Preparation and Characterization of Silymarin Vesicular System Customized for Wound Healing

Abstract

Preparation and Characterization of Silymarin Vesicular System Customized for Wound Healing

The cascade of healing is divided into four overlapping phases: Hemostasis, Inflammatory, Proliferative, and Maturation phases. Wound healing aims to repair the injured tissues, replace the damaged structures and prevent invasion of pathogens into the damaged tissues. Conventional treatment of wounds depends mainly on moisture intake in order to permit moist treatment of wounds. They often require long duration; do not provide optimal conditions to permit recovery of the wounds, and dressings usually cause injuries upon removal. Moreover, they require frequent application and wound coverage to ensure sterility of wound, therapeutic action of drugs and avoid wound drying; hence, they decrease patient compliance and acceptance. The therapeutic efficacy of a locally applied drug depends mainly on its ability to penetrate and permeate the skin. PEVs are deformable vesicles which improve ex- vivo cutaneous drug deposition more effectively than conventional vesicles. They are able to squeeze themselves through the intercellular regions of the stratum corneum so they are able to achieve higher skin penetration and permeation than conventional vesicles. Silymarin (SIL) is the active component of Milk thistle which was shown to suppress inflammation and oxidation, which are important in the process of wound healing. Additionally, lactic acid helps collagen synthesis, angiogenesis and stimulates endothelial cell proliferation and migration. CaCl2 has an effective role in wound healing as it augments the production of granulation tissue in early stages of wound healing and it stimulates clot formation. High molecular weight hyaluronic acid has anti-inflammatory and antioxidant activities. Also, it is able to increase

fibroblast cell proliferation and stimulate keratinocyte migration, proliferation and differentiation. Chitosan has antibacterial activity; it is able to absorb wound exudates, and to protect the wound. The purpose of the first chapter was to formulate these novel families of deformable vesicles; called penetration enhancer containing vesicles (PEVs) as carriers for enhanced topical delivery of silymarin for wound healing. Thin film hydration method was used to prepare SIL loaded PEVs consisting of soybean phosphatidylcholine (PC) as bilayer forming lipid and Transcutol® as penetration enhancer. The PEVs formulations were evaluated for their particle size, polydispersity, entrapment efficiency and zeta potential. Three formulae from the preliminary study were chosen in order to augment their wound healing activity through addition of functional additives like low molecular weight chitosan, high molecular weight hyaluronic acid, lactic acid and calcium chloride. All the optimized PEVs formulae were characterized for their particle size, polydispersity, zeta potential, entrapment efficiency, viscosity. Assessment of the physical stability of the vesicles was performed by monitoring the change in particle size, span index, zeta potential and entrapment efficiency when stored at (2-8°C) after six months. Ex vivo deposition on rat skin was conducted on the chosen formulae (P1, P5, and P11) and compared with silymarin suspension. Then, the selected formulation (P11) was tested for its morphology, antibacterial activity against staphylococcus aureus and pseudomonas aeruginosa compared to pure silymarin in DMSO and its plain counterpart formulation using DMSO as negative control. Results revealed that SIL could be successfully incorporated within PEVs. The prepared PEVs were nearly spherical, in the size ranged from 3.08-5.73 µm, depending on the type and amounts of additives added and all the prepared PEVs were charged (hyaluronic acid containing vesicles were negatively charged while chitosan containing vesicles were positively charged. PEVs were able to entrap SIL with percentages reaching 76.15%. The PEVs dispersions were found to be of higher viscosity than water and they displayed good

storage properties as manifested by the generally insignificant changes in particle size, SPAN index, zeta potential and EE% values compared to freshly prepared formulations after 6 months of storage. Ex vivo deposition of such vesicles proved their superiority in accumulating the drug in different skin layers with deep penetration potential (in the stratum corneum, epidermis and dermis) in comparison with the silymarin suspension. In the antibacterial assay, the zone of inhibition produced by formulation P11 against staphylococcus aureus was comparable to that produced by gentamicin, and was higher than both the plain formulation and silymarin drug. The zone of inhibition produced by formulation P11 against pseudomonas aerugionsa was larger than its plain counterpart. Finally, in the second chapter the selected PEVs formula (P11) was tested for its wound healing activity on rats with incisional wounds. In vivo studies were done in comparison to its plain counterpart formulation and healosol®. A three weeks (21 days) wound healing assessment study was carried out on different rat groups at 3,5,7,10,14 and 21 days after wound induction, rats were photographed using a digital camera and the diameter of the wound was measured in mm. Then, staining using hematoxylin and eosin (H&E) was performed for the obtained sections, followed by histopathological examination. A semi-quantitative method was used to evaluate the following: epithelization, PMNL (polymorphonuclear leucocytes) formation, fibroblasts formation, new vessels and collagen production. Measurement of dermal content of collagen at day 14 which expressed as MTC area % and measurement of angiogenesis through measuring VEGF expression by immune staining technique at day 21.