title: Molecular engineering of silk-elastinlike polymers for matrix-mediated gene delivery: biosynthesis and characterization.
abstract: The unique advantage of genetic engineering techniques for the design and development of polymers for controlled gene delivery lies in exquisite control over polymer structure. In this article we report the biosynthesis and characterization of a series of new silk-elastinlike protein polymers (SELPs), namely, SELP415K, with larger elastin blocks per monomer unit than SELP47K previously studied for matrix-mediated gene delivery. A new cloning strategy was used, where a block of eight elastin units (8E) was integrated into the existing DNA sequence of SELP47K monomer genes using appropriate restriction endonuclease recognition sites. Following random multimerization, multimer gene segments of desired size were selected, expressed, and purified on Ni-agarose columns. The molecular weight and sequence composition of the purified SELPs were determined by MALDI-TOF and amino acid analysis, respectively. The influence of structural changes on the rheological properties of the polymers was investigated. In addition, hydrogel disks were prepared from 47K and 415K-8mer polymer solutions, and the effects of cure time and environmental conditions on the hydrogel equilibrium swelling ratio as a function of polymer composition were studied. DNA sequencing and agarose gel electrophoresis confirmed the successful cloning of the monomer gene segment of SELP415K consisting of 312 bp. Random concatemerization of SELP415K monomer gene segments resulted in a library of SELP415K multimer sequences of 6, 8, and 10 repeats respectively, each yielding a polymer with exact molecular weight and sequence. Rheometric measurements showed that both complex shear modulus (G*) and gelation point were influenced by polymer composition. Equilibrium swelling studies on hydrogel disks prepared from 47K and 415K-8mer polymer solutions showed that changes in polymer composition resulted in different gelation patterns and increased sensitivity toward changes in temperature and ionic strength but not pH. Together these results demonstrate the potential of recombinant techniques in engineering polymers with defined structures which allows the study of the structural parameters affecting matrix-mediated delivery of genes and bioactive agents.
authors.affiliation: Department of Pharmaceutical Sciences, Greenebaum Cancer Center, University of Maryland, 20 North Penn Street, Baltimore, MD 21201, USA.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Haider
authors.forename: Vivian
authors.initials: V
authors.lastname: Leung
authors.forename: Franco
authors.initials: F
authors.lastname: Ferrari
authors.forename: John
authors.initials: J
authors.lastname: Crissman
authors.forename: James
authors.initials: J
authors.lastname: Powell
authors.forename: Joseph
authors.initials: J
authors.lastname: Cappello
authors.forename: Hamidreza
authors.initials: H
authors.lastname: Ghandehari
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001672:Biocompatible Materials / Q000737:chemistry*; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004549:Elastin / Q000737:chemistry* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry; D020100:Hydrogels / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008969:Molecular Sequence Data; D009532:Nickel / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry; D011108:Polymers / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012685:Sepharose / Q000737:chemistry; D017422:Sequence Analysis, DNA; D047011:Silk / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D013997:Time Factors
chemical_list: D001672:Biocompatible Materials; D020100:Hydrogels; D007477:Ions; D009841:Oligonucleotides; D011108:Polymers; D011994:Recombinant Proteins; D047011:Silk; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D009532:Nickel; D004247:DNA; D004549:Elastin; D012685:Sepharose