Evaluating the Efficacy of Polyglycolic Acid-Loading Tetrandrine Nanoparticles in the Treatment of Dry Eye

Abstract

Introduction: Dry eye disease (DED) is a multifactor-induced disease accompanied by increased osmolarity of the tear film and inflammation of the ocular surface. Traditional anti-inflammation agent corticosteroids applied in DED treatment could result in high intraocular pressure, especially in long-term treatment. Therefore, we explored a nano drug that aimed to block the formation pathway of DED which had anti-inflammatory, sustained release, and good biocompatibility characteristics in this study.

Methods: We prepared a novel nanomedicine (Tet-ATS@PLGA) by the thin film dispersion-hydration ultrasonic method and detected its nanostructure, particle size, and zeta potential. Flow cytometry was used to detect the cell survival rate of each group after 24 h of drug treatment on inflammed Statens Seruminstitut Rabbit Corneal (SIRC) cells. Observed and recorded corneal epithelial staining, tear film rupture time, and Schirmer test to detect tear secretion on the ocular surface of rabbits. The corneal epithelial thickness, morphology, and number of bulbar conjunctival goblet cells were recorded by H&E staining. Finally, we detected the expression of VEGF, IL-1β, PGE2, and TNF-α by cellular immunofluorescence staining and enzyme-linked immunosorbent assay (ELISA).

Results: The encapsulation efficiency and drug loading of Tet-ATS@PLGA were 79.85% and 32.47%, respectively. At eye surface temperature, Tet can easily release from Tet-ATS@PLGA while that it was difficult to release at storage temperature and room temperature. After 2 weeks medication, Tet-ATS@PLGA can effectively improve the tear film rupture time and tear secretion time in a DED model (p <0.05). Compared with the normal group (62.34 ± 4.86 mm), the thickness of corneal epithelium in ATS (29.47 ± 3.21 mm), Tet-ATS (46.23 ± 2.87 mm), and Tet-ATS@PLGA (55.76 ± 3.95 mm) gradually increased. Furthermore, the flow cytometry indicated that Tet-ATS@PLGA can effectively promote the apoptosis of inflammatory SIRC cells, and the cellular immunofluorescence and ELISA experiments showed that the expression intensity of inflammatory factors such as VEGF, IL-1β, PGE2, and TNF-α decreased in this process. Interestingly, Tet also had the effect of reducing intraocular pressure.

Conclusion: Tet-ATS@PLGA can effectively promote the apoptosis of inflammatory corneal epithelial cells, thus inhibiting the expression of inflammatory factors to block the formation of DED and improve the secretion of tear on the ocular surface.

Keywords: Dry eye disease; Inflammation; Nanomedicine; Polyglycolic acid; Tetrandrine.

Fig. 1.

a The TEM of Tet-ATS@PLGA. b Particle size distribution of PLGA (100 μg/mL). c Particle size distribution of Tet-ATS@PLGA (100 μg/mL). d The EE and LC of Tet-ATS@PLGA. e Zeta potential of @PLGA and Tet-ATS@PLGA. f In vitro cumulative release of Tet from Tet-ATS@PLGA in ATS solution at 4°C, 25°C, and 33°C. Linear regression, t test, and row mean with SD were performed for analysis (n = 3, p <0.05). TEM, transmission electron microscopy.

Fig. 2.

a The corneal-staining images with DED in the normal, control, ATS, Tet-ATS, and Tet-ATS@PLGA groups after 14 days treatment. b, c The results of tear film rupture time (BUT) and tear secretion test (Schirmer) of normal, control, ATS, Tet-ATS, and Tet-ATS@PLGA group after 2 weeks treatment. d The difference of intraocular pressure in the normal, control, ATS, Tet-ATS, and Tet-ATS@PLGA group after 2 weeks treatment (n = 5, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 3.

The corneal epithelial thickness analysis of normal, control, ATS, Tet-ATS, and Tet-ATS@PLGA observed by the corneal pathological section after 2 weeks treatment (n = 5; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 4.

The apoptotic rate of inflammatory SIRCs were detected in the control, ATS, Tet-ATS groups and Tet-ATS@PLGA group after incubation for 24 h (Tet: 5 µg/mL) (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 5.

a–d Representative immunofluorescence images of VEGF, IL-1β, PGE₂, and TNF-α antibodies in inflammed SIRC cells exposed to different therapeutic schedules. Scale bar = 100 μm. e–h The counts analysis of VEGF, IL-1β, PGE₂, and TNF-α antibody expression in inflammed SIRC cells by ImageJ software. Ordinary one-way ANOVA was performed (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 6.

The expression of VEGF, IL-1β, PGE₂, and TNF-α antibodies in the normal, control, ATS, Tet-ATS group and Tet-ATS@PLGA group after 2 weeks treatment in the model rabbits from the perspective of protein level (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).