pH-tunable oxidase-like activity of cerium oxide nanoparticles achieving sensitive fluorigenic detection of cancer biomarkers at neutral pH

The reliable and sensitive detection of cancer-specific biomarkers is important for the diagnosis and treatment of cancer. Hence, detection of these biomarkers has to be reliably and rapidly performed in diverse settings. A limitation of the conventional biomarker-screening method of enzyme-linked immunosorbent assay (ELISA) is the employment of labile components, such as hydrogen peroxide and horseradish peroxidase. Previously, we reported that nanoceria is able to oxidize various colorimertic dyes at acidic pH, such as 3,3',5,5'-tetramethylbenzydine (TMB) and 2,2-azinobis-(3-ethylbenzothizoline-6-sulfonic acid) (AzBTS), and an assay was designed for screening the folate receptor. Herein, we show that the ability of nanoceria to oxidize a substrate can be tuned by modulating the pH. Results showed that nanoceria can oxidize the nonfluorescent substrate ampliflu, either to the very stable fluorescent product resorufin at pH 7.0 or to the nonfluorescent resazurin at pH 4.0. On the basis of these findings, we conjugated Protein G to immobilize antibodies on the surface of nanoceria, in order to detect the expression of prototypic cancer biomarkers at pH 7.0, such as the folate receptor and EpCAM. We found that within 3 h, nanoceria identified the expression of the folate receptor and EpCAM on lung carcinoma and breast adenocarcinoma cells, respectively. Traditional ELISA had a readout time of 15 h and a higher detection threshold, while requiring multiple washing steps. Considering these results and nanoceria's ability to oxidize ampliflu to its stable fluorescent product at neutral pH, the use of antibody-carrying nanoceria in the lab and point-of-care molecular diagnostics is anticipated.     

Rare earth nanoparticles prevent retinal degeneration induced by intracellular peroxides

Photoreceptor cells are incessantly bombarded with photons of light, which, along with the cells' high rate of oxygen metabolism, continuously exposes them to elevated levels of toxic reactive oxygen intermediates (ROIs). Vacancy-engineered mixed-valence-state cerium oxide nanoparticles (nanoceria particles) scavenge ROIs. Our data show that nanoceria particles prevent increases in the intracellular concentrations of ROIs in primary cell cultures of rat retina and, in vivo, prevent loss of vision due to light-induced degeneration of photoreceptor cells. These data indicate that the nanoceria particles may be effective in inhibiting the progression of ROI-induced cell death, which is thought to be involved in macular degeneration, retinitis pigmentosa and other blinding diseases, as well as the ROI-induced death of other cell types in diabetes, Alzheimer's disease, atherosclerosis, stroke and so on. The use of nanoceria particles as a direct therapy for multiple diseases represents a novel strategy and suggests that they may represent a unique platform technology.     

Nanoceria coating imperfections and their effect on the high-temperature oxidation resistance of a 304 stainless steel

The microemulsion method was employed in this work for synthesizing nanocrystalline cerium oxide particles. Average nanoparticle sizes of 3.45 nm were produced by these means. XPS determinations indicated that both Ce³⁺ and Ce⁴⁺ are present in the synthesized nanoceria particles, with an average amount of 22.8 % of Ce³⁺ ions. It was found that the nanocrystalline cerium oxide coatings lead to significant improvements (of 1–2 orders of magnitude) in the high-temperature oxidation resistance of 304 SS. In addition, it was found that by decreasing the nanoparticle mean size from 10 to 3.45 nm, the effect of the coating protection was drastically improved. The experimentally determined parabolic rate constants k _p at 1073 and 1273 K for 304 SS indicated a reduction of up to two orders of magnitude when nanoceria coatings with 3.45 nm in mean size were applied. Also, the scale thickness was reduced from 15 to 5 μm when oxidized at 1073 K for 442 h. Coatings with purchased nanoceria particles (10 nm) were not as effective as the coatings with synthesized nanoceria. Apparently, at increasing nanoceria sizes, the oxidation protection is significantly reduced. In addition, it was found that the method of dipping for coating 304 SS does not provide a uniform coverage with nanoceria particles. Fe-rich ‘islands’ develop during high-temperature oxidation indicating that some regions do not exhibit the protection that nanoceria can provide. In contrast, relatively thick-coating regions on the steel substrate exhibit minimal oxidation, and the resultant scale is fine grained and uniform.     

Gold nano-popcorn attached SWCNT hybrid nanomaterial for targeted diagnosis and photothermal therapy of human breast cancer cells

Breast cancer presents greatest challenge in health care in today's world. The key to ultimately successful treatment of breast cancer disease is an early and accurate diagnosis. Current breast cancer treatments are often associated with severe side effects. Driven by the need, we report the design of novel hybrid nanomaterial using gold nano popcorn-attached single wall carbon nanotube for targeted diagnosis and selective photothermal treatment. Targeted SK-BR-3 human breast cancer cell sensing have been performed in 10 cancer cells/mL level, using surface enhanced Raman scattering of single walls carbon nanotube's D and G bands. Our data show that S6 aptamer attached hybrid nanomaterial based SERS assay is highly sensitive to targeted human breast cancer SK-BR-3 cell line and it will be able to distinguish it from other non targeted MDA-MB breast cancer cell line and HaCaT normal skin cell line. Our results also show that 10 min of photothermal therapy treatment by 1.5 W/cm(2) power, 785 nm laser is enough to kill cancer cells very effectively using S6 aptamer attached hybrid nanomaterials. Possible mechanisms for targeted sensing and operating principle for highly efficient photothermal therapy have been discussed. Our experimental results reported here open up a new possibility for using aptamers modified hybrid nanomaterial for reliable diagnosis and targeted therapy of cancer cell lines quickly.     

Selective cytotoxic effect of ZnO nanoparticles on glioma cells

In this study we examined the cytotoxic effect of ZnO nanoparticles on various human cancer and normal cells. We found that the ZnO nanoparticles exerted a cytotoxic effect on the human glioma cell lines A172, U87, LNZ308, LN18, and LN229, whereas no cytotoxic effect was observed on normal human astrocytes. Similarly, the ZnO nanoparticles induced cell death in breast and prostate cancer cell lines while no major effect was observed in the respective normal breast and prostate cell lines. Using the fluorescent dye 2,7-dichlorofluorescein diacetate, we found that treatment of the glioma cells with ZnO nanoparticles induced a large increase in the generation of reactive oxygen species (ROS) and treatment of the cells with N-acetyl cysteine decreased the cytotoxic effect of the ZnO nanoparticles. In contrast, a smaller effect on ROS generation was observed in the normal astrocytes. These results suggest that ZnO nanoparticles may be employed as a selective cytotoxic agent for the eradication of cancer cells.     

A Surface‐Charge Study on Cellular‐Uptake Behavior of F3‐Peptide‐Conjugated Iron Oxide Nanoparticles

Surface‐charge measurements of mammalian cells in terms of Zeta potential are demonstrated as a useful biological characteristic in identifying cellular interactions with specific nanomaterials. A theoretical model of the changes in Zeta potential of cells after incubation with nanoparticles is established to predict the possible patterns of Zeta‐potential change to reveal the binding and internalization effects. The experimental results show a distinct pattern of Zeta‐potential change that allows the discrimination of human normal breast epithelial cells (MCF‐10A) from human cancer breast epithelial cells (MCF‐7) when the cells are incubated with dextran coated iron oxide nanoparticles that contain tumor‐homing F3 peptides, where the tumor‐homing F3 peptide specifically bound to nucleolin receptors that are overexpressed in cancer breast cells.     

Green tea extract selectively targets nanomechanics of live metastatic cancer cells

Green tea extract (GTE) is known to be a potential anticancer agent (Yang et al 2009 Nat. Rev. Cancer 9 429-39) with various biological activities (Lu et al 2005 Clin. Cancer Res. 11 1675-83; Yang et al 1998 Carcinogenesis 19 611-6) yet the precise mechanism of action is still unclear. The biomechanical response of GTE treated cells taken directly from patient's body samples was measured using atomic force microscopy (AFM) (Binnig et al 1986 Phys. Rev. Lett. 56 930). We found significant increase in stiffness of GTE treated metastatic tumor cells, with a resulting value similar to untreated normal mesothelial cells, whereas mesothelial cell stiffness after GTE treatment is unchanged. Immunofluorescence analysis showed an increase in cytoskeletal-F-actin in GTE treated tumor cells, suggesting GTE treated tumor cells display mechanical, structural and morphological features similar to normal cells, which appears to be mediated by annexin-I expression, as determined by siRNA analysis of an in vitro cell line model. Our data indicates that GTE selectively targets human metastatic cancer cells but not normal mesothelial cells, a finding that is significantly advantageous compared to conventional chemotherapy agents.     

Vascular targeted single-walled carbon nanotubes for near-infrared light therapy of cancer

A new approach for targeting carbon nanotubes to the tumor vasculature was tested using human endothelial cells and MCF-7 breast cancer cells in vitro. Single-walled carbon nanotubes were functionalized with the F3 peptide using a polyethylene glycol linker to target nucleolin, a protein found on the surface of endothelial cells in the vasculature of solid tumors. Confocal microscopy and Raman analysis confirmed that the conjugate was internalized by actively dividing endothelial cells. Dividing endothelial cells were used to mimic these cells in the tumor vasculature. Incubation with the conjugate for 8?h or more caused significant cell death in both actively dividing endothelial cells and MCF-7 breast cancer cells, an effect that is hypothesized to be due to the massive uptake of the conjugate. This targeted cell killing was further enhanced when coupled with near-infrared laser treatment. For confluent (non-dividing) endothelial cells, no cytotoxic effect was seen for incubation alone or incubation coupled with laser treatment. These results are promising and warrant further studies using this conjugate for cancer treatment in vivo.     

Scanning electrochemical microscopy of living cells. 5. Imaging of fields of normal and metastatic human breast cells

Scanning electrochemical microscopy (SECM) was used to image fields of different types of human breast cells in monolayer culture. The goal of these experiments was to demonstrate the possibility of distinguishing between nontransformed human breast epithelial cells (MCF-10A) and metastatic breast cells (MDA-MB-231) by their redox activities. Imaging of densely packed cells by SECM requires approaches that differ from previously reported experiments with well-separated single cells. The combination of SECM with optical and fluorescence microscopies was used to locate individual cells in a homogeneous or heterogeneous field of cells. To establish that metastatic breast cells can be detected against a field of normal cells, the former were preloaded with fluorescent nanospheres and plated together with unlabeled MCF-10A cells. By matching SECM and fluorescence images of a selected group of metastatic cells, the level of discrimination and fidelity of the SECM signal could be shown. Several factors (distance between the electrode and the cells, cell density, choice of mediator, and its concentration) were identified that can be used to maximize the contrast between images of metastatic and nontransformed cells. These studies provide a framework for future analysis of malignant cells in human breast tissue samples.     

Tamoxifen-loaded solid lipid nanoparticles-induced apoptosis in breast cancer cell lines

An in vitro study was conducted to determine the apoptosis induced by tamoxifen (TAM) and TAM-loaded solid lipid nanoparticles (SLNs) in breast cancer cell lines, MCF-7 and MDA-MB231 cells. The effect of free drug and drug-loaded SLN on the cell lines was characterised by cell morphology and cell cycle distribution using phase contrast microscopy, nuclear morphology and flow cytometry, respectively. The results showed that TAM-loaded SLNs have an equally efficient cytotoxic activity against MCF-7 and MDA-MB231 cells, compared to free TAM, and the half maximal inhibitory concentration (IC₅₀) of TAM-loaded SLNs was generally lower than that of free TAM. In the presence of TAM and TAM-loaded SLN, the viability of the both cells diminishes and the cancer cells lose their normal morphological characteristics, detaches, aggregates and later develops apoptotic bodies. Flow cytometry analysis showed that TAM-loaded SLN like the free TAM caused a dose- and time-dependent apoptosis without cell cycle arrest of human breast cancer cells. Therefore, TAM-loaded SLN has great potential in human medicine for the treatment of breast cancers.     

The Fibrillar Matrix: Novel Avenues for Breast Cancer Detection and Treatment

Breast cancer is marked by large increases in the protein fibers around tumor cells. These fibers increase the mechanical stiffness of the tissue, which has long been used for tumor diagnosis by manual palpation. Recent research in bioengineering has led to the development of novel biomaterials that model the mechanical and architectural properties of the tumor microenvironment and can be used to understand how these cues regulate the growth and spread of breast cancer. Herein, we provide an overview of how the mechanical properties of breast tumor tissues differ from those of normal breast tissue and non-cancerous lesions. We also describe how biomaterial models make it possible to understand how the stiffness and viscosity of the extracellular environment regulate cell migration and breast cancer metastasis. We highlight the need for biomaterial models that allow independent analysis of the individual and different mechanical properties of the tumor microenvironment and that use cells derived from different regions within tumors. These models will guide the development of novel mechano-based therapies against breast cancer metastasis.     

Detection of a thousand copies of miRNA without enrichment or modification

We report direct quantitative analysis of multiple miRNAs with a detection limit of 1000 copies without miRNA enrichment or modification. A 300-fold improvement over the previously published detection limit was achieved by combining capillary electrophoresis with confocal time-resolved fluorescence detection through an embedded capillary interface. The method was used to determine levels of three miRNA biomarkers of breast cancer (miRNA 21, 125b, 145) in a human breast cancer cell line (MCF-7). A 30 pL volume of the cell lysate with approximately a material content of a single cell was sampled for the analysis. MiRNA 21, which is up-regulated in breast cancer, was detected at a level of approximately 12 thousand copies per cells. MiRNAs 125b and 145, which are down-regulated in breast cancer, were below the 1000-copy detection limit. This sensitive method may facilitate the analysis of miRNA in fine-needle-biopsy samples and even in single cells without enrichment or modification of miRNA. Advantageously, the instrumental setup developed here can be reproduced by others as it requires no sophisticated custom-made parts.     

Analysis of cell surface carbohydrate expression patterns in normal and tumorigenic human breast cell lines using lectin arrays

Cell surface carbohydrates play important roles in a wide variety of biological processes including cell adhesion, fertilization, differentiation, development, and tumor cell metastasis. Lectins are proteins of nonimmune origin which recognize and bind to specific carbohydrate structural epitopes. We have recently described the development and use of lectin arrays as tools for the elucidation of the carbohydrate structures expressed on cell surfaces. In the present work this technology is employed for the characterization of differences in carbohydrate expression patterns on normal and tumorigenic human breast cell lines, as well as on sublines differing in their tendency to "home" to different tissues during metastasis. Significant differences were observed, including changes that correlate with metastatic potential as well as with tissue-specific homing of metastatic cells.     

Tracking and Finding Slow‐Proliferating/Quiescent Cancer Stem Cells with Fluorescent Nanodiamonds

Quiescent cancer stem cells (CSCs) have long been considered to be a source of tumor initiation. However, identification and isolation of these cells have been hampered by the fact that commonly used fluorescent markers are not sufficiently stable, both chemically and photophysically, to allow tracking over an extended period of time. Here, it is shown that fluorescent nanodiamonds (FNDs) are well suited for this application. Genotoxicity tests of FNDs with comet and micronucleus assays for human fibroblasts and breast cancer cells indicate that the nanoparticles neither cause DNA damage nor impair cell growth. Using AS‐B145‐1R breast cancer cells as the model cell line for CSC, it is found that the FND labeling outperforms 5‐ethynyl‐2′‐deoxyuridine (EdU) and carboxyfluorescein diacetate succinimidyl ester (CFSE) in regards to its long‐term tracking capability (>20 d). Moreover, through a quantification of their stem cell activity by measuring mammosphere‐forming efficiencies (MFEs) and self‐renewal rates, the FND‐positive cells are identified to have an MFE twice as high as that of the FND‐negative cells isolated from the same dissociated mammospheres. Thus, the nanoparticle‐based labeling technique provides an effective new tool for tracking and finding slow‐proliferating/quiescent CSCs in cancer research.     

Fe3O4纳米粒子与细胞的相互作用研究

研究10 nm粒径的表面未修饰Fe3O4纳米粒子在体外对人正常肝细胞HL-7702及人肝癌细胞SMMC-7721的生长影响.通过倒置显微镜、透射电镜(TEM)观察加入Fe3O4纳米粒子后肝癌细胞和正常肝细胞的形态变化及磁性纳米粒子在细胞内分布状态.用CCK-8测定加入磁性纳米粒子培养后细胞增殖能力的变化.倒置显微镜、透...     

Nuclease resistance of telomere-like oligonucleotides monitored in live cells by fluorescence anisotropy imaging

Telomeres carry important biological functions such as the protection of chromosomes. In this paper, we have developed a fluorescence anisotropy imaging system for monitoring DNA digestion inside live cells. The nuclease-resistant capability of telomere-like ssDNAs in nuclei of human breast cancer cells is studied. We found that those oligonucleotides were clearly more stable than regular DNA sequences during the time course of the experiments. We conclude that the G-quadruplex structure of the telomere-like ssDNA makes it inherently more stable in intracellular environments than non-G-quadruplex structures. This will help us understand why the G-quadruplex forming telomere sequences were adopted by almost all eukaryotic cells to protect the ends of chromosomes. This is the first time such a phenomenon was observed in live cells. Our fluorescence anisotropy imaging provides an efficient way to directly monitor DNA digestion in any region of live cells in real time, providing insights into many important and related intracellular processes.     

Preparation and optimization of monodisperse polymeric microparticles using modified vibrating orifice aerosol generator for controlled delivery of letrozole in breast cancer therapy

Abstract

Letrozole (LTZ) is effective for the treatment of hormone-receptor-positive breast cancer in postmenopausal women. In this work, and for the first time, using vibrating orifice aerosol generator (VOAG) technology, monodisperse poly-ε-caprolactone (PCL), and poly (D, L-Lactide) (PDLLA) LTZ-loaded microparticles were prepared and found to elicit selective high cytotoxicity against cancerous breast cells with no apparent toxicity on healthy cells in vitro. Plackett–Burman experimental design was utilized to identify the most significant factors affecting particle size distribution to optimize the prepared particles. The generated microparticles were characterized in terms of microscopic morphology, size, zeta potential, drug entrapment efficiency, and release profile over one-month period. Long-term cytotoxicity of the microparticles was also investigated using MCF-7 human breast cancer cell lines in comparison with primary mammary epithelial cells (MEC). The prepared polymeric particles were monodispersed, spherical, and apparently smooth, regardless of the polymer used or the loaded LTZ concentration. Particle size varied from 15.6 to 91.6?µm and from 22.7 to 99.6?µm with size distribution (expressed as span values) ranging from 0.22 to 1.24 and from 0.29 to 1.48 for PCL and PDLLA based microparticles, respectively. Upon optimizing the manufacture parameters, span was reduced to 0.162–0.195. Drug entrapment reached as high as 96.8%, and drug release from PDLLA and PCL followed a biphasic zero-order release using 5 or 30% w/w drug loading in the formulations. Long-term in vitro cytotoxicity studies indicated that microparticles formulations significantly inhibited the growth of MCF-7 cell line over a prolonged period of time but did not have toxic effects on the normal breast epithelial cells.     

Drug Response Prediction of Liver Cancer Cell Line Using Deep Learning

Cancer is the second deadliest human disease worldwide with high mortality rate. Rehabilitation and treatment of this disease requires precise and automatic assessment of effective drug response and control system. Prediction of treated and untreated cancerous cell line is one of the most challenging problems for precise and targeted drug delivery and response. A novel approach is proposed for prediction of drug treated and untreated cancer cell line automatically by employing modified Deep neural networks. Human hepatocellular carcinoma (HepG2) cells are exposed to anticancer drug functionalized CFO@BTO nanoparticles developed by our lab. Prediction models are developed by modifying ResNet101 and exploiting the transfer learning concept. Last three layers of ResNet101 are re-trained for the identification of drug treated cancer cells. Transfer learning approach in an appropriate choice especially when there is limited amount of annotated data. The proposed technique is validated on acquired 203 fluorescent microscopy images of human HepG2 cells treated with drug functionalized cobalt ferrite@barium titanate (CFO@BTO) magnetoelectric nanoparticles in vitro. The developed approach achieved high prediction with accuracy of 97.5% and sensitivity of 100% and outperformed other approaches. The high performance reveals the effectiveness of the approach. It is scalable and fully automatic prediction approach which can be extended for other similar cell diseases such as lung, brain tumor and breast cancer.     

Augmented anticancer activity of naringenin-loaded TPGS polymeric nanosuspension for drug resistive MCF-7 human breast cancer cells

Naringenin (NAR) is a naturally occurring plant flavonoid, found predominantly in citrus fruits, possesses a wide range of pharmacological properties. However, despite the therapeutic potential of NAR, its clinical development has been hindered due to low aqueous solubility and inefficient transport across biological membranes resulting in low bioavailability at tumor sites. In our previous studies, nanosuspension of naringenin (NARNS) was prepared using high pressure homogenization method using different polymers. D-α-Tocopheryl polyethylene glycol succinate 1000 (TPGS) was added as a co-stabilizer. All formulation characterization studies were performed. As a continuation of our previous research, current study has further evaluated the ability of the TPGS-coated NARNS, to reverse drug-resistance of P-gp-over expressing MCF-7 human breast adenocarcinoma cell line and animal model. MTT-based colorimetric assay revealed higher cytotoxic efficacy of NARNS than free NAR in MCF-7 cells. NARNS treatment significantly increased intracellular ROS level, mitochondrial membrane potential, caspase-3 activity, lipid peroxidation status (TBARS) and decreased GSH levels when compared to free NAR treatment in MCF-7 cells. It has been also noticed that the presence of apoptotic indices (membrane blebbing, nuclear fragmentation) in NARNS treated cancer cells. Further, NARNS exhibited dose-dependent in vitro antitumor activity with DLA cells. A significant increase in the life span and a decrease in the cancer cell number and tumor weight were noted in the tumor-induced mice after treatment with NARNS.     

Functionalised gold nanoparticles for selective induction of in vitro apoptosis among human cancer cell lines

The interaction of citrate- and polyethylene imine (PEI)-functionalised gold nanoparticles (GNP) with cancer cell lines with respect to the cellular response was studied. It was found that GNP/citrate nanoparticles were able to induce apoptosis in human carcinoma lung cell lines A549, but GNP/PEI did not show any reduction in the viability of the cells in human breast cancer cell line MCF-7 and A549 cell lines. FACS data confirmed that the number of apoptotic cells increased with increase in the concentration of GNP/citrate nanoparticles. Decline in cellular expansion and changes in the nuclear morphology were noted after the treatment of GNP/citrate nanoparticles on A549 cell lines, which itself is a direct response for stress induction. The induction of cellular apoptosis was further confirmed by DNA fragmentation assay. These data confirm the potential of GNP/citrate nanoparticle to evoke cell-specific death response in the A549 cell lines.