siRNA Complexes Research Articles

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.

Targeting EZH2 for glioma therapy with a novel nanoparticle-siRNA complex.

BackgroundFor the past few years, gene-therapy has recently shown considerable clinical benefit in cancer therapy, and the applications of gene therapies in cancer treatments continue to increase perennially. EZH2, an ideal candidate for tumor gene therapy, plays an important role in the tumorigenesis.MethodsIn this study, we developed a novel gene delivery system with a self-assembly method by Methoxy polyethylene glycol-polycaprolactone (MPEG-PCL) and DOTAP(DMC). And EZH2si-DMC was used to research anti-glioma both in vitro and in vivo.ResultsDMC with zeta-potential value of 36.7 mV and size of 35.6 nm showed good performance in the delivery siRNA to glioma cell in vitro with high 98% transfection efficiency. EZH2si-DMC showed good anti-glioma effect in vitro through inducing cell apoptosis and inhibiting cell growth. What’s more, treatment of tumor-bearing mice with DMC-EZH2si complex had significantly inhibited tumor growth at the subcutaneous model in vivo by inhibiting EZH2 protein expression, promoting apoptosis and reducing proliferation.ConclusionThe EZH2 siRNA and DMC complex may be used to treat the glioma in clinical as a new drug.

Tumor Chemosensitization through Oncogene Knockdown Mediated by Unique α-Tocopherylated Cationic Geminis.

Herein, siRNA transfection efficiency of a unique set of α-tocopherylated gemini lipids has been established in vitro and in vivo. High efficacy of oncogene silencing achieved using the biomacromolecular assembly, formed from siRNA complexes of co-liposomes containing an α-tocopherylated gemini lipid, has been utilized for tumor regression via chemosensitization. Delivery studies with the gemini bearing hydroxyethyl headgroup with octamethylene spacer (TH8S) pointed to a higher siRNA transfection efficacy than its analog without hydroxyethyl group (T8S). Owing to p53 upregulation, transfected cells showed enhanced sensitivity to the chemotherapeutic agent, doxorubicin. Studies in murine model revealed significantly low levels of survivin mRNA in xenograft tumors injected with siRNA lipoplexes, leading to effective inhibition of tumor growth and an increase in sensitivity of the tumors toward doxorubicin. These findings enable us to propose the anti-survivin siRNA carrying TH8S co-liposomes as a potent member of cancer management strategies using suicide gene therapy.

Autophagy inhibitor Vacuolin-1 interferes with lipid-based small interference RNA delivery

Autophagy and endocytosis are important pathways regulating macromolecule recycling and regeneration. Small molecule inhibitors are utilized to modulate these pathways and to treat autophagy-related diseases. Vacuolin-1 is a small molecule that can potently and reversibly inhibit autophagy by activating Rab5. In addition, Vacuolin-1 can be applied to inhibit exocytosis in a variety of cell types. Here we report that Vacuolin-1 significantly reduces small interference RNA (siRNA)-mediated gene silencing delivered by liposome transfection reagent or lipid nanoparticles in Hela cells. Vacuolin-1 exhibits the strongest inhibition effect among a few autophagy inhibitors including Chloroquine, Wortmannin, and Bafilomycin A1. We found that siRNAs are over-accumulated intracellularly and colocalized with a late endosome marker Rab7 in Vacuolin-1 treated cells, suggesting Vacuolin-1 inhibits the cytoplasmic release of lipid siRNA complexes from late endosomes. We propose that Vacuolin-1 could potentially be used to control the effects of lipid nanoparticle-based RNAi and gene therapy drugs.

Delivery of BACE1 siRNA mediated by TARBP-BTP fusion protein reduces β-amyloid deposits in a transgenic mouse model of Alzheimer’s disease

Systemic delivery of nucleic acids to the central nervous system (CNS) is a major challenge for the development of RNA interference-based therapeutics due to lack of stability, target specificity, non-permeability to the blood-brain barrier (BBB), and lack of suitable carriers. Using a designed bi-functional fusion protein TARBP-BTP in a complex with siRNA, we earlier demonstrated knockdown of target genes in the brain of both AβPP-PS1 (Alzheimer's disease, AD) and wild-type C57BL/6 mice. In this report, we further substantiate the approach through an extended use in AβPP-PS1 mice, which upon treatment with seven doses of β-secretase AβPP cleaving Enzyme 1 (BACE1) TARBP-BTP:siRNA, led to target-specific effect in the mouse brain. Concomitant gene silencing of BACE1, and consequent reduction in plaque load in the cerebral cortex and hippocampus (greater than 60%) in mice treated with TARBP-BTP:siRNA complex, led to improvement in spatial learning and memory. The study validates the efficiency of TARBP-BTP fusion protein as an efficient mediator of RNAi, giving considerable scope for future intervention in neurodegenerative disorders through the use of short nucleic acids as gene specific inhibitors.

Фармакокинетика инновационного липосомального препарата для лечения гепатита C

Главными преимуществами использования препаратов на основе малых интерферирущих РНК (миРНК) являются их высокая специфичность и эффективность, так как вводимые миРНК способны действовать в крайне низких концентрациях. Возможность доставки миРНК в клетки достигается путём её заключения в комплекс с липопептидами. Сложность изучения фармакокинетики препаратов, содержащих миРНК и липопептиды, заключается в том, что в организме млекопитающих присутствует большое количество соединений, сходных по природе, что создает сложности для селективной детекции именно компонентов самого лекарственного средства. Авторами разработан подход к изучению фармакокинетики инновационного препарата для лечения вируса гепатита C, представляющего собой комплекс молекул миРНК и носителя – катионного липопептида, при нанесении флуоресцентных меток с различной длиной волны испускания.

A Systematic Comparison of Lipopolymers for siRNA Delivery to Multiple Breast Cancer Cell Lines: <i>In Vitro</i> Studies

The small interfering RNA (siRNA) therapy is a promising approach for treatment of a wide range of cancers, including breast cancers that display variable phenotypic features. To explore the general utility of siRNA therapy to control aberrant expression of genes in breast cancer, we conducted a detailed analysis of siRNA delivery and silencing response in vitro in 6 separate breast cancer cell models (MDA-MB-231, MDA-MB-231-KRas-CRM, MCF-7, AU565, MDA-MB-435 and MDA-MB-468 cells). Using lipopolymers for siRNA complexation and delivery, we found a large variation in siRNA delivery efficiency depending on the specific lipopolymer used for siRNA complexation and delivery. Some lipopolymers were effective in all cell types used in this study, indicating the possibility of universal carriers for siRNA therapy. The delivery efficiency for effective lipopolymers was not correlated with dextran uptake in all cells tested, which indicated a receptor-mediated internalization for siRNA complexes with lipopolymers, unlike fluid-phase transfer associated with dextran uptake. Consistent with this, specific inhibitors involved in clathrin- and caveolin-mediated endocytosis significantly (>50%) reduced the internalization of siRNA complexes in all cell types. Using JAK2 and STAT3 silencing in MDA-MB-231 and MDA-MB-468 cells, a general correlation between the uptake and silencing efficiency at the mRNA level was evident, but it appeared that the choice of the target rather than the cell type was more critical for consistent silencing. We conclude that siRNA therapy with well-characterized lipopolymers can be undertaken with multiple breast cancer cell phenotypes with similar efficiency, indicating the general applicability of non-viral RNAi in clinical management of molecularly heterogeneous breast cancers.

Graphene oxide as a 2D platform for complexation and intracellular delivery of siRNA.

The development of efficient and safe nucleic acid delivery vectors remains an unmet need holding back translation of gene therapy approaches to the bedside. Graphene oxide (GO) could help bypass such bottlenecks, thanks to its large surface area, versatile chemistry and biocompatibility, which could overall enhance transfection efficiency while abolishing some of the limitations linked to the use of viral vectors. Here, we aimed to assess the capacity of bare GO, without any further surface modification, to complex a short double-stranded nucleic acid of biological relevance (siRNA) and mediate its intracellular delivery. GO formed stable complexes with siRNA at 10 : 1, 20 : 1 and 50 : 1 GO : siRNA mass ratios. Complexation was further corroborated by atomistic molecular dynamics simulations. GO : siRNA complexes were promptly internalized in a primary mouse cell culture, as early as 4 h after exposure. At this time point, intracellular siRNA levels were comparable to those provided by a lipid-based transfection reagent that achieved significant gene silencing. The time-lapse tracking of internalized GO and siRNA evidenced a sharp decrease of intracellular siRNA from 4 to 12 h, while GO was sequestered in large vesicles, which may explain the lack of biological effects (i.e. gene silencing) achieved by GO : siRNA complexes. This study underlines the potential of non-surface modified GO flakes to act as 2D siRNA delivery platforms, without the need for cationic functionalization, but warrants further vector optimization to allow the effective release of the nucleic acid and achieve efficient gene silencing.

VEGFA Gene Silencing in CXCR4-Expressing Cells via siRNA Delivery by Means of Targeted Peptide Carrier.

Discovery of small interfering RNA as a tool for specific gene inhibition led to the development of new therapeutic strategy for the treatment of cancers. The efficacious delivery of therapeutic siRNAs into the cells is a crucial step in RNA interference (RNAi) application, but it remains challenging. Non-viral vectors can provide specific cellular uptake, stable siRNA complex formation, and intracellular siRNA release. Recently, we evaluated modular peptide carrier L1 bearing CXCR4 targeting ligand for its ability to condense siRNA and facilitate endosomal escape and VEGFA gene silencing in CXCR4-expressing endothelial and glioblastoma cells. The present chapter showcases the ability of L1 targeted peptide carrier to form complexes with siRNA and provide efficient VEGFA gene knockdown. We showed that siRNA delivery by means of L1 peptide carrier can result in significant decrease of VEGFA gene expression in A172 glioblastoma cells and in EA.hy 926 endothelial cells. Also, delivery of anti-VEGFA siRNA/peptide complexes led to significant inhibition of endothelial cell migration. Our results showed that L1 peptide carrier modified with CXCR4 ligand is a promising tool for targeted siRNA delivery into CXCR4-expressing cancer and endothelial cells.

A "self-accelerating endosomal escape" siRNA delivery nanosystem for significantly suppressing hyperplasia via blocking the ERK2 pathway.

Small interfering RNA (siRNA)-based therapy is an emerging treatment to address serious cardiovascular disease. It is essential to construct highly efficient vehicles for therapeutic siRNA intracellular delivery. Extracellular signal-regulated kinase-2 (ERK2) siRNA (abbreviated as ERK2-siRNA) is known as a type of siRNA to selectively silence the expression of ERK2. Herein, a type of ternary delivery system characterized by an endosome-selective-self-accelerating-escape ability was designed and prepared for the purpose of inhibiting the migration of vascular smooth muscle cells (VSMCs) in vitro. This system was called ternary ERK2-siRNA complexes (abbreviated as TRCs-Aco), which were fabricated via sequential electrostatic self-assembly of a star-shaped cell-penetrating peptide based on polyhedral oligomeric silsesquioxane (POSS-(C-G-R8-G-W)16), ERK2-siRNA and a pH-sensitive anionic polymer of cis-aconitic anhydride grafted ε-poly(l-lysine). Importantly, TRCs-Aco could break down the obstacle of biocompatibility-silencing efficiency. In comparison with the parent binary siRNA complexes (abbreviated as BRCs), which are composed of POSS-(C-G-R8-G-W)16 and ERK2-siRNA, our designed TRCs-Aco revealed more excellent biocompatibility including hemocompatibility and cytocompatibility. Unexpectedly, TRCs-Aco exhibited stronger ERK2 silencing efficiency at the level of mRNA and protein, which was mainly due to its remarkable self-accelerating endosomal escape. Definitive evidence demonstrated that this ternary ERK2-siRNA delivery system significantly prevented the migration of VSMCs and decreased the dermal thickness in bleomycin-treated mice. In brief, this unique structured system could provide a valuable nanoplatform for highly efficient siRNA delivery in VSMCs, and it might hold great potential in guiding ERK2-siRNA-based proliferative disease therapy.

Ultrasound-mediated delivery of siESE complexed with microbubbles attenuates HER2+/- cell line proliferation and tumor growth in rodent models of breast cancer.

The highly tunable, noninvasive and spatially targeted nature of microbubble-enhanced, ultrasound-guided (MB+US) drug delivery makes it desirable for a wide variety of therapies. In breast cancer, both HER2+ and HER2- type neoplasms pose significant challenges to conventional therapeutics in greater than 40% of breast cancer patients, even with the widespread application of biologics such as trastuzumab. To address this therapeutic challenge, we examined the novel combination of tumor-injected microbubble-bound siRNA complexes and monodisperse size-isolated microbubbles (4-µm diameter) to attenuate tumor growth in vivo, as well as MB+US-facilitated shRNA and siRNA knockdown of ESE-1, an effector linked to dysregulated HER2 expression in HER2+/- cell line propagation. We first screened six variants of siESE and shESE for efficient knockdown of ESE in breast cancer cell lines. We demonstrated efficient reduction of BT-474 (PR+, ER+, HER2+; luminal B) and MDA-MB-468 (PR-, ER-, HER2-; triple-negative) clonogenicity and non-adherent growth after knockdown of ESE-1. A significant reduction in proliferative potential was seen for both cell lines using MB+US to deliver shESE and siESE. We then demonstrated significant attenuation of BT-474 xenograft tumor growth in Nod/SCID female mice using direct injection of microbubble-adsorbed siESE to the tumor and subsequent sonication. Our results suggest a positive effect on drug delivery from MB+US, and highlights the feasibility of using RNAi and MB+US for breast cancer pathologies. RNAi coupled with MB+US may also be an effective theranostic approach to treat other acoustically accessible tumors, such as melanoma, thyroid, parotid and skin cancer.

Therapeutic effects of human monoclonal PSMA antibody-mediated TRIM24 siRNA delivery in PSMA-positive castration-resistant prostate cancer.

Background and Aims: Prostate specific membrane antigen (PSMA) is specifically expressed on prostate epithelial cells and markedly overexpressed in almost all prostate cancers. TRIM24 is also up-regulated from localized prostate cancer to metastatic castration-resistant prostate cancer (CRPC). Because of the high relevance of TRIM24 for cancer development and the universal expression of PSMA in CPRC, we investigated the efficacy of human monoclonal PSMA antibody (PSMAb)-based platform for the targeted TRIM24 siRNA delivery and its therapeutic efficacy in CRPC in vivo and in vitro.Methods: The therapeutic complexes were constructed by conjugating PSMAb and sulfo-SMCC-protamine, and encapsulating TRIM24 siRNA. Flow cytometry, immunofluorescence, and fluorescence imaging were performed to detect the receptor-binding, internalization, and targeted delivery of PSMAb-sulfo-SMCC-protamine (PSP)-FAM-siRNA complex (PSPS) in vitro and in vivo. CCK-8, plate-colony formation, apoptosis, cell cycle, and Transwell assays were performed to evaluate the therapeutic potential of the PSP-TRIM24 siRNA complex in vitro, whereas the in vivo therapeutic efficacy was monitored by small animal imaging, radiography, and micro CT.Results: We confirmed that PSP could efficiently protect siRNA from enzymatic digestion, enable targeted delivery of siRNA, and internalize and release siRNA into PSMA-positive (PSMA+) prostate cancer cells in vitro and in vivo. Silencing TRIM24 expression by the PSP-TRIM24 siRNA complex could dramatically suppress proliferation, colony-formation, and invasion of PSMA+ CRPC cells in vitro, and inhibit tumor growth of PSMA+ CRPC xenografts and bone loss in PSMA+ CRPC bone metastasis model without obvious toxicity at therapeutic doses in vivo.Conclusion: PSMAb mediated TRIM24 siRNA delivery platform could significantly inhibit cell proliferation, colony-formation, and invasion in PSMA+ CRPC in vitro and suppressed tumor growth and bone loss in PSMA+ CRPC xenograft and bone metastasis model.

Lipid-based Nanocarriers for siRNA Delivery: Challenges, Strategies and the Lessons Learned from the DODAX: MO Liposomal System.

The possibility of using the RNA interference (RNAi) mechanisms in gene therapy was one of the scientific breakthroughs of the last century. Despite the extraordinary therapeutic potential of this approach, the need for an efficient gene carrier is hampering the translation of the RNAi technology to the clinical setting. Although a diversity of nanocarriers has been described, liposomes continue to be one of the most attractive siRNA vehicles due to their relatively low toxicity, facilitated siRNA complexation, high transfection efficiency and enhanced pharmacokinetic properties. This review focuses on RNAi as a therapeutic approach, the challenges to its application, namely the nucleic acids' delivery process, and current strategies to improve therapeutic efficacy. Additionally, lipid-based nanocarriers are described, and lessons learned from the relation between biophysical properties and biological performance of the dioctadecyldimethylammonium:monoolein (DODAX: MO) system are explored. Liposomes show great potential as siRNA delivery systems, being safe nanocarriers to protect nucleic acids in circulation, extend their half-life time, target specific cells and reduce off-target effects. Nevertheless, several issues related to delivery must be overcome before RNAi therapies reach their full potential, namely target-cell specificity and endosomal escape. Understanding the relationship between biophysical properties and biological performance is an essential step in the gene therapy field.

Exosome-delivered TRPP2 siRNA inhibits the epithelial-mesenchymal transition of FaDu cells.

The prognosis for patients with head and neck cancer (HNC) remains poor, owing to uncontrolled tumor invasion and metastasis. Epithelial-mesenchymal transition (EMT) serves an important role in this invasion and metastasis, and transient receptor potential polycystic 2 (TRPP2) enhances metastasis and invasion by regulating EMT in human laryngeal squamous cell carcinoma. The present study examined whether exosomes/TRPP2 small interfering RNA (siRNA) complexes were able to reduce EMT by suppressing TRPP2 expression in FaDu cells, a cell line of human pharyngeal squamous cell carcinoma. Using agarose gel electrophoresis, it was determined that exosome/TRPP2 siRNA complexes were stable in the presence of nucleases and serum. A fluorescence assay and western blotting analysis was performed, and it was reported that the FaDu cells took up exosomes, the exosomes effectively delivered TRPP2 siRNA into FaDu cells and that exosome/TRPP2 siRNA complexes significantly suppressed TRPP2 protein expression levels in FaDu cells. Furthermore, expression levels of E-cadherin were significantly increased, whereas expression levels of N-cadherin and vimentin were significantly decreased in FaDu cells transfected with TRPP2 siRNA. Thus, exosome/TRPP2 siRNA complexes markedly suppressed TRPP2 expression and EMT in FaDu cells. These results suggested that further development of exosome/TRPP2 siRNA complexes for use as an RNA-based gene therapy in the treatment of HNC is warranted.

Local Silencing of Connective Tissue Growth Factor by siRNA/Peptide Improves Dermal Collagen Arrangements

Collagen organization within tissues has a critical role in wound regeneration. Collagen fibril diameter, arrangements and maturity between connective tissue growth factor (CTGF) small interfering RNA (siRNA) and mismatch scrambled siRNA-treated wound were compared to evaluate the efficacy of CTGF siRNA as a future implement for scar preventive medicine. Nanocomplexes of CTGF small interfering RNA (CTGF siRNA) with cell penetrating peptides (KALA and MPG∆NLS) were formulated and their effects on CTGF downregulation, collagen fibril diameter and arrangement were investigated. Various ratios of CTGF siRNA and peptide complexes were prepared and down-regulation were evaluated by immunoblot analysis. Control and CTGF siRNA modified cells-populated collagen lattices were prepared and rates of contraction measured. Collagen organization in rabbit ear 8mm biopsy punch wound at 1day to 8wks post injury time were investigated by transmission electron microscopy and histology was investigated with Olympus System and TS-Auto software. CTGF expression was down-regulated to 40% of control by CTGF siRNA/KALA (1:24) complexes (p < 0.01) and collagen lattice contraction was inhibited. However, down-regulated of CTGF by CTGF siRNA/MPG∆NLS complexes was not statistically significant. CTGF KALA-treated wound appeared with well formed-basket weave pattern of collagen fibrils with mean diameter of 128 ± 22nm (n = 821). Mismatch siRNA/KALA-treated wound showed a high frequency of parallel small diameter fibrils (mean 90 ± 20nm, n = 563). Controlling over-expression of CTGF by peptide-mediated siRNA delivery could improve the collagen orientation and tissue remodeling in full thickness rabbit ear wound.

Oral siRNA delivery using dual transporting systems to efficiently treat colorectal liver metastasis

Oral siRNA delivery is an ideal way to translate siRNA therapeutic effects in the clinic due to its ability to be administered in convenient and multiple dosages. However, an effective oral delivery system requires overcoming both a hostile gastrointestinal (GI) environment and non-specific targeting. Here, an HTsRP-NC system is a new oral siRNA delivery system consisting of a siRNA/protamine (sRP) nano-complex protected by a multi-functional hyaluronic acid-taurocholic acid (HA-TCA) conjugate. The HTsRP-NC promotes cell penetration and enhances endosomal escape in cancer cells. Moreover, protection of the sRP by HA-TCA from the hostile GI environment helps the AKT siRNA complex to reach the liver through the utilization of a TCA-mediated enterohepatic bile acid recycling system. AKT siRNA was released by 90% in presence of hyaluronidase in the tumor cells which indicate the potential use of HTsRP-NCs for siRNA delivery to treat tumor. After HA receptor (CD44)-mediated cellular uptake of the HTsRP-NC by the liver cancer cells, functional expression of AKT siRNA leads to the suppression of metastatic liver cancer growth in a colorectal liver metastasis (CLM) murine model. Tumor nodules were reduced by more than 1 mm size compared to control group and tumor cells were suppressed by 50% after HTsRP-NCs treatment with AKT siRNA. Overall, oral administration of the HTsRP-NC supports its potential in therapeutic applications for the effective treatment of CLM.

Bioreducible Polyspermine-Based Gene Carriers for Efficient siRNA Delivery: Effects of PEG Conjugation on Gene Silencing Efficiency

A polyspermine-derived bioreducible polymer was synthesized as efficient siRNA carriers. Spermine was polymerized using disulfide-bearing cross-linkers, resulting in poly(disulfide spermine) (PDS). PDS was also conjugated with poly(ethylene glycol) (PEG) to tailor siRNA condensation efficiency of the polymer. PEG-conjugated PDS (PEG-PDS) was able to condense siRNA into nano-sized polyplexes at high N/P ratios. siRNA condensation efficiency of PDS was reduced by PEGylation due to its weakened interaction with siRNA caused by its decreased cationic charge density. An ethidium bromide exclusion assay demonstrated that siRNA was more efficiently released from the less stable PEG-PDS polyplexes compared to stable PDS polyplexes. In addition, bioreducible siRNA/PEG-PDS polyplexes were efficiently disassembled in the reductive conditions, leading to facilitated siRNA release into the cytoplasm. The lowered siRNA condensation but efficient cytoplasmic release of siRNA by PEG-PDS induced higher gene silencing efficiency in comparison with PDS. Moreover, PEG-PDS exhibited lower cytotoxicity compared to PDS and non-biodegradable branched polyethylenimine. These results suggest that siRNA delivery efficiency of cationic gene carriers can be improved by tailoring their siRNA complexation through PEG conjugation. This study demonstrates that bioreducible PEG-PDS is a promising candidate for safe and efficient siRNA delivery.

Green Tea Catechin Dramatically Promotes RNAi Mediated by Low-Molecular-Weight Polymers.

Cytosolic delivery is the major challenge that limits the clinical translation of siRNA-based therapeutics. Although thousands of polymers have been developed for siRNA delivery, the efficiency–toxicity correlation is unsatisfactory. Here, we report a facile strategy to fabricate core–shell-structured nanoparticles with robust siRNA delivery efficiency. The nanoparticle is prepared by entropy-driven complexation of siRNA with a green tea catechin to yield a negatively charged core, followed by coating low-molecular-weight polymers to form the shell. This supramolecular strategy facilitates the polymers condensing siRNA into uniform nanoparticles. The nanoparticle specifically down-regulates target genes in vitro and in vivo, and efficiently attenuates chronic intestinal inflammation in an inflammatory bowel disease model. Notably, the highly efficient nanoparticles are applicable for various polymers with different topologies and chemical compositions, providing a versatile technique to break down the efficiency–toxicity correlation of cationic polymers. The proposed strategy in this study permits the development of a promising platform for polymer-mediated siRNA delivery.

Synergistic inhibition of aggressive breast cancer cell migration and invasion by cytoplasmic delivery of anti-RhoC silencing RNA and presentation of EPPT1 peptide on “smart” particles

Overexpression of RhoC protein in breast cancer patients has been linked to increased cancer cell invasion, migration, and metastases. Suppressing RhoC expression in aggressive breast cancer cells using silencing RNA (siRNA) molecules is a viable strategy to inhibit the metastatic spread of breast cancer. In this report, we describe the synthesis of a series of asymmetric pH-sensitive, membrane-destabilizing polymers engineered to complex anti-RhoC siRNA molecules forming “smart” nanoparticles. Using β-CD as the particle core, polyethylene glycol (PEG) chains were conjugated to the primary face via non-cleavable bonds and amphiphilic polymers incorporating hydrophobic and cationic monomers were grafted to the secondary face via acid-labile linkages. We investigated the effect of PEG molecular weight (2 & 5 kDa) on transfection capacity and serum stability of the formed particles. We evaluated the efficacy of EPPT1 peptides presented on the free tips of the PEG brush to function as a targeting ligand against underglycosylated MUC1 (uMUC1) receptors overexpressed on the surface of metastatic breast cancer cells. Results show that “smart” nanoparticles successfully delivered anti-RhoC siRNA into the cytoplasm of aggressive SUM149 and MDA-MB-231 breast cancer cells, which resulted in a dose-dependent inhibition of cell migration and invasion. Further, EPPT1-targeted nanoparticles demonstrate a synergistic inhibition of cell migration and invasion imparted via RhoC knockdown and EPPT1-mediated signaling via the uMUC1 receptor.

Targeted siRNA delivery to tumor cells by folate-PEG-appended dendrimer/glucuronylglucosyl-β-cyclodextrin conjugate

We previously reported the utility of 6-O-α-(4-O-α-d-glucuronyl)-d-glucosyl-β-cyclodextrin (GUG-β-CyD) conjugates with polyamidoamine dendrimer [GUG-β-CDE (generation 3; G3)] as siRNA carriers. In this study, to prepare GUG-β-CDE (G3) possessing a targeting ability to tumor cells overexpressing folate receptor-α (FR-α), we newly synthesized folate-polyethylene glycol (PEG)-appended GUG-β-CDEs (G3) [Fol-PEG-GUG-β-CDEs (G3)] having degrees of substitution of folate (DSF) of 3.9, 6.7 and 7.3, and evaluated their utility as tumor-selective siRNA carriers. Of various Fol-PEG-GUG-β-CDEs (G3), Fol-PEG-GUG-β-CDE (G3, DSF6.7) showed the highest siRNA transfection activity at a charge ratio of 50 (carrier/siRNA) in both 786-0-luc cells [FR-α (+)] and KB cells [FR-α (+)]. In addition, the cellular uptake of the complex was significantly decreased by an addition of folic acid in a concentration-dependent manner, suggesting its FR-α-mediated endocytosis pathway. Moreover, Fol-PEG-GUG-β-CDE (G3, DSF6.7)/siRNA complex induced a potent RNAi effect, comparable to Lipofectamine™ 2000/siRNA complex. Furthermore, Fol-PEG-GUG-β-CDE (G3, DSF6.7) complex with siRNA against Polo-like kinase 1 (siPLK1) showed a significant cytotoxic activity in KB cells. Thus, Fol-PEG-GUG-β-CDE (G3, DSF6.7) has the potential as the targeted siRNA delivery carrier for FR-α-overexpressing tumor cells.