CN113402661B

CN113402661B - Zwitterionic polymer-based nitric oxide-driven nano motor and preparation method and application thereof

Info

Publication number: CN113402661B
Application number: CN202110503975.6A
Authority: CN
Inventors: 毛春; 万密密; 陈焕; 陶莹芳; 陈城龙
Original assignee: Nanjing Normal University
Current assignee: Nanjing Normal University
Priority date: 2021-05-08
Filing date: 2021-05-08
Publication date: 2022-07-26
Anticipated expiration: 2041-05-08
Also published as: CN113402661A

Abstract

The invention discloses a preparation method and application of a zwitterionic polymer-based Nitric Oxide (NO) driven nano motor. The nanomotor disclosed by the invention can respond to the ROS concentration gradient to generate chemotactic behavior, and reacts with ROS in the nanomotor under the environment of tumor cells to generate NO to drive the NO to move and gradually degrade. The preparation method of the zwitterionic polymer-based NO driving nano motor is simple and efficient, has excellent biocompatibility, has active movement capability in a ROS microenvironment, and has a wide application prospect in the field of biomedicine.

Description

Zwitterionic polymer-based nitric oxide-driven nano motor and preparation method and application thereof

Technical Field

The invention belongs to a novel biomedical nano material, and particularly relates to a zwitterionic polymer based Nitric Oxide (NO) driven nano motor and a preparation method and application thereof.

Background

NO is a key signaling molecule in the cardiovascular, immune and central nervous systems and shows versatility in biomedical applications, such as playing a prominent role in regulating vasodilation, angiogenesis, chemosensitization, bactericidal and cancer-related physiological/pathophysiological activities. However, NO delivery techniques are still severely limited due to the physiological properties of NO, such as short half-life (<5s), short diffusion distance (20-160 μm), high reactivity with oxygen species, and concentration-dependent therapeutic outcome. To address these problems, a variety of NO-donor-based delivery systems have been developed for various NO-delivery platforms for cancer therapy, antimicrobial and cardiovascular therapy. Currently small molecule NO donors do not address the problem of sustained NO release. In the prior art, a post-load type NO driving nano motor with autonomous movement capability is constructed by utilizing a polymer and L-arginine electrostatic combination mode, and the problem of continuous NO release is solved, but the method of combining the polymer and the L-arginine by weak electrostatic action may face the problems of low load efficiency of L-arginine (fuel) and unstable load and possible shedding in the nano motor driving process, so that the release period and the release amount are not satisfactory. There is therefore a need to develop new types of NO-driven nanomotors with higher, high sustained NO release capabilities.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a zwitterionic polymer based Nitric Oxide (NO) driven nano motor, which mainly solves the problems that the existing NO driven nano motor is low in fuel load efficiency and the load is unstable and can fall off in the nano motor driving process, so that the release period and the release amount of NO are improved.

The invention also provides a preparation method and application of the zwitterionic polymer based Nitric Oxide (NO) driven nano motor.

The technical scheme is as follows: in order to achieve the purpose, the invention provides a zwitterionic polymer based Nitric Oxide (NO) driven nano motor which is formed by initiating a free radical polymerization reaction through an initiator by taking an L-arginine derivative as a monomer and a diselenide compound as a cross-linking agent; the L-arginine derivative is a compound with a carbon-carbon double bond at the chain end of a high-density active guanidyl crown functional group; the diselenide compound is a Reactive Oxygen Species (ROS) -responsive diselenide compound; the nanomotors can produce chemotactic behavior in response to ROS concentration gradients.

Wherein the structural formulas of the L-arginine derivative and the diselenide compound are respectively shown as follows:

preferably, the initiator is one of azobisisobutyronitrile, azobisisoheptonitrile or dimethyl azobisisobutyrate.

The preparation method of the zwitterionic polymer based Nitric Oxide (NO) driven nanomotor comprises the following steps:

(1) dissolving L-arginine in a mixed solvent of deionized water and 1, 4-dioxane, adding triethylamine, cooling the solution by using an ice-water bath, dropwise adding methacrylic anhydride while stirring, removing the ice-water bath, stirring for reaction, and precipitating a product in acetone to obtain an L-arginine derivative;

(2) adding 2,2' -diselenide diethyl bis (1-ethylamine) dihydrochloride and triethylamine into an anhydrous dichloromethane solution, and slowly adding methacryloyl chloride; then stirring the reaction mixture for reaction under the nitrogen atmosphere; extracting and drying the reaction system, and removing an organic phase; purifying to obtain a diselenide compound cross-linking agent with ROS response type;

(3) dissolving the L-arginine derivative obtained in the step (1) and the diselenide compound cross-linking agent with ROS response type obtained in the step (2) in a solvent, adding an initiator to perform polymerization reaction, and performing centrifugal washing to obtain the zwitterionic polymer-based NO driving nano motor.

Wherein the molar ratio of the L-arginine to the methacrylic anhydride in the step (1) is 0.5-1, the volume ratio of the deionized water to the 1, 4-dioxane is 1-5, the volume ratio of the 1, 4-dioxane to the triethylamine is 1-5, the stirring reaction temperature is 20-30 ℃, and the reaction time is 10-48 h.

Preferably, the molar ratio of the L-arginine to the methacrylic anhydride is 0.6; the volume ratio of the deionized water to the 1, 4-dioxane is 2.4; the volume ratio of the 1, 4-dioxane to the triethylamine is 1.9; the reaction temperature is 25 ℃, and the reaction time is 12 h.

Wherein the molar ratio of the 2,2' -diselenodiethylbis (1-ethylamine) dihydrochloride to the methacryloyl chloride in the step (2) is 0.1-0.5, the stirring reaction temperature is 0-30 ℃, and the reaction time is 10-48 h;

wherein the volume of the anhydrous dichloromethane is 30-100mL, and the dosage of the triethylamine is 1-5 mL.

Preferably, the molar ratio of 2, 2-diselenediylbis (1-ethylamine) dihydrochloride to methacryloyl chloride is 0.25; the volume of the anhydrous dichloromethane is 60 mL; the dosage of triethylamine is 3.6 mL; the reaction temperature is 25 ℃, and the reaction time is 24 h.

Further, the reaction system in the step (2) is extracted by deionized water to remove impurities, namely anhydrous Na ₂ SO ₄ Drying overnight, removing an organic phase by reduced pressure evaporation, and purifying by silica gel column chromatography with ethyl acetate/petroleum ether as a raw material to obtain the ROS-responsive diselenide compound crosslinking agent.

Preferably, the ratio of the purified separation solvent is ethyl acetate/petroleum ether volume ratio of 1:2, 1:1, 2: 1.

Wherein, in the step (3), the mol ratio of the L-arginine zwitterionic derivative to the diselenide compound cross-linking agent is 5-20, the mol ratio of the L-arginine zwitterionic derivative to the initiator is 1-20, the reacted solvent is one of acetonitrile or dimethyl sulfoxide, the dosage of the solvent is generally 10-50mL, the reaction temperature is 80-100 ℃, and the reaction time is 1-5 h.

Preferably, the molar ratio of the L-arginine zwitterion derivative to the diselenide compound cross-linking agent is 10; the molar ratio of the L-arginine zwitterionic derivative to the initiator is 16.7; the solvent of the reaction is dimethyl sulfoxide; the dosage of the solvent is 20 mL; the initiator is azobisisobutyronitrile; the reaction temperature is 90 ℃ and the reaction time is 4 h.

Wherein, the centrifugal washing in the step (3) is collecting the obtained nanogel through ultracentrifugation, washing and freeze-drying to obtain the zwitterionic polymer-based NO driving nano motor, the rotation speed of the ultracentrifugation is 10000-12000rpm, and the centrifugation time is 10-30 min.

Preferably, the ultracentrifugation is performed at 12000rpm for 10 min.

The invention relates to application of a zwitterionic polymer based Nitric Oxide (NO) driven nano motor in biomedical fields such as treatment of cardiovascular and cerebrovascular diseases, cancer and the like.

The invention relates to application of a zwitterionic polymer-based Nitric Oxide (NO) driven nano motor in preparation of a medicine for targeting cardiovascular diseases and tumor focuses. The nano motor can respond to ROS concentration gradient in a microenvironment and embody chemotactic behavior, and is expected to achieve the purpose of targeting diseases including cardiovascular diseases and tumors through microenvironment targeting.

The zwitterionic polymer based Nitric Oxide (NO) driven nano motor prepared by the invention is a polymer polymerized by taking an L-arginine derivative as a monomer, has high-density active guanidyl sites, and can efficiently react with ROS in a tumor microenvironment to generate NO which is used as a fuel for driving the motor to move and a therapeutic agent for playing a therapeutic role; and can respond to the concentration gradient of ROS to generate chemotactic behavior, namely, the nanomotor can generate self-diffusion electrophoresis movement towards a tumor part or an inflammation part with higher concentration of ROS. The nano motor can be broken and degraded in the presence of ROS due to the diselenide ether bond in the structure, and the nano motor can be broken into low molecular segments and can be eliminated by a human body through the metabolism of each organ.

In the invention, the nano motor is added into the culture environment of cancer cells, and the movement condition of the nano motor is tracked by using a laser confocal microscope and a fluorescence microscope; the effective demonstration is that: the nano motor can respond to the concentration gradient of ROS to generate chemotactic behavior, reacts with ROS in the tumor cell environment to generate NO to drive the tumor cell to move and gradually degrade, has excellent biocompatibility, has active movement capability in the ROS microenvironment, and has wide application prospect in the field of biomedicine.

The invention synthesizes a novel NO driving nano motor with high-density guanidyl active sites by a method of free radical polymerization and covalent grafting. Wherein, the monomer is the L-arginine derivative of the zwitterionic polymer group, so the monomer has good biocompatibility; the cross-linking agent is a diselenide compound with Reactive Oxygen Species (ROS) response type, so the cross-linking agent has response degradation performance, and the invention synthesizes a brand new NO driving nano motor by using the L-arginine derivative and the diselenide compound for the first time. Compared with electrostatic combination, the nano motor prepared by adopting the covalent combination mode has more abundant active guanidine-based sites capable of releasing NO, so that the nano motor has the performance of releasing NO in a more lasting and larger amount. Eventually providing faster motion speed to the nanomotor.

The invention adopts a covalent grafting method to prepare a nano particle with rich active guanidyl sites on the surface, but not a micromolecular NO donor, so the nano particle has longer NO release capacity than the donor. In addition, the monomers used for synthesizing the nano-motor are zwitterionic polymer-based L-arginine derivatives, so that the nano-motor has unique good biocompatibility of zwitterions, and the risk of rapid clearance, degradation and even acute poisoning in vivo can be avoided. The synthesized nano motor is prepared by combining free polymerization and covalent bonds, and has high-density guanidine active sites (sites for generating NO), so that the performance of generating NO is more stable and durable, a theoretical basis is provided for the nano motor to react with ROS in a tumor microenvironment to continuously release NO, and the problems of short half-life period and short release time of NO are solved.

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

1. according to the NO driving nano motor based on the zwitterionic polymer base, the material main body of the nano motor is an L-arginine derivative, so that the nano motor has good biocompatibility and dense guanidyl active sites; the diselenide compound with Reactive Oxygen Species (ROS) response type is a cross-linking agent, so that the diselenide compound has response degradation performance. Due to the fact that the high-density guanidino active sites provide a theoretical basis for the guanidino active sites to react with ROS in a tumor microenvironment to continuously release NO, the problems of short half-life period and short release time of the NO are solved. In addition, NO can be used as a driving force for driving the nano motor to move, and can also be used as a therapeutic agent for treating tumors.

2. The preparation method is simple and efficient, the synthesis conditions are mild, the material dispersion performance is good, and the synthesized nano motor has the following characteristics: (1) excellent biocompatibility, the zwitterionic polymer matrix material has low immunogenicity, which is beneficial to maintaining the bioavailability and the treatment effect of the material in the long-term administration process, and inhibits the activation of phagocytes by reducing protein endocytosis, interfering protein hydrolysis, physically blocking MHCII-epitope combination and the like, thereby weakening the immune response; (2) degrading the performance of nanomotors as required, the Se-Se bond has low energy, reacts even under mild stimuli, is particularly sensitive to reductive or oxidative stimuli, and can be cleaved in the presence of active oxygen, triggering the disintegration of the nanogels. The small molecular fragment can be metabolized by human organs and discharged out of the body, and has good biological safety.

3. The zwitterionic polymer-based Nitric Oxide (NO) driven nano motor prepared by the invention has the advantages of long NO release time, large release amount and the like, has spontaneous chemotactic performance, and can generate self-diffusion swimming movement and tumor killing performance towards a tumor part or an inflammation part with higher ROS concentration.

Drawings

FIG. 1 is a nuclear magnetic spectrum of a derivative of L-arginine of example 1;

FIG. 2 is a nuclear magnetic spectrum of the diselenide ether crosslinker of example 2;

FIG. 3 is a transmission electron micrograph (scale: 500nm) of the zwitterionic NO-driven nanomotor obtained in example 3;

FIG. 4 is a transmission electron micrograph of PAMSe in example 4 after incubation at 37 ℃ for 12h,24h and 48h in 0.002% hydrogen peroxide solution;

FIG. 5 is a graph showing the NO release amount of the nanomotor in the cancer cell environment in example 5;

FIG. 6 is the movement trace of the nanomotor in the cancer cell environment in example 6;

FIG. 7 is a Mean Square Displacement (MSD) quadratic fit curve of the nanomotor motion trajectory in the cancer cell environment of example 6;

FIG. 8 is a linear fit curve of Mean Square Displacement (MSD) of the nano-motor motion trajectory in the cancer cell environment in example 6;

FIG. 9 is a model of the nanomotor ROS concentration chemotaxis assay of example 7;

FIG. 10 is the fluorescence intensity of nanomotors in example 7 at b-terminal in chemotaxis model with/without ROS gradient

FIG. 11 is a graph showing the cell activity of the nanomotors incubated with endothelial cells at different concentrations for different periods of time in example 8 to evaluate the biocompatibility of the nanomotors;

FIG. 12 is the cell activity of the degraded nanomotor of example 8 after incubation with cancer cells for various times to evaluate the biocompatibility of the nanomotor;

FIG. 13 is an evaluation of the killing performance of the nanomotor on cancer cells in example 9;

FIG. 14 is a transmission electron micrograph of the nanomotor prepared in comparative example 1 (lower right angle scale: 500 nm);

FIG. 15 shows NO release amount of the supported PMSE/A nanomotor of comparative example 1 under a cell environment;

FIG. 16 is a graph of the movement of the supported PMSE/A nanomotor of comparative example 1 in a cellular environment;

fig. 17 is a MSD quadratic fit curve of the moving trajectory of the supported PMSe/a nanomotor of comparative example 1 in a cancer cell environment.

Detailed Description

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

Example 1

Preparation of L-arginine derivative:

(1) weighing 11.5mmol L-arginine, dissolving in a mixed solvent of deionized water (20mL) and 1, 4-dioxane (8.5mL), ultrasonically dispersing, and magnetically stirring at room temperature;

(2) adding 4.5mL of triethylamine into the reaction system in the step (1), and cooling the solution to 0 ℃ by using an ice water bath;

(3) adding 18.9mmol of methacrylic anhydride dropwise under stirring, then removing the ice water bath, and stirring for reaction, wherein the reaction system is 25 ℃, and the reaction time is 12 h.

(4) The solution obtained in step (3) is added dropwise to 400mL of acetone for precipitation, then the precipitate is re-dissolved in water and precipitated again in acetone, and the precipitation step is repeated twice to dissolve in water.

(5) Centrifuging the solution in (4) at 8000rpm for 15min, discarding the upper liquid layer, taking the lower layer precipitate, and vacuum drying at 60 deg.C to obtain L-arginine derivative: n- (2-methyl-1-OXO-2-propen-1-yl) -L-arginine.

FIG. 1 is a nuclear magnetic spectrum of N- (2-methyl-1-OXO-2-propen-1-yl) -L-arginine, ¹ H NMR(400MHz,D ₂ o) δ 5.61(s,1H),5.35(s,1H),4.13(dd, J ═ 8.0,5.1Hz,1H),3.09(t, J ═ 6.8Hz,2H),1.83(s,3H), 1.81-1.59 (m,2H), 1.56-1.46 (m, 2H). As a result, it was confirmed that the L-arginine derivative was successfully produced.

Example 2

Synthesis of diselenide compound cross-linking agent with Reactive Oxygen Species (ROS) -responsive type:

(1) 1.0g of 2,2' -diselenediylbis (1-ethylamine) dihydrochloride (CAS number 3542-13-0) and 3.6mL of triethylamine are weighed into 60mL of dichloromethane, and the mixture is cooled to 0 ℃.

(2) To the solution obtained in step (1), 1.3g of methacryloyl chloride was slowly added, followed by a reaction at a reaction temperature of 25 ℃ for 24 hours in a nitrogen atmosphere.

(3) And (3) extracting the solution reacted in the step (2) by using a large amount of deionized water to remove impurities, and collecting an organic solvent phase.

(4) The organic phase collected in step (3) was dried over anhydrous sodium sulfate overnight to give a crude product, and the organic phase was removed by evaporation under reduced pressure.

(5) And (3) respectively taking ethyl acetate/petroleum ether (volume ratio is 1:2, 1:1 and 2:1) with different proportions as raw materials for the crude product obtained in the step (4), and separating and purifying the raw materials through a silica gel column to obtain a product, namely light yellow solid powder and a diselenide compound with Reactive Oxygen Species (ROS) response type.

FIG. 2 is a nuclear magnetic spectrum of a synthesized diselenide compound having a Reactive Oxygen Species (ROS) response pattern. ¹ HNMR(400MHz,CDCl ₃ ) δ 6.54(s,2H),5.77(s,2H),5.43-5.34(m,2H),3.70(q, J ═ 6.5Hz,4H),3.16-3.04(m,4H),2.03-1.94(m,6H),1.68(s, 4H). The successful synthesis of a diselenide compound with Reactive Oxygen Species (ROS) responsiveness can be seen in fig. 2.

Example 3

Preparation of the NO-driven nanomotor based on the zwitterionic polymer group:

(1) weighing 0.5mmol of L-arginine derivative and 0.05mmol of active oxygen response type diselenide ether cross-linking agent in a three-neck flask;

(2) removing air in the flask in the step (1) by using a vacuum pump, and continuously introducing nitrogen;

(3) adding 20mL of dry dimethyl sulfoxide solution into the step (2), and performing ultrasonic dispersion and dissolution;

(4) weighing 0.03mmol of azodiisobutyronitrile, adding 500 mu L of dry dimethyl sulfoxide solution, performing ultrasonic dispersion and dissolution, and injecting the syringe into the reaction system in the step (3);

(5) transferring the three-neck flask into an oil bath kettle, magnetically stirring at 300rpm, and polymerizing at the reaction temperature of 90 ℃ for 4 hours in a nitrogen atmosphere to obtain a milky uniformly dispersed zwitterion nitric oxide nano motor solution;

(6) the obtained zwitterion-based NO-driven nano motor solution is placed in a high-speed centrifuge to be centrifuged at 12000rpm for 10min, the upper layer liquid is discarded, and the precipitate is repeatedly washed with deionized water for 3 times. And (4) freeze-drying the finally obtained precipitate in a freeze-dryer to obtain the final zwitterion-based nitric oxide nanomotor (PAMSe). As shown in fig. 3, the synthesized nanomotor has a particle size of about 230nm and appears as uniformly dispersed and irregular quasi-spherical nanoparticles.

Example 4

Zwitterionic NO drives degradation of the nanomotors:

(1) the PAMSe nanomotor prepared in example 3 was weighed and dispersed in 1mL of 0.002% hydrogen oxide solution to a final concentration of 200. mu.g mL ^-1 And reacting in a water bath environment at 37 ℃, and observing the morphology change of the nano motor by using a transmission electron microscope in 12h,24h and 48h respectively. As shown in FIG. 4, it can be seen that the PAMSe nanomotor is associated with H ₂ O ₂ (0.002%) incubation time was prolonged, the size of the nanoparticles gradually increased until finally the spheroidal particle structure completely collapsed and degraded, indicating that the PAMSe nanomotor of the present invention can be degraded in response in the ROS environment.

Example 5

Zwitterionic NO-driven nanomotor release of NO:

(1) 3mg of the zwitterionic-based NO nanomotor prepared in example 3 was added to MCF-7 cells seeded in 24-well plates, respectively. Cell seeding density of 5 x 10 ⁶ cell/well, complete medium volume of 1mL, cell 37 degrees C after 24h incubation and adding the nanometer motor.

(2) The cells were incubated with zwitter-ion based nitric oxide nanomotors (0, 1h, 3h, 6h, 9h, 12h,24h, 36h, 72h, 96h and 108h), respectively. After the reaction, the mixture was centrifuged at 12000rpm for 10min, and the supernatant was collected for further use.

(3) The obtained supernatants were each tested for the amount of NO produced using a nitric oxide kit (Biyun day). As shown in fig. 5, the nano-motor NO release amount is continuously increased with the time extension in the time range set by the experiment, i.e. the nano-motor can continuously release NO 96h, and the release amount is 4.7 μ M. The nano motor NO of the invention is proved to have long release time and high release amount.

Example 6

The zwitterionic NO drives the movement performance of the nano motor in the cancer cell environment to study:

(1) weighing 1mg of the nano motor prepared in the embodiment 3, adding 5mL of deionized water, and performing ultrasonic treatment for 20min to fully disperse the solution to obtain a uniformly dispersed nano motor solution of 200 mug/mL;

(2) breast cancer cells MCF-7 with 5 x 10 ⁵ Inoculating the cell/mL into a 14mm cell culture dish, wherein the volume of a complete culture medium is 1mL, and placing the cell culture dish into a constant-temperature incubator at 37 ℃ to allow the cells to adhere to the wall overnight;

(3) and taking 10 mu L of the uniformly dispersed nano motor solution of 200 mu g/mL, directly adding the solution into the adherent cell culture dish, and immediately observing and recording the motion state of the nano motor by using a fluorescence microscope.

(4) Fig. 6 is a motion trace of the nanomotor, which proves that the nanomotor can move in the cell environment. And the motion speed is calculated to be about 3.48 mu m/s according to the motion track, parabolic fitting and linear fitting are respectively carried out on the mean square displacement (fig. 7 and 8), as shown in fig. 7 and 8, the result shows high correlation with the parabola, and the motion mode of the nanomotor in the cell environment is proved to be self-driven rather than Brownian motion.

Example 7

Amphoteric ion-based NO-driven nanomotor chemotaxis study:

(1) weighing 1mg of the nanomotor prepared in example 3, adding 1mL of deionized water, and performing ultrasonic full dispersion to obtain 1mg mL ^-1 A uniformly dispersed nanomotor solution. Breast cancer cells MCF-7 with 5 x 10 ⁵ cell/mL was inoculated into the right chamber of the chemotaxis model apparatus shown in fig. 9, placed in a 37 ℃ incubator at a constant temperature to allow cells to adhere overnight, and a ROS concentration gradient was constructed in the apparatus from right to left with ROS concentration varying from high to low. As a control, cells were not seeded in the right chamber to provide an environment without ROS concentration gradient.

(2) To the left chamber of the two units with and without ROS concentration gradient was added 250. mu.L of 1mg m concentrationL ^-1 The zwitterionic NO-driven nanomotor solution of (a). After 20min, the solution in the right chamber of the model was pipetted 100. mu.L to measure its fluorescence intensity. As shown in fig. 10, the fluorescence intensity of the nanomotors detected in the model right chamber with ROS concentration gradient was about 8 times higher than that of the control group (without ROS concentration gradient), demonstrating that the zwitterionic NO-driven nanomotor chemotaxis has high ROS concentration chemotaxis.

Example 8

Evaluation of biocompatibility of NO-driven nanomotors of zwitterionic derivatives;

(1) endothelial Cells (HUVECs) were treated with 5X 10 ⁴ cell/mL was seeded in 96-well plate, after cell attachment, the nanomotors prepared in example 3 were dispersed in culture medium to prepare nanomotors (20,50, 100,150, 200. mu.g mL) of different concentrations ^-1 ) After incubation with HUVECs for different times (12h,24h,48h), cell activity was detected using MTT reagent;

(2) from fig. 11, it can be seen that the cell activities of HUVECs do not change significantly after incubation with HUVECs for different time periods (12h,24h and 48h) at the above concentration of nanomotors, which preliminarily proves the biocompatibility of nanomotors;

(3) then, 5mg nanomotor was added to 5mL of hydrogen oxide solution (0.002%) and reacted in a water bath environment at 37 ℃ for 48 hours sufficiently, and the degraded product in the lower layer was centrifuged to take out, at which time it was considered that NO was continuously generated in the cancer cell environment. Mixing MCF-7 with 5 x 10 ⁵ cells/mL were seeded in 96-well plates and after cells were attached, the degraded material was treated with different concentrations of product (20,50, 100,150, 200. mu.g mL) ^-1 ) Incubating the cells and the cancer cells in an incubator at 37 ℃ for different time (12h,24h and 48h) to check the activity of the cells;

(4) from fig. 12, it can be seen that the cancer cell activity was hardly affected by the increase of the material concentration (after degradation) and the extension of the incubation time, thus demonstrating that the zwitterionic substrate has good biocompatibility.

Example 9

Evaluation of anticancer Properties of NO-driven nanomotors of zwitterionic derivatives

(1) Mixing MCF-7 with 5 x 10 ⁵ cell/mLInoculating the cells in a 96-well plate, after the cells are attached to the wall, preparing the nano-motor prepared in example 3 into dispersion with complete culture medium according to different final concentrations (20,50, 100,150,200 mu g mL) ^-1 ) Detecting the cell activity of the cells after incubation with the cancer cells for different time periods (12h,24h and 48 h);

(2) it can be seen from fig. 13 that the killing performance of nanomotors against cancer cells is concentration and time dependent. Specifically, the cell activity of the cancer cells gradually decreased with the increase in the concentration of the material incubated with the cells at the same time, and the concentration of the material was adjusted from 20. mu.g mL at a time point of 48 hours ^-1 Increase to 200. mu.g mL ^-1 When the cell activity is reduced from 87.4% to 49.7%; and when the concentration of the nano motor is constant, the cell activity is gradually reduced along with the prolonging of the incubation time of the nano motor and the cell. The reason for the above results may be that the NO produced by the nanomotor in the cell environment has concentration and time dependency, and the killing ability to the cell is gradually enhanced as the concentration of the nanomotor is increased and the NO production amount is gradually increased with the prolongation of the cell incubation time.

Comparative example 1

Methacrylic acid with double bonds at the end groups is selected as a monomer and a diselenide compound is selected as a cross-linking agent to prepare the PMSE nanoparticles, and then the PMSE nanoparticles and L-arginine are combined with each other in a weak static combination manner to prepare the rear-loading type PMSE/A nano motor.

The specific experimental steps are as follows: methacrylic acid (500mg, 5.81mmol), AIBN (11.10mg, 0.07mmol) and diselenide crosslinker (55.60mg, 0.15mmol, prepared in inventive example 2) were dissolved in 40mL acetonitrile in a three-neck flask and kept under nitrogen atmosphere at reflux for 1 h. The resulting nanogels were collected by centrifugation (5000rpm, 10min), washed at least three times with deionized water and freeze-dried. Next, 50mg of the prepared product is weighed and dispersed in 5mL of aqueous solution, 5mg of L-arginine is added and stirred at room temperature for 24 hours, then the mixture is washed with distilled water for 3 times, and the product is obtained by centrifugation and dried, so that the supported NO-driven nanomotor is obtained, wherein a TEM image of the supported NO-driven nanomotor is shown in fig. 14, and the particle size of the supported NO-driven nanomotor is close to that of the nanomotor prepared in example 3.

The NO release amount and the moving speed were measured by the method of example 5, respectively, as shown in FIGS. 15 to 17, and as a result, it was found that the NO release amount of the supported PMSE/A nanomotor was 1.57. mu.M as compared with PAMSe (4.71. mu.M). The movement speed obtained by the movement track of the load type PMSE/A nano motor in the cell environment and MSD quadratic term fitting curve is about 1.8 +/-0.4 mu m/s, which is far less than the movement speed (3.48 +/-0.9 mu m/s) of the covalent grafting nano motor provided by the invention. Therefore, the PAMSe nano motor provided by the invention has higher fuel load efficiency, so that the release period and the release amount of NO are improved, and the movement speed of the nano motor is also improved.

Example 10

Example 10 preparation of a zwitterionic polymer based Nitric Oxide (NO) driven nanomotor. Wherein an L-arginine derivative was prepared by the method of example 1, except that: the mol ratio of L-arginine to methacrylic anhydride is 0.5, the volume ratio of deionized water to 1, 4-dioxane is 1, the volume ratio of 1, 1, 4-dioxane to triethylamine is 1, the stirring reaction temperature is 20 ℃, and the reaction time is 48 hours;

wherein diselenide compound cross-linking agent is prepared by the method of example 2, the difference is that: the molar ratio of 2, 2-diselenodiethylbis (1-ethylamine) dihydrochloride to methacryloyl chloride is 0.1, the reaction temperature is 0 ℃ under stirring, and the reaction time is 48 hours.

The method of example 3 was used to prepare a nanomotor, except that: the molar ratio of the L-arginine zwitterionic derivative to the diselenide compound cross-linking agent is 5, the molar ratio of the L-arginine zwitterionic derivative to the initiator is 1, the initiator is azodiisoheptanonitrile, the reaction solvent is acetonitrile, the reaction temperature is 80 ℃, the reaction time is 5h, the rotation speed of ultracentrifugation is 10000rpm, and the centrifugation time is 30 min.

Example 11

Example 11 preparation of a zwitterionic polymer based Nitric Oxide (NO) driven nanomotor. Wherein an L-arginine derivative was prepared by the method of example 1, except that: the mol ratio of L-arginine to methacrylic anhydride is 1, the volume ratio of deionized water to 1, 4-dioxane is 5, the volume ratio of 1, 4-dioxane to triethylamine is 5, the stirring reaction temperature is 30 ℃, and the reaction time is 10 hours;

wherein diselenide compound cross-linking agent is prepared by the method of example 2, the difference is that: the molar ratio of 2, 2-diselenide diethyl bis (1-ethylamine) dihydrochloride to methacryloyl chloride is 0.5, the stirring reaction temperature is 30 ℃, and the reaction time is 10 hours.

The method of example 3 was used to prepare a nanomotor, except that: the molar ratio of the L-arginine zwitterionic derivative to the diselenide compound cross-linking agent is 20, the molar ratio of the L-arginine zwitterionic derivative to the initiator is 20, the initiator is one of dimethyl azodiisobutyrate, the reaction temperature is 100 ℃, the reaction time is 1h, the ultracentrifugation rotating speed is 12000rpm, and the centrifugation time is 10 min.

Claims

1. A zwitterionic polymer-based NO-driven nano motor is characterized in that the zwitterionic polymer-based NO-driven nano motor is formed by initiating polymerization reaction through an initiator by taking an L-arginine derivative as a monomer and a diselenide compound as a cross-linking agent; the L-arginine derivative is a compound with a high-density active guanidyl functional group and a carbon-carbon double bond at the chain end; the diselenide compound is a Reactive Oxygen Species (ROS) -responsive diselenide compound;

the structural formulas of the L-arginine derivative and the diselenide compound are respectively shown as follows:

structural formula of L-arginine derivative

The structural formula of the diselenide compound.

2. The zwitterionic polymer-based nitric oxide-driven nanomotor of claim 1, wherein the initiator is one of azobisisobutyronitrile, azobisisoheptonitrile, or dimethyl azobisisobutyrate.

3. A method for preparing the zwitterionic polymer-based nitric oxide-driven nanomotor of claim 1, comprising the steps of:

(1) dissolving L-arginine in a mixed solvent of deionized water and 1, 4-dioxane, adding triethylamine, cooling the solution, dropwise adding methacrylic anhydride while stirring, and stirring for reaction to obtain an L-arginine derivative;

(2) adding 2,2' -diselenide diethyl bis (1-ethylamine) dihydrochloride and triethylamine into an anhydrous dichloromethane solution, slowly adding methacryloyl chloride, and stirring for reaction; extracting and drying the reaction system, and removing an organic phase; purifying to obtain a diselenide compound cross-linking agent with ROS response type;

(3) dissolving the L-arginine derivative obtained in the step (1) and the diselenide compound cross-linking agent with ROS response type obtained in the step (2) in a solvent, adding an initiator to carry out polymerization reaction, and carrying out centrifugal washing to obtain the zwitterionic polymer-based NO driving nanomotor.

4. The preparation method according to claim 3, wherein the molar ratio of the L-arginine to the methacrylic anhydride in the step (1) is 0.5-1, the volume ratio of the deionized water to the 1, 4-dioxane is 1-5, the volume ratio of the 1, 4-dioxane to the triethylamine is 1-5, the stirring reaction temperature is 20-30 ℃, and the reaction time is 10-48 h.

5. The preparation method of claim 3, wherein the molar ratio of 2,2' -diselenediylbis (1-ethylamine) dihydrochloride to methacryloyl chloride in the step (2) is 0.1 to 0.5, the reaction temperature is 0 to 30 ℃ and the reaction time is 10 to 48 hours under stirring.

6. The method according to claim 4, wherein the reaction system of step (2) is extracted with deionized water to remove impurities, anhydrous Na ₂ SO ₄ Drying overnight, removing an organic phase by reduced pressure evaporation, and purifying by silica gel column chromatography with ethyl acetate/petroleum ether as a raw material to obtain the ROS-responsive diselenide compound crosslinking agent.

7. The preparation method according to claim 3, wherein the molar ratio of the L-arginine zwitterionic derivative to the diselenide compound cross-linking agent in the step (3) is 5-20, the molar ratio of the L-arginine zwitterionic derivative to the initiator is 1-20, the reaction solvent is one of acetonitrile or dimethyl sulfoxide, the reaction temperature is 80-100 ℃, and the reaction time is 1-5 h.

8. The preparation method according to claim 3, wherein the centrifugal washing in step (3) is collecting the obtained nanogel by ultracentrifugation, and washing and freeze-drying to obtain the zwitterionic polymer-based NO-driven nanomotor, wherein the rotation speed of the ultracentrifugation is 10000-12000rpm, and the centrifugation time is 10-30 min.

9. Use of the zwitterionic polymer-based nitric oxide-driven nanomotor of claim 1 in the manufacture of a medicament for targeting a cardiovascular disease lesion or a tumor lesion.