Carbopol 940-based metronidazole nanogels: formulation, physicochemical characterization, in vitro drug release, and antimicrobial evaluation

1 Introduction

The development of novel drug delivery systems using nanotechnology has greatly contributed to modern pharmaceutical research because of increased drug solubility, controlled release pattern, superior performance of drug, and specific targeting ability. Nanogels have gained substantial interest due to high water content, polymeric network of three-dimensional nature, biocompatibility, modifiable physical and chemical properties, and ability to entrap hydrophilic as well as hydrophobic drugs. The small size and hydrated polymer matrix of nanogels facilitate better retention of drug, control of diffusion, and sustained release, making them ideal carriers for topical drug delivery (Oh et al., 2008; Hamidi et al., 2008; Ahmed, 2015; Soni and Yadav, 2016). Administration of topical drug is characterized by a number of benefits, including local drug effect, avoiding first pass effect, decreased adverse effects on whole body, better patient compliance, and convenience in administration. However, topical preparations like creams and gels may be characterized by limited residence time, lack of controlled drug release, instabilities, and requirement for repeated application, which may affect the therapeutic effectiveness. Therefore, significant amount of research has been devoted to polymeric nanogels with controlled drug release properties, long residence time at application site, and local drug delivery improvement (Sarkar et al., 2026; Peppas et al., 2000; Qiu and Park, 2012; Zhang et al., 2025; Li et al., 2026; Kumar et al., 2026). Metronidazole is a BCS Class I nitroimidazole antimicrobial agent with a molecular weight of 171.15 g/mol and a pKa of approximately 2.6. It is freely soluble in water and exhibits broad activity against obligate anaerobic bacteria and protozoa. Following intracellular reduction of its nitro group under anaerobic conditions, reactive intermediates are generated that interact with microbial DNA and inhibit nucleic acid synthesis, ultimately resulting in microbial cell death (Freeman et al., 1997). The antimicrobial drug is extensively used for the management of dermatological, periodontal, gastrointestinal, and gynecological infections caused by anaerobes. However, traditional topical preparations of metronidazole may suffer from limited residence time, lack of controlled drug release, and necessity for repeated administration. Encapsulation of metronidazole in polymeric nanogels could result in sustained drug release, local drug retention, and increased concentrations of drug at application site with minimum system-wide distribution (Chen et al., 2014; Oliveira et al., 2022). In the case of polymers used in topical formulations, Carbopol 940 is a polymer that has been extensively researched for the reason of its excellent swelling ability, bioadhesivity, biocompatibility, high viscosity, and excellent gelling ability. Carbopol 940 is a cross-linked poly (acrylic acid) polymer, which produces hydrating three-dimensional networks that control water absorption, drug diffusion, and provides favorable rheological property for the topical application of drugs. Previous works have revealed the favorable physicochemical properties, rheological behavior, and drug-release properties of Carbopol-based formulations, thus proving that such gels can be used in the topical drug delivery systems (Singh et al., 2014; Islam et al., 2004; Daood et al., 2019). Previous literature has indicated the use of Carbopol containing metronidazole formulations for the purpose of topical antimicrobial therapy. For instance, in a study by Chen et al., poly (methacrylic acid)-based metronidazole nanogels that have shown sustained drug release and good physicochemical stability have been developed (Chen et al., 2014). In another study, Singh et al. developed metronidazole bigels based on Carbopol showing good rheological properties suitable for topical application (Singh et al., 2014). Moreover, according to the work done by Daood et al., gelling agent selection is found to influence the viscosity, physicochemical properties, and drug-release properties of topical metronidazole formulations (Daood et al., 2019). Likewise, Roy et al. have observed that the Carbopol concentration greatly influences the viscosity and sustained drug release properties (Roy et al., 2022). However, most of the previous researches have mostly dealt with the conventional gel formulations. However, there is a lack of research in which the correlation between Carbopol 940 concentration and nanostructural characteristics, swelling, rheology, size distribution of particles, controlled drug release, antimicrobial activity, and formulation stability is thoroughly studied (Roy et al., 2022; Maru et al., 2012; Boateng et al., 2008; Dash et al., 2010). Additionally, in previous studies, none of them considered all these parameters in a single optimized formulation. Thus, this study was based on the hypothesis that change in the concentration of Carbopol 940 would affect the structure of the polymer matrix, hence the physiochemical characteristics, rheology, swelling property, size distribution of particles, controlled release of drugs, antimicrobial activity, and stability of the metronidazole loaded nanogels. Based on the hypothesis, the metronidazole-loaded carbopol 940 nanogels were synthesized using the homogenization assisted solvent evaporation method and tested to obtain an optimized formulation. The formulations were tested for their physicochemical characteristics, rheology, size distribution of particles, zeta potential, drug content, swelling index, in vitro drug release, antimicrobial activity, and short-term stability.

2 Materials and methods

2.1 Materials

The anti-microbial agent metronidazole was purchased from Aarti Drugs Pvt. Ltd., while carbopol 940 was bought from Sisco Research Laboratories Pvt. Ltd. These were used to formulate the nanogel as the model drug and polymer, respectively. The permeation enhancer propylene glycol was purchased from Loba Chemicals Pvt. Ltd. Triethanolamine was sourced from Sisco Research Laboratories Pvt. Ltd. and used for neutralization purpose. Glycerol and sodium benzoate were procured from Nice Chemicals Pvt. Ltd. Purified water was used for preparation of all formulations. All chemicals and reagents used in the study were of analytical grade and utilized without further purification.

2.2 Preparation of metronidazole-loaded carbopol nanogel

The Metronidazole loaded Carbopol nanogels (F1-F3) were prepared using homogenization facilitated polymer dispersion technique (Table 1). The exact amount of Carbopol 940 was slowly dispersed in distilled water through magnetic stirring to obtain an even polymer dispersion. The polymeric dispersion was allowed sufficient time to be hydrated completely. Metronidazole was dissolved in propylene glycol in a stirred manner to form a homogeneous drug solution. This formed drug solution was then slowly added into the previously created polymer dispersion with continued stirring to create a homogeneous distribution of the drug in the polymer matrix. The inclusion of glycerol into the formulation acted as a humectant to aid consistency and hydration. Sodium benzoate in the formulation acted as a preservative agent. The formulation was subjected to high-speed homogenization for 1 h at 8000 rpm to enable the nanoscale dispersion. A drop-wise addition of triethanolamine was done into the formulation to facilitate neutralization to the required p^H. The formulated nanogels of carbopol were left overnight at room temperature for equilibrium before further analysis.

TABLE 1. Composition of metronidazole-loaded carbopol nanogel formulations.

2.3 Fourier transform infrared spectroscopy (FTIR)

The Fourier transform infrared spectroscopy (FTIR) technique was used to investigate the interaction among metronidazole and other excipients used in the formation of Carbopol nanogels. The FTIR spectra for pure metronidazole, Carbopol 940, and optimized nanogel formulation were obtained using an FTIR spectrophotometer within the scanning range of 4,000–400 cm^-1 using the potassium bromide (KBr) pellet technique. The FTIR spectra obtained were then examined to determine specific absorption peaks that represented functional groups in both metronidazole and Carbopol 940. Changes in the shape, intensity, absence or appearance of any peaks in the spectrum would help to detect the possible interactions between the drug and other excipients.

2.4 Evaluation of metronidazole-loaded carbopol nanogels

2.4.1 Physical appearance and homogeneity

The physical appearance, clarity, consistency, homogeneity, and particulates of the formulated nanogels were analyzed.

2.4.2 Determination of pH value

To measure the pH values of the formulated nanogels, a calibrated digital pH meter was used. In this respect, 1 g of the formulation was diluted in distilled water, and then the electrode was placed in the solution for reading the value. Three repetitions of this procedure were carried out, and the mean value was considered.

2.4.3 Viscosity measurement

Viscosity values of the nanogels were measured by employing a Brookfield viscometer with proper spindles (models 12, Chiarentin et al., 2023; Dabbaghi et al., 2021; Huang, 2019; Jin et al., 2023; Welin-Berger et al., 2001).

2.4.4 Spreadability study

Spreadability study of the nano-gel formulations was performed using the glass slide method. An equal amount of the gel was kept between two glass slides, and an equal amount of weight was applied on the glass slides to make a thin layer of the gel. The time taken to detach the two glass slides was recorded and spreadability was determined using the following equation:

S = M × L T

Here, S represents spreadability, M represents the applied weight in relation to the upper slide, L represents the distance traveled by the glass slide, and T represents the time taken to separate the slides (Kryscio et al., 2008).

2.4.5 Extrudability study

The extrudability property of the formulations was studied in terms of the ease with which the formulation can be extruded out of a collapsible tube when a certain amount of pressure was applied to it. Comparative evaluation of the prepared formulations was done in terms of the amount of gel extruded out of the tube (Jones et al., 1997). The extrudability property can be defined as:

Extrudability % = ( Weight of extruded material / Total weight of material ) x 100

2.4.6 Drug content determination

Spectrophotometric measurement of the drug content uniformity was carried out. The precise weights of nanogels, which corresponded to a known amount of metronidazole, were dissolved in appropriate solvents and then filtrated. Proper dilutions were done, and absorbance values were recorded using the UV–vis spectrophotometer at the pre-set wavelength. Drug contents were analyzed by comparing with the standard calibration curve of metronidazole.

2.4.7 Swelling index

The swelling property of the Carbopol nanogels was studied to determine their capacity for hydration. A precise amount of nanogels was allowed to swell in the phosphate buffer solution for some pre-set time. The hydrated nanogels were weighed after removing excess surface moisture. The swelling index was determined using the formula:where W_t is the weight of swollen gel at time t, while W₀ represents the initial weight of the formulation.

S w e l l i n g I n d e x = W t − W 0 W 0 × 100

2.4.8 Particle size, polydispersity index, and zeta potential analysis

Dynamic light scattering technique was used to determine the particle size distribution, polydispersity index (PDI), zeta potential, and conductivity of the optimized Carbopol nanogels. The samples were diluted with distilled water before analysis to avoid scattering effects.

2.4.9 In-Vitro drug release study

In vitro drug release studies were carried out using a diffusion cell with continuous stirring. Phosphate buffer solution (pH 7.4) was used as the recipient media to keep sink condition during the test period. Pre-saturated cellulose dialysis membrane (molecular weight cutoff: 12–14 kDa) was used as the diffusion barrier between the donor and recipient phases. An exact amount of prepared nanogel formulations was loaded into the donor phase. Aliquots were withdrawn from the recipient phase at definite time intervals and immediately replaced with fresh phosphate buffer (pH 7.4) of the same volume to keep constant volume and sink conditions. The withdrawn samples were subjected to analysis by UV visible spectrophotometry at predetermined wavelength of metronidazole, and the percentage of drug released was determined for each sampling time point. Release studies were carried out in triplicates (n = 3).

2.4.10 Drug release kinetics

The drug release data obtained from in-vitro release study was fitted to zero-order, first order, Higuchi and Korsmeyer-Peppas kinetic models for investigating the mechanism of drug release from Carbopol nanogel formulations. These equations have been shown below:

The zero-order release model was expressed as:

Q t = Q 0 + k 0 t

The first-order release model was expressed as:

log ⁡ Q t = log ⁡ Q 0 − k 1 t 2.303

The Higuchi diffusion model was expressed as:

Q = k H t 1 / 2

The Korsmeyer–Peppas model was expressed as:

M t M ∞ = k t n

Wherein, Q_t = Amount of drug released at time t; Q₀ = Amount of drug released initially, and n is the diffusion exponent showing the mode of release (Dash et al., 2010; Higuchi, 1963; Korsmeyer et al., 1983).

2.4.11 Antimicrobial activity

Antimicrobial activity of the formulated metronidazole-loaded Carbopol nanogels was evaluated using paper disc diffusion assay against Gram-positive and Gram-negative microorganisms such as Staphylococcus aureus and Escherichia coli. A nutrient agar medium was prepared and autoclaved. Sterilized Petri dishes containing a layer of nutrient agar medium were inoculated separately with the bacterial cultures of Staphylococcus aureus and Escherichia coli under aseptic conditions in laminar flow hood. Sterile paper discs soaked with metronidazole-loaded nanogel formulations at a concentration of 100 μg/mL were placed on the inoculated nutrient agar plates. The diameter of the inhibition zone around each of the paper discs was taken after 24 h of incubation at 37 °C using a BOD incubator to assess the antimicrobial activity of the formulation. Minimum inhibitory concentration (MIC) was considered to be the lowest concentration of the formulation required to inhibit any visible bacterial growth. All antimicrobial experiments were performed in triplicates (Freeman et al., 1997; Chen et al., 2014; Oliveira et al., 2022).

2.4.12 Stability study

Accelerated stability studies were carried out on the developed metronidazole loaded Carbopol nanogel formulations as per International Conference on Harmonization (ICH). Formulations were kept under predetermined conditions of temperature and humidity for a specified period and tested for changes in their physical appearance, pH, viscosity, drug content, and drug release profile.

3 Results

3.1 Fourier transform infrared spectroscopy (FTIR)

The FTIR spectrum of pure metronidazole showed typical absorption peaks for the major functional groups (Figure 1; Table 2). The peaks present at 496.62 cm^-1 and 670.04 cm^-1 belonged to aromatic C-H rocking vibrations. The peaks present at 1,067.77 cm^-1 and 1,261.97 cm^-1 were due to C-O stretching vibrations from alcohol and ether functional groups. The peak present at 1,469.08 cm^-1 was because of C-H bending vibrations. An intense peak observed at 2,351.95 cm^-1 was for the vibration of the nitro group. The peaks at 2,822.78, 3,090.79, and 3,206.89 cm^-1 were for C-H stretching vibrations from aliphatic and aromatic rings. Broad absorption peaks at 3,610.34, 3,671.26, and 3,731.45 cm^-1 were attributed to O-H and N-H stretching vibrations, which suggested that there were hydroxyl groups and imidazole-functional groups of metronidazole. The FTIR spectrum of metronidazole loaded into carbopol 940 nanogels (Figure 2; Table 3) revealed typical absorption peaks for both metronidazole and carbopol 940. The peak present at 759.62 cm^-1 was for aromatic C-H rocking vibrations, while the peak at 919.43 cm^-1 was for aliphatic C-C stretching vibrations. The range 987.51–1,226.86 cm^-1 was for C-O stretching vibrations associated with polyacrylic acid structure of Carbopol and hydroxyl groups. The peak present at 1,454.74 cm^-1 was attributed to methyl group bending vibrations. The peak present at 1,641.07 cm^-1 was because of the C=O stretching vibrations of Carbopol 940 carboxylic acid groups. Broad absorption peaks observed within the range 2,892.29 and 3,285.00 cm^-1 were for C-H stretching vibrations. The peak present at 3,732.17 cm^-1 was for O-H stretching vibrations associated with the hydrated polymeric network. The nanogel formulation showed the characteristic peaks of metronidazole without any significant loss of major peaks of the drug or appearance of any new peaks. The absence of any shift in the peaks and appearance of absorption bands proves that there was no chemical incompatibility of metronidazole with Carbopol 940 in the formulation process. This shows that metronidazole is successfully formulated in Carbopol 940 polymer matrix system.

TABLE 2. IR spectrum peaks of metronidazole.

TABLE 3. IR spectrum peaks of metronidazole nanogel using carbopol 940 polymer.

3.2 Physical appearance and homogeneity

The physical attributes such as color, clarity, consistency, homogeneity, and particle size were determined for the prepared metronidazole loaded Carbopol nanogels (F1-F3). All formulations were homogeneous and consistent, free of visible lumps and devoid of any phase separation. Their color was found to be a pale yellowish straw like with a distinct odor. Microscopically all formulations proved to be free of coarse particles and there was no indication of formation of aggregates by the formulations in the gel matrices. The only problem observed was some entrapped air bubbles in the gel samples, which are usually formed during formulation and pose no harm to the appearance and the consistency of the formulations (Table 4).

TABLE 4. Results of evaluation parameter of metronidazole nanogels.

mean ± standard deviation (SD), n = 3.

3.3 pH determination

pH levels of metronidazole-loaded nanogels of Carbopol 940 have been determined in triplicates (n = 3) and shown as mean ± standard deviation (Table 4). It was found that formulations F1 and F2 have pH levels of 5.30 ± 0.02 and 5.30 ± 0.03 respectively; meanwhile, F3 had a bit higher value of pH of 5.50 ± 0.02. As can be seen, obtained pH levels (5.30–5.50) fall into physiological range of pH (around 4.5–6.5) and make these formulations suitable for topical administration. Preserving pH level within physiological limits is necessary for maintenance of skin integrity during topically delivered drugs and for minimizing chances of inducing skin irritation. Thus, it can be concluded that the developed Carbopol nanogels have appropriate pH levels and are not expected to cause pH related skin irritation.

3.4 Drug content uniformity

The drug content uniformity test was performed thrice (n = 3) in order to assess the drug distribution of metronidazole in the nanogels based on Carbopol 940 (Table 4). Drug content of the formulations varied from 94.70% ± 0.46% to 99.94% ± 0.21%, which showed satisfactory inclusion of metronidazole in the gel system. Among all the formulations, F3 showed the maximum drug content of 99.94% ± 0.21%, followed by F2, which had 98.63% ± 0.38%. In contrast to the above mentioned formulations, F1 had relatively lower drug content of 94.70% ± 0.46%. The relatively higher drug content of F2 and F3 could have been due to better drug entrapment in the hydrated network of carbopol and its more effective distribution in the formulation. All the formulations had drug content more than 90%, showing good drug loading and distribution.

3.5 Swelling index

The swelling behavior of carbopol 940 nanogel formulations was conducted in triplicate (n = 3) in order to evaluate the hydration capacity of the polymeric network (Table 4). The swelling index was found to be 30.00% ± 0.84% for formulation F1, 29.00% ± 0.79% for formulation F2, and 33.00% ± 0.91% for formulation F3. The formulation that showed the highest swelling capacity among all the formulations studied was formulation F3. The swelling behavior of formulations based on carbopol is mainly due to the ionization of carboxylic acid groups in the cross-linked poly (acrylic acid) network. On hydration, electrostatic repulsion of ionized polymer chains leads to the uptake of water and expansion of the gel matrix. Higher swelling will allow improved matrix hydration, diffusion of drug molecules through the hydrated matrix, and increase residence time of the formulation on the applied surface. The comparatively high swelling index of F3 formulation indicates favorable interplay between polymer concentration and hydration of the network, which may be the reason for the sustained release properties of the formulation and improved antimicrobial efficacy in the following experiments.

3.6 Viscosity and rheological behavior

The rheological characterization of the Carbopol nanogels’ formulations (F1-F3) reveals that these nanogels have pseudoplastic or non-Newtonian nature with the viscosity continuously decreasing with an increase in the speed of the spindle. All the formulations demonstrate shear thinning behavior, which is desirable in case of any topical drug delivery system as it allows easy application due to reduced viscosity but keeps good consistency when stored at rest. The higher viscosities at lower rotational speeds of F1 and F3 formulations suggest a better polymer network structure within the formulations. For instance, F1 and F3 formulations have viscosities of 73,600 cP and 72,000 cP, respectively, at 0.6 RPM, whereas with an increase in RPM, the viscosity continues to drop slowly. Despite being subjected to higher rotational speeds, the formulations still have fairly high viscosity values. This suggests good structural stability of the formulations when subjected to shear forces. The pseudoplastic behavior is quite desirable in this case as the formulations become more fluid on rubbing and, thus, easy to apply while becoming more viscous once applied (Table 5).

TABLE 5. RPM vs. viscosity of F1, F2, F3.

3.7 Particle size, polydispersity index, zeta potential, and conductivity

Nanosize particle distributions in the Carbopol gel systems were analyzed using the dynamic light scattering technique. The particle size distributions showed that among the gel systems, F3 had the minimum particle size (143.4 nm), whereas formulations F1 and F2 had maximum particle sizes of 484.3 nm and 323.5 nm, respectively. Particle size reduction favors higher diffusion and biological penetration. Zeta potential was measured between −11.0 mV to −12.3 mV for formulation systems F1 and F3, signifying moderate electrostatic stability of nanogels; F2 showed lesser values than F1 and F3. Surface negativity is due to the presence of ionized carboxylic groups on the polymeric structure of Carbopol. The polydispersity index was found to show moderate dispersion of particles in the formulations, signifying good homogeneity among the nanosystems. Further, conductivity results confirmed the ionic nature of the formulated nanogels and showed their good dispersion in an aqueous medium (Figure 3; Table 6).

TABLE 6. Particle size, zeta potential value, polydispersity index and conductivity values.

3.8 Spreadability study

Spreadability is one of the most important parameters that affect applicability and even distribution of topical formulations on the surface of the skin. The spreadability of nanogels with Carbopol 940 was determined in triplicates (n = 3) and expressed as average ± standard deviation (Table 7). The range of spreadability was 1.20 ± 0.03–1.68 ± 0.05 g·cm/s. As follows from analysis of the results, F1 formulation was characterized by the highest spreadability (1.68 ± 0.05 g·cm/s) and therefore, it had better spreading properties during topical application. Conversely, the lowest spreadability (1.20 ± 0.03 g·cm/s) was found for F3 formulation due to high viscosity and structurization of the polymeric matrix. An increase in polymer concentration in formulations leads to enhancement of intermolecular interaction in the hydrated gel structure, and therefore to higher viscosity and lower spreadability. Thus, the spreadability values obtained for different formulations demonstrate that polymer concentration and rheological characteristics significantly affect spreading property of nanogels. Though F3 formulation had comparatively lower spreadability values, it was within acceptable limits for topical semi-solid preparations.

TABLE 7. Evaluation of spredability study and extrudability study.

mean ± standard deviation (SD), n = 3.

3.9 Extrudability study

Extrudability is one of the vital quality attributes for topical semisolid products since it impacts dispensability, dosage accuracy, and usability of the formulation. The extrudability of the formulated nanogels based on Carbopol 940 is given in Table 7 (three replicate tests, n = 3) and varied within the range from 82.40% ± 1.12% to 86.66% ± 0.88%. As can be seen, among the investigated formulations, the highest extrudability had F3 (86.66% ± 0.88%), followed by F2 (85.90% ± 0.96%), whereas the lowest extrudability had F1 (82.40% ± 1.12%). The low extrudability of F1 could be explained by high viscosity and better-formed gel structure that may increase extrusion resistance from the collapsible tube. Despite these variations, the obtained results reveal that all formulations have adequate extrudability for topical administration. It means that formulated Carbopol nanogels demonstrate good dispensing characteristics that play an essential role in comfortable application and dose reproducibility.

3.10 In-Vitro drug release study

In vitro release studies of metronidazole loaded nanogels formulations (F1-F3) using a diffusion-based release model were done over an 8 h’ duration. All experiments were carried out in triplicate (n = 3) and results presented as means ± SD (Table 8; Figure 4). Results from the release profiles showed a biphasic release profile comprising of a burst release followed by a sustained release phase. Within the first hour, the cumulative drug release percentages achieved were 10.88% ± 0.42% for F1, 13.06% ± 0.51% for F2 and 12.22% ± 0.47% for F3. The burst release phase is ascribed to the quick diffusion of the drug molecules either associated or localized on the surface of the hydrated nanogel matrix. Such a release profile could be favorable for the antimicrobial formulation since it enables rapid attainment of therapeutic concentration levels at the site of application. After the burst phase, the three nanogels continued releasing drugs steadily during the entire study period. After 8 h of release, the cumulative drug release percentages achieved were 95.57% ± 1.84% for F1, 92.54% ± 1.76% for F2 and 97.03% ± 1.91% for F3. The F3 formulation exhibited the highest cumulative drug release percentage as compared to other formulations. The better release performance of F3 might be due to its smaller particle size (143.4 nm), increased diffusion area and good swelling property of the Carbopol polymeric matrix. Swelling of the cross-linked Carbopol matrix will enable diffusion of metronidazole through the aqueous channels formed within the matrix, hence sustained drug release. In summary, the release study demonstrates the ability of the nanogel formulations to deliver the prolonged release of metronidazole drug over the 8-h period. The high percentage release of F3 shows a favorable balance between the nanoscale features, polymer hydration and diffusion-controlled drug release process.

TABLE 8. In vitro cumulative drug release profile of Carbopol nanogel formulations (F1–F3).

mean ± standard deviation (SD), n = 3.

3.11 Drug release kinetic modeling

The in-vitro drug release results of carbopol-based nanogel formulation F1-F3 were fitted to several kinetic models like Zero-order, First-order, Higuchi, and Korsmeyer-Peppas models to analyze the mechanism of drug release (Table 9). Regression analysis provided values of coefficients of determination (R²) that showed that all nanogels follow Zero order kinetics based on higher R² values compared to other kinetic models used. F3 gave the highest value of Zero order (R² = 0.997) followed by F1 (R² = 0.994) and F2 (R² = 0.991). These higher values of Zero order show the predominance of Zero order kinetics in which release occurs independent of concentration in a controlled manner. The Higuchi model also gave high R² values, suggesting the significant role of diffusion from hydrated polymeric network in drug release process. On the other hand, low R² values obtained by using First order kinetic model indicate that release is independent of concentration. Korsmeyer-Peppas model analysis showed good linearity suggesting that anomalous/Non-Fickian diffusion was involved, which could be due to polymer chain relaxation/swelling effect. Overall, the kinetic study showed that nanogel formulations have sustained and controlled release nature, thus useful for prolonged topical antimicrobial therapy. From among all the formulations, F3 gives the most optimized result due to its structural features.

TABLE 9. Drug release kinetic modeling of metronidazole-loaded carbopol nanogels (F1–F3).

3.12 Antimicrobial activity

The effectiveness of metronidazole-loaded nanogels F1-F3 based on carbopol 940 was determined by the disc diffusion method for the gram-positive bacterium Staphylococcus aureus and the gram-negative bacterium Escherichia coli. Both of these bacteria were used as a model for antimicrobial activity testing because of their widespread use in studies of new topical antimicrobial products. All the experiments were done in triplicate (n = 3). The data are shown in Figure 5. As can be seen from the results presented in Figure 5 F3 had the highest antimicrobial activity against S. aureus since this formulation had significantly larger and consistent zones of inhibition than any other formulation. At the same time, F2 had comparatively lower antimicrobial activity, and F1 did not produce zones of inhibition at all in the experimental conditions. Thus, the higher efficacy of F3 in the inhibition of S. aureus growth can be explained by the smaller size of nanoparticles in F3, high swelling and release properties of this formulation that allow for maintaining the prolonged presence of metronidazole in the vicinity of the colonies. In spite of the fact that both formulations F1 and F2 had relatively high contents of drugs in amounts over 90% and high cumulative release over 90% after 8 h, the activity in the agar diffusion test is dependent not only on the total drug release from the formulation but also on the diffusion rate of the drug through the agar layer and local concentrations near the bacterial colonies. Thus, the comparatively smaller size of particles and good swelling of F3 could lead to efficient diffusion of metronidazole.

No inhibition zone was detected for the tested formulations against Escherichia coli. Such results correspond to the known antimicrobial spectrum of metronidazole that is characterized by high activity against obligate anaerobes. The absence of significant action against E. coli can be explained by natural resistance of aerobic Gram-negative bacteria due to the presence of outer membranes containing lipopolysaccharides that hinder the action of many antibacterial agents. In its turn, more penetrable cellular structure of S. aureus can contribute to higher activity of metronidazole. First of all, it should be highlighted that the conducted experiments were focused only on the disc diffusion test as the method of evaluation of antimicrobial activity. Quantitative analysis including determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill tests, and reduction of bacterial load were not conducted but they could serve as the subjects for further research. Additionally, since metronidazole is mostly effective against anaerobic microorganisms, antimicrobial activity against clinically important anaerobic microorganisms can provide more detailed information on the studied nanogel formulations. In summary, it can be stated that among the tested Carbopol 940 nanogels, the best antibacterial activity against Staphylococcus aureus was shown by formulation F3 while no activity was revealed for Escherichia coli.

3.13 Stability study

Stability studies on the optimized formulations of Carbopol 940 nanogels (F1-F3) were performed according to ICH guideline Q1A (R2) at three different storage conditions such as refrigerated condition (2 °C–8 °C), room temperature (28 °C–32 °C/60%–70% RH ± 5%), and accelerated condition (40 °C ± 2 °C/75% ± 5% RH) for 60 days. All evaluations were performed on days 15, 30, and 60, while all measurements were performed in triplicate (n = 3). Evaluated parameters included physical appearance, color, odor, pH, homogeneity, and content uniformity of the drug (Table 10). It can be stated that there were no any visible changes in the color, odor, phase separation, precipitation, and homogeneity of any of the formulations under the investigated storage conditions. Thus, all investigated formulations possessed the characteristic pale yellow color and gel-like consistency, which can indicate satisfactory physical stability of Carbopol matrix in the period of storage. The pH values showed insignificant variations during the 60-day period. In particular, the pH value of the formulation F1 varied from 5.30 ± 0.02 to 5.25 ± 0.05; of the formulation F2 – from 5.30 ± 0.03 to 5.24 ± 0.05; of the formulation F3 – from 5.50 ± 0.02 to 5.44 ± 0.05 under accelerated storage condition. Nonetheless, all the formulations were still in the limits of acceptable physiological pH range. Similarly, there was an insignificant drop in the drug content of the formulations during the storage period. When the formulations were stored under accelerated storage conditions, there was a decrease in the drug content from 94.70% ± 0.46% to 93.84% ± 0.74% in formulation F1, from 98.63% ± 0.38% to 97.71% ± 0.66% in formulation F2, and from 99.94% ± 0.21% to 98.87% ± 0.48% in formulation F3 within 60 days. This slight drop is not beyond the pharmaceutical standards and does not point out any drug degradation or any failure of the formulation structure. The slight variation in pH and drug content can be attributed to the variation in the analytical data and to the effect of temperature and humidity when exposed to accelerated storage conditions. From the stability results, one can deduce that the cross-linked three-dimensional structure of Carbopol 940 was capable of stabilizing the nanostructured matrix of the gel. Even though there is some promising stability data obtained in the current study within 60 days of storage period, longer duration stability studies and drug release study after storage will give a more holistic stability information.

TABLE 10. Stability study of optimized Carbopol nanogel formulations under different storage conditions.

mean ± standard deviation (SD), n = 3.

4 Discussion

The present study systematically evaluated the effects of varying concentrations of Carbopol 940 on the physicochemical properties, rheology, nanoscale characteristics, drug release pattern, antimicrobial efficacy, and stability of nanogels loaded with metronidazole for topical applications. Unlike most earlier Carbopol formulations of metronidazole which mainly focused on conventional gels and/or isolated properties of the formulation, the current study evaluated various important aspects of quality simultaneously in a particular formulation to find out the optimal system and to establish the correlation between the polymer concentration and performance of the formulation. Carbopol 940 was selected owing to its ability to form gels, swellability, bioadhesiveness, rheological stability, and suitability in topical pharmaceutical formulations (Singh et al., 2014; Islam et al., 2004; Daood et al., 2019; Roy et al., 2022). Polymer concentration affected the arrangement of the hydrated polymeric network resulting in variations in viscosity, swelling, particle size distribution, and drug release properties. Correlations between the polymer concentration, gel structure, and diffusion-dependent release of drug were also observed in Carbopol-containing semisolid systems (Singh et al., 2014; Islam et al., 2004; Daood et al., 2019; Roy et al., 2022). Some recent studies have also shown the potential of bioadhesive and biomimetic nanogel systems to improve the local drug delivery, drug retention, and biocompatibility (Zhang et al., 2025; Li et al., 2026; Kumar et al., 2026). All the formulations exhibited satisfactory appearance, homogeneity, and pH in the physiological range of the skin, and thus were suitable for topical application. No phase separation in any of the formulations during the study suggests good physical compatibility between metronidazole and Carbopol polymeric matrix. The swelling behavior was considerably affected by the hydration of the cross-linked Carbopol matrix. Ionization of the carboxylic acid groups facilitates water sorption and hydrated diffusion pathways. Formulation F3 showed the highest swelling index, implying good balance between the concentration of the polymer and the hydration of the network, which might favor the sustained diffusion of the drug from the matrix. The rheological test exhibited pseudoplastic (shear-thinning) flow behavior in all the formulations developed; this type of flow behavior is preferable in semisolid formulations because viscosity decreases with shear and then increases with the absence of shear, which might lead to a better formulation retention at the application site. Dynamic light scattering test showed nanometric particles for the formulated samples. According to the test results, Formulation F3 had the smallest average particle size (143.4 nm), along with a good polydispersity index and an adequate zeta potential, which means fairly uniform dispersion of particles and colloidal stability. However, additional morphological studies (for instance, SEM, TEM, or AFM) were not performed to confirm the structure of the nanogels in further studies. Release kinetics study showed biphasic drug release, including fast (burst) drug release and sustained drug release. The burst release can be explained by the rapid diffusion of the drug molecules adsorbed on the surface of the hydrated matrix, whereas the second phase (sustained release) can be caused by the diffusion of metronidazole molecules through the hydrated Carbopol gel network. While the kinetics of drug release was described the best with the zero order model among other models analyzed, the presence of the burst phase means that the drug release process was not completely constant during the whole drug release period. Hence, the zero order can be considered predominant after the diffusion phase. Antimicrobial evaluation showed that the F3 formulation exhibited superior antibacterial activity against Staphylococcus aureus compared to other formulations. The better activity of the formulation can be due to the synergistic action of small particles, swelling property, and drug release rate. As a result, the sustained drug availability near bacterial colonies was achieved during the course of the agar diffusion test. Inhibition was not found for Escherichia coli, which is in agreement with the known antimicrobial spectrum of metronidazole and natural resistance of aerobic Gram-negative bacteria to it under experimental conditions.

Stability studies carried out according to ICH Q1A (R2) demonstrated no visible changes in appearance, odour, homogeneity, or phase separation during 60 days of storage at refrigerated, room, and accelerated conditions. Small variations in pH and drug content were detected during the experiments, especially under accelerated conditions; however, all results are within acceptable pharmaceutical ranges and demonstrate sufficient physicochemical stability of Carbopol nanogel matrix. There are some limitations of the study. Morphological studies using SEM, TEM, or AFM methods have not been conducted. Skin permeation and penetration, cytocompatibility, skin irritation, and in vivo efficacy were not studied in the current research. For the antimicrobial evaluation, only the agar diffusion method was used; MIC, MBC, and killing rate have not been measured. Additionally, the process of formulations’ optimization was conducted only by comparing their properties without applying any statistical approaches such as DoE or RSM. From all together, the obtained data suggests that carbopol 940 concentration plays a significant role in physicochemical parameters and performance of metronidazole nanogels. From all tested formulations, F3 showed the optimal balance between size of particles, swelling, rheological properties, release of the drug, antimicrobial effect and stability. Although further preclinical studies should be done, it is considered as a good platform for further development and optimization of metronidazole nanogel formulations based on carbopol 940 for topical drug delivery.

5 Conclusion

The metronidazole-loaded nanogel formulations containing Carbopol 940 have been developed and investigated for topical drug delivery applications. The formulated products possessed acceptable physicochemical characteristics, including proper pH, homogeneity, spreadability, extrudability, and pseudoplasticity suitable for topical administration. It has been confirmed by dynamic light scattering analysis that there were nanoparticles with acceptable colloidal properties in all prepared formulations. Formulation F3 had the best combination of size of particles, swelling properties, viscosity, drug content, and in vitro drug release performance among the tested formulations. According to the release study, there were two stages of the process–initial burst release and sustained release over 8 h. According to the results of the kinetic analysis, it is possible to say that the mechanism of the process was the best described by the zero order kinetic model. Antimicrobial studies have shown better inhibitory effect of the formulation F3 on Staphylococcus aureus growth. It was found out that the formulations had no antimicrobial activity towards Escherichia coli bacteria. The performed short-term stability studies in compliance with ICH Q1A (R2) have shown preservation of the physicochemical characteristics of the formulation during 60 days at different temperatures. From the results, it can be concluded that tuning the optimal level of Carbopol 940 significantly affects the physicochemical, rheological, drug delivery, and antibacterial properties of metronidazole-loaded nanogels. Of all the formulations prepared, formulation F3 showed optimal performance in general; hence, it could be considered a suitable candidate for developing topical formulations containing metronidazole. However, more studies have to be conducted including morphology investigation, skin permeation studies, cytocompatibility study, skin irritation test, stability study, and efficacy study in vivo, before clinical application of these formulations.

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Footnotes

Edited by: Suman Basak, Dyson, United Kingdom

Reviewed by: Farhad Mohammadi, Shahid Sadoughi University of Medical Sciences and Health Services, Iran

Lakshmi Usha Ayalasomayajula, RAGHU College of Pharmacy, India