Near-Infrared-Triggered Photodynamic Therapy ... - ACS Publications

Mar 31, 2017 - School of Biology & Basic Medical Science, Medical College, Soochow University, Suzhou, Jiangsu 215123, China. •S Supporting Informat...
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Near-Infrared-Triggered Photodynamic Therapy with Multitasking Upconversion Nanoparticles in Combination with Checkpoint Blockade for Immunotherapy of Colorectal Cancer Jun Xu,† Ligeng Xu,*,† Chenya Wang,‡ Rong Yang,† Qi Zhuang,† Xiao Han,† Ziliang Dong,† Wenwen Zhu,† Rui Peng,*,† and Zhuang Liu*,† †

Institute of Functional Nano & Soft Materials (FUNSOM), Collaborative Innovation Center of Suzhou Nano Science and Technology, and ‡School of Biology & Basic Medical Science, Medical College, Soochow University, Suzhou, Jiangsu 215123, China S Supporting Information *

ABSTRACT: While immunotherapy has become a highly promising paradigm for cancer treatment in recent years, it has long been recognized that photodynamic therapy (PDT) has the ability to trigger antitumor immune responses. However, conventional PDT triggered by visible light has limited penetration depth, and its generated immune responses may not be robust enough to eliminate tumors. Herein, upconversion nanoparticles (UCNPs) are simultaneously loaded with chlorin e6 (Ce6), a photosensitizer, and imiquimod (R837), a Toll-like-receptor-7 agonist. The obtained multitasking UCNP-Ce6-R837 nanoparticles under near-infrared (NIR) irradiation with enhanced tissue penetration depth would enable effective photodynamic destruction of tumors to generate a pool of tumor-associated antigens, which in the presence of those R837containing nanoparticles as the adjuvant are able to promote strong antitumor immune responses. More significantly, PDT with UCNP-Ce6-R837 in combination with the cytotoxic Tlymphocyte-associated protein 4 (CTLA-4) checkpoint blockade not only shows excellent efficacy in eliminating tumors exposed to the NIR laser but also results in strong antitumor immunities to inhibit the growth of distant tumors left behind after PDT treatment. Furthermore, such a cancer immunotherapy strategy has a long-term immune memory function to protect treated mice from tumor cell rechallenge. This work presents an immune-stimulating UCNP-based PDT strategy in combination with CTLA-4 checkpoint blockade to effectively destroy primary tumors under light exposure, inhibit distant tumors that can hardly be reached by light, and prevent tumor reoccurrence via the immune memory effect. KEYWORDS: photodynamic therapy, upconversion nanoparticles, checkpoint blockade, immunotherapy, immune memory

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such effects usually are not strong enough to inhibit growth of remaining tumor cells in the body left behind after PDT.7 Upconversion nanoparticles (UCNPs) usually containing lanthanide ions are able to convert long-wavelength nearinfrared (NIR) light with greatly improved tissue penetration into short-wavelength light through a multiphoton absorption/ single-photon emission process.8−10 Taking advantages of such optical features of those optical nanomaterials, UCNPs have shown promising applications in biomedical sensing, imaging,

hotodynamic therapy (PDT) that kills cancer cells by utilizing photosensitizers to generate reactive oxygen species (ROS) such as singlet oxygen (1O2) under light irradiation is an effective therapeutic modality with advantages in great spatiotemporal selectivity and minimal invasiveness.1 In addition to direct killing of tumor cells, it has also been uncovered that PDT can promote antitumor immune responses by producing tumor-associated antigens from tumor cell residues.2,3 However, conventional photosensitizers used in PDT are often excited by visible light with limited tissue penetration, hampering the applications of PDT in treating deeply seated tumors.4−6 On the other hand, although PDT treatment may generate certain levels of immune responses, © 2017 American Chemical Society

Received: February 1, 2017 Accepted: March 31, 2017 Published: March 31, 2017 4463

DOI: 10.1021/acsnano.7b00715 ACS Nano 2017, 11, 4463−4474

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Figure 1. Characterization of UCNP-Ce6 and UCNP-Ce6-R837. (a) Schematic drawing showing the fabrication process of UCNP-Ce6-R837. Right on the surface of UCNPs and beneath the PEG coating, there is a hydrophobic layer available for loading of hydrophobic small molecules such as Ce6 and R837.16 (b) Transmission electron microscopy image of UCNP-Ce6-R837. (c) UV−vis absorbance spectra of Ce6, UCNP-Ce6, and UCNP-Ce6-R837. (d) Upconversion luminescence spectra of UCNP-Ce6 and UCNP-Ce6-R837 under 980 nm excitation recorded at the same UCNP concentration. The inset shows photographs of UCNP-Ce6-R837 nanoparticles exposed to the 980 nm laser. (e) Generation of singlet oxygen by Ce6, UCNP-Ce6, and UCNP-Ce6-R837 based on the fluorescence intensity changes of singlet oxygen sensor green (SOSG) under 980 nm light irradiation (0.5 W/cm2).

and therapies.11−15 In particular, it has been demonstrated in our’s and others’ previous reports that by coupling UCNPs with photosensitizers, NIR-triggered PDT with remarkably improved tissue penetration depth compared to that in conventional PDT could be realized in animal experiments.16−21 It has also been uncovered that those nanoparticles could be gradually excreted from the mouse body over months, without rendering significant toxicity to the treated animals.16 Recently, research on UCNP-based therapeutic applications has shifted from mono-PDT toward a combination of photothermal therapy, chemotherapy, radiotherapy, or gene therapy with PDT to further optimize the therapeutic outcomes in cancer treatment.22−29 However, the strategy of UCNP-based PDT in combination with immunotherapy has not yet been reported to our best knowledge. Checkpoint blockade as a promising type of cancer immunotherapy method has attracted tremendous interests in recent years.30−33 Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) blockade and programmed death 1/programmed death ligand 1 (PD-1/PD-L1) blockade, both which are able to modulate the immunosuppressive environments within tumors, have already been approved by the U.S. Food and Drug Administration (FDA) for treatment of several types of cancers.34−37 The combination of checkpoint blockade with other types of treatment strategies could offer additional therapeutic benefits in cancer treatment.38−41 Recently, we discovered that by combining a CTLA-4 checkpoint blockade with photothermal therapy using immune adjuvant nanoparticles, strong antitumor immunities can been generated to inhibit tumor metastasis.42 In this work, aiming at employing NIR-triggered deep PDT to trigger effective cancer immunotherapy, we design a multifunctional UCNP-based platform by coloading chlorin e6 (Ce6), a photosensitizer, and imiquimod (R837), a Tolllike-receptor-7 (TLR-7) agonist as an immune adjuvant, onto

polymer-coated UCNPs (Figure 1a). The formed UCNP-Ce6R837 nanoparticles under the 980 nm NIR laser excitation could trigger photodynamic destruction of primary tumors grown on mice. After PDT treatment with those nanoparticles, the released tumor-associated antigens with the assistance of R837-containing UCNPs as immune adjuvants could trigger antitumor immune responses, which could be further promoted by the anti-CTLA-4 checkpoint blockade to effectively eliminate both irradiated tumors and tumors grown on distant sites without laser-triggered PDT treatment. Furthermore, such a cancer immunotherapy strategy shows a strong long-term immune memory function to protect the treated mice against tumor cell rechallenge. Therefore, this work presents an immune-stimulating UCNP-based PDT strategy in combination with a CTLA-4 checkpoint blockade to effectively destroy primary tumors under NIR light exposure, inhibit distant tumors that can hardly be reached by light, and prevent tumor reoccurrence via the immune memory effect.

RESULTS AND DISCUSSION UCNP-Ce6-R837 nanoparticles were prepared beginning with the synthesis of UCNP cores followed by coating of amphiphilic polymers and loading with Ce6 and R837 (Figure 1a). UCNPs based on 20% Yb and 2% Er-doped NaYF4 nanoparticles were synthesized following a previously established method.43 The X-ray diffraction analysis of the asprepared UCNP nanoparticles showed that all peaks could be indexed to the hexagonal structure of NaYF4 (JCPDS card, No. 28-1192) (Figure S1a). To transfer those nanoparticles into the aqueous phase, an amphiphilic polymer, polyethylene glycol (PEG)-grafted poly(maleic anhydride-alt-1-octadecene) (C18PMH-PEG), was then used to modify as-prepared UCNPs,16 obtaining PEGylated UCNPs with great stability in physiological buffers. As uncovered in our previous reports, there would be a hydrophobic layer on top of the UCNP core 4464

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Figure 2. In vitro PDT and immune stimulation effect of UCNP-Ce6-R837. (a) Confocal fluorescence images of UCNP-Ce6 and UCNP-Ce6R837 incubated CT26 tumor cells after irradiation by the 980 nm laser (0.5 W/cm2) for 20 min. The cells were costained by calcein AM (green, live cells) and propidium iodide (red, dead cells) before imaging. Scale bar: 50 μm. (b,c) Flow cytometry quantification of CD86 and CD80 expressions, which are markers for DC maturation, after in vitro incubation of DCs with UCNP-Ce6, R837, or UCNP-Ce6-R837 for 20 h. (d) Secretion of IL-12p40 and TNF-α in DC suspensions measured by ELISA.

ured to be −13.1 and −12.9 mV, respectively (Supporting Figure S1c). At our optimized condition, the loading capacities of Ce6 and R837 on UCNP-Ce6-R837 nanoparticles were measured to be ∼10 and ∼1%, as determined by UV−vis absorbance spectra and high-performance liquid chromatography, respectively (Supporting Figure S2a and Figure 1c). The release profiles of Ce6 and R837 in UCNP-Ce6-R837 nanoparticles were also measured in PBS (pH 7.4). Relatively slow releasing rates were observed, with ∼15% of Ce6 and ∼10% of R837 detached from UCNPs after 48 h incubation at room temperature (Supporting Figure S2b). Under NIR excitation by the 980 nm laser, those UCNPs would exhibit two upconversion luminescence emission peaks at 550 and 660 nm, the latter of which could be employed to excite Ce6 molecules attached on the UCNP surface by the resonance energy transfer (Figure 1d). As determined by the singlet oxygen sensor green (SOSG) assay, both UCNP-Ce6 and UCNP-Ce6-R837, but not free Ce6, showed efficient generation of singlet oxygen under 980 nm NIR laser

and beneath the PEG shell to enable loading of hydrophobic molecules. For further loading with photosensitizing and immune-stimulating molecules on those nanoparticles, Ce6 in phosphate buffered saline (PBS, pH 7.4) and R837 in dimethyl sulfoxide were mixed with PEGylated UCNPs overnight. After removal of excess unloaded molecules by centrifugation at 14 800 rpm for 10 min and three PBS washes, Ce6 and R837 coloaded UCNPs (UCNP-Ce6-R837) were obtained and used for further experiments. The UCNP-Ce6 nanoparticles were obtained following the same method without addition of R837. We then carefully characterized the obtained UCNP-Ce6R837 nanoparticles. Transmission electron microscopy images clearly revealed that those UCNP-Ce6-R837 nanoparticles showed uniform sizes with an average diameter of ∼80 nm (Figure 1b). The hydrodynamic sizes of UCNP-Ce6-R837 and UCNP-Ce6 were slightly larger than that of PEGylated UCNPs before molecular loading as measured by dynamic light scattering (Supporting Figure S1b). The zeta-potentials of UCNP-Ce6 and UCNP-Ce6-R837 nanoparticles were meas4465

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Figure 3. In vitro transwell system experiment. (a) Scheme showing the design of the transwell system experiment. CT26 tumor cells and their residues were placed in the upper chamber, and DCs were cultured in the lower chamber. (b) Quantification of CD86 and CD80 expression by flow cytometry for various samples in the in vitro transwell system experiment. (c,d) Quantification of CD86 and CD80 on the surface of DCs (c) and the secretion of IL-12p40 and TNF-α in DC suspensions (d), after treatment for 20 h in the transwell system experiment.

irradiation (0.5W/cm2) (Figure 1e), enabling us to use such Ce6-loaded nanoparticles for NIR-triggered PDT. The in vitro photodynamic toxicities of UCNP-Ce6 and UCNP-Ce6-R837 to the mouse colon adenocarcinoma cell line (CT26) cells were then evaluated. Cell viability assays were carried out for CT26 cells after various treatments. While both UCNP-Ce6 and UCNP-Ce6-R837 without laser irradiation were found to not be notably toxic to cells even under high concentrations, the two types of nanoparticles showed concentration-dependent phototoxicities toward CT26 cells under NIR laser exposure (980 nm, 0.5 W/cm2, 20 min) (Supporting Figure S3a,b). Fluorescence staining of living/dead cells further proved the excellent cancer cell killing ability of NIR-induced PDT by both UCNP-Ce6 and UCNP-Ce6-R837 (Figure 2a). Dendritic cells (DCs), as a key type of antigen-presenting ̈ T cells. cells (APCs), are responsible for activating naive Immature DCs (iDCs) collect antigens from the surrounding fluid and then process them into peptides during migration from peripheral tissues to nearby draining lymph nodes, where they present the peptide major histocompatibility complex (MHC) to T cell receptors (TCR) for T cell activation.44,45 The up-regulation of co-stimulatory molecules (CD80, CD86)

as the typical markers on the surface of DCs could indicate the level of DC maturation. Thus, we cocultured UCNP-Ce6, UCNP-Ce6-R837, or free R837 with the bone-marrow-derived DCs harvested from C57bl/6 mice for 20 h. From flow cytometry analysis, the percentage of mature DCs (CD11c+CD86+ and CD11c+CD80+) treated by UCNP-Ce6R837 nanoparticles appeared to be obviously higher than those induced by the same dose of free R837, whereas UCNP-Ce6 nanoparticles without R837 loading triggered no appreciable immune responses to DCs (Figure 2b,c). Meanwhile, the DCsecreted immune-related cytokines such as interleukin 12 (IL12p40) and tumor necrosis factor α (TNF-α), which are also indicators of DC activation, were measured by enzyme-linked immune sorbent assay (ELISA).46 It was found that the secretion levels of IL-12p40 and TNF-α from DCs were obviously enhanced after UCNP-Ce6-R837 treatment, to levels higher than those post-treatment with free R837 (Figure 2d). Thus, UCNP-Ce6-R837 nanoparticles could act as a strong immune nanoadjuvant. Tumor residues post-PDT may act as tumor-associated antigens to trigger immune responses, which may be further enhanced by immune adjuvants.47 A transwell system was then employed to study such effect at the in vitro level. In our 4466

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Figure 4. UCNP-Ce6-R837-based in vivo PDT induces DC maturation and stimulates the expression of pro-inflammatory cytokines. (a) Schematic illustration of our experiment design to examine immune responses triggered by UCNP-Ce6-R837-based PDT. (b,c) DC maturation induced by UCNP-Ce6-R837-based PDT on mice bearing CT26 tumors. Cells in the tumor-draining lymph nodes were collected for assessment by flow cytometry after staining with CD11c, CD80, and CD86. (d−f) Cytokine levels of IL-12p40 (d), IFN-γ (e), and TNF-α (f) in sera from mice isolated on day 3 post-UCNP-Ce6-R837-based PDT. Three mice were measured in each group in (b−f). Error bars represent standard deviation (SD) of at least three replicates.

if the latter one (UCNP-Ce6-R837) was used (Figure 3b,c). Consistent with DC maturation data, we also found that CT26 tumor cell residues post-PDT treatment with UCNP-Ce6-R837 could trigger the highest levels of IL-12p40 and TNF-α secretions by DCs (Figure 3d). Our results taken together suggest that the tumor-associated antigens from tumor cell residues post-PDT, especially with help of R837-loaded nanoparticles as the adjuvant, could trigger effective DC maturation. Recent studies have shown that PDT treatment has the ability to activate the tumor-specific immune responses by

experiment, the upper chamber containing CT26 tumor cells after various treatments was placed into the transwell system, whose lower chamber was seeded with DCs (Figure 3a). The DC maturation and cytokine secretion from DCs were then evaluated by flow cytometry and ELISA, respectively. Compared to the DC maturation level after coculturing DCs with untreated CT26 cells, addition of UCNP-Ce6-R837 nanoparticles into this system resulted in a slight increase of DC maturation. On the other hand, if those CT26 cells were treated by PDT using UCNP-Ce6 or UCNP-Ce6-R837, their residues could remarkably promote DC maturation, particularly 4467

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Figure 5. Antitumor immune effects and the related mechanisms of UCNP-Ce6-R837-based PDT in combination with CTLA-4 checkpoint blockade. (a) Schematic illustration of our experiment design. Mice with CT26 tumors on both sides were used in our experiment. Tumors on the left side were designated as “primary tumors” for PDT treatment, and those on the right side were designated as “distant tumors” without PDT. (b,c) Growth curves for primary tumors (b) and distant tumors (c) on mice after various treatments indicated. (d,e) Proportions of tumor-infiltrating CD8+ killer T cells (d) and CD4+FoxP3+ effector T cells (Treg) (e) in the distant tumor collected on day 15. (f) CD8+ CTL to Treg ratios in distant tumors for different groups of mice on day 15. (g) IFN-γ cytokine levels in sera from mice isolated from different groups of mice on day 15. Six mice were measured in each group in (a−g). Error bars represent SD P values: *P < 0.05, **P < 0.01, ***P < 0.001.

PDT treatment (day 11), mice were then sacrificed to cut off the draining lymph nodes, which were used to assess the levels of DC maturation by flow cytometry analysis (Figure 4a). It was found that UCNP-Ce6-R837-based PDT therapy promoted a much higher level of DC maturation compared to that observed for PDT therapy with UCNP-Ce6 in the absence of R837, as well as those with UCNP-Ce6-R837 injection but in the absence of laser irradiation (Figure 4b,c). Meanwhile, we analyzed the changes of various cytokines including interleukin 12 (IL-12p40), interferon γ (IFN-γ), and TNF-α in sera from mice (day 11) after different treatments by ELISA assay. The secretion level of IL-12p40, which plays an important role in T-helper cell 1 type (Th1) immune response, for mice treated by UCNP-Ce6-R837-based PDT treatment appeared to be much higher than that in other control groups (Figure 4d). The other two types of important cytokines, IFN-γ and TNF-α, also showed significant up-regulation in mice after

producing tumor-associated antigens from tumor cell residues, which afterward may be processed by APCs such as DCs and then presented to T cells.48,49 We thus wonder whether PDT with UCNP-Ce6-R837, which could also act as an immune adjuvant, would be able to trigger strong immune responses in vivo. Although, in general, it would be better to deliver therapeutic agents via systemic administration for conventional cancer therapies, for immunotherapy, local injections are more commonly used in the clinic (e.g., for vaccination), partly to avoid the risk of a cytokine storm that may occur if antigens and adjuvants are systemically administered.50,51 In our experiments, when CT26 tumors grown on BALB/c mice reached ∼100 mm3, 50 μL of UCNP-Ce6 or UCNP-Ce6-R837 (20 mg/mL UCNP, ∼2 mg/mL Ce6, ∼0.2 mg/mL R837) was injected locally into those tumors. After 2 h, we exposed the mice to the 980 nm NIR laser at 0.5 W/cm2 for 30 min with 1 min interval for every 2 min of light exposure. Three days after 4468

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importance of R837 in our system as the adjuvant to boost antitumor immunities. Those results together suggest that UCNP-Ce6-R837-based PDT treatment combined with CTLA-4 blockade would offer a strong synergistic antitumor immunological effect to suppress the growth of tumor cells, even for those without direct treatment by PDT. Next, we ought to understand the mechanism of such antitumor immunological responses post-combination therapy. It is well-known that CD8+ T cells which bind to the antigenMHC I complex could kill cancer cells by releasing the cytotoxinsIFN-γ, perforin, granzymes, and granulysin.55−57 On the other hand, CD4+ T cells play important roles in the regulation of immune systems. Therefore, we collected the right flank tumor of all the mice on day 15 (day 7 post-PDT) to test the tumor-infiltrating CD8+ (CD3+CD4−CD8+) and CD4+ (CD3+CD4+CD8−) T cells within those tumors by flow cytometry. The percentage of CD8+ T cells in the right flank distant tumor after the combined UCNP-Ce6-R837-based PDT plus anti-CTLA-4 treatment was dramatically increased (by ∼3fold compared to the PBS control). In contrast, no significant enhancement of CD8+ T cell infiltration into the distant tumors was observed in other control groups (Figure 5d). The CD4+ helper T cells could be classified into effector T cells (Teff) which could stimulate and interact with other leukocytes by secreting cytokines, as well as regulatory T cells (T reg ) which instead are able to suppress antitumor immunity.58,59 With forkhead box P3-positive (Foxp3+) as the marker for Treg cells, we found that although UCNP-Ce6-R837based PDT treatment could result in tumor-antigen releases, maturation of DCs, as well as the increase in the percentage of CD4+ helper T cells infiltrated into distant tumors, most of those CD4+ T cells were immunosuppressive Treg cells (CD3+CD4+Foxp3+) (Figure 5e). Impressively, with the help of the anti-CTLA-4 antibody, the percentage of Treg cells in the distant tumors were all remarkably reduced, evidencing that CTLA-4 blockade could greatly abrogate the activity of Treg cells (Figure 5e). Therefore, the ratio of CD8+ CTLs to Treg cells was greatly enhanced in the distant tumors of mice after the CTLA-4 blockade, and the ratio of CD8+ CTLs to Treg cells after UCNP-Ce6-R837-based PDT plus anti-CTLA-4 treatment appeared to be the highest among all tested groups (Figure 5f). Meanwhile, the serum concentration of IFN-γ in the combination therapy group, UCNP-Ce6-R837-based PDT plus anti-CTLA-4, was also found to be much higher than that in the other control groups (Figure 5g). Generally speaking, after PDT treatment, the released tumor-associated antigens with the assistance by R837-containing UCNPs as immune adjuvants could amplify the antitumor immune responses. Meanwhile, anti-CTLA-4 antibody would effectively hamper the immune suppression activity of the Treg cells and increase the ratio of CTLs to Treg cells, favorable for promoting antitumor cellular immunity to attack tumors. It is known that the immunological memory response, which is the hallmark feature of adaptive immunities, plays crucial roles in protecting organisms from the second attack of pathogens including tumor cells.60−62 That is to say, upon a second encounter with the same pathogens, memory T cells can rapidly respond and mount faster and stronger immune responses than the first time the immune system responded. To further assess the immune memory effects of PDT combined with CTLA-4 blockade immunotherapy, we rechallenged mice with the second inoculation of CT26 cells (106 cells per mouse) on day 44, which was 36 days after surgery or UCNP-

UCNP-Ce6-R837-based PDT treatment on day 11 (Figure 4e,f), suggesting cellular immunity was successfully induced by our UCNP-Ce6-R837-based PDT system. Next, we wondered whether such robust immunological responses triggered by PDT with UCNP-Ce6-R837 would be able to inhibit the growth of tumor cells left behind after PDT at distant sites (e.g., metastatic sites). It has been well-studied that the blockade of cytotoxic CTLA-4 could inhibit the immune-suppressive regulator ability of regulatory T cells (Treg).52,53 UCNP-Ce6-R837-based PDT was then combined with CTLA-4 blockade therapy for in vivo cancer treatment. In our experiments, we subcutaneously (s.c.) inoculated 106 CT26 tumor cells into both the left and right flanks of each BABL/c mouse. The tumors in the left flanks as primary tumors (1#) were designed for UCNP-Ce6-R837-based PDT therapy, and the distant tumors in the right flanks (2#) were designed without direct treatment as an artificial model of abscopal tumors. When both tumors reached ∼100 mm3 on day 8, we would divide these mice into seven groups: (1) PBS; (2) UCNP-Ce6-R837; (3) UCNP-Ce6-R837 plus anti-CTLA-4 antibody; (4) UCNP-Ce6 with NIR irradiation; (5) UCNPCe6 with NIR irradiation plus anti-CTLA-4 antibody; (6) UCNP-Ce6-R837 with NIR irradiation; and (7) UCNP-Ce6R837 with NIR irradiation plus anti-CTLA-4 antibody. For each tumor on the left flank of a mouse in groups 2, 3, 6, and 7, 50 μL of UCNP-Ce6-R837 ([Ce6] = 2 mg/mL, [R837] = 0.2 mg/mL) was intratumorally injected. UCNP-Ce6 at the same dose was intratumorally injected to the left flank of each mouse in groups 4 and 5. Two hours after injection, the tumors with nanoparticle injection (left side) in groups 4−7 were exposed to the 980 nm NIR laser at 0.5 W/cm2 for 30 min with 1 min interval for every 2 min of light exposure. After irradiation, we intravenously (i.v.) injected anti-CTLA-4 (clone 9H10) antibody into mice for groups 3, 5, and 7 at 10 μg per mouse, a relatively low dose compared to that used in previous studies on checkpoint blockade therapy,42,54 for two times on days 9 and 13 (Figure 5a). The tumor sizes on both sides (primary tumors with direct PDT treatment and distant tumors without PDT treatment) were closely monitored afterward. For primary tumors, while UCNP-Ce6-R837 injection alone showed no appreciable effect on the tumor growth, PDT with UCNP-Ce6-R837 could significantly suppress their growth within the first 10 days posttreatment. However, those tumors after PDT treatment alone with UCNP-Ce6-R837 recovered later and grew into rather large tumors 1 month post-PDT (Figure 5b). Meanwhile, such a type of treatment by UCNP-Ce6-R837-based PDT could trigger significant immune responses as mentioned above (Figure 4b−d), it could only partially delay the tumor growth at the distant site (Figure 5c). Interestingly, whereas the CTLA-4 blockade by itself could only partially delay the tumor growth in group 3 on both sides (Figure 5b,c), the combination of UCNP-Ce6-R837-based PDT with CTLA-4 blockade could not only completely eliminate primary tumors but also strongly inhibit the growth of distant tumors (Figure 5b,c). Notably, in group 7 for those distant tumors without nanoparticle injection and direct PDT treatment, their sizes gradually shrunk after their primary tumors on the opposite side were treated by PDT and disappeared about 2 weeks later, with only one out of six tumors reoccurring after ∼50 days. In contrast, for mice with PDT treatment by UCNP-Ce6 (without R837) (groups 4 and 5), their distant tumors could not be effectively inhibited even in combination with the CTLA-4 blockade, highlighting the 4469

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Figure 6. Immune memory effects after UCNP-Ce6-R837-based PDT. (a) Scheme showing the experiment design to evaluate the immune memory effect. (b) Tumor growth curves of rechallenged tumors inoculated 36 days post-elimination of their first tumors by either surgery or PDT (n = 6 per group). (c) Survival curves of mice after various treatments as indicated in (b). (d,e) Proportions of effector memory T cells (TEM) in the spleen analyzed by flow cytometry. (f,g) Cytokine levels in sera from mice isolated 7 days after mice were rechallenged with secondary tumors. Error bars represent SD of at least three replicates. P values: *P < 0.05, **P < 0.01, ***P < 0.001.

Ce6-R837-based PDT plus anti-CTLA-4 treatment to remove their first tumors (Figure 6a). Specifically in our experiments, we divided these mice into four groups: (1) surgery + antiCTLA-4 (post); (2) surgery + UCNP-Ce6-R937 + anti-CTLA4 (pre and post); (3) PDT + anti-CTLA-4 (pre and post); and (4) PDT + anti-CTLA-4 (pre). Whereas “pre” refers to i.v. injection of anti-CTLA-4 (10 μg per mouse) on day 9 and day 13 (before inoculation of secondary tumors), “post” refers to i.v. injection of anti-CTLA-4 (10 μg per mouse) on day 45 and day 49 (after inoculation of secondary tumors). The PDT treatment was conducted on their first CT26 tumors with UCNP-Ce6-R837 using the same parameters as mentioned above. For mice with their first tumors removed by surgery, the growth of their secondary tumors appeared to be rather fast, even if those mice were pretreated with UCNP-Ce6-R937 and anti-CTLA-4 (group 2). Excitingly, the growth of secondary tumors in both groups 3 and 4 with their first tumors eliminated by PDT with UCNP-Ce6-R937 were remarkably

inhibited. While only 1 out of 6 mice in group 3 (with only preCTLA-4 blockade) showed reoccurring secondary tumors, all inoculated secondary tumors in group 4 (with both pre- and post-CTLA-4 blockade) failed to grow (Figure 6b and Supporting Figure S4). Moreover, all mice in group 4 survived over 50 days after inoculation of secondary tumors (Figure 6c), in marked contrast to the two surgery groups (groups 1 and 2) which showed average life spans of 18−19 days, demonstrating the excellent long-term immune memory protection effect induced by UCNP-Ce6-R837-based PDT plus anti-CTLA-4 treatment. To identify the antitumor immune memory responses, on day 43, we harvested the spleen from different groups of mice and measured the changes of central memory CD8+ T cells (TCM) and effector memory CD8+ T cells (TEM). TCM cells mainly locating in lymphoid tissues will expand, differentiate and migrate to infection sites within 1 week post attack. Distinguished from TCM, TEM cells, which reside in both 4470

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Figure 7. Scheme summarizing the mechanisms of combining NIR-mediated PDT with CTLA-4 checkpoint blockade for cancer immunotherapy. UCNP-Ce6-R837 nanoparticles under NIR light would enable effective photodynamic destruction of tumors. The generated tumor-associated antigens in the presence of those nanoparticles as the adjuvant are able to promote strong antitumor immune responses, which with the help of a CTLA-4 checkpoint blockade would eliminate primary tumors under direct NIR exposure, inhibit the growth of distant tumors left behind after PDT, and further yield a long-term immune memory to prevent tumor reoccurrence.

CONCLUSION In summary, in this work, we have rationally designed a type of multitasking nanoparticle based on UCNPs to trigger cancer immunotherapy by NIR-induced PDT. Our UCNP-Ce6-R837 nanoparticles could be used for NIR-induced PDT to directly destroy tumor cells and to stimulate immune responses by triggering the maturation of DCs and secretion of cytokines. Combined with the clinically approved CTLA-4 checkpoint blockade therapy to inhibit the activities of Treg cells, such UCNP-Ce6-R837-based PDT is able to efficiently eliminate primary tumors directly exposed to PDT and inhibit distant tumors left behind after PDT by employing the strong antitumor immune responses. Furthermore, the long-term immune memory effect induced by UCNP-Ce6-R837-based PDT in combination with CTLA-4 blockade would provide a strategy to protect the body from tumor reoccurrence. Therefore, this work has demonstrated the great potency of integrating UCNP-based PDT with cancer immunotherapy to realize a remarkable synergistic therapeutic outcome in eliminating primary tumors, inhibiting distant tumors, and preventing tumor relapse.

lymphoid and nonlymphoid tissues, can induce immediate immunities by producing multiple cytokines like TNF-α and IFN-γ upon second encounter with the same antigen.63−65 Intriguingly, from the flow cytometry data, the percentage of TEM cells (CD3+CD8+CD44+CD62L−) after UCNP-Ce6R837-based PDT plus anti-CTLA-4 antibody to remove the first tumors was much higher than that of the control group in which tumors were removed by surgery or the surgery group together with s.c. injection of nanoparticle and i.v. injection of anti-CTLA-4 (Figure 6d,e). In contrast, the percentage of TCM cells (CD3+CD8+CD44+CD62L+) in the combination therapy group was much lower than that of the two surgery groups (Supporting Figure S5). Furthermore, 1 week after mice were challenged with secondary tumors, the concentrations of both TNF-α and IFN-γ in mouse sera increased significantly in the mice after UCNP-Ce6-R837-based PDT plus CTLA-4 blockade (Figure 6f,g). Our data strongly evidenced that UCNPCe6-R837-based PDT plus CTLA-4 blockade could indeed boost a strong immune memory effect to effectively prevent reoccurrence of tumors. Generally speaking, the underlying mechanisms of the combination therapy with ideal inhibition activities on the growth of both primary and distant tumors, as well as the immune memory protection to prevent tumor relapse may be explained as follows (Figure 7). (1) The PDT destruction of primary tumors would generate a pool of tumor-associated antigens to trigger specific immune responses, which are then amplified by R837-loaded UCNP-Ce6-R837 nanoparticles as the immune adjuvant. (2) CTLA-4 checkpoint blockade strategy could effectively hamper the immunosuppression activity of Treg cells and increase the ratios of CTL to Treg cells to promote antitumor cell immunity. (3) UCNP-Ce6R837-based PDT combined with CTLA-4 blockade would effectively induce the generation of TEM-based immune memory response to prevent tumor relapse, similar to the functions of cancer vaccines.

EXPERIMENTAL SECTION Synthesis of UCNP-Ce6-R837 Nanoparticles. NaYF4 (Y/Yb/Er = 78:20:2) upconversion nanoparticles were synthesized by a typical thermal decomposition method following a well-established method.43 To transfer the hydrophobic as-made UCNPs into aqueous solutions, 2 mg of C18PMH−PEG polymer, which was synthesized following our previously reported protocol,16 was mixed with the 2.5 mg of UCNPs in 5 mL of chloroform and then stirred for 6 h after ultrasonication for 30 min. After chloroform was blown-dry, the obtained UCNP-PEG nanoparticles were dispersed in water. Ce6 and R837 loading onto PEGylated UCNPs was carried out by mixing 0.48 mg of Ce6 and 0.1 mg of R837 with 1 mg of UCNP-PEG in 1 mL of PBS at room temperature overnight. After centrifugation at 14 800 rpm for 10 min and being washed with PBS until no obvious color in the filtrate, the 4471

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ACS Nano obtained UCNP-Ce6-R837 nanoparticles were then suspended in deionized water at 4 °C. Characterization of UCNP-Ce6-R837 Nanoparticles. The morphology of nanoparticles was characterized by a FEI Tecnai F20 transmission electron microscope. The Zetasizer Nano-ZS (Malvern Instruments, UK) was used to measure the hydrodynamic diameters of nanoparticles. The concentrations of Ce6 in UCNP-Ce6-R837 samples were determined by a UV−vis−NIR spectrophotometer (PerkinElmer Lambda 750). The loading capacity of R837 was determined by a high-performance lipid chromatography (Agilent 1260) with a UV detector at 325 nm. The upconversion luminescence spectra of nanoparticles were measured by a FluoroMax 4 fluorometer using an external 980 nm laser as the excitation source. Detection of Singlet Oxygen. In our experiments, we used an external 980 nm laser to trigger photodynamic therapy. UCNP, UCNP-Ce6, and UCNP-Ce6-R837 samples were exposed to the 980 nm laser with the power density of 0.5 W/cm2 for 0, 10, 20, 30, 40, 50, and 60 min. SOSG (2.5 μM), which was highly sensitive to singlet oxygen, was employed to measure the singlet oxygen generation by Ce6 (2 μM). Typically, the fluorescence intensity of SOSG was acquired by a fluorometer with excitation at 494 nm. In Vitro Cellular Experiments. The CT26 murine colorectal cancer cell line was originally obtained from American Type Culture Collection (ATCC) and cultured in Roswell Park Memorial Institute (PRMI) 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C under 5% CO2. For the cell cytotoxicity assay, CT26 cells seeded into 96-well plates after adherence were mixed with UCNP-Ce6 and UCNP-Ce6-R837 at various concentrations for 24 h at 37 °C under 5% CO2. The standard thiazolyltetrazolium (MTT) test was then conducted to measure the relative cell viabilities. For in vitro photodynamic therapy, CT26 cells were seeded into 96well plates at 1 × 104/well until adherent and then incubated with series concentrations of UCNP-Ce6 or UCNP-Ce6-R837 for 2 h. After the culture medium was replaced with the fresh medium, cells were then irradiated by the 980 nm laser for 10 min at a power density of 0.5 W/cm2. After incubation for 24 h at 37 °C under 5% CO2, the standard MTT assay was conducted to determine the cell viabilities relative to the untreated cells. For confocal imaging, 24 h after irradiation by the 980 nm laser at the power density of 0.5 W/cm2 for 20 min, CT26 cancer cells were stained by calcein AM and PI and then imaged using a Leica SP5 laser scanning confocal microscope. Bone-marrow-derived dendritic cells were generated from the bone marrow of 8-week-old C57BL/6 mice from Nanjing Peng Sheng Biological Technology Co. Ltd. according to an established method.66 For in vitro DC stimulation experiments, we cocultured R837, UCNPCe6, or UCNP-Ce6-R837 nanoparticles ([UCNP] = 100 μg/mL, [Ce6] = 7.5 μg/mL, [R837] = 1 μg/mL) with 106 bone-derived DCs from C57bl/6 mice for 20 h. After various treatments, DCs stained with anti-CD11c-FITC, anti-CD86-PE, anti-CD11c-FITC, anti-CD80PE antibody were then analyzed by flow cytometry (BD FACSCalibur). For transwell experiments, residues of 4T1 cells after photodynamic treatment with either UCNP-Ce6 or UCNP-Ce6-R837 were also added into the 106 DC culture using the transwell system. In Vivo Animal Model. Female BALB/c mice (6−8 weeks) were purchased from Nanjing Peng Sheng Biological Technology Co. Ltd. and used under protocols approved by Soochow University Laboratory Animal Center. A total of 1 × 106 CT26 cells were subcutaneously injected into both left and right flanks of each mouse. When those tumors reached ∼100 mm3, UCNP-Ce6 or UCNP-Ce6-R837 nanoparticles were injected intratumorally into tumors at the UCNP dose of 0.8 mg per mouse. After 2 h, mice were irradiated with a 980 nm laser with a power density of 0.5 W/cm2 for 30 min with 1 min interval for every 2 min of light exposure. On day 1 and day 5 after irradiation, anti-CTLA-4 antibody was i.v. injected at a dose of 10 μg per mouse. Tumor sizes and mouse body weights were monitored every day. The tumor volume was calculated according to the following formula: width2 × length × 0.5. Flow Cytometry Analysis. To analyze the immune cells in the right flank tumors (distant tumors), those tumors were harvested from

mice and digested using 1500 U/mL collagenase (Sigma), 1000 U/mL hyaluronidase (Sigma), and Sigma DNase (Sigma) at 37 °C for 30 min. Cells were filtered through nylon mesh filters and washed with PBS containing 1% FBS. The single-cell suspension was incubated with anti-CD3-FITC (Biolegend), anti-CD8a-APC (Biolegend), and anti-CD4-PerCP (Biolegend) antibodies. Afterward, those cells were washed with PBS containing 1% FBS and analyzed using flow cytometry analysis. To analyze regulatory T cells, the single-cell suspension was stained with anti-CD3-FITC (eBioscience), anti-CD4PerCP (Biolegend), and anti-Foxp3-PE (eBioscience) antibodies. For analysis of memory T cells, spleen cells harvested from mice after various treatments were stained with anti-CD3-FITC (eBioscience), anti-CD8-PerCP-Cy5.5 (eBioscience), anti-CD62L-APC (eBioscience), and anti-CD44-PE (eBioscience) antibodies. Data analysis was carried out using FCS express software. Detection of Cytokines. The pro-inflammatory cytokines from sera and DC medium supernatants including TNF-α (eBiosciences), IFN-γ (eBiosciences), and IL-12p40 (eBiosciences) were determined by using enzyme-linked immunosorbent assay (ELISA) kits following standard protocols.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b00715. Figures S1−S5 (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Zhuang Liu: 0000-0002-1629-1039 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was partially supported by the National Research Programs from Ministry of Science and Technology (MOST) of China (2016YFA0201200), the National Natural Science Foundation of China (51525203 and 31300824), the Collaborative Innovation Center of Suzhou Nano Science and Technology, and a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions. REFERENCES (1) Dolmans, D. E. J. G. J.; Fukumura, D.; Jain, R. K. Photodynamic Therapy for Cancer. Nat. Rev. Cancer 2003, 3, 380−387. (2) Lu, K.; He, C.; Guo, N.; Chan, C.; Ni, K.; Weichselbaum, R. R.; Lin, W. Chlorin-Based Nanoscale Metal-Organic Framework Systemically Rejects Colorectal Cancers via Synergistic Photodynamic Therapy and Checkpoint Blockade Immunotherapy. J. Am. Chem. Soc. 2016, 138, 12502−12510. (3) 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, 12499. (4) Zhou, Z.; Song, J.; Nie, L.; Chen, X. Reactive Oxygen Species Generating Systems Meeting Challenges of Photodynamic Cancer Therapy. Chem. Soc. Rev. 2016, 45, 6597−6626. (5) Quail, D. F.; Joyce, J. A. Microenvironmental Regulation of Tumor Progression and Metastasis. Nat. Med. 2013, 19, 1423−1437. 4472

DOI: 10.1021/acsnano.7b00715 ACS Nano 2017, 11, 4463−4474

Article

ACS Nano (6) Wang, H.-W.; Putt, M. E.; Emanuele, M. J.; Shin, D. B.; Glastein, E.; Yodh, A. G.; Busch, T. M. Treatment-Induced Changes in Tumor Oxygenation Predict Photodynamic Therapy Outcome. Cancer Res. 2004, 64, 7553−7561. (7) Castano, A. P.; Mroz, P.; Hamblin, M. R. Photodynamic Therapy and Anti-Tumour Immunity. Nat. Rev. Cancer 2006, 6, 535−545. (8) Wang, F.; Liu, X. Recent Advances in theChemistry of Lanthanide-Doped Upconversion Nanocrystals. Chem. Soc. Rev. 2009, 38, 976−989. (9) Haase, M.; Schafer, H. Upconverting Nanoparticles. Angew. Chem., Int. Ed. 2011, 50, 5808−5829. (10) Zhou, J.; Liu, Z.; Li, F. Upconversion Nanophosphors for SmallAnimal Imaging. Chem. Soc. Rev. 2012, 41, 1323−1349. (11) Cheng, L.; Yang, K.; Li, Y.; Zeng, X.; Shao, M.; Lee, S. T.; Liu, Z. Multifunctional Nanoparticles for Upconversion Luminescence/MR Multimodal Imaging and Magnetically Targeted Photothermal Therapy. Biomaterials 2012, 33, 2215−2222. (12) He, F.; Feng, L.; Yang, P.; Liu, B.; Gai, S.; Yang, G.; Dai, Y.; Lin, J. Enhanced Up/Down-Conversion Luminescence and Heat: Simultaneously Achieving in One Single Core-Shell Structure for Multimodal Imaging Guided Therapy. Biomaterials 2016, 105, 77−88. (13) Schartner, E. P.; Jin, D.; Ebendorff-Heidepriem, H.; Piper, J. A.; Lu, Z.; Monro, T. M. Lanthanide Upconversion within Microstructured Optical Fibers: Improved Detection Limits for Sensing and the Demonstration of a New Tool for Nanocrystal Characterization. Nanoscale 2012, 4, 7448−7451. (14) Lu, Y.; Zhao, J.; Zhang, R.; Liu, Y.; Liu, D.; Goldys, E. M.; Yang, X.; Xi, P.; Sunna, A.; Lu, J.; Shi, Y.; Leif, R. C.; Huo, Y.; Shen, J.; Piper, J. A.; Robinson, J. P.; Jin, D. Tunable Lifetime Multiplexing Using Luminescent Nanocrystals. Nat. Photonics 2013, 8, 32−36. (15) Zhou, B.; Shi, B.; Jin, D.; Liu, X. Controlling Upconversion Nanocrystals for Emerging Applications. Nat. Nanotechnol. 2015, 10, 924−936. (16) Wang, C.; Tao, H.; Cheng, L.; Liu, Z. Near-Infrared Light Induced in vivo Photodynamic Therapy of Cancer Based on Upconversion Nanoparticles. Biomaterials 2011, 32, 6145−6154. (17) Wang, C.; Cheng, L.; Liu, Z. Upconversion Nanoparticles for Photodynamic Therapy and Other Cancer Therapeutics. Theranostics 2013, 3, 317−330. (18) Liu, K.; Liu, X.; Zeng, Q.; Zhang, Y.; Tu, L.; Liu, T.; Kong, X.; Wang, Y.; Cao, F.; Lambrechts, S. A. G.; Aalders, M. C. G.; Zhang, H. Covalently Assembled NIR Nanoplatform for Simultaneous Fluorescence Imaging and Photodynamic Therapy of Cancer Cells. ACS Nano 2012, 6, 4054−4062. (19) Lucky, S. S.; Muhammad Idris, N.; Li, Z.; Huang, K.; Soo, K. C.; Zhang, Y. Titania Coated Upconversion Nanoparticles for NearInfrared Light Triggered Photodynamic Therapy. ACS Nano 2015, 9, 191−205. (20) Sun, M.; Xu, L.; Ma, W.; Wu, X.; Kuang, H.; Wang, L.; Xu, C. Hierarchical Plasmonic Nanorods and Upconversion Core-Satellite Nanoassemblies for Multimodal Imaging-Guided Combination Phototherapy. Adv. Mater. 2016, 28, 898−904. (21) Lu, F.; Yang, L.; Ding, Y.; Zhu, J. J. Highly Emissive Nd3+Sensitized Multilayered Upconversion Nanoparticles for Efficient 795 nm Operated Photodynamic Therapy. Adv. Funct. Mater. 2016, 26, 4778−4785. (22) Tian, G.; Ren, W.; Yan, L.; Jian, S.; Gu, Z.; Zhou, L.; Jin, S.; Yin, W.; Li, S.; Zhao, Y. Red-Emitting Upconverting Nanoparticles for Photodynamic Therapy in Cancer Cells under Near-Infrared Excitation. Small 2013, 9, 1929−1938. (23) Yuan, Y.; Min, Y.; Hu, Q.; Xing, B.; Liu, B. NIR Photoregulated Chemo- and Photodynamic Cancer Therapy Based on Conjugated Polyelectrolyte-Drug Conjugate Encapsulated Upconversion Nanoparticles. Nanoscale 2014, 6, 11259−11272. (24) Fan, W.; Shen, B.; Bu, W.; Chen, F.; He, Q.; Zhao, K.; Zhang, S.; Zhou, L.; Peng, W.; Xiao, Q.; Ni, D.; Liu, J.; Shi, J. A Smart Upconversion-Based Mesoporous Silica Nanotheranostic System for Synergetic Chemo-/Radio-/Photodynamic Therapy and Simultaneous MR/UCL Imaging. Biomaterials 2014, 35, 8992−9002.

(25) Jayakumar, M. K. G.; Bansal, A.; Huang, K.; Yao, R.; Li, B. N.; Zhang, Y. Near-Infrared-Light-Based Nano-Platform Boosts Endosomal Escape and Controls Gene Knockdown in vivo. ACS Nano 2014, 8, 4848−4858. (26) Idris, N. M.; Jayakumar, M. K. G.; Bansal, A.; Zhang, Y. Upconversion Nanoparticles as Versatile Light Nanotransducers for Photoactivation Applications. Chem. Soc. Rev. 2015, 44, 1449−1478. (27) Punjabi, A.; Wu, X.; Tokatli-Apollon, A.; El-Rifai, M.; Lee, H.; Zhang, Y.; Wang, C.; Liu, Z.; Chan, E. M.; Duan, C.; Han, G. Amplifying the Red-Emission of Upconverting Nanoparticles for Biocompatible Clinically Used Prodrug-Induced Photodynamic Therapy. ACS Nano 2014, 8, 10621−10630. (28) Wu, X.; Zhang, Y.; Takle, K.; Bilsel, O.; Li, Z.; Lee, H.; Zhang, Z.; Li, D.; Fan, W.; Duan, C.; Chan, E. M.; Lois, C.; Xiang, Y.; Han, G. Dye-Sensitized Core/Active Shell Upconversion Nanoparticles for Optogenetics and Bioimaging Applications. ACS Nano 2016, 10, 1060−1066. (29) Yue, C. X.; Zhang, C. L.; Alfranca, G.; Yang, Y.; Jiang, X. Q.; Yang, Y. M.; Pan, F.; de la Fuente, J. M.; Cui, D. X. Near-Infrared Light Triggered ROS-activated Theranostic Platform based on Ce6CPT-UCNPs for Simultaneous Fluorescence Imaging and ChemoPhotodynamic Combined Therapy. Theranostics 2016, 6, 456−469. (30) Topalian, S. L.; Drake, C. G.; Pardoll, D. M. Immune Checkpoint Blockade: A Common Denominator Approach to Cancer Therapy. Cancer Cell 2015, 27, 450−461. (31) Melero, I.; Hervas-Stubbs, S.; Glennie, M.; Pardoll, D. M.; Chen, L. Immunostimulatory Monoclonal Antibodies for Cancer Therapy. Nat. Rev. Cancer 2007, 7, 95−106. (32) Walter, S.; Weinschenk, T.; Stenzl, A.; Zdrojowy, R.; Pluzanska, A.; Szczylik, C.; Staehler, M.; Brugger, W.; Dietrich, P.-Y.; Mendrzyk, R.; et al. Multipeptide Immune Response to Cancer Vaccine IMA901 after Aingle-Dose Cyclophosphamide Associates with Longer Patient Survival. Nat. Med. 2012, 18, 1254−1261. (33) Mellman, I.; Coukos, G.; Dranoff, G. Cancer Immunotherapy Comes of Age. Nature 2011, 480, 480−489. (34) Pardoll, D. M. The Blockade of Immune Checkpoints in Cancer Immunotherapy. Nat. Rev. Cancer 2012, 12, 252−264. (35) 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. (36) Topalian, S. L.; Taube, J. M.; Anders, R. A.; Pardoll, D. M. Mechanism-Driven Biomarkers to Guide Immune Checkpoint Blockade in Cancer Therapy. Nat. Rev. Cancer 2016, 16, 275−287. (37) Wang, C.; Sun, W.; Wright, G.; Wang, A. Z.; Gu, Z. Inflammation-Triggered Cancer Immunotherapy by Programmed Delivery of CpG and Anti-PD1 Antibody. Adv. Mater. 2016, 28, 8912−8920. (38) Vanneman, M.; Dranoff, G. Combining Immunotherapy and Targeted Therapies in Cancer Treatment. Nat. Rev. Cancer 2012, 12, 237−251. (39) Wang, C.; Ye, Y.; Hochu, G. M.; Sadeghifar, H.; Gu, Z. Enhanced Cancer Immunotherapy by Microneedle Patch-Assisted Delivery of Anti-PD1 Antibody. Nano Lett. 2016, 16, 2334−2340. (40) Ye, Y.; Wang, J.; Hu, Q.; Hochu, G. M.; Xin, H.; Wang, C.; Gu, Z. Synergistic Transcutaneous Immunotherapy Enhances Antitumor Immune Responses through Delivery of Checkpoint Inhibitors. ACS Nano 2016, 10, 8956−8963. (41) Acharya, A. P.; Sinha, M.; Ratay, M. L.; Ding, X.; Balmert, S. C.; Workman, C. J.; Wang, Y.; Vignali, D. A.; Little, S. R. Localized MultiComponent Delivery Platform Generates Local andSystemic AntiTumor Immunity. Adv. Funct. Mater. 2017, 27, 1604366. (42) 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, 13193. (43) Cheng, L.; Yang, K.; Li, Y.; Chen, J.; Wang, C.; Shao, M.; Lee, S. T.; Liu, Z. Facile Preparation of Multifunctional Upconversion 4473

DOI: 10.1021/acsnano.7b00715 ACS Nano 2017, 11, 4463−4474

Article

ACS Nano

(63) Sallusto, F.; Lenig, D.; Forster, R.; Lipp, M.; Lanzavecchia, A. Two Subsets of Memory T Lymphocytes with Distinct Homing Potentials and Effector Functions. Nature 1999, 401, 708−712. (64) Wherry, E. J.; Teichgräber, V.; Becker, T. C.; Masopust, D.; Kaech, S. M.; Antia, R.; Von Andrian, U. H.; Ahmed, R. Lineage Relationship and Protective Immunity of Memory CD8 T Cell Subsets. Nat. Immunol. 2003, 4, 225−234. (65) Kaech, S. M.; Wherry, E. J.; Ahmed, R. Effector and Memory Tcell Differentiation: Implications for Vaccine Development. Nat. Rev. Immunol. 2002, 2, 251−262. (66) Xu, L.; Liu, Y.; Chen, Z.; Li, W.; Liu, Y.; Wang, L.; Ma, L.; Shao, Y.; Zhao, Y.; Chen, C. Morphologically Virus-Like Fullerenol Nanoparticles Act as the Dual-Functional Nanoadjuvant for HIV-1 Vaccine. Adv. Mater. 2013, 25, 5928−5936.

Nanoprobes for Multimodal Imaging and Dual-Targeted Photothermal Therapy. Angew. Chem. 2011, 123, 7523−7528. (44) Palucka, K.; Banchereau, J. Cancer Immunotherapy via Dendritic Cells. Nat. Rev. Cancer 2012, 12, 265−277. (45) Janeway, C. A., Jr.; Bottomly, K. Signals and Signs for Lymphocyte Responses. Cell 1994, 76, 275−285. (46) Kasturi, S. P.; Skountzou, I.; Albrecht, R. A.; Koutsonanos, D.; Hua, T.; Nakaya, H. I.; Ravindran, R.; Stewart, S.; Alam, M.; Kwissa, M.; Villinger, F.; Murthy, N.; Steel, J.; Jacob, J.; Hogan, R. J.; GarciaSastre, A.; Compans, R.; Pulendran, B. Programming the Magnitude and Persistence of Antibody Responses with Innate Immunity. Nature 2011, 470, 543−547. (47) Firczuk, M.; Nowis, D.; Golab, J. PDT-Induced Inflammatory and Host Responses. Photoch. Photobio. Sci. 2011, 10, 653−663. (48) Gollnick, S. O.; Vaughan, L.; Henderson, B. W. Generation of Effective Antitumor Vaccines Using Photodynamic Therapy. Cancer Res. 2002, 62, 1604−1608. (49) Korbelik, M.; Sun, J.; Cecic, I. Photodynamic Therapy−Induced Cell Surface Expression and Release of Heat Shock Proteins: Relevance for Tumor Response. Cancer Res. 2005, 65, 1018−1026. (50) Kwong, B.; Gai, S. A.; Elkhader, J.; Wittrup, K. D.; Irvine, D. J. Localized Immunotherapy via Liposome-Anchored Anti-CD137+ IL-2 Prevents Lethal Toxicity and Elicits Local and Systemic Antitumor Immunity. Cancer Res. 2013, 73, 1547−1558. (51) Kwong, B.; Liu, H.; Irvine, D. J. Induction of Potent AntiTumor Responses While Eliminating Systemic Side Effects via Liposome-Anchored Combinatorial Immunotherapy. Biomaterials 2011, 32, 5134−5147. (52) Wing, K.; Onishi, Y.; Prieto-Martin, P.; Yamaguchi, T.; Miyara, M.; Fehervari, Z.; Nomura, T.; Sakaguchi, S. CTLA-4 Control over Foxp3+ Regulatory T Cell Function. Science 2008, 322, 271−275. (53) Sakaguchi, S. Naturally Arising Foxp3-Expressing CD25+CD4+ Regulatory T Cells in Immunological Tolerance to Self and Non-Self. Nat. Immunol. 2005, 6, 345−352. (54) Wang, C.; Xu, L.; Liang, C.; Xiang, J.; Peng, R.; Liu, Z. Immunological Responses Triggered by Photothermal Therapy with Carbon Nanotubes in Combination with Anti-CTLA-4 Therapy to Inhibit Cancer Metastasis. Adv. Mater. 2014, 26, 8154−8162. (55) Kagi, D.; Ledermann, B.; Burki, K.; Seiler, P.; Odermatt, B.; Olsen, K. J.; Podack, E. R.; Zinkernagel, R. M.; Hengartner, H. Cytotoxicity Mediated by T Cells and Natural Killer Cells is Greatly Impaired in Perforin-Deficient Mice. Nature 1994, 369, 31−37. (56) Smyth, M. J.; Trapani, J. A. Granzymes: Exogenous Porteinases that Induce Target Cell Apoptosis. Immunol. Today 1995, 16, 202− 206. (57) Stenger, S.; Hanson, D. A.; Teitelbaum, R.; Dewan, P.; Niazi, K. R.; Froelich, C. J.; Ganz, T.; Thoma-Uszynski, S.; Melián, A. n.; Bogdan, C.; Porcelli, S. A.; Bloom, B. R.; Krensky, A. M.; Modlin, R. L. An Antimicrobial Activity of Cytolytic T Cells Mediated by Granulysin. Science 1998, 282, 121−125. (58) Facciabene, A.; Motz, G. T.; Coukos, G. T-Regulatory Cells: Key Players in Tumor Immune Escape and Angiogenesis. Cancer Res. 2012, 72, 2162−2171. (59) Duraiswamy, J.; Kaluza, K. M.; Freeman, G. J.; Coukos, G. Dual Blockade of PD-1 and CTLA-4 Combined with Tumor Vaccine Effectively Restores T-Cell Rejection Function in Tumors. Cancer Res. 2013, 73, 3591−3603. (60) Kinjyo, I.; Qin, J.; Tan, S.-Y.; Wellard, C. J.; Mrass, P.; Ritchie, W.; Doi, A.; Cavanagh, L. L.; Tomura, M.; Sakaue-Sawano, A. RealTime Tracking of Cell Cycle Progression During CD8+ Effector and Memory T-Cell Differentiation. Nat. Commun. 2015, 6, 6301. (61) D’Souza, W. N.; Hedrick, S. M. Cutting Edge: Latecomer CD8 T Cells Are Imprinted with a Unique Differentiation Program. J. Immunol. 2006, 177, 777−781. (62) Teixeiro, E.; Daniels, M. A.; Hamilton, S. E.; Schrum, A. G.; Bragado, R.; Jameson, S. C.; Palmer, E. Different T Cell Receptor Signals Determine CD8+ Memory versus Effector Development. Science 2009, 323, 502−505. 4474

DOI: 10.1021/acsnano.7b00715 ACS Nano 2017, 11, 4463−4474