Shuk Han Cheng

Polymer-coated NaYF4: Yb3+, Er3+ upconversion nanoparticles for charge-dependent cellular imaging

Lanthanide-doped upconversion nanoparticles (UCNPs) are considered promising novel near-infrared (NIR) bioimaging agents with the characteristics of high contrast and high penetration depth. However, the interactions between charged UCNPs and mammalian cells have not been thoroughly studied, and the corresponding intracellular uptake pathways remain unclear. Herein, our research work involved the use of a hydrothermal method to synthesize polyvinylpyrrolidone-coated UCNPs (UCNP-PVP), and then a ligand exchange reaction was performed on UCNP-PVP, with the help of polyethylenimine (PEI) and poly(acrylic acid) (PAA), to generate UCNP-PEI and UCNP-PAA. These polymer-coated UCNPs demonstrated good dispersibility in aqueous medium, had the same elemental composition and crystal phase, shared similar TEM and dynamic light scattering (DLS) size distribution, and exhibited similar upconversion luminescence efficiency. However, the positively charged UCNP-PEI evinced greatly enhanced cellular uptake in comparison with its neutral or negative counterparts, as shown by multiphoton confocal microscopy and inductively coupled plasma mass spectrometry (ICP-MS) measurements. Meanwhile, we found that cationic UCNP-PEI can be effectively internalized mainly through the clathrin endocytic mechanism, as revealed by colocalization, chemical, and genetic inhibitor studies. This study elucidates the role of the surface polymer coatings in governing UCNP-cell interactions, and it is the first report on the endocytic mechanism of positively charged lanthanide-doped UCNPs. Furthermore, this study provides important guidance for the development of UCNPs as specific intracellular nanoprobes, allowing us to control the UCNP-cell interactions by tuning surface properties.

Robotic Cell Injection System With Position and Force Control: Toward Automatic Batch Biomanipulation

Biological cell injection is laborious work that requires lengthy training and suffers from a low success rate. In this paper, a robotic cell-injection system for automatic injection of batch-suspended cells is proposed. To facilitate the process, these suspended cells are held and fixed to a cell array by a specially designed cell-holding device, and injected one by one through an ldquoout-of-planerdquo cell-injection process. A micropipette equipped with a polyvinylidene fluoride microforce sensor to measure real-time injection force is integrated in the proposed system. Through calibration, an empirical relationship between the cell-injection force and the desired injector pipette trajectory is obtained in advance. Then, after decoupling the out-of-plane cell injection into a position control in the X - Y horizontal plane and an impedance control in the Z -axis, a position and force control algorithm is developed to control the injection pipette. The depth motion of the injector pipette, which cannot be observed by microscope, is indirectly controlled via the impedance control, and the desired force is determined from the online X - Y position control and cell calibration results. Finally, experimental results demonstrate the effectiveness of the proposed approach.

Reversible Accumulation of PEGylated Single-Walled Carbon Nanotubes in the Mammalian Nucleus

Carbon nanotubes (CNTs) have been shown to cross cell membranes and can mediate the internalization of macromolecules. These characteristics have constituted CNTs as an exciting new tool for drug delivery and biological sensing. While CNTs exhibit great potential in biomedical and pharmaceutical applications, neither the cell penetration mechanism of CNTs nor the intracellular fate of the internalized CNTs are fully understood. In this study, time-lapse fluorescence microscopy was used to investigate the intracellular distribution of FITC labeled PEGylated single-walled CNTs (FITC-PEG-SWCNTs) in living cells and shown that PEGylated SWCNTs entered the nucleus of several mammalian cell lines in an energy-dependent process. The presence of FITC-PEG-SWCNTs in the cell nucleus did not cause discernible changes in the nuclear organization and had no effect on the growth kinetics and cell cycle distribution for up to 5 days. Remarkably, upon removal of the FITC-PEG-SWCNTs from the culture medium, the internalized FITC-PEG-SWCNTs rapidly moved out of the nucleus and were released from the cells. Thus, the intracellular PEGylated SWCNTs were highly dynamic and the cell penetration of PEGylated SWCNTs appeared as bidirectional. These observations suggest SWCNTs may be used as an ideal nanovector in biomedical and pharmaceutical applications.

Acute and long-term effects after single loading of functionalized multi-walled carbon nanotubes into zebrafish (Danio rerio)

Carbon nanotubes (CNTs) are widely explored for biomedical applications, but there is very limited information regarding their in vivo biodistribution and biocompatibility. Here, we report the in vivo biodistribution and long-term effects of functionalized multi-walled carbon nanotubes (MWCNTs) in developing zebrafish. The fluorescent-labeled MWCNTs were introduced into zebrafish embryos at 1-cell stage and at 72 h post fertilization through microinjection. After single injection, both acute and long-term interactions between zebrafish and functionalized MWCNTs were studied. The injected FITC-BSA-MWCNTs (at 1-cell stage) were allocated to all blastoderm cells of the embryos through proliferation, and were distinctively excluded from the yolk cell. When introduced into the circulation system, FITC-BSA-MWCNTs moved easily in the compartments and finally were cleaned out by the body at 96 h after the loading. At early stages, the treated zebrafish embryos generated immune response by accumulating circulating white blood cells at the trunk region. Under transmission electron microscope, many lysosome-like vesicles were observed in the blastoderm cells of the treated embryos. The zebrafish loaded with MWCNTs had normal primordial germ cells at early stage and produced second generation later on. However, the larvae of the second generation had obviously lower survival rates as compared to the untreated groups, suggesting a negative effect on the reproduction potential. These results suggest that extensive purification and functionalization processes can help improve the biocompatibility of CNTs. This study also indicates that purified CNTs may have long-term toxicity effects when they were delivered into the body.

Gold-doxorubicin nanoconjugates for overcoming multidrug resistance

UNLABELLED Multidrug resistance (MDR) is a major clinical obstacle to the success of cancer chemotherapy. Here we developed a gold-doxorubicin (DOX) nanoconjugates system to overcome MDR. Gold nanoparticles (AuNPs) were first PEGylated as Au-PEG-NH(2), and DOX was then grafted onto AuNPs via a cleavable disulfide linkage (Au-PEG-SS-DOX). Confocal images revealed that the extent of intracellular uptake of Au-PEG-SS-DOX was greater than that of free DOX in the MDR cells, and inductively coupled plasma mass spectroscopy analysis further confirmed that AuNPs significantly increased the level of drug accumulation in MDR cells at a nanoparticles dose greater than 15 μM. The cytotoxicity study demonstrated that the Au-PEG-SS-DOX nanoconjugates system efficiently released the anticancer drug DOX and enhanced its cytotoxicity against MDR cancer cells. This study highlights the potential of using AuNPs for overcoming of MDR in cancer chemotherapy. FROM THE CLINICAL EDITOR This study demonstrates that gold nanoparticles can be successfully applied to overcome MDR in cancer chemotherapy.

Expression of two novel mouse Iroquois homeobox genes during neurogenesis

Members of the Drosophila Iroquois homeobox gene family are implicated in the development of peripheral nervous system and the regionalization of wing and eye imaginal discs. Recent studies suggest that Xenopus Iroquois homeobox (Irx) genes are also involved in neurogenesis. Three mouse Irx genes, Irx1, Irx2 and Irx3, have been previously identified and are expressed with distinct spatio-temporal patterns during neurogenesis. We report here the cloning and expression analysis of two novel mouse Irx genes, Irx5 and Irx6. Although Irx5 and Irx6 proteins are structurally more related to one another, we find that Irx5 displays a developmental expression pattern strikingly similar to that of Irx3, whereas Irx6 expression resembles that of Irx1. Consistent with the notion that Mash1 is a putative target gene of the Irx proteins, all four Irx genes display an overlapping expression pattern with Mash1 in the developing CNS. In contrast, the Irx genes and Mash1 are expressed in complementary domains in the developing eye and olfactory epithelium.

Mechanical Modeling of Biological Cells in Microinjection

Microinjection is an effective technique to introduce foreign materials into a biological cell. Although some semi-automatic and fully-automatic microinjection systems have been developed, a full understanding of the mechanical response of biological cells to injection operation remains deficient. In this paper, a new mechanical model based on membrane theory is proposed. This model establishes a relationship between the injection force and the deformation of biological cells with the quasi-static equilibrium equations, which are solved by the Runge-Kutta numerical method. Based on this model, other mechanical responses can also be inferred, such as the effect of the injector radius, the membrane stress and tension distribution, internal cell pressure, and the deformed cell shape. To verify the proposed model, experiments are performed on microinjection of zebrafish embryos at different developmental stages and medaka embryos at the blastula stage. It is demonstrated that the modeling results agree well with the experimental data, which shows that the proposed model can be used to estimate the mechanical properties of cell biomembranes. (In this paper, biomembrane refers to the membrane-like structures enveloping cells.)

A Force Control Approach to a Robot-assisted Cell Microinjection System

Robotic cell microinjection is a technique that utilizes automation technology to insert substances into a single living cell with a fine needle. Compared with manual microinjection, the main benefits of the robotic cell injection are quality, productivity and repeatability. In this paper we aim to control the penetration force during robotic cell injection to quantify the influence of the penetration force on cells. A force-control-based cell injection approach that is capable of regulating the penetration force in a desired force trajectory is developed. The proposed force control framework includes two control loops. The inner loop is an impedance control used to specify the interaction between the needle and the cell. The outer loop is a force tracking non-linear controller using a feedback linearization technique. The cell model is identified online with a least-squares parameter estimator. With the proposed force control approach, the penetration force can be regulated explicitly to follow the desired force trajectory during the cell injection process. Experiments performed on fish embryos verify the effectiveness of the proposed approach.

Uptake and internalisation of copper by three marine microalgae: comparison of copper-sensitive and copper-tolerant species

Although it has been well established that different species of marine algae have different sensitivities to metals, our understanding of the physiological and biochemical basis for these differences is limited. This study investigated copper adsorption and internalisation in three algal species with differing sensitivities to copper. The diatom Phaeodactylum tricornutum was particularly sensitive to copper, with a 72-h IC50 (concentration of copper to inhibit growth rate by 50%) of 8.0 microg Cu L(-1), compared to the green algae Tetraselmis sp. (72-h IC50 47 microg Cu L(-1)) and Dunaliella tertiolecta (72-h IC50 530 microg Cu L(-1)). At these IC50 concentrations, Tetraselmis sp. had much higher intracellular copper (1.97+/-0.01 x 10(-13)g Cu cell(-1)) than P. tricornutum (0.23+/-0.19 x 10(-13)g Cu cell(-1)) and D. tertiolecta (0.59+/-0.05 x 10(-13)g Cu cell(-1)), suggesting that Tetraselmis sp. effectively detoxifies copper within the cell. By contrast, at the same external copper concentration (50 microg L(-1)), D. tertiolecta appears to better exclude copper than Tetraselmis sp. by having a slower copper internalisation rate and lower internal copper concentrations at equivalent extracellular concentrations. The results suggest that the use of internal copper concentrations and net uptake rates alone cannot explain differences in species-sensitivity for different algal species. Model prediction of copper toxicity to marine biota and understanding fundamental differences in species-sensitivity will require, not just an understanding of water quality parameters and copper-cell binding, but also further knowledge of cellular detoxification mechanisms.

Visual-Based Impedance Control of Out-of-Plane Cell Injection Systems

In this paper, a vision-based impedance control algorithm is proposed to regulate the cell injection force, based on dynamic modeling conducted on a laboratory test-bed cell injection system. The injection force is initially calibrated to derive the relationship between the force and the cell deformation utilizing a cell membrane point-load model. To increase the success rate of injection, the injector is positioned out of the focal plane of the camera, used to obtain visual feedback for the injection process. In this out-of-plane injection process, the total cell membrane deformation is estimated, based on the X-Y coordinate frame deformation of the cell, as measured with a microscope, and the known angle between the injector and the X-Y plane. Further, a relationship between the injection force and the injector displacement of the cell membrane, as observed with the camera, is derived. Based on this visual force estimation scheme, an impedance control algorithm is developed. Experimental results of the proposed injection method are given which validate the approach.