CN114129764A

CN114129764A - Multifunctional hydrogel and preparation method thereof

Info

Publication number: CN114129764A
Application number: CN202111406746.9A
Authority: CN
Inventors: 周丽; 杨作婷; 郑华; 张秋禹; 张宝亮; 刘金保
Original assignee: Northwestern Polytechnical University; Guangzhou Medical University
Current assignee: Northwestern Polytechnical University; Guangzhou Medical University
Priority date: 2021-11-24
Filing date: 2021-11-24
Publication date: 2022-03-04

Abstract

The invention discloses a multifunctional hydrogel and a preparation method thereof, wherein the multifunctional hydrogel is prepared from an aqueous solution of an alcohol-cationic polymer, an aqueous solution of an aldehyde biomedical polymer and an aqueous solution of 3,3' -dithiobis (propionohydrazide). The multifunctional hydrogel disclosed by the invention can be self-healed, can quickly stop bleeding, has redox responsiveness and lower cytotoxicity, has stronger antibacterial property to escherichia coli, staphylococcus aureus and methicillin-resistant staphylococcus aureus, can effectively resist bacterial biofilm, can effectively promote the healing of the wound surface of mouse bacterial biofilm infected skin, is simple in preparation method, low in cost, environment-friendly and low in toxicity, is expected to become a novel multifunctional biological material capable of simultaneously realizing the stopping bleeding, resisting infection and promoting the healing of the wound surface, and has a good application prospect in the clinical treatment of chronic infectious skin wound surfaces.

Description

Multifunctional hydrogel and preparation method thereof

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a multifunctional hydrogel and a preparation method thereof.

Background

Bacterial infection is the most common problem of skin wound healing and regeneration, and serious inflammatory reaction caused by infection not only obviously increases the number of diseases induced by wound infection, but also prolongs the normal inflammatory period due to the release of harmful enzyme, oxygen free radical and inflammatory cells, finally causes tissue damage, further deteriorates the wound and is difficult to heal. In addition, bacterial biofilms are easily formed after the wound surface is infected by bacteria, and the bacteria in the biofilms wrap themselves in an Extracellular Polymer (EPS) shielding matrix. The EPS matrix can be used as a protective barrier, which not only protects bacteria from host innate immune cells, but also prevents penetration of antibacterial agents. It is therefore difficult to completely remove them with antibiotics, which only kill free bacteria on the surface of the capsule or in the blood that cause the onset of infection. When the body resistance is reduced, the bacteria living in the biofilm can be released again to cause infection again. Moreover, due to the encapsulation of EPS, the biofilm microenvironment is hypoxic, leading to anaerobic glycolysis, forming an acidic and highly reducing microenvironment. For example, the pH of the microenvironment of methicillin-resistant Staphylococcus aureus (MRSA) biofilms is 5.5 or less, and the concentration of reduced Glutathione (GSH) in E.coli biofilms is as high as 10 mM. The biofilm acts as a 'bacteria nest', so that the infection is repeatedly bought, a chronic infectious wound surface is formed, the biofilm can not be healed for a long time, great pain is brought to patients, and huge economic burden is caused to the society.

Currently, clinical treatments include the use of novel dressings, anti-biofilm therapy, negative pressure occlusive drainage, hyperbaric oxygen therapy, growth factor therapy, and stem cell therapy. Among the new types of dressings, hydrogels have become the most promising wound dressings due to their properties of providing a moist microenvironment, absorbing tissue penetration fluid, and resisting infection. However, most conventional hydrogels tend to lack multifunctional properties and requireOther auxiliary means are used to promote the healing of the infective wounds. An ideal hydrogel for healing of infectious wounds should have the following characteristics: proper mechanical performance, self-healing, rapid hemostasis, stronger tissue adhesion, antibacterial activity, good biocompatibility and the like. For example: the self-healing hydrogel can keep stable structure in the process of wound surface repair; the hydrogel with strong tissue adhesion can be well attached to the wound surface, does not fall off in the healing process, can keep the moist microenvironment of the wound surface in the whole healing process, and reduces the formation of scars; the rapid hemostatic ability of the hydrogel can rapidly stop bleeding in the early healing period of the wound surface, so that excessive tissue blood loss is avoided, and the wound surface is facilitated to heal; the hydrogel with antibacterial activity can effectively resist bacterial infection and reduce the formation of bacterial biofilm and inflammatory reaction of wound surface; good biocompatibility can reduce the cytotoxicity of the hydrogel and can also promote cell proliferation. Although a number of hydrogel wound dressings have emerged clinically, such as: kanghuier, Tegaderm^3MSkin disease, polyurethane gel, etc. but the price is high, and the key problems of breaking bacterial biofilm, improving wound microenvironment, poor anti-infection capability, weak tissue adhesion, poor healing, etc. when the medicine is used for treating infective wounds, the medicine can not be effectively solved.

Therefore, the development of novel hydrogels with multifunctional properties for the healing treatment of infectious wounds is imminent.

Disclosure of Invention

Based on the above, one of the objectives of the present invention is to provide a multifunctional hydrogel, which has the function of promoting the healing of infectious skin wounds, and also has the effects of hemostasis and bacterial infection resistance.

The specific technical scheme for realizing the aim of the invention comprises the following steps:

the multifunctional hydrogel is prepared from an aqueous solution of an alcohol-cationic polymer, an aqueous solution of an aldehyde biomedical polymer and an aqueous solution of 3,3' -dithiobis (propionohydrazide).

In some of these embodiments, the alcohol in the alcohol-cationic polymer is glycerol, pentaerythritol, or polyethylene glycol; and/or the cationic polymer in the alcohol-cationic polymer is epsilon-polylysine or carboxymethyl chitosan.

In some of these embodiments, the alcohol-cationic polymer is prepared by: reacting the alcohol compound with p-toluenesulfonyl chloride to prepare alcohol-Ots; alcohol-Ots is then reacted with the cationic polymer to produce an alcohol-cationic polymer.

In some embodiments, the aldehyde-based biomedical polymer is one or more of aldehyde-based Pluronic F127, aldehyde-based hyaluronic acid, aldehyde-based pullulan, and aldehyde-based dextran.

In some of these examples, the aldehydized Pluronic F127 was prepared by the following method: pluronic F127 was reacted with p-toluenesulfonyl chloride to prepare F127-Ots; the reaction of F127-Ots with 4-hydroxybenzaldehyde and potassium carbonate produced the aldehyde Pluronic F127.

In some embodiments, the aldehyde-modified hyaluronic acid is prepared by reacting hyaluronic acid with sodium periodate; the aldehyde pullulan polysaccharide is prepared by reacting pullulan polysaccharide with sodium periodate; the aldehyde dextran is prepared by reacting dextran with sodium periodate.

In some embodiments, the volume ratio of the aqueous solution of the alcohol-cationic polymer, the aqueous solution of the aldehyde-based biomedical polymer and the aqueous solution of the 3,3' -dithiobis (propionohydrazide) is 1-10: 16-30: 1-10%, wherein the mass concentration of the aqueous solution of the alcohol-cationic polymer is 2-30%, the mass concentration of the aqueous solution of the aldehyde biomedical polymer is 5-40%, and the mass concentration of the aqueous solution of the 3,3' -dithiobis (propionohydrazide) is 1-10%.

In some embodiments, the mass concentration of the aqueous solution of the alcohol-cationic polymer is 5% to 20%; and/or the mass concentration of the aqueous solution of the aldehyde biomedical polymer is 5-30 percent; and/or the mass concentration of the 3,3' -dithiobis (propionohydrazide) aqueous solution is 5-10%.

In some embodiments, the volume ratio of the aqueous solution of the alcohol-cationic polymer, the aqueous solution of the aldehyde-based biomedical polymer and the aqueous solution of the 3,3' -dithiobis (propionohydrazide) in the step (2) is 1-8: 18-24: 1 to 8.

The invention also provides a preparation method of the multifunctional hydrogel, and the specific technical scheme comprises the following steps:

a preparation method of multifunctional hydrogel comprises the following steps: and (2) uniformly mixing the aqueous solution of the alcohol-cationic polymer and the aqueous solution of the 3,3' -dithiobis (propionohydrazide), adding the mixture into the aqueous solution of the aldehyde biomedical polymer, and standing the mixture at room temperature for 5min to 24h to obtain the aldehyde biomedical polymer.

Compared with the prior art, the invention has the following beneficial effects:

(1) in the multifunctional hydrogel, the inventor modifies a proper alcohol compound and a cationic polymer with stronger antibacterial property into an alcohol-cationic polymer with better biocompatibility, the biomedical polymer is subjected to hydroformylation to obtain a macromolecular compound containing aldehyde groups, DTPH with disulfide bonds is added, and the multifunctional hydrogel (GAD) is obtained through Schiff base reaction.

(2) The multifunctional hydrogel disclosed by the invention can be self-healed, can quickly stop bleeding, has redox responsiveness and lower cytotoxicity, has stronger antibacterial property to escherichia coli, staphylococcus aureus and methicillin-resistant staphylococcus aureus, can effectively resist bacterial biofilm, can effectively promote the healing of the wound surface of mouse bacterial biofilm infected skin, is simple in preparation method, low in cost, environment-friendly (no organic solvent residue), and low in toxicity, is expected to become a novel multifunctional biomaterial capable of simultaneously realizing the hemostasis, the infection resistance and the wound surface healing promotion, and has a good application prospect in the clinical treatment of the chronic infectious skin wound surface.

Drawings

FIG. 1 shows one of the components of preparation of GEPL of GAD1 hydrogel in example 1 of the present invention¹H-NMR nuclear magnetic spectrum.

FIG. 2 shows one of the components of the preparation of GAD1 hydrogel of example 1 of the present invention, FCHO¹H-NMR nuclear magnetic spectrum.

FIG. 3 is a diagram showing the gelation process of the GAD1 hydrogel in example 1 of the present invention.

FIG. 4 is an IR spectrum of the components of the GAD1 hydrogel of example 1 of the present invention.

FIG. 5 is a graph showing the sol-gel transition of the redox stimulus response of the GAD1 hydrogel in the experimental examples of the present invention.

FIG. 6 shows the self-healing results of the GAD1 hydrogel in the experimental examples of the present invention.

FIG. 7 shows the results of the measurement of the tissue adhesiveness of the GAD2 hydrogel in the test examples of the present invention.

FIG. 8 shows the results of toxicity measurements of GAD3 and each fraction on L929 cells at different concentrations in the test examples of the present invention.

Fig. 9 shows the results of the anti-bacterial effects of the GAD4 and GAD5 hydrogels and the control group against escherichia coli (e.coli), staphylococcus aureus (s.aureus) and methicillin-resistant staphylococcus aureus (MRSA) in the test examples of the present invention.

FIG. 10 shows the result of measurement of hemostatic ability of GAD5 hydrogel in the test example of the present invention.

FIG. 11 shows the measurement results of the consumption of GSH in the biofilm by the GAD5 hydrogel in the test examples of the present invention.

FIG. 12 shows the results of repairing skin lesions infected with bacterial biofilm in mice with the GAD5 hydrogel and each control group in the experimental examples of the present invention.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In one aspect, the invention provides a multifunctional hydrogel, which is prepared from an aqueous solution of an alcohol-cationic polymer, an aqueous solution of an aldehyde-based biomedical polymer and an aqueous solution of 3,3' -dithiobis (propionohydrazide).

When in use, the cationic polymer is modified in consideration of both antibacterial effect and toxicity, wherein the alcohol in the alcohol-cationic polymer is selected from glycerol, pentaerythritol or polyethylene glycol (molecular weight is preferably 2000,5000 and 10000), and the cationic polymer is selected from epsilon-polylysine or carboxymethyl chitosan. The main structure of the hydrogel is combined with aldehyde biomedical polymers, so that the hydrogel has good biocompatibility, the tissue adhesion of the hydrogel can be enhanced, the hydrogel has the effects of quickly stopping bleeding, promoting angiogenesis and the like, DTPH is creatively introduced as one of the components of the hydrogel, amino at the tail end of the DTPH participates in the formation of a gel network through Schiff base reaction with aldehyde groups, the gel network structure is enabled to be more stable, disulfide bonds in the structure can endow the gel network with redox responsiveness, GSH in a biofilm microenvironment can be consumed, the wound microenvironment is improved, and wound healing is facilitated. Under the organic synergistic effect of the components, the hydrogel can obviously promote the healing of the infected skin wound, and has the functions of hemostasis and antibiosis.

Furthermore, the inventor of the present invention has screened the concentrations and volume ratios of the above three components, and the concentrations and volume ratios directly affect whether the hydrogel can be gelled and also affect the functions of the hydrogel. The strength, modulus, tissue adhesion, hemostatic ability, antibacterial activity and wound healing ability of the gel can be affected by the change of concentration and volume ratio. In consideration of toxicity and gel strength, the appropriate mass concentration of the alcohol-cationic polymer aqueous solution is 2-30%, the appropriate mass concentration of the aldehyde-based biomedical polymer aqueous solution is 5-40%, the appropriate mass concentration of the 3,3 '-dithiobis (propionohydrazide) aqueous solution is 1-10%, and the appropriate volume ratio of the alcohol-cationic polymer aqueous solution, the aldehyde-based biomedical polymer aqueous solution and the 3,3' -dithiobis (propionohydrazide) aqueous solution is 1-10: 16-30: 1 to 10.

The present invention will be described in further detail with reference to the following specific embodiments and the accompanying drawings.

EXAMPLE 1 multifunctional hydrogel and method for preparing the same

The multifunctional hydrogel of the present example was prepared from 5 μ L of an alcohol-cationic polymer aqueous solution (glycerol-polylysine polymer GEPL, 20 wt%), 90 μ L of an aldehyde-based biomedical polymer aqueous solution (F127-p-hydroxybenzaldehyde FCHO, 20 wt%) and 5 μ L of DTPH (3,3' -dithiobis (propionohydrazide)) aqueous solution (10 wt%).

The preparation method of the multifunctional hydrogel comprises the following steps:

(1) preparation of Pluronic F127-p-toluenesulfonate (F127-OTs)

6.2g of Pluronic F127 was dissolved in 60mL of anhydrous chloroform, followed by addition of 0.42mL of triethylamine followed by 0.57g of p-toluenesulfonyl chloride, and reacted at room temperature for 24 h. After the reaction was complete, the organic phase was washed with dilute hydrochloric acid and saturated sodium bicarbonate solution, respectively. After removal of excess organic solvent, it was concentrated in glacial ethyl ether and precipitated to give F127-OTs (86% yield).

(2) Preparation F127-p-hydroxybenzaldehyde (FCHO)

2g F127-OTs were dissolved in 40mL DMF and 0.09g 4-hydroxybenzaldehyde and 0.1g potassium carbonate (K) were added sequentially₂CO₃) The reaction was carried out at 80 ℃ for 72 h. After the reaction solution was cooled to room temperature, 50mL of water was added and the mixture was extracted with dichloromethane. After drying the organic phase over anhydrous magnesium sulfate, FCHO was obtained by precipitation in iced ether (91% yield).

FIG. 1 is a drawing of F127-p-hydroxybenzaldehyde (FCHO)¹H-NMR nuclear magnetic spectrum, from which it is known that characteristic peaks ascribed to F127 and p-hydroxybenzaldehyde are shown on the spectrum, indicating that FCHO was successfully synthesized.

(3) Preparation of Glycerol-p-toluenesulfonate (alcohol-Ots)

1g of glycerol was dissolved in 50mL of dichloromethane, and then 3mL of triethylamine and 4g of p-toluenesulfonyl chloride were sequentially added to react at room temperature for 24 hours. After the reaction was complete, the organic phase was washed with dilute hydrochloric acid and saturated sodium bicarbonate solution, respectively. The organic phase was rotary evaporated to afford glycerol-OTs (80% yield).

(4) Preparation of Glycerol-polylysine Polymer (GEPL)

0.31g of glycerol-OTs and 10g of EPL (. epsilon. -polylysine) were dissolved in 5mL of water and 100mL of DMSO, respectively, and then an aqueous solution of glycerol-OTs was added to the EPL solution to react at 60 ℃ for 72 hours. After the reaction is finished, dialyzing in distilled water for 3 days by using a dialysis bag with the molecular weight cutoff of 5000, and freeze-drying to obtain the GEPL polymer.

FIG. 2 is of a glycerol-polylysine polymer (GEPL)¹H-NMR nuclear magnetic spectrum shows that characteristic peaks attributed to glycerol and EPL (. epsilon. -polylysine) are shown on the spectrum, indicating that GEPL was successfully synthesized.

(5) Preparation of hydrogel (GAD1)

mu.L of a GEPL polymer (20 wt%) solution and 5. mu.L of an aqueous DTPH (3,3' -dithiobis (propionohydrazide)) solution (10 wt%) were mixed, and then added to 90. mu.L of an aqueous LFCHO solution (20 wt%), followed by mixing and standing at room temperature for 5min to obtain the GAD1 hydrogel of this example. FIG. 3 is a photograph showing the gelation process of the GAD1 hydrogel, wherein the gel state is obtained by adding GEPL and DTPH into FCHO solution for 5min, namely GAD1 (wherein G is alcohol-cationic polymer; A is aldehyde biomedical polymer; and D is 3,3' -dithiobis (propionohydrazide) hydrogel.

Sample GA1, prepared with 5 μ L of water instead of aqueous DTPH, was used as a control.

FIG. 4 is an IR spectrum of each component of the GAD1 hydrogel prepared in this example, showing that the C-H stretching vibration absorption peak of F127 is 2876cm^-1，1692cm^-1And 1600cm^-1The characteristic absorption peaks of the skeleton vibration and the aldehyde group on the FCHO benzene ring respectively show the successful synthesis of FCHO. 1692cm^-1Treatment of aldehydesExtinction of base peak and 1669cm^-1Stretching vibration of the imine bond indicates that the aldehyde group of FCHO and the amino group of GEPL react via schiff base to form a GAD1 hydrogel network. Furthermore, 3320cm^-1The disappearance of the characteristic absorption peak from the primary amine on DTPH indicates that the primary amine of DTPH also undergoes schiff base reaction with the aldehyde group of FCHO to participate in the formation of the GAD1 gel network.

EXAMPLE 2 multifunctional hydrogel and method for preparing the same

The multifunctional hydrogel of the embodiment is prepared from 30 μ L of an alcohol-cationic polymer aqueous solution (polyethylene glycol-polylysine polymer PEPL, 10 wt%), 120 μ L of an aldehyde-based biomedical polymer aqueous solution (F127-p-hydroxybenzaldehyde FCHO, 20 wt%) and 20 μ L of DTPH (3,3' -dithiobis (propionohydrazide)) aqueous solution (5 wt%).

(1) preparation of Pluronic F127-p-toluenesulfonate (F127-OTs)

Same as example 1, step (1).

(2) Preparation F127-p-hydroxybenzaldehyde (FCHO)

Same as example 1, step (2).

(3) Preparation of polyethylene glycol-p-toluenesulfonate (PEG-Ots)

5g of polyethylene glycol (molecular weight 2000) was dissolved in 100mL of methylene chloride, and then 1.4mL of triethylamine and 1.9g of p-toluenesulfonyl chloride were sequentially added and reacted at room temperature for 72 hours. After the reaction was complete, the organic phase was washed with dilute hydrochloric acid and saturated sodium bicarbonate solution, respectively. The organic phase was rotary evaporated to give polyethylene glycol-OTs (PEG-OTs) (83% yield).

(4) Preparation of polyethylene glycol-polylysine Polymer (PEPL)

1g of PEG-OTs and 2.5g of EPL (. epsilon. -polylysine) were dissolved in 20mL of water and 30mL of DMMSO, respectively, and then an aqueous solution of PEG-OTs was added to the EPL solution to react at 60 ℃ for 72 hours. After the reaction is finished, dialyzing in distilled water for 3 days by using a dialysis bag with the molecular weight cutoff of 6000, and freeze-drying to obtain the PEPL polymer.

(5) Preparation of hydrogel (GAD2)

After mixing 30. mu.L of PEPL polymer (10 wt%) solution and 20. mu.L of DTPH aqueous solution (5 wt%), the mixture was added to 120. mu.L of FCHO aqueous solution (20 wt%), and after mixing, the mixture was left at room temperature for 5min to obtain the GAD2 hydrogel of this example.

Sample GA2, prepared with 20 μ L of water instead of aqueous DTPH, was used as a control.

EXAMPLE 3 multifunctional hydrogel and method for preparing the same

The multifunctional hydrogel of the embodiment is prepared from 10 μ L of an alcohol-cationic polymer aqueous solution (pentaerythritol-polylysine polymer, TEPL, 10 wt%), 80 μ L of an aldehydized biomedical polymer aqueous solution (aldehydized hyaluronic acid, HA-CHO, 5 wt%) and 10 μ L of DTPH (3,3' -dithiobis (propionohydrazide)) aqueous solution (10 wt%).

(1) preparing aldehyde hyaluronic acid (HA-CHO)

1g Hyaluronic Acid (HA) was dissolved in 100mL distilled water, 210mg sodium periodate (NaIO) was added₄) The reaction was carried out at room temperature for 3h in the absence of light. After the reaction was completed, 500. mu.L of ethylene glycol was added to quench the reaction. Then, the reaction solution was dialyzed in distilled water for 3 days using a dialysis bag with a molecular weight cut-off of 14000, and lyophilized to obtain aldehyde-modified hyaluronic acid (HA-CHO) (yield 85%).

(2) Preparation of pentaerythritol-p-toluenesulfonate (alcohol-Ots)

1g of pentaerythritol was dissolved in 50mL of methylene chloride, and then 5mL of triethylamine and 6.7g of p-toluenesulfonyl chloride were sequentially added to react at room temperature for 24 hours. After the reaction was complete, the organic phase was washed with dilute hydrochloric acid and saturated sodium bicarbonate solution, respectively. The organic phase was rotary evaporated to obtain pentaerythritol-OTs (82% yield).

(3) Preparation of pentaerythritol-polylysine Polymer (TEPL)

0.2g of pentaerythritol-OTs and 10g of EPL (. epsilon. -polylysine) were dissolved in 5mL of water and 100mL of DMSO, respectively, and then an aqueous solution of pentaerythritol-OTs was added to the EPL solution to react at 60 ℃ for 72 hours. After the reaction is finished, dialyzing for 3 days in distilled water by using a dialysis bag with the molecular weight cutoff of 10000, and freeze-drying to obtain the TEPL polymer.

(4) Preparation of hydrogel (GAD3)

mu.L of TEPL polymer (10 wt%) solution and 10. mu.L of DTPH aqueous solution (10 wt%) were mixed, and then added to 80. mu.L of HA-CHO aqueous solution (5 wt%), and after mixing, the mixture was left at room temperature for 10min to obtain the GAD3 hydrogel of this example.

Sample GA3, prepared using 10. mu.L of water instead of aqueous DTPH, was used as a control.

EXAMPLE 4 multifunctional hydrogel and method for preparing the same

The multifunctional hydrogel of the embodiment is prepared from 5 μ L of an alcohol-cationic polymer aqueous solution (polyethylene glycol-carboxymethyl chitosan polymer PCS, 5 wt%), 90 μ L of an aldehyde-based biomedical polymer aqueous solution (aldehyde-based hyaluronic acid HA-CHO, 5 wt%) and 5 μ L of DTPH (3,3' -dithiobis (propionohydrazide)) aqueous solution (10 wt%).

(1) preparing aldehyde hyaluronic acid (HA-CHO)

Same as example 3, step (1).

(2) Preparation of polyethylene glycol-p-toluenesulfonate (PEG-Ots)

Same as example 2, step (3).

(3) Preparing polyethylene glycol-carboxymethyl chitosan Polymer (PCS)

1g of PEG-OTs and 2g of carboxymethyl chitosan were dissolved in 100mL of water and reacted at 60 ℃ for 72 hours. After the reaction, the PCS polymer is obtained by dialyzing the mixture in distilled water for 3 days by using a dialysis bag with the molecular weight cutoff of 14000 and freeze-drying the mixture.

(4) Preparation of hydrogel (GAD4)

mu.L of PCS polymer (5 wt%) solution and 5. mu.L of DTPH aqueous solution (10 wt%) were mixed, and then added to 90. mu.L of HA-CHO aqueous solution (5 wt%), and after mixing, the mixture was left at room temperature for 1 hour to obtain the GAD4 hydrogel of the present example.

Sample GA4, prepared with 5 μ L of water instead of aqueous DTPH, was used as a control.

EXAMPLE 5 multifunctional hydrogel and method for preparing the same

The multifunctional hydrogel of the embodiment is prepared from 5 μ L of an alcohol-cationic polymer aqueous solution (pentaerythritol-polylysine polymer TEPL, 20 wt%), 90 μ L of an aldehydized biomedical polymer aqueous solution (aldehydized pullulan Pu-CHO, 10 wt%) and 5 μ L of a DTPH (3,3' -dithiobis (propionohydrazide)) aqueous solution (5 wt%).

(1) preparing aldehyde pullulan (Pu-CHO)

Dissolving 1g pullulan (Pu) in 100mL distilled water, adding 855mg sodium periodate (NaIO)₄) The reaction was carried out at room temperature for 3h in the absence of light. After the reaction was completed, 500. mu.L of ethylene glycol was added to quench the reaction. Then, the reaction solution was dialyzed in distilled water for 3 days using a dialysis bag with a cut-off of 14000, and lyophilized to obtain aldehyde-modified pullulan (Pu-CHO) (yield 80%).

(2) Preparation of pentaerythritol-p-toluenesulfonate (alcohol-Ots)

Same as example 3, step (2).

(3) Preparation of pentaerythritol-polylysine Polymer (TEPL)

Same as example 3, step (3).

(4) Preparation of hydrogel (GAD5)

mu.L of a TEPL polymer (20 wt%) solution and 5. mu.L of an aqueous DTPH solution (5 wt%) were mixed together, and then added to 90. mu.L of Pu-CHO aqueous solution (10 wt%), followed by mixing and standing at room temperature for 10min to obtain the GAD5 hydrogel of this example.

Sample GA5, prepared with 5 μ L of water instead of aqueous DTPH, was used as a control.

EXAMPLE 6 multifunctional hydrogel and method for preparing the same

The multifunctional hydrogel of the embodiment is prepared from 5 μ L of an alcohol-cationic polymer aqueous solution (polyethylene glycol-carboxymethyl chitosan polymer PCS, 10 wt%), 90 μ L of an aldehyde-based biomedical polymer aqueous solution (aldehyde-based pullulan Pu-CHO, 10 wt%) and 5 μ L of a DTPH (3,3' -dithiobis (propionohydrazide)) aqueous solution (6 wt%).

(1) preparing aldehyde pullulan (Pu-CHO)

Same as example 5, step (1).

(2) Preparation of polyethylene glycol-p-toluenesulfonate (PEG-Ots)

Same as example 2, step (3).

(3) Preparing polyethylene glycol-carboxymethyl chitosan Polymer (PCS)

Same as example 4, step (3).

(4) Preparation of hydrogel (GAD6)

After 5. mu.L of PCS polymer (10 wt%) solution and 5. mu.L of DTPH aqueous solution (6 wt%) were mixed, 90. mu.L of Pu-CHO aqueous solution (10 wt%) was added, and after mixing, the mixture was left at room temperature for 20min to obtain the GAD6 hydrogel of the present example.

Sample GA6, prepared with 5 μ L of water instead of aqueous DTPH, was used as a control.

EXAMPLE 7 multifunctional hydrogel and method for preparing the same

The multifunctional hydrogel of the embodiment is prepared from 20 μ L of an alcohol-cationic polymer aqueous solution (polyethylene glycol-polylysine polymer PEPL, 10 wt%), 90 μ L of an aldehyde-based biomedical polymer aqueous solution (aldehyde-based dextran Dex-CHO, 10 wt%) and 15 μ L of DTPH (3,3' -dithiobis (propionohydrazide)) aqueous solution (5 wt%).

(1) preparation of aldehyde Glucan (Dex-CHO)

1g of dextran (Dex) was dissolved in 100mL of distilled water, and 991mg of sodium periodate (NaIO) was added₄) The reaction was carried out at room temperature for 3h in the absence of light. After the reaction was completed, 500. mu.L of ethylene glycol was added to quench the reaction. Then, the reaction solution was dialyzed in distilled water using a dialysis bag with a cut-off of 14000 for 3 days, and lyophilized to obtain aldehyde-converted dextran (Dex-CHO) (yield 87%).

(2) Preparation of polyethylene glycol-p-toluenesulfonate (PEG-Ots)

Same as example 2, step (3).

(3) Preparation of polyethylene glycol-polylysine Polymer (PEPL)

Same as example 2, step (4).

(4) Preparation of hydrogel (GAD7)

After 20. mu.L of PEPL polymer (10 wt%) solution and 15. mu.L of DTPH aqueous solution (5 wt%) were mixed well, 90. mu.L of Dex-CHO aqueous solution (10 wt%) was added, and after mixing well, the mixture was left at room temperature for 40min to obtain the GAD7 hydrogel of this example.

Sample GA7, prepared with 15 μ L of water instead of aqueous DTPH, was used as a control.

EXAMPLE 8 multifunctional hydrogel and method for preparing the same

The multifunctional hydrogel of the present example was prepared from 10 μ L of an aqueous solution of an alcohol-cationic polymer (glycerol-carboxymethyl chitosan polymer GCS, 10 wt%), 80 μ L of an aqueous solution of an aldehyde-modified biomedical polymer (aldehyde-modified dextran Dex-CHO, 10 wt%) and 10 μ L of an aqueous solution of DTPH (3,3' -dithiobis (propionohydrazide)) (5 wt%).

(1) preparation of aldehyde Glucan (Dex-CHO)

Same as example 7, step (1).

(2) Preparation of Glycerol-p-toluenesulfonate (Glycerol-Ots)

Same as example 1, step (3).

(3) Preparation of Glycerol-carboxymethyl Chitosan Polymer (GCS)

0.5g of glycerol-OTs and 1.5g of carboxymethyl chitosan were dissolved in 60mL of water and reacted at 60 ℃ for 72 hours. After the reaction is finished, dialyzing for 3 days in distilled water by using a dialysis bag with the molecular weight cutoff of 10000, and freeze-drying to obtain the GCS polymer.

(4) Preparation of hydrogel (GAD8)

mu.L of GCS polymer (10 wt%) solution and 10. mu.L of DTPH aqueous solution (5 wt%) were mixed, and then added to 80. mu.L of Dex-CHO aqueous solution (10 wt%), and after mixing, the mixture was left at room temperature for 20min to obtain the GAD8 hydrogel of this example.

Sample GA8, prepared using 10. mu.L of water instead of aqueous DTPH, was used as a control.

Test examples 1 to 8 Performance test of the hydrogel GAD obtained in examples 1 to 8

The performance of the hydrogel GAD prepared in examples 1 to 8 was tested.

The hydrogel GAD1 prepared in example 1 was subjected to a redox response test and a self-healing capability test. The hydrogel GAD2 prepared in example 2 was tested for tissue adhesion. The hydrogel GAD3 prepared in example 3 was subjected to cytotoxicity test. The hydrogel GAD4 obtained in example 4 was subjected to an antibacterial test. The hydrogel GAD5 prepared in example 5 was tested for antibacterial, hemostatic, GSH depleting capacity in biofilm and for promoting healing of skin wounds.

1. Redox responsiveness

The redox responsiveness of the GAD1 hydrogel was observed by adding the reducing agent Dithiothreitol (DTT) to the GAD1 hydrogel for 2 min. The results are shown in FIG. 5.

As can be seen from FIG. 5, the addition of the reducing agent Dithiothreitol (DTT) to the GAD1 hydrogel resulted in the GAD1 becoming in solution as the DTT breaks the disulfide bonds in the gel network; when adding oxidant hydrogen peroxide (H)₂O₂) After that, the disrupted disulfide bond is oxidized again to form GAD1, which is changed back to gel state, thus proving that GAD1 has redox responsiveness.

2. Self-healing capability

The self-healing process of the GAD1 hydrogel was observed. The results are shown in FIG. 6.

As can be seen from fig. 6, the pores in the gel gradually became smaller over time until they disappeared, demonstrating that GAD1 can self-heal.

3. Tissue adhesive capacity

The adhesion properties of the hydrogel to the skin were examined with fresh pig skin. The method comprises the following steps: the pigskin was first washed and excess fat removed and then cut into two small squares (10 mm. times.10 mm). Then two pigskins are respectively fixed on a detection platform and a parallel plate of the rheometer by double faced adhesive tapes, and the gap is 500 mu m. 100 μ L GAD2 hydrogel or control GA2, FCHO were added to the gap between the two pigskins, respectively. Subsequently, the two pigskins after the addition of the material were pressed at 37 ℃ for 2 minutes, and the reverse stress change of each group upon stretching was measured by a TA rheometer (DHR-2), thereby evaluating the tissue adhesiveness of the stent.

FIG. 7 shows the results of adhesion measurements of GAD2 and the control groups, which shows that GAD2 has the highest stress on fresh pig skin compared to GA2 and FCHO, indicating the strongest tissue adhesion.

4. Cytotoxicity assays

FIG. 8 shows the cytotoxicity results of GAD3 and each fraction incubated in L929 cells for 24h at different concentrations, and it can be seen from FIG. 8 that the cytotoxicity of GAD3 and each fraction is low in L929 cells, and the cell viability is above 80% even at a concentration of 25. mu.g/mL, indicating that the GAD3 hydrogel has good biocompatibility.

5. Test for antibacterial Properties

The antimicrobial performance of GAD hydrogels was evaluated by measuring the viability of escherichia coli (e.coli), staphylococcus aureus (s.aureus), and methicillin-resistant staphylococcus aureus (MRSA) after 2 hours incubation on the hydrogel surface. The method comprises the following specific steps:

e.coli, S.aureus and MRSA were inoculated separately in MHB medium and cultured on a shaker (200rpm) at 37 ℃ until the bacteria were in logarithmic growth phase. 400 μ L GAD4 hydrogel was prepared in 24-well plates. GA4, HCHO and ampicillin were used as controls. Then, the diluted bacterial solution (10. mu.L, 10 ml) was added⁶CFU mL^-1) Was added dropwise to the surfaces of GAD4 and control, respectively. After incubation at 37 ℃ for 2 hours, the cells were diluted 100-fold with PBS, and 10. mu.L of the suspension was inoculated on LB agar (Sigma) and incubated for 18 hours. The number of colonies in each dish was recorded by photography. The antimicrobial properties of GAD4 and the control were expressed as the relative size of the bacterial count (bacterial viability%) in each dish.

The antibacterial experiment of GAD5 was performed with vancomycin as a positive control, as described above.

Fig. 9 shows the antibacterial results of the GAD4(a) hydrogel and the control group, and the GAD5(B) hydrogel and the control group against escherichia coli (E, coli), staphylococcus aureus (S, aureus), and methicillin-resistant staphylococcus aureus (MRSA), and it can be seen that GAD4, GA4, GAD5, and GA5 all show higher antibacterial performance among the three bacteria, and the antibacterial ability thereof reaches 95% or more, indicating that the GAD hydrogel prepared by the present invention has stronger anti-infection ability.

6. Hemostatic ability measurement experiment

The hemostatic ability of GAD hydrogels was determined using a mouse model of bleeding liver (Kunming mouse, 20-30g, female). The method comprises the following steps:

the anesthetized mice were first opened ventrally to expose the liver and remove interstitial fluid around the liver. The liver was then placed on pre-weighed filter paper (W)₀) And the liver was bled with an 18G needle. Immediately thereafter, 50 μ L GAD5 was overlaid on the bleeding site. Bleeding from the bleeding sites of the liver was recorded by photographing at 0,5, 15, 30 and 60s, respectively, and mice with untreated bleeding sites were used as controls. The results are shown in FIG. 10.

As can be seen from fig. 10, the amount of bleeding in the control group significantly increased with time. After 5s of treatment with GAD5 hydrogel, bleeding at the site of liver bleeding was rapidly controlled.

7. Determination of GSH consumption Capacity in biofilms

MRSA (100. mu.L, 10)⁸CFU mL^-1) Inoculating the mixture into a 96-well plate added with MHB culture solution, placing the mixture in an incubator at 37 ℃ for incubation for 24h, replacing the old culture solution with fresh MHB every 24h, changing the solution twice, and continuing incubation for 24h to obtain the MRSA biofilm. PBS, GA5, and GAD5(100uL) were added to the MRSA biofilms, incubated for 1h, and the GSH concentration of each group was determined using the GSH detection kit according to the instructions. The results are shown in FIG. 11.

As can be seen from fig. 11, the lowest GSH concentration in the GAD5 group compared to the control group indicates that GSH in the biofilm microenvironment was consumed due to the presence of DTPH.

8. Test for promoting healing of skin wound

The in vivo antibacterial performance and healing capacity of the GAD hydrogel to infectious skin wounds were determined in a mouse full-thickness skin wound bacterial biofilm infection model. The method comprises the following steps:

BALB/c female mice (25-30g) were anesthetized and their backs shaved. After iodophor sterilization, a round full-thickness wound (diameter: 7mm) was made on the back of the mouse using a skin incision device, and 10. mu.L of MRSA (1X 10) was dropped on the wound site⁶CFU mL^-1) And (5) constructing an MRSA biofilm infection skin wound model by using the bacterial liquid. Mice were randomly divided into four groups (n ═ 6): control (blank), GA5, GAD5 and commercial wound adjuvant Tegaderm^3MAnd (4) grouping. Then, GA5 and GAD5(50 μ L) were applied to the wound separately, and hollow Tegaderm was applied to the wound^3MThe film is applied to the defect site to prevent wound shrinkage. All procedures were performed under sterile conditions, and the skin repair was recorded for each group by taking pictures on

days

0, 3, 7 and 14, respectively. The results are shown in FIG. 12.

As can be seen from fig. 12, the skin defect area of each group gradually decreased with time. Compared with a control group and a commercial wound adjuvant (3M), the GAD5 group shows more excellent wound repair effect, which indicates that the GAD5 hydrogel can promote the healing of infective wounds.

Test example results show that: the GAD hydrogel has the advantages of redox responsiveness, self-healing property, strong tissue adhesion, low cytotoxicity of the hydrogel and each component, promotion of cell proliferation and rapid hemostasis. In addition, the GAD hydrogel has strong antibacterial performance on escherichia coli, staphylococcus aureus and methicillin-resistant staphylococcus aureus, can effectively resist bacterial biofilms, consumes GSH in the biofilms, and effectively promotes healing of infectious skin wounds.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The multifunctional hydrogel is characterized by being prepared from an aqueous solution of an alcohol-cationic polymer, an aqueous solution of an aldehyde biomedical polymer and an aqueous solution of 3,3' -dithiobis (propionohydrazide).

2. The multifunctional hydrogel according to claim 1, wherein the alcohol in the alcohol-cationic polymer is glycerol, pentaerythritol or polyethylene glycol; and/or the cationic polymer in the alcohol-cationic polymer is epsilon-polylysine or carboxymethyl chitosan.

3. The multifunctional hydrogel according to claim 1, wherein the alcohol-cationic polymer is prepared by the following method: reacting the alcohol compound with p-toluenesulfonyl chloride to prepare alcohol-Ots; alcohol-Ots is then reacted with the cationic polymer to produce an alcohol-cationic polymer.

4. The multifunctional hydrogel according to claim 1, wherein the aldehyde-based biomedical polymer is one or more of aldehyde-based Pluronic F127, aldehyde-based hyaluronic acid, aldehyde-based pullulan and aldehyde-based dextran.

5. The multifunctional hydrogel according to claim 4, wherein the aldehyde-modified Pluronic F127 is prepared by the following method: pluronic F127 was reacted with p-toluenesulfonyl chloride to prepare F127-Ots; the reaction of F127-Ots with 4-hydroxybenzaldehyde and potassium carbonate produced the aldehyde Pluronic F127.

6. The multifunctional hydrogel according to claim 4, wherein the aldehyde-modified hyaluronic acid is prepared by reacting hyaluronic acid with sodium periodate; the aldehyde pullulan polysaccharide is prepared by reacting pullulan polysaccharide with sodium periodate; the aldehyde dextran is prepared by reacting dextran with sodium periodate.

7. The multifunctional hydrogel according to any one of claims 1 to 6, wherein the volume ratio of the aqueous solution of the alcohol-cationic polymer, the aqueous solution of the aldehyde-based biomedical polymer, and the aqueous solution of the 3,3' -dithiobis (propionohydrazide) is 1 to 10: 16-30: 1-10%, wherein the mass concentration of the aqueous solution of the alcohol-cationic polymer is 2-30%, the mass concentration of the aqueous solution of the aldehyde biomedical polymer is 5-40%, and the mass concentration of the aqueous solution of the 3,3' -dithiobis (propionohydrazide) is 1-10%.

8. The multifunctional hydrogel according to claim 7, wherein the mass concentration of the aqueous solution of the alcohol-cationic polymer is 5-20%; and/or the mass concentration of the aqueous solution of the aldehyde biomedical polymer is 5-30 percent; and/or the mass concentration of the 3,3' -dithiobis (propionohydrazide) aqueous solution is 5-10%.

9. The multifunctional hydrogel according to claim 7, wherein the volume ratio of the aqueous solution of the alcohol-cationic polymer, the aqueous solution of the aldehyde-based biomedical polymer and the aqueous solution of the 3,3' -dithiobis (propionohydrazide) in the step (2) is 1-8: 18-24: 1 to 8.

10. The method for preparing the multifunctional hydrogel according to any one of claims 1 to 9, comprising the steps of: and (2) uniformly mixing the aqueous solution of the alcohol-cationic polymer and the aqueous solution of the 3,3' -dithiobis (propionohydrazide), adding the mixture into the aqueous solution of the aldehyde biomedical polymer, and standing the mixture at room temperature for 5min to 24h to obtain the aldehyde biomedical polymer.