as-1411 and Glioma

Radioresistance significantly decreases the efficacy of radiotherapy, which can ultimately lead to tumor recurrence and metastasis. As a novel type of nano-radiosensitizer, silver nanoparticles (AgNPs) have shown promising radiosensitizing properties in the radiotherapy of glioma, but their ability to efficiently enter and accumulate in tumor cells needs to be improved. In the current study, AS1411 and verapamil (VRP) conjugated bovine serum albumin (BSA) coated AgNPs (AgNPs@BSA-AS-VRP) were synthesized and characterized. Dark-field imaging and inductively coupled plasma mass spectrometry were applied to investigate the accumulation of AgNPs@BSA-AS and AgNPs@BSA-AS-VRP mixed in different ratios in U251 glioma cells. To assess the influences of 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP on the P-glycoprotein (P-gp) efflux activity, rhodamine 123 accumulation assay was carried out. Colony formation assay and tumor-bearing nude mice model were employed to examine the radiosensitizing potential of 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP. Thioredoxin Reductase (TrxR) Assay Kit was used to detect the TrxR activity in cells treated with different functionally modified AgNPs. Characterization results revealed that AgNPs@BSA-AS-VRP were successfully constructed. When AgNPs@BSA-AS and AgNPs@BSA-AS-VRP were mixed in a ratio of 19:1, the amount of intracellular nanoparticles increased greatly through AS1411-mediated active targeting and inhibition of P-gp activity. In vitro and in vivo experiments clearly showed that the radiosensitization efficacy of 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP was much stronger than that of AgNPs@BSA and AgNPs@BSA-AS. It was also found that 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP significantly inhibited intracellular TrxR activity. These results indicate that 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP can effectively accumulate in tumor cells and have great potential as high-efficiency nano-radiosensitizers in the radiotherapy of glioma.

Topics: Aptamers, Nucleotide; Brain Neoplasms; Cell Line, Tumor; Glioma; Humans; Metal Nanoparticles; Oligodeoxyribonucleotides; Radiation Tolerance; Radiation-Sensitizing Agents; Silver; Verapamil

RNA interference is a promising therapy in glioma treatment. However, the application of RNA interference has been limited in glioma therapy by RNA instability and the lack of tumor targeting. Here, we report a novel DNA tetrahedron, which can effectively deliver small interfering RNA to glioma cells and induce apoptosis.. siRNA, a small interfering RNA that can suppress the expression of survivin in glioma, was loaded into the DNA tetrahedron (TDN). To enhance the ability of active targeting of this nanoparticle, we modified one side of the DNA nanostructure with aptamer as1411 (As-TDN-R), which can selectively recognize the nucleolin in the cytomembrane of tumor cells. The modified nanoparticles were characterized by agarose gel electrophoresis, dynamic light scattering, and transmission electron microscopy. The serum stability was evaluated by agarose gel electrophoresis. Nucleolin was detected by Western blot and immunofluorescence, and targeted cellular uptake was examined by flow cytometry. The TUNEL assay, flow cytometry, and Western Blot were used to detect apoptosis in U87 cells. The gene silencing of survivin was examined by qPCR, Western Blot, and immunofluorescence.. As-TDN-R alone showed better stability towards siRNA, indicating that TDN was a good siRNA protector. Compared with TDN alone, there was increased intercellular uptake of As-TDN-R by U87 cells, evidenced by overexpressed nucleolin in glioma cell lines. TUNEL assay, flow cytometry, and Western Blot revealed increased apoptosis in the As-TDN-R group. The downregulation of survivin protein and mRNA expression levels indicated that As-TDN-R effectively silenced the target gene.. The novel nanoparticle can serve as a good carrier for targeting siRNA delivery in glioma. Further exploration of the DNA nanostructure can greatly promote the application of DNA-based drug systems in glioma.

Topics: Apoptosis; Aptamers, Nucleotide; Cell Death; Cell Line, Tumor; Cell Survival; DNA; Down-Regulation; Endocytosis; Gene Silencing; Gene Transfer Techniques; Glioma; Human Umbilical Vein Endothelial Cells; Humans; Nanostructures; Nucleolin; Oligodeoxyribonucleotides; Phosphoproteins; RNA-Binding Proteins; RNA, Small Interfering; Survivin

In this study, we developed an AS1411 aptamer/hyaluronic acid-bifunctionalized microemulsion co-loading shikonin and docetaxel (AS1411/SKN&DTX-M). Such microemulsion was capable of penetrating the blood-brain barrier (BBB), targeting CD44/nucleolin-overexpressed glioma, and inhibiting the orthotopic glioma growth. AS1411/SKN&DTX-M showed a spherical morphology with a diameter around 30 nm and rapidly released drugs in the presence of hyaluronidase and mild acid. In the U87 cellular studies, AS1411/SKN&DTX-M elevated the cytotoxicity, enhanced the cellular uptake, and induced the cell apoptosis. In the artificial blood-brain barrier model, the transepithelial electrical resistance was decreased after the treatment with AS1411/SKN&DTX-M and thereby of increasing the apparent permeability coefficient. Furthermore, AS1411/SKN&DTX-M showed a strong inhibition against the formation of cancer stem cell-enriched U87 cell spheroids, in which the expression of CD133 was downregulated significantly. In the biodistribution studies, AS1411/SKN&DTX-M could selectively accumulate in the brains of orthotopic luciferase-transfected U87 glioma tumor-bearing nude mice. Importantly, AS1411/SKN&DTX-M exhibited the overwhelming inhibition of glioma growth of orthotopic luciferase-transfected U87 glioma models and reached the longest survival period among all the treatments. In summary, the codelivery of shikonin and docetaxel using bifunctionalization with hyaluronic acid and AS1411 aptamer offers a promising strategy for dual drug-based combinational antiglioma treatment.

Topics: Animals; Antineoplastic Agents; Apoptosis; Aptamers, Nucleotide; Cell Line; Cell Line, Tumor; Docetaxel; Drug Delivery Systems; Emulsions; Glioma; Humans; Hyaluronic Acid; Mice; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Naphthoquinones; Nucleolin; Oligodeoxyribonucleotides; Phosphoproteins; RNA-Binding Proteins; Tissue Distribution

The efficacy of radiotherapy for glioma is often limited by the radioresistance of glioma cells. The radiosensitizing effects of silver nanoparticles (AgNPs) on glioma were found in the previous studies of our group. In order to enhance the radiosensitivity of tumor cells and selectively kill them while reducing the side effects of irradiation therapy, targeted modification of AgNPs is urgently needed.. In the present study, AgNPs functionalized with polyethylene glycol (PEG) and aptamer As1411 (AsNPs) were synthesized and subsequently characterized by transmission electron microscopy, ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy. Then the targeting property of AsNPs was evaluated by dark-field imaging, confocal microscopy and in vivo imaging. Both colony formation assay and glioma-bearing mouse model were employed to study the radiosensitizing effect of AsNPs.. The characterization results revealed a spherical shape of AgNPs with an average diameter of 18 nm and the successful construction of AsNPs. AsNPs were confirmed to specifically target C6 glioma cells, but not normal human microvascular endothelial cells. Moreover, AsNPs could not only internalize into tumor cells, but also penetrate into the core of tumor spheroids. In vitro experiments showed that AsNPs exhibited a better radiosensitizing effect than AgNPs and PEGylated AgNPs (PNPs), inducing a higher rate of apoptotic cell death. In vivo imaging demonstrated that Cy5-AsNPs preferentially accumulated at the tumor site, and the ratio of fluorescence intensity of Cy5-AsNPs to that of Cy5-PNPs reached the maximum at 6 h post-systemic administration. Furthermore, the combination of AsNPs with irradiation significantly prolonged the median survival time of C6 glioma-bearing mice.. Our results indicated that AsNPs could be an effective nano-radiosensitizer for glioma targeting treatment.

Topics: Animals; Apoptosis; Aptamers, Nucleotide; Cell Line, Tumor; Disease Models, Animal; Endothelial Cells; Female; Glioma; Humans; Hydrodynamics; Metal Nanoparticles; Mice, Inbred BALB C; Mice, Nude; Oligodeoxyribonucleotides; Particle Size; Polyethylene Glycols; Radiation-Sensitizing Agents; Rats; Silver; Spheroids, Cellular; Static Electricity; Tissue Distribution

One of the major abundant proteins in the nucleous is nucleolin that overexpressed on the cytoplasmic membrane of malignant and endothelial cells and makes it as a promising condidate for targeted drug delivery.. In this study, doxorubicin (Dox) as a chemotherapy drug was entrapped into the Poly lacticco- glycolic acid (PLGA)-based nanoparticles (NPs). Then, the targeting ability of anti nucleolin AS1411 aptamer-targeted Dox-encapsulated PLGA-based NPs (AS1411-NPs) was investigated in high nucleolin-expressing C26 colon carcinoma and rat C6 glioma cell lines compared with low nucleolinexpressing mouse L929 cell line.. We recently first assessed the existence of cell surface nucleolin of these three different cell lines by immunocytochemistry method. We found that a large amount of nucleolin was localized in the cytoplasmic membrane of C26 and C6 cell lines, with a very smaller amount on the surface of L929 cell line.. As a result, more rapidly internalization of AS1411-NPs into the C26 and C6 cells compared with L929 cells was verified.. We think that AS1411-NPs, as a ligand, first bind to nucleolin, as a receptor, and then the receptor-ligand complex is more efficiently incorporated into the high nucleolin-expressing cell lines through receptor-mediated endocytosis pathway.

Topics: Animals; Antibiotics, Antineoplastic; Aptamers, Nucleotide; Cell Line; Cell Proliferation; Colonic Neoplasms; Doxorubicin; Drug Delivery Systems; Drug Screening Assays, Antitumor; Glioma; Humans; Ligands; Mice; Nanoparticles; Nucleolin; Oligodeoxyribonucleotides; Particle Size; Phosphoproteins; Polylactic Acid-Polyglycolic Acid Copolymer; Rats; RNA-Binding Proteins; Surface Properties

AS1411 binds nucleolin (NCL) and is the first oligodeoxynucleotide aptamer to reach phase I and II clinical trials for the treatment of several cancers. However, the mechanisms by which AS1411 targets and kills glioma cells and tissues remain unclear. Here we report that AS1411 induces cell apoptosis and cycle arrest, and inhibits cell viability by up-regulation of p53 and down-regulation of Bcl-2 and Akt1 in human glioma cells. NCL was overexpressed in both nucleus and cytoplasm in human glioma U87, U251 and SHG44 cells compared to normal human astrocytes (NHA). AS1411 bound NCL and inhibited the proliferation of glioma cells but not NHA, which was accompanied with up-regulation of p53 and down-regulation of Bcl-2 and Akt1. Moreover, AS1411 treatment resulted in the G2/M cell cycle arrest in glioma cells, which was however abolished by overexpression of NCL. Further, AS1411 induced cell apoptosis, which was prevented by silencing of p53 and overexpression of Bcl-2. In addition, AS1411 inhibited the migration and invasion of glioma cells in an Akt1-dependent manner. Importantly, AS1411 inhibited the growth of glioma xenograft and prolonged the survival time of glioma tumor-bearing mice. These results revealed a promising treatment of glioma by oligodeoxynucleotide aptamer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Aptamers, Nucleotide; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Glioma; Humans; Male; Mice; Mice, SCID; Middle Aged; NF-kappa B; Nucleolin; Oligodeoxyribonucleotides; Phosphoproteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; RNA-Binding Proteins; Signal Transduction; Survival Analysis; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

To evaluate the internalization and subcellular fate of AS1411 aptamer (for glioma targeting) and TGN peptide (for blood-brain barrier targeting)-modified nanoparticles (AsTNPs), which was important for optimizing targeted delivery systems and realizing the potential toxicity to cells.. Organelles were labelled with specific markers. Several uptake inhibitors were used to determine the endocytosis pathways. Transmission electron microscopy (TEM) was utilized to directly observe the endocytosis procedure and subcellular fate of AsTNPs.. Subcellular localization demonstrated that endosomes and mitochondria were involved in the uptake of AsTNPs by both C6 and bEnd.3 cells, however, lysosomes and Golgi apparatus were only involved in the internalization by C6 cells rather than bEnd.3 cells. Uptake mechanism study demonstrated the clathrin- and caveolae-mediated endocytosis were the main pathways in the uptake of AsTNPs by C6 and bEnd.3 cells. However, other pathways, including clathrin- and caveolae-independent endocytosis and macropinocytosis are also involved in the uptake by C6 cells and not by bEnd.3 cells. TEM directly demonstrated the involvement of these pathways. Particles could be found mostly in endosomes.. Compared to unmodified nanoparticles, AsTNPs displayed different internalization pathways involved in several cell organelles.

Topics: Animals; Aptamers, Nucleotide; Blood-Brain Barrier; Brain Neoplasms; Cell Line, Tumor; Drug Carriers; Endocytosis; Endothelial Cells; Glioma; Mice; Microscopy, Electron, Transmission; Nanoparticles; Oligodeoxyribonucleotides; Particle Size; Peptides; Subcellular Fractions; Surface Properties

Targeted delivery of therapeutic nanoparticles in a disease-specific manner represents a potentially powerful technology especially when treating infiltrative brain tumors such as gliomas. We developed a nanoparticulate drug delivery system decorated with AS1411 (Ap), a DNA aptamer specifically binding to nucleolin which was highly expressed in the plasma membrane of both cancer cells and endothelial cells in angiogenic blood vessels, as the targeting ligand to facilitate anti-glioma delivery of paclitaxel (PTX). Ap was conjugated to the surface of PEG-PLGA nanoparticles (NP) via an EDC/NHS technique. With the conjugation confirmed by Urea PAGE and XPS, the resulting Ap-PTX-NP was uniformly round with particle size at 156.0 ± 54.8 nm and zeta potential at -32.93 ± 3.1 mV. Ap-nucleolin interaction significantly enhanced cellular association of nanoparticles in C6 glioma cells, and increased the cytotoxicity of its payload. Prolonged circulation and enhanced PTX accumulation at the tumor site was achieved for Ap-PTX-NP, which eventually obtained significantly higher tumor inhibition on mice bearing C6 glioma xenografts and prolonged animal survival on rats bearing intracranial C6 gliomas when compared with PTX-NP and Taxol(®). The results of this contribution demonstrated the potential utility of AS1411-functionalized nanoparticles for a therapeutic application in the treatment of gliomas.

Topics: Animals; Aptamers, Nucleotide; Cell Proliferation; Drug Delivery Systems; Electrophoresis, Polyacrylamide Gel; Glioma; Humans; Inhibitory Concentration 50; Injections, Intravenous; Kaplan-Meier Estimate; Lactic Acid; Magnetic Resonance Spectroscopy; Mice; Mice, Nude; Nanoparticles; Oligodeoxyribonucleotides; Paclitaxel; Particle Size; Photoelectron Spectroscopy; Polyethylene Glycols; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Rats; Static Electricity; Surface Properties; Tissue Distribution; Treatment Outcome

The recent advances in molecular imaging techniques, using cancer-targeting nanoparticle probes, provide noninvasive tracking information on cancer cells in living subjects. Here, we report a multimodal cancer-targeted imaging system capable of concurrent fluorescence imaging, radionuclide imaging, and MRI in vivo.. A cobalt-ferrite nanoparticle surrounded by fluorescent rhodamine (designated MF) within a silica shell matrix was synthesized with the AS1411 aptamer (MF-AS1411) that targets nucleolin (a cellular membrane protein highly expressed in cancer) using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC). This purified MF-AS1411 particle was bound with 2-(p-isothio-cyanatobenzyl)-1,4,7-triazacyclonane-1,4,7-triacetic acid (p-SCN-bn-NOTA) chelating agent and further labeled with (67)Ga-citrate (MFR-AS1411). The shape and size distribution of MFR-AS1411 were characterized by transmission electron microscope (TEM). The cellular distribution of the nucleolin protein using the MFR-AS1411 nanoparticle was detected by fluorescence confocal microscopy. Phantom MR images were obtained as the concentration of MFR-AS1411 increased, using a 1.5-T MRI scanner. In vivo (67)Ga radionuclide imaging and MRI were performed using a gamma-camera and a 1.5-T MR imager, respectively.. TEM imaging revealed MF and MFR-AS1411 to be spheric and well dispersed. The purified MFR-AS1411 nanoparticle showed specific fluorescence signals in nucleolin-expressing C6 cells, compared with MFR-AS1411 mutant (MFR-AS1411mt)-treated C6 cells. The rhodamine fluorescence intensity and (67)Ga activity of MFR-AS1411 were enhanced in a dose-dependent manner as the concentration of MFR-AS1411 was increased. The (67)Ga radionuclide was detected in both thighs of the mice injected with MFR-AS1411, whereas the MFR-AS1411 mutant (MFR-AS1411mt) administration revealed rapid clearance via the bloodstream, demonstrating that MFR-AS1411 specifically targeted cancer cells. Bioluminescence images in the C6 cells, stably expressing the luciferase gene, illustrated the in vivo distribution. T2-weighted MR images of the same mice injected with MFR-AS1411 showed dark T2 signals inside the tumor region, compared with the MRI signal of the tumor region injected with MFR-AS1411mt particles.. We developed a nanoparticle-based cancer-specific imaging probe using the AS1411 aptamer in vivo and in vitro. This multimodal targeting imaging strategy, using a cancer-specific AS1411 aptamer, can be used as a versatile imaging tool for specific cancer diagnosis.

Topics: Animals; Aptamers, Nucleotide; Cell Line, Tumor; Citrates; Drug Design; Fluorescent Dyes; Gallium; Glioma; Magnetic Resonance Imaging; Mice; Mice, Nude; Microscopy, Confocal; Microscopy, Electron, Transmission; Nanoparticles; Neoplasms; Nucleolin; Oligodeoxyribonucleotides; Phantoms, Imaging; Phosphoproteins; Radionuclide Imaging; Radiopharmaceuticals; Rats; Rhodamines; RNA-Binding Proteins