Dermal delivery of Centella asiatica using hyaluronic acid niosomal system for wound healing

Abstract

Background and aims: The human skin is the primary barrier that protects internal tissues from mechanical injury, infection, extreme heat and is crucial for haemostasias. The skin is vulnerable to injury, which has many consequences for both individual patients and the healthcare economy. Skin injuries and wounds should eventually heal normally through the natural process of skin cell restoration. However, significant clinical challenges act as an obstacle for time-controlled clean wound healing. Asiaticoside (ASC) and asiatic acid (AA) are natural bioactive compounds from Centella asiatica. Many effects of these two compounds, including promoting fibroblast proliferation, increased epithelial cell migration, and new capillary formation, has been reported. Their efficacy has been demonstrated in in vitro and in vivo studies. However, these compounds are fragile and need to be protected from degradation. Taken together, it would be advantageous if the compounds were encapsulated, which would shield them from the external environment and also facilitate sustained release, enhancing their therapeutic effects. In addition, enriching a biocompatible hydrogel with drug-loaded niosomes will allow easy application, and the beneficial effects of the hydrogel will also enhance healing. Objective: The aim of this project was to develop a niosome loaded hydrogel system to deliver bioactive compounds to skin wounds to accelerate wound healing. The drug candidates are two compounds from Centella asiatica, namely, asiaticoside (ASC) and asiatic acid (AA). Methods: A high-performance liquid chromatography (HPLC) method was developed and validated to simultaneously quantify the two compounds; preformulation studies were performed to study their physicochemical properties. ASC-loaded and AA-loaded niosomes were prepared using film hydration method and the Design of Experiment strategy was applied to optimise the formulations. Optimised niosomes were coated with hyaluronic acid and subjected to characterisation studies to determine their morphology, particle size, entrapment efficiency, zeta potential, thermal activities, drug and excipient interactions and in vitro release profiles. The effects of the two drugs and the drug-loaded niosomes on human skin fibroblasts (Fbs), including cytotoxicity, uptake and wound healing were evaluated. A self-healing hydrogel was developed based on oxidised konjac glucomannan and carboxymethyl and was characterised. In vivo studies were carried out on Sprague Dawley rats using a full-thickness excisional wound model to examine the effects of the niosome loaded hydrogel. Results and discussion: The optimised hyaluronic acid-coated ASC niosome (HaASC) and AA niosome (HaAA) were spherical and with acceptable size in the nanoscale (234.60 ± 15.24 nm and 175.80 ± 13.54 nm, respectively), with entrapment efficiencies of 79.45 ±4.32 % and 94.18 ± 3.98 %, respectively. DSC and FTIR confirmed that the drugs were entrapped in niosomes in an amorphous state. The in vitro drug release study showed that both HaASC and HaAA could effectively release their drug in a sustained manner over 48 h prolonged period. The stability studies showed a protective effect of the niosomes on drugs, and they were relatively stable when stored between 2-8 °C for 90 days. Drug deposition in the skin layers was significantly enhanced by niosome carriers (p <0.05). The in vitro wound-healing assay showed that a combination of ASC and AA loaded niosomes enhanced cell migration and proliferation compared to free drugs. The niosome carrier was effective for delivering ASC and AA across the stratum corneum to the dermis layer and enhancing cellular uptake in vitro, improving their biological effects. A self-healing hydrogel based on carboxymethyl chitosan and oxidised konjac glucomannan was successfully prepared and enriched with drug-loaded niosomes. The self-healing properties were observed via a macroscopic self-healing test and a cyclic strain test. Following the addition of niosomes, the storage modulus was close to the blank hydrogel, suggesting that the inclusion of niosomes did not affect the self-healing properties. Drug release from hydrogel showed an initial fast release followed by a sustained release phase, which is ideal for wound healing applications as the frequency of dressing changes could be reduced. Neither blank nor niosome-enriched hydrogels affected the cell viability after incubation for 48 h. In the in vivo wound healing study, SD rats treated with HaASC+HaAA noisome-enriched hydrogel showed much faster-wound closure on day 3, 6, 9 and completely healed on day 14, which was significantly higher than the control (p < 0.01) and blank hydrogel (p < 0.05). Histological and immunohistochemical examination revealed that niosomes enriched hydrogel enhanced epithelialisation and collagen deposition, corresponding to accelerated healing compared with the other groups. Conclusion: The project has demonstrated that the development of niosomes improved the stability of ASC and AA and enhanced their uptake into cells. The research highlighted that niosomes are a potential carrier for ASC and AA for wound healing. The functionalised hydrogel served as a promising dressing for accelerating wound healing and could potentially treat wounds. This system may serve as a platform into which a range of compounds with wound healing properties can be loaded.