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dc.contributor.authorRaut, Sangram
dc.contributor.authorSabnis, Nirupama
dc.contributor.authorLacko, Andras
dc.creatorConjeevaram Nagarajan, Bhavani Saranya
dc.description.abstractPurpose: Antisense Oligonucleotides (ASO) are short single stranded DNA oligomers that represent a novel class of Nucleic acid therapeutics with high potential to treat a broad range of disorders including cancer by modulating gene expression. Phosphorodiamidate Morpholinos (PMOs) are third generation ASOs which are chemically modified to possess neutral charge, high stability and resisitivity to nucleases and low toxicity/immunogenicity. Despite the enhanced pharmacokinetics, the cellular internalization of PMO is very poor and necessitates the need for a delivery vehicle. Recently, we have developed a simple formulation using an Apo-A1 mimetic peptide conjugated to myristic acid (Myr5A) that self-assembles into nanoparticles (NP) and has same functional properties as endogenous high density lipoproteins (HDL). Hence, we hypothesize that Myr 5A, an HDL mimetic lipo-peptide can potentially be used as a delivery vehicle to entrap ASO. Methods: Myr5A PMO NPs were prepared using microfluidics based Nanoassemblr platform with various concentrations of Myr5A peptide (2.5 5,10 mg/ml) and carboxyfluorescein PMO 80µg/ml. Entrapment efficiency of PMO was determined by fluorescence spectrometry. Dynamic Light Scattering (DLS) was used to analyze the size and the zeta potential. Time resolved (TR) anisotropy was measured to confirm the binding of PMO. Additionally, size exclusion chromatography by FPLC was also carried out to determine the entrapment of PMO and the heterogeneity of the population (if any). Results: Based on our preliminary findings, formulations prepared with 10mg/ml Myr 5A and 80ug/ml PMO resulted in more homogenous particles with entrapment efficiency of 40%. This data was further supported by the TR anisotropy as values for Free PMO and Myr5A PMO NP were 0.09±0.01 and 0.12±0.01 respectively. Furthermore, DLS measurements revealed the presence of homogenous particles with a diameter of 4.98+0.9nm and zeta potential of 0.9mV. Additionally, FPLC has shown distinctive difference in the elution of Free Myr 5A and Myr 5A PMO nanoparticles as they eluted at 12.95 and 17.8 minutes respectively indicating the difference in molecular size. Conclusion: The studies show that HDL mimetic Myr 5A peptide can be a promising candidate for the entrapment of PMOs. However, our future experiments designed to determine the knockdown efficiency of these nanoparticles in-Vitro and in-vivo would hold definitive answers for determining the efficiency of Myr5A PMO NP.
dc.titleOptimization of Antisense Oligonucleotides (ASO) Entrapped HDL Mimetic Lipo-peptide Nanoparticles

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