CN109705369B - Sodium alginate-dopamine/polyvinyl alcohol hydrogel and preparation method and application thereof - Google Patents
Sodium alginate-dopamine/polyvinyl alcohol hydrogel and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a sodium alginate-dopamine/polyvinyl alcohol hydrogel and a preparation method and application thereof. The method is realized by the following scheme: dopamine (DA) is introduced into nerve conduction substances through chemical cross-linked grafted Sodium Alginate (SA) to communicate cells and transmit chemical substances, so that the effects of promoting wound healing, improving physical adhesion capacity and improving material biocompatibility are achieved, and then the dopamine is compounded with medical grade polyvinyl alcohol (PVA) which is good in biocompatibility, cheap and easily available, so that the physical property of the hydrogel is improved, and the hydrogel with higher mechanical strength is prepared. The hydrogel prepared by the method has good biocompatibility and excellent physical properties, and the reaction raw materials are cheap and easy to obtain, the reaction conditions are mild, and the operation steps are simple and convenient. Has wide application prospect in bedsore wound nursing and biomedicine.
Description
Technical Field
The invention belongs to the field of biological polymer materials, and particularly relates to a sodium alginate-dopamine/polyvinyl alcohol hydrogel and a preparation method and application thereof.
Background
Bedsore is also called pressure sore as a clinical common complication. The most prominent external causes of pressure sores are the pressure factors: the skin pressure is the vertical pressure (the local tissue is subjected to continuous vertical pressure such as the convex part of the bone of the body), the friction force (skin friction), and the shearing force (after an acting force is applied on an object, a plane sliding in the opposite direction is generated, for example, when the body slides down when a bed head is lifted in a flat lying state, the skin and the bed generate parallel friction force, and the gravity in the vertical direction of the skin is added, so that the shearing force is generated, and the blood circulation of the local skin is obstructed, so that the pressure sore is generated). Currently, clinical care is divided into two categories: firstly, nursing with traditional sterile gauze; secondly, the novel dressing wound nursing, the traditional sterile gauze nursing has the obvious defects that the barrier function of the gauze is poor, the possibility of bacteria invasion is high, granulation tissues of wound surfaces easily grow into meshes of the gauze, new tissues can be damaged during dressing change to cause pain, obvious promotion effect on wound surface healing is not provided, the wound is sticky, and the dressing wound nursing is not only painful but also can cause secondary wound during damaged wound surface change.
Researchers have attempted to solve the problem of preparing novel hydrocolloid dressings from natural and synthetic polymeric materials. However, most of the existing hydrocolloid dressings only consider the problems of biocompatibility and mechanical property during design, and a substance capable of promoting wound healing is not introduced, so that the dressing has obvious defects: for example, the problems of high preparation cost, complicated preparation process, difficulty in removing impurities, high reaction conditions, poor effect of promoting wound healing and the like cannot be solved relatively comprehensively. Therefore, there is an urgent need to further research materials with multiple functions and excellent biocompatibility, and to realize the purpose of solving the above problems at one time, and to apply them to clinical diagnosis and research as early as possible.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention mainly aims to provide the sodium alginate-dopamine/polyvinyl alcohol hydrogel.
The invention also aims to provide a preparation method of the sodium alginate-dopamine/polyvinyl alcohol hydrogel.
The invention further aims to provide application of the sodium alginate-dopamine/polyvinyl alcohol hydrogel in the biological field.
The purpose of the invention is realized by the following technical scheme:
a preparation method of sodium alginate-dopamine/polyvinyl alcohol hydrogel comprises the following steps:
(1) adding N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a Sodium Alginate (SA) aqueous solution with the concentration of 1-2% (namely 1-2 g/100ml), uniformly stirring, adding Dopamine (DA), and stirring at room temperature for reacting for 12-36 hours to obtain a sodium alginate-dopamine solution;
(2) adding the sodium alginate-dopamine solution obtained in the step (1) into a polyvinyl alcohol (PVA) aqueous solution with the concentration of 40-60 g/L, uniformly stirring, freezing, thawing and dialyzing to obtain an SA-DA/PVA hydrogel;
the molar ratio of the sodium alginate to the dopamine in the sodium alginate aqueous solution in the step (1) is 1: 1-2;
the molar ratio of the N-hydroxysuccinimide to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the step (1) is 1: 1; the concentration of the reaction system in the step (1) is 30-50 mmol/L;
the volume ratio of the sodium alginate-dopamine solution to the polyvinyl alcohol aqueous solution in the step (2) is 1:1 to 2.
Preferably, the molar ratio of sodium alginate to dopamine in the sodium alginate aqueous solution in the step (1) is 1: 1.
Preferably, the preparation method of the sodium alginate aqueous solution in the step (1) comprises the following steps: adding sodium alginate into distilled water, and then stirring at 500-800 rpm for 10-15 h to dissolve the sodium alginate.
Preferably, the stirring speed in the step (1) is 500-800 rpm.
Preferably, the reaction time in step (1) is 24 h.
Preferably, the sodium alginate-dopamine solution in the step (1) needs to be dialyzed with distilled water for 1 day after the reaction is finished so as to remove excessive NHS and EDC.
Preferably, the sodium alginate-dopamine solution in the step (1) is stored in a sealed manner at the temperature of 2-8 ℃, and the sodium alginate-dopamine is connected to a sodium alginate main chain by utilizing the reaction of amino in dopamine and carboxyl in sodium alginate through a chemical cross-linking agent.
Preferably, the stirring speed in the step (2) is 800-1000 rpm, and the stirring time is 12-36 h.
More preferably, the stirring time in step (2) is 24 h.
Preferably, the freezing-thawing method in the step (2) is as follows: freezing and unfreezing for 3-5 times at-20 ℃ and 4-8 ℃.
Preferably, the dialysis method in step (2) is: dialyzing with distilled water at room temperature for 3-4 days.
The sodium alginate-dopamine/polyvinyl alcohol hydrogel prepared by the method.
The application of the sodium alginate-dopamine/polyvinyl alcohol hydrogel in the biological field.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the sodium alginate is grafted with dopamine, so that the traditional introduction of nerve conduction substance communication is broken, and the expression of cytokines is promoted, so that the wound healing is promoted, the physical adhesion capacity is improved, and the biocompatibility of the material is improved.
2. The natural polymer material can be formed into gel by a composite freezing and thawing process with synthetic polymer medical grade polyvinyl alcohol, and the mechanical property of the natural polymer material can be improved.
3. The SA-DA/PVA hydrogel prepared by the invention has good antibacterial property and procoagulant blood, and has wide biomedical application prospect.
4. According to the invention, the nerve conduction substance dopamine is grafted to the sodium alginate under mild reaction conditions, cytokine expression is promoted by inducing cells to transmit signals, so that the effect of promoting wound healing is achieved, the prepared hydrogel can achieve a barrier effect, the possibility of bacterial invasion is low, the hydrogel is good and comfortable in water absorption when contacting with a wound, pain is not caused during dressing change, and simultaneously, the excellent mechanical property can bear external various pressure factors, so that secondary wound is not easily caused.
Drawings
FIG. 1 is an infrared spectrum of the sodium alginate-dopamine/polyvinyl alcohol hydrogel prepared in example 1.
FIG. 2 is a scanning electron microscope image of the sodium alginate-dopamine/polyvinyl alcohol hydrogel lyophilized sample prepared in example 1 at 1000 times.
FIG. 3 is a stress-strain plot of a blank polyvinyl alcohol hydrogel prepared in example 1.
FIG. 4 is a stress-strain diagram of the sodium alginate-dopamine/polyvinyl alcohol hydrogel prepared in example 1.
FIG. 5 is a graph of the relative proliferation rate (RGR) of mouse fibroblasts 3T3 obtained after 1 day treatment with sodium alginate-dopamine/polyvinyl alcohol hydrogel and sodium alginate-dopamine hydrochloride/polyvinyl alcohol hydrogel.
FIG. 6 is a graph of the relative proliferation rate (RGR) of mouse fibroblasts 3T3 obtained after treatment with sodium alginate-dopamine/polyvinyl alcohol hydrogel and sodium alginate-dopamine hydrochloride/polyvinyl alcohol hydrogel for 2 days.
FIG. 7 is a graph of the relative proliferation rate (RGR) of mouse fibroblasts 3T3 obtained after treatment with sodium alginate-dopamine/polyvinyl alcohol hydrogel and sodium alginate-dopamine hydrochloride/polyvinyl alcohol hydrogel for 3 days.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
Example 1
(1) Sterilizing treatment of experimental raw materials and instruments
Placing experimental raw materials in a superclean bench for ultraviolet irradiation for 1h for raw material sterilization, and placing a glass instrument in a high-pressure condition of 121 ℃ for sterilization for 20 minutes.
(2) Preparation of sodium alginate-dopamine
Adding 2g of Sodium Alginate (SA) into 100ml of distilled water, stirring and dissolving at 800rpm for 12 hours to prepare an SA solution for later use. To the SA solution were added 0.5755g of N-hydroxysuccinimide (NHS) and 0.9585g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in a molar ratio of 1:1 and stirred at 800rpm for 20min, followed by addition of 1.5473g of Dopamine (DA) and stirring at 800rpm at room temperature for 24 hours. After the reaction is finished, the prepared sodium alginate-dopamine (SA-DA) solution is stored in a sealed manner at the temperature of 2-8 ℃.
(3) Preparation of sodium alginate-dopamine/polyvinyl alcohol hydrogel
Uniformly stirring 30ml of SA-DA solution for later use, stirring 5g of polyvinyl alcohol (PVA) and dissolving in 100ml of distilled water to prepare PVA solution, mixing the SA-DA solution and the PVA solution according to the volume ratio of 1:1, stirring at room temperature and 800rpm for 24 hours, pouring into a prepared forming mold, and freezing and thawing at the temperature between minus 20 ℃ and 4 ℃ for three times to form hydrogel. And taking out the formed hydrogel, and dialyzing the formed hydrogel for 3 days by using distilled water at room temperature to obtain the sodium alginate-dopamine/polyvinyl alcohol (SA-DA/PVA) hydrogel.
The infrared spectrogram of the prepared sodium alginate-dopamine/polyvinyl alcohol hydrogel is shown in figure 1, the scanning electron microscope image of a freeze-dried sample under 1000 times is shown in figure 2, and the figures 1 and 2 show that the SA-DA/PVA hydrogel is successfully prepared.
The mechanical property experiment of the sodium alginate-dopamine/polyvinyl alcohol hydrogel prepared in this example is as follows:
(A) the sodium alginate-dopamine/polyvinyl alcohol solution prepared in the step (3) of example 1 and 5g/L of polyvinyl alcohol solution (used as a blank control group) are respectively poured into a cylindrical hydrogel forming mold with the groove depth of 10mm and the diameter of 12mm, and freezing-thawing is carried out at the temperature of-20 ℃ and 4 ℃ for three times to form hydrogel, so as to prepare test samples, and three same samples are prepared for the experimental group and the blank control group.
(B) Each sample was subjected to a compression test using an electronic universal tester (model ELF3220) with the following specific parameters: 1. the compression rate is 2 mm/min; 2. the compression was 60% length (i.e. 6mm per sample).
(C) And (3) taking a stress-strain diagram and obtaining a slope as the elastic modulus of the sample as shown in FIGS. 3 and 4.
From fig. 3, it can be seen that the elastic modulus of the polyvinyl alcohol hydrogel in the blank group is 0.3KPa, and from fig. 4, it can be seen that the elastic modulus of the sodium alginate-dopamine/polyvinyl alcohol hydrogel prepared by the present application is 17.2KPa, which is significantly superior to that of the polyvinyl alcohol hydrogel, so that the sodium alginate-dopamine/polyvinyl alcohol hydrogel prepared by the present application has excellent mechanical properties and can meet the requirements of mechanical properties of wound dressings.
Comparative example 1
(1) Sterilizing treatment of experimental raw materials and instruments
The same as in example 1.
(2) Preparation of sodium alginate-dopamine hydrochloride
Adding 2g of Sodium Alginate (SA) into 100ml of distilled water, stirring and dissolving at 800rpm for 12 hours to prepare an SA solution for later use. To the SA solution were added 0.5755g of N-hydroxysuccinimide (NHS) and 0.9585g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in a molar ratio of 1:1 and stirred at 800rpm for 20min, then 1.9156g of dopamine hydrochloride was added and stirred at room temperature and 800rpm for reaction for 24 hours. After the reaction is finished, the prepared sodium alginate-dopamine hydrochloride is stored in a sealed manner at the temperature of 2-8 ℃.
(3) Preparation of sodium alginate-dopamine hydrochloride/polyvinyl alcohol hydrogel
With reference to the steps (1) to (3) of example 1, except that dopamine was replaced by dopamine hydrochloride, the experimental raw materials and process parameter conditions were the same as those of example 1, and a sodium alginate-dopamine hydrochloride/polyvinyl alcohol hydrogel was prepared.
Example 2
Sodium alginate-dopamine/polyvinyl alcohol hydrogel in vitro cell experiment
The lyophilized hydrogel samples prepared in example 1 were sterilized by Co60 gamma irradiation prior to in vitro cytotoxicity experiments.
(1) NIH 3T3 cells (purchased from Wuhan Poncirus Tech., Ltd.) were removed from liquid nitrogen and resuscitated in DMEM medium.
(2) Subculturing with DMEM culture solution for several times, collecting cells in logarithmic growth phase, counting NIH 3T3 cells with cell counting plate until cell density is 5 × 104one/mL.
(3) Taking a 96-well plate and adding 100 μ L of the cell-containing solution of the step (2) having a cell density of 5X 10 to 30 wells of the 96-well plate4DMEM culture solution per mL is used as an experimental group; at the same time, 3 wells were set as blank control, i.e., 100. mu.L of DMEM culture solution (without cells) was added to the blank control, and the blank control was incubated at 37 ℃ with 5% CO2The cells were cultured in a cell incubator for 24 hours.
(4) After 24 hours, the cells were attached, the 96-well plate was removed and the DMEM medium in 30 wells of the experimental group of step (3) was aspirated, and 1 thereof was addedAdding 100 μ L of SA-DA/PVA hydrogel immersion fluid solution (DMEM medium immersed in SA-DA/PVA hydrogel) with concentration of 25mg/ml, 50mg/ml, 100mg/ml, 150mg/ml and 200mg/ml into 5 wells, and setting 3 wells for each concentration as experimental group; meanwhile, setting other 15 holes as a negative control group, respectively adding 100 microliter of sodium alginate/polyvinyl alcohol hydrogel immersion fluid solution with the concentration of 25mg/ml, 50mg/ml, 100mg/ml, 150mg/ml and 200mg/ml, and setting 3 holes at each concentration; then, the 96-well plate was placed at 37 ℃ with 5% CO2Culturing in a cell culture box for 24, 48 and 72 hours respectively;
the preparation method of the sodium alginate/polyvinyl alcohol hydrogel comprises the following steps: mixing a sodium alginate aqueous solution with the concentration of 2% (2g/100ml) and a polyvinyl alcohol aqueous solution with the concentration of 50g/L according to the volume ratio of 1:1, stirring at room temperature and 800rpm for 24 hours, pouring the mixture into a prepared forming mold, freezing and unfreezing the mixture for three times at the temperature between 20 ℃ below zero and 4 ℃ to form hydrogel, taking out the formed hydrogel, and dialyzing the formed hydrogel for 3 days by using distilled water at the room temperature to obtain the sodium alginate/polyvinyl alcohol hydrogel.
(5) Taking out 96-well plate, removing SA-DA/PVA hydrogel leachate solution in 15 wells of experimental group and sodium alginate/polyvinyl alcohol hydrogel leachate solution in 15 wells of negative control group, adding 10 μ L DMEM culture solution and 10 μ L CCK-8 solution into the 30 wells, respectively, placing 96-well plate at 37 deg.C and 5% CO2The cells were cultured in a cell incubator for 4 hours.
(6) After 4 hours, the 96-well plate was removed, and the absorbance of each well was measured using a microplate reader at λ 450nm, and the relative proliferation rate (RGR) of the cells was calculated according to the following formula:
RGR% (a experimental group-a blank)/(a 0 negative control group-a blank) × 100%.
(7) And (4) taking data to prepare a relative cell proliferation rate map.
Comparative example 2
Sodium alginate-dopamine hydrochloride/polyvinyl alcohol hydrogel in vitro cell experiment
Before in vitro cytotoxicity test, Co60 gamma-ray radiation sterilization treatment was carried out on the lyophilized hydrogel sample prepared in comparative example 1.
Refer to example 2 steps (1) - (3) to resuscitate and culture NIH 3T3 cells.
(4) After 24 hours, the cells adhere to the wall, a 96-well plate is taken out, DMEM culture solution in 30 holes of the experimental group in the step (3) is sucked and removed, then 100 mu L of sodium alginate-dopamine hydrochloride/polyvinyl alcohol hydrogel soaking solution (DMEM culture medium soaked with sodium alginate-dopamine hydrochloride/polyvinyl alcohol hydrogel) with the concentration of 25mg/ml, 50mg/ml, 100mg/ml, 150mg/ml and 200mg/ml is respectively added into 15 holes of the experimental group, and 3 holes are arranged in each concentration to serve as the experimental group; meanwhile, setting other 15 holes as a negative control group, respectively adding 100 microliter of sodium alginate/polyvinyl alcohol hydrogel immersion fluid solution with the concentration of 25mg/ml, 50mg/ml, 100mg/ml, 150mg/ml and 200mg/ml, and setting 3 holes at each concentration; then, the 96-well plate was placed at 37 ℃ with 5% CO2Culturing in a cell culture box for 24, 48 and 72 hours respectively;
the preparation method of the sodium alginate/polyvinyl alcohol hydrogel is the same as that of the example 2.
Data were obtained and a graph of relative cell proliferation rate was prepared by referring to the steps (5) to (7) of example 2.
Fig. 5, 6, and 7 show the relative proliferation rate (RGR) of the mouse fibroblast cells 3T3 obtained after 1, 2, and 3 days of treatment with the sodium alginate-dopamine/polyvinyl alcohol hydrogel and the sodium alginate-dopamine hydrochloride/polyvinyl alcohol hydrogel, respectively, and it can be seen from fig. 5 that the sodium alginate-dopamine hydrochloride/polyvinyl alcohol hydrogel group has no significant increase in cell proliferation speed of the sodium alginate-dopamine/polyvinyl alcohol hydrogel group on the first day. In fig. 6 and 7, it can be analyzed that the sodium alginate-dopamine hydrochloride/polyvinyl alcohol hydrogel extract solution has substantially no cytotoxicity but does not promote cell proliferation at low concentration, but shows a certain cytotoxicity once the concentration is increased, and the cell proliferation rate is lower than 100%. The sodium alginate-dopamine/polyvinyl alcohol hydrogel prepared by the method has a certain effect of promoting cell proliferation in low concentration in days 2 and 3; the hydrogel prepared by the method has small cell proliferation promoting effect at high concentration, but does not have cytotoxicity, and the biocompatibility and the cell proliferation promoting effect are outstanding. Through NIH 3T3 cell proliferation experiment of this application, can obtain the promotion mouse epidermis fibroblast growth that this application made to promote wound healing to reach faster one-step repair wound effect.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A preparation method of sodium alginate-dopamine/polyvinyl alcohol hydrogel is characterized by comprising the following steps:
(1) adding N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a sodium alginate aqueous solution with the concentration of 1-2 g/100mL, uniformly stirring, adding dopamine, and stirring at room temperature for reaction for 12-36 h to obtain a sodium alginate-dopamine solution;
(2) dissolving polyvinyl alcohol with the concentration of 40-60 g/L in water, adding the sodium alginate-dopamine solution obtained in the step (1), uniformly stirring, freezing, thawing and dialyzing to obtain sodium alginate-dopamine/polyvinyl alcohol hydrogel;
the molar ratio of the sodium alginate to the dopamine in the sodium alginate aqueous solution in the step (1) is 1: 1-2;
the molar ratio of the N-hydroxysuccinimide to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the step (1) is 1:1, and the concentration of the N-hydroxysuccinimide to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the reaction system in the step (1) is 30-50 mmol/L;
the volume ratio of the sodium alginate-dopamine solution to the polyvinyl alcohol aqueous solution in the step (2) is 1:1 to 2.
2. The method for preparing sodium alginate-dopamine/polyvinyl alcohol hydrogel according to claim 1, wherein the molar ratio of sodium alginate to dopamine in the sodium alginate aqueous solution in the step (1) is 1: 1.
3. The preparation method of the sodium alginate-dopamine/polyvinyl alcohol hydrogel as claimed in claim 1, wherein the stirring rotation speed in step (1) is 500-800 rpm.
4. The method for preparing sodium alginate-dopamine/polyvinyl alcohol hydrogel according to claim 1 or 3, wherein the reaction time in the step (1) is 24 h.
5. The preparation method of the sodium alginate-dopamine/polyvinyl alcohol hydrogel as claimed in claim 4, wherein the stirring speed in the step (2) is 800-1000 rpm; the stirring time is 12-36 h.
6. The method for preparing sodium alginate-dopamine/polyvinyl alcohol hydrogel according to claim 5, wherein the stirring time in the step (2) is 24 hours.
7. The method for preparing sodium alginate-dopamine/polyvinyl alcohol hydrogel according to claim 4, wherein the freezing-thawing method in the step (2) comprises the following steps: freezing and unfreezing for 3-5 times at-20 ℃ and 4-8 ℃.
8. The method for preparing the sodium alginate-dopamine/polyvinyl alcohol hydrogel as claimed in claim 4, wherein the sodium alginate-dopamine solution in the step (1) is dialyzed with distilled water for 1 day after the reaction;
the dialysis method in the step (2) comprises the following steps: dialyzing with distilled water at room temperature for 3-4 days.
9. A sodium alginate-dopamine/polyvinyl alcohol hydrogel prepared by the preparation method of any one of claims 1 to 8.
10. The use of a sodium alginate-dopamine/polyvinyl alcohol hydrogel as claimed in claim 9 in the biological field, excluding the treatment of disease.
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