Topical Ophthalmic Liposomes Dual-Modified with Penetratin and Hyaluronic Acid for the Noninvasive Treatment of Neovascular Age-Related Macular Degeneration

Abstract

Introduction: Since intrinsic ocular barrier limits the intraocular penetration of therapeutic protein through eye drops, repeated intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF) agents are the standard therapy for neovascular age-related macular degeneration (nAMD), which are highly invasive and may cause particular ocular complications, leading to poor patient compliance.

Methods: Using Penetratin (Pen) as the ocular penetration enhancer and hyaluronic acid (HA) as the retina-targeting ligand, a dual-modified ophthalmic liposome (Penetratin hyaluronic acid-liposome/Conbercept, PenHA-Lip/Conb) eye drop was designed to non-invasively penetrate the ocular barrier and deliver anti-VEGF therapeutic agents to the targeted intraocular tissue.

Results: PenHA-Lip effectively penetrates the ocular barrier and targets the retinal pigment epithelium via corneal and non-corneal pathways. After a single topical administration of conbercept-loaded PenHA-Lip (PenHA-Lip/Conb), the intraocular concentration of conbercept peaked at 18.74 ± 1.09 ng/mL at 4 h, which is 11.55-fold higher than unmodified conbercept. In a laser-induced choroidal neovascularization (CNV) mouse model, PenHA-Lip/Conb eye drops three times daily for seven days inhibited CNV formation and progression without any significant tissue toxicity and achieved an equivalent effect to a single intravitreal conbercept injection.

Conclusion: PenHA-Lip efficiently and safely delivered conbercept to the posterior eye segment and may be a promising noninvasive therapeutic option for nAMD.

Keywords: Penetratin; conbercept; dual-modified liposomes; hyaluronic acid; ocular drug delivery.

Graphical abstract

Figure 1

Schematic diagram of the preparation and action mechanism of PenHA-Lip/Conb eye drops.

Figure 2

Schematic diagram of the in vivo therapeutic procedure for laser-induced CNV mice (n = 3).

Figure 3

Physical characterization and ex vivo corneal penetration ability. (A) TEM image of PenHA-Lip/Conb (scale bar = 100 nm). (B) Drug release profiles of conbercept in different formulations at pH 7.4 and 6.3 (n = 3). (C) Permeability curves and (D) apparent permeability coefficients of macromolecular cargos (BSA) in different liposomes to cross the isolated rabbit corneas (n = 3). Data analyzed by ordinary one-way ANOVA. **p< 0.01, ***p<0.001.

Figure 4

In vitro cellular uptake and targeting ability of different liposomes. (A) Laser scanning confocal images of ARPE-19 cells after 4 h incubation with Nr-labeled liposomes (scale bar = 25 μm). (B) Flow cytometry results and (C) quantitative analysis of ARPE-19 cells after 4 h incubation with Nr-labeled liposomes (n = 3). Data analyzed by ordinary one-way ANOVA. **p< 0.01, ***p<0.001.

Figure 5

In vitro anti-angiogenic ability of drug-loaded liposomes under the induction of VEGF. (A) Proliferation curves of HUVEC cells treated by different conbercept formulations (n = 3). (B) Representative images of wound healing in HUVEC cells treated by different conbercept formulations (scale bar = 100 μm). (C) Representative images of tube formation in HUVEC cells treated by different conbercept formulations for 4 h (scale bar = 50 μm). (D) Quantitative analysis of wound closure in different groups after cell scratch (n = 3). (E) Quantitative analysis of relative tube length in different groups when compared with the control group (n = 3). Data analyzed by ordinary one-way ANOVA. **p< 0.01, ***p<0.001.

Figure 6

The ocular penetration path and RPE targeting study of PenHA-Lip. (A) Confocal images of corneal tissue after PenHA-Lip/FAM topical instillation for in vivo and ex vivo (scale bar = 50 μm). (B) Fluorescence distribution in the corneoscleral limbus after PenHA-Lip/FAM topical instillation in vivo (scale bar = 50 μm). (C) Fluorescence colocalization of PenHA-Lip/FAM (green fluorescence) and CD44 (red fluorescence) in RPE layer after PenHA-Lip/FAM topical instillation in vivo (scale bar = 50 μm).

Figure 7

The intraocular distribution of macromolecular cargos carried by different liposome eye drops. (A) Fluorescence distribution in the whole eye at 30 min (scale bar = 500 μm). (B) Fluorescence distribution in cornea and retina at 30 min (scale bar = 50 μm). (C) Dynamic fluorescence distribution in cornea and retina during 24 h (scale bar = 50 μm).

Figure 8

Ocular pharmacokinetic behavior and therapeutic effects of PenHA-Lip/Conb eye drops. (A) Ocular pharmacokinetic behavior of PenHA-Lip/Conb eye drops after a single topical instillation (n = 4). (B) The quantitative comparison of conbercept concentrations at 4 h in different groups (n = 4). (C) FFA images on day 7 in laser-induced CNV mice treated with eye drops t.i.d. or a single intravitreal injection (scale bar = 200 μm). (D) Quantitative statistics of fluorescence leakage areas in each group (n = 3). (E) Representative immunofluorescence images on day 7 in laser-induced CNV mice treated with eye drops t.i.d. or a single intravitreal injection (scale bar = 200 μm). (F) Quantitative statistics of CNV lesion areas in each group (n = 3). The black arrow represents the fluorescence leakage area; the white arrow represents the CNV lesion area. Data analyzed by ordinary one-way ANOVA. *p< 0.05, **p< 0.01, ***p< 0.001.

Figure 9

Biocompatibility evaluation of liposomes. Cell proliferation effects of different liposome concentrations on (A) HCEC cells and (B) ARPE-19 cells (n = 3). (C) Hydration value (n = 3) and (D) HE stained sections of isolated rabbit corneas after the corneal penetration test (scale bar = 50 μm). (E) Histological images of eye tissues on day 7 treated with eye drops t.i.d (scale bar = 50 μm). Data analyzed by ordinary one-way ANOVA.