Poloxamer 407, poloxamer 188, HA, CBD, and colloidal Ag (density, 10.49 g/cm³) were purchased from Sigma-Aldrich (Taufkirchen, Germany). Purified water was purchased from Merck Millipore (Darmstadt, Germany).

The study utilized several bacterial strains sourced from the American Type Culture Collection (ATCC; Manassas, VA, USA), including Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853), as well as a fungal strain, Candida albicans (ATCC 14053).

Primary normal human dermal fibroblasts (NHDFs) were obtained from the ATCC (Manassas, VA, USA). These cells were selected for their well-characterized phenotype and relevance as a model for dermal tissue studies. NHDFs were maintained under standard culture conditions at 37 °C with 5% carbon dioxide (CO₂) in a humidified incubator to ensure optimal growth and viability.

The culture medium consisted of Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Thermo Fisher Scientific, Leicestershire, UK) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific) to provide essential growth factors and nutrients. To prevent microbial contamination, the medium was further supplemented with 1% (v/v) antibiotic–antimycotic solution containing 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 0.25 μg/mL of amphotericin B (Sigma-Aldrich, St. Louis, MO, USA). Cells were routinely passaged at 80–90% confluence using 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA) (Gibco, Thermo Fisher Scientific) and seeded at a density of 5 × 10³ cells/cm² for experimental assays. All procedures were performed under sterile conditions in a Class II biosafety cabinet to ensure the reproducibility and reliability of the results.

The hydrogels were prepared under controlled low-temperature conditions to preserve the stability of thermolabile constituents and prevent nanoparticle (NP) aggregation. Cold purified water was pre-equilibrated and maintained at 4–8 °C in a refrigerated chamber. All subsequent steps, including HA incorporation and addition of the Ag-CBD dispersion, were carried out in an ice bath with continuous monitoring, ensuring that the temperature remained consistently below 15 °C throughout the process.

Poloxamer 407 and poloxamer 188 were accurately weighed and slowly dispersed in cold water under magnetic stirring to achieve complete solubilization while minimizing air entrapment. The solution was refrigerated overnight (≥12 h) to ensure full polymer hydration, yielding a clear, homogeneous base. HA was then gradually added under continuous slow stirring at low temperatures to prevent lump formation and ensure uniform distribution. Separately, CBD was dissolved in 4% (v/v) ethanol and gently mixed with colloidal AgNPs (0.02%) to form a stable Ag-CBD dispersion. This mixture was incorporated into the HA–poloxamer base at <15 °C, promoting even distribution, emulsification, and preservation of molecular integrity (Table 1; Figure 1a–c). The final hydrogel was transferred into opaque, airless containers to protect light-sensitive components and stored at 4–8 °C until use.

The CBD concentration (0.5%, w/v) was selected based on prior reports demonstrating therapeutic efficacy of topical CBD in the 0.1–1.0% range [17,18,19,20,21,26,27] while ensuring stability and compatibility with the HA–poloxamer–AgNP system. Preliminary optimization confirmed that 0.5% provided an optimal balance between antioxidant/anti-inflammatory activity and hydrogel integrity.

The incorporation of CBD and AgNPs into the HA–poloxamer hydrogel was confirmed through complementary characterization techniques: Fourier-transform infrared (FTIR) analysis to verify the presence of CBD functional groups within the gel matrix, scanning electron microscopy (SEM) imaging to confirm the homogeneous AgNP distribution, and dynamic light-scattering (DLS) measurements to display NP stability and preserved hydrodynamic size. These analyses were selected to support the successful loading and structural integrity of both actives, although quantitative release into biological media was not measured in this study.

All hydrogel batches were prepared following a controlled low-temperature protocol, with strict monitoring of temperature and mixing. Reproducibility was verified by assessing particle distribution and hydrogel morphology using SEM and DLS, ensuring consistent appearance and rheological behavior across independent replicates.

The pH of the final HA-Ag-CBD hydrogel was measured at room temperature (22–25 °C) using a calibrated WTW^® VARIO 2V00 digital pH meter (Xylem Analytics GmbH & Co. KG, Weilheim, Germany). Measurements were performed in triplicate on freshly prepared samples to ensure reproducibility. The hydrogel consistently exhibited a pH range of 6.8–7.4, confirming its compatibility with physiological and topical skin conditions [36,37].

FTIR spectra were acquired using a Shimadzu AIM-9000 spectrometer with attenuated total reflectance (ATR) accessories (Shimadzu Corp., Tokyo, Japan). Spectra were recorded over 20 scans at a resolution of 4 cm⁻¹ in the range of 4000–400 cm⁻¹. Wavelength assignments were based on the literature analysis.

Surface morphology was investigated using a JSM-IT200 InTouchScope™ scanning electron microscope (JEOL Ltd., Freising, Germany) equipped with a field emission gun (FEG) and an energy-dispersive X-ray spectroscopy (EDS) system. Samples were mounted on carbon tape and sputter-coated with a 10 nm gold layer using a Quorum Q150R ES sputter coater (Quorum Technologies Ltd., Laughton, UK). The coating chamber was evacuated to ~0.1 mbar, and sputtering was performed at 40 mA for 60 s to achieve a uniform conductive layer while preserving nanoscale surface features, optimizing resolution and contrast. Imaging was carried out at an accelerating voltage of 15 kV and a working distance of 10 mm, with magnifications ranging from 100× to 10,000×. EDS analysis was performed to quantify elemental distributions, with spectra collected from multiple regions to ensure representativeness.

Particle size distribution (PSD) of the hydrogel samples was measured using a Microtrac Nanotrac Wave II (Microtrac Retsch GmbH, Montgomeryville, PA, USA). Samples were dispersed in deionized water at a concentration of 0.1 mg/mL to minimize multiple scattering and ensure colloidal stability. Measurements were performed in batch mode at 23 °C, using a laser wavelength of 780 nm and a scattering angle of 180°, with each sample equilibrated for 2 min prior to acquisition. All measurements were conducted in triplicate, and results are reported as the mean hydrodynamic diameter and polydispersity index (PDI). This protocol ensures reproducible PSD and PDI across independent hydrogel batches.

The rheology of the liquid was investigated by using a Thermo Haake 500 viscosimeter (Thermo Fisher Scientific Inc., Waltham, MA, USA) with a thermostat in order to obtain the rheograms for each temperature. Measurement of viscosity was performed, and after 5 min, the corresponding temperature was obtained. Throughout the time needed to wait for the temperature to settle, the rotational speed of the rotor was maintained at very low revolutions per minute (RPM) in order to maintain a uniform distribution of the liquid properties. After the required temperature was obtained and the waiting period expired, the rotational speed was increased, and after 5 s, the rheological parameters were obtained. The rotational speed increased from the first rotational speed (5 RPM) to the last (600 RPM). The shear rate (1/s) for each rotational step was obtained from the instrument and used to create rheograms.

The in vitro release of CBD and AgNPs from the Ag-CBD complex and the HA-Ag-CBD hybrid hydrogel was investigated using a modified membraneless diffusion method [38,39].

For the hydrogel, 1.5 mL of the HA–poloxamer sol containing CBD and AgNPs was transferred into pre-weighed flat-bottomed vials, allowed to gel at 37 °C in a water bath, and overlaid with 2 mL of pre-heated phosphate-buffered saline (PBS; pH 7.4) containing 5% v/v ethanol to maintain sink conditions. For the Ag-CBD complex, 1.5 mL of colloidal dispersion (0.5% w/v CBD, 0.02% w/v AgNPs in 4% v/v ethanol) was placed in identical vials without gelation. All vials were incubated at 37 °C under shaking (100 ± 10 RPM).

At predetermined time points (0.5, 1, 2, 4, 8, 12, 24, and 48 h), aliquots (1.0 mL) of the release medium were withdrawn and replaced with an equal volume of fresh pre-warmed PBS/ethanol solution to maintain a constant volume and concentration gradient. The collected samples were centrifuged (10,000 RPM, 10 min, at 4 °C) to remove residual HA–poloxamer matrix fragments and analyzed by ultraviolet–visible (UV–Vis) spectrophotometry (CBD at 274 nm and AgNPs at 417 nm) using validated calibration curves prepared under identical solvent conditions (PBS containing 5% v/v ethanol) [40,41].

Hydrogel dissolution was quantified gravimetrically by weighing dried vials after complete release.

All experiments were conducted in triplicate (n = 3), and the results are presented as the mean ± standard deviation (SD). Cumulative release profiles were analyzed using the Korsmeyer–Peppas model to characterize the underlying release mechanism (Equation (1)):(1)MtM∞=ktn where MtM∞ is the fraction of the drug released at time t (h); k is the kinetic constant; and n is the release exponent, indicating the mechanism of drug transport.

The antimicrobial efficacy of the HA-Ag-CBD hybrid gel was evaluated using standardized in vitro assays against three bacterial reference strains—S. aureus (ATCC 29213), E. coli (ATCC 25922), and P. aeruginosa (ATCC 27853)—and one fungal reference strain, C. albicans (ATCC 14053). Bacterial and fungal inocula were adjusted to a turbidity equivalent of the 0.5 McFarland standard and uniformly spread onto Müller–Hinton agar plates within 15 min of preparation. Sterile 6 mm paper disks (Thermo Fisher Scientific Inc., Oxford, UK) were impregnated with test samples and aseptically placed onto the agar surface using sterilized forceps. Sterile water served as the negative control. The plates were incubated at 37 °C for 24 h. Antimicrobial activity was quantified by measuring the inhibition zone (IZ) diameters in millimeters, including the 6 mm disk.

Minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC) assays were conducted for CBD, colloidal Ag, the Ag-CBD complex, and the HA-Ag-CBD hydrogel. Stock solutions were prepared in the appropriate broth medium and serially diluted two-fold to achieve a concentration range of 1–400 μg/mL of active compound equivalents. The maximum tested concentration (400 μg/mL) ensured coverage above all inhibitory thresholds for the selected microbial strains. For disk diffusion assays, 10 μL of each solution was applied per disk, with the deposited amount of active compound calculated directly from the stock concentration. All reported concentrations for MIC, MBC/MFC, and disk diffusion refer exclusively to active compound equivalents rather than the total formulation mass. This strategy enables precise quantification of antimicrobial effects, ensures reproducibility, and facilitates direct comparison between individual agents and hybrid formulations.

To evaluate the potential intrinsic antimicrobial effects of the delivery vehicle, the HA–poloxamer gel matrix was included as a negative control in all assays. The blank gel was independently tested against Gram-positive, Gram-negative, and fungal strains using both disk diffusion and broth microdilution methods. These controls confirm that the antimicrobial effects observed in the HA-Ag-CBD formulation are attributable solely to the incorporated active agents and not to the gel matrix.

Assessing cytotoxicity on NHDFs is a critical step in evaluating the biocompatibility of compounds intended for topical delivery or skin-contact applications. NHDFs, as key mediators of skin homeostasis, collagen production, and tissue regeneration, provide a biologically relevant in vitro model for predicting cellular responses to candidate formulations.

Cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, measuring cell viability across a concentration range of 25–100 μg/mL at three exposure durations (24, 48, and 72 h). This time- and dose-dependent analysis enables comprehensive assessment of how the hydrogel and its individual components influence fibroblast viability under conditions simulating both acute and prolonged skin exposure.

Half-maximal inhibitory concentration (IC₅₀) values were calculated for each timepoint from triplicate measurements using nonlinear regression of the concentration–response curves, providing quantitative evaluation of cytotoxic potency. Although direct quantification of CBD and AgNP release from the hydrogel was not performed in this study, the selected concentration range corresponds to therapeutically relevant doses, based on prior in vitro antimicrobial efficacy and preliminary cytotoxicity screening.

These analyses allow direct comparison of biological safety profiles, identification of safe concentration thresholds, and evaluation of the potential formulation-dependent modulation of toxicity. Collectively, the cell viability and IC₅₀ data establish a robust framework for assessing the in vitro biocompatibility of both free and hydrogel-encapsulated active agents.

All experiments were performed in triplicate, with results reported as the mean ± SD. Statistical significance was determined using Student’s t-test for pairwise comparisons and one-way analysis of variance (ANOVA), followed by Tukey’s honestly significant difference (HSD) post hoc test for multiple pairwise comparisons, analyzed using Microsoft Excel 2019 version 16.31 (Microsoft Corporation, Redmond, WA, USA). A p-value < 0.05 was considered statistically significant.

FTIR spectroscopy was employed to rigorously assess the chemical integrity, molecular interactions, and structural stability of the newly prepared hydrogel system, which incorporates CBD and colloidal Ag within a HA gel (HA–poloxamer) matrix (Figure 2a). The spectral data presented in Figure 2b,c provide detailed evidence of characteristic vibrational bands, revealing shifts and interactions that confirm the successful integration of each component.

The FTIR spectrum of colloidal Ag (Figure 2b) displays a broad O–H stretching band at 3440 cm⁻¹, attributed to surface hydroxyl (–OH) groups or adsorbed water, typical of colloidal systems. Aliphatic C–H stretching vibrations appear at 2926 cm⁻¹ and 2855 cm⁻¹, while distinct asymmetric and symmetric carboxylate (–COO⁻) stretching bands at 1635 cm⁻¹ and 1383 cm⁻¹ suggest coordination of an organic stabilizing agent at the Ag surface. Additional peaks at 1111 cm⁻¹ (C–O stretching) and 618 cm⁻¹ (metal–oxygen interactions) further support the presence of functional groups contributing to the colloidal Ag’s surface chemistry and stability, in agreement with the literature data [42].

The FTIR spectrum of the Ag-CBD complex (Figure 2b) reveals notable spectral shifts and the emergence of characteristic bands, confirming the successful incorporation of CBD onto the colloidal Ag surface. The broad O–H stretching vibration shifts slightly to 3450 cm⁻¹, indicative of enhanced hydrogen bonding interactions, possibly between CBD’s –OH groups and the Ag surface. A weak aromatic C–H stretching band appears at 3006 cm⁻¹, while the aliphatic C–H asymmetric and symmetric stretching bands intensify at 2916 cm⁻¹ and 2855 cm⁻¹, respectively, corresponding to the hydrocarbon backbone of CBD. The carboxylate stretching vibrations also undergo a bathochromic shift, appearing at 1646 cm⁻¹ and 1424 cm⁻¹, which suggests specific coordination of CBD’s phenolic or carboxylic moieties with the Ag surface. The emergence of a new peak at 1323 cm⁻¹ is assigned to C–C aromatic stretching of the phenolic ring, further indicating CBD’s structural integrity within the complex. Additional fingerprint bands observed at 1162 cm⁻¹ and 1021 cm⁻¹ (C–O stretching), along with peaks at 953 cm⁻¹, 668 cm⁻¹, and 472 cm⁻¹, are consistent with the vibrational profile of CBD. Collectively, these spectral modifications, shifts, intensity changes, and new band appearances provide compelling evidence for the successful binding of CBD to the colloidal Ag surface while preserving its molecular framework.

The FTIR spectrum of the final HA-Ag-CBD hybrid gel (Figure 2c) displays the combined spectral features of all constituents, confirming their successful and cohesive integration within the hydrogel matrix. A broad O–H stretching band centered at 3433 cm⁻¹ persists, reflecting the presence of –OH groups and extensive hydrogen bonding within the hydrated polymeric network. Aliphatic C–H stretching bands appear at 2979 cm⁻¹, 2929 cm⁻¹, and 2868 cm⁻¹, characteristic of the polymer backbone and the CBD hydrocarbon moieties. The complete disappearance of the free HA carboxylic acid peak at 1748 cm⁻¹, along with the appearance of coordinated carboxylate bands at 1646 cm⁻¹ and 1457 cm⁻¹, strongly suggests the involvement of electrostatic interactions or hydrogen bonding between the carboxyl groups of HA and functional moieties of both colloidal Ag and CBD. The overlapping peaks at 1356 cm⁻¹, 1296 cm⁻¹, and 1255 cm⁻¹ are attributed to the combined contributions of the HA gel (HA–poloxamer) matrix and CBD, further supporting their structural association. Preservation of distinct fingerprint bands at 1104 cm⁻¹, 1024 cm⁻¹, 953 cm⁻¹, 711 cm⁻¹, and 476 cm⁻¹ confirms the chemical integrity of both CBD and HA within the new hybrid gel. Collectively, these spectral features provide robust evidence for the stable and homogeneous incorporation of colloidal Ag and CBD into the HA-based hydrogel matrix, forming a multifunctional network with retained molecular characteristics of all components [43,44,45].

SEM was employed to evaluate the surface morphology and nanoscale distribution of components within the new HA-Ag-CBD hybrid gel. This technique provides high-resolution imaging critical for confirming the successful incorporation of the Ag-CBD complex, assessing particle dispersion, and characterizing the structural integrity of the hydrogel matrix.

Figure 3a–d presents SEM micrographs highlighting the morphological profiles of CBD, Ag-CBD, the HA gel (HA–poloxamer) matrix, and the HA-Ag-CBD hybrid gel. These high-resolution images provide clear insight into the distinct micro- and nanoscale features of each component, revealing critical structural differences that reflect their individual compositions and interactions within the composite system.

SEM imaging of the CBD sample (Figure 3a) reveals an irregular, semi-amorphous surface morphology with heterogeneous structures at the nanoscale. The observed features likely reflect dried lipidic residues and dispersed organic phases characteristic of oil-based formulations, consistent with the encapsulated nature of the CBD product.

The micrograph of the Ag-CBD sample (Figure 3b) reveals a core–shell-like morphology, with a dark central region corresponding to AgNPs (50–150 nm), surrounded by bright, irregular, and fibrous surface structures attributed to the CBD sample.

The uniform particle distribution and well-defined architecture suggest controlled assembly and effective surface interaction.

This contrast indicates the successful association of CBD with the colloidal Ag surface. The relatively uniform particle distribution and defined architecture suggest effective surface interaction and stabilization. The coexistence of crystalline features within the CBD layer and semi-amorphous interfacial regions indicates strong interfacial interactions between the organic CBD layer and the inorganic Ag core, which enhance the stability of the new Ag-CBD complex. Overall, the SEM data support the formation of a nanoscale Ag-CBD complex with structural coherence.

Conversely, SEM analysis of the HA hydrogel (Figure 3c) reveals a porous nanoscale architecture composed of irregular semi-crystalline clusters and amorphous domains, corresponding to the HA and poloxamer regions, respectively. This well-organized network, stabilized by poloxamer micelles, exhibits particle sizes ranging from tens to hundreds of nanometers, indicating uniform dispersion and precise structural control. The resulting architecture supports high water retention, biocompatibility, and mechanical stability.

SEM analysis of the HA-Ag-CBD hybrid gel (Figure 3d) reveals a smooth, continuous matrix with sparsely distributed bright nanoscale deposits. These high-contrast features correspond to the Ag-CBD complex embedded within the HA–poloxamer network. Particle sizes range from approximately 20 to 100 nm, suggesting a heterogeneous yet controlled dispersion. The uniform background indicates a homogenous hydrogel structure without prominent porous features, characteristic of a well-integrated HA–poloxamer system. The discrete distribution of the Ag-CBD complex confirms successful incorporation with minimal aggregation, maintaining matrix integrity and structural uniformity.

The DLS analysis presented in the comparative plot (Figure 4) provides a detailed assessment of the PSD and hydrodynamic behavior of CBD, the Ag-CBD complex, the HA–poloxamer hydrogel matrix, and the newly formulated HA-Ag-CBD hybrid gel. Distinct differences in size profiles among the samples underscore their specific structural characteristics and varying degrees of colloidal stability.

The DLS comparison plot presented in Figure 4 illustrates the PSD of the base HA hydrogel, CBD, the Ag-CBD complex, and the final HA-Ag-CBD hybrid gel. The analysis provides insight into the colloidal behavior and dispersion stability of each system, highlighting the influence of component interactions on particle size heterogeneity.

The base hydrogel, composed of HA and the poloxamer, exhibited a primary particle size population within the nanometric range (~0.001–0.005 μm), suggesting a well-organized and uniformly hydrated polymeric network. This narrow size distribution is indicative of efficient physical crosslinking and structural homogeneity within the hydrogel matrix. The measured PDI (0.58) falls within the acceptable range for physically crosslinked hydrogels, confirming a consistent nanoscale formulation.

In contrast, CBD dispersed in an aqueous medium showed a broader PSD (~0.01–1 μm), reflecting a tendency to form micron-scale aggregates due to limited aqueous solubility. Despite a relatively low PDI (0.046), the presence of larger particles indicates a heterogeneous dispersion with reduced colloidal stability.

The Ag-CBD complex revealed a multimodal particle size profile, extending from ~0.01 to 1.2 μm, with multiple distinct peaks suggestive of hybrid aggregate formation. The corresponding PDI values (0.036, 1.473, and 0.131) point to a mixed population ranging from monodisperse to polydisperse domains. These results reflect dynamic interactions between AgNPs and CBD molecules, forming variably sized aggregates that confirm the successful formation of hybrid nanostructures.

The final HA-Ag-CBD hybrid gel exhibited a significantly refined and more homogeneous PSD, with a dominant peak in the low nanometer range (~0.001–0.01 μm). Compared to the individual and binary systems, this formulation demonstrated superior size uniformity and a notable reduction in aggregation. Although some higher PDI values (0.966, 52.686, and 7.077) were recorded, these were associated with minor secondary populations and did not impact the main nanometric fraction. The primary peak confirms the effective dispersion and stabilization of both the CBD and AgNPs within the HA–poloxamer matrix.

Overall, the DLS analysis demonstrates that the integration of the Ag-CBD complex into the HA hydrogel matrix results in a stable, nanoscale system with enhanced dispersion and minimized heterogeneity. This uniform particle profile is particularly advantageous for biomedical applications, where nanoscale dimensions and colloidal stability are essential for enhanced cellular uptake, improved bioavailability, and sustained release. The results confirm that the HA-Ag-CBD hybrid gel exhibits optimized physicochemical characteristics, making it a promising candidate for therapeutic topical delivery.

The DLS analysis demonstrates that the incorporation of the Ag-CBD complex into the HA–poloxamer matrix results in a well-dispersed nanoscale system with reduced particle aggregation and enhanced size uniformity. Compared to the individual and binary components, the final HA-Ag-CBD hybrid gel exhibits improved colloidal stability and homogeneity. The nanoscale PSD and consistent hydrodynamic behavior observed in the composite formulation reflect effective stabilization of the active components within the hydrogel network. These physicochemical characteristics are essential for biomedical applications, particularly in topical therapeutic delivery systems, where uniformity and stability influence performance, reproducibility, and safety.

The rheological behavior of the HA-Ag-CBD hybrid gel was systematically characterized to evaluate its suitability for topical or sprayable biomedical applications.

A temperature-controlled rheometry protocol was employed to monitor changes in viscosity, flow behavior, and structural stability across a physiologically relevant temperature range (16–36 °C). This approach allowed insights into the gel’s thermal responsiveness, flow stability, and shear-dependent behavior.

Figure 5 presents the rheograms of the HA-Ag-CBD hybrid gel measured across a range of temperatures, revealing a distinct temperature-dependent viscoelastic behavior.

At lower temperatures (16 °C and 22 °C), the gel displays Newtonian flow characteristics, as evidenced by the linear shear stress–shear rate relationships and excellent correlation coefficients (R² values 0.9992 and 0.9990, respectively). These profiles suggest a low-resistance, predictable flow regime ideal for sprayability and ease of application, especially under ambient or refrigerated conditions. However, as the temperature rises to 28 °C and 36 °C, the flow behavior shifts markedly. The flow curves deviate from linearity, and a pronounced yield stress emerges, reflected by intercepts on the shear stress axis. This indicates a transition to non-Newtonian plastic behavior, further corroborated by decreasing R² values (0.9779 at 28 °C and 0.8677 at 36 °C). These changes suggest the formation of internal structural interactions, likely due to thermal activation of polymer and NP interactions, promoting a cohesive gel state suitable for in situ film formation upon skin contact.

Figure 6 illustrates the temperature-dependent trends of viscosity and yield stress.

Below 22 °C, the gel exhibits negligible yield stress (<10 Pa) and maintains a low viscosity (<40 mPa·s), supporting a fluid-like consistency that facilitates uniform application over sensitive or irregular skin surfaces. Above 22 °C, both yield stress and viscosity rise significantly. Yield stress increases steeply to ~35 Pa at 26 °C and stabilizes around ~40 Pa between 28 °C and 36 °C, while viscosity peaks at 0.22 Pa·s at 28 °C. These findings define 22 °C as a functional transition point, beyond which the gel assumes a more structured, stable consistency. The presence of yield stress at these elevated temperatures implies the development of internal cohesion and network strength, which enhances topical retention and provides a stable physical barrier after application.

The establishment of measurable yield stress under these conditions suggests the development of internal network strength, promoting topical retention and physical barrier formation upon skin contact.

Figure 7 explores the effect of the shear rate on viscosity at different temperatures, simulating mechanical stresses encountered during spreading or movement.

At 16 °C and 22 °C, viscosity remains constant across shear rates, confirming Newtonian flow behavior. However, at 28 °C and 36 °C, viscosity becomes increasingly dependent on the shear rate. At low shear rates (<10 s⁻¹), viscosity rises sharply, and threshold shear stresses reach up to 50 Pa, consistent with the plastic-like behavior indicated in previous figures. This growing resistance to deformation with temperature suggests that the gel responds mechanically to skin contact or manipulation by reinforcing its structural integrity, an advantageous property for film-forming dermal applications.

Figure 8a–c presents the gel’s response to ramp-up and ramp-down shear tests at 16 °C, 22 °C, and 36 °C, designed to evaluate time-dependent behavior such as thixotropy or rheopexy.

At all three temperatures, the ascending and descending flow curves overlap, demonstrating the absence of hysteresis. This consistency indicates that the gel structure remains stable under mechanical stress and recovers without permanent breakdown or thickening, a crucial property for predictable performance during repeated application or wear. Furthermore, the gel transitions from dilatant at 16 °C to Newtonian at 22 °C and plastic at 36 °C, highlighting its thermally adaptive flow behavior across physiologically relevant conditions.

Figure 9 identifies an exception to the time-independent flow behavior.

At 24 °C, the gel displays a significant hysteresis loop between the ramp-up and ramp-down flow curves, indicative of thixotropic behavior. The thixotropic index, calculated at 83.78%, reflects a pronounced breakdown and subsequent recovery of the internal structure under shear. This finding suggests that 24 °C represents a metastable zone where the gel microstructure is particularly responsive to mechanical stimuli. Although not representative of the broader temperature profile, this localized anomaly may offer opportunities for tuning delivery properties through careful thermal or mechanical conditioning.

Collectively, the rheological results demonstrate that the HA-Ag-CBD hybrid gel exhibits a sharp, reproducible transition centered at 22 °C. Below this threshold, the gel behaves as a low-viscosity Newtonian fluid, ideal for spraying and smooth application. Above this temperature, it transitions into a semi-solid plastic material with increased viscosity and yield stress, supporting enhanced skin adherence and barrier functionality. The isolated thixotropic behavior at 24 °C adds nuance to its rheological profile, suggesting responsiveness that could be harnessed for tailored release or application strategies. Altogether, these results confirm the gel’s promise as a thermoresponsive topical system, combining ease of administration with robust performance at body temperature.

The in vitro release profiles of CBD and the AgNPs were assessed to compare the release kinetics from the non-gelled Ag-CBD complex and the HA-Ag-CBD hydrogel, thereby elucidating the role of the hydrogel matrix in achieving sustained and controlled delivery of both active components. The corresponding cumulative release data are shown in Figure 10.

The Ag-CBD complex exhibited the rapid release of both CBD and AgNPs. The release kinetics followed a predominantly diffusion-controlled mechanism, as expected for a non-gelled dispersion. Release data fitted to the Korsmeyer–Peppas model yielded the following (Equations (2) and (3)):(2)CBD=MtM∞=(0.068±0.003)t(0.62±0.03) (R2=0.987)

(3) AgNPs=MtM∞=(0.064±0.003)t(0.60±0.03) (R2=0.985)

The cumulative release of CBD increased from 5.12 ± 1.03% at 0.5 h to 95.34 ± 1.65% at 48 h, while AgNP release increased from 4.35 ± 0.95% to 92.15 ± 1.89% (Figure 10). The release profiles of CBD and the AgNPs were highly correlated (r = 0.97, p < 0.001), reflecting the stable co-dispersion of the active components. The Korsmeyer–Peppas exponents (n ≈ 0.60) confirm a Fickian diffusion-dominated release, consistent with the absence of a structured matrix to modulate transport.

In contrast, the HA–poloxamer hydrogel provided controlled and sustained release. The cumulative release of CBD increased from 2.87 ± 0.92% at 0.5 h to 90.12 ± 1.87% at 48 h, while AgNP release rose from 1.78 ± 0.76% to 85.67 ± 2.32% over the same period. Gel dissolution progressed from 3.65 ± 1.12% to 88.45 ± 2.10%, closely mirroring the release profiles. The release and dissolution data exhibited strong correlations for CBD (r = 0.96, p < 0.001) and for AgNPs (r = 0.95, p < 0.001), indicating that matrix erosion contributed to the release.

Korsmeyer–Peppas modeling confirmed anomalous transport (Equations (4) and (5)):(4)CBD=MtM∞=(0.045±0.002)t(0.78±0.02) (R2=0.994)

(5) AgNPs=MtM∞=(0.041±0.002)t(0.74±0.03) (R2=0.991)

The higher exponents (n = 0.74–0.78) indicate that release is governed by both diffusion and hydrogel matrix relaxation/erosion, highlighting the hydrogel’s capacity for prolonged, synchronized delivery of CBD and AgNPs.

Notably, the HA–poloxamer hydrogel significantly modulated the initial burst release, reducing the rapid spike observed in the Ag-CBD complex. This controlled release behavior is critical for maintaining therapeutic concentrations over extended periods, minimizing cytotoxic peaks, and sustaining antimicrobial efficacy.

Overall, these results demonstrate that the HA-Ag-CBD hydrogel provides a superior delivery platform to that of the unstructured Ag-CBD complex, enabling precise temporal control of co-delivered active agents, which is essential for antimicrobial and cytotoxic applications.

The antimicrobial efficacy of the CBD, colloidal Ag, their binary complex (Ag-CBD), and the newly formulated HA-Ag-CBD hybrid gel was systematically assessed against selected Gram-positive (S. aureus), Gram-negative (E. coli, P. aeruginosa), and fungal (C. albicans) pathogens. Antimicrobial performance was initially evaluated by agar disk diffusion, followed by MIC and MBC assays. Gentamicin (100 μg/mL) served as the positive control, while the HA gel matrix (10 μg/mL) was used as the negative control.

The comparative antimicrobial activities are summarized in Table 2 and Table 3.

Gentamicin was employed as a uniform positive control across all assays to ensure methodological consistency. Although it lacks intrinsic antifungal activity against C. albicans, its inclusion allowed standardized comparison, with future studies planned to incorporate dedicated antifungal control, such as fluconazole.

The HA–poloxamer gel matrix, evaluated independently against S. aureus, E. coli, P. aeruginosa, and C. albicans, produced no measurable IZs (0 mm) in the disk diffusion assay and exhibited MIC and MBC values exceeding 400 μg/mL in broth microdilution assays. These results confirm that the gel base itself lacks intrinsic antimicrobial activity, ensuring that the inhibitory effects observed in the HA-Ag-CBD formulations arise exclusively from the incorporated CBD, colloidal Ag, or their hybrid complex.

CBD demonstrated significant antimicrobial activity, with mean IZs of 9.07 ± 0.35 mm against S. aureus and 9.15 ± 0.46 mm against C. albicans. Colloidal Ag exhibited smaller IZs—6.32 ± 0.41 mm for S. aureus, 6.58 ± 0.37 mm for C. albicans, and 6.53 ± 0.44 mm for E. coli—highlighting its comparatively low activity, particularly against Gram-negative strains.

The Ag-CBD hybrid showed comparable or slightly enhanced IZs relative to CBD alone, e.g., 9.17 ± 0.49 mm for S. aureus, while incorporation into the HA–poloxamer hydrogel preserved antimicrobial activity, with IZs of 9.53 ± 0.30 mm for S. aureus and 9.27 ± 0.38 mm for C. albicans. No IZs were observed for P. aeruginosa for any formulation except gentamicin, consistent with its intrinsic resistance.

Notably, the HA-Ag-CBD formulation exhibited an unusually high MBC of 387.35 μg/mL against E. coli, while the MIC remained low (9.24 ± 0.48 μg/mL). This discrepancy reflects the inherent resistance of Gram-negative bacteria, whose lipopolysaccharide-rich outer membrane limits antimicrobial penetration, and may also indicate modulation of local release rates by the hydrogel, requiring higher concentrations to achieve bactericidal activity. Importantly, effective growth inhibition is still achieved at much lower concentrations. These data confirm that the HA-Ag-CBD system effectively inhibits bacterial growth while highlighting the necessity of evaluating both MIC and MBC to fully interpret antimicrobial efficacy.

In broth microdilution assays, CBD demonstrated the lowest MIC and MBC/MFC values among all tested samples, indicating strong bactericidal and fungicidal activity (MIC: 1.74 ± 0.25 μg/mL for S. aureus, 1.98 ± 0.31 μg/mL for C. albicans; MBC/MFC: 2.15 ± 0.34 μg/mL and 2.57 ± 0.29 μg/mL, respectively). Colloidal Ag exhibited higher MICs (e.g., 2.67 ± 0.36 μg/mL for S. aureus, 3.43 ± 0.38 μg/mL for E. coli) and MBCs up to 4.25 ± 0.43 μg/mL for E. coli. The Ag-CBD hybrid retained substantial antimicrobial potency (MIC: 2.06 ± 0.29 μg/mL for S. aureus, 2.35 ± 0.33 μg/mL for C. albicans), with MBC/MFC values similar to those of CBD alone, consistent with additive effects, and hydrogel encapsulation did not compromise activity. HA-Ag-CBD MICs were comparable: 2.18 ± 0.27 μg/mL for S. aureus and 2.45 ± 0.30 μg/mL for C. albicans.

All formulations exhibited MIC and MBC values > 8 μg/mL against P. aeruginosa, confirming resistance. The absence of measurable IZs and MIC/MBC values for P. aeruginosa reflects its well-known intrinsic resistance. This Gram-negative pathogen possesses a highly impermeable outer membrane, active efflux pumps, and biofilm-forming capabilities, all of which limit the uptake and efficacy of antimicrobial agents, including CBD, colloidal Ag, and their hybrid formulations. These features account for the lack of observable activity under the current assay conditions.

Statistical analysis confirmed that CBD significantly outperformed colloidal Ag across all susceptible strains (p < 0.01), while differences among CBD, Ag-CBD, and HA-Ag-CBD were not significant (p > 0.05), underscoring the preservation of CBD’s antimicrobial potency in the hybrid and hydrogel systems.

These findings are consistent with the existing literature, which recognizes CBD’s pronounced antimicrobial effects, particularly against Gram-positive bacteria and fungi, and confirms the moderate but measurable efficacy of colloidal Ag under similar experimental conditions. These MIC and MBC/MFC values align with previous reports, reinforcing the observed efficacy of CBD and colloidal Ag under in vitro conditions and validating the reliability of the findings.

Overall, these results demonstrate that both CBD and colloidal Ag possess antimicrobial properties, with CBD displaying superior efficacy, particularly against S. aureus and C. albicans. Incorporation of these agents into the Ag-CBD hybrid and subsequent integration into the HA–poloxamer hydrogel maintained their bioactivity without loss of efficacy, consistent with the existing literature [46,47,48,49,50,51], supporting their potential for topical antimicrobial applications.

The results, presented in Figure 11 and Figure 12, inform the selection of optimized concentrations for further formulation refinement and preclinical development in skin-focused biomedical applications.

Cytotoxicity assays on NHDFs revealed that both concentration (p < 0.0001) and incubation time (p < 0.0001) significantly influenced cell viability across all tested compounds. A significant interaction between concentration and time (p < 0.001) indicated that cytotoxic effects were enhanced synergistically with increasing dose and exposure duration. Tukey’s HSD post hoc analysis demonstrated that at concentrations ≥50 μg/mL, all active formulations except the HA–poloxamer gel matrix significantly reduced cell viability compared to untreated controls (p < 0.01), with notable differences among formulations.

CBD exhibited moderate, dose-dependent cytotoxicity, maintaining > 95% viability at 25 μg/mL across all timepoints, while at 100 μg/mL, viability declined to 65.2 ± 3.5%, 55.6 ± 4.1%, and 48.7 ± 4.3% at 24, 48, and 72 h, respectively. Colloidal Ag displayed slightly stronger cytotoxicity at higher concentrations, with 40.6 ± 4.8% viability at 72 h. Differences between CBD and Ag were not statistically significant (p > 0.05), consistent with the literature reporting mild-to-moderate dermal fibroblast cytotoxicity for both compounds at elevated doses [52,53,54,55,56,57].

The Ag-CBD complex exhibited the highest cytotoxicity among the active agents, with cell viability of 52.4 ± 4.0%, 43.6 ± 4.7%, and 35.2 ± 5.0% at 100 μg/mL across 24, 48, and 72 h, respectively. These values were significantly lower than those for CBD or Ag alone (p < 0.05), suggesting a synergistic cytotoxic effect likely mediated by CBD-facilitated AgNP uptake and intracellular Ag⁺ release.

Incorporation of the Ag-CBD complex into the HA–poloxamer hydrogel significantly mitigated cytotoxicity, maintaining viability above 75% at concentrations ≤ 50 μg/mL. At 100 μg/mL, the HA-Ag-CBD hydrogel yielded 61.5 ± 3.6%, 52.3 ± 4.3%, and 45.8 ± 4.6% viability at 24, 48, and 72 h, respectively, significantly higher than the Ag-CBD complex alone (p < 0.05). The HA–poloxamer matrix alone preserved > 92% viability across all concentrations and timepoints, confirming its inertness and excellent biocompatibility.

Dose- and time-dependent IC₅₀ values were derived using nonlinear regression from triplicate measurements. CBD IC₅₀ decreased from 112.3 ± 5.2 μg/mL (24 h) to 82.1 ± 4.5 μg/mL (72 h), while colloidal Ag ranged from 98.5 ± 5.0 μg/mL to 67.8 ± 4.2 μg/mL, reflecting slightly higher cytotoxicity. The Ag-CBD complex exhibited lower IC₅₀ values (85.6 ± 4.9 μg/mL at 24 h; 58.4 ± 4.0 μg/mL at 72 h; p < 0.01), consistent with synergistic cytotoxicity. Incorporation into the hydrogel significantly increased IC₅₀ values (105.7 ± 5.1 μg/mL at 24 h; 76.3 ± 4.4 μg/mL at 72 h; p < 0.01), confirming the protective effect of the HA–poloxamer matrix. The HA–poloxamer exhibited IC₅₀ values >200 μg/mL at all timepoints, with no difference from untreated controls (p > 0.05).

Overall, the HA-Ag-CBD hydrogel effectively mitigates the intrinsic cytotoxicity of the Ag-CBD complex while maintaining fibroblast viability above 70% at therapeutically relevant concentrations, fulfilling International Organization for Standardization (ISO) 10993-5:2009 [58] and European Medicines Agency (EMA) [59] guidelines for topical applications. These findings establish a clear therapeutic window and highlight the hydrogel’s potential as a safe, biocompatible delivery system for dermal applications.