Preparation and In Vitro Analysis of Dendrimer-encapsulated Liposomes
thesisposted on 24.10.2013 by Eri Iwasaki
In order to distinguish essays and pre-prints from academic theses, we have a separate category. These are often much longer text based documents than a paper.
Since most of currently available cancer chemotherapeutics often come with severe side effects caused by high toxicity to healthy cells and tissues, it is important to design a novel platform to deliver drugs specifically to the target tissue. In order to minimize these side effects, a number of nanoparticle-based therapeutic, including liposomes, polymeric nanoparticles, micelles, dendrimers, and metallic nanoparticles, has been developed in the last few decades. Consequently, application of these nanotechnologies in the medical field has enhanced the therapeutic activity and reduced the toxic side effects of cancer drugs. In this study, G2 PAMAM dendrimer conjugates (G2-RITC-NH2) were encapsulated into liposomes consisting of DMPC, DSPE-PEG-2000, and cholesterol using a lipid film hydration method. Three different liposomal formulations were prepared by varying the cholesterol content at 25, 50, and 75 mol%. The liposomes were then investigated in terms of (i) release kinetics of G2-RITC-NH2 from each liposome, (ii) cellular uptake kinetics, (iii) endocytosis mechanisms, and (iv) cytotoxicity. The release profiles of G2-RITC-NH2 from each liposome revealed that the stability of the liposomes is highly dependent on the cholesterol level in the lipid bilayer. Microscope observations and flow cytometry measurements also showed the different internalization kinetics of the three liposomal formulations from that of unencapsulated G2-RITC-NH2. In order to further investigate the endocytosis mechanisms of these materials, MCF-7 cells were pre-treated with 5 mM MβCD before being incubated with unencapsulated G2-RITC-NH2 and the liposomal formulations up to 24 h. The results obtained from the flow cytometry analysis indicated that G2-RITC-NH2 internalizes into the cells by a different mechanism compared to the liposomes. Finally, cytotoxicity of G2-RITC-NH2 and the three liposomal formulations was investigated, which showed no significant inhibition of cell proliferation up to 10 µM normalized based on the G2-RITC-NH2 concentration, indicating that the materials could be used as a drug delivery platform. In summary, the in vitro characterization experiments conducted in this study serve as a critical starting point to engineer effective drug delivery systems that combine the advantages of dendrimers and liposomes.