Multifunctional Calcium–Manganese Nanomodulator Provides Antitumor Treatment and Improved Immunotherapy via Reprogramming of the Tumor Microenvironment

Ions play a vital role in regulating various biological processes, including metabolic and immune homeostasis, which involves tumorigenesis and therapy. Thus, the perturbation of ion homeostasis can induce tumor cell death and evoke immune responses, providing specific antitumor effects. However, antitumor strategies that exploit the effects of multiion perturbation are rare. We herein prepared a pH-responsive nanomodulator by coloading curcumin (CU, a Ca2+ enhancer) with CaCO3 and MnO2 into nanoparticles coated with a cancer cell membrane. This nanoplatform was aimed at reprogramming the tumor microenvironment (TME) and providing an antitumor treatment through ion fluctuation. The obtained nanoplatform, called CM NPs, could neutralize protons by decomposing CaCO3 and attenuating cellular acidity, they could generate Ca2+ and release CU, elevating Ca2+ levels and promoting ROS generation in the mitochondria and endoplasmic reticulum, thus, inducing immunogenic cell death. Mn2+ could decompose the endogenous H2O2 into O2 to relieve hypoxia and enhance the sensitivity of cGAS, activating the cGAS-STING signaling pathway. In addition, this strategy allowed the reprogramming of the immune TME, inducing macrophage polarization and dendritic cell maturation via antigen cross-presentation, thereby increasing the immune system’s ability to combat the tumor effectively. Moreover, the as-prepared nanoparticles enhanced the antitumor responses of the αPD1 treatment. This study proposes an effective strategy to combat tumors via the reprogramming of the tumor TME and the alteration of essential ions concentrations. Thus, it shows great potential for future clinical applications as a complementary approach along with other multimodal treatment strategies.


Preparation of nanoplatform (CM NPs)
Firstly, B16F10 cancer cells were cultured in DMEM medium replenished with 10% fetal bovine serum and penicillin-streptomycin. To collect cellular membrane, the cells were fully collected with 2 mM EDTA in pre-cold PBS. After washing with precold PBS thrice by centrifugation, the cells were suspended and disrupted in a 10 mL hypotonic lysing buffer (10 mM Tris-HCl (pH 7.5), 10 mM KCl, 2 mM MgCl2 and protease inhibitor), and homogenized using a Dounce homogenizer. The homogenized solution was centrifuged at 3500 g for 5 min at 4 ℃ to collect the supernatant. The supernatant was then centrifuged at 20,000 g for 15 min, after which the pellets were discarded and the supernatant was centrifuged again at 100,000 g for 30 min. The plasma membrane pellets were then washed once in 10 mM Tris-HCl buffer, and resuspended in PBS and stored at -80 ℃ for further experiments. The protein content of obtained cellular membrane was quantified using a BCA kit (Beyotime).
To prepare cancer cell membrane vesicles, the membrane material (200 μg) and CaCO3-CU@MnO2 (5 mg) was physically extruded through a 450 nm polycarbonate membrane (Millipore, USA) for 30 passes. The obtained CM NPs was centrifuged at 12,000 g for 15 min to pellet the cell membrane coated CaCO3-CU@MnO2 rather than free vesicles. The same method was applied to obtain B@MnO2 (coined as MnO2) and B@CaCO3-CU (CaCO3@CU).

SDS PAGE protein analysis
Membrane proteins were analyzed via SDS-PAGE. Briefly, B16f10 cell membrane, MnO2 and CaCO3@CU without cell membrane coating, together CM NPs samples were suspended in 1 x SDS sample buffer at concentrations determined based on a BCA assay kit. Samples were then boiled to 100 °C for 10 min, after which 20 µg per sample was separated via 10% SDS-PAGE for 1.5 h at 120 V, with the resultant gels being stained using Coomassie Blue for 2 h, washed overnight with ddH2O, and imaged.
In vitro Ca 2+ and Mn 2+ release: CM NPs (100 μg/mL) was sufficiently dissolved into PBS buffer (pH 7.4 and pH 5.4) for different time points, respectively. Subsequently, the mixture was centrifuged at 13,000 rpm for 15 min, the supernatant was collected for detecting Ca 2+ and Mn 2+ concentrations by ICP-OES.

Stability Performance of NPs
The 100 μg/mL (Mn ion concentration) of CM NPs were dispersed in PBS solution with different pH value (pH 5.4 and pH7.4) at rt for 8 days. The change of CM NPs stability was evaluated using imaging and UV-Vis spectrum.

Quenching of H 2 O 2 by CM NPs
For the quenching experiment, CM NPs or as-prepared MnO2 NSs (50 μM Mn) was suspended in PBS, H2O2 (100 μM) was added to initiate the reaction. For in vitro reactivity of as-prepared NPs toward H2O2, the concentration of H2O2 was measured using Hydrogen Peroxide Assay Kit. For in vitro cellular reactivity of as-prepared NPs toward H2O2, RAW264.7 cells were pretreated with 25 ng/mL IL-4 to generate M2 macrophages, then incubated with 20 μg/mL of as-prepared NPs for 12 h. The amount of intracellular H2O2 was quantified using Hydrogen Peroxide Assay Kit.

Acidic Attenuation Effect of NPs in vitro
The as-prepared CM NPs (20 μg/mL) was suspended in PBS with different pH values (pH 5.4, 6.5 and 7.4), stirring at rt and evaluating the pH value using a pH meter at default timepoints (0, 0.5, 1, 2, 6, 12, 24 and 48 h).

Detection of GSH Depletion
Ellman's assay was employed to measure the depletion of GSH content by the as-

Generation of •OH by the CM NPs
First, the generation of •OH by a Mn 2+ -catalyzed Fenton-like reaction was evaluated. The 25 mM NaHCO3/5% CO2 buffer or PBS buffer solutions contained 10 μg/mL of MB, 20 mM of H2O2, and 20 μg/mL CM NPs were incubated at 37 °C for 60 min. The generation of •OH was revealed by increasing absorption of MB at 665 nm.
Next, the •OH generation ability of the CM NPs was investigated. The NPs was incubated with different concentrations (0, 1, 3 and 5 mg/mL) of GSH solution (NaHCO3/5% CO2 buffer) for 60 min. After centrifugation at 12,000 rpm for 10 min, the mixture of MB (10 μg/mL) and H2O2 (20 mM) in NaHCO3/5% CO2 buffer were added to the supernatant and incubated at 37 °C for 0.5 h in a shaker before recording the UV−Vis spectra. The CM NPs solution with the equivalent concentration was used as the control group.

Cellular toxicity assay
293T, HeLa and B16F10 cells were cultured on a 96-well plate (2 х 10 4 cells/well), the cells were incubated in a humid chamber of 5 % CO2 and 95% humidity at 37 °C.
After the confluence of cells in 96-well plate up to 60-65%, the culture medium was replaced with 200 μL of new full culture medium that contained the desired amount of CM NPs (0, 5 ,10, 20, 50 and 100 μg/mL). Six multiples were set for every sample and the cells were treated with samples for specific interval for 24 h. then 10 μL CCK-8 and 90 μL serum free culture medium mixed gently and added into each well, incubated for

Phagocytosis in vitro
First, B16F10 cells were cultured overnight into six-well plates with 2 × 10 5 cells/well seeding density. When the confluency was up to 70%, cells were treated with the following groups: MnO2 (10 μg/mL), CaCO3@CU (10 μg/mL), CM (10 μg/mL) and Control (PBS). After these treatments, the culture supernatant of B16F10 cells was collected for IFNγ detection. Meanwhile, these treated B16F10 cells were placed under the UV radiation (1500 J cm -2 ) to kill all the cells. Then, the dying B16F10 cells were collected and fed to RAW264.7 cells. After the phagocytosis of dying B16F10 cells, the culture supernatant of RAW 264.7 cells were collected and prepared for IFNγ detection.

Hemolysis assay
The blood samples were collected from C57BL/6J mice. In general, 1 mL blood sample was diluted with 5 mL of PBS, and then red blood cells (RBCs) were separated from the serum by centrifugation (2000 rpm,10 min). After washing with PBS six times, the RBCs were diluted with 10 mL of PBS. Then the RBCs were treated with different concentrations of CM NPs solution (5, 10, 20, 50 and 100 μg/mL), and the ddH2O and PBS were set as positive and negative controls to evaluate hemolysis ability. After incubated and placed in the table concentrator at 37 ℃ at a speed of 180 rpm for 3 h, the mixtures were centrifuged at 10,000 rpm for 10 min. Finally, the absorbances of supernatants at 570 nm were recorded by a microplate reader to calculate the percent hemolysis.

In vitro antitumor efficiency
To determine the antitumor efficiency in vitro toxicity of the nanoplatform,

Detection of ATP levels
B16F10 cells were seeded in 24-well plate at a density of 5 × 10 4 cells per well for 16 h (the confluence up to 80-85%), then the cell culture was replaced with fresh culture medium that containing various different concentration of NPs (50 μg/mL) and cultured for another 24 h, then the cells were collected and evaluated culture ATP content with ATP assay kit under the instruction of the provider's manual. The bioluminescence was detected and recorded using SPARK 10M plate reader.

Evaluation of ROS production
B16F10 cells were incubated with various concentration of CM NPs (50 μg/mL) for 12 h, then, culture medium was replaced with fresh serum-free medium containing 5 μM of DCFH-DA for a further incubation of 30 min without light. Then, CLSM were applied for ROS analysis.

Intracellular Ca 2+ concentration assay
The intracellular Ca 2+ concentration of Chromogenic Reagent and 75 μL Calcium Assay Buffer were added to the 50 μL lysates, followed by the 5 min incubation, the absorbance value at 575 nm were recorded using microplate reader.

Measurement of intracellular pH (pHi)
The intracellular pH was measured using BCECF-AM in a SPARK Immunoblotting bands were observed using ChemiScope Series 6000Touch(Clinx Science Instruments Co.,Ltd) after incubated with Pierce™ ECL Plus western blotting substrate (cat. no. 32132; Life).

Immunofluorescent staining
Cells or tissue slices samples were fixed with 4% paraformaldehyde for 15 min at rt. Next, the samples were permeabilized with 0.1% Triton X-100 PBS for 10 min and cultured with a blocking buffer containing 5% bovine serum albumin (BSA) for 1 hour at rt. Then, the samples were incubated overnight with specific primary antibodies at various dilution at 4 °C overnight, washed with PBS three times, and incubated in specific secondary antibody for 2 h at rt. Last, the sample were stained with DAPI, washed with PBS, and imaged using an Leica CLSM (SP8). For the determination of immune cell subpopulation, the specific antibody of canonical marker were listed in the section of results detailedly.

Animals and tumor models
Female BALB/c nude mice and C57BL/6J (5-6 weeks old) were purchased from GemPharmatech Co.,Ltd, All animals were housed in a specific pathogen-free (SPF) laboratory in the Animal Center of Shenzhen People's Hospital at 22 ± 1 °C temperature and 40-50% humidity under a 12 h light/dark cycle with free access to water and standard laboratory chow. All procedures were approved by the Institutional Ethics Committee for Animal Experimentation and were conducted in accordance with the Shenzhen People's Hospital. B16F10 cells bearing tumor models were established by subcutaneously injecting B16F10 cancer cells (1 × 10 5 cells suspended in 100 μL of PBS) or PANC-1 cancer cells (1 × 10 7 cells suspended in 100 μL of PBS) into the flank of each mouse. The tumors allowed to grow to ~150 mm 3 for further use. In the in vivo tumor inhibition experiment, mice were divided randomly into 4 groups, and each group contained 6 mice.

In vivo evaluation of tumor targetability and distribution
When the B16F10 tumor volume bearing in C57BL/6J mice reached ∼150 mm 3 , the mice were treated with the intravenous injection of Ce6 labelled CM NPs (100 μL).
Next, the mice were placed in IVIS imaging systems for the observation of the fluorescence images at default time (0 and 24 h). 24 h later, the mice were anesthetized, and the tumors and major organs (liver, heart, lung, spleen and kidney) were harvested to analyze the fluorescence distribution. The main organs, including heart, liver, spleen, lung, kindey and tumor were collected to measure the concentration of Mn element at day 0, 12 and 24 h. At same time, 30 μL blood was collected from tail vein and determined the concentration of Mn element to evaluate the blood circulation and metabolism of NPs. where VT is the tumor volume after the treatments, and VC is the tumor volume of the control group.

In vivo tumor inhibition
Two weeks later, the mice were sacrificed, and the main organs (heart, liver, spleen, lung, and kidney) and the tumors were collected for HE staining analysis, and the blood were also collected for blood routine and biochemical analysis. The routine blood measurement included white blood cell (WBC) counts, RBC counts, hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), and hematocrit