Nanoparticles for cancer imaging: The good, the bad, and the promise.

Recent advances in molecular imaging and nanotechnology are providing new opportunities for biomedical imaging with great promise for the development of novel imaging agents. The unique optical, magnetic, and chemical properties of materials at the scale of nanometers allow the creation of imaging probes with better contrast enhancement, increased sensitivity, controlled biodistribution, better spatial and temporal information, multi-functionality and multi-modal imaging across MRI, PET, SPECT, and ultrasound. These features could ultimately translate to clinical advantages such as earlier detection, real time assessment of disease progression and personalized medicine. However, several years of investigation into the application of these materials to cancer research has revealed challenges that have delayed the successful application of these agents to the field of biomedical imaging. Understanding these challenges is critical to take full advantage of the benefits offered by nano-sized imaging agents. Therefore, this article presents the lessons learned and challenges encountered by a group of leading researchers in this field, and suggests ways forward to develop nanoparticle probes for cancer imaging. Published by Elsevier Ltd.

Nanotechnology-based cancer therapeutics--promise and challenge--lessons learned through the NCI Alliance for Nanotechnology in Cancer.

The new generation of nanotechnology-based drug formulations is challenging the accepted ways of cancer treatment. Multi-functional nanomaterial constructs have the capability to be delivered directly to the tumor site and eradicate cancer cells selectively, while sparing healthy cells. Tailoring of the nano-construct design can result in enhanced drug efficacy at lower doses as compared to free drug treatment, wider therapeutic window, and lower side effects. Nanoparticle carriers can also address several drug delivery problems which could not be effectively solved in the past and include reduction of multi-drug resistance effects, delivery of siRNA, and penetration of the blood-brain-barrier. Although challenges in understanding toxicity, biodistribution, and paving an effective regulatory path must be met, nanoscale devices carry a formidable promise to change ways cancer is diagnosed and treated. This article summarizes current developments in nanotechnology-based drug delivery and discusses path forward in this field. The discussion is done in context of research and development occurring within the NCI Alliance for Nanotechnology in Cancer program.

Kinetic properties and functional dynamics of sodium channels during repetitive spiking in a slow pacemaker neuron.

We examined the kinetic properties of voltage-gated Na(+) channels and their contribution to the repetitive spiking activity of medullary raphé neurons, which exhibit slow pacemaking and strong spiking adaptation. The study is based on a combination of whole-cell patch-clamp, modeling and real-time computation. Na(+) currents were recorded from neurons in brain slices obtained from male and female neonatal rats, using voltage-clamp protocols designed to reduce space-clamp artifacts and to emphasize functionally relevant kinetic features. A detailed kinetic model was formulated to explain the broad range of transient and stationary voltage-dependent properties exhibited by Na(+) currents. The model was tested by injecting via dynamic clamp a model-based current as a substitute for the native TTX-sensitive Na(+) currents, which were pharmacologically blocked. The model-based current reproduced well the native spike shape and spiking frequency. The dynamics of Na(+) channels during repetitive spiking were indirectly examined through this model. By comparing the spiking activities generated with different kinetic models in dynamic-clamp experiments, we determined that state-dependent slow inactivation contributes significantly to spiking adaptation. Through real-time manipulation of the model-based current, we established that suprathreshold Na(+) current mainly controls spike shape, whereas subthreshold Na(+) current modulates spiking frequency and contributes to the pacemaking mechanism. Since the model-based current was injected in the soma, the results also suggest that somatic Na(+) channels are sufficient to establish the essential spiking properties of raphé neurons in vitro.

The NCI Alliance for Nanotechnology in Cancer: achievement and path forward.

Nanotechnology is a 'disruptive technology', which can lead to a generation of new diagnostic and therapeutic products, resulting in dramatically improved cancer outcomes. The National Cancer Institute (NCI) of National Institutes of Health explores innovative approaches to multidisciplinary research allowing for a convergence of molecular biology, oncology, physics, chemistry, and engineering and leading to the development of clinically worthy technological approaches. These initiatives include programmatic efforts to enable nanotechnology as a driver of advances in clinical oncology and cancer research, known collectively as the NCI Alliance for Nanotechnology in Cancer (ANC). Over the last 5 years, ANC has demonstrated that multidisciplinary approach catalyzes scientific developments and advances clinical translation in cancer nanotechnology. The research conducted by ANC members has improved diagnostic assays and imaging agents, leading to the development of point-of-care diagnostics, identification and validation of numerous biomarkers for novel diagnostic assays, and the development of multifunctional agents for imaging and therapy. Numerous nanotechnology-based technologies developed by ANC researchers are entering clinical trials. NCI has re-issued ANC program for next 5 years signaling that it continues to have high expectations for cancer nanotechnology's impact on clinical practice. The goals of the next phase will be to broaden access to cancer nanotechnology research through greater clinical translation and outreach to the patient and clinical communities and to support development of entirely new models of cancer care.

Strategic workshops on cancer nanotechnology.

Nanotechnology offers the potential for new approaches to detecting, treating, and preventing cancer. To determine the current status of the cancer nanotechnology field and the optimal path forward, the National Cancer Institute's Alliance for Nanotechnology in Cancer held three strategic workshops, covering the areas of in vitro diagnostics and prevention, therapy and post-treatment, and in vivo diagnosis and imaging. At each of these meetings, a wide range of experts from academia, industry, the nonprofit sector, and the U.S. government discussed opportunities in the field of cancer nanotechnology and barriers to its implementation.

Raphé neurons stimulate respiratory circuit activity by multiple mechanisms via endogenously released serotonin and substance P.

Brainstem serotonin (5-HT) neurons modulate activity of many neural circuits in the mammalian brain, but in many cases endogenous mechanisms have not been resolved. Here, we analyzed actions of raphé 5-HT neurons on respiratory network activity including at the level of the pre-Bötzinger complex (pre-BötC) in neonatal rat medullary slices in vitro, and in the more intact nervous system of juvenile rats in arterially perfused brainstem-spinal cord preparations in situ. At basal levels of activity, excitation of the respiratory network via simultaneous release of 5-HT and substance P (SP), acting at 5-HT(2A/2C), 5-HT(4), and/or neurokinin-1 receptors, was required to maintain inspiratory motor output in both the neonatal and juvenile systems. The midline raphé obscurus contained spontaneously active 5-HT neurons, some of which projected to the pre-BötC and hypoglossal motoneurons, colocalized 5-HT and SP, and received reciprocal excitatory connections from the pre-BötC. Experimentally augmenting raphé obscurus activity increased motor output by simultaneously exciting pre-BötC and motor neurons. Biophysical analyses in vitro demonstrated that 5-HT and SP modulated background cation conductances in pre-BötC and motor neurons, including a nonselective cation leak current that contributed to the resting potential, which explains the neuronal depolarization that augmented motor output. Furthermore, we found that 5-HT, but not SP, can transform the electrophysiological phenotype of some pre-BötC neurons to intrinsic bursters, providing 5-HT with an additional role in promoting rhythm generation. We conclude that raphé 5-HT neurons excite key circuit components required for generation of respiratory motor output.

Real-time kinetic modeling of voltage-gated ion channels using dynamic clamp.

We propose what to our knowledge is a new technique for modeling the kinetics of voltage-gated ion channels in a functional context, in neurons or other excitable cells. The principle is to pharmacologically block the studied channel type, and to functionally replace it with dynamic clamp, on the basis of a computational model. Then, the parameters of the model are modified in real time (manually or automatically), with the objective of matching the dynamical behavior of the cell (e.g., action potential shape and spiking frequency), but also the transient and steady-state properties of the model (e.g., those derived from voltage-clamp recordings). Through this approach, one may find a model and parameter values that explain both the observed cellular dynamics and the biophysical properties of the channel. We extensively tested the method, focusing on Na(v) models. Complex Markov models (10-12 states or more) could be accurately integrated in real time at >50 kHz using the transition probability matrix, but not the explicit Euler method. The practicality of the technique was tested with experiments in raphe pacemaker neurons. Through automated real-time fitting, a Hodgkin-Huxley model could be found that reproduced well the action potential shape and the spiking frequency. Adding a virtual axonal compartment with a high density of Na(v) channels further improved the action potential shape. The computational procedure was implemented in the free QuB software, running under Microsoft Windows and featuring a friendly graphical user interface.

Tumor-specific Th17-polarized cells eradicate large established melanoma.

CD4+ T cells can differentiate into multiple effector subsets, but the potential roles of these subsets in anti-tumor immunity have not been fully explored. Seeking to study the impact of CD4+ T cell polarization on tumor rejection in a model mimicking human disease, we generated a new MHC class II-restricted, T-cell receptor (TCR) transgenic mouse model in which CD4+ T cells recognize a novel epitope in tyrosinase-related protein 1 (TRP-1), an antigen expressed by normal melanocytes and B16 murine melanoma. Cells could be robustly polarized into Th0, Th1, and Th17 subtypes in vitro, as evidenced by cytokine, chemokine, and adhesion molecule profiles and by surface markers, suggesting the potential for differential effector function in vivo. Contrary to the current view that Th1 cells are most important in tumor rejection, we found that Th17-polarized cells better mediated destruction of advanced B16 melanoma. Their therapeutic effect was critically dependent on interferon-gamma (IFN-gamma) production, whereas depletion of interleukin (IL)-17A and IL-23 had little impact. Taken together, these data indicate that the appropriate in vitro polarization of effector CD4+ T cells is decisive for successful tumor eradication. This principle should be considered in designing clinical trials involving adoptive transfer-based immunotherapy of human malignancies.

Sodium currents in medullary neurons isolated from the pre-Bötzinger complex region.

The pre-Bötzinger complex (preBötC) in the ventrolateral medulla contains interneurons important for respiratory rhythm generation. Voltage-dependent sodium channels mediate transient current (I(NaT)), underlying action potentials, and persistent current (I(NaP)), contributing to repetitive firing, pacemaker properties, and the amplification of synaptic inputs. Voltage-clamp studies of the biophysical properties of these sodium currents were conducted on acutely dissociated preBötC region neurons. Reverse transcription-PCR demonstrated the presence of mRNA for Nav1.1, Nav1.2, and Nav1.6 alpha-subunits in individual neurons. A TTX-sensitive I(NaP) was evoked in all tested neurons by ramp depolarization from -80 to 0 mV. Including a constant in the Boltzmann equation for inactivation by estimating the steady-state fraction of Na+ channels available for inactivation allowed prediction of a window current that did not decay to 0 at voltages positive to -20 mV and closely matched the measured I(NaP). Riluzole (3 microM), a putative I(NaP) antagonist, reduced both I(NaP) and I(NaT) and produced a hyperpolarizing shift in the voltage dependence of steady-state inactivation. The latter decreased the predicted window current by an amount equivalent to the decrease in I(NaP). Riluzole also decreased the inactivation time constant at potentials in which the peak window/persistent currents are generated. Together, these findings imply that I(NaP) and I(NaT) arise from the same channels and that a simple modification of the Hodgkin-Huxley model can satisfactorily account for both currents. In the rostral ventral respiratory group (immediately caudal to preBötC), I(NaP) was also detected, but peak conductance, current density, and input resistance were smaller than in preBötC region cells.

Resurgent Na currents in four classes of neurons of the cerebellum.

Action potential firing rates are generally limited by the refractory period, which depends on the recovery from inactivation of voltage-gated Na channels. In cerebellar Purkinje neurons, the kinetics of Na channels appear specialized for rapid firing. Upon depolarization, an endogenous open-channel blocker rapidly terminates current flow but prevents binding of the "fast" inactivation gate. Upon repolarization, unbinding of the blocker produces "resurgent" Na current while allowing channels to recover rapidly. Because other cerebellar neurons, including granule cells, unipolar brush cells, and neurons of the cerebellar nuclei, also fire rapidly, we tested whether these cells might also express Na channels with resurgent kinetics. Neurons were acutely isolated from mice and rats, and TTX-sensitive Na currents were recorded under voltage clamp. Unlike Purkinje cells, the other cerebellar neurons produced only tiny resurgent currents in solutions optimized for voltage-clamping Na currents (50 mM Na+; Co2+ substitution for Ca2+). Under more physiological ionic conditions (155 mM Na+; 2 mM Ca2+ with 0.03 mM Cd2+), however, granule cells, unipolar brush cells, and cerebellar nuclear cells all produced robust resurgent currents. The increase in resurgent current, which was greater than predicted by the Goldman-Hodgkin-Katz equation, appeared to result from a combination of knock-off of open-channel blockers by permeating ions as well as relief of divalent block at negative potentials. These results indicate that resurgent current is typical of many cerebellar neurons and suggest that rapid open-channel block and unblock may be a widespread mechanism for restoration of Na channel availability in rapidly firing neurons.

Intrinsic bursting activity in the pre-Bötzinger complex: role of persistent sodium and potassium currents.

Computational models of single pacemaker neuron and neural population in the pre-Bötzinger Complex (pBC) were developed based on the previous models by Butera et al. (1999a,b). Our modeling study focused on the conditions that could define endogenous bursting vs. tonic activity in single pacemaker neurons and population bursting vs. asynchronous firing in populations of pacemaker neurons. We show that both bursting activity in single pacemaker neurons and population bursting activity may be released or suppressed depending on the expression of persistent sodium ( I(Na) P) and delayed-rectifier potassium ( I(K)) currents. Specifically, a transition from asynchronous firing to population bursting could be induced by a reduction of I(K) via a direct suppression of the potassium conductance or through an elevation of extracellular potassium concentration. Similar population bursting activity could be triggered by an augmentation of I(Na) P. These findings are discussed in the context of the possible role of population bursting activity in the pBC in the respiratory rhythm generation in vivo vs. in vitro and during normal breathing in vivo vs. gasping.

Sodium currents in neurons from the rostroventrolateral medulla of the rat.

Rapidly inactivating and persistent sodium currents have been characterized in acutely dissociated neurons from the area of rostroventrolateral medulla that included the pre-Bötzinger Complex. As demonstrated in many studies in vitro, this area can generate endogenous rhythmic bursting activity. Experiments were performed on neonate and young rats (P1-15). Neurons were investigated using the whole cell voltage-clamp technique. Standard activation and inactivation protocols were used to characterize the steady-state and kinetic properties of the rapidly inactivating sodium current. Slow depolarizing ramp protocols were used to characterize the noninactivating sodium current. The "window" component of the rapidly inactivating sodium current was calculated using mathematical modeling. The persistent sodium current was revealed by subtraction of the window current from the total noninactivating sodium current. Our results provide evidence of the presence of persistent sodium currents in neurons of the rat rostroventrolateral medulla and determine voltage-gated characteristics of activation and inactivation of rapidly inactivating and persistent sodium channels in these neurons.

The murine neurokinin NK1 receptor gene contributes to the adult hypoxic facilitation of ventilation.

Substance P and neurokinin-1 receptors (NK1) modulate the respiratory activity and are expressed early during development. We tested the hypothesis that NK1 receptors are involved in prenatal development of the respiratory network by comparing the resting respiratory activity and the respiratory response to hypoxia of control mice and mutant mice lacking the NK1 receptor (NK1-/-). In vitro and in vivo experiments were conducted on neonatal, young and adult mice from wild-type and NK1-/- strains. In the wild strain, immunohistological, pharmacological and electrophysiological studies showed that NK1 receptors were expressed within medullary respiratory areas prior to birth and that their activation at birth modulated central respiratory activity and the membrane properties of phrenic motoneurons. Both the membrane properties of phrenic motoneurons and the respiratory activity generated in vitro by brainstem-spinal cord preparation from NK1-/- neonate mice were similar to that from the wild strain. In addition, in vivo ventilation recordings by plethysmography did not reveal interstrain differences in resting breathing parameters. The facilitation of ventilation by short-lasting hypoxia was similar in wild and NK1-/- neonates but was significantly weaker in adult NK1-/- mice. Results demonstrate that NK1 receptors do appear to be necessary for a normal respiratory response to short-lasting hypoxia in the adult. However, NK1 receptors are not obligatory for the prenatal development of the respiratory network, for the production of the rhythm, or for the regulation of breathing by short-lasting hypoxia in neonates.

Respiratory rhythm generation: converging concepts from in vitro and in vivo approaches?

The timing and activation pattern of breathing movements are determined by the respiratory network. This network is amenable to a variety of in vivo and in vitro approaches, which offers a unique opportunity to investigate multiple organizational levels. It is only recently, however, that concepts obtained under in vivo and in vitro conditions are being integrated into a coherent model of breathing behavior. For example, the pre-Bötzinger complex as an essential site for rhythm generation was first identified in vitro, but has since been verified in vivo. Conversely, timing signals provided by other central and peripheral neuronal areas have so far been investigated in vivo, but it is now possible to address these issues with more complex in vitro preparations. Several key issues remain unresolved. For example, to what extent is the respiratory pattern controlled independently of the underlying rhythm? Answers to this and other questions require a dissection of mechanisms that is only possible through a complementary combination of experimental approaches.