Gene delivery mediated by recombinant silk proteins containing cationic and cell binding motifs.
Silk proteins are biodegradable and biocompatible, and can also be tailored to contain additional features via genetic engineering, suggesting utility for gene delivery. In the present study, novel silk-based block copolymers were bioengineered both with poly(L-lysine) domains to interact with plasmid DNA (pDNA) and RGD, to enhance cell-binding and transfection efficiency. Ionic complexes of these silk-polylysine-RGD block copolymers with pDNA were prepared, characterized and utilized for gene delivery to HeLa cells and human embryonic kidney (HEK) cells. The material systems were characterized by agarose gel electrophoresis, zeta-potentialmeter, atomic force microscopy, and dynamic light scattering. Sizes and charges of the pDNA complexes were regulated by the polymer/nucleotide molar ratio. Samples with 30-lysine residues and 11 RGD sequences, prepared at the ratio of number of amines/phosphates from pDNA (N/P) of 2, had an average solution diameter of 186 nm and showed the highest transfection efficiency. The intracellular distribution of complexes of Cy5-labeled pDNA was investigated by confocal laser scanning microscopy. The Cy5-labeled pDNA was distributed near the cell membrane and around the nuclei, indicating that the pDNA was transferred near the nucleus. The results demonstrated the potential of bioengineered silk proteins with additional functional features as a new family of highly tailored gene delivery systems.
Numata
Keiji
K
Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, Massachusetts 02155, USA.
Hamasaki
Juliana
J
Subramanian
Balajikarthick
B
Kaplan
David L
DL
D020816:Amino Acid Motifs; D002412:Cations; D003001:Cloning, Molecular; D004247:DNA / Q000008:administration & dosage / Q000235:genetics; D004337:Drug Carriers / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D005349:Fibroins / Q000737:chemistry* / Q000235:genetics; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D016023:Integrins / Q000096:biosynthesis / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics; D010957:Plasmids; D011107:Polylysine / Q000737:chemistry; D011485:Protein Binding; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D014162:Transfection
D002412:Cations; D004337:Drug Carriers; D016023:Integrins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C076815:spidroin 2; D011107:Polylysine; C047981:arginyl-glycyl-aspartic acid; D004247:DNA; D005349:Fibroins
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