Golgi apparatus-targeted AIEgens for effective photodynamic therapy through the crosstalk of Golgi apparatus and mitochondria

Golgi apparatus (GA) oxidative stress induced by in situ reactive oxygen species (ROS) could severely damage the morphology and function of GA, which may open up a new avenue for effective photodynamic therapy (PDT). However, due to the lack of effective design strategy, photosensitizers (PSs) with specic GA targeting ability have not been reported. Herein, we report aggregation-induced emission luminogen (AIEgen) based PSs that can effectively target to GA with Pearson correlation coecient (PCC) up to 0.98 and singlet oxygen generation rate up to 77.8%. GA fragmentation and cleavage of GA proteins (p115/GM130) were observed upon light irradiation. Meanwhile, the apoptotic pathway was activated through a crosstalk between GA oxidative stress and mitochondria in HeLa cells. Finally, TPE-PyT-CPS can effectively inhibited tumour growth in vivo with negligible adverse effect. This work provided a promising design strategy for the development of PSs with specic GA targeting ability, which is of great importance for precise and effective PDT. from its high singlet oxygen generation and effective GA targeting features, TPE-PyT-CPS signicantly inhibited the tumor growth of mice without obvious adverse effects on normal tissues in vivo after PDT. This work provided a reliable design strategy for the development of AIEgen based GA targeting PSs, which offered a new avenue for precise and ecient PDT through transferring stress signals from Golgi apparatus to mitochondria.


Introduction
Photodynamic therapy (PDT) is an attractive tumor treatment which could be spatially and temporally controlled by light. [1] It shows advantages such as minimal invasiveness, selective killing of tumor by light induced cytotoxic reactive oxygen species (ROS), particularly singlet oxygen ( 1 O 2 ), and repeated administration without drug resistance, which are problems related to the use of chemotherapeutic drugs (e.g., cisplatin). [2] However, traditional photosensitizers (PSs) tend to show diminished 1 O 2 quantum yield at aggregated states due to the enhanced non-radiative decay rate, which might decrease their PDT e ciency. [3][4][5] In this regard, PSs with aggregation induced emission (AIE) characteristics are superior to traditional PSs since they show both increased 1 O 2 and uorescence quantum yield upon aggregation. [6][7] In addition, AIE luminogens (AIEgens) with intramolecular charge transfer (ICT) effect could facilitate the intersystem crossing (ISC) by decreasing ΔE ST (energy gap between the lowest singlet state S 1 and the lowest triplet state T 1 ), which could be adopted for developing heavy-atom-free PSs that show minimum dark toxicity. [8][9] Thus, AIEgen based PSs could serve as appealing alternatives for traditional PSs and have attracted much attention for e cient PDT. [10][11][12] Studies have revealed that 1 O 2 shows very short lifetime (half-life: 0.03 to 0.18 ms) and narrow diffusion distance in biological systems (a radius of < 0.02 μm). [1] Hence, it is desirable to develop PSs that could target organelles and generate high dosage of 1 O 2 in situ to direct cause dysfunction of subcellular organelles and activate speci c cell death signals. Several PSs targeting mitochondria, [13][14][15] endoplasmic reticulum (ER), [16][17][18] lysosome [19][20] and cell membrane [21][22] have been reported and exhibited high PDT e ciency. However, due to the lack of effective Golgi apparatus (GA) targeting strategies, speci c GA targeting AIGgen based PSs have seldomly been reported. Golgi apparatus is a central node at the intersection point between the exocytic and endocytic channels in intracellular membrane transporting, which plays a pivotal role in the classi cation of newly synthesized and recycled proteins and lipids to their nal destinations. [23] Oxidative stress will induce signi cant damages on the structure and physiological function of GA. [24] These changes in GA may trigger and propagate downstream stress signals, leading to GA disruption or even apoptosis. [25][26][27][28] Therefore, GA targeting AIEgen based PSs could serve as good candidates for e cient PDT.
Herein, we report AIEgen based GA targeting PSs and their applications in e cient suppression of tumor cells by PDT induced GA oxidative stress ( Figure 1A). Among the AIEgens, TPE-PyT-CPS showed a high 1 O 2 quantum yield of 77.8 % and excellent GA targeting ability with a Pearson's correlation coe cient (PCC) of 0.98. Structure-property relationship studies suggested that the cyano-group played a key role for speci c GA targeting, while the strong ICT process and pyrene group contributed for high ISC rate and 1 O 2 generation quantum yield. GA suffered from severe oxidative stress and fragmentation after ROS was generated in situ upon PDT. As shown in Figure 1B, the structural protein p115 of GA was found to be cleaved into N-terminal and C-terminal fragments, the latter then translocated into nucleus, resulting up regulation of p53 and dysfunction of mitochondria and activation of apoptotic pathway. Finally, prominent inhibition of tumor cell growth was demonstrated without noticeable adverse effect. This study not only provided an e cient strategy for developing GA targeted PSs, but also offered new insights for e cient and precise treatment of cancers.

Results And Discussion
Design, synthesis, spectroscopic and ROS generation properties Traditional PSs usually have to face problems such as aggregation caused quenching (ACQ) and potential dark toxicity due to heavy atom modi cation. [29][30] AIEgen based PSs with a heavy atom free Dπ-A structure can e ciently separate the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) distribution, which is helpful to promote the rate of intersystem crossing (k ISC ) process and thus bene cial to increase 1 O 2 generation. [30] TPE-PyT-CPS was designed with an AIE active tetraphenylethene (TPE) derivative as the electron donating group (EDG) and a cyanopyridinium salt moiety act as the electron withdrawing group (EWG). To improve the ISC e ciency, a pyrene ring and thiophene group was introduced as π spacer for better separation of HOMO-LUMO and decreasing ΔE ST . The synthesis procedures of TPE-PyT-CPS and its control compounds were presented in Supporting Information. All the intermediates and target compounds were fully characterized by 1 H and 13 C NMR spectroscopy and HRMS.
The spectroscopic properties of all compounds in water were studied with the UV−vis and photoluminescence (PL) spectrometer ( Figure S17), in which the TPE-PyT-CPS shows a maximum absorption peak at 498 nm and an NIR emission at 693 nm, respectively. The Stokes shift was nearly 200 nm, which can reduce the interference of background signal and helps to improve the image-guided therapy. Moreover, compared with the TPE-PyT-PS and TPE-PyT-CP, the UV absorption spectra and uorescence emission spectra of TPE-PyT-CPS and TPE-T-CPS show a signi cant red shift, which is due to the stronger ICT effect caused by EDGs of cyano-group and pyridinium salt group. As depicted in Figure 2A and B, TPE-PyT-CPS in pure acetonitrile exhibited very weak emission, while with the water fraction increasing to 30%, the emission enhanced slightly. Further increasing the water fraction to 99%, lead to a distinct enhancement on PL intensity with a gradual red shift of the emission maximum to 680 nm, indicating typical AIE characteristics of TPE-PyT-CPS. The uorescence quantum yields of TPE-PyT-CPS in acetonitrile and water (with 1% acetonitrile, v/v) were 2.74% and 17.1%, respectively. In addition, The AIE features of the other three AIEgens were also con rmed using a mixed solvent (acetonitrile and water) system with different water fractions ( Figure S18, Table S1).
Next, dynamic light scattering (DLS) tests showed that the particle sizes of TPE-PyT-CPS in water and acetonitrile were around 410 nm and 1 nm, respectively ( Figure 2C). The results suggested that the TPE-PyT-CPS is almost monodisperse in acetonitrile but form aggregates  Table S1). TPE-PyT-PS showed much higher Φ Δ than TPE-PyT-CP, suggesting that the pyridinium group contributed more for ROS generation than the -CN group. The stronger electron withdrawing ability of pyridinium group than -CN group lead to stronger ICT effect and more e cient ISC of TPE-PyT-PS than TPE-PyT-CP. It is also reasonable that TPE-PyT-CP and TPE-PyT-PS showed lower ROS generation yields than TPE-PyT-CPS and TPE-T-CPS, since the former two showed weaker ICT effects due to the lack of EDGs of either pyridinium group or -CN group.
To understand the mechanism of higher ROS generation ability of TPE-PyT-CPS than that of TPE-T-CPS, which showed identical electron donor and acceptor but different π spacers, density functional theory (DFT) calculation was conducted. As shown in Figure 3A, although the HOMO and LUMO electronic distributions of TPE-T-CPS are well separated, there are some overlaps at the region of thiophene and the nearby phenyl ring of TPE-T-CPS. However, by introducing an additional large π spacer pyrene, the overlap between HOMO and LUMO of TPE-PyT-CPS was distinctly reduced. As a result, the ΔE ST value of TPE-PyT-CPS was calculated to be 0.20 eV, smaller than that of TPE-T-CPS, which was calculated 0.39 eV ( Figure 3B, Table S2). The DFT calculation was consistent with the experimental data that TPE-PyT-CPS showed higher 1 O 2 quantum yield than TPE-T-CPS in water, which veri ed our design strategy that the introduction of pyrene spacer can help improve the HOMO-LUMO separation within the ICT system, thus elevate the 1 O 2 generation ability.

Subcellular localization study
The intracellular localization of TPE-PyT-CPS was investigated by confocal laser scanning microscopy (CLSM). As shown in Figure 4, the Pearson's correlation coe cients (PCCs) between uorescence images of marked commercial dyes and the TPE-PyT-CPS were determined to be 0.36 for Mito, 0.54 for ER, 0.98 for GA and 0.61 for lysosome, respectively. The results indicated that TPE-PyT-CPS localized preferentially in GA with high speci city. It was strange that TPE-Py-CPS showed minimum localization in mitochondria, although it contained a cationic pyridinium group. Therefore, the structure-property relationships of the four compounds were investigated to gure out which moiety was responsible for the GA targeting ability ( Figure 1A). The PCC values between the uorescence images of GA marker and those of each compound were determined to be 0.98, 0.90, 0.89 and 0.68 for TPE-PyT-CPS, TPE-T-CPS, TPE-PyT-CP and TPE-PyT-PS, respectively ( Figure 4, Figure S21-S23). We also tested the lipophilicity of all compounds ( Figure S20, Table S2) because lipophilicity was reported to play an important role in the cell uptake and distribution of compounds in subcellular organelles. [31] The and TPE-PyT-PS, respectively. There seemed to be no direct link between lipophilicity and GA targeting ability of these AIEgens. However, it was obvious that the absence of the -CN group in TPE-PyT-PS made it a poor GA targeting dye, indicating that the -CN group played an essential role for GA targeting in this AIEgen system.

ROS generation and PDT in tumor cells
The high 1 O 2 quantum yield and excellent GA targeting ability of TPE-PyT-CPS encouraged us to investigate its ability to kill cancer cells through PDT. The in situ 1 O 2 generation capability was evaluated by detecting the uorescence intensity of Singlet Oxygen Sensor Green (SOSG). As shown in Figure  To study the cell death mechanism of tumor cells, Annexin V-FITC and propidium iodide (PI) were used to monitor the process of cell apoptosis induced by TPE-PyT-CPS mediated PDT. HeLa cells were incubated with TPE-PyT-CPS and irradiating with 532 nm laser (65 mW cm -2 , 2 min), followed by Annexin V-FITC and PI staining, then the uorescence images were collected using CLSM. As shown in Figure 5D, the green and red uorescence signals were hardly detected with irradiation alone or with TPE-PyT-CPS but without irradiation. However, after TPE-PyT-CPS incubation and light irradiation for 2 minutes, distinct green and red uorescence were observed in HeLa cells, suggesting the occurrence of cell apoptosis. In addition, compared with the control group, the cells treated with TPE-PyT-CPS and light irradiation, GA oxidative stress caused apoptosis pathway The morphological changes of Golgi apparatus can activate the related signal pathways and trigger cell repair or apoptosis. [34] P115 is a vesicle tethering protein, which functions in Golgi-vesicle tethering and Golgi-cisternal stacking by bridging GM130 and giantin. [35] To further ascertain whether these structural proteins were affected during GA-targeted PDT process, the expressions of p115 and GM130 in HeLa cells were evaluated by western blot. As shown in Figure 7A and B, the expression of GM130 and p115 in HeLa cells was gradually decreased with the extension of irradiation time during PDT, especially for GM130. In addition, we found two new fragments of 30 kD (C-terminal fragment) and 90 kD (N-terminal fragment) proteins which originated from p115 during this process, con rming that p115 was cleaved after GA oxidative stress. Moreover, the western blot experiments demonstrated that p53 and its phosphorylation derivative (Ser15) were signi cantly upregulated, which suggested that PDT induced GA stress was related to apoptosis.
Studies have suggested that the 30 kD residue of p115 can translocate into the nucleus through sumoylation and then interact with p53, leading to p53 phosphorylation, which leads to apoptosis pathway. [36][37] To verify this mechanism, we detected the level of p53 upregulated modulator of apoptosis (PUMA), which was located in the outer membrane of the mitochondria. [38] Indeed, the dramatically increased expression of pro-apoptotic protein PUMA and Bax was observed, accompanied with a down regulation of anti-apoptotic Bcl-2 in HeLa cells during PDT, resulting an increased Bax/Bcl-2 ratio ( Figure 7C, D). In addition, an obvious upregulation of cleaved caspase-3 (a mitochondria-apoptotic protein) was also observed.
The activation of Bax can cause its conformational change and then oligomerization in the outer membrane of mitochondria, which will reduce the mitochondrial membrane potential (MMP,ΔΨ m ). [39][40] Next, the MMP was evaluated through JC-1 staining assay. As shown in Figure 8, the HeLa cells after PDT treatment showed an evident increase in green uorescence of JC-1 monomers and a signi cant decrease in red channel of JC-1 aggregates, suggesting a distinct loss of MMP upon PDT. However, HeLa cells treated by light only, dark and "dark + AIEgen" showed intense red uorescence and weak green uorescence, indicating intact MMPs. The above results suggested that PDT induced GA oxidative stress could severely affect mitochondria homeostasis and active the apoptosis signal pathway (Figure 9).

GA-targeting mediated PDT in vivo
The in vivo PDT performance of TPE-PyT-CPS was evaluated in a subcutaneous HeLa tumor-bearing mouse model. All animal experiments were carried out in accordance with the regulations of the Institutional Animal Care and Use Committee (IACUC) of Nanjing University. Firstly, we evaluated the imaging ability of TPE-PyT-CPS in mice ( Figure 10A) and monitored the uorescence signals at speci ed intervals. Distinct uorescence signal in the tumor sites was observed after intratumoral injection of TPE-PyT-CPS (0.1 mM, 120 μL/200 mm 3 tumor) for 3 hours (Figure S25), and the uorescence intensity reached maximum after 18 hours. To gain the uorescence distribution image of the tumor and other major organs, the mice were sacri ced at 30 h after injection of TPE-PyT-CPS, and the ex vivo uorescence images of isolated organs were shown in Figure S25. The results showed that TPE-PyT-CPS could effectively retain in the tumor tissue and exhibit strong uorescence signal. Moreover, there was almost no uorescence signals originated from other major organs, including heart, spleen, lung and kidney, except for a weak uorescence in liver tissue. The results demonstrated that TPE-PyT-CPS could retain in tumor area and serve as an imaging agent.
Subsequently, the anticancer e cacy of TPE-PyT-CPS was assessed in vivo. Tumor (HeLa) bearing Balb/c mice were randomly divided into four groups (n = 5): saline group (saline light -), saline group with laser irradiation group (saline light +), AIEgen group (AIEgen light -) and AIEgen with laser irradiation group (AIEgen light +). After intratumoral injection of TPE-PyT-CPS (0.2 mM, 100 μL) for 18 h, the tumor area of the mice was irradiated with 532 nm laser of 35 mW cm -2 for 5 min, labeled as AIEgen (light +) groups. As can be seen from Figure 10B, for the control groups (saline light -, saline light+), the tumor volumes increased rapidly regardless of light or dark conditions, suggesting that the tumor growth was not affected in the absence of TPE-PyT-CPS. However, in the presence of our AIEgen, the tumor volumes and weight were effectively suppressed in the light group ( Figure 10C, D). Additionally, the body weights of mice in "AIEgen light +" group showed no obvious difference from those of control group during PDT ( Figure 10E), which clearly demonstrated the minimal side effects of "AIEgen light + " treatment. To understand and verify the different antitumor e cacy and whether TPE-PyT-CPS cause in vivo side toxicity of those treatments, tumors and major organs from the four groups are excised and appraised using H&E (hematoxylin and eosin) staining and TUNEL (terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling) assay for histological analysis. [41] The staining results showed that the TPE-PyT-CPS treated group with laser irradiation (AIEgen light +) exhibited obvious cell apoptosis with pyknotic cells with condensed nuclei ( Figure 10F, Figure S26), whereas the tumors tissues of mice that treated in control groups (AIEgen light -, saline + light and saline) had massive viable cancer cells. Similar results were obtained in the TUNEL assay, the tumors slice of mice treating with PDT exhibited signi cantly higher level of cells apoptosis than the control group. Meanwhile, there was no abnormal pathological morphology or prominent tissue damage in the heart, liver, spleen, lung and kidney of mice in all treatment groups ( Figure S27). Taken together, these results con rmed that TPE-PyT-CPS could e ciently suppress tumor growth by GA targeting mediated-PDT with negligible side effects.

Conclusions
In conclusion, we have designed and synthesized AIEgen based PSs with excellent GA targeting ability and PDT effect. Structure-property relationship studies revealed that the cyano-group played a decisive role in GA targeting, while the introduction of large π spacer pyrene group further enhanced the singlet oxygen quantum yield of the D-π-A system. Large morphological change of GA was observed upon the in situ generation of ROS during PDT. Moreover, we found the GA stress can trigger the mitochondria dysfunction during PDT. The GA-mitochondria crosstalk lead to the collapse of MMP and ultimately cell death. TPE-PyT-CPS showed excellent phototoxicity and negligible dark toxicity to HeLa cancer cells with a large PI over 1500. Finally, bene t from its high singlet oxygen generation and effective GA targeting features, TPE-PyT-CPS signi cantly inhibited the tumor growth of mice without obvious adverse effects on normal tissues in vivo after PDT. This work provided a reliable design strategy for the development of AIEgen based GA targeting PSs, which offered a new avenue for precise and e cient PDT through transferring stress signals from Golgi apparatus to mitochondria.

Methods
Materials and instrument. All chemicals for compounds synthesis were commercially available (Sigma-Aldrich, J&K, Sinopharm, Bidepharma-Tech) and used without further puri cation. The silica gel (300-400 mesh) was used for column chromatography. The 1 H and 13 C NMR spectra were recorded on a Bruker DRX 400 MHz or 600 MHz spectrometer. ESI-MS was determined on a LCQ electrospray mass spectrometer (Thermo Finnigan). High resolution mass spectra (HR-MS) were determined using an Agilent 6540Q-TOF HPLC-MS spectrometer. UV-visible absorption spectra were recorded on a PerkinElmer Lambda 35 spectrophotometer. The transmission electron microscopy (TEM) images were obtained using a JEOL JEM-1011 transmission electron microscope (Japan). Fluorescence confocal imaging was carried out on a laser scanning confocal imaging system (Olympus TH4-200) consisting of ZEISS Laser Scanning Microscope (LSM 710) and a 20-mW output 488 nm argon ion laser. Flow cytometry analysis was performed with a BD LSRFortessa Cell Analyzer.
Density functional theory (DFT) calculation. Theoretical calculations were adopted to rationalize the ISC process of the TPE-PyT-CPS and TPE-T-CPS using Gaussion 09w. The geometries of TPE-PyT-CPS and TPE-T-CPS were optimized based on the method TD-DFT//B3LYP/6-31G(d). TD-DFT was used to predict the excitation energies for the singlet and triplet excited states of TPE-PyT-CPS and TPE-T-CPS in tetrahydrofuran.
Morphology and Size. Dynamic light scattering (DLS) was used to monitor the size of the TPE-PyT-CPS in water, acetonitrile solution and the morphology was observed by Transmission Electron Microscope (TEM).
Singlet oxygen detection in solution. ABDA (9,10-Anthracenediyl-bis(methylene)-dimalonic acid) was used as an indicator to detect singlet oxygen production capacity since the absorbance of ABDA decreases upon reaction with singlet oxygen, and RB (Rose Bengal) was used as an internal reference. For singlet oxygen detection, the ABDA (50 μM) was mixed with the TPE-PyT-CPS in acetonitrile/water (1:99, v/v) and exposed to 532 nm laser irradiation (10 mW cm -2 ). The decomposition of ABDA was monitored by the absorbance decrease at 378 nm. The 1 O 2 quantum yield of the PS in water was calculated using the Subcellular organelle imaging. The HeLa cancer cells were used in the following experiments and cultured using DMEM culture medium containing 10% FBS and 1% penicillin-streptomycin in an arti cial environment (5% CO 2 at 37 o C). The HeLa cells were regularly checked for mycoplasma contamination and then conducted when the cells were grown to 80% con uence in the culture dish. The HeLa cancer cells were cultured in the special confocal chambers at a density of 10 5 cells mL -1 in culture medium. Intracellular ROS detection. The capability of TPE-PyT-CPS and TPE-T-CPS for intracellular ROS production were assessed using singlet oxygen sensor green (SOSG) as an ROS indicator. The HeLa cancer cells were cultured in the special confocal chambers and incubated with TPE-PyT-CPS (0.2 μM) or TPE-T-CPS (0.4 μM) for 6 h at 37 o C. Subsequently, the cells were washed three times with 1×PBS, followed by addition of singlet oxygen sensor green (SOSG, 15 μM) in FBS free culture medium and incubation for 30 min. The above-mentioned process was performed in dark. Then, the cells were exposed to white light irradiation (25 mW cm -2 ) for 2 min, followed by imaging with CLSM. For CLSM imaging, the excitation of SOSG was 488 nm with a collection of uorescence signal at 525 ± 20 nm.
Cytotoxicity Study. Brie y, 100 μL of HeLa cell suspension were added into each well of 96-well plate at a density of 8×10 4 cells mL -1 . After 80% con uence, fresh culture medium (the concentration of DMSO lower than 0.5%) containing a series of concentrations of TPE-PyT-CPS was added and incubated with the HeLa cancer cells for 6 h at 37 o C in dark. Subsequently, the cells were exposed to 532 nm laser irradiation (65 mW cm -2 ) for 2 min. Alternatively, the TPE-PyT-CPS-treated HeLa cancer cells were kept in dark without light exposure. At 24 h post irradiation, the wells were replaced with freshly prepared 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; 2.5 mg mL -1 in PBS) solution. After 4 h incubation, the solution in each well was carefully removed and 150 μL of DMSO was added to each well to dissolve the formazan. The plate was gently shaken for 10 min at room temperature and then the absorbance of MTT at 595 nm was monitored by the microplate reader (Thermo Scienti c Varioskan Flash) in order to determine the cell viability. Cell viability rates (%) and IC 50 values were calculated on the data of three parallel tests.  200, Abcam, ab184787) and Anti-GAPDH (1:10000, Abcam, ab8245) followed by incubation with the peroxidase-labeled goat antirabbit HRP secondary antibody. Western blots were visualized by enhanced chemiluminescence detection system. Relative grayscale was calculated by ImageJ.
Mitochondrial membrane potential (MMP) assay. MMP was evaluated by confocal imaging via JC-1 staining. HeLa cells were seeded in a glass bottom cell culture dish at 40% con uence and cultured in media containing TPE-PyT-CPS (0.2 μM) respectively. After the incubation of 6 h, then cells were stained with JC-1 (Beyotime Biotechnology) following the manufacture's protocol. After rinse three times with incomplete culture medium, the cells irradiated with a laser of 532 nm (25 mW cm -2 , 2 min), and confocal imaging was immediately carried out with Leica SP8. The imaging band path for green channel of JC-M was 520-550 nm (λ ex , 490 nm), while that for red channel of JC-A was 570-640 nm (λ ex , 525 nm).
In vivo uorescent imaging. In vivo uorescent imaging of TPE-PyT-CPS in HeLa tumor-bearing nude mice model was tested by using the IVIS Lumina III in vivo Imaging System (PerkinElmer). Nude mice at 4-5 weeks old were purchased at pushing medical. TPE-PyT-CPS (100 μM, 120 μL) was injected intratumorally and uorescent images were captured by the Lumina III at 0, 3, 6, 12, 18 and 30 hours after injections (Ex: 530 nm, Em:690 nm). The mice were sacri ced at designated time points (24 h) and the tissues including heart, liver, spleen, lung, kidney, were excised and imaged by IVIS ® Lumina III uorescence imaging system.
Histological analysis. All mice of different groups were sacri ced on day 22, and major organs and tumors were separated and made into slices for H&E or Tunel staining. [3] Major organs were collected and xed in 4% paraformaldehyde, which were then embedded into para n, sliced at thickness of 5 μm.
The tissue slices were stained with H&E or TUNEL and then imaged by optical microscopy and assessed by 3 independent pathologists.

Competing interests
The authors declare no competing nancial interest.          Proposed mechanism for the cell apoptosis through the crosstalk between Golgi apparatus and mitochondria in GA-targeted photodynamic therapy.