Generation of a Drug-incorporated Membrane for Biomedical Applications

Abstract

Current approach for ocular wound healing involves bandages combined with repeated administration of therapeutic eye drops. There is a need to develop ocular medicated bandages as a convenient and cost-effective strategy for wound healing. This project aimed to develop a controlled release biocompatible bandage consisting of 10-50 µm drug–incorporated poly (lactic-co-glycolic acid) (PLGA) microparticles (MP) and eggshell membrane (ESM) for ocular wound healing application. The eggshell membrane is a natural material with unique physical and biological properties. No studies have demonstrated nor reported its potential as a novel ocular wound dressing yet. ESM samples were isolated from the eggshell by either manual peeling (ESMstrip) or, via extraction, using in-house developed acetic acid or ethylenediaminetetraacetic acid, EDTA, protocols Energy-dispersive X-ray spectroscopy (EDS) confirmed that the ESM samples contained carbon, nitrogen, oxygen, and sulphur, with no traces of calcium residues from the extraction process. The extraction method (acetic acid and EDTA) did not alter the chemical bonding structures of the ESM, and the chemical composition of the fibrous proteins of the ESM was further clarified using Fourier transform infrared (FTIR) spectroscopy. The ESM has a three-layer composite structure, according to scanning electron microscopy (SEM) analyses: an inner layer that is continuous, dense, and non-fibrous (limiting membrane), a middle layer with a network of fibres (inner shell membrane), and an outer layer (outer shell membrane) with larger fibres. Furthermore, optical transparency, porosity, fluid absorption/uptake, thermal stability, mechanical/physical profiling of the ESM samples were performed and showed suitable profiles for translational applications. Biological in vitro studies using SV40 immortalised corneal epithelial cells (ihCEC) and corneal mesenchymal stromal cells (C-MSC) demonstrated excellent biocompatibility. A sub-study presented a design of experiment technique (DOE) known as Taguchi Design for the generation of a formulation model for optimizing the various factors shown to affect MP size when fabricated using the solvent emulsion technique and to produce the most appropriate MP for topical drug delivery. The first optimization step using the L12 design showed that all parameters significantly influenced the particle size of the fabricated MP with exception of the concentration of PVA in the hardening bath. In contrast, the L18 design results showed that the molecular weight of PLGA does not significantly affect the particle size. The optimized formulation was identified to be run 16 from the L18 OA design. The mean particle size was found to be 23.0 µm. that falls under the required range that is 10 – 50 µm. The results from this study showed that the application of Taguchi design can be used to predict and determine the best combination of process parameters that can provide the optimal response condition- in this case, to identify the most appropriate microparticle size for topical application. Additional studies were conducted with the formulation model to fabricate BSA-loaded PLGA-MP as well as their characterization using microscopy, size distribution and drug release profile. The MP were successfully attached onto the ESM using a chemical method (NaOH). The presence of the loaded MP did not compromise the transparency of the bandage. The FTIR spectra reveal the crosslink of the ESMs absorption peak with the absorption peak of the MP. The 2 in 1 bandage consisting of MP and ESM demonstrated high biocompatibility, durability, flexibility, fluid absorption capacity, thermal stability and a sustained drug release profile. These findings show that the ESM could be used in a variety of regenerative medical and/or biotechnological applications, such as ocular and skin wound dressings.