Influence of cationic lipid composition on gene silencing properties of lipid nanoparticle formulations of siRNA in antigen-presenting cells

Lipid nanoparticles (LNPs) are currently the most effective in vivo delivery systems for silencing target genes in hepatocytes employing small interfering RNA. Antigen-presenting cells (APCs) are also potential targets for LNP siRNA. We examined the uptake, intracellular trafficking, and gene silencing potency in primary bone marrow macrophages (bmMΦ) and dendritic cells of siRNA formulated in LNPs containing four different ionizable cationic lipids namely DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA. LNPs containing DLinKC2-DMA were the most potent formulations as determined by their ability to inhibit the production of GAPDH target protein. Also, LNPs containing DLinKC2-DMA were the most potent intracellular delivery agents as indicated by confocal studies of endosomal versus cytoplamic siRNA location using fluorescently labeled siRNA. DLinK-DMA and DLinKC2-DMA formulations exhibited improved gene silencing potencies relative to DLinDMA but were less toxic. In vivo results showed that LNP siRNA systems containing DLinKC2-DMA are effective agents for silencing GAPDH in APCs in the spleen and peritoneal cavity following systemic administration. Gene silencing in APCs was RNAi mediated and the use of larger LNPs resulted in substantially reduced hepatocyte silencing, while similar efficacy was maintained in APCs. These results are discussed with regard to the potential of LNP siRNA formulations to treat immunologically mediated diseases.     

Enhanced hepatic delivery of siRNA and microRNA using oleic acid based lipid nanoparticle formulations

Many cationic lipids have been developed for lipid-based nanoparticles (LNPs) for delivery of siRNA and microRNA (miRNA). However, less attention has been paid to “helper lipids”. Here, we investigated several “helper lipids” and examined their effects on the physicochemical properties such as particle size and zeta potential, as well as cellular uptake and transfection efficiency. We found that inclusion of oleic acid (OA), an unsaturated fatty acid, into the LNP formulation significantly enhanced the delivery efficacy for siRNA and miRNA. For proof-of-concept, miR-122, a liver-specific microRNA associated with many liver diseases, was used as a model agent to demonstrate the hepatic delivery efficacy both in tumor cells and in animals. Compared to Lipofectamine 2000, a commercial transfection agent, LNPs containing OA delivered microRNA-122 in a more efficient manner with a 1.8-fold increase in mature miR-122 expression and a 20% decrease in Bcl-w, a target of microRNA-122. In comparison with Invivofectamine, a commercial transfection agent specifically designed for hepatic delivery, LNPs containing OA showed comparable liver accumulation and in vivo delivery efficiency. These findings demonstrated the importance of “helper lipid” components of the LNP formulation on the cellular uptake and transfection activity of siRNA and miRNA. LNPs containing OA is a promising nanocarrier system for the delivery of RNA-based therapeutics in liver diseases.     

siRNA-containing liposomes modified with polyarginine effectively silence the targeted gene.

Development of RNA interference (RNAi) technology utilizing the short interfering RNA sequences (siRNA) based 'targeted' therapeutics has focused on creating methods for delivering siRNAs to cells and for enhancing siRNA stability in vitro and in vivo. Here, we describe a novel approach for siRNA cellular delivery using siRNA encapsulated into liposomes additionally bearing arginine octamer (R8) molecules attached to their outer surface (R8-liposomes). The R8-liposomal human double minute gene 2 (HDM2)-siRNA demonstrated a significant stability against degradation in the blood serum (siRNA-loaded R8-liposomes remained intact even after 24-h incubation), and higher transfection efficiency into all three tested lung tumor cell lines. siRNA delivery successfully proceeds in the presence of plasma proteins, and R8-liposomes demonstrate low non-specific toxicity. The mechanism of action of R8-liposome-encapsulated siRNA is associated with the RNAi-mediated degradation of the target mRNA. siRNA in R8-liposomes effectively inhibited the targeted gene and significantly reduced the proliferation of cancer cells. The approach offers the potential for siRNA delivery for various in vitro and in vivo applications.     

Noninvasive Imaging of Lipid Nanoparticle�CMediated 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.     

Effect of the array of amines on the transfection efficiency of cationic peptidomimetic lipid molecules into neural cells

The amino groups in the head group of a cationic lipid play a determinative role regarding the nucleic acid delivery efficiency of the LNP formulated from lipids. Herein, we designed four types of lipid bearing different amine-containing branched head groups to investigate the influence of type and number of amines on the neural cell targeted nuclei acid delivery. Conjugation of an ethylamino group at selected positions of a lysine-based cationic lipid resulted in 4 distinct lipids with 3 (denoted N3 lipid), 4 (denoted N4 lipid), 5 (denoted N5 lipid) and 6 (denoted N6 lipid) amino groups, respectively. Comparative analysis by flow cytometry revealed that the N3 lipid had the highest nucleic acid (plasmid and siRNA) transfection efficiency to neural cell lines (BV2 cells and N2a cells). Furthermore, the N3 lipid mediated delivery of siRNA against Toll Like Receptor 4 (TLR4) into oxygen glucose deprivation (OGD)-treated BV2 cells resulted in remarkable silencing of TLR4, inducing alternative polarization (M2) of the cells. Collectively, our data suggest that the N3 lipid is a promising siRNA delivery agent in neural cells.

The amino groups in the head group of a cationic lipid play a determinative role regarding the nucleic acid delivery efficiency of the LNP formulated from lipids.     

In vivo gene silencing in solid tumors by targeted electrically mediated siRNA delivery

RNA interference (RNAi)-mediated gene silencing approaches appear very promising for therapies based on the targeted inhibition of disease-relevant genes. The major hurdle to the therapeutic development of RNAi strategies remains, however, the efficient delivery of the RNAi-inducing molecules, the short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), to the target tissue. With respect to cancer treatment the development of efficient delivery methods into solid tumors appears as a critical issue. However, very few studies have addressed this problem. In this study we have investigated the contribution of electrically mediated delivery of siRNA into murine tumors stably expressing an enhanced green fluorescent protein (EGFP) target reporter gene. The silencing of EGFP gene expression was quantified over time by fluorescence imaging in the living animal. Our study indicates that electric field can be used as an efficient method for siRNA delivery and associated gene silencing into cells of solid tumors in vivo.     

RGD-based active targeting of novel polycation liposomes bearing siRNA for cancer treatment

For the purpose of systemic delivery of siRNA, we previously developed polycation liposomes (PCLs) containing dicetylphosphate-tetraethylenepentamine (DCP-TEPA) as an effective siRNA carrier. In the present study, to endow these PCLs (TEPA-PCL) actively target cancer cells and angiogenic vessels, we decorated the PCLs with cyclic RGD, by using cyclic RGD-grafted distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), and investigated the usefulness of this type of carrier (RGD-PEG-PCL) for active targeting. Firstly, the gene-silencing efficacy of siRNA for luciferase (siLuc2) formulated in RGD-PEG-PCL (RGD-PEG-PCL/siLuc2) was examined in vitro by using B16F10-luc2 murine melanoma cells stably expressing the luciferase 2 gene, where the siRNA was grafted with cholesterol at the 3'-end of the sense strand (siRNA-C) for the stable association of the siRNA with the PCL. RGD-PEG-PCL/siLuc2 showed high knockdown efficiency compared with siLuc2 formulated in PEGylated TEPA-PCL without cyclic RGD (PEG-PCL). Next, the gene-silencing efficacy of RGD-PEG-PCL/siLuc2 was examined in vivo by use of B16F10-luc2 lung metastatic model mice. The intravenous injection of RGD-PEG-PCL/siLuc2 showed high knockdown efficiency against metastatic B16F10-luc2 tumors in the lungs of the mice, as assessed with an in vivo imaging system. These data strongly suggest that systemic and active targeting siRNA delivery using RGD-PEG-PCL is useful for cancer RNAi therapy.     

Liposome-mediated, nonviral gene transfer induces a systemic inflammatory response which can exacerbate pre-existing inflammation

Cationic liposome and plasmid-mediated gene transfer has emerged as a novel technique for the targeted delivery of protein-based therapies in acute inflammatory diseases. However, concerns have arisen that cationic liposomes and plasmid DNA have inherent proinflammatory properties which could exacerbate pre-existing inflammatory processes. In healthy mice, intraperitoneal administration of cationic liposomes (200 nmol) complexed to plasmid DNA (100 microg) induced a proinflammatory response characterized by the induction of tumor necrosis factor alpha and interleukin-1beta mRNA expression. The plasma concentrations of the hepatic acute phase proteins interleukin-6, amyloid A, amyloid P, and seromucoid were also increased (P<0.05), and this response was seen in endotoxin-resistant (C3H/HeJ) mice. The inflammatory response associated with gene transfer increased the mortality and severity of experimentally induced sterile inflammation (pancreatitis). We conclude that systemic administration of cationic liposomes and plasmid DNA is associated with induction of the innate immune response which may exacerbate pre-existing inflammatory processes.     

An Amino Acid-based Amphoteric Liposomal Delivery System for Systemic Administration of siRNA

We demonstrate a systematic and rational approach to create a library of natural and modified, dialkylated amino acids based upon arginine for development of an efficient small interfering RNA (siRNA) delivery system. These amino acids, designated DiLA² compounds, in conjunction with other components, demonstrate unique properties for assembly into monodisperse, 100-nm small liposomal particles containing siRNA. We show that DiLA²-based liposomes undergo a pH-dependent phase transition to an inverted hexagonal phase facilitating efficient siRNA release from endosomes to the cytosol. Using an arginine-based DiLA², cationic liposomes were prepared that provide high in vivo siRNA delivery efficiency and are well-tolerated in both cell and animal models. DiLA²-based liposomes demonstrate a linear dose–response with an ED₅₀ of 0.1 mg/kg against liver-specific target genes in BALB/c mice.     

A New Potent Secondary Amphipathic Cell�Cpenetrating Peptide for siRNA Delivery Into Mammalian Cells

RNA interference constitutes a powerful tool for biological studies, but has also become one of the most challenging therapeutic strategies. However, small interfering RNA (siRNA)-based strategies suffer from their poor delivery and biodistribution. Cell-penetrating peptides (CPPs) have been shown to improve the intracellular delivery of various biologically active molecules into living cells and have more recently been applied to siRNA delivery. To improve cellular uptake of siRNA into challenging cell lines, we have designed a secondary amphipathic peptide (CADY) of 20 residues combining aromatic tryptophan and cationic arginine residues. CADY adopts a helical conformation within cell membranes, thereby exposing charged residues on one side, and Trp groups that favor cellular uptake on the other. We show that CADY forms stable complexes with siRNA, thereby increasing their stability and improving their delivery into a wide variety of cell lines, including suspension and primary cell lines. CADY-mediated delivery of subnanomolar concentrations of siRNA leads to significant knockdown of the target gene at both the mRNA and protein levels. Moreover, we demonstrate that CADY is not toxic and enters cells through a mechanism which is independent of the major endosomal pathway. Given its biological properties, we propose that CADY-based technology will have a significant effect on the development of fundamental and therapeutic siRNA-based applications.     

Cellular uptake and intracellular release are major obstacles to the therapeutic application of siRNA: novel options by phosphorothioate-stimulated delivery

The cellular uptake of oligomeric nucleic acid-based tools and drugs including small-interfering RNA (siRNA) represents a major technical hurdle for the biologic effectiveness and therapeutic success in vivo. Subsequent to cellular delivery it is crucial to direct siRNA to the cellular location where it enters the RNA interference pathway. Here the authors summarise evidence that functionally active siRNA represents a minor fraction in the order of 1% of total siRNA inside a given target cell. Exploiting possibilities of steering intracellular release or trafficking of siRNA bears the potential of substantially increasing the biological activity of siRNA. The recently described phosphorothioate stimulated cellular delivery of siRNA makes use of the caveolar system ending in the Golgi apparatus, which contrasts all other known delivery systems. Therefore, it represents an attractive alternative to study whether promoted intracellular release is related to increased target suppression and, thus, increased phenotypic biologic effectiveness.     

Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo.

Targeted silencing of disease-associated genes by synthetic short interfering RNA (siRNA) holds considerable promise as a novel therapeutic strategy. However, unmodified siRNA can be potent triggers of the innate immune response, particularly when associated with delivery vehicles that facilitate intracellular uptake. This represents a significant barrier to the therapeutic development of siRNA due to toxicity and off-target gene effects associated with this inflammatory response. Here we show that immune stimulation by synthetic siRNA can be completely abrogated by selective incorporation of 2'-O-methyl (2'OMe) uridine or guanosine nucleosides into one strand of the siRNA duplex. These noninflammatory siRNA, containing less than 20% modified nucleotides, can be readily generated without disrupting their gene-silencing activity. We show that, coupled with an effective systemic delivery vehicle, 2'OMe-modified siRNA targeting apolipoprotein B (apoB) can mediate potent silencing of its target mRNA, causing significant decreases in serum apoB and cholesterol. This is achieved at therapeutically viable siRNA doses without cytokine induction, toxicity, or off-target effects associated with the use of unmodified siRNA. This approach to siRNA design and delivery should prove widely applicable and represents an important step in advancing synthetic siRNA into a broad range of therapeutic areas.     

Triggered release of siRNA from poly(ethylene glycol)-protected, pH-dependent liposomes

The ability of small interfering RNA (siRNA) to regulate gene expression has potential therapeutic applications, but its use is limited by inefficient delivery. Triggered release of adsorbed poly(ethylene glycol) (PEG)-b-polycation polymers from pH-dependent (PD) liposomes enables protection from immune recognition during circulation (pH 7.4) and subsequent intracellular delivery of siRNA within the endosome (pH ~5.5). Polycationic blocks, based on either poly[2-(dimethylamino)ethyl methacrylate] (31 or 62 DMA repeat units) or polylysine (21 K repeat units), act as anchors for a PEG (113 ethylene glycol repeat units) protective block. Incorporation of 1,2-dioleoyl-3-dimethylammonium-propane (DAP), a titratable lipid, increases the liposome's net cationic character within acidic environments, resulting in polymer desorption and membrane fusion. Liposomes encapsulating siRNA demonstrate green fluorescent protein (GFP) silencing in genetically-modified, GFP-expressing HeLa cells and glyceraldehyde-3-phosphate dehydrogenase (GAPD) knockdown in human umbilical vein endothelial cells (HUVEC). Bare and PD liposomes coated with PEG113-DMA31 exhibit a 0.16 ± 0.2 and 0.32 ± 0.3 fraction of GFP knockdown, respectively. In contrast, direct siRNA administration and Oligofectamine complexed siRNA reduce GFP expression by 0.06 ± 0.02 and 0.14 ± 0.02 fractions, respectively. Our in vitro data indicates that polymer desorption from PD liposomes enhances siRNA-mediated gene knockdown.     

Targeted Small Interfering RNA-Immunoliposomes as a Promising Therapeutic Agent against Highly Pathogenic Avian Influenza A (H5N1) Virus Infection

This study describes a proof-of-concept study on the use of small interfering RNA (siRNA)-immunoliposomes as a therapeutic agent against H5N1 influenza virus infection. siRNA specific for influenza virus nucleoprotein (NP) mRNA was employed as the key antiviral agent to inhibit viral replication in this study. A humanized single-chain Fv antibody (huscFv) against the hemagglutinin (HA) of H5N1 highly pathogenic avian influenza virus (HPAI) was used as the targeting molecule to HA of H5N1 virus, which is abundantly expressed on the surface of infected cells (the HA target cells). The huscFv was applied to cationic polyethylene glycol-conjugated 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol–dioleoylphosphatidyl ethanolamine (PEGylated DC-Chol–DOPE) liposomes to generate immunoliposomes for siRNA delivery. The immunoliposomes were shown to specifically bind HA-expressing Sf9 cells and demonstrated enhanced siRNA transfection efficiency. The siRNA transfection efficiency was significantly reduced after preincubation of the HA target cells with an excess amount of free huscFv. These results therefore demonstrated that the enhanced siRNA delivery by use of immunoliposomes was mediated via targeting by huscFv. Furthermore, the siRNA silencing effect was more pronounced when the immunoliposomes were administered 6 to 12 h post-H5N1 infection in MDCK cells compared with the nontargeted liposomes. This proof-of-concept study may contribute to the future design and development of an siRNA delivery system for combating viral infectious diseases in humans.     

Liposome based systems for systemic siRNA delivery: stability in blood sets the requirements for optimal carrier design

siRNA therapeutics are currently regarded as promising candidates to make a leap forward in the search for treatments of various hard to cure diseases. In order to exploit the full potential of siRNA based therapeutics, development of delivery systems that can efficiently guide the siRNA molecules to their target without major side effects will be the key to success. Lipid based delivery systems, originating from earlier research in the fields of gene delivery, are the most studied candidates for siRNA delivery. Here we discuss the requirements that need to be met by these siRNA delivery systems to ensure adequate stability after systemic application and subsequent deposition in the target tissue. The encountered hurdles in the blood stream and the solutions proposed in literature are discussed.     

Update on reslizumab for eosinophilic asthma

The cellular uptake of oligomeric nucleic acid-based tools and drugs including small-interfering RNA (siRNA) represents a major technical hurdle for the biologic effectiveness and therapeutic success in vivo. Subsequent to cellular delivery it is crucial to direct siRNA to the cellular location where it enters the RNA interference pathway. Here the authors summarise evidence that functionally active siRNA represents a minor fraction in the order of 1% of total siRNA inside a given target cell. Exploiting possibilities of steering intracellular release or trafficking of siRNA bears the potential of substantially increasing the biological activity of siRNA. The recently described phosphorothioate stimulated cellular delivery of siRNA makes use of the caveolar system ending in the Golgi apparatus, which contrasts all other known delivery systems. Therefore, it represents an attractive alternative to study whether promoted intracellular release is related to increased target suppression and, thus, increased phenotypic biologic effectiveness.     

Tumor-targeting nanoimmunoliposome complex for short interfering RNA delivery

The potential of short interfering RNA (siRNA) to be developed for therapeutic use against cancer depends on the availability of an efficient tumor-specific delivery vehicle. We have previously shown that a nanoscale nonviral liposome-based complex that includes an anti-transferrin receptor single-chain antibody fragment as the targeting moiety can, when systemically administered, specifically and efficiently target primary and metastatic tumors and deliver molecules useful in gene medicine, including plasmid DNA and antisense oligonucleotides. Here we explore the ability of this complex to deliver a fluorescein-labeled siRNA to tumor cells in vivo and examine the intracellular localization in vitro by confocal microscopy. We show that the immunoliposome--siRNA complex maintains its nanoscale size and, using three separate tumor models, can efficiently and specifically deliver siRNA to both primary and metastatic disease after systemic delivery, thus increasing the possibility for translating the potent effects of siRNA observed in vitro into clinically useful therapeutics.     

Nonviral in vivo delivery of therapeutic small interfering RNAs

Since its discovery in the late 1990s, RNA interference (RNAi) has gained much attention as a powerful strategy for silencing activity. Instrumental for this naturally occurring targeting mechanism is the intracellular presence of a target gene-specific small interfering RNA (siRNA). Therefore, the in vivo delivery of highly specific siRNA molecules represents one major goal in the further development of RNAi-based approaches for clinical applications. For the non-viral delivery of siRNAs, except for local or topical administration, various routes of application and delivery vehicles/strategies have been explored so far, including the systemic injection of pure, unmodified or chemically modified siRNAs, physical methods such as hydrodynamic injection or electropulsation, encapsulation of siRNAs in liposomes, lipoplexes or cationic lipids, formation of nanoplexes through complexation of siRNAs in cationic or other carriers, or chemical coupling of siRNAs to specific carrier molecules. Therefore, approaches to establish the clinical application of RNAi may rely on a combination of biosciences and nanotechnology; in particular, for the identification of optimal siRNAs against optimal target molecules, and the development of sophisticated delivery systems.     

In vivo silencing of a molecular target by short interfering RNA electroporation: tumor vascularization correlates to delivery efficiency

Screening for a molecular target for cancer therapy requires multiple steps, of which an important one is evaluation of the knockdown effect of the target molecule on pregrown xenograft tumors. However, methods currently used for local administration of knockdown reagents, such as short interfering RNA (siRNA), are not satisfactory as to simplicity and efficiency. We established an electroporation method involving a constant voltage and "plate and fork" type electrodes and used it for in vivo delivery of siRNA. The delivery efficiency correlated to the electric current. The electric current correlated to the microvascular density and vascular endothelial growth factor (VEGF) expression and exhibited a threshold that guaranteed efficient delivery. Consequently, we showed that the vascularization and VEGF expression in tumors determined the efficiency of delivery of siRNA by electroporation. VEGF was chosen as a model target. VEGF siRNA electroporation suppressed the growth of tumors exhibiting high VEGF expression to less than 10% of the control level, but it had no effect on low VEGF-expressing tumors. Notably, a long interval (20 days) of electroporation was enough to obtain a satisfactory effect. Systemically injected siRNA could also be delivered into tumors by this method. Our data will provide the technical basis for in vivo electroporation, and this simple and efficient siRNA delivery method is applicable to in vivo comprehensive screening for a molecular target.