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smll.200800413C
full paper Nanoparticle bioeffects M. A. Malvindi, L. Carbone, A. Quarta, A. Tino, L. Manna, T. Pellegrino, C. Tortiglione* __________________ 1–9
Rod-Shaped Nanocrystals Elicit Neuronal Activity In Vivo
Asymmetrical CdSe/CdS quantum rods (QRs) elicit a coordinated motor behavior, namely tentacle writhing, when added to the culture medium of the small coelenterate, Hydra vulgaris (see image). This activity is Ca2þ dependent, and relies on the presence of tentacle neurons and of cell electrical coupling. Electrical properties of QRs, shape dependent, account for the neuronal stimulation.
Rod-Shaped Nanocrystals Elicit Neuronal Activity In Vivo
Nanoparticle bioeffects DOI: 10.1002/smll.200800413
Rod-Shaped Nanocrystals Elicit Neuronal Activity In Vivo Maria Ada Malvindi, Luigi Carbone, Alessandra Quarta, Angela Tino, Liberato Manna, Teresa Pellegrino, and Claudia Tortiglione*
The development of novel nanomaterials has raised great interest in efforts to evaluate their effect on biological systems, ranging from single cells to whole animals. In particular, there exists an open question regarding whether nanoparticles per se can elicit biological responses, which could interfere with the phenomena they are intended to measure. Here we report that challenging the small cnidaria Hydra vulgaris in vivo with rod-shaped semiconductor nanoparticles also known as quantum rods (QRs) results in an unexpected tentacle writhing behavior, which is Ca2þ dependent and relies on the presence of tentacle neurons. Due to the absence of surface functionalization of the QRs with specific ligands, and considering that spherical nanoparticles with same composition as the QRs fail to induce any in vivo behavior on the same experimental model, we suggest that unique shape-tunable electrical properties of the QRs may account for the neuronal stimulation. Our model system may represent a widely applicable tool for screening neuronal response to nanoparticles in vivo.
1. Introduction 1 2 3 4 5 6 7 8 9 10 11
At the crossroads of nanotechnology and biological functional, nanocrystals are being developed towards use in a number of biological applications, such as imaging, biosensing, and cancer diagnosis, by the fine tuning of their properties.[1–4] Due to their unique optoelectronic properties, which have already motivated their use in tunable polarized lasers and organic–inorganic hybrid solar cells, quantum rods (QRs) are expected to be even more promising than the widely used quantum dots (QDs).[5–8] In addition to QD properties, such as bright photoluminescence (PL), narrow emission spectra, and broad UV excitation, QRs have larger absorption
[] Dr. C. Tortiglione, Dr. M. A. Malvindi, Dr. A. Tino CNR, Istituto di Cibernetica ‘‘E Caianiello’’ Via Campi Flegrei 34, 80078 Pozzuoli (Italy) Fax: (þ39) 0818675326 E-mail:
[email protected] Dr. A. Quarta, Dr. L. Carbone, Dr. L. Manna, Dr. T. Pellegrino National Nanotechnology Laboratory of CNR-INFM Unita` di ricerca IIT and Scuola, Superiore ISUFI Via per Arnesano, 73100 Lecce (Italy) : Supporting Information is available on the WWW under http:// www.small-journal.com or from the author. small 2008, x, No. x, 1–9
Keywords:
biological activity hydra vulgaris nanocrystals nanorods
cross-sections,[9] which might allow improvement to certain biological applications, such as deep tissue and singlemolecule fluorescence imaging, where extremely high brightness and photostability are required. Nonetheless, the use of QRs in biology is still in its infancy,[10,11] and the effects of their intrinsic physical properties on cell/tissue response in vivo need to be evaluated prior to their employment &Q1OK?& as diagnosis, imaging, and biosensing tools. Noteworthy attention should be paid to the chemical composition of surfactant or polymer-coated nanoparticles in determining their stability in aqueous media,[12] or investigating their interaction with cells and intracellular localization &OK?&.[13,14] Here, we investigate the effect of a new generation of colloidal semiconductor nanoparticles, namely asymmetric core/shell CdSe/CdS nanorods (see Fig. 1), on a freshwater coelenterate, Hydra vulgaris (Cnidaria, Hydrozoa), which has been recently successfully approached by bioconjugated QDs.[15] The simple body plan, with a head and a foot at opposite ends of a hollow, two-layered, epithelial cylinder (see Figure S1 of the Supporting Information), the transparencies of the two epithelial layers, and the highly plastic and dynamic phenomena underlying developmental and regeneration programs[16–18] makes H. vulgaris a valuable model to be approached by fluorescent nanocrystals. Despite the simplicity of its nervous system, organized as a mesh-like network of neurons extending throughout the
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range of 24–30 nm) along with large PL quantum yields. Rods of length and diameter 35 2 nm and 4.2 0.4 nm, respectively, and a maximum emission wavelength of 605 nm, were transferred from chloroform to water by wrapping them within an amphiphilic polymer shell.[24] To these polymer-coated QRs (henceforth referred to as PC-QRs) polyethylene glycol (PEG) molecules can be bound (by exploiting an EDC&please provide definition of EDC&-catalyzed crosslinking scheme, see Section 5 and also previously published works).[25,26] The resulting PEGcoated QRs (from here simply referred to as QRs) were stable in aqueous solution and preserved their fluorescence (Figure 1). When challenging living H. vulgaris with QRs, but not with the vehicle solution, an unexpected animal behavior was observed which consisted of an intense tentacle writhing (Figure 2a and Video S1 of Supporting Information). The tentacle writhing activity was comprised of Figure 1. Characterization of QRs used in this study. a) A schematic representation of the contractions and bending along the axial CdSe/CdS rods, highlighting the asymmetrical shape derived from the synthesis length of each tentacle, independently from procedure. The rod samples are an average of 35 nm in length and 4 nm in diameter as the others, as shown in Figure 2b. No differconfirmed by b) the TEM image of the corresponding sample and c) the ‘‘mean dilatation ences in the response duration were observed image’’ obtained from high-resolution electron microscope image. Upon performing geometric phase analysis (GPA), information about the Fourier components of the HRTEM at QR concentrations ranging from 7 to 100 nM image can be obtained in order to detect variations in the lattice periodicities. Areas of the (Figure S2 of Supporting Information), while same color will correspond to regions with the same lattice parameters. This image shows concentrations lower than 7 nM initiated bioan area (red-region) with lattice constants different from the rest of the rod logical activity only in 30% of animals (data (green-region). This region should correspond to the CdSe original-seed. (Adapted from not shown); thereafter we assumed 7 nM as a Carbone, 2007). [23] d) Absorption (green curve) and PL (red curve) spectra of the PEG-coated suprathreshold concentration for all further CdSe/CdS rods. trials. The end of activity was indicated by a progressive tentacle relaxation and straightening &OK?& before a return to the normal animal, the complexity of the mechanisms underlying behavior of environmental exploration (see Figure S3 and neurotransmission resemble those of higher vertebrates, includ- Video S2 of Supporting Information). Although after an ing both classical and peptidergic neurotransmitters.[19–22] Thus, average time of 8 min most of the observed polyps stopped the studies on H. vulgaris may allow for in vivo testing of neuronal activity, periods of activity as long as 14 min were recorded responsiveness (neuronal function/toxicological effect) to (Fig. 3). Fluorescence stereomicroscope investigations on thornanoparticles. While studying QR influence on H. vulgaris, we observed the elicitation of a coordinated behavior, which oughly rinsed animals 24 h after treatment did not show any was characterized and explained as result of neuronal response body or tentacle fluorescent cells that could possibly indicate QR internalization, thus pointing to a QR action performed at to the electrical properties of QRs. the cell exterior. To establish whether QR-induced motor response was generated locally inside the tentacles, or trans2. Results mitted from the polyp body, whole animals were amputated subhypostomally, and QR response was monitored on 2.1. QRs Elicit a Specific Behavior in H. vulgaris amputated body regions. As summarized in Table 1, ampuCdSe/CdS core/shell QRs were synthesized according to a tated heads and excised tentacles, but not decapitated bodies, recently reported procedure.[23] This method involves co- were responsive to QR elicitation, indicating that the QRs act injecting Cd2þ and S2 precursors and preformed spherical directly on the tentacles. The epitheliomuscular cells present CdSe seeds into an environment of hot surfactants, well suited on the tentacles, referred to as battery cells, possess for the anisotropic growth of the second shell-material (CdS) contracting fibers extending parallel to the tentacle axis, on the first underlying core (CdSe). The procedure itself and contain embedded batteries of nematocytes (the stinging proves useful in controlling the homogeneity of growth as well cells involved in prey capture) and a sensory neuron. as the strong and tunable (from green to red) light emission.[23] Ultrastructural evidence suggests that the contractile activity The resulting nanocrystals are characterized by narrow PL of battery cells is controlled by neuronal cells, as they establish peaks (full widths at half maximum of the PL peaks are in the synaptic connection with them.[27] To determine whether QRs
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Rod-Shaped Nanocrystals Elicit Neuronal Activity In Vivo
Figure 2. Elicitation of tentacle writhing by QRs. The test was initiated by adding CdSe/CdS core/shell QRs to each well containing six polyps and motor activity was monitored by continuous video recording using a Camedia-digital camera (Olympus) connected to a cold-light Wild stereo microscope. a) Within seconds of addition of QRs to the culture medium the polyp’s tentacle begin to writhe, bending toward the mouth. Contractions are not synchronous for all tentacles (left). Untreated animals or treated with same volumes (0.7 mL) of vehicle solution show tentacles completely outstretched and nearly motionless, forming a ring around the hypostome (right). b) Dynamic of tentacle writhing in H. vulgaris treated with QRs. Images are extracted from a sequence of 532 frames (acquisition time: 75 ms) acquired during the first minute of QR administration. (1–6) correspond to frames 461, 479, 480, 482, 484, and 486, respectively. QR response is induced in the young (y), the adult (a), and the budding polyp (b). Each tentacle moves independently of the others. Black arrow shows the progressive contraction of a tentacle toward the mouth (1–3) and then a complete bending of its apical end (4–6). Similarly, it is possible to follow the various positions that each other tentacle takes place. Scale bars: 500 mm.
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induce tentacle writhing by directly activating epitheliomuscular or neuronal cells we tested the effect of QRs on epithelial H. vulgaris, i.e., polyps chemically depleted of interstitial stem cells and derivatives (neurons, nematocytes, gland cells) and thus presenting only epitheliomuscular cells.[28] Such nervefree animals, whose behavior to electrical stimuli has been characterized,[29] were unresponsive to QRs (Table 1) suggesting the involvement of tentacle excitable cells, namely neurons/nematocytes, in the QR evoked response. To investigate which molecules and ions could underlie such activation we performed QR bioassay, eliminating from the bathing medium (1 mM CaCl2 and 0.1 mM NaHCO3) either Ca2þ or Naþ. As reported in Table 1, in the absence of Ca2þ or in presence of Ca2þ chelators such as EGTA, the tentacle writhing activity did not set in, whereas Naþ depletion, on the other hand, did not interfere with the elicitation of the activity. Thus, the presence of Ca2þ in the medium is a necessary condition to initiate the activity and 104 M was calculated as the minimal concentration required for elicitation of QR response (see Figure S5 of Supporting Information). Following perception by neuronal cells, signals are propagated by a small 2008, x, No. x, 1–9
combination of chemical neurotransmitters and electrical signals carried by metal ions. Rapid syncytial communication within groups of cells is achieved in the animal kingdom by gap junctions (GJs), clusters of membrane channels between adjacent cells permeable to ions and small molecules. In H. vulgaris, the morphological characterization of GJs between adjacent epitheliomuscular cells as well as in the proximity of neuromuscular and neuronematocyte synapses has been followed by the molecular identification of innexins, which might indicate the presence of innexin-based GJ.[30,31] However, their functional role in mediating communication within groups of cells, including the spread of electrical waves, has not been demonstrated to date. Assuming that in H. vulgaris, as in other invertebrates, electrical coupling between neurons allows the fast propagation of impulses between cells,[32] we exploit the possibility that gap-junctional communication (GJC) may underlie QR signal transmission. Two GJC-blocking reagents were used to rapidly modify GJ states in living polyps, namely heptanol and anandamide. They have been tested on electrical and chemical GJC in both connexin and innexin-based GJ.[33–36] H. vulgaris were exposed for 1 h to drugs dissolved in the medium at working concentrations that were sufficiently low to cause no effects on the polyps’ physiology, as determined by macroscopic observation. As shown in Figure 4, treated animals were able to respond to QRs, but the evoked activity stopped within the first minute after QR administration. This behavior indicates the capability of animals to respond to, but not transmit, the QR signal, suggesting that cell electrical coupling is necessary for QR evoked signal transmission.
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2.2. Influence of Shape and Chemical Composition on QR Response
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In our previously published study,[15] PEG-coated CdSe/ ZnS QDs were unable to interfere with animal behavior or physiology when supplied to living polyps, unless they were functionalized with a specific ligand. Those nanocrystals differed, however, from those of the present study in both their shape (they were spherical, while the present ones are rod shaped), and in their chemical composition (they were made from a spherical CdSe inner core surrounded by a thin concentric shell made of ZnS, while in those described here the CdSe core is buried in a thick and asymmetric rod-shaped CdS shell), which makes a comparison between the two samples rather cumbersome. Therefore, in order to identify the possible role played by nanoparticle shape in the activity elicitation, we additionally prepared spherical CdSe/CdS nanocrystals in which the CdSe spherical core was buried in a thick spherical shell. The final average diameter of these spherical core/shell nanocrystals was equal to 23.9 3.4 nm (see Section 5 for further details). In Figure 5 we report the optical absorption and emission spectra of these spherical nanocrystals in solution, after they had been water-solubilized and functionalized with PEG molecules, as was done for the CdSe/CdS QRs.[24–26] These spherical nanocrystals did not induce tentacle writhing or any other behavior when added to the medium of living H. vulgaris, and this suggests that the shape anisotropy plays a role in the elicitation of the activity.
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QR response in culture solution
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different from mechano- or chemoreception underlie tentacle contraction. QR response was observed also in dim-light condition, in the absence of the microscope light, ruling out the involvement of the light not only in the elicitation of animal behavior, but also in the modification of QR physical properties.
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3. Discussion
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In this paper we described an in vivo model system used to evaluate the biological response to nanoparticles at a whole animal level. We report the unexpected 0 response of the small coelenterate H. vulgaris to rod-shaped semiconductor Time (min) nanoparticles and propose them as new Figure 3. Temporal dynamic of QR induced tentacle writhing in culture solution. Groups of six tools for neuronal stimulation. To date, the tentacle-writhing behavior has been H. vulgaris per well were challenged with QRs and the motor activity monitored simultaneously for all polyps and recorded at 1 min intervals for 10–15 min from the beginning of described as part of the feeding response, the experiment. Values indicate the mean plus/minus standard deviation &OK?& of six naturally occurring during prey capture.[37] independent experiments, each performed in triplicate (n ¼ 108). Periods of activity as long Induction of such behavior has never been as 14 min could be recorded. associated to mechanical or physical stimuli, or to the presence of organic– inorganic particles, but rather to chemoreception of specific molecules, mainly amino To rule out possible involvement of PEG molecules of the acids. The most extensively investigated chemostimulant for nanoparticle outer shell in the elicitation of the activity, the H. vulgaris and other cnidarians is the tripeptide glutathione QR bioassay was performed using PC-QRs, therefore missing (GSH).[38] Comparing the kinetics of GSH and QR induced PEG molecules at their surface. The addition of these responses, we suggest two different target molecules and nanoparticles to the culture medium induced polyps to writhe independent transduction pathways (see Table S1 of Supporttheir tentacles (Figure S4 of Supporting Information). The ing Information). These results prompted us to investigate, on activity, however, was soon ceased, probably due to QR one hand, the possible anatomical structures, cell types or ions precipitation in the H. vulgaris culture medium. QR aggre- employed by the animal to perceive and to transduce the QR gates, in fact, were easily detectable by fluorescence micro- elicitation, and on the other the possible chemical or physical scopy even at the lowest concentration (7 nM). The induction nature of the signal represented by QR external addition to of activity even by PC-QRs suggested that the ability to induce living animals. The characterization of H. vulgaris behavior in a motor response is related to nanoparticle per se and not to response to QRs led to the identification of the tentacles as their particular coating&OK?&, and that mechanisms minimal anatomical regions able to react to QRs, of neurons as 0: 00 0: 30 1: 00 1: 30 2: 00 2: 30 3: 00 3: 30 4: 00 4: 30 5: 00 5: 30 6: 00 6: 30 7: 00 7: 30 8: 00 8: 30 9: 3 10 0 :0 10 0 :3 11 0 :0 11 0 :3 12 0 :0 12 0 :3 13 0 :0 0
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Table 1. Evaluation of QR response in different ionic conditions and different anatomical regions. Whole animals
Excised heads
Excised tentacles
Decapitated bodies
Nerve-free polyps
þþ þþ
þþ þþ
þ þ
Sol Hy Ca2þ free Naþ free Sol Hy þEGTA
Whole adult animals or amputated body regions were challenged with 7 nM QRs in Sol Hy, Ca2þ-free or Naþ-free medium and the presence/absence of activity were monitored. All responsive specimens marked by þþ showed the same induced behavior, as duration, typology, and coordination of motor activity. Behavioral response of excised tentacles consisted in contractions and bending along the axial length and was recorded for shorter periods (indicated by þ). Decapitated bodies and nerve-free polyps, marked as minus () were unresponsive. Both in Ca2þ-free solution or in Sol Hy containing the Ca2þ chelator EGTA (4 mM) the motor activity in response to QRs was prevented. Data are means of three independent experiments, on a total number of 54 animals.
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the axon hillock voltage-sensitive ion channels, upon membrane depolarization, undergo conformational changes, allowing ion transit and the firing of an action potential. Following similar signaling mechanisms in H. vulgaris, the QR induced tentacle writhing might result from activation of appropriate receptors (i.e., voltage gated channels) present on tentacle excitable cells (sensory neurons or nematocytes) involved, in physiological conditions, in the initiation and propagation of action potentials. Because in the absence of Ca2þ ions the QR response is prevented, we tried to pharmacologically block voltage dependent Ca2þ channels (VDCC) using both organic (nifedipine) and inorganic (Ni2þ) blockers specifically developed to modulate vertebrate L-type and T-type calcium Figure 4. Exposure to drugs and inhibition of QR evoked response in channels, respectively.[40] Results indicated, in the first case, living H. vulgaris. GJ blockers heptanol (0.003%) and anandamide no differences in the QR evoked response between drug (1 mM) were independently bath applied 1 h before QR addition. Experiments were performed as described in Figure 3. The tentacle treated and untreated animals (data not shown), while Ni2þ writhing activity could be treatment was highly toxic to living animals. However, elicited but was soon abolished by both GJ blockers, indicating the need considering the electrophysiological and pharmacological of cell electrical coupling for QR signal transmission. Data are means of diversity reported between vertebrate and invertebrate three independent experiments, on a total number VDCC[41–43] the absence of pharmacological inhibition does of 54 animals. not exclude a possible involvement of VDCC in QR response, remanding to different approaches the identification of the QR molecular targets. In an attempt to investigate the specific cell targets, and of calcium ions as required elements plausible mechanism responsible for the propagation of QR for activity induction. According to the general model of stimuli between adjacent cells, which usually occurs by means stimulus perception, the energy contained in a stimulus is used of a combination of chemical neurotransmitters and electrical by the neuronal receptor to change the conductance of its signals carried by ions, we showed that electrical transmission membrane for one or more ions. These conductance changes is involved in the QR response. In fact, blockers of GJC, will cause the receptor’s membrane potential to change and, if widely conserved throughout vertebrates and invertebrates, above a threshold level, to initiate an action potential.[39] On inhibit tentacle writhing. While dictating the need of cell electrical coupling, this finding confirmed that a process of neuronal activation is induced by QRs, as at least one mechanism of neurotransmission underlies it. Considering the additional modulatory role played by anandamide on various vertebrate ion channels (i.e., its involvement in the inactivation of low voltage activated T-type Ca2þchannels)[44] we cannot rule out that the inhibition effect displayed by anandamide on QR evoked activity might result from the combined modulation of GJ and voltage gated Ca2þ channels. The remarkable result of the present work is the coordinated behaviors that can be evoked in a living animal by water soluble QRs. The experimental trials performed as controls, namely the addition of the same volumes of culture solution, or of PC-QRs, lead us to exclude the involvement of mechano- or chemoreception, respectively, in the mechanism of QR signaling, suggesting electrical sensory properties of tentacle neurons or nematocytes. This property has already been shown in H. vulgaris and other hydrozoan, through analyzing the mechanism Figure 5. Characterization of the near spherical nanocrystals used in this study; a) schematic representation of the CdSe/CdS seeded grown nanodots, sizing an average underling mechanoelectric transduction in nematocytes &OK?&.[45,46] In H. vulgaris, of 20 nm in diameter as confirmed by b) the TEM image and c) absorption (blue curve) and PL (pink curve) spectra of the PEG-coated CdSe/CdS dots. the process of nematocyst discharge (a small 2008, x, No. x, 1–9
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vesicular organelle present inside the nematocyte) can be induced by applying external electric fields to the nematocytes, which implicates electrical excitation as a possible trigger mechanism.[47] Nematocyte discharge was induced by depolarizing the apical membrane by about 25 mV (threshold). Thus, relying on these membrane properties, we suggest that the tentacle writhing activity results from neuron/nematocyte response to an electrical stimulus, represented by QRs. As Figure 6. A model of neuronal activation by QR on H. vulgaris tentacles. The electric spherical nanocrystals with analogous compofield associated with the QR dipole moment causes a change of membrane conductance sition are unable to induce a response, the for one or more ions. The conductance change causes the receptor membrane potential to shape of CdSe/CdS QRs may account for their change and, if above a threshold level, to fire an action potential. On the left a schematic effect on neuronal activation. illustration of the bilayered structure of a H. vulgaris tentacle is illustrated (en ¼ endoderm Several studies have targeted the origin and and ec ¼ ectoderm). Tentacle ectodermal cells are called battery cells (bc) and contain the estimation of the permanent dipole embedded several type of nematocytes (nem), one sensory neuron facing the exterior (sn), moment in colloidal spherical QDs and a ganglial neuron (gn) making connections both with other cells and to myonemes (myo), namely contracting fibers lying parallel to the tentacle length. On the right is a elongated QRs exhibiting a hexagonal wurtzite magnification of the circled area, showing QRs diversely oriented at the cell surface, in structure, such as the CdSe/CdS core/shell proximity of a neuronal membrane receptor (gray protein located in the phospholipidic cell samples investigated here.[48–51] The origin of membrane bilayer; amino acid voltage sensors are depicted as þ). QRs (orange cylinders an intrinsic dipole moment along the of length d) possess a dipole moment illustrated as charge separation (þ and ) at the c-crystallographic axis in wurtzite stacking of opposite poles, and an associated voltage potential field. Results from a geometrical atom planes relies on the lattice deviations approximation (see Supporting Information) shows that a QR voltage potential of sufficient intensity to stimulate a voltage gated ion channel can be produced at from the ideal wurtzite structure. Theoretical nanometric separation distances, regardless of its orientation at the cell surface. These results indicate that it is linearly dependence estimated values correspond to the distances at which biological molecules naturally [49] on the nanoparticle volume. High-tempera- interact each other, thus it is theoretically possible for QRs to elicit neuronal activity. ture seed-grown CdSe/CdS nanostructures have been shown to occur exclusively with the wurtzite structure, with internal CdSe dots localized in a region between one-third and one-fourth of the dependence of the QR electric dipole potential on its distance overall CdS rod length (Figure 1c).[23] According to the Nann with respect to the ion channel, regardless of its orientation, and Schneider model,[49] a QR (treated as a cylinder), which plays a less crucial role (see Table S2 and S3 of the composed only of CdS and of diameter and length equivalent Supporting Information and Fig. 6). Voltage potentials above to the QRs studied in this work, should posses a dipole threshold (i.e., 15 mV)[39,54] can be produced at very short moment of 1 350 D. Additionally, in the present case, as a distances (1 nm). In light of several physical-chemical consequence of the elastic surface strain between the core/ approximations (the actual action potential threshold V and shell interfaced materials (CdSe/CdS), a further contribution the intrinsic dipole moment p should be experimentally to an internal field should originate from a spontaneous measured) we consider it reasonable to use the magnitude piezoelectric polarization.[51,52] Based on these premises we order (within the nanometric range) of these estimated values, suggest that CdSe/CdS QRs, regardless of surface chemical as they correspond to the distances at which biological functionalization, generate local electric fields associated with molecules naturally interact with each other &OK?&. Thus, it their permanent dipole moments that are intense enough to appears theoretically possible for QRs to activate voltagestimulate voltage dependent ion channels, thus eliciting an sensitive membrane channels. For spherical CdSe/CdS nanocrystals, here synthesized, a action potential resulting in motor activity. The electric dipole potential V generated at a point of similar approximation cannot be made. In fact, several interest (i.e., an ion channel voltage sensor) by a QR dipole controversies surround the calculation of even a rough dipolemoment p may be estimated using the following equation:[53] moment value for spherical QDs. For instance, Nann and Schneider rely upon a dipole moment linear dependence on the volume,[49] exactly as for a QR except for a minor p r rþ V¼K (1) proportionality constant. In total conflict is the model of Shim d r rþ and Guyot-Sionnest, which uses a linear dependence on the nanosphere radius and not on the volume.[55] Therefore, where K ¼ 1/(4per) accounts for the vacuum permittivity, d is considering the present results and those formerly published the dipole length, and rþ and r are the distances of the point by our group on QDs,[15] we believe that, for spherical of interest from the dipole positive and negative charges, nanocrystals, an inadequate internal dipole moment is the respectively. We estimated the potentials created by a dipole most straightforward interpretation of their inactivity. Only moment of 1 350 D at various separation distances (see experimental measurement of the dipole moments generated Supporting Information). Our calculations show a strict by both nanoparticles will unravel this point.
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4. Conclusions The numerical values estimated in our approximation justify our hypothesis of QR electrical activation of ion channels and neuronal stimulation. The peculiarities of our biological model system, such as the low-ionic-strength culture media and the presence of excitable cells directly facing the outer media, allowed us to highlight the neuronal stimulation by a nanometric inorganic particle, which might be difficult to study in vivo in a more complex whole organism. Avoiding the difficulties in investigating vertebrate brain behavior in vivo, our cnidarian model organisms provided a simple, reliable, and fast system for screening nanoparticle activity in vivo on a functionally connected nerve net. On the other hand, the lack of available techniques for performing electrophysiological recording on H. vulgaris dissociated neurons leaves to a different model system the identification of the molecular actors underlying the QR response. Modulation of the electrophysiological properties of cultured cells or mammalian brain slices by QRs might indicate that QR response is not specific to our experimental model, casting light on a fascinating field deserving further investigation. Considering the challenges encountered in the design and synthesis of electrical nanodevices for neuronal stimulation,[56] we propose biocompatible, soluble QRs as a novel resource for neuronal devices, for diagnostic and therapeutic applications where noninvasive probing and fine tuning of neuronal activity is required.
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5. Experimental Section
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Synthesis of QRs and water-solubilization: CdSe/CdS nanorods were prepared by the seeded growth approach as recently reported.[23] The surfactant-coated colloidal QRs and QDs (for the synthesis of which see the paragraph below) were transferred from chloroform into water by implementing a previously developed polymer-coating procedure.[24] Further surface coating with diamino-PEG molecules was achieved by means of EDC crosslinking chemistry.[25,26] A diamino-PEG (molecular weight 897 g mol1) solution (21 mM) in borate buffer (pH 9, 6 mM) was added to a QR solution in the same buffer to reach a PEG/QRs ratio of 500. An amount of EDC (0.2 M) was added. The reaction mixture was incubated at room temperature under rigorously stirring for 3 h. After the reaction, to remove the excess EDC and unbound PEG molecules, the sample was washed twice on centrifuge filter with a membrane with a molecular weight cut off of 100 000 Da. The last washing step was performed with Milli-Q water. Synthesis of spherical seeded-grown CdSe/CdS nanocrystals: In a typical synthesis of CdSe/CdS nanodots via seeded growth, CdO (0.060 g, 99.5%; Sigma–Aldrich), were mixed in a 50 mL flask together with trioctylphosphine oxide (TOPO, 3 g, 99%; Strem Chemicals), octadecylphosphonic acid (ODPA, 0.291 g, 99%; PCI), and hexylphosphonic acid (HPA, 0.083 g, 99%; PCI). After pumping the flask to vacuum for about 1 h at 150 8C, the resulting solution was heated to 380 8C under nitrogen. At this step trioctylphosphine (TOP, 1.5 g, 97%; Strem Chemicals) was
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injected, after which the temperature was allowed to recover to the value required for the injection of the solution of sulfur precursor plus nanocrystals. Such solution was prepared by dissolving sulfur (S 0.070 g, 99%; Strem Chemicals) in TOP (0.870 g) and adding to this a solution (600 mL) of readily prepared CdSe dots dissolved in TOP (here, the concentration of dots in the TOP solution was always 500 mM).[23] The resulting solution was quickly injected in the flask. After injection, the temperature dropped to 320 8C and recovered within 2 min to the preinjection temperature. The nanocrystals were allowed to grow for about 8 min after the injection, after which the heating mantle was removed. Growth and maintenance of H. vulgaris: H. vulgaris (strain Zurich, originally obtained by P. Tardent) were asexually cultured in physiological solution (Sol Hy: 1 mM CaCl2, 0.1 mM NaHCO3, pH 7) using the method of Loomis and Lenhoff with minor modifications.[57] The animals were kept at 18 1 8C and fed three times per week with freshly hatched Artemia salina nauplii. Polyps from homogeneous populations, three weeks old and carrying one or two buds, were starved 2 days prior to the experiments. Animals depleted of their interstitial cells and derivatives of these stem cells (nematoblasts, nematocytes, and nerve cells) termed epithelial H. vulgaris, were generated by treatment with colchicine as described.[28] In vivo experiments: Groups of six animals were collected in plastic multiwells and allowed to equilibrate at room temperature in physiological solution (Sol Hy, 300 mL) buffered with Tris-HCl (1 mM, pH 7.4). The test was initiated by adding CdSe/CdS core/ shell QRs at different concentrations (ranging from 7 to 100 nM) to each well containing six polyps, and motor activity was monitored by continuous video recording using a Camedia-digital camera (Olympus) connected to a cold-light Wild stereo microscope (Olympus ZSX-RFL2). The time course of QR effect on H. vulgaris motility was quantified by counting the number of polyps showing motor activity at 1 min intervals for 10–15 min from the beginning of the experiments. Data were expressed as percent of moving animals. Experiments were performed in an air-conditioned environment at 22 8C, and repeated three times for each QR concentration and solution tested. Control experiments were performed by treating polyps with vehicle solution (Sol Hy) not containing QRs. When performing in vivo experiments with epithelial H. vulgaris, the control animals were polyps kept under the same conditions of starvation used to prepare nerve-free animals. The presence of fluorescent QR targeted H. vulgaris regions was evaluated under a stereomicroscope (Olympus ZSXRFL2) equipped with a fluorescence filter set (BP460-490/DM505/ LP510). H. vulgaris anatomical dissection and Ca2þ chelation: Intact H. vulgaris were bisected subhypostomally, and groups of six amputated heads or tentacles excised from six animals and placed in the culture medium for approximately 1 h were challenged with QRs as described above. The involvement of Ca2þ ions in the induction of biological activity was evaluated by performing experiments in Ca2þ-free medium (300 mL), namely in NaHCO3 (0.1 mM), buffered with Tris-HCl (1 mM, pH 7.4) or in Sol Hy modified with EGTA (4 mM) as a Ca2þ chelator &OK?&. The minimal amount of Ca2þ required for the biological activity was estimated by performing bioassays in culture solution containing
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NaHCO3 (0.1 mM) and decreasing the concentration of CaCl2 (1, 0.1, and 0.01 mM, or complete CaCl2 depletion). Drugs exposure: The effect of the GJ blockers on the modulation of biological activity was evaluated by adding drugs to Sol Hy culture medium, and allowing the polyps to equilibrate in this solution for 1 h before testing QR activity. Pure heptanol (heptan-1-ol), diluted in Sol Hy, was added at a final dilution equal to 3 105; ananadamide (arachidonoylethanolamide), kindly provided by Dr V. Di Marzo (Institute of Biomolecular Chemistry, Pozzuoli, Italy), was diluted to working concentrations (1 mM) from a stock (5 mM). The doses of the drugs used were within the range of magnitude of the doses used to modulate GJ in either vertebrate or invertebrate organisms[33–36]. In these set of experiments the effect of QRs at a single concentration (7 nM) was evaluated.
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Acknowledgements
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M. A. M. and L. C. contributed equally to this work. This work was supported by the Italian Ministry of Research (FIRB grants RBN01KJHT-007, RBIN048TSE, and RBLA03ER38), the European STREP project SA-NANO (contract number 013698), and the Italian National Research Council. We thank Bert Hobmayer for careful and critical reading of the manuscript.
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