Nanoreactor with anti-cancer effect as well as preparation method and application of nanoreactor

Abstract

The invention relates to the technical field of biomedical polymer materials, in particular to a multi-enzyme active nano-reactor with an anti-cancer effect and a preparation method and application of the multi-enzyme active nano-reactor. The nanoreactor has glucose oxidase activity, peroxidase activity, catalase activity and glutathione reductase activity, can be actively targeted to tumor tissues, and can be used for inducing ferroptosis and apoptosis of cells by improving the level of reactive oxygen species (ROS) and oxygen in the cells and reducing the level of glutathione (GSH) and the expression level of GPX4 protein. The anti-tumor effect is realized. The material can be obtained through a one-step assembly method at room temperature, raw materials are easy to obtain, the preparation method is simple, and the material has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of bio-nanomedicine, and in particular to a nanoreactor with anti-cancer efficacy and a preparation method and application thereof. Background Art

[0002] Cancer, as a major health challenge worldwide, is becoming increasingly harmful. Traditional cancer treatment strategies mainly rely on inducing apoptosis of tumor cells to achieve therapeutic effects. However, tumor cells may gradually develop resistance to apoptosis, resulting in unsatisfactory treatment effects. Studies have shown that the anti-tumor effect can be significantly improved by inducing combined treatment of cell ferroptosis and apoptosis. Ferroptosis is a newly discovered form of regulated cell death, which is different from apoptosis and is characterized by iron-dependent accumulation of reactive oxygen species and lipid peroxidation. On the one hand, excessive reactive oxygen species can damage DNA, proteins, and mitochondria, thereby inducing cell apoptosis; on the other hand, reactive oxygen species can enhance lipid peroxidation in tumor cells, leading to ferroptosis. However, glutathione peroxidase 4 (GPX4) can inhibit ferroptosis by mediating glutathione-dependent elimination of lipid hydroperoxides. Therefore, an ideal ferroptosis-apoptosis inducer should not only have the ability to increase reactive oxygen species (ROS), but also have the potential to consume high levels of intracellular glutathione (GSH) to induce GPX4 inactivation.

[0003] By mimicking the biological activities of natural enzymes such as peroxidase, catalase and glutathione peroxidase, nanozymes can effectively produce reactive oxygen species and consume glutathione in tumor cells at the same time, thereby achieving ferroptosis-apoptosis combined anticancer therapy. However, most nanozymes are composed of inorganic materials and have poor biosafety. G-quadruplex / hemin (DNA-G4 / hemin) complex is a special nanozyme composed of guanine-rich DNA segments and hemin, which has higher biosafety than traditional inorganic nanozymes. However, the high cost of DNA materials and their susceptibility to deoxyribonucleases hinder their further application. Therefore, it is urgent to develop a nanoreactor with low price, simple preparation method and multi-enzyme activity as a tumor cell ferroptosis-apoptosis inducer.

[0004] Guanosine is one of the four main nucleosides that make up RNA, consisting of a guanine base and a ribose molecule. The complex formed by the combination of nucleoside-G4 and hemin also has catalytic activity similar to peroxidase. Compared with DNA materials, guanosine is cheaper, but its water solubility is very poor, which limits the biomedical potential of guanosine-based nucleoside-G4 / hemin. So far, there has been no report on the preparation of nanoreactors using guanosine as raw material. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a multi-enzyme active nanoreactor with anti-cancer efficacy and its preparation method and application. The nanoreactor provided by the present invention has four enzyme catalytic activities, namely glucose oxidase (GOx), peroxidase (POD), catalase (CAT) and glutathione peroxidase (GPX). Its raw materials are easily available, the cost is low, the stability is high, and it has a high application prospect.

[0006] The invention provides a nanoreactor with multi-enzyme activity, the raw materials of which include hyaluronic acid modified by phenylboronic acid, guanosine, hemin chloride and glucose oxidase.

[0007] Experiments show that the nanoreactor made from the above raw materials can actively target tumor tissues, and can achieve anti-tumor effects by increasing the level of intracellular reactive oxygen species (ROS) and oxygen levels, reducing the level of glutathione (GSH), and the expression level of GPX4 protein, inducing cell ferroptosis and apoptosis. The raw materials of the nanoreactor are easily available, low in cost, and highly stable.

[0008] In the present invention, the molecular weight ratio of the phenylboronic acid-modified hyaluronic acid to guanosine is 1:(0.29-2.86), specifically 1:0.29, 1:0.72, 1:1.43, 1:2.15, 1:2.86, among which 1:1.43 is preferred.

[0009] In the present invention, each raw material (including phenylboronic acid-modified hyaluronic acid, guanosine, hemin and glucose oxidase) is prepared by self-assembly in an aqueous medium, wherein the aqueous medium is a buffer solution with a pH value of 8.0 containing 0.1M KCl; the buffer solution is selected from 0.1M Tris-HCl buffer, PBS or deionized water. In some specific embodiments, the aqueous medium is a buffer solution with a pH value of 8.0 containing 0.1M Tris-HCl and 0.1M KCl, and the solvent of the aqueous medium is deionized water.

[0010] The present invention provides a method for preparing the nanoreactor, which comprises:

[0011] The hyaluronic acid modified with phenylboronic acid is dissolved in an aqueous medium, and guanosine, hemin chloride and glucose oxidase are added in sequence, and a nanoreactor is obtained through a self-assembly process and dialysis.

[0012] In some embodiments, the guanosine, hemin and glucose oxidase added sequentially are all added in the form of solutions. Specifically, the preparation method comprises the following steps:

[0013] The hyaluronic acid modified with phenylboronic acid is dissolved in an aqueous medium, and then a guanosine solution, a hemin solution and a glucose oxidase solution are added, and the hyaluronic acid is dialyzed after a self-assembly process to obtain the hyaluronic acid.

[0014] According to the present invention, the aqueous medium is a buffer solution containing 0.1M Tris-HCl and 0.1M KCl with a pH value of 8.0, and the solvent is deionized water.

[0015] In the phenylboronic acid modified hyaluronic acid of the present invention, the molecular weight of the hyaluronic acid is 1 kDa to 50 kDa, specifically 1 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, preferably 10 kDa. The modification degree of phenylboronic acid modification is 40% to 70%, specifically 40%, 45%, 55%, 60%, 70%. In a specific embodiment of the present invention, the modification degree of phenylboronic acid is 55%.

[0016] The reaction concentration of the phenylboronic acid modified hyaluronic acid of the present invention is 0.1-5 mg / mL, specifically 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 / mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, preferably 1 mg / mL.

[0017] In the preparation method of the present invention, the concentration of the guanosine solution is 100mM. In some specific embodiments, the guanosine solution is obtained by dissolving 28.6mg guanosine and 5.6mg potassium hydroxide in 1mL deionized water. In the present invention, in the reaction system of the co-assembly reaction (including phenylboronic acid-modified hyaluronic acid, guanosine, hemin chloride and glucose oxidase), the reaction concentration of guanosine (i.e., assembly concentration) is 1 to 10mM, specifically 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, preferably 5mM.

[0018] In the preparation method of the present invention, the concentration of the hemin solution is 10mM; in some specific embodiments, the hemin solution is obtained by dissolving 6.52mg of hemin powder in 1mL of DMSO. In the present invention, in the reaction system of the co-assembly reaction (including phenylboronic acid-modified hyaluronic acid, guanosine, hemin and glucose oxidase), the reaction concentration of hemin (i.e., assembly concentration) is 0.1-1mM, specifically 0.1mM, 0.25mM, 0.5mM, 0.75mM, 1mM, preferably 0.25mM.

[0019] In the preparation method of the present invention, the concentration of the glucose oxidase solution is 1 mg / mL; in some specific embodiments, the glucose oxidase solution of the present invention is obtained by dissolving 1 mg of glucose oxidase in 1 mL of PBS. In the present invention, in the reaction system of the co-assembly reaction (including a system of hyaluronic acid, guanosine, hemin and glucose oxidase modified with phenylboronic acid), the reaction concentration of glucose oxidase (i.e., assembly concentration) is 1 to 50 μM, specifically 15 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, preferably 10 μM.

[0020] The reaction temperature is 4-25°C, specifically 4°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 25°C, among which 25°C is preferred.

[0021] The reaction time is 24 to 48 hours, specifically 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, preferably 24 hours.

[0022] The dialysis time is 48 to 72 hours, specifically 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, or 72 hours, preferably 48 hours.

[0023] The present invention has clearly described the above preparation method, which will not be repeated here.

[0024] The present invention also provides a multi-enzyme active nanoreactor with anti-cancer efficacy prepared by any one of the preparation methods described above.

[0025] Experiments show that the nanoreactor prepared by the invention has catalytic activities of glucose oxidase, peroxidase, catalase and glutathione peroxidase.

[0026] The present invention provides the use of the above nanoreactor or a multi-enzyme active nanoreactor with anti-cancer efficacy obtained by any one of the preparation methods as an anti-tumor drug.

[0027] In the above application, the nanoreactor has glucose oxidase activity, peroxidase activity, catalase activity and glutathione reductase activity, and can consume glucose, generate hydroxyl radicals, generate oxygen and / or consume glutathione.

[0028] The present invention provides a multi-enzyme active nanoreactor with anticancer efficacy, which is quickly obtained by a one-step assembly method in an aqueous medium through phenylboronic acid-modified hyaluronic acid, guanosine, hemin and glucose oxidase. The nanoreactor has glucose oxidase activity, peroxidase activity, catalase activity and glutathione reductase activity. Experiments show that the nanoreactor can effectively target tumor tissues through active targeting mediated by hyaluronic acid-CD44 receptors. After being taken up by tumor cells, the nanoreactor first oxidizes the excess glucose in the tumor cells in the presence of oxygen under the action of glucose oxidase activity to generate gluconic acid and hydrogen peroxide; then, using peroxidase activity, the nanoreactor converts the generated hydrogen peroxide into highly toxic hydroxyl free radicals, increasing the level of reactive oxygen in tumor cells, thereby causing nuclear DNA damage and inducing cell apoptosis; at the same time, under the action of catalase activity, the nanoreactor can convert the generated hydrogen peroxide into oxygen to supplement the oxygen consumption during the oxidative decomposition of glucose, further increasing the generation of reactive oxygen; finally, under the action of glutathione peroxidase activity, the nanoreactor can effectively reduce the level of glutathione in tumor cells, further reduce the activity of GPX4 protein, enhance the accumulation of lipid peroxidation in cells, and ultimately induce cell ferroptosis.

[0029] The multi-enzyme active nanoreactor with anti-cancer efficacy described in the present invention can be obtained by a one-step assembly method under room temperature conditions, the raw materials are easily available, the preparation method is simple, and it is expected to be mass-produced.

[0030] In the preparation method of the nanoreactor provided by the present invention, guanine-modified polymer chains are prepared using guanosine and hyaluronic acid, which are cheaper and more stable, to replace guanine-rich DNA segments, thereby reducing costs and is expected to broaden the application potential of biomedical materials constructed based on nucleosides. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a schematic diagram of the construction of the multi-enzyme activity nanoreactor of the present invention;

[0032] Figure 2 is the hydrodynamic diameter of the nanoreactor described in Examples 2 to 7 and Comparative Examples 1 to 2 of the present invention;

[0033] Figure 3 is a TEM image of the nanoreactor described in Example 3 of the present invention;

[0034] Figure 4 The glucose oxidase activity test of the nanoreactors described in Example 3, Comparative Example 1 and Comparative Example 2 of the present invention;

[0035] Figure 5The peroxidase catalytic activity test of the nanoreactors described in Example 3, Comparative Example 1 and Comparative Example 2 of the present invention;

[0036] Figure 6 It is a test of the cascade reaction capability of the nanoreactors described in Example 3, Comparative Example 1 and Comparative Example 2 of the present invention;

[0037] Figure 7 The catalase catalytic activity test of the nanoreactors described in Example 3, Comparative Example 1 and Comparative Example 2 of the present invention;

[0038] Figure 8 The glutathione peroxidase catalytic activity test of the nanoreactors described in Example 3, Comparative Example 1 and Comparative Example 2 of the present invention;

[0039] Fig. 9 The ability of the nanoreactors described in Example 3, Comparative Example 1 and Comparative Example 2 of the present invention to increase the level of intracellular active oxygen is tested;

[0040] Fig.10 The ability of the nanoreactors described in Example 3, Comparative Example 1 and Comparative Example 2 of the present invention to reduce the intracellular GSH level is tested;

[0041] Fig.11 The cytotoxicity of the nanoreactors described in Examples 2 to 9, Comparative Examples 1 and 2 of the present invention;

[0042] Fig.12 The nanoreactors described in Example 3, Comparative Example 1 and Comparative Example 2 of the present invention are tested for their ability to promote intracellular lipid peroxidation;

[0043] Fig.13 The nanoreactor described in Example 3, Comparative Example 1 and Comparative Example 2 of the present invention causes DNA damage in cells;

[0044] Fig.14 The in vivo distribution data of the nanoreactor described in Example 3 of the present invention;

[0045] Fig.15 These are in vitro photographs of tumor tissues after 14 days of treatment using the nanoreactors described in Examples 3, 8, 9, Comparative Examples 1 and 2 of the present invention as anti-tumor drugs. DETAILED DESCRIPTION

[0046] The present invention provides a multi-enzyme active nanoreactor with anti-cancer efficacy and a preparation method and application thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they all belong to the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0047] To further illustrate the present invention, a multi-enzyme active nanoreactor with anti-cancer efficacy, a preparation method thereof and an application thereof provided by the present invention are described in detail below in conjunction with examples.

[0048] Example 1 Preparation of Nanoreactor

[0049] 12.1 mg of tris(hydroxymethyl)aminomethane (Tris) and 7.41 mg of potassium chloride were dissolved in 1 mL of deionized water, and the pH was adjusted to 8.0 using a dilute hydrochloric acid solution to obtain a solution 1.

[0050] 28.6 mg of guanosine and 5.6 mg of potassium hydroxide were dissolved in 1 mL of deionized water to obtain solution 2.

[0051] 6.52 mg of hemin was dissolved in 1 mL of DMSO to obtain Solution 3.

[0052] 1 mg of GOx was dissolved in 1 mL of PBS to obtain solution 4.

[0053] Example 2 Preparation of Nanoreactor

[0054] The solution 1 to 4 of Example 1 is used for preparation, comprising:

[0055] 1 mg of phenylboronic acid-modified hyaluronic acid (Mw=5kD, modification degree 55%) was dissolved in solution 1 (0.915 mL), and then solution 2 (50 μL), solution 3 (25 μL) and solution 4 (10 μL) were slowly added thereto in sequence. The mixture was stirred at 25°C for 24 hours, and a multi-enzyme active nanoreactor (denoted as HPG@hemin-GOx) was obtained after dialysis.

[0056] Example 3 Preparation of Nanoreactor

[0057] The solution 1 to 4 of Example 1 is used for preparation, comprising:

[0058] 1 mg of phenylboronic acid-modified hyaluronic acid (Mw = 10 kDa, modification degree 55%) was dissolved in solution 1 (0.915 mL), and then solution 2 (50 μL), solution 3 (25 μL) and solution 4 (10 μL) were slowly added thereto in sequence. The mixture was stirred at 25°C for 24 hours, and a multi-enzyme active nanoreactor (denoted as HPG@hemin-GOx) was obtained after dialysis.

[0059] Example 4 Preparation of Nanoreactor

[0060] The solution 1 to 4 of Example 1 is used for preparation, comprising:

[0061] 1 mg of phenylboronic acid-modified hyaluronic acid (Mw=50 kDa, degree of modification 55%) was dissolved in solution 1 (0.915 mL), and then solution 2 (50 μL), solution 3 (25 μL) and solution 4 (10 μL) were slowly added thereto in sequence. The mixture was stirred at 25°C for 24 hours, and a multi-enzyme active nanoreactor was obtained after dialysis.

[0062] Example 5 Preparation of Nanoreactor

[0063] The solution 1 to 4 of Example 1 is used for preparation, comprising:

[0064] 1 mg of phenylboronic acid-modified hyaluronic acid (Mw=10 kDa, modification degree 55%) was dissolved in solution 1 (0.955 mL), and then solution 2 (10 μL), solution 3 (25 μL) and solution 4 (10 μL) were slowly added thereto in sequence. The mixture was stirred at 25°C for 24 hours, and a multi-enzyme active nanoreactor was obtained after dialysis.

[0065] Example 6 Preparation of Nanoreactor

[0066] The solution 1 to 4 of Example 1 is used for preparation, comprising:

[0067] 1 mg of phenylboronic acid-modified hyaluronic acid (Mw=10 kDa, degree of modification 55%) was dissolved in solution 1 (0.94 mL), and then solution 2 (25 μL), solution 3 (25 μL) and solution 4 (10 μL) were slowly added thereto in sequence. The mixture was stirred at 25°C for 24 hours, and a multi-enzyme active nanoreactor was obtained after dialysis.

[0068] Example 7 Preparation of Nanoreactor

[0069] The solution 1 to 4 of Example 1 is used for preparation, comprising:

[0070] 1 mg of phenylboronic acid-modified hyaluronic acid (Mw=10 kDa, modification degree 55%) was dissolved in solution 1 (0.865 mL), and then solution 2 (100 μL), solution 3 (25 μL) and solution 4 (10 μL) were slowly added thereto in sequence. The mixture was stirred at 25° C. for 24 hours, and a multi-enzyme active nanoreactor was obtained after dialysis.

[0071] Example 8 Preparation of Nanoreactor

[0072] The solution 1 to 4 of Example 1 is used for preparation, comprising:

[0073] 1 mg of phenylboronic acid-modified hyaluronic acid (Mw = 10 kDa, modification degree 55%) was dissolved in solution 1 (0.915 mL), and then solution 2 (50 μL), solution 3 (10 μL) and solution 4 (10 μL) were slowly added thereto in sequence. The mixture was stirred at 25°C for 24 hours, and a multi-enzyme active nanoreactor (denoted as HPG@hemin-GOx) was obtained after dialysis.

[0074] Example 9 Preparation of Nanoreactor

[0075] The solution 1 to 4 of Example 1 is used for preparation, comprising:

[0076] 1 mg of phenylboronic acid-modified hyaluronic acid (Mw = 10 kDa, modification degree 55%) was dissolved in solution 1 (0.915 mL), and then solution 2 (50 μL), solution 3 (25 μL) and solution 4 (5 μL) were slowly added thereto in sequence. The mixture was stirred at 25°C for 24 hours, and a multi-enzyme active nanoreactor (denoted as HPG@hemin-GOx) was obtained after dialysis.

[0077] Comparative Example 1 Preparation of Nanoreactor

[0078] The solution 1 to 3 of Example 1 is used for preparation, comprising:

[0079] 1 mg of phenylboronic acid-modified hyaluronic acid (Mw=10 kDa, modification degree 55%) was dissolved in solution 1 (0.925 mL), and then solution 2 (50 μL) and solution 3 (25 μL) were slowly added thereto in sequence. The mixture was stirred at 25°C for 24 hours, and a multi-enzyme active nanoreactor (denoted as HPG@hemin) was obtained after dialysis.

[0080] Comparative Example 2 Preparation of Nanoreactor

[0081] The solution 1, 2, 4 of Example 1 is used for preparation, comprising:

[0082] 1 mg of phenylboronic acid-modified hyaluronic acid (Mw=10 kDa, modification degree 55%) was dissolved in solution 1 (0.94 mL), and then solution 2 (50 μL) and solution 4 (10 μL) were slowly added thereto in sequence. The mixture was stirred at 25°C for 24 hours, and a multi-enzyme active nanoreactor (denoted as HPG@GOx) was obtained after dialysis.

[0083] Example 10

[0084] The size of the nanoreactors obtained in Examples 2 to 7 and Comparative Examples 1 to 2 was analyzed using a dynamic light scattering (DLS) instrument, and the particle sizes of the nanoreactors obtained in Examples 2 to 7 and Comparative Examples 1 to 2 were measured. Figure 2 The particle size statistics of the nanoreactors shown in Examples 2 to 7 of the present invention and Comparative Examples 1 to 2 are shown. According to the results, the size of the nanoreactor is related to the molecular weight of the hyaluronic acid modified with phenylboronic acid and the feed ratio of the hyaluronic acid modified with phenylboronic acid to guanosine. The particle sizes of Examples 4 to 7 are relatively large, which is not conducive to the EPR effect. In order to improve the EPR effect and exert the anti-tumor effect of the nanoreactor, the present invention selects Examples 2 to 3 and 8 to 9 with smaller particle sizes for subsequent testing, and Comparative Examples 1 to 2 are used as controls.

[0085] Embodiment 11

[0086] The morphology of the nanoreactor was analyzed using a transmission electron microscope. The nanoreactor obtained in Example 3 was dropped onto the surface of a copper mesh and tested after being naturally dried.

[0087] Results Reference Figure 3 , Figure 3 This is a transmission electron microscope image of the nanoreactor according to Example 3 of the present invention. According to the results, it can be concluded that the microscopic morphology of the nanoreactor according to the present invention is spherical.

[0088] Example 12 Evaluation of the catalytic activity of glucose oxidase in nanoreactors using the titanium sulfate (Ti(SO4)2) method

[0089] The nanoreactor described in Example 3 was added to a solution containing 2 g L -1 In the glucose solution, PBS, the nanoreactor described in Comparative Example 1 and the nanoreactor described in Comparative Example 2 or the same amount of GOx as that contained in the nanoreactor described in Example 3 were used as the control group. After reacting at 37°C for 30 minutes, 50 μL of the mixed solution was mixed with 100 μL of titanium sulfate solution for 10 minutes and then its absorbance at 410 nm was tested, and the hydrogen peroxide content in the mixed solution was converted using the standard curve method.

[0090] Results Reference Figure 4 , Figure 4The ability of the nanoreactors shown in Example 3, Comparative Examples 1 and Comparative Examples 2 of the present invention to generate hydrogen peroxide. According to the results, it can be concluded that Comparative Example 1 (HPG@hemin) without GOx loading does not have glucose oxidase catalytic activity; Example 3 (HPG@hemin-GOx) and Comparative Example 2 (HPG@GOx) loaded with GOx have glucose oxidase catalytic activity and can generate hydrogen peroxide in a solution containing glucose. And the amount of hydrogen peroxide generated by the nanoreactor is similar to that generated by free GOx, indicating that the nanoreactor of the present invention does not affect the glucose oxidase activity. It can be seen from Comparative Examples 1 and 2 that the glucose oxidase activity of the nanoreactor of the present invention is related to the GOx loaded in the nanoreactor, and Comparative Example 2 (HPG@GOx) also has glucose oxidase activity.

[0091] Example 13 Evaluation of Nanoreactor Peroxidase Catalytic Activity Using TMB Colorimetric Method

[0092] The nanoreactor described in Example 3 was added to a solution containing 2 mM hydrogen peroxide and 2 mM TMB, and PBS, the nanoreactor described in Comparative Example 1, and the nanoreactor described in Comparative Example 2 were used as control groups. After reacting at 37° C. for 1 hour, the ultraviolet absorption of the mixed solution at 500-800 nm was tested to evaluate the oxidation of TMB to oxTMB.

[0093] Results Reference Figure 5 , Figure 5 The ultraviolet absorption spectra of the nanoreactors shown in Example 3 (HPG@hemin-GOx), Comparative Example 1 (HPG@hemin) and Comparative Example 2 (HPG@GOx) of the present invention after reacting with TMB for 1 hour under the condition of containing hydrogen peroxide. According to the results, it can be concluded that the nanoreactor of the present invention has peroxidase activity, that is, it can catalyze hydrogen peroxide to oxidize TMB substrate and enhance its absorbance at 500-800nm. It can be seen from Comparative Examples 1 and 2 that the peroxidase activity of the nanoreactor of the present invention is related to the hemin loaded in the nanoreactor, and Comparative Example 1 (HPG@hemin) also has peroxidase activity.

[0094] Example 14 Evaluation of the cascade reaction between the catalytic activities of glucose oxidase and peroxidase in nanoreactors using TMB colorimetric method

[0095] In Example 3, the nanoreactor was added to a solution containing 2 g L -1 In the solution of glucose and 2 mM TMB, PBS, the nanoreactor described in Comparative Example 1, and the nanoreactor described in Comparative Example 2 were used as control groups. After reacting at 37° C. for 1 hour, the ultraviolet absorption of the mixed solution at 500-800 nm was tested to evaluate the oxidation of TMB to oxTMB.

[0096] Results Reference Figure 6 , Figure 6 This is the ultraviolet absorption spectrum of the nanoreactor shown in Example 3 of the present invention after reacting with TMB for 1 hour in the presence of glucose. According to the results, it can be concluded that the nanoreactor of the present invention can oxidize the TMB substrate through a cascade reaction between the glucose oxidase activity and the peroxidase catalytic activity, that is, the glucose oxidase activity first oxidizes glucose to generate hydrogen peroxide, and then the hydrogen peroxide generated by the peroxidase activity catalyzes the TMB substrate, thereby enhancing its absorbance at 500-800nm.

[0097] Example 15 Evaluation of the Catalytic Activity of Nanoreactor-type Catalase Using a Dissolved Oxygen Meter

[0098] The nanoreactor described in Example 3 was added to a solution containing 2 mM hydrogen peroxide, and PBS, the nanoreactor described in Comparative Example 1, and the nanoreactor described in Comparative Example 2 were used as control groups. After reacting for 30 minutes, the dissolved oxygen content in the mixed solution was measured using a portable dissolved oxygen tester.

[0099] Results Reference Figure 7 , Figure 7 This is Example 3 of the present invention, and the dissolved oxygen content generated by the nanoreactors shown in Comparative Examples 1 and 2 under conditions containing hydrogen peroxide. According to the results, it can be concluded that the nanoreactor of the present invention has catalase catalytic activity and can catalyze the oxidation of hydrogen peroxide to produce oxygen. It can be seen from Comparative Example 1 (HPG@hemin) and Comparative Example 2 (HPG@GOx) that the catalase activity of the nanoreactor of the present invention is related to the hemin loaded in the nanoreactor, and Comparative Example 1 (HPG@hemin) also has catalase activity.

[0100] Example 16 Evaluation of Glutathione Peroxidase-like Catalytic Activity of Nanoreactor Using DTNB Reagent

[0101] The nanoreactor described in Example 3 was added to a solution containing 2 mM glutathione, and PBS, the nanoreactor described in Comparative Example 1, and the nanoreactor described in Comparative Example 2 were used as control groups. After reacting for 2 hours, 50 μL of the mixed solution was mixed with 150 μL of DDTNB solution for 10 minutes, and its absorbance at 412 nm was tested, and the glutathione clearance rate in the mixed solution was converted using the standard curve method.

[0102] Results Reference Figure 8 , Figure 8This is an evaluation of the glutathione removal effect of the nanoreactors shown in Example 3, Comparative Example 1 and Comparative Example 2 of the present invention. According to the results, it can be concluded that the nanoreactor of the present invention has glutathione peroxidase catalytic activity and can effectively remove glutathione in the solution. It can be seen from Comparative Example 1 (HPG@hemin) and Comparative Example 2 (HPG@GOx) that the glutathione peroxidase activity of the nanoreactor of the present invention is related to the hemin loaded in the nanoreactor, and Comparative Example 1 (HPG@hemin) also has glutathione peroxidase activity.

[0103] According to Examples 12-16, the nanoreactor HPG@hemin-GOx of the present invention has glucose oxidase activity, peroxidase activity, catalase activity and glutathione peroxidase activity at the same time, and the multi-enzyme activities can generate hydrogen peroxide through a cascade reaction. Compared with the nanoreactor HPG@hemin-GOx of the present invention, Comparative Example 1 (HPG@hemin) does not have glucose oxidase activity; Comparative Example 2 (HPG@GOx) only has glucose oxidase activity.

[0104] Example 17 Evaluation of the generation of reactive oxygen species in cells by the nanoreactor using the DCFH-DA probe

[0105] 4T1 cells were inoculated in a sterile 96-well plate, with 10,000 cells planted in each well. After culturing in a 37°C incubator for 24 hours, 10 μL of the nanoreactor described in Example 3, Comparative Example 1 or Comparative Example 2 was added to each well, and the control group was an equal amount of PBS, and the culture was continued for 6 hours. The culture medium was then discarded, and serum-free culture medium containing DCFH-DA probes was added to each well. After culturing at 37°C for 1 hour, the intracellular fluorescence intensity level was observed using a fluorescence microscope.

[0106] Results Fig. 9 , Fig. 9 The intracellular reactive oxygen levels after treatment with the nanoreactors shown in Example 3 (HPG@hemin-GOx), Comparative Example 1 (HPG@hemin) and Comparative Example 2 (HPG@GOx) are shown. The results show that under the same concentration conditions (working concentration 6.8 μg mL -1 Hemin, 1 μg mL -1 GOx), the nanoreactor shown in Example 3 can effectively increase the level of intracellular reactive oxygen. On the contrary, Comparative Example 1 or Comparative Example 2 which do not have the same multiple enzyme activities cannot effectively increase the level of intracellular reactive oxygen.

[0107] Example 18 Intracellular GSH level test

[0108] 4T1 cells were inoculated in a sterile 96-well plate, with 10,000 cells planted in each well. After culturing in a 37°C incubator for 24 hours, 10 μL of the nanoreactor described in Example 3, Comparative Example 1 or Comparative Example 2 was added to each well, and the control group was an equal amount of PBS, and the culture was continued for 6 hours. The intracellular GSH level was then determined using a GSH detection kit.

[0109] Results Fig.10 , Fig.10 The intracellular GSH levels after treatment with the nanoreactors shown in Example 3 (HPG@hemin-GOx), Comparative Example 1 (HPG@hemin) and Comparative Example 2 (HPG@GOx) are shown. The results show that under the same concentration conditions (working concentration 6.8 μg mL -1 Hemin, 1 μg mL -1 GOx), the nanoreactor shown in Example 3 can most effectively reduce the intracellular GSH level. In contrast, Comparative Example 1 or Comparative Example 2, which do not have the same multiple enzyme activities, cannot effectively reduce the intracellular GSH level.

[0110] Example 19 Inhibitory Effect on Tumor Cells

[0111] 4T1 cells were inoculated in a sterile 96-well plate, 10,000 cells were planted in each well, and after re-cultivation in a 37°C incubator for 24 hours, 10 μL of the nanoreactor shown in Examples 2 to 9, Comparative Example 1 or Comparative Example 2 was added to each well, the negative control group was pure cell culture fluid, and the positive control group was sterile water, and culture was continued for 24 hours. The culture fluid was then discarded, and 100 μL of serum-free culture medium containing CCK8 was added to each well. After continuing to culture at 37°C for 1 hour, the absorbance at 450 nm was tested using an ELISA reader.

[0112] Cell survival rate % = (OD drug group - OD positive group) / (OD negative group - OD positive group) * 100%

[0113] Results Fig.11 , Fig.11 The cytotoxicity of the nanoreactors shown in Examples 2 to 9 (HPG@hemin-GOx), Comparative Example 1 (HPG@hemin) and Comparative Example 2 (HPG@GOx) is shown in Table 1. Under the same concentration conditions (working concentration 6.8 μg mL -1 Hemin, 1 μg mL -1 GOx), the nanoreactors (HPG@hemin-GOx) shown in Examples 2 to 9 can effectively inhibit the activity of tumor cells. On the contrary, Comparative Example 1 or Comparative Example 2 which do not have the same multiple enzyme activities cannot effectively inhibit the activity of tumor cells.

[0114] Example 20 Intracellular lipid peroxidation level test

[0115] 4T1 cells were inoculated in a sterile 96-well plate, with 10,000 cells planted in each well. After culturing in a 37°C incubator for 24 hours, 10 μL of the nanoreactor described in Example 3, Comparative Example 1 or Comparative Example 2 was added to each well, and the control group was an equal amount of PBS, and the culture was continued for 6 hours. The intracellular lipid peroxidation level was then determined using a lipid peroxidation MDA detection kit.

[0116] Results Fig.12 , Fig.12 The MDA levels in cells after treatment with the nanoreactors shown in Example 3 (HPG@hemin-GOx), Comparative Example 1 (HPG@hemin) and Comparative Example 2 (HPG@GOx) are shown in Table 1. The results show that under the same concentration conditions (working concentration 6.8 μg mL -1 Hemin, 1 μg mL -1 GOx), the nanoreactor shown in Example 3 can most effectively promote the lipid peroxidation process in tumor cells. On the contrary, Comparative Example 1 or Comparative Example 2, which do not have the same multiple enzyme activities, cannot effectively promote the lipid peroxidation process in tumor cells.

[0117] Example 21 Intracellular DNA damage test

[0118] 4T1 cells were inoculated in a sterile 96-well plate, with 10,000 cells planted in each well. After culturing in a 37°C incubator for 24 hours, 10 μL of the nanoreactor described in Example 3, Comparative Example 1 or Comparative Example 2 was added to each well, and the control group was an equal amount of PBS, and the culture was continued for 6 hours. Subsequently, a DNA damage kit was used to assess the intracellular DNA damage (DNA damage marker γ-H2AX was detected by immunofluorescence staining).

[0119] Results Fig.13 , Fig.13 The DNA damage in cells after treatment with the nanoreactors shown in Example 3 (HPG@hemin-GOx), Comparative Example 1 (HPG@hemin) and Comparative Example 2 (HPG@GOx) is shown in Figure 3. From the confocal imaging results, it can be seen that under the same concentration conditions (working concentration 6.8 μg mL -1 Hemin, 1 μg mL -1 GOx), the nanoreactor shown in Example 3 can effectively cause DNA damage in cells. In contrast, Comparative Example 1 or Comparative Example 2, which do not have the same multiple enzyme activities, cannot effectively cause DNA damage in cells.

[0120] Example 22 Tumor Targeting Test

[0121] 1×10 suspended in 0.1 mL PBS 64T1 cells were injected subcutaneously into the right abdomen of BALB / c mice. When the tumor volume reached about 100 mm 3 At 24 h after injection, in vivo imaging was performed using the Maestro in vivo imaging system (IVIS Lumina LT Series III, PerkinElmer, USA).

[0122] Results Fig.14 , Fig.14 This is the in vivo imaging of Example 3 (HPG@hemin-GOx) modified with Cy5 24 hours after tail vein injection. The results show that the nanoreactor shown in Example 3 can effectively target tumor tissue sites.

[0123] Example 23 Test for inhibiting tumor growth

[0124] Female BALB / c mice were injected subcutaneously in the right abdomen with 1×10 6 4T1 cells. When the tumor grows to 100 mm 3 The nanoreactors of Example 3, Example 8, Example 9, Comparative Example 1 or Comparative Example 2 were injected through the tail vein for 5 times, respectively, on days 0, 2, 4, 6 and 8, with an equal amount of PBS as the control group, and 5 mice in each treatment group. The mice were euthanized on day 14, and the tumors were analyzed.

[0125] The nanoreactors shown in Examples 3, 8 and 9 are used for anti-tumor drugs, which can effectively increase the level of reactive oxygen in tumor cells through the synergistic effect of multiple enzyme activities, reduce the level of glutathione, cause DNA damage, promote lipid peroxidation, and ultimately induce cell apoptosis and ferroptosis, thereby inhibiting tumor growth.

[0126] In contrast, comparative example 1 (HPG@hemin) with single loading of hemin or comparative example 2 (HPG@hemin-GOx) with single loading of GOx could not effectively inhibit tumor growth.

[0127] Some of the results can be found in Fig.15 , Fig.15 These are photos of in vitro tumor tissues of tumor-bearing mice treated with the nanoreactors described in the control group and Examples 3, 8, 9 and Comparative Examples 1 and 2 as anti-tumor drugs for 14 days.

[0128] The results showed that the tumor volume of mice in the nanoreactor treatment groups of Examples 3, 8 and 9 was significantly smaller than that in the comparative examples 1-2, indicating that the nanoreactors prepared in Examples 3, 8 and 9 of the present invention can effectively inhibit the growth of tumor volume, among which Example 3 has the best inhibitory effect. The tumor volumes of other mice in the treatment groups were Fig.15The results shown are similar.

[0129] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A nanoreactor with multi-enzyme activity, characterized in that: Its raw materials include phenylboronic acid-modified hyaluronic acid, guanosine, hemin and glucose oxidase.

2. The nanoreactor according to claim 1, characterized in that: The mass ratio of phenylboronic acid-modified hyaluronic acid to guanosine is 1:(0.29-2.86).

3. The nanoreactor according to claim 1 or 2, characterized in that: Each raw material is prepared by self-assembly in an aqueous medium; the aqueous medium is a buffer solution containing 0.1M KCl and having a pH value of 8.0; the buffer solution is selected from 0.1M Tris-HCl buffer, PBS or deionized water.

4. The method for preparing the nanoreactor according to any one of claims 1 to 3, characterized in that: include: The hyaluronic acid modified with phenylboronic acid is dissolved in an aqueous medium, and guanosine, hemin chloride and glucose oxidase are added in sequence, and a nanoreactor is obtained through a self-assembly process and dialysis.

5. The preparation method according to claim 4, characterized in that: The molecular weight of the phenylboronic acid modified hyaluronic acid is 1 kDa to 50 kDa.

6. The preparation method according to claim 4, characterized in that: The reaction concentration of the phenylboronic acid-modified hyaluronic acid is 0.1 to 5 mg / mL; The reaction concentration of guanosine is 1 to 10 mM; The reaction concentration of the hemin is 0.1-1 mM.

7. The preparation method according to claim 4, characterized in that: The temperature of the self-assembly reaction is 4 to 25° C. and the time is 24 to 48 hours; The dialysis time is 48 to 72 hours.

8. The nanoreactor prepared by the preparation method according to any one of claims 4 to 7.

9. Use of the nanoreactor according to any one of claims 1 to 3 and 8 or the nanoreactor prepared by the preparation method according to any one of claims 4 to 7 in the preparation of anti-tumor drugs, or in the preparation of combined anti-tumor drugs.

10. The use according to claim 9, characterized in that: The nanoreactor has glucose oxidase activity, peroxidase activity, catalase activity and glutathione reductase activity, and can consume glucose, generate hydroxyl free radicals, generate oxygen and / or consume glutathione.

Images