Daniella Yeheskely-Hayon

High levels of reactive oxygen species in gold nanoparticle-targeted cancer cells following femtosecond pulse irradiation

Cancer cells could be locally damaged using specifically targeted gold nanoparticles and laser pulse irradiation, while maintaining minimum damage to nearby, particle-free tissue. Here, we show that in addition to the immediate photothermal cell damage, high concentrations of reactive oxygen species (ROS) are formed within the irradiated cells. Burkitt lymphoma B cells and epithelial breast cancer cells were targeted by antibody-coated gold nanospheres and irradiated by a few resonant femtosecond pulses, resulting in significant elevation of intracellular ROS which was characterized and quantified using time-lapse microscopy of different fluorescent markers. The results suggest that techniques that involve targeting of various malignancies using gold nanoparticles and ultrashort pulses may be more effective and versatile than previously anticipated, allowing diverse, highly specific set of tools for local cancer therapy.

Modulation of P‐glycoprotein‐mediated multidrug resistance by acceleration of passive drug permeation across the plasma membrane

The drug concentration inside multidrug‐resistant cells is the outcome of competition between the active export of drugs by drug efflux pumps, such as P‐glycoprotein (Pgp), and the passive permeation of drugs across the plasma membrane. Thus, reversal of multidrug resistance (MDR) can occur either by inhibition of the efflux pumps or by acceleration of the drug permeation. Among the hundreds of established modulators of Pgp‐mediated MDR, there are numerous surface‐active agents potentially capable of accelerating drug transbilayer movement. The aim of the present study was to determine whether these agents modulate MDR by interfering with the active efflux of drugs or by allowing for accelerated passive permeation across the plasma membrane. Whereas Pluronic P85, Tween‐20, Triton X‐100 and Cremophor EL modulated MDR by inhibition of Pgp‐mediated efflux, with no appreciable effect on transbilayer movement of drugs, the anesthetics chloroform, benzyl alcohol, diethyl ether and propofol modulated MDR by accelerating transbilayer movement of drugs, with no concomitant inhibition of Pgp‐mediated efflux. At higher concentrations than those required for modulation, the anesthetics accelerated the passive permeation to such an extent that it was not possible to estimate Pgp activity. The capacity of the surface‐active agents to accelerate passive drug transbilayer movement was not correlated with their fluidizing characteristics, measured as fluorescence anisotropy of 1‐(4‐trimethylammonium)‐6‐phenyl‐1,3,5‐hexatriene. This compound is located among the headgroups of the phospholipids and does not reflect the fluidity in the lipid core of the membranes where the limiting step of drug permeation, namely drug flip‐flop, occurs.

Noninvasive imaging of flowing blood cells using label-free spectrally encoded flow cytometry

Optical microscopy of blood cells in vivo provides a unique opportunity for clinicians and researchers to visualize the morphology and dynamics of circulating cells, but is usually limited by the imaging speed and by the need for exogenous labeling of the cells. Here we present a label-free approach for in vivo flow cytometry of blood using a compact imaging probe that could be adapted for bedside real-time imaging of patients in clinical settings, and demonstrate subcellular resolution imaging of red and white blood cells flowing in the oral mucosa of a human volunteer. By analyzing the large data sets obtained by the system, valuable blood parameters could be extracted and used for direct, reliable assessment of patient physiology.

Controlled release of Rituximab from gold nanoparticles for phototherapy of malignant cells.

Releasing drug molecules at their targets with high spatial and temporal accuracy could aid numerous clinical applications which require low systemic damage and low side effects. Nano-carriers of drugs are an attractive solution for such task, allowing specific accumulation in tumors and gradual release of their payload. Here, we utilize gold nanospheres conjugated to Rituximab, an anti-CD20 monoclonal antibody-based drug, for carrying and releasing the drug upon irradiation of specifically tailored femtosecond laser pulses. The released anti-CD20 molecules retain their functionality and ability of triggering the complement-dependent cytotoxicity. This effect comes in addition to cell necrosis caused by the plasmonic nanometric shock waves emanating from the nanospheres and rupturing the plasma membranes. Main advantages of the presented technique include high spatial and temporal resolution, low toxicity and high repeatability and consistency due to the morphological stability of the nanospheres.

Optical Nanomanipulations of Malignant Cells: Controlled Cell Damage and Fusion

Specifically targeting and manipulating living cells is a key challenge in biomedicine and in cancer research in particular. Several studies have shown that nanoparticles irradiated by intense lasers are capable of conveying damage to nearby cells for various therapeutic and biological applications. In this work ultrashort laser pulses and gold nanospheres are used for the generation of localized, nanometric disruptions on the membranes of specifically targeted cells. The high structural stability of the nanospheres and the resonance pulse irradiation allow effective means for controlling the induced nanometric effects. The technique is demonstrated by inducing desired death mechanisms in epidermoid carcinoma and Burkitt lymphoma cells, and initiating efficient cell fusion between various cell types. Main advantages of the presented approach include low toxicity, high specificity, and high flexibility in the regulation of cell damage and cell fusion, which would allow it to play an important role in various future clinical and scientific applications.

Role of the plasma membrane leaflets in drug uptake and multidrug resistance

The present study aimed to investigate the role played by the leaflets of the plasma membrane in the uptake of drugs into cells and in their extrusion by P‐glycoprotein and multidrug resistance‐associated protein 1. Drug accumulation was monitored by fluorescence resonance energy transfer from trimethylammonium‐diphenyl‐hexatriene (TMA‐DPH) located at the outer leaflet to a rhodamine analog. Uptake of dye into cells whose mitochondria had been inactivated was displayed as two phases of TMA‐DPH fluorescence quenching. The initial phase comprised a rapid drop in fluorescence that was neither affected by cooling the cells on ice, nor by activity of mitochondria or ABC transporters. This phase reflects the association of dye with the outer leaflet of the plasma membrane. The subsequent phase of TMA‐DPH fluorescence quenching occurred in drug‐sensitive cell lines with a half‐life in the range 20–40 s. The second phase of fluorescence quenching was abolished by incubation of the cells on ice and was transiently inhibited in cells with active mitochondria. Thus, the second phase of fluorescence quenching reflects the accumulation of dye in the cytoplasmic leaflet of the plasma membrane, presumably as a result of flip‐flop of dye across the plasma membrane and slow diffusion from the inner leaflet into the cells. Whereas activity of P‐glycoprotein prevented the second phase of fluorescence quenching, the activity of multidrug resistance‐associated protein 1 had no effect on this phase. Thus, P‐glycoprotein appears to pump rhodamines from the cytoplasmic leaflet either to the outer leaflet or to the outer medium.

Competition between innate multidrug resistance and intracellular binding of rhodamine dyes

The present study aimed to elucidate the contribution of the intracellular binding of drugs to multidrug resistance. For this purpose, uptake of rhodamines was studied in cells whose mitochondria had been uncoupled with carbonyl cyanide m‐chlorophenylhydrazone. Surprisingly, in a variety of drug‐untreated cells, presumed to be sensitive to multidrug resistance‐type drugs, rhodamines were excluded from entering the cells. Thus, the amount of rhodamine 123 taken up into parental untreated K562 cells was less than the amount bound to the cell exterior. Rhodamine uptake was prevented by an active efflux pump. The efflux was inhibited by 4‐chloro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐Cl) and MK571 and, to a lesser extent, by ATP depletion, indomethacin, probenecid and vanadate. All the inhibitors, apart from NBD‐Cl, are known to modulate multidrug resistance‐associated protein (MRP) 1. Because MRP1 was expressed in all the cell lines tested and the efflux of rhodamines in MRP1 over‐expressing cells was abolished by NBD‐Cl, it appears that rhodamines are excluded from these cells by MRP1. On the other hand, the uptake of rhodamines into cells respiring with their coupled mitochondria demonstrated diminished sensitivity to NBD‐Cl and MK571. Thus, active pumping into the mitochondria allowed enhanced uptake into the cells, overcoming the innate resistance. The innate resistance provided by MRP1 to cells prevents rhodamine dyes, and possibly drugs such as doxorubicin, from achieving equilibration of their concentration in the cytoplasm with their concentration in the external medium. The protection provided to multidrug resistance cells by ABC transporters has to overcome competition by passive uptake of the drugs and binding/uptake of the drugs into intracellular targets.

Optically induced cell fusion using bispecific nanoparticles.

Redirecting the immune system to eliminate tumor cells is a promising alternative to traditional cancer therapies, most often requiring direct interaction between an immune system effector cell and its target. Herein, a novel approach for selective attachment of malignant cells to antigen-presenting cells by using bispecific nanoparticles is presented. The engaged cell pairs are then irradiated by a sequence of resonant femtosecond pulses, which results in widespread cell fusion and the consequent formation of hybrid cells. The dual role of gold nanoparticles as conjugating agents and fusion promoters offers a simple yet effective means for specific fusion between different cells. This technology could be useful for a variety of in vitro and in vivo applications that call for selective fusion between cells within a large heterogenic cell population.

Differential growth identified in salamander larvae half-sib cohorts: Survival strategy?

In this study we describe the growth of several different larval cohorts (i.e. half‐siblings of the same mother born on the same day) of a rare, xeric‐adapted salamander Salamandra s. infraimmaculata Martens, 1885, under constant density and food conditions from birth to metamorphosis. The larvae spend the critical first phase of their lives in water, mostly in temporary ponds. Age and weight at metamorphosis were highly affected by varying food conditions. We have identified six different growth modes that these larvae use, both fast growing and slow growing. Each larval cohort was found to use 2–4 different such growth modes regardless of their initial weight. Fast growing modes (I–III) will enable larvae to survive dry years, and metamorphose bigger. Slow growing modes (IV–VI), used by 8% of the larval population, will enable survival only in rainy years. These last growth modes effect differential temporal dispersal in wet years by delaying the emergence of postmetamorphs onto land. Distribution of growth modes in the larval population is affected by food but not by density conditions. Late‐born, fast‐growing larvae will have an advantage in dry years being able to metamorphose and disperse, whereas the slow‐growing larvae will survive only in wet years.

The Roles of the Catalytic and Noncatalytic Activities of Rpd3L and Rpd3S in the Regulation of Gene Transcription in Yeast

In budding yeasts, the histone deacetylase Rpd3 resides in two different complexes called Rpd3L (large) and Rpd3S (small) that exert opposing effects on the transcription of meiosis-specific genes. By introducing mutations that disrupt the integrity and function of either Rpd3L or Rpd3S, we show here that Rpd3 function is determined by its association with either of these complexes. Specifically, the catalytic activity of Rpd3S activates the transcription of the two major positive regulators of meiosis, IME1 and IME2, under all growth conditions and activates the transcription of NDT80 only during vegetative growth. In contrast, the effects of Rpd3L depends on nutrients; it represses or activates transcription in the presence or absence of a nitrogen source, respectively. Further, we show that transcriptional activation does not correlate with histone H4 deacetylation, suggesting an effect on a nonhistone protein. Comparison of rpd3-null and catalytic-site point mutants revealed an inhibitory activity that is independent of either the catalytic activity of Rpd3 or the integrity of Rpd3L and Rpd3S.