1. The engineered exosome nano motor is characterized in that a nitric oxide driving matrix with sulfhydrylation surface is used for carrying out surface modification on exosomes through a water-soluble cross-linking agent to form the engineered exosome nano motor.

2. The engineered exosome nanomotor according to claim 1, wherein the nitric oxide driving matrix is a zwitterionic polymer covalently bonded with L-arginine or a bowl-shaped mesoporous silicon nanomaterial loaded with L-arginine.

3. The engineered exosome nanomotor according to claim 1, wherein the water-soluble cross-linker is a maleimide cross-linker.

4. The engineered exosome nanomotor according to claim 1, wherein the source of exosomes comprises stem cells or macrophages.

5. A method of preparing the engineered exosome nanomotor of claim 1, comprising the steps of:

(1) carrying out mercaptopropionation surface modification on the nitric oxide driving matrix to obtain a NO driving matrix rich in sulfydryl;

(2) reacting the nitric oxide driving matrix rich in sulfydryl and obtained in the step (1) with a water-soluble cross-linking agent, and centrifugally washing to obtain the nitric oxide driving matrix rich in hydroxysuccinimide groups on the surface;

(3) and (3) mixing the nitric oxide driving matrix with the surface rich in the hydroxysuccinimide group obtained in the step (2) with the exosome at a low temperature, and separating and purifying to obtain the engineered exosome nano motor.

6. The method of claim 5, wherein the nitric oxide driver matrix of step (1) is a zwitterionic polymer of covalently bonded L-arginine, and is prepared by: reacting methacrylic anhydride with L-arginine in a mixed solvent of deionized water, 1, 4-dioxane and triethylamine, separating and purifying to obtain an arginine monomer; dissolving the obtained arginine monomer in deionized water, adding an initiator and a double bond cross-linking agent, performing ultrasonic dispersion reaction, and then separating and purifying to obtain the covalently bonded L-arginine zwitterionic polymer.

7. The preparation method according to claim 5, wherein the nitric oxide-driven matrix in step (1) is a bowl-shaped mesoporous silicon nanomaterial loaded with L-arginine, and the preparation process comprises: adding bis (gamma-triethoxysilylpropyl) tetrasulfide and ethyl orthosilicate into a mixed system which takes CTAB as a template agent, strong ammonia water as a catalyst and ethanol as a cosolvent, and forming a bowl-shaped mesoporous silicon nano material under the action of sodium hydroxide; and (3) forming the bowl-shaped mesoporous silicon nano material loaded with the L-arginine in a high-concentration arginine solution.

8. The method of claim 5, wherein the concentration of the surface modified nitric oxide-driven matrix that is mercaptopropionated in step (1) is 10, preferably when the nitric oxide-driven matrix is a zwitterionic polymer of covalently bonded L-arginine⁵-10¹²The amount of 3-mercaptopropyltriethoxysilane is 0.1-10mL per mL, the reaction temperature is 10-50 ℃, and the reaction time is 10-48 h; when the nitric oxide driving substrate is a bowl-shaped mesoporous silicon nano material loaded with L-arginine, the mass of the bowl-shaped mesoporous silicon nano material subjected to mercaptopropionation surface modification in the step (1) is 1-50mg, the using amount of 3-mercaptopropyltriethoxysilane is 0.01-1mL, the using amount of aminopropyltriethoxysilane is 0.01-1mL, the reaction temperature is 10-50 ℃, and the reaction time is 10-48 h; the concentration of the NO driving matrix rich in sulfhydryl in the step (2) is 10⁵-10¹²The concentration of the water-soluble cross-linking agent is 0.1-10mg/mL, the reaction temperature is 10-50 ℃, and the reaction time is 10-48 h; in the step (3), the number ratio of the nitric oxide driving matrix with the surface rich in the hydroxysuccinimide group to the exosome is 10/1-1/10, the reaction temperature is 0-10 ℃, and the reaction time is 1-24 h.

9. The method according to claim 6, wherein the molar ratio of the arginine monomer to the crosslinking agent is 1-20, the molar ratio of the initiator to the crosslinking agent is 0.1-1, the reaction time is about 0.5-5h, and the reaction temperature is 50-350 ℃.

10. The method according to claim 7, wherein the volume ratio of the bis (γ -triethoxysilylpropyl) tetrasulfide to the ethyl orthosilicate is 1: 1-1: 10, the reaction time is 12-36 h, and the reaction temperature is 25-50 ℃;the concentration of the sodium hydroxide is 0.1-1M, the action time is 10-60min, and the reaction temperature is 25-50 ℃; the concentration of the high-concentration arginine solution is 0.1-10mg mL^-1。

Background

Exosomes are extracellular vesicles that are secreted by many types of cells in the body, distributed approximately 30-150nm in diameter and in various body fluids. Exosomes are thought to contain most of the biological information (micro RNAs, proteins, lipids, etc.) in the maternal cell and are able to transmit this information to the recipient cell through cell membrane fusion. The novel intercellular information transmission system participates in information transmission among different cells, regulates intercellular signal transmission, influences the physiological state of the cells and is closely related to the occurrence and progress of various diseases.

The exosome with good biocompatibility has the advantages of long in-vivo circulation time, targeting to a focus part, repairing a damaged part and the like, thereby being used as a good drug delivery carrier. However, in the face of complicated and variable pathological mechanisms of diseases, simple exosomes are not capable of achieving drug delivery deep in patients due to lack of active motor ability when used as drug carriers, so that the treatment effect is not ideal, and therefore, the exosomes need to be engineered to impart the autonomous motor ability.

The Nitric Oxide (NO) nanomotor converts active oxygen and L-arginine into driving gas nitric oxide under the action of nitric oxide synthase in vivo based on human endogenous biochemical reaction. Besides being used as driving gas, the nitric oxide also has the effects of enhancing tissue permeability, promoting vascular endothelialization, improving anticancer efficiency and the like, and has potential biomedical application. More importantly, the nitric oxide nanomotor based on autonomous movement can better realize the targeting and aggregation at the disease part by utilizing the microenvironment of inflammation parts of patients or high ROS concentration. If the NO driving matrix can be modified on the surface of the exosome, the exosome is expected to be endowed with the autonomous motor capacity to realize the deep treatment of the diseased part, thereby improving the treatment effect. At present, no report on the engineering of exosomes by using a nano motor technology exists. Therefore, there is a need for new technology to develop and prepare an engineered exosome nanomotor.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides an engineered exosome nano motor which can effectively realize accurate targeting of exosomes to focus positions and repair damaged positions, so that exosomes can be used as a drug carrier to realize drug delivery in the depth of a patient and endow the patient with the autokinetic ability.

The invention also provides a preparation method of the engineered exosome nano-motor.

The technical scheme is as follows: in order to achieve the purpose, the engineered exosome nanomotor is formed by performing surface engineering modification on exosomes through a water-soluble cross-linking agent by driving a matrix through surface thiolated Nitric Oxide (NO). The engineering modification is to modify substances with certain specific functions to the exosomes through physical and chemical means to form an engineered exosome, and the exosome can play more functions in cooperation with the exosome. In the invention, the NO nanomotor is modified to an exosome through a cross-linking agent.

The nitric oxide driving matrix is a zwitterionic polymer covalently bonded with L-arginine or a bowl-shaped mesoporous silicon nano material loaded with L-arginine.

The covalent bonding L-arginine zwitterionic polymer is mainly formed by the reaction of methacrylic anhydride and L-arginine to form a monomer, and is polymerized with a cross-linking agent under the initiation of an initiator to form an arginine monomer; the cross-linking agent is a double bond cross-linking agent containing a disulfide bond, and can be N, N' -cysteamine bisacrylamide and the like; the initiator is a water-soluble initiator, such as azobisisobutyramidine hydrochloride and the like.

The bowl-shaped mesoporous silicon nanomaterial loaded with L-arginine is used as a Nitric Oxide (NO) driving substrate, CTAB is used as a template agent, strong ammonia water is used as a catalyst, ethanol is used as a cosolvent, a mixed precursor of bis (gamma-triethoxysilylpropyl) tetrasulfide and ethyl orthosilicate is subjected to dehydration condensation, and bowl-shaped mesoporous silicon dioxide is formed under the etching action of sodium hydroxide; the method for loading the L-arginine mainly utilizes the mesoporous confinement effect, and bowl-shaped mesoporous silicon dioxide is physically adsorbed in a high-concentration arginine solution to obtain the L-arginine-loaded bowl-shaped mesoporous silicon nanomaterial motor.

Preferably, the water-soluble crosslinking agent is a maleimide crosslinking agent, such as 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt.

Wherein the source of the exosome comprises stem cells, cancer cells or macrophages and the like, and the extraction method comprises a differential centrifugation method or a kit extraction method.

The preparation method of the engineered exosome nano-motor comprises the following steps:

(1) carrying out mercaptopropionation surface modification on the nitric oxide driving matrix to obtain a NO driving matrix rich in sulfydryl; dehydrating and combining the NO driving matrix and silicon hydroxyl in mercaptopropyltriethoxysilane to obtain a mercapto-rich NO driving matrix;

(2) carrying out addition reaction on the NO driving matrix rich in sulfydryl obtained in the step (1) and maleimide groups in a water-soluble cross-linking agent at a certain temperature for a period of time, and carrying out centrifugal washing to obtain the NO driving matrix rich in hydroxysuccinimide groups on the surface;

(3) and (3) mixing the NO driving matrix with the surface rich in the hydroxysuccinimide group obtained in the step (2) with the exosome at a low temperature, and separating and purifying to obtain the engineered exosome nano motor.

Wherein, when the nitric oxide driving matrix is a zwitterionic polymer covalently bonded with L-arginine, the concentration of the surface modified nitric oxide driving matrix subjected to mercaptopropionation in the step (1) is 10⁵-10¹²The amount of 3-mercaptopropyltriethoxysilane is 0.1-10mL per mL, the reaction temperature is 10-50 ℃, and the reaction time is 10-48 h; when the nitric oxide driving substrate is the bowl-shaped mesoporous silicon nano material loaded with L-arginine, the mass of the bowl-shaped mesoporous silicon nano material subjected to mercaptopropionation surface modification in the step (1) is 1-50mg, the using amount of 3-mercaptopropyltriethoxysilane is 0.01-1mL, the using amount of aminopropyltriethoxysilane is 0.01-1mL, the reaction temperature is 10-50 ℃, and the reaction time is 10-48 h.

Wherein the concentration of the NO driving matrix rich in sulfhydryl in the step (2) is 10⁵-10¹²The concentration of the water-soluble cross-linking agent is about 0.1-10mg/mL, the reaction temperature is 10-50 ℃, and the reaction time is 10-48 h.

Wherein, the number ratio of the NO driving matrix with the surface rich in the hydroxysuccinimide group to the exosome in the step (3) is 10/1-1/10, the reaction temperature is 0-10 ℃, and the reaction time is 1-10 h.

Preferably, the NO driving matrix in step (1) is carboxyl-rich zwitterionic polymer of covalently bonded L-arginine, and the preparation process is as follows: reacting methacrylic anhydride with L-arginine in a mixed solvent of deionized water, 1, 4-dioxane and triethylamine, separating and purifying to obtain an arginine monomer; dissolving the obtained arginine monomer in deionized water, adding an initiator and a double bond cross-linking agent, performing ultrasonic dispersion reaction, and then separating and purifying to obtain the covalently bonded L-arginine zwitterionic polymer.

Further, the molar ratio of the arginine monomer to the crosslinking agent is 1-20, the molar ratio of the water-soluble initiator azodiisobutymidine hydrochloride to the double bond crosslinking agent containing the disulfide bond is 0.1-1, the reaction time is 0.5-5h, and the reaction temperature is 50-350 ℃.

As another preferable mode, the NO-driven substrate in step (1) is a bowl-shaped mesoporous silicon nanomaterial rich in carboxyl and loaded with L-arginine, and the preparation process is as follows: adding a mixed precursor of bis (gamma-triethoxysilylpropyl) tetrasulfide and ethyl orthosilicate into a mixed system which takes CTAB as a template agent, strong ammonia water as a catalyst and ethanol as a cosolvent to perform dehydration condensation, and forming a bowl-shaped mesoporous silicon nano material under the etching action of sodium hydroxide; and carrying out physical adsorption on the obtained bowl-shaped mesoporous silicon nano material in a high-concentration arginine solution by utilizing a mesoporous confinement effect to form the bowl-shaped mesoporous silicon nano material loaded with L-arginine.

Further, the CTAB is 0.1-0.5g in mass, the volume ratio of the bis (gamma-triethoxysilylpropyl) tetrasulfide to the ethyl orthosilicate is 0.1-1, the reaction time is 12-36 h, and the reaction temperature is 25-50 ℃; the concentration of the sodium hydroxide is 0.1-1M, the etching time is 10-60min, and the reaction temperature is about 25-50 ℃; the concentration of the high-concentration arginine solution is 0.1-10mg mL^-1。

The invention relates to application of an engineered exosome nano-motor in the field of biological medicine.

The invention relates to an application of an engineered exosome nano-motor in preparing medicines for treating cancers and cardiovascular and cerebrovascular diseases.

The present invention will for the first time combine exosomes with NO-driven substrates by a specific method. The exosome carries a plurality of active substances, including DNA, RNA, protein, lipid, micromolecule metabolites and the like, so that a reaction solvent, temperature and pH are specially considered when the exosome is engineered, the engineering modification of the exosome is realized, the activity maintenance of the exosome in the engineering process is ensured, the engineered exosome cannot be obtained by a simple mixing method, and the engineering exosome is difficult to combine.

Compared with a pure NO driving matrix, the nano motor not only has the similar movement capacity and NO physiological function of the NO driving matrix, but also has the capacity of targeting an inflammation part possessed by an exosome, and simultaneously has the capacity of repairing injury possessed by the exosome.

Has the advantages that: compared with the prior art, the invention has the following advantages:

1. the engineered exosome nano motor is characterized in that a zwitterionic polymer with sulfhydrylation on the surface or a bowl-shaped mesoporous silicon nano material is combined with arginine as a Nitric Oxide (NO) driving matrix by a self-assembly or loading method, and the arginine is used for engineering modification of exosomes. Meanwhile, the zwitter-ion matrix is degraded under the action of reduced glutathione in a cell environment and can be eliminated by the metabolism of the liver and the kidney.

2. The preparation method is simple and efficient, the synthesis conditions are mild, the material dispersion performance is good, the synthesized engineered exosome nano-motor has excellent biocompatibility, the zwitterionic polymer has the bionic property of cell membranes, and has excellent nonspecific protein adsorption/adhesion resistance and low immunogenicity in vivo. The mesoporous silica has larger specific surface area and pore confinement effect, and can improve the cargo loading capacity and realize the controllable long-term release of the cargo. Meanwhile, L-arginine is a common amino acid molecule in vivo. Secondly, reaction products of the nano motor are used without waste materials, and a nitric oxide gas molecule which is one of catalytic products is a signal molecule in the body and can be used for treating inflammation or cancer; in addition, the exosome with good biocompatibility has the advantages of long in-vivo circulation time, targeting to a focus part, repairing a damaged part and the like, so that the exosome can be used as a good drug delivery carrier.

3. The engineered exosome nano motor prepared by combining exosomes and an NO driving matrix for the first time can be used for accurately targeting an infection focus part firstly, and then has active movement capacity in an inflammation and active oxygen microenvironment by utilizing the NO driving matrix and can target specific inflammatory cells, so that the step-by-step targeting effect and the treatment effect of the engineered exosome nano motor are realized, and the pure exosomes are ensured to be used as a drug carrier to realize drug delivery in the depth of a patient.

Drawings

FIG. 1 is a transmission electron micrograph of the mesenchymal stem cell exosome obtained in example 1;

FIG. 2 is a transmission electron micrograph of a covalently bonded zwitterionic polymer of L-arginine obtained in example 2;

FIG. 3 is a transmission electron micrograph of the engineered exosome nanomotor obtained in example 2;

FIG. 4 shows the surface potentials of different materials in the construction process of the engineered exosome nanomotor obtained in example 2;

FIG. 5 is a transmission electron microscope image of the bowl-shaped mesoporous silica nanomaterial obtained in example 4;

FIG. 6 is a distribution diagram of the pore size of the bowl-shaped mesoporous silica nanomaterial obtained in example 4;

FIG. 7 is a fluorescence diagram of an engineered exosome nanomotor;

FIG. 8 is a MSD fit curve of engineered exosome nanomotor motion;

FIG. 9 shows a graph of 5X 10 in example 2⁵cell/mL of a dry extracellular secretion (a) for inflammatory stimulation of neuroid cells, covalently bonded with L-arginineThe speed of movement of the zwitterionic polymer (b) and the engineered exosome nanomotor (c);

figure 10 is the cellular uptake efficiency of exosomes and engineered exosome nanomotors.

Detailed Description

The present invention is further illustrated by the following examples.

The experimental methods described in the examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

The cells of the invention are commercially available. For example, the human umbilical cord mesenchymal stem cells, the nerve-like cells SH-SY5Y and the endothelial cells HUVECs are all sold in the market, and the human umbilical cord mesenchymal stem cells can also be replaced by other stem cells such as skin stem cells, bone marrow stem cells, hematopoietic stem cells and the like.

Example 1

The preparation method of the exosome comprises the following steps:

(1) will 10⁵Inoculating the human umbilical cord mesenchymal stem cells/mL into a T75 culture bottle, adding a complete culture solution to 10 mL/bottle, and placing in CO₂Culturing in an incubator at 37 ℃ for 48 h; when the growth density of the cells in the culture bottle is about 70 percent, discarding the upper layer culture solution, and replacing 10 mL/bottle of the exosome-free serum culture solution (containing 20ng/mL of vascular endothelial cell adhesion molecule 1 (VCAM-1)); culturing at 37 deg.C for 48 hr, collecting upper layer cell culture fluid, and storing at-80 deg.C;

(2) obtaining exosome by adopting a differential centrifugation method, centrifuging the cell culture solution at 4 ℃ and 2000rpm for 20min, collecting the cell culture solution on the upper layer, and removing dead cells on the lower layer; centrifuging the upper layer culture solution at 4 deg.C at 10000rpm for 30min, collecting the upper layer cell culture solution, and discarding the lower layer cell debris; centrifuging the upper layer culture solution at 100000rpm at 4 deg.C for 120min, discarding the upper layer cell culture solution, collecting the lower layer exosome, dispersing in PBS (pH 7.5), and storing at-80 deg.C and 10 deg.C⁹one/mL. As shown in fig. 1, the obtained human umbilical cord mesenchymal stem cell exosome has a particle size of about 90nm and is presented as dispersed and regular spherical nanoparticles (EXO).

Example 2

A preparation method of a zwitterionic polymer engineered exosome nano-motor covalently bonded with L-arginine comprises the following steps:

(1) 2g L-arginine was weighed out and dissolved in a mixed solvent of 20mL of deionized water and 8.5mL of 1, 4-dioxane, and 4.5mL of triethylamine was added. The mixed solution was then cooled with an ice-water bath, stirred and 3mL of methacrylic anhydride was added dropwise over about 10 min. The ice-water bath was removed and the reaction was stirred at room temperature overnight. Precipitating the product with 400mL of acetone, slowly dropping acetone into the product, after the solution is layered, slightly sucking out the lower layer of white emulsion by using a suction pipe, centrifuging at 8000rpm for 10min, dissolving the obtained precipitate in a small amount of deionized water, precipitating in acetone again to obtain a white precipitate, and drying in vacuum at room temperature to obtain an arginine monomer;

(2) weighing 0.0624mmol of N, N' -bis (acryloyl) cystamine (cross-linking agent), adding 10.75mL of deionized water, performing ultrasonic treatment for 10min to dissolve, adding into a reaction flask, introducing condensed water, and adding into a reaction flask₂Purifying for 30min under atmosphere. 0.0184mmol of azobisisobutyramidine hydrochloride (initiator) was weighed, dissolved in 0.5mL of deionized water, and injected into the above reaction flask by syringe, and then an arginine monomer solution (0.252 mmol of arginine monomer obtained in step (1) dispersed in 11mL of deionized water) was added to the flask in N₂Reacting for 1h at 140 ℃ under protection. The product was centrifuged (10000rpm, 10min), precipitated and washed 3 times with water to give a covalently bonded L-arginine zwitterionic polymer. As shown in fig. 2, the synthetic covalently bonded L-arginine zwitterionic polymer appears as dispersed and regular spherical nanoparticles (PMA);

(3) blending the covalently bonded L-arginine zwitterionic polymer obtained in the step (2) with 1mL of 3-mercaptopropyltriethoxysilane at room temperature for 24h to obtain 10⁹Thiolated NO-driven matrix/mL, supernatant discarded by centrifugation, and solid product dispersed in 0.5mL of PBS (pH 7.5) buffer (PMA-SH, 2X 10)⁹one/mL) of the total solution, adding 0.5mg of a water-soluble crosslinking agent 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfonic group succinimide ester sodium salt, mixing for 12h at 25 ℃, centrifuging the product (10000rpm, 10min), washing the precipitate for 3 times with water to obtain the hydroxyl-enriched amber with the surfaceCovalently bonded L-arginine zwitterionic polymers of succinimide groups (PMA-SMCC, 10)⁹One).

(4) After the product of step (3) was ultrasonically dispersed in 0.5mL of PBS (pH 7.5), the human umbilical cord mesenchymal stem cell exosome (0.5mL, 10) obtained in example 1 was added⁹one/mL) at 4 deg.C for 24h, centrifuging the product (10000rpm, 10min), collecting precipitate, washing with PBS for 3 times to obtain engineered exosome nano-motor (PMA/EXO), dispersing in 1mL PBS at 10 concentration⁹one/mL. As shown in fig. 3, the prepared engineered exosome nanomotor appears as dispersed peanut-like nanoparticles.

Example 3

Surface potential detection of the engineered exosome nano-motor:

the human umbilical cord mesenchymal stem cell Exosomes (EXO) in example 1, the stepwise surface-modified NO nanomotors (PMA-SH) and (PMA-SMCC) obtained in example 2, and the engineered exosome nanomotors were configured to 10 using PBS⁷one/mL solution (NanoSight nanoparticle tracking analyzer), the above solutions of different materials were added to a potential cell, and the surface potentials of the different materials were measured using a NanoSight potential analyzer. As shown in fig. 4, the potential of human umbilical cord mesenchymal stem cell Exosome (EXO) in example 1 is negative because the cell membrane has electronegativity. In addition, the zwitterionic Polymer (PMA) obtained in example 2 is rich in carboxyl groups on the surface, so the surface potential of PMA is negative. Subsequently, PMA is gradually modified with negatively charged sulfhydryl (PMA-SH) and maleimide (PMA-SMCC) groups, and the surface potential of the material is gradually reduced. Finally, the electronegativity of the engineered exosome nanomotor (PMA/EXO) obtained in example 2 was further reduced due to the electronegativity of EXO. The engineered exosome nanomotor is formed by gradually modifying a maleimide cross-linking agent by a zwitterion-based NO nanomotor and then combining with an exosome. Due to the difference in electronegativity of the functional groups specific to the surface of each substance. Therefore, the potential detection in the embodiment proves that each step of the preparation process of the engineered exosome nano-motor is successfully synthesized.

Example 4

A preparation method of a bowl-shaped mesoporous silicon nanomaterial engineered exosome nano motor loaded with L-arginine comprises the following steps:

(1)0.16g CTAB dissolved in 75mL H₂Performing ultrasonic dispersion on O and 30mL of ethanol, and then adding 1mL (mass fraction is 25%) of ammonia water; the mixed precursor solution (0.1mL TESPTS with 0.25mL TEOS) was added and reacted for 24h at 35 ℃. Centrifuging and collecting the product, and washing the precipitate for 3 times by using ethanol and water respectively; dispersing in 63mL NaOH (0.48M) again, etching for 30min at room temperature, centrifugally collecting the product, washing the precipitate for 3 times with water, and removing the template agent CTAB by using a Soxhlet extraction device to obtain the bowl-shaped mesoporous silicon nanomaterial. As shown in fig. 5, the prepared mesoporous silicon nanomaterial is in the form of bowl-shaped nanoparticles with upward openings. In addition, the pore size distribution data of fig. 6 shows that the prepared bowl-shaped mesoporous silicon nanomaterial has a pore size of about 4 nm.

(2) Dissolving 20mg of the mesoporous silicon nano material obtained in the step (1) in 20mL of H₂O, 50. mu.L of aminopropyltriethoxysilane and 50. mu.L of 3-mercaptopropyltriethoxysilane were added to the mixture and mixed at 25 ℃ for 24 hours. Centrifuging the product (10000rpm, 10min), precipitating, washing with water for 3 times, dispersing in 5mL deionized water again, mixing with 200mg L-Arg at room temperature for 24h, centrifuging the product, removing the supernatant, and washing the precipitate with water for 3 times to obtain the bowl-shaped mesoporous silicon nanomaterial loaded with L-arginine, namely the NO driving substrate with thiolated surface;

(3) dispersing the bowl-shaped mesoporous silicon nanomaterial loaded with L-arginine in step (2) in 0.5mL of PBS (pH 7.5) buffer (concentration of 0.5 × 10)⁹one/mL) is added with 0.5mg of 4- (N-maleimide methyl) cyclohexane-1-carboxylic acid sulfonic group succinimide ester sodium salt and mixed for 12h at room temperature, the product is centrifuged (10000rpm, 10min), and the precipitate is washed for 3 times by water to obtain the bowl-shaped mesoporous silicon nano material (10) which is rich in hydroxyl succinimide group on the surface and is loaded with L-arginine (L-arginine-loaded bowl-shaped mesoporous silicon nano material)⁹One).

(4) After the product of step (3) was ultrasonically dispersed in 0.5mL of PBS (pH 7.5), the human umbilical cord mesenchymal stem cell exosome (0.5mL, 10) obtained in example 1 was added⁹piece/mL) at 4 ℃ for 24h, centrifuging the product (10000rpm, 10min), taking the precipitate and washing the precipitate for 3 times by PBS (phosphate buffer solution), obtaining the engineered exosome nano-motor, dispersing the engineered exosome nano-motorConcentration 10 in 1mL PBS (pH 7.5)⁹one/mL. The nano-motor prepared by the embodiment is peanut-shaped nano-particles formed by combining mesoporous silicon nano-materials and spherical exosomes.

Example 5

Fluorescence labeling characterization of engineered exosome nanomotors:

(1) 0.5mL of the human umbilical cord mesenchymal stem cell exosome (10) obtained in example 1 was added⁹piece/mL) and 0.5mL cell membrane dye DiO (20 mu M), dyeing for 10min at room temperature in the dark, and centrifuging at 4 ℃ (100000rpm, 120min) to obtain DiO fluorescence labeled human umbilical cord mesenchymal stem cell exosome.

(2) After 0.5mg of the zwitterionic polymer covalently bonded with L-arginine, which is the final product of the step (2) in the example 2, and 0.5mg of the mesoporous silicon nanomaterial loaded with L-arginine, which is the final product of the step (3) in the example 4, are respectively blended with 1mL of 3-mercaptopropyltriethoxysilane for 24 hours at room temperature, discarding the supernatant, centrifuging and dispersing the product in 0.5mL PBS (pH 7.5) buffer, adding 0.5mg of 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfonic acid group succinimide ester sodium salt and 10 μ L of Cy 5-maleimide (1mg/mL), mixing at room temperature for 12h, centrifuging the product (10000rpm, 10min), precipitating, and washing with water for 3 times to obtain Cy5 fluorescence labeled covalent bonding L-arginine zwitterionic polymer nanoparticles and Cy5 fluorescence labeled L-arginine loaded mesoporous silicon nanomaterial;

(3) and (3) carrying out reaction on the DiO fluorescence labeled exosome obtained in the step (1) and the Cy5 fluorescence labeled zwitter-ionic polymer covalently bonded with L-arginine or the mesoporous silicon nanomotor loaded with L-arginine, which is obtained in the step (2), according to the number ratio of 1: 1 at 4 ℃ for 24h, centrifuging the product (10000rpm, 10min), washing the precipitate for 3 times by PBS, and obtaining two fluorescence-labeled engineered exosome nanomotors.

(4) Dispersing the two fluorescence-labeled engineered exosome nanomotors obtained in the step (3) in a PBS buffer solution (the concentration is 10)⁹one/mL) was added dropwise to a 14mm cell culture dish containing a PBS solution in a volume of 1mL, and the binding of the fluorescently labeled engineered exosome nanomotors was immediately observed using a fluorescence microscope. Covalent bonding of fluorescent labels as shown in FIG. 7The red fluorescence-labeled nano particles and the green fluorescence-labeled exosomes in the L-arginine zwitterionic polymer engineered exosome nano motor are well overlapped, which indicates that the engineered exosome nano motor is successfully prepared; in the same way, the red fluorescence labeled nano particles and the green fluorescence labeled exosomes in the fluorescence labeled L-arginine-loaded bowl-shaped mesoporous silicon nano material engineering exosome nano motor also show the overlapping result.

Example 6

Engineered exosome nanomotors at 5 x 10⁵Study of motor performance in an inflammatory-stimulated neuronal (or endothelial) cell(s) environment at cell/mL density:

(1) the neuron-like cells SH-SY5Y or (endothelial cells HUVECs) are added at 5X 10⁵Inoculating the cell/mL into a 14mm cell culture dish, wherein the volume of a complete culture medium is 1mL, placing the cell culture dish in a constant-temperature incubator at 37 ℃, and after 24 hours, adhering the cell to the wall; adding inflammation stimulating factor (0.1mM lipopolysaccharide), and stimulating for 24 hr;

(2) take 10. mu.L of the mesenchymal stem cell exosome of example 1 (10)⁹one/mL, PBS formulation), zwitterionic polymer of covalently bound L-arginine (10) as the end product of step (2) of example 2⁹Per mL, PBS preparation), the final product of step (3) of example 4, L-arginine-loaded mesoporous silicon nanomaterial (10)⁹one/mL, PBS formulated), engineered exosome nanomotors (10) prepared in examples 2 and 4⁹one/mL, prepared with PBS), and directly adding into the adherent cell culture dish containing the 1mL culture solution, and immediately observing and recording the motion of the nanomotor in the cell environment by using a fluorescence microscope.

(3) The kinetic behavior of the umbilical cord mesenchymal stem cell exosomes according to example 1, the zwitterionic polymer covalently bonded with L-arginine according to example 2, and the engineered exosome nanomotors according to example 2 in a cell environment is analyzed. As shown in fig. 8, the motion trajectory of the engineered exosome nanomotor in a cell environment conforms to a quadratic function, which indicates that the motion behavior belongs to autonomous motion. In addition, the locomotor speeds of exosome, covalently bound zwitterionic polymer of L-arginine and engineered exosome nanomotors were calculated to be 1.34, 4.35, 2.63 μm/s, respectively (fig. 9). The result shows that compared with the movement speed of a pure exosome, the movement speed of the zwitterion-based nanomotor with the autonomous movement capability is obviously improved. The movement speed of the engineered exosome nanomotor is reduced to some extent, probably because the gravity of the material is increased, and is obviously faster than that of a pure exosome.

Similarly, the movement trajectory results of the umbilical cord mesenchymal stem cell exosome in example 1 and the mesoporous silicon nanomaterial loaded with L-arginine and the engineered exosome nanomotor in example 4 in a cell environment show that the movement speed of the engineered exosome nanomotor is reduced compared with that of the mesoporous silicon nanomaterial loaded with L-arginine, which may be caused by the fact that the movement speed of the material loaded with exosome is reduced due to the increase of the gravity of the material itself, but is obviously faster than that of the simple exosome.

The data show that the movement locus of the engineered exosome nano motor is in cells stimulated by inflammation (a microenvironment contains active oxygen with higher concentration), and the engineered exosome nano motor has better autonomous movement capability.

Example 7

The targeted performance of the engineered exosome nanomotor in an inflammatory cell model is studied:

(1) 200. mu.L of SH-SY5Y cells (cell density 1X 10)⁵one/mL) were inoculated in 6-well plates and incubated overnight at 37 ℃; followed by the addition of H₂O₂The final concentration is 0.1mM, and the culture is continued for 24 h; subsequently, 10. mu.L of the DiO fluorescently-labeled exosomes (10) of example 5 were added to the well plate⁹one/mL), Cy5 fluorescently labeled L-arginine covalently bonded zwitterionic polymer (10)⁹one/mL) and fluorescently labeled engineered nanomotors (10)⁹seed/mL), and culturing for 24 h;

(2) collecting all the supernatant in the pore plate into a centrifugal tube, washing the supernatant for three times by using 0.1mL PBS, collecting the washing liquid into the centrifugal tube, and keeping the volume of the washing liquid to 1mL by using the PBS and recording the volume as culture supernatant; then adding 0.1mL of pancreatin to digest for 10min at 37 ℃; adding 0.5mL PBS into the pore plate, transferring the cell suspension into a new centrifugal tube, and centrifuging to collect cells (1000rpm, 5 min); after the centrifugation, 0.1mL of cell lysate was added to the supernatant, and the volume of the cell lysate was adjusted to 1mL with PBS to obtain a cell lysate.

(3) The fluorescence intensities of the culture supernatant and the cell sap were measured using a fluorescence spectrophotometer, and the cell uptake efficiency of each sample was calculated using the formula of cell uptake efficiency (%) -fluorescence intensity of the cell sap/(fluorescence intensity of the cell sap + fluorescence intensity of the culture supernatant) × 100%. As shown in fig. 10, compared with the cellular uptake efficiency of EXO alone, the cellular uptake efficiency of PMA/EXO is significantly improved, which indicates that the nanomotor based on autonomous movement utilizes the cellular inflammation microenvironment to better achieve targeting and aggregation, and ensures that the EXO alone is used as a drug carrier to achieve drug delivery deep in a patient.

Example 8

Example 8 was prepared identically to example 2, except that: the molar ratio of the arginine monomer to the crosslinking agent is 1, the molar ratio of the initiator to the crosslinking agent is 0.1, the reaction time is about 0.5h, and the reaction temperature is 350 ℃; the concentration of the nitric oxide driving matrix for carrying out the mercaptopropionation surface modification is 10⁵The amount of 3-mercaptopropyltriethoxysilane is 0.1mL per mL, the reaction temperature is 10 ℃, and the reaction time is 48 hours; concentration of thiol-rich NO-driven substrate 10⁵The reaction time is 48 hours, the concentration of the water-soluble cross-linking agent is 0.1mg/mL, the reaction temperature is 10 ℃; the number ratio of the nitric oxide driving matrix with the surface rich in the hydroxysuccinimide group to the exosome is 10/1, the reaction temperature is 0 ℃, and the reaction time is 24 hours.

Example 9

Example 9 was prepared identically to example 2, except that: the molar ratio of the arginine monomer to the crosslinking agent is 20, the molar ratio of the initiator to the crosslinking agent is 1, the reaction time is about 5 hours, and the reaction temperature is 50 ℃; the concentration of the nitric oxide driving matrix for carrying out the mercaptopropionation surface modification is 10¹²The amount of the 3-mercaptopropyltriethoxysilane is 10mL, the reaction temperature is 50 ℃, and the reaction time is 10 hours; concentration of thiol-rich NO-driven substrate 10¹²The concentration of the water-soluble cross-linking agent is 10mg/mL, the reaction temperature is 50 ℃, and the reaction time is 10 hours; surface ofThe number ratio of the nitrogen monoxide driving matrix rich in the hydroxysuccinimide group to the exosome is 1/10, the reaction temperature is 10 ℃, and the reaction time is 1 h.

Example 10

Example 10 was prepared identically to example 4, except that: CTAB mass 0.1g, volume ratio of bis (gamma-triethoxysilylpropyl) tetrasulfide to ethyl orthosilicate 1: 1, the reaction time is 12h, and the reaction temperature is 50 ℃; the concentration of sodium hydroxide is 0.1M, the etching time is 60min, and the reaction temperature is about 50 ℃; the concentration of the high-concentration arginine solution is 0.1mg mL^-1(ii) a The mass of the bowl-shaped mesoporous silicon nano material subjected to mercaptopropylation surface modification is 1mg, the using amount of 3-mercaptopropyltriethoxysilane is 0.01mL, the using amount of aminopropyltriethoxysilane is 0.01mL, the reaction temperature is 10 ℃, and the reaction time is 48 h.

Example 11

Example 11 was prepared identically to example 4, except that: CTAB 0.5g, bis (gamma-triethoxysilylpropyl) tetrasulfide and ethyl orthosilicate in a volume ratio of 1: 10, the reaction time is 36h, and the reaction temperature is 25 ℃; the concentration of sodium hydroxide is 1M, the etching time is 10min, and the reaction temperature is about 25 ℃; the concentration of the high-concentration arginine solution is 10mg mL^-1(ii) a The mass of the bowl-shaped mesoporous silicon nano material subjected to mercaptopropylation surface modification is 50mg, the using amount of 3-mercaptopropyltriethoxysilane is 1mL, the using amount of aminopropyltriethoxysilane is 1mL, the reaction temperature is 50 ℃, and the reaction time is 10 hours.