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Hironori Uehara, Santosh Kumar Muddana, Xiaohui Zhang, Austin Bohner, Balamurali Ambati; Targeted delivery of sFLT-MO using cyclic RGD peptide suppresses laser CNV. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4034.
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© ARVO (1962-2015); The Authors (2016-present)
Soluble form Flt-1 (sFlt-1) is an endogenous anti-angiogenic factor by sequestering VEGF-A. Previously, we showed sFLT morpholino oligomer (sFLT-MO), which decreases membrane bound form Flt-1 (mbFlt-1) and increases sFlt-1 by controlling alternative splicing, suppresses laser induced choroidal neovascularization (CNV) in mouse via intravitreal injection. In this study, we examine whether cyclic RGD peptide conjugation can help sFLT-MO accumulation at CNV region via tail vein injection, and suppress CNV growth in mouse.
Cyclic RGD peptide was conjugated to fluorescence conjugated morpholino oligomer using SIAB reaction. To examine whether cRGD conjugation can enhance the entry of morpholino oligomer into endothelial cells, cRGD morpholino oligomer was added to mouse aortic endothelial cell culture. Fluorescence of morpholino oligomer was observed by fluorescence microscope the following day.Next, we examined whether cRGD morpholino can accumulate into laser induced CNV in mouse. After laser photocoagulation, 40 nmol (80mL) of cRGD-sFLT-MO-FITC, cRGD-STD-MO-FITC (control morpholino oligomer) or 80mL of DPBS was delivered through tail vein injection at 1 and 4 days post laser injury to fundus. On seven days post laser injury, we observed the fundus using autofluorescence. Laser CNV was stained with AlexaFluor647 conjugated with Isolectin GS-IB4, and the size of CNV was quantified by confocal fluorescence microscope.
The conjugation of cRGD peptide to the morpholino oligomer was confirmed with mass spectroscopy. 2µM of cRGD-sFLT-MO-FITC was enough to find FITC positive cells. But we could not detect any fluorescence from the unconjugated morpholino oligomer treated cells at even 40µM.On the seven day post laser injury, we found cRGD-STD-MO-FITC or cRGD-sFLT-MO-FITC accumulated at laser CNV region. cRGD-sFLT-MO-FITC treatments significantly suppressed laser CNV size (2.7x105 ± 0.3x105µm3, p<0.05 by student`s t-test) compared with the controls (DPBS: 5.1x105 ± 0.6x105µm3 and cRGD-STD-MO-FITC: 5.5x105 ± 0.8x105µm3).
In this study, we showed cyclic RGD peptide help to introduce morpholino oligomer to endothelial cells in vitro and in vivo. Tail vein injection of cRGD-sFLT-MO-FITC suppressed laser induced CNV. Thus, cRGD peptide can be a good candidate for morpholino oligomer delivery to endothelial cells in vitro and in vivo.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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