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Abstract The transdermal pathway appears to be one of the most promising drug delivery routes due to the merits it carries over other routes as it allows for the controlled and constant delivery of molecules especially in chronic diseases, avoiding firstpass effect and non-invasive application. Targeting solid tumors via the transdermal route has risen as an encouraging emerging approach. Though, the skin poses exceptional barrier properties, which challenge the delivery of all the therapeutic agents, generally and the lipophilic molecules, specifically, due to the high probability of their confinement and entrapment in the skin lipid layers. In transdermal delivery, the goal of dosage design is to maximize the flux through the skin into systemic circulation. Accordingly, various strategies have been implemented to augment the transdermal delivery of bioactives. Mainly, they include in one hand methods that act on the molecule or the barrier itself such as: iontophoresis, electroporation, sonophoresis and on the other hand, there are other methods that depend on formulation adjustment such as: the use of colloidal carriers like microemulsions, lipid-based nanoparticles and vesicular systems Microemulsions are nano-colloidal systems systems composed of oily and aqueous phases, together with surfactants and co-surfactants. Hydrophobic drugs that can be integrated (solubilized) in the oily phase and water phase hydrates the skin leading to increased permeability. The surfactants and co-surfactants act as penetration enhancers and can impact the skin barrier functions in many aspects. Microemulsions are isotropic, macroscopically homogeneous, kinetically thermodynamically stable, translucent and with low energy input method of preparation. On a microscopic level the surfactant molecules form an interfacial Summary 138 film separating the polar and the non-polar domains. This interfacial layer forms different microstructures ranging from droplets of oil dispersed in a continuous water phase (O/W-microemulsion) over a bicontinuous “sponge” phase to water droplets dispersed in a continuous oil phase (W/O microemulsion). The use of microemulsion as delivery systems can improve the efficacy of a drug, allowing the total dose to be reduced and thus minimizing side effects. Thereby, these systems were utilized in this study for the delivery of a phytochemical, namely Methyl Dihydrojasmonate (MDHJ). Methyl Dihydrojasmonte (MDHJ) is an oily derivative of jasmonates (which are a class of plant stress hormones) that is isolated from Jasmine plant (Jasminum officinale). In plant cells, upon wounding or during a pathogenic attack, methyl jasmonates (MJ) causes the induction and the accumulation of proteinase inhibitor as a response. They are also involved in the coordinated activation of programmed cell death (which resembles mammalian apoptosis) and defence. They were also reported to attain selective anticancer activities, which were exhibited recently in both in vitro and in vivo studies. Jasmonates and their derivatives managed to inhibit the proliferation of cancer cells in vitro and to provoke cell death in multiple human and murine cancer cell lines such as: breast, prostate, melanoma, cervix, colon, colorectal, gastric, hepatoma, lung, myeloid leukaemia, neuroblastoma, sarcoma, lymphoblastic leukaemia and lymphoma cells. They accomplish selective cytotoxicity against cancer cells even in the presence of normal cells. Various mechanisms of action are shown by the Jasmonates family to exhibit the anticancer activities, which include the bio-energetic mechanism, the re-differentiation mechanism and the reactive oxygen species (ROS)-mediated mechanism Summary 139 In this thesis attempts have been made to prepare and characterize novel and stable MDHJ microemulsion formulations aiming at obtaining optimum transdermal delivery system possessing high transdermal flux. This is attained via developing a novel method of specific and accurate analysis of MDHJ and through the preparation of different MDHJ microemulsion formulations. Furthermore, an experimental design; Simplex Lattice Mixture Design was used to select the best MDHJ formulation for transdermal delivery. The evaluation of the microemulsion formulations was performed using different methods such as: the measurement of particle size and poly dispersity indices, Transmission Electron Microscopy (TEM) imaging, ex-vivo permeation studies and cytotoxicity studies. Additionally, further in-vivo investigations were conducted to investigate the effect the optimum formulation via application on mice bearing Ehrlich ascites carcinoma (EAC) in solid form, with auxiliary histopathological examinations to the obtained samples from EAC mice model. The work in this thesis is divided into two chapters Chapter I: Development, optimization and ex vivo evaluation of Methyl Dihydrojasmonate microemulsion formulations. This chapter deals with the preparation of microemulsion formulations. Accordingly, pseudoternary phase diagrams were constructed using several combinations of oils, surfactants and co-surfactants and following the water titration method. The drug (MDHJ) was integrated in the oily phase either alone or in combination with other oils such as Oleic acid, Capryol 90®, isopropyl myristate (IPM), Labrafac PG®, Labrafac CC® and Labrafil M® in ratio 1:1. Also, Labrasol®, Summary 140 Transcutol P®, Tween 80® and Plurol Oleique® were employed as surfactants or co-surfactants. The mixing ratio of surfactant with cosurfactant was kept at 1:1. Nine systems were constructed and evaluated according to the largest domains of microemulsion. Two systems were selected and an experimental design(Simplex Lattice Mixture Design) was utilized to select twenty six formulations for further investigation using different weight percentages of oily phase, surfactant /cosurfactant and water as independent factors, while particle sizes and poly dispersity indicesas dependent (response) variable. Seven formulae were chosen for an ex-vivo permeation study through mouse skin. The best microemulsion formulae from each ME system were selected to be examined using TEM. The superlative formulation was further investigated on MCF-7 breast cancer cell lines. The work in this chapter comprised the following: 1. Analysis of MDHJ in phosphate buffer solution of pH 7.4 Methanol (70:30, v/v) using Ultra Performance Liquid chromatography (UPLC). 2. Constructing Pseudo-ternary phase diagrams. 3. Preparation of selected microemulsion formulae according to the Simplex lattice mixture experimental design. 4. Evaluation of the prepared formulation using particle size and poly disperisty index 5. Ex-vivo skin permeation of the selected MDHJ microemulsions formulae. 6. Transmission Electron Microscopy (TEM) 7. Cytotoxicity studies Summary 141 The result of this work revealed the following: 1. The isocratic elution based UPLC method showed sharp chromatograms with a good retention time of 1.6 ± 0.3 min using a mobile phase consisting of acetonitrile: triethylamine buffer (3 ml/L, pH 2.6) with a ratio of 70:30 v/v; a flow-rate of 0.5 ml/min and an injection volume of 10 μl and utilizing a detection wavelength of 190 nm. And according to ICH validation parameters, the UPLC assay method was proven to be highly sensitive and selective for the investigated drug; MDHJ. 2. Two systems S8 ((MDHJ :Capryol 90®)/ (Labrasol® : Transcutol®)/ water system) and S9 ((MDHJ: Oleic acid)/ (Labrasol®: Transcutol®)/ water system) were denoted as successful pertaining to their large microemulsion domains in the pseudo ternary phase diagram. 3. The data of S8 (MDHJ :Capryol 90®)/ (Labrasol® : Transcutol®)/ water system) formulations showed that the particle sizes ranged from 10 nm to 440 nm. 4. The data of S8 (MDHJ : Capryol 90®)/ (Labrasol® : Transcutol®)/ water system) formulations showed polydispersity indices ranging from 0.144 to 0.641 except for FC6,FC12 and FC13 which had a relatively high PDI value of 1. 5. The data obtained from system S9 ((MDHJ: Oleic acid)/ (Labrasol®: Transcutol®)/ water system) showed that the particle sizes ranged from 11 nm to 240 nm |