Oral monoclonal antibody micro-coated nano-polysaccharide microsphere preparation for targeted therapy of inflammatory bowel disease and preparation method of oral monoclonal antibody micro-coated nano-polysaccharide microsphere preparation

Abstract

The invention discloses an oral monoclonal antibody micro-coated nanopolysaccharide microsphere preparation for targeted therapy of inflammatory bowel diseases and a preparation method thereof. According to the method, oxidized mannan and an anti-TNF-alpha monoclonal antibody are mixed and cross-linked by a cross-linking agent containing a diselenide bond to form the drug-loaded nanoparticles. Mixing the drug-loaded nanoparticles with pectin, dropwise adding the mixture into a calcium chloride solution through an electrostatic spinning device, and carrying out ionic crosslinking to obtain the oral monoclonal antibody micro-coated nano polysaccharide microsphere preparation with the particle size range of 180-200 microns. The drug-loaded polysaccharide microsphere delivers the wrapped drug-loaded nanoparticles to a colitis disease part through electrostatic interaction. The drug-loaded nanoparticles are targeted to macrophages, release of the monoclonal antibody is accelerated under the triggering of high-concentration active oxygen at an inflammation part, and the oral stability and the treatment effect of the monoclonal antibody drug are improved. The polysaccharide is used as a monoclonal antibody targeted delivery carrier, raw materials are cheap and easy to obtain, the preparation process is simple and convenient, reaction conditions are mild, and application and development are easy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to an oral anti-TNF-α monoclonal antibody (mAb) microencapsulated polysaccharide microspheres for treating inflammatory bowel disease (IBD) and a preparation method thereof. BACKGROUND

[0002] IBD is a chronic intestinal inflammatory disease with unknown etiology, easy recurrence and incurable for a lifetime, mainly including ulcerative colitis (UC) and Crohn's disease (CD). The main treatment in clinic is chemical drugs, but long-term high-dose use of chemical drugs has great systemic toxicity and unsatisfactory curative effect. Anti-tumor necrosis factor alpha (TNF-α) is the most closely related inflammatory factor to the occurrence, development and outcome of IBD. Biological therapy targeting TNF-α provides a new choice for the treatment of moderate / severe IBD that is resistant or dependent on chemical drugs, and ineffective for traditional treatment. In recent years, anti-TNF-α monoclonal antibodies such as infliximab, adalimumab and certolizumab have been put on the market, but these monoclonal antibodies are for injection. Non-specific neutralization of TNF-α inevitably inhibits the immune system, which may lead to serious adverse reactions such as severe infection and malignant tumor, and the production of anti-drug antibodies may lead to ineffective treatment. Therefore, designing an oral mAb drug delivery system to reduce the side effects of mAb has broad application prospects.

[0003] Oral administration is the most ideal administration method for IBD treatment, which is simple, safe and has high patient compliance. However, protein drugs are easily degraded by digestion when taken orally, and they have high water solubility and large molecular weight, which cannot penetrate the intestinal mucus barrier to reach target cells, resulting in extremely low bioavailability. In addition, the concentration of reactive oxygen species (ROS) in the inflamed intestinal tissue is about 10-100 times higher than that in healthy intestinal tissue, and there are a large number of positively charged eosinophil cationic protein and transferrin in the inflamed intestinal tissue. Macrophages are the main inflammatory cells that secrete TNF-α and are also one of the targets of anti-TNF-α monoclonal antibodies. Designing a microenvironment-responsive carrier based on these characteristics is expected to improve the local drug concentration and improve the treatment effect. Micro / nano drug delivery systems based on polysaccharides are good carriers for oral delivery of mAbs. Polysaccharide carriers can prevent nucleic acid drugs from being destroyed by gastric acid or digestive enzymes, while improving targeting and transport efficiency. Polysaccharides containing mannose can be recognized by the mannose receptor on the surface of immune cells and can be used as specific targeting carriers. Pectin is a natural acidic heteropolysaccharide with a main chain of polygalacturonic acid. Negatively charged pectin gel microspheres can protect the encapsulated drugs from enzymatic degradation in gastric acid and can be degraded by colonic flora to release the encapsulated drugs, making it an ideal colon-targeted drug delivery carrier.

[0004] The present application utilizes diselenium bond cross-linking oxidized mannitol to load anti-TNF-alpha monoclonal antibody to form drug-loaded nanoparticles, and then forms micro-encapsulated nanocellulose hydrogel microspheres by ion cross-linking with pectin. The preparation can significantly improve the stability of the monoclonal antibody in the gastrointestinal environment, and position it to the colon site, slowly releasing the drug-loaded nanoparticles. The drug-loaded nanoparticles further target intestinal inflammation tissue macrophages, and respond to the release of monoclonal antibodies under the action of high-concentration ROS in the inflammatory tissue, thereby treating IBD. At present, there is no research report on oral preparations for targeting intestinal inflammation tissue to deliver monoclonal antibodies using pectin and mannose as micro-nano carriers. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides an oral monoclonal antibody micro-encapsulated nanopolysaccharide microsphere preparation for targeted treatment of inflammatory bowel disease and a preparation method thereof. The method is simple, the raw materials are cheap and easy to obtain, and is easy to apply and develop.

[0006] The technical scheme adopted by the present application is as follows: An oral monoclonal antibody micro-encapsulated nanopolysaccharide microsphere for targeted treatment of inflammatory bowel disease is obtained by the following method: first, oxidized mannitol with sodium periodate is mixed with anti-TNF-alpha monoclonal antibody, and cross-linked by a cross-linking agent containing a diselenium bond to form drug-loaded nanoparticles. Then the drug-loaded nanoparticles are mixed with pectin, and ion cross-linking is performed by dropping into a calcium chloride solution through an electrospinning device to obtain an oral monoclonal antibody micro-encapsulated nanopolysaccharide microsphere preparation.

[0007] A preparation method of an oral monoclonal antibody micro-encapsulated nanopolysaccharide microsphere preparation for targeted treatment of inflammatory bowel disease, the preparation steps are as follows: Preparation of drug-loaded nanoparticles: dissolve the mannose in deionized water, add a certain amount of sodium periodate solution drop by drop, avoid light reaction for a period of time, then add ethylene glycol to terminate the reaction. After dialysis of the reaction mixture for 3 days, freeze-drying is performed to obtain aldehyde-containing oxidized mannose. Dissolve the oxidized mannose in a phosphate buffer, slowly add the monoclonal antibody solution under stirring, and cross-link the aldehyde group with 2, 2'-diselenoic acid bis(ethane-1-amine). After adding a reducing agent and reacting for a period of time, ice bath ultrasonic, high-speed centrifugation, to obtain drug-loaded nanoparticles. The particle size of the nanoparticles is in the range of 100-200 nm; (2) Preparation of micro-encapsulated nanopolysaccharide microspheres: dissolve the pectin in deionized water, mix it evenly with the drug-loaded nanoparticle solution, and add it to a syringe with a metal needle. Add it to a calcium chloride solution through an electrospinning device. After stirring the obtained microsphere suspension at 150 rpm for 30 minutes, add it to a calcium chloride solution, stir for a period of time, centrifuge, and wash with water for 3 times to obtain a micro-encapsulated nanopolysaccharide microsphere solution loaded with monoclonal antibody. The particle size of the microspheres is in the range of 180-220 μm.

[0008] Preferably, the molecular weight of the mannose in step (1) is 10-100 kDa.

[0009] Preferably, the oxidation degree of the mannan in step (1) is 10-50%.

[0010] Preferably, the molar ratio of the aldehyde group of the oxidized mannan to 2,2'-diseleno bis (ethane-1-amine) in step (1) is 1:1-4:1.

[0011] Preferably, the reducing agent in step (1) is sodium borohydride or sodium cyanoborohydride.

[0012] Preferably, the mass ratio of the oxidized mannan to the monoclonal antibody in step (1) is 10-20:1, and the drug loading of the monoclonal antibody is 5%-20%.

[0013] Preferably, the monoclonal antibody drug in step (1) is infliximab, adalimumab or certolizumab.

[0014] Specifically, the period of the periodate oxidation reaction in step (1) is 3-24 hours, the pH of the phosphate buffer is 7.4, the reaction time is 3-6 hours, the reduction time is 12-48 hours, and the stirring speed is 600-1000 rpm.

[0015] Preferably, the mass ratio of the pectin to the drug-loaded nanoparticles in step (2) is 3-6:1.

[0016] Preferably, the concentration of calcium chloride in step (2) is 1-5%.

[0017] Specifically, the molecular weight of the pectin in step (2) is in the range of 50-200 kDa, the galacturonic acid content is greater than 70%, and the esterification degree is less than 50%.

[0018] Specifically, the metal needle in step (2) is 23G, the electrospinning flow rate is 0.5 mL / min, and the voltage is 10 kV.

[0019] The beneficial effects of the present application mainly include: (1) The present application utilizes the colon positioning of pectin and the macrophage targeting effect of mannan, and can transport the monoclonal antibody drug to the intestinal inflammatory site through oral administration, thereby avoiding the adverse reactions caused by injection of monoclonal antibodies and increasing the compliance of patients.

[0020] (2) The micro-encapsulated polysaccharide microspheres prepared by the present application can improve the drug loading efficiency of monoclonal antibodies and their stability in the gastrointestinal tract, increase the retention time of the drug in the intestine, and thereby enhance the efficacy of monoclonal antibodies.

[0021] (3) The microencapsulated polysaccharide microsphere preparation prepared by the present invention has a sustained release effect on monoclonal antibodies. When the diselenide bond is broken under the triggering of high concentrations of ROS in the colon inflammation site, the micro-nanostructure of the preparation is destroyed, and the drug can be released quickly, thus achieving a sustained and controlled release of the monoclonal antibody drug in the gastrointestinal tract.

[0022] (4) The present invention uses natural polysaccharides as carrier materials, which have high biocompatibility and biodegradability. The raw materials are cheap and easily available, the preparation process is simple, the reaction conditions are mild, and it is easy to apply and develop. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a scanning electron micrograph of drug-loaded mannan nanoparticles; Figure 2 This is a scanning electron micrograph of drug-loaded nanopolysaccharide microspheres; Figure 3 is the drug release curve of drug-loaded mannan nanoparticles; Figure 4 Figure 2 shows the changes in body weight of mice with ulcerative colitis during treatment in different experimental groups. Figure 5 The figure shows the changes in colon length after treatment of ulcerative colitis mice in different experimental groups; Figure 6 These are photos of the colon of mice with ulcerative colitis after treatment in different experimental groups. DETAILED DESCRIPTION

[0024] The following is a detailed description of the preparation method of an oral monoclonal antibody microencapsulated polysaccharide microsphere preparation for targeted treatment of inflammatory bowel disease provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention. Example 1

[0025] (1) Preparation of drug-loaded nanoparticles: 0.5 g of 50 kDa mannan was fully dissolved in 20 mL of deionized water, and sodium periodate solution was added dropwise to make the molar ratio of sodium periodate to mannose unit 1:1. After 12 hours of reaction in the dark, 10 mL of ethylene glycol was added and the reaction continued for 30 minutes. The reaction mixture was placed in a dialysis bag (molecular weight cutoff of 14 kDa) and dialyzed for 3 days, then freeze-dried to obtain oxidized mannan. 10 mg of mannan was dissolved in 5 mL of phosphate buffer (0.01 M, pH 7.4), and 0.2 mL of infliximab (5 mg / mL) solution was slowly added dropwise with stirring. 2, 2'-diselenidediylbis(ethane-1-amine) was added at a molar ratio of aldehyde to amino group of 2:1, and the cross-linking reaction was stirred at 4 degrees (600 rpm) for 4 hours. 0.2 g of sodium cyanoborohydride was added and the reaction was stirred at 4 degrees for 24 hours. After ice-bath ultrasonication (power 200 W), high-speed centrifugation (16000 rpm, 20 min) was performed to obtain drug-loaded nanoparticles.

[0026] (2) Preparation of micro-encapsulated polysaccharide microspheres: Pectin was dissolved in deionized water to obtain a pectin solution (10 mg / mL). The pectin solution was mixed with the drug-loaded nanoparticle solution (2 mg / mL) at a mass ratio of 4:1, and then added into a syringe with a 23G metal needle. The mixture was added dropwise into a 5% calcium chloride solution through an electrospinning device at a flow rate of 0.5 mL / min and a voltage of 10 kV. After stirring at 150 rpm for 30 minutes, the obtained microspheres were centrifuged and washed with water three times to obtain a solution of micro-encapsulated polysaccharide microspheres loaded with the monoclonal antibody. Example 2

[0027] (1) Preparation of drug-loaded nanoparticles: 0.25 g of mannitol with a molecular weight of 100 kDa was dissolved in 20 mL of deionized water, and a sodium periodate solution was added dropwise to make the molar ratio of sodium periodate to mannose units 1:1. After 24 hours of reaction in the dark, 10 mL of ethylene glycol was added and the reaction was continued for 30 minutes. The reaction mixture was placed in a dialysis bag (molecular weight cut-off 14 kDa) and dialyzed for 3 days, and then freeze-dried to obtain oxidized mannitol. 10 mg of oxidized mannitol was dissolved in 5 mL of phosphate buffer (0.01 M, pH 7.4), and 0.2 mL of infliximab (5 mg / mL) solution was added dropwise under stirring. According to the molar ratio of aldehyde groups to amino groups of 3:1, 2, 2'-diselenadiyl bis(ethane-1-amine) was added, and the crosslinking reaction was carried out at 4 degrees under stirring (600 rpm) for 4 hours. 0.2 g of sodium cyanoborohydride was added and the reaction was carried out at 4 degrees for 24 hours. After ice bath ultrasonication (power 200 W), high-speed centrifugation (16000 rpm, 20 minutes) was performed to obtain drug-loaded nanoparticles.

[0028] (2) Preparation of micro-encapsulated polysaccharide microspheres: Pectin was dissolved in deionized water to obtain a pectin solution (10 mg / mL). The pectin solution was mixed with the drug-loaded nanoparticle solution (2 mg / mL) at a mass ratio of 4:1, and then added into a syringe with a 23G metal needle. The mixture was added dropwise into a 5% calcium chloride solution through an electrospinning device at a flow rate of 0.5 mL / min and a voltage of 10 kV. After stirring at 150 rpm for 30 minutes, the obtained microspheres were centrifuged and washed with water three times to obtain a solution of micro-encapsulated polysaccharide microspheres loaded with the monoclonal antibody. Example 3

[0029] The morphology of the drug-loaded mannitol nanoparticles and polysaccharide microsphere preparations was observed by scanning electron microscopy. Figure 1 and Figure 2The electron micrographs of the drug-loaded nanoparticles and polysaccharide microspheres prepared in the manner of Example 1 are shown. The results show that the drug-loaded mannitol nanoparticles are ellipsoidal in shape and have a size of about 150-200 nm. The polysaccharide microspheres formed finally are spherical in structure and have a size of about 200 μm. Example 4

[0030] Drug-loaded mannitol nanoparticles and polysaccharide microspheres were prepared according to Example 1. The resulting preparation solution was incubated in simulated gastric fluid at pH 1.2 at 37°C and 100 rpm for 2 hours, centrifuged and then incubated in simulated small intestinal fluid at pH 6.8 for 4 hours, and then transferred into simulated colon fluid at pH 7.4 containing 100 nM hydrogen peroxide for 18 hours. The release solution was taken at specific time points, reacted with a known amount of TNF-α (1000 pg / mL) at room temperature for 1 hour, and the amount of TNF-α not neutralized by the monoclonal antibody was determined by ELISA. Free infliximab (40 ng / mL) was used as a positive control, in which the amount of neutralized TNF was 100%, and the cumulative release rate of the monoclonal antibody was calculated. As shown in Figure 3 As shown, the polysaccharide microspheres can reduce the premature release of the monoclonal antibody in simulated gastric fluid and small intestinal fluid, and the cumulative release rate is less than 25% within 6 hours. In simulated colon fluid containing hydrogen peroxide, the drug-loaded nanoparticles respond to the bond cleavage and the pectin microspheres swell, accelerating the release of the monoclonal antibody, and the cumulative release rate is greater than 85% within 24 hours. Example 5

[0031] BALB / C mice (body weight 18-20 g) were selected and acclimated for one week before the experiment. The mice were randomly divided into 4 groups, i.e. a control group, an intestinal inflammation model group, a monoclonal antibody group and a drug-loaded polysaccharide microsphere group, 6 mice in each group. Except that the control group drank distilled water, the other 2 groups drank 2% dextran sulfate sodium solution for 7 days in a normal environment. On the 7th day, the dextran sulfate sodium solution drunk by the mice in the model group and the administration group was replaced with distilled water, and the monoclonal antibody group and the drug-loaded polysaccharide microsphere group were given 0.1 mL of free monoclonal antibody or polysaccharide microsphere preparation by gavage for 3 consecutive days (the dose of the monoclonal antibody was 10 mg / kg). The body weight of the mice in each group was measured every day, and the ratio of the body weight on that day to the body weight on the 0th day was calculated. The mice were sacrificed on the 12th day, the colon was removed, and the colon length of each group was measured and photographed. The results are shown in Figure 4 and Figure 5 As shown, after the intestinal inflammation model mice were given the drug-loaded polysaccharide microsphere preparation by gavage, the symptoms of body weight loss, blood in stool and diarrhea caused by dextran sulfate sodium in the mice were significantly improved, and the colon length and morphology were close to normal. However, the oral anti-inflammatory efficacy of the same dose of free monoclonal antibody had no significant difference from that of the intestinal inflammation model group, indicating that the drug-loaded polysaccharide microspheres can significantly improve the oral stability and efficacy of the monoclonal antibody.

[0032] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. An oral mononucleosomal polysaccharide microsphere formulation for targeted treatment of inflammatory bowel disease and a method of preparation thereof, the main features of which include: The application discloses a preparation method of oral monoclonal antibody micro-oxypolysaccharide microspheres and application of the microspheres in targeted treatment of inflammatory bowel disease.

2. The oral monoclonal antibody micro-oxypolysaccharide microspheres for targeted treatment of inflammatory bowel disease and the preparation method thereof according to claim 1, wherein the monoclonal antibody drug is infliximab, adalimumab or certolizumab.

3. The oral monoclonal antibody micro-oxypolysaccharide microspheres for targeted treatment of inflammatory bowel disease and the preparation method thereof according to claim 1, wherein the oral monoclonal antibody micro-oxypolysaccharide microspheres for targeted treatment of inflammatory bowel disease have good activity on an intestinal inflammation mouse model, significantly improve symptoms such as weight loss, hematochezia and diarrhea of mice caused by dextran sulfate sodium, and make the colon length and morphology close to normal.

4. The oral mononuclear polysaccharide microspheres formulation for targeting the treatment of inflammatory bowel disease according to claim 1, and a method for preparing the same, characterized in that The preparation process specifically comprises the following steps: (1) preparing drug-loaded nanoparticles: sodium periodate solution is added dropwise into a mannose solution, and the reaction is carried out in the dark; glycol is added to terminate the reaction; the reaction mixture is dialyzed, and freeze-drying is carried out to obtain aldehyde group-containing oxidized mannose, which is re-dissolved in a phosphate buffer solution; monoclonal antibody solution is slowly added dropwise under stirring; 2, 2'-diseleno bis (ethane-1-amine) is added to crosslink the aldehyde group; a reducing agent is added to react; ice-bath ultrasonic treatment is carried out; and high-speed centrifugation is carried out to obtain drug-loaded nanoparticles; (2) preparing micro-oxypolysaccharide microspheres: pectin solution and drug-loaded nanoparticle solution are uniformly mixed, and the mixture is added dropwise into a calcium chloride solution through an electrospinning device; the obtained microsphere suspension is stirred at 150 rpm for 30 minutes, and then added dropwise into a calcium chloride solution for stirring, centrifugation and water washing three times to obtain a monoclonal antibody-loaded micro-oxypolysaccharide microsphere solution.

5. The oral monoclonal antibody micro-oxypolysaccharide microspheres for targeted treatment of inflammatory bowel disease and the preparation method thereof according to claim 4, wherein the mannose in step (1) is a commercially available product, the molecular weight range is 50-100 kDa, the oxidation degree is 10-50%, the molar ratio of mannose to sodium periodate is 1:1, the reaction time is 3-24 hours, the glycol addition amount is 0.5:1 of the volume ratio of the mannose solution, and the dialysis bag has a molecular weight cut-off of 14 kDa.

6. The oral monoclonal antibody micro-oxypolysaccharide microspheres for targeted treatment of inflammatory bowel disease and the preparation method thereof according to claims 4 and 5, wherein the pH of the phosphate buffer solution in step (1) is 7.4, the drug loading amount of the monoclonal antibody is 5%-20%, the mass ratio of the oxidized mannose to the monoclonal antibody is 10-20:1, the molar ratio of the aldehyde group of the oxidized mannose to 2, 2'-diseleno bis (ethane-1-amine) is 1:1-4:1, the reaction time is 3-6 hours, the reducing agent is sodium borohydride or sodium cyanoborohydride, the stirring speed is 600-1000 rpm, and the reduction time is 12-48 hours.

7. The oral monoclonal antibody micro-oxypolysaccharide microspheres for targeted treatment of inflammatory bowel disease and the preparation method thereof according to claims 4-6, wherein the nanoparticle in step (1) has a particle size range of 100-200 nm.

8. The preparation method of claim 4, wherein the mass ratio of pectin to drug-loaded nanoparticles in step (2) is 3-6:1, the concentration of calcium chloride is 1-5%, the molecular weight of pectin is 50-200 kDa, the galacturonic acid content is greater than 70%, the esterification degree is less than 50%, the metal needle is 23G, the electrospinning flow rate is 0.5 mL / min, and the voltage is 10 kV.

9. The preparation method of claim 4, wherein the particle size of the microspheres in step (2) is 180-220 μm.

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