Surface-Functionalized Modified Copper Sulfide Nanoparticles

Subscriber access provided by Drexel University Libraries

Biological and Medical Applications of Materials and Interfaces

Surface Functionalized Modified Copper Sulfide Nanoparticles Enhance Checkpoint Blockade Tumor Immunotherapy by Photothermal Therapy and Antigen-Capturing Ruoping Wang, Zhesheng He, Pengju Cai, Yao Zhao, Liang Gao, Wenzhi Yang, Yuliang Zhao, Xueyun Gao, and Fuping Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 26 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27 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

ACS Applied Materials & Interfaces

Surface Functionalized Modified Copper Sulfide Nanoparticles Enhance Checkpoint Blockade Tumor Immunotherapy by Photothermal Therapy and Antigen-Capturing Ruoping Wang,†, ‡ Zhesheng He,‡ Pengju Cai,‡ Yao Zhao, Yang, *, ‡ Yuliang Zhao, † Xueyun Gao, *, ⊥ Fuping Gao*, † †

‡

Liang Gao, ⊥ Wenzhi

CAS Key Laboratory for the Biological Effects of Nanomaterials and Nanosafety,

Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China ‡

School of Pharmacy, Hebei University, Baoding 071002, China

⊥

Department of Chemistry and Chemical Engineering, Beijing University of

Technology, Beijing 100124, China

A

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

Page 2 of 27

ABSTRACT: Nano-material-based tumor photothermal therapy (PTT) has attracted increasing attention and been a promising method for cancer treatment due to its low level of adverse effects and noninvasiveness. However, thermotherapy alone still cannot control tumor metastasis and recurrence. Here, we developed surface functionalized modified copper sulfide nanoparticles (CuS NPs). CuS NPs can not only be used as photothermal mediator for tumor hyperthermia, but also can adsorb tumor antigens released during hyperthermia as an antigen-capturing agent to induce antitumor immune response. We selected maleimide polyethylene glycol modified CuS NPs (CuS NPs-PEG-Mal) with stronger antigen adsorption capacity, in combination with immune checkpoint blocker (anti-PD-L1) to evaluate the effect of hyperthermia improving immunotherapy in 4T1 breast cancer tumor model. The results showed hyperthermia based on CuS NPs-PEG-Mal distinctly increased the levels of inflammatory cytokines in serum, leading to tumor immunogenic microenvironment. In cooperation with anti-PD-L1, photothermal therapy mediated by CuS NPs-PEG-Mal enhanced the number of tumor-infiltrating CD8+ T cells, inhibited the growth in primary and distant tumors sites of the 4T1 tumor model. The therapeutic strategies provide a simple and effective treatment option for metastatic and recurrent tumors. KEEYWORDS:

CuS

antigen-capturing agent,

NPs,

photothermal

anti-PD-L1,

therapy,

immune response

B


checkpoint-blockade,

Page 3 of 27 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


INTRODUCTION Recently, nano-material-based tumor photothermal therapy (PTT), as a new treatment for cancer causes high attention for its minimally invasive high-efficiency, low adverse reactions and inhibition of tumor metastasis. They use a special photothermal conversion material to absorb near-infrared light, then transform the absorbed near infrared light into heat,1-4

raising the temperature in tumor site, inducing cell

apoptosis or directly killing tumor cells.5,6 However, thermotherapy alone still cannot control tumor metastasis and recurrence. Tumor immunotherapy by stimulating the host immune system has showed capable of controlling metastatic tumor growth by generating an antitumor immune response.7,8 Photothermal therapy stimulates the host's immune system by causing tumor cell apoptosis and necrosis, which in turn releases tumor antigens into the tumor microenvironment, promotes the presentation of tumor-derived antigens to T cells.9-12 However, tumor cells can escape the recognition and response of the immune system by activating specific inhibitory signaling pathways of the tumor microenvironment. Among them, the signaling pathways of programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway arouse the largest attention of researchers.13-15 The PD-1/PD-L1 pathway is up-regulated in many tumor models, which causes tumor specific cytotoxic lymphocyte apoptosis, induces the tumor cells anti-apoptotic signal, inhibits effector T cells function to suppress the immune activation.16,17 The immune checkpoint inhibition of PD-1/PD-L1 pathway using specific antibody generated significant anti-tumor activity among the various tumor models.18-20 However, in clinical trials, except melanoma, the long-lasting effect of immunological checkpoint blockade therapy is still low, and individual differences among patients are relatively significant. Facing the current challenges, we hypothesize that combined nanomaterial-mediated photothermal tumor therapy with immunological checkpoint blockade treatment can further improve the therapeutic effect of tumors and reduce recurrence and metastasis. During photothermal tumor ablation, antigen presenting cells such as dendritic cells (DCs) can capture the abundant tumor-associated antigens released, after processing, present to lymphocytes to induce proliferation and C



activation of lymphocytes, which in turn produce corresponding anti-tumor immunity effect.21-24 Andrew Z. Wang et al. reported poly(lactic-co-glycolic acid) (PLGA) nanoparticles with different surface chemistry could be employed to improve the antitumor effect of immunotherapy and to induce the distal effect by capturing tumor-derived protein antigens (TDPAs) released in the tumor environment after receiving radiotherapy, then transporting them to antigen-presenting cells (APCs) and thereby improve tumor immunotherapy.25-27 However, in the research, PLGA nanoparticles only had a single function, namely antigen capture, which must be combined with additional radiotherapy to improve the effect of immunotherapy. In our study, we prepared copper sulfide nanoparticles (CuS NPs) with different surface chemistry. Here surface functionalized modified CuS NPs play two functions: (1) as a photothermal coupling agents for photothermic tumor ablation; and (2) as an antigen-capturing agent adsorbing antigens released during hyperthermia and transporting to dendritic cells (DCs). Combined with immunocheckpoint inhibitor, anti-PD-L1, we assessed the therapeutic effect of surface functionalized modified CuS nanoparticles-mediated hyperthermia to improve tumor immunotherapy ( Figure 1 ).

Figure 1. Schematic diagram of anti-tumor immune responses induced by CuS D


Page 4 of 27

Page 5 of 27 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


NPs-PEG-Mal based photothermal therapy plus immunological checkpoint inhibitor. RESULT AND DISCUSSION Characterization of Surface Functionalized Modified CuS Nanoparticles. CuS nanoparticles (CuS NPs) were modified with polyethylene glycol (PEG) with different functional end groups, maleimide polyethylene glycol (PEG-Mal), amine polyethylene

glycol

(PEG-NH2)

and

methoxypolyethylene

glycol

(mPEG),

respectively. The transmission electron microscope (TEM) images indicated the synthesized three PEG-modified CuS nanoparticles were in excellent dispersion, and the hydrodynamic sizes of the three prepared CuS nanoparticles were approximately 12 nm ( Figure 2a-c). As shown in Figure 2d-f,

the average hydrodynamic size of

CuS NPs-mPEG, CuS NPs-PEG-NH2, and CuS NPs-PEG-Mal were 10 ± 2 nm, 14 ± 2 nm, and 13 ± 2 nm, the zeta potential of CuS NPs-mPEG, CuS NPs-PEG-NH2, and CuS NPs-PEG-Mal were 25.47 mv, 34.37 mv, and 44.08 mv respectively (Table S1). As depicted in Figure 1g, the UV-vis-NIR absorption peaks at 980 nm ranging from 400 nm-1300 nm, illustrated the successful formation of CuS NPs.28-30 The three surface functionalized modified CuS NPs were very stable. The appearance and particle size hardly changed significantly after incubation in deionized water, pH 7.4 PBS and high glucose Dulbecco's Modified Eagle's Medium (DMEM) medium for 24 hours at 25 ℃ (Figure S1). To evaluate the photothermal conversion efficiency medicated by CuS NPs-PEG-Mal, CuS NPs-PEG-NH2 and CuS NPs-mPEG, the 808 nm near-infrared light was employed. After being irradiated at different output time, the temperature increased significantly, reaching about 80 ℃ in 8 min for three CuS NPs dispersed in deionized water, while there were no significant temperature changes for the pure water (Figure 2h and Figure S2), which demonstrated that temperature changes were mainly based on the absorption of CuS NPs rather than pure water. Apparent time-dependent and

concentration-dependent photothermal

effect were proved (Figure S3). These results demonstrate that the increase in temperature of PEG-coated CuS NPs after irradiation may can be introduced to photothermal therapy in vitro and in vivo. E



Figure 2. Characterization of CuS NPs and its photothermal effect. (a-c) Transmission electron microscopy (TEM) images of CuS NPs-mPEG, CuS NPs-PEG-NH2 and CuS NPs-PEG-Mal. (d-f) Hydrodynamic diameter distribution of CuS NPs-mPEG, CuS NPs-PEG-NH2, CuS NPs-PEG-Mal determined by dynamic light scattering (DLS). (g) UV-Vis-NIR absorption spectra of three types of PEG-coated CuS NPs. (h) The temperature changes of three types of PEG-coated CuS NPs, as exposed under NIR irradiation (808 nm, 2 W/cm2) lasting for different times. Cytotoxicity and Tumor Antigens Adsorption of PEG -coated CuS NPs. Prior to applications in vivo, standard CCK-8 was used to assess the biocompatibility of the PEG-coated CuS NPs in vitro. As shown in Figure 3a, no significant tumor cytotoxicity efficacy was observed in 4T1 cells after being incubated for 48 h, even at 240 μM of CuS NPs concentration. The results indicated that PEG-coated CuS NPs had good biocompatibility and low toxicity. However, compared with PEG-coated F


Page 6 of 27

Page 7 of 27 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


CuS NPs alone with laser, the cell survival rate of 4T1 cells exhibited a concentration-dependent inhibition under 808 nm laser at a power density of 2 W/cm2 for 5 min (Figure 3b). All results proved that PEG-coated CuS NPs had a outstanding efficacy in directly killing tumor cells under 808 nm laser irradiation. Here, we formulated CuS NPs modified with PEG with different functional end groups

on

the

surface.

The

tumor

protein

antigens

capture

depended

on the surface coating of CuS NPs. 4T1 cells were incubated with 240 μM of PEG-coated CuS NPs under irradiation in vitro (808 nm, 2 W/cm2, 5 min,). Zeta potential and particle size of the CuS NPs were changed after incubation (Table S2), which revealed the successful tumor protein antigens capture by the CuS NPs. CuS NPs-mPEG captured little protein at 0.20 mg/mL and tumor protein antigens captured by CuS NPs–PEG-NH2 were higher than mPEG-CuS NPs at 0.55 mg/mL. Obviously, the most amount of tumor protein antigens reached to 0.69 mg/mL was detected in the cells received with CuS NPs-PEG-Mal (Figure 3c). Therefore, CuS NPs-PEG-Mal were selected as the optimal material for the further study in vivo.

Figure 3. Cytotoxicity and effect of antigen uptake by the PEG-coated CuS NPs. (a) Cytotoxicity of 4T1 cells with various concentrations of PEG-coated CuS NPs without NIR irradiation. (b)

Cytotoxicity of 4T1 cells with different concentrations

of PEG-coated CuS NPs with NIR irradiation( 2 w/cm2 for 5 min ). (c) Amount of protein captured by three types of PEG-coated CuS NPs, as assessed by BCA. Data are presented as the mean ± s.d.(n=3); ***P ＜ 0.001, in comparation with the group of CuS NPs-mPEG; #P ＜0.05, compared with the group of CuS NPs-PEG-NH2. Tumor Ablation by PTT and Abscopal Effect in vivo. According to the previous experiment results in vitro, BALB/c mice-bearing 4T1 tumors were used to evaluate G



the photothermal effect in vivo. Mice were injected intratumorally 50 μL of CuS NPs-PEG-Mal with a concentration of 15 mM. Tumor sites were exposed under 808 nm laser with a power density of 0.45 W/cm2 for 5 min. We monitored the changes of temperature in tumor sites via infrared thermal imager. There were no significant temperature changes in PBS with laser group (the control group). While the tumor temperature of mice received CuS NPs-PEG-Mal with laser was higher than PBS group, increasing from 32 ℃ to 55 ℃ (Figure S4), which was high enough to effectively ablate tumor. Nanoparticles can enhance tumor immunotherapy by antigen capture and delivery to antigen presenting cells.31,32 Determining whether CuS NPs-PEG-Mal-mediated tumor photothermia combined with immunological checkpoint blocker, anti PD-L1, can further enhance tumor immunotherapy, we conducted a series of abscopal effect experiments in vivo. Mice were injected different numbers of 4T1 cells into the left and right flank regions at the same day. The right site was considered as primary tumor and the left site was as the distant tumor. We divided all mice into 5 groups at random as the primary tumor volume reached about 50 mm3. The mice received different treatments: PBS with laser, CuS NPs-PEG-Mal, CuS NPs-PEG-Mal with laser, anti-PD-L1 alone, and CuS NPs-PEG-Mal with laser plus anti-PD-L1. Mice received laser with a power density of 0.45 W/cm2 for 5 min under 808 nm. The primary tumors received treatment for photothermal therapy, and the distant tumors accepted no direct treatment. Anti-PD-L1 was injected intraperitoneally into mice at a dose of 50 μg per mouse on days 1, 4, 7 (i.p), while CuS NPs-PEG-Mal were injected intratumorally at a concentration of 15 mM on days 1, 3, 5, 7. Subsequently, the primary tumor sites were exposed under 808 nm laser and the individual growth of tumor was carefully monitored (Figure 4a,b). As expected, PTT based on CuS NPs-PEG-Mal could delay the growth of primary tumors. Tumor size in group of anti-PD-L1 alone was similar to CuS NPs-PEG-Mal with irradiation, which showed a moderate anticancer efficacy (Figure 4c). Significantly, the combination treatment using CuS NPs-PEG-Mal with irradiation plus anti-PD-L1 slowed down the tumor growth in primary and distant tumor, in marked contrast to mice received PBS and H


Page 8 of 27

Page 9 of 27 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


CuS NPs alone ( Figure 4c,d). At the end of the observation period, the primary tumor size in the combined treatment group was approximately 300 mm3, which was 3 times smaller than that of PBS and CuS NPs treatment groups. Anti-PD-L1 alone as a checkpoint inhibitor, caused a bit antitumor immunity effect, the average size of distant tumors was decreased (Figure 4d), which was smaller compared to CuS NPs-PEG-Mal without laser. Consequently, it was clearly that immune responses was triggered by photothermal ablation of CuS NPs-PEG-Mal in primary tumors. When combined with anti-PD-L1 therapy, this treatment program could effectively inhibit primary and metastatic tumor growth. All mice did not experience significant weight loss during treatment (Figure 4e).

I



Figure 4. CuS NPs–PEG-Mal promoted enhancement of immunotherapy and the abscopal effect in vivo. (a) Primary tumor growth and (b) Distant tumor growth curves of individual mice treated with PBS with laser, CuS NPs-PEG-Mal, Anti-PD-L1, CuS NPs-PEG-Mal with laser and CuS NPs-PEG-Mal with laser plus Anti-PD-L1 (n = 5). (c) Average primary tumor growth curves of mice (n = 10). (d) Average distant tumor growth curves of mice (n = 10). (e) Changes of body weight during 24 days treatment. J


Page 10 of 27

Page 11 of 27 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


(f-g) Representative photos of both primary (f) and distant tumor (g) of mice in different groups after treatment for 16 days (1. PBS + Laser; 2. CuS NPs-PEG-Mal; 3. Anti-PD-L1; 4. CuS NPs-PEG-Mal + Laser; 5. CuS NPs-PEG-Mal + Laser + Anti-PD-L1). Data are presented as the mean ± s.d. (n = 5); ***P ＜ 0.001; *P ＜ 0.05, compared with the group of PBS + Laser or CuS NPs-PEG-Mal; ###P ＜ 0.001, compared with the group of CuS NPs-PEG-Mal + Laser or Anti-PD-L1. Cytokine Analysis in vivo. The mice blood were collected after 4 times treatment to detect the changes in cytokines secretion. As key markers for immune responses, the amount of tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), interleukin-6 (IL-6) and interferon-γ (IFN-γ) were detected using enzyme-linked immune sorbent assay (ELISA). As revealed in Figure 5, there were no significant changes of TNF-α, IL-2, IL-6 and IFN-γ were tested in mice treated with PBS with laser and CuS NPs-PEG-Mal. However, a significant increase of TNF-α, IL-6, IL-2 and IFN-γ levels were proved in mice treated with CuS NPs-PEG-Mal with laser plus anti-PD-L1. More importantly, cytokines secretions of TNF-α triggered by CuS NPs-PEG-Mal with laser plus anti-PD-L1 was obviously higher compared to CuS NPs-PEG-Mal with laser and anti-PD-L1 alone, showing almost twice as much as that of the other two groups. Similar results were also observed in the other three cytokines secretion. All the studies suggested that CuS NPs-based photothermal therapy could successfully activate higher level of immune response and cause inflammation in vivo.

K



Figure 5. ELISA analysis of cytokine levels in sera isolated from mice after different treatments (PBS + Laser, CuS NPs-PEG-Mal, Anti-PD-L1 CuS NPs-PEG-Mal + Laser, and CuS NPs-PEG-Mal + Laser + Anti-PD-L1). (a) Analysis of TNF-α in the serum, (b) Analysis of IFN-γ in the serum, (c) Analysis of IL-2 in the serum, (d) Analysis of IL-6 in the serum. Data are presented as the mean ± s.d. (n = 3); * P ＜ 0.05; ** P ＜ 0.01; *** P ＜ 0.001, compared with the group of PBS + Laser or CuS–PEG-Mal;

##

P ＜

0.01;

###

P ＜

0.001 , compared with the group of

CuS-PEG-Mal + Laser or Anti-PD-L1. Potential Antitumor Immunity Treatment Mechanism of CuS NPs-PEG-Mal Based PTT. To verify the antitumor immunity mechanism induced by CuS NPs-PEG-Mal based PTT, we harvested the distant tumors and tumor-draining lymph nodes after BALB/c mice-bearing 4T1 tumors received 4 times treatments by PTT. As depicted in Figure 6a, a high level percentage of infiltration CD3+CD45+ T cells in group of combination treatment was presented by flow cytometry analysis, which was L


Page 12 of 27

Page 13 of 27 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


up to 21.9%, while in CuS NPs-PEG-Mal with laser and anti-PD-L1 alone treated groups, the percentage of CD3+CD45+ T cells were only 10% and 10.8% respectively. In the group of CuS NPs-PEG-Mal and PBS treatment, the percentage of CD3+CD45+ T cells were only 3.75% and 1.94 % respectively (Figure 6a and Figure S5a). Similar results were provoked in infiltration CD8+ T cells (Figure 6b and Figure S5b), it showed notably elevated infiltration of CD8+ T cells in the tumors treated with CuS NPs-PEG-Mal with laser plus anti-PD-L1, which was up to 45%, compared with the other four treatment groups. Furthermore, we harvested distant tumors to analyze the expression of PD-L1 and tumor-infiltrating CD8+ T cells by immunohistochemistry. As

shown

in

Figure

7a,

consistent

with

flow

cytometry

analysis,

immunohistochemistry staining revealed an accumulation of CD8+ T cells in distant tumors in the combination treatment group. While the other control group (PBS with laser and CuS NPs-PEG-Mal) showed no significant tumor-infiltrating CD8+ T cells (Figure 7a and Figure S7a). It was reported that 4T1 cells, as tumor model of triple-negative breast cancer, express low amounts of PD-L1.33 We found the up-regulation of PD-L1 expression in tumor tissues treated with CuS NPs with laser plus anti-PD-L1 compared with tumor tissues in PBS with laser or CuS NPs-PEG-Mal alone treatment group (Figure 7b and Figure S 7b). It is known IFN-γ can induce PD-L1 expression in tumor cells.34,35 The results verified that CuS NPs-PEG-Mal based tumor photothermal therapy could cause an inflamed and immunogenic tumor microenvironment. Therefore, CuS NPs-PEG-Mal-mediated photothermotherapy combined with immunological checkpoint blockers could produce greater anticancer efficacy compared with CuS NPs photohyperthermia or anti-PD-L1 alone. Histopathological examination revealed that tumor tissues of the mice treated with CuS NPs with laser plus anti-PD-L1 showed noticeable destruction, a large number of cells with apoptosis and necrosis (Figure 7c). TUNEL detection further confirmed that CuS NPs-PEG-Mal-mediated photothermotherapy combined with anti-PD-L1 triggered the highest amount of tumor cell apoptosis (Figure 7d and Figure S8). We thus hypothesized necrotic or apoptotic tumor cells induced by CuS NPs mediated M



photothermotherapy could released tumor antigens into tumor microenvironment, CuS NPs-PEG-Mal as a good antigen capture agent can transport tumor antigens into antigen-presenting cells, stimulate the proliferation of CD8+ T cells and infiltrate into tumor tissues, and generate anti-tumor immune effect. We therefore examined the Cu content in the tumor-draining lymph nodes by inductively coupled plasma mass spectrometry (ICP-MS) analysis. Interestingly, the Cu content of the tumor-draining lymph nodes in the mice treated with CuS NPs-PEG-Mal with laser (~69.58 ppm) was almost 8 times more than that of mice treated with CuS NPs-PEG-Mal alone (~8.55 ppm) (Figure S6 ). Collectively, these results suggest that CuS NPs-PEG-Mal serve as a dual function nano therapeutic formulation to enhance immune checkpoint blockade effect to inhibit tumor recurrence.

Figure 6.

Flow cytometry analysis of CD3+CD45+ T cells (a) and CD8+ T cells (b)

in distant tumors collected from various groups after treatment.

N


Page 14 of 27

Page 15 of 27 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


Figure 7. Tumor tissues analysis of the mice treated with different formulations. (a) Immunohistochemical analysis for tumor-infiltrating CD8+ T cells in distant tumor tissues obtained from various groups after treatment. (b) Immunohistochemical analysis for expression of PD-L1 in distant tumors after the indicated treatment. (c) H&E-stained images of distant tumor tissues obtained from five groups after treatment (PBS + Laser, CuS NPs, CuS NPs + Laser, Anti-PD-L1 and CuS NPs + Laser + Anti-PD-L1. (d) TUNEL staining of tumor tissues in various treatment groups. The Effect of Tumor Vaccination in vivo. To further confirmed that CuS NPs-PEG-Mal were not only used as a photothermal mediator in the study, but also capture antigens to improve immunotherapy response and abscopal effect, we detected the effects of direct management of CuS NPs-PEG-Mal adsorbed with protein antigens released by hyperthermia ex vivo to tumor-bearing mice. 4T1 cells were injected subcutaneously into the left flank regions of BALB/c mice on day 0, then we divided all mice into three groups, subcutaneously injected with PBS, CuS NPs-PEG-Mal and CuS NPs-PEG-Mal adsorbing protein antigens into the right flank regions of the mice respectively. All tumor-bearing mice received anti-PD-L1 immunotherapy treatment. As shown in Figure 8a, b, tumors in mice that received CuS NPs-PEG-Mal containing adsorption protein antigens clearly showed O



significantly slower growth. However, no appreciable tumor growth inhibition effect were showed in the other two groups, which indicated that CuS-NPs-PEG-Mal adsorbing protein antigens can stimulate the immune system to inhibits tumor growth.

Figure 8. Analysis of effect of CuS NPs-PEG-Mal adsorbing protein antigens as tumor vaccination. (a) Representative photos of the 4T1 tumor-bearing mice after treatment for 12 days. 1: PBS control group, 2: CuS NPs-PEG-Mal group, 3: CuS NPs-PEG-Mal adsorbing protein antigens group. (b) Photographs of the collected tumor tissues for each treatment group. 1: PBS control group, 2: CuS NPs-PEG-Mal group, 3: CuS NPs-PEG-Mal adsorbing protein antigens group. (c) Average tumor growth curves of mice. (d) Body weight changes of mice bearing 4T1 tumors over the course of treatments. Data are presented as the mean ± s.d. (n = 5); *** P ＜ 0.001, compared with the group of PBS + Anti-PD-L1 or CuS-PEG-Mal +Anti-PD-L1. CONCLUSION In summary, we have constructed surface functionalized modified CuS NPs with a facile method. The synthesized CuS NPs not only exhibited good photothermal conversion efficiency, but also had a good tumor protein antigen capture ability by the P


Page 16 of 27

Page 17 of 27 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


surface modified PEG terminal group. CuS NPs-PEG-Mal-based tumor photothermal therapy induced an immune microenvironment in the triple-negative breast cancer 4T1 tumor model, which prominently increased PD-L1 checkpoint blockade therapy by producing a systematic anti-tumor immunity. Photothermal therapy mediated by NPs-PEG-Mal in combination with anti-PD-L1 not only inhibited the growth of primary tumors, but also, more importantly, the growth of distant tumors in 4T1 tumor model. The therapeutic strategies currently designed in this study hold great promise for the treatment of tumors with extensive metastasis, while primary tumors can be achieved by photothermal therapy. MATERIAL AND METHODS Materials. We purchased sodium sulfide nonahydrate (Na2S·9H2O), Copper chloride dihydrate (CuCl2·2H2O) and sodium citrate from Shanghai Macklin Biotechnology.

Maleimide-polyethylene

glycol-thiol

(Mal-PEG5000-SH),

methoxy-polyethylene glycol-thiol (mPEG5000-SH), amine-polyethylene glycol-thiol (NH2-PEG5000-SH) were obtained from Seebio Biotechnology. Molecular weight of all polyethylene glycol were 5000 Da. Anti-PD-L1 (Biolegend 124328) was obtained from Biolegend. The phosphate buffer solution (PBS) and cell culture media RPMI 1640 were acquired from HyClone, USA. Trypsin-EDTA and fetal bovine serum were acquired from Gibco, USA. Ultrapure water (18 MΩ) was employed during the experiments. Synthesis of PEG-coated CuS NPs. Details synthesis of copper sulfide nanoparticles was as follows: 17.05 mg of CuCl2·2H2O and 20 mg of sodium citrate were mixed into a erlenmeyer, containing 100 mL of ultrapure water. Sodium sulphide (24 mg) was added into the reaction system slowly under gentle（480 rpm） stirring at 25 ℃ for 5 min. The color of solution would turn to dark-brown from pale-blue. The reaction mixture was then heated to 90 oC under stirring for 15 min. When the color of solution turned to dark-green, the formation of CuS NPs (Cit-CuS NPs) were completed. To coat the PEG, Mal-PEG5000-SH, NH2-PEG5000-SH and mPEG5000-SH (0.1 mM) were added into the above mixture under stirring overnight at 25oC. For experimental applications, the products were centrifuged at 8500 rpm for 8 Q



min, using ultrafiltration centrifuge tube (5 kD) and then washed the solution with deionized water. Above step was repeated for three times. Characterization of PEG-coated CuS NPs. Transmission electron microscopy (TEM) was used for the size and shape of CuS nanoparticles. The particle size distribution and zeta potential of nanoparticles were measured by dynamic light scattering (DLS). The absorption spectra of PEG-coated CuS nanoparitcle was identified by UV-Vis-NIR spectrophotometer. Stability of PEG-coated CuS NPs. To investigate the stabilities of PEG-coated CuS NPs in various media, PEG-coated CuS NPs were incubated in water, high glucose DMEM, and pH7.4 PBS at 25oC for 24 h. The appearance of precipitation was observed by visual inspection and the particle size and distribution of PEG-coated CuS Nanoparitcle were recorded by DLS after incubation for 24 h. Analysis of PEG-coated CuS NPs Concentration by ICP-MS. The concentration of CuS NPs were measured by an ICP-MS analysis system (Thermo Elemental X7, USA). 4 mL nitric acid was added into the 100 μL CuS NPs for digestion. After 24 h, the mixture was heated to 60oC and evaporated its nitric acid solution, till the final volume was about 1 mL. Then the mixture was diluted by 2% HNO3 36-37. Different concentrations of Cu standard (0.5, 1, 5, 10, 50 and 100 ng/mL) was measured to confirm the Cu calibration curve. Then, samples were measured by inductively coupled plasmamass spectrometry (ICP-MS, Thermo-X7). Photothermal Effect of CuS NPs in Aqueous Solution. The photothermal conversion efficiency of PEG-coated CuS NPs (1 mL) was detected by 808 nm laser irradiation. Various concentrations of CuS NPs were exposed under different output power densities and laser times. Temperature changes were displayed using the thermal imaging photograph by infrared thermal imager. Antigen Adsorption Analysis of CuS NPs. 4T1 cells (purchased from Chinese Academy of Medical Sciences Cancer Hospital) were seeded into 96-well plates, cultured in RPMI-1640 medium, which was added with 10 % fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 ℃ under 5% CO2 for 24 h. The fresh medium containing three types of CuS NPs at a concentration of 240 μM was used to R


Page 18 of 27

Page 19 of 27 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


replace the supernatant of the cells. Then 4T1 cells were exposed under laser irradiation (808 nm, 2 W/cm2, 5 min). Changes in size and zeta potential induced by protein antigen adsorption of CuS NPs were measured by DLS. The amount of proteins captured by CuS NPs were detected by bicinchoninic acid analysis (BCA). Cytotoxicity Analysis of CuS-PEG NPs. 4T1 cells with the density of 5000 cells/wells were seeded into two 96-well plates respectively and cultured for another 24 h at 37 °C under 5% CO2 , after discarding the supernatant, 4T1 cells were washed three times with PBS and then fresh medium containing different concentrations of CuS NPs (ranging from 0, 6, 12, 24, 36, 60, 120, 240 μM) was added. After further incubation for 24 h, 4T1 cells of each well were treated with a 808 nm laser one by one with a power density of 2 W/cm2 for 3min, then the 4T1 cells were further cultured for another 12 h. 4T1 cells without laser irradiation were used as controls. The viability of all cells was tested by CCK-8 assay. In Vivo Photothermal Effect and Abscopal Effect. For photothermal effect studies, 200 µL of pure RMPI-1640 medium containing 1 × 106 cells were injected subcutaneously into the right flank of female BALB/c mice. When the tumor volume reached about 50 cm3, CuS NPs-PEG-Mal (50 µL, 15 mM) was injected intratumorally. Then tumor sites were treated with laser irradiation (808 nm, 0.45 W/cm2, 5 min). The changes of tumor temperature were monitored by using infrared thermal imager. For abscopal effect studies, 200 µL of pure RMPI-1640 medium containing 1 × 106 cells were injected subcutaneously into the right flank of female BALB/c mice as the primary tumor and 100 µL of 5 × 105 cells were injected onto the left flank, considering as the distant tumor. When the primary tumor volume reached about 50 mm3, dividing mice randomly into following five groups (n = 10): PBS with laser, CuS NPs-PEG-Mal alone, CuS NPs-PEG-Mal with laser, Anti-PD-L1 alone and CuS NPs-PEG-Ma with laser plus Anti-PD-L1. CuS NPs were injected intratumorally on the primary tumor on days 1, 3, 5, 7 at a concentration of 15 mM, then mice were treated with a 808 nm laser at a power density of 0.45 W/cm2 for 5 min. Anti-PD-L1 was injected (i.p) immediately at a dose of 50 μg per mouse on days 1, 4, 7. As the S



volume of tumor-bearing mice reached about 2 cm3, mice would be euthanized. Caliper and electronic balance were used for monitoring the tumor volume and body weight of mice respectively. The calculation of tumor volume was followed the formula: length×width2/ 2. CuS NPs-PEG-Mal Adsorbing Protein Antigens as Tumor Vaccine. 1 × 106 cells in 200 µL of pure RMPI-1640 medium were injected subcutaneously onto the left flank of female BALB/c mice on day 0. Mice were divided into three group (n = 5) randomly: PBS, CuS NPs-PEG-Mal and CuS-NPs-PEG-Mal adsorbing protein antigens. For treatment, 4T1 cells containing 240 μM concentrations of CuS NPs-PEG-Mal were exposed under laser at a power density (808 nm, 2 W/cm2, 5 min). Supernatant containing CuS NPs was collected and concentrated by ultrafiltration with a cutoff of 100,000. PBS, CuS NPs-PEG-Mal and CuS-NPs-PEG-Mal adsorbing protein antigens were injected the right flank of mice on days 3, 5, 7. Anti-PD-L1 was injected (i.p.) immediately (dose: 50 μg per mouse) on days 3, 5, 7. We monitored the tumor growth and body weight of mice every two days. Flow Cytometry. To conducted the relative abundance of tumor-infiltrating T-cells in the 4T1 tumor, the mice were killed by dislocation on days 24 post-tumor inoculation and the distant tumors were dissected. After removing necrosis and connective tissue, the tumor were cut into 1-2 mm3 of small pieces, repeatedly ground and washed with PBS to form a single-cell suspensions. The cells were stained with anti-mouse CD8a-APC (Biolegend 100711), anti-mouse CD3-PE (Biolegend 100201) and anti-mouse CD45-FITC (Biolegend 147709). The samples was disclosed by flow cytometry (BD Accuri C6) for the number of infiltration CD3+CD45+ T cells and CD8+ T cells. Histopathology and Immunohistochemistry Analysis. For histopathological analysis, The distant tumors fixed in 10% formalin were embedded in paraffin, then microtomically sectioned into slices (5 μm) and stained with haematoxylin and eosin. For the immunohistochemistry analysis, the tumors were processed following the steps of above histopathological analysis. The 5-μm thick paraffin-embedded tumor sections were deparaffinized using xylene and rehydrated using ethanols according a T


Page 20 of 27

Page 21 of 27 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


graded method, then PBS was employed for rinsing. 3% H2O2 was used for the inactivation of endogenous peroxide. Using sodium citrate buffer for antigen repairing, tumor sections were heated by microwave oven for 20 min. 5% goat serum was used to block the sections, then the sections were incubated overnight with primary antibodies of CD8 (Bioss，bs-4790R) and PD-L1 (Bioss，bs-10159R) at 4 ℃. After rinsing extensively with PBS, the sections were further incubated with HRP-conjugated secondary antibody (Beyotime, China) for 30 min at 37 ℃. Then the tumor sections were dyed using diaminobenzidine (DAB) solution(time = 3 min) at 25oC, hematoxylin was used for counterstaining. Imaging was obtained using an Olympus optical microscope (CX31). IPWIN32 software was employed for quantity of positive staining, which was considered as the mean number of positive pixels/ tissue section. TUNEL Assay. We used terminal DNA transferase-mediated dUTP nick end labeling (TUNEL) assay to detect apoptosis of distant tumor tissue, following manufacturer's instructions of a commercial apoptosis detection kit. Apoptosis of tumor tissue was observed by using an olympus optical microscope by a researcher blind to the experiment detail. Cytokine Analysis. After completion of treatment, serum was isolated from mice in the different treatment groups. The concentrations of cytokine including IFN-γ, TNF-α, IL-6 and IL-2 were analyzed following the manufacturer's instructions of ELISA kits (Proteintech, USA). Statistical Analysis. All results are represented as the mean ± standard deviation. Statistical analyses were performed using SPSS software (SPSS version 22.0, USA). Statistical differences between PBS with laser and other groups was determined by one-way analysis of variance (ANOVA). P < 0.05 was considered as having statistically significant. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXX. The Supporting Information includes partical size and zeta U



potential; Infrared thermal images of CuS NPs in vitro and in vivo ; Temperature change of three PEG-coated CuS NPs at different concentrations under laser irradiation; and statistical analysis of infiltration CD3+CD45+ T cells and CD8+ T cells; Content of Cu in the organ of tumor-draining lymph nodes; Statistical analysis of immunohistochemistry analysis; and TUNEL analysis. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID Fuping Gao: 0000-0002-0287-9763 Xueyun Gao: 0000-0002-2267 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (31670976, 81472851, 21425522 and 21727817) and the Natural Science Foundation of Hebei Province (H2017201052, H2018201045). REFERENCES (1) Wei, C.W.; Liao, C. K.; Tseng, H. C.; Lin, Y. P.; Chen, C. C.; Li, P. C. Photoacoustic Flow Measurements with Gold Nanoparticles. IEEE Trans Ultrason Ferroelectr Freq Control. 2006, 53, 1955-1959. (2) Liang, L.; Peng, S.; Yuan, Z.; Wei, C.; He, Y.; Zheng, J.; Gu, Y.; Chen, H. Biocompatible Tumor-Targeting Nanocomposites Based on CuS for Tumor Imaging and Photothermal Therapy. RSC Advances 2018, 8 , 6013-6026. (3) Li, L.; Rashidi, L. H.; Yao, M.; Ma, L.; Chen, L.; Zhang, J.; Zhang, Y.; Chen, W. CuS Nanoagents for Photodynamic and Photothermal Therapies: Phenomena and Possible Mechanisms. Photodiagnosis Photodyn. Ther. 2017, 19, 5-14. (4) Yang, W.; Guo, W.; Le, W.; Lv, G.; Zhang, F.; Shi, L.; Wang, X.; Wang, J.; V


Page 22 of 27

Page 23 of 27 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


Wang, S.; Chang, J.; Zhang, B. Albumin-Bioinspired Gd:CuS Nanotheranostic Agent for In Vivo Photoacoustic/Magnetic Resonance Imaging-Guided Tumor-Targeted Photothermal Therapy. ACS Nano 2016, 10, 10245-10257. (5) Ali, M. R.; Ibrahim, I. M.; Ali, H. R.; Selim, S. A.; El-Sayed, M. A. Treatment of Natural

Mammary

Gland

Tumors

in

Canines

and

Felines

Using

Gold

Nanorods-Assisted Plasmonic Photothermal Therapy to Induce Tumor Apoptosis. Int. J. Nanomedicine 2016, 11, 4849-4863. (6) Yan, C.; Tian, Q.; Yang, S. Recent Advances in the Rational Design of Copper Chalcogenide to Enhance the Photothermal Conversion Efficiency for the Photothermal Ablation of Cancer cells. RSC Advances 2017, 7 , 37887-37897. (7) Emens, L. A.; Middleton, G. The Interplay of Immunotherapy and Chemotherapy: Harnessing Potential Synergies. Cancer Immunol. Res. 2015, 3, 436-443. (8) Wang, M.; Yin, B.; Wang, H. Y.; Wang, R. F. Current Advances in T-Cell-Based Cancer Immunotherapy. Immunotherapy 2014, 6, 1265–1278. (9) Sherlock, S. P.; Tabakman, S. M.; Xie, L.; Dai, H. Photothermally Enhanced Drug Delivery by Ultrasmall Multifunctional FeCo/Graphitic Shell Nanocrystals. ACS Nano 2011, 5, 1505–1512 . (10) Mroz, P.; Hashmi, J. T.; Huang, Y. Y.; Lange, N.; Hamblin, M. R. Stimulation of Anti-Tumor Immunity by Photodynamic Therapy. Expert. Rev. Clin. Immunol. 2011, 7, 75–91. (11) Garg, A. D.; Nowis, D.; Golab, J.; Agostinis, P. Photodynamic Therapy: Illuminating the Road from Cell Death Towards Anti-Tumour Immunity. Apoptosis 2010, 15, 1050-1071. (12) Krosll, G.; Korbelik, M.; Dougherty, G. J. Induction of Immune Cell Infiltration into Murine SCCVII Tumour by Photofrin-Based Photodynamic Therapy. Br. J. Cancer 1995, 71, 549-555. (13) Sharma, P.; Allison, J. P. The Future of Immune Checkpoint Therapy. Science 2015, 348, 56. (14) Pardoll, D. M. The Blockade of Immune Checkpoints in Cancer W



Page 24 of 27

Immunotherapy. Nat. Rev. Cancer 2012, 12 , 252-264. (15) Choo, Y. W.; Kang, M.; Kim, H. Y.; Han, J.; Kang, S.; Lee, J. R.; Jeong, G. J.; Kwon, S. P.; Song, S. Y.; Go, S.; Jung, M.; Hong, J.; Kim, B. S., M1 Macrophage-Derived Nanovesicles Potentiate the Anticancer Efficacy of Immune Checkpoint Inhibitors. ACS Nano 2018, 12, 8977-8993. (16) Brusa, D.; Serra, S.; Coscia, M.; Rossi, D.; D'Arena, G.; Laurenti, L.; Jaksic, O.; Fedele, G.; Inghirami, G.; Gaidano, G.; Malavasi, F.; Deaglio, S. The PD-1/PD-L1 Axis Contributes to T-cell Dysfunction in Chronic Lymphocytic Leukemia. Haematologica 2013, 98, 953-963. (17) Gordon, S. R.; Maute, R. L.; Dulken, B. W.; Hutter, G.; George, B. M.; McCracken, M. N.; Gupta, R.; Tsai, J. M.; Sinha, R.; Corey, D.; Ring, A. M.; Connolly, A. J.; Weissman, I. L. PD-1 Expression by Tumour-Associated Macrophages Inhibits Phagocytosis and Tumour Immunity. Nature 2017, 545, 495-499. (18) Sznol, M.; Chen, L. Antagonist Antibodies to PD-1 and B7-H1 (PD-L1) in the Treatment of Advanced Human Cancer. Clin. Cancer Res. 2013, 19, 1021-1034. (19) Daskivich, T. J.; Belldegrun, A. Words of Wisdom. Re: Safety, Activity, and Immune Correlates of Anti-PD-1 Antibody in Cancer. Eur. Urol. 2015, 67, 816-817. (20) Hamid, O.; Robert, C.; Daud, A.; Hodi, F. S.; Hwu, W. J.; Kefford, R.; Wolchok, J. D.; Hersey, P.; Joseph, R. W.; Weber, J. S.; Dronca, R.; Gangadhar, T. C.; Patnaik, A.; Zarour, H.; Joshua, A. M.; Gergich, K.; Elassaiss-Schaap, J.; Algazi, A.; Mateus, C.; Boasberg, P.; Tumeh, P. C.; Chmielowski, B.; Ebbinghaus, S. W.; Li, X. N.; Kang, S. P.; Ribas, A. Safety and Tumor Responses with Lambrolizumab (Anti-PD-1) in Melanoma. N. Engl. J. Med. 2013, 369, 134-144. (21) Palucka, K.; Ueno, H.; Fay, J.; Banchereau, J. Dendritic Cells and Immunity against Cancer. J. Intern. Med. 2011, 269 , 64-73. (22) Bae, M. Y.; Cho, N. H.; Seong, S. Y. Protective Anti-Tumour Immune Responses

by

Murine

Dendritic

Cells

Pulsed

with

Recombinant

Tat-Carcinoembryonic Antigen Derived from Escherichia coli. Clin. Exp. Immunol. 2009, 157, 128-138. X


Page 25 of 27 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


(23) Figdor, C. G.; de Vries, I. J.; Lesterhuis, W. J.; Melief, C. J. Dendritic Cell Immunotherapy: Mapping the Way. Nat. Med. 2004, 10, 475-480. (24) Bryson, P. D.; Han, X.; Truong, N.; Wang, P. Breast Cancer Vaccines Delivered by Dendritic Cell-Targeted Lentivectors Induce Potent Antitumor Immune Responses and Protect Mice from Mammary Tumor Growth. Vaccine 2017, 35, 5842-5849. (25) Min, Y.; Roche, K. C.; Tian, S.; Eblan, M. J.; McKinnon, K. P.; Caster, J. M.; Chai, S.; Herring, L. E.; Zhang, L.; Zhang, T.; DeSimone, J. M.; Tepper, J. E.; Vincent, B. G.; Serody, J. S.; Wang, A. Z. Antigen-Capturing Nanoparticles Improve the Abscopal Effect and Cancer Immunotherapy. Nat. Nanotechnol. 2017, 12, 877-882. (26) Fang, R. H.; Kroll, A. V.; Zhang, L. Nanoparticle-Based Manipulation of Antigen-Presenting Cells for Cancer Immunotherapy. Small 2015, 11, 5483-5496. (27) Goldberg, Michael S. Immunoengineering: How Nanotechnology Can Enhance Cancer Immunotherapy. Cell 2015, 161, 201-204. (28) Irvine, D. J.; Hanson, M. C.; Rakhra, K.; Tokatlian, T. Synthetic Nanoparticles for Vaccines and Immunotherapy. Chem. Rev. 2015, 115, 11109-11146. (29) Zhao, Y.; Pan, H.; Lou, Y.; Qiu, X.; Zhu, J. J.; Burda, C. Plasmonic Cu2-xS Nanocrystals: Optical and Structural Properties of Copper-Deficient Copper(I) Sulfides. J. Am. Chem. Soc. 2009, 131, 4253–4261. (30) Wang, S.; Riedinger, A.; Li, H.; Fu, C.; Liu, H.; Li, L.; Liu, T.; Tan, L.; Barthel, M. J.; Pugliese, G.; De Donato, F.; Scotto D'Abbusco, M.; Meng, X.; Manna, L.; Meng, H.; Pellegrino, T. Plasmonic Copper Sulfide Nanocrystals Exhibiting Near-Infrared Photothermal and Photodynamic Therapeutic Effects. ACS Nano 2015, 9, 1788-1800. (31) Li, A. W.; Sobral, M. C.; Badrinath, S.; Choi, Y.; Graveline, A.; Stafford, A. G.; Weaver, J. C.; Dellacherie, M. O.; Shih, T. Y.; Ali, O. A.; Kim, J.; Wucherpfennig, K. W.; Mooney, D. J. A Facile Approach to Enhance Antigen Response for Personalized Cancer Vaccination. Nat. Mater. 2018, 17, 528-534. (32) Hacohen, N.; Fritsch, E. F.; Carter, T. A.; Lander, E. S.; Wu, C. J., Getting Y



Personal with Neoantigen-Based Therapeutic Cancer Vaccines. Cancer Immunol. Res. 2013, 1, 11-15. (33) Kakavand, H.; Wilmott, J. S.; Menzies, A. M. PD-L1 Expression and Tumor-Infiltrating Lymphocytes Define Different Subsets of MAPK Inhibitor Treated Melanoma Patients[J]. Clin. Cancer Res. 2015, 21, 3140-3148. (34) Abiko, K.; Matsumura, N.; Hamanishi, J.; Horikawa, N.; Murakami, R.; Yamaguchi, K.; Yoshioka, Y.; Baba, T.; Konishi, I.; Mandai, M. IFN-γ from Lymphocytes Induces PD-L1 Expression and Promotes Progression of Ovarian Cancer. Br. J. Cancer 2015, 112, 1501-1509. (35) Mandai, M.; Hamanishi, J.; Abiko, K.; Matsumura, N.; Baba, T.; Konishi, I. Dual Faces of IFNγ in Cancer Progression: A Role of PD-L1 Induction in the Determination of Pro-and Antitumor Immunity. Clin. Cancer Res. 2016, 22, 2329-2334. (36) Zhang, X.; Liu, R.; Shu, Q.; Yuan, Q.; Xing, G.; Gao, X. Quantitative Analysis of Multiple Proteins of Different Invasive Tumor Cell Lines at the Same Single-Cell Level. Small 2018, 14, 1703684 (37) Zhang, X.; Liu, R.; Yuan, Q.; Gao, F.; Li, J.; Zhang, Y.; Zhao, Y.; Chai, Z.; Gao, L.; Gao, X. The Precise Diagnosis of Cancer Invasion/Metastasis via 2D Laser Ablation Mass Mapping of Metalloproteinase in Primary Cancer Tissue. ACS Nano 2018, 12, 11139-11151.

Graphic for manuscript Z


Page 26 of 27

Page 27 of 27


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 AA


Surface-Functionalized Modified Copper Sulfide Nanoparticles

Recommend Documents