Tool For siRNA Delivery Research Articles

Engineered ionizable lipid nanoparticles mediated efficient siRNA delivery to macrophages for anti-inflammatory treatment of acute liver injury

Small interfering RNA (siRNA) mediating specific gene silencing provides a promising strategy for anti-inflammatory therapy. However, the development of potent carriers for anti-inflammatory siRNA to macrophages remains challenging. With the aim of realizing potent delivery of siRNA to macrophages, we engineered ionizable lipid nanoparticles (LNPs) with the key component of synthetic lipid-like materials. By varying the amine molecules in the structure of synthetic lipid-like materials, a potent LNP (1O14-LNP) was identified, which exhibited efficient transfection of macrophages by facilitating efficient internalization and endosomal escape. The 1O14-LNP successfully delivered anti-inflammatory siRNA against interleukin-1β (siIL-1β) with more than 90% downregulation of IL-1β expression in LPS-activated macrophages. From in vivo studies, systemic administrated 1O14-LNP/siRNA mainly distributed in liver and efficiently captured by hepatic macrophages without notable sign of toxicity. Furthermore, LPS/d-GalN-induced acute liver injury model treated with 1O14-LNP/siIL-1β resulted in significant suppression of IL-1β expression and amelioration of liver tissue damage. These results demonstrate that the engineered ionizable LNP provides a powerful tool for siRNA delivery to macrophages and that the strategy of silencing of pro-inflammatory cytokines holds great potential for treating inflammatory diseases.

Oleyl Conjugated Histidine-Arginine Cell-Penetrating Peptides as Promising Agents for siRNA Delivery.

Recent approvals of siRNA-based products motivated the scientific community to explore siRNA as a treatment option for several intractable ailments, especially cancer. The success of approved siRNA therapy requires a suitable and safer drug delivery agent. Herein, we report a series of oleyl conjugated histidine–arginine peptides as a promising nonviral siRNA delivery tool. The conjugated peptides were found to bind with the siRNA at N/P ratio ≥ 2 and demonstrated complete protection for the siRNA from early enzymatic degradation at N/P ratio ≥ 20. Oleyl-conjugated peptide -siRNA complexes were found to be noncytotoxic in breast cancer cells (MCF-7 and MDA-MB-231) and normal breast epithelial cells (MCF 10A) at N/P ratio of ~40. The oleyl-R3-(HR)4 and oleyl-R4-(HR)4 showed ~80-fold increased cellular uptake in MDA-MB-231 cells at N/P 40. Moreover, the conjugated peptides-siRNA complexes form nanocomplexes (~115 nm in size) and have an appropriate surface charge to interact with the cell membrane and cause cellular internalization. Furthermore, this study provides a proof-of-concept that oleyl-R5-(HR)4 can efficiently silence STAT-3 gene (~80% inhibition) in MDA-MB-231 cells with similar effectiveness to Lipofectamine. Further exploration of this approach holds a great promise in discovering a successful in vivo siRNA delivery agent with a favorable pharmacokinetic profile.

Ultrasound-Responsive Nrf2-Targeting siRNA-Loaded Nanobubbles for Enhancing the Treatment of Melanoma.

The siRNA-mediated inhibition of nuclear factor E2-related factor 2 (Nrf2) can be an attractive approach to overcome chemoresistance in various malignant tumors, including melanoma. This work aims at designing a new type of chitosan-shelled nanobubble for the delivery of siRNA against Nrf2 in combination with an ultrasound. A new preparation method based on a water–oil–water (W/O/W) double-emulsion was purposely developed for siRNA encapsulation in aqueous droplets within a nanobubble core. Stable, very small NB formulations were obtained, with sizes of about 100 nm and a positive surface charge. siRNA was efficiently loaded in NBs, reaching an encapsulation efficiency of about 90%. siNrf2-NBs downregulated the target gene in M14 cells, sensitizing the resistant melanoma cells to the cisplatin treatment. The combination with US favored NB cell uptake and transfection efficiency. Based on the results, nanobubbles have shown to be a promising US responsive tool for siRNA delivery, able to overcome chemoresistance in melanoma cancer cells.

Preparation of siRNA\u2013PLGA/Fab\u02b9\u2013PLGA mixed micellar system with target cell-specific recognition

Small interfering RNAs (siRNAs) are susceptible to nucleases and degrade quickly in vivo. Moreover, siRNAs demonstrate poor cellular uptake and cannot cross the cell membrane because of its polyanionic characteristics. To overcome these challenges, an intelligent gene delivery system that protects siRNAs from nucleases and facilitates siRNA cellular uptake is required. We previously reported the potential of siRNA-poly(d,l-lactic-co-glycolic acid; PLGA) micelles as an effective siRNA delivery tool in a murine peritoneal dissemination model by local injection. However, there was no effective formulation for siRNA delivery to target cells via intravenous injection. This study aimed to prepare siRNA–PLGA/Fabʹ–PLGA mixed micelles for siRNA delivery to target floating cells and evaluate its formulation in vitro. As the target siRNA protein in CEMx174, CyclinB1 levels were significantly reduced when siRNA–PLGA/Fabʹ–PLGA mixed micelles were added to cells compared with siRNA–PLGA micelles. siRNA–PLGA/Fabʹ–PLGA mixed micelles have high cell permeability and high target cell accumulation by endocytosis because flow cytometry detected labeling micelles in target cells. This study supports siRNA–PLGA/Fabʹ–PLGA mixed micelles as an effective siRNA delivery tool. This formulation can be administered systemically in dosage form against target cells, including cancer metastasis or blood cancer.

Designing siRNA-conjugated plant oil-based nanoparticles for gene silencing and cancer therapy

In this study, the anticancer activities of two siRNA carriers were compared using a human lung adenocarcinoma epithelial cell line (A549). Firstly, poly(styrene)-graft-poly(linoleic acid) (PS-g-PLina) and poly(styrene)-graft-poly(linoleic acid)-graft-poly(ethylene glycol) (PS-g-PLina-g-PEG) graft copolymers were synthesized by free-radical polymerization. PS-PLina and PS-PLina-PEG nanoparticles (NPs) were prepared by solvent evaporation method and were then characterized. The size was found as 150 ± 10 nm for PS-PLina and 184 ± 6 nm for PS-PLina-PEG NPs. The NPs were functionalized with poly(l-lysine) (PLL) for c-myc siRNA conjugation. siRNA entrapment efficiencies were found in the range of 4–63% for PS-PLina-PLL and 6–42% for PS-PLina-PEG-PLL NPs. The short-term stability test was realised for 1 month. siRNA release profiles were also investigated. In vitro anticancer activity of siRNA-NPs was determined by MTT, flow cytometry, and fluorescence microscopy analyses. Obtained findings showed that both NPs systems were promising as siRNA delivery tool for lung cancer therapy.

Transportan-derived cell-penetrating peptide delivers siRNA to inhibit replication of influenza virus in vivo.

IntroductionIn this study, we report on the development of an effective delivery system for siRNAs; a novel cell-penetrating peptide (CPP), T9(dR), obtained from transportan (TP), was used for in vivo and in vitro testing.MethodsIn this study, toxicity of T9(dR) and TP and efficient delivery of siRNA were tested in 293T, MDCK, RAW, and A549 cells. Furthermore, T9(dR)- and TP-delivered siRNAs against nucleoprotein (NP) gene segment of influenza virus (siNP) were studied in both cell lines and mice.ResultsGel retardation showed that T9(dR) effectively condensed siRNA into nanoparticles sized between 350 and 550 nm when the mole ratio of T9(dR) to siRNA was ≥4:1. In vitro studies demonstrated that T9(dR) successfully delivered siRNA with low cellular toxicity into several cell lines. It was also observed that T9(dR)-delivered siRNAs inhibited replication of influenza virus more efficiently as compared to that delivered by TP into the MDCK and A549 cells. It was also noticed that when given a combined tail vein injection of siNP and T9(dR) or TP, all mice in the 50 nmol siNP group infected with PR8 influenza virus survived and showed weight recovery at 2 weeks post-infection.ConclusionThis study indicates that T9(dR) is a promising siRNA delivery tool with potential application for nucleotide drug delivery.

Strontium Sulfite: A New pH-Responsive Inorganic Nanocarrier to Deliver Therapeutic siRNAs to Cancer Cells.

Inorganic nanoparticles hold great potential in the area of precision medicine, particularly for treating cancer owing to their unique physicochemical properties, biocompatibility and improved pharmacokinetics properties compared to their organic counterparts. Here we introduce strontium sulfite nanoparticles as new pH-responsive inorganic nanocarriers for efficient transport of siRNAs into breast cancer cells. We employed the simplest nanoprecipitation method to generate the strontium sulfite nanoparticles (SSNs) and demonstrated the dramatic roles of NaCl and d-glucose in particle growth stabilization in order to produce even smaller nanosize particles (Na-Glc-SSN) with high affinity towards negatively charged siRNA, enabling it to efficiently enter the cancer cells. Moreover, the nanoparticles were found to be degraded with a small drop in pH, suggesting their potential capability to undergo rapid dissolution at endosomal pH so as to release the payload. While these particles were found to be nontoxic to the cells, they showed higher potency in facilitating cancer cell death through intracellular delivery and release of oncogene-specific siRNAs targeting ros1 and egfr1 mRNA transcripts, than the strontium sulfite particles prepared in absence of NaCl and d-glucose, as confirmed by growth inhibition assay. The mouse plasma binding analysis by Q-TOF LC-MS/MS demonstrated less protein binding to smaller particles of Na-Glc-SSNs. The biodistribution studies of the particles after 4 h of treatment showed Na-Glc-SSNs had less off-target distribution than SSNs, and after 24 h, all siRNAs were cleared from all major organs except the tumors. ROS1 siRNA with its potential therapeutic role in treating 4T1-induced breast tumor was selected for subsequent in vivo tumor regression study, revealing that ROS1 siRNA-loaded SSNs exerted more significant anti-tumor effects than Na-Glc-SSNs carrying the same siRNA following intravenous administration, without any systemic toxicity. Thus, strontium sulfite emerged as a powerful siRNA delivery tool with potential applications in cancer gene therapy.

Glypican-3 gene silencing for ovarian cancer using siRNA-PLGA hybrid micelles in a murine peritoneal dissemination model

Small interfering RNA (siRNA) has received much attention and for possible therapeutic applications to treat incurable chronic and genetic diseases, including cancer. However, the development of safe and efficient carriers for siRNA delivery still remains formidable hurdles for in vivo. The purpose of this study is to prepare siRNA–PLGA hybrid micelles to deliver the siRNA into the ovarian cancer cells and to evaluate of gene silencing effects in mice model. Here we focused on glypican-3 (Gpc3) gene silencing, which involved in tumor progression and inflammatory reaction, as a siRNA target in a murine ovarian cancer cells, HM-1. As a result, linear polyethyleneimine (LPEI)-coated siRNA–PLGA hybrid micelles were shown to effectively inhibit GPC3 expression in vitro in HM-1 cells, compared with siRNA in solution, because of their superior intracellular uptake and enhanced gene silencing effects. In addition, intraperitoneal administration of the cationic LPEI-coated siRNA–PLGA hybrid micelles decreased the number of tumor nodes in the mesentery, compared with the siRNA sole solution, in a HM-1 peritoneal dissemination model. These results suggested that siRNA–PLGA hybrid micelles could be an effective siRNA delivery tool in a murine ovarian cancer model, especially in case it targets molecules, such as Gpc3.

Preparation of siRNA encapsulated nanoliposomes suitable for siRNA delivery by simply discontinuous mixing

Previously a scalable and extrusion-free method has been developed for efficient liposomal encapsulation of DNA by twice stepwise mixing of lipids in ethanol and DNA solution using T-shape mixing chamber. In this study, we prepared nanoliposomes encapsulating siRNA by simply discontinuous mixing of lipids in ethanol/ether/water mixture and acidic siRNA solution without use of special equipment. The simple mixing siRNA/liposomal particles (siRNA/SMLs) prepared using ethanol/ether/water (3:1:1) mixture showed 120.4 ± 20.2 nm particle size, 0.174 ± 0.033 polydispersity and 86.5 ± 2.76% siRNA encapsulation rate. In addition, the SMLs almost completely protected the encapsulated siRNA from RNase A digestion. Coupling of anti-human epidermal growth factor receptor (EGFR) Fab′ to siRNA/SMLs enhanced EGFR-specific cell penetration of SMLs and induced siRNA dependent gene silencing. Unexpectedly, the Cy5.5-labeled Fab′ showed almost no in vivo targeting to the xenografted A549 tumors in SCID-NOD mice. However, multiple injection of the unmodified siRNA/SMLs accumulated in the tumors and induced siRNA-dependent in vivo gene silencing. These results demonstrate that the siRNA/SMLs can be used as a siRNA delivery tool for gene therapy.

Nanotechnology As Potential Tool for siRNA Delivery in Parkinson's Disease.

The lack of an outright treatment for Parkinson's disease (PD) is a pivotal concern in medicine and has driven the search for novel alternatives for treating the disease. Among the proposed approaches, small interfering RNA (siRNA)-based therapy is attracting significant attention as a potential method for the treatment of PD; however, siRNAs delivery possesses potential drawbacks, such as reduced stability in blood circulation and low capacity for reaching the target site. This review aims to explore siRNA-based approaches to PD and the latest advances for designing nanoparticles that effectively target siRNAs to the action site and that protect these against degradation in blood circulation. siRNA-based approaches provide an interesting option for designing new strategies for treating PD through the silencing of genes, whose abnormal expressions contribute to the pathophysiology of the disease; however, siRNA delivery to the brain is a key issue that remains unsolved to date. Current research efforts are focused on designing vectors that effectively transport and protect siRNAs. In this regard, nanoparticles are being developed as carriers for siRNAs with controlled delivery efficiency and low toxicity profiles, and these represent an alternative to common vectors. Identification of putative gene targets for siRNA therapy of PD has set the pace for researching non-viral vectors; however, the technological aspects for tackling the challenge that siRNAs targeting to the brain represents are essentials. In this respect, the formulation of siRNAs in nanoparticles would avoid harmful side effects, such as immunogenic and oncogenic drawbacks.

The AGMA1 polyamidoamine mediates the efficient delivery of siRNA

AGMA1, a prevailingly cationic, guanidine-bearing, linear, amphoteric polyamidoamine is an effective siRNA condensing agent. Here two AGMA1 samples of different molecular weight, i.e. AGMA1–5 and AGMA1–10 were evaluated as siRNA condensing agents and transfection promoters. AGMA1–10 formed stable polyplexes with a size lower than 50 nm and positive zeta potential. AGMA1–5 polyplexes were larger, about 100 nm in size. AGMA1–10 polyplexes, but not AGMA1–5 proved to be an effective intracellular siRNA carrier, able to trigger gene silencing in Hela and PC3 cell lines without eliciting cytotoxic effects. AGMA1–10 knocked down AKT-1 expression upon transfection with an AKT-1 specific siRNA. The polyplex entry mechanism was investigated and was mediated by macropinocytosis. In conclusion, AGMA1 has potential as an efficient, non-toxic tool for the intracellular delivery of siRNA and warrants further investigation.

Quantum dot-mediated delivery of siRNA to inhibit sphingomyelinase activities in brain-derived cells.

The use of RNAi to suppress protein synthesis offers a potential way of reducing the level of enzymes or the synthesis of mutant toxic proteins but there are few tools currently available for their delivery. To address this problem, bioconjugated quantum dots (QDs) containing a hydrophobic component (N-palmitate) and a sequence VKIKK designed to traverse across cell membranes and visualize drug delivery were developed and tested on cell lines of brain origin. We used the Zn outer shell of the QD to bind HIS6 in JB577 (W•G•Dap(N-Palmitoyl)•VKIKK•P9 •G2 •H6 ) and by a gel-shift assay showed that siRNAs would bind to the positively charged KIKK sequence. By comparing many peptides and QD coatings, we showed that the QD-JB577-siRNA construct was taken up by cells of nervous system origin, distributed throughout the cytosol, and inhibited protein synthesis, implying that JB577 was also promoting endosome egress. By attaching siRNA for luciferase in a cell line over-expressing luciferase, we showed 70% inhibition of mRNA after 24-48h. To show more specific effects, we synthesized siRNA for neutral (NSMase2), acid (lysosomal ASMase) sphingomyelinase, and sphingosine kinase 1 (SK1), we demonstrated a dose-dependent inhibition of activity. These data suggest that QDs are a useful siRNA delivery tool and QD-siRNA could be a potential theranostic for a variety of diseases.

Systemic Administration of siRNA via cRGD-containing Peptide.

Although small interfering RNAs (siRNAs) have been demonstrated to specifically silence their target genes in disease models and clinical trials, in vivo siRNA delivery is still the technical bottleneck that limits their use in therapeutic applications. In this study, a bifunctional peptide named RGD10-10R was designed and tested for its ability to deliver siRNA in vitro and in vivo. Because of their electrostatic interactions with polyarginine (10R), negatively charged siRNAs were readily complexed with RGD10-10R peptides, forming spherical RGD10-10R/siRNA nanoparticles. In addition to enhancing their serum stability by preventing RNase from attacking siRNA through steric hindrance, peptide binding facilitated siRNA transfection into MDA-MB-231 cells, as demonstrated by FACS and confocal microscopy assays and by the repressed expression of target genes. When RGD10 peptide, a receptor competitor of RGD10-10R, was added to the transfection system, the cellular internalization of RGD10-10R/siRNA was significantly compromised, suggesting a mechanism of ligand/receptor interaction. Tissue distribution assays indicated that the peptide/siRNA complex preferentially accumulated in the liver and in several exocrine/endocrine glands. Furthermore, tumor-targeted delivery of siRNA was also demonstrated by in vivo imaging and cryosection assays. In summary, RGD10-10R might constitute a novel siRNA delivery tool that could potentially be applied in tumor treatment.

Abstract 5519: Engineered protein nanocage for targeted delivery of siRNA to cancer cells

Abstract Considering the problems of small interfering RNA (siRNA) delivery using traditional viral and non-viral vehicles, a new siRNA delivery system to enhance efficiency and safety needs to be developed. Here we genetically engineered human ferritin based protein nanocage to simultaneously display various functional peptides on the surface of protein nanocage: cationic peptide to capture siRNA, tumor cell targeting and penetrating peptides, and enzymatically cleaved peptide to release siRNA inside tumor cell. In particular, we easily changed the tumor cell targeting peptide depending on target moiety [epidermal growth factor receptor or vimentin in this case] on the tumor cell surface. The polymerized siRNA (poly siRNA) tightly bound to the engineered protein nanocage and formed stable and condensed structure (poly siRNA-protein nanocage complex) without cytotoxicity problem. Furthermore, siRNAs in the condensed complex were effectively protected from endonuclease due to a shielding effect of protein nanocage. In the in vitro treatment of poly siRNA-protein nanocage complex, both of the tumor cell targeting and penetrating peptides were important for efficient delivery of siRNA, and the red fluorescent protein (RFP) expression in RFP-expressing tumor cells was notably suppressed by the delivered siRNA with the complementary sequence to RFP mRNA. It seems that the human ferritin-based protein nanocage is an efficient, stable, and safe tool for siRNA delivery, having a great potential for application to in vivo cancer treatment. The unique feature of protein nanocage is that multiple and functional peptides can be simultaneously and evenly placed and also easily switched on the protein nanocage surface through a simple genetic modification, which is likely to make protein nanocage appropriate for targeted delivery of siRNA to a wide range of cancer cells. Citation Format: Eun Jung Lee, Yoosoo Yang, In-san Kim, Kwangmeyung Kim, Jeewon Lee. Engineered protein nanocage for targeted delivery of siRNA to cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5519. doi:10.1158/1538-7445.AM2015-5519

Engineered Proteinticles for Targeted Delivery of siRNA to Cancer Cells

Considering the problems of small interfering RNA (siRNA) delivery using traditional viral and nonviral vehicles, a new siRNA delivery system to enhance efficiency and safety needs to be developed. Here human ferritin‐based proteinticles are genetically engineered to simultaneously display various functional peptides on the surface of proteinticles: cationic peptide to capture siRNA, tumor cell targeting and penetrating peptides, and enzymatically cleaved peptide to release siRNA inside tumor cell. In the in vitro treatment of poly‐siRNA‐proteinticle complex, both of the tumor cell targeting and penetrating peptides are important for efficient delivery of siRNA, and the red fluorescent protein (RFP) expression in RFP‐expressing tumor cells is notably suppressed by the delivered siRNA with the complementary sequence to RFP mRNA. It seems that the human ferritin‐based proteinticle is an efficient, stable, and safe tool for siRNA delivery, having a great potential for application to in vivo cancer treatment. The unique feature of proteinticles is that multiple and functional peptides can be simultaneously and evenly placed and also easily switched on the proteinticle surface through a simple genetic modification, which is likely to make proteinticles appropriate for targeted delivery of siRNA to a wide range of cancer cells.

A novel siRNA validation system for functional screening of effective RNAi targets in mammalian cells and development of a derivative lentivirus delivery system

RNA interference technology is a widely used tool for the regulation of gene expression at the post-transcriptional level. One major challenge is to find the effective short interfering (si)RNA for target gene rapidly and easily, and then to deliver the siRNA into cells or tissues with high efficiency. Here, we designed a novel siRNA validation vector using a dual luciferase reporter system for the functional screening of effective RNAi targets in mammalian cells. Then, based on a siRNA expression cassette, we developed a derivative lentivirus delivery system to infect the appropriate cells or tissues for the efficient knockdown of target gene expression. Based on this system, we used human IRF7 gene, a key regulatory factor for the differentiation of monocytes to macrophages, as an example. We screened for the optimal siRNA, then packaged it into a lentiviral siRNA expression system. Then, monocytes were infected and we confirmed the knockdown of IRF7 expression could inhibit the differentiation of monocytes to macrophages. To validate our method further, we also screened and identified optimal siRNA for human TLR4 gene. In summary, we developed a novel siRNA validation system to identify optimal siRNA to target genes rapidly. In addition, the lentivirus system is an efficient tool for siRNA delivery for the further study of target gene function. Taken together, this represents an efficient and user-friendly strategy to validate and deliver siRNAs.

Recombinant High Density Lipoprotein Nanoparticles for Target-Specific Delivery of siRNA

Regulation of gene expression using small interfering RNA (siRNA) is a promising strategy for treatments of numerous diseases. However, the progress towards broad application of siRNA requires the development of safe and effective vectors that target to specific cells. In this study, we developed a novel recombinant high density lipoprotein (rHDL) vector with high siRNA encapsulation efficiency. They were prepared by condensing siRNA with various commercial cationic polymers and coating the polyplex with a layer of lipids and apolipoprotein AI (apo AI). The rHDL nanoparticles were used to transfect SMMC-7721 hepatoma cells with stable luciferase expression. The uptake and intracellular trafficing of siRNA were also investigated. Characterization studies revealed these rHDL nanoparticles had similar physical properties as natural HDLs. The various rHDL formulations had high silencing efficiency (more than 70% knockdown) in hepatocytes with minimum cytotoxicity. Moreover, the uptake of rHDL by SMMC-7721 was confirmed to be mediated through the natural HDL uptake pathway. The work described here demonstrated the optimized rHDL nanoparticles may offer a promising tool for siRNA delivery to the liver.

Binary and ternary complexes based on polycaprolactone-graft-poly (N, N-dimethylaminoethyl methacrylate) for targeted siRNA delivery

Small interfering RNA (siRNA) is a powerful gene silencing tool and has promising prospects in basic research and the development of therapeutic reagents. However, the lack of an effective and safe tool for siRNA delivery hampers its application. Here, we introduced binary and ternary complexes that effectively mediated siRNA-targeted gene silencing. Both complexes showed excellent siRNA loading even at the low N/P/C ratio of 3:1:0. FACS and confocal microscopy demonstrated that nearly all cells robustly internalized siRNAs into the cytoplasm, where RNA interference (RNAi) occurred. Luciferase assay and Western blot verified that silencing efficacy reached >80%, and introducing folate onto the ternary complexes further enhanced silencing efficacy by about 10% over those without folate at the same N/P/C ratio. In addition, the coating of PGA-g-mPEG decreased the zeta potential almost to electroneutrality, and the MTT assay showed decreased cytotoxicity. In vivo distribution measurement and histochemical analysis executed in C57BL/6 and Hela tumor-bearing BALB/c nude mice showed that complexes accumulated in the liver, lungs, pancreas and tumors and were released slowly for a long time after intravenous injection. Furthermore, ternary complexes showed higher siRNA fluorescence intensity than binary complexes at the same N/P ratio in tumor tissues, those with folate delivered more siRNAs to tumors than those without folate, and more folate induced more siRNA transport to tumors. In addition, in vivo functional study showed that both binary and ternary complexes mediated down-regulation of ApoB in liver efficiently and consequently blocked the secretion of fatty acids into the blood, resulted in lipid accumulation in liver, liver steatosis and hepatic dysfunction. In conclusion, these complexes provided a powerful means of administration for siRNA-mediated treatment of liver-related diseases and various cancers, especial for pancreatic and cervical cancer.

Targeted Delivery of siRNA to Macrophages for Anti-inflammatory Treatment

Inflammation mediated by tumor necrosis factor-alpha (TNF-alpha) and the associated neuronal apoptosis characterizes a number of neurologic disorders. Macrophages and microglial cells are believed to be the major source of TNF-alpha in the central nervous system (CNS). Here, we show that suppression of TNF-alpha by targeted delivery of small interfering RNA (siRNA) to macrophage/microglial cells dramatically reduces lipopolysaccharide (LPS)-induced neuroinflammation and neuronal apoptosis in vivo. Because macrophage/microglia express the nicotinic acetylcholine receptor (AchR) on their surface, we used a short AchR-binding peptide derived from the rabies virus glycoprotein (RVG) as a targeting ligand. This peptide was fused to nona-D-arginine residues (RVG-9dR) to enable siRNA binding. RVG-9dR was able to deliver siRNA to induce gene silencing in macrophages and microglia cells from wild type, but not AchR-deficient mice, confirming targeting specificity. Treatment with anti-TNF-alpha siRNA complexed to RVG-9dR achieved efficient silencing of LPS-induced TNF-alpha production by primary macrophages and microglia cells in vitro. Moreover, intravenous injection with RVG-9dR-complexed siRNA in mice reduced the LPS-induced TNF-alpha levels in blood as well as in the brain, leading to a significant reduction in neuronal apoptosis. These results demonstrate that RVG-9dR provides a tool for siRNA delivery to macrophages and microglia and that suppression of TNF-alpha can potentially be used to suppress neuroinflammation in vivo.

Delivery of siRNA into the cytoplasm by liposomal bubbles and ultrasound

Small interfering RNA (siRNA) is expected to be a novel therapeutic tool, however, its utilization has been limited by inefficient delivery systems. Recently, we have developed novel polyethyleneglycol modified liposomes (Bubble liposomes; BL) entrapping an ultrasound (US) imaging gas, which can work as a gene delivery tool with US exposure. In this study, we investigated whether the BL were suitable for the delivery of siRNA. BL efficiently delivered siRNA with only 10 s of exposure to US in vitro. Specific gene silencing effects could be achieved well even in the presence of serum or with the disruption of endocytosis. We suggest that siRNA is directly introduced into the cytoplasm by the BL and US and the mechanism enables effective transfection within a short time and in the presence of high serum. Transfection of siRNA into the tibialis muscles with BL and US was also performed. The gene-silencing effect could be sustained for more than 3 weeks. Thus, BL could be a useful siRNA delivery tool in vitro and in vivo.