A kind of degradable immunomagnetic beads and preparation method thereof

Abstract

The present invention relates to the field of nanomagnetic materials and the field of biology, and discloses a method for preparing degradable immunomagnetic beads, including preparing degradable magnetic bead microsphere raw materials, using rapid membrane emulsification technology and the degradable magnetic bead microsphere raw materials prepared in step S1 to prepare alginate magnetic bead microspheres, and coupling alginate magnetic bead microspheres with antibodies; also discloses a degradable immunomagnetic bead; and also discloses the application of degradable immunomagnetic beads in products for separating human islet cell clusters. The immunomagnetic beads prepared by the present invention can specifically target islets in the human pancreas, can separate islet cell clusters from exocrine tissues, can greatly shorten the time of islet separation, improve separation efficiency, improve the activity and function of islet cell clusters, improve transplantation effects, and indirectly reduce the cost of islet transplantation surgery; the immunomagnetic beads prepared by the present invention have low toxicity, good magnetic response, good surface biocompatibility, and can be degraded when the solution pH is high.

Description

Technical Field

[0001] The invention relates to the field of nanomagnetic materials and biology, and in particular to degradable immunomagnetic beads and a preparation method thereof. Background Art

[0002] Islet transplantation refers to the separation and purification of islet cell clusters from the donor pancreas, which are usually infused and transplanted into the liver of diabetic patients through the portal vein. This surgical method, as one of the radical cures for type 1 diabetes, has also made many advances in recent years. At present, the following problems and confusions exist in the field of islet transplantation: (1) donor shortage, and a large number of islets required for transplantation (>11000 IEQ / kg body weight); (2) the difficulty of long-term survival of the transplanted organ in the host body, and it is difficult for the patient to achieve a long-term insulin-free state; (3) several problems with immunosuppression, etc. At present, the conventional clinical islet cell cluster separation process is time-consuming, taking about 5-8 hours, and requires 4-5 people to operate at the same time. The results of islet separation are often affected by factors such as the length of patient hospitalization, organ acquisition method, storage time and storage conditions, which often leads to a small density difference between exocrine tissue and islet tissue, making it difficult to achieve the separation of islet and exocrine gland tissue using the density gradient method.

[0003] Immunomagnetic microspheres (IMM) generally refer to uniform, spherical particles with superparamagnetism and protective shells. The basic principle of immunomagnetic cell separation is that magnetic beads can bind to active proteins (antibodies) and be attracted by magnets. After a certain treatment, specific monoclonal antibodies can be bound to magnetic beads, making them carriers of antibodies. After the antibodies on the magnetic beads bind to specific antigen substances, antigen-antibody-magnetic bead immune complexes are formed. When this complex is placed in a magnetic field, the complex will move mechanically due to the action of the magnetic force, so that the complex is separated from other substances, achieving the purpose of separating specific components. Among the various methods for preparing microspheres, the membrane emulsification method proposed by T.Nakashima, M.Shimizu, etc. in the early 1990s is undoubtedly recognized as the best method. The basic principle of the membrane emulsification process is: the two sides of the porous membrane are the dispersed phase and the continuous phase respectively. The dispersed phase is pressed into the continuous phase through the porous membrane at a certain pressure to form droplets. The droplets grow to a certain size on the porous membrane and then detach. If the pore spacing of the porous membrane is too small, the droplets will merge.

[0004] Sodium alginate is a byproduct of extracting iodine and mannitol from brown algae such as kelp or sargassum. Sodium alginate has obvious pH sensitivity. Sodium alginate can quickly form gels under extremely mild conditions. 2+ , Ba 2+ In the presence of cations, the Na on the G unit +Ion exchange reaction occurs with divalent cations, and G units are stacked to form a cross-linked network structure, thus forming a hydrogel. The conditions for sodium alginate to form a gel are mild, which can avoid the inactivation of active substances such as sensitive drugs, proteins, cells and enzymes.

[0005] HPi antibody is a mouse anti-human pancreatic islet cell membrane protein-specific monoclonal antibody developed by Novus Biologicals Inc and ThermoFisher, the world's top experimental material suppliers. It can be divided into HPi1, HPi2, HPi3, and HPi4. It can specifically bind to the surface membrane protein of human pancreatic islet cell clusters and almost does not bind to exocrine or ductal cells.

[0006] Therefore, it is extremely important to improve the method of isolating and extracting human pancreatic islets. Therefore, the inventors invented a degradable immunomagnetic bead and a preparation method thereof. Summary of the invention

[0007] Based on the above problems, the present invention provides a degradable immunomagnetic beads and a preparation method thereof. The immunomagnetic beads prepared by the present invention have low toxicity, good magnetic response, good surface biocompatibility, can effectively separate human islets, and greatly shorten the time for human islet separation.

[0008] In order to solve the above technical problems, the present invention provides a method for preparing degradable immunomagnetic beads, comprising the following steps:

[0009] S1: Preparation of biodegradable magnetic microsphere raw materials

[0010] Take 0.05-0.5g sodium alginate, add distilled water to 10mL, dissolve for 0.5-1h at room temperature and magnetic stirring to obtain 0.5-5% sodium alginate solution; take 5-10mL of the above-prepared sodium alginate solution, add 2-50μL of magnetic beads with a concentration of 50mg / mL and a particle size of 100-500nm, shake up and down to mix the magnetic beads and sodium alginate evenly, as the water phase; in addition, take 100-1000ml of liquid paraffin, add it to a beaker, and then add 0.5-5mL of Span 80 to the beaker and mix evenly, as the oil phase; then mix the water phase and the oil phase, stir evenly with a glass rod, and simply disperse the water phase with the oil phase to obtain a degradable magnetic bead microsphere raw material;

[0011] S2: using the rapid membrane emulsification technology and the degradable magnetic microsphere raw material prepared in step S1 to prepare alginate magnetic microspheres;

[0012] S3: Coupling of alginate magnetic microspheres with antibodies

[0013] Place the alginate magnetic microspheres prepared in step S2 in a test tube, wash them three times with 0.01mol / LPBS, and finally adjust the volume to 1-5mL, add 1-20mg EDC and 5-30mg suflo-NHS, mix well and react at room temperature for 5-30min, then add 2-20mg 6-aminohexanoic acid, rotate and stir at room temperature for 12-28h, and wash them three times with 0.01mol / L PBS; take the magnetic microspheres with 6-aminohexanoic acid "arm" molecules connected to the above-mentioned microspheres in an experimental tube, adjust the volume to 1-10mL with the above-mentioned 0.01mol / LPBS, add 1-20mg EDC and 5-30mg suflo-NHS, mix well and react at room temperature for 10-20 minutes, then add 200-800μg mouse anti-human pancreatic islet cell membrane protein monoclonal antibody hpi-2, mix well and rotate and stir at room temperature for 1-4h, add 0.05-0.5mol / L glycine solution, the glycine solution contains 0.2% human serum albumin or 0.2% bovine serum albumin, block for 10-20h, wash 3 times with 0.01mol / LPBS, add Hanks solution and store at 4℃.

[0014] Furthermore, the solution used for solidification in step S2 is any one or both of a CaCl2 solution with a concentration of 0.2-10% and a BaCl2 solution with a concentration of 0.2-10%.

[0015] Furthermore, the concentration of the sodium alginate solution in step S1 is 2%, the amount of 2% sodium alginate solution is 10 ml, the particle size of the added magnetic beads is 300 nm and the volume is 10 μL, the volume of liquid paraffin taken is 140 ml, and the volume of Span 80 added is 3.5 ml; the amount of alginate magnetic microspheres taken in step S3 is 2 mg, the fixed volume is 2 ml, and the masses of EDC and suflo-NHS added subsequently are 10 mg and 15 mg respectively. The reaction time at room temperature is 15 min, the volume of 6-aminohexanoic acid added is 10 mg, and then the fixed volume of 0.01 mol / L PBS is 2 ml. The masses of EDC and suflo-NHS added subsequently are 10 mg and 15 mg respectively, and the concentration of glycine solution added is 0.2 mol / L.

[0016] In order to solve the above technical problems, the present invention also provides degradable immunomagnetic beads.

[0017] In order to solve the above technical problems, the present invention also provides the use of degradable immunomagnetic beads in a product for separating human pancreatic islet cell clusters.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: the immunomagnetic beads prepared by the present invention are coated with islet cell membrane-specific antibodies, can specifically target islets in the human pancreas, and bind to islets during pancreatic digestion, and under the action of an external magnetic field, separation of islet cell clusters from exocrine tissues can be achieved, which can greatly shorten the time for islet separation, improve the efficiency of islet cell cluster separation, and improve the activity and function of islet cell clusters, which is beneficial to reducing the amount of islets required for transplantation, improving the transplantation effect, and indirectly reducing the cost of islet transplantation surgery; the immunomagnetic beads prepared by the present invention have low toxicity, good magnetic response, good surface biocompatibility, and can be degraded when the solution pH is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a particle size distribution diagram of the degradable microspheres of Example 2 of the present invention;

[0020] Figure 2 This is a particle morphology picture of the degradable microspheres of Example 2 of the present invention after freeze-drying under a cold field emission scanning electron microscope. DETAILED DESCRIPTION

[0021] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments and drawings. The exemplary embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0022] Embodiment 1:

[0023] This embodiment provides a method for preparing degradable immunomagnetic beads, comprising the following steps:

[0024] S1: Preparation of biodegradable magnetic microsphere raw materials

[0025] Take 0.05-0.5g sodium alginate, add distilled water to 10mL, dissolve for 0.5-1h at room temperature and magnetic stirring to obtain 0.5-5% sodium alginate solution; take 5-10mL of the above-prepared sodium alginate solution, add 2-50μL of magnetic beads with a concentration of 50mg / mL and a particle size of 100-500nm, shake up and down to mix the magnetic beads and sodium alginate evenly, as the water phase; in addition, take 100-1000mL of liquid paraffin, add it to a beaker, and then add 0.5-5mL of Span 80 to the beaker and mix evenly, as the oil phase; then mix the water phase and the oil phase, stir evenly with a glass rod, and simply disperse the water phase with the oil phase to obtain a degradable magnetic bead microsphere raw material;

[0026] S2: alginate magnetic microspheres are prepared by using rapid membrane emulsification technology and the degradable magnetic microsphere raw material prepared in step S1. The manufacturer of the membrane emulsifier used in the rapid membrane emulsification technology adopted in this embodiment is Zhongke Senhui Microsphere Technology (Suzhou) Co., Ltd. The model of the membrane emulsifier is FM0210-500M. The specific process is as follows: open the air supply for opening the valve and adjust the pressure reducing valve to 0.2-1.6Mpa; open the air supply for emulsification and adjust the pressure reducing valve to 0-3.0Mpa; place the dry membrane tube in the external phase solution of the emulsion for ultrasonication for 20-60min, and fill the bubbles in the membrane tube with the external phase solution; install the membrane tube on the membrane emulsifier in a wet state; turn on the power of the membrane emulsifier, click the touch screen, and produce magnetic microspheres according to the general membrane emulsification microsphere production process;

[0027] Solidification: slowly add any one of a 0.2-10% CaCl2 solution and a 0.2-10% BaCl2 solution or a mixture of the two to the magnetic bead microsphere solution obtained after membrane emulsification, and quickly stir the microsphere solution to solidify it for 5-50 minutes;

[0028] Separation and washing: The microspheres containing magnetic beads can be separated by super strong magnets, the paraffin in the supernatant is removed, isopropanol is added to wash the magnetic beads twice, and each time they are separated by super strong magnets, and then any one of 0.2-10% CaCl2 solution and 0.2-10% BaCl2 solution or a mixture of the two is used to store the alginate magnetic microsphere products, or freeze-dried in distilled water for storage; a laser particle size analyzer is used to analyze the particle size and distribution of the obtained magnetic microspheres, and the particle size is generally 0.5-50.0μm and the particle size distribution range is as small as possible; the freeze-dried microspheres can be subjected to cold field emission scanning electron microscopy to check the surface roundness of the microsphere particles;

[0029] S3: Coupling of alginate magnetic microspheres with antibodies

[0030] The alginate magnetic microspheres prepared in step S2 were placed in a test tube, washed three times with 0.01 mol / L PBS, and finally fixed to 1-5 mL, 1-20 mg of water-soluble carbodiimide (EDC) and 5-30 mg of N-hydroxysulfosuccinimide sodium salt (suflo-NHS) were added, mixed and reacted at room temperature for 5-30 min, and then 2-20 mg of 6-aminocaproic acid was added. After rotating and stirring at room temperature for 12-28 h, the alginate magnetic microspheres were washed three times with 0.01 mol / L PBS. The magnetic microspheres with the 6-aminocaproic acid "arm" molecules connected were placed in an experimental tube, fixed to 1-10 mL with the above 0.01 mol / L PBS, and 1-20 mg of EDC and 5-30 mg of suflo-NHS, mix well and react at room temperature for 10-20 minutes, then add 200-800μg mouse anti-human pancreatic islet cell membrane protein monoclonal antibody hpi-2, mix well and rotate and stir at room temperature for 1-4h, add 0.05-0.5mol / L glycine solution, the glycine solution contains 0.2% human serum albumin or 0.2% bovine serum albumin, block for 10-20h, wash 3 times with 0.01mol / LPBS, add Hanks solution and store at 4℃.

[0031] The degradable immunomagnetic beads prepared in this example can be applied to products for separating human pancreatic islet cell clusters.

[0032] Embodiment 2:

[0033] The formula of the degradable immunomagnetic microspheres of this embodiment is as follows:

[0034]

[0035] The preparation method of this embodiment is as follows:

[0036] S1: Weigh 0.2g of sodium alginate, add distilled water to 10ml, dissolve at room temperature and magnetic stirring for 0.5-1h, dissolve evenly, and obtain 2% sodium alginate solution; take 10ml of 2% sodium alginate solution, add 10μL of magnetic beads with a concentration of 50mg / mL and a particle size of 300nm, shake up and down to mix the magnetic beads and sodium alginate evenly, as the water phase (inner phase); in addition, take 140ml of liquid paraffin, add it to a beaker, add 3.5ml of Span 80, mix evenly, as the oil phase (external phase); add the water phase to the oil phase or add the oil phase to the water phase, stir evenly with a glass rod, and the oil phase simply disperses the water phase to obtain the degradable magnetic microsphere raw material;

[0037] S2: alginate magnetic microspheres with a diameter of about 1 μm are prepared by using the rapid membrane emulsification technology and the degradable magnetic microsphere raw material prepared in step S1. The manufacturer of the membrane emulsifier used in the rapid membrane emulsification technology adopted in this embodiment is Zhongke Senhui Microsphere Technology (Suzhou) Co., Ltd. The model of the membrane emulsifier is FM0210-500M. The specific process is as follows: open the air supply for opening the valve and adjust the pressure reducing valve to 0.4-0.6Mpa; open the air supply for emulsification and adjust the pressure reducing valve to 0-2.0Mpa; place the dry membrane tube in the external phase solution of the emulsion, ultrasonicate for more than 30 minutes, and fill the bubbles in the membrane tube with the external phase solution; install the membrane tube on the membrane emulsifier in a wet state; turn on the power of the membrane emulsifier, click the touch screen, process operation---confirm---go to the membrane emulsifier menu; click Click the air inlet valve to open, confirm the air inlet pressure of the emulsification air supply valve (generally the air inlet pressure is about 0.54Mpa) ---- open the exhaust valve --- open the feed valve --- stir the sodium alginate oil-water mixture and feed it --- close the exhaust valve --- close the feed valve --- open the air inlet valve --- open the emulsification valve, wait a few seconds after each valve is opened to allow the gas to fully exchange in and out, complete one microsphere preparation, close the air inlet valve --- close the emulsification valve; generally, the microsphere production needs to repeat the above E process for 3-5 rounds of feeding and discharging. If the results of 5 rounds of sampling are not ideal, it may be necessary to adjust the ratio of the oil-water phase and conduct another test; after the microsphere preparation is completed, close the air inlet valve --- close the emulsification valve --- open the exhaust valve --- close the exhaust valve; shutdown: exit the interface, turn off the power at the back, and end this microsphere preparation operation.

[0038] Solidification: Slowly add 2% CaCl2 solution to the magnetic bead microsphere solution obtained after membrane emulsification, and quickly stir the microsphere solution to fully solidify it (>10min);

[0039] Separation and washing: The microspheres containing magnetic beads can be separated by super strong magnets, the supernatant liquid paraffin is removed, isopropanol is added to wash the magnetic beads twice, each time separated by super strong magnets, and then the calcium alginate magnetic microspheres are preserved with 2% CaCl2 solution; or freeze-dried in distilled water for preservation; the particle size and distribution of the obtained magnetic microspheres are analyzed by laser particle size analyzer. The general particle size is 0.5-100.0μm, and the particle size distribution range is as small as possible, see attached Figure 1 After freeze-drying, cold field emission scanning electron microscopy was used to examine the surface roundness of the microsphere particles. The results are shown in the attached Figure 2 ;

[0040] S3: Coupling of alginate magnetic microspheres with antibodies

[0041] Take 2 mg of the degradable calcium alginate magnetic microspheres prepared in step S2 in a test tube, wash them three times with 0.01 mol / L PBS, and finally adjust the volume to 2 ml, add 10 mg EDC and 15 mg suflo-NHS, mix well and react at room temperature for 15 min, then add 10 mg 6-aminohexanoic acid, rotate and stir at room temperature for 18 h, and wash them three times with the above 0.01 mol / L PBS; take the above magnetic microspheres with 6-aminohexanoic acid "arm" molecules connected to the test tube, adjust the volume to 2 ml with the above 0.01 mol / L PBS, add 10 mg EDC and 15 mg suflo-NHS, mix well and react at room temperature for 15 minutes, then add 400 μg mouse anti-human pancreatic islet cell membrane protein monoclonal antibody hpi-2, mix well and rotate at room temperature for 2 hours, add 0.2 mol / L glycine solution (containing 0.2% human serum albumin or 0.2% bovine serum albumin) to block for 12 hours, wash three times with the above 0.01 mol / L PBS, add modified Hanks solution and store at 4°C.

[0042] In this embodiment, a special separation device for islet cell immunomagnetic beads (patent application number: 201910125465.2) is used for the separation of human islet cells with immunomagnetic beads. The human islets containing magnetic beads are washed twice with a 0.01 mol / L PBS solution at pH 7.4 to dissolve the magnetic beads and obtain human islet cell clusters without immunomagnetic beads.

[0043] When the immunomagnetic beads prepared in this embodiment are used to separate human islet cell clusters, the average time required for islet separation (from organ pruning to islet culture) is about 3.5 hours, which is much shorter than the time required by the conventional Ricordi semi-automatic human islet separation method (about 6.2 hours on average). The purity of most islets can reach more than 85%, which is much higher than that of the Ricordi semi-automatic human islet separation method (purity of 30%-100%). The average islet activity is 89.32%, which is no different from the islet activity obtained by the conventional Ricordi semi-automatic human islet separation method. The average amount of islets obtained is 8214 IEQ / g digested pancreatic tissue, which is much higher than that of the Ricordi semi-automatic human islet separation method (6135 IEQ / g digested pancreatic tissue on average).

[0044] The degradable immunomagnetic beads prepared in this example can be applied to products for separating human pancreatic islet cell clusters.

[0045] The above is an embodiment of the present invention. The above embodiments and the specific parameters in the embodiments are only for the purpose of clearly describing the invention verification process, and are not intended to limit the patent protection scope of the present invention. The patent protection scope of the present invention is still subject to its claims. Any equivalent structural changes made by using the contents of the description and drawings of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing degradable immunomagnetic beads, characterized in that: The steps include: S1: Preparation of biodegradable magnetic microsphere raw materials Take 0.05-0.5g sodium alginate, add distilled water to 10mL, dissolve for 0.5-1h at room temperature and magnetic stirring to obtain 0.5-5% sodium alginate solution; take 5-10mL of the above-prepared sodium alginate solution, add 2-50μL of magnetic beads with a concentration of 50mg / mL and a particle size of 100-500nm, shake up and down to mix the magnetic beads and sodium alginate evenly, as the water phase; in addition, take 100-1000ml of liquid paraffin, add it to a beaker, and then add 0.5-5mL of Span 80 to the beaker and mix evenly, as the oil phase; then mix the water phase and the oil phase, stir evenly with a glass rod, and simply disperse the water phase with the oil phase to obtain a degradable magnetic bead microsphere raw material; S2: using the rapid membrane emulsification technology and the degradable magnetic microsphere raw material prepared in step S1 to prepare alginate magnetic microspheres, the solution used for solidification is any one or both of a CaCl2 solution with a concentration of 0.2-10% and a BaCl2 solution with a concentration of 0.2-10%; S3: Coupling of alginate magnetic microspheres with antibodies Place the alginate magnetic microspheres prepared in step S2 in a test tube, wash them three times with 0.01 mol / L PBS, and finally adjust the volume to 1-5 mL. Add 1-20 mg EDC and 5-30 mg suflo-NHS, mix well and react at room temperature for 5-30 min, then add 2-20 mg 6-aminohexanoic acid, rotate and stir at room temperature for 12-28 h, and wash them three times with 0.01 mol / L PBS. Place the magnetic microspheres with the 6-aminohexanoic acid "arm" molecules connected to the above-mentioned microspheres in an experimental tube, adjust the volume to 1-10 mL with the above-mentioned 0.01 mol / L PBS, add 1-20 mg EDC and 5-30 mg suflo-NHS, mix well and react at room temperature for 10-20 minutes, then add 200-800μg mouse anti-human pancreatic islet cell membrane protein monoclonal antibody HPi2, mix well and rotate and stir at room temperature for 1-4h, add 0.05-0.5mol / L glycine solution, the glycine solution contains 0.2% human serum albumin or 0.2% bovine serum albumin, block for 10-20h, wash 3 times with 0.01mol / LPBS, add Hanks solution and store at 4℃.

2. The method for preparing degradable immunomagnetic beads according to claim 1, characterized in that: The concentration of the sodium alginate solution in step S1 is 2%, the amount of 2% sodium alginate solution is 10 ml, the particle size of the added magnetic beads is 300 nm, the volume is 10 μL, the volume of liquid paraffin taken is 140 ml, and the volume of Span 80 added is 3.5 ml; the amount of alginate magnetic microspheres taken in step S3 is 2 mg, the fixed volume is 2 ml, the masses of EDC and suflo-NHS added subsequently are 10 mg and 15 mg respectively, the reaction time at room temperature is 15 min, the volume of 6-aminohexanoic acid added is 10 mg, and then the fixed volume of 0.01 mol / L PBS is 2 ml, the masses of EDC and suflo-NHS added subsequently are 10 mg and 15 mg respectively, and the concentration of glycine solution added is 0.2 mol / L.

3. Degradable immunomagnetic beads prepared by the preparation method described in any one of claims 1-2.

4. Use of the degradable immunomagnetic beads according to claim 3 in the preparation of a product for separating human pancreatic islet cell clusters.

Images