Magnetic-driven porous spiral nano motor for active antigen delivery and preparation method and application of magnetic-driven porous spiral nano motor

Abstract

The invention discloses a magnetic drive porous spiral nano motor for active antigen delivery and a preparation method and application thereof. The magnetic drive porous spiral nano motor comprises a nickel layer, porous spiral silicon and surface antigen ovalbumin OVA, and the nickel layer modifies the surface antigen ovalbumin OVA on the surface of the porous spiral silicon. The preparation method comprises the following steps: S1, adding tetraethyl orthosilicate into a water solution containing sodium hydroxide, perfluorooctanoic acid and hexadecyl trimethyl ammonium bromide by adopting a sol-gel method, mixing, stirring and reacting, and calcining to obtain a spiral siliceous mesoporous material HR; s2, a metal nickel layer is formed on the surface of the spiral siliceous mesoporous material through magnetron sputtering, and the HR-Ni spiral magnetic drive motor is obtained; s3, surface antigen ovalbumin OVA is modified for the HR-Ni spiral magnetic drive motor, and therefore the magnetic drive porous spiral nano motor is obtained. The nano motor has high antigen loading efficiency and high cell uptake efficiency, and can enhance cross delivery of antigens.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and in particular to a magnetically driven porous spiral nanomotor for active antigen delivery, and a preparation method and application thereof. Background Art

[0002] As an important immunotherapy, tumor vaccines have broad prospects in tumor treatment due to their strong specificity, few side effects, and the ability to produce immune memory after treatment. However, exogenous antigens are usually internalized into lysosomes by antigen-presenting cells, reducing the immune activity of antigens. Therefore, it is necessary to develop an antigen delivery system that can enhance antigen cytoplasmic delivery and promote antigen cross-delivery.

[0003] Micro / nanomotors are artificial micro / nanoscale devices that can convert external energy (such as chemical energy, light energy, sound energy, magnetic energy, electrical energy, etc.) into their own mechanical energy to achieve autonomous movement. Due to the autonomous movement characteristics and ultra-small size of micro / nanomotors, they show great potential in environmental remediation and biomedical applications. Among the various driving modes of micro / nanomotors, magnetic drive overcomes the dependence of chemically driven nanomotors on fuel. At the same time, this fuel-free micro / nanomotor can control the direction of movement by an external magnetic field, and magnetic field drive is more non-invasive than other forms of driving methods. Therefore, magnetic field-driven micro / nanomotors have broad prospects in many biomedical fields. The helical magnetically driven nanomotor is a typical representative of magnetic field drive. Under a rotating magnetic field, it can imitate the movement of bacterial flagella and perform spiral movement. Compared with other magnetically driven nanomotors driven by magnetic field gradients or alternating magnetic fields, the helical magnetically driven micro / nanomotor adopts an efficient torque drive strategy, which enables it to achieve precise movement with a lower magnetic field strength.

[0004] In the preparation method of helical magnetically driven micro-nanomotors (Helical meso-porous silicon@Ni,HON), the biological template method, microfluidics method, etc. can usually only prepare micron-sized helical magnetically driven motors. However, micron-sized motors have the problem of difficulty in performing more delicate tasks. The electrodeposition method and grazing angle deposition method that can prepare nanoscale helical motors require complex process equipment support, and the prepared motors are mainly composed of metals, which have disadvantages such as no porosity, poor load capacity and biocompatibility.

[0005] Chinese patent document CN 116350792 A discloses a flexible nanomotor constructed of mesoporous organic silicon oxide material, and its preparation method and application; rigid mesoporous organic silicon oxide nanoparticles are prepared by sol-gel method, and after etching, mesoporous organic silicon oxide nanocapsules with flexible skeletons are obtained. After amino modification, hydrogen peroxide catalyst is modified on the surface of nanocapsules to catalyze the generated oxygen, drive the nanocapsules to move, and construct a flexible nanomotor responsive to hydrogen peroxide. The nanomotor has an adjustable pore size (2.1-7.8nm) and controllable flexibility. The flexible nanomotor is used to construct a drug delivery system for the treatment of tumors.

[0006] Chinese patent document CN 114522242 A discloses a magnetically driven spiral micro-nano motor and its preparation method and application. The preparation method of the magnetically driven spiral micro-nano motor comprises the following steps: preparing a spiral carbon nanocoil; acidifying the spiral carbon nanocoil, and loading a magnetic nanomaterial on the surface of the acidified spiral carbon nanocoil to obtain a CNCs / magnetic composite material; dissolving an anthracycline anticancer agent and the CNCs / magnetic composite material in a buffer solution, and mixing the anthracycline anticancer agent so that the anthracycline anticancer agent is bound to the surface of the spiral carbon nanocoil to obtain a magnetically driven spiral micro-nano motor of CNCs / magnetic material@anticancer agent.

[0007] At present, the existing technology uses traditional complex and expensive photolithography, grazing angle deposition and other methods to prepare nano, porous, spiral magnetic drive motors. Therefore, the development of a nano-scale, high-load performance, good biocompatibility, simple preparation, and economical spiral magnetic drive motor is of milestone significance for the future development of spiral magnetic drive motors in the biomedical field. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a magnetically driven porous helical nanomotor for active antigen delivery. The prepared porous helical magnetically driven nanomotor has high antigen loading efficiency, high cell uptake efficiency, and can enhance antigen cross-delivery.

[0009] In order to solve the above technical solution, the technical solution of the present invention is: the magnetically driven porous spiral nanomotor for active antigen delivery includes a nickel layer, porous spiral silicon and a surface antigen ovalbumin OVA, and the nickel layer modifies the surface antigen ovalbumin OVA on the surface of the porous spiral silicon.

[0010] Preferably, the helical silicon mesoporous material of the magnetically driven porous helical nanomotor for active antigen delivery is prepared by magnetron sputtering an asymmetric metal nickel layer on the surface of the porous helical silicon to obtain the porous helical magnetically driven nanomotor.

[0011] The technical problem that the present invention also aims to solve is to provide a method for preparing a magnetically driven porous spiral nanomotor for active antigen delivery. The preparation method is simple, has a low preparation cost, and is suitable for large-scale mass production. At the same time, the prepared porous spiral magnetically driven nanomotor has high antigen loading efficiency, high cell uptake efficiency, and can enhance antigen cross-delivery.

[0012] In order to solve the above technical solution, the technical solution of the present invention is: a method for preparing the magnetically driven porous spiral nanomotor for active antigen delivery, the specific steps are:

[0013] S1: Using the sol-gel method, tetraethyl orthosilicate is added to an aqueous solution containing sodium hydroxide, perfluorooctanoic acid, and hexadecyltrimethylammonium bromide, mixed and stirred for reaction, and then calcined to obtain the spiral siliceous mesoporous material HR;

[0014] S2: A metal nickel layer is deposited on the surface of the spiral siliceous mesoporous material by magnetron sputtering to obtain a HR@Ni spiral magnetic drive motor;

[0015] S3: The HR@Ni helical magnetically driven motor was modified with the surface antigen ovalbumin (OVA) to obtain a magnetically driven porous helical nanomotor for active antigen delivery.

[0016] Preferably, the specific steps of step S1 are:

[0017] S11: Synthesizing a spiral siliceous mesoporous material using perfluorooctanoic acid and hexadecyltrimethylammonium bromide as templates; dissolving hexadecyltrimethylammonium bromide in deionized water, and then adding NaOH solution and perfluorooctanoic acid to the solution, respectively, to obtain a mixed solution;

[0018] S12: heating the mixed solution, adding tetraethyl orthosilicate to the mixed solution, and continuing to stir the solution; after the reaction is completed, a white precipitate is obtained;

[0019] S13: The white precipitate was collected by centrifugation and washed several times to remove the residual reactants. After drying at room temperature 25°C, the template was removed by calcination to obtain the sample helical mesoporous silica HR.

[0020] Preferably, the specific steps of step S2 are:

[0021] S21: The prepared spiral mesoporous silicon HR is monodispersed in anhydrous ethanol, and then coated on a glass slide pre-cleaned by O2 plasma using a pipette gun, and the glass slide is placed in a vacuum chamber of magnetron sputtering and evacuated; after the sputtering is completed, the sputtering source is turned off, the vacuum is removed, and the sample is taken out;

[0022] S22: The sample prepared in step S21 is dispersed in deionized water by ultrasonication, and then centrifuged and dried to obtain a HR@Ni helical magnetic drive motor.

[0023] Preferably, the specific steps of step S3 are:

[0024] S31: dispersing the HR@Ni helical magnetic drive motor in ethylene glycol, adding γ-aminopropyltriethoxysilane APTES to react, and then centrifuging and washing to obtain a washed product;

[0025] S32: dispersing the washed product in N,N-dimethylformamide DMF, adding succinic anhydride SAA and triethylamine TEA, stirring and mixing, and then centrifuging and washing to obtain a product with a carboxyl group;

[0026] S33: The HR@Ni helical magnetically driven motor with carboxyl groups is dispersed in MES solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxysuccinimide NHS are added, and the mixture is stirred for reaction; the reaction product is then washed with PBS buffer for multiple times and dispersed in PBS buffer; the antigen ovalbumin OVA is then added for reaction; the supernatant is removed by centrifugation to obtain the product, the magnetically driven porous helical nanomotor HON.

[0027] Preferably, in the aqueous solution containing sodium hydroxide, perfluorooctanoic acid and hexadecyl trimethylammonium bromide in step S11, the mass ratio of perfluorooctanoic acid to hexadecyl group is in the range of about 0 to 0.1; 0.1 to 0.3 g of hexadecyl trimethylammonium bromide is added, and hexadecyl trimethylammonium bromide is dissolved in 90 to 100 g of deionized water, and then 0.65 to 0.8 mL of 2M NaOH solution and 0.005 to 0.015 g of perfluorooctanoic acid are added to the solution respectively; the volume of tetraethyl orthosilicate added in step S12 is 1 to 1.5 mL; wherein the mixing reaction is stirred at 80° C. for 1.5 to 2.5 h; in step S13, the white precipitate is centrifuged at 8000 rpm / min for 5 min to collect the precipitate, and then washed with deionized water and anhydrous ethanol for 5 times respectively to remove the residual reactants, dried at room temperature at 25° C., and calcined at 550° C. for 5 h to remove the template to obtain a sample of spiral mesoporous silicon HR.

[0028] Preferably, in step S21, the vacuum degree is maintained at 9.9×10 -4 Pa, the magnetron sputtering power is 85w, and the Ni layer is sputtered for 3min in an Ar atmosphere with an Ar gas flow rate of 45-50sccm, and the thickness of the obtained Ni layer is 30-50nm; in the step S22, the sample is collected by centrifugation at a speed of 8000rpm for 5min, and the obtained sample is placed in a vacuum drying oven at 60℃ for drying, and the drying time is 12h.

[0029] Preferably, the mass of the HR@Ni helical magnetic drive motor in step S31 is 3-6 mg, which is dispersed in 15-25 mL of ethylene glycol, and then 150-250 μL of γ-aminopropyltriethoxysilane APTES is added, and the mixture is reacted at room temperature of 25° C. for 12 hours. After the reaction is completed, the product is collected by centrifugation at 8000 rpm / min for 5 minutes, and then washed with ethanol 3 times;

[0030] In the step S32, 3-5 mg of the washed product is dispersed in 7-15 mL of N,N-dimethylformamide DMF, 40-60 mg of succinic anhydride SAA and 50-60 μL of triethylamine TEA are added, and stirred at room temperature at 25° C. for 12 h; the product with carboxyl groups is collected by centrifugation at 8000 rpm / min for 5 min, and washed with ethanol 3 times;

[0031] In the step S33, 3-5 mg of HR@Ni helical magnetically driven motor with carboxyl group is dispersed in MES solution, and then 2-4 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide EDC and 2-4 mg of N-hydroxysuccinimide NHS are added, and stirred for 1 hour; then, the reaction product is washed with PBS buffer for 3 times and dispersed in 2-3 mL of PBS buffer; finally, 0.5-1.5 mg of antigen ovalbumin OVA is added and reacted for 24 hours; then, centrifuged at 8000 rpm / min for 5 minutes, the supernatant is removed, and the product magnetically driven porous helical nanomotor HON is obtained, and it is dispersed in 1 mL of physiological saline and stored at 4°C.

[0032] The technical problem that the present invention also aims to solve is to provide an application of a magnetically driven porous spiral nanomotor for active antigen delivery. The magnetically driven porous spiral nanomotor for active antigen delivery and its preparation method provide a new research idea for the future development of cost-effective and immune response-enhancing efficient dynamic vaccine delivery systems.

[0033] In order to solve the above technical solution, the technical solution of the present invention is: the magnetically driven porous spiral nanomotor for active antigen delivery is used to promote antigen cross-delivery and enhance cellular immune response, and the cellular immune response is BMDC cells.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) For the first time, a nanometer, porous, helical magnetically driven nanomotor was constructed. That is, for the first time, a simple sol-gel method was used to replace complex processes such as grazing angle deposition and electrodeposition to prepare a nanometer, porous helical magnetically driven nanomotor. The Helical meso-porous silicon@Ni(HON) nanomotor prepared by the method of the present invention has flexible and controllable magnetic drive performance under a rotating magnetic field, can overcome the resistance of various biological fluids to achieve efficient helical motion, and its motion speed is proportional to the frequency of the magnetic field;

[0036] (2) The preparation method is simple, low in cost, and suitable for large-scale production. It also provides a new research idea for the future development of cost-effective and efficient dynamic vaccine delivery systems that enhance immune responses.

[0037] (3) The porous helical magnetically driven nanomotors prepared can also be used to promote antigen cross-delivery and enhance cellular immune responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The present invention is a scanning electron microscope (SEM) image of the magnetically driven porous spiral nanomotor for active antigen delivery and its corresponding element distribution map; wherein (a) is a scanning electron microscope (SEM) image of the HON magnetically driven nanomotor; (b) is an EDS image of Si corresponding to the magnetically driven porous spiral nanomotor HON; (c) is an EDS image of Ni corresponding to the magnetically driven porous spiral nanomotor HON; (d) is an EDS image of S corresponding to the magnetically driven porous spiral nanomotor HON;

[0039] Figure 2 Zeta potential diagram of the helical siliceous mesoporous material HR, the surface antigen ovalbumin OVA, HR@Ni obtained by sputtering a metal Ni layer on the surface of HR, and the magnetically driven porous helical nanomotor HON for active antigen delivery in the present invention;

[0040] Figure 3 Fourier infrared spectra of the magnetically driven porous helical nanomotor HON, the helical siliceous mesoporous material HR, and the surface antigen ovalbumin OVA used for active antigen delivery in the present invention;

[0041] Figure 4 XRD characterization diagram of the magnetically driven porous helical nanomotor HON and the helical siliceous mesoporous material HR for active antigen delivery in the present invention;

[0042] Figure 5 : is a hysteresis loop diagram of the magnetically driven porous helical nanomotor HON for active antigen delivery in the present invention;

[0043] Figure 6The N2 adsorption-desorption isotherms and BJH-voidity measurement (inset) of the magnetically driven porous helical nanomotor HON and the helical siliceous mesoporous material HR for active antigen delivery in the present invention;

[0044] Figure 7 This is a graph showing the cell survival rate of HONDC2.4 cells treated with different concentrations of the magnetically driven porous helical nanomotor for active antigen delivery in the present invention;

[0045] Figure 8 This is a diagram of the motion mechanism of the magnetically driven porous helical nanomotor HON for active antigen delivery in the present invention under a rotating magnetic field;

[0046] Fig. 9 is the movement speed of the magnetically driven porous helical nanomotor HON for active antigen delivery in the present invention at different magnetic field frequencies;

[0047] Fig.10 is the movement speed of the magnetically driven porous helical nanomotor HON for active antigen delivery in different biological media;

[0048] Fig.11 Figures 2 and 3 are motion trajectory diagrams of the magnetically driven porous spiral nanomotor HON for active antigen delivery in the present invention at different magnetic field frequencies; wherein (a) is a motion trajectory diagram of the magnetically driven porous spiral nanomotor HON3s when the magnetic field frequency is 5 Hz; (b) is a motion trajectory diagram of the magnetically driven porous spiral nanomotor HON3s when the magnetic field frequency is 7 Hz; (c) is a motion trajectory diagram of the magnetically driven porous spiral nanomotor HON3s when the magnetic field frequency is 9 Hz;

[0049] Fig.12 Figures 2 and 3 show the motion trajectory of the magnetically driven porous helical nanomotor HON for active antigen delivery in different biological media; (a) shows the motion trajectory of the magnetically driven porous helical nanomotor HON in PBS buffer with a pH of 7.4; (b) shows the motion trajectory of the magnetically driven porous helical nanomotor HON in cell culture medium; (c) shows the motion trajectory of the magnetically driven porous helical nanomotor HON in fetal bovine serum;

[0050] Fig.13 A trajectory diagram of the magnetically driven porous helical nanomotor HON for active antigen delivery in the present invention moving along a predetermined path;

[0051] Fig.14 This is a motion trajectory diagram of the magnetically driven porous helical nanomotor HON used for active antigen delivery in the present invention, which navigates to the target DC2.4 cell in a directional manner;

[0052] Fig.15 Figure 1 is the result of uptake efficiency in DC2.4 cells under different treatment groups; Hoechst is the fluorescence image of the cell nucleus labeled with Hoechst3342 dye, FITC is the fluorescence image of the antigen on the magnetically driven porous helical nanomotor HON labeled with fluorescein isothiocyanate FITC, Merge is the fluorescence distribution after the fusion of Hoechst fluorescence and FITC fluorescence, the first row HON+M is the fluorescence distribution of the magnetically driven porous helical nanomotor HON in the cell in the moving state, the second row HON is the fluorescence distribution of the magnetically driven porous helical nanomotor HON in the static state, and the third row OVA is the fluorescence distribution of the antigen OVA in the free state in the cell;

[0053] Fig.16 : The lysosome co-localization diagram of DC2.4 cells under different treatment groups; wherein DAPI is a fluorescent image of cell nuclei labeled with DAPI dye, LysoGreen is a fluorescent image of lysosomes labeled with LysoGreen dye, Cy5 is a fluorescent image of antigens on the magnetically driven porous helical nanomotor HON labeled with Cy5 dye, Merge is the fluorescence distribution after the fusion of DAPI fluorescence, LysoGreen fluorescence and Cy5 fluorescence, the first row is the localization of the antigen OVA in the free state in the cell, the second row is the localization of the magnetically driven porous helical nanomotor HON in the static state in the cell, and the third row HON+M is the localization of the magnetically driven porous helical nanomotor HON in the moving state in the cell;

[0054] Fig.17 It is a lysosome co-localization diagram of the magnetically driven porous helical nanomotor HON for active antigen delivery in the present invention over time; wherein DAPI is a fluorescent image of a cell nucleus labeled with DAPI dye, LysoGreen is a fluorescent image of a lysosome labeled with LysoGreen dye, Cy5 is a fluorescent image of an antigen on the magnetically driven porous helical nanomotor HON labeled with Cy5 dye, Merge is the fluorescence distribution after the fusion of DAPI fluorescence, LysoGreen fluorescence and Cy5 fluorescence, 6h is the localization of the magnetically driven porous helical nanomotor HON in cells after co-culturing the magnetically driven porous helical nanomotor HON with cells for 6h, and 24h is the localization of the magnetically driven porous helical nanomotor HON in cells after co-culturing the magnetically driven porous helical nanomotor HON with cells for 24h;

[0055] Fig.18The figures are the detection images of lysosome integrity of DC2.4 cells after being treated in different treatment groups for 24 hours; (a) is the acridine orange staining image of lysosomes after co-culture of free antigen OVA with cells for 24 hours, (b) is the acridine orange staining fluorescence image of lysosomes after co-culture of magnetically driven porous helical nanomotor HON with cells for 24 hours in a static state, (c) is the acridine orange staining fluorescence image of lysosomes after co-culture of magnetically driven porous helical nanomotor HON with cells for 24 hours in a moving state;

[0056] Fig.19 Figures 24 are the flow cytometry results of the expression of MHC I and MHC II molecules in BMDCs after 24 hours of treatment with different treatment groups; (a) is the flow cytometry result of the expression of MHC II molecules in blank BMDCs, (b) is the flow cytometry result of the expression of MHC II molecules in BMDCs after co-treatment with free antigen OVA for 24 hours, (c) is the flow cytometry result of the expression of MHC II molecules in static magnetically driven porous helical nanomotor HON for 24 hours, (d) is the flow cytometry result of the expression of MHC II molecules in moving magnetically driven porous helical nanomotor HON for 24 hours, (e) is the flow cytometry result of the expression of MHC II molecules in BMDCs after co-treatment with lipopolysaccharide LPS for 24 hours, (f) is the flow cytometry result of the expression of MHC II molecules in blank BMDCs, (g) is the flow cytometry result of the expression of MHC II molecules in free antigen OVA for 24 hours. (h) is the flow cytometry result of MHC II molecule expression after co-treatment of the magnetically driven porous helical nanomotor HON and BMDCs cells for 24 hours, (i) is the flow cytometry result of MHC II molecule expression after co-treatment of the magnetically driven porous helical nanomotor HON and BMDCs cells for 24 hours, (j) is the flow cytometry result of MHC II molecule expression after co-treatment of lipopolysaccharide LPS and BMDCs cells for 24 hours;

[0057] Fig. 20 The figures are flow cytometry statistical analysis results of BMDCs cells in different treatment groups, wherein (a) is a statistical analysis graph of MHC II molecule expression of BMDCs cells in different treatment groups, (b) is a statistical analysis graph of MHC I molecule expression of BMDCs cells in different treatment groups, (c) is a statistical analysis graph of CD40 molecule expression of BMDCs cells in different treatment groups, (d) is a statistical analysis graph of CD80 molecule expression of BMDCs cells in different treatment groups, and (e) is a statistical analysis graph of CD86 molecule expression of BMDCs cells in different treatment groups. DETAILED DESCRIPTION

[0058] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments are only used to explain the present invention and do not limit the protection scope of the present invention.

[0059] Embodiment: The magnetically driven porous spiral nanomotor for active antigen delivery comprises a nickel layer, porous spiral silicon and a surface antigen ovalbumin (OVA), wherein the nickel layer modifies the surface antigen ovalbumin (OVA) on the surface of the porous spiral silicon; the spiral siliceous mesoporous material of the magnetically driven porous spiral nanomotor for active antigen delivery is prepared by magnetron sputtering an asymmetric metal nickel layer on the surface of the porous spiral silicon to obtain a porous spiral magnetically driven nanomotor.

[0060] The preparation method of the magnetically driven porous spiral nanomotor for active antigen delivery comprises the following specific steps:

[0061] S1: Using the sol-gel method, tetraethyl orthosilicate is added to an aqueous solution containing sodium hydroxide, perfluorooctanoic acid, and hexadecyltrimethylammonium bromide, mixed and stirred for reaction, and then calcined to obtain the spiral siliceous mesoporous material HR;

[0062] The specific steps of step S1 are:

[0063] S11: using perfluorooctanoic acid and hexadecyltrimethylammonium bromide as templates to synthesize a spiral siliceous mesoporous material; dissolving hexadecyltrimethylammonium bromide in deionized water, and then adding NaOH solution and perfluorooctanoic acid to the solution respectively to obtain a mixed solution; the mass ratio of perfluorooctanoic acid to hexadecyl in the aqueous solution containing sodium hydroxide, perfluorooctanoic acid and hexadecyltrimethylammonium bromide in step S11 is about 0 to 0.1; 0.1 to 0.3 g of hexadecyltrimethylammonium bromide is added, and hexadecyltrimethylammonium bromide is dissolved in 90 to 100 g of deionized water, and then 0.65 to 0.8 mL of 2 M NaOH solution and 0.005 to 0.015 g of perfluorooctanoic acid are added to the solution respectively;

[0064] S12: heating the mixed solution, adding tetraethyl orthosilicate to the mixed solution, and then continuing to stir the solution; after the reaction is completed, a white precipitate is obtained; the volume of tetraethyl orthosilicate added in step S12 is 1 to 1.5 mL; wherein the mixed reaction is stirred at 80° C. for 1.5 to 2.5 hours;

[0065] S13: Collect the white precipitate by centrifugation, wash it several times to remove the residual reactants, dry it at room temperature at 25°C, and calcine it to remove the template, so as to obtain a sample of spiral mesoporous silicon HR; In the step S13, the white precipitate is centrifuged at 8000rpm / min for 5min to collect the precipitate, and then washed with deionized water and anhydrous ethanol for 5 times respectively to remove the residual reactants, dry it at room temperature at 25°C, and calcine it at 550°C for 5h to remove the template, so as to obtain a sample of spiral mesoporous silicon HR;

[0066] S2: A metal nickel layer is deposited on the surface of the spiral siliceous mesoporous material by magnetron sputtering to obtain a HR@Ni spiral magnetic drive motor;

[0067] The specific steps of step S2 are:

[0068] S21: The prepared spiral mesoporous silicon HR is dispersed in anhydrous ethanol, and then coated on a glass slide pre-cleaned by O2 plasma with a pipette gun, and the glass slide is placed in a vacuum chamber of magnetron sputtering and evacuated; after the sputtering is completed, the sputtering source is turned off, the vacuum is removed, and the sample is taken out; in the step S21, the vacuum is maintained at 9.9×10 -4 Pa, the magnetron sputtering power is 85w, and the Ni layer is sputtered for 3min in an Ar atmosphere with an Ar gas flow rate of 45-50sccm. The obtained Ni layer thickness is 30-50nm;

[0069] S22: dispersing the sample obtained in step S21 in deionized water by ultrasonication, and then centrifuging and drying to obtain a HR@Ni helical magnetic drive motor; in step S22, the sample is collected by centrifugation at a speed of 8000 rpm for 5 minutes, and the obtained sample is placed in a vacuum drying oven at 60° C. for drying for 12 hours;

[0070] S3: The HR@Ni helical magnetically driven motor was modified with the surface antigen ovalbumin (OVA) to obtain a magnetically driven porous helical nanomotor for active antigen delivery.

[0071] The specific steps of step S3 are:

[0072] S31: dispersing the HR@Ni helical magnetic drive motor in ethylene glycol, adding γ-aminopropyltriethoxysilane APTES to react, and then centrifuging and washing to obtain a washed product;

[0073] The mass of the HR@Ni helical magnetic drive motor in step S31 is 3-6 mg, which is dispersed in 15-25 mL of ethylene glycol, and then 150-250 μL of γ-aminopropyltriethoxysilane APTES is added, and the mixture is reacted at room temperature of 25°C for 12 hours. After the reaction is completed, the product is collected by centrifugation at 8000 rpm / min for 5 minutes, and then washed with ethanol three times;

[0074] S32: dispersing the washed product in N,N-dimethylformamide DMF, adding succinic anhydride SAA and triethylamine TEA, stirring and mixing, and then centrifuging and washing to obtain a product with a carboxyl group;

[0075] In the step S32, 3-5 mg of the washed product is dispersed in 7-15 mL of N,N-dimethylformamide DMF, 40-60 mg of succinic anhydride SAA and 50-60 μL of triethylamine TEA are added, and stirred at room temperature at 25° C. for 12 h; the product with carboxyl groups is collected by centrifugation at 8000 rpm / min for 5 min, and washed with ethanol 3 times;

[0076] S33: Disperse the HR@Ni helical magnetically driven motor with carboxyl groups in MES solution, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide EDC and N-hydroxysuccinimide NHS, and stir to mix and react; then wash the obtained reaction product with PBS buffer for multiple times and disperse it in PBS buffer; then add antigen ovalbumin OVA to react; centrifuge to remove the supernatant, and obtain the product magnetically driven porous helical nanomotor HON;

[0077] In the step S33, 3-5 mg of HR@Ni helical magnetically driven motor with carboxyl group is dispersed in MES solution, and then 2-4 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide EDC and 2-4 mg of N-hydroxysuccinimide NHS are added, and stirred for 1 hour; then, the reaction product is washed with PBS buffer for 3 times and dispersed in 2-3 mL of PBS buffer; finally, 0.5-1.5 mg of antigen ovalbumin OVA is added and reacted for 24 hours; then, centrifuged at 8000 rpm / min for 5 minutes, the supernatant is removed, and the product magnetically driven porous helical nanomotor HON is obtained, and it is dispersed in 1 mL of physiological saline and stored at 4°C.

[0078] The magnetically driven porous spiral nanomotor for active antigen delivery is used to promote antigen cross-delivery and enhance cellular immune response, wherein the cellular immune response is BMDC cells.

[0079] Specific Example 1: The preparation method of the magnetically driven porous spiral nanomotor for active antigen delivery comprises the following specific steps:

[0080] S1: Using the sol-gel method, tetraethyl orthosilicate is added to an aqueous solution containing sodium hydroxide, perfluorooctanoic acid, and hexadecyltrimethylammonium bromide, mixed and stirred for reaction, and then calcined to obtain the spiral siliceous mesoporous material HR;

[0081] The specific steps of step S1 are:

[0082] S11: using perfluorooctanoic acid and hexadecyltrimethylammonium bromide as templates to synthesize a spiral siliceous mesoporous material; dissolving hexadecyltrimethylammonium bromide in deionized water, and then adding NaOH solution and perfluorooctanoic acid to the solution respectively to obtain a mixed solution; the mass ratio of perfluorooctanoic acid to hexadecyl in the aqueous solution containing sodium hydroxide, perfluorooctanoic acid and hexadecyltrimethylammonium bromide in step S11 is 0.5; 0.2 g of hexadecyltrimethylammonium bromide is added, and hexadecyltrimethylammonium bromide is dissolved in 96 g of deionized water, and then 0.7 mL of 2 M NaOH solution and 10 mg of perfluorooctanoic acid are added to the solution respectively;

[0083] S12: heating the mixed solution, adding tetraethyl orthosilicate to the mixed solution, and then continuing to stir the solution; after the reaction is completed, a white precipitate is obtained; the volume of tetraethyl orthosilicate added in step S12 is 1.34 mL; wherein the mixed reaction is stirred at 80° C. for 2 h;

[0084] S13: The white precipitate is collected by centrifugation and washed several times to remove the residual reactants. After drying at room temperature, the template is calcined to obtain the sample helical mesoporous silica HR;

[0085] In the step S13, the white precipitate is centrifuged at 8000 rpm / min for 5 min to collect the precipitate, and then washed with deionized water and anhydrous ethanol for 5 times respectively to remove the residual reactants, and then dried at room temperature and calcined at 550° C. for 5 h to remove the template, thereby obtaining a sample of spiral mesoporous silicon HR;

[0086] S2: A metal nickel layer is deposited on the surface of the spiral siliceous mesoporous material by magnetron sputtering to obtain a HR@Ni spiral magnetic drive motor;

[0087] The specific steps of step S2 are:

[0088] S21: The prepared spiral mesoporous silicon HR was dispersed in anhydrous ethanol, and then coated on a glass slide pre-cleaned by O2 plasma using a pipette gun. The glass slide was placed in a vacuum chamber of magnetron sputtering and evacuated. When magnetron sputtering the metal Ni layer, the vacuum degree was maintained at 9.9×10 -4 Pa, sputter the Ni layer in Ar atmosphere for 3 min; after the sputtering is completed, turn off the sputtering source, remove the vacuum, and take out the sample;

[0089] S22: dispersing the sample obtained in step S21 in deionized water by ultrasonication, and then centrifuging and drying to obtain a HR@Ni helical magnetic drive motor;

[0090] In step S22, the sample is collected by centrifugation at 8000 rpm for 5 min, and the obtained sample is placed in a vacuum drying oven at 60° C. for drying for a period of time;

[0091] S3: The HR@Ni helical magnetically driven motor was modified with the surface antigen ovalbumin (OVA) to obtain a magnetically driven porous helical nanomotor for active antigen delivery.

[0092] The specific steps of step S3 are:

[0093] S31: dispersing the HR@Ni helical magnetic drive motor in ethylene glycol, adding γ-aminopropyltriethoxysilane APTES to react, and then centrifuging and washing to obtain a washed product;

[0094] The mass of the HR@Ni helical magnetic drive motor in step S31 is 4 mg, which is dispersed in 20 mL of ethylene glycol, and then 200 μL of γ-aminopropyltriethoxysilane APTES is added, and the reaction is carried out at room temperature of 25°C for 12 hours. After the reaction is completed, the product is collected by centrifugation at a speed of 8000 rpm / min for 5 minutes, and then washed with ethanol three times;

[0095] S32: dispersing the washed product in N,N-dimethylformamide DMF, adding succinic anhydride SAA and triethylamine TEA, stirring and mixing, and then centrifuging and washing to obtain a product with a carboxyl group;

[0096] In the step S32, 2 mg of the washed product was dispersed in 10 mL of N,N-dimethylformamide DMF, 50 mg of succinic anhydride SAA and 52.1 μL of triethylamine TEA were added, and the mixture was stirred at room temperature at 25° C. for 12 h; the product with carboxyl groups was collected by centrifugation at 8000 rpm / min for 5 min, and then washed with ethanol for 3 times;

[0097] S33: Disperse the HR@Ni helical magnetically driven motor with carboxyl groups in MES solution, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide EDC and N-hydroxysuccinimide NHS, and stir to mix and react; then wash the obtained reaction product with PBS buffer for multiple times and disperse it in PBS buffer; then add antigen ovalbumin OVA to react; centrifuge to remove the supernatant, and obtain the product magnetically driven porous helical nanomotor HON;

[0098] In the step S33, 5 mg of the HR@Ni spiral magnetically driven motor with a carboxyl group is dispersed in the MES solution, and then 1.5 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide EDC and 1.6 mg of N-hydroxysuccinimide NHS are added, and the mixture is stirred for 1 hour; then, the reaction product is washed three times with PBS buffer and dispersed in 1 mL of PBS buffer; finally, 1 mg of antigen ovalbumin OVA is added and the mixture is reacted for 24 hours; then, the mixture is centrifuged at 8000 rpm / min for 5 minutes, the supernatant is removed, and the product, the magnetically driven porous spiral nanomotor HON, is obtained, and the product is dispersed in 1 mL of physiological saline and stored at 4°C.

[0099] The magnetically driven porous helical nanomotor for active antigen delivery prepared in Specific Example 1 is characterized and its motion mechanism is studied.

[0100] (1) First, the structure and performance of the magnetically driven porous helical nanomotors for active antigen delivery were characterized, including SEM, BET, FTIR, Zeta potential and BET characterization.

[0101] Figure 1 (a) is a scanning electron microscope (SEM) image of the magnetically driven porous spiral nanomotor HON. It can be seen that the magnetically driven porous spiral nanomotor HON is a uniform spiral structure. The length of a single spiral motor (about 3 pitches) is 2 μm and the cross-sectional diameter is about 200 nm. Figure 1 (b), (c), and (d) are the EDS images of Si, Ni, and S corresponding to the magnetically driven porous helical nanomotor HON. Figure 1 (b), (c), and (d) show that the three elements Si, Ni, and S are evenly distributed on the surface of the HON magnetically driven nanomotor. The appearance of the S element indicates the successful modification of the antigen OVA, and the Ni element indicates that the magnetic Ni layer is successfully sputtered on the motor surface.

[0102] Figure 2 The Zeta potential diagram of HR, OVA, HR@Ni, and HON is shown in Figure 2. Figure 2 It can be seen that the potential of the bare siliceous mesoporous helical silicon material HR is about -36mV, and the potential of OVA is about -32mV. After sputtering a Ni layer on the HR surface to obtain HR@Ni, its surface potential increases to about -18mV. After the antigen OVA is modified on the HR@Ni surface, its potential decreases to about -24mV. The potential change of the magnetically driven porous helical nanomotor HON prepared by layer-by-layer modification indicates the successful modification of its surface antigen and magnetic Ni layer.

[0103] Figure 3 The test results of Fourier transform infrared spectroscopy (FTIR) of HR, OVA and HON are as follows: Figure 3 It can be seen that the magnetically driven porous helical nanomotor HON is at 1052 cm -1 The Si-O stretching vibration peak appeared at 1638 cm-1 due to the modification of OVA on the surface of HON. -1 The stretching vibration peak of the peptide bond C=O in the protein appeared at 1559 cm -1 The NH bending vibration of the peptide bond appeared at , indicating the successful modification of OVA.

[0104] Figure 4 XRD patterns of HON and HR. Figure 4 It can be seen that the characteristic peaks of Ni element appear in the magnetically driven porous spiral nanomotor HON, namely the (110), (200), and (220) crystal planes, indicating the successful sputtering of Ni on the HON surface. Figure 5 This is the hysteresis loop diagram of the magnetically driven porous helical nanomotor HON, which shows that the magnetically driven porous helical nanomotor HON has superparamagnetism and the saturation magnetization intensity is about 34emu / g, which indicates that the prepared magnetically driven porous helical nanomotor HON has magnetic responsiveness.

[0105] The N2 isothermal adsorption-desorption curves and pore size distributions of magnetically driven porous helical nanomotors HON and HR were determined by N2 isothermal adsorption-desorption spectroscopy (BET). Figure 6 The N2 adsorption-desorption isotherms of the magnetically driven porous helical nanomotor HON and HR are presented, both showing type IV isotherms with H1-type hysteresis loops, indicating that the magnetically driven porous helical nanomotor HON and HR have typical mesoporous structures. Among them, the specific surface area of ​​the magnetically driven porous helical nanomotor HON is smaller than that of HR, indicating the successful modification of the magnetic Ni layer and antigen. Figure 6 The inset in the figure shows the BJH-porosity measurement result, which further proves that the pore size in the magnetically driven porous helical nanomotor HON belongs to the mesoporous size.

[0106] Figure 7 The figure shows the cell survival rate of DC2.4 cells in different concentration treatment groups. The results show that the magnetically driven porous helical nanomotor HON has good biocompatibility. After the cells were cultured with HR, HR-Ni or magnetically driven porous helical nanomotor HON in the concentration range of 1-150μg / mL for 24h, there was no significant change in the viability of DC2.4 cells. When the concentration of magnetically driven porous helical nanomotor HON was 150μg / mL, the cell survival rate was still around 85%.

[0107] In summary, through the characterization results of SEM, XRD, FTIR, Zeta potential and BET, it can be known that the material prepared in this embodiment is Helical meso-porous silicon@Ni@OVA, a magnetically driven porous helical nanomotor HON, which has a porous structure and can load the antigen ovalbumin OVA.

[0108] (2) Study on the motion mechanism of magnetically driven porous helical nanomotor HON

[0109] Figure 8 The motion mechanism of the magnetically driven porous helical nanomotor HON under a rotating magnetic field is presented. Under the action of the surrounding rotating magnetic field and the fluid viscosity force, the helical magnetically driven motor will generate axial force and radial force along the wire diameter in each cylindrical micro-segment of the magnetic micro-helix. After synthesis, it will generate a magnetic torque rotating around its own axis and an axial force to move forward, so that the entire helical motor will spiral in like a cork. Because the helical magnetically driven motor adopts this efficient torque drive strategy, its motion mode is more flexible and precise.

[0110] To verify the above conclusions, the present invention explores the movement of the magnetically driven porous spiral nanomotor HON under a rotating magnetic field. Thanks to the magnetic metal Ni layer of the magnetically driven porous spiral nanomotor HON, it has excellent magnetic responsiveness under a rotating magnetic field. Therefore, the motion control ability of the magnetically driven porous spiral nanomotor HON under a rotating magnetic field generated by three orthogonal Helmholtz coils was explored. The movement ability of the magnetically driven porous spiral nanomotor HON in different biological fluids such as PBS solution, cell culture medium, and serum was explored.

[0111] Depend on Fig. 9 It can be seen that when the fixed magnetic field strength is 1mT, the motor's movement speed is proportional to the magnetic field frequency. The motor's maximum movement speed can reach 8μm / s. Its corresponding movement trajectory Fig.11 It can be seen that with the increase of magnetic field frequency, the movement displacement of the magnetically driven porous helical nanomotor HON becomes longer and longer, indicating that the movement speed of the motor can be controlled by adjusting the magnetic field frequency.

[0112] Fig.10is the movement speed of the magnetically driven porous helical nanomotor HON in different biological fluids at the same frequency, where PBS represents the average movement speed of the magnetically driven porous helical nanomotor HON in a PBS buffer environment with a pH of 7.4, cell culture medium represents the average movement speed of the magnetically driven porous helical nanomotor HON in a cell culture medium environment, and Fetal bovine serum represents the average movement speed of the magnetically driven porous helical nanomotor HON in fetal bovine serum. When the magnetic field frequency is fixed at 5 Hz, the average speed of the motor in PBS, cell culture medium, and serum is 6.5 μm / s, and its corresponding movement trajectory Fig.12 This shows that the motor can achieve effective displacement in different biological fluids. By adjusting the rotation direction of the magnetic field, the movement direction of the nanomotor can be well controlled.

[0113] like Fig.13 As shown in the figure, the motor can complete directional navigation motion along the predetermined path "N", indicating that the HON magnetic drive motor has flexible and controllable motion behavior. Fig.14 As shown, the magnetically driven porous helical nanomotor HON can utilize active magnetic driving capabilities to navigate to the target for targeted delivery of antigens.

[0114] In summary, it can be seen that the magnetically driven porous spiral nanomotor HON prepared in this embodiment can realize flexible and controllable spiral motion under a rotating magnetic field. The nanomotor can also use its own porous and nano characteristics to carry antigens to target cells. It is the first porous, nano, spiral motor that can load and actively deliver antigens.

[0115] (3) Study on the effect of magnetically driven porous helical nanomotor HON on DC2.4 cell uptake efficiency

[0116] The active movement ability of the magnetically driven porous helical nanomotor HON can improve the efficiency of cell uptake. The cell uptake of the magnetically driven porous helical nanomotor HON by DC2.4 cells was observed using an upright fluorescence microscope. Hoechst means that the cell nucleus is stained with Hoeschst 3342 dye, and the blue fluorescence image of the cell nucleus can be observed under an upright fluorescence microscope with laser excitation at a wavelength of 346nm. FITC means that the antigen OVA on HON is labeled with fluorescein isothiocyanate FITC, and the green fluorescence image of the magnetically driven porous helical nanomotor HON can be observed under an upright fluorescence microscope with laser excitation at a wavelength of 490nm. Merge means that the two aforementioned fluorescence images are processed and fused using the supporting software of the upright fluorescence microscope to form a fused fluorescence image.

[0117] Fig.15The graphs show the results of cell uptake after the cells were treated with different control groups for 6 hours. The first row shows the fluorescence distribution of the magnetically driven porous spiral nanomotor HON in the moving state, the second row shows the fluorescence distribution of the magnetically driven porous spiral nanomotor HON in the stationary state, and the third row shows the fluorescence distribution of the antigen OVA in the free state in the cells. Among them, the first row and the first column show the fluorescence images of the cells with the nucleus of HON+M stained with Hoeschst 3342 dye, the first row and the second column show the fluorescence images of the cells with the antigen OVA on HON labeled with fluorescein isothiocyanate (FITC) by HON+M. The first row and the third column show the fluorescence images of HON+M after the two aforementioned fluorescence images were fused using the supporting software of the upright fluorescence microscope. The first row and the first column show the fluorescence images of the magnetically driven porous spiral nanomotor HON stained with Hoeschst The second row and the second column are fluorescence images of cells with the cell nucleus stained with Hoeschst 3342 dye; the second row and the second column are fluorescence images of the magnetically driven porous helical nanomotor HON with the antigen OVA on HON labeled with fluorescein isothiocyanate FITC in the cells; the second row and the third column are fluorescence images of the magnetically driven porous helical nanomotor HON after the two aforementioned fluorescence images were processed and fused using the supporting software for an upright fluorescence microscope; the third row and the first column are fluorescence images of cells with the cell nucleus stained with Hoeschst 3342 dye for OVA, and the third row and the second column are fluorescence images of cells with the antigen OVA on HON labeled with fluorescein isothiocyanate FITC for OVA; the third row and the third column are fluorescence images of OVA after the two aforementioned fluorescence images were processed and fused using the supporting software for an upright fluorescence microscope; the results show that the average green fluorescence intensity of the actively moving magnetically driven porous helical nanomotor HON group in the cells is 1.4 times higher than that of the stationary magnetically driven porous helical nanomotor HON, and 7 times higher than that of the free OVA group, which indicates that the magnetically driven porous helical nanomotor HON with active and rapid driving capabilities has significant cell penetration ability. The active movement of the motor can increase the contact with DC2.4 cells, and then enter the DC2.4 cells through endocytosis, enhancing the cell's uptake capacity.

[0118] (4) Co-localization and escape of magnetically driven porous helical nanomotor HON into lysosomes in DC2.4 cells

[0119] The intracellular fate of the magnetically driven porous helical nanomotor HON was further studied by confocal microscopy CLSM to determine its ability to escape from lysosomes and achieve subsequent cross-expression. First, DC2.4 cells were co-cultured with free antigen OVA, static magnetically driven porous helical nanomotor HON, and HON nanomotor in motion with a magnetic field for 6 hours. The antigen on the magnetically driven porous helical nanomotor HON was stained with Cy5 dye, and the red fluorescence image of the magnetically driven porous helical nanomotor HON was observed in CLSM with laser excitation at a wavelength of 646nm. The cell nucleus was then stained with DAPI dye, and the blue fluorescence image of the cell nucleus was observed in CLSM under laser excitation at a wavelength of 346nm. Lysosomes were stained with LysoGreen green fluorescent probe, and the green fluorescence image of lysosomes was observed in CLSM under laser excitation at a wavelength of 577nm. Finally, the image processing software supporting CLSM was used to merge the three fluorescences mentioned above to analyze the co-localization of the magnetically driven porous helical nanomotor HON and lysosomes.

[0120] like Fig.16 As shown, the first row shows the localization of the antigen OVA in the free state in the cell, the second row shows the localization of the magnetically driven porous helical nanomotor HON in the static state in the cell, and the third row shows the localization of the magnetically driven porous helical nanomotor HON in the moving state in the cell; the results show that the red fluorescence in HON-Cy5 is well integrated with the green fluorescence of the lysosome, indicating that the magnetically driven porous helical nanomotor HON is first located in the lysosome after being taken up by the cell, and also indicates that the magnetically driven porous helical nanomotor HON is distributed in the cell, rather than adsorbed outside the cell. Compared with the static magnetically driven porous helical nanomotor HON group, the motor with active motion behavior in the magnetic field can increase the probability of contact with DC2.4 cells, thereby improving the cell's ability to take up the motor, resulting in stronger red fluorescence in the lysosome.

[0121] like Fig.17 As shown in the figure, the distribution of magnetically driven porous helical nanomotor HON in cells at different times further confirmed the ability of magnetically driven porous helical nanomotor HON to escape from lysosomes. The first line was treated for 6 hours, and it can be seen from the figure that the red fluorescence mainly overlaps with the green lysosomal fluorescence; the second line was treated for 24 hours, and it can be seen from the figure that the green fluorescence signal gradually disappeared and the red fluorescence signal gradually dispersed. This is because the magnetically driven porous helical nanomotor HON has the ability to actively move, which can increase the lysosomal escape rate of the motor.

[0122] In order to further verify the lysosomal escape ability of the magnetically driven porous helical nanomotor HON, the cells in different treatment groups were stained with acridine orange dye after 24 hours of treatment to detect the integrity of their lysosomes, where (a) is the acridine orange staining fluorescence image of the free antigen OVA and DC2.4 cells co-treated with 24 hours, (b) is the acridine orange staining fluorescence image of the static magnetically driven porous helical nanomotor HON and DC2.4 cells co-treated with 24 hours, (c) is the acridine orange staining fluorescence image of the moving magnetically driven porous helical nanomotor HON and DC2.4 cells co-treated with 24 hours; green fluorescence of acridine orange dye can be observed by an upright fluorescence microscope under laser excitation at a wavelength of 520nm, and red fluorescence can be observed by an upright fluorescence microscope under laser excitation at a wavelength of 650nm. Since acridine orange dye is a cationic dye, it can bind to low pH parts in the cell, such as lysosomes, and emit red fluorescence, and the intensity is proportional to the acidity. When the lysosomes rupture, the acidity decreases, the red fluorescence decreases, and the green fluorescence increases.

[0123] The results are as follows Fig.18 As shown, the green fluorescence signal of the moving magnetically driven porous helical nanomotor HON group was the strongest, indicating that the lysosomal rupture rate of DC2.4 cells was the highest under this treatment group, which means that the moving magnetically driven porous helical nanomotor HON helps to achieve lysosomal escape.

[0124] (5) Study on the immune activation of BMDC cells by magnetically driven porous helical nanomotor HON

[0125] The expression levels of various molecules in BMDCs cells after 24 hours of treatment in different treatment groups were analyzed by flow cytometry, among which (a) is the expression level of MHC II molecules in blank BMDCs cells after 24 hours, (b) is the expression level of MHC II molecules in BMDCs cells after co-culture of free antigen OVA with BMDCs cells for 24 hours, (c) is the expression level of MHC II molecules in BMDCs cells after co-culture of static HON nanoparticles with BMDCs cells for 24 hours, (d) is the expression level of MHC II molecules in BMDCs cells after co-culture of moving HON nanoparticles with BMDCs cells for 24 hours, (e) is the expression level of MHCII molecules in BMDCs cells after co-culture of lipopolysaccharide LPS with BMDCs cells for 24 hours, (f) is the expression level of MHC I molecules in blank BMDCs cells after 24 hours, and (g) is the expression level of MHC (h) is the expression level of MHC II molecules in BMDCs cells after co-culture of BMDCs with magnetically driven porous helical nanomotor HON in static state for 24 hours, (i) is the expression level of MHC II molecules in BMDCs cells after co-culture of BMDCs with BMDCs in dynamic state for 24 hours, and (j) is the expression level of MHC II molecules in BMDCs cells after co-culture of lipopolysaccharide LPS with BMDCs for 24 hours.

[0126] Fig.19 The flow cytometry analysis of the expression level of MHC II molecules in BMDCs cells shows that the expression level of the magnetically driven porous helical nanomotor HON in the moving state (d) is the highest, at 89.05%, which is 1.5 times the expression level of the magnetically driven porous helical nanomotor HON in the static state (c) at 44.13%, and 3 times the expression level of the antigen OVA group in the free state (b). In the flow cytometry analysis of the expression level of MHC I molecules in BMDCs cells, the expression level of the magnetically driven porous helical nanomotor HON in the moving state (i) is the highest, at 94.91%, which is 1.5 times the expression level of the magnetically driven porous helical nanomotor HON in the static state (h) at 75.45%, and 1.4 times the expression level of the antigen OVA group in the free state (g). In summary, the magnetically driven porous helical nanomotor HON with active movement ability under a magnetic field can enhance the expression of MHC molecules in BMDCs cells and enhance the immune response of BMDCs cells.

[0127] Fig. 20The figures are flow cytometry statistical analysis results after BMDCs cells were treated with different control groups, where (a) is the statistical analysis result of the expression level of MHC II molecules in BMDCs cells of each treatment group 24 hours after treatment, (b) is the statistical analysis result of the expression level of MHC I molecules in BMDCs cells of each treatment group 24 hours after treatment, (c) is the statistical analysis result of the expression level of CD40 molecules in BMDCs cells of each treatment group 24 hours after treatment, (d) is the statistical analysis result of the expression level of CD80 molecules in BMDCs cells of each treatment group 24 hours after treatment, (e) is the statistical analysis result of the expression level of CD86 molecules in BMDCs cells of each treatment group 24 hours after treatment. The statistical analysis results were obtained by using GraphPad Prism 5 software to perform statistical analysis on the obtained data, and the differences between the groups were analyzed by one-way analysis of variance. The data are expressed as mean ± SEM. p<0.05p<0.01, p<0.001 indicates significant difference. Fig. 20 The statistical analysis results of various co-stimulatory molecules show that the expression levels of MHC molecules, typical surface marker molecules CD86 and other types of co-stimulatory molecules of the magnetically driven porous helical nanomotor HON with active movement ability under magnetic field conditions are the highest, indicating that the magnetically driven porous helical nanomotor HON for active antigen delivery has the ability to actively deliver antigens to achieve antigen cross-presentation and enhance cellular immunity.

[0128] For ordinary technicians in this field, the embodiments are only illustrative descriptions of the present invention. It is obvious that the specific implementation of the present invention is not limited to the above-mentioned methods. As long as various non-substantial improvements are made using the method concept and technical solution of the present invention, or the concept and technical solution of the present invention are directly applied to other occasions without improvement, they are all within the protection scope of the present invention.

Claims

1. A magnetically driven porous helical nanomotor for active antigen delivery, characterized in that: The magnetically driven porous spiral nanomotor for active antigen delivery comprises a nickel layer, porous spiral silicon and a surface antigen ovalbumin (OVA). The nickel layer modifies the surface antigen ovalbumin (OVA) on the surface of the porous spiral silicon.

2. The magnetically driven porous helical nanomotor for active antigen delivery according to claim 1, characterized in that: The magnetically driven porous spiral nanomotor for active antigen delivery is made of a spiral silicon mesoporous material, and an asymmetric metal nickel layer is magnetron sputtered on the surface of the porous spiral silicon to obtain a porous spiral magnetically driven nanomotor.

3. A method for preparing a magnetically driven porous helical nanomotor for active antigen delivery, characterized in that: The specific steps are: S1: Using the sol-gel method, tetraethyl orthosilicate is added to an aqueous solution containing sodium hydroxide, perfluorooctanoic acid, and hexadecyltrimethylammonium bromide, mixed and stirred for reaction, and then calcined to obtain the spiral siliceous mesoporous material HR; S2: A metal nickel layer is deposited on the surface of the spiral siliceous mesoporous material by magnetron sputtering to obtain a HR@Ni spiral magnetic drive motor; S3: The HR@Ni helical magnetically driven motor was modified with the surface antigen ovalbumin (OVA) to obtain a magnetically driven porous helical nanomotor for active antigen delivery.

4. The method for preparing a magnetically driven porous helical nanomotor for active antigen delivery according to claim 3, characterized in that: The specific steps of step S1 are: S11: Synthesizing a spiral siliceous mesoporous material using perfluorooctanoic acid and hexadecyltrimethylammonium bromide as templates; dissolving hexadecyltrimethylammonium bromide in deionized water, and then adding NaOH solution and perfluorooctanoic acid to the solution, respectively, to obtain a mixed solution; S12: heating the mixed solution, adding tetraethyl orthosilicate to the mixed solution, and continuing to stir the solution; after the reaction is completed, a white precipitate is obtained; S13: The white precipitate is collected by centrifugation and washed several times to remove the residual reactants. After drying at room temperature, the template is calcined to obtain a sample of helical mesoporous silica HR.

5. The method for preparing a magnetically driven porous helical nanomotor for active antigen delivery according to claim 4, characterized in that: The specific steps of step S2 are: S21: The prepared spiral mesoporous silicon HR is monodispersed in anhydrous ethanol, and then coated on a glass slide pre-cleaned by O2 plasma using a pipette gun, and the glass slide is placed in a vacuum chamber of magnetron sputtering and evacuated; after the sputtering is completed, the sputtering source is turned off, the vacuum is removed, and the sample is taken out; S22: The sample prepared in step S21 is dispersed in deionized water by ultrasonication, and then centrifuged and dried to obtain a HR@Ni helical magnetic drive motor.

6. The method for preparing a magnetically driven porous helical nanomotor for active antigen delivery according to claim 5, characterized in that: The specific steps of step S3 are: S31: dispersing the HR@Ni helical magnetic drive motor in ethylene glycol, adding γ-aminopropyltriethoxysilane APTES to react, and then centrifuging and washing to obtain a washed product; S32: dispersing the washed product in N,N-dimethylformamide (DMF), adding succinic anhydride and triethylamine (TEA), stirring and mixing, and then centrifuging and washing to obtain a product with a carboxyl group; S33: dispersing the HR@Ni helical magnetic motor with carboxyl groups in MES solution, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC and N-hydroxysuccinimide NHS, and stirring to mix and react; then washing the obtained reaction product with PBS buffer for multiple times and dispersing it in PBS buffer; Then, the antigen ovalbumin OVA is added to react; the supernatant is removed by centrifugation to obtain the product, the magnetically driven porous helical nanomotor HON.

7. The method for preparing a magnetically driven porous helical nanomotor for active antigen delivery according to claim 5, characterized in that: The mass ratio of perfluorooctanoic acid to hexadecyl in the aqueous solution containing sodium hydroxide, perfluorooctanoic acid and hexadecyltrimethylammonium bromide in step S11 is about 0-0.1; 0.1-0.3 g of hexadecyltrimethylammonium bromide is added, and hexadecyltrimethylammonium bromide is dissolved in 90-100 g of deionized water, and then 0.65-0.8 mL of 2M NaOH solution and 0.005-0.015 g of perfluorooctanoic acid are added to the solution respectively; the volume of tetraethyl orthosilicate added in step S12 is 1-1.5 mL; wherein the mixing reaction is stirred at 80° C. for 1.5-2.5 h; in step S13, the white precipitate is centrifuged at 8000 rpm / min for 5 min to collect the precipitate, and then washed with deionized water and anhydrous ethanol for 5 times respectively to remove the residual reactants, and after drying at room temperature, calcined at 550° C. for 5 h to remove the template to obtain a sample of spiral mesoporous silicon HR.

8. The method for preparing a magnetically driven porous helical nanomotor for active antigen delivery according to claim 6, characterized in that: In step S21, the vacuum degree is maintained at 9.9×10 -4 Pa, the magnetron sputtering power is 85w, and the Ni layer is sputtered for 3min in an Ar atmosphere with an Ar gas flow rate of 45-50sccm, and the thickness of the obtained Ni layer is 30-50nm; in the step S22, the sample is collected by centrifugation at a speed of 8000rpm for 5min, and the obtained sample is placed in a vacuum drying oven at 60℃ for drying, and the drying time is 12h.

9. The method for preparing a magnetically driven porous helical nanomotor for active antigen delivery according to claim 6, characterized in that: The mass of the HR@Ni helical magnetic drive motor in step S31 is 3-6 mg, which is dispersed in 15-25 mL of ethylene glycol, and then 150-250 μL of γ-aminopropyltriethoxysilane APTES is added, and the mixture is reacted at room temperature of 25°C for 12 hours. After the reaction is completed, the product is collected by centrifugation at 8000 rpm / min for 5 minutes, and then washed with ethanol three times; In the step S32, 3-5 mg of the washed product is dispersed in 7-15 mL of N,N-dimethylformamide DMF, 40-60 mg of succinic anhydride SAA and 50-60 μL of triethylamine TEA are added, and stirred at room temperature at 25° C. for 12 h; the product with carboxyl groups is collected by centrifugation at 8000 rpm / min for 5 min, and washed with ethanol 3 times; In the step S33, 3-5 mg of HR@Ni helical magnetically driven motor with carboxyl group is dispersed in MES solution, and then 2-4 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide EDC and 2-4 mg of N-hydroxysuccinimide NHS are added, and stirred for 1 hour; then, the reaction product is washed with PBS buffer for 3 times and dispersed in 2-3 mL of PBS buffer; finally, 0.5-1.5 mg of antigen ovalbumin OVA is added and reacted for 24 hours; then, centrifuged at 8000 rpm / min for 5 minutes, the supernatant is removed, and the product magnetically driven porous helical nanomotor HON is obtained, and it is dispersed in 1 mL of physiological saline and stored at 4°C.

10. An application of a magnetically driven porous helical nanomotor for active antigen delivery, characterized in that: The magnetically driven porous helical nanomotor for active antigen delivery is used to promote antigen cross-delivery and enhance cellular immune response, wherein the cellular immune response is BMDC cells.

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