Water solubilization of nanoparticles is a fundamental prerequisite for many biological applications. To date, no single method has emerged as ideal, and several different approaches have been successfully utilized. These 'phase-transfer' strategies are reviewed, indicating key advantages and disadvantages, and a discussion of conjugation strategies is presented. Coating of hydrophobic nanoparticles with amphiphilic polymers provides a generic pathway for the phase transfer of semiconductor, magnetic, metallic, and upconverting nanoparticles from nonpolar to polar environments. Amphiphilic polymers that include maleimide groups can be readily functionalized with chemical groups for specific applications. In the second, experimental part, some of the new chemical features of such polymer-capped nanoparticles are demonstrated. In particular, nanoparticles to which a pH sensitive fluorophore has been attached are described, and their use for intracellular pH-sensing demonstrated. It is shown that the properties of analyte-sensitive fluorophores can be tuned by using interactions with the underlying nanoparticles.

IntroductionGhrelin and cannabinoids stimulate appetite, this effect possibly being mediated by the activation of hypothalamic AMP-activated protein kinase (AMPK), a key enzyme in appetite and metabolism regulation. The cannabinoid receptor type 1 (CB1) antagonist rimonabant can block the orexigenic effect of ghrelin. In this study, we have elucidated the mechanism of the putative ghrelin-cannabinoid interaction.MethodsThe effects of ghrelin and CB1 antagonist rimonabant in wild-type mice, and the effect of ghrelin in CB1-knockout animals, were studied on food intake, hypothalamic AMPK activity and endogenous cannabinoid content. In patch-clamp electrophysiology experiments the effect of ghrelin was assessed on the synaptic inputs in parvocellular neurons of the hypothalamic paraventricular nucleus, with or without the pre-administration of a CB1 antagonist or of cannabinoid synthesis inhibitors.Results and ConclusionsGhrelin did not induce an orexigenic effect in CB1-knockout mice. Correspondingly, both the genetic lack of CB1 and the pharmacological blockade of CB1 inhibited the effect of ghrelin on AMPK activity. Ghrelin increased the endocannabinoid content of the hypothalamus in wild-type mice and this effect was abolished by rimonabant pre-treatment, while no effect was observed in CB1-KO animals. Electrophysiology studies showed that ghrelin can inhibit the excitatory inputs on the parvocellular neurons of the paraventricular nucleus, and that this effect is abolished by administration of a CB1 antagonist or an inhibitor of the DAG lipase, the enzyme responsible for 2-AG synthesis. The effect is also lost in the presence of BAPTA, an intracellular calcium chelator, which inhibits endocannabinoid synthesis in the recorded parvocellular neuron and therefore blocks the retrograde signaling exerted by endocannabinoids. In summary, an intact cannabinoid signaling pathway is necessary for the stimulatory effects of ghrelin on AMPK activity and food intake, and for the inhibitory effect of ghrelin on paraventricular neurons.

The in vitro labeling of therapeutic cells with nanoparticles (NPs) is becoming more and more common, but concerns about the possible effects of the NPs on the cultured cells are also increasing. In the present work, we evaluate the effects of poly(methacrylic acid)-coated 4 nm diameter Au NPs on a variety of sensitive and therapeutically interesting cell types (C17.2 neural progenitor cells, human umbilical vein endothelial cells, and PC12 rat pheochromocytoma cells) using a multiparametric approach. Using various NP concentrations and incubation times, we performed a stepwise analysis of the NP effects on cell viability, reactive oxygen species, cell morphology, cytoskeleton architecture, and cell functionality. The data show that higher NP concentrations (200 nM) reduce cell viability mostly through induction of reactive oxygen species, which was significantly induced at concentrations of 50 nM Au NPs or higher. At these concentrations, both actin and tubulin cytoskeleton were deformed and resulted in reduced cell proliferation and cellular differentiation. In terms of cell functionality, the NPs significantly impeded neurite outgrowth of PC12 cells up to 20 nM concentrations. At 10 nM, no significant effects on any cellular parameter could be observed. These data highlight the importance of using multiple assays to cover the broad spectrum of cell-NP interactions and to determine safe NP concentrations and put forward the described protocol as a possible template for future cell-NP interaction studies under comparable and standardized conditions.

SummaryUsing dual-focus fluorescence correlation spectroscopy, we have analyzed the adsorption of three human blood serum proteins, namely serum albumin, apolipoprotein A-I and apolipoprotein E4, onto polymer-coated, fluorescently labeled FePt nanoparticles (~12 nm diameter) carrying negatively charged carboxyl groups on their surface. For all three proteins, a step-wise increase in hydrodynamic radius with protein concentration was observed, strongly suggesting the formation of protein monolayers that enclose the nanoparticles. Consistent with this interpretation, the absolute increase in hydrodynamic radius can be correlated with the molecular shapes of the proteins known from X-ray crystallography and solution experiments, indicating that the proteins bind on the nanoparticles in specific orientations. The equilibrium dissociation coefficients, measuring the affinity of the proteins to the nanoparticles, were observed to differ by almost four orders of magnitude. These variations can be understood in terms of the electrostatic properties of the proteins. From structure-based calculations of the surface potentials, positively charged patches of different extents can be revealed, through which the proteins interact electrostatically with the negatively charged nanoparticle surfaces.

Ion sensors based on colloidal nanoparticles (NPs), either as actively ion-sensing NPs or as nanoscale carrier systems for organic ion-sensing fluorescent chelators typically require a charged surface in order to be colloidally stable. We demonstrate that this surface charge significantly impacts the ion binding and affects the read-out. Sensor read-out should be thus not determined by the bulk ion concentration, but by the local ion concentration in the nano-environment of the NP surface. We present a conclusive model corroborated by experimental data that reproduces the strong distance-dependence of the effect. The experimental data are based on the capability of tuning the distance of a pH-sensitive fluorophore to the surface of NPs in the nanometer (nm) range. This in turn allows for modification of the effective acid dissociation constant value (its logarithmic form, pK(a)) of analyte-sensitive fluorophores by tuning their distance to the underlying colloidal NPs.