Cellular Imaging Applications Research Articles

Hydrophilic Cu2-xSe/reduced graphene oxide nanocomposites with tunable plasmonic properties and their applications in cellular dark-field microscopic imaging.

A facile aqueous chemical approach is developed for one-pot synthesis of the nanocomposites of heavily doped semiconductor nanoparticles (Cu2-xSe NPs) and reduced graphene oxide (rGO) at room temperature, wherein the reduction of GO and the in situ growth of Cu2-xSe NPs on rGO sheets occur simultaneously. The as-prepared Cu2-xSe/rGO nanocomposites exhibit a well-defined near-infrared (NIR) localized surface plasmon resonance (LSPR), which arises from free carriers (holes) and could be tuned from 1360 to 1050 nm by varying the reaction time, owing to the increase of the free carrier density in the valence band of Cu2-xSe NPs. This approach not only offers an efficient strategy to synthesize the self-doped Cu2-xSe/rGO nanocomposites with strong and tunable NIR absorption, but also develops new light scattering nanoprobes with good biocompatibility as well as unique optical properties for in vitro cellular dark-field microscopic imaging (iDFM).

One‐Pot Microwave Synthesis of Fluorescent Carbogenic Nanoparticles from Triton X‐100 for Cell Imaging

AbstractCarbon‐based fluorescent nanoparticles have attracted a lot of attention in the field of bioimaging as a result of their excellent emission properties along with their low toxicity. Fluorescent carbogenic nanoparticles (CNPs) were synthesized by a one‐pot microwave pyrolysis technique by using Triton X‐100 as a carbon source. The CNPs are spherical in shape with an average diameter of 5.5 nm, as characterized by transmission electron microscopy and atomic force microscopy. The as‐synthesized CNPs showed strong blue emission under UV illumination, excitation‐dependent emission properties, and chemical stability under different pH conditions. The graphitic nature of the CNPs was studied by using Raman and X‐ray photoelectron spectroscopy. Moreover, the toxicity of these particles was studied through an MTT assay, which showed no noticeable toxicity. The results suggest that the CNPs can be used as effective fluorescent probes for cellular imaging applications.

Synthesis, two-photon absorption properties and bioimaging applications of mono-, di- and hexa-branched pyrimidine derivatives

A series of mono-, di- and hexa-branched pyrimidine derivatives possessing two-photon absorption have been synthesized. The spectral features of the dyes showed solvatochromic with significant bathochromic shifting of the emission spectra and large Stokes shifts in more polar solvents mainly due to the intramolecular charge-transfer. The two-photon absorption cross-section values were measured by two-photon excited fluorescence and open aperture Z-scan methods. The results revealed that the two-photon absorption cross-section values were enhanced with increasing electron-donating strength of the end group and branch number. The photophysical properties of the dyes were further investigated with the aid of theoretical calculations. In addition, the di-branched dyes has the largest values of two-photon absorption cross-section per molecular weight, low cytotoxicity and can be internalized by human liver cancer cell line and accumulates in the cytoplasm, allowing for live cell two-photon fluorescence imaging, so it is promising for in vitro and vivo cellular imaging applications.

MBeRFP, an Improved Large Stokes Shift Red Fluorescent Protein

Herein, we describe the generation of a monomeric large Stokes shift (LSS) red fluorescent protein, mBeRFP, with excitation and emission peaks at 446 and 615 nm, respectively. Compared with two previously reported LSS-RFPs (mKeima and LSS-mKate2), mBeRFP is approximately three times brighter. In addition, mBeRFP is characterized by improved photostability, rapid maturation, an extended lifetime, and a monomeric nature. Additionally, mBeRFP can be paired with the Alexa 647 dye as a FRET donor to detect caspase 3 activity. This FRET pair has an extremely dynamic range and a large Förster radius (approximately 6.5 nm). To demonstrate the applicability of mBeRFP for imaging in living cells, we performed dual-color imaging of mBeRFP and CFP simultaneously excited by a single excitation source, and we demonstrated that these fluorescent proteins allow the clear visualization of the dynamics of Bax during cancer cell apoptosis. Thus, mBeRFP appears to be particularly useful for cellular imaging applications.

Enzyme-catalyzed oxidation facilitates the return of fluorescence for single-walled carbon nanotubes.

In this work, we studied enzyme-catalyzed oxidation of single-walled carbon nanotubes (SWCNTs) produced by the high-pressure carbon monoxide (HiPco) method. While oxidation via strong acids introduced defect sites on SWCNTs and suppressed their near-infrared (NIR) fluorescence, our results indicated that the fluorescence of SWCNTs was restored upon enzymatic oxidation, providing new evidence that the reaction catalyzed by horseradish peroxidase (HRP) in the presence of H2O2 is mainly a defect-consuming step. These results were further supported by both UV-vis-NIR and Raman spectroscopy. Therefore, when acid oxidation followed by HRP-catalyzed enzyme oxidation was employed, shortened (<300 nm in length) and NIR-fluorescent SWCNTs were produced. In contrast, upon treatment with myeloperoxidase, H2O2, and NaCl, the oxidized HiPco SWCNTs underwent complete oxidation (i.e., degradation). The shortened, NIR-fluorescent SWCNTs resulting from HRP-catalyzed oxidation of acid-cut HiPco SWCNTs may find applications in cellular NIR imaging and drug delivery systems.

Thermal and pH Responsive Polymer-Tethered Multifunctional Magnetic Nanoparticles for Targeted Delivery of Anticancer Drug

Targeted and efficient delivery of therapeutics to tumor cells is one of the key issues in cancer therapy. In the present work, we report a temperature and pH dual responsive core-shell nanoparticles comprising smart polymer shell coated on magnetic nanoparticles as an anticancer drug carrier and cancer cell-specific targeting agent. Magnetite nanoparticles (MNPs), prepared by a simple coprecipitation method, was surface modified by introducing amine groups using 3-aminopropyltriethoxysilane. Dual-responsive poly(N-isopropylacrylamide)-block-poly(acrylic acid) copolymer, synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization, was then attached to the amine-functionalized MNPs via EDC/NHS method. Further, to accomplish cancer-specific targeting properties, folic acid was tethered to the surface of the nanoparticles. Thereafter, rhodamine B isothiocyanate was conjugated to endow fluorescent property to the MNPs required for cellular imaging applications. The nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), selected area electron diffraction (SAED), field emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDX), thermogravimetric analysis (TGA), zeta potential, vibrating sample magnetometer (VSM), X-ray photoelectron spectroscopy (XPS) measurements, and FTIR, UV-vis spectral analysis. Doxorubicin (DOX), an anticancer drug used for the present study, was loaded into the nanoparticles and its release behavior was subsequently studied. Result showed a sustained release of DOX preferentially at the desired lysosomal pH and temperature condition. The biological activity of the DOX-loaded MNPs was studied by MTT assay, fluorescence microscopy, and apoptosis. Intracellular-uptake studies revealed preferential uptake of these nanoparticles into cancer cells (HeLa cells) compared to normal fibroblast cells (L929 cells). The in vitro apoptosis study revealed that the DOX-loaded nanoparticles caused significant death to the HeLa cells. These nanoparticles were capable of target specific release of the loaded drug in response to pH and temperature and hence may serve as a potential drug carrier for in vivo applications.

Size-Dependent Stability of Water-Solubilized CdTe Quantum Dots and Their Uptake Mechanism by Live HeLa Cells

Water-solubilized quantum dots have led to a promising application in cellular labeling and biological imaging. The physicochemical properties of water-solubilized quantum dots, particularly in a physiological environment, are strongly dependent on their size. In this paper, we systematically studied the stability of mercaptosuccinic acid-coated CdTe quantum dots (MSA-QDs) of about 2.3 and 5.4 nm diameters in various buffers with different pH values and under laser irradiation by fluorescence spectroscopy. It was found that larger MSA-QDs showed better stability. Size-dependent uptake of MSA-QDs by living HeLa cells was further investigated by confocal microscopy. In phosphate buffer solution, the larger MSA-QDs entered the cells mainly by endocytosis, and part of the smaller ones entered the cells by passive penetration. In cell culture medium, their uptake pathways could be changed due to the changes of their surface properties. The cytotoxicity of smaller and larger MSA-QDs was significantly decreased due to the adsorption of some biological components in the cell culture medium on the nanoparticles surface.

Facile preparation and cellular imaging of photoluminescent carbogenic nanoparticles derived from defoliations

1 Introduction In the past two decades,photoluminescent semiconductor quantum dots(QDs) have received intensive attention for a variety of promising properties and applications[1,2],especially for their potential application in cellular imaging[3-5].QDs with high quantum yield have been prepared based on cadmium(zinc) selenide(sulfide) and related core-shell composites[6-8].However,the release of Cd2+,S2-and Se2-ions inevitably causes serious health and environmental concerns.Therefore,many efforts have been devoted to developing benign alternatives with enhanced photoluminescence,such as silicon nanoparticles and nanowires[9,10].

Wavelength-tunable luminescent gold nanoparticles generated by cooperation ligand exchange and their potential application in cellular imaging

This work reports a simple and novel method for the synthesis of wavelength-tunable luminescent gold nanoparticles (NPs) based on a cooperation ligand exchange using 11-mercaptoundecanoic acid (11-MUA) and D-penicillamine (DPA). Next, the obtained luminescent gold NPs were used for cellular imaging, and the observations indicated that the wavelength-tunable luminescent gold NPs have potential applications in cellular luminescence labeling and imaging.

Emerging applications of conjugated polymers in molecular imaging.

In recent years, conjugated polymers have attracted considerable attention from the imaging community as a new class of contrast agent due to their intriguing structural, chemical, and optical properties. Their size and emission wavelength tunability, brightness, photostability, and low toxicity have been demonstrated in a wide range of in vitro sensing and cellular imaging applications, and have just begun to show impact in in vivo settings. In this Perspective, we summarize recent advances in engineering conjugated polymers as imaging contrast agents, their emerging applications in molecular imaging (referred to as in vivo uses in this paper), as well as our perspectives on future research.

Photoresponsive quinoline tethered fluorescent carbon dots for regulated anticancer drug delivery

A photoresponsive nano drug delivery system (DDS) was constructed using two new ingredients: fluorescent carbon dots and a quinoline based phototrigger. The strong fluorescent properties of carbon dots have been explored for in vitro cellular imaging application, and the phototrigger ability of quinoline was exploited for efficient anticancer drug release using both one-photon and two-photon excitation.

Self-Assembly of Conjugated Polymer on Hybrid Nanospheres for Cellular Imaging Applications

A new kind of hybrid core-shell nanosphere was fabricated by combining the in situ formation of Au nanoparticles and covalent cross-linking of biocompatible carboxymethyl starch dialdehyde (CMSD) and chitosan (CTS). When the fluorescent dye poly[9,9'-bis(6″-(N,N,N-trimethylammonium)-hexyl)fluorene-2,7-ylenevinylene-co-alt-1,4-phenylene dibromide] (PFV) was assembled on the surface of the hybrid nanospheres through electrostatic attraction, these biocompatible hybrid nanospheres exhibited metal-enhanced fluorescence effects. The fluorescence intensity of (CTS-Au)@CMSD/PFV hybrid nanosphere is 1.43 times that of CTS-CMSD/PFV hybrid nanospheres lacking Au nanoparticle. In addition, the (CTS-Au)@CMSD/PFV hybrid nanospheres exhibit excellent biodegradability upon exposure to enzymatic aqueous solution and good biocompatibility when cocultured with HeLa cervical carcinoma cells; these advantages make them attractive for cellular imaging and biological analysis and detection.

Stable water-soluble quantum dots capped by poly(ethylene glycol) modified dithiocarbamate

A new bifunctional ligand (IA-PEG-DTC), poly(ethylene glycol) (PEG) appended dithiocarbamate derived from iminodiacetic acid, has been designed and synthesized. The obtained IA-PEG-DTC ligand with the dithiocarbamate group strongly bonding to the surface atom of quantum dots (QDs), the PEG segment rendering QDs water-soluble, and the free carboxylic group available for further bioconjugation was used successfully to transfer initial oil-soluble QDs into water-soluble ones. The resulting IA-PEG-DTC capped water-soluble QDs have relatively small hydrodynamic size, and importantly, retain high fluorescence quantum yield of the original hydrophobic QDs. In addition, the obtained water-soluble QDs exhibit excellent colloidal stability under conditions of wide pH range (2–13), long-term UV-irradiation, the boiling state, and high salt concentrations. These results indicate that the water-soluble QDs coated by the bifunctional IA-PEG-DTC ligand have potential for applications in cellular imaging and biosensor.

Covalent Cross‐Linking: Stable Functionalization of Small Semiconducting Polymer Dots via Covalent Cross‐Linking and Their Application for Specific Cellular Imaging (Adv. Mater. 26/2012)

Covalent Cross‐Linking: Stable Functionalization of Small Semiconducting Polymer Dots via Covalent Cross‐Linking and Their Application for Specific Cellular Imaging (Adv. Mater. 26/2012)

Quantum Dot‐Based Screening System for Discovery of G Protein‐Coupled Receptor Agonists

Cellular imaging has emerged as an important tool to unravel biological complexity and to accelerate the drug-discovery process, including cell-based screening, target identification, and mechanism of action studies. Recently, semiconductor nanoparticles known as quantum dots (QDs) have attracted great interest in cellular imaging applications due to their unique photophysical properties such as size, tunable optical property, multiplexing capability, and photostability. Herein, we show that QDs can also be applied to assay development and eventually to high-throughput/content screening (HTS/HCS) for drug discovery. We have synthesized QDs modified with PEG and primary antibodies to be used as fluorescent probes for a cell-based HTS system. The G protein-coupled receptor (GPCR) family is known to be involved in most major diseases. We therefore constructed human osteosarcoma (U2OS) cells that specifically overexpress two types of differently tagged GPCRs: influenza hemagglutinin (HA) peptide-tagged κ-opioid receptors (κ-ORs) and GFP-tagged A3 adenosine receptors (A3AR). In this study, we have demonstrated that 1) anti-HA antibody-conjugated QDs could specifically label HA-tagged κ-ORs, 2) subsequent treatment of QD-tagged GPCR agonists allowed agonist-induced translocation to be monitored in real time, 3) excellent emission spectral properties of QD permitted the simultaneous detection of two GPCRs in one cell, and 4) the robust imaging capabilities of the QD-antibody conjugates could lead to reproducible quantitative data from high-content cellular images. These results suggest that the present QD-based GPCR inhibitor screening system can be a promising platform for further drug screening applications.

Stable Functionalization of Small Semiconducting Polymer Dots via Covalent Cross‐Linking and Their Application for Specific Cellular Imaging

A facile cross-linking strategy covalently links functional molecules to semiconducting polymer dots (Pdots) while simultaneously providing functional groups for biomolecular conjugation. In addition to greatly enhanced stability, the formed Pdots are small (<10 nm), which can be difficult to achieve with current methods but is highly desirable for most biological applications. These characteristics are significant for improving labeling efficiency and sensitivity in cellular assays that employ Pdots.

Nanoclusters of Iron Oxide: Effect of Core Composition on Structure, Biocompatibility, and Cell Labeling Efficacy

Inorganic nanocrystals have a variety of applications in medicine. They may serve as contrast agents, therapeutics, and for in vitro diagnostics. Frequently, the synthesis route yields hydrophobically capped nanocrystals, which necessitates their subsequent coating to render a water-soluble and biocompatible probe. Biocompatibility is crucial for cellular imaging applications, which require large quantities of diagnostically active nanoparticles to be loaded into cells. We have previously reported the design and synthesis of a fluorescent and magnetic resonance imaging-detectable core-shell nanoparticle that encapsulates hydrophobically coated iron oxide nanocrystals. The core of soybean oil and iron oxide is covered by a shell mixture of phospholipids, some of which contained polyethylene glycol. Despite the biocompatibility of these components, we hypothesize that we can improve this formulation with respect to in vitro toxicity. To this aim, we measured the effect of six different core compositions on nanoparticle structure, cell labeling efficacy, and cell viability, as well as cell tracking potential. We methodically investigated the causes of toxicity and conclude that, even when combining biocompatible materials, the resulting formulation is not guaranteed to be biocompatible.

Aptamer-Mediated Nanoparticle-Based Protein Labeling Platform for Intracellular Imaging and Tracking Endocytosis Dynamics

Although nanoparticles have been widely used as optical contrasts for cell imaging, the complicated prefunctionalized steps and low labeling efficiency of nanoprobes greatly inhibit their applications in cellular protein imaging. In this study, we developed a novel and general strategy that employs an aptamer not only as a recognizer for protein recognition but also as a linker for nanoreporter targeting to specifically label membrane proteins of interest and track their endocytic pathway. With this strategy, three kinds of nanoparticles, including gold nanoparticles, silver nanoparticles, and quantum dots (QDs), have been successfully targeted to the membrane proteins of interest, such as nucleolin or prion protein (PrP(C)). The following investigations on the subcellular distribution with fluorescent immunocolocalization assay indicated that PrP(C)-aptamer-QD complexes most likely internalized into cytoplasm through a classical clathrin-dependent/receptor-mediated pathway. Further single-particle tracking and trajectory analysis demonstrated that PrP(C)-aptamer-QD complexes exhibited a complex dynamic process, which involved three types of movements, including membrane diffusion, vesicle transportation, and confined diffusion, and all types of these movements were associated with distinct phases of PrP(C) endocytosis. Compared with traditional multilayer methods, our proposed aptamer-mediated strategy is simple in procedure, avoiding any complicated probe premodification and purification. In particular, the new double-color labeling strategy is unique and significant due to its superior advantages of targeting two signal reporters simultaneously in a single protein using only one aptamer. What is more important, we have constructed a general and versatile aptamer-mediated protein labeling nanoplatform that has shown great promise for future biomedical labeling and intracellular protein dynamic analysis.

Nanoparticle uptake and gene transfer efficiency for MSCs on chitosan and chitosan-hyaluronan substrates

Nanoparticles (NPs) are usually surface modified to increase endocytosis for applications in cellular imaging and gene delivery. The influence of cell culture substrates on endocytosis remains relatively unexplored. This study investigated the substrate-mediated effects on the uptake of NPs by mesenchymal stem cells (MSCs). Two types of NPs were employed, negatively charged paramagnetic iron oxide (Fe3O4) NPs (∼5 nm) and bare plasmid DNA pTRE-Tight-DsRED2 (3.3 kb, ∼5 nm), each of which were poorly endocytosed by the adipose-derived MSCs grown on tissue culture polystyrene (TCPS). When cells were cultured on chitosan or hyaluronan-modified chitosan (chitosan-HA) membranes, significant increases (>5-fold) in the intracellular uptake of Fe3O4 NPs as well as transfectability of plasmid DNA were demonstrated. The enhancement in transgene expression was more pronounced than that using the transfection agent. The beneficial effects were not caused by elevated proliferation or a change in the differentiation state of interacting MSCs. On chitosan and chitosan-HA, cells moved fast and formed spheroids. The cytoskeletal arrangement associated with the up-regulated RhoA activity during spheroid formation may have accounted for the increased endocytosis. Using different inhibitors, the endocytosis pathways were further clarified. Both Fe3O4 NPs and plasmid DNA were taken up primarily by clathrin-mediated endocytosis on chitosan (∼50%). The caveolae-mediated endocytosis on chitosan-HA was more evident (∼30–40%) than that on chitosan (<25%). For plasmid DNA but not Fe3O4 NPs, macropinocytosis also occurred on both substrates. Chitosan and chitosan-HA as cell culture substrates may activate different endocytic pathways of MSCs to increase NP internalization or plasmid transfection. The substrate-mediated endocytosis described here may represent a new and potentially attractive approach to facilitate stem cell labeling or to improve gene delivery efficiency without altering cell viability and differentiation.

Preparation of strongly fluorescent silica nanoparticles of polyelectrolyte-protected cadmium telluride quantum dots and their application to cell toxicity and imaging

Based on the polyelectrolyte-protected CdTe quantum dots (QDs), which were prepared by self-assembling of QDs and poly-diallyldimethylammonium chloride (PDADMAC) in the help of electrostatic attraction, the strong fluorescence silica nanoparticles (QDs-PDADMAC@SiO2) have been prepared via a water-in-oil reverse microemulsion method. Transmission electron microscopy and Zeta potential analysis were used to characterize the as-prepared nanoparticles. All of the particles were almost spherical and there is a uniform distribution of the particle size with the average diameter about 25nm. There is a large Zeta potential of −35.07mV which is necessary for good monodispersity of nanoparticles solution. As compared with the QDs coated by SiO2 (QDs@SiO2), the QDs-PDADMAC@SiO2 nanoparticles have much stronger fluorescence, and their fluorescence stability could be obviously improved. Moreover, QDs-PDADMAC@SiO2 exhibits good biological compatibility which promotes their application in cellular imaging.