Surface modification and evaluation of bacterial cellulose for drug delivery

International Journal of Biological Macromolecules 113 (2018) 526–533

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Surface modification and evaluation of bacterial cellulose for drug delivery Munair Badshah a, Hanif Ullah a, Abdur Rahman Khan b, Shaukat Khan c, Joong Kon Park c, Taous Khan a,⁎ a b c

Department of Pharmacy, COMSATS Institute of Information Technology, 22060 Abbottabad, Pakistan Department of Chemistry, COMSATS Institute of Information Technology, 22060 Abbottabad, Pakistan Department of Chemical Engineering, Kyungpook National University, Buk-ku Sankyuk-dong 1370, Daegu 41566, South Korea

a r t i c l e

i n f o

Article history: Received 22 September 2017 Received in revised form 19 February 2018 Accepted 21 February 2018 Available online 23 February 2018 Keywords: Bacterial cellulose BC-drug matrices Surface modification

a b s t r a c t The current study was designed to prepare surface modified BC matrices loaded with model drugs selected on the basis of their aqueous solubility, i.e., poorly water soluble famotidine and highly water soluble tizanidine. Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM) and thermogravimetric analysis (TGA) confirmed the successful drug loading and thermal stability of the BC matrices. Invitro dissolution studies using USP type-II dissolution apparatus showed that most of the drug was released in 0.5–3 h from famotidine loaded matrices and in 0.25–0.5 h from tizanidine loaded matrices. The chemical structure, concentration of the loaded drug, concentration of the surface modifier, and pre- and post-drug loading modifications altered the physicochemical properties of BC matrices, which in turn affected the drug release behavior. In general, surface modification of the BC matrices enhanced the drug release retardant properties in premodification drug loading. Surface modification was found to be effective for controlling the drug release properties of BC. Therefore, these modified BC matrices have the potential for applications in modified drug delivery systems. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Bacterial cellulose (BC) is a fascinating biopolymer produced by microorganisms such as oomycetes, green algae, and numerous bacterial species of the genera Agrobacterium, Rhizobium, Sarcina and Gluconacetobacter [1,2]. The chemical structure of BC is similar to plant-based cellulose (PC) [3–6]. However, BC possesses distinguished features such as high purity without lignin, pectin and hemicellulose components. In addition, BC has an ultrafine and well organized nanofibrillar network structure with high water holding capability, biodegradability and non-toxic nature. Furthermore, it has simple production and purification process, and is moldable into desired shapes during and after biosynthesis, which make it an attractive biopolymer [7,8]. BC has been studied in as-synthesized form for applications in electronics, food industry, tissue engineering and enzymes immobilization [9]. In addition to the above applications, BC has been studied for antibiotics delivery to wound, as scaffold for drug delivery in tissue engineering, molecularly imprinted polymeric matrix system for enantioselective drug delivery, support in transdermal drug delivery and residue absorption, and drug delivery agent in dental canal treatment [2,10]. Furthermore, BC has been studied for macromolecular ⁎ Corresponding author. E-mail address: [email protected] (T. Khan).

https://doi.org/10.1016/j.ijbiomac.2018.02.135 0141-8130/© 2017 Elsevier B.V. All rights reserved.

drug delivery and hydrogels due to the presence of higher number of surface hydroxyl groups [11]. Recently, BC has been studied as matrices and capsule shells for oral drug delivery [12,13]. Studies on modified forms of BC such as hydrogels, nano-composites and irradiated BC are limited to protein delivery to colon, transdermal drug delivery and drug diffusion mechanism. Chemically modified BC has been studied for potential applications in e-paper, electrical and display devices and speaker diaphragm. However, no studies have been reported regarding application of chemically modified BC matrices in oral drug delivery. In addition, the literature also lacks studies about the application of surface modified BC in the delivery of poorly water soluble or insoluble drugs for oral and site specific delivery to stomach [14–16]. Since non-modified BC has highly porous structure with inability to resist the free movement of gases, solvents and other small molecules, it is an inappropriate biopolymer for controlled drug delivery systems [13]. Therefore, the desired chemical modifications of BC become inevitable for successful applications in controlled drug delivery. Such alterations will lead to enhancement in the physico-mechanical and surface features of BC [13,14]. Different physical, chemical and mechanical modification techniques have been reported using polymers, solvents and biosynthetic pathways to prepare advanced BC-based functional materials with superior properties [15–21]. The reported processes for chemical modification of cellulose included esterification, oxidation, etherification, carbamation and amidation [22], which have been most

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frequently carried out by creating reactive functional and charged groups on the surface through utilization of free hydroxyl groups [23]. Among the reported BC chemical modification procedures, surface acetylation has displayed greater advantages with the ability to maintain a large modified surface area with controlled hydrophobic properties and compatibility with other composite materials [22,24]. Furthermore, acetylated BC showed enhanced biocompatibility and non-toxic nature [25], thus making it a biopolymer of immense interest for potential applications in cosmetics products, disinfectants and as a base for the synthesis of advanced BC-based polymeric materials with functionalized surfaces [26,27]. Therefore, an extensive research work is required on BC surface modification and to explore the potential for its application in oral drug delivery as matrices. In addition to the current work, we have previously explored the non-surface modified BC's potential for application in oral drug delivery as matrices [12] and capsule shells [13]. However, these devices showed immediate release properties, irrespective of the drugs' aqueous solubility and dose. Therefore, surface modified BC matrices have been prepared and evaluated for applications in drug delivery system in the current study. Famotidine and tizanidine were used as model drugs. Famotidine was selected for its poor water solubility and a larger dose i.e., 20 mg, while tizanidine was selected on the basis of its high water solubility and a smaller dose i.e., 6 mg. 2. Materials and methods 2.1. Materials Bacterial strain of Gluconacetobacter xylinus (ATCC No. 23768), anhydrous D-glucose (Scharlau, Barcelona, Spain), peptone (Oxoid, Hampshire, UK), yeast extract and sodium dihydrogen phosphate (Merck, Darmstadt, Germany), sodium hydroxide (Sigma Aldrich, Steinheim, Germany), acetic anhydride (Scharlau, Barcelona, Spain), citric acid (RDH, Seelze, Germany), tizanidine (JPN Pharma, Mumbai, India) and famotidine (Suleshvari Pharma, Mumbai, India). All the chemicals and solvents were of analytical grade and were used without further processing/analysis. 2.2. Methods 2.2.1. Production of BC BC production was carried out in Hestrin-Schramm (HS) liquid medium comprising of D-glucose 20 g L−1, peptone 5 g L−1, yeast extract 5 g L−1, sodium dihydrogen phosphate 2.7 g L−1, citric acid 1.15 g L−1, distilled water (pH 6), and sterilized at 121 °C for 20 min. Pre-culture (50 mL) containing colonies of G. xylinus was incubated in shaking orbital incubator (J.P. Selecta S.A, Barcelona, Spain) at 150 rpm and 30 °C for 24 h. Pre-culture (10 mL) was incorporated into a rectangular box (18 × 10 × 20 cm) containing 300 mL HS medium and incubated in static incubator (Memmert, 100–800, Schwabach, Germany) at 28 °C for 7–15 days. The biosynthesized BC sheets were removed and washed with distilled water to eliminate bacterial colonies and HS medium remnants. The sheets were further purified by immersion in 200 mL of 0.3 M NaOH and sterilized in an autoclave (HVA-85, Hirayama Co. Japan) followed by thorough washing with distilled water till neutral pH and stored at 4 °C for further use [6,9,28]. 2.2.2. Preparation of drug loaded BC matrices BC matrices (12 mm in diameter) were prepared by using a disc fabricator (locally made) and soaked in 50 mL of acetic acid: water (40:60, v/v %) for 2 h. The matrices were further treated with 50 mL acetic acid for 2 h to remove excess of water. In the start of experiment, BC matrices were immersed either in solution of famotidine (20 mg/mL) in acetic acid or tizanidine (6 mg/mL) in water: acetic acid (40:60, v/v %) for 8 h. The drug loaded matrices were transferred to a three-necked round bottom flask containing 24 mL acetic acid solutions with

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Table 1 An overview of various parameters for surface modification and drug loading of BC matrices. Formulation

Acetic anhydride (mL)

Final drying

Drug loading (% ± SD)

F0 F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15

– 2.5 5 7.5 5 7.5 10 5 7.5 10 2.5 5 7.5 5 7.5 10

Freeze drying Oven drying Oven drying Oven drying Oven drying Oven drying Oven drying Freeze drying Freeze drying Freeze drying Freeze drying Freeze drying Freeze drying Freeze drying Freeze drying Freeze drying

– 39.81 ± 5.78 38.31 ± 5.66 46.90 ± 1.14 11.61 ± 0.20 10.16 ± 0.58 15.91 ± 1.30 36.10 ± 0.30 50.25 ± 1.82 36.19 ± 3.04 32.15 ± 4.79 16.98 ± 0.03 23.74 ± 0.95 27.04 ± 1.29 13.83 ± 1.13 20.56 ± 1.44

F0, F1–F3, F7–F9 and F13–F15 were treated with 20 mg/mL of famotidine, while F4–F6 and F10–F12 are treated with 6 mg/mL of tizanidine.

dissolved famotidine (20 mg/mL) or tizanidine (6 mg/mL) at 60 °C. Then, 1 mL mixture of acetic acid and sulfuric acid (95:5, v/v %) was added to the flask followed by addition of acetic anhydride (ratio shown in Table 1). For each experiment, 24 mL of acetic acid was used. The reaction was allowed to continue for 24 h. The drug loaded and surface modified matrices were then immersed in 25 mL distilled water (four times each) for 2 h to remove the acid remnants and dried individually either in oven (SANFA, DHG-9202, Jiangsu Jinyi, China) at 50 °C for 24 h or freeze dryer (Cydos-50, Telstar, Terrassa, Spain) at −50 °C and 0.025 mBar for 6 h. In the second approach, BC matrices were surface modified initially by transferring into three-necked round bottom flask containing 24 mL acetic acid, followed by addition of 1 mL mixture of acetic acid and sulfuric acid (95:5, v/v%) and acetic anhydride (Table 1) at 60 °C. The reaction was allowed to continue for 24 h. The matrices were then washed with 25 mL distilled water (four times) for 2 h and freeze dried at −50 °C and 0.025 mBar for 6 h. These BC matrices were then immersed in famotidine (20 mg/mL) or tizanidine (6 mg/mL) solution for 8 h and dried in freeze dryer at −50 °C and 0.025 mbar for 6 h (Table 1) [8,10,13,29–34]. The final drying method was selected on the basis of their effect on the inter-fibrous network arrangement and expected influence on the BC swelling, solvent uptake and drug release from the matrices. Although the freezedrying procedure requires expensive machinery and more labor, it leads to more loosely packed BC with interconnected fibrous structure as compared to that of oven-dried. In contrast, oven-drying is simple and comparatively less expensive. A general scheme illustrating the important steps of BC surface modification and drug loading has been shown in Fig. 1. Images of the as-synthesized BC matrices, drug loaded and surface modified and dried (oven and freeze dried) BC matrices can be seen in Fig. 2(a–c). 2.3. Physicochemical characterization 2.3.1. Fourier transform-infrared spectroscopy (FTIR) FTIR spectrum of the samples were recorded on a Perkin Elmer FTIR spectrophotometer (Spectrum GX & Autoimage, USA, Spectral range: 4000–600 cm−1; beam splitter: coated on KBr; detector: DTGS; resolution: 0.25 cm−1 (step selectable). Prior to analysis, the samples were dried at 60 °C for 4 h, followed by mixing with KBr pellets (IR grade, Merck, Germany) to prepare discs and processed for obtaining IR spectra. 2.3.2. X-ray diffraction (XRD) XRD patterns of different samples were recorded using an X-ray diffractometer (X'Pert-APD, PANalytical, EA Almelo, The Netherlands)

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Fig. 1. Schematic diagram illustrating the overall process of surface modification and drug loading.

with an X-ray generator (3 kW) and anode (LFF Cu) at 30 mA and 40 kV. The angle used for scanning was from 10° to 60°. 2.3.3. Scanning electron microscopy (SEM) Images of the samples were taken with field emission scanning electron microscope (FE-SEM, Hitachi S-4800, Hitachi, Tokyo, Japan) to investigate the surface topography and morphology. The samples were coated with osmium tetra oxide (OsO4) via VD HPC-ISW osmium coater (Tokyo, Japan) after fixing on to a brass holder, prior to obtaining FESEM images. 2.3.4. Thermogravimetric analysis (TGA) Thermogram of the samples was recorded using thermogravimetric analyzer (Thermal Analysis, SDT Q 600, TA Instruments, USA) on a thermo balance. The thermogram was obtained in the temperature range of 35–800 °C with 10 °C/min increments.

withdrawn from the dissolution vessels at pre-determined time intervals (0, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8 and 10 h) and replenished the same with 5 mL of fresh dissolution medium. The concentration of drug released was analyzed using a UV-spectrophotometer (UV-3000, Labindia-Analytical, India) at a wavelength of 265 nm for famotidine and 319 nm for tizanidine. The amount of drug released was calculated using standard calibration curve of the respective drug. The results obtained in triplicate were averaged and presented as cumulative percent drug release (mean ± SD) as a function of time [35–37]. 2.5. Drug release kinetics In order to study the release mechanism of drugs from drug loaded BC matrices, various kinetic models were applied including zero order, first order, Higuchi and Korsmeyer-Peppas models [13,37].

2.4. In-vitro drug release studies

2.6. Statistical analysis

In-vitro drug release studies of the BC-drug matrices were carried out using USP dissolution apparatus furnished with type II standard paddle (Curio DL 2020, Pak) in 900 mL dissolution medium (0.1 N HCl, pH 1.2) at 50 rpm and 37 ± 0.5 °C. Aliquots of 5 mL were

The results obtained from three independent experiments were averaged and presented as mean ± SD. The analysis was executed using GraphPad Prism 5.0 software (GraphPad Software Inc. USA). The statistical analysis was performed using one way ANOVA with

Fig. 2. Images of BC matrices in (a) as-synthesized, (b) drug loaded, surface modified and oven dried and (c) drug loaded, surface modified and freeze dried form.

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post-hoc Tukey's test, keeping the level of significance with probabilities of *p b 0.05, **p b 0.01 and ***p b 0.001. 3. Results and discussion 3.1. Preparation of chemically modified matrices and drug loading In current study, the drug loaded surface modified BC discs were successfully prepared by acetylation process. Excess water was removed from the hydrated BC discs in order to achieve effective surface modification of the matrices. The presence of high number of free –OH groups on the surface of BC provides favorable environment for carrying out surface acetylation [2,26,33]. Drying method affects the BC swelling, solvents uptake and fibers aggregation due to the formation of strong inter-fibers hydrogen bonding [13]. Therefore, two drying methods were selected, i.e., oven drying and freeze drying. Different formulations of BC with model drugs were prepared and dried under various conditions, which have been explained in Table 1. Formulations F1, F2 and F3, i.e., drug loaded (famotidine 20 mg/mL), surface modified and oven dried BC matrices have relatively higher drug loading, i.e., 39.81 ± 5.78%, 38.31 ± 5.66%, and 46.90 ± 1.14%, respectively as compared to other formulations. The possible reasons for high drug loading may be the hydrophilic nature and highly porous structure of BC [13], and higher drug concentration [35], whereby the drug solution can easily penetrate the BC matrices [15,36–38]. In case of drug loaded (tizanidine 6 mg/mL), surface modified and oven dried formulations of F4, F5 and F6, the drug loading was 11.61 ± 0.20%, 10.16 ± 0.58% and 15.91 ± 1.30%, respectively, which is comparatively lower than it was in F1–F3. The possible reasons may be the exposure to lower drug concentration (in comparison to F1–F3) during the loading process [37–39]. Similar to F1–F3, surface modified and freeze dried formulations F7– F9 showed higher drug loading when the matrices were loaded with famotidine (20 mg/mL) (Table 1). In addition, the formulations F10– F12, where the surface modified and freeze dried matrices were loaded with tizanidine (6 mg/mL) showed higher drug loading onto BC matrices (Table 1) than formulations F4–F6 [35–39]. The possible reason for higher drug loading may be the large surface area maintained by the BC matrices during freeze drying whereby the expulsion of drug is reduced during the process of drying [35,40,41]. In general, the reason for lower drug loading onto BC matrices for all formulations could be the removal of drug from the matrices during post-modification washing process [40,41]. In case of formulations F13–F15, where the matrices were surface modified, freeze dried, loaded with famotidine (20 mg/mL) and finally freeze dried, the drug loading was comparatively lower than in any other formulation loaded with similar drug concentration (Table 1). The formation of a hydrophobic surface layer on the BC matrices due to acetylation [26] and reduced swelling abilities could explain this phenomenon [39].

Fig. 3. FTIR spectra of BC, surface modified BC, famotidine, surface modified BC-famotidine matrices, tizanidine and surface modified BC-tizanidine matrices.

in the spectrum of acetylated BC-tizanidine matrices indicated the surface modification of the BC matrices [26,33,41]. The broadening of absorption peak at 1650–1530 cm−1 indicated the presence of H\\O\\H and C\\C aromatic and C\\N groups of tizanidine, respectively [12].

3.3. X-ray diffraction (XRD) XRD patterns of the BC and drug loaded BC matrices have been shown in Fig. 4. The scanning angle was varied in the range of 10° to 60°. The distinct peaks observed in the diffractogram for BC at 14.12°, 16.8° and 22.72° demonstrated the crystalline structure of BC [7,9]. Surface modified BC have peaks in the XRD pattern similar to assynthesized BC with lower intensity, indicating reduction in the crystallinity as a result of acetylation [7,46,47]. The pattern for BC-famotidine matrices have strong peaks at 11.58° and 17.76°, which indicated the drug crystal growth and the short peaks at 26.90°, 28.54° and 29.82° showed weak famotidine crystals, confirming the entrapment of drug into matrices and the progress of acetylation [35,45,47,48]. The appearance of distinct peaks in the pattern for BC-tizanidine matrices at 25.06° and 26.46° showed lower crystal growth of the drug [46,47,49].

3.2. Fourier transform-infrared spectroscopy (FTIR) Fig. 3 demonstrates FTIR spectra for as-synthesized BC, surface modified BC, famotidine, BC-famotidine matrices, tizanidine and BCtizanidine matrices. The reduction in peak intensities at 3345 and 1665 cm−1 (\\OH) and appearance of peaks at 1747 cm−1 and 1736 cm−1 (C_O) indicated the successful acetylation of the BC matrices. The increase in band intensities at 1375 cm−1 (\\CH3) and 1240 cm−1 (C\\O) in surface modified BC before drug loading and BCfamotidine matrices spectra confirmed the progress of the acetylation process [26,41,42]. The characteristic bands at 1553 cm−1 (\\NH2 bending vibration) at 1315, 1160 and 1135 cm−1 (\\SO2) and at 895 cm−1 (N\\S groups) are distinctive of famotidine as shown in BC-famotidine spectrum [12,43–45]. The spectral band for BC-tizanidine showed broader absorption peak at 3500–3200 cm−1, which is attributed to \\OH and\\CH2, and at 2904 and 2895 cm−1 represent C\\H stretching vibration, respectively [12]. An absorption peak at 1731 cm−1 observed

Fig. 4. X-rays diffractogram of BC, surface modified BC, famotidine, BC-famotidine, tizanidine and BC-tizanidine matrices.

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Fig. 5. Field Emission–Scanning Electron Microscopy showing surface morphologies of (a) BC, (b) surface modified BC, (c) BC-famotidine and (d) BC-tizanidine matrices.

3.4. Scanning electron microscopy (SEM) SEM images of different matrices were recorded at various wavelengths to study changes occurred in the surface morphology. Fig. 5 displayed the SEM images of BC, surface modified BC, BC-famotidine and BC-tizanidine matrices. The image (Fig. 5a) showed a clear, densely arranged, well-organized, porous and interconnected fiber network of BC [9,12]. The surface modified BC prior to drug loading (Fig. 5b) revealed that as a result of surface modification, the micro-fibrils become thick and the pore size has been reduced [47] due to micro-fibril stiffness caused by acetylation and deformation during the drying process [26]. This finding was further supported by results published by Ifuko et al. (2007) that incorporation of acetyl group(s) into the microfibrils increases the thickness and developed mutual contact between the neighboring fibrils during drying [26,41,47]. The micrographs of the BC-famotidine matrices (Fig. 5c) showed reduced pore size and dense coagulates appearance on the surface with entrapped drug crystals. The micrographs for BC-tizanidine matrices (Fig. 5d) showed identical dense fiber network with native BC having drug crystals on the surface indicating successful loading of tizanidine into the matrices.

decomposition and improved the stability of BC [47]. Thermogram for surface modified BC˗famotidine matrices (Fig. 6c) revealed 3.56% weight loss at 35–110 °C. Similarly, 26.47% was observed at 110–350 °C probably due to the elimination of hydroxyl groups, evaporation of solvents and de-polymerization of BC carbon skeleton as a result of cleavage of glycosidic linkage [19]. The maximum degradation was observed at the temperature 350–800 °C accounting for 42.67% of the total weight loss [41] indicating higher thermal stability of acetylated BC [47]. The spectrum for BC-tizanidine (Fig. 6d) showed 3% weight loss at a temperature of 35–110 °C probably due to loss of water content [28,30]. Similarly, 50% weight loss was observed at a temperature of 110–230 °C, which is an indication of the surface hydroxyl groups' acetylation [50]. At the temperature of 300–800 °C, about 40% weight was lost [51].

3.5. Thermogravimetric analysis (TGA) TGA was carried out in order to determine the thermal stability of the matrices and the results are shown in Fig. 6. The thermogram for BC (Fig. 6a) revealed about 5% dry material loss at 35–220 °C due to evaporation of the adsorbed water. The highest weight loss (65%) occurred at 230–340 °C due to the elimination of polyhydroxyl groups. Similarly, the remaining content was produced as ash at 350–800 °C [28,30]. The surface modified BC prior to drug loading (Fig. 6b) showed about 10% weight loss at 110 °C due to evaporation of adsorbed water followed by a gradual loss of dry matter at 110–230 °C. This accounts for 40% of the total weight loss, indicating modification in surface properties due to acetylation. Similarly, 30% weight loss was observed at 250–800 °C. TGA data revealed that acetylation has delayed the thermal

Fig. 6. Thermal gravimetric analysis (TGA) curves of (a) BC, (b) surface modified BC, (c) BC-famotidine and (d) BC-tizanidine matrices.

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3.6. In-vitro drug release studies 3.6.1. Oven dried BC-drug matrices The in-vitro drug release data for drug loaded, surface modified, and oven dried formulations have been displayed in Fig. 7. The formulations F1–F3, respectively, released 83.57 ± 5.12%, 85.79 ± 3.37% and 91.41 ± 1.27% of the drug content within 0.25 h. Similarly, after 0.5 h, the drug release from these formulations was 86.71 ± 5.66%, 86.07 ± 4.38% and 96.32 ± 2.61%, respectively. Formulations F4–F6 also released N80% of the loaded drug during the initial 0.25 h. These results showed that most of the drug content (i.e., N80%) was released in b0.5 h in all formulations and thus satisfying the immediate release criteria (Fig. 7) [13]. Based on these results, no correlation was observed between surface modification and the drug release in case of relatively high loading of water insoluble drug possibly due to the migration of drug content to the surface of the matrices (especially for F3) as well as lower path length for drug release (due to lower swelling of oven dried matrices). Another possible explanation could be that the drug release creates more pores in matrices thus facilitating the quicker drug release [27,38,42]. Likewise, the soluble drug even at low drug loading was quickly released due to hydrophilic nature of the drug. 3.6.2. Freeze dried BC-drug matrices The in-vitro drug release data for the formulations loaded with drug, surface modified and finally freeze dried have been displayed in Fig. 8 and 9. The formulations F7, F8 and F9, i.e., drug loaded, surface modified and freeze dried (Table 1), displayed diverse drug release pattern with sustained effect as compared to rest of the formulations (Fig. 8). These formulations released 18.80 ± 4.58%, 34.81 ± 2.07% and 54.91 ± 10.61% drug in the initial 0.25 h (p b 0.001). Similarly, the respective drug release from these formulations was 33.61 ± 4.71%, 58.78 ± 2.15% and 77.54 ± 12.09% at 0.5 h. The drug release was significantly increased in case of acetylation with higher quantity of acetic anhydride (p b 0.001). Moreover, the drug release was 51.77 ± 4.57%, 72.90 ± 1.80% and 91.62 ± 8.36% for F7, F8 and F9, respectively, after 1.0 h (p b 0.001), and was 74.11 ± 1.60%, 85.88 ± 0.49% and 97.83 ± 2.00% for F7, F8 and F9, respectively, after 2 h (p b 0.001). For statistical calculations of the effect of surface modification and freeze drying on the drug release from the formulations F7, F8 and F9, drug release results from un-modified BC matrices (F0) were used as reference (Table 1 and Fig. 9). It is suggested that surface modification of BC-matrices altered the BC surface properties by creating a hydrophobic coat (as evident from Fig. 5b and c) [22,41,51]. Freeze drying at the final stage might have

Fig. 7. In-vitro drug release profile for BC-drug matrices, drug loaded, surface modified and oven dried. Formulations F1–F3 are loaded with famotidine and F4–F6 with tizanidine.

Fig. 8. In-vitro drug release profile for BC-drug matrices, drug loaded, surface modified and freeze dried. Formulations F7–F9 and F13–F15 are loaded with famotidine, and F10–F12 with tizanidine.

increased the mutual contact among the nanofibers (which become thicker due to acetylation) thus reducing the BC micro-fibrils' interstitial voids [26,41,47] and increasing the pathway for diffusivity of the dissolution medium into matrices. These factors might have hindered the liquid diffusion and thus delayed the drug release [26,52]. The drug release from these formulations is slower than that of F1–F3 and un-modified matrices reported in our previous study [12]. This suggests that acetylation of BC together with freeze-drying can delay the drug release [53–56]. In contrast, the drug loaded, surface modified and freeze dried formulations F10, F11 and F12 showed 88.28 ± 2.17%, 75.33 ± 8.46% and 76.67 ± 3.02% drug release, respectively (Table 1) in 0.25 h. The same formulations released 98.27 ± 1.05%, 86.32 ± 6.52% and 93.97 ± 1.77% of the loaded drug in 0.5 h, which fall in the immediate release category [13]. In addition, formulations F13, F14 and F15, where most of the drug (≥80%) was released during the initial 0.25 h, also complied with the criterion established for immediate release [13]. The possible reasons may be that surface modification caused reduction in drug loading due to hydrophobic nature of the modified BC, suggesting that the drug is merely present on the surface of the matrices that can be easily released.

Fig. 9. Comparison of drug release data at different time intervals: (a) denote formulations surface modified and oven dried, i.e., F1–F6, at 0.25 h and 0.5 h, (b) shows formulations surface modified and finally freeze dried, i.e., F7–F9 at 0.25, 0.5, 1.0 and 2.0 h, and (c) represent formulations surface modified and finally freeze dried 0.25 and 0.5 h, using one way ANOVA with post-hoc Tukey's test, keeping the level of significance with probabilities of *p b 0.05, **p b 0.01 and ***p b 0.001. Data is presented as mean ± SD (n = 3).

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Table 2 Drug release mechanism from the drug loaded BC matrices via different kinetics models. Formulation

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15

Zero order

First order

Higuchi

Korsmeyer-Peppas

R2

K0 h−1

R2

K1 h−1

R2

Kh−0.5 H

n

R2

−0.849 −1.285 −2.298 −0.991 −0.930 −0.963 0.242 −1.574 −3.033 −0.970 −0.300 −0.594 −0.684 −0.600 −0.659

68.962 68.839 71.016 68.952 68.157 71.594 22.270 12.390 15.515 71.063 68.325 72.320 72.426 72.291 72.741

0.950 0.862 0.839 0.974 0.960.21 0.994 0.984 0.971 0.994 0.979 0.969 0.995 0.997 0.998 0.998

6.205 7.783 9.539 12.031 9.384 98.845 0.695 1.423 2.947 23.981 4.218 5.761 9.270 7.463 8.424

0.421 0.225 −0.219 0.254 0.300 0.268 0.922 0.202 −0.511 0.240 0.670 0.547 0.444 0.501 0.465

87.944 87.674 91.214 89.432 88.136 92.649 45.550 39.529 46.183 92.135 86.232 92.005 92.854 92.412 93.203

0.067 0.05 0.024 0.012 0.037 0.004 0.413 0.179 0.101 0.004 0.137 0.108 0.039 0.064 0.050

0.987 0.941 0.854 0.999 0.998 0.999 0.954 0.934 0.929 0.999 0.998 0.981 0.999 0.995 0.996

3.7. Drug release kinetics In order to investigate the drug release mechanism from the formulations, different kinetics models including zero order, first order, Higuchi's model and Korsmeyer-Peppas model were applied. For all the formulations, the regression co-efficient (R2) value has been presented in Table 2. Data for all formulations, i.e., F1–F15, showed best fit for first order kinetics model with highest R2 value. Upon application of Korsmeyer-Peppas model to all formulations, the release exponent n value was b0.5, which indicates that drug was released through Fickian diffusion [57,58]. 4. Conclusion Surface modified BC matrices were evaluated for drug loading and release capabilities. The results showed that surface modification of BC matrices altered the surface properties. The drug loading condition, concentration of surface modifier, aqueous solubility of drugs, their concentration and the drying techniques all influence the drug loading and release behavior. It was observed that matrices (F1–F6) dried using oven technique (simple operation and low-cost) release most of the drug (in-spite of difference in aqueous solubility) in the initial 0.25 h, without showing any drug retention effect. In contrast, BC matrices dried via freeze drying technique, i.e., F7, F8 and F9 released most of the drug (≥80%) in 3 h, 2 h and 1 h, respectively. It was observed that freeze dried formulations have shown superior drug sustaining effect as compared to oven drying for relatively low aqueous soluble drug, i.e., famotidine. It is suggested that surface modification together with freeze drying is an effective method for altering the hydrophilic properties of BC matrices for controlled drug release. It was concluded that modified BC has the potential for applications in drug delivery systems, particularly prolonged and controlled drug delivery, provided that more efficient modification process is adopted. The more effective surface modification is expected to result in an enhanced drug sustaining effect. Acknowledgements The manuscript was reviewed for English by Tamas Kriska, PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin, Watertown Plank Road 8701, WI, USA. The authors would like to thank Tamas Kriska, PhD ([email protected]) for his assistance. References [1] R.V. Augimeri, A.J. Varley, J.L. Strap, Establishing a role for bacterial cellulose in environmental interactions: lessons learned from diverse biofilm-producing Proteobacteria, Front. Microbiol. 6 (2015) 1282.

[2] H. Ullah, H.A. Santos, T. Khan, Applications of bacterial cellulose in food, cosmetics and drug delivery, Cellulose 23 (4) (2016) 2291–2314. [3] J.H. Ha, N. Shah, M. Ul-Islam, T. Khan, J.K. Park, Bacterial cellulose production from a single sugar α-linked glucuronic acid-based oligosaccharide, Process Biochem. 46 (9) (2011) 1717–1723. [4] M. Ul-Islam, T. Khan, J.K. Park, Water holding and release properties of bacterial cellulose obtained by in situ and ex situ modification, Carbohydr. Polym. 88 (2) (2012) 596–603. [5] N. Halib, M.M. Amin, I. Ahmad, M. Abrami, S. Fiorentino, R. Farra, G. Grassi, F. Musiani, R. Lapasin, M. Grassi, Topological characterization of a bacterial celluloseacrylic acid polymeric matrix, Eur. J. Pharm. Sci. 62 (2014) 326–333. [6] W.A. Khattak, T. Khan, M. Ul-Islam, M.W. Ullah, S. Khan, F. Wahid, J.K. Park, Production, characterization and biological features of bacterial cellulose from scum obtained during preparation of sugarcane jaggery (gur), J. Food Sci. Technol. 52 (12) (2015) 8343–8349. [7] O. Shezad, S. Khan, T. Khan, J.K. Park, Physicochemical and mechanical characterization of bacterial cellulose produced with an excellent productivity in static conditions using a simple fed-batch cultivation strategy, Carbohydr. Polym. 82 (1) (2010) 173–180. [8] T. Mukherjee, M. Sani, N. Kao, R.K. Gupta, N. Quazi, S. Bhattacharya, Improved dispersion of cellulose microcrystals in polylactic acid (PLA) based composites applying surface acetylation, Chem. Eng. Sci. 101 (2013) 655–662. [9] F. Esa, S.M. Tasirin, N.A. Rahman, Overview of bacterial cellulose production and application, Agric. Agric. Sci. Procedia 2 (2014) 113–119. [10] J.M. Gutiérrez-Hernández, D.M. Escobar-García, A. Escalante, H. Flores, F.J. González, P. Gatenholm, G. Toriz, In vitro evaluation of osteoblastic cells on bacterial cellulose modified with multi-walled carbon nanotubes as scaffold for bone regeneration, Mater. Sci. Eng. C 75 (2017) 445–453. [11] A.J. Silvestre, C.S. Freire, C.P. Neto, Do bacterial cellulose membranes have potential in drug-delivery systems? Expert Opin. Drug Deliv. 11 (7) (2014) 1113–1124. [12] M. Badshah, H. Ullah, S.A. Khan, J.K. Park, T. Khan, Preparation, characterization and in-vitro evaluation of bacterial cellulose matrices for oral drug delivery, Cellulose 24 (11) (2017) 5041–5052. [13] H. Ullah, M. Badshah, E. Mäkilä, J. Salonen, M.A. Shahbazi, H.A. Santos, T. Khan, Fabrication, characterization and evaluation of bacterial cellulose-based capsule shells for oral drug delivery, Cellulose 24 (3) (2017) 1445–1454. [14] M. Ul-Islam, S. Khan, M.W. Ullah, J.K. Park, Bacterial cellulose composites: synthetic strategies and multiple applications in bio-medical and electro-conductive fields, Biotechnol. J. 10 (12) (2015) 1847–1861. [15] S.A. de Oliveira, B.C. da Silva, I.C. Riegel-Vidotti, A. Urbano, P.C. Sousa FariaTischer, C.A. Tischer, Production and characterizationof bacterial cellulose membranes with hyaluronic acid from chicken comb, Int. J. Biol. Macromol. 97 (2017) 642–653. [16] G.F. Picheth, C.L. Pirich, M.R. Sierakowski, M.A. Woehl, C.N. Sakakibara, C.F. Souza, A.A. Martin, R. Silva, R.A. Freitas, Bacterial cellulose in biomedical applications: a review, Int. J. Biol. Macromol. 104 (2017) 97–106. [17] M.S. Reid, M. Villalobos, E.D. Cranston, Benchmarking cellulose nanocrystals: from the laboratory to industrial production, Langmuir 33 (7) (2017) 1583–1598. [18] J. Huang, Y. Gu, Self-assembly of various guest substrates in natural cellulose substances to functional nanostructured materials, Curr. Opin. Colloid Interface Sci. 16 (6) (2011) 470–481. [19] E. Oledzka, M. Sobczak, Polymers in the pharmaceutical applications, natural and bioactive initiators and catalysts in the synthesis of biodegradable and bioresorbable polyesters and polycarbonates, Innovat. Biotechnol. InTech, Croatia 2012, pp. 139–160. [20] Q. Shi, Y. Li, J. Sun, H. Zhang, L. Chen, B. Chen, H. Yang, Z. Wang, The osteogenesis of bacterial cellulose scaffold loaded with bone morphogenetic protein-2, Biomaterials 33 (28) (2012) 6644–6649. [21] N. Shah, M. Ul-Islam, W.A. Khattak, J.K. Park, Overview of bacterial cellulose composites: a multipurpose advanced material, Carbohydr. Polym. 98 (2) (2013) 1585–1598.

M. Badshah et al. / International Journal of Biological Macromolecules 113 (2018) 526–533 [22] W. Hu, S. Chen, J. Yang, Z. Li, H. Wang, Functionalized bacterial cellulose derivatives and nanocomposites, Carbohydr. Polym. 101 (2014) 1043–1060. [23] H. Ullah, F. Wahid, H.A. Santos, T. Khan, Advances in biomedical and pharmaceutical applications of functional bacterial cellulose-based nanocomposites, Carbohydr. Polym. 150 (2016) 330–352. [24] S. Eyley, W. Thielemans, Surface modification of cellulose nanocrystals, Nano 6 (14) (2014) 7764–7779. [25] J. Cook, Amine functionalization of bacterial cellulose for targeted delivery applications, Electronic Thesis and Dissertation Repository 2013, p. 1442http://ir.lib.uwo. ca/etd/1442. [26] D.Y. Kim, Y. Nishiyama, S. Kuga, Surface acetylation of bacterial cellulose, Cellulose 9 (3) (2002) 361–367. [27] O.A. Saibuatong, M. Phisalaphong, Novo aloe vera–bacterial cellulose composite film from biosynthesis, Carbohydr. Polym. 79 (2) (2010) 455–460. [28] T. Khan, H. Khan, J.K. Park, Physical properties of a single sugar α-linkedglucuronic acid-based oligosaccharide produced by a Gluconacetobacter hanseniistrain, Process Biochem. 42 (2) (2007) 252–257. [29] M. Pohl, N. Michaelis, F. Meister, T. Heinze, Biofunctional surfaces based on dendronized cellulose, Biomacromolecules 10 (2) (2009) 382–389. [30] F. Chen, Z. Kan, S. Hua, Z. Liu, M. Yang, A new understanding concerning theinfluence of structural changes on the thermal behavior of cellulose, J. Polym. Res. 22 (11) (2015) 225. [31] K.Y. Lee, J.J. Blaker, A. Bismarck, Surface functionalisation of bacterial celluloseas the route to produce green polylactide nanocomposites with improvedproperties, Comput. Sci. Technol. 69 (15) (2009) 2724–2733. [32] J.F. Sassi, H. Chanzy, Ultrastructural aspects of the acetylation of cellulose, Cellulose 2 (2) (1995) 111–127. [33] H.S. Barud, A.M. Araújo Júnior, B.D. Santos, R. Assunção, C.S. Meireles, D.A. Cerqueira, G. Rodrigues Filho, C.A. Ribeiro, Y. Messaddeq, S.J. Ribeiro, Thermal behaviour of cellulose acetate produced from homogeneous acetylation of bacterial cellulose, Thermochim. Acta 471 (1) (2008) 61–69. [34] M.A. Hussain, K. Abbas, M. Sher, M.N. Tahir, M. Tremel, M.S. Iqbal, M. Amin, M. Badshah, Macromolecular prodrugs of aspirin with HPMC: a nano particulate drug design, characterization and pharmacokinetic studies, Macromol. Res. 19 (12) (2011) 1296–1302. [35] V. Gupta, S. Ain, K. Babita, Q. Ain, J. Dahiya, Solubility enhancement of the poorly water-soluble antiulcer drug famotidine by inclusion complexation, Int. J. Pharm. Sci. Nanotechnol. 6 (2013) 1983–1989. [36] A. Viridén, B. Wittgren, A. Larsson, The consequence of the chemical composition of HPMC in matrix tablets on the release behaviour of model drug substances having different solubility, Eur. J. Pharm. Biopharm. 77 (1) (2011) 99–110. [37] G. Murtaza, H. Ullah, S.A. Khan, S. Mir, A.K. Khan, B. Nasir, S. Azhar, M.A. Abid, Formulation and in vitro dissolution characteristics of sustained-release matrix tablets of tizanidine hydrochloride, Trop. J. Pharm. Res. 14 (2) (2015) 219–225. [38] P. Sriamornsak, J. Nunthanid, K. Cheewatanakornkool, S. Manchun, Effect of drug loading method on drug content and drug release from calcium pectinate gel beads, AAPS Pharm. Sci. Technol. 11 (3) (2010) 1315–1319. [39] M. Ul-Islam, W.A. Khattak, M. Kang, S.M. Kim, T. Khan, J.K. Park, Effect of postsynthetic processing conditions on structural variations and applications of bacterial cellulose, Cellulose 20 (1) (2013) 253–263. [40] W. Tang, S. Jia, Y. Jia, H. Yang, The influence of fermentation conditions and posttreatment methods on porosity of bacterial cellulose membrane, World J. Microbiol. Biotechnol. 26 (1) (2010) 125.

533

[41] S. Ifuku, M. Nogi, K. Abe, K. Handa, F. Nakatsubo, H. Yano, Surface modification of bacterial cellulose nanofibers for property enhancement of optically transparent composites: dependence on acetyl-group DS, Biomacromolecules 8 (6) (2007) 1973–1978. [42] M. Suetsugu, M. Kotera, T. Nishino, Cellulosic nanocomposite prepared by acetylation of bacterial cellulose using supercritical carbon dioxide, Conference Proceedings of the 17th International Conference of Composite Materials, Edinburgh, 2009. [43] C. Clasen, B. Sultanova, T. Wilhelms, P. Heisig, W.M. Kulicke, Effects of different drying processes on the material properties of bacterial cellulose membranes, Macromol. Symp. 244 (2006) 48–58. [44] S.K. Adesina, U. Ezeonyebuchi, E.O. Akala, The effect of formulation variables on drug loading of antitubercular drugs in nanoparticle formulations, Mat. Res. Express 2 (9) (2015), 095403. . [45] W.T. Cheng, S.Y. Lin, Famotidine polymorphic transformation in the grindingprocess significantly depends on environmental humidity or water content, Int. J. Pharm. 357 (1) (2008) 164–168. [46] W. Hu, S. Chen, Q. Xu, H. Wang, Solvent-free acetylation of bacterial cellulose under moderate conditions, Carbohydr. Polym. 83 (4) (2011) 1575–1581. [47] J.A.Á. Ramírez, C.G. Hoyos, S. Arroyo, P. Cerrutti, M.L. Foresti, Acetylation of bacterial cellulose catalyzed by citric acid: use of reaction conditions for tailoring the esterification extent, Carbohydr. Polym. 153 (2016) 686–695. [48] S. Sagdinc, S. Bayarı, Experimental and theoretical infrared spectra of famotidine and its interaction with ofloxacin, J. Mol. Struct. 744 (2005) 369–376. [49] M.N. Aamir, M. Ahmad, Production and stability evaluation of modified-release microparticles for the delivery of drug combinations, AAPS Pharm. Sci. Technol. 11 (1) (2010) 351–355. [50] R. Colussi, V.Z. Pinto, S.L.M. El Halal, N.L. Vanier, F.A. Villanova, R.M. e Silva, A.R.G. Dias, Structural, morphological, and physicochemical properties of acetylated high-, medium-, and low-amylose rice starches, Carbohydr. Polym. 103 (2014) 405–413. [51] M.B. Agustin, F. Nakatsubo, H. Yano, The thermal stability of nanocellulose and its acetates with different degree of polymerization, Cellulose 23 (1) (2016) 451–464. [52] M. Larsson, A. Johnsson, S. Gårdebjer, R. Bordes, A. Larsson, Swelling and mass transport properties of nanocellulose-HPMC composite films, Mater. Des. 122 (2017) 414–421. [53] Y. Azeh, G.A. Olatunji, C. Mohammed, P.A. Mamza, Acetylation of wood flour from four wood species grown in Nigeria using vinegar and acetic anhydride, Int. J. Carbohydr. Chem. 2 (10) (2013) 147–155. [54] J.H. Abdelkader, O.Y. Abdalla, H. Salem, Formulation of controlled-release baclofen matrix tablets II: influence of some hydrophobic excipients on the release rate and in vitro evaluation, AAPS Pharm. Sci. Technol. 9 (2) (2008) 675–683. [55] J. Wu, Y. Zheng, Z. Yang, Q. Cui, Q. Wang, S. Gao, X. Ding, Chemical modifications and characteristic changes in bacterial cellulose treated with different media, J. Polym. Res. 19 (9) (2012) 9945. [56] G. Ritu, B. Meenakshi, Influence of formulations parameters on tizanidine hydrochloride nanoparticles, Int. J. Pharm. Bio Sci. 4 (2) (2013) 1056–1078. [57] F. Nasir, Z. Iqbal, J.A. Khan, A. Khan, F. Khuda, L. Ahmad, A. Khan, A. Khan, A. Dayoo, Development and evaluation of diclofenac sodium thermorevesible subcutaneous drug delivery system, Int. J. Pharm. 439 (1) (2012) 120–126. [58] S. Abbasi, G. Yousefi, A.A. Ansari, S. Mohammadi-Samani, Formulation and in vitro evaluation of a fast-disintegrating/sustained dual release bucoadhesive bilayer tablet of captopril for treatment of hypertension crises, Res. Pharm. Sci. 11 (4) (2016) 274–283.