Harnessing the potential of antibiotic functionalized coir nanocellulose in sustainable wound management

Materials

Raw coir fiber was sourced from the Central Coir Research Institute, Coir Board, Kerala, India. All reagents used namely sodium hydroxide, sodium chlorite, oxalic acid, copper (II) sulfate pentahydrate and ethylenediamine obtained from Merck (Mumbai. India) were of analytical grade. Ciprofloxacin (≥98%, HPLC) and gentamicin solution (10 mg/ml) procured from Sigma Aldrich (India) were used for drug loading and antimicrobial studies. Staphylococcus aureus (MTCC#96), Escherichia coli (MTCC#68), Bacillus cereus (MTCC#1307), and Pseudomonas aeruginosa (MTCC#1688) were purchased from MTCC, Chandigarh. Nutrient agar, nutrient broth, Luria Bertani Agar and MacConkey Agar procured from Hi-Media (Mumbai, India) were used for bacterial studies. Standard antibacterial discs of ciprofloxacin (CIP 10 mcg, Hi-Media) and gentamicin (GEN 10 mcg, Hi-Media) were used as positive controls for agar disc-diffusion studies.

Methods

Preparation of coir-nanocellulose (CNC)

Coir Nanocellulose (CNC) was extracted from coir fibers according to a previous method reported by Abraham et al., with slight modifications. ²⁸ Briefly, coir fiber was soaked in 2% NaOH overnight, then subjected to steam explosion by autoclaving at 20 lbs and 121^o C for 1 h, which was further treated with 2-3% sodium chlorite (NaClO₂) and then exposed to the second step of steam explosion for bleaching. This bleaching process was repeated until the material got completely lightened and the resultant sample was acid hydrolyzed with 5% oxalic acid during the last phase of steam explosion. The acid-treated sample was then subjected to ultrasonication, followed by rinsing with distilled water until the pH reached 7.0. The washed neutral sample was then air-dried, and ground in a mixer grinder to obtain nanocellulose fibers.

P e r c e n t a g e Y i e l d = F i n a l w e i g h t ( g m ) I n i t i a l w e i g h t ( g m ) × 100

X-ray diffraction (XRD)

RIGAKU MINIFLEX 600 Benchtop Powder X-Ray Diffractometer (XRD) instrument with Cu tube (as the X-ray source, λ = 1.54 A^o) and a Hybrid pixel array detector (HPAD), was used to get X-ray diffraction profiles of the CNC, C-CNC and G-CNC using CuK radiation at operating voltage and current of 30 kV and 20 mA, respectively and the diffraction strengths were measured from 3° to 80° (2θ range). The Crystallinity Index values (CrI) has been calculated from the XRD Spectrum using the equation:

C r I = ( I 002 − I a m ) I 002 × 100

Where, I₀₀₂ is the high intensity peak around 22.8^o and I_am is the peak intensity around 18^o

Fluid absorption studies

The fluid absorption characteristics of CNC were measured according to a previously reported method. ³⁰ The dressing components were cut into 1 cm² squares and weighed, denoted as the dry weight (W_d). Phosphate Buffer Saline (pH 7.4) was used for conducting fluid absorption studies, in which, the sample was immersed for different time periods (0.5 h, 2 h, 4 h, 8 h, 12 h and 24 h) while being covered to stop evaporation. After the specific time period, samples were taken out from PBS and allowed to drip for 30 s, and the wet weight (W_w) was measured.

F l u i d A b s o r p t i o n ( g / g ) = W w − W d W d

Preparation of antibiotic loaded CNC and determination of percentage drug loading

A stock solution of ciprofloxacin, was prepared by dissolving 150 mg ciprofloxacin hydrochloride in 150 mL distilled water to obtain a final concentration of 1 mg/mL. Similarly, 1 mg/mL stock solution of gentamicin was prepared by dissolving 15 mL gentamicin sulfate solution (10 mg/mL) in 150 mL distilled water. To each of these stock solutions, 300 mg CNC, was introduced, maintaining a sample-to-drug ratio of 2:1, and the mixtures were stirred at 60 rpm for 48 h. The products obtained were washed with distilled water 2-3 times in order to remove any unbound drug, and the spent liquid was analyzed using UV-Visible spectrophotometry, to calculate the drug loading efficiency. ³¹ To obtain C-CNC and G-CNC, the samples were dried overnight in a hot air oven at 70°C.

The ratio of the difference between the total amount of drug used to prepare the sample and the amount of drug contained in the aqueous medium to the mass of the polymer was used to determine the percentage of drug loaded in the nanocellulose sample. ³²

P e r c e n t a g e d r u g l o a d i n g = M a s s o f t o t a l d r u g − M a s s o f f r e e d r u g M a s s o f t o t a l p o l y m e r × 100

The encapsulation efficiency was determined by calculating ratio of the difference between the total drug added and the free drug, to the total drug added.

P e r c e n t a g e D r u g E n t r a p m e n t = M a s s o f t o t a l d r u g − M a s s o f f r e e d r u g M a s s o f t o t a l d r u g × 100

Preparation of coir-nanocellulose (CNC)

Coir fibers were initially treated with sodium hydroxide to make the fibers more susceptible to bleaching and acid hydrolysis. Sodium chlorite aided in the elimination of residual lignin in steam-exploded, alkali-treated fiber. Bleaching is a critical step for removing lignin and other non-cellulosic components from lignocellulosic biomass. Bleaching parameters such as the time and number of steps help in the modulation of the relative lignin content in the fibers. ³⁸ Because acid hydrolysis was performed in a heterogeneous system, the yield was affected by a variety of factors such as pre-treatment, acid concentration and hydrolysis time. The intention of the pre-treatment method was to allow the hydrolysate to permeate the cellulosic fiber more easily. When oxalic acid is employed as a hydrolyzing agent, it combines with the cellulose surface hydroxyl groups, and pure cellulose is formed when oxalic acid reacts with the sodium derivative of the fiber. Under acid hydrolysis, the amorphous portions of cellulose present in coir fibers behave as structural defects and cause the microfibrils to split into nanofibers. ³⁹ In terms of particle morphology, the use of oxalic acid during hydrolysis appeared to be a superior option compared to strong mineral acids, to avoid extreme conditions that result in total cellulose hydrolysis.

For every 100 g of coir fiber, 37 g of coir-nanocellulose was obtained after chemical treatment and steam explosion. The yield of CNC from coir fiber is influenced by a multitude of factors, including fiber composition, presence of contaminants, pre-treatment methods, process parameters and purification techniques. A yield of 37% highlights the possibility of using coir fibers as an environmentally friendly resource for CNC production. Owing to its unique properties, including high strength, stiffness and cytocompatibility, CNC can be considered as a nanomaterial with a broad range of applications. The ability to obtain a relatively high yield of CNC from coir fiber makes it a promising feedstock for the production of this valuable material. A flow-chart of CNC production is depicted in Figure 1.OPEN IN VIEWER

There have been rigorous studies for extracting nanocellulose from various lignocellulosic biomass such as sugarcane bagasse, corn stover, almond shells and other available substrates. ⁴⁰ In a review by Hu et al., the yield of cellulose from different lignocellulosic biomasses under different processing conditions were considered and it was found that the yield varied from 50% in tea stalk with 65% H₂SO₄ to 32.2% in bleached bagasse pulp with 80% citric acid to 8.2% in sweet potato peels with 72% malic acid. ⁴¹ The yields reported in our study align with the reports from Silva et al., where coconut fibre residue yields around 30% of nanocellulose after delignification and bleaching. ⁴² Some studies on coconut cellulose also reports that the yield of nanocellulose is around 20%, depending on the amount of cellulose in the substrate used, the type and severity of the pretreatment used. ¹²

Chemical composition analysis

The chemical composition of the coir fibers at various stages of processing is summarized in Table 1. Cellulose content varied from 39.32 ± 2.35% in raw coir fiber to 98.82 ± 1.18% in the final CNC (Figure 2). From raw to bleached fiber, the moisture content gradually increased owing to the high concentration of cellulose with three free hydroxyl groups. These observations are consistent with the chemical composition of the coir fibers and the processing steps involved in CNC production. The significant increase in cellulose content from raw coir fibers to final CNC indicates the effectiveness of chemical treatments and steam explosion in removing non-cellulosic components, such as hemicellulose and lignin.

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Table 1.

Chemical composition at different stages of CNC production.

​	Cellulose (%)	Lignin (%)	Moisture content (%)
Raw coir fiber	39.32 ± 2.35	49.24 ± 1.57	9.42 ± 0.23
Alkali treated fiber	50.54 ± 1.91	38.86 ± 1.25	9.66 ± 0.52
Steam exploded fiber	57.46 ± 1.72	30.97 ± 0.92	9.78 ± 0.41
Bleached fiber	88.37 ± 1.45	0.32 ± 0.04	9.81 ± 0.85
Acid hydrolyzed fiber	98.78 ± 1.22	-	10.35 ± 0.36
Coir-nanocellulose	98.82 ± 1.18	-	11.54 ± 0.91

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Figure 2.

Chemical composition and moisture content at different stages of CNC production viz. Raw Coir fiber (RCF), alkali treated fiber (ATF), sodium chlorite treated fiber (SCT), Oxalic acid treated fiber (OAT) and Coir-Nanocellulose (CNC).

When treated with NaOH and subjected to steam explosion, the cellulose proportion increased. Lignin reacts with NaClO₂ to form lignin chloride. ⁴³ The percentage increase in pure cellulose content and decrease in lignin and hemicellulose contents with each processing step was another main observation, which in turn confirmed the effectiveness of the extraction process.

Scanning electron microscopy (SEM)

Scanning electron microscopy images at different stages of processing (Figure 3) evidences the transformation of coir fibers to nanocellulose having an average diameter of 50-100 nm with apparent variations in the surface structures and with individual random fibers presenting approximately 88 nm diameter. The coir fibers initially observed as thick individual strands, were converted to dense intricate networks, meticulously interwoven, forming an inter-connected mesh-like structure with numerous pores, clearly confirming the formation of microfibrils. The increase in the surface area and porosity aids in acquiring the unique properties of CNC, such as fluid absorption, which can also help serve as an appropriate framework for the incorporation of antibiotics.OPEN IN VIEWER

The surface roughness of the raw coir fiber can be attributed to the presence of polyphenolic chemicals, which cause its coarse granular morphology. ⁴⁴ It was found that the fibers got softened after steam explosion using sodium hydroxide. It has been previously demonstrated that majority of the lignin and hemicellulose can be eliminated by mercerization. The depolymerization and defibrillation that occurred during steam explosion, further eliminated the remaining amorphous elements of lignin, hemicellulose, tannin, pectin, etc. from the core of the fiber. ⁴⁵ When steam-exploded fibers were bleached with sodium chlorite, substantial changes in morphology were observed, the product became fibrillated, and the size was reduced to the nanoscale range, which reached a range of 80 ± 10 nm after acid hydrolysis and grinding, as evident from the scanning electron microscopic image. ⁴⁶

Zeta potential measurement

The surface charge and colloidal stability of pristine and drug loaded CNC were determined by zeta potential measurements. CNC exhibited a zeta potential of −37.2 ± 2.9 mV confirming the electrostatic stability of the dispersion, with absolute value higher than 25 mV. The highly negative surface charge is attributed to the presence of surface hydroxyl groups in CNC. ⁴⁷ For C-CNC, a significant reduction in zeta potential was observed following drug incorporation with a value of −26.7 ± 2.3 mV which may be due to the interaction between CNC matrix and ciprofloxacin causing partial neutralization of surface charges. The strong cationic nature of gentamicin further reduced the zeta potential of G-CNC to −15.4 ± 1.8 mV with its strong ionic interactions with CNC. The reduction in zeta potential for C-CNC and G-CNC confirms effective drug loading and surface modification, indicating moderate stability. Thus, the zeta potential measurements reveal that CNC-drug interactions substantially alter the surface characteristics of CNC which in turn modulate drug release and dispersion properties. ⁴⁸

Fourier transform infrared (FT-IR) spectroscopy

FTIR spectra of CNC along with coir fiber at various stages of treatment are shown in Figure 4. The -OH and C–O stretching from the ether groups were responsible for the prominent spectral bands at 3327 and 1024 cm^-1 respectively. C-H stretching of CH₂ groups resulted in a peak in the region of 2900 cm^-1. The vibration at 2850 cm^-1 due to C–H stretching in lignin is absent in the treated samples, confirming the removal of this fraction. ⁴⁹ The peak at 1641 cm^-1 represents adsorbed water (H–O–H deformation), in the extracted cellulose samples, while the minimal spectral intensity ensures the elimination of hemicellulose fractions during processing. The band at 1432 cm^-1 indicates C–H deformation in lignin, as well as deformation of this bond in carbohydrates in the presence of C = O as it moves down from its normal location. Similarly, after chemical and physical modifications, the peak at 1024 cm^-1 became sharper, and this band was associated with CH₂ rocking vibrations in cellulose.OPEN IN VIEWER

Because the treated material was first neutralized with NaOH, the acid-extracted product did not show bound sulfate groups at 1210 cm^-1, but showed absorption peaks at 1160, 1024, and 736 cm^-1, indicating enhanced asymmetric stretching of the ring and vibration of C–O in cellulose. ⁴⁹ In the majority of the spectral bands, the extracted CNC were identical to the previously reported reference cellulose samples, indicating a strong correlation.

FTIR analysis of drug-loaded-CNCs is shown in Figure 5. A distinct characteristic peak at 3301 cm^-1 was identified in the FTIR spectrum of C-CNC and was attributed to the OH stretching vibration. Another band at 2894 cm^-1 corresponds to aromatic C-H stretching and alkenes, and the peak at 1627 cm^-1 corresponds to the -C = O vibration. ⁵⁰ The peaks at 1319 cm^-1 reveal the bending vibration of the O-H group, which suggests the existence of carboxylic acid, while the bands at 1498 cm^-1 are related to C-H stretching. In addition, the C-F group showed a significant absorption peak at 1053 cm^-1 G-CNC ⁵¹ shows peaks at 1630 cm^-1, and 1316 cm^-1 corresponding to amide I and amide III of gentamicin respectively. A new peak at 1050 cm^-1 has emerged in C-CNC and G-CNC which may be attributed to the C-F stretching in ciprofloxacin and C-O stretching in gentamicin. The irregular peaks around 1400 cm^-1 indicates the cross linking of nanocellulose with the antibiotics. ⁵² OPEN IN VIEWER

The N-H bending vibration at 1631 cm^-1 can be attributed to the aromatic amine of gentamicin. ⁵³ Distinct N-H and alkyl C-H stretching vibrations are reflected as bands at 3394 and 2906 cm^-1, respectively. The S-O bending and stretching vibrations were observed at 660 and 1054 cm^-1 respectively, which are related to the sulfur content of gentamicin sulfate, whereas the HSO₄ vibrational band was observed at 1104 cm^-1.54

X-ray diffraction (XRD) analysis

The coir fibers have crystalline and amorphous regions, as indicated by the XRD graph. Alkali treatment removed cementing components such as lignin, hemicellulose, and pectin, causing the percentage crystallinity of the fiber to increase. The cellulose components tend to arrange themselves in an organized manner after lignin was gradually removed by various treatments. The lignin disintegrated during the bleaching process became lignin chloride, leaving the intact cellulose component behind.

In the XRD spectra of CNC, C-CNC, and G-CNC, three characteristic peaks at 2θ = 16.8°, 22.7°, and 34.8° were observed. These diffraction peaks are characteristic of native cellulose (cellulose I) and correspond to the (110), (200) and (004) crystallographic planes, respectively, indicating the presence of a highly crystalline cellulose structure. ⁵⁵ Zimowska et al. had also reported that these peaks are dominant in monoclinic crystal lattice structure with well-ordered cellulose. ⁵⁶ These peaks present in CNC, also exist in C-CNC and G-CNC which suggest that loading antibiotics does not change the crystallinity substantially. However a small increase in peak intensity has been observed after loading CNC with gentamicin, which aligns with the increase in crystallinity index calculated. The crystallinity index values for CNC, C-CNC and G-CNC are 51.85, 51.89 and 57.44 % respectively. The results are also in agreement with the reports by Costa et al., where nanocellulose particles showed a crystallinity index value of 55%. ⁵⁷ (Figure 6).OPEN IN VIEWER

Degree of polymerization (DP)

The intrinsic viscosity [η] of CNC was calculated to be 273 ± 8 mL.g^-1 and Degree of Polymerization (DP) of CNC were computed to be 205 ± 6. The characteristics of derived nanocellulose predominantly depends on the polymeric chain length in addition to the degree of polymerization. The different pretreatment and extraction methods cause isolation of pure and crystalline cellulose by confirming the breaking of bonds between cellulose and non-cellulosic components viz. lignin and hemicellulose. ⁵⁸ The different treatments have removed amorphous regions and retained the crystalline structure as evident from the crystallinity index of the synthesized CNC. These removal of amorphous regions is caused by the breakage of the polymeric chains present in the material, which ultimately causes reduction in the degree of polymerization which in turn increases the porosity, surface area and reactivity of nanocellulose. ⁵⁹

Fluid absorption studies

Although there are various processes for managing fluid via absorption, passive fluid uptake is the most frequently evaluated characteristic and is an important consideration for wound dressings. It regulates wound infection and aids in preventing excessive build-up of exudate at the wound site. ⁶⁰ The absorption measurement obtained using a standard immersion technique revealed that with time, the fluid absorption increased for a period of up to 4 h, and then remained constant, indicating a maximum absorption of 3.08 g/g in 4 h. Thus, the CNC sample absorbed up to 3.08 times its own weight during swelling in phosphate-buffered saline (Figure 7).OPEN IN VIEWER

Fluid absorption studies performed on CNC dressings revealed the efficient absorption of excess fluid, which can help prevent maceration and promote a healthy wound environment, as excessive exudate can create a favorable environment for bacterial growth. By minimizing the fluid buildup, CNC dressings can help reduce the risk of infection. Therefore, a well-managed wound environment can facilitate healing and reduce the risk of complications. Rapid and substantial absorbency and moisture management are major requirements in the development of appropriate dressing for exuding wounds. A fast-wetting quality enables dressing to quickly absorb exudate and prevent maceration of the surrounding skin, which was observed in the case of CNC. Furthermore, high absorbency is preferred for all liquids released by wounds. The frequency of changing the wound dressing can be significantly reduced with high absorbency, thereby minimising the chances of disturbing the wound. ⁶¹

The ability of a dressing material to absorb wound exudate while maintaining a moist environment is one of the most desirable and critical property of a wound dressing material. The abundant surface hydroxyl groups present on CNC might have contributed to the fluid absorption capacity which augments water absorption through hydrogen bonding. In a study conducted by Minsart et al., the swelling capacities of 11 commercially available wound dressings were reported and compared and was found to be ranged in between 1.5 and 23.2 g/g, depending on material type and morphology. ⁶² The obtained value of CNC falls within the clinically relevant range particularly intended for light to moderately exuding wounds. The relatively moderate swelling behaviour of CNC is due to the compact nanofibrillar structure and absence of any surface modifications. In most cases, moderate fluid absorption is preferred as excessive absorption may lead to mechanical disintegration of wound dressings and drying of wound environment.

For fluid absorption studies one-way ANOVA has been performed which revealed a significant effect of immersion time on fluid uptake and obtained a p-value of 0.0154, which indicates that the fluid absorption increased significantly with time, further implying the progressive swelling behaviour of CNC matrix. The initial rapid increase in absorption occurred due to the availability of hydroxyl groups in cellulose, which followed by a saturation of the same, resulted in a subsequent plateau. Thus, the fluid absorption profile exhibited by CNC indicates that it holds suitable moisture absorption capacity maintaining structural stability, making it appropriate for wound healing applications, specifically for light to moderately exuding wounds.

Drug loading studies

Drug loading and entrapment efficiency are essential metrices for drug delivery systems. Drug loading and entrapment studies were performed for ciprofloxacin and gentamicin. The percentage drug loading for ciprofloxacin and gentamicin is 17.68 ± 0.63% and 19.27 ± 0.96% respectively with drug entrapment efficiency as 42.97 ± 1.26% for ciprofloxacin and 47.76 ± 1.93% for gentamicin respectively. In the drug loading and drug entrapment studies, although gentamicin demonstrated slightly better results, both antibiotics were successfully loaded and entrapped within the delivery systems. The variability or uncertainty in the results is indicated by the standard deviations linked to each measurement. According to the work done by Zahra Sobhani et al., ciprofloxacin hydrochloride-loaded chitosan nanoparticles with 1:0.5 mass ratio, the encapsulation efficiency was found to be 23%. ⁶³ Another study indicated that the best encapsulation performance for gentamicin sulfate which is 45%, was achieved using PLGA 50:50 at 2% nominal loading and a buffer at pH 6.0. ⁶⁴

In a recent study by Roy et al., it was demonstrated that ciprofloxacin can be successfully loaded on to nanocellulose owing to the strong electrostatic and hydrogen bonding interactions among the cellulose functional groups and drug molecules and the percentage loading can be enhanced through surface functionalization like dialdehyde modification. ⁶⁵ Similarly, recent research which focused on the development of bacterial nanocellulose incorporating gentamicin-loaded chitosan nanoparticles as a promising multifunctional wound dressing biomaterial revealed a sustained release pattern with strong antibacterial activity and good biocompatibility. ⁶⁶

The interaction between cellulose and drug molecules occurs mainly through hydrogen bonding and surface adsorption and not through true encapsulation in to a porous nanocellulose matrix. The highly crystalline nature of cellulose limits free sites for drug loading and the hydrophilic nature of ciprofloxacin and gentamicin cause partial drug loss during purification and washing steps. Consequently, the loading efficiency got restricted to 17-19% and entrapment efficiency to 42-47%. Recent studies reported that the bonding between ciprofloxacin and unmodified CNC is through weak intermolecular interactions and surface modification can provide additional binding sites to improve ciprofloxacin loading through strong electrostatic interactions. ⁶⁵ Likewise, previous studies report that gentamicin loading on to cellulose carriers happens through adsorption and partial penetration and hence the drug release is mainly governed by a balance between absorbed and adsorbed drug fractions. ⁶⁷ In a study by Gulsu et al., it was reported that oxidised cellulose enhances the loading efficiency of gentamicin by the utilization of reactive functional groups. ⁶⁸ Unmodified CNC systems exhibit moderate loading but deliver sustained drug release over extended period of time, making it beneficial for wound healing applications. The dense hydrogen bonding network in CNC considerably reduces drug diffusion and enhance prolonged antimicrobial activity in spite of moderate loading efficiencies. ⁶⁹ Hence, the drug loading and drug entrapment efficiencies recorded in this study indicates the inherent characteristics of unmodified CNC system.

In vitro drug release studies

The rate of drug release from the nanofiber surface had a significant impact on the effectiveness of the wound dressing material. The cumulative release of ciprofloxacin and gentamicin from CNC under in vitro conditions at various time points during a 72 h period was determined (Figure 8). Drug dissolution occurs when an aqueous medium enters the CNC structure, causing pores between the fibers to allow diffusion of the dissolved drug out of the membrane. ⁷⁰ Both ciprofloxacin and gentamicin loaded CNC samples exhibited burst release of 70.8 ± 3.4% and 64.3 ± 2.9% respectively, within a period of 2 h. The release rate became stable after 10 h which was further maintained at ∼80-85% and in case of both C-CNC and G-CNC, 92 ± 2.6% of the drug got released by 72 h.OPEN IN VIEWER

There observed a significant difference in the release behaviour of C-CNC and G-CNC during the initial period up to 12 h, where the p-value obtained was 0.0167 (1 h), 0.0142 (2 h), 0.0106 (4 h), 0.0084 (6 h), 0.0121 (8 h), and 0.0465 (12 h). This indicates that the physicochemical properties of two antibiotics influenced their diffusion from the CNC matrix. In contrast, no significant difference was observed after 24 h with p-value >0.05, suggesting sustained release equilibrium caused by the diffusion of drug molecules from the internal porous CNC matrix.

The drug release rate plays a crucial role in determining the effectiveness of wound dressings. Previous research has demonstrated that an antibiotic, when delivered rapidly, helps in controlling the infection at the wound site, and when delivered gradually over the course of a couple of days, can regulate wound healing in a better way. ⁷¹ In our study, the initial rapid release might have resulted from the diffusion of drug molecules that were superficially bound to the outer surface of the C-CNC and G-CNC samples. Thus, an initial burst can provide a high concentration of antibiotics at the wound site to combat existing bacteria, whereas sustained release offers long-term protection. Controlled drug release can also help minimize the development of antibiotic resistance. Thus, the combination of burst release and sustained release can be beneficial for wound healing, as it allows for the rapid control of infection and long-term protection. As the envisioned application was for the development of antimicrobial wound dressings, it might be considered suitable for treating wounds and preventing infection at the wound site based on the drug release rate.

Drug release kinetics

The drug release data were fitted in to different models viz. Zero order, First order, Higuchi and Korsmeyer-Peppas for C-CNC (Figure 9(a)) and G-CNC (Figure 9(b)) and the results are consolidated in Table 2. Both C-CNC as well as G-CNC followed a two-stage release profile featuring an initial burst release followed by a sustained release pattern. The initial burst release may be associated with release of drug molecules loosely bound to the surface of nanocellulose. ⁷² The lower correlation coefficient values for Zero order, First Order indicate that the release mechanism does not follow constant-rate or a concentration dependent process. ⁷³ OPEN IN VIEWER

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Table 2.

Drug Release Kinetics Modelling by comparing values of correlation coefficient (R²) and kinetic rate constant (k).

Model	C-CNC k	C-CNC R2	G-CNC k	G-CNC R2
Zero order	0.48021	0.1718	0.57917	0.27389
First order	0.00615	0.53553	0.00822	0.79456
Higuchi	6.2915	0.36881	6.94405	0.4924
Korsmeyer-peppas	0.758	0.99436	0.714	0.98842

The zero order, first order and Higuchi models exhibited low correlation coefficient values for both C-CNC and G-CNC indicating that the drug release do not follow a constant rate or is not based on diffusion alone. Comparing the different release models, the Korsmeyer-Peppas model exhibited highest correlation coefficient having R² values of 0.99 for C-CNC and 0.98 for G-CNC confirming the best fit for the drug release mechanism to be Korsmeyer-Peppas model.

The release exponent (n) calculated from Korsmeyer-Peppas model for both systems were significantly below 0.45 and was approximately 0.11462 for C-CNC and 0.05316 for G-CNC confirming a Fickian diffusion-controlled release mechanism. ⁷⁴ The low n values indicate the presence of a highly restricted dense nanocellulose matrix and strong intermolecular interactions. Recent studies further states that nanocellulose based drug delivery systems permits tunable and sustained drug release making them promising candidate for advanced drug delivery applications. ⁷⁵ Thus, the initial burst release followed by the sustained release highlights the potential of C-CNC and G-CNC for prolonged antibacterial activity in wound healing applications.

Effect of C-CNC and G-CNC on the growth of microorganisms

A series of phases in the development of the bacterial culture that were characterized by differences in growth rate were observed in all four strains of tested bacteria: B. cereus, S. aureus, P. aeruginosa and E. coli (Figure 10(a)–(d)). A normal bacterial growth curve with lag, log, and stationary phases was observed for bacteria alone and with CNC inoculated nutrient broth. In contrast, the growth declined for both C-CNC and G-CNC sample treated cultures, from 0.5 h onwards. This indicates that the C-CNC and G-CNC samples have a growth-inhibiting effect on microbes even during their lag phase, suggesting that multiplication is averted. ⁷⁶ OPEN IN VIEWER

The bacterial growth curves were analyzed using two-way ANOVA and it was observed that compared with untreated bacterial control, both C-CNC as well as G-CNC exhibited significant inhibition of bacterial growth for all the tested strains of microorganisms across all time points with a p-value <0.0001 and also a statistically significant difference was observed between C-CNC and G-CNC with a p-value of 0.0016, indicating drug formulation dependent antimicrobial activity.

Antimicrobial studies

The presence or absence of inhibition zones were used to evaluate the antibacterial activity of all the samples, including PC (CIP-10 mcg disc), PC (GEN-10 mcg disc), NC (CNC), C-CNC (1.0, 0.5, 0.25 mg/mL) and G-CNC (1.0, 0.5, 0.25 mg/mL), against the four tested bacterial strains. Pure CNC used as the negative control, did not show any antimicrobial activity against the tested organisms. In contrast, the incorporation of different concentrations of Ciprofloxacin (1.0, 0.5 and 0.25 mg/mL) (Figure 11) and Gentamicin (1.0, 0.5 and 0.25 mg/mL) (Figure 12) on to the CNC resulted in significant inhibition of growth against the tested strains of E. coli, B. cereus, P. aeruginosa and S. aureus and was found to be proportionate with the concentration of the drug used with the highest ZoI measuring 2.7 ± 0.024 cm for E. coli, 2.3 ± 0.011 cm for S. aureus, 2.6 ± 0.037 cm for B. cereus and 2.8 ± 0.045 cm for P. aeroginosa for C-CNC with a concentration of 1 mg/mL which was comparable with positive control. Similarly, ZoI measuring 2.6 ± 0.042 cm for E. coli, 2.4 ± 0.031 cm for S. aureus, 2.7 ± 0.073 cm for B. cereus, and 2.8 ± 0.054 cm for P. aeruginosa was observed for G-CNC at a concentration of 1 mg/mL.OPEN IN VIEWER

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Figure 12.

Inhibitory effect of G-CNC on Muller-Hinton agar plates at varying concentrations (a-Negative Control, b- Positive control, c- G-CNC 1.0 mg/mL, d- G-CNC 0.5 mg/mL and e- G-CNC 0.25 mg/mL) for (a) P. aeruginosa, (b) E. coli, (c) S. aureus and (d) B. cereus.

Analysis of the data (Figure 13) indicated that both C-CNC and G-CNC samples had significant inhibitory effects against the tested strains of gram-positive and gram-negative bacteria, and the maximum growth reduction was observed at a concentration of 1 mg/mL.^77,78OPEN IN VIEWER

The healing process is frequently slowed down by wound infection caused by bacteria and other microbes, which can also result in potentially fatal consequences. To avoid infection and promote wound healing, the wound must be covered with a suitable dressing material that hinders microbial growth on the wound surface. ⁷⁹ CNC loaded with Ciprofloxacin and Gentamicin exhibited antibacterial activity against the tested bacterial strains, which are common causatives of skin and soft tissue infections, and the activity was proportional to the concentration of the incorporated antibiotic. By inhibiting bacterial growth, antibiotic-loaded CNC can help prevent wound infections and promote healing. The ability to adjust the antibiotic concentration allows for tailoring of the antibacterial activity to specific wound needs.

Cytocompatibility studies

MTT assay performed with L929 cells cultured in conditioned media extracted from CNC, C-CNC and G-CNC evaluated the impact of CNC and antibiotic incorporation on to CNC in altering cellular viability. The results exhibited good cytocompatibility with significant viability and proliferation. Compared to the control group, which received fresh DMEM F12 media, cells exposed to the conditioned media exhibited an approximately 80% increase in cell survival after 48 h as shown in Figure 14, which was further confirmed by the absorbance measurements at 570 nm.OPEN IN VIEWER

The statistical analysis performed using one-way ANOVA revealed that both C-CNC and G-CNC exhibited larger zones of inhibition compared to control with p-value <0.05 and also the inhibition was more significant with higher drug concentrations. This also confirms that the antimicrobial effect has initiated from the loaded antibiotics rather than the nanocellulose carrier.

Several studies have explored the biocompatibility of nanocellulose for various applications. Cellulose nanofibers extracted from lemongrass were found to be nontoxic in the L132 cell line and all other cell lines studied, when exposed to 0-1000 µg/mL. ⁸⁰ In another study, the cytotoxicity of CNC was tested in GM07492 A cells, and it was observed that CNF showed more than 70% viability. ⁸¹ Cell viability and proliferation of L929 cells cultured in CNC in comparison with C-CNC and G-CNC conditioned media revealed no significant difference in cell viability. This results in comparison with the control demonstrated the possibility of using CNC-based antibiotic loaded wound dressings to deliver a favorable microenvironment for cell proliferation, where prolonged contact with healthy tissue is essential.