In Vitro Tracking of Human Umbilical Vein Endothelial Cells Using Ultra-Sensitive Quantum Dot-Embedded Silica Nanoparticles.

The nanoscale spatiotemporal resolution of single-particle tracking (SPT) renders it a powerful method for exploring single-molecule dynamics in living cells or tissues, despite the disadvantages of using traditional organic fluorescence probes, such as the weak fluorescent signal against the strong cellular autofluorescence background coupled with a fast-photobleaching rate. Quantum dots (QDs), which enable tracking targets in multiple colors, have been proposed as an alternative to traditional organic fluorescence dyes; however, they are not ideally suitable for applying SPT due to their hydrophobicity, cytotoxicity, and blinking problems. This study reports an improved SPT method using silica-coated QD-embedded silica nanoparticles (QD2), which represent brighter fluorescence and are less toxic than single QDs. After treatment of QD2 in 10 µg/mL, the label was retained for 96 h with 83.76% of labeling efficiency, without impaired cell function such as angiogenesis. The improved stability of QD2 facilitates the visualization of in situ endothelial vessel formation without real-time staining. Cells retain QD2 fluorescence signal for 15 days at 4 °C without significant photobleaching, indicating that QD2 has overcome the limitations of SPT enabling long-term intracellular tracking. These results proved that QD2 could be used for SPT as a substitute for traditional organic fluorophores or single quantum dots, with its photostability, biocompatibility, and superior brightness.

Magnetic Nanoparticles.

Magnetic nanoparticles have been used in various fields such as data storage, biomedicine, or bioimaging with their unique magnetic property. With their low toxicity, the importance of magnetic nanoparticles keeps increasing especially in biological field. In this chapter, content suitable for scientific inquirers or undergraduates to acquire basic knowledge about nanotechnology is introduced and then recent research trends in nanotechnology are covered.

Synthesis and Application of Silica-Coated Quantum Dots in Biomedicine.

Quantum dots (QDs) are semiconductor nanoparticles with outstanding optoelectronic properties. More specifically, QDs are highly bright and exhibit wide absorption spectra, narrow light bands, and excellent photovoltaic stability, which make them useful in bioscience and medicine, particularly for sensing, optical imaging, cell separation, and diagnosis. In general, QDs are stabilized using a hydrophobic ligand during synthesis, and thus their hydrophobic surfaces must undergo hydrophilic modification if the QDs are to be used in bioapplications. Silica-coating is one of the most effective methods for overcoming the disadvantages of QDs, owing to silica's physicochemical stability, nontoxicity, and excellent bioavailability. This review highlights recent progress in the design, preparation, and application of silica-coated QDs and presents an overview of the major challenges and prospects of their application.

Stent coated with antibody against vascular endothelial-cadherin captures endothelial progenitor cells, accelerates re-endothelialization, and reduces neointimal formation.

OBJECTIVE: In contrast to CD34, vascular endothelial-cadherin (VE-cadherin) is exclusively expressed on the late endothelial progenitor cells (EPC) whereas not on the early or myeloid EPC. Thus, VE-cadherin could be an ideal target surface molecule to capture circulating late EPC. In the present study, we evaluated whether anti-VE-cadherin antibody-coated stents (VE-cad stents) might accelerate endothelial recovery and reduce neointimal formation through the ability of capturing EPC. METHODS AND RESULTS: The stainless steel stents were coated with rabbit polyclonal anti-human VE-cadherin antibodies and exposed to EPC for 30 minutes in vitro. The number of EPC that adhered to the surface of VE-cad stents was significantly higher than bare metal stents (BMS) in vitro, which was obliterated by pretreatment of VE-cad stent with soluble VE-cadherin proteins. We deployed VE-cad stents and BMS in the rabbit right and left iliac arteries, respectively. At 48 hours after stent deployment in vivo, CD-31-positive endothelial cells adhered to VE-cad stent significantly more than to BMS. At 3 days, scanning electron microscopy showed that over 90% surface of VE-cad stents was covered with endothelial cells, which was significantly different from BMS. At 42 days, neointimal area that was filled with smooth muscle cells positive for actin or calponin was significantly smaller in VE-cad stents than in BMS by histological analysis (0.95±0.22 versus 1.34±0.43 mm(2), respectively, P=0.02). Immuno-histochemical analysis revealed that infiltration of inflammatory cells was not significantly different between 2 stents. CONCLUSIONS: VE-cad stents captured EPC successfully in vitro, accelerated endothelial recovery on stent, and eventually reduced neointimal formation in vivo.

Assembly of Plasmonic and Magnetic Nanoparticles with Fluorescent Silica Shell Layer for Tri-functional SERS-Magnetic-Fluorescence Probes and Its Bioapplications.

In this study, we report on the fabrication of multilayered tri-functional magnetic-SERS-fluorescence nanoprobes (MF-SERS particles) containing clustered superparamagnetic Fe3O4 nanoparticles (NPs), silver NPs, and a fluorescent silica layer. The MF-SERS particles exhibited strong SERS signals from the silver NPs as well as both superparamagnetism and fluorescence. MF-SERS particles were uptaken by cells, allowing successful separation using an external magnetic field. SERS and fluorescence signals could be detected from the NP-containing cells, and CD44 antibody-conjugated MF-SERS particles selectively targeted MDA-MB-231 cells. Based on these properties, MF-SERS particles proved to be a useful nanoprobe for multiplex detection and separation of cancer cells.

Ultrasensitive NIR-SERRS Probes with Multiplexed Ratiometric Quantification for In Vivo Antibody Leads Validation.

Immunotargeting ability of antibodies may show significant difference between in vitro and in vivo. To select antibody leads with high affinity and specificity, it is necessary to perform in vivo validation of antibody candidates following in vitro antibody screening. Herein, a robust in vivo validation of anti-tetraspanin-8 antibody candidates against human colon cancer using ratiometric quantification method is reported. The validation is performed on a single mouse and analyzed by multiplexed surface-enhanced Raman scattering using ultrasensitive and near infrared (NIR)-active surface-enhanced resonance Raman scattering nanoprobes (NIR-SERRS dots). The NIR-SERRS dots are composed of NIR-active labels and Au/Ag hollow-shell assembled silica nanospheres. A 93% of NIR-SERRS dots is detectable at a single-particle level and signal intensity is 100-fold stronger than that from nonresonant molecule-labeled spherical Au NPs (80 nm). The result of SERRS-based antibody validation is comparable to that of the conventional method using single-photon-emission computed tomography. The NIR-SERRS-based strategy is an alternate validation method which provides cost-effective and accurate multiplexing measurements for antibody-based drug development.

Highly Sensitive Magnetic-SERS Dual-Function Silica Nanoprobes for Effective On-Site Organic Chemical Detection.

We report magnetic silver nanoshells (M-AgNSs) that have both magnetic and SERS properties for SERS-based detection. The M-AgNSs are composed of hundreds of Fe3O4 nanoparticles for rapid accumulation and bumpy silver shell for sensitive SERS detection by near-infrared laser excitation. The intensity of the SERS signal from the M-AgNSs was strong enough to provide single particle-level detection. We obtained much stronger SERS signal intensity from the aggregated M-AgNSs than from the non-aggregated AgNSs. 4-Fluorothiophenol was detected at concentrations as low as 1 nM, which corresponds to 0.16 ppb. The limit of detection for tetramethylthiuram disulfide was 10 µM, which corresponds to 3 ppm. The M-AgNSs can be used to detect trace amounts of organic molecules using a portable Raman system.

Double-Layer Magnetic Nanoparticle-Embedded Silica Particles for Efficient Bio-Separation.

Superparamagnetic Fe3O4 nanoparticles (NPs) based nanomaterials have been exploited in various biotechnology fields including biomolecule separation. However, slow accumulation of Fe3O4 NPs by magnets may limit broad applications of Fe3O4 NP-based nanomaterials. In this study, we report fabrication of Fe3O4 NPs double-layered silica nanoparticles (DL MNPs) with a silica core and highly packed Fe3O4 NPs layers. The DL MNPs had a superparamagnetic property and efficient accumulation kinetics under an external magnetic field. Moreover, the magnetic field-exposed DL MNPs show quantitative accumulation, whereas Fe3O4 NPs single-layered silica nanoparticles (SL MNPs) and silica-coated Fe3O4 NPs produced a saturated plateau under full recovery of the NPs. DL MNPs are promising nanomaterials with great potential to separate and analyze biomolecules.

Direct identification of on-bead peptides using surface-enhanced Raman spectroscopic barcoding system for high-throughput bioanalysis.

Recently, preparation and screening of compound libraries remain one of the most challenging tasks in drug discovery, biomarker detection, and biomolecular profiling processes. So far, several distinct encoding/decoding methods such as chemical encoding, graphical encoding, and optical encoding have been reported to identify those libraries. In this paper, a simple and efficient surface-enhanced Raman spectroscopic (SERS) barcoding method using highly sensitive SERS nanoparticles (SERS ID) is presented. The 44 kinds of SERS IDs were able to generate simple codes and could possibly generate more than one million kinds of codes by incorporating combinations of different SERS IDs. The barcoding method exhibited high stability and reliability under bioassay conditions. The SERS ID encoding based screening platform can identify the peptide ligand on the bead and also quantify its binding affinity for specific protein. We believe that our SERS barcoding technology is a promising method in the screening of one-bead-one-compound (OBOC) libraries for drug discovery.

Quantum dot-assembled nanoparticles with polydiacetylene supramolecule toward label-free, multiplexed optical detection.

Quantum dot (QD)-assembled silica nanoparticles bearing a polydiacetylene (PDA) supramolecule on their surface (SiO(2)@QDs@PDA NPs) were developed for label-free and multiplexed detection of biological molecules. Two types of QD-assembled silica NPs (SiO(2)@QDs NPs) were prepared and coated with the PDA supramolecule via photo-induced polymerization of 10,12-pentacosadiynoic acid. One of the SiO(2)@QDs NPs was embedded with blue-QDs, and the other was embedded with green-QDs for encoding. The resulting SiO(2)@QDs@PDA NPs showed discrete QD photoluminescence for encoding as well as PDA fluorescence for sensing a target without interference or overlap. Under heating stress of the SiO(2)@QDs@PDA NPs, the color of the PDA changed from blue to red, which allowed us to observe the fluorescence emitted from red PDA. The mixture of two different SiO(2)@QDs@PDA NPs, SiO(2)@QDs@blue-PDA NPs not emitting the fluorescence of PDA and SiO(2)@QDs@red-PDA NPs where stress was brought onto turn on the PDA fluorescence, was effectively imaged and readily distinguished via fluorescence microscopy, indicating their potential for label-free and multiplexed detection of target molecules.

Fabrication of biofunctional stents with endothelial progenitor cell specificity for vascular re-endothelialization.

Endothelial progenitor cells (EPCs) have been identified as a crucial factor for re-endothelialization after stenting, resulting in the prevention of stent thrombosis and neointimal hyperplasia. Because EPCs can be introduced by antibody-antigen interactions, the suitable choice of antibody and the biocompatible surface modification technology including antibody immobilization are essential for developing an EPC-capturing stent. In this study, we fabricated a biofunctional stent with EPC specificity by grafting a hydrophilic polymer and consecutively immobilizing the antibody against vascular endothelial cadherin (VE-cadherin) which is one of the specific EPC surface markers. The surface of a stainless steel stent was sequentially modified by acid-treatment, silanization and covalent attachment of polymers not only to improve biocompatibility but also to introduce functional groups on the stent surface. The surface-modified stent immobilized anti-VE-cadherin antibodies, and the EPCs were remarkably captured whereas THP-1s, human acute monocytic leukemia cells, were not adsorbed on the stent. Furthermore, we confirmed that the recruited EPCs developed the endothelial cell layers on the antibody-conjugated stent. These positive in vitro results will encourage the extensive application of biofunctional surface modification technology for a variety of medical devices.

Polymer-mediated formation and assembly of silver nanoparticles on silica nanospheres for sensitive surface-enhanced Raman scattering detection.

To impart a desired optical property to metal nanoparticles (NPs) suitable for surface-enhanced Raman scattering (SERS) applications, it is crucial to assemble them in two or three dimensions in addition to controlling their size and shape. Herein, we report a new strategy for the synthesis and direct assembly of Ag NPs on silica nanospheres (AgNPs-SiNS) in the presence of poly(ethylene glycol) (PEG) derivatives such as PEG-OH, bis(amino)-PEGs (DA-PEGs), and O,O'-bis(2-aminopropyl)PEG (DAP-PEG). They exhibited different effects on the formation of Ag NPs with variable sizes (10-40 nm) and density on the silica surface. As the molecular weight (MW) of DA-PEGs increased, the number of Ag NPs on the silica surface increased. In addition, DAP-PEG (MW of 2000), which has a 2-aminopropyl moiety at both ends, promoted the most effective formation and assembly of uniform-sized Ag NPs on a silica surface, as compared to the other PEG derivatives with the same molecular weight. Finally, we demonstrated that AgNPs-SiNS bearing 4-fluorobenzenethiol on its surface induced the strong SERS signal at the single-particle level, indicating that each hybrid particle has internal hot spots. This shows the potential of AgNPs-SiNS for SERS-based sensitive detection of target molecules.

Occurrence of Stolbur Phytoplasma Disease in Spreading Type Petunia hybrida Cultivars in Korea.

In January 2012, spreading type petunia cv. Wave Pink plants showing an abnormal growth habit of sprouting unusual multiple plantlets from the lateral buds were collected from a greenhouse in Gwacheon, Gyeonggi Province, Korea. The presence of phytoplasma was investigated using PCR with the primer pairs P1/P6, and R16F1/R1 for nested-PCR. In the nested PCR, 1,096 bp PCR products were obtained, and through sequencing 12 Pet-Stol isolates were identified. Comparison of the nucleotide sequences of 16S rRNA gene of the 12 Pet-Stol isolates with other phytoplasmas belonging to aster yellows or Stolbur showed that Pet-Stol isolates were members of Stolbur. The presence of phytoplasma in petunia was also confirmed by microscopic observation of the pathogens. In this study, Stolbur phytoplasma was identified from spreading type petunia cultivars by sequence analysis of 16S rRNA gene of phytoplasma and microscopic observation of phytoplasma bodies. This is the first report of Stolbur phytoplasma in commercial Petunia hybrida cultivars.

Comparison of endothelialization and neointimal formation with stents coated with antibodies against CD34 and vascular endothelial-cadherin.

Vascular endothelial-cadherin (VE-cadherin) is exclusively expressed on the late endothelial progenitor cells (EPC). Therefore, VE-cadherin could be an ideal target surface molecule to capture circulating late EPC. In the present study, we evaluated whether anti-VE-cadherin antibody-coated stents (VE-cad stents) might accelerate endothelial recovery and reduce neointimal formation more than anti-CD34 antibody-coated stents (CD34 stents) through the superior ability to capture the late EPC. The stainless steel stents were coated with anti-human VE-cadherin antibodies or anti-human CD34 antibodies under the same condition. In vitro, VE-cad stents showed higher number of adhering EPC (823.6 ± 182.2 versus 379.2 ± 137.2 cells per HPF, p < 0.001). VE-cad stents also demonstrated better specific capturing of cells with endothelial lineage markers than CD34 stents did in flow cytometric analysis. VE-cad stents showed more effective re-endothelialization after 1 h, 24 h, and 3 days in vivo. At 42 days, VE-cad stents demonstrated significantly smaller neointima area (0.92 ± 0.38 versus 1.24 ± 0.41 mm(2), p = 0.002) and significantly lower PCNA positive cells in neointima (1684.8 ± 658.8/mm(2) versus 2681.7 ± 375.1/mm(2), p = 0.008), compared with CD34 stents. In conclusion, VE-cad stents captured EPC and endothelial cells more selectively in vitro, accelerated re-endothelialization over stents, and reduced neointimal formation in vivo, compared with CD34 stents.