The Effects of Charge Separation in Quaternary Ammonium, DABCO-Containing Polymers on In Vitro Toxicity and Gene Deliver
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The Effects of Charge Separation in Quaternary Ammonium, DABCO-Containing Polymers on In Vitro Toxicity and Gene Delivery Theresa M. Reineke† and Mark E. Davis* Division of Chemistry and Chemical Engineering, California Institute of Technology Pasadena, CA 91107, U.S.A. † Current address: Department of Chemistry, University of Cincinnati, Cincinnati, OH 452210172, U.S.A. ABSTRACT Polycation materials have recently emerged as promising systems for the delivery of genetic material. In this study, several DABCO (1,4-diazabicyclo[2.2.2]octane) polymers are investigated for their ability to bind and deliver plasmid DNA (pDNA) into mammalian cells. The DABCO polymers are synthesized by copolymerization of DABCO with 1,3dibromopropane (D3), 1,4-dibromobutane (D4), 1,6-dibromohexane (D6), 1,8-dibromooctane (D8), and 1,10-dibromodecane (D10) to form a series of quaternary ammonium polymers with increasing charge separation. Gel retardation experiments reveal that each polymer (D3-D10) binds pDNA above a charge ratio of 1.0 (polymer + / pDNA -). The polycations are examined for in vitro transfection efficiency and toxicity in BHK-21 cells. Results of the transfection experiments indicate that the D6 polymer had the highest transfection efficiency. Although all of the polymers are shown to have some toxicity, the D8 and D10 polymers are more toxic to BHK21 cells; approximately 30% of the cells survive at a charge ratio of 5 +/- as compared to the D3, D4, and D6 polymers where survival rates are about 80%. INTRODUCTION Cationic polymers are currently being studied as alternatives to viral systems for the delivery of therapeutic genes. Advantages of using nonviral delivery vectors over their viral counterparts include low immunogenicity, noninfectivity, and virtually no limit to the size of the foreign gene that these vectors can carry [1-3]. Polycations have the ability to self-assemble with DNA and condense it into small particles that have been denoted as polyplexes. Polyplexes have been shown to deliver DNA into cultured cells through the endocytotic pathway. Several studies on polymeric delivery vectors have indicated that small changes in the structures of polymeric vectors play a significant yet undetermined role in the delivery efficiency and toxicity of these systems [4-7]. For example, previous experiments conducted by our group have indicated that the charge separation in β-cyclodextrin-containing polymers has a considerable effect on both the toxicity as well as the transfection efficiency of the delivery vectors in mammalian cell lines [6]. In the current study, several DABCO-containing polymers with increasing charge separations (Figure 1) have been created to further elucidate the effect of the charge separation within polycation delivery vectors. DABCO-based polymers have been formerly studied for other applications such as structure-directing agents in zeolite synthesis [8,9]. Here, their effectiveness as gene delivery agents is considered.
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