Harnessing the tunable cavity of nanoceria for enhancing Y-27632-mediated alleviation of ocular hypertension

Abstract

Background: Y-27632 is a potent ophthalmic drug for the treatment of ocular hypertension, a globally prevalent eye disease. However, the sustained delivery of Y-27632 by a therapeutic carrier to lesion sites located in the inner segments of the eye for effectively treating the ocular disorder still remains challenging. Methods: To realize the goal, a strategy based on solvothermal-assisted deposition/infiltration in combination with surface modification is utilized to synthesize hollow mesoporous ceria nanoparticles (HMCNs) with tailorable shell thicknesses and drug release profiles. The shell thickness of HMCNs is rationally exploited for achieving sustained drug release and advanced therapeutic benefits. Results: The shell thickness can regulate release profiles of Y-27632, displaying that thick and thin (~40 nm and ~10 nm) shelled HMCNs reveal burst release characteristics (within 2 days) or limited drug loading content (~10% for the 40 nm thick). As a compromise, the HMCNs with moderate shell thickness (~20 nm) possess the most sustained drug release over a period of 10 days. In a rabbit model of glaucoma, a single instillation of the optimized Y-27632-loaded HMCNs can effectively treat glaucoma for 10 days via simultaneously repairing the defected cornea (recovery of ~93% ATP1A1 mRNA levels), restoring the reduced thickness of outer nuclear layer to normal (~64 µm), and restoring ~86% of the impaired photoreceptor cells. Conclusion: A comprehensive study on the importance of HMCN shell thickness in developing long-acting nano eye drops for the efficient management of glaucoma is proposed. The findings suggest a central role of nanobiomaterial structural engineering in developing the long-life eye drops for pharmacological treatment of intraocular diseases.

Keywords: Y-27632; ceria nanostructure; hollow carrier system; ocular therapeutics; shell thickness.

Figure 1

Material characterization. (A) Representative TEM images, (B) DLS measurements, (C) XRD patterns, (D) BET surface areas, and (E) zeta potentials of surface-modified HMCNs fabricated using different solvothermal deposition and infiltration times. Values are mean ± SD (n = 5). ^*P < 0.05 vs all groups. Scale bars: 20 nm.

Figure 2

In vitro cellular studies. (A) Effect of different types of HMCNs on suppression of H₂O₂-induced intracellular ROS production in TM cells. TM cells were incubated with test HMCNs for 24 h and further exposure to H₂O₂ for 24 h; cells incubated with no materials served as Control. Scale bars: 100 µm. (B) Intracellular levels of ROS were measured by the fluorescence intensity of DCFH-DA, with a microplate reader. (C) Levels of IL-6 released from TM cultures after incubation with test samples for 3 days. Unstimulated and TGF-β-stimulated cells without contacting the test HMCNs were the negative control (NC) and positive control (PC). Values are mean ± SD (n = 6). ^*P < 0.05 vs NC, CNP20 nm and CNP40 nm groups; ^#P <0.05 vs PC and CNP10 nm groups. (D) TEER values of rabbit corneal endothelial cells exposed to different types of HMCNs for 24 h. Control: without materials. Values are mean ± SD (n = 6). ^*P < 0.05 vs all groups.

Figure 3

In vitro drug release and cellular regulation studies. (A) Y-27632 drug loading content. Data are mean ± SD (n = 5); ^*P < 0.05 vs all groups. (B) Cumulative drug release profiles from various types of HMCNs. (C) Immunofluorescence staining with DAPI (blue fluorescence), F-actin (red fluorescence) and FITC-HMCNs (green fluorescence) and (D) mean fluorescence intensity of the TM cells after incubation with test materials for 5 days. Scale bars: 10 µm. Values are mean ± SD (n = 6). ^#P < 0.05 vs PC, Y/CNP10 nm and Y/CNP40 nm groups; ^{^}P < 0.05 vs NC and Y/CNP20 nm groups.

Figure 4

In vitro therapeutic studies. (A) Western blot analysis of β-catenin, phosphorylated MLC2 (p-MLC2), phosphorylated cofilin (p-cofilin), and tubulin in healthy TM cells (lane 1, NC group) and TGF-β-stimulated TM cells untreated (lane 2, PC group) and treated (lane 3-5, test groups) with different types of Y-27632-loaded HMCNs. (B) Schematic model of TGF-β-stimulated ROCK signaling pathway and inhibition in TM cells by the Y-27632-loaded HMCNs with shell thickness of 20 nm.

Figure 5

In vivo studies on shell thickness effects on corneal structure of glaucomatous eyes. (A) Typical TEM images of corneal stroma from different rabbit eyes. Scale bars: 1 µm. (B) Young's modulus of corneal tissues; pink and blue dotted lines showing the values of Pre and GL groups, respectively. Values are mean ± SD (n = 6). ^#P < 0.05 vs Pre, GL and Y/CNP20 nm groups; ^{^}P < 0.05 vs GL, Ctrl, Y/CNP10 nm and Y/CNP40 nm groups. (C) Representative SEM images of endothelium of different rabbit eyes. Scale bars: 20 µm. (D) ATP1A1 mRNA levels from endothelium tissues of different rabbit eyes; pink and blue dotted lines showing the values of Pre and GL groups, respectively. Values are mean ± SD (n = 6). ^#P < 0.05 vs Pre, GL, and Y/CNP20 nm groups; ^{^}P < 0.05 vs GL, Ctrl, Y/CNP10 nm and Y/CNP40 nm groups. Ocular tissues were harvested from test rabbit eyes at 10 days post single administration (50 µL of Y-27632-loaded HMCN solution, a mixture of 2% w/v Y-27632 and 1 mg/mL of HMCNs).

Figure 6

In vivo studies on shell thickness effects on retinal structure of glaucomatous eyes. (A) Representative histological images of healthy (Pre), glaucomatous (GL), untreated (Ctrl), and treated (Y/CNP10 nm, Y/CNP20 nm, and Y/CNP40nm) glaucomatous retinas. Sections are stained with H&E. Scale bars: 50 µm. (B) Thickness of outer nuclear layer in different retinas; pink and blue dash lines showing the values of Pre and GL groups, respectively. Values are mean ± SD (n = 6). ^#P < 0.05 vs Pre, GL, and Y/CNP20 nm groups; ^{^}P < 0.05 vs GL, Ctrl, Y/CNP10 nm and Y/CNP40 nm groups. (C) Typical histological images of retinas from different rabbit eyes. Sections are stained with H&E and TUNEL. Red fluorescence is TUNEL positive nuclei staining. Scale bars: 80 µm. (D) Number of apoptotic TUNEL-positive cells/300 µm; pink and blue dotted lines showing the values of Pre and GL groups, respectively. Values are mean ± SD (n = 6). ^#P < 0.05 vs Pre, GL, CNP20 nm, and CNP40 nm groups; ^{^}P < 0.05 vs GL, Ctrl, CNP10 nm, and CNP40 nm groups; ^&P < 0.05 vs Pre, Ctrl, CNP10 nm, and CNP20 nm groups. Ocular tissues were harvested from test rabbit eyes at 10 days post single administration (50 µL of Y-27632-loaded HMCN solution, a mixture of 2% w/v Y-27632 and 1 mg/mL of HMCNs).