CN110755407A

CN110755407A - Manganese dioxide/glucose oxidase @ hyaluronic acid composite anti-cancer material and preparation and application thereof

Info

Publication number: CN110755407A
Application number: CN201911221953.XA
Authority: CN
Inventors: 卿志和; 杨荣华; 柏爱玲
Original assignee: Changsha University of Science and Technology
Current assignee: Changsha University of Science and Technology
Priority date: 2019-12-03
Filing date: 2019-12-03
Publication date: 2020-02-07
Anticipated expiration: 2039-12-03
Also published as: CN110755407B

Abstract

The invention belongs to the technical field of anti-cancer drugs, and particularly discloses a manganese dioxide/glucose oxidase @ hyaluronic acid composite anti-cancer material which comprises a core and a shell coating the core; the core comprises manganese dioxide nanosheets and glucose oxidase modified on the surfaces of the manganese dioxide nanosheets; the shell is hyaluronic acid. The invention also provides the preparation and application of the anti-cancer material. Researches show that the composite material can effectively improve the selectivity of cancer cells and normal cells in a synergistic manner through innovative components and the synergistic effect of the composite morphology of the components, can efficiently and specifically kill the cancer cells, and can induce a large amount of apoptosis of the cancer cells under low dosage; moreover, the material of the invention basically does not influence the metabolism of normal cells and has little toxic and side effect.

Description

Manganese dioxide/glucose oxidase @ hyaluronic acid composite anti-cancer material and preparation and application thereof

Technical Field

The invention belongs to the field of nanotechnology and cancer treatment, and relates to a nano composite medicine based on manganese dioxide nanosheets, glucose oxidase and hyaluronic acid.

Background

As is known from world health organization statistics in 2015, cancer dies in 880 ten thousand worldwide and has become the second-leading death disease worldwide. To date, cancer remains a major public health challenge. Conventional molecular therapies and chemotherapies based on tight ligand receptor interaction or nucleic acid modification have the limitations of poor stability, poor specificity, and severe toxic and side effects, and have been largely unable to meet the challenges due to the complexity of physical barriers of cancer cells, tumor heterogeneity, drug resistance, and metastasis. Since the beginning of the 50 s of the 20 th century, researchers in the relevant field have continuously developed many highly effective cancer treatment methods such as chemokinetic therapy, photodynamic therapy, photothermal therapy, gas therapy, starvation therapy, etc., which are applied to various animal models and even clinical trials and achieve significantly improved tumor drug delivery capacity and treatment efficacy.

One of the promising cancer treatment strategies based on the action of reactive oxygen species, chemokinetic Therapy (CDT), has attracted considerable attention and research in recent years. Chemokinetic treatment is the conversion of H by a metal ion mediated Fenton or Fenton-like reaction₂O₂The OH group is converted to OH, which induces intracellular oxidative stress, thereby effectively killing cancer cells. Compared to other reactive oxygen species (superoxide anion, singlet oxygen), OH is the one with the highest ability to kill cancer cells, and is based on the damage of intracellular biomolecules such as proteins, DNA, lipids, etc. to kill cancer cells. The common CDT is mainly based on Fe²⁺Catalyzing the reaction of H₂O₂Converted to OH, in addition to some metal ions (e.g. Cu)²⁺) Has Fenton-like activity. However, the currently reported chemokinetic treatment techniques have the defects of insufficient lethality to cancer cells, large side effects and the like.

Disclosure of Invention

In order to solve the technical defects of low anticancer activity and insufficient selectivity of the existing anticancer material, the invention provides a manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material, and aims to provide a brand new material with good solution stability, excellent selectivity and anticancer activity.

The second purpose of the invention is to provide a preparation method of the manganese dioxide/glucose oxidase @ hyaluronic acid composite anti-cancer material.

The third purpose of the invention is to provide the application in pharmacy.

The fourth purpose of the invention is to provide an anticancer drug containing the manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material.

Manganese dioxide/glucose oxidase @ hyaluronic acid composite anti-cancer material (also called MnO in the invention)₂a/GOx @ HA composite, or simply composite or TSEN), comprising a core and a shell coating the core; the core comprises manganese dioxide nanosheets and glucose oxidase modified on the surfaces of the manganese dioxide nanosheets; the shell is hyaluronic acid.

The invention provides a brand-new material with a core-shell structure, which innovatively takes manganese dioxide nanosheets modified with glucose oxidase on the surfaces as cores, and innovatively embeds the manganese dioxide nanosheets in hyaluronic acid to assemble the composite material with the core-shell structure. Researches show that the composite material can effectively improve the selectivity of cancer cells and normal cells in a synergistic manner through innovative components and the synergistic effect of the composite morphology of the components, can efficiently and specifically kill the cancer cells, and can induce a large amount of apoptosis of the cancer cells under low dosage; moreover, the material of the invention basically does not influence the metabolism of normal cells and has little toxic and side effect.

Preferably, the manganese dioxide nanosheet is in a single-layer sheet shape, and the particle size is preferably not more than 150nm, and more preferably 90-100 nm.

The research of the invention finds that the reasonable control of the proportion of the components of the composite material is beneficial to further improving the solution stability and the selective capture effect of cancer cells of the composite material, and can further improve the selectivity and the lethality of the composite material on the cancer cells.

Preferably, in the manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material, the mass ratio of the glucose oxidase, the manganese dioxide nanosheet and the hyaluronic acid is 1-2: 5-10: 60-120; preferably 1: 5: 60. It has been found that control within the preferred range helps control the particle size of the material and helps improve solution stability.

The hydration particle size of the manganese dioxide/glucose oxidase @ hyaluronic acid composite anti-cancer material is not higher than 500 nm; preferably 290 to 300 nm.

The invention also provides a preparation method of the manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material, which comprises the steps of dissolving and mixing manganese dioxide nanosheets and glucose oxidase in water, adding the mixture into a hyaluronic acid aqueous solution, stirring and assembling to obtain a suspension, then carrying out centrifugal treatment, washing and purifying, and carrying out freeze drying to obtain the manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material.

Preferably, the synthesis steps of the manganese dioxide nanosheet are as follows: obtaining a catalyst comprising H₂O₂And tetramethylammonium hydroxide, adding the mixed solution to Mn²⁺Stirring and reacting the manganese solution, separating to obtain a manganese dioxide body, dispersing the manganese dioxide body in water, and ultrasonically stripping to obtain manganese dioxide nanosheets.

Preferably, the concentration of glucose oxidase is 0.001-0.1 mg/mL^-1(ii) a Preferably 0.02 to 0.03 mg/mL^-1。

Preferably, the concentration of hyaluronic acid is 1-2 mg/mL^-1(ii) a Preferably 1.5 mg/mL^-1。

Preferably, the mass ratio of the glucose oxidase to the manganese dioxide nanosheet to the hyaluronic acid is 1-2: 5-10: 60-120; preferably 1: 5: 60.

The time for stirring assembly is preferably not less than 6 h.

The invention also discloses an application of the manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material in preparation of anticancer drugs.

Preferably, the composite material is used for preparing an anti-cancer drug for killing cancer cells with high expression of CD 44. The high expression refers to abnormal increase of hyaluronic acid receptor CD44 in cancer cells.

Further preferably, the application is to prepare the anti-cancer drug for killing Hela cells by using the composite material.

More preferably, the application is matched with pharmaceutically acceptable auxiliary materials to prepare pharmaceutically acceptable anticancer drugs of any administration route, preferably anticancer drugs for injection administration.

The invention also provides an anti-cancer drug which comprises a pharmaceutically effective amount of the manganese dioxide/glucose oxidase @ hyaluronic acid composite anti-cancer material.

Preferably, the anticancer drug further comprises pharmaceutically acceptable auxiliary materials.

Preferably, the anticancer drug is a drug in any pharmaceutically acceptable dosage form; preferably an injectable drug.

The medicine of the present invention can selectively permeate CD44 high expression cancer cell structure and in the cancer cell, HCO₃ ^-And inducing the composite material to generate a large amount of OH to induce cancer cell apoptosis. The composite material has the characteristics of low dosage, high targeting and high-efficiency treatment, can realize high-efficiency and accurate cancer treatment in vivo and in vitro, and has important basic research value and application prospect.

Compared with the prior art, the invention has the advantages that:

1. the invention successfully constructs a brand-new anticancer material, and finds that the material has the advantages of good selectivity, low toxic and side effects, low dosage of TSEN nano-drugs capable of inducing cancer cells to remarkably die and the like; in addition, the material of the invention has toxic and side effects on normal organs and tissues; realizes the high-efficiency and accurate cancer treatment in vivo and in vitro. Therefore, the method has good basic research value and application prospect.

2. The research of the invention finds that the control of the proportion of the components of the material is helpful for improving the stability of the solution and improving the selective killing effect of cancer cells.

Drawings

FIG. 1 is a synthetic characterization of TSEN nano-drugs in example 1; (A) exploring the optimal synthesis proportion of the TSEN nano system; under the condition of the optimal synthesis ratio, the (B1) hydrated particle size, (B2) electric potential and (B3) transmission electron microscope image of the TSEN nano system.

FIG. 2 is a solution stability study of the TSEN nano-drug of example 2; (Control: manganese dioxide nanosheet and glucose oxidase mixed system, no hyaluronic acid package)

FIG. 3 is a performance study of the synergistic OH production of TSEN nano-drugs in example 3; (A) enzyme-activated degradation experiments of TSEN nano systems; (B) and (5) carrying out performance study on the TSEN nano system. (Control probe: manganese dioxide nanosheet coated with hyaluronic acid, no glucose oxidase; MB as an indicator for OH generation, which can be degraded by OH and has an obvious response change in absorbance; the maximum absorption wavelength of MB is 665nm)

FIG. 4 is the MTT characterization in example 4 for the effectiveness of TSEN nanomedicines in killing cancer cells; (Control probe: consisting of manganese dioxide nanosheet coated with hyaluronic acid, no glucose oxidase)

FIG. 5 is the MTT characterization of the selectivity of TSEN nano-drugs for cancer cell killing in example 5;

FIG. 6 is a graph of fluorescence imaging characterization of TSEN nano-drugs inducing oxidative stress in cancer cells in example 6; (Control: blank, no drug treatment. DCF is. OH detection probe DCFH-DA fluorescent product)

FIG. 7 is a graph of fluorescence imaging characterization of TSEN nanomedicines selective for cancer cell treatment in example 7; (Control: blank. Calcein AM: live cell dye in green fluorescence. PI: dead cell dye in red fluorescence)

FIG. 8 is a flow cytometry demonstration of TSEN nanomedicines to selectively induce apoptosis in cancer cells in example 8; (Control: blank, wherein the regions Q1, Q2, Q3 represent necrotic, late apoptotic and early apoptotic cells, respectively, the region Q4 represents a viable cell)

FIG. 9 is the monitoring of the TSEN nano-drug for in vivo tumor treatment in example 9. (A) Taking a picture of a tumor-bearing nude mouse in real time; (B) weight change of nude mice over time; (C) tumor volume changes over time.

(Control probe: consisting of manganese dioxide nanosheet coated with hyaluronic acid, no glucose oxidase)

Detailed Description

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings: the embodiment is implemented on the premise of the technical scheme of the invention, and a detailed implementation manner and a process are given, so that the technical scheme features of the invention are easy to understand, and the protection scope of the invention is not limited at all. All the technical solutions formed by equivalent transformation or equivalent replacement fall within the protection scope of the present invention.

The glucose oxidase and hyaluronic acid were purchased from J & K carbofuran reagent.

The hydrogen peroxide, tetramethylammonium hydroxide, manganese chloride and methanol were purchased from national pharmaceutical group chemical agents, ltd.

The synthesis steps of the manganese dioxide nanosheet are as follows: first, 20mL of 3 wt% H was prepared₂O₂And 0.6M aqueous tetramethylammonium hydroxide. The solution was then added rapidly to 10mL of 0.3M manganese chloride solution over 15 s. The solution immediately turned dark brown and was stirred vigorously at room temperature overnight (24 h). The prepared manganese dioxide body is processed at 2000 r.min^-1Centrifuging for 20min, and washing for 2-3 times by using water and methanol. The dried product was placed in a vacuum oven at 60 ℃ and stored in centrifuge tubes for further experiments. To prepare manganese dioxide nanosheets, 10mg of manganese dioxide body was dispersed in 20mL of water and sonicated for about 10 hours. The resulting suspension was then concentrated at 2000 r.min^-1Centrifuge for 30min and retain supernatant for further use. The obtained manganese dioxide nanosheet is characterized by a transmission electron microscope to find that: it is in the form of ultra-thin single-layer sheet with particle size of about 100 nm. The solution concentration of the supernatant was 0.5 mg/mL^-1。

Synthetic characterization of TSEN Nanoparticulates in example 1

(1) Taking four groups of 500 mu L manganese dioxide nano-sheet original solution (the concentration is 0.5 mg/mL)^-1(ii) a Prepared by the above preparation example; the following experimental examples, all of which are solutions of this concentration unless otherwise stated), were separately mixed with 10. mu.L of glucose oxidase solution (5 mg. multidot.mL)^-1The following experimental cases, all solutions of this concentration unless otherwise stated);

(2) the mixture was added to 1.5, 2, 2.5, and 3mL of hyaluronic acid solution (2 mg. multidot.mL)^-1) Respectively labeled as group 1, group 2, group 3, group 4;

(3) magnetically stirring at room temperature overnight (about 12 h) to obtain suspension respectively;

(4) the suspension is stirred at 6000 r.min^-1Centrifuging for 20min at the rotating speed, and washing for 2-3 times by using ultrapure water;

(5) the hydrated particle size of the four groups of solutions treated by the above procedure was determined by a Nano ZS90 particle sizer.

And (4) analyzing results: from FIG. 1A, it is found that: the hydrated particle size of the composite nano-materials obtained from the three groups of group 2, group 3 and group 4 is above 600nm, while the hydrated particle size of the composite nano-material of group 1 is below 300 nm. Nanoparticles in the 300nm range have high permeability and high aggregation in the cancer cell region by the EPR effect. Therefore, the group 1 is selected as the optimal synthesis proportion, and the mass ratio of the glucose oxidase, the manganese dioxide nanosheet and the hyaluronic acid is 1: 5: 60.

(6) Selecting the components according to the optimal proportion (the mass ratio of the glucose oxidase to the manganese dioxide nanosheet to the hyaluronic acid is 1: 5: 60) according to the method to synthesize a TSEN nanometer system (group 1TSEN), centrifugally washing, freeze-drying, and dispersing in 2mL of ultrapure water for later use (the concentration is 2 mg. mL)^-1(ii) a In the following cases, the TSEN nanosystems are solutions of this concentration, unless otherwise stated);

(7) and (4) measuring the hydrated particle size and the potential of the TSEN Nano system obtained in the step (6) by a Nano ZS90 particle size analyzer. In addition, the morphology was observed by transmission electron microscopy, TEM.

And (4) analyzing results: under the condition of the optimal synthesis ratio (the mass ratio of the glucose oxidase, the manganese dioxide nanosheet and the hyaluronic acid is 1: 5: 60; in the following cases, except for special statement, the TSEN systems are all medicaments with the mass ratio), the synthetic characterization data (figure 1B) of the assembled TSEN shows that the hydrated particle size of the TSEN is 294.8nm, the potential is-59.4, and the particle sizes are uniform. The particle size of the synthesized TSEN favors its high penetration and aggregation in the cancer cell area by the EPR effect.

Solution stability study of TSEN Nanoparticulates in example 2

(1) Adding a mixed system of manganese dioxide nanosheets and glucose oxidase (in the same proportion as the step (1) in the example 1) and a TSEN nano system (prepared in the step (6) in the example 1) into four solution systems of deionized water (DI water), physiological Saline (Saline), Phosphate Buffer Solution (PBS) and 10% fetal bovine serum-containing cell culture Medium (10% FBS Medium) respectively;

(2) wherein, a mixed system of the manganese dioxide nanosheet and the glucose oxidase is set as a Control group;

(3) standing at room temperature for 30 days;

(4) changes in TSEN in each solution were observed and recorded.

And (4) analyzing results: it can be seen from the solution stability experiment of TSEN in fig. 2 that the manganese dioxide and glucose oxidase mixed system is deposited on the 1 st day of standing in each solution, and TSEN can be stably dispersed in deionized water, normal saline, phosphate buffer, cell culture medium containing 10% fetal calf serum, and the like after standing for 30 days. The TSEN has good solution stability.

Example 3 Performance study of synergistic production of OH by TSEN Nanoparticulates

25mM NaHCO was prepared₃/5％CO₂The buffer solution is ready for use.

(1) Experimental group a: TSEN nano system (prepared in step (6) of example 1, 2 mg. multidot.mL)^-1) Adding the mixture to the reactor with or without 120-150 U.mL^-125mM NaHCO of Hyaluronidase₃/5％CO₂Buffer solution (containing 10mM GSH, 10mM glucose);

(2) experimental group B: a Control probe system (consisting of manganese dioxide nano-sheets and hyaluronic acid) and a TSEN nano-system (2 mg. mL)^-1) Added to 25mM NaHCO respectively₃/5％CO₂Buffer solution (containing 120-150 U.mL)^-1Hyaluronidase, 10mM GSH, 10mM glucose);

(3) respectively placing the A, B two groups of experimental solution systems at 37 ℃ and shaking;

(4) centrifuging after 4h, and respectively taking supernate;

(5) then, the mixture was mixed with 10. mu.g/mL^-1Methylene Blue (MB) and 50. mu. M H₂O₂Adding the supernatant into the supernatants obtained in the two experiments;

(6) incubating for 30-60 min at 37 ℃;

(7) measuring the ultraviolet absorption change of MB at 665nm of the two groups of experimental solution systems;

(8) both experiments were performed at 10. mu.g/mL^-1MB solution was used as a blank comparison.

The synthetic process of the Control probe system comprises the following steps: adding the manganese dioxide nanosheet solution (same as in example 1) into the hyaluronic acid aqueous solution (same as in example 1), and magnetically stirring at room temperature overnight to obtain a suspension. The suspension is at 6000 r.min^-1Centrifuging for 10min at a rotating speed, washing with ultrapure water for 2-3 times, and after freeze drying, dispersing the Control probe system in ultrapure water with a certain volume for later use.

And (4) analyzing results: based on the properties that the MB dye can be degraded by OH and can be used as an indicator of OH generation and the like, as can be seen from the ultraviolet absorption change of MB in figure 3A, under the condition of the presence of hyaluronidase, TSEN can generate a certain amount of OH to degrade MB, so that the absorbance of MB is greatly reduced, which proves that TSEN is degraded in an activating manner by hyaluronidase; in addition, as can be seen from fig. 3B, the absorbance of MB was reduced after both the Control probe and TSEN solution systems were treated, and the ability of TSEN to generate OH was significantly stronger than the Control probe as can be seen from the degree of change in uv absorption of MB. Indicating that TSEN has the capability of producing OH in a synergistic manner.

MTT characterization in example 4 high efficiency of TSEN nano-drug in killing cancer cells

(1) Firstly, laying Hale cells in a 96-well plate, adding 200 mu L culture medium for incubation until the Hale cells grow to about 70-80% for later use;

(2) removing culture medium in a 96-well plate, respectively adding serum-free culture medium containing Control probe nano systems (composed of manganese dioxide nano-sheets and hyaluronic acid) or TSEN nano systems with different concentrations into the 96-well plate, wherein the concentration of each group is five times in parallel, and incubating for 24h at 37 ℃;

(3) then, 10. mu.L of a LMTT agent (5 mg. multidot.mL) was added to each well of the sample solution^-1) And incubating for 4 h;

(4) finally removing the solution in each hole, and adding 100 mu LDMSO;

(5) setting relevant parameter emission wavelength 490 nm; shaking for 10min, and performing MTT detection on the treated solution by using a microplate reader.

And (4) analyzing results: as can be seen from the MTT experimental data in FIG. 4, under the same experimental conditions, the killing ability of TSEN to Hela cells is obviously stronger than that of Control probe nano-system, and the dosage is low (30 mug. mL)^-1) TSEN in (b) can induce significant apoptosis of Hela cells. The method is based on the fact that TSEN enters Hela cells and is activated by endogenous high-expression hyaluronidase, and released glucose oxidase can consume glucose in the cells to generate enough H in situ₂O₂Promote the generation of a large amount of OH, further induce the Hela cell to be greatly apoptotic, which makes up for H in the killing process of the Control probe nano system on the Hela cell₂O₂And insufficient content. Therefore, TSEN has synergistic killing capacity on cancer cells as a nano-drug.

MTT characterization in example 5 TSEN Nanoparticulate selectivity for cancer cell killing

(1) Firstly, respectively paving Hale cells (cancer cells with highly expressed CD44 receptors) and HEK-293 cells (normal cells without highly expressed CD44 receptors) in a 96-well plate, respectively adding 200 mu L of culture medium for incubation until the growth rate reaches about 70-80% for later use;

(2) removing the culture medium in the 96-well plate, adding the serum-free culture medium of the TSEN nano system into the 96-well plate respectively, and incubating for 24 hours at 37 ℃ for five times in parallel;

(3) then, 10. mu.L of MTT reagent (5 mg. multidot.mL) was added to each well of the sample solution^-1) And incubating for 4 h;

(4) finally, removing the solution in each well, and adding 100 mu L DMSO;

And (4) analyzing results: from the MTT experimental data of fig. 5, it can be found that under the same experimental conditions, compared with the killing of TSEN nano-drug on Hela cells, the killing of HEK-293 cells, which are normal cells, can be ignored. This is because TSEN can be mediated by CD44 receptor overexpressed in Hela cells, selectively and efficiently enter the HeLa cells, and is activated by hyaluronidase expressed endogenously, so that GSH and glucose in the cells are consumed and Mn is produced²⁺And a large amount of H₂O₂Further in HCO₃ ^-In the presence of the protein, a large amount of OH is generated to induce Hela cell apoptosis. The TSEN nano-drug can specifically target cancer cells and can realize efficient and accurate killing of the cancer cells.

Fluorescence imaging characterization of TSEN Nanoparticulates induces oxidative stress in cancer cells in example 6

(1) Firstly, respectively paving Hale cells and HEK-293 cells in a confocal dish, adding 1mL of culture medium respectively, and incubating until the Hale cells and the HEK-293 cells grow to about 70-80% for later use;

(2) removing the culture medium from the dish, washing with PBS 2-3 times, and adding 30 μ g/mL^-1Adding serum-free culture medium of TSEN nano system, setting the cells without TSEN nano system as control group, and incubating at 37 deg.C for 4 h;

(3) then removing the dressing medium, washing with PBS 2-3 times, and adding 10 μ g/mL^-1Adding a serum-free culture medium of the DCFH-DA active oxygen probe into each confocal dish, and incubating for 15-30 min at 37 ℃;

(4) performing fluorescence imaging research on the sample by using a laser confocal imaging system; before imaging, washing the sample with PBS buffer solution for 2-3 times, and adding 1mLPBS buffer solution into a confocal dish for imaging.

(5) The principle of oxidative stress detection: DCFH-DA as an oxidative stress detection probe, after entering cells, through intracellular esterase deacetylation to obtain non-fluorescent substance DCFH, whereas DCFH can be OH oxidized into fluorescent substance DCF.

And (4) analyzing results: as can be seen from the fluorescence intensity change generated by the oxidative stress detection of the confocal laser imaging system in FIG. 6, the TSEN with low dose can generate a large amount of OH in Hela cells, while the OH generated in HEK-293 of normal cells can be ignored, because the TSEN can specifically and targetedly accumulate into Hela cells, is activated and released by endogenous high-expression hyaluronidase, and provides H in situ in glucose consumption cells₂O₂On the basis, a large amount of. OH is produced. The TSEN nano-drug has good cancer cell specific targeting capability, and further has obvious oxidative stress capability in cancer cells.

Fluorescence imaging characterization of TSEN Nanoparticulates for Selective treatment of cancer cells in example 7

(3) removing the dressing culture medium, washing with PBS for 2-3 times, adding serum-free culture medium containing CalceiNAM living cell dye/PI dead cell dye into each confocal dish, and incubating at 37 deg.C for 15-30 min;

And (4) analyzing results: from the live/dead cell staining fluorescence imaging measurements by confocal laser imaging system of fig. 7, it can be seen that low doses of TSEN have significant killing ability on Hela cells, while killing on HEK-293 normal cells is minimal. The experimental results prove that the TSEN medicament has good specific targeting capability and efficient killing capability on cancer cells.

Example 8 flow cytometry validation of TSEN Nanomedicines selectively induce apoptosis in cancer cells

(1) Firstly, laying Hale cells and HEK-293 cells in a 6-hole plate, adding 2mL of culture medium respectively, and incubating until the Hale cells and the HEK-293 cells grow to about 70-80% for later use;

(2) removing the culture medium from the dish, washing with PBS 2-3 times, and adding 30 μ g/mL^-1Serum-free culture medium of TSEN nano system is added into the culture medium, cells without TSEN nano system are set as a control group, and the cells are incubated for 4h at 37 ℃.

(3) Removing the dressing culture medium, and washing with PBS for 2-3 times respectively;

(4) then digested with pancreatin, collected in 0.5mL each EP tube at 1500 r.min^-1Centrifuging for 10min, and washing for 2-3 times by PBS;

(5) adding Annexin V-FITC/PI double-staining apoptosis detection kit to each EP intraductal cell, and incubating for 15 min;

(6) the apoptosis analysis was performed on each of the cells collected above using a flow cytometer.

And (4) analyzing results: as can be seen from the difference in the data of the flow cytometry analysis of FIG. 8 for each of the corresponding apoptosis, TSEN induced significant apoptosis in Hela cells, whereas under the same conditions, TSEN did not substantially kill normal cells HEK-293. The result is consistent with the imaging experiment result, and further embodies that the TSEN nano-drug can efficiently induce cancer cell apoptosis and has low toxic and side effects on normal cells.

Example 9 monitoring of tumor treatment in vivo with TSEN Nanoparticulates

(1) BALB/C female nude mice were randomly divided into three groups (3 mice per group) with tumor size of about 50mm³；

(2) Then, different medicines (PBS, Control probe nano system and TSEN nano medicine) are injected into the vein every other day for 16 days, wherein the equivalent dose of the manganese dioxide nano-sheet is 1.2 mg-kg^-1The equivalent dose of glucose oxidase is 0.24 mg/kg^-1；

(3) The Control probe nano system consists of hyaluronic acid and manganese dioxide nano sheets.

(4) Tumor length (L), tumor width (W) and body weight were measured and recorded before each injection of drug. Tumor volume (V) calculation formula: v ═ L × W²)/2. Relative tumor volume is V/V₀And V is the tumor volume per treatment, wherein V₀Is the initial volume before treatment.

And (4) analyzing results: as can be seen from the monitoring experiments of the TSEN nano-drug of FIGS. 9A and 9B on the change of the tumor in the body and the relative tumor volume of the mouse, the tumor in the body of the mouse treated by the Control probe nano-system does not have obvious inhibition effect; the result shows that the TSEN nano-drug remarkably enhances the corresponding tumor treatment effect through the synergistic chemokinetic treatment effect. From the trend of the relative mouse body weight change in fig. 9C, the body weight change of the mice in each experimental group was not significant, indicating negligible systemic toxicity of TSEN nano-drug.

Claims

1. The manganese dioxide/glucose oxidase @ hyaluronic acid composite anti-cancer material is characterized by comprising a core and a shell coating the core; the core comprises manganese dioxide nanosheets and glucose oxidase modified on the surfaces of the manganese dioxide nanosheets; the shell is hyaluronic acid.

2. The manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material as claimed in claim 1, wherein in the manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material, the mass ratio of the glucose oxidase, the manganese dioxide nanosheet and the hyaluronic acid is 1-2: 5-10: 60-120; preferably 1: 5: 60.

3. The manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material according to claim 1, wherein the manganese dioxide nanosheet is in the shape of a single-layer sheet, and has a particle size of not more than about 150 nm.

4. The manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material according to claim 1, wherein the particle size of the manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material is not higher than 500 nm; preferably 290 to 300 nm.

5. The preparation method of the manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material according to any one of claims 1-4 is characterized in that manganese dioxide nanosheets and glucose oxidase are dissolved and mixed in water, then added into a hyaluronic acid aqueous solution, stirred and assembled to obtain a suspension, and then subjected to centrifugal treatment, washing and purification and freeze drying to obtain the manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material.

6. The preparation method of the manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material according to claim 5, wherein the synthesis steps of the manganese dioxide nanosheets are as follows: obtaining a catalyst comprising H₂O₂And tetramethylammonium hydroxide, adding the mixed solution to Mn²⁺Stirring and reacting the manganese solution, separating to obtain a manganese dioxide body, dispersing the manganese dioxide body in water, and ultrasonically stripping to obtain manganese dioxide nanosheets.

7. The application of the manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material according to any one of claims 1 to 4 or the manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material prepared by the preparation method according to any one of claims 5 or 6 is characterized in that the manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material is used for preparing anticancer drugs.

8. The application of the manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material as claimed in claim 7, which is used for preparing an anticancer drug for killing cancer cells with high CD44 expression;

preferably used for preparing anti-cancer drugs for killing Hela cells;

further preferably, the compound is matched with pharmaceutically acceptable auxiliary materials to prepare the anticancer drug for injection administration.

9. An anticancer drug, which is characterized by comprising a pharmaceutically effective amount of manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material according to any one of claims 1 to 4, or manganese dioxide/glucose oxidase @ hyaluronic acid composite anticancer material prepared by the preparation method according to any one of claims 5 or 6.

10. The anti-cancer agent of claim 9, further comprising a pharmaceutically acceptable excipient;

preferably, the anticancer drug is a pharmaceutically acceptable drug in any dosage form.