CN116650705B - Self-gelling powder and application thereof - Google Patents
Self-gelling powder and application thereof Download PDFInfo
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- CN116650705B CN116650705B CN202310371394.0A CN202310371394A CN116650705B CN 116650705 B CN116650705 B CN 116650705B CN 202310371394 A CN202310371394 A CN 202310371394A CN 116650705 B CN116650705 B CN 116650705B
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0052—Mixtures of macromolecular compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/04—Materials for stopping bleeding
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention provides self-gelling powder and application thereof, wherein the self-gelling powder is prepared by mixing a solution A, a solution B and an alkylated chitosan solution to obtain a mixed solution, freeze-drying the mixed solution and grinding the mixed solution to obtain the self-gelling powder, wherein the solution A comprises polydiene dimethyl ammonium chloride solution and polyethyleneimine solution, and the solution B comprises sodium polystyrene sulfonate solution and polyacrylic acid solution. According to the invention, alkylated chitosan is selected as a raw material, and a specific solution A and a specific solution B are selected, and the three solutions are uniformly mixed and freeze-dried to obtain self-gelling powder, and experimental results show that the freeze-drying powder provided by the invention has the advantages of strong hemostatic effect, short gelation conversion time and good mechanical strength.
Description
Technical Field
The invention relates to the technical field of medical biological materials, in particular to self-gelation powder formed in situ and application thereof.
Background
Wound healing is a physiological process that spontaneously initiates in the body after a wound that plays an important role in maintaining skin integrity. It generally involves four sequential and overlapping physiological processes including hemostasis, inflammation, regeneration, and remodeling. Improper wound healing can lead to hypertrophic scars and ulcers, and even to infection and death. In recent years, intervention in wound healing has attracted considerable attention in the field of medical care such as biomaterials, tissue engineering, and the like. Therefore, an ideal wound healing therapeutic material should have excellent procoagulant and antibacterial capabilities, as well as good cell proliferation effects, enabling it to participate in each of the four phases. In recent years, various functional materials for wound therapy have been developed, including porous sponges, micro/nano hydrogel particles, biocompatible hydrogels, synthetic fibers, liposomes, and the like.
To date, conventional wound treatment materials in common use have more or less certain drawbacks, such as poor effect of conventional gauze, hemostatic powder, etc. in terms of wound moisturization. And some patch materials have some skin irritation when applied.
Polyethyleneimine (PEI) and polyacrylic acid (PAA) are common cationic and anionic polymers. PEI/PAA can quickly form viscous hydrogel through physical crosslinking when absorbing moisture, can absorb blood, can form a relatively stable physical barrier at a wound, and has good flexibility. Can provide a moist environment for the wound, is beneficial to the hemostasis of the wound, and is an ideal wound dressing. Polydiene dimethyl ammonium chloride (PDDA) and sodium polystyrene sulfonate (PSS) can form nanoparticles in situ in the polymer hydrogel framework, the nanoparticles and the polymer matrix have non-covalent interactions, and the tensile strength and the tensile toughness of the polymer material can be enhanced at the same time, so that the blood pressure resistance of the gel is obviously enhanced. However, the gel-type wound healing materials disclosed at present have poor hemostatic effects. Therefore, it is of great importance to provide a wound healing material which is not only good in mechanical strength but also good in hemostatic effect.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a self-gelling powder and its application, and the self-gelling powder provided in the present aspect has good hemostatic effect and good mechanical strength.
Compared with the prior art, the invention provides self-gelling powder and application thereof, wherein the self-gelling powder is prepared by mixing an A solution, a B solution and an Alkylated Chitosan (ACS) solution to obtain a mixed solution, freeze-drying the mixed solution and grinding the mixed solution to obtain the self-gelling powder, wherein the A solution comprises polydiene dimethyl ammonium chloride solution and polyethyleneimine solution, and the B solution comprises sodium polystyrene sulfonate solution and polyacrylic acid solution. According to the invention, alkylated chitosan is selected as a raw material, and a specific solution A and a specific solution B are selected, and the three solutions are uniformly mixed and freeze-dried to obtain self-gelling powder, and experimental results show that the freeze-drying powder provided by the invention has the advantages of strong hemostatic effect, short gelation conversion time and good mechanical strength.
Drawings
FIG. 1 is an infrared spectrum of alkylated chitosan prepared in example 1 of the present invention;
FIG. 2 is a graph showing the measurement of hydrogel modulus of the polyethyleneimine solution and the polyacrylic acid solution prepared in example 3 according to the present invention in different proportions;
FIG. 3 is a graph showing the modulus of hydrogel prepared in example 5 of the present invention with different alkylated chitosan contents;
FIG. 4 is a graph showing the viscosity test of hydrogels with different alkylated chitosan contents prepared in example 5 of the present invention;
FIG. 5 is a graph showing the modulus of hydrogel of different polydiene dimethyl ammonium chloride and sodium polystyrene sulfonate contents prepared in example 7 of the present invention;
FIG. 6 is a graph showing the viscosity test of hydrogels prepared in example 7 of the present invention with different polydiene dimethyl ammonium chloride and sodium polystyrene sulfonate contents;
FIG. 7 is a graph showing gel transition times for in situ self-gelling powders containing different alkylated chitosans;
FIG. 8 is a schematic diagram of hydrogel synthesis;
FIG. 9 is an image and a scanning electron microscope image of the in situ formed self-gelling powder prepared in example 11 of the present invention;
FIG. 10 shows the results of the hemolysis rate of the self-gelling powder and its components;
FIG. 11 shows the cell proliferation rate results of the self-gelling powder and its components;
FIG. 12 is a graph showing the bacteriostasis of the self-gelling powder and its components against E.coli and Staphylococcus aureus;
FIG. 13 is an in vitro clotting time of the self-gelling powder and its components prepared;
FIG. 14 is an in vitro thrombogram of the self-gelling powder and its components prepared;
Fig. 15 is a graph showing the results of a rat wound healing model experiment for preparing the self-gelled powder and its components.
Detailed Description
The invention provides self-gelling powder, which is prepared by mixing a solution A, a solution B and an alkylated chitosan solution to obtain a mixed solution, then freeze-drying the mixed solution, and grinding the mixed solution;
wherein the solution A comprises polydiene dimethyl ammonium chloride solution and polyethyleneimine solution;
the solution B comprises a sodium polystyrene sulfonate solution and a polyacrylic acid solution.
According to the invention, the alkylated chitosan is preferably one or more of dodecyl chitosan, tetradecyl chitosan, hexadecyl chitosan and octadecyl chitosan, more preferably dodecyl chitosan, tetradecyl chitosan, hexadecyl chitosan or octadecyl chitosan, and most preferably dodecyl chitosan, the initial concentration of the alkylated chitosan solution is 0.2-1 wt%, more preferably 0.4-0.8 wt%, most preferably 0.6wt%, and the alkylated chitosan solution preferably accounts for 8-15% of the total volume in the mixed solution, more preferably 10-13%;
in the present invention, the preparation method of the alkylated chitosan preferably comprises the following steps:
providing a chitosan solution;
dripping dodecanal, tetradecaldehyde, hexadecaldehyde and octadecanol into each group of chitosan solution respectively, and carrying out condensation reaction under an acidic condition to obtain four groups of imidized chitosan;
Mixing the four groups of imidized chitosan with a reducing agent for reduction reaction to obtain long-chain alkylated chitosan.
In the invention, the mass concentration of chitosan in each group of chitosan solution is preferably 10.0g/L, and the chitosan solution preferably comprises chitosan, acetic acid solution and ethanol. The concentration of the acetic acid solution is preferably 2wt%, and the volume ratio of the acetic acid solution to ethanol is preferably 1:1.
After the chitosan solution is obtained, the dodecaoctadecanol is dripped into each group of chitosan solution to carry out condensation reaction, so that different imidized chitosan is obtained. In the present invention, the acidic condition is provided by an acetic acid solution. The temperature of the chitosan solution is preferably 45 ℃ during the dropwise addition, and the dropwise addition is preferably dropwise addition. The ratio of the four fatty aldehydes to the chitosan is preferably 0.125-0.5:1, more preferably 0.125:1, 0.25:1 or 0.5:1, calculated according to the molar ratio of aldehyde groups in the fatty aldehydes to amino groups in the chitosan. The time of the condensation reaction is preferably 4 hours.
After the condensation reaction, the reaction solution is preferably cooled to room temperature to obtain imidized chitosan.
After four groups of imidized chitosan are obtained, the imidized chitosan and a reducing agent are mixed for reduction reaction, so that four groups of long-chain alkylated chitosan are obtained. In the present invention, the reaction solution of the condensation reaction is directly mixed with the reducing agent without post-treatment. In the present invention, the reducing agent is preferably sodium borohydride, and the molar ratio of the sodium borohydride to aldehyde groups in the fatty aldehyde is preferably 3:1. In the present invention, the reducing agent is preferably used in the form of a reducing agent solution, and the mixing is preferably to drop the reducing agent solution into four groups of imidized chitosan. The temperature of the reduction reaction is preferably room temperature, and the time of the reduction reaction is preferably 3 hours.
In the invention, chitosan is condensed with fatty aldehyde to obtain imidized chitosan, and then reduced by sodium borohydride to obtain alkylated chitosan.
After the reduction reaction, the invention preferably carries out post-treatment on the obtained reduction reaction product to obtain the long-chain alkylated chitosan. In the present invention, the post-treatment preferably includes the steps of:
regulating the pH value of the reduction reaction product to 10, and separating out solids;
Centrifugally washing the solid to obtain neutral solid;
Dissolving the neutral solid in acetic acid solution to obtain solution, preserving at low temperature, and then pressurizing and filtering to obtain impurity-removed solid;
adjusting the pH value of the impurity-removed solid to 10, and centrifugally separating and precipitating;
And washing the precipitate to neutrality, and freeze-drying to obtain four groups of long-chain alkylated chitosan.
The invention preferably adds sodium hydroxide solution to the reduction reaction product to adjust the pH, the concentration of the sodium hydroxide solution preferably being 4 wt.%.
In the invention, the centrifugal washing is preferably three times of methanol washing and three times of ethanol washing, and finally water washing is carried out until the washing is neutral;
after the neutral solid is obtained, the neutral solid is preferably dissolved in acetic acid solution with the concentration of 2wt%, the temperature of the low-temperature preservation is preferably 4 ℃, and the time of the low-temperature preservation is preferably 30min.
In the present invention, the pH of the impurity-removed solid is preferably adjusted by adding the impurity-removed solid to a sodium hydroxide solution, and adjusting ph=10.
Experimental researches show that the chitosan provided by the application introduces a carbon chain of long-chain alkyl on the amino of a chitosan side chain, can be embedded into a cell membrane to form a chitosan-blood cell gel network structure, and enhances the hemostatic effect of the powder.
According to the invention, in the solution A, the initial concentration of the polyethyleneimine solution is 5-8wt%, more preferably 6-7wt%, and the initial concentration of the polydiene dimethyl ammonium chloride solution is preferably 7-12wt%, more preferably 8-10wt%.
According to the invention, in the solution B, the initial concentration of the polyacrylic acid solution is preferably 5-8wt%, more preferably 6-7wt%, and the initial concentration of the sodium polystyrene sulfonate solution is 9-12wt%, more preferably 10-11wt%, and most preferably 10.3-10.5wt%.
According to the invention, in the mixed solution, the added polydiene dimethyl ammonium chloride solution preferably accounts for 3-7% of the total volume, more preferably 5-6%, the added polyethyleneimine solution preferably accounts for 38-42% of the total volume, more preferably 40-41%, the added polystyrene sodium sulfonate solution preferably accounts for 3-7% of the total volume, more preferably 5-6%, and the polyacrylic acid solution preferably accounts for 38-42% of the total volume, more preferably 40-41%. In the invention, the volume ratio of the added polyethyleneimine solution to the added polyacrylic acid solution is preferably (3-7): (7~3), preferably 3:7, 4:6, 5:5, 6:4, 7:3, more preferably 5:5, and the volume ratio of the added polydiene dimethyl ammonium chloride (PDDA) solution to the added sodium polystyrene sulfonate (PSS) solution is preferably 1:1.
According to the invention, the weight average molecular weight of the Polyethyleneimine (PEI) is preferably 65000-75000, more preferably 70000, the weight average molecular weight of the polyacrylic acid (PAA) is preferably 230000-250000, more preferably 240000, the weight average molecular weight of the polydiene dimethyl ammonium chloride (PDDA) is preferably 80000-150000, more preferably 100000, and the weight average molecular weight of the sodium polystyrene sulfonate (PSS) is preferably 65000-75000, more preferably 70000.
The application also provides application of the self-gelling powder in preparing a wound treatment material. The wound treatment material prepared from the self-gelling powder provided by the application has good hemostatic effect and good mechanical property.
It should be noted that the "initial concentration" in the present application means the concentration of the solution of each raw material to be prepared before mixing the solutions. The volume ratio of each solution in the mixed solution in the present application means the ratio of each solution containing the substance at a certain concentration in the mixed solution. In the present application, the solvent is not particularly specified in the solution, and the solvent is water.
The self-gelling powder is prepared by mixing an A solution, a B solution and an alkylated chitosan solution to obtain a mixed solution, freeze-drying the mixed solution and grinding the mixed solution to obtain the self-gelling powder, wherein the A solution comprises PDDA and PEI, and the B solution comprises PAA and PSS. According to the application, the alkylated chitosan solution is selected as a raw material, and the specific solution A and the specific solution B are selected, the three solutions are uniformly mixed and freeze-dried to obtain self-gelling powder, and experiments show that the alkylated chitosan provided by the application introduces a long-chain alkyl carbon chain on the amino group of a chitosan side chain, and the long carbon chain has similar polarity with a phospholipid bilayer of a cell membrane, so that the alkylated chitosan can be embedded into the cell membrane to form a chitosan-blood cell gel network structure, and the hemostatic effect of the powder is enhanced. Meanwhile, the PDDA and the PSS added in the application can form nano particles at the intersection point of the polymer frameworks, so that the hydrogel obtained from the prepared gelled powder has good mechanical strength, and the product is self-gelled powder which is convenient to carry and has good market application prospect.
The following description of embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is shown, however, only some, but not all embodiments of the invention are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The raw materials provided by the invention are PEI purchased from Shanghai Alasdine Biochemical technology Co., ltd, the weight average molecular weight is 70000, PDDA is purchased from Shanghai Michelin Biochemical technology Co., ltd, the weight average molecular weight is 100000-200000, PAA is purchased from Beijing carboline Biochemical technology Co., ltd, the weight average molecular weight is 240000, PSS is purchased from Shanghai Alasdine Biochemical technology Co., ltd, and the weight average molecular weight is 70000.
Example 1
Preparing twelve-octadecyl chitosan.
1. Accurately weighing 2.0g of four groups of CS powder, adding into 100mL of 2wt% acetic acid solution, adding 100mL of ethanol after chitosan is dissolved, placing into a 35 ℃ water bath kettle, and stirring and uniformly mixing to obtain chitosan solution;
2. Taking dodecanal-octadecanol, respectively dropwise adding the dodecanal-octadecanol into four groups of chitosan solutions according to the molar ratio of-NH 2:0.125, stirring and reacting for 4 hours, and cooling to room temperature;
3. Accurately weighing NaBH 3, dissolving in pure water according to the concentration of 0.1g/mL, slowly dripping NaBH 3 solution into the reaction solution obtained in the step 2 in a molar ratio of NaBH 3 -CHO=3:1 while continuously stirring, and stirring at room temperature for reaction for 3 hours;
4. Adding a 4wt% NaOH solution into the solution obtained in the step 3 to ensure that the pH value is=10, separating out a product, centrifugally washing at a rotating speed of 8000rad/min, washing with methanol for three times and ethanol for three times in sequence, and washing with water to be neutral;
5. dissolving the product obtained in the step 4 in 2wt% acetic acid, fully dissolving, then placing the mixture into a temperature of 4 ℃ for preservation for 30min, carrying out vacuum filtration to remove impurities, adding 4wt% NaOH solution for stirring until the pH value is=10, centrifugally separating and precipitating at a rotating speed of 8000rad/min, and washing the precipitate to be neutral;
6. And 5, freeze-drying the obtained product for 30 hours to obtain four groups of different alkylated chitosan, namely ACS-1, ACS-2, ACS-3 and ACS-4.
Example 2
The alkylated chitosan obtained in example 1 was measured by infrared spectroscopy by mixing the prepared alkylated chitosan with potassium bromide powder by KBr tabletting, grinding into fine powder, making into translucent flakes by a tabletting machine, and placing the flakes into an infrared spectrometer for testing, wherein the scanning range is 4000-400 cm -1. The infrared spectrograms of chitosan and alkyl chitosan are shown as1, FIG. 1 is an infrared spectrogram of the alkylated chitosan prepared in the embodiment 1 of the invention, FIG. 1 is a black spectrogram showing chitosan, and a red spectrogram is the alkylated chitosan prepared, and compared with chitosan, the infrared spectrogram shows that characteristic peaks representing amino groups of chitosan at 1590cm -1 after alkylation disappear, which indicates that substitution reaction occurs at the N position, and in addition, a new absorption peak appears at 1461cm -1, which indicates that-CH 2 -and-CH 3 groups are introduced into chitosan molecules. The above results demonstrate that the grafting reaction did occur on the amino groups of chitosan, and that the resulting product was alkylated chitosan.
Example 3
The effect of the ratio of Polyethylenimine (PEI) solution and polyacrylic acid (PAA) solution on gel strength was studied. PEI/PAA hydrogel is prepared, wherein the volume ratio of PEI solution to PAA solution is 3:7, 4:6, 5:5, 6:4, 7:3, PEI is purchased from Shanghai Alasdine Biotechnology Co., ltd, the weight average molecular weight is 70000, PAA is purchased from Beijing carboliner Biotechnology Co., ltd, and the weight average molecular weight is 240000.
1. 35ML, 30mL, 25mL, 20mL, 15mL of 5wt% PEI solution were separately prepared and placed in different beakers.
2. 15ML, 20mL, 25mL, 30mL, 35mL of 5wt% PAA solution were placed in different centrifuge tubes.
3. The magnet was placed in a beaker containing the PEI solution, which was placed on a magnetic stirrer.
4. Starting a magnetic stirrer, and slowly pouring the corresponding PAA solution into the mixture after stabilizing according to the volume ratio of the PEI solution to the PAA solution of 3:7, 4:6, 5:5, 6:4 and 7:3, so that the PEI solution and the PAA solution are uniformly mixed, and the hydrogel with certain strength is prepared.
5. The hydrogels were collected into different glass dishes, sealed with preservative film, and frozen overnight at-20 ℃.
6. The preservative film used for sealing was perforated, freeze-dried at-60 ℃ for a total of 30 hours.
7. The lyophilized xerogel was transferred to a mortar and ground into a fine powder.
Example 4
According to the technical scheme of example 3, 5 groups of PEI/PAA gel powders with different volume ratios are prepared.
PEI/PAA gel powder obtained in example 3 was measured by rheometer A Kinexus-type rheometer was used and a 20mm diameter plate was chosen to determine the powder modulus of each group. Each group was prepared by adding 1ml of distilled water to 0.2g of the powder, removing unabsorbed water, and placing on a test plate. The modulus test procedure was selected, and the test was performed at a fixed strain of 1.0% and a frequency of 1.000Hz with plates spaced 0.8mm apart and a set temperature of 37℃to determine the storage modulus (G ') and loss modulus (G') for each of the five groups of products. The results are shown in FIG. 2, and FIG. 2 is a graph showing the hydrogel modulus test of PEI solution and PAA solution prepared in example 3 according to the present invention at different ratios;
it can be seen from the figure that at a volume ratio of PEI solution to PAA solution of 5:5, the hydrogel formed has the highest storage modulus and the most optimal mechanical properties.
Example 5
The effect of alkylated chitosan content on gel hemostatic ability and mechanical strength was studied. Wherein the concentration of the alkylated chitosan is set to 0.2wt%, 0.4wt%, 0.6wt%, 0.8wt% and 1.0wt%, and the volume accounts for 10% of the total system. The PDDA solution concentration was fixed at 8wt% and the PSS solution concentration was fixed at 10.3wt%, each accounting for 5% of the total system. PEI was purchased from Shanghai Ala Biotechnology Co., ltd, the weight average molecular weight was 70000, PDDA was purchased from Shanghai Milin Biotechnology Co., ltd, the weight average molecular weight was 100000-200000, PAA was purchased from Beijing Barling Wikipedia, the weight average molecular weight was 240000, PSS was purchased from Shanghai Ala Biotechnology Co., ltd, the weight average molecular weight was 70000, and alkylated chitosan was dodecylchitosan prepared in example 1.
1. Five sets of mixed solutions containing 22.5ml of 5wt% PEI solution and 2.5ml of 8wt% PDDA solution were prepared and placed in a beaker as solution A.
2. Five sets of mixed solutions containing 22.5ml of 5wt% PAA solution and 2.5ml of 10.3wt% PSS solution were prepared and placed in centrifuge tubes as solution B.
3.5 ML of each of the alkylated chitosan solutions of 0.2wt%, 0.4wt%, 0.6wt%, 0.8wt% and 1.0wt% was prepared and placed in a centrifuge tube.
4. The magnet was placed in a beaker containing solution a and the whole was placed on a magnetic stirrer.
5. Starting a magnetic stirrer, and slowly pouring the solution B and the corresponding alkylated chitosan solution simultaneously after stabilizing to uniformly mix the solution B, the alkylated chitosan solution and the corresponding alkylated chitosan solution, so as to prepare the hydrogel with certain strength.
6. The hydrogel was collected in a glass dish, sealed with a preservative film, and frozen overnight at-20 ℃.
7. The preservative film used for sealing was perforated, freeze-dried at-60 ℃ for a total of 30 hours.
8. The lyophilized xerogel was transferred to a mortar and ground into a fine powder.
Example 6
Gel powders of different alkylated chitosan content were prepared as in example 5 and the modulus of the gel powder obtained in example 5 was determined by rheometer using a Kinexus-type rheometer, a 20mm diameter plate was selected for each set of powder modulus measurements. Each group was prepared by adding 1ml of distilled water to 0.2g of the powder, removing unabsorbed water, and placing on a test plate. The modulus test procedure was selected, and the test was performed at a fixed strain of 1.0% and a frequency of 1.000Hz with plates spaced 0.8mm apart and a set temperature of 37℃to determine the storage modulus (G ') and loss modulus (G') for each of the five groups of products. As a result, FIG. 3 is a graph showing the modulus of hydrogel prepared in example 5 according to the present invention, and it can be seen from the graph that the mechanical strength of the resulting hydrogel is decreased as the ACS content increases, as the alkylated chitosan content is inversely proportional to the storage modulus (G') of the hydrogel.
Gel powders with different alkylated chitosan contents were prepared according to example 5, and the viscosity stress of the gel powders obtained in example 5 was measured by a tensile tester, by selecting a tensile strength test mode using an intelligent electronic tensile tester, and testing the viscosity stress of each group of powders. Each group was prepared from 0.4g of the powder to give a hydrogel strip having a length of 4cm, a width of 1cm and a thickness of 2 mm. And (3) preparing the pig skin with the grease removed into the same size, adhering one end of the hydrogel to the outer side of the pig skin, overlapping the hydrogel and the pig skin into a strip shape, enabling the area of the overlapped part to be 1cm 2, and pressing a weight of 200g on the overlapped part of the hydrogel and the pig skin for 20min. Then, the test is carried out by a tensile testing machine, the fixed stretching speed is 25mm/min until the hydrogel and the pigskin are completely pulled apart, and the maximum breaking force is recorded. The viscosity stress is the maximum breaking force divided by the area of overlap between the hydrogel and the pigskin. The results are shown in FIG. 4, and FIG. 4 is a graph showing the viscosity test of the alkylated chitosan content according to the invention in example 5, wherein the alkylated chitosan content is proportional to the viscosity stress of the hydrogel, indicating that the adhesion of the hydrogel to skin increases with increasing alkylated chitosan content.
Conclusion 0.6wt% was chosen as the optimal concentration of alkylated chitosan solution in order to balance the mechanical strength, hemostatic ability and skin adhesion of the hydrogel.
Example 7
The influence of the content of PDDA and PSS on the hemostatic capacity and mechanical strength of the gel was studied. The 8wt% PDDA solution and 10.3wt% PSS solution each account for the same proportion in the total system, and five groups were set, 3%, 4%, 5%, 6%, 7%, respectively. PEI was purchased from Shanghai Ala Biotechnology Co., ltd, the weight average molecular weight was 70000, PDDA was purchased from Shanghai Milin Biotechnology Co., ltd, the weight average molecular weight was 100000-200000, PAA was purchased from Beijing Barling Wikipedia, the weight average molecular weight was 240000, PSS was purchased from Shanghai Ala Biotechnology Co., ltd, the weight average molecular weight was 70000, and alkylated chitosan was dodecylchitosan prepared in example 1.
1. Five 5wt% PEI solutions, each having a volume of 21mL, 20.5mL, 20mL, 19.5mL, and 19mL, were uniformly mixed with 8wt% PDDA solutions of 1.5mL, 2mL, 2.5mL, 3mL, and 3.5mL, respectively, and placed in a beaker as a solution A in total of 45 mL.
2. Five 5wt% PAA solutions, each having a volume of 19mL, 19.5mL, 20mL, 21.5mL, and 22mL, were uniformly mixed with 10.3wt% PSS solutions of 1.5mL, 2mL, 2.5mL, 3mL, and 3.5mL, respectively, and the total was 45mL, and placed in a centrifuge tube as solution B.
3. Five groups of 5ml of 0.6wt% alkylated chitosan solutions were prepared and placed in centrifuge tubes, respectively.
4. The magnet was placed in a beaker containing solution a and the whole was placed on a magnetic stirrer.
5. Starting a magnetic stirrer, and slowly pouring the corresponding solution B and the alkylated chitosan solution simultaneously after stabilizing, so that the three solutions are uniformly mixed, and the hydrogel with certain strength is prepared.
6. The hydrogels were collected into different glass dishes, sealed with preservative film, and frozen overnight at-20 ℃.
7. The preservative film used for sealing was perforated, freeze-dried at-60 ℃ for a total of 30 hours.
8. The lyophilized xerogel was transferred to a mortar and ground into a fine powder.
Example 8
Gel powders of different PDDA and PSS contents were prepared according to example 7, and the modulus of the gel powder obtained in example 7 was determined by rheometry using a Kinexus-type rheometer, and a 20mm diameter plate was selected for each group of powder modulus measurements. Each group was prepared by adding 1ml of distilled water to 0.2g of the powder, removing unabsorbed water, and placing on a test plate. The modulus test procedure was selected, and the test was performed at a fixed strain of 1.0% and a frequency of 1.000Hz with plates spaced 0.8mm apart and a set temperature of 37℃to determine the storage modulus (G ') and loss modulus (G') for each of the five groups of products. The results are shown in FIG. 5, and FIG. 5 is a graph showing the modulus of hydrogel prepared in example 7 according to the present invention with different PDDA and PSS contents;
It can be seen from the figure that the content of PDDA and PSS is proportional to the storage modulus (G') of the hydrogel, indicating that the mechanical strength of the formed hydrogel is enhanced as the content of PDDA and PSS increases.
Gel powders with different PDDA and PSS contents were prepared according to example 7, and the viscosity stress of the gel powder obtained in example 7 was measured by a tensile tester, namely, the tensile strength test mode was selected by using an intelligent electronic tensile tester, and the viscosity stress of each group of powders was tested. Each group was prepared from 0.4g of the powder to give a hydrogel strip having a length of 4cm, a width of 1cm and a thickness of 2 mm. And (3) preparing the pig skin with the grease removed into the same size, adhering one end of the hydrogel to the outer side of the pig skin, overlapping the hydrogel and the pig skin into a strip shape, enabling the area of the overlapped part to be 1cm 2, and pressing a weight of 200g on the overlapped part of the hydrogel and the pig skin for 20min. Then, the test is carried out by a tensile testing machine, the fixed stretching speed is 25mm/min until the hydrogel and the pigskin are completely pulled apart, and the maximum breaking force is recorded. The viscosity stress is the maximum breaking force divided by the area of overlap between the hydrogel and the pigskin. The results are shown in FIG. 6, which is a graph of viscosity test of the content of PDDA and PSS obtained in example 7 of the present invention, and the results show that the content of PDDA and PSS is inversely proportional to the viscosity stress of the hydrogel, indicating that the adhesion of the hydrogel to skin is reduced with the increase of the content of PDDA and PSS.
And finally, selecting the optimal scheme of the volume ratio of PDDA and PSS of 5% by combining the mechanical performance test result and the viscosity test result.
Example 9
According to the technical scheme of examples 1-8, four groups of self-gelling powder containing different alkylated chitosan in situ are prepared, wherein the four alkylated chitosan are dodecyl chitosan, tetradecyl chitosan, hexadecyl chitosan and octadecyl chitosan.
Wherein the concentration of PEI and PAA is 5wt%, the volume ratio is 5:5, and the PEI and PAA respectively account for 40% of the total system volume. The mass concentration of the different alkylated chitosan is 0.6wt percent, accounting for 10 percent of the total system volume, the mass concentration of PDDA is 8wt percent, and the mass concentration of PSS is 10.3wt percent, accounting for 5 percent of the total system volume respectively. PEI was purchased from Shanghai Ala Biotechnology Co., ltd, the weight average molecular weight was 70000, PDDA was purchased from Shanghai Milin Biotechnology Co., ltd, the weight average molecular weight was 100000-200000, PAA was purchased from Beijing Barling Wikipedia, the weight average molecular weight was 240000, PSS was purchased from Shanghai Ala Biotechnology Co., ltd, the weight average molecular weight was 70000, and alkylated chitosan was the four alkylated chitosan prepared in example 1.
1. Four sets of mixed solutions containing 22.5mL of 5wt% PEI solution and 2.5mL of 8wt% PDDA solution were prepared and placed in a beaker as solution A.
2. Four sets of mixed solutions containing 22.5mL of 5wt% PAA solution and 2.5mL of 10.3wt% PSS solution were prepared and placed in a centrifuge tube as solution B.
3. Four different sets of alkylated chitosan solutions were configured as 5mL solutions with a concentration of 0.6wt% and placed in centrifuge tubes, respectively.
4. The magnet was placed in a beaker containing solution a and the whole was placed on a magnetic stirrer.
5. Starting a magnetic stirrer, and slowly pouring the solution B and the corresponding alkylated chitosan solution simultaneously after stabilizing to uniformly mix the solution B, the alkylated chitosan solution and the corresponding alkylated chitosan solution, so as to prepare the hydrogel with certain strength.
6. The hydrogel was collected in a glass dish, sealed with a preservative film, and frozen overnight at-20 ℃.
7. The preservative film used for sealing was perforated, freeze-dried at-60 ℃ for a total of 30 hours.
8. The lyophilized xerogel was transferred to a mortar and ground into a fine powder.
Example 10
Four groups of in situ formed self-gelling powders containing different alkylated chitosans prepared as in example 9 were tested for gel conversion rate and grouped as ACS-1, ACS-2, ACS-3, ACS-4. The gel transition times of the different groups were determined by timing, 1.0g of powder per group was placed in a petri dish 3.0mm in diameter and the powder was spread evenly. Proper amount of physiological saline is taken by a pipette or a disposable rubber head dropper, added into a culture dish containing powder, and simultaneously precisely timed by a timer. Each set of samples was repeated three times.
As shown in Table 1 and FIG. 7, FIG. 7 shows the gel conversion time of in situ self-gelling powder containing different alkylated chitosan, and as can be seen from the graph and the table, the time of the gel conversion of the dodecylated chitosan group is fastest and faster than the time of the gel conversion of the octadecylated chitosan is slowest as reported in the prior literature due to the hydrophobic nature of the alkyl group and the length difference of the carbon chain.
TABLE 1 gel transition time of in situ self-gelling powder containing different alkylated chitosans
And finally selecting the dodecyl chitosan as an optimal scheme according to the test results of the gel conversion time of each group.
Example 11
According to the technical scheme of examples 1-10, self-gelling powder formed in situ is prepared as shown in fig. 8. FIG. 8 is a schematic diagram of hydrogel synthesis, wherein the concentration of PEI and PAA is 5wt%, the volume ratio is 5:5, the mass concentration of alkylated chitosan is 0.6wt%, the mass concentration of PDDA is 8wt%, the mass concentration of PSS is 10.3wt%, and the mass concentration of alkylated chitosan is 5% of the total system volume. PEI was purchased from Shanghai Ala Biotechnology Co., ltd, the weight average molecular weight was 70000, PDDA was purchased from Shanghai Milin Biotechnology Co., ltd, the weight average molecular weight was 100000-200000, PAA was purchased from Beijing Barling Wikipedia, the weight average molecular weight was 240000, PSS was purchased from Shanghai Ala Biotechnology Co., ltd, the weight average molecular weight was 70000, and alkylated chitosan was dodecylchitosan prepared in example 1.
1. A5 wt% PEI solution and an 8wt% PDDA solution were prepared and placed in a beaker as solution A with uniform mixing.
2.5 Wt% PAA solution and 10.3wt% PSS solution were prepared and mixed homogeneously as solution B in a centrifuge tube.
3. An alkylated chitosan solution of 0.6wt% was prepared and placed in centrifuge tubes, respectively.
4. And (3) putting the magnetons into a beaker containing the mixed solution A, integrally placing the beaker on a magnetic stirrer, starting the magnetic stirrer, slowly pouring the mixed solution B and the alkylated chitosan solution after the magnetons are stabilized, and uniformly mixing the mixed solution B and the alkylated chitosan solution to form the hydrogel.
5. The hydrogel was collected in a glass dish, sealed with a preservative film, and frozen overnight at-20 ℃.
6. The preservative film used for sealing was perforated, freeze-dried at-60 ℃ for a total of 30 hours.
7. The lyophilized xerogel was transferred to a mortar and ground into a fine powder.
According to the invention, the form and the internal structure of the self-gelation powder are observed by shooting a powder object and an SEM image, and the result is shown in FIG. 9, wherein FIG. 9 is an image of the self-gelation powder prepared in example 11 of the invention and a scanning electron microscope image, the left image in FIG. 9 is a gel powder image, and the right image is a scanning electron microscope image, and as can be seen from FIG. 9, the self-gelation powder prepared by the invention has a finer appearance, can be ground into finer powder, has a loose porous structure inside, and has good moisture absorption performance.
Test case
1. Evaluation of safety of self-gelling powder formed in situ
1.1, Hemolysis ratio experiment, in which anticoagulated whole blood was centrifuged at 3000rpm for 10min at 4℃to remove the upper plasma and pale yellow fraction, and then the lower erythrocytes were washed with PBS to the upper solution without color. After that, a volume fraction of 2% of red blood cell suspension was prepared with PBS. Respectively taking 2mgPEI/PAA gel powder, ACS powder, PEI/PAA/ACS gel powder, (PEI/PDDA)/(PAA/PSS)/ACS gel powder, placing into 4 EP tubes, respectively adding 1ml PBS, soaking at 37 ℃ for 24 hours in a shaking table at constant temperature, and taking out the leaching liquid for standby. mu.L of this 2wt% suspension of erythrocytes was mixed with equal volumes of PBS (negative control) and deionized water (positive control) and 4 sets of leachates, respectively, and incubated for 2h at 37 ℃. The incubated sample and the erythrocyte mixture are centrifuged at 3000rpm for 5min, the supernatant is collected, absorbance at 570nm is detected by an enzyme-labeled instrument, the test is repeated three times, and the hemolysis rate of each group is calculated according to the following formula.
Wherein Hemolysis is hemolysis rate, abs and Abs 0、Absl are absorbance values after 4 groups of samples, PBS, deionized water and red blood cells react respectively.
The experimental results are shown in Table 1 and FIG. 10, FIG. 10 shows the results of the hemolysis rate of the self-gelling powder and the components thereof, wherein the hemolysis rates of ACS powder and the self-gelling powder are both less than 2.5%, and the hemolysis rate of ACS powder and the self-gelling powder accords with the national pharmaceutical standards YBB00032003-2015 (the sample hemolysis rate is not more than 5%).
TABLE 2 test results of hemolysis ratio of self-gel powder and its components prepared in the examples of the present invention
1.2 Cytotoxicity experiments MTT assay was performed using mouse fibroblast L929 co-culture. Firstly preparing sample leaching solution by using a DMEM culture medium, taking 2mg of each of PEI/PAA gel powder, ACS powder, PEI/PAA/ACS gel powder, (PEI/PDDA)/(PAA/PSS)/ACS gel powder, and soaking each of 4 groups of samples in a culture medium of 1mLDMEM at 37 ℃ for 24 hours after the samples are sterilized by ultraviolet irradiation for 24 hours. The extract was removed, and after filtration sterilization with a 0.45 μm filter, 500 μl was added to an equal volume of DMEM medium containing 20% Fetal Bovine Serum (FBS). L929 cells were digested with 0.25wt% pancreatin at 37℃for 1min, resuspended in medium, the cell density was adjusted to 5X 10 4 cells/mL, each well was inoculated into a 96-well plate, incubated in an incubator containing 5% CO 2 by volume at 37℃for 24h, after 24h, 100. Mu.L of the sample extract was added to the medium, while a normal culture control group was set, incubated in the CO 2 incubator for 48h, after 48h, a portion of the cells were collected, the stock culture was aspirated, 100. Mu.L of sterile medium and 10. Mu.L of sterile MTT solution were added to the 96-well plate, respectively, the culture was continued in the incubator for 4h, the MTT and medium were aspirated, and after repeated dissolution of the pellet by addition of SDS lysate, the absorbance at 490nm was measured with an ELISA meter, and the proliferation rates of the cells of each group were calculated according to the following formula.
Wherein RGR is the relative proliferation rate of cells, A 0 is the absorbance value of a solvent control group, A 1 is the absorbance value of a normal culture control group, and A 2 is the absorbance value of an experimental group.
The results of the cell proliferation rate experiments are shown in Table 2 and FIG. 11, and FIG. 11 shows the results of the cell proliferation rate of the self-gelled powder and the components thereof, wherein the cell proliferation rate of the PEI/PAA gel powder, ACS powder, PEI/PAA/ACS gel powder, (PEI/PDDA)/(PAA/PSS)/ACS gel powder, 4 groups of cells are all more than 80%, and the cytotoxicity classification is 1 grade. The results show that PEI/PAA gel powder, ACS powder, PEI/PAA/ACS gel powder, (PEI/PDDA)/(PAA/PSS)/ACS gel powder all show good biocompatibility.
TABLE 3 results of cell proliferation rate test of self-gelling powder and components thereof prepared in the examples of the present invention
1.3, Antibacterial property evaluation, namely selecting staphylococcus aureus (S.aureus) and escherichia coli (E.coli) as two different model bacteria of gram-positive bacteria and gram-negative bacteria respectively for antibacterial tests. Firstly, the material was mixed with 5mL of bacterial suspension (1X 10 4 CFU/mL) in a sterile tube, incubated for 24h at 37℃with a shaker, the incubated bacterial suspension was diluted 10 6 -fold and spread on the surface of solid agarose medium, placed in a37℃incubator for 24h, and the number of surviving bacteria was recorded by counting colonies on the solid agarose medium. The antibacterial rate is calculated by taking bacteria which are not subjected to any treatment as a control group, the experimental results are shown in table 4 and fig. 12, the antibacterial effect of the positive medicine chitosan on staphylococcus aureus and escherichia coli is obvious, the antibacterial rate on escherichia coli and staphylococcus aureus is 94.64% and 98.26% respectively, the improved alkylated chitosan also shows good antibacterial effect, the antibacterial rate on escherichia coli and staphylococcus aureus is 86.51% and 93.66% respectively, the PEI/PAA gel powder has no antibacterial effect, but the powder has water absorption, the living environment of bacteria is changed, the growth rate of bacteria is slowed down to some extent, the colony number is lower than that of other groups, and the gel powder added with the alkylated chitosan shows excellent antibacterial effect, and the antibacterial rate is up to 99%.
TABLE 4 antibacterial test results of self-gelling powder and its components prepared in the examples of the present invention
1.4 Evaluation of hemostatic healing Effect
In vitro coagulation experiments, 5mg of sample is placed in a 96-well plate, 100 mu L of anticoagulated blood is added, 10 mu LCaCl 2 of solution (the concentration is 0.2 mol/L) is added, timing is started at the same time, PBS is sucked by a pipette for flushing every 30 seconds, coagulation conditions are observed until the blood is completely coagulated, and the coagulation time of the whole blood is recorded when the flushing is unchanged for multiple times. Each sample was repeated 3 times, and blood to which no sample was added was used as a control group. The results of the in vitro coagulation test are shown in Table 4 and FIGS. 13 and 14, FIG. 13 shows the in vitro coagulation time of the self-gelling powder and its components, and FIG. 14 shows the in vitro coagulation physical pattern of the self-gelling powder and its components. Compared with the blank, the PEI/PAA gel powder, ACS powder, PEI/PAA/ACS gel powder, (PEI/PDDA)/(PAA/PSS)/ACS gel powder prepared by the invention has improved coagulation effect compared with zeolite powder.
TABLE 5 in vitro coagulation test results of self-gelling powder and its components prepared in the examples of the present invention
1.5 Evaluation of wound healing Effect
Wound healing effect evaluation 20 SD rats were randomly divided into 4 groups and were acclimatized for one week. After anesthetizing rats with 10% chloral hydrate at a dose of 0.3mL/100g, the rats were fixed on an operating table, the hair on the backs of the rats was cleaned with depilatory cream, sterilized with 75% alcohol, and a back wound model of SD rats was prepared with a biopsy sampler having a diameter of 10 mm. The prepared bacterial liquid is smeared on the surface of the wound, different powders are applied on the wound surface for treatment, and PBS is used as a control group. On days 0, 2, 4, 7 and 10, the condition of wound healing was observed and photographed for each group. The experimental results are shown in fig. 15, the wound healing degree of the PEI/PAA powder group treatment is not significantly enhanced compared with the PBS group, while the wound area of the PEI/PAA/ACS gel powder treatment and the (PEI/PDDA)/(PAA/PSS)/ACS gel powder treatment are significantly reduced in the same time period, and the healing degree is enhanced.
The above description of the embodiments is only for aiding in the understanding of the method of the present invention and its core ideas. 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.
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