Structure‐Function Regulation of Proteolytically Stable Amphiphilic Peptide‐Based Soft Materials With Antimicrobial Efficacy Against Drug‐Resistant Strains and Anticancer Activity in Triple‐Negative Breast Cancer Cells

Abstract

The rapid progress of multidrug‐resistant (MDR) bacterial infections and the clinical aggressiveness of triple‐negative breast cancer (TNBC) possess a great problem to human beings. It necessitates the discovery and development of multifunctional therapeutic biomaterials. Herein, we report two enzymatically stable short cationic peptide hydrogelators comprising of N‐terminal lysine headgroups, non‐coded amino acid spacers, aromatic phenylalanine residues, and dodecylamine tails. These amphiphilic peptides are rapidly self‐assembled in Tris‐HCl buffer (pH 7.4) to form hydrogels with nanofibrillar network. Remarkably, both of these hydrogelators exhibit potent antibacterial activity against several Gram‐positive and Gram‐negative drug‐resistant strains, functioning through multitiered mechanism including membrane permeabilization, trans‐membrane depolarization, and intracellular reactive oxygen species (ROS) generation. Moreover, one of these peptide amphiphiles demonstrates a significant anticancer efficacy (with IC₅₀ value of 6.9 µM) against human TNBC cells by activating the extrinsic caspase‐8/caspase‐3‐driven apoptotic pathway and reversing epithelial‐to‐mesenchymal transition (EMT). Interestingly, the peptide with centrally located amino acid residue with lesser number of methylene units than that of the other peptide shows better anticancer efficacy and less antimicrobial activity. This study vividly demonstrates a tunable class of proteolytically stable amphiphilic peptide gelators with potential antimicrobial as well as anticancer activities establishing a structure‐function relationship of these bioactive peptide scaffolds suggesting a versatile blueprint for peptide‐based next‐generation soft bioactive materials.

Structure‐activity modulation of two proteolytically stable cationic peptide hydrogelators with dual therapeutic action: antibacterial activity against drug‐resistant strains and anticancer efficacy against triple negative breast cancer cells.

1. Introduction

The rapid expansion of multidrug‐resistant (MDR) microbial infection continues to undermine the clinical utility of conventional frontline therapeutics and possesses a formidable threat to public health [1]. In particular, the World Health Organization (WHO) has identified “ESKAPE” pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp.) as predominant prototypes causing majority of hospital‐acquired infections (HAIs) driving global rate of mortality and morbidity [2]. Among these pathogens, MDR variants of K. pneumoniae and methicillin‐resistant S. aureus (MRSA), two resilient phenotypes responsible for various lethal nosocomial infections, are continuously imposing clinical challenges even towards next‐generation antibiotics [3, 6]. In parallel, despite advances in molecularly guided and immune‐based therapies in different cancer lines [7, 11], durable response and acquired resistance remain pervasive obstacles [12]. In the realm of oncology, breast cancer remains one of the most frequent malignancies diagnosed in women worldwide [13, 14]. Incidence of highly aggressive multipotent subtype triple‐negative breast cancer (TNBC), characterized by absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expressions, underscores the demand for innovative therapeutic platforms capable of modulating cell death pathways while minimizing adverse side‐effects on non‐malignant, healthy tissues [15]. Moreover, the propensity of TNBC to metastasize into visceral organs, its immunologically complex tumor microenvironment, limited availability of actionable molecular targets and emergence of therapy‐resistant clones with intra‐tumoral heterogeneity further contribute to poor prognosis [16, 17]. The convergence of these two critical healthcare challenges necessitates the development of new, multidimensional therapeutic remedials that outsmart both bacterial resistance and the inherent survival strategies of cancer cells [18, 20].

Over the decades, peptide‐based biomaterials have emerged as promising chemotherapeutics due to their low fabrication cost, potential tunability and the capacity to generate spontaneous hierarchical supramolecular architectures [21, 26]. Especially, cationic amphiphilic peptide (CAP) moieties have been recognized as versatile bio‐functional platforms for bacterial [27, 31] and fungal infection prevention [32, 33], antiparasitic activity [34, 35], biofilm eradication [36, 37], wound healing [38, 39], targeted cargo delivery [40], sustained release of bio‐actives [41, 43], localized anticancer therapy [44, 45], cell culture and tissue engineering [46, 49]. The ability of low‐molecular‐weight gelators (LMWGs) to be self‐assembled into nanofibrillar network structures via various non‐covalent interactions provides a robust structural basis for programmable mechanical rigidity and stability under optimal physicochemical conditions [50, 57]. Their mechanism of action rooted in electrostatic interaction, hydrophobic insertion, and co‐operative self‐assembly which enables membrane disruption, metabolic perturbation. and oxidative stress induction, thereby circumventing the likelihood of conventional resistance development [44, 58]. Despite growing interest, few studies have explored the multidimensional action of peptide‐based bioactive materials that unify stability, biocompatibility, potent bactericidal efficacy along with mechanistically elucidated anticancer activity within a single molecular platform. Previously, Banerjee and co‐workers have reported a series of cationic peptide hydrogelator based antibacterial‐anticancer bi‐functional therapeutics with varying N‐terminal residues [20]. However, a major limitation of such traditional peptide‐based therapeutic scaffolds is their intrinsic susceptibility to undergo rapid degradation by endogenous proteases in biological environments, drastically restricting their half‐life and thus hampering practical in vivo applications [59]. Addressing these limitations, this current study aims to develop cationic peptide amphiphiles with the variation of core‐group sequences involving non‐coded amino acid residues to enhance the enzymatic stability against proteolysis. These structurally modified peptides self‐assembled into hydrogel matrices and facilitated bacterial death along with regression of breast cancer.

With cationic head groups, these two peptide amphiphiles (abbreviated as P1, P2) undergo gelation in Tris‐HCl buffer under physiological pH 7.4. These meticulously engineered molecular scaffolds with C‐terminal modification and presence of non‐proteinogenic amino acid residues in between two alpha‐amino acid building blocks successfully attenuated proteolytic stability. Supramolecular nanostructure of the gelators has been thoroughly examined with various microscopic (FE‐SEM, FEG‐TEM) and spectroscopic (FT‐IR, PXRD, CD) experiments while viscoelastic properties are testified via rheological experiments. Concerning the amphipathicity‐driven aggregation propensity, P1 and P2 gelators have been tested for antimicrobial activity where they have shown remarkable bactericidal as well as bacteriostatic potency against a wide range of clinically isolated MDR strains. Eventually, detailed studies regarding plausible mechanistic pathway of antibacterial action were explored in which extensive degradation of membrane integrity followed by trans‐membrane potential alteration was demonstrated which ultimately facilitated the elevation of intracellular ROS production within cytoplasmic matrix. Furthermore, beyond anti‐infective activity, these peptide moieties were scrutinized for growth inhibition of cancerous cells. Anticipating MTT assay analysis, pronounced cytotoxicity was obtained especially with peptide P1 against TNBC cells (MDA MB‐231) while it was found to be non‐cytotoxic towards normal mammalian cell line such as human peripheral blood mononuclear cells (PBMC). Elaborated studies revealed the probable mode of anticancer action emphasizing on apoptotic decay via extrinsic non‐mitochondrial pathway initiated by membrane‐bound receptor binding and was characterized by the activation of initiator caspase‐8 at the death‐inducing signaling complex (DISC) which subsequently cleaved and activated effector caspase‐3, leading to programmed cell death. Furthermore, P1 showed reversal of epithelial‐to‐mesenchymal transition (EMT)—a hallmark trait essential for invasive metastasis, marked by restoration of E‐cadherin (Ecad) mediated cell–to‐cell adhesion and suppression of N‐cadherin (Ncad) driven migratory signaling. Thus, the combination of caspase‐3/8 switching and cadherin reprogramming offers a mechanistically distinct strategy to outsmart chemo‐resistance of intrinsic pathway commonly observed in aggressive cancer phenotypes like TNBC and complements conventional approaches by restoring epithelial identity and enhancing therapeutic susceptibility. The insights presented herein broaden the functional landscape of peptide‐based soft biomaterials and substantiate their potential as dual‐action advanced therapeutics to tackle complex clinical applications.

Interestingly, P2 with a longer spacer, more tightly sequestered nanofiber packing and gel stiffness‐features that increased the persistence of cationic domains within the gelator network, promoted sustained interaction with anionic bacterial membranes and resulted in superior antibacterial efficacy in the self‐assembled state. In contrast, P1 with shorter molecular length, retained a higher population of soluble aggregates and monomers in molecular state that more effectively induced extrinsic caspase‐8/3 apoptosis and reversed EMT in TNBC cells. Collectively, these observations highlight subtle variations in spacer length within otherwise isostructural amphiphilic peptides can facilitate stark contrast in diverse biological functions and allow decisive molecular design parameters for independent tuning of antimicrobial activity and anticancer efficacy in the domain of multifunctional therapeutic platforms (Figure 1).

FIGURE 1.

Illustrates dual‐action therapeutic efficacy of peptide amphiphiles P1 and P2 where prominent molecular level anticancer activity has been observed in case of lesser aggregation‐prone P1 whereas antibacterial activities against Gram‐positive and Gram‐negative multidrug‐resistant strains have been obtained more efficiently in case of P2 in its self‐assembled dilute hydrogelator solution.

2. Materials and Methods

2.1. Procedure for Gel Preparation

Lyophilized peptides P1 and P2 were dispersed in Milli‐Q water or buffer systems of varying pH and subjected to sonication, followed by gentle heating up to 90 °C and cooling to room temperature. Both peptides formed stable hydrogels in Tris–HCl buffer (pH 7.2–8.0). To determine the gel‐to‐sol transition temperature (T _gel), hydrogels prepared at minimum gelation concentration (MGC) were placed in screw‐cap glass vials and gradually heated in a water bath.

2.2. Critical Aggregation Concentration (CAC) Measurements

To determine the CAC, 1 mM stock of hydrophobic fluorescent dye Nile Red (Excitation: 550 nm; Emission: 635 nm) in tetrahydrofuran (THF) was made from which in each of sixteen screw‐cap vials 10 μL of dye was taken and heated to evaporate THF. After cooling, peptide gelators in Tris‐HCl buffer (pH 7.4) solution with different concentrations were added followed by gradual heating up to 80°–90 °C and then again cooling back to room temperature. After keeping all the solutions in dark for 1h, fluorescence spectra were recorded with FluoroMax 3 spectrofluorometer (Horiba).

2.3. Disc‐Diffusion Assay

Overnight bacterial cultures were grown in LB medium at 37 °C, and their optical density was adjusted to 0.6 at 600 nm. Cultures were evenly spread on MH agar plates using sterile cotton swabs. Sterile discs (4 mm) were loaded with 20 μL of peptide solutions (100 μM) and placed on the plates. Following incubation for 16 h at 37 °C, the diameter of the inhibition zones (ZOI) was measured.

2.4. Broth Microdilution Assay

MIC values were determined using the standard broth microdilution method. Exponentially growing bacterial cells (OD 0.6 at 600 nm) were mixed with serially diluted peptide solutions in MH broth within sterile 96‐well plates. Plates were incubated for 16 h at 37 °C, and MIC values were obtained from absorbance readings at 600 nm. Each experiment was performed in triplicate.

2.5. Procedure for Enzymatic Stability Assay

P1 and P2 were separately incubated with chymotrypsin, proteinase‐K (in HEPES buffer of pH 7.46, 37 °C) and pepsin (in glycine‐HCl buffer of pH 2.0, 37 °C) with peptide concentrations of 50 µM each and digested for 36h. At different time intervals (6, 12, 18, 24, 30, 36h), aliquots were taken out and mass spectra (HRMS) were recorded in order to monitor enzymatic degradation over time for both P1, P2.

2.6. MTT Assay for Cell Viability

MDA‐MB‐231 cells were cultured in RPMI 1640 media supplemented with 10% FBS and 1% Penicillin‐streptomycin at 37 °C in a humidified atmosphere with 5% CO₂. Cells were regularly tested for contamination. MDA‐MB‐231 cells were plated in 96 well plates (3.0  ×  10³ per well) and allowed to adhere. The cells were then treated with increasing concentrations of cationic peptide amphiphiles P1, P2 for 24 h. After treatment, 20 μl of MTT was added to each well and incubated at 37 °C for 2–3 h. 150 μl DMSO was added to dissolve the formazan, developed in the reaction. The OD was taken in a microplate reader at 570 nm.

3. Results and Discussion

3.1. Synthesis and Self‐Assembly Studies

A set of two peptide based amphiphiles were synthesized via standard solution‐phase DCC/HOBt coupling procedure with a common structural motif: N‐terminal lysine residues as polar head groups, intervening non‐coded amino acids (4‐aminobutyric acid in P1 and 6‐aminocaproic acid in P2) conjugated with phenylalanine residues and finally terminated with long hydrocarbon chains of dodecylamine at C‐termini. Solubility and aggregation propensity of P1 and P2 in various aqueous buffer systems have been listed in Table S1. Interestingly, both peptides were self‐assembled into semi‐transparent hydrogels within 5–10 min in Tris‐HCl buffer solutions at pH range 7.2–8.0 which was further confirmed by vial inversion test (Figure 2A,B). At physiological pH 7.4 within Tris‐HCl buffer, minimum gelation concentrations (MGC) were found out to be 0.6% (W/V) for P1 and 0.4% (W/V) for P2. Thermal stability of the gel was procured by measuring gel‐melting temperature (T _gel). At MGC, gel‐to‐sol transition temperature values were recorded as 60 °C and 69.5 °C for P1, P2, respectively. Both of these hydrogels exhibited thermo‐reversible nature and thus can be stored for several months at room temperature without any notable change in their gel‐state.

FIGURE 2.

(A,B) Represent chemical structure of the cationic peptides (P1, P2) along with optical images of hydrogels at 0.8% (W/V) in Tris‐HCl buffer (pH—7.4) confirmed by vial‐inversion test; (C,E) represent frequency sweep rheological plots of P1, P2, respectively, at 0.1% constant strain; (D,F) represent amplitude sweep rheological plots of P1, P2, respectively, at 10 rad/s constant frequency.

Furthermore, to probe aggregation behavior, hydrophobic dye Nile Red (λ _ex = 550 nm; λ _em= 635 nm) was used to monitor the degree of encapsulation by virtue of its variation in fluorescence behavior depending on change in environment within aggregated system. With an increase in gelator amount for both P1, P2, rapid enhancement of emission intensity occurred after a certain concentration range at which self‐assembly began to allow the fluorescent probe to get entrapped within the hydrophobic interior in order to exhibit hyperchromic shift and that marked the CAC values—32.5 µM (for P1) and 21.8 µM (for P2) (Figure S1A,B). Thus, CAC values provided the quantitative onset limit of self‐aggregation propensities of P1 and P2.

3.2. Rheological Studies

In order to examine visco‐elastic responsiveness as well as mechanical stiffness, series of rheological experiments were executed with P1, P2 hydrogels. Both the gels were formed under identical condition with gelator concentrations of 0.8% (W/V) in pH = 7.4 Tris‐HCl buffer and were allowed to settle for 6h at room temperature. From amplitude‐sweep experiments with fixed angular frequency of 10 rad/s and varying strain in between 0.01% and 100%, linear viscoelastic (LVE) domain was procured by measuring variations of storage moduli (G’), loss moduli (G’’) values. From the graphical representation of both G’ and G’’ against mechanical strain, continuous decrease of G’’ as compared to G’ indicated the collapse of gel networks from crossover points at around shear strain of 0.5% for P1 and 0.3% for P2 which indicated stress‐tolerance limits of the hydrogels (Figure 2D,F).

3.3. Morphological Studies

To investigate the supramolecular morphologies of the hydrogels, field emission gun transmission electron microscopy (FEG‐TEM) and field emission scanning electron microscopic (FE‐SEM) experiments were carried out concurrently. Once again, identical gel preparation procedure was followed for both P1 and P2 in Tris‐HCl buffer (pH = 7.4) followed by corresponding dilution (final concentrations—50 μM) within deionized water to make gelator solutions ready for sampling to perform TEM and SEM analysis. From micrographs, inter‐twined nanofibrillar networks were observed for both P1 and P2 gelators (Figure 3A–D). Average diameters of these nanofibers were measured to be 16 nm for P1 and 18 nm for P2.

FIGURE 3.

(A,B) Correspond to FEG‐TEM (scale bar—250 nm) images of the peptide gelators formed by P1 and P2, respectively, (50 μM) in Tris‐HCl buffer (pH 7.4); (C,D) represent FE‐SEM (scale bar—200 nm) images of the peptide gelators (50 μM) of P1 and P2, respectively, in Tris‐HCl buffer (pH 7.4) at similar concentrations; (E,F) depict zeta potential plots from DLS experiments of P1, P2, respectively, at the same concentration 50 μM.

3.4. Dynamic Light Scattering (DLS) Experiment

To understated the nature of surface charge density in terms of zeta potential values within the self‐assembled gelator solutions of P1, P2, DLS experiments were performed. With gelator solutions of 50 μM concentration at room temperature (25 °C) under physiological pH, in case of both the peptides, zeta potential was 31.71 mV for P1 and 32.58 mV for P2 (Figure 3E,F). High positive zeta potential values of P1 and P2 convincingly established cationic surface charge distributions and colloidal dispersion stability via electrostatic interaction between gelator molecules.

3.5. FTIR and PXRD Analysis

In the secondary structures of these self‐assembled gelators, various non‐covalent interactions like H‐bonding, electrostatic interaction between polar head groups, Π‐Π stacking involving aromatic side‐chain residues, and hydrophobic interactions between hydrocarbon tails altogether contribute to stabilize the intermolecular packing arrangements that finally lead to promote gelation. To characterize various intermolecular interactions within gel network, FTIR spectroscopic studies and powder XRD analysis were performed with solid, lyophilized xerogels of P1 and P2. From FTIR spectra analysis, some signature bands were obtained such as 1628–1650, 2870–2945, and 3200–3450 cm⁻¹ for P1 and 1635–1648, 2890–2970, and 3190–3370 cm⁻¹ for P2 (Figure 4A,B). Broad bands around 3100–3400 cm⁻¹ were observed in case of both P1, P2 which indicated intermolecular H‐bonded and non‐H bonded N‐H stretching frequency of the free N‐terminal primary amines of lysine residues, respectively [20, 36]. Distinct bands around 1625–1650 cm⁻¹ represented amide‐I features (amide C = O stretching) of the peptide bonds [20]. Moreover, these bands at 1645 cm⁻¹ further indicated presence of interplanar β‐sheet like secondary structural features within gelator networks [36]. Lastly, symmetric and asymmetric aliphatic C–C stretching of methylene groups around 2870–2970 cm⁻¹ suggested highly ordered all‐trans conformations of P1, P2 gelator molecules in overall intermolecular stacking [20].

FIGURE 4.

(A,B) Show FTIR spectra of xerogel P1, P2, respectively, where region I represents 1625–1650 cm⁻¹ range, II represents 2870–2970 cm⁻¹ range, and III represents 3100–3400 cm⁻¹ in each spectral diagram; (C,D) depict wide‐angle powder XRD pattern recorded for the peptides P1 and P2 in xerogel state, respectively; (E) characteristic CD spectra of the peptide gelators in 50 μM concentrations.

With finely divided xerogel powders of P1 and P2, wide angle X‐ray diffraction was performed. From the spectral analysis, in case of both P1 and P2, Bragg’s spacing (d) values around 4.52 Å (2θ  = 20.9°) and 4.38 Å (2θ  =  20.9°), respectively, suggested inter‐strand separation between two adjacent β‐strands parallel to fiber axis which ultimately extended as β‐sheet like arrangements where inter‐sheet distances between two stacked antiparallel β‐sheet layers were indicated by d = 8.91 Å (2θ  = 10.5°; for P1) and d  =  8.77 Å (2θ  = 10.5°; for P2) [36]. Furthermore, d  = 3.63 Å (2θ  =  23.2°) and d = 3.56 Å (2θ  =  23.2°) in both P1 and P2, respectively, possibly suggested Π‐Π stacking interaction between aromatic side‐chain residues of phenylalanine moieties (Figure 4C,D) [20, 36].

3.6. Circular Dichroism (CD) Analysis

To further investigate the secondary structural configurations within the gelator scaffolds of P1 and P2 in Tris‐HCl buffer medium, CD studies were done at room temperature using dilute gelator solutions (50 μM). Following up the CD spectra, maxima (positive Cotton Effect) at 194–196 nm range and prominent minima (negative Cotton Effect) at 216–218 nm range have been obtained as signature bands for both P1, P2 gelators, which strongly supported the presence of β‐sheet like secondary structural features within gel networks (Figure 4E).

3.7. Antibacterial Studies

3.7.1. Zone of Inhibition (ZOI) Measurement

In order to explore antimicrobial activity of P1 and P2 gelators, first, bacterial growth inhibition efficacy was testified in terms of disc‐diffusion assay. Analyzing the observations of zone diameter values performed with a variety of Gram‐positive and Gram‐negative MDR clinically isolated bacteria strains [MRSA (ATCC BAA 2313), Bacillus substilis (ATCC 23 857), K. pneumoniae (ATCC BAA 1705), Escherichia coli (ATCC 25 922), P. aeruginosa (ATCC BAA 3104), A. baumannii (ATCC 19 606)], dilute gelator solutions of both P1 and P2 (concentrations—50 μM in Tris‐HCl buffer of pH 7.4) exhibited potential bacteriostatic activities against all of these strains. From the optical images of the plates, ZOI was measured as corresponding zone diameter values ranging from 14–19 mm (Figure 5A, Table S2).

FIGURE 5.

(A) Displays optical images of disc diffusion assay (on Mueller–Hinton agar plates) performed with dilute gelator solutions of P1, P2 against six bacterial strains to measure zone of inhibition diameter values; (B–G) show FE‐SEM images (scale bar‐1 μm) of Gram‐positive MRSA and Gram‐negative K. pneumoniae before (control) and after treatment (50 μM) with diluted gelator solutions of P1 and P2.

3.7.2. Minimum Inhibitory Concentration (MIC) Measurement

After primary screening of bacterial growth inhibition potency of P1 and P2 gelators against a broad spectrum of various MDR strains, serial broth micro‐dilution technique was opted to quantify MICs by measuring optical density at 600 nm (OD₆₀₀). From the calculated MIC dataset, it was further established that P2 exhibited better microbial growth inhibition activities against the six tested strains than that of P1. MIC values were obtained within the range of 25.2–42.8 µM and these are listed in Table S2. Among tested two Gram‐positive strains, better antibacterial efficacy was observed against MRSA (ATCC BAA 2313) with ZOI: 16 mm, MIC: 34.2 µM (for P1) and ZOI:20 mm, MIC: 25.2 µM (for P2) while among tested four Gram‐negative strains, the peptide gelators showed higher activity against K. pneumoniae (ATCC BAA 1705) with ZOI: 17 mm, MIC: 35.7 µM (for P1) and ZOI: 21 mm, MIC: 28 µM (for P2). These two MDR strains were chosen as inoculum to anticipate detailed in‐vitro mechanistic studies regarding plausible antibacterial pathway displayed by the self‐assembled peptide gelator scaffolds.

3.7.3. FE‐SEM Imaging Studies

FE‐SEM analyses were carried out to investigate the alteration in cellular surface morphologies of treated bacteria cells in comparison to untreated cells to probe primary bactericidal mechanism [60]. For this purpose, MRSA (ATCC BAA 2313) and K. pneumoniae (ATCC BAA 1705) were treated with diluted gelator solutions (50 μM each) of P1 and P2 in Tris‐HCl buffer of pH 7.4. From the micrographs, prominent deformation of cell morphologies with ruptured; wrinkled surfaces along with leakage of intracellular matrix were observed in case of post‐treatment bacteria cells whereas for live bacteria cells, smooth and intact surfaces were observed (Figure 5B–G). Moreover, the self‐assembled nanofibrillar aggregation of the gelators induced agglutination by entrapping bacterial cells within the fibrous network and thus promoted effective membrane disrupting cytolysis which was evident from the micrographic images.

3.7.4. Confocal Microscopic Studies

To correlate FE‐SEM data supporting membrane rupture leading to cellular decay, bacterial live/dead assay was conducted with MRSA (ATCC BAA 2313) and K. pneumoniae (ATCC BAA 1705) treating with the same concentration for P1, P2 gelator solutions. In this microscopic analysis, SYTO‐9 and Propidium Iodide (PI) were used as a combinational fluorescent counterstain to distinguish treated and untreated bacteria cells [61]. Live cell‐permeable, fluorescent dye SYTO‐9 with the ability to emit bright green fluorescence after binding to nucleic acids whereas cell impermeable PI had the selectivity to get intercalated into DNA within membrane compromised cells imparting intense red fluorescence. Thus, from confocal laser micrographs, bacterial cell degradation ability of self‐assembled gelator solutions of P1 and P2 has been further substantiated where control cells showed green emission due to SYTO‐9 with minimum red spots while treated, membrane compromised dead cells exhibited major red fluorescence of PI (Figure 6A–X).

FIGURE 6.

Confocal microscopic images for cellular live/dead fluorescence imaging assay of K. pneumoniae [(A–L)] and MRSA [(M–X)] dual stained with SYTO‐9 (green emission) and PI (red emission) along with merged images before (control) and after treatment with P1, P2 gelator solutions.

3.7.5. Outer and Inner Membrane Permeability Assay

To assess the dose‐dependent bactericidal actions of P1 and P2 gelators, quantitative bacterial cell membrane permeability assays were performed by measuring intensity variations using selected fluorescence probes. Gram‐negative bacteria contain well‐defined lipophilic outer membrane composed of LPS, teichoic acid residues while Gram‐positive analogs have no such additional outer membrane rather a thick, multilayered sheath of peptidoglycan cell wall. Both strains have selectively permeable phospholipid bilayers as inner plasma membrane. For outer membrane permeability assay, 8‐anilinonaphthalene‐1‐sulfonic acid (ANS) was used as fluorescence probe which is hydrophobic and thus unable to show fluorescence in aqueous environment but its emission intensity gets enhanced upon binding to lipid‐rich interior hydrophobic regions of damaged and exposed cell‐membrane components [36, 62]. From the intensity versus concentration plot, it was evident that in case of Gram‐negative K. pneumoniae (ATCC BAA 1705), with increase in dosages of P1, P2 gelators, significant enhancement of fluorescence intensity was observed that referred to outer membrane penetration capability of the peptide gelators (Figure 7A).

FIGURE 7.

(A) Demonstrates outer membrane permeability assay of P1, P2 gelators with Gram‐negative K. pneumoniae (ATCC BAA 1705) using ANS probe; (B,C) depict inner membrane permeability assay of P1, P2 using Propidium Iodide (PI) probe. For (A–C), Triton X‐100 treatment was used as positive control and untreated bacteria suspensions were used as negative control; (D,E) show trans‐membrane potential alteration ability of P1 and P2 gelators over time in dose‐dependent manner against K. pneumoniae, respectively, where DiSC₃(5) was used as fluorescent probe; (F,G) exhibit intracellular ROS generation ability of P1 and P2 gelators against K. pneumoniae, respectively, using fluorescent probe DCF (active form of DCFDA). Untreated bacteria solutions were used as control for these experiments (D–G). All the experiments (A–E) were triplicated.

In case of cytoplasmic inner membrane permeability assay, uptake of live cell‐impermeable nuclear counterstain propidium iodide (PI) was studied to rationalize membrane deformation and penetration with both the bacteria strains MRSA (ATCC BAA 2313) and K. pneumoniae (ATCC BAA 1705) [36, 62]. In this study, an increase in fluorescent intensity of PI was observed with the ascending gelator concentrations and it was apparent from the bar diagrams of Figure 7B,C. This can be due to the fact that PI would have intercalated into DNA strands after the treatment of antimicrobial peptides P1, P2 with bacterial cells causing damage to inner membranes of the bacterial cells. From the overall quantitative data, membrane degradation ability of P2 was found out to be comparatively higher than that of P1 which matched the primary screening results obtained from ZOI and MIC values.

3.7.6. Trans‐Membrane Depolarization Assay

Bacterial trans‐membrane potential regulated via proton motive force (PMF), is an important physiochemical biomarker of cell sustainability. Thus, loss of membrane integrity by therapeutics often affects this electrostatic potential resulting in notable change in membrane fluidity and bacterial homeostasis which can be monitored by the time dependent response of a potential sensitive fluorescent dye 3,3^′‐dipropylthiadicarbocyanine iodide [DiSC3(5)] [36, 62]. Upon exposure to cationic gelator solutions of P1, P2, effective increase in fluorescent intensity of the probe by virtue of charge depolarization was observed in a concentration‐dependent manner over time with respect to control that indicated cytoplasmic potential alteration leading to cellular death (Figures 7D,E and S2C,D).

3.7.7. Estimation of ROS Generation

The combined effect of membrane penetration, respiratory dysfunction caused by proton motive force (PMF) alteration and collapse of synchronized electron transport chain (ETC) system ultimately resulted in oxidative stress generation [63]. Thus, amphiphilic cationic antimicrobial therapeutics have been widely reported to induce various reactive oxygen species (ROS) formation, primarily in trans‐membrane junction and finally within cytoplasmic matrix. To explore such ROS generation ability of lipopeptides P1, P2 within treated cells of both the strains, a fluorescent probe 2^′, 7^′‐dichlorodihydrofluorescein diacetate (DCFDA) was used [20, 36]. DCFDA is deacetylated to DCFH via bacterial intracellular esterase which in turn reacts with cytoplasmic ROS (majorly with H₂O₂) in order to get transformed into fluorescent‐activated form DCF. In comparison to untreated bacteria cells, peptide‐treated bacteria portrayed significant increase in emission intensity dose‐dependently (Figures 7F,G and S2AB).

Thus, assessing the in vitro mechanistic details of bactericidal efficacy, it is evident that after primary attachment to anionic bacterial cell surface via electrostatic interaction, the self‐assembled nanofibrillar cationic peptide gelators ruptured outer and inner membranes, disrupted trans‐membrane potential which in turn produced extreme metabolic stress within intracellular matrix that led to overproduction of highly deleterious ROS.

3.7.8. Proteolytic Stability Assay

In the sequence of both P1 and P2, there exist non‐proteinogenic amino acid residues‐4‐aminobutyric acid and 6‐aminocaproic acid, respectively, in between two α‐amino acid units and C‐termini of these peptides were modified with long fatty acyl chains of dodecylamine. Such suitable molecular design allows absence of peptide linkages between two naturally coded L(α)‐amino acid residues and thus these peptides were expected to exhibit resistance towards proteolytic degradation by any exogenous or endogenous protease. To verify this, enzymatic stability assay was performed with a set of three endopeptidases—pepsin, chymotrypsin and proteinase‐K [31]. Under optimal pH and at physiological temperature (37 °C), mass spectrometric analysis was carried out with enzyme‐incubated peptide gelator solutions at different time intervals [31]. From the graphical representation of % enzymatic stability based on high‐resolution mass spectrometric (HRMS) data, no such degradation was observed in presence of these enzymes up to 36 h (Figure S3).

3.8. Anticancer Studies

3.8.1. MTT Cell Viability Assay

For evaluating the cytotoxicity effect for peptide amphiphiles P1 and P2 on TNBC cells (MDA‐MB 231) in vitro, MTT assay was performed. The % cell viability data showed that P1 had greater growth inhibitory effect on TNBC cells as compared to P2. While P1 showed IC₅₀ value of 6.9 μM, P2 showed IC₅₀ values of 19.25 μM after 24h treatment (Figure 8A). Consequently, further experiments regarding plausible mechanistic pathway of anticancer potency were carried out using P1 as treatment. Furthermore, to assess the biocompatibility of the peptides in normal eukaryotic cells, MTT assay was carried out with human peripheral blood mononuclear cells (PBMC) as well as with human embryonic kidney (HEK 293) cells in which both P1 and P2 exhibited no significant cytotoxicity within the therapeutic window. After 24h of incubation, around 90% PBMC cells were viable in case of P1 while around 85% PBMC cells were viable in case of P2 at 200 µM concentration and in case of HEK‐293, around 70% cells were viable in case of P1 while 67% cells were viable in case of P2 up to 200 µM concentration (Figures 8B and S4). This dosage tolerance limit (considering up to 200 µM) was found out to be well above the IC₅₀ values obtained in case of post‐treatment cancerous MDA‐MB 231 cells and also far beyond the MIC ranges considering antibacterial activities.

FIGURE 8.

(A,B) Depict in‐vitro anticancer activity of P1, P2 in MDA‐MB 231 cell line in terms of MTT assay where % cell viability was plotted against concentration; (C,D) show the early apoptotic decay of MDA‐MB 231 cells after treating with P1 in terms of FITC‐Annexin V/Propidium Iodide (PI) assay. Here, Comp‐FITC‐A as abscissa represents cell population with FITC‐Annexin V component and ordinate Comp‐PI represents Propidium Iodide component.

3.8.2. Apoptotic Assay

Apoptosis assay using FITC‐Annexin V/PI dual staining method showed significant increase (p < 0.01) in early apoptotic cells (Annexin V⁺/PI⁻) in case of MDA‐MB 231 upon treatment with P1. Early apoptosis has been marked with increase in phosphatidylserine externalization on outer part of exposed cell membrane where Annexin V can bind while nuclear counterstain PI remains unable to enter within the cells due to non‐significant membrane lysis [64]. Flow cytometry data revealed that P1 effectively triggers early apoptosis within the treated cell populations (39.5%) with the enhancement of FITC‐Annexin V binding cellular populations along with simultaneous decrease in PI binding cell populations as compared to control where only 9.72% was found to be early apoptotic cells (Figure 8C,D). The changes in late apoptosis remained non‐significant. These data suggested that the induction of early apoptosis in MDA‐MB‐231 cells may occur under the influence of P1 which ultimately led to programmed cell death (Figure S5A).

3.8.3. Caspase 3/8 Activity

To investigate whether the observed apoptosis was mediated by extrinsic receptor‐dependent pathway or intrinsic mitochondria‐dependent pathway, caspase activities were analyzed using flow cyclometric experiments. Elevation of caspase‐8 (Cas 8) and caspase‐3 (Cas 3) activity was observed after treatment with P1 which subsequently indicated receptor‐binding, non‐mitochondrial extrinsic apoptosis within the TNBC (MDA‐MB 231) cells in presence of P1 (Figures 9A,B and S5B,C). Upon primary interaction of the anionic cell membrane, cationic peptide amphiphile P1 might initiate engagement of membrane‐bound death receptors to culminate the activation of caspase cascade bypassing the mitochondrial involvement. Significant post‐treatment increase (34.9%) in initiator Cas 8 orchestrated the translation of death‐receptor signals into proteolytic cascades which in turn converged on concomitant activation of executioner Cas 3 (45.1% in treated cells). Thus, preferential activation of Cas 8/Cas 3 switch avoiding mitochondrial route by P1 may offer a promising therapeutic approach to overcome apoptotic evasion caused by mitochondrial rewiring, a hallmark often associated with chemoresistance in aggressive TNBC.

FIGURE 9.

(A) Demonstrate flow cytometry data of caspase‐3 enhancement and (B) represent caspase‐8 enhancement after treatment of MDA MB 231 cells with P1. Here, FITC‐A represents the positive cell population for Cas‐3 and Cas‐8 and FSC‐H indicates forward scattered height of the cells. Caspase8/3 activated apoptosis was rationalized by increase in percentage of FITC‐A positive cells.

3.8.4. EMT Reversal Assay

EMT plays a pivotal role in tumor invasiveness in aggressive cancer phenotypes like TNBC [12]. Biomarkers such as E‐cadherin/N‐cadherin switching regulate EMT in metastatic dissemination of cancer cells [65]. From flow cylometric data, P1 was shown to exhibit EMT reversal by up‐regulating epithelial marker E‐cadherin along with simultaneous down‐regulation of mesenchymal marker N‐cadherin expression and thus possibly assisted restoration of cell‐to‐cell adhesion along with reduction of migration and motility which supported the scratch assay observations (Figures 10A,B and S5D,E). This EMT reprogramming coupled with extrinsic apoptosis induced by peptide P1 may provide a distinct synergistic strategy to attenuate malignancy while fostering therapeutic susceptibility.

FIGURE 10.

(A,B) Represent the flow cyclometric data for epithelial‐to‐mesenchymal transition (EMT) reversal potency of P1 by E‐cadherin up regulation and N‐cadherin down regulation after treatment with P1. Here, FITC‐A represents the positive cell population for E‐cad and N‐cad and the ordinate FSC‐H indicates the forward scattered height of the cells.

3.8.5. In‐Vitro Scratch Assay

Scratch assay (wound healing assay) was performed in order to explore cellular migration inhibiting ability of P1 in TNBC MDA‐MB 231 cell line where it has been observed that the scratch length was significantly reduced (p < 0.001) in case of control untreated cells whereas in case of P1‐treated cells the scratch length reduced much lesser (p < 0.05) (Figure S6). These data showed that P1 significantly reduced cellular proliferation, adherence and migration of MDA‐MB 231 cells to enclose the scratch wound over the span of 24 h suggesting anti‐metastatic property of P1 in TNBC in vitro (Figure S6).

4. Conclusion

This study introduces a structurally robust class of amphiphilic cationic peptide‐based hydrogelators (P1, P2) that integrate enzymatic stability with diverse therapeutic activity. The nanofibrillar networks disrupt bacterial membranes through a co‐operative mechanism involving membrane permeabilization, depolarization and overproduction of ROS‐mediated oxidative stress, effectively eliminating both Gram‐positive and Gram‐negative MDR strains. Concurrently, one peptide (P1) exerts excellent anticancer activity against TNBC cells by activating the extrinsic caspase‐8/caspase‐3 apoptotic pathway and reversing EMT‐associated phenotypes, thereby suppressing hallmarks of metastasis. These findings highlight the ability of rational peptide‐based molecular design to couple self‐assembly with multifunctional therapeutic outcomes. Moreover, from the comparative evaluation of P1 and P2 amphiphiles, a critical structure–function paradigm was established: better supramolecular assembly favors antibacterial efficacy whereas lesser aggregation tendency promotes molecular‐level anticancer efficacy. P1, with a shorter spacer and lower aggregation propensity (CAC 32.5 µM), showed superior anticancer activity (IC₅₀ of P1 = 6.9 µM < IC₅₀ of P2 = 19.25 µM) compared to P2. This indicated an enhanced anticancer efficacy of P1 in the monomeric state. In contrast, P2, with a longer spacer and higher aggregation tendency (CAC 21.8 µM), exhibited better antibacterial activity (MIC range for P2 = 25.2 μM–38.2 μM < MIC range for P1 = 34.2 μM–42.8 μM) than P1. This is consistent with the better self‐assembly‐driven antimicrobial action of P2 than that of P1. Thus, this study offers a promising foundation for tailoring advanced soft biomaterials targeted toward infection‐associated malignancies.

Author Contributions

Supratim Bose: methodology, writing – original draft, data curation, validation, formal analysis. Ananya Das: methodology, formal analysis. Swapnendu Deb: methodology, data curation, writing – original draft, formal analysis. Tanushree Mondal: methodology, data curation, validation, formal analysis. Nabanita Chatterjee: supervision, methodology, validation, investigation, formal analysis. Arindam Banerjee : conceptualization, writing – review & editing, supervision, visualization, validation, formal analysis, investigation, project administration, resources.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supplementary Material

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.