Conjugation of Anti-EpCAM Antibody on Alginate–RIP MJ-30 Nanoparticle through Carbodiimide Reaction as a Model of Targeted Protein Therapy

Hilda Ismail,Ronny Martien,Ag Yuswanto,M Arief Nur Ikhsan,Ummi H Ciptasari,Sismindari Sismindari,Fidya Suryani

doi:10.14499/indonesianjpharm30iss1pp52

Hilda Ismail, Ronny Martien + Show 5 more

Open Access

https://doi.org/10.14499/indonesianjpharm30iss1pp52

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Abstract

Ribosome inactivating proteins from Mirabilis jalapa L. (RIP MJ) has shown higher cytotoxic activity when being formulated as a nanoparticle. However, the selectivity of the delivery system is also an important aspect when it comes to cytotoxic cell therapy. Epithelial cell adhesion molecule (EpCAM) is a monomeric glycoprotein which is overexpressed in epithelial cancer cells. This study aim was to develop a model of targeted protein delivery system by formulating the base fraction of RIP MJ (RIP MJ-30) into alginate nanoparticles and conjugating it with anti-EpCAM antibody. RIP MJ-30 was formulated in to nanoparticle using alginate and CaCl2 as cross-linker. Optimization of conjugation reaction condition was done in the pH variation of 4.5, 5.5, and 6.5. The success of conjugation was analyzed qualitatively using native polyacrylamide gel electrophoresis (native-PAGE) method and BCA assay. The optimum formula of RIP MJ-30 nanoparticles was produced using 0.3% alginate and 0.2% CaCl2. Results indicated that optimum conjugation reaction was carried out at pH level of 5.5. The optimum native-PAGE condition was by using 8% polyacrylamide gel in duration of 6h. Characterization of nanoparticle resulted in particle size of 205.0nm, zeta potential of -6.9mV, entrapment efficiency of 71.11±4.84%, and conjugation efficiency of 89.55±6.18%. It was concluded that RIP MJ-30 was successfully formulated into alginate nanoparticle and conjugated to anti-EpCAM antibody through carbodiimide reaction using 1-ethyl-(dimethylprophilamine) carbodiimide (EDAC).

Highlights

Ribosome Inactivating Proteins (RIP) are proteins that usually found in plants, and have the ability to irreversibly disturb protein synthesis process
Formulation of Ribosome inactivating proteins from Mirabilis jalapa L. (RIP MJ)-30 nanoparticle was conducted based on Saraei et al (2013) (Saraei et al, 2013)
Entrapment efficiency of RIP MJ-30 alginate nanoparticles was determined by measuring the un-reacted RIP MJ-C using BCA assay kit (Sigma Aldrich)

Summary

Introduction

Ribosome Inactivating Proteins (RIP) are proteins that usually found in plants, and have the ability to irreversibly disturb protein synthesis process. This process happened by ribosome inactivation through specific mechanism known as site-specific rRNA N-glikosidase activity (Barbieri et al, 1993; Stirpe et al, 2006). Locally known as ‘four o’clock plant’ in Indonesia, have been found to contain RIPs with anti-cancer properties (Sismindari et al, 2010). Ikawati et al (2006) has isolated RIP-like protein with molecular weight of 30 kDa from M. jalapa L. leaves, called MJ-30, which was toxic against T47D and SiHa cells (Ikawati et al, 2006). One potential strategy to overcome this challenge is through targeted nanoparticle formulation of the protein using biopolymer, and followed by conjugation of antibody as a cancer cell targeting molecule

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