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.

Graphical Abstract

Keywords:

• ocular drug delivery
• dual-modified liposomes
• Penetratin
• hyaluronic acid
• conbercept

Abbreviations

ARPE-19, human retinal pigment epithelium cells; BRVO, branch vein occlusion; BSA, bovine serum albumin; CCK-8, cell counting kit-8; CD44, homing cell adhesion molecule; CHOL, cholesterol; Chr, choroid; CNV, choroidal neovascularization; Conb, conbercept; CPP, cell-penetrating peptides; DAPI, 4.6-diamino-2-phenyl indole; DL, drug loading; DLS, dynamic light scattering; DPPE, dipalmitoyl phosphoethanolamine; DSPE, distearoyl phosphoethanolamine; DSPE-PEG₂₀₀₀, distearoyl phosphoethanolamine-polyethylene glycol₂₀₀₀; DSPE-PEG₂₀₀₀-Mal, 1.2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000]; DSPE-PEG₂₀₀₀-Pen, distearoyl phosphoethanolamine-PEG-Penetratin; ECM, endothelial cell medium; ED, eye drop; EDC, N-(3-Dimethylaminopropyl)-N´-ethylcarbodiimide hydrochloride; EE, entrapment efficiency; ELISA, enzyme-linked immunosorbent assay; Endo, corneal endothelium; EPC, egg yolk lecithin; Epi, corneal epithelial layer; ESI-MS, electrospray ionization mass spectrometry; FAM, carboxyfluorescein; FFA, fundus fluorescein angiography; GCL, ganglion cell layer; HA, hyaluronic acid; HA-Lip or PenHA-Lip, HA-modified liposomes; HCEC, human corneal epithelial cells; HE, hematoxylin–eosin staining; HUVEC, human umbilical vein endothelial cells; ILM, internal limiting membrane; INL = inner nuclear layer; IPL, inner plexiform layer; IVT, intravitreal injection; Lip, liposome; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MFI, mean fluorescence intensity; MW, molecular weight; nAMD, neovascular age-related macular degeneration; NHS, N-hydroxysuccinimide; Nr, Nile red; Papp, apparent permeability coefficient; ns, no significance; ONL, outer nuclear layer; OPL, outer plexiform layer; PBS, phosphate-buffered saline; PDT, photodynamic therapy; PEG, polyethylene glycol; Pen, Penetratin; Pen-Lip, Penetratin-modified liposomes; Pen-Lip/FAM, FAM-labeled liposomes; PFA, paraformaldehyde fix solution; PLT, photoreceptor layer thickness; PRN, pro re nata; q.w, quaque week; RPE, retinal pigment epithelium; RPE/BM, retinal pigment epithelium/Bruch’s membrane complex; t.i.d., three times a day; TEM, transmission electron microscope; VEGF, vascular endothelial growth factor; ΔH, hydration values.

Data Sharing Statement

All data and material are included in the article and its additional files.

Disclosure

The authors declare that they have no competing interests in this work.

Additional information

Funding

This work was supported by the Interdisciplinary Program of Shanghai Jiao Tong University (No. 0507N17014, China).