P-Hydroxybenzoic Acid And Myistic Acid Modified Polyethylenimine For Brain-targeted Gene Delivery

Blood brain barrier (BBB) is present in a complex cellular system at the level of the tight junctions between endothelial cells in brain. It can prevent most foreign substances from entering the brain from the blood circulation, including many beneficial therapeutics. Nearly 100% of large molecules and 98% of small molecules are forbidden access to the central nervous system (CNS) due to the poor permeability of the BBB. For this reason it acts as an obstacle for the treatment of cerebral diseases, such as brain tumors, Alzheimer’s disease and Parkinson’s disease. Therefore, the research of enhancement of brain drug delivery, especially development of novel brain targeting drug delivery system is on focusing in pharmaceutics. Glioblastoma multiforme (GBM) represents one of the most lethal cancers and accounts for approximately 40% among all primary malignant brain tumors. The conventional therapy for GBM includes surgical debulking of the accessible tumor, and chemotherapy is also essential in the treatment. The blood-brain barrier (BBB) is an established reason for the dismal prognosis of GBM.Receptor-mediated drug delivery system is a new focus of the brain targeting research and a lot of publications were reported using nano drug delivery systems modified with lectoferrin, transferrin, insulin and monoclonal antibody. Nevertheless, the stability, immunogenicity and preparation highly restricted the use of these high-molecular-weight biological molecules as targeting moieties.The objective of this study is to establish an efficient gene delivery system for brain targeting using the conjugation of p-HA and MC with PEI. PEI with different molecular weight was first conjugated with p-HA or MC separately, then labeled with radio nuclide, 125I, and infrared dye, IR820, for in vivo distribution and imaging study. In vitro and in vivo gene transfection efficiency was investigated as well as anti-glioblastoma effect in U87MG glioblastoma-bearing mice.In chapter one, synthesis and characterization of p-HA-PEI25K and MC-PEI10K was discussed.6-EACA-IR820 was synthesized to label the conjugations and in vivo imaging study indicated the brain targeting capability of both P-HA-PEI25K and MC-PEI10K-Biodistribution study of 125I labeled conjugation also proved that the conjugation can cross BBB and accumulate in brain. In vitro cytotoxicity was also evaluated using U87 and SH-SY5Y cells, neither MC-PEI10K nor p-HA-PEI25K exhibited higher toxicity than PEI25K. In several higher N/P ratio groups for both MC-PEI10K and P-HA-PEI25K, higher cell viability was observed compared with groups without modification.In chapter two, p-HA-PEI25K and MC-PEI10K was used to prepare complex with DNA. Both of them can condense DNA successfully according to argrose gel electrophoresis. The particle size and zeta potential of p-HA-PEI25K/DNA and MC-PEI10K/DNA complexes were determined using dynamic light scattering technique. p-HA-PEI25K/DNA nanopartiles have particle size around 100 nm and zeta potential 31 mV and for MC-PEI10K/DNA the particle size and zeta potential is 110 nm and 25.7 mV. pEGFP and pGL3 loaded nanoparticles were also prepared for in vitro transfectioin study. The expression of luciferase of p-HA-PEI25K/pGL3 treated group is almost 2 times as that of PEI25K/pGL3 treated group at N/P=10 in both 5Y and U87 cells. Higher expression of luciferase was also observed for MC-PEI10K/pGL3 treated group compared to PEI10K/pGL3 treated group.In chapter three, in vivo transfection study and anti-glioblastoma effect were evaluated. Both p-HA-PEI25K and MC-PEI10K can facilitate the expression of GFP and RFP in brain from confocal image of the brain sections. Anti-glioblastoma study showed that both MC-PEI10K/pTRAIL and p-HA-PEI25K/pTRAIL could elongate the lifetime of U87 grafted mice significantly compared to control groups.Small molecules were used for the preparation of PEI based brain targeted drug delivery systems. Both the in vivo and in vitro data showed that this system could be potentially used for brain tumor gene therapy.