CN112386602A - Drug-loaded nano robot and preparation method and application thereof - Google Patents
Drug-loaded nano robot and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a drug-loaded nano robot and a preparation method and application thereof, wherein the drug-loaded nano robot comprises: a nano-robot; the polymer brush comprises a hydrophilic end and a hydrophobic end, and the hydrophobic end of the polymer brush is grafted on the surface of the nano robot; a drug attached to the hydrophilic end of the polymer brush; in the drug-loaded robot, drugs are loaded on the nano robot by the polymer brush with better biocompatibility, so that the efficient treatment of tumors by the nano robot is realized, and the potential medical application of the nano robot is expanded; the drug-loaded nano robot can effectively target tumors and directly reach the depths of the tumors, so that the tumor focus points are more accurately and effectively targeted, and the functions of biodegradation and drug release in redox response are achieved; in the preparation process, the compound can be obtained by a simple method, so that the loss of the medicine can be reduced, the high load rate can be achieved, and the compound has a wide biological application prospect.
Description
Technical Field
The invention belongs to the field of robots, relates to a drug-loaded nano robot and a preparation method and application thereof, and particularly relates to a nano robot with redox response drug release and a preparation method and application thereof.
Background
The nano robot is widely applied by the advantages of small size, sensitive movement, strong function and the like, and particularly has very wide application prospect in biomedicine. At present, although the drug-loaded nanoparticles have certain clinical effect, the traditional clinical operation, radiotherapy and chemotherapy treatment means still have a plurality of defects, such as easy relapse, lack of targeting, multi-drug resistance and serious toxic and side effects, which limit the further development of the drug-loaded nanoparticles. Because the nanoparticles cannot effectively target tumors by means of circulation and osmosis in vivo alone, and cannot reach the deep parts of the tumors. As a new interdisciplinary field, the nano medical robot has an important role in the aspect of targeted drug delivery, and the magnetic nano robot can be more accurately and effectively targeted to a focus point under the action of a magnetic field.
In order to effectively bind anticancer drugs to carriers, methods developed at present mainly include strategies such as liposomes, nanoparticles, and polymeric micelles. The polymer micelle can be used as a storage space of a slightly soluble medicament and can avoid inactivation of the medicament, but complex surface functional modification is usually required to be carried out on the polymer micelle, and the polymer micelle and the medicament form chemical bond combination. In addition, the stimulus-responsive nano-drug carrier designed based on the tumor microenvironment receives wide attention in the anti-tumor drug delivery system. The difference between the hypoxia surrounding tumor tissue and the oxygen content of normal tissue is a powerful stimulus to regulate the rate of controlled drug release. The most representative one is a disulfide bond compound, and the method for introducing a disulfide bond generally includes 1) converting an amino group, a carboxyl group, etc. into a thiol group, and regenerating a disulfide bond; 2) the disulfide bond is introduced by crosslinking a system by using a crosslinking agent containing a disulfide bond, and the methods have complex systems, are difficult to avoid introducing impurities and have complicated processes.
Therefore, it is very necessary to provide a drug-loaded nano robot with a simple preparation method.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a drug-loaded nano robot, wherein drugs are loaded on the nano robot by a polymer brush with better biocompatibility, so that the efficient treatment of tumors by the nano robot is realized, and the potential medical application of the nano robot is expanded; the drug-loaded nano robot can effectively target tumors and directly reach the depths of the tumors, so that the tumor focus points are more accurately and effectively targeted, and the functions of biodegradation and drug release in redox response are achieved; in the preparation process, the compound can be obtained by a simple method, so that the loss of the medicine can be reduced, the high load rate can be achieved, and the compound has a wide biological application prospect.
One of the objectives of the present invention is to provide a drug-loaded nano robot, comprising:
a nano-robot;
the polymer brush comprises a hydrophilic end and a hydrophobic end, and the hydrophobic end of the polymer brush is grafted on the surface of the nano robot;
a drug attached to the hydrophilic end of the polymer brush.
In the drug-loaded robot, the polymer brush with good biocompatibility is used for loading drugs onto the nano robot, so that the nano robot can effectively treat tumors, and the potential medical application of the nano robot is expanded.
In the invention, the drug-loaded nano robot can effectively target tumors and directly reach the depths of the tumors, so that the tumor foci can be targeted more accurately and effectively, and the functions of biodegradation and redox response drug release are achieved.
In the present invention, the average particle diameter of the nano-robot is 10 to 1000nm, for example, 10nm, 50nm, 100nm, 300nm, 500nm, 800nm, 1000nm, etc.
In the present invention, the shape of the nano robot includes any one of an L-shape, a spiral shape, a spherical shape, or a crescent shape.
In the present invention, the drug comprises doxorubicin.
In the present invention, the drug and the hydrophilic end of the polymer brush are linked by intermolecular forces.
In the present invention, the polymer brush disulfide-polyamide polymer (SS-PAA) has a structure as shown below:
where, m is (1-3) to (7-9), (e.g., 1:9, 1.5:8.5, 2:8, 2.5:7.5, 3:7, etc.), and m is 1-50, (e.g., 1,3, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, etc.).
According to the invention, the polymer brush with the structure is stable under normal physiological conditions, is disintegrated due to the breakage of disulfide bonds under a reduction condition, releases a drug, senses a hypoxic microenvironment around a tumor, and releases the drug under a hypoxic condition, so that an anti-tumor effect is achieved.
In the present invention, the method for preparing the polymer brush comprises the steps of:
s1, adding a dichloromethane solution of acryloyl chloride and an alkali solution into a cystamine hydrochloride aqueous solution for reaction to obtain cystamine bisacrylamide;
s2, reacting the cystamine bisacrylamide obtained in the step S1, phenethylamine and ethanolamine to obtain the polymer brush.
In the present invention, the reaction equation of the polymer brush (SS-PAA) is as follows:
in the present invention, the concentration of the methylene chloride solution of acryloyl chloride described in step S1 is 1.0-1.5g/mL, for example, 1.0g/mL, 1.05g/mL, 1.1g/mL, 1.15g/mL, 1.2g/mL, 1.25g/mL, 1.3g/mL, 1.35g/mL, 1.4g/mL, 1.45g/mL, 1.5g/mL, etc.
In the present invention, the alkali solution described in step S1 is a sodium hydroxide solution having a concentration of 0.5 to 0.6g/mL (e.g., 0.5g/mL, 0.51g/mL, 0.52g/mL, 0.53g/mL, 0.54g/mL, 0.55g/mL, 0.56g/mL, 0.57g/mL, 0.58g/mL, 0.59g/mL, 0.6g/mL, etc.).
In the present invention, the manner of addition described in step S1 is to simultaneously add dropwise a methylene chloride solution of acryloyl chloride and an alkali solution to an aqueous cystamine hydrochloride solution using a syringe at a rate of 0.1-0.3mL/min (e.g., 0.1mL/min, 0.12mL/min, 0.15mL/min, 0.17mL/min, 0.2mL/min, 0.22mL/min, 0.25mL/min, 0.28mL/min, 0.3mL/min, etc.).
In the present invention, the concentration of the cystamine hydrochloride aqueous solution in step S1 is 0.1-0.5g/mL (e.g., 0.1mL/min, 0.12mL/min, 0.15mL/min, 0.17mL/min, 0.2mL/min, 0.22mL/min, 0.25mL/min, 0.28mL/min, 0.3mL/min, 0.32mL/min, 0.35mL/min, 0.37mL/min, 0.4mL/min, 0.42mL/min, 0.45mL/min, 0.48mL/min, 0.5mL/min, etc.).
In the present invention, the reaction in step S1 is performed in an ice bath for 12-24h (e.g., 12h, 14h, 16h, 18h, 20h, 22h, 24h, etc.).
In the present invention, the step S1 further includes sequentially separating, washing and drying the reactant obtained after the reaction.
In the present invention, the reaction of step S2 is performed in a vacuum reaction tube.
In the present invention, the reaction temperature in step S2 is 110-150 ℃ (e.g. 110 ℃, 115 ℃, 120 ℃, 125 ℃, 130 ℃, 135 ℃, 140 ℃, 145 ℃, 150 ℃, etc.), and the reaction time is 12-24h (e.g. 12h, 14h, 16h, 18h, 20h, 22h, 24h, etc.).
In the present invention, step S2 is performed under the protection of argon.
In the present invention, the step S2 further includes purifying the reactant obtained after the reaction.
In the present invention, the purification method is an anti-solvent purification method by methanol and diethyl ether.
The second purpose of the invention is to provide a preparation method of the drug-loaded nano robot, which comprises the following steps:
(1) dissolving the drug solution, the nano robot and the polymer brush in an organic solvent, and mixing to obtain an oil-water microemulsion;
(2) and (3) placing the oil-water microemulsion into a hydrophilic solution, and mixing to obtain the drug-loaded nano robot.
In the preparation process, the compound can be obtained by a simple method, so that the loss of the medicine can be reduced, the high load rate can be achieved, and the compound has a wide biological application prospect.
In the present invention, the preparation method of the drug solution in step (1) comprises: the drug is mixed with an aqueous solution of polyvinyl alcohol to obtain a drug solution.
In the present invention, the concentration of the drug in the drug solution is 30% -50%, such as 30%, 32%, 35%, 38%, 40%, 42%, 45%, 48%, 50%, etc.
In the present invention, the concentration of the polyvinyl alcohol in the aqueous solution of polyvinyl alcohol is 3 to 10%, for example, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%.
In the present invention, the amount of the nanoprobe added in step (1) is 1.5 to 4.5mg (e.g., 1.5mg, 2mg, 2.5mg, 3mg, 3.5mg, 4mg, 4.5mg, etc.) and the amount of the polymer brush added is 20 to 30mg (e.g., 20mg, 21mg, 22mg, 23mg, 24mg, 25mg, 26mg, 27mg, 28mg, 29mg, 30mg, etc.) based on 5 to 15mg of the drug added in the drug solution.
In the present invention, the organic solvent of step (1) comprises dichloromethane.
In the invention, the mixing mode in the step (1) is ultrasonic mixing, and the mixing time is 20-50min (for example, 20min, 25min, 30min, 35min, 40min, 45min, 50min, etc.).
In the present invention, the hydrophilic solution of step (2) includes a polyvinyl alcohol solution.
In the present invention, the concentration of the polyvinyl alcohol solution is 3 to 10% (e.g., 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc.).
In the present invention, the mixing time in step (2) is 4-24h, such as 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, etc.
In the present invention, the step (2) further comprises sequentially separating, washing and drying the mixed solution.
In the present invention, the separation is carried out by removing the organic solvent by evaporation.
In the present invention, the cleaning manner is centrifugation, the centrifugation rate is 8000- & ltSUB & gt 12000r/min (such as 8000r/min, 8500r/min, 9000r/min, 9500r/min, 10000r/min, 10500r/min, 11000r/min, 11500r/min, 12000r/min, etc.), and the centrifugation time is 5-15min (such as 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, etc.).
In the present invention, the drying method is freeze drying.
The invention also aims to provide an application of the drug-loaded nano robot in an anti-tumor drug delivery system.
Compared with the prior art, the invention has the following beneficial effects:
in the invention, the drug-loaded robot loads drugs onto the nano-robot by the polymer brush with better biocompatibility, so that the nano-robot can effectively treat tumors, and the potential medical application of the nano-robot is expanded; the drug-loaded nano robot can effectively target tumors and directly reach the depths of the tumors, so that the tumor focus points are more accurately and effectively targeted, and the functions of biodegradation and drug release in redox response are achieved; in the preparation process, the compound can be obtained by a simple method, so that the loss of the medicine can be reduced, the high load rate can be achieved, and the compound has a wide biological application prospect.
Drawings
Fig. 1 is a schematic diagram of the preparation of a drug-loaded robot in example 1;
FIG. 2 is a drug release profile of the drug-loaded robot prepared in example 1 under the action of reduced glutathione and without the action of reduced glutathione;
fig. 3 is a graph of cytotoxicity of the drug-loaded robot prepared in example 1 and free drug after 1 day incubation of human lung cancer cell H1299;
FIG. 4 is a graph showing drug release profiles of the drug-loaded robot prepared in example 2 under the action of reduced glutathione and without the action of reduced glutathione;
FIG. 5 is a graph showing drug release profiles of the drug-loaded robot prepared in example 3 under the action of reduced glutathione and without the action of reduced glutathione;
FIG. 6 is a graph showing drug release profiles of the drug-loaded robot prepared in example 4 under the action of reduced glutathione and without the action of reduced glutathione;
fig. 7 is a graph showing the killing ability of the drug-loaded robot prepared in example 2, example 3 and comparative example 1 to tumor cells after increasing the drug load.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
In the specific implementation mode, taking an L-shaped robot as an example, the medicine carrying is carried out, wherein the preparation method of the L-shaped robot comprises the following steps:
(1) and (3) evaporating a 150nm metal aluminum layer on an inch silicon wafer by adopting an electron beam evaporation method.
(2) Spin-coating an electron beam adhesive with the type AR-7520.07 on the prepared silicon wafer with the metal aluminum layer with the thickness of 150nm on the surface, spin-coating (the spin-coating speed is 3000r/min) for 60s, placing the obtained silicon wafer on a hot plate at the temperature of 90 ℃, and baking for 2 min.
(3) Putting the prepared silicon wafer sample into an electron beam exposure machine, and selecting an exposure parameter of 600uC/cm2。
(4) Taking out the written silicon wafer sample from the electron beam exposure machine, putting the silicon wafer sample into a glass ware filled with an electron beam glue developing solution, shaking the glass ware for 1min to 2min, taking out the sample, putting the sample on a hot plate, and baking the sample for 1.5min at 90 ℃.
(5) The sample obtained after development was placed in an ICP plasma etcher and etched at a power of 250w for 50 s.
(6) And putting the silicon chip sample obtained after etching into an electron beam evaporation machine, and evaporating a plurality of layers of metals, namely a titanium layer of 15nm, a nickel layer of 70nm and a titanium layer of 15nm in sequence.
(7) And putting the obtained silicon wafer sample into a sodium hydroxide solution with the concentration of 2mol/L, adding a sodium citrate solution, standing for about one hour, taking out the silicon wafer, and filtering the solution to obtain the required L-shaped robot.
In a specific embodiment, the polymer brushes are all SS-PAA, having the formula:
wherein, m is 1-50, n is 1-3, and m is 7-9.
Taking m: n as an example of 3:7, the preparation method of the SS-PAA comprises the following steps:
(1) preparing cystamine hydrochloride aqueous solution (5.8 g cystamine hydrochloride, 25ml water), pouring into a three-neck flask placed in an ice bath, preparing 0.15 mol acryloyl chloride/dichloromethane solution (10ml) and NaOH aqueous solution (4g NaOH, 10ml water) respectively, and slowly dripping the two latter solutions into the cystamine hydrochloride aqueous solution by using a syringe simultaneously with stirring, wherein the dripping speed is controlled to be 0.2 ml/min. Stirring at room temperature overnight, and vacuum drying the product to obtain white cystamine bisacrylamide powder.
(2) Adding cystamine bisacrylamide (1g), phenethylamine (0.39g) and ethanolamine (0.08g) into a vacuum reaction tube, stirring and reacting at 125 ℃ under Ar gas overnight, and purifying the product SS-PAA by dissolving with methanol and precipitating with diethyl ether to obtain the SS-PAA.
When m: n is 2:8 or 1:9, the preparation method of SS-PAA is the same as that described above, except that the amount of the reactants added is different.
Example 1
The embodiment provides a medicine carrying nano robot, includes:
a nano-robot;
the polymer brush comprises a hydrophilic end and a hydrophobic end, and the hydrophobic end of the polymer brush is grafted on the surface of the nano robot;
a drug attached to the hydrophilic end of the polymer brush;
wherein the robot is the prepared L-shaped robot, and the average grain diameter of the L-shaped robot is 800 nm; the medicine is adriamycin; the polymer brush is SS-PAA, and the structural formula is as follows:
wherein, m is n is 3:7, and m is 1-50.
The embodiment also provides a preparation method of the drug-loaded robot, and fig. 1 is a schematic preparation diagram of the drug-loaded robot, and the preparation method includes:
(1) ultrasonic dissolving adriamycin (10mg) in 5% (W/v) PVA water solution (2mL), dissolving L-shaped robot (3mg) and SS-PAA (25mg) in dichloromethane solution (10mL), mixing the two solutions, and ultrasonic dissolving for 30min to form O/W oil-water microemulsion system.
(2) Adding the O/W oil-water microemulsion system into a 5% (W/v) PVA aqueous solution (40mL) to form a W/O/W system, evaporating the organic solvent at room temperature overnight, centrifuging the precipitate (the centrifugation speed is 10000r/min, the centrifugation time is 10min), and freeze-drying to obtain the Ni/SS-PAA robot loaded with the adriamycin.
The drug-loaded robot in this embodiment was subjected to the following performance tests:
(1) drug release testing:
1mg of the drug-loaded nano-robot prepared above and 1mM of reduced Glutathione (GSH) were added to PBS buffer (1mL, pH 7.4), placed in a 37 ℃ water bath and shaken, centrifuged to collect supernatants for different times for testing, and simultaneously supplemented with an equal amount of fresh PBS buffer. The absorbance intensity at 480nm was measured by fluorescence and the drug release efficiency was calculated. The results are shown in FIG. 2. As can be seen from the figure, the amount of release of the sample group to which the GSH reducing agent was added was 2 times that of the control group.
(2) Cytotoxicity test:
the CCK8 reagent was used to test the toxicity of the robot on human lung cancer cells H1299. The logarithmic phase cell suspension was pipetted into a 96-well plate (4 × 104/well) at 200 μ l per well and cultured in a 5% CO2 cell culture chamber at 37 ℃ for 1 day. After cell attachment, 100 microliters of medium containing a robot was added, 7 concentration gradients were set, and 3 parallel wells were repeated for each concentration. After the culture was continued for 1 day, a culture medium containing 10% of CCK8 reagent was added, and after the culture was continued for 3 hours, absorbance at 450nm was measured by a microplate reader, and the measurement results are shown in FIG. 3 (in which 3 parallel samples were set for each data point). As can be seen from fig. 3, after 1 day incubation, the drug-loaded nano-robot prepared in this example and pure doxorubicin were both significantly inhibited in cell viability. With the increase of the concentration of adriamycin (DOX), the cytotoxicity of the drug-loaded nano robot sample group is obviously enhanced.
Example 2
The embodiment provides a medicine carrying nano robot, includes:
a nano-robot;
the polymer brush comprises a hydrophilic end and a hydrophobic end, and the hydrophobic end of the polymer brush is grafted on the surface of the nano robot;
a drug attached to the hydrophilic end of the polymer brush;
wherein the robot is the L-shaped robot prepared above, and the average particle size of the L-shaped robot is 1000 nm; the medicine is adriamycin; the polymer brush is SS-PAA, and the structural formula is as follows:
wherein, m is 2:8, and m is 1-50.
The embodiment also provides a preparation method of the drug-loaded robot, which comprises the following steps:
(1) dissolving adriamycin (5mg) in 3% (W/v) PVA water solution (2mL) by ultrasonic wave, dissolving an L-shaped robot (1.5mg) and SS-PAA (20mg) together in dichloromethane solution (10mL), mixing the two solutions, and performing ultrasonic wave for 20min to form an O/W oil-water microemulsion system.
(2) Adding the O/W oil-water microemulsion system into 3% (W/v) PVA aqueous solution (40mL) to form a W/O/W system, evaporating the organic solvent at room temperature overnight, centrifuging the precipitate (the centrifugation speed is 8000r/min, the centrifugation time is 15min), and freeze-drying to obtain the Ni/SS-PAA robot loaded with the adriamycin.
The drug-loaded robot obtained in the embodiment is subjected to performance test in the manner of embodiment 1, and it can be seen from fig. 4 and 7 that the disulfide bond-polymer brush functionalized nano robot has improved drug loading capacity after the drug loading proportion is increased, and has low-oxygen perception drug release capacity and better killing capacity on tumor cells.
Example 3
The embodiment provides a medicine carrying nano robot, includes:
a nano-robot;
the polymer brush comprises a hydrophilic end and a hydrophobic end, and the hydrophobic end of the polymer brush is grafted on the surface of the nano robot;
a drug attached to the hydrophilic end of the polymer brush;
wherein the robot is the L-shaped robot prepared above, and the average particle diameter of the L-shaped robot is 100 nm; the medicine is adriamycin; the polymer brush is SS-PAA, and the structural formula is as follows:
wherein, m is 1:9, m is 1-50.
The embodiment also provides a preparation method of the drug-loaded robot, which comprises the following steps:
(1) dissolving adriamycin (15mg) in 10% (W/v) PVA water solution (2mL) by ultrasonic wave, dissolving an L-shaped robot (4.5mg) and SS-PAA (30mg) together in dichloromethane solution (10mL), mixing the two solutions, and performing ultrasonic wave for 30min to form an O/W oil-water microemulsion system.
(2) Adding the O/W oil-water microemulsion system into 10% (W/v) PVA aqueous solution (40mL) to form a W/O/W system, evaporating the organic solvent at room temperature overnight, centrifuging the precipitate (the centrifugation speed is 12000r/min, the centrifugation time is 5min), and freeze-drying to obtain the Ni/SS-PAA robot loaded with the adriamycin.
The drug-loaded robot obtained in this example was subjected to performance tests in the same manner as in example 1, and it can be seen from fig. 5 and 7 that the drug release amount was significantly increased and the survival rate of tumor cells was decreased after the doxorubicin concentration was increased.
Comparative example 1
The only difference from example 1 is that the polymer brush SS-PAA is replaced with polyamide PAA without disulfide bond, and the rest of the composition and the preparation method are the same as those of example 1.
The drug-loaded robot obtained in the comparative example is subjected to performance tests in the manner of example 1, and it can be seen from fig. 6 and 7 that the polymer brush without disulfide bonds cannot release the drug substantially without effectiveness under the condition of a reducing agent, and has no obvious killing effect on cancer cells.
Comparative example 2
This comparative example provides a medicine carrying nano robot, and the preparation method is as follows: adv. Mater.2009,21, 3286-.
Compared with the comparative example 2 and the example 1, the drug-loaded nano robot can reduce the loss of the drug in the preparation process, can improve the load rate, can be prepared by a simple preparation method, and has a wide application prospect.
The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.
Claims (10)
1. The drug-loaded nano robot is characterized by comprising:
a nano-robot;
the polymer brush comprises a hydrophilic end and a hydrophobic end, and the hydrophobic end of the polymer brush is grafted on the surface of the nano robot;
a drug attached to the hydrophilic end of the polymer brush.
2. The drug-loaded nano-robot of claim 1, wherein the nano-robot has an average particle size of 10-1000 nm;
preferably, the shape of the nano robot comprises any one of an L shape, a spiral shape, a spherical shape or a crescent shape;
preferably, the drug comprises doxorubicin;
preferably, the drug and the hydrophilic end of the polymer brush are linked by intermolecular forces.
3. The drug-loaded nanocontologies of claim 1 or 2, characterized in that the polymer brush has a structure as shown below:
wherein, m is 1-50, n is 1-3, and m is 7-9.
4. The drug-loaded nano-robot of claim 3, wherein the preparation method of the polymer brush comprises the following steps:
s1, adding a dichloromethane solution of acryloyl chloride and an alkali solution into a cystamine hydrochloride aqueous solution for reaction to obtain cystamine bisacrylamide;
s2, reacting the cystamine bisacrylamide obtained in the step S1, phenethylamine and ethanolamine to obtain the polymer brush.
5. The drug-loaded nano robot of claim 4, wherein the concentration of the dichloromethane solution of acryloyl chloride of step S1 is 1.0-1.5 g/mL;
preferably, the alkali solution in the step S1 is a sodium hydroxide solution with the concentration of 0.5-0.6 g/mL;
preferably, the adding manner in step S1 is to simultaneously add dropwise a dichloromethane solution of acryloyl chloride and an alkali solution into the aqueous solution of cystamine hydrochloride by using a syringe, wherein the dropwise adding rate is 0.1-0.3 mL/min;
preferably, the concentration of the cystamine hydrochloride aqueous solution in step S1 is 0.1-0.5 g/mL;
preferably, the reaction of step S1 is performed in an ice bath for 12-24 h;
preferably, the step S1 further includes sequentially separating, washing and drying the reactant obtained after the reaction;
preferably, the reaction of step S2 is performed in a vacuum reaction tube;
preferably, the reaction temperature of the step S2 is 110-150 ℃, and the reaction time is 12-24 h;
preferably, step S2 is performed under the protection of argon;
preferably, the step S2 further comprises purifying the reactant obtained after the reaction;
preferably, the purification method is an anti-solvent purification method by methanol and diethyl ether.
6. The method for preparing a drug-loaded nano-robot according to any one of claims 1 to 5, wherein the method for preparing a drug-loaded nano-robot comprises the following steps:
(1) dissolving the drug solution, the nano robot and the polymer brush in an organic solvent, and mixing to obtain an oil-water microemulsion;
(2) and (3) placing the oil-water microemulsion into a hydrophilic solution, and mixing to obtain the drug-loaded nano robot.
7. The method for preparing a drug-loaded nano robot according to claim 6, wherein the method for preparing the drug solution in step (1) comprises: mixing a drug with an aqueous solution of polyvinyl alcohol to obtain a drug solution;
preferably, the concentration of the drug in the drug solution is 30% -50%;
preferably, the concentration of the polyvinyl alcohol in the aqueous solution of the polyvinyl alcohol is 3-10%;
preferably, the adding amount of the nano robot in the step (1) is 1.5-4.5mg and the adding amount of the polymer brush is 20-30mg based on the adding amount of the drug in the drug solution being 5-15 mg;
preferably, the organic solvent of step (1) comprises dichloromethane;
preferably, the mixing mode of the step (1) is ultrasonic mixing, and the mixing time is 20-50 min.
8. The method for preparing a drug-loaded nano-robot according to claim 6 or 7, wherein the hydrophilic solution of step (2) comprises a polyvinyl alcohol solution;
preferably, the concentration of the polyvinyl alcohol solution is 3-10%;
preferably, the mixing time of step (2) is 4-24 h.
9. The method for preparing a drug-loaded nano robot according to any one of claims 6 to 8, wherein the step (2) further comprises sequentially separating, cleaning and drying the mixed solution;
preferably, the separation is carried out by removing the organic solvent by evaporation;
preferably, the cleaning mode is centrifugation, the centrifugation speed is 8000-12000r/min, and the centrifugation time is 5-15 min;
preferably, the drying is by freeze drying.
10. The use of the drug-loaded nano-robot of any one of claims 1-5 in an anti-tumor drug delivery system.
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