Synergistic antimicrobial nanocomposite: Protamine-loaded PVP-capped silver nanoparticles for combating multidrug-resistant infections

Abstract

The emergence of multidrug-resistant (MDR) bacterial infections has become a critical global health concern, driving the urgent need for innovative therapeutic strategies beyond conventional antibiotics. In this study, we developed a novel nanotherapeutic platform comprising protamine-loaded poly(N-vinyl-2-pyrrolidone)-stabilized silver nanoparticles (PVP-AgNPs) designed to enhance antimicrobial efficacy while improving biocompatibility. AgNPs were synthesized via chemical reduction using silver nitrate and sodium borohydride, with PVP serving as a stabilizing and capping agent. Protamine sulfate, a cationic antimicrobial peptide, was subsequently incorporated to exploit its strong affinity for bacterial membranes and synergistic bactericidal effects. The resulting nanoparticles exhibited spherical morphology with an average diameter of 70.96 ± 0.27 nm, a PDI of 0.25 ± 0.002, a zeta potential of +10.4 ± 0.15 mV, and a protamine loading efficiency of 66.2% ± 3.2%, indicating excellent colloidal stability and drug incorporation. In vitro antimicrobial testing demonstrated a two- to four-fold reduction in minimum inhibitory concentration values compared to blank PVP–AgNPs and free protamine, with disk diffusion assays confirming significantly enhanced activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria. Furthermore, in vivo evaluation using a murine wound infection model showed effective bacterial clearance, accelerated wound closure, and improved tissue regeneration. These findings demonstrate that protamine-loaded PVP-AgNPs offer multimodal antibacterial activity, enhanced cytocompatibility, and the potential to overcome resistance mechanisms. Collectively, this work introduces a promising nanotherapeutic strategy for managing MDR infections and promoting wound healing, with significant implications for future biomedical applications.

Graphical abstract

1. Introduction

The rapid rise of multidrug-resistant (MDR) and extensively drug-resistant (XDR) bacterial pathogens has emerged as one of the most pressing threats to global public health [1]. These pathogens compromise the efficacy of frontline antibiotics, leading to prolonged infections, higher mortality rates and increased healthcare costs [2]. Bacterial resistance to antibiotics occurs through several mechanisms, such as modification of drug targets, enzymatic inactivation, decreased membrane permeability, and upregulation of efflux pumps. These strategies reduce the intracellular concentration of antibiotics and impair their effectiveness [3]. Despite the severity of this crisis, antibiotic discovery has stagnated over the past decades, underscoring the urgent need to redesign and optimize existing antibacterial agents into advanced therapeutic platforms capable of effectively combating resistant infections [4].

Nanomedicine has emerged as a promising strategy for combating MDR bacterial infections [5]. Metallic nanoparticles, particularly silver nanoparticles (AgNPs), have demonstrated remarkable antimicrobial potential due to their high surface area, enhanced reactivity and ability to disrupt essential bacterial structures and functions [6]. AgNPs exhibit broad-spectrum activity against both Gram-positive and Gram-negative bacteria through multiple mechanisms, including membrane disruption, inhibition of enzymatic and metabolic pathways, interference with protein and ribosomal function, ATP depletion, and generation of reactive oxygen species (ROS), ultimately leading to bacterial death [7]. Despite their strong antibacterial efficacy, conventional AgNP formulations face significant challenges such as colloidal instability, uncontrolled release, potential cytotoxicity and limited bioavailability, which restrict their clinical translation.

To overcome these drawbacks, stabilizers are often employed to enhance AgNP dispersion and biocompatibility. Poly(N-vinyl-2-pyrrolidone) (PVP) is one of the most widely used capping and stabilizing agents for AgNPs due to its amphiphilic structure, which regulates nanoparticle morphology, prevents aggregation, and improves colloidal stability [8]. PVP functions as a protective agent by forming coordination bonds between its nitrogen atoms and the silver surface. Furthermore, PVP exhibits strong potential for drug binding, serving as a molecular bridge between the drug molecules and the silver surface. This interaction primarily occurs through hydrogen bonding involving its two highly active sites, the nitrogen atom and the carbonyl (–C=O) group present on its polymeric backbone. This dual interaction enhances the stability and functionality of PVP-coated AgNPs, reducing MDR and XDR [9]. In this study, AgNPs were synthesized via chemical reduction using sodium borohydride (NaBH₄) and silver nitrate (AgNO₃), with PVP serving as both a stabilizing and capping agent to improve nanoparticle uniformity and enable surface functionalization for targeted bacterial interactions [10]. Nevertheless, even stabilized AgNPs often exhibit reduced antimicrobial efficacy against MDR pathogens, and concerns regarding toxicity remain unresolved. This highlights the need for further functionalization strategies that not only stabilize nanoparticles but also enhance their antimicrobial potency and safety profile.

Functionalization with bioactive molecules, particularly antimicrobial peptides (AMPs), has emerged as a promising approach to improve the therapeutic performance of AgNPs [11]. AMPs are natural defense molecules with broad-spectrum activity, acting as molecular “knives” that disrupt bacterial membranes, increase permeability, and allow intracellular entry of antibacterial agents [12]. Protamine sulfate, a cationic AMP derived from fish sperm, has demonstrated significant antimicrobial and wound-healing properties in both in vitro and in vivo models [13]. Its mode of action is largely driven by electrostatic interactions with negatively charged bacterial cell walls, leading to leakage of potassium ions, ATP and intracellular enzymes, ultimately resulting in bacterial death [14]. Recent studies have further highlighted that loading protamine into nanocarriers enhances its stability, reduces immunogenicity, and improves its bactericidal efficacy [15].

Despite these promising advances, little is known about the stability and antimicrobial performance of AgNP–AMP conjugates, and the synergistic integration of protamine with PVP-stabilized AgNPs has not been systematically explored. In this study, we developed a novel protamine PVP–AgNP nanocarrier and investigated its antibacterial efficacy against clinically relevant MDR pathogens, including Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus). The physicochemical properties, minimum inhibitory concentration (MIC), and disk diffusion profiles were analyzed to assess antimicrobial potency, while cytocompatibility was examined using human dermal fibroblasts (HDFs). Furthermore, therapeutic efficacy was validated in a murine wound infection model to evaluate the potential of the nanocomposite in accelerating wound healing. The synergistic protamine PVP–AgNP platform is expected to eradicate resistant bacteria through multiple complementary pathways while minimizing cytotoxic effects and enhancing nanoparticle stability. Collectively, this work introduces a promising nanotherapeutic strategy with significant potential to address the growing global challenge of antimicrobial resistance and to improve clinical outcomes in infected wound management.

2. Materials and methods

2.1. Materials

AgNO₃ (>99%) as a precursor, NaBH₄ (>99%) as a reducing agent, and PVP (MW 40,000 kDa) as a capping agent were used for AgNP synthesis, with all chemicals purchased from Alfa Aesar (Ward Hill, MA, USA). Methylcellulose (MC) and protamine sulfate salt from salmon (Grade X, amorphous powder, MW ∼5.1 kDa) were obtained from Sigma-Aldrich (USA) and used as received, with the protamine sulfate employed for surface loading onto the synthesized AgNPs. Microbial culture media Mueller Hinton Broth (MHB) and Mueller Hinton Agar (MHA) powder and cytotoxicity assay materials were purchased from HI Media Laboratories (USA) and used for inoculating and propagating organisms, conducting total viable counts, and cultivating stock cultures.

2.2. Synthesis of PVP–AgNPs

The synthesis of AgNPs was performed following a previously reported protocol with minor modifications [16]. Briefly, 1.0 ml AgNO₃ solution (100 mM) was added to 4.0 ml distilled water containing 4.0 ml PVP (0.02–0.2 mM) as a stabilizing agent, and the mixture was magnetically stirred at 1000 rpm for 30 min. Subsequently, 1.0 ml freshly prepared NaBH₄ solution (10–100 mM) was added dropwise at a rate of approximately one drop per second. After 15 min of continuous stirring, the reaction mixture developed a characteristic yellow-brown coloration, confirming the successful reduction of Ag⁺ ions and the formation of AgNPs. The resulting nanoparticles were then purified by centrifugation to remove excess PVP and any remaining impurities, following previously described procedures [17].

To prepare protamine-loaded PVP–AgNPs, protamine was first dissolved in distilled water at concentrations ranging from 0.062 to 1.0 mg/ml, and then incubated with the pre-synthesized AgNPs under gentle stirring to facilitate peptide adsorption. The resulting nanoparticle suspension was subsequently purified by ultrafiltration using Amicon Ultra-15 centrifugal filters (100 kDa NMWL) to remove unbound protamine. The purified protamine-loaded nanoparticles were stored at 4 °C until further physicochemical characterization.

2.2.1. Characterization of PVP–AgNPs

The physicochemical properties of the blank and protamine-loaded PVP–AgNPs were characterized using multiple analytical techniques. Particle size, polydispersity index (PDI) and zeta potential were measured using dynamic and electrophoretic light scattering (Zetasizer Nano ZS, Malvern Instruments, UK). UV–Visible spectroscopy (200–800 nm) was performed to confirm nanoparticle synthesis and assess optical properties related to surface plasmon resonance (SPR) [18]. The morphology and size distribution of the nanoparticles were further analyzed using transmission electron microscopy (TEM) (JEM 1011, JEOL) after staining with phosphotungstic acid and uranyl acetate. Fourier-transform infrared (FTIR) spectroscopy (VERTEX 80v, Bruker) was used to identify functional groups and possible interactions in the nanoparticle matrix. Thermal stability was evaluated by thermogravimetric analysis (TGA/DSC2, Mettler Toledo, Switzerland).

Loading efficiency (LE%) of protamine was determined via centrifugal ultrafiltration method followed by quantification using high-performance liquid chromatography (HPLC). Post-lyophilization stability of the nanoparticles was assessed through comparative analysis of UV–Vis spectra, particle size, and zeta potential before and after freeze-drying in the presence and absence of cryoprotectants.

2.3. Evaluation of antibacterial activity of AgNPs

2.3.1. Bacterial strains

The antimicrobial activity of the synthesized AgNPs was assessed against clinically relevant bacterial strains, including E. coli (extended-spectrum β-lactamase [ESBL] strain, ATCC 51446), methicillin-resistant (S. aureus) (MRSA), and P. aeruginosa (ATCC 27853), which were kindly provided by the University of Jordan Hospital. The preparation of test organisms followed a previously described protocol [19]. Briefly, bacterial strains were sub-cultured on MHA plates and incubated at 37 °C for 24 h. Colonies from freshly grown overnight cultures were then suspended in 5 ml sterile broth and incubated at 37 °C in a shaking incubator at 225 rpm for an additional 24 h. The bacterial suspension was standardized to the 0.5 McFarland turbidity standard, corresponding to approximately 1.5 × 10⁸ CFU/ml, by adjusting the optical density using a spectrophotometer at 600 nm (OD₆₀₀, absorbance range: 0.08–0.13). Positive and negative controls were included to ensure experimental reliability. Polymyxin B (10 µg) and vancomycin (5 µg) served as positive controls, whereas sterile distilled water was used as the negative control.

2.3.2. MIC and MBC assays

The minimum inhibitory concentration (MIC) of blank and protamine-loaded PVP–AgNPs was determined using the broth microdilution method in accordance with CLSI guidelines against clinical isolates of P. aeruginosa, S. aureus and E. coli. Bacterial strains were first cultured overnight in MHB at 37 °C. The resulting cultures were then diluted in fresh MHB and adjusted to the 0.5 McFarland standard (∼1 × 10⁸ CFU/ml) using a densitometer (DENSICHECK, bioMérieux, France). Equal volumes of the standardized bacterial suspension and a two-fold serial dilution series of the AgNP stock solution (0.1–0.015 mg/ml) were mixed in a 96-well microplate. MHB alone and untreated bacterial suspensions served as negative and positive controls, respectively. Wells containing AgNP dilutions without bacteria were included to account for background absorbance.

Bacterial growth was monitored by measuring OD₆₀₀ using a GloMax® microplate reader (Promega, USA). The MIC was defined as the lowest nanoparticle concentration that showed no visible turbidity beyond faint background cloudiness. All experiments were conducted in triplicate to ensure reproducibility and analytical reliability. Minimum bactericidal concentrations (MBCs) were determined by aseptically transferring 10 µl aliquots from wells exhibiting no visible growth onto MH agar plates, followed by incubation at 37 °C for 24 h. The MBC was recorded as the lowest concentration resulting in complete inhibition of colony formation.

2.3.3. Agar disk diffusion technique

The agar disk diffusion method was used to evaluate the antimicrobial activity of protamine-loaded PVP–AgNPs in comparison with blank PVP–AgNPs, free protamine, and to assess potential synergistic effects [20]. Gram-positive and Gram-negative bacterial strains were cultured in MHB at 37 °C for 24 h and subsequently adjusted to ∼1.0 × 10^⁷ CFU/ml. Sterile 6 mm disks were placed on MHA plates, and 200 µl of each test sample of free protamine, blank PVP–AgNPs, protamine PVP–AgNPs, and sterile distilled water (negative control) was applied. The plates were then incubated at 37 °C overnight, and the diameters of the resulting inhibition zones were measured using a digital vernier caliper.

2.4. Cytotoxicity assay

2.4.1. Cell culture

Human dermal fibroblast (HDF) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (100 U/ml). The cells were maintained at 37 °C in a humidified incubator with 5% CO₂. Subculturing was performed every 3 d using 0.05% trypsin once the cultures reached approximately 90% confluency.

2.4.2. Cell viability

The cytotoxicity of blank and protamine-loaded PVP–AgNPs against HDF cells was evaluated using the MTT assay [21]. HDF cells were seeded into 96-well plates at a density of 8 × 10³ cells per well in 100 µl of complete culture medium and incubated at 37 °C in a 5% CO₂ atmosphere for 24 h. Cells were then treated with serial dilutions of the nanoparticle formulations (0–160 mM) and incubated for additional 72 h, with untreated cells serving as the control. Following treatment, the medium was replaced with 15 µl MTT solution and 100 µl fresh medium, and the plates were incubated for 4 h to allow formation of formazan crystals. The crystals were subsequently solubilized by adding 50 µl DMSO, and absorbance was measured at 560 nm using a Glomax microplate reader (Promega, USA). The IC₅₀ values were calculated from the dose–response curves using nonlinear regression analysis. Cell viability was determined according to Eq. 1:

$$\begin{array}{ccc}\hfill \text{Cell}\text{Viability}(\%)& =& \text{Absorbance}\text{of}\text{treated}\text{cells}/\hfill \\ & & \text{Absorbance}\text{of}\text{untreated}\text{cells}\times 100\%\hfill \end{array}$$ (1)

2.5. In vivo study

2.5.1. Formulation of AgNPs into a topical gel

Methylcellulose (MC, 2%, w/v) was employed as a neutral carrier to formulate AgNP-loaded hydrogels intended for topical application. MC solution was mixed with blank PVP–AgNPs, free protamine and protamine PVP–AgNP colloidal dispersions to prepare the formulations for subsequent evaluation in an infected wound model. To prepare the MC hydrogel, 6 g MC was dissolved in 50 ml distilled water and heated to 80 °C under gentle stirring for 30 min. After cooling, 36 ml of cold, filtered water was added to induce hydrogel formation. The required amount of concentrated AgNO₃ dispersions or pre-synthesized PVP–AgNPs and protamine PVP–AgNPs (100 mM) was then incorporated dropwise into the liquid MC hydrogel to achieve a final nanoparticle concentration of 1 mM. The mixture was stirred for an additional 30 min to ensure uniform distribution of nanoparticles within the hydrogel matrix [22].

2.5.2. Rat model for infected wound preparation and treatment

Male Wistar rats (3 months old, 200–250 g) were housed under standard laboratory conditions with free access to food and water. All procedures were approved by the Universiti Sultan Zainal Abidin Animal Ethics Committee (UAPREC/008/013) and complied with the National Research Council’s guidelines. Animals were anaesthetized via intraperitoneal injection of ketamine (30 mg/kg), and dorsal hair was shaved. Skin irritation was induced by repeated application and removal of adhesive plaster (15–20 times) until swelling and erythema were observed, followed by creating an 8 mm circular abrasion using a stainless-steel tool. Wounds were immediately covered with dressings soaked in 1% povidone-iodine [20]. After 5 min, each wound was intradermally inoculated with 100 µl S. aureus suspension (1 × 10⁸ CFU/ml). Signs of localized infection, including purulent exudate, were observed after 24 h [23]. One day post-infection, rats were randomly divided into four groups (n = 5) and treated daily with 0.5 g of the test formulations (1 mM): Group I, untreated control; Group II, blank PVP–AgNPs hydrogel; Group III, protamine PVP–AgNPs hydrogel; and Group IV, free protamine hydrogel. Treatments were applied once daily for 16 d. At the end of the study period, animals were sacrificed in a CO₂ chamber (Model CGSCO2G, Beiramar), and wound tissues were excised with a 3 mm margin for subsequent analyses.

Wound healing progression was monitored by capturing photographic images at each designated time point using a 14-megapixel Sony Alpha 390 camera. Wound diameters were measured along the inner margins using a digital caliper on Day 0 (immediately post-injury), 3, 8, 11 and 16. Image analysis was performed using ImageJ® software. The wound contraction was calculated according to Eq. 2:

$$\begin{array}{ccc}& & \text{Wound}\text{Contraction}(\%)\hfill \\ & & =\text{(Initial}\text{area}\text{-}\text{Day}\text{area}\text{of}\text{measurement)}\times 100\%\hfill \end{array}$$ (2)

2.6. Histomorphology analysis

Specimen collection involved excising the healed wound area along with a 0.5 cm margin of surrounding intact skin, extending down to the first muscle layer. On Day 16, tissue samples were harvested and immediately fixed in 10% neutral buffered formalin (pH 7.4), followed by standard histological processing procedures. Tissue samples were sectioned using a microtome at a thickness of 4 µm, followed by staining with hematoxylin and eosin (H&E), Picrosirius red and Masson’s trichrome (MT) for histological evaluation [24].

2.7. Statistical analysis

The results were expressed as the mean ± standard deviation (SD) from three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test in GraphPad Prism 8 (GraphPad Software Inc., USA). A P-value < 0.05 was considered statistically significant.

3. Results and discussion

3.1. Effect of NaBH₄ and PVP concentrations

The synthesized PVP–AgNPs were systematically characterized in terms of particle size, PDI and zeta potential, as summarized in Table 1. The molar concentration of AgNO₃ was consistently fixed at 100 mM, while both PVP and NaBH₄ concentrations were varied to probe their individual and interactive effects on nanoparticle formation dynamics and stability. A two-factor analysis of variance (ANOVA) revealed that the molar ratio of reducing agent (NaBH₄) to precursor (AgNO₃), along with the concentration of the capping agent (PVP), significantly impacted the resulting AgNP size distribution and surface charge characteristics (P < 0.05). Notably, formulation F1, with the highest NaBH₄ and PVP concentrations (NaBH₄ = 100 mM, PVP = 0.2 mM), produced nanoparticles with the smallest mean diameter (45.23 ± 0.22 nm), narrow size distribution (PDI = 0.38 ± 0.01), and a pronounced negative zeta potential (−18.7 ± 0.29 mV), indicative of excellent colloidal stability. In contrast, formulation F9 characterized by the lowest reducing agent and capping concentrations (NaBH₄ = 10 mM, PVP = 0.02 mM), generated significantly larger particles (145.60 ± 2.65 nm) with elevated PDI (0.72 ± 0.13) and near-neutral zeta potential (−8.15 ± 0.29 mV), suggesting prevalent agglomeration and limited electrostatic stabilization.

Table 1.

Effect of NaBH₄ and PVP concentrations on the physicochemical properties of AgNPs.

Formulation	PVP (mM)	NaBH4 (mM)	Size (nm)	PDI	Zeta potential (mV)
F1	0.2	100	45.23 ± 0.22*	0.38 ± 0.01*	−18.70 ± 0.29*
F2	0.2	50	76.59 ± 0.94⁎⁎	0.57 ± 0.01⁎⁎⁎	−15.40 ± 0.89⁎⁎
F3	0.2	10	89.23 ± 1.71⁎⁎⁎	0.53 ± 0.03⁎⁎	−12.30 ± 0.50⁎⁎⁎
F4	0.04	100	60.46 ± 0.18*	0.45 ± 0.02⁎⁎	−16.90 ± 0.92⁎⁎
F5	0.04	50	86.70 ± 3.00⁎⁎	0.49 ± 0.05⁎⁎⁎	−12.30 ± 0.50⁎⁎⁎
F6	0.04	10	110.10 ± 0.25⁎⁎⁎	0.62 ± 0.07⁎⁎⁎	−10.30 ± 1.48⁎⁎⁎
F7	0.02	100	68.04 ± 1.26*	0.50 ± 0.00⁎⁎	−14.45 ± 0.46⁎⁎
F8	0.02	50	124.10 ± 1.62⁎⁎	0.66 ± 0.16⁎⁎⁎	−11.25 ± 1.86⁎⁎⁎
F9	0.02	10	145.60 ± 2.65⁎⁎⁎	0.72 ± 0.13⁎⁎⁎	−8.15 ± 0.29⁎⁎⁎

Note: Values sharing the same symbol within each column do not differ significantly (P > 0.05).

The observed trends align with existing literature, emphasizing that optimal reduction kinetics and adequate surface capping are essential for producing monodisperse and stable nanoparticles [25,26]. At low NaBH₄ concentrations, slower reduction kinetics result in heterogeneous nucleation and uncontrolled particle growth, ultimately generating larger and more polydisperse nanoparticles. Concurrently, insufficient PVP availability weakens steric stabilization, allowing van der Waals forces to dominate and promote particle aggregation due to inadequate electrostatic repulsion. This dual limitation promotes accelerated particle growth and destabilization, as reflected by the tendency of low-PVP formulations to form larger agglomerates and exhibit diminished negative surface charge.

Formulations containing intermediate levels of PVP and NaBH₄ (e.g., F4 and F5) exhibited moderate particle sizes and acceptable stability, highlighting the importance of achieving a precise balance between reducing-agent strength and capping-polymer concentration. The gradual increase in particle size and PDI observed under these conditions is consistent with a progressive reduction in PVP surface coverage, which diminishes steric stabilization efficiency. Zeta potential measurements further support steric hindrance as the primary stabilization mechanism, as most formulations displayed moderately negative surface potentials (−10 to −18 mV). Although these absolute zeta potential values suggest limited electrostatic stabilization, the sustained colloidal stability of formulations containing higher PVP concentrations underscores the effectiveness of PVP’s polymer chain entanglement and steric barrier in preventing nanoparticle aggregation.

3.2. SPR analysis of AgNP formation

The formation and stabilization of AgNPs were systematically investigated using UV–Vis spectroscopy, dynamic light scattering (DLS), and zeta potential analysis. Distinct surface plasmon resonance (SPR) absorption peaks in the range of 390–420 nm confirmed the successful synthesis of AgNPs [27]. Notably, the intensity and wavelength of the SPR peaks varied substantially with formulation parameters, as illustrated in Fig. 1A. These spectral variations closely mirrored the visible color differences observed among formulations F1–F9. Formulations containing higher NaBH₄ concentrations exhibited bright yellow coloration, characteristic of well-formed nanosilver with strong SPR activity, whereas insufficient NaBH₄ produced darker solutions with attenuated SPR signals, indicative of incomplete silver ion reduction [28].

Fig. 1.

UV–Vis spectra and characterization of PVP–AgNPs at varying NaBH₄ and PVP concentrations (AgNO₃ fixed at 100 mM): (A) SPR peak shifts and intensity changes; (B) UV–vis spectrum of the optimized formulation (F1); (C) Particle size distribution of F1; (D) Zeta potential of F1.

Quantitative analyses further supported these observations. Increasing the NaBH₄ concentration resulted in a progressive rise in SPR peak intensity (from 0.37 to 1.06 a.u.), indicating more efficient reduction of Ag⁺ ions and enhanced formation of metallic AgNPs. The strongest SPR absorption (1.06 a.u. at 399 nm) was recorded for formulation F1 (Fig. 1B), correlating with its superior particle uniformity and dispersion relative to the other formulations [29]. In contrast, formulations prepared with lower NaBH₄ concentrations exhibited diminished SPR intensities and broader spectral profiles, reflecting suboptimal nucleation and growth kinetics that yielded more heterogeneous nanoparticle populations [29].

AgNPs synthesized using higher NaBH₄/AgNO₃ ratios displayed substantially smaller hydrodynamic diameters (45.23 ± 0.22 nm for F1) and narrower size distributions (Fig. 1C), indicating that accelerated reduction kinetics effectively suppress particle overgrowth. Consistent with this trend, zeta potential analysis showed that increasing the NaBH₄ concentration generated more negatively charged surfaces (−18.7 ± 0.29 mV for F1) and reduced polydispersity indices (PDI = 0.38 ± 0.005), reflecting improved electrostatic repulsion and enhanced colloidal stability [30]. In contrast, formulations produced with lower NaBH₄ levels exhibited larger particle sizes, weaker surface charge (−12.3 ± 0.50 mV), and elevated PDI values, collectively suggesting increased susceptibility to aggregation and poorer dispersibility.

At constant AgNO₃ and NaBH₄ levels, the PVP concentration functioned as a key regulator of nanoparticle stability and optical characteristics. Suboptimal PVP levels produced weak SPR intensity (0.68 a.u.) and visible aggregation, accompanied by color changes from yellow to brown (Fig. 1A). Inadequate surface capping facilitates agglomeration, broad size distributions, and diminished optical performance [31]. Conversely, an optimal PVP concentration (0.2 mM) generated a sharp and well-defined SPR peak at 399 nm, reflecting the formation of monodisperse and highly stable AgNPs with no evidence of precipitation [25]. Increasing the PVP concentration beyond this optimal point caused a slight red shift of the SPR peak to 401 nm and produced smaller particles due to enhanced steric stabilization. However, excessively high polymer content may restrict further particle growth and limit tunability for application-specific requirements.

3.3. Influence of pH value on AgNPs synthesis

The stability of PVP-capped AgNPs was strongly pH-dependent. DLS analysis showed the smallest particle sizes (<100 nm) with narrow distributions at pH 7.5 and 9, indicating well-dispersed nanoparticles. In contrast, acidic conditions (pH 5 and 3) triggered rapid aggregation, yielding large clusters (∼600 nm) and confirming colloidal destabilization (Fig. 2A). Zeta potential analysis after 24 h confirmed these observations (Fig. 2B), with higher absolute negative charges at pH 7.5 and 9, confirming stronger electrostatic repulsion and greater stability. This behavior is linked to OH⁻ ions increasing the surface charge and preventing particles from sticking together. Under acidic conditions, excess H⁺ leads to protonation of the PVP pyrrolidone groups, reducing repulsion and speeding up aggregation [32]. Overall, neutral to mildly alkaline conditions favored stable, monodisperse AgNPs, with optimal synthesis achieved using 100 mM NaBH₄, 0.2 mM PVP,and pH 7.5.

Fig. 2.

Effect of pH on the physicochemical properties of PVP–AgNPs. (A) Z-average hydrodynamic diameter (d, nm) and (B) zeta potential at various pH values.

3.4. Formulation and optimization of protamine loaded PVP–AgNPs

Protamine-loaded PVP-AgNPs were optimized by systematically varying protamine concentrations using a two-fold serial dilution (0.062–1 mg/ml). Increasing protamine levels led to a progressive rise in particle size (Fig. 3A), consistent with previous findings that greater polycation adsorption induces a thicker nanoparticle corona [33]. The PDI showed a non-linear pattern, with the lowest values at 0.125 and 0.25 mg/ml, suggesting that these concentrations produced the most uniform and stable dispersions (Fig. 3B). LE, quantified by HPLC after ultrafiltration of unbound protamine, achieved its maximum at 0.25 mg/ml (66.2% ± 3.2%) and remained comparatively high at 0.5 mg/ml (62.8% ± 2.1%). However, beyond 0.25 mg/ml, LE declined, likely due to surface saturation effects, where excess protamine molecules were unable to bind effectively to the nanoparticle surface (Fig. 3C).

Fig. 3.

Effect of protamine concentration on (A) particle size, (B) PDI, (C) loading efficiency, and (D) zeta potential of protamine PVP–AgNPs.

As shown in Fig. 3D, the blank PVP–AgNPs (protamine-free) exhibited a pronounced negative surface charge. Following protamine incorporation, the zeta potential shifted markedly toward positive values, reflecting strong electrostatic interactions between the cationic amine groups of protamine and the negatively charged nanoparticle surface [34]. Furthermore, as shown in Fig. 3C and 3D, the zeta potential rose steadily with increasing protamine concentrations, from about +5.08 mV at 0.062 mg/ml to +10.39 mV at 0.25 mg/ml. At the same time, the protamine loading efficiency also increased over this range, from 39.52% to 66.20%, almost doubling in line with the change in surface charge. These results confirm that protamine binds effectively to the PVP–AgNPs and demonstrate a clear concentration-dependent adsorption pattern of the cationic peptide onto the nanoparticle surface. At this optimal protamine concentration (0.25 mg/ml), the nanoparticles exhibited a mean diameter of 70.96 ± 0.27 nm and a narrow PDI of 0.25 ± 0.002, signifying high colloidal stability and excellent uniformity. Therefore, 0.25 mg/ml protamine is identified as the optimal loading for high-performance protamine PVP–AgNPs formulations.

3.5. UV–Vis spectra studies of blank and protamine PVP–AgNPs

Fig. 4 shows the UV–Vis absorption spectra for blank PVP-AgNPs, free protamine and protamine-loaded PVP–AgNPs at different concentrations. Free protamine displayed a clear absorption peak at 214 nm (Fig. 4A), which is typical for its peptide backbone [35]. The PVP–AgNPs showed a strong SPR band at 399 nm, confirming successful AgNP formation (Fig. 4B) [36]. Upon incremental loading of protamine (0.062–1 mg/ml), the SPR band was observed to shift between 395 and 401 nm, accompanied by a visible color transition of the solution from bright to dark yellow [33]. Increasing protamine concentration also led to broadening of the SPR peak and a drop in its intensity, with absorbance decreasing from 1.06 to 0.45 a.u. These spectral changes, together with peak broadening, suggest partial nanoparticle agglomeration or stronger surface interactions, although no visible aggregates or precipitation were observed.

Fig. 4.

(A) UV–vis spectrum of free protamine, and (B) protamine PVP–AgNPs at various concentrations and blank PVP–AgNPs; (C) Corresponding photographs of AgNPs solutions at indicated protamine concentrations.

These optical modulations confirm the successful loading of protamine onto the PVP-AgNP surface and demonstrate that nanoparticle optical properties are tunable via surface functionalization. Based on these results, a protamine concentration of 0.25 mg/ml was identified as optimal for subsequent formulations and biological evaluation.

3.6. TEM

TEM supported by uranyl acetate and phosphotungstic acid negative staining was utilized to examine the particle dimensions, structural morphology and surface features of the blank formulations and the protamine PVP–AgNPs. Blank PVP-AgNPs displayed uniform spherical morphology (Fig. 5A) with particle diameters ranging from 6.24 to 14.83 nm and an average size of 10.49 ± 1.38 nm determined by size distribution analysis (Fig. 5C). These findings confirm a monodisperse population and structural uniformity, closely correlating with UV–Vis and DLS data. In line with established literature, particle sizes determined by TEM were consistently smaller than those from DLS, an observation that can be attributed to dehydration and sample preparation effects [37].

Fig. 5.

TEM micrographs of (A) PVP–AgNPs and (B) protamine PVP–AgNPs (scale bar: 200 nm); (C) Size distribution for blank PVP–AgNPs and (D) protamine-loaded PVP–AgNPs (>100 particles); (E) Surface visualization of protamine-loaded PVP–AgNPs with dual staining, arrows indicating adsorbed protamine layer (scale bar: 200 nm).

Upon protamine coating, TEM analysis revealed a distinct increase in particle size, ranging from 5.66 to 25.14 nm, with an average diameter of 12.25 ± 3.29 nm (Fig. 5B and 5D). This increment reflects the successful adsorption of a thin protamine shell (∼1–2 nm), visible by the enhanced contrast in micrographs stained with phosphotungstic acid (Fig. 5E, red arrows). The electron-dense regions correspond to the AgNP cores, while the lighter areas represent the less dense, hydrogen-rich protamine layer, consistent with differential staining patterns and previous reports [9]. Collectively, these TEM findings verify efficient protamine loading on PVP–AgNPs, retention of nanocrystalline characteristics, and low polydispersity attributes critical for reproducible biological activity and formulation stability. The agreement between TEM results and other characterization methods further supports the stable and well-defined structure of these biofunctional nanocomposites.

3.7. FTIR profiles

Fig. 6 depicts the FTIR spectra of pure PVP, PVP–AgNPs, protamine and protamine PVP–AgNPs. The spectrum of pure PVP exhibits characteristic aliphatic methylene (–CH₂–) stretching vibrations at 2982 and 2869 cm⁻¹, an O–H stretching band at 3526 cm⁻¹, a prominent carbonyl (C = O) stretch of the pyrrolidone ring at 1658 cm⁻¹, N–C = O amide stretching at 1433 cm⁻¹, and C–N stretching at 1276 cm⁻¹ [38]. In the PVP–AgNPs, the C = O peak shifts to 1651 cm⁻¹ and the C–N stretch shifts to 1269 cm⁻¹, while the –CH₂– stretches appear at 2968 and 2898 cm⁻¹, indicating coordination between silver and the nitrogen site of PVP. A broad O–H stretch at 3435 cm⁻¹ is also present, which is typical of hydrogen bonding and hydration [39]. The reduced intensity of the N–OH and amide peaks at 1431 and 1269 cm⁻¹ further suggests that the nitrogen group of PVP participates in AgNP binding [40].

Fig. 6.

FTIR studies of pure PVP, blank PVP–AgNPs, pure protamine, and protamine PVP–AgNPs.

The protamine spectrum shows a characteristic amide I band (mainly C = O stretching of the peptide bond) at 1628 cm⁻¹ and an amide II band (mainly N–H bending and C–N stretching) at 1526 cm⁻¹, which reflect the protein’s secondary structure [41]. A strong band at 1047 cm⁻¹ is assigned to arginine in protamine and corresponds to the stretching vibration of the –C–NH– (amine) group [42]. In the protamine PVP–AgNP spectrum, the O–H stretching band appears at 3297 cm⁻¹, the –CH₂– stretches at 2915 cm⁻¹, and the C = O/amide I band at 1649 cm⁻¹. The characteristic arginine band shifts slightly to 1043 cm⁻¹, indicating amino group interactions with the carbonyl oxygen of PVP through hydrogen bonding [43]. Collectively, these shifts and newly observed signals confirm successful protamine attachment to the PVP–AgNPs, mediated by hydrogen bonding and coordination interactions involving amide, amino (N–H), and carbonyl (C = O) groups.

3.8. TGA

Thermogravimetric analysis (TGA) and derivative thermogravimetric analysis (DTGA) curves for free PVP, PVP–AgNPs, free protamine and protamine PVPAgNPs are illustrated in Fig. 7. For both free PVP and PVP-AgNPs (Fig. 7A and 7B), two distinct mass loss steps were observed. The initial weight reduction of approximately 5.22% for free PVP and 7.75% for PVP–AgNPs occurred at around 140 °C and 80 °C, respectively, corresponding to the evaporation of residual, physically adsorbed water, which is consistent with the established thermal profiles of polymer-based systems [44]. DTGA analysis revealed that free PVP exhibited a two-step degradation at approximately 80 °C and 430 °C, while PVP-AgNPs displayed two distinct decomposition events at approximately 130 °C and 300 °C (Fig. 7B). The primary decomposition phase, spanning 250–695 °C for both free PVP and PVP–AgNPs, resulted in mass losses of approximately 68.03% and 83.91%, respectively. This phase reflects the structural degradation of the free PVP matrix and the loss of PVP bound organics for PVP–AgNPs [45].

Fig. 7.

TGA and DTGA profiles for (A, B) free PVP, PVP–AgNPs, and (C, D) free protamine, protamine PVP–AgNPs, showing water loss, main decomposition regimes, and comparative thermal stability (E, F).

Upon protamine functionalization, the thermal profiles showed small but important differences compared to blank PVP–AgNPs and free protamine. All samples showed an initial mass loss between 30 and 200 °C, attributed to the loss of residual moisture and hydration water, with weight losses of 5.22% for PVP–AgNPs, 9.54% for protamine, and 5.19% for protamine PVP–AgNPs. The protamine PVP–AgNPs (Fig. 7C and 7D) showed a slightly higher overall weight loss (69.29%) across the full range (30–800 °C), which is expected due to the extra organic content from the adsorbed protein [46]. The DTGA curve of the modified nanoparticles displayed two main decomposition steps: the first (∼5.19% between room temperature and 150 °C) linked to water loss, and a second major phase starting near 240 °C and peaking around 300 °C, corresponding to degradation of the protamine-modified polymer structure [47], Free protamine, in contrast, showed two separate decomposition events at ∼60 °C and ∼260 °C (Fig. 5F). Notably, the maximum decomposition temperature of the protamine-functionalized nanoparticles shifted to ∼300 °C, compared to ∼235 °C for unmodified PVP–AgNPs and ∼250 °C for free protamine (Fig. 7E and 7F). This increase indicates improved thermal stability, likely due to stronger intermolecular interactions and greater structural integrity at the nanoparticle surface [46]. Overall, these thermogravimetric results confirm successful protamine incorporation and highlight the corresponding improvements in thermal stability and structural organization of the nanocomposite.

3.9. Colloidal stability of lyophilized AgNPs

Freshly prepared protamine PVP–AgNP suspensions were lyophilized with and without the addition of cryoprotectants, specifically sorbitol and sucrose at a concentration of 5% (w/v), to assess their ability to preserve nanoparticle stability during freeze-drying. After rehydration, formulations were evaluated for changes in mean particle size, PDI and zeta potential (Fig. 8). The results clearly demonstrate that lyophilization in the presence of either sugar significantly reduced post-rehydration particle size and PDI compared to the samples without cryoprotectant. This indicates that both sucrose and sorbitol help prevent swelling and aggregation of the protamine PVP–AgNPs during rehydration, thereby preserving nanoparticle uniformity and structural integrity [48].

Fig. 8.

Effect of cryoprotectants on the freeze-dried protamine PVP–AgNPs: (A) UV–vis absorbance spectra, (B) mean hydrodynamic diameter, (C) PDI and (D) zeta potential after rehydration.

A comparison of the two cryoprotectants showed that sucrose provided superior protection. Formulations lyophilized with sucrose displayed particle size, PDI and zeta potential values that were not statistically different from the freshly prepared controls, indicating minimal aggregation and good preservation of colloidal properties (Fig. 8B–8D). In contrast, nanoparticles freeze-dried with sorbitol showed a small but significant increase in particle size and PDI, along with a noticeable broadening of the plasmon resonance band (Fig. 8A), suggesting some degree of aggregation and weaker protection. Visual inspection supported these results: sucrose-containing samples remained bright yellow and well-dispersed, whereas sorbitol-containing samples developed a slight brown color, indicating partial loss of colloidal stability.

Notably, freeze-dried samples without any cryoprotectant showed severe aggregation, reflected by a very large particle size, high PDI, and the complete loss of a visible plasmon peak in the UV–Vis spectrum. This extreme aggregation made the data unusable, so the spectrum was omitted from Fig. 8A for clarity. Overall, these findings identify sucrose as the more effective cryoprotectant compared to sorbitol, as it consistently preserved the nanoscale features and surface charge of the protamine PVP–AgNPs during freeze-drying and rehydration [49]. The stronger stabilizing effect of sucrose is especially important for long-term storage and pharmaceutical formulation of protein–nanoparticle systems.

3.10. Evaluation of in vitro antibacterial activity of AgNPs

3.10.1. MIC and MBC assays

Table 2 provides MIC and MBC values for the tested formulations against E. coli, P. aeruginosa and S. aureus (MRSA). Data were statistically analyzed with standard deviations and appropriate tests (SPSS). The antimicrobial efficacy of the prepared formulations was assessed against key Gram-positive and Gram-negative bacterial strains, with MIC and MBC results summarized in Table 2. PVP–AgNPs exhibited notable antibacterial activity against all tested strains, supporting previous literature reports of their broad-spectrum efficacy [50]. Their efficacy was especially notable against Gram-positive bacteria, exhibiting a MIC as low as 31.25 µg/ml for S. aureus (MRSA). This enhanced activity is consistent with established observations that Gram-negative bacteria, such as E. coli, frequently demonstrate higher inherent resistance to silver-based agents, likely due to structural barriers presented by their outer membranes [51]. Free protamine, by comparison, yielded MIC values of 125 µg/ml for Gram-negative strains, while Gram-positive strains were more susceptible, demonstrating MICs of 62.5 µg/ml [13].

Table 2.

MIC and MBC values of PVP–AgNPs, free protamine, and protamine PVP–AgNPs against selected bacterial strains.

Material	E. coli		P. aeruginosa		S. aureus (MRSA)
	MIC (µg/ml)	MBC (µg/ml)	MIC (µg/ml)	MBC (µg/ml)	MIC (µg/ml)	MBC (µg/ml)
Vancomycin	N.A.	N.A.	N.A.	N.A.	8	8
PMB	15.63	15.63	15.63	15.63	<125	<125
PVP–AgNPs	62.5	62.5	62.5	62.5	31.25	31.25
Protamine	125	125	125	125	62.5	62.5
Protamine PVP–AgNPs (1 mg/ml)	15.63	62.5	31.25	62.5	15.63	15.63
Protamine PVP–AgNPs (0.5 mg/ml)	15.63	15.63	31.25	62.5	15.63	15.63
Protamine PVP–AgNPs (0.25 mg/ml)	7.81	15.63	15.63	15.63	7.81	7.81

Note: N.A. = not applicable; Protamine concentrations are based on total conjugate content.

In contrast, the protamine PVP–AgNP formulations (1 to 0.25 mg/ml) showed a marked increase in antimicrobial activity. All loaded formulations produced lower MIC values than either free protamine or the blank PVP–AgNPs, indicating a clear synergistic effect after conjugation. Importantly, the conjugates overcame the reduced sensitivity shown by E. coli and P. aeruginosa toward free protamine, lowering their MICs to 7.81 µg/ml and 15.63 µg/ml, respectively. These improvements represent a two- to four-fold increase in antibacterial potency compared to both the unmodified nanoparticles and free protamine.

MBC values, although not detailed in the original text, were determined and shown in Table 2. Across all bacterial strains, protamine-loaded PVP–AgNPs consistently exhibited lower MBCs compared to controls. The 0.25 mg/ml formulation exhibited the highest bactericidal efficiency, comparable to positive controls, indicating full bacterial eradication at minimal concentrations [52]. The enhanced antimicrobial activity of the protamine PVP–AgNP conjugates is likely due to the combined actions of both components, including membrane disruption by silver ions and increased cell permeabilization mediated by the cationic peptide.

3.10.2. Antibacterial activity by agar disk diffusion

Free protamine, tested at 1, 0.5 and 0.25 mg/ml, showed no antibacterial activity against either Gram-positive or Gram-negative bacteria at the tested levels (Table 3). This indicates that protamine on its own has limited antimicrobial effectiveness. In contrast, combining protamine with PVP–AgNPs produced a strong synergistic effect, resulting in clear antimicrobial activity across all tested strains. The protamine PVP–AgNPs showed greater activity against S. aureus and E. coli than against P. aeruginosa, indicating species-dependent performance. The zones of inhibition (ZOI) produced by these conjugates were much larger than those produced by free protamine or blank PVP–AgNPs and were consistent with previous reports on the broad-spectrum effects of PVP–AgNPs [50]. As seen in earlier studies, the nanoparticles displayed stronger effects against Gram-positive bacteria, while some E. coli strains showed higher resistance [53]. Table 3 summarizes the inhibition zones for all formulations. PS-1, PS-2 and PS-3 correspond to protamine sulfate at 1, 0.5 and 0.25 mg/ml, respectively, while PS-AgNPs-1, PS-AgNPs-2 and PS-AgNPs-3 refer to protamine-loaded PVP-AgNPs at the same concentrations. Values are reported as mean ± SEM from triplicate experiments.

Table 3.

ZOI (mm) of PVP–AgNPs–MC, protamine and protamine PVP-AgNPs against tested strains.

Sample	E. coli	P. aeruginosa	S. aureus (MRSA)
PVP–AgNPs	62.5 ± 0.29	62.5 ± 0.29	62.5 ± 0.29
PS-1 (1 mg/ml)	0.00	0.00	0.00
PS-2 (0.5 mg/ml)	0.00	0.00	0.00
PS-3 (0.25 mg/ml)	0.00	0.00	0.00
PS-AgNPs-1 (1 mg/ml)	12.17 ± 0.18⁎⁎	7.87 ± 0.77*	7.87 ± 0.77*
PS-AgNPs-2 (0.5 mg/ml)	15.88 ± 0.84*	8.21 ± 0.58*	8.21 ± 0.58*
PS-AgNPs-3 (0.25 mg/ml)	15.77 ± 0.79*	10.34 ± 0.83⁎⁎	10.34 ± 0.83⁎⁎

Note: Values sharing the same symbol within each column do not differ significantly (P > 0.05).

The antibacterial activity of free protamine, blank PVP–AgNPs, and protamine loaded PVP–AgNPs was systematically evaluated using the agar disk diffusion method. As shown in Fig. 9A, free protamine sulfate at concentrations of 1, 0.5 and 0.25 mg/ml (labeled as PS-1, PS-2, PS-3) did not produce detectable ZOI against E. coli, S. aureus or P. aeruginosa, confirming the limited antimicrobial potency of protamine in its unbound state. In contrast, incorporation of protamine into PVP–AgNPs (formulations PS-AgNPs-1, −2, −3, corresponding to the same concentrations) significantly enhanced antibacterial performance against all tested strains.

Fig. 9.

Antibacterial activity of blank PVP–AgNPs, pure protamine (PS), and protamine PVP–AgNPs: (A) Inhibition zone plates, (B) Quantitative analysis of zone diameters. PS-1, −2, −3 = protamine at 1, 0.5, 0.25 mg/ml. PS-AgNPs-1, −2, −3 = corresponding protamine PVP–AgNPs; all experiments performed in triplicate, values reported as mean ± SD.

Quantitative analysis of inhibition zones (Fig. 9B) shows that protamine PVP–AgNPs significantly inhibited both Gram-positive and Gram-negative bacteria, with zones for E. coli and S. aureus approximately doubling compared to blank PVP–AgNPs (P < 0.001). For E. coli, the mean zone increased from ∼7 mm (PVP–AgNPs) to ∼15 mm (protamine PVP–AgNPs), while for S. aureus it increased from 8.8 mm to more than 16 mm. Although P. aeruginosa is known for its high resistance, the optimized formulation (PS-AgNPs-3, 0.25 mg/ml) still achieved a significantly larger inhibition zone (10.34 ± 0.83 mm, P < 0.001) than either blank nanoparticles (8.88 ± 0.29 mm) or free protamine (0 mm). Higher protamine concentrations (PS-AgNPs-1 and −2) did not show significant improvements over blank PVP–AgNPs for P. aeruginosa (P > 0.05), suggesting a plateau in response for this organism.

Overall, these results show that loading protamine within PVP–AgNPs can overcome the natural resistance of Gram-negative bacteria such as E. coli and P. aeruginosa to free protamine, while also greatly enhancing activity against the Gram-positive S. aureus. The strong and statistically supported increases in inhibition zones across triplicate experiments highlight the potential of protamine loaded PVP–AgNPs as broad-spectrum antimicrobial agents capable of targeting drug-resistant pathogens.

3.11. Cell viability assay

3.11.1. MTT assay

The cytocompatibility of protamine PVP–AgNPs was evaluated using the MTT viability assay in HDF cells after 72 h of exposure (Fig. 10). Protamine alone, known for its antimicrobial properties, exhibited minimal cytotoxicity on mammalian cells, with previous studies reporting no significant reduction in HDF viability at concentrations up to 500 µg/ml [54]. Consistent with these findings, our results demonstrated that protamine PVP–AgNPs maintained high cell viability, with over 80% of HDF cells remaining viable at protamine-equivalent concentrations up to 250 µg/ml. Importantly, at concentrations exceeding the minimum bactericidal levels required to inhibit P. aeruginosa and E. coli, the formulations preserved ∼80% cell viability, highlighting their favorable biocompatibility. This balance between potent antimicrobial activity and low cytotoxicity highlights the potential of these nanoconjugates for safe application in wound healing and dermatological treatments. Thus, protamine PVP–AgNPs represent a promising therapeutic strategy, combining effective bacterial inhibition with preserved mammalian cell viability.

Fig. 10.

Cell viability of HDF cells after 72 h treatment with protamine loaded PVP–AgNPs (protamine content: 250 µg/ml). Data shown as mean ± SD.

3.12. In vivo antibacterial activity study of AgNPs embedded MC hydrogel

3.12.1. UV–Vis analysis

One of the main challenges in biomedical use of AgNPs is their tendency to aggregate in colloidal suspensions, which can reduce both stability and therapeutic performance [55]. To address this, a stable hydrogel formulation was developed using MC as the matrix and incorporating protamine loaded PVP–AgNPs, with the goal of improving nanoparticle stability and supporting wound-healing applications. The resulting hydrogel showed a characteristic yellow color, indicating the presence of AgNPs. UV–Vis analysis confirmed successful nanoparticle incorporation. As shown in Fig. 11A, the protamine PVP–AgNPs–MC hydrogel exhibited a clear absorption peak at 399 nm, a well-known signature of spherical AgNPs in colloidal systems [56]. This result agrees with earlier studies and highlights the ability of MC to act as an effective stabilizing and supportive medium for AgNP dispersal [57]. A photographic comparison (Fig. 11B) further shows the visual differences among the PVP–AgNPs–MC hydrogel, the protamine PVP–AgNPs–MC hydrogel, and the plain MC hydrogel, confirming successful nanoparticle incorporation and overall formulation integrity.

Fig. 11.

(A) UV–Vis absorption spectra of protamine PVP–AgNPs embedded in MC hydrogel, showing a prominent peak at 399 nm; (B) Photographic comparison of protamine PVP–AgNPs–MC hydrogel, protamine PVP–AgNPs–MC hydrogel, and MC hydrogel.

3.12.2. Healing activity in vivo

Representative wound photographs for all treatment groups show the progression of wound healing in the rat model (Fig. 12). During the early inflammatory phase (Day 0–3), purulent exudate and abscess formation were observed in all groups, which is typical of an acute wound response. As healing advanced into the later stages (Day 11–16), no signs of abnormal or hypertrophic scarring were seen. The lack of self-inflicted injuries or aggressive behavior among animals also indicates that the handling and experimental procedures did not introduce stress or confounding factors.

Fig. 12.

Progression of wound healing over 16 d: (A) negative control, (B) blank PVP–AgNPs–MC hydrogel, (C) free protamine-MC hydrogel, and (D) protamine PVP–AgNPs–MC hydrogel.

Strikingly, animals treated with the protamine PVP–AgNPs–MC hydrogel achieved complete epithelialization and wound closure by 16 d, demonstrating visibly accelerated recovery compared to the untreated control group (Fig. 12D). This rapid healing response surpassed that observed in the control (Fig. 12A), blank PVP–AgNPs–MC (Fig. 12B), and free protamine groups (Fig. 12C), highlighting the additive benefit of nanoparticle-polymer conjugation. These findings support the therapeutic potential of protamine PVP–AgNPs–MC hydrogel as a bioactive wound dressing, capable of promoting efficient tissue regeneration without adverse effects on animal welfare.

Quantitative analysis of the wound retraction index (WRI) over the 16-d observation period is displayed in Fig. 13. By Day 3, the group treated with protamine PVP–AgNPs–MC demonstrated a WRI of 27.5% ± 1.79%, which was statistically comparable to the free protamine group (26.34% ± 0.24%, P > 0.05), but significantly greater than both the blank PVP–AgNPs (17.66% ± 2.11%) and untreated control (18.3% ± 2.36%, P < 0.001). These early differences highlight the potent effect of protamine PVP–AgNPs in accelerating wound contraction during the initial repair phase (Fig. 13A).

Fig. 13.

Sequential WRI in negative control, blank PVP–AgNPs–MC hydrogel, free protamine–MC hydrogel and protamine PVP–AgNPs–MC hydrogel treatment groups, measured at Day 3 (A), 8 (B), 11 (C), and 16 (D) to assess healing dynamics. Data expressed as mean ± SD; statistical comparisons are indicated.

A pronounced therapeutic advantage was evident at Day 8, as the protamine PVP–AgNPs group achieved a WRI of 76.26% ± 0.04%, greatly surpassing the control (31.79% ± 3.26%), blank PVP–AgNPs (43.11 ± 1.36%), and free protamine (50.6% ± 0.52%) groups (all P < 0.001, Fig. 13B). This rapid progression suggests that co-delivery of protamine and AgNPs enhances antimicrobial efficacy and tissue contraction, reflecting both synergistic action and robust wound bed remodeling.

From Day 8 onward, granulation tissue formation and upregulation of transforming growth factor-beta (TGF-β) further drove wound closure. By Day 11, the WRI in the protamine PVP–AgNPs group climbed to 97.58% ± 0.52%, significantly higher than the control (82.66% ± 3.98%, P < 0.001), blank PVP–AgNPs (91.75% ± 2.04%, P = 0.010), and free protamine (92.33% ± 0.58%, P = 0.020) groups (Fig. 13C). This sustained benefit persisted through Day 16, where maximal wound closure was observed, confirming the superior and enduring efficacy of the protamine PVP–AgNPs hydrogel in promoting organized tissue repair and scar resolution (Fig. 13D).

3.13. Histomorphology analysis

The wound healing process is characterized by sequential stages of inflammation, tissue regeneration and reconstruction, all of which are essential for successful repair [57]. On Day 16, histological evaluation revealed marked epithelialization, granulation tissue formation, and contraction of underlying connective tissue in all experimental groups, as illustrated in Fig. 14A. However, the extent and quality of tissue regeneration varied distinctly among the different treatments.

Fig. 14.

Histological sections of wound tissue from negative control, blank PVP–AgNPs-MC hydrogel, free protamine-MC hydrogel, and protamine–PVP AgNPs-MC hydrogel groups at Day 16 post-injury: (A) H&E staining, (B) Masson’s trichrome staining, (C) appendage formation and collagen deposition.

In the untreated negative controls, H&E staining showed many inflammatory cells at the wound margins (Fig. 14A, black arrows), indicating ongoing infection and poor healing. These wounds also displayed thin epithelial layers and lacked hair follicles and sebaceous glands, demonstrating incomplete restoration of normal skin structure. In contrast, wounds treated with the protamine PVP–AgNPs–MC hydrogel showed thick epithelial layers, strong neovascularization, and numerous newly formed hair follicles (Fig. 14A–14C), reflecting robust tissue regeneration and superior healing.

Groups treated with free protamine or blank PVP–AgNPs–MC showed moderate reductions in inflammation and some new formation of skin appendages, but the extent of recovery did not match the comprehensive restoration observed with the protamine PVP–AgNPs–MC hydrogel. Although full epithelialization was evident across all groups, only the protamine PVP–AgNPs–MC treated wounds showed widespread epithelial budding, consistent with active regeneration and early development of skin appendages (Fig. 14A, red arrows; Fig. 14C, white arrows).

Collagen deposition, examined by Masson’s trichrome staining (Fig. 14B), also showed clear treatment-related differences. Newly formed, lightly stained type III collagen was more evident in the non-optimized groups, while the protamine PVP–AgNPs–MC hydrogel group displayed dense, dark blue staining in the dermis, indicating increased deposition of mature, cross-linked type I collagen. Collagen fibers in the protamine- PVP–AgNPs–MC group showed a mixed, organized pattern, whereas the other groups exhibited more fascicular arrangements. Overall, these histological findings show that the protamine PVP–AgNPs–MC promotes faster and more complete wound repair, marked by advanced epithelialization, appendage formation, and mature collagen deposition.

4. Conclusion

Protamine loaded PVP stabilized AgNPs (PVP–AgNPs) were successfully synthesized via a chemical reduction method and functionalized with protamine through PVP-mediated interactions, as confirmed by UV–Vis spectroscopy, FTIR, TEM, zeta potential analysis and HPLC. The optimized nanocomposite exhibited spherical morphology, high colloidal stability, and efficient protamine loading, enabling enhanced antibacterial performance. In vitro assays demonstrated that protamine PVP–AgNPs achieved significantly greater antibacterial activity against both Gram-positive (S. aureus) and Gram-negative (E. coli, P. aeruginosa) strains compared to blank PVP–AgNPs and free protamine. Importantly, the nanocomposite showed no induction of bacterial resistance during repeated exposure studies. Furthermore, cytocompatibility tests using fibroblasts confirmed the safety of the formulation, while in vivo wound healing assessments revealed accelerated wound closure and improved tissue regeneration, as supported by histological analysis. Overall, this work demonstrates the potential of protamine loaded PVP–AgNPs as a promising nanotherapeutic platform for treating MDR bacterial infections and supporting wound healing. These findings suggest that the formulation may be further explored for future commercial development. Future research is recommended to include quantitative evaluation of the inflammatory response through pro-inflammatory cytokine measurements, along with investigation of potential silver accumulation in skin tissues.

CRediT authorship contribution statement

Mohammad Jaafreh: Writing – original draft, Methodology, Data curation. Walhan Alshaer: Supervision, Investigation, Conceptualization. Mahmoud Alkawareek: Validation, Resources. Zalina Zahari: Supervision, Investigation. Shrouq Alsotari: Software, Resources. Dana A. Alqudah: Data curation, Conceptualization. Hazem Choukaife: Writing – review & editing, Methodology. Manal A. Abbas: Resources, Formal analysis. Yasser Bustanji: Validation, Investigation. Mulham Alfatama: Writing – review & editing, Supervision, Project administration, Investigation, Conceptualization.

Conflicts of interest

The authors declare that there is no conflicts of interest.