Gene Transfer Using StarburstTM Dendrimers

Abstract

The new class of synthetic, highly branched, spherical Starburst™ polyamidoamine (PAMAM) dendrimer polymers is unique in structure. These molecules are uniform in size with a high density of charged primary amino groups restricted to the surface, as well as being highly soluble and stable in aqueous solution. The major structural differences in PAMAM dendrimers relate to the initiator molecule, either ammonia (NH 3 ) as trivalent or ethylenediamine (EDA) as a tetravalent core, that starts the stepwise polymerization process and determines the overall shape, density, and surface charge of the molecule. With each new layer, or generation, the molecular weight of the polymer more than doubles and the number of surface amino groups exactly doubles. Dendrimers range in size from 10 to 130 A, with each polymerization step adding approximately 10 Ä to the diameter of the molecule. At least 10 generations of both types of PAMAM dendrimers (NH 3 and EDA core) can be synthesized (Tomalia etai, 1990). Each final dendrimer preparation is purified using ultrafiltration and structurally characterized using a number of techniques including electrospray-ionization mass spectroscopy, I3 C and 'H nuclear magnetic resonance spectroscopy, size exclusion chromatography, capillary electrophoresis, high performance liquid chromatography (HPLC), and gel electrophoresis (Tomalia et al, 1990). Starburst™ PAMAM dendrimers are identified using a standard nomenclature; for example G10 EDA is the 10th generation of an EDA core dendrimer. PAMAM dendrimers are currently the only class of dendritic macromolecules that are reliably produced in large quantities and that can be precisely synthesized over a broad range of molecular weights. The defined size, structure, and large number of surface amino groups of PAMAM dendrimers have enabled polymers to be employed as a substrate for the attachment of antibodies, contrast agents, and radionuclides for applications in different areas of biology and medicine. Studies using antibody-dendrimer conjugates in vitro and in vivo in 029 242 SELF-ASSEMBLINC COMPLEXES FOR GENE DELIVERYta X r P^? T al a f malS haVC d0C r Cnted these con J'"gates to be nontoxic and able to target biological agents to specific cells (Wiener ctal., 1994; Barth etai, 1994) ammo™ etal 1996; B.elmska et al., 1996). Several models of gene delivery have been evaluated using dendrimers complex* with reporter plasmids, con LinTgenes of the ell XT 01 erem "^ Pr0m0terS ' and with -pect'to specific bioTog^ of the cell. The reporter genes included chloramphenicol acetyltransferase (CAT) ^galactosidase (/*gal) luciferase (luc), or green fluorescence protein GFP M^ of these vectors can also be used to determine the cellular localization of the expressed reporter gene product in vitro and in vivo (£gal, CAT, GFP) In vitro gene transferIn vitro gene transfer using synthetic dendritic polymers has recently experienced rapid growth The efforts to understand the structure of the UNASS hiTthn a 0 n i 0 ;v T aniSm ^t ranSfeCti0n -crucüü to ...

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