Abstract
Purpose: :
To investigate whether Nile Red (NR)-conjugated nanoparticles loaded with ptracer.Flt23k, an anti-angiogenic plasmid, can home to regions of laser-induced choroidal neovascularization (CNV) in a murine model.
Methods: :
Ptracer.Flt23k was purified after culturing a single colony in LB broth and conjugated with NR-nanoparticles. Poly-lactic-co-glycolic acid (PLGA) nanoparticles loaded with plasmid were prepared using the double emulsion solvent evaporation method and tagged with Nile Red, a fluorescent, lipophilic dye. CNV was induced in C57/BL6 mice using a diode pump argon laser with a power, duration and spot size of 150mW, 0.2s and 50um respectively Two weeks post laser injury, ptracerFlt23k-NR-PLGA nanoparticles (concentration of 1ug plasmid/ul DMSO) were delivered via tail vein. We observed the fundus at 5, 10 and 20 minutes post-injection with Spectralis SLO (Scanning Laser Ophthalmoscopy; Heidelberg Engineering) using a red-free filter. At 30 minutes post-injection the mice were sacrificed and choroidal flat mounts made. Tissue sections from organs receiving high blood flow were also made. Confocal microscopy was carried out at a laser wavelength of 594nm, with an HV, gain and offset of 465, 4 and 22 respectively.
Results: :
NR-Flt23k-PLGA nanoparticles successfully homed to focal areas of murine laser-induced CNV. CNV-specific fluorescence was seen on SLO and confirmed by confocal microscopy of the choroid. At 5 minutes post-injection, the fluorescence on SLO first appeared in the retinal vasculature followed by homogeneous localization to the CNV lesion at 20 mins. Retinal layers were negative for focal fluorescence. Liver sections displayed significant fluorescence, presumably due to tail vein clearance, whereas there was much less systemic fluorescence in skin and lung. Fluorescence in CNV has been detected upto 3 weeks after systemic administration of NR-ptracerFlt23k-PLGA nanoparticles.
Conclusions: :
NR-Flt23k-PLGA nanoparticles can be imaged in vivo in the posterior segment. This novel delivery system opens avenues for targeting and monitoring gene therapy with a nontoxic dye. Further studies investigating the long-term effects of this approach are currently underway.
Keywords: choroid: neovascularization • gene transfer/gene therapy • vascular endothelial growth factor