Ji-Xin Cheng

Gold Nanorods as Contrast Agents for Biological Imaging: Optical Properties, Surface Conjugation and Photothermal Effects†

Gold nanorods (NRs) have plasmon‐resonant absorption and scattering in the near‐infrared (NIR) region, making them attractive probes for in vitro and in vivo imaging. In the cellular environment, NRs can provide scattering contrast for darkfield microscopy, or emit a strong two‐photon luminescence due to plasmon‐enhanced two‐photon absorption. NRs have also been employed in biomedical imaging modalities such as optical coherence tomography or photoacoustic tomography. Careful control over surface chemistry enhances the capacity of NRs as biological imaging agents by enabling cell‐specific targeting, and by increasing their dispersion stability and circulation lifetimes. NRs can also efficiently convert optical energy into heat, and inflict localized damage to tumor cells. Laser‐induced heating of NRs can disrupt cell membrane integrity and homeostasis, resulting in Ca2+ influx and the depolymerization of the intracellular actin network. The combination of plasmon‐resonant optical properties, intense local photothermal effects and robust surface chemistry render gold NRs as promising theragnostic agents.

Overcoming the barriers in micellar drug delivery: loading efficiency, in vivo stability, and micelle–cell interaction

Importance of the field: Spontaneously constructed from block copolymers in aqueous media, the polymer micelle has been extensively studied as a potential carrier of poorly water-soluble drugs, but cellular uptake pathways and stability of micelles in blood have not yet been clearly understood. An in-depth insight into the physical and biological behaviors of polymer micelles is necessitated for designing next-generation micelles. Areas covered in this review: This review suggests possible solutions to improve micellar drug loading capacity, scrutinizes the parameters influencing the micelle stability in blood, and also discusses the fate of micelles in cellular and in vivo environment, respectively. Direct and indirect evidences from the literatures mostly published after 90s were collected, analyzed and summarized. What the reader will gain: A critical analysis of micelles stability in vivo and micelle-cell interaction is provided to highlight the key issues to be addressed to affirm that micelle can properly work as a drug carrier in clinical settings. Take home message: With a clear understanding of its behaviors in biological environment, the polymer micelle is a promising nanocarrier for chemotherapy.

Polarization coherent anti-Stokes Raman scattering microscopy

We report polarization coherent anti-Stokes Raman scattering (P-CARS) microscopy that allows vibrational imaging with high sensitivity and spectral selectivity. The nonresonant background signals from both Raman scatterers and the solvent are efficiently suppressed in P-CARS microscopy. We demonstrate P-CARS imaging of unstained cells based on the contrast of the protein amide I band.

Laser-scanning coherent anti-Stokes Raman scattering microscopy and applications to cell biology.

Laser-scanning coherent anti-Stokes Raman scattering (CARS) microscopy with fast data acquisition and high sensitivity has been developed for vibrational imaging of live cells. High three-dimensional (3D) resolution is achieved with two collinearly overlapped near infrared picosecond beams and a water objective with a high numerical aperture. Forward-detected CARS (F-CARS) and epi-detected CARS (E-CARS) images are recorded simultaneously. F-CARS is used for visualizing features comparable to or larger than the excitation wavelength, while E-CARS allows detection of smaller features with a high contrast. F-CARS and E-CARS images of live and unstained cells reveal details invisible in differential interference-contrast images. High-speed vibrational imaging of unstained cells undergoing mitosis and apoptosis has been carried out. For live NIH 3T3 cells in metaphase, 3D distribution of chromosomes is mapped at the frequency of the DNA backbone Raman band, while the vesicles surrounding the nucleus is imaged by E-CARS at the frequency of the C-H stretching Raman band. Apoptosis in NIH 3T3 cells is monitored using the CARS signal from aliphatic C-H stretching vibration.

Triacylglycerol Synthesis Enzymes Mediate Lipid Droplet Growth by Relocalizing from the ER to Lipid Droplets

Lipid droplets (LDs) store metabolic energy and membrane lipid precursors. With excess metabolic energy, cells synthesize triacylglycerol (TG) and form LDs that grow dramatically. It is unclear how TG synthesis relates to LD formation and growth. Here, we identify two LD subpopulations: smaller LDs of relatively constant size, and LDs that grow larger. The latter population contains isoenzymes for each step of TG synthesis. Glycerol-3-phosphate acyltransferase 4 (GPAT4), which catalyzes the first and rate-limiting step, relocalizes from the endoplasmic reticulum (ER) to a subset of forming LDs, where it becomes stably associated. ER-to-LD targeting of GPAT4 and other LD-localized TG synthesis isozymes is required for LD growth. Key features of GPAT4 ER-to-LD targeting and function in LD growth are conserved between Drosophila and mammalian cells. Our results explain how TG synthesis is coupled with LD growth and identify two distinct LD subpopulations based on their capacity for localized TG synthesis.

Release of hydrophobic molecules from polymer micelles into cell membranes revealed by Förster resonance energy transfer imaging

It is generally assumed that polymeric micelles, upon administration into the blood stream, carry drug molecules until they are taken up into cells followed by intracellular release. The current work revisits this conventional wisdom. The study using dual-labeled micelles containing fluorescently labeled copolymers and hydrophobic fluorescent probes entrapped in the polymeric micelle core showed that cellular uptake of hydrophobic probes was much faster than that of labeled copolymers. This result implies that the hydrophobic probes in the core are released from micelles in the extracellular space. Förster resonance energy transfer (FRET) imaging and spectroscopy were used to monitor this process in real time. A FRET pair, DiIC18(3) and DiOC18(3), was loaded into monomethoxy poly(ethylene glycol)-block-poly(d,l-lactic acid) micelles. By monitoring the FRET efficiency, release of the core-loaded probes to model membranes was demonstrated. During administration of polymeric micelles to tumor cells, a decrease of FRET was observed both on the cell membrane and inside of cells, indicating the release of core-loaded probes to the cell membrane before internalization. The decrease of FRET on the plasma membrane was also observed during administration of paclitaxel-loaded micelles. Taken together, our results suggest a membrane-mediated pathway for cellular uptake of hydrophobic molecules preloaded in polymeric micelles. The plasma membrane provides a temporal residence for micelle-released hydrophobic molecules before their delivery to target intracellular destinations. A putative role of the PEG shell in the molecular transport from micelle to membrane is discussed.

Theoretical and experimental characterization of coherent anti-Stokes Raman scattering microscopy

We present a systematic characterization of coherent anti-Stokes Raman scattering (CARS) microscopy. CARS signal generation in a heterogeneous sample under a tight-focusing condition is formulated by the Green’s function method. The CARS radiation pattern and the forward- and backward-detected CARS signals from a three-dimensional Raman scatterer are calculated. The coherent nature of CARS image formation and its consequences for image contrast and spatial resolution are investigated. Experimental implementations of CARS microscopy with collinearly copropagating and counterpropagating excitation beams, forward and backward data collection, and polarization-sensitive detection are described. Finally, CARS images of unstained live cells with forward detection, epidetection, and polarization-sensitive detection are presented and compared.

In vivo quantitation of rare circulating tumor cells by multiphoton intravital flow cytometry

Quantitation of circulating tumor cells (CTCs) constitutes an emerging tool for the diagnosis and staging of cancer, assessment of response to therapy, and evaluation of residual disease after surgery. Unfortunately, no existing technology has the sensitivity to measure the low numbers of tumor cells (<1 CTC per ml of whole blood) that characterize minimal levels of disease. We present a method, intravital flow cytometry, that noninvasively counts rare CTCs in vivo as they flow through the peripheral vasculature. The method involves i.v. injection of a tumor-specific fluorescent ligand followed by multiphoton fluorescence imaging of superficial blood vessels to quantitate the flowing CTCs. Studies in mice with metastatic tumors demonstrate that CTCs can be quantitated weeks before metastatic disease is detected by other means. Analysis of whole blood samples from cancer patients further establishes that human CTCs can be selectively labeled and quantitated when present at ≈2 CTCs per ml, opening opportunities for earlier assessment of metastatic disease.

Fast Release of Lipophilic Agents from Circulating PEG-PDLLA Micelles Revealed by in Vivo Förster Resonance Energy Transfer Imaging

Understanding the in vivo behavior of nanoparticles is critical for the translation of nanomedicine from laboratory research to clinical trials. In this work, in vivo Forster resonance energy transfer (FRET) imaging was employed to monitor the release of hydrophobic molecules from circulating poly(ethylene glycol)-poly( D, L-lactic acid) (PEG-PDLLA) micelles. A lipophilic FRET pair (DiIC(18) and DiOC(18)) was physically entrapped into micelle cores by mimicking the loading of hydrophobic drugs. The FRET efficiency was found significantly reduced within 15 min after intravenous injection, implying that DiIC(18) and DiOC(18) quickly escaped from the circulating micelles. FRET spectroscopy studies further demonstrated that alpha- and beta-globulins were major factors for the observed fast release, while gamma-globulins, albumin, and red blood cells played minor roles. These results provide useful information for developing blood-stable micelles to deliver hydrophobic drugs to the target site via prolonged circulation and extravasation from the vascular system.

Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging

Despite functional evidence for disulfide bond-reducing activity in endosomal compartments, the mechanistic details pertaining to such process (e.g., kinetics and sites of disulfide reduction) remain largely controversial. To address these questions directly, we have synthesized a previously uncharacterized fluorescent folate conjugate, folate-(BODIPY FL)-SS-rhodamine (folate-FRET), that changes fluorescence from red to green upon disulfide bond reduction. Using this construct, we have observed that disulfide reduction: (i) occurs with a half-time of 6 h after folate-FRET endocytosis, (ii) begins in endosomes and does not depend significantly on redox machinery located on the cell surface or within the lysosome or the Golgi apparatus, (iii) occurs independently of endocytic vesicle trafficking along microtubules, and (iv) yields products that are subsequently sorted into distinct endosomes and trafficked in different directions. Finally, colocalization of folate and transferrin receptors suggest that conclusions derived from this study may apply to other endocytic pathways.