CN113403270B - Engineering exosome nano motor and preparation method thereof - Google Patents

Engineering exosome nano motor and preparation method thereof Download PDF

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CN113403270B
CN113403270B CN202110503881.9A CN202110503881A CN113403270B CN 113403270 B CN113403270 B CN 113403270B CN 202110503881 A CN202110503881 A CN 202110503881A CN 113403270 B CN113403270 B CN 113403270B
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arginine
exosome
nitric oxide
engineering
bowl
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CN113403270A (en
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万密密
沈健
毛春
王琪
赵梓楠
谈开元
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Nanjing Normal University
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Abstract

The invention discloses an engineering exosome nano motor and a preparation method thereof, wherein the engineering exosome nano motor takes a zwitterionic polymer with surface sulfhydrylation covalent bonding L-arginine or bowl-shaped mesoporous silicon nano material loaded with L-arginine as a Nitric Oxide (NO) driving matrix and is used for engineering modification of exosome. The preparation method of the engineering exosome nano motor is simple and efficient, has excellent biocompatibility, has active motion capability in inflammation and active oxygen microenvironment, can effectively realize accurate targeting of exosome to focus positions and repair of damaged positions, enables the exosome to realize drug delivery in deep patients as a drug carrier, endows the exosome with autonomous motion capability, and has wide application prospect in the field of biological medicine.

Description

Engineering exosome nano motor and preparation method thereof
Technical Field
The invention belongs to novel biological nanometer materials, and in particular relates to an engineering exosome nanometer motor and a preparation method thereof.
Background
Exosomes are extracellular vesicles capable of being secreted by many types of cells in the body, with diameters ranging from about 30-150nm, and in various body fluids. Exosomes contain most of the biological information (micro RNAs, proteins, lipids, etc.) in the maternal cells and are able to transfer these information to the recipient cells by cell membrane fusion. The novel intercellular information transmission system participates in the information transmission among different cells, regulates the signal transmission among the cells, 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 focus positions, repairing damaged positions and the like, thereby being capable of being used as a good drug delivery carrier. However, in the face of complex and variable disease pathological mechanisms, pure exosomes cannot achieve drug delivery deep in the patient due to lack of active exercise capability when used as drug carriers, so that the therapeutic effect is not ideal enough, and engineering is required to give the drug an autonomous exercise capability.
Nitric Oxide (NO) nanomotors convert active oxygen and L-arginine into the driving gas nitric oxide under the action of nitric oxide synthase in vivo based on human endogenous biochemical reactions. Besides being used as driving gas, the nitric oxide has the functions 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 site by utilizing the disease inflammation site or the microenvironment with high ROS concentration. If the NO-driven matrix can be modified to the surface of the exosome, the exosome is hopeful to be endowed with the autonomous movement capability to realize the deep treatment of the diseased part, so that the treatment effect is improved. No engineering of exosomes using nanomotor technology has been reported. Therefore, a new technology is needed to develop and prepare an engineering exosome nano motor.
Disclosure of Invention
The invention aims to: aiming at the problems existing in the prior art, the invention provides the engineering exosome nano motor, which can effectively realize the accurate targeting of the exosome to the focus part and repair the damaged part, so that the exosome can realize the drug delivery in the deep part of a patient as a drug carrier and endow the exosome with the autonomous movement capability.
The invention also provides a preparation method of the engineering exosome nano motor.
The technical scheme is as follows: in order to achieve the above purpose, the engineering exosome nanomotor is formed by surface engineering modification of an exosome by a surface thiolated Nitric Oxide (NO) driven matrix through a water-soluble cross-linking agent. The engineering modification in the invention is to modify substances with specific functions to exosomes through physical and chemical means, so that an engineering exosome is formed, and more functions are exerted by the engineering modification in cooperation with the exosomes. In the invention, the NO nanomotor is modified to an exosome by a cross-linking agent.
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.
Wherein, the zwitterionic polymer of covalent bonding L-arginine mainly comprises an arginine monomer formed by reacting methacrylic anhydride with L-arginine and polymerizing with a cross-linking agent under the initiation of an initiator; the cross-linking agent is a double bond cross-linking agent containing disulfide bonds, and can be specifically N, N' -bisacryloylcystamine and the like; the initiator is a water-soluble initiator, such as azobisisobutyronium hydrochloride and the like.
The bowl-shaped mesoporous silicon nanomaterial loaded with L-arginine is used as a Nitric Oxide (NO) driving matrix, CTAB is used as a template agent, concentrated ammonia water is used as a catalyst, ethanol is used as a cosolvent, and a mixed precursor of bis (gamma-triethoxysilylpropyl) tetrasulfide and tetraethoxysilane is subjected to dehydration condensation, and under the etching action of sodium hydroxide, bowl-shaped mesoporous silicon dioxide is formed; the L-arginine loading method mainly utilizes the mesoporous confinement effect, and bowl-shaped mesoporous silicon dioxide is physically adsorbed in high-concentration arginine solution to obtain the L-arginine loaded bowl-shaped mesoporous silicon nanomaterial motor.
Preferably, the water-soluble cross-linking agent is a maleimide cross-linking agent, such as sodium salt of sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate.
Wherein the exosome source comprises stem cells, cancer cells or macrophages, and the extraction method comprises differential centrifugation or kit extraction.
The preparation method of the engineering exosome nano motor comprises the following steps:
(1) Performing thiolated surface modification on the nitric oxide driving matrix to obtain a NO driving matrix rich in sulfhydryl groups; the NO driving matrix is dehydrated and combined with silicon hydroxyl in mercaptopropyl triethoxysilane to obtain the NO driving matrix rich in mercapto;
(2) Carrying out addition reaction on the NO-driven matrix rich in sulfhydryl groups obtained in the step (1) and maleimide groups in the water-soluble cross-linking agent for a period of time at a certain temperature, and centrifugally washing to obtain the NO-driven matrix rich in hydroxysuccinimide groups on the surface;
(3) And (3) mixing the NO-driven matrix with the surface rich in the hydroxysuccinimide groups obtained in the step (2) with an exosome at a low temperature, and separating and purifying to obtain the engineering exosome nano motor.
Wherein when the nitric oxide driving matrix is a zwitterionic polymer with covalently bonded L-arginine, the concentration of the nitric oxide driving matrix subjected to the thiolated surface modification in the step (1) is 10 5 -10 12 The dosage of 3-mercaptopropyl triethoxysilane is 0.1-10mL, the reaction temperature is 10-50 ℃ and the reaction time is 10-48h; when the nitric oxide driving matrix is the bowl-shaped mesoporous silicon nanomaterial loaded with L-arginine, the mass of the bowl-shaped mesoporous silicon nanomaterial subjected to the mercaptopropylation surface modification in the step (1) is 1-50mg, the dosage of 3-mercaptopropyl triethoxysilane is 0.01-1mL, the dosage of aminopropyl triethoxysilane is 0.01-1mL, the reaction temperature is 10-50 ℃, and the reaction time is 10-48h.
Wherein the concentration of NO-driven matrix rich in sulfhydryl groups in the step (2) is 10 5 -10 12 The 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 10-48h.
Wherein the number ratio of the NO-driven matrix with the surface rich in the hydroxysuccinimide groups to the exosomes in the step (3) is 10/1-1/10, the reaction temperature is 0-10 ℃, and the reaction time is 1-10h.
Preferably, the NO-driven matrix of step (1) is a carboxyl-rich zwitterionic polymer of covalently bonded L-arginine, prepared by the steps of: 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 separating and purifying to obtain the covalent bonding L-arginine zwitterionic polymer.
Further, the mol ratio of the arginine monomer to the cross-linking agent is 1-20, the mol ratio of the water-soluble initiator azo diisobutylamidine hydrochloride to the double bond cross-linking agent containing disulfide bonds is 0.1-1, the reaction time is 0.5-5h, and the reaction temperature is 50-350 ℃.
As another preferable aspect, the NO-driven substrate in the step (1) is a carboxyl-rich bowl-shaped mesoporous silicon nanomaterial loaded with L-arginine, and the preparation process thereof is as follows: adding a mixed precursor of bis (gamma-triethoxysilylpropyl) tetrasulfide and tetraethoxysilane into a mixed system taking CTAB as a template agent, concentrated ammonia water as a catalyst and ethanol as a cosolvent to carry out dehydration condensation, and forming a bowl-shaped mesoporous silicon nanomaterial under the etching action of sodium hydroxide; and (3) physically adsorbing the obtained bowl-shaped mesoporous silicon nanomaterial in high-concentration arginine solution by utilizing a mesoporous confinement effect to form the bowl-shaped mesoporous silicon nanomaterial loaded with L-arginine.
Further, the CTAB mass is about 0.1-0.5g, the volume ratio of the bis (gamma-triethoxysilylpropyl) tetrasulfide to the tetraethoxysilane 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 engineering exosome nano motor is applied to the field of biological medicine.
The invention relates to an application of an engineering exosome nano motor in preparing a medicament for treating cancer and cardiovascular and cerebrovascular diseases.
The present invention will for the first time bind exosomes to the NO-driven matrix by a specific method. The exosomes carry a plurality of active substances including DNA, RNA, protein, lipid, micromolecular metabolites and the like, so that special consideration needs to be carried out on reaction solvents, temperature and pH when the exosomes are engineered, the engineering modification of the exosomes is realized, the activity of the exosomes in the engineering process is ensured to be maintained, and the engineered exosomes cannot be obtained by a simple mixing method, and the combination is difficult.
Compared with a simple NO driven matrix, the nano motor provided by the invention not only has similar movement capability and physiological function of NO driven matrix, but also has the capability of targeting an inflammation part of an exosome, and also has the capability of repairing damage of the exosome.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
1. the engineering exosome nano motor is prepared from a surface thiolated zwitterionic polymer or bowl-shaped mesoporous silicon nano material by combining arginine as a Nitric Oxide (NO) driving matrix by a self-assembly or loading method, and is used for engineering modification of exosome. Meanwhile, the zwitterionic matrix is degraded under the action of reduced glutathione in the cellular environment, and can be removed from human bodies through the metabolism of livers and kidneys.
2. The preparation method is simple and efficient, the synthesis condition is mild, the material dispersion performance is good, the synthesized engineering exosome nano motor has excellent biocompatibility, the zwitterionic polymer has bionic physical and chemical properties of cell membranes, the non-characteristic protein adsorption/adhesion resistance effect is excellent, and the engineering exosome nano motor has low immunogenicity in vivo. The mesoporous silica has larger specific surface area and pore canal limiting effect, and can improve the cargo loading capacity and realize the controllable long-term release of the mesoporous silica. Meanwhile, L-arginine is a common amino acid molecule in vivo. Secondly, the reaction products of the nanomotor are respectively useful, no waste materials exist, and nitric oxide gas molecules of one of the catalytic products are signal molecules in the organism 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 focus positions, repairing damaged positions and the like, thereby being capable of being used as a good drug delivery carrier.
3. The method for preparing the engineering exosome nano motor combines the exosome and the NO driving matrix for the first time, can precisely target to a focus part by utilizing the exosome, and can target to specific inflammatory cells by utilizing the active motion capability of the NO driving matrix in inflammatory and active oxygen microenvironment, thereby realizing the step-by-step targeting effect and improving the treatment effect of the engineering exosome nano motor and ensuring that the pure exosome is used as a drug carrier to realize drug delivery in the depth of a patient.
Drawings
FIG. 1 is a transmission electron microscope image of mesenchymal stem cell exosomes obtained in example 1;
FIG. 2 is a transmission electron microscope image of the covalently bonded L-arginine zwitterionic polymer obtained in example 2;
FIG. 3 is a transmission electron microscope image of the engineered exosome nanomotor obtained in example 2;
FIG. 4 shows the surface potential of different materials during the construction 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 graph showing pore size distribution of the bowl-shaped mesoporous silica nanomaterial obtained in example 4;
FIG. 7 is a fluorescent plot of an engineered exosome nanomotor;
FIG. 8 is a MSD fitted curve of engineered exosome nanomotor motion;
FIG. 9 is a graph of example 2 at 5X 10 5 cell/mL inflammatory stimulus neural cell lower stem cell exosomes (a), covalently bonded L-arginine zwitterionic polymer (b), and engineered exosome nanomotors (c);
fig. 10 is a graph of cell uptake efficiency of exosomes and engineered exosome nanomotors.
Detailed Description
The invention is further illustrated by the following examples.
The experimental methods described in the examples, unless otherwise specified, are all conventional; the reagents and materials, unless otherwise specified, are commercially available.
Cells of the invention are commercially available. For example, human umbilical cord mesenchymal stem cells, neural cells SH-SY5Y and endothelial cells HUVECs are all available on the market, and the human umbilical cord mesenchymal stem cells of the invention can 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 10 5 Inoculating individual/mL human umbilical cord mesenchymal stem cells into T75 culture flask, adding complete culture solution to 10 mL/flask, and placing into CO 2 Culturing in an incubator at 37 ℃ for 48 hours; when the growth density of the cells in the culture flask is about 70%, the upper layer culture solution is discarded, and 10 mL/flask of exosome-free serum culture solution (containing 20ng/mL of vascular endothelial cell adhesion molecule 1 (VCAM-1)) is replaced; culturing at 37deg.C for 48 hr, collecting upper cell culture solution, and storing at-80deg.C;
(2) Obtaining exosomes by adopting a differential centrifugation method, centrifuging the cell culture solution at 2000rpm for 20min at 4 ℃, collecting the cell culture solution at the upper layer, and discarding dead cells at the lower layer; centrifuging the upper layer culture solution at 10000rpm for 30min at 4deg.C, collecting the upper layer cell culture solution, and discarding the lower layer cell fragments; centrifuging the upper layer culture solution at 100000rpm at 4deg.C for 120min, discarding the upper layer cell culture solution, collecting lower layer exosomes, dispersing in PBS (pH=7.5) and preserving at-80deg.C at concentration of 10 9 And each mL. As shown in fig. 1, the obtained human umbilical cord mesenchymal stem cell exosomes have a particle size of about 90nm, and are presented as dispersed and regular spherical nanoparticles (EXO).
Example 2
A method for preparing a zwitterionic polymer engineering exosome nano-motor with covalently bonded L-arginine, which comprises the following steps:
(1) 2. 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 10min. The ice-water bath was removed and the reaction was stirred at room temperature overnight. Precipitating the product with 400mL of acetone, slowly dripping acetone into the product, slightly sucking out the lower white emulsion by using a suction pipe after the solution is layered, centrifuging at 8000rpm for 10min, dissolving the obtained precipitate in a small amount of deionized water, precipitating in acetone again to obtain white precipitate, and vacuum drying at room temperature to obtain arginine monomer;
(2) Weighing 0.0624mmol of N, N' -bis (acryl) cystamine (cross-linking agent), adding 10.75mL of deionized water, dissolving by ultrasonic for 10min, adding into a reaction flask, and introducingCondensed water, N 2 Purifying for 30min under atmosphere. Weighing 0.0184mmol of azodiisobutylamidine hydrochloride (initiator), adding 0.5mL of deionized water for dissolution, injecting into the reaction flask by syringe, adding arginine monomer solution (0.252 mmol of arginine monomer obtained in step (1) is dispersed in 11mL of deionized water), adding the mixture into the flask, adding the mixture into the mixture, and adding the mixture into the mixture 2 The reaction is carried out for 1h at 140 ℃ under protection. The product was centrifuged (10000 rpm,10 min), precipitated and washed 3 times with water to give a zwitterionic polymer of covalently bound L-arginine. As shown in fig. 2, the synthesized zwitterionic polymer of covalently bonded L-arginine appears as dispersed and regular spherical nanoparticles (PMA);
(3) Blending the zwitterionic polymer of the covalent bonding L-arginine obtained in the step (2) with 1mL of 3-mercaptopropyl triethoxysilane for 24 hours at room temperature to obtain 10 9 Thiol-modified NO-driven matrix per mL, supernatant was discarded by centrifugation, and the solid product was dispersed in 0.5mL PBS (pH=7.5) buffer (PMA-SH, 2X 10) 9 0.5mg of water-soluble cross-linking agent 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt is added and mixed for 12h at 25 ℃, the product is centrifuged (10000 rpm,10 min), and the precipitate is washed 3 times with water to obtain a covalent bond L-arginine zwitterionic polymer (PMA-SMCC, 10) with the surface rich in hydroxysuccinimide groups 9 And (c) a).
(4) After the product of step (3) was ultrasonically dispersed in 0.5mL of PBS (ph=7.5), the human umbilical mesenchymal stem cell exosomes (0.5 mL, 10) obtained in example 1 were added 9 After 24h of blending at 4 ℃ and centrifuging (10000 rpm,10 min), taking precipitate and washing 3 times with PBS to obtain engineering exosome nano motor (PMA/EXO), dispersing in 1mL PBS with concentration of 10 9 And each mL. As shown in fig. 3, the prepared engineered exosome nanomotors were presented as dispersed peanut-like nanoparticles.
Example 3
Surface potential detection of engineering exosome nanomotor:
the human umbilical mesenchymal stem cell Exosomes (EXO) obtained in example 1, the stepwise surface-modified NO nanomotors (PMA-SH) and (PMA-SMCC) obtained in example 2, and the engineered exosome nanomotorsConfigured as 10 using PBS 7 And (3) adding the solution of the different materials into a potential pool, and measuring the surface potential of the different materials by using a nano potential analyzer. As shown in fig. 4, the potential of human umbilical cord mesenchymal stem cell Exosomes (EXO) in example 1 is negative because the cell membrane has electronegativity. Further, the surface of the zwitterionic Polymer (PMA) obtained in example 2 was rich in a large amount of carboxyl groups, so the surface potential of PMA was negative. Subsequently, negative sulfhydryl groups (PMA-SH) and maleimide groups (PMA-SMCC) are gradually modified to PMA, and the surface potential of the material is gradually reduced. Finally, the electronegativity of the engineered exosome nanomotors (PMA/EXO) obtained in example 2 was further reduced due to the electronegativity of EXO. The engineering exosome nano motor is formed by gradually modifying maleimide cross-linking agent by amphoteric ion radical NO nano motor and then combining with exosome. The electronegativity due to the specific functional groups on the surface of each substance is different. Therefore, the potential detection in the embodiment proves that each step of the preparation process of the engineering exosome nano motor is successfully synthesized.
Example 4
A preparation method of an L-arginine-loaded bowl-shaped mesoporous silicon nanomaterial engineering exosome nano motor comprises the following steps:
(1) 0.16g CTAB in 75mL H 2 O and 30mL of ethanol, after ultrasonic dispersion, adding 1mL (mass fraction 25%) of ammonia water; the mixed precursor solution (0.1 mL TESPTS with 0.25mL TEOS) was added and reacted at 35℃for 24h. Centrifuging and collecting the product, and cleaning the precipitate with ethanol and water for 3 times respectively; dispersing in 63mL NaOH (0.48M), etching at room temperature for 30min, centrifugally collecting the product, washing the precipitate with water for 3 times, and removing the template 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 appears as bowl-shaped nanoparticles with upward openings. In addition, the pore size distribution data of fig. 6 shows that the pore size of the prepared bowl-shaped mesoporous silicon nanomaterial is about 4nm.
(2) Dissolving 20mg of mesoporous silicon nanomaterial obtained in step (1) in 20mL of H 2 To the mixture of O, 50. Mu.L of aminopropyl triethoxysilane was addedThe alkane was mixed with 50. Mu.L of 3-mercaptopropyl triethoxysilane at 25℃for 24h. Centrifuging (10000 rpm,10 min) the product, precipitating, washing 3 times with water, dispersing in 5mL of deionized water again, mixing with 200mg of L-Arg at room temperature for 24h, centrifuging the product, discarding the supernatant, washing 3 times with water to obtain the bowl-shaped mesoporous silicon nanomaterial loaded with L-arginine, namely the NO-driven matrix with thiolated surface;
(3) Dispersing the bowl-shaped mesoporous silica nanomaterial loaded with L-arginine in step (2) in 0.5mL PBS (pH=7.5) buffer (concentration of 0.5X10) 9 0.5mg of 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt is added and mixed for 12 hours at room temperature, the product is centrifuged (10000 rpm,10 min), the precipitate is washed with water for 3 times, and the bowl-shaped mesoporous silica nanomaterial (10) with the L-arginine loaded and rich in hydroxysuccinimide groups on the surface is obtained 9 And (c) a).
(4) After the product of step (3) was ultrasonically dispersed in 0.5mL of PBS (ph=7.5), the human umbilical mesenchymal stem cell exosomes (0.5 mL, 10) obtained in example 1 were added 9 individual/mL) and then blending for 24 hours at 4 ℃, centrifuging (10000 rpm,10 min) the product, taking the precipitate and washing with PBS for 3 times to obtain the engineering exosome nanomotor, dispersing in 1mL PBS (ph=7.5) with a concentration of 10 9 And each mL. The nanomotor prepared in this example is presented as peanut-shaped nanoparticles formed by combining mesoporous silicon nanomaterial with spherical exosomes.
Example 5
Fluorescence labeling characterization of engineered exosome nanomotors:
(1) 0.5mL of human umbilical cord mesenchymal stem cell exosomes obtained in example 1 (10 9 And 0.5mL of cell membrane dye DiO (20 mu M), dyeing for 10min at room temperature in a dark place, and centrifuging (100000 rpm,120 min) at 4 ℃ to obtain DiO fluorescent-labeled human umbilical mesenchymal stem cell exosomes.
(2) Mixing 0.5mg of the zwitterionic polymer of the covalent bonding L-arginine of the final product of the step (2) of the example 2 and 0.5mg of the mesoporous silicon nanomaterial loaded with L-arginine of the final product of the step (3) of the example 4 with 1mL of 3-mercaptopropyltriethoxysilane at room temperature for 24 hours, discarding the supernatant, centrifuging and dispersing the product in 0.5mL of PBS (pH=7.5) buffer solution, adding 0.5mg of sodium salt of sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate and 10 mu L of Cy 5-maleimide (1 mg/mL) at room temperature, mixing for 12 hours, centrifuging the product (10000 rpm,10 min), precipitating and washing 3 times with water to obtain Cy5 fluorescent-labeled zwitterionic polymer nanoparticles of covalent bonding L-arginine and Cy5 fluorescent-labeled mesoporous silicon nanomaterial loaded with L-arginine;
(3) The DiO fluorescent-marked exosome obtained in the step (1) and the Cy5 fluorescent-marked covalent bonding L-arginine zwitterionic polymer or the mesoporous silicon nanomotor loaded with L-arginine obtained in the step (2) are mixed according to the number ratio of 1:1 were blended for 24h at 4 ℃, the product was centrifuged (10000 rpm,10 min) and the pellet was washed 3 times with PBS to give two fluorescence labelled engineered exosome nanomotors.
(4) Dispersing the two fluorescence-labeled engineering exosome nanomotors obtained in the step (3) in PBS buffer solution (concentration is 10 9 and/mL), was added dropwise to a 14mm cell culture dish containing a PBS solution in a volume of 1mL, and the binding of the fluorescence-labeled engineered exosome nanomotor was observed immediately using a fluorescence microscope. As shown in fig. 7, the red fluorescent-labeled nanoparticles and the green fluorescent-labeled exosomes in the fluorescent-labeled covalently-bonded L-arginine zwitterionic polymer-engineered exosome nanomotors are better overlapped, indicating successful preparation of the engineered exosome nanomotors; the red fluorescent-labeled nanoparticles and the green fluorescent-labeled exosomes in the fluorescent-labeled L-arginine-loaded bowl-shaped mesoporous silicon nanomaterial-engineered exosome nanomotor also show the overlapping result.
Example 6
Engineered exosome nanomotor at 5 x 10 5 cell/mL density inflammation stimulates motor performance studies in the neuronal (or endothelial) environment:
(1) The neural cells SH-SY5Y or (endothelial cells HUVECs) are used for 5×10 5 Inoculating the cell/mL into a 14mm cell culture dish, placing the cell culture dish in a constant temperature incubator at 37 ℃ until the volume of the complete culture medium is 1mL, and adhering cells to walls after 24 hours; adding inflammatory stimulus factor (0.1 mM lipopolysaccharide), and stimulating 24h；
(2) 10. Mu.L of mesenchymal stem cell exosomes (10) in example 1 were taken 9 personal/mL, PBS), the final product of step (2) of example 2, a zwitterionic polymer of covalently bonded L-arginine (10) 9 personal/mL, PBS), mesoporous silica nanomaterial loaded with L-arginine as the final product of step (3) of example 4 (10) 9 personal/mL, PBS), engineered exosome nanomotors prepared in examples 2 and 4 (10 9 And (3) preparing the nano motor by using a fluorescence microscope, and immediately observing and recording the movement condition of the nano motor in a cell environment by directly adding the nano motor into the culture medium containing 1mL of the culture medium attached to a cell culture dish.
(3) Analysis of the locomotor behavior of umbilical mesenchymal stem cell exosomes according to example 1, the zwitterionic polymer of the covalent bonding of L-arginine of example 2, the engineered exosome nanomotors of example 2 in a cellular environment. As shown in fig. 8, the motion track of the engineering exosome nanomotor in the cellular environment conforms to a quadratic function, which indicates that the motion behavior of the engineering exosome nanomotor belongs to autonomous motion. In addition, the movement speeds of exosomes, zwitterionic polymers covalently bonded to 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 simple exosome, the movement speed of the zwitterionic group nano motor with autonomous movement capability is obviously improved. The movement speed of the engineering exosome nano motor is reduced, probably because the gravity of the material is increased, and the engineering exosome nano motor is obviously faster than that of a single exosome.
The results of the movement track of the umbilical mesenchymal stem cell exosomes in example 1 and the L-arginine-loaded mesoporous silicon nanomaterial and the engineered exosome nanomotor in example 4 in the cellular environment also show that the movement speed of the engineered exosome nanomotor is reduced compared with the movement speed of the L-arginine-loaded mesoporous silicon nanomaterial, probably because the gravity of the material itself is increased after the exosome is loaded, resulting in a slower movement speed, but also significantly faster than the simple exosome.
The data above demonstrate that the engineered exosome nanomotor motion profile is in inflammatory stimulated cells (microenvironment contains higher concentration of active oxygen), which have better autonomous motion capability.
Example 7
Targeted performance research of engineering exosome nanomotors in inflammatory cell models:
(1) mu.L of SH-SY5Y cells (cell density 1X 10) 5 individual/mL) was inoculated into 6-well plates and incubated overnight at 37 ℃; subsequent addition of H 2 O 2 The final concentration was set at 0.1mM, and the culture was continued for 24 hours; subsequently, 10. Mu.L of DiO fluorescent-labeled exosomes (10) of example 5 were added to the well plate, respectively 9 personal/mL), cy5 fluorescent-labeled zwitterionic polymer covalently bound to L-arginine (10) 9 personal/mL) and fluorescent-labeled engineered nanomotors (10) 9 Number of cells/mL), culturing for 24 hours;
(2) Collecting all supernatant in the pore plate into a centrifuge tube, cleaning three times by using 0.1mL of PBS, collecting washing liquid into the centrifuge tube, and fixing the volume to 1mL by using the PBS to obtain culture supernatant; adding 0.1mL pancreatin, and digesting at 37 ℃ for 10min; then 0.5mL PBS was added to the well plate, and the cell suspension was transferred to a new centrifuge tube for centrifugation (1000 rpm,5 min); after centrifugation, the supernatant was discarded, and 0.1mL of the cell lysate was added, and the volume was fixed to 1mL with PBS to obtain a cell lysate.
(3) The fluorescence intensities of the culture supernatant and the cell fluid were detected using fluorescence spectrophotometers, respectively, and the cell uptake efficiency of the different samples was calculated using the formula cell uptake efficiency (%) = fluorescence intensity of the cell fluid/(fluorescence intensity of the cell fluid + fluorescence intensity of the culture supernatant) ×100%. As shown in fig. 10, the cell uptake efficiency of PMA/EXO is significantly improved compared with that of exosome EXO alone, which indicates that the autonomous movement-based nanomotor utilizes cellular inflammation microenvironment to better achieve targeting and aggregation, and ensure that exosome alone achieves drug delivery deep in patients as a drug carrier.
Example 8
Example 8 was prepared in the same manner as example 2, except that: the molar ratio of the arginine monomer to the cross-linking agent is 1, the molar ratio of the initiator to the cross-linking 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 subjected to the thiolated surface modification is 10 5 The dosage of 3-mercaptopropyl triethoxysilane is 0.1mL, the reaction temperature is 10 ℃, and the reaction time is 48h; concentration of NO-driven matrix rich in thiol groups of 10 5 The concentration of the water-soluble cross-linking agent is 0.1mg/mL, the reaction temperature is 10 ℃, and the reaction time is 48 hours; the number ratio of the nitric oxide driving matrix with the surface rich in hydroxysuccinimide groups to the exosomes is 10/1, the reaction temperature is 0 ℃, and the reaction time is 24 hours.
Example 9
Example 9 was prepared in the same manner as example 2, except that: the molar ratio of the arginine monomer to the cross-linking agent is 20, the molar ratio of the initiator to the cross-linking agent is 1, the reaction time is about 5 hours, and the reaction temperature is 50 ℃; the concentration of the nitric oxide driving matrix subjected to the thiolated surface modification is 10 12 The dosage of 3-mercaptopropyl triethoxysilane is 10mL, the reaction temperature is 50 ℃, and the reaction time is 10h; concentration of NO-driven matrix rich in thiol groups of 10 12 The concentration of the water-soluble cross-linking agent is 10mg/mL, the reaction temperature is 50 ℃, and the reaction time is 10h; the number ratio of the nitric oxide driving matrix with the surface rich in hydroxysuccinimide groups to the exosomes is 1/10, the reaction temperature is 10 ℃, and the reaction time is 1h.
Example 10
Example 10 was prepared in the same manner as example 4, except that: CTAB mass is 0.1g, and the volume ratio of the bis (gamma-triethoxysilylpropyl) tetrasulfide to the tetraethoxysilane is 1:1, the reaction time is 12 hours, 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 The method comprises the steps of carrying out a first treatment on the surface of the The mass of the surface-modified bowl-shaped mesoporous silicon nanomaterial subjected to mercaptopropyl is 1mg, the dosage of 3-mercaptopropyl triethoxysilane is 0.01mL, the dosage of aminopropyl triethoxysilane is 0.01mL, the reaction temperature is 10 ℃, and the reaction time is 48h.
Example 11
Example 11 the same procedure was used as in example 4, except thatIn the following steps: CTAB mass is 0.5g, and the volume ratio of the bis (gamma-triethoxysilylpropyl) tetrasulfide to the tetraethoxysilane is 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 The method comprises the steps of carrying out a first treatment on the surface of the The mass of the surface-modified bowl-shaped mesoporous silicon nanomaterial subjected to mercaptopropyl is 50mg, the dosage of 3-mercaptopropyl triethoxysilane is 1mL, the dosage of aminopropyl triethoxysilane is 1mL, the reaction temperature is 50 ℃, and the reaction time is 10h.

Claims (8)

1. An engineering exosome nano motor is characterized in that the engineering exosome nano motor is formed by surface modification of a surface thiolated nitric oxide driving matrix through a water-soluble cross-linking agent; the nitric oxide driving matrix is a zwitterionic polymer with covalent bonding of L-arginine or a bowl-shaped mesoporous silicon nanomaterial loaded with L-arginine; the water-soluble cross-linking agent is 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt.
2. The engineered exosome nanomotor of claim 1, wherein the source of exosomes comprises stem cells or macrophages.
3. A method of preparing an engineered exosome nanomotor according to claim 1, comprising the steps of:
(1) Performing thiolated surface modification on the nitric oxide driving matrix to obtain a NO driving matrix rich in sulfhydryl groups;
(2) Reacting the nitric oxide driving matrix rich in sulfhydryl groups 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) Mixing the nitric oxide driving matrix with the surface rich in hydroxysuccinimide groups obtained in the step (2) with an exosome at a low temperature, and separating and purifying to obtain an engineering exosome nano motor;
the nitric oxide driving matrix is a zwitterionic polymer of which the surface is rich in hydroxysuccinimide groups and is covalently bonded with L-arginine or a bowl-shaped mesoporous silicon nanomaterial of which the surface is rich in hydroxysuccinimide groups and is loaded with L-arginine; the water-soluble cross-linking agent is 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt.
4. The method of claim 3, wherein the nitric oxide driving matrix of step (1) is a zwitterionic polymer of covalently bonded L-arginine, 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 separating and purifying to obtain the covalent bonding L-arginine zwitterionic polymer.
5. The preparation method of claim 3, wherein the nitric oxide driving matrix in the step (1) is a bowl-shaped mesoporous silicon nanomaterial loaded with L-arginine, and the preparation process comprises the following steps: adding bis (gamma-triethoxysilylpropyl) tetrasulfide and tetraethoxysilane into a mixed system taking CTAB as a template agent, strong ammonia water as a catalyst and ethanol as a cosolvent, and forming a bowl-shaped mesoporous silicon nanomaterial under the action of sodium hydroxide; and forming the bowl-shaped mesoporous silicon nanomaterial loaded with L-arginine in the high-concentration arginine solution.
6. The method according to claim 3, wherein when the nitric oxide-driven matrix is a zwitterionic polymer of covalently bonded L-arginine, the concentration of the nitric oxide-driven matrix subjected to the thiolation surface modification in step (1) is 10 5 -10 12 The dosage of 3-mercaptopropyl triethoxysilane is 0.1-10mL, the reaction temperature is 10-50 ℃ and the reaction time is 10-48h; when the nitric oxide driving matrix isWhen the bowl-shaped mesoporous silicon nanomaterial loaded with L-arginine is prepared, the mass of the bowl-shaped mesoporous silicon nanomaterial subjected to the mercaptopropyl surface modification in the step (1) is 1-50mg, the dosage of 3-mercaptopropyl triethoxysilane is 0.01-1mL, the dosage of aminopropyl triethoxysilane is 0.01-1mL, the reaction temperature is 10-50 ℃, and the reaction time is 10-48h; the concentration of NO-driven matrix rich in sulfhydryl groups in step (2) is 10 5 -10 12 The concentration of the water-soluble cross-linking agent is 0.1-10mg/mL, the reaction temperature is 10-50 ℃, and the reaction time is 10-48h; the ratio of the number of the nitric oxide driving matrix with the surface rich in the hydroxysuccinimide groups to the number of the exosomes in the step (3) is 10/1-1/10, the reaction temperature is 0-10 ℃, and the reaction time is 1-24 h.
7. The method of claim 4, wherein the arginine monomer is present in a molar ratio of 1 to 20, the initiator is present in a molar ratio of 0.1 to 1, and the crosslinker is present in a molar ratio of about 0.5 to 5h, and the reaction temperature is 50 to 350 ℃.
8. The method according to claim 5, wherein the volume ratio of bis (γ -triethoxysilylpropyl) tetrasulfide to tetraethyl orthosilicate is 1:1-1:10, the reaction time is 12h-36h, 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 。
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