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

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

doi:10.14499/indonesianjpharm30iss1pp52

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

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

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).

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References

Barbieri L., Batelli MG., and Stirpe F. 1993. Ribosome-inactivating Proteins from plants. Biochim Biophys Acta 1154: 237-82.

Chen Y., Cai J., Xu QC. and Chen ZW. 2004. Atomic force bio-analytics of po- lymerization and aggregation of phyco-erythrin-conjugated immunoglobulin G molecules. Mol Immunol 41:1247-52.

Ciptasari U., 2016, Skripsi. Conjugation Reaction of Anti-EpCAM Antibody on Nanoparticles of Base Fraction of Ribosome Inactivating Protein Mirabilis Jalapa L. (RIP MJ) Using EDAC as Catalyst, Yogyakarta; Faculty of Pharmacy, Universitas Gadjah Mada

Fang C., Shi B., Pei YY., Hong MH., Wu J., Chen HZ. 2006. In vivo tumor targeting of tumor necrosis factor-alpha-loaded stealth nanoparticles: effect of MePEG molecular weight and particle size. Eur J Pharm Sci 27:27-36.

Fujita M., Lee B., Khazenzon NM., Penichet ML., Wawrowsky KA., et al., 2007, Brain tumor tandem targeting using a combination of

Gupta RB. 2006. Fundamentals of drug nanoparticles in nanoparticle technology for drug delivery. In Gupta RB and Kompella UB (Eds), Nanoparticle Technology for Drug Delivery (pp 1-9). New York, NY: Taylor & Francis Group.

Hermanson TG. 1996. Bioconjugation Techniques. New York: Academic Press Inc. 3-4:169-70.

Ikawati Z., Sudjadi, Sismindari. 2006. Cytotoxicity against Tumor Cell Lines of a Ribosome-Inactivating Protein (RIP)-Like Protein Isolated from Leaves of Mirabilis jalapa L. Malays J Pharm Sci. 4:31–41.

Ikhsan AN. 2017. Skripsi. Effect of Medium pH and Reactant Concentration on Conjugation Reaction Between Sodium Alginate and Antibody Anti-EpCAM catalyzed by EDAC. Yogyakarta; Faculty of Pharmacy, Universitas Gadjah Mada.

Jin L., Hong Y., Pyo J., Song H., Kang JY., Lee SW., Yoon DS., Kim B., and No K. 2009. Monitoring of Swelling and Degrading Behavior of Alginate Beads using Optical Tweezers. Biochip Journal. 3: 213-8.

Lu J., Liong M., Zink JI., Tamanoi F. 2007. Mesoporous silica nanoparticles as a delivery system for hydrophobic anticancer drugs. SMALL.; 3:1341–1346.

Merril CR. 1990. Gel-staining techniques. In Deutscher MP (Ed) in Guide to Protein Purification. Methods Enzymol (182:477–88). California: Academic press Inc.

Mohanraj and Chen. 2006. Nanoparticles–a review. Trop J Pharm Res 5:561-73.

monoclonal antibodies attached to biopoly(β-l-malic acid). J Control Release 122:356-63

Pertiwi D., Martien R., Sismindari and Ismail H. 2018. Formulation of nanoparticles ribosome inactivating protein from Mirabilis jalapa L. (RIP MJ) conjugated with anti-EpCAM antibody using low chain chitosan-pectinand cytotoxcic acti-vity against breast cancer cell line. Pak. J. Pharm. Sci Vol 31(2), 379 -384

Ranjan AP., Mukerjee A., Helson L., Vishwanatha JK. 2012. Scale up, op-timization and stability analysis of Curcumin C3 complex-loaded nanopar-ticles for cancer therapy. J Nano-biotechnology 10:38.

Saraei, Dougighi FM., Zolfagharian N., Bidhendi HM., Khaki S., and Inanlou P. 2013. Design and Evaluate Alginate Nanoparticle as A Protein Delivery System. Archives of Razi Institute. 68:139-46.

Sismindari, Hartati MS., Adhyatmika. 2010. Cytotoxic selectivity of MJC0.3 and MJC0.5, acidic Ribosome-inactivating Proteins isolated from Mirabilis jalapa L. leaves against various cancer cell-lines, J Med Sci 42:39 – 43.

Stirpe F., and Batelli MG. 2006. Ribosome-Inactivating Proteins: Progress and Problems. Cell Mol Life Sci 63:1850–66.

Suryani F. 2016. Skripsi. Effect of Polyacrylamide Concentration and Developing Time on Electrophoresis Analysis for Nanoparticle RIP MJ-30 – BSA Conjugation. Yogyakarta; Faculty of Pharmacy, Universitas Gadjah Mada.

Torchilin and Lukyanov. 2003. Peptide and protein drug delivery to and into tumors: challenges and solutions. Drug Discov Today 15:259-66.

Vanparijs N., Maji S., Louage B., Voorhaar L., Laplace D., et al. 2015. Polymer-protein conjugation via a “grafting to” approach: a comparative study of the performance of protein-reactive RAFT chain transfer agents. Polym Chem 6:5602–14.

Wicaksono PA., Sismindari, Martien R., Ismail H. 2016. Formulation and cytotoxicity of ribosome-inactivating protein Mirabilis jalapa L. nanoparticles using alginate-low viscosity chitosan conjugated with anti-EpCAM antibodies in the T47D breast cancer cell line. Asian Pac J Cancer Prev 17:2277-84.

Wittig I. and Schägger H. 2005. Advantages and limitations of clear-native PAGE. Proteomics. 5: 4338–46.

DOI: http://dx.doi.org/10.14499/indonesianjpharm30iss1pp52