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A Near-Infrared Organoplatinum(II) Metallacycle Conjugated with Heptamethine Cyanine for Trimodal Cancer Therapy

2021; Chinese Chemical Society; Volume: 4; Issue: 6 Linguagem: Inglês

10.31635/ccschem.021.202100950

2096-5745

Baoxuan Huang, Xi Liu, Guoliang Yang, Jia Tian, Xiaogang Li, Yucheng Zhu, Xiaopeng Li, Guang‐Qiang Yin, Wei Zheng, Lin Xu, Weian Zhang,

Dendrimers and Hyperbranched Polymers

Open AccessCCS ChemistryRESEARCH ARTICLE6 Jun 2022A Near-Infrared Organoplatinum(II) Metallacycle Conjugated with Heptamethine Cyanine for Trimodal Cancer Therapy Baoxuan Huang†, Xi Liu†, Guoliang Yang†, Jia Tian, Zhiyong Liu, Yucheng Zhu, Xiaopeng Li, Guangqiang Yin, Wei Zheng, Lin Xu and Weian Zhang Baoxuan Huang† Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237 , Xi Liu† Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062 , Guoliang Yang† Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237 , Jia Tian Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237 , Zhiyong Liu Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237 , Yucheng Zhu Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237 , Xiaopeng Li College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen 518055 , Guangqiang Yin Department of Chemistry, University of South Florida, Tampa, FL 33620 , Wei Zheng Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062 , Lin Xu *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062 and Weian Zhang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237 https://doi.org/10.31635/ccschem.021.202100950 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Discrete Pt(II) metallacycles have attracted particular attention for the chemotherapeutic treatment of cancer. However, a single chemotherapy cannot simultaneously balance efficiency and safety because the continuous administration throughout the entire therapy period will lead to inefficient therapy and potentially long-term systemic toxicity. Therefore, the development of a novel organoplatinum(II) metallacycle with multimodal treatment capabilities is urgently needed to overcome these issues. Herein, a discrete Pt(II) metallacycle ( SCY) bearing the near-infrared (NIR) photosensitizer heptamethine cyanine was fabricated and further encapsulated by amphiphilic 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethyleneglycol) (DSPE-mPEG) to form [email protected] nanoparticles. Heptamethine cyanine, which has excellent photoconversion efficiency, can generate reactive oxygen species (ROS) and heat simultaneously, and the cyanine moiety can target mitochondria in cancer cells due to their quaternary ammonium salt cations, which improve the effect of phototherapy. Due to its excellent phototherapy and chemotherapy properties, [email protected] exhibited remarkable trimodal therapeutic effects [chemo-/photodynamic therapy (PDT)/photothermal therapy (PTT)] against cancer cells (HepG2 cells, MCF-7 cells, and 4T1 cells) in vitro. Furthermore, in vivo results also confirmed that [email protected] had superior antitumor properties with minimal side effects in the 4T1 tumor model. This work presents a practicable approach to develop a multifunctional organoplatinum(II) metallacycle for multimodal tumor therapy. Download figure Download PowerPoint Introduction In recent decades, supramolecular coordination complexes (SCCs), including discrete two-dimensional (2-D) metallacycles and three-dimensional (3-D) metallacages with various well-defined shapes, sizes, and geometries, have been constructed through coordination-driven self-assembly.1–6 These SCCs have received considerable attention for both their aesthetic features and their broad applications in molecular recognition, catalysis, energy transfer, and sensing.7–11 As a classic type of SCC, Pt(II) embedded in a well-defined metallacycle has attracted particular attention for the chemotherapeutic treatment of cancer.12–14 Compared with clinical platinum-based drugs (oxaliplatin, carboplatin, and cisplatin), discrete Pt(II) metallacycle-encapsulated nanomaterials exhibit low systemic toxicity and an enhanced ability to inhibit tumor growth.15 Although Pt(II) metallacycles have shown potential for application in cancer therapy, single chemotherapy cannot simultaneously balance efficiency and safety because continuous administration throughout the entire therapy period will lead to inefficient therapy and potentially to long-term systemic toxicity.16,17 Recently, the development of a multifunctional Pt(II) metallacycle has provided an ingenious strategy to overcome the above limitation and enhance the effect of tumor treatment.18–21 In recent years, phototherapy, mainly photodynamic therapy (PDT) and photothermal therapy (PTT), has attracted extensive attention in cancer treatment.22–26 During PDT or PTT, the light energy absorbed by the photosensitizer (PS) is converted to reactive oxygen species (ROS) or local hyperthermia, inducing cancer cell death.27–29 Phototherapy has become an excellent complement to traditional chemotherapy because of its minimal invasiveness, few side effects, negligible drug resistance, and minimal damage to normal tissues. Hence, it is important to develop organoplatinum(II)-based SCCs combined with PSs for multimodal cancer treatment. Especially in recent years, the design and preparation of multifunctional Pt(II) metallacycles, such as Ru-Pt(II) metallacycles,30 fullerene-functionalized Pt(II) metallacycles31 and BODIPY-Pt(II) metallacycles,32 for tumor therapy have received great attention by many groups. An ideal PS should have strong absorption in the near-infrared (NIR) region (700–900 nm), as NIR light has good tissue penetration, which is beneficial for the phototherapy of deep tumor tissues.33,34 Moreover, effective photoconversion efficiency is also an important indicator for PSs. Heptamethine cyanine, as an NIR PS, has excellent photoconversion efficiency since it generates ROS and heat simultaneously.35–38 Moreover, NIR stimulation of heptamethine cyanine can minimize scattering and improve the photon penetration depth. In addition, because of the lipophilic cationic structure of heptamethine cyanine, these agents possess a strong ability to target intracellular mitochondria,39–41 which are hypersensitive to phototherapy-mediated damage, leading to enhanced phototherapeutic efficacy.42–44 Herein, we designed and fabricated a multifunctional organoplatinum(II) metallacycle ( SCY) incorporating 120° diplatinum acceptors with cyanine-containing 120° donors ( CY) via coordination-driven self-assembly (Scheme 1). Then, hydrophobic SCY was encapsulated by amphiphilic DSPE-mPEG to form metallacycle-loaded nanoparticles ( [email protected]), which are beneficial for improving the stability and solubility of the treatment reagent. Upon internalization by cancer cells, Pt(II) moieties acting as chemotherapy drugs enter into nuclei and bind with DNA, resulting in chemotoxicity. In addition, CY moieties with a positive charge target mitochondria and subsequently produce ROS and heat in the context of 808 nm laser irradiation, leading to efficient phototherapy. Therefore, the newly developed CY-based Pt(II) metallacycle possesses the following advantages: (1) the excitation source of this metallacycle is NIR light, which provides good tissue penetration and therapeutic efficacy; (2) the CY PS conjugated with the metallacycle targets mitochondria due to its lipophilic cationic structure, and specific accumulation in mitochondria promotes phototherapeutic efficacy; and (3) an efficient trimodal therapy involving PDT, PTT, and chemotherapy could be achieved by this single metallacycle. Finally, the advantageous properties and trimodal therapeutic effects of [email protected] were evaluated against cancer cells (MCF-7, HepG2, and 4T1 cells) in vitro as well as against the 4T1 tumor model in vivo. We believe that this study can provide a reference for the design of photochemotherapy reagents in the future. Scheme 1 | Schematic diagram for the manufacture of [email protected], the process for chemotherapy and the combination of mitochondria-targeted PDT and PTT after [email protected] enters the cancer cell. Download figure Download PowerPoint Experimental Methods Preparation of [email protected] and [email protected] The [email protected] and [email protected] nanoparticles were prepared via the nanoprecipitation method. Briefly, 1 mg of SCY and 10 mg of DSPE-mPEG2000 were dissolved in 1 mL of mixed solvents (VTHF:VCP = 1:1), and the solution was added dropwise into 5 mL of ultrapure water with stirring. The solution was dialyzed in a dialysis bag (molecular weight cut-off (MWCO) = 3500) against ultrapure water to remove the organic solvents for 24 h at room temperature in the dark. Next, [email protected] nanoparticles were obtained after passing through a 0.45 μm syringe filter and stored at 4 °C for further use. [email protected] nanoparticles were prepared through the same method. In vitro light-triggered ROS detection 1,3-Diphenylisobenzofuran (DPBF) was employed to monitor ROS generation. One milligram of DPBF was dissolved in 1 mL of dimethyl sulfoxide (DMSO) to achieve a final concentration of 1 mg/mL DPBF stock. Twenty microlitres of DPBF stock solution was added to 2 mL of [email protected] or [email protected] solution (6 μg/mL of CY) before irradiation and UV–vis absorption measurement. The absorption of DPBF was measured after irradiation at 808 nm (1 W/cm2) for 0, 10, 20, 30, 40, 50, and 60 s. We compared the ROS generation efficiency of [email protected] and [email protected]. Cell culture MCF-7 cells, HepG2 cells, and 4T1 cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum and 1% antibiotics (penicillin/streptomycin, 100 U/mL). Confocal microscopy cell imaging Cells were seeded in 35 mm glass-bottomed dishes at a density of 3 × 105 cells per dish in culture medium. After overnight culture, 4T1 cells were incubated with different concentrations of [email protected] or [email protected] (0.5 or 4 μg/mL of CY) for 4 h. Then the cells were washed with phosphate-buffered saline (PBS), stained with Hoechst (2 μg/mL) for 20 min, and washed three times with PBS before fixing with 4% paraformaldehyde for 15 min. After washing again with PBS, fluorescence images were acquired by confocal microscopy. For flow cytometry, 4T1 cells (2 × 105) were seeded in 6-well plates and cultured for 48 h. Then, the medium was replaced with fresh medium containing [email protected] or [email protected] (4 μg/mL of CY). After different incubation times (1 and 4 h), cells were washed three times with PBS, digested by trypsin, and harvested by centrifugation. The fluorescence of histograms of CY was recorded by flow cytometry (CytoFLEX LX; Becton Dickinson, San Jose, CA). 4T1 cells were seeded in 35 mm glass-bottomed dishes at a density of 3 × 105 cells per dish in culture medium. After overnight culture, 4T1 cells were incubated with [email protected] or [email protected] (2 μg/mL of CY) for 4 h and treated with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μM) for 30 min. After washing with PBS, cells were irradiated with 808 nm light (1 W/cm2, 1 min), and fluorescence images were acquired by confocal microscopy. The subcellular localization of cyanine after [email protected] and [email protected] entered 4T1 cells was determined by staining with MitoTracker (Molecular Probes; Invitrogen, Co., Carlsbad, CA) containing the two kinds of nanoparticles and then imaging under a confocal microscope. Briefly, 3 × 105 4T1 cells were seeded in a 35 mm Petri dish and cultured overnight. Then the cells were stained first with [email protected] or [email protected] (2 μg/mL of CY) for 4 h at 37 °C and then with MitoTracker (1:6000 diluted with PBS) for another 15 min after washing with PBS. Finally, the fluorescence of the cells was recorded by confocal microscopy after rinsing with PBS. Cell viability The cytotoxicity of [email protected] and [email protected] against different kinds of cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay in a 96-well cell culture plate. The cells were seeded at a density of 5 × 103 cells/well in a 96-well plate in culture medium and incubated for 24 h for attachment. Cells were then incubated with fresh serum-supplemented DMEM with [email protected] or [email protected] at various concentrations as experimental groups or with only fresh serum-supplemented DMEM as blank control groups. After incubation for 4 h, each well in the irradiation groups was exposed to 808 nm laser irradiation (1 W/cm2, 3 min), while other cells were incubated in the dark. The incubation continued for an additional 20 h. Then, 200 μL of MTT solution (5 mg/mL) was added to each well and incubated at 37 °C for another 4 h in the dark. The medium was removed, and 150 μL of DMSO was added to each well to dissolve the formazan crystals. Absorbance was measured at 490 nm in an ELx800 reader (BioTek Instruments, Inc., Winooski, VT). Cell viability values were determined according to the following formula: cell viability (%) = absorbance of the experimental group/absorbance of the blank control group × 100%. In vivo therapy on 4T1 solid tumors All animal procedures were performed in accordance with Chinese legislation on the Use and Care of Research Animals (Document No. 55, 2001) and the institutional guidelines for the Care and Use of Laboratory Animals established by the East China University of Science and Technology Animal Studies Committee, and this committee approved the experiments. A 4T1 tumor model was established by subcutaneous injection of 4T1 cells (1 × 106) on the right back of hind leg positions of the mice. The tumors were allowed to grow to ∼80 mm3 before experimentation. The mice were divided into six groups, and each group included five mice: group 1, PBS as a control; group 2, PBS with laser; group 3, [email protected]; group 4, [email protected] with laser; group 5, [email protected]; group 6, [email protected] with laser. Then, 25 μL of PBS was intratumorally injected into each mouse of groups 1 and 2; 25 μL of [email protected] (0.75 mg/kg for CY) was intratumorally injected into each mouse of groups 3 and 4; 25 μL of [email protected] (0.75 mg/kg for CY) was intratumorally injected into each mouse of groups 5 and 6; 2 h after injection, groups 2, 4, and 6 received irradiation with an 808 nm laser (1 W/cm2) in the tumor site for 5 min. After various treatments, the lengths (L) and widths (W) of the tumors were measured by a digital caliper every 2 days for 14 days. The tumor volume was defined as follows: V = W2 × L/2. Relative tumor volumes were calculated as V/V0 (V0 was the tumor volume when the treatment was initiated). All mice were sacrificed after treatments, and the tumors and major organs were collected for further hematoxylin and eosin (H&E) staining. Photothermal performance of [email protected] and [email protected] For the in vitro photothermal study, 200 μL of [email protected] and [email protected] (15 or 30 μg/mL for CY) were added to a 1.5 mL centrifuge tube. Then, the two groups were irradiated by an 808 nm laser (1 W/cm2) for 3 min. An IR thermal camera was used to record the temperature variation of the solution at different time intervals. For the in vivo photothermal study, three 4T1 tumor-bearing mice were intratumorally injected with 25 μL of PBS (control group), [email protected], or [email protected] (0.75 mg/kg for CY). After 2 h, an 808 nm laser at a power density of 1 W/cm2 was used to irradiate the tumor sites of the mice for 5 min. Meanwhile, an IR thermal camera recorded the temperature variations. Results and Discussion To synthesize a cyanine-containing 120° donor, the NIR PS IR780 was first synthesized in three steps, as shown in Supporting Information Figure S1. The corresponding compounds were successfully synthesized, as confirmed by the NMR and MS spectra ( Supporting Information Figures S2–S7). In the presence of sodium hydride, the chlorine of IR780 was replaced by the phenolic hydroxyl group of compound 3 via nucleophilic substitution to obtain the 120° donor ligand CY, which was confirmed by the NMR and MS spectra ( Supporting Information Figures S8–S10). Moreover, a single crystal of ligand CY that was suitable for X-ray diffraction was grown by the diffusion of isopropyl ether into a dichloromethane solution of the compound. The Oak Ridge Thermal Ellipsoid Plot (ORTEP) of compound CY ( Supporting Information Figure S34) showed that the core skeleton of heptamethine cyanine lay in approximately the same plane. It also showed that the angle between the two pyridyl rings of compound CY was close to 120°, confirming it as a suitable candidate for the formation of the molecular hexagon according to the directional bonding model and the symmetry interaction model. Then, CY was used stirred with 120° diplatinum-(II) acceptor compound 5 at a 1:1 molar ratio in acetone solvent for 16 h to obtain the metallacycle SCY, which was confirmed by multinuclear NMR spectra ( Supporting Information Figures S11–S16). As shown in Supporting Information Figure S17, compared with those of the free ligand CY, the signals of pyridine Hα and Hβ in SCY were shifted downfield because coordination bonds were formed between platinum (Pt) and nitrogen (N). In addition, SCY exhibited an upfield shift of 6.46 ppm compared with Pt(II) precursor compound 5 in the 31P{1H} NMR spectrum, and there was a sharp singlet at 13.10 ppm, which was consistent with a single-phosphorus environment ( Supporting Information Figure S18). Moreover, the formation of the discrete hexagonal metallacycle SCY was further confirmed by electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). As shown in Supporting Information Figure S19, the mass spectrum of SCY exhibited two peaks at m/z = 1221.3416 and 831.6651, corresponding to different charge states resulting from the loss of the hexafluorophosphate counterion species [M-5PF6−]5+ and [M-7PF6−]7+, respectively. The isotopic resolutions of these peaks were in good agreement with their theoretical distributions, thereby providing evidence for the establishment of metallacycle SCY. The metallacycle SCY was then encapsulated with amphiphilic DSPE-mPEG for biological applications. [email protected] nanoparticles were easily obtained after the hydrophobic metallacycle SCY was loaded into the nanoparticles through a nanoprecipitation method. Additionally, [email protected] nanoparticles were fabricated as the control sample by using CY in the same way. As shown in Figure 1a, [email protected] and [email protected] displayed their NIR absorption maxima at 780 nm, which was consistent with the behavior of free CY and SCY dissolved in DMF ( Supporting Information Figure S20). However, there were obvious shoulder peaks after CY and SCY were loaded into the nanoparticles due to the H-aggregation formation of cyanine,45 indicating that the hydrophobic CY and SCY were encapsulated successfully. Then, [email protected] and [email protected] were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. As shown in Figure 1b, the main hydrodiameters of [email protected] and [email protected] were 139 and 145 nm, respectively, at pH 7.4. Spherical assemblies could be observed in TEM images (Figures 1c and 1d). The average diameter of [email protected] in the TEM image was much larger than the hydrodynamic diameter from the DLS result because the DLS result for [email protected] was the average hydrodynamic diameter of [email protected], which was contributed to by all nanoparticles in solution. However, the TEM image showed only part of the nanoparticle morphologies, especially for large nanoparticles. Small nanoparticles can also be identified in the TEM image, but they were not clear since their contrast was quite low in comparison with that of large particles.46,47 In addition, the stability of both kinds of nanoparticles was examined, and there was no obvious change in their hydrodiameters over 48 h at pH 7.4 ( Supporting Information Figure S21), demonstrating the good stability of both [email protected] and [email protected]. Figure 1 | (a) UV–vis absorption spectra of [email protected], and [email protected] in PBS, and of CY and SCY in DMF. (b) The diameters of [email protected] and [email protected] in PBS (pH 7.4). TEM images of (c) [email protected] and (d) [email protected]. (e) Heating curves of PBS, [email protected] and [email protected] at different concentrations with 808 nm laser irradiation (1 W/cm2); [email protected] and [email protected] ([IR780] = 15 μg/mL), [email protected] and [email protected] ([IR780] = 30 μg/mL). (f) Plots of the change in absorption at 420 nm of DPBF solutions with [email protected] and [email protected] upon irradiation at 808 nm (1 W/cm2). Download figure Download PowerPoint The photothermal properties of [email protected] and [email protected] were first investigated with 808 nm laser irradiation ( Supporting Information Figures S22 and S23). As shown in Figure 1e, the temperature in the PBS group increased by only 1.4 °C after irradiation, which indicated that PBS produced almost no heat under 808 nm laser irradiation. Compared with the temperature in the PBS group, a significant increase was observed in the [email protected] and [email protected] groups. When the concentration of IR780 for CY-15 was 15 μg/mL, the temperature in these groups increased by approximately 14 °C at the end of laser irradiation. A larger temperature increase (approximately 23 °C) was observed when the concentration of IR780 for CY was increased to 30 μg/mL. Furthermore, the photothermal conversion efficiencies of [email protected] and [email protected] were 8.05% and 6.82%, respectively ( Supporting Information Figure S24). Although the photothermal conversion efficiency of [email protected] was low compared with those of other organic PTT agents such as semiconducting polymer nanoparticles,48–50 [email protected] as a phototherapeutic agent achieved multiple simultaneous effects of PTT and PDT against cancer. PDT also requires a larger amount of energy for treatment, leading to the low photothermal conversion efficiency of [email protected]. Overall, the results demonstrated that these nanoparticles have potential for PTT. After the photothermal effect of the nanoparticles was evaluated, the release profiles of SCY and CY from their corresponding nanoparticles of [email protected] and [email protected] were traced by IR780 fluorescence spectra. From Supporting Information Figure S25, it can be seen that the release rate of SCY from [email protected] was clearly slower than that of CY from [email protected], possibly because the molecular size of SCY was larger than that of CY. The release of SCY from [email protected] was investigated at different pH values, and nearly 47.1% and 43.92% of SCY was released from [email protected] in 60 h at pH 5.4 and 7.4, respectively. It can be observed that SCY from [email protected] has a slightly higher release rate at pH 5.4 than at pH 7.4, indicating [email protected] that is stable under acidic conditions. Moreover, the stability of metallacycle SCY under acidic conditions was further investigated by multinuclear NMR (1H and 31P) experiments on SCY with the gradual addition of 6 equiv of CF3COOH. Both the 1H NMR and 31P NMR spectra showed no apparent change upon the addition of 6 equiv of CF3COOH into the solution of SCY ( Supporting Information Figure S26), which indicated that the metallacycle SCY was stable under acidic conditions. Additionally, it has been reported that the presence of halide anions such as chloride (Cl−) and bromide (Br−) could induce the disassembly of Pt–N coordination-based metallacycles due to the stronger nucleophilicity of Cl− and Br− to the platinum atom compared to the nitrogen atom.51,52 The stability of the metallacycle SCY was investigated by 1H NMR and 31P NMR spectra in the presence of Cl− and Br−. As shown in Supporting Information Figure S27, with the gradual addition of 6.0 equiv of Bu4NCl to the solution of SCY in CD2Cl2, the typical proton and phosphorus signals of metallacycle SCY disappeared, indicating the complete disassembly of metallacycle SCY. As expected, the addition of Bu4NBr to the solution of metallacycle SCY caused NMR resonance changes similar to those of Bu4NCl ( Supporting Information Figure S28). These results clearly demonstrated that the presence of Cl− and Br− could cause the disassembly of metallacycle SCY and that Pt2+ can be rapidly released at the tumor site. Furthermore, the ability of [email protected] and [email protected] to generate ROS was then evaluated by measuring the absorption of DPBF, as the degradation of DPBF was associated with the content of ROS. We detected changes in the absorption of DPBF in the presence of [email protected] and [email protected] with 808 nm laser irradiation for 10 s intervals ( Supporting Information Figure S29). As expected, the absorbance of DPBF in the presence of [email protected] and [email protected] decreased obviously with increasing laser irradiation time. In contrast, there was almost no change in the absorbance of DPBF in PBS with 808 nm illumination, indicating that the 808 nm laser had no impact on DPBF. Furthermore, the changes in DPBF absorption at 420 nm were compared across different samples, as shown in Figure 1f. The rate of decline in DPBF absorption in the presence of [email protected] was similar to that observed in the presence of [email protected], indicating that the ROS production capacities of these two kinds of nanoparticles were nearly identical. [email protected] and [email protected] can efficiently generate ROS and heat, which is promising for phototherapy in living cells. Before the therapeutic effects of these agents were examined in vitro, the cellular uptake of these nanoparticles was first investigated by confocal laser scanning microscopy (CLSM) and flow cytometry. Benefiting from the NIR fluorescence of the cyanine-based PS, the red fluorescence of these nanoparticles was observed. As demonstrated in Figure 2a, after 4T1 cells were incubated with [email protected] and [email protected] for 4 h, red fluorescence was observed inside the cells, and the fluorescence of both samples was enhanced with increasing concentrations. In addition, quantitative cellular uptake of [email protected] and [email protected] after different incubation times was studied by flow cytometry ( Supporting Information Figure S30), and the histogram shifted toward increased fluorescence intensity with a longer incubation time, indicating that more nanoparticles entered the cells over time. As [email protected] and [email protected] were endocytosed efficiently by cancer cells, the subcellular location of CY was evaluated. Figure 2b shows that the red fluorescence ( CY) in cells treated with [email protected] or [email protected] overlapped well with the green fluorescence (MitoTracker Green). Furthermore, the Pearson's colocalization coefficients of [email protected] and [email protected] were ca. 0.82 and 0.86, respectively, demonstrating that cyanine could target mitochondria. This phenomenon was caused by the lipophilic cationic structure of cyanine. Mitochondria play an important role in signaling and apoptotic pathways, and they are the centers of energy production in cells.53,54 In addition, these organelles are easily damaged by ROS, either directly by PDT or indirectly by endogenous ROS induced by PTT, leading to the death of cancer cells.55 Therefore, phototherapy integrating PDT and PTT based on cyanine with mitochondrial targeting will be beneficial for enhancing anticancer efficacy.