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Efficient Transfer of Genetic Material Into Mammalian Cells Using Starburst Polyamidoamine Dendrimers (Transfection/Polymers/Cationic Lipids/Cdna) JOLANTA F

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Efficient Transfer of Genetic Material Into Mammalian Cells Using Starburst Polyamidoamine Dendrimers (Transfection/Polymers/Cationic Lipids/Cdna) JOLANTA F Proc. Natl. Acad. Sci. USA Vol. 93, pp. 4897-4902, May 1996 Genetics Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers (transfection/polymers/cationic lipids/cDNA) JOLANTA F. KUKOWSKA-LATALLO*, ANNA U. BIELINSKA*, JENNIFER JOHNSON*, RALPH SPINDLERt, DONALD A. TOMALIAt, AND JAMES R. BAKER, JR.*t *Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109-0666; and tMichigan Molecular Institute, Midland, MI 48604 Communicated by J. L. Oncley, The University of Michigan, Ann Arbor, MI, December 21, 1995 (received for review September 7, 1995) ABSTRACT Starburst polyamidoamine dendrimers are a primary amino groups (13, 14, 15). Compared with many other new class of synthetic polymers with unique structural and types of dendritic macromolecules that have recently been physical characteristics. These polymers were investigated for synthesized, PAMAM dendrimers are the only class of mac- the ability to bind DNA and enhance DNA transfer and romolecules that are unidispersed and show high charge expression in a variety of mammalian cell lines. Twenty densities restricted to the surface of the molecule (13, 16, 17). different types of polyamidoamine dendrimers were synthe- Dendrimers have been reliably produced in large quantities sized, and the polymer structure was confirmed using well- and can be precisely synthesized over a range of molecular defined analytical techniques. The efficiency of plasmid DNA weights similar to that of proteins. The major structural transfection using dendrimers was examined using two re- differences in PAMAM dendrimers relate to the core mole- porter gene systems: firefly luciferase and bacterial 1B-galac- cule, either ammonia (NH3) as trivalent initiator core or tosidase. The transfections were performed using various ethylenediamine (EDA) as a tetravalent initiator core (14), dendrimers, and levels of expression of the reporter protein that starts the stepwise polymerization process and dictates were determined. Highly efficient transfection of a broad several structural characteristics of the molecule, including the range of eukaryotic cells and cell lines was achieved with overall shape, density, and surface charge. With each new layer minimal cytotoxicity using the DNA/dendrimer complexes. or generation, the molecular weight of the dendrimer more However, the ability to transfect cells was restricted to certain than doubles, and the number of surface amine groups exactly types of dendrimers and in some situations required the doubles. The defined structure and large number of surface presence of, additional compounds, such as DEAE-dextran, amino groups of PAMAM dendrimers have led to these that appeared to alter the nature of the complex. A few cell polymers being employed as a substrate for the attachment of lines demonstrated enhanced transfection with the addition of antibodies, contrast agents, and radiopharmaceuticals for ap- chloroquine, indicating endosomal localization of the com- plications in a number of different areas of biology and plexes. The capability of a dendrimer to transfect cells ap- medicine (15, 18, 19). Studies using. antibody/dendrimer con- peared to depend on the size, shape, and number of primary jugates in vitro and in vivo in experimental animals have amino groups on the surface of the polymer. However, the documented these conjugates to be nontoxic and able to target specific dendrimer most efficient in achieving transfection biologic agents to specific cells (15, 18, 19). varied between different types of cells. These studies demon- Given that PAMAM dendrimers contain defined numbers strate that Starburst dendrimers can transfect a wide variety of amino groups on the surface of the polymer that are ofcell types in vitro and offer an efficient method for producing positively charged at physiologic pH, it was hypothesized that permanently transfected cell lines. these molecules could interact with biologically relevant poly- anions, including nucleic acids. It was further postulated that The introduction of genetic material into eukaryotic cells has DNA/dendrimer complexes might be able to transfect cells in been a major technique to investigate gene function and the a manner similar to DNA/polylysine complexes (11), but with regulation of gene expression (1, 2). In addition, recent better efficiency, given the high solubility and defined archi- advances in detecting inherited or acquired genetic disorders tecture of the dendrimer. This report details that highly have provided the possibility of transferring recombinant efficient transfection can be achieved with DNA/dendrimer genes into somatic cells to correct missing or defective gene complexes formed with certain types of dendrimers. products (3). A variety of methods have been developed to accomplish gene transfer into eukaryotic cells. These tech- niques involve the direct physical -introduction of genetic MATERIALS AND METHODS material into cells (4), the disruption of cell membranes to Dendrimer Synthesis. Ten generations of two different allow the transfer of DNA (5, 6), the use of genetically types of PAMAM dendrimers (NH3 and EDA core) were modified viruses to deliver genetic material (7, 8), and the synthesized in a stepwise process as described in detail (14). formation of DNA complexes with either inorganic salts, Each final dendrimer preparation was purified using ultrafil- polycations, or lipids to transfer the DNA across cell mem- tration and structurally characterized using a number of tech- branes (9, 10, 11). There is great utility for these techniques, niques, including electrospray ionization mass spectroscopy, but there are limitations in target cell type and in the ability to 13C and IH nuclear magnetic resonance spectroscopy, size transfer different types of genetic material (10, 11, 12). exclusion chromatography, capillary electrophoresis, HPLC, Starburst polyamidoamine (PAMAM) dendrimers are a and gel electrophoresis (14, 20, 21). Starburst PAMAM den- new class of highly branched spherical polymers that are highly drimers are identified using a standard nomenclature; for soluble in aqueous solution and have a unique surface of example, G10 (EDA) is the 10th-generation EDA core den- drimer. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviations: PAMAM, polyamidoamine; EDA, ethylenediamine. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 4897 Downloaded by guest on October 3, 2021 4898 Genetics: Kukowska-Latallo et al. Proc. Natl. Acad. Sci. USA 93 (1996) Preparation of DNA/Dendrimer Complexes. Plasmid DNA Transfections were also performed with different enhancing was amplified in bacteria and then isolated by two cycles of agents by adding the DNA/dendrimer complexes to medium cesium chloride gradient centrifugation (22). Dendrimers were that contained the additional compound(s). Compounds used diluted to an appropriate concentration in buffer containing 20 in this manner included: 0.25-1.0 ,uM DEAE-dextran chloride mM Hepes (pH 7.9), 100 mM KCl, 0.2 mM EDTA, 0.5 mM (average Mr, 500,000; Sigma), 25-100 ,uM chloroquine diphos- DTT, and 20% glycerol, with all solutions stored at 4°C. phate (Sigma), and 2-10% glycerol (Sigma). When stable DNA/dendrimer complexes were formed by incubating the clones were generated, transfections were performed with G7 two components together in a buffer containing 100 mM NaCl, (NH3) dendrimer (without dextran) and pRSV,3gal-Neo 10 mM Tris (pH 7.5), 5 mM DTT, 4% glycerol, and 1 mM and/or pRSVLuc-Neo plasmid DNA. Clones were selected by EDTA for a minimum of 5 min at room temperature. Ratios incubation in neomycin (G418, Geneticin; Life Technologies) of nucleic acid to dendrimer were based on the electrostatic at 300 ,ug/ml for 3 weeks. Control transfections were per- charge present on each component: the number of phosphate formed with three cationic lipid preparations [TFX 50 (Pro- groups in the nucleic acid compared with the number of mega), Lipofectamine, and Lipofectin (Life Technologies)] terminal NH2 groups on a dendrimer. For example, given that according to the vendor's protocols. the number of bases in 1.0 jig of DNA is 1.71 x 1015, 1.71 Analysis of the Cellular Uptake of DNA/Dendrimer Com- x 1015 negative charges are present per 1.0 jig of DNA, plexes. Linearized and dephosphorylated DNA from a 2.9-kb whereas a G7 (NH3) dendrimer has -2.65 x 1015 charges per plasmid was radiolabeled with [_y-32P]ATP using T4 polynu- ,tg. Therefore, to obtain a 1:1 charge ratio, 1.0 ,ug of DNA was cleotide kinase. After purification, 2.0 ng of radiolabeled DNA mixed with 0.65 ,tg of dendrimer. DNA/dendrimer complexes (5.2 x 107 cpm/,g) mixed with an appropriate amount of were analyzed by electrophoretic mobility-shift assays in 1% nonlabeled plasmid DNA was complexed with G7 (EDA) agarose gel in the standard Tris-borate EDTA (TBE) buffer dendrimer to achieve a 1:10 DNA-to-dendrimer charge ratio. (pH 8.3), where DNA was identified by staining with 0.1 ,ug of Rat2 cells were plated at 2.0 x 105 cells per well in six-well ethidium bromide per ml. plates and washed with serum-free medium. DNA/dendrimer Cells and Media. The cell lines used in these studies are complexes were then added and incubated with the cells for 6 listed in Table 1. All cells were maintained in standard culture h, and sampled at specific time points.
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