Liposome-DNA/protein interactions : impacts on anticancer drug molecules

Abstract

Characterization, application and modification of the bio-inspired, bioengineered and biomimetic systems in modern gene and drug delivery have been paid remarkable attention in the past few decades. Several groups have conducted extensive studies on the organized assemblies which act as biomimetic systems having close resemblances with biological systems. It has been observed that the bioactive molecules confined in macromolecular assemblies such as liposomes, micelles, reverse micelles, microemulsion and protein aggregates etc. exhibit higher degree of organization and activity compared to that in homogeneous solution. The biomimetic systems are able to mimic some of the reactions occurred in the in vivo biosystems and also have great potential to act as host systems for several organic entities including important drug molecules. As the local properties e.g. polarity, viscosity, and pH in such nano or micro environments are vastly different from that of the bulk region, the structure, dynamics and reactivity of biomolecules at an interface differ noticeably from those observed in the bulk. Interestingly most natural and biological processes occur at such interfaces or inside the confined systems e.g., proteins, biomembranes and vesicles. Therefore, chemistry of a molecule in organized assemblies mimics the extremely efficient in vivo processes occurring in the biological systems. Keeping in mind the wide range of functions performed by biological membranes and membrane proteins, researchers are motivated to look for simple model systems that can mimic the physicochemical properties of the membrane architecture. Because of the widespread interest, the study of different kinds of organized assemblies has grown enormously over the last decade and it has become literally impossible to summarize all the updates in a single article. The interactions of drug molecules with various biological targets like proteins, lipids, DNA etc. are of great importance to evaluate the structural and functional features of the corresponding biomolecules. Therefore entrapment of different fluorescent anticancer drug molecules in the model biomimetic systems and their changes in dynamical and photophysical behavior within the complexes were studied through different spectroscopic techniques. The biomimetic systems studied in the thesis are small unilamellar vesicle (SUV), liposome-DNA complex (lipoplex) and liposomeprotein complex. The interactions of the anticancer drug molecules (doxorubicin and ellipticine) with lipid bilayers of varying surface charges, chain lengths in presence of different metal ions have been extensively studied by steady state, time resolved fluorescence spectroscopy, time resolved anisotropy, circular dichroism spectroscopy, confocal imaging, zeta measurements, AFM etc. The overall objective of the thesis work is to explore the interactions of two major anticancer drug molecules with different biomimetic systems such as liposomes, DNA, protein aggregates, liposomes-DNA and liposomes-protein systems. The study reveals the binding of the drug molecules and their bio-distribution in different aforementioned systems. The interaction of liposomes with DNA and protein has also been explored by this study. Two model anticancer drugs chosen were doxorubicin and ellipticine to investigate the above mentioned interactions. The phospholipids used were 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-Dioleoyl-snglycero- 3-phosphocholine (DOPC), 2-Oleoyl-1-palmitoyl-sn-glycero-3- phosphocholine (POPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) sodium salt (DMPG) and 1,2-dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP). As the cationic phospholipids are cytotoxic, zwitterionic phospholipids were used to prepare liposomes and bivalent metal ions (Ca2+/Mg2+/Zn2+) were selected to mediate the complexation between the liposomes and DNA (lipoplex). Lipoplexes of phospholipids of different phase transition temperatures (Tm) and charges were selected to deintercalate the DOX from the DOX-DNA complex. On the other hand, ellipticine, a very sparingly water soluble drug, was entrapped inside the bilayers of liposomes. Release of the drug from liposomes was studied in presence of different biomolecules (calf thymus DNA and lysozyme). The contents of each chapter included in the thesis are discussed briefly as follows: