Article pubs.acs.org/journal/apchd5
Subcellular Optogenetics Enacted by Targeted Nanotransformers of Near-Infrared Light Artem Pliss,†,‡ Tymish Y. Ohulchanskyy,†,‡,∥ Guanying Chen,†,‡ Jossana Damasco,‡ Caroline E. Bass,§ and Paras N. Prasad*,‡,∥ ‡
Institute for Lasers, Photonics, and Biophotonics and Department of Chemistry, and §Department of Pharmacology and Toxicology, Jacobs School of Medicine, University at Buffalo, State University of New York, Buffalo, New York 14214, United States ∥ College of Optoelectronic Engineering, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, Shenzhen University, 518060, Shenzhen, People’s Republic of China S Supporting Information *
ABSTRACT: Light activation of photoswitchable molecules enables control over the course of physicochemical processes with high spatiotemporal precision, offering revolutionizing potential in multiple areas of contemporary biomedicine. Yet, application of this technology in live organisms remains severely limited due to the reliance on visible light that has poor penetration in biological tissues. Herein, we introduce highly efficient upconversion nanoparticles (UCNPs) as photon nanotransformers that intracellularly convert tissuepenetrating, near-infrared light into visible light required for photoactivation. The core/shell nanoparticles described here are determined to be about six times brighter in the blue range than the canonical hexagonal (NaYF4:Yb3+30%/Tm3+0.5%)/NaYF4 core/shell UCNPs with record efficiency. An application of such efficient photon nanotransformers can significantly advance optogenetics technology, wherein the signaling of genetically modified neurons is controlled through interaction of visible light with optogenetic proteins inserted into the cell membranes. We demonstrate that our photon nanotransformers, targeted to cultured cells, enable optogenetic activation with incident nearinfrared light. The resulting membrane potential modulation by ion channel activity is probed by Ca 2+ sensitive dye. In contrast to conventional optogenetic approaches involving unselective activation of optogenetic proteins in the cellular volume with incident light irradiation, the upconverted light generated in situ by intracellular UCNPs, activates the optogenetic proteins in close vicinity to the nanoparticles, thus, providing a high subcellular precision of photoactivation. KEYWORDS: optogenetics, near-infrared light, upconversion nanoparticles, ChR2, Ca2+ imaging
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which offers unsurpassed spatiotemporal precision for control of live cell functioning.27,28 Among the most important and rapidly developing biomedical applications of photochemistry is optogenetics, which is a rapidly emerging photoactivation technology on the interface between neuroscience, molecular biology and biophotonics. It enables light control over the ability of neuronal cells to generate and transmit signals.29,30 Optogenetics has all inherent advantages of photoactivation techniques, including generally noninvasive properties, high spatial resolution, and capability to alter the membrane potential on a physiologically relevant milisecond time scale, at which the neuron naturally fires, thus, enabling precise and noninvasive control over the neuron signaling.31 Optogenetic technology involves the insertion of light-gated ion channel proteins called opsins, into cellular membranes.32 Upon light
ight-triggered activation of physicochemical processes in biological matter has received increased attention in recent years due to the advantage of highly specific spatial and temporal control.1−8 Ultraviolet (UV) or visible (VIS) lightmediated photochemical reactions play a critical role in a myriad of biomedical applications7−11 that range from advanced imaging12,13 to drug delivery, that is, on-demand drug release.14−17 Photoresponsive agents include but are not limited to fluorescence imaging reporters,12,13 nanocomplexes utilized for controlled release of photocaged bioactive materials,14−17 and photoactivatable pharmaceutical drugs used for in situ therapeutic treatments.18−24 In addition to imaging and therapy, UV−vis photoactivation also enables to probe and manipulate the structure, function and subcellular distribution of photoinducible biomolecules, thus, providing a powerful tool for a range of emerging biomedical disciplines, such as synthetic biology and cell engineering.25,26 Furthermore, a number of photoactivation approaches have been developed for on-demand manipulation of gene expression, © 2017 American Chemical Society
Received: July 8, 2016 Published: February 1, 2017 806
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Figure 1. Schematic representation of the optogenetic activation of neuronal signaling, wherein ion channel protein ChR2 is inserted into cellular membrane. ChR2 opens for intracellular influx of Ca2+ and Na+ upon activation with blue light (∼470 nm), which results in membrane depolarization and neuronal signal firing. (a) ChR2 channel is inactive at dark. (b) Activation of ChR2 channel for Ca2+ and Na+ import by blue light irradiation. (c) Photon nanotransformers, internalized into the cell, upconvert incident NIR light (980 nm) into blue light at ∼470 nm for activation of the ChR2 channel.
activation, different types of opsins open for influx of either positively or negatively charged ions, such as Na+, K+, Ca2+, or Cl−, leading to changes in cell membrane potential and thus inducing either activation or suppression of cellular signaling. For instance, a commonly used opsin called channelrhodopsin2 (ChR2) is activated by blue light at ∼470 nm, which results into opening of ion pore within cellular membrane for selective entrance of Na+ and Ca2+ into the cell, leading to a membrane depolarization and firing of neuronal impulse. This capability is especially valuable for the studies of brain, where neuronal cells are linked into intricate network circuitry, which carries out specific brain functions.33 One ultimate goal of optogenetics is to learn how to alter signaling between specific neurons, which would enable to decipher the complex organization of brain and approach treatment of neural disorders and diseases at the cellular level.34 However, the application of ChR2 and many common opsins is limited, as their photoactivation requires visible light, which poorly penetrates through biological tissues due to high absorption and scattering by biomolecules.35 A commonly used approach for overcoming these limitations employs the near-infrared (NIR) light to excite photoswitchable compounds through multiphoton, typically twophoton, excitation mechanism. The NIR light within the biological window of maximum optical transparency (∼700− 1000 nm) provides considerably deeper tissue penetration than the UV or visible light.36−39 In addition, compared with the single-photon approach, the two-photon approach significantly improves a three-dimensional precision of photoactivation. However, a common challenge for two-photon optogenetics exists: the need to enlarge the excitation surface. As ChR2 has low single channel conductance, depolarization induced by photostimulating ChR2 ion channels within a standard twophoton excitation spot can exceed the threshold for action potential generation only in neurons that express high levels of opsins in membranes. Moreover, the relatively high two-photon absorption cross section of ChR2 and the long lifetime of the excited states40 cause fast saturation of the channels, preventing the increment of neuronal depolarization by simply raising the excitation power. To improve the efficiency of two-photon stimulation, different solutions (i.e., scanning and parallel excitation techniques) are proposed to extend the excitation area, complicating the in vivo applications of two-photon optogenetics.41
Another rapidly emerging approach for photoactivation in biological tissues involves upconversion of NIR light to visible emission, using rare earth doped upconversion nanoparticles (UCNPs).36,42−44 They employ real ladder-like energy levels of trivalent rare-earth ions embedded in an appropriate inorganic host lattice to implement frequency conversion for the incident light. The efficiency of the upconversion (UC) process is typically several orders of magnitude higher than that of conventional multiphoton absorption induced fluorescence, which involves virtual intermediate energy levels. As a result, UCNPs can be efficiently excited by various sources, including a low-cost continuous-wave (CW) diode laser, providing a practical advantage over multiphoton excitation, which can be only performed with ultrashort pulsed lasers. The efficiency of current UCNPs is dictated by two factors: (i) The type and concentration of rare-earth dopants within one single nanoparticle, directly influencing energy transfer rates; (ii) Surfacerelated deactivations, which can produce pronounced quenching effects on the UC photoluminescence (PL). With regard to the latter factor, surface quenching is enhanced by the high surface-to-volume ratio at the nanometer scale, as rare-earth dopants on surface are exposed to the quenching factors in the surroundings.42−44 On the downside, the luminescence quantum yield of established UCNPs, for example, for the NIR-to-blue upconversion emission, does not exceed 3.5%,45 which limits the utility of UCNPs in biological systems. Evidently, engineering UCNPs with high upconversion photoluminescence (UCPL) efficiency provides an effective way to fuel the field of photocontrol of various physicochemical processes with a precisely defined manner. To overcome notorious limitations of multiphoton activation, UCNPs have been recently applied as component of cellular substrate, to upconvert NIR into visible light for activation of opsins46,47 and even for internalization in small organisms, in C. elegans model.48 In a different experimental system, UCNPs were tested for regulation of a complex fusion macromolecule composed of photoswitch protein LOV2 and Ca2+ membrane ion channel. It was shown that targeting of UCNPs to the extracellular domain of the transmembrane protein fusion permits to activate channel with NIR light and induce expression of Ca2+ responsive genes.49 Building on the recent accomplishments in nanotechnology and optogenetics, further ambitious goal is to internalize UCNPs into the cells to target 807
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Synthesis of β-Phase Core Nanoparticles. A total of 10 mL of oleic acid, 10 mL of 1-octadecene, 2 mmol sodium trifluoroacetate, and 1 mmol α-core were added into the flask. The resulting solution was then heated at 120 °C with magnetic stirring for 45 min to remove water and oxygen. The brown solution was then heated to 320 °C at a rate of about 12 °C per min under argon gas protection and kept at this temperature under vigorous stirring for 30 min. A syringe needle was used to let the argon gas out during the synthesis. The mixture was cooled to room temperature, precipitated by excess ethanol and collected by centrifugation. The precipitate was washed with ethanol several times, and the nanocrystals were dispersed in hexane. Synthesis of β-Phase Core−Shell Nanoparticles. The βphase core−shell nanoparticles were synthesized by a thermal decomposition method. A total of 0.5 mmol of RE2O3 (RE = Y) was dissolved in 10 mL of 50% trifluoroacetic acid at 95 °C in a three-neck flask. Then, the solutions were evaporated to dryness under an argon gas purge. Next, 10 mL of oleic acid, 10 mL of 1-octadecene, 2 mmol sodium trifluoroacetate, and 1 mmol β-core were added into the flask. The resulting solution was then heated at 120 °C with magnetic stirring for 45 min to remove water and oxygen. The brown solution was then heated to 320 °C at a rate of about 12 °C per min under argon gas protection and kept at this temperature under vigorous stirring for 30 min. A syringe needle was used to let the argon gas out during the synthesis. The mixture was cooled to room temperature, precipitated by excess ethanol and collected by centrifugation. The precipitate was washed with ethanol several times, and the nanocrystals were dispersed in hexane. Cell Culture, Transfection, and Optogenetic Control. HeLa cells were grown in glass-bottom dishes (Mattek, Ashland, U.S.A.) and cultured in Advanced DMEM (ThermoFisher, Grand Island, U.S.A.), supplemented with 2.5% fetal calf serum at 37 °C in a humidified atmosphere containing 5% CO2. The transfection of ChR2-YFP, was performed with 1 μg/mL of plasmid per 35 mm dish, using Lipofectamine Plus transfection reagent (ThermoFisher, Grand Island, U.S.A.). To make ChR2 sensitive to light activation, cells were incubated with 1 μM of all-trans-retinal overnight. To enable detection of intracellular Ca2+, cells were loaded with 2 μM of Asante Calcium Red (ACR) dye (Teflabs, Austin, U.S.A.). For upconversion experiments, transfected cells were loaded with UCNPs overnight. Prior to experiments, cells were extensively rinsed and imaged at 37 °C. During the light-induced activation of ChR2 opsins, cells were incubated either in DMEM or in a buffer containing 80 mM CaCl 2+, 5 mM Na+, 3 mM KCl, 135 mM N-methyl-D-glucamine, 10 mM Hepes, 20 mM glucose, adjusted at pH = 7.4. In negative control experiments cells were not incubated with of all-trans-retinal (Figure S6). Experiments on optogenetic activation were performed using a confocal/two-photon microscope (Leica TCS-SP2/AOBS) equipped with 980 nm CW laser diode. Light stimulation was applied through a 63× oil immersion or 20× air objective, with a laser scanning frequency of 1000 Hz. In control experiments cells were irradiated by 476 nm pulses excitation for gated ChR2 activation and 543 nm light for continuous excitation of ACR. In optogenetic experiments involving upconversion of NIR light, cells were irradiated with 980 nm light. In this case, the power of excitation laser was set to be 20 mW at the objective output. Correspondingly, the average excitation power density, used in experiments, was either ∼5 W/cm2
specific cellular compartments for selective regulation of local molecular processes. However, because of rather low efficiency of UCPL applications of UCNPs for intracellular regulation are still are at the early stage of development. In this study, we report a new type of core/shell UCNPs, which upconvert the incident NIR light into blue emissions with exceptional efficiency. The NaYbF4 core absorbs NIR light and the excitation energy migrates in the Yb3+ sublattice to reach the lanthanide emitters, ions of Tm3+, producing selective blue UCPL. An epitaxial shell layer of NaYF4 was deposited onto the core to passivate lattice defects on the surface and to spatially isolate the core nanoparticle from quenching centers in the surrounding medium. The UCPL output of the hexagonal core/shell (NaYbF4:Tm)/NaYF4 UCNPs presented here is exceptionally high, about 6× higher than the typically established hexagonal core/shell (NaYF4:30%Yb/0.5%Tm)/ NaYF4 UCNPs.45 Next, we demonstrated the breakthrough potential of novel UCNPs in optogenetics. We treated live cells expressing ChR2 with UCNPs conjugated with targeting ligand, folic acid (FA). After FA-conjugated UCNPs were internalized to produce upconversion, cells were irradiated with NIR light at 980 nm. We show that the upcoverted blue light activates opsins, allowing for controlling ion channel activity (Figure 1). Whereas, in previous studies, optogenetic control over the membrane potential was mediated by upconversion material present in the substrate of cultured cells,46,47 our study demonstrated that optogenetic control could be mediated by UCNPs specifically targeted to cells. Moreover, owing to their exceptional upconversion efficiency and cell targeting ability, UCNPs are shown to provide for localized activation of ChR2 with a high subcellular precision. While the direct photoactivation with 476 nm light activates a large pool of opsins in the cellular membranes, as illustrated by bursts of Ca2+ in the entire cellular volume, the upconverted light activates predominantly those opsins, which are localized closely to intracellular UCNPs. Moreover, the scale of activation is proportional to the amount of UCNPs taken up by cells. In perspective, the reported here nanophotonics approach can advance subcellular optogenetics, which is a rapidly emerging discipline focused on control of cellular functions,50 as well as provide unprecedented opportunities for noninvasive control over neuronal circuitry in central neural system of live animals.
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METHODS
Synthesis of Core−Shell UCNPs. Synthesis of α-Phase Core Nanoparticles. The α-phase core nanoparticles were synthesized by a thermal decomposition method. 0.5 mmol of RE2O3 (RE = Yb, Tm) was dissolved in 10 mL of 50% trifluoroacetic acid at 95 °C in a three-neck flask. Then, the solution was evaporated to dryness under argon gas purge. Next, 8 mL of oleic acid, 8 mL of oleylamine, 12 mL of 1octadecene, and 2 mmol sodium trifluoroacetate were added into the flask. The resulting solution was then heated at 120 °C with magnetic stirring for 45 min to remove water and oxygen. The brown solution was then heated to 300 °C at a rate of about 12 °C per min under argon gas protection and kept at this temperature under vigorous stirring for 30 min. A syringe needle was used to let the argon gas out during the synthesis. The mixture was cooled to room temperature, precipitated by excess ethanol and collected by centrifugation. The precipitate was washed with ethanol several times, and the nanocrystals were dispersed in hexane. 808
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(when 20× objective was used, scan field of 750 μm × 750 μm) or ∼35 W/cm2 (for 63× objective, 238 μm × 238 μm). Instrumentation. The size and morphology of the resulting core/shell nanoparticles were characterized by transmission electron microscopy (TEM) using a JEM-2010 microscope at an acceleration voltage of 200 kV. The powder X-ray diffraction (XRD) patterns were recorded by a Siemens D500 diffractometer, using Cu Kα radiation (λ = 0.15418 nm). The 2θ angle of the XRD spectra was recorded at a scanning rate of 5°/min. The contents of elements were analyzed with energydispersive X-ray spectroscopy (EDX) using a Hitachi SU70 Field Emission Scanning Electron Microscope with a Oxford Energy-Dispersive X-ray Spectrometer. They were more precisely quantified with an inductively coupled plasma mass spectrometry (ICP-MS) utilizing a Thermo Scientific XSERIES 2 ICPMS Single Quadrupole Mass Spectrometer with Inductively Coupled Plasma Source. Upconversion luminescence (UCL) spectra were recorded using a Fluorolog-3.11 Jobin Yvon spectrofluorometer. PL excitation was provided by a continuous-wave laser diode (Q-Photonics). All luminescence spectra have been corrected for the spectral sensitivity of the spectroscopy system using the Fluorolog spectrofluorometer calibration curve.
Figure 2. (a) Transmission electron microscopy image of the synthesized (NaYbF4:Tm3+ 0.5%)@NaYF4 core/shell UCNPs. (b) UCPL of the core NaYbF4:Tm3+ nanoparticles (black line), the designed in this study (NaYbF4:Tm3+ 0.5%)@NaYF4 core/shell UCNPs (blue line), as well as the referenced canonical hexagonal phase (NaYF4:Yb3+30%/Tm3+ 0.5%)/NaYF4 UCNPs (∼30−40 nm). Spectra were acquired under 980 nm excitation at ∼5 W/cm2.
due to its distinct growth dynamics from the hexagonal NaYF4 nanoparticles. To achieve this, we used a two-step thermolysis protocol adapted from a recent work.53 The first step is to prepare cubic phase irregular NaYbF4 nanoparticles, which are then converted into uniform hexagonal phase nanoparticles using the Ostwald-ripening confined process in the second step. A third seed-mediated epitaxial growth enables the preparation of the shell layer with the hexagonal phase. Indeed, the X-ray diffraction (XRD) investigation indicates that the as-prepared core, and the core/shell nanoparticles are both in the highly efficient hexagonal crystal phase (Supporting Information, Figure S1). Next, the optical properties of the obtained UCNPs were studied. The UC PL spectra of the resulting NaYbF4:Tm3+ 0.5% core, and core/shell (NaYbF4:Tm3+ 0.5%)@NaYF4 are displayed in Figure 2b, along with referenced canonical (NaYF4:Yb3+30%/Tm3+ 0.5%)/NaYF4 particles, which up to date have been considered to be the most efficient UCNPs, with an upconversion quantum yield of ∼3.5%.45 These nanoparticles were dispersed in hexane under identical concentrations for the comparison. The UC PL peaks at 358, 450, 470, 650, and 801 nm correspond to transitions between 1 I6 → 3F4, 1D2 → 3F4, 1G4 → 3H6, 1G4 → 3F4, and 3H4 → 3H6 of Tm3+ ions, respectively. As could be expected from the chemical formulation, the upconversion spectra of both types of UCNPs contained same peaks corresponding to the Tm3+ transitions. At the same time the upconversion efficiency of synthesized in our study UCNPs was significantly higher. Remarkably, the intensity of the 450/470 nm peaks from the core/shell (NaYbF4:Tm3+ 0.5%)@NaYF4 UCNPs was ∼1600× higher than that of the NaYbF4:Tm3+ 0.5% core particles (Supporting Information, Figure S2). We also documented a ∼6−8× higher levels of 450/470 nm UCPL in comparison with the canonical “gold standard” (NaYF4:Yb3+30%/Tm3+ 0.5%)/ NaYF4 particles (Figure 2b). We conclude that the high upconversion efficiency of the characterized here UCNPs arises from a combined effect of highly efficient energy transfers from the sensitizer Yb3+ (enhanced by Yb3+-induced to the activator Tm3+54), as well as an efficient suppression of surface-related quenching mechanisms. To confirm the later effect, the PL decay at 801 nm was measured for both samples shown in Figure 2b. In these experiments, the core/shell (NaYbF4:Tm3+ 0.5%)@NaYF4 were found to have the longest lifetime (Supporting Information, Figure S3).
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RESULTS AND DISCUSSION Characterization of UCNPs. The UCNPs produce several UCPL bands, which could be used for selective photoactivation as well as for bioimaging applications. The emission of UCNPs is determined by the selected type of lanthanide dopants or their combinations.51 In UCNPs, typically, the doped Yb3+ ions (sensitizers) absorb infrared radiation and nonradiatively transfer excitation to the doped activators X3+ (X = Er, Ho, Tm) to produce visible and ultraviolet upconversion luminescence (UCL). The NaYbF4:Tm3+ 0.5% is selected as the core nanocrystals, not only because the energy transfer efficiency between the sensitizer Yb3+ ion and the emitter Tm3+ ion is demonstrated to be the highest, but also since the NaYbF4 matrix can provide the most abundant Yb3+ ion concentration for strongest NIR excitation light harvesting at ∼980 nm, which can then transfer to Tm3+ ion to produce blue UC PL. The epitaxial β-phase (hexagonal phase) (NaYbF4:Tm3+ 0.5%)@NaYF4 core/shell UCNPs were synthesized as described in the Methods; the shell of NaYF4 was selected due to its low lattice mismatch to the NaYbF4 matrix. As shown in the transmission electron microscopic (TEM) images, the resulting UCNPs were uniform, of regular spherical shape, and ∼50 nm in the diameter (Figure 2a). Within each UCNP, a ∼30 nm electron dense core coated with a ∼10 nm light shell can be clearly resolved, demonstrating the formation of a core/shell nanostructure. We attribute this contrast to a large difference in the atomic number between the Yb3+ at the core (in the NaYbF4 host lattice) and the Y3+ at the shell (in the NaYF4 host lattice), resulting in different electron scattering cross-section. The crystallographic phase has a strong impact on the efficiency of UCPL. A lower crystal phase can favor higher UCPL efficiency, as a low symmetry crystal filed can relax more dipole forbidden nature of 4f−4f transitions. Indeed, it has been shown that NaYF4:Yb3+/Er3+ microsized particles of hexagonal phase are about ∼10× more efficient than its cubic form particles.52 Though the hexagonal phase NaYF4 nanoparticles have been prepared using various chemistry, it is nontrivial to prepare uniform Tm3+-doped hexagonal NaYbF4 nanoparticles 809
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Figure 3. Direct activation of ChR2 protein in cellular membranes with 476 nm light. (a) Cells expressing ChR2 in cellular membranes visualized by YFP tag. (b) Transmitted light images of the same cells. Influx of Ca 2+ in response of ChR2 activation with 476 nm light was measured in two rectangular Region of Interests, ROI 1 and ROI 2, as indicated. (c) Comparative measurement of fluorescence intensity in the ACR spectral region with 476 and 543 nm light on and off.
Applications of UCNPs for Optogenetic Activation of ChR2. Next, we explored the utility of the developed UCNPs for optogenetic applications, wherein a control over cellular signaling is executed via NIR-to-blue upconversion light from the UCNPs that were internalized into live cells (Figure 1c). In these experiments, cells were transfected with a genetic construct coding for fusion of ChR2 and Enhanced Yellow Fluorescent Protein (EYFP). Upon synthesis and maturation, this EYFP tethered opsin relocates to the cell membranes, hence, the successfully transfected cells could be identified by the fluorescence signal from EYFP. At the beginning, the functionality of ChR2 was validated under direct activation with blue light, using 476 nm laser source. To detect opening of ChR2 ion channels, cells were loaded with Asante Calcium Red AM (ACR), a cell permeable fluorescence indicator of Ca2+.55 ACR can be excited at ∼540 nm and generate, in the presence of Ca2+, a fluorescence emission peak at 650 nm, where no measurable interference from cellular autofluorescence is present. Changes in fluorescence intensity of Ca2+ were recorded and comparatively analyzed using two alternative techniques. In one approach, cells were placed in N-methyl-D-glucamine (NMDG) containing buffer, optimized for permeation of Ca2+ ions through ChR2 membrane channels.56 In another approach, cells remained in the culture medium during the light-induced activation of ChR2. In this approach, the physiological conditions are not altered, however, the readout of optogenetic response is not as sensitive as in the first technique. In our experiments, cells were simultaneously scanned with two laser beams, one at 476 nm to activate ChR2 channel, and the other at 543 nm to excite ACR. In this experimental design,
the 476 nm laser line was periodically switched on/off (Figure 3). The excitation pulses were spaced apart with lags of several seconds, for deactivation of ChR2 and recovery of the membrane potential. At the same time, cells were scanned continuously with the 543 nm laser line, to excite ACR for monitoring changes in the intracellular Ca2+ signal in response to the opening and closing of opsin channels. We observed that cells responded to irradiation in a timely manner with 476 nm light, demonstrating a corresponding increase of the Ca2+ signal within several tens of milliseconds (Figure 3), which is consistent with reported data.57 Furthermore, we addressed a common methodological problem of overlapping signals between most Ca2+ reporters and a broad spectrum from the YFP tag of ChR-2. Because of this overlap, the YFP signal typically contributes to the fluorescence obtained from Ca2+ dyes, which requires carrying stringent control experiments to verify that the measured changes in the fluorescence intensity exceed contribution from YFP. In our study, we performed control experiments by turning off 543 nm laser. Under these experimental conditions, the contribution of YFP signal to the spectral range (600−640 nm) of ACR emission was identified. The efficiency of light-induced activation varied from cell-to cell. Typically, the elevation of ACR signal correlates with the abundance of ChR2-YFP in the cellular membranes. At the same time, we found that changes in the intensity of ACR signal were significantly more pronounced for cells growing in the NMDG containing buffer. On the average, the changes in fluorescence signal from ACR were three to four fold higher than those from YFP, which confirmed the light-gated control of the ChR2 ion channel using 476 nm source (Figure 3). In 810
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Figure 4. Targeting UCNPs to cultured cells: FA-conjugated UCNPs (upper row) and nonconjugated UCNPs (bottom row). A striking difference between cellular uptake for the FA-conjugated and nonconjugated UCNPs is apparent.
Figure 5. (a) Activation of ChR2 in HeLa cells maintained in NMDG buffer by upconverted NIR light or by 476 nm excitation. (a−c) Microscopic images of HeLa cells expressing ChR2-YFP and loaded with UCNPs. Panels correspond to (a) ChR2-YFP, (b) UCNPs, and (c) transmitted light images, as indicated. Two regions of interest (ROI), with different amounts of UCNPs are marked as green and purple squares. The accumulation of UCNPs is seen to be much higher in ROI 1 than in ROI 2. (d, e) Activation of ChR2 by 980 nm light. (d) Changes in Ca2+ signal intensity in response to exposures to 980 nm light for two ROIs. (e) Upconversion photoluminescence measured in ROI 1 and ROI 2 simultaneously with (d). +
The upconverted PL peaks in (e) coincide with the peaks of Ca2 signal, marked by dashed lines in (d). (f) Activation of ChR2 by 476 nm light. Changes in the ACR intensity in the same cell, in response to 476 nm impulses.
cellular uptake of UCNPs, while almost no intracellular signal was found for nontargeted particles (Figure 4). These data indicate that functionalization of UCNPs for recognition of various cellular receptors may enable incorporation of nanoformulations into specific cellular types, such as various types of neurons.59,60 Using confocal laser scanning microscopy, we could resolve UCNPs inside the treated cells, at the same time cellular morphology was not noticeably affected (Figure 4). Consistently, we did not detect any significant impact of UCNPs on the cellular viability (data not shown) using standard MTS assay as described elsewhere.61,62
comparison, the cells, which were maintained in the culture medium during ChR2 activation, demonstrated 2-fold increase in ACR signal in response to 476 nm light pulses (Figure S4). Results obtained with direct ChR2 activation at 476 nm were used as a benchmark for the analysis of UCNPs mediated activation of ChR2 with NIR light in further experiments. At the following stage of our study, HeLa cells were targeted with phospholipid-coated UCNPs. To ensure an efficient uptake of UCNPs by the cells, folic acid (FA) was used as a ligand for specific targeting of cancer cells with enhanced expression of FA receptors.58 We found that FA-mediated targeting was highly effective, resulting into significant intra811
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Figure 6. Application of UCNPs in HeLa cells maintained in cell culture medium. (a−c) Microscopic panels correspond to ChR2-YFP, UCNPs, and transmission light images, as indicated. For measurements of Ca2+ influx were selected 3 ROIs in the same cell. ROI 1 and ROI 2 contained a significant amount of UCNPs, while ROI 3 was virtually devoid of UCNPs. For ROI 1 and ROI 2, an increase in signal from UCNPs leads to an increase in Ca2+-induced ACR fluorescence, which indicates ChR2 activation. In contrast, ROI 3, which almost does not contain UCNPs shows no changes in ACR fluorescence. (d, e) Measurements of Ca2+ influx in response to exposure to 980 nm light. (d) Measurements of ACR intensity. (e) Measurements of the gated upconverted light produced on UCNPs. The color-coded lines in (d) and (e) correspond to the signals from ROIs highlighted with a rectangle of the same color.
stimulation of the nontargeted cells is not expected. Furthermore, the specific targeting along with the excitation of lower power density may allow for use of UCNPs in unsaturated regime. As a result, multiphoton dependence on the excitation power can be exploited for UCPL, allowing to stimulate the selected cell populations in depth, similar to the two-photon induced optogenetics. Moreover, we found that the influx of Ca2+ ions in different parts of the cell correlated with the subcellular concentration of UCNPs (Figures 5 and S7). Changes in Ca2+ signal intensity were recorded only in proximity of UCNPs, while no activation of ChR2 was detected in the regions of the same cell, which did not contain a significant number of these nanoparticles (Figure 5d,e). In comparison with direct activation of ChR2 by blue light, which induces very significant changes in the ACR signal in entire cellular volume (Figure 5e), activation by upconverted PL was found to not as significant and limited to the cellular domains with UCNPs. It is worth noting, that during the activation of ChR2 in the cell, using laser scanning microscope, the laser excitation beam stays in every pixel for few microseconds. At the same time, UCNPs have luminescence rise and decay times in a millisecond range (Figure S3). Therefore, in the laser scanning experiment, the efficiency of UCNPs-mediated activation is inherently limited.
To activate ChR2 through upconversion of NIR light, cells were irradiated by a 980 nm CW laser source. As could be seen in Figure 2b, the emission of UCNPs peaked at 450/470 nm, falls within the maximum absorption of ChR2. Hence, we utilized these upconversion bands to activate ChR2 protein in membranes of live cells. Simultaneously, these cells were irradiated at 543 nm to monitor the changes in intensity of ACR signal in response activation/deactivation of ChR2. We found that the NIR excitation at 980 nm (gated by mechanical shutter), upconverted on UCNPs to blue light, timely coincided with the significant increases of ACR fluorescence. The highest fluorescence response was observed in the cells incubated in the NMDG buffer (Figure 5). Cells with the highest number of internalized UCNPs demonstrated nearly as high increase in ACR fluorescence as in the experiments with direct activation of ChR2 at 476 nm (Figure 3), which points toward high efficiency of UCNPs for activation of opsins. Meanwhile, cells with a lower number of targeted UCNPs demonstrated a correspondingly lower influx of Ca2+ upon excitation with NIR light (Figure 5d,e). Consistent with this, no changes in Ca2+ level were observed for nontargeted UCNPs, which do not efficiently incorporate in the cells (Figure S4). This finding suggests that optogenetic activation can be limited to populations of neurons by targeting UCNPs to specific cellular types,60 whereas any significant 812
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Dependence between the subcellular distribution of UCNPs was confirmed in the cells, which were incubated in the cell medium during the activation of ChR2. However, as could be expected from prior results (Figure 3), the changes in ACR fluorescence were less pronounced (Figures 6 and S5). We believe that high localization of UCNP-mediated photoactivation represents great potential for subcellular optogenetics, which is an emerging direction in studies of cellular signaling activities.50 Since NIR light is not significantly absorbed by cellular biomolecules, the incident irradiation in the absence of UCNPs is not likely to trigger any cellular response. Meanwhile, it is feasible to target UCNPs to specific cellular compartments, by functionalization with various ligands,63 which would enable for spatially precise activation of photoprocesses in the sites of their subcellular localization. Potential applications may include regulation of intracellular Ca2+ release from the endoplasmic reticulum, regulation of organelle-bound enzymes and control of gene activities.50 Furthermore, the versatility of UCNPs can also be leveraged in the creation of a multifunctional reagent that will deliver genetic material coding for optogenetic proteins to a target cell and then subsequently act to upconvert light to the appropriate opsin-activating wavelength. In summary, we describe photoactivation of optogenetic proteins inserted into the cell membranes enabled by new, highly efficient NIR-to-blue UCNPs as photon nanotransformers. We demonstrate that our photon nanotransformers target cultured cells through FA receptors and enable optogenetic control of ion channels with the highly penetrating NIR light. Moreover, the upconverted blue light generated in situ by intracellular UCNPs activates the optogenetic proteins in close vicinity of the nanoparticles, providing photoactivation with high subcellular precision. The results here constitute a major step toward NIR subcellular optogenetics that are important in studies of cellular signaling activities.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsphotonics.6b00475.
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Supplementary Figures S1−S7 (PDF).
AUTHOR INFORMATION
Corresponding Author
*E-mail: pnprasad@buffalo.edu. ORCID
Artem Pliss: 0000-0003-4867-4074 Guanying Chen: 0000-0002-4789-1176 Author Contributions †
These authors contributed equally to this work (A.P., T.Y.O., and G.C.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by grants from NIH (1R21EY026411-01) and from SUNY Brain Initiative (1127716-1-72697). 813
DOI: 10.1021/acsphotonics.6b00475 ACS Photonics 2017, 4, 806−814
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DOI: 10.1021/acsphotonics.6b00475 ACS Photonics 2017, 4, 806−814
