Biomed Res Int. 2013; 2013:712678.

Formulation Development and Evaluation of Hybrid Nanocarrier for Cancer Therapy: Taguchi orthogonal Array Based Design.

Rakesh K. Tekade and Mahavir B. Chougule

Department of Pharmaceutical Science, the Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, Hawaii-96720, USA

 

Abstract

Taguchi Orthogonal Array Design is a statistical approach that helps to overcome limitations associated with time consuming full factorial experimental design. In this study, the Taguchi Orthogonal Array Design was applied to establish the optimum conditions for bovine serum albumin (BSA) nanocarrier (ANC) preparation. Taguchi method with L9 type of robust orthogonal array design was adopted to optimize the experimental conditions. Three key dependent factors viz, BSA concentration (% w/v), volume of BSA solution to total ethanol ratio (v:v) and concentration of diluted ethanolic aqueous solution (%v/v) were studied at three levels 3, 4 and 5 % w/v; 1:0.75, 1:0.90 and 1:1.05 v/v; and 40, 70 and 100 %v/v, respectively. The ethanolic aqueous solution was used to impart less harsh condition for desolvation and attain controlled nanoparticle formation. The interaction plot studies inferred that the ethanolic aqueous solution concentration to be the most influential parameter that affects the particle size of nanoformulation. This method (BSA, 4% w/v; volume of BSA solution to total ethanol ratio, 1:0.90 v/v; concentration of diluted ethanolic solution, 70 %v/v) was able to successfully develop Gemcitabine (GEM, G) loaded modified albumin nanocarrier (M-ANC-G) of size 25.07±2.81 nm (ζ=-23.03±1.015 mV) as against to 78.01±4.99 nm (ζ=-24.88±1.37 mV) using conventional method albumin nanocarrier (C-ANC-G). Hybrid nanocarriers (HNC) were generated by chitosan layering (solvent gelation technique) of respective ANC’s to form C-HNC-G and M-HNC-G of sizes 125.29±5.62 nm (ζ=12.01±0.51 mV) and 46.28±2.21 nm (ζ=15.05±0.39 mV), respectively. Zeta potential, entrapment, in vitro release and pH based stability studies were investigated and influence of formulation parameters are discussed. Cell line based cytotoxicity assay (A549 and H460 cells) and cell internalization assay (H460 cell line) was performed to assess the influence bio-performance of these nanoformulations.

Keywords: Taguchi design, Hybrid nanocarrier, Cancer, Cytotoxicity, Cell internalization

PMID: 24106715, PMCID: PMC3784087

 

Supplement

The core objective of this work was to develop and investigate an effective drug delivery strategy for delivery of anticancer drugs and improve the therapeutic outcome. To achieve these objectives an experimental scheme were designed; a) to optimize the formulation of ANC by the Taguchi design method which show a controllable particle size of <50 nm and a narrow size distribution; b) to develop the hybrid nanocarriers by coating the preformed ANC by Chitosan for efficient tumor cell internalization. In addition, this study also intends to establish a rational basis for the controlled production and application of protein-based nanocarriers as drug carrier systems. A simple coacervation technique was employed for formulation of ANC as a colloidal drug delivery system and the key factors effecting formulation of ANC such as BSA concentration, ethanol addition etc. were considered in preparation of nanoformulation. Hybrid nanocarrier (HNC) was formulated by solvent gelation technique using chitosan polymer. Formulation parameters for HNC were optimized employing APAP as model drug, following these optimized conditions, GEM loaded HNC were formulated and characterized for particle size, zeta potential, in vitro release, stability, cell viability, and cell uptake to establish the suitability of this approach in drug delivery and therapy. The Taguchi design has been applied to predict the significant contribution of the design variable(s) and the optimum combination of each variable by conducting experiments on a real-time basis. The modeling that is performed in Taguchi design essentially relates signal-to-noise ratio to the control variables in a ‘main effect only’ approach. This approach enables both multiple response and dynamic problems to be studied by handling noise factors. Taguchi principles and concepts have made extensive contributions to industry by bringing focused awareness to robustness, noise and quality.

The results indicate that HNC approach significantly enhances the anticancer potential of loaded GEM. It is encouraging that our data suggest albumin and chitosan biodegradable and biocompatible polymer based HNC’s to be efficient in inhibiting cancer cells growth as compared to GEM solution and GEM loaded ANC. Most importantly, treatment of HNC’s with H460 cells showed significant improvement in cell uptake as compared to that of unmodified as well as conventionally produced nanocarriers. It is envisioned that these HNC’s can be utilized to selectively deliver the loaded therapeutics to result a high drug concentration at desired site of action. Furthermore, it is envisaged that other bioactive compounds (such as antibiotics, anticancer drugs, plasmids, gene etc) can be loaded into HNC’s employing the same formulation strategy as reported in this article. These results with HNC’s are promising, but further in vivo investigations to explore the efficacy and safety of nanocarriers are necessary to optimize their use in various pharmaceutical applications.

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