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Edited by: Ronan McCarthy, Brunel University London, United Kingdom

Reviewed by: Diego Mantovani, Laval University, Canada; Adil Denizli, Hacettepe University, Turkey

This article was submitted to Biomaterials, a section of the journal Frontiers in Bioengineering and Biotechnology

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Bacterial cellulose (BC) is a highly pure form of cellulose and possesses superior physico-mechanical properties with wide range of applications. These properties of BC can further be improved by various modifications, including its regeneration from the BC solution. In the current research work, regenerated BC (R-BC) matrices were prepared using N-methyl-morpholine-oxide (NMMO; 50% w/w solution in water) and loaded with model drugs, i.e., famotidine or tizanidine. The characterization of drug loaded regenerated BC (R-BC-drug) matrices was carried out using Fourier transform infrared spectroscopy (FTIR), x-ray diffraction (XRD) analysis, scanning electron microscopy (SEM) and thermogravimetric analysis (TGA), which revealed the stability of matrices and successful drug loading. Results of dissolution studies showed immediate (i.e., >90%) drug release in 30 min. The drugs release data was found to best fit into first order kinetics model having ^{2} values >0.99 for all the formulations. These results indicated that regenerated BC-based matrices had the ability to be used for delivery of orally administered drugs.

Cellulose is the most abundant, inexpensive, biodegradable, and renewable biomaterial obtained from cotton, wood, and other plant sources, which often contains pectin, lignin, and hemicellulose as biogenic contaminations (Khan et al.,

To meet the current research demand and to explore further potential applications of BC in various fields, several physical and chemical procedures have been reported in the literature for preparation of BC based nano-composites (Ullah et al.,

The process of BC regeneration is associated with certain shortcomings, such as its limited solubility in solvents (commonly used for plant cellulose) and inability to tailor the polymeric properties after regeneration (Reddy and Yang,

In the current research work, R-BC-drug (famotidine and tizanidine) matrices were prepared for the first time using NMMO as solvent. The matrices were characterized using FTIR, XRD, SEM and TGA. The matrices were evaluated (

Anhydrous D-glucose (Dae-Jung, Gyeonggi-do, Korea), agar and peptone (Oxide, Hants, UK), sodiumhydroxide (Sigma Aldrich, St. Louis, USA), citric acid monohydrate (RDH, Seelze, Germany), sodium dihydrogen phosphate and yeast extract (Merk, Darmstadt, Germany), NMMO 50% (w/w) aqueous solution (a kind gift from Amines and Plasticizer, Mumbai, India), tizanidine HCl (JPN Pharma, Mumbai, India), famotidine (Suleshvari Pharma, Gujarat, India), and hydrochloric acid (Fishers Chemicals Ltd, Loughborough, UK) were used. The solvents and chemicals received were used without further processing.

Hestrin Schramm (HS) liquid medium, containing glucose anhydrous 2% citric acid monohydrate 0.11%, yeast extract 0.5%, peptone 0.5%, NaH_{2}PO_{4} 0.27% and distilled-water, was prepared (pH 6.0) and sterilized (121°C for 20 min). For the preparation of the pre-culture, the colonies of

BC was dried at 60°C for 10 h in a heating oven (SANFA, DHG-9202, Jiangsu Jinyi, China), followed by grinding to convert it into powder form. Then, powdered BC (2 g) was gradually added to NMMO solution (50 g) in glass petri plates (90 mm × 10 mm) and heated at 70°C for 24 h to dissolve it. The BC solution was added with different concentrations of famotidine and tizanidine (

Summary of R-BC-drug matrices thickness, friability and drug loading data.

F1 | 1 : 0.25 | 3.50 | 0 | 22.97 ± 0.81 |

F2 | 1 : 0.50 | 3.25 | 0 | 24.62 ± 3.98 |

F3 | 1 : 0.75 | 3.20 | 0 | 27.70 ± 3.24 |

G1 | 1 : 0.085 | 2.50 | 0 | 17.65 ± 1.80 |

G2 | 1 : 0.17 | 2.45 | 0 | 24.79 ± 3.27 |

G3 | 1 : 0.25 | 2.65 | 0 | 28.32 ± 1.00 |

Schematic diagram showing the general process of BC regeneration, drug loading, and matrices preparation.

The samples were dried at 50°C for 24 h prior to measurement. FTIR spectra for R-BC, drugs and R-BC-drug matrices was recorded using FTIR spectrophotometer (Perkin-Elmer Frontier FTIR Spectrometer, USA) in the spectral range of 4,000–400 cm^{−1} at resolution rate of 4 cm^{−1}, with an ATR Pike Gladi ATR diamond crystal.

XRD measurements were employed by means of x-ray diffractometer (D8 ADVANCE, BRUKER, Co. USA) with radiation Cu Kα at 2.29 Å and operated at room temperature for the determination of crystallinity of R-BC, drugs and R-BC-drug matrices. The sample scanning speed was 6°/min and the angle for scanning (2θ) used was in the range of 10–60°.

The surface morphology analysis of R-BC and R-BC-drug matrices was carried out with field emission scanning electron microscopy (LEO Ultra 55, LEO Electron Microscopy Ltd, Cambridge, UK). The samples for cross section analysis were prepared under the liquid nitrogen conditions. All the samples were fixed on the SEM holder using adhesive tape prior to proceeding for analysis. The samples were exposed for 1 min for sputter coating with gold in an atmosphere provided with Argon (S150B Sputter Coater, Edwards, England) to determine the surface topography and morphology.

Thermal stability analysis of the R-BC-drug matrices was carried out with the help of thermogravimetric analyzer (TGA/DSC 3^{+}, Mettler Toledo, UK). Thermogram of samples was attained in the temperature range of 35 to 800°C with an increment of 10°C/min under the nitrogen atmospheric conditions.

The R-BC-drug matrices thickness was measured using a Vernier caliper (SparkFun, USA). Percent drug loading of the matrices were calculated by averaging the amount of drug released of all the formulations during dissolution. The following equation was used to calculate matrices percent drug loading.

Friability test of the matrices was performed using a Friability Tester (FT-L, Galvano Scientific, Pakistan) having speed of 25 rpm and time limit of 4 min (Badshah et al.,

Where W1 represents pre-test weight of matrices, W2 denotes the weight of matrices after test and x show the percent weight loss.

The release of drugs from the R-BC-drug matrices was performed in simulated gastric conditions, i.e., 0.1 N HCl solution (900 mL) maintained at 37 ± 0.5°C using USP type-I dissolution apparatus (Dissolutest, Prolabo, France). The paddle rotation speed was in tune of 50 rpm. Samples (5 mL) from the medium were withdrawn at designated time intervals and replaced with an equal amount of fresh medium. The amount of drug released was tested using UV-spectrophotometer (Cary 60 UV-Vis, Agilent Technologies, USA) at 265 nm and 320 nm for famotidine and tizanidine, respectively. All the drug release experiments were performed in triplicate. The data obtained was averaged and presented as cumulative percent release vs. time (Badshah et al.,

The mechanism for drug release from R-BC-drug matrices was studied by applying selected kinetics models including

Where “Qt” is the cumulative amount of drug release at time “t”

“Q_{0}” is the initial amount of drug at time “0,” “K_{0}” is zero order rate constant and “t” is the time.

Where “Q_{t}” is the cumulative amount of drug release at time “t”

“Q_{0}” is the initial amount of drug at time “0,” “K_{t}” is first order rate constant and “t” is the time.

Where “Q_{t}” is the cumulative amount of drug release at time “t” and “K_{H}” is Higuchi rate constant and “t” is the time,

and

Whereas “Q_{t}” is drug cumulative amount released at time “t,” “Q” is the total amount of drug in the dosage form, “K_{kp}” is Korsmeyer-Peppas rate constant, “t” is the time and “n” is diffusion or release exponent (Gouda et al.,

The results obtained from three independent replicate experiments were presented as mean ± SD. The results were analyzed using GraphPad Prism 5.0 software (GraphPad Software Inc. USA). The statistical analysis was performed using one way ANOVA with ^{*}^{**}^{***}

In the current study, BC was successfully dissolved in NMMO, incorporated with model drugs in various concentrations and finally regenerated. Several solvents can dissolve BC, however, NMMO was selected in the current study because it is recyclable and environment friendly solvent (Gao et al.,

The prepared matrices were subjected to various physical tests. The matrices thickness was observed to be directly proportional to the initial concentration of drug for loading (

FTIR technique was used to study the compatibility and structural changes of the formulations ingredients, i.e., R-BC and drug loaded R-BC. FTIR spectra for BC, R-BC, famotidine, R-BC-famotidine, tizanidine and R-BC-tizanidine have been shown in ^{−1} and at 1,160 and 1,068 cm^{−1}, which are assigned to OH stretching, OH wagging and C-O-C pyranose ring (Chen et al., ^{−1}, representing OH stretching due to breakage of inter and intra molecular hydrogen bonding. In addition, the appearance of peaks in as-synthesized and regenerated BC at 1,160 and 1,068 cm^{−1} represent the C-H scissor vibration (Gao et al., ^{−1}, which may arise due to the merger of –OH and NH_{2} groups of R-BC and famotidine, respectively. Similarly, the region of 2,850–2,950 cm^{−1} represents the C–H bending of R-BC vibration (Sagdinc and Bayari, ^{−1} represent NH_{2} group, while at 1,290 and 1,135 cm^{−1} revealed CH_{2}=S and SO_{2} groups of famotidine, respectively (Sagdinc and Bayari, ^{−1} represent bending vibration due to NH_{2} group of famotidine and CH_{2} group of R-BC (Sagdinc and Bayari, ^{−1} represent glycosidic linkage of R-BC and 850 cm^{−1} has been assigned to the CH_{2} skeleton of famotidine and R-BC (Sagdinc and Bayari,

FTIR spectrum of the prepared BC, R-BC, famotidine, R-BC-famotidine, tizanidine, and R-BC-tizanidine matrices for comparison.

The IR spectrum of R-BC-tizanidine IR displayed bands at 3,200–3,500 cm^{−1} that represent OH and NH_{2} groups of R-BC and tizanidine, respectively. Similarly, bands at 1,665 cm^{−1} indicates the C=C aromatic stretching of tizanidine (Aamir and Ahmad, ^{−1} indicates R-BC C–H stretching vibration. The C–N stretching was confirmed by peak at 1,290 and 1,187 cm^{−1}, while bands at 1,113 and 1,068 cm^{−1} confirm the C–Cl group of tizanidine (Aamir and Ahmad,

Change in the crystallinity of the R-BC and drug loaded matrices was evaluated using XRD technique. XRD patterns of BC, R-BC, famotidine, R-BC-famotidine, tizanidine HCl and R-BC-tizanidine HCl have been displayed in

XRD pattern of R-BC, famotidine, R-BC-famotidine, tizanidine, and R-BC-tizanidine.

SEM images of

Surface morphology of R-BC and the matrices with loaded drugs was studied using SEM. The SEM micrographs of surface and cross section of R-BC and R-BC-drugs matrices were obtained at varying magnifications.

In order to study the thermal stability of R-BC and drugs loaded matrices, thermal analysis was carried out. Thermogram of BC, R-BC, and R-BC-drugs matrices were obtained in order to study the thermal behavior of these samples (

Curves of the thermogravimetric analysis for the prepared matrices of R-BC, R-BC-famotidine, and R-BC-tizanidine.

The release of drugs from the R-BC-drug matrices was studied in simulated gastric fluid, i.e., 0.1 N HCl solution using USP type-I dissolution apparatus under predefined conditions. _{90%} (time in which 90% of the drug was released) is <1 h for both of the drug loaded matrices. The comparison of drug release from F1–F3 and G1–G3 show that famotidine loaded matrices have released higher concentrations of drug as compared to tizanidine in the initial 15 min. This might become possible due to larger exposed surface of R-BC for binding of the lower concentration of tizanidine as compared to famotidine loaded formulations (Kolakovic et al.,

Comparison of R-BC matrices drug release data at various intervals of time i.e.,

It is evident from our current and previous studies that in comparison to the existing conventional dosage forms, BC forms a single excipient based intact oral dosage form due its higher tensile strength. Moreover, the as-synthesized BC membrane has limited thickness and more time is required to obtain desired thickness (Badshah et al.,

The drug release kinetics studies revealed that the release of drugs from the R-BC-drug matrices was dependent on the drug concentration. The hydrophilic property of R-BC might facilitate the diffusion of the medium into the matrices. It was observed that drug release during dissolutions was best fitted into the first order kinetics model with ^{2} value more than 0.99. The release exponent “

Drug release kinetics of famotidine and tizanidine from the R-BC-drug matrices.

^{2} |
_{0}h^{−1} |
^{2} |
_{1}h^{−1} |
^{2} |
_{H}^{h−0.5} |
^{2} |
||
---|---|---|---|---|---|---|---|---|

F1 | 0.4896 | 72.276 | 0.9993 | 5.936 | 0.5722 | 91.901 | 0.101 | 0.9879 |

F2 | 0.3376 | 71.847 | 0.9997 | 4.935 | 0.6440 | 91.047 | 0.136 | 0.9771 |

F3 | 0.3753 | 72.206 | 0.9998 | 5.298 | 0.6208 | 91.750 | 0.122 | 0.9790 |

G1 | 0.5115 | 72.526 | 0.9980 | 6.025 | 0.5404 | 92.520 | 0.098 | 0.9757 |

G2 | 0.1349 | 71.326 | 0.9998 | 4.064 | 0.7337 | 89.766 | 0.182 | 0.9717 |

G3 | 0.2580 | 71.947 | 0.9978 | 4.510 | 0.6758 | 90.967 | 0.158 | 0.9626 |

The current research work was carried out for the first time to evaluate the potential applications of regenerated BC for drug delivery. The R-BC-drug matrices were prepared using NMMO as solvent. Characterization data showed that R-BC-drug matrices were chemically and thermally stable. The drug loading and

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

TK, FW, and FH conceived the project, supervised the research, and writing of the manuscript. MB carried out the research work and wrote the manuscript basic draft in collaboration with HU and UF. MA has contributed in the characterization of samples and reviewed the manuscript critically. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We thank Prof. Dr. Annette Larsson, Chalmers University of Technology, Sweden for assistance with characterization techniques and providing space in laboratory to carry out research work. We are also thankful to Mr. H. K. Ruia of Amines & Plasticizers Limited, India for providing NMMO as donation for this research work.

The Supplementary Material for this article can be found online at: