Cellular response to nanoscale elastin-like polypeptide polyelectrolyte multilayers.

Ionic elastin-like polypeptide (ELP) conjugates are a new class of biocompatible, self-assembling biomaterials. ELPs composed of the repeat unit (GVGVP)(n) are derived from the primary sequence of mammalian elastin and produced in Escherichia coli. These biopolymers exhibit an inverse transition temperature that renders them extremely useful for applications in cell-sheet engineering. Cationic and anionic conjugates were synthesized by the chemical coupling of ELP to polyethyleneimine (PEI) and polyacrylic acid (PAA). The self-assembly of ELP-PEI and ELP-PAA using the layer-by-layer deposition of alternately charged polyelectrolytes is a simple, versatile technique to generate bioactive and biomimetic surfaces with the ability to modulate cell-substratum interactions. Our studies are focused on cellular response to self-assembled multilayers of ionic (GVGVP)(40) incorporated within the polymeric sequence H(2)N-MVSACRGPG-(GVGVP)(40)-WP-COOH. Angle-dependent XPS studies indicated a difference in the chemical composition at the surface ( approximately 10A below the surface) and subsurface regions. These studies provided additional insight into the growth of the nanoscale multilayer assembly as well as the chemical environment that the cells can sense. Overall, cellular response was enhanced on glass substrata coated with ELP conjugates compared with uncoated surfaces. We report significant differences in cell proliferation, focal adhesions and cytoskeletal organization as a function of the number of bilayers in each assembly. These multilayer assemblies have the potential to be successfully utilized in the rational design of coatings on biomaterials to elicit a desired cellular response.

Recombinant polymer-protein fusion: a promising approach towards efficient and targeted gene delivery.

BACKGROUND: Synthetic vectors such as polymers have the potential to reduce the safety problems associated with viral vectors; however, their low transfection efficiency limits their clinical utility. To study the critical steps involved in an efficient transgene expression, there is a need for creative approaches that allow a systematic correlation between gene carrier structure and properties necessary for successful gene transfer. Using recombinant techniques a prototype vector comprised of tandem repeating units fused to a targeting moiety was biosynthesized to mediate gene transfer in mammalian cell lines. The carrier was designed to have the structure of (KHKHKHKHKK)6-FGF2 where lysine (K) residues would allow complexation with plasmid DNA, basic fibroblast growth factor (FGF2) to target cells over-expressing FGF2 receptors (FGFR), and histidine (H) residues to facilitate escape from the endosomal compartments. METHODS: The gene carrier was biosynthesized in E. coli, purified using a Ni-NTA column, characterized, complexed with pDNA, and the complexes were used to transfect NIH 3T3, T-47D and COS-1 mammalian cell types known to express FGFR. RESULTS: Results demonstrate the successful cloning and expression of the gene carrier with over 95% purity. The molecular weight of the gene carrier was determined by MALDI-TOF to be 27 402. Amino acid content analysis and Western blot confirmed the expression of the gene carrier in E. coli. The vector was able to condense pDNA, induce cell proliferation in NIH 3T3 fibroblasts, and mediate transgene expression in NIH 3T3, T-47D and COS-1 mammalian cell types. CONCLUSION: Genetic engineering techniques show promise for systematic investigation of structure-activity relationships of non-viral gene delivery vectors.