CN111053947A

CN111053947A - Konjac glucomannan/fish gelatin hydrogel as well as preparation method and application thereof

Info

Publication number: CN111053947A
Application number: CN201911221613.7A
Authority: CN
Inventors: 陈浩; 陈传钦; 周丽萍; 续庭玮; 闫吉成; 李鑫
Original assignee: Qingdao Zhibang Network Service Co Ltd
Current assignee: Qingdao Zhibang Network Service Co Ltd
Priority date: 2019-12-03
Filing date: 2019-12-03
Publication date: 2020-04-24

Abstract

The disclosure belongs to the technical field of hydrogel, and particularly relates to konjac glucomannan/fish gelatin hydrogel, and a preparation method and application thereof. The hydrogel is a medical dressing with good application prospect, and the konjac glucomannan and the gelatin can be used for preparing the wound dressing due to good biocompatibility, but the materials have the defects of insufficient mechanical properties and the like when being used independently. The present disclosure provides a konjac glucomannan/fish gelatin composite hydrogel, wherein the konjac glucomannan and the fish gelatin form a gel in a highly cross-linked form. Proved by verification, the hydrogel has good strength, toughness and water retention, is not easy to degrade in vitro, and is a good medical dressing. In addition, the hydrogel serving as a drug-carrying matrix has good encapsulation effect and blood compatibility, has good antibacterial performance on escherichia coli and staphylococcus aureus after matrine is encapsulated, and is expected to be popularized as an external drug for gynecological diseases.

Description

Konjac glucomannan/fish gelatin hydrogel as well as preparation method and application thereof

Technical Field

The disclosure belongs to the technical field of hydrogel, and particularly relates to konjac glucomannan/fish gelatin drug-loaded hydrogel, a preparation method of the hydrogel and application of the hydrogel as a medical dressing or a drug carrier.

Background

The information in this background section is only for enhancement of understanding of the general background of the disclosure and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

The medical dressing directly covers the surface of the wound to serve as a barrier between the wound and the external environment, so that the wound can be protected from being infected, and the environment required by wound healing is provided. Traditional wound dressings such as gauze, cotton velvet, plaster, bandage and the like need to be replaced after being used for a certain time, are easy to adhere to wounds, cause secondary injury, and most importantly, the dressings can not keep the wounds moist and can delay the healing of the wounds. More advanced modern dressings such as sponges, fibers, films, hydrogels, etc. are gradually being developed based on the insufficient performance of traditional dressings. The hydrogel can absorb tissue exudates, keep the wound surface moist, permeate oxygen and cool the wound surface, and relieve pain of patients, so that the hydrogel is more suitable for wound surface nursing. The moist environment is favorable for cell growth and can accelerate wound healing, but is also favorable for growth of harmful microorganisms, in addition, for severe infectious and difficult wound surfaces, the hydrogel has poor effect of promoting healing, and external bioactive preparations such as solution, ointment and the like have poor effect of delivering medicaments to wounds because the solutions can quickly absorb wound exudates and become flowable, so that the medicament-carrying hydrogel dressing is produced at will and becomes a new hotspot of wound repair research.

The hydrogel is a hydrophilic polymer with a three-dimensional network structure, has higher water content, porosity and softness, has a structure and a function similar to natural biological tissues, and has good biocompatibility, biodegradability and nontoxicity, so the hydrogel is concerned in the biomedical engineering fields of drug sustained and controlled release, tissue engineering scaffolds, wound dressings and the like. The drug-loaded hydrogel is a combination of a hydrogel-based carrier and a therapeutic drug, provides a repairing microenvironment for a wound while retaining the characteristics of the hydrogel, can control the release rate of the drug, and has a good effect on the healing of the wound surface.

Konjac glucomannan is a natural high molecular polysaccharide separated from konjak tubers, has high water absorption and swelling capacity, and can form a heat irreversible gel under alkaline and heating conditions. In recent years, konjac glucomannan is reported to be used for wound dressings due to the characteristics of high water absorption, promotion of wound healing and the like. Gelatin is a macromolecular protein extracted from connective tissues such as skin and bone of animals. It is used to produce wound dressing in clinic because of its biocompatibility, biodegradability and non-antigenicity, and its function of activating macrophage and stopping bleeding. The application of konjak and gelatin in the field of wound dressings separately has deeper research at home and abroad, but the single gel of konjak and gelatin has poorer mechanical property, so that the further application of konjak and gelatin is limited. Huiqun Yu et al provided a hydrogel formulation formed by crosslinking oxidized konjac glucomannan with gelatin, which was obtained by mixing the oxidized konjac glucomannan with a gelatin solution after periodic acid oxidation. The inventor thinks that the residual of strong oxidizing reagent is easily caused in the process of oxidizing konjac glucomannan by periodic acid in the research, and the konjac glucomannan has certain potential safety hazard when being used as dressing.

Matrine is the main active ingredient of Chinese herbal medicines of leguminous plants such as radix sophorae flavescentis, sophora alopecuroide, sophora subprostratae and the like. At present, the matrine has various pharmacological actions including bacteriostasis, anti-inflammation, anti-tumor and the like according to literature reports, and has been widely applied in clinic, for example, matrine suppository (gynecological suppository) is mainly used for treating trichomonas or monilial vaginitis, chronic cervicitis, senile vaginitis, pelvic inflammation and the like.

Disclosure of Invention

Based on the research background, the present disclosure provides a hydrogel prepared from Konjac Glucomannan (KGM)/Fish Gelatin (FG) and a corresponding preparation method. The hydrogel prepared by the method disclosed by the invention has good mechanical properties, and overcomes the technical defects of poor mechanical properties and easiness in crushing of konjac glucomannan and fish gelatin which are used singly. The hydrogel has good swelling capacity, can absorb wound exudate, has a good moisturizing effect, and is expected to be used as a medical dressing. The hydrogel is also researched aiming at the drug loading performance of the hydrogel, and the research result shows that the hydrogel has good entrapment effect and blood compatibility as a drug loading matrix and can also be used as an in-vitro drug delivery matrix.

According to the research results, the present disclosure provides the following technical solutions:

in a first aspect of the present disclosure, there is provided a hydrogel formed by crosslinking konjac glucomannan and fish gelatin.

The konjac glucomannan and the fish gelatin are in a double cross-linking form, the molecular combination form is different from that in the hydrogel in the background art, and the exposed hydroxyl groups of the konjac glucomannan and the carbonyl groups of the fish gelatin form a complex through the hydrogen bond effect, so that the cross-linking degree is increased, and the konjac glucomannan and the fish gelatin have good mechanical property and moisture retention property.

In a second aspect of the present disclosure, a method for preparing the hydrogel of the first aspect is provided, the method comprising: adding alkali liquor into fish gelatin solution to adjust pH, adding konjac glucomannan into the fish gelatin solution with adjusted pH under stirring to fully swell to obtain mixed sol, heating to obtain colloidal liquid, cooling to obtain gel, and washing with buffer solution to make pH neutral.

According to the preparation method provided by the disclosure, the alkali liquor is added to deacetylate the konjac glucomannan, and the molecular chain is completely exposed, so that the konjac glucomannan is easier to generate hydrogen bond bonding effect with gelatin. In addition, strong oxide and the like are not introduced in the method, the prepared gel is neutral, and the use safety is higher.

In a third aspect of the present disclosure, there is provided a use of the hydrogel of the first aspect as a medical dressing or a drug-loaded matrix.

The research of the present disclosure shows that the hydrogel preparation has good mechanical property and swelling property, is used as a medical dressing or an external medicine matrix, is not easy to break when placed on the surface of skin, and the swelling property of the hydrogel preparation determines that the medicine auxiliary material can absorb the tissue exudate and provides a good moisture-preserving environment for wound recovery.

In a fourth aspect of the present disclosure, a matrine hydrogel preparation is provided, which employs the hydrogel of the first aspect as a drug-carrying matrix.

In a fifth aspect of the present disclosure, there is provided a use of the matrine hydrogel preparation of the fourth aspect in the treatment of gynecological diseases.

Preferably, the gynecological disease is caused by staphylococcus aureus or escherichia coli.

Compared with the prior art, the beneficial effect of this disclosure is:

1. the single KGM gel has the characteristics of fast gelling, high equilibrium swelling degree and high equilibrium swelling water content, but is easy to break, the water absorption swelling performance of the gel is balanced by adding FG, the defects of fast water evaporation rate and slow in-vitro degradation of the single KGM gel are improved, and the single KGM gel is a complementary collocation form.

2. The disclosed research shows that the hydrogel as a drug carrier has an obviously improved Mat embedding effect along with the increase of FG concentration; the composite gel is used for embedding Mat with different concentrations, and the antibacterial activity and the blood compatibility of the prepared drug-loaded gel are improved along with the increase of the concentration of the Mat; the nano-particle is applied as a drug matrix, has good controllability in clinical use and meets individual use requirements.

3. Hydrogels prepared by the disclosed method do not incorporate strong oxidizing agents, cytotoxic chemicals. The konjac glucomannan and the gelatin used in the composite gel are natural polymer materials, so that the composite gel has good biocompatibility and biodegradability, high in-vitro degradation rate and better safety performance.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.

FIG. 1 is a schematic representation of KGM/FG hydrogels of example 1 at different ratios;

wherein, FIG. 1(1) is a real object diagram of KGM/FG hydrogel prepared from different raw materials; FIG. 1(2) is a diagram showing the effect of gel formation; FIG. 1(3) is a diagram showing the gel bending effect.

FIG. 2 is a graph of the G ', G' versus time at 80 ℃ and 1rad/s for the KGM solution in example 1;

FIG. 3 is a graph of the FG solution in example 1 at 80 ℃ under 1rad/s G ', G' versus time;

FIG. 4 is a graph of the G ', G' versus time at 80 ℃ and 1rad/s for a KGM/FG solution in example 1;

figure 5 is a histogram of gel content of complex gels of different FG concentrations in example 1 (. star represents P <0.05 compared to FG 0);

FIG. 6 is a line graph of the swelling ratios of composite gels of example 1 at different FG concentrations;

figure 7 is the equilibrium swelling degree of the complex gels at different FG concentrations in example 1 (P < 0.01, very significant difference compared to FG 0);

figure 8 is the equilibrium water content of the complex gels at different FG concentrations in example 1 (. star represents P < 0.01, very significantly different from FG 0);

FIG. 9 is the water evaporation rate of composite gels of example 1 at different FG concentrations;

FIG. 10 is the in vitro degradation rate of complex gels at different FG concentrations in example 1;

FIG. 11 is a standard curve of matrine in example 2;

FIG. 12 is a histogram of the embedding rate of complex gels of different FG concentrations in example 2; (. represents P < 0.01 compared to FG 0)

FIG. 13 is a histogram of drug loading for the different FG concentrations complex gels in example 2 (. dotted.) representing P < 0.01 compared to FG0

FIG. 14 is a graph showing the bacteriostatic effects of the composite gels with different Mat concentrations in example 2 on Escherichia coli (A) and Staphylococcus aureus (B);

wherein the numbers 0-4 respectively represent Mat concentrations of 0, 10, 20, 30 and 40mg/ml

FIG. 15 is a histogram of the size of zone of inhibition for complex gels with different matrine concentrations in example 2;

figure 16 is a plot of the hemolysis rate of complex gels of different matrine concentrations in example 2 (. and. represent P <0.05 and P < 0.01, respectively, compared to 0);

FIG. 17 is a graph showing the gelling effect of pH 11.94 in the experimental examples;

FIG. 18 shows the KGM 1%, FG 1.5% heating temperature at 45 ℃ without forming gel in the experimental example;

FIG. 19 is a gel plot of KGM 1% at various concentrations FG in the experimental examples;

FIG. 20 is a graph showing the gelling effect of pH 11.09 in the experimental examples

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. 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 disclosure belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As described in the background art, Konjac Glucomannan (KGM) and Fish Gelatin (FG) have a defect of insufficient mechanical strength when used alone as a conventional hydrogel dressing, and the present disclosure provides a hydrogel preparation compounded with konjac glucomannan and fish gelatin, having good mechanical properties and drug-loading properties.

Preferably, in the hydrogel, the cross-linking region is formed by hydrogen bonds.

Preferably, the hydrogel contains 84% to 93% of gel. When the amount of the fish gelatin added in the raw materials is 3%, the gel content of the composite gel is the highest and can reach 92.95%, which indicates that KGM and FG are almost completely crosslinked.

Preferably, the concentration (w/v) of the fish gelatin solution is 1.9-3.2%.

Preferably, the concentration (w/v) of the konjac glucomannan is 1.6-2.4%.

Preferably, in the preparation method, the preparation steps of the fish gelatin solution are as follows: weighing a certain mass of FG, adding the FG into water, swelling for 12-18 h at room temperature, and heating in a water bath at 40-50 ℃ for 20-40 min to fully dissolve.

Preferably, the pH is adjusted to 10-12 by adding alkali liquor.

Preferably, the mixed solution is defoamed and then heated in a water bath for a period of time to form a colloidal liquid.

Preferably, the buffer solution is a PBS buffer solution with the pH value of 5-6.

Preferably, the drug loading (w/w) of the matrine hydrogel preparation is 2-3%.

Preferably, the preparation method of the matrine hydrogel preparation comprises the following steps: adding matrine into fish gelatin solution with pH adjusted, and adding konjac glucomannan after matrine is completely dissolved.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.

Table 1 main materials and reagents used in the following examples

Example 1 preparation of KGM/FG hydrogel and evaluation of Properties

1.1 preparation of KGM/FG hydrogels

30ml of distilled water is weighed by a measuring cylinder and put into a beaker, a certain mass of FG is weighed according to the table 3 and put into the beaker to prepare FG solution with corresponding mass concentration, and after the FG solution swells overnight at room temperature, the FG solution is heated in a constant-temperature water bath kettle at 45 ℃ for 30min to be fully dissolved. To the FG solution was added dropwise a 0.5mol/L NaOH solution to adjust the pH to 11. Weighing KGM powder with a certain mass, adding FG solution while stirring, magnetically stirring for 2 hours at room temperature to fully swell the KGM, pouring the mixed sol into a culture dish, ultrasonically defoaming for 30 minutes, heating in a water bath at 60 ℃ for 4 hours until the sol is gelled, taking out the sol, cooling to room temperature with flowing tap water, soaking for 5 minutes with PBS (phosphate buffer solution) with pH of 5.8 until the pH value of the gel is reduced to neutrality, pouring the PBS buffer solution, and placing the gel in a refrigerator at 4 ℃ for refrigeration for 24 hours. Taking out the gel from the refrigerator, standing at room temperature for 1h to restore the gel temperature to room temperature, and performing sensory evaluation and performance measurement on the gel.

TABLE 2 composite gel sample numbers of different mass concentrations

1.2 evaluation of gel Properties

1.2.1 sensory evaluation of gels

The gel was taken out of the refrigerator and left at room temperature for 1 hour to return the gel temperature to room temperature, and then surface moisture was blotted with absorbent paper, and the gel was subjected to sensory evaluation as shown in Table 3.

TABLE 3 sensory evaluation method

As shown in FIG. 1, FIG. 1 shows composite gels prepared by adding FG at different mass concentrations to 2.0% (w/v) KGM. And (3) wiping water on the surface of the gel by using absorbent paper, evaluating the bleeding property of the gel, and finding that the bleeding property of the composite gel is obviously weakened and the bleeding phenomenon is less to occur compared with that of the single KGM gel. Comparing the transparency of the gels on white paper printed with black fonts, as shown in fig. 1(1), the composite gels all had good transparency, with a slight decrease in transparency with increasing FG concentration; FIG. 1(2) is a diagram showing the molding effect of gel, and it can be seen that the prepared gel has good moldability and a thickness of about 3 mm; as shown in fig. 1(3), the gel was folded in half, and the toughness and strength thereof were sensed, and it was found that as the FG concentration increased, the composite gel strength and toughness first increased and then decreased; the strength and toughness of the composite gel are strongest at FG concentrations of 2% and 3%.

1.2.2 determination of rheological Properties

The gelation process of KGM, FG and KGM/FG hydrogels was characterized by rheological tests. The hydrodynamic properties of the sample solutions were determined using a rheometer model hakke mars 3. 0.5% (w/v) KGM solution, 4% FG solution and a complex solution of 0.5% KGM + 4% FG (w/v) were prepared and the gel kinetics were investigated at a constant temperature of 80 ℃. The rotor model is P35TiL, the diameter of the parallel plates is 35mm, the distance between the parallel plates is 1mm, the shearing frequency is 1Hz, and the strain is 0.1%. A pipette is used to aspirate 2ml of sample onto the sample stage of the rheometer, which is covered at the edge with cedar oil to prevent evaporation of water.

The viscoelasticity of a polymer solution can be described by two parameters, the dynamic storage modulus G' and the loss modulus G ", representing the elasticity of the network structure and the local friction of the polymer chains, respectively. The intersection of G' and G "is reported as the gel point during the change in shear modulus with time. The gelation process of KGM, FG and KGM/FG hydrogels was characterized by rheological testing. FIGS. 2, 3 and 4 show the relationship of G 'and G' as a function of time at 80 ℃ and 1rad/s frequency for a 0.5% (w/v) KGM solution at pH 11, a 4% (w/v) FG solution and 0.5% KGM + 4% FG (w/v), respectively. From FIG. 3, it can be seen that the gel point of a single KGM solution is at t ═ 800 s. At t < 800s, G "> G' means that gelation has not occurred and the sample is in a solution state. Then both G 'and G' undergo a sharp increase in rate with G 'being faster than G' and G '> G', indicating that the sample is transitioning from a solution state to a gel state, i.e., this stage corresponds to the gel formation process. Then G' and G "enter a plateau indicating that the gelation process is complete when all crosslinks have formed. From FIG. 2 it can be seen that the G 'and G' changes very slightly with time at 80 ℃ and 1rad/s frequency for the single FG solution and that G 'is constantly less than G', indicating that the single FG solution does not form a gel during this process. From FIG. 2, it can be seen that the gelation process of the KGM/FG complex solution is similar to that of the single KGM solution. And the gel point of the composite solution is at the position of 930 s. The rate of increase of G' and G "during the formation of the composite solution was slower and longer lasting compared to the single KGM solution, indicating that the addition of FG slowed the rate of gelation and extended the time required for gel formation. However, the comparison shows that after the gel is formed, the G 'of the composite gel is 90Pa, while the G' of the single KGM is only 70Pa, which indicates that the addition of FG enhances the elasticity of the gel.

1.2.3 determination of gel content

Cutting 3mm thick hydrogel into 2 × 2cm pieces²Drying the small blocks in a 37 ℃ oven for 48h to constant weight, accurately weighing the mass of the small blocks, and then weighing the small blocksSoaking in distilled water at room temperature for 4 days to remove non-crosslinked gel matrix, drying the soaked gel in oven at 37 deg.C for 48 hr to balance weight, weighing the gel mass again, and calculating gel content (Gelfactor, GF) by the following formula:

in the formula:

W_iquality of the dried gel before soaking (g)

W_fGel mass (g) dried again after soaking

The weight ratio of the dried gel in the soak rinse and un-soak rinse conditions can be used as an indicator of the degree of crosslinking of the two matrices. The gel content of the hydrogels with different FG contents is shown in FIG. 5, and the sample numbers are shown in Table 2. The gel content of the single KGM gel was 83.9%. In the range of FG concentration of 1-4% (w/v), along with the increase of FG concentration, the gel content of the composite gel is 84.80-91.75%, the composite gel has a trend of rising first and then falling and changes remarkably (p is less than 0.05), and the gel content of FG and KGM composite gel samples with various concentrations is higher than that of single KGM gel. Thus, the addition of FG increases the gel content of the composite gel. When FG was added at 3%, the gel content of the composite gel was the highest at 92.95%, indicating almost complete crosslinking of KGM and FG. This result can be explained from the mechanism of gel formation: under strong alkaline condition, KGM takes place deacetylation reaction, makes KGM molecular chain become naked form, and partial intermolecular forms hydrogen bond and produces crystallization, takes this crystallization as the node to form network structure, namely forms KGM gel. Both KGM and gelatin gels are physical gels, with cross-linking regions formed by hydrogen bonds. KGM has a large number of hydroxyl groups and readily forms hydrogen bonds with KGM and FG molecules. The reason for forming hydrogen bonds between KGM and gelatin molecules is the action between hydroxyl groups of KGM and carbonyl groups of gelatin, and because the hydrogen bonds have saturation and directionality, the bond angle is generally about 180 degrees, so when the number of KGM and FG molecules reaches the optimal proportion, the degree of crosslinking of the complex formed by mutual entanglement of KGM and FG molecules through hydrogen bond interaction is the largest, and the gel content reaches the highest. The hydrogen bonding between the individual KGM molecules is weaker than that between the KGM and FG molecules, resulting in a weaker self-crosslinking ability of the individual KGM molecules and thus a lower degree of crosslinking of the formed gel.

1.2.4 determination of swelling behavior

Cutting 3mm thick hydrogel into 3 × 3cm pieces²The pieces were dried in an oven at 37 ℃ for 48h to constant weight and weighed accurately. The dried gel was swollen in physiological saline at 37 ℃. Samples were taken every 1h, blotted dry with filter paper and weighed accurately. When swelling to the point where the gel mass no longer increased, it was weighed accurately. The Swelling Degree (SD) of the hydrogel at each time point can be calculated from formula (2), the Swelling degree at the Equilibrium of Swelling, i.e., the Equilibrium Swelling degree (EDS), can be calculated from formula (3), and the water content at the Equilibrium of Swelling (EWC) can be calculated from formula (4):

in the formula:

m₁weight (g) of hydrogel after drying in an oven

m_tMass (g) of hydrogel after swelling in physiological saline

m₂Constant weight (g) of hydrogel after swelling in physiological saline

The swelling properties of the hydrogel exemplify the ability of the hydrogel to absorb wound exudate. The swelling rate of complex gels with different FG concentrations as a function of swelling time is shown in FIG. 6, and the sample numbers are shown in Table 2. All gels exhibited a faster swelling rate over the first 3h, indicating that these hydrogels all had the ability to swell rapidly. Fig. 7 and 8 show the effect of different FG additions on the equilibrium swelling degree and swelling equilibrium water content of the hydrogel, respectively. The gel has an equilibrium swelling degree of more than 250% and a swelling equilibrium water content of 73-82%. With the increase of FG concentration, the equilibrium swelling degree and swelling equilibrium water content of the hydrogel are increased after decreased, and the change is obvious (p < 0.05). The comparison of the gel content with the data of the equilibrium swelling degree and the swelling equilibrium water content shows that a logical relationship exists between the swelling characteristic of the gel and the gel content, namely, the higher the gel content is, the lower the equilibrium swelling degree or the swelling equilibrium water content is. This result is caused by the fact that the higher the gel content, i.e.the higher the degree of crosslinking of the two matrices KGM and FG, the more compact the arrangement between KGM and FG molecules and the smaller the pore size of the gel formed, leading to a reduced ability to swell with water. In addition, the addition of the fish gelatin increases the crosslinking points on KGM and FG molecular chains, thereby shortening the distance between adjacent crosslinking points and reducing the speed of chain segment relaxation between the crosslinking points, so that the chain segment relaxation factor has influence on the swelling process of the hydrogel. Although the swelling capacity of the composite gel is reduced compared to the single KGM gel, the swelling capacity is sufficiently high to be used as a suitable dressing for healing exuding wounds.

1.2.5 determination of the Water Evaporation Rate

Cutting 3mm thick hydrogel into 3 × 3cm pieces²The small blocks are accurately weighed. The gel sample was placed in an environment at 37 ℃ and 50% humidity, taken out every 1h, weighed accurately, and measured continuously for 24 h. The hydrogel is dried to constant weight and then weighed again. The water evaporation rate of the hydrogel was calculated according to the formula (5):

in the formula:

m₁initial weight of hydrogel (g)

m_tPeriodically measured hydrogel weight (g)

m₃Weight (g) of completely dried hydrogel

The moist environment may promote healing of the wound. Moist wound beds have been widely accepted as the most ideal wound healing environment. The wound dressing needs to be replaced repeatedly before healing of the wound clinically. The hydrogel dressing has a lower water evaporation rate, reduces the replacement times, enables the healing speed to be faster, reduces pain, and saves a large amount of cost, and the influence of different FG addition amounts on the water evaporation rate of the gel is shown in figure 9. The sample numbers are shown in Table 2. The evaporation rate of water from the gel decreased as a whole with increasing FG addition. This is because the hydrogen bonding in the composite gel system can bind water molecules inside the gel, and the higher the gelatin concentration is, the stronger the hydrogen bonding in the gel is, and the slower the water evaporation rate is. After 24 hours, only 3.1% water remained with the single KGM gel, while the composite hydrogel still retained 10-20% water. The results show that the composite gel has a better water evaporation rate than the single KGM gel.

1.2.6 determination of gel in vitro degradation

Cutting 3mm thick hydrogel into 2 × 2cm pieces²The small pieces were dried in an oven at 37 ℃ for 48 hours to a constant weight and accurately weighed, put in a beaker previously placed in a thermostatic waterbath at 37 ℃ with 20mL of PBS buffer (pH 7.4,0.1M), the gel was taken out every 4 days, placed in an oven at 37 ℃ for drying for 48 hours to a constant weight, and the mass was accurately weighed again. The gel degradation rate (degradation rate) is calculated as follows (6):

in the formula:

W₁initial mass of composite gel (g)

W_tGel mass (g) measured periodically

The in vitro degradation rate curves for the different gels are shown in figure 10. The results show that the FG ratio has a significant effect on the in vitro degradation rate of the complex gel, and that the in vitro degradation rate of the complex gel increases with the FG concentration in the range of 0% to 4%. The possible reasons are: with the increase of FG concentration, the number of semi-interpenetrating networks of a three-dimensional network structure formed by inserting formed KGM molecular chains into FG is increased, and the degradation rate of the scaffold depends on the degradation rate of the three-dimensional network, so that the degradation rate of the gelatin cross-linked network is far greater than that of the KGM cross-linked network, and the degradation rate of the composite gel is increased.

Example 2 preparation and Performance characterization of KGM/FG drug-loaded hydrogel

Preparation of 2.1 KGM/FG drug-loaded hydrogel

Measuring 30ml of distilled water by using a measuring cylinder, putting a certain mass of FG into a beaker according to the table 2 to prepare FG solution with corresponding mass concentration, swelling at room temperature overnight, and heating in a constant-temperature water bath kettle at 45 ℃ for 30min to fully dissolve. Dropwise adding 0.5mol/L NaOH solution into FG solution to adjust pH to 11, weighing a certain mass of Mat, dissolving in FG solution to make the concentration of 30mg/ml, weighing a certain mass of KGM powder according to table 2 after matrine (Mat) is completely dissolved, adding the Mat/FG mixed solution, magnetically stirring for 2h at room temperature to fully swell KGM, pouring the mixed sol into a culture dish, ultrasonically defoaming for 30min, heating in water bath at 60 ℃ for 4h to gelatinize, taking out, cooling with running water to room temperature, soaking with PBS buffer solution with pH of 5.8 for 5min until the pH value of gel is reduced to neutrality, pouring out the PBS buffer solution, and placing the gel in a refrigerator at 4 ℃ for refrigeration for 24 h. And taking out the gel from the refrigerator, standing at room temperature for 1h to restore the temperature of the gel to room temperature, and then carrying out drug loading effect measurement on the gel.

2.2 evaluation of gel drug Loading Performance

2.2.1 determination of matrine Standard Curve

Accurately weighing 10mg of matrine sample, and placing in a 100ml volumetric flask to prepare 0.1 mg/ml^-1The solution of (1). Sucking 200, 400, 600, 800 and 1000 mul of solution by using a pipette, respectively placing the solution into a 60ml separating funnel, and sequentially adding the solution into a 2X 10 separating funnel^-4mol·L^-16.0ml of buffer solution of bromothymol blue pH7.6, 6.0ml of chloroform, shaking for 2min, standing for 2h to completely clear the water layer and the chloroform layer, separating the chloroform layer, and measuring the absorbance at 420 nm. When the determination is carried out, 6.0ml of buffer solution of bromothymol blue with pH7.6 is used, 6.0ml of chloroform is added, the mixture is shaken as above, and a chloroform layer is separated out to be used as a colorimetric blank. Linearly regressing with the measured absorbance as ordinate and the matrine concentration as abscissa to obtain bitterThe results are shown in FIG. 11, where the fitting equation of the standard curve is y-0.05812 x-0.0628 and the degree of fit is R²＝0.9972。

2.2.2 measurement of embedding Rate and drug Loading

About 0.5g of the drug-loaded gel was accurately weighed and placed in a 100ml small beaker, the gel was ground, 50ml of PBS buffer (pH 7.4,0.1M) was added, and the gel was left to stand for 24 hours to sufficiently dissolve the matrine embedded in the gel into the PBS buffer. 100ul of the supernatant was taken and placed in a 60ml separatory funnel, followed by 6.00ml of 2X 10^-4And (3) shaking the buffer solution of the bromothymol blue with the mol/L concentration and the pH value of 7.6 and 6.00ml of chloroform for 2min, standing for 2h, separating a chloroform layer after the water and the chloroform are completely separated, and measuring the absorbance at the wavelength of 420nm by using an ultraviolet spectrophotometer. Combining the standard curve of matrine, the Entrapment Efficiency (EE) of the drug can be obtained by the formula (7), and the drug loading capacity (LE) can be obtained by the formula (8)

In the formula:

W_cmatrine content (g) of the embedding gel

W_o-the total amount of matrine (g) added during preparation

W_iMass of gel (g)

The matrine embedding rate test results are shown in fig. 12 and 13, and the sample numbers are shown in table 4. At a KGM concentration of 2% (w/v), both the embedding rate and the drug loading rate tended to increase and then decrease with increasing FG mass concentration, with the embedding rate reaching a maximum of 89.46% at a FG mass concentration of 3% (w/v), with the best embedding effect, and with a maximum drug loading rate of 2.8% (w/w). In addition, the embedding effect of each FG and KGM composite gel sample on matrine is superior to that of KGM single gel. The analysis result is presumably that the increased FG concentration and KGM cross-link to form a compact network structure, so that matrine can be more effectively trapped in the gel, which is consistent with the result of gel content.

2.2.3 determination of bacteriostatic Properties

The bacteriostatic activity of the loaded gel was determined using staphylococcus aureus (s. aureus) and escherichia coli (e. coli) to represent gram-positive and negative bacteria, respectively. Taking 2.5g of tryptone, 1.25g of yeast powder, 2.5g of NaCl and 2.5g of agar powder, adding 200mL of purified water for dissolving, adjusting the pH value to about 7.4, fixing the volume to 250mL, placing the prepared culture medium and required instruments in a high-pressure steam sterilization pot, sterilizing for 20min at 121 ℃, adding about 20mL of culture medium into an aseptic culture dish when the culture medium is cooled to 50-60 ℃, opening a cover for ultraviolet sterilization for 30min, and cooling to prepare the LB solid culture medium. The medium was stored upside down at 4 ℃ until use. Using sterile physiological saline to prepare various strains with the concentration of 1 × 10⁶～1×10⁷CFU/mL of suspension. 100 mul of the suspension liquid is sucked by a pipette gun and evenly smeared on the culture medium. Cutting the hydrogel into a disc shape with the diameter of 6mm, placing the disc shape on an LB solid culture medium coated with bacterial liquid, culturing the disc shape in a constant temperature incubator at 37 ℃ for 24 hours, and measuring the diameter of a bacteriostatic circle. In parallel, 3 experiments were performed and the average was taken.

Composite gels with different Mat embedding amounts are prepared for carrying out bacteriostasis tests on escherichia coli and staphylococcus aureus. The bacteriostatic effect is shown in fig. 14, and the size of the zone of inhibition when different Mat concentrations are embedded is shown in fig. 15. FIG. 14(A) shows the bacteriostatic effect of Escherichia coli, and FIG. (B) shows the bacteriostatic effect of Staphylococcus aureus. It can be seen from the figure that when the Mat concentration reaches 30mg/ml or more, the drug-loaded gel has obvious inhibition effect on both escherichia coli and staphylococcus aureus. When the Mat concentration reaches 40mg/m, the average inhibition zone diameters of the drug-loaded gel to escherichia coli and staphylococcus aureus are respectively 12mm and 11.5mm (the gel diameter is 6mm), which shows that the drug-loaded gel has good antibacterial performance.

2.2.4 determination of blood compatibility

2mL of heparin anticoagulated sheep whole blood is taken and diluted by adding 2.5mL of physiological saline for later use. Weighing a certain amount of drug-loaded hydrogel sample, placing the drug-loaded hydrogel sample in a test tube, grinding, adding 10mL of normal saline, taking 10mL of deionized water as a positive control, and taking 10mL of normal saline as a negative control. And (3) placing all the test tubes in a 37 ℃ constant-temperature water bath pan, preserving the heat for 30min, taking out the test tubes, respectively adding 0.1mL of diluted whole blood, uniformly mixing, and preserving the heat for 60min at 37 ℃. Taking out the test tube, centrifuging the mixed solution after warm bath for 20min at the rotating speed of 3000r/min, taking the supernatant, measuring the absorbance at the wavelength of 545nm, repeatedly measuring each group of samples for 3 times in parallel, taking the average value, and calculating the hemolysis rate according to the following formula (9). According to the hemolysis test result, the material is divided into three types according to hemolysis index, (a) hemolysis rate (%) of hemolysis material is larger than 5%, (b) hemolysis rate (%) of mild hemolysis material is between 2% and 5%, and (c) hemolysis rate (%) of non-hemolysis material is smaller than 2%

In the formula:

A₁absorbance of the sample set

A₂Absorbance of Positive control group

A₃Absorbance of negative control group

Hemolysis rate is a method of determining the anticoagulant properties of a material, and the results can be used to determine whether the test material can be used as a biological material. The hemolysis rate of complex gels loaded with different matrine content is shown in FIG. 16. The hemolysis rate of the composite gel is increased along with the increase of Mat content, the hemolysis rate of the composite gel is obviously changed after being reduced, and the hemolysis rate of all the gels is less than 2%, which indicates that the composite gel has very good blood compatibility. And Mat can remarkably improve the blood compatibility of KGM. The prepared composite drug-loaded gel has excellent blood compatibility because KGM and FG are biocompatible high molecular materials, and the preparation of the gel only relates to physical crosslinking in the research, so that the prepared gel has good anticoagulation performance.

2.3 data statistics and analysis

All figures were drawn using origine 8.5 software and data processed through Excel 2016 software. All samples were run in at least 3 replicates and tested more than 3 times. All indices were mean of 3 or more groups of data and were evaluated for significance differences between groups using One-Way ANOVA analysis in SPSS 21.0 software (p < 0.05).

Examples of the experiments

1.1 selection of gelatin

The content of proline and hydroxyproline in fish gelatin is much lower than that of animal gelatin, while the content of methionine is much higher than that of pig skin gelatin. In addition, fish skin gelatin has a lower intrinsic viscosity and a lower heat denaturation temperature than pig skin gelatin. The gel temperature range of the carp skin gelatin measured by using a controlled stress rheometer is 6-15.7 ℃, the melting temperature range is 17.9-23.7 ℃, the gel and melting temperature of the pig skin gelatin are relatively higher, the hydrogel prepared from the carp gelatin has better mechanical strength and thermal stability, the temperature is lower when the gelatin is converted from a liquid state to a gel state, and the embedding of the medicine is facilitated.

1.2 preparation of Single KGM gels

KGM with a certain mass is dissolved in 30ml of distilled water to prepare KGM solutions with concentrations (w/v) of 0.4%, 0.5%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.5% and 2.0%, respectively. And (3) dropwise adding 0.5mol/L NaOH solution into the solution to adjust the pH value to 9, stirring at normal temperature for 2h, ultrasonically defoaming for 15min, heating in a water bath at 90 ℃ for 2h, taking out and cooling.

The result shows that the gel is not formed, and the gel can be formed when the KMG concentration is more than 1.0%. The 2.0% KGM solution had a small amount of undissolved KGM powder in bulk form, which was not added too quickly. After ultrasonic treatment, bubbles of 0.5% and 1.0% solutions float on the surface of the solution, and the 1.5% and 2.0% solutions have no effect.

The experiment was repeated, the pH was adjusted up and the results are shown in table 4:

TABLE 4

The single KGM gel has concentration of 1.5% or more and pH value of 11 or more.

1.3 preparation of KGM/FG hydrogels

30ml of distilled water is weighed by a measuring cylinder and put into a beaker, a certain mass of FG is weighed and put into the beaker to prepare an FG solution with the mass concentration of 1.5 percent, and after the solution is swelled overnight at room temperature, the solution is heated in a constant temperature water bath kettle at 45 ℃ for 30min to be fully dissolved. Adding 0.5mol/L NaOH solution dropwise into FG solution to adjust pH, weighing KGM powder with a certain mass, and adding FG solution while stirring to make KGM concentration 1.50%. Magnetically stirring for 2h at room temperature to fully swell KGM, pouring the mixed sol into a culture dish, ultrasonically defoaming for 30min, heating in a water bath at 60 ℃ for 4h, taking out, cooling to room temperature with flowing tap water, and refrigerating for 24h in a refrigerator at 4 ℃. The results are shown in table 5 below:

TABLE 5

1.4 temperature optimization

KGM and FG with certain mass are dissolved in 30ml of distilled water to prepare 4 parts of FG solution with the concentration (w/v) of 1.5 percent, and the FG solution is swelled at room temperature overnight and then heated in a constant temperature water bath kettle at 45 ℃ for 30min to be fully dissolved. After stirring at room temperature for 2h, 0.5mol/L NaOH solution was added dropwise to the FG solution to adjust the pH to 11, a mass of KGM powder was added to a concentration of 1%, and the mixture was heated in a water bath at 50 ℃, 60 ℃, 70 ℃ and 80 ℃ for 3h, respectively, as shown in the following table 6:

TABLE 6

1.5 KGM concentration optimization

30ml of distilled water is weighed by a measuring cylinder and put into a beaker, a certain mass of FG is weighed and put into the beaker to prepare an FG solution with the mass concentration of 1.5 percent, and after the solution is swelled overnight at room temperature, the solution is heated in a constant temperature water bath kettle at 45 ℃ for 30min to be fully dissolved. Adding KGM powder with a certain mass, uniformly mixing, swelling, adjusting the pH value with 0.5mol/L NaOH solution, ultrasonically defoaming for 30min, heating in water bath at 80 ℃ for 2h, taking out and cooling, wherein the results are shown in Table 7:

TABLE 7

The heating time may be too short, no gel is formed, but No. 4, No. 5 and No. 6 have good formability, wherein No. 5 has the best formability, and the KGM concentration of the gel is 1.75% or more, and 2.0% KGM is the best.

1.6 FG concentration optimization

30ml of distilled water is weighed by a measuring cylinder and put into a beaker, a certain mass of FG is weighed and put into the beaker to prepare an FG solution with the mass concentration of 1.5 percent, and after the solution is swelled overnight at room temperature, the solution is heated in a constant temperature water bath kettle at 45 ℃ for 30min to be fully dissolved. Adding KGM powder with a certain mass, uniformly mixing, swelling, adjusting the pH value to 11 with 0.5mol/L NaOH solution, ultrasonically defoaming for 30min, heating in a water bath at 80 ℃ for 2h, taking out and cooling, wherein the results are shown in Table 8:

TABLE 8

1.7 KGM/FG hydrogel gel formation pH optimization

30ml of distilled water is weighed by a measuring cylinder and put into a beaker, a certain mass of FG is weighed and put into the beaker to prepare an FG solution with the mass concentration of 1.5 percent, and after the solution is swelled overnight at room temperature, the solution is heated in a constant temperature water bath kettle at 45 ℃ for 30min to be fully dissolved. 0.5mol/L NaOH solution is dripped into FG solution to adjust the pH, a certain mass of KGM powder is weighed, and the FG solution is added while stirring to ensure that the KGM concentration is 1.75%. Magnetically stirring for 2h at room temperature to fully swell KGM, pouring the mixed sol into a culture dish, ultrasonically defoaming for 30min, heating in a water bath at 60 ℃ for 4h, taking out, cooling to room temperature with flowing tap water, and refrigerating for 24h in a refrigerator at 4 ℃. The results are shown in table 9 below:

TABLE 9

The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.

Claims

1. A hydrogel, which is formed by crosslinking konjac glucomannan and fish gelatin.

2. The hydrogel of claim 1, wherein said hydrogel comprises cross-linked regions formed by hydrogen bonds.

3. The hydrogel of claim 1, wherein said hydrogel comprises 84% to 93% gel.

4. A method for preparing the hydrogel according to any one of claims 1 to 3, which comprises the steps of: adding alkali liquor into fish gelatin solution to adjust pH, adding konjac glucomannan into the fish gelatin solution with adjusted pH under stirring to fully swell to obtain mixed sol, heating to obtain colloidal liquid, cooling to obtain gel, and washing with buffer solution to make pH neutral.

5. The method for preparing the hydrogel according to claim 4, wherein the concentration (w/v) of the fish gelatin solution is 1.9 to 3.2%;

or the concentration (w/v) of the konjac glucomannan is 1.6-2.4%;

or in the preparation method, the preparation steps of the fish gelatin solution are as follows: weighing a certain mass of FG, adding the FG into water, swelling for 12-18 h at room temperature, and then heating in a water bath at 40-50 ℃ for 20-40 min to dissolve.

6. The method for preparing the hydrogel according to claim 4, wherein the pH is adjusted to 10-12 by adding an alkali solution;

or heating in water bath for a period of time after the mixed solution is defoamed to form colloidal liquid.

Or the buffer solution is PBS buffer solution with pH of 5-6.

7. Use of the hydrogel of any one of claims 1 to 3 as a medical dressing or drug-loaded matrix.

8. A matrine hydrogel formulation characterized in that the matrine hydrogel formulation employs the hydrogel according to any one of claims 1 to 3 as a drug-loaded matrix.

9. The aqueous matrine gel preparation of claim 8, wherein the drug-loading rate of the aqueous matrine gel preparation is 2 to 3%;

or the preparation method of the matrine hydrogel preparation comprises the following steps: adding matrine into fish gelatin solution with pH adjusted, and adding konjac glucomannan after matrine is dissolved.

10. Use of the matrine hydrogel formulation of claim 8 or 9 for the treatment of gynecological diseases.