A Lysosome-Assisted Mitochondrial Targeting Nanoprobe based on

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Biological and Medical Applications of Materials and Interfaces

A Lysosome-Assisted Mitochondrial Targeting Nanoprobe based on Dye-modified Upconversion Nanophosphors for Ratiometric Imaging of Mitochondrial Hydrogen Sulfide Xiang Li, Hui Zhao, Yu Ji, Chao Yin, Jie Li, Zhen Yang, Yufu Tang, Qichun Zhang, Quli Fan, and Wei Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b16818 • Publication Date (Web): 02 Nov 2018 Downloaded from http://pubs.acs.org on November 3, 2018

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ACS Applied Materials & Interfaces

A

Lysosome-Assisted

Dye-modified

Mitochondrial

Targeting

Nanoprobe

based

on

Upconversion Nanophosphors for Ratiometric Imaging of

Mitochondrial Hydrogen Sulfide ` Xiang Li, Hui Zhao, Yu Ji, Chao Yin, Jie Li, Zhen Yang, Yufu Tang, Qichun Zhang,* Quli Fan, * Wei Huang. Xiang Li, Hui Zhao, Yu Ji, Chao Yin, Jie Li, Zhen Yang, Yufu Tang, Quli Fan, Wei Huang Key Laboratory for Organic Electronics and Information Displays & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, 9 Wenyuan Road, Nanjing 210023, China. E-mail: [email protected]. Prof. Qichun Zhang School of Materials Science and Engineering Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore. E-mail: [email protected] Prof. W. Huang Shaanxi Institute of Flexible Electronics (SIFE), Northwestern Polytechnical University (NPU), Xi’an 710072, China Prof. W. Huang Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), Nanjing 211816, China Keywords: Hydrogen sulfide, mitochondria, ratiometric, upconversion luminescence, nanoprobe Abstract Hydrogen sulfide (H2S) is a versatile modulator in mitochondria and involved in numerous diseases caused by mitochondrial dysfunction. Therefore, many efforts have been made to develop fluorescent probes for mitochondrial H2S detection. However, these cationic small molecule probes are inapplicable for in vivo imaging

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because of the shallow tissue penetration and poor biostability. Herein, a ratiometric upconversion luminescence nanoprobe with the acid-activated targeting strategy is developed for detecting and bioimaging of mitochondrial H2S. The merocyanine TPAMC modified upconversion nanophosphors, acting as the targeting and response component, are encapsulated into a pH-sensitive husk, composed of DSPE-PEG and poly(L-histidine)-b-PEG, which improved the nanoprobe’s stability during transport in vivo. Under lysosomal pH, the PEG shell is interrupted and the targeting sites are exposed to further attach to mitochondria. Taking advantage of the LRET process between TPAMC and UCNPs, ratiometric detection of mitochondrial H2S can be achieved with high selectivity and sensitivity. Cellular testing reveals the precise targeting to mitochondria via a lysosme delivery process. Importantly, the nanoprobe can be used for monitoring of mitochondrial H2S levels in living cells and colon cancer mouse models.

1. Introduction Hydrogen sulfide (H2S), traditionally recognized to be a smelly toxic gas, was recently nominated as the third endogenous gasotransmitter.1-3 It is an essential modulator of

various physiological processes, including cytoprotection of

cardiovascular system,4 modulation of neuronal excitability,5 and suppression of oxidative stress.6,

7

Mitochondria are the major metabolic locus of H2S in living

organisms.8 They are the power plants of eukaryotic cells, and play a vital role in cellular signaling pathway9 and apoptosis.10 The endogenous H2S exerts regulating functions for energy production11 and redox homeostasis in mitochondria.12,


13

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Production deficiency of H2S is closely related to mitochondrial dysfunction which would lead to a variety of neurodegenerative diseases14 and cardiovascular diseases.15 In addition, the exogenous H2S also shows cytoprotection in lesion tissue by preservation of mitochondrial function.16,

17

Therefore, detecting and imaging of

mitochondrial H2S is of great importance for studying the physiological and pathological roles of H2S. For now, only a few fluorescent probes based on small molecule dyes have been reported for detection of mitochondrial H2S. The ratiometric fluorescence was generally adopted in these researches to achieve accurate detection with high fidelity. Compared with fluorescence turn-on method, ratiometric probes possess two or multiple emissions, building a self-calibration system that could minimize the environmental influences such as probe concentration and excitation light intensity.18-21 Chen et al. first developped a merocyanine derivative as ratiometric probe for H2S detection in mitochondria of live cells,22 in which the merocyanine group served as both target and response sites. This probe exhibited rapid and specific response to H2S via a nucleophilic addition mechanism. However, its application is limited by the shallow tissue penetration depth because of the short excitation wavelength. Subsequently, Bae et al. reported a near-infrared excited two-photon ratiometric probe, which was composed of the mitochondrial targeting unit triphenylphosphonium (TPP+) and the azide-bound two-photon chromophores.23 With the 750 nm femtosecond pulses excitation this probe was capable of detecting mitochondrial H2S in live-tissues with depth over 100 μm. However, it consumes a



long response time up to 1h, and these small molecules shows short blood circulation time that are unfit for long-term bioimaging in living body. In addition, the conventional cationic mitochondrial targeting systems suffer from limited biostability and specificity because they are easily absorbed with proteins and captured by phagocytes before reaching the target cells.24-26 Therefore, development of ratiometric probes with near infrared excitation, high biostability and photo-stability, and good mitochondrial targeting performance after cell internalization, for monitoring of mitochondrial H2S in vivo is still a big challenge. Upconversion nanophosphors (UCNPs) are a kind of excellent anti-stock emitting materials which can absorb NIR light and convert it to high energy lights with wavelengths range from ultraviolet (UV) to NIR.27-29 Compared with two-photon materials, it possesses much higher conversion efficiency for the existence of stable intermediate states, which also permit continuous-wave excitation with lower power density.30-32 UCNPs exhibit attractive advantages for bioimaging, including no photo-bleaching, high tissue penetration, resistance to autofluorescence interference and minimized photodamage.33, 34 To date, many upconversion luminescence (UCL) probes have been developped for detecting or bioimaging of small molecular,35-37 biomacromolecules,38,

39

organs40,

41

and tumors.42-44 Therefore, the UCL systems

would be a promising approach for constructing of novel mitochondrial targeting NIR probes for H2S imaging in cells and small animals. In this report, we developed a lysosome-assisted mitochondria-targeting probe based on merocyanine modified UCNPs (TPAMC-UCNPs@PEG, Scheme 1) for


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ratiometric detecting and imaging of mitochondrial H2S. Lanthanide-doped UCNPs (NaYF4: 20% Yb, 2% Er, 0.2% Tm) were used to construct a multiple-emissive UCL system with excitation at 980 nm. The molecular probe triphenylamine-merocyanine (TPAMC) was employed to functionalize the UCNPs (denoted as TPAMC-UCNPs) since TPAMC can respond to H2S via the nucleophilic addition on the indolenium C-2 atom.22, 45, 46 In addition, the merocyanine derivatives also exhibit mitochondrial targeting properties owing to the lipophilic cationic nature.47, 48 We further introduced a long carboxylic-terminated alkyl chain into TPAMC to promote efficient binding of dyes to the oleic acid (OA) capped UCNPs via Van der Waals force and hydrophobic interaction.49,

50

The absorption of TPAMC (530 nm) matched well with the green

UCL (540 nm), which caused a strong LRET effect from UCNPs to TPAMC. Thus, only weak UCL signal in green channel was observed for TPAMC modified UCNPs under 980 nm excitation. When reacted with H2S, the absorption of TPAMC would dramatically decline, therefore leading to recovery of the green UCL (Scheme 2). Utilizing the NIR UCL (800 nm) as an internal standard, we were able to construct a ratiometric nanoprobe with NIR excitation. In order to overcome the defects of cationic mitochondrial targeting systems and render the nanoprobe water-soluble for bio-applications, TPAMC-UCNPs were encapsulated with a polyethylene glycol (PEG) shell composed of mDSPE-PEG and poly(L-histidine)-b-PEG (PHIS-PEG). The PEG shell could block the adhesion of biomolecules and improve nanoprobes’ steric stability in blood circulation.51-53 However, after reaching the target cells, the acidic environment in the lysosome caused protonation of the unsaturated nitrogen in



PHIS-PEG, making it hydrophilic.54 The protonated PHIS-PEG would detach from the nanoprobes and hence lead to exposure of the previously hidden targeting groups, allowing for subsequent delivery of the nanoprobes to mitochondria. In vitro studies demonstrated the precise mitochondrial targeting and H2S response properties of the as-prepared nanoprobes, which enabled the ratiometric UCL imaging of mitochondrial H2S in living cells. Furthermore, in vivo experiments suggested that the TPAMC-UCNPs@PEG was capable of monitoring mitochondrial H2S in colon cancer mouse models.

2. Results and Discussion

2.1. Synthesis and Characterization of TPAMC-UCNPs@PEG TPAMC was mainly synthesized in two steps. First, the undecanoic acid side chain was introduced into trimethylindolenine by a substitution reaction and then combined with 4-formyltriphenylamine via condensation in ethanol solution to form a conjugated system. The pH-sensitive block copolymer PHIS-PEG was synthesized by conjugating PHIS with PEG2000-NHS via amidation reaction. Successful synthesis of organic components was confirmed by NMR and mass spectrum. The hexagonal UCNPs NaYF4: 20% Yb, 2% Er, 0.2% Tm were synthesized via the solvothermal method with surface ligands of oleic acid. TPAMC were attached on the UCNPs by intercalating to OA ligands. Both of them were then coated with the amphiphilic


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polymer mixture of DSPE-mPEG and PHIS-PEG (mass ratio, 1:1) to produce aqueous dispersed nanoparticles TPAMC-UCNPs@PEG. Transmission electron microscopy (TEM) images revealed the well dispersion of OA-capped UCNPs in cyclohexane with a uniform diameter of ∼18 nm (Figure 1a). The modifcation of TPAMC did not cause significant change to the particle size (Figure 1b). Wherear, an increased particle size to ∼24 nm was observed after enveloped with the PEG shell of DSPE-mPEG and PHIS-PEG (Figure 1c). The as-prepared TPAMC-UCNPs@PEG showed high dispersity and homogeneity in aqueous solution. A layer of organic moieties was presented in the magnified TEM image (Figure 1c insert), surrounding around the UCNPs core with the thickness of ∼3 nm. The high resolution TEM image revealed a crystal lattice distance of ∼0.51 nm (Figure 1d), which was expected for the lattice plane (100) of hexagonal NaYbF4 structure.55 This result demostrated the stable crystal structure of UCNPs in the nanoprobe. Dynamic light scattering (DLS) revealed a larger mean of ∼35 nm (Figure 1e) relative to that in TEM, which should be attributed to the more stretched PEG chains in the aqueous solution. The zeta potential of TPAMC-UCNPs@PEG was measured to be −9.65 mV. Although TPAMC possessing strong positive charges (+42.18 mV), the encapsulated nanoparticles showed a slightly negative potential, which indicated the well “shield effect” of the PEG shell. With these data in hand, we next studied the biostability of TPAMC-UCNPs@PEG. After 24 hours storage in PBS buffer with 10% fetal bovine serum (pH 7.4), no obvious size change was observed, which indicated the high stability of the nanoprobes in blood (Figure S5).



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Moreover, the structure of the nanprobe was further characterized by Fourier Transform infrared spectroscopy (FTIR). As Figure S6 showed, absorption peaks in OA-UCNPs FTIR spectrum were mainly derived from the oleic acid ligands. The peaks at 3431 and 1576 cm−1 were ascribed to σ (O−H) and σ (C＝O) of the carboxyl group. The peaks at 2926 and 2855 cm−1, associated with the asymmetric and symmetric stretching vibration of C−H in alkyl chain, also presented in the spectra of TPAMC and DSPE-PEG. Compared with OA-UCNPs, several new peaks appeared in the spectrum of TPAMC-UCNPs@PEG. The peaks at 1256 and 800 ∼ 650 cm−1 were attributed to δ (O−H) and δ (Ar−H) of TPAMC. A strong peak present at 1655nm belonged to σ (C＝C) of the amide group in PHIS-PEG, while another strong peak at 1113 cm−1 was associated with the symmetric C−O−C stretching vibration

in

DSPE-mPEG. The presence of these characteristic features further confirmed the successful assembly of TPAMC and the PEG shell on the surface of UCNPs. 2.2. Basic Optical Characterization of TPAMC-UCNPs@PEG The optical properties of the probe prepared were characterized by UCL emission and absorption spectroscopy. As Figure 1f showed, upon 980 nm excition, TPAMC-UCNPs@PEG mainly exhibited three emission bands: green UCL at 525 and 540 nm, red UCL at 660nm and NIR UCL at 800nm. Compared with OA-UCNPs, the green UCL emission, overlapping with the absorption band of TPAMC, was significantly quenched. The quenching coefficient was calculated to be 92.6 %, according to a formula.56 In contrast, the quenching coefficient in physically mixed solution of TPAMC and UCNPs was only 8.9%, under the same conditions,


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indicating that the quenching of green UCL was dominated by the LRET mechanism. The strong LRET effect should be attributed to the well matched optical spectra and tight binding (within 10 nm as shown in the TEM images) of the organic chromophores (acceptor) and UCNPs (donor). Far away from the absorption center of TPAMC, only slight attenuation was observed for the red emission (at 660 nm), while the near infrared channel was unaffected. Moreover, the UCL emissions of TPAMC-UCNPs@PEG showed no obvious changes in PBS buffers with different pH from 5.0 to 8.0 (Figure S8), indicating the nanoprobe was suitable for application in bio-conditions. TPAMC-UCNPs@PEG exhibited a broad absorption band at 530 nm, which was ascribed to the surface modified TPAMC. By comparing with the standard concentration-absorption curve, the loading capacity of TPAMC could be determined as ∼10.79 wt % (0.05 mg mL−1, ∼8.59 μM, Figure S9). And the loading density was calculated to be ∼2.06 × 103 units of TPAMC per TPAMC-UCNPs@PEG particle, considering the average diameter of 18 nm for UCNPs (hexagonal phase). To assess the photostability of the nanoprobe, TPAMC-UCNPs@PEG solutions were exposed to lights at 530 nm (0.3 mW cm−2) and 980 nm (13.6 mW cm−2) separately. Absorbance (530 nm) of the two groups was recorded every half hour. After 8h of continuous exposure, the absorbance reduced by 70.2% under visible light irradiation, while there was only a decrease of 7.9% for the NIR group (Figure S10). This result indicated that NIR excited UCL system could effectively improve the photostability of the probe.



2.3. pH-sensitive Characteristics of TPAMC-UCNPS@PEG To investigate acid-induced surface morphological and potential changes of TPAMC-UCNPs@PEG, size distribution and zeta potential of the nanoprobes in different pH environments were measured. In pH 7.4 PBS buffer, the nanoprobes showed zeta potential and average hydrodynamic diameter of −9.65 mV and 35.8 ± 2.1 nm (Figure 2) respectively, indicating the well shielding of positive mitochondrial targeting ligands TPAMC by PEG shell. When the pH dropped to 5.5 (endo/lysosome environment), the unsaturated nitrogen in PHIS-b-PEG was protonated, causing the hydrophilic transformation of the copolymer, thereby disturbing the PEG “shield”. As shown in Figure 2b, a dramatic increase of the zeta potential at pH 5.5, measured as +28.72 mV, was observed. This apparent reversal of the surface potential indicated that the targeting molecules were exposed due to the detaching of the protonated PHIS-b-PEG. Furthermore, the particle size also increased as pH dropped. A mean hydrodynamic diameter of 68.9

± 4.9 nm was observed at pH 5.5, indicating that a

certain degree of nanoparticle aggregation occurred under acid environment, which should be attributed to the enhanced surface hydrophobicity caused by the PHIS-b-PEG detaching. These results confirmed that the acid environment in endo/lysosome vessels would lead to the exposure of the hidden targeting sites.

2.4. H2S Sensing Capability of TPAMC-UCNPs@PEG The sensing capability of the designed nanoprobe for H2S was evaluated through UV-vis and UCL spectroscopy. With the addition of HS−, the maximum absorption


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band at 530 nm declined gradually. The reaction reached equilibrium when the amount of HS− increased to 40 μM and led to a visible color changing from red to colorless (Figure 3a). These phenomenon indicated the interruption of the intramolecular charge transfer of the organic dye in the sensing progress. The response mechanism was validated by analyzing the mass spectra of TPAMC before and after treated with NaHS. The MALDI-TOF mass spectra showed mass peaks at m/z 599.917 and m/z 656.402 for the precursor and NaHS-treated TPAMC (Figure S11), which were assigned to [TPAMC]+ and [TPAMC+ HS+Na]+, respectively. It confirmed that the organic dye reacted with H2S through a nucleophilic addition route in physiological conditions. This result was also confirmed by the 1H NMR characterization. Figure S3 showed the significant up-field shift of the signals from the protons in the conjugate system after H2S treatment, indicating the increase in electron cloud density caused by nucleophilic addition of HS−. Meanwhile, the singlet signal from 3-dimethyl group in indole turned to multiplets in TPAMC-SH, which demonstrated the addiition of HS− to the C=N+ bond to form a chirality center. Moreover, via the modulation of the LRET process, the UCL signal changed following the interaction between TPAMC and H2S. Upon the addition of HS−, a significant increase in the intensity of green UCL at 540 nm was presented. The intensity of the red UCL (660 nm) also showed a slight enhancement, while the NIR UCL (800 nm) remained constant (Figure 3b). Thus the NIR emission was used as an internal reference standard for ratiometric detection.

As shown in Figure 3c, the

intensity ratio of UCL540 nm / UCL800 nm showed a liner increase with the concentration



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of HS− (0−40 μM). The detection limited of the ratiometric signal was measured to be ∼0.22 μM (Figure S12), while that of the UCL (540 nm) intensity based detection system was measured to be ∼1.15 μM (Figure S13). Such lower detection limit should be attributed to the higher signal-to-noise ratio in the ratiometric detection system. The nanoprobe also showed a short response time of ≈ 110 s (Figure S14). These data indicated the property of the nanoprobe for real-time detection of H2S in physiological condition. The specificity of the nanoprobe to H2S was further investigated by UCL spectroscopy. Sulfur-containing anions (SCN−, SO32−, S2O3 2−), metal cations (K+, Na+, Ca2+, Fe3+), and reactive oxygen species (H2O2, CIO−, O2·−, NO2−) were introduced to react with the detection system in the same condition as HS−. As Figure 3d showed, no significant fluctuations were observed for the ratiometric signals (UCL540

nm

/

UCL800 nm) following addition of these analytes. The nanoprobes also exhibited higher selectivity for H2S over bio-thiols (glutathione, cysteine and homocysteine), which are the main interferences of H2S detection in physiological environments. This can be attributed to the stronger nucleophilicity of H2S (pKa = 7.0) than bio-thiols (pKa > 8.5).57 These data indicated that TPAMC-UCNPs@PEG is able to be used for detection of H2S in biological systems.

2.5. Mitochondria Localization of TPAMC-UCNPs@PEG The cytotoxicity of TPAMC-UCNPs@PEG was firstly evaluated via the MTT assay before iamging applications. HeLa cells were incabuted with different concentrations


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of nanoprobes in range of 1 to 800 μg mL−1 for 24 h (Figure S15). The cell viability remained higher than 80% after the exposure of 800 μg mL

−1

dose after 24 h,

indicating the low cytotoxicity of TPAMC-UCNPs@PEG. These data suggested that the nanoprobes were suitable for bioimaging application. To investigate the mitochondria-targeting capability of the nanoprbe, co-localization imaging experiments were performed with Mito-Tracker Green (MTG) as the mitochondrial label. Considering the quenching in green channel of the nanoprobe, red UCL (660 nm) was chose for subcellular imaging applications. TPAMC-UCNPs@PEG were incubated with HeLa or human breast cancer (MCF-7) cells for 12h, then mitochondrial costaining was performed with MTG before imaging experiments. To gain insight into the targeting function of the nanoprobe, Co-localization imaging were conducted with two independent fluorescent channels simultaneously. The multi-photon excited UCL emission at 660 nm (multi-photon channel) and the single-photon excited fluorenscence of surface modified TPAMC (single-photon channel) were used as illuminant source respectively. As shown in the confocal

images

of

HeLa

cells

(Figure

4),

the

fluorescence

of

TPAMC-UCNPs@PEG overlapped well with the signals from MTG in both single-photon and multi-photon channels. The changes in the intensity profiles of linear regions of interest (ROIs) between the signals of MTG and the fluorescence of TPAMC (Figure 4b) or the red UCL emission (Figure 4e) were also synchronous. The co-localization between TPAMC-UCNPs@PEG and MTG was further qualified by Pearson’s coefficients. The intensity of the correlation plot (Figure 4c, f) reflected



high Pearson’s coefficients in both single-photon (0.96) and multi-photon (0.92) channels. Furthermore, the co-localization imaging experiments performed in MCF-7 cells (Figure S16) also showed high synchronicity between the nanoprbe and MTG with the Pearson’s coefficients of 0.95 and 0.90 for single-photon and multi-photon imaging respectively. These results demonstrated the excellent mitochondrial specificity of TPAMC-UCNPs@PEG in living cells. The mitochondrial targeting capability was further confirmed by the results of mitochondrial separation experiments. As shown in Figure S17, the UCL emission spectrums revealed a much higher UCL intensity in the separated mitochondria than cytoplasm, demonstrating the accumulation of the nanoprobes in the mitochondria.

2.6. Intracellular Trafficking Profile To comprehend the mechanism underlying mitochondrial targeting, the intracellular trafficking profile of TPAMC-UCNPs@PEG was further monitored by confocal laser scanning microscope. The UCL imaging was conducted in red channel (660 nm). In order to avoid signal crosstalk between the organelle-trackers and TPAMC, which showed a broad emission band at 650-800 nm (Figure S7), Lyso-Tracker Green (Lyso-Tracker DND-26) and MTG were employed to label lysosome and mitochondria respectively.

Firstly, TPAMC-UCNPs@PEG was incubated with

HeLa cells at 37° C. After 4 hours, the surplus nanoprobes were removed by washing with PBS buffer followed by additional incubation of 8 h with fresh medium. Co-localization imaging experiments were conducted with lyso-tracker and


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miot-tracker at 2, 4, and (4+2) 6, (4+4) 8, (4+8) 12 h respectively. Figure 5 showed the confocal images of TPAMC-UCNPs@PEG colocalized with lyso-tracker (a) or miot-tracker (b) at different time points. The yellow color in the merge images of TPAMC-UCNPs@PEG and lyso-tracker indicated the accumulation of nanoprobes in endo/lysosome at 2 and 4 h. Whereas only slightly overlap between UCL emission and mito-tracker were observed in the first 4 hours. After removing the excess nanoprobes an obvious separation of the probes and lysosome was observed from 4 to 12 hours. Meanwhile, the overlap between the probes and mitochondria increased continuously. Clearly yellow color was presented in the merge images of TPAMC-UCNPs@PEG and mito-tracker after 8 h indicating the final trafficking of the nanoprobes to mitochondria. The co-localization effect was further quantified by the Pearson coefficient and presented as a time-dependent curve in Figure 5c. The overlap of TPAMC-UCNPs@PEG with lyso-tracker and mito-tracker were manifested as gradual decrease (from 0.76 to 0.15) and increase (from 0.20 to 0.92) respectively, as time went by, which described the intracellular trafficking pathway from lysosome to mitochondria of the nanoprobes. These data revealed a lysosomal-assisted mitochondrial targeting process. After endocytosed by the target cells, the nanoprobes were transported via the endo/lysosome vesicles,58, 59 in which the PEG shell was disrupted by the acidic environment. Then the “activated” TPAMC-UCNPs escaped from endo/lysosomes and further localized to mitochondria by the exposed targeting sites. The efficient release of the nanoprobes from lysosome



was attributed to the strong positive charge (~ +29 mV) of the deshielding TPAMC-UCNPs, which was known as the “proton sponge” effect.60 To confirm the effect of the TPAMC on mitochondrial-targeting profile of the nanoprobe, we also prepared TPAMC-free nanoparticles (noted as UCNPs@PEG) by encapsulating UCNPs with DSPE-mPEG and PHIS-b-PEG in the same conditions, for comparison. Hydrodynamic size and zeta potential of the resulting UCNPs@PEG were measured to be 39.6 ± 2.0 nm and −19.83 mV under pH 7.4, or 76.3 ± 4.3 nm and +5.08 mV under pH 5.5, respectively (Figure S18). The slight positive potential observed in acid condition should be attributed to the protonated nitrogen atoms in PHIS-b-PEG. Although UCNPs@PEG showed a zeta potential reversal and similar particle size characteristics to TPAMC-UCNPs@PEG, a rather low co-localization ratio within mitochondria was measured after incubation for 12 h (Pearson’s coefficient, 0.26, Figure S19), which indicated that the nanoparticles showed non-specificity to mitochondria in the absence of TPAMC. Therefore, it could be certain that TPAMC played a critical role in the mitochondrial localization progress as targeting site.

2.7. Ratiometric imaging of mitochondrial H2S in living cells In light of its excellent H2S response and mitochondrial targeting properties, the nanoprobe was next employed for ratiometric imaging of H2S in mitochondria of HeLa cells. Cysteine was reported to be a H2S precursor that produced endogenous hydrogen sulfide through enzymatic reaction in living cells. To mimic the intracellular


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H2S production, pretreatment of the cells with a thiol trapping reagent N-ethylmaleimide (NEM)61 were performed to remove free cellular thiols. The upconversion

luminescence

imaging

were

conducted

by

excitation

of

TPAMC-UCNPs@PEG at 980 nm leaser to detect the signals in mitochondria. After incubating the NEM-treated cells with the nanoprobe for 12 h, only weak UCL signals were detected for the green channel (Figure 6a), which indicated the suppression of H2S generation in mitochondria. Then the cells were supplemented with cysteine to stimulate the production of endogenous H2S. When incubated with cysteine (1mM) for 1h, a significant enhancement of UCL emission was observed for the green channel in probe labeled cells (Figure 6d), indicating the rise of endogenous H2S contents. Compared to the significantly enhanced UCL intensity in green channel, the red UCL at 660 nm remained almost unchanged. we further investigated the ratiometric UCL imaging of mitochondrial H2S level. The ratiometric luminescence signal of green to red channel was used to monitor the changes of endogenous H2S in mitochondria. The nanoprobes incubated with NEM-pretreated HeLa cells showed a UCL ratio less than 0.3 in the control group (Figure 6d). Nevertheless, a significant increase in the UCL ratio up to 0.7 was observed in the group further treeated with cysteine

(Figure

6h).

These

data

demonstrated

the

capability

of

TPAMC-UCNPs@PEG for monitoring cysteine-stimulated H2S production in mitochondria via ratiometric UCL imaging.

2.8. Monitoring of mitochondrial H2S in colorectal cancer mouse model



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As previously reported, nanoparticles with size between 30-200 nm are optimal for tumor accumulation by the enhanced permeability and retention (EPR) effects.62 In addition, PEG modification is believed to prolong the blood circulation time and promote the EPR effects of nanomaterials.63 Therefore, TPAMC-UCNPs@PEG should be suitable for imaging of tumor-related H2S in vivo. Human colorectal cancer tissue are reported to present a high content of H2S because of the overexpression of cystathionine-b-synthase (CBS), an important H2S synthetase.64 Recent researches have revealed that CBS is mostly localized to the mitochondria in colon cancer cells, increasing the production of mitochondrial H2S, which is closely related to the proliferation and bioenergetics of cancer tissue.65,

66

Herein, we employed

TPAMC-UCNPs@PEG as a UCL probe for monitoring of mitochondrial H2S in colorectal cancer mouse model. The enrichment of TPAMC-UCNPs@PEG at tumor site was firstly investigated. The HCT116 (human colorectal cancer cell line) tumor-bearing mice were intravenously injected with TPAMC-UCNPs@PEG and UCL imaging was conducted after designated time intervals on an in vivo imaging system (IVIS) equipped with a 980 nm semiconductor laser. As figure 7a showed, the UCL intensity in tumor region increased gently and reached the maximum at 8h, indicating the effective accumulation of the nanoprobes in tumor site. This result was also confirmed by the ex vivo quantification of UCL signals recorded tumor and organs excised from mice 24 h after injection (figure 7b). In addition to tumor, high uptake of TPAMC-UCNPs@PEG was ameasured in the liver and spleen, which was


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consistent with previous report that UCNPs were mainly cleared from the body through hepatobiliary excretion.29 To investigate the ability of the nanoprobe for monitoring of different mitochondrial H2S levels in colorectal cancer mouse model, the HTC116 tumor-bearing mice were pretreated with injection of TPAMC-UCNPs@PEG for 24 h to label the mitochondria of tumor cells. Subsequently, they were randomly divided into two groups. One group was treated with S-adenosyl-L-methionine (SAM), an allosteric CBS activator that could stimulate H2S production by CBS.65 The other group was treated with physiological saline as control. The UCL imaging was performed under 980 nm excitation with an emission window from 450-700 nm. The UCL signals collected enhanced about 15.3% in the group treated with SAM (Figure S20), indicating the exaltation of mitochondrial H2S in tumor tissues. The mice were then sacrificed and ratiometric imaging studies were executed on tumor slices of mice from two groups. Compared with the control group, the ratio signals of green to red UCL increased from 0.5 to more than 0.8 in SAM treated mice as described in the ratiometric images (Figure 7d). Imaging experiments with the nanoprobe suggested the elevation of mitochondrial H2S level caused by CBS activity upregulation in colon tumor. These results demonstrated the potential of TPAMC-UCNPs@PEG for UCL monitoring of mitochondrial H2S levels ex vivo and in vivo.

3. Conclusion



Page 20 of 46

In summary, we reported a novel strategy to construct mitochondria-targeted nanoprobes for detecting and imaging of hydrogen sulfide in mitochondria with ratiometric UCL method. This nanoprobe was composed of a sensing core of merocyanine dye TPAMC-modified UCNPs and a pH-sensitive PEG shell. The TPAMC acting as both mitochondrial targeting and H2S-response sites was initially hidden underneath the PEG shell and exposed though an acid-assisted activation process to management the targeting and detection functions. The strong FRET effect between UCNPs and TPAMC allowed sensitive detection of H2S with ratiometric UCL signals. It was uncovered that the nanoprobe was stable with compact PEG shell and negative surface potential under normal physiological pH at 7.4, while showed interruption of the PEG shell and exposure of recognition sites under low pH at 5.5 (endo/lysosome environment). Cell uptake experiments revealed an acid-activated mitochondria targeting process via lysosomal delivery. TPAMC-UCNPs@PEG was able to be used not only for ratiometric UCL imaging of mitochondrial H2S generation in living cells, but for monitoring mitochondrial H2S levels in colon cancer mouse models. This UCL nanoprobe is envisioned to be an effective tool for studies on the biological and pathological roles of mitochondrial H2S.

4. Experimental Section Materials. Ammonium fluoride (98%), ytterbium (III) chloride hydrate (99.9%), yttrium (III) chloride hydrate (99.9%), erbium (III) chloride hydrate (99.9%), thulium (III)

chloride

hydrate

(99.9%),

oleic

acid

(90%),


1-octadecene

(90%),

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2,3,3-trimethylindolenin, 4-formyltriphenylamine, 11-bromoundecanoic acid, and poly(L-histidine)

(PHIS)

Methoxy-(polyethylene

were

purchased

glycol)-2000-Succinimide

ester

from

Sigma-Aldrich.

(mPEG2000-NHS)

and

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000] (mDSPE-PEG) were purchased from ZZBIO. CO., LTD (Shanghai, China). Mito-Tracker Green and Lyso-Tracker DND-26 were purchased from Molecular Probes (Invitrogen, USA). All the reagents were sued as starting materials without further purification. Characterization. NMR spectra were recorded by a Bruker Ultra Shield Plus 400 MHz spectrometer using tetramethylsilane (TMS) as the internal reference. Mass analysis was performed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system (MALDI-TOF, Bruker AutoFlex). Transmission electron microscope (TEM) images were acquired on JEM 2010 operating with an acceleration voltage of 100 kV. HR-TEM were operated on a Tecnai G2 F20 microscope. The zeta potentials were determined by a zeta potential analyzer (ZetaPALS, Brookhaven Instruments).The hydrodynamic sizes of nanoparticles were measured on a 90 Plus particle size analyzer (Brookhaven Instruments). Fluorescence and UV-Vise absorption spectra were collected by RF-5301PC spectrofluorometer and Shimadzu UV-3600 ultraviolet-visible-near-infrared spectrophotometer severally. The UCL spectra were collected on Edinburgh FLSP920 fluorescence spectrophotometer equipped with 980 nm CW laser (2 W). All confocal images were acquired on Leica TCS SP5X Confocal Microscope System with imaging software Fluoview FV 1000.



Page 22 of 46

In vivo UCL imaging experiments were performed with IVIS Lumina K (Perkin Elimer) equipped with a 980 nm CW laser. Synthesize of N-(10-carboxydecyl)-2,3,3-trimethylindolenium bromide (1). In 50 ml flask equipped with a magnetic stirrer 11-bromoundecanoic acid (14.6g, 55mmol) and 2,3,3-trimethylindolenin (7.9g, 50mmol) were added into 10 mL acetonitrile solurtion and refluxed for 24h. After reaction was compelted, the solvent was removed by evaporation. The residual viscous oil was precipitated in diethyl ether. The obtained solid was further washed with ether for several times to provide a purple solid of 1. The products were used for the next step without characterization. Synthesize of triphenylamine-merocyanine (TPAMC). In 50 ml flask equipped with a magnetic stirrer, 4-formyltriphenylamine (175 mg, 0.6 mmol) and compound 1 (190 mg, 0.5 mmol) were dissolved in 10ml of absolutely ethanol. After 24h of refluxing, the ethanol was removed with rotary evaporation.

The obtained crud

product was purified by column chromatography (SiO2, methanol/DCM 1:30) and then washed with diethyl ether for three times to afford TPAMC as a purple solid (0.22g, yield 69%). 1H NMR (400 MHz, CDCl3, δ): 8.06–8.11 (m, 3H, Ar-H), 7.80 (d, J = 15.7 Hz, 1H; Ar-H), 7.49 (m, 4H; Ar-H), 7.36 (t, J = 7.6 Hz, 4H; Ar-H), 7.20 (m, 6H; Ar-H), 7.02 (d, J = 8.6 Hz, 2H; Ar-H), 4.95 (t, J = 7.2 Hz, 2H; -CH2-), 2.34 (t, J = 7.5 Hz, 2H; -CH2-), 1.92 (m, 2H; -CH2-), 1.90 (s, 6H; -CH3), 1.48–1.61 (m, 4H; -CH2-),1.24–1.33 (m, 12H; -CH2-); 13C NMR (100 MHz, CDCl3, δ): 180.25, 173.91, 154.47, 153.88, 145.27, 142.80, 141.15, 134.23, 129.89, 129.22, 128.51, 126.74,


Page 23 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


126.22, 126.03, 122.42, 119.24, 113.87, 108.7860.15, 51.49, 47.56, 34.35, 29.25, 29.15, 29.05, 27.53, 24.92, 14.26; MALDI-TOF-MS m/z: 599.917 [M]+. Synthesize of poly (L-histidine)-b-poly(ethylene glycol) (PHIS-PEG). The pH-sensitive block copolymer PHIS-PEG was synthesized according to the literature67 with some modification. In 25 mL flask equipped with a magnetic stirrer poly (L-histidine) (10 mg) and mPEG-NHS (2.4 mg) were dissolved in 5 mL DMSO. The reaction solution was stirred at RT for 24h under argon atmosphere, and dialyzed using a dialysis bag with the molecular weight cut-off (MWCO) of 5 kDa against deionized water for 3 days. Then the solvent was removed by freeze-drying to afford a white flocculent product (7.2 mg, yield 60%). 1H NMR (400 MHz, CDCl3, δ): 3.98 (m, -CH-), 3.66 (s, -CH2-O-), 1.87–2.05 (m, -CH2-). Synthesize of OA-UCNPs. The OA-coated up UCNPs were synthesized by solvothermal procedure in accord with the literature.68 In 100 ml three-necked flask YbCl3 •6H2O (0.20 mmol), YCl3 •6H2O (0.778 mmol), TmCl3 •6H2O (0.002 mmol) , and ErCl3 •6H2O (0.02 mmol), were added into 10 ml methanol and disperse by ultrasonication. Then methanol was removed by heating, 15 mL 1-octadecene and 7 mL oleic acid were added. The reaction mixture was first heated to 160 °C to obtained a homogeneous solution, then cooled down to RT. A solution of NH4F (4 mmol) and NaOH (2.5 mmol) was added slowly, which was then heated slowly to 300°C under argon environment for 1h. After the reaction solution was cooled down, ethanol was added. The OA-UCNPs were obtained by centrifugation and washed repeatedly with ethanol.



Page 24 of 46

Assembly of TPAMC-UCNPs@PEG. In a 25 mL flask UCNPs (5 mg) and TPAMC (5 mg) were added into 6 ml of chloroform, and ultrasonicated to form a dispersion. The obtained dispersion was vigorously stirred for 12h at RT. Subsequently, the mixture of DSPE-mPEG (5 mg) and PHIS-PEG (5 mg) was added, and continued to stir overnight. After removing the solvent, the solid was transferred in to water solution by ultrasonication and filtered with a 220nm syringe filter. The obtained transparent solution was centrifugated (10000 rpm, 10 min) to obtained purple precipitate, which was further washed with water/ethanol (volume ratio 9:1). The collected nanoprobes were stored at 4 °C. Cell Culture. human breast cancer (MCF-7) and HeLa cell lines were cultivated in Dulbecco’s Modified Eagles Medium (DMEM) containing 10% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin at 37 °C and 5% CO2 atmosphere.

The

cells were transferred into dishes at 80% confluency for imaging experiments. Cytotoxicity Assay. HeLa cell lines were used to assess the cytotoxicity of TPAMC-UCNPS@PEG by MTT assays.

The cells were seeded in 96-well plates

and incubated for 24 h in DMEM at 37 °C under 5% CO2. Then the cells were incubated with fresh medium containing different dose of TPAMC-UCNPs@PEG (0, 1, 10, 100, 200, 400, 800 μg mL−1) at 37 °C under 5% CO2. After 24h, the cells were treated with MTT solution (5 mg mL−1) for another 3h. Then the culture medium was washed and 200 μL of DMSO was added into each well. The absorbance of each well at 490 nm was recorded by a microplate reader (BioTek, PowerWave XS/XS2, U.S.). Each experimental group contained six wells, and the mean absorbance was used to


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calculate the cellular viability relative to the control group (0 μg mL−1 TPAMC-UCNPs@PEG), whose mean absorbance was designated as 100% cell viability. Mitochondrial colocalization imaging. HeLa Cells were plated into 35 mm glass-bottom dishes for 24h, then added 2 mL DMEM medium solution of TPAMC-UCNPs@PEG (0.1 mg mL−1) and further incubated under 37 °C and 5% CO2. The extracellular nanoprobes were removed by washing with PBS after 12 h. Mitochondria co-staining was performed with Mito-Tracker Green (20 nM) in fresh medium for 20 min before observed by confocal laser scanning microscope. The fluorescence of MTG was collected at 500-550 nm with excitation at 488nm, and the red UCL was collected at 620-680 nm by using a Ti-Sapphire laser excitation wavelength at 980 nm. Mitochondrial colocalization imaging in MCF-7 cells was done in the same procedure as above. Subcellular fractions separation. TPAMC-UCNPs@PEG (0.1 mg mL−1) was incubated with HeLa cells for 12h. Mitochondria of the probe labeled cells were isolated by using the mitochondria isolation kit (Beyotime, Shanghai, China) following

the manufacturer's instructions. The obtained lysates of mitochondrial and

cytoplasmic fractions were both diluted to 2 mL with PBS buffer. The distribution of TPAMC-UCNPs@PEG in each fraction were assessed using an Edinburgh FLSP920 fluorescence spectrophotometer with an external 3 W adjustable CW 980 nm laser. Intracellular trafficking monitoring. HeLa Cells were plated into 35 mm glass-bottom dishes, then divided into ten groups. After 24 h of incubation, the



nanoprobe TPAMC-UCNPs@PEG (0.1 mg mL−1) was added, and continued to be incubated under 37°C and 5% CO2. Two groups of cells were harvested at 2 h, the other 8 groups were washed with PBS to clear excess nanoprobes at 4 h and further incubated with fresh medium for 0, 2, 4, and 8 h. After each designated time interval, two groups of cells were harvested. They were then stained with Mito-Tracker Green (20 nM) and Lyso-Tracker DND-26 (50 nM) for 20 min, respectively, and used for subcellular localization imaging by a confocal laser scanning microscope. The imaging signals were captured at 505-555 nm (excitation 488 nm) for Lyso-Tracker, 500-550 nm (excitation 488 nm) for Mito-Tracker, and 620-680 nm (excitation 980 nm) for red UCL. Detecting mitochondrial H2S in living cells. HeLa cells were plated into glass-bottom dishes for 24h, then treated with NEM (500 μM) to clear the intracellular thiols beforehand. After 1h, the nanoporbes TPAMC-UCNPs@PEG (0.1 mg mL−1) were added and further incubated at 37 °C under 5% CO2 for 12h to label mitochondria. After removing the extracellular nanoprobes, the experimental group was further treated with cysteine (1 mM) for 1h. The UCL imaging signals were captured in two emission windows of 500-560 nm (green channel) and 600-680 nm (red channel), under 980 nm excitation. Mouse model. All animal experiments were carried out in accordance with the guidelines of the Laboratory Animal Center of Jiangsu KeyGEN Biotech Corp., Ltd. HCT116 tumor-bearing mice (KeyGEN Biotech, Nanjing, China) were used after 3 weeks of implantation. The mice were injected with the nanoprobes solution in


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physiological saline (2 mg mL−1, 150 μL) and observed with an in vivo imaging system

(IVIS)

(Lumina

K,

Perkin

Elmer).

The

UCL

signals

of

TPAMC-UCNPs@PEG were captured at 450-700 nm by excitated at 980 nm. After 12h, the probe labeled mice were further treated with subcutaneous injection of S-adenosyl-L-methionine or physiological saline the in tumor region for 4h. UCL images for the two groups of mice were recorded by the IVIS (Lumina K, Perkin Elimer), and then the mice were dissected to isolate the tumor tissues. The tumor slices were used for ratiometric UCL imaging on the CLSM. The imaging signals were collected under 980 nm excitation and emission windows of 500−560 nm (green UCL) and 600−680 nm (red UCL)..



Scheme

1.

Design

of

TPAMC-UCNPs@PEG,

Page 28 of 46

and

the

illustration

of

lysosome-assisted mitochondrial targeting process and UCL monitoring of mitochondrial H2S in cell.


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Scheme 2. The illustration of LRET processes from UCNPs to TPAMC in the absence and present of H2S.

Figure 1. TEM images of OA-UCNPs in cyclohexane (a), TPAMC-UCNPs in chloroform (b), and TPAMC-UCNPs@PEG in water (c), magnification inset shows the organic layer. (d) HR-TEM image of TPAMC-UCNPs@PEG. (e) Dynamic light scattering

(DLS)

result

of

TPAMC-UCNPs.

(f)

UCL

TPAMC-UCNPs@PEG (red line) and OA-UCNPs (blue line)

spectra

under 980 nm

excitation; Absorption spectrum of TPAMC-UCNPs@PEG (black line);


of


Page 30 of 46

Figure 2. (a) Particle size distribution of TPAMC-UCNPs@PEG in PBS buffers of pH 7.4 (blue line) and 5.5 (red line). (b) Zeta potential of TPAMC-UCNPs@PEG in PBS buffers of pH 7.4 (blue line) and 5.5 (red line), and zeta potential of TPAMC (black dash line).

Figure 3. (a) Gradual evolution of absorption spectra of TPAMC-UCNPs@PEG (0.05 mg mL−1) in PBS solution (pH 7.4) upon treatment with HS− (0-60 μM). Insert: photo of color change in the presence of HS−. (b) Gradual evolution of upconversion luminescence spectra of TPAMC-UCNPs@PEG (0.05 mg mL−1) in PBS solution (pH 7.4) upon treatment with HS− (0-52 μM). Insert: change of green UCL in the presence of HS−. (c) UCL emission intensity ratio at 540 to 800 nm (UCL540 / UCL800) as a function of the HS−concentration. (d) UCL signal (UCL 540 nm / 800 nm) changes of 0.05 mg mL−1 TPAMC-UCNPs@PEG upon treated with various analytes:

1) SCN−

(500 μM), 2) SO32− (500 μM), 3) S2O3 2− (500 μM), 4) H2O2 (200 μM), 5) CIO− (200 μM), 6) O2·− (200 μM), 7) NO2− (800 μM), 8) K+ (800 μM), 9) Na+ (800 μM), 10)


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Ca2+ (800 μM), 11) Fe3+ (800 μM), 12) Hcys (500 μM), 13) GSH (500 μM), 14) Cys (500 μM), 15) HS− (50 μM). Error bars indicate standard deviation (n = 3).

Figure 4 ．

Intracellular localization of TPAMC-UCNPs@PEG in Hella cells.

TPAMC-UCNPs@PEG were incubated with Hella cells for 12h followed by treatment with Mito Tracker. Colocalization imaging eperiments were performed in single-photon channel (a) and multi-photon channel (d) respectively: Green, MTG fluorescence; Red, TPAMC fluorescence (top, excitation: 559 nm, collection: 650−750 nm) and UCL emmision (buttom, excitation: 980 nm, collection: 600−680 nm); Yellow, merged signals. Intensity profile of ROIs between MTG and TPAMC-UCNPs@PEG single-photon channel (b) and multi-photon channel (e). Correlation plot of the intensities of Mito Tracker and TPAMC-UCNPs@PEG in single-photon channel (c) and multi-photon channel (f).



Figure 5. Intracellular trafficking of TPAMC-UCNPs@PEG. HeLa cells were first incubated with TPAMC-UCNPs@PEG (0.1 mg mL−1) for 4 h, then removed excess nanoprobes and further incubated with fresh media for another 2, 4 or 8 h. Co-localization imaging were performed with lyso-tracker (a) or miot-tracker (b) at different time points . The UCL emmisom was collected at 600−680 nm, under 980 nm excitation. The scale bars are 20 μm. (c) Pearson’s correlation between TPAMC-UCNPs@PEG and Lyso Tracker (black line) and Mito Tracker (Red line) at each time spot. Error bars indicate standard deviation (n = 3).


Page 32 of 46

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Figure 6. Ratiometric UCL imaging of mitochondrial H2S in HeLa cells. All the cells were pretreated with 500 μM NEM for 1h. (a-d) Cells incubated with TPAMC-UCNPs@PEG (0.1 mg mL−1) for 12h. (e-h) Cells incubated with TPAMC-UCNPs@PEG (0.1 mg mL−1) for 12h and then treated with 1mM Cys for another 1h. The emission signals were acquired by excitation at 980 nm, in collection windows of 500−560 nm (a, e) for green UCL and 600−680 nm (b,f) for red UCL. (c, g) Overlay images of green channel, red channel and bright-field. (d ,h) Ratiometric UCL images with ratio of green to red channels.



Figure 7. (a) In vivo UCL imaging of HCT116 tumor-bearing mouse after intravenous injection with TPAMC-UCNPs@PEG. (b) UCL images of tumor and main organs at 24h after intravenous injection with TPAMC-UCNPs@PEG. (c) The quantitative fluorescent radiance analysis of biodistribution of the nanoprobes in various organs of mice after 24 h intravenous injection. Error bars indicate standard deviation (n = 3). Images were acquired under 980 nm excitation and emission windows of 450−700 nm. (d) Ratiometric UCL images of tumor slices of mice injected intravenously with TPAMC-UCNPs@PEG for 24 h and further treated with physiological saline (control, top raw) and SAM (SAM+, button raw). Images were


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acquired under 980 nm excitation and emission windows of 500−560 nm (green UCL) and 600−680 nm (red UCL).

Supporting Information Supporting Information is available free of charge on the ACS Publications website

Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 21674048 and 21574064), the Jiangsu Province “333 high-level Personnel Training project” (No. BRA2016379) and the Primary Research & Development Plan of Jiangsu Province (No. BE2016770).

Author Contributions Q.F. and Q.Z. conceived and designed the research, supervised the project. X.L. performed most of the experiments and wrote the article. H.Z. synthesized the UCNPs and helped with the UCL characterizations. J.Y. carried out the in vivo experiments. C.Y. and J.L. helped with the fabrication of the nanoparticles. Z.Y., Y.T. and W.H. participated in the discussion of the data and the modification of the article.

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A Lysosome-Assisted Mitochondrial Targeting Nanoprobe based on

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