Human Glioblastoma Cell Line U87MG Research Articles

Chemical composition of essential and fixed oils of Tagetes erecta fruits (Iran) and their implications in inhibition of cancer signaling

The current research was conducted to explore, for the first time, Tagetes erecta L. (family Asteraceae) fruits from northwest Iran in terms of the chemical composition of essential and fixed oils, their cytotoxic activities, and the inhibitory effect of essential oil on the PI3K/AKT/mTOR signaling pathway. The volatile oil was obtained through hydrodistillation (Clevenger apparatus). According to gas chromatography–mass spectrometry analysis, the essential oil was rich in cyclic monoterpenoids, 2-isopropyl-5-methyl-3-cyclohexen-1-one (19.99%), d-limonene (12.75%), terpinolene (11.64%) and also the saturated fatty acid palmitic acid (19.09%). Furthermore, the seeds of T. erecta were extracted using hexane by the maceration method. The analysis of fatty acid profile of the fixed oil by gas chromatography-flame ionization detector (GC-FID) demonstrated that the most predominant fatty acids in fixed oil were linoleic acid (59.53%), palmitic acid (13.70%), stearic acid (10.20%), and oleic acid (9.20%). The cytotoxic activity of essential oil, crude oil, and fraction A (obtained from fixed oil) were evaluated by using the MTT assay on MCF7 (human breast cancer cell line), PC3 (human prostate cancer cell line), and U87MG (human glioblastoma cell line). Finally, the effect of essential oil on inhibiting the PI3K/Akt/mTOR signaling pathway was evaluated using real-time PCR. The essential oil exhibited vigorous cytotoxic activity on the U87MG cell line, with an IC50 value of 32.65 μg/mL. Interestingly, the essential oil significantly inhibited the PI3K/AKT/mTOR cascade compared to the non-treated group. Our results suggest that the essential oil holds promise as an anticancer agent for glioblastoma cell lines. To the best of our knowledge, this study is the first to report on the profile of the essential oil of T. erecta fruits and its implications for targeting the PI3K/AKT/mTOR signaling pathway.

Polymer-lipid hybrid nanomedicines to deliver siRNA in and against glioblastoma cells

Small interfering RNA (siRNA) holds great potential to treat many difficult-to-treat diseases, but its delivery remains the central challenge. This study aimed at investigating the suitability of polymer-lipid hybrid nanomedicines (HNMeds) as novel siRNA delivery platforms for locoregional therapy of glioblastoma. Two HNMed formulations were developed from poly(lactic-co-glycolic acid) polymer and a cationic lipid: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol). After characterization of the HNMeds, a model siRNA was complexed onto their surface to form HNMed/siRNA complexes. The physicochemical properties and siRNA binding ability of complexes were assessed over a range of nitrogen-to-phosphate (N/P) ratios to optimize the formulations. At the optimal N/P ratio of 10, complexes effectively bound siRNA and improved its protection from enzymatic degradation. Using the NIH3T3 mouse fibroblast cell line, DOTAP-based HNMeds were shown to possess higher cytocompatibility in vitro over the DC-Chol-based ones. As proof-of-concept, uptake and bioefficacy of formulations were also assessed in vitro on U87MG human glioblastoma cell line expressing luciferase gene. Complexes were able to deliver anti-luciferase siRNA and induce a remarkable suppression of gene expression. Noteworthy, the effect of DOTAP-based formulation was not only about three-times higher than DC-Chol-based one, but also comparable to lipofectamine model transfection reagent. These findings set the basis to exploit this nanosystem for silencing relevant GB-related genes in further in vitro and in vivo studies.

Comprehensive antifungal investigation of green synthesized silver nanoformulation against four agriculturally significant fungi and its cytotoxic applications

The present study reports the green synthesis of silver nanoparticles (AgNPs) in powder form using the leaf extract of Azadirachta indica. The synthesis of AgNPs was confirmed by UV–vis spectroscopy, FTIR, XRD, FESEM, and EDX. The synthesized AgNPs were in a powdered state and dispersed completely in 5% polyethylene glycol (PEG) and demonstrated prolonged shelf life and enhanced bioavailability over a year without any aggregation. The resulting silver nanoformulation demonstrated complete inhibition against Sclerotinia sclerotiorum and Colletotrichum falcatum and 68% to 80% inhibition against Colletotrichum gloeosporioides and Rhizoctonia solani respectively, at 2000 ppm. The EC50 values determined through a statistical analysis were 66.42, 157.7, 19.06, and 33.30 ppm for S. sclerotiorum, C. falcatum, C. gloeosporioides, and R. solani respectively. The silver nanoformulation also established significant cytotoxicity, with a 74.96% inhibition rate against the human glioblastoma cell line U87MG at 250 ppm. The IC50 value for the cancerous cell lines was determined to be 56.87 ppm through statistical analysis. The proposed silver nanoformulation may be used as a next-generation fungicide in crop improvement and may also find application in anticancer investigations. To the best of our knowledge, this is also the first report of silver nanoformulation demonstrating complete inhibition against the economically significant phytopathogen C. falcatum.

10073-CBMS-6 CURCUMIN ANALOG B EXHIBIT ANTI-TUMOR ACTIVITY AGAINST GLIOBLASTOMAAT LOWER CONCENTRATIONS THAN CURCUMIN

Abstract PURPOSE Glioblastoma (GBM) has a high risk of recurrence and a poor prognosis due to the difficulty of surgical resection and resistance to the standard treatment, temozolomide. Therefore, the development of noble therapeutic agents against GBM is required. This study investigated the anti-tumor activity of the curcumin (Cur) analog Compound B (ComB) against GBM. METHODS AND RESULTS To evaluate anti-tumor activity against GBM, we performed an MTT assay. The human glioblastoma cell lines U87-MG and U251 were pre-treated with Cur or ComB, and then cell viability was examined using a cell counting kit, and IC50 values were calculated. The IC50 value for U87-MG of Cur was 9.78 μM, and that of ComB was1.28 μM. The IC50 value for U251 of Cur was 9.50 μM, and that of ComB was 0.64 μM. To examine the effects of ComB on normal cells, the same MTT assay was performed on primary cultured astrocytes prepared from neonatal rats. ComB did not reduce cell viability in astrocytes at concentrations that had an anti-tumor effect on GBM cells. Next, a cell cycle analysis was performed with Propidium Iodide (PI) staining and an apoptosis assay with Annexin V/PI staining using flow cytometry. ComB induced cell cycle G2/M arrest and apoptosis at lower concentrations than Cur. qPCR showed that mRNA expression levels of CDK1 and CyclinB1, which play an essential role in the progression from the G2 phase to the M phase, decreased by ComB at lower concentrations than Cur. DISCUSSION These results suggest that ComB, at lower concentrations than Cur, has an anti-tumor effect without affecting normal cells by inducing cell cycle arrest and apoptosis against GBM. Further detailed analysis and in vivo studies are expected to lead to the development of novel therapeutic agents for GBM.

DDDR-16. CURCUMIN ANALOGS EXHIBIT ANTI-TUMOR ACTIVITY AGAINST GLIOBLASTOMA AT LOWER CONCENTRATIONS THAN CURCUMIN

Abstract PURPOSE Glioblastoma (GBM) has a high risk of recurrence and a poor prognosis due to the difficulty of surgical resection and resistance to the standard treatment, temozolomide. Therefore, the development of new therapeutic agents against GBM is required. This study investigated the anti-tumor activity of the curcumin (Cur) analogs Compound A (ComA) and B (ComB) against GBM. METHODS AND RESULTS To evaluate anti-tumor activity against GBM, we performed a MTT assay. The human glioblastoma cell lines U87-MG and U251 were pre-treated with Cur, ComA, or ComB, then cell viability was examined using a cell counting kit, and IC50 values were calculated. The IC50 values for U87-MG were: Cur, 9.78 µM; ComA, 2.42 µM; and ComB, 1.28 µM. Those for U251 were: Cur, 9.50 µM; ComA, 2.27 µM; ComB, 0.64 µM. To examine the effects of ComA and ComB on normal cells, the same MTT assay was performed on primary cultured astrocytes from neonatal rats. Neither ComA nor ComB resulted in reduced cell viability in astrocytes at concentrations that had an anti-tumor effect on GBM cells (ComA, 3 µM; ComB, 1.5 µM). Next, a cell cycle analysis was performed with PI staining and an apoptosis assay with Annexin V/PI staining using flow cytometry. qPCR was performed to examine the mRNA expression levels of the cell cycle-related proteins CDK1 and CyclinB1. Both ComA and ComB, at lower concentrations than Cur, induced 1) G2/M phase arrest due to a decrease in the mRNA expression levels of CDK1 and CyclinB1 and 2) apoptosis. DISCUSSION These results suggest that both ComA and ComB, at lower concentrations than Cur, have an anti-tumor effect without affecting normal cells by inducing cell cycle arrest and apoptosis against GBM. Further detailed analysis and in vivo studies are expected to lead to the development of novel therapeutic agents for GBM.

Preparation and Characterization of Hydrogel Films and Nanoparticles Based on Low-Esterified Pectin for Anticancer Applications.

Prospective adjuvant anticancer therapy development includes the establishing of drug delivery systems based on biocompatible and biodegradable carriers. We have designed films and nanoparticles (NPs) based on low-esterified pectin hydrogel using the ionic gelation method. We investigated morphology, nanomechanical properties, biocompatibility and anticancer activity. Hydrogel films are characterized by tunable viscoelastic properties and surface nanoarchitectonics through pectin concentration and esterification degree (DE), expressed in variable pore frequency and diameter. An in vitro study showed a significant reduction in metabolic activity and the proliferation of the U87MG human glioblastoma cell line, probably affected via the adhesion mechanism. Glioma cells formed neurosphere-like conglomerates with a small number of neurites when cultured on fully de-esterified pectin films and they did not produce neurites on the films prepared on 50% esterified pectin. Pectin NPs were examined in terms of size distribution and nanomechanical properties. The NPs' shapes were proved spherical with a mean diameter varying in the range of 90-115 nm, and a negative zeta potential from -8.30 to -7.86 mV, which indicated their stability. The NPs did not demonstrate toxic effect on cells or metabolism inhibition, indicating good biocompatibility. Nanostructured biomaterials prepared on low-esterified pectins could be of interest for biomedical applications in adjuvant anticancer therapy and for designing drug delivery systems.

MTOR Inhibition Is Effective against Growth, Survival and Migration, but Not against Microglia Activation in Preclinical Glioma Models.

Initially introduced in therapy as immunosuppressants, the selective inhibitors of mTORC1 have been approved for the treatment of solid tumors. Novel non-selective inhibitors of mTOR are currently under preclinical and clinical developments in oncology, attempting to overcome some limitations associated with selective inhibitors, such as the development of tumor resistance. Looking at the possible clinical exploitation in the treatment of glioblastoma multiforme, in this study we used the human glioblastoma cell lines U87MG, T98G and microglia (CHME-5) to compare the effects of a non-selective mTOR inhibitor, sapanisertib, with those of rapamycin in a large array of experimental paradigms, including (i) the expression of factors involved in the mTOR signaling cascade, (ii) cell viability and mortality, (iii) cell migration and autophagy, and (iv) the profile of activation in tumor-associated microglia. We could distinguish between effects of the two compounds that were overlapping or similar, although with differences in potency and or/time-course, and effects that were diverging or even opposite. Among the latter, especially relevant is the difference in the profile of microglia activation, with rapamycin being an overall inhibitor of microglia activation, whereas sapanisertib was found to induce an M2-profile, which is usually associated with poor clinical outcomes.

Inducible Knockout of 14-3-3β Attenuates Proliferation and Spheroid Formation in a Human Glioblastoma Cell Line U87MG

Glioblastomas (GBs) are the most common and malignant brain tumors in adults. A protein encoded by the gene YWHAB, 14-3-3β, is commonly found to be upregulated throughout the initiation and progression of GB. The 14-3-3β has oncogenic roles in several different types of cancer cells through interactions with proteins such as Bad, FBI1, Raf-1, Cdc25b, and others. Previous RNA interference studies have shown that 14-3-3β promotes proliferation, cell cycle progression, and migration and invasion of GB cells. However, despite the many oncogenic functions of 14-3-3β, a CRISPR/Cas9 knockout model of 14-3-3β has not been investigated. This study confirmed previous findings and showed that siRNA inhibition of 14-3-3β results in reduced cellular proliferation in a human glioblastoma cell line, U87MG. We also used a YWHAB Tet-On CRISPR/Cas9 U87MG cell line that, upon doxycycline induction, leads to robust Cas9 expression and subsequent knockout of 14-3-3β. Using this model, we show that loss of 14-3-3β significantly reduces cellular proliferation and spheroid formation of U87MG cells.

Nanoarchitectured conjugates targeting angiogenesis: investigating heparin-taurocholate acid conjugates (LHT7) as an advanced anti-angiogenic therapy for brain tumor treatment

BackgroundGlioblastoma is a highly malignant brain tumor associated with poor prognosis. Conventional therapeutic approaches have limitations due to their toxic effects on normal tissue and the development of tumor cell resistance. This study aimed to explore alternative mechanisms for glioblastoma treatment by targeting angiogenesis.MethodsThe study investigated the anti-angiogenic properties of heparin in glioblastoma treatment. To overcome the limitations of heparin, a heparin-taurocholate conjugate (LHT7) was synthesized by conjugating heparin to taurocholic acid. The study utilized the U87MG human glioblastoma cell line and human umbilical vein endothelial cells (HUVEC) as experimental models. Cell viability assays and sprouting assays were performed to assess the effects of LHT7. Additionally, phosphorylation of angiogenesis-related proteins, such as phospho-ERK and phospho-VEGFR2, was measured. The anti-angiogenic effects of LHT7 were further evaluated using a glioblastoma orthotopic mouse model.ResultsTreatment with LHT7 resulted in a dose-dependent reduction in cell viability in U87MG human glioblastoma cells. The sprouting of HUVEC cells was significantly decreased upon LHT7 treatment. Furthermore, LHT7 treatment led to a decrease in the phosphorylation of angiogenesis-related proteins, including phospho-ERK and phospho-VEGFR2. In the glioblastoma orthotopic mouse model, LHT7 exhibited anti-angiogenic effects, supporting its potential as a therapeutic agent.ConclusionsThe conjugation of heparin and taurocholic acid to create LHT7 offers several advantages over conventional therapeutic approaches for glioblastoma. LHT7 demonstrated anti-angiogenic properties, as evidenced by the reduction in cell viability and inhibition of endothelial cell sprouting. Moreover, LHT7 modulated the phosphorylation of angiogenesis-related proteins. These findings suggest that LHT7 holds promise as a medication for glioblastoma treatment, offering potential implications for improving patient outcomes.Graphical

Metabolic Adjustments following Glutaminase Inhibition by CB-839 in Glioblastoma Cell Lines.

Most tumor cells can use glutamine (Gln) for energy generation and biosynthetic purposes. Glutaminases (GAs) convert Gln into glutamate and ammonium. In humans, GAs are encoded by two genes: GLS and GLS2. In glioblastoma, GLS is commonly overexpressed and considered pro-oncogenic. We studied the metabolic effects of inhibiting GLS activity in T98G, LN229, and U87MG human glioblastoma cell lines by using the inhibitor CB-839. We performed metabolomics and isotope tracing experiments using U-13C-labeled Gln, as well as 15N-labeled Gln in the amide group, to determine the metabolic fates of Gln carbon and nitrogen atoms. In the presence of the inhibitor, the results showed an accumulation of Gln and lower levels of tricarboxylic acid cycle intermediates, and aspartate, along with a decreased oxidative labeling and diminished reductive carboxylation-related labeling of these metabolites. Additionally, CB-839 treatment caused decreased levels of metabolites from pyrimidine biosynthesis and an accumulation of intermediate metabolites in the de novo purine nucleotide biosynthesis pathway. The levels of some acetylated and methylated metabolites were significantly increased, including acetyl-carnitine, trimethyl-lysine, and 5-methylcytosine. In conclusion, we analyzed the metabolic landscape caused by the GLS inhibition of CB-839 in human glioma cells, which might lead to the future development of new combination therapies with CB-839.

Cancer Cytotoxicity of a Hybrid Hyaluronan-Superparamagnetic Iron Oxide Nanoparticle Material: An In-Vitro Evaluation.

While hyaluronic acid encapsulating superparamagnetic iron oxide nanoparticles have been reported to exhibit selective cytotoxicity toward cancer cells, it is unclear whether low-molecular-weight hyaluronic acid-conjugated superparamagnetic iron oxide nanoparticles also display such cytotoxicity. In this study, high-molecular-weight hyaluronic acid was irradiated with γ-ray, while Fe3O4 nanoparticles were fabricated using chemical co-precipitation. The low-molecular-weight hyaluronic acid and Fe3O4 nanoparticles were then combined according to a previous study. Size distribution, zeta potential, and the binding between hyaluronic acid and iron oxide nanoparticles were examined using dynamic light scattering and a nuclear magnetic resonance spectroscopy. The ability of the fabricated low-molecular-weight hyaluronic acid conjugated superparamagnetic iron oxide nanoparticles to target cancer cells was examined using time-of-flight secondary ion mass spectrometry and T2* weighted magnetic resonance images to compare iron signals in U87MG human glioblastoma and NIH3T3 normal fibroblast cell lines. Comparison showed that the present material could target U87MG cells at a higher rate than NIH3T3 control cells, with a viability inhibition rate of 34% observed at day two and no cytotoxicity observed in NIH3T3 normal fibroblasts during the three-day experimental period. Supported by mass spectrometry images confirming that the nanoparticles accumulated on the surface of cancer cells, the fabricated materials can reasonably be suggested as a candidate for both magnetic resonance imaging applications and as an injectable anticancer agent.

The effect of temozolomide on Hsp60 and Hsp70 expression in extracellular vesicles derived from U87MG glioma cells

Abstract Objectives Temozolomide (TMZ) is an effective drug for glioblastoma multiforme (GBM), but the mechanism underlying TMZ resistance is poorly understood. New evidence has revealed that the release of heat shock proteins (Hsps) derived from extracellular vesicles (EVs) play an important role in cancer progression by modulating tumor microenvironment and cellular cross-talk. This study aims to evaluate the effects of TMZ on the expression of EV-derived and cellular Hsps and cell motility in U87MG human glioblastoma cell line. Methods Glial-EVs were isolated from the culture medium and characterized by SEM and immunoblotting. The effect of TMZ treatments (25, 200 and 750 µM) on cell proliferation (MTT assay), migration (scratch assay), and Hsp60 and Hsp70 levels (immunoblotting) were evaluated. Results TMZ treatments led to an increase in intracellular Hsp70 while decreasing EV-derived Hsp70. Cellular Hsp60 level was elevated at the low dose of TMZ, but it reduced at higher TMZ concentrations. Hsp60 was also decreased in EVs secreted from TMZ-treated cells. Besides, TMZ treatment reduced the proliferation and migration of glioma cells in a dose-dependent manner. Conclusions Our results suggest that TMZ has the potential to target both EV-derived and cellular Hsps for GBM treatment, thus it may reduce cell motility.

Green Synthesis of Silver Nanoparticles Using Diospyros malabarica Fruit Extract and Assessments of Their Antimicrobial, Anticancer and Catalytic Reduction of 4-Nitrophenol (4-NP).

The green synthesis of silver nanoparticles (AgNPs) has currently been gaining wide applications in the medical field of nanomedicine. Green synthesis is one of the most effective procedures for the production of AgNPs. The Diospyros malabarica tree grown throughout India has been reported to have antioxidant and various therapeutic applications. In the context of this, we have investigated the fruit of Diospyros malabarica for the potential of forming AgNPs and analyzed its antibacterial and anticancer activity. We have developed a rapid, single-step, cost-effective and eco-friendly method for the synthesis of AgNPs using Diospyros malabarica aqueous fruit extract at room temperature. The AgNPs began to form just after the reaction was initiated. The formation and characterization of AgNPs were confirmed by UV-Vis spectrophotometry, XRD, FTIR, DLS, Zeta potential, FESEM, EDX, TEM and photoluminescence (PL) methods. The average size of AgNPs, in accordance with TEM results, was found to be 17.4 nm. The antibacterial activity of the silver nanoparticles against pathogenic microorganism strains of Staphylococcus aureus and Escherichia coli was confirmed by the well diffusion method and was found to inhibit the growth of the bacteria with an average zone of inhibition size of (8.4 ± 0.3 mm and 12.1 ± 0.5 mm) and (6.1 ± 0.7 mm and 13.1 ± 0.5 mm) at 500 and 1000 µg/mL concentrations of AgNPs, respectively. The anticancer effect of the AgNPs was confirmed by MTT assay using the U87-MG (human primary glioblastoma) cell line. The IC50 value was found to be 58.63 ± 5.74 μg/mL. The results showed that green synthesized AgNPs exhibited significant antimicrobial and anticancer potency. In addition, nitrophenols, which are regarded as priority pollutants by the United States Environmental Protection Agency (USEPA), can also be catalytically reduced to less toxic aminophenols by utilizing synthesized AgNPs. As a model reaction, AgNPs are employed as a catalyst in the reduction of 4-nitrophenol to 4-aminophenol, which is an intermediate for numerous analgesics and antipyretic drugs. Thus, the study is expected to help immensely in the pharmaceutical industries in developing antimicrobial drugs and/or as an anticancer drug, as well as in the cosmetic and food industries.

Abstract 2806: Development of an iRFP713-based orthotopic patient derived brain cancer model to study tumor development in vivo

Abstract While the use of bioluminescent proteins for molecular imaging is a powerful technology to support our knowledge of complex processes, with the advent of near infrared-labeling capabilities optical imaging can be pursuit with deeper tissue penetration, a better signal to background ratio and a quicker image acquisition without the need to inject substrates in preclinical oncology mouse models. This is specifically important in orthoptic brain tumors, when the substrate concentration in the tumor tissue might be influenced by the blood-brain barrier. In our recent study, we validated the feasibility of stable or transient transfection of human brain tumor cell lines and patient derived xenografts to determine tumor load in an intracranial setting. As a proof-of principle, we stably transfected the human glioblastoma cell line U87MG with an iRFP713 construct and followed tumor growth of intracranially implanted NSG mice. As a follow-up, five PDX of pediatric brain tumors (two Ependymoma, two Medulloblastoma, one high grade Glioblastoma) were transiently transfected with the same construct 24h before intracranial implantation. Tumor load was determined twice a week with the Pearl trilogy system (LiCor, Germany) and animals were examined for neurological symptoms every day. When the overall condition of the mice got deteriorated, animals were taken down and brains fixed in formalin. The tumor load and localization was confirmed by H&E and human-specific IHC for LamininB. The comparison between non-transfected and transfected lines of one specific tumor model, revealed that the tumor growth was very similar in both settings. The tumor take rate was as well not affected. In the subsequent analyses we could show that the tumor load linearly correlated with the imaging signal. Moreover, the iRFP713 signal was stable post-resection of the tumor. Thus, it was possible to correlate the tumor load quantification via iRFP713-Protein expression and LamininB-IHC within one slide. The transient transfection was successful for the five tested models. When termination criteria were reached, over 90% of the tumor tissue were expressing the iRFP713 protein. The more general applicability for larger cohorts of mice (> 20) and drug testing experiments will be evaluated soon. We believe the superior optical properties of iRFP713 will be a valuable asset to overcome some of the complications inherent to imaging live animals and a powerful tool for preclinical drug development in clinical relevant mouse models of brain cancer. Citation Format: Kanstantsin Lashuk, Jacqueline Bersano, Dorothee Lenhard, Eva Oswald, Kerstin Klingner, Julia Schüler. Development of an iRFP713-based orthotopic patient derived brain cancer model to study tumor development in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2806.

SN38 loaded nanostructured lipid carriers (NLCs); preparation and in vitro evaluations against glioblastoma

SN38 is the active metabolite of irinotecan with 1000-fold greater cytotoxicity compared to the parent drug. Despite the potential, its application as a drug is still seriously limited due to its stability concerns and low solubility in acceptable pharmaceutical solvents. To address these drawbacks here nanostructured lipid carrier (NLC) containing SN38 was prepared and its cytotoxicity against U87MG glioblastoma cell line was investigated. The formulations were prepared using hot ultrasonication and solvent evaporation/emulsification methods. NLCs with a mean size of 140 nm and particle size distribution (PDI) of 0.25 were obtained. The average loading efficiency was 9.5% and its entrapment efficiency was 81%. In order to obtain an accurate determination of released amount of SN38 a novel medium and extraction method was designed, which lead to an appropriate in vitro release profile of the drug from the prepared NLCs. The MTT test results revealed the significant higher cytotoxicity of NLCs on U87MG human glioblastoma cell line compared with the free drug. The confocal microscopy images confirmed the proper penetration of the nanostructures into the cells within the first 4 h. Consequently, the results indicated promising potentials of the prepared NLCs as a novel treatment for glioblastoma.

Enhancement of T2* Weighted MRI Imaging Sensitivity of U87MG Glioblastoma Cells Using γ-Ray Irradiated Low Molecular Weight Hyaluronic Acid-Conjugated Iron Nanoparticles.

IntroductionIt has been reported that low-molecular-weight hyaluronic acid (LMWHA) exhibits a potentially beneficial effect on cancer therapy through targeting of CD44 receptors on tumor cell surfaces. However, its applicability towards tumor detection is still unclear. In this regard, LMWHA-conjugated iron (Fe3O4) nanoparticles (LMWHA-IONPs) were prepared in order to evaluate its application for enhancing the T2* weighted MRI imaging sensitivity for tumor detection.MethodsLMWHA and Fe3O4 NPs were produced using γ-ray irradiation and chemical co-precipitation methods, respectively. First, LMWHA-conjugated FITC was prepared to confirm the ability of LMWHA to target U87MG cells using fluorescence microscopy. The hydrodynamic size distribution and dispersion of the IONPs and prepared LMWHA-IONPs were analyzed using dynamic light scattering (DLS). In addition, cell viability assays were performed to examine the biocompatibility of LMWHA and LMWHA-IONPs toward U87MG human glioblastoma and NIH3T3 fibroblast cell lines. The ability of LMWHA-IONPs to target tumor cells was confirmed by detecting iron (Fe) ion content using the thiocyanate method. Finally, time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging and in vitro magnetic resonance imaging (MRI) were performed to confirm the contrast enhancement effect of LMWHA-IONPs.ResultsFlorescence analysis results showed that LMWHA-FITC successfully targeted the surfaces of both tested cell types. The ability of LMWHA to target U87MG cells was higher than for NIH3T3 cells. Cell viability experiments showed that the fabricated LMWHA-IONPs possessed good biocompatibility for both cell lines. After co-culturing test cells with the LMWHA-IONPs, detected Fe ion content in the U87MG cells was much higher than that of the NIH3T3 cells in both thiocyanate assays and TOF-SIMs images. Finally, the addition of LMWHA-IONPs to the U87MG cells resulted in an obvious improvement in T2* weighted MR image contrast compared to control NIH3T3 cells.DiscussionOverall, the present results suggest that LMWHA-IONPs fabricated in this study provide an effective MRI contrast agent for improving the diagnosis of early stage glioblastoma in MRI examinations.

Graphene Oxide/Zinc Oxide Nanocomposite Displaying Selective Toxicity to Glioblastoma Cell Lines

Glioblastoma is considered the most aggressive and prevalent type of glioma. Resistance mechanisms, side effects, and the blood–brain barrier are factors that make its treatment difficult, requiring the development of alternative therapeutic strategies. Herein, we propose a nanocomposite composed of carboxylated graphene oxide nanosheets decorated with zinc oxide nanoparticles and post-functionalized with Pluronic (GOC-ZnO-P) for chemotherapy against U87MG and U138MG human glioblastoma cell lines. The GOC-ZnO-P formulation is fairly stable in the physiological medium (DMEM-FBS) and demonstrates selectivity toward tumor cell lines, even though it is less cytotoxic than free ZnO. Whereas the photothermal activity of the nanocomposite has a negligible cytotoxic effect, images obtained by scanning and transmission electron microscopies reveal that it induces changes in adhesion points and roughness of the tumor cell membrane, and it is uptaken through vesicles and capable of accumulating in the nucleus, a process that can induce cell death by apoptosis, as verified by flow cytometry. In summary, the GOC phase serves as an inert platform for transporting ZnO, which maintains its selectivity for tumor cells and does not interfere with the mechanisms for inducing apoptotic pathways. Therefore, the GOC-ZnO-P nanocomposite offers a strategy for glioblastoma treatment.

Health Informatics of Composite Nanoparticles as an Effective Contrast Agent in Magnetic Resonance Imaging and for Glioblastoma Radiosensitization

Background: Radiation therapy is a method using radiation to treat tumors, and has become commonly used and most effective treatments for malignant tumors. However, radiotherapy still has the shortcomings of high radiation dose, especially radiation resistance of tumor cells. Multifunctional nanoradiosensitizers can enhance the radiosensitivity of tumor cells and improving the effect of radiotherapy. Hyaluronic acid-functionalized ironbismuth composite particles are an effective contrast agent for magnetic resonance imaging (MRI), and can be used as radiosensitizers. Method: Hyaluronic acid-functionalized iron-bismuth composite nanoparticles HABilOPs can be prepared by a hydrothermal polyol method. In the cytotoxicity experiment, we took human glioblastoma cell line U87MG in logarithmic growth phase and rat vascular smooth muscle cells. We set different mass concentrations of 0, 15, 30, 45, 60, 100, and 300 mg/LHA-BMOPs was cultured for 48 h to calculate the cell proliferation rate. In the histocompatibility experiment, we injected HA-BilOPs solution into the tail vein of ICR mice to observe the pathological changes of mouse organs. In the radiosensitization experiment, we took U87MG cells in logarithmic growth phase for group culture. X-ray irradiation of 0, 3, 6, 9 Gy was given, and the HA-BilOPs group was added with culture medium of different mass concentration of LHA-BilOPs, and the combined group was added to the culture medium of different mass concentration of HA-BilOPs first, and then given X-ray irradiation of 0, 3, 6, 9 Gy. To analyse the features of glioblastoma, the conventional MRI manifestations of glioblastoma were examined. Results: We found that different concentrations of HA-BilOPs had no significant effect on the proliferation of vascular smooth muscle cells and U87MG cells. It was found that no pathological changes occurred in mouse organs after tail vein injection of HA-BilOPs solution. Nanoparticles can be phagocytosed by U87MG cells and it was found that U87MG cell proliferation rate had a significant linear negative correlation with HA-BilOPs mass concentration (0–200 mg/L) and radiation dose (0–9 Gy). At 6 Gy X Under irradiation, 200 mg/L HA-BilOPs reduced the cell proliferation rate to (41± 7)%. Using 100 mg/L HA-BilOPs and 6 Gy X-ray irradiation for colony formation experiments, the proliferation rate of U87MG cells in the combined group was statistically lower than that of the blank control group and radiotherapy group (P < 0.05). Conclusion: Hyaluronic acid-functionalized iron-bismuth composite nanoparticles have a significant radiosensitizing effect on glioblastoma and proved to be a good contrast agent used in MRI.

High-voltage 10 ns delayed paired or bipolar pulses for in vitro bioelectric experiments

Recent studies proved that classical bio-effects induced by nanosecond pulsed electric field (nsPEF) can be reduced by the delivery of a negative polarity pulse generated immediately after a positive polarity pulse. This phenomenon is known as “bipolar cancellation” and it was reported for a wide range of bipolar pulses with pulse duration from 2 ns to 900 ns. On the contrary, paired pulses, i.e., two identical pulses with the same polarity, increased traditional nsPEF outcomes. Herein, we propose a novel robust and flexible generator, based on the frozen-wave concept, able to produce a broad range of pulses with the duration of 10 ns and delay between 17 and 360 ns. Numerical simulations and experimental measurements were performed to fully characterize the proposed generator. YO-PROTM-1 uptake was investigated in the U87-MG human glioblastoma cell line as a marker of membrane permeabilization in response to 10 ns, 11.5MV/m nsPEF. Our results showed that bipolar cancellation occurred for delays of 0–30 ns and decreased as a function of the interphase interval. In addition, we observed that cellular response following the application of paired nsPEF was more than two-fold compared to the unipolar pulse response and was independent from the interphase interval.

CARMUSTINE LOADED NANOSIZE LIPID VESICLES SHOWED PREFERENTIAL CYTOTOXICITY AND INTERNALIZATION IN U87MG CELL LINE ALONG WITH IMPROVED PHARMACOKINETIC PROFILE IN MICE: A STRATEGY FOR TREATMENT OF GLIOMA

Objective: Successful treatment of glioma still remains a tough challenge. The present study aims at the development and evaluation of carmustine loaded nanosize phospholipid vesicles (CNLVs) for the treatment of glioma.
 Methods: The experimental NLVs were developed by conventional lipid layer hydration technique and were characterized by different in vitro tools such as diffraction light scattering (DLS), zeta potential, field emission scanning electron microscopy (FESEM), cryo-transmission electron microscopy (cryo-TEM), in vitro drug loading capacity, drug release study etc. In vitro cytotoxicity and cellular uptake of the optimized drug-loaded NLVs were carried out in U87MG human glioblastoma cell line. In vivo pharmacokinetic study was conducted in Swiss albino mice.
 Results: DLS data showed an average vesicle diameter of 92 nm with narrow size distribution. Optimized CNLVs were spherical in shape with a smooth surface as depicted from FESEM data. Cryo-TEM study confirmed formation of unilamellar vesicles with intact outer bilayer. A reasonable drug loading of 7.8 % was reported for the optimized CNLVs along with a sustained release of CS over a 48 h study period. In vitro cytotoxicity assay revealed a considerable higher toxicity of CNLVs than free drugs in the U87MG cells. Confocal microscopy showed a satisfactory internalization of the optimized drug-loaded NLVs in the tested cell line. Pharmacokinetic data demonstrated an enhanced mean residence time of optimized CNLVs in blood than free drug.
 Conclusion: Results depicted the potential of experimental CNLVs for the treatment of glioma after further in vivo tests.