Applications of siRNA for Cancer Gene Therapy

Gene therapy is a potent and versatile nano-medicine strategy in the treatment of cancer. Of the many tools currently used in this application, short-interfering RNA (siRNA) are among the most commonly employed due to their ability to silence oncogenic mRNA with high precision through the RNA interference (RNAi) pathway potentially leading to cancer cell death. Our work revolves around silencing the Glucose-Regulated Proteins (GRPs) whose expressions are upregulated in cancerous tissues and are implicated in the proliferative, pro-survival, and anti-apoptotic pathways that govern tumor biology. Here we present a variety of applications to improve the potency and functionality of GRP-targeting siRNAs while investigating the role of these oncoproteins in cancer cell cytotoxicity and adhesion. We’ve generated branched RNA templates that have the ability to self-assemble into higher order siRNA nanostructures and are capable of delivering multiple siRNAs that can synergistically silence the GRPs in endometrial, breast, and cervical cancer. From the branched RNA templates, second generation bioconjugates were developed to include fluorescently labeled and fatty acid conjugated siRNAs in an effort to expand their functionality into theranostic and self-delivery applications. Using our silencing strategy, we’ve also revealed a novel role of the master GRP regulator, GRP78, in modulating the expression of N-cadherin, a mesenchymal marker pivotal in the progression of metastatic tumor cell adhesion to the bone microenvironment. Silencing of GRP78 ultimately led to a decrease in prostate cancer adhesion in a model bone co-culture system. Moreover, our latest work highlights a method for developing cancer-targeting and cell penetrating peptides for the targeted delivery of siRNA to prostate cancer cells. Together, these studies have introduced new technologies that can further enhance the potency and applicability of siRNAs in cancer gene therapy

A Biologic-Device Combination Product Delivering Tumor-Derived Antigens Elicits Immunogenic Cell Death-Associated Immune Responses Against Glioblastoma

Background IGV-001 is a personalized, autologous cancer cell-based immunotherapy conceived to deliver a tumor-derived antigenic payload in the context of immunostimulatory signals to patients with glioblastoma (GBM). IGV-001 consists of patient-derived GBM cells treated with an antisense oligodeoxynucleotide against insulin-like growth factor 1 receptor (IGF1R) and placed in proprietary biodiffusion chambers (BDCs). The BDCs are then exposed to 5–6 Gy radiation and implanted at abdominal sites for ~48 hours. IGV-001 has previously been shown to be generally safe with promising clinical activity in newly diagnosed GBM patients. Methods Mouse (m) or human (h) variants of IGV-001 were prepared using GL261 mouse GBM cells or human GBM cells, respectively. BDCs containing vehicle or mIGV-001 were implanted in the flanks of C57BL/6 albino female mice in preventative and therapeutic experiments, optionally in combination with a programmed cell death 1 (PD-1) blocker. Bioactivity of the general approach was also measured against hepatocellular carcinoma Hepa 1–6 cells. Mice were followed for the growth of subsequently implanted or pre-existing tumors and survival. Draining lymph nodes from mice receiving mIGV-001 were immunophenotyped. mIGV-001 and hIGV-001 were analyzed for extracellular ATP and high mobility group box 1 (HMGB1) as indicators of immunogenic cell death (ICD), along with flow cytometric analysis of viability, surface calreticulin, and reactive oxygen species. Stress and cell death-related pathways were analyzed by immunoblotting. Results IGV-001 causes oxidative and endoplasmic reticulum stress in GL261 cells, resulting in a cytotoxic response that enables the release of antigenic material and immunostimulatory, ICD-associated molecules including ATP and HMGB1 from BDCs. Immunophenotyping confirmed that IGV-001 increases the percentage of dendritic cells, as well as effector, and effector memory T cells in BDC-draining lymph nodes. Consistent with these observations, preventative IGV-001 limited tumor progression and extended overall survival in mice intracranially challenged with GL261 cells, a benefit that was associated with an increase in tumor-specific T cells with effector features. Similar findings were obtained in the Hepa 1–6 model. Moreover, therapeutically administered IGV-001 combined with PD-1 delayed progression in GBM-bearing mice. Conclusions These results support treatment with IGV-001 to induce clinically relevant ICD-driven anticancer immune responses in patients with GBM

Membrane Interactome of a Recombinant Fragment of Human Surfactant Protein D Reveals GRP78 as a Novel Binding Partner in PC3, a Metastatic Prostate Cancer Cell Line

ICMR- National Institute for Research in Reproductive Health ICMR-NIRRH (Accession no. 921); ICMR- National Institute for Research in Reproductive Health ICMR-NIRRH-JRF; ICMR- National Institute for Research in Reproductive Health ICMR-SRF; Researchers Supporting Project (RSP-2020/26) King Saud University, Riyadh

Stabilizing metastable tetragonal HfO \u3c inf\u3e 2 using a non-hydrolytic solution-phase route: Ligand exchange as a means of controlling particle size

This journal is © 2016 The Royal Society of Chemistry. There has been intense interest in stabilizing the tetragonal phase of HfO2 since it is predicted to outperform the thermodynamically stable lower-symmetry monoclinic phase for almost every application where HfO2 has found use by dint of its higher dielectric constant, bandgap, and hardness. However, the monoclinic phase is much more thermodynamically stable and the tetragonal phase of HfO2 is generally accessible only at temperatures above 1720 °C. Classical models comparing the competing influences of bulk free energy and specific surface energy predict that the tetragonal phase of HfO2 ought to be stable at ultra-small dimensions below 4 nm; however, these size regimes have been difficult to access in the absence of synthetic methods that yield well-defined and monodisperse nanocrystals with precise control over size. In this work, we have developed a modified non-hydrolytic condensation method to precisely control the size of HfO2 nanocrystals with low concentrations of dopants by suppressing the kinetics of particle growth by cross-condensation with less-reactive precursors. This synthetic method enables us to stabilize tetragonal HfO2 while evaluating ideas for critical size at which surface energy considerations surpass the bulk free energy stabilization. The phase assignment has been verified by atomic resolution high angle annular dark field images acquired for individual nanocrystals

Improved Topotactic Reactions for Maximizing Organic Coverage of Methyl Germanane

The topotactic transformation of Zintl phases such as CaGe₂ into organic-terminated germanium graphane analogues using haloalkanes is a powerful route for generating new 2D optoelectronic and spintronic building blocks. However, uniform ligand coverage is necessary for optimizing the properties and stability of these single-atom-thick frameworks. Here, we compare the effectiveness of different topochemical methods to maximize methyl-termination in GeCH₃. We show that a previously developed CH₃I/H₂O phase transfer route produces a small percentage of partially oxidized germanane. The partially oxidized termination is readily removed upon HCl treatment, which leads to Ge–Cl termination, but rapidly reoxidizes after exposure to the ambient atmosphere. We then show that a one-pot route with CH₃I in distilled CH₃CN solvent and at least six equivalents of H₂O results in no oxidation. The GeCH₃ prepared from this one-pot route also has an increased −CH₃/–H ratio of termination from ∼90:10 to ∼95:5, is air-stable, has greater thermal stability, has a sharper absorption onset, and has more narrow band edge photoluminescence, all of which are signatures of a less defective semiconductor

GRP78 modulates cell adhesion markers in prostate Cancer and multiple myeloma cell lines

Abstract Background Glucose regulated protein 78 (GRP78) is a resident chaperone of the endoplasmic reticulum and a master regulator of the unfolded protein response under physiological and pathological cell stress conditions. GRP78 is overexpressed in many cancers, regulating a variety of signaling pathways associated with tumor initiation, proliferation, adhesion and invasion which contributes to metastatic spread. GRP78 can also regulate cell survival and apoptotic pathways to alter responsiveness to anticancer drugs. Tumors that reside in or metastasize to the bone and bone marrow (BM) space can develop pro-survival signals through their direct adhesive interactions with stromal elements of this niche thereby resisting the cytotoxic effects of drug treatment. In this study, we report a direct correlation between GRP78 and the adhesion molecule N-cadherin (N-cad), known to play a critical role in the adhesive interactions of multiple myeloma and metastatic prostate cancer with the bone microenvironment. Methods N-cad expression levels (transcription and protein) were evaluated upon siRNA mediated silencing of GRP78 in the MM.1S multiple myeloma and the PC3 metastatic prostate cancer cell lines. Furthermore, we evaluated the effects of GRP78 knockdown (KD) on epithelial-mesenchymal (EMT) transition markers, morphological changes and adhesion of PC3 cells. Results GRP78 KD led to concomitant downregulation of N-cad in both tumors types. In PC3 cells, GRP78 KD significantly decreased E-cadherin (E-cad) expression likely associated with the induction in TGF-β1 expression. Furthermore, GRP78 KD also triggered drastic changes in PC3 cells morphology and decreased their adhesion to osteoblasts (OSB) dependent, in part, to the reduced N-cad expression. Conclusion This work implicates GRP78 as a modulator of cell adhesion markers in MM and PCa. Our results may have clinical implications underscoring GRP78 as a potential therapeutic target to reduce the adhesive nature of metastatic tumors to the bone niche

Ferroelastic Domain Organization and Precursor Control of Size in Solution-Grown Hafnium Dioxide Nanorods

We demonstrate that the degree of branching of the alkyl (R) chain in a Hf(OR)₄ precursor allows for control over the length of HfO₂ nanocrystals grown by homocondensation of the metal alkoxide with a metal halide. An extended nonhydrolytic sol–gel synthesis has been developed that enables the growth of high aspect ratio monoclinic HfO₂ nanorods that grow along the [100] direction. The solution-grown elongated HfO₂ nanorods show remarkable organization of twin domains separated by (100) coherent twin boundaries along the length of the nanowires in a morphology reminiscent of shape memory alloys. The sequence of finely structured twin domains each spanning only a few lattice planes originates from the Martensitic transformation of the nanorods from a tetragonal to a monoclinic structure upon cooling. Such ferroelastic domain organization is uncharacteristic of metal oxides and has not thus far been observed in bulk HfO₂. The morphologies observed here suggest that, upon scaling to nanometer-sized dimensions, HfO₂ might exhibit mechanical properties entirely distinctive from the bulk