Fluorescent siRNA Research Articles

Co-delivery of PD-L1- and EGFR-targeting siRNAs by synthetic PEG12-KL4 peptide to the lungs as potential strategy against non-small cell lung cancer

BackgroundSmall interfering RNA (siRNA) holds great promise for treating various lung diseases, but the lack of safe and efficient pulmonary siRNA delivery systems has hindered its advance into the clinics. The epidermal growth factor receptor (EGFR) which promotes cell proliferation, and the programmed cell death ligand 1 (PD-L1) which plays a crucial role in suppressing cytotoxic T cells activity, are two important targets for treating non-small cell lung cancer (NSCLC). Here, we explored the potential of PEG12-KL4, a synthetic peptide, to deliver siRNA to various NSCLC cells and to lung tissues in mice. MethodsPEG12-KL4 was used to transfect siRNAs targeted at both EGFR and PD-L1 into NSCLC cells. Immunoblotting was used to evaluate the siRNA silencing effects in HCC827 and NCI-H1975 NSCLC cells. CD8+ T cell-mediated NSCLC cell killing was employed to demonstrate the functional effects of PD-L1 siRNA knock-down. Fluorescent siRNAs were used to visualise siRNA uptake in cells as well as to enable biodistribution studies in BALB/c mice. ResultsOur results showed that PEG12-KL4 was efficient in mediating siRNA knock-down of EGFR and PD-L1 in various NSCLC cells. Importantly, the PEG12-KL4 peptide enabled significantly better siRNA delivery than the commercial Lipofectamine 2000 reagent. We hypothesised that PEG12-KL4 peptide enabled siRNA to either escape from or bypass endosomal degradation as indicated by confocal fluorescence imaging. Notably, combined knock-down of EGFR and PD-L1 in NCI-H1975 cells resulted in better effector T cell-mediated cancer cell killing than knock-down of PD-L1 alone. Moreover, biodistribution of PEG12-KL4/siRNA complexes following intravenous administration revealed poor lung delivery with the fluorescent siRNA accumulating in the liver. In contrast, intratracheal delivery of PEG12-KL4/siRNA complexes resulted in the fluorescent siRNA to be detected in the lung with retarded renal excretion. ConclusionIn conclusion, we demonstrated that the co-delivery of siRNAs targeting EGFR and PD-L1 using PEG12-KL4 is feasible and represents a promising future strategy to treat NSCLC, whereby pulmonary siRNA delivery is favourable to intravenous administration.

Delivery of siRNAs Against Selective Ion Channels and Transporter Genes Using Hyaluronic Acid-coupled Carbonate Apatite Nanoparticles Synergistically Inhibits Growth and Survival of Breast Cancer Cells.

Dysregulated calcium homeostasis and consequentially aberrant Ca2+ signalling could enhance survival, proliferation and metastasis in various cancers. Despite rapid development in exploring the ion channel functions in relation to cancer, most of the mechanisms accounting for the impact of ion channel modulators have yet to be fully clarified. Although harnessing small interfering RNA (siRNA) to specifically silence gene expression has the potential to be a pivotal approach, its success in therapeutic intervention is dependent on an efficient delivery system. Nanoparticles have the capacity to strongly bind siRNAs. They remain in the circulation and eventually deliver the siRNA payload to the target organ. Afterward, they interact with the cell surface and enter the cell via endocytosis. Finally, they help escape the endo-lysosomal degradation system prior to unload the siRNAs into cytosol. Carbonate apatite (CA) nanocrystals primarily is composed of Ca2+, carbonate and phosphate. CA possesses both anion and cation binding domains to target negatively charged siRNA molecules. Hybrid CA was synthesized by complexing CA NPs with a hydrophilic polysaccharide - hyaluronic acid (HA). The average diameter of the composite particles was determined using Zetasizer and FE-SEM and their zeta potential values were also measured. The stronger binding affinity and cellular uptake of a fluorescent siRNA were observed for HA-CA NPs as compared to plain CA NPs. Hybrid CA was electrostatically bound individually and combined with three different siRNAs to silence expression of calcium ion channel and transporter genes, TRPC6, TRPM8 and SLC41A1 in a human breast cancer cell line (MCF-7) and evaluate their potential for treating breast cancer. Hybrid NPs carrying TRPC6, TRPM8 and SLC41A1 siRNAs could significantly enhance cytotoxicity both in vitro and in vivo. The resultant composite CA influenced biodistribution of the delivered siRNA, facilitating reduced off target distribution and enhanced breast tumor targetability.

Potentiation of anti-angiogenic eNOS-siRNA transfection by ultrasound-mediated microbubble destruction in ex vivo rat aortic rings.

Nitric oxide (NO) regulates vascular homeostasis and plays a key role in revascularization and angiogenesis. The endothelial nitric oxide synthase (eNOS) enzyme catalyzes NO production in endothelial cells. Overexpression of the eNOS gene has been implicated in pathologies with dysfunctional angiogenic processes, such as cancer. Therefore, modulating eNOS gene expression using small interfering RNAs (siRNAs) represents a viable strategy for antitumor therapy. siRNAs are highly specific to the target gene, thus reducing off-target effects. Given the widespread distribution of endothelium and the crucial physiological role of eNOS, localized delivery of nucleic acid to the affected area is essential. Therefore, the development of an efficient eNOS-siRNA delivery carrier capable of controlled release is imperative for targeting specific vascular regions, particularly those associated with tumor vascular growth. Thus, this study aims to utilize ultrasound-mediated microbubble destruction (UMMD) technology with cationic microbubbles loaded with eNOS-siRNA to enhance transfection efficiency and improve siRNA delivery, thereby preventing sprouting angiogenesis. The efficiency of eNOS-siRNA transfection facilitated by UMMD was assessed using bEnd.3 cells. Synthesis of nitric oxide and eNOS protein expression were also evaluated. The silencing of eNOS gene in a model of angiogenesis was assayed using the rat aortic ring assay. The results showed that from 6 to 24 h, the transfection of fluorescent siRNA with UMMD was twice as high as that of lipofection. Moreover, transfection of eNOS-siRNA with UMMD enhanced the knockdown level (65.40 ± 4.50%) compared to lipofectamine (40 ± 1.70%). Silencing of eNOS gene with UMMD required less amount of eNOS-siRNA (42 ng) to decrease the level of eNOS protein expression (52.30 ± 0.08%) to the same extent as 79 ng of eNOS-siRNA using lipofectamine (56.30 ± 0.10%). NO production assisted by UMMD was reduced by 81% compared to 67% reduction transfecting with lipofectamine. This diminished NO production led to higher attenuation of aortic ring outgrowth. Three-fold reduction compared to lipofectamine transfection. In conclusion, we propose the combination of eNOS-siRNA and UMMD as an efficient, safe, non-viral nucleic acid transfection strategy for inhibition of tumor progression.

RACK1 promotes porcine reproductive and respiratory syndrome virus infection in Marc-145 cells through ERK1/2 activation

Porcine reproductive and respiratory syndrome (PRRS) is an acute infectious disease that spreads rapidly among pigs and seriously threatens the pig industry. Activation of ERK1/2 is a hallmark of most viral infections. RACK1 interacts with a variety of kinases and membrane receptors that closely associated with viral infections and the development and progression of cancer. However, no studies have clearly defined whether RACK1 can regulate PRRSV infection through ERK1/2 activation. In our study, using RT-qPCR, immunoblotting, indirect fluorescent staining, siRNA knockdown and protein overexpression techniques, we found that downregulation of cellular RACK1 inhibited ERK1/2 activation and subsequently suppressed PRRSV infection, while overexpression of RACK1 enhanced ERK1/2 activation and PRRSV infection. Bioinformatic and Co-immunoprecipitation experimental analysis revealed that cellular RACK1 could interact with viral N protein to exert its function. We elaborated that RACK1 promoted PRRSV replication in Marc-145 cells through ERK1/2 activation. Our study provides new insights into regulating the innate antiviral immune responses during PRRSV infection and contributes to further understanding of the molecular mechanisms underlying PRRSV replication.

Design and Testing of a Disposable Flow Cuvette for Continuous Electroporation of a Bioreactor’s Initial Algae Cultivation

Electroporation is a technique applied both in biomedical and biotechnological fields which uses a high-voltage electric current to temporarily destabilize the plasma membrane of living cells, permitting the introduction of small molecules as well as nucleic acids into the cytosol. Besides viral and chemical transfections, this method is a common way to manipulate living cells. However, the majority of electroporation machines available on the market can only work using batch-based cuvettes treating only a few micrograms of cells. To transform cells in the order of several grams in the quickest possible way, it is necessary to use a continuous-flow method. In this work, we present the design, electric and fluid dynamics simulations, construction and testing of a flow cuvette that can adapt to standard electroporator systems. The flow cuvette connected with a peristaltic pump was able to successfully electroporate 20 mL of medium containing microalgae cells in less than 5 min. Microalgae Scenedesmus almeriensis cells were transfected with a fluorescent siRNA oligo as well as magnetically transformed by introducing magnetic nanoparticles in their cytoplasm. The flow cuvette presented here offers a valid tool for the high-throughput transformation/transfection/transfer of both prokaryotic and eukaryotic organisms, especially suitable for bioreactor cultivation and other industrial biotechnological contexts.

Design of Smart Nanodiamonds: Introducing pH Sensitivity to Improve Nucleic Acid Carrier Efficiency of Diamoplexes.

The mechanism of cellular uptake and intracellular fate of nanodiamond/nucleic acid complexes (diamoplexes) are major determinants of its performance as a gene carrier. Our group designed lysine-nanodiamonds (K-NDs) as vectors for nucleic acid delivery. In this work, we modified the surface of K-NDs with histidine to overcome endo-lysosomal entrapment diamoplexes, the major rate limiting step in gene transfer. Histidine is conjugated onto the NDs in two configurations: lysyl-histidine-NDs (HK-NDs) where histidine is loaded on 100% of the lysine moieties and lysine/lysyl-histidine-NDs (H50K50-NDs) where histidine is loaded on 50% of the lysine moieties. Both HK-NDs and H50K50-NDs maintained the optimum size distribution (i.e., <200 nm) and a cationic surface (zeta potential > 20 mV), similar to K-NDs. HK-NDs binds plasmid deoxyribonucleic acid (pDNA) and small interfering ribonucleic acid (siRNA) forming diamoplexes at mass ratios of 10:1 and 60:1, respectively. H50K50-NDs significantly improved nucleic acid binding, forming diamoplexes at a 2:1 mass ratio with pDNA and a 30:1 mass ratio with siRNA, which are at values similar to the K-NDs. The amount of histidine on the surface also impacted the interactions with mammalian cells. The HK-NDs reduced the cell viability by 30% at therapeutic concentrations, while H50K50-NDs maintained more than 90% cell viability, even at the highest concentrations. H50K50-NDs also showed highest cellular uptake within 24 h, followed by K-NDs and HK-NDs. Most functionalized NDs show cellular exit after 5 days, leaving less than 10% of cells with internalized diamonds. The addition of histidine to the ND resulted in higher transfection of anti-green fluorescent protein siRNA (anti-GFP siRNA) with the fraction of GFP knockdown being 0.8 vs. 0.6 for K-NDs at a mass ratio of 50:1. H50K50-NDs further improved transfection by achieving a similar fraction of GFP knockdown (0.8) at a lower mass ratio of 30:1. Overall, this study provides evidence that the addition of histidine, a pH-modulating entity in the functionalization design at an optimized ratio, renders high efficiency to the diamoplexes. Further studies will elucidate the uptake mechanism and intracellular fate to build the relationship between physicochemical characteristics and biological efficacy and create a platform for solid-core nanoparticle-based gene delivery.

Delivery of siRNA to the Eye: Protocol for a Feasibility Study to Assess Novel Delivery System for Topical Delivery of siRNA Therapeutics to the Ocular Surface.

Drug delivery to the eye remains a real challenge due to the presence of ocular anatomical barriers and physiological protective mechanisms. The lack of effective siRNA delivery mechanism has hampered the real potential of RNAi therapy, but recent literature suggests that nanocarrier systems show great promise in enhancing siRNA bioavailability and reducing the need for repeated intraocular injections. A diverse range of materials are under exploration worldwide, including natural and synthetic polymers, liposomes, peptides, and dendrimeric nanomaterials. This chapter describes a simple workflow for feasibility assessment of a proposed ocular surface siRNA delivery system. Gel retardation assay is used for investigation of optimal siRNA to carrier loading ratio. Fluorescent siRNA allows for initial in vitro testing of cellular uptake to corneal epithelial cells and investigation of in vivo siRNA delivery into mouse cornea by live animal imaging and fluorescence microscopy.

Abstract 2868: A novel fusogenic peptide for delivery of bioactive siRNAs targeting CSNK2A1 in ovarian cancer cells in vitro

Abstract Introduction: Ovarian cancer recurrence and survival rates have remained stagnant over time with current therapeutics displaying low efficacy and often rendered ineffective by degradation. This work presents novel fusogenic peptides that will electrostatically complex with siRNAs and cause a pH-responsive insertion into the endosomal membrane, resulting in destabilization and breakdown allowing for release of siRNAs into the cytoplasm of ovarian cancer cells. Delivery of bioactive siRNAs targeting the CSNK2A1 gene in ovarian cancer cells is hypothesized to reduce cell viability, migration, and invasion. Experimental Procedures: Peptide formulations were complexed with non-targeting siRNAs (siNT) and CSNK2A1 siRNAs (siCSNK2A1) at various N:P ratios for all procedures. Analysis of complex formation was completed through gel-shift assays and dynamic light scattering. After characterization of the complexes, each formulation without siRNA was delivered to OVCAR-3 and CAOV-3 cells for cytotoxicity analysis measured via MTS assay. Observation of cellular uptake and endosomal escape was completed through fluorescent imaging after delivering each peptide formulation complexed with Cy3-labeled siNT (Cy3-siNT) and performing early endosome antigen-1 staining. To confirm bioactivity of targeted siRNAs, complexes with siCSNK2A1 were delivered and gene silencing was analyzed via western blotting. Results: Complexation of siRNAs with each formulation was achieved at N:P ratios of 40:1 and greater. Each of the three peptide formulations were able to form uniform nanocomplexes, at 195 nm, 111 nm, and 59 nm with 8.25 mV, -14.56 mV, and -18.42 mV surface charges respectively, and did not display vehicle cytotoxicity at an 80:1 ratio. Each peptide also mediated siRNA uptake into ovarian cancer cells, with fluorescent siRNA signal increasing with increasing N:P ratios. Of note, we observed that Cy3-siRNA appeared separate from the endosome, indicative of endosomal escape, which was seen in several peptide formulations with both CAOV-3 and OVCAR-3 cell lines. Confirmation of bioactive delivery of siCSNK2A1 is shown through western blot imaging, demonstrating knockdown of CSNK2A1 in comparison to cells treated with siNT alone or left untreated. Conclusion: Our novel fusogenic peptides have shown the ability to complex with and deliver siRNAs into ovarian cancer cells. Each peptide mediates endosomal escape activity, with varying degrees of effectiveness, and silencing of CSNK2A1 as measured by western blot. Further study into downstream effects of CSNK2A1 is ongoing. Future work includes optimization of a single fusogenic sequence for optimal delivery of siRNAs and development of a novel targeting peptide to increase cancer targeting and uptake efficiency. Acknowledgements: This research was supported in part by the National Science Foundation EPSCoR Program under NSF Award # OIA-1655740. Citation Format: Timothy Samec, Jessica Boulos, Serena Gilmore, Angela Alexander-Bryant. A novel fusogenic peptide for delivery of bioactive siRNAs targeting CSNK2A1 in ovarian cancer cells in vitro [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2868.

Functionalized Spiky Particles for Intracellular Biomolecular Delivery.

The intracellular delivery of biomolecules is of significant importance yet challenging. In addition to the conventional delivery of nanomaterials that rely on biochemical pathways, vertical nanowires have been recently proposed to physically penetrate the cell membrane, thus enabling the direct release of biomolecules into the cytoplasm circumventing endosomal routes. However, due to the inherent attachment of the nanowires to a planar 2D substrate, nanowire cell penetrations are restricted to in vitro applications, and they are incapable of providing solution-based delivery. To overcome this structural limitation, we created polyethylenimine-functionalized microparticles covered with nanospikes, namely, “spiky particles”, to deliver biomolecules by utilizing the nanospikes to penetrate the cell membrane. The nanospikes might penetrate the cell membrane during particle engulfment, and this enables the bound biomolecules to be released directly into the cytosol. TiO2 spiky particles were fabricated through hydrothermal routes, and they were demonstrated to be biocompatible with HeLa cells, macrophage-like RAW cells, and fibroblast-like 3T3-L1 cells. The polyethylenimine-functionalized spiky particles provided direct delivery of fluorescent siRNA into cell cytosol and functional siRNA for gene knockdown as well as successful DNA plasmid transfection which were difficult to achieve by using microparticles without nanospikes. The spiky particles presented a unique direct cell membrane penetrant vehicle to introduce biomolecules into cell cytosol, where the biomolecules might bypass conventional endocytic degradation routes.

Hyaluronic acid/poly(ethylenimine) polyelectrolyte multilayer coatings for siRNA-mediated local gene silencing.

Local gene delivery systems utilizing RNA interference technology are a promising approach for therapeutic applications where site-specific release of agents is desired. Polyelectrolyte multilayers (PEMs) can be constructed using the layer-by-layer (LbL) technique and serve as a depot for bioactive substances, which can then be released in a controlled manner. Multilayers of hyaluronic acid/poly(ethylenimine) HA/PEI were built up with different numbers of bilayers and PEI-siRNA particles were embedded in bioactive layers for gene silencing. The increase of the bilayers and the release of siRNA particles were demonstrated by fluorescence intensity measurement with a fluorescence reader. Two different LbL techniques were tested for the reduction of ICAM–1 expression in EA.hy926: PEM build-up by dipping or drying steps, respectively. Herein, the drying technique of the bioactive layers with ICAM siRNA mediated a significant reduction of the ICAM–1 expression from 3 to 24 bilayers. The fluorescent siRNA release study and the re-culturing of the HA/PEI films demonstrated a release of the transfection particles within the first hour. The advantage of dried built-up PEMs compared to a dried monolayer of PEI-siRNA particles with the same siRNA concentration was a significant higher amount of viable cells.

Evaluation of Amino-Functional Polyester Dendrimers Based on Bis-MPA as Nonviral Vectors for siRNA Delivery.

Herein, we present the first evaluation of cationic dendrimers based on 2,2-bis(methylol)propionic acid (bis-MPA) as nonviral vectors for transfection of short interfering RNA (siRNA) in cell cultures. The study encompassed dendrimers of generation one to four (G1–G4), modified to bear 6–48 amino end-groups, where the G2–G4 proved to be capable of siRNA complexation and protection against RNase-mediated degradation. The dendrimers were nontoxic to astrocytes, glioma (C6), and glioblastoma (U87), while G3 and G4 exhibited concentration dependent toxicity towards primary neurons. The G2 showed no toxicity to primary neurons at any of the tested concentrations. Fluorescence microscopy experiments suggested that the dendrimers are highly efficient at endo-lysosomal escape since fluorescently labeled dendrimers were localized specifically in mitochondria, and diffuse cytosolic distribution of fluorescent siRNA complexed by dendrimers was observed. This is a desired feature for intracellular drug delivery, since the endocytic pathway otherwise transfers the drugs into lysosomes where they can be degraded without reaching their intended target. siRNA-transfection was successful in C6 and U87 cell lines using the G3 and G4 dendrimers followed by a decrease of approximately 20% of target protein p42-MAPK expression.

Abstract 132: Application of a Cryogel-Coated Prosthetic Vascular Graft Material for Delivery of Targeted Gene Therapies in a Rabbit Model

Objectives: Long-term success of prosthetic grafts (PG) in peripheral arterial disease is limited by development of anastomotic neointimal hyperplasia. We have constructed a bioactive prosthetic graft material (BPGM) capable of delivering biologic agents in vitro, and evaluate our BPGM in vivo with a rabbit carotid interposition bypass model. Methods: We synthesized our BPGM by cryopolymerization of RGD with methacrylated alginate and heparin, coating 1.5cm by 2mm electrospun PET (ePET) grafts, and dipcoating in fluorescent siRNA for 3 hours. Three rabbits received bare ePET and 3 received cryogel-coated ePET for a carotid interposition bypass (Figure 1). After 24 hours, bypass patency was assessed, and cell toxicity of the proximal anastomosis, mid-graft, and distal anastomosis examined with H&amp;E staining. Confocal microscopy was used to visualize fluorescence, correlating with ability to deliver siRNA in vivo. Results: Graft patency was equal between groups, with no increased cell toxicity in rabbits receiving cryogel-coated ePET. Confocal microscopy demonstrated no difference in retained fluorescence between rabbits receiving cryogel-coated or bare ePET, and no increased transfection of cells at 24 hours (Figure 2). Conclusion: Creation of the optimal PG demands a material that is biocompatible, responsive, and nonthrombogenic. We have constructed a modified PG capable of in vitro delivery of targeted gene therapies, with comparable patency and biocompatibility in our large animal model. Additional optimization to achieve predictable and sustained release is needed to validate this as an effective and practical method to deliver biologic agents in vivo.

Lipofection of siRNA into bovine 8-16-cell stage embryos using zona removal and the well-of-the-well culture system.

Bovine preimplantation embryos exhibit dramatic biological changes between before and after the 8-16-cell stage. Here we report a simple lipofection method to transfect siRNA into bovine 8-16-cell stage embryos using zona removal and the well-of-the-well (WOW) culture system. Bovine one-cell embryos produced in vitro were freed from the zona pellucida and cultured up to the 8-16-cell stage in WOW dishes. The 8-16-cell embryos were lipofected with siRNA and the transfection efficiency was assessed at 48 h of transfection. Lipofection with a red fluorescent non-targeting siRNA revealed the importance of zona removal for transfection of siRNA into embryos. Using this method, we knocked down the methionine adenosyltransferase 2A (MAT2A) gene, achieving a significant reduction in MAT2A expression (P < 0.05) concomitant with the marked inhibition of blastocyst development. Our proposed method, tentatively named ‘Octo-lipofection’, may be useful to analyze gene functions in bovine preimplantation embryos without expensive equipment and skill-intensive techniques.

Precision delivery of RAS-inhibiting siRNA to pancreatic cancer via peptide-based nanoparticles.

287 Background: Greater than 95% of pancreatic adenocarcinomas (PDACs) are driven by KRAS activation; yet, despite decades of work, no RAS inhibitors have reached the clinic. Furthermore, the delivery of therapeutic agents of any kind to PDAC has been hindered by the extensive desmoplasia that accompanies these tumors. Herein, we show that serum-stable and pH-sensing nanoparticles (NPs) are taken up by PDAC cells, can deliver KRAS-specific siRNA into the cytoplasm and inhibit KRAS expression, thereby causing cell death. We go on to use a spontaneous model of pancreas cancer to show that this system can effectively deliver siRNA to stroma-rich tumors. Methods: The murine PDAC cell line KP1 was tested for NP uptake in vitro utilizing fluorescent siRNA NPs (fNPs) in combination with confocal microscopy and flow cytometry. KP1 cells were treated with KRAS-siRNA NP, and KRAS expression and cell viability were assessed with RT-PCR and CellTiter-Glo, respectively. Mice bearing subcutaneous KP1 tumors and KPPC mice with spontaneous PDAC were injected with fNP, and tumor fluorescence was assessed using an in vivo imaging system and fluorescence microscopy. Results: KP1 cells take up fNP in vitro, with &gt; 99% of cells positive for fluorescent signal at 24 hours. Treatment with KRAS-siRNA NP of KP1 cells reduced KRAS expression by 69% (see Figure) and reduced cell viability by 45% compared to untreated and scramble-siRNA treated controls. Gemcitabine demonstrated an additive effect with anti-KRAS therapy. Tumors from KP1 cells grown in mice, and tumors from KPPC mice, were strongly fluorescent 24 hours after IV injection of fNP. Fluorescence microscopy showed successful delivery of fNP to tumors. Conclusions: Our NP system can precisely deliver siRNA to KP1 cells and spontaneous PDAC, overcoming the predominant stromal component in these tumors. KRAS-siRNA delivery downregulates KRAS expression, leading to cell death. This represents a novel treatment for PDAC. Furthermore, with its ability to deliver siRNA into the tumor microenvironment and suppress a known oncogene, this platform could be used to target other putative drivers of tumor progression across various cancer types.

Abstract B42: Silencing of DNA repair proteins with ECO/siRNA nanoparticles for the enhancement of radiation response in glioblastoma

Abstract Glioblastoma (GBM) is one of the most lethal human cancers due in large part to the presence of cancer stem cells which contribute to treatment resistance and disease recurrence. Radiation therapy remains a major component of the standard of care for GBM patients, fueling the development of modification and sensitization strategies that can treat cancer stem cell populations and enhance therapeutic outcome. One such strategy is targeting the DNA damage response (DDR) to interfere with DNA repair in response to ionizing radiation. Ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNApk) play critical roles in the DDR through the detection and subsequent repair of double-strand breaks (DSBs) via homologous recombination and non-homologous end-joining. Therapeutic attempts to target these proteins have suffered from poor bioavailability and specificity, spurring investigation into the use of nanoparticle delivery of siRNA to more specifically inhibit the DDR pathways. Previously, (1-aminoethyl)iminobis[N-(oleicylcysteinyl-1-amino-ethyl)-propionamide (ECO) nanoparticles have been shown to effectively deliver siRNA targeting pathways involved with tumor growth and metastasis. In this study we investigate the use of these nanoparticles to deliver siRNA to inhibit ATM and DNApk activity and enhance radiation response in both glioma and glioma stem cell lines. Established glioma (U251) and glioma stem cell (NSC11) lines were used to evaluate the effectiveness of ECO nanoparticle delivery of siRNA in vitro . Cellular uptake of ECO nanoparticles loaded with fluorescent siRNA was assessed using flow cytometry and fluorescent microscopy, demonstrating the rapid uptake of ECO/siRNA nanoparticles in comparison to commercially available transfection agents. Protein and mRNA analyses revealed the kinetics and degree of gene silencing with ECO nanoparticle delivery. The effect of ECO delivery of ATM and DNApk siRNA on radiation response was evaluated using clonogenic cell survival and γH2AX foci formation assays. In comparison to untreated cells, cells treated with the ECO nanoparticle/siRNA complex exhibited a delayed DNA repair response following irradiation. Our results demonstrate the potential of ECO nanoparticles to efficiently deliver siRNA targeting ATM and DNApk to glioma and glioma stem cells, resulting in an impaired ability to repair DNA damage and an enhanced response to radiation treatment. These results provide the basis for our future investigation into the use of ECO/siRNA complexes targeting ATM and DNApk to improve the radiation response of GBM in vivo . Citation Format: Jennifer A. Lee, Nadia Ayat, Anita Tandle, Zheng-Rong Lu, Kevin Camphausen. Silencing of DNA repair proteins with ECO/siRNA nanoparticles for the enhancement of radiation response in glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B42.

Abstract 1080: Kras downregulation by nanoparticle delivery of siRNA reduces tumor growth in mice

Abstract Introduction Small-interfering RNA (siRNA) is an attractive option for delivering precision therapy to cancer patients because of the potential for highly-specific control of gene expression. Attempts to utilize siRNA in vivo have been hampered by its short circulating half-life, limited cellular uptake, and cellular confinement within endosomes. To overcome these barriers, we packaged siRNA within serum-stable, cell-penetrating, and endosomolytic peptide-based nanoparticles (NPs) to deliver siRNA to human and mouse pancreas and colorectal cancers. In a small pilot study, twice weekly administration of KRAS siRNA-NP to mice bearing human colorectal cancers significantly reduced tumor growth compared to controls. Methods NP uptake was assessed in cell lines utilizing fluorescent siRNA-NPs in combination with fluorescence microscopy and flow cytometry. Cell lines were incubated with KRAS-siRNA NP and KRAS knockdown was assessed by quantitative PCR. Mice bearing tumors derived from these cell lines were injected intravenously with fluorescent NP, and localization and uptake were assessed with an in vivo imaging system (IVIS) and flow cytometry. Immune deficient mice were inoculated subcutaneously with a human colorectal cancer and half were treated for 4 weeks with twice weekly IV injections of KRAS siRNA-NP (1 nmol KRAS siRNA per dose). Results On average, fluorescent siRNA was detected in more than 93% of human and mouse pancreas and colorectal cancer cells (n = 5 cell lines) after 24 hours of incubation. Exposure of colorectal cancer cells to KRAS siRNA-NP in vitro produced a conservatively-estimated 70% downregulation of KRAS. Tumors derived from all cell lines were strongly fluorescent 2 hours after IV injection of fluorescent NP and signal persisted beyond 30 hours. The liver and kidneys also expressed fluorescent signal by IVIS. 86% of tumor cells derived from tumors exposed to fluorescent-NP by IV injection expressed fluorescent signal 24 hours post-injection. Only 18% and 14% of liver and kidney cells, respectively, were fluorescent. Compared to control mice, tumor size was reduced in mice bearing colorectal cancer that received KRAS-siRNA NP IV injections. Eighteen days after initiation of treatment, tumor volumes in the control and treatment groups were 229.5 mm3 and 460.25 mm3 respectively (p &amp;lt; 0.05). Hematologic, liver and kidney function tests revealed no differences between the treatment and control groups. Conclusions Peptide NPs are rapidly and efficiently taken up by human and mouse pancreas and colorectal cancer cells both in vitro and in vivo. Incubation of cancer cells with peptide NP-packaged KRAS-siRNA reduces KRAS expression in vitro. Treatment of tumor-bearing mice with KRAS-siRNA NP leads to a statistically significant reduction in tumor growth compared to control mice. This delivery platform overcomes traditional limitations of siRNA and may be generalizable to target other putative drivers of tumor progression. Citation Format: Matthew Strand, Hua Pan, Xiuli Zhang, Julie Grossman, Peter Goedegebuure, Timothy Fleming, William E. Gillanders, Samuel A. Wickline, Ryan C. Fields. Kras downregulation by nanoparticle delivery of siRNA reduces tumor growth in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1080.

Regression of Gastric Cancer by Systemic Injection of RNA Nanoparticles Carrying both Ligand and siRNA.

Gastric cancer is the second leading cause of cancer-related death worldwide. RNA nanotechnology has recently emerged as an important field due to recent finding of its high thermodynamic stability, favorable and distinctive in vivo attributes. Here we reported the use of the thermostable three-way junction (3WJ) of bacteriophage phi29 motor pRNA to escort folic acid, a fluorescent image marker and BRCAA1 siRNA for targeting, imaging, delivery, gene silencing and regression of gastric cancer in animal models. In vitro assay revealed that the RNA nanoparticles specifically bind to gastric cancer cells, and knock-down the BRCAA1 gene. Apoptosis of gastric cancer cells was observed. Animal trials confirmed that these RNA nanoparticles could be used to image gastric cancer in vivo, while showing little accumulation in crucial organs and tissues. The volume of gastric tumors noticeably decreased during the course of treatment. No damage to important organs by RNA nanoparticles was detectible. All the results indicated that this novel RNA nanotechnology can overcome conventional cancer therapeutic limitations and opens new opportunities for specific delivery of therapeutics to stomach cancer without damaging normal cells and tissues, reduce the toxicity and side effect, improve the therapeutic effect, and exhibit great potential in clinical tumor therapy.

Abstract 4460: Super carbonate apatite as simplified systemic nanoparticle carrier for therapy of solid tumors

Abstract Introduction: RNA interference technology is underway in clinical trials for limited diseases. RNAi technology for solid tumors, especially in systemic administration, still faces hurdles and has not emerged on the clinical stage. Here we introduce an in vivo pH sensitive delivery system of siRNA and microRNA using super carbonate apatite (sCA) nanoparticles (mean size: 10 nm), which simply consist of inorganic ions. In order to show the therapeutic superiority of the sCA system over currently available in vivo siRNA delivery systems, we perform further experiments comparing the sCA system with Invivofectamine 2.0 (IF) and Atelocollagen (Atelo). Finally we examine the toxicity of sCA nanoparticle system in mice and monkeys. Methods: To assess the bio-distribution of fluorescently labeled siRNA in normal organs and tumor tissues, we used the IVIS Spectrum CT system (PerkinElmer) and fluorescence microscopy. In addition, the multiphoton live imaging system (SP5; Leica) and light sheet fluorescence microscopy (Carl Zeiss) were employed for detailed analysis and clear visualization of extravasation and intracellular distribution of fluorescent siRNA in tumor cells. To examine therapeutic potential of the sCA system, we administered survivin siRNA in pre-established HCT116 tumor mice models. Results &amp; Discussions: Ex vivo imaging analysis using IVIS Spectrum CT showed that the fluorescence of sCA-siRNA was highly accumulated into the tumors as early as 90 min. Light sheet fluorescence microscopy revealed that sCA-siRNA accumulated in the cytoplasm of HCT116 cells with considerable green fluorescence signals at 4 h after intravenous injection. On the other hand, IF-siRNA and Atelo-siRNA did not accumulate in tumor cells. sCA-survivin-siRNA exhibited anti-tumor effects on day 11 and the tumor volume of mice treated with sCA-survivin-siRNA was significantly smaller than in mice treated with sCA-control-siRNA (P = 0.0008), IF-survivin-siRNA (P = 0.0028), or Atelo-survivin-siRNA (P = 0.0046) on day 18. For in vivo safety, we carried out a toxicity study by intravenous injection of sCA to mice. Blood chemistry tests and HE stained sections of the liver, kidney and spleen showed no physiologically significant difference between the saline and sCA treated groups. To aim the final goal of therapy and diagnosis of human tumors, we made up approximately 70-fold sCA of that administered in mouse per injection and administered it to monkeys on 4 consecutive days (n = 3). Consequently intravenous administer of sCA did not cause any serious adverse events in monkeys. Conclusion: We have demonstrated a highly efficient in vivo siRNA delivery system targeting solid tumor models, which is characterized as 10 nm minimal particle size and quick accumulation in tumors, thus it can realize to deliver abundant intact siRNA into tumor cells. Citation Format: Hirofumi Yamamoto, Xin Wu, Susumu Miyazaki, Mamoru Uemura, Taishi Hata, Junichi Nishimura, Ichiro Takemasa, Tsunekazu Mizushima, Yuichiro Doki, Masaki Mori. Super carbonate apatite as simplified systemic nanoparticle carrier for therapy of solid tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4460. doi:10.1158/1538-7445.AM2014-4460

A MicroRNA Processing Defect in Smokers' Macrophages Is Linked to SUMOylation of the Endonuclease DICER

Despite the fact that alveolar macrophages play an important role in smoking-related disease, little is known about what regulates their pathophysiologic phenotype. Evaluating smoker macrophages, we found significant down-regulation of multiple microRNAs (miRNAs). This work investigates the hypothesis that cigarette smoke alters mature miRNA expression in lung macrophages by inhibiting processing of primary miRNA transcripts. Studies on smoker alveolar macrophages showed a defect in miRNA maturation. Studies on the miRNA biogenesis machinery led us to focus on the cytosolic RNA endonuclease, DICER. DICER cleaves the stem-loop structure from pre-miRNAs, allowing them to dissociate into their mature 20-22-nucleotide single-stranded form. DICER activity assays confirmed impaired DICER activity following cigarette smoke exposure. Further protein studies demonstrated a decreased expression of the native 217-kDa form of DICER and an accumulation of high molecular weight forms with cigarette smoke exposure. This molecular mass shift was shown to contain SUMO moieties and could be blocked by silencing RNA directed at the primary SUMOylating ligase, Ubc9. In determining the cigarette smoke components responsible for changes in DICER, we found that N-acetylcysteine, an antioxidant and anti-aldehyde, protected DICER protein and activity from cigarette smoke extract. This massive down-regulation of miRNAs (driven in part by alterations in DICER) may be an important regulator of the disease-promoting macrophage phenotype found in the lungs of smokers.

CRGD grafted liposomes containing inorganic nano-precipitate complexed siRNA for intracellular delivery in cancer cells

Development of effective vector for intracellular delivery of siRNA has always been a challenge due to its hydrophilicity, net negative surface charge and sensitivity against nucleases in biological milieu. The present investigation was aimed to develop a novel non-viral liposomal carrier for siRNA delivery. Nano-precipitate of calcium phosphate was entrapped in liposomes composed of a neutral lipid (DPPC), a fusogenic lipid (DOPE), a PEGylated lipid (DSPE-mPEG2000) and cholesterol. siRNA was made permeable through liposomal bilayer and complexed to calcium phosphate precipitates inside the liposomes. siRNA entrapped liposomes were further grafted with cRGD to achieve targeting potential against cancer cells. More than 80% of siRNA was entrapped inside the liposomes having average particle size below 150nm. Cryo-transmission electron microscopy revealed the intra-liposomal calcium phosphate precipitation and unilamellar morphology of prepared liposomes. The viability of A549 lung cancer cells was significantly higher after treatment with siRNA entrapped liposomes as compared to Lipofectamine2000 complexed siRNA. Fluorescent intensity in lung carcinoma cells was significantly higher after exposure to fluorescent siRNA entrapped liposomes than with Lipofectamine2000, which were confirmed by both confocal microscopy and flow cytometry. Live imaging by confocal microscopy ascertained the targeting efficacy of cRGD grafted liposomes compared to naked siRNA and non-grafted liposomes. Developed liposomal formulation showed effective protection of siRNA against serum nucleases along with less haemolytic potential and excellent stability against electrolyte induced flocculation. At 5nM concentration gene expression of target protein was reduced up to 24.1±3.4% while Lipofectamine2000 reduced expression level up to 26.35±1.55%. In vivo toxicity in mice suggested admirable safety profile for developed lipid based delivery vector. These results advocate that prepared liposomal system would be of high value for intracellular delivery of siRNA.