Young-Woock Noh

Noninvasive imaging of dendritic cell migration into lymph nodes using near-infrared fluorescent semiconductor nanocrystals

Effective tracking of immunotherapeutic cells is essential for monitoring the migration of injected cells to the target tissue. Here we report the use of near‐infrared (NIR) ‐emitting fluorescent semiconductor nanocrystals, called quantum dots (QDs), for noninvasive in vivo tracking of dendritic cell (DC) migration into lymph nodes. The effect of QDs on DC viability and maturation was systematically investigated using MTT assays and FACS analysis. We found that the labeling of DCs with QDs had no effect on DC phenotype or maturation potential. Cytokine and migration assays revealed that there were no significant changes in either cytokine production or chemokine‐dependent migration of QD‐labeled DCs relative to unlabeled cells; in both labeled and unlabeled cells, cytokine production and migratory capacity was increased by stimulation with lipopolysaccharide. Furthermore, QDs did not influence allogenic naive T cell activation or uptake of exogenous antigens. Notably, we also demonstrated that it was possible to track QD‐labeled DCs injected into the footpad into popliteal and inguinal lymph nodes using NIR fluorescence. Taken together, our protocols establish the potential of noninvasive in vivo imaging of NIR‐emitting QDs for tracking immunotherapeutic cells.— Noh, Y.‐W., Lim, Y. T., Chung, B. H. Noninvasive imaging of dendritic cell migration into lymph nodes using near‐infrared fluorescent semiconductor nanocrystals. FASEB J. 22, 3908–3918 (2008)

Simultaneous in vivo tracking of dendritic cells and priming of an antigen-specific immune response

We report the fabrication of a one-pot antigen system that delivers antigen to dendritic cells (DCs) and tracks their in vivo migration after injection. Multifunctional polymer nanoparticles containing ovalbumin protein, magnetic resonance imaging contrast agents (iron oxide nanoparticles), and near-infrared fluorophores (indocyanine green, ICG), MPN-OVA, were prepared using a double emulsion method. The MPN-OVA was efficiently taken up by the dendritic cells and subsequently localized in the lysosome. Flow cytometry analysis revealed an increase in the uptake of OVA antigen by MPN-OVA at 37 °C, when compared with soluble OVA protein. We found that MPN-OVA had no effect on DC surface expression of MHC class I, costimulatory (CD80, CD86) or adhesion (CD54) molecules or the ability of DCs to mature in response to LPS. Following the uptake of MPN-OVA, exogenous OVA antigen was delivered to the cytoplasm, and OVA peptides were presented on MHC class I molecules, which enhanced OVA antigen-specific cross-presentation to OT-1 T cells and CD8OVA1.3 T cell hybridoma in vitro. The immunization of mice with MPN-OVA-treated DCs induced OVA-specific CTL activity in draining lymph nodes. The presence of MPN allowed us to monitor the migration of DCs via lymphatic drainage using NIR fluorescence imaging, and the homing of DCs into the lymph nodes was imaged using MRI. This system has potential for use as a delivery system to induce T cell priming and to image DC-based immunotherapies.

Multiplexed imaging of therapeutic cells with multispectrally encoded magnetofluorescent nanocomposite emulsions.

Here, we describe the fabrication of multispectrally encoded nanoprobes, perfluorocarbon (PFC)/quantum dots (QDs) nanocomposite emulsions, which could provide both multispectral MR and multicolor optical imaging modalities. Our strategy exploited the combination of the multispectral MR properties of four different PFC materials and the multicolor emission properties of three different colored CdSe/ZnS QDs. The PFC/QDs nanocomposite emulsions were fabricated by exchanging hydrophobic ligands coated onto CdSe/ZnS QDs using 1H,1H,2H,2H-perfluorooctanethiol, which renders the QDs to be dispersible in the PFC liquids. To provide biocompatibility, the PFC liquids containing QDs were emulsified into aqueous solutions with the aid of phospholipids. The distinct (19)F-based MR images of PFC/QDs nanocomposite emulsions were obtained by selective excitation of the nanocomposite emulsions with magnetic resonance frequency of each PFC, while a specific fluorescence image of them could be selected using appropriate optical filters. The uptake of PFC/QDs nanocomposite emulsions was high in phagocytic cells such as macrophages (90.55%) and dendritic cells (85.34%), while it was low in nonphagocytic T cells (33%). We have also shown that the nanocomposite emulsions were successfully applied to differentially visualize immunotherapeutic cells (macrophages, dendritic cells, and T cells) in vivo. The PFC/QDs nanocomposite emulsions are expected to be a promising multimodality nanoprobe for the multiplexed detection and imaging of therapeutic cells both in vitro and in vivo.

Growth mode and structural characterization of GaSb on Si (001) substrate: A transmission electron microscopy study

Growth mode and structural properties of GaSb layers grown on silicon substrate by molecular beam epitaxy method are investigated by transmission electron microscopy. It is found that the GaSb grows to three-dimensional islands and grains are tilted to reduce a lattice mismatch through twin boundaries when they are directly grown on Si substrate. A low-temperature (LT) AlSb buffer plays a key role in transferring the growth mode from a three-dimensional island to a layer-by-layer structure. When the LT AlSb layer is used as a buffer, 90° misfit dislocations, with the Burgers vector b of 1∕2a⟨110⟩, are observed on the interface.

Near-infrared emitting fluorescent nanocrystals-labeled natural killer cells as a platform technology for the optical imaging of immunotherapeutic cells-based cancer therapy

This study describes the development of near-infrared optical imaging technology for the monitoring of immunotherapeutic cell-based cancer therapy using natural killer (NK) cells labeled with fluorescent nanocrystals. Although NK cell-based immunotherapeutic strategies have drawn interest as potent preclinical or clinical methods of cancer therapy, there are few reports documenting the molecular imaging of NK cell-based cancer therapy, primarily due to the difficulty of labeling of NK cells with imaging probes. Human natural killer cells (NK92MI) were labeled with anti-human CD56 antibody-coated quantum dots (QD705) for fluorescence imaging. FACS analysis showed that the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 have no effect on the cell viability. The effect of anti-human CD56 antibody-coated QD705 labeling on the NK92MI cell function was investigated by measuring interferon gamma (IFN-gamma) production and cytolytic activity. Finally, the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 showed a therapeutic effect similar to that of unlabeled NK92MI cells. Images of intratumorally injected NK92MI cells labeled with anti-human CD56 antibody-coated could be acquired using near-infrared optical imaging both in vivo and in vitro. This result demonstrates that the immunotherapeutic cells labeled with fluorescent nanocrystals can be a versatile platform for the effective tracking of injected therapeutic cells using optical imaging technology, which is very important in cell-based cancer therapies.

Perfluorodecalin/[InGaP/ZnS quantum dots] nanoemulsions as 19F MR/optical imaging nanoprobes for the labeling of phagocytic and nonphagocytic immune cells.

Multimodal imaging contrast agents with unique magnetic resonance (MR) and optical imaging capabilities have great potentials in the diagnosis and therapy of disease. Using a rational materials design approach, the bimodal imaging contrast agent, perfluorodecalin (PFD)/[InGaP/ZnS quantum dots (QDs)] composite nanoemulsions is developed in this study. (19)F molecules in the PFD/[InGaP/ZnS QDs] nanoemulsions provide a (19)F-based MR imaging capability, while fluorescent QDs dispersed in PFD nanodroplets provide an optical imaging modality. This study also demonstrates that these bimodal imaging contrast agents can be delivered easily into both phagocytic and nonphagocytic immune cells. Internalization of multifunctional PFD/[InGaP/ZnS QDs] nanoemulsions into immunotherapeutic cells permits the labeled cells to be imaged by both magnetic resonance and fluorescence imaging with little effect on cell viability and function. The results of our study highlight the potential of PFD/[InGaP/ZnS QDs] nanoemulsion as a bimodal imaging nanoprobe for molecular imaging in immune cell-based cancer therapies.

Enhancement of the photostability and retention time of indocyanine green in sentinel lymph node mapping by anionic polyelectrolytes

Sentinel lymph node (SLN) biopsy techniques have been widely used in the diagnosis of cancer metastasis because lymph node metastasis is one of the most important prognostic signs. Indocyanine green (ICG) has potential application as a molecular imaging probe for SLN mapping due to its fluorescent properties emitting in the near-infrared (NIR) region, where light transmission through biological tissue is maximized. However, its low photostability in an aqueous solution at the physiological temperature and its rapid diffusion behavior through SLN into the second lymph node have limited its wide use in real clinical fields. In this study, we developed a new NIR imaging contrast system consisting of ICG and poly (γ-glutamic acid) (γ-PGA) polymers for efficient sentinel lymph node mapping. By a combination of clinically used ICG and the biocompatible anionic polyelectrolyte, γ-PGA, the photostabilities of aqueous ICG solutions at room and body temperatures were drastically enhanced. When the ICG/γ-PGA complex was injected subcutaneously into the front paw of a mouse, it entered the lymphatics and migrated to the axillary sentinel lymph node (SLN) within 2 min. Furthermore, the NIR fluorescent signal intensity and retention time of ICG/γ-PGA complex in lymph node were superior to those of ICG only. In addition, a histofluorescentstudy of the SLN resected under NIR imaging revealed that ICG and γ-PGA were co-localized in the lymph node.Taken together, the experimental results on the enhanced photostability and retention time of the ICG/γ-PGA complex provide strong evidence that it has promising potential for improved sentinel lymph node mapping.

Diagnosis and therapy of macrophage cells using dextran-coated near-infrared responsive hollow-type gold nanoparticles

We describe the development of hollow-type gold nanoparticles (NPs) for the photonic-based imaging and therapy of macrophage cells. The strong light-absorption and light-scattering properties of gold NPs render them to be useful as molecular imaging agents as well as therapeutic moieties. By controlling the geometry of the gold NPs, the optical resonance peak was shifted to around the near-infrared (NIR) region, where light transmission through biological tissue is known to be fairly high. Hollow-type gold NPs modified with dextran were phagocytosed by macrophage cells. Using dark-field microscopy, it was possible to image macrophage cells targeted with NPs. After NIR irradiation, macrophages labeled with NPs were selectively destroyed by the photothermal effect. FACS analysis revealed that the photothermal effect caused principally late apoptosis-related cell death or secondary necrosis. The experimental results showed that hollow-type gold NPs conjugated with dextran could be used not only as optical imaging contrast agents but also as a component of a novel anti-macrophage therapeutic strategy.

Anti-proliferative effect of honokiol in oral squamous cancer through the regulation of specificity protein 1.

Honokiol (HK), a novel plant-derived natural product, is a physiologically activated compound with polyphenolic structure, and has been identified to function as an anticancer agent. It has been widely used in several diseases as a traditional medicine for a long time. We investigated whether HK could show anticancer effects on two oral squamous cell lines (OSCCs), HN-22 and HSC-4. We demonstrated that HK-treated cells showed dramatic reduction in cell growth and apoptotic cell morphologies. Intriguingly, the transcription factor specificity protein 1 (Sp1) was significantly inhibited by HK in a dose-dependent manner. Furthermore, we checked changes in cell cycle regulatory proteins and anti-apoptotic proteins at the molecular level, which are known as Sp1 target genes. The important key regulators in the cell cycle such as p27 and p21 were up-regulated by HK-mediated down-regulation of Sp1, whereas anti-apoptotic proteins including Mcl-1 and survivin were decreased, resulting in caspase-dependent apoptosis. Taken together, results from this study suggest that HK could modulate Sp1 transactivation and induce apoptotic cell death through the regulation of cell cycle and suppression of anti‑apoptotic proteins. In addition, HK may be used in cancer prevention and therapies to improve the clinical outcome as an anticancer drug.

Multifunctional perfluorocarbon nanoemulsions for 19F-based magnetic resonance and near-infrared optical imaging of dendritic cells

A novel type of bimodal imaging nanoprobe based on (19)F-based magnetic resonance imaging and near-infrared optical imaging has been synthesized and applied for the labeling and imaging of dendritic cells both in vitro and in vivo.