Dokument

Summary:
The main theme of the present work was the development of a novel nanocarrier system that can deliver two different therapeutics modalities to the cancer cells. Such a nanocarrier is superior to conventional drug delivery system as it combines two different approaches (i.e. chemotherapy and photodynamic therapy) within one carrier system to treat the cancer. In this work, we were interested in lipid enveloped biodegradable nanoparticles termed as lipoparticles in which two hydrophobic drugs could be encapsulated in separate compartments, a chemotherapeutic agent (Pirarubicin, THP) in the nanoparticle core and a photosensitizer (Temoporfin, mTHPC) in the lipid bilayer shell. Such a system not only increases the therapeutic efficacy, circulating time and bioavailability of the drugs but also reduce the drug leakage and side effects to the other body tissues. In the introduction part of the thesis deals with back ground of the basic principles and detailed mechanism of the photodynamic therapy. The ideal properties of the photosensitizers were discussed. This was followed by a brief insight into the nanocarriers including the liposomes, polymeric nanoparticles and lipid-polymer hybrid nanoparticles. Different methods to prepare the lipoparticles have also been discussed. The methodology section of the thesis deals with the preparation of mTHPC loaded liposomes, THP loaded nanoparticles and consequently the lipid enveloped polymeric nanoparticles. The formed nanoformulations were then evaluated in terms of physicochemical characterizations, in vitro, in ovo, in vivo as well as the biocompatibility studies. In results section, the encapsulation of mTHPC, a potent 2nd generation PS in the liposomes was discussed. For this purpose, a broad range of lipid combination was explored. The physicochemical characterizations including size distribution, zeta potential and encapsulation efficiency was performed. Surface morphological studies using the at atomic force microscopy and cryogenic transmission electron microscopy was conducted. The results obtained from these studies were found to be in accordance with the previous results obtained from the zeta sizer measurement. Further investigations including quantitative assessment of the reactive oxygen species and cellular photodynamic therapy at different wavelengths and different light doses have been discussed. In order to minimize the unethical use of the animals, chick chorioallantoic membrane model (in ovo) as an alternative in vivo model was elaborated for the vascular targeted photodynamic therapy. The intracellular uptake studies using confocal laser scanning microscopy was performed and an effective cellular uptake in the perinuclear region have been shown. 92 In the next chapter of the results, the encapsulation of THP loaded PLGA nanoparticles have been discussed. Two different size of nanoparticles (200nm and 400nm) were prepared in order to have a comparative evaluation of the nanoparticles size, polydispersity index as well as the cell viability using dynamic light scattering and MTT assay respectively. In vitro drug release in simulated conditions revealed a biphasic drug release pattern with initial burst release phase followed by the sustained released pattern for the following days. The THP nanoparticles with smaller size (200nm) showed a higher drug release as compared to 400nm THP NP which was attributed to the larger available surface area for drug diffusion in the earlier case. Further in the results section, the preparation of the lipoparticles have been discussed. Based on the preliminary studies, two best performing liposomes DPPC/mPEG-DPPE5000 and DPPC/DPPG were combined to form a single liposome i.e. DPPC/DPPG/mPEG-DPPE5000 and was coated over the 200nm THP nanoparticles. The formed lipoparticles were also subjected to physicochemical characterizations. An increase in the lipoparticles size of 4-5 nm as compared to uncoated nanoparticles was perceived which was also confirmed with the surface morphological studies using AFM and TEM. The determination higher therapeutic efficacy of the lipoparticles by in vitro cytotoxicity synergism and ROS assay has also been described. The lipoparticles did not show any genotoxicity has been elaborated using single cell gel electrophoresis (comet assay). The stability of the lipoparticles was established in simulated physiological conditions (60% serum & PBS 7.4) and small reduction in particles size owing to the presence of protein corona was demonstrated. Biocompatibility studies were also performed to validate the compatibility of the lipoparticles with the blood components. Hemolysis assay, activated partial thromboplastin time and erythrocyte aggregation assay confirmed the non-toxic and biocompatible nature of the lipoparticles. After the evidencing the biocompatibility of the lipoparticles, the acute in vivo toxicity assessment was performed using BALB/c mice. No significant changes in the body visceral index and serum biomarkers was observed. Also, no significant changes in the tissue histopathology was observed. On the basis of all of these finding, it can be concluded that development of such a novel nanocarrier system can be employed for simultaneous delivery of the multiple drugs to the cancer cells with minimum toxicity. Future pharmacokinetic profiling with in vivo biodistribution and in vivo tumor models with targeting ligands (e.g. antibodies, aptamer etc.) attached on the lipoparticles can serve as a critical link for the pre-clinical studies.

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