is a well‐established methodology adopted in research to prevent bias due to subject selection, which may impact the result of the intervention/experiment being studied. It is one of the fundamental principles of an experimental study designs and ensures scientific validity. It provides a way to avoid predicting which subjects are assigned to a certain group and therefore, prevent bias on the final results due to subject selection. This also ensures comparability between groups as most baseline characteristics are similar prior to randomization and therefore helps to interpret the results regarding the intervention/experiment group without bias.

There are various ways to randomize and it can be as simple as a ‘flip of a coin’ to use computer software and statistical methods. To better describe randomization, there are three types of randomization: simple randomization, block randomization and stratified randomization.

Simple randomization

In simple randomization, the subjects are randomly allocated to experiment/intervention groups based on a constant probability. That is, if there are two groups A and B, the subject has a 0.5 probability of being allocated to either group. This can be performed in multiple ways, and one of which being as simple as a ‘flip of a coin’ to using random tables or numbers. 17 The advantage of using this methodology is that it eliminates selection bias. However, the disadvantage with this methodology is that an imbalance in the number allocated to each group as well as the prognostic factors between groups. Hence, it is more challenging in studies with a small sample size.

Block randomization

In block randomization, the subjects of similar characteristics are classified into blocks. The aim of block randomization is to balance the number of subjects allocated to each experiment/intervention group. For example, let's assume that there are four subjects in each block, and two of the four subjects in each block will be randomly allotted to each group. Therefore, there will be two subjects in one group and two subjects in the other group. 17 The disadvantage with this methodology is that there is still a component of predictability in the selection of subjects and the randomization of prognostic factors is not performed. However, it helps to control the balance between the experiment/intervention groups.

Stratified randomization

In stratified randomization, the subjects are defined based on certain strata, which are covariates. 18 For example, prognostic factors like age can be considered as a covariate, and then the specified population can be randomized within each age group related to an experiment/intervention group. The advantage with this methodology is that it enables comparability between experiment/intervention groups and thus makes result analysis more efficient. But, with this methodology the covariates will need to be measured and determined before the randomization process. The sample size will help determine the number of strata that would need to be chosen for a study.

Blinding is a methodology adopted in a study design to intentionally not provide information related to the allocation of the groups to the subject participants, investigators and/or data analysts. 19 The purpose of blinding is to decrease influence associated with the knowledge of being in a particular group on the study result. There are 3 forms of blinding: single‐blinded, double‐blinded and triple‐blinded. 1 In single‐blinded studies, otherwise called as open‐label studies, the subject participants are not revealed which group that they have been allocated to. However, the investigator and data analyst will be aware of the allocation of the groups. In double‐blinded studies, both the study participants and the investigator will be unaware of the group to which they were allocated to. Double‐blinded studies are typically used in clinical trials to test the safety and efficacy of the drugs. In triple‐blinded studies, the subject participants, investigators and data analysts will not be aware of the group allocation. Thus, triple‐blinded studies are more difficult and expensive to design but the results obtained will exclude confounding effects from knowledge of group allocation.

Blinding is especially important in studies where subjective response are considered as outcomes. This is because certain responses can be modified based on the knowledge of the experiment group that they are in. For example, a group allocated in the non‐intervention group may not feel better as they are not getting the treatment, or an investigator may pay more attention to the group receiving treatment, and thereby potentially affecting the final results. However, certain treatments cannot be blinded such as surgeries or if the treatment group requires an assessment of the effect of intervention such as quitting smoking.

Placebo is defined in the Merriam‐Webster dictionary as ‘an inert or innocuous substance used especially in controlled experiments testing the efficacy of another substance (such as drug)’. 20 A placebo is typically used in a clinical research study to evaluate the safety and efficacy of a drug/intervention. This is especially useful if the outcome measured is subjective. In clinical drug trials, a placebo is typically a drug that resembles the drug to be tested in certain characteristics such as color, size, shape and taste, but without the active substance. This helps to measure effects of just taking the drug, such as pain relief, compared to the drug with the active substance. If the effect is positive, for example, improvement in mood/pain, then it is called placebo effect. If the effect is negative, for example, worsening of mood/pain, then it is called nocebo effect. 21

The ethics of placebo‐controlled studies is complex and remains a debate in the medical research community. According to the Declaration of Helsinki on the use of placebo released in October 2013, “The benefits, risks, burdens and effectiveness of a new intervention must be tested against those of the best proven intervention(s), except in the following circumstances:

Where no proven intervention exists, the use of placebo, or no intervention, is acceptable; or

Where for compelling and scientifically sound methodological reasons the use of any intervention less effective than the best proven one, the use of placebo, or no intervention is necessary to determine the efficacy or safety of an intervention and the patients who receive any intervention less effective than the best proven one, placebo, or no intervention will not be subject to additional risks of serious or irreversible harm as a result of not receiving the best proven intervention.

Extreme care must be taken to avoid abuse of this option”. 22

Hence, while designing a research study, both the scientific validity and ethical aspects of the study will need to be thoroughly evaluated.

Bias has been defined as “any systematic error in the design, conduct or analysis of a study that results in a mistaken estimate of an exposure's effect on the risk of disease”. 23 There are multiple types of biases and so, in this review we will focus on the following types: selection bias, information bias and observer bias. Selection bias is when a systematic error is committed while selecting subjects for the study. Selection bias will affect the external validity of the study if the study subjects are not representative of the population being studied and therefore, the results of the study will not be generalizable. Selection bias will affect the internal validity of the study if the selection of study subjects in each group is influenced by certain factors, such as, based on the treatment of the group assigned. One of the ways to decrease selection bias is to select the study population that would representative of the population being studied, or to randomize (discussed in section “Randomization”).

Information bias is when a systematic error is committed while obtaining data from the study subjects. This can be in the form of recall bias when subject is required to remember certain events from the past. Typically, subjects with the disease tend to remember certain events compared to subjects without the disease. Observer bias is a systematic error when the study investigator is influenced by the certain characteristics of the group, that is, an investigator may pay closer attention to the group receiving the treatment versus the group not receiving the treatment. This may influence the results of the study. One of the ways to decrease observer bias is to use blinding (discussed in section “Blinding”).

Thus, while designing a study it is important to take measure to limit bias as much as possible so that the scientific validity of the study results is preserved to its maximum.

Overview of drug development in the United States of America

Now that we have reviewed the various clinical designs, clinical trials form a major part in development of a drug. In the United States, the Food and Drug Administration (FDA) plays an important role in getting a drug approved for clinical use. It includes a robust process that involves four different phases before a drug can be made available to the public. Phase I is conducted to determine a safe dose. The study subjects consist of normal volunteers and/or subjects with disease of interest, and the sample size is typically small and not more than 30 subjects. The primary endpoint consists of toxicity and adverse events. Phase II is conducted to evaluate of safety of dose selected in Phase I, to collect preliminary information on efficacy and to determine factors to plan a randomized controlled trial. The study subjects consist of subjects with disease of interest and the sample size is also small but more that Phase I (40–100 subjects). The primary endpoint is the measure of response. Phase III is conducted as a definitive trial to prove efficacy and establish safety of a drug. Phase III studies are randomized controlled trials and depending on the drug being studied, it can be placebo‐controlled, equivalence, superiority or non‐inferiority trials. The study subjects consist of subjects with disease of interest, and the sample size is typically large but no larger than 300 to 3000. Phase IV is performed after a drug is approved by the FDA and it is also called the post‐marketing clinical trial. This phase is conducted to evaluate new indications, to determine safety and efficacy in long‐term follow‐up and new dosing regimens. This phase helps to detect rare adverse events that would not be picked up during phase III studies and decrease in the delay in the release of the drug in the market. Hence, this phase depends heavily on voluntary reporting of side effects and/or adverse events by physicians, non‐physicians or drug companies. 2

We have discussed various clinical research study designs in this comprehensive review. Though there are various designs available, one must consider various ethical aspects of the study. Hence, each study will require thorough review of the protocol by the institutional review board before approval and implementation.

CONFLICT OF INTEREST

Chidambaram AG, Josephson M. Clinical research study designs: The essentials . Pediatr Invest . 2019; 3 :245‐252. 10.1002/ped4.12166 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]

• Open access
• Published: 19 April 2024

Applications of peptides in nanosystems for diagnosing and managing bacterial sepsis

• Mohammed A. Gafar 1 , 2 ,
• Calvin A. Omolo 1 , 3 ,
• Eman Elhassan 1 ,
• Usri H. Ibrahim 4 &
• Thirumala Govender 1  

Journal of Biomedical Science volume  31 , Article number:  40 ( 2024 ) Cite this article

154 Accesses

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Sepsis represents a critical medical condition stemming from an imbalanced host immune response to infections, which is linked to a significant burden of disease. Despite substantial efforts in laboratory and clinical research, sepsis remains a prominent contributor to mortality worldwide. Nanotechnology presents innovative opportunities for the advancement of sepsis diagnosis and treatment. Due to their unique properties, including diversity, ease of synthesis, biocompatibility, high specificity, and excellent pharmacological efficacy, peptides hold great potential as part of nanotechnology approaches against sepsis. Herein, we present a comprehensive and up-to-date review of the applications of peptides in nanosystems for combating sepsis, with the potential to expedite diagnosis and enhance management outcomes. Firstly, sepsis pathophysiology, antisepsis drug targets, current modalities in management and diagnosis with their limitations, and the potential of peptides to advance the diagnosis and management of sepsis have been adequately addressed. The applications have been organized into diagnostic or managing applications, with the last one being further sub-organized into nano-delivered bioactive peptides with antimicrobial or anti-inflammatory activity, peptides as targeting moieties on the surface of nanosystems against sepsis, and peptides as nanocarriers for antisepsis agents. The studies have been grouped thematically and discussed, emphasizing the constructed nanosystem, physicochemical properties, and peptide-imparted enhancement in diagnostic and therapeutic efficacy. The strengths, limitations, and research gaps in each section have been elaborated. Finally, current challenges and potential future paths to enhance the use of peptides in nanosystems for combating sepsis have been deliberately spotlighted. This review reaffirms peptides' potential as promising biomaterials within nanotechnology strategies aimed at improving sepsis diagnosis and management.

Graphical Abstract

• Due to their unique characteristics, Peptides hold significant promise as part of nanotechnology approaches for diagnosing and treating sepsis, a current leading global killer.

• Various diagnostic nanotools utilizing peptides as pathogen recognition moieties can improve the pathogen capturing efficiency for sepsis diagnosis.

• Nano-delivery can overcome the limitations of bioactive peptides and enhance their antibacterial and anti-inflammatory efficacy in sepsis management.

• Peptides offer significant capabilities as targeting moieties and nanocarriers to augment the effectiveness of antisepsis agents.

• Future research identified can potentiate the applications of peptides for the diagnosis and treatment of sepsis.

Introduction

Bacterial infections are still a major public health concern [ 1 ]. As per WHO reports, infections due to antimicrobial-resistant organisms resulted in 1.27 million deaths in the year 2019 and contributed to 4.95 deaths globally [ 2 ]. It is estimated that 700,000 people die each year worldwide, which is expected to rise to 10 million by 2050 [ 3 ]. The problem of mortality and morbidity of bacterial infections is made worse due to associated complications such as sepsis [ 4 ]. An estimated 48.9 million incident instances of sepsis were reported globally in 2017, leading to 11 million fatalities, accounting for 19.7% of all deaths worldwide [ 5 ]. With the emergence of the COVID-19 pandemic, antimicrobial resistance continues to gain ground and exacerbates bacterial sepsis, which is now the leading cause of death from infections [ 6 ]. If not detected and treated timely, sepsis can progress to septic shock, multiple organ failure, and death due to cardiovascular, coagulation, and endothelial dysfunction [ 7 ]. Accordingly, sepsis is a critical worldwide health problem with life-threatening implications, necessitating immediate attention to developing novel and powerful diagnostic and therapeutic strategies.

Nanoplatforms are providing new avenues for critical illnesses diagnosis and treatment [ 8 ]. These platforms offer cutting-edge approaches for disease diagnosis, enhancing sensitivity and decreasing processing time Without the necessity for specialized expertise [ 9 ]. Moreover, nanoplatforms can be fine-tuned to overcome conventional dosage forms' limitations by enhancing loaded drug pharmacokinetic and pharmacodynamic characteristics through disease site targeting, stimuli responsiveness, and mimicking disease pathophysiology [ 10 , 11 ]. These positive attributes enable the use of lower drug concentrations, co-loading of different drugs in the nanosystems, having multi-responsive systems that respond to different disease environments, hence reducing systemic toxicity and improving therapeutic effectiveness [ 12 ]. These distinguishing characteristics of nanoscale drug formulations make them promising candidates for enhancing the efficiency of existing conventional antibiotics against multidrug-resistant bacteria [ 13 ]. Compared to conventional preparations, nano-antimicrobial formulations have demonstrated superior outcomes in managing sepsis [ 14 , 15 ]. Therefore, nanotechnology-based systems provide efficient tools to decrease the burden of bacterial infections and sepsis.

The advancements and improvement of nanosystems' functionality require synthesizing bio-functional materials that can be employed in formulating them. Peptides are emerging as useful biomaterials for the formulation of nanosystems [ 16 ]. Peptides are a class of biological molecules composed of short chains of around 50 amino acids or less joined together by amide bonds [ 17 , 18 ]. Due to the infinite possibilities of joining amino acids, peptides can serve as pathogens and biomarkers capturing motifs, bioactive agents, or as excipients in making diagnostic biosensors and drug delivery systems. As components in nanotools for sepsis diagnosis, peptides can be designed to have specific binding with high affinity to causative pathogens and sepsis-released inflammatory biomarkers, thus making diagnostic procedures more efficient and prompter [ 19 ]. Furthermore, peptides can be incorporated within these nanoplatforms to improve the stability and binding of sepsis biomarkers-capturing immune-colloids to mesoporous nano-templates for sepsis immunoassays [ 20 ]. Therefore, peptide-based nanoplatforms hold promising potential in advancing sepsis diagnosis, allowing for efficient and rapid interventions that will improve patient outcomes.

In sepsis management, bioactive peptides have been found to exhibit therapeutic and protective properties against sepsis and so provide effective new treatment options for patients suffering from this deadly condition [ 21 , 22 ]. Among different classes of bioactive peptides, antimicrobial peptides (AMPs) are naturally existing peptides that have the ability to fight microbial infections and their related complications, such as sepsis [ 23 ]. Due to their novel antimicrobial modes of action, robust antimicrobial efficacy, minimal drug residual, and simplicity of production and modification, AMPs have held significant potential as a promising alternative to antibiotic therapy over decades [ 24 ]. More importantly, it is shown that antimicrobial resistance levels developed by AMPs are substantially lower as they target a variety of mechanisms that are not targeted by traditional antibiotics [ 25 ]. Additionally, anti-inflammatory peptides (AIPs) demonstrated beneficial effects in bacterial infections and sepsis management. The use of these peptides has been shown to help reduce inflammation by targeting various sites in the sepsis inflammatory cascade, thus reducing the amount of tissue and organ damage associated with sepsis and making the treatment more effective [ 26 , 27 , 28 , 29 , 30 ]. Overall, the unique properties and potential for applying AMPs and AIPs in bacterial infections and sepsis make them a promising area of research for developing new treatments. Nevertheless, bioactive peptides have certain drawbacks regarding bioavailability and tolerability (Teixeira et al. 2020), and research focuses on improving their potency and safety profile.

One of the ways the therapeutic profile of bioactive peptides is being improved is through encapsulation in nano-delivery systems [ 31 ]. Moreover, due to their diversity and ability to produce secondary nanostructures, bioactive peptides may be modified to form nanomaterials with enhanced characteristics [ 32 , 33 ]. Apart from their biological activity and due to their superior physical, chemical, and biological characteristics, peptides have emerged as a potential constituent for the development of nanosystems for targeted delivery of drugs and genes [ 34 , 35 , 36 ]. Peptides can be employed in drug delivery technologies as nanocarriers, cell penetration enhancers, and targeting agents [ 17 , 36 ]. Consequently, peptide-based nano-delivery systems have been developed and applied to treat a wide range of illnesses, such as sepsis, cancer, viral infections, and immune system disorders [ 37 , 38 , 39 , 40 , 41 , 42 ].

Numerous review articles have highlighted the application of peptides in nanotechnology-based bacterial infection management [ 43 , 44 ] and the use of nanotechnology for AMPs delivery against general bacterial infections [ 45 , 46 , 47 , 48 , 49 ]. Additionally, several publications have reviewed the use of nanotechnology to manage sepsis [ 14 , 19 , 50 , 51 ]. To the best of our knowledge, no review has discussed the various applications of peptides in nanosystems for diagnosing and managing bacterial sepsis.

Therefore, this review focuses on the numerous applications of peptides in nanosystems to identify and control bacterial sepsis. Initially, a theoretical background about the pathophysiology of sepsis, challenges associated with sepsis diagnosis and management, drug targets in sepsis, and peptides' physicochemical properties and their potential for application in nanotools against sepsis are presented. In addition, following a thorough search of many scientific databases, we discuss and critically analyze different applications of peptides in nanotechnology for sepsis diagnosis and management. The studies have been organized into two main sections, viz. diagnostic and management peptides-based nanosystems. We have further systematically categorized the nanosystems for sepsis management according to the role of peptides into: (i) nano-delivered bioactive peptides; (ii) peptides as targeting moieties on the surface of nanosystems; (iii) peptides as nanocarriers for antisepsis drug. Finally, this review highlights the challenges, gaps, and future perspectives to maximize the potential of applying peptides in nanotechnology tools to improve sepsis diagnosis and management.

A clear understanding of the pathophysiology of sepsis and potential drug targets is critical for developing new effective sepsis diagnostic and therapeutic tools. This section will discuss the pathophysiological background of sepsis, including the inflammatory pathways triggered by invading microorganisms and the consequences of that on the structure and function of body organs. The current trends in sepsis diagnosis and management and potential drug targets for sepsis management will also be discussed, accompanied by their challenges. Finally, the physicochemical and biological properties that make peptides a potential component of nanotools for sepsis diagnosis and management are covered.

Pathophysiology of sepsis

Sepsis is a medical emergency and a life-threatening condition associated with a global disease burden [ 52 ]. Despite all experimental and clinical research efforts, sepsis remains one of the leading causes of morbidity and mortality in critically ill patients [ 53 ]. In the Third International Consensus (Sepsis-3), sepsis is defined as "organ dysfunction caused by a dysregulated host response to infection", highlighting for the first time the critical role of immune responses in the establishment of the illness [ 7 ]. After the invasion of microorganisms into the body, an immune response is triggered to fight off the invading microorganisms. This causes inflammation, a normal and necessary response to promptly identify, eradicate, and keep the infection localized [ 54 ]. However, as shown in Fig. 1 , the immune response is exaggerated during sepsis, resulting in collateral damage and death of host cells and tissues, compromising both the affected and distant organs and leading to functional abnormalities and life-threatening multiorgan failure [ 55 ]. The pathophysiology of sepsis is generally defined as an early hyperinflammatory state that lasts many days, followed by a longer immunosuppressive state [ 56 ]. These two stages are connected with higher mortality, with the highest death rate in the early phase attributable to an enormous inflammatory response (cytokine storm) [ 14 ].

Immune responses in sepsis owing to infection. Illustration of converting to sepsis from infection. Immune cells activation results in the overproduction of inflammatory mediators that induce detrimental changes in cells and tissues, leading to multiorgan dysfunction and failure (SOFA: sequential organ failure assessment; EWS: early warning score; iNOS: inducible nitric oxide synthase; ARDS: acute respiratory distress syndrome) (Adopted with permission from [ 55 ]

The over-released inflammatory mediators during the cytokine storms lead to significant damage to the endothelium and disruption of it is barrier function, vasodilation, activation of coagulation pathways, platelet aggregation and adhesion, and mitochondrial dysfunction [ 57 , 58 ]. Overall, the dysregulated inflammatory-immune responses and their consequences mentioned above eventually lead to the formation of microvascular thrombi, hypotension, impaired cellular functions, local perfusion defects, tissue hypoxia, and progressive tissue damage, which finally cause refractory shock and multiorgan failure [ 59 , 60 , 61 , 62 ]. Cardiovascular Dysfunction, acute lung injury and acute respiratory distress syndrome, acute kidney injury, hepatic dysfunction, and CNS dysfunction and encephalopathy are well-known complications of sepsis, and their underlying mechanisms are reported in the literature [ 63 , 64 , 65 , 66 , 67 , 68 ]. These alterations and dysfunctions in the tissues and organs collectively contribute to much of the morbidity and mortality of sepsis [ 69 ].

Although the advancements in therapeutic approaches have enhanced the survival rate in the early phase of the exaggerated inflammatory response, current patterns in sepsis indicate that mortality arises during the subsequent stage of a compensatory immunosuppressive response when there is a shift toward an overall anti-inflammatory milieu [ 56 , 70 ]. This post-sepsis immune paralysis involves various quantitative and functional defects of immune cells as a result of uncontrolled apoptosis of lymphocytes and decreased immunoglobulin production, which is linked to an increased susceptibility to secondary infections and organ injury and/or failure [ 14 , 60 , 71 , 72 ]. Immunosuppression can last months after the septic event and is associated with increased mortality [ 73 , 74 ]. The detailed sepsis pathophysiology and different involved pathways have been widely discussed in the literature, and readers are referred to them for more details [ 55 , 67 , 69 , 73 , 75 , 76 , 77 ].

Sepsis diagnosis and management

Sepsis is considered a medical emergency that, if not diagnosed in its early stages, will result in a poor prognosis with increased morbidity and mortality [ 78 ]. The current diagnosis of sepsis relies on clinical evaluation, blood or urine cultures, and detection of inflammatory response biomarkers such as C-reactive protein (CRP), procalcitonin (PCT), and interleukin 6 (IL-6) [ 19 ]. However, the currently used biomarkers are not specific, and none have proven to be a specific sepsis indicator [ 79 ]. Moreover, the microbial cultures take a long time, and results may come out after 72 hours, making rapid sepsis diagnosis difficult [ 80 ]. Starting sepsis management as early as possible is critical to avoid complications and multiorgan failure [ 81 ]. As the current diagnostic tools for sepsis have such a delay, the empirical administration of intravenous broad-spectrum antibiotics is a usual initial intervention together with other additional therapies (e.g., anti-inflammatory (corticosteroids) and venous thromboembolism prophylactics) and measures for ventilation and hemodynamic stabilization (e.g., oxygen, albumin, and vasopressors administration and fluid resuscitation) [ 82 ]. However, the empirical use of broad-spectrum antibiotics with the uncertainty of diagnosis results and difficulties in differentiating infectious sepsis from noninfectious inflammations [ 83 ] will result in unwanted side effects for the already stressed patient and increased risk of antimicrobial resistance development [ 81 , 84 ]. Putting all these challenges together raises the urgent need for new and specific sepsis diagnostics and management approaches.

Drug targets in sepsis

As mentioned above, treating sepsis involves a combination of antibiotics to fight the underlying infection and supportive care to address the systemic inflammation and organ dysfunction that can occur because of the condition [ 85 ]. One of the key challenges in treating sepsis is identifying effective drug targets that can help reducing inflammation and tissue damage [ 86 ]. Besides targeting the invading microorganisms with antibiotics, several drug targets have been identified and studied in sepsis management [ 87 , 88 ]. One of the main drug targets in sepsis is the inhibition of inflammatory mediators that play a critical role in developing sepsis, including cytokines, chemokines, and other inflammatory signaling molecules [ 89 ]. Drugs that target these inflammatory mediators have been developed and tested as potential treatments for sepsis [ 90 ]. For example, monoclonal antibodies that neutralize TNF-α, such as infliximab and etanercept, have been shown to improve outcomes in patients with sepsis [ 91 ]. Similarly, drugs that inhibit the activity of IL-1 (e.g., IL-1 receptor antagonist), IL-6, and IL-8 have also been shown to improve outcomes in patients with sepsis [ 92 ].

The coagulation cascade is another critical therapeutic target in sepsis [ 93 ]. As sepsis is associated with a hypercoagulable state, targeting the coagulation process with drugs such as anticoagulants or clotting factor inhibitors can prevent micro-clots formation and improve outcomes in sepsis [ 94 ]. Drugs that target the coagulation cascade, such as activated protein C (APC) and thrombin inhibitors, have been studied as sepsis therapies and demonstrated to enhance sepsis outcomes [ 88 , 95 ]. Furthermore, the endothelial cells that line blood vessels play a crucial role in the body's reaction to inflammation and infection [ 96 ]. As a result, targeting the endothelium to manage sepsis is an active area of research [ 96 ]. In sepsis, the dysfunction of these cells can lead to increased permeability of blood vessels and decreased blood flow and so leakage of fluid and plasma protein into the tissues, resulting in hypotension and organ dysfunction [ 97 ]. One of the key pathways activated in the endothelium during sepsis is the nitric oxide (NO) pathway, leading to excessive vasodilation and decreased blood pressure [ 98 ]. Drugs that target the NO pathway, such as nitric oxide synthase inhibitors, have been investigated as potential treatments for sepsis [ 99 ]. Another promising strategy is the use of drugs that can improve endothelial function and reduce inflammation [ 100 ]. For example, endothelial protective agents, such as statins, have been shown to reduce inflammation and improve blood flow in sepsis [ 101 ]. In addition, new approaches, such as using extracellular vesicles as drug carriers for targeting the endothelium, are also being explored [ 102 ].

The complement system, a part of the immune system that helps identify and eliminate foreign invaders such as bacteria and viruses, is also one of the potential drug targets for sepsis management [ 103 ]. In sepsis, the complement system is overactivated, which can lead to inflammation and tissue damage [ 104 ]. As a result, targeting the complement system has been investigated for managing sepsis [ 105 ]. One approach to target the complement system in sepsis management is using complement inhibitors, which are drugs that block the activation of the complement system [ 106 ]. For example, eculizumab, a monoclonal antibody that targets the complement protein C5, has been shown to reduce the incidence of death and organ failure in patients with sepsis caused by meningococcal infections [ 107 ].

Bacterial toxins such as lipopolysaccharides (LPS) from Gram-negative bacteria, exotoxins from Gram-positive bacteria, and superantigens from both Gram-positive and Gram-negative bacteria are also potential targets in the management of sepsis [ 108 ]. Bacterial toxins can contribute to the development of sepsis by triggering the release of inflammatory cytokines and damaging vital organs and tissue, leading to septic shock and death [ 109 ]. Inhibiting the production or activity of these toxins can prevent toxicity and improve outcomes in sepsis management [ 108 ]. Various strategies are being studied, such as blocking toxins' binding to host cells, inhibiting their production, or neutralizing them using toxin-binding proteins or immunoglobulins [ 90 , 110 ]. Even though developing therapies that target bacterial toxins is a promising area of research for treating sepsis, more research needs to be done to understand how these drugs work fully and if they are safe to use in clinical settings.

Overall, managing sepsis remains a complex and challenging task, and there is currently no single drug that can effectively address all the different pathways and processes involved in the condition. Further research is needed to identify additional drug targets and to develop more specific and effective medicines for sepsis.

Potential of peptides for use in sepsis

Several drugs have failed in the treatment of sepsis. However, continued research and improved understanding of sepsis pathophysiology, including the complex interactions between inflammatory, coagulation, and fibrinolytic systems, has accelerated the development of novel treatments [ 88 , 111 ]. Some of these drugs being researched and developed are peptides that hold great promise as therapeutic agents for treating sepsis [ 112 , 113 , 114 ]. As amino acids can be used in infinite arrangements in peptide synthesis, then peptides can be designed to have a wide range of unique physicochemical and biological properties. These unique properties include: 1) High specificity: Peptides may be designed to specifically target specific pharmacological targets, making their action highly selective with reduced off-target effects [ 115 ]; 2) Ability to target multiple pathways: Peptides can target multiple pathways in the sepsis cascade, which can help reduce the chances of antimicrobial resistance [ 19 ]; 3) Biodegradability: Inside the body, peptides are broken down into smaller biocompatible components, making them biodegradable with fewer side effects than traditional small molecule drugs [ 116 ]; 4) Ease of synthesis: Peptides can be synthesized using modern techniques in a laboratory setting, making it possible to produce cost-effectively large quantities of specific peptides for drug development [ 116 ]; 5) Permeability and bioavailability: some peptides can cross cell membranes, which increases their bioavailability and allows them to target intracellular targets in sepsis pathophysiology [ 117 ].

The properties mentioned above make peptide synthesis a promising approach for developing new diagnostic and management tools that can efficiently diagnose sepsis and effectively target both the bacteria causing sepsis and the pathophysiological pathways involved in the disease. For example, peptides have been designed to have strong and specific binding affinity to certain pathogens and inflammatory biomarkers, making them excellent capturing motifs for the diagnosis of sepsis [ 118 ]. Moreover, antimicrobial peptides have been shown to have potent bactericidal activity against Gram-negative and Gram-positive bacteria that are commonly associated with sepsis [ 119 ]. In addition to their antibacterial properties, peptides can target the pathophysiological pathways involved in sepsis, such as inflammation, oxidative stress, complement system, and coagulation [ 114 , 120 ]. Furthermore, the excellent physicochemical and biological properties of peptides make them hold great potential as drug delivery systems construction materials for antisepsis drug delivery [ 121 ]. For example, peptides for drug delivery can be designed to respond to the characteristic microenvironment of sepsis, including acidity and high concentrations of reactive oxygen species (ROS) [ 121 ]. This allows for site-specific drug release, so good therapeutic outcomes with low side effects can be achieved with small doses of drugs that improve patient compliance and decrease the treatment cost. As a result, the application of peptides in sepsis diagnosis and management is an active area of research with promising outcomes that make them an attractive option in the battle against sepsis.

Application of peptides in nanotools for sepsis diagnosis and management

Sepsis continues to pose a significant global health challenge despite remarkable advancements in medical technology. Its high mortality rates and limited effective diagnosis and treatment approaches underscore the urgency to develop new, efficient, and novel techniques for accurate diagnosis and timely intervention [ 79 , 122 ]. The application of nanotechnology-based tools presents a multitude of opportunities for advancing sepsis diagnosis and management [ 14 ]. With their distinctive properties, peptides have emerged as promising candidates for developing nanotools to combat critical illnesses like sepsis [ 112 , 114 ]. Figure  2 provides a visual representation of the multiple roles peptides play as components within nanosystems for sepsis diagnosis and management. This section aims to explore and critically review the diverse range of research studies that have employed peptides as integral elements of nanotools for sepsis diagnosis and management, encompassing both pharmacological and pharmaceutical applications.

A schematic illustration of various applications of peptides in nanotools for sepsis diagnosis and management (NPs: Nanoparticles; AIPs: Anti-inflammatory Peptides; AMPs: Antimicrobial Peptides) (Created with BioRender.com)

Peptides in nanotechnology for sepsis diagnosis

The timely diagnosis of sepsis is crucial to ensure effective treatment outcomes [ 78 ]. Although they can offer a promising avenue for early detection of sepsis, the use of peptides in developing nanotechnology tools for sepsis diagnosis is still in its infancy. Table 1 summarizes various studies done on the utilization of peptides in nanotools for sepsis diagnosis, highlighting the type of nanosystem, the peptide sequence, the role of peptide in the nanosystem, the targeted microorganisms or biomarkers, the mechanism of detection, the mode of investigation (in vitro and/or in vivo), and the key findings. As shown in the table, peptides have been mainly used for two different roles. The major role was using peptides as pathogen recognition moieties conjugated to the surface of magnetic or fluorescent nanoparticles to allow the capturing of bacteria and then separation or imaging. The other role has been the utilization of peptide as a thiol-rich moiety to enhance the binding of immuno-colloidal metallic nanoparticles to the surface of a mesoporous Surface-enhanced Raman scattering (SERS) template. It is clear that there is great potential for further research to uncover additional roles that peptides can play in nanotools for sepsis diagnosis. Moreover, most studies have focused on Gram-positive bacteria, indicating the need to address Gram-negative bacteria, which significantly influence the development of bacterial sepsis. The studies will be discussed according to the role of peptides in the nanosystem in the following subsections.

Peptides as pathogen capturing motifs on nanoplatforms    

To date, various approaches for detecting, capturing, and separating bacteria from blood have been developed to improve the diagnosis and management of bloodstream infections [ 126 , 127 ]. Various pathogen recognition molecules, such as aptamers, antibodies, oligonucleotides, and carbohydrates, have been used to modify nanoplatforms for pathogens detection and separation [ 19 , 128 , 129 ]. Since the bacterial surface displays unique molecular compositions, a well-designed peptide can have a specific and robust interaction with an epitope or a receptor on the bacterial walls. When engineered on the surface of nanoparticles, these peptides will yield an efficient nanotool for pathogen capturing with improved efficacy in sepsis diagnosis [ 130 ]. The subsequent paragraphs will discuss the findings of various studies that have investigated the utilization of peptides as capturing motifs exhibited on magnetic nanoparticles or fluorescent quantum dots to fabricate nanoplatforms for pathogen detection and then isolation or imaging.

Magnetic nanoparticles are promising nanoplatforms that can be effectively engineered with peptides as pathogen-capturing motifs for detecting and separating bacteria from human samples in simple and controllable processes [ 79 , 131 , 132 ]. Among different magnetic nanoparticles, magnetic beads (MBs), known for their superparamagnetic characteristics, have demonstrated their versatility in detecting, purifying, and analyzing analytes from intricate matrices. In order to achieve selectivity, MBs can be complexed with ligands such as peptides, aptamers, and antibodies to target the concerned pathogen selectively [ 133 ]. To this end, Feng et al., as illustrated in Fig. 3 , have developed a vancomycin (Van)-modified magnetic nanoplatform composed of a dendrimer (G4 PAMAM) anchored with biotin and complexed with streptavidin-modified magnetic beads (MBs-S) to detect and isolate L. monocytogenes and S. aureus from human blood samples [ 123 ]. The glycopeptide antibiotic vancomycin (Van) can interact with the surface of the bacterial wall via hydrogen bonding and works as a pathogen recognition molecule [ 134 ].

Detection of S. aureus and L. monocytogenes using a two-step approach paired with m-qPCR, utilizing the MBs-S~Bio-den-Van~bacteria complex [ 123 ]

They combined the MBs-S~Bio-den-Van platform with a multiplex quantitative PCR (m-qPCR) to enrich and identify the isolated pathogens. The platform demonstrated rapid bacteria isolation within 2 minutes and exhibited a capturing efficiency of 93.14% for L. monocytogenes and 94.58% for S. aureus from spiked healthy donors’ whole blood. The platform showed high sensitivity with limits of detection of 32 and 41 CFUmL -1 for L. monocytogenes and S. aureus , respectively. Their findings exhibited several potential advantages for bacterial detection in septic patients, including processing simplicity, low cost, high stability and specificity, and low detection limit. However, it should be noted that Van has the ability to bind to different types of Gram-positive bacteria, but it cannot bind to Gram-negative bacteria because of the differences in the components of their outer cell wall. Therefore, we believe this nanoplatform efficiency would be limited in cases of Gram-negative or mixed infections due to the specificity of Van to Gram-positives bacteria.

Another type of magnetic nanoparticles that can be modified with peptides to capture and separate bacteria from human samples are superparamagnetic iron oxide nanoparticles (SPIONs). SPOINs' non-toxicity, controllable size, large surface area-to-volume ratio, and ability to be functionalized with targeting moieties make them promising tools for early detection and diagnosis of diseases [ 135 , 136 ]. For instance, Friedrich and coworkers developed a 3-aminopropyl triethoxysilane (APTES)-coated superparamagnetic iron oxide nanoparticles modified with bacterial cell wall-binding peptides for bloodstream bacterial pathogens separation [ 118 ]. The two peptide sequences (Pep1 (RKQGRVEVLYRASWGTV) and Pep2 (RKQGRVEILYRGSWGTVC)) were obtained from the salivary glycoprotein GP-340, which is known to interact with bacterial cell wall components [ 137 ]. A modified one-step coprecipitation approach was used to produce the SPION-APTES with a hydrodynamic diameter of 166 nm, which formed nanoparticle agglomerates of 1679 nm diameter after peptide functionalization.

The nanoparticles were found to be cyto- and hemo-compatible. As shown in Fig. 4 , whole blood samples from healthy volunteers spiked with Gram-negative ( E. coli , S. marcescens, S. enterica, S. enteritidis , and P. aeruginosa ) and Gram-positive ( S. aureus ) bacteria were used to test the separation efficiency. S. aureus had an above 60% removal rate, E. coli and S. marcescens over 50%, and P. aeruginosa separation was only 35%. Besides the diagnostic role, the system showed a strong inhibition of cytokines (TNF-α, IL-6, IL-1β, Il-10, and IFN-γ) release. It is worth noting that this dual and simple theranostic approach could hasten both diagnosis and management for patients suspected to have sepsis. However, as the platform showed reduced efficiency in Gram-negative bacteria, we are of the view that this will limit its applicability, especially in the cases of mixed infections. Moreover, the separation efficiency was found to be affected by the anticoagulant employed and the concentration of Ca 2+ ions in the blood collection tubes. As a result, it is imperative to underscore that this may affect the system's applicability in emergency situations where blood collection tubes with the suitable anticoagulant are unavailable.

Experimental setup of separation of bacteria from blood using SPION-APTES-Pep (Adopted from [ 118 ])

To further improve the performance of magnetic iron oxide nanoparticles, coating with a layer of polyethylene glycol (PEG) can be performed. PEGylation provides improved stability, biocompatibility, and magnetic properties. PEGylation also reduces non-specific binding and potential immunogenicity, making the magnetic nanoparticles more suitable for various biomedical applications such as diagnosis and targeted drug delivery [ 138 ]. On this point, Pan et al. developed a peptide-modified PEGylated-iron oxide composite nanoclusters (peptide@PEG@MNCs) to isolate and identify S. aureus form blood samples [ 124 ]. The peptide (SA5-1: VPHNPGLISLQG) was chosen from a bacteriophage display library for its ability to selectively attach to the cell surface of S. aureus . SA5-1 was chemically bonded to the PEGylated magnetic nanoclusters (PEG@MNCs), resulting in the formation of the peptide@PEG@MNCs particles with an average diameter of 150.8 ± 1.8 nm and good cytocompatibility and magnetic properties. Human serum spiked with different bacterial strains, including E. coli , P. aeruginosa , S. aureus (susceptible and resistant strain), and S. epidermidis, was utilized to demonstrate the capturing efficacy under conditions simulating sepsis. A capture efficiency of over 70% was achieved for all tested microorganisms within 10 minutes. When a rinsing step was introduced to the process, only S. aureus was detected, notably, indicating the system's ability to selectively capture S. aureus , which was anticipated to the strong affinity of the peptide to S. aureus pathogens. Nevertheless, as the capture efficiency declined with increasing the bacterial concentration, we believe this may affect the applicability of the platform if high bacterial load exists in patients' blood samples.

In summary, the findings from the three studies indicate that peptides-modified magnetic nanoparticles have significant promise as a rapid and effective diagnostic tool for bacterial sepsis. Regarding efficacy, Feng et al.'s system showed a significantly higher capturing efficiency of S. aureus compared to the other two systems by Friedrich et al. and Pan et al. Although the second study by Friedrich et al. included Gram-negative bacteria in their evaluations, their system’s capturing efficacy was again higher for Gram-positive than Gram-negative bacteria. Interestingly, Pan et al. in their study were successfully captured both Gram-positive and Gram-negative bacteria and, at the same time able to enhance the system’s specificity toward Gram-positive bacteria by introducing a rinsing step; consequently, we contend that their approach was more flexible and comprehensive for sepsis diagnosis applications. Notably, Friedrich et al.'s system stood out for its unique ability to inhibit the release of cytokines, which will add value to the therapy of septic patients, in addition to its diagnostic use. Overall, the advantages and limitations of each one of them should be considered when applying them in clinical practice.

While the other three studies have used peptides as capturing motifs expressed on magnetic nanoparticles for bacterial separation from blood samples, another study conducted by Shrivastava and coworkers [ 125 ] applied the pathogen-capturing properties of peptides for in vivo imaging of bacteria inside the body. Imaging techniques for bacteria play an essential role in various aspects of diagnosing infections and understanding their pathogenesis [ 139 ]. Using in vivo imaging techniques, researchers can gain insights into the spatial distribution of bacteria within the host, track their movement, and understand the dynamics of infections [ 140 ]. Various molecular probes have been employed to target biological processes during infections. Nonetheless, visualizing bacteria in vivo remains challenging due to the inherent difficulty of targeting the specified bacteria directly [ 141 ]. Hence, there exists a necessity to identify highly efficient materials capable of precisely targeting specific pathways within bacterial internal processes that play a crucial role in their pathogenicity and so allow for efficient in vivo imaging of bacterial pathogens.

A potential solution to enhance the in vivo imaging of bacteria lies in using biomaterials capable of selectively targeting the quorum sensing (QS) communication employed by many bacteria to coordinate the expression of virulence genes during infections [ 140 ]. Based on that, Shrivastava and colleagues have developed a new fluorescent quorum-based nano-bio probe (QNBP) to monitor the localization of multiple-drug resistant S. aureus (MRSA) bacteria in vivo. An auto-inducing peptide (AIPq) with the ability to target the MRSA accessory gene regulator (AGR) QS system has been conjugated onto a fluorescent quantum dot (QD) surface to develop the QNBP system [ 125 ]. The nano-bio probe was assessed in vitro for bacterial binding characteristics and in vivo for imaging the bacteria in a mouse model. The QNBPs showed higher selectivity in binding to AGR-positive virulent strains than the mutant strain, thus confirming its suitability for in vivo imaging of pathogenic S. aureus. The QNBP was also capable of penetrating MRSA biofilm and effectively image embedded colonies, giving a new approach for identifying MRSA embedded in biofilms [ 142 ]. When employed as a fluorescence probe for in vivo imaging of MRSA in a systemic infection mouse model, QNBP resulted in the detection of robust fluorescent signals in most infected organs and high-quality fluorescence images were acquired post-infection. It is to be noted that this peptide-based nano-bio probe can offer new perspectives into exploration of infection pathways in vivo and aid in the diagnosis and management of life-threatening infections such as MRSA-induced sepsis. However, we argue that the need for advanced instruments such as fluorescence spectrophotometer and confocal laser scanning microscopy for recording the results may restrict the practical application of this system.

Peptides as thiol-rich moiety for binding of metallic colloids to mesoporous templates

Surface-enhanced Raman spectroscopy (SERS) is a surface-sensitive technique that enhances Raman signals of molecules adsorbed on metallic nanostructures such as plasmonic-magnetic silica templates. SERS templates modified with SERS- tags specific to certain disease biomarkers can effectively detect and analyze these biomarkers, aiding in disease diagnosis [ 143 , 144 ]. Metallic colloids, such as gold nanoparticles, when functionalized with Raman dyes and disease biomarkers-specific antibodies and subsequently immobilized on plasmonic mesoporous templates, hold great promise as SERS-based diagnostic tools for the detection of biomarkers levels [ 145 , 146 , 147 ]. Bacteriophages have the potential for synthesizing mesoporous templates; however, to incorporate metallic colloids into them, their surfaces must be chemically modified with thiol donors, which affects their critical properties, such as assembly and binding [ 148 ]. To overcome this, thiol moieties, such as cysteine-rich peptides, can be displayed on the phage during synthesis without chemical modification [ 149 ]. In this regard, Nguyen and colleagues have developed a mesoporous SERS substrate based on M13KE phage displaying a cysteine-rich peptide (243bp: GBS101000616.1) as a template for sepsis biomarkers assay in human serum sample [ 20 ].

As shown in Fig. 5 , the surface of the template was magnetized with gold-coated magnetic nano-stars (Au-MNS) modified with a SERS-tag consisting of specific antibodies for three sepsis biomarkers (soluble Triggering Receptor Expressed on Myeloid cells-1 (sTREM1)), C-reactive protein (CRP), and procalcitonin (PCT) and a RAMAN dye for RAMAN signal amplification. The cysteine-rich peptide allowed strong binding of the Au-MNS when its thiol groups were reduced to active thiols. The SERS-based immunoassay was done on human serum samples, and the SERS spectra of the magnetically separated template exhibited characteristic peaks of the tags corresponding to the three biomarkers. The system demonstrated high sensitivity, excellent specificity, and low detection limits for CRP (27 pM), PCT (103 pM), and sTREM1 (78 pM). We believe this approach offers a potential alternate tool for the initial stages of sepsis monitoring. Despite this, it is to be mentioned that the fabrication complexity and risk of surface contaminants interference due to the high surface area of mesoporous templates could limit the scalability and reproducibility and interfere with the accuracy and reliability of SERS measurements in clinical settings.

Representation of the process from phage display to manufacturing of the SERS substrate. A Insertion of the cysteine-rich peptide into the major PV111 protein domain of the M13KE phage using two restriction enzymes (BtgZI and HinP1I). B The colony PCR analysis on a 1% agarose gel confirms the effective integration of the cysteine-rich peptide into the pVIII region of the M13KE plasmid. C Utilization of cysteine-rich peptide phage display for the production of SERS substrates. The phage was decorated with an immuno-colloid made of gold-coated magnetic nano-stars (Au-MNS) after treatment with tris (2-carboxyethyl) phosphine hydrochloride solution (TCEP) to activate the thiol groups. The phage was polymerized with silica precursor to give amorphous biomaterial gel and then calcinated to form the mesoporous template. After incubation of the template with a serum sample spiked with sepsis biomarkers, the complexes were separated using a magnet and subjected to Surface-enhanced Raman scattering (SERS) measurement (adopted from [ 20 ])

To conclude, the studies discussed in this section demonstrated the crucial role of peptides in sepsis diagnosis by either offering pathogen recognition capacities or enabling the binding of metallic nanoparticles to mesoporous templates. These approaches offer several advantages, including enhanced selectivity and sensitivity in detecting sepsis-causing pathogens, as well as the ability to provide rapid results. Nevertheless, relying on blood samples from healthy volunteers to assess the developed nanoplatforms may not accurately reflect the complexity of sepsis cases. Therefore, it is imperative to validate the clinical applicability of the developed nanoplatforms by testing them on blood samples obtained from sepsis patients. Such validation would ensure these nanoplatforms' accuracy, reliability, and effectiveness in real-world clinical settings.

Peptides in nanotechnology for sepsis management

Although numerous potential therapeutics for sepsis have been identified, achieving effective treatment remains a formidable challenge, and sepsis continues to threaten healthcare systems worldwide with its high mortality rates and complex pathophysiology [ 150 ]. In recent years, using peptides in nanotechnology has ignited new hopes for developing targeted and efficient therapeutic strategies for complex illnesses such as sepsis [ 151 ]. This section will delve into the developments and transformative potential of peptides in nanosystems to manage sepsis. The multifaceted applications of these peptides will be explored and discussed, including their utilization as bioactive agents with antimicrobial or anti-inflammatory properties delivered through nanosystems. Additionally, their role as nanosystem components, including nanocarriers for antisepsis drug delivery or as surface modifiers for nanosystems to target sepsis microenvironment or causative bacteria, will be highlighted and critically analyzed in the following subsections. An emphasis will be put on the manufacturing processes of nanosystems, key characterization and evaluation, and key findings of the studies.

Nano-delivered bioactive peptides

Antimicrobial peptides nanosystems.

Antimicrobial peptides (AMPs) are short chains of amino acids, typically ranging from 5 to 50 residues, which may be regarded as natural antibiotics synthesized by various organisms, including mammals, plants, protozoa, fungi, and bacteria. They may have amphipathic or cationic structural composition and can display a wide range of antimicrobial activity, targeting both Gram-positive and Gram-negative bacteria, fungi, viruses, and protozoa [ 152 ]. A complete understanding of the mechanism of action of AMPs remains elusive. Nevertheless, many mechanisms have been proposed, suggesting diverse interactions with phospholipid membranes of microorganisms [ 153 , 154 ]. In recent years, exploring AMPs as potential therapeutics has gained substantial momentum, driven by their remarkable antimicrobial activity and ability to overcome antimicrobial resistance [ 155 ]. However, the clinical translation of AMPs into effective sepsis treatments has been hindered by various challenges, including toxicity, limited stability and enzymatic degradation, and ununderstood pharmacokinetic profiles [ 156 ]. Therefore, robust delivery strategies are needed to allow effective use of AMPs in clinical settings.

The utilization of nanotechnology-based delivery systems has emerged as a promising trajectory for mitigating the aforementioned challenges and augmenting the therapeutic efficacy of AMPs. Literature has documented that nano-scaled delivered peptides exhibit diminished cytotoxicity, improved physiological stability, and increased efficiency at the desired target [ 157 ]. Hence, nano-delivery systems could enhance the physicochemical and pharmacological characteristics of AMPs, enabling their use as effective sepsis therapeutics in clinical practice. Table 2 illustrates different studies reported on nano-delivered AMPs for sepsis management, highlighting the type of nanosystem, the peptide sequence, the nano-delivery strategy, the targeted bacteria, the key evaluations, and the key findings. As illustrated, peptides have been either conjugated with other moieties to aid nanoscale self-assembly, encapsulated into nanosystems, or linked to the surface of nanoparticles. Evidently, most of the studies have focused on the self-assembly and nano-encapsulation of AMPs, indicating room for more research on the conjugation of AMPs to the surfaces of metallic and organic nanoparticles. The studies will be discussed in the following subsections according to the strategy of nano-delivery of AMPs.

Self-assembled AMP nanosystems

Designing peptides to self-assemble into nanostructures is a powerful strategy to bolster their stability, biosafety, and therapeutic outcomes, allowing their effective application in disease management [ 166 ]. The self-assembly of peptides is usually achieved by selecting amino acid series encompassing both hydrophilic and hydrophobic amino acids and linking them to yield amphiphilic structures capable of spontaneous self-assembly upon exposure to water. Alternatively, amphiphilic peptides can be engineered by conjugating them with hydrophobic motifs such as alkyl chains and fatty acids [ 166 ].

AMPs are among the bioactive peptides that can be designed to self-assemble and become more stable and effective [ 167 ]. In this regard, Lei et al. have successfully prepared self-assembled nanoparticles of the host-defense antimicrobial peptide, human alpha-defensin 5 (HD5), with improved stability, biosafety, and antibacterial effectiveness [ 158 ]. To introduce hydrophobicity and promote the nano-assembly of HD5, they conjugated myristic acid, a 14-carbon chain saturated fatty acid, to the C-terminus of HD5 to produce myristoylated HD5 (HD5-myr). HD5-myr spontaneously self-assembled in aqueous media, producing spherical-shaped nanoparticles -termed Nanobiotic- that were found to be hemocompatible and unlike the native HD5 resistant to proteolytic enzymes. The Nanobiotic exhibited significantly enhanced broad-spectrum bactericidal activity in vitro against different Gram-positive and Gram-negative bacterial strains, including S. aureus, MRSA , E. coli, A. baumannii, P. aeruginosa, and K. pneumoniae, compared to the free HD5 . The Nanobiotic was stable and retained the antibacterial efficacy even in the presence of proteolytic enzymes and high salt concentration, while the free HD5 underwent extensive hydrolysis within 24 hours. In the in vivo studies, the Nanobiotic exhibited protective effects, effectively rescued mice from E. coli -induced sepsis, and improved their survival rates by reducing the overall bacterial load in the body and preventing organ damage. Their outcomes have shown that the supramolecular assembly of AMP to make nanoparticles has tackled peptides' instability problem and achieved good antibacterial activity both in vitro and in vivo. We believe this work provides a promising candidate for treating bacterial sepsis that can be simply scaled up and manufactured.

Utilizing the same concept of peptide-conjugates assembly into nanoparticles, Tan and coworkers designed self-assembling chimeric peptide nanoparticles to treat bacterial infection and sepsis [ 159 ]. As shown in Fig. 6 , the AMP peptide sequence (PFPFPFP-KPKPKPKPKPKP-NH2) was linked to a 14-carbon alkyl chain to provide hydrophobic properties and modified with PEG domain at various locations to offer stealth effect and biocompatibility. The peptide amphiphiles self-assembled into nanoparticles of around 20-50 nm in diameter, which have shown good in vitro and in vivo biocompatibility. The self-assembled nanoparticles demonstrated broad-spectrum antibacterial activity against various strains of E. coli (MICs: 7.3 to 12.3 μM) and S. aureus (MICs: 5.3 to 10 μM) even in the presence of high concentrations of proteases and different salt conditions. Moreover, no spontaneous antimicrobial resistance to the peptide nanoparticles was detected when the E. coli ATCC25922 strain was subjected to sub-MIC dose treatment. In vivo, the nanoparticles have also demonstrated the ability to alleviate E. coli sepsis in mice and piglets and significantly reduced organs' bacterial load and the concentrations of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β). We argue that this approach provides an effective strategy to accelerate the clinical translation of newly developed peptides to meet the real need for effective sepsis therapies and to fight the growing antimicrobial resistance.

The structural design of self-assembling chimeric peptide. The peptide sequence comprises hydrophobic and cationic amino acids. The peptide is linked to the hydrophobic alkyl chain to enhance the self-assembly and the hydrophilic PEG unit to provide a stealth effect and improve biocompatibility (adopted from [ 159 ])

Carrying on with the application of peptide nano-assembly, Pan and colleagues reported two studies [ 22 , 160 ] on the development of co-assembled antibacterial peptide polymeric nanoparticles (AMPNP) for targeting bacteria and inflammation sites to combat bacterial sepsis. They synthesized antibacterial peptide (KR-12: KRIVKRIKKWLR)-grafted amphiphilic block copolymer and biotin grafted block copolymer, which were co-assembled in aqueous solution to produce the AMPNP. After that, as illustrated in Fig. 7 , they tried two different approaches to achieve AMP-targeted delivery against sepsis. In their first approach [ 22 ], they modified the AMPNP with an antibody against intercellular adhesion molecule-1 (anti-ICAM-1 antibody) to target the inflammation sites with over-expressed ICAM-1 receptor. In their second targeting approach [ 160 ], they coated the AMPNP with a macrophage membrane of the mouse leukemia cells of monocyte-macrophage (M) to achieve specific binding to bacteria through the bacterial recognition molecules (Toll-Like receptors) on the macrophage membrane. The anti-ICAM-1-AMPNP and the M-AMPNP have shown specific targeting and adhesion to the inflamed human cells and bacterial cells, respectively. The in vitro antibacterial activity of the anti-ICAM-1-AMPNP and M-AMPNP was evaluated against E. coli, S. aureus , and MRSA, and they both exhibited good antibacterial efficacy. Moreover, when evaluated in vivo on mice sepsis model, both nanosystems have demonstrated a superior effect over the uncoated AMPNP regarding the reduction in serum cytokines (IL-1β, TNF-α, and IL-6) levels and inflammatory cells tissue infiltration.

A Preparation of anti-ICAM-1-AMPNP to specifically target inflammation sites with overexpressed ICAM-1 receptor. B Preparation of macrophages membrane-coated AMPNP (M−AMPNP) to specifically target bacteria through the TLR2 and TLR4 on the macrophage membrane (Adopted from [ 22 , 160 ])

Overall, both nanosystems (anti-ICAM-1-AMPNP and M-AMPNP) are promising and suggest a potential efficacy in bacterial sepsis management by explicitly targeting the inflammation sites and the causative bacteria. It is worth noting that, although the in vitro antibacterial activities of anti-ICAM-1-AMPNP and M-AMPNP were comparable to the bare peptide when evaluated in vivo , the nanosystems' efficacies were superior, which could be due to the improved stability of nanoparticles against proteases enzymes and the specific targeting and delivery to the inflammation and bacterial infections sites. However, using macrophage membranes derived from cancerous mouse cells could raise safety and immunogenicity concerns. Therefore, we think this mouse macrophage coating should be carefully considered when the nanosystem is to be further taken for clinical translation.

Nano-encapsulated AMPs

Encapsulation into nanosystems is another effective strategy for nano-delivery of AMPs to fight bacterial sepsis. This nano-encapsulation improves the AMPs' stability, reduces systemic toxicity, and improves their therapeutic efficacy [ 157 ]. In this context, Saúde et al. have encapsulated the antimicrobial peptide Clavanin A in a polymeric matrix for bacterial sepsis control, aiming to improve its stability and therapeutic efficacy [ 161 ]. The Clavanin A peptide (VFQFLGKIIHHVGNFVHGFSHVF-NH2) was nanostructured in a mixture of the methacrylate polymers EUDRAGIT® L 100-55 and EUDRAGIT® RS 30 D to make a nano-antibiotic. The nano-antibiotic demonstrated a sustained release, with 69% of the loaded Clavanin A released after 48 hrs. The in vitro antibacterial assay showed that nanoparticles containing 12 µg of Clavanin A inhibited the growth of S. aureus by 91%, K. pneumoniae by 20%, P. aeruginosa by 39.8%, and no effect on E. coli . In vivo efficacy of the nano-antibiotic evaluated on polymicrobial sepsis model on mice showed a 100% survival rate under a sub-lethal dose of bacteria and a 40% survival rate with a lethal inoculum. It is worth mentioning that, although the peptide loading significantly reduced the stimulation of pro-inflammatory cytokines (TNF-α, IL-12, and IL-10) release in vitro compared to the blank nanoparticles, these nanoparticles-induced cytokines releases were still significant. Therefore, we believe more in vivo evaluations are needed in this regard, and it would be better to avoid using these thiolated methacrylate polymers for antisepsis drug delivery as they are known to induce inflammation and cytokines release [ 168 ].

Another study carried out by Hassan and coworkers also reported the nano-encapsulation of an AMP (Mastoparan (Mast)) in polymeric nanoparticles to improve its stability and efficacy in managing multidrug-resistant bacterial sepsis [ 162 ]. They nano-encapsulated Mast (INLKALAALAKKIL-NH2) by structuring it with chitosan to produce a chitosan–Mast nano-construct (Mast-Cs NC). Mast-Cs-NC's in vitro antibacterial activity against A. baumannii clinical isolates demonstrated a significantly lower MIC of 4 μg/mL compared to the bare-Mast, which got an MIC of 16 μg/mL. When evaluated for in vivo efficacy on an A. baumannii- induced mice sepsis model, Mast-Cs-NC improved the physical activity and significantly decreased the blood bacterial counts compared to the chitosan and bare-Mast treated groups. Their findings showed enhanced in vitro and in vivo activity; however, they didn't report evaluation of the peptide release behavior from the nanosystem, which we believe is a fundamental property that will affect the selection of dosing frequency in clinical settings.

While the other studies reported encapsulation of functional AMPs in nanoparticles, Hou et al. have used an alternative unique approach by encapsulating the mRNA of the AMP-IB367 ( RGGLCYCRGRFCVCVGR CONH2 ) linked to mRNA of cathepsin B (CatB) (AMP-CatB mRNA) in vitamin C lipid nanoparticles (VLNPs) [ 163 ]. They transfected the nanoparticles in macrophages, where the mRNA will be translated to functional AMP-IB367 and CatB. CatB is an endogenous lysosomal protein that assists in translocating AMP-IB367 inside the macrophages' lysosomes, resulting in macrophages containing antimicrobial peptides linked to cathepsin B in the lysosomes (MACs). Upon the adoptive transfer of MACs to animals infected with bacteria, the lysosomes will fuse with phagosomes encapsulating bacteria and effectively kill the bacteria through both AMP-IB367 and lysosomal antimicrobial constituents. In vitro, MACs showed strong bacterial growth inhibition of 87% when evaluated against multi-drug resistant S. aureus (MDRSA) intracellular infection on RAW264.7 cells. MACs also significantly decreased bacterial loads in blood and improved survival rates of MDRSA-induced septic mice. Their findings presented the applicability of using nano-delivered mRNA of AMPs to target intracellular bacterial infections and sepsis. However, this strategy is limited by the possibility of mRNA degradation during loading and transfection, the immunogenicity that may arise from the adoptive transfer of macrophages, and the difficulties of scaling up these complex nanosystems. Therefore, we think these limitations must be carefully addressed before the MACs can be used in clinical practice.

AMPs conjugated to nanoparticles surface

Covalent conjugation of AMPs to the surface of nanoparticles has been applied to achieve nano-delivery of these conjugated AMPs. The conjugation can be accomplished on various types of nanoparticles, including both organic and metallic nanoparticles [ 24 ]. This approach is beneficial, especially in the cases of organic nanoparticles where another antisepsis drug can be encapsulated in the nanosystem and achieve simultaneous multiple drug delivery to target various pathways involved in the complex pathophysiology of sepsis. So far, only two studies have been reported on the covalent conjugation of AMPs to the surface of nanoparticles to enhance the stability and efficacy against bacterial sepsis, that is, one has conjugated the AMP to gold nanoparticles [ 164 ], and the other conjugated AMP to the surface of liposomes loaded with antibiotic [ 165 ]. Therefore, this strategy has not been thoroughly investigated, providing an opportunity to efficiently administer AMPs alone or combined with other drugs to address bacterial sepsis.

Rai and colleagues conjugated Cecropin melittin-cysteine (CM-SH: KWKLFKKIGAVLKVLC) AMP to gold nanoparticles (Au NPs) to improve the physiological stability and therapeutic efficacy against bacterial sepsis [ 164 ]. The optimized AMP-conjugated gold nanoparticles (CM-SH-Au NPs) were produced in one-step synthesis and found to have high AMP concentration (50% per nanoparticle mass). The in vitro antibacterial activity of CM-SH-Au NPs against S. aureus and E. coli showed a 4-fold reduction in MIC compared to the free CM-SH peptide. Unlike the free CM-SH peptide, CM-SH-Au NPs were found to be resistant to degradation, retaining even in the presence of cell culture media, human serum, and proteolytic enzymes such as trypsin, S. aureus V8 protease, and human neutrophil elastase. To evaluate the antimicrobial resistance development, they exposed E. coli to a sub-MIC dose of CM-SH-Au NPs for 28 days and then evaluated their efficacy against the treated strain. CM-SH-Au NPs were found to be still effective against the sub-MIC-exposed E. coli strain with no resistance development, unlike the control drug, chloramphenicol, which developed resistance after only 3 days of exposure. The therapeutic potential of CM-SH-Au NPs was also evaluated against CLP mice model of sepsis and demonstrated a significant reduction in bloodstream bacterial count and IL-10 level compared to unconjugated Au-NPs and free-CM-SH. We contend that their findings are promising and provide an effective strategy for improving the physiological stability and therapeutic efficacy of AMPs. However, the use of metallic nanoparticles needs comprehensive toxicity evaluations as they are known to carry more risk of systemic toxicity [ 169 ] than organic nanoparticles.

In another study, Fan et al. linked the AMP S-thanatin (Ts) (GSKKPVPIIYCNRRSGKCQRM) to the surface of liposomes loaded with levofloxacin (Ts-LPs-LEV) to target K. pneumoniae induced sepsis [ 165 ]. As illustrated in Fig. 8 , Levofloxacin-loaded liposomes were prepared by thin film hydration method with the incorporation of Ts-PEG2000-DSPE to produce positively charged liposomes and Ts anchored on the surface. The incorporation of Ts synergistically improved levofloxacin's antimicrobial activity on sensitive K. pneumoniae and restored its sensitivity on multidrug-resistant clinical isolates. The calculated MICs of Ts-LPs-LEV were 2 to 8 folds less than that of LPs-LEV on different strains of K. pneumoniae. Moreover, when evaluated on mice sepsis model of MDR K. pneumonia clinical isolate, the anchoring of Ts peptide resulted in a significant difference in bacterial clearance from blood and mice's survival rates with a reduction in lethality from 73.3% to 6.7% compared to LPs-LEV. It should be pointed out that while the peptide linking synergistically improved the efficacy of LEV, the Ts-linked liposomes without loading of LEV showed no activity against the majority of tested bacterial isolates (16 out of 18 isolates). However, the free peptide was effective on those isolates; therefore, we think this peptide's activity loss needs to be addressed and investigated. Furthermore, they didn't examine the enzymatic stability of conjugated peptide compared to the free peptide, which we believe is one of the main advantages of incorporating AMPs into nanosystems. Besides, they didn't evaluate the release pattern of the Ts and LEV, which is critical in determining dosing frequencies in clinical settings.

Preparation of Ts-LPs-LEV. Adopted from [ 165 ]

In a nutshell, nano-delivery of AMPs has been proven to be an effective strategy to overcome the shortcomings of antimicrobial peptides, such as physiological instability and systemic toxicity, resulting in improved biosafety and efficacy against bacterial infections and sepsis both in vitro and in vivo. The AMPs have been nano-delivered through various strategies, including conjugation with hydrophobic moieties to allow their self-assembly into nanoparticles, encapsulation in organic nanosystems, and linking the surfaces of metallic and organic nanoparticles. Therefore, we believe that with more investigations, these nanomedicines could be scaled up and become available for clinical use to help combat the life-threatening bacterial sepsis and the growing antimicrobial resistance.

Anti-inflammatory peptides nanosystems

Recently, anti-inflammatory peptides (AIPs) nanosystems have shown excellent properties, making them exceptional therapeutic candidates for sepsis management [ 23 , 170 ]. These characteristics include potent neutralizing effects against pro-inflammatory molecules [ 171 , 172 , 173 ], improved biodegradability and biocompatibility [ 174 ], advanced delivery properties [ 175 ], and multiple actions across various intracellular inflammatory pathways [ 30 , 176 ]. This section covers all AIP nanosystems reported for sepsis management, including self-assembled nanostructured AIPs and other nanocarriers used to deliver AIPs.

Self-assembled nanostructured AIPs

Self-assembled nanostructured peptides have been introduced to improve the anti-inflammatory properties of LPS-binding proteins (Limulus anti-LPS factor, serum amyloid P, and bactericidal permeability-increasing protein) by Mas-Moruno et al. This study successfully synthesized and structurally characterized several N-acylated peptides derived from the above proteins with advanced anti-inflammatory activity against LPS-induced cytokines storm. In vitro investigations have been done to evaluate their biosafety profile against RAW 264.7 macrophages, and most of them were found to be biosafe and tolerable. Compared to their parent peptides, some N-acylated peptides showed up to 10-fold enhancement in the in vitro LPS neutralizing activity within their biosafe concentration ranges. This activity enhancement may be related to their ability to form fibril-like and micellar nanostructures, as shown during TEM imaging. Their findings are promising, and we suggest they can be taken for preclinical and clinical evaluations to prove these results and allow for application in clinical settings [ 171 ].

Later, Tram et al. designed a stimuli-responsive self-assembled nanostructured dual active peptides (anti-inflammatory and antimicrobial) as an effective antisepsis agent with an advanced ability to form amyloid-like nanostructured nets in response to LPS and other bacterial endotoxins contact [ 173 ]. These multifunctional positively charged synthetic β-hairpin peptides could efficiently exert their anti-inflammatory effect through selective entrapping of negatively charged pro-inflammatory molecules and cytokines such as TNF-α, IL-6, LPS, and lipoteichoic acid (LTA) and, therefore, inhibiting bacterial endotoxin-induced cytokine storm. On the other hand, they exerted their antimicrobial activity by physically trapping bacterial cells and lysing bacterial membranes through interaction with the negatively charged bacterial cell walls and membranes. In this study, the selected peptides have shown promising bacterial toxin-neutralizing activity, which has been evaluated in a bacterial toxin-challenged-murine macrophage cell line model (in vitro) and acute lung injury mice model (in vivo). Furthermore, this study has involved various techniques to show the selective trapping of negatively charged pro-inflammatory cytokines (TNF-α and IL-6). In summary, modifications that confer AIPs the ability to self-assemble into nanostructured systems significantly enhanced their neutralizing activity against pro-inflammatory molecules and overall antisepsis outcomes with improved stability and circulation time.

Conventional nanocarriers-delivered AIPs

Several nanocarriers, including polymeric-based [ 30 , 176 ], protein-based [ 174 ], and metallic-based nanosystems [ 172 ], have been employed to deliver AIPs in attempts to improve their antisepsis activity. Table 3 summarizes the studies reported on designing AIP-loaded nanocarriers for sepsis management, highlighting the utilized AIP, the type of nanocarrier, the preparation method, and the key advantages of AIP nano-delivery. As depicted, nano-delivery enhanced the peptide's anti-inflammatory activity, stability, circulation time, biodistribution, biodegradability, and hemocompatibility. Moreover, nanocarriers are involved in the AIPs' co-delivery with antibiotics and other antithrombotic peptides to develop innovative and comprehensive antisepsis therapies with promising clinical outcomes. The studies will be discussed in this subsection based on the type of constituents of the nanocarriers, including polymeric, metallic, and protein-based materials.

Firstly, modified polyethylene glycol and acrylate polymers have been used to fabricate biocompatible nanocarriers to improve AIPs delivery for sepsis treatment [ 30 , 176 , 177 ]. These polymer-based nanocarriers significantly increased the antisepsis activity of AIPs by enhancing their stability, anti-inflammatory activity, and circulation time. For instance, Sadikot et al. utilized distearoyl phosphatidyl-linked PEG (DSPE-PEG 2000 ) to form 15 nm-micelles as an innovative co-delivery system for the two AIPs; human glucagon-like peptide (GLP-1) and triggering receptor expressed on myeloid cells 1 (TREM-1) inhibitor peptide (LP17)) to treat sepsis-related acute lung injury. This phospholipid micellar system stabilized both peptides in their activated alpha helix form and conferred prolonged circulation and in vivo bioactivity time compared to their parent peptides. Similarly, Cheng et al. and colleagues used DSPE-PEG 2000 to develop highly loaded peptide nanoparticles to deliver anti-inflammatory/antithrombotic dual active peptide, MB2mP6 (Myr-FEKEKL), as a new thoughtful approach for sepsis treatment [ 30 ]. MB2mP6 nanoparticles effectively inhibited both thrombosis and inflammation with limited vascular leakage by targeting inflammatory and thrombotic pathways associated with integrin’s G-protein alpha subunit-13 (Gα13) interactions in leukocytes and platelets. Immediate and late administration of MB2mP6 nanoparticles after severe sepsis initiation significantly increased mice survival rate, reduced inflammatory and thrombosis mediators, and prevented tissue and organ damage.

Likewise, utilizing polymeric material for AIP delivery, Novoselova et al. used polyacrylate-modified polybutyl cyanoacrylate polymer to formulate thymulin (an AIP thymic peptide)-bound nanoparticles to efficiently treat chronic inflammation and sepsis [ 177 ]. The developed acrylate-based nanoparticles showed 90% entrapment efficiency, improving the delivery aspect of thymulin against sepsis, such as circulation time and biodegradability. Thymulin-loaded nanoparticles efficiently alleviated sepsis-induced cytokines storm, decreased heat shock proteins and TLR-4 expression, reduced apoptosis, and increased splenic cell counts in mice. In summary, the findings of the three studies [ 30 , 176 , 177 ] demonstrated that utilizing polymeric nanosystems significantly enhanced the therapeutic efficacy of AIPs. However, it is essential to highlight that they didn't report much characterization of the developed nanosystems, such as size, PDI, ZP, and release kinetics measurements, which are highly important in indicating nanomedicines' storage stability and dosing frequencies.

Secondly, two studies by Karawacka et al. and Piktel et al. have used magnetic metallic nanoparticles to immobilize anti-inflammatory peptides via electrostatic interaction to improve their stability, activity, and circulation time [ 172 , 178 ]. In their study, Karawacka and colleagues used a coated superparamagnetic iron oxide nanoparticle to bind and immobilize agglutinating salivary proteins-derived peptides (LPS-neutralizing peptides) via hetero functional linkers. This modification significantly enhanced the in vitro LPS-neutralizing activity of conjugated peptides by more than 3-fold when evaluated by the endotoxin binding assay [ 178 ]. In the second study, Piktel and coworkers developed an innovative iron oxide-based peptide nanosystem with advanced delivery properties. Synthetic pro-inflammatory molecules-neutralizing peptide PBP10 (synthetic rhodamine B-conjugated peptide, bioinspired from the naturally occurring protein human plasma gelsolin) and its derivatives were used in this study to functionalize iron oxide nanoparticles. These fabrications simultaneously enhanced the antibacterial activity of metallic nanoparticles, the anti-inflammatory properties of immobilized peptides, and their biocompatibility properties, promoting the promising potential of AIPs metallic nanosystems as effective antisepsis therapeutic agents [ 172 ].

Finally, ferritin-based nanocages are well-known nanocarriers that improve stability and overall activity for different types of drugs due to their excellent properties, such as inherent cavity sizes and biocompatibility [ 179 ]. With this regard, Wei and coworkers used an emulsification technique to formulate ferritin-based nanocages as an efficient nano drug delivery system for both anti-inflammatory peptide GF9 (a TREM-1 inhibitor) and the antibacterial agent streptomycin as a dual therapy against bacterial-induced sepsis [ 174 ]. Using an E. coli -induced sepsis mice model, this Antibacterial/ anti-inflammatory co-delivery successfully reduced bacterial burden, suppressed harmful inflammatory responses, prevented lungs from sepsis-associated tissue damages, and achieved better overall clinical outcomes and survival rates compared to monotherapies. Thus, we believe ferritin-based co-delivery of AIPs with antibacterial agents could be an efficient therapeutic strategy against sepsis.

Stimuli-responsive nanocarriers- delivered AIPs

Compared to conventional nanocarriers, stimuli-responsive nanocarriers have shown superior clinical outcomes due to their advanced release patterns that accumulate the loaded therapeutic agents in their site of action in response to distinguished pathophysiological changes [ 180 ]. Concerning this, a brilliant study by Lee et al. reported stimuli-responsive ferritin-based nanocages that simultaneously delivered two bioactive peptides, targeting two different intracellular pathways to improve sepsis control and reduce harmful side effects [ 175 ]. In this study, ferritin was Genetically modified by inserting the endothelial protein C receptor-targeting ligand (PC-Gla domain) and protease-activated receptor-1 activator (TRAP peptide) to form ferritin-based nanocarriers, which showed an advanced antisepsis activity. This promising activity has been further improved by inserting a matrix metalloproteinase-sensitive linker to confer PC-Gla domain a stimuli-response release pattern in response to metalloproteinase at the metalloproteinase-rich inflammatory sites. In vitro and in vivo sepsis models confirmed the stimuli-responsive PC-Gla release, significantly reducing inflammatory cells infiltration and lung injury scores and improving mice survival rates after CLP.

Later, Lui et al. reported pH-responsive nanoplexes that could target CD44-overexpressed cells as an efficient stimuli-responsive nanocarrier of SS-31 peptide (an AIP) against sepsis-induced acute kidney injury [ 181 ]. This innovative design perfectly overcame the poor pharmacokinetic characteristics of the loaded peptide SS-31, enhancing its activity and targetability. As presented in Fig. 9 , biocompatible polymers Hyaluronic acid and chitosan (CS) electrostatically interacted to form stable nanoplexes, promoting payload release via low pH condition destabilization. SS-31 loaded nanoplexes were stable at physiological pH with an average size of 53 nm, ZP of -20 mV, and PDI of 0.17. large surface charge conferred nanoplexes high stability to prevent aggregation and enhance accumulation at the site of action. In vitro drug release studies confirmed the pH-responsive release pattern with an approximately 10-fold higher drug release percentage at pH 4.5 compared to pH 7.4. SS-31 loaded nanoplexes showed an enhanced intracellular uptake and higher antioxidant and antiapoptotic properties compared to bare SS-31 peptides in both in vitro and in vivo studies. Furthermore, histopathological analysis revealed that treatment with SS-31-loaded Nanoplexes improved kidney functions and reduced sepsis-associated tissue damage and tubular injury.

Preparation and evaluation of SS-31 loaded nanoplexes. A The method for fabricating nanoplexes through electrostatic complexation; ( B ) and ( D ) DLS characterization; ( C ) TEM imaging; ( E ) pH-responsiveness; ( F ) release patterns at different pHs. (Taken from [ 181 ])

In conclusion, exploring nano-delivered AIPs to combat sepsis has promising outcomes. The improved neutralizing action of AIPs in nanosystems against pro-inflammatory cytokines opened avenues for further preclinical and clinical evaluations. In addition, as compared to free peptides, nano-delivered AIPs exhibited markedly enhanced stability, circulation time, and therapeutic effectiveness. Furthermore, stimuli-responsive nanocarriers demonstrated superior clinical outcomes with advanced release patterns, offering a targeted and efficient approach to sepsis treatment.

Overall, the studies discussed in the above two sections underscore the potential of nano-delivery systems to enhance bioactive peptides (AMPs and AIPs)' stability and therapeutic efficacy for effectively managing sepsis, presenting a compelling foundation for further exploration and clinical translation.

Peptides as nanocarriers components

Peptides as targeting moieties on nanoparticles’ surface.

The surface of nanoparticles can be modified using various techniques and modifying materials. This surface modification achieves several advantages, including targeted and site-specific drug delivery, improved cellular uptake of loaded drugs, and enhanced nanosystems' physicochemical, biological, and therapeutic properties [ 44 ]. Different materials, such as antibodies [ 182 ], oligonucleotides [ 183 ], polymers [ 184 ], and peptides [ 185 ], have been used for nanoparticles’ surface modifications. Table 4 presents a summary of peptides’ application for surface modification of nanoparticles delivered against bacterial sepsis, showcasing the type of the nanosystem, peptide sequence, biological target, loaded drugs, key characterizations, and key findings. As presented, peptides have been used as targeting moieties for specific delivery to inflammation sites, bacterial cells, and body organs. It is obvious that the most studied application was the use of peptides to target inflammation sites through the overexpressed receptors, enzymes, and other proteins. Within these, ICAM-1 was the most targeted inflammatory site components. This open avenues for more research on the application of peptides to target other sepsis inflammatory-microenvironment’s overexpressed components such as selectins, CD44, TREM-1, and protease-activated receptor-1 (PAR-1) [ 186 ]. Also, the of targeting invading bacteria and specific body organs that are at high risk of damage during sepsis are yet to be more explored. The studies will be discussed in the following subsections based on the biological target that has been utilized to achieve improved sepsis management.

Inflammation sites targeting peptides

Inflammation sites, such as those of sepsis, are characterized by an overexpression of certain proteins involved in the inflammation processes [ 188 , 191 ]. These up-regulated proteins can be exploited to develop smart nanosystems modified with targeting moieties (such as peptides) that bind specifically to these proteins, resulting in targeted drug release at the inflammation sites [ 195 ]. In this subsection, we discuss the utilization of peptides as surface modifiers for nanosystems to target various upregulated proteins at the sepsis’s inflammation sites. The studies will be organized according to the targeted protein.

ICAM-1 is a key adhesion molecule on epithelial cells that acts as a ligand to the integrins receptors on polymorphonuclear leukocytes, mediating their recruitment and migration into tissues. ICAM-1 is typically expressed at low levels; however, its expression is up-regulated during sepsis and inflammatory conditions, increasing the leukocytes adhesion [ 196 ]. Therefore, ICAM-1 targeting ligands such as peptides [ 187 ] and anti-ICAM-1 antibodies [ 22 ] have been used to modify nanoparticles to achieve inflammation sites targeted drug release. To this end, three studies [ 187 , 188 , 189 ] have reported the use of peptides for surface modification of Poly DL-lactic-co-glycolic acid (PLGA) nanoparticles targeting the inflammation-induced overexpression of ICAM-1. In their study, Zhang group reported a proof-of-concept investigation in which they decorated PLGA nanoparticles with cyclo (1,12) PenITDGEATDSGC (cLABL) peptide, which is proven to have a good binding affinity to the D1 domain of ICAM-1 [ 197 ], to target the overexpressed ICAM-1 on inflamed cells. cLABL-PLGA-NPs demonstrated rapid binding and internalization into human umbilical cord vascular endothelial cells (HUVECs) with up-regulated ICAM-1 induced by interferon -γ treatment. They found the binding of cLABL-PLGA-NPs to be inhibited by pretreatment with free cLABL, proving that the binding of nanoparticles was due to the peptide conjugation [ 187 ].

The other two studies by Yang et al. and Liu et al. utilized γ3 peptide (NNQKIVNLKEKVAQLEA) as an ICAM-1 ligand for surface modification of PLGA nanoparticles achieving targeted release of the loaded antisepsis agents at the sepsis’s inflamed sites. Yang and colleagues, as illustrated in Fig. 10 , utilized the γ3-PLGA-NPs for co-delivery of the antibiotic Sparfloxacin (SFX) and the anti-inflammatory/immunosuppressant Tacrolimus (TAC), resulting in γ3-PLGA-NPs@SFX/TAC. Conversely, Liu et al. applied a monotherapy strategy by loading only ciprofloxacin (CIP) in the γ3 modified PLGA-NPs. However, Liu’s group coated the PLGA-NP with red blood cells (RBC) membrane producing γ3-RBCNPs@CIP to avoid immune vigilance and provide prolonged circulation time. Both γ3-PLGA-NPs@SFX/TAC and γ3-RBCNPs@CIP showed good cytocompatibility, hemocompatibility, and low systemic toxicity. When evaluated on TNF-α activated HUVECs cell line, both nanosystems demonstrated significantly higher binding to the stimulated cells than the peptide-unconjugated nanoparticles. Furthermore, they both exhibited superior in vitro antibacterial efficacy.

Preparation and in vivo evaluation of γ3-PLGA NPs ( A ) Preparation of γ3-PLGA NPs loaded with Sparfloxacin and Tacrolimus ( B ) Effective treatment of lung-infected mice by specific targeting of the overexpressed ICAM-1 (Taken from [ 188 ])

In the in vivo therapeutic efficacy evaluated against acute lung infection mice models, both γ3-PLGA-NPs@SFX/TAC and γ3-RBCNPs@CIP achieved significantly higher lung tissue accumulation and significantly reduced the bacterial load, inflammatory cytokines level, and inflammatory cells infiltration. As a result, they improved the mice survival rates compared to the peptide-unmodified nanosystems, proving the enhanced release at the inflamed lung tissues due to the ICAM-1 targeting. Both nanosystems have been extensively characterized and evaluated in vitro and in vivo. It should be noted that while the nanosystem developed by Yang group was assessed against both Gram-positive and Gram-negative bacteria ( S. aureus and P. aeruginosa ), the efficacy of the one by Liu et al. was studied only against Gram-negative bacteria (K. pneumoniae). On the other hand, Liu’s group proved the ability of their system to avoid macrophages’ phagocytosis, which we consider an essential feature that improves and prolongs the in vivo activity. Overall, we believe using peptides for surface modification of nanoparticles as targeting ligands to the overexpressed ICAM-1 at the inflammatory sites is a promising strategy for effective sepsis management with minimized systemic toxicities, as evidenced by these reports.

Exploring the same ICAM-1/integrin ligand-receptor pair, Shi and coworkers have also designed a peptide-modified nanosystem for inflammation targeting. However, their approach targeted the integrin receptor on the immune cells instead of the ICAM-1 ligand. They anchored the Arginine-Glycine-Aspartic Acid (RGD) peptide, a well-known integrin receptor ligand, on curcumin (Cur)-loaded liposomes (RGD-lipo/Cur) to achieve macrophages targeted Cur release against sepsis-induced inflammation [ 190 ]. While there was no significant increase in the uptake of RGD-unmodified liposomes after LPS-stimulation, RGD-lipo/Cur demonstrated a significantly higher uptake by LPS-stimulated RAW264.7 cells compared to the unstimulated cells. Also, the fluorescence microscope imaging showed colocalization of the RGD-lipo/Cur and the fluorescently labeled integrin receptors, indicating the liposomes' internalization to be through the RGD/integrin interaction. As a result, RGD-lipo/Cur showed a superior reduction in intracellular ROS levels and significantly inhibited the high inflammatory lytic programmed cell death (pyroptosis) of LPS-activated RAW264.7 cells compared to the peptide-unmodified liposomes. In vivo, RGD-lipo/Cur significantly reduced the release of inflammatory cytokines (TNF-α and IL-6) and prevented sepsis-induced organ damage in the LPS-induced mice sepsis model. Their findings showed the promising potential of RGD modification of Cur-loaded liposomes to improve sepsis management. However, they did not report ZP measurement, which we believe is a critical property in determining the storage and physiological stability of the nanosystem.

Another up-regulated protein during sepsis inflammatory conditions is dipeptidase 1 (DPEP1), expressed on cells of key organs such as the kidney, liver, and lung. Similar to ICAM-1, DPEP1 is an adhesion molecule for the recruitment of leukocytes [ 198 , 199 ]. Therefore, DPEP1 ligands can be utilized for nanoparticles surface modification to accomplish targeted drug release at the inflamed tissues during sepsis. In this respect, Yan and colleagues exploited Cys-LSA peptide (CLSALTPSPSWLKYKAL), a DPEP1 ligand, for surface coating of hollow mesoporous polydopamine nanocarrier (HMPDA) specifically targeting inflammation sites for the management of sepsis. As depicted in Fig. 11 , HMPDA was prepared by soft template method, loaded with NAD + and BAPTA-AM, and grafted with LSA peptide to give HMPDA@BA/NAD + @LSA NPs [ 191 ].

Preparation and mechanism of action of HMPDA@BA/NAD + @LSA NPs ( A ) Preparation of HMPDA@BA/NAD + @LSA NPs. B Pharmacological effects of HMPDA@BA/NAD + @LSA NPs in an LPS-induced sepsis mice model (Adopted from [ 191 ]

Their approach was based on a triple therapy to prevent sepsis-induced cells, tissues, and organs damage with NAD + restoring the energy production and exerting anti-inflammatory effect, BAPTA-AM chelating the overloaded intracellular Ca ++ and PDA scavenging the intracellular ROS. When evaluated in vitro using H 2 O 2 -stimulated liver and kidney cells, HMPDA@BA/NAD + @LSA NPs demonstrated superior ability to restore mitochondrial function, Ca ++ hemostasis, and antioxidant system and so rescued endangered cells. Studied in LPS-induced mice sepsis model, the accumulation of the peptide-modified nanoparticles in the key organs was markedly higher than that of healthy mice. Moreover, peptide modification significantly increased the accumulation of nanoparticles in the liver, kidney, and lung (1.4, 1.6, and 1.5 times) compared to the peptide-unmodified nanoparticles, proving the targeting of the overexpressed DPEP1 at the inflamed organs. As a result, HMPDA@BA/NAD + @LSA NPs significantly reduced the sepsis-induced key organs damage and improved the survival rate of mice. We argue that this multifunctional nanosystem with inflammation-targeted drug release is a promising nanomedicine to help combat sepsis’s multiple pathogenesis with less systemic drug exposure.

In conclusion, these studies underscore the potential of utilizing peptides for surface modification of nanoparticles, serving as selective targeting moieties for the up-regulated proteins at inflammation sites for sepsis management. However, more avenues are available to explore other up-regulated proteins using various peptide sequences.

Organ targeting peptides

Among the strategies for incorporating peptides as targeting moieties on nanosystems surface, a notable approach is targeting specific organs’ tissues and cells. Organ-specific peptides allow for accurate drug delivery to particular organs during sepsis, offering a mechanism to rescue key organs at high risk of damage and failure [ 192 , 200 ]. In this regard, Huang et al. engineered zeolite imidazolate framework-8 nanoparticles coated with renal tubular epithelial cell membrane and modified with a kidney targeting peptide (KCSAVPLC) to promote specific drug uptake by renal tubular cells. This nano-construct, denoted as KMZ@FGF21, was loaded with the antioxidant/anti-inflammatory hormone, fibroblast growth factor 21 (FGF21), against sepsis-induced acute kidney injury (AKI) [ 193 ]. Peptide modification significantly increased the nanoparticles uptake by murine renal tubular epithelial cells (TCMK-1) in vitro and decreased intracellular oxidative stress, inflammation, and apoptosis compared to the peptide-unmodified nanoparticles. Moreover, when injected into mice, the fluorescently labeled KMZ@FGF21 favorably accumulated in kidney tissues. When evaluated in a sepsis-induced AKI mice model, KMZ@FGF21 exhibited superior antioxidant and anti-inflammatory efficacy, alleviated AKI, and improved renal function recovery.

Utilizing a similar approach, Ouyang and coworkers designed hollow mesoporous silica nanoparticles (PCM-MSN@LA) loaded with L-arginine (LA) as nitric oxide (NO)-releasing agent and modified with primary cardiomyocytes specific peptide (PCM) for heart tissues-targeted delivery to combat LPS-induced cardiac injury [ 192 ]. As illustrated in Fig. 12 , cardiac targeting was further increased by applying low-intensity focused ultrasound (LIFU). PCM-MSN@LA were found to have significantly higher localization and affinity to cardiomyocytes compared to the hepatoblastoma cell line (HepG2) as a control, suggesting PCM's cardiac selectivity. Likewise, when evaluated in vivo, the fluorescently labeled PCM-MSN@LA showed 60-fold higher fluorescence intensity in the hearts of mice compared to PCM-unmodified nanoparticles. In contrast, the other organs (kidney, spleen, lung, and liver) showed markedly less nanoparticles distribution. Moreover, the fluorescence intensity of PCM-MSN@LA in mice’s hearts increased by 7-fold upon application of LIFU. In vitro, PCM-MSN@LA and PCM-MSN@LA + LIFU improved the cell viability of LPS-treated cardiomyocytes from 62 to 72% and 80%, respectively. Furthermore, PCM-MSN@LA combined with LIFU significantly reduced inflammatory cells recruitment, mitochondrial dysfunction, and oxidative stress in LPS-induced septic mice's cardiac tissues, thus prevented myocardial injury and cardiac dysfunction. We believe their strategy is promising and holds great potential for preventing sepsis-induced cardiac dysfunction in clinical settings.

Design of heart-targeting L-arginine loaded mesoporous silica nanoparticles (PCM-MSN@LA) and its combined application with low-intensity focused ultrasound (LIFU) to prevent cardiac damage in mice [ 192 ]

Ultimately, the findings of these two studies by Huang et al. and Ouyang et al. show the potential of organs targeting peptides as surface modifiers of nanosystems loaded with cytoprotective agents to prevent organ damage and dysfunction associated with sepsis. However, more avenues are available to explore targeting of other vulnerable organs such as liver, spleen, and lung.

Bacterial cells targeting peptides

Beyond targeting specific human body organs and inflammation sites, nanosystems can be tailored to selectively target the causative bacteria, addressing sepsis at the early levels of the pathogen invasion. It is well demonstrated that short peptides possessing cationic or amphiphilic properties can be adsorbed to the negatively charged bacterial membranes, specifically targeting bacterial cells and surmounting cellular barriers [ 201 , 202 , 203 ]. However, only one study by Lee et al. has been retrieved about using peptides for nanoparticles surface modification to improve bacterial cell affinity for sepsis management [ 194 ]. They utilized the chiral dipeptide D-/L-Cys-Phe (CF) for surface modification of gold nano-bipyramids (GBPs) to improve adsorption to and targeting of bacterial cells.

As depicted in Fig. 13 , D-/L-CF peptides gave the GBPs a spike shape (sea cucumber-like morphology) with higher binding affinity to protein A of the S. aureus cell membrane. Fluorescence imaging showed a higher overlapping of D-GBPs with the fluorescence of S. aureus compared to L-GBPs and DL-GBPs, indicating the higher adsorption and interaction of D-GBPs with bacterial cells. D-/L-GBPs demonstrated good photothermal properties and efficient absorption of near-infrared light irradiation (NIR), raising temperatures above the human body, suggesting they could damage the bacterial cell wall and achieve photothermal antibacterial treatments. Both D-GBPs and L-GBPs showed superior antibacterial activity against S. aureus compared to the peptide-unmodified GBPs, with increased activity upon NIR irradiation. Notably, the activity of D-GBPs was substantially higher compared to that of L-GBPs. Evaluated in vivo using S. aureus -induced mice sepsis model, D-/L-GBPs treatment showed significant reduction in organs’ bacterial counts and recovery of serum white blood cells levels and animal weights, with the D-GBPs + NIR group being the most effective. It is worth mentioning that their findings prove the strong potential of peptides for coating metallic nanoparticles to achieve bacterial targeting and better antibacterial efficacy. Moreover, their findings shed light on the difference in activity between D- and L-chiral isomers of peptides, which we believe is of significant importance when designing peptides for different purposes.

Preparation and characterization of D-/L-GBPs. A Coating of GBPs with D-/L-CF ( B ) TEM images of peptide-unmodified Au NBPs ( C ) TEM and ( D ) SEM images of D-/L-GBPs (adopted from ([ 194 ])

In summary, the studies highlighted in this section contribute valuable insights into applying peptides as targeting moieties modified on the surface of nanoparticles for effective sepsis management. Three biological targets have been utilized, viz. inflammation sites, specific organs, and bacterial cells. With inflammation sites targeting being the most studied strategy, more avenues are available to unlock the full potential of peptides as targeting motifs for nanoparticles against sepsis, especially bacterial cells targeting.

Peptides as nanocarriers for antisepsis agents

Peptides can be designed to have varied surface charges, solubility, and other physicochemical properties that allow them to form stable nanosystems with excellent encapsulation and cargo delivery efficacy [ 204 ]. Therefore, Peptides could be used as nanocarriers to improve the antisepsis drugs' stability, cellular uptake, and therapeutic outcomes. Besides, the nanocarrier peptides can be designed to have adjuvant pharmacological activity toward sepsis management, such as antimicrobial and anti-inflammatory efficacy. So far, only three studies [ 205 , 206 , 207 ] have been reported on using peptides as nanocarriers for antisepsis agents, highlighting the potential for further exploration to leverage these excellent biomaterials for enhanced and targeted drug delivery against bacterial sepsis. This section will discuss the application of peptides as nanocarriers for antisepsis agents based on the therapeutic effect of the loaded agent, whether it was an anti-inflammatory or antibacterial effect.

As for this, He and co-authors designed polypeptide-based hybrid nanoparticles (HNPs) encapsulating the anti-inflammatory TNF-α small interfering RNA (TNF-α siRNA) to provide endosomal escape and cellular internalization against hepatic sepsis [ 205 ]. HNPs were designed from a combination of the cationic helical polypeptide (PPABLG) and the anionic polypeptide (PAOBLG-MPA). The helical structure of PPABLG is supposed to form pores on the cell membrane and provide strong membrane permeability, enhancing the TNF-α siRNA internalization. This intracellular delivery of TNF-α siRNA was evaluated in vitro in LPS-stimulated murine macrophages (RAW 264.7) cells, and the HNPs were found to markedly increase the cellular uptake of TNF-α siRNA, achieving 90% inhibition of TNF-α production, unlike the free TNF-α siRNA which showed a negligible gene-silencing effect. Moreover, HNPs mediated endosomal escape and significantly reduced the colocalization of TNF-α siRNA with the fluorescently labeled endosomes/lysosomes compared to free TNF-α siRNA. When evaluated in an LPS/D-galactosamine (D-GalN)-mice model of hepatic sepsis, the free TNF-α siRNA degraded within 2 hours while the HNPs-encapsulated siRNA remained stable and attained higher accumulation in the macrophage-rich organs such as liver, lung, and spleen. Furthermore, compared to the free siRNA, the HNPs demonstrated a stronger anti-inflammatory efficacy and significantly downregulated TNF-α, rescuing the mice from hepatic sepsis.

While in the previous study, a peptide with cell penetration enhancing properties was used as a nanocarrier for the anti-inflammatory agent, Chen et al. employed an antimicrobial peptide nanogel as a nanocarrier for the anti-inflammatory agent, TNF-Related Apoptosis-Inducing Ligand (TRAIL), for effective sepsis management [ 206 ]. The bactericidal cationic poly(L-lysine)-block-poly(L-threonine) co-polypeptide (PLL-b-PLT) was crosslinked to nanogel encapsulating TRAIL protein to accomplish dual antibacterial and anti-inflammatory efficacy to combat bacterial sepsis. The optimized TRAIL nanogel was found to have a spherical shape with size, PDI, ZP, and EE% of 295.3 ± 5.7 nm, 0.21 ± 0.01, 18.5 ± 2.1 mV, and 98.5 ± 2.1%, respectively. Evaluated in MLE-12 (mouse lung epithelial cell) and Raw264.7 (macrophage) cells, TRAIL nanogel exhibited a good biosafety profile against normal cells. However, it showed superior cytotoxicity and apoptosis induction to LPS-activated cells compared to free TRAIL and blank nanogel. Furthermore, TEM analyses of TRAIL nanogel-treated K. pneumoniae revealed significant cell membrane destruction and loss of integrity. In vivo, TRAIL-encapsulated nanogel significantly reduced TNF-α and IL-6 levels, blood bacterial load, and pulmonary leukocytes accumulation; thus, it protected mice against K. pneumoniae- induced sepsis and LPS-induced lung and kidney injury and prolonged their survival rates. It is noteworthy that their dual therapeutic approach holds great potential in targeting both infecting bacteria and the inflammatory syndrome of sepsis.

In contrast to the other studies in which anti-inflammatory agents have been loaded in peptides-based nanocarriers, Z. Chen and coworkers have encapsulated the antibacterial antisense oligonucleotides (ASOs) in dendritic polypeptides nanoparticles coated with DSPE-mPEG2000 (DP-AD7) against multidrug-resistant bacterial infections and sepsis [ 207 ]. The high molecular weight and hydrophilicity of ASOs hinder them from cellular penetration. However, their encapsulation into cationic nanoparticles can provide a solution [ 208 ]. While the free ASO could not penetrate bacterial cells, DP-AD7 achieved 90% uptake by S. aureus , MRSA, E. coli , and extended-spectrum beta-lactamases producing (ESBLs)- E. coli . As a result, DP-AD7 significantly inhibited the in vitro growth and the expression of target genes in these bacteria. Going further, when evaluated in septic mice model of ESBLs- E. coli, DP-AD7 significantly improved the survival rate and decreased the organs’ bacterial load.

Notably, the findings of the studies in this section underscore peptides as promising nanocarriers for antisepsis agents, including both anti-inflammatory and antibacterial agents. However, none of the discussed studies reported the evaluation of in vitro drug release . We believe it would have been worthwhile to investigate the release profiles as they are critical for the clinical use of the nanosystems in terms of dosing frequency and therapeutic efficacy. Overall, peptides exhibit the potential to overcome the inherent limitations that hinder the clinical translation of antisepsis agents, such as improving their stability and cellular internalization.

Conclusion, challenges and future perspectives

Peptides have been effectively utilized in nanotools against sepsis and have shown enormous potential to improve its diagnosis and management. This review highlighted and critically discussed various reports on using peptides in nanotechnology against sepsis with the capacity to attain prompt diagnosis and efficient management. Based on the reported findings, peptides demonstrated specific and robust interactions with bacterial cell membranes and inflamed tissues, enhancing the bacterial recognition in blood for diagnosis, cell penetration for intracellular nanosystems-payload delivery, membrane disruption for effective bacterial killing, and inflammation-targeted and organ-specific release of nano-delivered antisepsis agents. In addition, engineering within nanosystems significantly overcame the limitation of bioactive peptides (AMPs and AIPs) and enhanced their stability, biosafety, and efficacy. Moreover, peptides have been effectively applied as nanocarriers for antisepsis agents, improving their effectiveness, reducing systemic toxicity, and improving management outcomes. The potentiality of the reviewed nanosystems has been investigated both in vitro and in vivo and found to be superior to that of free bioactive peptides and the peptides-unmodified nanosystems. Cumulatively, these findings reveal diverse and prominent roles peptides can undertake as active agents or excipients in nanosystems for fighting sepsis.

For sepsis diagnosis, it is apparent that peptides were predominantly used as pathogen recognition moieties conjugated to nanoplatforms, focusing mainly on Gram-positive bacteria identification. Thus, more research is recommended to explore using peptides to capture other virulent bacteria that significantly contribute to sepsis development, such as E. coli and K. pneumoniae  [ 209 ]. Furthermore, the advancement of molecular sciences and increased understanding of sepsis pathophysiology continue to identify new biomarkers suited for sepsis diagnosis. Henceforth, further investigations to design peptides with high affinity to such biomarkers and engineering them within nanotools could significantly help to advance sepsis diagnosis techniques.

With respect to sepsis management, peptides have been mostly utilized as bioactive peptides that are nano-delivered through self-assembly, encapsulation, or conjugation to surfaces of nanoparticles. With self-assembly and nanoencapsulation being the prevalent explored strategies, linking bioactive peptides to the surface of metallic or organic nanoparticles awaits more investigations as a nano-delivery approach against sepsis. It is perceivable that the application of peptides as targeting moieties on nanosystems has been mostly directed to ICAM-1 targeting to achieve drug release at the inflamed tissues. However, with the development in identifying new proteins and receptors involved in sepsis pathogenesis, peptides could be more investigated to specifically target those proteins such as PAR-1, CD44, and TREM-1 [ 186 ], accomplishing improved management outcomes with less systemic toxicity. Moreover, peptides utilization as nanocarriers for antisepsis drugs was the least studied application in nanotechnology for sepsis management. This fosters opportunities for further utilization of these unique biomaterials to enhance antisepsis drug delivery and improve patients’ management outcomes. Finally, with the aid of molecular dynamics simulation and artificial intelligence, peptide sequences can be harnessed to have excellent and improved properties, such as having a stable nano-assembly and strong binding to biological targets and sepsis biomarkers, reducing the cost and accelerating the clinical translation to improve both diagnosis and management of sepsis.

Even with encouraging advancements in the utilization of peptides in sepsis diagnosis and management nanotools, the field is still emerging, and various challenges are encountering that hinder clinical translation. Primarily, these nanoplatforms' design and efficacy depend on the sepsis pathophysiology, which is very discrepant during different disease stages and from patient to patient. Moreover, all the discussed nanosystems have been evaluated in vitro or in vivo on mice models from which human sepsis pathogenesis, prognosis, and treatment response may vary significantly. Therefore, new sepsis models that mimic human responses and outcomes are of great importance in hastening the clinical adoption of the developed nanotools. Moreover, the fabrication of these nanoplatforms is complex, resulting in concerns regarding their reproducibility, scalability, stability, and cost-effectiveness. Additionally, the absence of official quality control guidance and recommendations for nanoplatforms characterization is further delaying their clinical utilization. Thus, optimization and comprehensive evaluation during development are critical to ensure reproducible and scalable manufacturing. Also, pharmacoeconomic and regulatory assessments are required to evaluate the cost-effectiveness of further clinical application of these nanosystems.

Even so, with the ongoing research and in-depth investigations, we envisage that the aforementioned challenges would be overcome and allow for clinical utilization of peptides-based nanosystems to enhance sepsis diagnosis and management practices, ultimately improving patient outcomes. Consequently, this review provides a foundation and comprehensive background for both academia and industry to advance and scale-up these nanoplatforms for efficient sepsis diagnosis and management. To bring it all together, peptides have shown promising potential as active principals and excipients in nanotools against sepsis, achieving rapid identification and on-time superior interventions to ensure better patient outcomes.

Availability of data and materials

Not applicable.

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Acknowledgments

The authors are thankful to the University of KwaZulu-Natal (UKZN), the National Research Foundation of South Africa (Grant Nos. 123162, 106040, 103664, and 11665), and the Medical Research Council (MRC) of South Africa for financial support.

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Mohammed A. Gafar, Calvin A. Omolo, Eman Elhassan & Thirumala Govender

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Calvin A. Omolo

Discipline of Human Physiology, School of Laboratory Medicine and Medical Sciences, College of Health Sciences, University of KwaZulu-Natal, Durban, South Africa

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MAG: Conceptualization; data curation; investigation; methodology; validation; visualization; writing-original draft; writing-review and editing; project administration. CAO: Conceptualization; validation; writing-review and editing; supervision. EE: Conceptualization; date curation; validation; writing-review and editing. UHI: Data curation; methodology; writing-original draft. TG: Conceptualization; validation; writing-review and editing; funding acquisition; project administration; supervision. All authors read and approved the final manuscript.

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Gafar, M.A., Omolo, C.A., Elhassan, E. et al. Applications of peptides in nanosystems for diagnosing and managing bacterial sepsis. J Biomed Sci 31 , 40 (2024). https://doi.org/10.1186/s12929-024-01029-2

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Received : 25 February 2024

Accepted : 10 April 2024

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DOI : https://doi.org/10.1186/s12929-024-01029-2

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