الفهرس 
General IntroductionOnly 14 pages are availabe for public view
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Abstract
When the skin is injuried through physical, chemical, mechanical, and/or thermal damage, the body sets into motion an automatic series of events, often referred to as the ―cascade of healing,‖ in order to repair the injured tissues, replace damaged structures and prevent invasion of pathogens into damaged tissues. The cascade of healing is divided into four overlapping phases: Hemostasis, Inflammatory, Proliferative, and Maturation phases. A number of commercial products are available such as gels, creams, ointments, dressings and solutions, which are mainly based on the principle of moisture intake that is intended to support tissue repair, yet they are not anti-infective and sometimes even cannot be used in the presence of a potential infection.
Additionally, healing of wounds with conventional treatments often requires longer 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.
Penetration enhancer containing vesicles have been intensively studied as carrier systems for topical delivery of drugs and cosmetic agents. They contain one or more penetration enhancers (PE) in their structure, such as oleic acid, transcutol® and labrasol®. They improve the accumulation of drugs in deeper skin layers as they penetrate intact down to the epidermis, followed by further penetration to deeper layers owing to the bilayers ’fluidity enhancement caused by the penetration enhancer. In addition, the free penetration enhancer exerts a synergistic effect through interaction with the skin lipids, which leads to the perturbation of the intercellular skin lipid pathway.
Silybum marianum L. is the scientific name for Milk thistle or St. Mary’s
thistle. Silymarin (SIL) is the active component of this herb. Silymarin was shown to suppress inflammation and oxidation, which are important in the process of wound healing. Reactive oxygen species (ROS) which are formed after cutaneous injury, may lead to destruction of lipids, proteins and extracellular matrix elements that contribute to delayed healing of wounds.
There are many functional materials that help healing of wound in optimum way as CaCl2, lactic acid, hyaluronic acid and Chitosan. CaCl2 has an effective role in wound healing as it augments the production of granulation tissue in early stages of wound healing through the Ca++- calmodulin system. Additionally, lactic acid helps in the synthesis of collagen, stimulates angiogenesis, endothelial cell proliferation and migration. Also, hyaluronic acid (HA) is the main polyanionic polysaccharide constituent of all connective tissues in the body, it increases fibroblast cell proliferation and stimulate keratinocyte migration, proliferation and differentiation. Chitosan is a polycationic aminopolysaccharide polymer that widely used in wound healing owing to its broad spectrum antibacterial activity, its ability to absorb wound exudates, and to protect the wound.
Therefore, the aim of the work in the current thesis was to formulate SIL loaded PEVs containing functional additives such as chitosan, hyaluronic acid, CaCl2, and lactic acid for wound healing.
The work in this thesis is divided into two chapters
Chapter I: Preparation and characterization of SIL loaded PEVs
The work in this chapter included the following:
1- Preparation of preliminary formulations (F1-F7) by thin film hydration technique in order to optimize the loading of SIL in the PEVs, a total amount of 900 mg of phosphatidylcholine and different amounts of silymarin (10, 20, 50, 100, 160, 180, 200 mg), using transcutol as penetration enhancer.
2- Characterization of the prepared SIL PEVs through the following studies:
a) Analysis of particle size, SPAN index of the freshly prepared SIL PEVs using mastersizer.
b) Analysis of zeta potential of the freshly prepared SIL PEVs formulations using zetasizer.
c) Analysis of EE% and loaded amount of SIL
3- Optimization of the selected SIL loaded PEVs formulations by adding functional additives namely lactic acid, calcium chloride, hyaluronic acid and chitosan.
4- Characterization of the optimized SIL PEVs through the following studies:
a) Analysis of particle size, SPAN index of the freshly prepared SIL PEVs using mastersizer.
b) Analysis of zeta potential of the freshly prepared SIL PEVs formulations using zetasizer.
c) Analysis of EE% and loaded amount of SIL.
d) Determination of the viscosity of the optimized SIL PEVs formulation using a viscometer.
e) Assessment of the physical stability of the SIL PEVs through monitoring the change in particle size, SPAN index, EE% and zeta potential after storage for 6 months at refrigeration temperature.
f) Ex-vivo deposition study of the selected formulae P1, P5, P11 and SIL SUSP.
g) Antimicrobial susceptibility assay of selected P11 formulation, its plain formula counterpart, SIL in DMSO using DMSO as negative control and gentamycin as standard against staphylococcus aureus and pseudomonas aeruginosa.
h) Examination of P11 morphology using transmission electron microscope.
The results of this work revealed the following:
11. PEVs containing transcutol as penetration enhancers were successfully prepared using the thin film hydration method.
12. PEVs particle size ranged from 3.08-5.73 µm, depending on the type and amounts of additives added.
13. All of the prepared PEVs were charged (hyaluronic acid containing vesicles were negatively charged while chitosan containing vesicles were positively charged).
14. The EE% of SIL in PEVs ranged from 42.65-76.15% owing to the hydrophobicity of the drug, suggesting its successful incorporation within the lipid bilayers.
15. An increase in the EE% of SIL within PEVs was achieved upon adding chitosan in the preparation of PEVs, correlating with the increased particle size of chitosan containing PEVs.
16. The PEVs dispersions were found to be of higher viscosity than water, owing to the presence of vesicular lamellar structures.
17. SIL loaded PEVs 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.
18. Ex vivo skin deposition experiments demonstrated the high potential of the selected PEVs formulations (P1, P5 and P11) in accumulating the drug into the deeper epidermal and dermal layers of the skin, compared to SIL suspension, with the chitosan containing formulation P11 being superior to P1 and P5.
19. Transmission electron microscopy of P11 displayed the sealed spherical PEVs of SIL.
20. 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. This delineates it as a promising formulation for wound healing.
Chapter 2: Assessment of wound healing activity of SIL loaded PEVs
The work in this chapter included the following:
1- Histopathological examination of wound parameters as re-epithalization, granulation tissue formation, angiogenesis, collagen fibers formation throughout treatment period (21 days) for the selected formula (P11), its plain counterpart (the same composition as P11 but without silymarin), compared to the marketed healosol® spray.
2- Measurement of wound diameters throughout treatment period (21 days).
3- Measurement of dermal content of collagen which expressed as MTC area %. 4- Measurement of VEGF expression which indicates angiogenesis process.
The results of this work revealed the following:
11. By measuring wound diameter, both P11 and plain formula received groups showed 50% reduction of wounds in 7 to 10 days which was faster than other experimental groups (untreated control group and Healosol® received group) that took 10 to 14 days to show 50% closure of wounds.
12. P11, Healosol® and plain formula received groups showed complete wound closure at the end of the treatment period 21 days but control group wasn’t able to be healed completely.
13. The examination of histological parameters showed that wound gaps in all the experimental groups at day 3 were covered with thick necrotic crust admixed with infiltration of acute inflammatory cells mainly neutrophils, edema and hemorrhages.
14. At day 5, both P11 formulation (the selected formulation) and Healosol® received groups showed the start of wound healing with decrease in inflammation compared to other groups (untreated control group and plain formula received group).
15. At day 7, the wound gap of P11 received group showed well vascularized granulation tissue which characterized by numerous newly formed blood capillaries (angiogenesis), but plain formula received group and control group showed less healing signs.
16. At day10, Both P11 formulation and Healosol® received groups showed marked progress in wound healing, whereas plain received group showed mild to moderate enhancement in wound healing compared to delayed healing in control group.
17. At day 14, complete epidermal covering with evidence of keratinization was observed in Healosol® and P11 received groups, however, P11 received group was superior to Healosol® received group in reduction of wound area.
18. Also at day 14, plain formula received group showed limited epidermal growth at the wound edges whereas control group showed delayed healing manifested by inflammation, edema, haemorrhage and absence of epidermal remodeling.
19. At day 21, P11, healosol® and plain formulation received groups showed complete epidermal covering with evidence of keratinization especially P11 and healosol® but control group missed epidermal coverage, the wound gap was filled with mild to moderate inflamed granulation tissue.
20. Measurement of collagen deposition in each group showed that The highest collagen content was detected in the dermis of rats from P11 treated group followed by Healosol® treated group then plain formula treated group then control group with MTC area % of 35±1.5%, 23±1.05%, 12.2±0.9% and 7.5±0.86% respectively.
21. Measurement of angiogenesis process through measurement of VEGF expression showed that the highest VEGF expression was found in P11 treated group followed by Healosol® treated group then plain formula treated group then control group with VEGF area % of 14±1.3%, 12.1±1%, 5.2±0.8% and 1.3±0.4% respectively.