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.

Targeted elastin-like polypeptide fusion protein for near-infrared imaging of human and canine urothelial carcinoma.

Cystoscopic visualization of bladder cancer is an essential method for initial bladder cancer detection and diagnosis, transurethral resection, and monitoring for recurrence. We sought to develop a new intravesical imaging agent that is more specific and sensitive using a polypeptide based NIR (near-infrared) probe designed to detect cells bearing epidermal growth factor receptors (EGFR) that are overexpressed in 80% of urothelial carcinoma (UC) cases. The NIR imaging agent consisted of an elastin like polypeptide (ELP) fused with epidermal growth factor (EGF) and conjugated to Cy5.5 to give Cy5.5-N24-EGF as a NIR contrast agent. In addition to evaluation in human cells and tissues, the agent was tested in canine cell lines and tissue samples with naturally occurring invasive UC. Flow cytometry and confocal microscopy were used to test cell-associated fluorescence of the probe in T24 human UC cells, and in K9TCC-SH (high EGFR expression) and K9TCC-Original (low EGF expression) canine cell lines. The probe specifically engages these cells through EGFR within 15 min of incubation and reached saturation within a clinically relevant 1 h timeframe. Furthermore, ex vivo studies with resected canine and human bladder tissues showed minimal signal from normal adjacent tissue and significant NIR fluorescence labeling of tumor tissue, in good agreement with our in vitro findings. Differential expression of EGFR ex vivo was revealed by our probe and confirmed by anti-EGFR immunohistochemical staining. Taken together, our data suggests Cy5.5-ELP-EGF is a NIR probe with improved sensitivity and selectivity towards BC that shows excellent potential for clinical translation.

Nanomedicine-Assisted Combination Therapy of NSCLC: New Platinum-Based Anticancer Drug Synergizes the Therapeutic Efficacy of Ganetespib.

Purpose: K-RAS is the most common mutated oncogene associated with Non-Small-Cell Lung Cancer (NSCLC). So far, there are no promising chemotherapies for the direct inhibition of K-RAS, and considered to be undruggable. In this work, we have introduced a new platinum-based cyanoximate complex, Pt(MCO)2, as an anti-cancer drug to enhance the therapeutic efficacy of Hsp90 inhibitor drug, ganetespib for the combination therapy of NSCLC. Methods: We have synthesized polyacrylic acid (PAA)-coated magnetic nanoparticles (MNPs) and used as drug delivery system. These MNPs were decorated with folic acid in order to target folate receptor-expressing NSCLC. The individual and combination of drugs as well as an optical dye DiI were co-encapsulated successfully inside the PAA-coating of MNPs to evaluate synergistic treatment option for NSCLC. The magnetic resonance (MR) and optical imaging modalities assisted for the monitoring drug loading and NSCLC treatment. Results: To evaluate the therapeutic efficacy of these customized MNPs, various cell-based assays including cell viability, apoptosis and necrosis, cell migration, comet and ROS experiments were performed. Results showed minimal toxicity for functional MNPs with no therapeutic drug and more than 60% cell death within 48 h of treatment, when single drug was encapsulated. Importantly, more than 90% cells were dead when both drugs were delivered. Overall, the results indicated that the Pt(MCO)2 drug enhances the therapeutic efficacy of ganetespib by more than 30% toxicity towards the targeted treatment of NSCLC, while showed minimal toxicity to the normal healthy tissues. Conclusion: We successfully developed new dual-modal magnetic nanomedicines for the rapid and controlled release of combination of drugs for the effective treatment of NSCLC. The MR and fluorescence modalities help monitoring the delivery of drugs, where the new platinum-based drug Pt(MCO)2 synergizes the therapeutic efficacy of ganetespib.