ZnO-based multifunctional nanocomposites to inhibit progression and metastasis of melanoma by eliciting antitumor immunity via immunogenic cell death

Rationale: The development of a highly effective and tumor-specific therapeutic strategy, which can act against the primary tumor and also condition the host immune system to eliminate distant tumors, remains a clinical challenge. Methods: Herein, we demonstrate a facile yet versatile ZnO-capping and Doxorubicin (DOX)-loaded multifunctional nanocomposite (AuNP@mSiO2@DOX-ZnO) that integrates photothermal properties of gold nanoparticles (NPs), pH-responsive properties and preferential selectivity to tumor cells of ZnO QDs and chemotherapeutic agent into a single NP. The photothermal performance, pH-triggered release and preferential phagocytic ability were assessed. The induced anti-tumor immunity was determined by analyzing immune cell profile in tumor in vivo and molecular mechanism were identified by detecting expression of immunogenic cell death (ICD) markers in vitro. Moreover, mice models of unilateral and bilateral subcutaneous melanoma and lung metastasis were established to evaluate the antitumor effects. Results: As an efficient drug carrier, ZnO-capped NPs guarantee a high DOX payload and an in vitro, efficient release of at pH 5.0. In murine melanoma models, the nanocomposite can significantly inhibit tumor growth for a short period upon low-power laser irradiation. Importantly, ZnO NPs not only demonstrate preferential selectivity for melanoma cells but can also induce ICD. Meanwhile, AuNP@mSiO2-based photothermal therapy (PTT) and DOX are directly cytotoxic towards cancer cells and demonstrate an elevated ICD effect. The induced ICD promotes maturation of dendritic cells, further stimulating the infiltration of effector T cells into tumor sites, preventing tumor growth and distant lung metastases. Conclusions: This study highlights the novel mechanism of ZnO-triggered anti-tumor immunity via inducing ICD. Additionally, we shed light on the multifunctionality of nanocomposites in delivering localized skin tumor therapy as well as inhibiting metastatic growth, which holds great promise in clinical applications.

Zn (CH3COO)2· 2H2O was obtained from Aladdin. Formaldehyde solution (37.0%), zincacetate dihydrate and Tetraethyl orthosilicate (TEOS) were purchased from sinopharm chemical reagent Co., Ltd. Deionized water (Millipore Milli-Q grade) with resistivity of 18.0 MΩ was used in all the experiments. All chemicals were of analytical grade and were used as received without further purification.

Photothermal performance of AuNP@mSiO2-ZnO.
The AuNP@mSiO2-ZnO aqueous solutions at various concentrations were irradiated with 655 nm laser with varied power densities and time. The solution temperatures were detected by thermocouples (SC-GG-K-30-36, Omega, USA) and PBS was used as control.

In-vivo distribution.
Mice model of subcutaneous melanoma was generated as previously described. PBS or Cy5-labeled AuNP@mSiO2-ZnO nanocomposites were intratumorally injected to tumor-bearing mice with a dose of 60 μg per mice. Animals were sacrificed (n = 5) at 1 h, 3 h, 6 h and 12 h after injection and the fluorescence images were obtained by a live small animal optical imaging system (IVIS Lumina XR; Caliper).

Immunofluorescence.
Tumors as mentioned above were collected and the frozen tissue sections were prepared. Then, the sections were air-dried, fixed in acetone and blocked in 1% bovine serum albumin for 30 minutes at 37 °C.

In-vivo biosafety analysis.
Healthy female C57 mice were intravenously injection with AuNP@mSiO2-ZnO at dosages from 65 to 200 μg (60 μg, 100 μg and 200 μg) per mice (n = 5). 15 days later, all the mice were sacrificed and blood samples were collected for blood routine analysis (Sysmex 1800i, Japan) and blood biochemistry test (Hitachi 7600, Japan). In addition, the major organs, including heart, liver, spleen, lung and kidney, were harvested and fixed with 4% paraformaldehyde for H&E staining. indicating a uniform mesopore. The BET surface area and total pore volume were calculated to be 686 m 2 /g and 0.9 cm 3 /g, respectively. (F) The wide-angle X-ray diffraction (XRD) pattern indicated the successful fabrication of AuNP@mSiO2-ZnO, compared with those of ZnO QDs and AuNP@mSiO2. (G) The photothermal heating curves of AuNP@mSiO2-ZnO solution irradiated with 2.0 W/cm 2 NIR for 5 min and turned off for 5 min for 5 cycles to examine the photothermal stability of AuNP@mSiO2-ZnO, and this process was repeated for continuous five cycles. We observed that the temperature of AuNP@mSiO2-ZnO did not decrease significantly, which indicated that the photothermal stability of AuNP@mSiO2-ZnO was high.  experiments. Data was given as mean ± SD. Statistical significance was calculated by the t-test. *P < 0.05, **P < 0.01. three independent experiments. Data was given as mean ± SD. Statistical significance was calculated by the t-test. *P < 0.05, ***P < 0.001, ns, not significant.  (B) CD4 + T cells (CD45 + CD3 + CD4 + PI − )/CD8 + T cells (CD45 + CD3 + CD8 + PI − ) and (C) activated CD8 + T cells (CD45 + CD3 + CD8 + CD44 + CD62L -PI − ). Results are representative of at least three independent experiments. S14 Figure S11. The tumor-bearing mice were treated as indicated and all the tumors were collected, sectioned and stained with DAPI (blue), CD3 (red) and CD8 (green). Representative CLSM images were showed after immunofluorescence staining (n = 5). Images are representative of 5 fields. The scale bars can be applied to all the other images. S15 Figure S12. Flow cytometric analysis of cell apoptosis and necrosis of B16/F10 melanoma cells after treatment as indicated for 24 h by staining with Annexin V-FITC and PI. Live cells were also counted. Results are representative of at least three independent experiments. Data were given as mean ± SD. Statistical significance was calculated by the t-test. Data was presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. respectively. Results are representative of at least three independent experiments. Data was presented as mean ± SD. Statistical significance was calculated by the t-test. *P < 0.05, **P < 0.01, ns, not significant. NAC: oxidative stress inhibitor Nacetyl L-cysteine. S19 Figure S16. Bilateral subcutaneous melanoma mice model was established. The photographs (A), tumor volumes (B) and tumor weight (C) of the primary tumors in each group were measured at the end of experiment (n=6). (D) Kaplan−Meier survival curves. There were five mice per group. Statistical significance with two groups was calculated by the t-test. Comparisons of survival curves were made using the long-rank test. Data was presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. S20 Figure S17. B16/F10 tumor cells were inoculated on the right flank of each mouse. A week later, a second tumor was i.v. injection of the same mouse as an artificial mimic of lung metastasis. In the following week, the 1 st tumor of all the groups received intratumoral treatments every three days for a total of three times. The photographs of the primary tumors (A) and lungs (B) in each group were measured at the end of experiment. Figure S18. Body weight of tumor-bearing mice receiving treatments as indicated. Data was presented as mean ± SD (n=5).    Table：   Table S1. Characteristics of the core-shell AuNP@mSiO2 and AuNP@mSiO2-ZnO from BET analysis. SBET and Vt represent surface area and pore volume, respectively