Care shouldn't start from the emergency room.

In recent years, following five decades of relative stability in the global nation-state system, the resurgence of insurgent elements and unpredicted catastrophes have challenged the prevailing notion of our era as a time of peace.

Frequent wars and natural disasters have profoundly impacted societies worldwide. Warfare, particularly in conflict regions such as Eastern Europe and the Middle East where casualties from destructive weapons continue to rise, exacerbates health risks for civilians and soldiers. The most recent and deadliest conflict, the Ukraine War, has caused approximately 500,000 deaths and injuries since the war began in February 2022. (Cooper, 2023) Meanwhile, the consecutive floods in Guangxi and the Dongting Lake in China in June and July this year exemplify how natural disasters such as floods, hurricanes, and earthquakes can also generate widespread casualties and property damage.

We aim to reduce the number of non-natural human deaths caused by catastrophes, but unfortunately, we cannot prevent the occurrence of wars and natural disasters. However, aside from deaths directly caused by destructive weapons and major disasters, infections are also major contributors to the rising death toll, especially in regions with limited medical resources and poor medical conditions. According to data from the World Health Organization (WHO, 2020), approximately 11 million people die each year from sepsis, often caused by untreated or poorly managed infections. Infections can lead to septic shock, multiple organ failure, and even loss of life, with survivors potentially facing lifelong disabilities. Given the rising numbers of deteriorating medical conditions in post-war and post-disaster regions, casualties due to infections will assuredly reach a new level if we do not interfere with the status quo.

Figure 1: Infections and onset period after natural disasters. Various infections may arise after natural disasters, with their nature and occurrence depending on the type and duration of the disaster, as well as the time elapsed since the onset.

The primary method for combating microbial infections today remains the use of antibiotics (Alyson Powell Key, 2024). A study conducted across 76 countries, published in the Proceedings of the National Academy of Sciences, revealed a 65% increase in global antibiotic usage between 2000 and 2015 (Briony Harris, 2018). However, the overuse of antibiotics has inevitably led to the rise of antibiotic-resistant microbes, posing a severe threat to global health (Sommer et al., 2017). In 2019 alone, bacterial resistance was associated with an estimated 1.27 million deaths, with major pathogens such as Gram-negative Acinetobacter baumannii and Gram-positive Staphylococcus aureus contributing to 929,000 of those fatalities. Without new and effective therapeutics, bacterial infections caused by "superbugs" could result in as many as 10 million deaths annually by 2050, with a projected economic cost of 100 trillion dollars per year (Zhang et al., 2023). These alarming statistics underscore the urgent need to find alternatives to antibiotics, leading to a growing interest in antimicrobial peptides (AMPs), which offer great potential in the fight against antibiotic resistance.

Figure 2: Statistics on antibiotic use by country (Briony Harris, 2018)

To address these challenges, we propose a new first aid kit combined with antimicrobial peptides (AMPs).

Traditional antibiotics, while still effective, are increasingly compromised by the rapid development of drug-resistant bacteria, making the need for innovative solutions more urgent than ever. CuraPack tackles this critical issue by harnessing the power of AMPs—naturally occurring molecules known for their potent antimicrobial properties. Unlike conventional antibiotics, AMPs offer a dual-action mechanism, combating bacteria through two approaches: bactericidal (membrane disruption causing cell lysis) and bacteriostatic (metabolic processes interference through nucleic acids binding and modulation of essential bacterial functions) (Madani et al., 2011) (Jenssen et al., 2006). These multifaceted mechanisms against infection signify that AMPs would have a lower likelihood of resistance development, which is one major advantage of AMPs over traditional antibiotics.

Current first aid kit products on the market are designed for different application scenarios, such as the home first aid kit for homes and travel first aid kit for travel. These kits typically include basic tools such as gloves, band-aids, tweezers, and sterile gauze pads. While some incorporate antibacterial drugs for infection prevention, none of the kits use AMPs to achieve their antibacterial function. Therefore, we wish to create a first aid kit incorporating AMPs that hold antimicrobial functions to save lives from the complications related to infections.

Through extensive literature review, we discovered that integrating defensins into surgical sutures can impart antibacterial properties. Thus, initially, we aimed to develop absorbable antibacterial sutures as a replacement for triclosan-coated sutures, common antibacterial sutures with coating material known for causing resistance and adverse effects such as endocrine disruption within the human body (Zhang & Lu, 2023). By fusing the collagen binding domain with antimicrobial peptides, we could bind these peptides to collagen-based materials. However, after consulting numerous medical professionals, particularly those with battlefield experience, we learned that wounds from combat are especially diverse and complex. Immediate priorities on the battlefield would be to stop bleeding, clean wounds, and evacuate patients to safety, often delaying the opportunity for suturing. During this evacuation process, infection prevention is crucial.

As a result, we shifted our focus from sutures to wound dressings. Collagen, particularly Type I collagen, was our original choice for sutures due to its absorbability, biocompatibility, and ability to promote wound healing. Further research revealed that collagen could serve not only as a suture material but also as an effective wound dressing. Building on this, we explored additional materials like chitosan, which offers moderate antibacterial properties, and alginate, a more environmentally friendly option. Additionally, inspired by the 2021 LINKS-China project, we identified cellulose-based gauze, commonly found in medical kits, as a potential material. By utilizing the CBM3 binding domain, we can effectively bind AMPs to cellulose, making it another viable candidate for our wound dressing.

Table 1: Binding domain proteins and their specialized usage

The table shows the selected binding domain proteins and their targeted binding material's specialized function in First Aid. We tested each binding domain protein's ability to bind to their target material by expressing a fusion protein composed of the binding domain and a chromoprotein. The test result is displayed in Figure 3. Look into Engineering Success for details.

Figure 3 (a) Binding efficiencies of cellulose binding domains CBM2 and CBM3 toward cellulose gauze (b) Binding efficiencies of collagen binding domains α1 and α2; chitosan binding domain CBM5; alginate binding domain CBMxx toward respective hydrogel materials. Each fusion protein can successfully bind to their target material.

In terms of antimicrobial agents, we remain committed to using defensins, guided by two considerations. First, many FDA-approved antimicrobial peptides are non-ribosomal peptides (NRAMPs), which would significantly extend our experimental timeline if synthesized de novo. Second, ribosomally synthesized antimicrobial peptides (RAMPs) from non-human sources may trigger undesired immunogenic responses. Therefore, we chose defensins, naturally occurring proteins in the human body, as our antimicrobial peptide to be incorporated into our first aid kit – the CuraPack.

Defensins are a class of antimicrobial peptides with immense potential. Found naturally in our innate immune system, they are applicable against a broad spectrum of pathogenic microorganisms, including Gram-positive and Gram-negative bacteria, fungi, and even some viruses (Zhen et al., 2019). Compared to traditional antimicrobial agents, defensins offer consistent antibacterial effects without engendering the concern of antibiotic resistance (Zhang et al., 2010).

In humans, alpha defensins include HNP1, HNP2, HNP3, HNP4, and HD5, while beta defensins include HBD1, HBD2, HBD3, and HBD4 (Huang et al., 2022). The mechanisms by which different defensins prevent the growth and proliferation of pathogens are similar: they cause the leakage of bacterial contents, leading to bacterial death or lysis (Huang et al., 2022). Their cationic nature allows them to bind to negatively charged components of the bacterial cell wall, such as the lipopolysaccharides of Gram-negative bacteria, disrupting their integrity. Alternatively, their hydrophobic regions insert into the lipid bilayer of Gram-positive bacteria, destabilizing it and promoting cell lysis. Additionally, defensins interfere with bacterial metabolism by blocking protein and nucleic acid synthesis, thereby halting bacterial growth and reproduction (Zhang et al., 2010).

Given their diverse mechanisms of action, defensins are less likely to induce antimicrobial resistance. Unlike traditional antimicrobial agents that target bacteria through a single main pathway, defensins attack from several different approaches, making it difficult for bacteria to develop resistance. This multi-faceted approach positions defensins as highly promising without intensifying antibiotic resistance (Huang et al., 2022).

Our AMP first aid kit utilizes human-derived defensins, specifically α-defensins HNP1, HNP4, and HD5, and β-defensin hBD3. These peptides exhibit superior antimicrobial efficacy compared to other defensins and are unlikely to cause adverse reactions.

We expressed and tested antibacterial ability of the four selected defensins, and optimized 3 deviations of HNP1 defensin through Modelling. During the assessment of antibacterial ability of the four selected defensins, the defensins are cleaved by Ulp1 enzyme from the original fusion protein of CBM3-sumo-defensin. Our result shows that the CBM3-sumo↓Defensins cleaved by Ulp1 enzyme exhibited antimicrobial activity against both E. coli and S. aureus, while the uncleaved CBM3-sumo-Defensins showed no antimicrobial activity. This suggests that we successfully produced active defensin molecules using the fusion protein cleavage approach. Look into Engineering Success for details.

Figure 4: Antimicrobial assays of four selected types of CBM3-SUMO↓Defensins against Escherichia coli and Staphylococcus aureus. -Ulp1 represents CBM3-SUMO-Defensins that have not been treated with Ulp1, +Ulp1 represents CBM3-SUMO↓Defensins cleaved by Ulp1.

In alignment with our combined domain design, our first aid kits will not only contain standard items like povidone-iodine and scissors, but will also feature advanced wound care materials tailored for use in environments with limited medical resources, such as battlefields and disaster zones. These include antibacterial cellulose gauze for both hemostasis and infection control, collagen dressings to promote wound healing, chitosan dressings specifically for severely infected wounds, and alginate dressings for broader wound care. This comprehensive outlook aims to reduce fatalities caused by infections in challenging, high-risk scenarios.

Figure 5: An example of how defensins affixed to gauze(cellulose domain) would function.

Through using defensins such as HNP1, HNP4, HBD3, and HD5 integrated into items within the kit by affixing them with four different binding domains, namely α1, CBM2, CBM3, and CBM5 which correspond to various textures using the SUMO enzyme, our kit represents a crucial advancement in wound care, providing a powerful, reliable defense against bacterial infections while mitigating the growing concern of antibiotic resistance.