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 ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b06395 • Publication Date (Web): 24 Oct 2017 Downloaded from http://pubs.acs.org on October 24, 2017

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Near-Infrared Light Triggered Upconversion Optogenetic Nanosystem for Cancer Therapy Bin Zheng, Ji,

†

†,#

Li Zhao,

†

Hanjie Wang, *, ,# Huizhuo Pan, ‡

Hongbin Chen,

†

†,#

Chao Liang,

Xiaoqun Gong,

†

‡

Wanying †

Xiaoli Wu, & 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. Address

correspondence

to

E-mail:

[email protected]

[email protected].

ACS Paragon Plus Environment

and

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ABSTRACT: In vivo the application of optogenetic manipulation in deep tissue is seriously obstructed by the limited penetration depth of visible light which has been continually applied to activate photoactuator. Herein, we designed a versatile upconversion optogenetic nanosystem based on a blue light–mediated heterodimerization module and rear-earth upconversion

nanoparticles

(UCNs).

The

UCNs

worked

as

a

nanotransducer to convert external deep-tissue-penetrating near infrared (NIR) light to local blue light to noninvasively activate photoreceptor for optogenetic manipulation in vivo. In this, we demonstrated that deeply penetrating NIR light could be used for control the apoptotic signaling pathway of cancer cell in both mammalian cells and mice by UCNs. We believe

that

this

interesting

NIR-light-responsive

upconversion

optogenetic nanotechnology will bring a lot of significant application potentials for both basic research and clinical applications in vivo. KEYWORDS: near infrared (NIR) light, optogenetic manipulation, upconversion nanoparticles, apoptosis, cancer therapy Optogenetic manipulation provides spatiotemporally precise control over molecular processes, cellular signals, and animal behavior by genetically encoded light-dependent receptor.1-3 In these applications, the majority photoreceptor can only be activated by visible light (such as blue light or yellow light), which makes it is essential to surgically implant LED in vivo because visible light is difficult to penetrate the epidermis.4 However,


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the LED implantation surgery inevitably causes healthy tissue damage and the penetration of the visible light emitted from LED is still poor in vivo, which lead to the low efficiency of optogenetic manipulation and seriously restrict the further application of optogenetics in vivo. Hence, how to avoid implanting LED is essential for further employment 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 nm-1,000 nm) light with the deep tissue penetrability and minimal invasiveness for organisms had already been successfully applied in the imaging and therapy in vivo.5 Based on the idea, some NIR light photosensitive proteins were found for making optogenetic system noninvasively used in in vivo, such as bacterial phytochrome BphP1 and its partner Ppsr2, which are sensitive to 740 nm 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 surgically implanting LED, 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.


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Interestingly, rear-earth upconversion nanoparticles (UCNs) can harvest highly penetrating near-infrared (NIR) light in vitro to visible light in vivo.13-15 On account of the fascinating photoluminescence characteristic, upconversion nanoparticles have been extensively used as NIR-triggered

mediators

for

photothermal

therapy

(PTT)

and

photodynamics therapy (PDT) in vivo.16,17 Therefore, the UCNs can act as the 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 (UCNs) to control protein interactions with NIR light. This noninvasive upconversion optogenetic nanosystem consisted of two parts. One was the Arabidopsis flavoprotein cryptochrome 2 (Cry2) and its interacting partner Cib1 plasmids, which could express blue light photoreceptor Cry2 and its partner Cib1. Another part was the upconversion nanoparticles which could 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 epidermis and stimulated UCNs emitting local blue light to noninvasively induce Cry2 and Cib1 interacting for activating apoptotic signaling pathway of cancer cell in vivo (as shown in Scheme 1). Hence, upconversion

optogenetic

nanosystem

was

expected


to

trigger

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conventional photoreceptors for both basic research and clinical applications in vivo without surgically implanting LED light source. RESULTS AND DISCUSSION Physicochemical Characterization of Upconversion Nanocarriers. The most attractive point of upconversion optogenetic nanosystem was able to convert external deep-tissue-penetrating NIR light to local blue light to activate photoreceptor without implanting LED light source in vivo. While the key of upconversion optogenetic manipulation was whether the upconversion nanoparticles (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, Tm core-shell upconversion nanoparticles were showed (Figure 1a) and its physicochemical properties such as morphology, nanostructure, optical performance and gene delivery efficiency were tested by TEM, energy-dispersive X-ray spectroscopy (EDX), fourier transform infrared spectroscopy (FTIR), fluorescence spectrometer and electrophoretic mobility shift assay. As seen in Figure 1b–1g, these nanoparticles had good monodispersity with an average diameter of 35 nm and very clear profile (Figure 1, Figure S1). The peaks of amino groups increased at 1580-1650 cm−1 nearby after that PEI was conjugated with UCNs (UCNs@PEI）in the FTIR spectra of the gene nanocarriers (Figure 1 h, Figure S3). Meanwhile, upon excited by 980 nm


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NIR laser, UCNs displayed a strong emission bands nearby 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 for expecting to realize the applications of optogenetics in vivo. To exam the complex formation of nanocarriers and plasmid DNA and gene delivery efficiency, Zeta potential， binding DNA property of UCNs nanocarriers and the biocompatibility of the upconversion gene nanocarriers were evaluated (Figure 1j, Figure S4-S7). Moreover, Hela cells were transfected with nanocarriers–DNA complex. The plasmid DNA of Cib1-EGFP-CAAX and Cry2-mCherry could be better expressed and the protein could locate to plasma membrane and cytoplasmic respectively (Figure 1k, Figure S8). The transfection efficiency of plasmid DNA were up to 61.0% and 54.5% detected by flow cytometry method (Figure 1l, Figure S9). The results showed that the gene nanocarriers

had

biocompatibility

been and

successfully

high

gene

prepared

delivery

with

efficiency

excellent for

blue

light–mediated heterodimerization module. Cellular

Protein–Protein

Interactions

by

NIR-Controlled

Upconversion Optogenetic Nanosystem. Upconversion nanoparticles


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(UCNs) 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 contained blue light–mediated heterodimerization module, Arabidopsis flavoprotein cryptochrome 2 (Cry2) and its interacting partner Cib1.7,8 They had been used to subcellular localization of protein in plasma membrane, mitochondrial and nuclear membrane by NIR with the help of UCNs. (Figure 2). Firstly, we fused Cib1 with EGFP and CAAX plasma membrane localization motif, and fused Cry2 with mCherry 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 (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 (Figure 2c, Figure

S11),

respectively.

Subsequently,

we

demonstrated

the

reversibility of the Cry2-mCherry back to the cytoplasm after removal NIR laser for 10 min (Figure 2a-2c, Figure S10-S13). Therefore, NIR


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light could activate photoactuators to induce cellular protein–protein interactions by UCNs. Then we need to exam whether it was still valid to activate the photoreceptor after NIR light through the deep skin. Herein, we selected a pigskin with thickness of 2 mm and covered on the glass bottom culture dish. Illumination of Hela cells with NIR laser outside of the pork tissue could also cause the translocation of Cry2-mCherry to cytomembrane (Figure 2d). Under the same conditions, it was hard to trigger the translocation of Cry2-mCherry to 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 protoplasm to the initial state after removal NIR light 10 min (Figure 2d). And 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 cytomembrane and back to the protoplasm for the second of 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-tissue-penetrating NIR light to blue light to activate photoreceptor for optogenetic manipulation through biological tissue using the upconversion optogenetic nanosystem.


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NIR-Triggered

Apoptosis

by

Upconversion

Optogenetic

Nanosystem. Upconversion optogenetic nanosystem could convert deep-tissue-penetrating NIR light to local blue light to activate photoreceptor as mentioned above. Whether this technology could be used for killing cancer cells need to be further discussed. Accordingly, we selected the classic apoptosis signal pathway molecules, Fas and its adaptor molecule FADD,24-27 for preparation Fas-Cib1-EGFP and Cry2-mCherry-FADD constructs to induce apoptosis. This FADD would combine with Fas intracellularly to triggered cell death after illumination with NIR laser by upconversion optogenetic nanosystem without exogenous Fas ligands. The apoptosis was detected by western blot analysis, CellTiter 96® AQueous one 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

firstly

examined

the

light-initiated

recruitment

of

Cry2-mCherry-FADD construct to Fas-Cib1-EGFP plasma membrane in Hela cells. There was an obvious translocation of FADD to the Fas on the plasma membrane and a distinctly 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 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,


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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 NIR-activated apoptosis signal pathway by upconversion optogenetic nanosystem, PARP cleavage was assayed by western blot analysis, and there were four groups experiments, including group one treated with only NIR laser irradiation for 2 h (10 s in every 1 min) (negative control group), group two treated with 1 uM staurosporine (STS), a broad spectrum protein kinase inhibitor that induced apoptosis, for 3 h (positive control group), group three treated with NIR laser irradiation for 2 h (10 s in every 1 min) through 2 mm tissue after UCNs cotransfection Cry2 -FADD and Fas-Cib1 constructs for 48 h (experimental group 1), and group four treated with 475 nm blue light irradiation for 2 h (10 s in every 1 min) through 2 mm tissue after UCNs cotransfection Cry2-FADD and Fas-Cib1 constructs for 48 h (experimental group 2). After treatment for 48 h post-light exposure, the result showed that the PARP cleavage was found in group three (Figure 3c line 3) and it was similar with positive control group (Figure 3c line 2). Under the same condition, PARP cleavage was not found after NIR laser irradiation alone (Figure 3c line 1) or treated with blue light irradiation (Figure 3c line 4). This result indicated that the apoptosis signal pathway could be activated by upconversion optogenetic nanosystem after NIR light illumination.


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We also examined the effect of NIR-induced Fas recruitment of FADD on long term cell viability (12, 24, 48 h) using solution cell proliferation assay and trypan blue exclusion assay for detection the cell activity28 (Figure 3d, Figure S17-S19). After NIR light exposure for 2 h, about 60% cells of the group three had stained at 48 h post-light exposure and it was similar with positive control group. By contrast, the rate of group one treated with NIR laser irradiation was less than 9 %, and group four was less than 16 %. Besides, Hoechst-PI double staining method was used for detecting the change of cell nucleus morphology of different treatment. It was obvious to nucleus condensation after treated 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 full for group one and group four (Figure 3e). Meanwhile, living cells and dying cells were dyed using calcein-AM/PI double stain kit. There were many dead cells for group three, while the cells were almost all alive for group one and group four (Figure 3f). In addition, annexin V-FITC/PI apoptosis detection assay and flow cytometry were applied to test apoptosis efficiency. The early apoptotic cells and late apoptotic cells were reached about 24.9 % and 30.9% for group three, while there were very low apoptotic efficiency for group one and group four (Figure 3f). These results showed that the apoptosis signaling pathway could be activated


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and induced cells death by NIR-controlled upconversion optogenetic nanosystem. In vivo Suppression of Tumor in Mice Using NIR-Controlled 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 by intratumoral （ Figure 4 ） . Owing to conventional optogenetic system lacked imaging property and it was difficult to be used in precisely guiding the irradiation position of light source. In order to realize visual guiding optogenetic manipulation in vivo, indocyanine green (ICG) as a common water soluble tricarbocyanine dye could be conjugated on the surface of upconversion nanoparticles （ UCNs@ICG ） . The UCNs@ICG aqueous solution of various concentrations were added into the 96-well plate and the fluorescence intensity was monitored by 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 if the concentration decreased to 0.0625 mg/ml. The in vivo imaging of mouse was detected after injection UCNs@ICG dissolved in PBS subcutaneously, and it showed intense fluorescence (Figure 4b, Figure S20 S21). The UCNs contained Gd element could be used in magnetic resonance imaging (MRI) (Figure


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S20). Hence, our upconversion optogenetic nanosystem could realize double modal imaging of fluorescence imaging and MIR for realizing the visual guiding cancer optogenetic therapy in vivo. The

NIR-controlled

Cry2-mCherry-FADD

translocation

to

Fas-Cib1-EGFP on plasma membrane was further evaluated in vivo for tumor-bearing

mice.

After

injection

the

Fas-Cib1-EGFP

and

Cry2-mCherry-FADD constructs （1:2 ratio） with UCNs for 48 h, 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 plasma membrane, while the blue light could hardly trigger the Fas-Cib1-EGFP translocation to plasma membrane on account of low penetrability (Figure 4c, Figure S22). Besides, after injection the Fas-Cib1 and Cry2-FADD constructs （1:2 ratio） with UCNs for 48 h, tumors were illuminated with blue light (4 W) or NIR laser (4 W) for 2 h (10 s in every 1 min) and the tumors were made into ultrathin section (50-80 nm) at 48 h post-light exposure for detecting the cell nucleus morphology and apoptotic body of different treatment. It was obvious to nucleus condensation after treated 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 full for irradiation with blue light. Similarly, there were many apoptotic body for NIR laser


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irradiation group (Figure 4c, Figure S22, S23). These results suggested that deeply penetrating NIR light could trigger photoreceptor to recruit the FADD to Fas on plasma membrane and activate apoptosis signaling pathway in vivo by 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, including received PBS intratumoral injection (group 1); got NIR laser irradiation for 2 h (10 s

in

every

1

min)

at

4

W

(group

2),

received

(UCNs@(Fas-Cib+Cry2-FADD)) intratumoral injected (group 3), and received the UCNs@(Fas-Cib+Cry2-FADD) intratumoral injection and irradiated with blue light for 2 h (10 s in every 1 min) at 4 W (group 4) and received the UCNs@(Fas-Cib+Cry2-FADD) intratumoral injection and irradiated by NIR laser for 2 h (10 s in every 1 min) at 4 W (group 5). After 4 weeks treatment, the 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 apparently 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 tumor apparently diminished for the group 5 after irradiating with NIR laser (Figure 4d). These results illustrated that UCNs


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could harvest NIR light into local blue light in vivo to active apoptosis pathway by upconversion optogenetic nanosystem and further killed the tumor cells. In addition, the excised tumor volume and weight of the five groups were also measured after euthanasia, and the group 5 showed smaller tumor volumes and weight of 0.25 g and 200 mm3 compared with another four groups after treatment for 4 weeks (Figure 4e, 4f). Besides, the volume changes of each group’s tumor was measured every other day during different treatment within 4 weeks. Compared with the tumor volumes of control groups, it was obviously suppressed within 200 mm3 for group 5 after 4 week treatment and it showed slower tumor growth (Figure 4f). Meanwhile, the survival curve was manufactured to the five groups. The curve result showed that the group 5 had a longer survival time than other group (Figure 4g). These results indicated that the NIR-controlled upconversion optogenetic nanosystem could successfully suppress the growth of tumor by activation apoptosis pathway in vivo. Furthermore, the Hela xenograft tumors were collected and the histological analyses were also performed using TUNEL staining. More significant tumor cell apoptosis could be observed in group 5, compared with another four groups (Figure 4h). These results certified the excellent anti-tumor ability of group 5 which received the UCNs@(Fas-Cib+Cry2-FADD) intratumoral injection and irradiated by NIR laser. During the animal


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experiments, weights of these tumor-bearing mice and histological analyses of main organs indicated no obvious difference in the five groups for suggesting excellently biocompatibility for the upconversion optogenetic nanosystem in vivo (Figure S25, S26). All these results illustrated that this NIR-triggered cancer optogenetic therapeutic method could successfully inhibit the growth of tumor in vivo by deeply penetrating NIR light.


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CONCLUSIONS In summary, we used blue light-sensitive pair photoactuator as an example to discussion and illustration that the deeply penetrating NIR light could trigger photoreceptor in vitro and in vivo. Our results constituted a basis for the development of a distinctive generation of cancer therapy nanosystem, which could be applied to activate the visible light sensitive optogenetic tools in vivo by NIR light. As the emission wavelength of upconversion nanoparticles can be readily adjusted from ultraviolet region to near-infrared region,28 we expect that it can be used to trigger the majority of photoreceptors, which excited by short wavelength light, such as Rh, ChR2, LOV (Supplementary Table 1). Besides, the versatile optogenetic therapy method can be used for exciting most of signaling pathway protein by NIR in vivo. Moreover, the surface of the 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 plenty of molecular processes and cellular signals to treat the disease by NIR light in vivo. METHODS


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Preparation of NaYF4: Yb/Tm (50/0.5 mol %) Core Nanocrystals. The aqueous solution containing YCl3 (0.495 mmol), YbCl3 (0.5 mmol), and TmCl3 (0.005 mmol) was added into round-bottom flask and keep them completely dry under stirring at 110 ℃.Then they were dissolve completely with a mixture of oleic acid (6 mL) and 1-octadecene (15 mL) to form luminous yellow lanthanide-oleate complexes at 140 ℃ for stirring 30 min. Thereafter, the methanol solution (6 mL) containing 0.148g NH4F and 0.1 g NaOH was added after cooling down to 60 ℃. Subsequently, the temperature was increased to 90 ℃ to adequately evaporate the methanol under vigorous stirring. After vacuuming for 20 minutes for the mixture solution, the reaction temperature was increased to 290 ℃ 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 round-bottom flask and keep them completely dry under stirring at 110 ℃ .Then they were dissolve completely using 6 mL oleic acid and 15 mL 1-octadecene to form luminous yellow lanthanide-oleate complexes at 140 ℃ for stirring 30 min. Thereafter, the methanol solution (6 mL) containing 0.148g NH4F and 0.1 g NaOH was added along with the as-prepared NaYF4: Yb/Tm core


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nanocrystals (5 mL in cyclohexane) after cooling down to 50 ℃ . Subsequently, the temperature was increased to 90 ℃ to adequately evaporate the methanol under vigorous stirring. After vacuuming for 20 minutes for the mixture solution, the reaction temperature was increased to ℃

290

for

1

h

under

NaYF4:Yb/Tm@NaGdF4:Yb

an

core-shell

argon

atmosphere.

nanocomplex

was

Then finally

obtained after washed twice times using ethanol and partially dispersed in cyclohexane for measurements. The

Surface

Modification

of

Core-Shell

Upconversion

Nanoparticle (UCNs) with PEI to Form Gene Nanocarriers. The as-prepared core-shell upconversion nanoparticles in 2 mL cyclohexane were settled down after added acetone in the same volume and centrifugation.

The

precipitation

was

dissolved

in

2

mL

1,

4-Dichlorobenzene and 2 mL N, N-Dimethylformamide (DMF). Meanwhile, 0.1 g citric acid was dissolved to 2 mL DMF and 2 mL 1, 4-Dichlorobenzene. And then these two kinds of solution were mixed to a 25 mL-flask for 120 ℃ stirring with 4 h. The resulting nanoparticles (UCNs@CA) were precipitated with ethanol and re-dispersed in DI water (2 mL). The poly (ethylene imine) (abbreviated as PEI) conjugation to the UCNs

(UCNs@PEI)

was

performed

based

on

1-ethyl-3-(3-dimethylaminopropyl) arbodiimide)/N-Hydroxysuccinimide (EDC/NHS) chemistry. The attaching methods of ICG to the PEI modified


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UCNs, the specific methods can refer to the relevant references.33 Characterization of UCNs. High Resolution Transmission Electron Microscopy (Tecnai G2 F20, FEI) was used to record morphology and element mapping with an operating voltage of 200 kV. X-ray diffraction (XRD) was measured by X-ray diffractometer (Bruker AXS, D8-Focus) with 4°/min scanning rate in 2θ range of 10-80°. Fourier transform infrared (FTIR) spectra were acquired by FTS 6000 spectrometer (Bio-Rad Company, Hercules, CA). Dynamic light scattering (DLS) sizes and zeta potential were determined by Nano-Zetesizer ZS 90 (England) at room temperature.

34,35

The fluorescence spectrophotometer (Cnilaser,

China) was employed to measure the fluorescence spectrum of UCNs using 980 nm laser. The concrete details about optical fiber were showed in Figure S27. Design of Mammalian Plasmids. Mammalian expression plasmids were constructed based on either pEGFP-N1 vector or mCherry-N1 (Clontech), with a standard CMV promoter. The 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


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(University

of

North

Tom20-Cib1-EGFP

Carolina,

USA),

and

expressed

Tom20-GGSGGS-Cib1-GGSGGSRSFEF-EGFP.36

the

construct

the

of

protein A

plasma

membrane-localization CAAX signal (-K-K-K-K-K-K-S-K-T-K-C-V-I-M) was inseted in C-terminal of Cib1-EGFP to form Cib1-EGFP-CAAX construct. The importin α（GenBank: U93240.1）was added in the N-terminal of Cib1-EGFP to form importin α-Cib1-EGFP construct, and it expressed the protein of importin α-GGSGGS-Cib1-GGSGGSRSFEFEGFP. The Fas (Sequence ID: XR_945732.2) was added in the N-terminal of Cib1-EGFP to form Fas-Cib1-EGFP construct, and it expressed the protein Fas-GGSGGS-Cib1-GGSGGSRSFEF-EGFP. The FADD (GenBank: LT735858.1) was added in the C-terminal of Cry2-mCherry to form Cry2-mCherry-FADD construct, and it expressed the protein Cry2-mCh-GGSGGG-FADD. All oligos used in this research showed in Supplementary Table 2. Cell Light Activation and Imaging. The details of Hela cells culture methods and biocompatibility assay could refer to relevant references. 37,38

In order to discuss the manipulation performance of optogenetics

system, the Hela cells were seeded into the glass bottom culture dish (2× 104 cells/dish) made by high transparent borosilicate glass and the glass diameter for 10 mm and the thickness of the glass bottom for 0.17 mm±0.02 mm (NEST). Then the Hela cells were transiently transfered


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the gene by nanocarriers-plasmid DNA (mass ratio=30:1).37,38 2 ug Cry2-mCherry plasmids and 1ug 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. For discussing lighttriggered 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 980 nm NIR laser (MDL-N-980 (Cnilaser, China)) or blue high power LED 475 nm (Cree Inc., USA) for 1 min. Cry2→Cib1 relocalization experiments were carried out at 25 ℃ . The 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 in every 1 min）using 4 W 980 nm NIR laser and were returned to 5 % CO2 atmosphere at 37 °C for incubation 48 h. Cell Viability Assay. To evaluate in vitro tumor cell destruction, transiently transfected cells were either kept in the dark or subjected to 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 post-irradiation, Hela cell apoptosis and viability was 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

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Scientific）,36, 39 CellTiter 96® AQueous one solution cell proliferation assay (Promega),17 Besides, Hoechest and propidium iodide (PI) dye were were used for detected apoptotic. Annexin V-FITC and PI were used for detected apoptotic and necrotic cells by flow cytometer (BD FACSCalibur). The cell viability was also evaluated by calcein-AM/PI double stain kit (Invitrogen). The specific methods can 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 Hela tumor model, 1×106 Hela cells (in 100 ul PBS) were injected subcutaneously (s.c.). The treatment was carried out when the tumor growth to 200 mm3. The upconversion optogenetic nanosystem contained 5 ug Fas-Cib1-EGFP and Cry2-mCherry-FADD plasmids (1:2 ratio）in 100 ul PBS was injected into the tumor. The fluorescence imaging of mouse were detected by in vivo imaging system under exciting for 645 nm.37 After injection for 48 h, mice were illuminated with 980 nm NIR laser or 475 nm blue LED for 2 h (10 s in every 1 min). For detection the relocalization in vivo, the tumors were immediately excised and cryosectioned at 10 µm thickness onto slides


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after illumination. EGFP and mCherry were visualized under confocal microscopy, respectively, through×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 in every 1 min) with 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 weeks 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 an Fluorescence Inversion Microscope System (Olympus, Japan). Statistical Analyses. Data were expressed as mean ± standard deviation (SD) of experiments and each experiment group contained 5 repeated samples. Data analysis was performed using OriginPro 8.0 and Microsoft Excel. The significance between groups were analyzed using


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unpaired two-tailed t-test (compared two groups) and one-way analysis of variance (ANOVA) (compared multiple groups) by Statistics Analysis System (* p < 0.05 and ** p < 0.01, respectively). p < 0.05 was considered as significant. 40 ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano. The authors declare no competing financial interest. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]. ORCID Jin Chang: 0000-0002-6752-8526 Hanjie Wang: 0000-0001-9400-814X Bin Zheng: 0000-0002-0369-5560 Author Contributions


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#

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These authors contributed equally.

■ ACKNOWLEDGMENTS We thank Robert M Hughes (University of North Carolina) for plasmids and Xue Li for 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), Tianjin

Natural Science Foundation

(15JCQNJC03100 and 13JCYBJC37200). REFERENCES 1. Tischer, D.; Weiner, O. D., Illuminating Cell Signalling with Optogenetic Tools. Nat. Rev. Mol. Cell Bio. 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. 3. Levskaya, A.; Weiner, O. D.; Lim, W. A.; Voigt, C. A., Spatiotemporal Control of Cell Signalling Using a Light-Switchable Protein Interaction. Nature 2009, 461, 997-1001. 4. Gautier, A.; Gauron, C.; Volovitch, M.; Bensimon, D.; Jullien, L.; Vriz, S., How to Control Proteins with Light in Living Systems. Nat. Chem. Bio. 2014, 10, 533-541.


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5. Weissleder, R.; Ntziachristos, V., Shedding Light onto Live Molecular Targets. Nat. Med. 2003, 9, 123-128. 6. Kaberniuk, A. A.; Shemetov, A. A.; Verkhusha, V. V., A Bacterial Phytochrome-Based Optogenetic System Controllable with Near-Infrared Light. Nat. Methods 2016, 13, 591-597. 7. Kennedy, M. J.; Hughes, R. M.; Peteya, L. A.; Schwartz, J. W.; Ehlers, M. D.; Tucker, C. L., Rapid Blue-Light-Mediated Induction of Protein Interactions in Living Cells. Nat. Methods 2010, 7, 973-975. 8. Lee, S.; Park, H.; Kyung, T.; Kim, N. Y.; Kim, S.; Kim, J.; Heo, W. D., Reversible Protein Inactivation by Optogenetic Trapping in Cells. Nat. Methods 2014, 11, 633-636. 9. Nihongaki, Y.; Kawano, F.; Nakajima, T.; Sato, M., Photoactivatable CRISPR-Cas9 for Optogenetic Genome Editing. Nat. Biotechnol. 2015, 33, 755-760. 10. Mottamena, L. B.; Reade, A.; Mallory, M. J.; Glantz, S.; Weiner, O. D.; Lynch, K. W.; Gardner, K. H., An Optogenetic Gene Expression System with Rapid Activation and Deactivation Kinetics. Nat. Chem. Biol. 2014, 10, 196-202. 11. Kawano, F.; Suzuki, H.; Furuya, A.; Sato, M., Engineered Pairs of Distinct Photoswitches for Optogenetic Control of Cellular Proteins. Nat. Commun. 2015, 6, 6256. 12. Ni, M.; Tepperman, J. M.; Quail, P. H., Binding of Phytochrome B to


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19. Endo, T.; Kohda, D., Functions of outer Membrane Receptors in Mitochondrial Protein Import. Biochim. Biophys. Acta 2002, 1592, 3-14. 20. Goldfarb, D. S.; Corbett, A. H.; Mason, D. A.; Harreman, M. T.; Adam, S. A., Importin Alpha: a Multipurpose Nuclear-Transport Receptor. Trends Cell Biol. 2004, 14, 505-514. 21. Juzenas, P.; Juzeniene, A.; Kaalhus, O.; Iani, V.; Moan, J., Noninvasive Fluorescence Excitation Spectroscopy During Application of 5-Aminolevulinic Acid in vivo. Photochem. Photobiol. Sci. 2002, 1, 745-748. 22. Wu, S.; Butt, H. J., Near-Infrared-Sensitive Materials Based on Upconverting Nanoparticles. Adv. Mater. 2016, 28, 1208-1226. 23. Nagarajan, S.; Zhang, Y., Upconversion Fluorescent Nanoparticles as a Potential Tool for in-Depth Imaging. Nanotechnology 2011, 22, 395101. 24. Boldin, M. P.; Varfolomeev, E. E.; Pancer, Z.; Mett, I. L.; Camonis, J. H.; Wallach, D., A Novel Protein that Interacts with the Death Domain of Fas/APO1 Contains a Sequence Motif Related to the Death Domain. J. Biol. Chem. 1995, 270, 7795-7798. 25. Kischkel, F. C.; Lawrence, D. A.; Tinel, A.; Leblanc, H.; Virmani, A.; Schow, P.; Gazdar, A.; Blenis, J.; Arnott, D.; Ashkenazi, A., Death Receptor Recruitment of Endogenous Caspase-10 and Apoptosis Initiation in the Absence of Caspase-8. J. Biol. Chem. 2001, 276, 46639-46646.


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26. Kischkel, F. C.; Hellbardt, S.; Behrmann, I.; Germer, M.; Pawlita, M.; Krammer, P. H.; Peter, M. E., Cytotoxicity-Dependent APO-1 (Fas/CD95)-Associated Proteins form a Death-Inducing Signaling Complex (DISC) with the Receptor. EMBO J. 1995, 14, 5579-5588. 27. Lee, E. W.; Seo, J.; Jeong, M.; Lee, S.; Song, J., The Roles of FADD in Extrinsic Apoptosis and Necroptosis. Bmb Reports 2012, 45, 496-508. 28. Deng, R.; Qin, F.; Chen, R.; Huang, W.; Hong, M.; Liu, X., Temporal Full-Colour Tuning through Non-Steady-State Upconversion. Nat. Nanotechnol. 2015, 10, 237-242. 29.Mitragotri, S.; Anderson, D. G.; Chen, X.; Chow, E. K.; Ho, D.; Kabanov, A. V.; Karp, J. M.; Kataoka, K.; Mirkin, C. A.; Petrosko, S. H., Accelerating the Translation of Nanomaterials in Biomedicine. Acs Nano 2015, 9, 6644-6654. 30. Hu, Q.; Sun, W.; Qian, C.; Bomba, H. N.; Xin, H.; Gu, Z., Relay Drug Delivery for Amplifying Targeting Signal and Enhancing Anticancer Efficacy. Adv. Mater. 2017, 29, 1605803-1605810. 31. Qian, R. C.; Cao, Y.; Zhao, L. J.; Gu, Z.; Long, Y. T., A Two‐Stage Dissociation System for Multilayer Imaging of Cancer Biomarker‐ Synergic Networks in Single Cells. Angew. Chem. Int. Edit. 2017, 56, 4802-4905. 32. Lu, Y.; Aimetti, A. A.; Langer, R.; Gu, Z., Bioresponsive Materials. Nat. Rev. Mater. 2016, 1, 16075.


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33 Deng, K.; Hou, Z.; Deng, X.; Yang, P.; Li, C.; Lin, J., Enhanced Antitumor Efficacy by 808 nm Laser‐Induced Synergistic Photothermal and Photodynamic Therapy Based on a Indocyanine-Green-Attached W18O49 Nanostructure. Adv. Funct. Mater. 2015, 25:7280-7290. 34.Zheng, B.; Gong, X.; Wang, H.; Wang, S.; Wang, H.; Li, W.; Tan, J.; Chang, J., A NIR-Remote Controlled Upconverting Nanoparticle: an Improved Tool for Living Cell Dye-Labeling. Nanotechnology, 2015, 26, 425102. 35. Zheng, B.; Chen, H. B.; Zhao, P. Q.; Pan, H. Z.; Wu, X. L.; Gong, X. Q.; Wang, H. J.; Chang, J., Persistent Luminescent Nanocarrier as an Accurate Tracker in vivo for Near Infrared-Remote Selectively Triggered Photothermal Therapy. Acs Appl. Mater. Inter. 2016, 8, 21603-21611. 36. Hughes, R. M.; Freeman, D. J.; Lamb, K. N.; Pollet, R. M.; Smith, W. J.; Lawrence, D. S., Optogenetic Apoptosis: Light-Triggered Cell Death. Angew. Chem. Int. Edit. 2015, 54, 12064-12068. 37 Zheng, B.; Wang, J.; Pan, H.; Chen, H.; Ji, W.; Liao, Z.; Gong, X.; Wang, H.; Chang, J., A Visual Guide to Gene/Optothermal Synergy Therapy Nanosystem Using Tungsten Oxide. J. Colloid. Interf. Sci. 2017, 506, 460-470. 38 Zheng, B.; Su, L.; Pan, H.; Hou, B.; Zhang, Y.; Zhou, F.; Wu, X.; Gong, X.; Wang, H.; Chang, J., NIR-Remote Selected Activation Gene Expression in Living Cells by Upconverting Microrods. Adv. Mater.


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Figures:

Scheme 1. The schematic diagram depicted the application of upconversion optogenetic nanosystem. In this schematic illustration, the Arabidopsis flavoprotein cryptochrome 2 (Cry2) is the photoreceptor of blue light and 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 photoreceptor for optogenetic manipulation in vivo, while it is hardly for blue light realizing this manipulation because of its low tissue penetrability. In this upconversion optogenetic nanosystem, external NIR light can penetrate epidermis and stimulate UCNs emitting blue light to noninvasively trigger photoactuators for activating apoptotic signaling pathway of cancer cell in vivo, which is unachievable for visible light.


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Figure 1. The preparation of gene nanocarriers. (a) The schematic diagram of 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 nanocrystal NaYF4: Yb, Tm@NaGdF4: Yb and (d) the UCNs were modified with PEI on the surface to form gene nanocarriers (UCNs@PEI). Scale bars, 100 nm. (e) The high-resolution TEM image of UCNs@PEI. Scale bar, 5 nm. (f) The STEM-HAADF test for inspection element distribution of UCNs@PEI. (g) The EDX test for element analysis of UCNs@PEI. (h) The FTIR spectra of upconversion nanoparticles (UCNs), citric acid modified upconversion nanoparticles (UCNs@CA) and PEI modified UCNs@CA (UCNs@PEI). (i) The emission spectra of UCNs (red line) and UCNs@CA (red line) and UCNs@PEI (purple line). Insert photograph was the laser beam traveled through the UCNs@PEI aqueous solutions. (j) The electrophoretic mobility shift assay of Cry2-mCherry and Cib1-EGFP-CAAX plasmids and UCNs@PEI. “1” means only 100 ng Cry2-mCherry plasmid DNA; “2” means only 100 ng Cib1-EGFP-CAAX plasmid DNA; “ 3 ” means 100 ng


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Cry2-mCherry plasmid DNA and 100 ng Cib1-EGFP-CAAX plasmid DNA; and “4” means 100 ng Cry2-mCherry plasmid DNA and 100 ng Cib1-EGFP-CAAX plasmid DNA and 6 ug UCNs@PEI. (k) Cells transfection experiment of preparation gene nanocarriers with Cib1-EGFP-CAAX and Cry2-mCherry plasmids （1:2 ratio）. The image of cells transfection was observed for EGFP and mCherry with 488 and 561 nm laser excitation under confocal microscopy, respectively, through×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

Near-Infrared Light Triggered Upconversion Optogenetic Nanosystem

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