Evaluation of formulations based on silk fibroin and insulin in the topical treatment of ocular wounds

Author(s)

Camila Cubayachi

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2019-02-28

Examining board members

Renata Fonseca Vianna Lopez; Marisa Masumi Beppu; Marlus Chorilli; Jayter Silva de Paula; Fabiana Testa Moura de Carvalho Vicentini

• Advisor: Renata Fonseca Vianna Lopez

Abstract

The corneal wound healing process in patients suffering of chronic diseases, like diabetes mellitus, is impaired, which can lead to recurrent erosions, opacity and even blindness, and usually does not respond to conventional treatments. Studies have demonstrated the potential use of insulin (INS) in wound healing, however its topical administration requires the use of delivery systems to ensure its stability and controlled release on the area to be regenerated. Thus, the objective of this work was to develop films based on silk fibroin (SF) containing INS and evaluate their potential on the topical treatment of ocular wounds. The SF films containing INS at 100 IU/cm2 and glycerin as plasticizer were obtained by casting. They were homogeneous, transparent, permeable to water vapor, with low swelling index and high mechanical resistance. Fourier transformed infrared spectroscopy and differential scanning calorimetry analyses suggested the ?-sheet/Silk II conformation of SF, besides the occurrence of non-covalent interactions between INS and SF, capable to promote the stabilization of film structure. An analytical method based on high performance liquid chromatograph was developed and validated, presenting selectivity, precision, accuracy and linearity in the range of 3 to 100 ?g/mL. In vitro drug release studies have demonstrated that the films were capable of releasing about 8% of INS in the first 30 minutes, reaching 22% in 12 hours, totaling 1 IU (3.5 ?g), without further release up to 24 hours. However, enzymatic degradation studies suggested that the proteinases present in the wounds would be capable of causing breaks in the film when applied, which could be an additional component in the INS release. The circular dichroism spectrum of the receptor solution samples demonstrated that the INS released from the films maintained their native conformation, in addition to conserving its biological activity in vivo, reducing the blood glycemia of Wistar rats. In vitro studies with human epithelial cornea cells (HCEC-SV40) showed that the treatment with the films were not cytotoxic. Furthermore, in an inflammatory model established by the cell stimulation with E. coli lipopolysaccharide, it was observed that the films were able to modulate the levels of inflammatory mediators of wound healing process, such as interleukin-6 and matrix metalloproteinase-9. Finally, in vivo wound healing studies using corneal epithelial debridement model of diabetic Wistar rats allowed the evaluation of INS release from the films to modulate the inflammatory process and regenerate the corneal epithelia, favoring the phenotypic characteristics of the epithelial cells. Therefore, SF films have been shown to be a promising INS delivery system for topical ocular administration and healing of corneal epithelial wounds. (AU)

• FAPESP's process: 14/19018-0 - Evaluation of formulations based on silk fibroin and insulin for topical treatment of ocular wounds
• Grantee: Camila Cubayachi
• Support Opportunities: Scholarships in Brazil - Doctorate