Small Interfering RNA Delivery System Research Articles

Nanoparticle delivery of CD40 siRNA suppresses alloimmune responses by inhibiting activation and differentiation of DCs and macrophages.

CD40 is an important costimulatory molecule expressed on antigen-presenting cells (APCs) and plays a critical role for APC activation, offering a promising therapeutic target for preventing allograft rejection. Here, we developed a biodegradable nanoparticle small interfering RNA delivery system (siCD40/NPs) to effectively deliver CD40 siRNA (siCD40) into hematopoietic stem cells (HSCs), myeloid progenitors, and mature dendritic cells (DCs) and macrophages. Injection of siCD40/NPs not only down-regulated CD40 expression in DCs and macrophages but also inhibited the differentiation of HSCs and/or myeloid progenitors into functional DCs and macrophages. Furthermore, siCD40/NPs treatment significantly prolonged allograft survival in mouse models of skin allotransplantation. In addition to reiteration of the role of CD40 in APC activation, our findings highlight a previously unappreciated role of CD40 in DC and macrophage differentiation from their progenitors. Furthermore, our results support the effectiveness of siCD40/NPs in suppressing alloimmune responses, providing a potential means of facilitating tolerance induction and preventing allotransplant rejection.

Recent Advances in Treatment of Lung Cancer: Nanoparticle-based Drug and siRNA Delivery Systems.

Lung cancer is the second most diagnosed cancer in both men and women worldwide. Considering the high mortality rate of lung cancer and inadequacy of conventional treatment methods such as surgical resection, chemotherapy and radiotherapy; new treatment strategies are an emerging area of interest. Nanoparticle-based drug and small interfering RNA delivery systems such as lipid, polymeric, inorganic, micellar and dendrimer nanoparticles are designed to enhance the bioavailability, stability and retention of anti-cancer drugs in the targeted regions of the lung. These nanoparticle-based delivery systems increase the active ingredient half-life and targeting efficiency while reducing the required dose of the drug. Hence, they have many advantages such as higher therapeutic efficacy and reducedside effects and adverse events. Combinations of active ingredients, anti-cancer agents and small interfering RNA can be formulated into nanoparticle-based delivery systems that can be administered by various routes including inhalation and intravenous. In this review, the development of lipidic and polymeric nanoparticle-based drug and small interfering RNA delivery systems used in the treatment of lung cancer is discussed.

Engineering magnetosomes with chimeric membrane and hyaluronidase for efficient delivery of HIF-1 siRNA into deep hypoxic tumors

Hypoxia-inducible factor-1 (HIF-1) plays a critical role in tumor development and chemotherapy resistance, and suppression of HIF-1 has emerged as a captivating antitumor approach. To efficiently silence HIF-1 in deep hypoxic tumors, we herein developed a versatile small interfering RNA delivery system that could overcome multiple physiological barriers. The system is comprised of an Fe3O4 magnetic nanocluster as the core, pre-engineered chimeric membrane for the cloak, and decorated hyaluronidase on the surface, which succeeded in prolonged circulation time, magnetic resonance imaging guidance, magnetic tumor accumulation, hypoxic site penetration, homotypic tumor targeting, and cytoplasmic trafficking. Such versatile and programmed delivery performance enabled the engineered magnetosomes to achieve the remarkable silencing of HIF-1, potent therapeutic effect and chemoresistance amelioration with few abnormalities, showing a promising modality for anticancer therapy.

Store-operated calcium entry suppressed the TGF-β1/Smad3 signaling pathway in glomerular mesangial cells.

Our previous study demonstrated that the abundance of extracellular matrix proteins was suppressed by store-operated Ca2+ entry (SOCE) in mesangial cells (MCs). The present study was conducted to investigate the underlying mechanism focused on the transforming growth factor-β1 (TGF-β1)/Smad3 pathway, a critical pathway for ECM expansion in diabetic kidneys. We hypothesized that SOCE suppressed ECM protein expression by inhibiting this pathway in MCs. In cultured human MCs, we observed that TGF-β1 (5 ng/ml for 15 h) significantly increased Smad3 phosphorylation, as evaluated by immunoblot. However, this response was markedly inhibited by thapsigargin (1 µM), a classical activator of store-operated Ca2+ channels. Consistently, both immunocytochemistry and immunoblot showed that TGF-β1 significantly increased nuclear translocation of Smad3, which was prevented by pretreatment with thapsigargin. Importantly, the thapsigargin effect was reversed by lanthanum (La3+; 5 µM) and GSK-7975A (10 µM), both of which are selective blockers of store-operated Ca2+ channels. Furthermore, knockdown of Orai1, the pore-forming subunit of the store-operated Ca2+ channels, significantly augmented TGF-β1-induced Smad3 phosphorylation. Overexpression of Orai1 augmented the inhibitory effect of thapsigargin on TGF-β1-induced phosphorylation of Smad3. In agreement with the data from cultured MCs, in vivo knockdown of Orai1 specific to MCs using a targeted nanoparticle small interfering RNA delivery system resulted in a marked increase in abundance of phosphorylated Smad3 and in nuclear translocation of Smad3 in the glomerulus of mice. Taken together, our results indicate that SOCE in MCs negatively regulates the TGF-β1/Smad3 signaling pathway.

Application of biodegradable dendrigraft poly-l-lysine to a small interfering RNA delivery system

Dendrigraft poly-l-lysine (DGL), including its central core, consists entirely of lysine, hence it is completely biodegradable. We applied DGL in a small interfering RNA (siRNA) delivery system. Binary complexes with siRNA and DGL had particle sizes of 23–73 nm and ζ-potentials of 34–42 mV. The siRNA-DGL complexes showed significant silencing effects in a mouse colon carcinoma cell line expressing luciferase (Colon26/Luc cells). The siRNA-DGL complexes induced slight cytotoxicity and hematological toxicity at a high charge ratio of DGL to siRNA, probably because of their cationic charges. Therefore, we recharged the siRNA-DGL complexes with γ-polyglutamic acid (γ-PGA), a biodegradable anionic compound, which was reported to reduce the cytotoxicity of cationic complexes. The ternary complexes showed particle sizes of 35–47 nm at a charge ratio of greater than 14 to siRNA with negative charges. Strong silencing effects of the ternary complexes were observed in Colon26/Luc cells without cytotoxicity or hematological toxicity. The cellular uptake and degradation of the binary and ternary complexes were confirmed by fluorescence microscopy. The ternary complexes suppressed luciferase activity in the tumor after direct injection into the tumors of mice bearing Colon26/Luc cells. Thus, a potentially important siRNA delivery system was constructed using biodegradable DGL.

Targeted siRNA Silencing of Indoleamine 2, 3-Dioxygenase in Antigen-presenting Cells Using Mannose-conjugated Liposomes

Indoleamine 2, 3-dioxygenase (IDO) expression in dendritic cells (DCs) leads to the inhibition of T-cell activation, induction of T-cell apoptosis, and promotion of T-cell differentiation into regulatory T cells. All of these could promote tumor escapement of the host's immune surveillance system. We hypothesized that DC-targeted gene silencing of IDO would enhance antitumor immunity and thus restrain tumor growth. Mannose receptors are highly expressed in antigen-presenting cells (APCs) including DCs. In this study, we developed a novel APC-targeted small interfering RNA delivery system using mannosed liposomes (Man-lipo) with encapsulated IDO small interfering RNA (Man-lipo-siIDO), which preferentially knocked down IDO expression in draining lymph node and spleen of melanoma-bearing mice. Mice treated with Man-lipo-siIDO displayed a delayed time of onset of implanted murine melanomas, increased survival time, reduced tumor size, and increased reactivity of T cells from spleen and lymph nodes against melanoma antigens. The enhanced antitumor immunity may be linked to inhibition of apoptosis in CD8 and CD4 T cells as well as Treg cells in spleen and lymph nodes. This study is the first to demonstrate that Man-lipo-siIDO can preferentially targets APCs and efficiently silence IDO expression in vitro and in vivo; events expected to enhance antitumor immune reactions against melanoma xenografts. This study supports the hypothesis that Man-lipo-siIDO may possess the potential for development as an immune-targeting therapeutic anticancer agent.

Enhanced Intrapulmonary Delivery of Anticancer siRNA for Lung Cancer Therapy Using Cationic Ethylphosphocholine-based Nanolipoplexes

Here, we report a cationic nanolipoplex as a pulmonary cellular delivery system for small-interfering RNA (siRNA). Six nanoliposomes differing in cationic lipids were formulated and screened in vitro and in vivo for cellular delivery functions in lung cells/tissues. Although the six nanoliposomes showed similar siRNA delivery efficiency in vitro, they exhibited significant differences in pulmonary cellular delivery functions in vivo. Among the various nanoliposomes, cationic dioleoyl-sn-glycero-3-ethylphosphocholine and cholesterol (ECL)-based nanoliposomes showed the highest pulmonary cellular delivery in vivo and the lowest cytotoxicity in vitro. The delivery efficiency of fluorescent siRNA in ECL nanoliposomes was 26.2-fold higher than that of naked siRNA in vivo. Treatment with Mcl1 (myeloid cell leukemia sequence 1)-specific siRNA (siMcl1) using ECL nanolipoplexes reduced target expression in B16F10 cell lines, whereas control, luciferase-specific siGL2 in ECL nanolipoplexes did not. In metastatic lung cancer mouse models induced by B16F10 or Lewis lung carcinoma (LLC) cells, intratracheal administration of siMcl1 in ECL nanolipoplexes significantly silenced Mcl1 mRNA and protein levels in lung tissue. Reduced formation of melanoma tumor nodules was observed in the lung. These results demonstrate the utility of ECL nanoliposomes for pulmonary delivery of therapeutic siRNA for the treatment of lung cancers and potentially for other respiratory diseases.

Nanoscale Small Interfering RNA Delivery Systems For Personalized Cancer Therapy

Summary: Nanoparticles represent particularly attractive delivery systems for small interfering RNA (siRNA) and may provide the foundation for rational design and formulation of RNAi-triggering nanomedicines. siRNA can be delivered with a therapeutic intent using lipid-based delivery platforms such as stable nucleic acid lipid particles (SNALP) with a lipid bilayer containing cationic as well as fusogenic lipids and a diffusible PEG-lipid coat, polymers, cationic complexes, recombinant fusion proteins, conjugates, or polyconjugates. Several investigators have reported preclinical or early clinical proof of concept studies demonstrating that systemic delivery of siRNA nanoparticles targeting specific gene transcripts can elicit biologic responses. Therapeutic nanoparticles containing siRNA targeting specific genes that contribute to the aggressiveness and/or radiochemotherapy resistance of cancer cells may facilitate a paradigm shift in modern cancer therapy.

Delivery of siRNA to the Mouse Lung via a Functionalized Lipopolyamine

We have designed a series of versatile lipopolyamines which are amenable to chemical modification for in vivo delivery of small interfering RNA (siRNA). This report focuses on one such lipopolyamine (Staramine), its functionalized derivatives and the lipid nanocomplexes it forms with siRNA. Intravenous (i.v.) administration of Staramine/siRNA nanocomplexes modified with methoxypolyethylene glycol (mPEG) provides safe and effective delivery of siRNA and significant target gene knockdown in the lungs of normal mice, with much lower knockdown in liver, spleen, and kidney. Although siRNA delivered via Staramine is initially distributed across all these organs, the observed clearance rate from the lung tissue is considerably slower than in other tissues resulting in prolonged siRNA accumulation on the timescale of RNA interference (RNAi)-mediated transcript depletion. Complete blood count (CBC) analysis, serum chemistry analysis, and histopathology results are all consistent with minimal toxicity. An in vivo screen of mPEG modified Staramine nanocomplexes-containing siRNAs targeting lung cell-specific marker proteins reveal exclusive transfection of endothelial cells. Safe and effective delivery of siRNA to the lung with chemically versatile lipopolyamine systems provides opportunities for investigation of pulmonary cell function in vivo as well as potential treatments of pulmonary disease with RNAi-based therapeutics.

Development of small interfering RNA delivery system using PEI-PEG-APRPG polymer for antiangiogenic vascular endothelial growth factor tumor-targeted therapy

BackgroundSmall interfering RNA (siRNA) can silence target genes in the cytoplasm and be a major tool in gene therapy. Vascular endothelial growth factor (VEGF), a potent regulator of angiogenesis, is overexpressed in most tumors and is closely associated with tumor growth and metastasis. It has been shown that inhibition of VEGF expression by siRNA is an effective and useful method for antiangiogenic tumor therapy.MethodsIn the present study, we synthesized a targeted delivery system of PEI-PEG-APRPG incorporating angiogenic vessel-homing Ala-Pro-Arg-Pro-Gly (APRPG) peptide into cationic polyethylenimine (PEI) via a hydrophilic poly(ethylene glycol) (PEG) spacer.ResultsPEI-PEG-APRPG effectively condensed siRNA into 20–50 nm nanoparticles with a positive surface charge using a suitable N/P ratio. The siRNA/PEI-PEG-APRPG complex effectively enhanced the stability of siRNA in RNase A, and improved the proliferation-inhibiting ability and transfection efficiency of siRNA in vitro and tumor accumulation in vivo. In addition, the siRNA/PEI-PEG-APRPG complex exhibited high efficiency as antitumor therapy with regard to tumor growth, microvessel density, and VEGF protein and mRNA levels.ConclusionThese findings suggest that PEI-PEG-APRPG effectively delivers siRNA to tumors overexpressing VEGF and thereby inhibits tumor growth.

Noninvasive Imaging of Lipid Nanoparticle–Mediated Systemic Delivery of Small-Interfering RNA to the Liver

Mouse models with liver-specific expression of firefly luciferase were developed that enable a noninvasive and longitudinal assessment of small-interfering RNA (siRNA)-mediated gene silencing in hepatocytes of live animals via bioluminescence imaging. Using these models, a set of lipid nanoparticles (LNPs) with different compositions of cationic lipids, polyethylene glycol (PEG), and cholesterol, were tested for their abilities in delivering a luciferase siRNA to the liver via systemic administration. A dose-dependent luciferase knockdown by LNP/siRNA assemblies was measured by in vivo bioluminescence imaging, which correlated well with the results from parallel ex vivo analyses of luciferase mRNA and protein levels in the liver. RNA interference (RNAi)-mediated target silencing was further confirmed by the detection of RNAi-specific target mRNA cleavage. A single dose of LNP02L at 3 mg/kg (siRNA) caused 90% reduction of luciferase expression and the target repression lasted for at least 10 days. With identical components, LNPs containing 2% PEG are more potent than those with 5.4% PEG. Our results demonstrate that these liver-luciferase mouse models provide a powerful tool for a high-throughput evaluation of hepatic delivery platforms by noninvasive imaging and that the molar ratio of PEG lipid can affect the efficacy of LNPs in silencing liver targets via systemic administration.

Structural features of glutamate‐based lipidic materials for small interfering RNA delivery system

Understanding of materials for small interfering RNA (siRNA) delivery system is essential for the effective design and selection of appropriate materials as potential applications in basic and therapeutic research. However, little attempt has been made to investigate how the structural features of lipidic materials are essential for delivery function. The purpose of this study was to identify the structural features of glutamate-based lipidic materials as potential siRNA carriers for intracellular delivery. Totally designed lipid didodecylacetylglutamatetrimethylammonium (TMA-C2-Glu-C12) and its analogs were synthesized and estimated by silencing effects of fluorescent protein (EGFP) expression in CHO-EGFP cells. Here, we demonstrate that TMA-C2-Glu-C12 among a series of lipidic materials is able to perform efficient siRNA delivery with low cytotoxicity into the cells; the transfer ability resulted in 92.2% +/- 1.16% of the transferred cells with Cy3-labeled siRNA. The chemical structure of TMA-C2-Glu-C12 having both hydrophobic chain (--CH(2)CO--) in head region, hydrophobic chain (--(CH(2))(11)CH(3)) in tail regions, and ethylene (--CH(2) CH(2)--) in connector region played a crucial role to enhance the siRNA transfer efficiency. Moreover, compared with commercial reagents, TMA-C2-Glu-C12 was found to have an effective activity for siRNA delivery. These results may improve the novel design and development of lipidic materials on siRNA delivery system.

CRE recombinase-inducible RNA interference mediated by lentiviral vectors

Recently, several systems designed to trigger RNA interference by using small hairpin RNA driven by polymerase III promoters have been described. Here, we report a lentiviral-mediated small interfering RNA delivery system that can be induced by CRE recombinase. The system consists of a lentiviral vector carrying a mouse U6 promoter that is separated from a small hairpin RNA by a random DNA stuffer sequence flanked by modified loxP sites. The silencing cassette is not expressed until activated by addition of CRE recombinase delivered by a lentiviral vector. We have used this system to show specific down-regulation of GFP and two endogenous genes (the tumor suppressor p53 and the NF-kappaB transcription factor subunit p65) in vitro. Furthermore, down-regulation of both p53 and p65 resulted in the expected effect on downstream genes and cellular phenotype. We foresee multiple applications of this system both in vitro and in vivo to down-regulate specific targets in a tissue-specific and localized manner.