Intelligent biosynthetic nanobiomaterials (IBNs) for hyperthermic gene delivery.
OBJECTIVE
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.
Chen
Tze-Haw Howard
TH
Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin 53705-2222, USA.
Bae
Younsoo
Y
Furgeson
Darin Y
DY
D001672:Biocompatible Materials; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004549:Elastin / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005240:Feasibility Studies; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008970:Molecular Weight; D049329:Nanostructures; D036103:Nanotechnology; D009690:Nucleic Acid Conformation; D010316:Particle Size; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D011107:Polylysine / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D044366:Transition Temperature
D001672:Biocompatible Materials; D010455:Peptides; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011107:Polylysine; D004247:DNA; D004549:Elastin