Kaushal Rege

Inorganic nanoparticles for cancer imaging and therapy.

Inorganic nanoparticles have received increased attention in the recent past as potential diagnostic and therapeutic systems in the field of oncology. Inorganic nanoparticles have demonstrated successes in imaging and treatment of tumors both ex vivo and in vivo, with some promise towards clinical trials. This review primarily discusses progress in applications of inorganic nanoparticles for cancer imaging and treatment, with an emphasis on in vivo studies. Advances in the use of semiconductor fluorescent quantum dots, carbon nanotubes, gold nanoparticles (spheres, shells, rods, cages), iron oxide magnetic nanoparticles and ceramic nanoparticles in tumor targeting, imaging, photothermal therapy and drug delivery applications are discussed. Limitations and toxicity issues associated with inorganic nanoparticles in living organisms are also discussed.

Simultaneous Enhancement of Photothermal Stability and Gene Delivery Efficacy of Gold Nanorods Using Polyelectrolytes

The propensity of nanoparticles to aggregate in aqueous media hinders their effective use in biomedical applications. Gold nanorods (GNRs) have been investigated as therapeutics, imaging agents, and diagnostics. We report that chemically generated gold nanorods rapidly aggregate in biologically relevant media. Depositing polyelectrolyte multilayers on gold nanorods enhanced the stability of these nanoparticles for at least up to 4 weeks. Dispersions of polyelectrolyte (PE)-gold nanorod assemblies (PE-GNRs) demonstrate a stable Arrhenius-like photothermal response, which was exploited for the hyperthermic ablation of prostate cancer cells in vitro. Subtoxic concentrations of PE-GNR assemblies were also employed for delivering exogenous plasmid DNA to prostate cancer cells. PE-GNRs based on a cationic polyelectrolyte recently synthesized in our laboratory demonstrated higher transfection efficacy and lower cytotoxicity compared to those based on polyethyleneimine, a current standard for polymer-mediated gene delivery. Our results indicate that judicious engineering of biocompatible polyelectrolytes leads to multifunctional gold nanorod-based assemblies that combine high stability and low cytotoxicity with photothermal ablation, gene delivery, and optical imaging capabilities on a single platform.

Spatiotemporal temperature distribution and cancer cell death in response to extracellular hyperthermia induced by gold nanorods.

Plasmonic nanoparticles have shown promise in hyperthermic cancer therapy, both in vitro and in vivo. Previous reports have described hyperthermic ablation using targeted and nontargeted nanoparticles internalized by cancer cells, but most reports do not describe a theoretical analysis for determining optimal parameters. The focus of the current research was first to evaluate the spatiotemporal temperature distribution and cell death induced by extracellular hyperthermia in which gold nanorods (GNRs) were maintained in the dispersion outside human prostate cancer cells. The nanorod dispersion was irradiated with near-infrared (NIR) laser, and the spatiotemporal distribution of temperature was determined experimentally. This information was employed to develop and validate theoretical models of spatiotemporal temperature profiles for gold nanorod dispersions undergoing laser irradiation and the impact of the resulting heat generation on the viability of human prostate cancer cells. A cell injury/death model was then coupled to the heat transfer model to predict spatial and temporal variations in cell death and injury. The model predictions agreed well with experimental measurements of both temperature and cell death profiles. Finally, the model was extended to examine the impact of selective binding of gold nanorods to cancer cells compared to nonmalignant cells, coupled with a small change in cell injury activation energy. The impact of these relatively minor changes results in a dramatic change in the overall cell death rate. Taken together, extracellular hyperthermia using gold nanorods is a promising strategy, and tailoring the cellular binding efficacy of nanorods can result in varying therapeutic efficacies using this approach.

Biogenic nanoporous silica-based sensor for enhanced electrochemical detection of cardiovascular biomarkers proteins

The goal of our research is to demonstrate the feasibility of employing biogenic nanoporous silica as a key component in developing a biosensor platform for rapid label-free electrochemical detection of cardiovascular biomarkers from pure and commercial human serum samples with high sensitivity and selectivity. The biosensor platform consists of a silicon chip with an array of gold electrodes forming multiple sensor sites and works on the principle of electrochemical impedance spectroscopy. Each sensor site is overlaid with a biogenic nanoporous silica membrane that forms a high density of nanowells on top of each electrode. When specific protein biomarkers: C-reactive protein (CRP) and myeloperoxidase (MPO) from a test sample bind to antibodies conjugated to the surface of the gold surface at the base of each nanowell, a perturbation of electrical double layer occurs resulting in a change in the impedance. The performance of the biogenic silica membrane biosensor was tested in comparison with nanoporous alumina membrane-based biosensor and plain metallic thin film biosensor. Significant enhancement in the sensitivity and selectivity was achieved with the biogenic silica biosensor, in comparison to the other two, for detecting the two protein biomarkers from both pure and commercial human serum samples. The sensitivity of the biogenic silica biosensor is approximately 1 pg/ml and the linear dose response is observed over a large dynamic range from 1 pg/ml to 1 microg/ml. Based on its performance metrics, the biogenic silica biosensor has excellent potential for development as a point of care handheld electronic biosensor device for detection of protein biomarkers from clinical samples.

A programmable microfluidic cell array for combinatorial drug screening

We describe the development of a fully automatic and programmable microfluidic cell culture array that integrates on-chip generation of drug concentrations and pair-wise combinations with parallel culture of cells for drug candidate screening applications. The device has 64 individually addressable cell culture chambers in which cells can be cultured and exposed either sequentially or simultaneously to 64 pair-wise concentration combinations of two drugs. For sequential exposure, a simple microfluidic diffusive mixer is used to generate different concentrations of drugs from two inputs. For generation of 64 pair-wise combinations from two drug inputs, a novel time dependent variable concentration scheme is used in conjunction with the simple diffusive mixer to generate the desired combinations without the need for complex multi-layer structures or continuous medium perfusion. The generation of drug combinations and exposure to specific cell culture chambers are controlled using a LabVIEW interface capable of automatically running a multi-day drug screening experiment. Our cell array does not require continuous perfusion for keeping cells exposed to concentration gradients, minimizing the amount of drug used per experiment, and cells cultured in the chamber are not exposed to significant shear stress continuously. The utility of this platform is demonstrated for inducing loss of viability of PC3 prostate cancer cells using combinations of either doxorubicin or mitoxantrone with TRAIL (TNF-alpha Related Apoptosis Inducing Ligand) either in a sequential or simultaneous format. Our results demonstrate that the device can capture the synergy between different sensitizer drugs and TRAIL and demonstrate the potential of the microfluidic cell array for screening and optimizing combinatorial drug treatments for cancer therapy.

The influence of mediators of intracellular trafficking on transgene expression efficacy of polymer–plasmid DNA complexes

Polymer-mediated gene delivery is an attractive alternative to viral vectors, but is limited by low efficacies of transgene expression. We report that polymers possess differential efficacies for transfecting closely related human prostate cancer cells, which correlates with dramatically different intracellular fate of nanoscale cargo in these cells. Sequestration of nanoscale cargo (27 nm quantum dots and 150-250 nm polyplexes) at a single location near the microtubule organizing compartment (MTOC) in PC3-PSMA human prostate cancer cells correlated with lower polymer-mediated transgene expression compared to PC3 cells, which showed distributed localization throughout the cytoplasm. We show, for the first time, that treatment with the histone deacetylase 6 (HDAC6) inhibitor tubacin, which acetylates tubulin of microtubules in the cytoplasm, abolished quantum dot and polyplex sequestration at the perinuclear recycling compartment/microtubule organizing center (PNRC/MTOC) and increased polymer-mediated transgene expression by up to forty-fold compared to cells not treated with the HDAC6 inhibitor drug. Treatment with the class I and II HDAC inhibitor trichostatin A (TSA) demonstrated similar levels of transgene expression enhancement. These results indicate that mediators of intracellular trafficking can be employed to modulate nanoparticle fate and enhance the efficacy of nanoscale therapeutics in cells. Simultaneous use of high-efficacy polymers along with mediators of intracellular trafficking is an attractive synergistic strategy for enhancing polymer-mediated transgene expression.

Parallel synthesis and screening of polymers for nonviral gene delivery

We describe the parallel synthesis and in vitro evaluation of a cationic polymer library for the discovery of nonviral gene delivery vectors. The library was synthesized based on the ring-opening polymerization reaction between epoxide groups of diglycidyl ethers and the amines of (poly)amines. Parallel screening of soluble library constituents led to the identification of lead polymers with high DNA-binding efficacies. Transfection efficacies of lead polymers were evaluated using PC3-PSMA human prostate cancer cells and murine osteoblasts in the absence and presence of serum. In vitro experiments resulted in the identification of a candidate polymer that demonstrated significantly higher transfection efficacies and lower cytotoxicities than poly(ethyleneimine) (pEI), the current standard for polymeric transfection agents. In addition, polymers that demonstrated moderately higher and comparable transfection efficacies with respect to pEI were also identified. Our results demonstrate that high-throughput synthesis and screening of polymers is a powerful approach for the identification of novel nonviral gene delivery agents.

Utilization of lysozyme charge ladders to examine the effects of protein surface charge distribution on binding affinity in ion exchange systems.

A lysozyme library was employed to study the effects of protein surface modification on protein retention and to elucidate preferred protein binding orientations for cation exchange chromatography. Acetic anhydride was used as an acetylating agent to modify protein surface lysine residues. Partial acetylation of lysozyme resulted in the formation of a homologous set of modified proteins with varying charge densities and distribution. The resulting protein charge ladder was separated on a cation exchange column, and eluent fractions were subsequently analyzed using capillary zone electrophoresis and direct infusion electrospray ionization mass spectrometry. The ion exchange separation showed a significant degree of variation in the retention time of the different variants. Several fractions contained coelution of variants, some with differing net charge. In addition, several cases were observed where variants with more positive surface charge eluted from the column prior to variants with less positive charge. Enzymatic digest followed by mass spectrometry was performed to determine the sites of acetylation on the surface of the variants eluting in various fractions. Electrostatic potential maps of these variants were then generated to provide further insight into the elution order of the variants.

Parallel screening of FDA-approved antineoplastic drugs for identifying sensitizers of TRAIL-induced apoptosis in cancer cells.

BackgroundTumor Necrosis Factor-α Related Apoptosis Inducing Ligand (TRAIL) and agonistic antibodies to death receptor 4 and 5 are promising candidates for cancer therapy due to their ability to induce apoptosis selectively in a variety of human cancer cells, while demonstrating little cytotoxicity in normal cells. Although TRAIL and agonistic antibodies to DR4 and DR5 are considered safe and promising candidates in cancer therapy, many malignant cells are resistant to DR-mediated, TRAIL-induced apoptosis. In the current work, we screened a small library of fifty-five FDA and foreign-approved anti-neoplastic drugs in order to identify candidates that sensitized resistant prostate and pancreatic cancer cells to TRAIL-induced apoptosis.MethodsFDA-approved drugs were screened for their ability to sensitize TRAIL resistant prostate cancer cells to TRAIL using an MTT assay for cell viability. Analysis of variance was used to identify drugs that exhibited synergy with TRAIL. Drugs demonstrating the highest synergy were selected as leads and tested in different prostate and pancreatic cancer cell lines, and one immortalized human pancreatic epithelial cell line. Sequential and simultaneous dosing modalities were investigated and the annexin V/propidium iodide assay, in concert with fluorescence microscopy, was employed to visualize cells undergoing apoptosis.ResultsFourteen drugs were identified as having synergy with TRAIL, including those whose TRAIL sensitization activities were previously unknown in either prostate or pancreatic cancer cells or both. Five leads were tested in additional cancer cell lines of which, doxorubicin, mitoxantrone, and mithramycin demonstrated synergy in all lines. In particular, mitoxantrone and mithramycin demonstrated significant synergy with TRAIL and led to reduction of cancer cell viability at concentrations lower than 1 μM. At these low concentrations, mitoxantrone demonstrated selectivity toward malignant cells over normal pancreatic epithelial cells.ConclusionsThe identification of a number of FDA-approved drugs as TRAIL sensitizers can expand chemotherapeutic options for combination treatments in prostate and pancreatic cancer diseases.

Discovery of Cationic Polymers for Non-Viral Gene Delivery Using Combinatorial Approaches

Gene therapy is an attractive treatment option for diseases of genetic origin, including several cancers and cardiovascular diseases. While viruses are effective vectors for delivering exogenous genes to cells, concerns related to insertional mutagenesis, immunogenicity, lack of tropism, decay and high production costs necessitate the discovery of non-viral methods. Significant efforts have been focused on cationic polymers as non-viral alternatives for gene delivery. Recent studies have employed combinatorial syntheses and parallel screening methods for enhancing the efficacy of gene delivery, biocompatibility of the delivery vehicle, and overcoming cellular level barriers as they relate to polymer-mediated transgene uptake, transport, transcription, and expression. This review summarizes and discusses recent advances in combinatorial syntheses and parallel screening of cationic polymer libraries for the discovery of efficient and safe gene delivery systems.