ABSTRACT
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.
Subject(s)
Biocompatible Materials , DNA/chemistry , Gene Transfer Techniques , Nanostructures , Nanotechnology , Peptides/chemistry , Transition Temperature , Cell Line, Tumor , Cell Survival/drug effects , DNA/metabolism , Elastin/chemistry , Electrophoresis, Polyacrylamide Gel , Feasibility Studies , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Kinetics , Molecular Weight , Nucleic Acid Conformation , Particle Size , Peptides/genetics , Peptides/metabolism , Peptides/toxicity , Polylysine/chemistry , Protein Binding , Recombinant Proteins/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
An intelligent biosynthetic nanobiomaterial (IBN) platform was explored for drug delivery applications for hyperthermic combination chemotherapy and thermal drug targeting. Geldanamycin (GA), a heat shock protein 90 inhibitor, was conjugated to novel thermosensitive poly(K)(8)-poly(VPGXG)(60) block copolymers [K(8)-ELP(1-60)] with guest residues as valine, alanine and glycine in a 5:2:3 ratio at the 'X' position. The conjugates were completely soluble in PBS and showed a characteristic thermosensitive inverse phase transition. [K(8)-ELP(1-60)]-GA conjugate nanoparticles showed a size ranging from 50 to 200 nm depending upon temperature. Relevant to systemic drug delivery in vivo, these IBNs stably disperse in aqueous solution. Cytotoxicity assays have shown that the IBN from [K(8)-ELP(1-60)]-GA conjugates exhibits effective hyperthermic combination chemotherapy with facile heat modulation.
