Reprogramming Tumor-Associated Macrophages by Nanoparticle

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Reprogramming Tumor-Associated Macrophages by Nanoparticle-based Reactive Oxygen Species Photogeneration Changrong Shi, Ting Liu, Zhide Guo, Rongqiang Zhuang, Xianzhong Zhang, and Xiaoyuan Chen Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b03568 • Publication Date (Web): 19 Oct 2018 Downloaded from http://pubs.acs.org on October 19, 2018

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Reprogramming Tumor-Associated Macrophages by Nanoparticle-based Reactive Oxygen Species Photogeneration Changrong Shi1#, Ting Liu1*#, Zhide Guo1, Rongqiang Zhuang1, Xianzhong Zhang1*, Xiaoyuan Chen2*

1

State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center for

Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen, 361102, China. 2

Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging

and Bioengineering, National Institutes of Health, Bethesda, MD 20892 *Correspondence: [email protected], [email protected], [email protected] # C.S. and T.L. contributed equally on this work.

ABSTRACT: Without coordinated strategies to mitigate the immunosuppressive nature of the tumor microenvironment, cancer immunotherapy generally offers limited clinical benefit for established tumors. Tumor associated macrophages (TAMs) are the critical driver of this immunosuppressive tumor microenvironment, which also promotes tumor metastasis. Here we successfully reprogrammed TAMs to an anti-tumor M1 phenotype using precision nanoparticlebased reactive oxygen species (ROS) photogeneration, which demonstrated superior efficiency and efficacy over lipopolysaccharide (LPS) stimulation. Meanwhile, antigen presentation and T cell-priming by TAMs were enhanced by inhibiting lysosomal proton pump and proteolytic activity or by promoting tumor associated antigen (TAA) release in the cytoplasm. The reprogrammed TAMs orchestrate cytotoxic lymphocyte (CTL) recruitment in the tumor and direct

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memory T cells toward tumoricidal responses. This strategy could effectively eradicate tumors, inhibit metastasis, and further prevent their recurrence, which holds tremendous promise to realize potent cancer immunotherapy. KEYWORDS: Tumor associated macrophages, Cancer immunotherapy, Reactive oxygen species, Photogeneration

Cancer immunotherapy is attracting more attention as a next generation cancer therapeutic strategy due to its ability to harness the innate immune system to attack tumor cells1. Several types of cancer immunotherapies including cytokine therapy, adoptive T-cell transfer, tumor-associated antigen (TAA), and checkpoint-blockade therapy, have been well studied in both preclinical and clinical settings2, 3. However, high numbers of activated tumor specific CD4+ Th1 and cytotoxic CD8+ T cells (CTL) still fail to reject established tumors due to the immunosuppressive nature of the tumor microenvironment which is resistant to effector T cell infiltration. The critical drivers of this immunosuppressive tumor microenvironment are the abundant tumor-associated macrophages (TAMs), which not only suppress T cell activation, but also promote tumor metastatic extravasation and impart resistance to cytotoxic therapies4-6. Thus, strategies that can eliminate the TAM suppression may offer great therapeutic potential for cancer. Macrophages are highly plastic, which may undergo classical M1 activation or alternative M2 activation in response to various stimulations7. M1 macrophages are extremely potent effector cells that can kill tumor cells via nitric oxide (NO) and tumor necrosis factor alpha (TNF-α). In contrast, M2 macrophages, such as TAMs, are involved in tumor progression by reciprocal interaction with malignant cells8. Moreover, the phenotype of polarized M1-M2 macrophages can be reversed in vitro and in vivo9-12. Therefore, therapeutics that could redirect M2 TAMs to M1


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phenotype would not only mitigate the TAM suppression, but also induce an antitumor innate response. Several studies demonstrated that activation of NF-κB pathway and modulation of STAT1 activity can repolarize the M2 macrophages toward an M1 antitumor phenotype by using toll-like receptor (TLR) agonists, such as IL-10 mAbs, anti-CD40 mAbs and IFN-γ9,

11, 13, 14.

However, these strategies require the combination of several of these agents to achieve rapid phenotype switching. Moreover, although reorienting and reshaping TAM polarization is the holy grail of macrophage-based cancer therapy, only activating innate immunity is difficult to achieve complete tumor rejection. Developing effective TAM-directed adaptive immunotherapeutic strategies would facilitate eradication of tumors, inhibition of metastasis, and further prevent tumor recurrence, achieving comprehensive cancer immunotherapy. As TAMs are highly infiltrative in the tumor microenvironment, once serving as antigen-presenting cells (APCs), they have more opportunities to capture, process, and present tumor associated antigen (TAA) to T cells, and activate specific anticancer defenses. However, although macrophages have the ability to activate memory T cells, they are not efficient in antigen presentation and naïve T cell activation due to enhanced lysosomal proteolytic activity and lack of cross-presentation15, 16, 17, 18, 19-22. Moreover, the lysosomes of macrophages have a low pH due to enhanced activity of the proton pumps, which further increase antigen degradation. Thus, precise control of proteolysis and the pH of lysosomes to preserve antigens from degradation is required for the loading of antigenic peptides onto MHC II. Enhancing antigen presentation of TAMs is essential for a successful TAM-directed cancer immunotherapy. Macrophage reprogramming toward M1 phenotype along with proinflammatory gene expression is mediated by activation of NF-κB signaling cascades23. Reactive oxygen species (ROS) serve as second messengers in M1 signal transduction, facilitating the regulation of


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downstream pathways of mitogen-activated protein kinase (MAPK) and NF-κB via the oxidation of cysteine residues of proteins24. In addition, the cytoplasmic ROS are reported to participate in adaptive immune response by enhancing the activation of proteasomes which are involved in the cross presentation of soluble antigens25, 26. Moreover, physiological ROS can temporarily modify the function and three-dimensional structure of biomolecules by redox reactions27, which carry the potential for modifying the action of proteases and H+ pumps in endosomes/lysosomes to change the environment of lysosomes, prevent antigen degradation, and enhance antigen-presentation of TAMs. Thus, we asked whether precision nanoparticle-based ROS photogeneration in TAMs would be an effective way to repolarize TAMs and enhance their antigen presentation by directly activating the M1 signaling pathway and inhibiting lysosomal proton pump and proteolytic activity as a strategy for cancer immunotherapy. Photosensitizers could generate multiple ROS types (1O2, HO·, H2O2) under light irradiation with specific wavelength, which can be controlled by turning on or off light28. Many types of ROS will not migrate far from their source of production (< 20 nm) because of their transient and reactive nature, and the redox-buffering capacity of cells27. In M1 signal transduction, ROS are generated in the endosomal membrane by NADPH oxidases in response to lipopolysaccharide (LPS) or interferon-γ (IFN-γ) stimulation. Upon entering the cytoplasm, they oxidize the catalytic cysteine of MAPK phosphatase, and trigger activation of MAPK cascades including JNK and p38 MAPK29, 30. Thus, initial ROS photogeneration in endosome/lysosome or cytoplasm might have different effects on activation of M1 signal transduction. Therefore, we coencapsulated photosensitizers indocyanine green (ICG) and titanium dioxide (TiO2) with or without ammonium bicarbonate (NH4HCO3) in mannose-modified PEGylated poly(lactic-coglycolic acid) (PLGA) nanoparticles for the delivery of photosensitizers to endosome/lysosome or


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cytoplasm of TAMs (Figure 1a). When nanoparticles are internalized by TAMs through mannose receptor-mediated endocytosis, NH4HCO3 in the core of nanoparticles could produce CO2 and NH3 to disrupt the endosome/lysosome membrane and release photosensitizers into the cytoplasm, upon endosome/lysosome acidification (pH ~5.0-6.0)31. This is based on the fact that TAMs overexpress mannose receptor, which can specifically bind with mannose on the surface of nanoparticles. To ensure that photosensitizer could be equally distributed in macrophages to generate ROS and limit ROS toxicity, photosensitizer loading capacity of nanoparticles were around 1% (Table S1). The formulated nanoparticles (MAN-PLGA (without NH4HCO3) and MAN-PLGA-N (with NH4HCO3)) showed well-defined spherical shape and homogenous sizes as revealed by the transmission electron microscope (TEM) images (Figure S1a). MAN-PLGA and MAN-PLGA-N had similar size, charge, and photosensitizer loading capacity. Characteristics of nanoparticles were shown in Figure S1 and Table S1. Flow cytometry analysis revealed the specificity of MAN-PLGA and MAN-PLGA-N toward M2 macrophages (Figure S2a). Moreover, after incubation of M2 macrophages with MAN-PLGA and MAN-PLGA-N at 37 °C, confocal microscopy showed that MAN-PLGA mostly colocalized with lysosomes up to 12 h, with increase in lysosome size (Figure S2b). In contrast, MAN-PLGA-N uptake was accompanied by disruption of lysosomes from 3 h, and most of the ICG escaped from the lysosome, with evident distribution throughout the cytoplasm, but a noticeable absence in the nucleus. These data demonstrated that MAN-PLGA

and

MAN-PLGA-N

nanoparticles

could

be

specifically

delivered

to

endosome/lysosome and cytoplasm, respectively. When nanoparticle labeled M2 macrophages were exposed to 808 nm laser (0.7 W/cm2), the fluorescence signals of pretreated ROS-sensitive fluorescent dye, 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) reactive to H2O2 and hydroxyl radical (HO·) or singlet oxygen sensor green (SOSG) responsive to singlet oxygen (1O2)


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gradually increased over time, which could be stopped by turning off the 808 nm laser (0.7 W/cm2), demonstrating that nanoparticle-based ROS photogeneration could be precisely controlled (Figure S2c,d). We detected intracellular calcium levels, an important second messenger to transduce signal, with a calcium-sensitive fluorescent dye, Fluo-4 AM, during illumination and found the changes in intracellular calcium levels were consistent with that of ROS photogeneration (Figure S2e). We optimized the nanoparticle concentration and illumination condition under which macrophages had high cell viability and low apoptosis (Figure S3 and S4). We then studied nanoparticle biodistribution in BALB/C mice bearing 4T1 breast cancer after i.v. administration, and found that tumor accumulation of MAN-PLGA and MAN-PLGA-N peaked at 6 h (Figure S5a,b), which was significantly higher than those in other organs by ways of ex vivo optical imaging and ICP-MS (Figure S5c-e). Collection and analysis of tumor samples at 6 h after nanoparticle injection by confocal microscopy and flow cytometry revealed that nanoparticles were mainly located in TAMs (CD206+F4/80+) with only few nanoparticles in M1 macrophages, tumor cells, DCs, T cells, granulocytes, and NK cells, which could be blocked by pretreatment with 300-fold excess of free mannose (Figure 2a and S6), demonstrating the specificity of MAN-PLGA and MAN-PLGA-N toward TAMs. Moreover, more spatiotemporal information provided by TEM revealed that MAN-PLGA nanoparticles were mainly located in the endosome/lysosome of macrophages at 6 h after nanoparticle injection, whereas most MANPLGA-N nanoparticles escaped from the disrupted endosome/lysosome and distributed in the cytoplasm (Figure 2b). To ensure that TAMs still had high cell viability after intracellular ROS photogeneration, we started illumination at 6 h after nanoparticle injection to generate ROS in TAMs to optimize the illumination condition by flow cytometry analysis of TAM apoptosis using Annexin V, caspase-3, casepase-8, and propidium iodide (PI) staining. We observed that MAN-


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PLGA or MAN-PLGA-N at a dose of 5, 20 or 50 µg/mL could not lead to TAM apoptosis (Figure S7). In addition, serum AST and ALT and mouse weight were examined to determine systemic toxicity and revealed nontoxic doses of MAN-PLGA and MAN-PLGA-N (5, 20 or 50 µg/mL) (Figure S8a, b). To investigate whether ROS photogeneration in endosome/lysosome or cytoplasm could induce phenotype switching of M2 macrophages, we firstly evaluated several known markers of M1/M2 macrophages at 1 h after illumination using flow cytometry in vitro. Interestingly, after 3 min of 808 nm (0.7 W/cm2) laser

illumination, both MAN-PLGA and MAN-PLGA-N

nanoparticle treatments promptly increased the expression of M1 markers iNOS and interleukin12 (IL-12), and reduced the levels of M2 markers CD206 and interleukin-10 (IL-10), in a nanoparticle concentration-dependent manner (Figure 1b, S9a and S10a,b). This phenomenon was observed not only in RAW264.7 cell line, but also in bone marrow-derived macrophages. Importantly, this was not a transient effect, the upregulation of M1 markers and downregulation of M2 markers could still be detected at 24 h after treatment (Figure S10a), while the pattern of M1/M2 marker expression after nanoparticle alone or laser alone treatment was not changed compared with that of standard M2 macrophages. These data indicated that intracellular ROS photogeneration could skew M2 macrophages to M1 phenotype. Interestingly, at the same level of ROS generation, MAN-PLGA-N treatment showed higher iNOS and IL-12 levels and lower CD206 and IL-10 levels than those after MAN-PLGA treatment. We further confirmed the repolarization of M2 macrophages by ROS photogeneration by using NAC (N-acetyl-L-cysteine), an inhibitor for ROS generation, which abolished the phenotype modification by ROS photogeneration (Figure 1b, S9 and S10a). It is worth noting that the induction of M2 macrophage repolarization by intracellular ROS photogeneration was more rapid and efficacious than that after


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LPS stimulation, which could skew M2 macrophages to M1 phenotype after at least 4 h incubation at the maximal nontoxic concentration (2 µg/mL). In addition, we observed that several M1 markers, including TNF-α, IFN-γ, MHC-II and CD86, were upregulated after intracellular ROS photogeneration (Figure S9b). To demonstrate that this is a universal phenomenon, TiO2, an oxygen independent nanophotosensitizer32, was used for ROS photogeneration under UV irradiation. ROS generation in M2 macrophages by TiO2 under 10 min of UV illumination similarly increased iNOS expression and decreased CD206 level, which was also nanoparticle concentration-dependent (Figure S10c). Moreover, we observed that treatment with low concentration of TiO2 (8.8 µg/mL) under 60 min of UV illumination could also induce M2 macrophage repolarization to M1 phenotype with high cell viability. Importantly, the temperature was not elevated during ROS photogeneration by TiO2 under UV illumination. And heat alone could not decrease IL-10 level (Figure S10d). These data indicated that M2 macrophage repolarization to M1 phenotype by nanoparticle treatment under illumination was directed by ROS photogeneration not by slight increase in temperature during illumination. We determined the gene expression profile of M2 macrophages after ROS stimulation and found that the macrophages treated with nanoparticles plus illumination displayed reduced expression of M2 phenotype related genes, including Arg1, Retnla, Ccl22 and IL-10. In contrast, M1 signature genes, such as Nos2, IL12a, Cxcl10 and IL-6 were upregulated (Figure S11a), which were not observed in the macrophages treated with nanoparticles alone, further indicating M2 phenotype switching to M1 by ROS photogeneration. To dig into the mechanism of M2 macrophage repolarization by intracellular ROS photogeneration, we analyzed signaling activation at 10, 60, and 240 min after ROS photogeneration in endosome/lysosome or in cytoplasm and compared results with LPS


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stimulation. Substantial increase in phosphorylation of p38, JNK MAP kinases, and ERK was observed as early as 10 min after ROS stimulation and sustained for up to 4 h, which was delayed and not significant until 1 h after LPS stimulation (Figure S11b). Increase in nuclear translocation of NF-κB, NF-κB phosphorylation, and activation of STAT1 were observed after illumination, suggesting M1 signaling activation by ROS photogeneration. The activation of M1 signaling pathway by ROS photogeneration in cytoplasm by MAN-PLGA-N was more rapid and effective and lasted for a longer period of time than that in endosome/lysosome by MAN-PLGA, which could be abolished by NAC, indicating that M2 phenotype switching by ROS photogeneration was mediated by directly phosphorylating M1 signal transduction proteins in the cytoplasm. Notch signaling plays a critical role in determination of M2 vs. M1 phenotype polarization through suppressors of cytokine signaling (SOCS) proteins, SOCS3 and SOCS1, by feedback inhibition of the JAK/STAT signaling pathway33. We observed that SOCS1 was upregulated after ROS photogeneration, whereas SOCS3 was downregulated, which further demonstrated that intracellular ROS photogeneration could skew M2 macrophages to M1 phenotype by activating the M1 signaling pathway. We then determined the TAM phenotype by flow cytometry analysis at 24 h after intravenous treatment with MAN-PLGA or MAN-PLGA-N at a dose of 5, 20 or 50 µg/mL in 4T1 tumor mice, followed by 3 min of 808 nm (0.7 W/cm2) laser illumination. Both MAN-PLGA and MAN-PLGA-N treatments plus illumination drastically increased iNOS expression in TAMs and changed their activity to produce IL-12, consistent with the M1 phenotype. Meanwhile the expression levels of M2 markers CD206 and IL-10 decreased, in a nanoparticle dose-dependent manner (Figure 2c,d and S12a), which was not observed after treatment with nanoparticles alone. Moreover, consistent with in vitro findings, under illumination, treatment with MAN-PLGA-N


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showed more effectiveness than MAN-PLGA administration. It is worth noting that we did not detect significant differences in the total number of macrophages at 24 h after treatment with nanoparticles plus illumination compared with that after saline treatment in tumors of the same volume (Figure S13). These data demonstrated that nanoparticle-based ROS photogeneration could skew TAMs to M1 phenotype in vivo, as opposed to recruitment of new M1 macrophages. We further verified TAM repolarization after treatment with nanoparticles plus illumination by immunostaining of tumor sections, and observed large number of iNOS+ macrophages in the tumor, especially around the necrotic region where CD206 expression significantly decreased, which was in contrast with that after saline treatment where CD206-expressing macrophages were most abundantly represented (Figure 2e). We also used FACS to isolate TAMs from tumors harvested at 24 h after nanoparticle administration and determined their gene expression profiles. TAMs from the tumors treated with nanoparticles plus laser displayed reduced expression of immunosuppressive molecules, including Arg1, Retnla, Ccl22, and IL-10. In contrast, anti-tumor immunity genes, such as Nos2, IL-12a, Cxcl10, and IL-6, were upregulated compared with those after saline treatment (Figure 2f), but not after treatment with nanoparticles alone. Additionally, we found that intracellular ROS photogeneration not only promptly switched infiltrating TAMs from M2 to M1 phenotype, but also triggered innate response to debulk tumor cells within 24 h. Enhanced phagocytosis of apoptotic tumor cells by TAMs was observed by TEM at 24 h after treatment (Figure S13a). Meanwhile, TNF-α and IFN-γ levels in TAMs were significantly increased (Figure S13b). These results demonstrated that intracellular ROS photogeneration could restore antitumor ability of TAMs to promote the innate antitumor response. It has been reported that singlet oxygen can inhibit activities of cysteine proteases and proton pump34, 35. And the cytoplasmic ROS could enhance the activation of proteasomes and stimulate


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autophagy, which could facilitate the cross presentation of soluble antigens36. To investigate whether modulation of the environment in the lysosome of TAMs by ROS photogeneration or endosomal escape of exogenous antigen and ROS would be efficient ways to improve antigen presentation of TAMs. We co-encapsulated TAA made from whole GFP+ tumor cell lysates in the core of MAN-PLGA and MAN-PLGA-N nanoparticles and delivered them to the endosome/lysosome or cytoplasm of cultured M2 macrophages or TAMs in tumor. After illumination, we observed that ROS photogeneration in endosome/lysosome by MAN-PLGATAA could increase the pH in endosome/lysosome and inhibit activities of cathepsins B, L, and S and proton pumps in both cultured M2 macrophages and TAMs in tumor, which could further be abolished by NAC pretreatment (Figure S14a,b). Moreover, we detected the tagged TAA in cytoplasm after MAN-PLGA-N-TAA administration by confocal imaging, and found the increased activity of proteasomes and stimulated autophagy in TAMs after illumination (Figure S14c-f). Induction of immune response involves several steps, including APC capture of antigens and migration into lymph nodes and spleen, where they present antigen peptides to CD4+ and CD8+ T cells and then trigger adaptive immune responses with the help of co-stimulatory molecules37, 38.

We observed that both MAN-PLGA and MAN-PLGA-N treatments plus 808 nm (0.7 W/cm2)

laser illumination could upregulate the expression of MHC I, MHC II, CD40, CD80, CD86, IL-12 and IFN-γ in TAMs, but not nanoparticle only treatment (Figure 3a and S15a). Moreover, the enhanced expression of chemotactic signal (CCR7) and rearrangement of the cytoskeleton (F-actin) in TAMs were found, which were responsible for APC migration (Figure 3a and S15a). To investigate whether intracellular ROS photogeneration could enhance TAM migration into lymph nodes and their antigen presentation, we exploited a foot pad tumor model which often shows


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draining lymph node metastasis 20 days after tumor inoculation39. We injected intratumorally with MAN-PLGA-TAA or MAN-PLGA-N-TAA at 7 days after tumor inoculation. Through optical imaging, we observed that TAMs with ICG migrated from tumor to popliteal lymph nodes at 48 h after 3 min of 808 nm (0.7 W/cm2) laser illumination (Figure 3b), which was not observed after nanoparticle only treatment. We further verified TAM migration by immunofluorescence staining of draining lymph node and quantified TAMs by flow cytometry. A large number of ICG-labeled macrophages situated in the medullary and T cell zone of popliteal lymph nodes, which were significantly more than those treated with nanoparticles alone (Figure 3c and S15b). Interestingly, we observed significant increase in Ki67+GFP+CD3+ T cells and vigorously stimulated CD4+ and CD8+ T cells producing IFN-γ (Figure 3d and S15c-e) and elevated levels of inflammatory cytokines IL-12, IL-4, IL-6 and IFN-γ in draining lymph nodes at day 2 (Figure S15f), which were not observed after treatment with nanoparticles alone, indicating a successful antigen-presentation to T cells by TAMs. Importantly, we could not detect drained nanoparticles in lymph node at day 1 after injection, indicating that antigen presentation and T cell priming were not induced by resident DCs that could capture nanoparticles in lymph nodes (Figure S16a). Moreover, at 48 h after treatment, the number of migratory dendritic cells (CD11c+CD8- DCs) in the draining lymph nodes did not increase and few ICG positive DCs were found in the draining lymph nodes by ways of immunostaining as well as flow cytometry analysis (Figure S16b-d), suggesting antigen presentation and T cell priming were not induced by migratory DCs homing to lymph nodes. These data demonstrated that intracellular ROS photogeneration could enhance antigen-presentation of TAMs which successfully primed CD4+ Th1 and CD8+ cytotoxic T lymphocyte responses. Interestingly, MAN-PLGA-TAA nanoparticles inclined to stimulate tumor specific CD4+ Th1, while MAN-PLGA-N-TAA nanoparticles tended to prime cytotoxic CD8+ T cell responses. To


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further confirm the adaptive immunity generated by repolarized TAMs by intracellular ROS photogeneration, 4T1 tumor-bearing mice were intratumorally treated with nanoparticles loaded with OVA257-264, followed by 808 nm (0.7 W/cm2) laser illumination. At 7 days after treatment, the lymph nodes were isolated and the OVAp-specific T cell response was measured by OVApspecific tetramers. The repolarized TAMs isolated from tumors were cocultured in vitro with T cells from nonimmunized mice for 3 days to further prove the generation of antigen-specific T cells. The percentages of OVAp-specific CD4+ and CD8+ T cells after treatment with nanoparticles loaded with OVA257-264 plus illumination were significantly higher than those of free OVAp or saline treatment (Figure S16e,f), indicating a specific T cell response generated by repolarized TAMs by intracellular ROS photogeneration. Moreover, to confirm that T cell response was induced by TAMs not DCs, we depleted TAMs with intravenous pretreatment with clodronate liposomes (CL) before nanoparticle treatment40, which didn’t reduce the number of DCs in the tumor (Figure S16g). We observed that after TAM depletion nanoparticle treatment plus illumination can’t prime T cell immunity, the percentages of OVAp-specific CD4+ and CD8+ T cells in lymph nodes after intratumoral treatment with nanoparticles loaded with OVA257-264 plus illumination were not higher than those of free OVAp treatment in TAM depletion group, indicating that T cell priming induced by intracellular ROS photogeneration was through manipulation of function of TAMs not DCs. To evaluate tumor specific T cell response, we intravenously injected MAN-PLGA-TAA or MAN-PLGA-N-TAA into BALB/C mice bearing 4T1 tumors, followed by 3 min of 808 nm (0.7 W/cm2) laser illumination. We observed that tumor progression was modestly reduced at day 7, accompanied by robust increases in CD3+CD8+ CTLs and CD3+CD4+ effector T cells, and significant decrease in CD3+CD4+Foxp3+ T regulatory cells (TReg) (Figure S17a,b), which were


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not observed in those after nanoparticle only treatment. Notably, the majority of tumor-infiltrating CD8+ CTLs and CD4+ effector T cells had IFN-γ activation. Moreover, we observed upregulation of cytokines responsible for stimulating anti-tumor immunity in tumor on days 1, 3, and 7 after treatment, including IL-12, IFN-γ, IL-6, and TNF-α, and down-regulation of cytokines associated with immune suppression, including cytotoxic T lymphocyte antigen 4 (CTLA4), programmed death 1 ligand 1 (PDL1), macrophage colony-stimulating factor (M-CSF), IL-10, and TGF-β (Figure S17c). To evaluate the therapeutic activity of intracellular ROS photogeneration in TAMs, we intravenously injected a mixture of MAN-PLGA-TAA and MAN-PLGA-N-TAA nanoparticles into normal or nude BALB/c mice bearing subcutaneous 4T1-luc breast murine tumors, followed by 3 min of 808 nm (0.7 W/cm2) laser illumination when tumors reached around 100 mm3. As shown in Figure 4, in BALB/c mice, treatment with nanoparticles plus illumination significantly inhibited tumor growth, extended survival, and resulted in durable cures for 87.5% of the mice, which was significantly more efficient than IFN-γ treatment that has been widely used in clinic. Treatment with MAN-PLGA and MAN-PLGA-N nanoparticles without TAA plus illumination slowed down tumor progression but was difficult to achieve complete inhibition, indicating only activating innate immunity is difficult to achieve complete tumor rejection. There were no significant difference in tumor growth and percent survival between treatment with MAN-PLGATAA and MAN-PLGA-N-TAA nanoparticles or laser alone and saline treatment. While the survival advantage provided by ROS photogeneration in TAMs was lost in nude mice which lack T-cells (Figure S18a), and there was no significant difference in inhibition of tumor growth between treatment with nanoparticles with and without TAA. These data demonstrated that intracellular ROS photogeneration in TAMs could induce an effective cancer immunotherapy


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which was achieved by eliciting a robust effector T-cell response. Moreover, we found that longterm immunity was acquired after treatment with MAN-PLGA-TAA and MAN-PLGA-N-TAA nanoparticles plus illumination. Mice previously cured of 4T1 tumors by treatment with MANPLGA-TAA and MAN-PLGA-N-TAA nanoparticle plus illumination were completely resistant to tumor engraftment when rechallenged with 4T1 cells (Figure S18b). Whereas the rejection of tumor engraftment was not observed in mice initially treated with MAN-PLGA and MAN-PLGAN nanoparticles without TAA plus illumination or IFN-γ, indicating ROS photogeneration in endosome/cytosome or cytoplasm of TAMs is essential to prime T cell immunity. The percentage of effector memory T cells (TEM, CD3+CD8+CD62L-CD44+) was much higher than central memory T cells (TCM, CD3+CD8+CD62L+CD44+) in the spleen after TAM-directed cancer immunotherapy with ROS photogeneration, which expressed high level of IFN-γ (Figure S18c). We also observed TAM repolarization and T cell priming by intracellular ROS photogeneration in large B16 melanoma (ca. 500 mm3) by utilizing Cerenkov radiation (CR) from clinically used radiotracer 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG) to activate TiO2 in TAMs. CR occurs when charged particles, such as positrons and electrons, travel faster than the speed of light in a given medium, emitting predominantly ultraviolet light that tails off to the visible spectrum (250–600 nm)41.

18F-FDG

is an ideal source for CR because of its high positron (β+)

emission decay and relatively short half-life32. TiO2 nanoparticle is an oxygen independent nanophotosensitizer that absorbs ultraviolet light (λ = 275–390 nm) with high efficiency to generate cytotoxic hydroxyl and superoxide radicals32, 42, 43. The hydroxyl radicals are produced through electron–hole transfer to chemisorbed H2O in an oxygen-independent process, whereas superoxide radical generation requires aerobic condition for the electron transfer to molecular oxygen. As a result of its high efficiency in harvesting ultraviolet light, where CR quantum


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efficiency is the highest, we explored the use of TiO2 nanoparticles as a nanophotosensitizer for ROS photogeneration. This strategy could overcome the relatively shallow penetration of 808 nm laser into tissues and the reliance on tissue oxygenation of ICG to generate ROS.

18F-FDG

(50

μCi/mouse) was intravenously administered into mice bearing B16 melanoma at 6 h after nanoparticle treatment. We observed that the dynamic

18F-FDG

accumulation in tumor was

associated with the kinetics of ROS photogeneration in TAMs, which peaked at 1 h after i.v. injection, followed by a rapid decay over time (Figure S8c,d). Treatment with optimized doses of nanoparticles plus

18F-FDG

(50 μCi/mouse), under which TAMs still had high cell viability,

similarly induced a rapid TAM repolarization at 24 h (Figure S12b). And treatment with nanoparticles coencapsulated TAA plus 18F-FDG (50 μCi/mouse) induced increase in IFN-γ+CD4+ and IFN-γ+CD8+ T cells, elevated levels of inflammatory cytokines IL-12, IL-6, TNF-α and IFNγ, and decreased IL-10 expression in the spleen at 48 h in the subcutaneous B16 melanoma model, which further demonstrated that intracellular ROS photogeneration in TAMs could enhance antigen presentation and T cell priming (Figure S19a,b). In the tumor, increase in percentages of IFN-γ+CD4+ and IFN-γ+CD8+ T cells and significantly improved effector-to-TReg and CTL-to-TReg ratios were found from day 3 (Figure S19c,d). We further explored these observations and reformulated PLGA nanoparticles with PEGshedding and in vivo TAA encapsulation capacity for potential clinical translation. Conventional methods usually incorporate TAA made from dissected tumor tissue into nanoparticles with low loading efficiency, which may lead to TAA instability during formulation. We herein reformulated PLGA nanoparticles by conjugating poly-histidine (Phis) between PEG and PLGA (MAN-PHPPLGA and MAN-PHP-PLGA-N) (Figure 5a), which could form complexes with proteins under mild acidic conditions (pH 4.5-7.0)44,

45.

There were abundant TAAs in the tumor


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microenvironment when the apoptosis of tumor cells was induced by NO and TNF-α released by repolarized TAMs. Once the nanoparticles arrive in the tumor region, they have the capacity to encapsulate TAA in the mild acidic tumor microenvironment (~ pH 6.8). Moreover, when TAA/Phis complex is exposed to neutral pH, it could dissociate and release the naïve proteins. Thus, by encapsulating ammonium chloride (NH4Cl) in the core of nanoparticles, the pH of lysosomes of TAMs can be neutralized, leading to the release of TAA from nanoparticles in the lysosomes and facilitating TAA loading onto MHC II. While by encapsulating NH4HCO3 in the core of nanoparticles (MAN-PHP-PLGA-N), TAA could be released from nanoparticles into the neutral cytoplasm, triggered by the interaction of NH4HCO3 with protons in lysosomes of TAMs to produce NH3 and CO2. Furthermore, to selectively deliver photosensitizers to TAMs and avoid uptake by macrophages in the reticuloendothelial system (RES) organs, we used sheddable PEG to modify the nanoparticles, which was synthesized by conjugating PEG with Phis using a hydrazone bond (PEG-hydrazone-Phis, PHP). When the nanoparticles are modified with both Mannose-Phis and PHP, the long PEG chains can shield the mannose and prevent its association with mannose receptors on the RES macrophages before arriving at the tumor region after i.v. injection. Once the nanoparticles accumulate in the acidic tumor microenvironment (~ pH 6.8), the hydrolysis of PHP can be catalyzed, and the mannose will be exposed on the surface of the nanoparticles, which allows recognition and uptake by TAMs via mannose receptor. Characteristics of MAN-PHP-PLGA and MAN-PHP-PLGA-N nanoparticles are shown in Figure S20 and Table S1. First, we determined the acid-sensitive shedding of PEG from the nanoparticles at pH 6.8 by an iodide staining method. MAN-PHP-PLGA and MAN-PHP-PLGAN nanoparticles shed around 20, 50, and 94% of the PEG after incubation at pH 6.8 for 2, 6, and 24 h, respectively. In contrast, no significant PEG shedding was observed even after 24 h of


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incubation at pH 7.4 (Figure S20b). The average size and charge of MAN-PHP-PLGA and MANPHP-PLGA-N were significantly influenced by varying the pH of medium due to PEG shedding and protonation of Phis (Figure 5b and Table S2). Next, we investigated the in vivo TAA encapsulation capacity of MAN-PHP-PLGA and MAN-PHP-PLGA-N nanoparticles. After incubation with 4T1 tumor lysates ex vivo at pH 6.8, both size and zeta potential of MAN-PHP-PLGA and MAN-PHP-PLGA-N changed in comparison to those without TAA incubation (Table S2), which indicated successful formation of the complex between TAA and Phis. We further confirmed the TAA complexation by quantifying the total amount of protein bound. Significantly more proteins were bound to MAN-PHP-PLGA and MANPHP-PLGA-N after incubation with TAA at pH 6.8, as compared to those incubated at pH 7.4 (Figure S20c). We then isolated and identified the proteins using mass spectrometry. Number of proteins bound to MAN-PHP-PLGA and MAN-PHP-PLGA-N was up to 3000, including some tumor-specific antigens such as Actn4 and Dag1 (Table S3, S4 and S5), which were quite different from non-specific TAA binding at pH 7.4. In addition, we investigated TAA release from MANPHP-PLGA at pH 5.0 to mimic lysosome. The amount of TAA released from MAN-PHP-PLGA was up to 80% at 24 h due to the NH4Cl neutralization (Figure S21b). TAA release from MANPHP-PLGA-N was more rapid than that from MAN-PHP-PLGA. The activity of TAA released from nanoparticles was retained as confirmed by the CD spectra (Figure S21c). High tumor accumulation of MAN-PHP-PLGA and MAN-PHP-PLGA-N and their specificity toward TAMs were observed (Figure S22). We investigated the ability of MAN-PHP-PLGA and MAN-PHP-PLGA-N in T cell priming after illumination, and found increase in IFN-γ+CD3+CD4+ T cells and IFN-γ+CD3+CD8+ T cells in lymph node on day 2 after treatment with MAN-PHP-PLGA and MAN-PHP-PLGA-N


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nanoparticles plus 808 nm (0.7 W/cm2) laser illumination in comparison to that after saline treatment (Figure S22f). We further investigated their therapeutic efficacy in inhibition of tumor growth and lung metastasis by systemic administration of a mixture of MAN-PHP-PLGA and MAN-PHP-PLGA-N nanoparticles in mice bearing orthotropic 4T1-luc breast tumors, followed by 18F-FDG (50 μCi/mouse), when tumors reached 100 mm3. Mice were sacrificed at day 32 after tumor inoculation and scored for lung metastases by H&E staining. We found that 8 days of TAMdirected cancer immunotherapy with ROS photogeneration reduced tumor growth and completely prevented metastasis (Figure 5c-f). Whereas without ROS photogeneration, even though MANPHP-PLGA and MAN-PHP-PLGA-N nanoparticles could deliver TAA to TAMs, they failed to impede tumor progression. In C57BL/6 mice bearing subcutaneously implanted B16 melanoma, we intravenously injected a mixture of MAN-PHP-PLGA and MAN-PHP-PLGA-N nanoparticles (1:1), followed by 808 nm (0.7 W/cm2) laser illumination. We observed significant reduction in tumor growth, and increase in mouse survival, resulting in an 75% cure rate (Figure S23). Moreover, we found that mice cured from B16 melanoma were resistant to tumor engraftment of B16 cells, indicating the acquisition of a long-term immunity (Figure S24). We have demonstrated that TAMs play an important role in T cell priming after ROS photogeneration by TAM depletion in 4T1 breast cancer model (Figure S16e). To investigate the role of TAMs in tumor eradication after intracellular ROS photogeneration and exclude the DCs’ contribution, we depleted TAMs with CL before treatment with nanoparticles plus illumination in C57BL/6 mice bearing B16 melanoma. We observed that the advantages in tumor growth inhibition and high survival rate provided by ROS photogeneration in TAMs were lost in the mice pretreated with CL, indicating that intracellular ROS photogeneration induced inhibition of tumor progression was mediated by manipulation of TAM function, which was not directed by DCs.


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Photodynamic therapy (PDT) involves the use of photochemical reactions mediated through activation of photosensitizing agents under light with specific wavelength in the presence of oxygen to generate a great amount of ROS for treatment of malignant diseases. The ROS generated during PDT could directly kill tumor cells, which was much higher than ROS photogeneration for manipulation of the function of TAMs. To differentiate with PDT, we intravenously injected ICG (4 mg/kg) to C57BL/6 mice bearing B16 melanoma and illuminated with 808 nm laser (1.5 W/cm2) for 15 min in the same schedule. PDT with ICG didn’t show as efficient as ROS photogeneration in TAMs for cancer immunotherapy in inhibition of tumor growth and extension of survival (Figure S23). Moreover, at day 40, we removed the initial B16 melanoma treated with PDT and rechallenged with B16 melanoma cells. We observed that the tumor engraftment of B16 cells grew quickly, indicating that PDT fails to acquire a long-term immunity (Figure S24). In addition, we observed normalization and remodeling of the tumor vasculature at 7 days after treatment with nanoparticles plus 3 min of 808 nm (0.7 W/cm2) laser illumination, which was reflected by a reduction in the CD31+ vessel area, an increase of the vessel circularity index, and decrease in hypoxia regions in most tumors (Figure S25a,b). Such vascular normalization would benefit effective T cell immigration into tumors, which occurs through interaction with normal endothelial cells. In contrast, after PDT, a significant reduction in the CD31+ vessel area and decrease in the vessel circularity index, accompanied with significant increase in hypoxia region. These data demonstrated that our strategy of manipulating TAM function with ROS photogeneration for cancer immunotherapy is vitally different from PDT which kills tumor cells and destroys tumor vascular system in the meantime. Moreover, increased hypoxia by PDT will decrease therapeutic efficacy of chemotherapy, radiotherapy as well as PDT itself. Furthermore, the destroyed vasculatures would reduce T cell immigration into the tumor


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region, which is not in favor of cancer immunotherapy. Importantly, PDT itself does not acquire a long-term immunity. One limitation of this study is the rapid attenuation of light within tissue. For example, 808 nm laser of 1 W/cm2 at skin surface would be weakened to only 1 mW/cm2 at 3.4 cm tissue depth46. Although promising, the tumor must be “reachable” by the irradiation. Future studies would focus on how to enhance deep tissue penetration for the photogeneration of ROS. In conclusion, TAM-directed cancer immunotherapy with nanoparticle-based ROS photogeneration efficiently stopped tumor progression and prevented recurrence by skewing TAMs to M1 phenotype and enhanced their antigen-presentation and T cell-priming ability, which has tremendous promise to finally make successful immunotherapy of cancer a reality.

ASSOCIATED CONTENT Supporting Information Additional details on materials, synthesis of MAN-PLGA, MAN-PLGA-N, MAN-PLGA-TAA, MAN-PLGA-N-TAA,

MAN-PHP-PLGA

and

MAN-PHP-PLGA-N

nanoparticles,

flow

cytometric analysis, RT-qPCR, immunohistology, nanoparticle Biodistribution, cytokine analyses, western blot analysis, measurement of Cathepsin B, Cathepsin L, Cathepsin S, H+-ATPase, and proteasome activities, immunization and antigen-specific T cell detection, tumor growth and survival, TAM depletion

COMPETING FINANCIAL INTERESTS The authors of this manuscript declare no competing financial interests.


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ACKNOWLEDGMENTS This work was supported by the National Key Basic Research Program of China (2014CB744503); the National Natural Science Foundation of China (81501533); the Intramural Research Program of the National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health (ZIA EB000073); and partially by the Scientific Research Foundation of the State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics (2016ZY002).

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Figure 1. ROS photogeneration in endosome/lysosome or cytoplasm skews M2 macrophages to M1 phenotype. (a) Schematic illustrating the composition/structure of MAN-PLGA and MAN-


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PLGA-N nanoparticles and their mechanism of action. TAMs overexpress mannose receptor that could mediate endosomal internalization of nanoparticles, and NH4HCO3 in the core of MANPLGA-N nanoparticles could produce CO2 and NH3 to disrupt the endosome/lysosome membrane and release photosensitizers into the cytoplasm, under acidic conditions (pH ~5.0-6.0). Subcellular photosensitizers could generate ROS under 808 nm (0.7 W/cm2) laser illumination, which activate the M1 signal transduction and then repolarize M2 macrophages to M1 phenotype. (b) Flow cytometry analysis of the expression of M1 and M2 markers in M2 macrophages derived from RAW264.7 cells at 1 h after treatment with 20 μg/mL MAN-PLGA or MAN-PLGA-N nanoparticles plus 3 min of 808 nm laser (0.7 W/cm2). MAN-PLGA and MAN-PLGA-N nanoparticle treatment plus illumination significantly upregulated iNOS expression and downregulated CD206 expression compared with that after PBS treatment, which could be abolished by pretreatment with NAC (N-acetyl-L-cysteine). Lipopolysaccharide (LPS) treatment (2 μg/mL, 4 h) was less effective than that after ROS stimulation. Comparison of multiple groups for statistical significance was determined using Tukey’s post hoc test (n = 5 per group; **P < 0.01, *P < 0.05)


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Figure 2. Intracellular ROS photogeneration reprograms TAMs to M1 phenotype in vivo. (a) Immunofluorescence staining of tumor sections at 6 h after intravenous treatment with MANPLGA or MAN-PLGA-N nanoparticles (50 μg/mL, 200 μL) showed accumulation of MANPLGA and MAN-PLGA-N nanoparticles in TAMs. (scale bar 100 µm) (b) TEM examination of the distribution of MAN-PLGA and MAN-PLGA-N nanoparticles in tumors at 6 h after intravenous treatment with MAN-PLGA and MAN-PLGA-N nanoparticles. MAN-PLGA nanoparticles were mainly located in the endosome/lysosome of macrophages at 6 h after nanoparticle injection, whereas most MAN-PLGA-N nanoparticles escaped from the disrupted endosome/lysosome and distributed in the cytoplasm. (c-f) Examination of the expression of M1 and M2 markers in TAMs of tumors intravenously treated with MAN-PLGA or MAN-PLGA-N nanoparticles (50 μg/mL, 200 μL) plus 3 min of 808 nm (0.7 W/cm2) laser illumination by flow cytometry (c, d), RT-qPCR (e) and immunostaining (f). (scale bar 100 µm) The expression of M1 markers was significantly increased, whereas remarkable decrease in levels of M2 markers after ROS stimulation was observed. P value were calculated by Tukey’s post hoc test (**P < 0.01, *P < 0.05) by compared other groups with saline treatment.


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Figure 3. ROS photogeneration enhances antigen-presentation of TAMs and their T cell-priming ability. (a) Flow cytometric analysis of the expression of CCR7, CD80, CD86, MHC-II and MHCI in TAMs separated from tumors treated with MAN-PLGA or MAN-PLGA-N nanoparticles (50 μg/mL, 200 μL) plus 3 min of 808 nm (0.7 W/cm2) laser illumination or nanoparticles alone. Nanoparticle treatment plus illumination significantly increase the expression of CCR7, CD80, CD86, MHC-II, and MHC-I in TAMs, but not with the treatment of nanoparticles alone. (n = 4 per group; **P < 0.01, *P < 0.05 compared with saline) (b) Optical images of BALB/c mice bearing 4T1 tumors in the foot pad taken on days 1 and 2 after intratumoral administration of MAN-PLGATAA or MAN-PLGA-N-TAA nanoparticles (150 μg/mL, 40 μL) plus 3 min of 808 nm (0.7 W/cm2) laser illumination or nanoparticles alone. Arrows indicate the migration of TAMs to the draining lymph node. (c) Immunostaining of the draining lymph nodes harvested on day 2 after treatment. (scale bar 100 µm) (d) Flow cytometry analysis of IFN-γ expressing CD4+ and CD8+ T cells in draining lymph nodes harvested on day 2 after treatment. (n = 5 per group; **P < 0.01, *P < 0.05 compared with saline)


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Fig. 4 TAM-directed cancer immunotherapy with intracellular ROS photogeneration inhibits tumor growth. (a) Schematic of TAM-directed cancer immunotherapy with nanoparticle-based ROS photogeneration. ROS photogeneration in both endosome/lysosome and cytoplasm can repolarize TAMs to an anti-tumor M1 phenotype and enhance their antigen presentation. The reprogrammed TAMs by ROS photogeneration not only are capable of trafficking to draining lymph nodes to present antigens to T cells and prime T cell responses, but also orchestrate CTL recruitment and reduce Treg cells in tumor. (b) Bioluminescence images of BALA/c mice bearing Luc-expressing 4T1 tumors in different groups taken on days 0, 5 and 12 after various treatment. Treatment with a mixture of MAN-PLGA-TAA and MAN-PLGA-N-TAA nanoparticles (1:1, total 50 μg/mL, 200 μL) plus 808 nm laser (0.7 W/cm2) significantly inhibited tumor growth. (c) Tumor growth curves of different groups. Treatment with nanoparticles plus illumination significantly inhibited tumor growth. (d) Percent survival of different groups of mice-bearing 4T1 tumors after various treatments (n = 7 per group). Treatment with nanoparticles plus illumination significantly extended survival.


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Nano Letters


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Fig. 5 Reformulated nanoparticles with in vivo TAA encapsulation capacity for TAM-directed cancer immunotherapy with ROS photogeneration. (a) Schematic of MAN-PHP-PLGA and MAN-PHP-PLGA-N nanoparticles. Conjugating poly-histidine (Phis) between PEG and PLGA (MAN-PHP-PLGA and MAN-PHP-PLGA-N) could form complexes with proteins and encapsulate TAA in the acidic tumor microenvironment (~ pH 6.8). By encapsulating ammonium chloride (NH4Cl) in the core of nanoparticles, the pH of lysosomes of TAMs can be neutralized, leading to the release of TAA from nanoparticles in the lysosomes and facilitating TAA loading onto MHC II. While by encapsulating NH4HCO3 in the core of nanoparticles (MAN-PHP-PLGAN), TAA could be released from nanoparticles into the neutral cytoplasm, triggered by the interaction of NH4HCO3 with protons in lysosomes of TAMs to produce NH3 and CO2. Furthermore, to selectively deliver photosensitizers to TAMs and avoid macrophage uptake in reticuloendothelial system (RES) organs, sheddable PEG was developed by modifying the nanoparticles using a hydrazone bond (PEG-hydrazone-Phis, PHP), which could shield the mannose and prevent its association with mannose receptors on the RES macrophages before arriving at the tumor region. Once the nanoparticles accumulate in the acidic tumor microenvironment (~ pH 6.8), the hydrolysis of PHP can be catalyzed, and the mannose will be exposed on the surface of the nanoparticles. (b) TEM and size distribution plots from dynamic light scatting (DLS) of MAN-PHP-PLGA and MAN-PHP-PLGA-N nanoparticles incubated at pH 6.8 or 7.4. The nanoparticle sizes were increased at pH 6.8 compared with those at pH 7.4. (c) Bioluminescence images of mice (n = 5 per group) bearing orthotopically implanted Lucexpressing 4T1 tumors received nanoparticles (MAN-PHP-PLGA and MAN-PHP-PLGA-N, 1:1, total 22 μg TiO2/mL, 200 μL) plus 18F-FDG (50 μCi/mouse), nanoparticles, or 18F-FDG alone. (d, e) Mice were sacrificed at 32 days and the number of lung metastases was determined after various treatments by hematoxylin/eosin (H&E) staining. TAM-directed cancer immunotherapy with ROS photogeneration completely prevented tumor metastasis. (e) Shown are the lung sections stained with H&E with a 20-fold magnification at 32 days after various treatments. (scale bar, 100 µm) (f) Tumor growth curves of different groups. Treatment with nanoparticles plus illumination significantly inhibited tumor growth.


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