PURPOSE: Intelligent biosynthetic nanobiomaterials (IBNs) were constructed as recombinant diblock copolymers, notated as K8-ELP(1-60), containing a cationic oligolysine (VGK8G) and a thermosensitive elastin-like polypeptide (ELP) block with 60 repetitive pentapeptide units [(VPGXG)60; X is Val, Ala and Gly in a 5:2:3 ratio]. METHODS: K8-ELP(1-60) was synthesized by recursive directional ligation for DNA oligomerization. Purity and molecular weight of K8-ELP(1-60) were confirmed by SDS-PAGE and mass spectrometry. DNA polyplexes were prepared from K8-ELP(1-60) and pGL3-Control (pGL3-C) plasmid DNA (pDNA) and stability was evaluated by gel retardation, DLS, and DNA displacement with heparin. Thermal transition profiles were studied by measuring the turbidity change at 350 nm and the polyplexes were used to transfect MCF-7 cells with a concomitant cytotoxicity assay. RESULTS: SDS-PAGE and MALDI-TOF studies showed highly pure copolymers at the desired molecular weight. K8-ELP(1-60) condensed pDNA at a cation to anion (N/P) ratio above 0.25 with a tight distribution of particle size ranging from 115.5-32.4 nm with increasing N/P ratio. Thermal transition temperatures of K8-ELP(1-60)/pDNA and K8-ELP(1-60) alone were 44.9 and 71.5 degrees C, respectively. K8-ELP(1-60)/pDNA complexes successfully transduced MCF-7 cells with qualitative expression of enhanced green fluorescent protein (EGFP) and minimal cytotoxicity compared to branched poly(ethyleneimine) controls. CONCLUSIONS: K8-ELP(1-60) was successfully designed and purified through recombinant means with efficient and stable condensation of pDNA at N/P ratios>0.25 and polyplex particle size<115 nm. MCF-7 cells successfully expressed EGFP with minimal cytotoxicity compared to positive controls; moreover, polyplexes retained sharp, thermotransitive kinetics within a narrow Tt range at clinically relevant hyperthermic temperatures, where the decrease of Tt was due to the increased hydrophobicity upon charge neutralization.
PURPOSE: Intelligent biosynthetic nanobiomaterials (IBNs) were constructed as recombinant diblock copolymers, notated as K8-ELP(1-60), containing a cationic oligolysine (VGK8G) and a thermosensitive elastin-like polypeptide (ELP) block with 60 repetitive pentapeptide units [(VPGXG)60; X is Val, Ala and Gly in a 5:2:3 ratio]. METHODS: K8-ELP(1-60) was synthesized by recursive directional ligation for DNA oligomerization. Purity and molecular weight of K8-ELP(1-60) were confirmed by SDS-PAGE and mass spectrometry. DNA polyplexes were prepared from K8-ELP(1-60) and pGL3-Control (pGL3-C) plasmid DNA (pDNA) and stability was evaluated by gel retardation, DLS, and DNA displacement with heparin. Thermal transition profiles were studied by measuring the turbidity change at 350 nm and the polyplexes were used to transfect MCF-7 cells with a concomitant cytotoxicity assay. RESULTS:SDS-PAGE and MALDI-TOF studies showed highly pure copolymers at the desired molecular weight. K8-ELP(1-60) condensed pDNA at a cation to anion (N/P) ratio above 0.25 with a tight distribution of particle size ranging from 115.5-32.4 nm with increasing N/P ratio. Thermal transition temperatures of K8-ELP(1-60)/pDNA and K8-ELP(1-60) alone were 44.9 and 71.5 degrees C, respectively. K8-ELP(1-60)/pDNA complexes successfully transduced MCF-7 cells with qualitative expression of enhanced green fluorescent protein (EGFP) and minimal cytotoxicity compared to branched poly(ethyleneimine) controls. CONCLUSIONS: K8-ELP(1-60) was successfully designed and purified through recombinant means with efficient and stable condensation of pDNA at N/P ratios>0.25 and polyplex particle size<115 nm. MCF-7 cells successfully expressed EGFP with minimal cytotoxicity compared to positive controls; moreover, polyplexes retained sharp, thermotransitive kinetics within a narrow Tt range at clinically relevant hyperthermic temperatures, where the decrease of Tt was due to the increased hydrophobicity upon charge neutralization.
Authors: Greta Gronau; Sreevidhya T Krishnaji; Michelle E Kinahan; Tristan Giesa; Joyce Y Wong; David L Kaplan; Markus J Buehler Journal: Biomaterials Date: 2012-08-28 Impact factor: 12.479
Authors: Roelof Schipperus; Rosalie L M Teeuwen; Marc W T Werten; Gerrit Eggink; Frits A de Wolf Journal: Appl Microbiol Biotechnol Date: 2009-06-30 Impact factor: 4.813