Abstract:
Background: Skin ageing is a cumulative result of oxidative stress, which is mainly caused by reactive oxygen species (ROS) formed from respiration, pollutants, toxins or ultraviolet A (UVA) irradiation. 80% of skin damage conditions by extrinsic factors are caused by UVA irradiation. Antioxidants are considered to be useful compounds for the removal of ROS, for the prevention of skin ageing and maintenance of skin health. Recently, L-Glutathione (GSH), the mother of all antioxidants, is of great interest for combating skin ageing, due to its superior antioxidant activities against UVA irradiation induced oxidative stress. Although the most efficient route of administration for skin related conditions is topical, the main challenges of GSH for topical delivery are due to its poor stability in aqueous conditions and poor penetration through the stratum corneum (SC) resulting from its high hydrophilicity. A dual drug delivery system (DDS) combining solid lipid nanoparticles (SLNs) and hydrogel can enhance skin penetration and improve the stability of GSH. In DDS, SLNs are the nanoscale vesicles with solid lipids base, which can provide a lipophilic carrier system for the delivery of GSH, while hydrogels are serving as the suitable dispersing medium, which allows for the ease of application and skin hydration. Aim: The aim of this project is to develop a GSH-SLNs enriched hydrogel system (DDS) to enhance the physicochemical stability of GSH, and improve its skin penetration to exert its antioxidant effect in deeper viable skin layers for the purpose of anti-ageing. Methods: A high performance liquid chromatography (HPLC) method was modified to quantify the amount of GSH and validated for linearity, accuracy, precision, sensitivity, and repeatability. Preformulation studies were performed to investigate the physicochemical properties of GSH, such as partition coefficient, aqueous solubility and stability (in acidic, basic, oxidative, thermal, and photolytic conditions). The degradation products of GSH were identified by liquid chromatography-mass spectrometry (LC-MS). GSH loaded SLNs (GSHSLNs) were prepared and developed by double emulsion method based on factorial design (including screening test and optimisation design) to create the formulations with most desirable properties. The optimised SLNs were then dispersed into a carbopol based hydrogel system to form the DDS. The physicochemical properties of the developed DDS were characterised with respect to particle size, size distribution, zeta potential, morphology, entrapment efficiency (EE%), drug loading, FTIR analysis, mechanical properties, and rheological properties. In vitro drug release profiles of DDS were investigated using Franz diffusion cells for three days, and its release mechanism were deteremined by modeling into several release models. Stability studies (4℃, 25℃, and 40℃) of the DDS was also carried out by analysing key characterisation parameters and drug content at different time intervals over three months. The changes in permeation of GSH across the skin barrier and the GSH deposition in the different skin layers were determined using Franz diffusion cell after 48 hours of study on human skin samples. The in vitro cytotoxicity and antioxidant effects of GSH formulations were evaluated on human skin fibroblasts cell line using MTT assay and UVA irradiation assay. Results and discussion: A rapid, simple, and reliable HPLC analytical method for GSH was optimised and validated. The preformulation studies showed that GSH exhibits column shaped crystalline structure with a high aqueous solubility (252.7 mg/mL) and a low Log P of -3.1. GSH was found to be unstable, and it followed the first order degradation kinetics in most tested stress conditions, especially oxidative, in which the main degradation product is glutathione disulphide. The optimum GSH-SLNs formulation was obtained at the following conditions: 0.6 g stearic acid with surfactants ratio at 1 : 10 (Span® 85: Tween® 80); chitosan polymer concentration was 0.1% (w/v); two sonication time ratio was 1 : 2 (first emulsion: second emulsion); GSH concentration was 40 mg/mL in GSH-SLNs; and 45% sonication amplitude for both two sonications. GSH-SLNs were produced with the desirable particle size (305 ± 0.6 nm) and zeta potential (+20.1 ± 9.5 mV) for topical delivery. The GSH-SLNs demonstrated a high drug EE% (72.0 ± 0.5%) and an acceptable drug loading capacity (3.0 ± 0.2%). The best DDS was GSH-SLNs enriched 3% carbopol hydrogel, in which gel strength (5.1 ± 0.5 g) and spreadability (33.6 ± 1.9 g sec) were comparable with a commercial gel (Merino® 97% Pure Aloe Vera Gel, NZ). Pseudoplastic, thixotropic and viscoelastic properties were found in the final DDS, indicating the good rheological profiles and the ease of application for topical administration. The DDS demonstrated sustained release over 72 hours, which followed Korsmeyer-Peppas model, indicating the release mechanism was complicated (a combination of diffusion and erosion). The DDS also showed a 3.7-fold improvement of GSH penetration across SC on human skin model when compared with the GSH aqueous solution and GSH hydrogel. The distribution of GSH in skin layers released from the DDS were detected to be around 20% permeated full thickness skin, 55% deposited in the viable epidermis, and 25% entrapped in SC and skin surface. No cytotoxicity was observed when fibroblast cells were subjected to GSH formulations (containing 0.2 mg/mL GSH). The high antioxidant activity (UVA rescue) of GSH in DDS was found to protect fibroblast cells from UVA induced death and promote cell growth. Conclusion: This research has illustrated that a DDS combining SLNs and hydrogels, was able to enhance the physicochemical stability of GSH; improve GSH skin penetration; achieve the drug deposition in the viable epidermis and dermis layers of the skin; and protect human skin fibroblast cells against oxidative stress caused by UVA irradiation. This project also highlighted that DDS is a successful topical delivery system for dermal delivery of water soluble antioxidant. This delivery system of GSH shows promise as an anti-ageing complex for improving skin health, and may serve as a topical delivery platform for other bioactive compounds with similar physiochemical properties in future.