Year 1: Nanoassemblies for Gene Silencing

Project: Research project

Description

All Applicants are required to submit an abstract of the proposed activity suitable for publication. The abstract should be informative to other persons working in the same or related fields and, insofar as possible, understandable to a scientifically or technically literate lay reader. The abstract shall not include any proprietary or sensitive business information as it may be made available to the public. The abstract shall not exceed 200 words in length. Summary. Hyperthermia (elevated temperatures) is a novel therapeutic approach for cancer diseases, particularly in cases where localized administration is possible. However, cancer cells overexpress heat shock proteins (HSPs) that enable survival at elevated temperatures. We propose a nanoassembly platform that can simultaneously deliver shRNA-expressing plasmids for silencing (knocking down) HSPs, as well as near infrared (NIR) light-triggered hyperthermia (photothermal effect) to cancer cells. This 1-2 punch is anticipated to enhance the ablation of malignant cells due to synergy between the two treatments; silencing of HSPs sensitizes cancer cells to nanoparticle-induced hyperthermia. Gold nanorods (GNRs) are coated with polymers (polyaminoethers or PAEs), unique to the PIs laboratory, resulting in the formation of PAE-GNR nanoassemblies. Plasmid shRNA and siRNA targeting HSPs (e.g. 90, 72 or 27) will be loaded on PAE-GNR nanoassemblies and delivered to cancer cells. HSP knockdown kinetics will be studied in order to appropriately time the second (hyperthermia) hit using NIR lasers. The proposed research is anticipated to result in novel combination treatments for cancer cell ablation, non-viral methods for gene silencing, and fundamental advancements in nanoscale and molecular therapeutics. Existing and proposed outreach activities from the Rege lab will enhance STEM engagement of high- and middle-school students in Arizona. Introduction and Background. The discovery of RNA interference (RNAi) and gene silencing phenomena by double stranded RNA has opened new vistas in therapeutics / medicine, biotechnology / synthetic biology, and cell biology. Short interfering RNA or siRNA are exogenous double-stranded strands 21-22 nucleotides in length that are delivered to cells using viral or non-viral methods. Short hairpin RNA or shRNA can also be used for silencing, and can expressed by delivering a plasmid DNA (pDNA) that expresses shRNA (shRNA plasmids). While siRNA based silencing is more widely investigated, particularly with non-viral delivery vehicles, shRNA plasmid delivery, although under-investigated, has several advantages for gene silencing, and will be a key focus of the proposed research. Cationic polymers and lipids are widely investigated for nucleic acid delivery, including transformative advancements on polyaminoethers or PAEs from the PIs laboratory. However, these delivery vehicles are not multifunctional and cannot be directly used for combination therapeutics. We therefore propose the use of PAE-GNR nanoassemblies with an eye towards facilitating nucleic acid (shRNA plasmid as well as siRNA) delivery, coupled with nanoparticle-induced hyperthermia for synergistic ablation of cancer cells. Furthermore, the optical properties of gold nanorods can be used for 2-photon and X-Ray CT imaging studies in future applications. Proposed Research in the One-Year Performance Period. In the one-year performance period of this proposal, we will focus on two goals (a) discovery of nanoassemblies with high efficacies for gene silencing, and (b) nanoassembly mediated synergistic ablation of cancer cells using NIR-induced hyperthermia and HSP knockdown. (a) Discovery of high-efficacy nanoassemblies (Months 0-5). The first part of the proposed research will focus on the discovery of nanoassemblies that result in>80% gene silencing in case of both, luciferase (reporter protein), as well as HSPs 90, 72, and 27 using shRNA plasmids and siRNA against the respective targets. Twenty-five different PAEs synthesized in the PIs laboratory will be coated on GNRs using methods previously established by us. These PAE-GNR nanoassemblies will be loaded with shRNA plasmids as well as siRNA against the four protein targets. Luciferase knockdown will be investigated in constitutively expressing 22Rv1-luc and PC3-luc prostate cancer cells. The same cells will be investigated for knockdown of the HSP targets above; in addition, PC3, PC3-PSMA, 22Rv1, and LNCaP cells will also be employed. Luciferase knockdown will be investigated using luminescence assays, while HSP knockdown will be verified using Western blots. Efficacy of silencing will be correlated to nanoassembly size, zeta potential, PAE-chemistry, shRNA plasmid or siRNA binding efficacy, and cellular uptake (details not provided for brevity). The toxicity of these nanoassemblies will be determined using the MTT metabolic assay, and silencing efficacies will be normalized relative to cell viability following PAE-GNR treatment. We will identify those shRNA plasmid and siRNA-loaded nanoassemblies for inducing>80% silencing of luciferase and HSPs 90, 72, and 27. These effective nanoassemblies will be employed in subsequent investigations. (b) Nanoassembly-mediated synergistic ablation of cancer cells (Months 4-12). We have previously demonstrated the colloidal stability as well as photothermal efficacy of polymer-coated GNRs for ablation in several peer-reviewed publications. We will characterize the ability of PAE-GNRs for inducing moderately hyperthermic temperatures (i.e. 45oC) APPLICATION PACKET ATTACHMENT A RFGA NO. ADHS14-00003606 following irradiation with NIR lasers. We will investigate the temperature response as a function of time, laser power, and type (i.e. continuous vs. pulsed; Ultrafast laser facility, ASU). Those conditions that result in moderately hyperthermic temperatures at lower GNR concentrations will be identified. shRNA plasmid and siRNA release from nanoassemblies will be investigated as a function of the parameters described above. Finally, GNR-induced hyperthermia and HSP knockdown will be investigated for synergistic ablation of cancer cells using cell viability and apoptosis assays. Efficacy of the combination treatment will be compared to the individual treatments.
StatusFinished
Effective start/end date10/23/1410/22/16

Funding

  • Arizona Biomedical Research Commission: $100,000.00

Fingerprint

Gene Silencing
Small Interfering RNA
Nanotubes
Gold
Plasmids
Heat-Shock Proteins
Induced Hyperthermia
Neoplasms
Luciferases
HSP90 Heat-Shock Proteins
Lasers
Temperature
HSP72 Heat-Shock Proteins
HSP27 Heat-Shock Proteins
Polymers
Fever
Therapeutics
RNA Interference
Research
Nanoparticles