A cascade catalytic nanotherapeutic agent and its preparation method and application

Abstract

The present invention discloses a cascade catalytic nanotherapeutic agent and a preparation method and application thereof. The cascade catalytic nanotherapeutic agent comprises: a polymer carrier, and glucose oxidase, lactate oxidase and catalase located inside the polymer carrier. The cascade catalytic nanotherapeutic agent of the present invention is a complex formed by a polymer carrier and glucose oxidase, lactate oxidase and catalase, wherein the polymer carrier is combined with glucose oxidase, lactate oxidase and catalase through hydrophobic interaction and electrostatic interaction, and the cascade catalytic nanotherapeutic agent can simultaneously cut off multiple nutrient sources of tumor cells and alleviate the hypoxia condition at the tumor site, thereby achieving a powerful starvation treatment for malignant tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedical nanomaterials, and in particular to a cascade catalytic nano therapeutic agent and a preparation method and application thereof. Background Art

[0002] The occurrence of tumors depends on the reprogramming of cell metabolism. A common feature of tumor metabolism is the ability to obtain essential nutrients from a nutrient-poor environment and use these nutrients to maintain survival and generate new cancer cells. As one of the main nutrients that support survival and biosynthesis in mammalian cells, glucose metabolism maintains the generation of various carbon intermediates, which are the cornerstones of various macromolecular assemblies. Unlike normal cells, tumors use aerobic glycolysis with extremely low efficiency to produce adenosine-5'-triphosphate (ATP) to maintain life activities. Therefore, in order to meet the rapidly growing needs of bioenergy and biosynthesis, cancer cells are abnormally dependent on glucose. Based on this, cutting off glucose-related energy metabolism pathways can effectively inhibit tumor growth and has little toxicity and side effects on the body, so it has received widespread attention from scientists in recent years. Among them, glucose oxidase (GOx), as a type of oxidase, can efficiently decompose glucose and has received widespread attention in the context of tumor treatment. However, the energy metabolism regulation network of tumor cells is complex, and glucose is not the only energy source. Cutting off glucose-related metabolic pathways alone cannot achieve true starvation therapy, and the interior of tumors is mostly anoxic, which greatly limits the catalytic performance of GOx on glucose. In addition, as a biomacromolecule, GOx is easily degraded, and its surface negative charge also severely limits its entry into cells.

[0003] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a cascade catalytic nanotherapeutic agent and a preparation method and application thereof, aiming to solve the problems of single target and hypoxia in the existing glucose oxidase-mediated starvation therapy.

[0005] The technical solution of the present invention is as follows:

[0006] In a first aspect of the present invention, a cascade catalytic nanotherapeutic agent is provided, which comprises: a polymer carrier (FP), and glucose oxidase (GOx), lactate oxidase (LOx) and catalase (CAT) located inside the polymer carrier.

[0007] Optionally, the ratio of the mass of the polymer carrier to the total mass of the three enzymes (ie, the glucose oxidase, lactate oxidase, and catalase) is 0.25-4.

[0008] Optionally, the mass ratio of the glucose oxidase, lactate oxidase and catalase is 1:1:1.

[0009] Optionally, the cascade catalytic nanotherapeutic agent is a spherical particle with a diameter of 40 to 70 nm.

[0010] Optionally, the polymer carrier is selected from polyethyleneimine modified with a perfluorocarbon chain.

[0011] The second aspect of the present invention provides a method for preparing the cascade catalytic nanotherapeutic agent described in the present invention, which comprises the steps of: mixing a polymer carrier aqueous solution with glucose oxidase, lactate oxidase, and catalase, and stirring at room temperature to obtain the cascade catalytic nanotherapeutic agent.

[0012] Optionally, the stirring time at room temperature is 2 to 12 hours.

[0013] The third aspect of the present invention provides a use of the cascade catalytic nanotherapeutic agent of the present invention in the preparation of a tumor treatment preparation.

[0014] Optionally, the treatment is intensive starvation treatment.

[0015] Beneficial effects: The cascade catalytic nanotherapeutic agent of the present invention is a nanomedicine capable of continuously supplying oxygen for strong starvation therapy of tumors. Among them, the glucose oxidase and lactate oxidase are used as nutritional blockers to achieve efficient starvation therapy. Catalase decomposes hydrogen peroxide to produce oxygen to overcome the limitations of tumor hypoxia on glucose oxidase and lactate oxidase-mediated tumor starvation therapy, thereby achieving strong inhibition of tumor growth. The cascade catalytic nanotherapeutic agent of the present invention can simultaneously cut off the nutritional sources of tumor cells in multiple ways and produce a large amount of reactive oxygen, effectively curbing tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a TEM image of FPGLC formed by combining glucose oxidase, lactate oxidase, catalase and a polymer carrier in Example 1 of the present invention.

[0017] Figure 2 This is a comparison chart of hydrogen peroxide produced by GOx, FPG, FPGL and FPGLC in Example 2 of the present invention.

[0018] Figure 3 This is a comparison chart of hydrogen peroxide produced by LOx, FPL, FPGL and FPGLC in Example 3 of the present invention.

[0019] Figure 4 This is a comparison chart of oxygen produced by CAT, FP and FPGLC in Example 4 of the present invention.

[0020] Figure 5 This is a graph of glucose concentration in the cell culture medium of different treatment groups in Example 5 of the present invention.

[0021] Figure 6 This is a graph of lactic acid concentration in the cell culture medium of different treatment groups in Example 6 of the present invention.

[0022] Figure 7 This is a graph of intracellular ATP concentrations in different treatment groups in Example 7 of the present invention.

[0023] Figure 8 This is a graph showing cell survival rates of different treatment groups under hypoxic conditions in Example 8 of the present invention.

[0024] Fig. 9 This is a tumor-targeted fluorescence imaging image of mice in different treatment groups in Example 9 of the present invention.

[0025] Fig.10 This is a graph showing the improvement of tumor blood oxygen saturation in different treatment groups in Example 10 of the present invention.

[0026] Fig.11 This is a diagram showing the inhibitory effects of different treatment groups on tumor growth in Example 11 of the present invention. DETAILED DESCRIPTION

[0027] The present invention provides a cascade catalytic nanotherapeutic agent and a preparation method and application thereof. To make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0028] The embodiment of the present invention provides a cascade catalytic nano therapeutic agent, which includes: a polymer carrier, and glucose oxidase, lactate oxidase and catalase located inside the polymer carrier.

[0029] The cascade catalytic nanotherapeutic agent described in this embodiment is a complex formed by glucose oxidase, lactate oxidase, catalase and a polymer. The polymer is used as a carrier and is combined with glucose oxidase, lactate oxidase and catalase through hydrophobic interaction and electrostatic interaction. The three enzymes (glucose oxidase, lactate oxidase and catalase) are located inside the polymer carrier.

[0030] In this embodiment, the cascade catalytic nanotherapeutic agent is a nanomedicine for strong starvation therapy of tumors that can continuously supply oxygen. Among them, the glucose oxidase and lactate oxidase are used as nutrient blockers to achieve efficient starvation therapy. Catalase decomposes hydrogen peroxide to produce oxygen to overcome the limitation of tumor hypoxia on glucose oxidase and lactate oxidase-mediated tumor starvation therapy, thereby achieving strong inhibition of tumor growth. The cascade catalytic nanotherapeutic agent can simultaneously achieve targeted delivery and strong starvation therapy of tumors.

[0031] Furthermore, the ratio of the mass of the polymer carrier to the total mass of the three enzymes is 0.25-4, so as to improve the delivery efficiency of the three enzymes and thus achieve a good therapeutic effect.

[0032] Furthermore, the mass ratio of the glucose oxidase, lactate oxidase and catalase is 1:1:1.

[0033] Furthermore, the cascade catalytic nanotherapeutic agent is a spherical particle with a diameter of 40 to 70 nm.

[0034] In this embodiment, the polymer carrier can also deliver one or more of superoxide dismutase, uricase, oxalate oxidase, thrombin, protein kinase, hyaluronidase, ferritin, collagen, silk protein, lipoprotein and recombinant protein.

[0035] Furthermore, the polymer carrier is selected from polyethyleneimine modified with a perfluorocarbon chain. The preparation method of the polyethyleneimine modified with a perfluorocarbon chain may include the following steps: mixing polyethyleneimine and 3-(perfluoro-n-hexyl)propylene oxide in methanol, and then stirring at room temperature; after dialysis and freeze-drying, the polyethyleneimine modified with a perfluorocarbon chain is obtained.

[0036] The present invention provides a method for preparing the cascade catalytic nanotherapeutic agent as described above, which comprises the steps of: mixing a polymer carrier aqueous solution with glucose oxidase, lactate oxidase, and catalase, and stirring at room temperature (15-25° C.) to obtain the cascade catalytic nanotherapeutic agent. It should be noted that the water used in this embodiment is sterile water, which does not need to be removed and can be used directly.

[0037] In this embodiment, the three enzymes are stirred with the polymer at room temperature, and the hydrophobicity and positive charge of the polymer are used to combine with the negatively charged three enzymes. The polymer carrier encapsulates the three enzymes into nanoparticles to form a self-oxygenated cascade catalytic nanotherapeutic agent. Due to the high permeability and retention effect of tumor tissue, the cascade catalytic nanotherapeutic agent can be passively targeted to the tumor site, dissociated under the stimulation of the tumor microenvironment, and achieve a strong starvation treatment of the tumor. At the same time, the cascade catalytic nanotherapeutic agent is simple to prepare and easy to operate, does not require complex and expensive equipment, can be mass-produced, and is easy to industrialize, and has good application prospects in the field of tumor treatment.

[0038] The embodiment of the present invention also provides a use of the cascade catalytic nanotherapeutic agent as described above in the preparation of a tumor treatment preparation.

[0039] In this embodiment, the three enzymes are all natural enzymes. Among the three enzymes, glucose oxidase is a drug that has been used in clinical practice and has good biocompatibility. Lactate oxidase and catalase also have good biocompatibility and biodegradability. The three enzymes are encapsulated into nanoparticles using polymer carriers, which not only reduces the toxicity of the enzymes, but also prolongs the circulation time of the drugs (i.e., the three enzymes) in the blood, facilitates the accumulation of drugs in the tumor site, promotes the consumption of glucose and lactic acid, better cuts off the supply of nutrients to tumor cells, and starves more tumor cells to death, thereby achieving an efficient tumor treatment effect.

[0040] In this embodiment, the cascade catalytic nanotherapeutic agent can achieve a strong tumor starvation treatment, and therefore has a good application prospect in the field of tumor treatment. At the same time, the preparation process of this embodiment is simple, easy to operate, does not require complex and expensive equipment, and is easy to achieve industrial production.

[0041] The present invention is further described below by means of specific examples.

[0042] Example 1: Synthesis of Cascade Catalytic Nanotherapeutic Agent (FPGLC)

[0043] Polyethyleneimine (30.0 mg) and 3-(perfluoro-n-hexyl)propylene oxide (16.0 mg) were mixed in methanol and then stirred at room temperature for 48 h. After dialysis (MWCO 3500 Da) and freeze-dried, the polymer carrier FP was obtained. In the FP aqueous solution, equal amounts of GOx, LOx and CAT (the mass ratio of the three enzymes to FP was 1:1.5) were added in sequence and stirred at room temperature for 2 h to obtain the FPGLC cascade catalytic nanotherapeutic agent.

[0044] TEM image of the synthesized FPGLC cascade catalytic nanotherapeutic agent Figure 1 As shown. Figure 1It can be seen that the synthesized FPGLC cascade catalytic nanotherapeutic agent is spherical, and the particle size of the cascade catalytic nanotherapeutic agent is 60 nm.

[0045] Comparative Example 1: Synthesis of FPGL

[0046] In the FP aqueous solution, equal masses of GOx and LOx (the mass ratio of the dual enzyme to FP was 1:1.5) were added in sequence and stirred at room temperature for 2 h to obtain the FPGL nanotherapeutic agent.

[0047] Comparative Example 2: Synthetic FPG

[0048] An equal amount of GOx as that in control example 1 (the mass ratio of GOx to FP was 1:1.5) was added to the FP aqueous solution and stirred at room temperature for 2 h to obtain the FPG nanotherapeutic agent.

[0049] Comparative Example 3: Synthetic FPL

[0050] An equal amount of LOx as that in control example 1 (the mass ratio of LOx to FP was 1:1.5) was added to the FP aqueous solution and stirred at room temperature for 2 h to obtain the FPG nanotherapeutic agent.

[0051] Comparative Example 4: Synthesis of Free GOx

[0052] In the aqueous solution, GOx of the same mass as that of Control Example 1 was added to obtain Free GOx.

[0053] Comparative Example 5: Synthesis of Free LOx

[0054] In the aqueous solution, add LOx of the same mass as that of Control Example 1 to obtain Free LOx.

[0055] Example 2: Evaluation of GOx enzyme activity of FPGLC cascade catalytic nanotherapeutics

[0056] Different concentrations of glucose solution were selected as the reaction substrate, namely 0, 0.5, 1, 2, 4 and 8 mM. Free GOx and FPG, FPGL and FPGLC with the same GOx enzyme concentration were added, and the hydrogen peroxide content produced was measured using a hydrogen peroxide kit after 2 hours. Figure 2 It can be seen that compared with Free GOx, the amount of hydrogen peroxide produced by glucose solutions treated with FPG and FPGL is basically the same, indicating that the GOx enzyme activity of the prepared FPGLC cascade catalytic nanotherapeutic agent is basically maintained. Since CAT can decompose the produced hydrogen peroxide, the hydrogen peroxide produced by glucose solutions treated with FPGLC is less than that of FPG and FPGL.

[0057] Example 3: Evaluation of LOx enzyme activity of FPGLC cascade catalytic nanotherapeutics

[0058] Different concentrations of lactic acid solution were selected as the reaction substrate, namely 0, 0.05, 0.1, 0.2 and 0.4 mM. Free LOx and FPL, FPGL and FPGLC with the same LOx enzyme concentration were added, and the hydrogen peroxide content produced was measured using a hydrogen peroxide kit after 2 hours. Figure 3 It can be seen that compared with Free LOx, the amount of hydrogen peroxide produced by lactic acid solutions treated with FPL and FPGL is basically the same, indicating that the LOx enzyme activity of the prepared FPGLC cascade catalytic nanotherapeutic agent is basically maintained. Since CAT can decompose the produced hydrogen peroxide, the hydrogen peroxide produced by lactic acid solutions treated with FPGLC is less than that of FPL and FPGL.

[0059] Example 4: Evaluation of CAT enzyme activity of FPGLC cascade catalytic nanotherapeutic

[0060] Add Free CAT enzyme, FP, CAT enzyme, and FPGLC with the same CAT enzyme concentration to 8 mL of hydrogen peroxide solution, and measure the oxygen content in the solution every 15 seconds. Figure 4 It can be seen that there is basically no difference in the activity of FPGLC and pure catalase, which indicates that the CAT enzyme activity of the prepared FPGLC cascade catalytic nanotherapeutic agent is basically maintained.

[0061] Example 5: Evaluation of the effects of different treatments on glucose levels in cell culture medium

[0062] 4T1 cells were cultured at 1×10 5 The cells were inoculated into a 12-well plate at a density of 10 μg / mL and incubated at 37°C and 5% CO2 for 24 h. Then, the old culture medium in the 12-well plate was aspirated and fresh culture medium containing PBS, GOx, FPG, FPGL, and FPGLC with the same GOx enzyme concentration was added. After 8 h of incubation, the blood glucose concentration in the culture medium was measured using a blood glucose monitor. The test results are shown in Table 1. Figure 5 As shown, after 8 h of FPGLC treatment of cells, the glucose concentration in the culture medium was the lowest, indicating that the FPGLC cascade catalytic nanotherapeutic agent consumed more glucose.

[0063] Example 6: Evaluation of the effect of different treatments on lactate levels in cell culture medium

[0064] 4T1 cells were cultured at 1×10 5 The cells were inoculated into 12-well plates at the same density and incubated at 37°C and 5% CO2 for 24 hours. Then, the old culture medium in the 12-well plates was aspirated, and PBS and fresh culture medium containing Free LOx, FPL, FPGL, and FPGLC with the same LOx concentration were added. After culturing for 8 hours, the concentration of lactic acid in the culture medium was determined using a lactate kit. The test results are shown in Figure 2. Figure 6 As shown, after 8 h of FPGLC treatment of cells, the concentration of lactate in the culture medium was the lowest, indicating that the FPGLC cascade catalytic nanotherapeutic agent consumed more lactate.

[0065] Example 7: Evaluation of the effects of different treatments on intracellular ATP levels

[0066] 4T1 cells were cultured at 1×10 5 The cells were inoculated into 12-well plates at a density of 10 μg / mL and incubated at 37°C and 5% CO2 for 24 h. Then, the old culture medium in the 12-well plates was aspirated, and fresh culture medium containing PBS and FPL, FPG, FPGL, and FPGLC with the same GOx and LOx concentrations was added. After culturing for 24 h, the intracellular ATP concentration was measured using an ATP kit. The test results are shown in Table 1. Figure 7 As shown, after 24 h of FPGLC treatment, the intracellular ATP concentration was the lowest, indicating that the FPGLC cascade catalytic nanotherapeutic agent can effectively block the production of intracellular ATP.

[0067] Example 8: Evaluation of the effect of starvation treatment on the survival rate of 4T1 cells

[0068] The standard MTT method was used to evaluate the effects of different drugs on the survival rate of 4T1 cells. 4T1 cells were plated at 5×10 3 Density was inoculated into a 96-well plate and incubated at 37°C and 5% CO2 for 24 hours. Next, the old culture medium in the 96-well plate was aspirated, and DMEM culture medium with different enzyme concentrations of 0.025, 0.05, 0.1, 0.2 and 0.4 μg / mL of FPL, FPG, FPGL, and FPGLC was added. After culturing for 24 hours under hypoxic conditions, the old culture medium in the 96-well plate was aspirated, and 100 μL of culture medium solution containing 5 mg / mL MTT was added to each well, and the culture was continued for 4 hours. The OD value of each well was then detected on a Synergy H1 microplate reader (the detection wavelength was 490 nm), and the cell survival rate was calculated using the following formula. Cell survival rate (%) = (OD value of sample 490 Value / BlankOD 490 Value) × 100%. The test results are as follows Figure 8As shown in the figure, the cytotoxicity of FPL is relatively low, and the toxicity comes from the cell death caused by hydrogen peroxide produced by the oxidation of intracellular lactate. The toxicity of FPG nanotherapeutic agent increased significantly, and the survival rate of 4T1 cells decreased. At the same time, FPGL can consume the glucose and lactate of cells at the same time and produce a large amount of hydrogen peroxide, so the survival rate of the FPGL group is lower than that of the FPG group. For FPGLC, on the one hand, hydrogen peroxide is decomposed and the cell survival rate increases; on the other hand, the oxygen produced by the decomposition of hydrogen peroxide can promote the consumption of glucose and lactate, and the cell survival rate decreases. Therefore, FPGLC exhibits higher cytotoxicity.

[0069] Example 9: Evaluation of the accumulation effect of cascade catalytic nanotherapeutics in 4T1 subcutaneous tumors

[0070] Female Balb / c mice (3 weeks, 15-20 g) were injected subcutaneously in the right hind leg with 2×10 6 4T1 tumor cells were used to establish a mouse subcutaneous tumor model. When the tumor volume reached 100 mm 3 Fluorescence imaging was performed when the IR800 fluorescent probe was covalently modified on the polymer carrier to perform in vivo fluorescence imaging of the FPGLC cascade catalytic nanotherapeutic agent to observe its accumulation in the tumor. Fig. 9 As shown in the figure, within 2 hours of tail vein administration, FPGLC began to accumulate in the tumor, indicating that the cascade catalytic nanotherapeutic agent can be targeted to the tumor site. However, since GOx and LOx produce a large amount of hydrogen peroxide when consuming glucose and lactate, which will destroy the IR800 molecules, the fluorescence of the tumor site began to weaken in the following time. Therefore, the cascade catalytic nanotherapeutic agent (FPGLC) has tumor targeting and can produce a large amount of hydrogen peroxide at the tumor site.

[0071] Example 10: Effect of cascade catalytic nanotherapeutic agent (FPGLC) on blood oxygen saturation of 4T1 subcutaneous tumor

[0072] Female Balb / c mice (3 weeks, 15-20 g) were injected subcutaneously in the right hind leg with 2×10 6 4T1 tumor cells were used to establish a mouse subcutaneous tumor model. When the tumor volume reached 100 mm 3 Photoacoustic imaging was performed at 0, 2, 4, 8, 12 and 24 hours after the tail vein injection of different treatments in tumor-bearing mice. The blood oxygen saturation of tumor tissue was analyzed by photoacoustic imaging system at 0, 2, 4, 8, 12 and 24 hours. The test results are shown in Fig.10As shown in the figure, after tail vein administration, since GOx and LOx consume glucose and lactate and need to consume oxygen in the tumor, the blood oxygen saturation gradually decreases, and the oxygen content in the tumor site decreases over time. Since CAT catalyzes the decomposition of hydrogen peroxide to produce oxygen, the oxygen content in the FPGLC group gradually increases after 8 hours, while the oxygen in the FPGL group continues to decrease at this time, indicating that FPGLC has a certain improvement effect on tumor hypoxia in the process of consuming glucose and lactate.

[0073] Example 11: Inhibitory effect of cascade catalytic nanotherapeutic agents on the growth of 4T1 subcutaneous tumors

[0074] Female Balb / c mice (3 weeks, 15-20 g) were injected subcutaneously in the hind legs with 2×10 6 4T1 tumor cells were used to establish a mouse subcutaneous tumor model. When the tumor volume reached 60 mm 3 Treatment testing was performed at 1:10 pm. In the case of unilateral tumors, the tumor-bearing mice were randomly divided into 5 groups: (1) PBS group; (2) FPL group; (3) FPG group; (4) FPGL group; (5) FPGLC group. Tumor volume was measured with a vernier caliper every other day, and the weight of the mice was monitored at the same time. The formula V = AB 2 / 2 to calculate the tumor volume, where A is the long diameter of the tumor (mm) and B is the short diameter of the tumor (mm). Fig.11 Injection of FPGLC can significantly inhibit tumor growth.

[0075] In summary, the cascade catalytic nanotherapeutic agent of the present invention can simultaneously cut off multiple nutrient sources inside the tumor and effectively curb tumor growth. At the same time, the catalase delivered by the cascade catalytic nanotherapeutic agent of the present invention can catalyze excess hydrogen peroxide to produce sufficient oxygen, effectively overcome the limitations of the tumor hypoxic microenvironment on starvation therapy, and achieve long-term starvation therapy for tumors, which will have good application prospects in the field of tumor treatment. At the same time, the preparation process of the present invention is simple, easy to operate, does not require complex and expensive equipment, and is easy to achieve industrial production.

[0076] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A cascade catalytic nanotherapeutic agent, characterized in that: The invention comprises a polymer carrier, and glucose oxidase, lactate oxidase and catalase located inside the polymer carrier; the polymer carrier is selected from polyethyleneimine modified with a perfluorocarbon chain; the mass ratio of the glucose oxidase, lactate oxidase and catalase is 1:1:1; the cascade catalytic nanotherapeutic agent is a spherical particle with a diameter of 40 to 70 nm; the ratio of the mass of the polymer carrier to the total mass of the glucose oxidase, lactate oxidase and catalase is 1.5:1; The preparation method of the cascade catalytic nanotherapeutic agent comprises the steps of: Mixing polyethyleneimine and 3-(perfluoro-n-hexyl)propylene oxide in methanol and stirring at room temperature; after dialysis and freeze-drying, obtaining the perfluorocarbon chain-modified polyethyleneimine; The perfluorocarbon chain-modified polyethyleneimine aqueous solution is mixed with glucose oxidase, lactate oxidase and catalase, and stirred at room temperature for 2 to 12 hours to obtain the cascade catalytic nanotherapeutic agent.

2. Use of the cascade catalytic nanotherapeutic agent according to claim 1 in the preparation of a tumor treatment preparation.

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