A Cu9S5 nanoparticle-based CpG delivery system for synergistic photothermal-, photodynamic- and immunotherapy

Despite its great potential in cancer therapy, phototherapy, including photothermal therapy (PTT) and photodynamic therapy (PDT), often cause metastasis of tumors. Immunotherapy has revolutionized the cancer treatment owing to the capability of activating immune system to eliminate tumors. However, the integration of phototherapy and immunotherapy in a single nanoagent for cancer therapy is still a challenging task. Here, we fabricated (Cu9S5@mSiO2-PpIX@MnO2@CpG (CSPM@CpG)) as a synergistic therapeutic model for phototherapy enhanced immunotherapy. The intracellular uptake of cytosine-phosphate-guanine (CpG) promoted the infiltration of cytotoxic T lymphocytes (CTLs) in tumor tissue, further stimulating the production of interferon gamma (IFN-γ) and remarkably elevating the immune response level. Excellent anti-tumor effects have been achieved by synergistic PTT/PDT/immunotherapy. The metastasis of tumors was effectively inhibited by the immune response of CpG. Thus, our proposed work provides a strategy to combine phototherapy with immunotherapy to enhance the therapeutic efficiency and further inhibit metastasis of tumors.


Reporting Summary
Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, see our Editorial Policies and the Editorial Policy Checklist.

Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted

Software and code
Policy information about availability of computer code Data collection Powder X-ray diffraction (XRD) patterns were conducted by a D8 advance (Bruker, Germany). The sizes and morphologies of samples were determined by a transmission electron microscope (TEM) at an accelerating voltage of 200 KV (JEM-2100, JEOL, Japan). The specific surface area and pore volume of the products were determined by Brunauer-Emmett-Teller (BET) and Barett-Joyner-Halenda (BJH) methods (TriStar 3020, Micromeritics, America). X-ray-photoelectron spectroscopy (XPS) analysis was examined with kratos Axis ultra dld. UV-visible absorption spectra were recorded by a Hitachi U-2900 Spectrophotometer. Inductively coupled plasma optical emission spectroscopy (ICP-OES, PE 8300) was used to validate the content of copper ions intracellular uptake by cells. The Zetasizer Nano Z (Malvern, Britain) was selected to measure zeta potential.2D imaging data were obtained through Confocal Laser Scanning Microscope (Nikon, Japan). Flow cytometry (BD FACS Aria II, USA) is used to collect the flow cytometry data.
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability All other remaining data are available within the article and supplementary files, or available from the authors upon request.

nature research | reporting summary
April 2020 Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
The tumor diameters were measured by vernier caliper for three times to get mean value.
Data exclusions Abnormal Data obtained from enzyme linked immunosorbent assay were excluded .

Replication
Measurement of Cytokines (such as TNF-α, IL-12 and INF-γ) were taken by protocols.
Randomization To evaluate the anti-tumor effects in our tumor model, BALB/c mice were injected s.c. with 4T1 mammary tumor cells or PBS and randomly assigned to either of the study groups (n = 5).

Blinding
The investigators were blinded to group allocation during data collection and analysis.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Authentication Cell lines were authenticated by ATCC and the Chinese Academy of Sciences cell library.

Mycoplasma contamination
The cell lines were not tested for mycoplasma contamination.
Commonly misidentified lines (See ICLAC register) No misidentified cell lines were found in this study.
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals
Four-to-five-week-old female Balb/C mice were purchased from Shanghai Laboratory Animal Center (SLAC, shanghai, China), and were bred in a sterilized, specific pathogen-free (SPF) Lab of Tongji University.

Wild animals
The study was not involved in wild animals.
Field-collected samples The study did not involve samples collected from the field.

Ethics oversight
Animal study protocols were approved by Tongji University Experimental Animal Center.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Sample preparation
All tissues were obtained from Balb/C mice in this study. The tissues inflated in digestion solution and incubated for 30 mins at 37 °C with continuous shaking every 5-8 min and gently grinded to acquire single-cell suspensions after passing the digested organs through a 70 μm strainer.

Instrument
Flow cytometry (BD FACS Aria II, USA) is used to collect the flow cytometry data.
Software Flowjo(10) was used to analyze the flow cytometry data.

Cell population abundance
The amounts of cells in flow tube tested by flow cytometry (BD FACS Aria II, USA) are 5*10^5 /tube.

Gating strategy
The preliminary FSC-A/SSC-A gates of the starting cell population were 50K-150K/10^3-10^5. Cells were further stained by immunophenotyping antibodies with FITC-CD8, PE-Granzyme B. Subsequently, cells were stained with Fixable Viability Dye eFluor 450 and observed using BD flow cytometry. the cells that FITC-CD8, PE-Granzyme B and Dye eFluor 450 positive were cytotoxic T lymphocytes (CTLs).
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.