Iron Nanoparticles for Low-Power Local Magnetic Hyperthermia in

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Letter Cite This: Nano Lett. 2019, 19, 4287−4296

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Iron Nanoparticles for Low-Power Local Magnetic Hyperthermia in Combination with Immune Checkpoint Blockade for Systemic Antitumor Therapy Yu Chao, Guobin Chen, Chao Liang, Jun Xu, Ziliang Dong, Xiao Han, Chao Wang, and Zhuang Liu* Institute of Functional Nano and Soft Materials (FUNSOM), Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou, Jiangsu 215123, China Downloaded via UNIV OF SOUTHERN INDIANA on July 18, 2019 at 10:48:44 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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ABSTRACT: Magnetic hyperthermia (MHT) utilizing heat generated by magnetic nanoparticles under alternating magnetic field (AMF) is an effective local tumor ablation method but can hardly treat metastatic tumors. In this work, we discover that pure iron nanoparticles (FeNPs) with high magnetic saturation intensity after being modified by biocompatible polymers are stable in aqueous solution and could be employed as a supereffective MHT agent to generate sufficient heating under a lowpower AFM. Effective MHT ablation of tumors is then achieved, using either locally injected FeNPs or intravenously injected FeNPs with the help of locally applied tumor-focused constant magnetic field to enhance the tumor accumulation of those nanoparticles. We further demonstrate that the combination of FeNP-based MHT with local injection of nanoadjuvant and systemic injection of anticytotoxic Tlymphocyte antigen-4 (anti-CTLA4) checkpoint blockade would result in systemic therapeutic responses to inhibit tumor metastasis. A robust immune memory effect to prevent tumor recurrence is also observed after the combined MHT-immunotherapy. This work not only highlights that FeNPs with appropriate surface modification could act as a supereffective MHT agent but also presents the great promises of combining MHT with immunotherapy to achieve long-lasting systemic therapeutic outcome after local treatment. KEYWORDS: Iron nanoparticles, magnetic hyperthermia therapy, low power, immune checkpoint blockade

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oxidation in the aqueous solution.16 To stabilize FeNPs, surface passivation by coating the iron core with an inorganic shell (e.g., gold or iron oxide) has been explored by many groups to fabricate iron-containing core−shell nanoparticles for diverse biomedical applications.17−19 Nevertheless, the use of pure FeNPs for in vivo MHT ablation of tumors has been rarely reported to our best knowledge. Cancer immunotherapy20 such as immune checkpoint blockade (ICB),21 by activating the patient’s own immune system to attack tumor cells has demonstrated tremendous promises in recent years.22 Recently, many research groups including ours have discovered that cancer cell debris generated after various types of local tumor treatment (e.g., photothermal therapy,23−25 photodynamic therapy,26−28 radiotherapy29−31) could trigger robust systemic antitumor immune responses, particularly with the help of immune-adjuvant and in combination with ICB therapy.32 However, the combination of MHT therapy with ICB immunotherapy has not yet been explored by surveying the literature.

agnetic hyperthermia (MHT) therapy is a kind of technology which can treat a tumor with heat generated by magnetic nanoparticles under a strong alternating magnetic field (AMF).1,2 Unlike widely explored photothermal hyperthermia using light to heat up tumors, MHT can deal with deep tumors in various organs because of the excellent tissue penetration ability of AMF.3,4 In practice, magnetic nanoparticles are directly administrated into the tumor so that only the tumor would be heated within the AMF.5,6 Although MHT has already been tested in the clinic as a minimally invasive method for local treatment of solid tumors,7 there are a number of limitations for current MHT.8 While superparamagnetic iron oxide nanoparticles (SPIONs) with high biocompatibility are often used as the MHT agent,9 the magnetic heating efficiency determined by the specific absorption rate (SAR) of SPIONs is poor because of the low inherent saturation magnetization (Ms) (60 emu/g) of iron oxide.10 Therefore, local injection of SPIONs with high concentrations together with high-power AMF (Happl × fappl > 3 × 109 A m−1 s−1) is often required for effective MHT ablation of tumors.11,12 Moreover, as a local heating treatment, MHT therapy can hardly manage the spreading metastatic tumors.13 Nanoparticles of pure iron (FeNPs) may be an effective MHT agent as the Ms of metal iron at 218 emu/g is much higher than that of iron oxide.14,15 However, iron is easily susceptible to © 2019 American Chemical Society

Received: February 8, 2019 Revised: May 23, 2019 Published: May 27, 2019 4287

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Figure 1. Preparation and characterization of PEGylated FeNPs. (a) Schematic illustration for the synthesis and surface modification of PEGylated FeNPs. (b) A TEM image of PEGylated FeNPs. (c) The corresponding magnetic hysteresis loops at T = 300 K for FeNPs and PEGylated FeNPs. (d) Temperature-dependent magnetization of FeNPs, IONC, CIONP, and IONP. (e) SAR measured at 100 kHz of FeNPs, IONC, CIONP and IONP. (f,g) Real time in vivo IR thermal imaging and temperature rise curves of FeNPs, IONC, CIONP, and IONP.

demonstrated in multiple tumor models. Thus, our work not only presents PEGylated FeNPs as a promising therapeutic candidate with great performance for MHT therapy but also claims a new strategy by combining MHT with ICB immunotherapy to achieve whole-body therapeutic responses after local MHT treatment. Preparation and Characterization of PEGylated FeNPs. FeNPs were synthesized by a chemical reduction method from iron chloride (FeCl3) reduced by sodium borohydride (NaBH4) under room temperature following a literature protocol.33,34 In this method, the growth of FeNPs would happen on the surface of bubbles constructed by sodium borohydride and foaming agent F127 (Figure 1a). Such a hubble-bubble synthetic procedure protected the whole synthesis from traditionally employed harsh conditions. Through this method, we were able to obtain a large amount of FeNPs product (about 1 g) from a single synthesis (Figure S1). Afterward, a polymer by cografting poly(acrylic acid) (PAA) with DA and amine-terminated PEG (5 kDa) was synthesized and used to modify as-made FeNPs. In this system, DA with two phenol groups could strongly coordinate to the Fe atom on the surface of FeNPs by Fe−O bonds. Such PEG surface modification was able to stabilize FeNPs in various physiological solutions and prevent their aggregation (Figure S2). Impor-

In this work, pure iron nanoparticles (FeNP) are prepared by a chemical reduction method33,34 and then functionalized with polyethylene glycol (PEG)/dopamine (DA) cografted polymer.35 The obtained PEGylated FeNPs could be well dispersed in aqueous solutions with good stability and may be stored in the lyophilized form with a long shelf life before being redispersed and used. Such PEGylated FeNPs show much higher Ms and SAR than other types of commonly used magnetic nanomaterials including our homemade iron oxide nanoparticles (IONPs), iron oxide nanoclusters (IONCs), and commercial SPIONs (USPIO-30).36 After local injection of our FeNPs into tumors, effective MHT heating of tumors could be achieved under AMF with a relative low power at the Happl of 12 kA m−1 and fappl of 100 kHz (Happl × fappl = 1.2 × 109 A m−1 s−1), resulting in the complete ablation of those tumors. Meanwhile, those PEGylated FeNPs after intravenous (i.v.) injection under magnetic tumor targeting show highly specific tumor accumulation, which would be sufficient for effective MHT tumor ablation. We further combine such FeNP-based MHT with locally applied nanoadjuvant and systemically administrated anti-Cytotoxic Tlymphocyte associated protein 4 (anti-CTLA4) checkpoint blockade antibody. The vaccine-like systemic immune responses triggered by such MHT-immunotherapy are able to effectively inhibit tumor metastasis and prevent tumor recurrence, as 4288

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Figure 2. In vivo magnetic hyperthermia (MHT). (a) Schematic illustration of in vivo MHT post local injection of FeNPs. (b) Photographs of the magnetorheological machine. (c,d) Real time in vivo IR thermal imaging (c) and temperature rise curves (d) of mice under AMF. (e,f) Tumor growth curves (e) and mouse survival data (f) of different groups of mice after various treatments indicated (n = 6). (g) Schematic illustration of in vivo MHT post systemic injection with the help of magnetic tumor targeting. (h) Optical fluorescence imaging of Cy5.5 labeled FeNPs through i.v. injection. The right side of tumor was attached with a magnet (M+) post injection of FeNPs. (i) Blood circulation curve profile of Cy5.5-labeled FeNPs in mice post i.v. injection. (j) Biodistribution profile of FeNPs in mice. (k,l) Real time in vivo IR thermal imaging and tumor temperature rise curves of mice. (m,n) Tumor growth curves and percent survival after various treatments as indicated (n = 5). FeNPs used here were PEGylated nanoparticles. Statistical significance was calculated via one-way ANOVA with a Tukey posthoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

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abnormality (Figure 2e,f, Figure S11). Our results suggest that PEGylated FeNPs could serve as an extremely effective MHT agent for tumor ablation under a low-power AMF. MHT treatments were typically carried out through local injection of magnetic nanoparticles directly into tumors, mainly owing to the limited magnetic heating efficiency of conventionally used iron oxide nanoparticles, as well as the insufficient tumor accumulation of MHT nanoagents post systemic i.v. injection. FeNPs with super high Ms and SAR may solve this difficult problem. 4T1 cells were inoculated on both flanks of each mouse, which was injected with PEGylated FeNPs (4 mg/ mL, 200 μL) labeled with a near-infrared (NIR) fluorescence dye, Cy5.5. After that, a neodymium magnet (NE036) was placed nearby one tumor on each mouse for 6 h (Figure 2g). The mice were imaged by an IVIS in vivo fluorescence imaging system. Interestingly, we found that Cy5.5 fluorescence signals from FeNPs in tumors with the magnet attached were 3-fold higher than that in tumors without magnetic targeting (Figure 2h). Quantitative analysis by measuring Fe levels in blood and other organs using inductively coupled plasma mass spectrometer (ICP-MS) further confirmed the prolonged blood half-life of PEGylated FeNPs, as well as the significantly enhanced accumulation of FeNPs in tumors with magnetic targeting (Figure 2i,j); the tumor uptake of FeNPs increased from 4.7 ± 0.6% of injected dose per gram tissue (% ID/g) to as high as 15.8 ± 0.6% ID/g with the help of tumor-focused magnetic targeting. Owning to the magnetic-targeting (MT)-enhanced tumor uptake of FeNPs, in vivo magnetic thermal therapy through i.v. injection of PEGylated FeNPs were carried out. Mice bearing 4T1 tumors were randomly divided into five groups (n = 6): 1, Saline; 2, saline + AMF; 3, FeNP; 4, FeNP + AMF; and 5, FeNP + AMF + MT. In group 5, the tumor on the right flank of each mouse was attached with a magnet. FeNPs were i.t. injected at doses of 50 μg per mouse. At 24 h post nanoparticle injection, tumors were also heated by the same portable device and imaged by infrared imaging camera for groups 2, 4, and 5. Temperature of tumors with magnetic targeting under AMF could rise to ∼50−52 °C, much higher than that without targeting (Figure 2k,l). Therefore, tumors could be completely ablated by magnetic-targeting-assisted MHT using i.v. injected PEGylated FeNPs as the MHT agent, and all treated mice survived for over 60 days after treatment (Figure 2m,n, Figure S12). By contrast, the tumor growth of mice in the other four groups, including the group with i.v. injection of FeNP−PEG and AMF treatment but no tumor-focused magnetic targeting, showed no obvious delay. Generally speaking, PEGylated FeNPs with strong magnetism and long circulation time provided the possibility of magnetic tumor targeting to enable in vivo MHT ablation of tumors through intravenous injection of those nanoparticles. In vivo MHT-Immunotherapy to Achieve the Abscopal Effect for the CT26 Tumor Model. It has been reported that local tumor therapy (e.g., by photothermal therapy,23−25 photodynamic therapy,26,27,39 or radiotherapy29−31) by triggering immunogenic cell death would provide tumor-associated antigens to induce tumor-specific immune responses.40 The combination of those local tumor therapies with immunotherapy (e.g., ICB therapy) could not only destroy local solid tumors but also help to attack distant metastatic tumors as an abscopal effect. Therefore, we tested the combination of FeNP-based MHT therapy with immunotherapy in the following studies. In order to boost the immunogenicity of tumor-associated antigens in tumor debris after MHT, we encapsulated imiquimod41 (R837), a toll-like receptor 7 agonist as a clinically approved

tantly, those PEGylated FeNPs could be stored in the form of lyophilized powder for months and used later after being redispersed in aqueous solution. We then carefully characterized the obtained PEGylated FeNPs. As observed under transmission electron microscopy (TEM) (Figure 1b), the obtained FeNP−PEG nanoparticles with individual sizes at 30−50 nm showed worm-like aggregated structure. The structure of a single particle was characterized by TEM imaging at a higher resolution (Figure S3). As determined by dynamic light scattering (DLS), the hydrodynamic size of PEGylated FeNP−PEG was about 100 nm, indicating that those ferromagnetic nanoparticles may form small aggregates owing to their interparticle magnetic interactions (Figure S4). The zeta potential of as-made FeNPs was negative before modification and turned into neutral after modification by PEG (Figure S5). X-ray diffraction (XRD) showed that the structure of asobtained FeNPs to be amorphous (Figure S6). Furthermore, Xray photoelectron spectroscopy (XPS) results confirmed that the obtained nanoparticles were made up of iron (0) and were not oxidized (Figure S7a). XPS data of FeNPs stored in the form of lyophilized powder for 60 days also confirmed the feasibility of freeze-drying preservation to prevent oxidization of those PEGylated FeNPs by air. In addition, quantitative energy dispersive spectrometer (EDS) analysis of FeNPs with or without PEGylation after incubation in aqueous solution for 1 h indicated that the DA−PAA−PEG polymer coating could effectively prevent oxidation of FeNPs (Figure S7b). The magnetic properties of PEGylated FeNPs were then studied. Those FeNP−PEG nanoparticles showed a slender hysteresis loop, suggesting their ferromagnetic property (Figure 1c). The Ms and SAR of FeNP−PEG were much higher than other magnetic nanoparticles including iron oxide nanoparticles (IONPs), iron oxide nanoclusters (IONCs), and commercial iron oxide nanoparticles (USPIO-30) (Figure 1d,e). Afterward, the magnetic heating efficiency was evaluated under thermal imaging by placing virus samples inside a magnetic coil with AMF. Notably, the temperature of the FeNP−PEG sample at 2 mg/mL increased by as much as 45 °C in 5 min in marked contrast to 0.5 °C, 6.6 °C, and 13.3 °C of temperature increase for IONPs, USPIO-30, and IONCs, respectively (Figure 1f,g). Importantly, the power of the magnetorheological machine as just a portable device used in this work was quite low at the Happl of 12 kA m−1 and fappl of 100 kHz (Happl × fappl = 1.2 × 109 A m−1 s−1). Furthermore, FeNP−PEG showed a concentrationdependent MHT heating effect (Figure S8). In Vivo MHT Tumor Ablation with PEGylated FeNPs Post Local Injection or Systemic Injection. Being aware of the high magnetic-induced heating efficiency of PEGylated FeNPs, the therapeutic efficacy of in vivo MHT was evaluated (Figure 2a). First, those FeNPs with surface PEG coating showed no appreciable in vitro cytotoxicity to cells (Figure S9). The instrument we used for in vivo experiments was also a portable device and its introduction heating power (Happl × fappl = 1.2 × 109 A m−1 s−1) was pretty low compared to that of previously used devices in literature (Happl × fappl > 3 × 109 A m−1 s−1) (Figure 2a and Figure S10).37,38 Mice bearing 4T1 tumors were intratumorally (i.t.) injected with saline or PEGylated FeNPs (2 mg/mL, 25 μL) and the tumors were put into the magnetic coil for 5 min. The temperature of tumors injected with FeNP−PEG increased by 30 °C and maintained at 55 °C for 3 min (Figure 2c,d). After MHT treatment with FeNPs, tumors on those mice could be completely ablated, and those mice survived for 60 days after treatment without any death or 4290

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Figure 3. In vivo MHT-immunotherapy to achieve the abscopal effect for the CT26 tumor model. (a) Schematic illustration of MHT plus CTLA4 blockade to inhibit tumor growth at distant sites. (b,c) Tumor growth curves of primary and distant tumors after various treatments as indicated on CT26 tumor model (n = 6). (d) Percent survival of different groups of tumors after various treatments as indicated on CT26 tumor model (n = 6). (e) Representative flow cytometry plots showing different groups of T cells in secondary tumors. Tumor cell suspensions were analyzed by flow cytometry for T-cell infiltration (gated on CD3+ T cells) of CD4+CD8+ T cells. (f) Representative flow cytometry plots showing percentages (gated on CD4+ cells) of CD4+FoxP3+ T cells in secondary tumors after various treatments indicated. (g,h) Proportions of tumor-infiltrating CD8+ killer T cells and CD4+ FoxP3+ regulatory T cells within the tumor. (i) CD8+ CTLs: Tregs ratios in the secondary tumors upon various treatments to remove the first tumors. (j) Cytokine levels in sera from mice isolated at 1, 5, and 9 d post different treatments. Statistical significance was calculated via one-way ANOVA with a Tukey posthoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

nanoadjuvant would impose risks in cytokine storms and thus local injection of immune adjuvant is generally preferred for tumor immunotherapy.42 Therefore, we chose local injection of the mixture of FeNPs and PR nanoadjuvant directly into tumors for the MHT-immunotherapy treatment. According to our

immune adjuvant molecule, into poly(lactic-co-glycolic) acid (PLGA), obtaining PLGA-R837 (PR) nanoparticles as a robust nanoscale immune-adjuvant.25 Although MHT ablation of tumors could be realized by i.v. injected FeNPs with the help of magnetic tumor targeting, systemic administration of 4291

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Figure 4. Immune-memory effect. (a) The experimental design to evaluate the immune memory effect post MHT-immunotherapy. (b,c) Tumor growth curves of different groups after various treatments as indicated (n = 5). (d) Percent survival of different groups after various treatments as indicated (n = 5). (e) Proportions of effector memory T cells (TEM) in the spleen analyzed by flow cytometry (gated on CD3+CD8+T cells) at day 40, right before rechallenging mice with secondary tumors. (f) Cytokine levels in sera from mice isolated 5 days after mice were rechallenged with secondary tumors. (g) The mechanism of antitumor immune responses induced by FeNP/PR-based MHT in combination with checkpoint-blockade. Statistical significance was calculated via one-way ANOVA with a Tukey posthoc test. *P < 0.05; **P < 0.01; ***P < 0.001. 4292

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7 (when they ∼50 mm3 in size) to test the tumor-infiltrating CD8+ (CD3+CD4−CD8+) and CD4+ (CD3+CD4+CD8−) T cells by flow cytometry (n = 5) (Figure 3e,f). The magnetic thermal ablation of primary tumors indeed resulted in obvious infiltration of both CD8+ and CD4+ T cells in the abscopal tumors, especially in combination with immune-adjuvant PR (Figure 3g and Figure S14). However, the percentages of Treg cells (CD3+CD4+Foxp3+), which are immunosuppressive T cells, also increased substantially post MHT (Figure 3h). Notably, the CTLA-4 blockade could effectively suppress the percentages of Tregs in the respective groups. As a result, the percentage of CD8+ CTLs, as well as the ratio of CTLs/Tregs (Figure 3i), appeared to be the highest in group 7 posting the combined MHT plus PR plus anti-CTLA4 therapy. Importantly, the percentage of cytotoxic CD8+ T cells with IFN-γ secretion was also found to be the highest in group 7 (Figure S15). Furthermore, remarkable infiltration of CD8+ T cells and decrease of Tregs within distant tumors in group 7 were observed by immunohistochemistry staining (Figure S16). In addition to T cells in the tumors, cytokines including TNF-α and IFN-γ in the serum samples of all mice on day 1, 5, and 9 were tested by enzyme-linked immuno sorbent assay (ELISA). Both TNF-α and IFN-γ levels in group 7 were the highest among all groups (Figure 4jand Figure S17), further indicating the robust immune responses established after the combined MHTimmunotherapy. To evaluate the therapeutic effect of MHT-immunotherapy in theabsence of T cells, T cell blocking experiments were conducted using anti-CD4 and anti-CD8 antibodies. Mice bearing CT26 tumors were randomly divided into four groups (n = 6) and i.v. injected with anti-CD4, anti-CD8, or mouse IgG (as control) (20 μ g per mouse) on days 0 and 5 post MHTimmunotherapy (refer to group 7 in Figure 3). Compared to mouse IgG group, the abscopal therapeutic effect of our combination therapy was remarkably inhibited by blocking of either CD8+ T cells or CD4+ T cells (Figure S18), indicating that the adoptive immune responses through both CD4+ and CD8+ T cells are important to the antitumor abscopal effect. Therefore, after local MHT treatment of primary tumors, tumor-associated antigen in the tumor debris in the presence of adjuvant nanoparticles would trigger robust immune responses, such as greatly enhanced infiltration of T cells (both CTLs and Tregs) into distant tumors. However, the up-regulated Tregs in the distant tumor would partly compensate the antitumor immune responses of CTLs. Therefore, checkpoint blockade with anti-CTLA4 should be introduced to inhibit immunosuppressive Tregs and further boost the systemic antitumor immune attack of abscopal distant tumors. Long-Term Immune Memory Effect after MHTImmunotherapy. It is known that effector T cells will turn into memory T cells in order to have a long-term protection.46 The whole procedure in order to further assess the immune memory effect of our MHT-immunotherapy is shown in Figure 4a. First, 20 mice bearing CT26 tumors were randomly divided into two groups (n = 20) and the first inoculated tumors were removed by surgery or MHT therapy. For the latter group, tumors were injected FeNPs and PR at the doses of 50 μg (in term of Fe) and 100 μg (in terms of R837) per mouse, respectively, before being treated by AMF (Happl × fappl = 1.2 × 109 A m−1 s−1). Later on, mice were rechallenged with the second tumors on day 40. Subsequently, the two groups of mice were randomly divided into four groups (n = 10): 1. surgery; 2. surgery + anti-CTLA4; 3. FeNP + PR + AMF; and 4, FeNP + PR

previous reports, CTLA-4 blockade could greatly abrogate regulatory T cells at distant tumors after ablation of primary tumors.24,43 Thus, anti-CTLA4 as a clinically used checkpoint inhibitor was intravenously (i.v.) injected to further help the immune attack of abscopal tumors. To confirm immunogenic cell death (ICD) triggered by FeNP-based MHT, CT26-tumor-bearing mice were randomly divided into three groups (n = 3) and intratumorally (i.t.) injected with saline, FeNPs, and the mixture of FeNPs and PR. FeNPs and nanoadjuvant PR were i.t. injected at doses of 50 and 100 μg, respectively. One day after MHT, the leftover tumors were harvested to analyze high mobility group box-1 (HMGB1) expression, a marker of ICD, by flow cytometry. It was found that HMGB1 was significantly up-regulated for tumor cells in the FeNP-based MHT group compared to the control group, indicating immunogenic cell death induced by FeNP-based MHT (Figure S13). In our experiments, both the left and right flanks of each BABL/c mouse were subcutaneously (s.c.) inoculated with CT26 murine colon tumor cells. The tumors in left flanks as primary tumors were designed for FeNP-based MHT and the distant tumors in right flanks were designed without direct MHT treatment as the model of abscopal tumors. Mice were divided into seven groups (n = 6): 1, saline; 2, FeNP + PR; 3, FeNP + PR + anti-CTLA4; 4, FeNP + AMF; 5, FeNP + AMF + anti-CTLA4; 6, FeNP + PR + AMF; 7, FeNP + PR + AMF + anti-CTLA4. FeNPs and nanoadjuvant PR were mixed together for i.t. injection at doses of 50 and 100 μg, respectively. MHT was carried out by a portable device (SP-15A) and its introduction heating power (Happl × fappl = 1.2 × 109 A m−1 s−1). Afterward, mice in groups 3, 5, and 7 were i.v. injected with anti-CTLA4 (clone 9H10) antibody at 10 μg per mouse three times on day 1, 5, and 9 (Figure 3a). Both primary tumors with direct MHT treatment and abscopal tumors in the absence of MHT were measured afterward (Figure 3b,c). As expected, FeNP nanoparticles without alternating magnetic field showed no inhibition of both primary and distant tumors in group 2. Owing to the nonspecific immune responses elicited by adjuvant nanoparticles and anti-CTLA4, the tumor growth was partly delayed in group 3. For group 4, FeNP-based MHT could only ablate primary tumors but could not effectively inhibit distant tumors. Moreover, combining FeNP-based MHT with either immune adjuvant PR alone in group 6 or CTLA-4 blockade therapy alone in group 5 could only offer moderate abscopal effect in inhibiting the growth of distant tumors. Notably, the combination of FeNP-based MHT (to treat primary tumors) together with both immune adjuvant PR nanoparticles and CTLA-4 blockade in group 7 resulted in a remarkably synergistic effect to eliminate abscopal tumors, whose sizes gradually shrunk after their primary tumors on the opposite side were treated by MHT, and disappeared about 1 week later. All the mice in group 7 survived for 60 days (Figure 3d). Next, we need to investigate the mechanism of the abscopal effect obtained by such combined MHT-immunotherapy. It is well-known that in the process of cancer immunotherapy, cytotoxic T lymphocytes (CTLs)44 play a positive role in attacking tumor cells, while regulatory T Cells (Tregs)45 with the primary function to maintain immune homeostasis would play a negative role by suppressing the immune attack of tumor cells. It is known that CTLA-4 blockade would effectively hamper the immune-suppression activity of the Treg cells. Therefore, the abscopal tumors of all mice were collected on day 4293

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Figure 5. MHT plus CTLA4 blockade on orthotopic murine cancer models. (a) Schematic illustration of MHT plus CTLA4 blockade to inhibit distant tumor in the orthotopic B16 melanoma tumor model. (b,c) Tumor growth curves of primary and distant tumors after various treatments as indicated (n = 6). (d) Percent survival of different groups of tumors after various treatments as indicated (n = 6). (e) Schematic illustration of MHT plus CTLA4 blockade to inhibit spontaneous tumor metastases in the orthotopic 4T1 breast tumor model. (f) In vivo bioluminescence images to track the spreading and growth of fLuc-4T1 cells in mice after various treatments to eliminate their primary tumors. (g) Survival of mice bearing orthotopic 4T1 tumors with spontaneous metastases after various treatments indicated to eliminate their primary breast tumors (n = 10 per group).

+ AMF + anti-CTLA4. Mice in groups 2 and 4 were i.v. injected with anti-CTLA4 antibody at 10 μg per mouse three times on day 41, 45, and 49. The growth of the second tumors in surgery groups appeared to be rather fast in group 1 and CTLA4 blockade alone could only slightly delay the growth of second tumors in group (2). Importantly, the tumor growth of mice in group 3 were remarkably inhibited, whereas no tumor growth was observed in group 4 (Figure 4b,c). All the mice in group 4 and 60% of mice in group 3 survived for 90 days in marked

contrast to mice in group 1 and 2, which all died within 70 days (Figure 4d). Furthermore, spleens of mice were collected on day 40 before inoculation of the second tumors in order to further evaluate effector T memory (TEM) cells (CD3+CD8+CD44+CD62L−) by flow cytometry (n = 5). For mice with their first tumors ablated by FeNP-based MHT therapy together with PR nanoadjuvant, 80.7% of CD8+ T cells turned into TEM cells in their spleens. This ratio appeared to be much higher than 16.9% in mice undergoing surgery (Figure 5e). Serum cytokine such as 4294

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Nano Letters

of PEGylated FeNPs and their strong magnetism to allow effective magnetic targeting enhanced tumor accumulation of FeNPs, MHT tumor ablation could also be achieved by systemically administrated FeNPs, also under a low-power AMF. Therefore, this work presents a unique type of MHT agent based on pure iron NPs with appropriate surface coating that can enable safe and highly effective MHT tumor therapy and is superior to other existing MHT nanoagents. As a local tumor ablation method, MHT is not able to treat whole-body spreading metastatic tumors. In our work, the combination of FeNP-based MHT together with locally administrated immune adjuvant and systemically administrated checkpoint blockade antibody (anti-CTLA4) would be able to trigger strong systemic antitumor immune responses. After MHT tumor ablation with locally injected FeNPs and nanoadjuvant PR, tumor-associated antigens would be exposed in the generated tumor debris and interact with nanoadjuvant, so as to offer tumor-vaccine-like functions. Owing to the enhanced infiltration of CLTs into distant tumors after FeNP/PR-based MHT, as well as the function of anti-CTLA4 to suppress Tregs, robust abscopal antitumor immunotherapeutic effects are achieved, as evidenced by experiments in a subcutaneous tumor model and two orthotopic tumor models with distant metastasis. Furthermore, the MHT-immunotherapy could induce strong immune memory effect to prevent tumor recurrence. Notably, we expect that combining FeNP/PRbased MHT with anti-PD1/PD-L1, another widely applied immune checkpoint blockade therapy strategy, may also achieve synergistic antitumor effect, which however remains to be evidenced in further studies. In summary, FeNPs with PEGylation are developed in this work as a super effective MHT agent that allows complete tumor ablation under a low-power AMF using a portable device. The combination of MHT with immunotherapy is also demonstrated for the first time. Such MHT-immunotherapy after local tumor ablation is able to trigger systemic tumor-specific immune responses to attack whole-body spreading metastatic tumors and also results in long-term immune memory to prevent tumor recurrence. Our finding would possibly extend the applications of MHT for future clinical treatment of later stage cancer patients with distant metastases, which cannot be handled by conventional MHT. As Fe is an abundant element in the body, our PEGylated pure FeNPs that can be easily prepared and stored (in the power lyophilized form) may indeed have significant potential in clinical translation, for either MHT ablation therapy or combined MHT-immunotherapy against cancer.

TNF-α and IFN-γ levels in groups 3 and 4 were found to be much higher than that in groups 1 and 2 (Figure 5f). All those results evidenced that MHT treatment with FeNPs together with nanoadjuvant PR would be able to elicit robust immune memory effect. In Vivo MHT-Immunotherapy to Treat Orthotopic Tumor Models. In addition to the subcutaneous CT26 tumor model, we next evaluated the therapeutic responses of such MHT-immunotherapy against two types of orthotopic tumor models. In the first orthotopic tumor model, murine B16 skin melanoma cells (2 × 106) were subcutaneous (s.c.) injected into back skin of C57 mice (n = 6) (Figure 5a). While FeNP-based MHT had no inhibition of distant tumors, FeNP-based MHT with the help of either PR or anti-CTLA4 could notably inhibit the growth of distant melanoma tumors. Consistent to the previous CT26 tumor model, the combination of FeNP MHT with the help of both immune adjuvant PR and CTLA-4 checkpoint blockade resulted in the strongest abscopal effect to completely inhibit the growth of distant tumors (Figure 5b,c). All the mice in group 5 survived for 90 days in marked contrast to mice in groups 1−4, which all died within 26 days (Figure 5d). To further confirm the efficacy of MHT-immunotherapy, treatment on an orthotopic murine breast cancer model with spontaneous metastasis was carried out. Firefly luciferase (fLuc)-expressing 4T1 murine breast cancer cells were inoculated into the mouse breast pad of each Balb/C mouse. Fifteen days later, when tumor metastasis should have occurred, FeNP/PR-based MHT therapy or surgery was carried out to remove the primary tumor on each mouse and anti-CTLA4 was further systemically administrated for certain groups of mice (n = 10) (Figure 5e). Next, the in vivo bioluminescence imaging was used to track the metastases of fLuc-4T1 tumor cells after different treatments. While FeNP-based MHT had no inhibition of tumor metastasis, the combination of FeNP-based MHT with CTLA-4 blockade, or FeNP-based MHT with the help of nanoadjuvant PR, also could not effectively suppress tumor metastasis at later time points. Excitingly, no detachable signals from metastatic fLuc-4T1 cells were found within 50 days for mice in group 5, for which orthotopic breast tumors were ablated by FeNP-based MHT with the help of PR nanoadjuvant and anti-CTLA-4 blockade therapy (Figure 5f). Different from all other control groups, in which all mice died in 30−35 days, the combination of FeNP/PR-based magnetic thermal therapy with anti-CTLA-4 therapy resulted in 80% of animal survival in group 5 within our observation period of 90 days (Figure 5g). Those results strongly evidenced that FeNP-based MHT with the help of immune adjuvant and CTLA4 checkpoint blockade would trigger robust systemic antitumor immune responses to effectively eliminate cancer metastasis and prolong the survival of mice bearing orthotopic tumors with whole-body spreading tumor cells. In our work, pure iron NPs were synthesized by a chemical reduction method and functionalized with PEG to offer those nanoparticles high stability in physiological solutions. Such PEGylated FeNPs could be stored in the form of lyophilized powder for months and used later after being redispersed in aqueous solutions. Owing to superior inherent Ms of metallic iron, the Ms and SAR of FeNPs appears to be much higher than other types of magnetic iron oxide nanoparticles, making FeNPs a highly efficient MHT agent that could enable in vivo MHT tumor ablation using a portable AMF device with a pretty low introduction heating power (Happl × fappl = 1.2 × 109 A m−1 s−1). Moreover, taking advantages of the long blood circulation time

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.9b00579. Detailed synthesis and characterization, experimental procedures, and supporting Figures S1−S18 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: +86-512-65882036. ORCID

Chao Wang: 0000-0002-8054-3472 Zhuang Liu: 0000-0002-1629-1039 4295

DOI: 10.1021/acs.nanolett.9b00579 Nano Lett. 2019, 19, 4287−4296

Letter

Nano Letters Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by the National Basic Research Programs of China (973 Program) (2016YFA0201200), the National Natural Science Foundation of China (51525203, 51761145041), a Collaborative Innovation Center of Suzhou Nano Science and Technology, a “111” program from the Ministry of Education of China, and a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

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DOI: 10.1021/acs.nanolett.9b00579 Nano Lett. 2019, 19, 4287−4296