Identification of PHB2 as a Potential Biomarker of Luminal A Breast Cancer Cells Using a Cell-Specific Aptamer

Precise diagnosis of breast cancer molecular subtypes remains a great challenge in clinics. The present molecular biomarkers are not specific enough to classify breast cancer subtypes precisely, which requests for more accurate and specific molecular biomarkers to be discovered. Aptamers evolved by the cell-systematic evolution of ligands by exponential enrichment (SELEX) method show great potential in the discovery and identification of cell membrane targets via aptamer-based cell membrane protein pull-down, which has been regarded as a novel and powerful weapon for the discovery and identification of new molecular biomarkers. Herein, a cell membrane protein PHB2 was identified as a potential molecular biomarker specifically expressed in the cell membranes of MCF-7 breast cancer cells using a DNA aptamer MF3Ec. Further experiments demonstrated that the PHB2 protein is differentially expressed in the cell membranes of MCF-7, SK-BR-3, and MDA-MB-231 breast cancer cells and MCF-10A cells, and the binding molecular domains of aptamer MF3Ec and anti-PHB2 antibodies to the PHB2 protein are different due to there being no obvious competitions between aptamer MF3Ec and anti-PHB2 antibodies in the binding to the cell membranes of target MCF-7 cells. Due to those four cells belonging to luminal A, HER2-positive, and triple-negative breast cancer cell subtypes and human normal mammary epithelial cells, respectively, the PHB2 protein in the cell membrane may be a potential biomarker for precise diagnosis of the luminal A breast cancer cell subtype, which is endowed with the ability to differentiate the luminal A breast cancer cell subtype from HER2-positive and triple-negative breast cancer cell subtypes and human normal mammary epithelial cells, providing a new molecular biomarker and therapeutic target for the accurate and precise classification and diagnostics and personalized therapy of breast cancer

Chemiluminescent Labels Released from Long Spacer Arm-Functionalized Magnetic Particles: A Novel Strategy for Ultrasensitive and Highly Selective Detection of Pathogen Infections

Previously, the unique advantages provided by chemiluminescence (CL) and magnetic particles (MPs) have resulted in the development of many useful nucleic acid detection methods. CL is highly sensitive, but when applied to MPs, its intensity is limited by the inner filter-like effect arising from excess dark MPs. Herein, we describe a modified strategy whereby CL labels are released from MPs to eliminate this negative effect. This approach relies on (1) the magnetic capture of target molecules on long spacer arm-functionalized magnetic particles (LSA-MPs), (2) the conjugation of streptavidin-alkaline phosphatase (SA-AP) to biotinylated amplicons of target pathogens, (3) the release of CL labels (specifically, AP tags), and (4) the detection of the released labels. CL labels were released from LSA-MPs through LSA ultrasonication or DNA enzymolysis, which proved to be the superior method. In contrast to conventional MPs, LSA-MPs exhibited significantly improved CL detection, because of the introduction of LSA, which was made of water-soluble carboxymethylated β-1,3-glucan. Detection of hepatitis B virus with this technique revealed a low detection limit of 50 fM, high selectivity, and excellent reproducibility. Thus, this approach may hold great potential for early stage clinical diagnosis of infectious diseases

Multifunctional Yolk–Shell Mesoporous Silica Obtained via Selectively Etching the Shell: A Therapeutic Nanoplatform for Cancer Therapy

Herein, we fabricated a new yolk–shell-structured mesoporous silica nanoparticle (YMSN) with multifunctionalities of fluorescence imaging, photothermal therapy (PTT), and drug delivery by using a fluorescein isothiocyanate-doped silica nanoparticle partially covered by patchy gold as the core. Different from the conventional selective etching procedure, the multifunctional silica core is left intact, and the alkali etching mainly occurs in a hexadecyl trimethyl ammonium bromide/silica hybrid layer, which leads to the formation of the void space in YMSNs. In addition, the utilization of patchy gold as the PTT agent can avoid the shield against the outer irradiation on the core. Results show that the as-prepared YMSNs have a good biocompatibility in the concentration of 0–1000 μg·mL^–1 and a high doxorubicin-loading capability (8.04 wt %). In vitro and in vivo antitumor experiments reveal that the resulting YMSNs can be utilized for chemo- and photothermic combination therapy as well as optical imaging

Action of Gold Nanospikes-Based Nanoradiosensitizers: Cellular Internalization, Radiotherapy, and Autophagy

A major challenge to achieve effective X-ray radiation therapy is to use a relatively low and safe radiation dose. Various radiosensitizers, which can significantly enhance the radiotherapeutic performance, have been developed. Gold-based nanomaterials, as a new type of nanoparticle-based radiosensitizers, have been extensively used in researches involving cancer radiotherapy. However, the cancer therapeutic effect using the gold nanoparticle-based radiotherapy is usually not significant because of the low cellular uptake efficiency and the autophagy-inducing ability of these gold nanomaterials. Herein, using gold nanospikes (GNSs) as an example, we prepared a series of thiol-poly­(ethylene glycol)-modified GNSs terminated with methoxyl (GNSs), amine (NH₂-GNSs), folic acid (FA) (FA-GNSs), and the cell-penetrating peptide TAT (TAT-GNSs), and evaluated their effects on X-ray radiotherapy. For the in vitro study, it was found that the ionizing radiation effects of these GNSs were well correlated with their cellular uptake amounts, with the same order of GNSs < NH₂-GNSs < FA-GNSs < TAT-GNSs. The sensitization enhancement ratio (SER), which is commonly used to evaluate how effectively radiosensitizers decrease cell proliferation, reaches 2.30 for TAT-GNSs. The extremely high SER value for TAT-GNSs indicates the superior radiosensitization effect of this nanomaterial. The radiation enhancement mechanisms of these GNSs involved the increased reactive oxygen species (ROS), mitochondrial depolarization, and cell cycle redistribution. Western blotting assays confirmed that the surface-modified GNSs could induce the up-regulation of autophagy-related protein (LC3-II) and apoptosis-related protein (active caspase-3) in cancer cells. By monitoring the degradation of the autophagy substrate p62 protein, GNSs caused impairment of autolysosome degradation capacity and autophagosome accumulation. Our data demonstrated that autophagy played a protective role against caner radiotherapy, and the inhibition of protective autophagy with inhibitors would result in the increase of cell apoptosis. Besides the above in vitro experiments, the in vivo tumor growth study also indicated that X-ray + TAT-GNSs treatment had the best tumor growth inhibitory effect, which confirmed the highest radiation sensitizing effect of TAT-GNSs. This work furthered our understanding on the interaction mechanism between gold nanomaterials and cancer cells and should be able to promote the development of nanoradiosensitizers for clinical applications

Selection of HBsAg-Specific DNA Aptamers Based on Carboxylated Magnetic Nanoparticles and Their Application in the Rapid and Simple Detection of Hepatitis B Virus Infection

Aptamers are short single-stranded DNA or RNA oligonucleotides and can be selected from synthetic combinatorial libraries in vitro. They have a high binding affinity and specificity for their targets. Agarose gels, nitrocellulose membranes, and adsorptive microplates are often used as carriers to immobilize targets in the SELEX (systematic evolution of ligands by exponential enrichment) process, but the subsequent separation step is tedious and time-consuming. Therefore, we used magnetic nanoparticles (MNPs) as carriers to immobilize the target, hepatitis B surface antigen (HBsAg), which is convenient for fast magnetic separation. In this study, we first selected DNA aptamers against HBsAg by immobilizing HBsAg on the surface of carboxylated MNPs. The ssDNA library of each selection round was prepared by asymmetric PCR amplification for the next selection round. To obtain aptamer sequences, the final selected products were purified by gel electrophoresis, then cloned, and sequenced. DNA aptamers that specifically bind to HBsAg were successfully obtained after 13 selection rounds. The selected aptamers were used to construct a chemiluminescence aptasensor based on magnetic separation and immunoassay to detect HBsAg from pure protein or actual serum samples. There was a linear relationship between HBsAg concentration and chemiluminescent intensity in the range of 1–200 ng/mL. The aptasensor worked well even in the presence of interfering substances and was highly specific in the detection of HBsAg in serum samples, with a detection limit 0.1 ng/mL lower than the 0.5 ng/mL limit of an ELISA in use at the hospital. This aptasensor can contribute to better detection of hepatitis B virus infection

Effective Integration of Targeted Tumor Imaging and Therapy Using Functionalized InP QDs with VEGFR2 Monoclonal Antibody and miR-92a Inhibitor

Rapid diagnosis and targeted drug treatment require agents that possess multiple functions. Nanomaterials that facilitate optical imaging and direct drug delivery have shown great promise for effective cancer treatment. In this study, we first modified near-infrared fluorescent indium phosphide quantum dots (InP QDs) with a vascular endothelial growth factor receptor 2 (VEGFR2) monoclonal antibody to afford targeted drug delivery function. Then, a miR-92a inhibitor, an antisense microRNA that enhances the expression of tumor suppressor p63, was attached to the VEGFR2–InP QDs via electrostatic interactions. The functionalized InP nanocomposite (IMAN) selectively targets tumor sites and allows for infrared imaging in vivo. We further explored the mechanism of this active targeting. The IMAN was endocytosed and delivered in the form of microvesicles via VEGFR2–CD63 signaling. Moreover, the IMAN induced apoptosis of human myelogenous leukemia cells through the p63 pathway in vitro and in vivo. These results indicate that the IMAN may provide a new and promising chemotherapy strategy against cancer cells, particularly by its active targeting function and utility in noninvasive three-dimensional tumor imaging

Coating Carbon Nanosphere with Patchy Gold for Production of Highly Efficient Photothermal Agent

Gold- or carbon-based photothermal therapy (PTT) agents have shown encouraging therapeutic effects of PTT in the near-infrared region (NIR) in many preclinical animal experiments. It is expected that gold/carbon hybrid nanomaterial will possess combinational NIR light absorption and can achieve further improvement in photothermal conversion efficiency. In this work, we design and construct a novel PTT agent by coating a carbon nanosphere with patchy gold. To synthesize this composite particle with Janus structure, a new versatile approach based on a facile adsorption–reduction method was presented. Different from the conventional fabrication procedures, the formation of patchy gold in this approach is mainly a thermodynamics-driven spontaneous process. The results show that when compared with the conventional PTT agent gold nanorod the obtained nanocomposites not only have higher photothermal conversion efficiency but also perform more thermally stable. On the basis of these outstanding photothermal effects, the in vitro and in vivo photothermal performances in a MCF-7 cells (human breast adenocarcinoma cell line) and mice were investigated separately. Additionally, to further illustrate the advantage of this asymmetric structure, their potential was explored by selective surface functionalization, taking advantage of the affinity of both patchy gold and carbon domain to different functional molecules. These results suggest that this new hybrid nanomaterial can be used as an effective PTT agent for cancer treatment in the future

Efficient and Facile Synthesis of Gold Nanorods with Finely Tunable Plasmonic Peaks from Visible to Near-IR Range

Although gold nanorods (GNRs) have been prepared with a wide range of methods for their uses as novel diagnostic and therapeutic agents, the synthesis of monodispersed GNRs with high yields and size tunability still requires further improvements. We report on a simple one-pot method for preparing highly monodispersed GNRs using phenols (e.g., hydroquinone, 1,2,3-trihydroxybenzene, and 1,2,4-trihydroxybenzene) as the reducing agent and NaBH₄ as the initiating reactant. Fine-tuning of the LSPR peak position of phenols-reduced GNRs from 550 to 1150 nm is accomplished by regulating the silver ion concentrations. The size of GNRs produced via phenols reduction can also be controlled by changing the NaBH₄ concentration. By systematically optimizing the concentrations of the reagents involved in the one-pot synthesis of GNRs, the yield (in many cases exceeding 90%) is significantly higher than that prepared with the commonly used reductant (e.g., ascorbic acid). The improved efficiency and controllability cut down the cost and time involved in GNRs production

Intrinsic, Cancer Cell-Selective Toxicity of Organic Photothermal Nanoagent: A Simple Formulation for Combined Photothermal Chemotherapy of Cancer

Nano-agent-mediated photothermal therapy (PTT) combined with chemotherapy has been proposed as an effective strategy against cancer. However, chemotherapeutic agents often cause serious side effects. Herein, a novel PTT nanoagent (Cy5.5–MSA–G250) with unanticipated intrinsic tumor-selective cytotoxicity is developed. The Cy5.5–MSA–G250 nanoparticles (NPs) are created by mixing mouse serum albumin (MSA) and coomassie brilliant blue (G250) and then conjugated with cyanine 5.5 (Cy5.5). As expected, Cy5.5–MSA–G250 NPs can efficiently kill cancer cells in vitro and in vivo by PTT. Meanwhile, we accidentally discover that Cy5.5–MSA–G250 have intrinsic specific cytotoxicity against tumor cells but not against normal cells. Moreover, the tumor-specific cytotoxicity of Cy5.5–MSA–G250 is much stronger than that of cytarabine, an FDA-approved anticancer drug. In vivo experiments also prove that Cy5.5–MSA–G250 NPs can effectively eliminate residual tumor cells and prevent metastasis. Further study indicates that selective induction of G1 cell cycle arrest and inhibition of DNA duplication in tumor cells may be the possible mechanism of the tumor cell-selective cytotoxicity of Cy5.5–MSA–G250 NPs. In addition, direct visualization, low systematic toxicity, good biodegradation, and efficient body excretion further make Cy5.5–MSA–G250 NPs attractive for in vivo applications. Taken together, Cy5.5–MSA–G250 NPs are proven to be a promising platform for combined photothermal chemotherapy

Influence factors evaluation to Combined Paternity Index (CPI) based on real sample sequencing data.

<p>(A) The number of effective SNPs and the logarithm of CPI did not relate with the fetal fraction when fetal fraction over 10%. (B) There was no obvious correlation between Paternity Index of each effective SNPs and fetal fraction. (C) The logarithm of CPI had a significant positive correlation with effective sequencing depth, while the number of effective SNPs did not relate with effective sequencing depth. (D) The logarithm of PI had a positive correlation with the effective sequencing depth.</p