273. MMPs-Responsive Release of siRNA from 4-Arm PEG siRNA Conjugate

Abstract

Small interfering RNA (siRNA) has drawn much attention for its unique and effective gene silencing mechanism although its poor cellular membrane permeability, limited stability and low flexibility still remain major concerns. In particular, when siRNAs are complexed with the cationic carriers, the resultant complexes shows much lower physical stability than those prepared with plasmid DNA due to the low charge density of siRNA. Thus, we conjugated siRNAs to 4-arm polyethylene glycol (PEG) via a matrix metalloproteinases (MMPs)-cleavable peptide to enhance complexation with linear polyethyleneimine (LPEI). In addition, release of siRNA/LPEI complex from 4-arm PEG-siRNA/LPEI and subsequent gene silencing efficacy were investigated in diabetic ulcers. After synthesis of 4PEG-siRNA conjugate, the conjugation amount of siRNA per 4-arm PEG was 2.6 (mol/mol). According to gel retardation assay, 4PEG-siRNA/LPEI showed superior association to siRNA/LPEI at the same N/P ratio, which suggests the flexible structure of 4PEG-siRNA conjugate is more favorable for complexation with LPEI than naked siRNA. The particle size and zeta potential of 4PEG-siRNA/LPEI was 1415.5±73.1nm and 13.8±1.9mV, respectively. However, those values were changed similarly to those of siRNA/LPEI in the presence of MMP-2 because the MMPs-cleavable linker between 4-arm PEG and siRNA was digested by MMP-2. In addition, 4PEG-siRNA/LPEI complexes could not be uptaken by cells before dissociation by MMP-2 due to their huge size. In diabetic ulcers, 4PEG-siRNA/LPEI was rapidly cleaved and separated to siRNA/LPEI fraction and showed higher gene silencing of MMP-2 for 14 days. Thus, wound recovery was subsequently enhanced by inhibiting MMP-2 expression in diabetic ulcers. View Large Image | Download PowerPoint Slide