Effects of retinoid-loaded hyaluronic acid nanomicelles on vaginal epithelium in a murine menopause model

Abstract

Purpose: This study aimed to develop hyaluronic acid (HA)-based, retinoic acid (RA)-containing nanomicelles and to investigate the effects of these newly developed nanomicelles on regeneration of the vaginal epithelium and aquaporin 3 (AQP3) expression in a murine menopause model.

Materials and methods: The HA-based, RA-loaded nanomicelles were developed, and the RA-loading rate, encapsulation efficiency, and hydrodynamic diameter were measured. Female BALB/c mice (8 weeks; n=30) were divided into control and experimental groups. Menopause was established in the experimental group by removing both ovaries. The experimental group was further divided into an ovariectomy group, an HA-C18 vehicle group, and an HA-C18-RA group (2.5 µg per mouse); vaginal administration of HA-C18 or HA-C18-RA was performed once daily. After 4 weeks of treatment, murine vaginal tissue was removed, and histological analysis was performed.

Results: Three drug-loaded nanomicelles were synthesized: the RA content in HA-C18-RA-10, HA-C18-RA-20, and HA-C18-RA-30 was 3.13%, 2.52%, and 16.67%, respectively, and the RA encapsulation efficiency was 95.57%, 83.92%, and 93.24%, respectively. In the experimental versus control group, serum estrogen levels were significantly reduced, and the vaginal mucosal epithelial layer was significantly thinner. After 4 weeks of treatment, the thickness of the vaginal mucosal epithelial layer and AQP3 expression was increased in the HA-C18-RA group compared with the HA-C18 vehicle group.

Conclusions: The newly developed HA-based nanomicelles containing RA resulted in vaginal epithelial recovery and increased AQP3 expression. The results may contribute to the development of functional vaginal lubricants or moisturizers for the treatment of vaginal dryness.

Keywords: Aquaporins; Epithelium; Hyaluronic acid; Regeneration; Vagina.

Fig. 1. Physicochemical characterization of HA-C18-RA. (A) Mean hydrodynamic diameter. (B) Mean zeta potential and ultraviolet spectra of HA-C18-RA. (C) Ultraviolet-visible spectroscopy of RA in dimethyl sulfoxide and HA-C18-RA in distilled water. HA, hyaluronic acid; RA, retinoic acid; OD, optical density; DMSO, dimethyl sulfoxide.

Fig. 2. Immunofluorescence results of aquaporin 3 (AQP3) in vaginal tissue from animals in the control (CON), ovariectomized (OVX), ovariectomized plus HA-C18 (HA-C18), and ovariectomized plus HA-C18-RA (HA-C18-RA) groups. AQP3 is mainly expressed throughout the epithelium of vaginal tissue. AQP3 levels decreased in ovariectomized mice compared to controls but were restored by HA-C18-RA treatment. Immunofluorescence images were acquired by confocal microscopy. Data represent at least three independent experiments. DAPI, 4′,6-diamidino-2-phenylindole; HA, hyaluronic acid; RA, retinoic acid.

Fig. 3. Immunoblotting results of aquaporin 3 (AQP3) in vaginal tissue from animals in the control (CON), ovariectomized (OVX), ovariectomized plus HA-C18 (HA-C18), and ovariectomized plus HA-C18-RA (HA-C18-RA) groups. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hyaluronic acid; RA, retinoic acid. ^*p<0.05 vs. control. ^#p<0.05 vs. OVX.