Development of an Elastin-like Polypeptide-Based Nucleic Acid Delivery System Targeted to EGFR+ Bladder Cancer Cells Using a Layer-by-Layer Approach.

Nucleic acid (NA)-based therapies are revolutionizing biomedical research through their ability to control cellular functions at the genetic level. This work demonstrates a versatile elastin-like polypeptide (ELP) carrier system using a layer-by-layer (LbL) formulation approach that delivers NA cargos ranging in size from siRNA to plasmids. The components of the system can be reconfigured to modulate the biochemical and biophysical characteristics of the carrier for engaging the unique features of the biological target. We show the physical characterization and biological performance of LbL ELP nucleic acid nanoparticles (LENNs) in murine and human bladder tumor cell lines. Targeting bladder tumors is difficult owing to the constant influx of urine into the bladder, leading to low contact times (typically <2 h) for therapeutic agents delivered via intravesical instillation. LENN complexes bind to bladder tumor cells within 30 min and become rapidly internalized to release their NA cargo within 60 min. Our data show that a readily adaptable NA-delivery system has been created that is flexible in its targeting ability, cargo size, and disassembly kinetics. This approach provides an alternative path to either lipid nanoparticle formulations that suffer from inefficiency and physicochemical instability or viral vectors that are plagued by manufacturing and immune rejection challenges. This agile ELP-based nanocarrier provides an alternative route for nucleic acid delivery using a biomanufacturable, biodegradable, biocompatible, and highly tunable vehicle capable of targeting cells via engagement with overexpressed cell surface receptors.

Unravelling the Mechanism of Elastin-like Polypeptide-Enzyme Fusion Stabilization in Organic Solvents.

Elastin-like polypeptides (ELP) are a class of materials that are widely used as purification tags and in potential therapeutic applications. We have used the hydrophobic nature of ELP to extract them into organic solvents and precipitate them to obtain highly pure materials. Although many different types of ELP have been rapidly purified in this manner, the underlying mechanism for this process and its ability to retain functional proteins within organic phase-rich media has been unclear. A cleavable ELP-Intein construct fused with the enzyme chorismate mutase (ELP-I-Cm2) was used to better understand the organic solvent extraction process for ELP and the factors impacting the retention of enzyme activity. Our extraction studies indicated that a cell lysis step was essential to stabilize the ELP-I-Cm2 in the organic phase, prevent intein cleavage, and extract the fusion protein with high efficiency and retained activity. Circular dichroism and infrared spectroscopic characterization of ELP-I-Cm2 in organic solvents and aqueous solutions of the extracted and precipitated material indicated that the ELP secondary structure was retained in both environments. Atomic force microscopy and negative stain transmission electron microscopy imaging of ELP-I-Cm2 in organic solvents revealed highly regular circular features that were ∼50 nm in diameter, in contrast to larger (>100 nm) irregular features found in aqueous solutions. Since reverse micelles have often been used in catalytic processes, we evaluated the enzymatic activity of the ELP-I-Cm2 reversed micelles in different organic solvent mixtures and found that Cm2-mediated reactions in organic media were of comparable rate and efficiency to those in aqueous media. Based on these findings, we report an exciting new opportunity for ELP-enzyme fusion applications by exploiting their ability to form catalytically active reverse micelles in organic media.