339. Development of Multifunctional Nanoparticles for Targeted Gene Delivery
Authors: Rajendra N. Mitra, Min Zheng, Zongchao Han
Journal: Molecular Therapy (2016)

Purpose: Progress in gene therapy is determined by the highly efficient delivery strategies of therapeutic genes to the targeted tissues. The purpose of this study was to develop a nano-delivery system that has the capability of being functionalized to carry multiple imaging and targeting moieties, with the goal of developing rationally decorated multifunctional nanoparticles (NPs) for the delivery of therapeutic genes to the targeted site of ocular tissues.Methods: Primarily polymeric CK30PEG and the antioxidant nanoceria, which have a proven history of safe use for the intended application, were fabricated using standard lab protocols. The compounds were further conjugated with folic acid (FA)-NHS and amine reactive DyLight (DL) 550 NHS ester derivative by standard bioconjugation technique (FA-CK30PEG-DyLight550 and FA-nanoceria-DyLight550). In addition, the FA-CK30PEG-DyLight550 was taken as acetate salt and used to compact with plasmid DNA into NP formulation as described previously. The integrity of NP formulations were further characterized by TEM, DLS, FT-IR, and UV-vis. To determine the targeted efficiency and specificity, the NPs were tested in tumor cell lines (KB cells, neuroblastoma, and Y79-retinoblastoma) known to overexpress FA receptors, and cell lines (NIH 3T3, HTB and ARPE19) that have a low level of FA receptor expression. Western blot and confocal microscopy were performed to detect the FA protein level and to visualize the intracellular trafficking of NPs. Results: In line with previous reports, the UV absorbance spectra determined the formation of the conjugations, which were further confirmed by FT-IR analysis compared with the control folic acid molecules. In terms of CK30 NPs, the intact plasmid DNA came out from the digested NP formulation by trypsinsation and reflected the integrity of plasmid DNA inside formulation state. We observed a significant number of labelled NPs (red) into the nucleus (labelled with DAPI). Earlier it was observed that CK30PEG compacted NPs can traffic to the nucleus via nucleolin receptors. Therefore, in our current findings, we can consider that FA conjugation helped the NPs to enter inside the cell via folate receptor mediated endocytosis followed by nucleolin mediated uptake inside the nucleus. When we compared this result with FA-nanoceria-DyLight550 compacted DNA under the same condition, we found a promising result that nanoceria-DyLight550 compacted NPs (labelled red) only showed up inside the cytosol but not in the nucleus (Fig. 1). Therefore, our initial finding demonstrates that FA conjugation could boost up the CK30PEG-FA and nanoceria conjugated NPs to cross the cell membrane barriers for targeted delivery and express gene of interest. Conclusions: We explored a smart NP gene delivery system with multifunctionalities. The design of multifunctional NP complexes in this study can be custom-built and functionalized to target different diseases by binding the surface of NPs with a cell-specific targeting molecule and therapeutic specificity. We are now conducting these approaches by using different bio-markers in mouse models in vivo for targeted delivery. Project success will provide attractive tools and templates with broad potential for precision medicine.