Safe siRNA Delivery Research Articles

Two-Photon Light Trigger siRNA Transfection of Cancer Cells Using Non-Toxic Porous Silicon Nanoparticles.

The concept of using two-photon excitation in the NIR for the spatiotemporal control of biological processes holds great promise. However, its use for the delivery of nucleic acids has been very scarcely described and the reported procedures are not optimal as they often involve potentially toxic materials and irradiation conditions. This work prepares a simple system made of biocompatible porous silicon nanoparticles (pSiNP) for the safe siRNA photocontrolled delivery and gene silencing in cells upon two-photon excitation. PSiNP are linked to an azobenzene moiety, which possesses a lysine group (pSiNP@ICPES-azo@Lys) to efficiently complex siRNA. Non-linear excitation of the two-photon absorber system (pSiNP) followed by intermolecular energy transfer (FRET) to trans azobenzene moiety, result in the photoisomerization of the azobenzene from trans to cis and in the destabilization of the azobenzene-siRNA complex, thus inducing the delivery of the cargo siRNA to the cytoplasm of cells. Efficient silencing in MCF-7 expressing stable firefly luciferase with siRNAluc against luciferase is observed. Furthermore, siRNA against inhibitory apoptotic protein (IAP) leads to over 70% of MCF-7 cancer cell death. The developed technique using two-photon light allows a unique high spatiotemporally controlled and safe siRNA delivery in cells in few seconds of irradiation.

MC3/SAINT-O-Somes, a novel liposomal delivery system for efficient and safe delivery of siRNA into endothelial cells

Increased understanding of chronic inflammatory diseases and the role of endothelial cell (EC) activation herein, have urged interest in sophisticated strategies to therapeutically intervene in activated EC to treat these diseases. Liposome-mediated delivery of therapeutic siRNA in inflammation-activated EC is such a strategy. In this study, we describe the design and characterisation of two liposomal siRNA delivery systems formulated with the cationic MC3 lipid or MC3/SAINT mixed lipids, referred to as MC3-O-Somes (MOS) and MC3/SAINT-O-Somes (MSS). The two formulations showed comparable physicochemical properties, except for better siRNA encapsulation efficiency in the MSS formulation. Antibody-mediated VCAM-1 targeting (AbVCAM-1) increased the association of the targeted MOS and MSS with activated EC, although the targeted MOS showed a significantly higher VCAM-1 specific association than the targeted MSS. AbVCAM-1 MSS containing RelA siRNA achieved significant downregulation of RelA expression, while AbVCAM-1 MOS containing RelA siRNA did not downregulate RelA expression in activated EC. Additionally, AbVCAM-1 MSS containing RelA siRNA showed low cytotoxicity in EC and at the same time prohibited endothelial inflammatory activation by reducing expression of cell adhesion molecules. The AbVCAM-1 MSS formulation is a novel siRNA delivery system based on a combination of the cationic lipids MC3 and SAINT, that shows good physicochemical characteristics, enhanced endothelial cell association, improved transfection activity, low toxicity and significant anti-inflammatory effect, thereby complying with the requirements for future in vivo investigations.

SiRNA Functionalized Lipid Nanoparticles (LNPs) in Management of Diseases.

RNAi (RNA interference)-based technology is emerging as a versatile tool which has been widely utilized in the treatment of various diseases. siRNA can alter gene expression by binding to the target mRNA and thereby inhibiting its translation. This remarkable potential of siRNA makes it a useful candidate, and it has been successively used in the treatment of diseases, including cancer. However, certain properties of siRNA such as its large size and susceptibility to degradation by RNases are major drawbacks of using this technology at the broader scale. To overcome these challenges, there is a requirement for versatile tools for safe and efficient delivery of siRNA to its target site. Lipid nanoparticles (LNPs) have been extensively explored to this end, and this paper reviews different types of LNPs, namely liposomes, solid lipid NPs, nanostructured lipid carriers, and nanoemulsions, to highlight this delivery mode. The materials and methods of preparation of the LNPs have been described here, and pertinent physicochemical properties such as particle size, surface charge, surface modifications, and PEGylation in enhancing the delivery performance (stability and specificity) have been summarized. We have discussed in detail various challenges facing LNPs and various strategies to overcome biological barriers to undertake the safe delivery of siRNA to a target site. We additionally highlighted representative therapeutic applications of LNP formulations with siRNA that may offer unique therapeutic benefits in such wide areas as acute myeloid leukaemia, breast cancer, liver disease, hepatitis B and COVID-19 as recent examples.

Influence of protein corona on the interaction of glycogen-siRNA constructs with ex vivo human blood immune cells.

Glycogen-nucleic acid constructs i.e., glycoplexes are emerging promising platforms for the alteration of gene expression and transcription. Understanding the interaction of glycoplexes with human blood components, such as serum proteins and peripheral blood mononuclear cells (PBMCs), is important to overcome immune cell activation and control biodistribution upon administration of the glycoplexes in vivo. Herein, we investigated the interactions of polyethylene glycol (PEG)ylated and non-PEGylated glycoplexes carrying siRNA molecules with PBMCs isolated from the blood of healthy donors. We found that both types of glycoplexes were non-toxic and were primarily phagocytosed by monocytes without triggering a pro-inflammatory interleukin 6 cytokine production. Furthermore, we investigated the role of the protein corona on controlling the internalization efficiency in immune cells - we found that the adsorption of serum proteins, in particular haptoglobin, alpha-1-antitrypsin and apolipoprotein A-II, onto the non-PEGylated glycoplexes, significantly reduced the uptake of the glycoplexes by PBMCs. Moreover, the non-PEGylated glycoplexes were efficient in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) knockdown in monocytic THP-1 cell line. This study provides an insight into the rational design of glycogen-based nanocarriers for the safe delivery of siRNA without eliciting unwanted immune cell activation and efficient siRNA activity upon its delivery.

The effects of fluoroalkyl chain length and density on siRNA delivery of bioreducible poly(amido amine)s

Fluorination is an attractive strategy for the improvement of transfection efficiency of nucleic acid delivery vectors. Bioreducible poly(amido amine)s (bPAAs) are an important class of biomaterials exhibited to effectively deliver multiple nucleic acids. However, still, the effects of fluoroalkyl chain length and density of bPAA on siRNA delivery are unveiled. Here, we synthesized bPAAs and grafted with different chain lengths and densities of fluorocarbon compounds. Furthermore, we prepared a library of complexes of fluorinated bPAA and siRNA, and investigated the effects of fluorination on the siRNA delivery in vitro and in vivo. We found that all the synthesized bPAAs readily formed complexes with siRNA and the fluorinated complexes considerably achieved improved gene silencing efficacies both in vitro and in vivo. Dramatically, the gene silencing efficacy was increased with increasing fluorine contents. Heptafluorobutyric anhydride (HF) modified bPAAs achieved better gene silencing efficacy when compared with bPAAs fluorinated by trifluoroacetic anhydride (TF) and pentafluoropropionic anhydride (PF) providing the evidence for choosing of best one among fluorocarbon compounds. In addition, a combination of fluorination with bioreducibility enables efficient and safe siRNA delivery.

PH-sensitive zwitterionic polycarboxybetaine as a potential non-viral vector for small interfering RNA delivery.

Small interfering RNA (siRNA) has great potential for the treatment of various diseases. However, its intrinsic deficiencies seriously limit its application. Herein, pH-sensitive zwitterionic polymer polycarboxybetaine (PCB) was developed as a non-viral vector for siRNA. The PCB could be protonated in an acidic environment and become positively charged from a cancer site. After protonation, PCB could complex siRNA via electrostatic interaction, and its loading ability was enhanced with a decrease of pH value. Compared with the PEI 10k, PCB50 with a similar molecular weight had comparable siRNA loading ability and lower cytotoxicity. Besides, siRNA loaded by PCB50 could escape from endosomes and reduce the loss of drugs, and based on the excellent uptake and obvious apoptotic effect on HeLa cells, the pH-sensitive PCB with low cytotoxicity could be used as a non-viral vector for safe siRNA delivery for cancer treatment.

Lipid nanoparticle-mediated siRNA delivery for safe targeting of human CML in vivo.

Efficient and safe delivery of siRNA in vivo is the biggest roadblock to clinical translation of RNA interference (RNAi)-based therapeutics. To date, lipid nanoparticles (LNPs) have shown efficient delivery of siRNA to the liver; however, delivery to other organs, especially hematopoietic tissues still remains a challenge. We developed DLin-MC3-DMA lipid-based LNP-siRNA formulations for systemic delivery against a driver oncogene to target human chronic myeloid leukemia (CML) cells in vivo. A microfluidic mixing technology was used to obtain reproducible ionizable cationic LNPs loaded with siRNA molecules targeting the BCR-ABL fusion oncogene found in CML. We show a highly efficient and non-toxic delivery of siRNA in vitro and in vivo with nearly 100% uptake of LNP-siRNA formulations in bone marrow of a leukemic model. By targeting the BCR-ABL fusion oncogene, we show a reduction of leukemic burden in our myeloid leukemia mouse model and demonstrate reduced disease burden in mice treated with LNP-BCR-ABL siRNA as compared with LNP-CTRL siRNA. Our study provides proof-of-principle that fusion oncogene specific RNAi therapeutics can be exploited against leukemic cells and promise novel treatment options for leukemia patients.

Biomimetic Dextran-Peptide Vectors for Efficient and Safe siRNA Delivery.

Small interfering RNA (siRNA)-based therapeutics have the potential to treat a series of hereditary and acquired diseases. However, one serious obstacle for siRNA therapy is the lack of an efficient strategy to transport the siRNA to the targeted organ/cell with minimal toxicity. To take advantage of the good biocompatibility and degradability of natural polymers, and to understand how the peptide sequence affects the properties of the vector, four biomimetic vectors (D10-K3H7, D10-R3H7, D20-K3H7, and D20-R3H7) were designed and synthesized by conjugating the peptide (K3H7 or R3H7) and dextran with a molecular weight of 10 or 20 kDa. Taking the commercial cellular transfection reagent Lipofectime RNAiMAX as a control, dextran-peptide/siRNA complexes exhibited smaller particle sizes, lower ζ potentials, and lower toxicity with the same value of N/P ratio. To evaluate the potential of this system for therapeutics, siRNA targeting the mRNA of the PCSK9 gene was chosen as a gene drug model to knock down the PCSK9 expression in the HepG2 cell line. Dextran-peptide/siRNA complexes exhibit a more consistent and higher knockdown efficiency than Lipofectamine RNAiMAX/PCSK9 siRNA complexes in a medium with 20% fetal bovine serum (FBS). D20-R3H7/PCSK9 siRNA complexes could knock down the level of PCSK9 mRNA by 85.2%, and they demonstrated a higher efficiency than Lipofectamine RNAiMAX, having 70.5% knockdown in the medium with 20% FBS at the PCSK9 siRNA concentration of 100 nM. These results suggest that the dextran-peptide-based vector has more efficient therapeutic agent properties for a siRNA-based drug transporter.

Magnetically responsive hybrid nanoparticles for in vitro siRNA delivery to breast cancer cells

Short interfering RNA (siRNA) showed to be a viable alternative to a better prognosis in cancer therapy. Nevertheless, the successful application of this strategy still depends on the development of nanocarriers for the safe delivery of siRNA into the diseased tissue, which mostly occurs by passive accumulation. When an external magnetic field is applied, magnetic nanoparticles biodistribution is partially modulated to favor accumulation in a target tissue. In this work we designed a novel magnetic responsive siRNA nanocarrier. The new delivery system is composed of superparamagnetic iron oxide nanoparticles (SPIONs) coated with calcium phosphate (CaP) and PEG-polyanion block copolymers, which are known to be biocompatible. The nanoparticles presented rounded shape with small size and narrow distribution suitable for biomedical applications. TEM images showed dark spheres in the core surrounded by a lower electron density material in the corona. The X-ray photoelectron spectra (XPS) confirmed CaP-polymer coating of the magnetic core. In addition, the coating procedure did not affect the superparamagnetic property as showed using a vibrating sample magnetometer (VSM). With a high loading efficiency (80%), the nanoparticles enhanced vascular endothelium growth factor (VEGF) silencing in breast cancer cells in vitro, at gene and protein levels (~60% and 40%, respectively), without associated toxicity. Iron and siRNA quantification showed that the novel nanoparticles move towards a magnetic source carrying siRNA molecules. Therefore, these novel nanoparticles are a promising tool for cancer therapy based on RNAi effect, added by a magnetic capability to further modulate siRNA accumulation in the target tissue.

The development of tertiary amine cationic lipids for safe and efficient siRNA delivery.

Cationic liposomes have shown great potential in efficient siRNA delivery, and their positive charge is crucial for tight extracellular siRNA binding, effective intracellular siRNA disassembly and physiological toxicity. Thus, the development of novel cationic lipids with a suitable positive charge is desirable for safe and efficient siRNA delivery. Herein, we fabricated a library of 21 tertiary amine-derived cationic lipids (TA) to achieve a balance between effectiveness and safe siRNA delivery. The screened TA13 liposomes, which consisted of TA13 and helper lipid DOPE at a mole ratio of 1 : 1, readily condensed siRNA to form lipoplexes (TA13 LPs), achieving stronger gene silencing in diverse cells than the commercially available vector Lipo2000. Moreover, the TA13 LPs demonstrated effective in vivo gene silencing and good safety in normal mice. The improved gene silencing efficiency of the TA13 LPs is ascribed to their capability of sequentially conquering the barriers met by in vivo siRNA delivery. Notably, the TA13 LPs delivered ApoB-siRNA and obviously decreased ApoB mRNA expression in the liver and the total cholesterol and low-density lipoprotein in the serum of hypercholesterolemia mice, indicating a potential siRNA therapeutic for hypercholesterolemia treatment. It is anticipated that these novel tertiary amine-based liposomes can provide a simple and widely-used platform for the safe and effective delivery of siRNA, and their structure-activity relationships can aid in the further development of effective cationic lipids.

Targeted siRNA delivery using aptamer‐siRNA chimeras and aptamer‐conjugated nanoparticles

The sequence-specific gene-silencing ability of small interfering RNA (siRNA) has been exploited as a new therapeutic approach for the treatment of a variety of diseases. However, efficient and safe delivery of siRNA into target cells is still a challenge in the clinical development of siRNA-based therapeutics. Recently, nucleic acid-based aptamers that target cell surface proteins have emerged as a new class of targeting moieties due to their high specificity and avidity. To date, various aptamer-mediated siRNA delivery systems have been developed to enhance the RNA interference (RNAi) efficacy of siRNA via targeted delivery. In this review, we summarize recent advances in developing aptamer-mediated siRNA delivery systems for RNAi therapeutics, mainly aptamer-siRNA chimeras and aptamer-functionalized nanocarriers incorporating siRNA, with a focus on their molecular designs and formulations. In addition, the challenges and engineering strategies of aptamer-mediated siRNA delivery systems for clinical translation are discussed. This article is categorized under: Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Development of fluorinated polyplex nanoemulsions for improved small interfering RNA delivery and cancer therapy

We report the development of a small interfering RNA (siRNA) delivery vector based on cationic perfluorocarbon nanoemulsions. We have prepared perfluorodecalin (PFD) emulsions with a positive surface charge provided by a fluorinated poly(ethylenimine) (F-PEI). The fluorinated emulsion (F-PEI@PFD) reduced cytotoxicity of F-PEI and demonstrated effective binding with siRNAs to form nanosized emulsion polyplexes. The prepared emulsion polyplexes enhanced cellular uptake and improved endosomal escape of the siRNA. In addition to increased reporter gene silencing in multiple cancer cell lines, when compared with control F-PEI and PEI polyplexes, the siRNA emulsion polyplexes showed an excellent resistance to serum deactivation and maintained high activity, even in high-serum conditions. The F-PEI@PFD emulsion polyplexes carrying an siRNA to silence the expression of Bcl2 gene induced apoptosis and inhibited tumor growth in a melanoma mouse model in vivo and showed potential for in vivo ultrasound imaging. This study demonstrates the potential of F-PEI@PFD emulsions as a multifunctional theranostic nanoplatform for safe siRNA delivery, with integrated ultrasound imaging functionality.

Highly Efficient and Safe Delivery of VEGF siRNA by Bioreducible Fluorinated Peptide Dendrimers for Cancer Therapy.

RNA interference (RNAi) has a great promise in treating various acquired and hereditary diseases. However, it remains highly desirable to develop new delivery system to circumvent complex extra- and intracellular barriers for successful clinical translation. Here, we report on a versatile polymeric vector, bioreducible fluorinated peptide dendrimers (BFPD), for efficient and safe small interfering RNA (siRNA) delivery. In virtue of skillfully integrating all of the unique advantages of reversible cross-linking, fluorination, and peptide dendrimers, this novel vector can surmount almost all extra- and intracellular barriers associated with local siRNA delivery through highly improved physiological stability and serum resistance, significantly increased intratumoral enrichment, cellular internalization, successful facilitation of endosomal escape, and cytosolic siRNA release. BFPD polyplexes, carrying small interfering vascular endothelial growth factor (siVEGF), demonstrated excellent VEGF silencing efficacy (∼65%) and a strong capability for inhibiting HeLa cell proliferation. More importantly, these polyplexes showed superior performance in long-term enrichment in the tumor sites and had a high level of tumor growth inhibition. Furthermore, these polyplexes not only exhibited excellent in vivo antitumor efficacy but also demonstrated superior biocompatibility, compared with LPF2000, both in vivo and in vitro. These findings indicate that BFPD is an efficient and safe siRNA delivery system and has remarkable potential for RNAi-based cancer treatment.

Relationship Between the Physicochemical Properties of Lipid Nanoparticles and the Quality of siRNA Delivery to Liver Cells

While a variety of short interfering RNA (siRNA) delivery compounds have been developed, a deep understanding of the key parameters that determine the quality of siRNA delivery are not known with certainty. Therefore, an understanding of the factors required for the efficient, selective, and safe delivery of siRNA is a great challenge for successful siRNA delivery. Herein, we report on the development of two pH-sensitive cationic lipids and their use in examining the impact of the acid dissociation constant (pKa) value, lipase sensitivity and the size of lipid nanoparticles on the biodistribution, and efficiency and cell specificity of gene silencing in the liver. An increase in the pKa value resulted in a significant change in the intrahepatic localization of siRNA and gene-silencing efficiency in hepatocytes and liver sinusoidal endothelial cells (LSECs). The sensitivity of the pH-sensitive cationic lipid to lipases was a major factor in achieving hepatocyte-specific gene silencing. Increasing the particle size can improve the LSEC specificity of gene silencing. As a consequence, we succeeded in developing both a highly efficient, hepatocyte-specific formulation, and the most efficacious LSEC-targeted formulation reported to date. These findings will facilitate the development of more sophisticated siRNA delivery systems.

Receptor‐targeted liposome‐peptide‐siRNA nanoparticles represent a novel and efficient siRNA delivery system to prevent conjunctival fibrosis

PurposeGlaucoma is the leading cause of irreversible blindness worldwide and fibrosis is the main cause of failure of glaucoma surgery. We have previously described how the Myocardin‐related transcription factor/Serum response factor (MRTF/SRF) pathway is intricately linked to all the key pathways in ocular fibrosis. Our aim was to develop a novel liposome‐peptide‐siRNA (LYR) nanoparticle as an efficient delivery system for MRTF siRNA in conjunctival fibrosis.MethodsThe LYR nanoparticles were characterised with regard to particle size and zeta potential. Real‐time qPCR and western blotting were used to compare the silencing efficiency in human Tenon's fibroblasts using different MRTF siRNA concentrations, targeting peptides, and liposomes. The cytotoxicity of the LYR nanoparticles was assessed using the MTT cell assay. Three‐dimensional fibroblast‐populated collagen matrices were also used as a functional assay to measure contraction in vitro.ResultsAll LYR nanoparticles were strongly cationic with sizes around 100 nm and PDIs < 0.4. The LYR nanoparticles efficiently silenced the MRTF gene by 76% and 84% using 50 nM and 100 nM siRNA respectively. The MRTF gene was also efficiently silenced by 76% and 75% using the targeting peptides Y and ME27 respectively. The MRTF protein expression was significantly decreased by the LYR nanoparticles. The non‐PEGylated liposome formulations showed higher silencing efficiency than the cationic PEGylated formulations. The MRTF nanoparticles were also not cytotoxic at 50 nM siRNA concentration and prevented matrix contraction after a single transfection treatment.ConclusionsThis is the first study to show that receptor‐targeted liposome‐peptide‐siRNA nanoparticles represent an efficient and safe siRNA delivery system that could be used to prevent fibrosis after glaucoma surgery.

Receptor‐targeted liposome‐peptide‐siRNA nanoparticles represent a novel and efficient siRNA delivery system to prevent conjunctival fibrosis

PurposeGlaucoma is the leading cause of irreversible blindness worldwide and fibrosis is the main cause of failure of glaucoma surgery. We have previously described how the Myocardin‐related transcription factor/Serum response factor (MRTF/SRF) pathway is intricately linked to all the key pathways in ocular fibrosis. Our aim was to develop a novel liposome‐peptide‐siRNA (LYR) nanoparticle as an efficient delivery system for MRTF siRNA in conjunctival fibrosis.MethodsThe LYR nanoparticles were characterised with regard to particle size and zeta potential. Real‐time qPCR and western blotting were used to compare the silencing efficiency in human Tenon's fibroblasts using different MRTF siRNA concentrations, targeting peptides, and liposomes. The cytotoxicity of the LYR nanoparticles was assessed using the MTT cell assay. Three‐dimensional fibroblast‐populated collagen matrices were also used as a functional assay to measure contraction in vitro.ResultsAll LYR nanoparticles were strongly cationic with sizes around 100 nm and PDIs < 0.4. The LYR nanoparticles efficiently silenced the MRTF gene by 76% and 84% using 50 nM and 100 nM siRNA respectively. The MRTF gene was also efficiently silenced by 76% and 75% using the targeting peptides Y and ME27 respectively. The MRTF protein expression was significantly decreased by the LYR nanoparticles. The non‐PEGylated liposome formulations showed higher silencing efficiency than the cationic PEGylated formulations. The MRTF nanoparticles were also not cytotoxic at 50 nM siRNA concentration and prevented matrix contraction after a single transfection treatment.ConclusionsThis is the first study to show that receptor‐targeted liposome‐peptide‐siRNA nanoparticles represent an efficient and safe siRNA delivery system that could be used to prevent fibrosis after glaucoma surgery.

Improved islet transplantation outcome by the co-delivery of siRNAs for iNOS and 17β-estradiol using an R3V6 peptide carrier

Silencing target genes such as inducible nitric oxide synthase (iNOS) using small interfering double-stranded RNA (siRNA) in islet cells has been attempted to enhance the survival of transplanted islets. However, the efficient and safe delivery of siRNA into intact islets is challenging. Here, we prepared R3V6 peptides containing a three-arginine stretch and a six-valine stretch, which form micelles with hydrophobic valine cores and cationic arginine surfaces in aqueous solution, to co-deliver siRNA and cytoprotective hydrophobic drugs to islet cells. The cationic surfaces bound to the negatively charged iNOS siRNA, and the hydrophobic core was loaded with 17β-estradiol (E2), which exerts anti-apoptotic effects on the islet cells. The E2-loaded R3V6 peptide micelles delivered siRNA-5′-FITC to mouse islets more efficiently than did Lipofectamine 2000, and without cytotoxicity. The micelle complexes containing siRNA-iNOS knocked down the expression of iNOS mRNA by >60% in islets and reduced cytokine-induced apoptotic cell death in vitro significantly. The delivery of siRNA-iNOS and E2 simultaneously using E2-loaded R3V6 peptide micelles improved the diabetes reversal rate of marginal mass islet transplantation into the renal subcapsular space of diabetic syngeneic mice significantly compared with the siRNA-iNOS and E2 alone treatment control groups. Our results demonstrated that the co-delivery of siRNA and a cytoprotective drug within a single non-toxic carrier, R3V6 peptide micelles, provides a novel rational strategy for combined ex vivo islet therapy to improve the outcome of islet transplantation.

Nanocarriers for siRNA delivery to overcome cancer multidrug resistance

Multidrug resistance (MDR) is an extremely complexed phenomenon in tumor chemotherapy and is one of the major obstacles for successful treatment. Discovery of RNA interference (RNAi) offers a new strategy with great potential to reverse MDR. Specific genes, which contribute to the emerging of MDR, can be silenced by RNAi. However, many natural barriers have to be overcome for efficient and safe delivery of siRNA. In recent years, the various kinds of nanocarriers, such as liposomes, cationic polymers, and inorganic materials, have been developed to deliver siRNA and show good results in reversing MDR. This review mainly summarizes the barriers in siRNA delivery and recent progress in designing nanotechnology-based siRNA delivery systems to overcome tumor MDR.

Self-assembling Modified β-Cyclodextrin Nanoparticles as Neuronal siRNA Delivery Vectors: Focus on Huntington’s Disease

Huntington's disease (HD) is a rare autosomal dominant neurodegenerative disease caused by the expression of a toxic Huntingtin (HTT) protein. The use of short interfering RNAs (siRNAs) to silence the mutant protein is one of the most promising therapeutic strategies under investigation. The biggest caveat to siRNA-based approaches is the lack of efficient and nontoxic delivery vectors for siRNA delivery to the central nervous system. In this study, we investigated the potential of modified amphiphilic β-cyclodextrins (CDs), oligosaccharide-based molecules, as novel siRNA neuronal carriers. We show that CDs formed nanosize particles which were stable in artificial cerebrospinal fluid. Moreover, these complexes were able to reduce the expression of the HTT gene in rat striatal cells (ST14A-HTT120Q) and in human HD primary fibroblasts. Only limited toxicity was observed with CD·siRNA nanoparticles in any of the in vitro models used. Sustained knockdown effects were observed in the striatum of the R6/2 mouse model of HD after single direct injections of CD·siRNA nanoparticles. Repeated brain injections of CD·siRNA complexes resulted in selective alleviation of motor deficits in this mouse model. Together these data support the utility of modified β-CDs as efficient and safe siRNA delivery vectors for RNAi-based therapies for neuropsychiatric and neurodegenerative disorders.

Efficient and safe delivery of siRNA using anionic lipids: Formulation optimization studies

Novel formulations based on physiologically occurring anionic lipids have been designed to achieve safe and efficient siRNA delivery. Anionic liposomes (DOPG/DOPE) were complexed with siRNA using calcium ion bridges to prepare anionic lipoplexes. Various formulation parameters (liposome composition, lipid and calcium concentration) were evaluated and optimized to achieve efficient silencing and high cell viability in breast cancer cells. The optimal anionic lipoplexes composed of 1μg/mL lipid (40:60 (DOPG/DOPE m/m)), 2.4mM calcium and 10nM siRNA, showed maximum silencing (∼70% knockdown) without being cytotoxic. These lipoplexes also showed stability and high efficiency in the presence of serum. Additionally, optimal anionic lipoplexes showed efficient intracellular uptake and endosomal escape. Characterization studies indicated the optimal anionic formulations were 324.2±19.6nm with a surface charge of (−22.9±0.1)mV and 98.5±1.4% encapsulation efficiency. Control cationic lipoplexes (Lipofectamine 2000) showed silencing comparable to the anionic lipoplexes but were highly cytotoxic as indicated by IC50 values (cationic – 22.9μg/mL, compared to anionic – greater than 107μg/mL). Calcium–siRNA complexes (without liposomes) showed low efficiency (∼50% silencing), and highly variable results. The optimized anionic formulations may offer a safer alternative to conventional cationic based systems for efficient in vitro as well as in vivo delivery of therapeutic siRNAs.