CN110314242B - Preparation method and application of controlled-release antibiotic composite hydrogel - Google Patents

Abstract

The invention provides a composite antibiotic hydrogel based on aminoglycoside antibiotics and ornidazole controllable release, which is formed by cross-linking oxidized natural polysaccharide macromolecules, aminoglycoside antibiotics and ornidazole modified by 1 generation polyamidoamine dendrimer with an amino terminal through acid-sensitive Schiff base bonds. The preparation method is simple, the cost is low, the strength of the gel, the shape of the gel, the degradation of the gel, the release rate of the drug and the like can be controlled according to the content of the antibiotic in the prepared controlled-release composite antibiotic hydrogel, the hydrogel has very broad-spectrum efficient antibacterial performance, and the antibacterial effect is superior to that of various commercial antibacterial gels on the market. The hydrogel is expected to be made into external dressing, ointment preparation, implant, coating on medical apparatus, etc., for resisting infection of gram negative bacteria, gram positive bacteria, anaerobic bacteria, etc.

Description

A kind of preparation method of controllable release antibiotic composite hydrogel and use thereof

technical field

The present invention relates to the technical field of polymer chemistry and biological materials. In particular, it relates to a medical antibiotic composite hydrogel, in particular, the linking agent is an aminoglycoside antibiotic drug and ornidazole itself modified by a first-generation dendrimer polyamidoamine with an amino terminal, and the linking bond is an acid-sensitive chemical bond . The hydrogel can be degraded in the acidic environment generated by bacterial proliferation, thereby releasing drugs for broad-spectrum sterilization, and at the same time realizing the on-demand release of aminoglycoside antibiotics and ornidazole, and the drug release rate can be self-regulated.

Background technique

Despite the rapid development of medicine in recent years, human beings still face many difficulties in the prevention and treatment of bacterial infections. For example: (1) Military aspects: high-energy weapons in modern warfare often lead to a variety of compound injuries, and a large number of pathogenic bacteria are brought into the wound with shrapnel, which often leads to compound infections including anaerobic infection in the wound; Due to its particularity and danger, it is impossible for the wounded to receive timely antibiotics and effective debridement. These factors often lead to complex infections and even life-threatening war wounds; (2) Civil trauma first aid: due to the high risk of bacterial infection in trauma patients, Clinically, open clean wounds generally require debridement within 6 hours after injury to close the wound in one stage, while wounds that exceed 6 hours and contaminated wounds need to be sutured in the second stage after debridement. For patients with open fractures, regardless of whether the injury time exceeds 6 hours, external fixation is generally required first, followed by internal fixation and wound closure in the second stage. (3) Special infections: infections involving internal plants and bedsore infections in paralyzed patients often lead to prolonged infection, extensive drug resistance and mixed infection of various bacteria. (4) In daily life, although the wound on the body surface does not affect the life, it often causes obvious scar healing due to local infection, which affects the appearance.

Scientists have recently invented gels coated with antibiotic drugs, which have gradually become an important means of treating local infections. However, the antibiotic hydrogels currently in clinical use are all encapsulated in the gel and released from the pores of the gel network by physical diffusion, such as traditional erythromycin gel, ofloxacin gel, Clindamycin gel, etc., are prepared by directly mixing antibiotic drugs with some macromolecular matrix and other excipients. The drugs in such gels cannot be released according to the actual demand, and their release kinetics cannot be adjusted. In view of the above deficiencies, our research team has recently successfully prepared an antibiotic gel with high-efficiency, broad-spectrum antibacterial effect and release in response to infection stimuli using aminoglycoside antibiotics and oxidized polysaccharides as raw materials. The principle is that the aldehyde groups on the surface of oxidized polysaccharides and the amino groups of aminoglycoside antibiotics are cross-linked through acid-sensitive Schiff base bonds to form gels. The Schiff base bond is a dynamic equilibrium chemical bond, which can be decomposed under the stimulation of acidic conditions and restored to amino and aldehyde groups, and the hydrogel degrades to release antibiotics and highly biocompatible polysaccharides. It is known that bacterial growth produces substances such as formic acid, propionic acid, butyric acid, lactic acid, etc., which make the surrounding environment appear acidic. Therefore, this antibiotic hydrogel is different from the traditional antibiotic drug gel. The aminoglycoside antibiotics in this type of gel are cross-linked in the network as a gel component. If there is no acid stimulation, its properties are very stable and do not It will be released out of the system by physical diffusion. The acidic environment generated during bacterial infection stimulates the hydrogel with acid, breaking the Schiff base bonds between aminoglycoside antibiotics and oxidized polysaccharide molecules, thereby realizing the responsive release of antibiotic drugs.

However, this type of gel has limited therapeutic effect on mixed anaerobic bacterial infection. Therefore, in clinical application, aminoglycoside antibiotic-oxidized polysaccharide hydrogel still has certain defects in mixed infection of mixed anaerobic bacterial infection.

SUMMARY OF THE INVENTION

The invention overcomes the deficiencies of the existing traditional gels, and innovatively proposes a drug-on-demand gel that can precisely control the release of aminoglycoside antibiotics and ornidazole. The azoles are cross-linked by acid-sensitive Schiff base bonds. Since bacterial proliferation will generate an acidic environment, the Schiff base bond that builds the gel will be broken and the gel will be degraded. At the same time, aminoglycoside antibiotics and ornidazole will be released on demand for sterilization. Different from the traditional gel that releases the drug through passive diffusion, the drug gel gradually releases the drug through the degradation of the gel, which avoids the initial burst release of the drug, and can make the drug release rate consistent with the degradation rate of the gel. . The invention utilizes oxidized natural polysaccharide and aminoglycoside polysaccharide antibiotics and G1-ornidazole covalently linked to form a gel through a Schiff base bond, and obtains a drug gel that can release antibiotics on demand according to the degree of infection, and has the advantages of simple synthesis and broad antibacterial spectrum. , the antibacterial efficiency is high, and the strength of the gel and the release rate of the drug can be adjusted by the content of the drug.

The present invention is achieved through the following technical solutions:

A preparation method of a controllable-release antibiotic composite hydrogel, comprising the following steps:

Ornidazole is modified with a first-generation polyamide-amine dendrimer compound with an amino terminal to obtain the modified ornidazole;

Oxidize natural polysaccharides to obtain natural polysaccharides containing aldehyde groups on the surface;

The aminoglycoside antibiotic, the modified ornidazole and the natural polysaccharide containing an aldehyde group on the surface are cross-linked through a Schiff base bond to obtain the controlled release antibiotic hydrogel.

The oxidized natural polysaccharide polymer and aminoglycoside antibiotics, G1 ornidazole are cross-linked through Schiff base bonds to form a gel. The cross-linking mechanism is as follows:

In the present invention, the temperature of gel formation is room temperature.

In the present invention, the gelation time of the hydrogel can be adjusted by adjusting the contents of aminoglycoside antibiotics and G1 ornidazole, the natural polysaccharide-G1 ornidazole hydrogel, natural polysaccharide-aminoglycoside antibiotic- The gelation time of the G1 ornidazole composite hydrogel is between 1 second and 60 minutes, preferably between 10 seconds and 15 minutes; more preferably, it is 1 minute. The gelation time is related to the hydroformylation ratio of the polysaccharide polymer and the number of amino groups in the aminoglycoside antibiotic molecule and G1-ornidazole structure. The higher the formylation ratio, the shorter the gelation time; the amino group in the antibiotic molecule The higher the number, the shorter the gelation time.

The present invention also proposes a natural polysaccharide-G1 ornidazole hydrogel, a natural polysaccharide-aminoglycoside antibiotic-G1 ornidazole composite hydrogel prepared by the above preparation method, and the hydrogel is composed of aminoglycoside The antibiotic-like, G1 ornidazole is in situ cross-linked with oxidized natural polysaccharide macromolecules through Schiff base bonds, and the obtained hydrogel exhibits an obvious microscopic porous structure.

In the present invention, the mechanical strength of the natural polysaccharide-G1 ornidazole hydrogel, natural polysaccharide-aminoglycoside antibiotic-G1 ornidazole composite hydrogel can be changed by changing the polysaccharide macromolecule and aminoglycoside antibiotic or G1 ornidazole. The mechanical strength of the hydrogel can be adjusted by adjusting the concentration ratio of nitazole, and the mechanical strength of the hydrogel can also be adjusted by adjusting the content of aminoglycoside antibiotics and G1 ornidazole; the storage modulus of the hydrogel is between 10 Pa and 10,000 Pa. time, preferably between tens of Pascals and thousands of Pascals, can be used to prepare a variety of dosage forms.

In the present invention, the prepared natural polysaccharide-G1 ornidazole hydrogel and natural polysaccharide-aminoglycoside antibiotic-G1 ornidazole composite hydrogel have good tissue adhesion properties and are very suitable for skin dressings and the like. application in tissue engineering.

In the present invention, the prepared natural polysaccharide-G1 ornidazole hydrogel and natural polysaccharide-aminoglycoside antibiotic-G1 ornidazole composite hydrogel can be degraded in response to the acidic environment generated by bacterial proliferation, Thereby, antibiotics are released, and the release rate can be adjusted by changing the mass percentage of aminoglycoside antibiotics and G1-ornidazole in the gel, that is, by adjusting the aminoglycoside antibiotics, G1-ornidazole. The degradation rate of the hydrogel can be adjusted by adjusting the content of the hydrogel, and the release rate of the drug can also be adjusted by adjusting the content of aminoglycoside antibiotics and G1-ornidazole. The release half-cycle of aminoglycoside antibiotics and G1-ornidazole can be adjusted from 1 hour to several months, which is suitable for a variety of different administration situations.

As a preferred solution, the natural polysaccharide macromolecule is selected from glucan, chitosan, alginic acid, hyaluronic acid, cellulose, lignin, chondroitin, glycosaminoglycan, starch, pectin, and mannan. at least one of.

Preferably, the natural polysaccharide is glucan, and its chemical structural formula is shown in formula II:

In formula (II), n is the number of repeating units of the polysaccharide polymer, and is 1 to 100,000, more preferably 300 to 400.

In the present invention, the way to obtain the oxidized natural polysaccharide macromolecule is that the natural polysaccharide macromolecule is oxidized to oxidized natural polysaccharide macromolecule in the presence of an oxidizing agent, so that an aldehyde group is formed in its molecular structure, wherein the oxidizing agent For sodium periodate and so on.

The structure of oxidized dextran is shown in formula (III):

In formula III, x is the ratio of aldehyde formation, which is 5-95%, more preferably 40-60%.

As a preferred solution, the aminoglycoside antibiotic drug is selected from netilmicin, isopamicin, capreomycin, ribomycin, sisomicin, apramycin, amikacin, kanamycin , at least one of gentamicin, paromomycin, tobramycin, and neomycin.

In the present invention, the aminoglycoside antibiotics are glycoside antibiotics formed by connecting aminosugar and aminocycloalcohol through an oxygen bridge, and the molecular structure contains at least 2 amino groups, and the general formula is shown in formula IV:

In formula I, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ are H or alkyl, R ₆ , R ₇ , R ₈ are hydroxyl or alkyl hydroxyl, preferably, the aminoglycoside antibiotic is selected from Netilmicin, Isopamicin, Capreomycin, Ribomycin, Sisomicin, Apramycin, Amikacin, Kanamycin, Gentamicin, Paromomycin, Tobu At least one of neomycin and neomycin.

As a preferred solution, the first-generation polyamide-amine dendrimer compound with an amino terminal and ornidazole are covalently linked through a substitution reaction between an amino group and benzyl bromide.

As a preferred solution, the gelling concentration of the natural polysaccharide containing aldehyde groups on the surface is 30-200 mg/mL, the aldehyde grouping ratio is 5-95%, and the mass percentage of aminoglycoside antibiotics is 0.1-20%.

As a preferred solution, the mass concentration of the modified ornidazole is 5.38%, wherein the molar ratio of the first-generation polyamidoamine dendrimer at the amino end to the ornidazole is 1:1.

A controllable-release antibiotic hydrogel obtained by the aforementioned preparation method.

Application of the aforementioned controlled-release antibiotic hydrogel in the preparation of a medicament for inhibiting bacterial infection.

As a preferred solution, the controlled-release antibiotic hydrogel is used for preparing external dressings, ointment preparations, implants or coatings in medical devices.

As a preferred embodiment, the bacteria are selected from one or more of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, anaerobes Clostridium sporogenes and Bacteroides fragilis.

In order to make up for the defects of the prior art and make its antibacterial properties better and broad-spectrum, the present invention further introduces ornidazole against anaerobic bacteria on the basis of the aminoglycoside antibiotic-oxidized polysaccharide hydrogel developed by our team, thereby constructing a broad-spectrum ornidazole. A novel aminoglycoside antibiotic-ornidazole-oxidized polysaccharide composite antibiotic hydrogel against gram-positive bacteria, gram-negative bacteria and anaerobic bacteria. The specific scheme and principle are: pretreating ornidazole and first-generation polyamidoamine dendrimer with amino terminal (G1 for short) to form G1-ornidazole, and then pass through the amino group on the surface of G1 and the aldehyde on the surface of the oxidized polysaccharide. The groups are cross-linked by Schiff base bonds to form a hydrogel. In this invention, aminoglycoside antibiotics and G1-ornidazole are connected with oxidized polysaccharides by Schiff base bonds, and both are gel components themselves, and hydrogels of different shapes and properties can be constructed by adjusting the content of antibiotics . According to this feature, products with various properties such as dressings, ointments, coatings or implants can be produced according to specific clinical uses. For example: for daily life and ordinary war wounds, this material can be prepared into self-adhesive dressings, which can be equipped in individual first aid kits and household daily first aid kits. ; For deep pollution wounds such as abdominal intestine, limb fractures, and complex wounds such as bullet wounds, the above hydrogel ointment can be injected into the wound after simple hemostasis; Prevent infection while reducing water loss from wound surface. Therefore, the material of the invention can be widely used in daily life and the treatment of local complex infections, especially for war wounds. In addition, G1-ornidazole alone can form hydrogels with oxidized polysaccharides exclusively for anaerobic infection.

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

1. Compared with the traditional antibiotic gel that uses physical entrapment to load drugs, aminoglycoside antibiotics and G1 ornidazole drug molecules in the present invention can be very stably cross-linked in the gel network, avoiding the passive diffusion of drugs. resulting in a detonation;

2. The Schiff base bonds forming the natural polysaccharide-G1 ornidazole hydrogel and natural polysaccharide-aminoglycoside antibiotic-G1 ornidazole composite hydrogel have acid sensitivity. The base bond is broken to release drugs, which realizes the on-demand release of aminoglycoside antibiotic drugs and G1 ornidazole at the site of bacterial infection;

3. The release rate of drug molecules and various properties of the gel can be adjusted by adjusting the mass percentage of aminoglycoside antibiotic drugs and G1-ornidazole in the gel.

Description of drawings

Other features, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments with reference to the following drawings:

Fig. 1 is the synthetic route diagram of G1-ornidazole in embodiment 1;

Fig. 2 is the preparation route map of oxidized dextran-tobramycin-G1 ornidazole composite hydrogel in embodiment 2;

Figure 3: is a schematic diagram of the thixotropic properties of the oxidized dextran-tobramycin-G1 ornidazole composite hydrogel in Example 3;

4 is an injectable schematic diagram of the oxidized dextran-tobramycin-G1 ornidazole composite hydrogel in Example 4;

5 is a graph of pH change during bacterial proliferation in Example 5;

Fig. 6 is the antibiotic release diagram of oxidized dextran-tobramycin-G1 ornidazole hydrogel in response to pH in Examples 6 and 7;

Fig. 7 is the pH response graph of oxidized dextran-tobramycin-G1 ornidazole hydrogel modulus in Example 8;

8 is a graph of the toxicity test data of oxidized dextran-tobramycin-G1 ornidazole hydrogel to NIH3T3 cells in Example 9;

9 is a graph of the hemolysis test of the oxidized dextran-tobramycin-G1 ornidazole hydrogel in Example 10;

Figure 10 is a graph of the in vitro antibacterial effect of the oxidized dextran-tobramycin-G1 ornidazole composite hydrogel A in Example 11;

11 is a graph showing the in vitro antibacterial effect of oxidized dextran-tobramycin-G1 ornidazole composite hydrogel B in Example 12;

12 is a graph showing the in vitro antibacterial effect of the oxidized dextran-tobramycin-G1 ornidazole composite hydrogel C in Example 13.

Detailed ways

The present invention will be described in detail below with reference to specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

Example 1: Preparation of G1-ornidazole

250 mg of G1 and 153.6 mg of ornidazole were formulated into 100 mg/mL and 50 mg/mL DMSO solutions, respectively, and the ornidazole solution in DMSO was added dropwise to G1, followed by 115.8 mg of potassium carbonate. After that, it was stirred at a constant speed for two days at 60°C, and the solvent was removed by lyophilization. Subsequently, the lyophilized product was dissolved in 2 mL of methanol, the unreacted small molecule drug was removed by ether precipitation, and further filtered and vacuum dried to obtain a brown-yellow powder product, which was a dendrimer prodrug of ornidazole. The synthetic route of G1-ornidazole is shown in Figure 1.

Example 2: Preparation of Oxidized Dextran-Tobramycin-G1 Ornidazole Antibacterial Hydrogel

The oxidized polysaccharide in this embodiment is mainly represented by oxidized dextran as a polysaccharide macromolecule, and the aminoglycoside antibiotic is mainly represented by tobramycin. Combine aminoglycoside antibiotics, G1 ornidazole, and oxygenated dextran. The synthetic route is shown in Figure 2.

Mix 25 μL of oxidized dextran solution (30% oxidation degree, 75 mg/mL), 12.5 μL of tobramycin solution (20 mg/mL) and 12.5 μL of G1-ornidazole solution (50 mg/mL), and it will be formed for about 5 minutes at room temperature. The gel is defined as oxidized dextran-tobramycin-G1 ornidazole composite hydrogel A in the present invention.

Mix 25 μL of oxidized dextran solution (30% degree of oxidation, 50 mg/mL), 12.5 μL of tobramycin solution (50 mg/mL) and 12.5 μL of G1-ornidazole solution (100 mg/mL), at room temperature for about 5 minutes. The gel is defined as oxidized dextran-tobramycin-G1 ornidazole composite hydrogel B in the present invention.

Mix 30 μL of oxidized dextran solution (50% degree of oxidation, 50 mg/mL), 5 μL of tobramycin solution (20 mg/mL) and 15 μL of 1-ornidazole solution (200 mg/mL), and gelatinize about 5 minutes at room temperature. In the present invention, it is defined as oxidized dextran-tobramycin-G1 ornidazole composite hydrogel C.

Mix 25 μL of oxidized dextran solution (30% degree of oxidation, 75 mg/mL), 12.5 μL of amikacin solution (20 mg/mL) and 12.5 μL of G1-ornidazole solution (50 mg/mL), and form a solution for about 5 minutes at room temperature. The glue is defined as oxidized dextran-amikacin-G1 ornidazole composite hydrogel D in the present invention.

Mix 25 μL of oxidized dextran solution (30% degree of oxidation, 75 mg/mL), 12.5 μL of netilmicin solution (20 mg/mL) and 12.5 μL of G1-ornidazole solution (50 mg/mL), and form a solution for about 5 minutes at room temperature. The glue is defined as oxidized dextran-netilmicin-G1 ornidazole composite hydrogel E in the present invention.

The experimental results show that, after the polysaccharide polymer such as dextran is oxidized to contain aldehyde groups in its molecular structure, it can efficiently cross-link with aminoglycoside antibiotics and G1 ornidazole to form hydrogels.

In the following examples, the oxidized dextran-tobramycin-G1 ornidazole hydrogel is mainly used as an example to illustrate its performance in terms of gel morphology, gel formation time, mechanical strength, drug release, and antibacterial activity. Effect.

Example 3: Schematic diagram of thixotropic properties of oxidized dextran-tobramycin-G1 ornidazole hydrogel

Preparation of oxidized dextran-tobramycin-G1 ornidazole hydrogel:

Mix 300 μL of oxidized dextran solution (50% oxidation degree, 50 mg/mL), 50 μL of tobramycin solution (20 mg/mL) and 150 μL of G1-orni solution (200 mg/mL), and gelatinize about 5 minutes at room temperature. The thixotropy of the gel was tested by transferring the gel to a plate of a multi-function rheometer at 37 °C, setting the strain rate to 1% and 200%, and performing time-dependent modulus measurements alternately and repeated 3 times. performance. As shown in Figure 3, it can be seen that the composite gel still maintains a high storage modulus after three times of failure-recovery experiments, indicating that the gel has good thixotropic properties.

Example 4: Injectable properties of the gel

180 μL of oxidized dextran solution (50% degree of oxidation, 50 mg/mL), 30 μL of tobramycin solution (20 mg/mL) and 90 μL of G1-orni solution (200 mg/mL) were mixed and aspirated into a syringe. As shown in Figure 4, the gel can be extruded under the thrust of the syringe, and can be used for clinical injection.

Example 5: pH changes during bacterial proliferation

The proliferation of bacteria will produce lactic acid, carbon dioxide and other substances, which will cause the surrounding environment to be acidic. We take Escherichia coli and Clostridium sporogenes as the representatives of aerobic and anaerobic bacteria respectively, and simulate the proliferation process of bacteria in vitro to observe the change of pH value of bacterial solution.

Escherichia coli in logarithmic phase was diluted to 10 ⁶ CFU/mL, one milliliter of bacterial solution was taken out every half an hour, and 3 μL of bromothymol blue (1%) was added to observe the color change of the bacterial solution. For Clostridium sporogenes, the initial concentration was also set to 10 ⁶ CFU/mL, and it was divided into about 10 portions, and the measurement was started after 12 hours of incubation. At 6, 12, 24 and 32 hours, 1 mL of bacterial solution was taken out, and 3 μL of bromothymol blue (1%) was added to observe the color of the bacterial solution. As shown in Figure 5, with the proliferation of bacteria, the pH value of the bacterial solution gradually decreased, indicating that an acidic environment was gradually generated.

Example 6: Degradation behavior of oxidized dextran-tobramycin-G1 ornidazole hydrogel in pH 5.0 buffer

To study the degradation behavior of oxidized dextran-tobramycin-G1 ornidazole hydrogel. The specific method is: add 500 μL of oxidized dextran-tobramycin-G1 ornidazole hydrogel (5.38% G1-ornidazole, 0.45% tobramycin) into the dialysis bag, and then put it into 50 mL of buffer solution (pH 5.0) in vials with gentle and constant agitation of the external solution. At intervals, 3 mL of the solution was withdrawn from the external solution, and the absorbance at 315 nm was measured, denoted as y1. The concentration of G1-ornidazole was obtained according to the scale y ₁ =4.8441× ₁ +0.0306 of the absorbance value at 315 nm of the sample and the concentration, thereby drawing the release curve of G1-ornidazole. On the other hand, the sample taken out was subjected to the ortho-phthalaldehyde derivatization method to measure the absorbance Y of the sample at 333 nm, and the theoretical absorbance value y2 at 333 nm was obtained through the standard curve after derivatization of the sample. Substitute Y-y2 as y3 into the standard curve y ₃ =0.9305x ₃ +0.0297 at 333 nm after aminoglycoside derivatization, so as to calculate the theoretical release amount of tobramycin and obtain the release curve of tobramycin.

The steps of the ortho-phthalaldehyde derivatization method are as follows: 536 mg of ortho-phthalaldehyde (OPA), 20 mL of methanol, 2.8 mL of thioglycolic acid, and 77.2 mL of boric acid buffer at pH 10.5 are mixed to prepare a derivatized OPA reagent. 600 μL was added for derivatization, and after 20 minutes, the UV absorption of sample z at a wavelength of 333 nm was detected and analyzed.

As shown in Figure 6, the release rate of oxidized dextran-tobramycin-G1 ornidazole hydrogel in pH 5.0 buffer solution tobramycin within 24 hours is about 94.08%. The release rate is about 92.28%.

Example 7: Release behavior of oxidized dextran-tobramycin-G1-orni hydrogel to tobramycin and G1-ornidazole in pH 7.4 buffer

The specific method was as described in Example 6, 500 μL of oxidized dextran-tobramycin-G1 ornidazole hydrogel (5.38% G1-ornidazole, 0.45% tobramycin) was added into the dialysis bag, and then put into a vial containing 50 mL of buffer (pH 5.0) with gentle and constant agitation of the outer solution. The method for determining the drug content in the samples is the same as that described in Example 6. After analysis, the release rate of the drug in pH7.4 buffer is shown in Figure 6. The release rate of tobramycin within 24 hours is about 25.67%, and the release rate of G1-ornidazole is about 20.45%. , significantly lower than the release in pH 5.0 buffer.

Examples 6 and 7 illustrate that the oxidized dextran-tobramycin-G1 ornidazole hydrogel is very stable in a neutral environment, but responds rapidly in an acidic environment, and the Schiff base bonds that constitute the gel network are broken and released. The release of antibiotics can realize the on-demand release of antibiotic drugs.

Example 8: pH responsiveness of oxidized dextran-tobramycin-G1 ornidazole hydrogel modulus

The oxidized dextran-tobramycin-G1-ornidazole hydrogels in Examples 6 and 7 were also prepared, soaked in buffer solutions of pH 5.0 and pH 7.4 for one hour respectively, and the gel was transferred to a multi-kinetic rotational rheometer Time-dependent modulus measurements were performed on the plate. After measurement, the modulus becomes 229.25pa and 144.48pa, respectively, as shown in Figure 7. It indicated that the oxidized dextran-tobramycin-G1 ornidazole hydrogel had pH responsiveness, and the gel became soft after the antibiotic was released.

Example 9: Toxicity of Oxidized Dextran-Tobramycin-G1 Ornidazole Hydrogel on Cells

To evaluate the cytotoxicity of the oxidized dextran-tobramycin-G1 ornidazole hydrogel, 200 μL of the gel was soaked in 2 mL of DMEM medium without bovine serum albumin for 24 hours, and the leachate was subsequently collected and supplemented with 10% Bovine serum albumin was prepared into different concentrations and added to NIH3T3 cells (mouse embryonic fibroblasts) for incubation, and then the cell survival rate was counted by MTT method. The experimental results show that the oxidized dextran-tobramycin-G1 ornidazole hydrogel has no obvious toxicity to NIH3T3 cells and has excellent biocompatibility, as shown in Figure 8.

Example 10: Hemolysis test of oxidized dextran-tobramycin-G1 ornidazole hydrogel

The blood safety of the gel was further evaluated by hemolysis. Fresh blood of Balb/c mice about 8 weeks old was collected, and red blood cells were collected by centrifugation, and prepared into a red blood cell suspension of about 2%. 20 μL of hydrogel was added to 1 mL of red blood cell suspension and incubated at 37°C for 1 hour. At the same time, Triton X-100 (0.5%) and PBS (pH 7.4) were respectively added to 1 mL of red blood cell suspension for incubation as positive and negative controls. The red blood cell suspension treated with the material was centrifuged at 2000 r/min for 5 minutes, the supernatant was collected, and its absorption at 540 nm was detected. Triton X-100 was set to 100% hemolysis. The experimental results show that the oxidized dextran-tobramycin-G1 ornidazole hydrogel has no obvious hemolysis and has excellent biocompatibility, as shown in Figure 9.

Example 11: In vitro antibacterial effect of oxidized dextran-tobramycin-G1 ornidazole composite hydrogel A.

In the present invention, Staphylococcus aureus (S. aureus) is used as the aerobic bacteria model, and C. sporogenes is used as the anaerobic bacteria model to verify the oxidized dextran-tobramycin-G1 ornidazole water. The in vitro antibacterial effect of the gel on aerobic and anaerobic bacteria, respectively.

50 μL of oxidized dextran-tobramycin-G1 ornidazole hydrogel A was added to the 96-well plate, and G1 ornidazole hydrogel and tobramycin hydrogel containing the same drug content were prepared as controls. And the medium without bacteria and the bacteria liquid without drug treatment were used as blank control and positive control, respectively. Staphylococcus aureus and Clostridium sporogenes were cultured to the logarithmic growth phase, and prepared to an initial concentration of 1.0×10 ⁶ CFU/mL, and 100 μL of bacterial solution was added to each well, incubated for 24 hours, and then analyzed by absorbance method. The inhibition efficiency in the treated bacterial liquid was counted.

As shown in Figure 10, the inhibitory efficiencies of tobramycin hydrogel and G1 ornidazole hydrogel against Staphylococcus aureus and Clostridium sporogenes were 1.08% and 1.27%, respectively, indicating that they were effective against aerobic bacteria and Anaerobic bacteria have a good inhibitory effect. However, the inhibition efficiency of G1 ornidazole hydrogel against Staphylococcus aureus was only 24.86%, and the inhibition rate of tobramycin hydrogel against Clostridium sporogenes was only 29.20%, indicating that these two drugs are respectively effective against anaerobic bacteria. And aerobic bacteria have no obvious inhibitory effect. However, the inhibition rates of oxidized dextran-tobramycin-G1 ornidazole composite hydrogel A on Staphylococcus aureus and Clostridium sporogenes reached 0.52% and 2.08%, indicating that the composite hydrogel A can inhibit aerobic bacteria and anaerobic bacteria. Oxygen bacteria have very good bacteriostatic effect.

Example 12: In vitro antibacterial effect of oxidized dextran-tobramycin-G1 ornidazole composite hydrogel B

50 μL of oxidized dextran-tobramycin-G1 ornidazole hydrogel B was added to the 96-well plate, and G1 ornidazole hydrogel and tobramycin hydrogel containing the same drug content were prepared as controls. And the medium without bacteria and the bacteria liquid without drug treatment were used as blank control and positive control, respectively. Staphylococcus aureus and Clostridium sporogenes were cultured to the logarithmic growth phase, and prepared to an initial concentration of 1.0×10 ⁶ CFU/mL, 100 μL of bacterial solution was added to each well, incubated for 24 hours, and then counted by plate The bacterial survival in the treated bacterial liquid was counted by the method.

As shown in Figure 11, the survival amount of Staphylococcus aureus and Clostridium sporogenes treated with tobramycin hydrogel and G1 ornidazole hydrogel respectively were 3.67E ⁷ CFU/mL and 3.3E ⁴ CFU /mL, indicating that they have a good inhibitory effect on aerobic bacteria and anaerobic bacteria respectively. The survival amount of Staphylococcus aureus treated with G1 ornidazole hydrogel was 2.67E ¹⁰ CFU/mL, and the survival amount of C. sporogenes treated with tobramycin hydrogel was 2.3E ⁶ CFU/mL. mL, indicating that the two drugs had no obvious inhibitory effect on anaerobic bacteria and aerobic bacteria, respectively. However, Staphylococcus aureus and Clostridium sporogenes treated with oxidized dextran-tobramycin-G1 ornidazole composite hydrogel B were 3.0E ⁷ CFU/mL and 3.0E ⁴ CFU/mL, respectively, indicating that the composite water Gel B has very good bacteriostatic effect on both aerobic and anaerobic bacteria.

Example 13: In vitro antibacterial effect of oxidized dextran-tobramycin-G1 ornidazole composite hydrogel C.

50 μL of oxidized dextran-tobramycin-G1 ornidazole hydrogel C was added to the 96-well plate, and G1 ornidazole hydrogel and tobramycin hydrogel containing the same drug content were prepared as controls. And the medium without bacteria and the bacteria liquid without drug treatment were used as blank control and positive control, respectively. Staphylococcus aureus and Clostridium sporogenes were cultured to the logarithmic growth phase, and prepared to an initial concentration of 1.0×10 ⁶ CFU/mL, 100 μL of bacterial solution was added to each well, incubated for 24 hours, and then counted by plate The bacterial survival in the treated bacterial liquid was counted by the method.

As shown in Figure 12, the survival amount of Staphylococcus aureus and Clostridium sporogenes treated with tobramycin hydrogel and G1 ornidazole hydrogel respectively was 1.3E ⁸ CFU/mL and 3.0E ³ CFU /mL, indicating that they have a good inhibitory effect on aerobic bacteria and anaerobic bacteria respectively. The survival amount of Staphylococcus aureus treated with G1 ornidazole hydrogel was 4.0E ¹⁰ CFU/mL, and the survival amount of C. sporogenes treated with tobramycin hydrogel was 6.3E ⁸ CFU/mL. mL, indicating that the two drugs had no obvious inhibitory effect on anaerobic bacteria and aerobic bacteria, respectively. However, Staphylococcus aureus and Clostridium sporogenes treated with oxidized dextran-tobramycin-G1 ornidazole composite hydrogel C were 2.0E ⁸ CFU/mL and 2.0E ³ CFU/mL, respectively, indicating that the composite water Gel C has a very good bacteriostatic effect on both aerobic and anaerobic bacteria.

Examples 11-13 illustrate that the oxidized dextran-tobramycin-G1 ornidazole composite hydrogel not only has a good inhibitory effect on aerobic bacteria, but also has significant antibacterial properties on anaerobic bacteria, which is successful to a certain extent The antibacterial spectrum of the aminoglycoside antibiotic hydrogel has been broadened, and it has a certain potential application value in the treatment of clinical infections, especially complex war-wound infections during military operations.

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and those skilled in the art can make various variations or modifications within the scope of the claims, which do not affect the essential content of the present invention.

Claims (7)

1. a preparation method of the antibiotic compound hydrogel of controllable release, is characterized in that, comprises the steps: Ornidazole is modified with a first-generation polyamide-amine dendrimer compound with an amino terminal to obtain the modified ornidazole; Oxidize natural polysaccharides to obtain natural polysaccharides containing aldehyde groups on the surface; cross-linking the aminoglycoside antibiotic, the modified ornidazole and the natural polysaccharide containing an aldehyde group on the surface through a Schiff base bond to obtain the controlled release antibiotic hydrogel; The natural polysaccharide macromolecule is selected from at least one of glucan, chitosan, alginic acid, hyaluronic acid, cellulose, lignin, chondroitin, glycosaminoglycan, starch, pectin, and mannan ; The aminoglycoside antibiotic drug is selected from netilmicin, isopamicin, capreomycin, ribosomycin, sisomicin, apramycin, amikacin, kanamycin, gentamycin at least one of pyridoxine, paromomycin, tobramycin, and neomycin; The gelling concentration of the natural polysaccharide containing aldehyde groups on the surface is 30-200 mg/mL, the aldehyde grouping ratio is 5-95%, and the mass percentage of aminoglycoside antibiotics is 0.1-20%. 2. the preparation method of the antibiotic hydrogel of controllable release as claimed in claim 1, is characterized in that, described band amino terminal 1 generation polyamide-amine dendrimer and ornidazole pass through amino and benzyl Substitution reactions between bromines are covalently linked. 3. the preparation method of the antibiotic hydrogel of controllable release as claimed in claim 1, is characterized in that, the concentration of the ornidazole after described modification is 5.38%, wherein, the 1st generation polyamidoamine tree of amino terminal The linking ratio of the macromolecule compound and ornidazole is 1:1. 4. A controlled-release antibiotic hydrogel obtained by the preparation method according to any one of claims 1 to 3. 5. The application of the controllable-release antibiotic hydrogel of claim 4 in the preparation of a medicament for inhibiting bacterial infection. 6. The use according to claim 5, wherein the controlled release antibiotic hydrogel is used for preparing external dressings, ointment preparations, implants or coatings in medical devices. 7. The application according to claim 6, wherein the bacteria are selected from Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, anaerobic bacteria Clostridium sporogenes and Bacteroides fragilis.

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