Yung Doug Suh

Nanogap-engineerable Raman-active nanodumbbells for single-molecule detection

Surface-enhanced Raman scattering (SERS)-based signal amplification and detection methods using plasmonic nanostructures have been widely investigated for imaging and sensing applications. However, SERS-based molecule detection strategies have not been practically useful because there is no straightforward method to synthesize and characterize highly sensitive SERS-active nanostructures with sufficiently high yield and efficiency, which results in an extremely low cross-section area in Raman sensing. Here, we report a high-yield synthetic method for SERS-active gold-silver core-shell nanodumbbells, where the gap between two nanoparticles and the Raman-dye position and environment can be engineered on the nanoscale. Atomic-force-microscope-correlated nano-Raman measurements of individual dumbbell structures demonstrate that Raman signals can be repeatedly detected from single-DNA-tethered nanodumbbells. These programmed nanostructure fabrication and single-DNA detection strategies open avenues for the high-yield synthesis of optically active smart nanoparticles and structurally reproducible nanostructure-based single-molecule detection and bioassays.

Highly uniform and reproducible surface-enhanced Raman scattering from DNA-tailorable nanoparticles with 1-nm interior gap

An ideal surface-enhanced Raman scattering (SERS) nanostructure for sensing and imaging applications should induce a high signal enhancement, generate a reproducible and uniform response, and should be easy to synthesize. Many SERS-active nanostructures have been investigated, but they suffer from poor reproducibility of the SERS-active sites, and the wide distribution of their enhancement factor values results in an unquantifiable SERS signal. Here, we show that DNA on gold nanoparticles facilitates the formation of well-defined gold nanobridged nanogap particles (Au-NNP) that generate a highly stable and reproducible SERS signal. The uniform and hollow gap (∼1 nm) between the gold core and gold shell can be precisely loaded with a quantifiable amount of Raman dyes. SERS signals generated by Au-NNPs showed a linear dependence on probe concentration (R(2) > 0.98) and were sensitive down to 10 fM concentrations. Single-particle nano-Raman mapping analysis revealed that >90% of Au-NNPs had enhancement factors greater than 1.0 × 10(8), which is sufficient for single-molecule detection, and the values were narrowly distributed between 1.0 × 10(8) and 5.0 × 10(9).

Theranostic Probe Based on Lanthanide-Doped Nanoparticles for Simultaneous In Vivo Dual-Modal Imaging and Photodynamic Therapy

Dual-modal in vivo tumor imaging and photodynamic therapy using hexagonal NaYF(4):Yb,Er/NaGdF(4) core-shell upconverting nanoparticles combined with a photosensitizer, chlorin e6, is reported. Tumors can be clearly observed not only in the upconversion luminescence image but also in the magnetic resonance image. In vivo photodynamic therapy by systemic administration is demonstrated under 980 nm irradiation.

Long-Term Real-Time Tracking of Lanthanide Ion Doped Upconverting Nanoparticles in Living Cells†

Recently, there has been great interest in employing nanoparticles for various biological applications. Nanoparticles can be synthesized in a controlled manner such that they have desirable sizes, shapes, and optical or magnetic properties. In addition, one may provide nanoparticles with biological functions through chemical surface modifications and conjugation of ligands. Such intrinsic and extrinsic properties of nanoparticles enable them to be used as excellent biological imaging probes and diagnostic/therapeutic agents at the cellular level. Among the various nanoparticle systems developed thus far, semiconductor nanocrystals or quantum dots (QDs) are most widely used. QDs are extremely bright and photostable, and exhibit excellent spectral properties (i.e., broad absorption and narrow emission bands) suited for multicolor detection. However, the drawbacks such as photoblinking, the presence of nonradiant dark particles, and potential cytotoxicity limit their applicability. In recent years, several alternative types of luminescent nanoparticles have been introduced for biological applications. For example, nanodiamonds (NDs) with nitrogen vacancy centers were found to be highly photoluminescent while exhibiting no photoblinking and photobleaching, 11] and even useful as the imaging probe for super-resolution optical microscopy. However, applying NDs for biological imaging has limitations, especially in the case of long-term tracking studies, since the excitation in the blue or green region (typically 488 or 532 nm) might result in fatal photodamage to cells or low penetration depth into tissues. In contrast, single-walled carbon nanotubes (SWNTs) were shown to be appropriate for biological imaging in that the excitation and emission lie in the near-infrared (NIR) spectral range. However, being longer than 100 nm typically, SWNTs are considered to be too large to be used as biolabels. Meanwhile, lanthanide ion doped upconverting nanoparticles (UCNPs), which emit in the visible range upon absorption of NIR photons, have attracted great attention owing to their unique optical properties. First, two-photon upconversion of NIR excitation to the emission of a visible photon is so efficient that a tiny continuous-wave (CW) diode laser (980 nm) with the output of tens of milliwatts is sufficient as the excitation source. Second, by employing NIR excitation, one can suppress cellular autofluorescence, induce little photodamage to living cells, and achieve relatively deep penetration into tissues. Finally, UCNPs exhibit neither photoblinking on the millisecond and second time scales nor photobleaching even with hours of continuous excitation, 21] their cytotoxicity is very low, 22] and the inclusion or doping of Gd ions in the host materials endows UCNPs with an additional modality for magnetic resonance imaging (MRI). 23] As a result, UCNPs became one of the most promising nanoparticle systems for biological imaging and there are continuing efforts to improve their properties (e.g., increasing luminescence intensity and reducing the particle size) by designing new synthetic strategies. Herein, we report the first real-time tracking study with UCNPs at the single vesicle level in living cells. Thanks to the remarkable photostability of UCNPs and the noninvasiveness of NIR excitation, we were able to visualize the intracellular movements of UCNPs for as long as 6 h without interruption. We first assessed the benefits of using NIR radiation as the excitation source to demonstrate the feasibility of long-term live-cell imaging with UCNPs. The UCNPs (hexagonal-phase NaYF4 co-doped with Yb 3+ and Er, ca. 30 nm in diameter) coated by amphiphilic PEG–phospholipids (PEG = poly(ethylene glycol)) were internalized into HeLa cells and imaged on a home-made epi-fluorescence microscope setup (Methods section and Figures S1 and S2 in the Supporting [*] Dr. S. H. Nam, Y. M. Bae, Dr. H. M. Kim, Dr. K. T. Lee, Dr. Y. D. Suh Laboratory for Advanced Molecular Probing (LAMP) NanoBio Fusion Research Center, Korea Research Institute of Chemical Technology, Daejeon 305-600 (Korea) Fax: (+ 82)42-860-7164 E-mail: ktlee@krict.re.kr ydsuh@krict.re.kr Y. I. Park, Dr. J. H. Kim, Prof. Dr. T. Hyeon National Creative Research Initiative Center for Oxide Nanocrystalline Materials, World Class University (WCU) Program of Chemical Convergence for Energy & Environment (C2E2) School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744 (Korea) Y. M. Bae, Prof. Dr. J. S. Choi Department of Biochemistry, Chungnam National University Daejeon 305-764 (Korea) [] These authors contributed equally to this work.

Hypoxia-responsive polymeric nanoparticles for tumor-targeted drug delivery

Hypoxia is a condition found in various intractable diseases. Here, we report self-assembled nanoparticles which can selectively release the hydrophobic agents under hypoxic conditions. For the preparation of hypoxia-responsive nanoparticles (HR-NPs), a hydrophobically modified 2-nitroimidazole derivative was conjugated to the backbone of the carboxymethyl dextran (CM-Dex). Doxorubicin (DOX), a model drug, was effectively encapsulated into the HR-NPs. The HR-NPs released DOX in a sustained manner under the normoxic condition (physiological condition), whereas the drug release rate remarkably increased under the hypoxic condition. From in vitro cytotoxicity tests, it was found the DOX-loaded HR-NPs showed higher toxicity to hypoxic cells than to normoxic cells. Microscopic observation showed that the HR-NPs could effectively deliver DOX into SCC7 cells under hypoxic conditions. In vivo biodistribution study demonstrated that HR-NPs were selectively accumulated at the hypoxic tumor tissues. As consequence, drug-loaded HR-NPs exhibited high anti-tumor activity in vivo. Overall, the HR-NPs might have a potential as nanocarriers for drug delivery to treat hypoxia-associated diseases.

Tuning and Maximizing the Single-Molecule Surface-Enhanced Raman Scattering from DNA-Tethered Nanodumbbells

We extensively study the relationships between single-molecule surface-enhanced Raman scattering (SMSERS) intensity, enhancement factor (EF) distribution over many particles, interparticle distance, particle size/shape/composition and excitation laser wavelength using the single-particle AFM-correlated Raman measurement method and theoretical calculations. Two different single-DNA-tethered Au-Ag core-shell nanodumbbell (GSND) designs with an engineerable nanogap were used in this study: the GSND-I with various interparticle nanogaps from ∼4.8 nm to <1 nm or with no gap and the GSND-II with the fixed interparticle gap size and varying particle size from a 23-30 nm pair to a 50-60 nm pair. From the GSND-I, we learned that synthesizing a <1 nm gap is a key to obtain strong SMSERS signals with a narrow EF value distribution. Importantly, in the case of the GSND-I with <1 nm interparticle gap, an EF value of as high as 5.9 × 10(13) (average value = 1.8 × 10(13)) was obtained and the EF values of analyzed particles were narrowly distributed between 1.9 × 10(12) and 5.9 × 10(13). In the case of the GSND-II probes, a combination of >50 nm Au cores and 514.5 nm laser wavelength that matches well with Ag shell generated stronger SMSERS signals with a more narrow EF distribution than <50 nm Au cores with 514.5 nm laser or the GSND-II structures with 632.8 nm laser. Our results show the usefulness and flexibility of these GSND structures in studying and obtaining SMSERS structures with a narrow distribution of high EF values and that the GSNDs with < 1 nm are promising SERS probes with highly sensitive and quantitative detection capability when optimally designed.

Thiolated DNA-based chemistry and control in the structure and optical properties of plasmonic nanoparticles with ultrasmall interior nanogap.

The design, synthesis and control of plasmonic nanostructures, especially with ultrasmall plasmonically coupled nanogap (∼1 nm or smaller), are of significant interest and importance in chemistry, nanoscience, materials science, optics and nanobiotechnology. Here, we studied and established the thiolated DNA-based synthetic principles and methods in forming and controlling Au core-nanogap-Au shell structures [Au-nanobridged nanogap particles (Au-NNPs)] with various interior nanogap and Au shell structures. We found that differences in the binding affinities and modes among four different bases to Au core, DNA sequence, DNA grafting density and chemical reagents alter Au shell growth mechanism and interior nanogap-forming process on thiolated DNA-modified Au core. Importantly, poly A or poly C sequence creates a wider interior nanogap with a smoother Au shell, while poly T sequence results in a narrower interstitial interior gap with rougher Au shell, and on the basis of the electromagnetic field calculation and experimental results, we unraveled the relationships between the width of the interior plasmonic nanogap, Au shell structure, electromagnetic field and surface-enhanced Raman scattering. These principles and findings shown in this paper offer the fundamental basis for the thiolated DNA-based chemistry in forming and controlling metal nanostructures with ∼1 nm plasmonic gap and insight in the optical properties of the plasmonic NNPs, and these plasmonic nanogap structures are useful as strong and controllable optical signal-generating nanoprobes.

Bioreducible core-crosslinked hyaluronic acid micelle for targeted cancer therapy.

For drug delivery nanocarriers to be a safe and effective therapeutic option, blood stability, tumor-targetability, and intracellular drug release features should be considered. In this study, to develop a potent drug delivery carrier that can meet the multiple requirements, we engineered a bioreducible core-crosslinked polymeric micelle based on hyaluronic acid (CC-HAM) by a facile method using d,l-dithiothreitol in aqueous conditions. The CC-HAM exhibited enhanced structural stability under diluted conditions with PBS containing FBS or sodium dodecyl sulfates. We also successfully encapsulated doxorubicin (DOX), chosen as a hydrophobic anti-cancer drug, in CC-HAMs with high loading efficiency (>80%). The drug release rate of CC-HAMs was rapidly accelerated in the presence of glutathione, whereas the drug release was significantly retarded in physiological buffer (pH7.4). An in vivo biodistribution study demonstrated the superior tumor targetability of CC-HAMs to that of non-crosslinked HAMs, primarily ascribed to robust stability of CC-HAMs in the bloodstream. Notably, these results correspond with the improved pharmacokinetics and tumor accumulation of DOX-loaded CC-HAMs as well as their excellent therapeutic efficacy. Overall, these results suggest that the robust, bioreducible CC-HAM can be applied as a potent doxorubicin delivery carrier for targeted cancer therapy.

Single-molecule and single-particle-based correlation studies between localized surface plasmons of dimeric nanostructures with ~1 nm gap and surface-enhanced Raman scattering.

Understanding the detailed electromagnetic field distribution inside a plasmonically coupled nanostructure, especially for structures with ~ 1 nm plasmonic gap, is the fundamental basis for the control and use of the strong optical properties of plasmonic nanostructures. Using a multistep AFM tip-matching strategy that enables us to gain the optical spectra with the optimal signal-to-noise ratio as well as high reliability in correlation measurement between localized surface plasmon (LSP) and surface-enhanced Raman scattering (SERS), the coupled longitudinal dipolar and high-order multipolar LSPs were detected within a dimeric structure, where a single Raman dye is located via a single-DNA hybridization between two differently sized Au-Ag core-shell particles. On the basis of the characterization of each LSP component, the distinct phase differences, attributed to different quantities of the excited quadrupolar LSPs, between the transverse and longitudinal regimes were observed for the first time. By assessing the relative ratio of dipolar and quadrupolar LSPs, we found that these LSPs of the dimer with ~ 1 nm gap were simultaneously excited, and large longitudinal bonding dipolar LSP/longitudinal bonding quadrupolar LSP value is required to generate high SERS signal intensity. Interestingly, a minor population of the examined dimers exhibited strong SERS intensities along not only the dimer axis but also the direction that arises from the interaction between the coupled transverse dipolar and longitudinal bonding quadrupolar LSPs. Overall, our high-precision correlation measurement strategy with a plasmonic heterodimer with ~ 1 nm gap allows for the observation of the characteristic spectral features with the optimal signal-to-noise ratio and the subpopulation of plasmonic dimers with a distinct SERS behavior, hidden by a majority of dimer population, and the method and results can be useful in understanding the whole distribution of SERS enhancement factor values and designing plasmonic nanoantenna structures.

Bioreducible Carboxymethyl Dextran Nanoparticles for Tumor-Targeted Drug Delivery

Bioreducible carboxymethyl dextran (CMD) derivatives are synthesized by the chemical modification of CMD with lithocholic acid (LCA) through a disulfide linkage. The hydrophobic nature of LCA allows the conjugates (CMD-SS-LCAs) to form self-assembled nanoparticles in aqueous conditions. Depending on the degree of LCA substitution, the particle diameters range from 163 to 242 nm. Doxorubicin (DOX), chosen as a model anticancer drug, is effectively encapsulated into the nanoparticles with high loading efficiency (>70%). In vitro optical imaging tests reveal that the fluorescence signal of DOX quenched in the bioreducible nanoparticles is highly recovered in the presence of glutathione (GSH), a tripeptide capable of reducing disulfide bonds in the intracellular compartments. Bioreducible nanoparticles rapidly release DOX when they are incubated with 10 mm GSH, whereas the drug release is greatly retarded in physiological buffer (pH 7.4). DOX-loaded bioreducible nanoparticles exhibit higher toxicity to SCC7 cancer cells than DOX-loaded nanoparticles without the disulfide bond. Confocal laser scanning microscopy observation demonstrate that bioreducible nanoparticles can effectively deliver DOX into the nuclei of SCC7 cells. In vivo biodistribution study indicates that Cy5.5-labeled CMD-SS-LCAs selectively accumulate at tumor sites after systemic administration into tumor-bearing mice. Notably, DOX-loaded bioreducible nanoparticles exhibit higher antitumor efficacy than reduction-insensitive control nanoparticles. Overall, it is evident that bioreducible CMD-SS-LCA nanoparticles are useful as a drug carrier for cancer therapy.