Recent Studies on Metal-Embedded Silica Nanoparticles for Biological Applications.

Recently, silica nanoparticles (NPs) have attracted considerable attention as biocompatible and stable templates for embedding noble metals. Noble-metal-embedded silica NPs utilize the exceptional optical properties of novel metals while overcoming the limitations of individual novel metal NPs. In addition, the structure of metal-embedded silica NPs decorated with small metal NPs around the silica core results in strong signal enhancement in localized surface plasmon resonance and surface-enhanced Raman scattering. This review summarizes recent studies on metal-embedded silica NPs, focusing on their unique designs and applications. The characteristics of the metal-embedded silica NPs depend on the type and structure of the embedded metals. Based on this progress, metal-embedded silica NPs are currently utilized in various spectroscopic applications, serving as nanozymes, detection and imaging probes, drug carriers, photothermal inducers, and bioactivation molecule screening identifiers. Owing to their versatile roles, metal-embedded silica NPs are expected to be applied in various fields, such as biology and medicine, in the future.

Recent Advances in Surface-Enhanced Raman Scattering Magnetic Plasmonic Particles for Bioapplications.

The surface-enhanced Raman scattering (SERS) technique, that uses magnetic plasmonic particles (MPPs), is an advanced SERS detection platform owing to the synergetic effects of the particles' magnetic and plasmonic properties. As well as being an ultrasensitive and reliable SERS material, MPPs perform various functions, such as aiding in separation, drug delivery, and acting as a therapeutic material. This literature discusses the structure and multifunctionality of MPPs, which has enabled the novel application of MPPs to various biological fields.

Evaluation of Sterilization Performance for Vaporized-Hydrogen-Peroxide-Based Sterilizer with Diverse Controlled Parameters.

Hydrogen-peroxide-based low-temperature sterilization is a new sterilization technology for temperature-dependent medical devices. The effect of the process parameters of hydrogen-peroxide-based sterilizer on the sterilization performance of process challenge devices (PCDs) needs to be investigated. Sterilant amount, operating temperature, vacuum pressure, diffusion time, and chamber loading of the sterilizer on the sterilization performance of PCDs were adjusted. Seven PCDs with various morphologies and material containing biological indicators (BI) (EZTest, Geobacillus stearothermophilus) were used to evaluate the sterilization performance. The sterilization success rates of PCDs were 86, 71, and 57% with controlled temperature and pressure, diffusion time, and sterilant volume injection, respectively. The PCD material and structure also obviously affected sterilization performance. The sterilization of PCD A is the least successful for all parameters. Meanwhile, the sterilization of PCD B was influenced by the diffusion time and the sterilant injection volume. PCD B and PCD C were successfully sterilized by controlling the temperature and pressure. The weights and volume of the sterilization loading chamber resulted in a different sterilization performance. Sterilization performances of PCD 1, PCD 2, and PCD 3 were <70, <90, and 100%, respectively. Sterilant volume, sterilant diffusion time, pressure, temperature, PCD types, and chamber loading were proven to be important process parameters of sterilizer that affect the sterilization performance of vaporized-hydrogen-peroxide-based sterilizers.

Functionalized ß-Cyclodextrin Immobilized on Ag-Embedded Silica Nanoparticles as a Drug Carrier.

Cyclodextrins (CDs) have beneficial characteristics for drug delivery, including hydrophobic interior surfaces. Nanocarriers with ß-CD ligands have been prepared with simple surface modifications as drug delivery vehicles. In this study, we synthesized ß-CD derivatives on an Ag-embedded silica nanoparticle (NP) (SiO2@Ag NP) structure to load and release doxorubicin (DOX). Cysteinyl-ß-CD and ethylenediamine-ß-CD (EDA-ß-CD) were immobilized on the surface of SiO2@Ag NPs, as confirmed by transmission electron microscopy (TEM), ultraviolet-visible (UV-Vis) spectrophotometry, and Fourier transform infrared (FTIR) spectroscopy. DOX was introduced into the ß-CD on the SiO2@Ag NPs and then successfully released. Neither cysteinyl-ß-CD and EDA-ß-CD showed cytotoxicity, while DOX-loaded cysteinyl-ß-CD and EDA-ß-CD showed a significant decrease in cell viability in cancer cells. The SiO2@Ag NPs with ß-CD provide a strategy for designing a nanocarrier that can deliver a drug with controlled release from modified chemical types.

Target-specific near-IR induced drug release and photothermal therapy with accumulated Au/Ag hollow nanoshells on pulmonary cancer cell membranes.

Au/Ag hollow nanoshells (AuHNSs) were developed as multifunctional therapeutic agents for effective, targeted, photothermally induced drug delivery under near-infrared (NIR) light. AuHNSs were synthesized by galvanic replacement reaction. We further conjugated antibodies against the epidermal growth factor receptor (EGFR) to the PEGylated AuHNS, followed by loading with the antitumor drug doxorubicin (AuHNS-EGFR-DOX) for lung cancer treatment. AuHNSs showed similar photothermal efficiency to gold nanorods under optimized NIR laser power. The targeting of AuHNS-EGFR-DOX was confirmed by light-scattering images of A549 cells, and doxorubicin release from the AuHNSs was evaluated under low pH and NIR-irradiated conditions. Multifunctional AuHNS-EGFR-DOX induced photothermal ablation of the targeted lung cancer cells and rapid doxorubicin release following irradiation with NIR laser. Furthermore, we evaluated the effectiveness of AuHNS-EGFR-DOX drug delivery by comparing two drug delivery methods: receptor-mediated endocytosis and cell-surface targeting. Accumulation of the AuHNS-EGFR-DOX on the cell surfaces by targeting EGFR turned out to be more effective for lung cancer treatments than uptake of AuHNS-EGFR-DOX. Taken together, our data suggest a new and optimal method of NIR-induced drug release via the accumulation of targeted AuHNS-EGFR-DOX on cancer cell membranes.

One-step synthesis of silver nanoshells with bumps for highly sensitive near-IR SERS nanoprobes.

A seedless, one-step synthetic route to uniform bumpy silver nanoshells (AgNSs) as highly NIR sensitive SERS substrates is reported. These substrates can incorporate Raman label compounds and biocompatible polymers on their surface. AgNS based NIR-SERS probes are successfully applied to cell tracking in a live animal using a portable Raman system.

Aerosol delivery of beclin1 enhanced the anti-tumor effect of radiation in the lungs of K-rasLA1 mice.

Radiotherapy alone has several limitations for treating lung cancer. Inhalation, a non-invasive approach for direct delivery of therapeutic agents to the lung, may help to enhance the therapeutic efficacy of radiation. Up-regulating beclin1, known as a tumor suppressor gene that plays a major role in autophagy, may sensitize tumors and lead to tumor regression in lungs of K-ras(LA1) lung cancer model mice. To minimize the side-effects of radiotherapy, fractionated exposures (five times, 24-h interval) with low dose (2 Gy) of radiation to the restricted area (thorax, 2 cm) were conducted. After sensitizing the lungs with radiation, beclin1, complexed with a nano-sized biodegradable poly(ester amine), was prepared and delivered into the murine lung via aerosol three times/week for four weeks. In a histopathological analysis, animals treated with beclin1 and radiation showed highly significant tumor regression and low progression to adenocarcinoma. An increase in the number of autophagic vacuoles and secondary lysosomes was detected. Dissociation of beclin1-bcl2 stimulated autophagy activation and showed a synergistic anti-tumor effect by inhibiting the Akt-mTOR pathway, cell proliferation and angiogenesis. The combination of radiation with non-invasive aerosol delivery of beclin1 may provide a prospect for developing novel therapy regimens applicable in clinics.

Surface-enhanced Raman scattering-active nanostructures and strategies for bioassays.

Surface-enhanced Raman scattering (SERS) techniques offer a number of advantages in molecular detection and analysis, particularly in terms of the multiplex detection of biomolecules. So far, many new SERS-based substrates and analytical techniques have been reported. For easy understanding, various SERS techniques are classified into the following four categories: adsorption-mediated direct detection; antibody- or ligand-mediated direct detection; binding catalyzed indirect detection; and tag-based indirect detection. Among these, recent successes of SERS tagging/encoding (nano/micro) materials and detection methods are highlighted, including our recent works. Some novel SERS-based strategies for the detection of several biological molecules are also introduced.

Suppression of A549 lung cancer cell migration by precursor let-7g microRNA.

Let-7g miRNAs, short non-coding RNAs approximately 21 nucleotides long, repress protein translation by binding to the 3'UTR of target mRNAs. Aberrant expression of let-7g is associated with the poor prognosis of lung cancer patients. Compared to normal lung cells, let-7g expression is absent in non-small cell lung cancer (NSCLC) cells. Furthermore, K-Ras and HMGA2 are well known as targets of let-7g. In this study, we evaluated the potential role of precursor (pre)-let-7g in lung cancer cell metastasis, focusing on the two targets of let-7g, HMGA2 and K-Ras. We found that pre-let-7g inhibited the migration of A549 lung cancer cells through HMGA2-mediated E2F1 down-regulation. Thus, our results suggest that pre-let-7g could be used as a suitable target for the suppression of lung cancer cell migration.

Multifunctional silver-embedded magnetic nanoparticles as SERS nanoprobes and their applications.

In this study, surface-enhanced Raman spectroscopy (SERS)-encoded magnetic nanoparticles (NPs) are prepared and utilized as a multifunctional tagging material for cancer-cell targeting and separation. First, silver-embedded magnetic NPs are prepared, composed of an 18-nm magnetic core and a 16-nm-thick silica shell with silver NPs formed on the surface. After simple aromatic compounds are adsorbed on the silver-embedded magnetic NPs, they are coated with silica to provide them with chemical and physical stability. The resulting silica-encapsulated magnetic NPs (M-SERS dots) produce strong SERS signals and have magnetic properties. In a model application as a tagging material, the M-SERS dots are successfully utilized for targeting breast-cancer cells (SKBR3) and floating leukemia cells (SP2/O). The targeted cancer cells can be easily separated from the untargeted cells using an external magnetic field. The separated targeted cancer cells exhibit a Raman signal originating from the M-SERS dots. This system proves to be an efficient tool for separating targeted cells. Additionally, the magnetic-field-induced hot spots, which can provide a 1000-times-stronger SERS intensity due to aggregation of the NPs, are studied.

Low dietary inorganic phosphate affects the lung growth of developing mice.

Inorganic phosphate (Pi) plays a critical role in diverse cellular functions, and regulating the Pi balance is accomplished by sodium-dependent Pi co-transporter (NPT). Pulmonary NPT has recently been identified in mammalian lungs. However, to date, many of the studies that have involved Pi have mainly focused on its effect on bone and kidney. Therefore, current study was performed to discover the potential effects of low Pi on the lung of developing transgenic mice expressing the renilla/firefly luciferase dual reporter gene. Two-weeks old male mice divided into 2 groups and these groups were fed either a low PI diet or a normal control diet (normal: 0.5% Pi, low: 0.1% Pi) for 4 weeks. After 4 weeks of the diet, all the mice were sacrificed. Their lungs were harvested and analyzed by performing luciferase assay, Western blotting, kinase assay and immunohistochemistry. Our results demonstrate that low Pi affects the lungs of developing mice by disturbing protein translation, the cell cycle and the expression of fibroblast growth factor-2. These results suggest that optimally regulating Pi consumption may be important to maintain health.

Magnetic surface-enhanced Raman spectroscopic (M-SERS) dots for the identification of bronchioalveolar stem cells in normal and lung cancer mice.

Bronchioalveolar stem cells (BASCs) play an important role in the development of cancer. To study the characterization of BASCs, their isolation and purification are important. However, the cells are very rare in tissues and the available methods of isolating them are limited. The current study was performed to isolate BASCs in the murine lung using magnetic nanoparticle-based surface-enhanced Raman spectroscopic dots (M-SERS Dots). We used K-ras(LA1) mice, a laboratory animal model of non-small cell lung cancer of human, and C57BL/6 mice having the same age as a control. We compared the BASCs between 2 species by FACS analysis with 4 markers of BASCs, CCSP, SP-C, CD34, and Sca-1. We found that BASCs were more abundant in the K-ras(LA1) mice than in the C57BL/6 mice. Also, the M-SERS Dot-mediated positive selection of the CD34(pos) cells enabled the BASCs to be enriched to an approximately 4- to 5-fold higher level than that in the case without pre-separation. In summary, our study demonstrates the potential of using M-SERS Dots as a sorting system with very effective isolation of BASCs and multiplex targeting probe, showing that they may play an effective role in the study of BASCs in the future.

Protein separation and identification using magnetic beads encoded with surface-enhanced Raman spectroscopy.

This article presents a prototype of a surface-enhanced Raman spectroscopy (SERS)-encoded magnetic bead of 8mum diameter. The core part of the bead is composed of a magnetic nanoparticle (NP)-embedded sulfonated polystyrene bead. The outer part of the bead is embedded with Ag NPs on which labeling molecules generating specific SERS bands are adsorbed. A silica shell is fabricated for further bioconjugation and protection of SERS signaling. Benzenethiol, 4-mercaptotoluene, 2-naphthalenethiol, and 4-aminothiophenol are used as labeling molecules. The magnetic SERS beads are used as substrates for protein sensing and screening with easy handling. As a model application, streptavidin-bound magnetic SERS beads are used to illustrate selective separation in a flow cytometry system, and the screened beads are spectrally recognized by Raman spectroscopy. The proposed magnetic SERS beads are likely to be used as a versatile solid support for protein sensing and screening in multiple assay technology.

Multiplex immunoassay using fluorescent-surface enhanced Raman spectroscopic dots for the detection of bronchioalveolar stem cells in murine lung.

Immunoassays using nanomaterials have been rapidly developed for the analysis of multiple biomolecules. Highly sensitive and biocompatible surface enhanced Raman spectroscopy-active nanomaterials have been used for biomolecule analysis by many research groups in order to overcome intrinsic problems of conventional immunoassays. We used fluorescent surface-enhanced Raman spectroscopic dots (F-SERS dots) to detect biomolecules in this study. The F-SERS dots are composed of silver nanoparticle-embedded silica nanospheres, organic Raman tagging materials, and fluorescent dyes. The F-SERS dots demonstrated highly sensitive, selective, and multifunctional characteristics for multiplex targeting, tracking, and imaging of cellular and molecular events in the living organism. We successfully applied F-SERS dots for the detection of three cellular proteins, including CD34, Sca-1, and SP-C. These proteins are simultaneously expressed in bronchioalveolar stem cells (BASCs) in the murine lung. We analyzed the relative expression ratios of each protein in BASCs since external standards were used to evaluate SERS intensity in tissue. Quantitative comparisons of multiple protein expression in tissue were first attempted using SERS-encoded nanoprobes. Our results suggested that immunoassays using F-SERS dots offered significant increases in sensitivity and selectivity. Such immunoassays may serve as the primary next-generation labeling technologies for the simultaneous analysis of multiple biomolecules.

Serum immunoglobulin fused interferon-alpha inhibited tumor growth in athymic mice bearing colon 26 adenocarcinoma cells.

Interferon (IFN) has therapeutic potential for a wide range of infectious and proliferative disorders. However, the half-life of IFN is too short to have a stable therapeutic effect. To overcome this problem, serum immunoglobulin has been fused to IFN. In this study, the efficacy of serum immunoglobulin fused INFs (si-IFN1 and si-IFN2) was evaluated on athymic mice bearing colon 26 adenocarcinoma cells. Seven days after the implantation of tumor cells, each group of mice was injected once a week with si-IFN1 and si-IFN2 at two different concentrations (10 x : 30 microg/kg and 50 x : 150 microg/kg). A slight anti-tumoral effect was observed in all 10 x groups compared to the control. In the 50 x groups, however, si-IFN1 and si-IFN2 showed significant anti- tumoral effects compared to the control. To gain more information on the mechanisms associated with the decrease of tumor size, a Western blot assay of apoptosis-related molecules was performed. The protein expression of cytochrome c, caspase 9, 6, and 3 were increased by si-IFN1 and si-IFN2. These 2 IFNs also increased the expressions of p53, p21, Bax and Bad. Interestingly, si-IFN1 and si-IFN2 decreased the expression of VEGF-beta. Taken together, serum immunoglobulin fused IFNs increased therapeutic efficacy under current experimental condition.

Effects of 7-hydroxy-3-methoxycadalene on cell cycle, apoptosis and protein translation in A549 lung cancer cells.

Previously we reported that cadalene extracted from Zelkova serrata inhibited lung tumorigenesis in mice. However, the precise mechanism has not yet investigated. Here, we examined the effects of cadalene on signal pathways important for apoptosis, cell cycle, and protein translation in lung cancer cells. Our results showed that cadalene suppressed the expression of Akt and its phosphor-forms through controlling PI3K and PTEN. Cadalene also induced apoptosis through facilitating pro-apoptotic protein expression. In addition, cadalene caused cell cycle arrest and decreased mTOR-mediated protein translation. Taken together, cadalene may be developed as a lung cancer therapeutic agent in the future.

Multiplex targeting, tracking, and imaging of apoptosis by fluorescent surface enhanced Raman spectroscopic dots.

We have developed multifunctional fluorescent surface enhanced Raman spectroscopic tagging material (F-SERS dots) composed of silver nanoparticle-embedded silica spheres with fluorescent organic dye and specific Raman labels for multiplex targeting, tracking, and imaging of cellular/molecular events in the living organism. In this study, F-SERS dots fabricated with specific target antibodies (BAX and BAD) were employed for the detection of apoptosis. The F-SERS dots did not show any particular toxicity in several cell lines. The F-SERS dots could monitor the apoptosis effectively and simultaneously through fluorescent images as well as Raman signals in both cells and tissues with high selectivity. Our results clearly demonstrate that F-SERS dots can be easily applicable to multiplex analysis of diverse cellular/molecular events important for maintaining cellular homeostasis.

Cellular uptake of magnetic nanoparticle is mediated through energy-dependent endocytosis in A549 cells.

Biocompatible silica-overcoated magnetic nanoparticles containing an organic fluorescence dye, rhodamine B isothiocyanate (RITC), within a silica shell [50 nm size, MNP@SiO2(RITC)s] were synthesized. For future application of the MNP@SiO2(RITC)s into diverse areas of research such as drug or gene delivery, bioimaging, and biosensors, detailed information of the cellular uptake process of the nanoparticles is essential. Thus, this study was performed to elucidate the precise mechanism by which the lung cancer cells uptake the magnetic nanoparticles. Lung cells were chosen for this study because inhalation is the most likely route of exposure and lung cancer cells were also found to uptake magnetic nanoparticles rapidly in preliminary experiments. The lung cells were pretreated with different metabolic inhibitors. Our results revealed that low temperature disturbed the uptake of magnetic nanoparticles into the cells. Metabolic inhibitors also prevented the delivery of the materials into cells. Use of TEM clearly demonstrated that uptake of the nanoparticles was mediated through endosomes. Taken together, our results demonstrate that magnetic nanoparticles can be internalized into the cells through an energy-dependent endosomal-lysosomal mechanism.