CN115778983B

CN115778983B - Selenium-doped manganese phosphate nanoparticle as well as preparation method and application thereof

Info

Publication number: CN115778983B
Application number: CN202211514946.0A
Authority: CN
Inventors: 张扬; 秦环龙; 裴曼曼; 陈�峰
Original assignee: Shanghai Tenth Peoples Hospital
Current assignee: Shanghai Tenth Peoples Hospital
Priority date: 2022-11-30
Filing date: 2022-11-30
Publication date: 2024-02-09
Anticipated expiration: 2042-11-30
Also published as: CN115778983A

Abstract

The invention belongs to the field of anti-tumor drug research, and particularly discloses selenium-doped manganese phosphate nano particles, and a preparation method and application thereof. The selenium-doped manganese phosphate nano-particle is prepared by mixing an organic phosphorus source and selenite, performing enzymatic reaction at pH of 8.0-8.5, and adding manganese salt in the reaction process. The selenium-doped manganese phosphate nano-particles with good biocompatibility are prepared through biomimetic mineralization catalyzed by alkaline phosphatase, and the selenium-doped manganese phosphate nano-particles can effectively reverse multi-drug resistance of colorectal cancer cells.

Description

Selenium-doped manganese phosphate nanoparticle as well as preparation method and application thereof

Technical Field

The invention belongs to the field of anti-tumor drug research, and particularly discloses selenium-doped manganese phosphate nano particles, and a preparation method and application thereof.

Background

Colorectal cancer (CRC) is one of the most common and deadly malignant tumors of the digestive system. The number of new cases of colorectal cancer worldwide in 2020 is ranked third according to data published by the world health organization's international cancer research institute (WHO IARC). Currently, non-metastatic colorectal cancer is commonly treated with colectomy and adjuvant chemotherapy. Chemotherapy is one of the most widespread and effective treatments for colorectal cancer, and although the combined use of drugs including 5-fluorouracil, oxaliplatin and folinic acid has been used as standard chemotherapy, many patients will die from the disease. In addition to the side effects of chemotherapy, drug resistance (MDR) is also the biggest obstacle affecting chemotherapy in cancer patients and is one of the main causes of chemotherapy failure.

MDR is the reason for low response rate of solid tumors of patients treated by anti-tumor drugs to chemotherapeutic drugs for a long time, and limits the effectiveness of clinical chemotherapy. This clinical phenomenon is due to the ability of cancer cells to develop resistance to many structurally and functionally diverse drugs simultaneously in anticancer therapy. Once a multi-drug therapy (MDR) is developed on a tumor, the drug cannot exert an anticancer effect, which can lead to recurrence of the cancer and ineffective subsequent cancer chemotherapy. Oxaliplatin (oxaliplatin) -based chemotherapy plays an important role in the treatment of colorectal cancer. However, multi-drug resistance (MDR) due to long-term treatment severely impairs the therapeutic effect. Overexpression of ABC transporter is the primary mechanism by which cancer cells produce MDR, and increased drug efflux mediated by ABC transporter results in decreased intracellular drug accumulation, and thus decreased bioavailability.

Many documents report that nanoparticles can reverse multi-drug resistance of colorectal cancer cells, and the nanostructure delivery system (NDDS) has the following characteristics: 1. EPR effect, i.e. high permeability and retention effect of solid tumors; 2. biodegradable; 3. good biocompatibility; 4. the PH sensitivity/photo-thermal is utilized to promote the drug release; 5. reversing the multidrug resistance of cells. NDDS should satisfy the following conditions: first, the payload and effective endocytosis of NDDS should be good enough that premature drug/RNA leakage during circulation can lead to treatment failure, leading to off-target effects; second, NDDS should be biologically safe. Many NDDS are composed of materials that are not present in the human body, which can pose a potential hazard, particularly for materials for which biological safety is not yet clear. Therefore, developing an NDDS fabricated similar to the composition of the target tissue is an ideal approach to solve this problem.

Disclosure of Invention

In view of the above technical problems, the present invention provides the following technical solutions:

the invention provides a preparation method of selenium-doped manganese phosphate nano particles, which comprises the steps of mixing an organic phosphorus source with selenite, carrying out enzymatic reaction at pH of 8.0-8.5, and adding manganese salt in the reaction process to obtain the selenium-doped manganese phosphate nano particles.

Preferably, the enzymatic reaction is carried out at 37 ℃ under alkaline phosphatase.

Preferably, the molar ratio of the organic phosphorus source to the selenite is 18-25:1;

the molar ratio of the organic phosphorus source to the manganese salt is 1.5-2.5:1.

Preferably, the volume ratio of alkaline phosphatase solution with concentration of 20U/mL to organophosphorus source solution with concentration of 0.0165g/mL is 1:38-45.

Preferably, the organic phosphorus source is disodium fructose diphosphate; the selenite is sodium stannate; the manganese salt is manganese chloride.

The invention also provides the selenium-doped manganese phosphate nano-particles prepared by the method.

The invention also provides application of the selenium-doped manganese phosphate nano-particles in preparing antitumor drugs.

Preferably, the selenium-doped manganese phosphate nanoparticle is used for preparing a drug for reversing multi-drug resistance of colorectal cancer cells.

Preferably, the selenium-doped manganese phosphate nanoparticle is used for preparing a medicament for inducing caspase-mediated colorectal cancer cell apoptosis.

The invention also provides an anti-tumor drug, which comprises the selenium-doped manganese phosphate nano-particles and pharmaceutically acceptable auxiliary materials or carriers.

Compared with the prior art, the invention has the beneficial effects that:

1. the selenium-doped manganese phosphate nano particles (Se-MnP NPs) with good biocompatibility are prepared through biomimetic mineralization catalyzed by alkaline phosphatase, and the Se-MnP NPs show good Fenton reaction activity in chemical kinetics treatment due to the existence of manganese ions. Se-MnP NPs are used as effective drug carriers of Oxaliplatin (OX), can obviously inhibit the growth of OX-resistant HCT116 tumors in nude mice, reverse the multi-drug resistance of colorectal cancer cells, induce apoptosis of colorectal cancer cells mediated by kappa enzymes, and show higher anti-tumor performance.

2. In vitro and in vivo studies show that Se-MnP NPs can reverse the multidrug resistance of colorectal cancer by down-regulating the expression of multidrug resistance-associated ABC transporter ABCB1, ABCC1 caused by postoperative chemotherapy of colorectal cancer, and that ABC family protein expression in drug resistant cells is significantly up-regulated and Caspase3, caspase8 and Caspase9 expression is significantly increased following ox@se-MnP treatment. The result of the invention shows that the nanometer particles OX@Se-MnP can be an effective way for treating the ABC protein family mediated tumor multi-drug resistance.

Drawings

FIG. 1 is a structural characterization diagram of Se-MnP nanoparticles; a. scanning electron microscope image, b, transmission electron microscope image;

FIG. 2 is a scanning electron microscope (SEM, A) and a transmission electron microscope (TEM, B) of MnP;

FIG. 3 is Na before and after drug loading ₂ SeO ₃ FTIR spectra of MnP control sample, se-MnP microspheres;

FIG. 4 is an elemental mapping and corresponding merged image of Se-MnP nanoparticles;

FIG. 5 is Na ₂ SeO ₃ XRD patterns of MnP, se-MnP, OX@Se-MnP;

FIG. 6 is XPS spectrum of OX@Se-MnP;

FIG. 7 is XPS spectrum of Se3d in OX@Se-MnP;

FIG. 8 is XPS spectrum of Pt4f (G) in OX@Se-MnP;

FIG. 9 is the Zeta potential of MnP, se-MnP, OX, OX, OX@Se-MnP;

FIG. 10 is a thermogravimetric analysis of SeMnP and OX@SeMnP;

FIG. 11 is a hydrodynamic diameter distribution of MnP, se-MnP, OX@Se-MnP;

FIG. 12 is the relative viability of HCT116/DR cells incubated with OX (20. Mu.g/mL), se-MnP (80. Mu.g/mL), OX@Se-MnP (100. Mu.g/mL) for 48 hours;

FIG. 13 is a graph showing the relative viability of HCT116/DR cells after 48 hours of co-incubation with different concentrations of OX;

FIG. 14 is the relative cell viability of HCT116/DR cells after 48h incubation with OX and OX@Se-MnP nanoparticles;

FIG. 15 is an image processing of CAM and PI stained HCT116/DR cells;

FIG. 16 is a fluorescence microscope observation after incubation of OX@Se-MnP with HCT116/DR cells;

FIG. 17 is a flow cytometry analysis of HCT116/DR cells after 24h of differential treatment such as PBS, OX, HCT/DR cells, se-MnP, OX@Se-MnP, etc.;

FIG. 18 is a fluorescence image of HCT116/DR cells stained with DCFH-DA;

FIG. 19 is a fluorescence image of HCT116/DR cells stained with JC-1;

FIG. 20 shows the expression levels of ABCC1 and ABCG2 in HCT116/DR cells, HCT116 cells and HIEC cells;

FIG. 21 is the expression levels of ABCC1 and ABCG2 in HCT116/DR cells;

FIG. 22 shows tumor morphology and weight in mice treated differently; A. tumor morphology and size; B. tumor weight;

FIG. 23 is tumor volume and mouse body weight; A. tumor volume, B, mouse body weight;

FIG. 24 is a histological analysis of major organs of untreated mice (PBS) and OX, se-MnP, OX@Se-MnP treated mice;

FIG. 25 is a histological analysis of tumor tissue stained with hematoxylin and eosin, ki-67, caspase-3;

FIG. 26 is a histological analysis of tumor tissue stained with ABCB1, ABCC1 and ABCG 2;

FIG. 27 is a flow chart of a process for preparing Se-MnP.

Detailed Description

The invention will now be described in further detail with reference to the drawings and examples. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention. The experimental methods for which specific conditions are not specified in the examples are generally as described in conventional conditions or as recommended by the manufacturer.

Example 1

The selenium-doped manganese phosphate nano particle is prepared by the following steps:

will be 0.66g Na ₂ FDP solublenessSolution in 40mL deionized water, 0.02gNa ₂ SeO ₃ (Sigma-aldrich co., US) in 40mL deionized water, mixed, stirred for 5 minutes, at which time the pH of the solution was about 6.5; then, 1mL of an aqueous ALP (20U/mL) solution was added to the mixed solution for enzymatic reaction, the resulting reaction solution was magnetically stirred, and the temperature was controlled in a water bath at 37 ℃. At this time, 0.19g of MnCl ₂ ·4H ₂ O (A Ding Shenghua Co., ltd., china) was dissolved in 20mL of water and added dropwise to the above solution. During the enzymatic reaction, the phosphate ions are taken from Na ₂ The FDP biomolecules are released, and the pH value of the reaction solution gradually decreases. Thus, the pH of the reaction solution was maintained between 8.0 and 8.5 throughout the reaction by the continuous addition of dilute aqueous NaOH (0.5M). And washing the prepared sample twice with ethanol and twice with deionized water, centrifugally collecting, and freeze-drying to obtain the selenium-doped manganese phosphate nano particles.

Example 2

will be 0.63g Na ₂ FDP was dissolved in 40mL deionized water, 0.02g Na ₂ SeO ₃ Dissolving in 40mL deionized water, mixing, stirring for 5 minutes, wherein the pH of the solution is about 6.5; then, 1mL of an aqueous ALP (20U/mL) solution was added to the mixed solution for enzymatic reaction, the resulting reaction solution was magnetically stirred, and the temperature was controlled in a water bath at 37 ℃. At this time, 0.19g of MnCl ₂ ·4H ₂ O was dissolved in 20mL of water and added dropwise to the above solution. During the enzymatic reaction, the phosphate ions are taken from Na ₂ The FDP biomolecules are released, and the pH value of the reaction solution gradually decreases. Thus, the pH of the reaction solution was maintained between 8.0 and 8.5 throughout the reaction by the continuous addition of dilute aqueous NaOH (0.5M). And washing the prepared sample twice with ethanol and twice with deionized water, centrifugally collecting, and freeze-drying to obtain the selenium-doped manganese phosphate nano particles.

Example 3

will be 0.88g Na ₂ FDP was dissolved in 40mL deionized water, 0.02g Na ₂ SeO ₃ Dissolving in 40mL deionized water, mixing, stirring for 5 minutes, wherein the pH of the solution is about 6.5; then, 1mL of an aqueous ALP (20U/mL) solution was added to the mixed solution for enzymatic reaction, the resulting reaction solution was magnetically stirred, and the temperature was controlled in a water bath at 37 ℃. At this time, 0.19g of MnCl ₂ ·4H ₂ O was dissolved in 20mL of water and added dropwise to the above solution. During the enzymatic reaction, the phosphate ions are taken from Na ₂ The FDP biomolecules are released, and the pH value of the reaction solution gradually decreases. Thus, the pH of the reaction solution was maintained between 8.0 and 8.5 throughout the reaction by the continuous addition of dilute aqueous NaOH (0.5M). And washing the prepared sample twice with ethanol and twice with deionized water, centrifugally collecting, and freeze-drying to obtain the selenium-doped manganese phosphate nano particles.

Example 4

will be 0.66g Na ₂ FDP was dissolved in 40mL deionized water, 0.02g Na ₂ SeO ₃ Dissolving in 40mL deionized water, mixing, stirring for 5 minutes, wherein the pH of the solution is about 6.5; then, 1mL of an aqueous ALP (20U/mL) solution was added to the mixed solution for enzymatic reaction, the resulting reaction solution was magnetically stirred, and the temperature was controlled in a water bath at 37 ℃. At this time, 0.15g of MnCl ₂ ·4H ₂ O was dissolved in 20mL of water and added dropwise to the above solution. During the enzymatic reaction, the phosphate ions are taken from Na ₂ The FDP biomolecules are released, and the pH value of the reaction solution gradually decreases. Thus, the pH of the reaction solution was maintained between 8.0 and 8.5 throughout the reaction by the continuous addition of dilute aqueous NaOH (0.5M). And washing the prepared sample twice with ethanol and twice with deionized water, centrifugally collecting, and freeze-drying to obtain the selenium-doped manganese phosphate nano particles.

Example 5

will be 0.66g Na ₂ FDP was dissolved in 40mL deionized water, 0.02g Na ₂ SeO ₃ Dissolving in 40mL deionized water, mixing, stirring for 5 minutes, wherein the pH of the solution is about 6.5; then, 1mL of an aqueous ALP (20U/mL) solution was added to the mixed solution for enzymatic reaction, the resulting reaction solution was magnetically stirred, and the temperature was controlled in a water bath at 37 ℃. At this time, 0.25g of MnCl ₂ ·4H ₂ O was dissolved in 20mL of water and added dropwise to the above solution. During the enzymatic reaction, the phosphate ions are taken from Na ₂ The FDP biomolecules are released, and the pH value of the reaction solution gradually decreases. Thus, the pH of the reaction solution was maintained between 8.0 and 8.5 throughout the reaction by the continuous addition of dilute aqueous NaOH (0.5M). And washing the prepared sample twice with ethanol and twice with deionized water, centrifugally collecting, and freeze-drying to obtain the selenium-doped manganese phosphate nano particles.

Since the properties of the selenium-doped manganese phosphate nanoparticles prepared in examples 1 to 5 are substantially the same, effects will be described below using only the selenium-doped manganese phosphate nanoparticles prepared in example 1 as an example.

Experimental example 1

The selenium-doped manganese phosphate nanoparticle prepared in example 1 was used as a subject for structural characterization.

The preparation method of the MnP particles comprises the following steps: will be 0.66g Na ₂ FDP was dissolved in 80mL deionized water, 1mL ALP (20U/mL) in water was added, the solution was magnetically stirred, and the temperature was controlled at 37℃in a water bath. At this time, 0.19g of MnCl ₂ ·4H ₂ O was dissolved in 20mL of water and added dropwise to the above solution. The pH of the reaction solution was maintained between 8.0 and 8.5 throughout the reaction by the continuous addition of dilute aqueous NaOH (0.5M). And washing the prepared sample with ethanol for 2 times, washing with deionized water for 2 times, centrifugally collecting, and freeze-drying to obtain the MnP nanosheets.

The x-ray powder diffraction (XRD) pattern was recorded using a ka-ray diffractometer (RigakuD/max 2550V, cuka radiation, λ= 1.54178A). Fourier Transform Infrared (FTIR) spectra were obtained by FTIR spectroscopy (FTIR-7600, lambda science, australia). Scanning Electron Microscopy (SEM) was performed using a scanning electron microscope (hitachi S4800, japan). Transmission Electron Microscopy (TEM) and Energy Dispersive Spectroscopy (EDS) analysis were performed using a FEITecnaiG2F20 field emission transmission electron microscope. Thermogravimetric (TG) analysis was performed using a STA-409/PC simultaneous thermal analyzer (Netzsch, germany). The elemental content of MnP or Se-MnP was analyzed by inductively coupled plasma emission spectrometry (ICP-OES, JY2000-2, horiba).

By means of fructose disodium diphosphate (C) ₆ H ₁₂ O ₁₂ P2，Na ₂ FDP) as a source of organic phosphorus. The FDP (fructose 1,6 diphosphate) biomolecules are gradually hydrolyzed to F6P (fructose-6-phosphate), fructose and dissociated PO ₄ ^3- Solution of ions in an aqueous ALP solution. At the same time, free Mn ²⁺ The ions react with the hydrolyzed phosphate and react with SeO ₃ ^2- The ions form a precipitate of Se-MnP clusters. In this process, the biomolecules of FDP and their dephosphorylated products bind to Se-MnP clusters to prevent crystallization and growth of the clusters.

Scanning Electron Microscopy (SEM) (fig. 1 a) and Transmission Electron Microscopy (TEM) (fig. 1 b) of Se-MnP nanoparticles show one nanoparticle with a diameter of about 50-100 nm. Adding SeO ₃ ^2- The size of the Se-MnP nanoparticles prepared in the reaction solution is smaller than that of Mnp nanoplatelets (FIG. 2). Due to the small size, the amorphous phase is stable, and the organic/inorganic hybrid Se-MnP clusters are further formed.

FTIR spectra revealed the presence of FDP biomolecules and their derivatives. The results show that the prepared Se-MnP cluster is an inorganic-organic complex (FIG. 3).

The success of Se-MnPNPs synthesis in situ was confirmed by element mapping of Mn, P, O, se and C elements (FIG. 4).

In the XRD patterns of Se-MnP and OX@Se-MnP samples (FIG. 5), there was a broad hump at a 2 theta value of about 30 deg., and no diffraction peaks appeared.

The valence state of OX@Se-MnP was analyzed by x-ray photoelectron spectroscopy (XPS), clearly showing that manganese (Mn),Signals of selenium (Se) and platinum (Pt) (FIG. 6), se ₃ The binding energy of d at 51.2eV corresponds to that of metallic selenium (Se) pair, as shown in FIG. 7, (XPS grids show Se3d of 55.1 Evhttps:// www.thermofisher.cn/cn/zh/home/industrial/electrolytic-technical-analysis/surface-analysis/xpssibility-stability/html), and Pt4f peak at 72.6eV is attributed to PtO (FIG. 8), further confirming the presence of OX@Se-MnPNPs.

As shown in FIG. 9, the zeta potential was varied from-11.3 mV to-11.8 mV due to the negative potential (-7.48 mV) after the OX loading.

FIG. 10 shows the results of thermogravimetric analysis of SeMnP and OX@SeMnP.

Dynamic Light Scattering (DLS) measurements showed that the hydrodynamic diameter of the obtained Se-MnPNPs was about 92nm (FIG. 11), consistent with the observation of TEM images.

Experimental example 2

MDR of Se-MnP reversed HCT116/DR cell

1. Cell culture

Human colorectal adenocarcinoma cells HCT116 were purchased from american culture collection and cultured in 1640 medium containing 10% fetal bovine serum, 100 units/mL penicillin and 100mg/mL streptomycin, with medium changed every 2 days.

Oxaliplatin-resistant human colorectal cancer cells HCT116/DR, available from Meixuan Biotechnology Inc. Shanghai. By exposing drug sensitive HCT116 cells to increasing doses of OX, a gradual production occurs. The surviving cells were then preserved in conditioned medium containing 1 μg/mL OX to maintain their drug resistant phenotype.

2. Cell viability assay

HCT116 cells or OX-resistant HCT116/DR cells were treated at 8X 10 ³ Cell/well density was seeded in 96-well plates and cultured for 24h. OX, se-MnP and OX@Se-MnP nanoparticles were added to each well and incubated for 24 and 48 hours. With oxaliplatin as positive control, pure medium without any compound added was designed as a blank control. Cell viability was measured using a CCK-8 kit and optical density values were measured using an enzyme-labeled instrument (Eon, bioTek, USA) at a wavelength of 450 nm.

Values of half-inhibitory concentration (half-maximal inhibitory concentration) of OX and Se-MnP on HCT116 cells and HCT116/DR are shown in Table 1.

TABLE 1 cytotoxicity of Se-MnP and OX

RI (resistance index): ratio of half-inhibitory concentration (HCT 116/DX) to half-inhibitory concentration (HCT 116).

OX acts as a cytotoxic drug, interfering with DNA replication, inducing tumor cell apoptosis. However, OX may cause serious side effects such as neurotoxicity caused by nonspecific uptake, and it is less effective on tumor cells with MDR. In co-culturing free OX, free Se-MnP and OX@Se-MnP with colorectal cancer cells alone for 48 hours, it was apparent that OX@Se-MnP had superior cytotoxicity to HCT116/DR as compared to free OX or Se-MnP (FIG. 12). The use of free OX (20. Mu.g/ml) or Se-MnP alone (80. Mu.g/ml) reduced the cell viability of HCT116/DR cells to about 63% and 42%, respectively, while OX@Se-MnP reduced the cell viability to about 12%.

As shown in FIG. 13, OX@Se-MnP had a higher content, and after 48 hours of co-culture with HCT116/DR cells, the concentration resulted in higher cytotoxicity. The use of OX (20 μg/mL) has an antitumor effect (cell viability 9%) compared to the same concentration of Se-MnP (cell viability 72%) or free OX (cell viability 68%).

As shown in FIG. 14, at the same concentration, OX@Se-MnP resulted in higher cell damage than free OX alone.

FIG. 15 shows that PBS (Control) and OX are less cytotoxic, se-Mnp and OX@Se-Mnp are more capable of promoting apoptosis of colorectal cancer resistant HCT116/DR cells, consistent with the detection results of CCK-8.

The results of Confocal Laser Scanning Microscopy (CLSM) showed that intracellular absorption of FITC-ox@se-MnP from FITC-labeled green fluorescence allowed tracking of NPs cells (fig. 16), confirming that ox@se-MnP could be treated by HCT116/DR cells, especially at 8-10h and accumulated in tumor tissue.

In addition, apoptosis was verified by flow cytometry using an Annexin-V-FITC and PI staining assay (fig. 17, this flow q2+q4 was not greater than 50%).

3. ROS production

ROS production was determined by intracellular conversion of dichlorofluorescein diacetate (DCFH/DA) to the fluorescent product DCF. HCT116/DR cells were plated in 6-well plates at a density of 1X 10 ⁶ After 24h incubation, cells/well were incubated with OX (20. Mu.g/mL), se-MnP (80. Mu.g/mL), OX@Se-MnP (100. Mu.g/mL), respectively, for 12h. Then, DCFH/DA (1. Mu.M) (beyotidme, china) was added to each well, and the wells were incubated at 37℃for 30min, and the cells were isolated. Cells were collected by centrifugation, washed 2 times with PBS and resuspended in PBS. ROS production in each group of cells was observed by confocal.

The results show little or no significant DCFH detection by oxaliplatin. However, DCFH was significantly increased in HCT116/DR cells cultured with Se-MnP and OX@Se-MnP (FIG. 18). These results are consistent with the results of CCK8 detection and flow cytometry, indicating that ox@se-MnP induced ROS overproduction has a significant inhibitory effect on cancer cells.

Furthermore, mitochondrial membrane potential (Δψm) was further studied using JC-1 fluorescent probe, which can change from red to green fluorescence during Δψm deletion. From this, ox@se-MnP significantly induced loss of Δψm, as shown in fig. 19, demonstrating that ox@se-MnP may induce mitochondrial dysfunction, thereby activating excessive ROS production, leading to apoptosis.

The ABC transporter plays an important role in MDR of cancer cells, the invention further evaluates the inhibition of the ABC transporter by Se-MnP and analyzes the expression level of ABC family proteins in HCT116/DR cells, HCT116 cells and HIEC cells.

As shown in FIG. 20, the expression levels of ABCC1 and ABCG2 in HCT116/DR cells were higher than those of HCT116 cells and HIEC cells. Notably, the expression levels of ABC family proteins (ABCC 1 and ABCG 2) in HCT116/DR cells were significantly lower than OX at the time of collection of Se-MNP and ox@se-MNP samples, fig. 21.

Experimental example 3

Evaluation of in vivo anti-osteosarcoma Activity

1. Construction of animal models

A colorectal cancer model is built by adopting BALB/c nude mice (4-6 weeks), and the in vivo anti-tumor activity of Se-MnP and OX@Se-MnP is studied. Animal proposals were approved by the institutional animal care and use committee of the tenth, shanghai, people's hospitals. Injection of 5X 10 on left shoulder vaccinates osteosarcoma model ⁶ HCT116/DR cell/site. Tumor volume of approximately 100mm 14 days after cell implantation ³ Mice were randomly divided into 4 groups (5 per group) including OX (4 mg/kg), se-MnPs (16 mg/kg), OX@Se-MnPs (20 mg/kg) and saline placebo. Each mouse was injected with 100 μl of nanoparticles and PBS.

2. Method of

Mice were treated repeatedly every 2 days, weighed every 2 days, and tumor volumes were measured with vernier calipers: volume (mm) ³ ) = (length x width ² )/2. On day 14, all mice were euthanized and necropsied. Tumors were isolated, weighed, and fixed with 10% neutral formalin. Tumor tissues were paraffin-embedded, sectioned 5 μm, and mounted on slides for histological examination. The growth state of the tumor and lesions of the organ tissue were observed by HE staining. The Ki-67 method is adopted to detect the apoptosis of osteosarcoma cells in tumor tissues. Caspase-3 is analyzed by immunohistochemistry. The expression of MDR proteins ABCB1, ABCC1 and ABCG2 is detected by adopting an immunohistochemical analysis method.

3. Statistical analysis

Single factor anova was performed using SPSS 21.0 and Tukey multiple comparison tests to determine significant differences between three or more groups. Significance levels were set to p <0.05, p <0.01, p <0.001.

4. Results

As shown in FIGS. 22 and 23A, at the end of treatment (day 14), OX@Se-MnP greatly reduced tumor volume from 0.66.+ -. 0.14 to 0.12.+ -. 0.22cm ³ . Tumor weights ranged from 0.77.+ -. 0.27 to 0.15.+ -. 0.08g compared to control. After 6d of treatment, OX@Se-MnP has obvious inhibition effect on tumor growth and along with treatment timeIncreasing, its inhibition was progressively more pronounced (fig. 23A). Compared with OX or Se-MnP, the OX@Se-MnP has obvious inhibition effect on tumor growth.

All mice survived to the experimental endpoint, and the body weight of mice treated with Se-MnP or OX@Se-MnP was not different from that of the control group (FIG. 23B).

Hematoxylin and eosin (H & E) staining was performed after heart, liver, spleen, lung and kidney sections to assess nanoparticle toxicity and treatment performed. The results of the H & E staining experiments showed that the tissue morphology of the treated group was normal, indicating no obvious organ damage (fig. 24).

Tumor samples were pathologically analyzed using hematoxylin-eosin (HE) staining and Ki67, caspase-3, ABCB1, ABCC1 and ABCG 2. As shown in FIGS. 25-26, control HCT116/DR tumor cells grew well, the nuclei were large and oval, and the intratumoral injection of OX was not very inhibitory. In contrast, tumors treated with Se-MnP and OX@Se-MnP exhibit typical apoptotic tumor tissue, with nuclear chromatin concentration. We then analyzed the levels of Ki67 and caspase-3 expression in tumor samples. Ki67 is a well-known marker of cell proliferation, while caspase-3 is generally thought to be a central regulator of apoptosis. Compared with the control group, the expression of Ki67 of Se-MnP group and OX@Se-MnP group is obviously reduced, and the expression of caspase-3 is obviously increased. These results indicate that Se-MnP and OX@Se-MnP inhibit proliferation of tumor cells by decreasing Ki67 expression, and promote apoptosis of tumor cells by activating caspase-3 signaling pathway.

The preparation process flow of Se-MnP in the invention is shown in FIG. 27.

The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that various changes and modifications can be made herein without departing from the principles of the invention, which would otherwise fall within the scope of the invention as defined in the appended claims.

Claims

1. The preparation method of the selenium-doped manganese phosphate nano-particle is characterized in that an organic phosphorus source and selenite are mixed, enzymatic reaction is carried out at pH of 8.0-8.5, and manganese salt is added in the reaction process, so that the selenium-doped manganese phosphate nano-particle is obtained.

2. The method according to claim 1, wherein the enzymatic reaction is carried out at 37 ℃ under alkaline phosphatase.

3. The method according to claim 2, wherein,

the molar ratio of the organic phosphorus source to the selenite is 18-25:1;

4. The method according to claim 3, wherein the volume ratio of the alkaline phosphatase solution having a concentration of 20U/mL to the organic phosphorus source solution having a concentration of 0.0165g/mL is 1:38-45.

5. The method of claim 1, wherein the source of organophosphorus is disodium fructose diphosphate; the selenite is sodium selenite; the manganese salt is manganese chloride.

6. Selenium-doped manganese phosphate nanoparticle prepared by the method of any one of claims 1-5.

7. Use of the selenium-doped manganese phosphate nanoparticle of claim 6 in the preparation of an antitumor drug.

8. The use according to claim 7, wherein the selenium doped manganese phosphate nanoparticle is used for the preparation of a multi-drug resistant drug for reversing colorectal cancer cells.

9. The use according to claim 7, wherein the selenium doped manganese phosphate nanoparticle is used for the preparation of a medicament for inducing caspase-mediated apoptosis of colorectal cancer cells.

10. An antitumor drug comprising the selenium-doped manganese phosphate nanoparticle of claim 6 and a pharmaceutically acceptable adjuvant or carrier.