Topical Application of RNAi Therapy Using Surface-Modified Liposomes for Treating Retinal-Vein Occlusion

Abstract

Retinal diseases can result in blindness and visual impairment. They represent a significant medical burden and adversely affect life expectancy. Recently, antibody- and nucleic acid-based pharmaceuticals have increasingly been used to treat retinal diseases, with improvement or cure as the goal; however, these drugs are currently only administered by intravitreal injection. In this study, we present a novel approach to treating retinal diseases using eye drops that contain PnkRNA, a single-stranded RNA nucleic acid. PnkRNA-loaded liposomes were shown to effectively deliver retinal drugs and significantly inhibit retinal thickening in a mouse retinal-vein occlusion model. Cationic modification of the liposome surface enhanced the delivery of nucleic acids and therapeutic efficacy. Moreover, to reduce the frequency of eye-drop administration, liposomes were incorporated into the thermoresponsive gels. This formulation provided sustained retinal delivery and exhibited superior therapeutic efficacy compared with liposomal eye drops. This nucleic acid retinal delivery technology represents a significant advancement in drug-delivery technology, offering a safe and simple treatment for retinal diseases.

Keywords: drug delivery; eye drop; nucleic acid; retina; retinal-vein occlusion; thermoresponsive gel.

Figure 1

Mouse retinal delivery of PnkRNA through eye-drop administration. GCL, ganglion cell layer; IPL, inner plexiform layer; Lip, liposome; and R8-Lip, stearoyl-octa-arginine (R8) modified liposome. (a) Epifluorescence microscopic images of the mouse retina 30 min after TAMRA-labeled PnkRNA eye-drop administration. (b) Fluorescence intensity in the mouse IPL after 30 min of eye-drop administration (n = 6, mean ± standard error of the mean [SEM]). * p < 0.05 vs. PnkRNA solution, and † p < 0.05 vs. Unmodified-Lip (one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test).

Figure 2

Thickness of the inner nuclear layer (INL) following definite intervals of eye-drop administration in retinal-vein occlusion (RVO) model mice. Lip, liposome; R8-Lip, stearoyl-octa-arginine (R8)-modified liposome. Each value represents the mean ± standard error of the mean (SEM) of eight measurements. * p < 0.05 vs. untreated (one-way ANOVA followed by Tukey’s multiple comparison test).

Figure 3

Mouse retinal delivery of coumarin 6 (C6) by single eye-drop administration of stearylamine-modified liposome (SA-Lip)-loaded thermoresponsive gels. GCL, ganglion cell layer; IPL; inner plexiform layer; SC, sodium citrate; SR, sorbitol; and TG, thermoresponsive gel. (a) Epifluorescence microscopic images of the mouse retina following the administration of eye-drop-loaded thermoresponsive gels. Scale bar: 50 μm. (b) Fluorescence intensity in the mouse IPL after eye-drop administration of the SA-Lip-loaded thermoresponsive gels (mean ± standard error of the mean [SEM], n = 6), * p < 0.05 vs. SA-Lip-loaded SC-TG (Aspin−Welch’s t test).

Figure 4

Fluorescence images of coumarin 6 (C6)-loaded liposomes in the thermoresponsive gel. SC, sodium citrate; SR, sorbitol; and TG, thermoresponsive gel. (a) Stearylamine-modified liposome (SA-Lip). (b) SA-Lip-loaded SR-TG. (c) SA-Lip-loaded SC-TG. Scale bar: 50 μm. The fluorescence derived from C6 was observed by fluorescence microscopy after heating each eye-drop formulation to 37 °C.

Figure 5

Liposome retention study of the thermoresponsive gels. MC, methylcellulose; SA-Lip, stearylamine-modified liposome; SR, sorbitol; and TG, thermoresponsive gel. (a) Composition and viscosity of the TG and high-viscosity solution. (b) Retention test of liposomes versus temperature. Rate of liposomes in the supernatant of the thermoresponsive gel or high-viscosity solution after centrifugation at various temperatures. (c) Retention of low-molecular-weight drugs (fluorescein) versus temperature. Rate of fluorescein in the supernatant of the thermoresponsive gel or high-viscosity solution after centrifugation at various temperatures. Each value represents the mean ± standard error of the mean (SEM) of three measurements. * p < 0.01 vs. SA-Lip-loaded high-viscosity solution (Aspin−Welch’s t test). N.S.: not significant.

Figure 6

Mouse retinal delivery of coumarin 6 (C6) via single eye-drop administration of a stearylamine-modified liposome (SA-Lip). GCL, ganglion cell layer; IPL, inner plexiform layer; SR, sorbitol; and TG, thermoresponsive gel. (a) Time course of the epifluorescence microscopy images of the mouse retina. (b) Time course of the fluorescence intensity in the IPL. Each value represents the mean ± standard error of the mean (SEM) of four measurements. * p < 0.01 vs. SA-Lip, and † p < 0.05 vs. SA-Lip-loaded high-viscosity preparation (Aspin−Welch’s t test).

Figure 7

Thickness of the inner nuclear layer (INL) after specific intervals of eye-drop administration in retinal-vein occlusion (RVO) model mice. Lip, liposome; R8-Lip, stearoyl-octa-arginine (R8) modified liposome; SR, sorbitol; and TG, thermoresponsive gel. Each value represents the mean ± standard error of the mean (SEM) of eight measurements. * p < 0.05 vs. untreated, and † p < 0.05 vs. R8-Lip (one-way ANOVA followed by Tukey’s multiple comparison test). Note: the surface properties of the R8-Lip-loaded SR-TG are summarized in Table S2.