A three-dimensional micro-nano structure and preparation method thereof

Abstract

The present invention provides a three-dimensional micro-nano structure and a preparation method thereof, which belongs to the technical field of biological separation and purification. The preparation method comprises the following steps: adding polysaccharide and water into a reaction container, controlling the temperature and stirring to obtain a polysaccharide aqueous solution; adding a porogen into the polysaccharide aqueous solution, controlling the temperature and stirring; stopping stirring and cooling to obtain a polysaccharide three-dimensional structure frame; after cleaning the polysaccharide three-dimensional structure frame, successively adding a sodium hydroxide solution and epichlorohydrin to react; after cleaning, a three-dimensional micro-nano structure is obtained. The method of the present invention is conducive to the preparation of a three-dimensional micro-nano structure with a larger appearance size and a wider adjustable pore size range. The obtained micro-nano structure can effectively improve the industrial production efficiency in biological separation, and provides a new choice for efficient and large-scale biological separation and purification.

Description

Technical Field

[0001] The present invention relates to the technical field of biological separation and purification, and in particular to a three-dimensional micro-nano structure and a preparation method thereof, and application thereof in the separation and purification of biological materials such as exosomes and viruses. Background Art

[0002] At present, in the large-scale production of exosomes, viruses and other materials, agarose microspheres are mainly used for exclusion mode chromatography purification, specifically separation through the gaps between microspheres and the pores inside the microspheres. For example, when exosomes are separated and purified using agarose microspheres, the exosomes can only come out of the external water volume through the gaps between the microspheres, while impurities will enter the pores inside the microspheres and come out from the internal water volume. According to the different order of coming out, the effective separation of exosomes and impurities is achieved.

[0003] The advantage of agarose microspheres is that they do not affect the biological activity of materials such as exosomes and viruses, and can maximize the stability of the separated materials. However, the disadvantage of agarose microspheres is also obvious, that is, the industrial production efficiency is low. For example, when isolating exosomes, the exosomes need to be concentrated 20-50 times before loading, and the loading amount is less than 10% of the column volume. Moreover, agarose microsphere column loading and column efficiency detection require 2-3 days or even more than a week in large-scale production. At present, due to the limitations of the structural morphology of agarose microspheres, their internal pore size is usually controlled below 100nm. In addition, agarose microspheres are mostly prepared by inverse emulsion polymerization to obtain microsphere droplets and then solidify to obtain microspheres (such as patent CN101715364A). This method requires a large amount of organic solvent as the oil phase, which increases the production cost. The pressurization during the emulsification process leads to a higher risk in the production process. When the emulsion is squeezed through the hydrophobic porous membrane, the fragility of the hydrophobic porous membrane increases the instability of the quality of the microspheres.

[0004] Therefore, developing a safe and economical preparation method for separation materials with a larger pore size range, no sample loading limit, and no need for column packing and column efficiency testing is of great significance to enterprises' efficient and large-scale separation and purification. Summary of the invention

[0005] In view of this, the present invention provides a three-dimensional micro-nanostructure and a preparation method thereof to address the deficiencies of existing separation methods and provide a new option for efficient and large-scale separation and purification of exosomes, viruses and other biological materials.

[0006] In a first aspect, the present invention provides a method for preparing a three-dimensional micro-nano structure, which comprises the following steps: S1, adding polysaccharide and water into a reaction vessel, controlling the temperature and stirring to obtain a polysaccharide aqueous solution; S2, adding a porogen to the polysaccharide aqueous solution, and stirring under controlled temperature; S3, stopping stirring and cooling to obtain a three-dimensional polysaccharide structure framework; S4, after cleaning the polysaccharide three-dimensional structure frame, adding sodium hydroxide solution and epichlorohydrin successively to react; S5. After cleaning, a three-dimensional micro-nano structure is obtained.

[0007] Preferably, in the above preparation method, the polysaccharide is one or more of chitosan, agarose, cellulose, and dextran, and the mass concentration of the polysaccharide aqueous solution is 1-20%.

[0008] Preferably, in the above preparation method, the porogen is one or more of polyoxyethylene fatty acid esters, polysorbate, and polyethylene glycol, and the mass ratio of the porogen to the polysaccharide is 1:(5-100).

[0009] Preferably, in the above preparation method, the reaction temperature of steps S1 and S2 is controlled at 50-90°C, and the temperature of step S3 is reduced to 10-25°C.

[0010] The porogen in the present invention has strong interfacial activity in the polysaccharide aqueous solution, and forms a molecular film on the inner and outer surfaces of the polysaccharide aqueous solution, thereby changing the energy state of the surface of the polysaccharide aqueous solution, making it easy to be eroded, and then forming mesh holes inside the polysaccharide aqueous solution, obtaining a liquid form with a mesh porous structure inside, and finally obtaining a three-dimensional mesh porous skeleton structure of the polysaccharide by cooling and solidification, and then removing the porogen by diffusion cleaning.

[0011] Preferably, in the above preparation method, the mass ratio of polysaccharide to sodium hydroxide and epichlorohydrin is 1:(0.1-10):(0.1-10).

[0012] In the method of the present invention, the hydroxyl groups on the polysaccharide undergo a ring-opening reaction with the epoxy groups of epichlorohydrin under alkaline conditions, so that the adjacent hydroxyl groups are connected through the ring-opening of the epoxy groups, thereby strengthening the three-dimensional structural framework, improving its pressure resistance and flow rate, and obtaining a hollow network structure with sufficient strength. It should be noted that experiments have shown that when the sodium hydroxide in the method of the present invention is replaced with other bases, it cannot provide continuous and reliable alkaline support for the reaction, or when the ratio of sodium hydroxide to epichlorohydrin is not appropriate, the three-dimensional structural framework of the polysaccharide cannot be fully strengthened, and the final structure has problems of unevenness and insufficient structural strength.

[0013] Preferably, in the above preparation method, step S4 controls the reaction temperature at 20-30° C. by a water bath. The ring-opening reaction between epichlorohydrin and hydroxyl is an exothermic reaction, and the structure prepared by the present invention has a significantly larger size than the polysaccharide beads, so the reaction conditions are required to be higher; the reaction process needs to be continuously cooled to achieve a temperature control state, otherwise the continuous heat release of the reaction will verify and affect the uniformity of the internal hollow network structure.

[0014] Preferably, in the above preparation method, the specific operation of cleaning is: soaking in water for multiple times and then changing the liquid.

[0015] In a second aspect, the present invention provides a three-dimensional micro-nano structure, which is prepared according to the method provided by the present invention. It is understandable that the appearance and size of the three-dimensional micro-nano structure provided by the present invention depend on the reaction container. For example, in some embodiments of the present invention, a glass beaker is used as a reaction container, and the three-dimensional micro-nano structure finally prepared is in the shape of a cylinder.

[0016] In a third aspect, the present invention provides the application of the above three-dimensional micro-nano structure in biological separation, such as separation and purification of exosomes, separation and purification of viruses, etc. For example, in one embodiment of the present invention, the above three-dimensional micro-nano structure is used to separate parvoviruses, and the experimental data show that it can not only effectively separate, but also has a good separation efficiency.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The method of the present invention is to first change the physical state of the polysaccharide, directly obtain a solidified three-dimensional micro-nano structure base by dissolving the powder, and then obtain a high-strength three-dimensional micro-nano structure through a chemical reaction. The entire preparation process is completed at room temperature and pressure, and is safer, more environmentally friendly and economical.

[0018] The appearance of the product prepared by the present invention can be achieved at the centimeter, decimeter or even meter level, effectively meeting the different needs from laboratory process development to workshop production and preparation; moreover, by adjusting the ratio of polysaccharides and porogens, the hollow mesh pore size of the product can be achieved at the nanometer level, micrometer level or even millimeter level, and the pore size can be adjusted specifically according to the size and shape of the target object in the application scenario; in addition, the continuous pore structure inside the product can achieve high flow rate and low back pressure, better meeting the process requirements of large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 Flow chart of the method for preparing a three-dimensional micro-nano structure according to an embodiment of the present invention; Figure 2 This is a morphological diagram of the appearance of the three-dimensional micro-nano structure prepared in an embodiment of the present invention; Figure 3 This is a microscope image of a partial network structure of a three-dimensional micro-nano structure prepared in an embodiment of the present invention. DETAILED DESCRIPTION

[0021] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the terms "including" and "having" in the specification and claims of the present invention and the above-mentioned figure descriptions and any variations thereof are intended to cover non-exclusive inclusions.

[0023] In view of the problems that the products used for biological separation of exosomes, viruses, etc. in the prior art have a single appearance, a narrow adjustable range of size and pore size, and low industrial production efficiency, the present invention provides a three-dimensional micro-nano structure and a preparation method thereof, which provides a new option for large-scale biological separation. Among them, in the preparation method of the present invention, polysaccharide powder is heated and dissolved in an aqueous solution to obtain a long chain of polysaccharide macromolecules in a sol state, and a porogen is added to obtain a liquid structure with an internal structure of a polysaccharide three-dimensional structure, and then the three-dimensional structural frame with an internal micro-nano hollow network structure is obtained by cooling and solidification. Finally, under alkaline conditions, the hydroxyl groups on the polysaccharide react with the epoxy groups on the epichlorohydrin to open the ring, so that the similar hydroxyl groups are connected through the opening of the epoxy groups, thereby strengthening the three-dimensional structural frame, improving its pressure resistance and use flow rate, and finally obtaining a three-dimensional micro-nano structure with a hollow network structure.

[0024] like Figure 1 As shown, the three-dimensional micro-nano structure in the embodiment of the present invention is prepared by the following process: S1, adding polysaccharide and water into a reaction vessel, controlling the temperature and stirring to obtain a polysaccharide aqueous solution; S2, adding a porogen to the polysaccharide aqueous solution, and stirring under controlled temperature; S3, stopping stirring and cooling to obtain a three-dimensional polysaccharide structure framework; S4, after cleaning the polysaccharide three-dimensional structure frame, adding sodium hydroxide solution and epichlorohydrin successively to react; S5. After cleaning, a three-dimensional micro-nano structure is obtained.

[0025] Although the present invention still uses polysaccharides to construct three-dimensional micro-nano structures, the appearance and size of the products prepared by the method of the present invention can be achieved by using different reaction vessels, and the appearance of the products can be achieved at the centimeter, decimeter or even meter level; in actual production, the appearance of the products can be produced according to the requirements of the workshop site and the equipment used, which can effectively reduce the supporting use of some equipment. In addition, the hollow mesh apertures required for different application scenarios are different, and the hollow mesh apertures of the products of the present invention can be achieved at the nanometer level, micrometer level or even millimeter level, so customized production can also be carried out according to actual needs.

[0026] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0027] Example 1 This example provides a method for preparing a three-dimensional micro-nano structure, comprising the following steps: (1) Weigh 50 g of cellulose powder and add it into a 1000 ml glass beaker, then weigh 500 g of purified water and add it into the glass beaker; place the glass beaker in a water bath and heat it to 90°C, and stir at 300 rpm for 24 hours to obtain a completely dissolved polysaccharide aqueous solution.

[0028] (2) Add 10 g of polysorbate to the polysaccharide aqueous solution, maintain it in a water bath at 90 °C and stir at 300 rpm for 1 h.

[0029] (3) Stop stirring and cool the glass beaker containing the polysaccharide aqueous solution to 25° C. in a water bath to obtain a polysaccharide three-dimensional structural framework.

[0030] (4) Take 300 g of purified water and add it to a glass beaker to clean the polysaccharide three-dimensional structure frame, and repeat the cleaning 10 times; add 300 g of 10 wt% sodium hydroxide aqueous solution to the glass beaker, control the temperature in a water bath to 25°C, and then add 50 g of epichlorohydrin to the glass beaker and soak for 24 hours.

[0031] (5) Take 300 g of purified water to wash the polysaccharide three-dimensional structure, and repeat the washing 10 times to obtain a polysaccharide three-dimensional micro-nano structure with a cylindrical appearance.

[0032] Example 2 This example provides a method for preparing a three-dimensional micro-nano structure, comprising the following steps: (1) Weigh 50 g of agarose powder and add it to a 1000 ml glass beaker. Then weigh 500 g of purified water and add it to the glass beaker. Place the glass beaker in a water bath and heat it to 90°C. Stir at 300 rpm for 24 h to obtain a completely dissolved polysaccharide aqueous solution.

[0033] (2) Add 10 g of polysorbate to the polysaccharide aqueous solution, maintain it in a water bath at 90 °C and stir at 300 rpm for 1 h.

[0034] (3) Stop stirring and cool the glass beaker containing the polysaccharide aqueous solution to 25° C. in a water bath to obtain a polysaccharide three-dimensional structural framework.

[0035] (4) Take 300 g of purified water and add it to a glass beaker to clean the polysaccharide three-dimensional structure frame, and repeat the cleaning 10 times; add 300 g of 10 wt% sodium hydroxide aqueous solution to the glass beaker, control the temperature in a water bath to 25°C, and then add 50 g of epichlorohydrin to the glass beaker and soak for 24 hours.

[0036] (5) Take 300 g of purified water to wash the polysaccharide three-dimensional structure, and repeat the washing 10 times to obtain a polysaccharide three-dimensional micro-nano structure with a cylindrical appearance.

[0037] Example 3 This example provides a method for preparing a three-dimensional micro-nano structure, comprising the following steps: (1) Weigh 50 g of agarose powder and add it to a 1000 ml glass beaker. Then weigh 500 g of purified water and add it to the glass beaker. Place the glass beaker in a water bath and heat it to 90°C. Stir at 300 rpm for 24 h to obtain a completely dissolved polysaccharide aqueous solution.

[0038] (2) Add 10 g of polyethylene glycol 4000 to the polysaccharide aqueous solution, maintain it in a water bath at 90 °C and stir at 300 rpm for 1 h.

[0039] (3) Stop stirring and cool the glass beaker containing the polysaccharide aqueous solution to 25° C. in a water bath to obtain a polysaccharide three-dimensional structural framework.

[0040] (4) Take 300 g of purified water and add it to a glass beaker to clean the polysaccharide three-dimensional structure frame, and repeat the cleaning 10 times; add 300 g of 10 wt% sodium hydroxide aqueous solution to the glass beaker, control the temperature in a water bath to 25°C, and then add 50 g of epichlorohydrin to the glass beaker and soak for 24 hours.

[0041] (5) Take 300 g of purified water to wash the polysaccharide three-dimensional structure, and repeat the washing 10 times to obtain a polysaccharide three-dimensional micro-nano structure with a cylindrical appearance.

[0042] Example 4 This example provides a method for preparing a three-dimensional micro-nano structure, comprising the following steps: (1) Weigh 10 g of agarose powder and add it to a 1000 ml glass beaker. Then weigh 500 g of purified water and add it to the glass beaker. Place the glass beaker in a water bath and heat it to 90°C. Stir at 300 rpm for 24 h to obtain a completely dissolved polysaccharide aqueous solution.

[0043] (2) Add 1 g of polysorbate to the polysaccharide aqueous solution, maintain it in a water bath at 90 °C and stir at 300 rpm for 1 h.

[0044] (3) Stop stirring and cool the glass beaker containing the polysaccharide aqueous solution to 25° C. in a water bath to obtain a polysaccharide three-dimensional structural framework.

[0045] (4) Take 300 g of purified water and add it to a glass beaker to clean the polysaccharide three-dimensional structure frame, and repeat the cleaning 10 times; add 300 g of 10 wt% sodium hydroxide aqueous solution to the glass beaker, control the temperature in a water bath to 25°C, and then add 50 g of epichlorohydrin to the glass beaker and soak for 24 hours.

[0046] (5) Take 300 g of purified water to wash the polysaccharide three-dimensional structure, and repeat the washing 10 times to obtain a polysaccharide three-dimensional micro-nano structure with a cylindrical appearance.

[0047] Figure 2 The appearance of the three-dimensional micro-nano structure prepared in the above examples is shown in Figure 2. Since the containers used in Examples 1-4 are all beakers, the appearance of the obtained products is cylindrical. Figure 1 It can be seen that the product has sufficient supporting strength and will not collapse or deform.

[0048] The three-dimensional micro-nano structures prepared in Examples 1-4 were sampled, their network structures were observed under a microscope, and their network pore sizes were measured respectively. Figure 3 It is a microscopic image of a partial mesh structure of a three-dimensional micro-nanostructure, wherein ad corresponds to the products obtained in Examples 1-4 respectively. Table 1 is the mesh pore size related data of each product in Examples 1-4. Through the comparative analysis of the mesh pore size test results of Examples 1-4, the main factor affecting the pore size is the content of polysaccharides such as agarose. The lower the content of polysaccharides such as agarose, the larger the pore size; the main factors affecting the specific surface area and pore volume are different polysaccharide materials such as cellulose and agarose and the type of porogen.

[0049] Table 1 Mesh aperture test results

[0050] Example 5 This example uses viruses as an example to verify the effectiveness of the three-dimensional micro-nanostructure provided by the present invention in biological separation, including the following steps: Use the open end of a 50ml centrifuge tube to insert part of the three-dimensional structure of the cylindrical product prepared in Example 2, and place it in the middle of the centrifuge tube. Take 30ml of inactivated mouse parvovirus MVM strain culture concentrate and add it to the upper end of the centrifuge tube. Under the action of gravity, effluent appears below the three-dimensional structure. When the effluent is about 15ml, take out the virus solution at the upper and lower ends of the centrifuge tube for detection, and the test results are shown in Table 2. The above test results show that filtering the inactivated virus liquid through the three-dimensional structure can effectively reduce the loss of the virus, and at the same time has a higher ability to remove protein impurities. It can be seen that the three-dimensional micro-nano structure provided by the present invention can be used for the separation and preparation of macromolecules such as viruses and exosomes.

[0051] Table 2 Analysis of the separation effect of three-dimensional micro-nanostructures on inactivated mouse parvovirus MVM strain

[0052] In summary, the present invention provides a new option for successful bioseparation. Its preparation method is safer, more environmentally friendly and economical. It can provide a pore size range from nanometers to micrometers or even millimeters. It can realize continuous loading without limiting the sample amount, and does not require column loading and column efficiency testing, thus meeting the needs of large-scale production in enterprises.

[0053] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. A method for preparing a three-dimensional micro-nano structure, characterized in that: The following steps are involved: S1, adding polysaccharide and water into a reaction vessel, controlling the temperature and stirring to obtain a polysaccharide aqueous solution; S2, adding a porogen to the polysaccharide aqueous solution, and stirring under controlled temperature; S3, stopping stirring and cooling to obtain a three-dimensional polysaccharide structure framework; S4, after cleaning the polysaccharide three-dimensional structure frame, adding sodium hydroxide solution and epichlorohydrin successively to react; S5. After cleaning, a three-dimensional micro-nano structure is obtained.

2. The preparation method according to claim 1, characterized in that: The polysaccharide is one or more of chitosan, agarose, cellulose and dextran, and the mass concentration of the polysaccharide aqueous solution is 1-20%.

3. The preparation method according to claim 2, characterized in that: The porogen is one or more of polyoxyethylene fatty acid ester, polysorbate, and polyethylene glycol, and the mass ratio of the porogen to the polysaccharide is 1:(5-100).

4. The preparation method according to claim 1, characterized in that: The reaction temperature of steps S1 and S2 is controlled at 50-90°C.

5. The preparation method according to claim 1, characterized in that: The temperature of step S3 is reduced to 10-25°C.

6. The preparation method according to claim 1, characterized in that: The reaction temperature of step S4 is 20-30°C.

7. The preparation method according to claim 2, characterized in that: The mass ratio of the polysaccharide to sodium hydroxide and epichlorohydrin is 1:(0.1-10):(0.1-10).

8. The preparation method according to claim 1, characterized in that: The cleaning is performed by soaking in water for multiple times and then changing the liquid.

9. The three-dimensional micro-nano structure obtained according to the preparation method according to any one of claims 1 to 8.

10. Use of the three-dimensional micro-nanostructure as claimed in claim 9 in separation of exosomes or viruses.

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