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Intracellularly generated immunological gold nanoparticles for combinatorial photothermal therapy and immunotherapy against tumor Dan Zhang, Tingting Wu, Xianya Qin, Qi Qiao, Lihuan Shang, Qingle Song, Conglian Yang, and Zhiping Zhang Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b02903 • Publication Date (Web): 08 Aug 2019 Downloaded from pubs.acs.org on August 8, 2019

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Intracellularly generated immunological gold nanoparticles for combinatorial photothermal therapy and immunotherapy against tumor Dan Zhang1, 2, Tingting Wu1, 3, Xianya Qin1, Qi Qiao1, Lihuan Shang1, Qingle Song1, Conglian Yang1, and Zhiping Zhang1, 4, 5*

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Tongji School of Pharmacy Department of Pharmacy, Wuhan First Hospital 3 Department of Pharmacy, Union Hospital 4 National Engineering Research Center for Nanomedicine 5 Hubei Engineering Research Center for Novel Drug Delivery System Huazhong University of Science and Technology, Wuhan 430030, China E-mail: [email protected] Telephone: +86-027-83601832 2

Abstract Gold nanoparticle (AuNP) has been widely used in cancer photothermal therapy (PTT) for ablating accessible tumor, while it is insufficient for inhibiting tumor metastasis and relapse in current stage. Here, we first developed a novel immunological AuNP through intracellular generation and exocytosis for combinatorial PTT and immunotherapy. Melanoma B16F10 cells were employed to generate AuNPs firstly and then shed nanoparticle trapped vesicles to extracellular environment with retained tumor antigens (AuNP@B16F10). By further introducing the nanoparticles into dendritic cells (DCs), DCderived AuNPs (AuNP@DCB16F10) were generated with enhanced biosafety, which can induce hyperthermia and provoke antitumor immune responses. This immunological nanoplatform demonstrated efficient inhibition or even eradication of primary tumor, tumor metastasis as well as tumor relapse, with significantly improved overall survival of mice. With our design, the intracellularly generated AuNPs with immunological property could act as an effective treatment modality for cancer.

Keywords: intracellular generation, photothermal therapy, immunotherapy, gold nanoparticles, cancer therapy

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Introduction Gold nanoparticles (AuNPs) are a kind of promising candidate for biomedical applications, which have been widely studied as drug delivery system or mediator for photothermal therapy (PTT) and theranostics14

. As a classical photothermal agent, AuNPs have shown great potential in cancer treatment as efficient

light-to-heat conversion, good photostability, low toxicity, well-defined physicochemical properties and tunable surface functionalities5-9. Conventional AuNPs were mostly synthesized using chemical method, while it was hard to get rid of using the capping agents and reducing agents which were toxic, as well as the high temperature or high pressure condition which was dangerous and complicated during the process. Certain transition metals can also be generated in cell system such as fungi, bacteria and actinomycete1012

. It could be resulted from the carbohydrates and redox enzymes of cell membrane and cytoplasm which

could interact with positive charged metal ions and help reduce them into metal nanoparticles13. On account of this, the growth of AuNP is supposed to be a green and facile approach without other chemical agents. Moreover, in virtue of cellular environment, the nanoparticles could be endowed with bioinformation of parent cells. Almost all cells can secrete vesicles which participate in a variety of biological communication as a miniature of parent cells14. Through shedding or budding, vesicles were released to extracellular environment, consisting of proteins, cell-specific antigens, cytosolic elements and other contents. It has been reported that vesicles can serve as cancer vaccines, therapeutics to regulate systemic immunity or drug delivery vehicles to fight against cancer15, 16. Tumor derived vesicles can convey tumor antigens to dendritic cells (DCs) and achieve the processing and presentation of antigens to T cells for inducing subsequent immune response17, 18. DC derived vesicles can serve as miniature of DCs, improve the biocompatibility and maintain the capability of boosting immune response19-21. Vesicles can also act as a

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tool to discharge foreign substances of cells22. Especially, bioinspired vesicles can convey metal nanoparticles exocytosed by cells with biological information23,

24

. We thus speculated that AuNPs

generated in cells could be exocytosed as nanoparticle trapped vesicles containing similar information as original cells and further serving as a role in intercellular communication. Here we developed a novel kind of immunological AuNP with core-shell structure. The nanoparticles were intracellularly generated, followed by exocytosis from murine melanoma B16F10 cells with retained tumor antigens (AuNP@B16F10), then further internalized by DCs and secreted as DCs derived vesicles (AuNP@DCB16F10). The biological camouflage from DCs could improve the immunological property, confer biocompatibility and stealth to AuNPs as well as get rid of the possibility of metastasis induced by tumor derived vesicles1616, 25. The generated immunological AuNPs could thus realize the combination of AuNPs mediated PTT and tumor antigens based immunotherapy in one nanoplatform. It could not only eradicate primary tumor but also stimulate systemic immunity to attack tumor cells, clear tumor residues and guard against tumor metastasis and recurrence (Scheme 1). To the best of our knowledge, it is the first time to develop intracellularly generated immunological nanoparticles serving as a platform for synergistic antitumor efficacy.


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Scheme 1. Schematic preparation of AuNP@DCB16F10 and mechanism of AuNP@DCB16F10 mediated combinational treatment modality. AuNPs were intracellularly generated in B16F10 cells and further shed to extracellular environment as AuNP@B16F10. Under co-incubation with DC2.4 and exposure to UV irradiation, immunological AuNP@DCB16F10 were obtained. AuNP@DCB16F10 can migrate to both tumor and lymph nodes after administration. With near infrared (NIR) laser irradiation, AuNP@DCB16F10 at tumor site generated hyperthermia, resulting in the apoptosis and necrosis of primary tumor and release of tumor antigens which can be taken up and processed by DCs. Upon activation, mature DCs can present antigens to T cells and trigger subsequent antitumor immune response. This intracellularly generated nanoplatform can mediate efficient combination of PTT and immunotherapy with an obvious effect on the elimination of primary tumor, as well as rejection of distant tumor and rechallenged tumor cells.


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Results and Disccussion Intracellular formation and characterization of AuNP@DCB16F10. AuNPs were generated intracellularly using B16F10 cells. Briefly, 1 mM HAuCl4 was added to 5×106 B16F10 cells in phosphate buffered saline (PBS). B16F10 cells were then starved to facilitate the exocytosis of vesicles together with the generated AuNPs 15, 16, 26. After 4 days incubation, the color of medium turned purple indicating the generation of AuNPs. As shown in the transmission electron microscopic (TEM) images of B16F10 cells, the intracellular generation of AuNPs displayed a time dependent increase of size and change of morphology. With time extended, the AuNPs generated in cytoplasm were migrated to cell membrane and then shed outside (Figure 1A and Figure S1). The supernatant was then collected and centrifuged to obtain the secreted nanoparticles, AuNP@B16F10. The nanoparticles showed regular morphology with particles size around 30 nm and were trapped in vesicles with membrane thickness of 4 - 6 nm (Figure 1B). The Au element detected in energy dispersive X-ray spectroscopy (EDX) technique indicated the successful generation of AuNPs, and the existence of O element further confirmed the outer coating structure27 (Figure 1C). These results demonstrated the formation of AuNPs inside cells and generation of AuNP@B16F10. Vesicles derived from DCs pulsed with tumor antigens could mimic parent DCs to serve as a trigger of systemic antitumor immune response. And they showed better biocompatibility and safety compared with tumor derived vesicles16, 25. There for to prevent the possibility of metastasis induced by tumor derived AuNP@B16F10, promote the antigen presenting capability and improve biocompatibility of the nanoparticles, we further developed DC derived AuNPs (AuNP@DCB16F10). AuNP@B16F10 prepared above were introduced to DC2.4, followed by UV irradiation and starvation to facilitate the secretion of vesicles and then generate AuNP@DCB16F10 with concentration of 3.41 μg Au /μg protein (Figure 1D). As shown in Figure S2, 3, AuNP@B16F10 were internalized and transported to cytoplasm after incubation with DC2.4 for 6 h. After UV irradiation, the AuNPs were exocytosed outside as DCs

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derived EVs. The obtained AuNP@DCB16F10 were observed by TEM which showed particles size around 40 nm and a core-shell structure with thickness around 6 - 8 nm (Figure 1E), which was consistent with the thickness of lipid bilayer28. The nanoparticles exhibited good stability with no aggregation or flocculation, and no significant change of size and zeta potential during storage in PBS (Figure S4A, B). AuNP@DCB16F10 exhibited broad band and Au concentration dependent absorbance in near infrared region, which indicated the potential light-to-heat conversion of AuNP@DCB16F1029 (Figure 1F). The photothermal effect of AuNP@DCL929 and AuNP@DCB16F10 was detected by IR thermal camera. AuNP@DCL929 were prepared in similar method as AuNP@DCB16F10 by replacing B16F10 cells with fibroblast L929 cells, which served as a non-immunogenic control without tumor associated antigens30-32. As shown in Figure 1G, the temperature of AuNP@DCB16F10 was increased to 45.7 oC in only 20 s and up to 58.4 oC in 60 s after irradiation with an 808 nm NIR laser. It rose up to 73.5 oC at 5 min with temperature change near 50 oC, which demonstrated the photothermal effect of AuNP@DCB16F10. AuNP@DCL929 also exhibited rapid temperature rise as AuNP@DCB16F10, revealing that cell derived AuNPs can effectively convert light to heat with good photothermal efficiency. However, chemically synthesized AuNP with similar size rose less than 3 oC in 5 min under similar condition for lack of absorbance in NIR region (Figure S5). Furthermore, SERS spectrum of bare AuNPs, cells and AuNP@DCB16F10 were also detected by 532 nm light as shown in Figure S6. It showed that from 600-2500 nm, bare AuNPs and AuNP@DCB16F10 exhibited similar Raman shift while AuNP@DCB16F10 displayed a special SERSgenerating location at around 2900 nm, which may also be the reason that AuNP@DCB16F10 can achieve transformation of light-to-heat. The intracellularly generated AuNPs also showed good photostability and achieved repeatable temperature increase. As Figure 1H showed, ΔT can reach to 33.4 oC, 34.6 oC and 34.8 oC for AuNP@DCB16F10 after three cycles of irradiation, respectively. There was a similar tendency


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for AuNP@DCL929. The results suggested that the AuNPs generated from cells can achieve expected photothermal therapeutic efficacy.

Figure 1. Intracellular formation and characterization of AuNP@DCB16F10. A) Typical TEM images of AuNPs generation and discharge from B16F10 cells in 4 days. The scale bar is 200 nm. The insert image with orange border is the magnification of shed nanoparticles. Red arrows, AuNP trapped vesicles. Blue arrows, blank vesicles. Green arrows, AuNPs. B) TEM images of AuNP@B16F10. The scale bar is 500 nm and 50 nm, respectively. C) EDX spectrum of AuNP@B16F10. D) Cryo-TEM images of AuNP@DCB16F10 shed from DC2.4. The scale bar is 200 nm and 1 μm, respectively. E) TEM images of AuNP@DCB16F10. The scale bar is 200 nm and 50 nm, respectively. F) UV absorption of AuNP@DCB16F10 with different concentrations. The insert image showed the absorption at range of 700-810 nm. G) IR images and corresponding temperature profiles of chemically synthesized AuNP, AuNP@DCL929 and AuNP@DCB16F10 during 5 min laser irradiation (808 nm, 2.0 W·cm-2). H) Temperature change (ΔT) of


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chemically synthesized AuNP, AuNP@DCL929 and AuNP@DCB16F10 over repeated laser irradiation which was measured by thermocouple.

Proteomics analysis of nanoparticles. To explore the properties of outer shell, proteins on nanoparticles were isolated and identified using an online liquid chromatography-mass spectrometry (LC-MS) system. Proteomics data showed that AuNP@B16F10 and AuNP@DCB16F10 carried 644 and 553 proteins, respectively. And the two nanoparticles exhibited 466 and 400 common proteins respectively compared with their parent cells, which was in accordance with the property of vesicles carrying bioinformation of parent cells (Figure 2A). There were 305 common proteins in the two nanoparticles and it signified that other than the 249 intrinsic proteins of DC2.4, AuNP@DCB16F10 also carried 56 proteins from AuNP@B16F10. The proteins of AuNP@DCL929 were also compared with AuNP@B16F10 and AuNP@DCB16F10. There were few common proteins except for the 131 major constituent proteins of cells such as skeleton proteins (Figure 2B, C). AuNP@B16F10 displayed 56 and 41 common proteins with AuNP@DCB16F10 and AuNP@DCL929, respectively (Figure 2D and table S1, 2). Notably, most of the 41 common proteins between AuNP@B16F10 and AuNP@DCL929 were skeleton proteins such as ribosomal proteins and transferases. As shown in Figure 2E, neoantigens expressed on B16F10 cells which can be identified by immune cells were presented in AuNP@B16F1033, 34. Some damage-associated molecular pattern proteins (DAMPs), which can also induce immune response and mediate immunogenic cell death, were also carried such as high mobility group box 1 (Hmgb1), heat shock proteins (HSPs), and histone proteins35, 36. Importantly, AuNP@DCB16F10 retained much information of B16F10 after being processed in DC2.4, which facilitated AuNP@DCB16F10 to induce enhanced antitumor immunity. However, there was almost no apparent expression of neoantigens and DAMPs in AuNP@DCL929 (table S3). What’s more, tumor associated antigens such as glycoprotein 100 (gp100, 43.4% of parent cells) and tyrosinase-related


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protein 2 (TRP 2, 70.9% of parent cells) were retained in AuNP@B16F10 by western blot analysis. MHC II, marker of mature DC37 , TSG 101, protein involved in multivesicular body formation38 and the two tumor associated antigens mentioned above were found in AuNP@DCB16F10 (Figure 2F and Figure S7, 8). The results above indicated that the two nanoparticles can be successfully derived from parent cells with retained vital information. AuNP@B16F10 can be internalized and degraded by DCs, and the peptides can be further processed for presentation in DCs and secreted as DC-derived EVs (AuNP@DCB16F10)25.


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Figure 2. Proteomics analysis of nanoparticles. A) Comparison of proteins lysed from nanoparticles and parent cells. B) Comparison of proteins lysed from AuNP@B16F10, AuNP@DCL929 and their parent cells. C) Comparison of proteins lysed from the three different nanoparticles. D) Heatmap of 56 common proteins between AuNP@B16F10 and AuNP@DCB16F10, and 41 common proteins between AuNP@B16F10 and AuNP@DCL929. E) The comparison of neoantigens and DAMPs acquired from AuNP@B16F10, AuNP@DCB16F10 and AuNP@DCL929. F) Western blot analysis of tumor antigens.

In vitro cytotoxicity and in vivo characterization of AuNP@DCB16F10. In vitro cytotoxicity and therapeutic efficacy of AuNP@DCB16F10 was further evaluated on B16F10 cells. Laser irradiation exhibited the concentration dependent phototoxicity of nanoparticles with significantly low cell viability (Figure 3A). The cell mortality reached to 77.4% at the Au concentration of 100 μg·mL-1. The results indicated that AuNP@DCB16F10 can efficiently kill tumor cells with laser irradiation while nanoparticles or laser alone cannot. The cytotoxicity was further observed with LIVE/DEAD Viability/Cytotoxicity assay. Cells treated with different formulations (100 μg·mL-1Au) were stained with Calcein-AM and PI to identify the live and dead cells, respectively. The cells treated with laser or nanoparticles alone showed almost no apparent death, while AuNP@DCB16F10 + NIR displayed full red fluorescence with negligible green fluorescence (Figure 3B). It was in good accordance with the results of MTT assay. It visually confirmed that AuNP@DCB16F10 achieved good photothermal therapeutic effect on tumor cells through external control. To study the biodistribution of AuNP@DCB16F10 in tumor bearing mice, DiR labeled AuNP@DCB16F10 were subcutaneously injected and the fluorescence images at different time interval were captured by IVIS (Figure 3C). The intensity of fluorescence in tumor was increased gradually and reached to the maximum at 24 h after administration. The images revealed significant accumulation of AuNP@DCB16F10 at tumor


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site compared free DiR (Figure S9). The tumor accumulation of AuNP@DCB16F10 was increased with time prolonging, and reached the peak at 24 h. We also compared the tumor targeting ability between AuNP@B16F10 and AuNP@DCB16F10 through IVIS and ICP-MS (Figure S9, 10). Both AuNP@B16F10 and AuNP@DCB16F10 showed good accumulation in tumor. AuNP@DCB16F10 exhibited better tumor biodistribution which may be attributed to the tumor homing effect. Interestingly, we found that AuNP@DCB16F10 could drain to inguinal lymph nodes (iLNs) rapidly and then to axillary lymph nodes (aLNs) on the same flank with injection site in a short time. Then the nanoparticles gradually drained to the opposite lymph nodes with increasing fluorescence intensity over time (Figure 3D). As shown in Figure S11, AuNP@DCB16F10 can accumulate to lymph nodes more quickly than AuNP@B16F10. The results indicated that AuNP@DCB16F10 could efficiently traffick to local lymph nodes, thus speculating the following antitumor immunity. To further evaluate the photothermal effect in vivo, the temperature in tumor region with various treatments was recorded by an IR thermal camera (Figure 3E, F). At 24 h post administration, the tumor bearing mice were anaesthetized and irradiated with NIR laser (808 nm, 2.0 W·cm-2). Upon irradiation, the temperature of mice treated with AuNP@DCB16F10 was increased rapidly from ~32 oC to higher than 42 oC in the first 40 s and reached around 50 oC at 60 s, which was enough for hyperthermia to induce tumor ablation. It revealed that AuNP@DCB16F10 can convert light to hyperthermia in vivo efficiently. AuNP@DCL929 exhibited similar temperature increase profile as AuNP@DCB16F10, indicating that the photothermal effect of intracellularly generated AuNP was not affected by parent cells.


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Figure 3. In vitro cytotoxicity and in vivo characterization of AuNP@DCB16F10. A) Cell viability of B16F10 cells treated with AuNP@DCB16F10 with or without NIR after incubation for 24 h. B) The photothermal cytotoxicity images of B16F10 cells. Scale bar = 20 μm. C) In vivo distribution of AuNP@DCB16F10 labeled with DiR in tumor bearing mice at different time points (n=3). D) Fluorescence images of nanoparticles migration to iLNs and aLNs. The LNs on the left indicated the aLNs and iLNs at same flank of tumor and the right ones indicated the opposite flank. E, F) IR thermal images and corresponding temperature profiles of tumor bearing mice injected with PBS, AuNP@DCL929 and AuNP@DCB16F10 respectively with laser irradiation (n=3).


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Antitumor effect of synergistic PTT and immunotherapy in murine melanoma model. As described above, AuNP@DCB16F10 can induce hyperthermia and be retained at tumor and lymph nodes. In vivo antitumor effect of the nanoparticles was further investigated. When the tumor volume reached to ~50 mm3, the mice were subcutaneously injected with various formulations (1.35 mg·kg-1 Au) and irradiated at tumor site after 24 h (808 nm, 1 min). The administration was repeated for 3 times with 3 days interval. As presented previously, tumor derived EVs could induce tumor metastasis16, 39-41. We firstly evaluated the tumorigenesis and metastasis with treatment of AuNP@B16F10, AuNPs generated in DCs (AuNP@DC), AuNP@DCL929 and AuNP@DCB16F10 (Figure 4A, B and Figure S12, 13). It showed that without NIR, AuNP@B16F10 could not inhibit tumor growth efficiently. What’s more, obvious lung nodules appeared in AuNP@B16F10 treated mice. Compared with AuNP@DCL929 and AuNP@DC without transferred tumor antigens, which could not induce effective antitumor immunity, AuNP@DCB16F10 displayed better tumor suppression. In consideration of the possibility of tumor metastasis caused by AuNP@B16F10, we only evaluated the antitumor effect of AuNP@DCB16F10 in the following experiments. The antitumor effect of AuNP@DCB16F10 with different laser power was further investigated. As Figure 4C showed, compared with PBS and PBS + NIR, AuNP@DCB16F10 + NIR exhibited significant inhibition on tumor growth. However, the tumor volume of mice treated with 1.5 W·cm-2 laser power boosted which could be caused by the tumor residue. Some mice treated with 2.5 W·cm-2 were suffered from serious inflammation and wound fester caused by high power irradiation. The treatment with laser power of 2.0 W·cm-2 showed satisfied tumor growth inhibition with minimal damage to mice, which was chosen for the following studies. The combinatorial photothermal and immunotherapeutic effect of AuNP@DCB16F10 was then evaluated (Figure 4D). AuNP@DCL929 + NIR and AuNP@DCB16F10 were designed as the respective controls for single PTT and immunotherapy. As shown in Figure 4D, AuNP@DCL929 + NIR showed tumor growth inhibition with an inhibitory rate of 69.3%


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compared to PBS. AuNP@DCB16F10 also displayed modest antitumor effect with an inhibitory rate of 61.6%, which demonstrated the potential antitumor immunity induced by AuNP@DCB16F10. However, the tumor volume of monotherapy boosted rapidly since tumor was not eradicated after the last treatment. It is worth to note that AuNP@DCB16F10 + NIR exhibited significant tumor growth suppression with the inhibitory rate of 96.7%, which demonstrated nearly complete elimination of tumor. Mice were sacrificed at 21th day and tumors were harvested, photographed and weighed (Figure 4E, F). Notably, AuNP@DCB16F10 + NIR showed complete tumor elimination on three of seven mice. Moreover, AuNP@DCB16F10 + NIR exhibited serious nuclei dissolve and disappeared cell morphology from H&E staining (Figure 4G). AuNP@DCL929 + NIR and AuNP@DCB16F10 also displayed moderate apoptosis or necrosis compared to PBS with intact nuclei morphology. Taken together, AuNP@DCB16F10 can serve as an effective nanoplatform with synergistic photothermal and immunotherapeutic effect.


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Figure 4. Antitumor effect of synergistic photothermal and immunotherapy in murine melanoma model. A) Tumor growth curve treated with PBS, AuNP@DC, AuNP@B16F10, AuNP@DCL929 and AuNP@DCB16F10 (n=9). B) Representative H&E sections of lung nodules (red circles) of different groups. C) Tumor growth inhibition with different power of laser irradiation (n=7). D) Tumor growth curves after different treatments with or without laser irradiation of 2.0 W·cm-2 (n=7). The arrows represent the injection time. E) Tumor weight of the sacrificed mice at 21th day (n=7). F) Mice were sacrificed after 21 days and tumors were harvested for imaging. G) H&E sections of tumor with various treatments. (*p< 0.05, **p< 0.01)

Mechanism study of synergistic PTT and immunotherapy in murine melanoma model.

The

interaction of AuNP@DCB16F10 with the immune system was further investigated. In vitro immune response of AuNP@DCB16F10 was firstly evaluated on the proliferation of T cells (Figure 5A). Bone marrow dendritic cells (BMDCs) were exploited to simulate the antigen presenting cells (APCs) mediated


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activation of T cells. It showed that AuNP@DCB16F10 significantly augmented T cells proliferation through both indirect mediation by APCs and direct interaction with T cells, which can be attributed to the introduction of DC2.4. However, AuNP@B16F10 only induced slight augmentation via APCs mediated pathway. The mechanism of inducing antitumor immune response was then investigated. Tumor bearing mice were injected with various formulations and irradiated with NIR laser. At 48 h post the final administration, lymphocytes were harvested from aLNs, iLNs and tumors, stained with antibodies of CD11c, the specific marker of DCs, and CD86, the co-stimulatory molecule as hallmarks of DC maturation, and then assessed by flow cytometer. As shown in Figure 5B-D and Figure S14A, AuNP@DCB16F10 and AuNP@DCB16F10 + NIR promoted DC maturation in aLNs, iLNs and tumor tissue. As previous reports demonstrated, the laser irradiation can also make a contribution to the stimulation of antitumor immune response42, 43, it was found that AuNP@DCL929 + NIR showed modest DC maturation serving as control of single PTT. The mature DCs can initiate primary T cell response, which is important for attacking tumor cells. To detect the proliferation and activation of T cells, tumor infiltrating lymphocytes were stained with anti-CD3, anti-CD4, and anti-CD8 for flow cytometry assay. AuNP@DCB16F10 + NIR showed the highest infiltration of CD4+ and CD8+ T cells among all treatments. AuNP@DCB16F10 also exhibited relatively high proportion. AuNP@DCL929 + NIR showed slightly increased infiltration of T cells, which might be resulted from a relatively weak immune response induced by PTT (Figure 5E, F and Figure S14B). To further validate the activation of cytotoxic T cells, CD8+CD69+, CD8+CD107+ and CD8+ Granzyme B+ T cells were then analyzed. CD69 is an early marker of T cells activation and proliferation44. CD107, a marker of degranulation enzyme, is also an essential factor for T cells to kill tumor cells45. Expression of Granzyme B represents the secretion of granules from cytotoxic T cells, which mediates the apoptosis of tumor cells46. As shown in Figure 5G-I and Figure S14C, D, AuNP@DCB16F10 and AuNP@DCB16F10 + NIR significantly promoted the activation of CD4+


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and CD8+ T lymphocytes in tumor while AuNP@DCL929 + NIR showed much weaker activation. The results evidenced that AuNP@DCB16F10 with or without NIR laser could elicit effective antitumor immune responses compared to single PTT, PBS or PBS+NIR. Moreover, the secretion of various cytokines was also investigated, including interferon γ (IFN-γ) for Th1 immune response, tumor necrosis factor α (TNFα) for cellular immunity and interleukin 6 (IL-6) for humoral immunity. It was found that IFN-γ and TNFα levels in AuNP@DCB16F10 + NIR and AuNP@DCB16F10 were increased, maintained for a period and then decreased gradually. However, there was no apparent change with the treatment of PBS, PBS+NIR or PTT alone. The level of IL-6 also exhibited significant elevation in the two groups treated with AuNP@DCB16F10 compared with the control and monotherapy. AuNP@DCB16F10 with or without laser exhibited enhanced cytokines secretion, signifying the immune capability of AuNP@DCB16F10 to provide a pro-inflammatory immune environment (Figure 5K). Splenocytes were also harvested 48 h after the last treatment to evaluate the rejection of tumor cells. AuNP@DCB16F10 exhibited more obvious killing of tumor cells in comparison with other treatments (Figure S15). What’s more, the mechanism was explored by blocking CD4+ (αCD4) or CD8+ (αCD8) T cells on tumor bearing model (Figure 5K). It was found that AuNP@DCB16F10 could still prime the antitumor immune response after blocking of CD4+ T cells. However the nanoparticles mediated tumor growth suppression was inhibited due to the absence of CD8+ T cells. It showed that CD8+ T cells could play a major role in AuNP@DCB16F10 triggered antitumor immunity. Collectively, AuNP@DCB16F10 can be a prominent immunological nanoplatform for amplifying the immune response and the combination with NIR irradiation could further enhance antitumor immunity.


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Figure 5. Mechanism study of synergistic photothermal and immunotherapy in murine melanoma model. A) Proliferation of CD3+ T cells after co-incubation with nanoparticles (AuNP@B16F10 and AuNP@DCB16F10) or nanoparticles pulsed BMDC (n=3). B-D) The maturation of DC in B) aLNs, C) iLNs, and D) tumor. E-I) Lymphocytes in tumor. The proportion of E) CD4+ T cells and F) CD8+ T cells, and activated lymphocytes G) CD3+CD8+CD107+, H) CD3+CD8+CD69+ and I) CD3+CD8+Granzyme B+ T cells after the third injection of different formulations (n=3). J) Cytokine levels in serum from tumorbearing mice isolated at 24, 48, and 72 h after the last injection (n=3). K) Tumor growth curves of B16F10


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bearing mice treated with AuNP@DCB16F10 in the presence or absence of CD4+ (αCD4)/CD8+ (αCD8) T cells, plus corresponding control groups (n=8). (+) represented with NIR and (-) without NIR. (*p< 0.05, **p< 0.01)

Abscopal effect of AuNP@DCB16F10 combined with NIR irradiation. Metastasis is one of the leading causes for cancer death, which usually cannot be cured with traditional antitumor therapy such as surgery and radiotherapy47,

48

. Here a bilateral subcutaneous tumor model was established to mimic tumor

metastasis49. To determine the antimetastasis efficiency of AuNP@DCB16F10 combined with PTT, C57BL/6 mice were subcutaneously inoculated with 5 × 104 cells on left flank as primary tumor and 1 × 104 cells on right flank as distant tumor. When primary tumor volume reached ~50 mm3, the mice received various treatments only on primary tumor. As Figure 6A displayed, the growth curve of primary tumor was with similar tendency as the antitumor effect described above. The treatment of AuNP@DCB16F10 + NIR significantly delayed the occurrence of distant tumor with 50% mice of tumor free at 19th day, while 83% mice in PBS showed palpable tumor at 15th day (Figure S16A, B). Moreover, the distant tumor growth was efficiently inhibited by combinatorial treatment (Figure 6B). The distant tumors were also collected for analysis at 48 h after the last administration. AuNP@DCB16F10 + NIR significantly elicited the maturation of DCs and activation of T cells in distant tumor compared with monotherapy (Figure 6CG and Figure S17). The immunofluorescence assay showed similar enhancement of CD3+CD8+ T cells (Figure 6H). The secretion of cytokines in tumor was also detected using confocal microscope (Figure 6H). AuNP@DCB16F10 + NIR enhanced the secretion of IFN-γ compared with other groups and also induced the release of TNF-α and IL-6, which can prime adaptive immune response. The combination of immunotherapy and PTT based on AuNP@DCB16F10 can efficiently inhibit the growth of distant tumor


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owing to the abscopal effect, indicating that AuNP@DCB16F10 based combinatorial therapy owned great potential to be an effective strategy to treat cancer metastasis. To monitor long term antitumor effect, the overall survival of mice after tumor inoculation was then evaluated and the schedule was shown as Figure S18A. Compared with other treatments, mice treated with AuNP@DCB16F10 + NIR exhibited overall survival of 91.7% for more than 35 days with no palpable tumor (Figure 6I). Representative images of treated mice displayed the recovery of escharosis in AuNP@DCB16F10 + NIR at 34th day. Then the tumor free mice were rechallenged at 35th day to monitor the long term protective effect of nanoparticles on tumor recurrence. Even at 50th day, the rechallenged mice did not occur with tumor (Figure S18B). At 59th day, the tumor began to recur while 5/11 mice were still free of tumor at 70th day (the insert graph in Figure 6I). The combinatorial therapy showed a long term effect on tumor inhibition and recurrence, which may be attributed to the antitumor immunity induced by AuNP@DCB16F10.


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Figure 6. Abscopal effect of AuNP@DCB16F10 combined with NIR irradiation. The bilateral tumor model was developed by subcutaneously injecting B16F10 cells into both left and right flanks of mice. The left tumor was treated with laser as primary tumor, and the right tumor served as distant tumor without


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any treatment (n=12). A) Growth curves of primary tumor. B) Weight of the distant tumor harvested at 19th day. C-G) Infiltration of C) CD11C+CD86+ DCs, D) CD3+CD4+ T cells, E) CD3+CD8+ T cells, F) activated CD4+ T cells and G) activated CD8+ T cells in distant tumor (n=3). H) Immunofluorescence staining of CD3+CD8+ T cells and cytokines expressed in distant tumor, scale bar = 50 μm. I) Survival rate of mice with various treatments (n=12). And the percentage of tumor free mice rechallenged with B16F10 cells at 35th day was shown in the insert image (n=11). (*p< 0.05, **p< 0.01)

Antimetastasis of AuNP@DC4T1 in 4T1 tumor model. To further explore applicability of immunological nanoparticles, AuNP@DC4T1 were developed with similar method by replacing B16F10 cells with breast cancer cells 4T1. As shown in Figure 7A, AuNP@DC4T1 displayed similar core-shell structure with AuNP@DCB16F10. A 4T1 metastatic tumor model was then developed by inoculating 2 × 105 4T1 cells into the second mammary fat pads, which can induce lung metastasis easily. The power of laser was optimized considering the difference between Balb/c and C57BL/6 mice (Figure 7B). Compared with laser power of 2 W·cm-2 and 4 W·cm-2, mice treated with 3 W·cm-2 showed obvious tumor eradication with no perceptible lesions (Figure 7C). The therapeutic effect of immunological nanoparticles was further confirmed. As shown in Figure 7D, AuNP@DC4T1 exhibited similar antitumor effect with AuNP@DCB16F10. The lungs were harvested to investigate the lung metastasis. The calculated pulmonary nodules, images of bovin’s solution stained lungs and H&E sections showed that aggressive lung metastasis occurred in PBS and PBS + NIR. Lung nodules were also observed in AuNP@DCL929 while nearly no appreciable metastases was found in AuNP@DC4T1 and AuNP@DC4T1 + NIR, validating the antimetastasis effect of immunological nanoparticles (Figure 7E-G).


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Figure 7. Antimetastasis of AuNP@DC4T1 in 4T1 tumor model. A) TEM of AuNP@DC4T1. The insert image was the magnification. Scale bar is 200 nm and 50 nm, respectively. B) Growth curves of 4T1 tumor treated with AuNP@DC4T1 at different power of laser (n=3). C) Representative images of mice with different treatments. D) Tumor growth curve (n=8). E). Number of pulmonary nodules after different treatments. F) Representative images of stained lungs of different groups. The arrows refer to the tumor nodules on surface. G) Representative H&E sections of lung nodules of different groups.

In vivo safety evaluation. To examine the in vivo toxicity, histopathological assays were conducted. The major tissues were observed through H&E staining. There was no obvious inflammatory infiltrates and pathological changes in all organs after various treatments, indicating the nontoxic property and good


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biocompatibility of AuNP@DCB16F10 with or without NIR (Figure S19A). Moreover, the blood was collected for serum biochemistry assay. As shown in Figure S19B, the levels of liver and kidney function markers were all within normal range, indicating that AuNP@DCB16F10 based combinational therapy can be a safe, nontoxic strategy for cancer treatment. Conclusion In summary, here we developed a novel intracellularly generated immunological nanoplatform for combinatorial antitumor effects of PTT and immunotherapy. Taking advantage of cellular properties, AuNPs can be generated intracellularly and then exocytosed as nanoparticle trapped vesicles with retained original bioinformation, which can be a green and facile method without any chemical agents. After further introduction to DCs, DCs derived immunological nanoparticles can enhance the biocompatibility and activate the immune response, promoting DCs maturation, multiple cytokines secretion and T cells activation. The induced antitumor immunity together with the AuNP based PTT can realize not only efficient tumor eradication but also sufficient prevention on tumor metastasis and relapse. Therefore, we provided a promising strategy for efficient cancer therapy through intracellularly generated immunological nanoparticles. The nanoparticles mediated cancer therapy exhibited broad applicability in multiple tumor models, indicating the flexibility of the nanoplatform. By further exploiting specific intracellular environment and metabolism, the nanoplatform can be generated aiming to different diseases with controllable bioinformation.

Acknowledgements: This work was supported by National Natural Science Foundation of China (81673374 and 81872810) and Program for HUST Academic Frontier Youth Team (2018QYTD13). We thank the Analytical and Testing Center of Huazhong University of Science & Technology for performing the TEM, and Pei Zhang and Anna Du from The Core Facility and Technical Support, Wuhan Institute of


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Virology for performing the cryoTEM. We also appreciated Dr. Honghao Sun (School of Food and Pharmaceutical Engineering, Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Hubei University of Technology) and Dr. Haifang Yin (Department of Cell Biology and Research Centre of Basic Medical Science, Tianjin Medical University) for providing material and instrument, Dr. Zhifeng Du (Tongji School of Pharmacy, Huazhong University of Science and Technology) and Dr. Honghuang Lin (Cardiology Section, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA) for proteomics analysis, as well as Tetsuro Sasada (Kanagawa Cancer Center Research Institute) for analysis of immune mechanisms. Supporting Information Supporting Information is available in the online version of the paper. For Table of Contents Only

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Scheme 1. Schematic preparation of AuNP@DCB16F10 and mechanism of AuNP@DCB16F10 mediated combinational treatment modality. 276x194mm (150 x 150 DPI)


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Figure 1. Intracellular formation and characterization of AuNP@DCB16F10. A) Typical TEM images of AuNPs generation and discharge from B16F10 cells in 4 days. The scale bar is 200 nm. The insert image with orange border is the magnification of shed nanoparticles. Red arrows, AuNP trapped vesicles. Blue arrows, blank vesicles. Green arrows, AuNPs. B) TEM images of AuNP@B16F10. The scale bar is 500 nm and 50 nm, respectively. C) EDX spectrum of AuNP@B16F10. D) Cryo-TEM images of AuNP@DCB16F10 shed from DC2.4. The scale bar is 200 nm and 1 μm, respectively. E) TEM images of AuNP@DCB16F10. The scale bar is 200 nm and 50 nm, respectively. F) UV absorption of AuNP@DCB16F10 with different concentrations. The insert image showed the absorption at range of 700-810 nm. G) IR images and corresponding temperature profiles of chemically synthesized AuNP, AuNP@DCL929 and AuNP@DCB16F10 during 5 min laser irradiation (808 nm, 2.0 W•cm-2). H) Temperature change (ΔT) of chemically synthesized AuNP, AuNP@DCL929 and AuNP@DCB16F10 over repeated laser irradiation which was measured by thermocouple. 488x280mm (150 x 150 DPI)


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Figure 2. Proteomics analysis of nanoparticles. 439x412mm (150 x 150 DPI)


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Figure 3. In vitro cytotoxicity and in vivo characterization of AuNP@DCB16F10. 437x339mm (150 x 150 DPI)


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Figure 4. Antitumor effect of synergistic photothermal and immunotherapy in murine melanoma model. 146x82mm (220 x 220 DPI)


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Figure 5. Mechanism study of synergistic photothermal and immunotherapy in murine melanoma model. A) Proliferation of CD3+ T cells after co-incubation with nanoparticles (AuNP@B16F10 and AuNP@DCB16F10) or nanoparticles pulsed BMDC (n=3). B-D) The maturation of DC in B) aLNs, C) iLNs, and D) tumor. E-I) Lymphocytes in tumor. The proportion of E) CD4+ T cells and F) CD8+ T cells, and activated lymphocytes G) CD3+CD8+CD107+, H) CD3+CD8+CD69+ and I) CD3+CD8+Granzyme B+ T cells after the third injection of different formulations (n=3). J) Cytokine levels in serum from tumor-bearing mice isolated at 24, 48, and 72 h after the last injection (n=3). K) Tumor growth curves of B16F10 bearing mice treated with AuNP@DCB16F10 in the presence or absence of CD4+ (αCD4)/CD8+ (αCD8) T cells, plus corresponding control groups (n=8). (+) represented with NIR and (-) without NIR. (*p< 0.05, **p< 0.01) 845x918mm (150 x 150 DPI)


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Figure 6. Abscopal effect of AuNP@DCB16F10 combined with NIR irradiation. 764x899mm (150 x 150 DPI)


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Figure 7. Antimetastasis of AuNP@DC4T1 in 4T1 tumor model. 166x122mm (220 x 220 DPI)


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