Intravenous transferrin, RGD peptide and dual-targeted nanoparticles enhance anti-VEGF intraceptor gene delivery to laser-induced CNV

Abstract

Choroidal neovascularization (CNV) leads to loss of vision in age-related macular degeneration (AMD), the leading cause of blindness in adult population over 50 years old. In this study, we developed intravenously administered, nanoparticulate, targeted nonviral retinal gene delivery systems for the management of CNV. CNV was induced in Brown Norway rats using a 532 nm laser. We engineered transferrin, arginine-glycine-aspartic acid (RGD) peptide or dual-functionalized poly-(lactide-co-glycolide) nanoparticles to target delivery of anti-vascular endothelial growth factor (VEGF) intraceptor plasmid to CNV lesions. Anti-VEGF intraceptor is the only intracellularly acting VEGF inhibitory modality. The results of the study show that nanoparticles allow targeted delivery to the neovascular eye but not the control eye on intravenous administration. Functionalizing the nanoparticle surface with transferrin, a linear RGD peptide or both increased the retinal delivery of nanoparticles and subsequently the intraceptor gene expression in retinal vascular endothelial cells, photoreceptor outer segments and retinal pigment epithelial cells when compared to nonfunctionalized nanoparticles. Most significantly, the CNV areas were significantly smaller in rats treated with functionalized nanoparticles as compared to the ones treated with vehicle or nonfunctionalized nanoparticles. Thus, surface-functionalized nanoparticles allow targeted gene delivery to the neovascular eye on intravenous administration and inhibit the progression of laser-induced CNV in a rodent model.

Figure 1

(a) Schematic representation of functionalized nanoparticles. (b) Schematic representation of choroidal neovascularization induction in Brown Norway rats using laser. (c) Schematic representation of potential mechanism of gene delivery to the retina by intravenously administered functionalized nanoparticles.

Figure 2

Intravenously administered nanoparticles are delivered to laser-treated (right) eye but not control (left) eye. Nanoparticles (10 mg) were administered on day 14 after laser treatment (eight spots; 150 mW, 100 ms, 100 µm) by injection into tail vein of Brown Norway (BN) rats. The rats were euthanized 24 h after nanoparticles injection. Both right and left eyes were enucleated and fixed in 4% paraformaldehyde for 1 h. The posterior segment (retina–choroid–sclera) of the eye was flatmounted. The flatmounts were stained for cell nuclei using 4′,6-diamidino-2-phenylindole (DAPI, blue) and analyzed by confocal microscopy in multitrack mode at a magnification of × 20. Images (7–8) were obtained so as to photograph the entire flatmount all around the optic nerve for each eye. (a) Representative image of right and left eye of the same rat showing the flatmount all around the optic nerve. The full flatmount image has been created by placing 7–8 partial confocal images in a jigsaw puzzle manner, to show distribution of nanoparticles in the flatmount. Nanoparticles (red) can be clearly seen in the right eye flatmount, but not in the left eye flatmount. (b) Representative confocal images of flatmounts of right eye (top) and left eye (bottom) obtained in various groups of rats all across the study. In each group, nanoparticles (red) were observed in the laser-treated (right) eye but not the left eye. Blank-NP, blank Nile red PLGA nanoparticles; Flt23K-NP, Flt23K-plasmid-loaded Nile red PLGA nanoparticles; Tf-Flt23K-NP, transferrin-conjugated Flt23K-plasmid-loaded Nile red PLGA nanoparticles; RGD-Flt23K-NP, RGD-peptide-conjugated Flt23K-plasmidloaded Nile red PLGA nanoparticles; RGD-Tf-Flt23K-NP, RGD-peptide- and transferrin-conjugated Flt23K-plasmid-loaded Nile red PLGA nanoparticles.

Figure 3

Representative confocal microscopy images of posterior segment flatmounts of laser-treated (right) eye at 24 h after intravenous nanoparticle administration. Eight laser burns (spot size 100 µm; power, 150 mW; duration, 0.1 s) were made in Brown Norway (BN) rats’ right eye only. On day 14 after laser treatment rats (N = 6) were administered one of the following types of nanoparticles by injection into the tail vein: unconjugated Flt23K-plasmid-loaded nanoparticles (Flt23K-NP), transferrin-conjugated Flt23K-plasmid-loaded nanoparticles (Tf-Flt23K-NP), RGD-peptide-conjugated Flt23K-plasmid-loaded nanoparticles (RGD-Flt23K-NP) or dual (RGD peptide and transferrin)-conjugated Flt23K-plasmid-loaded nanoparticles (RGD-Tf-Flt23K-NP). All the particles contained Nile red to enable their tracking in vivo. The rats were euthanized 24 h after nanoparticle injection, both eyes were enucleated and fixed in 4% paraformaldehyde for 1 h. The flatmounts of posterior segment (retina–choroid–sclera) were stained with 4′,6-diamidino-2-phenylindole (DAPI). The flatmounts were subsequently scanned using × 20 objective and three channels in multitrack mode: red (nanoparticles containing Nile red as a tracking dye), green (green fluorescent protein expression on transfection of retinal cells) and blue (cell nuclei stained with DAPI) channels.

Figure 4

Surface functionalization with RGD peptide and transferrin increases retinal delivery of PLGA nanoparticles on intravenous administration. At 24 h after nanoparticle administration the Nile red intensity in each image of right eye flatmount of each Brown Norway (BN) rat was quantified using LSM image examiner. The average Nile red intensity was compared across various groups in the study. The data are expressed as mean ± s.d. for N = 3. *P<0.05 as compared to nonfunctionalized Flt23K-plasmid-loaded nanoparticles (Flt23K-NP).

Figure 5

Biodistribution of nonfunctionalized and functionalized nanoparticles. Day-14 laser-treated Brown Norway (BN) rats were administered Nile-red-loaded PLGA nanoparticles (NP), transferrin functionalized NP (Tf-NP), RGD-peptide-functionalized NP (RGDNP) or RGD-peptide- and transferrin-functionalized NP (RGD-Tf-NP) intravenously. The animals were euthanized and various tissues were isolated at 24 h after nanoparticles injection. Nile red fluorescence in tissues was measured by spectrofluorometry at an excitation and emission wavelength of 544 and 590 nm, respectively. (a) Amount of nanoparticle in the entire tissue. (b) Amount of nanoparticles per gram of the tissue. Data are expressed as mean±s.d. for N = 6.

Figure 6

Functionalized nanoparticles, but not nonfunctionalized nanoparticles, transfect retinal pigment epithelial (RPE) cell layer in vivo. Frozen sections (10 µm thick) of the laser-treated eye were obtained at 48 h after nanoparticle administration. The sections were stained for cell nuclei with 4′,6-diamidino-2-phenylindole (DAPI) and analyzed by confocal microscopy in multitrack mode using × 40 objective. The images were obtained using differential interference contrast (DIC), blue (DAPI-stained cell nuclei), green (GFP expression) and an overlay of all the three channels. Blank-NP, blank nanoparticles; Flt23K-NP, Flt23K-plasmid-loaded nanoparticles; Tf-Flt23K-NP, transferrin-conjugated Flt23K-plasmid-loaded nanoparticles; RGD-Flt23K-NP, RGD-peptide-conjugated Flt23K-plasmid-loaded nanoparticles; RGD-Tf-Flt23K-NP, RGD-peptide- and transferrin-conjugated Flt23K-plasmid-loaded nanoparticles.

Figure 7

Functionalized nanoparticles reduce (a) retinal and (b) choroid-RPE vascular endothelial growth factor (VEGF) levels. On day 14 after choroidal neovascularization (CNV) induction the rats were administered one of the following treatments by injection into the tail vein: (1) vehicle, (2) naked Flt23K plasmid, (3) blank nanoparticles, (4) unconjugated Flt23K-plasmid-loaded nanoparticles (Flt23K-NP), (5) transferrin-conjugated Flt23K-plasmid-loaded nanoparticles (Tf-Flt23K-NP), (6) RGD-peptide-conjugated Flt23Kplasmid-loaded nanoparticles (RGD-Flt23K-NP), (7) dual (RGD peptide and transferrin)-conjugated Flt23K-plasmid-loaded nanoparticles (RGD-Tf-Flt23K-NP). The rats were euthanized 48 h after nanoparticle injection. Both the eyes were enucleated, snap frozen and stored at −80°C. Neural retina and choroid-RPE were dissected out and homogenized in phosphate-buffered saline (PBS, pH 7.4). The VEGF levels were quantified by sandwich enzyme-linked immunosorbent assay (ELISA). *P <0.05 as compared to vehicle, naked Flt23K, blank nanoparticles and nonfunctionalized nanoparticle-treated groups.

Figure 8

Functionalized nanoparticles reduce laser-induced choroidal neovascular area on histopathologic examination. On day 14 after choroidal neovascularization (CNV) induction the rats were administered one of the following treatments intravenously: (1) vehicle, (2) naked Flt23K plasmid, (3) blank nanoparticles, (4) unconjugated Flt23K-plasmid-loaded nanoparticles (Flt23K-NP), (5) transferrin-conjugated Flt23K-plasmid-loaded nanoparticles (Tf-Flt23K-NP), (6) RGD-peptide-conjugated Flt23K-plasmid-loaded nanoparticles (RGD-Flt23K-NP), (7) dual (RGD peptide and transferrin)-conjugated Flt23K-plasmid-loaded nanoparticles (RGD-Tf-Flt23K-NP). The rats were euthanized 2 weeks after nanoparticle injection. The eyes were enucleated and fixed in 4% paraformaldehyde overnight. Hematoxylin-and-eosin (H&E)-stained paraffin sections of the eye were subjected to histopathologic examination by a masked pathologist. (a) Representative light microscopic images of the CNV lesions (marked by *) from various groups of rats. (b) Quantitative comparison of CNV areas of various groups. CNV areas were measured using ImageJ software. *P < 0.05 as compared to naked plasmid group. wP<0.05 as compared to nonfunctionalized nanoparticles.

Figure 9

Functionalized nanoparticles reduce laser-induced choroidal neovascular lesion areas in fluorescein isothiocyanate (FITC)-dextran perfused full choroidal flatmounts. On day 14 after horoidal neovascularization (CNV) induction the rats were administered one of the following treatments intravenously: (1) vehicle, (2) naked Flt23K plasmid, (3) blank nanoparticles (blank NP), (4) unconjugated Flt23K-plasmid-loaded nanoparticles (Flt23K-NP), (5) transferrin-conjugated Flt23K-plasmid-loaded nanoparticles (Tf-Flt23K-NP), (6) RGD-peptide-conjugated Flt23K-plasmid-loaded nanoparticles (RGD-Flt23K-NP), (7) dual (RGD peptide and transferrin)-conjugated Flt23K-plasmid-loaded nanoparticles (RGD-Tf-Flt23K-NP). The rats were euthanized 2 weeks after nanoparticle injection. The rats after euthanasia were infused with phosphate-buffered saline (PBS, pH 7.4), 4% paraformaldehyde and 50 mg ml⁻¹ FITC-dextran 2× 10⁶ Da solution. The eyes were enucleated. Choroidal flatmounts were prepared and analyzed by a masked ophthalmic pathologist. (a) Representative confocal images of the CNV lesions in choroidal flatmounts from various groups of rats. (b) Quantitative comparison of CNV areas of various groups. CNV areas were measured using ImageJ software. *P <0.05 as compared to naked plasmid group. wP <0.05 as compared to nonfunctionalized nanoparticles.