阅读说明：本技术 低分散度壳聚糖载黄芪多糖纳米粒及制备方法、用途 (Low-dispersion chitosan-carried astragalus polysaccharide nanoparticles, and preparation method and application thereof ) 是由 王铮 姜明燕 李凯 万龙 于 2020-12-25 设计创作，主要内容包括：本发明属于药品制备技术领域,具体提供一种低分散度壳聚糖载黄芪多糖纳米粒及制备方法、用途,采用喷雾干燥的方法进行制备,具体包括称取黄芪多糖,溶于乙酸溶液中,超声后,一定转速下搅拌一定时间,得到黄芪多糖-乙酸混合液；将混合液在一定转速下离心,取上清液,称取壳聚糖加入上清液中,超声后一定转速下搅拌一定时间,得低分散度壳聚糖载黄芪多糖溶液；将低分散度壳聚糖载黄芪多糖溶液进行喷雾干燥,得到低分散度壳聚糖载黄芪多糖纳米粒。本发明提供的低分散度壳聚糖载黄芪多糖纳米粒增加了黄芪多糖的吸收,并且具有免疫系统增强和免疫系统纠正的作用。(The invention belongs to the technical field of medicine preparation, and particularly provides a chitosan-loaded astragalus polysaccharide nanoparticle with low dispersity, a preparation method and application thereof, wherein the chitosan-loaded astragalus polysaccharide nanoparticle is prepared by adopting a spray drying method, and the preparation method specifically comprises the steps of weighing astragalus polysaccharide, dissolving the astragalus polysaccharide in an acetic acid solution, carrying out ultrasonic treatment, and stirring for a certain time at a certain rotating speed to obtain an astragalus polysaccharide-acetic acid mixed solution; centrifuging the mixed solution at a certain rotating speed, taking supernatant, weighing chitosan, adding into the supernatant, and stirring for a certain time at a certain rotating speed after ultrasonic treatment to obtain a low-dispersion chitosan-carried astragalus polysaccharide solution; and (3) carrying out spray drying on the low-dispersion-degree chitosan-carried astragalus polysaccharide solution to obtain the low-dispersion-degree chitosan-carried astragalus polysaccharide nanoparticles. The chitosan-loaded astragalus polysaccharide nanoparticles with low dispersity provided by the invention increase the absorption of astragalus polysaccharide, and have the functions of enhancing and correcting an immune system.)

1. The preparation method of the chitosan-carried astragalus polysaccharide nanoparticles with low dispersity is characterized by adopting a spray drying method for preparation, and specifically comprises the following steps:

1) weighing 1-2.831 g of astragalus polysaccharide, dissolving the astragalus polysaccharide in 2000-2200 ml of 0.5-0.65% acetic acid solution, carrying out ultrasonic treatment for 10-15 min, and stirring at 500-600 rpm for 10-12 hours to obtain astragalus polysaccharide-acetic acid mixed solution;

2) centrifuging the mixed solution at 4000-4500 rpm for 10-15 min, taking supernatant, weighing 5-6 g of chitosan, adding into the supernatant, performing ultrasonic treatment for 10-15 min, and stirring at 600-650 rpm for 10-12 hours to obtain a low-dispersion chitosan astragalus polysaccharide-loaded solution;

3) carrying out spray drying on the low-dispersity chitosan-loaded astragalus polysaccharide solution prepared in the step 2), and setting parameters as inlet temperature: 120-125 ℃, sample injection speed of a peristaltic pump: 3-4 mL/min, air compressor sprayer flow: 650-700L/h, aspirator power: and (3) 90-95% of chitosan-loaded astragalus polysaccharide nanoparticles with low dispersity are obtained.

2. The method for preparing low-dispersity chitosan-supported astragalus polysaccharide nanoparticles as claimed in claim 1, wherein in the step 1), 2.831g of astragalus polysaccharide is weighed and dissolved in 2000ml of 0.5% acetic acid solution.

3. The method for preparing low-dispersity chitosan-supported astragalus polysaccharide nanoparticles as claimed in claim 1, wherein in the step 3), a spray head of a spray dryer is 0.7 mm.

4. The chitosan-astragalus polysaccharide nanoparticles with low dispersity prepared by the preparation method of the chitosan-astragalus polysaccharide nanoparticles with low dispersity as claimed in claim 1, 2 or 3.

5. The use of the chitosan-supported astragalus polysaccharide nanoparticles with low dispersity prepared by the preparation method of the chitosan-supported astragalus polysaccharide nanoparticles with low dispersity of claim 1, 2 or 3 in the enhancement and correction of the immune system.

6. The use of claim 5, wherein said enhancement of the immune system comprises enhancement of the hypoimmunity resulting from the treatment of immunodeficiency disorders, chronic infectious diseases and cancer; the immune system correction includes use in the treatment of autoimmune disease.

Technical Field

The invention belongs to the technical field of medicine preparation, and particularly provides a chitosan-loaded astragalus polysaccharide nanoparticle with low dispersity, a preparation method and application thereof.

Background

The morbidity, mortality and recurrence rate of cancer are high, and the health of people is seriously threatened. Many patients are difficult to diagnose early, the patients are in the advanced stage during the treatment, the curative effect of the operation is poor or the chance of the operation is lost, and the postoperative chemical drug treatment plays an important role in the comprehensive treatment of the cancer. However, most of the existing chemotherapeutic drugs cause the destruction and damage of other histiocytes and the degeneration and necrosis of local histiocytes while inhibiting and killing tumor tissues, and the inflammatory reaction occurs. The traditional Chinese medicine is used for treating the gastric cancer patient in a combined way, so that the toxic and side effects can be reduced while the effect of the chemotherapeutic drug is ensured, the immune function of the organism is improved, and the inflammatory reaction is relieved, which becomes the current research hotspot and achieves remarkable effect. The traditional Chinese medicine radix astragali is capable of tonifying qi and deficiency, and is suitable for tumor patients with leucopenia and hypoimmunity after chemotherapy. The astragalus polysaccharide is the main effective component of astragalus and plays a better role in treating tumors. However, reports on whether astragalus polysaccharide can improve the postoperative inflammatory reaction of cancer are few, and the research and development of medicaments and clinical application are not mature.

The treatment effect is influenced by factors such as overlarge molecular weight, poor water solubility, poor absorption and low bioavailability of Astragalus Polysaccharide (APS), and is easily influenced by gastric acid (gastric soluble type), so that the Astragalus polysaccharide is also a main factor for limiting the wide application of the Astragalus polysaccharide to the clinical treatment of cancers at present.

Disclosure of Invention

In order to solve the technical problems, the invention provides a chitosan-loaded astragalus polysaccharide nanoparticle with low dispersity, a preparation method and application thereof.

The invention is realized in such a way, and provides a preparation method of chitosan-loaded astragalus polysaccharide nanoparticles with low dispersity, which is prepared by adopting a spray drying method and specifically comprises the following steps:

1) weighing 1-2.831 g of astragalus polysaccharide, dissolving the astragalus polysaccharide in 2000-2200 ml of 0.5-0.65% acetic acid solution, carrying out ultrasonic treatment for 10-15 min, and stirring at 500-600 rpm for 10-12 hours to obtain astragalus polysaccharide-acetic acid mixed solution;

2) centrifuging the mixed solution at 4000-4500 rpm for 10-15 min, taking supernatant, weighing 5-6 g of chitosan, adding into the supernatant, performing ultrasonic treatment for 10-15 min, and stirring at 600-650 rpm for 10-12 hours to obtain a low-dispersion chitosan astragalus polysaccharide-loaded solution;

3) carrying out spray drying on the low-dispersity chitosan-loaded astragalus polysaccharide solution prepared in the step 2), and setting parameters as inlet temperature: 120-125 ℃, sample injection speed of a peristaltic pump: 3-4 mL/min, air compressor sprayer flow: 650-700L/h, aspirator power: and (3) 90-95% of chitosan-loaded astragalus polysaccharide nanoparticles with low dispersity are obtained.

Preferably, in the step 1), the astragalus polysaccharide with the mass of 2.831g is weighed and dissolved in 2000ml of 0.5% acetic acid solution.

Further preferably, in the step 3), a spray head of the spray dryer is 0.7 mm.

The invention also provides the low-dispersion chitosan-supported astragalus polysaccharide nanoparticles prepared by the preparation method.

The invention also provides application of the low-dispersion chitosan astragalus polysaccharide nanoparticles prepared by the preparation method of the low-dispersion chitosan astragalus polysaccharide nanoparticles in immune system enhancement and immune system correction.

Preferably, the enhancement of the immune system comprises enhancement of the immune system hypofunction caused in the course of treating immunodeficiency diseases, chronic infectious diseases and cancer; the immune system correction includes use in the treatment of autoimmune disease.

Compared with the prior art, the invention has the advantages that:

the chitosan with good biocompatibility is selected as the astragalus polysaccharide nano-carrier, so that the stability of the astragalus polysaccharide can be improved, and the chitosan is used as a cationic polymer, so that the close connection of intestinal tracts can be opened due to the unique biological property of the chitosan, and the oral absorption of the astragalus polysaccharide is further improved. The immunoregulation function of astragalus polysaccharide can control the balance of Th1/Th2 cell ratio, so that the astragalus polysaccharide can be used for enhancing immune system and treating diseases with hyperfunction of immune system. Immunopotentiation is used for the treatment of immunodeficiency diseases, chronic infections and as an adjuvant treatment for cancer. The immune correction effect can be used for treating autoimmune diseases such as rhinitis and lupus erythematosus.

Drawings

FIG. 1 is a photograph showing the appearance of chitosan-loaded astragalus polysaccharide nanoparticles with low dispersion degree prepared by the spray drying method provided by the present invention;

FIG. 2 is a transmission electron micrograph of the low-dispersity chitosan-supported astragalus polysaccharide nanoparticles shown in FIG. 1;

FIG. 3 is a photograph showing the appearance of the low-dispersity chitosan-supported astragalus polysaccharide nanoparticles prepared by the freeze-drying method in example 4;

FIG. 4 is a transmission electron micrograph of the low-dispersity chitosan-loaded astragalus polysaccharide nanoparticles shown in FIG. 3;

FIG. 5 shows the effect of low-dispersity chitosan-loaded astragalus polysaccharide nanoparticles on spleen index of gastric cancer mice;

FIG. 6 shows the effect of low-dispersity chitosan-loaded astragalus polysaccharide nanoparticles on thymus index of gastric cancer mice;

FIG. 7 shows the effect of low-dispersity chitosan-loaded astragalus polysaccharide nanoparticles on the percentage of gastric cancer mouse T lymphocytes;

FIG. 8 shows the effect of low-dispersity chitosan-loaded astragalus polysaccharide nanoparticles on the ratio of CD4+/CD8+ of gastric cancer mice;

FIG. 9 shows the effect of low-dispersity chitosan-loaded astragalus polysaccharide nanoparticles on the regulation of the levels of plasma cytokines IFN-gamma and IL-4 in gastric cancer mice;

FIG. 10 shows the effect of low-dispersity chitosan-loaded astragalus polysaccharide nanoparticles on the ratio of IFN-gamma/IL-4 of gastric cancer mice.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Examples 1,

1) Weighing 1g of astragalus polysaccharide, dissolving the astragalus polysaccharide in 2000ml of 0.5% acetic acid solution, carrying out ultrasonic treatment for 10min, and stirring at 500rpm for 10-12 hours to obtain astragalus polysaccharide-acetic acid mixed solution;

2) centrifuging the astragalus polysaccharide-acetic acid mixed solution at 4000rpm for 10min, taking supernatant, removing precipitate, weighing 5g of chitosan, adding into the supernatant, performing ultrasonic treatment for 10min, and stirring at 600rpm for 10-12 h to obtain low-dispersion chitosan-loaded astragalus polysaccharide solution;

3) transferring the low-dispersion chitosan-loaded astragalus polysaccharide solution to a sample stage of a spray dryer B-290 for later use, and selecting the smallest spray head (0.7mm) as the spray nozzle. Instrument parameter settings were inlet temperature (inlet temperature): 120 ℃, peristaltic pump feed rate (feed rate): 3mL/min (12.4%), air compressor nebulizer flow (spray air flow): adjusting to 4cm high (about 667L/h), and setting the aspirator power (asparator flow) to 90% to obtain chitosan-loaded astragalus polysaccharide nanoparticle powder with low dispersity.

4) Accurately weighing 50mg of dry constant-weight Astragalus polysaccharides, placing in a 100mL volumetric flask, adding water to dissolve, diluting to scale, and shaking to obtain test solution (containing 0.50mg of Astragalus polysaccharides per 1 mL). The total sugar content is determined by applying an anthrone-sulfuric acid method, 0.5mL of test solution is taken, 0.5mL of water is added, the test tube is placed in an ice water bath, 4.0mL of anthrone reagent is added, and the test tube is shaken up. Boiling water bath for 15min, standing for 10min to room temperature, adjusting to zero with blank tube to determine absorbance, and substituting the absorbance into standard curve regression equation of D-glucose reference substance to calculate total sugar content. The obtained astragalus polysaccharide has the percentage content of 50 percent.

Examples 2,

1) 1.5g of astragalus polysaccharide is weighed, dissolved in 2000ml of 0.5 percent acetic acid solution, and stirred for 10-12 hours at 500rpm after ultrasonic treatment for 10 min.

2) Centrifuging the mixed solution of astragalus polysaccharide and acetic acid at 4000rpm for 10min, collecting supernatant, discarding precipitate, weighing 5g chitosan, adding into the supernatant, performing ultrasonic treatment for 10min, and stirring at 600rpm for 10-12 hr to obtain low-dispersion chitosan-loaded astragalus polysaccharide solution.

3) Transferring the low-dispersity chitosan-carried astragalus polysaccharide solution to a sample table of a spray dryer B-290 for later use. The smallest nozzle (0.7mm) was selected. Instrument parameter settings were inlet temperature (inlet temperature): 120 ℃, peristaltic pump feed rate (feed rate): 3mL/min (12.4%), air compressor nebulizer flow (spray air flow): the height was adjusted to 4cm (about 667L/h) and the aspirator power (asparator flow) was set at 90%. Obtaining the chitosan-carried astragalus polysaccharide nano particle powder with low dispersion degree.

4) Accurately weighing 50mg of dry constant-weight Astragalus polysaccharides, placing in a 100mL volumetric flask, adding water to dissolve, diluting to scale, and shaking to obtain test solution (containing 0.50mg of Astragalus polysaccharides per 1 mL). The total sugar content is determined by applying an anthrone-sulfuric acid method, 0.5mL of test solution is taken, 0.5mL of water is added, the test tube is placed in an ice water bath, 4.0mL of anthrone reagent is added, and the test tube is shaken up. Boiling water bath for 15min, standing for 10min to room temperature, adjusting to zero with blank tube to determine absorbance, and substituting the absorbance into standard curve regression equation of D-glucose reference substance to calculate total sugar content. The percentage content of the obtained astragalus polysaccharide, namely the drug loading rate, is 60 percent.

Examples 3,

1) Weighing 2.831g Astragalus polysaccharides, dissolving in 2000ml 0.5% acetic acid solution, ultrasonic treating for 10min, and stirring at 500rpm for 10-12 hr.

2) Centrifuging the low-dispersion chitosan-carrying astragalus polysaccharide solution at 4000rpm for 10min, taking the supernatant, and discarding the precipitate. And 5g of chitosan is weighed and added into the supernatant, ultrasonic treatment is carried out for 10min, stirring is carried out at 600rpm overnight, and the low-dispersion degree chitosan astragalus polysaccharide-loaded solution is obtained.

3) Transferring the low-dispersity chitosan-carried astragalus polysaccharide solution to a sample table of a spray dryer B-290 for later use. The smallest nozzle (0.7mm) was selected. Instrument parameter settings were inlet temperature (inlet temperature): 120 ℃, peristaltic pump feed rate (feed rate): 3mL/min (12.4%), air compressor nebulizer flow (spray air flow): the height was adjusted to 4cm (about 667L/h) and the aspirator power (asparator flow) was set at 90%. Obtaining the chitosan-carried astragalus polysaccharide nano particle powder with low dispersion degree.

4) Accurately weighing 50mg of dry constant-weight Astragalus polysaccharides, placing in a 100mL volumetric flask, adding water to dissolve, diluting to scale, and shaking to obtain test solution (containing 0.50mg of Astragalus polysaccharides per 1 mL). The total sugar content is determined by applying an anthrone-sulfuric acid method, 0.5mL of test solution is taken, 0.5mL of water is added, the test tube is placed in an ice water bath, 4.0mL of anthrone reagent is added, and the test tube is shaken up. Boiling water bath for 15min, standing for 10min to room temperature, adjusting to zero with blank tube to determine absorbance, and substituting the absorbance into standard curve regression equation of D-glucose reference substance to calculate total sugar content. The obtained astragalus polysaccharide has the percentage content of 80 percent.

Example 4

1) Weighing 2.831g Astragalus polysaccharides, dissolving in 2000ml 0.5% acetic acid solution, ultrasonic treating for 10min, and stirring at 500rpm for 10-12 hr.

2) Centrifuging the low-dispersion chitosan-carrying astragalus polysaccharide solution at 4000rpm for 10min, taking the supernatant, and discarding the precipitate. And 5g of chitosan is weighed and added into the supernatant, the mixture is subjected to ultrasonic treatment for 10min and stirred at 600rpm for 10-12 hours, and the low-dispersion chitosan astragalus polysaccharide-loaded solution is obtained.

3) Samples were dispensed into petri dishes and frozen overnight in a deep low temperature freezer (-80 ℃). The next day the frozen samples were transferred to the freeze compartment baffle of the lyophilizer. And opening a freezing switch until the temperature of the freezing chamber is lower than-40 ℃, opening a vacuum pump switch, and waiting for 15min until the system pressure is lower than 100 millitorr. And (4) freeze-drying the sample for 72h to obtain a freeze-dried LCA nanoparticle sample.

4) Accurately weighing 50mg of dry constant-weight Astragalus polysaccharides, placing in a 100mL volumetric flask, adding water to dissolve, diluting to scale, and shaking to obtain test solution (containing 0.50mg of Astragalus polysaccharides per 1 mL). The total sugar content is determined by applying an anthrone-sulfuric acid method, 0.5mL of test solution is taken, 0.5mL of water is added, the test tube is placed in an ice water bath, 4.0mL of anthrone reagent is added, and the test tube is shaken up. Boiling water bath for 15min, standing for 10min to room temperature, adjusting to zero with blank tube to determine absorbance, and substituting the absorbance into standard curve regression equation of D-glucose reference substance to calculate total sugar content. The obtained astragalus polysaccharide has the percentage content of 70 percent of drug-loading rate.

Test examples,

The experimental mice were provided by Beijing Huafukang Biotechnology Ltd.

1. Morphological characterization

An appropriate amount of the low-dispersion chitosan-supported astragalus polysaccharide nanoparticles prepared in examples 1 to 4 was diluted with double distilled water, and the solution was dropped on a copper mesh covered with a carbon film, and then a drop of sodium phosphotungstate solution was added for negative dyeing, and the preparation was naturally dried at room temperature, and the morphology was observed under a transmission electron microscope. The spray-dried low-dispersion-degree chitosan-loaded astragalus polysaccharide nanoparticles are light yellow, uniform in particle and fluffy in surface (as shown in figure 1), and the low-dispersion-degree chitosan-loaded astragalus polysaccharide nanoparticles are uniform and consistent in shape, spherical, good in dispersion, free of adhesion and nano-sized in diameter (as shown in figure 2) observed by a transmission electron microscope. The lyophilized low-dispersity chitosan-loaded astragalus polysaccharide nanoparticles are golden, flocculent, coiled and tough (as shown in figure 3), and the lyophilized low-dispersity chitosan-loaded astragalus polysaccharide nanoparticles are disordered and inhomogeneous in shape and are locally adhered (as shown in figure 4) under the observation of a transmission electron microscope

2. Encapsulation efficiency and drug loading measurements

Since the invention uses spray drying, the default formulation is entirely encapsulated, so that the encapsulation efficiency is not needed to be determined.

Accurately weighing 50mg of dry constant-weight Astragalus polysaccharides, placing in a 100mL volumetric flask, adding water to dissolve, diluting to scale, and shaking to obtain test solution (containing 0.50mg of Astragalus polysaccharides per 1 mL). The total sugar content is determined by applying an anthrone-sulfuric acid method, 0.5mL of test solution is taken, 0.5mL of water is added, the test tube is placed in an ice water bath, 4.0mL of anthrone reagent is added, and the test tube is shaken up. Boiling water bath for 15min, standing for 10min to room temperature, adjusting to zero with blank tube to determine absorbance, and substituting the absorbance into standard curve regression equation of D-glucose reference substance to calculate total sugar content. Finally, the percentage content of the astragalus polysaccharide, namely the drug loading rate, can be obtained according to the following formula.

The drug loading capacity (%) is APS mass/total mass of nanoparticles X100%

The results are shown in Table 1

	Drug loading (%)
		Example 1	50
Example 2	60
		Example 3	80
Example 4	70

3. In vivo experiments

And (3) inoculating 0.2mL of MFC gastric cancer cell suspension in logarithmic growth phase to the right axilla subcutaneous layer of the mouse, and dividing 40 tumorigenic Balb/c mice into 6 groups by using a random digital table method when tumors grow to be about 1cm (about 2-3 weeks) in diameter, wherein each group comprises 6 males and females. Setting as a negative control group; a group of cyclophosphamide compounds; LCA nanoparticles low, medium and high dose + cyclophosphamide group; lentinan positive control traditional Chinese medicine + cyclophosphamide group.

The LCA nanoparticle low, medium and high dose plus cyclophosphamide groups are respectively gavaged to administer astragalus polysaccharide 200mg/kg, 400mg/kg and 800mg/kg on the grouping day, 1 time per day and 0.2mL, and the administration is continuously carried out for 14 days; the lentinan positive control traditional Chinese medicine + cyclophosphamide group is administered with lentinan 400mg/kg by intragastric administration for 1 time per day and 0.2mL for 14 days. Wherein, the cyclophosphamide is administrated by intraperitoneal injection of 30mg/kg and 0.2mL, the administration period is 5-14 days, and the administration is once a day; the negative control group is administered with 0.2mL of water by intragastric administration once a day, and is injected with 0.2mL of normal saline by intraperitoneal injection once a day on the 5 th to 14 th days. 24h after the last administration, on day 15, the mice were blood-collected by eye-picking, EDTA anticoagulated, a portion of the whole blood was subjected to T lymphocyte subpopulation analysis, a portion of the whole blood was centrifuged at 1000g for 30min to obtain a plasma sample, which was stored at-20 ℃ for plasma cytokine determination. After the cervical dislocation was sacrificed, the mice were quickly dissected, spleen and thymus wet weights were measured, and spleen and thymus indices were calculated, which are organ mass (mg)/body weight (g), and the results showed that LCA had a significant effect on the spleen and thymus indices (see fig. 5 and 6).

The result of the measurement of the immune related indexes shows that an immunosuppressed mouse tumor forming model is caused by intraperitoneal injection of cyclophosphamide and subcutaneous tumor formation, and LCA treatment is given, and the result shows that LCA can increase the percentage of T lymphocytes and improve the ratio of CD4+/CD8+, and the balance of Th1 and Th2 cells is adjusted by adjusting the levels of plasma cytokines IFN-gamma and IL-4 and the proportion of IFN-gamma/IL-4, so that the immune function of an organism is improved. The low-dispersion chitosan-carried astragalus polysaccharide nanoparticles are proved to be capable of remarkably improving the immune function of mice with hypoimmunity caused by chemotherapeutic drugs and have remarkable auxiliary treatment effect on the mice with gastric cancer chemotherapy (see figures 7, 8, 9 and 10).