HMPB / FA nano system with multi-enzyme activity and preparation method of HMPB / FA nano system

Abstract

The invention discloses an HMPB / FA nano system with multienzyme activity and a preparation method of the HMPB / FA nano system, and belongs to the technical field of biological medicines. The method comprises preparation of mesoporous prussian blue PB, hollow mesoporous prussian blue HMPB and an HMPB / FA nano system with multienzyme activity, and is simple in preparation, high in synthesis efficiency, low in cost, good in repeatability and efficient in drug utilization rate. The HMPB / FA nano-system prepared by the invention takes HMPB with a hollow mesoporous structure as a carrier, ferulic acid FA is loaded by a solution impregnation method, and the HMPB / FA nano-system has good multi-enzyme activity and has no obvious cytotoxicity to C2C12 and RAW264.7 cells. The HMPB / FA nano system prepared by the invention is applied to treatment of lower limb ischemia diseases, can obviously improve related symptoms (including weight loss and blood perfusion) of lower limb ischemia, and solves the problems of various inflammatory reactions caused by lower limb ischemia, defects in the prior art and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a preparation method and application of a HMPB / FA nanosystem with multi-enzyme activity. Background Art

[0002] Critical limb ischemia (CLI) is a severe and terminal stage of peripheral artery disease (PAD), which can cause severe symptoms such as limb rest pain, ulcers, and gangrene. If revascularization is not performed in time, the amputation rate is about 25% within 1 year after diagnosis, which greatly affects the patient's quality of life and poses a great threat to the patient's limbs and even life. At present, surgical bypass or endovascular treatment is the clinical first choice for CLI patients, but about 30% of patients are still not suitable for surgery or endovascular treatment due to severe complications. Therefore, improving limb blood perfusion and skeletal muscle function in CLI patients through non-surgical treatment and reducing amputation rate and mortality are clinical problems that need to be solved urgently. Therefore, it is necessary to develop new safe and effective biomaterials for the treatment of lower limb ischemia.

[0003] Studies have shown that excessive reactive oxygen species (ROS) and reduced redox capacity lead to progressive dysfunction of the antioxidant defense system, which is closely related to the prognosis of CLI. Ischemia-reperfusion increases the production of ROS by mitochondria in PAD patients, and the increase in oxidative stress leads to ischemic muscle damage and a decrease in the cross-sectional area of ​​muscle fibers.

[0004] Therefore, reducing oxidative stress, inhibiting inflammation, and improving the local ischemic microenvironment and skeletal muscle function are of great significance in reducing the incidence of PAD, reducing CLI mortality, and improving the quality of life of patients.

[0005] Studies have shown that ferulic acid (FA) is an active ingredient mainly found in Chinese medicinal materials Angelica sinensis and Ligusticum chuanxiong, and has a wide range of pharmacological activities. However, its application as a therapeutic drug is limited due to its low solubility and bioavailability. Hollow mesoporous Prussian blue (HMPB) as a nanocarrier can significantly improve the bioavailability of FA, and HMPB itself has enzyme-like activity, thereby achieving a synergistic therapeutic effect on lower limb ischemia.

[0006] Therefore, designing and developing HMPB / FA nanosystems with multi-enzyme activity will hopefully significantly improve the therapeutic effects of lower limb ischemia.

[0007] So far, there are no reports on the hollow mesoporous Prussian blue (HMPB)-loaded ferulic acid (FA) nanosystem with multi-enzyme activity. Summary of the invention

[0008] In view of this, the first object of the present invention is to provide a HMPB / FA nanosystem with multi-enzyme activity, which effectively solves the problems of the insufficiency of the prior art, the low bioavailability of FA, and the lack of drugs combined with nanozymes for synergistic treatment.

[0009] The second object of the present invention is to provide a method for preparing a HMPB / FA nanosystem with multi-enzyme activity at a low preparation cost.

[0010] The third object of the present invention is to provide an application of an HMPB / FA nanosystem with multi-enzyme activity in the treatment of lower limb ischemia, so as to solve various inflammatory reactions caused by lower limb ischemia.

[0011] The present disclosure provides a method for preparing a HMPB / FA nanosystem with multi-enzyme activity, comprising the following steps:

[0012] Step (1), dissolving 4.5-5.0 g PVP and 198-220 mg K3[Fe(CN)6] in 60-65 mL 0.01 M HCl solution, and obtaining mesoporous Prussian blue PB powder by a hydrothermal method in an oven;

[0013] Step (2), dissolving 30-35 mg of mesoporous Prussian blue PB and 150-175 mg of PVP in 30-35 mL of 1.0 M HCl solution, and performing a hydrothermal method in an oven to obtain a hollow mesoporous Prussian blue HMPB powder having multiple enzyme activities;

[0014] Step (3), dissolving ferulic acid FA in anhydrous ethanol to obtain solution A, and dispersing HMPB in ultrapure water by ultrasonication to obtain solution B;

[0015] Step (4), mixing solution A and solution B, stirring them evenly, washing them by centrifugation, and freeze-drying them to obtain a HMPB / FA nanosystem with multi-enzyme activity.

[0016] Furthermore, in step (4), the stirring temperature is 20 to 26° C., and the stirring time is 12 to 24 hours.

[0017] Furthermore, the time of centrifugal washing in step (4) is 15 to 20 minutes, and the rotation speed is 10000 to 11000 r / min.

[0018] Furthermore, in step (3), the particle size of HMPB is 140 to 220 nm.

[0019] Furthermore, in the step (3), the mass ratio of FA to HMPB is 1:1.

[0020] Furthermore, the HMPB / FA nanosystem with multiple enzyme activities is a drug that uses HMPB as a carrier and is loaded with FA. The FA in the HMPB / FA nanosystem with multiple enzyme activities will not affect the activities of multiple enzymes of HMPB.

[0021] Furthermore, the load rate of the FA is 32.43% to 36.71%.

[0022] Furthermore, the HMPB comprises multiple enzymes.

[0023] Furthermore, the multi-enzyme is one or a combination of catalase-like, superoxide dismutase-like and / or peroxidase-like.

[0024] Furthermore, the HMPB / FA nanosystem with multi-enzyme activity is a drug used to treat lower limb ischemia diseases.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The preparation method of the present invention is simple, and the HMPB / FA nanosystem with multi-enzyme activity has high synthesis efficiency, low preparation cost, good repeatability and high drug utilization rate.

[0027] 2. The HMPB / FA nanosystem with multi-enzyme activity prepared by the present invention has excellent multi-enzyme-like activity, reduces inflammation and has no obvious cytotoxicity.

[0028] 3. The HMPB / FA nanosystem with multi-enzyme activity prepared by the present invention can significantly improve the symptoms related to lower limb ischemia (including weight loss and blood perfusion), and can be used as a new potential drug for the treatment of lower limb ischemia, thereby solving the problems of various inflammatory reactions caused by lower limb ischemia and the deficiencies of existing technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a transmission electron microscopy (TEM) image of the HMPB / FA nanosystem with multi-enzyme activity prepared in Example 1 of the present invention;

[0030] Figure 2 The infrared spectra (a) and ultraviolet spectra (b) of HMPB, FA and HMPB / FA nanosystem with multi-enzyme activity prepared in Example 1 of the present invention;

[0031] Figure 3 These are three enzyme activity test graphs of the HMPB / FA nanosystem with multi-enzyme activity prepared in Example 1 of the present invention, wherein: a is peroxidase-like activity, b is superoxide dismutase-like activity, and c is catalase-like activity;

[0032] Figure 4 Schematic diagram of the cytotoxic activity of the HMPB / FA nanosystem with multi-enzyme activity prepared in Example 1 of the present invention on C2C12 and RAW264.7 cells;

[0033] Figure 5 This is a diagram showing the blood perfusion of mice in different groups in the lower limb ischemia model in Example 1 of the present invention;

[0034] Figure 6 Figure 1 shows the weight change data (a) and blood perfusion data (b) of different groups of mice in the lower limb ischemia model in Example 1 of the present invention;

[0035] Figure 7 is a flow chart of the steps of the method for preparing the HMPB / FA nanosystem with multi-enzyme activity in Example 1 of the present invention; DETAILED DESCRIPTION

[0036] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. Preferred embodiments of the present invention are provided in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0037] It should be noted that when an element is referred to as being "fixed to" another element, it may be directly on the other element or there may also be an element centered thereon. When an element is considered to be "connected to" another element, it may be directly connected to the other element or there may be an element centered thereon at the same time. The terms "vertical", "horizontal", "left", "right", "up", "down", "front", "rear" and similar expressions used herein are for illustrative purposes only.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0039] The present application provides a HMPB / FA nanosystem with multi-enzyme activity and a preparation method thereof, as well as its application in lower limb ischemia.

[0040] The HMPB / FA nanosystem with multi-enzyme activity has the advantages of multi-enzyme activity, low preparation cost, low degree and high efficiency.

[0041] The technical solution of the present application will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0042] Embodiment 1, as Figure 7 As shown:

[0043] (1) Preparation of mesoporous Prussian blue PB: 4.5 g PVP and 198 mg K3[Fe(CN)6] were dissolved in 60 mL 0.01 M HCl solution and stirred magnetically until fully dissolved. The resulting mixed solution was transferred to an oven and reacted at 80 °C for 24 h. After the reaction, the mixture was cooled to room temperature, and the mesoporous PB nanoparticles were collected by centrifugation, washed with water and ethanol, and dried at 60 °C for 5 h to obtain mesoporous Prussian blue PB powder.

[0044] (2) Preparation of hollow mesoporous Prussian blue HMPB: 35 mg of PB and 175 mg of PVP were dissolved in 35 mL of 1.0 M HCl solution, stirred at room temperature for 4 h, and the resulting mixed solution was transferred to a hydrothermal reactor and reacted at 140 °C for 3 h; after the reaction, the mixture was cooled to room temperature, and HMPB nanoparticles were collected by centrifugation, washed with water and ethanol, and dried at 60 °C for 5 h to obtain hollow mesoporous Prussian blue HMPB powder;

[0045] (3) Preparation of FA solution and HMPB solution: 5 mg FA was added to 200 μL anhydrous ethanol and fully dissolved to obtain solution A; 5 mg HMPB was added to 5 mL ultrapure water and ultrasonically dispersed to obtain solution B;

[0046] (4) Preparation of HMPB / FA nanosystem with multi-enzyme activity: Solution A and solution B were mixed, stirred at 600 r / min for 24 h at room temperature in the dark, washed with water in sequence, then centrifuged at 11000 r / min for 20 min, and freeze-dried to obtain HMPB / FA nanosystem with multi-enzyme activity.

[0047] Figure 1 TEM image of the HMPB / FA nanosystem prepared in this embodiment; Figure 1 It can be seen that HMPB has a hollow mesoporous structure, and the loading of FA does not change the structure of HMPB; Figure 2 The infrared spectrum (a) and ultraviolet spectrum (b) of the sample prepared in this example show that the HMPB / FA nanosystem has typical infrared peaks and ultraviolet peaks of HMPB and FA, proving the successful preparation of the nanosystem.

[0048] In order to demonstrate the advantages of the technical solution provided by this application, a test example of the technical solution provided by this application is given below:

[0049] Test Example 1:

[0050] Example 1 of the present invention provides a method for testing the activity of multiple enzymes in a HMPB / FA nanosystem, the steps comprising:

[0051] The HMPB / FA nanosystems of different concentrations provided in Example 1 were placed in an acetic acid-sodium acetate buffer, and TMB and H2O2 were added. The reaction was allowed to proceed for 2 min, and the absorbance at 300-800 nm was measured. The POD mimetic enzyme catalyzed the oxidation reaction of TMB, which simulated the role of the POD enzyme in clearing peroxides in vivo.

[0052] The HMPB / FA nanosystems of different concentrations provided in Example 1 were placed in a detection solution prepared by sequentially adding riboflavin, NBT, and EDTA·Na2 to PBS, and the mixed solution was irradiated under a xenon lamp for 3 minutes, and the absorbance at the wavelength corresponding to the absorption peak of the sample before and after illumination was measured. This simulates the SOD enzyme capturing electrons from the excited riboflavin, thereby reducing NBT to a purple compound;

[0053] The HMPB / FA nanosystem provided in Example 1, as well as PB and HMPB, were placed in deionized water and ultrasonically homogenized, and H2O2 was added. The generation of bubbles was observed and recorded at 0-5 min, which simulated the reaction of CAT enzyme with H2O2 to generate water and oxygen;

[0054] The results of various enzyme activity tests are as follows Figure 3 As shown, from Figure 3 It can be seen that the HMPB / FA nanosystem provided by the present invention has excellent multi-type enzyme activities.

[0055] Test Example 2:

[0056] The cytotoxic activity of the HMPB / FA nanosystem provided in Example 1 on C2C12 and RAW264.7 cells was tested. The cytotoxicity test process included: after incubating the cells (3000 cells / well) in a 96-well plate for 24 hours, adding different concentrations of the HMPB / FA nanosystem thereto and incubating for another 24 hours, and detecting cell viability with a CCK8 reagent.

[0057] The experimental test results are as follows Figure 4 As shown, from Figure 4 It can be seen that the HMPB / FA nanosystem provided by the present invention has no obvious cytotoxicity to C2C12 and RAW264.7 cells.

[0058] Test Example 3:

[0059] In Example 4 of the present invention, the HMPB / FA nanosystem prepared in Example 1 was subjected to a mouse hind limb ischemia experiment test. The tested mice were 6-8 week old C57BL / 6 male mice. A mouse hind limb ischemia model was constructed by femoral artery ligation interruption method. The test indicators included changes in mouse weight and blood perfusion in the lower limbs.

[0060] Among them, the process of testing the weight change and lower limb blood perfusion of mice included randomly dividing the mice into 5 groups: sham operation group, PBS+operation group, HMPB+operation group, FA+operation group, and HMPB / FA+operation group. The drug administration group was given multi-point injection therapy (0.5 mg / kg) to the surgical muscle of mice on the 1st, 3rd, and 5th days, and the mice were killed on the 21st day. The weight of each mouse was recorded on the 1st, 3rd, 5th, 7th, 14th, and 21st days after surgery, and the blood perfusion of mice was recorded by laser Doppler blood flow imaging.

[0061] The results of the test of HMPB / FA nanosystem in treating lower limb ischemia in mice are shown in Figure 5 , 6 As shown in the figure, it can be seen that the HMPB / FA nanosystem provided by the present invention can improve the relevant symptoms of lower limb ischemia in mice (including weight loss and blood perfusion of mice), and is expected to become a new potential drug for the treatment of lower limb ischemia.

[0062] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a HMPB / FA nanosystem with multi-enzyme activity, characterized in that: The method comprises the following steps: Step (1), dissolving 4.5-5.0 g PVP and 198-220 mg K3[Fe(CN)6] in 60-65 mL 0.01 M HCl solution, and obtaining mesoporous Prussian blue PB powder by a hydrothermal method in an oven; Step (2), dissolving 30-35 mg of mesoporous Prussian blue PB and 150-175 mg of PVP in 30-35 mL of 1.0 M HCl solution, and performing a hydrothermal method in an oven to obtain a hollow mesoporous Prussian blue HMPB powder having multiple enzyme activities; Step (3), dissolving ferulic acid FA in anhydrous ethanol to obtain solution A, and dispersing HMPB in ultrapure water by ultrasonication to obtain solution B; Step (4), mixing solution A and solution B, stirring them evenly, washing them by centrifugation, and freeze-drying them to obtain a HMPB / FA nanosystem with multi-enzyme activity.

2. The method for preparing a HMPB / FA nanosystem with multi-enzyme activity according to claim 1, characterized in that: The stirring temperature in step (4) is 20 to 26° C., and the stirring time is 12 to 24 hours.

3. The method for preparing a HMPB / FA nanosystem with multi-enzyme activity according to claim 1, characterized in that: The time of centrifugal washing in step (4) is 15 to 20 minutes, and the rotation speed is 10000 to 11000 r / min.

4. The method for preparing a HMPB / FA nanosystem with multi-enzyme activity according to claim 1, characterized in that: The particle size of HMPB in the step (3) is 140 to 220 nm.

5. The method for preparing a HMPB / FA nanosystem with multi-enzyme activity according to claim 1, characterized in that: The mass ratio of FA to HMPB in the step (3) is 1:

1.

6. The HMPB / FA nanosystem with multi-enzyme activity prepared by the preparation method of claim 1, characterized in that: The HMPB / FA nanosystem with multiple enzyme activities uses HMPB as a carrier and is loaded with FA as a drug. The FA in the HMPB / FA nanosystem with multiple enzyme activities will not affect the activities of multiple enzymes of HMPB.

7. The HMPB / FA nanosystem with multi-enzyme activity according to claim 6, characterized in that: The loading rate of the FA is 32.43% to 36.71%.

8. The HMPB / FA nanosystem with multi-enzyme activity according to claim 6, characterized in that: The HMPB comprises multiple enzymes.

9. The HMPB / FA nanosystem with multi-enzyme activity according to claim 8, characterized in that: The multi-enzyme is one or a combination of catalase-like, superoxide dismutase-like and / or peroxidase-like.

10. The HMPB / FA nanosystem with multi-enzyme activity according to claim 6, characterized in that: The HMPB / FA nanosystem with multi-enzyme activity is a drug used for treating lower limb ischemia diseases.

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