Design and Performance of a Chemically Crosslinked CMC/PVP Hydrogel for Drug Release

Rice husk was collected from a local rice mill in Bangladesh and used as the raw biomass material. Sodium hydroxide pellets (NaOH, 98.7% of purity), glacial acetic acid (CH₃COOH, 99% of purity), absolute ethanol (C₂H₅OH, 99.5% of purity), and hydrochloric acid (HCl, 37% of purity) were purchased from Merck, Darmstadt, Germany. Sodium chlorite (NaClO₂) and sulfuric acid (H₂SO₄) were used as received. Monochloroacetic acid (CH₂ClCOOH, 99% of purity) was obtained from Qualikems Fine Chem Pvt. Ltd., India. Polyvinylpyrrolidone (C₆H₉NO)_x was supplied by Sisco Research Laboratories Pvt. Ltd., Mumbai, India, while glutaraldehyde (C₅H₈O₂, 25% of purity) was procured from Finar Ltd., Gujarat, India. Merck in Darmstadt, Germany, provided standard buffer solutions with pH values of 4.0, 7.2, and 9.4. All compounds were analytical grade and utilized without additional purification.

Rice Husk (sample) was collected and washed with hot water and then distilled water, respectively. Then the washed sample was dried into hot air oven at 800C until it become dry and grinded finely thereafter. It was mixed with 20% NaOH in a 250 mL beaker and transferred to a Teflon-lined hydrothermal reactor. The reactor was heated at 170°C for 90 minutes, after which the sample was filtered, washed to neutrality, and dried at 80°C for 5 hours to obtain cellulose residue. The dry cellulose was bleached using a 2.2% sodium chlorite solution at a weight ratio of 1:0.5 powder to sodium chlorite. Acetic acid was added to the solution to reduce its pH to 3.7, followed by a 4-hour reaction at 70°C. The pure cellulose was recovered by filtration, washed repeatedly until it was free of acid, and lastly dried to achieve a constant weight [27]. The total process was repeated twice to four times for better removal of lignin. A schematic diagram of cellulose extraction process from rice husk has been shown in scheme-1.

NaOH was added 50 minutes after cellulose was added to a 30% ethanol aqueous solution while being stirred magnetically. At 300ºC, the alkalization (steeping) process was carried out. A 120% (w/v) aqueous monochloroacetic acid solution was added dropwise at 650ºC and agitated for three hours following the alkalization reaction. Carboxymethylation is the term for this process. After neutralizing the solution with HCl, it was filtered. The residue dried until its weight remained constant [28].

A 20 mL solution of 5% (w/v) CMC in water was prepared in a beaker. To this CMC solution, 5 mL of PVP solution (containing 0.25 and 1 g of PVP to achieve CMC: PVP ratios of 80:20 and 50:50) was added drop wise with continuous stirring to ensure a homogeneous mixture. Glycerin was then added to the CMC-PVP blend while stirring to prevent brittleness in the hydrogel. A glutaraldehyde reagent (HCl + GA) was introduced as a crosslinking agent, and the resulting dispersion was stirred at room temperature for two hours to allow proper reaction between CMC and PVP. The thick dispersions obtained were then converted into hydrogel membranes using conventional methods. The hydrogel membranes were left to dry at room temperature for 72 hours. Once dried, the membranes were peeled off and thoroughly washed with distilled water to remove any residual HCl and glutaraldehyde (GA). The resulting hydrogel membranes were labeled as HGEL-1 and HGEL-2. The resulting hydrogel possible reaction mechanism is given in scheme 2.

The Fourier Transform Infrared (FTIR) spectrophotometer-3000 Hyperion Microscope Vertex 80 was used to examine the different functional groups of Na-CMC. FTIR spectra with a resolution of 1 cm⁻¹ were acquired from KBr pressed pellets in the 4000-400 cm⁻¹ range. The morphology (granule surface and shape) of the produced hydrogel was examined using scanning electron microscopy (SEM). Scanning electron microscopy (SEM) was used to examine the morphology of produced cellulose (Model: ZEISS EVO 18).

Hydrogel samples after extraction were dried to a constant weight and immersed in distilled water at room temperature. At first, weight of swelled gel was taken 1, 3, 7, 12 and 15 hours and it was continued after 24, 48 and 72 hours. After completion of absorbing period, the hydrogels were taken out and adhering water was removed using tissue paper. It was then weighed. The water absorption (Wa) can be calculated as g H₂O/g dry copolymer using the following equation:

Where, m2 is the weight of sample with absorbed water and m1 is the weight of dried sample.

The dried hydrogel membranes (HGEL-1 and HGEL-2) were immersed directly in buffer solutions of pH 1.2, 5.4, 7.4 and 9.4 at room temperature for 72 h. The equilibrium percentage of swelling (% swelling) of the product was calculated as

Where W_S is the weight of the product after hydration for 72 h and W_D is the initial weight of dried hydrogel membrane [29].

The hydrogel sample was dried to a constant weight. The gel content in the dried sample was estimated by measuring its insoluble part in dried sample after immersion in deionized water for 48h at room temperature [30].

The gel fraction was calculated as follows,

Where, W_i is the initial weight of dry gel and W_d is the weight of dried insoluble part of sample after extraction with water.

Studies on the hydrogel samples' degradation were carried out in sandy and clay soils. Samples were taken out on a regular basis, cleaned with distilled water, and dried until their weight remained consistent. The following formula can be used to determine the hydrogel's biodegradability:

Salicylic acid (SA) was used as a model drug to investigate drug loading in the crosslinked CMC/PVP hydrogel membrane. Diffusion was used to add SA to the hydrogel membrane. The SA solution in acetone (1 g SA in 10 ml acetone) was used to submerge the CMC/PVP hydrogel membrane for five hours. To remove the medication that had adhered to the surface of the membrane, distilled water was used to wash the drug-loaded hydrogel membrane. Drug concentrations were evaluated both before and after the hydrogel membrane was submerged in the staining solution [31].

A magnetic stirrer was used to mechanically agitate the drug-loaded CMC/PVP hydrogel membrane (HGEL-2) in a beaker filled with a 100 ml pH 7.4 solution. Five milliliters of releasing medium were collected in the glass vial and replaced with five milliliters of stock solution at equal intervals of twenty minutes. Using the same technique, the drug release investigation was also conducted at SGF (stimulated gastric fluid pH 1.2) and distilled water. Samples were then subjected to spectrophotometric analysis using a UV-Vis spectrophotometer at a wavelength of 294 nm [31].

Antimicrobial activity of the compounds was measured by observing the growth response of Staphylococcus aureus (a gram positive model) and Escherichia coli (a gram negative model). The susceptibility of such growth rate of microorganism was measured in vitro by disc diffusion method [32]. The steps were conducted in a laminar air flow cabinet under aseptic conditions. Freshly developed bacterial cultures were used to create nutrient agar plates, which were then left to solidify. Kanamycin (30 µg/disc) was employed as the reference antibiotic, and sterile (BBL, U.S.A.) filter paper discs (5 mm diameter) impregnated with the test samples were placed on the inoculated agar plates. The plates were incubated for 12–18 hours at 37 °C after being held at 4 °C for diffusion. The diameter of the zones of inhibition (mm) was used to calculate the antimicrobial activity.

Figure 1 shows the FT-IR peaks for CMC-Na, HGEL-1, and HGEL-2. The FT-IR of CMC-Na reveals a peak at 3451 cm⁻¹, which illustrates the stretching of the –OH group in carboxylic acid. The signal at 2937 cm⁻¹, on the other hand, demonstrated C-H stretching of alkane. The peak for C=O stretching vibrations caused by amide (I) was at 1641 cm⁻¹. The peaks at 1423 and 1329 cm⁻¹ could be due to the bending modes of CH₂ and CH₃ in alkanes, respectively. The peak at 1142 cm⁻¹ was thought to be caused by C-N stretching vibrations because it had aromatic amine groups. The signal at 1027 cm⁻¹ demonstrated that carboxylic acids were stretching C-O. The signal at 535 cm⁻¹ showed that alkynes C-H bending was present. The FTIR spectra of produced CMC/PVP blended hydrogel membranes (HGEL-1, HGEL-2) exhibit similar peaks between 1650–1660 cm⁻¹, corresponding to amide (I). The creation of hydrogen bonds decreased the strength of the carbonyl bond in PVP in all of the hydrogel membranes and moved the carbonyl stretching to a lower frequency. The FTIR spectra of CMC/PVP hydrogel membranes show that when PVP is mixed with CMC, the –OH stretching vibration peak of CMC at 3451 cm⁻¹ moves to lower frequencies of 3430 and 3399 cm⁻¹. The presence of hydrogen bonding in the hydrogel membranes can explain why the –OH groups' frequency has gone down. The peaks between 2,930 and 2,955 cm⁻¹showed that there was a peak for the –CH stretching vibration. The peaks between 1740 and 1760 cm⁻¹ are caused by the carboxymethyl groups (–CH₂OCH₂COOH) in the hydrogel membranes. The peaks at 1030 and 1042 cm⁻¹ reveal that the hydrogel membranes have a stretching vibration peak for –CN. The peaks suggest that the hydrogel was made by successfully combining CMC and PVP.

The effect of amount of crosslinker on swelling of the hydrogel was studied varying amount of crosslinker (5-14%) in Figure 2. The maximum swelling was obtained at 10% crosslinker with respect to CMC. Above or below this amount of crosslinker, the swelling percent of hydrogel was decreased. Below this amount proper reaction cannot be formed and excess crosslinker can cause strong bond formation in the hydrogel which cause less swelling of hydrogel. From this, hydrogels are synthesized with 10% crosslinker.

Figure 3 shows how hydrogels in water swell over time. All hydrogel reacted with water in different ways. Over time, the hydrogels showed an increase in swelling. Because more PVP may result in more hydrogen bonding between CMC and PVP, an increase in PVP ratio in membranes raises the swelling ratio of hydrogel. A 50:50 ratio of CMC to PVP can result in the greatest swelling. Because hydrogel has certain hydrophilic groups, it absorbs water. Water was absorbed by it until those groups were saturated. By forming hydrogen bonds with the molecules, it also took up water.

The crosslinked CMC/PVP hydrogel membranes (HGEL-1, HGEL-2) were immersed in buffer solutions with pH values of 1.2, 4, 7.2, and 9.4 to observe their swelling behavior. Figure 4 illustrates the swelling patterns of these hydrogel membranes. The results show that swelling increased with pH for all CMC/PVP hydrogel compositions (HGEL-1, HGEL-2). The maximum swelling degree was observed at pH 9.4 (513%, 1022%), in contrast to the swelling at pH 1.2. Since the apparent pKa value of CMC is 4.756, at pH values lower than this, the carboxymethyl groups (–CH2COOH) of CMC collapse, leading to a lower swelling degree. Additionally, hydrogen bonding occurs between the hydroxyl group (–OH) of CMC and the carbonyl group (–C=O) of PVP, which further reduces swelling at these lower pH values. However, at higher pH values (above the pKa value), the hydrogel's swelling increases due to the dissociation of the hydrogen bonds between CMC's hydroxyl group and PVP's carbonyl group. Similar findings have been reported in studies on the swelling behavior of crosslinked PVA/PVP and PVP/AAc hydrogels in different pH buffer solutions [33]. At the basic pH of 9.4, much higher than CMC's pKa, the dissociation of hydrogen bonds and the ionization of –COOH groups in CMC contribute to an increase in the swelling rate. The hydrogel films demonstrated pH-responsive swelling, showing sensitivity to small pH changes, which could lead to alterations in the hydrogel's properties. Among the prepared hydrogel formulations, HGEL-2 demonstrated superior swelling performance in water as well as enhanced pH-responsive swelling behavior across the investigated pH range. Owing to its higher swelling ratio and sensitivity to pH variations, HGEL-2 was considered the optimized formulation and was chosen for subsequent studies.

The gel fraction of hydrogel is found approximately 91% by calculating the value from table 1.

The fabricated hydrogel (HGEL-2) is mainly composed of CMC. The monomers of CMC are linked via glycosidic linkages which can be easily broken by various enzymes resulting in small polysaccharide chains. These chains are further broken down. Figure 5, it can be seen that, biodegradability of the hydrogel is increased with increasing time. From the above figure, it can also be seen that the minimum biodegradability of hydrogel is 7.25% at the time 3 days and the maximum biodegradability of hydrogel is 72.65% at 15 days. So, we can say that hydrogel is a biodegradable polymer.

Scanning electron microscopy (SEM) offers several advantages for morphological analysis and has been widely used in fields such as metallurgy, natural and modified fibers, polymers, biomaterials, and composites [34]. Figures 6(a) and 6(b) show the surface structures of the synthesized CMC and the hydrogel derived from CMC, respectively, at 5000x magnification. As depicted in Figure 6, hydrogels with larger pore sizes exhibit denser network structures. It can be concluded that hydrogels with larger pores tend to have lower density due to the greater void volume fraction. Additionally, finer pores are visible in the hydrogel. The smooth, uniform appearance of the films indicates a compact structure with a clear porous surface. The analysis suggests that CMC, PVP, and their blends were chemically crosslinked with GA. The CMC surface appears rough, granular, and uniform, while the hydrogel SEM images reveal a greater number of pores and a more textured surface. These larger pores reduce diffusion resistance and enhance mass transfer, thanks to the increased internal surface area. This structure also provides a higher drug-delivery capacity, as confirmed by SEM analysis, which shows a significant surface area.

By measuring the inhibition zones that developed surrounding the hydrogel, the antibacterial activity was evaluated. Figure 7 displays the hydrogels' (HGEL-1, HGEL-2) antibacterial activity, and Table 2 contains the inhibition zone sizes (d in mm). Using the disc diffusion method and air as the negative control, the antibacterial effectiveness of hydrogels against Escherichia coli and Staphylococcus aureus was investigated.

The hydrogels possibly broke up the cell surface and made the outermost parts of the cells uneven. This stopped microbes from growing near the hydrogel samples (Figure 7). When bacterial cells came in contact with CMC, the -COOH group lost an H+ ion and was changed into a -COO− ion. The bacteria's pH was affected by the H+ ions, which split the cell wall. Also, the carboxylate ions disrupted up the work that was going on in the cells by connecting to things that were positively charged in the bacterial cells [35]. This behavior suggests that these hydrogels could enhance the antimicrobial effectiveness of antibiotics, making them suitable for use in wound healing applications.

Water-soluble drugs that are trapped in hydrogels don't come out until water gets into the polymer networks, which makes the drug swell and dissolve. After this, the drug moves through the water channels and reaches the surface of the device [36]. In contrast to pH 1.2, where a total of 51% of the drug was released in 20 minutes, a total of 67% of the drug was released in 20 minutes. The SA release profile of the drug-loaded CMC/PVP hydrogel membrane (HGEL-2) in various release media, including pH 1.2, pH 7.4, and pure water, as shown in Figure 8. 67% of the drugs were released in 20 minutes, according to the hydrogel membrane's percent release profile at pH 7.4. At pH 1.2, only 51% of the drugs were released in the same amount of time. The release pattern shows that after three hours, 92% of the medicine was released at a pH of 7.4, and then 82% of the medicine was released at a pH of 1.2. Based on the results, we can guess that the pH 7.4 medium released more SA than the pH 1.2 medium and distilled water. You can connect this event to how the CMC/PVP hydrogel membranes swell at different pH levels (Fig. 8), where the swelling got bigger when the medium's pH went from mostly acidic to slightly basic. The carboxymethyl group in the CMC/PVP complex becomes ionized when the pH is high. Because of this, the –COO- groups push against each other, which makes the hydrogels that have already formed swell more and release more SA.