Nanoscale Reduced Graphene Oxide-Mediated Photothermal

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Biological and Medical Applications of Materials and Interfaces

Nanoscale reduced graphene oxide-mediated photothermal therapy together with IDO inhibition and PD-L1 blockade synergistically promote antitumor immunity Mengmeng Yan, Yijia Liu, Xianghui Zhu, Xiaoli Wang, Lanxia Liu, Hongfan Sun, Chun Wang, Deling Kong, and Guilei Ma ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18751 • Publication Date (Web): 24 Dec 2018 Downloaded from http://pubs.acs.org on December 25, 2018

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ACS Applied Materials & Interfaces

Nanoscale reduced graphene oxide-mediated photothermal therapy together with IDO inhibition and PD-L1 blockade synergistically promote antitumor immunity

Mengmeng Yan#, Yijia Liu#, Xianghui Zhu, Xiaoli Wang, Lanxia Liu, Hongfan Sun, Chun Wang, Deling Kong, Guilei Ma*

The Tianjin Key Laboratory of Biomaterials, Institute of Biomedical Engineering, Peking Union Medical College & Chinese Academy of Medical Sciences, Tianjin, China

#

Mengmeng Yan and Yijia Liu contributed equally to this work.

*Correspondence

to: Guilei Ma (E-mail: [email protected])

ACS Paragon Plus Environment

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

Abstract Despite the potential efficacy of immune checkpoint blockade for effective treatment of cancer, this therapeutic modality is not generally curative and only a fraction of patients respond. Combination approaches provide strategies to target multiple antitumor immune pathways to induce synergistic antitumor immunity. Here, a multi-combination immunotherapy including photothermal therapy (PTT), indoleamine-2,3-dioxygenase (IDO) inhibition and programmed cell death-ligand 1 (PD-L1) blockade is introduced for inducing synergistic antitumor immunity. We designed a multifunctional IDO inhibitor (IDOi)-loaded reduced graphene oxide (rGO)-based nanosheets (IDOi/rGO nanosheets) with the properties to directly kill tumor cells under laser irradiation and in situ trigger antitumor immune response. In vivo experiments further revealed that the triggered immune response can be synergistically promoted by IDO inhibition and PD-L1 blockade; the responses included the enhancement of tumor-infiltrating lymphocytes (TILs) including CD45+ leukocytes, CD4+ T cells, CD8+ T cells and NK cells, the inhibition of the immune suppression activity of regulator T cells (Tregs) and the production of INF-. We also demonstrate that the three combinations of PTT, IDO inhibition and PD-L1 blockade could effectively inhibit the growth of both irradiated tumors and tumors in distant sites without PTT treatment. This work can be thought as an important proof-of-concept to target multiple antitumor immune pathways to induce synergistic antitumor immunity.

Keywords: combinatorial immunotherapy, PD-L1, IDO inhibition, photothermal therapy, reduced graphene oxide


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1. Introduction Cancer immunotherapy has been recently proven to be a promising way to control cancer, which can induce the inherent immunological system of the body to kill tumor cells,.1-3 Of particular interest for the immunotherapy is the blockade of immune checkpoint that down-modulate immune functions, which has demonstrated clinical activity and provided a new strategy to treat cancer.4-6 The interaction between PD-1 (programmed cell death-receptor 1) and PD-L1 (programmed cell death-ligand 1) is a potent negative regulator of T cell and can be manipulated by tumor cells to escape immune attack.7,8 Blocking PD-1/PD-L1 pathway with monoclonal antibodies has provided significant advances in various cancer treatments. However, anti-PD-L1-based therapies are not generally curative, and have partial antitumor activity in a fraction of cancer patients. Sufficient T cell infiltration in immunogenic tumor microenvironment is critical for an effective PD-L1 blockade therapy.9,10 In this context, it is envisioned that combination therapy to target both promotion of lymphocytic infiltration and induction of an immunogenic tumor microenvironment will be required for maximal benefit of anti-PD-L1 therapy. Photothermal therapy (PTT) to treat local tumors using photothermal agents under laser irradiation has been widely studied recently.11-14 PTT can directly kill tumor cells and the resulted tumor-derived antigens can recruit and activate antigen-presenting cells in the ablated tumor site, thus inducing the antigen-specific antitumor immune response.15,16 However, The “abscopal effect” induced by PTT usually is not strong enough to suppress the growth of tumors without PTT treatment. More and more studies showed that a combination of PTT therapy with checkpoint blockade therapy demonstrated potential in cancer therapy.17-19 For example, a combination of PTT treatment with indoleamine-2,3-dioxygenase (IDO) inhibition have a synergetic effect on inducing systemic antitumor immunity and preventing cancer metastasis.20 IDO is an immune checkpoint, which is over-expressed in tumor cells and catalyzes the oxidative catabolism of tryptophan to kynurenine.21 The depletion of



tryptophan can prevent the clonal expansion of T cells through the induction of immune tolerance. IDO inhibitors such as INCB24360 and NLG919 can effectively block tryptophan catabolism and augment T cell expansion. The research reported by Lu et al. showed that a chlorin-based metal-organic framework by co-delivery of INCB24360 can achieve a combination of photodynamic therapy and IDO inhibition.17 Peng et al. also developed a NLG919/IR780 nanosystem with a combined PTT and IDO inhibition.18 These studies suggested that regulation of IDO functions in tumor has great significance in the modulation of anti-tumor immune response. Therefore, it is hypothesized that the efficacy of PD-L1 blockade can be improved when in combination with PTT-based treatment to create an immunogenic tumor microenvironment and IDO inhibition to promote T cell activity. The individual and combined impact of PTT, IDO inhibition and PD-L1 blockade in tumor-bearing mice has been shown to enhance efficiency of cancer therapy.20-22 The impact of simultaneously targeting all three pathways in tumor-bearing mice has not been explored until now. Herein, we have started with PTT as an effective direct approach of killing tumor, along with IDO inhibition in tumor sites and PD-L1 blockade. We design a multifunctional reduced graphene oxide (rGO)-based nanosheets containing a small molecule IDO inhibitors (IDOi, Epacadostat), which enable a novel cancer treatment strategy by combining near infrared fluorescence (NIR)-triggered PTT to kill tumor cells and IDO inhibitor-based immunotherapy in tumor sites. rGO nanosheets are funtionalized with polyethylene glycol (PEG) and folic acid (FA) to increase the stability, blood circulation time and tumor accumulation of rGO nanosheets and IDOi. The formed PEG-rGO-FA-IDOi nanosheets under NIR laser irradiation could trigger PTT destruction of primary tumors and release tumor-associated antigens which are presented to antigen presentation cells. Meanwhile, the IDOi released from PEG-rGO-FA-IDOi nanosheets in tumor sites modulates tryptophan catabolism to enhance antitumor immune response, which could be further promoted by anti-PD-L1 blockade to effectively eliminate both irradiated tumors and tumors without PTT treatment (Scheme 1). In this study, we aim to investigate the impact of the strategy


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of multi-combination immunotherapy on the inhibition of tumor growth and the promotion of antitumor immunity.

Scheme 1. The mechanism of PEG-rGO-FA-IDOi nanosheets-mediated PTT therapy together with IDO inhibition and PD-L1 blockade for synergistically effective antitumor immunity.

2. Experimental Section 2.1 Synthesis of FA conjugated rGO (rGO-FA) nanosheets For the synthesis of rGO-FA conjugates, FA was dissolved into MilliQ water and reacted with hydrazine hydrate and GO at 80 ℃ for 1 h.23 Then a black resulting product was obtained and purified using an Amicon ultra-4 centrifugal filter (10 kDa MWCO) to remove excess hydrazine hydrate and FA. The collected product was freeze-dried for further use.

2.2 Preparation of PEG modified rGO-FA (PEG-rGO-FA) nanosheets



To improve the instability in the saline solution of rGO-FA, PEG grafted C18PMH (PEG-C18PMH) was synthesized as described elsewhere.24 Briefly, PEG-C18PMH was synthesized by mixing PEG and MHC18 monomer at a molar ratio of 1:1 in dichloromethane. The mixture was reacted with EDC and triethylamine under stirring for 24 h. Dichloromethane was removed by the means of rotary evaporation till the solid product was separated out. The obtained product was then dialyzed against water and was lyophilized and storied at 4 ℃ for further use. To prepare PEG-rGO-FA, C18PMH-PEG was added to rGO-FA in MilliQ water. The mixture was ultra-sonicated and then ultra-centrifuged to remove the excess C18PMH-PEG.

2.3 Characterization of the prepared rGO nanosheets To confirm conjugation of FA and PEG to GO nanosheets, the prepared GO, rGO-FA, PEG-rGO-FA nanosheets were analyzed by fourier transform infrared (FTIR) spectra and raman spectroscopy, respectively. A 90Plus particle size (Brookhaven) was used to determine the average particle size of the prepared GO, rGO-FA, PEG-rGO-FA nanosheets, respectively. The morphology of the prepared nanosheets was visualized by using atomic force microscopy (AFM, Veeco Instruments). The absorption spectra of ultraviolet, visible and NIR (UV-vis-NIR) range absorption spectroscopy from 200 nm to 900 nm were detected to determine whether the prepared nanosheets we designed have the significant photothermal effect. The photothermal conversion capability of the prepared GO nanosheets was evaluated under laser irradiation at 808 nm (1 W/cm2). The temperature change during laser irradiation was determined by a FLIR Ax5 camera every 60 s for 8 min.

2.4 Preparation and characterization of IDOi-loaded PEG-rGO-FA (PEG-rGOFA-IDOi) nanosheets For IDOi loading, PEG-rGO-FA was incubated with IDOi in DMSO for 12 h. The reaction mixture was added 10 mL of distilled water and then ultra-centrifuged to remove free IDOi and DMSO. The collected product was freeze-dried for further use. The amount of free IDOi was determined in the supernatant by HPLC with UV


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detection at 280 nm using supernatant of non-loaded nanosheet as basic correction (Figure S1). The loading capacity of IDOi on the PEG-rGO-FA nanosheet was calculated according equation: ((the total amount of IDOi － the free amount of IDOi)/the weight of PEG-rGO-FA-IDOi nanosheet) 100%. Release behaviour of IDOi from PEG-rGO-FA-IDOi nanosheet was studied in PBS (pH 7.4) at 37 °C.

2.5. In vitro photothermal toxicity of the prepared rGO nanosheets The in vitro photothermal toxicity was measured by CT26 (murine colon carcinoma) cell lines. The cells was seeded into 96-well plate (1×104 cells/well) and incubated with PEG-rGO-FA or PEG-rGO at the same GO concentration of 20 g/mL. After 6 h incubation, the cells were exposed to a 1W/cm 2 808 nm laser every 60 s for 8 min. To incubate for 24 h continuously, the photothermal toxicity was examined by a CCK-8 assay kit.

2.6 Targeted uptake of the prepared rGO nanosheets by CT26 cells To investigate FA receptor-mediated cellular internalization, CT26 cells were treated with PEG-rGO-Cy7, PEG-rGO-FA-Cy7 or PEG-rGO-FA-Cy7 with free FA (1×10−3 M, as an inhibitor) for 6 h. The cellular uptake of Cy7-labelled rGO nanosheets was analysed by BD Accuri™ C6 software. To evaluate the intracellular distribution of the prepared nanosheets, the cells were labelled with DAPI to identify nuclei. Finally, cells were observed using confocal laser scanning microscopy (CLSM).

2.7 In vivo biodistribution PEG-rGO and PEG-rGO-FA, CT26 cells (1×106 cells/mouse) were inoculated subcutaneously into the right flank of female BALB/c mice (6-8 weeks old). When the tumor reached ~150 mm3, the mice were injected via the tail vein with 100 μl of PEG-rGO-FA-Cy7 or PEG-rGO-Cy7 (at the same GO concentration of 200 g/100 L). The Maestro imaging system (CRI, USA) was used to monitor the fluorescent signals of Cy7 (745 nm excitation and 774 nm filter) at 4, 8, 12, 16 and 24 h after



injection. The mice were sacrificed 24 h after injection and the organs were collected for imaging. For quantitative biodistribution of IDOi, FITC was covalently coupled to IDOi, and FITC-labeled IDOi (FITC-IDOi) was then loaded onto PEG-rGO-FA. Mice bearing CT26 tumors mice as decribed above were injected via the tail vein with 100 μL of PEG-rGO-FA-Cy3-IDOi or free Cy3-IDOi (at the same IDOi concentration of 30 g/100 L). The mice were sacrificed at 2, 12, and 24 h after injection and excised the organs. The amount of FITC-IDOi was determined by fluorescent spectrometer. Detailed protocols are provided in Supplementary Materials. 2.8 Activation of dendritic cells (DCs) induced by PTT Mice bearing subcutaneous CT26 tumors were injected via the tail vein with 100L of PEG-rGO, PEG-rGO-FA or PEG-rGO-FA-IDOi (at the same GO concentration of 200 g/100 L) as described above. 12 h after injection, the mice were irradiated with an 808nm NIR laser at 1W/cm2. The temperature change in tumor sites was studied using a FLIR Ax5 camera. To study activation of DCs induced by PTT, the inguinal lymph nodes near the tumor sites were harvested from mice in different groups 96 h after treatment. Then, single cell suspension prepared from the lymph nodes was stained with fluorescein-labeled anti-mouse CD86 and CD80 and analysed using flow cytometry.

2.9 Tumor challege in vivo 1×106 CT26 cells were inoculated subcutaneously into the right flank and 2×105 CT26 cells into the left flank of each female BALB/c mouse (6-8 weeks old). The left tumors as primary tumors (1#) were treated by local NIR irradiation and the right tumor as distant (abscopal) tumors (2#) without direct treatment. When the primary tumor reached ~50 mm3, the mice were subcutaneously injected at their tail-base site with 100 μl of PBS, PEG-rGO or PEG-rGO-FA-IDOi (at the same GO concentration of 200 g/100 μl) with or without laser irradiation. Twelve hours after injection, laser irradiation (808 nm, 1 W/cm2) was applied to irradiate the tumor site for 8 min. For


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the combination immunotherapy of PD-L1 checkpoint blockade, mice were intraperitoneally injected with 200 μg anti-PD-L1 antibody on day 1, 3, and 5 after the right tumor was under irradiation. Tumour volume was measured every 2-3 days. When the primary tumor reached ~2000 mm3, all the mice were sacrificed. To study immune cells in the distant tumor, cell suspensions of tumor was stained with fluorescein-labeled antibodies: CD3, CD4, CD8a, CD45, Foxp3, and NKp46. Serum samples were isolated from mice after various treatments and diluted for analysis of IFN-γwith ELISA kits (eBiosciences).

2.10 Statistical analysis All data were expressed as means ± standard deviation (SD) or means ± standard errors (SEM). All figures shown in this article were obtained from at least three independent experiments (as detailed throughout the paper) with similar results. Differences between groups were determined using one-way ANOVA followed by Tukey’s post-test (GraphPad Prism 5.0, GraphPad software, CA, USA). Statistical significance is denoted by p < 0.05, p < 0.01 and p < 0.001.

3. Results and Discussion 3.1 Preparation and characterization of the prepared rGO nanosheets The routes of synthesis and surface modifications of PEG-rGO-FA nanosheets are shown in Figure 1A. rGO-FA was first synthesized as reported by Park et al.23 PEG-C18PMH is modified onto the rGO-FA surface via the strong hydrophobic interactions between C18 hydrocarbon chains in the PEG-C18PMH and the hydrophobic rGO nanosheets.24 The formed PEG-rGO-FA nanosheets can be further applied to load IDOi, Epacadostat, via π - π stacking interaction.25,26 The chemical structure of rGO-FA and PEG-rGO-FA was first confirmed by Raman, FTIR and UV-Vis-NIR adsorption spectra. Raman spectrum of rGO-FA showed increased intensity ratio between D and G peaks (ID/IG) of 2.37 compared with 0.85 of GO, indicating a conclusive evidence of GO reduction (Figure 1B). Figure 1C shows the



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FT-IR spectra of rGO-FA and PEG-rGO-FA. The stretching peaks of carboxylic acid at 1722 cm-1 disappeared while the strong stretching peak of aromatic C=C at 1627 cm-1 preserved. Furthermore, after PEG-C18PMH coating on the rGO-FA surface via the strong hydrophobic interactions between C18PMH and rGO, strong C-H (2886 cm-1) and O-H (1097 cm-1) stretch peaks emerged in the FT-IR of PEG-rGO-FA. Figure 1D shows the UV-vis-NIR range absorption spectra of the prepared rGO nanosheets at the same GO concentration. At 808 nm, the absorbance of rGO-FA and PEG-rGO-FA showed an approximately 6-fold increase compared to that of GO. These data suggest that rGO-FA and PEG-rGO-FA nanosheets are more effective than GO nanosheets in light-to-heat conversion under NIR laser irradiation.27,28 The photothermal potential of the prepared rGO nanosheets was evaluated by NIR laser irradiation

at

808

nm.

As

shown

in

Figure

1E,

the

temperature

of

PEG-rGO-FA-containing solutions increased to 53 °C within 8 min of irradiation, which can result in irreversibly destroying to cells.29,30 AFM image showed a well distributed size of rGO-FA, PEG-rGO-FA and PEG-rGO-FA-IDOi nanosheets with mean sizes less than 200 nm, which is in accordance with the result of size measurements (Figure 1F and Figure S2). The sheet thickness of PEG modified rGO nanosheets was much higher than that of rGO nanosheets, indicating the successful coating of PEG on rGO. The stability of PEG-rGO-FA and PEG-rGO-FA-IDOi nanosheets was tested by monitoring changes in the average hydrodynamic diameter in saline and serum at 4 ℃, respectively. As shown in Figure 1G, PEG-rGO-FA and PEG-rGO-FA-IDOi nanosheets were relatively stable over 7 days in the above mentioned condition. The prepared PEG-rGO-FA-IDOi nanosheets owns IDOi loading efficiency of >99% determined by HPLC. The high loading efficiency of IDOi onto the prepared rGO nanosheets was likely due to π–π stacking and hydrophobic interactions between IDOi and rGO nanosheets. The results of in vitro release suggested that PEG-rGO-FA-IDOi nanosheets exhibited a sustained IDOi release over 22 days (Figure 1H).


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Figure 1. A) Strategic illustration of synthesis of PEG-rGO-FA-IDOi nanosheets. B) Raman spectra, C) FTIR spectra and D) UV-Vis-NIR adsorption spectra of the prepared different GO nanosheets. E) Photothermal effect of the prepared different GO nanosheets suspensions with laser irradiation. F) AFM images of the prepared different GO nanosheets. (G) The stability of PEG-rGO-FA-IDOi nanosheets over time in saline and serum at 4 ℃. (H) In vitro release of IDOi from PEG-rGO-FA-IDOi nanosheets.

3.2 FA receptor-mediated cellular uptake of PEG-rGO-FA nanosheets by CT26



cells Cellular uptake of PEG-rGO-FA nanosheets was evaluated in vitro. CLSM images in Figure 2A show that PEG-rGO-FA-treated cells exhibited significantly strong red fluorescence compared to cells treated with PEG-rGO. In contrast, treatment with excessive free FA led to the decrease of red fluorescence in cells incubated with PEG-rGO-FA nanosheets. Figure 2B shows the quantitative results determined by flow cytometry. The mean fluorescence intensities of PEG-rGO-FA nanosheets reached approximately 2.2-fold and 2.8-fold of that for PEG-rGO nanosheets and PEG-rGO-FA nanosheets treated with free FA, respectively. The results demonstrated FA receptor-mediated intracellular uptake of PEG-rGO-FA nanosheets. The photothermal toxicity of PEG-rGO-FA and PEG-rGO nanosheets in cultured CT26 cells was evaluated. As shown in Figure 2C, the enhanced photothermal toxicity with the increased irradiation time was observed in all cases and nearly 80 % cells treated with PEG-rGO-FA and PEG-rGO nanosheets were dead under irradiation under laser irradiation for 6 min.

Figure 2. (A) CLSM images and of cellular uptake of Cy7-labeled PEG-rGO and PEG-rGO-FA by CT26 cells. (B) Flow cytometry assay of cellular uptake of


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Cy7-labeled PEG-rGO and PEG-rGO-FA by CT26 cells. (C) Survival of CT26 cells incubated with PEG-rGO and PEG-rGO-FA under laser irradiation for different time.

3.3 PEG-rGO-FA-IDOi nanosheets show high tumor-specific targeting Tumor-specific accumulation of Cy7-labeled nanosheets was determined in the CT26-tumor bearing mice intravenously injected with PEG-rGO-FA or PEG-rGO. Figure 3A shows that PEG-rGO-FA nanosheets had a rapid initial fluorescence as early as 4 h post-injection, while PEG-rGO nanosheets did not display obvious fluorescence in tumor area at that time point, which could be due to the enhanced accumulation of PEG-rGO-FA nanosheets in tumor via FA-mediated active targeting. At 12 h post-injection, the tumor fluorescence of PEG-rGO-FA group reached the maximum, which was still stronger than that of non-targeting PEG-rGO group. In particular, ex vivo fluorescence images of organs excised from mice (sacrificed at 24 h post-injection) also demonstrated the superior tumor-targeting efficiency of PEG-rGO-FA nanosheets (Figure 3B and 3C). For the PEG-rGO-FA group, decreased fluorescence signals could be detected in the liver, lung and kidney compared to PEG-rGO, respectively. However, the average fluorescence intensity of the PEG-rGO-FA nanosheets in the tumor was greatly enhanced, which could be helpful for the PTT treatment of the tumor. The tumor targeting ability of PEG-rGO-FA-IDOi nanosheets also affect the consequent immune modulation by IDOi in tumor site. To better demonstrate the tumor targeting of IDOi, ex vivo quantitative biodistribution of IDOi was carried out in different organs at different time points. As shown in Figure 3D, the biodistribution of PEG-rGO-FA-IDOi showed the highest accumulations in the tumors at different time intervals, whereas the accumulation of free IDOi was mainly in the liver and kidney. The content of IDOi in the tumor after intravenously injection of PEG-rGO-FA-IDOi was enhanced by 7-fold at 2h, 38-fold at 12 h and 4-fold at 24h compared

to

the

free

IDOi,

respectively.

These

results

indicated

that

PEG-rGO-FA-IDOi nanosheets have greater potential to target tumor and enhanced the accumulation of rGO nanosheets and IDOi in the tumor site.



Surface coatings with biocompatible polymer such as PEG could improve the biocompatibility of GO-based nanosheets.31 In this study, major organs (heart, liver, spleen, lung and kidney) from the prepared PEG-rGO nanosheets treated mice were collected at 1 day, 7 days and 18 days post-injection (Figure S3 and Figure S4). Hematoxylin and eosin (H&E) stained tissue slices showed that the prepared PEG-rGO nanosheets may not be obviously toxic to mice.

Figure 3. (A) In vivo NIR fluorescence imaging of the CT26 tumor-bearing mice after intravenous injection with Cy7-labeled PEG-rGO-FA nanosheets at different time interval. (B) Ex vivo imaging and (C) Semi-quantitative biodistribution of Cy7-labeled PEG-rGO-FA nanosheets in different organs at 24 h post-injection. (D) Ex vivo quantitative biodistribution of FITC-IDOi in different organs at different time points after intravenous injection of free FITC-IDOi and PEG-rGO-FA-FITC-IDOi. The data are presented as the mean ± SD (n = 3) ( ***p < 0.001).


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3.4 PEG-rGO-FA-IDOi-mediated PTT activates systematic antitumor immune response -mediated PTT induces the activation of dendritic cells (DCs) in vivo

Figure 4. The prepared rGO nanosheets-mediated PTT induces the activation of DCs in vivo. (A) Infrared thermographic maps of mice-bearing CT26 tumors intravenously injected with the prepared rGO nanosheets were determined during the 8 min laser irradiation (808 nm, 1 W/cm2). (B) Temperature changes of irradiated areas of mice-bearing CT26 tumors intravenously injected with the prepared rGO nanosheets or saline were measured during the 8 min laser irradiation (808 nm, 1 W/cm2). (C)-(E) Expression of the co-stimulatory molecules CD86 and CD80 on CD11c+ DCs in lymph node determined by flow cytometry. The data are presented as the mean ± SD (n = 3) (*p < 0.05; **p < 0.01; ***p < 0.001).

Activation of DCs is prerequisite to initiate immune responses.32 Here, we assessed whether PEG-rGO-FA-IDOi mediated PTT influenced the activation of DCs. Mice-bearing subcutaneous CT26 tumors were intravenously injected with PEG-rGO, PEG-rGO-FA or PEG-rGO-FA-IDOi and then treated with laser irridiation. The tumour temperature of mice treated with PEG-rGO-FA had a maximum temperature



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of ~53 °C, significantly higher than PEG-rGO group (~46 °C) and saline group (~42 °C) (Figure 4A and 4B,). The higher tumor temperature observed in PEG-rGO-FA group may be due to the FA-mediated accumulation in tumor, which exceeded the destroy thershold needed to effectively ablate tumors. Similar to the PEG-rGO-FA-treated group, a maximum temperature of ~53 °C could be detected in the mice treated with PEG-rGO-FA-IDOi, indicating that IDOi loading had no effect on the photothermal potential of PEG-rGO-FA. Maturated DCs can exhibit an enhancement in the surface expression of costimulatory molecules CD86 and CD80. After 24 h post-NIR laser irradiation, expression of CD86 and CD80 on DCs in the inguinal lymph nodes near the tumor sites collected from mice was determined using flow cytometry. Figure 4C-4E show that treatment with PEG-rGO-FA with NIR laser irradiation could significantly promote DC maturation compared to that of PEG-rGO-FA without NIR laser irradiation. Moreover, PEG-rGO-FA-mediated PTT treatment induced significantly upregulation of CD86 and CD80 on DCs compared to that resulted from PEG-rGO with PTT treatment, likely due to the enhanced tumor-specific accumulation and tumor cell uptake. Studies have shown that tryptophan usage in the microenvironment is essential for DC maturation, and high expression of IDO in regulatory DCs inhibits DC maturation.33 As shown in Figure 4E and S5, increased percentage of CD86+CD80+

DCs

was

PEG-rGO-FA-IDOi-mediated

detected PTT

in

(27.77%),

the

group

significantly

treated higher

than

with the

PEG-rGO-FA-mediated PTT (22.73%) and the PEG-rGO-mediated PTT (18.4%). These results demonstrated that PEG-rGO-FA-IDOi-based PTT treatment could efficiently promote the activation of DCs and initiate the induction of immune responses.

3.5 PEG-rGO-FA-IDOi-mediated PTT activates systematic antitumor immune response Next, we assessed whether PEG-rGO-FA-IDOi-mediated PTT treatment and immunotherapy could be applied to enhance the anticancer efficacy and the


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immunological response using a bilateral CT26 model in mice. The left tumors as primary tumors (1#) were treated by local NIR irradiation and the right tumor as distant (abscopal) tumors (2#) without direct treatment (Figure 5A). Mice were randomly divided into four groups: (1) PBS, (2) PEG-rGO-FA with NIR irradiation, (3) PEG-rGO-FA-IDOi and (4) PEG-rGO-FA-IDOi with NIR irradiation. Mice were intraveneously injected with the above indicated formulations. For PTT treatment, the tumor site was irradiated with NIR laser (808 nm, 1 W/cm2, 8 min) 12 h after injection. As shown in Figure 5B, PTT treatment with both PEG-rGO-FA and PEG-rGO-FA-IDOi led to near elimination of the primary tumors. Regarding the distant tumor, PEG-rGO-FA-IDOi-mediated PTT treatment showed no significant inhibition in the tumor growth compared with the PEG-rGO-FA with NIR irradiation and PEG-rGO-FA-IDOi without NIR irradiation, but nonetheless the tumor growth was decreased significantly compared with the PBS control group (Figure 5C). We then investigated the abilityt of PEG-rGO-FA-IDOi-mediated PTT treattment to induce the immunological response. IFN-γ is essential for developing antitumor immunogical response.34 The results herein showed that PEG-rGO-FA-IDOimediated PTT treatment exhibited higher IFN-γ than did all other groups. We further determined infiltrating lymphocytes in the distant tumor 16 days post PTT treatment. As shown in Figure 5F, 5G, S6 and S7, in mice treated with PEG-rGO-FA-IDOi with PTT, increased proportion of CD3+ CD4+ T cells, and CD3+ CD8+ T cells in the distant tumor was observed, significantly higher than all other groups. Moreover, the proportion of CD45+ cells in the distant tumour also increased after the treatment with the PEG-rGO-FA-IDOi under NIR irradiation (46.84%), significantly higher than the PEG-rGO-FA-IDOi-mediated PTT group (29.44%) and the PBS control group (21.72%) (Figure 5E and S8). Pprevious reports have reported that IDO is known to downregulate the function of NK cells. We further profiled NK cells in the distant tumors. As shown Figure 5H, elevated NK cells were also observed in the distant tumors after the treatment with the PEG-rGO-FA-IDOi under NIR irradiation (16.12%), significantly higher than the PEG-rGO-FA-IDOi-mediated PTT group (10.66%) and the PBS control group (3.58%). These results imply that the synergistic



therapy of PEG-rGO-FA-IDOi-mediated PTT treatment and IDO inhibition elicits a more effective TILs response that may aid in the distant tumor inhibition. It was reported that IDO inhibition can downregulate the function of regulatory T cells (Treg).35,36 Treg are able to hamper effective antitumor immunity, in contrast to CD4+ effector T cells (CD4+ Teff ) and CD8+ T cells which play critical rols in the promotion of antitumor immune responses. In this study, PEG-rGO-FA-IDOi-based PTT-treated group enhanced the ratio of both CD4+ Teff and CD8+ to Treg cells compared to PBS control group but had no significant difference from that of PEG-rGO-FA-IDOi group without PTT and PEG-rGO-FA with PTT group (Figure S9).

Figure 5. PEG-rGO-FA-IDOi-mediated PTT activates systematic antitumor immune response. (A) Scheme illustration of PEG-rGO-FA-IDOi-mediated PTT. Tumor growth for (B) primary tumor (1#) and (C) distant tumor (2#). (D) The production of INF- in serum was determined by ELISA. Percentages of (E) CD45+ leukocytes, (F) CD3+ CD4+ T cells, (G) CD3+ CD8+ T cells and (H) NK cells (CD45+CD3-NKp46+) in the distant tumor. The data are presented as the mean ± SD (n = 5) (*p < 0.05; **p < 0.01; ***p < 0.001).


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3.6 PEG-rGO-FA-IDOi-mediated PTT plus anti-PD-L1 combination therapy inhibits tumor growth and enhances tumor-infiltrating lymphocytes (TILs) The above results indicated that CT26 tumor showed no significant effective response to PEG-rGO-FA-IDOi-mediated PTT treatment, possibly due to CT26 as an immunogenic colon tumor model that demonstrates PD-L1 expression on tumor cells in vivo.37 Previous studies reported that the interaction between PD-1 and PD-L1 is a potent negative regulator of T cell and can be manipulated by cancer cells to evade immune attack.7,8 In this study, we further investigated whether the antitumor efficacy of PEG-rGO-FA-IDOi-mediated PTT treatment can be improved when used in combination with PD-L1 blockade therapy. We treated bilateral CT26 tumor-bearing mice as shown in Figure 6A. Mice were randomly divided into five groups: (1) PBS, (2) anti-PD-L1, (3) PEG-rGO-FA-IDOi with NIR irradiation, (4) PEG-rGO-FA-IDOi plus anti-PD-L1, (5) PEG-rGO-FA-IDOi plus anti-PD-L1 with NIR irradiation. Mice were intraveneously injected with the above indicated formulations and irradiated with NIR laser as described above. After irradiation, anti-PD-L1 antibody was i.p. injected into mice on day 1, 3, 5 (100 g/mouse each). As shown in Figure 6B, anti-PD-L1 alone had no apparent effect on the inhibition of tumor growth on both sides. PEG-rGO-FA-IDOi with irradiation led to a complete ablation of the primary tumors but exhibited no significantly inhibition of the distant tumors. Interestingly, PEG-rGO-FA-IDOi plus anti-PD-L1 without NIR irradiation could partially confer tumor growth control on both sides. However, the combination of PEG-rGO-FA-IDOi plus anti-PD-L1 with NIR irradiation significantly augmented the antitumor efficacy, could not only efficiently eradicate primary tumors but also strongly inhibit distant tumors progression. These results together suggest excellent antitumor abilitiy of PEG-rGO-FA-IDOi-mediated PTT treatment combined with PD-L1 blockade, which could be attributable to strong synergistic antitumor immunity induced by targeting multiple antitumor immune pathways. Moreover, during the course of treatment, the drop of animal body weight was not observed (Figure S10). To address the cellular mechanisms mediating the excellent antitumor abilitiy in this system, the propotion of TILs including CD45+ leukocytes, CD4+ T cells and



CD8+ T cells in the distant tumors was further analyzed using flow cytometry. Impressively, treatment with the combined PEG-rGO-FA-IDOi-mediated PTT plus PD-L1 antibody led to the remarkably increased propotion of CD45+ leukocytes, CD4+ T cells and CD8+ T cells in the distant tumors and approximately 1.8-4 fold enhancement compared to treatment with the single PEG-rGO-FA-IDOi-mediated PTT (Figure 6C-6E). Moreover, recent data have shown that PD-L1 plays a key role in the conversion of native T cells to Tregs.9,38 The decrease in immunosupressive Treg cell infiltration is critical following antitumor immune activation, which could be reflected in the increase in CD8+ and CD4+ Teff to Treg cell ratios. As shown in Figure 6F, With the help of the PD-L1 blockade, PEG-rGO-FA-IDOi-based PTT-treated group significantly enhanced the ratio of both CD4+ Teff and CD8+ to Treg cells in the distant tumor compared to the all other groups, which correlated with antitumor cellular immunity to attack tumors. Meanwhile, treatment with the combined PEG-rGO-FA-IDOi-mediated PTT plus PD-L1 antibody exhibited the higher serum concentration of IFN-γ than did all other groups (Figure 6G). Taken together, the combination of PTT, IDO inhibition and anti-PD-L1 effectively hampered the immune suppression and promoted antitumor immune responses, which are primarily responsible for the efficient inhibition of tumor growth.


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Figure 6. PEG-rGO-FA-IDOi-mediated PTT plus anti-PD-L1 combination therapy inhibits

tumor

growth

and

enhances

TILs.

(A)

Scheme

illustration

of

PEG-rGO-FA-IDOi-mediated PTT plus anti-PD-L1 combination therapy. (B) Individual tumor growth for primary tumor (1#) and distant tumor (2#). Percentages of (C) CD45+ leukocytes, (D) CD3+ CD4+ T cells, (E) CD3+ CD8+ T cells, (F) CD3+ CD4+ Foxp+ effector T cells (CD4+ Teff): CD3+ CD4+ Foxp- regulator T cells (Treg) ratios and CD3+ CD8+ T cells: Treg ratios in the distant tumor. (G) The production of INF- in serum was determined by ELISA.The data are presented as the mean ± SD



(n = 5) (*p < 0.05; **p < 0.01; ***p < 0.001).

4. Conclusion Our studies suggested that the three combinations of PEG-rGO-FA-based PTT treatment, PD-L1 blockade and IDO inhibition are attractive for synergistically enhancing antitumor immunity and suppressing the growth of the tumor. Generally speaking, after PEG-rGO-FA-based PTT treatment, the released tumor-associated antigens could efficiently promote the activation of antigen presenting cells and trigger antitumor immune responses. Meanwhile, the triggered antitumor immune response can be synergistically promoted by IDO inhibition and PD-L1 blockade; the responses included the enhancement of tumor-infiltrating lymphocytes (TILs) including CD45+ leukocytes, CD4+ T cells, CD8+ T cells and NK cells, the inhibition of the immune suppression activity of Tregs and the production of INF-. We also demonstrate that the three combinations of immunotherapies could effectively inhibit the growth of both irradiated tumors and tumors in distant sites without PTT treatment. This work can be thought as an important proof-of-concept to target multiple antitumor immune pathways to induce synergistic antitumor immunity. The present three combinations of PTT, IDO inhibition and PD-L1 blockade therapy strategy offer clinically valuable therapeutic advantages over monotherapy in cancer treatment, which may, in the future, have a good chance for clinical translation.

Supporting Information The supporting information is available on line for materials and reagents, corresponding experimental processes and supplementary figures (quantification of IDOi in organs, size distribution of rGO-based nanosheets, HPLC spectrum of FA and epacadostat, H&E stained images, representative FACS plots of CD3+CD8+ T cells, representative FACS histograms of CD4+ Teff:Treg ratios and CD8+ T:Treg ratios, and relative weight during the treatment course).


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Acknowledgements We acknowledge financial support from National Natural Science Foundation of China (No. 81673027, 31670948 and 31600743), Tianjin Natural Science Fund for Distinguished Young Scholars (17JCJQJC46400), CAMS Innovation Fund for Medical Sciences (CAMS-I2M-3-026) and Specific Program for High-Tech Leader&Team of Tianjin Government.

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Abstract Graphic


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Nanoscale Reduced Graphene Oxide-Mediated Photothermal

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