Concurrent photothermal therapy and photodynamic therapy for cutaneous squamous cell carcinoma by gold nanoclusters under a single NIR laser irradiation

Pei Liu a, Weitao Yang ab, Lei Shi a, Haiyan Zhang a, Yan Xu ab, Peiru Wang a, Guolong Zhang a, Wei R. Chen c, Bingbo Zhang *ab and Xiuli Wang *a
aInstitute of Photomedicine, Shanghai Skin Disease Hospital, Tongji University School of Medicine, Shanghai, 200443, P. R. China. E-mail:;
bTongji University Cancer Center, The Institute for Biomedical Engineering & Nano Science, Tongji University School of Medicine, Shanghai, 200092, P. R. China
cBiophotonics Research Laboratory, Center for Interdisciplinary Biomedical Education and Research, University of Central Oklahoma, Edmond, OK 73034, USA

Received 27th July 2019 , Accepted 28th September 2019

First published on 7th October 2019

Phototherapy, particularly photothermal therapy (PTT) and photodynamic therapy (PDT), has become a promising therapeutic technique for the treatment of skin cancers because of its minial invasiveness, high efficacy, and low side effects. Nevertheless, single modality therapy, either PTT or PDT, has limited clinical effectiveness in treating skin cancers. Thus, combined applications of PTT and PDT have been frequently reported; however, PTT and PDT often require their respective photoagents and excitation light sources, resulting in challenges in clinical transformation. In this study, to address these issues, we report the use of biocompatible gold nanoclusters Au25(Capt)18 for the concurrent PTT and PDT treatment of cutaneous squamous cell carcinoma (cSCC) using an 808 nm near-infrared (NIR) laser. Utilizing their high light-thermal conversion efficiency, potent generation of singlet oxygen, and strong photothermal stability, Au25(Capt)18 nanoclusters potentiated a significant proliferation suppression of cSCC XL50 cells in vitro and the inhibition of cSCC tumors on SKH-1 mice in vivo. In particular, under 808 nm light irradiation, the tumor-cell-killing contributions of PTT and PDT were estimated to be 28.86% and 71.14%, respectively, by using an ROS scavenger to quench the PDT effect. Tumor-infiltrating CD4+ T and CD8+ T cells were observed after one course of concurrent PTT and PDT. Preliminary toxicity studies indicated low adverse effects of the Au25(Capt)18 nanoclusters. Through this study, we report the use of a simple nanostructure for simultaneous PTT and PDT applications to effectively kill cSCC and to induce anti-tumor immune responses. Our study could lead to the development of effective photoagents for current, synergistic applications of different phototherapies with targeted immunological responses for the treatment of cancers.

1. Introduction

Cutaneous squamous cell carcinoma (cSCC) is one of the most common skin cancers, resulting from the uncontrolled growth of squamous cells in the epidermis.1–3 The special predilection sites of cSCC are the head and face due to their overexposure to ultraviolet irradiation (UV).4,5 For the treatment of cSCC, traditional surgery often causes scars and even other sequelae-like malformations.6,7 Therefore, alternative treatments are urgently needed, especially for elderly patients who are unable to tolerate surgery.

Photodynamic therapy (PDT), a form of phototherapy involving light irradiation and a photosensitizer, is frequently used for the treatment of noninvasive cSCC in clinics as a potent alternative to chemotherapy, radiotherapy, and surgery due to its relative non-invasiveness, superior efficacy, and low adverse events.8–10 The injected photosensitizers are activated by visible light to generate reactive oxygen species (ROS), such as singlet oxygen (1O2), which can elicit phototoxicity and kill target cells.11–13 Unlike radiation therapy and chemotherapy that inevitably damage neighboring normal tissues, PDT shows good cancer selectivity because of the targeting of the photosensitizers and the locally applied light irradiation.14,15 Unfortunately, clinical data shows that the recurrence rate is still high after PDT, which is mainly attributed to the poor tissue penetration of red light and the limited lethality of singlet oxygen on tumor cells.10,16,17 As such, combined therapy, like PDT with photothermal therapy (PTT), could enhance therapeutical outcomes.18

PTT, another form of phototherapy, has similar advantages as PDT but works via a different mechanism. In PTT, the injected photothermal agent converts photo-energy into thermal energy under light excitation, leading to the thermal ablation of cancer cells.19–21 Both of these two modalities involve light irradiation and the use of photosensitive drugs. Similarly, a single PTT treatment usually cannot achieve good therapeutic effect due to its limited photothermal conversion efficiency, the poor tissue penetration of light, and low photothermal stability.22,23 These unfavorable factors usually lead to cSCC relapsement.24,25 Because of their different therapeutical mechanisms, in most cases, combined PTT and PDT requires two different wavelength lights and their own drugs, which causes complexity and impedes its use in clinics. Therefore, there remains a strong need for a single agent that could be used as both a PTT and PDT agent and that could particularly work under single wavelength light irradiation.

In very recent years, a few photosensitive materials have been synthesized with PTT and PDT features with an aim to enhance therapeutic efficacy and reduce the recurrence rates of tumors. For instance, Tian et al. synthesized polyethylene glycol (PEG)-functionalized graphene as a multifunctional nanocarrier to load the photosensitizer Chlorin e6 (Ce6) and to realize photothermally enhanced photodynamic therapy.26 Wang et al. prepared a multifunctional nanoplatform by grafting GO with UCNPs for the loading of the photosensitizer ZnPC to realize a combination of PTT with PDT.27 Yudasaka et al. loaded zinc phthalocyanine (ZnPc) onto single-wall carbon nanohorns to achieve PTT–PDT double therapy under 670 nm laser irradiation.28 Sun et al. anchored minute quantities of the photosensitizer Ce6 onto amino-rich red emissive carbon dots to possess superior PTT and PDT under 671 nm laser irradiation.29 As shown in these papers, various structures have been fabricated and shown effectiveness in PTT–PDT combined therapy. However, these reported concurrent PTT–PDT therapy systems have been generally based on the physical combinations of photosensitizers and photothermal agents, and most of them require different excitation lights to separately trigger PTT and PDT, which makes the setup complicated and difficult to be used in clinics. An ideal concurrent PTT and PDT photoagent should at least possesses the following features: (1) robust photothermal conversion efficiency for PTT; (2) potent ROS generation capability for PDT without the interference of PTT; (3) simultaneous action of PTT and PDT under the same light irradiation; (4) good photostability with a clear and simple structure; and (5) good biocompatibility. Through literature research, it is still highly desirable to develop such agents with the above features at the same time.

In this work, we report an ultrasmall gold nanostructure, namely captopril-stabilized Au nanoclusters Au25(Capt)18 for the concurrent PTT and PDT treatment of cSCC under a single 808 nm NIR light irradiation. Compared to previously reported photosensitive agents which were partially mentioned above, the prepared Au25(Capt)18 has a clear, stable, and simple structure, and particularly provides good photothermal stability and a high production of 1O2 under the excitation of an 808 nm NIR laser.30–33 This NIR irradiation allows deeper tissue penetration for treating invasive skin cancers compared to the clinically used red laser treatment. To the best of our knowledge, Au25(Capt)18 has not been used for the treatment of cSCC. In this study, we investigated the concurrent effects of PTT and PDT and estimated their cell killing contributions by using an ROS inhibitor to quench PDT during the light irradiation. Furthermore, tumor-infiltrating CD4+ T and CD8+ T cells were found by immunohistochemical examination after one course of Au25(Capt)18 nanoclusters-based concurrent PTT and PDT treatment, and the potential immunogenic response induced by Au25(Capt)18 is discussed herein Scheme 1.

image file: c9tb01573f-s1.tif
Scheme 1 Schematic diagram of the concurrent photothermal and photodynamic therapy mechanism of cutaneous squamous cell carcinoma by single drug Au25(Capt)18 nanoclusters under a single NIR laser irradiation.

2. Experimental section

2.1. Materials

All the chemical reagents for Au25(Capt)18 synthesis in this study were purchased from Aladdin Co. Ltd without further purification, including tetrachloroauric(III) acid (HAuCl4·3H2O, 99.99%), sodium borohydride (NaBH4, 99.99%), tetraoctylammonium bromide (TOABr, >98%), and captopril. DMEM cell culture medium, ethanol, phosphate buffered saline (PBS), penicillin/streptomycin, and trypan blue, and the cell counting kit-8 (CCK8) were obtained from Hyclone (Thermo Scientific). Fetal bovine serum (FBS) was obtained from GIBCO. Rabbit antimouse monoclonal antibody against CD4/CD8 and 3,3′-diaminobenzidine (DAB) chromogen were obtained from Abcam Technology. The murine cutaneous squamous cell carcinoma cell line XL50 was established from UV-induced squamous cell carcinomas in SKH-1 hairless mice and stored at China Center for Type Culture Collection (CCTCC no: C201827, Wuhan, China).34 Male SKH-1 hairless mice aged 5–8 weeks old were recruited from Shanghai Public Health Clinical Center. All the animal experiments were performed in compliance with the approved protocol of the Animal Ethics Committee of Shanghai Skin Disease Hospital.

2.2. Synthesis of Au25(Capt)18

Au25(Capt)18 was synthesized according to the published procedure with slight modifications.30 Briefly, HAuCl4·3H2O (0.20 mmol, 78.7 mg) and TOABr (0.23 mmol, 126.8 mg) were dissolved in 10 mL of methanol, followed by vigorous stirring. After 20 min, the solution color changed from yellow-orange to deep red. Then, captopril (1 mmol, 217.2 mg) was dissolved in 5 mL of methanol, and quickly added into the above reaction system, and the solution color immediately turned to white. After stirring at 1000 rpm for 30 min, NaBH4 (2 mmol, 75.6 mg) dissolved in 5 mL of ice water was quickly added into the above reaction mixture. The solution color immediately turned to black-brown. The reaction was continuously stirred for 8 h. Then the solution was centrifuged to remove the unreacted Au(I) conjugates. The supernatant was collected by rotary evaporation under reduced pressure. The precipitate was dissolved with ethanol, and the mixture was extracted with minimum amounts of methanol several times. Then the cluster solution was precipitated again by ethanol. Finally, 1 mL of DI water was added to obtain a final aqueous solution of Au25(Capt)18.35

2.3. Characterizations

Transmission electron microscopy (TEM) images of Au25(Capt)18 were obtained on a FEI Tecnai G2 F20 S-TWIN operated at 200 kV to observe the size and shape of the clusters. The hydrodynamic size and distribution of Au25(Capt)18 were determined by using a Zetasizer Nano-ZS (Malvern, NanoZS) at 25 °C. UV-vis spectra were recorded on Cary 60 UV-visible spectrophotometer (Agilent Technologies). The inductively coupled plasma (ICP) method was used to determine the Au concentration.

2.4. Stability analysis

Au25(Capt)18 was dispersed in 1 mL of DI water, PBS, and DMEM, respectively. Then, the colloidal stability of Au25(Capt)18 was analyzed by monitoring the changes of UV-vis absorption at different incubation times (2, 24, 48, 72, and 96 h).

2.5. Photothermal conversion effect of Au25(Capt)18 irradiated by an 808 nm NIR laser

The photothermal effect of Au25(Capt)18 was evaluated by using a thermal infrared imager to record the temperature change of Au25(Capt)18 aqueous solution at different concentrations under 808 nm laser irradiation (2 W cm−2, 5 min). Cyclic experiments were carried out to further evaluate the photothermal stability of Au25(Capt)18. Briefly, a solution of Au25(Capt)18 at 0.5 mg mL−1 (Au) was irradiated by an 808 nm laser (2 W cm−2) until the temperature reached 60 °C, and then the laser was removed immediately. The heated solution was monitored and allowed to cool to its starting temperature. Then, another round of irradiation was applied. In total, 4 rounds of irradiation were operated to evaluate the photostability. To further evaluate the photothermal conversion efficiency (η) of Au25(Capt)18, 1 mL of Au25(Capt)18 aqueous solution was added into a standard quartz cell (5.76 g) and given continuous 808 nm laser exposure (2 W cm−2) for 15 min. Then, the laser was removed immediately, and the solution was allowed to cool naturally to its starting temperature. As a control, 1 mL of DI water was used in the same process to obtain the heat absorbed by the quartz cell and water (QDis). The thermal infrared imager was used to record the temperature change of Au25(Capt)18 solution and DI water during the whole process. The value of η was calculated according to equations reported by Roper et al.36

2.6. Singlet oxygen generation of Au25(Capt)18 irradiated by an 808 nm NIR laser

The photodynamic effect of Au25(Capt)18 induced by 808 nm laser irradiation (1 W cm−2) was verified by detecting 1O2 using a singlet oxygen sensor green (SOSG) reagent. Herein, 1O2 detection was carried out at different time points during the irradiation. Furthermore, the photodynamic effect in a hyperpyrexia environment was evaluated by the detection of 1O2 generation under increasing laser power density (0.5, 1, 2 W cm−2).

2.7. In vitro dark-cytotoxicity study of Au25(Capt)18

The murine squamous cell carcinoma XL50 cell line was used for the cytotoxicity tests. The CCK8 assay was used to evaluate the in vitro dark-cytotoxicity. XL50 cells were cultured with DMEM medium, containing 10% FBS and 1% penicillin/streptomycin antibiotics at 37 °C with 5% CO2. For the cytotoxicity tests, the cells were seeded in a 96-well plate at a density of 10[thin space (1/6-em)]000 cells per well. After overnight incubation, the cells were divided into 6 groups, each group containing 6 wells. The supernatant was replaced with 100 μL of fresh medium containing Au25(Capt)18 at different concentrations (0, 20, 40, 60, 80, and 100 μg mL−1 of Au). Another group of 6 wells with pure medium was set as the blank. After 24 h of dark incubation, the culture media was discarded and replaced by fresh media containing 10% of CCK-8 solution. After another 2 h of culture, the absorbance values at 450 nm wavelength were determined by a microplate reader.

2.8. Concurrent PTT and PDT of XL50 cells by Au25(Capt)18

XL50 cells were seeded into a 96-well plate at a concentration of 10[thin space (1/6-em)]000 cells each well with fresh DMEM. After 24 h of incubation, the cells were divided into 4 groups: Au25(Capt)18 + laser, Au25(Capt)18 only, laser only, and the control. The supernatant in the Au25(Capt)18 + laser group and Au25(Capt)18 group were replaced with 100 μL of fresh medium containing Au25(Capt)18 with the concentration equal to the maximum non-cytotoxic concentration obtained in the above test. After 2 h of dark-culture at 37 °C with 5% CO2, the cells in the Au25(Capt)18 + laser group were irradiated by an 808 nm laser for 5 min at 0.6 W cm−2. In comparison, the cells in the Au25(Capt)18 and laser groups were only treated by Au25(Capt)18 incubation or by laser illumination. The cells in the control group were not applied any process. After another 24 h of incubation, the CCK8 assay was used to determine the cell viability of each group. At the same time, during the irradiation, the temperature changes of the cells in the Au25(Capt)18 + laser and laser groups were recorded by a thermal imager.

To demonstrate the cellular PDT effect, another four groups of cells (Au25(Capt)18 + laser, laser, Au25(Capt)18, the control) were set. After 24 h of incubation, 100 μL of SOSG (10 μM) diluted with DMEM was added and incubated for 30 min. The fluorescence intensity at 530 nm wavelength was measured. Pure SOSG solution with the same concentration was set for the control. Furthermore, the proportions of PTT and PDT were determined in terms of the contribution of the cell-killing effect by Au25(Capt)18 under the laser irradiation with the help of NAC (N-acetyl-L-cysteine), an ROS scavenger that was used to quench the PDT contribution. Briefly, after 2 h of Au25(Capt)18 incubation, NAC was added into the cells. The laser irradiation experiment was the same as mentioned above. The cell survival rates of Au25NCS + laser + NAC were determined by the CCK8 assay and then compared with the other four groups above.

2.9. In vivo toxicology analysis of Au25(Capt)18

Routine blood cell tests, blood biochemistry analysis, and H&E staining were carried out to evaluate the in vivo toxicology of Au25(Capt)18. Female healthy SKH-1 hairless mice (6 weeks) were divided into two groups: Au25(Capt)18 injection group and the control group (n = 5). The mice in the Au25(Capt)18 group were intravenously injected with 100 μL of Au25(Capt)18 solution at a dose of 20 mg Au per kg weight, while the mice in the control group were treated with PBS. The behaviors of the mice were monitored during the treatment. Two weeks later, blood samples were collected from the mice by removing the eyeballs for the routine blood test and blood biochemical examinations. Then, the mice were sacrificed after euthanasia to dissect them and collect the heart, liver, spleen, lung, and kidney. The main organs were fixed in 10% formalin and paraffin-embedded, followed with H&E staining. The H&E staining images were taken using an optical microscope.

2.10. In vivo concurrent PTT and PDT of cSCC

Twenty female SKH-1 hairless mice approximately 6-weeks old were subcutaneously inoculated with XL 50 cells (5 × 106/L) near the neck to establish the cSCC xenograft tumor model. When the diameter of the tumor had grown to about 5–7 mm, the mice were randomly divided into four groups: Au25(Capt)18 + laser (a), Au25(Capt)18 only (b), laser only (c), and the control group (d) (n = 5). The detailed treatment procedures were the following: (a) intratumoral injection of Au25(Capt)18 (100 μL, 2.5 mg mL−1) and application of tumor laser irradiation (1 W cm−2, 10 min) at 4 h after the injection; (b) intratumoral injection of Au25(Capt)18 only (100 μL, 2.5 mg mL−1); (c) laser irradiation (1 W cm−2, 10 min) only; (d) no treatment applied for the control. All the treatments were only given once. The temperature changes of the tumor were recorded by a thermal imager during the irradiation. Tumor volumes (mm3) were recorded by measuring two perpendicular diameters (mm) using a Vernier caliper (“a” stands for the longer diameter, while “b” stands for the shorter one. V = ab2/2). At the same time, the body weight was monitored during the experiments. After 20 days of treatment, except for the three mice in the Au25(Capt)18 + laser group chosen randomly for continuous observation, the remaining mice in the four groups were euthanized.

In order to confirm the in vivo generation of 1O2, another two tumor-bearing mice were given the following treatment: (a) intratumoral injection of Au25(Capt)18 (100 μL, 2.5 mg mL−1) and SOSG (100 μL, 50 μmol L−1) plus laser irradiation (1 W cm−2, 10 min), (b) intratumoral injection with SOSG only (100 μL, 50 μmol L−1) plus laser irradiation (1 W cm−2, 10 min). Then, the mice were put in the IVIS Lumina K Series III imaging system, and the fluorescence signals of the tumor sites were recorded to detect 1O2.

2.11. Preliminary study of the immune responses induced by PTT and PDT

To investigate whether the concurrent PTT and PDT based on Au25(Capt)18 under laser irradiation could induce immunological responses, 8 of the SKH 1 tumor-bearing mice were randomly divided into four groups: Au25(Capt)18, laser, Au25(Capt)18 + laser, and the control. At different time points before and after treatment (before treatment, 3 days, and 7 days after treatment), the mice were euthanized and the tumor tissues were taken for immunohistochemical examination. The tumor slides (n = 3) were stained with antibodies against CD4 and CD8 to detect the presence of CD4+ and CD8 +T cells in the local cancerous lesions.

2.12. Statistical analysis

GraphPad Prism 6.0 software was used to perform the statistical tests. One-way analysis of variance (ANOVA) was used to compare cell viabilities, tumor volumes, temperature changes, and 1O2 fluorescence intensities. Independent t-tests were used to compare the differences between two groups. A p-value <0.01 was considered as statistically significant.

3. Results and discussion

3.1. Synthesis and characterizations of Au25(Capt)18

As shown in Fig. 1A, the TEM image shows a uniform size of Au25(Capt)18 with a mean diameter of 1.38 ± 0.17 nm. Compared to the glutathione (GSH)-capped Au25(SG)18, Au25(Capt)18 nanoclusters were found to have higher thermal stability besides a small size and good water solubility, which indicate this material is suitable for photomedicine applications.30Fig. 1B shows a larger hydration particle size of the Au25(Capt)18 nanoclusters, which was most likely due to the captopril encapsulation and the water around them.37 The inset image in Fig. 1B shows a dark brown color of Au25(Capt)18 water solution and no stratification phenomenon. After complete reaction, the optical properties of Au25(Capt)18 were measured and the results showed fluorescence centered at ∼650 nm (Fig. 1C) and the quite prominent characteristic absorption peaks at 670 and 450 nm (Fig. 1D). In particular, the absorption in the range of 800–900 nm wavelength is also remarkable, favoring NIR photomedicine. These results are basically consistent with previous reports.30,35 Furthermore, the colloidal stability was assessed by monitoring the UV-vis absorption spectra of Au25(Capt)18 incubated with DI water, PBS, and DMEM over 96 h. As presented in Fig. S2 (ESI), the shape of the UV-vis absorption spectra and absorption intensity at 808 nm remained almost stable over 2, 24, 48, 72, and 96 h. These results suggest the good colloidal stability of the Au25(Capt)18.
image file: c9tb01573f-f1.tif
Fig. 1 Characterizations of Au25(Capt)18. (A) TEM image (scale bar = 10 nm); (B) hydrodynamic diameter and size distribution in water, and the inset is the digital image of Au25(Capt)18 water solution; (C) fluorescence spectrum (Ex = 514 nm) and (D) UV-vis absorption spectrum of Au25(Capt)18.

3.2. Photothermal and photodynamic effects of Au25(Capt)18 irradiated by an NIR laser

First, the photothermal conversion effect of Au25(Capt)18 was verified. Considering the absorption of Au25(Capt)18 and the tissue penetration wavelength, an 808 nm laser was selected for the light irradiation. Au25(Capt)18 aqueous solution at different concentrations (0.9 mg mL−1, 0.45 mg mL−1, 0.225 mg mL−1, 0.1125 mg mL−1) and deionized water were exposed to the 808 nm laser at a power density of 2 W cm−2 for 5 min. As shown in Fig. 2A–C, the temperature of the Au25(Capt)18 aqueous solution of 0.9 mg mL−1 quickly increased above 60 °C within 5 min, while the temperature of deionized water showed only a slight increase under the same irradiation condition, while the photothermal conversion effect was concentration dependent.

Furthermore, the photothermal stability of Au25(Capt)18 was assessed and the heating–cooling cycles are shown in Fig. 2D, indicating its superior stability, thus favoring phototherapy in vivo. This high thermal stability of Au25(Capt)18 is mainly attributed to the ligand stability of the captopril. In contrast, the glutathione ligand in glutathione-capped gold nanoclusters tends to gradually degrade under light irradiation.30,38 As shown in the UV-vis absorption spectrum, the absorption value of the Au25(Capt)18 at 808 nm was prominent but lower than that at 650 nm. Further, the concrete photothermal conversion efficiency (η) of Au25(Capt)18 at 808 nm was calculated to be 41.1%, which was higher than that of some other gold nanoparticles.39,40 The detailed calculation is shown in the ESI (Fig. S3). The relatively high η value of Au25(Capt)18 might be attributed to the super small diameter and the surface captopril ligand with good stability protecting the metal core, thus improving the plasmon-based photothermal conversion.41–43

image file: c9tb01573f-f2.tif
Fig. 2 Photothermal and photodynamic effects of Au25(Capt)18. (A) Thermal images of Au25(Capt)18 aqueous solution at different Au concentrations (0.9 mg mL−1, 0.45 mg mL−1, 0.225 mg mL−1, 0.1125 mg mL−1) under laser irradiation (808 nm, 2 W cm−2, 5 min); also, DI water subjected to laser irradiation under the same condition was set as the control; (B) temperature curves in (A); (C) temperature changes in (A); (D) photostability evaluation by measuring the temperature curves of 0.5 mg mL−1 Au25(Capt)18 aqueous solutions after 4 heating–cooling cycles under repeated laser irradiation (2 W cm−2, 5 min for each); (E) generation of 1O2 by Au25(Capt)18 (0.1 mg mL−1) measured using singlet oxygen sensor green (SOSG) reagent via recording the fluorescence emission spectra at 530 nm subjected to laser irradiation at different time points (1 W cm−2, 1–70 min); (F) fluorescence intensities with irradiation time increasing in (E); (G) generation of 1O2 by Au25(Capt)18 (0.2 mg mL−1) measured by SOSG via recording the fluorescence intensities at 530 nm under laser irradiation with different power densities for 5 min (0.5 W cm−2, 1 W cm−2, 2 W cm−2). The corresponding temperatures were recorded by a thermal imager. The excitation wavelength for fluorescence emission measurement was 488 nm.

The 1O2 generation by Au25(Capt)18 was further measured using singlet oxygen sensor green (SOSG) reagent via recording the fluorescence emission spectra at 530 nm subjected to laser irradiation at different time points (1 W cm−2, 1–70 min). Fig. 2E and F show the time-dependent fluorescence intensity at 530 nm during laser illumination, indicating continuous 1O2 generation of Au25(Capt)18. After just 1 min of laser exposure, prominent fluorescence intensity at 530 nm could be observed indicating significant 1O2 generation (Fig. 2E and F). Over 1 h of irradiation, an increase in 1O2 generation could still be noticed (Fig. 2F), suggesting a good photostability. The potent 1O2 generation of Au25(Capt)18 is critical to the following PDT treatment of cSCC.

As is well known, photosensitizers in their excited triplet state undergo two kinds of reactions. In order to know whether there are hydroxyl radicals (˙OH) involved in the type I pathway, we used 3,3,5,5-tetramethylben-zidine (TMB) to capture the ˙OH generated during laser irradiation. However, we did not see the absorption peak of oxidized TMB by ˙OH at 654 nm with increasing laser irradiation time (1–5 min) (Fig. S4, ESI) and it is worth mentioning that the absorption peak at 670 nm is the characteristic absorption peak of Au25(Capt)18. According to the results, we think the pathway for PDT induced by Au25(Capt)18 was the type II photochemical reaction to generate 1O2.

Considering the prominent photothermal conversion efficiency of Au25(Capt)18, the effect of hyperpyrexia on the quenching of the 1O2 generation was further investigated. As shown in Fig. 2G, the SOSG fluorescence signal at 530 nm increased with the increase in temperature or the power density. This data clearly indicates that heating to around 50 °C cannot massively quench the 1O2 generation of Au25(Capt)18. In other words, Au25(Capt)18 can exert PTT and PDT simultaneously in this study. It has been proved that ROS generated by PDT can increase cytotoxicity induced by PTT, while on the contrary, heat induced by PTT can improve blood flow, thus increasing oxygen supply in the tumors to improve the generation of 1O2.44,45 Therefore, PDT/PTT synergistic treatment induced by Au25(Capt)18 has a better therapeutic outcome than the respective PTT or PDT alone might for the same reasons.

3.3. In vitro dark-cytotoxicity study of Au25(Capt)18

Before the cell experiments, the dark-cytotoxicity of Au25(Capt)18 was investigated by CCK8 assay to determine whether the Au25(Capt)18 would affect the viability of the XL50 cells. Fig. 3A shows that the percentages of the viable cells were all over 80% when the Au concentration was under 80 μg mL−1 (80 μg mL−1 included), suggesting the low cytotoxicity of Au25(Capt)18.
image file: c9tb01573f-f3.tif
Fig. 3 In vitro dark-cytotoxicity and concurrent PTT and PDT treatment of skin cancer cells by Au25(Capt)18 irradiated by an 808 nm NIR laser. (A) Viability of XL50 cells incubated with Au25(Capt)18 at different concentrations (0 μg mL−1, 20 μg mL−1, 40 μg mL−1, 60 μg mL−1, 80 μg mL−1 and 100 μg mL−1 of Au) for 24 h; (B) viability of XL50 cells of different groups: control, Au25(Capt)18, laser, Au25(Capt)18 + laser, and Au25(Capt)18 + laser + NAC (Au concentration: 80 μg mL−1, laser: 0.6 W cm−2, irradiation time: 5 min); temperature curves (C) and temperature changes (D) of the cells treated in different groups in B. (E) Thermal images of the cells during the laser irradiation with or without Au25(Capt)18 incubation (Au concentration: 80 μg mL−1, laser: 0.6 W cm−2, irradiation time: 5 min). (F) In situ detection of 1O2 generation in cells via recording the fluorescence signals by using SOSG reagent during the treatment in the control, Au25(Capt)18, laser, and Au25(Capt)18 + laser groups (Au concentration: 80 μg mL−1, laser: 0.6 W cm−2, irradiation time: 5 min); and (G) the quantified histogram of F. Error bars are based on triplet measurements (*p < 0.01). Au is short for Au25(Capt)18 and L is short for laser, which are the same below.

3.4. Concurrent PTT and PDT treatment of skin cancer cells using Au25(Capt)18 under NIR laser irradiation

After confirming the photothermal conversion and 1O2 generation capabilities of Au25(Capt)18, we used PTT and PDT on skin cancer cells. We chose 80 μg mL−1 as the working concentration of Au25(Capt)18 for concurrent PTT and PDT since this concentration has been proven to be safe for cells without light irradiation. When exposed to an 808 nm laser, the survival rates of XL50 cells in the Au25(Capt)18 + laser group dropped to 44.30% ± 9.30%, which was much lower than those of the control, the Au25(Capt)18 (94.80% ± 2.65%) and the laser (94.23% ± 6.98%) groups (Fig. 3B). The corresponding cell morphology in each group was photographed (Fig. S1, ESI), which showed that after 5 min of irradiation, the cells in the laser group and Au25(Capt)18 group (80 μg mL−1) grew in a good way without morphology changes. However, in the Au25(Capt)18 + laser group, the cells shrank and became spherical in shape. These results collectively indicate the strong cellular lethality of Au25(Capt)18 under laser irradiation.

With the photothermal conversion and 1O2 generation capabilities of Au25(Capt)18in vitro shown in Fig. 2, the significant cell death could be attributed to the hyperthermia and 1O2 both generated by Au25(Capt)18 with laser irradiation. Furthermore, to differentiate the contributions of PTT and PDT to the cell killings, NAC, a scavenger for ROS, was added to quench the effect of PDT during the treatment. As shown by the results in Fig. 3B, the cell viability of Au25(Capt)18 + laser + NAC was higher than that in the Au25(Capt)18 + laser group, but lower than that of the control, which means the proliferation of cells after the interference of Au25(Capt)18 + laser can be partly recovered by the elimination of ROS by NAC. The experimental data indicated that the cell killing of Au25(Capt)18 triggered by the 808 nm laser involved a concurrent photothermal and photodynamic effect. We estimated that the cell killing contribution ratios of PTT and PDT were 28.86% and 71.14%, respectively, according to the cell viability values. To the best of our knowledge, this is the first time that the contributions of PTT and PDT in a single concurrent cancer treatment have been assessed.

The results in Fig. 3C–E show that the cells in the Au25(Capt)18 + laser group had the fastest temperature increase. It took only 5 min of irradiation for the cells to reach 44 °C, which is sufficient for cell killing.32 In comparison, the temperature of the cells in the laser only group increased to 25 °C (Fig. 3C), which is much lower than the cell death temperature (40–60 °C).46 A thermal imager was used to record the thermal images of the laser and Au25(Capt)18 + laser groups at different time points during the irradiation (Fig. 3E). The thermal image patterns were consistent with the results shown in Fig. 3C and D. According to the previous studies, the temperature of 44 °C can trigger immune responses, in which photothermal effect could not only cause coagulative necrosis, but also increase antigen uptake and upregulate the expression of MHC class II, CD80, CD86, and CD40 and induce DCs maturation to inspire a non-adaptive immune response.47–49 Also, for the adaptive immune response, Elizabeth et al. showed that hyperthermia above 39.5 °C could also enhance the effector T cell function, i.e., increase the expression of CD95L in CTLs and augment NK cell migration and lysis.50

Moreover, intracellular 1O2 levels were determined. Cancer cells were treated with SOSG before adding Au25(Capt)18 or before exposure to irradiation. From the results in Fig. 3F and G, the fluorescence intensity in the Au25(Capt)18 + laser group was significantly higher than for any other group. Although 1O2 could be induced from cells with laser illumination or Au25(Capt)18 alone, the intensities in those cells were quite weak. These results in this part prove the significant 1O2 generation capability of Au25(Capt)18 under laser irradiation in cells, and together with the temperature increase, a potent cell killing capacity could be achieved.

3.5. Toxicity study of Au25(Capt)18in vivo

Before treating tumor-bearing mice, the in vivo toxicity of Au25(Capt)18 was investigated. Blood biochemistry tests and H&E staining of the main organs were performed on day 14 after vein injection with Au25(Capt)18. Mice with PBS injection were set as control. Fig. 4A and B show the data of the liver function markers of alkaline phosphates (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), and the kidney function indicators of blood urea nitrogen (BUN) and serum creatinine (SCr). It can be seen that the values in the Au25(Capt)18-treated mice were consistent with those of the control healthy mice injected with PBS (p > 0.01), suggesting there was no obvious hepatotoxicity or nephrotoxicity induced by Au25(Capt)18.
image file: c9tb01573f-f4.tif
Fig. 4 In vivo toxicity study with intravenous injection of Au25(Capt)18. (A and B) Blood biochemistry data: (A) ALP, ALT, and AST; (B) BUN and SCr; (C) routine blood analysis: WBC, RBC, PLT, HGB, HCT, MCV, MCH, MCHC, NEUT, LYMPH, MONO; and (D) H&E staining images of tissue sections harvested from the heart, liver, spleen, lung, and kidney at day 14 post intravenous injection of Au25(Capt)18. Intravenous injection with equivalent PBS was set as a control.

The hematologic toxicity of Au25(Capt)18 was further evaluated. The commonly used hematology markers were selected, including white blood cells (WBCs), red blood cells (RBCs), hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), hematocrit (HCT), neutrophil (NEUT), lymphocyte (LYMPH), and monocytes (MONO).51 The results in Fig. 4C show that the values in the Au25(Capt)18-treated mice were similar with that in the control groups. Furthermore, neither obvious apoptosis nor noticeable pathological changes could be detected in the heart, liver, lung, spleen, and kidney in the groups receiving Au25(Capt)18 injections (Fig. 4D). These results collectively suggest the low toxicity of Au25(Capt)18in vivo.

3.6. In vivo concurrent PTT and PDT of cSCC

After confirming the cell therapy efficacy and biocompatibility of Au25(Capt)18, in vivo concurrent PTT and PDT treatment of cSCC on tumor-bearing SKH 1 mice was performed. Fig. 5A shows the temperature changes of the tumors, which were monitored by thermal imaging. With the extension of the illumination time, mice in the Au25(Capt)18 + laser group exhibited a rapid and sustainable increase in the tumor temperature, reaching to ∼60 °C after 10 min of laser exposure. While in the laser only group, although there was a rapid temperature increase in the first 2 min of illumination, the rate of increase slowed down and then kept stable. Fig. 5B shows the histogram of the tumor temperature changes after 10 min of illumination in both groups. It can be seen that the tumor temperature increased by 28.1 °C ± 6.76 °C after 10 min of irradiation in the Au25(Capt)18 + laser group, while the tumor temperature in the laser only group increased by only 8.36 °C ± 2.11 °C, significantly lower than that in the Au25(Capt)18 + laser group (t = 6.24, p < 0.01). According to the previous studies, the hyperpyrexia could directly induce tumor cells damage, and promote the release of a large number of damage-associated molecular patterns (DAMPs) at the same time, which could in turn activate cellular immunity to clear residual tumor cells.46,52
image file: c9tb01573f-f5.tif
Fig. 5 In vivo concurrent PTT and PDT treatment of cSCC tumor-bearing mice with Au25(Capt)18 under NIR laser irradiation. (A) Thermal images of mice in the Au25(Capt)18 + laser group and the laser group (Au25(Capt)18[thin space (1/6-em)]:[thin space (1/6-em)]2.5 mg mL−1, 100 μL; laser: 1 W cm−2, 10 min); (B) the temperature changes of tumors in A; (C) In vivo1O2 generation detection by using SOSG reagent via recording the fluorescence in different treatment groups, including the blank (1 and 2), SOSG, SOSG + laser, Au25(Capt)18, Au25(Capt)18 + SOSG, and Au25(Capt)18 + SOSG + laser; (D) Images of mice in the four groups, including Au25(Capt)18 + laser, Au25(Capt)18, laser and the control before and at 3, 7, 10, 15, 20, 40, and 90 days after treatment; (E) tumor volume curves in D; and (F) mice weight during the treatment. Error bars were based on triplet measurements (n = 5, *: p < 0.01, ϕ: the mice were euthanized on day 20 in the cases of tumor grow oversize).

PDT is an efficient treatment modality for selectively inducing oxidative damage in cancer cells. 1O2 and other reactive oxygen species (ROS), including hydroxyl radicals (˙OH), alkoxyl radicals (RO˙), and superoxide anions (O2˙), are the main killers of cancer cells induced by PDT.53 Kawasaki reported that Au25(Capt)18 could efficiently generate 1O2 under visible/NIR (532, 650, and 808 nm) irradiation, which was also confirmed in our in vitro study (Fig. 2E and 3F).35 As shown in Fig. 5C, there was no SOSG fluorescence in the tumor of mice after the intratumoral injection of Au25(Capt)18 or laser irradiation alone. However, in the Au25(Capt)18 + laser group, SOSG fluorescence was observed. This result indicates that under the excitation of an 808 nm laser, the Au25(Capt)18 nanoclusters taken up by tumor cells could produce 1O2, which is the key contribution to the photodynamic effect. As for the control, there was no obvious fluorescence observed in the tumor of mice with SOSG injection alone or SOSG injection plus laser irradiation. We verified the production of 1O2in vivo, aiming to show that near-infrared light can penetrate the skin surface to activate the PDT effect of Au25(Capt)18 which are then taken up by the tumor cells.18,54

The tumor volumes were measured and calculated every day during the treatment. As the results show in Fig. 5D and E, before the treatment (day 0), the starting volumes of the tumors in the 4 groups were 57.89 ± 17.42 mm3 (Au25(Capt)18 + laser group), 61.29 ± 16.16 mm3 (laser group), 59.95 ± 19.81 mm3 (Au25(Capt)18 group), and 55.96 ± 8.43 mm3 (the control group). ANOVA showed that there was no significant difference between the starting tumor volumes of these 4 groups (F = 0.11, p > 0.01). On the contrary, the tumor sizes of the mice in the control group showed a sustained increase and reached 398.76 ± 146.46 mm3 on average at day 20. Also, the tumors of the mice treated with the laser only or Au25(Capt)18 only showed continuous growth and the sizes grew up to 588.39 ± 448.44 mm3 and 482.27 ± 196.60 mm3, respectively, on average at day 20. Both of these were slightly larger than that in the control group, but not significantly different (F = 0.52, p > 0.01). Inspiringly, the t-test showed that the tumor size in the Au25(Capt)18 + laser group was significantly smaller than that in the other groups (Au25(Capt)18 + laser vs. laser group: t = 2.93, p < 0.01; Au25(Capt)18 + laser vs. Au25(Capt)18 group: t = 5.49, p < 0.01; Au25(Capt)18 + laser vs. control group: t = 6.09, p < 0.01).

During the treatment, as shown in Fig. 5F, the body weights of the mice were stable and no signs of appetite loss, physical activity decrease, or other behavioral changes were observed for the mice, suggesting the biosafety of Au25(Capt)18 without obvious systemic side effects.55 It should be noted that day 20 was set as the observing endpoint; except for 2 mice randomly chosen in Au25(Capt)18 + laser group to be further observed, all the other mice were euthanized before the tumor size exceeded 20 mm in any direction.56

It should be noted that necrosis was observed on the solid tumors of the Au25(Capt)18 + laser group on day 3 after the treatment. Escharosis of the original tumor tissue occurred on or around day 7. Finally, the local eschar of the solid tumor on most mice in the Au25(Capt)18 + laser group gradually disappeared, and no tumor recurrence was found on day 90. However, we noticed a case of recurrence near the eschar on one mouse in the Au25(Capt)18 + laser group on day 8 after treatment, which could be attributed to the revival of the tumor cells caused by inhomogeneous PTT or PDT. Surprisingly, the recurrence tumor disappeared on day 16 without any further treatment (Fig. 6C). This could indicate an immune response induced by PTT and PDT.

image file: c9tb01573f-f6.tif
Fig. 6 T cells infiltration study on the SKH-1 tumor-bearing mice. Tumors were biopsied on day 3 (A) and day 7 (B) after the different therapies. Biopsies were sectioned and stained for immunohistochemistry analysis of CD4 and CD8. Arrows identify the positive stained cells. (C) Photos of one mouse in the Au25(Capt)18 + laser group showing tumor inhibition, recurrence, and finally cured after one treatment.

3.7. Preliminary study of immune responses induced by PTT and PDT

CD4+ helper T cells and CD8+ cytolytic T cells are reported to play significant roles in immunotherapy, killing tumor cells and preventing tumor recurrence.57,58 In this study we used SKH-1 hairless mice with complete immune systems. Our immunohistochemical examination results in Fig. 6 show a high expression of CD4+ T cells and CD8+ T cells on day 3 (Fig. 6A) and day 7 (Fig. 6B) in the tumors of the Au25(Capt)18 + laser group mice, while no significant positive T cells were found in the laser, Au25(Capt)18, or the control groups. Compared to the results on day 3, T cells infiltration on day 7 became more pronounced. This result suggests a strong elicitation of specific cellular immunity against cSCC induced by Au25(Capt)18-mediated phototherapy. The concurrent PTT and PDT with Au25(Capt)18 may promote T cell activation and enhance adaptive antitumor immunity, which can contribute to the prevention of recurrence.

Interestingly, more CD8+ T cells were found than CD4+ T cells in the infiltrated area (Fig. 6). This phenomenon is consistent with previous reports: cytotoxic CD8+ T cells are essential to the immunotherapy outcome, whereas the CD4+ T cells play a supportive role.58–60 Therefore, PTT and PDT using Au25(Capt)18 under laser irradiation could not only destroy primary tumors by producing heat and 1O2 but could also stimulate the immune system to further destroy residual tumor cells and to block recurrence.59

4. Conclusions

In summary, we demonstrated Au25(Capt)18 can generate potent 1O2 and also produce heat through the direct photosensitization under NIR 808 nm laser irradiation. Au25(Capt)18 possesses the main features desired of agents for photomedicine with a clear and simple structure, water solubility, and good colloidal stability and photostability. The therapeutic efficacy of Au25(Capt)18 was verified on the cSCC cells and a mouse model. We demonstrated that the effect of concurrent PTT and PDT using Au25(Capt)18 during laser irradiation, and for the first time estimated their contribution ratios to the cell death. Moreover, local infiltration of CD4+ T cells and CD8+ T cells were found post one treatment of Au25(Capt)18 + laser and these T cells could potentiate immune therapy. Our results indicate that Au25(Capt)18 nanoclusters could be a promising nanomaterial for concurrent PTT and PDT to treat skin cancers.

Conflicts of interest

There are no conflicts to declare.


We thank the Financial support from the National Natural Science Foundation of China (NSFC 81872212, 81871399, 81571742, 81801823, 81601601), the Fundamental Research Funds for the Central Universities, the Science and Technology Innovation Plan of Shanghai Science and Technology Committee (19441904200).


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Electronic supplementary information (ESI) available. See DOI: 10.1039/c9tb01573f

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