Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle

Potent sequence selective gene inhibition by siRNA ‘targeted’ therapeutics promises the ultimate level of specificity, but siRNA therapeutics is hindered by poor intracellular uptake, limited blood stability and non-specific immune stimulation. To address these problems, ligand-targeted, sterically stabilized nanoparticles have been adapted for siRNA. Self-assembling nanoparticles with siRNA were constructed with polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached at the distal end of the polyethylene glycol (PEG), as a means to target tumor neovasculature expressing integrins and used to deliver siRNA inhibiting vascular endothelial growth factor receptor-2 (VEGF R2) expression and thereby tumor angiogenesis. Cell delivery and activity of PEGylated PEI was found to be siRNA sequence specific and depend on the presence of peptide ligand and could be competed by free peptide. Intravenous administration into tumor-bearing mice gave selective tumor uptake, siRNA sequence-specific inhibition of protein expression within the tumor and inhibition of both tumor angiogenesis and growth rate. The results suggest achievement of two levels of targeting: tumor tissue selective delivery via the nanoparticle ligand and gene pathway selectivity via the siRNA oligonucleotide. This opens the door for better targeted therapeutics with both tissue and gene selectivity, also to improve targeted therapies with less than ideal therapeutic targets.     

An RGD-Modified MRI-Visible Polymeric Vector for Targeted siRNA Delivery to Hepatocellular Carcinoma in Nude Mice

RNA interference (RNAi) has significant therapeutic promise for the genetic treatment of hepatocellular carcinoma (HCC). Targeted vectors are able to deliver small interfering RNA (siRNA) into HCC cells with high transfection efficiency and stability. The tripeptide arginine glycine aspartic acid (RGD)-modified non-viral vector, polyethylene glycol-grafted polyethylenimine functionalized with superparamagnetic iron oxide nanoparticles (RGD-PEG-g-PEI-SPION), was constructed as a magnetic resonance imaging (MRI)-visible nanocarrier for the delivery of Survivin siRNA targeting the human HCC cell line Bel-7402. The biophysical characterization of the RGD-PEG-g-PEI-SPION was performed. The RGD-modified complexes exhibited a higher transfection efficiency in transferring Survivin siRNA into Bel-7402 cells compared with a non-targeted delivery system, which resulted in more significant gene suppression at both the Survivin mRNA and protein expression levels. Then, the level of caspase-3 activation was significantly elevated, and a remarkable level of tumor cell apoptosis was induced. As a result, the tumor growth in the nude mice Bel-7402 hepatoma model was significantly inhibited. The targeting ability of the RGD-PEG-g-PEI-SPION was successfully imaged by MRI scans performed in vitro and in vivo. Our results strongly indicated that the RGD-PEG-g-PEI-SPION can potentially be used as a targeted non-viral vector for altering gene expression in the treatment of hepatocellular carcinoma and for detecting the tumor in vivo as an effective MRI probe.     

A robust in vivo positive-readout system for monitoring siRNA delivery to xenograft tumors

Delivering small interfering RNA (siRNA) to tumors is the major technical hurdle that prevents the advancement of siRNA-based cancer therapy. One of the difficulties associated with the development of clinically relevant delivery systems is the lack of reliable tools for monitoring siRNA delivery to tumors in vivo. We describe here a novel, positive-readout system where siRNA-mediated target knockdown elicits a rapid and robust increase of reporter activity. Using the positive-readout system, we created (1) β-galactosidase-based tumor models that allow the detection of target knockdown in 1%-2% of tumor cells and can distinguish between tumor areas where effective target knockdown occurs versus tumor areas that are not accessible to delivery, and (2) luciferase-based tumor models that allow the quantitative assessment of a large number of delivery systems. Using these positive-readout models, we screened a number of literature-described siRNA delivery systems and identified lipid nanoparticles as a promising delivery platform for siRNA-based cancer therapy.     

Optimisation of Dex-GMA nanoparticles prepared in modified micro-emulsion system: Physical and biologic characterization

In recent years, utilizing nanoparticles as delivery system for drug targeting delivery has raised increasing interest. In this study, glycidyl methacrylate derivatized dextran (Dex-GMA) nanoparticles encapsulating basic fibroblast growth factor (bFGF) have been prepared inside the aqueous cores of sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/n-hexane reverse micelles. These nanoparticles were identified to be uniformly spherical in shape with an average size of 109.57 ± 2.09 nm. And 90.2% of the nanoparticles were in a narrow size range of 80–110 nm. The release of bFGF from the nanoparticles is completely and sustained as long as 35 days. The impact of the nanoparticles on mouse bone marrow mesenchymal stem cells (BMSCs) was assessed with cell cytotoxicity/viability and adhesion assay. Those studies show that the Dex-GMA nanoparticles prepared by water-in-oil micro-emulsion systems with aprotic solvent adding are novel, effective and biocompatible delivery system for bioactive protein.     

siRNA associated with immunonanoparticles directed against cd99 antigen improves gene expression inhibition in vivo in Ewing's sarcoma

Ewing's sarcoma is a rare, mostly pediatric bone cancer that presents a chromosome abnormality called EWS/Fli‐1, responsible for the development of the tumor. In vivo, tumor growth can be inhibited specifically by delivering small interfering RNA (siRNA) associated with nanoparticles. The aim of the work was to design targeted nanoparticles against the cell membrane glycoprotein cd99, which is overexpressed in Ewing's sarcoma cells to improve siRNA delivery to tumor cells. Biotinylated poly(isobutylcyanoacrylate) nanoparticles were conceived as a platform to design targeted nanoparticles with biotinylated ligands and using the biotin–streptavidin coupling method. The targeted nanoparticles were validated in vivo for the targeted delivery of siRNA after systemic administration to mice bearing a tumor model of the Ewing's sarcoma. The expression of the gene responsible of Ewing's sarcoma was inhibited at 78% ± 6% by associating the siRNA with the cd99‐targeted nanoparticles compared with an inhibition of only 41% ± 9% achieved with the nontargeted nanoparticles. Copyright © 2013 John Wiley & Sons, Ltd.     

Multifunctional Nanoparticles Delivering Small Interfering RNA and Doxorubicin Overcome Drug Resistance in Cancer

Drug resistance is a major challenge to the effective treatment of cancer. We have developed two nanoparticle formulations, cationic liposome-polycation-DNA (LPD) and anionic liposome-polycation-DNA (LPD-II), for systemic co-delivery of doxorubicin (Dox) and a therapeutic small interfering RNA (siRNA) to multiple drug resistance (MDR) tumors. In this study, we have provided four strategies to overcome drug resistance. First, we formed the LPD nanoparticles with a guanidinium-containing cationic lipid, i.e. N,N-distearyl-N-methyl-N-2-(N′-arginyl) aminoethyl ammonium chloride, which can induce reactive oxygen species, down-regulate MDR transporter expression, and increase Dox uptake. Second, to block angiogenesis and increase drug penetration, we have further formulated LPD nanoparticles to co-deliver vascular endothelial growth factor siRNA and Dox. An enhanced Dox uptake and a therapeutic effect were observed when combined with vascular endothelial growth factor siRNA in the nanoparticles. Third, to avoid P-glycoprotein-mediated drug efflux, we further designed another delivery vehicle, LPD-II, which showed much higher entrapment efficiency of Dox than LPD. Finally, we delivered a therapeutic siRNA to inhibit MDR transporter. We demonstrated the first evidence of c-Myc siRNA delivered by the LPD-II nanoparticles down-regulating MDR expression and increasing Dox uptake in vivo. Three daily intravenous injections of therapeutic siRNA and Dox (1.2 mg/kg) co-formulated in either LPD or LPD-II nanoparticles showed a significant improvement in tumor growth inhibition. This study highlights a potential clinical use for the multifunctional nanoparticles with an effective delivery property and a function to overcome drug resistance in cancer. The activity and the toxicity of LPD- and LPD-II-mediated therapy are compared.     

Local delivery of chitosan/VEGF siRNA nanoplexes reduces angiogenesis and growth of breast cancer in vivo

Vascular endothelial growth factor (VEGF) is the important angiogenic factor associated with tumor growth and metastasis in a wide variety of solid tumors. The aim of this study is to investigate the tumor suppressive effect of chitosan/small interfering RNA (siRNA)-VEGF nanoplexes in the rat breast cancer model. Chitosan/siRNA nanoplexes (siVEGF-A, siVEGFR-1, siVEGFR-2) and NRP-1 were prepared in a 15 to1 ratio and injected (intratumorally) into the breast-tumor-bearing Sprague-Dawley rats. Tumor volumes were measured during 21 days. To investigate the effect of chitosan/siRNA nanoplexes on VEGF expression in tumors, VEGF was analyzed with immunohistochemistry and western blotting. The mRNA levels of VEGF in tumor samples were determined with real-time PCR (RT-PCR). After siRNA treatment, a marked reduction in tumor volumes was measured in complex-injected rats (97%). Free siRNA injection showed lower tumor inhibition. Reduction of VEGF protein was also shown with western blotting and immunohistochemistry. Similar results were obtained with RT-PCR also. These results indicate that the chitosan/siRNA targeting to VEGF nanoplexes have a remarkably suppressive effect on VEGF expression and tumor volume in breast cancer model of rats.     

Nanomaterials for cancer therapy and imaging

A variety of organic and inorganic nanomaterials with dimensions below several hundred nanometers are recently emerging as promising tools for cancer therapeutic and diagnostic applications due to their unique characteristics of passive tumor targeting. A wide range of nanomedicine platforms such as polymeric micelles, liposomes, dendrimers, and polymeric nanoparticles have been extensively explored for targeted delivery of anti-cancer agents, because they can accumulate in the solid tumor site via leaky tumor vascular structures, thereby selectively delivering therapeutic payloads into the desired tumor tissue. In recent years, nanoscale delivery vehicles for small interfering RNA (siRNA) have been also developed as effective therapeutic approaches to treat cancer. Furthermore, rationally designed multi-functional surface modification of these nanomaterials with cancer targeting moieties, protective polymers, and imaging agents can lead to fabrication versatile theragnostic nanosystems that allow simultaneous cancer therapy and diagnosis. This review highlights the current state and future prospects of diverse biomedical nanomaterials for cancer therapy and imaging.     

Stability,Intracellular Delivery,and Release of siRNA from Chitosan Nanoparticles Using Different Cross-Linkers

Chitosan (CS) nanoparticles have been extensively studied for siRNA delivery; however, their stability and efficacy are highly dependent on the types of cross-linker used. To address this issue, three common cross-linkers; tripolyphosphate (TPP), dextran sulphate (DS) and poly-D-glutamic acid (PGA) were used to prepare siRNA loaded CS-TPP/DS/PGA nanoparticles by ionic gelation method. The resulting nanoparticles were compared with regard to their physicochemical properties including particle size, zeta potential, morphology, binding and encapsulation efficiencies. Among all the formulations prepared with different cross linkers, CS-TPP-siRNA had the smallest particle size (ranged from 127 ± 9.7 to 455 ± 12.9 nm) with zeta potential ranged from +25.1 ± 1.5 to +39.4 ± 0.5 mV, and high entrapment (>95%) and binding efficiencies. Similarly, CS-TPP nanoparticles showed better siRNA protection during storage at 4˚C and as determined by serum protection assay. TEM micrographs revealed the assorted morphology of CS-TPP-siRNA nanoparticles in contrast to irregular morphology displayed by CS-DS-siRNA and CS-PGA-siRNA nanoparticles. All siRNA loaded CS-TPP/DS/PGA nanoparticles showed initial burst release followed by sustained release of siRNA. Moreover, all the formulations showed low and concentration-dependent cytotoxicity with human colorectal cancer cells (DLD-1), in vitro. The cellular uptake studies with CS-TPP-siRNA nanoparticles showed successful delivery of siRNA within cytoplasm of DLD-1 cells. The results demonstrate that ionically cross-linked CS-TPP nanoparticles are biocompatible non-viral gene delivery system and generate a solid ground for further optimization studies, for example with regard to steric stabilization and targeting.     

5-Fluorouracil Nanoparticles Inhibit Hepatocellular Carcinoma via Activation of the p53 Pathway in the Orthotopic Transplant Mouse Model

Biodegradable polymer nanoparticle drug delivery systems provide targeted drug delivery, improved pharmacokinetic and biodistribution, enhanced drug stability and fewer side effects. These drug delivery systems are widely used for delivering cytotoxic agents. In the present study, we synthesized GC/5-FU nanoparticles by combining galactosylated chitosan (GC) material with 5-FU, and tested its effect on liver cancer in vitro and in vivo. The in vitro anti-cancer effects of this sustained release system were both dose- and time-dependent, and demonstrated higher cytotoxicity against hepatic cancer cells than against other cell types. The distribution of GC/5-FU in vivo revealed the greatest accumulation in hepatic cancer tissues. GC/5-FU significantly inhibited tumor growth in an orthotropic liver cancer mouse model, resulting in a significant reduction in tumor weight and increased survival time in comparison to 5-FU alone. Flow cytometry and TUNEL assays in hepatic cancer cells showed that GC/5-FU was associated with higher rates of G0–G1 arrest and apoptosis than 5-FU. Analysis of apoptosis pathways indicated that GC/5-FU upregulates p53 expression at both protein and mRNA levels. This in turn lowers Bcl-2/Bax expression resulting in mitochondrial release of cytochrome C into the cytosol with subsequent caspase-3 activation. Upregulation of caspase-3 expression decreased poly ADP-ribose polymerase 1 (PARP-1) at mRNA and protein levels, further promoting apoptosis. These findings indicate that sustained release of GC/5-FU nanoparticles are more effective at targeting hepatic cancer cells than 5-FU monotherapy in the mouse orthotropic liver cancer mouse model.     

Preparation and characterization of novel albumin-sericin nanoparticles as siRNA delivery vehicle for laryngeal cancer treatment

Abstract

Small interfering RNA (siRNA)-based gene silencing strategy has high potential on suppressing specific molecular targets, involved in cancer progression. However, the lack of an effective nanocarrier system that safely delivers siRNA to its target still limits the clinical applications of siRNA. This study aimed to develop albumin-sericin nanoparticles (Alb-Ser NPs) as a novel siRNA delivery system for laryngeal cancer treatment. Nanoparticle formulations composed of albumin and sericin at different ratios (1:1, 2:1, 1:2 w/w) were synthesized by desolvation method. The nanoparticles were modified with poly-L-lysine (PLL) for siRNA binding and decorated with hyaluronic acid (HA) to target laryngeal cancer cell line, Hep-2. HA/PLL/Alb-Ser NPs were individually loaded with siRNAs for casein kinase 2 (CK2), Absent, Small, or Homeotic-Like (ASH2L), and Cyclin D1 genes, which are overexpressed in Hep-2 cells. Downregulation of genes was confirmed by real-time PCR (RT-PCR). Size, morphological, and thermogravimetric characterizations revealed that Alb-Ser NPs having 2:1 (w/w) ratio are the most optimized formulation. Between 36.8 and 61.3% of siRNA entrapment efficiencies were achieved. HA/PLL-siRNA/Alb-Ser (2:1) NPs-mediated gene silencing resulted in a significant inhibition of cell growth and induction of apoptosis in cells. Our findings showed that HA/PLL/Alb-Ser (2:1) NPs were promising as a siRNA carrier.     

Polyethylenimine/small interfering RNA‐mediated knockdown of vascular endothelial growth factor in vivo exerts anti‐tumor effects synergistically with Bevacizumab

Background

RNA interference is a powerful method for the knockdown of pathologically relevant genes. The in vivo delivery of siRNAs, preferably through systemic, nonviral administration, poses the major challenge in the therapeutic application of RNAi. Small interfering RNA (siRNA) complexation with polyethylenimines (PEI) may represent a promising strategy for siRNA‐based therapies and, recently, the novel branched PEI F25‐LMW has been introduced in vitro. Vascular endothelial growth factor (VEGF) is frequently overexpressed in tumors and promotes tumor growth, angiogenesis and metastasis and thus represents an attractive target gene in tumor therapy.

Methods

In subcutaneous tumor xenograft mouse models, we established the therapeutic efficacy and safety of PEI F25‐LMW/siRNA‐mediated knockdown of VEGF. In biodistribution and siRNA quantification studies, we optimized administration strategies and, employing chemically modified siRNAs, compared the anti‐tumorigenic efficacies of: (i) PEI/siRNA‐mediated VEGF targeting; (ii) treatment with the monoclonal anti‐VEGF antibody Bevacizumab (Avastin®); and (iii) a combination of both.

Results

Efficient siRNA delivery is observed upon systemic administration, with the biodistribution being dependent on the mode of injection. Toxicity studies reveal no hepatotoxicity, proinflammatory cytokine induction or other side‐effects of PEI F25‐LMW/siRNA complexes or polyethylenimine, and tumor analyses show efficient VEGF knockdown upon siRNA delivery, leading to reduced tumor cell proliferation and angiogenesis. The determination of anti‐tumor effects reveals that, in pancreas carcinoma xenografts, single treatment with PEI/siRNA complexes or Bevacizumab is already highly efficacious, whereas, in prostate carcinoma, synergistic effects of both treatments are observed.

Conclusions

PEI F25‐LMW/siRNA complexes, which can be stored frozen as opposed to many other carriers, represent an efficient, safe and promising avenue in anti‐tumor therapy, and PEI/siRNA‐mediated, therapeutic VEGF knockdown exerts anti‐tumor effects. Copyright © 2010 John Wiley & Sons, Ltd.     

Induction of apoptosis in murine neuroblastoma by systemic delivery of transferrin-shielded siRNA polyplexes for downregulation of Ran

The polymer, OEI-HD, based on beta-propionamide-cross-linked oligoethylenimine and its chemical transferrin conjugate were evaluated for siRNA delivery into murine Neuro2A neuroblastoma cells in vitro and in vivo. An 80% silencing of luciferase expression in neuroblastoma cells, stably transfected with a luciferase gene, was obtained using standard OEI-HD polyplexes or transferrin-conjugated shielded OEI-HD polyplexes. The Ras-related nuclear protein Ran was selected as a therapeutically relevant target protein. Systemic delivery of transferrin-conjugated OEI-HD/RAN siRNA formulations (three intravenous applications at 3 days interval) resulted in >80% reduced Ran protein expression, apoptosis, and a reduced tumor growth in Neuro2A tumors of treated mice. The treatment was not associated with signs of acute toxicity or significant changes in weight, hematology parameters, or liver enzymes (AST, ALT, or AP) of mice. All our results demonstrate that OEI-HD/siRNA formulations can knockdown genes in tumor cells in vitro and in vivo in mice in the absence of unspecific toxicity.     

In vivo imaging of siRNA delivery and silencing in tumors

With the increased potential of RNA interference (RNAi) as a therapeutic strategy, new noninvasive methods for detection of siRNA delivery and silencing are urgently needed. Here we describe the development of dual-purpose probes for in vivo transfer of siRNA and the simultaneous imaging of its accumulation in tumors by high-resolution magnetic resonance imaging (MRI) and near-infrared in vivo optical imaging (NIRF). These probes consisted of magnetic nanoparticles labeled with a near-infrared dye and covalently linked to siRNA molecules specific for model or therapeutic targets. Additionally, these nanoparticles were modified with a membrane translocation peptide for intracellular delivery. We show the feasibility of in vivo tracking of tumor uptake of these probes by MRI and optical imaging in two separate tumor models. We also used proof-of-principle optical imaging to corroborate the efficiency of the silencing process. These studies represent the first step toward the advancement of siRNA delivery and imaging strategies, essential for cancer therapeutic product development and optimization.     

Cellular Uptake and Antitumor Activity of DOX-hyd-PEG-FA Nanoparticles

A PEG-based, folate mediated, active tumor targeting drug delivery system using DOX-hyd-PEG-FA nanoparticles (NPs) were prepared. DOX-hyd-PEG-FA NPs showed a significantly faster DOX release in pH 5.0 medium than in pH 7.4 medium. Compared with DOX-hyd-PEG NPs, DOX-hyd-PEG-FA NPs increased the intracellular accumulation of DOX and showed a DOX translocation from lysosomes to nucleus. The cytotoxicity of DOX-hyd-PEG-FA NPs on KB cells was much higher than that of free DOX, DOX-ami-PEG-FA NPs and DOX-hyd-PEG NPs. The cytotoxicity of DOX-hyd-PEG-FA NPs on KB cells was attenuated in the presence of exogenous folic acid. The IC₅₀ of DOX-hyd-PEG-FA NPs and DOX-hyd-PEG NPs on A549 cells showed no significant difference. After DOX-hyd-PEG-FA NPs were intravenously administered, the amount of DOX distributed in tumor tissue was significantly increased, while the amount of DOX distributed in heart was greatly decreased as compared with free DOX. Compared with free DOX, NPs yielded improved survival rate, prolonged life span, delayed tumor growth and reduced the cardiotoxicity in tumor bearing mice model. These results indicated that the acid sensitivity, passive and active tumor targeting abilities were likely to act synergistically to enhance the drug delivery efficiency of DOX-hyd-PEG-FA NPs. Therefore, DOX-hyd-PEG-FA NPs are a promising drug delivery system for targeted cancer therapy.     

MAT2A基因小干扰RNA诱导人肝癌细胞凋亡的分子机制

为探讨甲硫氨酸腺苷转移酶2A(MAT2A)小干扰RNA对人肝癌细胞生长和细胞凋亡的影响及其机 制,采用脂质体转染法将MAT2A小干扰RNA质粒表达载体转染人肝癌细胞系Bel 7402细胞、HepG 2细胞和 HepG3B细胞.半定量RT PCR检测MAT2A mRNA表达,Western印迹检测MAT2A 蛋白质表达, M TT法观察MAT2A小干扰RNA对肝癌细胞生长的影响,流式细胞仪及DAPI染色检测siRNA对肝癌细 胞凋亡的影响.为探讨其作用机制, 进一步检测转染后肝癌细胞MAT的活性、MAT1A mRNA表 达及SAM、SAH含量.结果发现, MAT2A小干扰RNA特异性抑制人肝癌细胞MAT2A mRNA和蛋白质 的表达, 刺激MAT表达由MAT2A向MAT1A转变, 降低了肝癌细胞中MATⅡ活性（P<005） ,从而诱导肝癌细胞凋亡; MAT2A小干扰RNA诱导Bel－7402细胞、HepG 2细胞、 Hep 3B细胞凋亡 指数分别为19．3%±2．8%、22．8%±3．5%、21．8%±4．2%, 较对照组siRNA(凋亡指数为5 2%±19%)具有明显差异(P<005).DAPI染色显示, MAT2A小干扰RNA转染组可见多个细胞核 浓缩、碎裂成蓝色的小块状,染色质凝聚,形成典型的凋亡小体, 而对照siRNA转染组未发现典型的 凋亡小体.肝癌细胞的生长也受到抑制,MAT2A小干扰RNA转染Bel 7402细胞、HepG 2细胞 、HepG3B细胞72 h后,细胞生长抑制率达高峰,分别为39．62%、41．27%、38．84%.肝癌细胞 中SAM含量明显升高(P<001),而SAH含量改变不明显, SAM/SAH变化伴随SAM含量变化而改 变.提示靶向MAT2A基因的siRNA通过升高肝癌细胞中SAM含量,刺激MAT表达由MAT2A向MAT1A转变, 从而诱导肝癌细胞凋亡,抑制肝癌细胞生长.     

A therapeutic trial of human melanomas with combined small interfering RNAs targeting adaptor molecules p130Cas and paxillin activated under expression of ganglioside GD3

We previously demonstrated that focal adhesion kinase (FAK), p130Cas and paxillin are crucially involved in the enhanced malignant properties under expression of ganglioside GD3 in melanoma cells. Therefore, molecules existing in the GD3-mediated signaling pathway could be considered as suitable targets for therapeutic intervention in malignant melanoma. The aim of this study was to determine whether blockade of p130Cas and/or paxillin by RNAi suppresses melanoma growth. We found a suitable dose (40 μM siRNA, 25 μl/tumor) of the siRNA to suppress p130Cas in the xenografts generated in nu/nu mice. Based on these results, we performed intratumoral (i.t.) treatment with anti-p130Cas and/or anti-paxillin siRNAs mixed with atelocollagen as a drug delivery system in a xenograft tumor of a human melanoma cell line, SK-MEL-28. Mixture of atelocollagen (1.75%) and an siRNA (500 or 1000 pmol/tumor) was injected into the tumors every 3 days after the first injection. An siRNA against human p130Cas markedly suppressed tumor growth of the xenograft in a dose-dependent manner, whereas siRNA against human paxillin slightly inhibited the tumor growth. A control siRNA against firefly luciferase showed no effect. To our surprise, siRNA against human p130Cas (500 or 1000 pmol/tumor) combined with siRNA against human paxillin dramatically suppressed tumor growth. In agreement with the tumor suppression effects of the anti-p130Cas siRNA, reduction in Ki-67 positive cell number as well as in p130Cas expression was demonstrated by immunohistostaining. These results suggested that blockade of GD3-mediated growth signaling pathways by siRNAs might be a novel and promising therapeutic strategy against malignant melanomas, provided signaling molecules such as p130Cas and paxillin are significantly expressed in individual cases. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.     

EpCAM aptamer mediated cancer cell specific delivery of EpCAM siRNA using polymeric nanocomplex

Background

Epithelial cell adhesion molecule (EpCAM) is overexpressed in solid tumors and regarded as a putative cancer stem cell marker. Here, we report that employing EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) dual approach, for the targeted delivery of siRNA to EpCAM positive cancer cells, efficiently inhibits cancer cell proliferation.

Results

Targeted delivery of siRNA using polyethyleneimine is one of the efficient methods for gene delivery, and thus, we developed a novel aptamer-PEI-siRNA nanocomplex for EpCAM targeting. PEI nanocomplex synthesized with EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) showed 198 nm diameter sized particles by dynamic light scattering, spherical shaped particles, of 151 ± 11 nm size by TEM. The surface charge of the nanoparticles was −30.0 mV using zeta potential measurements. Gel retardation assay confirmed the PEI-EpApt-SiEp nanoparticles formation. The difference in size observed by DLS and TEM could be due to coating of aptamer and siRNA on PEI nanocore. Flow cytometry analysis revealed that PEI-EpApt-SiEp has superior binding to cancer cells compared to EpApt or scramble aptamer (ScrApt) or PEI-ScrApt-SiEp. PEI-EpApt-SiEp downregulated EpCAM and inhibited selectively the cell proliferation of MCF-7 and WERI-Rb1 cells.

Conclusions

The PEI nanocomplex fabricated with EpApt and siEp was able to target EpCAM tumor cells, deliver the siRNA and silence the target gene. This nanocomplex exhibited decreased cell proliferation than the scrambled aptamer loaded nanocomplex in the EpCAM expressing cancer cells and may have potential for EpCAM targeting in vivo.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0108-9) contains supplementary material, which is available to authorized users.