CN110585122B

CN110585122B - Injectable natural triterpenoid antibacterial hydrogel and preparation method thereof

Info

Publication number: CN110585122B
Application number: CN201911063092.7A
Authority: CN
Inventors: 胡君; 张�浩; 吴剑桥; 于霞
Original assignee: Beijing University of Chemical Technology
Current assignee: Beijing University of Chemical Technology
Priority date: 2019-11-04
Filing date: 2019-11-04
Publication date: 2021-03-02
Anticipated expiration: 2039-11-04
Also published as: CN110585122A

Abstract

The invention relates to the field of biological antibacterial hydrogel materials, in particular to an injectable natural triterpene compound antibacterial hydrogel and a preparation method thereof. The hydrogel glycyrrhizic acid and pentacyclic triterpene compound or tetracyclic triterpene compound mixture disclosed by the invention is prepared by taking Phosphate Buffered Saline (PBS) as a gel solvent and utilizing non-covalent bond acting force formed between the mixtures through a micromolecule self-assembly mechanism, and the prepared hydrogel can express antibacterial property without adding antibacterial drugs and obviously improves the mechanical properties of the hydrogel. The preparation method of the hydrogel is simple, not only can simplify the preparation process of the injectable antibacterial hydrogel, but also expands the application field of triterpenoids. Making it possible to produce new antibacterial materials with biomedical application properties and with mechanical strength injectable.

Description

Injectable natural triterpenoid antibacterial hydrogel and preparation method thereof

The technical field is as follows:

the invention belongs to the field of biological antibacterial hydrogel materials, and particularly relates to an injectable natural triterpene compound antibacterial hydrogel.

Background art:

low molecular weight hydrogels (simply hydrogels) are generally obtained by non-covalent interactions of small molecular compounds in water to form a relatively stable three-dimensional network structure. Among them, the hydrogel having injectability is widely used in the fields of tissue engineering, drug delivery, wound healing, and the like. However, since the hydrogel is often applied in a humid environment, if it has no antibacterial property, it is easily infected with bacteria, and finally, the target site is seriously infected. Therefore, the preparation of an injectable antibacterial hydrogel becomes a research hotspot in the field of biomaterials.

In view of the particularity of the injectable antibacterial hydrogel in performance and application scenarios, some basic requirements, such as antibacterial property, biocompatibility, rapid sol-gel state transition, good mechanical strength retention rate after injection, etc., need to be satisfied in the preparation. There are few injectable antimicrobial hydrogels currently available, subject to the harsh preparation conditions. There are two main methods for the reported injectable antibacterial hydrogels. Firstly, the antibacterial agent is added into the hydrogel matrix to form a composite hydrogel. However, excessive amounts of antimicrobial agents can cause phase separation of the hydrogel matrix, resulting in reduced injectability and mechanical properties of the hydrogel. Secondly, synthesizing a small molecule with gelling ability and antibacterial effect, and further preparing the hydrogel with inherent antibacterial activity. However, this method often requires complicated procedures for synthesis and purification of gel molecules, resulting in increased preparation cost and low practical application value. Therefore, finding a directly injectable hydrogel with intrinsic antibacterial properties without the need for complex synthesis and purification processes is of great interest in the field of biomaterials.

The triterpenoid is a natural resource with wide sources in nature, has wide pharmacological action and important biological activity, and particularly shows interesting pharmacological properties in the aspects of antibiosis, anti-tumor, anti-inflammation and the like of a decomposed product. Not only does this, triterpenoids have a unique molecular structure that allows self-assembly processes to occur in water, forming aggregates that are relatively stable and have regular shapes. Apparently, the excellent bioactivity, assemblability and abundant content make natural triterpenoids ideal small molecules for preparing injectable antibacterial hydrogels.

In view of the above, the invention screens out appropriate natural triterpene compounds through research, and is used for preparing injectable hydrogel with inherent antibacterial activity, so that the preparation process of the injectable antibacterial hydrogel can be simplified, the application field of the triterpene compounds is expanded, the triterpene compounds are not only used as antibacterial drugs, but also can be prepared into novel antibacterial materials with biomedical application characteristics.

The invention content is as follows:

the invention aims to overcome the defects of the prior art and provide an injectable natural triterpene compound antibacterial hydrogel and a preparation method thereof, wherein the hydrogel can simultaneously have antibacterial property, injectability and various functional indexes, and can be widely used in medical materials.

The gel molecules of the injectable natural triterpene compound antibacterial hydrogel provided by the invention are a mixture consisting of glycyrrhizic acid and one or more of pentacyclic triterpene compounds and tetracyclic triterpene compounds except glycyrrhizic acid, and a gel solvent is a PBS buffer solution, wherein the mixing concentration of the mixture consisting of glycyrrhizic acid and one or more of pentacyclic triterpene compounds and tetracyclic triterpene compounds except glycyrrhizic acid and the gel solvent is 1-8 mM.

Wherein in the mixture consisting of glycyrrhizic acid and one or more of pentacyclic triterpene compounds and tetracyclic triterpene compounds except glycyrrhizic acid, the molar fraction of glycyrrhizic acid is 60-90%, and the balance is one or more of pentacyclic triterpene compounds and tetracyclic triterpene compounds except glycyrrhizic acid.

Wherein the pH range of the PBS buffer solution is 7.0-7.4.

Wherein the pentacyclic triterpene compound comprises: ursolic acid, oleanolic acid, saikoside, sanguisorbin, betulin, lupeol, and triptonide.

Wherein the tetracyclic triterpene compounds comprise: ginsenoside, cycloastragenol and its saponin, ganoderic acid, cucurbitacin, and cucurbitacin.

Meanwhile, the invention also provides a preparation method of the injectable natural triterpene compound antibacterial hydrogel, which comprises the following steps:

preparing a mixture suspension solution consisting of 1-8mM glycyrrhizic acid and one or more of pentacyclic triterpene compounds and tetracyclic triterpene compounds except glycyrrhizic acid by using a PBS buffer solution;

ultrasonically treating the suspension for 2-15min at 37 +/-1 ℃ to obtain uniformly dispersed mixture suspension;

thirdly, placing the mixture suspension in an environment with the temperature of 90-97 ℃ for processing for 100-150 min;

and fourthly, placing the mixture suspension liquid treated in the third step in an environment with the temperature of 25-37 ℃ until hydrogel is formed, and obtaining the injectable natural triterpene compound antibacterial hydrogel.

Wherein the pH range of the PBS buffer solution is 7.0-7.4.

Has the advantages that:

firstly, the network structure of the stable and injectable hydrogel is formed by the non-covalent bond interaction between the pentacyclic triterpene compound or the tetracyclic triterpene compound and glycyrrhizic acid molecules, and no other impurity or other functional group is introduced, so that the possible biological toxicity caused by residual micromolecular by-products after chemical crosslinking reaction can be effectively avoided. Therefore, as can be seen from verification experiments, the hydrogel prepared by the invention has lower toxicity than PBS buffer solution, and can be used as a filling material for medical wounds.

Secondly, the invention solves the problem that the antibacterial effect of glycyrrhizic acid hydrogel on gram-negative bacteria is not obvious by mixing pentacyclic triterpene compounds (except glycyrrhizic acid) or tetracyclic triterpene compounds with glycyrrhizic acid, and after the mixed hydrogel is formed, the antibacterial rate on gram-negative bacteria exceeds 96%, and the mixed hydrogel can play a good antibacterial effect under the condition of not adding any antibacterial agent, thereby effectively avoiding the influence of additionally added antibacterial agents on the mechanical strength, transparency and other properties of the hydrogel. The hydrogel has the antibacterial condition that the concentration of the gel molecule mixture suspension is within the range of 1-8mM on the premise of ensuring that cytotoxicity and hemolysis are not generated.

Thirdly, the network structure of the hydrogel is formed by the non-covalent bond interaction between the pentacyclic triterpene compound or the tetracyclic triterpene compound and glycyrrhizic acid molecules, so that the problems of poor mechanical property and insufficient hydrogel strength caused by independently preparing the hydrogel by glycyrrhizic acid are solved, and the hydrogel with high mechanical strength is provided for the medical field.

Fourthly, the hydrogel of the invention has good shear thinning characteristic and shape plasticity performance, thus having certain injectability and being capable of showing good injectability and shape plasticity within the scope of the invention. Experiments show that the concentration range of the gel molecule mixture suspension with the injectability and shape plasticity of the hydrogel is 0.5-8 mM on the premise of ensuring that the antibacterial property is not influenced. Can be widely applied to the field of biological materials.

Fifthly, the preparation method is simple, convenient and fast to operate and accurate in conditions; the hydrogel is formed at room temperature, the condition is mild, the requirements on the concentration of gel molecules and the temperature condition are low, the formed gel has high mechanical strength, is safe and nontoxic, has transparent color, and has good development prospect in the field of medical biomaterials.

Description of the drawings:

FIG. 1 is a general structural diagram of a part of triterpene compounds used in the present invention.

FIG. 2 is a diagram showing the structural formulae of four triterpene compounds used in examples of the present invention.

FIG. 3 shows the microstructure of the hydrogel of four triterpene compounds in the examples of the present invention.

Detailed Description

The present invention will be described in detail with reference to the following embodiments and the accompanying drawings. The structural formula of the four compounds adopted in the embodiment of the invention is shown in figure 2, and in figure 2, (1) is glycyrrhizic acid; (2) is glycyrrhetinic acid; (3) is ursolic acid; (4) is ginsenoside.

Example 1

The inventor conducts intensive research to prepare a triterpene compound hydrogel which has inherent antibacterial property and can be injected. The invention fully utilizes the antibacterial property and the amphiphilic molecular structure of the natural triterpene compound, and selects the hydrogel with injectability and antibacterial property and the preparation method thereof through the gel forming experiment and the experimental research on the antibacterial property, the cell activity and the hemolysis of the hydrogel.

This example used a mixture of glycyrrhizic acid and glycyrrhetinic acid in pentacyclic triterpene compounds as gel molecules to prepare a hydrogel, and PBS buffer of pH =7 as a solvent. Wherein, the glycyrrhizic acid mole fraction is 70%, the glycyrrhetinic acid mole fraction is 30%, the preparation method comprises the following steps:

firstly, preparing a glycyrrhizic acid and glycyrrhetinic acid suspension solution with the concentration of 3.5mM in a container, wherein the glycyrrhizic acid mole fraction is 70%; then carrying out ultrasonic treatment on the suspension for 10 min at 37 ℃ to promote glycyrrhizic acid and glycyrrhetinic acid molecules to be uniformly dispersed in PBS buffer solution; then placing the suspension solution in an oven at 95 ℃ to heat for 100 min, wherein the solution is changed from a turbid state to a transparent state; finally, the transparent solution was placed in an environment of 37 ℃ until hydrogel formation, and the injectable natural triterpene compound antibacterial hydrogel of the present example was obtained.

Example 2

This example used a mixture of glycyrrhizic acid and ursolic acid in the pentacyclic triterpene compound as gel molecules to prepare a hydrogel, and PBS buffer of pH =7.4 as a solvent. Wherein, the glycyrrhizic acid mole fraction is 60%, the ursolic acid mole fraction is 40%, the preparation method comprises the following steps:

firstly, preparing a glycyrrhizic acid and ursolic acid suspension solution with the concentration of 5mM in a container, wherein the molar fraction of glycyrrhizic acid is 60 percent, and the molar fraction of ursolic acid is 40 percent; then carrying out ultrasonic treatment on the suspension for 2 min at the temperature of 37 +/-1 ℃ to promote glycyrrhizic acid and ursolic acid molecules to be uniformly dispersed in the aqueous solution; then, the suspension solution is placed in an oven at 97 ℃ to be heated for 150 min, and the solution is changed from a turbid state to a transparent state; finally, the transparent solution was placed in an environment of 25 ℃ until hydrogel formation, to obtain the injectable natural triterpene compound antibacterial hydrogel of this example.

Example 3

This example used a mixture of glycyrrhizic acid and ginsenoside in tetracyclic triterpene compounds as gel molecules to prepare a hydrogel, and PBS buffer of pH =7.2 as a solvent. Wherein, the glycyrrhizic acid mole fraction is 90%, the ginsenoside mole fraction is 10%, the preparation method comprises the following steps:

firstly, preparing a mixed suspension solution of glycyrrhizic acid and ginsenoside with the concentration of 2mM in a container by using PBS (phosphate buffer solution) with the pH =7.2, wherein the molar fraction of the glycyrrhizic acid is 90 percent, and the molar fraction of the ginsenoside is 10 percent; then, carrying out ultrasonic treatment on the suspension for 15min at the temperature of 37 ℃ to promote the two gel molecules to be uniformly dispersed in the aqueous solution; then, the suspension solution is placed in an oven at 90 ℃ to be heated for 120 min, and the solution is changed from a turbid state to a transparent state; finally, the transparent solution was placed in an environment of 25 ℃ until hydrogel formation, to obtain the injectable natural triterpene compound antibacterial hydrogel of this example.

Example 4

Experiments show that the injectable natural triterpene compound antibacterial hydrogel with the antibacterial effect of more than 99.9 percent can be prepared after the mixture of the glycyrrhizic acid and the tetracyclic triterpene compound, such as cycloastragenol and the saponin thereof, the ganoderic acid, the cucurbitacin and the cucurbitacin in the glycyrrhizic acid and the tetracyclic triterpene compound is mixed according to the proportion that the molar fraction of the glycyrrhizic acid is 60-90 percent and the molar fraction of the total of one or more tetracyclic triterpene compounds is 10-40 percent, and the mixing molar ratio range of gel molecules and PBS buffer solution is 1-8 mM.

The antibacterial hydrogel containing the injectable natural triterpenoid and having the antibacterial effect of more than 99.9 percent can be prepared by mixing oleanolic acid, saikoside, sanguisorbin, betulin, lupeol, triptonide and the like in glycyrrhizic acid and pentacyclic triterpenoid according to the proportion that the molar fraction of glycyrrhizic acid is 60-90 percent and the molar fraction of pentacyclic triterpenoid is 10-40 percent, and the mixing molar ratio range of gel molecules and PBS buffer solution is 1-8 mM.

Through theoretical research, the network structure of the stable and injectable hydrogel is formed by virtue of non-covalent bond interaction between the pentacyclic triterpene compound or the tetracyclic triterpene compound and glycyrrhizic acid molecules, and no other impurities or other functional groups are introduced, so that possible biological toxicity caused by residual micromolecular byproducts after chemical crosslinking reaction can be effectively avoided.

When pentacyclic triterpene compounds (excluding glycyrrhizic acid) or tetracyclic triterpene compounds are used alone, the same similar method is difficult to form hydrogel, and is not injectable and antibacterial.

Comparative example

A single-component hydrogel is prepared by using glycyrrhizic acid in a pentacyclic triterpene compound as a gel molecule, and a PBS (phosphate buffered saline) with pH =7.2 as a solvent. Firstly, preparing a glycyrrhizic acid suspension solution with the concentration of 1.5mM in a container; then ultrasonically treating the glycyrrhizic acid suspension for 5min at 37 deg.C to promote glycyrrhizic acid molecules to be uniformly dispersed in the water solution; then placing the glycyrrhizic acid suspension solution in an oven at 90 ℃ for heating for 120 min, and then converting the solution from a turbid state to a transparent state; and finally, placing the transparent glycyrrhizic acid solution in an environment at 37 ℃ until hydrogel is formed, so as to obtain the hydrogel.

Microscopic morphology testing method of hydrogel

Microscopic morphologies of the injectable antibacterial hydrogel of natural triterpene compound prepared in the example were observed by SEM and TEM. SEM images were taken on a field emission scanning electron microscope (JSM-7610F) with an acceleration voltage of 10 kv. A sample preparation process: the hydrogel is lyophilized under vacuum at-60 deg.C, and then the lyophilized gel is subjected to gold spraying treatment. The TEM image was obtained using a Hitachi H-7650B electron microscope (Hitachi Co., Ltd.) at an acceleration voltage of 80 kv. A sample preparation process: after diluting the hydrogel 20 times, the sol was drop-coated on a copper mesh with a carbon coating, left to stand for 1 minute, and excess sample solution was wiped off with filter paper, dried in the air, and then observed.

Hydrogel rheological property testing method

The triterpene hydrogel prepared in the examples was tested for rheological properties by a rotational rheometer. First, a shear strain (strain) is set to 1%, an angular frequency range is set to 0.1-10 Hz, and a storage modulus of a sample is recorded (G') And loss modulus: (G'') Curve with frequency. Setting the angular frequency to 1 HzThe shear strain was determined to be in the range of 0.1-30%, and the samples were recordedG'AndG''curve with shear strain.

Antibacterial experiment method of hydrogel

The bacteriostatic activity of the triterpene compound hydrogel is evaluated by the bacteriostatic rate, and staphylococcus aureus (A), (B), (C) and (D) are respectively usedS. aureus) Escherichia coli (E.coli)E. coli) Representing gram-positive and gram-negative bacteria. First, a single seed was inoculated into 5 mL of LB broth at 37 ℃, shaken at 200 rpm and incubated overnight; after a one-night preculture, the bacterial suspension was diluted to 10 in LB medium⁶cfu mL^-1Then adding the prepared triterpene compound hydrogel; finally, the mixed culture was gently shaken overnight at 37 ℃. Measuring the absorbance (OD) of the solution (sample) after the triterpene compound hydrogel is cultured by using a Synergy H1 microplate reader at the wavelength of 600 nm, and recording the change of the absorbance of the bacterial culture solution along with the time. Meanwhile, the change of the bacteriostatic rate was calculated by using a culture solution to which no triterpene compound hydrogel was added as a control group (positive). The calculation formula is as follows:

cell activity test method of hydrogel

As an antibacterial biological material, the evaluation of the cytotoxicity of the triterpene compound hydrogel has important clinical application value. The cytotoxicity of the triterpene compound hydrogel is determined by performing cell activity measurement on L929 fibroblasts. First, at 37 ℃ and 5% CO₂L929 cells were cultured in medium containing 10% bovine fetal serum under atmosphere. After overnight, the cultured cells were transferred to a medium containing a triterpene compound hydrogel and incubated for 24 hours. Then, 100. mu.L of the culture broth was transferred to a medium containing 10% CellTiter-Blue^TMThe culture was continued at 37 ℃ for 2 hours in a cuvette of (Promega). Measuring different triterpene compound water congelation by using Synergy H1 enzyme-linked immunosorbent assay instrument at an excitation wavelength of 550 nm and an emission wavelength of 590 nmFluorescence intensity of the gel-treated culture broth. The relative viability of the cells was compared by comparison with the fluorescence intensity of the L929 cell culture medium (standard) after PBS treatment. If the fluorescence intensity of the culture solution treated by the triterpene compound hydrogel is not lower than that of the culture solution treated by PBS, the hydrogel is proved to have no toxicity to cells.

Hemolytic test method for hydrogel

Firstly, centrifuging for 5min at 2000 rpm by using a centrifuge, separating to obtain red blood cells in rat blood, washing and precipitating for 3 times by using PBS for later use. The red blood cells were suspended in PBS to obtain a solution with a volume concentration of 2%, and then different triterpene compound hydrogels were added to the solution to obtain a sample group (sample). PBS-treated erythrocytes (positive) and water-treated erythrocytes (negative) were used as control groups, respectively. After incubation for 2 hours at 37 ℃, the treated erythrocytes were centrifuged for 5min at 2000 rpm. The absorbance (OD) of the supernatant at 545 nm was measured with a microplate reader. The lysis destruction rate of the water, PBS and triterpene hydrogel to erythrocytes was calculated by using the Hemolysis rate (Hemolysis ratio) formula. The hemolysis rate is formulated as follows:

OD represents the absorbance of the sample at 545 nm.

The microstructure of the hydrogel of examples 1-3 and comparative example is shown in FIG. 3, wherein (A) is the comparative example; (B) example 1 was used; (C) example 2 was used; (D) example 3 was used.

The bacteriostatic ratios of the hydrogels of examples 1-3 and comparative example 8h are compared in the following table.

The rheological properties and biological activities of the hydrogels of examples 1-3 and comparative examples are shown in the table below.

As can be seen from the above data, the glycyrrhizic acid of comparative exampleG'Are all smaller than the others and are,G''the mechanical properties of the hydrogel are poor and cannot meet the strength requirement of the medical hydrogel, and after the pentacyclic triterpene compound (except glycyrrhizic acid) or the tetracyclic triterpene compound is added, the mechanical strength of the hydrogel is remarkably improved, and the mechanical properties of the hydrogel are remarkably enhanced.

According to the data of the antibacterial rate of the hydrogel for 8h, the antibacterial effect of glycyrrhizic acid on gram-negative bacteria is poor and is only 40 +/-5%, the growth of the gram-negative bacteria cannot be substantially inhibited, the antibacterial rate of glycyrrhizic acid hydrogel on the gram-negative bacteria is rapidly improved to 98 +/-2% after the glycyrrhetinic acid, the ursolic acid and the ginsenoside are added, and the antibacterial property of the glycyrrhizic acid hydrogel is also solved.

The standard group is a group of red blood cells treated by pure PBS buffer solution, no hydrogel is added, the PBS buffer solution can well keep the red blood cells from breaking, the hemolysis rate of the standard group can be seen to be 2.2%, if the red blood cells are treated by hydrogel, the hemolysis phenomenon, namely the erythrocyte breaking phenomenon, finally the measured fluorescence intensity is lower than that of the standard group, and as can be seen from the fluorescence intensity, the hydrogel prepared by glycyrrhizic acid is non-toxic and harmless, but has higher hemolysis rate, the high hemolysis rate means that the hydrogel can not be used in the medical industry, if the hydrogel is used as a wound, the high hemolysis rate can damage the red blood cells of the wound, the wound healing can not be promoted, and the wound is subjected to secondary injury.

Claims

1. An injectable natural triterpene compound antibacterial hydrogel, which is characterized in that: the gel molecules of the injectable natural triterpene compound antibacterial hydrogel are a mixture of glycyrrhizic acid and one or more selected from ursolic acid, glycyrrhetinic acid and ginsenoside, and the gel solvent is a PBS buffer solution with the pH value of 7.0-7.4, wherein in the mixture of glycyrrhizic acid and one or more selected from ursolic acid, glycyrrhetinic acid and ginsenoside, the mole fraction of glycyrrhizic acid is 60-90%, and the mixing concentration of the mixture of glycyrrhizic acid and one or more selected from ursolic acid, glycyrrhetinic acid and ginsenoside and the gel solvent is 1-8 mM.

2. A method for preparing the injectable antibacterial hydrogel of natural triterpene compound of claim 1, which comprises the following steps:

preparing a mixture suspension solution consisting of glycyrrhizic acid and one or more of ursolic acid, glycyrrhetinic acid and ginsenoside with the concentration of 1-8mM by using a PBS buffer solution with the pH value of 7.0-7.4;

thirdly, the mixture suspension is placed in an environment with the temperature of 90-97 ℃ for treatment for 100-150 min;

placing the mixture suspension liquid treated in the step (III) in an environment with the temperature of 25-37 ℃ until hydrogel is formed, and obtaining the injectable natural triterpene compound antibacterial hydrogel.