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Abstract Quercetin [3,3‘,4‘, 5, 7-pentahydroxyflavone] (QT) is a polyphenolic flavonoid that has been recently extensively investigated for its biological activities, such as anti-inflammatory, antioxidant, hepatoprotective activities, antitumor and antiproliferative effects on a wide range of human cancer cell lines. Its antitumor activity was found to be through inhibiting glycolysis and macromolecules synthesis in addition to some enzymes such as matrix metalloproteinases (MMPs), NaıKıATPase, protein kinase C, tyrosine kinases, and pp60ı kinase. It is considered a promising candidate for clinical trials but its extreme water insolubility hinders its introduction in clinical trials. It is usually administered in dimethyl sulfoxide (DMSO) as a vehicle which carries risks of vasoconstriction and neurological, liver and kidney toxicity. Therefore, the work in this thesis aimed at formulating QT in injectable polymeric nanocapsules with an oil core in which the drug is soluble which would result in high encapsulation of the drug hence overcoming its extreme water insolubility and allowing its i.v. administration in a stable nanoform. Polymeric nanocapsules offers the advantages of the possibility of loading high amount of water insoluble drug molecules into the oil core, their physicochemical stability, and protection against enzymatic degradation due to the presence of the polymeric wall. Moreover, their subcellular size allows relatively higher intracellular uptake. Furthermore, attaching a targeting moiety (passive or active) to these nanocapsules would enable targeted delivery of the drug enhancing the efficiency of treatment through concentrating the drug at the tumor site and hence decreasing its systemic side effects which was tried in this thesis as well. In the first chapter, QT loaded PLGA polymeric nanocapsules (PNCs) were prepared and characterized. To determine the oil to be used in preparation of the nanocapsules, the saturated solubility of QT in different oils namely; castor oil, ethyl oleate, soybean oil, mygliol, labrafac propylene glycol (Labrafac PG), oleic acid, olive oil and sesame oil was determined at 25°C. QT polymeric nanocapsules (QT-PNCs) were then prepared using the nanoprecipitation technique utilizing the oil selected from solubility study. A preliminary study was done on some factors to select the suitable conditions to formulate QT-PNCs of appropriate size range. The studied factors were; the lipophilic emulsifier type and organic solvent composition, oil and drug concentration, Soybean Lecithin Type and PLGA Type. For further optimization of particle size (PS), a full factorial design experiment was built up to study the effect of three factors namely; Tween 80 concentration, polymer concentration and soybean lecithin concentration using the PS as response and performing check point analysis. The selected formulations with optimum PS were characterized through studying their zeta potential, EE%, invitro drug release and physical stability. Results showed that castor oil dissolved the highest amount of QT so it was selected as the oil core for preparation of QTPNCs. Most suitable formulation conditions among the studied ones to produce QT-PNCs of PS lower than 200 nm were found to be: Epikuron E145V soybean lecithin as an emulsifier with acetone: ethanol 6:4, 3% w/v castor oil, 5 mg QT and PLGA 7502 A as a polymer. The results of factorial design experiment showed that increasing both Tween 80 and E145V concentration significantly decreased PS of QT-PNCs (p˂0.0001) while polymer concentration didn‘t have a significant effect on PS in the studied range. Moreover, a significant two way interaction was found between Tween 80 and soybean lecithin concentrations (p˂0.0001). QT-PNCs carried a highly negative charge due to soybean lecithin and carboxylic groups of PLGA which resulted in high stability of PNCs. The EE% of QT in PNCs was between 94 -98% which provides the advantage of formulating QT in a nanoparticulate system allowing its parenteral administration in the required dose. In-vitro release studies in 70:30 ethanol: water at 37oC showed a biphasic pattern of QT release from PNCs; an initial burst release, 50% in 24 h, which was still lower than that obtained from other studies, followed by sustainment of release over a long period (3 days). Non-significant change in PS and zeta potential of selected formulations was observed over 6 months indicating its suitable stability. In the second chapter, the composition (3% castor oil, 0.5% soybean lecithin, 0.5% PLGA 7502 A polymer and 0.2% Tween 80) was selected for modification by pegylation and conjugation of folic acid (FA) moiety for passive and active targeting, respectively. For this aim, PLGA conjugates were synthesized; PLGAPEG conjugate for passive targeting and PLGA-PEG-FA for active targeting. PLGA-PEG-FA was synthesized using two synthetic methods which were compared. The synthesized conjugates were characterized by FTIR and H1-NMR, furthermore, polyethylene glycol (PEG) and FA content were quantified using colorimetric and spectrophotometric assay, respectively. QT-PNCs were then prepared using the synthesized conjugates and characterized by determination of their PS, zeta potential, EE%, in-vitro release study and physical stability. Morphological examination was done by Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM) and Cryo-Transmission Electron Microscopy (Cryo-TEM). The chemical conjugation and formation of PLGA-PEG and PLGA-PEG-FA conjugates was confirmed by FTIR and 1H-NMR. All conjugates showed high PEG content of 14-16%. Moreover, the FA content of PLGA-PEG-FA synthesized by both tried synthetic methods was found to be similar. PNCs formulated using PLGA-PEG showed significantly smaller size and lower zeta potential than non pegylated ones. PLGA-PEG-FA NCs size and zeta potential were close to that with PLGA only. No difference was found in EE% among the PNCs formulated with the three different polymers. AFM and TEM confirmed the spherical shape of PNCs and confirmed the presence of polymer shell around the oil core. The release profile of the three formulations, NC1 (PLGA), NC3 (PLGA-PEG), NC5 (PLGA-PEG-FA) in phosphate buffer saline (PBS) was similar with a biphasic pattern of burst release followed by sustained one. In presence of serum, NC3 and NC5 showed higher stability than NC1 due to presence of PEG that provides more protection from destabilization by serum proteins. No significant change in size and zeta potential of particles were observed after 90 days showing high shelf life stability. Therefore, NC1, NC3 and NC5 were selected for further cytotoxicity and in-vivo studies. In the third chapter, In-vitro cytotoxicity and cellular uptake of QT loaded PNCs was divided into two sections; cytotoxicity studies and cellular uptake ones. The cytotoxicity studies were done using the MTT assay. First, the cytotoxicity of free QT in DMSO solution was determined on four different cell lines, namely; C6, B16F10, CT26 and HeLa, to determine those of highest sensitivity to QT. CT26 and HeLa cells showed the highest sensitivity to QT followed by B16F10 with C6 glioma cell line showing least sensitivity.Therefore, CT26 and HeLa cells were selected for further studies. The next step was to test the cytotoxicity of QT versus QT loaded PNCs, NC 1, NC 3 & NC 5, on the selected cell lines, CT26 and HeLa, to determine the effect of drug encapsulation on its cytotoxicty. To test selectivity and active targeting of PNCs, cytotoxicity studies were done on folate expressing HeLa cells in folate free and folate enriched media (200 μm). For further confirmation, similar experiment was done on non-folate expressing CT26 cells. It was found that encapsulation of QT in PNCs didn‘t compromise its in-vitro cytotoxicity on both CT26 and HeLa cell lines. FA-targeted PNCs, NC 5, showed highest cytotoxicity, 56.63%, on Hela cells compared to QT, NC 1 & NC 3 at 10 μM after 24 h in folate free medium due to their uptake by folate recpetors, whereas, in the presence of excess FA, it showed equal cytotoxicity to other formulations due to saturation of folate receptors by free folic acid in the medium. No difference in the cytotoxicity of the targeted and non-targeted PNCs was seen on CT26 in both folate free and folate enriched medium further confirming the results. In the second part of this chapter, the uptake of PNCsin HeLa cells was studied by fluorescence labeling of PNCs through incorporation of DiI in the oil core of PNCs. The uptake of fluorescently labeled PNCs in HeLa cells, at 10 ug/ml after 1 & 4 h in folate free and folate enriched medium, was quantified by spectrofluorimetry and examined by Confocal Laser Scanning Microscopy (CLSM). NC5 was found to have higher uptake than NC 1& NC 3 in folate free medium after 1h and 4 h incubation. Higher uptake was found after 4h than 1h. In presence of excess FA in the medium, all PNCs formulations showed similar uptake. Similar results were shown by CLSM. Therefore, both cytotoxicity and cellular uptake studies proved superiority and active targeting of FA-targeted PNCs, NC 5 than non-FA ones. In the fourth chapter, the three PNCs formulations, NC1, NC 3 & NC 5, were selected for in vivo uptake studies which were done using two different cell lines. The first cell line was the CT26 which was selected with the aim of studying and proving the passive targeting of PNCs in case of non folate expressing tumors. This was done utilizing a radiolabeling technique. In this study PLGA-PEG-DTPA conjugate was synthesized for incorporation in the polymeric shell of PNCs and then chelation with radiotracer, In111. Its chemical conjugation and formation was confirmed by FTIR and H1-NMR. It was then incorporated in the polymeric shell of PNCs in different ratios with the polymer (PLGA or PLGA-PEG or PLGAPEG- FA) forming the coat (1-50%) to find the optimum ratio to be used for efficient radiolabeling without affecting the properties of the PNCs optimized previously. The efficiency and stability of radio-labeling was tested using thin layer chromatography (TLC) technique. 5% w/w PLGA-PEG-DTPA was found to be efficient for labeling. Furthermore, labeled PNCs showed stability after incubation with PBS (pH 7.4) and serum for 24 h. No significant change on the PS or zeta potential of PNCs (NC 1, NC 3 & NC 5) was observed after incorporation of the 5% PLGA-PEG-DTPA. After proving the efficiency and stability of labeled PNCs, they were injected in CT26 tumor bearing mice for studying tumor uptake by live animal SPECT/CT imaging and tissue biodistribution was determined quantitatively by Gamma Scintigraphy. In the SPECT/ CT, animals were imaged at 3 time points, 0.5, 4 & 24 h. Similar organ biodistribution was observed in case of NC 3 and NC 5 formulations with obvious prolonged blood circulation profile than NC 1 which showed fast accumulation in the liver and spleen (~90% ID) even within the first 30 min confirming the stealth characteristics of the PEGylated PNCs (NC 3& NC 5). Therefore, NC 1 was excluded from further studies. In the quantitative biodistribution studies, NC 5 showed lower blood % and lower % injected dose/ g (ID/g) of CT26 tumor than NC 3 by gamma counting due to uptake of particles by folate receptors in the liver. The % ID/g of tumor accounted for 3.9% in case of pegylated PNCs, NC 3, which proves passive targeting of these particles and high accumulation in tumors. For further confirmation an in vivo tumor growth delay study was carried out on CT26 tumor bearing BALB/cs. When the tumor reached appropriate size (200 mm 3), PNCs, NC 3 & NC 5, were injected i.v. for a total of 4 doses over 12 days; 50 mg/ kg QT every 3 days, and tumor size was measured using a vernier caliper. NC 3 showed significant tumor size reduction starting 3rd day after the start of treatment. This proves the efficiency of the pegylated NCs, NC 3, in targeting QT and increasing its uptake to the tumors, hence increasing its therapeutic effect. The second part of the work was done on folate expressing tumors, HeLa and IGROV1, to prove the active targeting of NC 5. This was done through using fluorescence labeling of PNCs, NC 3 & NC 5, through incorporation of DiR fluorescent dye in oil core of PNCs. HeLa and IGROV-1 bearing animals were kept on folate free diet one week before and during the period of the experiment. PNCs were injected in animals and they were imaged at different time points, 1, 4 and 24 h, using IVIS® Lumina Series III in Vivo Imaging System. Organs and tumor uptake was imaged and uptake quantified using Living Image® software. Tumors were frozen and sections were cut by a cryostat and examined by CLSM. Both NC 3 and NC 5 showed similar tumor accumulation (%ID per gram of tumor) in HeLa and IGROV-1 folate expressing tumor models due to their uptake by EPR effect although uptake of NC 5 may occur by folate receptors in liver as mentioned before. Moreover, the selectivity of uptake of folate bearing PNCs, NC 5, was further shown by altered intra-tumoral distribution and better association with cancer cells thus confirming the in vitro results. Therefore from the work in this thesis, a stable intravenously administered nanopaticulate system of QT of optimum size for tumor targeting (˂200μm) was successfully prepared. Conjugation of PEG and PEG-FA moieties for both passive and active targeting was achieved. The efficiency of the prepared particles for both passive and active targeting of QT was proved both in-vitro and in-vivo. |