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Abstract Title of thesis: Lipid Polymer Hybrid Nanoparticles as an Efficient Hepatic Drug Delivery System Summary In spite of the great development in the field of hepatology, CHB, is a major public health problem worldwide. Unfortunately, CHB is a major public health challenge in Egypt where 1.4% of the total population were diagnosed with HBV and millions are undiagnosed or at risk. E, a nucleoside reverse transcriptase inhibitor, is one of the first line potent antiviral therapies for HBV which could reduce the risk of virus progression to liver cirrhosis and hepatocellular carcinoma. Although its proved efficacy, E is accompanied with some drawbacks as high risk of resistance and serious adverse effects as neuropathy, skeletal and cardiac myopathy, nephrotoxicity, pancreatitis and lactic acidosis. Moreover, the high rate of non-compliance in patients with chronic diseases and long-life treatment are serious healthcare concerns that pose a great challenge to the successful delivery of pharmaceuticals. To tackle these problems, intramuscular administration of E has been attempted. To provide a suitable delivery system, we hypothesized that LPH will be able to control the hydrophilic drug release in a sustained manner. Accordingly, the ultimate goal of this study was to control E release and improve its macrophage uptake. A special focus will be given to the effects of E loading technique, the tuning of the lipid corona as well as vitamin E coating on its release and uptake. To fulfill the above goal, the work in this thesis was divided into three chapters: Summary 146 I. Preparation and characterization of Entecavir Lipid Polymer Hybrid Nanoparticles. II. Preparation and characterization of Entecavir-Hyaluronate conjugate Lipid Polymer Hybrid Nanoparticles. III. Biological Studies on Different Entecavir Lipid polymer Hybrid Nanoparticles. Chapter I Preparation and characterization of Entecavir Lipid Polymer Hybrid Nanoparticles. In this chapter we aimed to prepare LPH with high drug EE% and PS < 200 nm suitable for the controlled delivery of E, to the liver. Four studies were performed: study I: A preliminary study to set the surfactant concentration and time of stirring which deemed to be of crucial importance during nanoprecipitation. Study II, where optimization of different critical process parameters on E LPH was done to achieve the quality target product profile. Thirdly the release of the optimized LPH, with the highest EE% and minimum PS<200 nm, was modified using different shell lipid with the evaluation of their different physicochemical characteristics. Finally, the effect of vitamin E coating on the characteristics of the optimally lipid modified LPH was investigated. PS and EE % could be manipulated by changing the polymer, lipid and/ or E amount as well as the stirring speed. The optimized formula were selected depending on the maximum EE % and PS < 200 nm with the following composition: F1-LEC: 5 mg PLGA, 2.85 mg lecithin and 13.33 mg E prepared at 900 rpm. F2-LEC: 15 mg PLGA, 1 mg lecithin and 9.45 mg E prepared at 900 rpm. Summary 147 F3-LEC: 5 mg PLGA, 2.93 mg lecithin and 9.45 mg E prepared at 1000 rpm. All the prepared formulae showed a negative zeta potential ranged from -14.51±1.85to -25.51±2.62 depending on E amount and lecithin content. The in vitro release study showed biphasic drug release with T100% achieved in 12-18 h. The in vitro release depended directly on the drug amount and inversely polymer to lecithin ratio. In our study, using lecithin as the sole component in the lipid shell prolonged the release for less than 24 h. Hence, it deemed necessary to modify the lipid shell in order to, provide more control over the E release from the proposed LPH. Different lipid mixtures were tried to control drug release namely; lecithin in combination with cholesterol or glyceryl monostearate in different ratios (1:1, 1:1.5, 1:2) while keeping the total lipid amount constant during the fabrication. All the tried lipid mixture ratios resulted in significant increase in PS. However and as previously highlighted, a PS below 200 nm is crucial for hepatic passive targeting. Therefore, E LPH prepared using lecithin to cholesterol ratio (1:1.5) and lecithin to GMS (1:1) were elected for further studies, viz. zeta potential, drug loading efficiency and in vitro drug release. Moreover, the prepared lipid shell modified LPH showed a significant increase in zeta potential. Both cholesterol based and GMS based LPH extended E release for 36-48 h and 72-120 h respectively. A further coat with vitamin E extended the release for one week, while keeping the PS ≤ 200 nm. conclude that the modification of lipid shell and vitamin E coating of E loaded LPH have been provided a lucrative option to control the E release over a period of one week. Such a control release is efficient to achieve patient compliance and would be suitable for patients with renal impairment. In the next chapter attempts were made for to prolong drug release for liver patients without any accompanied diseases. Summary 148 Chapter II Preparation and characterization of Entecavir-Hyaluronate conjugate Lipid Polymer Hybrid Nanoparticles. Moreover, the impact of drug incorporation technique in the LPH on E release was studied. In this chapter, the preparation of vitamin E coated E-HA LPH was exploited in order to achieve a controlled release of E. The drug was first conjugated with HA and the resultant compound was characterized. An indepth study of all structural alterations occurred during conjugation process was assessed by UV spectrophotometric analysis, 1H NMR, 13C NMR and Mass spectroscopy. The synthetized conjugate was further incorporated in LPH using the optimized lipid shell composition and vitamin E coating based on the results of chapter I. The primary purpose of using a drug-polymer conjugate for nanocarriers’ drug delivery is the modulation of drug’s release and kinetics towards controlled one and ideally, zero order release kinetics. Therefore, the effect of using E-HA conjugate in vitamin E coated LPH fabrication was studied by drug association, PS and zeta potential. AFM and TEM were used to give an insight on the morphological characteristics of the proposed system. Moreover, the feasibility of controlling the release of Ewas tested by in vitro drug release study and comparing the results to vitamin E coated LPH with passively loaded; unconjugated E. In this study, the grafting of E with HA was based on the carbodiimide chemistry with conjugation efficiency up to ≈ 30%. The prepared E-HA conjugate was successfully used to fabricate LPH with also the same QTPP (PS < 200 nm and maximum association efficiency). The obtained results showed that using E-HA conjugate led to controlled release over a period of one month. The conjugation of E to HA prior to fabrication the LPH as well as the customizable lipid shell could provide an efficient way to control E release. The proposed system showed good stability in the presence of serum as well as shelf Summary 149 life stability. Further work will be conducted in the next chapter to evaluate the feasibility of E-HA to improve drug cellular internalization. Chapter III Biological Studies on Different Entecavir Lipid polymer Hybrid Nanoparticles In this chapter, the cytocompatibility of the developed LPHs was evaluated by testing its hemolysis effect on red blood cells and cytotoxicity on macrophage J774 cells. The feasibility of vitamin E coating to improve the macrophage uptake of different E LPHs was evaluated. The in vivo biodistribution of the intramuscularly injected DiR labelled EE-HA LPH mice were assessed. Furthermore, the body weight change, vital organ weight and histopathological examination of liver, spleen, lung, kidney and intestine tissues were evaluated as toxicity markers. The results of the toxicity studies were compared to those of PBS injected mice. The biocompatibility and cytocompatibility of either E LPH or E- HA LPH were proven by low haemolytic effect on rats’ RBCs (< 5%) and negligible effect on macrophage J774 cells viability. Moreover, the negative charge of the proposed systems was able to evade the adsorption of plasma proteins and subsequently kept the system stable under physiological conditions. The second target of this study focused on the assessment of the LPH ability on cellular retention in J774 macrophages cells. The cellular uptake of E LPH was confirmed qualitatively and quantifiably by confocal laser microscope and flow cytometry respectively. The presence of vitamin E coat on both types of fabricated LPH significantly improved the macrophage uptake in comparison to uncoated ones. Long muscular residence and high liver uptake were confirmed Summary 150 by in vivo imaging of the prepared E-HA based LPH which is positively correlated with the in vitro release results. |