Trilysinoyl oleylamide-based cationic liposomes for systemic co-delivery of siRNA and an anticancer drug

Oligolysine-based cationic lipid derivatives were synthesized for delivery of siRNA, and formulated into cationic liposomes. Among various oligolysine-based lipid derivatives differing in lysine residue number and lipid moiety, trilysinoyl oleylamide (TLO)-based liposomes (TLOL) showed the highest delivery efficiency combined with minimal cytotoxicity. Delivery of siRNA using TLOL silenced target genes both in vitro and in vivo. In green fluorescent protein (GFP)-expressing tumor tissue, a significant reduction of fluorescence was observed after intratumoral administration of siGFP using TLOL compared with control siGL2. Intravenous administration of siMcl1 employing pegylated TLOL (pTLOL) reduced the expression of human Mcl1 protein in KB-xenografted tumor tissue. Despite the reduction in target protein Mcl1 expression following such systemic delivery, tumor growth was only slightly reduced compared to a siGL2-treated control group. To potentiate the anticancer activity of siMcl1, the anticancer drug suberoylanilide hydroxamic acid (SAHA) was additionally encapsulated in pTLOL. After intravenous administration of siMcl1 using SAHA-loaded pTLOL (pSTLOL), a significant reduction in tumor growth was observed compared to that seen in animals treated with free SAHA or siGL2 complexed with pSTLOL. The results indicate that pTLOL could be further developed as a systemic delivery system for synergistic anticancer siRNA and a drug.     

Design of an inhalable dry powder formulation of DOTAP-modified PLGA nanoparticles loaded with siRNA

Matrix systems based on biocompatible and biodegradable polymers like the United States Food and Drug Administration (FDA)-approved polymer poly(DL-lactide-co-glycolide acid) (PLGA) are promising for the delivery of small interfering RNA (siRNA) due to favorable safety profiles, sustained release properties and improved colloidal stability, as compared to polyplexes. The purpose of this study was to design a dry powder formulation based on cationic lipid-modified PLGA nanoparticles intended for treatment of severe lung diseases by pulmonary delivery of siRNA. The cationic lipid dioleoyltrimethylammoniumpropane (DOTAP) was incorporated into the PLGA matrix to potentiate the gene silencing efficiency. The gene knock-down level in vitro was positively correlated to the weight ratio of DOTAP in the particles, and 73% silencing was achieved in the presence of 10% (v/v) serum at 25% (w/w) DOTAP. Optimal properties were found for nanoparticles modified with 15% (w/w) DOTAP, which reduced the gene expression with 54%. This formulation was spray-dried with mannitol into nanocomposite microparticles of an aerodynamic size appropriate for lung deposition. The spray-drying process did not affect the physicochemical properties of the readily re-dispersible nanoparticles, and most importantly, the in vitro gene silencing activity was preserved during spray-drying. The siRNA content in the powder was similar to the theoretical loading and the siRNA was intact, suggesting that the siRNA is preserved during the spray-drying process. Finally, X-ray powder diffraction analysis demonstrated that mannitol remained in a crystalline state upon spray-drying with PLGA nanoparticles suggesting that the sugar excipient might exert its stabilizing effect by sterical inhibition of the interactions between adjacent nanoparticles. This study demonstrates that spray-drying is an excellent technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue.     

Nanostructured lipid carriers as multifunctional nanomedicine platform for pulmonary co-delivery of anticancer drugs and siRNA

We developed, synthesized, and tested a multifunctional nanostructured lipid nanocarrier-based system (NLCS) for efficient delivery of an anticancer drug and siRNA directly into the lungs by inhalation. The system contains: (1) nanostructured lipid carriers (NLC); (2) anticancer drug (doxorubicin or paclitaxel); (3) siRNA targeted to MRP1 mRNA as a suppressor of pump drug resistance; (4) siRNA targeted to BCL2 mRNA as a suppressor of nonpump cellular resistance and (5) a modified synthetic analog of luteinizing hormone-releasing hormone (LHRH) as a targeting moiety specific to the receptors that are overexpressed in the plasma membrane of lung cancer cells. The NLCS was tested in vitro using human lung cancer cells and in vivo utilizing mouse orthotopic model of human lung cancer. After inhalation, the proposed NLCS effectively delivered its payload into lung cancer cells leaving healthy lung tissues intact and also significantly decreasing the exposure of healthy organs when compared with intravenous injection. The NLCS showed enhanced antitumor activity when compared with intravenous treatment. The data obtained demonstrated high efficiency of proposed NLCS for tumor-targeted local delivery by inhalation of anticancer drugs and mixture of siRNAs specifically to lung cancer cells and, as a result, efficient suppression of tumor growth and prevention of adverse side effects on healthy organs.     

Poly(oligo--arginine) With Internal Disulfide Linkages as a Cytoplasm-sensitive Carrier for siRNA Delivery

Small interfering RNA (siRNA) has emerged as a therapeutic strategy for various diseases due to its target-specific gene silencing; however, its relatively high molecular weight, negative charge, and low stability hamper in vitro and in vivo applications. Approaches to overcome those drawbacks have relied on nonviral siRNA carriers based on cationic polymers or peptides. Nevertheless, cationic polymer-based siRNA carriers have yet to resolve intrinsic problems such as cytotoxicity and immunogenicity. An environment-sensitive carrier was recently proposed to enhance siRNA bioactivity and to reduce the carrier safety issues. Only a few studies, however, have shown cytoplasm-sensitive dissociation of the polyplex. In the present study, we clearly demonstrated decondensation of siRNA/poly(oligo--arginine) polyplex in the cytoplasm in response to intracellular glutathione (GSH) and the enhanced bioactivity of siRNA against VEGF (siVEGF) used as a model both in vitro and in an animal model. Reducible poly(oligo--arginine) (rPOA) rapidly dissociated in the cytoplasm, resulting in fast siRNA release to its target location while maintaining siRNA bioactivity both in vitro and in vivo.     

Drug Delivery�Cmediated Control of RNA Immunostimulation

RNA interference (RNAi) has generated significant interest as a strategy to suppress viral infection, but in some cases antiviral activity of unmodified short-interfering RNA (siRNA) has been attributed to activation of innate immune responses. We hypothesized that immunostimulation by unmodified siRNA could mediate both RNAi as well as innate immune stimulation depending on the mode of drug delivery. We investigated the potential of immunostimulatory RNAs (isRNAs) to suppress influenza A virus in vivo in the mouse lung. Lipidoid 98N12-5(1) formulated with unmodified siRNA targeting the influenza nucleoprotein gene exhibited antiviral activity. Formulations were optimized to increase antiviral activity, but the antiviral activity of lipidoid-delivered siRNA did not depend on sequence homology to the influenza genome as siRNA directed against unrelated targets also suppressed influenza replication in vivo. This activity was primarily attributed to enhancement of innate immune stimulation by lipidoid-mediated delivery, which indicates increased toll-like receptor (TLR) activation by siRNA. Certain chemical modifications to the siRNA backbone, which block TLR7/8 activation but retain in vitro RNAi activity, prevented siRNA-mediated antiviral activity despite enhanced lipidoid-mediated delivery. Here, we demonstrate that innate immune activation caused by unmodified siRNA can have therapeutically relevant effects, and that these non-RNAi effects can be controlled through chemical modifications and drug delivery.     

Vaginal delivery of siRNA using a novel PEGylated lipoplex-entrapped alginate scaffold system

Sustained vaginal delivery of siRNA has been precluded by the mucosal barrier lining the vaginal tract. In contrast to prior reports, we showed that conventional lipoplexes administered intravaginally are unable to reach the vaginal epithelium under normal physiological conditions. Here we have developed a novel alginate scaffold system containing muco-inert PEGylated lipoplexes to provide a sustained vaginal presence of lipoplexes in vivo and to facilitate the delivery of siRNA/oligonucleotides into the vaginal epithelium. These PEGylated lipoplex-entrapped alginate scaffolds (PLAS) were fabricated using a freeze-drying method and the entrapment efficiency, release rate, and efficacy were characterized. We demonstrated that the PLAS system had an entrapment efficiency of ~ 50%, which released PEGylated lipoplexes gradually both in vitro and in vivo. While the presence of alginate diminished the cell uptake efficiency of PEGylated lipoplexes in vitro, as expected, we showed a six-fold increase their uptake into the vaginal epithelium compared to existing transfection systems following intravaginal administration in mice. A significant knockdown of Lamin A/C level was also observed in vaginal tissues using siLamin A/C-containing PLAS system in vivo. Overall, our results indicated the potential of the biodegradable PLAS system for the sustained delivery of siRNA/oligonucleotides to vaginal epithelium.     

Design and evaluation of new pH-sensitive amphiphilic cationic lipids for siRNA delivery

Synthetic small interfering RNA (siRNA) has become the basis of a new generation of gene-silencing cancer therapeutics. However, successful implementation of this novel therapy relies on the ability to effectively deliver siRNA into target cells and to prevent degradation of siRNA in lysosomes after endocytosis. In this study, our goal was to design and optimize new amphiphilic cationic lipid carriers that exhibit selective pH-sensitive endosomal membrane disruptive capabilities to allow for the efficient release of their siRNA payload into the cytosol. The pH sensitive siRNA carriers consist of three domains (cationic head, hydrophobic tail, amino acid-based linker). A library of eight lipid carriers were synthesized using solid phase chemistry, and then studied to determine the role of (1) the number of protonable amines and overall pK_a of the cationic head group, (2) the degree of unsaturation of the hydrophobic tail, and (3) the presence of histidine residues in the amino acid linker for transfection and silencing efficacy. In vitro screening evaluation of the new carriers demonstrated at least 80% knockdown of a GFP reporter in CHO cells after 72 h. The carriers ECO and ECLn performed the best in a luciferase knockdown study in HT29 human colon cancer cells, which were found to be more difficult to transfect. They significantly reduced expression of this reporter to 22.7 ± 3.31% and 23.5 ± 5.11% after 72 h post-transfection, better than Lipofectamine RNAiMax. Both ECO and ECLn carriers caused minimal cytotoxicity, preserving relative cell viabilities at 87.3 ± 2.72% and 88.9 ± 6.84%, respectively. A series of hemolysis assays at various pHs revealed that increasing the number of amines in the protonable head group, and removing the histidine residue from the linker, both resulted in improved membrane disruptive activity at the endosomal pH of 6.5. Meanwhile, the cellular uptake into HT29 cancer cells was improved, not only by increasing the amines of the head group, but also by increasing the degree of unsaturation in the lipid tails. Due to flexibility of the synthetic procedure, the delivery system could be modified further for different applications. The success of ECO and ECLn for in vitro siRNA delivery potentially makes them promising candidates for future in vivo studies.     

Chitosan-based siRNA delivery systems

Recently, chitosan has attracted significant attention in the formulation of small interfering RNA (siRNA). Because of its cationic nature, chitosan can easily complex siRNA, thus readily forming nanoparticles. Moreover, chitosan is biocompatible and biodegradable, which make it a good candidate for siRNA delivery in vivo. However, chitosan requires further development to achieve high efficiency. This review will describe the major barriers that impair the efficiency of the chitosan-based siRNA delivery systems, including the stability of the delivery system in biological fluids and endosomal escape. Several solutions to counteract these barriers have been developed and will be discussed. The parameters to consider for designing powerful delivery systems will be described, particularly the possibilities for grafting targeting ligands. Finally, optimized systems that allow in vivo therapeutic applications for both local and systemic delivery will be reviewed. This review will present recent improvements in chitosan-based siRNA delivery systems that overcome many of these system's previous pitfalls and pave the way to a new generation of siRNA delivery systems.     

Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function.     

Influence of lipoplex surface charge on siRNA delivery: application to the in vitro downregulation of CXCR4 HIV-1 co-receptor

Objective: Cationic lipidic formulations have been successfully used to deliver small interfering RNA (siRNA) into cells but they show limitations for in vivo application due to their cytotoxicity and instability in the presence of serum. To overcome these limitations, the authors developed an anionic lipid-based carrier named Neutraplex (Nx). Here, they wanted to investigate the influence of the lipoplex (Lx) surface charge on cytotoxicity, delivery and silencing activity of siRNAs.

Methods: The efficiency of three Nx formulations (cationic, close to neutrality and anionic) to deliver anti-CXCR4 siRNAs in MAGI cells was investigated and compared with the cationic commercial transfection reagent Lipofectamine RNAiMAX. Cellular uptake and intracellular localization of a fluorescent siRNA was monitored in live cells using fluorescence microscopy and silencing activity was measured by flow cytometry and RT-PCR analysis.

Results: The authors found that the Lx surface charge influenced cellular uptake and silencing activity of siRNA in cell cultures. Although cationic Lx formulations were the most efficient carriers to deliver active silencing siRNAs, negatively charged lipoplexes were taken up by cells, delivered active siRNAs and presented low cytotoxicity.

Conclusions: Altogether, the findings support further investigation for in vivo delivery of therapeutic siRNAs using Nx. Furthermore, this study indicates that anionic delivery systems may have potential for in vivo RNAi therapeutics.     

Ultrasound and Microbubble-Induced Local Delivery of MicroRNA-Based Therapeutics

MicroRNAs are involved in many pathologic processes and are a promising target for therapeutic intervention. However, successful, localized delivery of microRNA-based therapeutics is lacking. In this study, cationic ultrasound-responsive microbubbles (MBs) were used to deliver microRNA blockers and mimics in vitro and in vivo. Cationic MBs successfully delivered microRNA blockers to human endothelial cells on ultrasound (US) exposure in vitro. This in vitro US protocol did not successfully deliver microRNA mimics to skeletal muscle of mice, whereas an US protocol that is routinely used for contrast imaging did. Additionally, we used cationic MBs and US to locally deliver antimiR and antagomiR molecules with US causing inertial cavitation. Delivery of antimiR to the extracellular compartments of the muscle was only slightly increased, whereas delivery of antagomiR to the capillaries, myocytes and extracellular space was significantly increased. AntagomiR seems to be a more suitable microRNA blocker than antimiR for use in combination with MBs and US for local delivery.     

Identification of Protein Cofactors Necessary for Sequence-specific Plasmid DNA Nuclear Import

Although transfections are routinely used in the laboratory, the mechanism(s) by which exogenous DNA is transported into the nucleus is poorly understood. By improving our understanding of how vectors circumvent the numerous cellular barriers to gene transfer, more efficient gene delivery methods can be devised. We have begun to design plasmid constructs that enter the nucleus of specific cell types in the absence of cell division, thereby enhancing levels of expression. We have shown that inclusion of specific DNA sequences in plasmid constructs mediates nuclear import both in vitro and in vivo. Here, we use plasmid affinity chromatography, mass spectrometry (MS), and live-cell pulldowns of transfected plasmid constructs to identify protein cofactors that interact in a sequence-specific manner with these DNA nuclear targeting sequences (DTSs). Importin β₁, importin 7, and the small guanosine triphosphatase Ran all demonstrate DTS-specific interaction in both MS and pull-down assays, consistent with our model of plasmid nuclear import. In addition, knockdown of importin β₁ with small interfering RNA (siRNA) abrogates plasmid nuclear import, indicating that it is a necessary cofactor. Our discovery that specific karyopherins mediate plasmid nuclear import can be used to design more effective vectors for gene delivery.     

RNAi-mediated CCR5 Silencing by LFA-1-targeted Nanoparticles Prevents HIV Infection in BLT Mice

RNA interference (RNAi)–mediated knockdown of gene expression offers a novel treatment strategy for human immunodeficiency virus (HIV) infection. However, the major hurdle for clinical use is a practical strategy for small interfering RNA (siRNA) delivery to the multiple immune cell types important in viral pathogenesis. We have developed a novel immunoliposome method targeting the lymphocyte function–associated antigen-1 (LFA-1) integrin expressed on all leukocytes and evaluated it for systemic delivery of siRNA in a humanized mouse model. We show that in vivo administration of the LFA-1 integrin–targeted and stabilized nanoparticles (LFA-1 I-tsNPs) results in selective uptake of siRNA by T cells and macrophages, the prime targets of HIV. Further, in vivo administration of anti-CCR5 siRNA/LFA-1 I-tsNPs resulted in leukocyte-specific gene silencing that was sustained for 10 days. Finally, humanized mice challenged with HIV after anti-CCR5 siRNA treatment showed enhanced resistance to infection as assessed by the reduction in plasma viral load and disease-associated CD4 T-cell loss. This study demonstrates the potential in vivo applicability of LFA-1-directed siRNA delivery as anti-HIV prophylaxis.     

Targeted Delivery of siRNA to Macrophages for Anti-inflammatory Treatment

Inflammation mediated by tumor necrosis factor-α (TNF-α) and the associated neuronal apoptosis characterizes a number of neurologic disorders. Macrophages and microglial cells are believed to be the major source of TNF-α in the central nervous system (CNS). Here, we show that suppression of TNF-α 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-α siRNA complexed to RVG-9dR achieved efficient silencing of LPS-induced TNF-α production by primary macrophages and microglia cells in vitro. Moreover, intravenous injection with RVG-9dR-complexed siRNA in mice reduced the LPS-induced TNF-α 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-α can potentially be used to suppress neuroinflammation in vivo.     

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.     

miR-29 Inhibits Bleomycin-induced Pulmonary Fibrosis in Mice

Loss of microRNA-29 (miR-29) is known to be a mechanism of transforming growth factor-β (TGF-β)-mediated pulmonary fibrosis, but the therapeutic implication of miR-29 for pulmonary fibrosis remains unexplored. The present study investigated whether miR-29 had therapeutic potential for lung disease induced by bleomycin in mice. In addition, the signaling mechanisms that regulated miR-29 expression were investigated in vivo and in vitro. We found that miR-29 was a downstream target gene of Smad3 and negatively regulated by TGF-β/Smad signaling in fibrosis. This was evidenced by the findings that mice or pulmonary fibroblasts null for Smad3 were protected against bleomycin or TGF-β1-induced loss of miR-29 along with fibrosis in vivo and in vitro. Interestingly, overexpression of miR-29 could in turn negatively regulated TGF-β and connective tissue growth factor (CTGF) expression and Smad3 signaling. Therefore, Sleeping Beauty (SB)-mediated miR-29 gene transfer into normal and diseased lung tissues was capable of preventing and treating pulmonary fibrosis including inflammatory macrophage infiltration induced by bleomycin in mice. In conclusion, miR-29 is negatively regulated by TGF-β/Smad3 and has a therapeutic potential for pulmonary fibrosis. SB-mediated miR-29 gene therapy is a non-invasive therapeutic strategy for lung disease associated with fibrosis.     

Co-delivery of anti-PLK-1 siRNA and camptothecin by nanometric polydiacetylenic micelles results in a synergistic cell killing

Recently, it has been shown that the efficiency of antitumoral drugs can be enhanced when combined with therapeutic siRNAs. In the present study, an original platform based on polydiacetylenic micelles containing a cationic head group able to efficiently deliver a small interfering RNA (siRNA) targeting the PLK-1 gene while offering a hydrophobic environment for encapsulation of lipophilic drugs such as camptothecin is developed. We demonstrate that the co-delivery of these two agents with our micellar system results in a synergistic tumor cell killing of cervical and breast cancer cell lines in vitro. The combined drugs are active in a subcutaneous in vivo cancer model. Altogether, the results show that our nanometric micellar delivery system can be used for the development of new drug–siRNA combo-therapies.

Recently, it has been shown that the efficiency of antitumoral drugs can be enhanced when combined with therapeutic siRNAs.