Date of Award

5-1-2015

Degree Type

Thesis

Degree Name

Master of Science

Field of Study

Biomedical Sciences

Department

Graduate School of Biomedical Sciences

First Advisor

Andras G. Lacko

Second Advisor

David P. Cistola

Third Advisor

Raghu R. Krishnamoorthy

Abstract

Nucleic acid therapeutics involves the use of polynucleotides (DNA, RNA) as novel therapeutic agents for the treatment of a wide range of diseases including cancer and several metabolic and genetic disorders. However, the highly unstable nature of RNA molecules necessitates the use of drug carriers to prevent them from nuclease degradation and facilitate targeted delivery in vivo. Hence, this study was conducted to optimize the preparation of nanoparticle carriers in order to improve the stability of the polynucleotides (siRNA and mRNA). Additionally, as heterogeneity and stability of nanoparticle formulations are major issues preventing the clinical approval of therapeutic formulations this study was also focused on improving the homogeneity and the stability of the nanoparticles. In the siRNA study, reconstituted high density lipoprotein (rHDL) nanoparticles were used as the delivery vector. Optimization of siRNA-rHDL formulation was attempted with respect to homogeneity, size of the nanoparticle and entrapment efficiency of siRNA. The results showed that the inclusion of the lyophilization step in the preparation of nanoparticles resulted in a marginal increase in the homogeneity. The size analysis of siRNA rHDL nanoparticles using AFM and TEM imaging revealed the presence of spherical nanoparticles in the range of 10-16nm. Optimization studies with mRNA peptide nanoparticle formulation were conducted using a combination of cationic detergents and peptides at varied concentrations. The particle size analysis via Dynamic Light Scattering (DLS) detector revealed the presence of 268 nm diameter particles as the major component of the mRNA nanoparticle formulation that involved the combination of DOTAP (neutralizer) and Myr-5A (Apo A-I mimetic peptide). Further optimization of this formulation will be required to improve the homogeneity of the nanoparticles.

Comments

Conjeevaram Nagarajan,Bhavani Saranya. Characterization and Optimization of Nanoparticles for Polynucleotide delivery. Master of Science (Biomedical Sciences), April, 2015. 68 pp., 5 tables, 28 illustrations, bibliography, 47 titles.

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