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Abstract English summary Preparation and characterization of nano-formulations for acne treatment from the plethora of research lately, many novel drug delivery systems were discovered, enhancing the drug’s skin deposition, retention, and permeation. Several delivery systems have been exploited for such purpose, among which are the lipid nanoparticles, such as the solid lipid nanoparticles (SLNs), the nanostructured lipid carriers (NLCs), and the nano-emulsions, as well as vesicular systems, such as liposomes, niosomes, ethosomes, transfersomes, and penetration enhancer vesicles (PEVs). New delivery systems have emerged, such as aspasomes consisting of a bilayer forming material ascorbyl palmitate and cholesterol as a stabilizing agent, enclosing an aqueous environment, where they offer the advantage of having an antioxidant property. Nanofibers is another delivery system consisting of fiber threads with diameter in the nm range prepared by electrospinning technique, where their morphology depends on three main process parameters; solution flow rate, distance, and electrical voltage. Both systems offer many advantages in the topical delivery of drugs. Quercetin is a natural flavanol, which gained much interest in the last period due to its tremendous number of actions regarding its anti-cancer, anti-bacterial potential against P. acne, anti-inflammatory, and anti-oxidant potential, where it acts as a topical anti-oxidant due to its ability to scavenge the free radicals. It also inhibits the tumor necrosis factor alpha (TNF-α) production, responsible for chronic inflammatory diseases such as acne. Therefore, the aim of this work was to encapsulate quercetin as a bacteriostatic and anti-inflammatory herbal compound, in English summary P a g e | 161 non-traditional nano-delivery systems such as aspasomes and nanofibers, to be characterized and tested for their potential in the treatment of acne upon topical application. The work in this thesis was divided into three chapters: Chapter I: Preparation and characterization of quercetin-loaded aspasomes This chapter involved the formulation, and characterization of quercetinloaded aspasomes. Aspasomes were prepared by the thin film hydration technique and characterized for their particle size, zeta potential, entrapment efficiency (EE%), stability when stored at a temperature of 4-8° C for three months, their ex-vivo skin deposition/permeation, their anti-oxidant potential, their morphology using the transmission electron microscope (TEM), and their thermal properties using differential scanning calorimetry (DSC). The work in this chapter included the following: 1. Preparation of the aspasomes by the thin film hydration technique, using different concentrations of the lipidic mixture to formulate plain vesicles, where they were first prepared without quercetin, nor any oils for optimization purposes, then the optimized formula was selected for incorporation of the quercetin and different types of oils namely tea tree oil, neem oil and cinnamon oil at different amounts. Ingredients were dissolved in the organic solvent mixture, which was evaporated till the formation of the thin film of dry lipid. The dry film was then rehydrated with phosphate buffer saline (PBS pH 7.4), and the dispersion was rotated to allow the rehydration and maturation of vesicles. Finally, the aspasomal formulations were tested for further investigations. English summary P a g e | 162 2. characterization of the prepared aspasomes was done through the following studies: a) Particle size, PDI and surface charge using the Zeta-sizer Nano b) Entrapment efficiency percentage (EE%) of the aspasomal formulae via an ultrafiltration method using the viva-spin tubes. c) Physical stability study on the selected aspasomal formulae by the assessment of the effect of storage on the particle size, charge, PDI, and EE% of the nanoparticles. d) Ex vivo-deposition on rat skin using Franz diffusion apparatus e) Measurement of the anti-oxidant potential of the selected aspasomal formulae was carried out using theDiphenyl-1-picrylhydrazyl (DPPH) anti-oxidant assay f) Morphology of the selected formula using the transmission electron microscope (TEM). g) Thermal analysis using the differential scanning calorimetry (DSC). The results of this work revealed the following: 1. Quercetin was successfully loaded into aspasomes, using the thin film hydration technique. 2. Aspasomes preparation necessitated the presence of three main components ascorbyl palmitate, cholesterol, and dicetyl phosphate. Ascorbyl palmitate was used as the bilayer vesicle forming material, while the cholesterol was used as the main stabilizing component, and the dicetyl phosphate acted as a charge inducer. 3. Particle size of the selected formulae (F10, F13, and F16) ranged from (125-184 nm) depending on the presence of cholesterol, the presence of neem oil, tea tree oil, or their combination together. Some formulae were excluded due to their micrometer size, and others were excluded due to the presence of oil droplets. English summary P a g e | 163 4. All of the prepared aspasomes were charged, with charges ranging from (-79.9 to -100 mV) suggesting that they are stable against vesicle aggregation and fusion. 5. The EE% of the selected formulae (F10, F13, and F16) ranged from (96.13- 99.81%) owing to the hydrophobicity of the drug, suggesting its successful incorporation within the lipidic bilayers of the aspasomes. 6. Different EE% values were obtained for quercetin depending on the composition of vesicles, the presence of cholesterol formulating them, and the presence of neem oil, where the maximum entrapment was shown for F13 and F16 99.81%, and 99.63% respectively. 7. Stability studies for the selected formulae suggested that minor changes in particle size, PDI, zeta potential and EE% occurred, indicating adequate stability. 8. The Ex-vivo deposition of quercetin from the selected formulae (F13, and F16) was 27.10% and 40.93% respectively, which showed that the aspasomes acted as a good nanoparticulate system loading quercetin allowing its good skin retention, deposition, and exhibiting low transdermal permeation rates. 9. DPPH assay suggested that quercetin showed an anti-oxidant activity when loaded into aspasomes, where the quercetin solution, and the selected best formulae (F13and F16) showed a better anti-oxidant activity compared to the ascorbic acid reference. 10. Transmission electron microscope of the selected formula F16 demonstrated the sealed spherical nature of quercetin vesicular system. 11. DSC charts showed the absence of the endothermic peak of quercetin, which ensures the complete drug encapsulation within the lipid bilayers. 12. The selected formula F16 was selected for conduction of further experiments. English summary P a g e | 164 Chapter II: Preparation and characterization of quercetin-loaded nanofibers This chapter dealt with the formulation, and characterization of quercetinloaded nanofibers, where preparation of both PVA/aspasomes composite NFs and PVA/quercetin/essential oils NFs using PVA as a fiber forming polymer was attempted. characterization of the nanofibers included visual examination, morphological examination using the scanning electron microscope (SEM), ex-vivo deposition/permeation using Franz diffusion apparatus, physical integrity of the nanofibers using the water-retention test, the thermal properties by DSC and chemical interaction using Fourier transform infrared spectroscopy (FT-IR). The work in this chapter included the following: 1. Preparation of the nanofibers using a horizontal set up type electrospinner, where 10% PVA was used as the basic polymeric solution. Firstly, four trials for preparation of PVA/aspasomes composite nanofibers were attempted by mixing an aliquot of aspasomal formulation F16 with 10% PVA solution by different ratios, followed by electrospinning. Secondly, another four trials were attempted, by mixing different amounts of quercetin and neem/tea tree oils with the 10% PVA solution, to yield PVA/QC/EOs nanofibers upon electrospinning. 2. characterization of the prepared nanofibers was done through the following studies: a) selection by visual examination b) Morphological examination of the nanofibers using the scanning electron microscope (SEM) c) Ex-vivo deposition/permeation using Franz diffusion apparatus over a period of 6 hours d) Fourier transform infrared spectroscopy (FT-IR) experiments to examine any chemical interaction occurring within the nanofiber mats English summary P a g e | 165 e) Thermal analysis using the differential scanning colorimetry (DSC) f) Physical integrity (water-retention test) to ensure the mechanical strength of the nanofibers The results of this work revealed the following: 1. Quercetin was successfully loaded in the nanofibers, using the electrospinning technique, operated by a horizontal type electrospinner. 2. The electrospinning process was found to be affected by many process parameters which control the morphology and the average diameter for the nanofibers. Those parameters are the electrical voltage, the solution flow rate, and the distance. 3. The optimum parameters for the plain nanofibers P1 were a flow rate of 3 mL/hr, distance of 10 cm, and 20 KV as voltage. As for the PVA/Asp composite nanofibers (N1, N2, N3, and N4), they were excluded from further characterization steps, owing to the high percentage of beads occurring as shown in the SEM images, and the incidence of dropping. 4. SEM analysis showed that nanofibers were homogenously dispersed, where the best formula H2 was homogenous and yielded an average fiber diameter of 313.08 nm, and it was considered beadless and showed no dropping. 5. The Ex-vivo deposition of quercetin from the selected nanofiber formulae H2 was 28.24%± 0.012, which showed that the nanofibers acted as a good nano-system for the quercetin loading, allowing its good skin retention, deposition, and drug accumulation on skin. 6. Permeation experiments showed very low in vitro permeated percent of quercetin (less than 1%) which is favorable to produce its topical action on the acne pimples. English summary P a g e | 166 7. The FT-IR charts revealed broadening of the peaks which may be due to the principle of superimposition which occurred between the PVA and the other components, or a chemical interaction between different groups of the polymeric PVA, and the quercetin and the two oils, and this is ascribed to the fact of having a large number of hydroxyl groups in the composite nanofiber system. 8. DSC charts showed the absence of the endothermic peak for the quercetin, which ensures the complete drug encapsulation within the nanofiber mat. 9. Water retention test revealed that the nanofiber mat H2 was proven to exhibit high mechanical strength and good physical integrity, which hence qualifies it to be used in topical drug delivery. 10. The selected nanofiber mat H2 was selected for further clinical investigations, based on all the previous results. Chapter III: Safety, efficacy and clinical applicability of the selected quercetin loaded aspasomes and nanofibers formulations The aim of the current chapter was to test the antibacterial properties and safety of the selected aspasomes and nanofibers formulations, in addition to testing their clinical efficacy on acne patients when compared to Panthenol® cream as placebo. English summary P a g e | 167 The work in this chapter included the following: 1. An anti-bacterial assay was conducted on the selected aspasomal and nanofiber formulations to test their activity against Propionibacterium acne (P. acne). 2. The safety was evaluated by examining the viability percentage of the 3T3 CCL92 skin fibroblastic cells when both best formulae were applied, using the neutral red cytotoxicity assay. 3. The clinical efficacy was evaluated where the study included 40 patients suffering from mild or moderate acne vulgaris. group I patients were instructed to apply a thin film of quercetin aspasomal formula F16, whereas, group II patients were instructed to apply wet squared shaped quercetin nanofiber patches (H2) of dimensions 1 cm x 1 cm, the placebo formulation was Panthenol®. The treatment period continued up to 8 weeks, and patients were instructed to report any discomfort or irritation encountered during the study. All patients were photographed every 2 weeks and evaluated clinically after 8 weeks on both sides of the face through the counting of comedones, inflammatory and total acne lesions and a reduction percentage was calculated for all types of lesions. The results of this work revealed the following: 1. The best aspasomal formula F16, and the nanofiber patch H2 were tested for their anti-bacterial activity against Propionibacterium acne, their safety on the 3T3 CCL92 skin fibroblastic cells, and clinical applicability on different patients. 2. F16 displayed an average inhibition zone of 15±1.53 mm which was considered significantly higher when compared to the quercetin alone (8.25± 2.08 mm), and this was attributed to the formula ingredients; tea tree oil, neem oil, and the bilayer forming material “ascorbyl palmitate”, showing synergistic antibacterial activity with quercetin. English summary P a g e | 168 3. H2 showed an average inhibition zone of 18± 0.01 mm which was significantly larger when compared to the aspasomal formula F16 (15±1.53 mm), and the quercetin (8.25± 2.08 mm). 4. The aspasomal and nanofibers formulations in addition to quercetin solution all displayed considerable safety on 3T3 CCL92 skin fibroblastic cells, owing to the safe nature of all the ingredients used in the formulae preparation. 5. The clinical applicability of the aspasomal formula F16, and the nanofiber patch H2 was evaluated, where they displayed considerable percentages reduction for inflammatory acne lesions and total acne lesions, which could be ascribed to the presence of the anti-inflammatory drug quercetin, and the anti-inflammatory ingredients tea tree oil, neem oil, and the ascorbyl palmitate. Results of this thesis delineate that quercetin delivery systems adopted in the current thesis (aspasomes and nanofibers) are both promising topical nanoformulations which can be used in the treatment of acne vulgaris. |