Nanoparticle formulation enhances the delivery and activity of a vascular endothelial growth factor antisense oligonucleotide in human retinal pigment epithelial cells

Abstract

The objective of this study was to investigate the delivery and activity of a vascular endothelial growth factor (VEGF) antisense oligonucleotide in a human retinal pigment epithelial cell line (ARPE-19) using a biodegradable nanoparticulate delivery system. A 19-mer antisense phosphorothioate oligonucleotide (PS-ODN) complementary to bases 6-24 relative to the translational start site of the VEGF mRNA, a sense PS-ODN and a mismatch PS-ODN were examined for the inhibition of secretion and mRNA expression of VEGF using an enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction, respectively. Nanoparticles of the antisense oligonucleotides were formulated using a poly(lactide-co-glycolide) (50:50) copolymer using a double emulsion solvent evaporation method. After preparing nanoparticles, drug loading, encapsulation efficiency and particle size were determined. The cells were exposed to either plain solution of oligonucleotide or nanoparticles of oligonucleotide from Day 3 through Day 6. Alternatively, the cells were incubated with PS-ODNs and lipofectin for 4 h on Day 4. In all studies, VEGF secretion and mRNA expression were determined on Day 6. The particle size, drug loading and encapsulation efficiency were 252 nm, 5.5% and 16.5%, respectively. The antisense PS-ODN inhibited VEGF mRNA and protein secretion when delivered using nanoparticles or lipofectin but not in its free form. This was consistent with the ability of nanoparticles and lipofectin to elevate the cellular uptake of the oligonucleotide by 4-fold and 13-fold, respectively. Neither mismatch nor sense oligonucleotides inhibited VEGF secretion. In conclusion, biodegradable nanoparticles enhance cellular delivery of a VEGF antisense oligonucleotide and inhibit VEGF secretion and mRNA expression in a human retinal pigment epithelial cell line.

Antiapoptotic Properties of α-Crystallin–Derived Peptide Chaperones and Characterization of Their Uptake Transporters in Human RPE Cells

“…This is consistent with our previous findings that ARPE-19 cells can internalize nano-as well as microparticles through nonsaturable processes involving phagocytosis or pinocytosis, depending on particle size. 38,39 Both of these mechanisms were also shown for RPE cells in vivo. 40 Furthermore, encapsulation of a 19-mer oligonucleotide by poly (D-Llactide-co-glycolic acid) nanoparticle enhanced uptake of the oligopeptide.…”

“…40 Furthermore, encapsulation of a 19-mer oligonucleotide by poly (D-Llactide-co-glycolic acid) nanoparticle enhanced uptake of the oligopeptide. 39 Further studies will be needed to fully elucidate the mechanism of entry of PCL particles into hfRPE cells. We found that both aA and aB-crystallin mini-chaperones protected RPE cells from H 2 O 2 -induced cell death, and inhibited caspase-3 activation as was previously shown for the parent protein aB-crystallin.…”

Inhibitory efficacy of hypoxia-inducible factor 1α short hairpin RNA plasmid DNA-loaded poly (D, L-lactide-co-glycolide) nanoparticles on choroidal neovascularization in a laser-induced rat model

“…74 Therefore, RPE cells are especially of our interest as they are a potential target for HIF-1a-directed siRNA transfer, which aims at arresting or reversing the processes leading to CNV. RPE cells are able to ingest NPs or microparticles in vitro 40,42,75 or after in vivo subretinal injection. 43 In vivo, it was reported that PLGA NPs injected into the vitreous of rat eyes followed a 'trans-retinal pathway' and underwent a rapid internalization into the RPE cells layer.…”

Basic Research and Clinical Application of Drug Delivery Systems for the Treatment of Age-Related Macular Degeneration

“…Another therapeutic approach is the inhibition of gene expression using antisense oligonucleotides, aptamers and siRNA (Fattal & Bochot, 2006;Fattal & Bochot, 2008;Tanito, Li, Elliott, et al 2007). Aukunuru et al showed that nanoparticles formulated using a PLGA (50:50) copolymer could deliver VEGF antisense oligonucleotide to the human ARPE-19 cell line, and inhibit VEGF secretion and mRNA expression (Aukunuru, Ayalasomayajula & Kompella, 2003). In a study performed by Carrasquillo et al (Carrasquillo, Ricker, Rigas, et al 2003) and summarized by Moshfeghi and Peyman (Moshfeghi & Peyman, 2005), the anti-VEGF RNA aptamer (EYE001, Macugen®, OSI Pharmaceuticals, NY, USA) was incorporated into PLGA microspheres to develop a sustainedrelease inhibition of VEGF for the treatment of neovascular AMD.…”