A drying method of magnetic microspheres

Abstract

The application relates to the technical field of magnetic microspheres, and relates to a drying method of magnetic microspheres. The drying method of the magnetic microspheres comprises the following steps: S1, adding a solvent and a surfactant into the magnetic microspheres, uniformly mixing, and obtaining a mixed solution; S2, pre-freezing the mixed solution at a temperature of 0-18 DEG C for 2-10 h; S3, carrying out two-stage vacuum drying on the pre-frozen mixed solution; the temperature of the first-stage vacuum drying is 0-18 DEG C, and the vacuum degree is 100-300 mbr; the temperature of the second-stage vacuum drying is 0-18 DEG C, and the vacuum degree is 0.1-10 mbr; the solvent is dimethyl sulfoxide; and the surfactant is a non-ionic surfactant. The non-surfactant and the solvent are added to play the dispersing and skeleton supporting roles, and provide a volatile ice crystal skeleton between the magnetic microspheres, so that the magnetic beads are not prone to aggregation in the freeze-drying process, and the damage of the magnetic beads is avoided.

Description

Technical Field

[0001] This invention relates to the field of magnetic microsphere technology, and specifically to a method for drying magnetic microspheres. Background Technology

[0002] Magnetic microspheres are tiny, magnetic spheres formed by combining magnetic inorganic particles with organic polymers through methods such as emulsion polymerization, dispersion polymerization, suspension polymerization, and seed polymerization. Their diameter typically ranges from nanometers to micrometers. They possess many of the properties of ordinary polymer microspheres and also exhibit magnetic responsiveness. The main materials of magnetic microspheres include iron oxides, nickel, and cobalt, which endow them with unique magnetism. Compared to ordinary polymer microspheres, they not only have better monodispersity but also improved biocompatibility. They have been widely applied in targeted drug delivery, enzyme immobilization, removal of heavy metal ions and phenol from wastewater, nuclear magnetic resonance imaging, protein purification, and cell separation.

[0003] However, the drying and preservation technology of magnetic microspheres has not been extensively studied. Magnetic microspheres are prone to aggregation, damage at low temperatures, and resolvability. Most existing technologies preserve magnetic microspheres in buffer solutions or organic solvents. However, this liquid preservation method severely affects the shelf life of the microspheres and presents transportation difficulties. Furthermore, the organic solvents used for preservation also pose certain hazards. Therefore, there is an urgent need to provide a novel drying and preservation technology. Summary of the Invention

[0004] The purpose of this invention is to provide a drying method for magnetic microspheres based on vacuum drying technology, so as to solve the defects existing in the current liquid preservation methods.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for drying magnetic microspheres includes the following steps:

[0007] S1. Add solvent and surfactant to magnetic microspheres and mix thoroughly to obtain a mixed solution;

[0008] S2. Pre-freeze the mixed solution at a temperature of 0-18℃ for 2-10 hours;

[0009] S3. Perform two-stage vacuum drying on the pre-frozen mixed solution; the temperature of the first stage vacuum drying is 0-18℃ and the vacuum degree is 100-300mbr; the temperature of the second stage vacuum drying is 0-18℃ and the vacuum degree is 0.1-10mbr.

[0010] The solvent is dimethyl sulfoxide; the surfactant is a nonionic surfactant.

[0011] Dimethyl sulfoxide can crystallize at 0-10℃ and is highly volatile, providing a volatile ice crystal framework between magnetic microspheres and preventing damage to the magnetic beads.

[0012] Nonionic surfactants generally have better dispersibility due to their ability to form steric hindrances, reduce van der Waals forces, and improve their own stability. At the same time, they have a lower critical micelle concentration, making micelles easier to form and resulting in a larger micelle aggregation number. Therefore, they have the strongest solubilizing effect on hydrocarbons and polar organic compounds.

[0013] In one preferred embodiment, the surfactant is one or more of Tween or PEG.

[0014] Through multiple studies, this invention has found that adding a certain proportion of nonionic surfactants to magnetic microspheres can achieve the functions of dispersion and skeletal support, while also having a certain solubilizing effect during reconstitution.

[0015] In one preferred embodiment, the Tween is one or more of Tween 20, Tween 40, Tween 60, and Tween 80.

[0016] In one preferred embodiment, the PEG is one or more of PEG2000, PEG3350, PEG4000, and PEG6000.

[0017] In one preferred embodiment, the PEG is one or both of PEG3350 and PEG4000.

[0018] In one preferred embodiment, the microspheres include one or more of the following: agarose magnetic microspheres, viral nucleic acid extraction magnetic microspheres, silanol magnetic microspheres, carboxyl magnetic microspheres, Oligo(dT) magnetic microspheres, streptavidin magnetic microspheres, cationic magnetic microspheres, affinity ligand magnetic microspheres, and toluenesulfonyl magnetic microspheres.

[0019] In one preferred embodiment, the amount of solvent added is 3-10% of the mass of the microspheres.

[0020] In one preferred embodiment, the amount of surfactant added is 3-10% of the mass of the microspheres.

[0021] In one preferred embodiment, the pre-freezing time of the mixed solution in S2 is 2-4 hours.

[0022] In one preferred embodiment, the temperature of the mixed solution in S2 is 0-10°C.

[0023] In one preferred embodiment, the temperature of the two vacuum drying stages in S3 is 0-10°C.

[0024] In one preferred embodiment, in S3, the vacuum degree of the first stage of vacuum drying is 100-150 mbr.

[0025] In one preferred embodiment, in S3, the vacuum degree of the second stage of vacuum drying is 0.1-5 mbr.

[0026] In one preferred embodiment, in step S3, the first stage of vacuum drying takes 2-4 hours, and the second stage of vacuum drying takes 30-40 hours.

[0027] This invention provides a freeze-drying process for magnetic microspheres. By adding non-surfactant and solvent components, dispersion and skeletal support are achieved, providing a volatile ice crystal framework between the magnetic microspheres. This prevents the magnetic beads from agglomerating during freeze-drying, thus avoiding damage. Simultaneously, rapid dissolution in pure water achieves a gel-like transformation, and desolvation is completed under vacuum freeze-drying, transforming the magnetic microspheres from a gel solution to a solid powder state. This facilitates the storage and transportation of the magnetic microspheres while retaining most of their load-bearing capacity. The process of this invention is simple, controllable, and easily scalable for large-scale industrial production. The freeze-dried magnetic microspheres of this invention retain their complete structure, show no agglomeration, exhibit rapid reconstitution time, and have minimal organic solvent residue, making it a drying method conducive to the long-term preservation of magnetic microspheres. Attached Figure Description

[0028] Figure 1 The microstructure of the dried magnetic microspheres prepared in Example 1;

[0029] Figure 2 This is a resolubilized gel image of the dried magnetic microspheres prepared in Example 1. Detailed Implementation

[0030] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0031] Materials and equipment

[0032] The magnetic microspheres used in this experiment were provided by Suzhou Weidu Biotechnology Co., Ltd., and included: agarose magnetic microspheres, viral nucleic acid extraction magnetic microspheres, silanol magnetic microspheres, carboxyl magnetic microspheres, Oligo(dT) magnetic microspheres, streptavidin magnetic microspheres, cationic magnetic microspheres, affinity ligand magnetic microspheres, and toluenesulfonyl magnetic microspheres. The lyophilization equipment was a Changsha Kaipu Instrument LA2 in-situ lyophilizer. All reagents and instruments used, unless otherwise specified, were commercially available products. Unless otherwise specified, all percentages in this invention are by mass.

[0033] Example 1

[0034] A vacuum freeze-drying method for magnetic microspheres

[0035] Step 1: Add 5% DMSO and 5% PEG3350 powder (by weight of the microspheres) to agarose magnetic microspheres stored in an organic solvent (such as acetone or chloroform) and stir until homogeneous to obtain a magnetic microsphere solution. Add 0.1g DMSO solution and PEG3350 powder to a 5mL vial containing 2g of agarose magnetic microsphere solution and stir until homogeneous to obtain another magnetic microsphere solution.

[0036] Step 2: Place the magnetic microsphere solution obtained in Step 1 into an in-situ vacuum freeze dryer with a partition temperature of 4°C and pre-freeze for 2 hours.

[0037] Step 3: The pre-frozen magnetic microsphere solution is kept at a partition temperature of 4°C and subjected to a first vacuum drying at 100 mbr. After the vacuum stabilizes and stops changing, the drying time is 2-4 hours. The vacuum is then adjusted to 0.1 mbr for a second vacuum drying, which takes 30-44 hours to obtain the dried magnetic microspheres.

[0038] The dried magnetic microspheres prepared in Example 1 were tested. After gold sputtering, the dried magnetic microspheres were observed using a scanning electron microscope at 20 KeV, 8 kx, and 10 μm. The microstructure of the microspheres was as follows. Figure 1 As shown, the microstructure of the magnetic microspheres was preserved intact after freeze-drying, with no obvious damage to the spherical structure.

[0039] The dried magnetic microspheres from Example 1 were reconstituted as follows: 2 mL of pure water was added to the freeze-dried magnetic microspheres in a vial, mixed thoroughly, and the reconstituted state was observed. The resulting reconstituted gel image is shown below. Figure 2 As shown, the dried magnetic microspheres prepared in Example 1 were reconstituted into a very uniform gel-like liquid.

[0040] Example 2

[0041] Based on Example 1, step 1 was adjusted as follows: 5% DMSO was added to the magnetic microspheres stored in an organic solvent and stirred until homogeneous to obtain a magnetic microsphere solution. Other steps were the same as in Example 1, resulting in dried magnetic microspheres.

[0042] Example 3

[0043] Based on Example 1, step 1 was adjusted as follows: 10% DMSO was added to the magnetic microspheres stored in an organic solvent and stirred until homogeneous to obtain a magnetic microsphere solution. The other steps were the same as in Example 1 to obtain dried magnetic microspheres.

[0044] Example 4

[0045] Based on Example 1, step 1 was adjusted as follows: 30% DMSO was added to the magnetic microspheres stored in an organic solvent and stirred until homogeneous to obtain a magnetic microsphere solution. Other steps were the same as in Example 1, resulting in dried magnetic microspheres.

[0046] Example 5

[0047] Based on Example 1, step 1 was adjusted as follows: 5% PEG3350 was added to the magnetic microspheres stored in an organic solvent and stirred until homogeneous to obtain a magnetic microsphere solution. Other steps were the same as in Example 1, resulting in dried magnetic microspheres.

[0048] Example 6

[0049] Based on Example 1, step 1 was adjusted as follows: 10% PEG3350 was added to the magnetic microspheres stored in an organic solvent and stirred until homogeneous to obtain a magnetic microsphere solution. Other steps were the same as in Example 1, resulting in dried magnetic microspheres.

[0050] Example 7

[0051] Based on Example 1, step 1 was adjusted as follows: 30% PEG3350 was added to the magnetic microspheres stored in an organic solvent and stirred until homogeneous to obtain a magnetic microsphere solution. Other steps were the same as in Example 1, resulting in dried magnetic microspheres.

[0052] Example 8

[0053] Based on Example 1, step 1 was adjusted as follows: 5% Tween 20 was added to the magnetic microspheres stored in the organic solvent and stirred until homogeneous to obtain a magnetic microsphere solution. Other steps were the same as in Example 1, resulting in dried magnetic microspheres.

[0054] Example 9

[0055] Based on Example 1, step 1 was adjusted as follows: 10% Tween 20 was added to the magnetic microspheres stored in the organic solvent and stirred until homogeneous to obtain a magnetic microsphere solution. The other steps were the same as in Example 1 to obtain dried magnetic microspheres.

[0056] Example 10

[0057] Based on Example 1, step 1 was adjusted as follows: 30% Tween 20 was added to the magnetic microspheres stored in the organic solvent and stirred until homogeneous to obtain a magnetic microsphere solution. Other steps were the same as in Example 1, resulting in dried magnetic microspheres.

[0058] Example 11

[0059] Based on Example 1, step 1 was adjusted as follows: 5% DMSO and 5% Tween 20 powder were added to the magnetic microspheres stored in an organic solvent and stirred until homogeneous to obtain a magnetic microsphere solution. Other steps were the same as in Example 1, resulting in dried magnetic microspheres.

[0060] Example 12

[0061] Based on Example 1, step 1 was adjusted as follows: 5% Tween 20 and 5% PEG3350 powder were added to the magnetic microspheres stored in an organic solvent and stirred until homogeneous to obtain a magnetic microsphere solution. Other steps were the same as in Example 1, resulting in dried magnetic microspheres.

[0062] Example 13

[0063] Based on Example 1, step 1 was adjusted as follows: 5% mannitol was added to the magnetic microspheres stored in an organic solvent and stirred until homogeneous to obtain a magnetic microsphere solution. Other steps were the same as in Example 1, resulting in dried magnetic microspheres.

[0064] Example 14

[0065] Based on Example 1, step 1 was adjusted as follows: 5% aromatic alkyloxy ether was added to the magnetic microspheres stored in an organic solvent and stirred until homogeneous to obtain a magnetic microsphere solution. Other steps were the same as in Example 1, resulting in dried magnetic microspheres.

[0066] Example 15

[0067] Based on Example 1, step 1 is adjusted as follows: the magnetic microspheres stored in an organic solvent are used as a magnetic microsphere solution for subsequent steps. Other steps are the same as in Example 1, resulting in dried magnetic microspheres.

[0068] The magnetic microspheres stored in organic solvent and the dried magnetic microspheres prepared in Examples 1-15 were subjected to reconstitution and load-bearing tests. The quality of the selected excipients was judged by the reconstitution time and the load-bearing capacity of the magnetic microspheres.

[0069] The method for the reconstitution test is as follows: Add 2 mL of pure water to the freeze-dried magnetic microspheres in the vial, mix well, and observe the time required for it to reconstitute into a gel.

[0070] The method for determining protein binding capacity is as follows:

[0071] S1. Take 2 mL of cation exchange medium and pack it into a 2 mL pre-packed column. Connect the packed column to the AKTA protein purification system.

[0072] S2. IgG solution with a sample concentration of 5 mg / ml was sampled onto the pump head at a flow rate of 2.5 mL / min. Buffer A contained 0.02 M NaAC and 40 M NaCl, pH = 4.5. Buffer B contained 0.02 M NaAC and 1 M NaCl, pH = 4.5. The sample was sampled onto the pump head until 50% flow-through of the protein solution was achieved. Buffer B was then used to elute and collect the protein load.

[0073] The results are as follows:

[0074] Table 1. Effects of different excipients on the reconstitution time and load-bearing capacity of magnetic microspheres.

[0075]

[0076]

[0077] The results showed that the differences in dispersibility due to different excipients and concentrations, stemming from varying degrees of structural support, resulted in different dissolution times and microsphere damage, ultimately leading to differences in microsphere resolution and carrying capacity. DMSO provided some protection for the vacuum freeze-drying of magnetic microspheres, but the resolution time after DMSO treatment alone was relatively long, while the carrying capacity of microspheres treated with PEG3350 alone decreased significantly. The combined effects of DMSO and PEG3350 were superior to those of either treatment alone.

[0078] Example 16

[0079] Based on Example 1, the temperature of the partition in steps 2 and 3 was adjusted to 10°C. Other steps were the same as in Example 1, resulting in dried magnetic microspheres.

[0080] Example 17

[0081] Based on Example 1, the temperature of the partition in steps 2 and 3 was adjusted to 8°C. Other steps were the same as in Example 1, resulting in dried magnetic microspheres.

[0082] Example 18

[0083] Based on Example 1, the temperature of the partition in steps 2 and 3 was adjusted to 6°C. Other steps were the same as in Example 1, resulting in dried magnetic microspheres.

[0084] Example 19

[0085] Based on Example 1, the temperature of the partition in steps 2 and 3 was adjusted to 2°C. Other steps were the same as in Example 1, resulting in dried magnetic microspheres.

[0086] The dried magnetic microspheres prepared in Examples 1, 16-19 were compared in terms of drying cycle, reconstitution, and carrier. The quality of the selected excipients was judged by the reconstitution time and the carrying capacity of the magnetic microspheres. The test methods were the same as above. The results are as follows:

[0087] Table 2 Comparison of drying time, reconstitution time, and magnetic microsphere carrying capacity at different drying temperatures.

[0088]

[0089] At higher temperatures, the microsphere solution cannot solidify. Around 18℃, DMSO easily dissolves, increasing the difficulty of drying and extending the drying cycle by tens of hours. Below 0℃, the microspheres themselves will break down and aggregate. It can be seen that the 2-10℃ range has little impact on drying time, reconstitution time, and microsphere carrying capacity, with 4℃ showing the best results.

[0090] Example 20

[0091] Based on Example 1, the vacuum level in step 3 was adjusted to 10 mbr. The other steps were the same as in Example 1, resulting in dried magnetic microspheres.

[0092] Example 21

[0093] Based on Example 1, the vacuum level in step 3 was adjusted to 5 mbr. Other steps were the same as in Example 1, resulting in dried magnetic microspheres.

[0094] The dried magnetic microspheres prepared in Examples 1 and 20-21 were compared in terms of drying cycle, reconstitution test, and carrier test. The quality of the selected excipients was judged by the reconstitution time and the carrying capacity of the magnetic microspheres. The test methods were the same as above. The results are as follows:

[0095] Table 3 Comparison of drying time, resolution time, and magnetic microsphere carrying capacity under different drying vacuum levels.

[0096]

[0097] The first stage of vacuum is mainly to provide a vacuum environment for the evaporation of residual organic solvents, ensuring a uniform evaporation rate. The second stage of vacuum is to ensure compliance with the saturated vapor pressure curve and prevent the product from melting.

[0098] It is evident that a drying vacuum of 0.1-5 mbr is beneficial for reducing the drying cycle of magnetic microspheres and improving drying efficiency.

[0099] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A method for drying magnetic microspheres, characterized in that, Includes the following steps: S1. Add solvent and surfactant to magnetic microspheres and mix thoroughly to obtain a mixed solution; S2. Pre-freeze the mixed solution at 0-10℃ for 2-10 hours; S3. Perform two-stage vacuum drying on the pre-frozen mixed solution; the temperature of the first stage vacuum drying is 0-10℃ and the vacuum degree is 100-300 mbr; the temperature of the second stage vacuum drying is 0-10℃ and the vacuum degree is 0.1-10 mbr. The solvent is dimethyl sulfoxide; the surfactant is a nonionic surfactant. The microspheres include one or more of the following: agarose magnetic microspheres, viral nucleic acid extraction magnetic microspheres, silanol magnetic microspheres, carboxyl magnetic microspheres, Oligo magnetic microspheres, streptavidin magnetic microspheres, cationic magnetic microspheres, affinity ligand magnetic microspheres, and toluenesulfonyl magnetic microspheres. The amount of solvent added is 3-10% of the mass of the microspheres, and the amount of surfactant added is 3-10% of the mass of the microspheres. The surfactant is one or more of Tween or PEG; The Tween is one or more of Tween 20, Tween 40, Tween 60, and Tween 80; The PEG is one or more of PEG2000, PEG3350, PEG4000, and PEG6000.

2. The drying method according to claim 1, characterized in that, In S2, the pre-freezing time of the mixed solution is 2-4 hours.

3. The drying method according to claim 1 or 2, characterized in that, The vacuum degree of the first stage of vacuum drying is 100-150 mbr; the vacuum degree of the second stage of vacuum drying is 0.1-5 mbr.

4. The drying method according to claim 1 or 2, characterized in that, In S3, the first stage of vacuum drying takes 2-4 hours, and the second stage of vacuum drying takes 30-40 hours.

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