No abstract

Intraocular drug delivery is extraordinarily hampered by the impermeability of defensive barriers of the eye. In this study, the ocular permeability of fluorophore-labeled cell-penetrating peptides (CPPs), including penetratin, TAT, low molecular weight protamine, and poly(arginine)8, was investigated based on multilevel evaluations. The human conjunctival epithelial cell (NHC) was exposed to various CPPs to determine the cytotoxicity and cellular uptake. Ex vivo studies with rabbit cornea were performed using side-by-side diffusion chambers to evaluate the apparent permeability coefficients and acute tissue tolerance of the CPP candidates. Among all examined CPPs, penetratin shows an outstanding cellular uptake, by increasing more than 16 and 25 times at low and high concentrations, compared to the control peptide poly(serine)8 respectively. Additionally, the permeability of penetratin across excised cornea is 87.5 times higher in comparison with poly(serine)8. More importantly, after instilled in the conjunctival sac of rat eyes, fluorophore-labeled penetratin displayed a rapid and wide distribution in both anterior and posterior segment of the eye, and could be observed in the corneal epithelium and retina lasting for at least 6 h. Interestingly, penetratin showed the lowest ocular cell and tissue toxicities among all examined CPPs. The high ocular permeability of penetratin could be attributed to its amphipathicity and spatial conformation determined by circular dichroism. Taken together, these data demonstrate that penetratin is potentially useful as an absorption enhancer for intraocular drug delivery.

Gene delivery to the posterior segment of the eye is severely hindered by the impermeability of defensive barriers; therefore, in clinical settings, genomic medicines are mainly administered by intravitreal injection. We previously found that penetratin could transport the covalently conjugated fluorophore to the fundus oculi by topical instillation. In this study, gene delivery systems enabled by penetratin were designed based on electrostatic binding to target the retina via a noninvasive administration route and prepared with red fluorescent protein plasmid (pRFP) and/or poly(amidoamine) dendrimer of low molecular weight (G3 PAMAM). Formulation optimization, structure confirmation, and characterization were subsequently conducted. Penetratin alone showed limited ability to condense the plasmid but had powerful uptake and transfection by corneal and conjunctival cells. G3 PAMAM was nontoxic to the ocular cells, and when introduced into the penetratin-incorporated complex, the plasmid was condensed more compactly. Therefore, further improved cellular uptake and transfection were observed. After being instilled in the conjunctival sac of rats, the intact complexes penetrated rapidly from the ocular surface into the fundus and resided in the retina for more than 8 h, which resulted in efficient expression of RFP in the posterior segment. Intraocular distribution of the complexes suggested that the plasmids were absorbed into the eyes through a noncorneal pathway during which penetratin played a crucial role. This study provides a facile and friendly approach for intraocular gene delivery and is an important step toward the development of noninvasive gene therapy for posterior segment diseases.