Gene transfer using micellar nanovectors inhibits choroidal neovascularization in vivo

Abstract

Purpose: Age-related macular degeneration caused by choroidal neovascularization (CNV) remains difficult to be treated despite the recent advent of several treatment options. In this study, we investigated the in vivo angiogenic control by intravenous injection of polyion complex (PIC) micelle encapsulating plasmid DNA (pDNA) using a mice CNV model.

Methods: The transfection efficiency of the PIC micelle was investigated using the laser-induced CNV in eight-week-old male C57 BJ/6 mice. Firstly, each mouse received intravenous injection of micelle encapsulating pDNA of Yellow Fluorescent Protein (pYFP) on days 1,3 and 5. The expression of YFP was analyzed using fluorescein microscopy and western blotting analysis. In the next experiments, each mouse received intravenous injection of micelle encapsulating pDNA of soluble Fms-like tyrosine kinase-1 (psFlt-1) 1,3 and 5 days after the induction of CNV and the CNV lesion was analyzed by choroidal flatmounts on day 7.

Results: Fluorescein microscopy and western blotting analysis revealed that the expression of YFP was confirmed in the CNV area after injection of the PIC micelle, but the expression was not detected neither in mice that received naked pDNA nor those without CNV. Furthermore, the CNV area in the mice that received intravenous injection of the psFlt-1-encapsulated PIC micelle was significantly reduced by 65% compared to that in control mice (p<0.01).

Conclusions: Transfection of sFlt-1 with the PIC micelle by intravenous injection to mice CNV models showed significant inhibition of CNV. The current results revealed the significant potential of nonviral gene therapy for regulation of CNV using the PIC micelle encapsulating pDNA.

Figure 1. Preparation of PIC micelles encapsulating pDNA.

Figure 2. Targeted gene expression in the CNV area after intravenous injection of pYFP-PM.

a: Choroidal flatmount and histological analysis demonstrated YFP fluorescence in the CNV area after intravenous injection of pYFP-PM. b: Western blotting analysis demonstrated that YFP protein was detected in the eyes with CNV, after intravenous injection of pYFP-PM. YFP expression was detected neither after generation of CNV alone nor after intravenous injection of pYFP-PM without CNV. c: The results of immunohistochemistry demonstrated that the expression of F4/80 was partially overlapping the expression of YFP, indicating that YFP protein expressed in F4/80-positive macrophage. CNV: choroidal neovascularization, pYFP-PM: the PIC micelles encapsulating pYFP (yellow fluorescent protein), PC: photocoagulation, bar:100 um.

Figure 3. Intravenous injection of psFlt-1-PM decreased the area of CNV.

Quantification of the CNV lesion demonstrated that the neovascularized area in the mice that received psFlt-1-PM was significantly reduced by 60% than that of control mice (n = 7, p<0.01), whereas the administration of pYFP-PM had no significant effects on the neovascularized area. The lower panels show representative micrographs. Arrows indicate the CNV lesion. CNV: choroidal neovascularization, psFlt-1-PM: the PIC micelle encapsulating psFlt-1 (fms-like tyrosine kinase-1), pYFP-PM: the PIC micelles encapsulating pYFP (yellow fluorescent protein).