A. Sakhrie, A. Kalluri, C.V. Kumar, M.I. Khan
University of Connecticut, CT,
United States
Keywords: nanoparticle, EGFP mRNA, nanocomplex, cationic BSA nanocomplex
Summary:
Albumin–drug conjugates/complexes have been exploited for decades to use as drug delivery as they have the advantage of being biodegradable, nonantigenic and metabolizable. In our study, the COOH groups of aspartic and glutamic acid side chains of BSA were modified systematically by covalent attachment of polyamines using EDC chemistry and then coupled with polyethylenehexamine (PEHA) which resulted in a decrease in the negative charge of the protein. The nanoparticle was characterized by using DLS, zetasizer and TEM techniques. The size of the nanoparticle was shown to be 10-14 nm and the charge was found to be +45 ±3. Polyamines inherently bind strongly to polynucleotides and stabilize both secondary and tertiary structure.Therefore, the resulting cationic BSA-polyamine nanocomplex has the ability to bind with nucleic acids. The cytotoxicity and the cellular uptake of the nanoparticle was evaluated using Vero cells and HeLa cells. The binding and the expression of the nanoparticle with the nucleic acid was studied using in-vitro transcription-messenger RNA (IVT- mRNA) encoding enhanced green fluorescent protein (EGFP). The binding study by gel electrophoresis showed that the migration of the cationic BSA nanoparticle with the EGFP mRNA nanocomplex is altered. The cationic BSA nanoparticle now migrates towards the positive pole and when the molar ratio of the nanoparticle was increased, the nanocomplex formed became more cationic. The intracellular delivery and expression of EGFP mRNA will be evaluated in the HeLa cells. The successful delivery and expression of the mRNA using the cationic BSA nanoparticle will demonstrate its potential use in therapeutics due to the low cost of manufacture and simplicity of designing the mRNA.