Formulation of Medium Viscosity Chitosan-Pectin –MJ Protein Nanoparticles Conjugated with Anti-Ep-CAM and Its Cytotoxicity Against T47D Breast Cancer Cell Lines

Abstract

Chitosan nanoparticle could become potential formula to protect protein degradation during therapy, since chitosan nanoparticles have “proton sponge hypothesis” mechanism on its protection. Chitosan and pectin is used as basic formula of drug delivery because of its biodegradable and biocompatible properties. Chitosanpectin nanoparticles can be formulated by polyelectrolit complex. EpCAM showed excessive expression in epithelial cancer cells thus can be used as a therapeutic biomarker. MJ protein, a Ribosome-Inactivating Proteins (RIPs) isolated from Mirabilis jalapa L had a higher cytotoxicity on malignant cells than normal cells. MJ protein need to be formulated to protect from proteosome degradation in endosome. The aim of this research was to develop MJ protein-chitosan-pectin nanoparticles and conjugated with anti EpCAM for breast cancer therapy. Mj protein was extracted from M.jalapa leaves. RIPs activity was assayed by supercoiled DNA cleavage. MJ protein were loaded into chitosan nanoparticles using medium viscous chitosan and pectin as cross-linker with polyelectrolit complex method. Anti EpCAM was conjugated to MJ protein-chitosan-pectin nanoparticles by carbodiimide reaction and characterized for its entrapment efficiency, morphology by transmission electron microscope, particles size, and zeta potential. MJ protein nanoparticles conjugated anti EpCAM and without anti EpCAM were cytotoxicity assayed toward T47D and Vero cell lines. MJ protein was able to cleave the supercoiled DNA into linear and nicked-circular ones. The nanoparticles optimal concentration of medium viscous chitosan: MJ protein: pectin was 0.01%: 0.01%: 1% (m/v). A high entrapment efficiency of MJ protein nanoparticles was 98.97 ± 0.07%. Morphology nanoparticles showed an amorphic structure with 200.00 nm particles size. The nanoparticles conjugated anti EpCAM showed average particles size 367.67nm, polydispersity index 0.332, and zeta potential +39.97mV. MJ protein-chitosan-pectin nanoparticles conjugated anti EpCAM and unconjugated both had higher cytotoxicity with the IC50 57.64 μg/mL and 46.84 μg/mL respectively against T47D and 99.38 μg/mL and 111.34 μg/mL against Vero cell lines compared to MJ protein with IC50 of 3075.61 μg/mL against T47D and 3286.88 μg/mL against Vero cell lines. Both MJ protein-nanoparticles could increase the cytotoxicity effects about 50 times compared to the unformulated MJ protein activity, however had less specificity toward T47D and Vero cell lines.

Keywords

• Mirabilis jalapa L.
• RIPs
• nanoparticle
• pectin
• chitosan
• anti EpCAM