Cu2-xS Nanocrystals Cross-Linked with Chlorin e6-Functionalized

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Biological and Medical Applications of Materials and Interfaces

Cu2-xS Nanocrystals Cross-Linked with Chlorin e6Functionalized Polyethylenimine for Synergistic Photodynamic and Photothermal Therapy of Cancer Huijing Xiang, Fengfeng Xue, Tao Yi, Huijun Phoebe Tham, Jin-Gang Liu, and Yanli Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018

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ACS Applied Materials & Interfaces

Cu2-xS

Nanocrystals

Cross-Linked

with

Chlorin

e6-Functionalized

Polyethylenimine for Synergistic Photodynamic and Photothermal Therapy of Cancer Huijing Xiang,†,‡,∆ Fengfeng Xue,§,∆ Tao Yi,*,§ Huijun Phoebe Tham,† Jin-Gang Liu*,‡ Yanli Zhao*,†,¶

†

Division of Chemistry and Biological Chemistry, School of Physical and Mathematical

Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371, Singapore. Email: [email protected] ‡

Key Laboratory for Advanced Materials, School of Chemistry and Molecular Engineering,

East China University of Science and Technology, Shanghai 200237, China. Email: [email protected] §

Department of Chemistry and Collaborative Innovation Center of Chemistry for Energy

Materials, Fudan University, 220 Handan Road, Shanghai 200433, China. Email: [email protected] ¶

School of Materials Science and Engineering, Nanyang Technological University, 50

Nanyang Avenue, Singapore 639798, Singapore

Keywords: Mitochondria-targeting, Multifunctional platform,

Photodynamic therapy,

Photothermal therapy, Synergistic therapy

Abstract: Achieving an integrated system for combinational therapy of cancer with enhanced efficacy is always a challenge. A multifunctional system (CCeT NPs) for synergistic photodynamic and photothermal cancer therapy was successfully developed. This system is composed with Cu2-xS nanoclusters functionalized with chlorin e6 (Ce6)-conjugated branched 1 Environment ACS Paragon Plus

ACS Applied Materials & Interfaces 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

polyethylenimine

(PEI-Ce6)

and

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mitochondria-targeting

3-(carboxypropyl)triphenylphosphonium bromide (TPP-COOH). The colocalization of the resulted CCeT NPs inside mitochondria of cancer cells was proven. CCeT NPs exhibited significant photodynamic therapy (PDT) efficacy due to efficient singlet oxygen (1O2) generation triggered by a 630 nm laser. This system also showed excellent photothermal conversion capability upon the irradiation of 808 nm laser for photothermal therapy (PTT). In particular, the platform achieved nearly 100% inhibitory rate of the tumor growth in vivo through combinational PDT and PTT. Thus, CCeT NPs could efficiently inhibit the tumor growth in vitro and in vivo by combinational PDT and PTT, offering synergistic therapeutic efficiency as compared to PTT or PDT alone.

INTRODUCTION Phototherapy, represented by photothermal therapy (PTT) and photodynamic therapy (PDT), is minimally invasive and highly efficient therapeutic technique for the cancer treatment.1-3 PDT is a useful therapeutic modality for numerous nonmalignant and malignant diseases.4 In the process of PDT, photosensitizers upon light irradiation transfer photon electrons to produce cytotoxic singlet oxygen (1O2) for irreversible destruction of diseased tissues.5-7 Chlorin e6 (Ce6) is a very promising photosensitizer because of its low utilization dosage with high efficacy.8 In addition, Ce6 could be also employed as a bioimaging agent to assist fluorescence imaging-guided therapy.9,10 On the other hand, the use of Ce6 as a photosensitizer is often involved with several limitations including poor water solubility, low stability, and lack of selectivity, hindering its administration and treatment efficiency. Therefore, developing stable Ce6 delivery vehicles and integrating it with other efficient therapeutic approaches in a single system hold a great potential to overcome its PDF limitations, thus achieving enhanced anticancer efficacy.11-16 Among various types of

2 Environment ACS Paragon Plus

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combinational therapeutics delivered by nanoscale theranostic agents, the integration of PDT and PTT has aroused tremendous research interests.17,18

Scheme 1. Schematic illustration for the synthesis of CCeT NPs and their in vitro and in vivo anticancer applications in mitochondria-targeted synergistic PDT and PTT under NIR light irradiation.

Up to now, several photothermal agents including gold (Au),19,20 carbon (C),21,22 and palladium (Pd) nanostuctures23 have been applied to load photosensitizers for combinational PTT and PDT of cancer. The combination of PTT and PDT is useful to improve the accuracy of diagnosis and the efficiency of therapy in order to overcome the drawbacks of single-agent cancer treatment. PTT has gained a lot of attention as a powerful modality in the cancer treatment,24,25 which often uses photothermal agents to convert near-infrared (NIR) light energy into heat for inducing the cancer cell death.26-29 Most of photothermal agents, for example, indocyanine green (ICG),30 IR825,31 carbon-based nanomaterials,32-35 and Au-based nanostructures are prone to photobleaching with low photothermal conversion efficiency,



limiting their PTT applications.36 Alternatively, nanocrystals such as Cu2-xS with characteristics including low toxicity, good stability, and high photothermal conversion efficacy present a promising potential as photothermal agents.37-41 In the light of above considerations, multifunctional theranostic platform (CCeT NPs) was constructed by integrating Cu2-xS nanocrystals with Ce6-conjugated branched polyethylenimine (PEI-Ce6), followed by conjugating mitochondria-targeted molecule TPP-COOH for synergistic PDT and PTT. The TPP-COOH moiety could specifically guide the obtained CCeT NPs to mitochondria of cancer cells, and the internalized CCeT NPs could efficiently produce 1O2 upon 630 nm laser illumination and heat under 808 nm NIR laser irradiation, resulting in the cancer cell death. Both in vitro and in vivo studies demonstrated that this multifunctional platform showed significantly enhanced tumor inhibition induced by synergistic PDT and PTT.

RESULTS AND DISCUSSION Synthetic procedure of CCeT NPs is shown in Scheme 1. First, heating up Cu(II) acetylacetonate and sulphur powder in a mixture of 1-dodecanethiol/oleic acid (1/2 v/v) resulted in monodispersed uniform Cu2-xS nanoclusters with a diameter of approximately 5-6 nm (Figures S1 and S2). To enhance the water solubility, Cu2-xS nanoclusters dispersed in CHCl3 were dropwise added to an aqueous solution containing PEI-Ce6, and obtained mixture was ultrasonicated to afford stable oil-in-water emulsion. Cu2-xS@PEI-Ce6 (CCe NPs) was obtained after evaporating residual solvent within droplets. Mitochondria-targeting group, TPP-COOH, was covalently conjugated with amino groups on the surface of CCe NPs to afford mitochondria-targeted CCeT NPs. While PEI is a hydrophilic polymer, the photosensitizer Ce6 and mitochondria targeting ligand TPP-COOH are hydrophobic, making the CCeT NPs hydrophobic and less soluble in water. In addition, the CCeT NPs were highly


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stable in phosphate-buffered saline (PBS) solution. The morphology and size of CCeT NPs were characterized by transmission electron microscopy (TEM). As presented in Figures S2, S3 and 1A, CCeT NPs exhibited a spherical morphology with diameters of about 6-7 nm. The hydrodynamic diameter of CCeT NPs in water was determined to be 11 nm by dynamic light scattering (Figure S3), consistent with the TEM measurement. The surface charge variations of CCeT NPs during the modification process were evaluated by zeta potential measurements (Figure 1B). Free Ce6 showed a zeta potential of -28 mV and became +43 mV after conjugating with PEI. Due to negatively charged nature of Cu2-xS nanoclusters and TPP-COOH, zeta potentials of CCe NPs and CCeT NPs are +24.2 mV and -9 mV, respectively. The changes in the zeta potential of the Cu2-xS nanoclusters after the modification confirmed the successful construction of the CCeT NPs. Fourier transform infrared spectroscopy (FTIR) was conducted to further confirm the formation of CCeT NPs. As demonstrated in Figure 1C, a typical peak at 1650 cm-1 is assigned to the formation of amide bonds between PEI and Ce6 or TPP-COOH. Obvious peaks at 750, 1210, and 3015 cm-1 are attributed to stretching vibrations of Cu2-xS nanoclusters in CCeT NPs. In addition, the band centered at 2900 cm-1 corresponds to the -CH2 stretching vibration of PEI in CCeT NPs. The oxidation states of Cu, S and N in CCeT NPs were investigated by X-ray photoelectron spectroscopy (XPS). The Cu2p spectrum consists of two peaks at binding energies of 932.5 eV and 952.2 eV assigning to the Cu2p3/2 and Cu2p1/2 core electrons, respectively (Figure 1D-F). The splitting of the two peaks by 19.7 eV is a character of the Cu(I) oxidation state. The Cu(II) oxidation state normally presents a satellite peak at 942 eV for Cu2p3/2. Meanwhile, the binding energies of S2p and N1s were observed at 164.2 and 399.6 eV, respectively. The chemical composition of this nanoplatform was also determined by energy disperse spectroscopy (EDS), giving the Cu/S ratio of 1.88 (Figure S4).



Figure 1 (A) TEM images of CCeT NPs. (B) Zeta potential values of Ce6, PEI-Ce6, CCe NPs, and CCeT NPs in water. (C) FTIR spectra of Cu2-xS nanoclusters, PEI-Ce6, CCe NPs, and CCeT NPs. XPS spectra of (D) Cu2p, (E) S2p, and (F) N1s for CCeT NPs.

The photophysical properties of CCeT NPs were investigated by UV-Vis and fluorescence spectroscopy (Figure 2A). As compared to the absorption of Cu2-xS nanocrystals, CCeT NPs showed a new absorption peak at 658 nm assigning to the Ce6 unit, indicating the successful introduction of Ce6 onto the nanocrystals. In addition, the absorption intensity of CCeT NPs from 450 nm to 1000 nm was higher than that of PEI-Ce6 or free Ce6 in PBS, attributing to the additive effect of the absorbance from Cu2-xS nanoclusters. The Ce6 loading amount was determined to be 4.73 µg per 100 µg CCeT NPs according to UV-vis spectral measurements (Figure S7). The fluorescence emission intensity of CCeT NPs at 675 nm was weaker than that of PEI-Ce6 or free Ce6 under the same conditions, ascribing to the quenching effect of Cu2-xS nanocrystals (Figure 2B).


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PEI-Ce6

A

Ce 6

Fl. Intensity (a.u.)

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2.0

CCeT

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Ce6 CCeT

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1.0 C/C0@658 nm

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ABMDA

0.3 0.0

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0.0

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Absorbance (a.u.)

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Cu2-xS Ce6 0

CCeT 2

Ce6 PEI-Ce6 CCeT

0.4 0.2

4 6 Time (min)

8

10

0

3

6 9 Time (min)

12

15

Figure 2. (A) UV-Vis absorption spectra and (B) fluorescence spectra of Cu2-xS nanoclusters, free Ce6, PEI-Ce6 and CCeT NPs in PBS. (C) Normalized absorbance decay of ABMDA absorbance at 400 nm in the absence and the presence of Cu2-xS nanoclusters, Ce6, and CCeT NPs under laser irradiation (630 nm, 40 mW/cm2, 10 min) for different irradiation time. (D) Normalized absorbance decay of PEI-Ce6, Ce6, and CCeT NPs at 658 nm under laser irradiation (630 nm, 40 mW/cm2, 15 min) for different irradiation time.

The 1O2 generation ability of CCeT NPs upon the irradiation of 630 nm laser was evaluated by recording the time-dependent photo-degradation of 9,10-anthracenediyl bis(methylene) dimalonic acid (ABMDA), a widely used 1O2 trapping agent. Figure 2C shows the absorption intensity changes of ABMDA in the presence of CCeT NPs, Cu2-xS nanoclusters, and Ce6 solution under different irradiation time. As a control, undetectable change of the ABMDA absorption intensity under the same conditions was observed in PBS. In the presence of CCeT



NPs, the ABMDA absorbance at 400 nm decreased significantly when exposed to 630 nm laser for 10 min. In addition, the absorption intensity of ABMDA showed a negligible change in the presence of Cu2-xS nanocrystals, suggesting highly efficient generation of 1O2 by CCeT NPs. The capability of 1O2 generation by CCeT NPs was weaker as compared with that of free Ce6 due to the fact that Cu2-xS could absorb part of irradiation light (Figure S8). The 1O2 generation quantum yield of CCeT NPs under 630 nm irradiation was calculated to be 0.22, using methyl blue in DMF as the standard (Figure S9). The photostability of free Ce6, PEI-Ce6, and CCeT NPs after exposing to 630 nm laser at 40 mW/cm2 for 10 min was then investigated. After the laser irradiation, the absorption intensity of free Ce6 at 658 nm decreased about 57.4%, while the absorbance of PEI-Ce6 and CCeT NPs was almost unchanged (Figure 2D), indicating that the irradiation energy generated by 630 nm laser could cause the photobleaching to free Ce6, and CCeT NPs and PEI-Ce6 exhibit excellent photostability under the same conditions (Figure S11). The photothermal effect of CCeT NPs with different concentrations upon the irradiation of 808 nm laser was then evaluated. The temperature of water as a control only increased by 2.0 o

C from the room temperature, while the temperature of CCeT NPs (10 µg/mL) rose by 5.3 oC

after the irradiation for 10 min (Figure 3A). When increasing the concentration of CCeT NPs from 20 to 200 µg/mL, the solution temperature went up by 8.0, 13.5, 19.5, and 27.7 oC, respectively (Figure 3B). In addition, Cu2-xS nanoclusters, PEI-Ce6, and CCeT NPs with the same amount of Cu2-xS or Ce6 were irradiated by 808 nm laser for 10 min, and the temperature variations were recorded. The cooling curve was shown in Figure S12, from which the conversion efficiency was calculated with photothermal conversion efficiency of 10.6%. A similar temperature increase trend was observed from Cu2-xS nanoclusters and CCeT NPs, while PEI-Ce6 exhibited a slight change in temperature (Figure 3C). The results demonstrated that Cu2-xS nanoclusters and CCeT NPs could effectively convert laser energy


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into heat. Furthermore, the photostability of CCeT NPs was also studied by means of exposing CCeT NPs to 808 nm laser irradiation. Six laser irradiation on/off cycles were performed by irradiating the solution of CCeT NPs for 10 min, followed by naturally cooling the solution down without the laser irradiation for 15 min. The temperature elevation of 22.2 o

C was obtained after the first laser irradiation at the concentration of 100 µg/mL (Figure 3D).

No significant attenuation in the temperature increase was observed in six cycles, indicating good NIR photostability of this nanoplatform for PTT. 0 10 µg/mL 20 µg/mL 50 µg/mL 100 µg/mL 200 µg/mL

T (oC)

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PEI-Ce6

CCeT

50 100 150 Concentration (µg/mL)

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40

36 30

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4 6 Time (min)

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80 Time (min)

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Figure 3. (A) Temperature change curves of deionized water and CCeT NPs at different concentrations upon the laser irradiation (808 nm, 0.5 W/cm2) as a function of time. (B) Temperature change curves of CCeT NPs as a function of concentration upon the laser irradiation (808 nm, 0.5 W/cm2, 10 min). (C) Temperature increase curves of free Ce6, Cu2-xS nanocrystals, and CCeT NPs with the same amount of Cu2-xS or Ce6 upon the laser irradiation



(808 nm, 0.5 W/cm2). (D) Temperature change curves of CCeT NPs (100 µg/mL) over six laser irradiation on/off cycles (808 nm, 0.5 W/cm2).

Figure 4. Confocal fluorescence images of HeLa cells (A) incubated with CCeT NPs for 4 h, and then treated with the laser irradiation (630 nm, 40 mW/cm2, 5 min), (B) irradiated by the laser irradiation (630 nm, 40 mW/cm2, 5 min), and (C) incubated with CCeT NPs without laser irradiation for 4 h. Blue and green fluorescence images were gained with excitation wavelengths at 405 and 488 nm, respectively. Scale bar: 50 µm.

Intracellular reactive oxygen species (ROS) generation of CCeT NPs upon 630 nm laser was monitored via an ROS probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA). The probe is almost nonfluorescent and emits green fluorescence after the reaction with ROS.42 As shown in Figures 4 and S13, a slight green fluorescence signal was observed when HeLa cells were incubated with CCeT NPs without laser irradiation, suggesting that CCeT NPs could only


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produce weak ROS. In addition, HeLa cells exhibited negligible ROS generation when they were illuminated by 630 nm laser for 5 min. Interestingly, strong green fluorescence was visualized when HeLa cells were incubated CCeT NPs under 630 nm laser irradiation for 5 min, which could be ascribed to the reaction of the probe DCFH-DA with the ROS generated in HeLa cells. The merged image in Figure 4A presented low overlapping between Hoechst 33342 and DCFH-DA fluorescence with a Pearson’s colocalization coefficient (0.18), clearly indicating that the internalized CCeT NPs were specifically located in the cytoplasm. Taken together, CCeT NPs possess high ROS generation capability in HeLa cells after the irradiation by 630 nm laser for 5 min.

dark

PTT + PDT

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Cell viability (%)

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laser irradiation

100 80 60 40

20 10 20 50 Concentration((µg/mL) ∝g/mL) Concentration

20 Control

Ce6 PDT

Cu2-xS CCeT PTT PTT + PDT

Figure 5 Cell viability of (A) HeLa cells and (B) MCF-7 cells treated with different concentrations of CCeT NPs under PTT treatment (808 nm, 0.5 W/cm2, 5 min) followed by PDT (630 nm, 40 mW/cm2, 10 min). (C) Cell viability of HeLa cells treated with different concentrations of CCeT NPs under different laser irradiation (630 nm, 40 mW/cm2, 10 min;



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808 nm, 0.5 W/cm2, 5 min). (D) Cell viability of HeLa cells treated with 2.37 µg/mL Ce6 under PDT treatment (630 nm, 40 mW/cm2, 10 min), 50 µg/mL Cu2-xS under PTT treatment (808 nm, 0.5 W/cm2, 5 min), and 50 µg/mL CCeT NPs under combined PTT (808 nm, 0.5 W/cm2, 5 min) and PDT (630 nm, 40 mW/cm2, 10 min) treatment.

To investigate the subcellular localization of CCeT NPs in MCF-7 and HeLa cells, the colocalization experiments were conducted by co-staining cells with Mito-Tracker green, a commercially available mitochondrial dye. The intracellular localization of CCeT NPs and Mito-tracker Green could be recorded by using confocal microscopy under two different fluorescence channels. As seen from Figure S14, upon excitation wavelength at 405 nm, targeted CCeT NPs exhibited bright fluorescence signals in the red channel (640-690 nm). Strong fluorescence emission in the green channel (500-550 nm) was also observed for Mito-tracker Green with excitation wavelength at 488 nm. After 4 h incubation, the fluorescence of Mito-tracker Green was perfectly colocalized with the red fluorescence of CCeT NPs, as displayed in the merged image (Figure S14C,F). In order to evaluate the feasibility of CCeT NPs as a dual modal PTT/PDT agent for cancer therapeutics,

the

cell

cytotoxicity

was

measured

via

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. HeLa, MCF-7, HepG2, and 4T1 cells were chosen as cell models. The cell viability of HeLa and MCF-7 cells was quantified after incubating with different concentrations of CCeT NPs for 12, 24, and 48 h, respectively. Encouragingly, the cell viability after treating with CCeT NPs still kept above 80% even at a high concentration of 100 µg/mL when the incubation time was over 48 h (Figure S15), indicating that CCeT NPs have a low cytotoxicity within a concentration range of 0-100 µg/mL. In addition, the laser itself without any CCeT NPs did not induce any cytotoxicity. The efficiency of CCeT NPs in killing cancer cells under NIR lasers irradiation


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was then investigated. Upon combinational laser irradiation with 630 nm (40 mW/cm2) and 808 nm (0.5 W/cm2), the cell viability of HeLa, MCF-7, HepG2, and 4T1 cells treated with CCeT NPs was dramatically decreased, showing a dose-dependent effect (Figures 5A,B and S16). When the concentration of CCeT NPs reached 100 µg/mL, the cell viability of HeLa and MCF-7 cells declined to 16.4% and 22.3%, respectively (Figure 5A,B). As shown in Figure 5C, combined PTT and PDT treatment induced remarkably higher cytotoxicity than that of PTT or PDT alone. For instance, after the treatment with CCeT NPs at 50 µg/mL, the cell viability for PDT, PTT, and combined PDT and PTT was 61.3%, 44.2%, and 27.3%, respectively. Meanwhile, a moderate PTT effect was determined when HeLa cells were incubated with Cu2-xS nanoclusters under the irradiation of 808 nm laser, and 42.4% HeLa cells survived with the concentration of Cu2-xS nanoclusters at 50 µg/mL (Figure 5D). Approximately 62% HeLa cells remained alive when they were treated with an equivalent concentration of Ce6 under 630 nm laser irradiation for PDT. These results indicate that a synergistic therapeutic effect could be achieved through combinational PTT and PDT provided by CCeT NPs. To demonstrate the feasibility of CCeT NPs for in vivo fluorescence imaging, imaging experiments on HeLa tumor bearing mice were conducted. CCeT NPs were administered into nude mice by subcutaneous injection because of their relatively low water solubility. The mice were then imaged with an excitation wavelength at 405 nm. As shown in Figure 6A, strong fluorescence signals resulted from CCeT NPs were clearly observed in the tumor of the mice after 1 h post injection, indicating high tissue penetrability of the fluorescence emitted by CCeT NPs. In order to monitor the bio-distribution of the CCeT NPs in the animal model, the whole-body fluorescence image was measured after 24 h of intratumoral injection (Figure S18). The mice only exhibited strong signals assigning to the fluorescence of the CCeT NPs in the tumor but not from the entire animal body, implying that most of CCeT NPs were still



in the tumor site even after 24 h of the injection. In addition, in vivo photothermal effect was investigated on HeLa tumor bearing mice. The time-dependent thermographic images and temperature increasing curves were shown in Figure 6B,C. Obviously, the mouse group treated with CCeT NPs exhibited significant hyperthermia effect after 808 nm laser irradiation for 5 min, when compared to the group without the treatment of CCeT NPs under the same experimental conditions. Subsequently, combinational PDT and PTT were investigated in vivo. The mice were randomly divided into six groups for different treatments. Tumor volumes of the groups after the treatment with PBS, lasers (630 and 808 nm), and CCeT NPs increased rapidly during a 20-day treatment period (Figure 6D). Nevertheless, the group treated with CCeT NPs under PDT exhibited a minor inhibiting effect on the tumor growth on account of the generation of 1O2. Meanwhile, the tumor growth was suppressed slowly after the treatment with CCeT NPs under PTT. Significantly, the tumors treated with CCeT NPs under combinational PDT and PTT disappeared, and there was no tumor recurrence throughout the entire treatment period, indicating that CCeT NPs exhibited synergetic PTT/PDT efficiency upon laser irradiation (630 and 808 nm) to efficiently inhibit the tumor growth (Figure S19). The body weights of mice under different treatments were recorded in Figure 6E. A slight increase in mouse body weights was observed for all groups during the treatment period, suggesting excellent biocompatibility of CCeT NPs in vivo (Figure S20). The tumor inhibitory rate was calculated using tumor volumes (Figure 6F). In contrast to the PBS-treated group, the inhibitory rate of CCeT NPs under combinational PDT and PTT was nearly 100%. This rate is also significantly higher than that of other treatment groups. The in vivo experimental results indicate that synergetic PDT and PTT efficacy by CCeT NPs was the highest among all therapeutic groups.


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(A) A

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Figure 6. (A) Fluorescence images of HeLa tumor bearing nude mice after the injection of CCeT NPs (20 mg/kg) in vivo. (B) Thermographic images of tumors after the treatment with PBS and CCeT NPs under laser irradiation (808 nm, 1.5 W/cm2, 2 min) with different irradiation time. (C) Temperature variation curves in tumors as a function of irradiation time depicted in (B). Quantitative measurements of (D) tumor volumes and (E) body weights of mice after different treatments with PBS (control), laser irradiation (630 nm, 0.5 W/cm2, 2 min and 808 nm, 1.5 W/cm2, 2 min), CCeT NPs, CCeT NPs plus PDT (630 nm, 0.5 W/cm2, 2 min), CCeT NPs plus PTT (808 nm, 1.5 W/cm2, 2 min), and CCeT NPs plus PTT (808 nm, 1.5 W/cm2, 2 min) + PDT (630 nm, 0.5 W/cm2, 2 min). (F) Tumor inhibitory rate after the treatment with PBS (control), laser irradiation (630 and 808 nm), CCeT NPs, CCeT NPs plus PDT (630 nm, 0.5 W/cm2, 2 min), CCeT NPs plus PTT (808 nm, 1.5 W/cm2, 2 min), and CCeT NPs plus PTT (808 nm, 1.5 W/cm2, 2 min) + PDT (630 nm, 0.5 W/cm2, 2 min).

Finally, the antitumor efficacy of various treatment approaches was evaluated by histological analyses. As depicted in Figure S21, the hematoxylin and eosin (H&E) stained slices pretreated with PBS, lasers (630 and 808 nm), or CCeT NPs only presented obvious



membrane and nuclear structures due to rapid tumor growth. On the contrary, apparent extensive cancer necrosis was recorded in the tumor slices of the mice treated with CCeT NPs under combinational PDT and PTT. For comparison, the tumors of mice received CCeT NPs-based PDT or PTT alone still showed large areas of living cancer cells. Furthermore, TUNEL staining was employed to assess the anticancer effect in various treatment groups. As shown in Figure S12, severe destruction of tumor tissues was obtained in the case of CCeT NPs under combinational PDT and PTT. By contrast, a very few of tumor tissues could be stained in the group treated with PBS, lasers, or CCeT NPs only. All these results demonstrate that CCeT NPs could provide synergistic PDT and PTT to afford much higher anticancer efficacy as compared to single PDT or PTT in vivo.

CONCLUSIONS In conclusion, we have successfully developed a multifunctional system (CCeT NPs) by incorporating Ce6 with Cu2-xS nanocrystals, followed by the modification with mitochrondria-targeting molecule TPP-COOH on the nanoparticle surface. The resulted CCeT NPs show high 1O2 production efficiency under irradiating with 630 nm laser, and an obvious photothermal effect upon 808 nm laser illumination. The colocalization experiments have demonstrated that CCeT NPs could be localized within mitochondria after their internalization into cells. The potential of CCeT NPs as a bioimaging agent has been confirmed by recording its strong fluorescence signal in vitro and in vivo. As compared with individual PDT or PTT, this therapeutic system exhibits significant antitumor efficiency in vitro and in vivo owing to the synergistic PDT and PTT effect upon the laser irradiation. Therefore, the constructed system for fluorescence imaging-guided synergistic PDT/PTT shows a promising potential for next generation cancer theranostics.


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METHODS Cu2−xS (x > 0) nanoclusters. In a typical procedure, Cu(acac)2 (210 mg, 0.4 mmol) and sulfur powder (12.8 mg, 0.2 mmol) were mixed with DDT (4 mL) and OA (8 mL) in a three-neck round-bottom flask and degassed under vacuum at 100 °C for 30 min. After this process, the flask was backfilled with N2 and heated up to 240 °C. The mixture was stirred at 240 °C for 30 min, resulting in a black colloidal solution. After cooling the system down to room temperature, the resulted solution was precipitated using excess EtOH and treated by centrifugation. The obtained precipitate was redispersed in CHCl3 and centrifuged for 5 min to remove larger particles as the byproduct. The purified samples were redispersed in CHCl3 for further characterizations. 3-(Carboxypropyl)triphenylphosphonium

bromide

(TPP-COOH).

A

mixture

of

4-bromobutanoic acid (3.5 g, 21 mmol) and triphenylphosphine (5.5 g, 21 mol) was refluxed in MeCN for 12 h. After the system was cooled down to room temperature, white solid precipitate was collected by filtration, and then crystallized from diethyl ether/ethyl acetate mixture. Yield: 7.22g, 80%. PEI-Ce6 conjugate. Typically, Ce6 (10 mg, 0.02 mmol) was dissolved in DMSO (0.5 mL), followed by adding EDC (20 mg, 0.10 mmol) and NHS (20 mg, 0.17 mmol) to activate for 4 h. After this process, an aqueous solution (20 mg, 1 mL) of PEI was added to the solution, and the obtained mixture was reacted at room temperature for 12 h. Unreacted Ce6 was removed by dialyzing against deionized water for 48 h (Mw cutoff of 30 kDa). The product PEI-Ce6 was dispersed in ultrapure water for characterizations. Cu2-xS@PEI-Ce6 (CCe NPs). Oleic acid-coated Cu2-xS nanoclusters (10 mg) dispersed in CHCl3 (1 mL) were added to deionized water (10 mL) containing PEI-Ce6 (30 mg). After the sonication for 10 min to form oil-in-water (O/W) emulsion, the solvent was evaporated, and unreacted Cu2-xS nanoclusters were removed by dialyzing against deionized water for 48 h



(Mw cutoff of 30 kDa). The purified CCe NPs were dispersed in fresh deionized water for further uses. Cu2-xS@PEI-Ce6@TPP (CCeT NPs). The CCeT NPs were synthesized using a modified EDC-NHS reaction. Typically, TPP-COOH (10 mg, 0.02 mmol) was dissolved in DMSO (0.5 mL), followed by adding EDC (20 mg, 0.10 mmol) and NHS (20 mg, 0.17 mmol) to activate for 4 h. CCeT NPs (20 mg) in aqueous solution were then added to the solution, and the obtained mixture was reacted at room temperature for 24 h. Unreacted TPP-COOH was removed by dialyzing against deionized water for 48 h (Mw cutoff of 30 kDa). The resulting CCeT NPs were dispersed in ultrapure water for characterizations.

ASSOCIATED CONTENT Supporting Information. Experimental details, TEM images, size distribution, EDX, UV-Vis spectra, confocal images, cell viability, in vivo fluorescence images, relative tumor volumes, body weights, and H&E and TUNEL stained images. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; [email protected]; [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ∆

These authors contributed equally.


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Notes The authors declare no competing financial interest.

Acknowledgements We are grateful for the financial support from the National Nature Science Foundation of China (No. 21571062, 21628401 and 21671043), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning, the Fundamental Research Funds for the Central Universities (No. 222201717003), and the Singapore National Research Foundation Investigatorship (No. NRF-NRFI2018-03). H. X. acknowledges the scholarship support from the Chinese Scholarship Council (No. 201506740024).

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Table of Contents


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Cu2-xS Nanocrystals Cross-Linked with Chlorin e6-Functionalized

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