CN105943497B - A kind of self-assembled nanometer hemostat, preparation method and application - Google Patents

Abstract

The invention discloses a self-assembled nanohemostatic agent, its preparation method and application. The nanohemostatic agent is composed of a hydrophilic cationic polymer and a carboxyl-containing hydrophobic compound, wherein the hydrophilic cationic polymer and carboxyl-containing The mass ratio of the hydrophobic compound is 1:0.1-1:5, and the particle diameter of the nano-hemostatic agent is 20nm-800nm. During preparation, the hydrophilic cationic polymer is dissolved in a strong polar solvent, the carboxyl-containing hydrophobic compound is dissolved in a water-miscible organic solvent, and then the organic solution in which the carboxyl compound is dissolved is slowly added to the polymer under the action of ultrasound. solution, put the mixed solution in a dialysis bag, and dialyze in deionized water under magnetic stirring to remove the organic solvent. The nanometer hemostatic agent can not only stop bleeding locally by dripping or smearing on tissues, but also prevent bleeding by injection. The nanometer hemostatic agent has simple synthesis, low price, simple and easy self-assembly method, easy removal of organic solvent, good stability and no immunogenicity.

Description

A self-assembled nanohemostatic agent, its preparation method and application

technical field

The invention relates to the field of nano-hemostatic materials, in particular to a self-assembled nano-hemostatic agent, a preparation method of the hemostatic agent and its application in preventing and treating body hemorrhage.

Background technique

In trauma and clinical operation, massive hemorrhage of the body is one of the main factors causing death. Blood clotting is the first step in wound healing after trauma. In the case of less severe damage, such as the destruction of some small blood vessels, the body will be stimulated to produce physiological hemostasis. But if it is a relatively serious body injury, a series of measures must be taken to achieve rapid and effective hemostasis. At present, the hemostasis methods commonly used in clinical operations and trauma treatment mainly include chemical hemostasis, heat hemostasis and mechanical hemostasis.

The chemical method mainly uses chemical reagents to change the coagulation activity of blood. For example, vasoconstrictors (thromboxane A2) can cause vasoconstriction and reduce blood flow, thereby promoting coagulation. Thermal coagulation hemostasis includes burning hemostasis and laser hemostasis. Mechanical hemostasis is mainly through pressing, ligation and using bandages to stop bleeding. At present, chemical and mechanical methods are combined to stop bleeding, and fibrin glue formed by fibrinogen, thrombin and calcium ions is often used as a platelet substitute. Nevertheless, this method is still subject to many limitations, such as some reagents are solid, can not reach the wound to play the hemostatic effect; some liquid hemostatic agents (such as cyanoacrylates) need to be in a dry environment to play the hemostatic effect; and some Hemostatic materials are prone to immunogenicity or graft-versus-host disease, causing secondary damage to the body, or their own survival time is short, so they need to be prepared immediately before use.

In order to overcome the above problems, some new hemostatic technologies have been gradually developed in recent years, including the use of functional liposomes, block copolymers, synthetic platelets, and nanomaterials. Among them, nanohemostatic agents have great clinical application value due to their good hemostatic effect and flexible use, and have attracted the attention of researchers. For example, some polypeptide self-assembled nanofibers can form a barrier similar to extracellular matrix, and at the same time decompose into natural amino acids, thereby promoting wound recovery and improving hemostasis efficiency. And studies have shown that carbon nanoparticles and Clottocytes nanoparticles can activate platelets and reduce the bleeding time of the body. A block copolymer of poly(lactic acid-glycolic acid) and polylysine (PLGA-PLL), through the introduction of polyethylene glycol (PEG) in which a distally bonded RGD short peptide is self-assembled to form nano This type of nanoparticle can activate platelets to promote blood coagulation, so it is called "artificial platelets". RGD binds to platelet receptor glycoprotein Ⅱb-Ⅲa and integrin α _v β ₃ , activates platelets and promotes platelet aggregation.

Although the above-mentioned research on nanohemostatic agents has made some progress, hemostasis through nanoparticles is still subject to many limitations, such as short circulation time in the body, nanotoxicity itself, irreversible damage to the body, and increased risk of thrombosis-related diseases. . In addition, the currently researched hemostatic agents still have problems such as poor storage stability, complex synthesis process, and high preparation cost.

Contents of the invention

The purpose of the present invention is to provide the composition and preparation method of a self-assembled nano-hemostat, and to verify the effect of the nano-hemostat in different bleeding models.

The present invention constructs a novel nanohemostatic agent by self-assembly mediated by multiple non-covalent bond forces (including electrostatic interaction, hydrogen bond interaction and hydrophobic interaction) between hydrophilic cationic polymers and carboxyl-containing hydrophobic compounds, by promoting small The constriction of blood vessels promotes platelet aggregation and the activation of the blood coagulation system, and combines with iron ions in red blood cells to form clots to reduce the bleeding time of the body. And because the surface of the nanoparticle is positively charged, it can interact and combine with negatively charged white blood cells, red blood cells, and platelets in the blood to form a thrombus, and finally exert its hemostatic effect, greatly improving the hemostatic efficiency. Compared with other hemostatic agents, the nano-hemostatic agent prepared by self-assembly of the present invention has many advantages, and thus has broad application prospects.

In view of this, the technical scheme adopted in the present invention is as follows: a self-assembled nano-hemostatic agent consists of a hydrophilic cationic polymer and a carboxyl-containing hydrophobic compound, wherein the mass of the hydrophilic cationic polymer and the carboxyl-containing hydrophobic compound The ratio is between 1:0.1 and 1:5, and the particle size of the nanohemostatic agent is between 20nm and 800nm. Wherein the hydrophilic cationic polymer is selected from polyethyleneimine (including branched polyethyleneimine and linear polyethyleneimine), poly(L-lysine) hydrobromide and poly(L-arginine) Hydrochloride, the molecular weight is between 500Da and 50000Da; the hydrophobic compound containing carboxyl group is selected from Cholic acid, Ursodeoxycholic acid, Chenodeoxycholic acid, Hyodeoxycholic acid ( Hyodeoxycholic acid), Lithocholic acid, Deodeoxycholic acid, Dehydrocholic acid, Ibuprofen, Ketoprofen, Fenoprofen , Flurbiprofen, Oxaprozin, Naproxen, Indomethacin, Sulindac, Etodolac, Mefenamic acid ( Mefenamic acid), Meclofenamic acid, Flufenamic acid, Tolfenamic acid, Lumiracoxib, Licofelone, Diflunisal ), Aspirin, Gemfibrozil, Ciprofloxacin, Norfloxacin and Ofloxacin.

The present invention also provides a preparation method of a self-assembled nanohemostatic agent, comprising the following steps: first dissolving a hydrophilic cationic polymer in a certain amount of strong polar solvent to obtain a polymer solution, dissolving a carboxyl-containing hydrophobic compound In a water-soluble organic solvent, the organic solution containing a carboxyl-containing hydrophobic compound is slowly added to the polymer solution under the action of ultrasound, and then the above-mentioned mixed solution is placed in a dialysis bag, and is dialyzed in deionized water under magnetic stirring to remove The self-assembled nanometer hemostatic agent can be obtained by using an organic solvent.

Wherein, the above-mentioned strong polar solvent is selected from deionized water, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-dimethylacetamide. The water-soluble organic solvent is selected from acetone, acetonitrile, ethanol, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, acetic acid.

The concentration of the above polymer solution is between 1 mg/mL and 100 mg/mL, and the concentration of the organic solution of the carboxyl-containing hydrophobic compound is between 1 mg/mL and 100 mg/mL.

The present invention further provides the application of the self-assembled nano-hemostat, including applying the nano-hemostat to the local tissue (including spraying, smearing or externally applying it to the tissue), or using the nano-hemostat for intravenous injection to achieve hemostasis or prevention of bleeding. purpose of bleeding.

The present invention has the following advantages:

1) The hydrophilic cationic polymers used in the present invention are all commercial products, which are simple to synthesize and relatively cheap, so it is easy to realize the industrialization of corresponding preparations.

2) The self-assembly method adopted in the present invention is simple and easy, and the organic solvent used is easy to remove, which ensures the feasibility and safety of the final application of the nanohemostatic agent.

3) The particle size of the nanohemostatic agent prepared by the present invention can be regulated by the preparation process parameters.

4) The nanohemostatic agent prepared by the present invention is a liquid, which is flexible and convenient to use.

5) The nanohemostatic agent prepared by the present invention has good stability and no immunogenicity, which ensures the safety in vivo.

6) The nanohemostatic agent prepared by the present invention can be applied locally or injected intravenously.

Description of drawings

Figure 1 is a transmission electron microscope image of nanoparticles with a mass ratio between 1:1 and 1:4 prepared by self-assembly of branched polyethyleneimine with a molecular weight of 25,000 Da and ursodeoxycholic acid.

Fig. 2 is a phase-contrast microscope image of platelet aggregation promoted by the blank control group (0.9% normal saline) and the treatment group (nanohemostatic agent), where the scale bar is 10 μm.

Figure 3 is a confocal image of platelet aggregation promoted by the blank control group (0.9% normal saline) and the treatment group (nanohemostatic agent), where the scale bar is 5 μm.

Figure 4 is a section of femoral artery with intravenous injection of A (0.9% normal saline), B (Cy5-labeled ursodeoxycholic acid/polyethyleneimine nanoparticles, no treatment) and C (Cy5-labeled ursodeoxycholic acid) into the femoral artery of rats. Cholic acid/polyethyleneimine nanoparticles, local puncture of a section of femoral artery) solution, in vivo imaging to observe blood agglutination.

Fig. 5 shows that the nanohemostatic agent prepared by branched polyethyleneimine with a molecular weight of 25000Da and ursodeoxycholic acid in a mass ratio of 1:1 to 1:4 has the effect on mouse tail, rat femoral artery, liver and intravenous injection of nanometer Effect of hemostatic agents on the bleeding time of the femoral artery.

Fig. 6 is the effect of different concentrations (1, 2 and 4mg/mL) of nanohemostatic agents on mouse tail, rat femoral artery, liver, and the bleeding time of femoral artery after intravenous injection of nanohemostatic agents.

Fig. 7 is different polyethylenimine (molecular weight is the branched polyethyleneimine PEI1800 of molecular weight 1800Da and molecular weight is the linear polyethyleneimine PEI10000 of 10000Da) and ursodeoxycholic acid preparation nano-hemostatic agent to mouse tail, rat Effect of femoral artery, liver, and femoral artery bleeding time after intravenous administration of nanohemostats.

Detailed ways

The content of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the embodiments of the present invention are only used to illustrate the present invention and not to limit the present invention. Without departing from the technical idea of the present invention, various replacements and changes can be made according to common technical knowledge and conventional means in the field. All should be included within the scope of the present invention.

The present invention will be described in detail below in conjunction with non-limiting examples. During the self-assembly process, the particle size of the nanohemostatic agent can be controlled between 20nm and 800nm.

Example 1

Dissolve 5 mg of branched polyethyleneimine with a molecular weight of 25,000 Da in 5 mL of deionized water, and the concentration of the polymer solution is 1 mg/mL; dissolve 25 mg of ursodeoxycholic acid in 1 mL of dimethyl sulfoxide, in which ursodeoxy The concentration of cholic acid is 25 mg/mL; under the action of ultrasonic waves, slowly add 1 mL of ursodeoxycholic acid in acetone solution to the polymer solution; Dialyze in deionized water at a temperature of 20°C, and replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing a self-assembled nanohemostatic agent, and the particle size of the nanohemostatic agent is between 200 and 500nm.

Example 2

Dissolve 10 mg of branched polyethyleneimine with a molecular weight of 25,000 Da in 1 mL of dimethyl sulfoxide, and the concentration of the polymer solution is 10 mg/mL; dissolve 1 mg of cholic acid in 1 mL of acetonitrile, where the concentration of cholic acid is 1 mg/mL mL; under the action of ultrasonic waves, slowly add 1 mL of acetonitrile solution of cholic acid into the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 7000 Da, dialyze in deionized water under magnetic stirring, at a temperature of 20 °C, every Replace the deionized water in 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agent, the particle size of the nanohemostatic agent is between 200-400nm.

Example 3

Dissolve 10 mg of branched polyethyleneimine with a molecular weight of 25000 Da in 1 mL of N,N-dimethylformamide, and the concentration of the polymer solution is 10 mg/mL; dissolve 2 mg of chenodeoxycholic acid in 1 mL of ethanol, where The concentration of chenodeoxycholic acid is 2 mg/mL; under the action of ultrasonic waves, slowly add 1 mL of chenodeoxycholic acid ethanol solution into the polymer solution; Dialyze in deionized water at a temperature of 20°C, and replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agent, and the particle size of the nanohemostatic agent is between 150-300nm.

Example 4

Dissolve 10 mg of branched polyethyleneimine with a molecular weight of 25,000 Da in 1 mL of N,N-dimethylacetamide, and the concentration of the polymer solution is 10 mg/mL; dissolve 5 mg of hyodeoxycholic acid in 1 mL of dimethyl sulfoxide , wherein the concentration of hyodeoxycholic acid is 5 mg/mL; under the action of ultrasonic waves, slowly add 1 mL of dimethyl sulfoxide solution of hyodeoxycholic acid into the polymer solution; In the bag, dialyze in deionized water under magnetic stirring at a temperature of 20°C, and replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agent, the particle size of the nanohemostatic agent is between 100- Between 300nm.

Example 5

Dissolve 10 mg of branched polyethyleneimine with a molecular weight of 1800 Da in 1 mL of N,N-dimethylacetamide, and the concentration of the polymer solution is 10 mg/mL; dissolve 10 mg of lithocholic acid in 1 mL of N,N-dimethylacetamide In dimethyl formamide, the concentration of lithocholic acid is 10 mg/mL; under the action of ultrasonic waves, slowly add 1 mL of lithocholic acid N,N-dimethylformamide solution into the polymer solution; In a dialysis bag with a molecular weight of 3500Da, dialyze in deionized water under magnetic stirring, at a temperature of 20°C, and replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agent, nanohemostatic agent The particle size is between 100-200nm.

Example 6

Dissolve 10 mg of branched polyethyleneimine with a molecular weight of 800 Da in 1 mL of DMSO, and the concentration of the polymer solution is 10 mg/mL; dissolve 20 mg of deoxycholic acid in 1 mL of DMSO, in which The acid concentration is 20 mg/mL; under the action of ultrasonic waves, slowly add 1 mL of deoxycholic acid dimethyl sulfoxide solution into the polymer solution; Dialyze in deionized water at a temperature of 20°C, and replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agents. The particle size of nanohemostatic agents is between 200-300nm.

Example 7

Dissolve 10 mg of branched polyethyleneimine with a molecular weight of 500 Da in 1 mL of dimethyl sulfoxide, and the concentration of the polymer solution is 10 mg/mL; dissolve 30 mg of dehydrocholic acid in 1 mL of dimethyl sulfoxide, wherein The concentration of hydrocholic acid is 30 mg/mL; under the action of ultrasound, slowly add 1 mL of dehydrocholic acid dimethyl sulfoxide solution into the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 3500 Da, and magnetic Dialyze in deionized water under stirring at a temperature of 20°C, and replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agent, and the particle size of the nanohemostatic agent is between 150-250nm.

Example 8

Dissolve 10 mg of linear polyethyleneimine with a molecular weight of 2500 Da in 1 mL of deionized water, the concentration of the polymer solution is 10 mg/mL; dissolve 20 mg of ibuprofen in 1 mL of acetone, and the concentration of ibuprofen is 20 mg/mL; Under the action of ultrasonic waves, slowly add 1 mL of ibuprofen in acetone solution to the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 3500 Da, dialyze in deionized water under magnetic stirring, at a temperature of 20 ° C, every 30 min Replace the deionized water; collect the dialysate after 24 hours to obtain an aqueous solution containing the self-assembled nanohemostatic agent, and the particle size of the nanohemostatic agent is between 200-300nm.

Example 9

Dissolve 10 mg of linear polyethyleneimine with a molecular weight of 5000 Da in 1 mL of deionized water, and the concentration of the polymer solution is 10 mg/mL; dissolve 20 mg of ketoprofen in 1 mL of acetonitrile, wherein the concentration of ketoprofen is 20 mg/mL; Under the action of ultrasonic waves, slowly add 1 mL of ketoprofen acetonitrile solution into the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 3500 Da, dialyze in deionized water under magnetic stirring, at a temperature of 20 ° C, every 30 min Replace the deionized water; collect the dialysate after 24 hours to obtain an aqueous solution containing the self-assembled nanohemostatic agent, and the particle size of the nanohemostatic agent is between 100-200nm. .

Example 10

20 mg of linear polyethyleneimine with a molecular weight of 5000 Da was dissolved in 1 mL of deionized water, and the concentration of the polymer solution was 20 mg/mL; 60 mg of fenoprofen was dissolved in 1 mL of dimethyl sulfoxide, wherein the concentration of fenoprofen The concentration is 60 mg/mL; under the action of ultrasonic waves, slowly add 1 mL of fenoprofen dimethyl sulfoxide solution into the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 3500 Da, and remove Dialyze in deionized water at a temperature of 20°C, and replace deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agents, and the particle size of nanohemostatic agents is between 200-350nm.

Example 11

Dissolve 20 mg of linear polyethyleneimine with a molecular weight of 20,000 Da in 1 mL of deionized water, and the concentration of the polymer solution is 20 mg/mL; dissolve 100 mg of flurbiprofen in 1 mL of dimethyl sulfoxide, where the concentration of flurbiprofen The concentration is 100 mg/mL; under the action of ultrasonic waves, slowly add 1 mL of flurbiprofen dimethyl sulfoxide solution into the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 3500 Da, and remove Dialyze in deionized water at a temperature of 20°C, and replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agents. The particle size of nanohemostatic agents is between 300-450nm.

Example 12

Dissolve 20 mg of linear polyethyleneimine with a molecular weight of 10,000 Da in 1 mL of deionized water, and the concentration of the polymer solution is 20 mg/mL; dissolve 80 mg of oxaprozin in 1 mL of dimethyl sulfoxide, of which The concentration is 80 mg/mL; under the action of ultrasound, slowly add 1 mL of oxaprozin dimethyl sulfoxide solution into the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 3500 Da, and remove Dialyze in deionized water at a temperature of 20°C, and replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agents. The particle size of nanohemostatic agents is between 300-480nm.

Example 13

50 mg of branched polyethyleneimine with a molecular weight of 25000 Da was dissolved in 1 mL of deionized water, and the concentration of the polymer solution was 50 mg/mL; 50 mg of naproxen was dissolved in 1 mL of dimethyl sulfoxide, and the concentration of naproxen was 50 mg/mL; under the action of ultrasonic waves, slowly add 1 mL of naproxen dimethyl sulfoxide solution into the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 3500 Da, and dissolve it in deionized water under magnetic stirring For dialysis, the temperature is 20°C, and the deionized water is replaced every 30 minutes; after 24 hours, the dialysate is collected to obtain an aqueous solution containing a self-assembled nanohemostatic agent, and the particle size of the nanohemostatic agent is between 100-200nm.

Example 14

80 mg of branched polyethyleneimine with a molecular weight of 1800 Da was dissolved in 1 mL of deionized water, and the concentration of the polymer solution was 80 mg/mL; 40 mg of indomethacin was dissolved in 1 mL of N,N-dimethylacetamide, wherein The concentration of indomethacin is 40 mg/mL; under the action of ultrasonic waves, slowly add 1 mL of indomethacin N,N-dimethylacetamide solution into the polymer solution; In the dialysis bag, dialyze in deionized water under magnetic stirring at a temperature of 20°C, and replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agent, the particle size of the nanohemostatic agent is 150 Between -250nm.

Example 15

Dissolve 100 mg of branched polyethyleneimine with a molecular weight of 50,000 Da in 1 mL of deionized water, and the concentration of the polymer solution is 100 mg/mL; dissolve 100 mg of sulindac in 1 mL of dimethyl sulfoxide, wherein the concentration of sulindac 100 mg/mL; under the action of ultrasonic waves, slowly add 1 mL of sulindac dimethyl sulfoxide solution into the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 3500 Da, and dissolve it in deionized water under magnetic stirring For dialysis, the temperature is 20°C, and the deionized water is replaced every 30 minutes; after 24 hours, the dialysate is collected to obtain an aqueous solution containing a self-assembled nanohemostatic agent, and the particle size of the nanohemostatic agent is between 100-200nm.

Example 16

10mg of branched polyethyleneimine with a molecular weight of 1800Da was dissolved in 1mL of dimethyl sulfoxide, and the concentration of the polymer solution was 10mg/mL; 30mg of etodolac was dissolved in 1mL of acetone, and the concentration of etodolac was 30 mg/mL; under the action of ultrasonic waves, slowly add 1 mL of etodolac acetone solution into the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 3500 Da, and dialyze in deionized water under magnetic stirring at a temperature of 20 °C , replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agent, the particle size of the nanohemostatic agent is between 150-250nm.

Example 17

Dissolve 10 mg of branched polyethyleneimine with a molecular weight of 1800 Da in 1 mL of dimethyl sulfoxide, and the concentration of the polymer solution is 10 mg/mL; dissolve 25 mg of mefenamic acid in 1 mL of acetonitrile, wherein the concentration of mefenamic acid is 25 mg/mL; under the action of ultrasonic waves, slowly add 1 mL of mefenamic acid in acetonitrile solution to the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 3500 Da, dialyze in deionized water under magnetic stirring, and the temperature is 20 °C , replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agent, the particle size of the nanohemostatic agent is between 150-250nm.

Example 18

10 mg of branched polyethyleneimine with a molecular weight of 25000 Da was dissolved in 1 mL of dimethyl sulfoxide, and the concentration of the polymer solution was 10 mg/mL; 25 mg of meclofenamic acid was dissolved in 1 mL of dimethyl sulfoxide, wherein The concentration of meclofenamic acid is 25mg/mL; under the action of ultrasonic waves, slowly add 1mL of meclofenamic acid dimethyl sulfoxide solution into the polymer solution; put the above mixed solution in a dialysis bag with a molecular weight cut-off of 3500Da Dialyze in deionized water under magnetic stirring at a temperature of 20°C, and replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agent, the particle size of nanohemostatic agent is 140-200nm between.

Example 19

Dissolve 10 mg of poly(L-lysine) hydrobromide with a molecular weight of 2000 Da in 1 mL of deionized water, and the concentration of the polymer solution is 10 mg/mL; dissolve 20 mg of flufenamic acid in 1 mL of dimethyl sulfoxide , wherein the concentration of flufenamic acid is 20 mg/mL; under the action of ultrasonic waves, slowly add 1 mL of flufenamic acid dimethyl sulfoxide solution into the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 3500 Da Dialyze in deionized water under magnetic stirring at a temperature of 20°C, and replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agent, the particle size of nanohemostatic agent is 170-230nm between.

Example 20

10 mg of poly(L-lysine) hydrobromide with a molecular weight of 5000 Da was dissolved in 1 mL of deionized water, and the concentration of the polymer solution was 10 mg/mL; 10 mg of trofenamic acid was dissolved in 1 mL of dimethyl sulfoxide, Wherein the concentration of trofenamic acid is 10 mg/mL; under the action of ultrasonic waves, 1 mL of trofenamic acid dimethyl sulfoxide solution is slowly added to the polymer solution; Dialyze in deionized water under stirring at a temperature of 20°C, and replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing a self-assembled nanohemostatic agent, and the particle size of the nanohemostatic agent is between 200-400nm.

Example 21

Dissolve 10 mg of poly(L-lysine) hydrobromide with a molecular weight of 15,000 Da in 1 mL of deionized water, and the concentration of the polymer solution is 10 mg/mL; dissolve 15 mg of lumiracoxib in 1 mL of dimethyl sulfoxide , wherein the concentration of lumiracoxib is 15 mg/mL; under the action of ultrasound, slowly add 1 mL of dimethyl sulfoxide solution of lumiracoxib into the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 7000Da Dialyze in deionized water under magnetic stirring at a temperature of 20°C, and replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing a self-assembled nanohemostatic agent, and the particle size of the nanohemostatic agent is 180-230nm between.

Example 22

Dissolve 10 mg of poly(L-arginine) hydrochloride with a molecular weight of 15000 Da in 1 mL of deionized water, and the concentration of the polymer solution is 10 mg/mL; The concentration of Feilong is 15 mg/mL; under the action of ultrasonic waves, slowly add 1 mL of dimethyl sulfoxide solution of Likfeilong into the polymer solution; Dialyze in deionized water at a temperature of 20°C, and replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agents. The particle size of nanohemostatic agents is between 230-300nm.

Example 23

20 mg of poly(L-arginine) hydrochloride with a molecular weight of 15000 Da was dissolved in 1 mL of deionized water, and the concentration of the polymer solution was 20 mg/mL; 20 mg of diflunisal was dissolved in 1 mL of dimethyl sulfoxide, The concentration of diflunisal is 20 mg/mL; under the action of ultrasonic waves, slowly add 1 mL of diflunisal dimethyl sulfoxide solution into the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 7000 Da , dialyze in deionized water under magnetic stirring, temperature 20°C, replace deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agent, the particle size of nanohemostatic agent is between 300-400nm between.

Example 24

10 mg of poly(L-arginine) hydrochloride with a molecular weight of 15000 Da was dissolved in 1 mL of deionized water, and the concentration of the polymer solution was 10 mg/mL; 50 mg of aspirin was dissolved in 1 mL of dimethyl sulfoxide, wherein aspirin The concentration is 50 mg/mL; under the action of ultrasonic waves, slowly add 1 mL of aspirin dimethyl sulfoxide solution into the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 7000 Da, and dialyze in deionized water under magnetic stirring , the temperature is 20°C, and the deionized water is replaced every 30 minutes; after 24 hours, the dialysate is collected to obtain an aqueous solution containing self-assembled nanohemostatic agent, and the particle size of the nanohemostatic agent is between 200-300nm.

Example 25

Dissolve 10 mg of branched polyethyleneimine with a molecular weight of 25,000 Da in 1 mL of deionized water, and the concentration of the polymer solution is 10 mg/mL; dissolve 20 mg of gemfibrozil in 1 mL of dimethyl sulfoxide, where gemfibrozil The concentration is 20 mg/mL; under the action of ultrasound, slowly add 1 mL of gemfibrozil dimethyl sulfoxide solution into the polymer solution; Dialyze in deionized water at a temperature of 20°C, and replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agents, and the particle size of nanohemostatic agents is between 180-220nm.

Example 26

Dissolve 10 mg of branched polyethyleneimine with a molecular weight of 25,000 Da in 1 mL of deionized water, and the concentration of the polymer solution is 10 mg/mL; dissolve 10 mg of ciprofloxacin in 1 mL of acetic acid, wherein the concentration of ciprofloxacin is 10 mg /mL; Under the action of ultrasonic waves, slowly add 1 mL of ciprofloxacin acetic acid solution into the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 7000 Da, and dialyze in deionized water under magnetic stirring at a temperature of 20 °C , replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agent, the particle size of the nanohemostatic agent is between 180-250nm.

Example 27

10 mg of branched polyethyleneimine with a molecular weight of 25000 Da was dissolved in 1 mL of deionized water, and the concentration of the polymer solution was 10 mg/mL; 15 mg of norfloxacin was dissolved in 1 mL of acetic acid, and the concentration of norfloxacin was 15 mg /mL; Under the action of ultrasonic waves, slowly add 1 mL of norfloxacin acetic acid solution into the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 7000 Da, dialyze in deionized water under magnetic stirring, and the temperature is 20 °C , replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agent, the particle size of the nanohemostatic agent is between 200-260nm.

Example 28

Dissolve 10 mg of branched polyethyleneimine with a molecular weight of 25000 Da in 1 mL of deionized water, and the concentration of the polymer solution is 10 mg/mL; dissolve 20 mg ofloxacin in 1 mL of acetic acid, and the concentration of ofloxacin is 20 mg /mL; Under the action of ultrasonic waves, slowly add 1 mL ofloxacin acetic acid solution into the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 7000 Da, and dialyze in deionized water under magnetic stirring at a temperature of 20 °C , replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agent, the particle size of the nanohemostatic agent is between 180-160nm.

Example 29

Dissolve 10 mg of branched polyethyleneimine with a molecular weight of 25,000 Da in 1 mL of dimethyl sulfoxide, and the concentration of the polymer solution is 10 mg/mL; dissolve 15 mg of indomethacin in 1 mL of dimethyl sulfoxide, where indomethacin The concentration of indomethacin is 15 mg/mL; under the action of ultrasound, slowly add 1 mL of indomethacin dimethyl sulfoxide solution into the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 3500 Da, magnetic Dialyze in deionized water under stirring at a temperature of 20°C, and replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agent, the particle size of the nanohemostatic agent is between 20-60nm.

Example 30

Dissolve 10 mg of branched polyethyleneimine with a molecular weight of 25,000 Da in 1 mL of deionized water, and the concentration of the polymer solution is 10 mg/mL; dissolve 20 mg of dehydrocholic acid in 1 mL of dimethyl sulfoxide, wherein dehydrocholic acid The concentration is 20mg/mL; under the action of ultrasonic waves, slowly add 1mL of dehydrocholic acid dimethyl sulfoxide solution into the polymer solution; Dialyze in deionized water at a temperature of 20°C, and replace the deionized water every 30 minutes; collect the dialysate after 24 hours to obtain an aqueous solution containing self-assembled nanohemostatic agents. The particle size of nanohemostatic agents is between 500-600nm.

Example 31

Dissolve 10 mg of branched polyethyleneimine with a molecular weight of 25,000 Da in 1 mL of deionized water, and the concentration of the polymer solution is 10 mg/mL; dissolve 30 mg of lithocholic acid in 1 mL of dimethyl sulfoxide, and the concentration of lithocholic acid is 30 mg/mL; under the action of ultrasound, slowly add 1 mL of lithocholic acid dimethyl sulfoxide solution into the polymer solution; place the above mixed solution in a dialysis bag with a molecular weight cut-off of 7000 Da, and dissolve it in deionized water under magnetic stirring For dialysis, the temperature is 20°C, and the deionized water is replaced every 30 minutes; after 24 hours, the dialysate is collected to obtain an aqueous solution containing a self-assembled nanohemostatic agent, and the particle size of the nanohemostatic agent is between 700-800nm.

The transmission electron microscope image of nanoparticles in the nanohemostatic agent prepared according to the above method is shown in FIG. 1 .

Fig. 2 is a phase-contrast microscope image of platelet aggregation promoted by the blank control group (0.9% normal saline) and the treatment group (nanohemostatic agent), where the scale bar is 10 μm. Take blood from Sprague-Dawley male rats (230-250g) after anesthesia, centrifuge at 1000rpm for 10min at room temperature, take the supernatant to obtain platelet-rich plasma (PRP), take 100μL PRP and put it in a 96-well plate, add 10μL ursodeoxycholic acid (UDCA) and molecular weight of 25000Da branched polyethyleneimine self-assembly (mass ratio of 2:1) nanoparticle solution as a precoagulation stimulus (Fig. 2 experimental group) or add normal saline as a blank control (Fig. 2 blank control group ). The sample was fixed by adding an equal volume of 2% glutaraldehyde for 1 h, and after incubation at room temperature for 10 min, the aggregation state of platelets was observed under a phase-contrast microscope (Fig. 2).

Figure 3 is a confocal image of platelet aggregation promoted by the blank control group (0.9% normal saline) and the treatment group (nanohemostatic agent), where the scale bar is 5 μm. Precisely measure 1mL of 20mg/mL branched polyethyleneimine PEI (MW=25000) in DMSO, add 1mL of 2.36mg/mL Cy5NHS ester in DMSO, and react at 40°C for 12 hours in the dark to obtain PEI-Cy5 solution . Then, absorb 1 mL of PEI-Cy5 solution and mix with 1 mL of 20 mg/mL UDCA in DMSO, place in a dialysis bag with a molecular weight of 3500, and dialyze with pure water for 24 hours to obtain Cy5-labeled nanoparticles. Add 500 μL of PRP to 3 mL of THB (bench-top solution), label with 50 μL of CD61/FITC, incubate at room temperature for 40 min, centrifuge at 1600 g for 5 min, and dilute with 1.5 mL of PPP to obtain reconstituted platelets. Take 150 μL of reconstituted platelets and add 15 μL of Cy5-modified PEI/UDCA (10:20) nanoparticle solution or 15 μL of THB as a blank control, then add 15 μL of formaldehyde to fix; confocal observation of platelet aggregation state (Figure 3).

Figure 4 is a section of femoral artery with intravenous injection of A (0.9% normal saline), B (Cy5-labeled ursodeoxycholic acid/polyethyleneimine nanoparticles, no treatment) and C (Cy5-labeled ursodeoxycholic acid) into the femoral artery of rats. Cholic acid/polyethyleneimine nanoparticles, local puncture of a section of femoral artery) solution, in vivo imaging to observe blood agglutination. After Sprague-Dawley male rats (230-250g) were anesthetized, 0.5mL Cy5-PEI-UDCA nanoparticles or normal saline were injected into the tail vein, and the femoral artery of the rat was exposed after 5min, and the femoral artery was punctured with a needle of 0.45mm. After hemostasis, the femoral artery was cut to observe the fluorescence distribution.

Fig. 5 shows that the nanohemostatic agent prepared by branched polyethyleneimine with a molecular weight of 25000Da and ursodeoxycholic acid in a mass ratio of 1:1 to 1:4 has the effect on mouse tail, rat femoral artery, liver and intravenous injection of nanometer Effect of hemostatic agents on the bleeding time of the femoral artery.

Fig. 6 is the effect of different concentrations (1, 2 and 4mg/mL) of nanohemostatic agents on mouse tail, rat femoral artery, liver, and the bleeding time of femoral artery after intravenous injection of nanohemostatic agents.

Fig. 7 is different polyethylenimine (molecular weight is the branched polyethyleneimine PEI1800 of molecular weight 1800Da and molecular weight is the linear polyethyleneimine PEI10000 of 10000Da) and ursodeoxycholic acid preparation nano-hemostatic agent to mouse tail, rat Effect of femoral artery, liver, and femoral artery bleeding time after intravenous administration of nanohemostats.

The different bleeding models described in Figures 5-7 were established and evaluated as follows:

1) Mouse Tail Bleeding Model and Evaluation

20-25g C57BL mice, after anesthesia, use sharp scissors to cut transversely at 0.5cm from the tail tip of the anesthetized mouse. When the first drop of blood flows out automatically, suck it up with filter paper, and place the tail tip of the mouse in normal saline, PEI25000 , PEI1800, PEI10000 aqueous solutions and different ratios (10:10, 10:20, 10:30 and 10:40), different concentrations (1, 2 and 4mg/mL) and different molecular weight polyethyleneimine (bPEI1800 and bPEI10000) For the nanoparticle suspension, after the tail of the mouse was in full contact with the nanoparticle suspension, take it out and start timing, suck the blood with filter paper every 15 seconds until the bleeding stops completely, and record the bleeding time.

2) Rat femoral artery hemorrhage model and evaluation

Take 250-300g Sprague-Dawley rats, after anesthesia, unfold the hind limbs to expose the inner thigh, cut the skin, cut the overlying muscle, and expose the femoral artery and sciatic nerve. The femoral artery injury produces high pressure, which causes the mouse to bleed a lot. The femoral artery After the injury, normal saline, PEI25000, PEI1800 and PEI10000 aqueous solutions were added to the arterial injury, with different ratios (10:10, 10:20, 10:30 and 10:40), different concentrations (1, 2 and 4 mg/mL) and 300 μL of polyethyleneimine (bPEI1800, bPEI10000) nanoparticle suspension with different molecular weights was used to record the bleeding time.

3) Liver hemorrhage model and evaluation

Take 250-300g Sprague-Dawley rats, after anesthesia, incise the abdomen with a scalpel to expose the liver, and make a transverse incision in the middle lobe of the liver from the rostral to caudal direction, so that a wound of about 4mm is formed in the liver, and the damaged parts of the liver are respectively Add physiological saline, PEI25000, PEI1800 and PEI10000 aqueous solutions, different ratios (10:10, 10:20, 10:30 and 10:40), different concentrations (1, 2 and 4 mg/mL) and different molecular weight polyethyleneimine (bPEI1800 and bPEI10000) 300 μL of nanoparticle suspension, and record the bleeding time.

4) Bleeding of femoral artery after intravenous injection of nanohemostatic agent

Take 250～300g Sprague-Dawley rats, after anesthesia, inject 0.5mL normal saline, PEI25000, PEI1800 and PEI10000 aqueous solutions, different ratios (10:10, 10:20, 10:30 and 10:40), different concentrations (1, 2 and 4 mg/mL) with different molecular weight polyethyleneimine (bPEI1800 and bPEI10000) nanoparticle suspensions. After 5 minutes, the rat femoral artery was exposed, and a 0.45mm needle was used to puncture the femoral artery, and the bleeding time was recorded.

main reference

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Claims (6)

1. The application of a self-assembled nanohemostatic agent in the preparation of hemostatic medicine, characterized in that: the nanohemostatic agent is composed of a hydrophilic cationic polymer and a carboxyl-containing hydrophobic compound, wherein the hydrophilic cationic polymer The mass ratio to the carboxyl-containing hydrophobic compound is between 1:0.1 and 1:5, the hydrophilic cationic polymer is selected from branched or linear polyethyleneimine, and the carboxyl-containing hydrophobic compound is selected from cholic acid , ursodeoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, lithocholic acid, deoxycholic acid, dehydrocholic acid, the particle size of the nano-hemostatic agent is between 20 nm and 800 nm. 2. according to claim 1, the application of a kind of self-assembled nano-hemostatic agent in the preparation of hemostatic medicine is characterized in that: the preparation method of the self-assembled nano-hemostatic agent is as follows: first, the hydrophilic cationic polymer is dissolved in a certain amount of In a strong polar solvent, obtain a polymer solution, make the concentration of the polymer solution between 1 mg/mL to 100 mg/mL, dissolve the carboxyl-containing hydrophobic compound in a water-soluble organic solvent, and make the carboxyl-containing hydrophobic compound The concentration of the organic solution is between 1 mg/mL and 100 mg/mL, and then the organic solution containing the carboxyl-containing hydrophobic compound is slowly added to the polymer solution under the action of ultrasound, and then the resulting mixture is placed in a dialysis bag , dialyzed in deionized water to remove the organic solvent under magnetic stirring, and then the self-assembled nanohemostatic agent can be obtained. 3. According to claim 2, the application of a self-assembled nano-hemostatic agent in the preparation of hemostatic medicine is characterized in that: the strong polar solvent is selected from deionized water, dimethyl sulfoxide, N,N-dimethyl Formamide, N,N-Dimethylacetamide. 4. According to claim 2, the application of a self-assembled nano-hemostatic agent in the preparation of hemostatic medicine is characterized in that: the water-soluble organic solvent is selected from acetone, acetonitrile, ethanol, dimethyl sulfoxide, N,N-di Methylformamide, N,N-dimethylacetamide, acetic acid. 5. The application of a self-assembled nano-hemostatic agent in the preparation of hemostatic medicine according to claim 1, characterized in that: the nano-hemostatic agent is used for local tissue, or the nano-hemostatic agent is used for intravenous injection. 6. The application of a self-assembled nanohemostatic agent in the preparation of hemostatic medicine according to claim 5, characterized in that: said external use includes spraying, smearing or external application.