CN117695436A - Gallic acid modified bletilla striata polysaccharide hydrogel and preparation method and application thereof - Google Patents
Gallic acid modified bletilla striata polysaccharide hydrogel and preparation method and application thereof Download PDFInfo
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- Materials For Medical Uses (AREA)
Abstract
The invention relates to the technical field of hydrogel, in particular to a gallic acid modified bletilla striata polysaccharide hydrogel, a preparation method and application thereof, wherein gallic acid is grafted on sugar chains of the bletilla striata polysaccharide to prepare a GA-BSP polymer through chemical modification, mussel mucin is connected with the GA-BSP polymer through electrostatic action, and Fe is used for preparing the gel 3+ Is used for preparing GA-BSP hydrogel through physical crosslinking. The hydrogel not only endows the bletilla striata polysaccharide with better physical and chemical properties such as structure, mechanics, tissue adhesion, photo-thermal and the like, but also has better photo-thermal antibacterial effect. The hydrogel has good biocompatibility, low cytotoxicity and no immunogenicity, can absorb a large amount of wound exudate, and can keep the wound moistThe porous structure is favorable for proliferation and migration of epidermal cells, the spongy porous structure is favorable for removal of wound secretions, bacterial breeding is avoided, infection of microorganisms to the wound is effectively prevented, and healing of the wound is promoted. The invention can be used for acute wound surface including burn, abrasion, operation wound, laceration, battlefield penetration wound and other diseases, chronic wound and other diseases.
Description
Technical Field
The invention relates to the technical field of hydrogels, in particular to a gallic acid modified bletilla striata polysaccharide hydrogel, and a preparation method and application thereof.
Background
Chronic skin wounds, especially full thickness skin wounds, with bacterial infections often lead to abscesses and pain, even amputation and death, which certainly has become a challenge for the medical industry. At present, medical materials used in domestic wound treatment and clinical operation have the defects of poor immunogenicity and mechanical property, inconvenient use and the like. Similar products sold in foreign markets have high price and lower cost performance, and are difficult to popularize and use in China. The main strategy for clinically treating infected wounds remains the use of antibiotics. However, overuse of antibiotics often leads to the emergence of resistant strains, which makes the treatment of contaminated wounds increasingly unsatisfactory. There remains a need in the market today for wound dressings with antimicrobial activity and a strong regeneration capacity.
Therefore, how to enhance the healing promoting and antibacterial capabilities of medical materials is a technical problem that needs to be solved in the art. At present, the patent CN 104436277A is prepared by extracting bletilla striata polysaccharide from bletilla striata tubers, and preparing a loose porous sponge material for traumatic hemostasis through vacuum freeze drying; the patent CN 107349460A is prepared by spraying a layer of bletilla striata polysaccharide extracted from bletilla striata on the surface of a high-expansion sponge; patent CN108606971a uses the monomer compound bletilla striata glycoside separated from bletilla striata to prepare hemostatic tablet for oral administration, injection or external use for hemostasis; the patent CN 106267317A is prepared by mixing and crosslinking bletilla striata polysaccharide, chitosan and acetic acid solution, and freeze drying.
However, the existing medical materials using bletilla striata as raw materials are generally prepared from bletilla striata polysaccharide, have single action, and have certain limitations in the aspects of adhesion, dissolution, successful use of wound parts and the like.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the gallic acid modified bletilla striata polysaccharide hydrogel which has better physical and chemical properties such as structure, mechanics, tissue adhesion, photo-thermal and the like, has better photo-thermal antibacterial effect, has good biocompatibility and low cytotoxicity, has no immunogenicity, can absorb a large amount of wound exudate, and effectively promotes the healing of wound surfaces.
The aim of the invention is achieved by the following technical scheme:
a gallic acid modified bletilla striata polysaccharide hydrogel takes bletilla striata polysaccharide, gallic acid and mussel mucin as main raw materials.
A preparation method of gallic acid modified bletilla striata polysaccharide hydrogel comprises the following preparation steps:
(1) Carrying out an activation reaction on gallic acid to prepare and obtain DMA (direct-access memory access) ester;
(2) Dissolving bletilla striata polysaccharide in DMSO to prepare a bletilla striata polysaccharide solution;
(3) Mixing the bletilla striata polysaccharide solution with DMA (direct memory access) ester, standing, performing transesterification reaction on hydroxyl groups in the bletilla striata polysaccharide solution and ester groups of the DMA ester, and dialyzing and freeze-drying to obtain a GA-BSP polymer freeze-dried sample;
(4) Dissolving the GA-BSP polymer freeze-dried sample in PBS buffer solution, adding mussel mucin, mixing, and then adding FeCl 3 The solution is continuously stirred to react to form hydrogel.
Specifically, the activation reaction in the step (1) is performed according to the following steps:
(1.1) dissolving gallic acid, DMAP and EDC.HCl in DMSO, and adding 0.5-3g gallic acid, 0.5-3g DMAP and 1-5g EDC.HCl into every 100ml DMSO to prepare a mixed solution;
(1.2) carrying out an activation reaction under the light-shielding condition, wherein the temperature of the activation reaction is 15-40 ℃, and the reaction time is 0.5-3h, so as to obtain the DMA ester.
In edc.hcl mediated coupling reactions, gallic acid GA first reacts with edc.hcl to form an O-acyl acid intermediate that is highly unstable in aqueous solution and has a short lifetime. To increase the grafting efficiency of edc.hcl mediated reactions, DMAP was added to the reaction system. DMAP reacts with the O-acyl acid intermediate to form a DMA ester that is less susceptible to hydrolysis than the O-acyl acid.
Specifically, in the step (2), the bletilla striata polysaccharide is dissolved in DMSO, 3-8g of the bletilla striata polysaccharide is added into every 100ml of DMSO, and the solution is dissolved for 24-72 hours to prepare the bletilla striata polysaccharide solution.
Specifically, the preparation of the GA-BSP polymer freeze-dried sample in the step (3) is carried out according to the following steps:
(3.1) mixing the bletilla striata polysaccharide solution with the DMA ester, standing, and carrying out transesterification between hydroxyl groups of the bletilla striata polysaccharide and ester groups of the DMA ester, wherein the transesterification is carried out at a temperature of 15-60 ℃ for 12-36h, and the volume ratio of DMSO in the DMA ester to DMSO in the bletilla striata polysaccharide solution is (0.5-1): 1, mixing the materials in proportion;
(3.2) dialyzing and freeze-drying the solution of the transesterification reaction to obtain a freeze-dried GA-BSP polymer sample.
Specifically, when the bletilla striata polysaccharide solution and the DMA ester are mixed in the step (3.1), slowly dripping the bletilla striata polysaccharide solution into the DMA ester, and stopping stirring after dripping, wherein the stirring speed is 100-300r/min;
specifically, in the step (3.2), a dialysis bag with the molecular weight cut-off of 7000D is used in the dialysis, the dialysis treatment time is 2-4 days, and water is changed 3-5 times per day in the dialysis process.
Specifically, the preparation of the hydrogel in the step (4) is performed according to the following steps:
(4.1) dissolving a lyophilized sample of 0.05-0.3. 0.3gGA-BSP polymer in 100ml PBS buffer to obtain a GA-BSP polymer solution;
(4.2) adding 0.1-0.5g mussel mucin into the GA-BSP polymer solution in the above (4.1), and stirring and mixing uniformly to obtain a mixed solution;
(4.3) 20ml FeCl 3 The solution is added into the mixed solution of (4.2), and the reaction is continued with stirring, thus obtaining the hydrogel.
The hydrogel passes through the step (4) through hydrogen bonds (polysaccharide and phenolic acid) and ionic bonds (Fe 3+ ) A physically crosslinked hydrogel is formed.
Specifically, the pH of the PBS buffer solution in the step (4.1) is 6-8; step (4.3) FeCl 3 The concentration of the solution was: 0.01-0.05mol/L.
A use of gallic acid modified rhizoma Bletillae polysaccharide hydrogel for treating chronic wound, acute wound including burn, abrasion, operation wound, lacerated wound and battlefield penetration wound is provided.
Preferably, the hydrogels of the present invention are used by exposing them to near infrared laser (NIR) light at the time of use.
In particular, when the hydrogel of the present invention is used for chronic wounds and acute wounds including burns, abrasions, surgical wounds, lacerations, battlefield penetrations, it is more effective to use the hydrogel in combination with near infrared laser (NIR).
The Bletilla Striata Polysaccharide (BSP) in the bletilla striata has various pharmacological effects of promoting wound healing, stopping bleeding, inhibiting bacteria and the like, and is a natural biological material with good biocompatibility and degradability. BSP is a glucomannan, which is mainly polymerized from glucose and mannose in beta glycosidic linkages, and, due to the structure of glucomannan, BSP has also been shown to be involved in intercellular or intracellular signal transduction, exhibiting a typical ability to enhance immune activity. However, it is notable that the use of natural polysaccharides is incomparable with synthetic biopolymers due to the lack of sufficient functional groups.
Gallic acid GA is known for its powerful antibacterial, anti-inflammatory, angiogenic and antioxidant activities. The GA modified polysaccharide can show better water solubility, thermal stability, crystallization characteristics and antibacterial activity. In addition, phenolic acid compounds such as GA can provide strong tissue adhesion under humid conditions due to the presence of various types of interfacial interactions. This is because the catechol or pyrogallol functional groups have a high affinity for various nucleophiles (e.g., amines, thiols, imidazoles, etc.). Therefore, grafting GA to the sugar chains of BSP by chemical modification can also improve tissue adhesion and enhance the ability to apply to wound healing of infectious bacteria.
Mussel Mucin (MFP) is a raw material with good biocompatibility and particularly strong adhesiveness. Mussel mucin has positive charges, has good antibacterial and antioxidant properties, and can be widely applied to the research and development of adhesive materials such as tissue adhesives. Specifically, mussel protein is added, so that on one hand, the mussel protein has good biocompatibility, and in the use process, the mussel protein can absorb water so as to enable the bletilla polysaccharide component to exert the drug effect; secondly, mussel protein has good adhesiveness and plays a role in continuously repairing wound surfaces; third, mussel proteins contain 20% lysine, making mussel mucins a very small number of basic proteins (PI 9.5), with a large number of positive charges in the molecule in a physiological environment. The existence of a large amount of positive charges can not only enable the gel to be assembled with the bletilla striata polysaccharide modified by GA electrostatically, so that the formed hydrogel is more stable, but also enable potential difference to exist among cell molecules due to the existence of a large amount of positive charges, electron flow conduction of intermolecular potential is formed, signal transmission of a human neural network is generated, potential balance between healthy and sick cells is improved, the sick cells are recovered in time, an antipruritic effect is achieved, nerve endings can be passivated through electrostatic effect, and an antipruritic effect is achieved.
Hydrogels are three-dimensional hydrophilic polymer networks that are structurally similar to the extracellular matrix (ECM), which confers it excellent biocompatibility. At the same time, it can also reduce the risk of wound infection by absorbing wound exudates and maintaining a moist environment, which offers a good potential for improving repair of damaged tissue. During the hydrogel formation process, fe 3+ Physical crosslinking is an important form for forming phenolic acid grafted hydrogel, because metal ions not only can form a crosslinked network with macromolecular compounds to promote the formation of the hydrogel, but also can improve the toughness of the hydrogel, and the higher the valence state of the metal ions, the more crosslinking sites are, the more stable the formed hydrogel is, so that the invention adopts the method of preparing the phenolic acid grafted hydrogel with Fe 3+ The cross-linking mode improves the success rate of preparing the hydrogel. More importantly, part of the polyphenol compound can react with Fe 3+ The black chelate complex is formed by complexing, and the black chelate complex has stronger absorption in the near infrared region, so that the near infrared photothermal effect is generated, and the hydrogel has better photothermal antibacterial effect to a certain extent.
The invention has the beneficial effects that: the invention is modified by chemistryPreparing GA-BSP polymer by grafting gallic acid onto sugar chain of rhizoma Bletillae polysaccharide, connecting mussel mucin to GA-BSP polymer by electrostatic action, and passing through Fe 3+ Is used for preparing GA-BSP hydrogel through physical crosslinking. The hydrogel not only endows the bletilla striata polysaccharide with better physical and chemical properties such as structure, mechanics, tissue adhesion, photo-thermal and the like, but also has better photo-thermal antibacterial effect. The hydrogel has good biocompatibility, low cytotoxicity and no immunogenicity, can absorb a large amount of wound seepage, keeps the wound surface moist, is beneficial to proliferation and migration of epidermal cells, and the spongy porous structure is beneficial to the elimination of wound surface secretion, so that bacterial breeding is avoided, the infection of microorganisms on the wound surface is effectively prevented, and the healing of the wound surface is promoted. The invention can be used for acute wound surface including burn, abrasion, operation wound, laceration, battlefield penetration wound and other diseases, chronic wound and other diseases.
Drawings
Fig. 1: cross-section scanning electron microscope pictures of hydrogels and comparative examples of the invention;
fig. 2: the tissue adhesion properties of the hydrogels of the present invention and comparative examples;
fig. 3: the photo-thermal properties of the hydrogels of the invention and comparative examples;
fig. 4: the hydrogel has in-vitro near infrared photo-thermal antibacterial performance;
fig. 5: the hydrogel has the effect of promoting healing of bacteria-infected wounds.
Detailed Description
The invention will be further described with reference to the following examples.
Example 1.
A gallic acid modified bletilla striata polysaccharide hydrogel takes bletilla striata polysaccharide, gallic acid and mussel mucin as main raw materials.
A preparation method of gallic acid modified bletilla striata polysaccharide hydrogel comprises the following preparation steps:
(1) Carrying out an activation reaction on gallic acid to prepare and obtain DMA (direct-access memory access) ester;
(2) Dissolving bletilla striata polysaccharide in DMSO to prepare a bletilla striata polysaccharide solution;
(3) Mixing the bletilla striata polysaccharide solution with DMA (direct memory access) ester, standing, performing transesterification reaction on hydroxyl groups in the bletilla striata polysaccharide solution and ester groups of the DMA ester, and dialyzing and freeze-drying to obtain a GA-BSP polymer freeze-dried sample;
(4) Dissolving the GA-BSP polymer freeze-dried sample in PBS buffer solution, adding mussel mucin, mixing, and then adding FeCl 3 The solution is continuously stirred to react to form hydrogel.
Specifically, the activation reaction in the step (1) is performed according to the following steps:
(1.1) dissolving gallic acid, DMAP and EDC.HCl in DMSO, and adding 0.5g gallic acid, 0.5g DMAP and 1 EDC.HCl into every 100ml DMSO to prepare a mixed solution;
(1.2) carrying out an activation reaction under the light-shielding condition, wherein the temperature of the activation reaction is 15 ℃, and the reaction time is 3 hours, so as to obtain the DMA ester.
Specifically, in the step (2), the bletilla striata polysaccharide is dissolved in DMSO, 3g of the bletilla striata polysaccharide is added into every 100ml of DMSO, and the solution is dissolved for 24 hours to prepare the bletilla striata polysaccharide solution.
Specifically, the preparation of the GA-BSP polymer freeze-dried sample in the step (3) is carried out according to the following steps:
(3.1) mixing the bletilla striata polysaccharide solution with the DMA ester, standing, and carrying out transesterification reaction on hydroxyl groups of the bletilla striata polysaccharide and ester groups of the DMA ester, wherein the transesterification reaction temperature is 15 ℃, and the reaction time is 36h, and the volume ratio of DMSO in the DMA ester to DMSO in the bletilla striata polysaccharide solution is 0.5 during mixing: 1, mixing the materials in proportion;
(3.2) dialyzing and freeze-drying the solution of the transesterification reaction to obtain a freeze-dried GA-BSP polymer sample.
Specifically, when the bletilla striata polysaccharide solution and the DMA ester are mixed in the step (3.1), slowly dripping the bletilla striata polysaccharide solution into the DMA ester, and stopping stirring after dripping, wherein the stirring speed is 100r/min;
specifically, in the step (3.2), a dialysis bag with a molecular weight cut-off of 7000D is used in the dialysis, the dialysis treatment time is 2 days, and water is changed for 5 times a day in the dialysis process.
Specifically, the preparation of the hydrogel in the step (4) is performed according to the following steps:
(4.1) dissolving a lyophilized sample of 0.05gGA-BSP polymer in 100ml PBS buffer to obtain a GA-BSP polymer solution;
(4.2) adding 0.1g of mussel mucin into the GA-BSP polymer solution in the step (4.1), and stirring and mixing uniformly to obtain a mixed solution;
(4.3) 20ml FeCl 3 The solution is added into the mixed solution of (4.2), and the reaction is continued with stirring, thus obtaining the hydrogel.
Specifically, the pH of the PBS buffer in step (4.1) is 6; step (4.3) FeCl 3 The concentration of the solution was: 0.01mol/L.
Example 2.
A gallic acid modified bletilla striata polysaccharide hydrogel takes bletilla striata polysaccharide, gallic acid and mussel mucin as main raw materials.
A preparation method of gallic acid modified bletilla striata polysaccharide hydrogel comprises the following preparation steps:
(1) Carrying out an activation reaction on gallic acid to prepare and obtain DMA (direct-access memory access) ester;
(2) Dissolving bletilla striata polysaccharide in DMSO to prepare a bletilla striata polysaccharide solution;
(3) Mixing the bletilla striata polysaccharide solution with DMA (direct memory access) ester, standing, performing transesterification reaction on hydroxyl groups in the bletilla striata polysaccharide solution and ester groups of the DMA ester, and dialyzing and freeze-drying to obtain a GA-BSP polymer freeze-dried sample;
(4) Dissolving the GA-BSP polymer freeze-dried sample in PBS buffer solution, adding mussel mucin, mixing, and then adding FeCl 3 The solution is continuously stirred to react to form hydrogel.
Specifically, the activation reaction in the step (1) is performed according to the following steps:
(1.1) dissolving gallic acid, DMAP and EDC.HCl in DMSO, and adding 2g gallic acid, 2g DMAP and 3g EDC.HCl into every 100ml DMSO to prepare a mixed solution;
(1.2) carrying out an activation reaction under the light-shielding condition, wherein the temperature of the activation reaction is 28 ℃, and the reaction time is 2 hours, so as to obtain the DMA ester.
Specifically, in the step (2), the bletilla striata polysaccharide is dissolved in DMSO, 6g of the bletilla striata polysaccharide is added into every 100ml of DMSO, and the solution is dissolved for 48 hours to prepare the bletilla striata polysaccharide solution.
Specifically, the preparation of the GA-BSP polymer freeze-dried sample in the step (3) is carried out according to the following steps:
(3.1) mixing the bletilla striata polysaccharide solution with the DMA ester, standing, and carrying out transesterification reaction on hydroxyl groups of the bletilla striata polysaccharide and ester groups of the DMA ester, wherein the transesterification reaction temperature is 50 ℃, and the reaction time is 24 hours, and the volume ratio of DMSO in the DMA ester to DMSO in the bletilla striata polysaccharide solution is 0.6 during mixing: 1, mixing the materials in proportion;
(3.2) dialyzing and freeze-drying the solution of the transesterification reaction to obtain a freeze-dried GA-BSP polymer sample.
Specifically, when the bletilla striata polysaccharide solution and the DMA ester are mixed in the step (3.1), slowly dripping the bletilla striata polysaccharide solution into the DMA ester, and stopping stirring after dripping, wherein the stirring speed is 200r/min;
specifically, in the step (3.2), a dialysis bag with a molecular weight cut-off of 7000D is used in the dialysis, the dialysis treatment time is 3 days, and water is changed for 4 times a day in the dialysis process.
Specifically, the preparation of the hydrogel in the step (4) is performed according to the following steps:
(4.1) dissolving a lyophilized sample of 0.2. 0.2gGA-BSP polymer in 100ml PBS buffer to obtain a GA-BSP polymer solution;
(4.2) adding 0.3g of mussel mucin into the GA-BSP polymer solution in the above (4.1), and stirring and mixing uniformly to obtain a mixed solution;
(4.3) 20ml FeCl 3 Adding the solution into the mixed solution of (4.2), and continuing stirring to react to obtain the hydrogel, namely the GA-BSP hydrogel.
Specifically, the pH of the PBS buffer in step (4.1) is 7; step (4.3) FeCl 3 The concentration of the solution was: 0.03mol/L.
Example 3.
A gallic acid modified bletilla striata polysaccharide hydrogel takes bletilla striata polysaccharide, gallic acid and mussel mucin as main raw materials.
A preparation method of gallic acid modified bletilla striata polysaccharide hydrogel comprises the following preparation steps:
(1) Carrying out an activation reaction on gallic acid to prepare and obtain DMA (direct-access memory access) ester;
(2) Dissolving bletilla striata polysaccharide in DMSO to prepare a bletilla striata polysaccharide solution;
(3) Mixing the bletilla striata polysaccharide solution with DMA (direct memory access) ester, standing, performing transesterification reaction on hydroxyl groups in the bletilla striata polysaccharide solution and ester groups of the DMA ester, and dialyzing and freeze-drying to obtain a GA-BSP polymer freeze-dried sample;
(4) Dissolving the GA-BSP polymer freeze-dried sample in PBS buffer solution, adding mussel mucin, mixing, and then adding FeCl 3 The solution is continuously stirred to react to form hydrogel.
Specifically, the activation reaction in the step (1) is performed according to the following steps:
(1.1) dissolving gallic acid, DMAP and EDC.HCl in DMSO, and adding 3g gallic acid, 3g DMAP and 5g EDC.HCl into every 100ml DMSO to prepare a mixed solution;
(1.2) carrying out an activation reaction under the light-shielding condition, wherein the temperature of the activation reaction is 40 ℃, and the reaction time is 3 hours, so as to obtain the DMA ester.
Specifically, in the step (2), the bletilla striata polysaccharide is dissolved in DMSO, 8g of the bletilla striata polysaccharide is added into every 100ml of DMSO, and the solution is dissolved for 72 hours to prepare the bletilla striata polysaccharide solution.
Specifically, the preparation of the GA-BSP polymer freeze-dried sample in the step (3) is carried out according to the following steps:
(3.1) mixing the bletilla striata polysaccharide solution with the DMA ester, standing, and carrying out transesterification reaction on hydroxyl groups of the bletilla striata polysaccharide and ester groups of the DMA ester, wherein the transesterification reaction temperature is 60 ℃ and the reaction time is 12h, and the volume ratio of DMSO in the DMA ester to DMSO in the bletilla striata polysaccharide solution is 1:1, mixing the materials in proportion;
(3.2) dialyzing and freeze-drying the solution of the transesterification reaction to obtain a freeze-dried GA-BSP polymer sample.
Specifically, when the bletilla striata polysaccharide solution and the DMA ester are mixed in the step (3.1), slowly dripping the bletilla striata polysaccharide solution into the DMA ester, and stopping stirring after dripping, wherein the stirring speed is 300r/min;
specifically, in the step (3.2), a dialysis bag with a molecular weight cut-off of 7000D is used in the dialysis, the dialysis treatment time is 4 days, and water is changed 3 times a day in the dialysis process.
Specifically, the preparation of the hydrogel in the step (4) is performed according to the following steps:
(4.1) dissolving a lyophilized sample of 0.3. 0.3gGA-BSP polymer in 100ml PBS buffer to obtain a GA-BSP polymer solution;
(4.2) adding 0.5g of mussel mucin into the GA-BSP polymer solution in the step (4.1), and stirring and mixing uniformly to obtain a mixed solution;
(4.3) 20ml FeCl 3 The solution is added into the mixed solution of (4.2), and the reaction is continued with stirring, thus obtaining the hydrogel.
Specifically, the pH of the PBS buffer in the step (4.1) is 8; step (4.3) FeCl 3 The concentration of the solution was: 0.05mol/L.
Comparative example
Weighing 8.04g of bletilla striata polysaccharide BSP into 120ml of DMSO, stirring for 48 hours for dissolution, and obtaining a BSP solution; and 0.3g carbomer 940 is added to the solution, heated to 60 ℃ for 5 hours and a proper amount of triethanolamine is added to adjust the pH to about 7.4, thus obtaining the BPS hydrogel.
Experimental part
The hydrogel obtained in example 2 of the present invention (hereinafter described as GA-BSP hydrogel) and the BSP hydrogel obtained in comparative example were experimentally confirmed.
1. Morphological study
The hydrogel samples were subjected to freeze-drying, cutting and gold plating, and the cross-sectional morphology of the hydrogel samples was observed by a scanning electron microscope (SEM, JSM 6390, °c JEOL, japan).
SEM images of hydrogel samples recorded at 100x magnification are shown in fig. 1, with the cross section of the BSP hydrogel biased toward the layered structure and without significant pore structure. Compared with BSP hydrogel, the GA-BSP hydrogel has a more communicated porous structure, smaller pore diameter and more uniform distribution. The variation in pore size and structure is mainly due to the difference in crosslink density in the hydrogel network. Thus, GA-BSP hydrogels have a tighter network structure, while their porous structure can provide access to water vapor, facilitating nutrient and oxygen supply and waste removal required for cell viability.
2. Tissue adhesion Properties
The tissue adhesion properties of hydrogels were evaluated by lap shear tests. Measurements were made using a texture analyzer and adhesion data of the hydrogels were recorded. Each sample was tested in duplicate 3 times.
The experimental results are shown in FIG. 2, in which GA-BSP hydrogels showed better tissue adhesion properties than BSP. Abundant mussel mucin L-3, 4-dihydroxyphenylalanine group and pyrogallol-Fe 3+ Coordination and derived from GA-BSP/Fe 3+ The flexible polymer chains of the network all contribute to the high interfacial adhesion capability of the GA-BSP hydrogel adhesive. An adhesive hydrogel dressing that adheres completely to and bonds with tissue can prevent wound infection, facilitate sealing of skin wounds, and provide a good healing microenvironment.
3. Photothermal properties
The hydrogel was placed at room temperature on the bottom of the bottle, which was then exposed to a near infrared laser (NIR) (wavelength 808nm, irradiation intensity 2W/cm 2 ) The thermal infrared imager is used to detect temperature changes (Δt) in the process.
FIG. 3 is a graph of the delta T-NIR radiation time profile for BSP hydrogels and GA-BSP hydrogels.
The experimental results show that there is no significant change in temperature of the BSP hydrogel after 10min of NIR irradiation. However, the temperature of the hydrogel GA-BSP reached above 40℃in the course of NIR irradiation for 10min, which suggests that the GA-BSP hydrogel has a good photo-thermal effect. More importantly, as can be seen from the figure, the temperature of the hydrogel is changed along with the change of the irradiation time. This also shows that by adjusting the irradiation time, the hydrogel can be warmed up only to the appropriate temperature required, so that a better photothermal effect is achieved without burning the tissue.
4. In vitro near infrared photothermal antibacterial properties of hydrogels
The near infrared photothermal antibacterial properties of the hydrogels were evaluated using staphylococcus aureus (Staphylococcus aureus, s.aureus) and Escherichia coli (e.coli) as model strains.
The specific operation is as follows:
1. 200mg of sterilized hydrogel (sterilized 3 times at 80 ℃ C.) was placed at the bottom of a 48-well plate, and 0.5mL of S.aureus (or E.coli) bacterial suspension (10) 6 CFU·mL -1 ) Added to this well;
2. the control group was supplemented with only 0.5ml of s.aureus (or e.coli) bacterial suspension (10 6 CFU·mL -1 ）；
3. Blank groups were supplemented with only 0.5. 0.5mL sterile medium.
4. All experimental groups were equally divided into two groups, one group was exposed to NIR for 3 min and the other group was not treated at all.
5. 0.5mL sterile culture medium was added to each well to re-suspend any surviving bacteria.
All samples were incubated in an incubator at 37℃for 12 hours. After the end of the incubation, bacterial numbers were studied by dilution spread plate counting and bacterial viability was calculated. Each experimental group was repeated at least 5 times in parallel. The survival rate of bacteria was calculated by the following equation: bacterial viability (%) = (number of surviving bacteria of experimental group)/(number of surviving bacteria of control group) x 100%.
The experimental results are shown in FIG. 4, and after 3 minutes of NIR irradiation, the bacterial kill rates of GA-BSP photo-thermal hydrogel on S.aureus and E.coli were 90.27 + -0.23% and 75.94 + -0.54%, respectively, and the significance difference analysis showed that there was a significant difference between this group and the other groups (p<0.05). Meanwhile, the control+nir group showed no significant bacterial reduction compared to the hydrogel+nir group, indicating that pure NIR has no significant damage to bacteria. Likewise, the bacteria of the Hydrogel-NIR group are significantly reduced in number compared to the Control-NIR and Control +NIR groups, mainly due to BSP, GA and Fe 3+ Also has certain antibacterial activity. But in contrast to the Hydrogel+NIR group, HThe bacterial kill rate of the ydrogel-NIR group was far from sufficient, which also demonstrates that GA-BSP hydrogels do not perform well in terms of their antimicrobial effect when used alone. All the above results show that GA-BSP hydrogels have excellent NIR assisted photothermal antibacterial activity against both gram positive and gram negative bacteria, which shows great potential for protecting wounds from contamination.
5. Healing experiments of contaminated wounds
Compared with the common wound surface, the bacteria-infected wound surface has high bacterial infection rate, which greatly delays the healing of the wound. Thus, the bacterial wound healing capacity of the hydrogels was assessed by an s.aureus infected rat full thickness skin defect model. First, SD rats (200-250 g, male rats) adapted to a one-week living environment were anesthetized and the hairs on the backs were removed, and 4 circular full thickness wounds of 8mm diameter were made on the backs of the rats. Next, 50. Mu.L of S.aureus suspension (10 8 CFU·mL -1 ) Dropping onto the wound, covering the wound with a sterile closed PU film for 24 hours, and removing the PU film to obtain a wound model of bacterial infection. At this time, the prepared hydrogel sample (300 mg, diameter about 8mm, height about 3 mm) was used to treat the contaminated full thickness wound. The 4 wounds per mouse were treated as follows:
1. no treatment (Control-NIR);
2. exposure to NIR light alone for 2min (control+nir);
3. only with Hydrogel (Hydrogel-NIR);
4. the Hydrogel was placed and exposed to NIR light for 2min (Hydrogel+NIR).
Thereafter, the wounds of the rats were monitored periodically for any discomfort and inflammation. And simultaneously observing the healing condition of the wound, and photographing at intervals.
The experimental results are shown in fig. 5, with successful molding wounds showing significant pathogen contamination, all wounds inflamed and with pale yellow exudate (Day 0). It is also shown that all wounds treated with the 4 methods showed significant shrinkage on days 5, 10, and 15. Wherein hydrogel-treated wounds exhibited higher wound shrinkage than the blank (Control) group, with or without NIR irradiation. This shows that the hydrogel has a relatively strong wound healing effect, which is also mainly beneficial to bletilla striata polysaccharide, gallic acid and mussel protein which are used as hydrogel forming materials, and has good antibacterial and tissue repair promoting capabilities. But more importantly, the wound healing effect of the hydro-gel + NIR group performed better than that of the hydro-gel-NIR group and there was a large amount of hair coverage, which also represented the importance of photothermal antimicrobial treatment to some extent.
Claims (10)
1. A gallic acid modified bletilla striata polysaccharide hydrogel is characterized in that: it uses bletilla striata polysaccharide, gallic acid and mussel mucin as main raw materials.
2. A preparation method of gallic acid modified bletilla striata polysaccharide hydrogel is characterized by comprising the following steps: the preparation method comprises the following preparation steps:
(1) Carrying out an activation reaction on gallic acid to prepare and obtain DMA (direct-access memory access) ester;
(2) Dissolving bletilla striata polysaccharide in DMSO to prepare a bletilla striata polysaccharide solution;
(3) Mixing the bletilla striata polysaccharide solution with DMA (direct memory access) ester, standing, performing transesterification reaction on hydroxyl groups in the bletilla striata polysaccharide solution and ester groups of the DMA ester, and dialyzing and freeze-drying to obtain a GA-BSP polymer freeze-dried sample;
(4) Dissolving the GA-BSP polymer freeze-dried sample in PBS buffer solution, adding mussel mucin, mixing, and then adding FeCl 3 The solution is continuously stirred to react to form hydrogel.
3. The method for preparing the gallic acid modified bletilla striata polysaccharide hydrogel, which is characterized in that: the activation reaction in the step (1) is carried out according to the following steps:
(1.1) dissolving gallic acid, DMAP and EDC.HCl in DMSO, and adding 0.5-3g gallic acid, 0.5-3g DMAP and 1-5g EDC.HCl into every 100ml DMSO to prepare a mixed solution;
(1.2) carrying out an activation reaction under the light-shielding condition, wherein the temperature of the activation reaction is 15-40 ℃, and the reaction time is 0.5-3h, so as to obtain the DMA ester.
4. The method for preparing the gallic acid modified bletilla striata polysaccharide hydrogel, which is characterized in that: in the step (2), the bletilla striata polysaccharide is dissolved in DMSO, 3-8g of the bletilla striata polysaccharide is added into every 100ml of DMSO, and the solution is dissolved for 24-72 hours to prepare the bletilla striata polysaccharide solution.
5. The method for preparing the gallic acid modified bletilla striata polysaccharide hydrogel, which is characterized in that: the preparation of the GA-BSP polymer freeze-dried sample in the step (3) is carried out according to the following steps:
(3.1) mixing the bletilla striata polysaccharide solution with the DMA ester, standing, and carrying out transesterification between hydroxyl groups of the bletilla striata polysaccharide and ester groups of the DMA ester, wherein the transesterification is carried out at a temperature of 15-60 ℃ for 12-36h, and the volume ratio of DMSO in the DMA ester to DMSO in the bletilla striata polysaccharide solution is (0.5-1): 1, mixing the materials in proportion;
(3.2) dialyzing and freeze-drying the solution of the transesterification reaction to obtain a freeze-dried GA-BSP polymer sample.
6. The method for preparing the gallic acid modified bletilla striata polysaccharide hydrogel according to claim 5, which is characterized in that: and (3.1) slowly dripping the bletilla striata polysaccharide solution into the DMA ester when the bletilla striata polysaccharide solution is mixed with the DMA ester, wherein the process requires low-speed stirring, and the stirring is stopped after the dripping is finished, and the stirring speed is 100-300r/min.
7. The method for preparing the gallic acid modified bletilla striata polysaccharide hydrogel according to claim 5, which is characterized in that: and (3.2) using a dialysis bag with the molecular weight cut-off of 7000D in the dialysis, wherein the dialysis treatment time is 2-4 days, and water is changed 3-5 times per day in the dialysis process.
8. The method for preparing the gallic acid modified bletilla striata polysaccharide hydrogel, which is characterized in that: the hydrogel prepared in the step (4) is prepared according to the following steps:
(4.1) dissolving a lyophilized sample of 0.05-0.3. 0.3gGA-BSP polymer in 100ml PBS buffer to obtain a GA-BSP polymer solution;
(4.2) adding 0.1-0.5g mussel mucin into the GA-BSP polymer solution in the above (4.1), and stirring and mixing uniformly to obtain a mixed solution;
(4.3) 20ml FeCl 3 The solution is added into the mixed solution of (4.2), and the reaction is continued with stirring, thus obtaining the hydrogel.
9. The method for preparing the gallic acid modified bletilla striata polysaccharide hydrogel, which is characterized in that: the pH of the PBS buffer solution in the step (4.1) is 6-8; step (4.3) FeCl 3 The concentration of the solution was: 0.01-0.05mol/L.
10. Use of a gallic acid modified bletilla striata polysaccharide hydrogel according to any one of claims 1-9, characterized in that: it is used for chronic wounds, including burns, abrasions, surgical wounds, lacerations, and acute wounds including battlefield penetration wounds.
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