CN114931663B - Bionic antibacterial hemostatic microsphere with erythroid structure and preparation method thereof - Google Patents
Bionic antibacterial hemostatic microsphere with erythroid structure and preparation method thereof Download PDFInfo
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Classifications
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- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
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- A61L2400/00—Materials characterised by their function or physical properties
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Abstract
The invention provides a bionic antibacterial hemostatic microsphere with a erythroid structure and a preparation method thereof. The preparation method provided by the invention comprises the following steps: a) Mixing carboxymethyl chitosan solution and gelatin solution to obtain mixed solution; b) Placing the mixed solution into a syringe to obtain FeCl 3 The solution is a receiving solution, and carboxymethyl chitosan-gelatin microspheres are formed by an electrostatic instillation method; the voltage in the electrostatic instillation method is 7-9 KV; the distance between the nozzle of the injector and the surface of the receiving liquid is 4-7 cm; the temperature of the mixed solution is 20-40 ℃. The preparation method is simple and efficient, can improve the uniformity of the microspheres, and is safe and nontoxic; the prepared microsphere has a bionic appearance structure similar to red blood cells, has excellent water absorption performance, can recover the original microsphere shape after swelling by absorbing water, and has excellent hemostatic property, antibacterial property and biodegradability.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a bionic antibacterial hemostatic microsphere with a erythroid structure and a preparation method thereof.
Background
The amino group on carboxymethyl chitosan (CMCM) has positive charge, can be combined with red blood cells with negative charge, so that the cells can be aggregated and coagulated quickly, and has good biocompatibility, biodegradability and spectral antibacterial property. Gelatin (Gel) is a natural polymer material which has a structure similar to that of a living body tissue and good biocompatibility, and is used as a natural water-soluble biodegradable polymer material, and degradation products are easy to be absorbed without producing inflammatory reaction. Therefore, the carboxymethyl chitosan and the gelatin are compounded to form the microsphere, which is beneficial to cell proliferation and in vivo hemostasis.
Microspheres are currently widely used in various fields. Wherein, the preparation method of the carboxymethyl chitosan-gelatin microsphere is mainly an emulsion crosslinking method. The emulsion crosslinking method is to add one or two mixed solutions to an oil phase (containing an emulsifier such as a surfactant) and then stir or ultrasonically treat the mixture to form a water-in-oil emulsion. And adding different doses of cross-linking agents into the emulsion according to the required cross-linking density, and filtering or centrifuging to form the required microspheres. For example, zhang Weiguang in 2018, an emulsion crosslinking method was used to prepare lincomycin carboxymethyl chitosan-gelatin composite microspheres for slow release of lincomycin. The chitosan/gelatin microsphere is prepared by using Wen Liu as a cross-linking agent by an inverse emulsion cross-linking method and using genipin as a cross-linking agent. Chitosan/gelatin microspheres were prepared for large scale culture of hepatocytes.
However, the above-mentioned methods for preparing carboxymethyl chitosan-gelatin microspheres using the emulsion crosslinking method have the following problems: (1) The cross-linking agent which is not crosslinked in the solution is difficult to remove, and has certain toxicity and side effects on human bodies. (2) the experimental process has complicated operation and complex operation; (3) The size of the formed microspheres is different, and the size of the microspheres is difficult to control.
Disclosure of Invention
In view of the above, the present invention aims to provide a bionic antibacterial hemostatic microsphere with a erythroid structure and a preparation method thereof. The preparation method is simple and efficient, can improve the uniformity of the microspheres, and is safe and nontoxic; the prepared microsphere has a bionic appearance structure similar to red blood cells, has excellent water absorption performance, can recover the original microsphere shape after swelling by absorbing water, and has excellent hemostatic property, antibacterial property and biodegradability.
The invention provides a preparation method of a bionic antibacterial hemostatic microsphere with a erythroid structure, which comprises the following steps:
a) Mixing carboxymethyl chitosan solution and gelatin solution to obtain mixed solution;
b) Placing the mixed solution into a syringe to obtain FeCl 3 The solution is a receiving solution, and carboxymethyl chitosan-gelatin microspheres are formed by an electrostatic instillation method;
the voltage in the electrostatic instillation method is 7-9 KV;
the distance between the nozzle of the injector and the surface of the receiving liquid is 4-7 cm;
the temperature of the mixed solution is 20-40 ℃.
Preferably, in the step a), the FeCl 3 The mass concentration of the solution is 3-5%.
Preferably, in the step a), the mass concentration of the carboxymethyl chitosan solution is 6% -10%;
the mass concentration of the gelatin is 3% -5%.
Preferably, the volume ratio of the carboxymethyl chitosan solution to the gelatin solution is (1-4) to 1.
Preferably, in the step b), the extrusion pressure in the electrostatic instillation method is 0.01 to 0.10cm/s.
Preferably, in the step b), in the electrostatic instillation method, a positive electrode of the electrostatic generator is connected with a nozzle of the injector, and a negative electrode of the electrostatic generator is connected with the copper sheet and placed in the receiving liquid.
Preferably, in the step b), the specification of the syringe is: a 27G syringe with an inner diameter of 210 μm.
The invention also provides the bionic antibacterial hemostatic microsphere with the erythroid structure prepared by the preparation method in the technical scheme.
The preparation method provided by the invention comprises the steps of mixing carboxymethyl chitosan solution and gelatin solution to prepare mixed solution, controlling the concentration and mixing ratio of the two solutions, and placing the obtained mixed solution into a syringe to prepare FeCl 3 The solution is a receiving solution, mixed liquid drops are injected into the receiving solution by an electrostatic instilling method, and the condition of electrostatic instilling is controlled, so that uniform spherical particles are obtained, the particle size of the particles is ensured to be moderate, and the liquid drops fall into FeCl 3 After the solution, at Fe 3+ Under the action, the carboxymethyl chitosan and the gelatin are chemically crosslinked and are combined with Fe 3+ Chemical coordination occurs to form carboxymethyl chitosan-gelatin crosslinked microspheres. The preparation method is simple and efficient, the microsphere size is uniform and controllable, and the problems of non-uniform size, complex process, toxicity caused by incomplete volatilization of the organic solvent and the like of the microsphere prepared by the emulsion method are avoided. In addition, the microsphere prepared by the invention has a bionic appearance structure extremely similar to red blood cells, has excellent water absorption performance, and can recover the original microsphere after swelling by water absorptionBall shape, and at the same time, has excellent hemostatic, antibacterial and biodegradability.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the overall structure of an apparatus used in an electrostatic instillation method;
FIG. 2 is an enlarged view of a portion of the apparatus used in the electrostatic instillation method;
FIG. 3 is a schematic illustration of the operation and reaction process after electrostatic instillation;
FIG. 4 is a graph of morphology and particle size characterization of various groups of samples;
FIG. 5 is an SEM and elemental analysis map of a dried microsphere sample;
FIG. 6 is a graph of the results of optical density measurements for each set of microsphere samples;
FIG. 7 is a graph of degradation testing of each set of microsphere samples;
FIG. 8 is a graph of the water absorption swelling ratio test for each set of microsphere samples;
FIG. 9 is a graph of the adsorption of fibronectin, platelets, and red blood cells by various groups of microsphere samples;
FIG. 10 is a graph of hemostasis time test for each set of microsphere samples;
FIG. 11 is a graph of the antimicrobial effect of each set of microsphere samples on E.Coli and S.aureus;
FIG. 12 is a graph of quantitative antibacterial effect of each microsphere sample on E.coli;
FIG. 13 is a graph of quantitative antimicrobial efficacy of various groups of microsphere samples against Staphylococcus aureus S.aureus;
FIG. 14 is a statistical graph of liver hemostasis time;
FIG. 15 is a schematic diagram showing the process of hemostasis of the liver by microsphere sample CMG 1;
FIG. 16 is a statistical plot of femoral artery hemostasis time;
FIG. 17 is a schematic diagram of the process of hemostasis of femoral artery by microsphere sample CMG 1;
FIG. 18 is a graph showing the effect of in vivo degradation test.
Detailed Description
The invention provides a preparation method of a bionic antibacterial hemostatic microsphere with a erythroid structure, which comprises the following steps:
a) Mixing carboxymethyl chitosan solution and gelatin solution to obtain mixed solution;
b) Placing the mixed solution into a syringe to obtain FeCl 3 The solution is a receiving solution, and carboxymethyl chitosan-gelatin microspheres are formed by an electrostatic instillation method;
the voltage in the electrostatic instillation method is 7-9 KV;
the distance between the nozzle of the injector and the surface of the receiving liquid is 4-7 cm;
the temperature of the mixed solution is 20-40 ℃.
The preparation method provided by the invention comprises the steps of mixing carboxymethyl chitosan solution and gelatin solution to prepare mixed solution, controlling the concentration and mixing ratio of the two solutions, and placing the obtained mixed solution into a syringe to prepare FeCl 3 The solution is a receiving solution, mixed liquid drops are injected into the receiving solution by an electrostatic instilling method, and the conditions of electrostatic instilling (such as voltage, receiving distance, temperature of the mixed solution and the like) are controlled, so that uniform spherical particles are obtained, the particle size of the particles is controlled to be moderate, and the liquid drops fall into FeCl 3 After the solution, at Fe 3+ Under the action, the carboxymethyl chitosan and the gelatin are chemically crosslinked and are combined with Fe 3+ Chemical coordination occurs to form carboxymethyl chitosan-gelatin crosslinked microspheres. The preparation method is simple and efficient, the microsphere size is uniform and controllable, and the problems of non-uniform size, complex process, toxicity caused by incomplete volatilization of the organic solvent and the like of the microsphere prepared by the emulsion method are avoided. In addition, the microsphere prepared by the invention has a bionic appearance structure very similar to red blood cells, has excellent water absorption performance, and can recover the original microsphere shape after swelling after water absorption, and is the same asAlso has excellent hemostatic, antibacterial and biodegradability.
[ about step a ]:
a) And mixing the carboxymethyl chitosan solution and the gelatin solution to obtain a mixed solution.
In the invention, the carboxymethyl chitosan solution is an aqueous solution of carboxymethyl chitosan. In the invention, the mass concentration of the carboxymethyl chitosan solution is 6% -10% (w/v%), and can be specifically 6%, 7%, 8%, 9% and 10%; if the concentration of the carboxymethyl chitosan solution is too low or too high, it is eventually difficult to form spherical particles. The mass concentration of the carboxymethyl chitosan solution is more preferably 8%.
In the invention, the gelatin solution is an aqueous gelatin solution. In the invention, the mass concentration of the gelatin solution is 3% -5%, and can be 3%, 4% and 5%; if the concentration of the gelatin solution is too low, the resulting microsphere product does not have elastic memory restorability, and if the concentration is too high, the microsphere product is easily swelled and broken when water swells to restore shape memory. The mass concentration of the gelatin solution is more preferably 4%.
In the invention, the volume ratio of the carboxymethyl chitosan solution to the gelatin solution is preferably (1-4) to 1, and can be specifically 1:1, 2:1, 3:1 and 4:1. By combining the concentration of the two solutions and controlling the volume ratio, the spherical particles can be smoothly aligned, and the microsphere product is ensured to have elastic memory restorability and not to be broken during swelling. The volume ratio is most preferably 1:1.
In the invention, the mass concentration of the carboxymethyl chitosan solution is 8%, the mass concentration of the gelatin solution is 4%, and when the volume ratio of the carboxymethyl chitosan solution to the gelatin solution is 1:1, the water absorption swelling property, the antibacterial property, the hemostatic property and other properties of the finally obtained microsphere are optimal.
In the present invention, the mixing means is preferably magnetic stirring mixing. The rotation speed of the mixing is preferably 300-700 rpm, and can be specifically 300rpm, 400rpm, 500rpm, 600rpm and 700rpm; the mixing time is preferably 10 to 14 hours, and may be specifically 10 hours, 11 hours, 12 hours, 13 hours, or 14 hours. After mixing, a uniform and transparent mixed solution is obtained.
In the present invention, the temperature of the mixed solution is controlled to 20 to 40℃and specifically may be 20℃21℃22℃23℃24℃25℃26℃27℃28℃29℃30℃31℃32℃33℃34℃35℃36℃37℃38℃39℃40℃40 ℃.
[ concerning step b ]:
b) Placing the mixed solution into a syringe to obtain FeCl 3 The solution is a receiving solution, and carboxymethyl chitosan-gelatin microspheres are formed by an electrostatic instillation method.
In the present invention, the syringe preferably has the following specifications: a 27G syringe with an inner diameter of 210 μm. The injector with the specification is favorable for smoothly balling and ensures that the particle size of the spherical particles is not too large.
In the present invention, the FeCl 3 The solution is FeCl 3 An aqueous solution. In the present invention, the FeCl 3 The mass concentration of the solution is 3% -5%, and can be 3%, 4% and 5%; if the concentration is too low, the crosslinking degree of the microspheres is poor, the microspheres are easy to deform or destroy, if the concentration is too high, the surface of the microspheres is easy to excessively crosslink, and the internal crosslinking of the microspheres is insufficient, so that the microspheres are uneven and unstable in properties. The FeCl 3 The mass concentration of the solution is more preferably 4%.
In the electrostatic instillation method, the positive electrode of the electrostatic generator is connected with the nozzle of the injector, and the negative electrode of the electrostatic generator is connected with the copper sheet and placed in the receiving liquid. Referring to fig. 1-2, fig. 1 is a schematic view of the overall structure of an apparatus used in the electrostatic instillation method, and fig. 2 is a partially enlarged view of the apparatus used in the electrostatic instillation method; wherein a is a constant speed propeller, b is an electrostatic generator, c is a stepping motor for controlling the constant speed propeller, d is a temperature controller, and e is a heating module.
In the invention, the voltage in the electrostatic instillation method is 7-9 KV, and can be specifically 7.0KV, 7.5KV, 8.0KV, 8.5KV and 9.0KV; if the voltage is too low, the spherical particles are formed into balls by gravity, and if the voltage is too high, the spherical particles are difficult to form balls. The voltage is more preferably 7.5KV. In the present invention, the extrusion pressure in the electrostatic instillation method is preferably 0.01 to 0.10cm/s, and specifically may be 0.01cm/s, 0.02cm/s, 0.03cm/s, 0.04cm/s, 0.05cm/s, 0.07cm/s, 0.08cm/s, 0.09cm/s, 0.10cm/s; under the pressure, the balling and uniformity can be ensured. The voltage is more preferably 0.05cm/s.
In the invention, the distance (namely the receiving distance) between the nozzle of the injector and the surface of the receiving liquid is 4-7 cm, and the distance can be specifically 4.0cm, 4.5cm, 5.0cm, 5.5cm, 6.0cm, 6.5cm and 7.0cm; under the control of the distance, the balling is ensured, the particle diameter of the spherical particles is ensured to be 80-320 mu m, and the spherical diameter is not excessively large. More preferably, the distance is 5.5cm.
In the invention, the mixed solution obtained in the step a) is completely introduced into FeCl by an electrostatic instillation method 3 After the solution was received in the liquid, stirring and mixing were performed. The stirring and mixing speed is preferably 300rpm, and the time is preferably 12h. During this period, in Fe 3+ Under the action, the carboxymethyl chitosan and the gelatin are chemically crosslinked and are combined with Fe 3+ Chemical coordination occurs to form carboxymethyl chitosan-gelatin crosslinked microspheres. The operation and reaction process are shown in fig. 3, and fig. 3 is a schematic diagram of the operation and reaction process after electrostatic instillation.
The main structure of the molecules of the obtained microsphere is as follows:
in the present invention, after the above treatment, it is preferable to further carry out: and (3) carrying out solid-liquid separation on the receiving liquid after receiving the materials, so that the formed microspheres are separated from the liquid, and the carboxymethyl chitosan-gelatin crosslinked microspheres are obtained. The solid-liquid separation mode is not particularly limited, and is a conventional separation mode in the field, such as filtration and the like. The particle size of the microsphere obtained by solid-liquid separation is 80-320 mu m. In the present invention, the solid-liquid separation is preferably followed by further drying to obtain dry microspheres. The drying temperature is preferably 20 to 30 ℃. In the present invention, the particle size of the dried microspheres obtained after drying is 35 to 115. Mu.m. The wet microspheres obtained after solid-liquid separation and the dried microspheres after drying can be sold as products directly after proper sterilization treatment, wherein the wet microspheres are required to be stored in absolute ethyl alcohol.
In the preparation method provided by the invention, carboxymethyl chitosan solution and gelatin solution are mixed to prepare mixed solution, the concentration and the mixing proportion of the two solutions are controlled, and the obtained mixed solution is placed in a syringe to prepare FeCl 3 The solution is a receiving solution, mixed liquid drops are injected into the receiving solution by an electrostatic instilling method, and the condition of electrostatic instilling is controlled, so that uniform spherical particles are obtained, the particle size of the particles is ensured to be moderate, and the liquid drops fall into FeCl 3 After the solution, at Fe 3+ Under the action, the carboxymethyl chitosan and the gelatin are chemically crosslinked and are combined with Fe 3+ Chemical coordination occurs to form carboxymethyl chitosan-gelatin crosslinked microspheres. The preparation method is simple and efficient, the microsphere size is uniform and controllable, and the problems of non-uniform size, complex process, toxicity caused by incomplete volatilization of the organic solvent and the like of the microsphere prepared by the emulsion method are avoided. In addition, the microsphere prepared by the invention has a bionic appearance structure extremely similar to red blood cells, has excellent water absorption performance, can recover the original microsphere shape after swelling by water absorption, and has excellent hemostatic property, antibacterial property and biodegradability.
For a further understanding of the present invention, preferred embodiments of the invention are described below in conjunction with the examples, but it should be understood that these descriptions are merely intended to illustrate further features and advantages of the invention, and are not limiting of the claims of the invention.
Examples 1 to 4 and comparative example 1
1. Preparation of samples
a) Carboxymethyl chitosan solution (mass concentration 8%, w/v%) and gelatin solution (mass concentration 4%, w/v%) are mixed according to a certain volume ratio, wherein the volume ratio is 1:0 (i.e. gelatin solution is not added), 1:1, 2:1, 3:1, 4:1 (corresponding products are respectively marked as CMCM, CMG1, CMG2, CMG3 and CMG 4), magnetic stirring is carried out, the rotating speed is 300rpm, the time is 12 hours, uniform transparent solution is obtained, and the temperature is controlled to 25 ℃.
b) The resulting solution was placed in a syringe (gauge: 27G, inner diameter 210 μm), feCl 3 The water solution (the mass concentration is 5%, w/v%) is the receiving solution, the receiving distance between the nozzle of the injector and the receiving solution is controlled to be 5.5cm, the positive electrode of the static generator is connected to the nozzle, the negative electrode is connected to the copper sheet and is placed in the receiving solution (the device diagram is shown in figure 1), the voltage is set to be 7.5KV, and the extrusion pressure is set to be 0.05cm/s, so that static instillation is carried out.
c) And after the electrostatic instillation is finished, filtering the receiving liquid to obtain carboxymethyl chitosan-gelatin microspheres (wet microspheres). And then drying the microspheres to obtain the dried microspheres. 5 microsphere samples were designated CMCM (as control, i.e., comparative example 1 product), CMG1, CMG2, CMG3, CMG4, respectively.
2. Characterization and testing
(1) Morphology and particle size analysis
And (3) performing optical lens morphology and particle size analysis on the microspheres filtered in the step c) (i.e. the microspheres before drying), the microspheres dried in the step c) and the microspheres swelled by water absorption respectively. The results are shown in fig. 4, and fig. 4 is a graph of morphology and particle size characterization of each group of samples; wherein group a represents the morphology and particle size distribution of the microspheres after filtration in step c), group b represents the morphology and particle size distribution of the microspheres after drying in step c), and group c represents the morphology and particle size distribution of the microspheres after swelling by water absorption. The test procedure for water swelling is described in the following item (4).
Meanwhile, carrying out scanning electron microscope characterization and element analysis on the microspheres dried in the step c), wherein the results are shown in fig. 5, and fig. 5 is an SEM and element analysis diagram of a dried microsphere sample; wherein the first behavior is an SEM image of dry microspheres, the second behavior is an enlarged SEM image of dry microspheres, and the third behavior is an elemental analysis image of dry microspheres.
As can be seen from fig. 4, the samples CMG1, CMG2, CMG3, CMG4 all exhibited spherical morphology and particles were relatively uniform before and after drying; wherein, in the sample of group a (i.e. the microspheres before drying), the particle size of the sample CMG1 is 200.5-282.3 μm, and the average particle size is about 248 μm; the particle size of the sample CMG2 is 251.2-311.1 mu m, and the average particle size is about 285.6 mu m; the particle size of the sample CMG3 is 199.5-281.9 mu m, and the average particle size is about 152.9 mu m; the sample CMG4 particles had a particle size of 200-304 μm and an average particle size of about 253. Mu.m. In the samples of group b (i.e., dried microspheres), the sample CMG1 particles had a particle size of 39.7-110.7 μm and an average particle size of about 69.9 μm; the particle size of the sample CMG2 is 54.6-105.6 mu m, and the average particle size is 79.0 mu m; the particle size of the sample CMG3 is 54.6-100.1 mu m, and the average particle size is about 69.4 mu m; the sample CMG4 particles had a particle size of 50.4 to 112 μm and an average particle size of about 75.4. Mu.m. In the samples of group c (i.e., the microspheres after water swelling), the particle size of the sample CMG1 is 1633.4-2749.7 μm, and the average particle size is about 2174.0 μm; the particle size of the sample CMG2 is 989.4-2909 mu m, and the average particle size is about 1780.2 mu m; the particle size of the sample CMG3 is 1044.1-3219.9 mu m, and the average particle size is about 2097.0 mu m; the particle size of the sample CMG4 was 864.7-2540.1 μm and the average particle size was about 1806.2. Mu.m.
As can be seen from the sample morphology graph in fig. 4, in the samples of group b (i.e. the dried microspheres), the samples CMG1, CMG2, CMG3, CMG4 are substantially in a erythroid structure, and this structure can be more clearly shown in combination with fig. 5. In the samples of group c (i.e., the microspheres after water swelling), the samples CMCM were broken and the samples CMG1, CMG2, CMG3, CMG4 recovered the microsphere morphology. Wherein, the sample CMG1 (namely, when the volume ratio of carboxymethyl chitosan solution to gelatin solution is=1:1) has the dry microsphere closest to the disc-shaped bionic appearance of the red blood cells and has good swelling recovery performance.
(2) Compatibility test
Murine embryonic fibroblasts (BALB) were co-cultured with 5 samples of dry microspheres and the optical density values (OD) were measured at 1 day, 3 days, and 7 days of culture, respectively 450 ) The results are shown in fig. 6, and fig. 6 is a graph of the results of the optical density value test for each set of microsphere samples. Wherein, the higher the optical density value, the more the cell number is represented, which indicates that the cell proliferation effect is good.
It can be seen that each group of samples exhibited a certain compatibility, among which the cell compatibility of sample CMG1 was optimal.
(3) In vitro degradability test
The experimental procedure was as follows: weigh CMCM and CMG&(&The initial mass of the dry microspheres =1, 2,3, 4) is noted as W 0 . The microspheres were then soaked in 10mL centrifuge tubes filled with PBS. In a shaking table at 37 DEG CAnd (5) carrying out degradation experiment test at 100 revolutions. The degradation time of the microspheres was set to 1 week, 2 weeks and 3 weeks, respectively. The PBS solution was removed by the indicated time and the sample was gently rinsed 3 times with deionized water to remove residual phosphate. The sample was then freeze-dried until the mass of the multiple weighed samples was unchanged, designated W n . Each group was at least five replicates. The degradation rate calculation formula is as follows: degradation rate (%) = (W) 0 -W n )/W 0 X100％。
Referring to fig. 7, fig. 7 is a degradation chart of each microsphere sample, and it can be seen that each microsphere can degrade with time, and the microspheres significantly degrade after 21 days, and the microspheres remain. The microsphere obtained by the invention has good degradability.
(4) Test of Water swelling Property
The experimental procedure was as follows: CMCM and CMG & (& = 1,2,3, 4) dry microspheres weighed 0.1g each. Samples were placed in cell culture dishes and incubated at 37℃with 10mL PBS solution. The observation was performed every 5 minutes until the microspheres swelled to a spherical shape. The sample was taken out of the PBS solution and excess water was sucked up with filter paper, and the mass of the swollen microspheres was weighed and recorded as W. And the diameter of the microspheres after swelling was counted using ImageJ software. The water absorption swelling ratio is calculated as: water absorption swelling ratio (%) = (W-0.1)/0.1X100%.
Referring to fig. 8, fig. 8 is a graph showing the water absorption swelling ratio of each microsphere sample, and it can be seen that each microsphere sample has a swelling water absorption ratio of more than 800% and shows a certain water absorption swelling property, wherein the water absorption swelling ratio of the sample CMG1 is the highest. Meanwhile, according to the test results of the (1) shown in the fig. 4-5, the sample CMCM is swelled and ruptured, and the samples CMG1, CMG2, CMG3 and CMG4 recover the microsphere morphology after swelling, so that better stability is shown.
(5) Adhesion test of fibronectin, platelets and Red Blood Cells (RBC) on microsphere sample surfaces
The experimental procedure was as follows: proper amount of fresh rabbit blood was centrifuged for 10min (800 rpm) to obtain erythrocytes and platelet-rich PRP. The red blood cells were repeatedly washed and centrifuged three times to remove the excess platelets. 15mg of the dried microspheres were placed in 48 well plates, 100. Mu.L of red blood cells and PRP were added to each well plate and incubated at 37℃for 60min. The samples were washed with PBS to remove non-adherent erythrocytes, platelets and fibronectin by a preset time. Then fixing with 2.5% glutaraldehyde solution at 4deg.C for 24 hr, gradient dehydrating with 20%, 40%, 60%, 80% and 100% ethanol solution, naturally drying, and observing adhesion of erythrocyte, platelet and fibronectin on microsphere surface under SEM, wherein the platelet and fibronectin morphology is very different, and distinguishing and recording can be identified.
Referring to fig. 9 to 10, fig. 9 is a graph showing the adsorption of fibronectin, platelets, and erythrocytes by each microsphere sample, and fig. 10 is a graph showing the hemostatic time of each microsphere sample (blank group, i.e., blank group, is also provided). As can be seen from the SEM image of fig. 9, each microsphere sample has a certain adsorptivity to fibronectin, platelets and erythrocytes, wherein the sample CMG1 has the greatest adsorptivity, and the best adsorptivity is achieved; as can be seen from fig. 10, the hemostatic time of each microsphere sample was reduced compared to the blank, wherein the hemostatic time required for sample CMG1 was the shortest, demonstrating the strongest hemostatic capacity.
(6) Antibacterial test
The experimental procedure was as follows: CMCM and CMG&(&Antibacterial properties of microspheres=1, 2,3, 4), the study was performed using escherichia coli (e.coli) and staphylococcus aureus (s.aureus) as model bacteria. The dried microspheres were spread over the bottom of a 48-well plate, then 1mL of 75% alcohol was added to each well and sterilized by irradiation with ultraviolet light for 2 hours. Residual ethanol was removed by washing 3 times with sterile PBS, followed by addition of 500. Mu.L of prepared E.coli and Staphylococcus aureus (1X 10) 5 CFU/ml). Incubate at 37℃for 3, 6 and 12 hours, to a preset time, transfer 150. Mu.L of the wash solution to a 96-well plate, and measure absorbance of the supernatant at 600nm by using a microplate reader. The blank group was not sampled, and 5 parallel experiments were performed for each group.
Bacterial viability was tested using a live/dead bacterial viability detection kit. Living bacteria were stained green with fluorescent probe (SYTO 9) and dead bacteria were stained red with fluorescent probe (propidium iodide, PI). After 12 hours of co-cultivation of E.coli and Staphylococcus aureus with the microspheres, the medium was discarded and gently washed 3 times with PBS. Equal amounts of SYTO 9 and Propidium Iodide (PI) were mixed in a microcentrifuge tube. mu.L of the mixed dye was added to each well and incubated for 15 minutes in the dark. And observed under a fluorescence microscope. The green fluorescence was recorded using FITC filters (λex/λem=488/500 nm) and the red fluorescence was recorded using TRICT filters (λex/λem=550/600 nm).
The test results are shown in FIGS. 11-13, and FIG. 11 is a graph showing the antibacterial effect of each set of microsphere samples on E.Coli and S.aureus, red for dead bacteria and green for viable bacteria; FIG. 12 is a graph of quantitative antibacterial effect of each microsphere sample on E.coli (with a blank control set); FIG. 13 is a graph of quantitative antimicrobial effect of each microsphere sample on Staphylococcus aureus S.aureus (with a blank control).
It can be seen that each group of microsphere samples has a certain antibacterial effect on E.Coli and S.aureus of staphylococcus aureus, wherein the sample CMG1 has the lowest test value on E.Coli and S.aureus of staphylococcus aureus and shows the best antibacterial effect.
(7) Hemostatic test
The experimental procedure was as follows:
the in vivo hemostatic effect of CMCM and CMG1 microspheres was studied by SD rat liver injury model. SD male rats were used as animal models (200 g,6 weeks old) in this experiment. SD rats were randomly divided into 4 groups of 6. Animals were anesthetized and fixed on surgical boards by injection of 10% chloral hydrate (0.3 mL/100 g). The liver was exposed by abdominal dissection and a bleeding wound was made using a sterile 26G needle with an outer diameter of 460 μm. The prepared commercial chitosan powder (CHP), CMCM and CMG1 microspheres, each 0.3g, were individually blocked at the site of liver injury. SD rats without any treatment after liver injury were used as control group. Wound hemostasis was observed every 10 seconds during hemostasis until the wound stopped bleeding recording hemostasis time.
The in vivo hemostatic effect of CMCM and CMG1 microspheres was further studied by SD rat leg artery rupture model. Male SD rats (200 g,6 weeks old) were divided into 4 groups, each group being divided equally into 5 at random. Mice were anesthetized by injection of 10% chloral hydrate (0.3 mL/100 g) and fixed to a wood board. The femoral artery was dissected from the leg of the SD rat and a filter paper prepared in advance was placed under the artery. The arteries were ruptured with medical scissors, followed by plugging the arterial lesion site with 1g of commercial chitosan powder (CHP), CMCM and CMG1 microspheres, respectively, prepared in advance, under gentle pressure. The hemostatic time was recorded during hemostasis.
The experimental tests were performed using the commercial chitosan hemostatic powder CHP, the dry microsphere sample CMCM, and the dry microsphere sample CMG1 as test subjects, respectively, while a blank control group was set. Referring to fig. 14 to 17, fig. 14 is a statistical chart of hemostasis time of liver, fig. 15 is a schematic diagram of a process of performing hemostasis effect of microsphere sample CMG1 on liver, fig. 16 is a statistical chart of hemostasis time of femoral artery (wherein blank group is bleeding all the time and hemostasis time cannot be counted), and fig. 17 is a schematic diagram of a process of performing hemostasis effect of microsphere sample CMG1 on femoral artery.
As can be seen from fig. 14 and 16, compared with the blank control group, the commercial chitosan hemostatic powder CHP and the microsphere sample CMCM, the liver hemostatic time and the femoral hemostatic time of the microsphere sample CMG1 group are both obviously reduced, which proves that the microsphere sample CMG1 remarkably improves the hemostatic effect.
(8) In vivo degradability test
The experimental procedure was as follows: using SD male rats as animal models (200 g,6 weeks old), animals were anesthetized and fixed on surgical plates by injection of 10% chloral hydrate (0.3 mL/100 g). A1 cm incision was made in the back of the rat, and 10mg of commercial chitosan powder (CHP), CMCM and CMG1 dry microspheres were implanted into subcutaneous tissue, respectively. The wounds were then opened after 9, 14 and 21 days, the degradation of the samples was recorded and the incision without any implant was used as a blank.
Test results referring to fig. 18, fig. 18 is a graph showing the effect of in vivo degradability test. It can be seen that after 28 days, the microsphere sample CMG1 group was almost completely degraded, showing the best in vivo degradability.
The principles and embodiments of the present invention have been described herein with reference to specific examples, the description of which is intended only to aid in understanding the method of the invention and its core concept, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims. The scope of the patent protection is defined by the claims and may include other embodiments that occur to those skilled in the art. Such other embodiments are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims.
Claims (4)
1. The preparation method of the bionic antibacterial hemostatic microsphere with the erythroid structure is characterized by comprising the following steps of:
a) Mixing carboxymethyl chitosan solution and gelatin solution to obtain mixed solution;
the mass concentration of the carboxymethyl chitosan solution is 6% -10%;
the mass concentration of the gelatin solution is 3% -5%;
the volume ratio of the carboxymethyl chitosan solution to the gelatin solution is (1-4) to 1;
the rotation speed of the mixing is 300-700 rpm, and the time is 10-14 h;
the temperature of the mixed solution is 20-35 ℃;
b) Placing the mixed solution into a syringe to obtain FeCl 3 The solution is a receiving solution, and carboxymethyl chitosan-gelatin microspheres are formed by an electrostatic instillation method;
the FeCl 3 The mass concentration of the solution is 3% -5%;
the voltage in the electrostatic instillation method is 7-9 KV;
the distance between the nozzle of the injector and the surface of the receiving liquid is 4-7 cm;
the extrusion pressure in the electrostatic instillation method is 0.01-0.10 cm/s.
2. The method according to claim 1, wherein in the step b), the positive electrode of the electrostatic generator is connected to the nozzle of the syringe, and the negative electrode is connected to the copper sheet and placed in the receiving liquid.
3. The method according to claim 1, wherein in the step b), the syringe has the following specifications: a 27G syringe with an inner diameter of 210 μm.
4. A biomimetic antibacterial hemostatic microsphere with a erythroid structure made by the method of any one of claims 1-3.
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| CN105106967A (en) * | 2015-08-07 | 2015-12-02 | 四川大学 | Chitosan microparticles with shapes and sizes identical to lurid cells and preparation method of chitosan microparticles |
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| CN105106967A (en) * | 2015-08-07 | 2015-12-02 | 四川大学 | Chitosan microparticles with shapes and sizes identical to lurid cells and preparation method of chitosan microparticles |
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