Development of novel combined nano delivery system to improve cellular uptake of weakly basic anticancer drugs and cell imaging

In this dissertation, different types of nanoconstructs, including MSNPs and liposomes were fabricated for the development of an advanced drug delivery system which can deliver both hydrophilic and hydrophobic drugs to the tumor cells with better efficacy and biocompatibility and minimized side effects to healthy tissues. The candidature of MSNPs as nanocarriers with potential features, supported with lipid layer was explained in introduction part along with their merits and demerits. The combined effects of liposomes and MSNPs in sustained release or stimuli responsive drug delivery systems equipped with literature were highlighted. This project was mainly divided in to four major parts where nanocarriers were characterized and further evaluated for in-vitro analysis under different conditions such as normoxia and hypoxia. In the first part, MSNPs of different molar mass ratios of surfactant/silica were fabricated. The parameters which can affect the particle size distribution, were evaluated. These MSNPs for their physicochemical properties were initially characterized by Dynamic Light Scattering (DLS) for size distribution and Laser Doppler Velocimetry (LDV) for surface charge. The removal of surfactant for the generation of porous structure, was evaluated with elemental analysis and surface charge. Surface area characterization of MSNPs were evaluated with nitrogen sorption desorption method, where the suface area and surface volume were influence by duration of extraction for surfactant removal. The morphological structures were characterized by different microscopies involving Atomic Force Microscopy and Electron microscopies. The morphological studies performed with AFM were in accordance to the size distribution obtained by DLS. The porous structure of MSNPs was observed with electron microscopies. Tranmission electron microscopy (TEM) has shown that the MSNPs are more or less round in shape with ordered porous structure. The pore size evaluated with TEM was the same as it was characterized with sorption-desorption pore size measurement. For further confirmation of porous structure of MSNPs, Scanning Transmission Electron Microscopy (STEM) was performed and particle size and porous distribution were evaluated. In the work described in second chapter, a lipid coated MSNPs system was designed and characterized for further investigations. Cationic liposomes prepared with thin layer hydration technique and its coating to MSNPs was characterized with hydrodynamic size, surface charge and FTIR spectrum. Lipid coating of MSNPs were further visualized with Cryo-TEM & TEM micrographs. Higher surface area of MSNPs with higher drug loading capacity made them suitable candidate for the delivery of large amounts of drugs. In-vitro drug release profile has shown sustained release effects where the rate of drug release from bared drug loaded MSNPs was faster than lipid coated ones. The lipid coating to MSNPs with reduced drug leakage under inert conditions addressed the issue of premature drug leakage. In a comparative study of in-vitro cytotoxic profile, lipid coated MSNPs due to higher biocompatibility delivered higher concentrations of Dox and has shown more cytotoxic effects as compared to bared MSNPs. These higher cytotoxic effects with lipid coated MSNPs were further evaluated with confocal laser scanning microscope where higher internalization of Dox in the cells was evident for the higher uptake of the carriers. In the third chapter, we have successfully developed a stimuli responsive smart drug delivery system composing of lipid coated MSNPs as a carrier, Dox as a model drug, US as a stimulus and PFP as a stimuli responsive agent. By combining MSNPs with liposomes, very significant results have been produced due to the morphology and unique structure of the MSNPs along with biocompatibility and gatekeeping effects of liposomes. PFP was successfully incorporated inside the pores of MSNPs by capillary filling and very stable nanocarriers were produced. The US-irradiation, with FDA approved specifications, has produced very satisfactory triggered release effects due to rupturing of lipid layer by the gaseous pressure of vaporized PFP inside pores. In-vitro cell culture experiments have shown higher internalization of carrier due to biocompatibility of lipid layer and higher cytotoxic effects due to triggered release by US-irradiations. These smart nanocarriers, can be used as drug delivery system for many chemotherapeutics for site-specific triggered release of drug to enhance the efficacy and to avoid undesired side effects to healthy tissues. The fourth chapter of this study was based on the development of a co-delivery system of carbonic anhydrase IX inhibitor and Dox, where carbonic anhydrase inhibitor loaded liposomes were coated to Dox loaded MSNPs. In-vitro cytotoxic experiments were performed under normoxia and hypoxia and it was evaluated that Carbonic anhydrase IX enzyme was overexpressed under hypoxic conditions. The enzyme inhibitor can be effective only when enzyme is overexpressed in hypoxia. Furthermore, the combined effects of enzyme inhibitor and Dox has produced synergistic cytotoxic effects under hypoxia due to inhibition of carbonic anhydrase IX enzyme. Better inhibition effects of carbonic anhydrase IX inhibitor loaded liposomes were observed as compared to pure enzyme inhibitor, although these findings are on the initial stage and need further to be investigated. In future, the aim of the study will be to perform in-vivo evaluation of drug delivery carriers, involving biodistribution of drug and carriers in different organs. The in-vivo studies will also involve the stimuli US-responsive triggered release in animal model. As for MSNPs as drug carriers are concerned we are looking forward to develop new drug delivery system for another drug like photosensitizer. This system will be a stimuli responsive advanced drug delivery system where US would trigger the release of drug and light will activate the drug to produce cytotoxic effects. As hypoxic experiments are in early stages, so we will extend our studies in hypoxic condition for further validation of already developed system and to evaluate photodynamic effects under oxygen deprived environment