Therapeutic method for preventing thrombosis by protecting endothelial cells

Abstract

The invention provides an application of catalase nanoparticles in preparation of a medicine for preventing or treating thrombus. A method for preparing the catalase nanoparticles comprises the following steps: 1) reacting dopamine with melanin to generate melanin modified by the dopamine; 2) preparing catalase with carboxyl activated by EDC; and 3) in the presence of NHS, mixing the melanin modified by dopamine with the catalase with carboxyl activated by EDC, stirring overnight, then adding amino methoxy polyethylene glycol (mPEG-NH2), and continuing to react for 3 hours to obtain a product, namely the melanin-dopamine-catalase nanoparticles. The catalase nanoparticles provided by the invention can maintain the biological activity of melanin and catalase for a long time, and can avoid the risk of bleeding when preventing or treating thrombus.

Description

TECHNICAL FIELD

[0001] The present application relates to the use of catalase nanoparticles, in particular in the prevention and treatment of thrombosis. BACKGROUND

[0002] Melanin (Mel) exists in most organisms as an endogenous polymer, showing good biocompatibility and biodegradability, and contains rich antioxidant groups, which is an effective antioxidant for treating a series of ROS-related diseases such as ischemic stroke, acute peritonitis, acute lung injury, etc. [1]. Currently, there have been related studies using Mel as a drug carrier, but its antioxidant treatment potential has not been fully explored [2]. In addition, directional migration in response to specific signals is very important in biological systems [3]. Catalase (CAT) coated liposomes prepared in this way have been confirmed to be able to move directionally to areas with higher substrate concentration, showing positive chemotaxis (Hofmeister effect), which can play a role in the directional transport of biological molecules and natural vesicles in a physiological environment, and achieve directional control of movement [4].

[0003] Currently, nanozymes, as a class of simulated enzymes with both unique properties of nanomaterials and catalytic functions, have attracted the attention of scientists [5]. However, we should also note that compared with natural enzymes, they may have problems of biocompatibility, excretion and metabolism, and do not have the advantage of high selectivity relative to natural enzymes [6]. Especially, the precise delivery of nanozymes can be a technical challenge.

[0004] Arterial thrombosis includes ischemic stroke and coronary artery embolism, and its incidence and mortality are positively correlated with age [7]. The risk of arterial thrombosis recurrence after treatment is very high, because with increasing age, the expression and activity of glutathione peroxidase decrease, leading to an increase in the production of reactive oxygen species (ROS) such as superoxide anion and H2O2, and the expression of inflammatory proteins mediated by them can further promote thrombosis [8]. Therefore, effective clearance of ROS and resistance to oxidative stress-mediated pathological processes play a key role in changing the vascular microenvironment and inhibiting thrombosis recurrence [9].

[0005] Antithrombotic drugs are divided into three categories: antiplatelet drugs, anticoagulants, and fibrinolytic agents. Because platelets are the main component in arterial thrombosis, antiplatelet drugs are the main means of prevention. The commonly used antithrombotic drugs in clinical practice are aspirin and clopidogrel, but all current antithrombotic drugs increase the risk of bleeding, because platelets and cross-linked fibrin are important components involved in protecting the vascular system from leaking blood

[10] . SUMMARY

[0006] In one aspect, provided herein is a use of a catalase nanoparticle in the preparation of a medicament for preventing or treating thrombosis, wherein the method of preparing the catalase nanoparticle comprises:

[0007] 1) reacting dopamine with melanin to produce dopamine-modified melanin;

[0008] 2) preparing carboxyl EDC-activated catalase; and

[0009] 3) mixing the dopamine-modified melanin with the carboxyl EDC-activated catalase in the presence of NHS, adding aminomethoxy polyethylene glycol (mPEG-NH2) after stirring overnight, continuing the reaction for 3 h, and obtaining the product melanin-dopamine-catalase nanoparticle.

[0010] In some embodiments, the ratio of the amount (wt) of melanin and dopamine used in the method is 2: 1.

[0011] In some embodiments, the ratio of the amount (wt) of melanin, dopamine, and catalase used in the method is 2: 1: 3.

[0012] In some embodiments, the ratio of the amount (wt) of melanin, dopamine, catalase, and mPEG-NH2 used in the method is 2: 1: 3: 1.

[0013] In some embodiments, step 1) is performed in a Tris-HCl buffer at pH 8.5.

[0014] In some embodiments, the molecular weight of the mPEG-NH2 used in step 3) is about 2000.

[0015] In some embodiments, step 3) is performed at pH 8.

[0016] In some embodiments, the method further comprises step 4): centrifuging the product of step 3) and collecting the precipitate.

[0017] In some embodiments, the method comprises: weighing 10 mg of melanin and dissolving it in 5 mL of Tris-HCl (0.1 M) with pH of 8.5, adding 5 mg of dopamine, stirring at 500 rpm for 3 h, then placing the obtained liquid in a 1000 Da dialysis bag and dialyzing overnight in an external PBS environment to obtain a liquid containing dopamine-modified melanin (MD); weighing 15 mg of catalase and dissolving it in PBS, stirring at 500 rpm, adding EDC to activate the carboxyl end of catalase after 3 h, adding the liquid containing MD and NHS and adjusting the pH to 8 after 20 min of continuous stirring, and stirring overnight; adding 5 mg of m-PEG-NH2, stirring for 3 h, and then centrifuging at 10000 rpm to obtain the melanin-dopamine-catalase nanoparticles (nanonized enzyme, MDCP).

[0018] In another aspect, the present disclosure provides a pharmaceutical composition comprising the catalase nanoparticles prepared by the above method and a pharmaceutically acceptable carrier.

[0019] The catalase nanoparticles provided herein can maintain the biological activity of melanin and catalase for a long time, and can avoid the risk of bleeding when preventing or treating thrombosis. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The synthesis process of MDCP of the present disclosure is shown in the schematic diagram, and the related characterization data are shown in (a) synthesis process of nanonized enzyme; (b) transmission electron microscopy results of MDCP; (c) element mapping face scanning results of MDCP; (d) transmission electron microscopy images of melanin and dopamine-modified melanin; (e) zeta potential of MDCP; (f) particle size distribution graph of MDCP; (g) surface potential graphs of melanin, dopamine-modified melanin, catalase and nanonized catalase, respectively; (h) MDCP catalase loading amount when two input amounts of catalase; (i) Fourier infrared spectra of melanin, dopamine, dopamine-modified melanin, catalase and nanonized catalase; (j) effects of different concentrations of catalase on the survival rate of HUVEC cells; (k) effects of different concentrations of MD on the survival rate of HUVEC cells; (l) effects of different concentrations of MDCP on the survival rate of HUVEC cells.

[0021] Figure 2The stability of MDCP and related performance data are shown. (a) Stability of MDCP surface potential; (b) Stability of MDCP particle size within 7 days of observation; (c) Inhibition rate of MDCP, CAT, MD on superoxide anion; (d) We detected the stability of hydrogen peroxidase enzyme activity of MD, CAT, MDCP; (e) Xanthine oxidase (XOD) can induce cells to produce reactive oxygen species, and the phagocytosis rate of MDCP by cells not induced by XOD is significantly lower than that of HUVEC incubated with xanthine oxidase. After XOD induction, the phagocytosis of HUVEC cells to MDCP and CAT is increased, and after comparison of MDCP with CAT of the same concentration, it is found that HUVEC phagocytizes the former faster, indicating that MDCP has a certain safety, i.e. promoting the phagocytosis of ROS-damaged HUVEC to MDCP. The figure is Figure 2 (f) Quantitative statistical graph of f; (f) Positive chemotaxis of nano hydrogen peroxidase to hydrogen peroxide, i.e. cells producing more ROS phagocytize MDCP faster and more.

[0022] Figure 3 Schematic diagram of MDCP preventing thrombosis.

[0023] Figure 4 MDCP protects endothelial cells by consuming excess ROS. (a) The control group is the normal control group, xanthine oxidase (XOD), hydrogen peroxidase plus 3-amino-1, 2, 4-triazole (CAT+blocker), dopamine modified melanin group (MD), hydrogen peroxidase group (CAT) and nano hydrogen peroxidase group (MDCP), wherein the control group and XOD group are only added with normal complete medium during treatment, and after 12 hours, except the control group, other groups are treated with 0.08 U / mL of xanthine oxidase overnight to observe whether the cells can produce ROS, and the DCFH-DA staining results show that the MDCP group can prevent the production of ROS and can protect endothelial cells; (b) The average fluorescence intensity statistical graph of DCFH-DA staining results is analyzed by image j; (c) The HUVEC cells are treated according to the foregoing method, and then stained with an apoptosis staining kit to observe the apoptosis of the cells, and it is found that MDCP has a protective effect on the cells; (d) The HUVEC cells are mixed with matrigel and then laid in eight wells, and treated according to the foregoing grouping and method, and the CellTracker TM Red staining results show that the number of cells in the XOD group is the least, and there is no difference in the number of cells between the MDCP and control groups, indicating that MDCP has a protective effect on the cells and can prevent the anokis of the cells; (e) is the CellTracker TMStatistical results of Red staining; (f) We planted cells in 96-well plates, and after the operation based on the method, we used CCK8 to detect the relative number of living cells and made statistics, and the results were consistent with the results of fluorescence staining. The number of living cells in the XOD and CAT+Blocker groups was the least, and it also showed that MDCP had a protective effect on cells and could prevent the anoikis of cells; (g) In addition to the apoptosis results of the cells, mitochondria is an important part of ROS production in cells. We studied the mitochondria of the cells. First, we detected the mitochondrial membrane potential, which is also an indicator of early apoptosis. The results showed that most of the mitochondria in the MDCP group presented in aggregate form, indicating that the number of apoptotic cells in this group was small; (h) In addition to observing the indicators of cell mitochondria, we also observed the morphology of endothelial cell mitochondria. After the above treatment, we can observe that the morphology of mitochondria in the XOD and CAT+Blocker groups is irregular, indicating that the state of the cells is not very good, which is consistent with the results of mitochondrial membrane potential.

[0024] Figure 5 MDCP mediated endothelial cell protection and prevented the release of corresponding inflammatory factors by removing excess ROS. (a) Flow cytometry results of HUVEC cell apoptosis, Figure 4 d is its statistical result; (b) After the above treatment, the ATP content of the cells, because ATP is an important indicator of oxidative phosphorylation in cells, if a large amount of ROS is produced in cells, the production of ATP will be relatively insufficient, which will lead to the continuous production of excess ROS in cells, and eventually lead to cell apoptosis; (c) Flow cytometry results of mitochondrial membrane potential, statistical chart is Figure 4 g; (d) After the above treatment, we collected the supernatant of the cells and detected the content of the inflammatory factor TNF-α in the cells; (e) The content of the inflammatory factor IL1-β in the cells; (f) The content of the inflammatory factor MMP-2 in the cells; (g) The content of the inflammatory factor MMP-9 in the cells; (h) Statistical results of Mitosox-Red staining, which is an indicator for detecting superoxide anion. The more superoxide anion in cells, the brighter the red fluorescence; (i) Confocal results of Mitosox-Red stained cells.

[0025] Figure 6In vivo study of MDCP preventing thrombosis by protecting endothelial cells.(a) Because our technology can prevent thrombosis while not affecting coagulation function, we did thrombelastography, and after 5 days of administration, we found that the amplitude of the aspirin and clopidogrel groups decreased; (b) The alpha angle decreased, suggesting that the aspirin and clopidogrel groups had a low coagulation bleeding risk compared to the other groups, while the MDCP and control groups had no statistical difference; (c) After completing the coagulation function evaluation, we studied the anti-thrombotic effect of MDCP. We observed the prevention of thrombosis on the MR. Four hours after the injection of MDCP into the tail vein for 5 consecutive days, we incubated the abdominal aorta with 10% ferric chloride for 5 minutes to observe whether the abdominal aorta thrombosis occurred to determine whether the prevention experiment was successful; (d) After processing in figure c, we scanned the abdominal aorta region of the rat with MR to observe whether thrombosis occurred. After repeated experiments on 7 rats in each group, we made statistics; (e) The results of MR detection of the abdominal aorta of rats showed that there was no thrombosis in the MDCP group of rats; (f) We cut the blood vessels along the sagittal plane and observed the intima of the blood vessels. Scanning electron microscopy showed that the endothelium of the control Saline group had damage and adhered to fibrin, red blood cells and platelets. The endothelium of each group was damaged, but the Saline group was the most severe, followed by the MD group; (g) We extracted the modeling abdominal aorta. We prepared the frozen section of the modeling abdominal aorta. DHE section showed that MDCP produced the least ROS; (h) We made statistics on the DHE fluorescence of g figure and found that the average fluorescence intensity of the Saline group was the largest; (i) Then we detected the hydrogen peroxide content of the tissue. It can be observed that the hydrogen peroxide content of the MDCP group is the lowest among other groups, which also indicates that MDCP has a good effect on removing hydrogen peroxide; (j) We extracted the ferric chloride modeling vascular tissue and detected the total sulfhydryl content of the tissue with a total sulfhydryl kit. The total sulfhydryl content is positively correlated with the antioxidant effect. We observed that the total sulfhydryl content of the MDCP group was the most, which also indicated that the group had a good antioxidant effect; (k) Then we observed two ELISA detection indicators related to vascular function. The ADMA indicator decreased in the MDCP detection, indicating that MDCP indeed has a protective effect on blood vessels; (l) L-arginine increased in the MDCP group, indicating that there is a byproduct L-arginine produced in the process of MDCP antioxidant, which also indicates the protective effect of MDCP; (m) After the above detection, we extracted the vascular tissue of MDCP and Saline to detect the metabolomics. The expression of dozens of amino acids, fatty acids including citrulline increased, indicating the protective effect of MDCP; (n) The metabolic indicators expressed differently in the MDCP and Saline groups. Their enriched pathways indicate that the MDCP group may be mainly related to the following three pathways: arginine biosynthesis, arginine and proline metabolism, and glutathione metabolism.

[0026] Figure 7 MDCP-mediated ROS scavenging can protect endothelial cells and prevent platelet activation. (a) After the foregoing studies, we collected the blood of rats, extracted the supernatant of the blood, and studied the apoptosis of endothelial cells. It can be observed that the endothelial microparticles representing endothelial cell apoptosis are the most in the Saline group; (b) On the pathological section, we can observe that the expression of proteins related to apoptosis in the Saline group is different, including increased expression of Caspase3, decreased expression of BCL-2, and no significant difference between BAX groups; after confirming that MDCP can protect endothelial cells, we detected two ELISA kits related to endothelial collagen exposure. We observed that the expression of tissue factor (TF) (c) vWF (d) in the MDCP group decreased compared with the Saline group, indicating that the protection of endothelial cells by MDCP can prevent the exposure of subendothelial collagen, thereby reducing the release of tissue factor and vWF; (e) In vivo, we evaluated the indicators of platelet activation and found that the activated platelets in the MDCP group were the least; statistically, the difference between the MDCP group and the Saline group was significant, p<0.0001, (f) In the platelet adhesion experiment, we found that the adhesion of platelets and leukocytes was the least in MDCP, which also indicated that MDCP can achieve the purpose of preventing platelet activation by protecting endothelial cells. DETAILED DESCRIPTION

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0028] The term "comprising" or "including" means including said elements, integers, or steps but does not exclude other elements, integers, or steps. In the present document, when using the term "comprising" or "including", it is intended that the elements, integers or steps stated in the claim are included, unless the context clearly indicates otherwise. For example, when referring to a method "comprising" several steps, it is intended that the method consist of these specific steps, unless the context clearly indicates otherwise.

[0029] The term "about" means any value within ±10% of a given value.

[0030] A "subject" includes an animal, such as a mammal, including, but not limited to, primates, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets. A subject can be male or female and can be any age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some examples, a subject refers to an individual in need of a diagnosis or treatment for a disease or condition. In some examples, a subject receiving a diagnosis or treatment can be a patient who has a condition associated with the diagnosis or treatment, or who is at risk of developing the condition. In particular examples, a subject is a human, such as a human patient. This term is often used interchangeably with "patient," "test subject," "treatment subject," and the like.

[0031] "Pharmaceutically acceptable carrier" as used with reference to a pharmaceutical composition refers to a solid or liquid diluent, filler, antioxidant, stabilizer, etc., that can be safely used in administration to humans and / or animals without undue toxicity, and is suitable for maintaining the integrity of the drug or active agent therein. Commonly used carriers include, but are not limited to, sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils such as castor oil, synthetic oils, polyols, alginic acid, phosphate buffered saline, emulsifiers, isotonic saline, and / or pyrogen-free water, and the like. The pharmaceutical compositions provided herein can be formulated into clinical acceptable dosage forms such as powders, injections, and the like. Any appropriate route of administration can be used to administer the pharmaceutical compositions of the present application to a subject, for example, oral, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, or via inhalation, transdermal, and the like.

[0032] "Prevention" refers to avoiding, reducing, or delaying the appearance of a particular disease or disease-related symptom in a subject, and such disease or disease-related symptom has not yet appeared prior to the administration of the relevant drug. "Prevention" does not require complete prevention of the appearance of a disease or disease-related symptom, for example, reducing the risk of a subject developing a particular disease or disease-related symptom after the administration of the relevant drug, or lessening the severity of the relevant symptom when it later appears, can both be considered as "preventing" the appearance or development of the disease. "Treatment" refers to alleviating, reducing, ameliorating, or inhibiting (e.g., arresting the development of) a disease that has already manifested or has occurred in a subject. In terms of a particular disease, "treatment" can include "curing" the disease, but in most cases does not require complete elimination of all symptoms thereof, for example, a subject can be considered to be treated if the administration of the relevant drug results in at least one symptom being reduced (e.g., the extent of a thrombus is reduced) or eliminated.

[0033] In the present application, we combine natural melanin with catalase, which can well solve the problem of biocompatibility, and by using Hofmeister effect, catalase can positively chemotaxis to the area where the substrate is more distributed, so as to achieve the purpose of precise drug delivery.

[0034] The method of the present application can prolong the blood circulation time of catalase by nanometerizing catalase, improve the biological safety of catalase, and reduce the production of inflammatory factors and protect cells by converting superoxide anion to hydrogen peroxide and converting hydrogen peroxide to water and oxygen.

[0035] In the method of the present application, since melanin has hydrophobicity, we use dopamine to react with natural melanin to enhance the water solubility of melanin and retain its natural properties; free form catalase has a short blood circulation time, and nanometerized catalase can prolong the blood circulation time and has good stability; the mild reaction of carbodiimide method can maximize the protection of the enzyme activity of catalase during preparation; the selected raw materials all have good biocompatibility, so the nanometerized enzyme has a relatively wide application range in biological medicine; the entire reaction is prepared without using organic solution, so the preparation method is safe; the prepared melanin-dopamine-catalase nanoparticles (nanometerized enzyme, MDCP) can accumulate in the lesion site with high hydrogen peroxide content by using the substrate specificity of catalase to hydrogen peroxide.

[0036] The present technology protects endothelial cells, prevents endothelial cell anoikis, and further prevents platelets from contacting collagen or vWF, thereby preventing platelet activation without affecting the function of platelets, so that thrombosis can be prevented without causing bleeding side effects.

[0037] In the cell experiment, we set the control group as the normal control group, xanthine oxidase (XOD), catalase plus 3-amino-1,2,4-triazole (CAT+blocker), dopamine-modified melanin group (MD), catalase group (CAT) and nanometerized catalase group (MDCP), except the control group, other groups are treated with xanthine oxidase overnight to produce ROS, and then washed with PBS, and then added according to the grouping. Then we did the experiment about the effect of MDCP on ROS consumption and the experiment of protecting endothelial cells.

[0038] We did the above verification in vivo, and added the relevant research of platelet activation to explore whether the protection of endothelial cells and the prevention of endothelial cell anoikis can prevent the activation of platelets, so as to ultimately prevent thrombosis. It was found that the positive chemotaxis of nano-catalase to hydrogen peroxide can make MDCP target the site of vascular injury, so as to ensure safety and a certain treatment efficiency; the first step of thrombosis is vascular injury, that is, endothelial cell injury, MDCP can prevent endothelial cell apoptosis by enriching in damaged endothelium and consuming excess ROS, and ultimately prevent thrombosis, which can not cause the side effect of bleeding.

[0039] The present application is further illustrated by the following specific examples.

[0040] Example 1 Preparation of MDCP

[0041] The preparation process is described in Figure 1 a.

[0042] 10 mg of melanin (Mel) (Sigma, item number M8631) was weighed and dissolved in 5 ml of Tris-HCl (0.1 M) with pH 8.5, 5 mg of dopamine was added and stirred at 500 rpm for 3 h, then the obtained liquid was placed in a 1000 Da dialysis bag and dialyzed in a PBS external environment overnight, the liquid was taken out, and melanin (MD) containing dopamine modification was obtained, and after freeze-drying, about 10 mg of MD was weighed. 15 mg of catalase was dissolved in PBS, stirred at 500 rpm, 3 h later, 1.15 mg (100 times molar excess, 1.2 mM) EDC (1-(3-dimethylaminopropyl)-3-ethyl carbodiimide) was added to activate the carboxyl end of catalase, and after continuous stirring for 20 min, MD and 0.345 mg of NHS (N-hydroxysuccinimide, 0.6 mM) were added, and the pH value was adjusted to 8, and after continuous stirring overnight, 5 mg of m-PEG2000-NH2 was added, stirred for 3 h, and then centrifuged at 10000 rpm to obtain the precipitate, which was melanin-dopamine-catalase nanoparticles (nano-enzyme, MDCP).

[0043] Example 2 Characterization and function of MDCP

[0044] The morphology of MDCP was observed by transmission electron microscopy, which was generally spherical with a diameter of about 100 to 200 nm Figure 1 b).

[0045] Elemental mapping face scanning (JEOL JEM-F200) was performed on MDCP, and iron and sulfur elements were observed, which were characteristic elements of catalase, indicating that catalase and melanin particles were successfully prepared into a nano-enzyme Figure 1 c).

[0046] The raw material melanin and the dopamine-modified melanin were observed by transmission electron microscopy, and the diameters of the two were not significantly different, indicating that the dopamine-modified melanin had little effect on the structure of the melanin Figure 1 d).

[0047] The potential of MDCP was detected, and it was found that it was mainly negative Figure 1 e), such a negative potential cannot increase the adsorption of the protein crown, and can increase the blood circulation time.

[0048] The particle size distribution of MDCP is shown in Figure 1 f.

[0049] The surface potential of melanin, dopamine-modified melanin, catalase, and nano-catalase (MDCP) was compared Figure 1 g).

[0050] MDCP was prepared according to the method described in Example 1, and the amount of catalase loaded in the prepared MDCP was compared when the two input amounts of catalase (15 mg and 10 mg) Figure 1 h). Therefore, we selected 15 mg of CAT as the amount of CAT used in the MDCP synthesis system, and calculated that the amount of 10 mg of MDCP loaded with CAT was about 2.2882 mg. In subsequent experiments, we used 2.2882 mg as the drug dosage of the CAT group, and 10 mg of MDCP as the control.

[0051] Fourier infrared detection was performed on melanin, dopamine, dopamine-modified melanin, catalase, and nano-catalase (MDCP), and the results are shown in Figure 1 i. As can be seen from the figure, the synthesis process of MDCP is a Schiff base reaction between the carbonyl group of melanin (1720.35) and the amino group of dopamine (N-H deformation vibration at 1617.46, C-H stretching vibration peak at 1502.3), and the generated MD increases the water solubility of melanin due to the presence of phenolic hydroxyl group (1393.16), while preserving the characteristics of melanin. Then CAT is connected to MD through the carbodiimide method. First, EDC activates the carboxyl terminal of CAT (1637.35), and then an amide bond is formed between the secondary amino group of MD (1643.76). Finally, m-PEG2000-NH2 is added to connect with the remaining carboxyl group of CAT, and MDCP is finally generated.

[0052] The effects of different concentrations of catalase on the survival rate of HUVEC cells, different concentrations of MD on the survival rate of human umbilical vein endothelial cells (HUVEC cells), and different concentrations of MDCP on the survival rate of HUVEC cells were detected, and the results are shown in Figure 1j-1l. Within a considerable concentration range, MDCP did not substantially affect cell viability.

[0053] The stability of MDCP surface potential was detected, and the results are shown in Figure 2 a. Within the 7-day observation period, there was no statistical difference in the surface potential of MDCP, indicating that the potential of MDCP was stable for 7 days.

[0054] The stability of the particle size of MDCP was detected within the 7-day observation period, wherein the average particle size on day 0 was about 144.067 nm, and no significant change in particle size was observed in the subsequent 7 days Figure 2 b).

[0055] Since melanin has the function of superoxide dismutase, we detected the inhibition rate of MD, CAT, and MDCP on superoxide anion, and this result indicates that the nano-catalase still retains the characteristics of MD Figure 2 c).

[0056] We detected the catalase enzyme activity stability of MD, CAT, and MDCP, and found that the nano-catalase (MDCP) has better enzyme activity stability than the free catalase, and the enzyme activity stability begins to decrease on the fifth day Figure 2 d).

[0057] The nano-catalase has a positive chemotaxis to hydrogen peroxide. We studied the phagocytosis of free catalase and nano-catalase by HUVEC treated or not treated with xanthine oxidase, and found that the nano-catalase is phagocytosed more by HUVEC than the free catalase, and after the HUVEC is treated with xanthine oxidase, i.e. after the production of superoxide anion and hydrogen peroxide, the phagocytosis of MDCP by HUVEC is accelerated, indicating that MDCP has a protective effect on cells, i.e. only the cells damaged by ROS will phagocytose more MDCP Figure 2 e, 2f).

[0058] The nano-catalase has a positive chemotaxis to hydrogen peroxide, and the damaged endothelial cells contain excessive superoxide anion and hydrogen peroxide, so MDCP can reach the damaged endothelial cells and be phagocytosed by them; MDCP consumes the ROS (superoxide anion and hydrogen peroxide) of the endothelial cells, protects the endothelial cells, prevents the anoikis of the endothelial cells, prevents the exposure of the collagen under the endothelium, and prevents the release of vWF under the endothelium, which prevents the activation and adhesion of platelets, and ultimately prevents the formation of thrombus.

[0059] During the entire prevention process, there is no direct impact on the function of platelets, so the coagulation system can be protected, and potential bleeding side effects can be prevented.

[0060] Example 3 MDCP protects endothelial cells by scavenging excess ROS

[0061] Figure 3 Schematic diagram of MDCP preventing thrombosis.

[0062] In Figure 4 a, the control group is the normal control group, and the xanthine oxidase group (XOD), the catalase plus 3-amino-1, 2, 4-triazole group (CAT+blocker), the dopamine-modified melanin group (MD), the catalase group (CAT), and the nano-catalase group (MDCP) are set up. Except for the control group, the other groups are treated with xanthine oxidase overnight to produce ROS in the cells. The DCFH-DA staining results show that the MDCP group can prevent the production of ROS and protect endothelial cells. Figure 4 b is a statistical chart of the average fluorescence intensity of the DCFH-DA staining results, analyzed by image j. The HUVEC cells are treated according to the foregoing method, and then stained with an apoptosis staining kit to observe the apoptosis of the cells. It is found that MDCP has a protective effect on the cells Figure 4 c). The HUVEC cells are mixed with Matrigel and then plated in eight-well dishes, treated according to the foregoing grouping and method, and the CellTracker TM Red staining results show that the number of cells in the XOD group is the least, and there is no difference in the number of cells between the MDCP and control groups, indicating that MDCP has a protective effect on the cells and can prevent the anoikis of the cells Figure 4 d). The statistical results of CellTracker TM Red staining are shown in Figure 4 e. We planted the cells in 96-well plates, and performed the basic operation according to the method of Figure 4 c. After that, we detected the relative number of living cells by CCK8 and made statistics, and the results were consistent with those of fluorescence staining. The number of living cells in the XOD and CAT+Blocker groups was the least, indicating that MDCP has a protective effect on the cells and can prevent the anoikis of the cells Figure 4 f). In addition to the apoptosis results of the cells, mitochondria are important sites for the production of ROS in cells. We studied the mitochondria of the cells. First, we detected the mitochondrial membrane potential, which is an early indicator of apoptosis. The results show that most of the mitochondria of the cells in the MDCP group present in the form of aggregates, indicating that the number of apoptotic cells in this group is small Figure 4g) In addition to the observation of the indicators of cell mitochondria, we also observed the morphology of endothelial cell mitochondria. After the above-mentioned treatment, we can observe that the mitochondria of the XOD group and the CAT+Blocker group are irregular in shape, indicating that the state of the cells is not very good, which is consistent with the result of the mitochondrial membrane potential Figure 4 h).

[0063] We found that MDCP mediates endothelial cell protection and prevents the release of corresponding inflammatory factors by removing excess ROS. The results are shown in Figure 5 . Figure 5 a shows the flow cytometry results of HUVEC cell apoptosis, Figure 4 d is the statistical result thereof. Figure 5 b shows the ATP content of the cells after the foregoing treatment, because ATP is an important indicator of cell oxidative phosphorylation. If the cells produce a large amount of ROS, the production of ATP will be relatively insufficient, which will lead to the continuous production of excess ROS by the cells, and eventually lead to cell apoptosis. Figure 5 c is the flow cytometry result of the mitochondrial membrane potential, and the statistical graph is Figure 4 g. After the foregoing treatment of the cells, we collected the supernatant of the cells to detect the content of the inflammatory factor TNF-α of the cells Figure 5 d), the content of the inflammatory factor IL1-β of the cells Figure 5 e), the content of the inflammatory factor MMP-2 of the cells Figure 5 f), and the content of the inflammatory factor MMP-9 of the cells Figure 5 g). The statistical result of Mitosox-Red staining is shown in Figure 5 h, which is an indicator for the detection of superoxide anions. The more superoxide anions the cells have, the brighter the red fluorescence is. The confocal result of Mitosox-Red stained cells is shown in Figure 5 i.

[0064] We conducted an in vivo study of MDCP preventing thrombosis by protecting endothelial cells. Our technology can prevent thrombosis without affecting coagulation function, so we did thromboelastography. After 5 days of drug administration, we found that the amplitude of the aspirin and clopidogrel groups was smaller Figure 6 a); the alpha angle was reduced Figure 6 b), indicating that the aspirin and clopidogrel groups had a low bleeding risk of coagulation compared with the other groups, and MDCP had no statistical difference from the control group. After evaluating the coagulation function, we studied the anti-thrombotic effect of MDCP. We observed the prevention of thrombosis on the MR. At 4 hours after the injection of MDCP into the tail vein for 5 consecutive days, we incubated the abdominal aorta of the rats with 10% ferric chloride for 5 min to observe whether there was thrombosis in the abdominal aorta, so as to determine whether the prevention experiment was successfulFigure 6 c). After the above treatment, we used MR to scan the abdominal aorta area of ​​the rats ( Figure 6 d) to observe whether there is thrombosis. After repeated experiments with 7 rats in each group, we made statistics. The results of MR examination of the abdominal aorta of rats are shown in Figure 6 In the figure, no thrombosis was observed in the MDCP group. We cut the blood vessels along the sagittal plane and observed the endothelium. Scanning electron microscopy revealed that the endothelium of the control group (Saline) was damaged and adhered with fibrin, red blood cells, and platelets. Endothelial damage was present in all groups, but was most severe in the Saline group, followed by the MD group ( Figure 6 f). We extracted the abdominal aorta from the model, made frozen sections of the abdominal aorta, and made DHE sections, which showed that MDCP produced the least ROS ( Figure 6 g). We performed statistics on the DHE fluorescence in Figure 6f and found that the average fluorescence intensity of the Saline group was the largest ( Figure 6 h). We then tested the hydrogen peroxide content in the tissues and found that the MDCP group had the lowest hydrogen peroxide content compared to the other groups, indicating that MDCP has a better effect in removing hydrogen peroxide ( Figure 6 i). We extracted vascular tissue from the ferric chloride model and used a total thiol kit to detect the total thiol content of the tissue. The total thiol content was positively correlated with the antioxidant effect. We observed that the MDCP group had the highest total thiol content, which also indicates that this group has a better antioxidant effect ( Figure 6 j). We then observed two ELISA test indicators related to vascular function. The ADMA index decreased during MDCP testing, indicating that MDCP does have a protective effect on blood vessels ( Figure 6 k); L-arginine production increased in the MDCP group, indicating that during the MDCP antioxidant process, a byproduct of L-arginine was produced, which also indirectly illustrates the protective effect of MDCP ( Figure 6 l). After completing the above tests, we extracted vascular tissues from MDCP and Saline and performed metabolomics tests. The expression of dozens of amino acids and fatty acids, including citrulline, increased, indicating the protective effect of MDCP ( Figure 6 m). Figure 7 Figure 4 shows the metabolic indicators with different expression between the MDCP and Saline groups. Their enriched pathways indicate that the effects of the MDCP group may be mainly related to these three pathways: arginine biosynthesis, arginine and proline metabolism, and glutathione metabolism.

[0065] We found that MDCP-mediated ROS removal can protect endothelial cells and prevent platelet activation. After the above studies, we collected blood from rats, extracted the blood supernatant, and studied the apoptosis of endothelial cells. It can be observed that the endothelial cell microparticles representing endothelial cell apoptosis are the most abundant in the Saline group ( Figure 7 a). In the pathological sections, we can observe that the expression of apoptosis-related proteins in the Saline group is different, including increased expression of Caspase3 and decreased expression of BCL-2. No significant difference was observed between the BAX groups ( Figure 7 b). After confirming that MDCP can protect endothelial cells, we used two ELISA kits for detection of subendothelial collagen exposure and observed that the expression of tissue factor (TF) and vWF in the MDCP group was lower than that in the Saline group ( Figure 7 c and 7d), indicating that MDCP protects endothelial cells by preventing the exposure of subendothelial collagen, thereby reducing the release of tissue factor and vWF. In vivo, we evaluated platelet activation indicators and found that the MDCP group had the least activated platelets. Statistical analysis showed that the MDCP group was significantly different from the Saline group (p < 0.0001). Figure 7 e). In the platelet adhesion experiment, we found that the adhesion between platelets and leukocytes was the least in the presence of MDCP, which also shows that MDCP can prevent platelet activation by protecting endothelial cells ( ​ f).

[0066] References:

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[0076]

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Claims

1. Use of catalase nanoparticles in the preparation of a medicament for preventing or treating thrombosis in a subject, wherein the method for preparing the catalase nanoparticles comprises: 1) allowing dopamine to react with melanin to generate dopamine-modified melanin; 2) preparing catalase with carboxyl groups activated by EDC; as well as 3) Mixing the dopamine-modified melanin with the catalase whose carboxyl group is activated by EDC in the presence of NHS, stirring overnight, adding aminomethoxy polyethylene glycol (mPEG-NH2), and continuing the reaction for 3 hours to obtain the product, which is melanin-dopamine-catalase nanoparticles.

2. The method according to claim 1, wherein the ratio of melanin to dopamine (by weight) is 2:

1.

3. The method according to claim 2, wherein the ratio of melanin, dopamine and catalase used in the method (by weight) is 2:1:

3.

4. The use according to claim 3, wherein the ratio (wt) of melanin, dopamine, catalase and mPEG-NH2 used in the method is 2:1:3:

1.

5. The method according to claim 1, wherein step 1) is carried out in a Tris-HCl buffer solution at pH 8.

5.

6. The method according to claim 1, wherein the molecular weight of the mPEG-NH2 used in step 3) is 2000.

7. The method according to claim 1, wherein step 3) is carried out at a pH of 8.

8. The use according to claim 1, wherein the method further comprises step 4): centrifuging the product of step 3) and collecting the precipitate.

9. The method according to claim 1, wherein the method comprises: 10 mg of melanin was weighed and dissolved in 5 mL of Tris-HCl (0.1 M) with a pH of 8.

5. 5 mg of dopamine was added and magnetically stirred at 500 rpm for 3 hours. The obtained liquid was placed in a 1000 Da dialysis bag and dialyzed overnight in a PBS external environment to obtain a liquid containing dopamine-modified melanin (MD); 15 mg of catalase was weighed and dissolved in PBS and stirred at 500 rpm. After 3 hours, EDC was added to activate the carboxyl end of the catalase. After continuous stirring for 20 minutes, the MD-containing liquid and NHS were added and the pH value was adjusted to 8. After continuous stirring overnight, 5 mg of m-PEG-NH2 was added. After stirring for 3 hours, the mixture was centrifuged at 10,000 rpm. The resulting precipitate was melanin-dopamine-catalase nanoparticles (nanoenzyme, MDCP).

10. A pharmaceutical composition comprising the catalase nanoparticles according to claim 1 and a pharmaceutically acceptable carrier.

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