Role of antimicrobial peptide-based biomaterials in respiratory tract infections control

Abstract

In experimental models, such as those of bacterial pneumonia and tuberculosis, AMP-containing systems promote bacterial clearance and reduce inflammatory responses more effectively compared to free AMPs. The use of AMP-containing biomaterials for treating infections offers several benefits over traditional antibiotics. Isolated AMPs are less likely to develop resistance due to their multiple mechanisms of action. Additionally, AMPs kill pathogens quickly and are likely more effective when combined with the body's defense systems. Commonly used antibiotics often lead to the development of resistant bacterial strains and typically target only a limited range of Gram-negative or Gram-positive bacteria. Families of AMPs may exhibit broad-spectrum activity against bacteria, viruses, and fungi, but with limited or no cytotoxicity at therapeutic concentrations. Challenges with AMP therapies include production costs, potential immunogenicity, and instability; however, some of these issues could be addressed through encapsulation in biomaterials. Future directions would involve optimizing hybrid therapies that combine AMPs with antibiotics or nanomaterials for individualized treatment of RTIs. Currently, AMP-based biomaterials offer new solutions to address the challenging problem of multidrug-resistant infections that still rely on earlier drug regimens, as well as to enhance clinical outcomes. AMPs may be more effective when incorporated into biomaterials designed to facilitate delivery, enhance efficacy, and/or maintain activity. This review provides an overview of the use of AMP-based biomaterials to control RTIs, focusing on their mechanisms of action, applications, and potential benefits. Antimicrobial peptides target bacterial membranes, prevent biofilm formation, and can modulate host immune response, demonstrating effectiveness against common RTI pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, and Mycobacterium tuberculosis. However, biomaterial platforms can facilitate controlled release, improved stability, and targeted administration to the respiratory mucosa, thereby overcoming rapid clearance and enzymatic degradation.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-026-05110-8.

Introduction

The global health burden from respiratory tract infections (RTIs) is considerable. It encompasses a range of diseases from upper RTIs — including the common cold and sinusitis — to lower RTIs — including bronchitis, pneumonia, and exacerbations of chronic disease states such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) [1–3]. RTIs that cause morbidity and sometimes mortality due to bacterial pathogens, such as Streptococcus pneumoniae, P. aeruginosa, and Klebsiella pneumoniae, as well as viruses like influenza, respiratory syncytial virus (RSV), and SARS‑CoV‑2, and occasionally fungi [4–6]. In 2019, RTIs caused 2.5 million deaths, and lower RTIs were responsible for a significant number amongst at-risk individuals such as the immunocompromised, elderly, and ventilated patients [7]. The impact of antimicrobial resistance (AMR) is contributing to severe RTIs as bacteria resist multiple antibiotics such as methicillin‑resistant Staphylococcus aureus (MRSA [8, 9]), multidrug-resistant Pseudomonas aeruginosa [10] and carbapenem resistant Acinetobacter baumannii [11], which leaves the clinician with few to no treatment options, contributing to lengthened hospital stays, increased healthcare costs, and decreased survival. If effective solutions to address AMR are not found, we expect more than 10 million deaths/year by 2050, which would obviously require new treatment pathways; therefore, we must change the treatment paradigm [12, 13].

Antimicrobial peptides (AMPs) or host defense peptides (HDPs) are part of the innate immune system in our body. They are small chains of amino acids with a positive charge that can bind to the negatively charged membranes of pathogens, allowing them to target bacteria, viruses, fungi, and parasites without readily developing resistance [14–16]. In the respiratory tract, airway and lung epithelial cells release AMPs, such as LL-37 and defensins [17, 18]. AMPs can directly target pathogens and also serve a secondary role in regulating the inflammatory response and recruiting immune cells to the site of infection. AMPs are effective against a broad range of microbes through various mechanisms, ultimately disrupting membranes and targeting specific biological processes within the intracellular space. These AMPs can be expressed constitutively or inducibly in airway epithelial, neutrophil, and macrophage cells, and they confoundingly create a chemical barrier in mucosal secretions [19–22].

Despite their purported advantages, AMPs present several serious drawbacks in a therapeutic context. Natural peptides are in low abundance and are quickly degraded and eliminated after delivery (i.e., after passing through the gastrointestinal tract or other bodily fluids). The rapid host degradation process subsequently limits peptide availability for optimal absorption and distribution, and their inherently flexible conformations lead to undesired interactions with other substances, which may result in adverse effects [23, 24]. The DRAMP database notes that ~ 67% of all described AMPs are > 20 amino acids, and an even more astonishing ~ 78% of human AMPs are also > 20 amino acids. An increase in amino acids also increases the complexity and difficulty of synthesis and applications. Moreover, these peptides are often enriched in complex cationic and hydrophobic residues [25–28], which is a consideration for production costs and creates significant technical challenges in delivering peptides to patients [29].

In response to these problems, researchers have begun examining AMP-based biomaterials, which utilize peptides delivered via novel modes of delivery. Interest in biomaterials (nanoparticles, hydrogels, liposomes, dendrimers, and self-assembling nanostructures) is being explored as a possible strategy to overcome these challenges [29, 30]. However, the success of the co-delivery system will depend on its ability to utilize non-covalent interactions to form and/or self-assemble into a specific structure. Self-assembling lipopeptide C16—3RP forms nanoparticles that reduce bacterial load in animal models of sepsis and pneumonia. For RTIs, AMP-based biomaterials have potential for other indications in mechanical ventilation, such as ventilator-associated pneumonia (VAP), a type of pneumonia caused by MDR gram-negative bacilli, which are often consulted for ICU patients, and we hope will translate safely and effectively for preventive use. Similarly, their noted success will depend on the construct's ability to protect AMPs while offering stimulus-responsive release [30–32].

Biomaterials can significantly enhance antibiotic therapies by reducing adverse effects through reduced systemic dosing and minimizing off-target effects. Biomaterials facilitate the use of AMPs to treat infections and mitigate the adverse effects of conventional treatment therapies. By tethering AMPs to drug delivery systems, their performance and duration of action are enhanced. The biomaterial used is critical to the delivery/efficacy. For RTIs, AMPs are essential due to their intrinsic antimicrobial potential and immunomodulating roles in innate immunity [33–35].

This review aims to provide a comprehensive account of the use of AMPs and their function as alternatives to conventional antibiotics, while examining their mechanisms of antimicrobial action, their ability to modulate the immune system, and methods to enhance their antimicrobial efficacy. This review will also address opportunities for using AMPs in biomaterials for biomedical applications, as well as the limitations and prospects of AMP development. We will also consider evidence that these peptides are natural antibiotics against respiratory viral and fungal infections.

Importance of antimicrobial peptides against respiratory tract infections

Despite the esteem attributed to AMPs as a fundamental element of the innate immune system and a first line of defense in the respiratory tract, there are caveats to their use. AMPs are produced in the airway mucosa by neutrophils, epithelial cells, and other types of immune cells, and although they do exhibit broad-spectrum antimicrobial activity, often, claims that AMPs kill or otherwise inhibit a wide range of pathogens—specifically, influenza viruses, coronaviruses, fungi (to include Candida albicans), bacteria (to include MRSA and P. aeruginosa), and parasites—rarely consider a host of important caveats. Although the mechanisms by which AMPs disrupt microbial membranes in vitro [such as barrel-stave, toroidal-pore, or carpet-like models] are known, bactericidal activity may not occur consistently in vivo, nor will they demonstrate robust in vivo efficacy [36–38].

In addition, while AMPs can inhibit cell wall formation or protein synthesis in microbes and can even influence immunity by recruiting immune cells, the effect of AMPs on the outcome of infection remains debated. Furthermore, in the absence of clinical evidence, the potential for LPS neutralization and for topical/in vivo wound-healing properties is often overstated. More realistically, AMPs are usually required at concentrations that cannot be achieved at the site of infection because of rapid degradation and/or inhibition by physiologic factors. In conclusion, although AMPs can show dysfunctional properties as immunotherapies and topical agents, the impact felt in people's daily care is unrealistically exaggerated, even in light of our current research [39–41].

Although AMPs are often credited with providing crucial defenses against RTI, their efficacy is not absolute. Although AMPs are secreted into the airway surface liquid (ASL) to maintain sterility, their activity can be severely compromised by factors such as ASL acidification and increased salinity. Losing AMP functionality can then permit the persistence of pathogens such as S. aureus and P. aeruginosa, thus calling into question the protective function that AMPs are purported to possess. Furthermore, altered expression of AMPs during disease states (such as tuberculosis) may confer susceptibility to infection and fail to provide their protective effects. Proponents of AMPs will point to the multi-target mechanisms of AMPs and their potential to synergize with antibiotics, particularly against drug-resistant bacteria, but such claims remain largely theoretical, or at most, limited to early-stage research [38, 42–44]. Claims of low resistance development also require substantial clinical research evidence before they can be accepted without incredulity. Even though AMPs are present in respiratory secretions and could contribute to host defense under ideal conditions, their actual capacity to prevent infection and treat RTIs will be limited by these physiological factors and disease states. It is best to scrutinize their potential for therapy until sufficient relevant evidence supports implementation in a clinical setting [44, 45]. Table 1.

Table 1.

Major antimicrobial peptides/factors are produced in the lung and respiratory tract

Antimicrobial peptides Factors	Source/Actions	Distribution in the Lung	Infection preventing	Source of Infections	Refs
Defensins (α and β defensins 1–4)	Human/Defensins combat both enveloped and non-enveloped viruses directly while modulating the immune system	Lung epithelium, airways, neutrophils	Viral infections Bacterial infections	Gram-negative bacteria, Gram-positive bacteria, COVID-19,	[36, 44]
Cathelicidins (LL-37)	Human Regulation of its expression involves growth hormones, cytokines, and, particularly, vitamin D	Lung epithelium, airways, respiratory sub-mucosal glands, neutrophils	Viral infections Bacterial infections	Gram-negative bacteria, Gram-positive bacteria, Rhinovirus and RSV	[36, 46]
Histatins	Human/Located only in the mouth, they function as the first line of defense against infections in the respiratory system	Existing naturally in saliva, the others are their proteolytic derivatives	Viral infections Bacterial infections Fungal infections	Gram-negative bacteria, Gram-positive bacteria, COVID-19, Candida spp.	[36]
Surfactant protein-A and surfactant protein-D (SP-A, SP-D)	Human/Alter the native immune system's activity, given its propensity to attach to bacterial lipopolysaccharide	Lung epithelium	Viral infections Bacterial infections Fungal infections	Gram-negative bacteria, Gram-positive bacteria, COVID-19, A. fumigatus and C. albicans	[36, 47]
Lysozyme	Human/Eliminates bacteria by disrupting the glycosidic link in bacterial cell walls	Tracheobronchial sub-mucosal glands, tracheal and bronchial surface epithelium, neutrophils, alveolar macrophages, monocytes	Viral infections Bacterial infections	Gram-negative bacteria, Gram-positive bacteria, COVID-19,	[36, 48]
Secretory leukocyte proteinase inhibitor (SLPI)	Human/SLPI inhibits serine proteases, protecting against neutrophil elastase during infection and inflammation	Respiratory sub-mucosal glands, monocytes, alveolar macrophages, and neutrophils	Viral infections Bacterial infections	Gram-negative bacteria, Gram-positive bacteria,	[36, 49]
Lactoferrin	Human/Oral intake can reduce lung cell infiltration during pneumonia by blocking IL-8 production and altering it	Tracheobronchial sub-mucosal glands, tracheal surface epithelium, and neutrophils	Viral infections Bacterial infections	Gram-negative bacteria, Gram-positive bacteria, HIV, CMV, Hepatitis B/C	[36, 48]
Phospholipase A2	Human/Combats bacteria through the breakdown of their membranes, a process driven by its positive charge and enzymatic capabilities	Lung epithelium, neutrophils	Bacterial infections	Gram-positive bacteria,	[36, 50]
Lactoperoxidase	Human/LPO facilitates iodine oxidation, reportedly enhancing the antiviral response of respiratory mucosal tissues	Airways mucosal membrane	Viral infections	Influenza virus	[36, 51]
CCL20	Human/It is involved in innate and adaptive immunity, affected by airway inflammatory mediators, and produced by neutrophils influenced by cytokines like IL-1 and TNF- via the NFkB pathway	Airways epithelium	Viral infections Bacterial infections	Gram-negative bacteria, Gram-positive bacteria, HIV-1, COVID-19	[36, 52]

Antimicrobial peptides classification

The heterogeneity of natural AMPs complicates their classification. AMPs can be classified in the following methods: (1) source, (2) structural characteristics, (3) amino acid-rich species, and (4) activity. Table 2.

Table 2.

Advantages and disadvantages of integrational methods of design of antimicrobial peptides-based biomaterials

Method	Description	Advantages	Disadvantages	Examples	Ref
Covalent Bonding	Chemical attachment of AMPs to biomaterial surfaces or matrices, often via amidic bonds, linkers, or functional groups like thiol or amino	High stability with no leakage; sustained activity against pathogens like E. coli and S. aureus	Potential reduction in AMPs' flexibility and activity; complex synthesis requiring specific conditions	UPy-modified AMPs (e.g., LASIO III) covalently linked to polycaprolactone (PCL) for elastomeric films	[53]
Physical Incorporation	Mixing or adsorbing AMPs into the biomaterial matrix without chemical bonds, allowing modular assembly	Simple preparation; dynamic release; easy to vary AMPs types	Risk of leakage (up to 3% in some cases), leading to reduced long-term efficacy and potential toxicity	Blending unmodified AMPs like L9K6 into UPy-functionalized PCL for supramolecular materials	[53]
Self-Assembly	AMPs form nanostructures (e.g., nanofibers, micelles, nanogels) through hydrophobic and electrostatic interactions, often enhanced by modifications like fatty acid conjugation	Improved stability and targeted delivery; mimics natural structures	Dependent on the AMPs sequence, it may require optimization for physiological conditions	Cholesterol or myristic acid-modified AMPs form micelles for wound healing	[33]
Conjugation with Nanoparticles	Coupling AMPs to metal or polymer nanoparticles using surface chemistry	Enhanced bactericidal synergy; protection from degradation	Potential cytotoxicity from nanoparticles; scalability issues	AMPs bound to gold nanorods (AuNRs) for photothermal-antimicrobial therapy against MRSA
Loading into Porous Materials	Encapsulating AMPs in mesoporous structures for controlled release	High loading capacity; protease protection	Limited to specific pore sizes; risk of burst release	AMPs loaded into silica or TiO2 nanotubes for implant coatings	[54]
Polymer Coupling	Linking AMPs to polymers like PEG or chitosan to form conjugates	Increased solubility and circulation time; reduced immunogenicity	May alter AMPs charge or amphipathicity	PEGylated AMPs like CT9W1000 for enhanced activity against P. aeruginosa	[55]
Hydrogel Formation	Incorporating AMPs into self-assembled or carrier-based hydrogels for sustained release	Biocompatible; suitable for topical applications; responsive to stimuli	Swelling or degradation in vivo; variable release profiles	Chitosan-hyaluronic acid hydrogels with AMPs for skin infections	[53]

Classification of AMPs based on sources

In terms of sources, AMPs are classified into mammalian, amphibian, microbial, and insect groups, with a large proportion of human host defense peptides documented in APD3 data. AMPs from ocean sources have also garnered broad attention. [56].

Mammalian antimicrobial peptides

AMPs are found across a wide range of mammals, including humans, sheep, cattle, and other vertebrates. The primary classes of AMPs are cathelicidins and defensins. Defensins can be identified by their specific structures, including the disulfide bonds of α-, β-, and θ-defensins. Human host defense peptides are designed to combat microbial infections; however, all AMPs are expressed at different stages of human development. The cathelicidin LL-37 is expressed in infant skin, whereas older adults express human beta-defensin 2 (hBD-2). AMPs exist in many areas of the body, such as skin, eyes, ears, mouth, respiratory tract, lungs, intestine, urethra, and during breastfeeding to minimize sickness and death in breastfed infants. Milk enzymatic hydrolysis forms AMPs, a significant class of antimicrobial peptides (AMPs) found in dairy products. AMPs have been located in fractions of α-lactalbumin, β-lactoglobulin, lactoferrin, and casein. While AMPs exert antimicrobial activity, human defensins have also been shown to regulate innate immunity, apoptosis, and healing [56, 57].

Amphibian-derived antimicrobial peptides

AMPs in amphibians play a crucial role in mitigating the effects of pathogens that threaten to cause widespread declines in amphibian populations. Frogs are the primary source of amphibian AMPs, with magainins representing the most well-studied peptide. Skin secretions of frogs in four genera, the Pipidae, are rich in AMPs. Moreover, cancrin (GSAQPYKQLHKVVNWDPYG) was identified as the first AMP from the sea amphibian Rana cancrivora, suggesting that there may be an even broader source of AMPs in amphibians [58–60].

Insect-derived antimicrobial peptides

The majority of insect AMPs are produced in blood cells and fat bodies, which play crucial roles in their adaptation and survival strategies. One of the best-studied families of cecropin AMPs is found in animals such as guppies, silkworms, BEE species, and the fruit fly (Drosophila). The action of AMPs against bacteria is well established, but not all species have demonstrated the same level of protection, as evidenced by the varying levels of AMPs across insects [61]. Cecropin A has been shown to have activity against various inflammatory diseases and cancers; however, this is not the case for all AMPs. Furthermore, the number of AMPs can vary significantly among species. Examples include the invasive harlequin ladybird beetle (Harmonia axyridis) and the black soldier fly (Hermetia illucens), among species that contain over 50 AMPs, compared with the pea aphid (Acyrthosiphon pisum), which does not contain any AMPs. Jellein, a peptide found in bee royal jelly, has shown interesting effects against multiple bacterial and fungal infections, but remains confined to laboratory settings. Further research has shown that this lauric acid-conjugated peptide alters the growth of the Leishmania major parasite; however, it does not provide a more effective treatment for parasitic infections [61–63].

Microorganisms-derived antimicrobial peptides

AMPs can be derived from actinobacteria, bacteria, fungi, and plants, with notable peptide types of AMPs found in Lactococcus lactis (nisin and lacticin 3147), B. subtilis (gramicidin), and Bacillus brevis. The difficulty of chemically synthesizing AMPs for in vivo studies has led to widespread investigation into biological expression. Several expression systems have been developed using specific yeast species (e.g., Pichia pastoris and Saccharomyces cerevisiae), bacteria (e.g., E. coli and Bacillus subtilis), and plants. However, the growing interest in prokaryotic and eukaryotic systems is complicated by purification issues, proteolytic degradation, and potential toxicity associated with E. coli AMPs, necessitating the use of fusion tags. Importantly, many AMPs have been isolated and purified from plant stems, seeds, and leaves, and can generally be classified as thionins, defensins, or snakins based on their structures. Furthermore, the increased attention on marine resources has led to the discovery of more marine-derived AMPs [64, 65].

Many AMPs of marine origin that have been described or tested in vitro have demonstrated potential in living organisms. As-CATH4 has been shown to activate immunity in living organisms, unlike standard antibiotics, which may enhance the anti-infective properties of other antibiotics when compared with fluid therapy alone. Myticusin-beta, an immune-related AMP from Mytilus couscous, may offer potential as an alternative to antibiotics. Furthermore, GE33, also known as pardaxin and an AMP of marine origin, has demonstrated the potential to enhance antitumor immunity in a vaccine approach in mice [64, 65].

Classification of AMPs based on amino acid-rich species

Proline-rich peptides (PrAMPs)

Proline serves as a non-polar amino acid, which is typical of the group of amino acids of which it is a member. However, PrAMPs exhibit distinct behavior compared to other AMPs. They do not cause membrane collapse; instead, a specific bacterium can be targeted after translocation into the cytoplasm via the inner membrane transporter SbmA. After translocation, PrAMPs interact with ribosomes, inhibiting protein biosynthesis. The mechanism of action occurs at the PTC by either obstructing the binding of aminoacyl-tRNA or by preventing the release of decoding release factors upon termination of translation [66]. A member of the Ortho-family of AMPs, Tur1A, which is a homologous AMP of the bovine PrAMP Bac7, discovered from the Tursiops truncatus, severs the transition from the initiation phase to the extension phase of protein biosynthesis by binding to bacterial ribosomes [67, 68].

Furthermore, PrAMPs often differ in sequence and do not necessarily exhibit high amino acid sequence similarity. However, most PrAMPs have 2 to 3 short motifs with shared sequence patterns, containing repeating proline and arginine residues, such as -PPXR- in Bac5 or -PRPX- in Bac7. Most PrAMPs interact with Gram-positive bacteria, but the proline-rich AMP pPR-AMP1 identified from the crab Scylla paramamosain has vigorous antibiotic activity against both Gram-positive and Gram-negative bacteria. Studies also reported that PrAMPs exhibit in vivo immunostimulatory properties [67, 68].

Tryptophan- and arginine-rich antimicrobial peptides

The non-polar amino acid tryptophan (Trp) has a significant effect on the lipid bilayer's interface region, while the basic amino acid arginine supplies charge to the peptide and hydrogen bonding interactions, which are essential for the coupling to the bacterial membrane's abundant anionic component. Trp residues likely act as natural aromatic enhancers of Arg-rich AMPs through ion-pair-π interactions, thereby enhancing peptide-membrane interactions. In addition to indolicidin and trippticin, which have been extensively shown to be Arg- and Trp-rich AMP, Octa 2 is also a typical AMP that is rich in Trp and Arg that inhibits Gram-negative E. coli and P. aeruginosa as well as Gram-positive S. aureus. Likewise, short AMPs rich in Trp and Arg, designed based on bovine and murine lactoferricin as models, have been shown to inhibit bacteria significantly [69, 70].

Histidine-rich peptides

Histidine is an essential basic amino acid, and AMPs rich in histidine exhibit enhanced membrane permeability. HV2 is a histidine-rich AMP that is modelled on RR(XH)2XDPGX(YH)2RR–NH2, where X is I, W, V, and F. This peptide increases the permeability of bacterial cell membranes, leading to a disruption of the cell membrane and subsequent cell death. Furthermore, HV2 exhibits concentration-dependent suppression of bacterial movement, in addition to effectively reducing inflammation by suppressing tumour necrosis factor α (TNF-α), a pro-inflammatory cytokine. Research has indicated that an AMP designed according to the principles of Octa 2 exhibits good therapeutic potential when intended to replace some arginine residues with histidine. Additionally, a compound, L4H4, was derived from a linear cationic amphiphilic peptide, magainin, which exhibited effective antibacterial activity and cellular permeation due to the introduction of four histidine residues among leucine and alanine residues [69, 70].

Glycine-rich antimicrobial peptides

Categorizing glycine as a nonpolar amino acid is an oversimplification from a biological standpoint. It is noteworthy to mention that glycine-rich antimicrobial polypeptides, such as attacins and diptericins, have been identified in nature and consist of a sufficiently large number of glycine residues. Glycine residues influence the tertiary structure of the polypeptide chain. Glycine-rich AMPs of salmonid cathelicidins are also believed to trigger a phagocyte-mediated microbe killing mechanism that is different from that of AMPs. This finding highlights glycine-rich AMPs as a distinct class. The glycine-rich complex-symmetrical (GG3) has been proposed as a candidate for a commercial drug against clinical Gram-negative bacteria. The classification is based on the provided activity and the potential therapeutic benefit [71–73].

Classification based on activity

The activity of AMPs can be categorised into 18 groups according to ADP3 database statistics. These categories can be encapsulated as antibacterial, antiviral, antifungal, antiparasitic, anti-human immunodeficiency virus (HIV), and anti-tumor peptides [74–76].

Antibacterial peptides

AMPs are a significant portion of antibacterial peptides that have a broad-spectrum inhibitory impact against a wide range of pathogenic bacteria, such as vancomycin-resistant enterococci (VRE), Acinetobacter baumannii, methicillin-resistant S. aureus (MRSA) in the clinical setting, S. aureus, Listeria monocytogenes, and E. coli on food surfaces, as well as Salmonella and Vibrio parahaemolyticus in aquatic foods. Several natural and synthetic AMPs, such as nisin, cecropins, and defensins, have demonstrated effective inhibitory properties against Gram-positive and Gram-negative bacteria. Previous studies have also reported that the AMPs P5 and P9, derived from interferon-I in Aristolochia nobilis, have potential inhibitory effects against MRSA with reduced toxicity to other host cells [68, 73, 77, 78].

Antifungal peptides (AFPs)

Antifungal peptides (AFPs), a subgroup of AMPs, target specific fungal infections that are further aggravated by increasing rates of drug resistance. Numerous AFPs demonstrate exceptional antifungal activity against common pathogenic fungi in the clinical world, including Aspergillus and Candida albicans, as well as yeast and filamentous fungi (such as Aspergillus flavus) in the food and agricultural sectors, and mould. Many synthetic peptides, excluding brevinin, ranatuerin, and cecropins, also possess antifungal properties. AurH1, a peptide derived from aurein 1.2, shows great potential for managing infections caused by C. albicans, a pathogen with a 40% mortality rate among infected individuals. Furthermore, aflatoxin, a carcinogenic produced by the fungus A. flavus, is harmful to the human body, and many AFPs possess the ability to inhibit the growth of A. flavus. One example includes an antifungal peptide, FPSHTGMSVPPP, which inhibited the growth of A. flavus MD3. Also derived from Lactobacillus plantarum TE10, 37 antifungal peptides and combination treatments can inhibit A. flavus spore formation on fresh maize seeds. Finally, two radish AMPs were chemically synthesized and displayed potent inhibition and antifungal ability against numerous yeast species, specifically Zygosaccharomyces bailii and Zygosaccharomyces rouxii [79–82].

Antiviral peptides

Antiviral peptides exert their potent killing action on viruses through three pathways (1), such as inhibiting the virus from binding to and fusing into the host cell membrane, (2) destroying the viral envelope, and (3) inhibiting viral replication. It has been demonstrated that AMP Epi-1 mediates the inactivation of virus particles and exhibits effective antiviral activity against the foot-and-mouth disease virus in animals [83, 84]. Additionally, a study showed that swine intestinal AMP (SIAMP) administered as an inoculum with infectious bronchitis virus (IBV) significantly reduced mortality in chick embryos compared with the IBV-only group, indicating SIAMP's potent antiviral activity against IBV. Antiviral peptides are also a subclass of AMP. Anti-HIV peptides such as defensins (including α defensins and β defensins, which act in distinct ways), LL-37, gramicidin D, caerin 1, maximin 3, magainin 2, dermaseptin-S1, dermaseptin-S4, siamycin-I, siamycin-II, and RP 71955 are examples of this subclass of peptides. The antiviral peptide FuzeonTM (enfuvirtide) has been commercialized as an anti-HIV drug [85–87].

The coronavirus is a significant global public health issue; therefore, it is imperative to discuss antiviral peptides that could be considered for preventing SARS-CoV-2 transmission during the COVID-19 pandemic. We also should recognize the potential limitations of antiviral peptides. They focus on the spike protein, which generates infectious viral particles. Antiviral peptides have been identified against potential viral infections, such as EK1C4, a fusion inhibitor against COVID-19, and temporin, an antiviral peptide against MERS-CoV. However, the antiviral work has been limited to small-animal testing and has not progressed to human clinical trials. Similarly, using molecular docking analysis to inhibit the interaction between COVID-19 and ACE2 shows promise. However, it also has limitations and requires further clinical trials, as it has been studied only in spontaneous claims and small-animal studies [88, 89]. Figure 1.

Fig. 1.

Overview of the production of various human antimicrobial peptides against respiratory tract infections. The notably significant and extensive area presented by the anatomical structures of the respiratory system invariably serves as the very first point of entry and direct interaction for a diverse range of microorganisms and other non-indigenous, foreign particulates. The entirety of the respiratory lining, often referred to as the epithelium, is consistently enveloped by a delicate film composed of various secretions originating from both the airways and the alveoli. Among the antimicrobial components expressed in the respiratory tract, human antimicrobial peptides, such as defensins and cathelicidins, are the subject of the most significant research and are found within respiratory secretions. Within human physiology, AMPs play a crucial role in the initial immune response, combating harmful microorganisms. Within human physiology, AMPs play a crucial role in the initial immune response, combating harmful microorganisms. Host peptides are generated by epithelial and immunological cells within the pulmonary surface fluids, where they function as intrinsic antimicrobial agents in the respiratory system

Challenges and potential solutions in using antimicrobial peptides

Resistance to antiviral peptides

Antiviral peptides (AVPs) have emerged as a promising class of therapeutics for treating viral infections, offering a viable alternative to traditional small-molecule antivirals, which can quickly fail due to resistance. AVPs can be derived from natural sources (defensins, cathelicidins, and cyclotides) or synthesized and comprise peptides that typically range from 10 to 100 amino acids. AVPs also exhibit amphipathic and cationic properties and often possess a myriad of mechanisms of action, including membrane disruption, inhibition of entry, prevention of fusion, targeting of viral enzymes, or immunomodulatory activities [68, 90]. AVPs can target enveloped viruses, such as HIV, influenza, SARS-CoV-2, and herpes simplex virus, while inducing disturbances in viral envelopes, binding to glycoproteins (gp41 or hemagglutinin), or modulating receptor engagement to interrupt the early stages of replication. However, despite their promise, viruses can develop resistance to AVP activity through mutagenesis under selective pressure from the antibiotic; this type of resistance primarily develops against targeted inhibitors. Resistance to enfuvirtide, a synthetic peptide fusion inhibitor used in the treatment of HIV, is thought to arise from mutations in the gp41 heptad repeat 1 (HR1) region, thereby decreasing the binding and efficacy of the fusion inhibitor to HIV-infected cells. Resistance to enfuvirtide in HIV requires multiple mutations, with costs to overall viral fitness, including infectivity/replication capacity, and fusogenicity [91, 92].

Similarly, influenza viruses may develop resistance to peptides like P9 via adaptations in hemagglutinin, but optimized variants such as P9R, with enhanced positive charge through arginine substitutions, demonstrate no detectable resistance even after 40 serial passages in vitro. Factors that exacerbate resistance include high viral mutation rates, incomplete viral suppression due to suboptimal dosing or poor adherence, and baseline polymorphisms that confer cross-resistance to antiretroviral therapies. Broad-spectrum AVPs that disrupt membranes are less prone to resistance, as altering envelope composition incurs significant evolutionary penalties [93]. To mitigate resistance, strategies focus on rational biodesign, targeting conserved viral structures using structural data from tools like AlphaFold. This approach incorporates noncanonical amino acids or cyclization for stability and combines AVPs with other antivirals to achieve synergistic effects that raise genetic barriers [90, 94, 95].

Additionally, host-targeted approaches or lethal mutagenesis can complement AVPs, reducing the likelihood of escape mutants. Overall, while resistance poses challenges, the multifaceted actions and design flexibility of AVPs position them as vital tools in combating emerging viral threats, potentially outpacing the evolution of resistant strains through innovative engineering and combination therapies. Cross-resistance between drug classes further limits available therapy options for clinicians, and structural adaptations at the target site are allowing viruses to outstrip our treatment options [96, 97]. The rapid recombination of viral genomes also enables evolution, as seen with the continually emerging genetic variants of influenza. A chronic infection like HIV can result in the emergence of multiple drug-resistant subpopulations in part due to genomic mutations, but also because the infection can induce increased efflux of the drug away from the target site. Dosage changes and other alterations are merely additional obstacles to our treatment regimens, reflecting fundamental deficiencies in our approach to treating and managing viral diseases effectively. This is a very real and discouraging truth that we must recognize and reassess if we are to prevent pathogens, in their evolutionary journey, from rendering us permanently vulnerable [90, 95].

Resistance to antibacterial peptide

AMPs, also known as antibacterial peptides, pose a growing threat in the management of bacterial infections because they are naturally occurring or synthetic molecules that can disrupt bacterial membranes, inhibit protein synthesis, or alter immune processes to limit pathogen growth. Bacteria have developed various mechanisms to resist AMPs, which, interestingly, occur at a slower rate than antibiotic resistance, but that also have critical ramifications for therapeutic use. The primary mechanism is to adjust the bacterial cell envelope to limit AMP attachment; for example, gram positive bacteria such as S. aureus may change their teichoic acids or modify the peptidoglycan layer to add positive charge to amino acids to repel cationic AMPs, while gram negative bacteria such as P. aeruginosa alter the lipopolysaccharide (LPS) layer of the bacterial envelope by adding aminoarabinose or phosphoethanolamine to reduce the electrostatic interaction. A second intrinsic mechanism of resistance involves enzymatic degradation, in which bacterial pathogens release proteases that cleave AMPs into non-inhibitory fragments, which are then degraded by various broad-spectrum proteases. Additionally, efflux pumps, a common mechanism in the resistance-nodulation-division (RND) family, are known to export AMPs from the cell. Biofilm formation provides additional physical resistance mechanisms that could impede the ability of some AMPs to penetrate the biofilm and reach the underlying viable cells, locking them in the extracellular matrix [98–101].

Studies from an evolutionary perspective suggest that resistance can develop under selective pressure, as observed in studies of S. aureus evolving in response to AMPs, leading to pharmacodynamic changes and cross-resistance to host defenses. In Gram-negative bacteria, the development and emergence of colistin resistance not only provide tolerance to this last-resort class of antibiotics and additional stability to human innate immune peptides, such as LL-37, but also demonstrate how the anthropogenic use of antibiotics enhances their inherent defenses against AMPs. Overall, while AMP resistance still occurs, it appears to happen less frequently than antibiotic resistance, which may be due to the multiple potential modes of action of the peptides themselves, presenting the potential for multifaceted mechanisms that make even complete evasion difficult. Despite resistance emerging to AMPs, concern remains about their ability to serve as long-term alternatives to antibiotics, which has spurred research into engineered AMPs with reduced susceptibility to resistance mechanisms. Ultimately, whether discussing resistance mechanisms or other mechanisms, a thorough understanding of these mechanisms will inform the development of the next generation of antimicrobials that can remain one step ahead of antimicrobial evolution, thereby continuing to work against multidrug-resistant human and agricultural pathogens into the future. If AMPs remain active at a quasi-simultaneous complete wake mechanism for killing bacteria, and that action is not based entirely on targeting multiple targets, then they will likely have a lower degree of susceptibility to change due to microbial adaptation; yet, microbes manage to evolve new mechanisms to survive these mechanisms [98, 102].

Moreover, this implies our potential inability to improve infectious disease control due to the significant turnover in microbial adaptation induced by AMP use. Our respect for microbes' imagination underscores our fragile dependence on AMPs and highlights the daunting task of staying ahead of bacterial evolution in the fight against infectious diseases [101–103].

Resistance to antifungal peptide

The increasing prevalence of antifungal peptide resistance has become another obstacle in combating fungal infections, as antifungal peptides, also known as natural defensins or synthetic analogs, target fungal membranes or cellular processes to compromise cellular integrity. To overcome antifungal peptides, fungi employ various resistance strategies, often similar to those mechanisms fungi have developed for conventional antifungal agents. However, they now utilize those same strategies against antifungal peptides, leveraging their multifaceted activity. Perhaps one of the major antifungal peptide strategies is membrane remodeling, which can occur in various pathogens, including Candida albicans—a common fungal pathogen—that modifies lipid rafts composed of ergosterol and sphingolipids to lower the binding affinity of antifungal peptides and reduce peptide-mediated membrane permeabilization [104, 105]. Efflux mechanisms also play a significant role in several antifungal peptides, as MDR proteins contribute to the increased extrusion of antifungal peptides [106]. In allogeneic studies, increased efflux of aureobasidin A was observed in the Aspergillus species, attributed to the increased expression of MDR proteins. Finally, fungi have also adapted to synthesize and secrete proteases that degrade antifungal peptides, thereby disrupting their accumulation at the target protein site. In addition to the aforementioned multidrug-resistant mechanisms, some fungi have also developed resistant strategies at the genomic level, using mutations in specific genes that encode targets of antifungal peptides (e.g., FKS1/FKS2) to modify glucan synthase activity, which lowers the efficacy of echinocandin class antifungals and ultimately produces acquired resistance in, for example, Candida glabrata [104, 107, 108].

Epigenetic changes and cellular stress responses further enhance the concept of tolerance; chitin synthesis is upregulated in response to peptide-induced cell wall damage, primarily through the PKC or calcineurin pathways, which restores some degree of growth under stress. Biofilm formation supports tolerance as well, not only through the physical barriers they form, but also by promoting persister cells, which can fill microenvironments with diluted concentrations of peptides [109, 110] . Overall, antifungal peptides tend to have a lower rate of resistance development than one-target antifungals, because they have a broader spectrum, some with rapid-killing pharmacodynamics and a limited mutant-selection window, which makes it more difficult for resistant mutants to develop or be selected for. However, as with azoles and polyenes to some extent, and with echinocandins in hospital settings, the continuous use of antifungal peptides could lead to rapid resistance. Combination therapies—combining a peptide with an azole or polyene, for example—can be employed to utilize synergies and reduce the emergence of resistant mutants. Additionally, developing a multi-target or multi-component peptide that targets different fungal components can also delay adaptation [108, 111, 112]. Figure 2.

Fig. 2.

The challenges associated with drug-resistant antimicrobial peptides (AMPs), including their inherent toxicity (1), insufficient stability, and attenuated antibacterial efficacy (2), along with their high production cost (3), necessitate the development of effective mitigation strategies. Protease degradation affects AMPs, as do serum elements, salt, and pH levels. The ionic strength of a solution can affect the electrostatic interaction between peptides and bacterial membranes. High salt levels, such as those in saline, can alter the stability and activity. The interaction of salt ions with the charged amino acids of AMPs can alter their structure, influencing their antimicrobial action. AMPs' solubility and aggregation are modified by salt and hydrophobicity. Another element that significantly influences the stability and activity of AMPs is environmental pH. Optimal antimicrobial action by AMPs occurs at neutral or slightly acidic pH levels, which are typical of many body tissues

New research has highlighted the importance of careful surveillance for resistance, particularly in immunocompromised populations at risk for invasive candidiasis and aspergillosis. Overall, we need to understand these resistance pathways to develop new antifungal peptides effective against evolving fungal threats [81, 113].

When used in biologically relevant materials, such as nanoparticles, hydrogels, polymers, or liposomes, they can enhance peptide stability, reduce toxicity, and enable controlled release and targeted delivery. This is especially advantageous for pulmonary infections through inhalation or aerosol delivery. These materials attempt to address issues associated with MDR bacterial, viral, or fungal pathogens responsible for pneumonia, CF exacerbations, or invasive aspergillosis. The following sections categorize the materials based on biomaterial platform and present data on their potential (applications) in pulmonary infections, with examples, mechanism of action, and efficacy data if known [114, 115].

Integration of antimicrobial peptides into biomaterials

Natural AMPs are prone to proteolytic degradation in the protease-loaded environment of the lung and have short half-lives. Biomaterials are recommended as protective matrices that enable prolonged release and localized delivery of a therapeutic agent. The incorporation of AMPs into biomaterials addresses key limitations, including low in vivo stability, rapid proteolytic degradation, and nonspecific toxicity, thereby enabling localized delivery, sustained release, and improved efficacy. This is particularly relevant for specific biomedical applications, such as minimizing implant-associated infections, promoting wound healing, and treating localized bacterial infections. For utilization through the respiratory system, biomaterials should be biocompatible, aerosolizable, and capable of penetrating the mucus barrier [99].

Types of antimicrobial peptides-based biomaterials

Hydrogels and gels have been used to create antimicrobial barriers, such as self-assembling peptide hydrogels, including those composed of AMPs with lipid tails. The practical utility of such barriers is unclear. AMP-functionalized mesoporous hydrogels have been utilized to combat biofilms associated with P. aeruginosa infections; however, it remains uncertain whether these solutions apply to other types of infections. Inhaled hydrogels, such as the spherical hydrogel inhalation for increased lung defense (SHIELD), create bioadhesive barriers against SARS-CoV-2 [100], but there is some uncertainty about their long-term efficacy. Polymeric nanoparticles, such as PLGA-based nanoparticles and other nanocarriers, are used to encapsulate AMP in AMP-controlled release systems, which is another method for sustaining AMP release. However, the kinetics and drug release may not be suitable for all types of infections [32, 33]. Modified copper nanozymes with 4-mercaptobenzoic acid have also shown potential for treating bacterial infections and immune system overactivation in the lungs, although their specificity to the target pathogens remains unclear. In addition, self-assembled nanopeptides have been shown to enter cells and target intracellular bacteria; however, the mechanism of action remains unknown. Liposomes and lipopeptides have also been used to deliver AMPs; however, the pulmonary deposition of these formulations may not be consistent across patient types [116–118]. Table 3.

Table 3.

Different Classification of Antimicrobial Peptides

Basis of Classification	Subcategories	Description	Examples	Ref
Source	Mammalian	Derived from humans, sheep, cattle, and other vertebrates; often from families like cathelicidins and defensins (subdivided into α-, β-, and θ-defensins based on disulfide bonds). Involved in immune regulation, apoptosis, and wound healing	Cathelicidin LL-37 (human skin), human beta-defensin 2 (hBD-2), lactoferricin B (LfcinB) from dairy sources, Casein201 from human breast milk	[56, 57]
	Amphibian	Primarily from frogs and sea amphibians; protect against pathogens	Magainin (frogs), cancrin (GSAQPYKQLHKVVNWDPYG from Rana cancrivora)	[58–60]
	Insect	Synthesized in fat bodies and blood cells; vary by species (e.g., up to 50 in some beetles)	Cecropin (e.g., Cecropin A from silkworms, bees, Drosophila; active against inflammation and cancer), jellein (from bee royal jelly; inhibits bacteria, fungi, and parasites when conjugated)	[61–63]
	Microorganisms (including plants and marine sources)	From bacteria, fungi, plants (e.g., stems, seeds, leaves), and marine organisms	Nisin and gramicidin (from Lactococcus lactis, B. subtilis/brevis), thionins and defensins (plants), As-CATH4 and myticusin-beta (marine; immunity stimulation and antibiotic alternatives)	[64, 65]
Activity	Antibacterial	Broad inhibition of pathogenic bacteria (e.g., ~ 60% of AMPs); target Gram-positive and Gram-negative strains like MRSA, E. coli, and Salmonella	Nisin, cecropins, defensins, synthetic P5 (YIRKIRRFFKKLKKILKK-NH₂) and P9 (SYERKINRHFKTLKKNLKKK-NH₂)	[74–76]
	Antifungal	Target fungal infections (e.g., ~ 26% of AMPs); effective against Candida, Aspergillus, and molds	Brevinin, ranatuerin, cecropins, AurH1 (from aurein 1.2; treats C. albicans), FPSHTGMSVPPP (inhibits A. flavus), radish AMPs (against Zygosaccharomyces)	[68, 73, 77, 78]
	Antiviral	Inhibit viruses by blocking attachment, destroying envelopes, or halting replication (e.g., ~ 2—5% of AMPs); includes anti-HIV and anti-coronavirus activities	Epi-1 (foot-and-mouth disease virus), SIAMP (avian influenza), defensins/LL-37 (anti-HIV), EK1C4 (COVID-19 fusion inhibitor), RTD-1 (SARS-CoV immunomodulation)	[85–89]
	Antiparasitic	Kill parasites causing diseases like malaria and leishmaniasis (e.g., ~ 2—5% of AMPs); often via membrane destruction	Cathelicidin, temporins-SHd, Epi-1 (Trichomonas vaginalis), jellein and KDEL (Leishmania)	[68, 80, 119]
	Anticancer	Recruit immune cells, induce apoptosis/necrosis, inhibit angiogenesis (e.g., ~ 2—5% of AMPs); balance of charge and hydrophobicity is key	Tritrpticin analogs (toxic to Jurkat cells), indolicidin, puroindoline A	[120, 121]
Structural Characteristics	Linear α-helical	Adopt α-helical conformation in membranes	LL-37	[67, 68, 122, 123]
	β-sheet	Stabilized by disulfide bonds; form sheet structures	Gomesin
	Linear extension	Lack defined 3D structure; extended conformation	Indolicidin
	Both α-helix and β-sheet	Combined helical and sheet elements	α1-Purothionin
	Other (e.g., cyclic, complex)	Emerging topologies like lasso or thioether-bridged	Various synthetic or natural variants
Amino Acid-Rich Species	Proline-rich (PrAMPs)	Non-polar; enter cells via transporters and target ribosomes (motifs like -PPXR- or -PRPX-); also immunostimulatory	Tur1A (from dolphins; inhibits protein synthesis), Bac7 (bovine), pPR-AMP1 (from crab; active against Gram-positive/negative bacteria)	[69–73]
	Tryptophan- and arginine-rich	Tryptophan aids membrane interaction; arginine provides cationic charge for anionic binding	Indolicidin, tritrpticin, Octa 2 (RRWWRWWR; inhibits E. coli, P. aeruginosa, S. aureus), lactoferricin-derived peptides	[69–73]
	Histidine-rich	Basic histidine enhances membrane permeation and anti-inflammatory effects	HV2 (RR(XH)₂XDPGX(YH)₂RR–NH₂; inhibits TNF-α), histidine-substituted Octa 2, L4H4 (magainin-based; antibacterial and cell-penetrating)	[69–73]
	Glycine-rich	High glycine content (14–22%) affects tertiary structure; activate phagocytes	Attacins, diptericins, glycine-rich cathelicidins (from salmonids), GG3 (against Gram-negative bacteria)	[69–73]

Research has shown that liposomal formulations enhance pulmonary deposition, and lipopeptides (AMPe + fatty acid) are effective in facilitating cell membrane entry against Gram-negative bacterial infections. However, the clinical significance of these observations remains unresolved [55]. In vitro, AMP-coated implants or tubes have become a strategy to reduce biofouling and albumin amyloid coatings, as well as vancomycin, which targets and kills bacterial biofilms on implants. However, the stability of these coatings has not been confirmed under practical settings. Administration of nebulized AMP therapy, specifically colistin or daptomycin, has been used to address MDR bacterial infections associated with ventilator-associated pneumonia (VAP). However, the therapeutic value of these treatments has been called into question due to exposure and the risk of developing resistant bacterial strains [123, 124]. Recently, advancements have been made in dry powder inhalers of AMPs; however, it remains to be seen whether these products will be commercially viable. Likewise, questions regarding the efficacy of methods that enhance pharmacokinetics (conjugation, encapsulation, and/or co-assembly) remain unanswered. Peptidomimetics have been developed to provide greater resistance to degradation, but the clinical relevance of these findings remains uncertain. AMPs can be developed using biomaterials with various methods (covalent, physical, or advanced nanostructured), but the resulting materials may not exhibit ideal biocompatibility and release properties for all applications [32, 117, 125].

Self-assembled peptide nanostructures in antimicrobial peptide-based biomaterials

A balance between the characteristics of amino acid residues controls the self-assembly of these systems. The core is composed of hydrophobic residues, including valine and phenylalanine, whereas charged residues, such as lysine and arginine, facilitate electrostatic interactions and membrane targeting. Assembly of AMP-based nanostructures usually begins with amphiphilic structures, in which a hydrophilic, bioactive head (such as an AMP sequence) is linked to a hydrophobic tail (e.g., an alkyl chain or lipid), resulting in micelles, fibers, or hydrogels once the critical aggregation concentration (CAC) is exceeded. The key mechanisms involve membrane disruption, in which cationic AMP nanostructures target negatively charged bacterial membranes, forming pores (such as barrel-stave, toroidal, or carpet models) or causing lysis through electrostatic disruption, osmotic imbalance, or autolytic enzyme activation [126].

After studying Polymyxin B (PMB), which is a lipopeptide antibiotic that is successful against MDR Gram Negative Bacteria (MDR GNB), the amphiphilic polypeptide NPs (Nano Particles) formed from Poly (L-glutamic Acid [-co-D-Phenylalanine]) have been used to create NPs with a higher Encapsulation Efficiency (EE) through both Electrostatic and Hydrophobic Interactions. The spherical NPs (approximately 162 nm) provide a controlled release profile that reduces Nephrotoxicity, increases plasma stability, and maintains Antimicrobial Activity against Pseudomonas Aeruginosa (Microbiological Inhibitory Concentration of 4 µg/mL). Together with PMB, the peptide hydrogels will be triggered to deliver sustained, localized release to support Wound Healing and Infection Control. Comparatively, several other examples can be used to illustrate the responses of β-Lactamase (Bla), an enzyme that provides resistance to antibiotics (Bacteria), to other responsive peptides [127, 128].

An example of these peptides is BLAP, which mimics the Human Defensin-6 peptide. However, incorporating the cephem structure (cephems are a group of β-lactams consisting of an oxime and a phenyl-β-lactam) as part of the structure of BLAP results in the cleavage of the cephem nucleus by Bla, leading to a structural change from Random Coils to β-Sheet Nanofibers, effectively creating a "nanoscale" net to restrict bacterial invasion and limit access to host cells (e.g., 293 T) and prevent abscess formation in murine abscess models, while decreasing levels of Inflammatory Cytokines (IL-6, TNF-α) that lead to the induction of Resistance. This type of nanostructure can increase the antimicrobial activity of therapeutic agents by allowing them to respond to enzymes produced by bacteria, thereby preventing the Spread of Infection [128].

Antimicrobial peptides-based biomaterials against viral respiratory infections

As shown in Table 3, for viral lung infections (e.g., influenza, RSV, SARS-CoV-2), AMPs-based biomaterials focus on immunomodulation and direct viral inhibition, often via inhalable formulations that target the respiratory epithelium [36]. In nanoparticle-based systems, the goal is to disrupt viral envelopes in a manner that intentionally affects viral entry. Examples of these systems include niclosamide- and lysozyme-based particles, as well as gold nanoparticles conjugated to lactoferrin or LL-37. These systems generally block viral entry, prevent replication, or down-modulate inflammation. The sustained release, facilitated by the nanoparticles, is a key feature of these particles for airway treatment. Niclosamide-lysozyme microspheres have shown effectiveness in lung models and are under investigation for the treatment of disseminated coronavirus infections via inhalation [129]. Clinical trials with lactoferrin have shown a reduction in RTIs. Hydrogel-based systems have also been evaluated for their ability to leverage mucosal host defense mechanisms to reduce viral infections. The LL-37/chitosan hydrogels or hydrogels encapsulated with exosomes/AMPs to decrease infections. These systems have been enhanced by upregulating AMP expression and balancing cytokine levels. Additionally, hydrogels have been shown to protect against viral adherence. In lung models, LL-37 administered at 1 μg/day/hr protected against respiratory syncytial virus (RSV) and rhinovirus, resulting in decreased inflammation in mice [129–131].

In polymer conjugate systems, nutritional components are used as molecular adducts that are purported to enhance the secretion of host-originated antifungal and antibacterial peptides. Some of these components include vitamin D molecules, which are purported to act as catalysts for increasing LL-37 and defensin secretion, and polymerized short fatty acids, such as butyrate, believed to act as catalysts that can help boost cathelicidin secretion. These actions are reportedly mediated by modulation of pathways such as NF-κB, followed by modulation of JAK/STAT pathways that facilitate the secretion of antifungal and bacterial peptides. These molecules are also supposed to enhance pulmonary bioavailability. In respiratory model systems, 1,25-Dihydroxyvitamin D3 has been shown to improve the secretion of the antifungal peptide LL-37, produced by bronchial cells, during rhinovirus infections [132, 133]. In liposome formulations, target-site delivery has been enabled by incorporating SLPI or lactoperoxidase into the liposomes. These actions reportedly involve the production of antifungal agents, such as hypothiocyanite, and the modulation of the redox balance. In experimental lung models, lactoperoxidase has demonstrated its ability to reduce Influenza entry into mouse lungs, while simultaneously decreasing neutrophil aggregation, a function performed by SLPI. In this section above, we have covered some of the most significant antifungal peptides-conjugate biomaterial systems used against viral RTI infections [134, 135].

Anti-influenza virus and SARS-CoV-2

In polymer-based delivery systems, P9, a derivative of mouse β-defensin-4, and its stable formulations, liposomes or micelles, are being explored as intranasal delivery systems. They target hemagglutinin (HA), inhibiting RNA release by preventing endosomal acidification, having an IC50 of 1.2—4.8 µg/mL. In vivo studies demonstrated a 100% survival rate in mouse H1N1 models when used as a pre-exposure prophylactic treatment. However, their effects on the immune system and their ability to modulate immune responses in other disease models remain unclear. Esculentin-1GN and urumin target viral entry through HA binding, through the same type of polymer as above. They focus on the cationic and amphipathic properties of AMPs, which provide low toxicity and high target specificity. Currently, these peptides are in clinical trials under the name Flufirvitide, with unpublished results, and their clinical application in settings remains unclear. They target the entry, envelope, and receptor (ACE2 or other coronavirus receptors), and have been proven effective against all types of coronaviruses due to their enveloped viral nature. However, its target specificity and potential off-target effects remain a concern and need to be addressed promptly [136–138].

In layer-by-layer (LbL) coatings, LL-37 is combined with chitosan to form LbL structures on hydroxyapatite or glass substrates. They target S1 subunits by inhibiting the ACE2 receptor and microthrombosis, achieving > 95% inhibition at concentrations > 25 µg/mL; however, the effect and potential duration of this inhibition in clinical practice remain unclear. Human defensin-5 and neutrophil α-defensin, HNP-1, target ACE2 receptor saturation through the same coatings as before. However, their effectiveness in preventing the spread of coronavirus has not been proven, and their impact on patients' immune and other physiological responses remains unclear; this needs to be addressed as soon as possible. In nano-material enhancements, P9 is used as a nanocarrier that targets S2 glycoproteins by inhibiting endosomal acidification, leading to a drastic decrease in viral load in the SARS-CoV mouse model. However, these studies should not be considered typical human responses, and their effects on patients' immune responses remain unclear and require prompt attention. EK1C4 fusion-inhibiting peptides are delivered via PEG or hyperbranched polyglycerol (HPG) attachments on nano-materials, potentially increasing their stability and target sensitivity. However, their effects on patients' autoimmune and other responses remain unclear and require immediate attention [139].

Nevertheless, its actual effectiveness and potential side effects on other targets are points of concern that have yet to be examined. Cellulose and copper nanoparticles reduce infectivity by physically inhibiting the infectivity process. Nevertheless, its target specificity and actual effectiveness as a coronavirus inhibitor are unknown and warrant further exploration. Additionally, hydrogels and nanogels consisting of lactoferrin and plitidepsin, when incorporated into hyaluronic acid nanogels, target ACE2 entry and replication through eEF1A interaction, offering superior bioavailability and low cytotoxicity, particularly in inhibiting inflammation associated with COVID-19. Nevertheless, its chronic effects on the immune system and its potential role as a coronavirus inhibitor remain unknown and warrant further exploration [139].

Anti-respiratory syncytial virus (RSV), anti-human adenovirus

There is limited information available on AMP-based biomaterials against RSV, but broad-spectrum AMPs exert inhibitory effects by disrupting cell membranes and inhibiting binding. There is no doubt that this is expected based on general AMP principles. When used as nanoparticles, LL-37 conjugates silver nanoparticles (AgNPs), producing reactive oxygen species (ROS) that destroy RSV cell membranes and inhibit binding, effects expected. GO nanoparticles pierce lipid membranes, often in combination with AMPs, providing enhanced viral destruction effects that are more a result of synergy than innovation. Surface coatings have LL-37 covalently attached and immobilized on textiles or surfaces, inhibiting viral entry by destroying viral envelopes, effects that can be considered straightforward applications of AMP principles. Efficacy has been inferred from in vitro studies that demonstrate prolonged respiratory delivery effects, which require final validation. Efficacy has been observed against human adenovirus, targeting non-enveloped viruses by stabilizing viral capsids or inhibiting entry, and inhibiting the entry of various types, including HAdV-5, effects that could be expected based on AMP principles [140, 141].

In textiles and surface coatings, bovine lactoferricin (bLfcin) is used as a boron-triclosan mixture on textiles or as a coating on glass and stainless steel through covalent conjugates, binding glycosaminoglycans, inhibiting entry, and blocking antigen synthesis, effects that can be considered somewhat arbitrary. Layer-by-layer (LbL) assembly is used for defensins, providing controlled delivery, stabilizing viral capsids, blocking interactions, and exerting greater control over delivery than innovation. In nanoparticles, conjugates of Mel4 (a synthetic AMP) and mimic-1083 target viral capsids, inhibiting cell growth by up to 50% with an IC50 of approximately 47 µM. These effects are considered somewhat impressive and require final validation. LL-37 is used on polymers, inhibiting cell membranes, effects that are as expected based on AMP principles. They have observed low exploration of non-enveloped viruses, whereas exploration of enveloped viruses is relatively low, as expected based on AMP principles [43, 141].

Anti-human rhinovirus (HRV) and anti-human metapneumovirus (hMPV)

Biomaterials tend to interact directly with Human Rhinovirus (HRV) and inhibit its protease activity, although they have been largely untested in real-world settings. In covalent surface coatings and peptide delivery systems, the peptide LL-37 is immobilized via covalent or electrostatic stabilization on titanium or fabric. This mechanism acts on the virus before infection by dampening metabolic activity, though it is more effective as a functional treatment than as a therapeutic mechanism [142, 143].

Similar applications of coatings were made with ovine SMAP-29 and porcine Protegrin-1, which showed in vitro activity; however, it is unclear whether these in vitro protection results can be replicated in vivo. In a nanoparticle system, a 6-amino-acid peptide, LVLQTM, was used in micelles or hydrogels to inhibit 2 A protease activity and, consequently, prevent replication in A549 cells and mice; however, its specificity was limited to a particular cell line and mouse model. Cathelicidins are an example of AMPs that exhibit conserved activity and can be modified; however, there is limited specific data on their effect against hMPV, which is typically inferred from the effect of broad-spectrum AMPs against related paramyxoviruses. In general, coatings and nanoparticles, such as LL-37 and defensins in layer-by-layer coatings or Ag/GO nanoparticles, are likely to alter membranes, blocking receptor activity, as observed with RSV treatment; however, the exact mechanism remains unknown. TL derivatives also have potential for immune modulation in polymeric systems, but their efficacy and safety are unknown [144, 145]. Table 4.

Table 4.

Different antimicrobial peptide-based biomaterials against viral respiratory infections

Biomaterial/Delivery System	AMPs(s) Incorporated	Targeted Virus(es)	Mechanism	Details/Efficacy	Ref
Niclosamide-lysozyme particles	Lysozyme	SARS-CoV-2	Protects against infection and inflammation by combining lysozyme's antiviral properties with niclosamide for broad-spectrum activity; inhibits viral replication in epithelial cells	Inhalable composite particles developed as patient-adaptable treatment; show potential for reducing coronavirus sequelae in human corneal epithelial cell models; efficacy includes protective effects against infection and reduced inflammation	[36]
Human mesenchymal stem cell (hMSC) therapy	Defensins (α, β, θ types, e.g., HD5, hBD-2, hBD-3), LL-37	SARS-CoV-2	Releases AMPs to inhibit viral entry by binding to spike protein receptor-binding domain (RBD) and cloaking ACE2 receptor; modulates inflammatory responses and directly kills microbes	hMSCs release AMPs in supernatants, effective in treating severe COVID-19 patients; reduces lung pathology and mortality (e.g., 100% survival in SARS-CoV-infected mice with θ-defensin RTD-1); used in cystic fibrosis models for antiviral effects	[146]
Intranasal aerosol delivery	LL-37, peptide derivatives (e.g., P9, P9R)	SARS-CoV-2, MERS-CoV	Binds to viral S2 domain or RBD to prevent endosomal acidification and viral RNA release; blocks ACE2 interaction; administered as prophylactic aerosols to ramp up AMPs levels in nasal cavities	Dose-dependent suppression of pseudovirion infection (IC50 ~ 1.05 μM for LL-37); P9R shows IC50 of 0.9 μg/ml against SARS-CoV-2; protects mice from pulmonary infection; proposed for early-stage prophylaxis	[146, 147]
Synthetic defensin mimetics (e.g., Brilacidin)	Defensin mimetics (non-peptide, e.g., Brilacidin)	SARS-CoV-2	Mimics defensin structure to inhibit viral entry in ACE2-positive lung cells; increases cationicity and amphipathicity for enhanced stability and membrane disruption	Inhibits SARS-CoV-2 in vitro; in phase 2 clinical trials (NCT04784897); modifications like D-amino acids improve metabolic stability; broad potential for respiratory infections	[146]
Nebulization delivery	LL-37	IAV, RSV, HRV	Interacts with viral envelopes to inhibit post-entry replication; reduces viral load and proinflammatory cytokines; enhances neutrophil responses	Nebulized in mice prior to IAV infection, increasing survival and reducing severity; reduces RSV titers and HRV load in respiratory cells; up to 85—90% reduction in replication for related defensins	[146, 147]
Polymeric nanoparticles (e.g., PLGA, hyaluronic acid nanogels)	LL-37, other AMPs (e.g., DJK-5)	IAV, RSV (potential for respiratory applications)	Protects AMPs from degradation; targeted delivery to lung infection sites; disrupts viral envelopes and modulates immune responses	Self-assembling HA nanogels reduce cytotoxicity (fourfold vs. free peptides); used in Pseudomonas models but adaptable for viral respiratory delivery, enhancing bioavailability for lung targeting	[147, 148]
Lipopeptide conjugates (e.g., EK1C4)	EK1C4 (lipopeptide)	SARS-CoV-2, other coronaviruses	Acts as a fusion inhibitor by disrupting the viral envelope and interfering with host cell entry; it has been validated in mouse models	Potent against SARS-CoV-2; reduces viral load; part of nanotechnology strategies to improve stability and targeted delivery	[147, 148]
Polymer conjugation (e.g., PEG, HPG)	Various AMPs (e.g., 73-derived peptides, lactoferrin)	Influenza viruses, SARS-CoV-2	Covalent attachment enhances stability and antimicrobial activity (2—eightfold increase); disrupts viral attachment and replication; and modulates immune responses	Eradicates biofilms in bacterial models and is applicable to viruses. Lactoferrin conjugates reduce viral load in H5N1-infected mice, addressing limitations of AMPs, such as toxicity and costs	[147]

Pulmonary delivery physics and quantitative comparisons

The delivery of drugs by inhalation is a complex integration of physics, physiology, and pharmacology that allows drugs to be delivered directly to the lungs for treatment of respiratory diseases as well as systemically, taking advantage of the large surface area of the lungs (approximately 70 to 140 m2), thin epithelial barrier, and extensive vascular supply [149]. Inhalation also has the benefit of not undergoing first pass metabolism and achieving a peak concentration very quickly (for example, for small drugs, within 15 to 60 min) with a higher bioavailability; however, the success of this delivery option is dependent on overcoming several barriers such as mucociliary escalator function, alveolar macrophage clearance, and differing airway geometry. In this review, we discuss the underlying physics of pulmonary drug delivery in detail, using computational modeling, experimental data, and clinical evidence. We also make quantitative comparisons between different inhalation methods, different sizes of particles, and different formulations [149, 150].

Core physical principles in pulmonary delivery

Fluid mechanics and particle aerodynamics govern how air travels through the respiratory system and how aerosols are transmitted through the respiratory tract. The airflow that enters the lungs creates velocity gradients during inspiration: high velocity in the trachea (Reynolds number is greater than 2000, turbulent) and low velocity in alveoli (near-zero velocities, laminar) [151, 152]. The three primary methods of deposition (i.e., how an aerosol or particle is retained within a specific part of the lungs) are as follows:

1. Inertial impaction: Particles with larger momentum (larger size, higher velocity) will not deviate from the direction of the airflow due to their momentum, therefore depositing in "upper" parts of the airways (generations 0 to 10). Inertial impaction is the primary mechanism of deposition for particles greater than 5—10 microns at inspiratory flow rates greater than 30 L/min. As a result, aerosols can incur oropharyngeal losses of as much as 90 percent when using devices that have not been appropriately optimized [153].

2. Gravitational sedimentation: Mid-sized particles will begin to settle due to the force of gravity under reduced airflow conditions (generations 11 to 16) leading to deposition in the bronchioles. The rate of sedimentation is governed by Stokes' Law, where the settling velocity is proportional to the density and the square of the size of the particle [154].

3. Diffusion (Brownian motion): Small particles deposit in small spaces in the alveoli, due to multiple random collisions with other molecules and due to there being negligible airflow in these spaces. The diffusion coefficient of any particle in the alveoli is inversely proportional to the size of the particle as governed by Einstein's relation [154].

The aerodynamic diameter (d_aer = d_geo × √ρ), where d_geo represents the geometric diameter and ρ represents density, is a quantifiable measurement of techniques where shape and density are taken into account. An ideal d_aer from 1—5 microns (µm) produces a 50—60% deposition rate directly into the alveolus (deep lung zone) with approximately 28% of aerosols that reach the bronchial zone of the trachea under normal respiration conditions. Because the relative humidity of the lungs is 99.5%, hygroscopic (water attracted) growth can increase particle size by 2—3 (≥ 2 µm) and 5 (< 1 µm) fold, resulting in a shift east/west of the central deposition point to an impaction > (> 1 µm), as deposition occurs centrally [155].

There are currently ways that breathing patterns can change these things. More inhales with longer durations (T_b) allow for a reduction of the Strouhal number (St_a), which will improve the ability for deep penetration of the lung. The amount of medication delivered into the lungs through aerosolized medication will be increased by a factor equal to the square root of 2 (doubling T_b) or there is a 100% increase in the mass of drug being deposited in the deep lung when T_b is doubled. Computational models can provide predictions based on the airway-mucous coupled flow using Peclet numbers (Pe_a ~ 10^6 to 10^11 for aerosols; Pe_d ~ 4.56 × 10^7 for dissolved drugs); therefore a high Peclet number (Pe_a) will promote advection over diffusion with the peak deposition in the deep lungs at approximately at 1.59 × 10^9 for aerosols [156].

Mucociliary clearance adds a convective layer: mucus velocity (V_m ~ ε^N, where ε is scaling factor) clears upper airways rapidly (hours), but ceases in alveolated regions (generations ≥ 18), prolonging retention via diffusion alone. Alveolar macrophages phagocytose insoluble particles over weeks, while soluble drugs absorb rapidly if < 40 kDa. Disease alters physics—e.g., COPD increases central:peripheral deposition ratios due to narrowed airways, reducing FEV₁-correlated penetration. Electrostatic forces offer enhancement: charged particles improve deposition via image forces, potentially reducing upper airway losses, though regulatory aspects emphasize charge characterization to avoid aggregation [156].

Aerosol characteristics and formulation impacts

Aerosol characteristics control their behaviour: whether they have monodisperse or polydisperse characteristics (geometric standard deviation) will affect their uniformity; the hygroscopic nature of the aerosol will determine the amount of growth (i.e. NaCl aerosols increase in size by approximately 2 times). The use of dry powder inhalers (DPIs) requires the patient to have a flow rate of approximately 30—60 L/min in order to achieve deaggregation; pressurised metered-dose inhalers (pMDIs) use pressurised gases to atomise the drug resulting in much finer drug particles (MMAD, 1.3 μm in HFA vs. CFC) and increasing the fine particle fraction by 1.94 times [157, 158].

Nanomedicines (e.g. liposomes, dendrimers) have a size-dependent endocytic uptake for active targeting (enhanced permeability through inflamed tissue) when the particles are < 200 nm; the addition of specific surface ligands (e.g. mannose to target macrophages) will increase the uptake by 20—100 times. Passive targeting can occur by charge and by the use of mucoadhesives (e.g. chitosan) to avoid clearance. Hybrid lipid-polymer nano-particles have the potential for controlled release. Larger porous particles (= 0.1 g/cm³,d_geo > 5 μm) decouple their geometric size from their aerodynamic size: this results in d_aer = 3 μm which allows for deep lung delivery and reduces the potential for phagocytosis because of the large particle size. This allows for sustained release; insulin-loaded porous PLGA particles having a sustained effectiveness of 96 h (as opposed to 4 h with a non-porous particle) and a tenfold increase in bioavailability compared to testosterone [159, 160].

Experimental and computational models

In vitro models, including Calu-3 monolayers, can be used to evaluate the permeability of different size particles (i.e., < 100nm) through transepithelial electrical resistance (TEER) methods that show that smaller sizes have increased diffusion rates; whereas ex vivo perfused lungs are used to determine the amount of drug that is deposited (i.e., 20–40% efficiency). Pharmacokinetic studies in rodents demonstrate that Cmax, Tmax and area under the curve (AUC) are dependent upon size in that nanocrystals increase dissolution rates thus equating to a greater bioavailability [161, 162].

Thus, computational tools, such as CymCip and Gastroplus, can be used to simulate deposition based upon size (i.e., optimal 1–5 μm) and airflow simulations indicate that approximately 60% of monodisperse aerosols deposited in alveoli. Furthermore, coupling these models demonstrates that the increased time of exposure through inhalation will increase the deep mass of the drug received. Toxicity models such as the MTT assay demonstrate that cationic NPs have an increased ability to be internalized by cells but may have a potential to cause an inflammatory response as documented by the assessment of apoptosis rates (p < 0.05) and the smaller and similar tumor reduction seen with the use of the targeted NP (compared to the free drug) [162, 163].

Quantitative comparisons in delivery methods and efficacy

The methods of delivery have shown comparative efficiencies. The following are the methods of administration of the tested compounds: Intratracheal instillation yields the greatest lung deposition (greater than 95%) with the disadvantage of localized, inhomogeneous deposition in the lung lobes, simplicity of methodology, inexpensive, less physiologic than the other methods, and is used in the preclinical pharmacokinetic study with accurate dosing, but not transferable to human administration. There was a similar percentage of lung deposition (greater than 85%); however, deposition was more uniform than with instillation; both methods had inhomogeneous deposition in the lungs, and the amount of drug deposited in the lungs was less than that with inhalation alone [164, 165].

For intranasal instillation, approximately 28% of the drug is deposited in the lungs, but the remainder is lost to the stomach and the variability between subjects was the most pronounced of the methods. Intranasal instillation is not a useful method for delivering drugs to the lungs. Inhalation is more effective than the other methods because only 20% of the drug is lost from the oral cavity versus 85% of the drug being lost from the nasal cavity; there is more variation in the amount of drug deposited in the lungs when compared to other administration methods due to the delivery device (20%−60%); the relative uniformity of the delivery of the drug allows for more accurate delivery than does the other administrations methods [166, 167].

Radiolabeled NP studies confirm instillation/spraying superiority for preclinical, but all show lobe variability, with intranasal least reliable. Table S1.

Antimicrobial Peptides-based biomaterials against bacterial respiratory infections

AMP-based biomaterials are a viable candidate for exploring new therapeutics to treat bacterial respiratory infections, given their diverse properties. Cathelicidins, one of the subgroups within the family of AMPs, are generated as pre-peptide and pro-peptide sequences in mammals. Although the N-terminal is invariant, the C-terminal portion of the AMP is highly variable and is responsible for its antimicrobial activity. The defensins comprise the second group of AMPs and are a large family of small peptides characterized by a β-sheet structure and six cysteine residues interconnected by disulfide bonds. The defensins are named according to their relationship with host immunity, and there are three structural classes of defensins in mammals: α-defensins, β-defensins, and θ-defensins [168, 169]. Figure 3.

Fig. 3.

Antimicrobial peptide-based biomaterials play a crucial role in the fight against antibiotic-resistant bacteria, employing various mechanisms to compromise bacterial membranes and induce cell death. These peptides employ different strategies depending on the type of bacteria—namely, gram-positive or gram-negative—due to structural differences in their cell membranes. Among the commonly accepted hypotheses for how AMPs disrupt bacterial membranes are models such as the Carpet Model, Barrel-Stave Model, Toroidal Pore Model, and Detergent-Like Mechanism. In the Carpet Model, AMPs coat the surface of a bacterial membrane like a carpet; upon reaching a critical concentration, they cause membrane disintegration by forming pores. The Barrel-Stave Model describes AMPs inserting into the membrane and aligning to form barrel-shaped pores that allow leakage of essential cellular components

Bacterial pneumonia

Bacterial pneumonia can be classified by the timeframe of infection and the location of acquisition into three categories: CA pneumonia (CAP), HA pneumonia (HAP), and VA pneumonia (VAP). The most common method of treatment is antibiotics; however, antibiotic resistance has made it more common to see cases with Multi-Drug Resistant (MDR) strains of pathogens such as P. aeruginosa that can become antibiotic resistant, as well as Methicillin Resistant S. aureus (MRSA) strains, thus resulting in increased mortality rates and Extended Length of Hospitalization (ELH). Scientists are finding that AMPs can offer a positive alternative to conventional treatment for bacterial pneumonia in both severely infected patients and those with antibiotic-resistant bacteria [170–173].

In animal models of bacterial pneumonia, AMPs have shown significant ability to reduce bacterial load and improve survival of infected animals. An example of a peptide that had this effect is ESC (1—21), a plant-derived peptide shown to exhibit high bactericidal activity against Pseudomonas aeruginosa in vitro and to increase survival in mice with pneumonia. A study of an AMP, CAMA, a hybrid antibiotic peptide with activity against MRSA, demonstrated in vitro efficacy in eradicating MRSA biofilms on lung tissue. The data suggest that AMPs have strong efficacy in treating lung bacterial infections [174, 175].

However, relatively few studies have specifically used AMP therapy for pneumonia due to challenges in delivering the peptides directly to the lungs. Historically, most early studies on AMPs used systemic administration; however, that route is not a realistic means of treating pneumonia. There is currently a strong interest in the use of inhalation to deliver AMPs directly to the lungs. A current example of an investigational study evaluating the use of inhaled antibiotics is the treatment of non-tuberculous mycobacterial lung disease with antibiotics encapsulated within advanced drug delivery technologies, such as liposomes, with products such as amikacin liposome inhalation (ALIS) [176, 177]. The product has been developed to include both antibiotic (amikacin) and liposome technology (liposomes); consequently, while the product does not contain a peptide AMP, the use of this product demonstrates a method to achieve significantly greater concentration of an antibiotic in the lungs via inhalation. When combined with liposomal delivery technology, other inhaled antibiotics, such as colistin and aztreonam, are being studied for the treatment of resistant bacterial pneumonia. The results of these studies support the concept that using local delivery systems can enhance the efficacy of antibiotics in treating pneumonia by increasing local exposure of antibiotics to the site of infection and decreasing systemic toxicity [178].

Current research includes ongoing clinical trials evaluating the safety and efficacy of peptide-based therapies for lung infections. One such trial, NCT02991859, is currently in Phase 1 in healthy volunteers to assess the safety of inhalation-based AMPs before progressing to patients with a respiratory infection. This trial may enable the development of later-stage clinical trials for patients with pneumonia. One strategy is to co-administer an AMP and a conventional antibiotic, and previous in vitro studies indicate that some AMPs enhance the effectiveness of antibiotics against resistant microorganisms, thereby allowing lower antibiotic dosages [179]. In addition, animal models of pneumonia have shown that co-administration of AMP and an antibiotic results in synergistic killing of microorganisms and decreased lung inflammation. These combination strategies may lead to shorter antibiotic treatment regimens and a reduced risk of antibiotic resistance. Nonetheless, any combination of AMP use in humans will require careful risk assessment due to the potential off-target activity of the AMPs themselves [180].

Tuberculosis

Mycobacterium tuberculosis (M. tuberculosis) is the primary pathogen responsible for the chronic lung infection known as Tuberculosis (TB). The standard treatment for TB includes antibiotics for 6 to 9 months. Drug-resistant TB strains present a therapeutic challenge and are often treated through complex, prolonged, and toxic protocols. The use of AMPs has been studied as a potential enhancement for the treatment of TB, either by directly killing M. tuberculosis or by enhancing the host's immune response to it [181, 182]. In vitro studies of select AMPs demonstrated the ability to kill M. tuberculosis, including drug-resistant strains. A peptide, Mu1140, derived from a streptococcal AMP, was shown to treat drug-resistant TB in mice effectively. In addition to killing microorganisms, AMPs can enhance the functions of host immune cells. Some AMPs stimulate macrophage production of antimicrobial substances and enhance immune cells' ability to engulf and destroy M. tuberculosis. Therefore, AMPs may benefit TB treatment by assisting the host in eliminating M. tuberculosis [181].

Clinical trials for AMPs as a treatment modality for TB have been relatively few compared to their preclinical and in vitro successes. This is evidenced by the major limitation in the systemic delivery of AMPs; the administration of AMPs to an infected lung (i.e. where TB bacterium resides) will be much less efficient than the delivery of AMPs to the lung through inhalation or local injection because TB bacterium often resides within immune cells called macrophages, and may form dormancy and develop into persistent or less susceptible bacterium. Based on this reasoning, the development of inhaled formulations of AMPs for TB remains of great interest to researchers [146]. Moreover, studies have demonstrated significant reductions in bacterial load in the lungs of laboratory animals when aerosolized AMPs were delivered to the TB bacterium residing there. Another possible method for delivering AMPs to the TB bacterium is to incorporate them into nanoparticles that can be absorbed through the bacteria's cell wall, or to target M. tuberculosis-infected macrophages. These nanocarriers can release AMPs directly into the bacteria or infected macrophages. A preclinical study demonstrated that a nanoparticle containing a peptide enhanced the antimicrobial activity of a TB antibiotic and reduced the burden of the TB bacterium in the lungs of infected mice [68, 146].

As of 2025, no AMP-based treatments are approved for tuberculosis. However, there is ongoing research at this time looking into different aspects of AMPs as complementary treatments to standard TB treatment methods. Clinical trials are being done assessing the effect of an AMP as a co-treatment with currently available TB medication, specifically looking into combining a peptide and the currently available TB medication on patients with ATB to see whether this combination can decrease the duration of treatment and/or increase the patient’s outcome from the TB treatment. Although no detailed information has been released regarding the results of these clinical trials, the preliminary results have been encouraging [183]. Another treatment method being explored is gene therapy to generate AMPs in the lung. The gene therapy method under investigation involves inhalation of a gene therapy that delivers genetic material (DNA) coding for an AMP (such as LL-37) to the lung, allowing the body to produce LL-37 peptide within the lung. Research on animals has shown that gene therapy can increase AMP production in the lungs and enhance the body's ability to clear TB bacteria from the lungs. If these methods are confirmed to be valid in human studies, they may serve as a new treatment modality for TB, working with the body’s innate defenses against the disease [184].

To summarize, AMP-based biomaterials have been demonstrated to be a potential treatment for TB, either supporting current TB treatment (i.e., antibiotics) or serving as an alternative when antibiotic treatment fails. At this point, the evidence supporting this is limited, but preclinical data and clinical research suggest that AMP's role in the future of TB is likely to be important when used with current treatments.

Cystic fibrosis lung infections

Cystic fibrosis (CF) is a hereditary ailment that leads to persistent respiratory infections, the most common of which are caused by P. aeruginosa and S. aureus. These viral and bacterial infections have caused people with cystic fibrosis to develop extreme difficulties with their breathing and physical activity. Antibiotics are a standard treatment for these infections, but as many CF patients develop multidrug resistance and have difficulty shaking their infections, antibiotic treatments are not always effective. Due to their broad antimicrobial action (including resistance), AMPs have been viewed as a potential treatment for CF. The body produces equal amounts of AMPs, β-defensins, IL-37, and others, all of which are naturally occurring antimicrobial agents that have been demonstrated to possess anti-inflammatory effects. Due to reduced levels of these AMPs in the lungs of CF patients, the body is more vulnerable to bacterial and viral infections [175].

Many clinical studies and trials have examined the use of AMPs in treating lung infections in CF patients. An early study evaluated over 150 AMPs against bacterial isolates obtained from CF patients, including mucoid P. aeruginosa and MRSA. The study identified several peptides with potent antimicrobial activity when exposed to CF sputum and salt concentrations. In this study, the peptides were evaluated in a rat model of chronic lung infection, resulting in significant reductions in lung bacterial burden compared with untreated controls. Additionally, three of the four effective peptides demonstrated anti-inflammatory effects in mice, suggesting they may be effective against both CF lung infections and CF lung inflammation. Evidence suggests that AMPs could be used as a potential treatment for cystic fibrosis by killing bacterial pathogens and reducing the inflammatory response associated with the disease [1, 175, 185, 186].

Specific AMPs have been researched in the context of CF. CAMA is a hybrid of peptides known to target MRSA and other pathogens isolated from people with CF. In addition, CAMA demonstrated good in vitro efficacy against CF isolates from patients. However, the presence of high levels of protease and mucus in CF sputum reduced the bactericidal activity of CAMA, underscoring the urgent need for suitable delivery methods to protect AMPs. When CAMA was assessed in an in vitro model simulating the CF lung using human bronchial epithelial cells and CF sputum, it showed reduced ability to kill MRSA but retained some activity. Therefore, it may be possible to enhance the bactericidal properties of CAMA by using delivery methods that maintain its integrity and protect it from the hostile lung environment in individuals with CF (e.g., by coating or encapsulating it). Therefore, research is currently underway to develop such delivery methods for CAMA, as well as for other AMPs that may be useful for treating individuals with CF [187, 188].

Clinical investigations have also been conducted to evaluate the use of AMPs in the treatment of CF. The first of these (NCT02991859) was designed as a Phase 2a study to evaluate the tolerability and pharmacokinetics of an inhaled AMP therapy in patients with CF. The results indicated that the AMP was well tolerated, with no serious adverse effects; furthermore, the peptide reached its target site, i.e., the lungs. Although the primary endpoints of the study were not published, the findings provide evidence that an AMP can be delivered to patients with CF via inhalation in a safe manner, thereby setting the stage for programs to establish the therapeutic efficacy of AMP. A second approach being investigated is the use of AMPs in combination with other agents used to treat patients with CF.

An example is azithromycin; although azithromycin has been used in the treatment of patients with CF for several years due to its anti-bacteriogenic and anti-inflammatory properties, researchers have been examining the utility of combining azithromycin with an AMP in CF animal models. The results demonstrate that this combination enhanced bacterial clearance and reduced inflammation. Should these combinations prove to be effective, they may ultimately become part of the standard therapeutic regimen for CF [1, 175, 187].

The clinical potential of AMPs as therapies for CF is reflected in growing evidence from numerous studies documenting their efficacy in killing CF pathogens in vitro and in various rodent models, as well as an initial inquiry into the safety profile of inhaled AMPs in a small number of human patients. Thus, the next series of studies will require conducting larger clinical trials to examine whether AMP-based therapies can reduce bacterial burden in CF, enhance lung function, and reduce exacerbations among CF patients. If successful, the use of AMPs as part of a new class of CF drugs will help combat infections and reduce inflammatory activity; both have the potential to delay the progression of lung disease in CF.

Ventilator-associated pneumonia

The VAP is one of the most prevalent types of HAIs documented as it develops in patients who have been placed on mechanical ventilators. Often, VAP develops from bacteria colonizing the endotracheal tube or airway of an intubated patient. As a result of this epidemiological factor, more extended hospital stays and higher mortality rates have been observed, and antimicrobial resistance has emerged. To help prevent VAP, one method that can be used is coating ETTs with appropriate antimicrobial coatings to kill bacteria that May come into contact with them. The use of Biomaterial-based coatings incorporating AMPs has been studied for this application; for example, using AMPs in conjunction with biocompatible polymers generally cationic peptides or defensin 189, 190]. The use of AMPs on ETTs enables continuous peptide release, inhibits biofilm formation, and eliminates bacteria that come into contact with the ETT in a laboratory environment. Researchers developed ETTs coated with a biocompatible polymer containing an AMP, such as a cationic peptide or defensin. Laboratory testing of these coated tubes has demonstrated a significant reduction in bacterial colonization by several pathogens, such as P. aeruginosa and MRSA. Nanoparticles have also been incorporated into studies on coating ETTs. In one study, ETTs were coated with nanoparticles loaded with AMPs and exhibited sustained antimicrobial activity against bacteria for several days. This suggests that the use of AMP-coated devices has the potential to substantially reduce VAP incidence by preventing bacterial attachment and biofilm formation on the ETT [190, 191].

Clinical trials of AMP applied to endotracheal tube coatings have just begun. Data collected during a pilot study identified several patients whose experience with an ETT coated with a cationic-derived peptide suggested the potential for this method to reduce infections and improve outcomes in patients receiving it. This finding, however, must be corroborated by larger-scale randomized, placebo-controlled studies before it will be possible to fully ascertain whether using AMP-coated tubes will be effective in preventing Ventilator-Associated Pneumonia. Nevertheless, manufacturers are starting to look at the possibility of developing an antimicrobial ETT, and one company, for instance, has already developed an ETT that is coated with an AMP and has begun conducting clinical trials to determine the safety and efficacy of this product in reducing the incidence of VAP [192, 193].

In addition to coating materials, other types of biomaterials can be used for VAP. Hydrogels or sponges placed adjacent to the cuff of the endotracheal tube have been used to deliver local AMPs and prevent bacteria from translocating into the lungs. Another modality is nebulization of AMPs to intubated patients on ventilators; however, the intent here is primarily to treat existing infection rather than prevent it. In all VAP interventions, the intent is to minimize the need for antibiotics by preventing bacterial adhesion and infection. From a safety perspective, any intervention aimed at VAP with AMPs must be biocompatible with the airway and not compromise its integrity [194]. In preliminary trials of peptide-coated ETTs, no significant toxicity has been observed with these materials, nor have any adverse effects been reported in patients using them to date. The concentrations of AMPs used in these investigations appear to be safe; however, continuous assessment of systemic toxicity and long-term safety is required to determine the potential for systemic absorption of peptide-based interventions. Currently, no documented major adverse events have been reported in any of the small clinical trials completed, lending credence to the idea that these biomaterial-based intervention methods for VAP will prove safe [190].

AMP-derived biomaterials offer a promising avenue for the prevention and treatment of VAP by inhibiting bacterial colonization of mechanical ventilation devices. Although the bulk of adult clinical data remains limited, encouraging results from initial in vitro observations and previous small scale human studies appear to suggest that, if proved efficacious, AMP Coated Endotracheal Tubes may become universally accepted as a method for prevention of VAP in the Intensive Care Unit (ICU) settings while lessening the number of people who develop VAP and reduce the need for antibiotics in the treatment of these patients [190, 195].

Antimicrobial peptides-based biomaterials against fungal respiratory infections

Nanoparticles, liposomes, hydrogels, and coatings are often considered biomaterials in the context of invasive infections caused by Aspergillus (aspergillosis), Cryptococcus (cryptococcosis), Histoplasma (histoplasmosis), and Candida (candidiasis). The development of these infections often occurs in individuals with compromised immunity, and they are frequently associated with high mortality rates in the lungs. Biomaterials derived from AMPs have been shown to serve as an alternative to antifungal treatments, addressing the issue of resistance. Nevertheless, while data are extensive for Aspergillus and Candida, in raw cases, there is limited data for Cryptococcus and Histoplasma in the lungs [76, 196].

In NP339, formulated for nebulization or inhalation, it reduces lung fungal burden in murine models of invasive pulmonary aspergillosis, synergistically with amphotericin B. It also has potential applications in allergic bronchopulmonary aspergillosis (ABPA) and CF-related infections. In Liposomal formulations, enhance the stability of peptides like lactoferrin for aerosol delivery, thereby improving lung penetration. In Hydrogels and nanoparticles, photopolymerized ε-poly-L-lysine hydrogels or gold/silver nanohybrids coated with cationic peptides inhibit adhesion and growth on surfaces, making them adaptable for pulmonary stents or inhalable particles. In some in vitro and in vivo studies, dose-dependent reductions in metabolic activity have been observed across clinical and environmental isolates, with no resistance linked to azole profiles. In vivo, Nebulized NP339 in neutropenic mice resulted in reduced A. fumigatus lung loads. Combinations with micafungin in silkworm and guinea pig models demonstrated synergy. Challenges: Environmental isolates are more susceptible than clinical isolates; optimized dosing is needed to avoid growth stimulation (e.g., LL-37 at low concentrations) [76, 196].

Aspergillus. spp

Aspergillus fumigatus is often implicated in cases of invasive pulmonary aspergillosis. Lysozyme breaks down the β−1,4 linkages of chitin and degrades the cell walls of A. fumigatus. Lysozyme reduces fungal metabolic activity in a dose-dependent manner but has a narrow therapeutic window. Histones inhibit conidial germination and hyphal growth, possibly by disrupting membranes as components of neutrophil extracellular traps (NETs) [197]; however, the precise mechanisms of histone action remain unclear. Beta-defensin-1 inhibits outgrowth of germinating conidia, but only during early stages of growth, and does not affect mature hyphae. The anti-fungal efficacy of beta-defensin-1 against A. fumigatus is unknown. Lactoferrin prevents spore germination by binding iron, and its demonstrated effectiveness against A. fumigatus in vitro is at concentrations of 0.25–2.5 µM; its relevance is currently unestablished. NP339 is a synthetic AMP that has cationic properties similar to β-defensins that damage the fungal membranes and kill fungal cells. NP339 is effective against Aspergillus species, but the mechanism by which it damages the majority of populations in the long term remains unknown [198, 199].

Human α-defensins, particularly HNP-1, HNP-2, and HNP-3, are essential components of neutrophil granules and important members of the innate immune system, possessing potent antimicrobial activity. However, the activity of human α-defensins against various species of fungal pathogens is inconsistent. HNP-1 and HNP-2 are effective against Candida albicans at 50 μg/ml, whereas HNP-3 is ineffective [200]. The reasons for differences in human defensins are unknown. Moreover, rabbit NP-1 is considerably more effective than HNPs against C. albicans, with a potency 10—20 times greater. Does it have clinical relevance? Treatment with HNP-1, HNP-2, and HNP-3 at the same concentration causes significant inhibition of C. neoformans growth after four hours of exposure, reducing colony-forming units by more than 1000 per milliliter. This work may suggest possible new antifungal therapeutics; however, additional studies are required to confirm their efficacy. The use of bovine tracheal AMP helps target the yeast form of C. albicans at 400 µg per milliliter and worked better than either magainin II or amphotericin B for A. fumigatus at targeting hyphal fungal forms; however, the long-term effects on fungal populations from bovine tracheal AMP are unknown [201, 202].

Cryptococcus. spp

Cryptococcus neoformans is a fungus that typically causes pulmonary cryptococcosis, which frequently disseminates to the central nervous system. AMPs target vulnerabilities in the fungus's capsule and membrane. NaD1, a plant-derived AMP, enters the fungus's cytoplasm, disrupting the cell membrane and generating reactive oxygen species (ROS). Another AMP, Histatin-5, binds to a component of the fungal cell wall, β−1,3-glucan, leading to potassium ion loss, ROS production, and depletion of the cell's energy source, ultimately causing the cell to die. NP339 inflicts damage to the cell membrane and exerts a broad range of activity against various species of Cryptococcus. Cathelicidins, such as LL-37, cause the cell to rupture and induce oxidative stress, although they have been less studied in the context of Cryptococcus. β-peptides are utilised in polymer multilayers to prevent biofilm formation on medical devices, such as catheters, which is relevant to infections involving Cryptococcus in ventilator patients [199].

In studies using mouse models of lung infections, inhaling NP339 has been shown to reduce the burden of fungal infection. This device is suitable for use in nebulizers for patients with weakened immune systems. Nanoparticles coated with LL-37 have been used to deliver the AMP directly to the site of infection, thereby enhancing its effectiveness and demonstrating potential for treating cryptococcal pneumonia. Research has found that NaD1 is effective against Cryptococcus neoformans at low concentrations, and histatin-5 prevents biofilm formation at concentrations ranging from 1.7 to 62.5 micromolars. Studies in mouse models of disseminated cryptococcosis have demonstrated that furanone derivatives, inspired by AMPs, exhibit immunomodulatory properties and reduce the fungal burden in the lungs [76, 203, 204]. Table 5.

Table 5.

Different antimicrobial peptides-based biomaterials against fungal respiratory infections

Type of Biomaterial	Examples	Mechanisms	Efficacy in Lung Models	Ref
Nanoparticle-Based Systems	Gold nanoparticles with indolicidin; chitosan nanoparticles with TistH; silver nanoparticles (AgNPs) with protegrin-1 or histatin 5; cubosomes with LL-37	Disrupt fungal membranes (e.g., toroidal pore model), induce ROS, inhibit hyphal growth; nanoparticles reduce toxicity and enhance penetration	VL-2397 (aluminum-chelated) reduces the lung fungal burden in Aspergillosis mouse models; indolicidin-AgNPs are effective against Candida biofilms and adaptable for use in the lungs. TistH-chitosan NPs improve anti-Candida activity in biofilms	[203]
Hydrogel-Based Systems	Self-assembling hydrogels with LL-37 or histatin 5; microgels with defensins	Controlled release via pH/enzyme triggers; disrupt cell walls (e.g., inhibiting β-glucan synthase) and induce apoptosis	Psoriasin is effective in mouse lung aspergillosis; LL-37 inhibits A. fumigatus hyphae dose-dependently	[204]
Polymer Conjugate Systems	PEGylated echinocandins (e.g., anidulafungin, caspofungin); chitosan-histatin conjugates	Bind mannans or inhibit chitin synthesis; conjugates improve stability for inhalation	Caspofungin (intravenous) treats invasive aspergillosis; nikkomycin Z synergizes with echinocandins against A. fumigatus	[203]
Liposomal Systems	Liposomes with indolicidin or aureobasidin A	Inhibit sphingolipid biosynthesis or nucleic acids; liposomes enable aerosol delivery	Indolicidin liposomes demonstrate in vivo activity against disseminated candidiasis and have potential for pulmonary applications	[203, 205]

Histoplasma. spp

Histoplasma capsulatum causes histoplasmosis, primarily pulmonary in endemic areas. Data on AMPs are sparse compared to those on other fungi. Lysozyme: Inhibits at 0.06 µM via chitin hydrolysis. Lactoferrin and β-defensins: Iron sequestration and membrane permeabilization; activity inferred from related yeasts. Limited specifics: Some plant defensins (e.g., HsAFP1) exhibit broad antifungal effects, but have not been extensively tested against Histoplasma [205]. In general formulations, pulmonary aerosols of antifungal agents (e.g., patent on lipid-based carriers for amphotericin B analogs, adaptable for AMPs) for targeted lung delivery. In Nanoparticles: Could incorporate lysozyme for inhalable therapy, but no Histoplasma-specific biomaterials have been reported. In some in vitro and in vivo studies, lysozyme is effective against H. capsulatum, while histones inhibit related yeasts at a concentration of 26 µg/mL. Minimal data; animal models of histoplasmosis primarily focus on conventional drugs, with the potential of AMPs in the lungs unexplored. However, a lack of dedicated research often results in activity being extrapolated from Candida or Aspergillus studies [203–205].

While there is considerable literature on AMP activity, only limited knowledge of the efficacy of AMP activity against this fungus exists when compared to other fungal types. Research has demonstrated that lysozyme can inhibit Histoplasma capsulatum at a concentration of 0.06 μmol by chitin hydrolysis, although this finding is based on limited evidence and warrants further research. Lactoferrin and beta-defensins have also been shown to be effective against the fungus; however, this finding was based on research with related yeasts, which may not apply to Histoplasma capsulatum. Plant defensins, such as HsAFP1, have been shown to have antifungal activity; however, their efficacy against Histoplasma capsulatum has not been thoroughly studied [205, 206]. Formulations of antifungal agents delivered directly to the lungs via aerosols have been developed; however, these formulations could potentially be adapted for use with AMPs, although this remains speculative at present. Lysozyme could be included in inhalable particles for treating histoplasmosis; however, no specific biomaterials have been developed to target Histoplasma capsulatum. Additionally, some studies have yielded conflicting findings: lysozyme is effective against the fungus in some instances, whereas histones have been shown to inhibit related yeasts at a concentration of 26 µg per milliliter. A notable deficiency exists in the dedicated research on using AMPs to treat histoplasmosis, and, consequently, activity is frequently inferred from studies on other fungi, such as Candida and Aspergillus, which presents a significant constraint in the field [206–208].

Candida. spp

LL 37 can stimulate oxidative damage to the cell membrane and reduce biofilm formation at levels below the MIC. However, the long-term outcomes of LL 37 are not known. Nucleated peptides such as hBD2 and hBD3 disrupt the ability of cells to synthesise ATP and the integrity of the cell membrane at concentrations of 0.3—13.8 µM. However, the specificity of these nucleated peptides is not known. Melittin and gomesin can induce apoptosis in cells via calcium-dependent pathways. However, these methods may cause collateral damage to surrounding cells due to the cytotoxic nature of the compounds. Different formulations of AMPs have been developed to improve dosing; however, their clinical efficacy has yet to be established [76, 209, 210].

Dipeptide-capped hybrids and magnetic LL 37 nanoparticles have been developed to inhibit biofilm formation on endotracheal tubes and similar medical devices. However, the long-term efficacy of these nanoparticles also remains unknown. While antifungal peptides have been engineered for pulmonary delivery, none have yet been tested against Candida. The evaluation of the potential of compounds to disrupt mature biofilms was conducted using a combination of LL-37 and caspofungin. In vitro testing showed that the combination of these agents can reduce mature biofilm levels by 70—90%. In vivo studies using mouse oral/oropharyngeal and rat catheter models showed that AMPs reduce Candida invasion; however, the clinical significance of their use remains unclear [209, 211].

Overall, AMPs-based biopolymers hold potential for pulmonary delivery, with NP339 exhibiting multi-fungal activity. While recent developments, such as biomimetic designs that mimic natural host defenses, have emerged, clinical translation will only be possible after addressing scalability and resistance issues [199]. Figure 4.

Fig. 4.

Antimicrobial peptides (AMPs) utilize several mechanisms to combat microbial cells, including the toroidal-pore, carpet, and aggregate models. In the toroidal-pore model, AMPs integrate into the membrane and interact with lipid molecules to form toroidal pore complexes. This involves a local concentration of AMPs that induces bending in lipid molecules, embedding peptides and lipid head groups within the membrane's hydrophobic center. The carpet model operates differently; it requires a high concentration of AMPs to form micelles that disrupt microbial membranes through surfactant-like action without penetrating the hydrophobic center or forming channels. This results in the partial or complete lysis of cell membranes, leading to subsequent cell death. The aggregate model enables AMPs to bind with anionic cytoplasmic membranes, forming peptide-lipid complex micelles that create channels for ion leakage and the release of intracellular content. These channels enable AMPs' entry into the cytoplasm, where they can target intracellular substances, adding another layer of antimicrobial action beyond merely compromising membrane integrity. Each model highlights distinct yet overlapping strategies employed by AMPs as crucial components of innate immune defense systems against microbial pathogens

Clinical evidence of efficacy and safety of antimicrobial peptides

As of now, no AMP-based medicinals are available through government-approved regulators for treating respiratory infections. However, many initial clinical studies have shown benefits, and preclinical data indicate that AMP-based products are effective against various respiratory infections. Below are summaries of the literature evidence on the effectiveness and safety of AMP-Based Biomaterials [212].

Efficacy evidence

Preclinical studies consistently demonstrate the efficacy of AMPs against bacterial pneumonia. An example is Esc (1–21), a peptide that significantly reduced bacterial counts in the lungs and increased survival in mice with Pseudomonas aeruginosa pneumonia. Another study demonstrated that the antimicrobial peptide CAMA eliminated MRSA biofilms from lung tissues, suggesting that AMPs may offer a practical approach for clearing chronic bacterial infections. The results of these studies provide support for the notion that antimicrobial peptides can be used effectively against the most common bacterial pathogens that cause pneumonia. However, although there is limited clinical evidence supporting the use of antimicrobial peptides, a novel formulation of inhaled antibiotic nanoparticles has demonstrated positive clinical outcomes in patients with pneumonia [212–214]. One study involving patients with non-tuberculous mycobacterial lung disease showed that those receiving inhaled amikacin (in liposomes) had lower bacterial loads and improved pulmonary function compared with those receiving traditional oral amikacin. The success of inhaled products containing conventional antibiotics provides a rationale for developing similar strategies using antimicrobial peptides to treat bacterial pneumonia. Additionally, clinical trials are currently underway to determine whether dry-powder microwave-dried lung inhalation of antimicrobial peptides will achieve therapeutic concentrations in the lungs and reduce the severity of bacterial infections in patients [212].

Clinical trials have demonstrated that several types of AMP kill M. tuberculosis and stimulate host immune responses against it. In particular, the peptide called Mu1140 was effective against drug-resistant TB in a mouse model. Clinical trials have assessed the potential for AMPs to be used as adjunctive therapies to standard TB treatment. One small clinical trial using a peptide in combination with conventional TB medications in patients with MDR showed evidence that the peptide reduced time to culture conversion in some participants. Although the trial was small and not placebo-controlled, it provides proof of concept that AMPs may offer TB patients better treatment options. Furthermore, a strategy is to use gene therapy to deliver AMPs directly to the lungs. Studies in animals suggest that using gene therapy to express LL-37 in the lungs could reduce the TB bacterial burden. If the above-mentioned strategies can be applied safely to human subjects, TB treatment could be made more effective in humans suffering from drug-resistant TB strains [215, 216].

Clinical research on the effectiveness of AMPs in CF is beginning. A Phase 2a clinical research project (NCT02991859) designed to test the potential of inhaled AMP is currently underway with participants with CF. While complete study data are not yet available, this research provides preliminary evidence of the tolerability of inhaled AMP and its ability to reach the lungs of patients with CF. The results of this study will guide further research to determine whether the use of AMPs can reduce the frequency of bacterial exacerbations or improve lung function in CF patients. In addition, a research study using AMPs and azithromycin in a CF mouse model demonstrated more effective bacterial clearance and reduced overall inflammation than either agent alone [175]. If the results of this and other studies to follow continue to verify the effectiveness of AMPs in human patients, a dual treatment approach combining AMPs with azithromycin will be a new treatment for lung infections caused by CF. In summary, there is growing evidence supporting the efficacy of AMP-based treatments for CF. There is some in vitro and animal model evidence supporting the effectiveness of multiple types of AMPs, and early human studies are beginning [175, 216].

Although AMP-coated ETTs decrease biofilm development and bacterial colonization associated with VAP, as shown in preclinical research, a small randomized controlled feasibility study found that patients who received an AMP-coated ETT had a lower infection rate than those who received a standard ETT. Despite the lack of statistical analysis to confirm this finding, these preliminary data support the use of AMP-coated devices as a possible means of preventing VAP. However, further confirmation of their efficacy requires larger, randomized controlled trials; an AMP-coated ETT for the prevention of VAP is currently being studied in a larger, well-controlled trial. Should this study confirm that AMP-coated ETTs significantly reduce the incidence of VAP, it would represent an important advancement in the prevention of nosocomial infections [189].

AMPs have shown promising results in various animal models, suggesting their potential for clinical safety and effectiveness in complex biological systems. One area that has been studied in murine models of fungal lung disease is IPA, in which intratracheal administration of the AMP LL-37 on Days 2 and 7 post-infection reduced yeast/mold levels in the lung by 90% compared to the control group. Upon histological evaluation, LL-37-treated lungs showed only fungal colonization of the bronchi. In contrast, control animal lungs showed highly inflamed alveolar elements and fungal hyphae in the lung parenchyma (air-filled spaces within the lung), leading to severe pneumonitis and abnormal structural lung damage [36, 217]. Importantly, LL-37 exhibited protective effects against A. fumigatus infections in immunosuppressed animals, indicating that LL-37's antifungal properties are a major contributor to its demonstrated efficacy in vivo and that this type of protection is independent of any immunomodulatory actions of the AMP. Furthermore, transgenic LL-37 mice showed reduced susceptibility to A. fumigatus infection, providing strong evidence for the protective properties of endogenous LL-37. The ability of LL-37 to inhibit the expression of pro-inflammatory cytokines (TNF-α and IL-6) in the lungs also indicates that the AMP has dual functions in the clearance of pathogens and the regulation of inflammatory responses [217, 218].

While most in vivo studies of AMPs focused on their effects against bacterial respiratory disease, their results remain informative and valuable. Researchers have developed a D-amino acid peptide, termed AMP 1003, which displays high stability and have tested this drug in various mouse pneumonia models induced by multiple strains of antibiotic-resistant Bacteria, including MRSA and Klebsiella pneumoniae. Subcutaneous administration of AMP 1003 significantly reduced bacterial loads in the lungs of these mice, similar to the effects observed with vancomycin or polymyxin B, and simultaneously significantly ameliorated lung pathology associated with bronchial damage and alveolar hemorrhage. Additionally, this AMP has demonstrated a strong ability to reduce lung inflammation in a model of LPS-induced acute lung injury [219].

Delivery systems improve in vivo activity. An albumin-based nanodrug delivery system for LL-37, administered intratracheally, demonstrated improved bacterial clearance in a mouse model of a P. aeruginosa lung infection compared with free LL-37. Likewise, in a murine model of COPD-type lung infection, inhaled immunoantimicrobial nanoparticles (IAMs) containing AMPs significantly decreased airway bacterial load and lung inflammation when nebulized. These findings provide a proof-of-concept for further research into the use of inhaled AMP-based biomaterials to treat fungal respiratory infections [45, 220].

Although clinical studies of the use of AMPs for treating viral respiratory infections are still in their infancy, several small-scale studies have evaluated the potential benefits of inhaled AMPs in patients with COVID-19. One pilot clinical trial demonstrated that inhaled AMP administration was associated with improved oxygen saturation and a trend toward lower viral load in patients with severe COVID-19. However, the limitations of small sample sizes and the lack of placebo controls make these studies inconclusive. Further studies will be required to assess the therapeutic potential of AMPs for viral pneumonia definitively. AMP formulations have also been trialed in the development of nasal sprays for potential use in preventing viral infection; one study in patients with influenza indicated that the use of AMP-based nasal sprays reduced the duration of illness in the enrolled subjects, but again, more research will need to take place actually to confirm the efficacy of this approach. Therefore, while the idea of developing AMP therapies for viral infections appears promising, further research is needed before clinical efficacy can be established [221]. Table S2.

Safety evidence

General safety

Harrison discovered that in vitro studies using the LL-37 peptide in monolayer cultures demonstrated little, if any, toxicity. In response to growing evidence supporting the biological activity of AMP-1, there is an urgent need for clinical trials to verify these findings in animal studies. There are several reasons why AMPs are viewed as promising candidates for developing an innovative form of therapy. The favorable safety profile of AMPs is one of the reasons clinical researchers have selected this type of antimicrobial therapy for further development and investigation. The clinical trials have shown that the safety profile of AMPs is similar across multiple clinical settings. Additionally, data from clinical trials on the safety and tolerability of AMPs have confirmed that their low toxicity is consistent across multiple studies. In a Phase 1 clinical trial in patients with CF, no serious adverse events were reported due to AMPs. Studies using AMP-coated endotracheal tubes (ETTs) have shown no significant airway irritation or other adverse effects in patients. Thus, the current body of evidence indicates that AMPs can be safely administered to humans, based on the available clinical data [175, 221].

Systemic toxicity

AMPs have been shown to cause systemic toxicities when they enter the bloodstream. Because AMPs are administered only locally via inhalation, device coatings, etc., this possibility is minimal. When AMPs have been administered systemically in a small number of studies on the subject, the results indicate that AMPs can be tolerated quite well. An early study of protegrin, a peptide with antibacterial activity, found that therapeutic doses were associated with minimal systemic effects. However, ensuring that the peptide does not accumulate in the body is crucial. This issue has been partially resolved by using biodegradable systems that deliver the peptide to the infected site, where it is released over time and then degrades. As demonstrated by animal studies, biodegradable systems are safe, and so far, there is no evidence from preliminary human studies to suggest that the peptides themselves cause any systemic toxicity [68, 222].

Immune modulation

AMPs can influence the body's ability to fight infections and can either enhance or suppress the immune response. In the case of LL-37, it has been shown to facilitate the production and release of both chemokines and cytokines in the bodies of infected individuals, which can assist the immune response in eliminating invading organisms; however, it may contribute to inflammatory damage. To effectively eliminate pathogens from the lungs, moderate immune responses are required; however, excessive inflammation can cause tissue damage. Preclinical studies indicate that AMPs can enhance or inhibit the inflammatory response, depending on the pathogen type and the dose administered. Regarding the clinical use of AMPs, it is essential to carefully consider potential adverse side effects and an overwhelming inflammatory response. To date, preliminary anecdotal evidence suggests that aerosolized AMPs do not induce marked hyperinflammatory responses in the lungs. On the contrary, they may modulate the inflammatory response. In one study using a mouse model of acute lung inflammatory disease, the AMP significantly decreased the production of pro-inflammatory cytokines while maintaining bacterial lethality. The combination of bacterial killing and reduced inflammation may be beneficial for many respiratory diseases [221, 223].

Allergic or hypersensitivity reactions

There is little chance of developing an allergy to AMPs for humans because AMPs are composed of amino acids created within the human body, meaning that our bodies have had sufficient time to learn about these naturally occurring molecules, so they do not create a typical allergic reaction as would drugs that have not been introduced into the human body before. However, since they are manufactured rather than occurring naturally, some individuals may develop an immune response. To date, no severe allergic reactions have been reported in clinical studies involving their use. Additionally, regarding the use of Gene Therapy to produce an AMP, while the vector and/or the AMP may cause an individual to develop an immune reaction to them, there has yet to be a definitive conclusion on whether this will occur [224, 225].

Long-term safety

Safety data for AMP-based therapeutic drugs over extended time periods are still emerging, and most studies in this area have short follow-up periods and are relatively recent. The Phase I study of an inhaled AMP was completed only recently. There will be follow-up in the future as these studies become established to determine any potential long-term impacts of AMP therapy, such as effects on lung function and/or alterations in the immune system, and to assess interactions with other drug classes. AMP therapy modulates the immune response and will therefore need to be studied alongside any immune-modulating drug to assess potential positive or negative interactions [115].

Antimicrobial peptides in clinical trials

Bacterial lung infections: advances and key candidates

Bacterial lung infections (pneumonia, VABP, HABP, CF exacerbations, and TB) have been the target of clinical research for AMP in clinical development. Most of these infections are caused by resistant organisms, such as MDR and Pseudomonas aeruginosa, Klebsiella pneumoniae and Mycobacterium tuberculosis, which do not respond to the standard classes of antibiotics [180, 226].

Murepavadin (POL7080; a synthetic cyclic peptide analogue of protegrin-1) is a targeted treatment for gram-negative bacteria, specifically Pseudomonas aeruginosa, that inhibits the transport of lipopolysaccharide (LPS) by targeting the outer membrane protein LptD. Murepavadin has undergone Phase II clinical trials for the treatment of CF and non-CF bronchiectasis, and has demonstrated efficacy against gram-negative bacterial infections. While research continues into the applications of Murepavadin against multidrug-resistant strains of bacteria, the concern regarding its nephrotoxic effects in the IV route of administration has prompted a focus on the development of an inhaled formulation [227, 228]. Iseganan (IB-367; an analogue of protegrin-1) has broad-spectrum antibacterial activity through its ability to disrupt membranes. A large multi-center Phase III trial (NCT00118781) was conducted to evaluate its efficacy for the prevention of ventilator-associated pneumonia (VAP) in mechanically ventilated patients; isean was given as a topical application to the oropharynx. The results of this double-blind placebo-controlled trial did not indicate that ise was effective in reducing the incidence of VAP, lowering mortality, or decreasing the number of ventilatory days in comparison with placebo, and therefore this product was removed from further development. Critics have suggested that ethical issues regarding the use of placebo controls were not sufficiently considered in the trial design and point to potential alternative delivery methods that might rekindle interest [229].

Colistin (Polymyxin E) is an AMP via a cationic cyclic peptide structure which acts to disrupt the integrity of the bacterial outer membrane and therefore is reserved as last resort therapy for MDR Gram negative bacterial infections. Colistin has been studied for many different applications in the lungs, including trialing inhaled formulations of colistin in patients with pneumonia, bronchiectasis and those experiencing an acute exacerbation of their chronic lung disease (e.g., NCT02918409 comparing IV colistin to tobramycin in those with CFs). The inhaled route is also likely to be advantageous over IV colistin in reducing the renal toxicity associated with IV colistin and is effective against multidrug resistant organisms such as P. aeruginosa and Acinetobacter species. The question of the potential for development of resistance as well as the variability of colistin pharmacokinetics within the lungs are still being actively debated [226, 230, 231]. Polymyxin B, which is similar to colistin, has also been studied for VAP (e.g., NCT02134106, a randomized trial studying the combination of polymyxin B and other antibiotics for the treatment of patients with MDR infections). Their use as a nebulized product has been studied, and the evidence comparing their safety and efficacy over colistin is currently inconclusive [232, 233]. Capreomycin and Enviomycin are two examples of cyclic peptide antibiotics that inhibit ribosome function within M. tuberculosis. Capreomycin is included in the treatment regimens for MDR TB, and there are studies that have recently evaluated injectable formulations of capreomycin for the treatment of pulmonary TB. Enviomycin is an analog of viomycin and is approved for TB in Japan but has yet to be approved for use globally. There have not been any recent large-scale clinical trials conducted on the efficacy and safety of envimycin; however, there is some evidence from preclinical studies that envimycin has activity against TB [234, 235].

There are several other promising but still in development AMPs for lung infections caused by bacteria such as AMP 1003 (active against MRSA, K. pneumoniae) and A20L (active against K. pneumoniae), as well as novel peptide-based AMPs delivered via mRNA, which demonstrate potent bactericidal and immunologic activity against MDR pneumoniae. Other examples of AMPs derived from the host, such as the three human beta defensin isoforms (HBD1—3) or cathelicidin LL-37 are shown to be upregulated during respiratory tract infections associated with CF and TB. Ongoing clinical trials which examine the potential of vitamin D supplementation in relation to LL-37 include NCT05431218, conducted on patients suffering from COPD [180, 236, 237].

Fungal lung infections: limited progress

There are very few AMP candidates currently under investigation for treatment of fungal lung infections (e.g., invasive aspergillosis and candidiasis) in immunocompromised individuals, and no clinical trials have been completed. NP213 (Novexatin) is a synthetic peptide that originates from host defense mechanisms has successfully completed Phase 2a clinical studies for the treatment of onychomycosis. However, although it has demonstrated antifungal action against dermatophytes, no studies have been conducted to investigate the efficacy of this compound against lung-specific fungi. Lactoferrin and secretory leukocyte protease inhibitor (SLPI) have demonstrated antifungal effects in vitro, however, their clinical application is directed toward non-pulmonary indications such as the treatment of diabetic foot ulcerations. Some of the preclinical AMPs that are being repurposed against the top 10 fungi listed by the WHO have shown promise, however, no advanced clinical investigations have been performed on pulmonary fungal infections; likely due to the concentration of research efforts on the top 10 bacterial priorities [40, 203, 237, 238].

Viral lung infections: preclinical emphasis

While no clinical studies have investigated the use of AMPs for viral respiratory infections, including SARS-CoV-2, influenza, and RSV, AMPs possess several unique antiviral properties that could be used as treatment options (e.g., blocking viral entry into host cells, immunomodulation). The ability of LL-37 to inhibit both RSV and rhinovirus infections has been demonstrated in vitro model systems and through indirect clinical trials that increase the levels of LL-37 by administering vitamin D3 to mechanically-ventilated patients (NCT01372995). The α and β-types of defensins (α and β) inhibit the entry of SARS-CoV-2 into host cells, while lactoferrin has also been shown in preventive randomized controlled trials (RCTs) to significantly reduce the risk of respiratory tract infections (e.g., through immunomodulatory effects). In observational studies (e.g., NCT02464059), SLPI and lysozyme have demonstrated anti-inflammatory activities in the treatment of pneumonia. It is expected that future therapeutic strategies will explore AMP master regulators and nutraceuticals [36, 146, 237]. Table S3.

Future perspectives and limitations

AMP‑directed biomaterials show promise against MDR pathogens such as P. aeruginosa and S. aureus, but instability, toxicity, delivery barriers, and cost hamper clinical translation. AMPs degrade rapidly in physiological conditions and in sputum, limiting their efficacy in the lung, and inhalation requires large doses that raise systemic toxicity and immunogenicity. Producing sub‑5 µm aerosols is difficult and can damage peptides, as evidenced by lysozyme loss during nebulisation. Only a handful of inhaled AMPs have FDA approval; Iseganan failed Phase III trials for biofilm eradication. Production costs, scalability, and even low‑level resistance remain concerns, while embedding AMPs in hydrogels or nanoparticles often diminishes activity, and long‑term biocompatibility data are lacking [78, 239, 240].

Despite significant advancements and breakthroughs in research, limitations still exist that prevent its clinical use. The primary limitation to date is proteolytic instability—the degradation of peptides and linkages in biological fluids occurs quickly. Scientists have identified modifications (e.g., D-amino acids or cyclization) in the literature that can slow this degradation, but this approach may increase the cost of goods sold. In broad-spectrum CPPs, inadequate tissue-selective delivery and the lack of distinct receptors for targeting specific tissues can lead to toxic side effects in healthy, uninfected cells. Consequently, clean peptides may have the potential to induce immunogenicity. The mechanisms for entering cells, specifically the escape from endosomes and the evasion of lysosomal degradation, remain underspecified and unsecured for efficacy in vivo. Like all therapeutics, the rapid mutation of viruses and the heterogeneity of viral species can pose a risk of resistance, especially to single-target peptides. However, delivery mechanisms that utilize detachment of functionally similar mechanisms to evaluate cell risk and observe a level of protection against resistance have been developed [241, 242].

High production costs and scalability challenges limit accessibility, particularly in low-resource areas at higher risk of RTI outbreaks. The compromised, fragile environment can affect biomaterials, leading to premature release or reduced overall effectiveness under physiologic conditions. There is currently a lack of clinical safety data, as most evidence is derived from cell culture or animal studies, and only a small number of peptides have progressed beyond preclinical studies regarding RTI. Furthermore, the safety of peptides is complicated by the possibility of cytotoxic effects (or disruption of the host microbiota) in the luminal tissues, making sufficient safety assessments necessary. The opportunity to overcome these challenges will depend substantially on an interdisciplinary approach that harnesses the synergistic potential of an investigative platform combining chemistry, biology, and engineering to optimize biomaterials for the management of RTI [243].

The expanding field of antiviral peptide‑based biomaterials aims to fill unmet therapeutic gaps for RTIs caused by influenza, SARS‑CoV‑2, and RSV, with researchers honing cell‑penetrating peptide conjugates that boost bioavailability, target the lungs via phage display linked to antibodies or folic acid, and activate only in infected tissues; liposomes and nanoparticles are employed to enhance stability, provide controlled release, shield against enzymatic degradation, and amplify immunomodulation to reduce RTI‑related inflammation; advanced AI and machine‑learning tools accelerate the design of peptide drug conjugates and predict activity against evolving viral strains, supporting multivalent peptides that disrupt viral envelopes or block ACE2 entry, thereby complementing existing antivirals and vaccines for high‑risk populations; self‑assembling antimicrobial peptide hydrogels are being explored as inhalation therapies that localize to the respiratory tree and may overcome biofilms; early animal trials of AMPs such as Plitidepsin and EK1C4 have shown reduced viral loads, laying groundwork for combination therapies and future variant strategies, while peptide assemblies also facilitate nucleic‑acid diagnostics that feed into broader pandemic readiness and mitigation plans [33, 125, 148, 204].

Despite recent advances, clinical use of peptide‑based RTI therapeutics remains limited by several key challenges. Proteolytic instability leads to rapid degradation of peptides and linkages in biological fluids, necessitating costly modifications that increase production costs. Broad‑spectrum cell‑penetrating peptides lack tissue‑specific receptors, leading to off‑target toxicity and potential immunogenicity, while the mechanisms of endosomal escape and lysosomal avoidance are poorly understood and critically affect in vivo efficacy. Viral heterogeneity and high mutation rates pose a resistance risk, especially for single‑target peptides; membrane‑disrupting entry strategies may offer some protection. High manufacturing costs and scalability issues restrict accessibility, particularly in low‑resource settings prone to RTI outbreaks. Environmental factors can destabilize biomaterials, causing premature release or reduced potency. Clinical safety data are sparse, with most evidence from cell culture or limited animal studies, and only a few peptides have advanced beyond preclinical stages. Cytotoxicity and disruption of host luminal microbiota further complicate safety assessments. Addressing these hurdles will require an interdisciplinary, integrated approach that combines chemistry, biology, and engineering to fully realize the therapeutic potential of these biomaterials for RTI control [148, 244, 245].

The use of antifungal peptide‑based biomaterials offers a promising alternative for treating RTIs caused by drug‑resistant pathogens such as Candida albicans and Aspergillus fumigatus. Future strategies focus on advanced pulmonary delivery—self‑assembling peptide hydrogels (e.g., RADA16, PAF26) or nanoparticles that mimic innate AMPs by targeting pathogens, providing sustained release, attacking membranes, and stimulating immune responses. Inhalable micelles or aerosols loaded with AMPs can reduce biofilm formation and inflammation in cystic fibrosis patients and may improve outcomes in bacterial-fungal coinfections. Peptide engineering enhances protease resistance and multifunctionality, enabling antifungal and anti‑inflammatory actions. Hybrid biomaterials, such as silica‑nanoparticle‑functionalized AMPs or chitosan‑AMP composites, offer pH‑modulated, on‑demand release, lower systemic exposure, and resistance prevention. Gene therapy delivering AMP genes to the airway epithelium could provide long‑lasting protection against RTIs in immunocompromised individuals [148, 246].

The successful clinical translation of RADA16-based PuraSinus has been demonstrated in post-nasal surgery healing, suggesting that the development of clinical treatments for fungal sinusitis and fungal pneumonia, and potentially broader antibiotics as an effector for lung infection management, is anticipated next. Combination approaches with AMPs and conventional antifungals or immunomodulatory scaffolds to reduce dependency on antibiotics, and to consider the need for lower resistance potentials. A higher likelihood of overcoming polymicrobial and complex mechanisms of airway epithelium modification facilitates the consideration of inhalers and customized lung-regeneration scaffolds. Further advancements in specialties may deliver hope through individualized inhalers and lung-regeneration graphene scaffolds, potentially linking preventive, therapeutic, and tissue-repair approaches to airway epithelium to reduce or eradicate RTIs [32, 247].

Despite promising biology, antifungal peptide biomaterials have limited clinical utility because of instability (enzymatic degradation), physicochemical challenges (pH, salt, surfactants in CF sputum), toxicity that harms the gut microbiome, heightens immunogenicity, accelerates clearance, and reduces efficacy, and high production costs. In vitro efficacy is often overstated; in vivo biofilms and pathogen adaptability—such as fungal resistance to membrane‑disrupting biocides—far exceed laboratory results. Clinical trials have stalled—e.g., Iseaganan never reached Phase III for ventilator‑associated pneumonia due to failure to eradicate infection and adverse effects—and few studies examine peptide antifungal activity in RTIs, with most focusing on bacteriocins. Overcoming these hurdles requires rigorous preclinical work, strict adherence to biomaterial regulations, and engineering advances in production. Collaboration with research facilities will unlock the transformative potential of antifungal peptides for RTIs and other health systems [247, 248].

Chemical modification strategies

AMPs have the potential to be an alternative to Conventional Antibiotics. While AMPs are produced naturally from many species and can also be designed synthetically, in the face of increasing Antimicrobial Resistance around the world.' Other factors will impede their potential efficacy. These include degradation of AMPs by proteolytic enzymes in biological fluids, Short Half-life in the circulation, poor bioavailability, and the potential to elicit an immune response and, ultimately, produce Toxic Effects on Host Cells. Accordingly, over the last decade, many AMS Researchers worldwide have made concerted efforts to develop Chemical Modifications that increase (improve) AMPs' stability, Efficacy, and Specificity while minimizing adverse effects. Examples of Chemical Modifications made to AMPs include: 1. The Introduction of D-amino acid substitutions 2. Lipidation 3. PEGylation 4. Glycosylation 5. Stapling. The five chemical modifications of AMPs and their corresponding Mechanisms of Action and AMPs Benefits/Limitations will be examined based on Recent Research [249].

D-amino acid substitution

D-amino acid substitution involves the partial to full substitution of naturally occurring L-amino acids present within the peptide with their corresponding D-enantiomers. This modification exploits the stereospecificity of proteolytic enzymes, which predominantly recognize L-amino acids, to render peptides more resistant to proteolysis under physiological conditions, such as plasma and the gastrointestinal tract. This greatly improves the proteolytic stability of the peptides without affecting their charge or hydrophobicity, and it often enhances and maintains their antimicrobial properties. These also show reduced immunogenicity due to decreased antibody production against PEG, enabling innovative approaches to phage display, including mirror-image phage display. AMPs retain their efficacy against MDR Gram-negative and Gram-positive bacteria, and, in some cases, show reduced cytotoxicity. Nevertheless, high substitution could destabilize secondary structures (α-helices), potentially reducing bioactivity and immunomodulation due to the loss of D-form-specific binding to target receptors. Certain residues, such as lysine, are more affected, and high substitution rates could reduce efficacy against a target microorganism [249, 250].

The D-enantiomer of polybia-MPI, an AMP extracted from wasp venom, possesses strong protease resistance and retains activity against both Gram-positive and Gram-negative bacteria and yeast/fungal organisms; however, the lysine stereochemistry affects the potency of polybia-MPI. The antimicrobial activity of an AMP can be maintained by partial D-substitution of the lysine residue at position W3R6; however, full D-W3R6 shows a reduction in potency and stability. The D-amino substitution of the bee venom peptide HYL, combined with glycosylation, results in the increased antitumor and antibacterial properties of this peptide. Specifically, unnatural D-amino acid analogs, such as those found in Pepo5 derivative of norleucine or α-aminoisobutyric acid (Aib), increase the stability of Pepo5 and decrease the hemolysis associated with peptides. In summary, AMP modifications are beneficial against antibiotic-resistant strains by conjugating to fatty acids bound to the D-amino acid side chain of a peptide that inhibits MDR bacteria [251].

Lipidation

Lipidation refers to the covalent addition of fatty acids to either the N- or C-terminus, or both, or to specific amino acids located along the length of AMPs via either amide bonds, ester bonds, or thiol ether bonds. Lipidation promotes self-assembly (due to hydrophobicity) and interaction with microbial membranes, associated with the increased hydrophilicity of the AMP; lipidation increases membrane permeability for antimicrobial action (including biofilms and resistant strains) by forming stable secondary structure when inserted into lipidic environments. The addition of lipids also prolongs the half-life of the AMP by binding to albumin and creates new opportunities for integration into delivery systems. Because they require only minimal amounts of lipidic material for effective activity, ultrashort lipopeptides may provide an effective, cost-efficient, and broad-spectrum alternative in many cases. However, excessively long chains may lead to premature self-assembly of the lipopeptide in solution, thereby limiting its ability to target specific membranes and increasing the likelihood of hemolytic toxicity to mammalian cells. Additionally, if lipids are incorporated into an AMP and the overall charge is reduced from the addition of the lipid(s), selectivity may decline, resulting in off-target effects [252].

Daptomycin, a naturally adipogenic antimicrobial peptide from the Streptomyces genus with a C10 carbon chain, has received FDA approval to treat gram-positive bacterial infections by disrupting bacterial membranes. Bacterial Surfactin (C16-β-hydroxy fatty acid) disrupts the bacterial membrane by depolarising and possesses both Antimicrobial and anti-viral properties. Temp L derivatives with a combination of Pro3, DLeu9, DLys10, as well as a C5–C13 carbon chain, remain active against Staphylococcus aureus as well as Pseudomonas aeruginosa, provided the charge of the Temp L is not altered. Peptides containing branched-chain lipids, such as S-8, have demonstrated the ability to inhibit the growth of MRSA Biofilms with minimal hemolysis, even in the presence of bacterial persisters. Glyco-lipos now increase the clinical effectiveness of the ESKAPE group of bacteria, but vary in Cytotoxicity. For example, Lipidation/linkage of multiple other modifications to achieve balanced activity, such as the Stearic Acid of D-NO3-D-NO3 increased 14 times, compared with the half-life of bivalirudin [253, 254].

PEGylation

PEGylation is the process of attaching polyethylene glycol (PEG) polymers—linear or branched, usually ranging from 5 to 40 kDa—to an AMP, commonly occurring at either the N- or C-terminal ends of the AMP to create a larger, more hydrophilic molecule. In addition to reducing renal clearance and increasing the duration of time that AMPs remain in circulation (i.e., extending their serum half-lives), PEGylation provides attributes such as improved solubility and resistance to enzymatic breakdown. In addition to protecting against cytotoxicity and hemolysis, the biocompatibility conferred by PEGylation on AMPs enables their use in many therapeutic areas, including pulmonary delivery. PEGylation has been shown to increase the selectivity of an AMP for bacterial cells; however, steric hindrance from the PEG chains can also reduce affinity for the intended target, impede membrane penetration, and ultimately reduce the overall potency of AMPs with longer PEG chains. In some situations, the C-terminal PEGylation of AMPs allows for the preservation of their activity to a greater extent than with N-terminal PEGylation [255].

PEG-CaLL is a hybrid of cecropin A and LL-37. This molecule has been shown to reduce hemolysis, protect the compound from serum degradation, and enhance treatment in cases of bacterial pneumonia. KYE28, when PEGylated, reduces antimicrobial potency; however, PEGylated KYE28 has significantly less hemolysis than non-PEGylated KYE28, indicating a higher bacterial selectivity. When administered into the airway, PEGylated AMPs minimize lung toxicity while preserving their efficacy against pulmonary infections. When C-terminally PEGylated, SAAP-148 displays enhanced stability as well as reduced cytotoxicity in both wound models of healing. Modularly designed PEGtides with different PEG backbones also exhibited vigorous bactericidal activity and specificity toward bacteria. LyeTx I-b, when PEGylated, maintained its three-dimensional structure while increasing activity against multidrug-resistant strains of both Gram-positive and Gram-negative bacteria [255, 256].

Glycosylation

Glycosylation is the covalent addition of carbohydrate residues, such as monosaccharides (including glucose, GalNAc, and others), often polysaccharides, to the AMPs' side chains. These are typically carried out by forming O, N, S, and/or C bonds, often via solid-phase chemistry and/or the "click reaction." Glycosylation significantly increases hydrophilicity, bioavailability, and resistance to proteolytic and glycosidase enzymes, and fixes structure while increasing membrane disruption, immunomodulation, and other activities. Higher stability is achieved by S-glycosylation than by either O-/or N-linkages. In antimicrobial peptides, glycosylation has been shown to improve antifungal and anti-biofilm activities, lower toxicity, and enhance delivery (such as crossing the blood–brain barrier). Inhibitory effects against hydrophobicity and basic charges are also possible, and may affect the ability to bind to negatively charged bacteria. Outcomes depend on glycan chain structure and length, stereochemistry (where β-linkages are potentially superior to α-linkages for unknown reasons), and methods of synthesis, which could induce an immune reaction [254, 256].

O-glycosylation with GalNAc on Thr11 decreases MIC to 75nM against Escherichia coli for Drosocin derived from Drosophila. Further, when deglycosylation is performed, the antimicrobial activity is diminished by as little as 5—10x. N-glycosylation of tyrocidine A analogue molecules containing monosaccharides increases their activity against MRSA and Vancomycin-Resistant Enterococcus (VRE), respectively. S-glycosylated sublancin analogues (including GalNAc) retain their antibacterial activity while demonstrating increased stability against Lactobacilli species. O-glycosylation of indolicidin reduces its hemolytic cytotoxicity without compromising its ability to bind LPS. C-Glycosylation (GlcNAc) of Hylaseptins-P1 produces more substantial antifungal effects against Candida due to the inhibition of the ergosterol biosynthetic pathway [256].

Stapling

A method of peptide stapling involves intramolecular cross-linking (or stapling) between side chains, typically achieved using all-hydrocarbon bridge methods such as ring-closing metathesis, triazole linkages, or N-alkylation. These cross-links create additional conformational rigidity in AMPs, forming two-dimensional, helical, or cyclic arrangements, e.g., i, i + 4; i, i + 7. Increased conformational rigidity provides greater proteolytic resistance, increased permeability through cellular membranes, and enhanced antimicrobial potency due to the ability to insert into cell membranes; stapled AMPs show greater activity against Gram-positive organisms and biofilm formation and may also result in an improved therapeutic index through increased selectivity. The increased hydrophobicity of staple peptides may consequently lead to increased haemolytic or cytotoxic potential. Enhanced activity is peptide-specific and may not necessarily correlate with helicity; although the solubility of stapled AMP may decrease, design efforts will require careful experimentation to prevent excessive rigidity or loss of activity [257].

All hydrocarbons by Polybia-MPI Stap led to 7x-23 × greater gram-positive bacterial inhibition and 68 × less degradation than the original MPs, but they showed even higher hemolytic activity. The double-stapled Ac-DS-14W exhibits increased gram-positive antimicrobial activity, greater stability, and reduced hemolysis due to a Trp substitution. Staple Aurein 1.2 has been shown to have increased resistance to fungi within some types of staples. Amongst the i, i + 4 staple type derivatives of MAG2 (e.g., Peptide 2), you can find examples that completely lack hemolytic activity at their Minimum Inhibitory Concentrations (MICs) while providing effective activity against resistant strains (MAG-1, MAG-3), against pneumoniae, and MAG-2 against Staphylococcus spp. Cyclic Cathelicidin BF15-A3 (Disulfide-Stapled) targets PCR and does so with low concentrations of hemolytic activity. Triazole-stapled C-MPI-1 provides only a slight increase in helical conformation but hardly an increase in activity yet [258].

High local concentrations, sub-MIC pockets and strategies to optimize encapsulation

Biomaterial encapsulation, such as in hydrogels, nanoparticles, or implant coatings, facilitates the controlled release of antibiotics directly at infection sites. This creates zones of elevated drug levels—often 1000—5000 times the MIC—that enable rapid eradication of both planktonic and biofilm-associated bacteria. For instance, ultrasound-triggered systems release a bolus of antibiotics like vancomycin, achieving supra-therapeutic concentrations that disrupt bacterial colonization and prevent persistence. Such high exposures minimize evolutionary opportunities for resistance by overwhelming bacterial defenses before adaptations can occur, reducing systemic toxicity and improving patient compliance. Research indicates that maintaining concentrations several times above MIC for extended periods maximizes therapeutic efficacy while curbing the emergence of resistant strains [167, 259].

Conversely, sub-MIC pockets arise when release is uneven or prolonged at low levels, exposing bacteria to subinhibitory doses. These conditions foster tolerance—extended lag phases allowing survival—and accelerate resistance evolution through selective pressure on mutants. In biofilms, low concentrations shield dormant cells, promoting genetic exchanges that spread resistance genes. Prophylactic applications in uninfected tissues are particularly risky, as sub-MIC elution may enhance biofilm formation and select for multidrug-resistant variants without proven efficacy. Evidence from laboratory evolution studies shows that sub-MIC environments enrich for mutations increasing resistance, emphasizing the need for precise dosing to avoid these evolutionary traps [260].

To balance these dynamics, advanced biomaterials like nanomaterials enable sustained release above MIC thresholds, bypassing traditional resistance pathways and reducing required dosages. Extended-release formulations, such as those using polymers for antibiotic delivery, maintain constant plasma levels over MIC, minimizing under-dosing that induces resistance. However, designs must prioritize short-duration bursts in high-risk scenarios to limit sub-lethal exposures, with ongoing research focusing on stimuli-responsive systems (e.g., enzyme-triggered) to adapt release profiles dynamically [261, 262].

These strategies are often combined (e.g., lipidation with D-substitution) to synergistically optimize AMPs for clinical translation, as highlighted in ongoing research toward novel anti-infectives.

Machine learning in antimicrobial peptides-based biomaterials

Machine Learning (ML) Algorithms such as Generative Adversarial Networks (GANs) and Variational Autoencoders (VAEs) use a variety of methods to generate and optimize AMPs for specific requirements (such as high bactericidal activity, low cytotoxicity, and resistance to degradation) at high speed. These newly created AMPs can be incorporated into Functional Polymers to enhance therapeutic applications across broader scenarios. However, challenges such as a lack of high-quality datasets for artificial intelligence (AI) training and the need to conduct further empirical testing of the created AMPs to determine whether they are compatible with living systems still remain. There are opportunities for the continued advancement of AI in predicting the interaction between peptides and biomaterials, leading to the development of more personalized treatment methods [115, 263].

The emergence of AI technologies for the development of antimicrobial peptide-based materials is transforming how antibiotic resistance is addressed in the field of infectious diseases. Using tools such as generative models, including latent diffusion models and large language models, AI can quickly and easily discover and optimize large volumes of potential new AMPs from very broad candidate sequence sets and facilitate predictions of key AMP properties, such as potent activity, low toxicity, and the ability to disrupt membrane function. Increasingly, AI-generated AMPs are being integrated into new biomaterials, including metal nanoparticles that enhance AMP stability, hydrogels designed for sustained release in wound-healing settings, and liposomes for targeted delivery of AMPs to pathogens associated with lung infections. Numerically, AI-based design algorithms (e.g., AMP-Diffusion) generate thousands of AMP candidates, and in many cases, incorporating thiol-modified AMPs into AI-guided hydrogel designs enables precise antibacterial therapies. These novel technologies are accelerating the development of AMPs while also decreasing the likelihood of antimicrobial resistance and demonstrating high in vivo efficacy in animal models of infection; however, there are still challenges regarding the generation of clinical-quality data and the eventual clinical translation of these technologies. Overall, the application of AI technologies to material development provides a bridge between the speed of computational processes and the innovation required to produce scalable solutions to global public health problems [264, 265].

Concerns and recommendations regarding addressing AMP resistance in the lung infections

Concerns in lung infections

AMP resistant strains of the pathogen P. aeruginosa lead to chronic infection, biofilm production, and increased inflammatory response in patients with CF. The resultant airway mucus obstruction impairs the ability of the host to clear bacteria from the lungs. In pneumonia, S. aureus (e.g., MRSA or Klebsiella pneumoniae) can evade host AMPs, leading to higher rates of mortality, decreased ability to phagocytize bacteria, and increased levels of pro-inflammatory cytokines. Chronic inflammation and tissue destruction results from the disruption of the immune balance by AMPs’ resistance, leading to increased recruitment of neutrophils and increased levels of oxidative stress. This synergy with antibiotic resistance complicates treatment due to the fact that many AMP-resistant strains are also resistant to antibiotics, thereby exacerbating the problems associated with VAP and nosocomial pneumonia for vulnerable hosts, e.g., immunocompromised individuals or those with COPD, increasing the likelihood of both increased healthcare costs and increased global burden of AMR. The occurrence of low levels of AMPs (induced resistance) following laboratory passage raises concerns regarding future viability of AMB therapy in the long term. In comparison to antibiotics, the incidence of AMP resistance in respiratory pathogens is lower due to multiple target mechanisms; however, current increase in prevalence of AMP resistance amongst respiratory pathogens warrants greater vigilance than previously [100, 180].

Recommendations and strategies to address AMP resistance

Overcoming resistances in AMPs requires creative and comprehensive approaches that focus on the design and delivery of peptides. Regarding the Synthetic and Modified AMPs category—engineering AMPs using D-amino acid residues or cyclization for greater protease resilience—Esc(1—21) acts against P. aeruginosa-causing pulmonary infection (MIC < 8 μmol/L) with efficacy established in murine models. Computer-aided design techniques like the Joker algorithm allow the development of narrow-spectrum AMPs (for example, PaDBS1R6) that selectively target Gram-negative bacteria without interfering with the host microbial flora [174, 180]. AMP combinations combined with standard antibiotics could prevent bacterial resistances, and a linear peptide like AMP 1003 (containing a structure composed of D-Trp-D-Arg-D-Lys residues) co-synergistically targets methicillin-resistant Staphylococcus aureus and Klebsiella pneumoniae (FICI ≤ 1.0) using ciprofloxacin or imipenem. It reduces the MIC values and functions as an efflux pump inhibitor. This synergistic action is associated with a less than fourfold increase in MIC after 14 rounds of bacterial passaging and does not augment the cytokines involved during lipopolysaccharides-induced pulmonary injury. Delivering these peptides via advanced pulmonary carrier formulations like aerosols, dry powder, and nanoparticles made of materials like poly(lactide co-glycolic acid) for Esc(1—21) will provide better tissue target specificities and penetrate deeper into the mucus present during cystic fibrosis or pulmonary infection. These technologies improve the degradation issues associated with the design and targeting of AMPs [174, 180].

Targeting bacterial resistances mediated by regulators like PhoPQ and efflux pumps is another area that could be exploited. AMP 1003 acts on membranes and generates ROS along with the ability to bind DNA. Bacterial recognition motifs (for example, the enterocecat pheromon) could be appended on AMPs for species-specific targeting. Other areas that require comprehensive development are the use of nanotechnology or the implementation of CRISPR-Cas systems for locus-specific modifications during bacterial resistances. Other areas that require coordination at the SAR front are quorum sensing inhibitors and the development of AMPs that do not require toxic materials [180, 266, 267].

Conclusions

In summary, AMP-based biomaterials exemplify a novel paradigm for RTI management, specifically designed to address the threat posed by multidrug-resistant organisms, including P. aeruginosa, S. aureus, and A. baumannii. These biomaterials exist in the form of self-assembling nanostructures, hydrogels, nanoparticle conjugates, and AMP-polymers hybrids that enhance the inherent stability, localized release, and biofilm-modulating potential of AMPs. AMP-based biomaterials outperformed conventional antibiotics, demonstrating greater therapeutic efficacy and continued reduced risk of pathogen resistance. Key in how innovative approaches across the drug development trichotomy can inspire AMPs. In optimized AMP animal models of lung infections, WLBU2 and HnMc micelles show significant reductions in bacterial load, inflammatory cytokines (TNFa, IL6), and tissue damage, as well as increased survival rates. These formulations (inhalation or infusion) effectively accumulate in the lungs to reduce bacterial load by altering the viscoelastic properties of biofilm membranes, while maintaining immune responses, which are of considerable importance for treating RTI complications involving biofilm in pneumonia, cystic fibrosis, and ventilator-associated conditions.

The incorporation of AMPs into biomaterials helps overcome the inherent limitations of natural peptides, such as rapid clearance and susceptibility to proteases, while facilitating a slow-release mechanism and multifunctionality, both of which are necessary for combinatorial therapies. This will not only address bacterial RTIs but may also contribute to the prevention of viral co-infection, as broad-spectrum activity has been afforded by AMPs such as LL-37 through disruption of the envelope and inhibition of entry, thereby lessening the risk of subsequent exacerbations of respiratory viral disease. The clinical development of murepavadin (Phase III for the treatment of nosocomial pneumonia) as a candidate therapy supports translational efforts to develop AMPs as alternatives to existing therapies that may preserve the host microbiome while minimizing toxicity. However, despite the advancements, challenges remain: the in vivo toxicity of AMP, high production costs, the immunogenicity of carriers (e.g., PEG), and the inability to address polysaccharides and eradicate complex biofilms. As there remain many opportunities to improve biomaterials, important future considerations should prioritize the use of AI-enhanced design to develop protease-resistant AMPs, a robust and comprehensive approach to conducting evidence-based research using in vivo databases, and the use of hybrid, engineered systems that combine antibiotics or immunomodulators to increase biocompatibility and efficacy. Thus, ultimately, AMP-based biomaterials promote novel approaches to the management of RTIs and can provide resilience to superior therapeutic approaches in a globally interdependent environment where antibiotic killing, cross-continental drug resistance, and microbial evolution pose threats to global societal health.

Abbreviations

AMPs

Antimicrobial peptides

RTI

Respiratory tract infections

MDR

Multidrug resistance

PGA

Poly-L-glutamic acid

PLL

Poly-L-lysine

LbL

Layer-by-layer

eDNA

Extracellular DNA

AI-2

Autoinducer-2

AHL

Acylated homoserine lactone

QS

Quorum-sensing

BAIs

Biomaterials

MRSA

Methicillin-resistant Staphylococci

GAS

Group A Streptococcus

CF

Cystic fibrosis

RSV

Respiratory syncytial virus

hMPV

Human metapneumovirus

HRV

Heart rate variability

HRVs

Human rhinoviruses

HAdV

Human adenovirus

LTB4

Leukotriene B4

RSV

Respiratory Syncytial Virus

ACE2

Angiotensin Converting Enzyme 2

TL

Temporin L

GPCR

G Protein-Coupled Receptor

CCR6

Chemokine Receptor 6

PLA₂

Phospholipase A2

LPO

Lactoperoxidase

SLPI

Secretory Leucoprotease Inhibitor

CAMPs

Cathelicidin antimicrobial peptide

CCL20

Chemokine ligand 20

LPS

Lipopolysaccharide

HBDs

Human β defensins

HDPs

Host defense peptides

CAP

Community-acquired pneumonia

VAP

Ventilator-associated pneumonia

ETTs

Inhaled endotracheal tubes

IAMs

Immunoantimicrobial nanoparticles

ICU

Intensive care unit

ALIS

Amikacin liposome inhalation

ELH

Extended length of hospitalization

MRSA

Methicillin-resistant s. Aureus

MDR

Multi-drug resistant

Aib

α-Aminoisobutyric acid

VRE

Vancomycin-Resistant Enterococcus

ML

Machine Learning

VAEs

Variational Autoencoders

AI

Artificial intelligence

Authors’ contributions

Conceptualization and design, M.R.A. and H.T.; first draft, H.T. and M.K.; writing—original draft preparation, M.K. and H.T.; writing—original draft preparation, software and design figures, H.T. and M.R.A.; data curation, writing—review and editing, M.R.A.; supervision and project administration. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.