Magnetic Cationic Amylose Nanoparticles Used to Deliver Survivin-Small Interfering RNA for Gene Therapy of Hepatocellular Carcinoma In Vitro.
ABSTRACT: Amylose is a promising nanocarrier for gene delivery in terms of its good biocompatibility and high transfection efficiency. Small interfering RNA against survivin (survivin-siRNA) can cause tumor apoptosis by silencing a hepatocellular carcinoma (HCC)-specific gene at the messenger RNA level. In this study, we developed a new class of folate-functionalized, superparamagnetic iron oxide (SPIO)-loaded cationic amylose nanoparticles to deliver survivin-siRNA to HCC cells. The cellular uptake of nanocomplexes, cytotoxicity, cell apoptosis, and gene suppression mediated by siRNA-complexed nanoparticles were tested. The results demonstrated that folate-functionalized, SPIO-loaded cationic amylose nanoparticles can mediate a specific and safe cellular uptake of survivin-siRNA with high transfection efficiency, resulting in a robust survivin gene downregulation in HCC cells. The biocompatible complex of cationic amylose could be used as an efficient, rapid, and safe gene delivery vector. Upon SPIO loading, it holds a great promise as a theranostic carrier for gene therapy of HCC.
Project description:Folate-targeted cationic magnetoliposomes (FTMLs) have been prepared with coencapsulated doxorubicin (DOX) and anionic superparamagnetic iron oxide (SPIO) nanoparticles (NPs) with 5 nm ?-Fe(2)O(3) cores and 16 nm hydrodynamic diameters. NP encapsulation (89%) was confirmed by cryogenic transmission electron microscopy (TEM), and the presence of the oppositely charged NPs did not cause liposome aggregation. The FTMLs had an average diameter of 174 ± 53 nm and existed as unilamellar and cup-shaped liposomes, which was attributed to dissimilar lipid packing parameters and the presence of PEG-lipids. A 3-fold increase in DOX release was achieved over 2 hours when the encapsulated SPIO NPs were heated by an alternating current electromagnetic field operating at radio frequencies (RF). Results with human cervical cancer cells (HeLa), which have been shown to exhibit high folate receptor (FR) expression, confirmed FTML surface binding and cellular uptake. In contrast, no uptake was observed for lower FR-expressing human breast carcinoma cells (ZR-75-1).This study discusses the design and cellular uptake of multifunctional folate-targeted cationic magnetoliposomes enabling doxorubicin delivery and SPIO labeling.
Project description: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.
Project description:Patients with hepatocellular carcinoma (HCC) are prone to folate deficiency (FD). Here we showed that, in cell line-specific manner, FD caused resistance to FD-induced oxidative stress and multi-drug resistance (MDR). This resistance was due to upregulation of glucose-regulated protein 78 (GRP78) and Survivin. Using siRNA and Epigallocatechin gallate (EGCG), we found that GRP78 and Survivin cooperatively conferred MDR by decreasing FD-induced ROS generation. Our data showed that FD increases GRP78 and Survivin, which serve as ROS inhibitors, causing MDR in HCC. We suggest that folate supplementation may enhance the efficacy of chemotherapy.
Project description:A combination of chemotherapeutic drugs and siRNA is emerging as a new modality for cancer therapy. A safe and effective carrier platform is needed for combination drug delivery. Here, a functionalized mixed micelle-based delivery system was developed for targeted co-delivery of methotrexate (MTX) and survivin siRNA. Linolenic acid (LA) was separately conjugated to branched polyethlenimine (b-PEI) and methoxy-polyethyleneglycol (mPEG). MTX was then conjugated to LA-modified b-PEI (MTX-bPEI-LA) to form a functionalized polymer-drug conjugate. Functionalized mixed micelles (M-MTX) were obtained by the self-assembly of MTX-bPEI-LA and LA-modified mPEG (mPEG-LA). M-MTX had a narrow particle size distribution and could successfully condense siRNA at an N/P ratio of 16/1. M-MTX/siRNA was selectively taken up by HeLa cells overexpressing the folate receptor (FR) and facilitated the release of the siRNA into the cytoplasm. In vitro, M-MTX/siRNA produced a synergy between MTX and survivin siRNA and markedly suppressed survivin protein expression. In tumor-bearing mice, M-MTX/Cy5-siRNA showed an elevated tumor uptake. In addition, M-MTX/siRNA inhibited tumor growth. Immunohistochemistry and a western blot analysis showed a significant target gene downregulation. In conclusion, M-MTX/siRNA was highly effective as a delivery system and may serve as a model for the targeted co-delivery of therapeutic agents.
Project description:Lipid nanoparticles (LNP) are the most potent carriers for the delivery of nucleic acid-based therapeutics. The first FDA approved a short interfering RNA (siRNA) drug that uses a cationic LNP system for the delivery of siRNA against human transthyretin (hTTR). However, preparation of such LNP involves tedious multi-step synthesis with relatively low yields. In the present study, we synthesized cationic peptidomimetic functionalized cholesterol (denote Chorn) in straightforward chemical approaches with high yield. When formulated with helper lipids, Chorn LNPs complexed with siRNA to form nanoparticles with an average diameter of 150 nm to 200 nm. Chorn LNP mediated transfection of a green fluorescence protein (GFP) expressing plasmid resulted in 60% GFP positive cells. Moreover, Chorn LNP delivered siRNA against polo-like kinase 1 (Plk1), a disease related gene in cancer cells and efficiently suppressed the expression of the gene, resulting in significant morphological changes in the cell nuclei. Our data suggested that cholesterol based cationic LNP, prepared through a robust chemical strategy, may provide a promising siRNA delivery system.
Project description:Ovarian cancer is a highly metastatic and lethal disease, making it imperative to find treatments that target late-stage malignant tumors. The packaging RNA (pRNA) of bacteriophage phi29 DNA-packaging motor has been reported to function as a highly versatile vehicle to carry small interference RNA (siRNA) for silencing of survivin. In this article, we explore the potential of pRNA as a vehicle to carry siRNA specifically targeted to metallothionein-IIa (MT-IIA) messenger RNA (mRNA), and compare it to survivin targeting pRNA. These two anti-apoptotic cell survival factors promote tumor cell viability, and are overexpressed in recurrent tumors. We find that pRNA chimeras targeting MT-IIA are processed into double-stranded siRNA by dicer, are localized within the GW/P-bodies, and are more potent than siRNA alone in silencing MT-IIA expression. Moreover, knockdown of both survivin and MT-IIA expression simultaneously results in more potent effects on cell proliferation in the aggressive ovarian tumor cell lines than either alone, suggesting that therapeutic approaches that target multiple genes are essential for molecular therapy. The folate receptor-targeted delivery of siRNA by the folate-pRNA dimer emphasizes the cancer cell-specific aspect of this system. The pRNA system, which has the capability to assemble into multivalent nanoparticles, has immense promise as a highly potent therapeutic agent.
Project description:Nanotechnology offers many benefits, and here we report an advantage of applying RNA nanotechnology for directional control. The orientation of arrow-shaped RNA was altered to control ligand display on extracellular vesicle membranes for specific cell targeting, or to regulate intracellular trafficking of small interfering RNA (siRNA) or microRNA (miRNA). Placing membrane-anchoring cholesterol at the tail of the arrow results in display of RNA aptamer or folate on the outer surface of the extracellular vesicle. In contrast, placing the cholesterol at the arrowhead results in partial loading of RNA nanoparticles into the extracellular vesicles. Taking advantage of the RNA ligand for specific targeting and extracellular vesicles for efficient membrane fusion, the resulting ligand-displaying extracellular vesicles were capable of specific delivery of siRNA to cells, and efficiently blocked tumour growth in three cancer models. Extracellular vesicles displaying an aptamer that binds to prostate-specific membrane antigen, and loaded with survivin siRNA, inhibited prostate cancer xenograft. The same extracellular vesicle instead displaying epidermal growth-factor receptor aptamer inhibited orthotopic breast cancer models. Likewise, survivin siRNA-loaded and folate-displaying extracellular vesicles inhibited patient-derived colorectal cancer xenograft.
Project description:Bladder cancer is the most common malignancy of the urinary tract for which the accurate measurement of minimal residual disease is critical to treatment and determining prognosis. Although cystoscope examination and voided urine cytology remain the current standard of care for detecting residual disease, these approaches are limited by mechanical trauma and lack sensitivity. To develop a new accurate noninvasive method, we developed a novel contrast agent where the surface of superparamagnetic iron oxide (SPIO) nanoparticles is functionalized with a bladder cancer-specific fluorescein isothiocyanate (FITC) labeled cell penetrating peptide (CPP)-polyarginine peptides (R11) for active targeting and imaging. The stable nanoparticles have an average hydrodynamic diameter of 51 nm, surface charge of -21 mV and MRI r2 relaxivity 135 mM-1s-1. In vitro cell studies demonstrated that the R11-conjugated SPIO (SPIO-R11) nanoparticles were taken up by bladder cancer cells (T24) in a dose-dependent manner, which was higher than unconjugated SPIO. TEM showed that SPIO-R11 was mainly concentrated on cell vesicle and lysosome, not in cell nucleus, and no obvious damage was seen on cell ultrastructure. Moreover, uptake of the nanoparticles showed significantly more SPIO-R11 accumulation in bladder cancer cells than in immortalized bladder epithelial cells unlike control SPIO. Further, SPIO-R11 was compatible with immortalized bladder epithelial cells at all tested concentrations up to 200 ?g/mL after 72 h incubation. Moreover, SPIO-R11 decreased the magnetic resonance T2 relaxation time by 73% in tumors cells in vitro compared to 12% with SPIO. These results indicate great potential of SPIO-R11 as contrast agent to target bladder cancer for diagnostic and therapeutic applications.
Project description:The co-delivery of chemotherapeutic agents and small interfering RNA (siRNA) within one cargo can enhance the anticancer outcomes through its synergistic therapeutic effects.We prepared smart polymeric nanoparticles (NPs) with pH-responsive and poly(ethylene glycol) (PEG)-detachable properties to systemically co-deliver paclitaxel (PTX) and siRNA against survivin gene for lung cancer therapy. The cationic polyethyleneimine-block-polylactic acid (PEI-PLA) was first synthesized and characterized, with good biocompatibility. PTX was encapsulated into the hydrophobic core of the PEI-PLA polymers by dialysis, and then the survivin siRNA was loaded onto the PTX-loaded NPs (PEI-PLA/PTX) through electrostatic interaction between siRNA and PEI block. Finally, the negatively charged poly(ethylene glycol)-block-poly(L-aspartic acid sodium salt) (PEG-PAsp) was coated onto the surface of NPs by electrostatic interaction to form final smart polymeric NPs with mean particle size of 82.4 nm and zeta potential of 4.1 mV. After uptake of NPs by tumor cells, the PEG-PAsp segments became electrically neutral owing to the lower endosome pH and consequently detached from the smart NPs. This process allowed endosomal escape of the NPs through the proton-sponge effect of the exposed PEI moiety.The resulting NPs achieved drug loading of 6.04 wt% and exhibited good dispersibility within 24 h in 10% fetal bovine serum (FBS). At pH 5.5, the NPs presented better drug release and cellular uptake than at pH 7.4. The NPs with survivin siRNA effectively knocked down the expression of survivin mRNA and protein owing to enhanced cell uptake of NPs. Cell counting kit-8 (CCK-8) assay showed that the NPs presented low systemic toxicity and improved antiproliferation effect of PTX on A549 cells. Moreover, in vivo studies demonstrated that accumulated NPs in the tumor site were capable of inhibiting the tumor growth and extending the survival rate of the mice by silencing the survivin gene and delivering PTX into tumor cells simultaneously.These results indicate that the prepared nano-vectors could be a promising co-delivery system for novel chemo/gene combination therapy.
Project description:Atherosclerosis is at the onset of the cardiovascular diseases that are among the leading causes of death worldwide. Currently, high-risk plaques, also called vulnerable atheromatous plaques, remain often undiagnosed until the occurrence of severe complications, such as stroke or myocardial infarction. Molecular imaging agents that target high-risk atheromatous lesions could greatly improve the diagnosis of atherosclerosis by identifying sites of high disease activity. Moreover, a "theranostic approach" that combines molecular imaging agents (for diagnosis) and therapeutic molecules would be of great value for the local management of atheromatous plaques. The aim of this study was to develop and characterize an innovative theranostic tool for atherosclerosis. We engineered oil-in-water nano-emulsions (NEs) loaded with superparamagnetic iron oxide (SPIO) nanoparticles for magnetic resonance imaging (MRI) purposes. Dynamic MRI showed that NE-SPIO nanoparticles decorated with a polyethylene glycol (PEG) layer reduced their liver uptake and extended their half-life. Next, the NE-SPIO-PEG formulation was functionalized with a fully human scFv-Fc antibody (P3) recognizing galectin 3, an atherosclerosis biomarker. The P3-functionalized formulation targeted atheromatous plaques, as demonstrated in an immunohistochemistry analyses of mouse aorta and human artery sections and in an <i>Apoe</i><sup>-/-</sup> mouse model of atherosclerosis. Moreover, the formulation was loaded with SPIO nanoparticles and/or alpha-tocopherol to be used as a theranostic tool for atherosclerosis imaging (SPIO) and for delivery of drugs that reduce oxidation (here, alpha-tocopherol) in atheromatous plaques. This study paves the way to non-invasive targeted imaging of atherosclerosis and synergistic therapeutic applications.