Mitochondria-Mediated Anticancer Effects of Non-Thermal Atmospheric Plasma.

Non-thermal atmospheric pressure plasma has attracted great interest due to its multiple potential biomedical applications with cancer treatment being among the most urgent. To realize the clinical potential of non-thermal plasma, the exact cellular and molecular mechanisms of plasma effects must be understood. This work aimed at studying the prostate cancer specific mechanisms of non-thermal plasma effects on energy metabolism as a central regulator of cell homeostasis and proliferation. It was found that cancer cells with higher metabolic rate initially are more resistant to plasma treated phosphate-buffered saline (PBS) since the respiratory and calcium sensitive signaling systems were not responsive to plasma exposure. However, dramatic decline of cancer oxidative phosphorylation developed over time resulted in significant progression of cell lethality. The normal prostate cells with low metabolic activity immediately responded to plasma treated PBS by suppression of respiratory functions and sustained elevation of cytosolic calcium. However, over time the normal cells start recovering their mitochondria functions, proliferate and restore the cell population. We found that the non-thermal plasma induced increase in intracellular ROS is of primarily non-mitochondrial origin. The discriminate non-thermal plasma effects hold a promise for clinical cancer intervention.

Surface-enhanced Raman spectroscopy-active substrates: adapting the shape of plasmonic nanoparticles for different biological applications.

We discuss the relationship between the shape of plasmonic nanoparticles and the biological surface-enhanced Raman spectroscopy (SERS) applications which they can enable. As a step forward in developing SERS-active substrates adapted to a particular application, we demonstrate that a modification of the widely used protocol for the sodium citrate mediated reduction of chloroauric acid, which is typically employed only for obtaining spherical gold nanoparticles, can yield flat polygonal nanoparticles at room temperature and a decreased amount of the reducing agent. The significant advantage of the described approach is that it allows for synthesis of nanoparticles with different geometries using a well-established synthesis protocol without the need for any additional chemicals or special synthesis apparatus. By contrasting spherical and anisotropically shaped nanoparticles, we demonstrate that multifaceted nanoparticles with sharp edges are better suitable for SERS analysis of low concentration analytes requiring strong SERS enhancement. On the other hand, gold nanoparticles with isotropic shapes, while giving a smaller enhancement, can provide a more reproducible SERS signal. This is important for analytical applications of complex biological systems where large SERS enhancement may not always be required, whereas data reproducibility and minimal false positive rate are imperative. Using a SERS-active substrate comprising isotropically shaped gold nanoparticles, we demonstrate the differences between Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria, attributable to the outer membrane and peptidoglycan layer, with the level of detail which has not been previously reported with optical spectroscopic techniques.

One-dimensional nanoprobes for single-cell studies.

Owing to variation of individual cells within a population, single-cell studies are of great interest to researchers. Recent developments in nanofabrication technology have made this area increasingly attractive as one-dimensional (1D) nanoscale probes can be manufactured with increasing accuracy. Here, we provide an overview and description of the major designs that have been reported to date. For more details of what applications could be realized and how, based on the probe shapes and designs, we summarize the most recently reported performances of 1D single-cell probes with their advantages and limitations. Minimally invasive probes are required for long-term experiments on single cells. Carbon nanotubes with their unique properties and structure are excellent candidates for multitask robotic intracellular probes. Carbon nanotube-tipped cellular endoscopes are less invasive compared with pipettes or cantilever tips. Advances in nanofabrication techniques have made it possible to produce more consistent nanoscale cellular probes that can capture a variety of information from optical, electrical and chemical signals. In addition, these tools can transfer tiny amounts of fluids and molecular materials in a highly localized fashion for the purpose of analyzing or stimulating a variety of responses at the level of individual cells and even cellular organelles. We conclude with a critical analysis of the current state of the field as well as the major obstacles for further probe development of minimally invasive probes and their widespread use in cell biology.

Stimuli-responsive magnetic nanomicelles as multifunctional heat and cargo delivery vehicles.

Hybrid nanoarchitectures are among the most promising nanotechnology-enabled materials for biomedical applications. Interfacing of nanoparticles with active materials gives rise to the structures with unique multiple functionality. Superparamagnetic iron oxide nanoparticles particles SPION are widely employed in the biology and in developing of advanced medical technologies. Polymeric micelles offer the advantage of multifunctional carriers which can serve as delivery vehicles carrying nanoparticles, hydrophobic chemotherapeutics and other functional materials and molecules. Stimuli-responsive polymers are especially attractive since their properties can be modulated in a controlled manner. Here we report on multifunctional thermo-responsive poly(N-isopropylacrylamide-co-acrylamide)-block-poly(ε-caprolactone) random block copolymer micelles as magnetic hyperthermia-mediated payload release and imaging agents. The combination of copolymers, nanoparticles and doxorubicin drug was tailored the way that the loaded micelles were cable to respond to magnetic heating at physiologically-relevant temperatures. A surface functionalization of the micelles with the integrin ß4 antibody and consequent interfacing of the resulting nanobio hybrid with squamous head and neck carcinoma cells which is known to specifically over-express the A9 antigen resulted in concentration of the micelles on the surface of cells. No inherent cytotoxicity was detected for the magnetic micelles without external stimuli application. Furthermore, SPION-loaded micelles demonstrate significant MRI contrast enhancement abilities.

Microfabricated magnetic structures for future medicine: from sensors to cell actuators.

In this review, we discuss the prospective medical application of magnetic carriers microfabricated by top-down techniques. Physical methods allow the fabrication of a variety of magnetic structures with tightly controlled magnetic properties and geometry, which makes them very attractive for a cost-efficient mass-production in the fast growing field of nanomedicine. Stand-alone fabricated particles along with integrated devices combining lithographically defined magnetic structures and synthesized magnetic tags will be considered. Applications of microfabricated multifunctional magnetic structures for future medicinal purposes range from ultrasensitive in vitro diagnostic bioassays, DNA sequencing and microfluidic cell sorting to magnetomechanical actuation, cargo delivery, contrast enhancement and heating therapy.

Physiological validation of cell health upon probing with carbon nanotube endoscope and its benefit for single-cell interrogation.

New-generation nanoscale devices for single-cell study are intensively being developed. As has been shown, nanodevices are minimally invasive because of their order-of-magnitude smaller size in comparison to conventional glass pipettes. However, in most studies the evaluation of the nanodevice impact on cell health has not extended to their effects on cell metabolic integrity. In this work we evaluated the degree to which the insertion of a carbon-based nanotube endoscope into a cell induces mechanical and biochemical stress, and affects cellular key metabolic systems. The effects of insertion of the nanotube endoscope on cell morphological and physiological modulations were monitored and compared to those of glass micropipettes. We report that nanotube endoscope insertion does not significantly modulate the plasma membrane and actin network. The cell metabolic mechanisms such as energy production and inositol 1,4,5-trisphosphate-dependent calcium signaling remain preserved for prolonged endoscope presence within a cell. FROM THE CLINICAL EDITOR: In this basic science study, the effects of insertion of carbon nanotube endoscope on cell morphological and physiological modulations were monitored and compared to those of glass micropipettes. Nanotube endoscope insertion is truly minimally invasive: it does not significantly modulate the plasma membrane and actin network; the energy production and inositol 1,4,5-trisphosphate-dependent calcium signaling also remain preserved during prolonged endoscope presence within a cell.

Multifunctional ferromagnetic disks for modulating cell function.

In this work, we focus on the methods for controlling cell function with ferromagnetic disk-shaped particles. We will first review the history of magnetically assisted modulation of cell behavior and applications of magnetic particles for studying physical properties of a cell. Then, we consider the biological applications of the microdisks such as the method for induction of cancer cell apoptosis, controlled drug release, hyperthermia and MRI imaging.

Multifunctional carbon-nanotube cellular endoscopes.

Glass micropipettes, atomic force microscope tips and nanoneedles can be used to interrogate cells, but these devices either have conical geometries that can damage cells during penetration or are incapable of continuous fluid handling. Here, we report a carbon-nanotube-based endoscope for interrogating cells, transporting fluids and performing optical and electrochemical diagnostics at the single organelle level. The endoscope, which is made by placing a multiwalled carbon nanotube (length, 50-60 µm) at the tip of a glass pipette, can probe the intracellular environment with a spatial resolution of ∼100 nm and can also access organelles without disrupting the cell. When the nanotube is filled with magnetic nanoparticles, the endoscope can be remotely manoeuvered to transport nanoparticles and attolitre volumes of fluids to and from precise locations. Because they are mounted on conventional glass micropipettes, the endoscopes readily fit standard instruments, creating a broad range of opportunities for minimally invasive intracellular probing, drug delivery and single-cell surgery.

Surface-enhanced Raman spectroscopy as a tool for detecting Ca2+ mobilizing second messengers in cell extracts.

Understanding of calcium signaling pathways in cells is essential for elucidating the mechanisms of both normal cell function and cancer development. Calcium messengers play the crucial role for intracellular Ca(2+) release. We propose a new approach to detecting the calcium second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) in cell extracts using surface-enhanced Raman spectroscopy (SERS). Currently available radioreceptor binding and enzymatic assays require extensive sample preparation and take more than 12 h. With a SERS sensor, NAADP can be detected in less than 1 min without any special sample preparation. To the best of our knowledge, this is the first demonstration of using SERS for calcium signaling applications.

In situ intracellular spectroscopy with surface enhanced Raman spectroscopy (SERS)-enabled nanopipettes.

We report on a new analytical approach to intracellular chemical sensing that utilizes a surface-enhanced Raman spectroscopy (SERS)-enabled nanopipette. The probe is comprised of a glass capillary with a 100-500 nm tip coated with gold nanoparticles. The fixed geometry of the gold nanoparticles allows us to overcome the limitations of the traditional approach for intracellular SERS using metal colloids. We demonstrate that the SERS-enabled nanopipettes can be used for in situ analysis of living cell function in real time. In addition, SERS functionality of these probes allows tracking of their localization in a cell. The developed probes can also be applied for highly sensitive chemical analysis of nanoliter volumes of chemicals in a variety of environmental and analytical applications.