Multifunctional micro-nano carrier as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological materials, in particular to a multifunctional micro-nano carrier and a preparation method and application thereof. The micro-nano carrier comprises raspberry-like silicon dioxide microspheres and a composite coating coated on the surfaces of the silicon dioxide microspheres, and the composite coating comprises poly (carboxylic acid betaine methyl methacrylate), tetramethoxyazobenzene, carboxymethyl-beta-cyclodextrin and folic acid. One end of the poly (carboxylic acid betaine methyl methacrylate) is grafted on the surface of the silicon dioxide microsphere, a chain segment of the poly (carboxylic acid betaine methyl methacrylate) is fixedly connected with one end of the tetramethoxyazobenzene, and the other end of the tetramethoxyazobenzene is embedded at one end of the carboxymethyl-beta-cyclodextrin; the folic acid is embedded at the other end of the carboxymethyl-beta-cyclodextrin. The micro-nano carrier realizes efficient capture and lossless release of CTCs through multiple synergy of structural design, chemical grafting, chemical modification and biological interface matching and fusion of material chemical and biological targeting.

Description

Technical Field

[0001] This application relates to the technical field of biomaterials, and particularly relates to a multifunctional micro-nano carrier, a preparation method thereof, and an application thereof. Background Art

[0002] Circulating tumor cells (CTCs) are a special type of viable tumor-derived cells that shed from the primary tumor or metastatic foci into the peripheral blood circulation system and play a crucial role in tumor metastasis; therefore, CTCs are an important non-invasive biomarker that can be enriched and analyzed without complex invasive tumor biopsies. Detecting CTCs is of great significance for early cancer diagnosis, dynamic monitoring, and prognostic treatment. However, CTCs are rare in peripheral blood, with approximately one to a dozen CTCs per 1 mL of blood, and containing approximately billions of blood cells. Therefore, CTCs are in a state of low abundance in peripheral blood. Considering the low abundance, fragility of CTCs, and the complexity of the blood environment, traditional cell detection methods based on size, density, and flow cytometry are no longer applicable. At present, many methods have been developed to eliminate the interference of the blood environment and improve the separation and enrichment purity of CTCs, and these methods include: single anti-fouling molecules, microfiltration, and negative enrichment. However, these methods all have problems such as low capture efficiency, low purity, and low specificity of CTCs.

[0003] Regarding the problem of low specificity of CTCs, most separation methods can use epithelial cell adhesion molecule (EpCAM) to improve the specificity of CTCs. However, EpCAM is severely restricted by the heterogeneity of CTCs. For example, the loss of EpCAM expression caused by epithelial-mesenchymal transition (EMT) will seriously affect the separation methods that rely on the expression of EpCAM. In addition, the high cost and poor stability of the corresponding EpCAM antibodies or aptamers will further hinder the clinical application of CTCs. In addition, the fragile nature of CTCs makes it difficult to meet the requirement of releasing CTCs without damage while ensuring a high CTC capture efficiency. Regarding the fragility of CTCs, a variety of release methods (such as enzymatic release, competitive release, and temperature-controlled release) have been used at present. Although these release methods have certain positive effects, these release methods will have a negative impact on cell viability. Summary of the Invention

[0004] This application provides a multifunctional micro-nano carrier, a preparation method thereof, and an application thereof to solve the following technical problems: how to improve the capture efficiency of CTCs on the premise of ensuring the non-destructive release of CTCs.

[0005] In a first aspect, an embodiment of the present application provides a multifunctional micro-nano carrier. The micro-nano carrier includes a biomimetic raspberry-shaped silica microsphere and a composite coating grafted on the surface of the silica microsphere. The composite coating includes poly(carboxybetaine methacrylate), tetramethoxyazobenzene, carboxymethyl-β-cyclodextrin, and folic acid. One end of the poly(carboxybetaine methacrylate) is grafted on the surface of the silica microsphere, a segment of the poly(carboxybetaine methacrylate) is fixedly connected to one end of the tetramethoxyazobenzene, the other end of the tetramethoxyazobenzene is embedded in one end of the carboxymethyl-β-cyclodextrin, and the folic acid is embedded in the other end of the carboxymethyl-β-cyclodextrin.

[0006] Optionally, the mass m1 of the silica microsphere, the mass m2 of the poly(carboxybetaine methacrylate), the mass m3 of the tetramethoxyazobenzene, the mass m4 of the carboxymethyl-β-cyclodextrin, and the mass m5 of the folic acid satisfy the relational expression: m1:m2:m3:m4:m5 = 1:1.11:0.00008:0.0002:0.04.

[0007] Optionally, the average particle size of the silica microsphere is 3 μm to 10 μm.

[0008] In a second aspect, an embodiment of the present application provides a method for preparing the micro-nano carrier described in the first aspect. The method includes:

[0009] Mixing the biomimetic raspberry-shaped silica microsphere with a dispersion solvent to obtain a silica microsphere dispersion liquid;

[0010] Spraying the silica microsphere dispersion liquid onto the surface of a semi-cured substrate and heating and curing it to obtain a silica microsphere nano-substrate;

[0011] Making the silica microsphere nano-substrate adsorb a benzophenone solution to obtain an adsorbed substrate;

[0012] Immersing the adsorbed substrate in a carboxybetaine methacrylate solution and performing a photo-crosslinking reaction to obtain a grafted substrate grafted with poly(carboxybetaine methacrylate);

[0013] Modifying the grafted substrate with tetramethoxyazobenzene to obtain a modified substrate;

[0014] Performing second carboxyl activation on the modified substrate to obtain an activated modified substrate;

[0015] Immersing the activated modified substrate in a carboxymethyl-β-cyclodextrin solution to graft the carboxymethyl-β-cyclodextrin onto the surface of the silica microsphere nano-substrate to obtain a primary functional modified substrate;

[0016] Soak the primary functional modified substrate with folic acid solution to obtain an intermediate functional modified substrate;

[0017] Subject the intermediate functional modified substrate to blocking treatment and first carboxyl activation to obtain a micro-nano carrier.

[0018] Optionally, the mass concentration of the silica microsphere dispersion is 0.1 g / mL; and / or

[0019] The mass of benzophenone in the benzophenone solution is 1.8% of the mass of the benzophenone solution; and / or

[0020] The mass of carboxybetaine methacrylate in the carboxybetaine methacrylate solution is 10% of the mass of the carboxybetaine methacrylate solution; and / or

[0021] The mass concentration of the tetramethoxyazobenzene solution is 0.08 mg / mL; and / or

[0022] The mass concentration of the carboxymethyl-β-cyclodextrin solution is 0.2 mg / mL; and / or

[0023] The mass concentration of the folic acid solution is 40 mg / mL.

[0024] Optionally, the light source wavelength of the photocrosslinking reaction is 315 nm to 450 nm, and the optical density of the photocrosslinking reaction is ≥40 mW / cm 2 .

[0025] Optionally, before mixing the biomimetic raspberry-like silica microspheres with the dispersion solvent to obtain a silica microsphere dispersion, the steps include:

[0026] Mix polyvinyl alcohol and cetyltrimethylammonium bromide to obtain a first mixed solution;

[0027] Carry out a complexation reaction on methanol, ammonia water and the first mixed solution to obtain a second mixed solution; wherein, the time of the complexation reaction is 15 min;

[0028] Add (3-chloropropyl)trimethoxysilane to the second mixed solution for a nucleation reaction to obtain biomimetic raspberry-like silica microspheres; wherein, the time of the nucleation reaction is 24 h.

[0029] Optionally, the molecular weight of the polyvinyl alcohol is 9 kDa to 10 kDa, and the mass of the ammonium ion in the ammonia water is 1.4% of the mass of the ammonia water; and / or

[0030] The mass m6 of the polyvinyl alcohol, the mass m7 of the cetyltrimethylammonium bromide, the volume V1 of the methanol, the volume V2 of the ammonia water, and the volume V3 of the (3-chloropropyl) trimethoxysilane satisfy the relational expression: m7:m6:V1:V2:V3 = 0.25:0.1:8:2:0.5; if the units of m6 and m7 are g, then the units of V1, V2, and V3 are mL.

[0031] In a third aspect, an embodiment of the present application provides a reagent for capturing and releasing cells, and the reagent includes the micro-nano carrier described in the first aspect.

[0032] In a fourth aspect, an embodiment of the present application provides a method for capturing and non-destructively releasing circulating tumor cells for non-diagnostic and non-therapeutic purposes, and the method includes:

[0033] Mixing the in vitro plasma containing circulating tumor cells with the micro-nano carrier described in the first aspect to adsorb the tumor cells through the micro-nano carrier to obtain an adsorbate;

[0034] Fixing the adsorbate with glutaraldehyde to obtain a fixed adsorbate;

[0035] Performing gradient dehydration treatment on the fixed adsorbate to obtain a capture substrate;

[0036] Incubating the capture substrate to obtain captured cells;

[0037] Irradiating the captured cells with a near-infrared light source to release the circulating tumor cells.

[0038] The above technical solutions provided by the embodiments of the present application have the following advantages compared with the prior art:

[0039] A multifunctional micro-nanocarrier provided by an embodiment of the present application. The micro-nanocarrier is based on a biomimetic raspberry-shaped silica microsphere and uses a composite coating including poly(carboxybetaine methacrylate), tetramethoxyazobenzene, carboxymethyl-β-cyclodextrin, and folic acid. The high specific surface area and multi-level hierarchical structure of the biomimetic raspberry-shaped silica microsphere provide more active sites, and these active sites can bind multiple poly(carboxybetaine methacrylate) polymer brushes. The abundant carboxyl functional groups on the poly(carboxybetaine methacrylate) polymer brushes enhance their chemical fixation ability. Through their synergistic effect, the fixation density of subsequent folic acid is improved. In addition, since most tumor cells overexpress folate receptors on their surfaces, the nano-carrier can precisely bind circulating tumor cells through the receptor-ligand interaction between the folate receptor and folic acid. Therefore, the specific and efficient capture of circulating tumor cells is achieved through the synergistic effect of the biomimetic raspberry-shaped silica microsphere, poly(carboxybetaine methacrylate) polymer brush, and folic acid. In addition, based on the good hydrophilicity and electro-neutrality characteristics of poly(carboxybetaine methacrylate), it has good anti-fouling performance and can avoid non-specific adsorption of the nano-carrier, thus ensuring the high purity of the nano-carrier in capturing circulating tumor cells. In addition, based on the matching and mismatching of the host-guest interaction between tetramethoxyazobenzene and carboxymethyl-β-cyclodextrin, the controlled release of the photo-responsive host-guest complex can be achieved, and thus the non-destructive release of circulating tumor cells can be realized. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The drawings herein are incorporated into the specification and constitute a part of this specification, showing embodiments consistent with the present application and used together with the specification to explain the principles of the present application.

[0041] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0042] Figure 1 Schematic diagram of the structure of a multifunctional micro-nanocarrier provided by an embodiment of the present application;

[0043] Figure 2 Comparison chart of the characterization results of the micro-nanocarriers of Example 1 and Comparative Example 1 of the present application, where Figure 2 A is the characterization result diagram of the micro-nanocarrier of Comparative Example 1, Figure 2 B is the characterization result diagram of the micro-nanocarrier of Example 1;

[0044] Figure 3 Schematic diagram of the process for preparing a micro-nanocarrier provided by an embodiment of the present application;

[0045] Figure 4 Schematic diagram of the detailed process of a method for preparing micro-nano carriers provided by an embodiment of the present application;

[0046] Figure 5 Schematic diagram of the process of a method for capturing and non-destructively releasing circulating tumor cells for non-diagnostic and non-therapeutic purposes provided by an embodiment of the present application;

[0047] Figure 6 Contact angle result diagram of a multifunctional micro-nano carrier provided by Embodiment 1 of the present application;

[0048] Figure 7 Anti-platelet adhesion test results of micro-nano carriers provided by Embodiment 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 of the present application;

[0049] Figure 8 Comparison diagram of the capture effects of micro-nano carriers on circulating tumor cells provided by Embodiment 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 of the present application;

[0050] Figure 9 Comparison diagram of the release effects of circulating tumor cells of a multifunctional micro-nano carrier provided by Embodiment 1 of the present application under light response and dark conditions;

[0051] Figure 10 Cell viability result diagram of the cells released by a multifunctional micro-nano carrier provided by Embodiment 1 of the present application. Detailed implementation manners

[0052] To make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are some, but not all, of the embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts shall fall within the scope of protection of the present application.

[0053] The various embodiments of the present application may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be construed as a rigid limitation on the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and individual values within that range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and individual numbers within that range, such as 1, 2, 3, 4, 5, and 6, and this applies regardless of the range; in addition, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0054] In this document, terms including "comprising" and the like mean "including but not limited to". Relative terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. "And / or" describes the association relationship of associated objects and indicates that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, and B exists alone; where A and B can be singular or plural. "At least one" means one or more, and "a plurality" means two or more; "at least one kind", "at least one of the following items (pieces)" or similar expressions refer to any combination of these items, including any combination of single items (pieces) or plural items (pieces); for example, "at least one of a, b, or c", or, "at least one of a, b, and c" can both mean: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or multiple respectively. The "parts representation method" such as parts by weight, parts by mass, etc. represents the proportional relationship between each component. In the proportional relationships involved in this document, the parameters that need to be described by proportion should be understood as the antecedents of the proportion formula in the order of description, and the proportional numbers should be understood as the consequents of the proportion formula. For example, if the mass ratio of substance A, substance B, and substance C is 1:2:3, then substance A, substance B, and substance C should correspond one by one with the proportional numbers in the proportion formula in the order of description, that is, the mass of substance A: the mass of substance B: the mass of substance C = 1:2:3.

[0055] Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments, equipment, etc. used in this document can be obtained through market purchase or can be prepared by existing methods.

[0056] It should be noted that in view of the problems of low specificity and fragility of CTCs, the inventors have found that a bio-friendly micro / nano structured platform can effectively capture rare CTCs from complex blood, and this micro / nano structured platform can gently release these rare CTCs from the capture substrate to maintain the integrity and viability of these rare CTCs. Therefore, this property of this micro / nano structured platform is crucial for the development of CTC-sensitive enrichment materials.

[0057] Figure 1 Exemplarily shows a schematic structural diagram of a multifunctional micro-nano carrier provided by an embodiment of the present application;

[0058] Figure 2 Exemplarily shows a comparison chart of the characterization results of the micro-nano carriers of Example 1 and Comparative Example 1 of the present application;

[0059] As Figure 1 and Figure 2 shown, an embodiment of the present application provides a multifunctional micro-nano carrier, the micro-nano carrier includes a biomimetic raspberry-shaped silica microsphere, and a composite coating grafted on the surface of the silica microsphere, the composite coating includes poly(carboxybetaine methacrylate), tetramethoxyazobenzene, carboxymethyl-β-cyclodextrin and folic acid, one end of the poly(carboxybetaine methacrylate) is grafted on the surface of the silica microsphere, a chain segment of the poly(carboxybetaine methacrylate) is fixedly connected to one end of the tetramethoxyazobenzene, the other end of the tetramethoxyazobenzene is embedded in one end of the carboxymethyl-β-cyclodextrin, and the folic acid is embedded in the other end of the carboxymethyl-β-cyclodextrin.

[0060] It should be noted that an embodiment of the present application provides a multifunctional micro-nano carrier. This nano-carrier constructs a multifunctional micro-nano carrier with high capture ability and non-destructive release for circulating tumor cells (CTCs) based on raspberry-shaped silica microspheres (SoS) and a composite coating. This property of this nano-carrier is mainly achieved through the following multi-level cooperative mechanism:

[0061] 1. Structural advantages of the biomimetic raspberry-shaped silica microsphere:

[0062] (1) Increase in specific surface area: The surface roughness of the raspberry-shaped silica microsphere is significantly increased, which can provide more binding sites and enhance the physical contact probability between the nano-carrier and CTCs.

[0063] (2) Hydrodynamic optimization: The biomimetic raspberry-shaped structure can reduce the influence of shear force in blood flow on the raspberry-shaped silica microsphere, making it easier for the nano-carrier to retain and capture CTCs.

[0064] 2. Folate (FA)-mediated active targeting:

[0065] (1) Specific recognition: Folate receptors (FRs) are overexpressed on the surface of most tumor cells. FA precisely binds to CTCs through receptor-ligand interaction, significantly enhancing the capture specificity of the nanocarrier.

[0066] (2) Reducing non-target adsorption: The targeting effect reduces the non-specific capture of normal cells, thereby improving the overall capture efficiency of the nanocarrier.

[0067] 3. Dual functions of the PCBMA anti-fouling coating:

[0068] (1) Anti-non-specific adsorption: Poly(carboxybetaine methacrylate) (PCBMA) has superhydrophilicity and electrical neutrality, which can effectively resist the adhesion of non-target substances such as plasma proteins and blood cells, reduce background interference, and improve the capture purity of the nanocarrier for CTCs.

[0069] (2) Providing a large number of active sites: The abundant carboxyl functional groups in the PCBMA chain provide more active sites for the subsequent immobilization of tetramethoxyazobenzene, increasing the density of tetramethoxyazobenzene. These high-density tetramethoxyazobenzenes can link more carboxymethyl-β-cyclodextrin and folic acid, thereby increasing the capture efficiency of the nanocarrier for circulating tumor cells.

[0070] 4. Photo-responsive host-guest complex controlled release:

[0071] Interaction between tetramethoxyazobenzene (mAzo) and carboxymethyl-β-cyclodextrin (CM-β-CD):

[0072] (1) Capture state: The trans structure of mAzo can stably bind to the cavity of CM-β-CD, fixing folic acid on the surface of the carrier.

[0073] (2) Release trigger: Near-infrared light irradiation can isomerize mAzo into the cis structure, which dissociates from the CM-β-CD cavity, resulting in the dissociation of the composite coating. CTCs thus lose the targeting anchor point and are gently released.

[0074] (3) Guarantee of non-damage: Photo-responsive release does not require chemical digestion or mechanical peeling, avoiding damage to the cell membrane integrity and maintaining the activity of CTCs.

[0075] 5. Multi-level synergistic effect:

[0076] (1) Targeting-anti-fouling synergy: While folic acid actively captures CTCs, PCBMA inhibits the non-specific adsorption of the nanocarrier, thereby doubling the capture efficiency and specificity of the nanocarrier.

[0077] (2) Dynamic reversible binding: The photo-responsive mechanism enables precise switching between the capture and release of CTCs, taking into account both high efficiency and the retention of cell viability.

[0078] 6. Comparative advantages over traditional methods:

[0079] Traditional capture (e.g., antibody-modified magnetic beads): It relies on enzymatic digestion or strong magnetic field separation, which is prone to damaging cells and has limited efficiency.

[0080] Advantages of the nanocarriers of this application: Through a trinity design of targeting - anti-fouling - photo-responsiveness, efficient (specific binding), non-destructive (photo-controlled release), and high-purity (anti-fouling coating) capture of CTCs is achieved.

[0081] 7. Potential application value:

[0082] (1) Liquid biopsy: The non-destructively released CTCs can be used for subsequent gene sequencing, drug sensitivity tests, etc., facilitating precision medicine.

[0083] (2) Dynamic monitoring: Photo-controlled release can be adapted to microfluidic systems, enabling real-time capture and analysis of CTCs.

[0084] In summary, a multifunctional micro-nanocarrier provided in the embodiments of this application. Through multiple synergies of structural design (high specific surface area of raspberry-like silica microspheres), chemical grafting of poly(carboxybetaine methacrylate) polymer brushes, chemical modification (multi-ligands of mAzo / CM-β-CD / FA), and biological interface matching (multivalence + morphology complementarity), through the ingenious integration of material chemistry and biological targeting, efficient capture of CTCs is achieved, solving the problem of balancing the capture efficiency of CTCs and cell viability; at the same time, this nanocarrier has the potential for integrated diagnosis and treatment, providing an innovative strategy for tumor liquid biopsy and treatment.

[0085] In some alternative embodiments, the mass m1 of the silica microspheres, the mass m2 of the poly(carboxybetaine methacrylate), the mass m3 of the tetramethoxyazobenzene, the mass m4 of the carboxymethyl-β-cyclodextrin, and the mass m5 of the folic acid satisfy the relationship: m1:m2:m3:m4:m5 = 1:1.11:0.00008:0.0002:0.04.

[0086] In these embodiments, the mass m1 of the silica microspheres, the mass m2 of poly(carboxybetaine methacrylate), the mass m3 of tetramethoxyazobenzene, the mass m4 of carboxymethyl-β-cyclodextrin, and the mass m5 of folic acid may satisfy the relationship: m1:m2:m3:m4:m5 = 1:1.11:0.00008:0.0002:0.04, such that the multifunctional micro-nanocarrier has sufficient silica microspheres, poly(carboxybetaine methacrylate), tetramethoxyazobenzene, carboxymethyl-β-cyclodextrin, and folic acid. Through the synergy of structural design, chemical grafting, chemical modification, and biological interface matching among the sufficient silica microspheres, poly(carboxybetaine methacrylate), tetramethoxyazobenzene, carboxymethyl-β-cyclodextrin, and folic acid, efficient capture of CTCs can be achieved.

[0087] In some alternative embodiments, the average particle size of the silica microspheres is 3 μm to 10 μm.

[0088] In these embodiments, the average particle size of the silica microspheres can be 3 μm to 10 μm, such that the raspberry-like silica microspheres have a sufficient specific surface area, enabling the silica microspheres to provide sufficient active sites for poly(carboxybetaine methacrylate), facilitating subsequent connection of tetramethoxyazobenzene, carboxymethyl-β-cyclodextrin, and folic acid through the molecular chains of poly(carboxybetaine methacrylate), and the silica microspheres can also enhance the adhesion ability of the micro-nanocarrier to CTCs through physical adsorption.

[0089] The average particle size of the silica microspheres can be 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, or 10 μm.

[0090] Figure 3 Exemplarily shown is a schematic flow chart of a method for preparing a micro-nanocarrier provided by an embodiment of the present application;

[0091] As Figure 3 shown, based on a general inventive concept, an embodiment of the present application provides a method for preparing the micro-nanocarrier, the method comprising:

[0092] S1. Mixing the biomimetic raspberry-like silica microspheres with a dispersion solvent to obtain a silica microsphere dispersion;

[0093] S2. Spraying the silica microsphere dispersion onto the surface of a semi-cured substrate and heating and curing to obtain a silica microsphere nano-substrate;

[0094] S3. Allowing the silica microsphere nano-substrate to adsorb a benzophenone solution to obtain an adsorbed substrate;

[0095] S4. Immerse the adsorption substrate in a carboxybetaine methacrylate solution and carry out a photo-crosslinking reaction to obtain a grafted substrate grafted with poly(carboxybetaine methacrylate);

[0096] S5. Modify the grafted substrate with tetramethoxyazobenzene to obtain a modified substrate;

[0097] S6. Perform second carboxyl activation on the modified substrate to obtain an activated modified substrate;

[0098] S7. Immerse the activated modified substrate in a carboxymethyl-β-cyclodextrin solution to graft carboxymethyl-β-cyclodextrin onto the surface of the silica microsphere nanosubstrate to obtain a primary functional modified substrate;

[0099] S8. Immerse the primary functional modified substrate in a folic acid solution to obtain an intermediate functional modified substrate;

[0100] S9. Perform a blocking treatment and first carboxyl activation on the intermediate functional modified substrate to obtain a micro-nano carrier.

[0101] This method is for the preparation method of the above micro-nano carrier. The specific structure and composition of the micro-nano carrier can refer to the above embodiments. Since this method adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought by the technical solutions of the above embodiments, which will not be elaborated here one by one.

[0102] It should be noted that the preparation method of the semi-cured substrate can be:

[0103] Dissolve 3.8 mL of trimethylolpropane triglycidyl ether and 2.4 mL of polyetheramine D-230 in 1.5 mL of ethyl acetate solution to obtain a mixed solution;

[0104] Drop the mixed solution on a glass slide and heat it to a semi-cured state at 120 °C to obtain a semi-cured substrate.

[0105] It should be noted that the specific semi-curing time is confirmed by the operator, and the semi-cured substrate generally refers to a substrate in a non-flowing but still viscous state.

[0106] It should be noted that in this method, silica microspheres are first mixed with a dispersion solvent, which can promote the uniform dispersion of silica microspheres in various dispersion solvents (such as ethanol) to improve their degree of uniform dispersion. Then, these uniformly dispersed silica microspheres are sprayed on the surface of the semi-cured substrate to fix the silica microspheres on the semi-cured substrate. Then, poly(carboxybetaine methacrylate), tetramethoxyazobenzene, carboxymethyl-β-cyclodextrin, and folic acid are grafted onto the silica microsphere nanosubstrate. Finally, through a blocking treatment and a first carboxylic acid activation, the poly(carboxybetaine methacrylate), tetramethoxyazobenzene, carboxymethyl-β-cyclodextrin, and folic acid on the surface of the silica microspheres are stably combined, and the nanocarrier is in an activated state.

[0107] In some alternative embodiments, the mass concentration of the silica microsphere dispersion is 0.1 g / mL; and / or

[0108] The mass of benzophenone in the benzophenone solution is 1.8% of the mass of the benzophenone solution; and / or

[0109] The mass of carboxybetaine methacrylate in the carboxybetaine methacrylate solution is 10% of the mass of the carboxybetaine methacrylate solution; and / or

[0110] The mass concentration of the tetramethoxyazobenzene solution is 0.08 mg / mL; and / or

[0111] The mass concentration of the carboxymethyl-β-cyclodextrin solution is 0.2 mg / mL; and / or

[0112] The mass concentration of the folic acid solution is 40 mg / mL.

[0113] In these embodiments, the mass content of silica microspheres in the silica microsphere dispersion can be 0.1 g / mL, the mass of benzophenone in the benzophenone solution can be 1.8% of the mass of the benzophenone solution, the mass of carboxybetaine methacrylate in the carboxybetaine methacrylate solution can be 10% of the mass of the carboxybetaine methacrylate solution, the mass concentration of the tetramethoxyazobenzene solution is 0.08 mg / mL, the mass concentration of the carboxymethyl-β-cyclodextrin solution can be 0.2 mg / mL, and the mass concentration of the folic acid solution can be 40 mg / mL, so that the multifunctional micro-nanocarrier has sufficient silica microspheres, poly(carboxybetaine methacrylate), tetramethoxyazobenzene, carboxymethyl-β-cyclodextrin, and folic acid. Through the synergy of structural design, chemical grafting, chemical modification, and biological interface matching among sufficient silica microspheres, poly(carboxybetaine methacrylate), tetramethoxyazobenzene, carboxymethyl-β-cyclodextrin, and folic acid, efficient capture of CTCs can be achieved.

[0114] In some alternative embodiments, the light source wavelength of the photo-crosslinking reaction is 315 nm to 450 nm, and the optical density of the photo-crosslinking reaction is ≥ 40 mW / cm 2 。

[0115] In these embodiments, the light source wavelength of the photo-crosslinking reaction can be 315 nm to 450 nm, and the optical density of the photo-crosslinking reaction is ≥ 40 mW / cm 2 so that the photo-crosslinking reaction proceeds sufficiently to achieve the polymerization reaction of the carboxybetaine methacrylate solution under the action conditions of the benzophenone solution, and a grafted substrate grafted with poly(carboxybetaine methacrylate) is obtained.

[0116] The light source wavelength of this photo-crosslinking reaction can be 315 nm, 320 nm, 325 nm, 330 nm, 335 nm, 340 nm, 345 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, or 450 nm.

[0117] Figure 4 Exemplarily shows a detailed process schematic diagram of a method for preparing a micro-nanocarrier provided by an embodiment of the present application;

[0118] In some alternative embodiments, as Figure 4 shown, before mixing the biomimetic raspberry-shaped silica microspheres with the dispersion solvent to obtain a silica microsphere dispersion, the steps include:

[0119] S101. Mix polyvinyl alcohol and cetyltrimethylammonium bromide to obtain a first mixed solution;

[0120] S102. Carry out a complexation reaction on methanol, ammonia water and the first mixed solution to obtain a second mixed solution; wherein, the time of the complexation reaction is 15 min;

[0121] S103. Add (3-chloropropyl)trimethoxysilane to the second mixed solution to carry out a nucleation reaction to obtain biomimetic raspberry-shaped silica microspheres; wherein, the time of the nucleation reaction is 24 h.

[0122] In these embodiments, polyvinyl alcohol and cetyltrimethylammonium bromide are mixed, and then methanol and ammonia water are added, so as to facilitate the subsequent regulation of the nucleation reaction process of (3-chloropropyl)trimethoxysilane, so as to promote the formation of raspberry-shaped silica microspheres by (3-chloropropyl)trimethoxysilane.

[0123] In some alternative embodiments, the molecular weight of the polyvinyl alcohol is 9 kDa to 10 kDa, and the mass of the ammonium ion in the ammonia water is 1.4% of the mass of the ammonia water; and / or

[0124] The mass m4 of the polyvinyl alcohol, the mass m5 of the cetyltrimethylammonium bromide, the volume V1 of the methanol, the volume V2 of the ammonia water and the volume V3 of the (3-chloropropyl)trimethoxysilane satisfy the relational expression: m4:m5:V1:V2:V3 = 0.25:0.1:8:2:0.5; if the units of m4 and m5 are g, then the units of V1, V2 and V3 are mL.

[0125] In these embodiments, the molecular weight of the polyvinyl alcohol can be 9 kDa to 10 kDa, and the mass of the ammonium ion in the ammonia water can be 1.4% of the mass of the ammonia water. In addition, the mass m4 of the polyvinyl alcohol, the mass m5 of the cetyltrimethylammonium bromide, the volume V1 of the methanol, the volume V2 of the ammonia water and the volume V3 of the (3-chloropropyl)trimethoxysilane can satisfy the relational expression: m4:m5:V1:V2:V3 = 0.25:0.1:8:2:0.5. Based on the complexation reaction of polyvinyl alcohol, cetyltrimethylammonium bromide, methanol and ammonia water, the nucleation reaction process of (3-chloropropyl)trimethoxysilane can be regulated to promote the formation of raspberry-shaped silica microspheres by (3-chloropropyl)trimethoxysilane.

[0126] Based on a general inventive concept, the embodiments of the present application provide a reagent for capturing and releasing cells, and the reagent includes the micro-nano carrier.

[0127] This reagent is realized based on the above-mentioned micro-nano carrier. For the specific composition and structure of the micro-nano carrier, reference can be made to the above-mentioned embodiments. Since this reagent adopts some or all of the technical solutions of the above-mentioned embodiments, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be elaborated one by one here.

[0128] Figure 5 Exemplarily shown is a schematic flowchart of a method for capturing and non-destructively releasing circulating tumor cells for non-diagnostic and non-therapeutic purposes provided by an embodiment of the present application;

[0129] Based on a general inventive concept, as Figure 5 shown, an embodiment of the present application provides a method for capturing and non-destructively releasing circulating tumor cells for non-diagnostic and non-therapeutic purposes, the method comprising:

[0130] S1. Mix the in vitro plasma containing circulating tumor cells with the micro-nano carrier to adsorb the tumor cells through the micro-nano carrier, obtaining an adsorbate;

[0131] S2. Fix the adsorbate with glutaraldehyde to obtain a fixed adsorbate;

[0132] S3. Perform gradient dehydration treatment on the fixed adsorbate to obtain a capture substrate;

[0133] S4. Incubate the capture substrate to obtain captured cells;

[0134] S5. Irradiate the captured cells with a near-infrared light source to release the circulating tumor cells.

[0135] This method is realized based on the above-mentioned micro-nano carrier. For the specific composition and structure of the micro-nano carrier, reference can be made to the above-mentioned embodiments. Since this method adopts some or all of the technical solutions of the above-mentioned embodiments, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be elaborated one by one here.

[0136] The following further elaborates the present application in conjunction with specific embodiments. For the experimental methods without specific conditions indicated in the following embodiments, they are generally determined according to national standards / industry standards; if there are no corresponding national standards / industry standards, they are carried out according to general international standards, conventional conditions or the conditions recommended by the manufacturer.

[0137] Example 1

[0138] As Figure 1 and Figure 2As shown, a multifunctional micro-nano carrier, the micro-nano carrier includes a biomimetic raspberry-shaped silica microsphere, and a composite coating coated on the surface of the silica microsphere. The composite coating includes poly(carboxybetaine methacrylate), tetramethoxyazobenzene, carboxymethyl-β-cyclodextrin and folic acid. One end of poly(carboxybetaine methacrylate) is grafted on the surface of the silica microsphere, the other end of poly(carboxybetaine methacrylate) is grafted to one end of tetramethoxyazobenzene, the other end of tetramethoxyazobenzene is embedded in one end of carboxymethyl-β-cyclodextrin, and folic acid is embedded in the other end of carboxymethyl-β-cyclodextrin.

[0139] The mass m1 of the silica microsphere, the mass m2 of poly(carboxybetaine methacrylate), the mass m3 of tetramethoxyazobenzene, the mass m4 of carboxymethyl-β-cyclodextrin and the mass m5 of folic acid satisfy the relational expression: m1:m2:m3:m4:m5 = 1:1.11:0.00008:0.0002:0.04.

[0140] The average particle size of the silica microsphere is 3 μm to 10 μm.

[0141] As Figure 4 shown, a method for preparing a micro-nano carrier includes:

[0142] S101. Mix polyvinyl alcohol and cetyltrimethylammonium bromide to obtain a first mixed solution;

[0143] S102. Carry out a complexation reaction on methanol, ammonia water and the first mixed solution to obtain a second mixed solution; wherein, the time of the complexation reaction is 15 min;

[0144] S103. Add (3-chloropropyl)trimethoxysilane to the second mixed solution to carry out a nucleation reaction to obtain a biomimetic raspberry-shaped silica microsphere; wherein, the time of the nucleation reaction is 24 h;

[0145] S1. Mix the biomimetic raspberry-shaped silica microsphere with a dispersion solvent to obtain a silica microsphere dispersion;

[0146] S2. Spray the silica microsphere dispersion onto the surface of a semi-cured substrate and carry out heat curing to obtain a silica microsphere nano-substrate;

[0147] S3. Make the silica microsphere nano-substrate adsorb a benzophenone solution to obtain an adsorption substrate;

[0148] S4. Immerse the adsorption substrate in a carboxybetaine methacrylate solution and carry out a photo-crosslinking reaction to obtain a grafted substrate grafted with poly(carboxybetaine methacrylate);

[0149] S5. Modify and graft the substrate with tetramethoxyazobenzene to obtain a modified substrate;

[0150] S6. Perform second carboxyl activation on the modified substrate to obtain an activated modified substrate;

[0151] S7. Immerse the activated modified substrate in a carboxymethyl-β-cyclodextrin solution to graft carboxymethyl-β-cyclodextrin onto the surface of the silica microsphere nanosubstrate, obtaining a primary functional modified substrate;

[0152] S8. Immerse the primary functional modified substrate in a folic acid solution to obtain an intermediate functional modified substrate;

[0153] S9. Perform blocking treatment and first carboxyl activation on the intermediate functional modified substrate to obtain a micro-nano carrier.

[0154] The mass concentration of the silica microsphere dispersion is 0.1 g / mL;

[0155] The mass concentration of the tetramethoxyazobenzene solution is 0.08 mg / mL;

[0156] The mass concentration of the carboxymethyl-β-cyclodextrin solution is 0.2 mg / mL;

[0157] The mass concentration of the folic acid solution is 40 mg / mL.

[0158] The mass of benzophenone in the benzophenone solution is 1.8% of the mass of the benzophenone solution;

[0159] The mass of carboxybetaine methacrylate in the carboxybetaine methacrylate solution is 10% of the mass of the carboxybetaine methacrylate solution.

[0160] The molecular weight of polyvinyl alcohol is 9 kDa - 10 kDa, and the mass of ammonium ions in ammonia water is 1.4% of the mass of ammonia water;

[0161] The mass m4 of polyvinyl alcohol, the mass m5 of cetyltrimethylammonium bromide, the volume V1 of methanol, the volume V2 of ammonia water, and the volume V3 of (3-chloropropyl)trimethoxysilane satisfy the relationship: m4:m5:V1:V2:V3 = 0.25:0.1:8:2:0.5; if the units of m4 and m5 are g, then the units of V1, V2, and V3 are mL.

[0162] The specific process of the above method is as follows:

[0163] (1) Synthesis of SoS silica microspheres: 0.25 g of polyvinyl alcohol and 0.1 g of hexadecyltrimethylammonium bromide were weighed and dissolved in 5 mL of RO water, and 8 mL of methanol and 2 mL of ammonia water were added respectively. After the complexation reaction lasted for 15 min, 0.5 mL of (3-chloropropyl)trimethoxysilane was added dropwise in mid-air. The addition process was completed within 15 s. The mixture was stirred at 600 rpm for 24 h, and then centrifuged and washed with anhydrous ethanol to obtain bionic raspberry-shaped silica microspheres.

[0164] (2) Preparation of functionalized nanosubstrates:

[0165] The SoS silica microspheres were dispersed in an ethanol solution to form an ethanol dispersion of the SoS silica microspheres with a mass concentration of 0.1 g / mL.

[0166] 3.8 mL of trimethylolpropane triglycidyl ether and 2.4 mL of polyetheramine D-230 were taken, and then the two were mixed in 1.5 mL of ethyl acetate to obtain a second mixed solution. The second mixed solution was evenly dropped on a clean round glass slide with a diameter of 14 mm, heated at 120°C until semi-solidified (not flowing but sticky), the semi-solidified substrate was removed, and then 10 mL of ethanol dispersion of SoS silica microspheres was evenly sprayed, and then continued to heat at 120°C for a period of time until the semi-solidified substrate was completely solidified. After cooling, the film was ultrasonically rinsed in an ethanol solution to remove loose SoS particles, and vacuum dried at 25°C to 37°C and 25kPa to obtain a SoS nanosubstrate (g@SoS).

[0167] The silica microsphere nano-substrate was immersed in a 1.8% benzophenone solution, and after adsorption for 1.5 hours, it was vacuum dried at 25°C to 37°C and 25kPa. Then, the adsorbed substrate was immersed in a 10% carboxylic acid betaine methyl methacrylate (CBMA) solution with a deoxygenated mass concentration, and ultraviolet light (the wavelength of ultraviolet light was 315nm to 450nm, the main wavelength was 365nm, and the optical density of ultraviolet light was 40mW / cm 2 ) After irradiation for 20min, a substrate grafted with PCBMA (g@SoS-P) was obtained.

[0168] The g@SoS-P substrate was subjected to second carboxyl activation for 3 h using a mixed PBS buffer (10 mM, pH = 6) of 1 M 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 M N-hydroxysuccinimide (NHS), then washed three times with water. A 0.08 mg / mL solution of tetramethoxyazobenzene (mAzo) was added, and then the mixture was placed on a shaker and reacted overnight to obtain the mAzo-modified substrate (g@SoS-PA). It was then repeatedly rinsed with water and vacuum dried at 25 °C to 37 °C and 25 kPa for later use. Then the g@SoS-PA substrate was immersed in a 0.2 mg / mL solution of carboxymethyl-β-cyclodextrin (CM-β-CD) and reacted on a shaker overnight to obtain the primary functionalized modified substrate (g@SoS-PAC) co-modified with mAzo and CM-β-CD.

[0169] The conjugate of the g@SoS-PAC substrate was blocked with 1 M ethanolamine for 10 min. After washing with water, it was placed in a mixed PBS buffer (10 mM, pH = 6) of 1 M EDC and 2 M NHS for first carboxyl activation for 3 h, then washed three times with water. A 40 mg / mL solution of folic acid (FA) was added, and then the mixture was placed on a shaker and reacted overnight to obtain the final multifunctional micro-nanocarrier (g@SoS-PACF).

[0170] Comparative Example 1

[0171] Based on the content disclosed in Example 1, the following further modifications were made:

[0172] Directly use the SoS nano-substrate.

[0173] Comparative Example 2

[0174] Based on the content disclosed in Example 1, the following further modifications were made:

[0175] Directly use the substrate grafted with PCBMA (g@SoS-P)

[0176] Comparative Example 3

[0177] Based on the content disclosed in Example 1, the following further modifications were made:

[0178] Do not add folic acid and directly use the primary functionalized modified substrate (g@SoS-PAC).

[0179] Related experiments and effect data:

[0180] The micro-nanocarriers of Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3 were applied as follows:

[0181] (1) After immersing the g@SoS-PACF substrate in platelet-rich plasma for several hours, it was gently rinsed with sterile PBS cell buffer and fixed with 2.5% glutaraldehyde by mass concentration, and then gradient dehydration treatment was carried out with ethanol solutions of different concentrations (30% - 100%) (according to the gradient concentrations of 30%, 50%, 75%, 90% and 100%, soaking for about 10 minutes at each concentration). The dehydrated substrate complex was subjected to the above vacuum drying, and the substrate was characterized by SEM, and the results are as Figure 2 shown. According to Figure 2 it can be seen that the SoS microspheres in Comparative Example 1 showed a uniform raspberry shape, while the morphology of g@SoS-PACF in Example 1 was uniform and regular, and there was no obvious change compared with the SoS microspheres.

[0182] (2) The wetting performance of the g@SoS-PACF substrate in Example 1 was tested using the static sessile drop method, and the specific steps were as follows:

[0183] The g@SoS-PACF substrate was fixed on the sample stage, and 5 μL of deionized water was dropped onto the surface of the @SoS-PACF substrate through a microsyringe. When the liquid drop was stable, the liquid drop was photographed by a CCD camera and the hydrophobic interface of the @SoS-PACF substrate was fitted and calculated using software to obtain the contact angle (WCA) data of the sample. During the detection process, it was required that each sample be randomly tested 3 times at different positions, and then the average value of the WCA data of these three times was taken as the final test result, and the results are as Figure 6 shown. From Figure 6 it can be seen that the WCA of the g@SoS-PACF substrate was close to 30°, indicating that the g@SoS-PACF substrate still maintained good hydrophilicity during the multi-step modification process, and this characteristic was beneficial to subsequent biological applications.

[0184] (3) The non-specific anti-platelet adhesion test and SEM characterization of g@SoS-PACF were carried out, and the specific steps were as follows:

[0185] Referring to the steps in experiment (1), after vacuum drying the substrates of Example 1, Comparative Example 1 and Comparative Example 2 respectively, gold spraying treatment was carried out, and then observation and photography were carried out by scanning electron microscopy, and the number of platelet adhesions in the observation area was counted, and the results are as Figure 7 shown. From Figure 7 it can be seen that compared with the g@SoS substrate, the number of platelet adhesions on the surfaces of the g@SoS-P and g@SoS-PACF substrates was reduced by more than 90%. This shows that the PCBMA polymer coating of g@SoS-PACF can effectively resist the non-specific adhesion of blood cells.

[0186] (4) Capture and release of CTCs in PBS:

[0187] 1) Comparison of capture ability:

[0188] EpCAM-negative HeLa cells resuspended in sterile PBS buffer were counted using a cell counter. Then, the sterile PBS cells were diluted with the buffer to obtain a 500 μL cell suspension. These cell suspensions were incubated with the g@SoS-PACF substrate in a shaker at 37 °C for 60 min. Then, the supernatant was aspirated, and the substrate was gently rinsed three times with PBS buffer to remove the uncaught cells. Finally, the cells were counted using a cell counter, and the cell capture efficiency of the g@SoS-PACF substrate was calculated using the following formula.

[0189] Capture efficiency = (Total number of cells - Number of uncaught cells in the supernatant) / Total number of cells × 100%.

[0190] The results are as Figure 8 shown. It can be seen from Figure 8 that the capture efficiency of the g@SoS-PACF substrate for cells is 92.7%, while the capture efficiencies of the g@SoS and g@SoS-P substrates for cells are only 53.1% and 8.2%, respectively. This indicates that the introduction of FA can enhance the capture ability of the g@SoS-PACF substrate for CTCs as a specific ligand.

[0191] 2) Comparison of release ability:

[0192] The captured cells and the g@SoS-PACF substrate were resuspended in PBS buffer, and then irradiated with infrared light at a wavelength of 808 nm for 8 min. The supernatant was aspirated, and the substrate was gently rinsed three times with PBS buffer. Then, the number of cells released into the solution system was counted using a cell counter, and the cell release efficiency of the g@SoS-PACF substrate was calculated using the following formula.

[0193] Release efficiency = Number of cells released in the supernatant / Number of captured cells × 100%.

[0194] The released cells were cultured in high-glucose DMEM medium for 6 h, and then the viability of the released cells was evaluated by the CCK-8 assay.

[0195] The results are as Figure 9 shown. It can be seen from Figure 9 that after light treatment, the release efficiency of the cells captured by the g@SoS-PACF substrate is as high as 96.1%. In contrast, under the condition of no light, the cell release rate of the g@SoS-PACF substrate is only 5.1%.

[0196] (5) Test of cell viability after release:

[0197] Based on the above release test, the activity of the cells released by the g@SoS-PACF substrate under light conditions was investigated by the CCK-8 assay. The specific steps are as follows:

[0198] Transfer the cells released by light to a new 24-well plate for re-culture. The normally cultured cells that were not exposed to light were inoculated in the well plate in the same number as the control group. After culturing for 6 h, gently rinse 3 times with PBS buffer to obtain the sample to be measured. Then use the CCK-8 kit to measure the sample to be measured, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value of the sample to be measured at 450 nm. The cell activity was calculated according to the following formula:

[0199] Cell viability = (OD 实验组 - OD 空白 ) / (OD 对照组 - OD 空白 ) × 100%.

[0200] The measurement results are Figure 10 as shown. It can be seen from Figure 10 that the survival rate of the cells released by the g@SoS-PACF substrate within 6 h is as high as 89%, which indicates that the CTCs cells released by light stimulation response can maintain good physiological activity.

[0201] In summary, a multifunctional micro-nanocarrier provided by an embodiment of the present application realizes the efficient capture of CTCs through multiple synergies of structural design (high specific surface area of raspberry-like silica microspheres), chemical grafting of PCBMA polymer brushes, chemical modification (mAzo / CM-β-CD / FA multi-ligands), and biological interface matching (multivalent + morphological complementarity), and at the same time has the potential of integrated diagnosis and treatment, providing an innovative strategy for tumor liquid biopsy and treatment.

[0202] The above are only specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown in the present application, but will conform to the widest scope consistent with the principles and novel features claimed in the present application.

Claims

1. A multifunctional micro-nano carrier, comprising bionic raspberry-shaped silica microspheres and a composite coating grafted on the surface of the silica microspheres, wherein the composite coating comprises poly(carboxylic acid betaine methyl methacrylate), tetramethoxyazobenzene, carboxymethyl-β-cyclodextrin and folic acid, one end of the poly(carboxylic acid betaine methyl methacrylate) is grafted on the surface of the silica microspheres, a chain segment of the poly(carboxylic acid betaine methyl methacrylate) is fixedly connected to one end of the tetramethoxyazobenzene, the other end of the tetramethoxyazobenzene is embedded in one end of the carboxymethyl-β-cyclodextrin, and the folic acid is embedded in the other end of the carboxymethyl-β-cyclodextrin.

2. According to the micro-nanocarrier according to claim 1, the mass m1 of the silica microspheres, the mass m2 of the poly(carboxybetaine methyl methacrylate), the mass m3 of the tetramethoxyazobenzene, the mass m4 of the carboxymethyl-β-cyclodextrin and the mass m5 of the folic acid satisfy the relationship: m1:m2:m3:m4:m5=1:1.11:0.00008:0.0002:0.

04. 3 . The micro-nano carrier according to claim 1 , wherein the average particle size of the silica microspheres is 3 μm to 10 μm.

4. A method for preparing the micro-nano carrier according to any one of claims 1 to 3, the method comprising: mixing the bionic raspberry-shaped silica microspheres with a dispersion solvent to obtain a silica microsphere dispersion; Spraying the silica microsphere dispersion onto the surface of the semi-cured substrate and heating and curing the substrate to obtain a silica microsphere nano-substrate; Allowing the silica microsphere nano-substrate to absorb the benzophenone solution to obtain an adsorption substrate; The adsorption substrate is immersed in a carboxylic acid betaine methyl methacrylate solution, and subjected to a light-irradiated cross-linking reaction to obtain a grafted substrate grafted with poly(carboxylic acid betaine methyl methacrylate); Modifying the grafted substrate using tetramethoxyazobenzene to obtain a modified substrate; Activating the modified substrate by a second carboxyl group to obtain an activated modified substrate; soaking the activated modified substrate in a carboxymethyl-β-cyclodextrin solution to graft carboxymethyl-β-cyclodextrin onto the surface of the silica microsphere nano-substrate to obtain a primary functional modified substrate; soaking the primary functional modified substrate in a folic acid solution to obtain an intermediate functional modified substrate; The intermediate functional modified substrate is subjected to sealing treatment and first carboxyl activation to obtain a micro-nano carrier.

5. The method according to claim 4, wherein the mass concentration of the silica microsphere dispersion is 0.1 g / mL; and / or The mass of benzophenone in the benzophenone solution is 1.8% of the mass of the benzophenone solution; and / or The mass of carboxylic acid betaine methyl methacrylate in the carboxylic acid betaine methyl methacrylate solution is 10% of the mass of the carboxylic acid betaine methyl methacrylate solution; and / or The mass concentration of the tetramethoxyazobenzene solution is 0.08 mg / mL; and / or The mass concentration of the carboxymethyl-β-cyclodextrin solution is 0.2 mg / mL; and / or The mass concentration of the folic acid solution is 40 mg / mL.

6. The method according to claim 4, wherein the wavelength of the light source for the photo-crosslinking reaction is 315nm to 450nm, and the optical density of the photo-crosslinking reaction is ≥40mW / cm 2 .

7. The method according to claim 4, wherein the bionic raspberry-shaped silica microspheres are mixed with a dispersion solvent to obtain a silica microsphere dispersion, comprising the steps of: Mixing polyvinyl alcohol and hexadecyltrimethylammonium bromide to obtain a first mixed solution; The methanol, ammonia water and the first mixed solution are subjected to a complex reaction to obtain a second mixed solution; wherein, The complex reaction time is 15min; (3-chloropropyl)trimethoxysilane is added to the second mixed solution to carry out a nucleation reaction to obtain bionic raspberry-shaped silica microspheres; wherein the nucleation reaction time is 24 hours.

8. The method according to claim 7, wherein the molecular weight of the polyvinyl alcohol is 9 kDa to 10 kDa, and the mass of the ammonium ions in the ammonia water is 1.4% of the mass of the ammonia water; and / or The mass m6 of the polyvinyl alcohol, the mass m7 of the hexadecyltrimethylammonium bromide, the volume V1 of the methanol, the volume V2 of the ammonia water and the volume V3 of the (3-chloropropyl)trimethoxysilane satisfy the relationship: m6:m7:V1:V2:V3=0.25:0.1:8:2:0.5; if the units of m6 and m7 are g, the units of V1, V2 and V3 are mL.

9. A reagent for capturing and releasing cells, comprising the micro-nano carrier according to any one of claims 1 to 3.

10. A method for capturing and non-destructively releasing circulating tumor cells for non-diagnostic and therapeutic purposes, the method comprising: Mixing the ex vivo plasma containing circulating tumor cells with the micro-nano carrier according to any one of claims 1 to 3, so that the tumor cells are adsorbed by the micro-nano carrier to obtain an adsorbate; Fixing the adsorbate using glutaraldehyde to obtain a fixed adsorbate; The fixed adsorbent is subjected to a gradient dehydration treatment to obtain a capture substrate; incubating the capture substrate to obtain captured cells; The captured cells are irradiated with a near-infrared light source to achieve the release of circulating tumor cells.

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