CN115531344B - Multifunctional calcium-manganese nano modulator for resisting tumor and enhancing immunotherapy by remodelling tumor microenvironment - Google Patents

Abstract

The invention discloses a multifunctional calcium-manganese nano modulator for resisting tumor and enhancing immunotherapy by remodelling tumor microenvironment. The invention provides a nanoparticle which consists of a cell membrane and a calcium ion enhancer, calcium carbonate and manganese dioxide which are wrapped in the cell membrane. The invention constructs a double ion overload nano therapeutic system (B16F10@CaCO3-CU@MnO2, named CM NPs) by a biomineralization method, and the CM NPs are obtained by wrapping B16F10 cell membranes. The ion-based tumor micro-environment remodelling agent has higher tumor targeting capability, so that the ion-based tumor micro-environment remodelling agent can accumulate in tumor tissues, and has higher drug loading rate and pharmacokinetics due to self drug composition, so that the ion-based tumor micro-environment remodelling agent can synergistically enhance the tumor treatment effect.

Description

Multifunctional calcium-manganese nano modulator for resisting tumor and enhancing immunotherapy by remodelling tumor microenvironment

Technical Field

The invention belongs to the technical field of biology, and relates to a multifunctional calcium-manganese nano modulator for antitumor and immunity enhancement treatment by remodelling tumor microenvironment.

Background

Tumors are a serious life-threatening disease and are considered to be an important impediment to the continued development of human society. It is estimated that by 2040 years, there will be two thousand eight million tumor patients worldwide. Currently, conventional therapeutic methods (surgery, medicine and radiotherapy) and emerging therapeutic methods (immunotherapy, photothermal therapy, photodynamic therapy, gene therapy, sonodynamic therapy, etc.) have been developed and widely used. However, the therapeutic effect of these treatments is still unsatisfactory. Among these methods, immunotherapy methods for achieving antitumor purposes by training the immune system of the body have great advantages and good application prospects, and in particular, monoclonal antibody drugs have shown great potential for immune checkpoint therapies. Similarly, immunotherapy has significant drawbacks such as lower therapeutic responsiveness and concomitant serious side effects. Intensive studies have found that the tumor microenvironment is the main cause of poor therapeutic effects. Wherein, the tumor microenvironment has the characteristics of low pH, hypoxia, higher active oxygen, abnormal metabolic activity and the like. This is mainly due to the fact that tumor tissue has high heterogeneity, including various cell compositions such as tumor stem cells, stromal cells, tumor-associated fibroblasts, tumor cells, stromal cells, immune cells, etc., and the high heterogeneity of tumor tissue results in remodelling microenvironments and functions that are different from normal cells, promoting the development of tumors. Therefore, metabolism in the tumor microenvironment has become an effective tumor therapeutic target.

Under normal physiological conditions, cellular metabolic homeostasis is regulated by a complex and variable signal regulation network. The function of the key proteins in these signal pathways is regulated by biological ions, such as ca2+, mn2+, fe2+/3+, cu+/2+, zn2+, mg2+, etc., which have both their own biological and nutritional functions, regulate the growth and survival of cells, and when the ion content is imbalanced, often lead to fatal consequences, even cell death. More importantly, a portion of the ions have been shown to be involved in regulating the progression of diseases in which the immune system of the body is retarded. Thus, induction of intracellular ion overload has been explored as an effective tumor treatment. Among many ions, ca2+ as a major element necessary for the body has been demonstrated to have a variety of different biological functions in the body, for example, regulation of intracellular signaling pathways, cell homeostasis, cell death, and the like. Based on these considerations, oscillations in intracellular ca2+ concentration will result in changes in a range of biological processes, including altering the charge of the phospholipid bilayer, thereby promoting T cell activation and immune responses. When the content of calcium ions in dendritic cells is excessive, the calcium stores in the cells are destroyed, endogenous signal molecules are formed, autophagy is induced, antigen presenting process is promoted, and immune system is finally induced to generate immune response. Therefore, calcium ion-based tumor treatment methods have achieved good therapeutic effects in a variety of tumors. For example, a deformable core-shell nanosensitizer (tio2@cap) degrades its CaP shell under acidic tumor microenvironment and sonication conditions, releasing calcium ions, enhancing ROS production efficiency, leading to mitochondrial dysfunction and immunogenic cell death, and in combination with PD1 antibody drug therapy, effectively recruiting T cells and infiltration, activating systemic antitumor effects, inhibiting distant tumor growth and lung metastasis. In addition to calcium ions, manganese ions are also of great concern due to their enzymatic activity and the important role in the immune system activation process. Manganese ions can enhance the sensitivity of cGAS to damaged DNA, promote cGAMP synthesis and enhance the ability to de-bind STING, resulting in overactivation of intracellular cGAS-STING signaling pathways and activation of the immune system. Manganese ions can also directly activate cGAS, induce the activation of an inherent immune system, promote the antigen presentation process of macrophages and dendritic cells, promote the infiltration and killing capacity of toxic T lymphocytes, promote the formation of immune memory and enhance the immunological detection capacity of organisms. Thus, manganese ions can significantly improve the immunotherapeutic effect and have been considered as an effective immunoadjuvant. Based on this, manganese ions have shown great potential in the field of disease treatment. In addition, some studies have shown that manganese ions can remodel the tumor microenvironment to alter the therapeutic effect.

However, to date, no methods of tumor treatment for dual ion overload modulation have been seen. Ion overload at tumor sites by targeted delivery of tumors is still an urgent problem to be solved by tumor therapy.

Disclosure of Invention

The invention aims to provide a nanoparticle which is a multifunctional calcium-manganese nano modulator.

In a first aspect, the present invention provides a nanoparticle comprising a cell membrane and a calcium ion enhancer, calcium carbonate and manganese dioxide entrapped therein.

In the above, the cell membrane is a B16F10 cell membrane.

In the above, the calcium ion enhancer is curcumin.

The nanoparticle may also include a fluorescent molecule, in embodiments of the invention, specifically ce6.

In an embodiment of the invention, the nanoparticle consists of a cell membrane and a calcium ion enhancer, calcium carbonate and manganese dioxide encapsulated therein. Or, the nano-particles are composed of cell membranes, calcium ion enhancers, calcium carbonate, manganese dioxide and fluorescent molecules ce6 which are wrapped in the cell membranes.

Above, the cell membrane: curcumin: calcium carbonate: the proportion of manganese dioxide is 170-230 mug: 0.35-0.41mg:2.7-3.3mg:3mg;

Or, the cell membrane: curcumin: calcium carbonate: the proportion of manganese dioxide is 200 mug: 0.38mg:3mg:3mg.

In the above, the particle size of the nanoparticle is 450nm or less.

In a second aspect, the present invention provides a method of preparing the nanoparticle of the first aspect, comprising the steps of:

1) Preparation of CaCO ₃ -CU@MnO ₂ And a cell membrane;

said preparing CaCO ₃ -CU@MnO ₂ The method of (2) is as follows: firstly, manganese dioxide nano-sheet and CaCl ₂ Mixing with curcumin in water, and adding Na ₂ CO ₃ Mixing again, collecting precipitate to obtain CaCO ₃ -CU@MnO _2；

The CaCl ₂ The adding mass ratio of the curcumin to the manganese dioxide nano-sheet is 2.7-3.3:0.7-1.3:3;

in an embodiment of the invention, the CaCl ₂ The adding mass ratio of the curcumin to the manganese dioxide nano-sheet is 3mg:1mg:3mg:

the Na is ₂ CO ₃ In an amount such that the mass of the catalyst in the system is greater than the CaCl ₂ In the examples of the present invention, na ₂ CO ₃ The addition amount of (2) is more than or equal to 3mg;

stirring at room temperature for 6h, and rapidly stirring overnight; the collecting sediment is centrifugal collecting sediment;

at the bookIn examples of the invention, caCO was prepared ₃ -CU@MnO ₂ The method of (2) is as follows: 10mL of the above-mentioned nano-sheet (MnO) containing 3mg of manganese dioxide prepared in 1 was first prepared ₂ NSs) of manganese dioxide nanosheets to 3mg CaCl ₂ And 1mg Curcumin (CU) (CaCl) ₂ CU and MnO ₂ NSs mass ratio 3:1:3), stirring at room temperature for 6h, and adding 500 μg/mL excessive Na (6 mL or more, 6mL added at this time) ₂ CO ₃ The aqueous solution was stirred rapidly overnight. Centrifuging 16437g for 10min, collecting precipitate to obtain CaCO ₃ -CU@MnO ₂ ；

The cell membrane is obtained by lysing B16F10 cells;

in the examples of the present invention, the above cell membrane was prepared as follows:

1) -1, lysing B16F10 cells, and collecting the lysate;

1) -2, after homogenizing the lysate, collecting the homogenized solution;

1) -3, centrifuging 3500g of the homogenized solution for 5min, and collecting the supernatant;

1) 4, centrifuging the supernatant obtained in the steps 1-3 for 15min at 20 g, collecting the supernatant,

1) -5, centrifuging the supernatant obtained in 1) -4 at 100 g for 30min, and collecting the precipitate to obtain cell membranes;

2) The CaCO is processed by ₃ -CU@MnO ₂ After being uniformly mixed with the cell membrane, the mixture is extruded through the membrane and collected to obtain the nano-particles in the claims 1-4;

said CaCO ₃ -CU@MnO ₂ And the proportion of the cell membrane is 4.7-5.3mg:200 μg;

in an embodiment of the invention, the CaCO ₃ -CU@MnO ₂ And the proportion of the cell membrane is 5mg:200 μg;

the CaCO described above ₃ -CU@MnO ₂ And the ratio of the CaCO to the cell membrane is that ₃ -CU@MnO ₂ And the mass ratio of proteins of the cell membrane.

The pore diameter of the membrane is 450nm.

In the examples of the present invention, CM NPs were prepared: will 100 mu.L of the B16f10 cell membrane suspension prepared above and having a concentration of 2mg/ml and 2.5ml of CaCO having a concentration of 2mg/ml ₃ -CU@MnO ₂ The solution was mixed well (cell membrane (protein embodiment) and CaCO ₃ -CU@MnO ₂ The mass ratio of (2) is 200 mug: 5 mg), and then physically extruding through a 450nm polycarbonate membrane (Millipore, YS-TB-GTTP 09030), repeating for 30 times, collecting the extruded solution, centrifuging for 15min with 14005g, and collecting the precipitate to obtain CM NPs, namely the cell membrane coated nano material named CM NPs.

In a third aspect, the use of the nanoparticle of the first aspect for the preparation of an anti-tumour product is also within the scope of the present invention.

In an embodiment of the present invention, the tumor may specifically be melanoma or the like.

In a fourth aspect, the use of the nanoparticle of the first aspect for the preparation of a remodelled tumor microenvironment tumor product.

In the above, remodelling the tumor microenvironment is particularly manifested in that the addition of the nanoparticles of the first aspect results in a decrease in intracellular acidity, a modulation of the degree of hypoxia in the tumor cells, a significant increase in intracellular calcium ion content, an increase in ROS production, induction of immunogenic cell death in the tumor cells and/or induction of immune system activation.

In a fifth aspect, the use of the nanoparticle of the first aspect for the preparation of a product for enhancing the effect of immunotherapy on tumors is also within the scope of the invention.

In a sixth aspect, the use of the nanoparticle and immunotherapeutic agent of the first aspect in the preparation of an immunotherapeutic tumor or an enhanced immunotherapeutic tumor product is also within the scope of the invention.

In a seventh aspect, the present invention provides a product comprising the nanoparticle of the first aspect and an immunotherapeutic agent;

the product has at least one of the following functions:

1) An anti-tumor;

2) Improving the immune treatment effect;

3) And (5) performing immunotherapy.

In the above, the immunotherapeutic agent is an immune checkpoint (PD 1) inhibitor, in particular αpd1.

The novel calcium-manganese nanomaterial is synthesized, has high tumor targeting capability, can accumulate in tumor tissues, has high drug loading rate and pharmacokinetics due to self drug composition, and has a synergistic enhancement effect on tumor treatment effect based on ion tumor microenvironment remodeling capability. Based on the biological functions of calcium and manganese ions, it is inferred that organic binding of calcium and manganese ions across the load enables effective antitumor and enhanced immunotherapy. Notably, due to the higher metabolic demands, tumor cells are able to accumulate more ions. Therefore, the tumor treatment strategy based on the double ions has better tumor treatment effect.

The pH responsive nano-drug (CM NPs) of the invention is coated with curcumin (CU, a calcium ion enhancer), calcium carbonate and manganese dioxide, and utilizes cell membrane encapsulation to improve tumor targeting efficiency. CM NPs aim to remodel tumor microenvironment and anti-tumor effects by ion concentration oscillations. CM NPs are capable of neutralizing the low pH in cells by way of calcium carbonate breakdown, weakening the acidity in cells, releasing curcumin and calcium ions simultaneously, and calcium ion concentration oscillations cause calcium pool damage and ROS increase in mitochondria and endoplasmic reticulum, leading to cell immunogenic cell death. Meanwhile, manganese ions generated by degradation catalyze hydrogen peroxide endogenous to cells to generate oxygen so as to reduce the hypoxia degree in tumor cells, improve the sensitivity of cGAS to free nucleotide, activate a cGAS-STING signal channel and further activate the immune response of organisms. Meanwhile, by remodelling the microenvironment, the macrophage can be polarized, the effect of antigen presenting cells is increased, the activation and maturation of an immune system are promoted, and tumor cells are effectively killed. And the responsiveness of the tumor to PD1 can be improved, and the enhanced immunotherapy is realized. Therefore, the study has a good clinical application study foundation, and can be used with other treatment methods to realize multi-mode tumor combined treatment.

The invention constructs a double ion overload nano therapeutic system (B16F10@CaCO3-CU@MnO2, named CM NPs) by a biomineralization method, and the CM NPs are obtained by wrapping B16F10 cell membranes. In the tumor microenvironment, calcium ions are overloaded by the decomposition of calcium carbonate and calcium ion enhancers (curcumin, CU), and MnO2 degradation overloads manganese ions. CM NPs achieve antitumor effects through multiple effects: i) B16F10 cell membrane encapsulation ensures passive targeting of CM NPs to tumor tissue, leading to rapid uptake and accumulation of tumor cells; ii) calcium ion and CU produced by h+ response and depletion leads to intracellular calcium ion overload, increased ROS production and immunogenic cell death; iii) Manganese ions reduce the intracellular hypoxia and enhance the ROS production rate, promoting proliferation and maturation of immune cells. By combining the nanometer treatment system prepared in the research, the double ion overload tumor treatment method obtains good anti-tumor effect, and enhances the immune treatment effect and the responsiveness of immune checkpoint inhibitor.

In summary, CM NPs nanodrug therapeutic systems were prepared, CM NPs having pH-responsive drug release capabilities and tumor microenvironment remodelling through multiple steps. In tumor cells, CM NPs overload calcium ions, induce immunogenic cell death, and reverse the immunosuppressive state of the tumor microenvironment. The co-loaded CU is precipitated on the surface of the manganese dioxide nano-sheet and is coated by tumor cell membranes to facilitate NPs delivery, reduce the organism clearance rate and improve the tumor targeting capability. Because calcium carbonate can effectively react with protons, CM NPs can act as an effective proton scavenger and consume intracellular protons, reverse tumor acidity and raise pH, favoring ROS production. Manganese ions generated by manganese dioxide consume hydrogen peroxide and induce ROS production, and overloaded calcium ions destroy calcium stores in mitochondria and endoplasmic reticulum, induce an increase in intracellular oxidative stress, combine biological functions of manganese ions, induce immunogenic cell death, and activate immune responses. The tail vein injection nanometer material can effectively strengthen CM NPs at tumor position, has good biological safety, and has anti-tumor effect and anti-tumor immunotherapy effect on melanoma. In addition, CM NPs enhanced the anti-tumor responsiveness produced by αpd 1. However, it is desirable to detect the antitumor effect of higher concentrations of the prepared nanomaterial. In summary, the results of this study provide a new strategy for reprogramming tumor microenvironments by altering the levels of essential ions in the body and achieving antitumor effects in the body. In addition, this strategy can also serve as a great potential as a complementary approach, enhancing the efficacy of other therapies, particularly in immunotherapy of cancer, and providing multi-modal clinical treatments in the future.

Drawings

FIG. 1 is a graph showing the characterization and detection of CM NPs, a is TEM imaging, b is SDS analysis result, c is particle size detection of CM NPs, d is zeta potential detection, e is UV-Vis-NIR spectrum detection, f is apparent graph of degradation of CM NPs in PBS solutions with different pH values, g is degradation rate result of CM NPs in PBS solutions with different pH values, and h is ICP-OES detection of calcium and manganese content.

FIG. 2 shows the detection result of the in vitro remodelling tumor microenvironment of the CM NPs, wherein a is the detection result of the in vitro acid attenuation of the CM NPs, b is the apparent hydrogen peroxide decomposition diagram of the CM NPs, c is the detection of the hydrogen peroxide decomposition content of the CM NPs, and d is the detection of the condition of inducing ROS (reactive oxygen species) by the CM NPs in hydrogen peroxide solution.

FIG. 3 shows the results of in vitro cell uptake and therapeutic effect detection of CM NPs, a is the result of detection that Ce6 can be successfully loaded to CM NPs, B is the result of detection that CM NPs are taken up by B16F10 cells at different times, c is the result of statistics of detection that CM NPs are taken up by B16F10 cells at different times, d and e are the results of manganese and calcium uptake in B16F10 and RAW264.7 cells, respectively, F is the hemolysis ratio detection of CM NPs, g-i is the survival rate detection of CM NPs added with 293T, hela and RAW264.7, respectively, j is the in vitro cell therapeutic effect of CM NPs detected by Calcein AM/PI fluorescent staining, and k is the in vitro cell therapeutic effect of CM NPs detected by Annecin V-C/PI staining flow cytometry.

FIG. 4 shows MnO ₂ STEM-EDS analysis result diagram of NSs, a is a white light diagram, a Mn element diagram, an O element diagram and a Merge diagram respectively from left to right, and b is an element content statistical diagram.

FIG. 5 is a STEM-EDS analysis result diagram of CM NPs, a is a white light diagram, mn, C, S, N, O, ca, N, b is an element content statistical diagram in sequence.

FIG. 6 shows quantitative statistics of fluorescence intensity of changes in pH in cells detected by BCECF.

FIG. 7 is a schematic representation of fluorescence imaging of a hypoxia probe after addition of cobalt chloride to a B16F10 cell culture medium to simulate a hypoxic environment.

FIG. 8 shows the results of intracellular tumor microenvironment detection of CM NPs, a and b are phenotypes of the intracellular plasma reticulum and the intracellular calcium ion content of mitochondria treated by CM NPs, and c is the calcium ion content of CM NPs treated cells and medium, respectively; d is CM NPs to induce intracellular production of ROS; e is hydrogen peroxide degradation detection in CM NPs induced cells, F is marker for western blotting detection of CM NPs immunogenic cell death, g is CM NPs induced ATP release detection, h is CM NPs treatment resulting in increased IFN gamma content in B16F10 cells, and i is CM NPs treatment resulting in increased IFN gamma content in RAW264.7 cells.

FIG. 9 shows the results of different nanomaterial-processed macrophage polarization assays, a is a schematic immunofluorescence imaging of macrophage polarized CD80 and CD206, and b is M1 macrophage hydrogen peroxide production.

Fig. 10 shows in vivo targeting detection of CM NPs, a is detection of tumor sites by a fluorescence imaging system after CM NPs are injected into mice, b is fluorescence intensity of tumor sites after CM NPs are injected into mice, c is in vitro fluorescence imaging of organs after CM NPs are injected into mice, wherein Lv represents liver, lu represents lung, he represents heart, sp represents spleen, ki represents kidney, tu represents tumor, d is detection of in vitro fluorescence intensity of organs after CM NPs are injected into mice, e is concentration of Mn element in blood after CM NPs are injected into mice, and f is concentration of manganese element in different tissues at different times.

FIG. 11 shows the detection of antitumor effect of CM NPs in Balb/c nude mice, a is the tumor size after CM NPs are injected into mice, b is the tumor volume after CM NPs are injected into mice, c is the tumor weight after CM NPs are injected into mice, d is the detection of ROS in tumor tissue after CM NPs are injected into mice, and e is the staining of CM NPs injected into mouse tumor tissue HE, ki67 and Caspase-3.

FIG. 12 shows the results of body weight and survival rate of CM NPs in Balb/c nude mice in vivo biosafety test, a is the statistical result of body weight change of mice; b is the survival rate statistics of the mice.

FIG. 13 shows the results of Balb/c nude mice in vivo safety assessment blood routine. (a) blood normative index WBC, RBC, HGB, MCV, MCHC, PLT, HCT; (b) biochemical indicators of blood. ALT, AST, TP, GLB, TBIL, UREA, CREA, ALB; group 1 control group, group 2 MnO ₂ Group 3 CaCO ₃ @ CU, group 4:CM.

FIG. 14 shows in vivo toxicity by HE staining of major organs after Balb/c nude mic.

FIG. 15 shows body weight measurement of CM NPs injected with C57BL/6J mice.

FIG. 16 shows the routine and biochemical blood tests of CM NPs injected into C57BL/6J mice, (a) routine blood index WBC, RBC, HGB, MCV, MCHC, PLT, HCT; (b) biochemical indicators of blood. ALT, AST, TP, GLB, TBIL, UREA, CREA, ALB; group 1 control group, group 2 MnO ₂ Group 3 CaCO ₃ @ CU, group 4:CM, group 5 αPD1 and group 6:CM+αPD1.

FIG. 17 shows in vivo toxicity by HE staining after C57BL/6J treatment for the detection of major organ heart, liver, spleen, lung and kidney tissue structure changes.

FIG. 18 shows the detection of the immune activation effect of CM NPs on C57BL/6J mice, wherein a is the tumor size of the CM NPs injected into the mice, b is the tumor volume of the CM NPs injected into the mice, C is the tumor weight of the CM NPs injected into the mice, d is the detection of ROS content and HE staining of the tumors to detect cell death, and e is the staining of anti-tumor immunoreactive tissue sections of the CM NPs.

FIG. 19 is a schematic diagram showing CM NPs synthesis and anti-tumor activity. .

Detailed Description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Part of the procedure in the examples below is as follows:

cell culture in the following examples: culture medium of 293T, hela and RAW 264.7 cells is DMEM+10% FBS+1% penicillin and cultured at 37deg.C and 5% CO ₂ In a cell incubator.

PBS in the examples below was used at a concentration of 0.01 and M, pH at a value of 7.4, available from Whansai Weibull Biotechnology Co., ltd, catalog number G4202.

TUNEL analysis in the following examples: TUNEL detection was performed using TUNEL detection kit (GDP 1042, wuhansai wil biotechnology limited), with apoptotic nuclei marked red and imaged by fluorescence microscopy.

Analysis of data in the following examples: all data in this study are mean price ± SEM. Significance of the analytical differences was detected by GpaphPad Prime 8.0 single-factor anova and HSD differential analysis, and the significance differences were expressed as: * p <0.05, < p <0.01, < p <0.001and p <0.0001.

EXAMPLE 1 preparation of double ion-overloaded nanoparticle CM NPs

1. Preparation of CM NPs

The synthetic route of CM NPs is shown in detail in fig. 19, wherein TMAH: tetramethyl ammonium hydroxide; CU is curcumin; ICD: immunogenic cell death; CDN: cyclic dinucleotides; APC: an antigen presenting cell; FL: fluorescence imaging. I, ii and iii in the figure are as follows:

i) The increase of the pH value improves the survival rate of dendritic cells;

ii) calcium accumulation, pH increase, hypoxia reduction promotes macrophage polarized survival M1 is a cell;

iii) (i) and (ii) synergistically enhance the antigen presentation process.

1. Manganese dioxide nano-sheet (MnO) ₂ NSs) synthesis method

Manganese dioxide nanoplatelets are commercially available or can be synthesized as follows: reference is made to earlier literature [ Q.Ouyang, L.Wang, W.Ahmad, Y.Rong, H.Li, Y.Hu, Q.Chen, food chemistry 2021,349,129157]The method specifically comprises the following steps: 18mL of 0.5mM tetramethylammonium hydroxide solution was added to 10mL of 0.2mM MnCl ₂ To the aqueous solution, uniformly mixed, and then 2mL of hydrogen peroxide was rapidly added dropwise to the above mixed solution until the solution became brown. The brown solution was further spun at room temperature for 24h and centrifuged at 13000rpm for 10min to collect the precipitate, which was then quenched with absolute ethanol and ddH ₂ O is washed three times respectively, and is centrifuged at 13000rpm for 10min to collect manganese dioxide nano-sheets (MnO) obtained by precipitation ₂ NSs, particle size 191.33 nm);

3mg manganese dioxide nanoplatelets were suspended in 10mL ddH ₂ O, obtain manganese dioxide nano-sheetSuspension (denoted as MnO) ₂ NSs solution, 3mg/ml concentration) was placed at 4℃for further use.

2. Preparation of CaCO ₃ -CU@MnO ₂

CaCO ₃ -CU@MnO ₂ : 10mL of the above-mentioned nano-sheet (MnO) containing 3mg of manganese dioxide prepared in 1 was first prepared ₂ NSs) of manganese dioxide nanosheets to 3mg CaCl ₂ And 1mg Curcumin (CU) (CaCl) ₂ CU and MnO ₂ NSs mass ratio 3:1:3), stirring at room temperature for 6h, and adding 500 μg/mL excessive Na (6 mL or more, 6mL added at this time) ₂ CO ₃ The aqueous solution was stirred rapidly overnight. Centrifuging 16437g for 10min, collecting precipitate to obtain CaCO ₃ -CU@MnO ₂ ；

ddH ₂ After O is washed 3 times, caCO ₃ -CU@MnO ₂ Heavy suspension in ddH ₂ O to obtain CaCO ₃ -CU@MnO ₂ Solution (concentration 2 mg/ml).

CU@CaCO ₃ Nano material: according to CaCO ₃ -CU@MnO ₂ Except that 10mL of the above-mentioned 2-prepared nanosheets (MnO) containing 3mg of manganese dioxide ₂ NSs) of manganese dioxide nanosheet suspension was replaced with 10ml of water; obtaining CU@CaCO ₃ A nanomaterial. CU@CaCO ₃ Dissolving the nano material in water to obtain CU@CaCO 3mg/ml ₃ A solution.

3. Preparation of CM NPs

1 x 10 to be overgrown ⁷ B16F10 cells (Shanghai Fu He Biotechnology Co., ltd., FH 0361) were washed with PBS, the cells were collected by adding PBS containing 2mM EDTA, washed three times with PBS, and resuspended in 10mL of cell lysate (10 mM Tris-HCl (pH 7.5), 10mM KCl,2mM MgCl) ₂ And protease inhibitor (Biyun Tian, protease inhibitor cocktail (general type, 100X), cat# P1005), balance water), homogenizing in a Dounce homogenizer, centrifuging 3500g at 4deg.C for 5min, collecting supernatant, centrifuging at 20 g for 15min, collecting supernatant, centrifuging at 100 g for 30min, collecting precipitate, re-suspending and washing 10mM Tris-HCl buffer once, re-suspending in PBS solution to obtain solution of B16f10 cell membrane, and storing at-80deg.C Is used. The cell membrane content in the solution of B16f10 cell membrane is represented by protein content measured by BCA kit, and the concentration is 2mg/ml.

In the subsequent experiment, the solution of the B16f10 cell membrane was centrifuged at 100 g for 30min, the precipitate was collected and resuspended in water to obtain a B16f10 cell membrane suspension for the experiment.

Preparation of CM NPs: mu.L of the B16f10 cell membrane suspension prepared above and having a concentration of 2mg/ml was mixed with 2.5ml of CaCO having a concentration of 2mg/ml ₃ -CU@MnO ₂ The solution was mixed well (cell membrane (protein embodiment) and CaCO ₃ -CU@MnO ₂ The mass ratio of (2) is 200 mug: 5 mg), and then physically extruding through a 450nm polycarbonate membrane (Millipore, YS-TB-GTTP 09030), repeating for 30 times, collecting the extruded solution, centrifuging for 15min with 14005g, and collecting the precipitate to obtain CM NPs, namely the cell membrane coated nano material named CM NPs. After the CM NPs were suspended in water, a CM NPs suspension was obtained at a concentration of 2mg/ml.

In the CM NPs prepared by the method, the ratio of each substance is cell membrane: curcumin: calcium carbonate: manganese dioxide 200 μg:0.38mg:3mg:3mg.

Preparation of B@MnO ₂ : 100. Mu.L of the B16f10 cell membrane suspension prepared above at a concentration of 2mg/ml and 2.5. Mu.L of MnO at a volume concentration of 2mg/ml ₂ Mixing NSs solution uniformly (the mass ratio of cell membrane and manganese dioxide nano-sheet is 200 μg:5 mg), physically extruding through 450nm polycarbonate membrane, repeating for 30 times, collecting the extruded solution, centrifuging for 15min with 14005g, collecting precipitate to obtain B@MnO ₂ 。

Preparation of B@CaCO ₃ -CU: mu.L of the B16f10 cell membrane suspension prepared above at a concentration of 2mg/ml was mixed with 2.5. Mu.L of CU@CaCO at a volume concentration of 2mg/ml ₃ The solution was mixed well (cell membrane and CU@CaCO) ₃ The mass ratio of (2) is 200 mug: 5 mg), physically extruding through a 450nm polycarbonate film, repeating for 30 times, collecting the extruded solution, centrifuging for 15min with 14005g, and collecting the precipitate to obtain B@MnO ₂ 。

2. Detection of CM NPs

1. TEM imaging

MnO prepared by the method ₂ NSs、CaCO3@CU and CM NPs were subjected to TEM imaging (JEM-3200 FS Transmission Electron Microscope).

As a result, as shown in FIG. 1a, TEM imaging shows the obtained MnO ₂ NSs (fig. 1a, left), caco3@cu (fig. 1a, middle) and CM NPs (fig. 1a, right) each have a uniform structure.

2. EDS analysis

MnO is added to ₂ The NSs and CM NPs were subjected to EDS analysis (D8 Advance (Bruker, germany, voltage 40kV,10 DEG 2Theta 80 DEG)).

EDS analysis shows that the manganese dioxide nanoplatelets have only manganese and oxygen elements (fig. 4a is a white light plot, a Mn element plot, an O element plot, and a Merge plot, respectively, from left to right, and fig. 4b is an element content statistical plot).

EDS analysis showed that the surface of CM NPs had Mn, C, O, ca, N and S element signals (fig. 5a is a white light plot, mn, C, S, N, O, ca, N, fig. 5b is an element content statistical plot, in order).

3. SDS analysis

SDS analysis: the B16f10 cell membrane suspension prepared in the above item 3, the MnO prepared in the above item 1 ₂ NSs and CaCO prepared by one of the above 2 ₃ -CU@MnO ₂ And the CM NPs prepared in 3 of the above were resuspended in 1 XSDS loading buffer after being quantified by BCA kit, boiled at 100℃for 10min, subjected to protein electrophoresis separation (120V, 1.5 h) by 10% SDS-PAGE, stained with Coomassie brilliant blue for 2h, ddH ₂ O was washed and imaged.

The results are shown in FIG. 1B, where 1 and 6 are markers, 2 is the B16f10 cell membrane, 3 is MnO ₂ NSs, 4 is CaCO ₃ -CU@MnO ₂ And 5 is CM NPs, and the fact that the lanes 2 and 5 have the same protein shows that the cell membrane of B16F10 is successfully coated on the surface of CaCO3-CU@MnO2, so that the CM NPs are successfully prepared.

4. DLS analysis

MnO is added to ₂ NSs (shown as MnO 2) and CaCO ₃ -CU@MnO ₂ And CM NPs (denoted as CM in the figure) were subjected to DLS analysis (Malven, ZETASIZER)

The DLS analysis showed that the particle sizes of MnO2, caCO3-CU@MnO2 and CM NPs were 191.33nm,345.66nm,and 392.70nm (FIG. 1 c), and zeta potentials were +0.38mV, -4.46mV and-17.43 mV, respectively (FIG. 1 d). The CM NPs were shown to have good dispersibility.

5. UV-Vis-NIR spectroscopic detection

MnO is added to ₂ NSs (shown as MnO 2) and CaCO ₃ -CU@MnO ₂ And CM NPs (denoted CM in the figure) were subjected to UV-Vis-NIR spectroscopy (SPARK 10M).

The UV-Vis-NIR spectrum results show that the prepared nanomaterial has no obvious characteristic peak except CU at 434nm (FIG. 1 e).

According to the characteristic absorption peak at 434nm, the load factor of the CU in CM was 38.91%, which is much higher (7.08%) than in CaCO3@CU.

The above results demonstrate successful preparation of CM NPs.

7. CM NPs degradation in PBS solutions of different pH values

The CM NPs prepared in the above were uniformly dispersed in PBS solutions (pH 7.4 and 5.4) at 100. Mu.g/mL, allowed to stand at room temperature for eight days, and the color of the solutions after the 0 th day and 8 th days of standing was observed.

The results are shown in FIG. 1f, and it can be seen that the CM NPs became transparent after 8 days of standing, indicating degradation of the CM NPs.

The change in absorbance was detected by UV-Vis-NIR spectroscopy and characterizes the material stability over 8 days of the above-described standing. The absorbance was calculated as degradation rate.

UV-Vis-NIR spectra showed that CM NPs were able to degrade in PBS (FIG. 1 g).

8. Calcium and manganese ion release rate detection

The CM NPs prepared above were uniformly dispersed in PBS solution (pH 7.4 and 5.4) at 100. Mu.g/mL, and at a preset time point, the solution was centrifuged at 13000rpm for 15min, and the supernatant was collected and assayed for calcium and manganese content by ICP-OES.

The ICP-OES is to digest the nano material with concentrated nitric acid, evaporate nitric acid by heating, dissolve ddH2O, and detect with inductively coupled plasma mass spectrometer (JY 2000-2,Horiba Jobinyvon,USA) instrument.

As a result, as shown in FIG. 1h, it was confirmed that the degradation rates of the elements in the degraded supernatants were further confirmed by ICP-OES detection, and the degradation rates of the calcium ions and the manganese ions in the solutions at pH5.4 and pH7.4 were 91.20%,65.88%,33.67% and 32.95%, respectively.

These results demonstrate that CM NPs were successfully produced and that they were characterized by low pH response and decomposition.

Example 2 application of CM NPs in vitro remodelling tumor microenvironment

Tumor microenvironments have been demonstrated to be immunosuppressive microenvironments with low pH, hypoxia, and high hydrogen peroxide content, among others.

The ability of CM NPs to remodel tumor microenvironment was tested in vitro as follows:

1. CM NPs have good acid attenuation capacity in vitro

The CM NPs prepared in example 1 above were dispersed in PBS solution at pH5.4,6.5 and 7.4 at 20. Mu.g/mL, stirred at room temperature, and the pH of the solution was measured at a predetermined time point (0,0.5,1,2,6,12,24 and 48 h) using pH.

As a result, as shown in fig. 2a, CM NPs were able to significantly raise the solution pH by the following reaction process: (i) (ii)/>and(iii)/>After 48h, the pH of the solution increased to 6.49,6.88 and 7.57, respectively, meaning that CM NPs had good acid weakening capacity in vitro, which contributed to CU release.

2. CM NPs hydrogen peroxide decomposition capability

CM NPs prepared in example 1 (referred to as CM in the figure) or MnO prepared in example 1 ₂ NSs (50. Mu.M Mn, designated MnO in the figure) ₂ ) Resuspended in a containing 100. Mu. M H ₂ O ₂ Through the PBS solution at a preset time pointHydrogen oxide analysis kit (Biyun Tian, S0038) for detecting residual H in solution ₂ O ₂ The content is as follows. In cells, H was detected according to the hydrogen peroxide assay kit detection instructions ₂ O ₂ The content is as follows.

As shown in FIGS. 2b and 2c, the imaging showed that the addition of CM NPs formed distinct bubbles, indicating the occurrence of hydrogen peroxide decomposition to oxygen (FIG. 2 b), and the quantitative analysis showed that both manganese dioxide and CM NPs rapidly decomposed hydrogen peroxide (FIG. 2 c), and that hydrogen peroxide decomposition was able to reduce the degree of hypoxia in the cells.

3. ROS production

Manganese dioxide passing through MnO in the presence of hydrogen peroxide ₂ +2H ⁺ ＝Mn ²⁺ 2H ₂ O+ ^1/2 O ₂ ROS production is induced. In tumor cells, hypoxia impedes the therapeutic effect of tumors. Thus, CM NPs were tested herein for induction of ROS production in hydrogen peroxide solution.

mu.M ROS probe DCFH-DA (Biyun Tian, S0033M) was combined with 50. Mu. M H ₂ O ₂ The mixture was added to CM NPs to a concentration of 20. Mu.g/mL, and the fluorescence intensity was measured by a fluorescence spectrophotometer.

As a result, the fluorescence intensity gradually increased with increasing time within 60min, and then the fluorescence intensity gradually decreased (30 min, 60min, 10min, 120min, 240min in this order from top to bottom in FIG. 2 d), the fluorescence intensity increased due to the increase in the generation of ROS, and the fluorescence intensity decreased due to the catalytic decomposition of the hydrogen peroxide content in the solution with increasing time.

These results demonstrate that CM NPs are able to remodel tumor microenvironments, induce an increase in intracellular oxidative stress, achieve chemo-kinetic therapy, and facilitate tumor immunotherapy.

Example 3 in vitro cell uptake and therapeutic Effect detection of CM NPs

To investigate the therapeutic effect of CM NPs, ce 6-labeled CM NPs were examined for uptake by cells.

1. Preparation of Ce 6-tagged CM NPs

The preparation of CM NPs was carried out according to one of the preparation methods of example 1, except that CaCO was prepared in step 2 ₃ -CU@MnO ₂ In the preparation, 10mL of the 3mg manganese dioxide nano-sheet (MnO) prepared in the above 1 was added ₂ NSs) of manganese dioxide nanosheets to 3mg CaCl ₂ 1mg curcumin and 1mg Ce6 (CU for short) (CaCl) ₂ ,CU、MnO ₂ The mass ratio of NSs to Ce6 is 3:1:3: 1) The rest are the same, and CM NPs marked by Ce6 are obtained.

In the prepared Ce 6-marked CM NPs, the ratio of each substance is the cell membrane: curcumin: calcium carbonate: manganese dioxide: ce6 is 200 μg:0.38mg:3mg;3mg;

Detection of example 1 preparation of B@CaCO using a fluorescence spectrophotometer ₃ CU (denoted CaCO in the figure) ₃ @CU)、B@MnO ₂ (shown as MnO) ₂ ) CU, ce6 and Ce 6-tagged CM NPs (denoted CM-Ce6 in the figure).

As a result, ce6 can be successfully loaded into CM NPs with a higher load factor (15.71%) as shown in fig. 3 a.

2. Cell uptake assay

Will be 1X 10 ⁵ B16F10 cells were inoculated in 35mm laser confocal dishes for overnight incubation, medium was removed, 2mg/mL of CM NPs suspension (CM NPs final concentration in the system 50. Mu.g/mL) was added for different times, medium was removed, PBS was washed, fluorescent microscopy was used for imaging, cells were collected, and cell fluorescence intensity was measured to characterize cell uptake.

The results are shown in FIGS. 3B and 3c, and it can be seen that CM NPs can be rapidly taken up by B16F10 cells.

Meanwhile, after B16F10 and RAW264.7 cells were incubated with 50. Mu.g/mL CM NPs for different times, respectively, using the above method, the levels of calcium and manganese ingested were detected by ICP-OES.

The analysis showed that CM NPs were taken up by two cells, and the detection showed that the calcium and manganese contents in B16F10 and RAW264.7 cells were 37.38, 41.29,8.90, 11.68. Mu.M/5 x 10, respectively ⁵ cells (fig. 3d,3 e), and with time, the cellular uptake content increased.

3. Blood compatibility test

Since biocompatibility and safety are unavoidable issues of nanomaterials, the biocompatibility and safety of CM NPs are then examined. Based on tumor cell membrane, the tumor targeting can be avoided and the body clearance can be improved.

The blood compatibility test uses the ratio of hemolysis to test: 1ml of C57BL/6J blood was collected by taking blood from eyeballs of C57BL/6J mice, diluted to 5ml with PBS, centrifuged at 2000rpm for 10min, and red blood cells were isolated 6 times. CM NPs (designated CM in the figure) were added to PBS solutions containing erythrocytes at various concentrations (5,10,20,50 and 100. Mu.g/mL), and the supernatant was collected by centrifugation at 10min at 10 rpm for 3h at 37℃on a shaker and absorbance at 570nm was measured using an enzyme-labeled instrument. Dispersing erythrocytes in ddH ₂ O served as positive control.

As a result, as shown in FIG. 3f, although the concentration of CM NPs was increased, no apparent red color was observed in the supernatant, indicating that CM NPs did not cause a significant hemolysis phenomenon.

4. Security detection

Cytotoxicity analysis: 293T (China academy of sciences cell bank GNHu 17), hela (China academy of sciences cell bank TCHu 187) and RAW 264.7 (China academy of sciences cell bank TCM 13) cells were cultured according to 2 х 10 ⁴ cells/well were inoculated in 96-well plates and cultured overnight, when the cell confluence reached 60-65%, 200. Mu.L of fresh medium containing CM NPs (designated CM in the figure) at different concentrations (0,5,10,20,50 and 100. Mu.g/mL) was added instead for further culture for 24 hours, each group was repeated six times, the medium was removed, CCK-8 detection reagent (10. Mu.L CCK-8+90. Mu.L serum-free DMEM medium) was added, and the mixture was allowed to stand in a 37℃incubator for 1 hour, and the absorbance at 450nm was measured by an enzyme-labeled instrument to calculate the cell activity characteristic material toxicity.

As shown in FIG. 3g, it can be seen that CM NPs do not affect cell growth.

5. In vitro cell therapeutic effects of CM NPs

In vitro tumor treatment was detected by CCK-8, calcein AM/PI fluorescent staining and Annexin V-FITC/PI staining flow cytometry as follows:

cells 293T, heLa and B16F10, which were not used, were seeded in 6-well plates, 200. Mu.L of CM NPs (designated CM in the figure) containing different concentrations (0,5,10,20,50 and 100. Mu.g/mL), B@CaCO were added instead ₃ CU (denoted CaCO in the figure) ₃ @ CU) and B @ MnO ₂ (shown as MnO) ₂ ) The culture is continued for 24 hours, the cell viability is detected by cck-8, and the anti-tumor effect is detected by Calcein-AM/PI fluorescent staining.

The results are shown in FIGS. 3g-3h (3 g 293 cells, 3h hela cells, 3i raw264.7 cells), and can be seen to correspond to B@CaCO ₃ -CU and B@MnO ₂ In contrast, after 24h incubation of CM NPs with different cells, the normal cell (293T) assay showed a slight inhibition of viability at higher concentrations, whereas CM NPs showed a significant inhibition in HeLa and B16F10 cells. For example, at a concentration of 50 μg/mL, about 50% of the cells die, confirming that overload with calcium and manganese ions is effective in inducing tumor suppression.

Similarly, the results of Calcein AM/PI fluorescent staining (FIG. 3 j) and Annexin V-FITC/PI stained flow cytometry (FIG. 3 k) also showed that CM NPs and B@CaCO ₃ -CU and B@MnO ₂ Compared with the traditional Chinese medicine, the traditional Chinese medicine has good treatment effect.

In conclusion, CM NPs can effectively kill tumor cells and have good biocompatibility.

Example 4 intracellular tumor microenvironment remodeling detection

Proton neutralization and manganese dioxide nanoenzyme properties informed by intracellular calcium carbonate were examined at the cellular level for their ability to remodel the tumor microenvironment, including intracellular acidity, hypoxia, calcium ion content, ROS content, hydrogen peroxide and GSH content.

1. Intracellular pH detection

The change in intracellular pH was detected by BCECF AM as an indicator probe as follows:

1×10 ⁴ B16F10 cells were inoculated in 48-well plates, cultured overnight, and added with various nanomaterial CM NPs (denoted as CM in the figure), B@CaCO ₃ CU (denoted CaCO in the figure) ₃ @ CU) and B @ MnO ₂ (shown as MnO) ₂ ) Incubation for 6h, adding 1. Mu.M pH probe BCECF-AM (Biyun Tian, S1006), cell incubator continued to culture for 15min, washed 3 times with PBS, fluorescence microscopy imaging and fluorescence spectroscopyThe intensity of fluorescence is detected by a densitometer.

The results are shown in FIG. 6, which shows the results with B@CaCO ₃ -CU and B@MnO ₂ In contrast, when CM NPs were incubated with B1F10 cells for 12h, the fluorescence intensity in the cells increased significantly, indicating CaCO ₃ The @ CU and CM NPs are effective in causing a decrease in intracellular acidity.

2. Degree of hypoxia

Changes in intracellular pH can lead to changes in intracellular ion flow rates and levels, which in turn lead to cell membrane scalability, and also to changes in organelle structure and function and communication between organelles, as well as intracellular free radical levels and cell survival.

B16F10 cells under hypoxic conditions contained different concentrations (0,5,10,20,50 and 100. Mu.g/mL) of CM NPs (denoted CM in the figure), B@CaCO at 200. Mu.L ₃ CU (denoted CaCO in the figure) ₃ @ CU) and B @ MnO ₂ (shown as MnO) ₂ ) The culture was performed in a medium (100 uM of cobalt chloride was added to the medium to simulate a hypoxia environment) for 1,2,6 hours.

The degree of hypoxia utilized a hypoxia probe Image-iT Green Hypoxia Reagent as an indicator.

As shown in FIG. 7, normoxia is a control with CoCl2 added, and the control means a control with no CoCl2 added, and it can be seen that the control is equivalent to B@CaCO ₃ Compared with CU, B@MnO ₂ And the degree of hypoxia within tumor cells was significantly reduced following CM NPs treatment.

These results demonstrate that manganese dioxide and CM NPs are effective in modulating the degree of hypoxia in tumor cells, whereas CaCO ₃ The @ CU had no significant effect on the degree of hypoxia in the cells.

3. Intracellular calcium ion content

Mitochondria and endoplasmic reticulum are intracellular calcium stores and ROS production sites, and have a critical role in the signaling molecules and redox state of biological processes within cells, while calcium ions have a critical role in the structure and function of mitochondria and endoplasmic reticulum. Therefore, the calcium ion content in the cells after different treatments was then detected using a calcium ion probe (Fluo-4 AM, biyun, S1060) and the calcium ion content was quantitatively obtained using Calcium Colorimetric Assay Kit (Biyun, S1063S).

5*10 ⁴ The amount of B16F12 cells was inoculated into a 12-well plate, followed by culturing for 18-20 hours, incubating with CM NPs (designated as CM in the figure) at a concentration of 50. Mu.g/mL for 12 hours, adding 3. Mu.M Fluo-4 AM, and detecting the fluorescence intensity of the probe by laser confocal imaging and a fluorescence spectrophotometer. Cells were collected and assayed for intracellular calcium ion content according to Calcium Colorimetric Assay Kit instructions.

As shown in fig. 8a (endoplasmic reticulum) and 8b (mitochondria), CM NPs resulted in a significant increase in intracellular calcium ion content, disrupting intracellular calcium ion homeostasis.

The quantification results showed a significant increase in calcium ion content in CM NPs treated cells and medium (fig. 8 c).

4. ROS detection

Tumor cells establish a tumor microenvironment suitable for their proliferation and metastasis, accompanied by abnormal cellular metabolism, e.g., increased hydrogen peroxide production rates in tumor cells, which in turn leads to increased ROS production.

The hydrogen peroxide and ROS content changes in the cells were detected by hydrogen peroxide kit (bi yun, S0038) and ROS probe (bi yun, S0033M), specifically as follows:

different nanomaterial CM NPs (denoted as CM in the figure), B@CaCO ₃ CU (denoted CaCO in the figure) ₃ @ CU) and B @ MnO ₂ Adding 50 mug/mL into B16F10 cell culture medium, incubating B16F10 cells in the culture medium for 12h, removing the culture medium, adding fresh culture medium containing 5 mug DCFH-DA, culturing in a cell culture box for 30min, and detecting ROS by laser confocal imaging.

The assay results show that manganese dioxide and CM NPs are able to significantly induce hydrogen peroxide decomposition within cells (fig. 8 e), and that increased ROS content results in increased oxidative stress within cells leading to cell death, especially ROS are prone to damage to mitochondrial structures releasing damaged DNA or Cyclic Dinucleotides (CDNs) and other damaged cellular components, and release into the cytosol matrix inducing cascade of immunogenic cell death.

5. Western immunoblotting

Different nanomaterial CM NPs (denoted as CM in the figure), B@CaCO ₃ CU (denoted CaCO in the figure) ₃ @ CU) and B @ MnO ₂ The cells were collected by adding 50. Mu.g/mL to B16F10 cell culture medium, incubating the B16F10 cells in the medium for 12 hours.

Extracting cell total protein by using a 1x SDS loading buffer solution, spotting and placing the cell total protein in an SDS-PAGE gel with the concentration of 8-15%, sealing the cell total protein in a transfer paper PVDF membrane by using a sealing solution (Beyotime Biotechnology, cat.no. P0023B), keeping the cell total protein at room temperature for 1h, adding a specific primary antibody, using beta-actin as a loading internal reference, washing the membrane for 3 times by using PBST for 5 min/time, adding secondary antibody anti-rabit IgG, HRP-linked second antibody secondary antibodies (cat.no. 7074; CST), incubating the membrane for 2h at room temperature, washing the membrane for 3 times by using PBST for 5 min/time, and performing imaging detection after adding a chemiluminescent solution.

Markers for detection of immunogenic cell death by western blotting include HMGB1, cGAS, STING and CRT.

As shown in fig. 8F, after B16F10 cells were treated with different nanomaterials, HMGB1, cGAS, STING and CRT were significantly increased, indicating that induction of immunogenic cell death occurred, activation of cGAS-STING signaling pathway, and further immune response, leading to dendritic cell proliferation and maturation and T cell activation.

6. ATP content detection

Following detection of other indicators of immunogenic cell death, the released ATP.

B16F10 cells were cultured according to 5X 10 ⁴ Inoculating cells/well into 24-well plate, culturing for 16 hr until cell confluence reaches 80-85%, removing old culture frame, adding CM NPs (CM) and B@CaCO containing different nanomaterials of 50 μg/mL ₃ CU (denoted CaCO in the figure) ₃ @ CU) and B @ MnO ₂ Incubating for 24h, collecting cell culture medium and cells, and detecting ATP content by chemiluminescence according to the instructions of ATP detection kit (Biyun Tian, S0026).

The results are shown in figure 8g, CM NPs were able to significantly induce ATP release into the cell culture medium.

Meanwhile, due to the important role of ifnγ in cGAS-STING signaling pathway. The ifnγ content in B16F10 cells after the above incubation was detected by ELISA method (Biolegend, 430807).

The results showed that CM NPs treatment resulted in an increase in ifnγ content in B16F10 cells (fig. 8 h).

7. Immune system activation detection

Immune system activation begins with antigen presentation by antigen presenting cells, including macrophages and dendritic cells. Therefore, RAW264.7 cells were selected to explore immune system activation.

B16F10 cells were incubated with high concentration CM NPs (200 μg/mL) to completely kill tumor cells, dead cells were collected, incubated with RAW264.7 cells, constituents within dead cells including damaged DNA, etc., activated STING signaling pathway, and induced ifnγ production. The method comprises the following steps:

According to 2X 10 ⁵ cell/well density B16F10 cells were seeded in 6-well plates and when cell confluence reached 70%, different nanomaterials were added: CM NPs (denoted as CM in the figure, 10. Mu.g/mL), B@CaCO ₃ CU (denoted CaCO in the figure) ₃ @ CU, 10. Mu.g/mL) and B @ MnO ₂ (shown as MnO) ₂ 10. Mu.g/mL) for 12h, and the cell culture medium was collected to detect IFNγ concentration.

At the same time, these cells were treated with UV irradiation (1500J cm ^-2 ) Killing tumor cells, collecting the treated B16F10, adding the B16F10 into RAW264.7 cells, and collecting the RAW264.7 cells to detect IFN gamma concentration.

The results of the assay are shown in FIGS. 8h and 8i, which show that the IFNγ content in nanomaterial-treated cells is much higher than in the control group, especially the CM NPs group.

In the tumor cell microenvironment, macrophages are usually polarized into M1 and M2 types, and these two types of macrophages have opposite biological functions.

Adding 25ng/mL IL-4 or lipopolysaccharide (1 μg/mL induced polarization process is converted into M2 and M1 macrophage) into RAW264.7 cell culture medium, culturing RAW264.7 in the culture medium for 24 hr to obtain M2 and M1 macrophage, and continuously adding CM NPs (shown as CM,10 μg/mL), B@CaCO ₃ CU (denoted CaCO in the figure) ₃ @ CU, 10. Mu.g/mL) and B @ MnO ₂ (shown as MnO) ₂ 10. Mu.g/mL) for 12h.

Polarized megaphagy was identified by marker proteins on the surface of the cell membrane.

The identification result of the immunofluorescence staining method is shown in fig. 9a, and M2 macrophages are incubated with different nano materials for 12 hours to induce the conversion of the M2 macrophages into M1, so that calcium and manganese are proved to be capable of inducing macrophage polarization.

Meanwhile, M1 macrophages are accompanied by the generation of a large amount of hydrogen peroxide, and the result is shown in FIG. 9b, it can be seen that M2 macrophages are also treated with different nanomaterials for 12h, at MnO ₂ ,CaCO ₃ The hydrogen peroxide content in the @ CU and CM NPs groups increased significantly.

Although CaCO alone ₃ The @ CU NPs had no significant effect on hydrogen peroxide production, but manganese dioxide and CM NPs had a more pronounced effect on hydrogen peroxide production after long-term treatment compared to short-term treatment (1 hour). Activated macrophages produce hydrogen peroxide that can release oxygen under the catalysis of manganese dioxide to reduce the level of hypoxia. In addition, the combination of remodelled TME with increased pH and hypoxia and the biological function of calcium and manganese results in efficient polarization of M1 macrophages that can evoke activation of the immune system. Also, calcium ion accumulation can enhance dendritic cell viability and improve antigen presentation. The synergistic effect of dendritic cells and macrophages in antigen cross presentation can induce activation of immune responses, recruiting effector T cells to the tumor site to kill cancer cells.

Taken together, these results indicate that CM NPs can induce ICD and activate the immune system.

Example 5 in vivo targeting detection of CM NPs

The biodistribution and clearance rate of nanotherapeutic drugs in vivo are critical to their potential biomedical applications. Thus, it was further evaluated whether CM NPs could target and accumulate at tumor sites.

1. Animal and tumor model establishment

Female Balb/C and C57BL/6J mice were purchased from Ji-Cuff, all kept in SPF animal houses at the temperature: 22+ -1deg.C, wetDegree: 40-50%, and 12-12h of daytime and night circulation, and enough feed and drinking water are maintained. According to 1x 10 ⁵ The cell/mouse B16F10 mice were inoculated on the right side of the back leg of the mice to establish tumor models. The mice were randomly divided into 4 groups of 6 mice each at a tumor volume of about 150mm 3.

2. In vivo tumor targeting and distribution detection

When the tumor size is about 150mm ³ At this time, 100. Mu.L of Ce 6-labeled CaCO was injected intravenously at the tail of the mouse ₃ -CU@MnO ₂ (methods refer to Ce 6-labeled CM NPs, curcumin was replaced with Ce 6-labeled curcumin alone) and Ce 6-labeled CM NPs were injected at a dose of 15mg/kg, and nanomaterial targeting was detected by a small animal in vivo imaging system at preset time points (0 and 24 h).

In vivo fluorescence imaging system of IVIS Spectrum of small animal living imager for detecting targeting and distribution (CaCO) of C57BL/6J mouse model after intravenous injection of Ce 6-labeled nanomaterial ₃ -CU@MnO ₂ (1) group and CM NPs, (2) group, 15 mg/kg).

After 24h of injection, the tumor sites showed enhanced Ce6 fluorescence signal (fig. 10 a). The fluorescence intensity of tumor sites of CM NPs-injected mice is significantly higher than that of CaCO-injected mice ₃ -CU@MnO ₂ Fluorescence intensity at the tumor site in mice indicated that B16F10 cell membrane encapsulation enhanced the targeting ability of NPs (fig. 10B).

After 24h imaging, the mice are anesthetized, tumors and major organs are separated, and the in-vivo distribution of the nano material is detected by imaging. Meanwhile, at 0,12 and 24 hours, tumors and main organs are taken, and after digestion by nitric acid, the content of manganese element is detected by ICP-OES to represent the in-vivo distribution and metabolism condition of the material. At a preset time point, 30 mu L of whole blood is taken through a tail vein, and after digestion, the content of manganese element is detected through ICP-OES to represent the in-vivo metabolism condition.

Mice were sacrificed under anesthesia and organ ex vivo fluorescence imaging was performed to examine the in vivo distribution of major organs. The fluorescence intensity in tumor tissue was highest, also significantly higher than that of liver tissue (fig. 10c and 10 d). And the fluorescence intensity of tumor tissue of mice injected with CM NPs is far higher than that of mice injected with CaCO ₃ -CU@MnO ₂ Mouse tumor groupFluorescence intensity of the fabric.

2. Immunofluorescent staining

Staining the tumor tissue after anesthesia:

cells or tissue sections were fixed with 4% paraformaldehyde at room temperature for 15min,0.1% Triton X-100PBS was allowed to pass through for 10min, 5% BSA solution was added, blocked at room temperature for 1h, specific primary antibody was added, incubated overnight at 4℃and washed 3 times with PBS, fluorescent-labeled secondary antibody was added, incubated at room temperature for 2h, and confocal laser imaging was performed.

Immunofluorescent staining demonstrated an increase in targeting ability of CM NPs.

3、Mn ²⁺ Concentration of

Analysis of circulating Process in blood of NPs injected mice Using ICP-OES and Mn in blood ²⁺ Concentration.

CaCO ₃ -CU@MnO ₂ And the circulation and elimination half-lives of CM NPs in C57BL/6J mice were 1.09 and 15.22h,1.63 and 16.19h, respectively (fig. 10 e). The half-life detection result shows that NPs coated by tumor cell membranes can reduce the clearance rate of the body.

Measurement of Mn in major organs and tumors by using ICP-OES ²⁺ Concentration characterization CM NPs in vivo biodistribution (1, 12 and 24 h).

The results of the study showed that CM NPs can accumulate gradually at the tumor site (fig. 10 f), probably due to the targeting ability provided by the cancer cell membrane and EPR effect, while accumulation in spleen and liver is a result of the capturing of nanomaterials by reticuloendothelial system.

Thus, these results indicate that CM NPs have good tumor targeting ability and pharmacokinetic profile, have longer blood circulation and high tumor accumulation efficiency.

Example 6 detection of antitumor Effect of CM NPs in vivo

CM NPs can effectively remodel the intratumoral environment, calcium ion overload has good therapeutic effect, and manganese ions can reverse immunosuppression, and CM NPs have good tumor targeting ability and accumulate in a large amount in tumor tissues.

1. CM NPs antitumor effect was tested in vivo.

B16F10 was transplanted subcutaneously on the dorsal side of the right hind limb of BALB/c nude mice (Balb/c nude mice), when the tumor volume size was 180mm ³ Mice were randomly divided into four groups as follows: (1) Control; (2) B@MnO ₂ (shown as MnO) ₂ )；(3)B@CaCO ₃ The @ CU (noted CU in the figure); and (4) CM NPs (denoted CM in the figure) of 6 mice per group: (1) Group injection of PBS, (2) - (4) B@CaCO respectively ₃ -CU(15mg/kg)，B@MnO ₂ (15 mg/kg) and CM NPs.

The sustained therapeutic effect of the nanomaterial against tumors was measured by measuring tumor size, body weight and production changes every two days.

The volume and the weight of the mice are measured once every two days, the survival condition of the mice is counted, and the calculation formula of the tumor volume is as follows: 0.5 length width ² After 14 days, mice were anesthetized, tumor tissue was isolated, and weighed. Tumor inhibition rate calculation formula: tgi= (V _C -V _T )/V _C ×100％，V _T For treating terminal tumor volume, V _C To size of tumor volume at the beginning of treatment.

At the end of treatment, whole blood and serum as well as major organs were collected and biosafety was detected by blood routine, blood biochemistry and HE staining.

The results showed MnO ₂ And CaCO (CaCO) ₃ The @ CU had some degree of tumor inhibition (FIGS. 11a, b), and CM NPs showed potent anti-tumor effects with tumor inhibition rates of-655.40%, -469.79%, -385.09% and +6.15%, respectively, for groups (1) - (4). At the end of the experiment, mice were sacrificed under anesthesia and tumor tissues were isolated and weighed, and CM NPs significantly inhibited tumor growth as shown in fig. 11a and 11 c.

The possible reason why the experiment failed to have a stronger tumor suppression effect compared to the previous study is that the injected nanomaterial dose was too small.

The isolated tumors were sectioned for detection of ROS content (as before) and cell death. As shown in fig. 11d, in the (2) - (4) groups, significant ROS production and apoptosis were observed as compared to the control group. HE staining (fig. 11 e) results showed a loose distribution of cells in group (4), an increase in nuclear debris, indicating that CM NPs induced tumor cell death, and significantly superfluous groups (2) and (3). Similarly, IHC results showed that (2) - (4) induced stronger tumor suppression compared to the control group. For example, ki67 expression is reduced and Caspase-3 expression is increased. The above results demonstrate that CM NPs have good anti-tumor effects.

In vivo biosafety is an important aspect of nanotherapeutic system investigation.

Throughout the treatment period, there was no significant change in the body weight of mice in groups (1) - (4) (FIG. 12 (a) statistical results of body weight changes of mice; and (b) statistical results of survival rate of mice), and only one mouse died spontaneously in the control group, indicating MnO ₂ 、CaCO ₃ The @ CU and CM did not cause significant toxicity. Likewise, mnO was further certified by blood routine (FIG. 13 a), blood biochemistry (FIG. 13 b) and organ HE staining (FIG. 14) ₂ 、CaCO ₃ The @ CU and CM did not cause significant toxicity.

Thus, the prepared pH-responsive CM NPs remodel TME by overloading calcium ions and manganese ions in tumor cells, inhibiting tumor progression. In addition, CM NPs have good biosafety.

2. CM NPs immune activation effect

CM NPs have previously been shown to have good anti-tumour effects and to be able to induce immunogenic cell death which is able to induce a transition of the immune system from "cold" to "hot" and thus achieve an anti-tumour effect. Thus, the immunotherapeutic anti-tumor effect was investigated in C57BL/6J mice.

Mice were randomly divided into (1) controls (Control group); (2) MnO (MnO) ₂ ；(3)CaCO ₃ The @ CU; (4) CM, 4 groups of 6 mice each: (1) Group injection PBS, (2) - (4) group injections B@MnO respectively ₂ (shown as MnO) ₂ )(15mg/kg)，B@CaCO ₃ CU (denoted CaCO in the figure) ₃ @ CU,15 mg/kg), and CM NPs (denoted CM in the figure).

Biosafety in vivo is an important aspect of nanotherapeutic system investigation.

The body weight of the mice in groups (1) - (4) did not change significantly throughout the treatment(FIG. 15). Blood convention (FIG. 16 a), blood biochemistry (FIG. 16b, group 1: control group, group 2: mnO ₂ Group 3 CaCO ₃ Group 4: cm. Group 5 is αpd1 and group 6: cm+αpd1) further certificate MnO for organ HE staining (fig. 17) ₂ 、CaCO ₃ The @ CU and CM have good biosafety.

The sustained therapeutic effect of the nanomaterial against tumors was measured by measuring tumor size, body weight and production changes every two days. The results showed MnO ₂ And CaCO (CaCO) ₃ The @ CU had some degree of tumor inhibition (FIGS. 18a, b), and CM NPs showed potent anti-tumor effects with tumor inhibition rates of-403.89%, -203.78%, -238.15% and +20.09% for groups (1) - (4), respectively. At the end of the experiment, mice were sacrificed under anesthesia and tumor tissues were isolated and weighed as shown in fig. 18a and 18c, CM NPs significantly inhibited tumor growth. The separated tumors were sectioned for ROS content and cell death. As shown in fig. 18d, in the (2) - (4) groups, significant ROS production and apoptosis were observed as compared to the control group. HE staining results showed a loose distribution of cells in group (4), an increase in nuclear debris, indicating that CM NPs induced tumor cell death, and significant redundancy in groups (2) and (3). Similarly, IHC results showed (Figure S16) that (2) - (4) induced stronger tumor suppression compared to the control group. For example, ki67 expression is reduced and Caspase-3 expression is increased.

The above results demonstrate that CM NPs have good anti-tumor effects.

In more detail, anti-tumor immune responses of CM NPs were detected. Tumor tissues were isolated and sectioned, and immunofluorescent staining labeled for different immune cells: m1 macromeans (CD 11 b) ⁺ /F4/80 ⁺ /CD80 ⁺ ),M2 macrophages(CD11b ⁺ /F4/80 ⁺ /CD206 ⁺ ),Tregs(CD3 ⁺ /CD4 ⁺ /Foxp3 ⁺ ),MDSCs(CD45 ⁺ /CD11b ⁺ /Gr-1 ⁺ ),total T cells(CD3 ⁺ ),CD4 ⁺ T cells,and cytotoxic T cells(CD3 ⁺ CD8 ⁺ ). In this study, calcium ion overload resulted in enhanced lymphocyte degranulation, cytokine production and target cell lysis, and manganese ions activated and matured the immune system. Staining of tissue sectionsAs shown in fig. 18e, CM NPs resulted in an increase in M1 in tumor tissue, and immunosuppressive immune cells, including M2 macrophages, tregs and MDSCs, were significantly reduced. Meanwhile, the detection result also shows that the content of mature T cells, effector T cells and cytotoxic T cells is obviously increased, and the content of the (4) group is far higher than that of the (2) group and the (3) group. These results demonstrate that CM NPs are able to activate anti-tumor immunotherapy. At the same time, the therapeutic responsiveness of immune checkpoint (PD 1) inhibitors was also examined. 12 mice were randomly divided into two groups, (5) αpd1 (Bioxcell, BE0089, 100 μg/mouse) and (6) cm+αpd1, αpd1 was administered by intraperitoneal injection on days 1,4 and 8 (αpd1 was injected one day after nanomaterial injection at an injection dose of 100 μg/mouse).

As shown in fig. 18a-c, αpd1 was able to inhibit tumor growth, but its tumor-inhibiting effect was poor due to lack of targeting specificity. And CM+aPD 1 can obviously inhibit tumor growth, which proves that CM NPs can promote the anti-tumor responsiveness of the aPD 1.

Likewise, ROS and TUNEL staining, as well as HE and IHC staining, both demonstrated that CM NPs were able to boost αpd1 anti-tumor responsiveness. These results demonstrate that CM NPs can promote αpd1 anti-tumor responsiveness, enabling synergistic treatment.

Claims (10)

1. A nanoparticle comprising a cell membrane and CaCO entrapped therein ₃ -CU@MnO ₂ ；

The CU is curcumin;

the cell membrane is a B16F10 cell membrane;

the MnO ₂ Is manganese dioxide nano-sheet.

2. The nanoparticle according to claim 1, wherein: the cell membrane: CU: caCO (CaCO) ₃ ：MnO ₂ The mixture ratio of (1) is 170-230 mug: 0.35-0.41 mg:2.7-3.3 mg:3 mg.

3. The nanoparticle of claim 2, wherein:

the cell membrane: CU: caCO (CaCO) ₃ ：MnO ₂ The proportion of (1) is 200 mug：0.38 mg：3 mg：3 mg。

4. A nanoparticle according to any one of claims 1 to 3, wherein: the particle size of the nano particles is less than or equal to 450nm.

5. A method of preparing the nanoparticle of claims 1-4, comprising the steps of:

1) Preparation of CaCO ₃ -CU@MnO ₂ And a cell membrane;

said preparing CaCO ₃ -CU@MnO ₂ The method of (2) is as follows: firstly, manganese dioxide nano-sheet and CaCl ₂ Mixing with CU in water, adding Na ₂ CO ₃ Mixing again, collecting precipitate to obtain CaCO ₃ -CU@MnO _2；

The CaCl ₂ The adding mass ratio of the CU to the manganese dioxide nano-sheets is 2.7-3.3:0.7-1.3:3;

the Na is ₂ CO ₃ In an amount such that the mass of the catalyst in the system is greater than the CaCl ₂ ，

The cell membrane is obtained by lysing B16F10 cells;

2) The CaCO is processed by ₃ -CU@MnO ₂ After being uniformly mixed with the cell membrane, the mixture is extruded through the membrane and collected to obtain the nano-particles in the claims 1-4;

said CaCO ₃ -CU@MnO ₂ And the proportion of the cell membrane is 4.7-5.3 mg: 200. μg;

or, the pore diameter of the membrane is 450 nm.

6. The method according to claim 5, wherein:

in step 1), the CaCl ₂ The adding mass ratio of the CU to the manganese dioxide nano-sheets is 3:1:3;

in step 2), the CaCO ₃ -CU@MnO ₂ And the proportion of cell membrane is 5 mg: 200. mu g.

7. Use of the nanoparticle according to any one of claims 1 to 4 for the preparation of an anti-tumour product.

8. Use of the nanoparticle of any one of claims 1-4 for the preparation of a remodelled tumor microenvironment product.

9. Use of the nanoparticle of any one of claims 1-4 for the preparation of a product for enhancing the effect of immunotherapy on tumors;

Or, the use of the nanoparticle of any one of claims 1 to 4 and an immunotherapeutic agent in the preparation of an immunotherapeutic tumor product or a product with an improved immunotherapeutic tumor effect.

10. A product comprising the nanoparticle of any one of claims 1-4 and an immunotherapeutic agent;

the product has at least one of the following functions:

1) An anti-tumor;

2) Improving the immune treatment effect;

3) And (5) performing immunotherapy.