Photothermal-Activatable Fe3O4 Superparticle Nanodrug Carriers

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Photothermal-Activatable Fe3O4 Superparticle Nanodrug Carriers with PD-L1 Immune Checkpoint Blockade for Anti-metastatic Cancer Immunotherapy Rui Ge,† Cangwei Liu,‡ Xue Zhang,† Wenjing Wang,† Binxi Li,† Jie Liu,‡ Yi Liu,† Hongchen Sun,‡ Daqi Zhang,*,§ Yuchuan Hou,*,∥ Hao Zhang,*,† and Bai Yang† †

State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, P. R. China ‡ Department of Oral Pathology, School and Hospital of Stomatology, Jilin University, Changchun 130021, P. R. China § Department of Thyroid Surgery, China-Japan Union Hospital, Jilin University, Changchun 130033, P. R. China ∥ Department of Urinary Surgery, The First Hospital of Jilin University, Changchun 130021, P. R. China S Supporting Information *

ABSTRACT: Checkpoint blockade immunotherapy has shown great potential in clinical cancer therapy, but the body’s systemic immune must be fully activated and generates a positive tumor-specific immune cell response. In this work, we demonstrate the design of the immune-adjuvant nanodrug carriers on the basis of poly(ethylene glycol)-block-poly(lacticco-glycolic acid) copolymer-encapsulated Fe3O4 superparticles (SPs), in which imiquimod (R837), a kind of Toll-like receptor 7 agonist, is loaded. The nanodrug carriers are defined as Fe3O4-R837 SPs. The multitasking Fe3O4-R837 SPs can destroy the 4T1 breast tumor by photothermal therapy (PTT) under near-infrared laser irradiation to generate the tumor-associated antigens because of the high efficiency of tumor magnetic attraction ability and photothermal effect. The PTT also triggers the release of R837 as the adjuvant to trigger a strong antitumor immune response. By further combining with the checkpoint blockade adjusted by programmed death ligand 1 (PD-L1) antibody, the Fe3O4-R837 SP-involved PTT cannot only eliminate the primary tumors but also prevent tumor metastasis to lungs/liver. Meanwhile, this synergistic therapy also shows abscopal effects by completely inhibiting the growth of untreated distant tumors through effectively triggering the tumors infiltrated by CD45+ leukocytes. Such findings suggest that Fe3O4-R837 SP-involved PTT can significantly potentiate the systemic therapeutic efficiency of PD-L1 checkpoint blockade therapy by activating both innate and adaptive immune systems in the body. KEYWORDS: Fe3O4 superparticles, cancer immunotherapy, photothermal-activatable, checkpoint blockade therapy, anti-metastasis

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apy,12−15 cytokine therapy,16,17 dendritic cell (DC)-based immunotherapy,18,19 cancer vaccines and so forth,20−23 have been established and achieved exciting results in clinical trials. Despite cancer immunotherapy is far from being satisfactory, the continuous improvements of this strategy will finally build an alternative for cancer treatment.24,25 Among the aforementioned cancer immunotherapies, checkpoint-blockade therapy, which utilizes a series of antibodies that can target to the overexpressed T-cell suppressor checkpoint signaling pathways, is a promising strategy to solve the adaptive immune evasion in the tumor and has been approved by the U.S. Food and Drug Administration (FDA) for cancer treatment.26,27

INTRODUCTION Cancer has become one of the major threats to human life for centuries.1 Because of its high metastasis and recurrence, the conventional treatments, such as surgery, chemotherapy and radiotherapy, are difficult to cure thoroughly.2,3 Accordingly, massive efforts are being devoted to the development of new and effective cancer treatments with low side effects, high specificities, and anti-metastasis.4 Recently, along with the deep understanding of cancer and their relationship with the immune system, cancer immunotherapy by stimulating and training the body’s immune system to actively attack tumor cells for the purpose of controlling metastatic tumor’s growth has attracted great attention and become one of the most promising treatments for cancer.5−8 Several kinds of cancer immunotherapies, such as chimeric antigen receptor/T-cell receptorengineered T-cell therapy,9−11 checkpoint-blockade ther© XXXX American Chemical Society

Received: April 11, 2018 Accepted: May 31, 2018

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DOI: 10.1021/acsami.8b05876 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Schematic illustration of Fe3O4-R837 SP PTT with PD-L1 checkpoint blockade for cancer immunotherapy. With the help of tumor magnetic attraction, Fe3O4-R837 SPs can destroy the tumors effectively under NIR irradiation. In the presence of Fe3O4-R837 SPs, the generated tumorassociated antigens can induce strong antitumor immune responses. By further combining with PD-L1 checkpoint blockade, this method can eliminate primary tumors, prevent the metastasis to lungs/liver, and further inhibit the distant tumor growth after PTT.

delivery system has not been reported because of the difficulty in choosing proper NPs. In this scenario, Fe3O4 NPs have been approved by FDA for the clinical treatment with the function of magnetic targeting and T2-weighted magnetic resonance imaging (MRI).49 However, individual Fe3O4 NPs show poor photothermal performance due to the low molar extinction coefficient, making them useless in PTT. In our previous works, spherical superparticles (SPs) of Fe3O4 NPs have been fabricated by oil-inwater (O/W) microemulsion template method, which significantly improves the photothermal performance and makes Fe3O4 a good agent for PTT.50 Meanwhile, the SPs can also load drugs to perform controlled drug release, thus acting as multifunctional nanodrug carriers for tumor theranostics. In this work, to utilize near-infrared (NIR)-triggered PTT to induce effective cancer checkpoint blockade therapy, Fe3O4based multifunctional nanodrug carriers are fabricated from three FDA-approved agents, namely, poly(ethylene glycol)-blockpoly(lactic-co-glycolic acid) copolymer (mPEG-PLGA), Fe3O4 NPs, and imiquimod (R837). The nanodrug carriers are defined as Fe3O4-R837 SPs. Fe3O4 SPs act as the NIR photothermal, magnetic targeting, and T2-weighted contrast agents, and mPEGPLGA is an amphiphilic copolymer for encapsulating the SPs. The loaded immune adjuvant R837, a kind of Toll-like receptor 7 (TLR7) agonist, can promote dendritic cells (DCs), phagocytize tumor-associated antigens, and become mature, thus enhancing the activation and proliferation of antigen-specific lymphocytes in draining lymph nodes (DLNs). The tumor retention rate of the SPs with external magnetic field is as high as 14.8%ID/g and under NIR irradiation, the Fe3O4-R837 SPs can effectively eliminate the primary tumors and release R837. After PTT, with the help of immune adjuvant R837, the released tumorassociated antigens can stimulate strong systemic antitumor immune response, which can be promoted by PD-L1 checkpoint blockade therapy to prevent the lungs/liver metastasis in a 4T1

Adaptive immune evasion mechanism is a self-protecting behavior of tumor cells to defend against the body’s immune response.28 Programmed death 1 (PD-1) and its ligand, programmed death ligand 1 (PD-L1), are two important immune checkpoint molecules related to immune resistance.29 PD-1, expressed on T cells surface, interacts with PD-L1 expressed on tumor cells, terminating immune responses by inhibiting cytotoxic/effector T-cell function and delivering antiapoptotic signals to tumors.30 The elimination of either one can lead to cytotoxic/effector T cells normal operation without inhibition.31 Hence, antibody-mediated PD-1/PD-L1 pathway specific blockade can enhance the potent antitumor activity of the body.32 However, the clinical trials show that if the body’s immune system cannot be fully activated and produce large numbers of immune cells, the durable responses generated by checkpoint blockade in most kinds of tumor cells are still very low.33−35 Therefore, combining checkpoint blockade therapy with other types of treatments that can effectively activate the systemic immune responses to enhance T-cell infiltration may enhance the antitumor response rates and broaden the application of immunotherapy in metastatic tumor.36,37 Photothermal therapy (PTT) is a new type of cancer treatment strategy developed in recent years and has been proved to kill tumor cells by thermal ablation and produce the tumor-associated antigens from ablated tumor cell residues.38−40 The thermal ablation process can also activate the host immune system and trigger acute inflammation in the tumor area, thereby increasing the presentation of tumor-associated antigens to T cells.41 A variety of inorganic nanoparticles (NPs), such as noble metals,42−45 metal chalcogenides,46,47 metal oxides, and so on,48 have been used for the delivery of PTT, showing obvious tumor ablation in primary animal experiments. However, the strategy for the combination of the PTT of inorganic NPs with checkpoint blockade therapy by constructing a novel drug B


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Figure 2. Characterization of Fe3O4 and Fe3O4-R837 SPs. (a) Schematic illustration of the synthesis of Fe3O4-R837 SPs. TEM (b) and SEM (c) images of Fe3O4-R837 SPs. Inset in (b): high-magnification TEM image of one SP. The scale bar represents 50 nm. (d) T2 relaxation rates and concentrationdependent T2-weighted MRI at pH 7.4 under 1.5 T magnetic field (inset) for the aqueous solution of Fe3O4-R837 SPs. (e) Temperature variation of PBS, 150 μg/mL Fe3O4 SPs, and Fe3O4-R837 SPs irradiated by a 1.0 W/cm2 808 nm laser. (f) UV−vis absorption spectra of Fe3O4 and Fe3O4-R837 SPs at the concentration of 150 μg/mL. (g) Fourier-transform infrared (FTIR) spectra of Fe3O4 NPs, mPEG-PLGA, Fe3O4 SPs, R837, and Fe3O4-R837 SPs.

prepared (Figure S1a).54 Then, O/W microemulsion is generated by mixing mPEG-PLGA chloroform solution, Fe3O4 NPs, R837 in dimethyl sulfoxide (DMSO), and water under vigorous stirring. The evaporation of chloroform generates Fe3O4-R837 SPs. The Fe3O4 SPs are synthesized with the same method but without R837. The obtained Fe3O4-R837 SPs are well dispersed in water and appear as quasispheres with good monodispersity under transmission electron microscopy (TEM) (Figure 2b). The size distribution of Fe3O4-R837 SPs is calculated from TEM observation, which shows the mean diameter of 149.8 nm (Figure S1b). The average number of Fe3O4 NPs in a superparticle is 1.27 × 104 (Calculation S1). The scanning electron microscopy (SEM) observation also shows the homogeneous size distribution of the SPs (Figure 2c). Fe3O4R837 SPs exhibit an average hydrodynamic diameter of 157.3 ± 3.0 nm, a ζ potential of −26.9 ± 2.8 mV, and a polydispersity

metastatic triple-negative breast cancer murine model. Besides, the synergistic therapy has an abscopal effect to attack and inhibit the nonirradiated distant tumors remaining in the mouse body. In vitro and in vivo experiments indicate that immunogenic Fe3O4-R837 SP PTT in combination with PD-L1 blockade therapy has significant efficacy in PTT-activated cancer immunotherapy (Figure 1).

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RESULTS AND DISCUSSION Preparation and Characterization of Fe3O4-R837 SPs. Fe3O4-R837 SPs are synthesized by assembling Fe3O4 NPs in O/ W microemulsion templates with the presence of mPEG-PLGA amphiphilic block copolymer and loading with R837 (Figure 2a).51−53 According to the conventional thermal decomposition method, 5.8 nm oleic acid (OA)-stabilized Fe3O4 NPs are first C


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Figure 3. In vitro transwell coculture system experiments. (a) Schematic illustration of the design of the transwell coculture system experiments. DCs are placed in the lower compartment, and 4T1 cells are cultured in the upper compartment. (b−h) The percentage of mature DCs (CD11c+CD80+CD86+) is analyzed by flow cytometry after different treatments. (i) Quantification of the level of DC maturation after different treatments in the transwell system experiments. “−” and “+” in the figure means treatment without or with irradiation, respectively. Error bars are based on standard deviations of three parallel samples.

55.4 °C under the irradiation of 1.0 W/cm2 808 nm laser (Figure 2e). The photothermal conversion efficiency of Fe3O4-R837 SPs is 68.2% (Figure S2 and Calculation S2). Both the UV−vis absorption and Fourier-transform infrared (FTIR) spectra of Fe3O4-R837 SPs show the characteristic absorption peak of R837, indicating the effective encapsulation of R837 in the SPs (Figure 2f,g). In water, the R837 concentration and the absorbance in 2.5−10 μg/mL maintain a linear relation (Figure S3a). The regression equation is y = 0.0698x − 0.03415 (R2 = 0.999) (Figure S3b). Thus, the R837 loading is calculated by UV−vis absorption spectrum, and the relationship between encapsulation/loading efficiency and the dosage of R837 solution in the reaction system are shown in Figure S3c,d. Considering the encapsulation efficiency and safe dose in vivo (0.4 mg/kg), the optimal feeding concentration of R837 is 200 μg/mL.57 In our system, R837 is loaded in mPEG-PLGA. Upon NIR laser irradiation, the system temperature increases due to the photothermal effect of Fe3O4-R837 SPs. According to the previous literature,58 the temperature increment promotes the degradation of PLGA-based NPs. So, NIR irradiation leads to rapid R837 release from Fe3O4-R837 SPs. Figure S4a shows the release of R837 with and without irradiation. Without irradiation,

index (PDI) of 0.05 ± 0.016, as measured by dynamic light scattering (DLS) (Figure S1c). The structural stability of Fe3O4R837 SPs is favorable in a physiological environment, as proved by stable size and PDI when dispersed in phosphate-buffered saline (PBS, pH 7.4) for up to 7 days (Figure S1d). According to the previous reports,55 such SPs with negative surface charge, high stability, and hydrophilic “stealth” polymer surface possess prolonged blood circulation and efficiently accumulate into tumor tissues by enhanced permeability and retention effect, potentially applicable for the living body. Recent studies have shown that the assembly of inorganic NPs into SPs can significantly enhance MRI and the photothermal performance.50 The self-assembly of NPs into Fe3O4-R837 SPs does not change the superparamagnetic behavior of Fe3O4 NPs in comparison with the magnetic curve of Fe3O4 NPs (Figure S1e). The r2 of Fe3O4-R837 SPs is 178.1 mM−1 s−1 (Figure 2d), which is higher than that of commercial MRI T2-weighted contrast agents (Feridex, 93 mM−1 s−1; Resovist, 143 mM−1 s−1).56 The high r2 allows Fe3O4-R837 SPs to act as an effective T2-weighted contrast, with fewer doses getting darker contrast (Figure 2d inset). Compared with the final temperature of PBS solution (32.9 °C), the temperature of SP solution increases to D


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Figure 4. In vivo tumor magnetic attraction experiments. (a) Schematic illustration of the design of in vivo tumor magnetic attraction. (b) A photograph showing how the magnet is applied during magnetic attraction. (c) Blood circulation kinetics of Fe3O4-R837 SPs without magnetic attraction. (d) Blood circulation kinetics of Fe3O4-R837 SPs with magnetic attraction. (e) Biodistribution of Fe3O4-R837 SPs in 4T1-bearing mice at 24 h p.i. administration with or without magnetic attraction. Error bars are based on standard deviations of three parallel samples. (f) T2-weighted MRI images of mice for control, Fe3O4-R837 SPs and Fe3O4-R837 SPs with magnetic attraction. The red dotted lined circle points to tumors.

necrosis more efficiently when the cells are irradiated under the 808 nm laser. The 24 h cytotoxicity of Fe3O4 SPs and Fe3O4-R837 SPs is investigated by standard methyl thiazolyltetrozolium (MTT) assay. Even when incubated with 400 μg/mL Fe3O4 SPs, the 4T1 cells’ viability is still maintained at a high level (>80%), (Figure S6a). Although R837 can induce apoptosis in cancer cells, the cell viability is still higher (>70%). Meanwhile, the cytotoxicity of Fe3O4-R837 SPs incubated with 293 cells (human embryo kidney cells) shows that the SPs also have low cytotoxicity in normal cells. The Fe3O4-R837 SPs can effectively induce the photothermal ablation of 4T1 cells in vitro (Figure S6b). 4T1 cells are cultured with Fe3O4-R837 SPs and then irradiated with an 808 nm laser at different power densities for different times. The cell viability is evaluated by MTT assay. The cell viability decreases with the increment of laser power and irradiation time. When the laser power density reaches 2 W/cm2 and the irradiation time is 20 min, less than 5% of the 4T1 cells survive.

the released R837 in water keeps a low concentration. Whereas, a high release rate is achieved and maintained under 1 W/cm2 808 nm laser. The released percentage is nearly 34% in 28 min, implying that R837 can be constantly released from Fe3O4-R837 SPs (Figure S4b). With higher laser power density, Fe3O4-R837 SPs release more R837 (Figure S4c). PTT-Induced Apoptosis and/or Necrosis in Vitro. 4T1 metastatic triple-negative breast cancer cells are used to test the photothermal therapy of Fe3O4-R837 SPs. Figure S5a shows that Fe3O4 SPs/Fe3O4-R837 SPs are rapidly engulfed by 4T1 cells, with most uptake occurring within 4 h and then remaining stable within 24 h. High cellular uptake and low efflux ensure the high cellular accumulation of SPs (Figure S5b). In the control group, the structure of 4T1 cells is intact and smooth. With the increment of incubation time, even though the cells maintain structural integrity, more and more SPs are adhered and internalized by the cells (Figure S5c). The high cellular accumulation of SPs is beneficial to induce cells apoptosis/ E


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Figure 5. In vivo Fe3O4-R837 SP PTT triggers DC maturation. (a) Schematic illustration showing the design of experiments to examine the immune responses induced by Fe3O4-R837 SP PTT. (b) IR thermal images of 4T1 tumor-bearing mice performed by an IR camera. The tumors are irradiated by a 0.33 W/cm2 808 nm laser for 5 min. “−” and “+” in the figure mean treatment without or with irradiation, respectively. (c) Quantification of the level of DC maturation induced by control, Fe3O4 SPs, and Fe3O4-R837 SP group with or without irradiation on 4T1-bearing mice. (d−i) The percentage of DC maturation after different treatments. After staining with CD11c, CD80, and CD86, cells in the tumor-draining lymph nodes are assessed by flow cytometry. Error bars are based on standard deviations of three parallel samples.

Then, 4T1 cells are stained with fluorescein diacetate (FD) and propidium iodide (PI), as the living cells can be stained into green and apoptotic cells into red.50 Figure S6c shows the confocal fluorescence and bright microscope images of 4T1 cells incubated with Fe3O4-R837 SPs after irradiation for 0, 5, 10, and 20 min. The decrease in green and increase in red along with the irradiation time indicate the effective photothermal therapy of

SPs in vitro. To investigate the contribution of Fe3O4-R837 SPs during the apoptosis/necrosis process, the apoptosis process on PBS, R837, Fe3O4 SPs, and Fe3O4-R837 SP group is monitored by flow cytometric analysis. As shown in Figure S7a−h, the cells in upper left, upper right, bottom left, and bottom right areas represent necrosis, late apoptosis, viability, and early apoptosis, respectively. In comparison with the control group, R837 cannot F


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extend the blood circulation half-life (from 1.94 ± 0.16 to 2.48 ± 0.23 h), the tumor retention rate of the magnetic attraction group increases almost 68% (from 8.8 to 14.8 of the injected dose per gram tissue (%ID/g)) at 24 h postinjection (p.i.) administration (Figure 4e). Compared with control and Fe3O4-R837 SP group, T2-weighted MRI reveals a darkening effect with the magnet attached, also suggesting the effective accumulation of SPs in the tumor. Such high tumor retention caused by magnetic attraction can make a contribution to improve PTT efficiency. On the basis of in vitro experiments, the 4T1 tumor cell residues post NIR-induced PTT with Fe3O4-R837 SPs can enhance the level of DC maturation, higher than individual Fe3O4-R837 SPs, or cell residues ablated by Fe3O4 SPs without R837. The results suggest that R837-loaded SPs serve as the adjuvant to induce the immune response from the tumorassociated antigen in the cell residues. In further in vivo experiments (Figure 5a), when the 4T1 tumors grown on Balb/c mice reach ∼200 mm3 on day 10, the mice are iv injected with PBS, Fe3O4 SPs, or Fe3O4-R837 SPs and attached to a magnet. After 24 h on day 11, the tumors are exposed to a 0.33 W/cm2 808 nm NIR laser for 30 min. The infrared (IR) thermal images show that the temperature increment caused by Fe3O4-R837 SP PTT is sufficient to ablate the cells and prevent their malignant proliferation (Figure 5b). To investigate the levels of DC maturation by flow analysis, the draining lymph nodes of mice are removed 3 days after PTT. Compared with the PBS without irradiation group, the percentage of matured DCs increases from 6.58 to 31.7% after PTT with Fe3O4-R837 SPs (Figure 5d,i). For the group of Fe3O4 SPs with PTT or Fe3O4-R837 SPs without PTT, the percentages of DC maturation only increase to 17.6 and 22.8%, respectively (Figure 5h,f). These mean that after Fe3O4-R837 SP PTT treatment for tumors, more DCs are recruited to the tumor site as APCs to engulf the tumorassociated antigens released by apoptotic tumor cells, then transported to the nearby lymph nodes and matured with the help of released R837 (Figure 5c). The secretion of cytokine is another marker of immune activation and indicates acute inflammation, a crucial mechanism to evoke antitumor immunity.21 Hence, the changes of various cytokines, including IL-6 (a kind of marker in the activation of humoral immunity), TNF-α (a kind of marker in the activation of cellular immunity), and interferon γ (IFN-γ) (a kind of marker in the activation of innate immunity) in sera after different treatments are analyzed by ELISA (Figure S9).21 Similarly, even though the individual Fe3O4-R837 SPs or PTT with Fe3O4 SPs can promote the secretion of cytokine, the increment of Fe3O4-R837 SPs PTT group is the highest, which is most favorable for inducing antitumor immune responses. Such results suggest that with the help of R837-loaded SPs, the tumorassociate antigens released from tumor ablation therapy can induce the systemic innate and adaptive immune systems effectively in vivo. In Vivo Fe3O4-R837 SP PTT with PD-L1 Blockade To Eradicate Primary Tumor and Prevent Lungs/Liver Metastasis. The main reason of a high cancer fatality rate is that it is prone to metastasis, which is difficult to treat with conventional treatments once they have metastasized.2,3 Hence, we investigate if PTT-activatable immunotherapy with Fe3O4R837 SPs can make a contribution to treat the metastatic tumor. As approved by FDA for clinically used immunotherapy, PD-L1 plays a pivotal role for tumor cells to evade the host’s immune system and can be blockaded by antibodies (anti-PD-L1).31 Therefore, in the animal experiments, PD-L1 blockade therapy is

cause cell necrosis distinctly but can lead to partial cell apoptosis (Figure S7b,f). It is consistent with the results of the MTT assay that the cell viability of Fe3O4-R837 SPs is a little lower than that of Fe3O4 SPs. Figure S7c,d,g,h shows that both Fe3O4 SPs and Fe3O4-R837 SPs have low toxicity and can induce the early apoptosis of 4T1 cells effectively after PTT (1 W/cm2, 10 min). The apoptosis ratios are as high as 68.7 and 71.6%, respectively. Fe3O4-R837 SP-Induced DC Maturation in Vitro. As one of the most crucial antigen presenting cells (APCs), DCs provide a pivotal functional link between innate and adaptive immune responses.18 The immature DCs can engulf and process antigens in the tissue and peripheral blood, then undergo maturation and migrate to lymphoid nodes where they present the antigens to ̈ T cells. The mature DCs can upregulate the costimulatory naive molecules such as CD80 and CD86 on the APC surface to ̈ CD8+ cytotoxic T cells and naive ̈ CD4+ helper activate both naive T cells.18 Hence, the immunological effects of Fe3O4-R837 SPs toward bone-marrow-derived DCs by analyzing the upregulation of CD80 and CD86 are first investigated. A transwell system is introduced to investigate the effect at the in vitro level. The upper compartment contains 4T1 cells after different treatments, whereas the lower compartment is placed with bone-marrowderived DCs from Balb/c mice (Figure 3a). It is found that in the absence of R837, compared with the 4T1 cells cocultured with Fe3O4 SPs without irradiation (Figure 3e), the released tumorassociated antigens after PTT can notably enhance the DC maturation from 26.3 to 39.2% (Figure 3g) and with the help of R837, Fe3O4-R837 SPs with irradiation (89.6%, Figure 3h) further promote in vitro DC maturation even higher than free R837 at the same dose (75.7%, Figure 3d). Note that although free R837 works well for DC maturation in vitro, the low solubility in water greatly limits the applications for a living body. Therefore, it must be loaded into amphiphilic drug carriers like Fe3O4 SPs. Meanwhile, the DC-secreted immune related cytokines, such as tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6), which are also indicators of DC maturation, are measured by enzyme-linked immunosorbent assay (ELISA) kits.18 The secretion levels of TNF-α and IL-6 from DCs are distinctly enhanced after Fe3O4-R837 SP PTT and higher than that in the treatment with free R837 (Figure S8). Both the flow cytometry and ELISA data prove that the tumor-associated antigens released by tumor cells after PTT, especially with the help of R837-loaded SPs adjuvant, can induce DC maturation effectively. In Vivo Magnetic Attraction-Enhanced Photothermal Tumor Ablation for Immune System Activation. The above results indicate that Fe3O4-R837 SPs are an effective immune stimulating reagent in vitro. Furthermore, the PTT of Fe3O4R837 SPs is considered to induce enhanced tumor-specific immune responses in vivo. Before this, because of the superparamagnetism of Fe3O4-R837 SPs, we test if adding an external magnetic field can effectively enhance tumor accumulation of SPs, thereby increasing the treatment efficiency of PTT. The blood circulation kinetics and biodistribution studies are performed on orthotopic 4T1 tumor-bearing Balb/c mice. When the 4T1 tumors grown on Balb/c mice reach ∼200 mm3 on day 10, Fe3O4-R837 SPs are intravenously (iv) injected into the mice. The mice are divided into two groups. The tumors of one group are attached to a magnet for 24 h (Figure 4a,b). Blood samples are collected at different time intervals and decomposed in aqua regia solution to determine the Fe3+ concentration by inductively coupled plasma optical emission (ICP-OES). As shown in Figure 4c,d, although adding the magnetic field does not significantly G


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Figure 6. In vivo Fe3O4-R837 SP PTT with PD-L1 blockade eliminates primary tumors and prevents lungs/liver metastasis. (a) Schematic illustration showing the design of experiments. (b−e, j−m) Photographs showing the tumor nodules in the lungs/livers. (f−i, n−q) Representative lungs/liver sections stained with H&E. The scale bar is 200 μm.

introduced to enhance the antitumor immunotherapy efficacies generated by PTT with Fe3O4-R837 SPs of the primary tumor. Figure 6a shows the experimental design. 4T1 cells are orthotopically injected into the right mammary fat pads, and they can spontaneously metastasize to lungs or liver.59,60 When the volume of tumor reaches ∼200 mm3, the mice are i.v. injected with Fe3O4-R837 SPs and attached to a magnet every 3 days three times (on day 10, 13, and 16). Twenty four hours p.i., tumors are exposed to a 808 nm NIR laser at 0.33 W/cm2 for 30 min. After laser irradiation, anti-PD-L1 antibody are i.v. injected into the mice at a dose of 75 μg/mouse. As shown in Figure S10, compared with the control group, PTT with Fe3O4-R837 SPs can eliminate the primary tumor and PD-L1 blockade therapy alone fails to delay the tumor proliferation. On day 24, tumor-bearing mice are all sacrificed to evaluate the extent of metastasis by checking the tumor nodules in the lungs and liver. The dehydrated tissues are imaged by digital camera and further sectioned and stained with hematoxylin−eosin (H&E) staining. For the control group, Figure 6b,f,j,n shows that there are many

tumor nodules in lungs and large area of edge necrosis in liver. Anti-PD-L1 blockade therapy alone shows little effect on preventing lungs/liver metastasis (Figure 6c,g,k,o). Although the primary tumor elimination by PTT with Fe3O4-R837 SPs can reduce the possibility of tumor metastasis and does not cause a local tissue necrosis, there are still some tumor nodules in lungs and obvious metastasis around the blood vessels in liver (Figure 6d,h,l,p). Only by combining the two treatments can the tumor metastasis be effectively eliminated (Figure 6e,i,m,q). Such results are consistent with the previous literature in that the individual anti-PD-L1 blockade therapy has almost no effect on 4T1 tumor cells and only once the immune system is activated by other immunogenic therapies, the efficiency of blockade therapy can be greatly enhanced.35 Therefore, we demonstrate that the tumor-specific immunity activated by PTT with immune adjuvants can enhance the effect of checkpoint blockade therapy, leading to the elimination of primary tumor and the prevention of lungs/liver metastasis. H


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Figure 7. In vivo Fe3O4-R837 SP PTT with PD-L1 blockade eliminates primary 4T1 tumors and inhibits the growth of distant tumors. (a) Schematic illustration showing the experiment’s design. 4T1 tumors are planted on both sides of the mice. The right tumors are designed as “primary tumors” for PTT, and the left tumors are designed as “distant tumors” without PTT. (b, c) The primary and distant tumor volume growing trend for different groups. (d, e) Tumor weights at the end point of different groups. Arrows represent the time of materials injection (black) and irradiation (red). “−” and “+” mean treatment without or with irradiation, respectively. Error bars are based on standard deviations of five parallel samples.

Fe3O4-R837 SP PTT Plus PD-L1 Blockade To Inhibit the Growth of Distant Tumors. In practical therapies, the metastatic tumors may have already existed so the studies on the inhibition of metastatic tumor growth is necessary. To facilitate the demonstration and further study that synergistic therapy can induce immune cells to specifically kill 4T1 tumor cells and thus prevent metastasis, the larger pre-existing simulated distant tumors are constructed for the analysis of immune cell content. A bilateral orthotopic 4T1 tumor model is introduced into the experiments to prove that the synergistic therapy cannot only prevent the tumor metastasis but also inhibit the pre-existing metastatic tumors (Figure 7a). The first tumor is inoculated into the right mammary fat pads as the primary tumor, and the second tumor is inoculated into the left mammary fat pads as an artificial mimic of distant metastatic tumor. After the SPs are systemically injected, the primary tumors are eliminated by PTT or not. Then,

anti-PD-L1 antibody are i.v. injected into the mice at a dose of 75 μg/mouse three times on day 11, 14, and 17. The sizes of the primary and distant tumors in different groups are measured by a digital caliper every other day. Figure 7b,c shows that anti-PD-L1 alone contributes minimally to the inhibition of both the primary and the distant tumors. Fe3O4 SPs/Fe3O4-R837 SPs injection alone shows almost no effect on the primary or distant tumor growth. PTT with Fe3O4 SPs/Fe3O4-R837 SPs in the absence of anti-PD-L1 three times can eradicate the primary tumor effectively but does not inhibit the growth of the distant tumor significantly. The distant tumor growth of Fe3O4-R837 SP PTT group is only partially delayed because the immune system can be successfully activated, as mentioned above. Only the combination of Fe3O4-R837 SP PTT and anti-PD-L1 treatment can completely eradicate the primary tumor and control the growth of the distant tumor. In addition, the average weight of mice in all groups are monitored. As indicated in Figure S11, no significant I


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Figure 8. Mechanism study of inhibiting the metastasis. (a) Schematic illustration showing the design of experiments. The distant tumors are collected for flow analysis; the percentages of CD45+ leukocytes (CD45+PI−) (b), NK cells (CD45+CD3ε−NKp46+PI−) (c), B cells (CD45+CD3ε−B220+PI−) (d), CD8+ T cells (CD45+CD3ε+CD8+PI−) (e), and CD4+ T cells (CD45+CD3ε+CD4+PI−) (f) in the tumors are measured. “−” and “+” in the figure mean treatment without or with irradiation, respectively. Error bars are based on standard deviations of five parallel samples.

To investigate the mechanism of synergistic antitumor immune responses triggered by Fe3O4-R837 SP PTT plus antiPD-L1 therapy, the distant 4T1 tumors are excised to test the immune cells on day 20 (Figure 8a). Immune cells, which can also be called leukocytes, include all cells that are involved in or associated with the immune response.27 Since CD45+ molecules are expressed on all leukocytes, known as leukocyte common antigen, we measure the percentage of the infiltrating leukocytes in the distant tumors. Compared to the control group (14.94 ± 1.84%), the percentage of CD45+ leukocytes increase almost 34 and 46% in anti-PD-L1 therapy alone (20.03 ± 1.29%) and Fe3O4-R837 SP PTT alone (21.84 ± 2.18%) (Figure 8b). For the subspecies of leukocytes, such as NK cells, compared with the control group (6.52 ± 0.58%), the increase in anti-PD-L1 group is about 19% (7.80 ± 0.63%) and in Fe3O4-R837 SP PTT group, about 47% (9.62 ± 0.98%) (Figure 8c). Similarly, for B cells, compared with the control group (3.81 ± 0.44%), the increase in anti-PD-L1 group is about 14% (4.36 ± 0.25%) and in Fe3O4R837 SP PTT group, about 58% (6.04 ± 0.15%) (Figure 8d). Such results indicate that compared with anti-PD-L1 alone, the percentage of NK cells and B cells is more affected by Fe3O4R837 SP PTT group. However, for CD8+ T cells, compared with the control group (0.28 ± 0.06%), the increase in anti-PD-L1 group is about 239% (0.95 ± 0.06%) and in Fe3O4-R837 SP PTT

weight loss is observed in either the PTT treatment, anti-PD-L1 alone, or the synergistic therapy of two methods, indicating the safety of the therapies. Meanwhile, considering the full activation of the immune system may be harmful to the organs, serum biochemistry assay is performed in 4T1 tumor-bearing mice on day 24 after the synergistic therapy and the healthy mice (Table S1). All parameters of the treatment group are consistent with those of the healthy one, indicating that such a full activation of the immune system by synergistic therapy can be tolerated by the mice. Mechanism Study of Synergistic Therapy To Inhibit the Metastasis. Before studying the mechanism of synergistic therapy, it must be ensured that the systemic antitumor immune response has been activated in the body, which makes sense for the immune cell test. Therefore, we evaluate the change of proinflammatory cytokines, including IL-6, TNF-α, and IFN-γ, in sera by ELISA kits within 7 days after a group of treatment (Figure S12). Although anti-PD-L1 alone or Fe3O4-R837 SP PTT alone can increase the secretion of proinflammatory cytokines, the secretions induced by the synergistic therapy are the highest, favorable for triggering antitumor immune response. However, on day 7, after PTT treatment, all cytokine levels drop back to the baseline levels, indicating that the inflammation induced by different kinds of therapy is merely an acute response. J


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and kept at 80 °C for 20 min under vacuum and vigorous stirring. Afterward, the mixture was raised to 200 °C with the rate of 20 °C/min, kept for 30 min at this temperature under nitrogen atmosphere, and then refluxed under 265 °C for another 30 min. Then, the reaction solution was cooled to room temperature naturally. With the help of a magnet, Fe3O4 NPs were extracted and washed by adding hexane and ethanol three times and finally dissolved in chloroform. Preparation of mPEG-PLGA-Capped Fe3O4 SPs. mPEG-PLGAcapped Fe3O4 SPs were synthesized by an emulsification method.51−53 OA-stabilized Fe3O4 NPs and mPEG-PLGA were dissolved in chloroform with the concentration of 20 and 200 mg/mL, respectively. Under a nitrogen atmosphere and mechanical stirring at room temperature, 0.5 mL of OA-stabilized Fe3O4 NPs, 50 μL of mPEGPLGA, and 0.5 mL of DMSO were injected dropwise into 10 mL of deionized water. After the ultrasonic treatment, the emulsion was further stirred to evaporate organic solvent for 4 h. The SPs were washed by centrifugation (6000 rpm, 10 min) three times and dialyzed (molecular weight cut-off (MWCO): 7 kDa) against deionized water for 24 h, which were defined as Fe3O4 SPs. Preparation of mPEG-PLGA-Capped Fe3O4-R837 SPs. The synthesis of mPEG-PLGA-capped Fe3O4-R837 SPs was similar to that of mPEG-PLGA-capped Fe3O4 SPs. R837 was dissolved in DMSO with the concentration of 4 mg/mL. Under a nitrogen atmosphere and mechanical stirring at room temperature, 0.5 mL of OA-stabilized Fe3O4 NPs, 50 μL of mPEG-PLGA, and 0.5 mL of R837 were injected dropwise into 10 mL of deionized water. After the ultrasonic treatment, the emulsion was further stirred to evaporate the organic solvent for 4 h. The products were washed by centrifugation (6000 rpm, 10 min) three times and dialyzed (MWCO: 7 kDa) against deionized water for 24 h, which were defined as Fe3O4-R837 SPs. R837 Loading Standard Curve and Releasing Experiments. R837 solution was prepared in DMSO with the concentration of 1 mg/ mL and then diluted into 2.5, 5, 7.5, and 10 μg/mL standard solution by water. The absorbance of R837 solution with different concentrations at 322 nm was measured by UV−vis absorption spectra. According to the absorbance, a standard curve was obtained. The encapsulation efficiency and loading efficiency of Fe3O4-R837 SPs were calculated from the UV− vis absorption spectra against the standard curve. For R837 release, the SPs were irradiated under NIR laser and centrifuged to get the supernatant. The released concentration and percentage can also be calculated from the absorbance at 322 nm according to the standard curve. Cellular Uptake and Efflux. 4T1 cells seeded in six-well plates (2 × 105 cells/well) were incubated with Fe3O4 SPs/Fe3O4-R837 SPs at a concentration of 150 μg/mL for 2, 4, 12, and 24 h. Then, cells were collected and washed three times with PBS, counted with a hemocytometer, and lysed with aqua regia (HCl/HNO3 = 3:1) to test the Fe3+ concentration with ICP-OES. The uptake level was expressed as the amount of Fe3O4 SPs (nmol) per 104 cells. To investigate the efflux of SPs, 4T1 cells were incubated with Fe3O4 SPs/Fe3O4-R837 SPs at a concentration of 150 μg/mL for 4 h. Then, the culture medium was discarded and the 4T1 cells were washed with PBS three times. Two milliliters of fresh culture medium was added to each well, and cells were further cultured for 2, 4, 12, and 24 h. Thereafter, the cells were collected and washed three times with PBS, counted with a hemocytometer, and lysed with aqua regia to test the Fe3+ concentration with ICP-OES. Results were expressed as the percent of the difference value of SPs being retained to the 4 h cellular uptake amount. The bright field images of internalization were also observed under a fluorescence microscope. Fe3O4-R837 SPs were incubated with 4T1 cells for 2, 4, 12, and 24 h and washed with PBS three times, fixed with 4% paraformaldehyde, and observed under the fluorescence microscope. Cytotoxicity Assay in Vitro. 4T1 cells were seeded in 96-well plates at a density of 4 × 103 cells per well. Different concentrations of Fe3O4 SPs/Fe3O4-R837 SPs were incubated with cells for 24 h. The cell viability was detected by standard MTT assay according to the manufacturer’s instructions. The experiment was repeated five times. Apoptosis Analysis in Vitro. 4T1 cells seeded in six-well plates (2 × 105 cells/well) were treated with Fe3O4-R837 SPs at a concentration of 150 μg/mL for 4 h and then irradiated with 808 nm NIR laser at

group, about 200% (0.84 ± 0.09%) (Figure 8e). For CD4+ T cells, compared with the control group (0.77 ± 0.18%), the increase in anti-PD-L1 group is about 179% (2.15 ± 0.26%) and in Fe3O4-R837 SP PTT group, about 156% (1.97 ± 0.18%) (Figure 8f). Such results suggest that anti-PD-L1 may have more influence on promoting the accumulation of CD8+ and CD4+ T cells in the distant tumor sites. Although the previous data demonstrate that Fe3O4-R837 SP PTT can activate the systemic immune response, the increments of CD8+ and CD4+ T cells are not significant. The reason is that Fe3O4-R837 SP PTT activates large numbers of negatively regulatory T cells while activating systemic immunity, resulting in a small increase in the total number of T cells. Therefore, we suppose only through the synergistic therapy, the activation of systemic immunity while inhibiting negatively regulatory T cells, the treatment efficiency can be maximized. This consideration is further proved by the experimental data. Compared to that in the control group (14.94 ± 1.84%), the total percentage of CD45+ leukocytes increases almost 70% in the synergistic therapy group (25.47 ± 2.09%). Similarly, the percentage of NK cells (11.24 ± 0.70%), B cells (6.54 ± 0.19%), CD8+ T cells (1.39 ± 0.07%), and CD4+ T cells (2.76 ± 0.30%) significantly increase in comparison with not only the control group but also the anti-PD-L1 alone or Fe3O4R837 SP PTT alone.

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CONCLUSIONS In summary, immunogenic Fe3O4-R837 SPs have been designed for the effective treatment of metastatic cancer by combining PTT and PD-L1 checkpoint blockade therapy. With the help of an external magnetic field, Fe3O4-R837 SPs can directly destroy the tumors upon NIR irradiation, activating the immune system by inducing DCs’ maturation and secretion of cytokines. Further in combination with the PD-L1 checkpoint blockade therapy, Fe3O4-R837 SPs cannot only eradicate the primary tumors directly exposed to PTT but also prevent the lungs/liver metastasis and inhibit the pre-existing distant tumors left after PTT. Our studies show the great potential of integrating Fe3O4based PTT with checkpoint blockade therapy to realize a remarkable synergistic therapeutic outcome and broaden the application of cancer immunotherapy in vivo.

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EXPERIMENTAL SECTION

Materials. Iron acetylacetonate (Fe(acac)3, 99.9+%), benzyl ether (99%), oleic acid (OA, 90%), oleyamine (OLA, 70%), 1,2hexadecanediol (90%), Imiquimod (R837), and fluorescein diacetate (FD) were purchased from Sigma-Aldrich. Poly(ethylene glycol)-blockpoly(lactic-co-glycolic acid) copolymer (mPEG-PLGA, 50:50 w/w, Mw ∼ 2000:5000 Da) was purchased from Daigang Biomaterial Co., Ltd. Propidium iodide (PI) was purchased from Invitrogen. Dulbecco’s modified Eagle’s medium with fetal bovine serum and high glucose was purchased from Gibco. Chloroform and dimethyl sulfoxide (DMSO) were of analytical grade. Deionized water and absolute ethanol were used as received. Antiprogrammed death ligand 1 used in vivo was obtained from BioXCell (α-PD-L1, clone: 10F.9G2, catalog no. BE0101). Anti-CD11c (N418), anti-CD80 (16-10A1), anti-CD86 (GL-1), anti-CD16/32 (93), anti-CD45 (30-F11), anti-CD3ε (1452C11), anti-CD4 (RM4-5), anti-CD8a (53-6.7), anti-NKp46 (29A1.4), and anti-B220 (RA3-6B2) antibodies were purchased from Biolegend. Mouse interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ) ELISA Kit were purchased from Dakewe biotech. Preparation of OA-Stabilized Fe3O4 NPs. OA-stabilized Fe3O4 NPs were prepared by thermal decomposition route following the previous literature.54 Briefly, 2 mmol Fe(acac)3, 6 mmol OLA, 6 mmol OA, 5 mmol 1,2-hexadecanediol, and 20 mL of benzyl ether were mixed. The mixture was then cycled between vacuum and nitrogen three times K


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where L and D (mm) represented the tumor lengths of long and short axes, respectively. At the end of the experiments, mice were sacrificed and tumors were excised and weighed. Lungs and livers were harvested, observed for the tumor nodules, or sectioned and stained with H&E. Abscopal Effect on Bilateral 4T1 Model. BALB/c mice were injected with 1 × 106 4T1 cells into the right mammary fat pads (primary tumor) and 2 × 105 4T1 cells into the left mammary fat pads (distant tumor). When the primary tumor reached ∼200 mm3, mice were randomly divided into eight groups (n = 5): control, anti-PD-L1, Fe3O4 SPs, Fe3O4 SPs PTT, Fe3O4-R837 SPs, Fe3O4-R837 SPs PTT, Fe3O4R837 SPs plus anti-PD-L1, and Fe3O4-R837 SPs PTT plus anti-PD-L1. The mice were i.v. injected with the materials and attached to the magnets every 3 days for a total of three injections. After 24 h of each injection, mice were anesthetized and irradiated with NIR laser, followed by i.v. injection with PD-L1 antibody at a dose of 75 μg/mouse. The primary and distant tumor volumes and body weights were monitored over 24 days. After 24 days, all mice were sacrificed and both the primary and distant tumors were excised and weighed. The distant tumors were harvested, treated with 1 mg/mL collagenase I for 1 h, and ground with the end of a syringe rubber. Cells were filtered, incubated in red blood cell lysis buffer on ice for 5 min, and washed with PBS three times. To reduce nonspecific binding to fragment crystallizable receptors (FcRs), the single-cell suspension was incubated with anti-CD16/32.27 Cells were further stained with fluorochrome-conjugated antibodies: antiCD45, anti-CD3ε, anti-CD4, anti-CD8a, anti-NKp46, anti-B220, and PI and analyzed by flow cytometry. Blood was also collected, and the serum IL-6, TNF-α, and IFN-γ were determined by using ELISA kits to evaluate the acute inflammation evoked by the treatment. Characterization. Transmission electron microscopy (TEM) images were measured by a Hitachi H-800 electron microscope. Scanning electron microscopy (SEM) images were acquired with a SU8020 Hitachi Company scanning electron microscope. Dynamic light scattering (DLS) measurements were measured by Zetasizer NanoZS, Malvern Instruments. UV−vis absorption spectra were performed with Shimadzu 3600 UV−vis−IR spectrophotometer. Fourier-transform infrared (FTIR) spectra were obtained by Bruker IFS66 V instrument. T2 relaxation time was measured by a Bruker AVANCE III 500 NMR spectroscope. An 808 nm diode laser (LEO photonics Co. Ltd.) with tunable output power densities was employed to study the photothermal effect. Fluorescent images of 4T1 cells were obtained by an Olympus IX71 inverted fluorescence microscope. The Fe3+ concentration was assessed by PerkinElmer Optima 3300DV inductively coupled plasma optical emission (ICP-OES) measurements. Infrared thermal images were acquired by a Fluke infrared (IR) thermal camera. Flow cytometric analysis was implemented on the BD Biosciences FACSCalibur flow cytometer. T2-weighted MRI images were performed by GE Signa 1.5 T unit, General Electric, Milwaukee, WI. The photographs of mice and lungs/liver were taken by macroscopic digital imaging workstation for histopathology.

different power densities for different times. The cell viability was evaluated by MTT assay. For fluorescence imaging, the treated cells were fixed with 4% paraformaldehyde and stained with 1 μg/mL PI and FD to visualize living cells (green fluorescence) and cell apoptosis (red fluorescence). For flow cytometric analysis, treated cells were harvested, washed with ice-cold PBS three times, stained with Alexa Fluor 488Annexin V and PI in the dark for 20 min at room temperature, and then analyzed by flow cytometry. In Vitro DC Stimulation Transwell Experiments. DCs were isolated from the bone marrow of ∼8 week-old BALB/c mice. The mode of transwell is the cocultivation system, and the pore diameter is 0.4 μm. First, 1 × 105 4T1 cells were incubated with R837, Fe3O4 SPs, or Fe3O4R837 SPs in the upper compartment and irradiated with 808 nm NIR laser or not. The residues of 4T1 cells were added into the 5 × 105 DC culture in the lower compartment using the transwell system. After various treatments, DCs stained with anti-CD11c, anti-CD80, and antiCD86 antibody were analyzed by flow cytometry. The proinflammatory cytokines in DC medium suspensions, including IL-6 and TNF-α, were determined by ELISA kits following standard protocols. Magnetic Attraction-Enhanced Pharmacokinetics, Biodistribution, and MRI. The 4T1 tumor-bearing mice were intravenously (i.v.) administrated with Fe3O4-R837 SPs at a dose of 6 mg/kg and attached to the magnets. Animals were sacrificed (n = 3) at the indicated time points to collect the same dose of blood from each mouse. Then, the blood was immediately centrifuged at 3000 rpm for 5 min to harvest plasma samples and dissolved in aqua regia to analyze the total amount of Fe3+ with ICP-OES. Major organs and tumors were also collected after 24 h, and the concentration of Fe3+ was measured with ICP-OES. Before collecting the organs, the mice need cardiac perfusion with physiological saline and paraformaldehyde to remove the effects of blood in organs. The T2-weighted MRI images were acquired after 24 h p.i. using a 1.5 T human clinical scanner. The organs’ and tumors’ retention rates were calculated by the formula ID%/g = (drug content in organs /total amount of drug injections)/organ weight Apoptosis, DC Stimulation, and Cytokines Release in Vivo. Female BALB/c mice (6−8 weeks) were divided into groups randomly. The first 4T1 tumor model was planted by an orthotopic injection of 1 × 106 4T1 cells into the right mammary fat pads of the mice, and tumors were allowed to grow until ∼200 mm3 before experiments. Then, the mice were i.v. injected with Fe3O4 SPs/Fe3O4-R837 SPs at a dose of 6 mg/kg and attached to the magnets. According to the previous literature,21,27 the laser irradiation after 24 h of injection of NPs is an ideal condition that has been optimized and verified. In addition, according to the blood circulation kinetics of Fe3O4-R837 SPs in vivo and 24 h tumor retention rate, it can be seen that the high drug accumulation in the tumor area after 24 h is beneficial to maximize the efficiency of photothermal therapy. Therefore, 24 h after i.v. injection is chosen as the time point for further experiments. After 24 h, mice were anesthetized and tumors were irradiated with an 808 nm NIR laser at 0.33 W/cm2 for 30 min. Three days later, lymph nodes were excised and analyzed by flow cytometry. At the same time, blood was collected and the serum IL-6, TNF-α, and IFN-γ were determined by using ELISA kits to evaluate the acute inflammation evoked by the treatment. Antitumor and Anti-metastatic Activity on Orthotopic 4T1 Model. Tumor-bearing mice were randomly divided into four groups (n = 5): control, anti-PD-L1, Fe3O4-R837 SP PTT, and Fe3O4-R837 SP PTT plus anti-PD-L1. The mice were i.v. injected with the materials and attached to the magnets every 3 days three times. After 24 h of each injection, mice were anesthetized and irradiated with NIR laser, followed by i.v. injection with PD-L1 antibody at a dose of 75 μg/mouse. Tumor volumes were monitored and recorded over 24 days. The volume of tumor was calculated by the equation

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b05876. Additional structural characterizations, the encapsulation and loading efficiency, cellular uptake and efflux, the release of cytokine, the trend body weight of mice and serum biochemistry (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (D.Z.). *E-mail: [email protected] (Y.H.). *E-mail: [email protected]. Fax: +86 431 85193423 (H.Z.). ORCID

Yi Liu: 0000-0003-0548-6073 Hongchen Sun: 0000-0002-5572-508X

1 V = L × D2 2 L


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(16) Matsuo, Y.; Takeyama, H.; Guha, S. Cytokine Network: New Targeted Therapy for Pancreatic Cancer. Curr. Pharm. Des. 2012, 18, 2416−2419. (17) Frick, S. U.; Domogalla, M. P.; Baier, G.; Wurm, F. R.; Mailänder, V.; Landfester, K.; Steinbrink, K. Interleukin-2 Functionalized Nanocapsules for T Cell-Based Immunotherapy. ACS Nano 2016, 10, 9216− 9226. (18) Sabado, R. L.; Balan, S.; Bhardwaj, N. Dendritic Cell-Based Immunotherapy. Cell Res. 2017, 27, 74−95. (19) Kim, S.-Y.; Phuengkham, H.; Noh, Y.-W.; Lee, H.-G.; Um, S. H.; Lim, Y. T. Immune Complexes Mimicking Synthetic Vaccine Nanoparticles for Enhanced Migration and Cross-Presentation of Dendritic Cells. Adv. Funct. Mater. 2016, 26, 8072−8082. (20) Zhu, G.; Zhang, F.; Ni, Q.; Niu, G.; Chen, X. Efficient Nanovaccine Delivery in Cancer Immunotherapy. ACS Nano 2017, 11, 2387−2392. (21) Chen, Q.; Xu, L.; Liang, C.; Wang, C.; Peng, R.; Liu, Z. Photothermal Therapy with Immune-Adjuvant Nanoparticles Together with Checkpoint Blockade for Effective Cancer Immunotherapy. Nat. Commun. 2016, 7, No. 13193. (22) Carreno, B. M.; Magrini, V.; Becker-Hapak, M.; Kaabinejadian, S.; Hundal, J.; Petti, A. A.; Ly, A.; Lie, W.-R.; Hildebrand, W. H.; Mardis, E. R.; Linette, G. P. A Dendritic Cell Vaccine Increases the Breadth and Diversity of Melanoma Neoantigen-Specific T Cells. Science 2015, 348, 803−808. (23) Fang, R. H.; Hu, C.-M. J.; Luk, B. T.; Gao, W.; Copp, J. A.; Tai, Y.; O’Connor, D. E.; Zhang, L. Cancer Cell Membrane-Coated Nanoparticles for Anticancer Vaccination and Drug Delivery. Nano Lett. 2014, 14, 2181−2188. (24) Ledford, H. Immunotherapy’s Cancer Remit Widens. Nature 2013, 497, 544. (25) Wang, R.-F. A Special Issue on Cancer Immunotherapy. Cell Res. 2017, 27, 1−2. (26) Webster, R. M. The Immune Checkpoint Inhibitors: Where Are We Now? Nat. Rev. Drug Discovery 2014, 13, 883−884. (27) Duan, X.; Chan, C.; Guo, N.; Han, W.; Weichselbaum, R. R.; Lin, W. Photodynamic Therapy Mediated by Nontoxic Core-Shell Nanoparticles Synergizes with Immune Checkpoint Blockade To Elicit Antitumor Immunity and Antimetastatic Effect on Breast Cancer. J. Am. Chem. Soc. 2016, 138, 16686−16695. (28) Drake, C. G.; Jaffee, E.; Pardoll, D. M. Mechanisms of Immune Evasion by Tumors. Adv. Immunol. 2006, 90, 51−81. (29) Liu, Z.; Ravindranathan, R.; Kalinski, P.; Guo, Z. S.; Bartlett, D. L. Rational Combination of Oncolytic Vaccinia Virus and PD-L1 Blockade Works Synergistically to Enhance Therapeutic Efficacy. Nat. Commun. 2017, 8, No. 14754. (30) Taube, J. M.; Klein, A.; Brahmer, J. R.; Xu, H.; Pan, X.; Kim, J. H.; Chen, L.; Pardoll, D. M.; Topalian, S. L.; Anders, R. A. Association of PD-1, PD-1 Ligands, and Other Features of the Tumor Immune Microenvironment with Response to Anti-PD-1 Therapy. Clin. Cancer Res. 2014, 20, 5064−5074. (31) Errico, A. PD-1-PD-L1 Axis: Efficient Checkpoint Blockade Against Cancer. Nat. Rev. Clin. Oncol. 2015, 12, 63. (32) Topalian, S. L.; Hodi, F. S.; Brahmer, J. R.; Gettinger, S. N.; Smith, D. C.; McDermott, D. F.; Powderly, J. D.; Carvajal, R. D.; Sosman, J. A.; Atkins, M. B.; Leming, P. D.; Spigel, D. R.; Antonia, S. J.; Horn, L.; Drake, C. G.; Pardoll, D. M.; Chen, L.; Sharfman, W. H.; Anders, R. A.; Taube, J. M.; McMiller, T. L.; Xu, H.; Korman, A. J.; Jure-Kunkel, M.; Agrawal, S.; McDonald, D.; Kollia, G. D.; Gupta, A.; Wigginton, J. M.; Sznol, M. Safety, Activity, and Immune Correlates of Anti-PD-1 Antibody in Cancer. N. Engl. J. Med. 2012, 366, 2443−2454. (33) Tumeh, P. C.; Harview, C. L.; Yearley, J. H.; Shintaku, I. P.; Taylor, E. J.; Robert, L.; Chmielowski, B.; Spasic, M.; Henry, G.; Ciobanu, V.; West, A. N.; Carmona, M.; Kivork, C.; Seja, E.; Cherry, G.; Gutierrez, A. J.; Grogan, T. R.; Mateus, C.; Tomasic, G.; Glaspy, J. A.; Emerson, R. O.; Robins, H.; Pierce, R. H.; Elashoff, D. A.; Robert, C.; Ribas, A. PD-1 Blockade Induces Responses by Inhibiting Adaptive Immune Resistance. Nature 2014, 515, 568−571.

Hao Zhang: 0000-0002-2373-1100 Bai Yang: 0000-0002-3873-075X Author Contributions

H.Z. proposed and supervised the project. H.Z., R.G., X.Z., Y.L., H.S., D.Z., Y.H., and B.Y. designed and performed the experiments and co-wrote the manuscript. C.L., B.L., J.L., and W.W. participated in most experiments. All authors discussed the results and commented on the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NSFC (51603084 and 51425303), JLU Science and Technology Innovative Research Team 2017TD-06, and the Special Project from MOST of China.

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REFERENCES

(1) Siegel, R. L.; Miller, K. D.; Jemal, A. Cancer Statistics, 2016. CA Cancer J. Clin. 2016, 66, 7−30. (2) Couzin-Frankel, J. Cancer Immunotherapy. Science 2013, 342, 1432−1433. (3) Gotwals, P.; Cameron, S.; Cipolletta, D.; Cremasco, V.; Crystal, A.; Hewes, B.; Mueller, B.; Quaratino, S.; Sabatos-Peyton, C.; Petruzzelli, L.; Engelman, J. A.; Dranoff, G. Prospects for Combining Targeted and Conventional Cancer Therapy with Immunotherapy. Nat. Rev. Cancer 2017, 17, 286−301. (4) Jiang, W.; von Roemeling, C. A.; Chen, Y.; Qie, Y.; Liu, X.; Chen, J.; Kim, B. Y. S. Designing Nanomedicine for Immuno-Oncology. Nat. Biomed. Eng. 2017, 1, No. 0029. (5) Shao, K.; Singha, S.; Clemente-Casares, X.; Tsai, S.; Yang, Y.; Santamaria, P. Nanoparticle-Based Immunotherapy for Cancer. ACS Nano 2015, 9, 16−30. (6) Rosenberg, S. A.; Yang, J. C.; Restifo, N. P. Cancer Immunotherapy: Moving Beyond Current Vaccines. Nat. Med. 2004, 10, 909−915. (7) Grivennikov, S. I.; Greten, F. R.; Karin, M. Immunity, Inflammation, and Cancer. Cell 2010, 140, 883−899. (8) Irvine, D. J.; Hanson, M. C.; Rakhra, K.; Tokatlian, T. Synthetic Nanoparticles for Vaccines and Immunotherapy. Chem. Rev. 2015, 115, 11109−11146. (9) Curran, K. J.; Brentjens, R. J. Chimeric Antigen Receptor T cells for Cancer Immunotherapy. J. Clin. Oncol. 2015, 33, 1703−1706. (10) Kalos, M.; June, C. H. Adoptive T Cell Transfer for Cancer Immunotherapy in the Era of Synthetic Biology. Immunity 2013, 39, 49−60. (11) Restifo, N. P.; Dudley, M. E.; Rosenberg, S. A. Adoptive Immunotherapy for Cancer: Harnessing the T Cell Response. Nat. Rev. Immunol. 2012, 12, 269−281. (12) Gubin, M. M.; Zhang, X.; Schuster, H.; Caron, E.; Ward, J. P.; Noguchi, T.; Ivanova, Y.; Hundal, J.; Arthur, C. D.; Krebber, W.-J.; Mulder, G. E.; Toebes, M.; Vesely, M. D.; Lam, S. S.; Korman, A. J.; Allison, J. P.; Freeman, G. J.; Sharpe, A. H.; Pearce, E. L.; Schumacher, T. N.; Aebersold, R.; Rammensee, H. G.; Melief, C. J.; Mardis, E. R.; Gillanders, W. E.; Artyomov, M. N.; Schreiber, R. D. Checkpoint Blockade Cancer Immunotherapy Targets Tumour-Specific Mutant Antigens. Nature 2014, 515, 577−581. (13) Pardoll, D. M. The Blockade of Immune Checkpoints in Cancer Immunotherapy. Nat. Rev. Cancer 2012, 12, 252−264. (14) Wang, C.; Sun, W.; Wright, G.; Wang, A. Z.; Gu, Z. InflammationTriggered Cancer Immunotherapy by Programmed Delivery of CpG and Anti-PD1 Antibody. Adv. Mater. 2016, 28, 8912−8920. (15) Wang, D.; Wang, T.; Liu, J.; Yu, H.; Jiao, S.; Feng, B.; Zhou, F.; Fu, Y.; Yin, Q.; Zhang, P.; Zhang, Z.; Zhou, Z.; Li, Y. Acid-Activatable Versatile Micelleplexes for PD-L1 Blockade-Enhanced Cancer Photodynamic Immunotherapy. Nano Lett. 2016, 16, 5503−5513. M


Research Article


Feasible Approach for Clinics. J. Am. Chem. Soc. 2015, 137, 15145− 15151. (47) Zhang, S.; Sun, C.; Zeng, J.; Sun, Q.; Wang, G.; Wang, Y.; Wu, Y.; Dou, S.; Gao, M.; Li, Z. Ambient Aqueous Synthesis of Ultrasmall PEGylated Cu2‑xSe Nanoparticles as a Multifunctional Theranostic Agent for Multimodal Imaging Guided Photothermal Therapy of Cancer. Adv. Mater. 2016, 28, 8927−8936. (48) Shen, S.; Wang, S.; Zheng, R.; Zhu, X.; Jiang, X.; Fu, D.; Yang, W. Magnetic Nanoparticle Clusters for Photothermal Therapy with NearInfrared Irradiation. Biomaterials 2015, 39, 67−74. (49) Lin, L.-S.; Cong, Z.-X.; Cao, J.-B.; Ke, K.-M.; Peng, Q.-L.; Gao, J.; Yang, H.-H.; Liu, G.; Chen, X. Multifunctional Fe3O4@Polydopamine Core-Shell Nanocomposites for Intracellular mRNA Detection and Imaging-Guided Photothermal Therapy. ACS Nano 2014, 8, 3876− 3883. (50) Ge, R.; Li, X.; Lin, M.; Wang, D.; Li, S.; Liu, S.; Tang, Q.; Liu, Y.; Jiang, J.; Liu, L.; Sun, H.; Zhang, H.; Yang, B. Fe3O4@Polydopamine Composite Theranostic Superparticles Employing Preassembled Fe3O4 Nanoparticles as the Core. ACS Appl. Mater. Interfaces 2016, 8, 22942− 22952. (51) Xia, Y.; Tang, Z. Monodisperse Inorganic Supraparticles: Formation Mechanism, Properties and Applications. Chem. Commun. 2012, 48, 6320−6336. (52) Zhuang, J.; Wu, H.; Yang, Y.; Cao, Y. C. Controlling Colloidal Superparticle Growth Through Solvophobic Interactions. Angew. Chem., Int. Ed. 2008, 47, 2208−2212. (53) Bai, F.; Wang, D.; Huo, Z.; Chen, W.; Liu, L.; Liang, X.; Chen, C.; Wang, X.; Peng, Q.; Li, Y. A Versatile Bottom-up Assembly Approach to Colloidal Spheres from Nanocrystals. Angew. Chem., Int. Ed. 2007, 46, 6650−6653. (54) Sun, S.; Zeng, H. Size-Controlled Synthesis of Magnetite Nanoparticles. J. Am. Chem. Soc. 2002, 124, 8204−8205. (55) Wang, S.; Huang, P.; Chen, X. Hierarchical Targeting Strategy for Enhanced Tumor Tissue Accumulation/Retention and Cellular Internalization. Adv. Mater. 2016, 28, 7340−7364. (56) Rohrer, M.; Bauer, H.; Mintorovitch, J.; Requardt, M.; Weinmann, H.-J. Comparison of Magnetic Properties of MRI Contrast Media Solutions at Different Magnetic Field Strengths. Invest. Radiol. 2005, 40, 715−724. (57) Miller, R. L.; Gerster, J. F.; Owens, M. L.; Slade, H. B.; Tomai, M. A. Imiquimod Applied Topically: a Novel Immune Response Modifier and New Class of Drug. Int. J. Immunopharmacol. 1999, 21, 1−14. (58) Kim, H. J.; Lee, S.-M.; Park, K.-H.; Mun, C. H.; Park, Y.-B.; Yoo, K.-H. Drug-loaded Gold/Iron/Gold Plasmonic Nanoparticles for Magnetic Targeted Chemo-photothermal Treatment of Rheumatoid Arthritis. Biomaterials 2015, 61, 95−102. (59) Wong, C. W.; Song, C.; Grimes, M. M.; Fu, W.; Dewhirst, M. W.; Muschel, R. J.; Al-Mehdi, A.-B. Intravascular Location of Breast Cancer Cells after Spontaneous Metastasis to the Lung. Am. J. Pathol. 2002, 161, 749−753. (60) Aslakson, C. J.; Miller, F. R. Selective Events in the Metastatic Process Defined by Analysis of the Sequential Dissemination of Subpopulations of a Mouse Mammary Tumor. Cancer Res. 1992, 52, 1399−1405.

(34) Rizvi, N. A.; Hellmann, M. D.; Snyder, A.; Kvistborg, P.; Makarov, V.; Havel, J. J.; Lee, W.; Yuan, J.; Wong, P.; Ho, T. S.; Miller, M. L.; Rekhtman, N.; Moreira, A. L.; Ibrahim, F.; Bruggeman, C.; Gasmi, B.; Zappasodi, R.; Maeda, Y.; Sander, C.; Garon, E. B.; Merghoub, T.; Wolchok, J. D.; Schumacher, T. N.; Chan, T. A. Mutational Landscape Determines Sensitivity to PD-1 Blockade in Non-Small Cell Lung Cancer. Science 2015, 348, 124−128. (35) He, C.; Duan, X.; Guo, N.; Chan, C.; Poon, C.; Weichselbaum, R. R.; Lin, W. Core-Shell Nanoscale Coordination Polymers Combine Chemotherapy and Photodynamic Therapy to Potentiate Checkpoint Blockade Cancer Immunotherapy. Nat. Commun. 2016, 7, No. 12499. (36) Zheng, D.-W.; Chen, J.-L.; Zhu, J.-Y.; Rong, L.; Li, B.; Lei, Q.; Fan, J.-X.; Zou, M.-Z.; Li, C.; Cheng, S.-X.; Xu, Z.; Zhang, X.-Z. Highly Integrated Nano-Platform for Breaking the Barrier between Chemotherapy and Immunotherapy. Nano Lett. 2016, 16, 4341−4347. (37) Bauleth-Ramos, T.; Shahbazi, M.-A.; Liu, D.; Fontana, F.; Correia, A.; Figueiredo, P.; Zhang, H.; Martins, J. P.; Hirvonen, J. T.; Granja, P.; Sarmento, B.; Santos, H. A. Nutlin-3a and Cytokine Co-loaded Spermine-Modified Acetalated Dextran Nanoparticles for Cancer Chemo-Immunotherapy. Adv. Funct. Mater. 2017, 27, No. 1703303. (38) Pelaz, B.; Alexiou, C.; Alvarez-Puebla, R. A.; Alves, F.; Andrews, A. M.; Ashraf, S.; Balogh, L. P.; Ballerini, L.; Bestetti, A.; Brendel, C.; Bosi, S.; Carril, M.; Chan, W. C.; Chen, C.; Chen, X.; Chen, X.; Cheng, Z.; Cui, D.; Du, J.; Dullin, C.; Escudero, A.; Feliu, N.; Gao, M.; George, M.; Gogotsi, Y.; Grunweller, A.; Gu, Z.; Halas, N. J.; Hampp, N.; Hartmann, R. K.; Hersam, M. C.; Hunziker, P.; Jian, J.; Jiang, X.; Jungebluth, P.; Kadhiresan, P.; Kataoka, K.; Khademhosseini, A.; Kopecek, J.; Kotov, N. A.; Krug, H. F.; Lee, D. S.; Lehr, C. M.; Leong, K. W.; Liang, X. J.; Ling Lim, M.; Liz-Marzan, L. M.; Ma, X.; Macchiarini, P.; Meng, H.; Mohwald, H.; Mulvaney, P.; Nel, A. E.; Nie, S.; Nordlander, P.; Okano, T.; Oliveira, J.; Park, T. H.; Penner, R. M.; Prato, M.; Puntes, V.; Rotello, V. M.; Samarakoon, A.; Schaak, R. E.; Shen, Y.; Sjoqvist, S.; Skirtach, A. G.; Soliman, M. G.; Stevens, M. M.; Sung, H. W.; Tang, B. Z.; Tietze, R.; Udugama, B. N.; VanEpps, J. S.; Weil, T.; Weiss, P. S.; Willner, I.; Wu, Y.; Yang, L.; Yue, Z.; Zhang, Q.; Zhang, Q.; Zhang, X. E.; Zhao, Y.; Zhou, X.; Parak, W. J. Diverse Applications of Nanomedicine. ACS Nano 2017, 11, 2313−2381. (39) Zhou, F.; Wu, S.; Song, S.; Chen, W. R.; Resasco, D. E.; Xing, D. Antitumor Immunologically Modified Carbon Nanotubes for Photothermal Therapy. Biomaterials 2012, 33, 3235−3242. (40) Tao, Y.; Ju, E.; Ren, J.; Qu, X. Immunostimulatory Oligonucleotides-Loaded Cationic Graphene Oxide with Photothermally Enhanced Immunogenicity for Photothermal/Immune Cancer Therapy. Biomaterials 2014, 35, 9963−9971. (41) Guo, L.; Yan, D. D.; Yang, D.; Li, Y.; Wang, X.; Zalewski, O.; Yan, B.; Lu, W. Combinatorial Photothermal and Immuno Cancer Therapy Using Chitosan-Coated Hollow Copper Sulfide Nanoparticles. ACS Nano 2014, 8, 5670−5681. (42) He, J.; Huang, X.; Li, Y.-C.; Liu, Y.; Babu, T.; Aronova, M. A.; Wang, S.; Lu, Z.; Chen, X.; Nie, Z. Self-Assembly of Amphiphilic Plasmonic Micelle-Like Nanoparticles in Selective Solvents. J. Am. Chem. Soc. 2013, 135, 7974−7984. (43) Wang, J.; Liu, J.; Liu, Y.; Wang, L.; Cao, M.; Ji, Y.; Wu, X.; Xu, Y.; Bai, B.; Miao, Q.; Chen, C.; Zhao, Y. Gd-Hybridized Plasmonic AuNanocomposites Enhanced Tumor-Interior Drug Permeability in Multimodal Imaging-Guided Therapy. Adv. Mater. 2016, 28, 8950− 8958. (44) Du, Y.; Jiang, Q.; Beziere, N.; Song, L.; Zhang, Q.; Peng, D.; Chi, C.; Yang, X.; Guo, H.; Diot, G.; Ntziachristos, V.; Ding, B.; Tian, J. DNA-Nanostructure-Gold-Nanorod Hybrids for Enhanced In Vivo Optoacoustic Imaging and Photothermal Therapy. Adv. Mater. 2016, 28, 10000−10007. (45) Li, Y.; Shi, Q.; Zhang, P.; Xiahou, Y.; Li, S.; Wang, D.; Xia, H. Empirical Structural Design of Core@Shell Au@Ag Nanoparticles for SERS Applications. J. Mater. Chem. C 2016, 4, 6649−6656. (46) Riedinger, A.; Avellini, T.; Curcio, A.; Asti, M.; Xie, Y.; Tu, R.; Marras, S.; Lorenzoni, A.; Rubagotti, S.; Iori, M.; Capponi, P. C.; Versari, A.; Manna, L.; Seregni, E.; Pellegrino, T. Post-Synthesis Incorporation of 64Cu in CuS Nanocrystals to Radiolabel Photothermal Probes: A N


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