In vitro cytotoxicity and in vivo efficacy of 5-fluorouracil-loaded enteric-coated PEG-crosslinked chitosan microspheres in colorectal cancer therapy in rats

Abstract

Purpose: Microspheres of chitosan (CS) crosslinked with polyethylene glycol (PEG) were prepared by emulsion crosslinking followed by solvent evaporation technique. The formulations were characterized and subjected to in vitro and in vivo tests to assess cell growth, changes in cell morphology and activities by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on human HT-29 colon cancer cell lines.

Methods: In vivo activity was evaluated for dimethyl hydrazine-induced colorectal cancer in albino male Wistar rats. Biochemical and histological parameters were evaluated to understand their effectiveness for colon cancer therapy.

Results: The 5-FU immediate release (IR) formulations suspended in sodium carboxymethyl cellulose (SCMC) produced an immediate cytotoxic effect, whereas microspheres inhibited the proliferation of tumor cells to induce apoptosis over an extended time. Minimum inhibitory concentration (IC₅₀) values for both standard plain 5-FU and 5-FU-loaded microspheres were, respectively, 5.00 ± 0.004 µg/mL and 165 ± 1.9 µg/mL which showed the improved safety profile of the microsphere formulation. Tissue distribution showed high concentration of 5-FU in colon that was higher than IC₅₀ required to stop the growth or death of colon cancer cells from the colonic dysplasia in Duke’s Stage A. Significant reduction in tumor volume and multiplicity was observed with increased levels of liver enzymes in animals when treated with standard 5-FU formulation compared to 5-FU-loaded microspheres. Elevated levels of serum albumin, creatinine, leukocytopenia and thrombocytopenia were observed in animals for the standard 5-FU formulation.

Conclusion: The PEG-crosslinked CS microspheres of this study slowly released 5-FU up to 24 h to colonic region and enhanced the antitumor activity.

• Chitosan
• colorectal cancer
• 5-fluorouracil
• microsphere
• poly(ethyleneglycol)

Acknowledgements

Authors duly acknowledge the help of Dr. S.D. Kholkute and RMRC, Belgaum, in this study.

Declaration of interest

Mr. Kuntal Ganguly thank the Council of Scientific and Industrial Research (CSIR) [grant no. 08/558(0001)/2010-EMRI], New Delhi, India, for providing a research fellowship and financial support. Professor T. M. Aminabhavi acknowledges the All India Council for Technical Education (AICTE), New Delhi, India [grant no. AICTE. F.No. 1-51/RIFD/EF(13)/2011-12] for Emeritus Fellowship. Authors are thankful to VGST, Govt of Karnataka for financial support under K-FIST Level 2.