Compound polysaccharide microspheres capable of releasing NO as well as preparation method and application of compound polysaccharide microspheres

Abstract

The invention discloses a preparation method of compound polysaccharide microspheres capable of releasing NO. The preparation method comprises the following steps: cross-linking swallow polysaccharide with L-ARG, and preparing L-ARG-containing swallow polysaccharide hydrogel through a Mannich reaction; then loading EGCG (epigallocatechin gallate) onto the L-ARG-containing swallow polysaccharide hydrogel through a Schiff base reaction, and obtaining the compound polysaccharide microspheres capable of releasing NO by utilizing an emulsification method. The invention also discloses an application thereof. The hydrogel microsphere capable of targeting the colon is prepared, and the hydrogel microsphere can be accurately exploded at the colon part after being swallowed. A dextran sodium sulfate (DSS)-induced IBD mouse model is established to test the drug effect, and the result shows that the compound polysaccharide microspheres have a remarkable treatment effect on DSS-induced ulcerative colitis, can obviously improve the symptoms of ulcerative colitis, and provides a theoretical basis and a clinical basis for new drugs for clinically preventing and treating ulcerative colitis.

Description

Technical Field

[0001] The invention relates to a composite polysaccharide microsphere capable of releasing NO and a preparation method and application thereof, belonging to the technical field of biomedical materials. Background Art

[0002] Ulcerative colitis (UC) is a chronic intestinal inflammatory disease with an unknown etiology. Typical symptoms include bloody diarrhea, abdominal pain, fecal urgency, and tenesmus, which seriously affect the patient's health and quality of life. The pathogenesis of UC involves multiple factors, such as the external environment, intestinal microorganisms, mucosal immunity, and genetics. At present, the main clinical treatment for UC is drug therapy, including aminosalicylic acids, glucocorticoids, immunomodulators, and biological agents. However, these drugs are usually expensive, and patients often experience a variety of adverse reactions during use, which reduces patient compliance and thus affects the treatment effect.

[0003] Epigallocatechin gallate (EGCG) is an ingredient extracted from Chinese green tea and is the most abundant catechin monomer substance in green tea polyphenols. Previous studies have shown that EGCG has very strong antioxidant activity and can increase the activity of superoxide dismutase (SOD) in the body, thereby removing superoxide anion free radicals in the body and protecting cells from oxidative damage. At the same time, EGCG can inhibit the expression of inflammatory factors such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), and has excellent anti-inflammatory effects. However, EGCG has a short gastrointestinal retention time and poor permeability in the body, and is easily affected by the gastrointestinal environment and becomes unstable. How to improve its stability in the intestine is the key to give full play to its excellent performance. Snow swallow, also known as karaya gum, is a natural colloidal secretion containing a variety of active ingredients such as flavonoids and polysaccharides. It has the effects of moistening the lungs and promoting fluid production, beautifying the skin, and moisturizing the intestines and laxatives. Studies have shown that intestinal microecological imbalance may damage the intestinal barrier function, and snow fungus polysaccharide can indirectly regulate the composition of intestinal flora by repairing the intestinal barrier. At the same time, snow fungus polysaccharide can promote the growth and repair of intestinal epithelial cells, enhance the integrity of the intestinal barrier, and reduce the invasion of harmful substances, thereby providing a good living environment for beneficial bacteria. Snow fungus has been consumed as a precious tonic for more than 1,000 years. However, there is currently very little research on snow fungus polysaccharide as a monomer carrier of traditional Chinese medicine, especially in improving intestinal health. Therefore, the use of EGCG-loaded snow fungus polysaccharide microspheres to treat UC is a further study of its mechanism of action and application prospects.

[0004] Microspheres are spherical or quasi-spherical particles made by encapsulating or adsorbing drugs with suitable polymer materials as carriers. Their particle size is very small, generally 1 to 500 μm. Microspheres are widely used in the field of biology. For example, in the detection of tumor markers, functionalized polystyrene microspheres have high sensitivity and selectivity. Using lateral flow immunoassay, markers such as alpha-fetoprotein can be detected in real time. Microspheres can also be used as drug carriers to achieve local delivery of drugs, inhibit the release and action of inflammatory factors, and exert anti-inflammatory effects.

[0005] Nitric oxide (NO) is a small gas molecule with multiple physiological functions, and plays an important role in the anti-inflammatory process. Studies have shown that low doses of NO can inhibit the release of inflammatory factors and chemokines, inhibit pro-inflammatory effects and reduce the aggregation of inflammatory factors to the site of inflammation, thereby reducing the inflammatory response. At the same time, NO is an important vasodilator factor. During the inflammatory process, it can dilate blood vessels and improve local blood circulation, which helps the exchange of substances at the site of inflammation, accelerates the removal of inflammatory cells and mediators, and also provides good nutrition and oxygen supply for tissue repair. As an important signaling molecule, NO can activate guanylate cyclase and participate in regulating the activity of multiple protein kinases and ion channels, thereby participating in the healing process of the disease. Summary of the invention

[0006] The purpose of the present invention is to provide a composite polysaccharide microsphere capable of releasing NO, in order to provide a theoretical basis and clinical foundation for the study of new drugs for preventing or treating ulcerative colitis.

[0007] The technical solution adopted by the present invention is:

[0008] A method for preparing composite polysaccharide microspheres capable of releasing NO, characterized in that the steps include:

[0009] (1) Snow fungus polysaccharide was cross-linked with L-ARG, and the snow fungus polysaccharide hydrogel containing L-ARG was prepared by Mannich reaction; Snow fungus polysaccharide is rich in aldehyde groups, and the molecular weight of the polysaccharide is 3k Da-2063k Da.

[0010] (2) EGCG was loaded onto the L-ARG-containing snow fungus polysaccharide hydrogel by Schiff base reaction, and the composite polysaccharide microspheres capable of releasing NO were obtained by emulsification method. The prepared composite polysaccharide microspheres had a particle size of 1-5 μm and a negatively charged surface.

[0011] Preferably, step (1) is specifically as follows: preparing a snow nut polysaccharide solution, then slowly dripping the L-Arg solution into the snow nut polysaccharide solution, then placing the solution in a dark environment at room temperature to react, and then lyophilizing the solution after dialyzing with running water to obtain a snow nut polysaccharide hydrogel containing L-ARG.

[0012] Preferably, the concentration of the snow fungus polysaccharide solution is 5 mg / ml, the concentration of the L-Arg solution is 5 mg / ml, the mass ratio of snow fungus polysaccharide to L-Arg is 20:1-40:1, the reaction time is 8-24 hours, and the dialysis time is 3-5 days.

[0013] Preferably, step (2) is specifically as follows: adding water to the snow fungus polysaccharide hydrogel containing L-ARG to form a solution, then dropping the EGCG solution into the snow fungus polysaccharide solution containing L-ARG to react as the water phase, adding an emulsifier to the paraffin liquid as the oil phase, stirring to make the oil phase uniform, dropping the water phase into the oil phase, stirring evenly, adding glutaraldehyde solution and stirring overnight to obtain composite polysaccharide microspheres.

[0014] Preferably, the concentration of the snow swallow polysaccharide solution containing L-ARG is 5 mg / mL, and then a 25 mg / mL EGCG solution is added dropwise to the snow swallow polysaccharide solution to react for more than 8 hours. At the same time, 2 mL of Span-80 emulsifier is added to 100 mL of paraffin liquid and stirred at 60 ° C for 1 hour to make the oil phase uniform. Then, the EGCG / derivatized snow swallow polysaccharide solution is slowly and evenly dripped into the oil phase using a microinjection pump and stirred slowly. After stirring for 2 hours, the solution is moved to a 4 ° C cold room, and 500 μL of glutaraldehyde solution is added and stirred overnight. Cross-linking obtains EGCG-loaded composite polysaccharide microspheres.

[0015] Preferably, the snow fungus polysaccharide can be commercially available or prepared by itself. Generally speaking, the preparation method of the snow fungus polysaccharide can be to purify the crude snow fungus polysaccharide by conventional water extraction and alcohol precipitation method.

[0016] Preferably, the preparation method of snow fungus polysaccharide is as follows: weigh and dissolve crude snow fungus polysaccharide, ultrasonicate at 60°C for 2h, centrifuge at low speed to discard the supernatant, take the precipitate and add anhydrous ethanol for alcohol precipitation, the final concentration of the alcohol precipitation is 90%, after standing at 4°C for 24h, separate the precipitate, and add Sevag reagent at a volume ratio of 1:4 to remove protein, after 72 hours of running water dialysis, freeze-dry to obtain the gelatinizable snow fungus polysaccharide.

[0017] The composite polysaccharide microspheres capable of releasing NO prepared by the above preparation method.

[0018] Application of the above-mentioned composite polysaccharide microspheres capable of releasing NO in the preparation of drugs for treating inflammatory bowel disease.

[0019] Preferably, the inflammatory bowel disease refers to ulcerative colitis or Crohn's disease.

[0020] The present invention uses C57BL / 6J mice as experimental animals and uses dextran sodium sulfate (DSS) to induce an ulcerative colitis (UC) model. The results of the study showed that the composite polysaccharide microspheres significantly reversed the weight loss of mice caused by DSS. In addition, the colon length measurement results showed that the composite polysaccharide microspheres were able to significantly restore the colon length of DSS mice. Overall, the composite polysaccharide microspheres showed significant therapeutic effects and significantly improved the pathological symptoms of UC mice. The drug described in the present invention is an effective content of negatively charged composite polysaccharide microspheres, which can target positively charged inflammatory sites, and the surface functional groups are rich and easy to modify, and can be used as a carrier of nanomaterials, suitable for clinical administration.

[0021] The present invention also provides the use of the composite snow fungus polysaccharide microspheres in the preparation of a preparation for treating ulcerative colitis. Preferably, the composite snow fungus polysaccharide microspheres of the present invention have the following excellent effects compared with the prior art:

[0022] (1) The present invention provides a method for preparing composite polysaccharide microspheres capable of releasing NO for treating ulcerative colitis. The method is based on the traditional sol-gel method, and L-ARG and EGCG are added step by step. EGCG is covalently bound to snow fungus polysaccharide through Mannich reaction and Schiff base reaction. Subsequently, the composite snow fungus polysaccharide microspheres prepared by the emulsification method have uniform particle size and good dispersibility.

[0023] (2) The present invention protects EGCG through functionalized composite snow swallow polysaccharide microspheres to achieve its efficient utilization, and at the same time uses the negative charge characteristics of the microspheres to target the inflammatory site, thereby playing a role in alleviating inflammation. Therapeutic experiments in a mouse colitis model showed that the composite snow swallow polysaccharide microspheres can accurately "explode" in the colon after oral administration, and can prolong the intestinal residence time to a certain extent, enhance the intestinal mucosal barrier function, and effectively relieve intestinal inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the relative molecular mass of the polysaccharide precursor prepared in Example 1 of the present invention;

[0025] Figure 2 is a graph of the aldehyde content of the polysaccharide precursor prepared in Example 1 of the present invention;

[0026] Figure 3 is the Zeta potential measured for the composite polysaccharide microspheres prepared in Example 1 and Example 2 of the present invention;

[0027] Figure 4 is a SEM image of the composite polysaccharide microspheres prepared in Example 2 of the present invention;

[0028] Figure 5is a particle size distribution diagram of the composite polysaccharide microspheres prepared in Example 2 of the present invention;

[0029] Figure 6 is the NO release amount of the composite polysaccharide microspheres prepared in Example 2 of the present invention;

[0030] Figure 7 This is the in vivo imaging after administration of composite polysaccharide microspheres to different groups;

[0031] Figure 8 The changes in colon length of mice in different experimental groups (n=5; *, P<0.05; **, P<0.01;

[0032] ***, P < 0.001, mean ± SEM);

[0033] Fig. 9 These are H&E images of the pathological structures of the colon of mice in different experimental groups. Specific implementation methods

[0035] The present invention is further described below in conjunction with embodiments, but the description of the embodiments does not impose any limitation on the protection scope of the present invention.

[0036] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention, and the terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. In addition, although demonstrations of parameters including specific values ​​may be provided herein, it should be understood that the parameters do not need to be exactly equal to the corresponding values, but may be approximated to the corresponding values ​​within an acceptable error tolerance or design constraint.

[0037] Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels.

[0038] Example 1 Extraction of Snow Fungus Polysaccharide and Preparation of Derivatized Snow Fungus Polysaccharide

[0039] The steps of preparing snow fungus polysaccharide by water extraction and alcohol precipitation method of the present invention are as follows: first, weigh 5g of snow fungus, perform ultrasonic extraction at 60°C for 2 hours, the ratio of extract to solid is 1:1g·L, extract twice in total, then centrifuge at 6000rpm / min for 10 minutes, take the precipitate and add anhydrous ethanol for alcohol precipitation, the final concentration of ethanol in the alcohol precipitation is 90%, after standing at 4°C for 48 hours, centrifuge at 6000rpm / min for 10 minutes to separate the precipitate. Subsequently, the obtained alcohol precipitate is dissolved in deionized water, centrifuged to remove the supernatant. Take the precipitate and use Sevag reagent (n-butanol: chloroform = 1:4) to remove protein in a volume ratio of 1:4, and repeat this step until there is no protein content. Then, collect the polysaccharide solution, after 48 hours of running water dialysis, freeze-dry, and finally obtain freeze-dried snow fungus polysaccharide, named snow fungus polysaccharide. Determine its relative molecular mass ( Figure 1 ), and its aldehyde content ( Figure 2 ).

[0040] The steps of preparing the derivatized snow fungus polysaccharide of the present invention are as follows: first, dissolving the snow fungus polysaccharide with ultrapure water to make the concentration of the snow fungus polysaccharide solution 5 mg / mL, then slowly dripping 5 mg / mL of L-Arg solution into the snow fungus polysaccharide solution (volume ratio is 1:10-40), and then placing it in a dark environment to react for 8-12 hours. The solution is placed in a dialysis bag for 3 days, and the water is changed every morning and evening. Then the sample is collected and freeze-dried in a freeze dryer for 2 days to obtain a derivatized snow fungus polysaccharide gel for the connection of L-Arg.

[0041] Example 2 Preparation of EGCG-loaded composite polysaccharide microspheres

[0042] The derivatized snow fungus polysaccharide was dissolved in ultrapure water at a concentration of 5 mg / mL, and then 5 mg / mL EGCG solution was slowly dripped into the snow fungus polysaccharide solution, and the reaction was carried out in the dark for 8-12 hours with magnetic stirring, wherein the volume ratio of EGCG solution to derivatized snow fungus polysaccharide was 1:10-40.

[0043] Add 2mL of Span-80 emulsifier to 100mL of paraffin liquid and stir at 60℃ for 1h to make the oil phase uniform. Then slowly drop the EGCG / derivatized snow swallow polysaccharide solution into the oil phase and stir slowly. After stirring for 2 hours, move the solution into a 4℃ cold room, add 500μL of glutaraldehyde solution and stir overnight. Cross-link to obtain EGCG-loaded composite polysaccharide microspheres. Add acetone to the solution and wash three times to wash off the oil phase, then wash three times with anhydrous ethanol to wash off the acetone, and then wash three times with deionized water to remove the anhydrous ethanol. Freeze-dry to obtain the final composite polysaccharide microspheres. Its potential ( Figure 3 ), take transmission electron microscope photos of microspheres ( Figure 4 ), measure the particle size distribution of microspheres ( Figure 5) and microsphere NO release ( Figure 6 ).

[0044] Example 3 Establishment of ulcerative colitis model and therapeutic effect of composite polysaccharide microspheres

[0045] First, 15 experimental mice were adaptively fed for one week and then randomly divided into 3 groups, with 5 mice in each group. The 3 groups were blank control group, model group and treatment group (complex polysaccharide microspheres). During the experiment, the mice in the blank control group drank sterile water freely and were given sterile water by gavage every day. The mice in the other groups drank 2.5% DSS (dextran sulfate sodium) solution freely for 5 days to establish an ulcerative colitis mouse model. The model group was given an equal volume of sterile water by gavage during the administration period. The treatment group was gavaged with 5 mg / mL complex polysaccharide microspheres starting on the 6th day after the model was established, and continued to be administered for 3 days. During the treatment period, the incidence of mice was checked every day and the weight changes were recorded, and the pathological characteristics of each mouse were scored. After the experiment, the colon morphology and length of each group of mice were compared, and pathological histology was observed to clarify the therapeutic effect of complex polysaccharide microspheres on ulcerative colitis mice.

[0046] C57BL / 6 male wild-type mice aged 8-10 weeks and weighing 18-22 g were used in the experiment. Mice were housed in a specific pathogen-free environment throughout the adaptation period and the study, following a 12-hour light-dark cycle (temperature 21±2°C, relative humidity 45±10%). During the experiment, mice had free access to food and water. All animal experiments were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and have been approved by the Laboratory Animal Ethics Committee of Nanjing University.

[0047] Conclusion: The composite polysaccharide microsphere solution was used to perform in vivo imaging of DSS-induced ulcerative colitis, and a blank control was set up to evaluate the treatment of DSS-induced ulcerative colitis by composite polysaccharide microspheres. The results showed that composite polysaccharide microspheres had a certain targeting effect on ulcerative mice ( Figure 7 ), and the colon length of mice after treatment ( Figure 8 ) and pathological results ( Fig. 9 ) has some relief.

[0048] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the contents of the present invention and implement them accordingly. They cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing composite polysaccharide microspheres capable of releasing NO, characterized in that: The steps include: (1) cross-linking snow nut polysaccharide with L-ARG to obtain snow nut polysaccharide hydrogel containing L-ARG through Mannich reaction; (2) EGCG was loaded onto the L-ARG-containing snow fungus polysaccharide hydrogel through the Schiff base reaction, and the composite polysaccharide microspheres that can release NO were obtained by the emulsification method.

2. The preparation method according to claim 1, characterized in that: Step (1) specifically comprises: preparing a snow fungus polysaccharide solution, then dropping an L-Arg solution into the snow fungus polysaccharide solution, then placing the solution in a dark environment at room temperature to react, and then dialysis with running water and freeze-drying to obtain a snow fungus polysaccharide hydrogel containing L-ARG.

3. The preparation method according to claim 2, characterized in that: The concentration of the snow fungus polysaccharide solution is 5-10 mg / ml, the concentration of the L-Arg solution is 1-5 mg / ml, the mass ratio of the snow fungus polysaccharide to L-Arg is 20:1-40:1, the reaction time is 8-24 hours, and the dialysis time is 3-5 days.

4. The preparation method according to any one of claims 1 to 3, characterized in that Step (2) is specifically as follows: adding water to the snow fungus polysaccharide hydrogel containing L-ARG to prepare a solution, then dropping the EGCG solution into the snow fungus polysaccharide solution containing L-ARG to react as the water phase, adding an emulsifier to the paraffin liquid as the oil phase, stirring to make the oil phase uniform, dropping the water phase into the oil phase, stirring evenly, adding glutaraldehyde solution and stirring overnight to obtain composite polysaccharide microspheres.

5. The preparation method according to claim 4, characterized in that: The solution concentration of the snow fungus polysaccharide hydrogel containing L-ARG is 5 mg / mL, the EGCG solution concentration is 25 mg / mL, the mass ratio of snow fungus polysaccharide to EGCG is 20:1-40:1, and the reaction time is 8-24 hours; the emulsifier is Span-80, the volume ratio of the emulsifier to the paraffin liquid is 1:50, the concentration of the glutaraldehyde solution is 50%, and the temperature of the reaction system during the glutaraldehyde cross-linking reaction is 4°C.

6. The preparation method according to claim 5, characterized in that: The preparation method of snow fungus polysaccharide is water extraction and alcohol precipitation.

7. The preparation method according to claim 6, characterized in that: The preparation method of snow fungus polysaccharide is specifically as follows: weigh and dissolve crude snow fungus polysaccharide, ultrasonicate at 60°C for 2h, centrifuge at low speed to discard the supernatant, take the precipitate and add anhydrous ethanol for alcohol precipitation, the final concentration of the alcohol precipitation is 90%, after standing at 4°C for 24h, remove the supernatant, add Sevag reagent, remove protein at a volume ratio of 1:4 between snow fungus polysaccharide and Sevag reagent, and after 72 hours of running water dialysis, freeze-dry to obtain the gelatinizable snow fungus polysaccharide.

8. The NO-releasing composite polysaccharide microspheres prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the NO-releasing composite polysaccharide microspheres according to claim 8 in the preparation of a drug for treating inflammatory bowel disease.

10. The use according to claim 9, characterized in that: The inflammatory bowel disease refers to ulcerative colitis or Crohn's disease.

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