Reducible siRNA dimeric conjugates for efficient cellular uptake and gene silencing

In this study, dimerized siRNAs linked by a cleavable disulfide bond were synthesized for efficient intracellular delivery and gene silencing. The reducible dimerized siRNAs showed far enhanced complexation behaviors with cationic polymers as compared to monomeric siRNA at the same N/P ratio, as demonstrated by microscopic techniques and gel characterization. Dimerized siRNAs targeting green fluorescent protein (GFP) or vascular endothelial growth factor (VEGF) were complexed with linear polyethylenimine (LPEI), and treated to various cell lines to examine gene transfection efficiencies. In comparison to monomer siRNA/LPEI complexes, dimeric siRNA/LPEI complexes showed greatly enhanced cellular uptake and gene silencing effects in vitro. These results were mainly due to the higher charge density and promoted chain flexibility of the dimerized siRNAs, providing more compact and stable siRNA complexes. In addition, the conjugation strategy of reducible siRNA dimers was further extended: poly(ethylene glycol) (PEG)-modified dimerized siRNAs and heterodimers of siRNAs targeting two different genes (e.g., GFP and VEGF) were synthesized, and their gene silencing efficiencies were characterized. The dimerized siRNA complex system holds great potential for in vivo systemic gene therapy.     

Self-crosslinked and reducible fusogenic peptides for intracellular delivery of siRNA

A novel self-crosslinked and reducible peptide was synthesized for stable formation of nanoscale complexes with an siRNA-PEG conjugate to enhance transfection efficiency in serum containing condition without compromising cytotoxicity. A fusogenic peptide, KALA, with two cysteine residues at both terminal ends was crosslinked via disulfide linkages under mild DMSO oxidation condition. The reducible crosslinked KALA (cl-KALA) was used to form nano-complexes with green fluorescent protein (GFP) siRNA. Size and morphology of various polyelectrolyte complexes formulated with KALA and cl-KALA were comparatively analyzed. cl-KALA exhibited more reduced cell cytotoxicity and formed more stable and compact polyelectrolyte complexes with siRNA, compared with naked KALA and polyethylenimine (PEI), probably because of its increased charge density. The extent of gene silencing was quantitatively evaluated using MDA-MB-435 cells. cl-KALA/siRNA complexes showed comparable gene silencing efficiency with those of cytotoxic PEI. In a serum containing medium, cl-KALA/siRNA-PEG conjugate complexes exhibited superior gene inhibition because of the shielding effect of PEG on the surface. The formulation based on the self-crosslinked fusogenic peptide could be used as a biocompatible and efficient nonviral carrier for siRNA delivery. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 881-888, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Controlled delivery of plasmid DNA and siRNA to intracellular targets using ketalized polyethylenimine

A new polyethylenimine (PEI)-derived biodegradable polymer was synthesized as a nonviral gene carrier. Branches of PEI were ketalized, and capabilities of nucleic acid condensation and delivery efficiency of the modified polymers were compared with ones of unketalized PEI. Ketalized PEI was able to efficiently compact both plasmid DNA and siRNA into nucleic acids/ketalized PEI polyplexes with a range of 80-200 nm in diameter. Nucleic acids were efficiently dissociated from the polyplexes made of ketalized PEI upon hydrolysis. In vitro study also demonstrated that ketalization enhanced transfection efficiency of the polyplexes while reducing cytotoxicity, even at high N/ P ratios. Interestingly, transfection efficiency was found to be inversely proportional to molecular weights of ketalized PEI, while RNA interference was observed in the opposite way. This study implies that selective delivery of plasmid DNA and siRNA to the nucleus and the cytoplasm can be achieved by tailoring the structures of polymeric gene carriers.     

siRNA delivery by a transferrin-associated lipid-based vector: a non-viral strategy to mediate gene silencing

BACKGROUND: RNA interference provides a powerful technology for specific gene silencing. Therapeutic applications of small interfering RNA (siRNA) however require efficient vehicles for stable complexation, protection, and extra- and intracellular delivery of these nucleic acids. Here, we evaluated the potential of transferrin (Tf)-associated liposomes for siRNA complexation and gene silencing. METHODS: Cationic liposomes composed of DOTAP : Cholesterol associated with or without transferrin (Tf) were complexed with siRNA at different lipid/siRNA charge ratios. Complexation and protection of siRNA from enzymatic degradation was assessed with the PicoGreen intercalation assay and gel electrophoresis. Cellular internalization of these siRNA Tf-lipoplexes was detected by confocal microscopy. Luciferase assay, immunoblot and fluorescence-activated cell sorting (FACS) analysis were used to evaluate reporter gene silencing in Huh-7 hepatocarcinoma and U-373 glioma cells. c-Jun knockdown in HT-22 cells was evaluated by quantitative real-time polymerase chain reaction (RT-PCR). Cytotoxicity of the siRNA complexes was assessed by Alamar blue, lactate dehydrogenase and MTT assays. RESULTS: Complexation of siRNA with the cationic liposomes in the presence of Tf results in the formation of stable particles and prevents serum-mediated degradation. Confocal microscopy showed fast cellular internalization of the Tf-lipoplexes via endocytosis. In the GFP glioma cells Tf-lipoplexes showed enhanced gene silencing at minimum toxicity in comparison to Tf-free lipoplexes. Targeting luciferase in the hepatocarcinoma cell line resulted in more than 70% reduction of luciferase activity, while in HT-22 cells 50% knockdown of endogenous c-Jun resulted in a significant protection from glutamate-mediated toxicity. CONCLUSIONS: Cationic liposomes associated with Tf form stable siRNA lipoplexes with reduced toxicity and enhanced specific gene knockdown activity compared to conventional lipoplexes. Thus, such formulations may constitute efficient delivery systems for therapeutic siRNA applications.     

Gene silencing activity of siRNA polyplexes based on thiolated N,N,N-trimethylated chitosan

N,N,N-Trimethylated chitosan (TMC) is a biodegradable polymer emerging as a promising nonviral vector for nucleic acid and protein delivery. In the present study, we investigated whether the introduction of thiol groups in TMC enhances the extracellular stability of the complexes based on this polymer and promotes the intracellular release of siRNA. The gene silencing activity and the cellular cytotoxicity of polyplexes based on thiolated TMC were compared with those based on the nonthiolated counterpart and the regularly used lipidic transfection agent Lipofectamine. Incubation of H1299 human lung cancer cells expressing firefly luciferase with siRNA/thiolated TMC polyplexes resulted in 60-80% gene silencing activity, whereas complexes based on nonthiolated TMC showed less silencing (40%). The silencing activity of the complexes based on Lipofectamine 2000 was about 60-70%. Importantly, the TMC-SH polyplexes retained their silencing activity in the presence of hyaluronic acid, while nonthiolated TMC polyplexes hardly showed any silencing activity, demonstrating their stability against competing anionic macromolecules. Under the experimental conditions tested, the cytotoxicity of the thiolated and nonthiolated siRNA complexes was lower than those based on Lipofectamine. Given the good extracellular stability and good silencing activity, it is concluded that polyplexes based on TMC-SH are attractive systems for further in vivo evaluations.     

Multimerized siRNA cross-linked by gold nanoparticles

In this study, siRNAs terminated with thiol groups were multimerized and cross-linked using ～5 nm gold nanoparticles (AuNPs) via Au-S chemisorption that can be intracellularly reduced. AuNPs immobilized with single-stranded antisense siRNA were assembled with those with single-stranded sense siRNA via complementary hybridization or assembled with those with single-stranded dimeric sense siRNA. The multimerized siRNA cross-linked by AuNPs showed increased charge density and enhanced enzymatic stability, and exhibited good complexation behaviors with a polycationic carrier, linear polyethylenimine (L-PEI). The resultant multi-siRNA/AuNPs/L-PEI polyelectrolyte complexes exhibited far greater gene silencing efficiencies of green fluorescent protein (GFP) and vascular endothelial growth factor (VEGF) compared to naked siRNA complexes. They could also be visualized by micro-CT imaging. The results suggest that AuNP-mediated multimerization of siRNAs could be a rational approach to achieve both gene silencing and imaging at a target tissue simultaneously.     

Antisense oligodeoxynucleotide-conjugated hyaluronic acid/protamine nanocomplexes for intracellular gene inhibition

Green fluorescent protein (GFP) antisense oligodeoxynucleotide (ODN) was covalently conjugated to hyaluronic acid (HA) via a reducible disulfide linkage, and the HA-ODN conjugate was complexed with protamine to increase the extent of cellular uptake and enhance the gene inhibition efficiency of GFP expression. The HA-ODN conjugate formed more stable polyelectrolyte complexes with protamine as compared to naked ODN, probably because of its increased charge density. The higher cellular uptake of protamine/HA-ODN complexes than that of protamine/naked ODN complexes was attributed to the formation of more compact nanosized complexes (approximately 200 nm in diameter) in aqueous solution. Protamine/HA-ODN complexes also showed a comparable level of GFP gene inhibition to that of cytotoxic polyethylenimine (PEI)/ODN complexes. Since both HA and protamine are naturally occurring biocompatible materials, the current formulation based on a cleavable conjugation strategy of ODN to HA could be potentially applied as safe and effective nonviral carriers for ODN and siRNA nucleic acid therapeutics.     

Effective Non-Viral Delivery of siRNA to Acute Myeloid Leukemia Cells with Lipid-Substituted Polyethylenimines

Use of small interfering RNA (siRNA) is a promising approach for AML treatment as the siRNA molecule can be designed to specifically target proteins that contribute to aberrant cell proliferation in this disease. However, a clinical-relevant means of delivering siRNA molecules must be developed, as the cellular delivery of siRNA is problematic. Here, we report amphiphilic carriers combining a cationic polymer (2 kDa polyethyleneimine, PEI2) with lipophilic moieties to facilitate intracellular delivery of siRNA to AML cell lines. Complete binding of siRNA by the designed carriers was achieved at a polymer:siRNA ratio of ～0.5 and led to siRNA/polymer complexes of ～100 nm size. While the native PEI2 did not display cytotoxicity on AML cell lines THP-1, KG-1 and HL-60, lipid-modification on PEI2 slightly increased the cytotoxicity, which was consistent with increased interaction of polymers with cell membranes. Cellular delivery of siRNA was dependent on the nature of lipid substituent and the extent of lipid substitution, and varied among the three AML cell lines used. Linoleic acid-substituted polymers performed best among the prepared polymers and gave a siRNA delivery equivalent to better performing commercial reagents. Using THP-1 cells and a reporter (GFP) and an endogenous (CXCR4) target, effective silencing of the chosen targets was achieved with 25 to 50 nM of siRNA concentrations, and without adversely affecting subsequent cell growth. We conclude that lipid-substituted PEI2 can serve as an effective delivery of siRNA to leukemic cells and could be employed in molecular therapy of leukemia.     

RNA interference-mediated simultaneous silencing of four genes using cross-shaped RNA

The structural flexibility of RNA interference (RNAi)-triggering nucleic acids suggests that the design of unconventional RNAi trigger structures with novel features is possible. Here, we report a cross-shaped RNA duplex structure, termed quadruple interfering RNA (qiRNA), with multiple target gene silencing activity. qiRNA triggers the simultaneous down-regulation of four cellular target genes via an RNAi mechanism. In addition, qiRNA shows enhanced intracellular delivery and target gene silencing over conventional siRNA when complexed with jetPEI, a linear polyethyleneimine (PEI). We also show that the long antisense strand of qiRNA is incorporated intact into an RNA-induced silencing complex (RISC). This novel RNA scaffold further expands the repertoire of RNAi-triggering molecular structures and could be used in the development of therapeutics for various diseases including viral infections and cancer.     

基于小鼠MMP-9基因的siRNA的筛选

通过GenBank数据库检索,基于小鼠MMP-9基因设计特异性的siRNA干扰靶点,克隆至pGCsi-U6/Neo/GFP载体中,使用PEI衍生物包裹,转染小鼠黑色素瘤B16细胞,流式细胞仪和激光扫描共聚焦显微镜分析细胞转染情况,RT-PCR检测24,48 h后MMP-9基因转录水平变化,筛选最佳的重组质粒和干扰靶点。结果显示：成功设计和构建了3个MMP-9-siRNA干扰质粒,3个重组质粒对B16细胞的转染率分别为60.04%、63.93%和56.27%,且3个重组质粒均能有效干扰B16细胞MMP-9 mRNA的表达,其中MMP-9-siRNA-2干扰效率最高（63%）,可持续干扰MMP-9基因表达。这些结果提示,MMP-9-siRNA-2为沉默小鼠黑色素瘤细胞MMP-9基因最优的siRNA重组质粒。     

Multifunctional siRNA delivery system: Polyelectrolyte complex micelles of six‐arm PEG conjugate of siRNA and cell penetrating peptide with crosslinked fusogenic peptide

For therapeutic applications of small interfering RNA (siRNA), serum stability, enhanced cellular uptake, and facile endosome escape are key issues for designing carriers. In this study, green fluorescent protein (GFP) siRNA was conjugated to a six‐arm polyethylene glycol (PEG) derivative via a reducible disulfide linkage (6PEG‐siRNA). The 6PEG‐siRNA conjugate was also functionalized with a cell penetrating peptide, Hph1 to enhance its cellular uptake property (6PEG‐siRNA‐Hph1). The 6PEG‐siRNA‐Hph1 conjugate was electrostatically complexed with cationic self‐crosslinked fusogenic KALA peptide (cl‐KALA) to form multifunctional polyelectrolyte complex micelles for gene silencing. The resultant siRNA complex formulation with multiple PEG chains showed superior physical stability and resistance to enzymatic degradation. The 6PEG‐siRNA‐Hph1/cl‐KALA complexes exhibited enhanced GFP gene silencing efficiency for MDA‐MB‐435 cells in the serum containing condition. The current reducible and multifunctional polyelectrolyte complex micelles are expected to have high potential for efficient delivery of therapeutic siRNA. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Polycation liposome-mediated gene transfer in vivo

The polycation liposome (PCL), a recently developed gene transfer system, is simply prepared by a modification of liposomes with cetylated polyethylenimine (PEI), and shows remarkable transgene efficiency with low cytotoxicity. In the present study, we investigated the applicability of PCLs for in vivo gene transfer, since the PCL-mediated transgene efficiency was found to be maintained in the presence of serum. PCLs composed of dioleoylphosphatidylethanolamine (DOPE) with 5 mol% cetyl PEI (PEI average mr. wt. 1800), were superior for transfection to those of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (2:1 as molar ratio) with 5 mol% cetyl PEI in vitro, although the latter PCLs were more efficient for gene transfer in vivo. PCL-DNA complexes were injected into mice via a tail or the portal vein, with the DNA being a plasmid encoding green fluorescent protein (GFP) or luciferase; and the expression was monitored qualitatively or quantitatively, respectively. Tail vein injection resulted in high expression of both GFP and luciferase genes in lung, and portal vein injection resulted in high expression of both genes in the liver. Concerning the gene delivery efficiency, the PCL was found to be superior to PEI or cetyl PEI alone. The optimal conditions for in vivo transfection with PCLs were also examined.     

Polycation liposome-mediated gene transfer in vivo

The polycation liposome (PCL), a recently developed gene transfer system, is simply prepared by a modification of liposomes with cetylated polyethylenimine (PEI), and shows remarkable transgene efficiency with low cytotoxicity. In the present study, we investigated the applicability of PCLs for in vivo gene transfer, since the PCL-mediated transgene efficiency was found to be maintained in the presence of serum. PCLs composed of dioleoylphosphatidylethanolamine (DOPE) with 5 mol% cetyl PEI (PEI average mr. wt. 1800), were superior for transfection to those of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (2:1 as molar ratio) with 5 mol% cetyl PEI in vitro, although the latter PCLs were more efficient for gene transfer in vivo. PCL-DNA complexes were injected into mice via a tail or the portal vein, with the DNA being a plasmid encoding green fluorescent protein (GFP) or luciferase; and the expression was monitored qualitatively or quantitatively, respectively. Tail vein injection resulted in high expression of both GFP and luciferase genes in lung, and portal vein injection resulted in high expression of both genes in the liver. Concerning the gene delivery efficiency, the PCL was found to be superior to PEI or cetyl PEI alone. The optimal conditions for in vivo transfection with PCLs were also examined.     

Delivery of siRNA using hyaluronic acid-guided nanoparticles for downregulation of CXCR4

In this study, effective transport of small interfering RNAs (siRNAs) via hyaluronic acid (HA) receptor was carried out with biodegradable HA and low-molecular weight polyethyleneimine (PEI)-based transport systems. Gold nanoparticles (AuNPs) capable of giving photothermal response, and their conjugates with PEI and HA, were also added to the structure. Thus, a combination of gene silencing, photothermal therapy and chemotherapy, has been accomplished. The synthesized transport systems ranged in size, between 25 and 690 nm. When the particles were applied at a concentration of 100 μg mL⁻¹ (except AuPEI NPs) in vitro, cell viability was above 50%. Applying radiation after the conjugate/siRNA complex (especially those containing AuNP) treatment, increased the cytotoxic effect (decrease in cell viability of 37%, 54%, 13%, and 15% for AuNP, AuPEI NP, AuPEI-HA, and AuPEI-HA-DOX, respectively) on the MDA-MB-231 cell line. CXCR4 gene silencing via the synthesized complexes, especially AuPEI-HA-DOX/siRNA was more efficient in MDA-MB-231 cells (25-fold decrease in gene expression) than in CAPAN-1 cells. All these results demonstrated that the synthesized PEI-HA and AuPEI-HA-DOX conjugates can be used as siRNA carriers that are particularly effective, especially in the treatment of breast cancer.     

A unique and highly efficient non-viral DNA/siRNA delivery system based on PEI-bisepoxide nanoparticles

Delivery of DNA and siRNA into mammalian cells is a powerful technique in treating various diseases caused by single gene defects. Herein, we report a highly efficient delivery system using 1,4-butanediol diglycidyl ether (bisepoxide) crosslinked polyethylenimine (PEI) nanoparticles (PN). The nanoparticle/DNA complexes (nanoplexes) exibited approximately 2.5- to 5.0-fold gene transfer efficacy and decreased cytotoxicity in cultured cell lines, compared to the native PEI (25 kDa) (gold standard) and commercially available transfection agents such as Lipofectamine 2000 and Fugene. The bisepoxide crosslinking results in change in amine ratio in PEI; however, it retains the net charge on PN unaltered. A series of nanoparticles obtained by varying the degree of crosslinking was found to be in the size range of 69-77 nm and the zeta potential varying from +35 to 40 mV. The proposed system was also found to deliver siRNA efficiently into HEK cells, resulting in approximately 70% suppression of the targetted gene (GFP).     

Amphiphilic peptides with arginine and valine residues as siRNA carriers

An efficient and safe delivery carrier is required for the therapeutic application of siRNA. In this research, amphiphilic peptides with arginine and valine residues were evaluated as siRNA carriers. The peptides were composed of 1-4 arginine-blocks and 6 valine-blocks. In the aqueous solution, the arginine-valine peptides (RV peptides) formed micelles with hydrophobic cores comprised of a valine block and a cationic surface comprised of an arginine block. In a gel retardation assay, the RV peptides completely retarded siRNA at a 1:10 weight ratio (siRNA:peptide). A heparin competition assay suggested that the RV peptides formed more stable complexes with siRNA than they did with polyethylenimine (25 kDa, PEI25k). In an in vitro silencing assay, a dual luciferase expression (Renilla and firefly luciferases) vector, psiCHECK2, was co-transfected into human embryonic kidney 293 cells with Renilla-siRNA using the RV peptides. The specific silencing effect of Renilla luciferase was analyzed in reference to firefly luciferase. The results showed that the R3V6 peptide was more efficient than the R1V6, R2V6, and R4V6 peptides in silencing Renilla luciferase. In the flow cytometry and in vitro silencing studies, the R3V6 peptide delivered Renila-siRNA as efficiently as PEI25k. The siVEGF/R3V6 peptide also reduced endogenous vascular endothelial growth factor (VEGF) expression in CT27 cells as efficiently as PEI25k. A cytotoxicity assay showed that RV peptides did not cause any cytotoxicity. Therefore, RV peptides may be useful for the development of a safe and efficient delivery carrier of siRNA.     

Hyaluronic acid-polyethyleneimine conjugate for target specific intracellular delivery of siRNA

A novel target specific small interfering RNA (siRNA) delivery system was successfully developed using polyethyleneimine (PEI)-hyaluronic acid (HA) conjugate. Anti-PGL3-Luc siRNA was used as a model system suppressing the PGL3-Luc gene expression. The siRNA/PEI-HA complex with an average size of ca. 21 nm appeared to be formed by electrostatic interaction between the negatively charged siRNA and the positively charged PEI of PEI-HA conjugate. The cytotoxicity of siRNA/PEI-HA complex to B16F1 cells was lower than that of siRNA/PEI complex according to the MTT assay. When B16F1 and HEK-293 cells were treated with fluorescein isothiocyanate (FITC) labeled siRNA/PEI-HA complex, B16F1 cells, with a lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), showed higher green fluorescent intensity than HEK-293 cells because of the HA receptor mediated endocytosis of the complex. Accordingly, the PGL3-Luc gene silencing of anti-PGL3-Luc siRNA/PEI-HA complex was more efficient in B16F1 cells than in HEK-293 cells. In addition, the inhibited PGL3-Luc gene silencing effect in the presence of free HA in the transfection medium revealed that siRNA/HA-PEI complex was selectively taken up to B16F1 cells via HA receptor mediated endocytosis. All these results demonstrated that the intracellular delivery of anti-PGL3-Luc siRNA/PEI-HA complex could be facilitated by the HA receptor mediated endocytosis.     

Folate receptor targeted delivery of polyelectrolyte complex micelles prepared from ODN-PEG-folate conjugate and cationic lipids

A polyelectrolyte complex micelle (PECM)-based delivery system for targeting folate (FOL) receptor overexpressing tumor cells is demonstrated using poly(ethylene glycol) (PEG)-conjugated oligonucleotide (ODN). The tumor targeting property was conferred to the PECM by tethering a folate moiety to the distal end of the PEG segment in an anti-sense green fluorescent protein (GFP) ODN-PEG conjugate. Nanoscale PECMs were spontaneously produced from ionic interactions between the ODN-PEG-FOL conjugate and a cationic lipid, lipofectamine (Lf). When treated with FOL receptor overexpressing cells (KB), the PCEMs caused a significant reduction in GFP expression in a dose-dependent manner. This effect was not observed in FOL receptor deficient cells (A549). The enhanced transfection of ODN-PEG-FOL/Lf PECMs to KB cells was caused by FOL receptor mediated endocytosis. The efficiency of target-specific gene suppression by ODN-PEG-FOL/Lf PECMs was maintained even in the presence of 10% fetal bovine serum in the transfection medium.