Oral microsphere for immunomodulation, preparation method therefor, and use thereof

Abstract

An oral microsphere for immunomodulation, a preparation method therefor, and use thereof. A microsphere-loaded small molecule drug is released to the colon to regulate bile acid metabolized by bacteria, and the microsphere releases an immunomodulator, which is absorbed by the small intestine, thereby improving the immune system of an organism. The oral microsphere for immunomodulation comprises: a biological protein, a macromolecular degradable material, a small molecule drug gut restrict-7, and an immune activator.

Description

An oral microsphere for immune regulation, preparation method and application Technical Field

[0001] The present invention relates to the technical field of nanomaterials and bioanalysis, and in particular to an immunomodulatory oral microsphere, a preparation method and application of the immunomodulatory oral microsphere. Background Art

[0002] Liver cancer is currently one of the leading causes of cancer-related deaths, and it has brought a huge health burden to the world. We urgently need to understand the pathogenesis of liver cancer and develop more effective treatment strategies. Due to its unique location and blood supply, the liver is exposed to a large number of food antigens and microbial antigens from the intestine. In order to maintain the body's homeostasis and avoid excessive inflammation, the liver must maintain a balance between immune response and tolerance. At the same time, the liver is also a metabolically active organ. For example, the liver is the central hub of bile acid metabolism. Bile acids not only play an important role in the absorption and metabolism of lipids, but also act as endocrine signaling molecules to regulate various biological processes, including immune responses. The enterohepatic circulation is the main channel for communication between the liver and the small intestine. The significant role of the ternary regulatory relationship between intestinal flora, bile acid metabolism, and tumor immunity in the formation of liver cancer can provide a theoretical basis for new strategies for immunotherapy of liver cancer.

[0003] The only existing drug that can regulate bile acids in vivo is the bile acid sequestrant cholestyramine, an anionic resin. Its disadvantage is that it can only passively adsorb excess bile acids through the porous structure of the chelator, and cannot address the immune problems caused by excessive bile acids from the root of bacterial metabolism. On the other hand, combined with immune agonists, it can further activate the liver's immune microenvironment and induce the body to produce tumor immune memory, which is expected to greatly improve anti-tumor immune efficacy, inhibit tumor growth, and reduce tumor recurrence. Therefore, it is necessary to design an oral microsphere that can simultaneously regulate the hepatoenteroidal bile acid cycle and deliver immune agonists.

[0004] Summary of the Invention

[0005] In order to overcome the defects of the prior art, the technical problem to be solved by the present invention is to provide an immune-regulating oral microsphere and a preparation method thereof, which releases small molecule drugs loaded on the microspheres into the colon, regulates bile acid metabolism of bacteria, and releases immunomodulators from the microspheres, which are absorbed through the small intestine and improve the body's immune system.

[0006] The technical solution of the present invention is: the oral microspheres for immune regulation include: biological protein, high molecular degradable material, small molecule drug gut restrict-7, and immune activator.

[0007] A method for preparing immunomodulatory oral microspheres comprises the following steps:

[0008] (1) adding a certain concentration of poly(lactic acid-co-glycolic acid) dichloromethane solution to a certain concentration of GR-7 dimethyl sulfoxide (DMSO) solution, stirring at room temperature to mix them uniformly, to obtain a first solution;

[0009] (2) the first solution is introduced into the oil phase channel of the microfluidic chip at a certain flow rate, and a polyvinyl alcohol (PVA) aqueous solution of a certain concentration is introduced into the water phase channel of the microfluidic chip at a certain flow rate; the liquid in the outflow channel of the microfluidic chip is collected and precipitated by centrifugation to obtain GR-7 loaded microspheres, which are called PLGA microspheres;

[0010] (3) adding a certain volume of Tris-HCl solution to a certain mass of Bacillus subtilis spores, adding crushing beads, and using a tissue homogenizer to crush the spore solution;

[0011] (4) The supernatant after homogenization was centrifuged to obtain the precipitate, which was then washed with Tris-HCl solution. A lysozyme solution of a certain concentration preheated at 37 degrees Celsius was added to the homogenized precipitate, shaken for 10 minutes, and then centrifuged to retain the precipitate.

[0012] (5) Add a certain concentration of sodium chloride solution (NaCl) to the precipitate, repeat the centrifugation step, and wash twice;

[0013] (6) Add pure water to the precipitate and centrifuge repeatedly for three times to obtain the biological protein.

[0014] (7) After a certain concentration of biological protein is treated with a cell disruptor, a certain concentration of α-GalCer solution is added, stirred overnight, and centrifuged to retain the precipitate. The precipitate is fully mixed with the PLGA microsphere solution, stirred at room temperature for 30 minutes, and the precipitate obtained after centrifugation is the oral microsphere.

[0015] Compared with the bile acid sequestrant cholestyramine, the oral immunomodulatory microspheres of the present invention include: biological protein, polymer degradable material, small molecule drug gut restrict-7, and immune activator. The small molecule drug loaded on the microspheres is released into the colon, regulating the bile acid metabolism of bacteria. The microspheres release the immunomodulator, which is absorbed through the small intestine and improves the body's immune system.

[0016] Also provided is the immunomodulatory oral microspheres for: transmission electron microscopy imaging characterization, scanning electron microscopy imaging characterization, or determining the co-localization of components of the microspheres by fluorescent dyes. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG1 is a TEM image of the oral microspheres prepared in Example 1 of the present invention.

[0018] FIG2 is a SEM image of the oral microspheres prepared in Example 2 of the present invention.

[0019] FIG3 is a flow chart of a method for preparing immunomodulatory oral microspheres according to the present invention. DETAILED DESCRIPTION

[0020] The oral immunomodulatory microspheres include biological proteins, high-molecular-weight degradable materials, small-molecule drug gut restrict-7, and immune activators.

[0021] Preferably, the mass ratio of the polymer degradable material to the biological protein is 1:a, where a is 1 to 100; the mass ratio of the polymer degradable material to the small molecule drug is 1:b, where b is 10 to 200; and the ratio of the biological protein to the immune activator is 1:c, where c is 10 to 200.

[0022] Preferably, the biological protein is spore protein of Bacillus subtilis; the high molecular biodegradable material is polylactic acid-glycolic acid copolymer; and the immune activator is galactosylceramide.

[0023] Preferably, the particle size is 1 to 8 microns.

[0024] As shown in FIG3 , a method for preparing the immunomodulatory oral microspheres is also provided, which comprises the following steps:

[0025] (1) adding a certain concentration of poly(lactic acid-co-glycolic acid) dichloromethane solution to a certain concentration of GR-7 dimethyl sulfoxide (DMSO) solution, stirring at room temperature to mix them uniformly, to obtain a first solution;

[0026] (2) the first solution is introduced into the oil phase channel of the microfluidic chip at a certain flow rate, and a polyvinyl alcohol (PVA) aqueous solution of a certain concentration is introduced into the water phase channel of the microfluidic chip at a certain flow rate; the liquid in the outflow channel of the microfluidic chip is collected and precipitated by centrifugation to obtain GR-7 loaded microspheres, which are called PLGA microspheres;

[0027] (3) adding a certain volume of Tris-HCl solution to a certain mass of Bacillus subtilis spores, adding crushing beads, and using a tissue homogenizer to crush the spore solution;

[0028] (4) The supernatant after homogenization was centrifuged to obtain the precipitate, which was then washed with Tris-HCl solution. A lysozyme solution of a certain concentration preheated at 37 degrees Celsius was added to the homogenized precipitate, shaken for 10 minutes, and then centrifuged to retain the precipitate.

[0029] (5) Add a certain concentration of sodium chloride solution (NaCl) to the precipitate, repeat the centrifugation step, and wash twice;

[0030] (6) Add pure water to the precipitate and centrifuge repeatedly for three times to obtain the biological protein.

[0031] (7) After a certain concentration of biological protein is treated with a cell disruptor, a certain concentration of α-GalCer solution is added, stirred overnight, and centrifuged to retain the precipitate. The precipitate is fully mixed with the PLGA microsphere solution, stirred at room temperature for 30 minutes, and the precipitate obtained after centrifugation is the oral microsphere.

[0032] Compared with the bile acid sequestrant cholestyramine, the oral immunomodulatory microspheres of the present invention include: biological protein, polymer degradable material, small molecule drug gut restrict-7, and immune activator. The small molecule drug loaded on the microspheres is released into the colon, regulating the bile acid metabolism of bacteria. The microspheres release the immunomodulator, which is absorbed through the small intestine and improves the body's immune system.

[0033] Preferably, in step (1), the concentration of the PLGA dichloromethane solution is 1-10%, and the concentration of the GR-7 dimethyl sulfoxide (DMSO) solution is 10-100 mg / ml;

[0034] In the step (2), the concentration of the PVA aqueous solution is 2-10%, and the flow rate ratio of the first solution and the PVA aqueous solution is 1-d, where d is 1-10;

[0035] In step (3), the mass of the spores is 0.1 to 100 mg; the concentration of the Tris-HCl solution is 1 to 100 mmol per liter; the parameters of the tissue homogenizer are set as follows: operating temperature of 4 degrees, speed of 6000 rpm, and operating time of 20 minutes;

[0036] In the step (4), the concentration of the lysozyme solution is 10 to 100 μg / ml; the centrifugal speed is 10,000 g, and the centrifugal time is 5 to 15 minutes.

[0037] Preferably, in step (5), the concentration of the NaCl solution is 0.5 to 2 mol / L; the centrifugal speed is 10,000 g, and the centrifugal time is 5 to 15 minutes.

[0038] Preferably, in step (6), pure water is added to make the mass concentration of the precipitate be 1 to 10 mg / ml; the centrifugal speed is 10000 g, and the centrifugal time is 5 to 15 minutes.

[0039] Preferably, in step (7), the working parameters of the cell disruptor are set as follows: power 100-300W, working time 30-60 minutes; centrifugal speed 200-300g, centrifugal time 3-10 minutes.

[0040] Also provided is the immunomodulatory oral microspheres for: transmission electron microscopy imaging characterization, scanning electron microscopy imaging characterization, or determining the co-localization of components of the microspheres by fluorescent dyes.

[0041] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0042] Example 1 Preparation of oral microspheres and TEM characterization

[0043] A) adding a 1% poly(lactic-co-glycolic acid) (PLGA) solution in dichloromethane to a 100 mg / ml GR-7 solution in dimethyl sulfoxide (DMSO), stirring at room temperature to mix uniformly, to obtain a first solution;

[0044] B) The first solution was introduced into the oil phase channel of the microfluidic chip at a flow rate of 3 μl / min, and a 2% polyvinyl alcohol (PVA) aqueous solution was introduced into the aqueous phase channel of the microfluidic chip at a flow rate of 6 μl / min. The liquid in the outflow channel of the microfluidic chip was collected and the precipitate was collected by centrifugation to obtain GR-7-loaded microspheres, referred to as PLGA microspheres.

[0045] C) 1 ml of a 10 mmol / L Tris-HCl solution was added to 1 mg of Bacillus subtilis spores, disrupting beads were added, and the spore solution was disrupted using a tissue homogenizer.

[0046] D) Centrifuge the homogenized supernatant to remove the precipitate, then add Tris-HCl solution to wash. Add 50 μg / ml lysozyme solution preheated at 37°C to the homogenized precipitate, shake for 10 minutes, and centrifuge to retain the precipitate.

[0047] E) Add 1 M sodium chloride solution (NaCl) to the pellet and repeat the centrifugation step to wash twice.

[0048] F) Add pure water to the precipitate and centrifuge repeatedly for three times to obtain the biological protein.

[0049] G) The obtained biological protein was treated with a cell disruptor, and then 1 mg / ml α-GalCer solution was added. The mixture was stirred overnight and centrifuged to obtain a precipitate. The precipitate was thoroughly mixed with the PLGA microsphere solution and stirred at room temperature for 30 minutes. The precipitate obtained after centrifugation was the oral microspheres.

[0050] H) Disperse the microspheres in pure water, take 10 μl and drop it onto a copper mesh. After the sample is dry, observe and photograph the morphology of the microspheres under a transmission electron microscope.

[0051] See Figure 1. Figure 1 is a TEM image of the oral microspheres prepared in Example 1 of the present invention.

[0052] Example 2 SEM Characterization of Oral Microspheres

[0053] A) The oral microspheres prepared in Example 1 were dispersed in pure water, and 10 μl was added dropwise onto a silicon wafer. After the sample dried, the microspheres were observed and photographed under a scanning electron microscope to determine their morphological characteristics.

[0054] See Figure 2. Figure 2 is a SEM image of the oral microspheres prepared in Example 2 of the present invention.

[0055] Example 3 Co-localization Verification of Fluorescently Dyed Oral Microspheres

[0056] A) adding a 1% poly(lactic-co-glycolic acid) (PLGA) solution in dichloromethane to a 1 mmol / L fluorescein isothiocyanate (FITC) dye solution in dimethyl sulfoxide (DMSO), and stirring at room temperature to mix the mixture uniformly to obtain a first solution;

[0057] B) The first solution was introduced into the oil phase channel of the microfluidic chip at a flow rate of 3 μl / min, and a 2% polyvinyl alcohol (PVA) aqueous solution was introduced into the aqueous phase channel of the microfluidic chip at a flow rate of 6 μl / min. The liquid in the outflow channel of the microfluidic chip was collected and the precipitate was collected by centrifugation to obtain FITC-loaded microspheres, referred to as PLGA microspheres.

[0058] C) 1 ml of a 10 mmol / L Tris-HCl solution was added to 1 mg of Bacillus subtilis spores, disrupting beads were added, and the spore solution was disrupted using a tissue homogenizer.

[0059] D) Centrifuge the homogenized supernatant to remove the precipitate, then add Tris-HCl solution to wash. Add 50 μg / ml lysozyme solution preheated at 37°C to the homogenized precipitate, shake for 10 minutes, and centrifuge to retain the precipitate.

[0060] E) Add 1 M sodium chloride solution (NaCl) to the pellet and repeat the centrifugation step to wash twice.

[0061] F) Add pure water to the precipitate and centrifuge repeatedly for three times to obtain the biological protein.

[0062] G) The obtained biological protein was treated with a cell disruptor, and then 1 mmol / L DiD dye solution was added. The mixture was stirred overnight, centrifuged to obtain a precipitate, and then thoroughly mixed with the PLGA microsphere solution. The mixture was stirred at room temperature for 30 minutes. The precipitate obtained after centrifugation was the oral microspheres.

[0063] H) Disperse the microspheres in pure water, take 200 μl and drop it into a confocal glass dish. After the sample settles, observe and photograph the morphology of the microspheres under a laser confocal microscope.

[0064] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of protection of the technical solution of the present invention.

Claims

1. An orally administered microsphere for immunomodulation, characterized in that: It includes: Biological protein, polymer biodegradable material, small molecule drug gut restrict-7, immune activator.

2. The immunomodulatory oral microspheres according to claim 1, wherein: The mass ratio of the polymer biodegradable material to the biological protein is 1:a, where a is 1 to 100; the mass ratio of the polymer biodegradable material to the small molecule drug is 1:b, where b is 10 to 200; the ratio of the biological protein to the immune activator is 1:c, where c is 10 to 200.

3. The immunomodulatory oral microspheres according to claim 2, wherein: The biological protein is the spore protein of Bacillus subtilis; the polymer biodegradable material is poly(lactic-co-glycolic acid); the immune activator is galactosylceramide.

4. The immunomodulatory oral microspheres according to claim 3, wherein: Its particle size is 1 to 8 microns.

5. The preparation method of the immunomodulatory oral microspheres according to claim 4, wherein: It includes the following steps: (1) Add a dichloromethane solution of poly(lactic-co-glycolic acid) at a certain concentration to a dimethyl sulfoxide (DMSO) solution of GR-7 at a certain concentration, and stir at room temperature to mix evenly to obtain a first solution; (2) Let the first solution enter the oil phase channel of the microfluidic chip at a certain flow rate, and let an aqueous solution of polyvinyl alcohol (PVA) at a certain concentration enter the aqueous phase channel of the microfluidic chip at a certain flow rate; collect the liquid flowing out of the microfluidic chip, centrifuge to collect the precipitate, and obtain microspheres loaded with GR-7, called PLGA microspheres; (3) Add a certain volume of Tris-HCL solution to a certain mass of Bacillus subtilis spores, add crushing beads, and use a tissue homogenizer to crush the spore solution; (4) Centrifuge the supernatant after homogenization to obtain the precipitate, and then add Tris-HCL solution for washing; add a lysosome solution at a certain concentration preheated in an environment at 37 degrees Celsius to the homogenized precipitate, shake in a shaker for 10 min, and then centrifuge to leave the precipitate; (5) Add a sodium chloride (NaCl) solution at a certain concentration to the precipitate, repeat the centrifugation step, and wash twice; (6) Add pure water to the precipitate, repeatedly centrifuge and wash, repeat three times, and the obtained precipitate is the biological protein; (7) After treating a certain concentration of biological protein with a cell disruptor, add a certain concentration of α-GalCer solution, stir overnight, centrifuge to leave the precipitate, fully mix the precipitate with the PLGA microsphere solution, stir at room temperature for 30 minutes, and the precipitate obtained after centrifugation is the oral microsphere.

6. The preparation method of the immunomodulatory oral microspheres according to claim 5, wherein: In the step (1), the concentration of the PLGA dichloromethane solution is 1 to 10%, and the concentration of the GR-7 dimethyl sulfoxide (DMSO) solution is 10 - 100 mg / ml; In the step (2), the concentration of the PVA aqueous solution is 2 to 10%, and the flow rate ratio of the first solution to the PVA aqueous solution is 1 to d, where d is 1 to 10; In the step (3), the mass of the spores is 0.1 to 100 mg; the concentration of the Tris-HCL solution is 1 to 100 mmol / L; the parameter settings for crushing with the tissue homogenizer are: working temperature is 4 degrees, rotation speed is 6000 rpm, and working time is 20 min; In the step (4), the concentration of the lysosome solution is 10 to 100 μg / ml; the rotation speed for centrifugation is 10000g, and the centrifugation time is 5 to 15 minutes.

7. The preparation method of the immunomodulatory oral microspheres according to claim 6, characterized in that: In the step (5), the concentration of the NaCl solution is 0.5 to 2 moles per liter; the centrifugation speed is 10,000 g, and the centrifugation time is 5 to 15 minutes.

8. The preparation method of the immunomodulatory oral microspheres according to claim 7, characterized in that: In the step (6), pure water is added such that the mass concentration of the precipitate is 1 to 10 mg per milliliter; the centrifugation speed is 10,000 g, and the centrifugation time is 5 to 15 minutes.

9. The preparation method of the immunomodulatory oral microspheres according to claim 8, wherein: In the step (7), the operating parameters of the cell disruptor are set as follows: power 100 to 300 W, working time 30 to 60 minutes; the centrifugation speed is 200 to 300 g, and the centrifugation time is 3 to 10 minutes.

10. The orally administered microspheres for immunomodulation according to claim 1 are used for: transmission electron microscopy imaging characterization, scanning electron microscopy imaging characterization, or determining the co-localization of the components of the microspheres by fluorescent dyes.

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