CN109045347B

CN109045347B - Degradable drug-loaded hemostatic microsphere and preparation method thereof

Info

Publication number: CN109045347B
Application number: CN201810944692.3A
Authority: CN
Inventors: 石长灿; 刘雯; 吴存造; 席光辉; 冯亚凯; 陈浩; 杨啸
Original assignee: First Affiliated Hospital of Wenzhou Medical University; Wenzhou Institute of Biomaterials and Engineering
Current assignee: First Affiliated Hospital of Wenzhou Medical University; Wenzhou Institute of UCAS
Priority date: 2018-08-19
Filing date: 2018-08-19
Publication date: 2021-05-28
Anticipated expiration: 2038-08-19
Also published as: CN109045347A

Abstract

The invention discloses a degradable drug-loaded hemostatic microsphere and a preparation method thereof, wherein the preparation method comprises the following steps: (1) preparing a hydrophobic artificially synthesized medical high-molecular compound spinning film; (2) preparing an ammonolysis product; (3) preparing ammonolysis product solution, sequentially adding a coupling agent and a hydrophilic natural high molecular compound aqueous solution, and reacting; (4) putting into dialysis bag, dialyzing in water solution, and vacuum freeze drying; obtaining microspheres; (5) and spraying the medicine water solution onto the microspheres by a solvent volatilization method, and drying to obtain the degradable medicine-carrying hemostatic microspheres. The invention has the advantages of low cost, controllable process and low production cost. The hemostatic microspheres formed by amphiphilic degradable macromolecule hydrophilic-hydrophobic self-assembly avoid the use of a cross-linking agent, and improve the safety of the hemostatic material. The hemostatic microspheres can simultaneously have the functions of hemostasis, antibiosis and promotion of wound healing, and are widely applicable to various bloody wounds.

Description

Degradable drug-loaded hemostatic microsphere and preparation method thereof

Technical Field

The invention belongs to the field of biomedical materials and medical instrument combination, and particularly relates to a degradable drug-loaded hemostatic microsphere and a preparation method thereof.

Background

In emergency situations such as battlefields, car accidents, natural disasters, etc., uncontrolled massive blood loss is a major cause of death of personnel. According to statistics, in 2011-2013, about 60 thousands of people die from traffic accidents every year in China, 85% of people die due to massive bleeding caused in the initial stage of trauma, and if the bleeding situation can be controlled within 30 minutes after the accidents occur, the survival rate of the injured people can be improved by over 40%. Common capillary vessel hemorrhage or arteriovenous hemorrhage can be effectively stopped by adopting conventional methods such as electrocoagulation, compression, suturing and the like. However, the traditional hemostatic methods cannot achieve satisfactory results for bleeding sites such as head, trunk, and visceral injuries. When the bleeding volume of the injured person reaches 20% (about 800mL), shock symptoms occur, which in turn endangers the life of the patient. In response to these injuries, the use of hemostatic products to accelerate hemostasis is currently a widely used clinical treatment.

The current clinical commonly used hemostatic products can be divided into three types: the first is to provide a clotting component to accelerate clotting (e.g., thrombin-like hemostatic materials); the second is that the gel material can stop bleeding by sealing the damaged tissue and preventing the loss of blood components such as red blood cells, blood platelets and the like (such as alpha cyanoacrylate hemostatic materials). Thirdly, the coagulation factors are coagulated to accelerate the coagulation of the wound part through the concentration effect of the hemostatic material on blood (such as hemostatic powder hemostatic materials); however, different types of hemostatic materials have a number of bottlenecks.

Blood products such as blood clotting components suffer from short supply, short shelf life and risk of adverse side effects such as febrile nonhemolytic reactions, immunogenicity and acute lung inflammation. The gel hemostatic products have the main defects of slow degradation speed, certain biotoxicity of the degradation products and certain potential safety hazard. The hemostatic powder product is particularly suitable for irregular wounds, and can absorb water in blood and concentrate blood coagulation components of damaged wounds so as to effectively stop bleeding. The typical hemostatic powder is Arista produced in the United states^TMAlthough the hemostatic powder has good hemostatic effect, there are still some problems to be solved: firstly, the product adopts small-molecule epichlorohydrin as a cross-linking agent, and has potential biological toxicity; the other is bleeding wound surface, which is a warm and humid environment, and the wound surface is easy to accumulate bacterial colony to cause wound infection. Therefore, wound infection should be avoided while hemostasis is achieved. The product has no antibacterial and healing promoting functions, can only complete primary hemostasis on the damaged part, and does not have the functions of resisting bacteria and promoting healingCan accelerate the later period of wound healing; thirdly, the product is expensive, not only is the clinical use limited, but also the product can not be used as an emergency reserve material in the aspects of military affairs, natural disasters and the like. Therefore, the development of a novel hemostatic material with low cost, multiple functions of hemostasis, antibiosis and healing promotion can better meet the use requirement of clinical treatment.

Chinese patent 201810124860.4 discloses an absorbable gelatin hemostatic powder and a preparation method thereof, the patent enables a cross-linking agent to have no residue in the hemostatic powder by optimizing the process, avoids the irritant influence on organisms caused by the existence of the cross-linking agent, and improves the safety of the product, but the prepared hemostatic powder only has a single hemostatic function and has no antibacterial effect. Chinese patent 201611121260.X discloses a sustained antibacterial hemostatic powder and its preparation method. However, the hemostatic powder takes the nano-silver as an antibacterial agent, although the nano-silver is a long-acting antibacterial agent, the pharmacokinetics of the hemostatic powder is not clear, and the nano-silver entering blood has potential reproductive toxicity and genetic toxicity, so that the clinical use risk is increased. Chinese patent CN201610952251.9 discloses an O-quaternary ammonium salt-N-alkylated chitosan and a preparation method and application thereof, although the modified chitosan shows good antibacterial effect, the chitosan hemostatic product is not suitable for wound surfaces with more bleeding due to limited water absorption capacity.

The hydrophobic artificially synthesized medical high molecular compound is not only suitable for large-scale production, but also has better biocompatibility and degradability, and is widely applied to the biomedical field, such as surgical sutures, tissue repair materials and drug controlled release systems, however, due to the hydrophobic property of the compound, the application of the compound to the hemostasis field is rarely reported.

In conclusion, a degradable hemostatic material which is low in cost, suitable for industrial production, and has the functions of hemostasis, antibiosis and wound healing and high biological safety is lacking in the current market.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a degradable drug-loaded hemostatic microsphere.

The second purpose of the invention is to provide a preparation method of the degradable drug-loaded hemostatic microspheres.

The technical scheme of the invention is summarized as follows:

a preparation method of degradable drug-loaded hemostatic microspheres comprises the following steps:

(1) dissolving a hydrophobic artificially synthesized medical high molecular compound in a first polar organic solvent to prepare a spinning solution with the concentration of 5% -12%, and preparing the spinning solution into a hydrophobic artificially synthesized medical high molecular compound spinning membrane by using an electrostatic spinning process under the conditions of positive voltage of 5-25 KV and negative voltage of (-2) (-0.2) KV;

(2) taking 0.1-10 parts by mass of the spinning membrane obtained in the step (1), placing the spinning membrane in 5-50 parts by mass of 0.1-50% ammonolysis solution, ammonolysis for 0.5-72 h at 4-60 ℃, centrifuging for 5-60 min at 500-5000 rpm, discarding supernatant, collecting solids, and drying to obtain ammonolysis products;

(3) dissolving 0.1-10 parts of aminolysis product in a second polar organic solvent to prepare 0.01-10% of aminolysis product solution, sequentially adding 0.01-1 part of coupling agent and 0.5-5 parts of hydrophilic natural high molecular compound aqueous solution with mass concentration of 0.01-10% into the aminolysis product solution, and reacting at 4-80 ℃ for 0.5-48 h;

(4) putting the product obtained in the step (3) into a dialysis bag with the molecular weight cutoff of 2000-10000, dialyzing in aqueous solution for 1-7 d, and then carrying out vacuum freeze drying; obtaining microspheres;

(5) and (3) spraying 1-20 parts of a 1-15% mass concentration aqueous solution of the medicine onto 20-800 parts of the microspheres obtained in the step (4) by using a solvent volatilization method, and drying at 25-80 ℃ for 0.5-48 h to obtain the degradable medicine-carrying hemostatic microspheres.

Preferably, the hydrophobic synthetic medical polymer compound is at least one of polylactic acid with the number average molecular weight of 100000-250000, poly (lactic acid-glycolic acid) with the number average molecular weight of 100000-250000, polycaprolactone with the intrinsic viscosity number of 0.40-2.7 dL/g and polydioxanone with the intrinsic viscosity number of 1.5-2.2 dL/g.

The first polar organic solvent is preferably: at least one of chloroform, methanol, dimethyl sulfoxide, dichloromethane, acetone, isopropanol, toluene, tetrahydrofuran and N, N-dimethylformamide.

The ammonolysis solution is preferably: at least one of an ethylene diamine aqueous solution, a 1, 3-propanediamine aqueous solution, a 1, 6-hexanediamine aqueous solution, a 1, 7-heptanediamine aqueous solution, a 1, 8-octanediamine aqueous solution and a 1, 9-nonanediamine aqueous solution.

The second polar organic solvent is preferably: at least one of dimethyl sulfoxide, N-dimethylformamide, trifluoroacetic acid, cyclohexane, p-xylene and pyridine.

The coupling agent is preferably: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, N' -diisopropylcarbodiimide and N-hydroxysuccinimide.

The hydrophilic natural polymer compound is preferably: at least one of starch, sodium alginate, carboxymethyl chitosan, dextran, hyaluronic acid, chondroitin sulfate and heparin.

The medicine is preferably: at least one of amoxicillin sodium, penicillin sodium, ceftriaxone sodium, kanamycin sulfate, vancomycin hydrochloride, gentamicin sulfate, aclacinomycin hydrochloride, erythromycin lactobionate, epidauriccin hydrochloride, metronidazole and ciprofloxacin lactate.

The degradable drug-loaded hemostatic microspheres prepared by the method.

The invention has the advantages that:

(1) the invention takes the hydrophobic artificially synthesized medical high molecular compound with low cost and controllable process as the raw material, expands the application of the hydrophobic artificially synthesized medical high molecular compound in the hemostasis field and reduces the production cost of the hemostasis material.

(2) The hemostatic microspheres formed by amphiphilic degradable macromolecule hydrophilic-hydrophobic self-assembly avoid the use of cross-linking agents, solve the problem of the use of various cross-linking agents in the prior art and improve the safety of hemostatic materials.

(3) The degradable drug-loaded hemostatic microspheres prepared by the invention have the functions of hemostasis, antibiosis and wound healing promotion, are widely applicable to various bloody wounds and have good clinical application prospects.

Drawings

FIG. 1 shows that the obtained nanofiber membrane is subjected to ammonolysis by using an ethylenediamine aqueous solution as an ammonolysis solution, and the ammonolysis product is a short rod-shaped product after purification and observation by a scanning electron microscope.

FIG. 2 shows the morphology of the microsphere, which is observed by a scanning electron microscope, wherein the overall morphology of the microsphere is micron-sized spheres, the surface is smooth, and the particle size distribution is uniform.

FIG. 3 is a scanning electron microscope picture of the morphology of drug-loaded microspheres, which shows that the microspheres still maintain spherical morphology and the particle size is not much different from that before drug loading.

Fig. 4 is a drug release profile of drug-loaded microspheres. After two weeks of release, the cumulative release amount of the drug can reach 30 percent, which shows that the obtained microspheres have long-acting slow release function to the drug.

Fig. 5 shows the antibacterial effect of the drug-loaded hemostatic microspheres, and gram-positive bacteria (staphylococcus aureus as an example) are adopted to investigate the antibacterial effect of the drug-loaded hemostatic microspheres, so that an obvious bacteriostatic ring can be formed after the drug-loaded hemostatic microspheres are added.

Fig. 6 shows the in vitro hemostatic effect of the degradable drug-loaded hemostatic microspheres. In which anticoagulated whole blood was dissolved in deionized water as a control.

FIG. 7 is a graph showing the cytotoxicity results of drug-loaded hemostatic microspheres. Culturing normal-growth logarithmic phase cells in a fresh sterile RAPI1640 culture medium containing 10% fetal calf serum to serve as a negative control, culturing normal-growth logarithmic phase cells in a 6% phenol solution to serve as a positive control, and culturing normal-growth logarithmic phase cells in a degradable drug-loaded hemostatic microsphere leaching liquor to serve as an experimental group.

Fig. 8 is a representation of the hemostatic performance of the drug-loaded hemostatic microspheres, and the hemostatic efficacy of the material of the invention is proved to have a rapid hemostatic effect by an SD rat liver hemorrhage hemostatic model. After applying the drug-loaded hemostatic microspheres to the bleeding part, the bleeding wound surface completes the hemostatic process within a short time (<5 s).

Detailed Description

The present invention will be further illustrated by the following specific examples. The examples are for the purpose of providing a more complete understanding of the present invention to those of ordinary skill in the art and are not intended to limit the invention in any way. In the following examples, unless otherwise specified, the experimental methods used were all conventional methods, and materials, reagents and the like used were all available from biological or chemical companies.

Example 1

(1) dissolving a hydrophobic artificially synthesized medical high molecular compound in a first polar organic solvent to prepare a spinning solution with the concentration of 5%, and preparing the spinning solution into a hydrophobic artificially synthesized medical high molecular compound spinning membrane by utilizing an electrostatic spinning process under the conditions of a positive voltage of 5KV and a negative voltage of (-0.2) KV;

the hydrophobic artificially synthesized medical high molecular compound is polylactic acid with the number average molecular weight of 100000. The first polar organic solvent is a chloroform/methanol (3:1, v/v) composite solution;

(2) taking 0.1g of the spinning membrane obtained in the step (1), putting the spinning membrane into 5g of ammonolysis solution with the mass concentration of 0.1%, ammonolysis for 0.5h at 4 ℃, centrifuging for 60min at 5000rpm, discarding supernatant, collecting solids, and drying to obtain ammonolysis products, wherein the ammonolysis solution is ethylenediamine aqueous solution;

(3) dissolving 0.1g of aminolysis product in a second polar organic solvent to prepare 0.01% aminolysis product solution, sequentially adding 0.01g of coupling agent and 0.5g of 0.01% hydrophilic natural high molecular compound aqueous solution into the aminolysis product solution, and reacting at 4 ℃ for 0.5 h;

the second polar organic solvent is dimethyl sulfoxide;

the coupling agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride;

the hydrophilic natural polymer compound is sodium alginate;

(4) putting the product obtained in the step (3) into a dialysis bag with molecular weight cutoff of 2000, dialyzing in aqueous solution for 1d, and then carrying out vacuum freeze drying; obtaining microspheres;

(5) and (3) spraying 1g of amoxicillin sodium aqueous solution with the mass concentration of 1% onto 20g of the microspheres obtained in the step (4) by using a solvent evaporation method, and drying at 25 ℃ for 0.5h to obtain the degradable drug-loaded hemostatic microspheres.

Morphology observation experiment

Preparing a sample support, sticking double-sided conductive copper adhesive on the support, naturally bonding a small amount of aminolysis product prepared respectively according to the embodiment 1, the microspheres obtained in the step (4) and the degradable drug-loaded hemostatic microspheres finally obtained with the conductive copper adhesive, performing surface gold plating treatment on the sample by using a sputtering instrument (Hitachi, E-1045, Japan), and observing the surface appearance by using a scanning electron microscope (SEM, Hitachi S-4800, Japan).

The picture of the scanning electron scanning microscope of the ammonolysis product is shown in figure 1, and the overall appearance of the ammonolysis product is a short rod-shaped appearance which is not the appearance of the nano-fiber before ammonolysis any more;

and (5) observing the morphology of the microspheres obtained in the step (4) by using a scanning electron microscope, wherein the overall morphology of the microspheres is micron-sized microspheres, the surfaces of the microspheres are smooth, and the particle sizes of the microspheres are uniformly distributed, as shown in fig. 2.

The morphology of the degradable drug-loaded hemostatic microspheres is observed by a scanning electron microscope, as shown in fig. 3, the microspheres still keep spherical morphology, and the particle size is not much different from that before the drug is loaded.

Drug Release test

Preparing a series of amoxicillin aqueous solutions with different concentration gradients of 0.0625mg/mL,0.125mg/mL,0.25mg/mL,0.5mg/mL,1mg/mL and 2 mg/mL.

The maximum absorption peaks of drugs with different concentrations were measured by uv-visible absorption spectroscopy, and a standard curve was drawn, and a certain amount of the degradable drug-loaded hemostatic microspheres prepared in example 1 was weighed and placed in an EP tube, added to 2mL of PBS solution (pH 7.4), and placed in a shaker at room temperature. The 1mL PBS solution was taken every 1d,7d,14d,21d and 1mL of fresh PBS solution was added to the original EP tube. And measuring the absorbance of the taken 1ml PBS solution by using an ultraviolet-visible absorption spectrum, calculating the cumulative release amount of the drug at different time points according to a standard curve, and drawing a drug release curve.

The experimental result shows that the cumulative release amount of the drug can reach 30% after the release of the drug for two weeks as shown in fig. 4, which indicates that the degradable drug-loaded hemostatic microspheres have a long-acting slow-release function on the drug.

Antibacterial experiments

Gram-positive bacteria (taking staphylococcus aureus as an example) are adopted to examine the antibacterial effect of the drug-loaded microspheres.

A suspension of Staphylococcus aureus bacteria (commercially available) was prepared at a concentration of 10⁵and/mL (using BP culture medium), after the agar plate is solidified, 0.2mL of bacterial liquid is dripped, the bacterial liquid is uniformly coated, the bacterial liquid is placed in an incubator at 37 ℃ for 24 hours and then taken out, the degradable drug-loaded hemostatic microspheres prepared in example 1 are placed on the bacterial plate, the bacterial plate is cultured at 37 ℃ for 24 hours, and the existence of the bacteriostatic zone is observed. The experimental result shows that the degradable drug-loaded hemostatic microspheres have obvious antibacterial function. The results are shown in FIG. 5.

Whole blood coagulation test

10mg of the degradable drug-loaded hemostatic microspheres prepared according to example 1 were weighed out and placed inside a petri dish and cultured at 37 ℃ for 5min, then 200ul of anticoagulated blood was slowly added dropwise to the surface of the sample, followed by addition of 20ul of 0.2mol/L calcium chloride solution and continued culture at 37 ℃ for 5 min. Afterwards, 10ml of distilled water is carefully added into the watch glass and is subjected to shake culture for 10min at the rotating speed of 30rpm in a shaking table, and meanwhile, the steps are repeated by taking the degradable drug-loaded hemostatic microspheres as a control group, and the adsorption effect of the degradable drug-loaded hemostatic microspheres on erythrocytes is observed. The experimental result shows that the degradable drug-loaded hemostatic microspheres prepared in example 1 have a good erythrocyte adsorption effect and a hemostatic effect. As shown in FIG. 6, the hemostatic microspheres were not added to the A-plate, and the erythrocytes were dispersed in distilled water after the anticoagulation. Because the hemostatic microspheres are added into the B disk, under the same condition, the red blood cells in the B disk are adsorbed to the surfaces of the microspheres, so that the phenomenon of aggregation of the red blood cells on the surfaces of the microspheres occurs.

Cytotoxicity test

The experimental cells used were L929 cell line cells (commercially available) that grew vigorously for 48-72 hours. The medium was RAPI1640 supplemented with 10% (V/V) fetal bovine serum.

Negative control group (fresh sterile RAPI1640 medium containing 10% fetal bovine serum);

an experimental group (0.5 g of the degradable drug-loaded hemostatic microspheres of example 1 is extracted with 10mL of fresh sterile RAPI1640 medium containing 10% fetal calf serum at 37 ℃ for 24h to obtain an extract);

a positive control group (5% phenol solution by mass);

the assay was performed on 96-well plates. The cell density of the initial culture medium was about 5 ten thousand per mL, and 200. mu.L of the cell culture medium was added to each well, so that the number of cells in each well was about 10000. Each well was repeated three times, incubated at 37 ℃ in a 5% (V/V) carbon dioxide/air incubator for 24h, and then the original medium was discarded.

Adding 200 μ L of fresh sterile RAPI1640 culture medium containing 10% fetal calf serum into the negative control group;

adding 10 μ L, 20 μ L, 30 μ L, 40 μ L and 50 μ L leaching solution into the experimental group, and adding negative control solution to make the volume of the solution in each well 200 μ L;

the positive control group was added with 10. mu.L, 20. mu.L, 30. mu.L, 40. mu.L, and 50. mu.L of 5% phenol solution, respectively, and then the negative control solution was added to make the volume of the solution in each well 200. mu.L.

Subsequently, the 96-well plate was placed in a 37 ℃ incubator and cultured for another 48 hours, and then the plate was taken out, the solution in the plate was discarded, 20. mu.L of MTT solution was added to each well, the culture was continued for 4 hours, then the stock solution was aspirated, 150. mu.L of dimethyl sulfoxide was added, shaking was carried out for ten minutes, and the absorbance value (ABS) was measured at a wavelength of 570nm on an immunomicroplate reader.

Relative cellular activity (%) ═ ABS_{570 test group}/ABS_{570 negative control}×100％

According to GB/T16886.5-2003 in vitro cytotoxicity judgment standards, the degradable drug-loaded hemostatic microsphere leaching solution disclosed in example 1 has 0-grade cytotoxicity at 2.5-12.5 mu g/mL, and shows good biocompatibility. The results are shown in FIG. 7.

The cytotoxicity of the drug-loaded hemostatic microspheres is considered, the normal-growth logarithmic phase cells are cultured in a fresh sterile RAPI1640 culture medium containing 10% fetal calf serum as a negative control, the normal-growth logarithmic phase cells are cultured in a 6% phenol solution as a positive control, and the normal-growth logarithmic phase cells are cultured in a degradable drug-loaded hemostatic microsphere leaching liquor to form an experimental group. Under the current experimental conditions, the cytotoxicity of the drug-loaded hemostatic microspheres is more than 70%, and no obvious cytotoxicity is shown.

Liver hemostasis test

After SD rats (with similar body weight) are anesthetized, the abdominal cavity is opened, a bleeding wound with the depth of 0.2cm and the length of 0.5cm is made on the right middle lobe of the liver by using a scalpel, degradable drug-loaded hemostatic microspheres with the minimum dose of 20mg are uniformly sprayed, timing is started after no treatment (blank control) or commercially available gold wound hemostatic powder (positive control), bleeding conditions are observed every 20s, and no excessive blood oozes out of the surface of the liver to serve as bleeding time. The experimental result shows that the degradable drug-loaded hemostatic microspheres prepared according to example 1 have good hemostatic efficacy, the hemostatic time is about 20s, the hemostatic time of the corresponding blank control is about 220s, and the hemostatic time of the commercially available Jinchuang hemostatic powder is about 180 s. The results are shown in FIG. 8. Wherein, the A picture is a blank control group, the B picture is a picture obtained by adding Jinchuang hemostatic powder (a commercial product) to a bleeding wound surface, and the C picture is a picture of the hemostatic effect after adding the hemostatic microspheres prepared in example 1.

A body surface hemostasis model is adopted, the hemostasis performance of the drug-loaded hemostasis microspheres is systematically characterized, and the SD rat liver bleeding hemostasis model is used for proving that the hemostasis effect of the material disclosed by the invention has a rapid hemostasis effect. The results are shown in fig. 8, after applying the drug-loaded hemostatic microspheres to the bleeding site, the bleeding wound completes the hemostatic process in a short time (<5 s).

Example 2

(1) dissolving a hydrophobic artificially synthesized medical high molecular compound in a first polar organic solvent to prepare a spinning solution with the concentration of 12%, and preparing the spinning solution into a hydrophobic artificially synthesized medical high molecular compound spinning membrane by using an electrostatic spinning process under the conditions of positive electricity of 25KV and negative voltage (-2) KV; the hydrophobic artificially synthesized medical high molecular compound is polylactic acid with the number average molecular weight of 250000; the first polar organic solvent is tetrahydrofuran/N, N-dimethylformamide (4:1, v/v) composite solution;

(2) taking 10g of the spinning membrane obtained in the step (1), putting the spinning membrane into 50g of ammonolysis solution with the mass concentration of 50%, ammonolysis for 72h at 60 ℃, centrifuging for 5min at 500rpm, removing supernatant, collecting solids, and drying to obtain ammonolysis products; the ammonolysis solution is a 1, 3-propane diamine aqueous solution;

(3) dissolving 10g of aminolysis product in a second polar organic solvent to prepare 10% aminolysis product solution, sequentially adding 1g of coupling agent and 5g of 10% hydrophilic natural high molecular compound aqueous solution to the aminolysis product solution, and reacting at 80 ℃ for 48 hours;

the second polar organic solvent is N, N-dimethylformamide;

the coupling agent is N, N' -diisopropylcarbodiimide;

the hydrophilic natural high molecular compound is chondroitin sulfate;

(4) putting the product obtained in the step (3) into a dialysis bag with the molecular weight cutoff of 10000, dialyzing in aqueous solution for 7 days, and then carrying out vacuum freeze drying; obtaining microspheres;

(5) and (3) spraying 20g of vancomycin hydrochloride aqueous solution with the mass concentration of 15% onto 800g of the microspheres obtained in the step (4) by using a solvent volatilization method, and drying at 80 ℃ for 48 hours to obtain the degradable drug-loaded hemostatic microspheres.

Experiments prove that the polylactic acid with the number average molecular weight of 250000 in the embodiment is replaced by poly (lactic acid-glycolic acid) with the number average molecular weight of 100000, poly (lactic acid-glycolic acid) with the number average molecular weight of 250000, poly (p-dioxanone) with the intrinsic viscosity of 1.5dL/g or poly (p-dioxanone) with the intrinsic viscosity of 2.2dL/g, and the properties and the effects of the prepared degradable drug-loaded hemostatic microspheres are similar to those of the degradable drug-loaded hemostatic microspheres prepared in the embodiment in other same embodiments.

Experiments prove that dimethyl sulfoxide, acetone, isopropanol or toluene are respectively used for replacing the first polar organic solvent (tetrahydrofuran/N, N-dimethylformamide (4:1, v/v) composite solution) in the embodiment, and the properties and effects of the degradable drug-loaded hemostatic microspheres prepared in the same embodiment are similar to those of the degradable drug-loaded hemostatic microspheres prepared in the embodiment.

Experiments prove that the properties and the effects of the degradable drug-loaded hemostatic microspheres prepared by using 1, 7-heptanediamine aqueous solution or 1, 9-nonanediamine aqueous solution to respectively replace the ammonolysis solution (1, 3-propanediamine aqueous solution) in the embodiment are similar to those of the degradable drug-loaded hemostatic microspheres prepared in the embodiment.

Experiments prove that the properties and the effects of the degradable drug-loaded hemostatic microspheres prepared by the method are similar to those of the degradable drug-loaded hemostatic microspheres prepared by the embodiment by using cyclohexane or pyridine to replace N, N-dimethylformamide in the embodiment.

Experiments prove that the properties and the effects of the degradable drug-loaded hemostatic microspheres prepared by using dextran or hyaluronic acid to respectively replace chondroitin sulfate in the embodiment are similar to those of the degradable drug-loaded hemostatic microspheres prepared in the embodiment.

Experiments prove that the gentamicin sulfate, the aclacinomycin hydrochloride, the erythromycin lactobionate, the epidaunorubicin hydrochloride or the metronidazole are respectively used for replacing the vancomycin hydrochloride aqueous solution in the embodiment, and the properties and the effects of the prepared degradable drug-loaded hemostatic microspheres are similar to those of the degradable drug-loaded hemostatic microspheres prepared in the embodiment in the same way as in the embodiment.

Example 3

(1) dissolving a hydrophobic artificially synthesized medical high molecular compound in a first polar organic solvent to prepare a spinning solution with the concentration of 8%, and preparing the spinning solution into a hydrophobic artificially synthesized medical high molecular compound spinning membrane by utilizing an electrostatic spinning process under the conditions of positive voltage of 18KV and negative voltage of (-1.2) KV; the hydrophobic artificially synthesized medical high molecular compound is polylactic acid with the number average molecular weight of 150000; the first polar organic solvent is chloroform;

(2) putting 6g of the spinning membrane obtained in the step (1) into 30g of ammonolysis solution with the mass concentration of 10%, ammonolysis for 24h at 20 ℃, centrifuging for 40min at 3000rpm, discarding supernatant, collecting solid, and drying to obtain ammonolysis products; the ammonolysis solution is 1, 8-octanediamine aqueous solution.

(3) Dissolving 5g of aminolysis product in a second polar organic solvent to prepare an aminolysis product solution with the mass concentration of 6%, sequentially adding 0.5g of coupling agent and 2g of hydrophilic natural high molecular compound aqueous solution with the mass concentration of 6% into the aminolysis product solution, and reacting at 40 ℃ for 24 hours;

the second polar organic solvent is trifluoroacetic acid;

the coupling agent is N-hydroxysuccinimide;

the hydrophilic natural polymer compound is heparin;

(4) putting the product obtained in the step (3) into a dialysis bag with the molecular weight cutoff of 5000, dialyzing in an aqueous solution for 3 days, and then carrying out vacuum freeze drying; obtaining microspheres;

(5) and (3) spraying 10g of ciprofloxacin lactate aqueous solution with the mass concentration of 5% onto 400g of microspheres obtained in the step (4) by using a solvent volatilization method, and drying at 50 ℃ for 12h to obtain the degradable drug-loaded hemostatic microspheres.

Example 4

(1) dissolving a hydrophobic artificially synthesized medical high molecular compound in a first polar organic solvent to prepare a spinning solution with the concentration of 12%, and preparing the spinning solution into a hydrophobic artificially synthesized medical high molecular compound spinning film by utilizing an electrostatic spinning process under the conditions of positive voltage of 22KV and negative voltage of (-2) KV;

the hydrophobic artificially synthesized medical high molecular compound is polycaprolactone with the intrinsic viscosity number of 2.7 dL/g; the first polar organic solvent is dichloromethane;

(2) taking 5g of the spinning membrane obtained in the step (1), putting the spinning membrane into 10g of ammonolysis solution with the mass concentration of 10%, ammonolysis for 36h at 25 ℃, centrifuging for 15min at 1000rpm, discarding supernatant, collecting solid, and drying to obtain ammonolysis products; the ammonolysis solution is a 1, 6-hexanediamine aqueous solution;

(3) dissolving 3g of aminolysis product in a second polar organic solvent to prepare an aminolysis product solution with the mass concentration of 5%, sequentially adding 0.6g of coupling agent and 4g of hydrophilic natural high molecular compound aqueous solution with the mass concentration of 5% into the aminolysis product solution, and reacting at 35 ℃ for 12 hours;

the second polar organic solvent is dimethyl sulfoxide;

the coupling agent is dicyclohexylcarbodiimide;

the hydrophilic natural polymer compound is carboxymethyl chitosan;

(4) putting the product obtained in the step (3) into a dialysis bag with the molecular weight cutoff of 6000, dialyzing in an aqueous solution for 2 days, and then carrying out vacuum freeze drying; obtaining microspheres;

(5) and (3) spraying 5g of a 5% drug aqueous solution by using a solvent volatilization method onto 100g of the microspheres obtained in the step (4), and drying at 40 ℃ for 18h to obtain the degradable drug-loaded hemostatic microspheres. The drug is ceftriaxone sodium/kanamycin sulfate (1:1, m/m).

Example 5

(1) dissolving a hydrophobic artificially synthesized medical high molecular compound in a first polar organic solvent to prepare a spinning solution with the concentration of 6%, and preparing the spinning solution into a hydrophobic artificially synthesized medical high molecular compound spinning membrane by utilizing an electrostatic spinning process under the conditions of positive voltage of 15KV and negative voltage of (-1.2) KV; the hydrophobic artificially synthesized medical high molecular compound is polycaprolactone with the intrinsic viscosity number of 0.40 dL/g; the first polar organic solvent is dichloromethane;

(2) putting 4g of the spinning membrane obtained in the step (1) into 10g of ammonolysis solution with the mass concentration of 20%, ammonolysis for 22h at 30 ℃, centrifuging for 5min at 5000rpm, discarding supernatant, collecting solid, and drying to obtain ammonolysis products; the ammonolysis solution is an ethylene diamine aqueous solution;

(3) dissolving 8g of aminolysis product in a second polar organic solvent to prepare 8% aminolysis product solution, and reacting 1g of coupling agent and 5g of 6% hydrophilic natural high molecular compound aqueous solution in the aminolysis product solution at 40 ℃ for 28 hours;

the second polar organic solvent is p-xylene;

the hydrophilic natural polymer compound is starch;

(4) putting the product obtained in the step (3) into a dialysis bag with the molecular weight cutoff of 5000, dialyzing in an aqueous solution for 5 days, and then carrying out vacuum freeze drying; obtaining microspheres;

(5) and (3) spraying 10g of 8% ceftriaxone sodium aqueous solution onto 50g of the microspheres obtained in the step (4) by using a solvent volatilization method, and drying at 50 ℃ for 48h to obtain the degradable drug-loaded hemostatic microspheres.

Experiments prove that the overall morphology of the ammonolysis products obtained in the step (2) of the embodiments 2 to 5 is short rod-shaped, the microspheres obtained in the step (4) are micron-sized microspheres, the surfaces of the microspheres are smooth, and the particle size distribution of the microspheres is uniform. The morphology of the degradable drug-loaded hemostatic microspheres is observed by a scanning electron microscope, and the spherical morphology is kept. Experiments prove that the degradable drug-loaded hemostatic microspheres prepared in each of examples 2 to 5 have similar drug long-acting slow release function, antibacterial function, erythrocyte adsorption function, biocompatibility and hemostatic efficacy to the degradable drug-loaded hemostatic microspheres prepared in example 1.

Claims

1. A preparation method of degradable drug-loaded hemostatic microspheres is characterized by comprising the following steps:

(1) dissolving a hydrophobic artificially synthesized medical high molecular compound in a first polar organic solvent to prepare a spinning solution with the concentration of 5-12%, and preparing the spinning solution into a hydrophobic artificially synthesized medical high molecular compound spinning membrane by utilizing an electrostatic spinning process;

2. The method according to claim 1, wherein the hydrophobic synthetic polymer compound is at least one of polylactic acid having a number average molecular weight of 100000 to 250000, poly (lactic-co-glycolic acid) having a number average molecular weight of 100000 to 250000, polycaprolactone having an intrinsic viscosity of 0.40 to 2.7dL/g, and polydioxanone having an intrinsic viscosity of 1.5 to 2.2 dL/g.

3. The method according to claim 1, wherein the first polar organic solvent is at least one of chloroform, methanol, dimethylsulfoxide, dichloromethane, acetone, isopropanol, toluene, tetrahydrofuran, and N, N-dimethylformamide.

4. The production method according to claim 1, characterized in that the aminolysis solution is at least one of an aqueous ethylenediamine solution, an aqueous 1, 3-propanediamine solution, an aqueous 1, 6-hexanediamine solution, an aqueous 1, 7-heptanediamine solution, an aqueous 1, 8-octanediamine solution and an aqueous 1, 9-nonanediamine solution.

5. The method according to claim 1, wherein the second polar organic solvent is at least one of dimethyl sulfoxide, N-dimethylformamide, trifluoroacetic acid, cyclohexane, p-xylene, and pyridine.

6. The production method according to claim 1, characterized in that the coupling agent is at least one of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, N' -diisopropylcarbodiimide and N-hydroxysuccinimide.

7. The method according to claim 1, wherein the hydrophilic natural polymer compound is at least one of starch, sodium alginate, carboxymethyl chitosan, dextran, hyaluronic acid, chondroitin sulfate and heparin.

8. The method according to claim 1, wherein the drug is at least one of amoxicillin sodium, penicillin sodium, ceftriaxone sodium, kanamycin sulfate, vancomycin hydrochloride, gentamicin sulfate, aclacinomycin hydrochloride, erythromycin lactobionate, epidauriccin hydrochloride, metronidazole, or ciprofloxacin lactate.

9. Degradable drug-loaded hemostatic microspheres made by the method of any one of claims 1-8.