Modified Bacterial Cellulose-Based Composite Profile for Drug Release of Tetracycline Hydrochloride

Abstract


INTRODUCTION
Drug release is the process of transferring drug solute from a material medium to the release medium 1 .To ensure effective drug releases, factors such as drug loading ratio and controlled release rate are crucial 2 .The main challenge lies in selecting a safe, non-toxic, and eco-friendly material that can maintain good biological activity.Controlled drug-release systems can use various materials, including polymers, where the drug-release rate depends on the matrix's structure and properties 3 .The release of drugs with polymer-based can be achieved by forming a composite.Composite is a combination of two or more materials that exhibit desirable properties due to the combination of constituent materials used.
Natural polymers like cellulose, starch, and glycogen have been researched for their potential in drug release applications 4 .Studies have shown cellulosebased materials, especially bacterial cellulose (BC), have several benefits in biomedical fields, including drug release 5 .Bacterial cellulose is a biodegradable natural polymer with high mechanical strength, biocompatibility, and non-toxicity.It has a high-water content (98-99%) and can be sterilized without affecting its properties.However, BC lacks antibacterial activity, which limits its use in biomedical applications.Therefore, in-situ or ex-situ modification of BC-based composites is necessary 6 .BC-based composites can be created by filling the porous BC matrix with particle solutions or suspensions through physical absorption.
BC-based composites have demonstrated effective antibacterial activity, which can be further enhanced by incorporating inorganic and organic antibacterial agents and antibiotics 6 .Research has shown that BC/G composites, which combine BC with graphite, exhibit high biocompatibility and improved mechanical properties while also providing mechanical inhibitory properties against bacterial growth by damaging bacterial cell membranes when exposed to graphite sheets 7,8 .However, the non-homogeneous distribution of graphite within the composite matrix and weak interfacial bonding between graphite and the matrix are crucial challenges to obtaining such functional composites for the biomedical field 9 .Furthermore, BC/PVA composites, created by combining BC with PVA, have been synthesized and found to have small molecular permeability, low interfacial tension, and the ability to release antibiotics in a controlled manner 10 .PVA also enhances the adhesion of BC and improves the interaction between the matrix and filler, making the filler distribution in the polymer matrix homogeneous and strengthening the mechanical effect of inhibiting bacterial growth 11,12 .However, PVA has weak mechanical properties 13 .
It has been found that the antibacterial properties of BC-based composites can be enhanced by incorporating antibiotics.Tetracycline hydrochloride (TCH) is an antibiotic that can target gram-negative and gram-positive bacteria and possesses a controlled drug release profile 14 .
Considering this, the novelty of this research lies in modifying BC-based composites with additional G and PVA fillers.The antibiotic TCH incorporated into the composite determines the drug release profile and kinetics.The addition of fillers graphite and PVA is expected to be a promising combination to obtain materials suitable for biomedical applications.Since drug release depends on many factors, this paper formulates a good drug release profile of TCH in BC/G/PVA composites using mathematical models including zero-order, first-order, Higuchi, Hixon-Crowell, and Korsmeyer-Peppas 15 .Antibacterial activity testing was conducted against S. aureus to observe its antibacterial performance when applied in the biomedical field.

Purification of Bacterial Cellulose (BC)
Purification was achieved by repeatedly washing BC with hot water and cooling it.The resulting material was soaked in 0.5 M NaOH for 24 hours and rinsed with distilled water until the pH reached 7 16 .

Composite Fabrication
Based on a previous method with modifications, graphite (0.5% w/v) was calcined (1000 ˚C, 5 min) and then homogenized using ultrasonication for 7 hours with the addition of CTAB (0.3% w/v) as a surfactant 7 .The BC hydrogel was added to the graphite solution, which had a known moisture content and weight, and homogenized by ultrasonication for 6 hours.Next, the composite with reduced water content was reacted in a PVA solution (0.03 wt%), to which a K2S2O8 initiator (0.04 wt%) was added and homogenized by ultrasonication at 55 ˚C for 3 hours.The selection of potassium persulfate as the initiator is because of its solubility in water and aims to trigger polymerization.The monomer will react with the initiator to form free radicals in the solution system, followed by the reaction between monomers to form homopolymers 17 .Finally, the obtained composite was dried for two days at 25 ˚C 12 .The illustration for fabrication of composite is presented in Figure 1.

Characterization SEM-EDS Analysis
The JEOL SEM-EDS instrument was utilized to analyze the morphology and atomic composition of the composite at the integrated laboratory of the State Islamic University of Mataram, Indonesia.Operating condition EDS was landing at an acceleration voltage of 15 kV, working distance of 12.7 mm, magnification ×2,000, and vacuum mode high.

Fourier Transform Infra-Red (FTIR) Analysis
The functional groups of the composite structure were analyzed using FTIR (Perkin-Elmer) at the integrated laboratory of the State Islamic University of Mataram, Indonesia.Sample configured with Attenuated Total Reflection FTIR (ATR-FTIR) at room temperature and carried out at wave numbers 4000 to 500 cm -1 with a scanning resolution of 2 cm -1 .

Physical and Mechanical Characterization
Physical characterization included thickness and porosity tests.Thickness measurements were taken at ten different points in each sample using a digital screw micrometer (T&E CR1632) and calculated with equation 1 18 .ṫ = t 1 +t 2 +…t n n (1) The value of t represents the thickness (in 10 -3 cm), n represents the number of data, and ṫ represents the average thickness (in 10 -3 cm).Porosity was measured by immersing the sample in 80% v/v n-butanol and then calculated using equation 2 19 .
The value of ɸ is porosity (%), Mk is the dry mass of the sample (g), Mb is the wet mass of the sample (g), ρB is the density of n-butanol (g/cm³), Vk is the dry volume of the sample (cm³).Mechanical characterization includes tensile strength and young modulus.A tensile strength test was conducted using a tension tool (RTG-1310).The tensile strength can be calculated based on equation 3 20 .
The value σt is the tensile strength (MPa), Ft is the maximum stress (N), and At is the cross-sectional area of the composite subjected to stress (mm 2 ).

Swelling Rate
The swelling ratio was evaluated by immersing the weighed and dried samples at room temperature until reached equilibrium and calculate using equation 4 20 .
Ws is the wet sample weight, Wd is the dry sample weight, and WAC is the swelling ratio.

Drug Release Kinetics
The assay was carried out in pH 6.86 medium (4 hours) at 30-minute intervals and analyzed using a UV-Vis spectrophotometer (Thermofisher) at the maximum wavelength of TCH.The data obtained were fitted to the kinetic model using equations 5, 6, 7, 8, and 9, representing zero-order, first-order, Higuchi, Hixson-Crowell, and Korsmeyer-Peppas, respectively 15 .
The Q value is the concentration of the drug released, k is the drug release rate constant, t is the drug release time, Mt/M∞ is the fractional release of the drug, and n is the diffusion exponent.

Antibacterial Activity
The study tested the effectiveness of a composite material in killing S. aureus bacteria using the disc diffusion method.The Advanced Biology Laboratory at FMIPA University of Mataram provided the bacterial strains.The composite was applied onto the media containing the bacterial strains and incubated for 24 hours at 37 ˚C.The diameter of the zone where the composite inhibited the growth of S. aureus was then measured.

Morphology and Chemical Analysis of Composite Film
According to Figure 2, the morphology of pure BC is smoother and flatter than that of BC/G/PVA.The darker color in BC/G/PVA indicates the presence of G and PVA absorbed in BC.Similar research was experienced during the synthesis of BC-based composite with the addition the chitosan, resulting in a grey coloration.This is caused by the presence of chitosan during the composite synthesis process 21 .Graphite is characterized by flat flakes with sharp corners, while PVA appears evenly dispersed in the composite 22,23 .The interactions between G and PVA fillers in the BC matrix are attributed to hydrophobic and electrostatic interactions with CTAB as a surfactant.CTAB is a cationic surfactant with an ammonium (N + ) head group and a hydrophobic alkyl chain tail composed of cetyl groups 7 .Hydrophobic interactions occur between the alkyl tails of CTAB and G.In contrast, electrostatic interactions occur between hydroxy groups on BC and PVA with quaternary ammonium groups (N + ) on CTAB, which are hydrophilic 24 .The presence of CTAB can reduce the hydrophobic nature of G, allowing it to spread in the BC matrix.
According to the EDS test results shown in Figure 3, the composition of the BC/G/PVA composite is the same as that of pure BC but with different masses and atomic compositions.The increase in the number of C atoms in BC/G/PVA is due to the addition of filler G and PVA, which are absorbed into the cellulose fibers during ultrasonication.A similar effect was observed in a previous study when researchers added G to the BC matrix 7 .The addition of G and PVA can reduce the composition of O atoms, which is reflected in the molecular formula of each component: BC (C6H10O5), G (consisting mainly of C atoms), and PVA (C2H4O).Comparing the elements in each component reveals that the number of C atoms is significantly higher than that of O atoms.Similar research was experienced during the synthesis of BC/PVA and BC/PVA/TiO2, resulting in the differences in composition of O atoms which were not much different, only 0.1% before and after the addition of TiO2 23 .

Identification of Functional Groups of Composite Film
The FTIR spectra demonstrate that all BC, G, and PVA characteristic bands overlap.Although the FTIR spectra in Figure 4 do not show any addition of new functional groups, there is a shift towards lower wave numbers in the strain vibration peak and a change in the intensity of the C-O-C absorption band in the BC/G/PVA composite.This shift and change in intensity are due to the interaction between each composite component, shown in Figure 2. The results obtained indicate that the interactions that occur are only physical interactions 20,25 .
The absorption bands at 3342 and 3338 cm -1 suggest the presence of O-H stretching, supported by the absorption bands at 1324 and 1322 cm -1, indicating the presence of C-O.These bands are also characteristic of BC and acetyl groups in PVA 20,26,27

Physical and Mechanical Properties
Table 1 shows that the thickness of the composite increased with the addition of G and PVA fillers.Filler added as a modifying agent has an impact on the composite thickness.Fillers increase the composite density, which can fill the cavities in the BC fiber.However, as the filler increases, the porosity of the composite decreases 29 .These results were obtained from Tanpichai et al. ( 2019) research, where cellulose nanofibrils as fillers increased the composite thickness and reduced its porosity 30 .The PVA has a biocompatible ability to form films, which can be used as a composite binder to minimize pores and increase composite density 11 .
It was discovered that the tensile strength of the composite decreased as the filler increased.This opposes previous research that suggested adding PVA to the cellulose matrix would increase the tensile strength value of the composite 28 .The decrease in tensile strength value is due to an increase in filler load caused by a reduction in matrix cross-section and an increase in stress concentration 29 .Adding G to the composite can similarly decrease the tensile strength value by adding

Swelling Rate
The results of the swelling test, shown in Figure 5A, demonstrate that adding filler to a composite reduces its swelling ratio.This is due to the low porosity of the composite, which makes it difficult for water to fill the cavities, thus decreasing the swelling ratio 29 .The size of the swelling value is an essential parameter for drug release 21 .The composite reached its maximum swelling state at 50 minutes.It began to experience a decrease in the swelling ratio at 60 minutes due to environmental exposure or mechanical stress, which can cause degradation of the composite.Degradation can occur due to breaking glycosidic bonds in BC during hydrolysis 12,31 .This is characterized by dissolved graphite and PVA particles, leaving parts of BC uncoated with fillers, leading to degradation.However, this degradation can be an advantage in the biomedical field 32 , such as drug release.An illustration of composite degradation is presented in Figure 5B.
Figure 6 presents the swelling ratio of a composite material at different pH levels.The results show that the composite is sensitive to pH changes, with the highest swelling ratio occurring at pH 7.4 and the lowest at pH 1.2.The hydrophilic nature of PVA is affected by ionization caused by acidic or alkaline pH levels, as PVA has a hydroxyl group (-OH) that can be ionized.PVA is neutral at a pH of 7.4, making it more hydrophilic.The swelling ratio can be used to determine the suitability of drug release in specific organs.The maximum PVA filler composite with dexamethasone drug was released at pH 6.8-7.4,which is compatible with intestinal fluids 31 .

Drug Release Profile of Composite
According to Figure 7, the different drug release rates in the composites during the first and second stages are due to the formation of drug-polymer structural units of various sizes 15 .The porosity of the composite can have an impact on the adsorption and release of TCH drugs.The release of TCH drugs is also influenced by the competition between the affinity of TCH for the adsorbent surface and its solubility in the release medium 33 .This mathematical drug release model has different regression coefficient (R 2 ) values in the release medium of pH 6.86, shown in Table 2.The R 2 value is the most reliable way to describe the drug release profile of a composite 12 .
Table 2 shows BC has drug release in the first and second stages following a zero-order kinetics model.The R 2 values for the two stages are 0.9609 and 1, respectively.Zero-order drug release means that the release does not depend on the concentration of the drug   34 .The high porosity of BC allows the entry of fillers and medicines, which can affect the release kinetics model of these substances 6 .Drug release of TCH on BC/G/PVA composite followed the Korsmeyer-Peppas kinetics model in the first stage (30 to 120 minutes) and the Hixson-Crowell model in the second stage (150 to 240 minutes).The R 2 values for the two stages are 0.8925 and 0.9039, respectively.The Hixson-Crowell model indicates that the mechanism of drug release from the composite is influenced by its surface area, and the dissolution rate of the drug particles affects the drug release.The addition of fillers G and PVA caused a compact composite structure with small pore sizes.So that facilitating slow and controlled drug release.The biomedical effectiveness of the Hixon-Crowell kinetic model enables to absorb and release of drugs for application in transdermal patches 21 .
Furthermore, drug release at the first stage plotted with the Korsmeyer-Peppas model tends to experience a diffusion process, with a linear regression equation of y = 0.2189x + 1.3406.The diffusion exponent (n) value is 0.2189, indicating that the diffusion mechanism of the composite includes Quasi-Fickian diffusion because n is less than 0.5 35 .Quasi-Fickian diffusion, like Fickian diffusion, describes drug release depending on polymer relaxation and material degradation 36 .

Antibacterial Activity of Composite
The antibacterial properties of a composite material can be measured by determining the zone of bacterial inhibition against the composite.Figure 8 shows that BC and BC/G/PVA composites have different inhibition zones against S. aureus.Adding filler G and PVA can enhance the antibacterial activity of BC-based composites.Graphite's sharp edges can break the bacterial membrane, providing mechanical inhibition.Part of the composite will interact with the bacterial membrane, consisting of lipopolysaccharides and phospholipids, thus penetrating several functional groups in bacteria to inhibit bacterial growth 12 .Adding G and PVA can also improve the matrix's interaction and Copyright©2024, Published By Jurnal Kimia Valensi dispersion of antibacterial agents, allowing for better contact between the antibacterial agent and bacteria.This results in increased antibacterial effectiveness of the composite 37 .
Adding antibiotics can help to identify signs of bacterial resistance to composite 21 .Figure 9 shows that adding antibiotics to BC and BC/G/PVA led to higher bacteria inhibition than before the addition and the TCHpositive control.BC-based composites with G and PVA fillers, as well as antibiotics, have the potential to be antibacterial membranes with an average inhibition zone of 15 mm.

CONCLUSION
The BC/G/PVA composite has been successfully synthesized, and the drug release of TCH on the composite followed the Korsmeyer-Peppas kinetics model in the first stage and Hixson-Crowell in the second stage.Adding filler G and PVA increased the composite's cohesiveness, making it suitable for use as a drug-release formulation material.Furthermore, adding fillers and antibiotics increased the composite's antibacterial activity against S. aureus.The composite has shown an average inhibition zone of 15 mm, indicating that it has the potential to be used in the field of biomedicine.

Figure 1 .
Figure 1.Schematic representation of a method for fabrication of BC/G/PVA composite

Figure 2 .Figure 3 .
Figure 2. Illustration of graphite and PVA filler in BC matrix . The absorption bands at 2914 and 2900 cm -1 confirm the presence of C-H stretching, reinforced by C-H bending at 1442 and 1437 cm -1 .The C-O-C absorption band, which indicates the antisymmetric stretching of 1,4-β-D-glucoside in BC, has a broader intensity and shifts to a lower wave number in BC/G/PVA, possibly due to C-O-H vibrations.This effect could be attributed to the addition of PVA, as supported by research by Vo et al. (2019), during FTIR characterization of PVA, C-O-H vibrations were observed around the wave number 1090 cm -1 28 .

Figure 4 .
Figure 4. FTIR spectra of BC and BC/G/PVA

Figure 5 .
Figure 5. (A) Swelling graph with time variation; (B) Schematic representation of the swelling process BC/G/PVA composite

Table 1 .
Physical and mechanical properties of composite