Review pubs.acs.org/molecularpharmaceutics
Nonlinear Nanomedecine: Harmonic Nanoparticles toward Targeted Diagnosis and Therapy Luigi Bonacina* GAP-Biophotonics, University of Geneva, 22 chemin de Pinchat, CH-1211 Geneva 4, Switzerland ABSTRACT: Harmonic nanoparticles were first introduced in 2006 as biomarkers for nonlinear imaging. This review provides a general explanation of the physical mechanism at the basis of this novel approach, highlighting its benefits and the complementarity to fluorescent/luminescent labels. A series of application examples from the very recent literature are reported, ranging from in vitro cell monitoring to the first proofs of in vivo imaging and rare event detection in physiological fluids. KEYWORDS: nonlinear microscopy, nanoparticles, optical markers, theranostic
■
■
INTRODUCTION
Some Useful Definitions. Nonlinear Optical Response. We can describe a light wave as a sinusoidal electric field oscillating at frequency ω or, equivalently, characterized by a wavelength λ = 2πc/ω, c being the speed of light. When this wave impinges on a medium, it causes its response, the induced polarization P. In most cases, we can state that P ∝ χ(ω)E, which tells us that the intensity of the response is linearly proportional to the intensity of the wave’s electric field, and that it depends on its specific frequency via the linear susceptibility χ(ω). The latter determines the linear index of refraction and the linear (one-photon) absorption of the system when the frequency ω is resonant to the energy transition between two states. The situation described holds until the strength of the wave’s electric field becomes comparable or exceeds the interatomic fields in the medium, a condition which can be nowadays easily achieved by pulsed lasers. Under these conditions, the description of the induced polarization has to be modified, by including additional nonlinear terms depending on higher powers of the wave electric field: P(t) ∝ χ(ω)E(t) + χ(2)(ω)E2(t) + χ(3)(ω)E3(t) ... Higher-order terms χ(n≥2) give rise to nonlinear effects which can result, for example, in the creation of radiation fields propagating at new frequencies (≠ω) or in the simultaneous absorption of multiple photons possibly followed by fluorescence emission. The latter two phenomena are the most widespread in imaging applications and, if we limit ourselves to two-photon processes, the most likely to happen. They assume the names of two-photon excited fluorescence and second harmonic (SH) generation. Their
Nanoparticles (NPs) are being increasingly applied as contrast agents for various imaging techniques (optical microscopy, MRI, PET, X-ray, computed tomography, and so on) or as treatment agents exploiting their preferential accumulation in targeted structures for drug delivery or their response to external stimuli (magnetothermal therapy, laser activation, and so on). Concerning optical properties, fluorescence/luminescence is by far the most exploited observable, followed by plasmonic response in metal NPs. One very recent development, yet little-known and strongly related to the evolution of laser technology, is the use of so-called harmonic nanoparticles (HNPs). (Given the novelty of this approach, a consensus has not yet been found on the name for these NPs. Some authors use SHRIMPs, others second harmonic probes or nanodoublers. We prefer to adopt HNPs, because it has a more general scope, not being limited to the second harmonic.) HNPs are optically active NPs exhibiting a strong and selective response to nonlinear optical excitation. This focused review will highlight the advantages of this novel approach, describing a series of promising demonstrations which recently appeared in the specialized literature. This paper is addressed to a converging audience, interested in the latest developments of nanotechnology with prospective use in medicine. It is assumed that the reader possesses a biomedical background with little knowledge in optics and material science. Therefore, the very concept of nonlinear optics, emphasizing and explaining its advantages for imaging in terms of tissue penetration and decreased photodamage, will be first introduced. On this ground, HNPs will be presented, as unique, inherently nonlinear NPs, and the main findings in this field, since their introduction in 2006, summarized. Considering the similarity (at least in terms of underlying motivations) of the HNP approach to that of upconverting NPs, which has been extensively covered by other review articles,1,2 a short critical comparison between the two techniques will be provided. © XXXX American Chemical Society
NONLINEAR OPTICS
Special Issue: Theranostic Nanomedicine with Functional Nanoarchitecture Received: September 17, 2012 Revised: November 7, 2012 Accepted: November 15, 2012
A
dx.doi.org/10.1021/mp300523e | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
Review
Figure 1. (a) One-photon excited fluorescence. (b) Linear scattering. (c) Two-photon-excited fluorescence. (d) Second harmonic generation. (e) Sequential absorption and upconverted emission. (f) Principle of molecular bleaching showing intersystem crossing upon one- or two-photon absorption.
of a cell. Such symmetry-breaking conditions can be realized much more efficiently in some specific crystal structures. And in fact, macroscopic noncentrosymmetric crystals are commonly used in optics to frequency double laser lights (e.g., in green laser pointers). However, when dealing with macroscopic crystals the SH generation efficiency is limited to a very narrow frequency range so that each crystal is specific to a defined laser source. Coherence. In optics two signals are said to be coherent if they possess a defined phase relationship and thus can generate interferences when they are superposed. Laser light is coherent, but this coherence is not conserved in the fluorescence which follows the absorption of a laser photon by a molecule (Figure 1a,c). On the contrary, scattering and nonlinear harmonic generation (Figure 1b,d) are coherent processes and the phase information is fully conserved in the emitted signal. Nonlinear Imaging. As pointed out by several authors, since the pioneering works of W. Webb at Cornell, nonlinear excitation leading to two-photon excited fluorescence and SH generation presents straightforward advantages for scanning microscopy including (i) increased spatial resolution, (ii) deeper imaging penetration, and (iii) reduced sample photodegradation.3,4 The first benefit descends directly from the aforementioned S = I2 dependence, which spatially constrains the nonlinear interaction to an effective interaction region smaller than the diffraction limited spot and allows the performing of three-dimensional sectioning without a confocal pinhole. Increased imaging penetration is guaranteed by the possibility to work at longer wavelengths, fully exploiting the transmission window of biological tissue (700−1000 nm) for effectively exciting dyes, fluorescent NPs, and autogenous fluorophores which mostly absorb in the ultraviolet−visible region, around 400 nm. Finally, the interplay of limited spatial interaction region and possibility to work with weak linearly absorbed photons imply that less energy is deposited on the sample, decreasing photodegradation. Microscopy is sensitive to the scattering of photons along the excitation path. Scattering, by modifying photon trajectories, spoils the spatial and temporal structure of the excitation pulse before it reaches the focal plane, decreasing, for instance, the possibility of obtaining two photons at the same position within an ultrashort time span. As the probability of a scattering event increases with the distance traveled by a photon, the nonlinear signal presents a characteristic exponential dependence as a function of the tissue depth z:
signals present a characteristic dependence on the second power of the excitation light intensity: S ∝ I2. The schemes in Figure 1 summarize a series of interactions between incoming photons and a medium. (a) One-photon excited fluorescence. The frequency of the photon matches the difference between two levels of a molecule in the medium. The photon is absorbed, promoting an electron to an excited state. Upon some reorganization following this sudden change (vibrational relaxation), and a time-varying permanence on the excited state, the electron falls back to the ground state, emitting in a randomized direction a fluorescence photon at frequency ωfluo < ω. (b) Linear scattering. The incoming photon has a frequency which does not correspond to any specific absorption in the medium. It simply collides with a particle and deviates from its original trajectory with no change in frequency. The interaction is represented by the dashed line, a virtual energy state, not associated with any stable molecular excitation, which indicates just the instantaneous response of the system to the perturbation at frequency ω. (c) Two-photon excited fluorescence. A flux of photons coming from an intense source create the conditions to have two photons interacting with the medium at the very same time and position in space. Each photon has a frequency ω/2 such that the sum of the two matches the difference between two molecular levels. The two photons are simultaneously absorbed. The subsequent fate of the system brought onto the excited state follows what is described in list item a. (d) SH scattering. As in the previous case, two photons at ω/ 2 coincide in time and space in the medium but their frequencies do not add up to an existing transition of the system. Under some special conditions, the two photons can generate together an instantaneous response of the system which combines their frequency emitting a new photon exactly at ω = (ω/2) + (ω/2). Concerning the last point, an essential aspect has to be clarified: SH scattering can only take place in noncentrosymmetric media. By oversimplifying, we can explain this property as the absence of symmetry on a microscopic scale. As a matter of fact, SH can be observed at interfaces, for example, cell membranes, where a sudden change occurs in the optical properties of the medium going from the inside to the outside B
dx.doi.org/10.1021/mp300523e | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics S(z) = exp−2z / Lscatt(ω)
Review
(1)
Lscatt(ω) is the average distance traveled by a photon at frequency ω between two successive scattering events. This parameter decreases with increasing ω, which implies that it is advantageous to use longer wavelength (i.e., smaller frequency) light to increase imaging penetration. Remark about the Equipment Required for Nonlinear Microscopy. For nonlinear techniques to be effective, an ultrafast laser, with typical pulse duration 20K euros) can enable a 10fold lowering of laser price. In a few years, thanks also to increased demand, many experts foresee that the price difference between a confocal and a nonlinear microscopy platform will be significantly less.8
Figure 2. Absence of bleaching. Image of a cell culture labeled by HNPs at the beginning (a) and end (b) of a 10 min long raster scan at relatively high laser intensity. Red: Two-photon excited fluorescence of FM1-43FX cell membrane. Blue: SH from NPs. In panel b, the fluorescence signal has completely disappeared with no recovery possibility, while HNPs remain unaltered upon irradiation. (c) Signal intensity as a function of the irradiation time for the two-photon fluorescence and SH channel corresponding to the images in panels a and b. (d) Long-term stability of SH from HNPs, no signal degradation is observed upon hours of constant irradiation.
■
time scan on the bottom right indicates that even after several hours of continuous irradiation the intensity of the HNP signal remains unchanged. This property is particularly appealing for long-term cell tracking applications, to follow the evolution of living samples on time scales going from a few hours to several days. Excitation-Wavelength Tunability. Contrary to resonant processes like one- and two-photon excited fluorescence, and also different to “standard” SH generation in bulk nonlinear crystals, tailored to effectively work in very narrow wavelength intervals, HNPs do not present any specific wavelength dependence. In the recent past, the possibility to frequency double without spectral limitation laser pulses from the ultraviolet to the infrared has been demonstrated.20−22 Figure 3 is quite comprehensive: the black dashed lines display the absorption coefficients of the most prominent tissue molecules (water, melanin, oxygenated and deoxygenated hemoglobin, and so on) and the green dotted line displays the scattering efficiency (via the reduced scattering coefficient, which is inversely proportional to the scattering length Lscatt introduced above) of skin. These optical properties are directly compared to the pulse spectra of different models of ultrafast lasers that can be used for SH generation (erbium ion (1550 nm), chromium-forsterite (1250 nm), wavelength tunable Ti:sapphire (700−1000 nm)) and to the corresponding experimental SH signals generated by HNPs.20,22,23 The comparison highlights the advantages of wavelength tunability for imaging applications. The excitation wavelength can be freely tuned to best match the optical properties of the sample, for example, to avoid autofluorescence and to increase penetration depth by minimizing absorption and scattering, or, conversely, to selectively photointeract with some specific tissue components (proteins, DNA, and so on). Increased penetration depth remains, at present, the most straightforward advantage of the HNP approach. In Figure 4 are presented the results of ad hoc numerical calculations of light transport by Extermann et al.20 These curves were
WHAT ARE HARMONIC NANOPARTICLES? After the overview about nonlinear optical phenomena given in the previous section, HNPs can be easily introduced. These NPs have been developed in recent years to address some specific issues relating to bioimaging performances of fluorescent markers. They represent a novel family of inherently nonlinear, nonfluorescent NPs, specifically conceived and realized for SH generation. HNPs are based on various inorganic crystals, including iron iodate (Fe(IO3)3),9,10 potassium niobate (KNbO3),11 lithium niobate (LiNbO3),12 barium titanate (BaTiO3),13,14 potassium titanyl phosphate (KTiOPO4, KTP),15−17 zinc oxide (ZnO),18,19 and SiC.14 As a consequence of their noncentrosymmetric structures, they all possess a large SH response. Photostability. Bleaching and blinking are well-known causes of image degradation. The mechanism of bleaching of molecular fluorescence is illustrated in Figure 1e. A fluorescent molecule, once it has reached the excited state upon photon absorption, possesses a nonnegligible probability to hop onto another nonfluorescing excited state. Clearly, this transition is associated with the presence of real energy levels and does not occur when the contrast mechanism of the marker is based on pure SH generation (see Figure 1d). Similarly, the intensity fluctuations affecting the emission from quantum dots (blinking), derived from the trapping of photoexcited electrons on the NPs’ surfaces, cannot be observed in SH. In Figure 2a,b, one can observe two microscopy images taken respectively at the beginning and at the end of a 10 min raster scan of a cell culture sample. The color red corresponds to fluorescence from a membrane dye, while blue dots originate from the SH emission of individual HNPs. Comparing the two images, one can notice a dramatic decrease of fluorescence intensity in contrast to the stability of the HNP signal. This observation is further confirmed by the quantitative comparison reported in the two plots below. In particular, the long-term C
dx.doi.org/10.1021/mp300523e | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
Review
Figure 3. Excitation wavelength flexibility. Experimental laser spectra (red) and corresponding SH from HNPs (blue) superimposed to the absorption spectra of major tissue constituents (black dotted lines, left axis, logarithmic scale) and scattering properties of skin (green dotted line, right axis, logarithmic scale). Data from refs 20, 22, and 23.
Table 1. Normalized Hyperpolarizabilities of Different Inorganic Nanomaterials Used for HNPs (Top; Data from Ref 23) and Efficiency of the Two-Photon Excited Response for Different Kind of Emitters (Bottom) HNP crystal ⟨d⟩
KNbO3
LiNbO3
3.4 ± 1.1 emitter
4.8 ± 1.6
Rhodamine 6G enhanced GFP quantum dot BaTiO3 HNP (120 nm)
BaTiO3
KTP
4.6 ± 0.7 1.4 ± 0.3 σ2P [GM] 150 75 47 × 103 25 × 103
ZnO 1.9 ± 0.6 ref 27 28 29 13, 30
luminescence efficiency of typical fluorescent molecules and quantum dots.13 These figures overall indicate that, with respect to intensity, a quantum dot is preferable. However, the unique benefits brought by the HNP approach can compensate for this signal intensity reduction in a series of contexts where imaging sensitivity is not the only priority. In fact, very often problems related to signal specificity and image contrast are more difficult to overcome than detection sensitivity, and the interplay between excitation wavelength flexibility, narrow emission bandwidths, and photostability assured by HNPs may yet provide the optimum solution. Additional Optical Properties. In contrast to fluorescent labels, HNPs convey additional information in their signals, related to their coherence and polarization properties. In particular the orientation of individual HNPs with respect to the polarization of the exciting laser field can modulate the intensity of the SH response.9 This characteristic has been used to optically retrieve the orientation of individual HNPs and, via the presence of a permanent dipole moment connected to the crystal structure, can be a priori exploited to investigate the local electric field in biological samples (e.g., cell membranes, synapses). Similarly, the coherent emission of HNPs has enormous potential in imaging applications. The information contained in the interference patterns generated among HNPs (see Figure 5) and with an external reference laser can be used to extract depth information in a scanningless fashion13 or counteract effects of sample turbidity for improving imaging contrast.31−33 Finally, it has been shown that adding a metal layer of tailored thickness to HNPs can lead to an enhancement of their SH response by several orders of magnitude.34 The plasmon-driven enhancement in core−shell NPs is limited to specific wavelength ranges depending on shell thickness. Cytotoxicity. One of the fundamental motivations shared by the research groups who introduced and promoted HNPs is
Figure 4. Imaging penetration depth. Results of numerical calculations, showing the expected normalized epi-detected SH signal generated by HNPs embedded in murine liver tissue at different depths for various excitation wavelengths. The calculations fully take into account the optical properties of the tissue (absorption, scattering) and the detection geometry. Data from ref 20.
validated against measurements based on SH excitation and detection of HNPs embedded in murine liver tissue. What emerges from a comparison of the plot traces is that the scattering along the excitation path assumes a more important role than the absorption properties of the sample, and that the longer wavelengths, although absorbed by water, systematically perform better in terms of penetration and nonlinear efficiency than those in the 750−1000 nm range, where water absorption is at its minimum. A similar scenario has also been confirmed by analogous experiments and calculations by Grange et al.24 and Balu et al.25 Signal Intensity. The nonlinear efficiency of the inorganic noncentrosymmetric nanocrystals used for HNPs are relatively similar, as one can appreciate from the comparison of their normalized hyperopolarizabilities in the first row of Table 1 derived from ensemble measurements.23,26 Therefore, as recently pointed out, the selection of HNPs does not critically rely on the nanomaterial of choice but should be based on other properties, mainly biocompatibility.23 Importantly, as SH intensity grows with the square of the HNP volume, for HNPs, as opposed to other fluorescent probes, size really matters. As also reported in Table 1, the Psaltis group has performed an insightful absolute quantitative derivation of the SH efficiency of an individual BaTiO3 HNP. The value is expressed in Göppert−Mayer units (1 GM = 10−50 cm4 s photon−1) to be directly compared with the two-photon excited fluorescence/ D
dx.doi.org/10.1021/mp300523e | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
Review
tested, leaving a sufficiently high number of living cells for performing cellular assays and biological evaluations. KTP caused a time-dependent decrease in cell survival in all cell lines, an effect even more pronounced in nontumoral BEAS-2B cells. Conversely, cell exposure to ZnO HNPs was found highly cytotoxic, after just 5 h incubation. The effects on cell survival after 72 h exposure of KNbO3, LiNbO3, and BaTiO3 NPs at 50 μg/mL indicate that these HNPs do not exhibit a major impact on cell survival even with a long exposure. Very similar trends were extracted by testing the same HNP series on humanderived breast cancer cells (MDA-MB-231, MCF-7, MDA-MB436) and MCF-10A human-derived nontumoral cells.39 The HNPs under scrutiny hemolyze human red blood cells in a range of 4.6% to 7%, KNbO3 HNPs being the least hemolytic of the series. For a comparison, HNPs were found to be less hemolytic than amorphous silica NPs,40 their effect being comparable to that of mesoporous silica nanoparticles (MSN).41
Figure 5. The interference pattern generated by simultaneously exciting the SH of two neighboring HNPs is a clear proof of their coherent response. Data from ref 17.
■
UPCONVERSION NPS, NOBLE METAL NPS, AND HNPS In the light of the increasing interest in the bionanotechnological approach of upconverting nanoparticles2 (UNPs) and to avoid any confusion for the reader, it is important at this stage to establish the main differences between HNPs and UNPs. The basic structure of a UNP is that of an inorganic lattice doped with rare-earth ions. The simplest photoexcitation diagram of such a system is reported in Figure 1e. It comprises two sequential (one-photon) absorption events promoting an electron into real levels of the rare-earth dopant.1 Strictly speaking, and in contrast to what has been stated in some publications, this process cannot be considered nonlinear as it is not governed by χ(n≥2) response. The involvement of real energy levels implicitly spoils this process for many of the characteristics discussed here for HNPs, in particular the properties related to wavelength flexibility and coherence. On the other hand, these NPs can be excited at lower power densities, not necessarily by ultrafast lasers, because the simultaneous presence of two photons is not required for their sequential two-step excitation. UNPs are usually excited around 980 nm giving rise to sharp (10−20 nm bandwidth)
providing nanomaterials with limited toxicity and good biocompatibility. By a rapid inspection of the chemical compositions of the nanomaterials so far proposed, the absence of heavy metals like cadmium seems to favor HNPs with respect to semiconductor NPs. To quantitatively assess their biocompatibility, Staedler et al. carried out a thorough biological evaluation, including cytotoxicity and hemolysis assays. The viability of cells exposed to different HNPs was assessed by MTT (3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide) test. The latter was applied on three human lung cancer cell lines, one human adenocarcinoma cell line (A549) derived from alveolar epithelial type II cells, one adenosquamous carcinoma cell line (HTB-178), and one lung squamous carcinoma cell line (HTB-182).35−37 Finally, nontumoral BEAS-2B cells were also tested.38 The results in Table 2 indicate that BaTiO3, KNbO3, and LiNbO3 HNPs show a weak cytotoxicity in all the samples after 5 or 24 h exposure. Globally, HNPs at 50 μg/mL cause a 20− 30% decrease in cell survival in the majority of the cell lines
Table 2. Cytotoxic Effect after 5 h and 24 h Exposure of Human Lung-Derived Cells to KNbO3, LiNbO3, BaTiO3, KTP, and ZnO Nanoparticles (50 μg/mL)a 5 h Exposure cell survival, % KNbO3 A549 HTB-182 HTB-178 BEAS-2B
81.2 81.6 74.3 84.9
± ± ± ±
4.4 6.7 2.1 5.3
LiNbO3
BaTiO3
± ± ± ±
91.0 ± 5.3 87.1 ± 9.4 87.7 ± 12.7 93.1 ± 0.6
78.6 82.6 90.7 87.9
3.7 2.7 8.8 7.1
KTP 76.3 67.8 78.4 63.2
± ± ± ±
4.2 2.3 6.2 5.6
ZnO 57.7 41.6 27.8 29.9
± ± ± ±
7.8 7.8 6.7 6.7
24 h Exposure cell survival, % KNbO3 A549 HTB-182 HTB-178 BEAS-2B
82.0 73.7 74.2 81.7
± ± ± ±
3.6 6.7 1.0 4.0
LiNbO3 84.7 80.9 83.6 93.9
± ± ± ±
BaTiO3
5.7 3.5 3.3 4.9
84.7 85.5 91.8 92.1
± ± ± ±
3.8 1.3 1.7 3.7
KTP 67.8 61.4 76.8 55.8
± ± ± ±
ZnO 2.5 6.0 7.7 5.3
17.7 ± 5.3 4.5 ± 1.0 6.8 ± 1.2 9.9 ± 3.3
A549, HTB-182, and HTB-178: human lung cancer cells. BEAS-2B: nontumoral lung-derived cells. Results are the mean ± SD of triplicates of two independent experiments. Data from ref 23.
a
E
dx.doi.org/10.1021/mp300523e | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
Review
over longer times.50,51 The cells tested look healthy, corroborating the results determined by MTT. Relatively large SiC NPs (50−80 nm) have been assessed as HNPs by the Fraser group.14 In other studies not specifically focused on their nonlinear optical properties, Alain Geloen and co-workers have shown that ≤5 nm SiC NPs do not remain at the membranes but they tend to penetrate inside cells. Interestingly, the behavior of these small SiC NPs exhibits a very strong dependence on the particle surface charge.52 In a series of experiments, the authors have modified the value and sign of the NP surface charge by covalent grafting of amino groups onto the carboxylic acid functionalities which naturally cover their surface. As reported in Figure 7, they showed that
emission features in the visible region of the spectrum, depending on the specific rare-earth element employed and its concentration. Finally, a complete overview on the use of NPs for nonlinear bioimaging cannot overlook the works on noble metal NPs. These particles, which are of paramount importance for sensing applications,42 are usually imaged by detecting their linear scattering by dark field microscopy43 or by photothermal imaging.44 Since 2000, they have also been proposed as nonlinear microscopy markers. The nonlinear response, which can be exploited using ultrafast pulse excitation, in this case is mostly a surface effect, with minor bulk contributions.45 The nonlinear signal is enhanced in correspondence with NP surface plasmon resonance (SPR), an aspect which limits their efficient nonlinear excitation to the SPR spectral region. In the context of nonlinear bioimaging, noble metal NPs have been used to resonantly enhance the fluorescence response of neighboring molecular probes46 or used directly as third harmonic emitters by Yelin et al.47 The interest of this approach for cancer cell detection48 is motivated also by the possibility of a concomitant use of metal NPs as photothermal therapy agents.49
■
HNP APPLICATION EXAMPLES AND PROSPECTIVE BIOMEDICAL USE In the following section, some of the latest demonstrations of HNPs for bioimaging and diagnostics are reviewed and their potential for therapy is briefly discussed. The reader should be aware that the HNP approach is very recent and the first biomedical-oriented applications date back only a few years. Cell Culture Labeling. One possible application of HNPs is as biomarkers for cell cultures. Figure 6 displays BEAS-2B
Figure 7. Fluorescence and visible microscopy images of 3T3-L1 cells: not labeled (a), labeled with negatively charged SiC-CO2H SiC NPs (zeta potential −30 mV) (b), labeled with quasi neutral SiC-NH2 SiC NPs (zeta potential +4 mV) (c), and labeled with positively charged SiC-NH2 SiC NPs (zeta-potential +100 mV) (d). Reprinted with permission from ref 52. Copyright 2012 IOP Publishing.
Figure 6. Sliced views from a z-stack of BEAS-2B cells exposed to KNbO3 HNPs. The green-yellow channel corresponds to the fluorescence of FM1-43FX membrane dye; the blue spots are individual HNPs. Data from ref 23.
negatively charged SiC NPs concentrate inside cell nuclei, close to neutrally charged SiC NPs that are present in both cytoplasm and nuclei, while positively charged SiC NPs are exclusively localized in the cytoplasm. In a second publication, the same research team provided evidence that the uptake of NPs and their intranucleus concentration depend on cell proliferation. This is a promising observation toward increased specificity for SiC cancer treatment or drug delivery.53 Clearly, to gather molecular selectivity HNPs need an ad hoc surface functionalization. Generally speaking, the surface functionalization of HNPs does not differ from that of other types of nanoparticles. A common preparation step shared by various protocols is to first coat by a silane layer the HNP
cells exposed for 5 h to PEG-stabilized KNbO3 at 50 μg/mL concentration. Even in the absence of a specific functionalization of the HNP surface, one can observe a reasonable association with cells of the HNPs, the latter have the tendency to remain confined at the membranes, as confirmed by the slice views, apart from a few exceptions of HNPs within the cytoplasm. This preferential association with the membranes is consistent with the short contact time, as it is known that uptake by endocytosis of solid core NPs typically takes place F
dx.doi.org/10.1021/mp300523e | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
Review
surface. Hsieh et al. demonstrated the surface functionalization of BaTiO3 HNPs by covalently binding IgG secondary antibodies to amine groups immobilized on their surface.54 The authors used this preparation to label specific primary antibodies. They observed less than 5% nonspecific labeling on a primary-antibody microarray and subsequently performed specific marking of cell membrane proteins. Very recently, a detailed protocol describing the coating of BaTiO3 with poly(ethylene glycol) and cross-linking to a biotin-linked moiety using click chemistry has been published by the Pantazis group.55 Stem Cell Tracking. The greatest advantages of HNPs over fluorescent labels comprise the possibility to work in the infrared and the striking photostability over long periods of these imaging markers. The integration of stem cells into transplanted tissues is a hot topic in regenerative medicine research, specifically requiring long-term monitoring of thick tissue samples. Magouroux et al. have recently established a protocol, to expose mouse embryonic stem cells (line CGR8) to HNPs. Cell differentiation was performed using the hanging drop method: embryoid bodies were formed within hanging drops of BHK21 medium, then collected, cultured, and finally plated onto gelatin-coated glass coverslips.56 Two different alternatives to expose the samples to HNPs were tested: in one case, embryonic stem cells were loaded with the HNPs at the beginning of their differentiation (formation of hanging drops); in the other one, the embryoid bodies were incubated with HNPs at day four of differentiation. In both cases, it was observed that cells in contact with HNPs were able to differentiate and could turn into neuronal rosettes as well as into contractile cardiac clusters displaying rhythmic autonomous contractions. No difference was detected in terms of percentage of beating clusters compared to unlabeled samples.57,58 In the same study, the rhythmic cluster movements were filmed at high speed (4 frames/s) over an area of 640 × 640 μm2 detecting SH radiation from isolated HNPs. The resulting movie provided three-dimensional information at sub-micrometric resolution about the contraction pattern of the cardiomyocytes. Starting from these observations, a thorough analysis of the HNP individual movements was carried over: first the time-varying displacement of each individual particle during the contractions was extracted. The arrows in panel b of Figure 8 indicate direction and length of in-plane movements. The absolute distances traveled by several HNPs during the rhythmic embryoid body contractions are plotted as a function of time in panel c, which shows a clear periodic and synchronous pattern for all HNPs. The out-of-plane motion acquired from the three-dimensional sectioning capability of nonlinear microscopy is displayed in panel b. In-plane movements were estimated to be between 1 and 5 μm, outof-plane displacements typically 1.2 μm light, followed by the HNP-mediated activation of the sensitizer molecules using visible and near-infrared wavelengths, ≤800 nm.
Figure 10. HNP detection in fetal calf serum. Cross-correlation curves (simultaneous second and third harmonic detection) for KNbO3 particles (blue line) and pure water, and microspheres in serum (flat response, overlapping traces). The graph demonstrates the excellent specificity to HNPs of this combined measurement. Data from ref 22.
simultaneous detection of second and third harmonics from microsphere-containing serum bears no signal as indicated by the flat line at 0, as compared to very distinct and strong response by HNPs plotted in blue. Toward Theranostics Applications. The therapeutical applications of HNPs can be exploited following two very different strategies. A first possible, relatively standard, approach is based on the specific (biochemical) interaction of some nanomaterials with living cells. The second one is based on the unique physical properties of HNPs and relies on the possibility of generating in situ new wavelengths which can interact with cells or selectively photoactivating target molecules at the surface of the NPs. Among the list of materials used for HNPs, so far two have shown strong effects on cells: SiC and ZnO. (The reader should note that most of the works on SiC and ZnO reviewed in this section were not focused on the nonlinear optical response of these materials.) A study has investigated the cellular localization and influence on viability and proliferation of the former nanomaterial on oral squamous carcinoma (AT84 and HSC) and immortalized cell lines (S-G). It was observed that SiC localize into the nuclei, and that the presence of these NPs in culture medium provoke morphological changes in cultured cells.60 The authors interestingly observed dose- and time-dependent selective cytotoxicity on cancer versus immortalized cells in vitro. As discussed in a previous section, the same research group has proven that the intracellular localization of SiC NPs can be tuned by modifying their surface charge.52 Compared to SiC, the literature concerning biological effects of ZnO NPs is much richer, and a complete survey goes beyond the scope of this review paper.61,62 According to various authors, ZnO cytotoxic effect is related to its partial dissolution and induction of oxidative stress. This explanation is in full agreement with a series of experimental findings indicating the absence of NPs after a few hours incubation with cells, as reported in Figure 11b. In this
■
CONCLUSIONS HNPs have been proposed very recently as biomarkers for nonlinear imaging. Thanks to the benefits associated with their optical contrast mechanism, which include wavelength flexibility, low cytotoxicity, and long-term photostability, they can complement fluorescent/luminescent nanoprobes in several demanding applications. In this focused review, some demonstrations provided by a restricted number of research groups worldwide, active in this developing field, have been highlighted, spanning from in vitro cell monitoring to the first proofs of in vivo imaging. Although these works have proven that HNPs are already mature for specific applications in biological research and nanomedecine, the deployment of their full potential is yet to come. The next task for physicists and H
dx.doi.org/10.1021/mp300523e | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
Review
(11) Nakayama, Y.; Pauzauskie, P. J.; Radenovic, A.; Onorato, R. M.; Saykally, R. J.; Liphardt, J.; Yang, P. D. Tunable nanowire nonlinear optical probe. Nature 2007, 447, 1098−1101. (12) Aufray, M.; Menuel, S.; Fort, Y.; Eschbach, J.; Rouxel, D.; Vincent, B. New Synthesis of Nanosized Niobium Oxides and Lithium Niobate Particles and Their Characterization by XPS Analysis. J. Nanosci. Nanotechnol. 2009, 9, 4780−4785. (13) Hsieh, C. L.; Grange, R.; Pu, Y.; Psaltis, D. Three-dimensional harmonic holographic microcopy using nanoparticles as probes for cell imaging. Opt. Express 2009, 17, 2880−2891. (14) Pantazis, P.; Maloney, J.; Wu, D.; Fraser, S. E. Second harmonic generating (SHG) nanoprobes for in vivo imaging. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 14535−14540. (15) Sandeau, N.; Le Xuan, L.; Chauvat, D.; Zhou, C.; Roch, J. F.; Brasselet, S. Defocused imaging of second harmonic generation from a single nanocrystal. Opt. Express 2007, 15, 16051−16060. (16) Le Xuan, L.; Zhou, C.; Slablab, A.; Chauvat, D.; Tard, C.; Perruchas, S.; Gacoin, T.; Villeval, P.; Roch, J. F. Photostable secondharmonic generation from a single KTiOPO4 nanocrystal for nonlinear microscopy. Small 2008, 4, 1332−1336. (17) Baumner, R.; Bonacina, L.; Enderlein, J.; Extermann, J.; FrickeBegemann, T.; Marowsky, G.; Wolf, J. P. Evanescent-field-induced second harmonic generation by noncentrosymmetric nanoparticles. Opt. Express 2010, 18, 23218−23225. (18) Johnson, J. C.; Yan, H. Q.; Schaller, R. D.; Petersen, P. B.; Yang, P. D.; Saykally, R. J. Near-field imaging of nonlinear optical mixing in single zinc oxide nanowires. Nano Lett. 2002, 2, 279−283. (19) Kachynski, A. V.; Kuzmin, A. N.; Nyk, M.; Roy, I.; Prasad, P. N. Zinc oxide nanocrystals for nonresonant nonlinear optical microscopy in biology and medicine. J. Phys. Chem. C 2008, 112, 10721−10724. (20) Extermann, J.; Bonacina, L.; Cuna, E.; Kasparian, C.; Mugnier, Y.; Feurer, T.; Wolf, J. P. Nanodoublers as deep imaging markers for multi-photon microscopy. Opt. Express 2009, 17, 15342−15349. (21) Extermann, J.; Bejot, P.; Bonacina, L.; Mugnier, Y.; Le Dantec, R.; Mazingue, T.; Galez, C.; Wolf, J. P. An inexpensive nonlinear medium for intense ultrabroadband pulse characterization. Appl. Phys. B 2009, 97, 537−540. (22) Geissbuehler, M.; Bonacina, L.; Shcheslavskiy, V.; Bocchio, N. L.; Geissbuehler, S.; Leutenegger, M.; Marki, I.; Wolf, J. P.; Lasser, T. Nonlinear Correlation Spectroscopy (NLCS). Nano Lett. 2012, 12, 1668−1672. (23) Staedler, D.; et al. Harmonic Nanocrystals for Biolabeling: A Survey of Optical Properties and Biocompatibility. ACS Nano 2012, 6, 2542−2549. (24) Grange, R.; Lanvin, T.; Hsieh, C. L.; Pu, Y.; Psaltis, D. Imaging with second-harmonic radiation probes in living tissue. Biomed. Opt. Express 2011, 2, 2532−2539. (25) Balu, M.; Baldacchini, T.; Carter, J.; Krasieva, T. B.; Zadoyan, R.; Tromberg, B. J. Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media. J. Biomed. Opt. 2009, 14 (1), 010508. (26) Le Dantec, R.; Mugnier, Y.; Djanta, G.; Bonacina, L.; Extermann, J.; Badie, L.; Joulaud, C.; Gerrmann, M.; Rytz, D.; Wolf, J. P.; Galez, C. Ensemble and Individual Characterization of the Nonlinear Optical Properties of ZnO and BaTiO3 Nanocrystals. J. Phys. Chem. C 2011, 115, 15140−15146. (27) Albota, M. A.; Xu, C.; Webb, W. W. Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm. Appl. Opt. 1998, 37, 7352−7356. (28) Heikal, A. A.; Hess, S. T.; Webb, W. W. Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid-base specificity. Chem. Phys. 2001, 274, 37−55. (29) Larson, D. R.; Zipfel, W. R.; Williams, R. M.; Clark, S. W.; Bruchez, M. P.; Wise, F. W.; Webb, W. W. Water-soluble quantum dots for multiphoton fluorescence imaging in vivo. Science 2003, 300, 1434−1436.
optical engineers is to develop new detection techniques based on the wealth of information conveyed by their coherent response, which can lead to methods with enhanced sensitivity and selectivity in optical imaging. On the other hand, the expertise of biochemists and biologists is necessary for establishing protocols for targeted HNP localization and NPmediated photoactivation of drug molecules, exploiting the possibility of using different excitation wavelengths for combining imaging and for triggering of the photosensitizers.
■
AUTHOR INFORMATION
Corresponding Author
*University of Geneva, Applied Physics, 22 chemin de Pinchat, CH-1211 Geneva 4, Switzerland. E-mail: luigi.bonacina@unige. ch. Tel: +41 22 379 05 08. Fax: +41 22 379 05 59. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The author is very grateful to all the scientific partners of European FP7 NAMDIATREAM project (http://www. namdiatream.eu), and in particular to A. Rogov, Th. Magouroux, D. Staedler, J. Extermann, and Mary Matthews for helping with the image preparation and critical reading of the manuscript and to Rachel Grange (University of Jena) and Vladymir Lysenko (INSA Lyon) for kindly accepting the reproduction of their published material. The financial support of the European Commission under Project NMP4-LA-2010246479 is also acknowledged.
■
REFERENCES
(1) Wang, F.; Banerjee, D.; Liu, Y. S.; Chen, X. Y.; Liu, X. G. Upconversion nanoparticles in biological labeling, imaging, and therapy. Analyst 2010, 135, 1839−1854. (2) Haase, M.; Schafer, H. Upconverting Nanoparticles. Angew. Chem., Int. Ed. 2011, 50, 5808−5829. (3) Denk, W.; Strickler, J. H.; Webb, W. W. 2-Photon Laser Scanning Fluorescence Microscopy. Science 1990, 248, 73−76. (4) Perry, S. W.; Burke, R. M.; Brown, E. B. Two-Photon and Second Harmonic Microscopy in Clinical and Translational Cancer Research. Ann. Biomed. Eng. 2012, 40, 277−291. (5) Durfee, C. G.; Storz, T.; Garlick, J.; Hill, S.; Squier, J. A.; Kirchner, M.; Taft, G.; Shea, K.; Kapteyn, H.; Murnane, M.; Backus, S. Direct diode-pumped Kerr-lens mode-locked Ti:sapphire laser. Opt. Express 2012, 20, 13677−13683. (6) Aviles-Espinosa, R.; Filippidis, G.; Hamilton, C.; Malcolm, G.; Weingarten, K. J.; Sudmeyer, T.; Barbarin, Y.; Keller, U.; Santos, S.; Artigas, D.; Loza-Alvarez, P. Compact ultrafast semiconductor disk laser: targeting GFP based nonlinear applications in living organisms. Biomed. Opt. Express 2, 739−747. (7) Roth, P. W.; Maclean, A. J.; Burns, D.; Kemp, A. J. Directly diodelaser-pumped Ti:sapphire laser. Opt. Lett. 2009, 34, 3334−3336. (8) Sibbett, W.; Lagatsky, A. A.; Brown, C. T. A. The development and application of femtosecond laser systems. Opt. Express 2012, 20, 6989−7001. (9) Bonacina, L.; Mugnier, Y.; Courvoisier, F.; Le Dantec, R.; Extermann, J.; Lambert, Y.; Boutou, V.; Galez, C.; Wolf, J. P. Polar Fe(IO3)(3) nanocrystals as local probes for nonlinear microscopy. Appl. Phys. B 2007, 87, 399−403. (10) Ladj, R.; El Kass, M.; Mugnier, Y.; Le Dantec, R.; Fessi, H.; Galez, C.; Elaissari, A. SHG Active Fe(IO3)3 Particles: From Spherical Nanocrystals to Urchin-Like Microstructures through the AdditiveMediated Microemulsion Route. Cryst. Growth Des. 2012, 12 (11), 5387−5395. I
dx.doi.org/10.1021/mp300523e | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
Review
spheres in photothermal cancer therapy. Lasers Surg. Med. 2007, 39, 747−753. (50) Cengelli, F.; Voinesco, F.; Juillerat-Jeanneret, L. Interaction of cationic ultrasmall superparamagnetic iron oxide nanoparticles with human melanoma cells. Nanomedicine 2010, 5, 1075−1087. (51) Kenzaoui, B. H.; Bernasconi, C. C.; Hofmann, H.; JuilleratJeanneret, L. Evaluation of uptake and transport of ultrasmall superparamagnetic iron oxide nanoparticles by human brain-derived endothelial cells. Nanomedicine 2012, 7, 39−53. (52) Serdiuk, T.; Alekseev, S. A.; Lysenko, V.; Skryshevsky, V. A.; Geloen, A. Charge-driven selective localization of fluorescent nanoparticles in live cells. Nanotechnology 2012, 23 (31), 315101. (53) Serdiuk, T.; Lysenko, V.; Mognetti, B.; Skryshevsky, V.; Géloën, A. Impact of cell division on intracellular uptake and nuclear targeting with fluorescent SiC-based nanoparticles. J. Biophotonics 2012, DOI: 10.1002/jbio.201200066. (54) Hsieh, C. L.; Grange, R.; Pu, Y.; Psaltis, D. Bioconjugation of barium titanate nanocrystals with immunoglobulin G antibody for second harmonic radiation imaging probes. Biomaterials 2010, 31, 2272−2277. (55) Culić-Viskota, J.; Dempsey, W. P.; Fraser, S. E.; Pantazis, P. Surface functionalization of barium titanate SHG nanoprobes for in vivo imaging in zebrafish. Nat. Protoc. 2012, 7, 1618−1633. (56) Magouroux, T.; Extermann, J.; Hoffmann, P.; Mugnier, Y.; Le Dantec, R.; Jaconi, M. E.; Kasparian, C.; Ciepielewski, D.; Bonacina, L.; Wolf, J.-P. High-Speed Tracking of Murine Cardiac Stem Cells by Harmonic Nanodoublers. Small 2012, 8, 2752−2756. (57) Li, J.; Puceat, M.; Perez-Terzic, C.; Mery, A.; Nakamura, K.; Michalak, M.; Krause, K. H.; Jaconi, M. E. Calreticulin reveals a critical Ca(2+) checkpoint in cardiac myofibrillogenesis. J. Cell Biol. 2002, 158, 103−113. (58) Maltsev, V. A.; Wobus, A. M.; Rohwedel, J.; Bader, M.; Hescheler, J. Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents. Circ. Res. 1994, 75, 233−244. (59) Williams, R. M.; Zipfel, W. R.; Webb, W. W. Interpreting second-harmonic generation images of collagen I fibrils. Biophys. J. 2005, 88, 1377−1386. (60) Mognetti, B.; Barberis, A.; Marino, S.; Di Carlo, F.; Lysenko, V.; Marty, O.; Geloen, A. Preferential Killing of Cancer Cells Using Silicon Carbide Quantum Dots. J. Nanosci. Nanotechnol. 2010, 10, 7971−7975. (61) Hanley, C.; Layne, J.; Punnoose, A.; Reddy, K. M.; Coombs, I.; Coombs, A.; Feris, K.; Wingett, D. Preferential killing of cancer cells and activated human T cells using ZnO nanoparticles. Nanotechnology 2008, 19 (29), 295103. (62) Taccola, L.; Raffa, V.; Riggio, C.; Vittorio, O.; Iorio, M. C.; Vanacore, R.; Pietrabissa, A.; Cuschieri, A. Zinc oxide nanoparticles as selective killers of proliferating cells. Int. J. Nanomed. 2011, 6, 1129− 1140. (63) Li, J. Y.; Guo, D. D.; Wang, X. M.; Wang, H. P.; Jiang, H.; Chen, B. A. The Photodynamic Effect of Different Size ZnO Nanoparticles on Cancer Cell Proliferation In Vitro. Nanoscale Res. Lett. 2010, 5, 1063−1071. (64) Ungun, B.; Prud’homme, R. K.; Budijono, S. J.; Shan, J. N.; Lim, S. F.; Ju, Y. G.; Austin, R. Nanofabricated upconversion nanoparticles for photodynamic therapy. Opt. Express 2009, 17, 80−86. (65) Wang, C.; Tao, H. Q.; Cheng, L.; Liu, Z. Near-infrared light induced in vivo photodynamic therapy of cancer based on upconversion nanoparticles. Biomaterials 2011, 32, 6145−6154. (66) Zhou, A. G.; Wei, Y. C.; Wu, B. Y.; Chen, Q.; Xing, D.; Pyropheophorbide, A. and c(RGDyK) Comodified Chitosan-Wrapped Upconversion Nanoparticle for Targeted Near-Infrared Photodynamic Therapy. Mol. Pharmaceutics 2012, 9, 1580−1589.
(30) Hsieh, C. L.; Grange, R.; Pu, Y.; Psaltis, D. Three-dimensional harmonic holographic microcopy using nanoparticles as probes for cell imaging: erratum. Opt. Express 2010, 18, 3456−3457. (31) Hsieh, C. L.; Pu, Y.; Grange, R.; Laporte, G.; Psaltis, D. Imaging through turbid layers by scanning the phase conjugated second harmonic radiation from a nanoparticle. Opt. Express 2010, 18, 20723−20731. (32) Kanga, S.-Y.; Shinb, J.; Jeonga, K.-Y.; Leea, C.-M.; Yeec, K.-J.; Leea, Y.-H. Metallic nanocavity-enhanced second harmonic generation from a KNbO3 nonlinear nanowire. Photonics Nanostruct.: Fundam. Appl. 2012, 10, 534−539. (33) Richter, J.; Steinbrück, A.; Pertsch, T.; Tünnermann, A.; Grange, R. Plasmonic Core-Shell Nanowires for Enhanced Second-Harmonic Generation. Plasmonics 2012, DOI: 10.1007/s11468-012-9429-2. (34) Pu, Y.; Grange, R.; Hsieh, C. L.; Psaltis, D. Nonlinear Optical Properties of Core-Shell Nanocavities for Enhanced Second-Harmonic Generation. Phys. Rev. Lett. 2010, 104. (35) Hukkanen, J.; Lassila, A.; Paivarinta, K.; Valanne, S.; Sarpo, S.; Hakkola, J.; Pelkonen, O.; Raunio, H. Induction and regulation of xenobiotic-metabolizing cytochrome P450s in the human A549 lung adenocarcinoma cell line. Am. J. Respir. Cell Mol. Biol. 2000, 22, 360− 366. (36) Hellmann, G. M.; Fields, W. R.; Doolittle, D. J. Gene expression profiling of cultured human bronchial epithelial and lung carcinoma cells. Toxicol. Sci. 2001, 61, 154−163. (37) Yang, F.; Xiao, Z. Q.; Zhang, X. Z.; Li, C.; Zhang, P. F.; Li, M. Y.; Chen, Y.; Zhu, G. Q.; Sun, Y.; Liu, Y. F.; Chen, Z. C. Identification of tumor antigens in human lung squamous carcinoma by serological proteome analysis. J. Proteome Res. 2007, 6, 751−758. (38) Nichols, W. K.; Terry, C. M.; Cutler, N. S.; Appleton, M. L.; Jesthi, P. K.; Yost, G. S. Oxidation at C-1 Controls the Cytotoxicity of 1,1-Dichloro-2,2-Bis(P-Chlorophenyl)Ethane by Rabbit and Human Lung-Cells. Drug Metab. Dispos. 1995, 23, 595−599. (39) Staedler, D. Unpublished results. 2012. (40) Slowing, I. I.; Wu, C. W.; Vivero-Escoto, J. L.; Lin, V. S. Y. Mesoporous Silica Nanoparticles for Reducing Hemolytic Activity Towards Mammalian Red Blood Cells. Small 2009, 5, 57−62. (41) He, Q.; Zhang, J.; Shi, J.; Zhu, Z.; Zhang, L.; Bu, W.; Guo, L.; Chen, Y. The effect of PEGylation of mesoporous silica nanoparticles on nonspecific binding of serum proteins and cellular responses. Biomaterials 2010, 31, 1085−1092. (42) Stewart, M. E.; Anderton, C. R.; Thompson, L. B.; Maria, J.; Gray, S. K.; Rogers, J. A.; Nuzzo, R. G. Nanostructured plasmonic sensors. Chem. Rev. 2008, 108, 494−521. (43) Wax, A.; Sokolov, K. Molecular imaging and darkfield microspectroscopy of live cells using gold plasmonic nanoparticles. Laser Photonics Rev. 2009, 3, 146−158. (44) Govorov, A. O.; Richardson, H. H. Generating heat with metal nanoparticles. Nano Today 2007, 2, 30−38. (45) Bachelier, G.; Butet, J.; Russier-Antoine, I.; Jonin, C.; Benichou, E.; Brevet, P. F. Origin of optical second-harmonic generation in spherical gold nanoparticles: Local surface and nonlocal bulk contributions. Phys. Rev. B 2010, 82. (46) Peleg, G.; Lewisyk, A.; Bouevitchy, O.; Loewx, L.; Parnasz, D.; Linialz, M. Gigantic optical non-linearities from nanoparticle-enhanced molecular probes with potential for selectively imaging the structure and physiology of nanometric regions in cellular systems. Bioimaging 1996, 4, 215−224. (47) Yelin, D.; Oron, D.; Thiberge, S.; Moses, E.; Silberberg, Y. Multiphoton plasmon-resonance microscopy. Opt. Express 2003, 11, 1385−1391. (48) Tai, S. P.; Wu, Y.; Shieh, D. B.; Chen, L. J.; Lin, K. J.; Yu, C. H.; Chu, S. W.; Chang, C. H.; Shi, X. Y.; Wen, Y. C.; Lin, K. H.; Liu, T. M.; Sun, C. K. Molecular imaging of cancer cells using plasmonresonant-enhanced third-harmonic-generation in silver nanoparticles. Adv. Mater. 2007, 19, 4520. (49) Huang, X.; Qian, W.; El-Sayed, I. H.; El-Sayed, M. A. The potential use of the enhanced nonlinear properties of gold nanoJ
dx.doi.org/10.1021/mp300523e | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
