CN108287234A - Nano immune magnetic bead and its preparation method and application - Google Patents

Abstract

The invention discloses a nano-immune magnetic bead, a preparation method and application thereof. The preparation method of nano-immunomagnetic beads of the present invention can prepare 50nm _- level dextran-coated _Fe3O4 and/or _γFe2O3 nano-immune-magnetic beads, the obtained nano-immune-magnetic beads have a _small particle size, and The particle size distribution is uniform and the stability is good, which can avoid the aggregation and sedimentation of magnetic beads. In addition, the antibody-coupled nano-immunomagnetic beads obtained through synthesis can ensure that the amount of antibodies on the surface of each batch of magnetic beads is basically the same, and the repeatability is good. During the specific sorting and isolation of cells. The preparation method of the nano-immune magnetic beads is quick, simple and low in cost, and can be widely popularized and applied.

Description

Nano-immunomagnetic beads and its preparation method and application

technical field

The invention relates to the field of cell engineering, in particular to a nano-immune magnetic bead and its preparation method and application.

Background technique

Cell sorting plays a very important role in clinical diagnosis and cell immunotherapy. In terms of clinical diagnosis, for example: fetal cell separation to determine whether the fetus has genetic defects; cancer cell separation to achieve early diagnosis and treatment of cancer; hematopoietic stem cell transplantation can be used as the main measure of hematopoietic support after high-dose radiotherapy and chemotherapy for malignant tumors. In terms of cellular immunotherapy, such as isolating immune cells from peripheral blood mononuclear cells of patients, using cytokines in vitro to activate them into cytokine-induced killer cells, such as DC-CIK cells, or using genetic engineering technology to T cells The cells add a chimeric antibody that can recognize tumor cells and activate T cells at the same time, so that they can be reinfused into the body after massive expansion in vitro, so as to kill tumor cells with corresponding specific antigens, such as CAR-T cells.

There are two main categories of cell sorting methods: one is density gradient centrifugation based on differences in sedimentation coefficients between cell populations; the other is based on immune recognition characteristics, including fluorescence Activated cell sorting (Fluorescence-activated cell sorting, FACS) and magnetic activated cell sorting (also known as magnetic bead sorting, Magnetic-activated cell separation, MACS). Density gradient centrifugation is simple and easy, but the purity of the isolated cells is low, and the markers on the cell surface are not clear, and the specificity is poor. Therefore, this method has been seldom used at present. The cells obtained by flow cytometry have high purity, high recovery rate, and are not easy to be contaminated, but the equipment required by this method is expensive, time-consuming, stimulates the cells greatly, and requires high-level technical support and professional operators. Additionally, the method can only sort one cell sample at a time.

Magnetic bead sorting is an excellent cell sorting technique developed in the 1970s. The immunomagnetic beads involved are magnetic particles coated with monoclonal antibodies, which can bind to antigens on the surface of target cells. In the external magnetic field, the target cells connected to the magnetic beads through the antibody are adsorbed and retained in the sorting column. Cells without such surface antigens have no magnetism because they cannot be connected with the magnetic beads and cannot stay in the magnetic field, thus reaching the cells. The purpose of enrichment and purification. The current commercial magnetic bead products for cell sorting mainly include superparamagnetic magnetic beads coated with nano-sized dextran from Miltenyi in Germany and micron-sized polystyrene magnetic beads from Dynal in the United States. Compared with Dyno micron-sized polystyrene magnetic beads, Miltenyi's magnetic beads of about 50nm have the following advantages: 1. After the magnetic beads are combined with cells, there is no need to dissociate the magnetic beads, and it is not necessary for cell physiology and No effect on activity; 2. Dextran-coated nano magnetic beads are degradable, have good biocompatibility, and have no toxicity to cells; 3. Target cells attached to small magnetic beads can be analyzed by flow cytometry without being too large 4. The sorted target cells are of high purity, which can avoid the interference of other non-specific cells.

Although Miltenyi's 50nm magnetic beads have better performance, they are expensive, and domestic research institutions and enterprises basically rely on foreign imports, which also discourages small and medium-sized enterprises. Although magnetic beads for cell analysis and protein purification have been developed in China, the particle size of these magnetic beads is mainly distributed in microns, and the coating layer is mainly concentrated in organic and inorganic substances. The biocompatibility and stability of the obtained magnetic beads The degradability is relatively poor, and it is difficult to apply in vivo. And some coated carbohydrates are wrapped by a two-step method. First, the nano-bare magnetic beads are synthesized by co-precipitation, and then wrapped with dextran on the surface, which will make the obtained nanoparticles easy to aggregate and disperse. Relatively poor, as the method mentioned in the patent application whose application number is CN200710176211; and the nanoparticles wrapped with one-step dextran, although the stability obtained is better, but in the group modification process, cyanogen bromide is used, However, the high toxicity of cyanogen bromide also limits its application (such as Clonis YD, Small DAP. High-performance dye-ligand Chromatograph. In Reactive in Protein and Enzyme Technology (M). London: Macmillan Press, 1987.87-100).

Contents of the invention

Based on this, it is necessary to provide a nano-immunomagnetic bead with small particle size, good stability and simple preparation method, its preparation method and application.

The technical solution of the present invention to solve the above-mentioned technical problems is as follows.

A preparation method of nano-immunomagnetic beads, characterized in that, comprising the steps of:

Step 1: preparing an anaerobic reaction solution, adding dextran, Fe ²⁺ and Fe ³⁺ to the anaerobic reaction solution, wherein the sum of the concentrations of Fe ²⁺ and Fe ³⁺ is 0.03mol/l-1mol /l, the molar ratio of Fe ³⁺ to Fe ²⁺ is 0.25:1～4:1, the ratio of the sum of the amount of Fe ²⁺ and Fe ³⁺ to the amount of dextran is 90:1～ 15:1, the dextran is hydroxyl dextran or carboxy dextran;

Step 2: Stir the anaerobic reaction solution at 50°C at 500-1200rpm/min for 10min, then add ammonia water to adjust the pH to 9.0-11, and continue stirring at 50-100°C for 30min-2h;

Step 3: Cool to room temperature after the reaction, centrifuge to remove larger particles in the solution, adjust the pH of the collected solution to neutral with acid, and then filter with a 0.22 μm hydrophilic filter membrane to further remove impurities, and purify the obtained filtrate After that, the nano-immunomagnetic beads coated with Fe ₃ O ₄ and/or γFe ₂ O ₃ by dextran are obtained.

When configuring the solution in step 1, oxygen can be removed from the solvent by passing nitrogen gas first, and the subsequent preparation process should be carried out under anaerobic conditions, such as nitrogen or an inert gas atmosphere.

In one of the embodiments, the molecular weight of the dextran is 20kDa-100kDa.

Further, in one of the embodiments, the molecular weight of the dextran is 20kDa, 40kDa or 100kDa.

In one embodiment, the sum of the concentrations of Fe ²⁺ and Fe ³⁺ is 0.09 mol/l.

In one embodiment, the molar ratio of Fe ³⁺ to Fe ²⁺ is 2:1.

In one of the embodiments, the ratio of the sum of the amounts of Fe ²⁺ and Fe ³⁺ to the amount of dextran is 90:1, 45:1, 30:1, 18:1 or 15:1 . Further, in one of the embodiments, the ratio of the sum of the amounts of Fe2+ and Fe3+ to the amount of dextran is 45:1.

In one embodiment, in the second step, the anaerobic reaction solution is stirred and reacted at 50°C at 1000rpm/min for 10min, then ammonia water is added to adjust the pH to 10, and the stirring is continued at 70°C Reaction 1h. Ammonia water can choose concentrated ammonia water with a concentration of 5mol/L～15mol/L. The amount of concentrated ammonia water can be added according to 8ml～50ml per 100ml anaerobic reaction solution, such as 8ml, 12ml, 16ml, 24ml or 50ml can be selected, In one embodiment, the amount of concentrated ammonia water can be added in an amount of 12ml per 100ml of anaerobic reaction solution.

In one embodiment, in the third step, the rotation speed of the centrifugation is 3000 rpm, and the centrifugation time is 5 minutes.

In one embodiment, in the third step, the pH is adjusted using HCl or glacial acetic acid with a concentration of 0.1M-2.0M, for example, HCl with a concentration of 1.0M can be used to adjust the pH to 7.0.

In one of the embodiments, in the third step, the purification is purification with a separation column (Miltenyi, Germany, MS separation column) to remove free dextran therein.

A nano-immunomagnetic bead, which is prepared by using the method for preparing nano-immunomagnetic beads described in any of the above embodiments.

The prepared nano-immunomagnetic beads can be adjusted to a concentration of 6 mg/ml and stored at 4° C. for future use.

Application of the above-mentioned nano-immunomagnetic beads in cell sorting.

As in one of the embodiments, the application includes combining nano-immunomagnetic beads with specific antibodies and using nano-immuno-magnetic beads bound with specific antibodies to target cells through specific antibody-target cell surface antigen binding, and in a magnetic field in the filtering steps. For carboxy dextran, it can be directly combined with specific antibodies; for hydroxy dextran, the hydroxy dextran can be oxidized to formaldehyde dextran first, and then the formaldehyde dextran can be combined with the specific antibody Combine.

In one of the embodiments, for the nano-immunomagnetic beads coated with hydroxydextran, the hydroxydextran can be oxidized to formyl dextran by the following method:

Add sodium periodate to the nano-immune magnetic bead solution coated with hydroxydextran, wherein, the addition of sodium periodate meets: nano-immune magnetic bead: sodium periodate=0.125～8:1 (mass ratio) , stirring and reacting at room temperature in the dark for 30 minutes to 3 hours. After the reaction, the free dextran is removed by column separation and purification, and washed with 10mM to 100mM sodium borate solution with a pH of 7.0 to 9.0 to obtain the product. Further, the concentration of the aldehyde dextran-coated nano-immunomagnetic beads can also be adjusted to 4 mg/ml and stored for later use.

Further, in one of the embodiments, the mass ratio of nano-immunomagnetic beads to sodium periodate can be 8:1, 4:1, 2:1, 1:1, 1:2, 1:3, 1:4 Or 1:8, wherein 1:1 is the preferred mass ratio.

Furthermore, in one of the embodiments, the reaction time is 1 h while avoiding light and stirring.

Still further, in one of the embodiments, 20 mM sodium borate solution with a pH of 8.5 was used to wash 3 times.

The binding of nano-immune magnetic beads coated with aldehyde dextran or carboxy dextran to specific antibodies can be carried out according to but not limited to the following steps. Nano-immune magnetic beads coated with dextran or carboxy-dextran can be taken as 100μl, diluted with 20mM sodium borate with a pH of 8.5, and then added according to the ratio of nano-immune magnetic beads:antibody mass ratio of 1 to 100:1 Antibody, react at room temperature and shake and mix for 1h~12h. After the reaction is over, add excess _NaBH4 to the reaction system for 30min~3h, separate and purify through the column, wash with PBS buffer for several times, add phosphate buffer at 4℃ Save it for later use. Other volumes may be adjusted proportionally.

Further, in one of the embodiments, the antibody with a concentration of 5 mg/ml can be added according to the ratio of nano-immunomagnetic beads:antibody mass ratio of 5:1, 15:1, 30:1 or 100:1. Wherein, the nano-immune magnetic beads:antibody mass ratio is preferably 15:1, and the time for shaking and mixing at room temperature is preferably 6h.

Furthermore, in one embodiment, NaBH ₄ with a concentration of 0.25M can be added according to the mass ratio of nano-immunomagnetic beads:NaBH ₄ being 20:1, 10:1, 1:10 or 1:20. Wherein, the preferred ratio is 10:1, and the preferred reaction time is 1 h.

The nano-immuno-magnetic beads coated with _Fe3O4 and/or _γFe2O3 by 50nm-level dextran can be prepared by the above-mentioned preparation method of nano-immune-magnetic beads. The obtained nano-immune-magnetic beads have a _small particle _size and The diameter distribution is uniform and the stability is good, which can avoid the aggregation and sedimentation of magnetic beads. In addition, the antibody-coupled nano-immunomagnetic beads obtained through synthesis can ensure that the amount of antibodies on the surface of each batch of magnetic beads is basically the same, and the repeatability is good. During the specific sorting and isolation of cells. The preparation method of the nano-immune magnetic beads is quick, simple and low in cost, and can be widely popularized and applied.

Description of drawings

Fig. 1 is the particle size distribution detection result figure of the nano-immunomagnetic beads prepared in embodiment 1;

Fig. 2 is the SEM picture of the hydroxyl nano-immunomagnetic beads that embodiment 4 makes;

FIG. 3 is a graph showing the results of fluorescent staining of CD4 T cells sorted in Example 5. FIG.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the associated drawings. Preferred embodiments of the invention are shown in the accompanying drawings. However, the present invention can be embodied in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field of the invention. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Example 1 Synthesis of nano-immunomagnetic beads coated with 50nm hydroxydextran

First weigh 8.0g of hydroxydextran (molecular weight 40kDa), dissolve it in 100ml of deionized water, pass nitrogen and deoxygenate for 10min, then add 1.62g FeCl ₃ .6H ₂ O and 0.62g FeCl ₂ .4H ₂ O, 1000rpm/min Stir and heat in a water bath at 50°C for 10 minutes, add 12ml of concentrated ammonia water with a concentration of 15mol/L to adjust the pH to 10.0; raise the temperature to 70°C and keep it warm for 1 hour, then stop the reaction and cool to room temperature; centrifuge at 3000rpm for 5 minutes to remove large particles in the solution After being removed by centrifugation, use 1.0M HCl to adjust the pH to neutral, and then filter with a 0.22 μm hydrophilic filter membrane, and use the German Miltenyi MS separation column to remove the free dextran in the solution; take 6mg/ Dilute 50nm hydroxydextran nano-magnetic beads with ultrapure water to 1mg/ml, characterize the particle size by DLS, calculate the average value three times, and obtain 50nm hydroxy-dextran-coated magnetic nano-beads, store at 4°C.

As shown in Figure 1, the obtained hydroxydextran-coated nano magnetic beads have an average particle size of 59.50 nm, and the hydroxy magnetic beads present a spherical single particle distribution with uniform size.

Table 1 below shows the stability results of the obtained hydroxydextran-coated nano-magnetic beads.

Table 1 Detection of particle size of hydroxyl magnetic beads at different times

The average particle size Peak particle size PDI Place 1 month 59.50 72.2 0.172 Place March 58.65 71.5 0.174 Place June 60.12 72.1 0.171

It can be seen from Table 1 that the hydroxyl magnetic beads can be stored at 4°C for more than half a year without aggregation between nanoparticles and affecting the performance of nanoparticles, which provides a stability guarantee for subsequent cell sorting applications.

Example 2 Dextran: Ferric ion/ferrous ion is 1:90 synthesis of 50nm hydroxyl dextran-coated nano-immune magnetic beads

First weigh 4.4g of hydroxydextran (molecular weight 40kDa), dissolve it in 100ml of deionized water, pass nitrogen to remove oxygen for 10min, then add 1.62g FeCl ₃ .6H ₂ O and 0.62g FeCl ₂ .4H ₂ O, 1000rpm/min Stir and heat in a water bath at 50°C for 10 minutes, add 12ml of concentrated ammonia water with a concentration of 15mol/L to adjust the pH to 10.0; raise the temperature to 70°C and keep it warm for 1 hour, then stop the reaction and cool to room temperature; centrifuge at 3000rpm for 5 minutes to remove large particles in the solution After being removed by centrifugation, use 1.0M HCl to adjust the pH to neutral, and then filter with a 0.22 μm hydrophilic filter membrane, and use the German Miltenyi MS separation column to remove the free dextran in the solution; take 6mg/ Dilute 1 mL of hydroxydextran nano-magnetic beads with ultrapure water to 1 mg/ml, characterize the particle size by DLS, and calculate the average value of three times to obtain 58nm hydroxy-dextran-coated nano-magnetic beads, which are stored at 4°C.

Example 3 Dextran: Synthesis of 50nm hydroxydextran-coated nano-immune magnetic beads with iron ion/ferrous ion ratio of 1:15

First weigh 24g of hydroxydextran (molecular weight: 40kDa), dissolve it in 100ml of deionized water, pass nitrogen to remove oxygen for 10min, add 1.62g FeCl ₃ .6H ₂ O and 0.62g FeCl ₂ .4H ₂ O, stir at 1000rpm/min After heating in a water bath at 50°C for 10 minutes, add 12ml of concentrated ammonia water with a concentration of 15mol/L to adjust the pH to 10.0; raise the temperature to 70°C and keep it warm for 1 hour, then stop the reaction and cool to room temperature; centrifuge at 3000rpm for 5 minutes to centrifuge the large particles in the solution After removal, use 1.0M HCl to adjust the pH to neutral, then filter with a 0.22 μm hydrophilic filter membrane, and use the German Miltenyi MS separation column to remove the free dextran in the solution; take 6mg/mL Hydroxydextran nano-magnetic beads were diluted to 1mg/ml with ultrapure water, and the particle size was characterized by DLS, and the average value was calculated three times to obtain 55nm hydroxy-dextran-coated nano-magnetic beads, which were stored at 4°C.

Example 4 SEM image of nano-magnetic beads coated with 50nm hydroxydextran

Take 10 μL of 0.1 mg/mL hydroxyl nano-immune magnetic beads (Example 1, dextran: ferric ion/ferrous ion ratio of 1:45) diluted with water and drop it on the copper grid. The characterization results are shown in Figure 2: from the left picture, we can see that the hydroxyl magnetic beads are uniformly distributed in granular form, and from the right picture, we can see that most of the magnetic beads have a particle size of about 60nm, which is basically consistent with the DLS test results.

Example 5 Nano-immunomagnetic bead-coupled antibody

Get 500 μ l of the 6mg/ml hydroxydextran-coated nano-immune magnetic beads prepared in Example 1 in an EP tube, add sodium periodate solution according to the mass ratio of sodium periodate:nano-immune magnetic beads=1:1 , after shaking and reacting for 1 hour at room temperature in the dark, the impurities carried by the nanoparticles were removed by magnetic separation and column method, and finally eluted with 20mM sodium borate solution with a pH of 8.5, and the concentration of nanoimmunomagnetic beads was adjusted to 5mg/mL.

According to the mass ratio of nano-immune magnetic beads: specific antibody = 15:1, the specific antibody is added to the magnetic bead solution, shaken and mixed for 6 hours, and then excessive sodium borohydride is added, such as according to the specific antibody: borohydride Sodium=1:1 mass ratio was added, and the specific antibody-coupled nano-immune magnetic beads were obtained by reduction with sodium borohydride, and the specific antibody-coupled magnetic beads were purified by column method again, and stored at 4°C.

Example 6 CD4 antibody-coupled magnetic beads for extraction of human CD4 T cells

1. 20ml of people's fresh blood that adds anticoagulant is diluted 2 times with PBS (phosphate buffered saline), according to blood: the volume ratio of lymphatic separation liquid=1:1 will spread the diluted blood on the lymphatic separation liquid; Peripheral blood mononuclear cells were obtained by centrifugation, and the cell concentration was adjusted to 3×10 ⁷ cells/ml;

2. Slurry 100 μl of 3× ¹⁰⁷ cells/ml of cell suspension and 5 μl of CD4 antibody-coupled nano-immune magnetic beads (prepared using the method used in Example 2, using CD4 antibody as the specific antibody) with a pipette After mixing with the gun, let it stand at 4°C for 15 minutes;

3. Centrifuge at 1200rpm for 5min to discard the supernatant, add 500μl buffer to the cell pellet to resuspend;

4. Wash the cells with Miltenyi separation column for 3 times, after washing each time with 500 μl of buffer, add 1ml of buffer to wash the cells and collect them into EP tubes;

5. Centrifuge the cells at 1200 rpm for 5 minutes to discard the supernatant, add 100 μl of buffer to the cell pellet to resuspend, add 10 μl of PE-labeled CD4 antibody and incubate at 4°C for 5 minutes;

6. Centrifuge at 1200 rpm for 5 minutes to discard the supernatant, add 100 μl PBS buffer to the cell pellet to resuspend, and observe the cell fluorescence with a fluorescence microscope.

The results are shown in FIG. 3 . It can be seen from FIG. 3 that after the cells sorted by immunomagnetic beads are fluorescently labeled, 90% of the cells with fluorescence can be seen under the microscope.

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (10)

1. a kind of preparation method of nano immune magnetic bead, which is characterized in that include the following steps：

Step 1：Anaerobic reaction solution is prepared, being added in the anaerobic reaction solution has glucan, Fe²⁺And Fe³⁺, wherein Fe²⁺ And Fe³⁺The sum of concentration be 0.03mol/l~1mol/l, Fe³⁺With Fe²⁺Molar ratio be 0.25:1~4:1, Fe²⁺And Fe³⁺'s The ratio of the sum of amount of substance and the amount of the substance of glucan is 90:1~15:1, the glucan is hydroxyl glucan or carboxyl Glucan；

Step 2：The anaerobic reaction solution is stirred to react 10min with 500~1200rpm/min at 50 DEG C, adds ammonia It is 9.0~11 that water, which adjusts acid-base value to pH, continues to be stirred to react 30min~2h at 50~100 DEG C；

Step 3：It is cooled to room temperature after reaction, larger particle, the solution of collection are adjusted with acid acid in centrifugation removal solution Then it is poly- up to Portugal after purification to filter the filtrate obtained further to clean to neutrality with 0.22 μm of hydrophilic filter membrane for basicity Glycolyx Fe₃O₄And/or γ Fe₂O₃The nano immune magnetic bead.

2. the preparation method of nano immune magnetic bead as described in claim 1, which is characterized in that the molecular weight of the glucan is 20kDa~100kDa.

3. the preparation method of nano immune magnetic bead as described in claim 1, which is characterized in that the molecular weight of the glucan is 20kDa, 40kDa or 100kDa.

4. the preparation method of nano immune magnetic bead as described in claim 1, which is characterized in that Fe²⁺And Fe³⁺The sum of concentration be 0.09mol/l。

5. the preparation method of nano immune magnetic bead as described in claim 1, which is characterized in that Fe³⁺With Fe²⁺Molar ratio be 2: 1。

6. the preparation method of nano immune magnetic bead as described in claim 1, which is characterized in that Fe²⁺And Fe³⁺Substance amount it Ratio with the amount of the substance with glucan is 90:1、45:1、30:1、18:1 or 15:1.

7. the preparation method of nano immune magnetic bead as described in claim 1, which is characterized in that in the step 2, be by The anaerobic reaction solution at 50 DEG C with 1000rpm/min be stirred to react 10min be and then added ammonium hydroxide adjust acid-base value to PH is 10, continues to be stirred to react 1h at 70 DEG C.

8. the preparation method of nano immune magnetic bead as described in claim 1, which is characterized in that described in the step 3 The rotating speed of centrifugation is 3000rpm, and the time is 5min.

9. a kind of nano immune magnetic bead, which is characterized in that using such as nano immune magnetic according to any one of claims 1 to 8 The preparation method of pearl is prepared.

10. application of the nano immune magnetic bead as claimed in claim 9 in cell sorting.