Guisheng Zhang

Synthesis of galactosyl compounds for targeted gene delivery.

Cell-specific DNA delivery offers a great potential for targeted gene therapy. Toward this end, we have synthesized a series of compounds carrying galactose residues as a targeting ligand for asialoglycoprotein receptors of hepatocytes and primary amine groups as a functional domain for DNA binding. Biological activity of these galactosyl compounds in DNA delivery was evaluated in HepG2 and BL-6 cells and compared with respect to the number of galactose residues as well as primary amine groups in each molecule. Transfection experiments using a firefly luciferase gene as a reporter revealed that compounds with multivalent binding properties were more active in DNA delivery. An optimal transfection activity in HepG2 cells requires seven primary amine groups and a minimum of two galactose residues in each molecule. The transfection activity of compounds carrying multi-galactose residues can be inhibited by asialofetuin, a natural substrate for asialoglycoprotein receptors of hepatocytes, suggesting that gene transfer by these galactosyl compounds is asialoglycoprotein receptor-mediated. These results provide direct evidence in support of our new strategy for the use of small and synthetic compounds for cell specific and targeted gene delivery.

Synthesis and evaluation of vitamin D-based cationic lipids for gene delivery in vitro.

A new panel of steroidal cationic lipids has been synthesized for gene delivery. Using commercially available vitamin D2 (calciferol) or vitamin D3 (cholecalciferol) as hydrophobic motifs and a variety of cationic head groups as binding sites for negatively charged phosphate groups in DNA, we demonstrated that the transfection activity of the synthetic vitamin D-based cationic lipids 1d, 2d formulated with dioleoylphosphatidylethanolamine (DOPE) as a co-lipid is comparable to that of 3-(-[N-N,N-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol). These synthetic lipids are effective in transfecting a variety of cell lines. These results suggest that vitamin D-based cationic lipids are useful transfection reagents for in vitro gene transfer studies.

Synthesis of bifunctional cationic compound for gene delivery

Bifunctional cationic compound carrying trivalent galactosides as the cell targeting ligand and DAB-dendr-(NH2)8 (generation 2.0) as the DNA binding domain was synthesized for gene delivery to hepatocytes. DAB-dendr-(NH2)4 (generation 1.0) conjugated with a hydrocarbon chain was used as a scaffold for the attachment of three galactosides, while the other hydrocarbon end was linked with DAB-dendr-(NH2)8 (generation 2.0) through a carbamate bond. This design provided an effective entry for the synthesis of a polyamine compound having the cell targeting galactosyl ligand. Preliminary in vitro transfection results demonstrated that the bifunctional cationic compound could effectively deliver the gene into HepG2 cells.

Synthesis and Characterization of Aromatic Ring-Based Cationic Lipids for Gene Delivery In Vitro and In Vivo

A new series of cationic lipids has been synthesized for gene delivery using 3,5-dihydroxybenzyl alcohol as the backbone and starting material. Using CMV driven expression system and luciferase gene as a reporter, we demonstrated that the transfection activity of these new lipids when formulated with Tween 80 as co-lipid is comparable to that of DOTAP, one of the most commonly used cationic lipids for transfection. Among the four different cell lines tested including murine melanoma BL-6 cells, human embryonic kidney 293 cells, HepG2 and HeLa cells, the highest transgene expression was seen in 293 cells. Results from in vivo experiments using mice as an animal model show that these cationic lipids preferentially transfect the cells in the lung upon tail vein administration. The cationic lipid, N,N,N-trimethyl-N-[3,5-bis(tetradecyloxy)benzyl] ammonium bromide 4c(di-C14:0) with two 14-hydrocarbon chains exhibits the best transfection activity. These results suggest that these new aromatic ring-based cationic lipids are useful transfection reagents for both in vitro and in vivo gene transfer studies.

TAT and TAT-Like Peptides for Protein Transduction and Intracellular Drug Delivery

Advances in biomedical research have significantly enhanced our capability to identify drug targets for medical intervention. At same time, major technological breakthroughs such as combinatorial chemistry, computer-assisted drug design, robotic screening, and molecular cloning have made thousands of small compounds and macromolecules such as proteins and nucleic acids available for therapeutic consideration. Despite this progress, two major obstacles need to be overcome before we are able to move these products from basic research into clinical application. The first of these obstacles is the lack of specificity of drug compounds toward target cells. Although exposure of intended cellular targets to a drug results in a therapeutic effect, exposure of unintended nontarget cells often results in undesirable toxic effects. The other obstacle is the inability of most drugs, especially those that are hydrophilic and those of high molecular weight, to pass through the cell membrane and enter cells. The cell membrane imposes a significant physical barrier to the entrance of drug molecules into cells. The current strategy for overcoming the first obstacle is to develop drugs that are cell specific, or pro-drugs that remain inactive until they are inside the intended target cells. A common approach to resolve the second limitation is to employ receptor-mediated endocytosis, pinocytosis, or phagocytosis to enhance drug internalization. Despite being extensively studied and well-documented, these approaches have nevertheless had limited success and have met with serious obstacles, including the lack of proper receptors, insufficient endosomal release or avoidance of lysosomal degradation, and low delivery efficiency. At present, only a handful of delivery systems have the potential to overcome these obstacles and none are capable of high-efficiency delivery of hydrophilic molecules, especially macromolecules. Therefore, development of a highly effective carrier for intracellular drug delivery has been the goal of much research effort in both the pharmaceutical industry and academic institutions.