Hydrogel Formulations Incorporating Drug Nanocrystals Enhance the Therapeutic Effect of Rebamipide in a Hamster Model for Oral Mucositis

Abstract

A mouthwash formulation of rebamipide (REB) is commonly used to treat oral mucositis; however, this formulation does not provide sufficient treatment or prevention in cases of serious oral mucositis. To improve treatment, we attempted to design a hydrogel incorporating REB nanocrystals (R-NPs gel). The R-NPs gel was prepared by a bead mill method using carbopol hydrogel, methylcellulose and 2-hydroxypropyl-β-cyclodextrin, and another hydrogel incorporating REB microcrystals (R-MPs gel) was prepared following the same protocol but without the bead mill treatment. The REB particle size in the R-MPs gel was 0.15-25 μm, and while the REB particle size was 50-180 nm in the R-NPs gel. Next, we investigated the therapeutic effect of REB nanocrystals on oral mucositis using a hamster model. Almost all of the REB was released as drug nanocrystals from the R-NPs gel, and the REB content in the cheek pouch of hamsters treated with R-NPs gel was significantly higher than that of hamsters treated with R-MPs gel. Further, treatment with REB hydrogels enhanced the healing of oral wounds in the hamsters. REB accumulation in the cheek pouch of hamsters treated with the R-NPs gel was prevented by an inhibitor of clathrin-dependent endocytosis (CME) (40 μM dynasore). In conclusion, we designed an R-NPs gel and found that REB nanocrystals are taken up by tissues through CME, where they provide a persistent effect resulting in an enhancement of oral wound healing.

Keywords: endocytosis; hydrogel; nanocrystals; oral mucositis; rebamipide.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflicts of interest.

Figures

Figure 1

Condition of rebamipide (REB) in REB hydrogels. (A) Photographs of the R-MPs gel (hydrogel with incorporated rebamipide microcyrstals) and R-NPs gel (hydrogel with incorporated rebamipide nanocrystals). (B,C) Particle size in the R-MPs (B) and R-NPs (C) gels. (D) AFM image of the R-NPs gel. Bead mill treatment decreased the particle size of REB to the range of 50–180 nm.

Figure 2

Characteristics of REB in the R-MPs and R-NPs gels. (A) Non-uniformity in REB particle distribution in the R-MPs and R-NPs gels. (B) Solubility of REB in the R-MPs and R-NPs gels. (C) XRD pattern of REB particles after bead mill treatment. N = 7. * p < 0.05 vs. R-MPs for each category. The mill-treated REB retained its crystal structure, but the uniformity of REB distribution in the R-NPs gel was higher than the non-milled REB in the R-MPs gel. Moreover, solubility of REB was increased by bead mill treatment.

Figure 3

Drug release from R-MPs and R-NPs gels through a 220-nm pore membrane. (A) Release behavior of REB from R-MPs and R-NPs gels through a membrane. (B) and (C) Size distribution (B) and number (C) of REB nanocrystals in the reservoir chamber 24 h after R-NPs application. n = 7. N.D., not detectable. * p < 0.05 vs. R-MPs gel for each category. REB was released from the R-NPs gel in the form of nanocrystals.

Figure 4

Changes in REB content in the cheek pouch of hamsters treated with REB hydrogels for oral mucositis. (A) REB contents in the cheek pouch of hamsters 2 and 8 h after treatment with R-MPs and R-NPs gels. (B,C) REB contents in the cheek pouch of hamsters treated with endocytosis inhibitors 2 h (B) and 8 h (C) after the application of R-MPs and R-NPs gels. Control—R-NPs-treated hamster. Nystatin—nystatin-treated hamster treated with R-NPs. Dynasore—dynasore-treated hamster treated with R-NPs. Rottlerin—rottlerin-treated hamster treated with R-NPs. Cytochalasin D—cytochalasin D-treated hamster treated with R-NPs. n = 5–7. * p < 0.05, vs. R-MPs for each category. #p < 0.05 vs. Control for each category. REB content in hamsters treated with R-NPs gel was higher than in those treated with R-MPs gel; the CME pathway appears to be related to the penetration of REB into the cheek pouch tissues from the hydrogel formulations.

Figure 5

Therapeutic effect of R-NPs gel on wounds in the cheek pouch of hamsters. (A) Representative images of the cheek pouch of the hamster model for oral mucositis 0 and 3 d after treatment with REB hydrogels. (B), Wound area in the cheek pouch of the hamster model 0–3 d after treatment with REB hydrogels. n = 5–8. * p < 0.05, vs. Vehicle for each category. #p < 0.05 vs. R-MPs gel for each category. Treatment with REB hydrogel enhanced the therapeutic effect on oral mucositis. The wound areas in hamsters treated with R-NPs gel were significantly smaller than in hamsters treated with R-MPs gel.

Figure 6

Microscopic effects of the R-NPs gel on oral mucositis. (A) Images of H&E-stained cheek pouch tissue specimens from a hamster with oral mucositis three days after treatment with REB hydrogels (4× objective lens; bars indicate 200 μm). (B) High magnification images in the areas delineated by the squares in Figure A (10× objective lens; bars indicate 100 μm). (C) Images of immunostaining for multi-cytokeratin in the serial sections shown in Figure A (4× objective lens; bars indicate 200 μm). (D) High magnification images of the areas delineated by the squares in Figure C (10× objective lens; bars indicate 100 μm).

Figure 7

Drug delivery pathway and therapeutic effect of R-NPs gel in the cheek pouch of the hamster model for oral mucositis. N.D., not detectable.