Topical Application of Cell-Penetrating Peptide Modified Anti-VEGF Drug Alleviated Choroidal Neovascularization in Mice

Abstract

Background: Age-related macular degeneration (AMD) stands as the foremost cause of irreversible central vision impairment, marked by choroidal neovascularization (CNV). The prevailing clinical approach to AMD treatment relies on intravitreal injections of anti-vascular endothelial growth factor (VEGF) drugs. However, this method is encumbered by diverse complications, prompting exploration of non-invasive alternatives such as ocular administration via eye drops for anti-VEGF therapy.

Methods: Two complexes, 5-FITC-CPP-Ranibizumab (5-FCR) and 5-FITC-CPP-Conbercept (5-FCC), were synthesized by incorporating the anti-VEGF drugs Ranibizumab (RBZ) or Conbercept (CBC) with cell-penetrating peptide (CPP). Circular dichroism spectrum (CD) facilitated complexes characterization. Eye drops was utilized to address laser-induced CNV in mice. Fluorescein fundus angiography (FFA) observe the CNV lesion, while FITC-dextran and IB4 dual fluorescent staining, along with hematoxylin-eosin (HE) staining, assessed in lesion size. Tissue immunofluorescence examined CD31 and VEGF expression in choroidal/retinal pigment epithelial (RPE) tissues. Biocompatibility and biosafety of 5-FCR and 5-FCC was evaluated through histological examination of various organs or cell experiments.

Results: Both 5-FCR and 5-FCC exhibited favorable biocompatibility and safety profiles. VEGF-induced migration of Human umbilical vein endothelial cells (HUVECs) significantly decreased post-5-FCR/5-FCC treatment. Additionally, both complexes suppressed VEGF-induced tube formation in HUVECs. FFA results revealed a significant improvement in retinal exudation in mice. Histological examination unveiled the lesion areas in the 5-FCR and 5-FCC groups showed a significant reduction compared to the control group. Similar outcomes were observed in histological sections of the RPE-choroid-sclera flat mounts.

Conclusion: In this study, utilizing the properties of CPP and two anti-VEGF drugs, we successfully synthesized two complexes, 5-FCR and 5-FCC, through a straightforward approach. Effectively delivering the anti-VEGF drugs to the target area in a non-invasive manner, suppressing the progression of laser-induced CNV. This offers a novel approach for the treatment of wet AMD.

Keywords: age-related macular degeneration; cell-penetrating peptide; choroidal neovascularization; conbercept; drug delivery; ranibizumab.

Graphical abstract

Figure 1

Synthesis and characterization of 5-FITC-CPP, 5-FCR, and 5-FCC. (A) mean particle size of 5-FITC-CPP, 5-FCR, 5-FCC. (B) Zeta surface potential of 5-FITC-CPP, 5-FCR, 5-FCC. (C) CD data of RBZ with increasing amounts of CPP in the complex. (D) CD data of CBC with increasing amounts of CPP in the complex.

Figure 2

Cell viability effects of 5-FITC-CPP, 5-FCR, and 5-FCC on ARPE-19 cells and HUVECs were analyzed using the CCK-8 assay. (A) The activity of ARPE-19 cells under the intervention of different concentrations of 5-FITC-CPP. (B) The activity of HUVECs under the intervention of different concentrations of 5-FITC-CPP. (C) The activity of ARPE-19 cells under the intervention of different concentrations of 5-FCR and 5-FCC. (D) The activity of HUVECs under the intervention of different concentrations of 5-FCR and 5-FCC. NS: no significance.

Figure 3

5-FITC was delivered by CPP to ARPE-19 cells. Scale bar: 100 µm.

Figure 4

5-FCR or 5-FCC attenuated VEGF-induced migration of HUVECs, which was measured by wound-healing assay. (A) After treatment with 20 ng/mL of VEGF with or without 5-FCR or 5-FCC for 24h and 48 h, the migration of HUVECs with different treatments were detected via wound-healing assay analysis. Scale bar: 200 μm. Migrated cells at 24 h (B) and 48 h (C) were quantified by counting three random vision fields under a microscope. Group 1: control, Group 2: 20 ng VEGF treatment, Group 3: 20 ng VEGF with 150 μg/mL CPP treatment, Group 4: 20 ng VEGF with 150 μg/mL CPP+10 μg/mL RBZ treatment, Group 5: 20ng VEGF with 150 μg/mL CPP+100 μg/mL RBZ treatment, Group 6: 20ng VEGF with 150 μg/mL CPP+10 μg/mL CBC treatment, Group 7:20ng VEGF with 150 μg/mL CPP+100 μg/mL CBC treatment. **P<0.01, ***P<0.001, **** P<0.0001.

Figure 5

5-FCR or 5-FCC attenuated VEGF-induced HUVECs tube formation. (A) The tube formation was stimulated by VEGF, and inhibited by 5-FCR/5-FCC incubation. Scale bar: 200 μm (B) The numbers of nodes, junctions and segments were quantified by imageJ software. (C) The relative tube length (%) was quantified by imageJ software. Group 1: control, Group 2: 20 ng VEGF treatment, Group 3: 20 ng VEGF with 150 μg/mL CPP treatment, Group 4: 20 ng VEGF with 150 μg/mL CPP+10 μg/mL RBZ treatment, Group 5: 20ng VEGF with 150 μg/mL CPP+100 μg/mL RBZ treatment, Group 6: 20ng VEGF with 150 μg/mL CPP+10 μg/mL CBC treatment, Group 7:20ng VEGF with 150 μg/mL CPP+100 μg/mL CBC treatment. **P<0.01, ***P<0.001, **** P<0.0001.

Figure 6

FFA was performed to observe the CNV lesion. Fluorescein leakage was marked by red arrow.

Figure 7

The CNV areas were stained and measured by HE assays. (A) The retina and choroid were examined using HE staining to visualize their structure. The presence of black spots indicated the areas of CNV. Scale bar: 100 µm. (B) The CNV length, thickness and areas were quantified in HE staining via ImageJ software. **P<0.01, ***P<0.001.

Figure 8

RPE-choroid-sclera flat mounts were prepared by perfusion with FITC-dextran and IB4 staining to measure CNV areas. (A) CNV areas with FITC-dextran and IB4 staining were captured by fluorescence microscope in non-treatment and RBZ treatment groups. (B) CNV areas with FITC-dextran and IB4 staining were captured by fluorescence microscope in CBC treatment groups. Scale bar: 200 µm. (C) CNV areas were quantified via ImageJ software. ***P<0.001.

Figure 9

Double immunostainings with endothelial marker CD31 and VEGF in the retina/choroid cryosections were performed in nine groups and images were recorded by fluorescence microscope. Scale bar: 100 µm.

Figure 10

Biosafety analysis of 5-FCR and 5-FCC eye drops. (A) Results of fluorescein sodium staining of the cornea of the mice on the 28th day of the intervention. (B) Results of HE staining of the cornea and retina. Scale bar: 100 μm. (C) Results of HE staining of different internal organ. Scale bar: 100 μm.