阅读说明：本技术 一种白藜芦醇原位凝胶制备及其表征方法 (Resveratrol in-situ gel preparation and characterization method thereof ) 是由 李磊 范郁琳 梁梦秋 鞠婉婷 于 2021-07-21 设计创作，主要内容包括：本发明提供一种白藜芦醇原位凝胶制备及其表征方法。涉及一种药用凝胶制备及其表征方法,该方法在配置凝胶溶液时,以泊洛沙姆407、泊洛沙姆188为载体材料,并以溶液的胶凝温度以及载药凝胶的胶凝温度为参数,对空白凝胶的处方和工艺进行优化,得到最优处方空白凝胶为3.705ml、浓度为21.4%的泊洛沙姆407,以及1.295ml、浓度为5%的泊洛沙姆188,制得凝胶温度接近于体温,并基于最优处方空白凝胶制得白藜芦醇原位凝胶,弥补了白藜芦醇水溶性差的缺点,同时,表征证明制得的白藜芦醇原位凝胶在临床治疗中更适于应用。(The invention provides a preparation method and a characterization method of resveratrol in-situ gel. The method comprises the steps of optimizing a prescription and a process of the white gel by using poloxamer 407 and poloxamer 188 as carrier materials and using the gelling temperature of the solution and the gelling temperature of the medicine-carrying gel as parameters when a gel solution is prepared, obtaining the poloxamer 407 with the concentration of 21.4% and the poloxamer 188 with the concentration of 5% in an optimal prescription, wherein the blank gel is 3.705ml, the temperature of the prepared gel is close to the body temperature, and the resveratrol in-situ gel is prepared based on the blank gel in the optimal prescription, so that the defect of poor water solubility of the resveratrol is overcome.)

1. The preparation method of the resveratrol in-situ gel is characterized by comprising the following preparation steps:

weighing 1.25g of poloxamer 188, and dissolving in a 25ml beaker;

weighing 10.7g of poloxamer 407 and dissolving in a 50ml beaker;

after the poloxamer 188 and the poloxamer 407 are completely dissolved respectively, fixing the volume and swelling;

1.295ml of poloxamer 188 with the concentration of 5 percent and 3.705ml of poloxamer 407 with the concentration of 21.4 percent are respectively measured by a pipette and mixed in a 7ml penicillin bottle;

under the ice bath condition, stirring poloxamer 188 and poloxamer 407 in a penicillin bottle by adopting a heat-collecting constant-temperature heating magnetic stirrer, uniformly mixing the poloxamer 188 and the poloxamer 407, and standing to obtain blank gel with an optimal prescription;

respectively weighing 0.0005g, 0.0010g and 0.0015g of resveratrol in a 7ml penicillin bottle;

respectively adding 5ml of the blank gel with the optimal prescription into the resveratrol, stirring the resveratrol in a penicillin bottle and the blank gel with the optimal prescription by adopting a heat collection type constant temperature heating magnetic stirrer under the ice bath condition, uniformly mixing the resveratrol and the blank gel with the optimal prescription, and standing to respectively obtain resveratrol in-situ gel with the concentration of 0.0001g/ml, 0.0002g/ml and 0.0003 g/ml.

2. The method for preparing resveratrol in-situ gel according to claim 1, wherein poloxamer 188 and poloxamer 407 are fully dissolved respectively, then the volume is determined and the swelling is carried out, and the swelling is carried out for 12h at 4 ℃.

3. The method for preparing resveratrol in-situ gel according to claim 1, characterized in that in the step of stirring poloxamer 188 and poloxamer 407 in a penicillin bottle by using a heat-collecting constant-temperature heating magnetic stirrer under an ice bath condition, so that poloxamer 188 and poloxamer 407 are uniformly mixed and stand to obtain blank gel, poloxamer 188 and poloxamer 407 in a penicillin bottle are stirred for 15min at 20r/min by using a heat-collecting constant-temperature heating magnetic stirrer under an ice bath condition.

4. The method for preparing resveratrol in-situ gel according to claim 1, characterized in that 5ml of blank gel is added into the resveratrol respectively, the resveratrol in a penicillin bottle and the blank gel with the optimal prescription are stirred by a heat collection type constant temperature heating magnetic stirrer under ice bath condition, so that the resveratrol and the blank gel with the optimal prescription are uniformly mixed and stand to obtain the resveratrol in-situ gel of 0.0001g/ml, 0.0002g/ml and 0.0003 g/ml respectively, and the resveratrol in the penicillin bottle and the blank gel with the optimal prescription are stirred by the heat collection type constant temperature heating magnetic stirrer at 20r/min for 15min under ice bath condition.

5. A detection method of in situ resveratrol gel, which is applied to the in situ resveratrol gel of any one of claims 1-4, and is characterized in that the detection method comprises the following processes: setting a constant-temperature water bath kettle, placing a penicillin bottle in the water bath kettle, adjusting the water temperature to rise by 1 ℃, inclining the penicillin bottle to observe the flowability of the resveratrol in-situ gel until the resveratrol in-situ gel does not flow any more, namely the gelation temperature; measuring the gel temperature of blank gel and resveratrol in-situ gel with the content of 0.0001g/ml, 0.0002g/ml and 0.0003 g/ml, recording the temperature, measuring each sample for 3 times, and taking the average value;

placing a penicillin bottle containing blank gel with an optimal prescription and resveratrol in-situ gel with the resveratrol content of 0.0003 g/ml in a constant-temperature water bath kettle at 25 ℃, quickly transferring to a constant-temperature water bath kettle at 37 ℃ after the penicillin bottle is stabilized, starting timing, recording the time required by the resveratrol in-situ gel from flowing to completely non-flowing, namely the gelling time, measuring each sample for 3 times, and taking an average value;

preparing 25ml of blank gel with an optimal prescription and resveratrol in-situ gel with the resveratrol content of 0.0003 g/ml, stirring for 15min under the ice bath condition by a heat collection type constant temperature heating magnetic stirrer at 20r/min, uniformly mixing the blank gel and the resveratrol, standing, extending a probe of a pH value measuring instrument into the solution, and reading data after the pH value is stable.

6. The characterization method of in-situ resveratrol gel according to claim 5, characterized in that 10.0mg of resveratrol standard is weighed and placed in a 50mL volumetric flask, dissolved and diluted to a scale with methanol, shaken up, 3mL of diluted solution is taken and put in a 25mL volumetric flask, constant volume is performed with methanol to obtain a first standard solution, 0, 1, 2, 3, 4 and 5mL of the first standard solution are absorbed, constant volume is performed with 25mL again to obtain 5 groups of second standard solutions, the light absorption values of the 5 groups of second standard solutions at 306nm are measured one by one, 3 times are measured, the average value is calculated, and a standard curve is drawn.

7. The characterization method of in situ resveratrol gel according to claim 6, wherein 1.5g of the in situ resveratrol gel is taken in a 50ml volumetric flask, dissolved in methanol and diluted to a scale, and mixed uniformly and shaken; taking 3mL of the solution after shaking up, putting the solution in a 25mL volumetric flask, diluting to a constant volume with methanol to obtain a third standard solution, sucking 2mL of the third standard solution, diluting to a constant volume of 25mL again, dissolving with methanol and diluting to a scale; and measuring the absorbance at the wavelength of 306nm, substituting into a standard curve regression equation, and calculating the content of the resveratrol.

8. The method for characterizing the in-situ gel of resveratrol as claimed in claim 7, wherein the method uses an NDJ-8S viscometer with a number 3 rotor, a rotation speed of 60rpm/min, a viscosity range: 10-200000mPa.s, respectively placing the blank gel and the resveratrol in-situ gel of the optimal formula into a sample bottle to be measured, keeping the liquid level of the solution to be submerged to the scale mark of the liquid level of the rotor, measuring the viscosity at 25 ℃ and 37 ℃, measuring each sample for 3 times, and taking an average value.

9. The characterization method of in situ resveratrol gel according to claim 8, characterized in that the method comprises weighing an empty vial of 10mL penicillin bottles, adding 5g of in situ resveratrol gel, and placing in a water bath constant temperature oscillator at 37.0 ± 0.1 ℃ for balancing for 10min to allow the polymer solution to completely form gel; taking out the gel, adding 1mL phosphate buffer solution PBS at 37 ℃, taking the pH =7.4 as a release medium, then putting the gel into a water bath constant temperature oscillator, oscillating at the temperature of 37.0 +/-0.1 ℃ at the speed of 40r/min, pouring out all the release medium within a set time, wiping the bottle dry and weighing; then putting into a water bath constant temperature oscillator again for balancing for 10min, then supplementing 1mL of PBS solution at 37 ℃, repeating the steps until the amount of the remaining gel is less than 10% of the initial amount, setting 3 parallel groups for each experiment, recording the erosion time, and taking the average value.

10. The method for characterizing the in-situ gel of resveratrol according to claim 9, wherein 2 parts of the in-situ gel of resveratrol with a resveratrol content of 0.0003 g/ml are respectively stood at 25 ℃ and 37 ℃ for 15min, and then centrifuged at 2000r/min for 15min to observe whether unstable phenomena such as layering occur.

Technical Field

The invention relates to preparation of medicinal gel and a characterization method thereof, in particular to preparation of resveratrol in-situ gel and a characterization method thereof.

Background

Lung cancer is a tumor that poses a great threat to humans, and among many cancers, lung cancer is the leading cause of death in humans. According to survey data, the incidence and fatality rates of the disease are increasing year by year. The data show that the occurrence and close relationship of long-term smoking and lung cancer. Meanwhile, high-risk occupations are closely related to lung cancer, and the patients are more likely to have extrapulmonary lesions and respiratory diseases, so that the treatment is more difficult and the life and health are seriously harmed. At present, chemotherapy, radiotherapy, surgery and the like are the main means for treating lung cancer, but the prognosis is not satisfactory. At present, the incidence and the fatality rate of lung cancer are the first in the world.

Hydrogels have good biocompatibility because they are structurally similar to soft tissue and can absorb large amounts of interstitial fluid and water. The temperature-sensitive hydrogel is one of in-situ hydrogels which are widely researched at present, can respond to the change of the external environment temperature in time, realizes the phase transformation of solution-gel, and has important application value in the aspects of biological medicine, tissue engineering, cell engineering and the like. Particularly in the aspect of drug controlled release, the three-dimensional network structure of the temperature-sensitive hydrogel is beneficial to improving the drug loading capacity. With the degradation or erosion of the gel in vivo, the drug is slowly released, the first pass effect of the drug can be avoided, and the residence time of the drug in vivo is prolonged, thereby achieving the purpose of continuous administration. In addition, some protein drugs are easily degraded by enzymes after entering an organism, so that the drug effect cannot be fully exerted, and the temperature-sensitive hydrogel is used as a carrier of the protein drugs, so that the degradation speed of the protein drugs can be remarkably slowed down, and the bioavailability of the drugs is improved by virtue of a slow release effect.

Resveratrol (Resveratrol, Res) is a non-flavonoid polyphenol, exists in various plants, has a remarkable curative effect particularly in the aspect of treating tumors, also has biological pharmacological activities such as mutation resistance, antibiosis and anti-inflammation, cell apoptosis induction and the like, and has a wide clinical application prospect. However, resveratrol is poorly water soluble, which limits its use in clinical therapy.

Disclosure of Invention

The invention aims to provide a preparation method and a characterization method of resveratrol in-situ gel.

The purpose of the invention is realized by the following technical scheme:

a method for preparing resveratrol in-situ gel, the method comprises the following steps:

weighing 1.25g of poloxamer 188, and dissolving in a 25ml beaker;

weighing 10.7g of poloxamer 407 and dissolving in a 50ml beaker;

after the poloxamer 188 and the poloxamer 407 are completely dissolved respectively, fixing the volume and swelling;

1.295ml of poloxamer 188 with the concentration of 5 percent and 3.705ml of poloxamer 407 with the concentration of 21.4 percent are respectively measured by a pipette and mixed in a 7ml penicillin bottle;

under the ice bath condition, stirring poloxamer 188 and poloxamer 407 in a penicillin bottle by adopting a heat-collecting constant-temperature heating magnetic stirrer, uniformly mixing the poloxamer 188 and the poloxamer 407, and standing to obtain blank gel with an optimal prescription;

respectively weighing 0.0005g, 0.0010g and 0.0015g of resveratrol in a 7ml penicillin bottle;

respectively adding 5ml of the blank gel with the optimal prescription into the resveratrol, stirring the resveratrol in a penicillin bottle and the blank gel with the optimal prescription by adopting a heat collection type constant temperature heating magnetic stirrer under the ice bath condition, uniformly mixing the resveratrol and the blank gel with the optimal prescription, and standing to respectively obtain resveratrol in-situ gel with the concentration of 0.0001g/ml, 0.0002g/ml and 0.0003 g/ml.

According to the preparation method of the resveratrol in-situ gel, poloxamer 188 and poloxamer 407 are respectively completely dissolved, then the volume is determined, and swelling is carried out at 4 ℃ for 12 hours in the steps of volume determination and swelling.

According to the preparation method of the resveratrol in-situ gel, under the ice bath condition, the poloxamer 188 and the poloxamer 407 in the penicillin bottle are stirred by adopting a heat collection type constant temperature heating magnetic stirrer, so that the poloxamer 188 and the poloxamer 407 are uniformly mixed and stand to obtain a blank gel, and under the ice bath condition, the poloxamer 188 and the poloxamer 407 in the penicillin bottle are stirred by adopting the heat collection type constant temperature heating magnetic stirrer at the speed of 20r/min for 15 min.

The method for preparing the resveratrol in-situ gel comprises the steps of respectively adding 5ml of blank gel into resveratrol, stirring the resveratrol in a penicillin bottle and the blank gel with the optimal prescription by adopting a heat collection type constant temperature heating magnetic stirrer under an ice bath condition, uniformly mixing the resveratrol and the blank gel with the optimal prescription, standing, and respectively obtaining the resveratrol in-situ gel with the concentration of 0.0001g/ml, 0.0002g/ml and 0.0003 g/ml, wherein in the step of stirring the resveratrol in the penicillin bottle and the blank gel with the optimal prescription for 15min by adopting the heat collection type constant temperature heating magnetic stirrer at 20r/min under the ice bath condition.

A resveratrol in-situ gel characterization method applied to the resveratrol in-situ gel of any one of claims 1-4, the method comprising the following processes: setting a constant-temperature water bath kettle, placing a penicillin bottle in the water bath kettle, adjusting the water temperature to rise by 1 ℃, inclining the penicillin bottle to observe the flowability of the resveratrol in-situ gel until the resveratrol in-situ gel does not flow any more, namely the gelation temperature; measuring the gel temperature of blank gel and resveratrol in-situ gel with the content of 0.0001g/ml, 0.0002g/ml and 0.0003 g/ml, recording the temperature, measuring each sample for 3 times, and taking the average value;

placing a penicillin bottle containing blank gel with an optimal prescription and resveratrol in-situ gel with the resveratrol content of 0.0003 g/ml in a constant-temperature water bath kettle at 25 ℃, quickly transferring to a constant-temperature water bath kettle at 37 ℃ after the penicillin bottle is stabilized, starting timing, recording the time required by the resveratrol in-situ gel from flowing to completely non-flowing, namely the gelling time, measuring each sample for 3 times, and taking an average value;

preparing 25ml of blank gel with an optimal prescription and resveratrol in-situ gel with the resveratrol content of 0.0003 g/ml, stirring for 15min under the ice bath condition by a heat collection type constant temperature heating magnetic stirrer at 20r/min, uniformly mixing the blank gel and the resveratrol, standing, extending a probe of a pH value measuring instrument into the solution, and reading data after the pH value is stable.

Weighing 10.0mg of a resveratrol standard substance, placing the weighed substance in a 50mL volumetric flask, dissolving the substance with methanol, diluting the substance to a scale, shaking the substance uniformly, taking 3mL of diluted solution, placing the solution in a 25mL volumetric flask, metering the volume with methanol to obtain a first standard solution, sucking 0, 1, 2, 3, 4 and 5mL of the first standard solution, metering the volume with 25mL again to obtain 5 groups of second standard solutions, measuring the light absorption values of the 5 groups of second standard solutions at 306nm one by one, measuring the values for 3 times, calculating the average value, and drawing a standard curve.

The characterization method of the resveratrol in-situ gel comprises the steps of filling 1.5g of the resveratrol in-situ gel in a 50ml volumetric flask, dissolving the resveratrol in methanol, diluting the dissolved resveratrol to a scale, and uniformly mixing and shaking the dissolved resveratrol and the scale; taking 3mL of the solution after shaking up, putting the solution in a 25mL volumetric flask, diluting to a constant volume with methanol to obtain a third standard solution, sucking 2mL of the third standard solution, diluting to a constant volume of 25mL again, dissolving with methanol and diluting to a scale; and measuring the absorbance at the wavelength of 306nm, substituting into a standard curve regression equation, and calculating the content of the resveratrol.

The resveratrol in-situ gel characterization method adopts an NDJ-8S viscometer, adopts a rotor No. 3, has the rotating speed of 60rpm/min and the viscosity range: 10-200000mPa.s, respectively placing the blank gel and the resveratrol in-situ gel of the optimal formula into a sample bottle to be measured, keeping the liquid level of the solution to be submerged to the scale mark of the liquid level of the rotor, measuring the viscosity at 25 ℃ and 37 ℃, measuring each sample for 3 times, and taking an average value.

Weighing an empty bottle of a 10mL penicillin bottle, adding 5g of resveratrol in-situ gel, and placing the resveratrol in-situ gel in a water bath constant temperature oscillator at 37.0 +/-0.1 ℃ for balancing for 10min to enable a polymer solution to completely form gel; taking out the gel, adding 1mL phosphate buffer solution PBS at 37 ℃, taking the pH =7.4 as a release medium, then putting the gel into a water bath constant temperature oscillator, oscillating at the temperature of 37.0 +/-0.1 ℃ at the speed of 40r/min, pouring out all the release medium within a set time, wiping the bottle dry and weighing; then putting into a water bath constant temperature oscillator again for balancing for 10min, then supplementing 1mL of PBS solution at 37 ℃, repeating the steps until the amount of the remaining gel is less than 10% of the initial amount, setting 3 parallel groups for each experiment, recording the erosion time, and taking the average value.

The method for characterizing the resveratrol in-situ gel comprises the steps of standing 2 parts of resveratrol in-situ gel with the resveratrol content of 0.0003 g/ml for 15min at the temperature of 25 ℃ and 37 ℃, centrifuging at 2000r/min for 15min, and observing whether unstable phenomena such as layering exist.

The invention has the advantages and effects that:

according to the preparation method of the resveratrol in-situ gel, when a gel solution is prepared, poloxamer 407 and poloxamer 188 are used as carrier materials, the formula and the process of the blank gel are optimized by taking the gelation temperature of the solution and the gelation temperature of the drug-loaded gel as parameters, the poloxamer 407 with the concentration of 21.4% and the poloxamer 188 with the concentration of 5% are obtained, the optimal formula blank gel is 3.705ml, the prepared gel temperature is close to the body temperature, the resveratrol in-situ gel is prepared based on the optimal formula blank gel, the defect of poor water solubility of resveratrol is overcome, and meanwhile, the prepared resveratrol in-situ gel is proved to be more suitable for application in clinical treatment by characterization.

Drawings

FIG. 1 is a flow chart of the preparation method of resveratrol in-situ gel of the invention;

FIG. 2 shows the concentration of poloxamer 407 of the present invention versus the gelling temperature;

FIG. 3 is a graph of the effect of the independent variable A, B versus the dependent variable gel temperature of the present invention;

FIG. 4 is a line contour plot of independent A, B versus dependent gel temperature for the present invention;

FIG. 5 is a graph of the gelling temperature for different dosages of the present invention;

fig. 6 is a line graph of a standard curve of resveratrol according to the invention.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention. The present invention provides the following preferred embodiments.

As shown in fig. 1, an embodiment of the present invention provides a method for preparing resveratrol in-situ gel, including the following steps:

step one, 1.25g of poloxamer 188 was weighed and dissolved in a 25ml beaker.

Step two, weighing 10.7g of poloxamer 407 and dissolving in a 50ml beaker.

And step three, after the poloxamer 188 and the poloxamer 407 are completely dissolved respectively, fixing the volume and swelling for 12 hours at 4 ℃.

And step four, respectively measuring 1.295ml of poloxamer 188 with the concentration of 5% and 3.705ml of poloxamer 407 with the concentration of 21.4% by using a pipette, and mixing the measured substances in a 7ml penicillin bottle.

And fifthly, stirring poloxamer 188 and poloxamer 407 in the penicillin bottle for 15min at 20r/min by adopting a heat-collecting constant-temperature heating magnetic stirrer under the ice bath condition, uniformly mixing the poloxamer 188 and the poloxamer 407, and standing to obtain blank gel with the optimal prescription.

And step six, respectively weighing 0.0005g, 0.0010g and 0.0015g of resveratrol in a 7ml penicillin bottle.

And seventhly, respectively adding 5ml of the blank gel with the optimal prescription into the resveratrol, stirring the resveratrol in a penicillin bottle and the blank gel with the optimal prescription for 15min at 20r/min by adopting a heat collection type constant temperature heating magnetic stirrer under the ice bath condition, uniformly mixing the resveratrol and the blank gel with the optimal prescription, and standing to obtain the resveratrol in-situ gel with the concentration of 0.0001g/ml, 0.0002g/ml and 0.0003 g/ml.

According to the preparation method of the resveratrol in-situ gel provided by the embodiment of the invention, poloxamer 407 and poloxamer 188 are used as carrier materials, the prescription and the process of the blank gel are optimized, poloxamer 407 with the optimal prescription blank gel of 3.705ml and the concentration of 21.4% and poloxamer 188 with the concentration of 1.295ml and the concentration of 5% are obtained, the prepared gel has the temperature close to the body temperature, the resveratrol in-situ gel is prepared based on the optimal prescription blank gel, the defect of poor water solubility of resveratrol is overcome, and the resveratrol is more suitable for application in clinical treatment.

Wherein the optimal prescription blank gel is obtained by the following experiment:

and (4) performing preliminary experiments. Sample preparation: at present, the common method for preparing the gel is a cold dissolving method, and specifically, the method comprises the steps of grinding the medicine, mixing the ground medicine with distilled water, stirring the mixture in an ice-water mixture by using a magnetic stirrer, and placing the mixture in an environment with a constant volume of 4 ℃ until the solution is clarified after the medicine is completely dissolved. Firstly, 3.75g, 4.50g, 5.00g, 5.25g, 6.00g and 6.25g of poloxamer 407 are precisely weighed and dissolved in distilled water in a small 25ml beaker to prepare poloxamer 407 solution with the concentration of 15%, 18%, 20%, 21%, 24% and 25%, the medicine is dissolved according to the above operation, the volume is determined after the medicine is completely dissolved, the gelling temperature is measured after the medicine is placed in a refrigerator for refrigeration for one night, and the relationship between the concentration of the poloxamer 407 and the gelling temperature is shown in figure 2.

Secondly, accurately weighing 0.25g, 0.75g, 1.00g, 1.25g, 1.50g and 1.75g of poloxamer 188, dissolving the poloxamer 188 in distilled water in a small 25ml beaker to prepare a poloxamer 188 solution with the concentration of 1%, 3%, 4%, 5%, 6% and 7%, dissolving the medicine according to the above operation, fixing the volume after the medicine is completely dissolved, and placing the solution in a refrigerator for refrigeration overnight.

And (3) determining the gelling temperature: in evaluating temperature sensitive in situ gels, the temperature of the gel is an indispensable reference data. At present, researchers often use common visual methods and rheology as a method of determining the gel temperature. Visual inspection means that the vial is tilted with a slow increase in temperature, when the contents are not flowable, at which point the temperature is the gelation temperature. While this method is simple and intuitive, it is very limited because the solution-gel transition needs to occur over a period of time, and the final measured gelation temperature may be higher than the actual gelation temperature due to the gelation time.

According to the invention, a stirrer is not added when the gelation temperature is measured in an experiment, a penicillin bottle containing 5ml of gel solution is placed under a water bath to be heated slowly, the penicillin bottle is inclined at the temperature of 1 ℃ every time the penicillin bottle is raised, whether gel is formed or not is observed, and the gelation temperature is recorded by taking the temperature when the content cannot flow as the gelation temperature. Because the influence of observation factors is large in the experimental process, each sample is measured for three times, and the average value is taken, so that the error is reduced.

Single factor experiments. According to the experimental data, the gel temperature of poloxamer 407 is shown to decrease when the concentration of poloxamer 407 increases, which is a concentration-dependent trend. When the poloxamer 407 solution is used alone, the gel temperature is too low, the solution is not in a flowing liquid state at room temperature and is in a semisolid state at the body temperature, which is required by experiments, and the gel carrying the medicine is used at a focus when the temperature is lower, so that the human body is stimulated, and therefore, the poloxamer 188 which is the homologue thereof needs to be added to adjust the gel temperature to prepare blank gel with proper gel temperature so as to carry the medicine.

Concentration of poloxamer 188:

TABLE 1 gelling temperature at varying concentrations of Poloxamer 188

Concentration of poloxamer 407:

TABLE 2 gelling temperature at varying concentrations of poloxamer 407

Volume of poloxamer 188:

according to the two groups of single-factor experiments, the gelling temperature of the mixture of 21% poloxamer 407, 5% poloxamer 188 and 3% poloxamer in volume ratio is closest to the body temperature, the concentrations of the poloxamer 407 and the poloxamer 188 are fixed in the following single-factor experiments, the volume ratio of the poloxamer 407 to the poloxamer 188 is changed, and the optimal volume ratio is determined.

TABLE 3 gelling temperature with modification of poloxamer 188 concentration

TABLE 4 gelling temperature at fixed poloxamer 188 volumes, modified volume ratios

Total volume of poloxamer 407 and poloxamer 188:

in order to ensure that the phase transition temperature, the gelling time and the like of the gel are not influenced by the volume of the solution, the total volume of the fixed solution is 5ml, and the volume ratio of poloxamer 407 to poloxamer 188 is changed.

TABLE 5 Total volume fixed

Response surface experiment: design Expert 10 software was used to analyze the response surface data in this experiment. Design Expert 10 is a specialized piece of experiment Design software with a high level of expertise. By selecting a proper design method, the research and development efficiency can be improved. And (3) optimizing the concentration of the prepared in-situ temperature-sensitive gel on three factors by using a Design Expert 10 experimental Design software and the gel temperature as a response value according to a single-factor experimental result.

TABLE 6 gelation temperature measured in response surface experiments

Determination of optimal prescription blank gel: from the single factor and response surface experimental data, the blank gel formula with the gel temperature closest to body temperature was 1.295ml of 5% poloxamer 188 and 3.705ml of 21.4% poloxamer 407. 1.25g of poloxamer 188 and 10.7g of poloxamer 407 are precisely weighed and respectively dissolved in 25ml and 50ml beakers, the volume is determined after the medicine is completely dissolved, and the medicine is swelled at 4 ℃ for 12 h. Respectively measuring the solutions according to the optimal prescription by using a pipette, mixing the solutions in 7ml penicillin bottles, stirring the solution for 15min under the ice bath condition under the condition of 20r/min by using a heat collection type constant temperature heating magnetic stirrer, uniformly mixing the two solutions, standing the mixture, and measuring the gelation temperature to be 35.9 ℃ to meet the experimental requirements, so that blank gel of the prescription is used in the next experiment.

Preparation of drug-loaded gel: setting the dosage of each 5ml of resveratrol as 0.0005, 0.0010 and 0.0015g, respectively precisely weighing 0.0005, 0.0010 and 0.0015g of resveratrol in a 7ml penicillin bottle, adding 5ml of blank gel with an optimal prescription, uniformly mixing the two according to the above method, and standing to obtain the resveratrol in-situ gel with the dosage of 0.0001, 0.0002 and 0.0003 g/ml.

The invention also provides a characterization method of the resveratrol in-situ gel, which is applied to the resveratrol in-situ gel, and the method comprises the following steps:

and (3) determining the gelling temperature: setting a constant-temperature water bath kettle, placing a penicillin bottle in the water bath kettle, adjusting the water temperature to rise by 1 ℃, inclining the penicillin bottle to observe the flowability of the resveratrol in-situ gel until the resveratrol in-situ gel does not flow any more, namely the gelation temperature; measuring the gel temperature of blank gel with optimal prescription and resveratrol in-situ gel with resveratrol content of 0.0001g/ml, 0.0002g/ml, and 0.0003 g/ml, recording temperature, measuring each sample for 3 times, and averaging.

And (3) measuring the gel time: placing a penicillin bottle containing blank gel with an optimal prescription and resveratrol in-situ gel with the resveratrol content of 0.0003 g/ml in a constant-temperature water bath kettle at 25 ℃, quickly transferring to a constant-temperature water bath kettle at 37 ℃ after the penicillin bottle is stabilized, starting timing, recording the time required by the resveratrol in-situ gel from flowing to completely non-flowing, namely the gelation time, measuring each sample for 3 times, and taking an average value.

Determination of gel pH: preparing 25ml of blank gel with an optimal prescription and resveratrol in-situ gel with the resveratrol content of 0.0003 g/ml, stirring for 15min under the ice bath condition by a heat collection type constant temperature heating magnetic stirrer at 20r/min, uniformly mixing the blank gel and the resveratrol, standing, extending a probe of a pH value measuring instrument into the solution, and reading data after the pH value is stable.

Preparation of a standard curve: weighing 10.0mg of resveratrol standard substance, placing in a 50mL volumetric flask, dissolving with methanol and diluting to a scale, shaking up, then taking 3mL of diluted solution, placing in a 25mL volumetric flask, metering the volume with methanol to obtain a first standard solution, sucking 0, 1, 2, 3, 4 and 5mL of the first standard solution, metering 25mL of the first standard solution respectively to obtain 5 groups of second standard solutions, measuring the light absorption values of the 5 groups of second standard solutions at 306nm one by one, measuring for 3 times, calculating the average value, and drawing a standard curve.

And (3) determination of sample content: filling 1.5g of resveratrol in-situ gel in a 50ml volumetric flask, dissolving the gel in methanol and diluting the gel to a scale, and uniformly mixing and shaking the gel; taking 3mL of the solution after shaking up, putting the solution in a 25mL volumetric flask, diluting to a constant volume with methanol to obtain a third standard solution, sucking 2mL of the third standard solution, diluting to a constant volume of 25mL again, dissolving with methanol and diluting to a scale; and measuring the absorbance at the wavelength of 306nm, substituting into a standard curve regression equation, and calculating the content of the resveratrol.

And (3) measuring the gel viscosity: an NDJ-8S viscometer is used, a No. 3 rotor is adopted, the rotating speed is 60rpm/min, and the viscosity range is as follows: 10-200000mPa.s, respectively placing the blank gel and the resveratrol in-situ gel of the optimal formula into a sample bottle to be measured, keeping the liquid level of the solution to be submerged to the scale mark of the liquid level of the rotor, measuring the viscosity at 25 ℃ and 37 ℃, measuring each sample for 3 times, and taking an average value.

Measuring the gel erosion time: weighing an empty vial of 10mL penicillin bottles, adding 5g of resveratrol in-situ gel, and placing in a water bath constant temperature oscillator at 37.0 +/-0.1 ℃ for balancing for 10min to enable a polymer solution to completely form gel; taking out the gel, adding 1mL phosphate buffer solution PBS at 37 ℃, taking the pH =7.4 as a release medium, then putting the gel into a water bath constant temperature oscillator, oscillating at the temperature of 37.0 +/-0.1 ℃ at the speed of 40r/min, pouring out all the release medium within a set time, wiping the bottle dry and weighing; then putting into a water bath constant temperature oscillator again for balancing for 10min, then supplementing 1mL of PBS solution at 37 ℃, repeating the steps until the amount of the remaining gel is less than 10% of the initial amount, setting 3 parallel groups for each experiment, recording the erosion time, and taking the average value.

And (3) carrying out stability investigation on the resveratrol in-situ gel: standing 2 parts of resveratrol in-situ gel with resveratrol content of 0.0003 g/ml at 25 deg.C and 37 deg.C for 15min, centrifuging at 2000r/min for 15min, and observing whether unstable phenomena such as delamination occur.

And (5) analyzing experimental data.

Response surface data:

according to the experimental data, Design Expert 10 software predicts that the optimal prescription is 1.295ml of 5% poloxamer 188 and 3.705ml of 21.4% poloxamer 407. The formula is as follows: gel temperature = +37.84+0.75A-6.75B +3.00C-0.10AB-0.20AC-0.65BC +0.26A2+3.11B2-0.25C 2. FIG. 3 is a graph of the effect of independent A, B versus dependent gel temperature. Fig. 4 is a contour plot of independent A, B versus dependent gel temperature.

Gel phase transition temperature:

experimental data show that the gel with the resveratrol content of 0.0015mg has proper phase transition temperature, and meets the requirement that the gel is a flowing liquid at room temperature and is subjected to phase deformation to form gel at body temperature. FIG. 5 is a graph of the gelling temperature at different dosages.

TABLE 7 gelation temperature at different dosages

Gel time:

the gel time of the blank gel is respectively 262s, 259s and 264s, and the gel time of the gel with the resveratrol dosage of 0.0015g is respectively 230s, 229s and 232 s.

Gel pH:

the blank gel has a pH value of 7.15, the gel with the resveratrol content of 0.0075 has a pH value of 7.12, and the gel meets the condition of being applied to a human body, so the gel obtained in the experiment can be used as a good drug administration matrix.

The determination result of the resveratrol standard curve is as follows: fig. 6 is a line graph of a standard curve of resveratrol. From FIG. 6, the regression equation of the standard curve of resveratrol is A =0.1283C +0.4682 (R2 = 0.9927), and the linear range is 0.5-5.0 μ g/ml.

Measurement results of gel drug loading:

the absorbance of the gel with resveratrol content of 0.0015g measured at 306nm was brought into the standard curve, and the drug loading of the gel was calculated to be 0.09% and the encapsulation efficiency was calculated to be 60%.

Drug loading = total resveratrol in gel/gel volume × 100%

Entrapment rate = actual drug load/theoretical drug load × 100%

Measurement results of gel viscosity:

TABLE 8 viscosity at different temperatures

Measurement of gel erosion time:

the in vitro erosion time of the gel was 5.6h and the erosion rate was 91.1% as determined by the membraneless dissolution method.

And (3) analyzing experimental phenomena:

the appearance of the resveratrol-loaded in-situ gel is white transparent liquid state at room temperature, and is white transparent gel state after reaching the gelation temperature.

Stability of resveratrol-loaded in situ gel:

the resveratrol in-situ gel solution does not generate phenomena of demixing, turbidity and the like after being gelled at 25 ℃ and 37 ℃ through centrifugation, and the preparation has good stability.

In conclusion, resveratrol has a good therapeutic effect on lung cancer, but the defect of poor water solubility of resveratrol causes that resveratrol cannot be well applied to clinical treatment. The gel prepared by taking various high polymer materials as the matrix is used for encapsulating resveratrol, and poloxamer is used as the matrix of the slow-release gel, so that the gel has low toxicity, good biocompatibility and the like. The optimal prescription of the drug-loaded gel is determined through a single-factor experiment and a response surface experiment, the dosage is determined by referring to relevant documents of resveratrol for inhibiting tumor cells, and the prescription is as follows: 3.705ml of 21.4 percent poloxamer 407 and 1.295ml of 5 percent poloxamer 188, and the content of the resveratrol is 0.3 mg/ml. The prepared gel has the gelling temperature of 36.4 ℃ and the gelling time of 230 s.

The pH value and the gelling temperature of the gel meet the requirements of being applied to human bodies.

The present invention is not limited to the above preferred embodiments, and any modifications, equivalent replacements, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.