阅读说明：本技术 载抗肿瘤药物纳米机器人及其制备方法 (Anti-tumor drug-loaded nano robot and preparation method thereof ) 是由 不公告发明人 于 2020-11-10 设计创作，主要内容包括：本发明属于纳米材料领域,尤其涉及一种载抗肿瘤药物纳米机器人及其制备方法。本发明提供的制备方法包括以下步骤：将顺磁性金属靶材置于溅射室内,向溅射室内通入氩气和氧气,启动溅射室电源；磁控溅射结束后,向溅射室内通入乙炔气体；通乙炔气体结束后,向溅射室内通入保护气体,并对溅射室进行快速降温,得到内包顺磁性金属富勒烯颗粒；将内包顺磁性金属富勒烯颗粒、硬脂酸、抗肿瘤药物、挥发性有机溶剂和水混合,然后对混合液进行急速冷却,并搅拌挥去挥发性有机溶剂,离心分离,得到载抗肿瘤药物纳米机器人。本发明提供的制备方法生产工艺稳定,采用该方法制备得到的载抗肿瘤药物纳米机器人具有良好的尺寸均匀性、顺磁性和抗肿瘤功能。(The invention belongs to the field of nano materials, and particularly relates to an anti-tumor drug-loaded nano robot and a preparation method thereof. The preparation method provided by the invention comprises the following steps: placing a paramagnetic metal target material in a sputtering chamber, introducing argon and oxygen into the sputtering chamber, and starting a power supply of the sputtering chamber; after the magnetron sputtering is finished, introducing acetylene gas into the sputtering chamber; after the acetylene gas is introduced, introducing protective gas into the sputtering chamber, and rapidly cooling the sputtering chamber to obtain the fullerene particles internally coated with the paramagnetic metal; mixing the fullerene particles internally coated with paramagnetic metal, stearic acid, the anti-tumor drug, the volatile organic solvent and water, rapidly cooling the mixed solution, stirring to volatilize the volatile organic solvent, and performing centrifugal separation to obtain the anti-tumor drug-loaded nano robot. The preparation method provided by the invention has a stable production process, and the anti-tumor drug-loaded nano robot prepared by the method has good size uniformity, paramagnetism and anti-tumor functions.)

1. A preparation method of an anti-tumor drug-loaded nano robot comprises the following steps:

a) placing a paramagnetic metal target material in a sputtering chamber, introducing argon and oxygen into the sputtering chamber, and starting a power supply of the sputtering chamber to perform magnetron sputtering; in the magnetron sputtering process, the temperature of a cavity of the sputtering chamber is 800-1200 ℃;

b) after the magnetron sputtering is finished, maintaining the temperature of a cavity of the sputtering chamber at 800-1200 ℃, and introducing acetylene gas into the sputtering chamber;

c) after the acetylene gas is introduced, introducing protective gas into the sputtering chamber, and reducing the temperature of the cavity of the sputtering chamber to 100-300 ℃ within 5-15 min to obtain the internally-wrapped paramagnetic metal fullerene particles;

d) mixing the coated paramagnetic metal fullerene particles, stearic acid, an anti-tumor drug, a volatile organic solvent and water to obtain a mixed solution;

e) rapidly cooling the mixed solution, and stirring to volatilize the volatile organic solvent to obtain a suspension;

f) and carrying out centrifugal separation on the suspension, and discarding the supernatant to obtain the antitumor drug-loaded nano robot.

2. The method according to claim 1, wherein in step a), the paramagnetic metal target comprises one or more of iron oxide, titanium oxide and zirconium oxide.

3. The preparation method of claim 1, wherein in the step a), during the magnetron sputtering process, the vacuum degree of the sputtering chamber is 10-30 Pa; the gas inflow rate of the argon is 40-50 sccm; the air inflow rate of the oxygen is 10-15 sccm.

4. The manufacturing method according to claim 1, wherein in the step b), the degree of vacuum of the sputtering chamber is 10 to 30 Pa; the gas inflow rate of the acetylene gas is 80-120 sccm.

5. The method according to claim 1, wherein in step c), the particle size of the encapsulated paramagnetic metal fullerene particles is 1 to 100 nm.

6. The method according to claim 1, wherein the antitumor drug in step d) comprises one or more of paclitaxel, dexamethasone, and ranopharyn.

7. The method according to claim 1, wherein the mixed solution further contains folic acid in step d).

8. The method according to claim 1, wherein step e) comprises in particular:

and dripping the mixed solution into a liquid cooling medium, and stirring to volatilize the volatile organic solvent to obtain a suspension.

9. The method according to claim 8, wherein the dropping speed is 1 to 5 mL/min; the temperature of the liquid cooling medium is-10 to 5 ℃; the stirring speed is 100-300 r/min; the stirring time is 15-30 min.

10. The anti-tumor drug-loaded nano robot prepared by the preparation method according to any one of claims 1 to 9.

Technical Field

The invention belongs to the field of nano materials, and particularly relates to an anti-tumor drug-loaded nano robot and a preparation method thereof.

Background

Tumor tissue is actually a complete ecosystem formed by tumor cells and tumor blood vessels, and contains not only tumor cells but also abundant tumor blood vessels inside. Modern biomedical research has demonstrated that tumor vessels are structurally very different from normal vessels. In general, normal blood vessels take a year to grow and are three-layer compact structures consisting of intima, media and adventitia, whereas tumor vessels can be formed in only 4 days and are structurally single-layer thin films consisting of endothelial cells. However, because the endothelial cells constituting the tumor vessels have large gaps and incomplete structures, the tumor vessels usually contain a large number of small pores with nanometer scale, so that small molecules and some nanoparticles can pass through the pores.

When the size of the nanoparticles is a proper size (for example, 50-200 nm), it takes several minutes or even several tens of minutes to pass through the gaps of the tumor vessels, and in this process, the nanoparticles are tightly surrounded by the endothelial cells of the tumor vessels, so that the tumor vessels can be specifically destroyed by proper design. Researchers in the chemical institute firstly use magnetic metal fullerene to design water-soluble nanoparticles with the size of about 150 nanometers, and the nanoparticles can improve internal energy through absorbing radio frequency, and have phase change due to the rise of the internal energy after several minutes to dozens of minutes, and the internal energy is accompanied with the violent expansion of the volume by about 50 percent. Then the magnetic metal fullerene nano-particles are injected into the body of the mouse intravenously, and after a few minutes, the nano-particles reach the tumor position and are stuck on the vessel wall for a long time. The mice were then subjected to radio frequency to "detonate" the nanoparticles. Research results show that the magnetic metal fullerene nano-particles embedded on the wall of the tumor vessel are exploded to effectively destroy the tumor vessel, then the nutrition supply to the tumor is rapidly blocked, and the tumor cells can be completely starved within a few hours.

At present, most reported methods for preparing magnetic metal fullerene still stay in the experimental stage, and have the problems of poor stability of the preparation process, poor tumor treatment effect, difficult adjustment of the size of a product, poor size uniformity of the product and the like, thereby seriously influencing the application of the magnetic metal fullerene in the field of tumor treatment.

Disclosure of Invention

In view of the above, the present invention aims to provide an anti-tumor drug-loaded nano robot and a preparation method thereof, the preparation method provided by the present invention has a stable production process, the size of the nano robot to be prepared can be freely adjusted according to clinical requirements, and the anti-tumor drug-loaded nano robot prepared by the method has good size uniformity, paramagnetism and anti-tumor functions, and has a wide application prospect in the field of tumor treatment.

The invention provides a preparation method of an anti-tumor drug-loaded nano robot, which comprises the following steps:

a) placing a paramagnetic metal target material in a sputtering chamber, introducing argon and oxygen into the sputtering chamber, and starting a power supply of the sputtering chamber to perform magnetron sputtering; in the magnetron sputtering process, the temperature of a cavity of the sputtering chamber is 800-1200 ℃;

b) after the magnetron sputtering is finished, maintaining the temperature of a cavity of the sputtering chamber at 800-1200 ℃, and introducing acetylene gas into the sputtering chamber;

c) after the acetylene gas is introduced, introducing protective gas into the sputtering chamber, and reducing the temperature of the cavity of the sputtering chamber to 100-300 ℃ within 5-15 min to obtain the internally-wrapped paramagnetic metal fullerene particles;

d) mixing the coated paramagnetic metal fullerene particles, stearic acid, an anti-tumor drug, a volatile organic solvent and water to obtain a mixed solution;

e) rapidly cooling the mixed solution, and stirring to volatilize the volatile organic solvent to obtain a suspension;

f) and carrying out centrifugal separation on the suspension, and discarding the supernatant to obtain the antitumor drug-loaded nano robot.

Preferably, in step a), the paramagnetic metal target includes one or more of iron oxide, titanium oxide, and zirconium oxide.

Preferably, in the step a), the vacuum degree of the sputtering chamber is 10-30 Pa in the magnetron sputtering process; the gas inflow rate of the argon is 40-50 sccm; the air inflow rate of the oxygen is 10-15 sccm.

Preferably, in the step b), the vacuum degree of the sputtering chamber is 10-30 Pa; the gas inflow rate of the acetylene gas is 80-120 sccm.

Preferably, in the step c), the particle size of the encapsulated paramagnetic metal fullerene particles is 1-100 nm.

Preferably, in step d), the anti-tumor drug comprises one or more of paclitaxel, dexamethasone and ranophanin.

Preferably, in step d), the mixed solution further contains folic acid.

Preferably, step e) specifically comprises:

and dripping the mixed solution into a liquid cooling medium, and stirring to volatilize the volatile organic solvent to obtain a suspension.

Preferably, the dropping speed is 1-5 mL/min; the temperature of the liquid cooling medium is-10 to 5 ℃; the stirring speed is 100-300 r/min; the stirring time is 15-30 min.

The invention provides an anti-tumor drug-loaded nano robot prepared by the preparation method according to the technical scheme.

Compared with the prior art, the invention provides an anti-tumor drug-loaded nano robot and a preparation method thereof. The preparation method provided by the invention comprises the following steps: a) placing a paramagnetic metal target material in a sputtering chamber, introducing argon and oxygen into the sputtering chamber, and starting a power supply of the sputtering chamber to perform magnetron sputtering; in the magnetron sputtering process, the temperature of a cavity of the sputtering chamber is 800-1200 ℃; b) after the magnetron sputtering is finished, maintaining the temperature of a cavity of the sputtering chamber at 800-1200 ℃, and introducing acetylene gas into the sputtering chamber; c) after the acetylene gas is introduced, introducing protective gas into the sputtering chamber, and reducing the temperature of the cavity of the sputtering chamber to 100-300 ℃ within 5-15 min to obtain the internally-wrapped paramagnetic metal fullerene particles; d) mixing the coated paramagnetic metal fullerene particles, stearic acid, an anti-tumor drug, a volatile organic solvent and water to obtain a mixed solution; e) rapidly cooling the mixed solution, and stirring to volatilize the volatile organic solvent to obtain a suspension; f) and carrying out centrifugal separation on the suspension, and discarding the supernatant to obtain the antitumor drug-loaded nano robot. The preparation method comprises the steps of firstly preparing small-size paramagnetic metal-encapsulated fullerene particles with good paramagnetism and particle size uniformity by utilizing magnetron sputtering equipment under specific process conditions, then mixing the paramagnetic metal-encapsulated fullerene particles, an anti-tumor drug, stearic acid and a solvent to prepare a mixed solution, then rapidly cooling the mixed solution, converting stearic acid from a liquid state to a solid state in the rapid cooling process, adhering the metal fullerene particles and the anti-tumor drug in the mixed solution to form a plurality of microspheres formed by adhering the stearic acid, the metal fullerene particles and the anti-tumor drug, and finally separating the microspheres by adopting a centrifugal separation mode to obtain the anti-tumor drug-loaded nano robot. According to the preparation method provided by the invention, the wrapped paramagnetic metal fullerene with good paramagnetism is added into the nano robot, so that the nano robot can show good paramagnetism; meanwhile, the anti-tumor drug is loaded in the nano robot, so that the nano robot can show more excellent tumor treatment effect; in addition, the preparation method can also realize the regulation and control of the size of the prepared nano robot by changing the technological condition parameters of rapid cooling. The anti-tumor drug-loaded nano robot prepared by the method has good size uniformity, paramagnetism and anti-tumor functions, and controllable size, and can efficiently block tumor blood vessels in a targeted manner under the assistance of radio frequency to kill tumor cells; meanwhile, after the tumor cells are killed, the medicine can be completely degraded or absorbed by a human body within 3-5 weeks, and has no side effect. The preparation method provided by the invention has stable and controllable production process, is suitable for industrialization, and has wide application prospect in the field of tumor treatment.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a preparation method of an anti-tumor drug-loaded nano robot, which comprises the following steps:

a) placing a paramagnetic metal target material in a sputtering chamber, introducing argon and oxygen into the sputtering chamber, and starting a power supply of the sputtering chamber to perform magnetron sputtering; in the magnetron sputtering process, the temperature of a cavity of the sputtering chamber is 800-1200 ℃;

b) after the magnetron sputtering is finished, maintaining the temperature of a cavity of the sputtering chamber at 800-1200 ℃, and introducing acetylene gas into the sputtering chamber;

c) after the acetylene gas is introduced, introducing protective gas into the sputtering chamber, and reducing the temperature of the cavity of the sputtering chamber to 100-300 ℃ within 5-15 min to obtain the internally-wrapped paramagnetic metal fullerene particles;

d) mixing the coated paramagnetic metal fullerene particles, stearic acid, an anti-tumor drug, a volatile organic solvent and water to obtain a mixed solution;

e) rapidly cooling the mixed solution, and stirring to volatilize the volatile organic solvent to obtain a suspension;

f) and carrying out centrifugal separation on the suspension, and discarding the supernatant to obtain the antitumor drug-loaded nano robot.

In the preparation method provided by the invention, a paramagnetic metal target is firstly placed in a sputtering chamber. Wherein, the paramagnetic metal target material preferably comprises one or more of iron oxide, titanium oxide and zirconium oxide. Then, argon and oxygen are introduced into the sputtering chamber, and a power supply of the sputtering chamber is started to carry out magnetron sputtering. Wherein the flow rate of the argon gas is preferably 40-50 sccm, and specifically can be 40sccm, 41sccm, 42sccm, 43sccm, 44sccm, 45sccm, 46sccm, 47sccm, 48sccm, 49sccm or 50 sccm; the aeration time of the argon is consistent with the time of magnetron sputtering; the flow rate of the inlet gas of the oxygen is preferably 10-15 sccm, and specifically 10sccm, 11sccm, 12sccm, 13sccm, 14sccm or 15 sccm; the aeration time of the oxygen is preferably 3-5 min, and specifically can be 3min, 3.5min, 4min, 4.5min or 5 min; the power supply is preferably a Direct Current (DC) power supply; the voltage of the power supply is preferably 330-420V, and specifically can be 330V, 340V, 350V, 360V, 370V, 380V, 390V, 400V, 410V or 420V; the power of the power supply is preferably 2400-3600W, and specifically can be 2400W, 2500W, 2600W, 2700W, 2800W, 2900W, 3000W, 3100W, 3200W, 3300W, 3400W, 3500W or 3600W; in the magnetron sputtering process, the vacuum degree of the sputtering chamber is preferably controlled to be 10-30 Pa, and specifically can be 10Pa, 12Pa, 15Pa, 20Pa, 25Pa or 30 Pa; in the magnetron sputtering process, the temperature of the cavity of the sputtering chamber is preferably controlled at 800-1200 ℃, and specifically can be 800 ℃, 850 ℃, 900 ℃, 950 ℃, 980 ℃, 1000 ℃, 1050 ℃, 1100 ℃, 1150 ℃ or 1200 ℃; the magnetron sputtering time is preferably 3-8 min, and specifically can be 3min, 3.5min, 4min, 4.5min, 5min, 5.5min, 6min, 6.5min, 7min, 7.5min or 8 min.

In the preparation method provided by the invention, acetylene gas is introduced into the sputtering chamber after the magnetron sputtering is finished. Wherein, in the process of introducing the acetylene gas, the temperature of the cavity of the sputtering chamber is maintained at 800-1200 ℃, and specifically can be 800 ℃, 850 ℃, 900 ℃, 950 ℃, 980 ℃, 1000 ℃, 1050 ℃, 1100 ℃, 1150 ℃ or 1200 ℃; the vacuum degree of the sputtering chamber is preferably controlled to be 10-30 Pa, and specifically can be 10Pa, 12Pa, 15Pa, 20Pa, 25Pa or 30 Pa; the inflow rate of the acetylene gas is preferably 80-120 sccm, and specifically may be 80sccm, 85sccm, 90sccm, 95sccm, 100sccm, 105sccm, 110sccm, 115sccm or 120 sccm; the preferable aeration time of the acetylene gas is 5-15 min, and specifically can be 5min, 6min, 7min, 8min, 9min, 10min, 10.5min, 11min, 11.5min, 12min, 12.5min, 13min, 13.5min, 14min, 14.5min or 15 min.

In the preparation method provided by the invention, after the acetylene gas is introduced, protective gas is introduced into the sputtering chamber, and the sputtering chamber is rapidly cooled. The vacuum degree of the sputtering chamber is preferably controlled to be 10-30 Pa, and specifically can be 10Pa, 12Pa, 15Pa, 20Pa, 25Pa or 30 Pa; the protective gas is preferably argon and/or nitrogen; the flow rate of the shielding gas is preferably 700 to 1000sccm, and specifically 700sccm, 750sccm, 800sccm, 850sccm, 900sccm, 950sccm or 1000 sccm. In the invention, the process of rapidly cooling is preferably to reduce the temperature of the cavity of the sputtering chamber to 100-300 ℃ within 5-15 min, and the time consumed by rapidly cooling is more preferably 8-10 min, specifically 8min, 8.5min, 9min, 9.5min or 10 min; the temperature of the cavity after the rapid cooling is more preferably 150-250 ℃, and specifically can be 150 ℃, 160 ℃, 170 ℃, 180 ℃, 190 ℃, 200 ℃, 210 ℃, 220 ℃, 230 ℃, 240 ℃ or 250 ℃. After the rapid cooling is finished, obtaining inner-wrapped paramagnetic metal fullerene particles, wherein the inner-wrapped paramagnetic metal fullerene particles comprise paramagnetic metal particles and carbon spheres which wrap the paramagnetic metal particles and have fullerene shapes; the paramagnetic metal particles preferably comprise one or more of Fe, Zr, and Ti; the particle size of the fullerene particles containing the paramagnetic metal is preferably 1-100 nm, more preferably 10-30 nm, and specifically 10nm, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm or 100 nm.

In an embodiment provided by the present invention, the internally-encapsulated paramagnetic metal fullerene particles are prepared in a magnetron sputtering apparatus provided with a wafer inlet chamber, a sputtering chamber and a wafer outlet chamber, and the prepared internally-encapsulated paramagnetic metal fullerene particles are loaded by using a work holder, specifically including the following steps:

cleaning a workpiece clamp, then installing the workpiece clamp on a moving track of a magnetron sputtering device, enabling the workpiece clamp to enter a wafer inlet chamber along the track, closing a vacuum valve of the wafer inlet chamber, vacuumizing the wafer inlet chamber, opening the vacuum valve of the sputtering chamber after the vacuum degree meets the requirement, and enabling the workpiece clamp to enter the sputtering chamber along the track; the preparation of the fullerene particles containing the paramagnetic metal is carried out in the sputtering chamber, and the specific process is described above and is not described again; the encapsulated paramagnetic metal fullerene particles generated by rapid cooling are adsorbed around the workpiece clamp; the working clamp moves to the sheet outlet chamber along the operation track and is naturally cooled to room temperature under the protective gas atmosphere; and opening a vacuum valve of the sheet outlet chamber, enabling the working clamp to leave the sheet outlet chamber along the rail, and then collecting the paramagnetic metal fullerene particles internally wrapped on the working clamp.

In the invention, taking paramagnetic metal as Fe as an example, the physical and chemical processes involved in the preparation process of the fullerene particle containing the paramagnetic metal are as follows: after a power supply of the sputtering chamber is started, positive argon ions generated by ionization bombard the iron target to sputter Fe particles from the target, and the sputtered Fe particles are in an oxygen atmosphere, so that 2P of iron can be obtained due to the fact that the oxygen atoms are atoms with larger electronegativity¹And 2P³The characteristic peak moves to the direction with higher binding energy, so that Fe particles exist in the form of iron oxide; then leading acetylene gas into the sputtering chamber to surround and coat Fe particles, and reacting at high temperature to generate a carbon tube prototype; and finally, splitting the carbon tube prototype by rapid cooling to form carbon spheres coated with Fe particles, and generating fullerene morphology, namely the fullerene particles (Fe @ fullerene) coated with paramagnetic metal prepared by the invention.

In the preparation method provided by the invention, after the coated paramagnetic metal fullerene particles are obtained, the coated paramagnetic metal fullerene particles, stearic acid, an anti-tumor drug, a volatile organic solvent and water are mixed to obtain a mixed solution. Wherein the volatile organic solvent includes, but is not limited to, acetone; the antineoplastic medicine comprises one or more of Paclitaxel (PTX), Dexamethasone (DXM) and ranafol; the liquid mixture preferably further contains folic acid. In the invention, stearic acid is in a liquid state at the preparation temperature of more than 60 ℃, is degradable in vivo, has good biocompatibility and no toxicity to human bodies, and can make the drugs have slow release, controlled release and targeting effects after the stearic acid is cooled and solidified and is adhered with the anti-tumor drugs. In the invention, folic acid can specifically react with a folic acid receptor on the cell surface to form a compound, and the binding force of the folic acid and the compound is very strong, has high selectivity on tumors, and can be used as a modified compound of a tumor-targeted drug.

In one embodiment of the present invention, the mixed solution is preferably prepared according to the following steps:

i) mixing the fullerene particles internally coated with paramagnetic metal, the antitumor drug and the volatile organic solvent to obtain an organic solvent mixed system, namely a mixed system A;

mixing stearic acid and water to obtain a mixed system of stearic acid and water, namely a mixed system B;

ii) mixing the mixed system A and the mixed system B to obtain the mixed solution.

In the step of preparing the mixed solution provided in the above embodiment of the present invention, when the mixed system a is prepared, the mass ratio of the encapsulated paramagnetic metal fullerene particles to the antitumor drug is preferably (0.5-5): 3, specifically 0.5:3, 1:3, 1.5:3, 2:3, 2.5:3, 3:3, 3.5:3, 4:3, 4.5:3 or 5: 3; the mass ratio of the antitumor drug to the volatile organic solvent is preferably (0.5-5): 5, specifically 0.5:5, 1:5, 1.5:5, 2:5, 2.5:5, 3:5, 3.5:5, 4:5, 4.5:5 or 5: 5.

In the step of preparing the mixed solution provided in the above embodiment of the present invention, when the mixed system a is prepared, preferably, the anti-tumor drug and the volatile organic solvent are mixed to obtain a drug solution, and then the drug solution is mixed with the encapsulated paramagnetic metal fullerene particles to obtain the mixed system a. The second mixing step is preferably carried out under the conditions of ultrasound and stirring, and the rotating speed of the stirring is preferably 200-300 r/min, and specifically can be 200r/min, 250r/min or 300 r/min.

In the mixed liquid preparation step provided in the above embodiment of the present invention, when preparing the mixed system B, the mass ratio of stearic acid to water is preferably (2 to 6): 6, specifically 2:6, 2.5:6, 3:6, 3.5:6, 4:6, 4.5:6, 5:6, 5.5:6 or 6: 6; the mixing temperature is preferably controlled to be a temperature at which stearic acid can be kept in a liquid state, and particularly can be controlled to be about 60 ℃; the mixing mode is preferably stirring mixing, the stirring speed of the stirring mixing is preferably 500-800 r/min, specifically 500r/min, 550r/min, 600r/min, 650r/min, 700r/min, 750r/min or 800r/min, and the time of the stirring mixing is preferably 60-120 min, specifically 60min, 70min, 80min, 90min, 100min, 110min or 120 min.

In the step of preparing the mixed solution provided in the above embodiment of the present invention, in the step ii), the volume ratio of the mixed system a to the mixed system B is preferably (4 to 8): 3, specifically 4:3, 5:3, 6:3, 7:3 or 8: 3; the mixing temperature is preferably controlled to be 55-60 ℃; the mixing mode is preferably stirring mixing, the stirring speed of the stirring mixing is preferably 400-500 r/min, specifically 400r/min, 450r/min or 500r/min, and the stirring mixing time is preferably 20-30 min, specifically 20min, 25min or 30 min. In the present invention, if the mixed solution further contains folic acid, folic acid is added to the mixed solution after the mixed solution a and the mixed solution B are uniformly mixed. The addition amount of the folic acid is preferably 5-15 wt% of the total mixed mass of the mixed system A and the mixed system B, and specifically can be 10 wt%; the temperature of the continuous mixing is preferably 35-45 ℃.

In the preparation method provided by the invention, after a mixed solution is obtained, the mixed solution is rapidly cooled, and the volatile organic solvent is stirred and volatilized to obtain a suspension, wherein the preparation method specifically comprises the following steps:

and dripping the mixed solution into a liquid cooling medium, and stirring to volatilize the volatile organic solvent to obtain a suspension.

In the process of preparing the suspension provided by the invention, the dropping speed is preferably 1-5 mL/min, and specifically can be 1L/min, 1.5L/min, 2L/min, 2.5L/min, 3L/min, 3.5L/min, 4L/min, 4.5L/min or 5L/min; the liquid medium refers to liquid which can still keep a flowing state at low temperature; the temperature of the system in the stirring process is preferably controlled to be-10-5 ℃, and specifically can be-10 ℃, -9 ℃, -8 ℃, -7 ℃, -6 ℃, -5 ℃, -4 ℃, -3 ℃, -2 ℃, -1 ℃, 0 ℃, 1 ℃, 2 ℃, 3 ℃, 4 ℃ or 5 ℃; the stirring speed is preferably 100-300 r/min, and specifically can be 100r/min, 120r/min, 150r/min, 180r/min, 200r/min, 230r/min, 250r/min, 270r/min or 300 r/min; the stirring time is preferably 15-30 min, and specifically can be 15min, 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min or 30 min.

In the preparation method provided by the invention, after the suspension is obtained, the suspension is subjected to centrifugal separation. Wherein the rotating speed of the centrifugal separation is preferably 8000-10000 r/min, and specifically 8000r/min, 8500r/min, 9000r/min, 9500r/min or 10000 r/min; the time of the centrifugal separation is preferably 15-30 min, and specifically can be 15min, 18min, 20min, 23min, 25min, 27min or 30 min. And after the centrifugal separation is finished, removing the supernatant to obtain the antitumor drug-loaded nano robot. In the invention, the surface of the prepared antitumor drug-loaded nano robot can be better smooth and has better compactness through centrifugal separation.

The preparation method comprises the steps of firstly preparing small-size paramagnetic metal-encapsulated fullerene particles with good paramagnetism and particle size uniformity by utilizing magnetron sputtering equipment under specific process conditions, then mixing the paramagnetic metal-encapsulated fullerene particles, an anti-tumor drug, stearic acid and a solvent to prepare a mixed solution, then rapidly cooling the mixed solution, converting stearic acid from a liquid state to a solid state in the rapid cooling process, adhering the metal fullerene particles and the anti-tumor drug in the mixed solution to form a plurality of microspheres formed by adhering the stearic acid, the metal fullerene particles and the anti-tumor drug, and finally separating the microspheres by adopting a centrifugal separation mode to obtain the anti-tumor drug-loaded nano robot. According to the preparation method provided by the invention, the wrapped paramagnetic metal fullerene with good paramagnetism is added into the nano robot, so that the nano robot can show good paramagnetism; meanwhile, the anti-tumor drug is loaded in the nano robot, so that the nano robot can show more excellent tumor treatment effect; in addition, the preparation method can also realize the regulation and control of the size of the prepared nano robot by changing the technological condition parameters of rapid cooling. The anti-tumor drug-loaded nano robot prepared by the method has good size uniformity, paramagnetism and anti-tumor functions, and controllable size, and can efficiently block tumor blood vessels in a targeted manner under the assistance of radio frequency to kill tumor cells; meanwhile, after the tumor cells are killed, the medicine can be completely degraded or absorbed by a human body within 3-5 weeks, and has no side effect. The preparation method provided by the invention has stable and controllable production process, is suitable for industrialization, and has wide application prospect in the field of tumor treatment.

The invention also provides the anti-tumor drug-loaded nano robot prepared by the method of the technical scheme. The anti-tumor drug-loaded nano robot provided by the invention has good size uniformity, paramagnetism and anti-tumor functions, and controllable size, and can efficiently block tumor blood vessels in a targeted manner under the assistance of radio frequency to kill tumor cells; meanwhile, after tumor cells are killed, the nano-robot can be completely degraded or absorbed by a human body within 3-5 weeks without any side effect, so that the nano-robot carrying the anti-tumor drug has a wide application prospect in the field of tumor treatment. In addition, the metal fullerene has excellent free radical scavenging effect and has the function of resisting oxidation damage and repairing various cells, so the nano robot carrying the anti-tumor medicament provided by the invention can also be used as a medicament and has good treatment effect on various diseases.

For the sake of clarity, the following examples are given in detail.

Example 1

The preparation method of the Fe @ fullerene particles with the particle size of 20nm comprises the following specific steps:

1) cleaning: firstly, wiping a glass workpiece clamp with alcohol, then putting the workpiece clamp into an ultrasonic cleaning machine, carrying out deionized water ultra-cleaning and alcohol ultra-cleaning, and finally putting the workpiece clamp into alcohol steam for drying;

2) pasting a protective film: sticking a polyethylene film on the cleaned workpiece clamp, protecting the cleanliness of the workpiece clamp and protecting the workpiece clamp from being damaged;

3) installing the workpiece clamp pasted with the film on a running track of a magnetron sputtering device, and tearing off the protective film before entering a film inlet chamber;

4) the working clamp moves to a film feeding chamber of the magnetron sputtering device, then a vacuum gate valve is closed, and a vacuum pump is started to keep the vacuum degree at 12 Pa;

5) opening a vacuum gate valve of a sputtering chamber of the magnetron sputtering device, and enabling the workpiece clamp to enter the fixed position of the sputtering chamber through a track;

6) introducing argon and oxygen into the sputtering chamber, starting a DC power supply (360V, 3100W) of the sputtering chamber, bombarding an iron target (ferric oxide) for magnetron sputtering, and keeping for 3 min; wherein the argon flow is 40sccm, and the ventilation time is consistent with the time for performing magnetron sputtering; the oxygen flow is 10sccm, and the ventilation time is 3 min; during magnetron sputtering, the temperature of the cavity of the sputtering chamber is maintained at 1000 ℃;

7) after the magnetron sputtering is finished, maintaining the temperature of a cavity of the sputtering chamber to be 1000 ℃, and introducing acetylene gas into the sputtering chamber, wherein the flow rate of the acetylene gas is 80sccm, and the introduction time is 8 min;

8) after the acetylene gas is introduced, introducing argon as a protective gas, introducing the amount of the argon of 900sccm, and simultaneously starting a cooling device to ensure that the temperature of the cavity of the sputtering chamber is up to 200 ℃ within 10min to obtain carbon sphere particles adsorbed around the working clamp, namely the internally-wrapped paramagnetic metal fullerene particles prepared by the embodiment;

9) then, the working clamp moves to a film discharging chamber of the magnetron sputtering device along the operation track, protective gas argon is introduced into the film discharging chamber, the introduction amount is 300sccm, and the film discharging chamber is naturally cooled to 25 ℃;

10) and opening a vacuum valve of the sheet outlet chamber, enabling the working clamp to leave the sheet outlet chamber along the rail, and then collecting the internally-wrapped paramagnetic metal fullerene particles on the working clamp to obtain the internally-wrapped paramagnetic metal fullerene particles.

The particle size, particle size uniformity, purity, graphitization degree, saturation magnetization, residual magnetization, and coercive force of the encapsulated paramagnetic metal fullerene particles prepared in this example were measured.

Wherein the particle size and the particle size are uniformThe fullerene particle size uniformity is obtained by analyzing the morphology and crystal morphology of the prepared fullerene particles of the internally-wrapped paramagnetic metal by using a JEM-2010 high-resolution transmission electron microscope, and the calculation formula of the particle size uniformity is as follows: (D)_{Big (a)}-D_Small)/(D_{Big (a)}+D_Small)×100％，D_{Big (a)}Denotes the maximum diameter value, D, measured from the granules prepared_SmallRepresents the smallest diameter measured from the prepared particles;

the purity and graphitization degree are obtained by performing water solubility analysis on the prepared inner-wrapped paramagnetic metal fullerene particles by using an FES165 Fourier infrared spectrometer (FT-IR) and a cary-300VARIAN ultraviolet visible spectrum analyzer;

the magnetization and coercive force were obtained by performing magnetic property analysis of the prepared internally-wrapped paramagnetic metal fullerene particles using a Lakeshore 7410 vibrating sample magnetometer.

The measurement results are as follows: the grain size is 20nm, the grain size uniformity is less than 6.2%, the purity is more than 99.9%, the graphitization degree is more than 93%, the saturation magnetization is 19.589emu/G, the residual magnetization is 4.4632emu/G, and the coercive force is 561.36G.

Example 2

The preparation method of the Fe @ fullerene particles with the particle size of 25nm comprises the following specific steps:

1) cleaning: firstly, wiping a glass workpiece clamp with alcohol, then putting the workpiece clamp into an ultrasonic cleaning machine, carrying out deionized water ultra-cleaning and alcohol ultra-cleaning, and finally putting the workpiece clamp into alcohol steam for drying;

2) pasting a protective film: sticking a polyethylene film on the cleaned workpiece clamp, protecting the cleanliness of the workpiece clamp and protecting the workpiece clamp from being damaged;

3) installing the workpiece clamp pasted with the film on a running track of a magnetron sputtering device, and tearing off the protective film before entering a film inlet chamber;

4) the working clamp moves to a film feeding chamber of the magnetron sputtering device, then a vacuum gate valve is closed, and a vacuum pump is started to keep the vacuum degree at 15 Pa;

5) opening a vacuum gate valve of a sputtering chamber of the magnetron sputtering device, and enabling the workpiece clamp to enter the fixed position of the sputtering chamber through a track;

6) introducing argon and oxygen into the sputtering chamber, starting a DC power supply (400V and 3200W) of the sputtering chamber, bombarding an iron target (ferric oxide) for magnetron sputtering, and keeping for 6 min; wherein the argon flow is 48sccm, and the ventilation time is consistent with the time for performing magnetron sputtering; the oxygen flow is 12sccm, and the ventilation time is 4 min; during magnetron sputtering, the temperature of the cavity of the sputtering chamber is maintained at 950 ℃;

7) after the magnetron sputtering is finished, maintaining the temperature of a cavity of the sputtering chamber at 950 ℃, and introducing acetylene gas into the sputtering chamber, wherein the flow rate of the acetylene gas is 110sccm, and the introduction time is 10 min;

8) after the acetylene gas is introduced, introducing argon gas as a protective gas, introducing the amount of 800sccm, and simultaneously starting a cooling device to ensure that the temperature of the cavity of the sputtering chamber is up to 200 ℃ within 10min to obtain carbon sphere particles adsorbed around the working clamp, namely the internally-wrapped paramagnetic metal fullerene particles prepared by the embodiment;

9) then, the working clamp moves to a film discharging chamber of the magnetron sputtering device along the operation track, protective gas argon is introduced into the film discharging chamber, the introduction amount is 300sccm, and the film discharging chamber is naturally cooled to 25 ℃;

10) and opening a vacuum valve of the sheet outlet chamber, enabling the working clamp to leave the sheet outlet chamber along the rail, and then collecting the internally-wrapped paramagnetic metal fullerene particles on the working clamp to obtain the internally-wrapped paramagnetic metal fullerene particles.

The particle size, particle size uniformity, purity, graphitization degree, saturation magnetization, residual magnetization, and coercive force of the encapsulated paramagnetic metal fullerene particles prepared in this example were measured.

The particle size and the particle size uniformity are obtained by analyzing the morphology and the crystallization morphology of the prepared inner-wrapped paramagnetic metal fullerene particles by using a JEM-2010 high-resolution transmission electron microscope, and the calculation formula of the particle size uniformity is as follows: (D)_{Big (a)}-D_Small)/(D_{Big (a)}+D_Small)×100％，D_{Big (a)}Denotes the maximum diameter value, D, measured from the granules prepared_SmallRepresents the smallest diameter measured from the prepared particles;

the purity and graphitization degree are obtained by performing water solubility analysis on the prepared inner-wrapped paramagnetic metal fullerene particles by using an FES165 Fourier infrared spectrometer (FT-IR) and a cary-300VARIAN ultraviolet visible spectrum analyzer;

the magnetization and coercive force were obtained by performing magnetic property analysis of the prepared internally-wrapped paramagnetic metal fullerene particles using a Lakeshore 7410 vibrating sample magnetometer.

The measurement results are as follows: the grain size is 25nm, the uniformity of the grain size is less than 5.9%, the purity is more than 99.9%, the graphitization degree is more than 95%, the saturation magnetization is 19.629emu/G, the residual magnetization is 4.4872emu/G, and the coercive force is 566.56G.

Example 3

The preparation method of the Fe @ fullerene particles with the particle size of 30nm comprises the following specific steps:

1) cleaning: firstly, wiping a glass workpiece clamp with alcohol, then putting the workpiece clamp into an ultrasonic cleaning machine, carrying out deionized water ultra-cleaning and alcohol ultra-cleaning, and finally putting the workpiece clamp into alcohol steam for drying;

2) pasting a protective film: sticking a polyethylene film on the cleaned workpiece clamp, protecting the cleanliness of the workpiece clamp and protecting the workpiece clamp from being damaged;

3) installing the workpiece clamp pasted with the film on a running track of a magnetron sputtering device, and tearing off the protective film before entering a film inlet chamber;

4) the working clamp moves to a film feeding chamber of the magnetron sputtering device, then a vacuum gate valve is closed, and a vacuum pump is started to keep the vacuum degree at 15 Pa;

5) opening a vacuum gate valve of a sputtering chamber of the magnetron sputtering device, and enabling the workpiece clamp to enter the fixed position of the sputtering chamber through a track;

6) introducing argon and oxygen into the sputtering chamber, starting a DC power supply (420V, 3600W) of the sputtering chamber, bombarding an iron target (ferric oxide) for magnetron sputtering, and keeping for 8 min; wherein the argon flow is 50sccm, and the ventilation time is consistent with the time for performing magnetron sputtering; the oxygen flow is 15sccm, and the ventilation time is 5 min; during magnetron sputtering, the temperature of the cavity of the sputtering chamber is maintained at 980 ℃;

7) after the magnetron sputtering is finished, maintaining the temperature of a cavity of the sputtering chamber to be 980 ℃, and introducing acetylene gas into the sputtering chamber, wherein the flow rate of the acetylene gas is 120sccm, and the introduction time is 15 min;

8) after the acetylene gas is introduced, introducing argon gas as a protective gas, introducing the amount of 800sccm, and simultaneously starting a cooling device to ensure that the temperature of the cavity of the sputtering chamber is up to 200 ℃ within 10min to obtain carbon sphere particles adsorbed around the working clamp, namely the internally-wrapped paramagnetic metal fullerene particles prepared by the embodiment;

9) then, the working clamp moves to a film discharging chamber of the magnetron sputtering device along the operation track, protective gas argon is introduced into the film discharging chamber, the introduction amount is 300sccm, and the film discharging chamber is naturally cooled to 25 ℃;

10) and opening a vacuum valve of the sheet outlet chamber, enabling the working clamp to leave the sheet outlet chamber along the rail, and then collecting the internally-wrapped paramagnetic metal fullerene particles on the working clamp to obtain the internally-wrapped paramagnetic metal fullerene particles.

The particle size, particle size uniformity, purity, graphitization degree, saturation magnetization, residual magnetization, and coercive force of the encapsulated paramagnetic metal fullerene particles prepared in this example were measured.

The particle size and the particle size uniformity are obtained by analyzing the morphology and the crystallization morphology of the prepared inner-wrapped paramagnetic metal fullerene particles by using a JEM-2010 high-resolution transmission electron microscope, and the calculation formula of the particle size uniformity is as follows: (D)_{Big (a)}-D_Small)/(D_{Big (a)}+D_Small)×100％，D_{Big (a)}Denotes the maximum diameter value, D, measured from the granules prepared_SmallRepresents the smallest diameter measured from the prepared particles;

the purity and graphitization degree are obtained by performing water solubility analysis on the prepared inner-wrapped paramagnetic metal fullerene particles by using an FES165 Fourier infrared spectrometer (FT-IR) and a cary-300VARIAN ultraviolet visible spectrum analyzer;

the magnetization and coercive force were obtained by performing magnetic property analysis of the prepared internally-wrapped paramagnetic metal fullerene particles using a Lakeshore 7410 vibrating sample magnetometer.

The measurement results are as follows: the grain size is 30nm, the grain size uniformity is less than 5.2%, the purity is more than 99.9%, the graphitization degree is more than 96%, the saturation magnetization is 19.821emu/G, the residual magnetization is 4.4956emu/G, and the coercive force is 568.69G.

Example 4

The preparation method of the anti-tumor drug-loaded nano robot comprises the following specific steps:

1) dissolving paclitaxel in acetone, stirring for 10-15 min until completely dissolved, and adding into the above-mentioned solvent

1-3 weight ratio of prepared Fe @ fullerene particles (paclitaxel, acetone and Fe @ fullerene particles is

3:5:2), stirring and ultrasonic treatment are carried out, and the stirring speed is 200r/min, so as to obtain a mixed system A.

2) Preparing a certain amount of purified water, heating to 60-70 ℃, adding stearic acid (the mass ratio of the purified water to the stearic acid is 6:4), stirring for 60min at a stirring speed of 500r/min, and maintaining the temperature at 58-61 ℃ during stirring to obtain a mixed system B.

3) Slowly pouring the mixed system A into the mixed system B according to the volume ratio of 6:3 of the mixed system A to the mixed system B, continuously stirring at the speed of 400r/min, controlling the stirring temperature to be 55-60 ℃, stirring for 20min, then adding folic acid accounting for 10 wt% of the total mass of the mixed system, and continuously stirring, wherein the stirring temperature is controlled to be 35-45 ℃.

4) Dripping the mixed solution prepared in the step 3) into a liquid cooling medium (ice water doped with an antifreezing agent), continuously stirring to volatilize the organic solvent, and stirring for 30min to obtain a suspension.

5) Placing the suspension prepared in the step 4) in a centrifuge tube, centrifuging at 9000r/min for 15min to obtain the antitumor drug-loaded nano robot.

The prepared anti-tumor drug-loaded nano-robot is observed by a transmission electron microscope (JEM-100SX transmission electron microscope, JEOL company, Japan), and the result shows that: the anti-tumor drug-loaded nano-robot prepared by the embodiment has a round and uniform spherical particle in most parts and has no adhesion.

In this embodiment, by using Fe @ fullerene particles with different particle sizes and changing the condition parameters of step 4), the anti-tumor drug-loaded nano-robots with different particle sizes can be obtained, as shown in table 1:

TABLE 1 particle size of the anti-tumor drug-loaded nano-robot prepared under different process parameters

Serial number	Fe @ fullerene particles	Dropping speed	Temperature of agitation	Stirring speed	Particle size of nano robot
						1	20nm	1mL/min	-10℃	300r/min	50～100nm
2	25nm	3mL/min	-2℃	200r/min	100～200nm
						3	30nm	5mL/min	5℃	100r/min	200～300nm

It can be seen from table 1 that the smaller the Fe @ fullerene particles, the slower the dropping speed, the lower the temperature, the faster the stirring speed, the smaller the particles of the drug-loaded nano robot, and vice versa.

Comparative example 1

The preparation of fullerene comprises the following steps:

the method adopts a thermal evaporation method, uses natural graphite as a raw material, and synthesizes fullerene from evaporated carbon atoms in a non-oxidizing atmosphere at a very high temperature.

The saturation magnetization, residual magnetization and coercive force of the fullerene prepared in the comparative example were measured, and the results were: saturation magnetization of 40X 10^-3emu/g, remanent magnetization 8.5862X 10^-3emu/G and coercive force 162.8G.

Evaluation of Effect

1) Tumor treatment

1.1) introduction of radio frequency heating technology and heating principle:

1.1.1) radio frequency heating technique: the tissue to be heated is placed between a pair of capacitance plates (the electrodes are not in contact with the human body), and radio frequency voltage is applied between the electrodes (capacitance field method), so that the tissue body between the capacitance plates absorbs electric field energy and converts the electric field energy into heat energy during thermotherapy, thereby raising the temperature of the tissue. Generally, the radio frequency band is 13.56MHz, and the power is 0-800W.

1.1.2) heating principle: the nanometer robot carrying the antitumor drug absorbs the energy of electromagnetic waves under the action of an alternating magnetic field to generate vibration motion, paramagnetic metal fullerene particles contained in the nanometer robot generate heat due to hysteresis loss, and tumor tissues accumulated by the paramagnetic metal fullerene particles also generate heat due to heat transfer. The tumor tissue has the advantages of distorted and expanded blood vessels, large blood resistance, unhealthy vascular receptors, poor temperature sensitivity, difficult heat dissipation under the action of high temperature, easy heat accumulation, quick temperature rise, formation of a huge heat storage reservoir, 5-10 ℃ temperature difference with normal tissue, and severe volume expansion of about 30-60% of paramagnetic metal fullerene particles contained in the nano robot. If the temperature of the tumor tissue is made to reach 43 ℃ or the tumor blood vessels burst, the tumor cells are overheated or cut off the nutrition to die.

1.2) tumor treatment procedure:

1mg of the anti-tumor drug-loaded nano-robot with the particle size of 50-100 nm prepared in the embodiment 4 is diluted into physiological saline, the diluted nano-robot is intravenously injected into a mouse, and the nano-robots reach the tumor position and are clamped on the blood vessel wall for a long time after 30-60 min (because the endothelial cells forming the tumor blood vessel have large gaps and incomplete structures, the tumor blood vessel usually comprises a large number of small holes with nanometer scales, so that small molecules and some nano-particles can penetrate through the small holes, and when the anti-tumor drug-loaded nano-robot passes through the gaps of the tumor blood vessel, the anti-tumor drug-loaded nano-robot can be tightly surrounded by the endothelial cells of the tumor blood vessel). After the excessive anti-tumor drug-loaded nano robots are confirmed to flow away through CT scanning, radio frequency is applied to the mice to detonate the anti-tumor drug-loaded nano robots. After several minutes to dozens of minutes, the internal energy rises to generate phase change, and the volume is expanded by about 30-60% along with the violent expansion, so that the anti-tumor drug-loaded nano robots embedded on the walls of the tumor blood vessels explode to effectively destroy the tumor blood vessels, quickly block the nutrition supply to the tumor, and completely starve the tumor cells within several hours.

1.3) evaluation of biocompatibility and degradation:

the final products prepared in examples 1 to 4 and comparative example 1 were used as test samples, the tumor treatment procedure of step 1.2) was performed, and then the biocompatibility and degradation condition of each test sample in vivo were identified by CT image, and the results are shown in table 2:

TABLE 2 biocompatibility and degradation of different test samples

As can be seen from the data in Table 2, the anti-tumor drug loaded nano robot is completely degraded or absorbed by the human body within 3-5 weeks after killing tumor cells, and has no side effect.

2) Other effects

The metal fullerene has excellent free radical scavenging effect and has the function of resisting oxidation damage and repairing various cells, so the fullerene nano robot provided by the invention can be used as a medicine and has good treatment effect on various diseases.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.