Near-Infrared Light Triggered Upconversion Optogenetic Nanosystem

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Near-Infrared Light Triggered Upconversion Optogenetic Nanosystem for Cancer Therapy Bin Zheng,†,# Hanjie Wang,*,†,# Huizhuo Pan,†,# Chao Liang,‡ Wanying Ji,† Li Zhao,‡ Hongbin Chen,† Xiaoqun Gong,† Xiaoli Wu,† and Jin Chang*,† †

School of Life Sciences, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, China Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin 300070, China

‡

S Supporting Information *

ABSTRACT: In vivo the application of optogenetic manipulation in deep tissue is seriously obstructed by the limited penetration depth of visible light that is continually applied to activate a photoactuator. Herein, we designed a versatile upconversion optogenetic nanosystem based on a blue-light-mediated heterodimerization module and rare-earth upconversion nanoparticles (UCNs). The UCNs worked as a nanotransducer to convert external deeptissue-penetrating near-infrared (NIR) light to local blue light to noninvasively activate photoreceptors for optogenetic manipulation in vivo. In this, we demonstrated that deeply penetrating NIR light could be used to control the apoptotic signaling pathway of cancer cells in both mammalian cells and mice by UCNs. We believe that this interesting NIR-light-responsive upconversion optogenetic nanotechnology has significant application potentials for both basic research and clinical applications in vivo. KEYWORDS: near-infrared (NIR) light, optogenetic manipulation, upconversion nanoparticles, apoptosis, cancer therapy

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surgically implanting LEDs, such as BLUF (blue light using FAD) domain proteins,7−9 LOV (light−oxygen−voltage) domain proteins,10,11 and plant phytochromes.3,12 Hence, it is urgent to find an approach that can effectively convert NIR light to visible light to noninvasively utilize these conventional photoactuators in vivo. Interestingly, rare-earth upconversion nanoparticles (UCNs) can harvest highly penetrating NIR light in vitro to visible light in vivo.13−15 On account of their fascinating photoluminescence characteristics, upconversion nanoparticles have been extensively used as NIR-triggered mediators for photothermal therapy (PTT) and photodynamic therapy (PDT) in vivo.16,17 Therefore, the UCNs can act as nanotransducers that absorb NIR light with deep tissue penetrability and minimal invasiveness and thus convert it into local blue light or other visible light used in optogenetic manipulation. In this work, we set out to combine photoreceptor and upconversion nanoparticles to control protein interactions with NIR light. This noninvasive upconversion optogenetic nanosystem consists of two parts. One is the Arabidopsis flavoprotein cryptochrome 2 (Cry2) and its interacting partner Cib1 plasmids, which can express blue light photoreceptor Cry2 and its partner Cib1. The other part is the upconversion

ptogenetic manipulation provides spatiotemporally precise control over molecular processes, cellular signals, and animal behavior by genetically encoded light-dependent receptors.1−3 In these applications, the majority of photoreceptors can only be activated by visible light (such as blue or yellow light), which makes it essential to surgically implant LEDs in vivo because the epidermis is difficult to penetrate with visible light.4 However, the LED implantation surgery inevitably causes healthy tissue damage, and the penetration of the visible light emitted from LEDs is still poor in vivo, which leads to the low efficiency of optogenetic manipulation and seriously restricts the further application of optogenetics in vivo. Hence, how to avoid implanting LEDs is essential for further employment of optogenetic manipulation in vivo. Many researchers have been looking for alternative ways to solve the serious invasiveness problem for in vivo organisms. In recent years, near-infrared (NIR, 700−1000 nm) light with deep tissue penetrability and minimal invasiveness for organisms had already been successfully applied in imaging and therapy in vivo.5 Based on this idea, some NIR-lightphotosensitive proteins were found for making a noninvasive optogenetic system that can be used in vivo, such as bacterial phytochrome BphP1 and its partner Ppsr2, which are sensitive to 740 to 780 nm NIR light.6 Although this phytochrome can implement optogenetic manipulation, it is still hard for plenty of more efficient photoactuators to be used in vivo without © 2017 American Chemical Society

Received: September 8, 2017 Accepted: October 24, 2017 Published: October 24, 2017 11898

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Scheme 1. Schematic diagram depicting the application of an upconversion optogenetic nanosystem. In this schematic illustration, Arabidopsis flavoprotein cryptochrome 2 (Cry2), the photoreceptor of blue light, will quickly interact with its partner Cib1 after irradiating by blue.7,8 The upconversion nanoparticles (UCNs) can deliver plasmid DNA into the cell and then work as a nanotransducer to convert external deep-tissue-penetrating near-infrared (NIR) light to local blue light to noninvasively activate photoreceptors for optogenetic manipulation in vivo, whereas it is hard for blue light to effect this manipulation because of its low tissue penetrability. In this upconversion optogenetic nanosystem, external NIR light can penetrate the epidermis and stimulate UCNs emitting blue light to noninvasively trigger photoactuators for activating apoptotic signaling pathways of cancer cells in vivo, which is unachievable with visible light.

nanostructure, optical performance, and gene delivery efficiency were tested by TEM, energy-dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), fluorescence spectrometry, and electrophoretic mobility shift assay. As seen in Figure 1b−g, these nanoparticles had good monodispersity with an average diameter of 35 nm and a very clear profile (Figure 1, Figure S1). The peaks of amino groups increased at 1580−1650 cm−1 after poly(ethylene imine) (PEI) was conjugated with the UCNs (UCNs@PEI) in the FTIR spectra of the gene nanocarriers (Figure 1 h, Figure S3). Meanwhile, upon excitation by a 980 nm NIR laser, the UCNs displayed a strong emission band near 475 nm, which could provide the light source for optogenetic manipulation (Figure 1i). These results illustrated that upconversion nanoparticles had been successfully prepared and had excellent upconversion performance, which could convert NIR light to blue light with the expectation of realizing the applications of optogenetics in vivo. To examine the complex formation of nanocarriers and plasmid DNA and gene delivery efficiency, the zeta potential, binding DNA property of UCN nanocarriers, and the biocompatibility of the upconversion gene nanocarriers were evaluated (Figure 1j, Figures S4−S7). Moreover, HeLa cells

nanoparticles, which can deliver plasmid DNA and emit local blue light after excitation by 980 nm NIR light at the same time. In this upconversion optogenetic nanosystem, external NIR light could penetrate the epidermis and stimulate UCNs emitting local blue light to noninvasively induce Cry2 and Cib1 interaction to activate an apoptotic signaling pathway of cancer cells in vivo (as shown in Scheme 1). Hence, the upconversion optogenetic nanosystem was expected to trigger conventional photoreceptors for both basic research and clinical applications in vivo without surgically implanting an LED light source.

RESULTS AND DISCUSSION Physicochemical Characterization of Upconversion Nanocarriers. The most attractive point of the upconversion optogenetic nanosystem is the ability to convert external deeptissue-penetrating NIR light to local blue light to activate photoreceptors without implanting an LED light source in vivo. The key of upconversion optogenetic manipulation is whether the UCNs coud effectively convert NIR light to blue light and successfully deliver plasmids to cells. In Figure 1, the synthesis process of lanthanide-doped NaYF4:Yb, Tm@NaGdF4:Yb, and Tm core−shell upconversion nanoparticles is shown (Figure 1a), and their physicochemical properties such as morphology, 11899

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Figure 1. Preparation of gene nanocarriers. (a) Schematic diagram of the preparation process for gene nanocarriers. The TEM picture of (b) the lanthanide-doped NaYF4:Tm,Yb upconverting nanocrystal cores, (c) the epitaxial core−shell upconversion nanocrystals NaYF4:Yb and Tm@NaGdF4:Yb, and (d) the UCNs modified with PEI on the surface to form gene nanocarriers (UCNs@PEI). Scale bars, 100 nm. (e) Highresolution TEM image of UCNs@PEI. Scale bar, 5 nm. (f) STEM-HAADF test for inspection of the element distribution of UCNs@PEI. (g) EDX test for element analysis of UCNs@PEI. (h) FTIR spectra of upconversion nanoparticles (UCNs), citric-acid-modified upconversion nanoparticles (UCNs@CA), and PEI-modified UCNs@CA (UCNs@PEI). (i) Emission spectra of UCNs (red line), UCNs@CA (blue line), and UCNs@PEI (purple line). Inset photograph is the laser beam traveling through the UCNs@PEI aqueous solutions. (j) Electrophoretic mobility shift assay of Cry2-mCherry and Cib1-EGFP-CAAX plasmids and UCNs@PEI. “1” means only 100 ng of Cry2-mCherry plasmid DNA; “2” means only 100 ng of Cib1-EGFP-CAAX plasmid DNA; “3” means 100 ng of Cry2-mCherry plasmid DNA and 100 ng of Cib1EGFP-CAAX plasmid DNA; and “4” means 100 ng of Cry2-mCherry plasmid DNA, 100 ng of Cib1-EGFP-CAAX plasmid DNA, and 6 μg of UCNs@PEI. (k) Cell transfection experiment of the preparation of gene nanocarriers with Cib1-EGFP-CAAX and Cry2-mCherry plasmids (1:2 ratio). The image of cell transfection was observed for EGFP and mCherry with 488 and 561 nm laser excitation under confocal microscopy, respectively, through a 100× oil immersion objective. Scale bars, 10 μm. (l) The gene expression efficiency was quantified by flow cytometry (FCM) for per 10 000 cells. Data represent mean ± SD (n = 5); *P < 0.05 and **P < 0.01 (one-way ANOVA).

Cellular Protein−Protein Interactions by the NIRControlled Upconversion Optogenetic Nanosystem. Upconversion nanoparticles could effectively convert NIR light to blue light as previously stated. Nevertheless, whether NIR light could trigger the conventional photoactuators by UCNs was essential in our next work. So we constructed three pairs of plasmid DNA containing a blue-light-mediated heterodimerization module, Arabidopsis flavoprotein cryptochrome 2 (Cry2) and its interacting partner Cib1.7,8 They were used for the subcellular localization of protein in the plasma,

were transfected with a nanocarrier−DNA complex. The plasmid DNA of Cib1-EGFP-CAAX and Cry2-mCherry could be better expressed and the protein could locate to the plasma membrane and cytoplasmic, respectively (Figure 1k, Figure S8). The transfection efficiency of plasmid DNA was up to 61.0% and 54.5%, detected by the flow cytometry method (Figure 1, Figure S9). The results showed that the gene nanocarriers had been successfully prepared with excellent biocompatibility and high gene delivery efficiency for blue-light-mediated heterodimerization. 11900

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Figure 2. Near-infrared-light-activated translocation of Cry2 in mammalian cells. (a) After the Cib1-EGFP-CAAX (located in the plasma membrane) and Cry2-mCherry plasmids (1:2 ratio) were cotransfection in HeLa cells using UCNs for 48 h, they were irradiated using nearinfrared light (980 nm, 4 W). The EGFP and mCherry were observed at various time points under confocal microscopy, through a 100× oil immersion objective. Scale bars, 10 μm. (b) After the Tom20-Cib1-EGFP (located in the mitochondria) and Cry2-mCherry constructs (1:2 ratio) were cotransfection in HeLa cells using UCNs for 48 h, they were irradiated using NIR light (980 nm, 4 W). Scale bars, 5 μm. (c) After the importin α-Cib1-EGFP (located in the nuclear membrane) and Cry2-mCherry constructs (1:2 ratio) were cotransfected in HeLa cells using UCNs for 48 h, they were irradiated using NIR light (980 nm, 4 W). Scale bars, 10 μm. (d) After the Cib1-EGFP-CAAX and Cry2mCherry constructs (1:2 ratio) were cotransfected in HeLa cells using UCNs for 48 h, they were irradiated outside of 2 mm pork tissue using near-infrared light (980 nm, 4 W). Scale bars, 10 μm. (e) After the Cib1-EGFP-CAAX and Cry2-mCherry plasmids (1:2 ratio) were cotransfected in HeLa cells using UCNs for 48 h, they were irradiated outside of 2 mm pork tissue using blue light (475 nm, 4 W). Scale bars, 10 μm.

mitochondrial, and nuclear membranes by NIR with the help of UCNs (Figure 2).

First, we fused Cib1 with EGFP and CAAX plasma membrane localization motifs and fused Cry2 with mCherry 11901

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Figure 3. NIR-induced apoptosis signaling pathway by the upconversion optogenetic nanosystem. (a) Schematic of NIR-triggered apoptosis by the upconversion optogenetic nanosystem. (b) After the Fas-Cib1-EGFP and Cry2-mCherry-FADD constructs (1:2 ratio) were cotransfected in HeLa cells using UCNs for 48 h, they were irradiated outside of 2 mm pork tissue using NIR light (980 nm, 4 W). The time course of Cry2-mCherry-FADD recruitment to the Fas-Cib1-EGFP on the plasma membrane was observed under confocal microscopy, through a 100× oil immersion objective. Scale bars, 25 μm. (c) Western blots of light-triggered formation of the cleaved PARP fragment in HeLa cells at 48 h post-light exposure after treatment with a 4 W NIR laser or blue LED irradiation for 2 h (10 s every 1 min). (d) The cell viability was detected by CellTiter 96 AQueous One solution cell proliferation assay at 48 h post-light exposure (n = 5). Data represent mean ± SD; *P < 0.05 and **P < 0.01 (one-way ANOVA). (e) The Hoechst−PI double staining method was used for detecting changes in the cell nucleus of apoptotic cells at 48 h post-light exposure. Scale bars, 10 μm. (f) The living and dying cells were dyed using calcein-AM/PI double stain assay at 48 h post-light exposure. Scale bars, 100 μm. (g) The annexin V-FITC/PI apoptosis detection assay was also applied to test apoptosis efficiency at 48 h post-light exposure by flow cytometry (FCM) per 10, 000 cells.

for cytoplasmic expression. After 4 W, 980 nm NIR laser exposure for 1 min, the Cry2-mCherry aggregated to the plasma membrane from the cytoplasmic membrane (Figure 2a). By the same method, we selected the outer mitochondrial membrane (Tom20)18,19 and outer nuclear membrane (importin α)20 anchored protein to further discuss the subcellular localization performance under NIR light exposure. After NIR light irradiation, the Cry2-mCherry successfully aggregated to the mitochondrial (Figure 2b, Figure S10) and nuclear membrane from the cytoplasmic membrane (Figure 2c, Figure S11), respectively. Subsequently, we demonstrated the reversibility of the Cry2-mCherry back to the cytoplasm after removal of the NIR laser for 10 min (Figure 2a−c, Figure S10−

S13). Therefore, NIR light could activate photoactuators to induce cellular protein−protein interactions by UCNs. Then we examined whether it was still valid to activate the photoreceptors after NIR light exposure through deep skin. Herein, we selected pig skin with a thickness of 2 mm and placed on a glass-bottomed culture dish. Illumination of HeLa cells with an NIR laser outside of the pork tissue could also cause the translocation of Cry2-mCherry to the cytomembrane (Figure 2d). Under the same conditions, it was hard to trigger the translocation of Cry2-mCherry to the cytomembrane after blue light illumination, as it was difficult for blue light to penetrate through the skin (Figure 2e, Figure S14).21−23 Subsequently, dark incubation restored Cry2-mCherry in the 11902

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Figure 4. In vivo imaging and suppression of tumor using the NIR-controlled upconversion optogenetic nanosystem. (a) Schematic of NIRtriggered apoptosis by the upconversion optogenetic nanosystem in vivo. (b) An aqueous solution of UCNs@ICG of various concentrations was added to a 96-well plate, and the fluorescence intensity was monitored using an in vivo imaging system in the left image. The fluorescence of the mouse was also acquired after injection of UCNs@ICG in 100 μL of PBS into the BALB/C nude mouse tumor model. (c) Forty-eight hours after injection of the Fas-Cib1-EGFP and Cry2-mCherry-FADD constructs (1:2 ratio) with UCNs, tumors were illuminated with blue light (4 W) or an NIR laser (4 W) for 1 min, and the tumors were immediately excised and cryosectioned at 10 μm thickness onto slides. EGFP and mCherry were detected using 488 and 561 nm laser excitation under confocal microscopy, respectively, through a 100× oil immersion objective. Scale bars, 25 μm. After 48 h post-light exposure with NIR laser irradiation, the tumors were cut into ultrathin sections (50−80 nm) for detecting the cell nucleus morphology and apoptotic bodies after different treatments by TEM imaging. Scale bars, 2 um. (d) Tumor size in different experimental groups after 4 weeks. (e) Tumor weight in different experimental groups after 4 weeks (n = 5). (f) Tumor volume changes in different experimental groups within 4 weeks (n = 5). (g) Survival curve of tumor-bearing mice in different experimental groups (n = 5). (h) TUNEL staining of HeLa xenograft tumors with different treatments. Scale bars, 100 μm. Data represent mean ± SD; *P < 0.05 and **P < 0.01 (one-way ANOVA).

NIR-Triggered Apoptosis by the Upconversion Optogenetic Nanosystem. The upconversion optogenetic nanosystem could convert deep-tissue-penetrating NIR light to local blue light to activate photoreceptors as mentioned above. Whether this technology could be used for killing cancer cells need to be further explored. Accordingly, we selected the classic apoptosis signal pathway molecule Fas and its adaptor molecule FADD24−27 for preparation of Fas-Cib1-EGFP and Cry2mCherry-FADD constructs to induce apoptosis. FADD could combine with Fas intracellularly to trigger cell death after illumination with an NIR laser by the upconversion optogenetic nanosystem without exogenous Fas ligands. The apoptosis was detected by Western blot analysis, CellTiter 96 AQueous One

protoplasm to the initial state after removal of the NIR light for 10 min (Figure 2d). We also proved that the two factors of upconversion nanoparticles and near-infrared light were both vital for controlling the translocation of Cry2-mCherry (Figure S15). We displayed the reversibility of Cry2-mCherry migration to the cytomembrane and back to the protoplasm for the second NIR illumination for 1 min followed by 10 min of dark relaxation (Figure S16 and Supplementary Video 1). These results suggested that the UCNs could convert deep-tissuepenetrating NIR light to blue light to activate photoreceptors for optogenetic manipulation through biological tissue using the upconversion optogenetic nanosystem. 11903

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death by the NIR-controlled upconversion optogenetic nanosystem. In Vivo Suppression of Tumor in Mice Using the NIRControlled Upconversion Optogenetic Nanosystem. The upconversion optogenetic nanosystem has been proved to activate HeLa cell apoptosis and induce cell death in vitro. To further discuss whether it could suppress tumor growth in vivo by NIR laser irradiation, the upconversion optogenetic nanosystem was injected intratumorally (Figure 4) owing to the conventional optogenetic system lacking imaging properties and the difficulty of using it in precisely guiding the irradiation position of a light source. In order to realize visual guiding optogenetic manipulation in vivo, indocyanine green (ICG), as a common water-soluble tricarbocyanine dye, was conjugated on the surface of upconversion nanoparticles (UCNs@ICG). UCNs@ICG aqueous solutions of various concentrations were added into a 96-well plate, and the fluorescence intensity was monitored by an in vivo imaging instrument. As shown in Figure 4b, 1 mg/mL UCNs@ICG had very intense fluorescence, and fluorescence still could be detected even when the concentration was decreased to 0.0625 mg/mL. The in vivo imaging of a mouse was detected after injection of UCNs@ICG dissolved in PBS subcutaneously, and it showed intense fluorescence (Figure 4b, Figures S20, S21). UCNs containing Gd element could be used in magnetic resonance imaging (MRI) (Figure S20). Hence, our upconversion optogenetic nanosystem could realize double modal imaging of fluorescence imaging and MRI for realizing visually guided cancer optogenetic therapy in vivo. The NIR-controlled Cry2-mCherry-FADD translocation to Fas-Cib1-EGFP on the plasma membrane was further evaluated in vivo for tumor-bearing mice. Forty-eight hours after injection of the Fas-Cib1-EGFP and Cry2-mCherry-FADD constructs (1:2 ratio) with UCNs, tumors were illuminated with blue light (4 W) or NIR laser (4 W) for 1 min, and the tumors were immediately excised and cryosectioned. The NIR laser illumination caused the translocation of Cry2-mCherry-FADD to the Fas-Cib1-EGFP on a plasma membrane, while the blue light could hardly trigger the Fas-Cib1-EGFP translocation to the plasma membrane on account of its low penetrability (Figure 4c, Figure S22). In addition, 48 h after injection of the Fas-Cib1 and Cry2-FADD constructs (1:2 ratio) with UCNs, tumors were illuminated with blue light (4 W) or NIR laser (4 W) for 2 h (10 s every 1 min), and the tumors were cut into ultrathin sections (50−80 nm) at 48 h post-light exposure to detect the cell nucleus morphology and apoptotic bodies after different treatments. Nucleus condensation was obvious after treatment with UCNs-coated Fas-Cib1 and Cry2-FADD constructs (UCNs@(Fas-Cib1+Cry2-FADD)) and NIR laser irradiation for 2 h, while the nucleus was round and full after irradiation with blue light. Similarly, there were many apoptotic bodies in the NIR laser irradiation group (Figure 4c, Figure S23). These results suggest that deeply penetrating NIR light could trigger photoreceptors to recruit FADD to Fas on the plasma membrane and activate the apoptosis signaling pathway in vivo by the NIR-triggered upconversion optogenetic nanosystem. To further discuss whether it could suppress tumor growth in vivo by NIR laser irradiation, we set up five pairs of trials: PBS intratumoral injection (group 1); NIR laser irradiation for 2 h (10 s every 1 min) at 4 W (group 2); UCNs@(Fas-Cib+Cry2FADD) intratumoral injection (group 3); UCNs@(Fas-Cib +Cry2-FADD) intratumoral injection and irradiation with blue

solution cell proliferation assay, trypan blue exclusion assay, Hoechst-PI double staining method, calcein-AM/PI double stain kit, and flow cytometer (Figure 3). We first examined the light-initiated recruitment of the Cry2mCherry-FADD construct to the Fas-Cib1-EGFP plasma membrane in HeLa cells. There was an obvious translocation of FADD to the Fas on the plasma membrane and a distinct increase in the plasma membrane within 1 min after 4 W NIR laser illumination through the 2 mm skin (Figure 3b). We also assessed whether the anticipated apoptotic signal could be activated after FADD recruitment to the Fas on the plasma membrane by NIR light exposure. After the apoptosis signal pathway activation, poly(ADP-ribose) polymerase (PARP) cleavage was thought to be an important indicator of apoptosis and was also commonly considered a marker of apoptosis activation. To verify the effect of the NIR-activated apoptosis signal pathway by the upconversion optogenetic nanosystem, PARP cleavage was assayed by Western blot analysis. There were four groups experiments: group 1 was treated with only NIR laser irradiation for 2 h (10 s every 1 min) (negative control group); group 2 was treated with 1 μM staurosporine (STS), a broad-spectrum protein kinase inhibitor that induces apoptosis, for 3 h (positive control group); group 3 was treated with NIR laser irradiation for 2 h (10 s every 1 min) through 2 mm tissue after UCN cotransfection with Cry2-FADD and FasCib1 constructs for 48 h (experimental group 1); and group 4 was treated with 475 nm blue light irradiation for 2 h (10 s every 1 min) through 2 mm tissue after UCN cotransfection with Cry2-FADD and Fas-Cib1 constructs for 48 h (experimental group 2). Forty-eight hours post-light exposure, the result showed that PARP cleavage was found in group 3 (Figure 3c line 3), and it was similar to the positive control group (Figure 3c line 2). Under the same conditions, PARP cleavage was not found after NIR laser irradiation alone (Figure 3c line 1) or treatment with blue light irradiation (Figure 3c line 4). This result indicates that the apoptosis signaling pathway could be activated by the upconversion optogenetic nanosystem after NIR light illumination. We also examined the effect of NIR-induced Fas recruitment of FADD on long-term cell viability (12, 24, 48 h) using a solution cell proliferation assay and the trypan blue exclusion assay for detection of cell activity28 (Figure 3d, Figures S17− S19). After NIR light exposure for 2 h, about 60% of cells of group 3 were stained at 48 h post-light exposure, similar to the positive control group. By contrast, the rate of group 1 treated with NIR laser irradiation was less than 9%, and that of group 4 was less than 16%. In addition, the Hoechst−PI double staining method was used for detecting the change in cell nucleus morphology after different treatments. Nucleus condensation was obvious after treatment with UCNs-coated Cry2-FADD and Fas-Cib constructs and NIR laser irradiation for 2 h (the position indicated by the arrow), while the nucleus was round and full for group 1 and group 4 (Figure 3e). Meanwhile, living and dying cells were dyed using calcein-AM/PI double stain kit. There were many dead cells for group 3, while the cells were almost all alive for group 1 and group 4 (Figure 3f). In addition, an annexin V-FITC/PI apoptosis detection assay and flow cytometry were applied to test apoptosis efficiency. The early and late apoptotic cells were reached at about 24.9% and 30.9% for group 3, while there was a very low apoptotic efficiency for group 1 and group 4 (Figure 3f). These results showed that the apoptosis signaling pathway could be activated and induced cell 11904

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nanoparticles can be modified in various ways to improve the targeting effect of particles, to further increase the controlled release of nucleic acid, to enhance specific fluorescence imaging performance, and to improve the substitutability with better biodegradable materials in vivo.29−32 Hence, this optogenetic therapy system is hopeful to noninvasively activate many molecular processes and cellular signals to treat diseases with NIR light in vivo.

light for 2 h (10 s every 1 min) at 4 W (group 4); and UCNs@(Fas-Cib+Cry2-FADD) intratumoral injection and irradiation by an NIR laser for 2 h (10 s every 1 min) at 4 W (group 5). After 4 weeks of treatment, group 4 had almost no obvious tumor suppression effect compared to group 3, indicating that the mice treatment with the UCNs@(Fas-Cib +Cry2-FADD) intratumoral injection and irradiated by blue light could hardly lead to apparent tumor suppression (Figure 4d, S24). This result explained that the optogenetic system of light-triggered apoptosis could hardly be activated by blue light in vivo owing to the weak penetrability, which was consistent with Figure 3. However, the tumors apparently diminished for group 5 after irradiating with NIR laser (Figure 4d). These results illustrated that UCNs could harvest NIR light into local blue light in vivo to activate the apoptosis pathway by the upconversion optogenetic nanosystem and further kill the tumor cells. In addition, the excised tumor volume and weight of the five groups were also measured after euthanasia, and group 5 showed the smallest tumor volumes of 200 mm3 and a weight of 0.25 g compared with the other four groups after treatment for 4 weeks (Figure 4e,f). Besides, the volume change of each group’s tumors was measured every other day during different treatment within 4 weeks. Compared with the tumor volumes of the control groups, it was obviously suppressed within 200 mm3 for group 5 after 4 weeks of treatment and it showed slower tumor growth (Figure 4f). Meanwhile, the survival curves were obtained for the five groups. The curve results showed that group 5 had a longer survival time than the other groups (Figure 4g). These results indicated that the NIRcontrolled upconversion optogenetic nanosystem could successfully suppress the growth of tumors by activation of the apoptosis pathway in vivo. Furthermore, the HeLa xenograft tumors were collected and histological analyses were also performed using TUNEL staining. More significant tumor cell apoptosis could be observed in group 5 compared with the other four groups (Figure 4h). These results certified the excellent antitumor ability of group 5, which received the UCNs@(Fas-Cib+Cry2-FADD) intratumoral injection and irradiation by a NIR laser. During the animal experiments, the weights of these tumor-bearing mice and histological analyses of the main organs indicated no obvious difference in the five groups, suggesting excellently biocompatibility for the upconversion optogenetic nanosystem in vivo (Figures S25, S26). All these results illustrated that this NIR-triggered cancer optogenetic therapeutic method could successfully inhibit the growth of tumors in vivo by deeply penetrating NIR light.

METHODS Preparation of NaYF4:Yb/Tm (50/0.5 mol %) Core Nanocrystals. An aqueous solution containing YCl3 (0.495 mmol), YbCl3 (0.5 mmol), and TmCl3 (0.005 mmol) was added into a round-bottom flask and kept completely dry under stirring at 110 °C. Then it was dissolved completely with a mixture of oleic acid (6 mL) and 1octadecene (15 mL) to form luminous yellow lanthanide-oleate complexes at 140 °C after stirring for 30 min. Thereafter, a methanol solution (6 mL) containing 0.148 g of NH4F and 0.1 g of NaOH was added after cooling to 60 °C. Subsequently, the temperature was increased to 90 °C to adequately evaporate the methanol under vigorous stirring. After vacuuming the mixture solution for 20 min, the reaction temperature was increased to 290 °C for 1 h under an argon atmosphere. The obtained nanoparticles were washed twice using ethanol and partially dispersed in cyclohexane for measurements. Preparation of NaYF4:Yb/Tm@NaGdF4:Yb (90/10 mol %) Core−Shell Nanocrystals. An aqueous solution of GdCl3 (0.9 mmol) and YbCl3 (0.1 mmol) was added into a round-bottom flask and kept completely dry under stirring at 110 °C. Then it was dissolved completely using 6 mL of oleic acid and 15 mL of 1octadecene to form luminous yellow lanthanide-oleate complexes at 140 °C after stirring for 30 min. Thereafter, a methanol solution (6 mL) containing 0.148 g of NH4F and 0.1 g of NaOH was added along with the as-prepared NaYF4:Yb/Tm core nanocrystals (5 mL in cyclohexane) after cooling to 50 °C. Subsequently, the temperature was increased to 90 °C to adequately evaporate the methanol under vigorous stirring. After vacuuming the mixture solution for 20 min, the reaction temperature was increased to 290 °C for 1 h under an argon atmosphere. Then the NaYF4:Yb/Tm@NaGdF4:Yb core−shell nanocomplex was finally obtained after washing twice using ethanol and partially dispersed in cyclohexane for measurements. Surface Modification of Core−Shell Upconversion Nanoparticles with PEI to Form Gene Nanocarriers. The as-prepared core−shell upconversion nanoparticles in 2 mL of cyclohexane settled after adding acetone in the same volume and centrifugation. The precipitate was dissolved in 2 mL of 1,4-dichlorobenzene and 2 mL of N,N-dimethylformamide (DMF). Meanwhile, 0.1 g of citric acid was dissolved in 2 mL of DMF and 2 mL of 1,4-dichlorobenzene. Then these two solutions were mixed in a 25 mL flask at 120 °C with stirring for 4 h. The resulting nanoparticles (UCNs@CA) were precipitated with ethanol and redispersed in DI water (2 mL). The PEI conjugation to the UCNs (UCNs@PEI) was performed based on 1-ethyl-3-(3(dimethylamino)propyl)arbodiimide)/N-hydroxysuccinimide (EDC/ NHS) chemistry. For the specific methods for attaching ICG to the PEI-modified UCNs, refer to ref 33. Characterization of UCNs. High-resolution transmission electron microscopy (Tecnai G2 F20, FEI) was used to record the morphology and element mapping with an operating voltage of 200 kV. X-ray diffraction (XRD) was measured by an X-ray diffractometer (Bruker AXS, D8-Focus) with a 4°/min scanning rate in the 2θ range of 10− 80°. FTIR spectra were acquired by an FTS 6000 spectrometer (BioRad Company, Hercules, CA, USA). Dynamic light scattering (DLS) sizes and zeta potential were determined by a Nano-Zetesizer ZS 90 (England) at room temperature.34,35 A fluorescence spectrophotometer (Cnilaser, China) was employed to measure the fluorescence spectrum of UCNs using a 980 nm laser. The concrete details about the optical fiber are shown in Figure S27. Design of Mammalian Plasmids. Mammalian expression plasmids were constructed based on either the pEGFP-N1 vector or mCherry-N1 (Clontech), with a standard CMV promoter. The

CONCLUSIONS In summary, we used a blue-light-sensitive pair of photoactuators as an example to discuss and illustrate that deeply penetrating NIR light could trigger photoreceptors in vitro and in vivo. Our results constitute a basis for the development of a distinctive generation of cancer therapy nanosystem, which could be applied to activate visible-light-sensitive optogenetic tools in vivo by NIR light. As the emission wavelength of upconversion nanoparticles can be readily adjusted from the ultraviolet region to the near-infrared region,28 we expect that it can be used to trigger the majority of photoreceptors, which are excited by short-wavelength light, such as Rh, ChR2, and LOV (Supplementary Table 1). In addition, this versatile optogenetic therapy method can be used for exciting most signaling pathway proteins by NIR in vivo. Moreover, the surface of the 11905

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ACS Nano

min). For detection of relocalization in vivo, the tumors were immediately excised and cryosectioned at 10 μm thickness onto slides after illumination. EGFP and mCherry were visualized under confocal microscopy, respectively, through a 100× oil immersion objective. In Vivo Antitumor Assessment. Tumor size was monitored by vernier caliper, and tumor volume (V) was calculated as V = Length × Width × (Length + Width)/2. When tumor volume reached about 200−250 mm3, mice were randomly distributed into five groups: PBS, NIR laser, plasmid, plasmid+blue, plasmid+NIR. Each experimental group contained five mice. Exposure groups were irradiated for 2 h (10 s every 1 min) with a 980 nm NIR laser or 475 nm blue LED at 4 W. Subsequently, tumor size and body weight of each mouse were recorded every other day. At week 4, some mice were sacrificed and tumors were collected. Then photos of tumors were taken by a digital camera (Nikon, Japan). After that, tumors were washed with saline three times and fixed in 10% neutral-buffered formalin. For the hematoxylin and eosin (H&E) and TUNEL assay ((Roche, Switzerland), paraffin tumor sections were stained and observed by a Fluorescence Inversion Microscope System (Olympus, Japan). Statistical Analyses. Data were expressed as mean ± standard deviation (SD) of experiments, and each experiment group contained five repeated samples. Data analysis was performed using OriginPro 8.0 and Microsoft Excel. The significance between groups was analyzed using an unpaired two-tailed t test (comparing two groups) and one-way analysis of variance (ANOVA) (comparing multiple groups) by Statistics Analysis System (*P < 0.05 and **P < 0.01, respectively). P < 0.05 was considered significant.40

cryptochrome 2 (Cry2) gene (GenBank: NM_179257.2) and cryptochrome-interacting basic-helix−loop−helix 1 (CIB1) gene (GenBank: NM_119618.3) of Arabidopsis thaliana were kindly provided by Robert M. Hughes (University of North Carolina, USA). Cry2-mCherry and Tom20-Cib1-EGFP constructs were kindly provided by Robert M. Hughes, and the construct of Tom20-Cib1EGFP expressed the protein Tom20-GGSGGS-Cib1GGSGGSRSFEF-EGFP.36 A plasma-membrane-localization CAAX signal (−K-K-K-K-K-K-S-K-T-K-C-V-I-M) was inseted at the Cterminal of Cib1-EGFP to form the Cib1-EGFP-CAAX construct. Importin α (GenBank: U93240.1) was added at the N-terminal of Cib1-EGFP to form the importin α-Cib1-EGFP construct, and it expressed the protein importin α-GGSGGS-Cib1-GGSGGSRSFEFEGFP. Fas (Sequence ID: XR_945732.2) was added at the N-terminal of Cib1-EGFP to form the Fas-Cib1-EGFP construct, and it expressed the protein Fas-GGSGGS-Cib1-GGSGGSRSFEF-EGFP. FADD (GenBank: LT735858.1) was added at the C-terminal of Cry2mCherry to form the Cry2-mCherry-FADD construct, and it expressed the protein Cry2-mCh-GGSGGG-FADD. All oligomers used in this research are shown in Supplementary Table 2. Cell Light Activation and Imaging. For details of the HeLa cell culture methods and biocompatibility assay refer to refs 37 and 38. In order to discuss the manipulation performance of the optogenetics system, HeLa cells were seeded onto a glass-bottom culture dish (2 × 104 cells/dish) made of high-transparent borosilicate glass with a glass diameter of 10 mm and a thickness of the glass bottom of 0.17 mm ± 0.02 mm (NEST). Then the HeLa cells were transiently transferred to the gene by nanocarriers−plasmid DNA (mass ratio = 30:1).37,38 A 2 μg amount of Cry2-mCherry plasmids and 1 μg of Cib1-EGFP-CAAX (located in the cytomembrane), Tom20-Cib1-EGFP (located in the mitochondrial outer membrane), and importin α-Cib1-EGFP (located in the outer nuclear membrane) were cotransfected, respectively. To determine light-triggered apoptosis, HeLa cells were cotransfected with Fas-Cib1-EGFP and Cry2-mCherry-FADD or Fas-Cib1 and Cry2-mCherry (1:2 ratio). After transfection for 48 h, HeLa cells were exposed to a 980 nm NIR laser (MDL-N-980 (Cnilaser, China)) or a blue high-power LED of 475 nm (Cree Inc., USA) for 1 min. Cry2 → Cib1 relocalization experiments were carried out at 25 °C. A laser scanning confocal microscope (PerkinElmer, USA) was employed to observe live-cell imaging. For light-activated apoptosis, the HeLa cells in 24-well plates were irradiated for 2 h (10 s every 1 min) using a 4 W, 980 nm NIR laser and were returned to a 5% CO2 atmosphere at 37 °C for incubation for 48 h. Cell Viability Assay. To evaluate in vitro tumor cell destruction, transiently transfected cells were either kept in the dark or subjected to a 980 nm NIR laser or 475 nm blue LED exposure (10 s flash every 1 min) for 2 h through 2 mm skin tissue. At 48 h postirradiation, HeLa cell apoptosis and viability were determined by Western blot analysis (rabbit antibody to PRAP; mouse antibody to β-actin, abcam),36 MTT assay (Sigma-Aldrich), trypan blue exclusion assay (Thermo Fisher Scientific),36,39 and CellTiter 96 AQueous One solution cell proliferation assay (Promega).17 In addition, Hoechst and propidium iodide (PI) dye were were used to detect apoptosis. Annexin V-FITC and PI were used to detect apoptotic and necrotic cells by flow cytometer (BD FACSCalibur). The cell viability was also evaluated by the calcein-AM/PI double stain kit (Invitrogen). For the specific methods refer to the relevant references. Mouse Models and in Vivo Imaging. Healthy female BALB/C nude mice of 20−25 g body weight were purchased from HFK Technology Co., Ltd. (Beijing). Animal experiments were performed in accordance with the statutory requirements of People’s Republic of China (GB14925-2010). To develop the HeLa tumor model, 1 × 106 HeLa cells (in 100 μL of PBS) were injected subcutaneously (s.c.). The treatment was carried out when the tumor growth reached 200 mm3. The upconversion optogenetic nanosystem containing 5 μg of Fas-Cib1-EGFP and Cry2-mCherry-FADD plasmids (1:2 ratio) in 100 μL of PBS was injected into the tumor. The fluorescence imaging of the mice was performed with an in vivo imaging system under 645 nm excitation.37 Forty-eight hours after injection, mice were illuminated with a 980 nm NIR laser or 475 nm blue LED for 2 h (10 s every 1

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b06395. Additional figures and tables (PDF) Supporting video (AVI)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Bin Zheng: 0000-0002-0369-5560 Hanjie Wang: 0000-0001-9400-814X Jin Chang: 0000-0002-6752-8526 Author Contributions #

B. Zheng, H. Wang, and H. Pan contributed equally.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank Robert M. Hughes (University of North Carolina) for plasmids and Xue Li for the MRI animal imaging instrument (The Second Hospital of Tianjian Medical University). This work was sponsored by National Key Research and Development Program of China (2017YFA0205104), National Natural Science Foundation of China (51373117, 51573128, 81771970, and 31371329), and Tianjin Natural Science Foundation (15JCQNJC03100 and 13JCYBJC37200). REFERENCES (1) Tischer, D.; Weiner, O. D. Illuminating Cell Signalling with Optogenetic Tools. Nat. Rev. Mol. Cell Biol. 2014, 15, 551−558. (2) Kramer, R. H.; Mourot, A.; Adesnik, H. Optogenetic Pharmacology for Control of Native Neuronal Signaling Proteins. Nat. Neurosci. 2013, 16, 816−823. 11906

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