Optimizing indomethacin-loaded chitosan nanoparticle size, encapsulation, and release using Box–Behnken experimental design

https://doi.org/10.1016/j.ijbiomac.2016.02.033Get rights and content

Abstract

Indomethacin chitosan nanoparticles (NPs) were developed by ionotropic gelation and optimized by concentrations of chitosan and tripolyphosphate (TPP) and stirring time by 3-factor 3-level Box–Behnken experimental design. Optimal concentration of chitosan (A) and TPP (B) were found 0.6 mg/mL and 0.4 mg/mL with 120 min stirring time (C), with applied constraints of minimizing particle size (R1) and maximizing encapsulation efficiency (R2) and drug release (R3). Based on obtained 3D response surface plots, factors A, B and C were found to give synergistic effect on R1, while factor A has a negative impact on R2 and R3. Interaction of AB was negative on R1 and R2 but positive on R3. The factor AC was having synergistic effect on R1 and on R3, while the same combination had a negative effect on R2. The interaction BC was positive on the all responses. NPs were found in the size range of 321–675 nm with zeta potentials (+25 to +32 mV) after 6 months storage. Encapsulation, drug release, and content were in the range of 56–79%, 48–73% and 98–99%, respectively. In vitro drug release data were fitted in different kinetic models and pattern of drug release followed Higuchi-matrix type.

Introduction

Chitosan is a cationic hydrophilic linear polysaccharide macromolecule of biological origin. This biocompatible/environment-friendly compound is composed of deacetylated unit (β-(1–4)-linked d-glucosamine) and acetylated unit (N-acetyl-d-glucosamine). Chitosan has mucoadhesive and membrane-permeability enhancing properties [1]. It has numerous advantages for mucosal delivery such as biodegradability and low toxicity [2]. It is being used as an excipient in various formulations including micro- and nanoparticles [3]. Multiple methods were reported to prepare chitosan nanoparticles including self-assembly technique through chemical modification, complex coacervation process, emulsion-droplet coalescence, ionotropic gelation and other techniques. Among these techniques, ionotropic gelation [4] is preferred for its relative simplicity, convenience, and the rid of high temperature and organic solvents; hence, sufficient encapsulation of therapeutic agents such as doxorubicin [5], cyclosporine-A [6] as well as proteins [7] could be possible.

Phytochemicals like catechins have been fabricated and encapsulated in chitosan-TPP nanoparticles with up to 53% encapsulation efficiency [8]. Chen and Subirade developed chitosan and TPP based nanoparticles to encapsulate a vitamin riboflavin [9]. Also, Desai et al. developed sustained release microspheres of cross linked chitosan loaded with vitamin-C by spray drying [10]. Moreover, the coating of alginate beads with chitosan to encapsulate the living microbial supplements (probiotics) was developed by Le-Tien et al. [11]. During encapsulation of Lactobacillus acidophilus and Lactobacillus casei in microspheres, chitosan coated alginate beads provided better protection for these two probiotics as compared to poly-L-lysine-coated alginate beads [12]. Similarly, L. acidophilus-547 and L. casei-01 strains were best protected by chitosan-coated alginate beads [13]. Unsaturated fatty acids functions as neuroprotective, antioxidant, and anti-inflammatory, but they are highly susceptible to oxidative rancidity; therefore, chitosan could be used to stabilized the o/w-emulsions by getting adsorbed on to the oil droplets and form a protective layer by reducing the interfacial tension. Spray dried tuna o/w-emulsion was stabilized by chitosan-lecithin and found oxidative stable as compared to buck oils, hence were found an excellent ω-3 fatty acid components for the functional foods [14]. Immobilization carriers for enzymes were possible by formulating the chitosan based macro-, micro- and nano-sized particles by precipitation, emulsification, and ionotropic gelation methods, respectively. For example, the highest activity and excellent storage stability of β-galactosidase was found when they were immobilized on chitosan-nanoparticles prepared by ionotropic gelation technique, where sodium sulfate was the gelation agent [15]. Flavors like citral and limonene in emulsion forms were stabilized by sodium dodecyl sulfate-chitosan and were found more effective at hindering the citral oxidation product formation than gum Arabica- stabilized emulsions. Similarly, emulsion of limonene was stabilized by sodium dodecyl sulfate-chitosan, and formation of limonene oxide and carvone were found very low as compared to gum Arabica-stabilized emulsions at pH 3.0. The sodium dodecyl sulfate-chitosan multilayer has ability to form a thick cationic emulsion droplet interface that inhibits the oxidative deterioration of citral and limonene [16].

In this study, we applied ionotropic gelation technique for the preparation of indomethacin-loaded chitosan nanoparticles. Due to the formation of inter- and intramolecular cross-linkages, chitosan gelation occurs when it comes in contact with certain polyanions [5]. In our study, tripolyphosphate was used a cross linker. Although ionotropic gelation of chitosan with tripolyphosphate was firstly reported by Bodmeier et al. [17], our main intention is to apply Box–Behnken design for the optimization of chitosan nanoparticles while assessing the effects of different preparation parameters on their physicochemical characteristics viz. particle size, encapsulation and release drug. In this study indomethacin was used as a model drug, however the developed nanoparticles can be used, in principle, to encapsulate hydrophilic or hydrophobic therapeutic agents, bacteria, food, or biochemical ingredients.

Section snippets

Materials

Indomethacin (Lot No. 134737/42) was purchased from its manufacturer Winlab, UK. High purity, molecular weight 140K-220K, deacetylated chitin (chitosan) and the cross linker tripolyphosphate (TPP) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Acetic acid glacial was purchased from BDH Limited (Poole, England). Dichloromethane and acetonitrile (HiPerSolv CHROMANORM for HPLC grade) were purchased from BDH, PROLABO®, LEUVEN, EC. Purified water was obtained by Milli-Q® water purifier

Results and discussions

In the present study we have chosen a lipophilic drug, indomethacin, to encapsulate into chitosan-TPP nanoparticles as a model drug, and evaluated the morphology of the produced nanoparticles by transmission electron microscopy (TEM), particle size, polydispersity, zeta potential, encapsulation efficiency, drug loading, swelling behavior of chitosan nanoparticles in water, and in-vitro drug release. Our results have given indications that the developed nanoparticles by ionic gelation method

Conclusions

Using ionotropic gelation method, good average sized positively charged chitosan-nanoparticles with high encapsulation efficiency; satisfactory drug release and good storage stability properties could be produced and optimized by using a three-factor, three-level Box–Behnken design. High magnitudes of zeta-potential of the NPs indicated a stable dispersive nature of the developed chitosan-NPs.The quantitative effect of the chosen independent factors at different levels to achieve minimum

Conflict of interest

The authors declare no competing financial interest.

Acknowledgment

This project was funded by the Research Groups Program (Research Group number RG-1436-027), Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia.

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