Study of Gelatin-grafted-2-Acrylamido-2-methylpropane sulfonic acid hydrogels as a controlled release vehicle for amorphous solid dispersion of Tripterygium Wilfordii bioactive constituents

2.1 Materials

Gelatin from Porcine skin (Gel; MW: 40,000 to 50,000 Da) was obtained from the Tianjin Fuchen chemical reagents factory, China. 2-Acrylamido -2-methyl-1-propanesulfonic acid (AMPS; MW: 207.25 g/moL), ammonium persulfate (APS), and ethylene glycol dimethacrylate (EGDMA; MW: 198.22 g/moL), were obtained from Sigma-Aldrich, Saint Louis, USA. Sodium bisulfite (SHS) was purchased from Shanghai Aladdin biochemical technology, China. ABTS, DPPH, and Cefepime (HCL) were procured from Meilune biological company (China). Tripterygium Wilfordii was acquired from Jiangxi Jiangzhong Traditional Chinese Medicine Co., Ltd., whose authenticity was certified by Prof. Deng Kezhong (School of Pharmacy, Jiangxi University of Chinese Medicine; voucher number: J20221014).

Bacterial strains such as Staphylococcus aureus (S. aureus: ATCC25923HBJZ005), Pseudomonas aeruginosa (P. aeruginosa: ATCC27853HBJZ017), and Escherichia coli (E. coli: ATCC25922HBJZ087) were procured from Qingdao Hope Biotechnology, Co, Ltd., Shandong, China.

2.2 Preparation of TWHF-SD

TWHF (80 g) was added to ultra-pure water (800 mL) in an electric heating sleeve, soaked for 30 min, and then subjected to heated reflux extraction to obtain the extract. The extract was then filtered through a 200-mesh screen. The filtered extract was then concentrated by rotating vacuum evaporators (Xiamen Jingyi Xingye Technology Co., LTD). Firstly, the extract was refrigerated at −20 °C for 24 h, followed by lyophilization for 2–3 days (Ningbo Xinzhi Biotechnology Co., LTD) to obtain freeze-dried TWHF-SD (Guan et al., 2023). The total yield of the TWHF extracts powder obtained was 9.75%.

2.3 Fabrication of Gelatin-g-AMPS hydrogels

Free radical polymerization was used to prepare the hydrogels, with a few modifications (Zang et al., 2022). Briefly, specified quantities of gelatin, APS/SHS, AMPS and EGDMA were weighed carefully and placed in separate glass bottles for each formulation (Table 1). In each labelled vial, sufficient water was added and stirred continuously until a clear solution was obtained. Gelatin was continuously stirred at 50 °C to ensure its complete dissolution. These labelled solutions were then mixed according to the order in which they had been labelled. Briefly, APS/SHS was added to AMPS and mixed evenly before adding the gelatin, thereby eliminating any lumps in the mixture. After ultrasonication, the resulting homogeneous solution was bubbled with nitrogen for 30 min to remove bubbles. EGDMA was then introduced dropwise into the mixture as a final step. In the next step, the final solution was transferred to a 50 °C constant temperature water bath. The water bath temperature was gradually increased to 65 °C. After 24 h, the hydrogels were formed and cut into 8 mm discs and vacuum dried for a week at 40 °C. Fig. 1 presents a proposed chemical structure of the synthesized Gelatin-g-AMPS hydrogel.

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Fig. 1

The proposed chemical structure of Gelatin-g-AMPS hydrogels.

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2.4 TWHF-SD loading in hydrogels

A swelling-diffusion technique was used to load TWHF-SD into Gelatin-g-AMPS hydrogels (Yu et al., 2022). Briefly, TWHF-SD was dissolved in pH 7.4 buffer, and a 1% solution of it was prepared, and then pre-weighed dried hydrogel was soaked in the solution for three days while being stirred slowly. After three days, the hydrogel discs were removed from the solution, dried completely, and weighed. The amount of TWHF-SD-loaded hydrogel was then determined by the weight subtraction method. Additionally, the TWHF-SD loading was confirmed by extracting all the TWHF with water and analyzing it using ultraviolet spectrometry (wavelength of 271 nm).

2.5 Solid-state characterization of hydrogels

2.6 Hydrogel polymer network variables

Hydrogel structures and properties can be analyzed using the following major indicators (Singh and Singh 2020).

2.7 Biodegradation analysis

The biodegradation of hydrogels was assessed in a 7.4 pH phosphate buffer solution at 37 °C ± 0.5 °C. Specifically, weighed discs of dry hydrogel were immersed over a period of time in a buffer solution with a pH of 7.4. These discs were then dehydrated in a vacuum oven at 40 °C, weighed again, and placed in a buffer solution with a pH of 7.4. The procedure was repeated for a week. For the calculation of hydrogel degradation, the following equation was used (Tapdiqov et al., 2022).

D stands for degradation, C1 stands for dry weight, and C2 stands for the weight of the sample after immersion at a specific time (t).

2.8 Antioxidant activities determination

2.9 Antibacterial assessment

TWHF-SD-loaded hydrogels were tested against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli using the disk diffusion method. Each bacterial suspension with a cell density of approximately 3 × 10⁸ CFU·mL⁻¹ was cultured on an agar medium. Hydrogel samples were cut into small pieces and sterilized to prevent microbial contamination. Each hydrogel was then placed on cultured agar plates and incubated at 37 °C for 24 h. The bacterial inhibition zones were then determined (Abbasi et al., 2020).

2.10 Statistical analysis

Data were expressed as mean ± standard deviation. The statistical difference between adjacent data was determined using two-way ANOVA and Tukey's post-hoc test. The significance of the difference was determined using p-values of p < 0.05, *p < 0.01, and ***p < 0.001.

3.1 Identifying chemical constituents in TWHF-SD using UHPLC-Q-TOF-MS

The chromatograms in Fig. 2 depict the base peak intensities of the sample in both positive and negative ion modes. The identification of the major chemical components of TWHF was carried out using mass spectrometry software (MassLynx v4.1). Table 2 presents 48 chemical compounds identified from the results of MS and MS/MS analyses, reference literature, and relevant scientific literature. Most of these compounds are diterpenes, triterpenes, and alkaloids. It has been demonstrated that these ingredients possess a variety of biological properties and are often used to treat conditions such as rheumatoid arthritis, lupus erythematosus, and other autoimmune diseases. According to some studies, these components are responsible for the primary therapeutic effects of TWHF.

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Fig. 2

TWHF-SD chromatograms obtained using UHPLC-Q-TOF-MS in positive (A) and negative ionization modes (B).

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3.2 FTIR spectroscopy

FTIR spectra of gelatin, AMPS, EGDMA, TWHF-SD, unloaded, and TWHF-SD loaded hydrogels are shown in Fig. 3. In the infrared spectrum of TWHF-SD, the band at 3463 cm⁻¹ belongs to the –OH stretching vibration, the band at 2940 cm⁻¹ and 2846 cm⁻¹ belongs to the C–H stretching vibration, and the band at 1607 cm⁻¹ belongs to the C–C stretching vibration above the benzene ring (Chen et al., 2014). In the infrared spectrum of gelatin, the band at 1630 cm⁻¹ corresponds to the absorption band of C = O, 1525 cm⁻¹ corresponds to C-N stretching vibration and 1239 cm⁻¹ corresponds to the N–H bending vibration, and these findings are consistent with those of other researchers (Aguilar-Méndez et al., 2020). Based on the FTIR spectrum of AMPS, a band was observed at 1461 cm⁻¹ associated with the binding vibration of CH₂. The band at 1360 cm⁻¹ represents the -C-O stretching vibration, whereas 2981 cm⁻¹ reflects the –CH stretching of –CH₂, and 1230 cm⁻¹ reflect the symmetrical stretching of the S = O functional group. FTIR spectrum of EGDMA revealed a band at 1713 cm⁻¹, indicative of stretching vibrations of C = O, along with bands at 1633, 1291 and 1153 cm⁻¹ corresponding to C = C and C–O stretching vibrations in both symmetric and asymmetric esters. Unloaded hydrogels display a different spectrum than their parent components. The band at 1458 cm⁻¹ is attributed to the –CH₂ binding vibration in AMPS. The band at 1630 cm⁻¹ is associated with the absorption band of C = O in gelatin. These bands and functional groups indicate that the polymers were successfully cross-linked. Additionally, FTIR analysis of TWHF-SD-loaded hydrogels was performed to confirm the presence of the TWHF-SD. The bands at 1453 cm⁻¹ and 1610 cm⁻¹ are representative of the TWHF-SD spectrum. The appearance of these functional bands of TWHF-SD demonstrated that TWHF-SD was successfully incorporated into the hydrogel.

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Fig. 3

FTIR spectra of TWHF-SD, gelatin, AMPS, EGDMA, unloaded and TWHF-SD-loaded hydrogels.

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3.3 TGA study

TGA was performed in order to assess the thermal integrity of all reagents and hydrogel systems (Fig. 4). Gelatin thermogravimetry results can be divided into three general stages. First, there is a weight loss of 5.8% between 30 and 95 °C, which may be attributed to water loss. The weight loss in the second stage reached 34.66% at 265–324 °C, which was attributed to gelatin degradation. In the 320–540 °C range, the weight loss was 69.25% due to residual material combustion (Ullah et al., 2019). TGA analysis of AMPS revealed a 6% weight loss at 208 °C and a further 20% between 210 and 250 °C, indicating AMPS dehydration. Furthermore, the sulfonic acid group began to decompose at 340 °C, and a weight loss of further 20% was observed within a temperature range of 250 to 340 °C (Naeem et al., 2023). TGA analysis of TWHF-loaded hydrogels revealed that an initial weight loss of 18% was caused by dehydration at 30 to 183 °C, followed by a 45% weight loss due to bond breakage between polymers at 183 to 331 °C. A gradual degradation of the polymer network occurred at 331 °C and continued until the polymer skeleton was completely degraded. The initial weight loss of 17% in the unloaded gel was caused by dehydration in the temperature range of 30–342 °C, followed by a 19% weight loss due to the breaking of bonds between the polymers in the temperature range of 342–367 °C. The polymer network began to decompose gradually after 367 °C and continued until the polymer backbone was fully decomposed. Based on the thermal profile of hydrogels with high residual weight, it can be concluded that the polymeric matrix provides greater stability against thermal degradation over a wide temperature range. The developed hydrogel also exhibits a slower weight loss rate as a result of stronger interaction between the polymer and monomer, as compared to the individual reactants. The development of rigid networks as well as the shifting of endothermic peak temperatures, can be attributed to a higher level of thermal stability (Liu et al., 2023a, 2023b).

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Fig. 4

TGA thermograms of gelatin, AMPS, TWHF-SD, EGDMA, unloaded and TWHF-loaded hydrogels.

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3.4 DSC study

Gelatin showed a strong endothermic peak at 119.62 °C in the DSC analysis, which is consistent with previous studies (Rahman et al., 2008). A DSC analysis of TWHF-SD revealed that there was a large endothermic peak at 81.02 °C, which was similar to the results obtained by other researchers. The AMPS analysis revealed an endothermic peak at 202 °C, indicating dehydration. One exothermic peak was observed around 315 °C in the developed hydrogel. Hydrogels at these temperatures exhibit exothermic peaks, indicating cross-linking of polymers which provides thermal stability to the newly formed network (Fig. 5). DSC analysis of synthesized hydrogels indicates that the formulations are more compatible with each other and form rigid networks by forming greater hydrogen bonds between the molecules indicating increased compatibility among the components. Therefore, the developed polymeric network is more thermally stable.

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Fig. 5

DSC thermogram of gelatin, AMPS, TWHF-SD, unloaded and TWHF-loaded hydrogels.

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3.5 XRD studies

XRD analysis confirmed the physical state of TWHF-SD, gelatin, and the developed hydrogels, as shown in Fig. 6. The diffraction pattern of gelatin exhibited an extensive broadening peak between 15.0 and 25.0°, resulting from its a-helix and triple-helical structure (Shi et al., 2018). The XRD diffractogram of unloaded hydrogel exhibits a broader peak at 2θ = 21.32°, indicating that the formulation is amorphous. Similarly, TWHF-SD shows a broader amorphous peak at 2θ = 21.22°. The diffractogram of TWHF-SD loaded hydrogels, however, shows a wider peak at 2θ = 21.12°; however, no other intense peak of TWHF was observed at its respective region, possibly due to physical interactions between TWHF and polymeric network.

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Fig. 6

XRD patterns of gelatin, TWHF-SD, unloaded and TWHF-loaded hydrogels.

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3.6 Morphological studies

Fig. 7 demonstrates that the hydrogels exhibited porous and compact surfaces that were somewhat coarse in texture. This morphological structure may indicate successful crosslinking between polymers and monomers. These properties are observed in hydrogels designed for biomedical purposes (Gu et al., 2020). The porous surface of the hydrogel allows water to penetrate directly into the spaces and fluid to diffuse within the network, thereby enhancing swelling characteristics. In addition, these pores allow drugs to attach to the surface or load inside the hydrogels and are released at the site where they are needed. The hydrogel can absorb aqueous fluid in large quantities through these pores. The durability of the hydrogel system depends on its solid mass and smooth surface. The microstructure of the hydrogel before and after drug loading was analyzed by scanning electron microscopy. Compared with the unloaded hydrogel, the surface of the drug-loaded hydrogels showed a porous structure with the drug attached to the surface and loaded inside the hydrogel through the porous network.

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Fig. 7

Morphological characteristics of unloaded hydrogels at 6000^× (A), 12000^× (B) and TWHF-SD-loaded hydrogels at 5000^× (C) and 5000^× (D).

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3.7 Mechanical properties of hydrogels

Hydrogels should be tested for their mechanical properties, including tensile strength (TS) and elongation at break (EAB), before they can be used for biomedical applications. The mechanical properties of the hydrogel gradually improved as the concentration of the gelatin was increased, and our results are consistent with those of other researchers (Table 3) (Biswal et al., 2016). AMPS is an anionic monomer, which results in a decrease in the mechanical strength of the hydrogel which may be attributed to an increase in electrostatic repulsion and osmotic pressure. The tensile strength also increased as the EGDMA content increased.

3.8 Sol-gel studies

Hydrogels are obtained through the polymerization of crosslinkers, monomers, and polymers. “Sol fractions” are non-crosslinked portions of hydrogels, while “gel fractions” are crosslinked portions of hydrogels. The lack of reactive sites during the polymerization process of hydrogels results in uncrosslinking of some parts (sol fraction) in the case of a large number of single or multiple ingredients being used. Thus, the crosslinking percentage of the hydrogel must be determined (Naeem et al., 2022a, 2022b, 2022c). The sol–gel properties of all hydrogel formulations were tested (Fig. 8A, B, and C). Sol-gel evaluation is principally used to assess the degree of uncrosslinking among polymers within a system. Depending on the material ratio in the hydrogel formulations, the gel fraction percentage ranges from 80.50% to 96.30%. The increase in gelatin concentration may result in a gradual increase in gel fraction, possibly due to the increase in biopolymer concentration. The findings of this study are in agreement with those of other researchers (Tytgat et al., 2018). Additionally, since AMPS are hydrophilic monomers, they allow more room for chemical reactions to take place. As AMPS concentration increases, the gel fraction increases. Gel fractions increase when the amount of EGDMA in the hydrogel increases, as EGDMA contributes to their formation as a result of its crosslinking properties.

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Fig. 8

Effect of gelatin (A), AMPS (B), and EGDMA (C) on sol–gel and porosity of hydrogel.

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3.9 Porosity study

The porosity of hydrogels influences several of their properties, including swelling, loading, and release of drugs. Pore size affects swelling, which contributes to greater loading and release of drugs. The high viscosity of the reaction mixture results in a higher porosity, inhibiting the egress of bubbles. As a result, the material becomes porous due to a large number of interconnected channels. Porosity percentages varied from 41.81% to 91.62% at different reagent ratios. Gradually, the porosity of the hydrogel increased with an increase in the gelatin ratio. SEM images of the newly synthesized hydrogel show that it has rough and porous properties, which is consistent with other researchers' findings (Elblbesy et al., 2020). EGDMA leads to the formation of tight junctions and crosslinked bulk densities, resulting in a decrease in porosity. A higher concentration of AMPS increases porosity since the sulfonate groups generate more electrostatic forces. AMPS contains a hydrophobic alkyl group, which can reduce hydrogen bond interactions by forming microregions of hydrophobicity. So, in the hydrogel preparation, the pores and network sizes are enhanced, which agrees with other studies (Khan et al., 2022).

3.10 Biodegradation studies

Fig. 9 depicts the results of a biodegradation experiment conducted on the prepared hydrogel to determine its degradation rate. Biodegradation studies were conducted in order to determine the degradation rate of the hydrogel prepared at various material ratios. Different weight ratios will likely have a significant impact on the degradation rate of the hydrogel. Hydrogels of different proportions showed varying rates of degradation between 5.6% and 9.3%. There was a positive correlation between the amount of gelatin and the rate of weight loss of the composite hydrogel (Hu et al., 2011). It was found that as the EGDMA ratio increased in the material, the degradation speed of hydrogels slowed down. It may be due to the generation of functional groups, which resulted in the production of a large number of free radicals. In addition to strengthening hydrogel crosslinks, these free radicals also play a significant role in the polymerization reaction, which slows down the degradation process.

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Fig. 9

Effect of gelatin (GAE-7,8,9) (A), AMPS (GAE-4,5,6) (B), and EGDMA (GAE-1,2,3) (C) on biodegradation of hydrogel.

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3.11 Hydrogel network properties

A number of structural parameters, including the average molecular weight between crosslinks Mc (degree to which the polymer has been crosslinked), the polymer volume fraction V2,s (the amount of fluid absorbed and retained by the network), the solvent interaction parameter χ, and the number of repeats between crosslinks N, were calculated for the synthesized hydrogels. The values of various structural parameters are shown in Table 4. These parameters are important for hydrogels because they indicate the compatibility of the solvent with the polymers and, thus, the maximum capacity for uptake and holding. V2, s and χ values increased as EGDMA concentration was increased, suggesting that hydrogel structures became tighter and stiffer, consistent with other studies (Jalil et al., 2017). Mc and N values also decreased with an increase in the concentration of EGDMA. The decrease in Mc and N values is due to the increased crosslinking density caused by the increasing amount of EGDMA utilized. Crosslinking density correlates with a decrease in swelling properties, while V2,s correlates with an increase in swelling properties.

3.12 Swelling studies

The swelling ratios of hydrogels in different media were examined using different ratios of polymer, monomer and crosslinker (gelatin, AMPS, and EGDMA). Fig. 10A and B illustrate the dried hydrogels' physical appearance. Due to the hydrophilic nature of the polymers and monomers used in the preparation of hydrogels, the bonds between them begin to break when they are soaked in a variety of media. This results in the swelling of the hydrogels. Fig. 10C and D illustrate variations in the swelling rate of hydrogels with respect to pH (Jayaramudu et al., 2019). It was found that the swelling of the hydrogel was slightly higher at pH 1.2 and less at pH 7.4. Water in the environment caused the ionization of the hydroxyl (–OH) functional groups of the hydrogel, resulting in higher swelling. It was found that the swelling degree of hydrogel ranged between 21.75% and 60.84% at pH 1.2. The maximum percentage of the swelling degree of hydrogel was observed in GAE-6 at pH = 1.2 (60.84%), while the lowest percentage was found in GAE-3 at pH = 1.2 (21.75%). The percentage of the swelling degree of the hydrogel at pH = 7.4 was determined to be between 15.53% and 46.46%. GAE-6 had the highest swelling degree at pH = 7.4 (46.46%), and GAE-3 had the lowest swelling degree at pH = 7.4 (15.53%). Hydrogel swelling increased with the increase in gelatin ratio. The results of our study are in accordance with the findings of other researchers. A higher concentration of AMPS monomer will increase the equilibrium swelling degree of the hydrogel, given that AMPS contains abundant –CONH₂ and -SO₃OH groups. The more of these groups present, the more likely they are to bind to water molecules, which are highly absorbent, so the more of these groups present, the higher the degree of absorption. AMPS improves the crosslinking density and storage modulus of hydrogel, enabling an optimal network structure to be formed. AMPS content resulted in an increase in equilibrium swelling ratios and swelling rate constants for hydrogel, with more hydrogen bonds forming between water molecules and hydrogel, resulting in an increase in non-frozen and frozen bound water, thereby improving hydrogel water retention and improving swelling. Other researchers have also reported similar results (Meng and Ye 2017). As the concentration of EGDMA increased, there was a decrease in swelling. When EGDMA concentration is increased in hydrogels, the packing density increases, and the porosity decreases. Thus, increased EGDMA concentration reduces water penetration into the hydrogel network, resulting in a decrease in swelling (Suhail et al., 2021).

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Fig. 10

The appearance of hydrogels after swelling at a pH of 1.2 (A) versus a pH of 7.4 (B). The swelling curves for hydrogels at 1.2 pH (C) and 7.4 pH (D) over time.

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3.13 Release and kinetic modelling analysis

The percentage of drug release in the buffer at pH of 1.2 ranged from 56.01% to 85.08%, as shown in Fig. 11. GAE-6 had the highest drug release rate at pH = 1.2 (85.08%), while GAE-3 had the lowest drug release rate at pH = 1.2 (56.01%). The drug release rate was 50.02%–77.03% in the buffer with pH of 7.4. GAE-6 had the highest drug release (77.03%), while GAE-3 had the lowest drug release (50.02%). The release curve indicates that the release of TWHF was different in different pH buffers, with the maximum release occurring in pH buffer of 1.2. The maximum release rate was 85.08% after 48 h.

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Fig. 11

The release of TWHF from hydrogels at pH 1.2 (A) and pH 7.4 (B) in 48 h.

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Water molecules diffuse into polymer networks when hydrogel discs are immersed in water. As a result of the diffusion of water, the hydrogel discs expand, causing channels to open, which in turn release the drug-loaded within them (Tabassum et al., 2022). Accordingly, we assessed the most appropriate model based on the regression coefficient value close to 1(Rashidi et al., 2022). A regression coefficient (r) was calculated for different concentrations of gelatin (GAE-7, GAE-8, GAE-9), which indicates that they follow Higuchi release kinetics as their regression coefficient values are closer to 1 than those of the Zero and First order models. Table 5 presents the regression coefficient values of samples (GAE-1, GAE-2, GAE-3) containing various concentrations of the crosslinker EGDMA. Since the regression coefficients of these samples were close to 1, they also followed Higuchi's release kinetics. Additionally, regression coefficient (r) values for all samples (GAE-4, GAE-5, GAE-6) with varying concentrations of AMPS showed that drug release is controlled by diffusion. According to the Korsmeyer-Peppas model, controlled drug delivery devices usually show two types of diffusion: Fickian and non-Fickian. The Fickian diffusion mechanism occurs when the “n” value is 0.45 < n < 0.50 for the cylindrical form of a controlled release device. As all the hydrogel formulations were used in cylindrical form, the hydrogels showed non-Fickian diffusion because the “n” values of all formulations ranged from 0.750 to 0.836.

3.14 Assessment of antioxidant potential

ABTS and DPPH were used as scavenging agents to evaluate the antioxidant activity of hydrogels, as shown in Fig. 12. Four formulations (GAE-6, GAE-9, GAE-1, and GAE-7) displayed higher antioxidant activity than the others. All of these formulations showed higher swelling and release. The percentage of antioxidant activity determined by DPPH ranged from 48.6% to 73.3%, of which GAE-6 had the highest antioxidant activity of 73.3%. The percentage of antioxidant activity measured by ABTS ranged from 42.8% to 71.2%, with GAE-6 having the maximum antioxidant activity of 71.2%. Celastrol, for instance, is one of the primary active ingredients in TWHF, and it is an antioxidant, anti-inflammatory, and anti-cancer compound (Li et al., 2017). Gelatin is a collagen derivative that absorbs water well (Nicholas et al., 2016). Our experiments showed that antioxidant properties were enhanced with gradual increases in gelatin content. Other studies have shown that the use of an adhesive film containing gelatin increases the antioxidant capacity of the film (Liu et al., 2015). We found that TWHF-SD-loaded hydrogels exhibited strong antioxidant properties depending on their proportions.

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Fig. 12

Antioxidant properties of hydrogels as measured against DPPH (A) and ABTS (B).

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3.15 Evaluation of antimicrobial activity

Fig. 13 illustrates the zone of inhibition for Gram-positive (E. coli and S. aureus) and Gram-negative (P. aeruginosa) bacteria. In the negative control and blank hydrogel groups, no zones were observed, whereas clear zones were observed in the positive control hydrogel (32 mm, 33 mm, and 35 mm) and TWHF-SD-loaded hydrogel (19 mm, 24 mm, and 27 mm), respectively, against S. aureus, P. aeruginosa, and E. coli. Accordingly, the percent bacterial inhibition rates of the TWHF-loaded hydrogels against S. aureus, P. aeruginosa, and E. coli were 59.37%, 72.72% and 77.14%, respectively. Antibiotics like cefepime have been proven to be effective against both Gram-positive and Gram-negative bacteria. During the testing of cefepime against selected bacteria strains, a smaller antibacterial zone was observed for Gram-negative bacteria than for Gram-positive bacteria. The structure of the bacteria's cell wall may be responsible for this behaviour. Gram-negative bacteria have an outer membrane, an inner membrane, and a peptidoglycan wall (Kamaci and Kaya 2018). In contrast to Gram-negative bacteria, Gram-positive bacteria lack an outermost membrane, even though their cell walls are thick. Gram-negative bacteria's outer membrane protects them from the outside environment. Consequently, Gram-positive bacteria have a larger inhibitory zone, allowing the tested compound to exert a greater antibacterial effect (Liu et al., 2022, Liu et al., 2023a, 2023b).

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Fig. 13

The zones of inhibition against E. coli, S. aureus, and P. aeruginosa of negative control, positive control (cefepime), blank and TWHF-SD-loaded hydrogels.

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