Structure–Activity Relationship and Biosafety of Linear Pentapeptide Analogs Derived from Battacin for Antimicrobial Development

TL;DR

Scientists modified a natural antimicrobial peptide to create safer, more effective versions that can fight drug-resistant bacteria and biofilms.

Contribution

The study introduces crosslinked nanostructures of modified pentapeptides with enhanced antimicrobial efficacy and biosafety.

Findings

Lauric acid-modified and crosslinked lipoic acid-modified pentapeptides effectively target MRSA and biofilms.

Modified peptides showed strong synergy with ciprofloxacin against resistant strains.

The physical mode of action prevents resistance development in sequential passaging assays.

Abstract

Background: Natural antimicrobial peptides (AMPs) present a promising solution to address the global threat of drug-resistant infections; however, their clinical translation is challenged by limitations in stability, cytotoxicity, and production costs. Methods: In the present study, a linear Battacin-derived peptide (DDLFD) was modified at the N-terminus with lipid chains, cinnamic acid, or lipoic acid. The lipoic acid-modified variant was further crosslinked by UV irradiation to form stable nanoparticles. The antibacterial performance against planktonic and biofilm bacteria was systematically evaluated in vitro. Results: The results demonstrated that lauric acid-modified pentapeptide (C12-5) and crosslinked lipoic acid-modified pentapeptide (cLA-5) exhibited potent and rapid-acting effects against various pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Moreover, they showed enhanced efficacy in eradicating bacterial biofilms. Biosafety assessments based on hemolysis and cytotoxicity assays indicated favorable biocompatibility profiles of cLA-5. Mechanistic investigations confirmed that the modified pentapeptides retained a membrane-targeting mode of action characteristic of natural AMPs, involving membrane depolarization and increased permeability. This physical mechanism effectively prevented the development of resistance in sequential passaging assays and showed strong synergistic effects with ciprofloxacin against ciprofloxacin-resistant strains, effectively restoring their antibiotic susceptibility. Conclusions: Together, these findings underscore the strategic potential of rational structural modification, especially the crosslinked nanostructure, in advancing engineered AMPs toward clinical application.

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1. Introduction

The global antimicrobial resistance (AMR) crisis poses a critical threat to public health [1]. Multidrug-resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter cloacae) exhibit complex resistance mechanisms [2]. These include drug-inactivating enzymes [3], target mutations and efflux pumps [4,5], reduced membrane permeability and target alteration [6,7], which lead to high rates of treatment failure in infections such as pneumonia and bloodstream infections. Developing novel antimicrobial agents that exhibit both rapid bactericidal activity and a low propensity for resistance development represents a key strategy in combating resistant pathogens.

Antimicrobial peptides (AMPs) are broad-spectrum agents of innate immunity that act primarily by disrupting bacterial membranes, a physical mechanism that reduces the risk of resistance [8,9]. In order to enhance the stability and selectivity of natural antimicrobial peptides, researchers have developed numerous semi-synthetic analogs. These analogues have been shown to retain the high bactericidal efficiency and low resistance potential of antimicrobial peptides while exhibiting superior pharmaceutical properties, thus garnering significant attention [10]. Lipopeptide antibiotics feature a characteristic structure comprising hydrophobic fatty acyl chains linked to a hydrophilic peptide moiety [11]. The lipid tail mediates membrane anchoring, while the cationic peptide segment facilitates initial binding and subsequent disruption [11,12]. This amphipathic architecture, which frequently incorporates non-proteinogenic amino acids or other unique constituents, underlies their considerable structural and functional diversity. Vogel et al. replaced all lysines with the shorter, non-proteinogenic 2,3-diaminopropanoic acid (Dap) in a Trp-rich peptide [13,14]. The resulting amphipathic sequence showed a four-fold increase in anti-E. coli activity and markedly higher protease stability [15]. This demonstrates that non-canonical residues can precisely tune both the structure and bactericidal function of antimicrobial peptides. Recent studies also reveal that structural modifications in lipopeptides can substantially alter their antibacterial mechanisms [11]. Strategies like amino acid substitution and cyclization have markedly improved efficacy in systems such as Aurein 1.2 derivatives, enhancing selectivity (~10-fold) and reducing MICs (up to 8-fold)for E. coli and S. aureus [16]. However, practical application remains constrained by complex sequences and laborious synthesis. Developing structurally simplified lipopeptides that retain potent activity has emerged as a critical research priority [17].

Battacin, a natural lipopeptide derived from Brevibacillus laterosporus [18], retains polymyxin-like α,γ-diaminobutyric acid (Dab) residues and a fatty acid chain. Despite similar amino acid compositions, Battacin exhibits a 2.3-fold higher LD_50_ in mice than polymyxin B, demonstrating that structural refinement reduces acute toxicity [19]. Luka Posa et al. engineered a derivative designated peptide 2.1 (vinylacetic acid-conjugated DDLFD) [20], utilizing the linear truncated pentapeptide scaffold DDLFD (Dab-Dab-Leu-Phe-Dab) as the template. This modified compound exhibited a MIC of 197 µM (128 µg/mL), representing a significant improvement over the unmodified parental scaffold DDLFD, which possessed a MIC of 1100 µM (786 µg/mL) [21,22]. Therefore, simplifying the natural lipopeptide backbone is a highly viable approach, as it significantly reduces synthetic complexity and allows subsequent optimization to enhance antimicrobial activity.

In this study, Battacin-derived linear pentapeptides were engineered through N-terminal modifications. Specifically, we designed a series of analogs modified with a long aliphatic chain (lauric acid), a short-branched chain (5-methylhexanoic acid), an intrinsically antimicrobial cinnamic acid derivative, and the most significant, a lipoic acid moiety. The lipoic acid modified peptide was further crosslinked via its five-membered ring to form nanoparticles. The potent analogs were subsequently subjected to a comprehensive evaluation, including MICs, time-kill kinetics, antibiofilm assays, membrane permeability studies, scanning electron microscopy, and biocompatibility analysis. A systematic evaluation was conducted on how these structural changes modulate the balance between antimicrobial activity and biocompatibility, providing a rational design framework for safer lipopeptide based antibiotics.

2. Results

2.1. Peptide Synthesis and Characterization

Previous structural simplification studies have identified the linear pentapeptide sequence (Dab-Dab-Leu-Phe-Dab, DDLFD) as a potent scaffold that functions effectively without cyclic constraints [19]. In this study, the pentapeptide DDLFD was strategically modified with four distinct moieties to investigate the structure–activity relationships. Specifically, lauric acid (CH_3_(CH_2_)_10_COOH) was conjugated to the N-terminus to introduce a long, straight aliphatic chain, thereby mimicking the amphiphilic architecture of lipopeptides. 5-methylhexanoic acid (MH) was employed as a shorter-chain analog to serve as a control, investigating the effect of the lipid chain length on peptide properties. In addition, cinnamic acid (CA) was incorporated to replace the conventional lipid chain with a bio-active moiety known for its intrinsic antimicrobial activity [23]. Furthermore, lipoic acid (LA) was attached to the pentapeptide to introduce a cyclic disulfide, which was subsequently crosslinked to form nanoparticles (Figure 1).

The products were successfully synthesized and characterized by ESI-MS and RP-HPLC. Analytical data confirmed that the molecular weights of all analogs correlated well with their theoretical values, and purities were greater than 90%. Representative chromatograms and mass spectra are provided in the Supplementary Materials File S1.

2.2. Antimicrobial Activity

The antimicrobial efficacy of the modified pentapeptide derivatives was determined by the MIC against a panel of representative microbial strains, including E. coli, S. aureus, MRSA, and C. albicans (as shown in Table 1).

C_12_-5 exhibited superior activity against S. aureus, including the resistant strain MRSA, with an MIC of 4 µg/mL, while remaining highly active against the E. coli and C. albicans (MIC of 8 µg/mL). However, its shorter-chain analog, MH-5 exhibited significantly reduced activity, with MICs ranging from 128 to 256 µg/mL. Although CA-5 displayed the lowest activity (MICs of 256–512 µg/mL) among the derivatives, it still represents a modest improvement over unmodified cinnamic acid (>500 µg/mL) [4], consistent with the intrinsic activity of its moiety.

The lipoic acid-modified pentapeptide LA-5 demonstrated a superior MIC compared to MH-5. Unexpectedly, the crosslinked LA-5 (cLA-5) achieved the lowest MIC among the modified pentapeptide derivatives tested, reducing it by 16-fold against bacterial strains and 8-fold against C. albicans. This notable enhancement may be attributed to the unique nanostructure formed after crosslinking (Figure 2), which promotes membrane targeting and further disruption. Among the tested analogs, C_12_-5 and cLA-5 nanoparticles exhibited the most potent and broad-spectrum activity. The results indicated that lipid chain length and nanostructure as key factors determining antimicrobial efficacy.

The antibacterial efficacy of lipid-modified analogs was systematically evaluated under conditions that are relevant to the body’s physiological processes(as shown in Table 2 and Table 3). All modified pentapeptides exhibited remarkable salt tolerance, with minimal fluctuations in MICs against E. coli and S. aureus, remaining within a range of 2-fold. In the presence of serum (3–10% v/v), C_12_-5, cLA-5, and LA-5 exhibited minimal alterations in their MIC (≤2-fold variation), while MH-5 and CA-5 exhibit serum concentration dependent susceptibility characteristics: under 3% serum conditions, changes are ≤2-fold, while under 10% serum conditions, MIC increases ≥4-fold. It is noteworthy that both C_12_-5/cLA-5 and polymyxin B exhibited excellent serum stability, with their MIC values increasing by less than 2-fold in serum. These results confirm that strategic lipid modifications confer dual resistance to ionic and serum interference, thereby enhancing therapeutic potential in physiological environments.

2.3. Hemolytic Activity

The hemolytic activity of modified pentapeptides against rabbit erythrocytes was evaluated to assess peptide toxicity towards eukaryotic cells. As shown in Table 4, both CA-5 and MH-5 induced negligible hemolysis (<5%) across the concentration range tested, with the only exception that MH-5 slightly exceeded the 5% threshold (reaching 5.12%) at the highest concentration of 512 μg/mL. The most pronounced hemolysis was observed for C_12_-5 and LA-5, which exhibited hemolysis ratios exceeding 10% at concentrations above 128 μg/mL. However, due to the low MIC value of C_12_-5 against various bacteria, it still showed a relatively low hemolysis ratio (<5%) at 2× MIC (8 μg/mL). The hemolytic activity of LA-5 was significantly improved after crosslinking. For example, at a concentration of 64 μg/mL, the hemolysis rate of LA-5 was 8.87%, while that of cLA-5 was only 2.24%. cLA-5 demonstrated superior hemocompatibility to the control polymyxin B, maintaining a hemolysis ratio below 5% at all concentrations up to 128 μg/mL.

2.4. Cytotoxicity

The cytotoxicity of the peptides against L929 cells was evaluated using a CCK-8 assay. As shown in Figure 3, cLA-5 (IC_50_ = 477.5 μg/mL) and MH-5 (IC_50_ = 414 μg/mL) exhibited lower cytotoxicity, with cLA-5 demonstrating optimal cytocompatibility at 256 μg/mL but significant viability reduction at 512 μg/mL. CA-5 (IC_50_ = 282 μg/mL) displayed intermediate toxicity, showing a concentration-dependent decline in cell viability at higher concentrations similar to cLA-5. In contrast, C_12_-5 (IC_50_ = 147.7 μg/mL) and LA-5 exhibited relatively higher cytotoxicity and limited biocompatibility, with C_12_-5 being the most toxic and displaying the highest hemolytic activity among the modified peptides. Notably, despite C_12_-5 showing relatively poorer hemocompatibility within the series, all modified peptides demonstrated significantly lower hemolysis rates compared to the positive control polymyxin.

2.5. Bactericidal Kinetics

The bactericidal kinetics of the modified pentapeptides were evaluated against E. coli, S. aureus, C. albicans, and MRSA using time-kill assays (Figure 4). Using E. coli as an example, C_12_-5 demonstrated rapid, concentration-dependent killing, reducing the microbial population by over 3 orders of magnitude (killing rate of 99.9%) within 0.5 h at 2× MIC (16 μg/mL) against all tested pathogens. The complete eradication of microbes was observed after 4 h, corresponding to a killing rate greater than 99.999%. cLA-5 exhibited slightly delayed kinetics, requiring 2 h at 2× MIC (8 μg/mL) to achieve an equivalent log-reduction (killing rate of 99.9%), though it also reached full sterilization within 4 h.

In contrast, CA-5 and MH-5 displayed significantly reduced bactericidal efficacy. At their respective 2× MICs (512 μg/mL for CA-5 and 256 μg/mL for MH-5), neither compounds reduced bacterial suspension concentration below 10^5^ CFU/mL after 4 h exposure.

The bactericidal kinetics of the modified pentapeptides against other bacterial strains were similar to those observed against E. coli. These kinetic differences highlight the crucial influence of lipid modification on achieving rapid bactericidal action in the pentapeptide system.

2.6. Drug Resistance

The potential of C_12_-5 and cLA-5 to induce resistance in E. coli and S. aureus was evaluated through serial passage experiments (Figure 5). During the course of 16 generations of passage, no significant resistance to either modified short peptide or polymyxin B was observed. Conversely, ciprofloxacin (MIC_0_ of 1 μg/mL for both strains) induced significant resistance in both strains, with a 32-fold increase in MIC after 9 generations of passage.

The combinatory effects of C_12_-5 or cLA-5 with ciprofloxacin were further evaluated against ciprofloxacin-resistant strains. When administered as a monotherapy against these pathogens, a concentration of 64 μg/mL of ciprofloxacin yielded only modest bacterial growth inhibition, as evidenced by the dark red/blue turbidity indicative of high bacterial density (Figure 6). In combination with C_12_-5, the effective dose of ciprofloxacin against E. coli was markedly reduced, achieving similar inhibition with combinations of 1 μg/mL:16 μg/mL or 2 μg/mL:8 μg/mL (C_12_-5: ciprofloxacin) as with 64 μg/mL of ciprofloxacin alone. A comparable synergy was observed for cLA-5 in combination with ciprofloxacin. However, a slightly higher concentration of cLA-5 was required to achieve an equivalent antibacterial activity. As shown in Figure 6B,D, the inhibitory effect against S. aureus was similar to that observed against E. coli. Complete inhibition of both strains was achieved with C_12_-5 and cLA-5 at a concentration of 4 μg/mL, as indicated by pale red/blue coloration.

The synergistic effect was further quantitatively confirmed by the fractional inhibitory concentration index (FICI). The combinations of C_12_-5/cLA-5 with ciprofloxacin yielded FICI values below 0.5 (Figure S1), demonstrating clear synergistic effects against the resistant strains.

2.7. Anti-Biofilm Activity

Bacteria embedded within a biofilm matrix usually exhibit an antibiotic susceptibility profile that is up to 1000-fold reduced in comparison with their planktonic counterparts [25,26], underscoring the need to evaluate the efficacy of modified pentapeptides in both eradicating biofilms and preventing their formation.

As demonstrated by the biofilm inhibition assay in Figure 7 (yellow column), C_12_-5 and cLA-5 suppressed more than 88% of biofilm formation across all tested pathogens at their respective 2× MIC doses. This resulted in a reduction of residual biomass to approximately 10%. MH-5 and CA-5 demonstrated substantially lower inhibition ratios of 65–70% and species-dependent inhibition, proving least effective against MRSA and most effective against C. albicans.

The biofilm eradication potency of the modified pentapeptides after 5 h treatment at 2× MIC was also shown in Figure 7. C_12_-5 (at concentration of 16 μg/mL) achieved 82% clearance across multiple bacterial species, while cLA-5 (at concentration of 8 μg/mL) performed even better, reaching 85% eradication. MH-5 (at concentration of 256 μg/mL) was limited to moderate and variable bacterial clearance (50–65%). CA-5, while similarly effective against bacterial biofilms (65% clearance), demonstrated superior activity against fungal biofilms (achieving 80% eradication).

CLSM directly visualized the mechanism of action by revealing intense propidium iodide (PI) fluorescence in E. coli and S. aureus biofilms [20]. As shown in Figure 8, treatment with C_12_-5 or cLA-5 dramatically reduced the thickness and density of the preformed biofilms. The result indicated that the potent antibiofilm activity was driven by the loss of membrane integrity.

2.8. Antimicrobial Mechanisms of Peptides

2.8.1. Membrane Depolarization

The membrane depolarization activity of modified pentapeptides was evaluated in hyperpolarized E. coli DC2 using the voltage-sensitive probe DiSC_3_(5). As shown in Figure 9A, MH-5 and CA-5 at 2× MIC induced only a gradual fluorescence increase over 600 s, whereas LA-5 exhibited accelerated signal accumulation. Furthermore, C_12_-5 and cLA-5 triggered immediate and robust fluorescence surges, reaching significantly higher intensities than those induced by LA-5 within the first 60 s. The results confirm that C_12_-5 and cLA-5 are highly effective at rapidly disrupting the bacterial membrane potential.

2.8.2. Outer Membrane Permeability

The hydrophobic fluorophore NPN was utilized to evaluate alterations in outer membrane (OM) permeability in E. coli DC2. This probe is virtually non-fluorescent in aqueous solution but exhibits strong emission upon integration into hydrophobic membrane compartments.

As demonstrated in Figure 9B, all modified pentapeptides induced time-dependent fluorescence accumulation. However, at concentrations of 2× MIC, MH-5, CA-5, and LA-5 exhibited modest fluorescence enhancement. cLA-5 and C_12_-5 at considerably lower 2× MIC doses (8/4 μg/mL) induced stronger NPN fluorescence, indicating superior OM disruption capacity.

The results demonstrate that the bactericidal efficacy of cLA-5 and C_12_-5 stems primarily from their membrane-targeting activity. The mechanism is characterized by rapid plasma membrane depolarization coupled with concurrent outer membrane disruption. These synergistic perturbations compromise membrane integrity, leading to ion leakage and ultimately cell death [27].

2.8.3. SEM Observation

The morphological changes in bacterial cells after treatment with modified pentapeptides were visualized by SEM. The results revealed time-dependent membrane damage in both E. coli and S. aureus treated with C_12_-5 or cLA-5 (Figure 10). Untreated controls displayed intact, smooth surfaces, whereas a 0.5 h treatment at 2× MIC induced substantial surface perturbations, including irregular contraction and textural roughening. The damage was exacerbated after a 4 h exposure, resulting in pore formation and cytoplasmic leakage. These progressive alterations demonstrate the membranolytic action of C_12_-5 and cLA-5, providing direct morphological evidence for their rapid membrane-disruption mechanism.

3. Discussion

The escalating global threat of drug-resistant pathogens underscores the urgent need for novel antimicrobial agents. The present study employed structural simplification and rational design to develop cost-effective analogs of natural antimicrobial peptides (AMPs) with enhanced antimicrobial activity and improved biosafety. Linear DDLFD pentapeptide was modified at the N-terminus by introduction of (1) a lauric acid long chain to mimic the amphiphilic architecture of natural lipopeptides; (2) 5-methylhexanoic acid as a short-chain control to investigate the effect of lipid chain length; (3) cinnamic acid (known for its intrinsic antimicrobial activity) to explore the potential of bioactive pharmacophores; (4) lipoic acid to provide a crosslink able site; (5) crosslinking of the lipoic acid-modified peptide to form stable nanoparticles. The design strategy centered on precisely modulate the molecular hydrophobicity, amphipathic, and spatial conformation, thereby optimizing interactions with microbial cell membranes. The selection of C_12_ as the lipid moiety was guided by the optimal balance between hydrophobic membrane insertion and hemolytic safety, as established in previous studies [28]. Specifically, lipid chains longer than C_14_ lead to significantly enhanced hemolysis, which is attributed to excessive hydrophobicity that reduces bacterial membrane selectivity [29]. This choice is also consistent with clinically approved lipopeptide antibiotics, including daptomycin (C_10_–C_13_ lipid tail) and polymyxins (C_8_–C_10_ fatty acyl group), whose lipid tails enable effective membrane targeting while maintaining a favorable therapeutic window [30]. UV-induced crosslinking of lipoic acid-modified pentapeptides was employed to construct stable spherical nanoparticles. This nanoconfinement strategy serves three key functional purposes: first, it enhances physiological stability of the peptide; second, it promotes multivalent membrane interactions at the bacterial surface, enabling cooperative membrane disruption; third, it reduces non-specific aggregation in solution and minimizes off-target interactions with eukaryotic membranes, thereby improving biosafety [31,32].

Among the derivatives, C_12_-5 (long-chain lipid-modified) and cLA-5 (crosslinked nanoparticles) performed most prominently, exhibiting potent activity against a range of pathogens including MRSA and C. albicans, with MICs as low as 4–8 µg/mL. The potency of C_12_-5 was enhanced by more than 16-fold compared to MH-5 and LA-5, demonstrating the significant benefit of its specific chain length. However, this benefit was constrained by the finding that extending the lipid chain beyond 14 carbons led to a sharp increase in hemolysis [33,34]. Together, these results confirm that an optimized degree of hydrophobicity is crucial for effective membrane insertion and disruption [35]. Notably, the nanostructure formed by crosslinking in cLA-5 likely promotes localized concentration accumulation at the membrane surface and enables more efficient multivalent membrane disruption. In contrast, the weaker activity of CA-5 reveals a fundamental conflict between its rigid planar structure and the structural adaptability required to form highly effective membrane-disruptive nanoaggregates [36].

Both hemolytic and cytotoxicity results revealed the importance of balancing antimicrobial activity with physiological safety [37]. CA-5 and MH-5 demonstrated the most favorable overall biosafety profiles among the tested peptides. They induced negligible hemolysis (<5%) across the entire concentration range tested and exhibited cytotoxicity against L929 cells comparable to the control polymyxin B. In contrast, C_12_-5 presented the highest biosafety risk, while its overall biocompatibility was the most limited. This is conclusively evidenced by its pronounced hemolytic tendency (>10% at concentrations > 128 μg/mL) coupled with its very low cytotoxicity IC_50_ value (64.79 μg/mL). This profile suggests that its long-chain lipid modification, while conferring potent antibacterial effects, also enhances non-selective membrane disruption against eukaryotic cells. Interestingly, while CA-5 and MH-5 were safe, they exhibited poor antimicrobial activity, with MICs as high as 256/512 μg/mL. Although C_12_-5 caused significant hemolysis at higher concentrations, its effective antimicrobial concentration was very low (4–8 μg/mL). Within this effective range, both the hemolysis rate and cytotoxicity of C_12_-5 were low, indicating an acceptable safety profile.

The selectivity of C_12_-5 and cLA-5 is rooted in the fundamental structural differences between bacterial and mammalian cell membranes: bacterial surfaces exhibit anionic properties, which mediate the electrostatic attraction of cationic peptides, whereas mammalian membranes are zwitterionic and enriched in cholesterol, thereby conferring resistance to peptide insertion [38]. C_12_-5 exerts potent antimicrobial activity via direct hydrophobic insertion at its minimum inhibitory concentration (MIC, 4–8 µg/mL), a concentration substantially lower than its hemolytic threshold [39]. Notably, the cLA-5 nanostructure further enhances biosafety through a multivalent avidity effect this structure facilitates the concentration of multiple peptide units on bacterial membranes while imposing steric hindrance to prevent penetration into eukaryotic cells [40].

The results for LA-5 and its crosslinked derivative, cLA-5, most clearly illustrate the decisive role of rational structural optimization in improving safety. While LA-5 itself exhibited high hemolytic activity and cytotoxicity, the formation of a crosslinked nanostructure in cLA-5 led to a qualitative leap in its biocompatibility. Its hemolysis ratio remained below 5% at concentrations up to 128 μg/mL, and it demonstrated excellent cytocompatibility with L929 cells (IC_50_ = 427.8 μg/mL). Its overall safety profile even surpassed that of polymyxin B. This transformation underscores the remarkable success and unique advantage of the cyclization and crosslinking strategy in balancing potent antimicrobial activity with host cell safety [41].

The overall biosafety ranking of these peptides can be approximated as: CA-5/MH-5 > cLA-5 > LA-5 > C_12_-5. Among them, cLA-5, through its rational structural design, successfully combines potent antibacterial efficacy with outstanding biocompatibility, representing the most promising therapeutic candidate with high clinical translation potential.

Experiments employing DiSC_3_(5) for membrane depolarization assessment, NPN uptake for outer membrane permeability, and SEM observation consistently confirmed that the core mechanism of action for C_12_-5 and cLA-5 remains membrane targeting. The potent bactericidal efficacy of the modified pentapeptides stems from the rapid, synergistic physical disruption of the bacterial cell membrane. SEM images visually demonstrated time-dependent morphological changes, progressing from membrane contraction and surface roughening to eventual pore formation and cytoplasmic leakage. This confirms that the modified pentapeptides retain the membrane-acting mechanism consistent with natural AMPs, which is associated with low resistance induction. Serial passage experiments confirmed that neither C_12_-5 nor cLA-5 induced detectable resistance, in sharp contrast to the rapid development of high-level resistance observed with ciprofloxacin. Furthermore, when combined with ciprofloxacin, both peptides demonstrated significant synergistic effects, substantially reducing the MIC of ciprofloxacin against resistant strains and effectively suppressing further resistance development. This promotes the considerable potential of these membrane-targeting peptides to act as resistance breakers or sensitizing agents in combination therapies with conventional antibiotics.

4. Materials and Methods

4.1. Materials

The test strains Escherichia coli ATCC 25922 (E. coli), E. coli DC 2 ATCC 8739 (E. coli DC 2), Staphylococcus aureus ATCC 25923 (S. aureus), Candida albicans ATCC 10231 (C. albicans), and methicillin-resistant Staphylococcus aureus ATCC 43300 (MRSA) were obtained from the Luria-Berta Broth (LB), Tryptone Soy Broth (TSB), Schar’s Dextrose Liquid Medium (SDB), Schar’s Dextrose Agar Medium (SDA), Nutrient Agar Medium (NAFM) and Mueller–Hinton Broth (MHB) were purchased from the Shanghai Biological Resource Conservation Center (SHBCC) (Shanghai, China). Broths (MHB) were purchased from Hopebiol (Qingdao, China).

Ethanol, iodinated 3,3-dipropylthiodicarbocyanine (DiSC_3_(5)), N-phenyl-1-naphthylamine (NPN), crystal violet, dimethyl sulfoxide (DMSO), CCK-8 Cell Proliferation and Cytotoxicity Assay Kit and other chemicals were purchased from Aladdin Co (Nanjing, China). NCTC clone 929 (L929 cells), PBS (Phosphate-Buffered Saline), Dulbecco’s Modified Eagle’s Medium high glucose (DMEM) and Fetal Bovine Serum (FBS) were obtained from KeyGEN BioTECH (Nanjing, China).

4.2. Peptide Synthesis

The peptides designed in this study were synthesized by solid-phase methods using standard Fmoc chemistry on Rink amide MBHA resin as previously described [42,43]. Lipid moieties (lauric acid, 5-methylhexanoic acid, cinnamic acid, and lipoic acid) were covalently conjugated to the N-terminus via identical condensation chemistry used for amino acid coupling [19]. The final peptides were purified and analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC, Waters, Milford, MA, USA) by addition of 95% trifluoroacetic acid after cleavage (TFA), 2.5% H_2_O, and 2.5% TIS, and the final peptides were extracted and analyzed by electrospray ionization mass spectrometry (EI-MS/MS). The results are summarized in Table 5.

4.3. Minimum Inhibitory Concentration (MIC) Experiment

The MICs of the antimicrobial peptides (C_12_-5, MH-5, CA-5, LA-5 and cLA-5) were determined against E. coli, S. aureus, MRSA, and C. albicans using the broth microdilution method. Mid-logarithmic phase bacteria, prepared by overnight culture and washed three times with PBS, were adjusted to 10^5^ CFU/mL. In a 96-well plate, two-fold serial dilutions of each peptide (starting at 512 μg/mL in MHB) were incubated with the bacterial suspension at 37 °C for 18 h [44]. The MIC was recorded as the lowest concentration with no visible growth. All experiments were performed in triplicate.

4.4. Stability Assessment

Physiological salts interfere with the electrostatic interactions between AMP and bacterial membranes, thus affecting the antibacterial activity of AMPs. The MICs of various salts (150 mM NaCl, 4.5 mM KCl, 1 mM MgCl_2_, 8 μM ZnCl_2_) against E. coli and S. aureus were determined using the MIC assay. Each experiment was repeated at least three times.

Different concentrations (3%, 10%) of serum were prepared using MHB medium. Antimicrobial peptides of 2× MIC were added to the serum solution and incubated at 37 °C for 6 h. After that, the MIC results determined by E. coli and S. aureus were used. Three independent experiments were performed in triplicate.

4.5. Drug Resistance Test

To assess the potential for drug resistance development, E. coli and S. aureus were subjected to serial passage in the presence of different modified lipopeptides, respectively. Bacteria survived after 24 h of incubation in the presence of a sub-inhibitory concentration (0.5× MIC) were transferred to fresh medium [45]. This serial passage was repeated for 15–23 cycles, and the MIC was re-evaluated after each passage to monitor for changes. Furthermore, the final passaged strains were tested for resistance to the modified lipopeptide using the same MIC assay. The experiment was independently performed in triplicate.

The combined antimicrobial effect of C_12_-5 and cLA-5 with ciprofloxacin was determined using a checkerboard assay. C_12_-5 and cLA-5 were diluted to concentrations of 1×, 1/2×, 1/4×, 1/8×, and 1/16× MIC, respectively. The diluted solutions were added vertically to a 96-well plate, followed by the horizontal addition of an equal volume of ciprofloxacin solution. Subsequently, 100 μL suspensions of ciprofloxacin-resistant E. coli and S. aureus (1 × 10^6^ CFU/mL) were added to each well. After incubation at 37 °C for 18 h, the OD at 600 nm was measured using a microplate reader [46].

The fractional inhibitory concentration index (FICI) was calculated as follows: FICI = (MIC of peptide in combination/MIC of peptide alone) + (MIC of antibiotic in combination/MIC of antibiotic alone). According to established criteria, synergy is defined as FICI ≤ 0.5, additivity as 0.5 < FICI ≤ 1.0, indifference (no significant effect) as 1.0 < FICI < 4.0, and antagonism as FICI ≥ 4.0 [47].

4.6. Time-Kill Kinetics Test

The bactericidal kinetics of C_12_-5, MH-5, CA-5, and cLA-5 against planktonic bacteria were evaluated using E. coli as an exemplar. Bacteria were cultured to mid-log phase in LB medium at 37 °C, washed with PBS, and diluted to 10^6^ CFU/mL in PBS. Peptides were added at 2× MIC final concentrations; control groups received PBS only. Mixtures (5 mL) underwent shaking incubation (37 °C, 200 rpm). Viability was assessed at 30, 60, 120, and 240 min by plating 100 µL aliquots on agar. Plates were spread evenly, dried, and incubated (37 °C, 14–16 h) for colony counting. Identical protocols were applied to S. aureus, MRSA, and C. albicans. The experiment was repeated at least 3 times.

4.7. Biofilm Resistance Measurement

E. coli was cultivated and washed following the method described above. A suspension of E. coli adjusted to 10^6^ CFU/mL was transferred to a 24-well plate and incubated at 37 °C for 72 h to induce biofilm formation [48]. The biofilm was then washed twice with PBS to eliminate non-adherent bacteria. Next, MHB medium supplemented with the antimicrobial peptide at 2× MIC was added to each well; wells containing only MHB medium served as blank controls. All treatments were performed in triplicate per well, and incubation was continued at 37 °C for an additional 5 h.

As previously reported, crystal violet (CV) staining can be used to assess biofilm biomass [49]. After discarding the supernatant from the antimicrobial peptide-bacteria co-incubation, the wells were washed twice with PBS. One milliliter of methanol fixative was added to each well, followed by staining with 0.1% crystal violet solution for 5 min. Finally, 1 mL of 33% acetic acid solution was added to solubilize the bound crystal violet, and the absorbance at 590 nm was measured using a microplate reader. Higher absorbance values correspond to greater bacterial biofilm biomass.

The ability of the peptide to inhibit bacterial biofilm formation was also evaluated. A 10^6^ CFU/mL bacterial suspension was mixed with the peptide at 2× MIC, and the mixture was added to a 24-well plate. This mixture was incubated at 37 °C for 72 h, with a 10^6^ CFU/mL bacterial suspension without peptide as the control group. Staining and absorbance detection were conducted as outlined in the preceding section.

Biofilm density was calculated using the formula below:

$$[eqn]$$

Each experiment was repeated at least three times per well, and bacterial biofilm content was determined as described above. The identical experimental protocol was used for S. aureus, MRSA, and C. albicans.

4.8. Biocompatibility Assays

The hemolytic activity of the peptide was assessed by monitoring the amount of hemoglobin released from rabbit RBCs, as described previously. Fresh rabbit RBCs were washed three times with PBS and diluted in PBS to a final concentration of 5% (v/v). Subsequently, equal volumes of the RBC suspension were mixed with a twofold serial dilution of the peptide solution (2–512 μg/mL) in a 96-well plate and incubated at 37 °C for 3 h. RBC suspension treated with 1% Triton X-100 and PBS served as positive and negative controls, respectively [50]. After centrifugation, the absorbance (OD) of the supernatant was measured at 455 nm using a microplate reader (Model 680, Bio-Rad, Hercules, CA, USA). The hemolysis rate (%) was calculated using the following formula:

$$[eqn]$$

Peptide cytotoxicity was evaluated using the CCK-8 assay with L929 fibroblasts. Cells were seeded at 5 × 10^4^ cells/well in 96-well plates and incubated overnight at 37 °C (5% CO_2_) to allow attachment [51]. After 24 h peptide treatment at varying concentrations, 10 μL of CCK-8 reagent was added to each well. Following a 2 h incubation, absorbance was measured at 450 nm to determine cell viability relative to untreated controls.

4.9. Outer Membrane Permeability Assay

The outer membrane permeability of E. coli was determined using the hydrophobic probe NPN (Aladdin Biochemical Technology Co., Ltd., Shanghai, China). Logarithmic growth phase E. coli was diluted in PBS to OD 600 = 0.2. Approximately 1.8 mL of bacterial suspension and 200 μL of 100 μM NPN solution were taken in a cuvette [52]. Next, 1× MIC of antimicrobial peptide was added. Then, the bacterial suspension and peptide solution were quickly mixed well. Fluorescence values were measured at an excitation wavelength of 350 nm and an emission wavelength of 420 nm with a scanning range of 360–600 nm. Each test was repeated independently at least three times.

4.10. Inner Membrane Potential Assay

Fluorescent probe DiSC_3_(5) was used to measure membrane potential changes in E. coli [53]. Bacteria in the logarithmic growth phase were diluted in PBS buffer to an OD 600 of 0.1. Subsequently, 10 mL of bacterial suspension was taken, DiSC_3_(5) dye was added to a final concentration of 0.8 μM, and the mixture was incubated in the dark for 90 min. Then, 500 μL of 4 mM KCl solution was added to the bacterial suspension. Subsequently, antimicrobial peptides at 1× MIC were added to the mixture. Immediately after mixing, the fluorescence emission spectrum of the solution was measured (excitation wavelength 622 nm, emission wavelength 670 nm). Changes in fluorescence intensity over time were recorded and the curve was plotted [54].

E. coli and S. aureus suspensions at 10^8^ CFU/mL were co-cultured with C_12_-5 and cLA-5 for 60 min and 240 min, respectively. The samples were fixed overnight with 2.5% glutaraldehyde, and dehydration was performed with an ethanol gradient. Dehydration was performed using an ethanol gradient [55]. Morphological changes before and after short peptide treatment were observed by SEM (Thermo Fisher Scientific, Hillsboro, OR, USA).

5. Conclusions

The linear Battacin-derived pentapeptide (DDLFD) was successfully modified with fatty chains, cinnamic acid, or lipoic acid, yielding a series of novel engineered lipopeptides. Evaluation of these compounds revealed that long fatty chain modification was the most effective strategy for enhancing antimicrobial activity. It was evidenced by the superior performance of lauric acid-modified pentapeptide (C_12_-5) over its 5-methylhexanoic acid-modified counterpart (MH-5) in both MIC and bactericidal kinetics. In contrast, modifications with cinnamic acid or lipoic acid (CA-5/LA-5) did not significantly improve antibacterial activity, despite conferring good biocompatibility. Notably, the lipoic acid-crosslinked nanoparticle (cLA-5) demonstrated the most potent antimicrobial activity comparable to C_12_-5, characterized by rapid bactericidal kinetics, excellent physiological stability (in saline and serum), and strong biofilm disruption capabilities. Furthermore, cLA-5 successfully resolved the common dilemma of balancing high activity with low toxicity, thus achieving a high safety threshold. Specifically, cLA-5 demonstrated a hemolysis rate of less than 5% at 128 μg/mL, while its MIC against most bacterial strains was only 4–8 μg/mL. The superior performance of C_12_-5 reflects an optimal C_12_ chain length that balances hydrophobic membrane insertion with minimal hemolysis, while cLA-5 operates through a unique nanostructure-mediated mechanism that promotes localized accumulation and multivalent membrane disruption. Although the core mechanism of action for C_12_-5 and cLA-5 remains membrane-targeting, the nanostructure of cLA-5 promotes localized accumulation at the membrane surface and more efficient multivalent disruption. This mechanism makes them less prone to inducing bacterial resistance and enables them to effectively reverse bacterial resistance to ciprofloxacin in combination therapy. These attributes make cLA-5 a promising therapeutic candidate that embodies a novel strategy for combating biofilm-associated and drug-resistant bacterial infections.