Pepholin, a bacteriophage holin-derived antimicrobial peptide with membrane-disruptive activity and therapeutic efficacy in MDR Pseudomonas aeruginosa burn wounds

Purushotham RV; Sameer Shrivastava; Sonal Saxena; Sabapathi Nagappan; Pradeep Kumar; Saminathan M; Abhinav Kumar; Sanjay Kumar Singh; Satyabrata Dandapat

doi:10.1039/d6ra03599j

. 2026 Jul 9;16(37):39479–39504. doi: 10.1039/d6ra03599j

Pepholin, a bacteriophage holin-derived antimicrobial peptide with membrane-disruptive activity and therapeutic efficacy in MDR Pseudomonas aeruginosa burn wounds

Purushotham RV ^a, Sameer Shrivastava ^a,^✉, Sonal Saxena ^a, Sabapathi Nagappan ^a, Pradeep Kumar ^a, Saminathan M ^b, Abhinav Kumar ^c, Sanjay Kumar Singh ^d, Satyabrata Dandapat ^a

PMCID: PMC13348281 PMID: 42427398

Abstract

The emergence of multidrug-resistant (MDR) Pseudomonas aeruginosa poses a serious threat to burn wound healing, necessitating the development of alternative therapeutics beyond conventional antibiotics. In this study, an artificial intelligence guided design strategy was employed to identify a novel antimicrobial peptide (AMP), Pepholin (12 amino acids, net charge +5) derived from the holin protein of a Pseudomonas bacteriophage, highlighting holins as an underexplored scaffold for AMP discovery. Pepholin demonstrated strong antibacterial and antibiofilm activities against MDR P. aeruginosa, achieving complete bacterial eradication within 90 minutes. Mechanistic investigations confirmed that Pepholin exerts its bactericidal action by permeabilizing bacterial membranes and disrupting membrane integrity in a concentration and time dependent manner. The peptide exhibited high hemocompatibility and minimal cytotoxicity in mammalian cells, resulting in selectivity indices greater than 8. Pepholin retained its antibacterial activity under physiological conditions of salt, serum, and temperature. To explore its therapeutic potential, Pepholin was incorporated into a carbomer-based hydrogel and evaluated in a murine burn wound infection model caused by MDR P. aeruginosa. Topical treatment significantly enhanced bacterial clearance, accelerated wound healing, promoted collagen deposition, and modulated cytokine responses. Collectively, these findings demonstrate that bacteriophage holin derived peptides represent a promising new class of antimicrobial agents and establishes Pepholin as a stable, biocompatible, and mechanistically defined AMP with dual antimicrobial and wound-healing activity, supporting its development as a next-generation therapeutic for infected wound management.

AI-guided identification of the holin-derived antimicrobial peptide, Pepholin with membrane-disruptive antibacterial activity, excellent biocompatibility, and enhanced healing of MDR Pseudomonas aeruginosa burn wounds.

1. Introduction

Antimicrobial resistance (AMR) has emerged as a major public health concern that intersects with environmental, animal, and human domains. Multidrug-resistant (MDR) bacterial infections are currently widespread in hospitals, persisting for extended durations within the healthcare facilities.¹ Almost 50–80% of burn wound patients acquire nosocomial infection, out of which 20–80% of these cases are by MDR infections. Pseudomonas aeruginosa is a major cause of nosocomial infection that belong to World Health Organisation (WHO) priority pathogen group ESKAPE family i.e., Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter spp.² It has the ability to rapidly disseminate from the site of infection and produce a wide range of virulence factors that contribute to tissue damage, endotoxic shock and potentially fatal sepsis. Furthermore, the rapid evolution of intrinsic and acquired resistance mechanisms in P. aeruginosa has rendered multiple antibiotic classes ineffective, underscoring the urgent need for novel antimicrobial agents with improved pharmacokinetic and safety profiles.^3,4

The escalating AMR crisis has sparked interest in antimicrobial peptides (AMPs) as potential therapeutic candidates for infectious diseases due to their minimal potential for resistance and broad-spectrum antimicrobial action.^5,6 AMPs are short, amphipathic peptides (6–100 amino acids) that primarily exert their activity through membrane permeabilization or pore formation, resulting in ion efflux, membrane depolarization, and cell death.^7–9 In addition to their antibacterial properties, many AMPs also exhibit immunomodulatory effects, such as regulating gene expression, chemoattraction of immune cells, inducing chemokine production, and promoting angiogenesis, wound healing, and antiadhesion responses.^5,10 Although significant progress has been made, the lack of clinical approval is primarily attributed to proteolytic degradation, low stability, and toxicity of AMPs.^11,12 To tackle the challenges posed by natural AMPs, designing novel synthetic peptides can overcome these issues by regulating their structural and physicochemical parameters, impacting potency, stability, toxicity, and antimicrobial activity spectrum.¹³

Recent advances in peptide engineering and computational prediction tools have significantly accelerated the discovery of synthetic AMPs with optimized physicochemical properties. Artificial intelligence based prediction platforms enable identification of bioactive peptide segments within larger proteins, thereby expanding the repertoire of antimicrobial scaffolds beyond naturally occurring AMPs. Bacteriophage proteins represent an underexplored but promising source of AMPs, owing to their natural specificity for bacterial targets and potent lytic mechanisms.¹⁴ Certain phage-derived proteins, especially from Gram-negative phages, contain amphipathic, cationic segments that can interact with negatively charged bacterial membrane components such as lipopolysaccharides.^9,15,16 Previous studies have explored phage-derived enzymes like endolysins (e.g., LysAB2, Pfly307) for antibacterial use;^9,17 however, phage holin proteins, which form membrane lesions during phage lysis, remain largely uncharacterized as AMP templates.

Ensuring sustained local delivery and stability is critical for harnessing the therapeutic potential of AMPs in wound healing.¹⁸ Hydrogels can be an ideal formulation for the topical delivery of AMPs, providing moisture retention while enabling controlled AMP release through mechanisms such as covalent conjugation, electrostatic interactions, and degradation.^19,20 In recent years, antimicrobial wound dressings have emerged as a viable solution for minimising microbial colonisation and infection in burn wounds.^21,22 In this study, we report the AI-guided design and synthesis of a novel AMP, Pepholin, derived from a bacteriophage holin protein. The peptide exhibits antibacterial and antibiofilm activity against MDR P. aeruginosa, operates via a membrane-disruptive mechanism, and demonstrates significant wound-healing efficacy when incorporated into a carbomer-based hydrogel. To the best of our knowledge, this work represents the first report of a holin-derived AMP exhibiting both antimicrobial and wound healing properties, expanding the therapeutic potential of phage-inspired peptides for the treatment of drug-resistant infections.

2. Materials and methods

2.1. Chemicals, media and bacterial strains

For peptide synthesis, the Rink amide MBHA resin (4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucyl-methylbenzhydrylamine hydrochloride resin, loading capacity 0.8 mmol g⁻¹), along with N,N′-diisopropylcarbodiimide (DIPC) and hydroxybenzotriazole (HOBT), as well as Fmoc-protected amino acid derivatives, were procured from NovaBiochem. The solvents used, including piperidine, trifluoroacetic acid (TFA), triisopropylsilane (TIPS), thioanisole, 1,2-ethanedithiol (EDT), phenol, and HPLC-grade water, were purchased from Merck, Germany.

Bacterial culture media, including Pseudomonas isolation agar (PIA) medium, Mueller-Hinton Agar (MHA), Mueller-Hinton Broth (MHB), Tryptone Soy Broth (TSB) and Brain Heart Infusion (BHI) broth, along with HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid) buffer, standard antibiotic discs and reference strains of ESKAPE group of bacteria such as Enterobacter cloacae (ATCC 13047), Acinetobacter baumannii (ATCC 19606), Staphylococcus aureus (ATCC 29213), Klebsiella pneumoniae (ATCC 13883), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 29522), and Enterococcus faecalis (ATCC 14506) were procured from HiMedia, India. Clinical isolates of P. aeruginosa were obtained from mastitic bovine milk samples collected at the Veterinary Clinical Complex, ICAR-IVRI.

2.2. Design, synthesis and purification of peptide

The novel AMP investigated in this study was derived from the holin protein of Pseudomonas bacteriophage Iggy using an artificial intelligence (AI) assisted in silico screening and prioritization strategy. The full length holin protein sequence was screened using the Deep-ABPpred web server, a deep learning-based predictor specifically developed for identifying antibacterial peptides within protein sequences. Deep-ABPpred employs a bidirectional long short-term memory (BiLSTM) architecture combined with word2vec-based amino acid embeddings, enabling the model to capture both local and long-range sequence dependencies relevant to antibacterial activity. The Deep-ABPpred model was trained and validated on large, curated datasets comprising experimentally confirmed antibacterial peptides and non-antibacterial peptides and has been shown to outperform several existing AMP prediction tools. Importantly, the server outputs a probability score (ranging from 0 to 1) that reflects the likelihood of a given peptide sequence exhibiting antibacterial activity, thereby enabling objective prioritization of candidate peptides for experimental validation.²³

Peptide sequences within the holin protein exhibiting high predicted antibacterial probability were shortlisted and further refined using rational physicochemical criteria established for potent activity against Gram-negative bacteria, particularly P. aeruginosa. These criteria included a net positive charge to promote electrostatic interaction with the anionic outer membrane, moderate hydrophobicity to facilitate membrane insertion while limiting host-cell toxicity, predicted amphipathic α-helical structure, and an optimal peptide length consistent with previously reported anti-P. aeruginosa AMPs. The final peptide sequence, designated Pepholin, was selected based on a balanced integration of high Deep-ABPpred probability scores and favorable physicochemical attributes rather than reliance on any single parameter.

Sequence alignment between Pepholin and its parent holin protein was performed using Clustal Omega (EMBL-EBI). The alignment output was visualized and annotated using Jalview to identify residue conservation and physicochemical features. Membrane topology and transmembrane regions of the holin protein were predicted using the Phobius server to determine the positional relationship of Pepholin to membrane-associated domains. Subsequent in silico analyses were conducted to further characterize Pepholin. Three-dimensional structure prediction was performed using PEP-FOLD3. Key physicochemical parameters, including molecular weight, net charge, hydrophobicity (GRAVY index), and solubility, were calculated using Innovagen PepCalc and APD3 calculator.

Pepholin was synthesized by solid-phase peptide synthesis (SPPS) employing standard fluorenylmethoxycarbonyl (Fmoc) chemistry on Rink amide MBHA resin using an automated peptide synthesizer (CS336X).²⁴ Following cleavage and deprotection, the purity of the synthesized peptide was assessed by reversed-phase high-performance liquid chromatography (RP-HPLC) (Shimadzu, Japan) using a C18 column with a linear gradient of solvent B (0.1% trifluoracetic acid in acetonitrile) against solvent A (0.1% trifluoracetic acid in water) and peptide elution was monitored at 220 nm, and the injection volume was 20 µL.

2.3. Secondary structure prediction and determination

The secondary structural organization and amphipathic nature of the peptide were predicted using the NetWheels server. Residues were classified as polar or nonpolar based on physicochemical properties, and the final helical wheel was annotated to visualize residue segregation and the direction of the hydrophobic moment (µH). The secondary structure was further analysed using circular dichroism (CD) spectroscopy, CD spectra were recorded at 25 °C using a J-810 spectropolarimeter (Jasco, Japan). Measurements were performed using a quartz cuvette with a path length of 1.0 mm over a wavelength range of 190–260 nm, with a scanning speed of 100 nm min⁻¹, bandwidth of 1 nm, and response time of 1 s. Peptide samples were prepared at a final concentration of 67 µM (0.1 mg) in different environments to mimic physiological conditions including deionized water, 50% trifluoroethanol (TFE) and 10 mM sodium dodecyl sulfate (SDS) micelles.

2.4. Determination of antibacterial and anti-biofilm activity of pepholin

In vitro antibacterial activity of the novel synthetic peptide was evaluated against WHO priority pathogens such as ESKAPE group of bacteria and clinical MDR Pseudomonas isolates. The genotypic and phenotypic characterisation of the isolates were done using Polymerase chain reaction (PCR) and Antibiotic sensitivity test (ABST) respectively. Details of characterisation and antibiotic resistance profiling are provided in the SI (Fig. S3 and S4). Minimum inhibitory concentration (MIC) was determined using the standard broth dilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines (2025). Minimum bactericidal concentration (MBC) was determined through the spread plating method, as described by ref. 25. Briefly, bacteria were inoculated in MHB and subsequently sub cultured until they reached the logarithmic growth phase. Bacteria at a concentration of 1 × 10⁶ CFUs per mL were incubated with peptides at final concentrations ranging from 15 µg mL⁻¹ to 400 µg mL⁻¹, then incubated at 37 °C for 18 hours in a humidified chamber and the plate was measured by microplate reader at OD₆₀₀. The MBC against P. aeruginosa (ATCC 27853) was determined by plating 10 µL of the incubated suspension on MHA for 18 hours.

The kill kinetics assay against P. aeruginosa (ATCC 27853) was conducted following the procedure described by ref. 26. Briefly, an overnight bacterial culture grown to mid-log phase and diluted to 1 × 10⁶ cells per ml. Peptide was then added to this bacterial suspension at final concentrations of 1×, 3×, and 6× MIC. Untreated bacteria was used as the negative control and gentamicin was used at 2 µg mL⁻¹ as a standard antibiotic control under identical experimental conditions. The samples were incubated at 37 °C for 2 hours, with aliquots taken at 15, 30, 60, 90, and 120 minutes intervals. These aliquots were serially diluted tenfold, and 50 µl of each dilution was plated onto MHA plates. After overnight incubation at 37 °C, the number of viable colonies was counted. The results were compared across time points to determine the kill kinetics of Pepholin at different concentrations. All experiments were performed in triplicate, with the results expressed as mean CFU per mL ± SD.

Anti-biofilm efficacy of peptide was assessed based on the protocol described by ref. 27 with minor adjustments. Briefly, 200 µl of P. aeruginosa (10⁶ CFU per mL) was cultured in 2% glucose-supplemented TSB in a 96-well microtiter plate at 37 °C for 48 hours to form mature biofilms. Wells were washed twice with PBS to remove planktonic bacteria. Then, 200 µl of fresh MHB with different MIC folds were added to each well in triplicate, followed by overnight incubation at 37 °C. Biofilms were fixed with 200 µl methanol for 20 min, then stained with 200 µl 0.2% crystal violet for 10 min. Bound crystal violet was dissolved in 33% acetic acid, and OD₅₇₀ nm was measured using a microtiter plate reader. Biofilm mass percentage was calculated by comparing to control wells treated with sterile MHB.

2.5. Outer and inner membrane permeability assays

The permeability of the outer membrane was analysed using the fluorescent probe 1 N-phenylnapthylamine (NPN) uptake assay.²⁸ Briefly, P. aeruginosa (ATCC 27853) cells were incubated to mid-log phase in MHB at 37 °C, harvested by centrifugation at 4000g for 10 min, washed thrice, and diluted to 10⁶ CFU per mL with 5 mM HEPES buffer (pH 7.2) containing 5 mM glucose. Then, the cells were mixed with 20 µM NPN, and treated with of 0.5×, 1×, 2×, 4× MIC concentration of peptide. 0.5% v/v Triton-X was be used as positive control and HEPES buffer taken as buffer control. NPN fluorescence was measured at excitation and emission wavelengths of 350 and 420 nm, respectively in fluorescence spectrometer (PerkinElmer, USA) for every 5 minutes for 2 hours. The permeability of the inner membrane was analysed using the SYTOX™ Green uptake assay.¹⁷ Briefly, overnight grown P. aeruginosa (ATCC 27853) cells were, washed thrice, and diluted to 10⁶ CFU per mL with phosphate buffered saline (PBS) (pH 7.4) buffer. Then, the cells were mixed with 500 nM Sytox green, and treated with peptides of final concentration like 0.5×, 1×, 2×, 4× MIC. 0.5% v/v Triton-X was included as positive control and PBS taken as buffer control. For every 10 minutes for 3 hours the fluorescence is measured at an excitation wavelength of 480 nm and an emission wavelength of 522 nm. Data is represented as normalised relative fluorescence intensity (RFU) with respect to initial (0 min) readings. The increased fluorescence indicates the disintegration of the bacterial membrane.

2.6. Flow cytometry

Flow cytometry was used to quantitatively assess peptide-induced bacterial membrane permeability over time, as described by ref. 29 with slight modifications. P. aeruginosa (ATCC 27853) were grown to the log phase, harvested, and subsequently washed thrice with PBS and diluted to 10⁶ CFU per mL in the same buffer. The bacterial suspension was incubated with the peptide (1×, 5× and 10× MIC) and PBS (negative control). Samples were incubated 37 °C for 15 min, 30 min, 60 min, 90 min, 120 min, 150 min and 180 min. Following incubation, Propidium iodide (PI) dye (0.1 mg mL⁻¹) was added and incubated at 37 °C for 15 minutes. Subsequently, the mixture was centrifuged, washed to remove unbound dye, and finally suspended in PBS. A CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) was used to obtain the data with a PI fluorescence using a 660 nm longpass filter.

2.7. Toxicity studies of peptide

2.7.1. Haemolytic assay

The peptide's hemolytic activity against erythrocytes was assessed by optical absorption assay as per ref. 30. Briefly, fresh blood from various species (Rabbit, Mice, Cattle, Buffalo, Sheep, Dog, and Human) were treated with alsever's solution (pH 6.1), washed thrice, and then centrifuged at 1000 g for 5 minutes. The red blood cells (RBCs) were suspended in alsever's solution to achieve a 4% (v/v) dilution. Subsequently, these RBCs were exposed to serially diluted peptide for 1 hour at 37 °C, with peptide concentrations up to 10× MIC. Erythrocytes were then centrifuged at 1000 g for 5 min at 4 °C. Supernatant was transferred to a new 96-well microtiter plate and haemoglobin release was measured by OD₅₇₆ nm. Untreated RBC suspension used as negative control, and RBCs treated with 0.1% Triton X-100 as positive control. All experiments were performed in triplicate and percent hemolysis was calculated using the following equation:% Haemolysis = [(OD₅₇₆ sample − OD₅₇₆ blank)/(OD₅₇₆ Triton − OD₅₇₆ blank)] × 100

2.7.2. Cytotoxicity assay

The cytotoxicity of the peptide towards VERO cells was estimated by MTT 3-(4,5-dimethylthiazol-2-yl)-(2,5-diphenyltetrazolium bromide) assay following.³¹ Briefly, 1 × 10³ cells per well in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum were seeded into 96-well plates and incubated at 37 °C in 5% CO₂ for 24 hours, peptide at varying concentrations were added, with untreated cells as controls. After 24 hours incubation, MTT (50 µL, 5 mg ml⁻¹) was added to each well and kept for incubation for 4 hours at 37 °C, then mixtures were centrifuged at 1000 g for 5 minutes. Supernatants were removed, and formazan crystals were dissolved by adding 150 µl of dimethyl sulfoxide (DMSO). OD was then measured at 570 nm using a microplate reader. Cytotoxicity as a percentage was determined using the formula, % cytotoxicity = (100 − percent viability).

To further evaluate the cytocompatibility of Pepholin, VERO cells and Bubaline Mammary Epithelial (BuMEC) cells were treated with Pepholin and analysed by PI-based flow cytometry using CytoFLEX flow cytometer.³² The percentage of PI-positive cells were quantified as an indicator of membrane integrity. Detailed experimental procedures are provided in the SI file.

2.7.3. In vivo toxicity studies in Galleria mellonella larvae

G. mellonella larvae were considered to be widely proven in vivo model to assess efficacy and toxicity of novel antimicrobial agents. The subsequent methodology was employed by ref. 33. In brief, healthy larvae of pre-pupa stage (250–300 mg) were selected and grouped for the experiment (n = 6). Selected larvae were starved and stored at 37 °C for 24 h in dark. Each larvae received 10 µl of peptide at final concentration of 5×, 10×, 15×, and 20× MIC using a hamilton's syringe. 10 µl of PBS and 50% Triton-X 100 were injected in the negative and positive control group, respectively. Followed by larvae were incubated in the dark at 37 °C and observed for larval mortality for 5 days. Toxicity was determined by mortality, identified by melanisation of larval cuticle.

2.7.4. Selectivity index

The half-maximal cytotoxic concentration (CC₅₀) and half-maximal hemolytic concentration (HC₅₀) were estimated from cytotoxicity and hemolysis assays, respectively. The selectivity index was calculated using the following equations:SI_CC50 = CC₅₀/MICSIH_C50 = HC₅₀/MICwhere MIC represents the minimum inhibitory concentration of the peptide against P. aeruginosa ATCC 27853.³⁴

2.8. Stability studies of peptide

The stability of Pepholin under physiologically relevant conditions was evaluated by assessing its antibacterial activity in the presence of salts, serum, and different temperature treatments using modified broth microdilution protocols. Salt sensitivity of the peptide was evaluated using a modified method as described by ref. 29. P. aeruginosa (ATCC 27853) at 10⁶ CFU per mL were treated with physiological concentrations of serum salts (150 mM NaCl, 4.5 mM KCl, 6 mM NH₄Cl, 8 mM ZnCl₂, 1 mM MgCl₂, and 4 mM FeCl₃). Subsequent procedures followed MIC determination protocol. The serum sensitivity of the peptides was assessed using a previously described method by ref. 29. P. aeruginosa (ATCC 27853) at 10⁶ CFU per mL was incubated in the presence of final concentrations of 10% and 20% serum obtained from bovine calf, followed by standard MIC determination procedures. The robustness of peptide against different temperature conditions was explored, employing the procedural modifications outlined by ref. 35. Briefly, peptide at concentrations of 1×, 2×, and 4× MIC were incubated at various temperatures (−80 °C, −20 °C, 4 °C, 37 °C, 50 °C, and 100 °C) for an hour. P. aeruginosa (ATCC 27853) at 10⁶ CFU per mL was then added and incubated overnight at 37 °C. Subsequent steps followed MIC determination protocols.

2.9. Preparation and characterisation of peptide-carbomer hydrogel

Carbopol® 940 polymer (pioneer chemical co), triethanolamine (Rankem, Avantor performance material India ltd) and aloe vera were used to prepare the hydrogel. Different concentrations of carbomer and triethanolamine were tested to optimize the hydrogel for good releasing ability, consistency, and application suitability. After the optimisation of hydrogel, peptide was incorporated to the carbomer hydrogel. Briefly, peptide was added to the 10 ml of sterile distilled water to make the final concentration of 10× MIC (1 mg ml⁻¹) and 0.5% carbomer, 2% aloe vera were added to the solution and kept for continuous stirring on magnetic stirrer till the carbomer completely dissolved. Following that 0.05% triethanolamine was added and the solution was kept for gel formation in room temperature.

The inverting tube method was used to determine the gelation time of optimised carbomer hydrogel.³⁶ Gelation time was determined by observing visible gel formation by inverting the tubes at 5 minutes intervals. In vitro drug releasing abilities of the hydrogel was assessed by using congo red and methylene blue dyes following previously described by ref. 37 with minor adjustments. Briefly, dye concentrations of 1 mg ml⁻¹ were combined with carbomer solution. The amount of dye released after gel formation was determined by adding 200 µl of PBS (pH 7.4) and incubated at 37 °C without stirring. At regular 30 minutes interval, 200 µl of PBS was drawn out and replaced with fresh PBS. The absorbance of the samples was measured at 660 nm for methylene blue dye and 490 nm for congo red dye using a microplate reader. Percentage of dye release was calculated according to the standard curve of each dye. To determine the antibacterial activity of hydrogel, 200 µl of hydrogel containing peptide were prepared and tested using the spot-on-the-lawn method. 100 µl of the potent MDR P. aeruginosa isolate's overnight culture was spreaded on an MHA plate, and 50 µl of plain carbomer hydrogel, peptide incorporated hydrogel, aloe vera and sterile disc was spotted on the lawn and incubated at 37 °C for overnight.

2.10. In vivo evaluation of peptide's therapeutic potential

2.10.1. Bacterial strain

A clinically characterised MDR P. aeruginosa-1 strain was used for burn wound infection studies. Briefly, the bacterial cells were cultured in MHB at 37 °C for the 16–18 h at 160 rpm in a shaker incubator. Centrifuged at 8000 rpm for 5 minutes, washed twice with sterile PBS. Cells were diluted to 1 × 10⁵ cells per 100 µl using OD₆₀₀ nm and used for wound infection.

2.10.2. Experimental animal

Animal experiments were performed in accordance with approval from the Institute Animal Ethics Committee, India. Swiss Albino mice, aged 4–6 weeks and weighing 20–25 g, were housed in clean polycarbonate cages under a 12 hours day/night cycle at a temperature of 30 ± 2 °C. Standard mice food was provided, and water was accessible ad libitum throughout the duration of the experiment. The mice were acclimated for one week before the study, after which 75 mice were randomly divided to five experimental groups (n = 15 per group), each receiving a different treatment formulation as described in Table 3. Burn wound induction and infection were performed following a protocol as described by ref. 26 with minor modifications. Briefly, aseptic preparation of the dorsum of each mouse was done by shaving and cleaning with antiseptic solution. Xylazine (Xylaxin®) and ketamine (Ketamin®) of dose 10–12.5 mg kg⁻¹ and 80–100 mg kg⁻¹ respectively were used to anaesthetize the mice and custom-made copper rod which is heated to 100 °C for 10 min were used to create 1 cm × 1 cm burn wound. The heated rod was applied with no pressure on the skin for 10 s to create the burn wound. The burn wound will be infected with 100 µl (1 × 10⁵ CFU) of MDR P. aeruginosa bacteria while the animal is under anaesthesia. After 3 hours of bacterial injection, 100 µg different treatments (100 µl PBS for control) were administered topically twice daily for 5 days. Each mouse was housed individually with separate food and water tanks to prevent skin contact with other mice. Three mice per group were sacrificed, and tissues were collected on days 1, 3, 7, 14, and 21 post-treatments to evaluate treatment efficacy.

Table 3. Experimental design and respective treatments.

Animal groups	Number of animals	Burn wound	Bacterial infection	Treatments
I	15	+	—	Sterile PBS (pH 7.4)
II	15	+	+	Sterile PBS (pH 7.4)
III	15	+	+	Carbomer hydrogel
IV	15	+	+	Pepholin hydrogel
V	15	+	+	Antibiotic (sulfadiazine)

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2.10.3. Wound size reduction (%)

Wound progression was documented through photographs and wound margins were traced on transparent sheets at intervals of 1st, 3rd, 7th, 14th, and 21st day of the experiment, then the wound sizes were measured in millimeter scale using ImageJ software.³⁸% Wound contraction = (initial wound size − wound size on specific day/initial wound size) × 100

2.10.4. In vivo antimicrobial efficacy of peptide against MDR P. aeruginosa

Mice were sacrificed at specified intervals (days 1, 3, 7, 14, and 21) post-burn and burn wound areas were aseptically excised, washed, and homogenized in sterile PBS (pH 7.4). Tissue weight was measured, and PBS was added accordingly to achieve a uniform concentration (1 mg ml⁻¹). Then PBS was serially diluted up to 10⁴ and 10⁵ times and 100 µl of diluted samples were plated onto PIA plates using sterile glass beads and incubated at 37 °C for 12–14 hours. Bacterial colonies were counted the following day and the experiment was performed in duplicate. CFU per gm = (Number of colonies × dilution factor)/(volume of sample pipetted on to plate (ml) × tissue weight (gm).

2.10.5. Quantitation of cytokines expression using qRT-PCR

The gene expression of pro-inflammatory cytokines and anti-inflammatory cytokines were evaluated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Skin tissues that were collected from mice on day 3 and day 7 were preserved in RNAlater® (Ambion, USA) and total RNA was extracted using TRIzol™ (Invitrogen™, ThermoFisher Scientific, Carlsbad, CA, USA) according to the manufacturer's protocol. RNA concentration and purity were analysed by spectrophotometry (Bio spectrometer, Eppendorf, Germany). Complementary DNA (cDNA) synthesis was performed by Prime Script reagent kit (Takara Bio, USA) following the manufacturer's instructions. The cDNA was subjected to amplification of the target genes, namely, Tumour necrosis factor (TNF)-α, Interleukin (IL)-1β, Epidermal Growth Factor (EGF), and Transforming Growth Factor (TGF)-β using gene-specific primers with glyceraldehyde-3 phosphate dehydrogenase (GAPDH) gene as an endogenous control. The qRT-PCR was performed using TB Green® Premix Ex Taq™ II kit as per manufacturer protocol (Takara Bio, Japan). All reactions were performed in triplicate on 7500 RT-PCR system (Applied Biosystems, Stockholm, Sweden). Results were expressed as cycle threshold (CT) and relative expression of target genes was computed using a comparative delta cycle threshold (CT) method, and the relative fold change values were derived from average normalized CT values (delta CT).

2.10.6. Histomorphologic analysis

The skin tissue was fixed in 10% neutral buffer formalin. After fixation, tissues were sliced into 2–3 mm sections, dehydrated with ascending grades of ethanol, and embedded in paraffin. Tissues sections were cut into 4µ sections and stained with haematoxylin and eosin (H & E) stain as per standard procedure. The grading of histopathological lesions was done based on the following parameters including necrosis, infiltration of inflammatory cells, regenerative process, and collagen deposition. The lesions were graded on a 0 to 3 scale: 0: absent, 1: mild, 2: moderate, and 3: severe across different layers of skin like epidermis, dermis, hypodermis, panniculus carnosus, and adventitia in mice.

Masson's trichrome staining was done to assess the collagen deposition in infected burn wounds according to the standard procedure. Grading of collagen deposition was done on a – to +++ scale: −: absent, +: mild, ++: moderate, and +++: severe collagen deposition across different layers of skin.

2.11. Statistical analysis

Quantitative data presented as mean ± standard deviation (SD) was analysed using GraphPad Prism 7.0 software (GraphPad Software, CA, USA). One-way ANOVA, unpaired t test, multiple comparison test determined intergroup differences and significance levels were denoted as ^nsp > 0.05, *p < 0.05; **p < 0.01; and ***p < 0.001.

3. Results and discussion

3.1. Design, synthesis, and characterization of peptide

Traditional AMPs discovery relies primarily on rational design based on physicochemical characters or extensive high throughput experimental screening. While rational design offers interpretability, it is often limited by heuristic assumptions and may overlook sequence activity relationships. Conversely, high throughput screening enables unbiased discovery but is resource intensive and experimentally demanding.^39–41 In the present study, novel peptide sequence was predicted from the holin protein of the Pseudomonas phage Iggy, by AI-assisted peptide prediction web servers to complement biological rationale by enabling rapid prioritization of peptide candidates based on learned sequence and activity patterns derived from experimentally validated datasets. Unlike purely rational design, this approach allows identification of candidate sequences that satisfy antibacterial activity likelihood while retaining biologically relevant features.^42,43 This integrated strategy combines the efficiency of AI-based prioritization with the mechanistic interpretability of rational design, while avoiding the experimental burden of large-scale high-throughput screening.

Holins are bacteriophage encoded membrane proteins that play a central role in host cell lysis by forming pores in bacterial membranes, making them attractive templates for the development of membrane active AMPs.^44,45 Sequence alignment analysis indicates that Pepholin corresponds to a contiguous C-terminal segment of the parent holin protein with complete sequence identity. Phobius topology prediction identified multiple transmembrane helices within the holin protein, including a transmembrane region spanning residues 76–98 followed by a cytoplasmic domain (99–112) (SI Fig. S1). The Pepholin sequence maps to residues 95–106, partially overlapping the C-terminal end of the transmembrane helix and extending into the adjacent cytoplasmic region. This region is enriched in hydrophobic and cationic residues, indicating retention of a membrane associated motif relevant to holin mediated lytic activity. Holins mediate bacterial cell lysis through membrane associated domains that destabilize the cytoplasmic membrane rather than through enzymatic activity.⁴⁴ Preservation of a hydrophobic cationic residue pattern characteristic of holin lytic segments provides a mechanistic basis for the observed membrane disruptive antibacterial activity of Pepholin and supports the biological validity of its designation as a holin derived peptide.^45,46

The complete amino acid sequence of Pepholin, along with its predicted physicochemical characteristics, is presented in Table 1. Pepholin has a molecular weight of 1472.8 g mol⁻¹, a net positive charge of +5, hydrophobicity of 42%. These parameters fall within the optimal range reported for biologically active cationic AMPs, where a net charge between +4 and +6 is considered critical for efficient electrostatic interaction with negatively charged bacterial membranes while limiting nonspecific cytotoxicity.^47,48 In addition, positively charged residues and overall charge density are known to be key determinants of antibacterial potency in membrane-active peptides.^29,39 The positive net charge of Pepholin is expected to promote preferential interaction with bacterial membranes, which are enriched in anionic phospholipids. In contrast, eukaryotic cell membranes are predominantly composed of zwitterionic lipids and contain sterols that enhance membrane stability, thereby reducing susceptibility to cationic peptides.⁴⁹ However, it is important to note that membrane-active AMPs are not absolutely selective, and their activity is primarily governed by electrostatic interactions and membrane composition rather than specific receptor binding. This inherent limitation is common to most cationic AMPs and necessitates careful evaluation of cytotoxicity across different biological systems.⁵⁰

Table 1. Physiochemical parameters of Pepholin.

S. No.	Parameter	Value
1	Amino acid sequence	NH₂-KVVYKRLGIIRK-CONH₂
2	Molecular formula	C69H125N21O14
3	Length	12
4	Molecular weight (g mol⁻¹)	1472.86
5	Net charge (pH 7)	+5
6	Hydrophobic ratio	42%
7	Hydrophobic moment (µH)	0.442
8	GRAVY score	−0.10
9	Amphipathicity	Yes
10	Iso-electric point	11.52
11	Extinction coefficient	1280 M cm⁻¹
12	Estimated solubility	Good water solubility

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Pepholin was synthesized by solid-phase peptide synthesis and RP-HPLC analysis at 220 nm showed a single dominant peak at a retention time of 36.82, indicating chromatographic homogeneity and purity exceeding 90% (Fig. 1), confirming its suitability for subsequent biological evaluation. The retention time, detailed gradient program, and individual peak area percentages are provided in the SI (Fig. S1 and Table S1). Overall, the design of Pepholin integrates holin-derived membrane activity, optimal charge-hydrophobic balance, and amphipathic features that are consistently associated with effective AMPs while maintaining a short sequence length that may offer advantages in synthesis, formulation, and translational development.

3.2. Structural characterization of Pepholin reveals a membrane-induced amphipathic α-helix

Secondary structure prediction and helical wheel projection demonstrated that Pepholin adopts an amphipathic α-helical conformation, with spatial segregation of hydrophobic residues on one face and positively charged residues on the opposite face (Fig. 1). Amphipathicity is a defining structural feature of most membrane-active AMPs, as it governs peptide partitioning into lipid bilayers, membrane insertion, and subsequent destabilization. The annotated helical wheel projection of the present peptide demonstrates a well-defined amphipathic α-helix, with cationic residues oriented to facilitate electrostatic attraction to bacterial membranes and hydrophobic residues positioned to promote membrane insertion and disruption,^40,51 the strong concordance between predicted amphipathic architecture and experimental functional outcomes supports the biological relevance of the proposed structure.⁵² Similar amphipathic architectures have been reported for several potent anti-Pseudomonas peptides, including synthetic and phage-derived AMPs that act via rapid membrane permeabilization rather than intracellular targeting.⁵³

To experimentally validate the predicted secondary structure, the conformational behavior of Pepholin was analyzed using CD spectroscopy under aqueous and membrane-mimicking conditions. Quantitative estimation was performed using the BeStSel deconvolution algorithm. In water, the spectra of the new peptides were characteristic of unordered conformations. However, a clear structural transition was observed when the peptide was analyzed in membrane-mimicking environments (Fig. 1). In the presence of 10 µM SDS micelles, which mimic the negatively charged surface of bacterial membranes, the CD spectrum displayed the typical α-helical signature characterized by two negative minima around 208 and 222 nm and a strong positive band near 190 nm. Quantitative deconvolution of the spectrum revealed that the peptide adopts a highly α-helical conformation under these conditions, with approximately 80.2% helicity, indicating strong helix induction upon interaction with membrane-like environments. A comparable, although slightly lower, helical content was observed in the presence of 50% trifluoroethanol (TFE), a solvent commonly used to stabilize intramolecular hydrogen bonding and promote α-helix formation in peptides. Under these conditions, Pepholin exhibited approximately 75.4% α-helical structure, further confirming the inherent helical propensity of the peptide.

The environment dependent structural transition, from a relatively disordered conformation in aqueous solution to a highly α-helical structure in membrane-mimicking environments strongly supports the predicted amphipathic architecture of Pepholin. Such conformational adaptability is a hallmark of membrane-active AMPs, which often remain disordered in aqueous environments but adopt amphipathic α-helical structures upon interaction with microbial membranes, thereby enhancing membrane binding and disruption.^53–55 In this conformation, hydrophobic residues orient toward the lipid acyl chains, whereas cationic residues interact electrostatically with negatively charged phospholipid head groups, ultimately leading to membrane destabilization through mechanisms such as pore formation or carpet-like disruption. This structural behavior is consistent with the observed antibacterial activity against Gram-negative pathogens and supports a mechanism of action primarily based on membrane disruption rather than intracellular enzymatic inhibition.^40,51

3.3. Pepholin shows pronounced antibacterial and antibiofilm effect against P. aeruginosa in vitro

The antibacterial activity of Pepholin was initially evaluated against the ESKAPE group of bacteria. MIC values ranged from 100–400 µg mL⁻¹ (Table 2), indicating that the peptide retains activity against multiple clinically relevant Gram-negative pathogens. Although the spectrum was not uniformly broad, such variability in MIC values is commonly reported for short cationic AMPs and reflects pathogen specific differences in outer membrane composition, surface charge density, and extracellular polymeric substances.^40,52 Among these, Pepholin showed effective antibacterial activity against P. aeruginosa, with an MIC of 100 µg mL⁻¹ and an MBC of 300 µg mL⁻¹, reflecting bactericidal activity at three times the inhibitory concentration. To further evaluate the clinical relevance of the peptide, its antibacterial activity was tested against multiple MDR P. aeruginosa clinical isolates. Pepholin retained antibacterial activity against these isolates, with MIC values ranging from 100–200 µg mL⁻¹ (Table 2). The comparable MIC values observed between the reference strain and MDR isolates suggest that the peptide maintains activity against antibiotic-resistant P. aeruginosa strains. Although the MIC values of Pepholin were observed to be relatively higher (>100 µg mL⁻¹) compared with conventional antibiotics, such activity profiles are not uncommon among AMPs derived from natural proteins. Several studies have reported that host-derived or phage-derived antimicrobial peptides often exhibit moderate MIC values while retaining significant biological functionality, including membrane disruption, antibiofilm activity, and therapeutic efficacy in infection models.^41,56

Table 2. Minimum inhibitory concentration (MIC) of Pepholin against ESKAPE pathogens and clinical MDR P. aeruginosa isolates.

Isolate	MIC (µg mL⁻¹)
Pseudomonas aeruginosa (ATCC 27853)	100
MDR P. aeruginosa-1	150
MDR P. aeruginosa-2	100
MDR P. aeruginosa-3	180
MDR P. aeruginosa-4	150
MDR P. aeruginosa-5	200
MDR P. aeruginosa-6	150
MDR P. aeruginosa-7	100
Enterobacter cloacae (ATCC 13047)	>200
Staphylococcus aureus (ATCC 29213)	200
Klebsiella pneumoniae (ATCC 13883)	150
Acinetobacter baumannii (ATCC 19606)	>200
Escherichia coli (ATCC 25922)	100
Enterococcus faecalis (ATCC 29212)	200

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Pepholin also demonstrated rapid bactericidal kinetics against P. aeruginosa (ATCC 27853) as shown in Fig. 2. At 3× MIC concentration, Pepholin was able to completely eliminate all bacteria within 90 minutes. Additionally, Pepholin exhibited dose dependent bactericidal kinetics, as increasing the concentration from 3× to 6× MIC shortened the killing time to 60 min, comparable to the bactericidal kinetics of gentamicin (2 µg mL⁻¹), which taken as the antibiotic control. This concentration dependent killing pattern is characteristic of membrane disruptive peptides and contrasts with the slower, growth dependent killing typically observed with conventional antibiotics. This highlights the peptide's strong antibacterial effectiveness, considering past findings on P. aeruginosa's protective mechanisms against AMPs through capsular polysaccharides and alginate secretion.²⁷ Based on these observations, subsequent experiments focused on further evaluating the activity of Pepholin against P. aeruginosa, rather than positioning it as a universally broad-spectrum agent. P. aeruginosa can produce dense, persistent biofilms which pose a major challenge for antimicrobial treatments, contributing to over 90% of chronic wound infections and hindering wound healing.⁵⁷ Given the substantial role of Pseudomonas biofilms in fostering recurrent infections, we explored the efficacy of novel peptide in disrupting mature P. aeruginosa biofilms. Peptide concentrations of 8× and 4× MIC, effectively eradicated 24 hours mature P. aeruginosa biofilms, leading to a substantial reduction of 85% and 80% in biofilm mass respectively and it was observed that concentrations of 2× and 1× MIC resulted in a decrease in biofilm mass by 55% and 20%, respectively (Fig. 2). This suggests that biofilms may adsorb peptides, impeding their access to bacteria, necessitating higher peptide concentrations to achieve biofilm disruption.⁵⁸ Pepholin exhibited relatively modest activity against certain Gram-negative bacteria such as A. baumannii and E. faecium when compared with traditional antibiotics and some hybrid AMPs, suggesting that membrane composition and surface charge variation significantly influence its spectrum of activity. Nevertheless, these findings highlight the potential of AMPs as effective alternatives to traditional antibiotics against resistant biofilm-forming bacteria.

3.4. Pepholin disrupts bacterial membrane integrity

The primary mechanism of action for AMPs is altering the bacterial membrane, particularly in Gram-negative bacteria where the outer membrane acts as a protective barrier.⁵⁹ This was confirmed by evaluating the permeability of the outer membrane of P. aeruginosa using NPN dye. NPN dye, a hydrophobic fluorescent probe, undergoes fluorescence quenching in aqueous conditions but exhibits intense fluorescence in hydrophobic environments. As shown in Fig. 3A, Pepholin rapidly permeabilized the outer membranes of bacteria at concentrations ranging from 50 µg mL⁻¹ to 400 µg mL⁻¹. Treatment with 2× and 4× MIC of Pepholin resulted in increased membrane permeability within 20 min, with 50% dye uptake observed after 90 min. These findings indicate Pepholin effectively compromises the integrity of the outer membrane, thereby facilitating access to deeper membrane layers.

To determine Pepholin effects on bacterial inner membrane permeability, SYTOX green dye was used, which selectively enters cells with damaged inner membranes and binds to nucleic acids. Pepholin of concentrations 4× and 2× MIC exhibited much permeability to dye and fluorescence uptake was evident within 10 minutes, signifying a rapid disruption of the cell membrane. At the 4× concentration, the fluorescence intensity was highest, demonstrating approximately twofold increase compared to the positive control, 1% Triton X within an hour (Fig. 3B). Pepholin maintained clear inner membrane permeabilization even at 1× and 0.5× MIC also. When compared with other short cationic peptides evaluated against MDR Gram-negative bacteria, the membrane disruptive behaviour of Pepholin is broadly consistent with previously reported findings. For example, a peptide tested against MDR P. aeruginosa and A. baumannii was shown to induce membrane damage, although higher concentrations were required to achieve comparable bactericidal effects, particularly against biofilm embedded cells.⁶⁰ In this context, Pepholin demonstrates the advantage of inducing rapid membrane permeabilization in planktonic P. aeruginosa at relatively low multiples of MIC, which aligns with its rapid killing kinetics observed in time kill assay. Moreover, while Triton X-100 produced lower fluorescence signals in bacterial membrane assays, this does not imply superior lytic activity of Pepholin toward eukaryotic membranes. Triton X-100 acts through nonspecific lipid solubilization, whereas Pepholin interacts preferentially with anionic bacterial membranes via electrostatic attraction and structured insertion.^49,51 Overall, the membrane permeabilization data support a mechanism whereby Pepholin initially binds to bacterial surfaces via electrostatic interactions, targeting lipopolysaccharide or teichoic acids. Then, hydrophobic residues penetrate the membrane, causing disruption through permeabilization, depolarization, or surface insertion. This leads to membrane thinning, integrity disruption, and cytoplasmic outflow, ultimately causing bacterial death.⁵⁹

3.5. Flow cytometric analysis confirms membrane damage induced by Pepholin

To quantitatively evaluate membrane damage induced by Pepholin, flow cytometric analysis using PI staining was performed at various time points to quantitatively assess the bacterial membrane integrity. PI is a membrane impermeable nucleic acid dye that enters only cells with compromised cytoplasmic membranes and is widely used as an indicator of irreversible membrane damage and cell death.⁶¹ The untreated control showed a 2.2% PI fluorescence at 0 minutes of incubation, reaching a maximum of 7% over 3 hours, indicating the presence of viable cell membranes. At 10× MIC and 6× MIC, PI fluorescence exceeded 90% and 70%, respectively, within 15 minutes. Across all intervals, PI uptake was in a concentration-dependent manner, exhibiting intensified fluorescence at higher peptide concentrations and prolonged incubation of peptide with bacteria led to a progressive increase in membrane disruption (Fig. 4). These findings substantiate the time-dependent and concentration-dependent capability of the peptide to disrupt bacterial membranes. Similar time and concentration dependent PI uptake profiles have been reported for AMP, acting primarily through membrane targeting mechanisms, where rapid PI entry at higher concentrations is associated with efficient killing, while delayed uptake near MIC reflects partial membrane destabilization.^62,63 These results further support the membrane disruptive mechanism suggested by NPN and SYTOX permeability assays.

Antimicrobial mechanism of the novel peptide involves membrane interference at lower concentrations and membrane destruction at higher concentrations, consistent with the carpet model of AMP mechanisms, in which amphipathic peptides accumulate on the bacterial surface and, upon reaching a threshold concentration, cause extensive membrane disintegration.^63–65 An advantage of Pepholin highlighted by this analysis is its ability to induce rapid and extensive membrane damage in P. aeruginosa at higher multiples of MIC, in agreement with its rapid killing kinetics. However, flow cytometry primarily reports membrane integrity and does not directly assess metabolic activity or intracellular targets. Complementary assays examining cellular respiration or ATP depletion would further strengthen the mechanistic conclusions. Nevertheless, the present data clearly demonstrate that Pepholin induced membrane disruption progresses to bacterial death in a concentration and time dependent manner.

3.6. Pepholin exhibits low haemolytic and cytotoxic effects

To be developed as effective therapeutic agents, AMPs must demonstrate non-toxic properties. Hemolysis and cytotoxicity are widely acknowledged as significant hurdles in AMP development and serve as key parameters for assessing their safety.⁶⁶ The haemolytic activity of the Pepholin was assessed using RBCs from seven different species at higher concentrations like 4×, 6×, 8× and 10× MIC. The Pepholin showed negligible or no haemolytic activity (<3%) across all concentrations, including the highest concentration of 10× MIC where haemolysis observed as 2.4%, 1.3%, 4.2%, 2.6%, 1.73%, 1.04%, and 1.4% in sheep, dogs, mice, rabbits, humans, buffalo, and cattle red blood cells (RBC), respectively (Fig. 5A and S5). The haemolysis assay serves as a reliable approach to explore the potential of an AMP to disrupt mammalian membranes and its biocompatibility. Similar low hemolytic profiles have been reported for AMPs with optimized charge, hydrophobic balance, whereas peptides with excessive hydrophobicity often exhibit pronounced hemolysis even near MIC levels.^67,68In vitro cytotoxicity of Pepholin towards VERO cells was evaluated by MTT reduction assay, an approach widely employed in AMP studies for preliminary safety evaluation.^67,69 As shown in Fig. 5B, there was no cytotoxicity was observed in 1× MIC, while peptide at 2× MIC and 4× MIC concentration demonstrated 7% and 15% of cytotoxicity, respectively. The highest level of cytotoxicity observed was 20% at an 8× MIC concentration, which still remains significantly lower compared to other synthetic AMPs, which frequently exceed 30–50% cytotoxicity at comparable multiples of MIC.^70,71 The additional PI-based flow cytometric analysis further confirmed the low cytotoxic potential of Pepholin towards mammalian cells by demonstrating minimal membrane damage with PI-positive cells remaining below 7% at 4× MIC in both VERO and BuMEC cell lines (Fig. S7).

Recent studies have highlighted that strong membrane active AMPs often lack sufficient selectivity between bacterial and mammalian membranes, resulting in elevated cytotoxicity that limits their translational potential.^67,69 In contrast, the favourable cytocompatibility profile of Pepholin suggests an improved balance between antibacterial potency and host cell selectivity. We also acknowledge that the future studies incorporating skin relevant cell lines such as human keratinocytes (HaCaT) and fibroblasts would further strengthen translational relevance.^31,72 To complement in vitro toxicity assessments, the in vivo toxicity of Pepholin was evaluated using the Galleria mellonella larvae model. Pepholin exhibited no toxicity towards G. mellonella larvae, even at the very high concentration of peptide (20× MIC). There was no mortality reported in all the tested groups except in positive control Triton-X (50%) where 100% mortality was observed (Fig. 6). G. mellonella larvae are a versatile model for toxicity and infection studies, providing a convenient approach to assess in vivo toxicity with outcomes correlating strongly to mammalian systems.⁷³ To further evaluate the selectivity of Pepholin toward bacterial cells, the selectivity index (SI) was calculated. Based on the experimental results, the estimated CC₅₀ value was greater than 800 µg mL⁻¹, while the HC₅₀ exceeded 1000 µg mL⁻¹. Using the MIC value of 100 µg mL⁻¹ against P. aeruginosa, the calculated SI_CC50 and SI_HC50 values were >8 and >10, respectively, indicating favorable selectivity toward bacterial membranes.

Consistent with previous studies, increased hydrophobicity, rather than α-helical propensity, may predominantly drive hemolytic activity and cytotoxicity in AMPs. Therefore, optimizing hydrophobicity is critical for designing synthetic peptides with effective antimicrobial activity while minimizing toxicity to mammalian cells.^67,70,74 Although numerous antimicrobial peptides demonstrate potent antibacterial activity, their clinical translation is frequently limited by hemolysis and mammalian cytotoxicity at therapeutically relevant concentrations. Hybrid peptides derived from cecropin and melittin, while effective against Gram-negative pathogens, have been reported to induce substantial hemolysis exceeding 20–40% at higher MIC multiples, reflecting poor membrane selectivity.⁴⁸ Importantly, previous studies have proposed that AMPs exhibiting less than 5% hemolysis at effective concentrations can be considered highly selective toward bacterial membranes.⁷⁵ The safety profile of Pepholin therefore compares favorably with many reported antimicrobial peptides, indicating an improved selectivity index and supporting its suitability for topical therapeutic applications.

3.7. Pepholin retains antibacterial activity under physiological conditions

The antimicrobial activity of many cationic peptides is strongly influenced by physiological conditions such as ionic strength, serum components, and temperature, which often represent major barriers to their clinical translation.^76,77 Therefore, the stability of the Pepholin under different conditions like salt, serum, and temperature was evaluated by broth microdilution assay according to the MIC concentration of peptide against P. aeruginosa (ATCC 27853). In the presence of monovalent cations NaCl, bacterial cell inhibition was decreased by 23% and 7% at 1× and 2× MIC, respectively. In contrast, KCl and NH₄Cl inhibition decreased by only 3% and 2% at the same concentrations. Divalent cations ZnCl₂ resulted in a 21% and 19% decrease at 1× and 2× MIC, while MgCl₂ showed a decrease of 16% and 13%. Notably, inhibition decreased by 55% at MIC with trivalent cations like FeCl₃, but only by 16% at 2× MIC. As per the proposed mechanism, antimicrobial activity depends on electrostatic interactions between peptides and bacterial membranes. Cationic charge screening attenuates these interactions, hindering peptide binding to bacterial membranes and reducing antimicrobial activity.^59,78 Monovalent cations displayed less antagonistic effects than trivalent cation, suggesting that excessive cations progressively increase the bacterial membrane rigidity via electrostatic interactions with negatively charged phospholipids. This rigidity hindered pore formation and marginally reduced antimicrobial activity.^59,79 Divalent and multivalent cations were observed to influence activity by competing for membrane binding with peptides. Conversely, at lower concentrations, divalent cations facilitated peptide-membrane binding.²⁹ Although divalent and trivalent cations competed with Pepholin for membrane binding, the peptide retained measurable activity across all tested ionic conditions, and modest increases in concentration largely compensated for ion-mediated attenuation. Several AMPs, including LL-37 and other peptides lose most of their activity under physiological salt concentrations, which has been identified as a critical limitation for in vivo application.^41,61 In comparison, Pepholin exhibited comparatively better salt tolerance, representing an advantage for applications in ion-rich environments such as infected wounds.

Serum can hinder the effectiveness of AMPs; this hindrance occurs primarily through proteolytic degradation facilitated by various proteases or by binding to protein and lipid components.⁸⁰ In the presence of 10% serum, Pepholin maintains its antibacterial effectiveness at 0.5×, 1×, 2×, and 4× MIC. However, as the serum concentration increased to 20%, bacterial inhibition of Pepholin at 1× MIC was slightly decreased by 10%, with no changes observed at other peptide concentrations. Serum induced loss of activity is a common and often severe limitation reported for many AMPs, with some peptides showing near to complete inactivation at serum concentrations below 20%.^41,70 We also examined the impact of maintaining a stable secondary structure on the effectiveness of AMPs. Except for a slight decrease in bacterial inhibition at 100 °C, there were no differences in bacterial inhibition across various peptide concentrations at different temperatures. Extreme temperature can disrupt peptide structural stability by affecting bond energy and chemical bonding.⁸¹ This shows the thermal stability of the peptide and its consistent antibacterial effectiveness across different temperature conditions. The observed thermal resilience of Pepholin therefore represents an additional practical advantage. It is notable that many synthetic peptide actions are impeded due to their stability issues, potentially hindering peptides' advancement as viable replacements for antibiotics.⁸² While multivalent cations and higher serum concentrations partially reduce activity an expected limitation shared by most cationic AMPs the overall stability profile of Pepholin compares favourably with many previously reported AMPs. This balance between antimicrobial potency and environmental robustness supports the further development of Pepholin, particularly for localized applications such as infected wound environments. The results of stability studies are presented in Fig. 7.

3.8. In vivo evaluation of peptide's therapeutic potential by formulating in carbomer hydrogel

The limited efficacy of natural AMPs in vivo and their systemic toxicity present significant obstacles to their development as antimicrobial agents. So further in vivo assessment of novel Pepholin is imperative to address these challenges and mouse model of infected burn wounds was chosen for this evaluation. Persistent colonization of burn wounds by P. aeruginosa can cause severe infections, prolonged hospital stays, increased antibiotic use, delayed healing, and higher drug resistance risk. Thus, there is a need to develop effective therapies to combat P. aeruginosa infections, especially in burn wounds.⁸³ Optimized delivery and local application of AMPs is a highly effective approach for wound treatment, resulting in high concentration at the site of action and minimal systemic side effects.^8,18 Natural polymers stand out as preferred biomaterials for dressings, offer biocompatibility, inherent antibacterial properties, degradability, and diverse healing pathways. Hydrogels, especially, excel by boosting stability and prolonging contact time at the wound site when formulated with AMPs.^65,84,85 Carbomer-based hydrogels are widely used for transdermal drug delivery due to their many benefits like high viscosity, tissue compatibility, stable temperature, and compatibility with various medications.⁸⁶

A homogenous hydrogel with optimal consistency was prepared using Carbopol 940 polymer (0.5%) and triethanolamine (0.05%) along with 2% aloe vera to enhance wound-healing properties. Triethanolamine facilitated crosslinking and thickening by neutralizing carbomer's carboxy groups, ensuring polymer stability and many studies have shown that treatment with aloe vera gel and its extracts significantly accelerates wound healing through anti-inflammatory, antioxidant, and fibroblast-stimulating effects.⁸⁷ Gelation was occurred at 1.05 ± 0.30 minutes with a pH of 7.2 ± 0.25. To test the drug releasing ability of carbomer hydrogel, dye release assays were done using methylene blue and congo red dye separately. The initial 30 minutes exhibited the highest dye release, accounting for 40% of methylene blue and 85% of congo red. Subsequently, the complete dyes were released within 5 hours (Fig. 8). Such release kinetics are desirable for infected wounds, as they provide an early high local antimicrobial concentration followed by prolonged exposure.⁸⁸ A zone of inhibition was observed when Pepholin–carbomer hydrogel was spotted onto MDR P. aeruginosa spreaded MHA plate which confirms the antibacterial efficacy of peptide incorporated carbomer hydrogel (Fig. 9). Comparable observations have been reported for other AMP based hydrogel systems. PXL150, a synthetic AMP, when combined with hydroxypropyl cellulose (HPC) gel, demonstrated antibacterial activity against P. aeruginosa both in vitro and in infected murine wounds.⁵ So, the Pepholin-carbomer hydrogel offers the advantage of combining antimicrobial activity with a simple, clinically familiar polymer platform and additional wound-healing support from aloe vera.

The clinical isolate was positive for the P. aeruginosa identification genes such as 16SrRNA, oprL and gyrB (Fig. S1). This genotypically characterised isolate showed no zone of inhibition for more than eight antibiotics as determined by the disc diffusion method. Thus, the clinical isolate was characterized as multi-drug resistant, displaying resistance to more than three classes of antibiotics as summarised in Table S2 and illustrated in Fig. S4. In this study, an experimental mouse model of superficial skin wound infection with a MDR P. aeruginosa strain was established to evaluate the efficacy of a Pepholin incorporated with hydrogel in reducing the infection and promoting wound healing using various parameters (Fig. 10A). There are relatively limited data on treatment of topical Pseudomonas infections, thus this experimental model may provide an opportunity to evaluate novel treatment strategies.

3.8.1. Morphological analysis of wound

The wound healing is a complex and dynamic process is influenced by the nature and severity of burn injuries, individual healing rates, and various biological factors.³⁸ In burn wound infections, persistent colonization by pathogens such as P. aeruginosa disrupts normal healing by prolonging inflammation and impairing epithelial regeneration. Macroscopic parameters such as wound contraction and epithelialization are therefore widely used as reliable indicators of healing progression in experimental wound models.^89,90 The wound contraction was quantified by comparing the wound area at each time point to the initial area on day 0. A two-way ANOVA statistical test revealed significant differences among the five groups (F (3,12) = 51.07, p < 0.0001) as well as across various time points (F (4,12) = 0.894, P < 0.001) indicating that both treatment modality and duration significantly influenced wound healing outcomes. Variations in wound healing were observed among different treatment groups, with the Pepholin hydrogel demonstrating superior progress. There was no significant difference between Group I and Group II, confirming that the experimental procedure itself did not impair healing in the absence of infection. However, by the 21st day, Group II showed the smallest wound contraction rate, underscoring the detrimental effect of MDR P. aeruginosa infection on tissue repair. By day 3, Groups III, IV, and V demonstrated significantly greater wound contraction than Group II (p < 0.05), with Group IV exhibiting the most pronounced contraction rate throughout the study (p < 0.005). A significant difference of p < 0.005 was found between Group III and Group IV, indicating reduced healing efficacy of carbomer hydrogel alone, which shows the essential contribution of the Pepholin in controlling infection and enabling tissue repair. Interestingly, no significant difference in wound contraction rate was observed between Group V and Group IV except at 21st day (p < 0.005). This suggests that Pepholin hydrogel achieved wound healing outcomes comparable to standard topical antibiotic therapy in an MDR infection setting. Comparable in vivo outcomes have been reported for other AMP based wound treatments, where they demonstrated enhanced wound contraction and epithelialization in infected skin wound models by simultaneously reducing bacterial burden and promoting tissue repair.^91,92 Peptide hydrogel systems have also been reported to improve wound healing kinetics, where sustained local delivery of AMPs facilitated infection control and supported re-epithelialization and granulation tissue formation.^69,93 Beyond peptide only systems, AMP loaded hydrogel and nanocomposite dressings have demonstrated comparable trends.^94,95 In comparison with these studies, the Pepholin–carbomer hydrogel exhibited a similarly robust wound contraction profile despite being challenged with a clinically relevant MDR P. aeruginosa isolate, supporting its therapeutic relevance. An advantage of the Pepholin hydrogel observed in this study is its ability to promote rapid wound contraction while maintaining antibacterial efficacy, suggesting a synergistic effect between infection control and tissue repair. However, it should be acknowledged that wound contraction alone does not fully reflect the quality of regenerated tissue. Therefore, histopathological evaluation and collagen deposition analysis, presented in subsequent sections, are essential to corroborate the morphological findings and assess tissue remodelling. Fig. 10B and 11A represent the wound contraction rate of all the groups.

3.8.2. Pepholin effectively treated MDR P. aeruginosa-infected burn wounds

The in vivo antibacterial efficacy of AMPs is a critical determinant of their translational potential, particularly in burn wound infections where bacterial persistence and biofilm formation severely impair healing.^69,76 Our study revealed a number of significant findings on ability of novel peptide to inhibit the growth of bacteria in vivo. Quantitative analysis of bacterial burden revealed a substantial increase in CFU at the wound site except group I, which showed no CFU throughout. Statistical analysis using a two-way ANOVA revealed significant differences among the five groups (F (4, 38) = 29.01, p < 0.0001), indicating that treatment modality strongly influenced bacterial clearance. Except for day 21, no significant difference was observed between the hydrogel group and the PBS treated group, confirming that the polymer matrix alone lacked intrinsic anti-pseudomonal activity. On the 14th and 21st days, there was a significant difference (p < 0.0001) between Group IV and Group III, indicating the absence of anti-pseudomonas activity in carbomer gel alone. The CFU reduction was significant (p < 0.001) in both Group V and Group IV compared to Group II across all study intervals (Fig. 11B and S6). An interesting observation was the lack of a significant difference in CFU reduction between the standard and AMP gel-treated groups at any interval, suggesting peptide's equivalent bacterial inhibition potential to standard antibiotic in vivo. Similar levels of bacterial reduction have been reported for other AMP-based topical therapies, such as the peptide PXL150, EPL and LL-37 based formulations, which demonstrated antibiotic comparable efficacy in murine burn and wound infection models when delivered locally via hydrogel systems.^5,20,96 In comparison, Pepholin demonstrated a similar magnitude of bacterial reduction while maintaining a favourable safety and stability profile, supporting its relevance as a topical therapeutic candidate. The predominant microbes identified at the wound site included S. aureus, P. aeruginosa, K. pneumoniae, and C. albicans. Among them, P. aeruginosa has demonstrated resistance to many potent antibiotics, continually evolving new mechanisms to withstand their effects, making it a leading cause of treatment failure in burn wound infections.^38,97 Considering these factors, the novel peptide has shown promising therapeutic potential in reducing MDR Pseudomonas infections in burn wounds.

3.8.3. Quantitation of cytokines using qRT-PCR

The expression of cytokines in the wound site was studied in all groups on the 3rd and 7th days after post treatment, as inflammation is prominent in the first week of injury or infection. After a burn infection, a systemic inflammatory response will be triggered, characterized by the release of pro-inflammatory cytokines. Subsequently, anti-inflammatory cytokines are deployed to mitigate the effects of the inflammatory cascade. This cytokine interplay unfolds promptly post-burn, preceding alterations in metabolic processes.⁹⁸ Inflammatory response in animals is highly regulated by the activity of proinflammatory cytokines like IL-1β and TNF-α, primarily released by activated macrophages.^98,99 In this study, we observed a significant elevation in the expression of these cytokines in group V compared to Group II (p < 0.01) on the 3rd day and exhibited the highest upregulation of these genes. Group IV showed significant downregulation compared to group II (p < 0.01). Furthermore, on the 7th day, TNF-α expression was notably downregulated in Group III, Group IV, and Group V compared to Group II (p < 0.01), indicating progression toward inflammatory resolution. The expression of the IL-1β gene was downregulated across all experimental groups, with no notable variances (p < 0.01) among the groups on the 7th day. The reduction of pro-inflammatory cytokines is widely acknowledged as a pivotal marker of the wound healing process and reflects a transition toward tissue repair rather than persistent immune activation.¹⁰⁰

Inflammation plays a crucial role in the body's response to injury, but prolonged inflammation can be detrimental to healing. Anti-inflammatory cytokines are crucial for moderating this response and facilitating the healing process.⁹⁹ Two key anti-inflammatory cytokines, EGF and TGF-β, cytokines expression showed significant upregulation in Groups III, IV, and V compared to the PBS-treated groups (p < 0.01) on both the 3rd and 7th days. Specifically, the expression of TGF-β and EGF in Groups IV and V was significantly (p < 0.0001) elevated on the 7th day compared to the 3rd day, indicating a temporal shift from inflammation toward regeneration. The Group IV exhibited the highest levels of TGF-β and EGF expression compared to the PBS-treated group on the 7th day. The results of cytokines gene expression were presented in Fig. 12. Studies indicate that the presence of EGF significantly contributes to the process of reepithelialisation in acute wounds by enhancing the proliferation of keratinocytes and promoting cell migration.^101,102 In a study where innate defense regulator peptides such as IDR-1018 have been shown to significantly accelerate wound closure in murine models by modulating inflammatory responses rather than acting solely through direct bactericidal mechanisms.^103,104 Importantly, the analysis of the pro inflammatory and anti-inflammatory cytokines expression in the Group IV revealed similar trend as Group V with significantly up regulation of anti-inflammatory cytokines and down regulation of pro inflammatory cytokines in 7th day compared to the 3rd day (p < 0.001). This pattern suggests that Pepholin not only controls bacterial burden but also favourably modulates the host immune response, facilitating timely resolution of inflammation and progression toward tissue repair. Such dual antimicrobial and immunomodulatory activity has been increasingly recognized as a desirable property for anti-infective therapeutics in burn wound management.^61,76

3.8.4. Histomorphologic analysis

Histomorphological analyses revealed significant disparities in the healing process among the experimental groups. In wound without infection and not treated group, epidermis showed homogenous eosinophilic areas of coagulative necrosis with pyknotic and fragmented nuclei. Dermis showed necrosis, congested blood vessels and infiltration of inflammatory cells especially neutrophils on 1 DPW (Fig. 13A). On 3 DPW, epidermis and dermis showed homogenous basophilic areas of coagulative necrosis and marked infiltration of neutrophils (Fig. 13B). On 7 DPW, epidermis showed necrosis with basophilic nuclear debris and dermis showed marked necrosis, infiltration of inflammatory cells, and mild regenerative process with proliferation of fibroblasts (Fig. 13C). On 14 DPW, epidermis and dermis showed marked necrosis with clumps of basophilic nuclear debris, infiltration of inflammatory cells with degenerated neutrophils, mild collagen deposition and moderate proliferation of fibroblasts (Fig. 13D). Inflammation plays a crucial role in the initial phases of healing by combating infections and initiating the proliferation stage, the progression of healing occurs only after inflammation is regulated.⁹⁰ On 21 DPW, incomplete epidermal epithelialization and dermis showed neovascularization, few regenerated hair follicles, moderate infiltration of inflammatory cells with collagen deposition and proliferation of fibroblasts (Fig. 13E).

In wound infected with P. aeruginosa and without treatment group, epidermis showed coagulative necrosis with basophilic shrunken pyknotic and fragmented nuclei. Dermis showed necrosis, congestion and infiltration of neutrophils on 1 DPW (Fig. 13F). On 3 DPW, epidermis and dermis showed homogenous basophilic areas of necrosis across all skin layers, detachment of the epidermal layer, and abscess formation with marked infiltration of degenerating neutrophils (Fig. 13G). On 7 DPW, epidermis and dermis showed diffuse area of necrosis and pyogranulomatous reaction with marked infiltration of neutrophils, absence of collagen deposition and fibroblasts (Fig. 13H). On 14 DPW, epidermis and dermis showed coagulative necrosis in all skin layers and infiltration of inflammatory cells especially multinucleated giant cells with absence of dermal collagen deposition (Fig. 13I). On 21 DPW, moderate necrosis, absence epidermal epithelialization and dermis showed marked inflammation with infiltration of multinucleated giant cells with mild collagen deposition and proliferation of fibroblasts (Fig. 13J).

In infected wound treated with hydrogel group, epidermis showed moderate coagulative necrosis and dermis showed moderate infiltration of neutrophils on 1 DPW (Fig. 13K). On 3 DPW, epidermis and dermis showed moderate necrosis and infiltration of neutrophils with regenerative progress of mild collagen deposition and fibroblast proliferation (Fig. 13L). On 7 and 14 DPW, mild epidermal epithelialization with hyperkeratinisation and dermis showed mild to moderate necrosis, infiltration of inflammatory cells, collagen deposition and proliferation of fibroblasts (Fig. 13M and N). Similar findings are observed in previous studies which highlighted the biodegradability, fibroblast proliferation, haemostatic properties, and collagen deposition capabilities of hydrogel.¹⁰⁵ On 21 DPW, moderate to marked epidermal epithelialization and dermis showed moderate infiltration of inflammatory cells with moderate to marked organized collagen fibers (Fig. 13O).

In infected wound treated with peptide hydrogel group, epidermis showed mild necrosis and dermis showed absence of infiltration of inflammatory cells on 1 DPW (Fig. 13P). On 3 DPW, mild early signs of tissue regenerative process of epidermal epithelialization and dermis showed mild necrosis and mild infiltration of neutrophils with moderate collagen deposition and fibroblast proliferation (Fig. 14Q). Accelerated tissue remodelling suggests rapid infection control combined with favourable modulation of the wound microenvironment. [101] On 7 DPW, moderate regenerative process of epidermal epithelialization and dermis showed regenerative process in hair follicles, mild necrosis and infiltration of inflammatory cells, marked collagen deposition and proliferation of fibroblasts (Fig. 14R). Moreover, angiogenesis is a critical process in the wound healing, marked by a significant increase in endothelial cell proliferation at the burn site, facilitating the formation of new blood vessels.¹⁰⁶ On 14 DPW, marked regenerative process of epidermal epithelialization with hyperkeratinisation and dermis showed regenerating hair follicles, mild inflammatory cells, and marked collagen remodelling where dense thick bundles were replaced by loosely arranged thin strands (Fig. 14S). On 21 DPW, completely developed epidermal layer and hyperkeratinisation with normal histological architecture and dermis showed fully developed and regenerating hair follicles, and marked collagen deposition with proper arrangement (Fig. 14T). Marked angiogenesis, organized collagen remodelling, and restoration of hair follicles by 14–21 DPW indicate advanced wound maturation, which has not been consistently achieved in antibiotic only treatments due to their reported inhibitory effects on fibroblast and keratinocyte proliferation.^107,108 Compared to previously reported AMP loaded hydrogels, which often show delayed collagen maturation or irregular fiber organization, the Pepholin hydrogel demonstrated dense yet well-organized collagen architecture, indicative of functional tissue regeneration rather than fibrotic repair.⁹³

In infected wound treated with antibiotic group, epidermis and dermis showed moderate necrosis and infiltration of inflammatory cells on 1 DPW (Fig. 14U). On 3 DPW, mild regenerative process in epidermis and dermis, mild necrosis and infiltration of neutrophils with mild collagen deposition and fibroblast proliferation were noticed (Fig. 14V). On 7 DPW, epidermis showed regenerative process; however, dermis showed abscess with marked infiltration of degenerating neutrophils with necrosis and proliferation of fibroblasts (Fig. 14W). On 14 DPW, moderate to marked regenerative changes of epidermal epithelialization with hyperkeratinisation and epidermal pegs, and dermis showed regenerating hair follicles, moderate inflammatory cells, and granulation tissue with a few newly formed blood vessels (Fig. 14X). On 21 DPW, completely developed epidermal layer with hyperkeratinisation; however, dermis showed ongoing healing processes with number of regenerating hair follicles were less, and inflammatory cells and granulation tissue were more when compared to peptide treated group (Fig. 14Y). Although antibiotic treatment reduced infection and promoted epithelialization, persistent dermal inflammation, abscess formation, and reduced hair follicle. The regeneration observed in this group are consistent with reports indicating delayed dermal remodelling following silver sulfadiazine therapy.^107,108 In contrast, the Pepholin treated group exhibited earlier resolution of inflammation and superior dermal maturation, highlighting a key advantage of peptide-based therapy over conventional topical antibiotics. The grading of histopathological lesions was summarized in Table 4.

Table 4. Grading of histopathological lesions in infected burn wounds following different treatment regimens.

Skin layers	Scoring criteria	DPW in group I					DPW in group II					DPW in group III					DPW in group VI					DPW in group V
Skin layers	Scoring criteria	1	3	7	14	21	1	3	7	14	21	1	3	7	14	21	1	3	7	14	21	1	3	7	14	21
Epidermis	Necrosis	2	1	2.5	2.5	2	2	3	3	3	2.5	2	2	3	3	1	1	1	1	1	0	1	1	2	1	0
	Infiltration of inflammatory cells	0	0	1	2	0	1	2	3	2.5	1.5	1	1.5	2	2	1	0	1	1	1	0	0	1	2	1	0
	Regenerative/healing process	0	0	0	2	2	0	0	0	1	0	0	0	1	2.5	0	1	1.5	1	3	3	0	1	1	1	3
Dermis	Necrosis	2	1	2.5	2.5	2	2	3	3	3	2.5	2	2.5	3	2	1	1	1	1	1	0	1	1	3	1	0
	Infiltration of inflammatory cells	0	1	2	2.5	1	1	2.5	3	2.5	1.5	1	2	2	1.5	1	0	1	2.5	1	1	0	1	2.5	1.5	1
	Regenerative/healing process	0	0	1	2	2	0	0	0	0	1	0	0	1	1.5	2.5	0	1	1	2.5	3	0	1	1	2	3
Hypodermis	Necrosis	1	1	2	2	1	1.5	2.5	3	3	1.5	2	2	2	1.5	1	0	1	2	1	0	1	1	2.5	1	1
	Infiltration of inflammatory cells	1	1	2	1.5	1.5	2	2	3	3	2	2	1.5	2	2	1.5	0	1	1.5	2.5	3	0	1	1.5	2	2.5
	Regenerative/healing process	0	0	1	1	2	0	0	0	0	1	0	0	1.5	1.5	2.5	0	1	1.5	2.5	3	0	1	1.5	2	2.5
Panniculus carnosus	Necrosis	1.5	1	3	2	1	2	3	3	3	2	2	2	2	1.5	1	0	1	3	1	0	1	1	3	1	0
	Infiltration of inflammatory cells	1	2	2	1	2	1.5	2.5	3	2.5	1.5	1	2	1.5	2	1.5	0	1	2.5	1.5	1	1	1	2.5	1.5	1
	Regenerative/healing process	0	0	1	2	2	0	0	0	1	1	0	0	1.5	2	2.5	0	1	2	2.5	3	0	1	1	2	2.5
Adventitia	Necrosis	1.5	1	3	2	0	2	3	3	2.5	2	2	2	2	1.5	0	0	1	3	1	0	0	1	3	1	0
	Infiltration of inflammatory cells	1	1	1	1	1	1.5	2	2.5	2	1.5	1	1.5	1.5	1.5	1	0	1	1.5	1	1	0	1	2	1	1
	Regenerative/healing process	0	0	1	2	2	0	0	0	1	1	0	0	1.5	1.5	2.5	0	1	1.5	2	3	0	1	1	2	3

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The Mason trichrome stain revealed red or purple colour for burn-damaged skin tissue (including burn-damaged coagulated collagen fibers), epithelium, keratin, muscle fibers, hair follicles, fibrin and erythrocytes; blue colour for normal collagen fibers; and black colour for nuclei. In wound without infected and not treated group, burn-damaged epidermis, coagulated collagen fibers and necrosed muscle fibres appeared red colour and without normal blue colour collagen on 1 and 3 DPW (Fig. 14A and B). Mild, moderate and marked newly formed immature collagen depositions of light blue colour were observed on 7, 14 and 21 DPW, respectively (Fig. 14C–E). In wound infected with P. aeruginosa and without treatment group, burn-damaged epidermis, coagulated collagen fibers and necrosed muscle fibres appeared red colour and without normal blue colour collagen on 1 and 3 DPW (Fig. 14F and G). Mild (7 and 14 DPW) and moderate (21 DPW) immature collagen depositions of light blue colour were observed (Fig. 14H–J). In infected wound treated with hydrogel group, no collagen deposition on 1 DPW (Fig. 14K) and mild (3 DPW), moderate (7 DPW) and marked (14 and 21 DPW) collagen deposition was noticed (Fig. 14L–O). In infected wound treated with peptide hydrogel group, mild newly formed immature collagen deposition of light blue colour was observed on 1 DPW (Fig. 14P). Moderate (3 DPW) and marked (7, 14 and 21 DPW) mature collagen depositions of dark blue colour with red colour regenerating and new hair follicles were noticed (Fig. 14Q–T). In infected wound treated with antibiotic hydrogel group, absence or mild immature collagen deposition was observed on 1 DPW (Fig. 14U). Moderate (3 and 7 DPW) and marked (14 and 21 DPW) mature collagen depositions with red colour regenerating hair follicles were noticed (Fig. 14V–Y). Early appearance of mature, dark-blue collagen fibers in the Pepholin treated group indicates accelerated extracellular matrix maturation, whereas antibiotic and hydrogel treated groups predominantly exhibited immature collagen for longer durations, a trend also reported in other burn wound studies.^91,92,102 So, it confirms significant collagen formation in the AMP-treated group, indicating comprehensive healing across skin layers. The grading of collagen deposition was summarized in Table 5.

Table 5. Grading of collagen deposition in infected burn wounds following different treatment regimens^a.

Groups	Days post wounding (DPW)
Groups	1	3	7	14	21
Group I	—	—	+ (IMC)	++ (IMC)	+++ (IMC)
Group II	—	—	+ (IMC)	+ (IMC)	++ (IMC)
Group III	—	+ (MC)	++ (IMC)	+++ (IMC)	+++ (IMC)
Group IV	+ (IMC)	++ (MC)	+++ (MC)	+++ (MC)	+++ (MC)
Group V	−/+ (IMC)	++ (MC)	++ (IMC)	+++ (MC)	+++ (MC)

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Negative; ±: negative or mild; +: mild; ++: moderate; +++: severe; IMC: immature collagen; MC: mature collagen.

4. Conclusion

In this study, a novel AMP, Pepholin, derived from the holin protein of a Pseudomonas bacteriophage, was successfully designed, synthesized, and characterized. The peptide exhibited potent antibacterial and antibiofilm activity against MDR P. aeruginosa, along with rapid bactericidal kinetics. Mechanistic investigations demonstrated that Pepholin kills bacteria through a membrane-permeabilization mechanism, causing loss of membrane integrity and cell death. The peptide showed selectivity toward bacterial cells over mammalian cells, with negligible hemolytic and cytotoxic effects, and retained its stability under physiological salt, serum, and temperature conditions. When incorporated into a carbomer-based hydrogel, Pepholin significantly accelerated the healing of MDR P. aeruginosa-infected burn wounds in mice by reducing bacterial burden, modulating inflammatory cytokines, and enhancing collagen deposition. While the present work demonstrates efficacy in murine models, further studies addressing peptide stability in physiological fluids, formulation strategies for systemic delivery, and resistance evolution potential are warranted to fully establish clinical utility. Collectively, our results identify Pepholin as the novel holin-derived AMP with dual antibacterial and wound-healing properties, representing a stable, biocompatible, and mechanism driven macromolecular therapeutic candidate. These comprehensive in vitro and in vivo findings establish Pepholin as a promising candidate for topical antimicrobial therapy and demonstrate the potential of phage-derived peptides as next-generation therapeutics for drug-resistant wound infections.

Ethical statement

All experiments were performed in accordance with the Guidelines of Committee for Control and Supervision of Experiments on Animals (CCSEA), Ministry of Fisheries, Animal Husbandry and Dairying, Government of India and experiments involving animals were approved by the Institute Animal Ethics Committee (IAEC) at “ICAR-Indian Veterinary Research Institute, Izatnagar, India (Approval No F. No. 26–1/2023–24/JD(R)/IAEC dated 20th February, 2024).

The study related to collection of human blood sample was conducted in accordance with the ethical principles. The protocol was reviewed and approved by the Institutional Ethics Committee (Intramural), Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India (Approval No INT/IEC/2025/SPL-402) [Reference No: IEC-INT/2024/PhD-2092]. Informed consent was obtained from human participant prior to any blood collection. The blood sampling was performed by qualified medical personnel using sterile, single-use equipment to ensure minimal risk and discomfort to the participant.

Consent for publication

All authors have given their consent to publish this article.

Author contributions

Purushotham RV: conceptualisation, writing – original draft, investigation, formal analysis, data curation. Sameer Shrivastava: conceptualisation, writing – review and editing, investigation, data curation, validation, supervision, project administration, funding acquisition. Sonal Saxena: visualisation, formal analysis. Sabapathi Nagappan: software, methodology. Pradeep Kumar: data curation. Saminathan M.: investigation, formal analysis. Abhinav Kumar: software, formal analysis. Sanjay Kumar Singh: software, formal analysis. Satyabrata Dandapat: supervision, formal analysis.

Conflicts of interest

There are no conflicts to declare.

Supplementary Material

RA-016-D6RA03599J-s001

RA-016-D6RA03599J-s001.pdf^{(1.6MB, pdf)}

Acknowledgments

The authors are thankful to Director, ICAR-IVRI, Izatnagar, for providing the necessary facilities and support. The study was conducted with support from grants received under National Agricultural Science Fund through the Project “Detection of peptide biomarkers and development of synthetic antimicrobial peptide hydrogels for bovine mastitis” (Project Grant No. NASF/ABA-6014/2016–17/367) and “Developing Novel Therapeutic Strategies for Mitigating Antimicrobial Resistance” sanctioned vide F. No. NASF/BGAM-9006-2022-23. Authors would like to acknowledge the help and support provided by Dr A. K. Mohanty, Director, Central Institute for Research on Cattle, Meerut, India for providing the Bubaline Mammary Epithelial Cells (BuMEC) for cytotoxicity analysis. We are also thankful to Ms Ritika Harchand, PhD Scholar and Dr Shivaprakash M Rudramurthy, Professor, Post Graduate Institute of Medical Education & Research, Chandigarh, India for facilitating in collecting blood sample for testing the toxicity of peptide molecule on human RBCs.

Data availability

All data supporting the findings of this study are available within the article and its supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d6ra03599j.

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Associated Data

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PERMALINK

Pepholin, a bacteriophage holin-derived antimicrobial peptide with membrane-disruptive activity and therapeutic efficacy in MDR Pseudomonas aeruginosa burn wounds

Purushotham RV

Sameer Shrivastava

Sonal Saxena

Sabapathi Nagappan

Pradeep Kumar

Saminathan M

Abhinav Kumar

Sanjay Kumar Singh

Satyabrata Dandapat

Abstract

1. Introduction

2. Materials and methods

2.1. Chemicals, media and bacterial strains

2.2. Design, synthesis and purification of peptide

2.3. Secondary structure prediction and determination

2.4. Determination of antibacterial and anti-biofilm activity of pepholin

2.5. Outer and inner membrane permeability assays

2.6. Flow cytometry

2.7. Toxicity studies of peptide

2.7.1. Haemolytic assay

2.7.2. Cytotoxicity assay

2.7.3. In vivo toxicity studies in Galleria mellonella larvae

2.7.4. Selectivity index

2.8. Stability studies of peptide

2.9. Preparation and characterisation of peptide-carbomer hydrogel

2.10. In vivo evaluation of peptide's therapeutic potential

2.10.1. Bacterial strain

2.10.2. Experimental animal

Table 3. Experimental design and respective treatments.

2.10.3. Wound size reduction (%)

2.10.4. In vivo antimicrobial efficacy of peptide against MDR P. aeruginosa

2.10.5. Quantitation of cytokines expression using qRT-PCR

2.10.6. Histomorphologic analysis

2.11. Statistical analysis

3. Results and discussion

3.1. Design, synthesis, and characterization of peptide

Table 1. Physiochemical parameters of Pepholin.

3.2. Structural characterization of Pepholin reveals a membrane-induced amphipathic α-helix

3.3. Pepholin shows pronounced antibacterial and antibiofilm effect against P. aeruginosa in vitro

Table 2. Minimum inhibitory concentration (MIC) of Pepholin against ESKAPE pathogens and clinical MDR P. aeruginosa isolates.

3.4. Pepholin disrupts bacterial membrane integrity

3.5. Flow cytometric analysis confirms membrane damage induced by Pepholin

3.6. Pepholin exhibits low haemolytic and cytotoxic effects

3.7. Pepholin retains antibacterial activity under physiological conditions

3.8. In vivo evaluation of peptide's therapeutic potential by formulating in carbomer hydrogel

3.8.1. Morphological analysis of wound

3.8.2. Pepholin effectively treated MDR P. aeruginosa-infected burn wounds

3.8.3. Quantitation of cytokines using qRT-PCR

3.8.4. Histomorphologic analysis

Table 4. Grading of histopathological lesions in infected burn wounds following different treatment regimens.

Table 5. Grading of collagen deposition in infected burn wounds following different treatment regimensa.

4. Conclusion

Ethical statement

Consent for publication

Author contributions

Conflicts of interest

Supplementary Material

Acknowledgments

Data availability

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Table 5. Grading of collagen deposition in infected burn wounds following different treatment regimens^a.