Development and Characterization of Chitosan–Polyvinylpyrrolidone Nanoparticles for Antimicrobial Drug Delivery Applications

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The developed CS-PVP nanoparticles serve as biocompatible carriers for the controlled delivery of synthetic and natural antimicrobial agents, offering a promising platform for treating microbial infections while minimizing cytotoxicity and enhancing drug stability.

Abstract

Chitosan (CS) and polyvinylpyrrolidone (PVP)-based nanoparticles (NPs) are promising carriers for drug delivery due to their biocompatibility, biodegradability, and intrinsic antimicrobial properties. This study explores CS-PVP NPs for the encapsulation and controlled release of synthetic compounds (bis-THTT, JH1, JH2) and natural antimicrobials (honey and propolis). NPs were synthesized via ionic gelation, optimizing CS:PVP and CS-PVP:sodium tripolyphosphate (TPP) ratios. The optimal formulation (CS:PVP 1:0.5) produced stable, homogeneous NPs. Characterization was performed using FTIR, TGA, XRD, and AFM. Encapsulation efficiencies ranged from 44–60%. Antimicrobial activity was evaluated against Escherichia coli and Staphylococcus aureus, showing significant inhibition for JH1-, JH2-, honey-, and propolis-loaded NPs against E. coli. Cytotoxicity assays on 3T3 fibroblasts confirmed the biocompatibility of all formulations at 5 and 10 µg/mL. In vitro release studies in artificial gastric fluid (pH 1.78) demonstrated sustained drug release over 180 min. These results confirm that CS-PVP NPs can effectively encapsulate and protect both synthetic and natural bioactive compounds, enhancing their therapeutic potential. The developed nanosystems represent a versatile and safe platform for antimicrobial drug delivery and may support future applications in biomedical therapies.

1. Introduction

Nanotechnology has transformed the landscape of modern medicine, offering innovative solutions for drug delivery systems (DDSs) [1]. Among these, nanoparticles (NPs) have gained prominence due to their unique properties, such as biocompatibility, controlled release, and enhanced targeting capabilities [2]. The integration of biopolymers like chitosan (CS) and polyvinylpyrrolidone (PVP) into nanoparticle systems further expands their potential applications in therapeutic and antimicrobial treatments [3,4]. Despite the advantages, challenges such as poor solubility and stability of drugs continue to hinder effective therapeutic outcomes, necessitating new delivery systems to address these limitations [5,6].

The controlled release of antimicrobial agents using nanoparticle-based delivery systems has garnered significant attention for improving therapeutic efficacy and reducing dosing frequency. This strategy offers several advantages, including maintaining drug concentrations within the therapeutic window over extended periods and minimizing side effects associated with peak concentrations [7]. Biopolymer-based nanoparticles, in particular, have shown promise in achieving controlled and sustained release of a wide range of antimicrobial compounds, including both synthetic drugs and natural extracts [8,9]. This controlled-release approach not only improves patient compliance but also helps mitigate the development of antimicrobial resistance by avoiding subtherapeutic dosing.

On the other hand, natural products, such as honey bee and propolis, and synthetic compounds like bis-tetrahydro-1,3,5-thiadiazine-2-thione (bis-THTT, JH1, and JH2) are known for their biological activity, including antimicrobial and antimalarial effects [10,11]. However, their clinical application is often limited by rapid degradation and suboptimal bioavailability [12]. Encapsulating these compounds in biopolymer-based nanoparticles can significantly enhance their stability, targeted delivery, and therapeutic efficacy [13,14].

In this context, CS and PVP have individually been used in drug-delivery systems [15,16], and their combination has also been explored previously. CS:PVP studies generally evaluate single cargo classes or present limited cross-comparisons across chemically diverse agents [17,18]. In contrast, here we wish to establish a single, optimized CS:PVP platform that encapsulates both synthetic bis-THTT derivatives (JH1, JH2) and natural apitherapy products (honey, propolis) and interrogates, under uniform synthesis and testing conditions, the impact of cargo chemistry on encapsulation (EE/LC), release in artificial gastric fluid, antimicrobial performance, and 3T3 cytocompatibility. This design allows for a direct, controlled comparison between synthetic and natural agents within the same CS–PVP matrix, clarifying how matrix–cargo interactions govern particle formation, stability, and function. By aligning materials optimization with comparative physicochemical characterization and biological readouts, our study delivers an integrated view of how CS–PVP can serve as a versatile, biocompatible carrier for chemically distinct antimicrobials, addressing an underexplored area in the CS:PVP literature.

The current study addresses these challenges by exploring CS-PVP-based nanoparticles as carriers for both synthetic and natural compounds. Previous research highlights the potential of CS for its mucoadhesiveness and antimicrobial activity and PVP for its stabilizing properties, yet the synergistic combination of these polymers remains underexplored.

This work aims to synthesize, optimize, and characterize CS-PVP-based nanoparticles for the encapsulation of bis-THTTs (JH1, JH2) and natural products (honey bee and propolis). We investigate their physicochemical properties, antimicrobial activity, cytotoxicity, and drug release profiles. This study could establish a robust platform for developing multifunctional drug delivery systems (DDSs), advancing treatments for microbial infections and malaria, and demonstrating the potential of integrating natural resources into cutting-edge nanotechnology [19,20].

2. Materials and Methods

Raw materials used for the preparation of NPs include low molecular weight chitosan (LMWCS, CAS 9012-76-4, Product Number 448869) and high molecular weight chitosan (HMWCS, CAS 9012-76-4, Product Number 419439) from Sigma Aldrich (St. Louis, MO, USA). Glacial acetic acid (AA) ACS grade 100% pure (CAS 64-19-7, Product Number S25118B, Fisher Chemical, Waltham, MA, USA), polyvinylpyrrolidone (PVP, CAS 9003-39-8, Product Number PVP40) (M.W.: 40,000 kDa), sodium hydroxide (NaOH) pellets (CAS 1310-73-2, Product Number 567530, Sigma Aldrich, USA), sodium tripolyphosphate (TPP, CAS 7758-29-4, Product Number 238503, Sigma Aldrich), and type I distilled water were used in this research. LMWCS:PVP weight-to-weight (w/w) ratios of 1:0.5, 1:0.75, 1:1, and 1:1.25 were used to optimize chitosan (CS)–polyvinylpyrrolidone (PVP) based NPs. JH1 and JH2 (Figure 1) were synthesized in the Organic Synthesis Laboratory at the University of Havana (La Habana, Cuba) and kindly donated by Professors Hortensia Rodriguez and Julieta Coro. Honey bees and propolis were sourced from the community of Iruguincho, located in San Miguel de Urcuquí, Ecuador.

2.1. Chitosan (CS)-Based Nanoparticles (CS-NPs)

The CS/TPP nanoparticles were synthesized by using the ionic gelation method with minor modifications [21,22]. Both high- and low-molecular-weight CS were separately dissolved in 10 mL of a 10% acetic acid solution under magnetic stirring. The resulting solution was then diluted with type I water to obtain a final chitosan concentration of 0.2% (w/v) [23]. The pH of the chitosan solution was adjusted to 5.85 using 2 M sodium hydroxide (NaOH). A 0.07% (w/v) TPP solution was prepared and added dropwise to the chitosan solution at volumetric ratios of CS:TPP of 1:0.5, 1:0.75, 1:1, and 1:1.25 under magnetic stirring at 1000 rpm for 2 h at room temperature. The stirring duration was chosen based on preliminary trials showing that it led to optimal nanoparticle stability and uniformity. The ionic gelation process was initiated by the addition of TPP, leading to the formation of CS NPs. The NPs were then separated by centrifugation at 60,000 rpm for 30 min. The centrifugation conditions used were based on optimizing pellet formation and removing unreacted material [21]. The pellet was resuspended, ultra-frozen at −80 °C, and freeze-dried for 72 h.

2.2. Chitosan (CS)–Polyvinylpyrrolidone (PVP)-Based Nanoparticles (CS-PVP NPs)

LMWCS (M.W.: 50-200 kDa, 76% deacetylation degree, Sigma Aldrich, USA) was mixed with PVP at the previously mentioned LMWCS:PVP ratios. The mixture was dissolved in 10 mL of a 10% acetic acid solution and further diluted with type I water to reach a final concentration of 0.2% (w/v). The pH was adjusted to 5.79 with 2 M NaOH. A 0.07% (w/v) TPP solution was added in a volumetric ratio of 1:0.5 under continuous stirring at 10,000 rpm for 1.5 h at room temperature, providing stable and reproducible nanoparticle formation. The centrifugation conditions used were based on optimizing pellet formation and removing unreacted material. [21] The nanoparticles were separated by centrifugation and processed as described above.

2.3. Encapsulation of Bioactive Compounds

To encapsulate cargo, the proportion of LMWCS-PVP: TPP was previously optimized to 5:2. The synthetic derivatives of bis-THTT (JH1 and JH2) were pre-dissolved in DMSO to enhance their solubility in the acidic CS-PVP solution, whereas the natural products (honey bee and propolis) were directly dissolved into the solution before the ionic gelation method [24,25]. The final concentration of each compound was 0.1% (w/v). TPP was added to the solution at a 1:1 volumetric ratio under continuous stirring at 10,000 rpm for 1.5 h. This 1:1 (v/v) ratio was selected to achieve an optimal balance between encapsulation efficiency and nanoparticle stability, ensuring sufficient cross-linking while avoiding particle aggregation. The NPs were separated by centrifugation and freeze-dried as described above.

2.4. Nanoparticles Characterization

Fourier-Transform Infrared Spectroscopy (FTIR): FTIR spectra were recorded using a Spectrum Two FTIR spectrometer (PerkinElmer, Inc., Waltham, MA, USA) with a diamond ATR accessory. The spectra were collected in the range of 4000 to 650 cm⁻¹.

Thermogravimetric Analysis (TGA): TGA was performed using a TGA 55 thermogravimetric analyzer (TA Instruments, New Castle, DE, USA) with a heating rate of 20 °C per minute from room temperature to 850 °C in a nitrogen atmosphere.

X-ray Diffraction (XRD): XRD analysis was conducted using a Minifelx 600 diffractometer (Rigaku Analytical Instruments, Woodlands, TX, USA) with CuKα radiation. The diffraction pattern was recorded in the 2θ range from 20° to 90°.

Atomic Force Microscopy (AFM): AFM imaging was performed using an AFM NX7 (Park Systems, Suwon, Republic of Korea) to study the surface morphology of the NPs. Samples were prepared at a concentration of 0.0001% and deposited onto 1 cm² silicon plates.

Dynamic light scattering (DLS) and zeta potential measurements were not available during this study; therefore, nanoparticle morphology and structural confirmation were assessed using AFM and XRD.

2.5. Antimicrobial Activity

The antimicrobial activity of the NPs was evaluated using the disk diffusion method. Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were cultured in nutrient broth and plated on Mueller–Hinton agar. The nanoparticles were applied to sterile paper disks, and inhibition zones were measured after 24 h of incubation at 35 °C. The positive controls used were ampicillin for E. coli and vancomycin for S. aureus.

2.6. Cytotoxicity Assay

Cytotoxicity was evaluated using the MTT colorimetric assay, following the instructions of the CellTiter 96^® Non-Radioactive Cell Proliferation Assay [26] in the fibroblast mouse cell line 3T3. Cell line was cultured in a 96-well microplate and incubated with NP solutions at concentrations of 5 μg/mL and 10 μg/mL. The cell culture plates were incubated for 72 h at 37 °C with 5% CO₂. After incubation, 20μl of MTT reagent (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide, Promega) was added to each well and incubated for three hours at 37 °C. Each hour, absorbance was measured at 490 nm at 0, 1, 2, and 3 h. A total of 200 μL of Dulbecco’s modified Eagle’s medium (DMEM) was used as negative control, and 50 μL of cells were added to 150 μL of DMEM as positive control.

2.7. In Vitro Controlled-Release Study in Gastric Fluid

The controlled release of the encapsulated compounds was studied by dispersing 15 mg of lyophilized NP in 15 mL of artificial gastric fluid (AGF), simulating gastrointestinal conditions. The release profile was monitored by measuring the absorbance of the solution at 280 nm using a NanoDrop spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA).

2.8. Statistical Analysis

All experiments were performed in triplicate, and data were analyzed using one-way ANOVA. The Shapiro–Wilk test was used to assess the normality of the data. Statistical significance was set at a p-value of 0.05.

3. Results

Polymer nanoparticles were prepared using CS and PVP as scaffold structures. The synthetical procedures were optimized to establish better options related to CS/TPP and CS/PVP ratios. In addition, NPs encapsulating bis-THTT (JH1 and JH2), honey bees, and propolis were prepared to test the potential of CS- and CS-PVP-based nanoparticles to be efficient carriers for synthetic compounds (JH1 and JH2) and natural products (honey bees and propolis).

3.1. CS-PVP-Based NP Synthesis and Cargo Encapsulation

LMWCS was selected to optimize CS-PVP-based NPs. HMWCS was also tested, exhibiting early material agglomerations and phase separation that was visibly evident during the nanoparticle formation step, particularly after the addition of TPP and before or during centrifugation, when precipitation was observed at the bottom of the beaker, signaling poor stability and inhomogeneity of the HMWCS-based system and leading to its exclusion in this research. The CS-PVP-based NPs were prepared using the ionic gelation method, which is based on ionic cross-linking in the presence of the inversely charged groups of the protonated amino groups of CS and negatively charged groups of the TPP (Figure 2), as it has been previously used [27]. In this context, different CS:PVP ratios were tested, starting from 1:0.5 and increasing the PVP proportion to 1:0.75, 1:1, and 1:1.25. It was found that the CS:PVP ratio that gives the best result was 1:0.5.

Chitosan (CS) and polyvinylpyrrolidone (PVP) interact primarily through hydrogen bonding between the hydroxyl and amino groups of CS and the carbonyl groups of PVP. These interactions improve the stability and solubility of the composite nanoparticles. Additionally, electrostatic interactions can occur between the positively charged amino groups of chitosan (under acidic conditions, i.e., HOAc (10%)) and any polar or partially negative regions in PVP. This synergistic interaction promotes homogeneous nanoparticle formation and reduces aggregation. Also, different ratios between the CS-PVP solution and the cross-linking agent (TPP) were tested, finding that the CS-PVP:TPP ratio of 1:0.5 shows the best results in terms of NP formation and quantity (Figure 3).

As the CS:TPP ratio increases, the resulting NPs exhibit larger sizes and a propensity to form agglomerates that precipitate at the bottom of the reaction vessel, consistent with previously reported findings [28].

To leverage the synergistic properties of CS and PVP, NPs loaded with cargos (JH1, JH2, honey, and propolis) were prepared (Figure 4). To encapsulate cargo, the proportion of LMWCS-PVP: TPP was previously optimized. It was observed that as the LMWCS-PVP:TPP ratio increases, the synthesis of nanoparticles becomes unfeasible since the particle size obtained is larger, promoting its separation from the aqueous phase as a precipitate. The dissolution of the cargos in the CS-PVP matrix caused the cross-linking agent ratio to also change, obtaining a new optimal CS-PVP:TPP ratio of 5:2. This is because the addition of the new molecules into the CS-PVP matrix also implies more interactions among the different functional groups present in the chemical structure of the cargos.

3.2. CS-PVP NPs Characterization

3.2.1. Fourier-Transform Infrared Spectroscopy (FTIR) Analysis

FTIR analysis confirmed the presence of characteristic bands of the precursors, CS and PVP (Figure 5), and the encapsulated compounds: bis-THTTs (JH1 NPs and JH2 NPs) (Figure 6), honey bee NPs, and propolis NPs (Figure 7). For the CS-PVP NPs encapsulating cargos, it is observed that there is a change in intensity of all bands with the addition of each cargo (JH1, JH2, honey bee, and propolis). Because the bis-THTTs are introduced during the ionic gelation cross-linking to the JH1 NP and JH2 NP formation, some modifications in the position and the intensity signals in the FTIR profile of JH1 NPs and JH2 NP compared with empty LMWCS-PVP NPs would be expected to confirm the successful encapsulation. Bands attributed to the amide bond (-NH) and hydroxyl groups (-OH) were observed in the range of 3200–3400 cm⁻¹ [29], indicating hydrogen bond interactions. The specific bands of PVP, such as those corresponding to the C=O groups, were found at 1660–1680 cm⁻¹ [30], confirming the integration of PVP into the NP matrix (Figure 5). On the other hand, the spectra of bis-THTT NPs (JH1 NPs and JH2 NPs) showed characteristic peaks [31,32] of the C-N bond at 1520 cm⁻¹ and the C-S bond at 700–750 cm⁻¹ [33], indicating their incorporation into the NPs (Figure 6).

When the honey was encapsulated to obtain honey bee NPs, the FTIR spectrum of NPs (Figure 7, Green line) showed some small shifts and intensity variations in the signals from CS-PVP NPs (Figure 7, Black line) and non-encapsulated honey (Figure 7, Fuchsia line). Because the honey bee NPs were previously lyophilized, the higher frequency zone of the NPs in the FTIR spectrum is more defined, presenting bands at 3200–3300 cm⁻¹, associated with hydroxyl groups (-OH) [34], and peaks at 1400–1600 cm⁻¹, corresponding to phenols and flavonoids [35], which confirmed their encapsulation (Figure 7).

The propolis is one of the best-known honey bee products. Organic compounds that have been identified in propolis are polyphenols, terpenes, esters, amino acids, vitamins, minerals, and sugars [36]. In our case, the raw propolis was previously extracted with ethanol to be encapsulated. In this context, for propolis (Figure 7, blue line), the FTIR spectrum shows the presence of several signals. A broad band at 3283 cm⁻¹ could be assigned to O-H bond stretching related to the alcohols, phenolic acids, and other organic compounds in the propolis. The bands at 2934 cm⁻¹ correspond to the stretching of C–H bonds in alkyl side chains of aliphatic and aromatic organic compounds. The peak at 1610 cm⁻¹ is a typical signal to C=O (carbonyl) bonds found in carboxylic acids and esters present in propolis [37]. C-O-C (ether) bonds are identified at 1258 cm⁻¹, present in ethers and glycosides in the propolis structure [38,39,40]. At 1518 cm⁻¹, the deformation of N-H bonds in amino groups is detected, which may be present in proteins and amino acids in propolis [41]. At 1031 cm⁻¹ is located a peak corresponding to C-O (stretch) bonds present in ethers and alcohols of phenolic derivatives, flavonoids, and other organic components. The FTIR spectrum of propolis NPs (Figure 7, red line) showed behavior similar to that of the previously discussed honey NPs. All mentioned signals related to propolis (Figure 7, blue line) and CS-PVP NPs (Figure 7, black line) are found in the propolis encapsulated NPs, with slight shifts and changes in the intensity of signals. Similar to honey NPs, the higher frequency region is more defined, in comparison with the non-encapsulated propolis. Signals at 3416, 3280, and 3175 cm⁻¹ were assigned to the O-H and N-H bond stretching of these functional groups in the polymers (CS and PVP) and propolis.

3.2.2. Thermogravimetric Analysis (TGA)

The thermogravimetric behavior of starting materials CS and PVP, as well as the thermogravimetric behavior of CS-PVP NPs, was studied. In the case of CS, an initial mass loss of 10–12% was observed between 50–100 °C (Figure 8a, black line), attributed to the removal of absorbed water [41]. The main decomposition occurred in the range of 250–350 °C, associated with the degradation of the polysaccharide (Figure 8a, black line) [42]. On the other hand, PVP exhibited a gradual weight loss between 150–350 °C, with a significant degradation peak at approximately 360 °C, due to the breaking of C-N bonds and the decomposition of its polymeric structure (Figure 8a, red line) [43,44]. CS-PVP NPs showed an improvement in thermal stability compared to the individual components (Figure 8a, blue line). Water loss occurred at 50–100 °C, while the main degradation was observed in the range of 398–473 °C, reflecting a synergistic interaction between chitosan and PVP. On the other hand, the cargos (JH1, JH-2, honey bee, and propolis) induce a lower thermal stability in the CS-PVP NPs [45] (Figure 8b).

3.2.3. X-Ray Diffraction (XRD)

Consistent with its amorphous nature [46,47], CS and PVP displayed a broad, diffuse diffraction pattern (Supplementary Materials, Figure SIB(a)). A notable increase in intensity at 2θ was observed in the range of 20–30°, attributed to the presence of semicrystalline regions within the CS and PVP structure, respectively [48]. A significant deviation in the XRD pattern was observed for CS-PVP NPs compared to their polymeric components [49]. The presence of multiple sharp and well-defined peaks, notably at 2θ in the range of 15–30°, provided evidence for the increased structural ordering in NPs compared to the individual polymers [25,50]. These results suggest that hydrogen bond formation between the pyrrolidine rings of PVP and the amino and hydroxyl groups of CS during NP formation facilitated an increase in the degree of order and crystallinity within the material [51].

The XRD results of cargos JH1 and JH2 showed that both are predominantly amorphous materials. JH1 exhibits a characteristic diffraction pattern of amorphous materials (Supplementary Materials, Figure SIB(b)) and JH2 presents some weak peaks, indicating a certain degree of crystalline order (Supplementary Materials, Figure SIB(c)). Upon incorporating JH1 and JH2 into the NPs, a decrease in the intensity of the characteristic peaks of CS-PVP NPs and a slight shift towards higher 2θ angles was observed. As was expected, the presence of the synthetic cargo reduces the overall crystallinity of the system [52,53].

XRD analysis of loaded NPs with natural products (honey bee NPs and propolis NPs) also revealed a decrease in their crystallinity compared to empty CS-PVP NPs (Supplementary Materials, Figure SIB(d)). While both honey bee NPs and propolis NPs presented multiple peaks at 2θ in the range of 15–50°, the intensity and definition of these peaks were lower. This reduction in crystallinity is attributed to the presence of amorphous components and the more disordered nature of honey and propolis, in addition to the water content in these natural products. When comparing both, propolis NPs showed a higher signal intensity, indicating a slightly higher degree of crystallinity compared to honey bee NPs.

3.2.4. Atomic Force Microscopy (AFM)

Unloaded CS-PVP NPs, at a concentration of 0.0001%, allowed clear AFM images (Figure 9), exhibiting a three-peak structure with an apparent height of approximately 8 nm and a width of 0.3 µm in the 3D model. However, a closer examination of the full scan area showed that this larger structure represents an aggregate of multiple smaller nanoparticles, rather than a single particle. Numerous smaller, individual nanoparticles are also visible across the scanned surface. Therefore, the 8 nm height measurement should be interpreted as representative of a nanoparticle cluster rather than a discrete particle. This aggregation behavior is likely due to residual interactions during drying and deposition on the silicon substrate, a phenomenon commonly observed in polysaccharide-based nanoparticles [54].

JH1 NPs showed a smaller size compared to CS-PVP NPs, with an average height of 0.4 nm and a width of 40 nm, suggesting that interactions between components during ionic gelation reduced the particle size. This size reduction may be attributed to the strong interaction between the thiadiazine derivative and the chitosan-PVP matrix, which could induce tighter particle packing during ionic cross-linking. A similar trend has been reported by Nematollahi et al. [55], who observed size reduction in drug-loaded chitosan nanoparticles due to drug–polymer interactions that alter micelle formation.

On the other hand, JH2 NPs presented an approximate height of 6 nm and a width of 100 nm, which was also corroborated by the 3D model. The greater uniformity in JH2 NPs may be related to improved compatibility of JH2 in the polymeric network, facilitating more symmetrical nanoparticle formation.

The NPs encapsulating natural products showed more variable morphologies. Honey bee NPs formed agglomerates due to the moist nature of honey, although isolated N Ps with heights of 0.37 nm and 0.39 nm and widths of 0.23 µm and 0.15 µm were identified. Propolis-loaded NPs also exhibited some aggregation, which was attributed to the sugars and phenolic compounds present, but three isolated nanoparticles with heights of 14.5 nm, 15 nm, and 17 nm and widths of 0.10 µm, 0.15 µm, and 0.12 µm, respectively, were also identified.

In general, the AFM-derived dimensions of our nanoparticles were lower than the optimized nanoparticle sizes typically reported in the literature for biomedical applications, which range between 50 and 200 nm [56]. AFM analysis confirmed the presence and morphology of the CS–PVP nanoparticles after deposition on mica substrates. The particles appeared well distributed and exhibited nanometric features consistent with their expected dimensions. However, AFM height values are typically lower than true hydrodynamic diameters because soft polymeric nanoparticles tend to flatten during drying and due to tip–sample interactions, while lateral widths are often exaggerated by tip convolution effects. Therefore, AFM is best interpreted as qualitative confirmation of nanoparticle formation, morphology, and relative nanoscale distribution, rather than as an absolute measure of particle size. In the present study, AFM observations were complemented by XRD and FTIR analyses, which together confirmed successful nanoparticle formation and matrix–cargo interactions.

3.3. Antimicrobial Activity

The antimicrobial activities against S. aureus 25923 and E. coli ATCC 25922 were established through the disk diffusion method. The antimicrobial evaluation in this study was limited to E. coli ATCC 25922 (Gram-negative) and S. aureus ATCC 25923 (Gram-positive), which are internationally recognized reference strains and commonly used to provide a baseline comparison across bacterial classes. The CS-PVP-based nanoparticles did not display antibacterial activity toward S. aureus 25923, probably due to the high resistance of this strain [57,58]. On the other hand, the prepared NPs displayed antibacterial activity toward E. coli ATCC 25922 (Figure 10). In general, the prepared NPs demonstrated higher efficacy than non-encapsulated cargos [59], although ampicillin as positive control and chloroquine (CQ) were the most effective materials (Figure 10).

JH1 NPs showed an inhibition halo of 0.52 mm (vs. 0.48 mm to JH1) and JH2 NPs showed an inhibition halo of 0.51 mm (vs. 0.49 mm for JH2). In contrast, honey bee NPs, with an inhibition halo of 0.46 mm, and Propolis NPs, with an inhibition halo of 0.43 mm, were less effective than their non-encapsulated forms (0.47 mm and 0.49 mm, respectively) (Figure 10).

The data followed a normal distribution according to the Shapiro–Wilk test (p > 0.05). ANOVA analysis revealed significant differences between at least nine types of nanoparticles (p < 0.05), confirmed in some cases by the Tukey test.

3.4. Cytotoxic Evaluation

Cytotoxicity analysis demonstrated that all investigated NPs have low cytotoxicity and maintain high cell viability in 3T3 mouse fibroblasts at 5 and 10 μg/mL, with the same trend, regardless of concentration. Throughout the 3 h of incubation after MTT addition, JH1 NPs presented the highest average absorbances. JH2 NPs showed absorbances slightly lower than those of JH1 NPs, supporting their low cytotoxicity. The honey bee NPs and propolis NPs also present high absorbances at 3 h, indicating high cell viability and low cytotoxicity, although slightly lower than the JH1 NPs and JH2 NPs (Figure 11).

Although absorbances decreased a little at higher concentrations, all loaded NPs outperformed the positive control, confirming their safety for therapeutic applications [60,61,62]. Empty NPs (CS-PVP NPs) showed very high absorbances, similar to the loaded ones, indicating that the CS and PVP matrix is not cytotoxic on its own and contributes to the biocompatibility of the evaluated systems. ANOVA statistical analysis at 2 h of incubation revealed significant differences between JH1 NPs and honey bee NPs at 5 µg/mL (p = 0.035), while at 10 µg/mL, no significant differences were found between any of the nanoparticles (p > 0.05) (Figure 11).

3.5. Encapsulation Efficiency (EE, %), Load Capacity (LC, %), and Cumulative Release

Propolis NPs stood out with an EE of 60% and an LC of 28%, being the highest (Figure 12). JH2 NPs reached an EE of 55% and an LC of 25%, while JH1 NPs presented the lowest values, with an EE of 44% and an LC of 22%. Honey bee NPs had an EE of 51% and a LC of 26%, reflecting good chemical compatibility with the polymeric matrix.

In the release study (Figure 13), JH2 NPs released approximately 20% of the encapsulated compound after 180 min, showing a more controlled and sustained release in gastric fluid profile compared to propolis NPs and honey bee NPs. Shapiro–Wilk statistical analysis confirmed a normal distribution of the data (p > 0.05). ANOVA and Tukey test revealed significant differences in EE and LC between most nanoparticles (p < 0.05), except between JH2 NPs and honey bee NPs (p = 0.22) and between propolis NPs and honey bee NPs (p = 0.10), where no significant differences were found (Figure 13).

NPs encapsulating natural products showed higher cumulative release in gastric fluid. Propolis NPs achieved 28% release and honey bee NPs achieved 26% at the end of the 180 min (Figure 13).

In NPs encapsulating synthetic compounds, JH2 NPs presented a higher cumulative release of 20%, while JH1 NPs, which had the lowest encapsulation efficiency (EE) and loading capacity (LC) values, achieved only 15% cumulative release (Figure 13).

4. Discussion

4.1. NPs Synthesis and Characterization

CS-PVP NPs encapsulating both synthesized compounds (bis-THTT, JH1, and JH2), and natural products (honey bee and propolis) were obtained, and their full physicochemical characterization was carried out to corroborate their formation and main features. The CS-PVP-based NPs can leverage the advantages of both materials, resulting in NPs with enhanced properties. The biocompatibility, mucoadhesiveness, and antimicrobial activity of CS complement the solubility and stability provided by PVP.

The NP characterization was carried out through FTIR, TGA, DRX, and AFM.

FTIR results supported successful cargo integration, with a slight shift in signals. The observed frequency changes are associated with the interaction between the polymers (CS and PVP) and cargos, confirming the successful cargo encapsulation.

TGA analysis revealed improved thermal stability of loaded NPs, suggesting that the polymer matrix offers protective effects against thermal degradation. The enhanced thermal properties of JH1 and JH2 NPs, and even more so for honey and propolis NPs, highlight the stabilizing role of the CS-PVP matrix. For JH1, the main decomposition range was 145–178 °C, while in JH1 NPs it was considerably broadened to 135–225 °C, indicating enhanced low-temperature resistance (Figure 8b). The maximum decomposition temperature increased from 321 °C in JH1 to 456 °C in JH1 NPs, reflecting a significant improvement in thermal stability. For JH2, the initial decomposition range was 135–175 °C, while in JH2 NPs it shifted to a higher range of 410–480 °C, evidencing significant thermal stabilization. The decomposition temperature decreased from 521 °C in JH2 to 323 °C in JH2 NPs, which may be a result of chemical interactions between the compound and the NP matrix [63]. Also, the residual mass increased from 0% in non-encapsulated compounds (JH1 and JH2) to 38% in loaded NPs, suggesting protection of the encapsulated material (Figure 8b) [64] (Supporting Information, Figure SI-A). For honey bees, the main decomposition range was 170–260 °C, while in honey bee NPs, it was extended to 400–470 °C, showing an improvement in thermal stability. The maximum decomposition temperature increased from 225 °C in honey bees to 541 °C in honey bee NPs. The residual mass increased from 0% in pure honey to 36% in NPs, demonstrating effective protection against complete decomposition. For propolis, the main decomposition range was 170–240 °C, while in propolis NPs, it shifted to 420–480 °C. The decomposition temperature increased from 268 °C in propolis to 719 °C in propolis NPs, and the residual mass also increased from 1% to 41%. Empty CS-PVP NPs showed the highest thermal stability (Supplementary Materials, Figure SIA).

XRD showed reduced crystallinity in loaded NPs. The decrease in crystallinity of CS-PVP NPs containing cargos (synthetic products JH1 and JH2 and natural products such as honey bee and propolis) could be attributed to the increased structural disorder due to the complexity of the cargos and their interactions with the CS and PVP during the formation of the NPs. The XRD patterns of CS–PVP nanoparticles revealed broad peaks indicative of partial structural ordering, likely due to hydrogen bonding between chitosan and PVP chains. While this represents increased organization relative to the individual polymers, the system remains predominantly semi-crystalline/amorphous, consistent with previous reports on CS–PVP blends [65,66,67,68,69].

Micrographs obtained by AFM (Figure 9) confirmed the successful formation of nanoparticles by the ionic gelation method and revealed significant morphological differences based on the encapsulated material. The previously described morphology is characteristic of well-formed polymeric nanoparticles stabilized by electrostatic and hydrogen-bonding interactions between chitosan and polyvinylpyrrolidone (PVP) [70]. The relatively smooth and continuous surface supports the notion that PVP contributes to improved nanoparticle uniformity and prevents aggregation, consistent with previous findings [71]. Specifically, the honey bee NP agglomeration observed is likely due to the hygroscopic and viscous nature of honey, which complicates uniform particle drying and dispersion on the silicon AFM substrate. Sharaf et al. [72] similarly reported aggregation in honey-based formulations due to their high sugar content and moisture retention. The larger dimensions of propolis NPs are probably due to the presence of flavonoids and phenolic compounds, which participate in secondary interactions—such as hydrogen bonding and π-π stacking—with the chitosan and PVP chains. Salatin et al. [73] reported a comparable increase in nanoparticle dimensions when incorporating phenolic-rich extracts into chitosan-based carriers.

AFM and XRD were employed to confirm nanoparticle morphology and structural organization, and future work will incorporate DLS and surface charge analysis to complement and strengthen the physicochemical characterization.

These results show the formation of NPs and the interaction between the components, highlighting the role of CS and PVP in the stabilization and encapsulation of the investigated compounds. Prior CS:PVP reports tend to focus on a single cargo class [17,18], while our study systematically unifies synthetic and natural antimicrobials in one optimized platform, enabling mechanistic comparisons under consistent synthesis/characterization/biological testing.

4.2. Biological Approach

The antimicrobial assays emphasized the efficacy of JH1 and JH2 NPs against E. coli, confirming the retained and possibly enhanced bioactivity after encapsulation. Honey and propolis NPs also showed activity, though slightly reduced compared to their free forms, likely due to slower diffusion from the NP matrix.

The enhanced antibacterial activity of the encapsulated formulations against E. coli ATCC 25922 compared to their free counterparts can be attributed to the protective and stabilizing effect of the CS:PVP matrix. Encapsulation likely improves solubility and prevents premature degradation of the cargo molecules, thereby facilitating a more sustained release and prolonged local availability at the bacterial surface. Additionally, chitosan itself is known to possess intrinsic antimicrobial properties through interactions with negatively charged bacterial membranes, which may synergize with the encapsulated compounds to enhance efficacy [74,75,76]. Nevertheless, the inhibition zones remained smaller than those produced by standard antibiotics such as ampicillin or chloroquine, which is expected given the potent, targeted activity of these clinical drugs compared to the broader, controlled-release action of the nanoparticle systems [75,76]. This observation is consistent with the limited diffusion of nanoparticle-based and controlled-release systems through agar, which restricts halo size even in the presence of antimicrobial activity [77,78]. The fact that encapsulated cargos produced slightly but statistically significant larger halos than their free counterparts (i.e., JH1 NPs at 0.52 mm vs. free JH1 at 0.48 mm) indicates that encapsulation improves solubility and stability of the active agents and may benefit from the intrinsic antimicrobial effect of chitosan [79]. While disk diffusion thus provides a conservative estimate of activity, complementary assays such as MIC determination and time-kill kinetics will be required in future work to fully establish the biological significance of these effects [78].

Future studies will therefore extend testing to a broader range of clinically relevant microorganisms such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Candida albicans to better define the antimicrobial spectrum of the CS–PVP nanoparticle systems.

Cytotoxicity results demonstrated the biocompatibility of all formulations, making them promising in terms of therapeutic use. The tested concentrations (5 and 10 μg/mL) confirmed high cell viability. Future work will therefore include higher NP concentrations and dose–response analyses to more precisely define the safety margin and potential therapeutic window.

These findings suggest that while CS–PVP NPs are less potent than standard antibiotics in terms of absolute inhibition, their combination of moderate antimicrobial activity, low cytotoxicity, and controlled-release profile highlights their potential as safer, biocompatible alternatives for long-term antimicrobial therapy.

4.3. Encapsulation Metrics and Controlled Release in Gastric Fluid

The release profiles were obtained in artificial gastric fluid (pH 1.78) over 180 min, which was chosen to simulate gastric residence times (typically 1–3 h). This assay thus served to evaluate nanoparticle stability and protection of the encapsulated cargos under acidic conditions.

The NPs showed moderate encapsulation efficiencies (EE) and loading capacities (LC) according to the literature [79]. Encapsulation metrics and sustained release profiles confirmed that these nanoparticles are capable of prolonged and efficient delivery, particularly for natural products. This behavior is related to the chemical composition of natural products: the multiple phenolic and flavonoid compounds in propolis, together with the sugars, proteins, and phenolic compounds in honey bees [79,80], facilitate their encapsulation and controlled release. The higher release observed in honey and propolis NPs reflects the solubility and interaction of their multiple functional groups with the matrix.

These findings collectively support CS-PVP NPs as versatile platforms for controlled and biocompatible drug delivery

5. Conclusions

Chitosan–polyvinylpyrrolidone (CS-PVP)-based nanoparticles (NPs) were successfully synthesized by ionic gelation method, using TPP as a cross-linking agent. LMWCS was selected for nanoparticle synthesis due to its superior performance in terms of stability and reduced agglomeration, as compared to HMWCS, which showed early phase separation and material precipitation during the ionic gelation process. The optimal proportions to control the size and avoid agglomeration of the NPs were CS:PVP (1:0.5) and CS-PVP:TPP (5:2). Characterization by FTIR, TGA, XRD, and AFM confirmed the formation of the NPs and the effective encapsulation of bis-THTT (JH1 and JH2) and natural antimicrobials (honey bee and propolis).

FTIR evidenced interactions between polymers and encapsulated compounds, while TGA showed higher thermal stability in encapsulated NPs, especially for natural products (honey and propolis), which outperformed synthetic compounds (JH1 and JH2) in stability before 450 °C. XRD analysis revealed more ordered systems after NP formation, although some loss of order was observed upon incorporation of compounds. AFM micrographs validated the morphological formation of NPs.

In terms of antimicrobial activity, NPs showed moderate efficacy against E. coli ATCC 25922 and none against S. aureus 25923, probably due to the higher intrinsic resistance of the latter. Chloroquine exhibited superior activity, while the inhibition zones observed for the NPs and their encapsulated compounds were significantly lower than those of the positive control.

Cytotoxicity assessment (MTT assay) confirmed that the NPs are not cytotoxic at concentrations of 5 and 10 µg/mL in 3T3 fibroblasts over 3 h of incubation, indicating their biocompatibility. Release studies demonstrated encapsulation efficiencies (44–60%) and loading capacities (22–28%), with propolis NPs standing out in both parameters. The cumulative release did not exceed 30% in 180 min, which positions them as promising systems for the sustained release of therapeutic compounds.

As a conclusion, CS-PVP cross-linked with TPP-based NPs shows promising properties for encapsulating other therapeutic molecules. It could be envisaged that the encapsulation of more biologically active molecules will allow us to highlight the benefits of these NPs.