Development of lipid nanodispersions containing insulin for the topical treatment of dry eye disease

Author(s)

Francieli Pereira

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2017-08-25

Examining board members

Renata Fonseca Vianna Lopez; Monica Alves; Karin Silva Caumo

• Advisor: Renata Fonseca Vianna Lopez

Abstract

Dry eye disease (DED) is a multifactorial disorder related to an inflammatory process in ocular structures. It affects tear film production and results in corneal wounds which are difficult to heal. Recent studies have shown that insulin (INS) facilitates the healing process of wounds and restores tear film production in diabetic rats. However, INS is a protein that degrades easily in biological environment, requiring strategies that improve its bioavailability in ocular delivery. In this context, liposomes may be a promising vehicle for ocular topical delivery of INS due to its biocompatibility, low toxicity and high residence time on the ocular surface. Thus, the aim of the present work was to develop liposomes containing INS for topical treatment of DED. Liposomes containing soy phosphatidylcholine (PC), sodium cholate (NaC) and poloxamer 188 in 10 mM phosphate buffered saline (PBS) pH 7.4 were prepared by the thin lipid film hydration method followed by high pressure homogenization. The INS was added to the PBS buffer used for lipid film hydration at a concentration of 0.4 mg/mL. The liposomes were sterilized using 0.22 µm filter and their particle size, polydispersity index (PdI), zeta potential (ZP), pH, encapsulation efficiency (EE%) and drug recovery (DR,%) were determined. Liposomes particle size was 125 ± 3 nm, PdI was 0.27 ± 0.01, ZP was -11 ± 1.5 mV, pH was 7.1 while EE and DR were 32 ± 2% and 82 ± 12%, respectively. Transmission electron and atomic force microscopy showed spherical and slightly asymmetric liposome vesicles in the presence of INS with a mean diameter varying from 120 to 150 nm. Small angle X-ray scattering (SAXS) studies showed symmetry in lipid bilayer of INS-loaded liposomes, with 42 Å thickness, and INS dispersed in populations varying from 6 to 15 monomers. Liposomes maintained their physical characteristics when stored at 4°C for up to 60 days while at room temperature, there was no INS degradation for at least 72 h. The in vitro ocular irritation assay HET-CAM showed low irritation potential for the formulations containing or not INS. Immortalized human corneal epithelial (HCE) cells culture demonstrated that formulations treatment containing 1 IU of INS were non-cytotoxic and did not increase MMP-9 levels compared to controls. However, increased IL-6 production has been observed, which may contribute to wound healing. Therefore, liposomes seem to be a promising delivery system for topical ocular delivery of INS for dry eye disease and wound healing. (AU)

• FAPESP's process: 15/04008-1 - Development of lipid nanodispersions containing insulin for the topical treatment of dry eye disease
• Grantee: Francieli Pereira
• Support Opportunities: Scholarships in Brazil - Master