Preparation method, product and application of local drug sustained-release carrier

Abstract

The invention relates to the technical field of preparation of local drug sustained-release carriers, in particular to a preparation method, a product and application of a local drug sustained-release carrier. The local drug sustained-release carrier is prepared from the following raw materials in percentage by mass: 0.5%-5% of gellan gum, 0.5%-3% of carbomer, 0.5%-5% of carboxylated chitosan, 0.01%-0.3% of cationic salt, 0.02%-0.2% of a neutralizing agent and the balance of water, the cationic salt is one of calcium chloride, potassium chloride, sodium chloride and magnesium chloride; the neutralizer is one of ammonia water, triethanolamine, sodium hydroxide and sodium bicarbonate. The invention provides a local sustained-release drug delivery scheme, which solves the problems of low adhesion and too fast drug release in local drug delivery, and the local drug sustained-release carrier has high adhesion in skin mucous membranes or tissues, can slowly release drugs, and promotes continuous and controllable release of drugs in target parts.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation of a topical drug sustained-release carrier, and in particular to a preparation method, a product and application of a topical drug sustained-release carrier. Background Art

[0002] Hydrogel is a type of extremely hydrophilic three-dimensional network structure gel that swells rapidly in water and can retain a large volume of water without dissolving in this swollen state. Any water-soluble or hydrophilic polymer can form a hydrogel through certain chemical or physical crosslinking. Hydrogels are widely used in the biomedical field due to their ability to maintain high water content and simulate natural extracellular matrix. On the one hand, the moist and soft properties of hydrogels can avoid friction with surrounding tissues and show a positive effect on the healing of injured tissues. Thanks to the high water content, hydrogels also soothe the injured area by reducing the temperature of the wound. On the other hand, hydrogels have a loose and porous internal structure, which not only absorbs wound exudates, but also helps the exchange of O2, H2O and nutrients, and promotes cell adhesion, migration and extracellular matrix deposition in the wound area. Compared with the many disadvantages of systemic drug administration (rapid drug excretion and liver metabolism leading to low bioavailability, and drugs with short biological half-lives requiring frequent administration), local drug delivery systems have relative advantages in medical applications, including direct application to specific sites to avoid gastrointestinal problems and first-pass metabolism, controlled drug release to improve efficacy and minimize systemic side effects, and reduced administration frequency to improve patient compliance.

[0003] However, current topical drug administration generally suffers from the problems of low adhesion and too-fast drug release. Therefore, it is of great significance to the art to provide a topical drug sustained-release carrier with high adhesion and the ability to slowly release drugs. Summary of the invention

[0004] Based on the above content, the present invention provides a preparation method, product and application of a local drug sustained-release carrier.

[0005] To achieve the above object, the present invention provides the following solutions: One of the technical solutions of the present invention is a topical drug sustained-release carrier, wherein the raw materials include, by mass percentage: 0.5% to 5% of gellan gum, 0.5% to 3% of carbomer, 0.5% to 5% of carboxylated chitosan, 0.01% to 0.3% of cationic salt, 0.02% to 0.2% of neutralizer and the balance of water; The cationic salt is one of calcium chloride, potassium chloride, sodium chloride and magnesium chloride; The neutralizing agent is one of ammonia water, triethanolamine, sodium hydroxide and sodium bicarbonate.

[0006] The second technical solution of the present invention is a method for preparing the above-mentioned local drug sustained-release carrier, comprising the following steps: Adding gellan gum to a portion of water to dissolve it to obtain a gellan gum solution, and adding carboxylated chitosan to the gellan gum solution to obtain component A; After adding carbomer to the remaining water for swelling, component B is obtained; The component A and the component B are mixed evenly, and then the cationic salt and the neutralizer are added and mixed evenly to obtain the topical drug sustained-release carrier; When the raw material contains epigallocatechin gallate, epigallocatechin gallate and carboxylated chitosan are added to the gellan gum solution at the same time to obtain component A.

[0007] The third technical solution of the present invention is the use of the above-mentioned local drug sustained-release carrier in the preparation of sustained-release drugs.

[0008] A fourth technical solution of the present invention is a method for preparing a sustained-release drug, comprising mixing a drug solution with the above-mentioned local drug sustained-release carrier and then shaking at a constant temperature to obtain the sustained-release drug; the drug solution is a ciprofloxacin solution.

[0009] A fifth technical solution of the present invention is a sustained-release drug prepared by the above-mentioned preparation method.

[0010] The present invention discloses the following technical effects: The present invention proposes a local sustained-release drug administration scheme, which solves the problems of low adhesion and too fast drug release in local drug administration. The local drug sustained-release carrier of the present invention has high adhesion to the skin mucosa or tissue, can slowly release the drug, and promotes the continuous and controllable release of the drug at the target site. It is a hydrogel drug carrier with high biocompatibility and high stability.

[0011] The invention optimizes the sustained-release performance of the hydrogel, increases the tissue adhesion rate, and improves the mechanical properties and stability while maintaining the biocompatibility of similar gels. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 The photos are the results of the inverted vial test of the hydrogels (local drug sustained-release carriers) prepared in Examples 1 and 2; wherein (a) is Example 1 and (b) is Example 2.

[0014] Figure 2 This is a scanning electron microscope photograph of the hydrogel prepared in Example 2.

[0015] Figure 3 The rheological properties of the hydrogels prepared in Examples 1 and 2; wherein (a) is Example 1, and (b) is Example 2.

[0016] Figure 4 The sustained-release effect of sustained-release drugs prepared by the hydrogels prepared in Examples 1 and 2 is shown; wherein (a) is the ultraviolet absorption spectrum of ciprofloxacin hydrochloride; (b) is the standard curve of ciprofloxacin hydrochloride; (c) is the release curve of the sustained-release drug prepared by the hydrogel in Example 1 in PBS at 37°C; (d) is the release curve of the sustained-release drug prepared by the hydrogel in Example 2 in PBS at 37°C.

[0017] Figure 5 The MTT test results of the hydrogel prepared in Example 2; wherein (a) represents the effect of the simple hydrogel extract prepared in Example 2 with concentrations of 0, 12.5%, 25%, 50%, and 100% on the survival rate of NIH-3T3; (b) represents the effect of the drug-loaded hydrogel extract with concentrations of 0, 10%, 20%, 30%, 40%, and 50% on the survival rate of NIH-3T3.

[0018] Figure 6 The results of cell scratch test of the hydrogel prepared in Example 2; wherein, (a) and (c) respectively represent the cell migration rates of NIH-3T3 cells co-cultured with 0, 12.5%, 25%, 50%, and 100% simple hydrogel extract for 24 hours; (b) and (d) respectively represent the cell migration rates of NIH-3T3 cells co-cultured with 0, 10%, 20%, 30%, 40%, and 50% drug-loaded hydrogel extract for 24 hours.

[0019] Figure 7 The hemolysis test results of the hydrogel prepared in Example 2.

[0020] Figure 8 9.4T MRI imaging of mouse gums; (a) is the MRI image of mouse gums; (b) is the quantitative image of mouse gum thickness.

[0021] Fig. 9 Evaluation of periodontal bone defects and H&E staining of gingival tissue under a stereomicroscope; (a) is a schematic diagram of the alveolar bone; (b) is the H&E staining of gingival tissue; (c) is a quantitative image from the lowest point of the alveolar bone to the cementoenamel boundary; the shooting magnification is 20X and the scale is 200μm.

[0022] Fig.10 It is used to evaluate the safety of various organs (heart, liver, spleen, lung, kidney, and intestine) of mice. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] The "%" described in the present invention, unless otherwise specified, refers to mass percentage.

[0029] The first aspect of the present invention provides a topical drug sustained-release carrier (hydrogel), wherein the raw materials include, by mass percentage: 0.5%-5% gellan gum, 0.5%-3% carbomer, 0.5%-5% carboxylated chitosan, 0.01%-0.3% cationic salt, 0.02%-0.2% neutralizer and the balance water.

[0030] In some embodiments of the present invention, the raw materials of the topical drug sustained-release carrier (hydrogel) include, by mass percentage: 0.5% to 1.5% gellan gum, 1% to 2% carbomer, 0.5% to 1.5% carboxylated chitosan, 0.035% to 0.045% cationic salt, 0.03% to 0.05% neutralizer and the balance water.

[0031] In the present invention, if the concentration of the cationic salt is too high, the hydrogel will solidify too quickly, the network structure will not be fully formed, and it will be unfavorable for the sustained release of the drug; if the concentration of the cationic salt is too low, the gel cannot be formed.

[0032] The present invention also verifies that, when the total concentration of carboxylated chitosan and gellan gum is fixed, the ratio of carboxylated chitosan and gellan gum is adjusted to 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1. The results show that when the ratio of carboxylated chitosan and gellan gum is 1:9, 2:8, 3:7, 4:6, uneven polyelectrolyte formation occurs due to electrostatic interaction, and when the ratio of carboxylated chitosan and gellan gum is 8:2, 9:1, gel cannot be formed. Only when the ratio of carboxylated chitosan and gellan gum is 5:5, 6:4, 7:3, a relatively stable hydrogel can be obtained.

[0033] In some embodiments of the present invention, the cationic salt is at least one of calcium chloride, potassium chloride, sodium chloride and magnesium chloride.

[0034] In some embodiments of the present invention, the neutralizing agent is one of triethanolamine, sodium hydroxide and sodium bicarbonate.

[0035] In some embodiments of the present invention, the raw material further comprises 0.1% to 0.3% epigallocatechin gallate, calculated by mass percentage.

[0036] In the present invention, chitosan and gellan gum are natural cationic polysaccharides or cationic modified polysaccharides, and hydrogen bonds or covalent bonds can be formed between the two, which can produce electrostatic adsorption with mucosal skin tissue. Hydrogen bonding is the main reason for the adhesion of most polysaccharide molecules to mucosa. There are a large number of hydroxyl groups, carboxyl groups and amino groups on its molecules, which can form hydrogen bonds with tissue surface mucins and glycoproteins. Among them, gellan gum is divided into high acyl gellan gum and low acyl gellan gum. High acyl gellan gum is more brittle and easy to break than low acyl gellan gum, while low acyl gellan gum is more resilient than high acyl gellan gum. According to the requirements of the present invention, low acyl gellan gum with more resilient and stronger compressive mechanical strength is selected. Carboxylated chitosan has its own antibacterial and anti-inflammatory effects, and carbomer is a high molecular polymer hydrogel. The combination with polysaccharide hydrogel makes its structure more stable, improves the mechanical properties to a certain extent, and makes it difficult to break and cause drug burst release when facing pH, ionic strength, and temperature changes. Cationic salts and neutralizers can adjust pH to trigger gel formation.

[0037] In a further preferred technical solution of the present invention, the raw materials also include epigallocatechin gallate (EGCG). The EGCG molecule contains a large number of hydroxyl groups, which can form hydrogen bonds with mucin and glycoprotein on the tissue surface to further improve the adhesion of the hydrogel. Its combination with chitosan and gellan gum can further improve the sustained release effect of the drug.

[0038] The invention can form a hydrogel under the triggering of appropriate cation and pH conditions and has a microscopic porous structure.

[0039] The second aspect of the present invention provides a method for preparing the above-mentioned local drug sustained-release carrier, comprising the following steps: Adding gellan gum to a portion of water to dissolve it to obtain a gellan gum solution, and adding carboxylated chitosan to the gellan gum solution to obtain component A; After adding carbomer to the remaining water for swelling, component B is obtained; The component A and the component B are mixed evenly, and then the cationic salt and the neutralizer are added and mixed evenly to obtain the topical drug sustained-release carrier; When the raw material contains epigallocatechin gallate, epigallocatechin gallate and carboxylated chitosan are added to the gellan gum solution at the same time to obtain component A.

[0040] In some embodiments of the present invention, the temperature for dissolving gellan gum by adding it into a portion of water is 90°C.

[0041] The third aspect of the present invention provides a use of the above-mentioned local drug sustained-release carrier in the preparation of sustained-release drugs.

[0042] The fourth aspect of the present invention provides a method for preparing a sustained-release drug, comprising mixing a drug solution with the above-mentioned local drug sustained-release carrier and then shaking at a constant temperature to obtain the sustained-release drug.

[0043] In some embodiments of the present invention, the volume ratio of the drug solution to the local drug sustained-release carrier is 1:1; and the concentration of the drug solution is 3 mg / mL.

[0044] The solvent of the drug solution is PBS buffer (pH range 7.0-7.4); The drug in the drug solution can be an antibiotic drug, such as ciprofloxacin hydrochloride.

[0045] In some embodiments of the present invention, the temperature of the constant temperature shaking is 37° C. and the time is 24 hours.

[0046] The fifth aspect of the present invention provides a sustained-release drug prepared by the above preparation method.

[0047] Unless otherwise specified, the raw materials and reagents used in the examples of the present invention can be obtained through commercial sources.

[0048] The carboxylation degree of the carboxylated chitosan used in the examples of the present invention is 80%.

[0049] The gellan gum used in the embodiments of the present invention is low acyl gellan gum powder.

[0050] The cationic salt used in the embodiment of the present invention is calcium chloride.

[0051] The neutralizing agent used in the embodiment of the present invention is sodium hydroxide.

[0052] The pH value of the PBS buffer used in the embodiments of the present invention is 7.0.

[0053] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0054] Example 1

[0055] A topical drug sustained-release carrier comprises, by weight percentage, 1% gellan gum, 1.5% carbomer, 1% carboxylated chitosan, 0.04% cationic salt, 0.04% neutralizer and the balance deionized water.

[0056] The preparation method of the above-mentioned local drug sustained-release carrier comprises the following steps: Disperse low acyl gellan gum powder in 1 / 2 volume of deionized water, heat to 90°C until completely dissolved, disperse carboxylated chitosan in the above solution, stir thoroughly to dissolve, and obtain component A; Disperse carbomer evenly in the remaining amount of deionized water, fully swell and stir thoroughly to obtain component B; Components A and B are mixed and stirred evenly to obtain a mixed solution; a neutralizer and a cationic salt are added to the mixed solution and stirred thoroughly, and the mixed solution changes from a liquid to a gel, thereby obtaining a local drug sustained-release carrier.

[0057] Comparative Example 1 The only difference from Example 1 is that the added amount of gellan gum is 0.5%, and the added amount of carbomer is 0.5%; the remaining steps and parameters are the same as those in Example 1.

[0058] Comparative Example 2 The only difference from Example 1 is that the added amount of carbomer is 0.5%; the remaining steps and parameters are the same as those in Example 1.

[0059] Comparative Example 3 The only difference from Example 1 is that the added amount of gellan gum is 1.5%, and the added amount of carbomer is 0.5%; the remaining steps and parameters are the same as those in Example 1.

[0060] Example 2

[0061] A topical drug sustained-release carrier comprises, by weight percentage, 1% gellan gum, 1.5% carbomer, 1% carboxylated chitosan, 0.04% cationic salt, 0.04% neutralizer, 0.2% EGCG and the remainder deionized water.

[0062] The preparation method of the above-mentioned local drug sustained-release carrier comprises the following steps: Disperse low acyl gellan gum powder in 1 / 2 amount of deionized water, heat to 90°C until completely dissolved, disperse carboxylated chitosan and EGCG in the above solution, stir thoroughly to dissolve, and obtain component A; Disperse carbomer evenly in the remaining amount of deionized water, fully swell and stir thoroughly to obtain component B; Components A and B are mixed and stirred evenly to obtain a mixed solution; a neutralizer and a cationic salt are added to the mixed solution and stirred thoroughly, and the mixed solution changes from a liquid to a gel, thereby obtaining a local drug sustained-release carrier.

[0063] Figure 2 The SEM image of the local drug sustained-release carrier prepared in this example is Figure 2 It can be seen that the pore size of the local drug sustained-release carrier prepared in this example is about 6 μm.

[0064] Effect verification example The effects of the local drug sustained-release carriers (hydrogels) prepared in Example 1 and Example 2 were verified as follows: 1. Inverted vial test Place the hydrogel in a glass bottle and tilt it to observe the degree of solidification of the hydrogel.

[0065] The results show that when the addition amount of gellan gum and carbomer is 0.5% (i.e., Comparative Example 1), the raw material concentration is low, the prepared hydrogel is in a flowing state, and the hydrogel is not solidified; when the gellan gum concentration is 1% and 1.5%, and the carbomer concentration is 0.5% (i.e., Comparative Example 2, Comparative Example 3), the hydrogel is in a semi-solidified state, and the mechanical strength of the hydrogel is weak.

[0066] The hydrogels prepared in Example 1 and Example 2 solidified completely and had good mechanical strength. Figure 1 As shown; Figure 1 (a) is the hydrogel prepared in Example 1, Figure 1 (b) is the hydrogel prepared in Example 2.

[0067] 2. Rheological test The measurements were carried out using a MARS 40 rheometer.

[0068] Determination mode: Rotational continuous mode scanning. Spread the sample hydrogel on the lower plate with a plate diameter of 20 mm, adjust the plate spacing to 1 mm, test temperature to 25°C, and test frequency to 1-100 / s. Draw a graph of hydrogel viscosity changes with frequency as the horizontal axis and viscosity as the vertical axis.

[0069] Measurement mode: Oscillation frequency mode scanning. The sample hydrogel is spread flat on the lower plate with a plate diameter of 20 mm, the plate spacing is adjusted to 1.0 mm, the test temperature is 25°C, and the test frequency is 1-10 rad / s. The modulus diagram of the hydrogel is plotted with frequency as the horizontal axis and storage modulus (G′) and loss modulus (G″) as the vertical axis. As the shear rate increases, the viscosity of the hydrogel gradually decreases, indicating that the hydrogel has shear-thinning and injectable properties.

[0070] The storage modulus (G′) represents the elastic properties of the hydrogel, and the loss modulus (G″) represents the viscosity. At the same frequency, the G′ value is much higher than the G″ value, which is an inherent property of the hydrogel and indicates that the gel has been formed. Usually within the frequency range, G′ is much larger than G″ when the system is slightly deformed, and G′ is independent of the frequency. When the concentration of gellan gum and carbomer is 0.5%, G′<G″, indicating that the hydrogel is not formed at this concentration and the system is in a liquid state. A comprehensive comparison of Example 1, Example 2 and Comparative Examples 1-3 showed that the mechanical strength of the hydrogel was stronger when the concentration of gellan gum was 1% and the concentration of carbomer was 1.5%, so subsequent experiments were based on this concentration.

[0071] The rheological properties of the hydrogels prepared in Example 1 and Example 2 are as follows: Figure 3 As shown; wherein, (a) is Example 1, and (b) is Example 2.

[0072] 3. Degradation in the body A volume of 200 μL of hydrogel was injected subcutaneously on the back of mice. The weight of the hydrogel was first evaluated by measuring the difference in weight of the syringe before and after injection. After 3 and 14 days, the skin in contact with the hydrogel was peeled off and the remaining hydrogel sample was weighed.

[0073] Remaining weight ratio = (remaining hydrogel after degradation / initial hydrogel) × 100% The hydrogel prepared in Example 1 had a residual weight ratio of 47.1% on the 3rd day and 25.2% on the 14th day. The hydrogel prepared in Example 2 had a residual weight ratio of 46.4% on the 3rd day and 20.7% on the 14th day. This indicates that the hydrogel has good in vivo degradation ability. Comparing Example 1 and Example 2, it can be seen that the hydrogel with EGCG added in Example 2 has better degradation ability in vivo, which may be due to the presence of EGCG promoting the degradation of the hydrogel.

[0074] 4. In vitro swelling rate of hydrogel Considering that a large swelling rate of the hydrogel may cause tissue compression, the present invention tested the swelling rates of the hydrogels prepared in Example 1 and Example 2. The test method is as follows: (1) The prepared hydrogel was transferred into an EP tube, and the initial hydrogel weight Wi was evaluated by measuring the weight difference of the EP tube before and after loading the hydrogel. Then, PBS was added and the tube was placed in a 37 °C constant temperature oven. (2) After 6, 12, 24, and 48 hours, the PBS liquid in the tube was removed, and the surface water of the hydrogel was absorbed as much as possible, and then Wt was weighed, and fresh PBS was replaced regularly. The swelling rate (%) of the hydrogel was calculated using the following formula: Swelling rate (%) of the hydrogel = (Wt-Wi) / Wi×100%. The results showed that at 25 min, the swelling rate of the hydrogel prepared in Example 1 was 11.5%, and the swelling rate of the hydrogel prepared in Example 2 was 7.3%. The results showed that the overall swelling rate of the hydrogels prepared in Examples 1 and 2 was low, which can reduce the adverse effects of water swelling after the gel is used in the humid environment of the oral cavity.

[0075] 5. Drug release The steps for preparing the hydrogel loaded with ciprofloxacin (sustained release drug) are as follows: Ciprofloxacin hydrochloride was dissolved in PBS buffer to prepare a 3 mg / mL ciprofloxacin hydrochloride solution; the ciprofloxacin hydrochloride solution was mixed with the hydrogels prepared in Example 1 and Example 2 at a volume ratio of 1:1, and then placed on a magnetic stirrer for stirring and mixing, and then shaken in a 37° C. incubator for 24 hours; and a hydrogel loaded with ciprofloxacin was obtained.

[0076] Take 0.5 mL of ciprofloxacin-loaded hydrogel and place it in 20 mL of PBS. Place it on a shaker at 37°C and 100 rpm and shake slowly. Take 0.5 mL of the supernatant at 1, 2, 4, 6, 12, 24, and 48 hours, add the same amount of fresh PBS, and use UV-Vis to detect the drug release. Data processing is performed according to the following formula: , Q: cumulative drug release; Cn: drug concentration at time t; Vt: total volume of release medium; Vs: volume of medium removed each time.

[0077] Figure 4 The sustained release of the sustained-release drug, wherein (a) is the ultraviolet absorption spectrum of ciprofloxacin hydrochloride; (b) is the standard curve of ciprofloxacin hydrochloride; (c) is the release curve of the sustained-release drug prepared by the hydrogel in Example 1 in PBS at 37°C; (d) is the release curve of the sustained-release drug prepared by the hydrogel in Example 2 in PBS at 37°C. Figure 4It can be seen that the sustained-release drugs prepared using the hydrogels in Examples 1 and 2 both have good sustained-release effects, and the sustained-release effect of Example 2 is better than that of Example 1.

[0078] 6. Experiments at the cell level The hydrogel used in the cell level related experiments is the hydrogel prepared in Example 2; the drug-loaded hydrogel is the hydrogel in Example 2 loaded with ciprofloxacin, and the preparation method is the same as the above-mentioned "5. Drug release".

[0079] 6.1 MTT assay Good cell compatibility of materials is a prerequisite for biomedical applications. In order to verify the safety of the extract containing or not containing ciprofloxacin hydrochloride hydrogel (hydrogel prepared in Example 2) on NIH-3T3 cell line, the extracts of different concentrations were incubated with NIH-3T3 for 24 hours. The results are shown in Figure 2. Figure 5 As shown, Figure 5 (a) shows the effect of the simple hydrogel extract prepared in Example 2 on the survival rate of NIH-3T3 at concentrations of 0, 12.5%, 25%, 50%, and 100% respectively; Figure 5 (b) shows the effects of drug-loaded hydrogel extracts at concentrations of 0, 10%, 20%, 30%, 40%, and 50% on the survival rate of NIH-3T3. Figure 5 It can be seen that the cell survival rates of the 100% hydrogel extract group and the 50% drug-loaded hydrogel extract group were both over 90%.

[0080] 6.2 Cell scratch assay The cell scratch test is a simple method to measure cell migration and repair ability. The test method used is as follows: (1) Marking the culture plate: Use a marker pen and a ruler to draw horizontal lines on the back of the 6-well plate. Make at least 5 lines across each well, and each line should be even and parallel.

[0081] (2) Cell plating: Take NIH-3T3 cells in the logarithmic growth phase, digest the cells, centrifuge them, and resuspend them at 5-10×10 5 The cells were inoculated into 6-well plates at a density of 100 cells / well, 2 mL of complete medium was added to each well, and the cell culture plates were placed in a 37°C, 5% CO2 incubator for overnight culture.

[0082] (3) Cell streaking: When the cells in each well are evenly attached to the wall and cover the bottom of the well, use a sterilized 200 μl pipette tip to draw a line perpendicular to the black marking line on the back of the well plate, so that the scratch intersects with the marking line. Note: It is best to use the same pipette tip for different wells and keep the force consistent. When streaking, the pipette tip should be vertical and not tilted.

[0083] (4) Wash the cells and remove the scratched cells: After the streaking is completed, wash the cells 2-3 times with sterile PBS. Be careful to add the solution slowly to avoid breaking up the monolayer of adherent cells. Remove the scratched cells and make the gaps left clearly visible to the naked eye.

[0084] (5) Drug treatment: Add 2 mL of a mixed solution of hydrogel extract with or without ciprofloxacin and serum-free culture medium at different concentrations to each well.

[0085] (6) Photo observation at 0 hours: Observe the cell scratch state at 0 hours after drug addition under an inverted microscope, and then place the cell culture plate in an incubator at 37°C and 5% CO2 for 24 hours.

[0086] (7) 24-hour photographic observation: After the hydrogel extract and cells were incubated for 24 hours, the cells were observed and photographed under an inverted microscope.

[0087] (8) Data analysis: The scratch area at 0 hours and 24 hours was compared. The images were analyzed and processed using Image J software, and the calculation formula was as follows: Cell migration rate (%) = (0 hour scratch area - 24 hour scratch area) / 0 hour scratch area × 100%.

[0088] The results are as follows Figure 6 As shown, Figure 6 (a) and (c) show the cell migration rate of NIH-3T3 cells co-cultured with 0, 12.5%, 25%, 50%, and 100% simple hydrogel extract for 24 hours, respectively; Figure 6 (b) and (d) show the cell migration rate of NIH-3T3 cells co-cultured with drug-loaded hydrogel extract at concentrations of 0, 10%, 20%, 30%, 40%, and 50% for 24 hours. The shooting magnification is 4X, and the scale bar is 200 μm. Figure 6 It can be seen that 100% simple hydrogel extract and 50% drug-loaded hydrogel extract had no significant effect on the migration of NIH-3T3 cells, and there was no statistically significant difference in the cell migration rate among the groups (P>0.05), indicating that the hydrogel had no side effects on the migration and healing of NIH-3T3 cells over time.

[0089] 6.3 Hemolysis test To further verify the biosafety of the hydrogel, whole blood from healthy mice was collected and subjected to hemolytic analysis. Figure 7 As shown, from left to right are the positive control group, negative control group, 12.5%, 25%, and 50% extract groups. From the digital photos, it can be seen that the blood cells in the hydrogel extract group did not undergo hemolysis, and the hemolysis rate was less than 5%. The above results all confirm that both the drug-loaded hydrogel and the simple hydrogel exhibit good biocompatibility.

[0090] 7. Animal-related experiments The drug-loaded hydrogel used in this animal-related experiment is the same as the drug-loaded hydrogel in “6. Cell-level related experiments”.

[0091] 7.1 9.4T MRI imaging of mouse gingiva In order to evaluate the efficacy of drug-loaded hydrogels in vivo, a periodontitis model was established by ligating non-absorbable surgical sutures on the maxillary second molars of mice. From the results of "5. Drug release", it can be seen that the drug-loaded hydrogels showed the ability of sustained release in vitro in a simulated human environment. Therefore, the anti-inflammatory effects of Hydrogel+CIP and CIP in mice were evaluated by local administration. A major feature of periodontitis is gingival edema, and the edema is indirectly reflected by measuring the thickness of the gums. MRI scans were performed on days 0, 14, and 35 of mouse modeling. Taking into account the differences in initial gingival thickness of different mice, the gingival thickness on day D0 was recorded as 100%, and days D14 and D35 were compared with D0. The results are shown in Figure 8 As shown in the figure (in the figure, Hydrogel+CIP represents drug-loaded hydrogel; CIP represents ciprofloxacin; control represents the healthy control experimental group; Periodontitis represents the periodontitis modeling experimental group). The operation method is as follows: The experimental mice in the Control group, Periodontitis group, Hydrogel+CIP group and CIP group were subjected to MRI imaging scans on days 0, 14 and 35 after modeling. Periodontitis can cause gingival swelling. The thickness of the gums is a surrogate indicator of edema. The level of inflammation is assessed by measuring the thickness of the gums. (1) Bruker 9.4 T Micro MR is started and the 1H magnetic resonance body coil is prepared. The respiratory gating, automatic temperature control system and small animal anesthesia machine are turned on; (2) The mouse is placed in a prone position, the upper jaw of the mouse is placed in the center of the coil, and the tongue of the mouse is fixed to avoid blocking the area of ​​interest. During the scanning process, the mice were continuously inhaled of isoflurane anesthetic to maintain an anesthetized state, with body temperature controlled at 37°C and respiratory rate at 20-30 times / min. (3) The experimental mice were continuously scanned in the axial position using T1WI and T2WI sequences, and the thickness of the gums on both sides of the maxillary molars was measured after the images were acquired.

[0092] like Figure 8 As shown in (a) and (b), on the 35th day, the gingival thickness of the periodontitis group was 170.67% of the initial thickness, and the gingival thickness of the Hydrogel+CIP group was 103.67% of the initial thickness, which can effectively reduce gingival edema, indicating that the local delivery system can increase the local drug concentration, prolong the sustained release of drugs, and increase the therapeutic efficacy.

[0093] 7.2 Stereomicroscope evaluation of periodontal bone defects and H&E staining of gingival tissue Periodontitis is essentially an inflammatory bone defect disease caused by bacteria. Bone defects were induced by tying a silk ligature on the maxillary second molar. The mice were killed on the 35th day, and the maxillary periodontal tissues on both sides were taken and the surrounding soft tissues were removed. The distance from the lowest point of the alveolar bone to the enamel-cementum junction (tooth neck) was measured under a stereomicroscope to evaluate the periodontal bone loss of the maxillary second molar. Fig. 9 As shown in (a) and (c), the distance from the lowest point of the alveolar bone to the cementoenamel junction in the periodontitis group was approximately 0.549±0.04782 mm, while that in the Hydrogel+CIP group was 0.3413±0.03109 mm. This indicates that the Hydrogel+CIP group can reduce local inflammation and delay bone loss. H&E staining was performed to further evaluate the inflammatory response of each experimental group from a histological perspective. Fig. 9 (b) shows the H&E staining of gingival tissue. The periodontitis group showed changes in epithelial structure. The epithelial spikes extended toward the connective tissue and were serrated. A large number of inflammatory cells infiltrated the connective tissue of the lamina propria. In the treatment group, the epithelial spikes did not extend significantly and the number of inflammatory cells was also relatively small.

[0094] 7.3 Biosafety Assessment In order to evaluate the biosafety of the drug-loaded hydrogel, the important organs of the mice were removed from each experimental group after treatment and analyzed by H&E staining. Fig.10 As shown in the results of H&E staining of tissue sections of the Control group, Periodontitis group, Hydrogel+CIP group and CIP group, no obvious organ damage and pathological changes were found in the heart, liver, spleen, lung, kidney and intestine of mice, indicating that local administration of drug-loaded hydrogel had no obvious damage to various organs of mice and had good biosafety. These in vivo safety tests all show that the injectable drug delivery system has good biocompatibility and biosafety.

[0095] 8. Antibacterial test The hydrogel after UV sterilization (hydrogel prepared in Examples 1 and 2) or the hydrogel loaded with ciprofloxacin (sustained-release drug prepared using the hydrogel in Examples 1 and 2, the preparation method is the same as "5. Drug release") was soaked in PBS at 37°C for 24 hours to obtain a hydrogel extract containing ciprofloxacin or not containing ciprofloxacin (200 mg / mL). The experiment was divided into 5 groups: Control group, simple hydrogel group in Example 1, simple hydrogel group in Example 2, ciprofloxacin-containing drug group in Example 1, and ciprofloxacin-containing drug group in Example 2. Then, the drug-containing filter paper was made, and 20 μl of the extract of different components was dropped on the blank drug-sensitive paper, and it was dried after being fully absorbed; the bacterial solution was dipped with a sterile cotton swab, and a zigzag line was drawn along the starting position above the LB solid culture medium to evenly apply the bacterial solution. After the bacterial solution was slightly dried, the dried drug-sensitive paper was attached to the pre-marked position and gently pressed to prevent the paper from falling after inversion. The plate was then inverted and cultured in a 37°C incubator for 16-20 hours. The presence of an inhibition zone around the filter paper was observed and the diameter of the inhibition zone was measured using a ruler. The results are summarized in Table 1.

[0096] Table 1 , The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A local drug sustained-release carrier, characterized in that: Calculated by mass percentage, the raw materials include: 0.5% to 5% of gellan gum, 0.5% to 3% of carbomer, 0.5% to 5% of carboxylated chitosan, 0.01% to 0.3% of cationic salt, 0.02% to 0.2% of neutralizer and the balance of water; The cationic salt is one of calcium chloride, potassium chloride, sodium chloride and magnesium chloride; The neutralizing agent is one of ammonia water, triethanolamine, sodium hydroxide and sodium bicarbonate.

2. The topical drug sustained-release carrier according to claim 1, characterized in that: Calculated by mass percentage, the raw materials also include 0.1% to 0.3% of epigallocatechin gallate.

3. A method for preparing the local drug sustained-release carrier according to claim 1 or 2, characterized in that: The following steps are involved: Adding gellan gum to a portion of water to dissolve it to obtain a gellan gum solution, and adding carboxylated chitosan to the gellan gum solution to obtain component A; After adding carbomer to the remaining water for swelling, component B is obtained; The component A and the component B are mixed evenly, and then the cationic salt and the neutralizer are added and mixed evenly to obtain the topical drug sustained-release carrier; When the raw material contains epigallocatechin gallate, epigallocatechin gallate and carboxylated chitosan are added to the gellan gum solution at the same time to obtain component A.

4. Use of the local drug sustained-release carrier according to claim 1 or 2 in the preparation of sustained-release drugs.

5. A method for preparing a sustained-release drug, characterized in that: The drug solution is mixed with the local drug sustained-release carrier according to claim 1 or 2 and then shaken at a constant temperature to obtain the sustained-release drug; the drug solution is a ciprofloxacin solution.

6. The sustained-release drug prepared by the preparation method according to claim 5.

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