Step-by-step targeted placenta trophoblast micro-nano robot as well as preparation method and application thereof

Abstract

The invention discloses a step-by-step targeting placenta trophoblast micro-nano robot as well as a preparation method and application thereof. The micro-nano robot comprises a yeast cell wall of a bag-shaped structure, bio-enzyme unevenly distributed on the surface of the yeast cell wall and targeted placenta trophoblast drug-loaded nanoparticles located in the yeast cell wall. The surfaces of the targeted placenta trophoblast drug-loaded nanoparticles are connected with placenta-like chondroitin sulfate A binding polypeptides. Inflammation placenta tissue can be targeted through macrophages, the medicine is accurately delivered to placenta trophoblastic cells through the medicine-carrying nano-particles connected with the targeting peptide, step-by-step accurate targeting is achieved, the medicine does not penetrate through the placenta barrier to reach the body of a fetus, and high biological safety is achieved for pregnancy administration.

Description

Technical Field

[0001] The present invention relates to the technical field of drug carriers, and particularly relates to a step-by-step targeted placental trophoblast micro-nanorobot, a preparation method thereof, and an application thereof. Background Art

[0002] Inflammation plays a key role in the development of placenta-related diseases. The latest research shows that the inflammatory response is not only a symptom of placental diseases, but may also be a triggering factor for these pathological conditions. The imbalance of inflammatory factors, such as cytokines, chemokines, and inflammatory mediators, during pregnancy may lead to abnormal placental function, and further cause diseases such as placental abruption, placental insufficiency, abnormal placental implantation, and preeclampsia. These inflammatory responses can interfere with the normal communication between the placenta and the mother and fetus, affect the fetal nutrient supply and oxygen transport, and thus lead to adverse pregnancy outcomes. In addition, studies have also found that the chronic inflammatory state may increase the incidence of placental diseases, which further emphasizes the importance of controlling inflammation during pregnancy to improve maternal and fetal health outcomes. Placenta-targeted drugs are an important treatment means. By concentrating the drugs to the placental lesion site, the treatment effect can be maximized, the distribution of drugs in other parts of the mother can be reduced, and thus the potential toxicity to the mother and fetus can be reduced. However, most of the existing placenta-targeted nano-drugs need to be delivered by intravenous injection. This method has high requirements for professional operation environments and personnel, increases the complexity and cost of treatment, requires professional medical operations, and has potential infection risks.

[0003] Therefore, in order to deliver drugs to the target organ more safely, there is an urgent need to develop a simple, low-cost, large-scale synthesis, high drug-loading capacity, and excellent biocompatibility micro-nano bio-robot. Summary of the Invention

[0004] Aiming at the defects in the prior art, the present invention provides a step-by-step targeted placental trophoblast micro-nanorobot, a preparation method thereof, and an application thereof. It can utilize raw materials such as glucose, fat, and urea to generate autonomous movement, improve the targeting of drug delivery, achieve step-by-step targeting and precise targeting, and has significant display significance and practical value.

[0005] The present invention provides a step-by-step targeted placental trophoblast micro-nanorobot, which includes a yeast cell wall with a sac-like structure, bioenzymes unevenly distributed on the surface of the yeast cell wall, and a drug-loaded nanoparticle targeting placental trophoblasts located inside the yeast cell wall;

[0006] The surface of the drug-loaded nanoparticle targeting placental trophoblasts is connected with a placental-like chondroitin sulfate A-binding polypeptide, and its amino acid sequence is as shown in SEQ ID NO.1.

[0007] In some embodiments, the yeast cells in the yeast cell wall of the sac-like structure are any one or more of Saccharomyces cerevisiae, Hanseniaspora uvarum, Issatchenkia orientalis, Kluyveromyces pichia, Pichia membranifaciens, Metschnikowia pulcherrima, Rhodosporidium toruloides, and Candida.

[0008] In some embodiments, the bioenzymes include any one or more of glucose oxidase, catalase, lipase, and trypsin; preferably, the bioenzymes are a combination of glucose oxidase and catalase; preferably, the mass ratio of glucose oxidase to catalase is (2-3):1, more preferably 3:1. The present invention discovers through research that by adjusting the bioenzymes, especially the ratio of catalase to glucose oxidase, the movement performance of the micro-nano bio-robots can be significantly affected; when the ratio of catalase to glucose oxidase is 3:1, the robots exhibit a higher movement rate and a longer movement distance.

[0009] In some embodiments, the drug-loaded nanoparticles targeting placental trophoblast cells include any one of lipid polymer nanoparticles, liposomes, and polymer nanoparticles.

[0010] In some embodiments, the drug-loaded nanoparticles targeting placental trophoblast cells include any one or more of small molecule drugs, polypeptides, macromolecule drugs, gene drugs, and mitochondria.

[0011] The present invention also provides a method for preparing the step-by-step targeted placental trophoblast cell micro-nano robot, comprising the following steps:

[0012] (1) Obtain a sac-like structure yeast cell wall to obtain a drug-loaded yeast microcapsule;

[0013] (2) Add an activator to the drug-loaded yeast microcapsule in step (1) to obtain a drug-loaded yeast microcapsule with a completely activated surface;

[0014] (3) Co-incubate one or more bioenzymes with the drug-loaded yeast microcapsule in step (2) to obtain a yeast biomimetic micro-nano robot precursor with asymmetric surface modification of bioenzymes;

[0015] (4) Prepare drug-loaded nanoparticles and connect chondroitin sulfate A-binding polypeptide-like placenta;

[0016] (5) Embed the drug-loaded nanoparticles obtained in step (4) into the yeast biomimetic micro-nano robot precursor in step (3) by electrostatic deposition;

[0017] (6) Purify the yeast biomimetic micro-nano robot precursor prepared in step (5) to obtain the step-by-step targeted placental trophoblast cell micro-nano robot.

[0018] In some embodiments, the mass ratio of the drug-loaded yeast microcapsules to the activator in step (2) is 1:(40 - 80).

[0019] In some embodiments, the mass ratio of the yeast bionic micro-nano robot precursor to the drug-loaded nanoparticles in step (5) is 1:(10 - 50).

[0020] In some embodiments, the activator in step (2) is a hydroxyl activator;

[0021] The hydroxyl activator is any one of tosyl chloride, cyanogen bromide, disuccinimidyl carbonate, N-hydroxysuccinimidyl chloroformate, carbonyldiimidazole, sodium periodate, N-acetyl-D-galactosamine, galactose oxidase, chloroacetic acid, and isothiocyanate.

[0022] The present invention also provides the application of the step-by-step targeted placental trophoblast micro-nano robot or the step-by-step targeted placental trophoblast micro-nano robot obtained by the preparation method in drug transportation.

[0023] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0024] 1. The present invention can target the inflamed placental tissue through macrophages, and accurately deliver the drug to the placental trophoblasts through the drug-loaded nanoparticles connected with the targeting peptide, realizing step-by-step precise targeting.

[0025] 2. The step-by-step targeted placental trophoblast micro-nano robot of the present invention can tolerate the acidic environment of the gastrointestinal tract, successfully pass through the gastrointestinal barrier and achieve effective absorption, and the oral route avoids the operation complexity brought by intravenous injection.

[0026] 3. The present invention can accurately target the placental trophoblasts without crossing the placental barrier to reach the fetal body, and has strong biological safety for drug administration during pregnancy.

[0027] 4. The yeast vector of the present invention is extracted from food-grade yeast cells, has low cost, simple preparation method, can be synthesized on a large scale, and has high biocompatibility; the surface-modified bioenzymes of the yeast microcapsules used are catalase, glucose oxidase, etc., and use raw materials such as glucose and urea in the body to provide driving force, and have high safety.

[0028] 5. By optimizing the bioenzyme ratio, the present invention can improve the movement efficiency of the robot, provide important guidance for its application in the biomedical field, and realize more efficient and precise biomedical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the accompanying drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as a limitation of the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0030] Figure 1 Schematic diagram of the structure of the step-by-step targeted placental trophoblast micro-nanorobot according to the embodiment of the present invention;

[0031] Figure 2 Scanning electron microscope comparison diagram of the yeast cell wall microcapsule and the double-enzyme modified yeast biomimetic micro-nanorobot according to the embodiment of the present invention, where (1) is the transmission electron microscope image and (2) is the scanning electron microscope image;

[0032] Figure 3 Fluorescence image of the asymmetric distribution of surface enzymes of the step-by-step targeted placental trophoblast by the double-enzyme modification according to the embodiment of the present invention;

[0033] Figure 4 Fluorescence image of the step-by-step targeted placental trophoblast micro-nanorobot being taken up by macrophages according to the embodiment of the present invention;

[0034] Figure 5 Fluorescence imaging diagram of the co-localization of the step-by-step targeted placental trophoblast micro-nanorobot with lysosomes in macrophages according to the embodiment of the present invention;

[0035] Figure 6 Fluorescence image of the drug-loaded particles targeting trophoblasts after the step-by-step targeted placental trophoblast micro-nanorobot ruptures at the placental site according to the embodiment of the present invention;

[0036] Figure 7 Fluorescence image of the drug distribution after oral administration of curcumin and the placental trophoblast-targeted drug-loaded yeast biomimetic micro-nanorobot in the test example of the present invention;

[0037] Figure 8 Movement trajectory of the micro-nano bio-robot transported under different ratios of catalase and glucose oxidase at the same glucose concentration according to the present invention;

[0038] Figure 9 Difference in the movement rate of the micro-nano bio-robot transported under different ratios of catalase and glucose oxidase at the same glucose concentration according to the present invention. Detailed implementation manners

[0039] To enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0040] The schematic diagram of the preparation process of the yeast biomimetic micro-nanorobot of the present invention is as Figure 1 shown. First, yeast is treated with acid and alkali to extract the yeast cell wall, and then after activating its surface, the activated yeast cell wall microcapsules are subjected to a linking reaction with catalase and glucose oxidase to obtain the yeast biomimetic micro-nanorobot. Then, the placenta-targeted drug-loaded nanoparticles are loaded into the yeast biomimetic micro-nanorobot by electrostatic adsorption to obtain the placenta-targeted drug-loaded yeast biomimetic micro-nanorobot.

[0041] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0042] Example 1

[0043] The preparation method of the yeast biomimetic immune micro-nanobiobot in this example includes the following steps:

[0044] (1) Extraction: Dissolve 100 mg of Saccharomyces cerevisiae in 10 mL of 1 M NaOH solution, incubate at 80 °C for 1 hour, then centrifuge at 3000 rmp for 10 min, discard the supernatant, wash twice with double-distilled water, add 10 mL of 1 M HCL, incubate at 60 °C for 1 hour, centrifuge at 3000 rpm for 10 min, discard the supernatant, wash twice with double-distilled water, add 45 mL of isopropanol and wash 4 times, wash twice with acetone and then discard the supernatant, and obtain the negatively charged yeast cell wall after vacuum drying.

[0045] (2) Activation: Prepare carbodiimide, 1,1'-carbonyldiimidazole (CDI) using ultradry DMSO at a concentration of 50 mg / mL, add it to the extracted yeast cell wall to activate the hydroxyl groups on the cell wall, and activate 0.5 mg of yeast microcapsules with every 1 mL of CDI; after activation, centrifuge at 1000 rpm for 15 min, discard the supernatant, and wash 3 times with double-distilled water.

[0046] (3) Enzyme linking: Add glucose oxidase and catalase in a ratio of 3:1, and incubate at 4 °C overnight or at room temperature for 6 hours.

[0047] (4) Preparation of curcumin-loaded cationic nanoparticles: Dissolve 750 μg of curcumin in water, add 3 mL of 4% ethanol solution, 90 μg of soybean lecithin solution, and 210 μg of Streptavidin-PEG(2000)-DSPE as the aqueous phase. Use 2 mg of PLGA dissolved in acetonitrile as the organic phase, and prepare curcumin-loaded nanoparticles using a nanoparticle synthesizer.

[0048] (5) Linking the targeting peptide: After dialysis of the nanoparticles obtained in step (4), add the placenta-targeting peptide biotin-plCSA. The targeting peptide sequence is EDVKDINFDTKEKFLAGCLIVSFHEGKC (SEQ ID NO.1). Add 25 μg of biotin-plCSA to every 1 mL of nanoparticles, incubate at 37 °C for 4 hours, and then dialyze again to obtain purified placenta-targeted drug-loaded nanoparticles.

[0049] (6) Preparation of drug-loaded yeast cell wall microcapsules: Dissolve 1 mg of empty yeast cell wall microcapsules in 0.1 M sodium bicarbonate buffer, add 10 mg of drug-loaded cationic nanoparticles, incubate at 37 °C for 6 h. The placenta-targeted drug-loaded nanoparticles are adsorbed and deposited into the yeast cell wall. Wash 4 times with ultrapure water to obtain trophoblast-targeted drug-loaded yeast biomimetic micro-nano robots.

[0050] (7) Administer 2 mg orally each time (the oral dose can be 0.5 - 2 mg) of the step-by-step targeted placenta trophoblast micro-nano robots, which are taken up by macrophages in the Peyer's patches of the intestine, reach the placenta site to release the yeast microcapsules, and the yeast microcapsules rupture in the inflammatory environment to release the drug-loaded nanoparticles, which precisely target trophoblasts through the targeting peptide.

[0051] The transmission electron microscopy and scanning electron microscopy images of the drug-loaded yeast biomimetic robots are as Figure 2 shown, and the asymmetric distribution of glucose oxidase and catalase on the surface of the drug-loaded yeast biomimetic micro-nano robots can be observed. The fluorescence imaging images of the drug-loaded yeast biomimetic micro-nano robots are as Figure 3 shown, and it can be seen that there is asymmetric modification of glucose oxidase and catalase on the surface of the yeast biomimetic micro-nano robots.

[0052] Through fluorescence imaging of the step-by-step targeted placenta trophoblast micro-nano robots, as Figure 4 shown, (1) is the macrophage nucleus stained with DAPI, (2) is the macrophage marker labeled with F4 / 80, (3) is the drug-loaded yeast biomimetic micro-nano robot labeled with green fluorescence, and (4) is the fluorescence imaging image of the co-localization of macrophages and yeast biomimetic micro-nano robots. It can be seen from the fluorescence imaging that macrophages can take up the micro-nano biological robots.

[0053] Through the co-localization fluorescence imaging of the drug-loaded yeast biomimetic micro-nanorobot and macrophage lysosomes, the results are as follows Figure 5 shown. (1) is the macrophage nucleus stained with DAPI, (2) is the macrophage lysosome labeled with a lysosome fluorescent probe, (3) is the drug-loaded yeast biomimetic micro-nanorobot, and (4) is the fluorescence imaging map of the co-localization of the drug-loaded yeast biomimetic micro-nanorobot and lysosomes. It can be seen that the drug-loaded yeast biomimetic micro-nano biological robot co-localizes with the lysosomes of macrophages inside the macrophages.

[0054] Figure 6 is the fluorescence imaging map of the micro-nanorobot targeting trophoblasts step by step to the placental trophoblasts. (1) is the trophoblast nucleus stained with DAPI, (2) is the trophoblast cytoskeleton stained with phalloidin, (3) is the placenta-targeted curcumin-loaded nanoparticles, and (4) is the fluorescence imaging map of the placenta trophoblast targeting the curcumin-loaded nanoparticles targeting trophoblasts. Fluorescence imaging indicates that the curcumin nanoparticles aggregate on the trophoblast cell membrane and partially enter the cells, indicating that the curcumin-loaded nanoparticles have good trophoblast targeting after being released in the inflammatory microenvironment.

[0055] Example 2

[0056] The preparation method of the yeast biomimetic immune micro-nano biological robot in this example includes the following steps:

[0057] (1) Extraction: Dissolve 100 mg of Saccharomyces cerevisiae in 10 mL of 1 M NaOH solution, incubate at 80 °C for 1 hour, then centrifuge at 3000 rmp for 10 min, discard the supernatant, wash twice with double-distilled water, add 10 mL of 1 M HCl, incubate at 60 °C for 1 hour, centrifuge at 3000 rpm for 10 min, discard the supernatant, wash twice with double-distilled water, add 45 mL of isopropanol and wash 4 times, wash twice with acetone and then discard the supernatant, and obtain a negatively charged yeast cell wall after vacuum drying.

[0058] (2) Activation: Prepare carbodiimide, 1,1'-carbonyldiimidazole (CDI) using ultra-dry DMSO at a concentration of 50 mg / mL, add it to the extracted yeast cell wall to activate the hydroxyl groups on the cell wall, and add 0.5 mg of yeast microcapsules to 1 mL of CDI for activation; after activation, centrifuge at 1000 rpm for 15 min, discard the supernatant, and wash 3 times with double-distilled water.

[0059] (3) Enzyme connection: Add glucose oxidase and catalase in a ratio of 2:1, and incubate at 4 °C overnight or at room temperature for 6 hours.

[0060] (4) Preparation of curcumin-loaded cationic nanoparticles: Dissolve 750 μg of curcumin in water, add 3 mL of 4% ethanol solution, 90 μg of soybean lecithin solution, and 210 μg of Streptavidin-PEG(2000)-DSPE as the aqueous phase. Use 2 mg of PLGA dissolved in acetonitrile as the organic phase. Prepare curcumin-loaded nanoparticles using a nanoparticle synthesizer.

[0061] (5) Linking the targeting peptide: After dialysis of the nanoparticles obtained in step (4), add the placental targeting peptide biotin-plCSA. Add 25 μg of biotin-plCSA to every 1 ml of nanoparticles. Incubate at 37 °C for 4 hours and then dialyze again to obtain purified placental targeting drug-loaded nanoparticles.

[0062] (6) Preparation of drug-loaded yeast cell wall microcapsules: Dissolve 1 mg of empty yeast cell wall microcapsules in 0.1 M sodium bicarbonate buffer, add 10 mg of drug-loaded cationic nanoparticles, and incubate at 37 °C for 6 h. The placental targeting drug-loaded nanoparticles are adsorbed and deposited into the yeast cell wall. Wash 4 times with ultrapure water to obtain trophoblast-targeting drug-loaded yeast biomimetic micro-nanorobots.

[0063] Experimental Example 1

[0064] Cage 8-week-old mice. When a vaginal plug is detected, it is marked as E0.5. Start injecting LPS (50 μg / kg) at E8.5 and inject continuously for three days to construct a placental inflammation model. From gestational day 10.5, divide the mice into three groups: a control group, a curcumin-loaded nanoparticle yeast microcapsule group, and a stepwise placental trophoblast-targeting curcumin-loaded nanoparticle yeast biomimetic robot group. According to the different groups, give 2 mg of the drug orally every two days. Observe the in vivo drug distribution at gestational day 18.5.

[0065] Figure 7 This is the drug distribution diagram. After oral administration of curcumin and fluorescence imaging in the placental trophoblast-targeting drug-loaded yeast biomimetic micro-nanorobot group, observe the distribution of curcumin in the placenta and fetus of mice by small animal imaging. (1) shows the fluorescence imaging of the placenta and fetus of mice in the oral curcumin group; (2) shows the oral trophoblast-targeting micro-nanorobot group. It can be seen that there is no fluorescence in the fetus and placenta in the oral curcumin group, and there is a strong fluorescence signal in the placenta in the oral trophoblast-targeting micro-nanorobot group, and no fluorescence signal in the fetus, indicating that the micro-nano biological robot has good placental targeting.

[0066] Experimental Example 2

[0067] The synthesized micro-nano robots in Examples 1 and 2 were respectively dropped on glass slides and photographed using a high-speed inverted microscope. Each field of view was photographed for 20 s, the movement trajectories of individual micro-nano robots were traced, and the movement trajectories were extracted using Image J and the movement rates were analyzed.

[0068] The results are as Figure 8 shown, demonstrating the influence of different ratios of glucose oxidase (GOx) and catalase (Cat) on the movement trajectories of micro-nano bio-robots at the same glucose concentration. (1) and (2) in the figure represent the ratios of Example 2 and Example 1 respectively. It can be observed that as the ratio of GOx and Cat changes, the movement trajectories of the micro-nano bio-robots also change significantly. At the ratio of GOx:Cat = 3:1, the movement trajectory of the robot is significantly longer than that at GOx:Cat = 2:1, indicating a farther movement distance. By Figure 9 further quantifying the average movement rates of micro-nano bio-robots under different enzyme ratios. The results show that at the ratio of GOx:Cat = 3:1, the average movement rate of the robot is significantly higher than that at GOx:Cat = 2:1, and the specific rates are approximately 1.5 um / s and 0.8 um / s respectively, indicating that the enzyme ratio has a significant influence on the movement performance of the robot.

[0069] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A micro-nano robot that targets placental trophoblast cells step by step, characterized in that: It comprises a yeast cell wall with a sac-like structure, a biological enzyme unevenly distributed on the surface of the yeast cell wall, and a placenta trophoblast-targeted drug-loaded nanoparticle located inside the yeast cell wall; The surface of the placental trophoblast targeted drug-loaded nanoparticles is connected to a placental-like chondroitin sulfate A binding polypeptide, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. The step-by-step targeted placental trophoblast micro-nano robot according to claim 1, characterized in that: The yeast cells in the yeast cell wall of the capsule structure are any one or more of Saccharomyces cerevisiae, Hansenula sporea, Issatchenko mycelium orientalis, Pichia Kluyveromyces, Pichia pastoris, Maggimyces maggi, Rhodosporidium sporea and Candida.

3. The step-by-step targeted placental trophoblast micro-nano robot according to claim 1, characterized in that: The biological enzyme includes any one or more of glucose oxidase, catalase, lipase and trypsin.

4. The step-by-step targeted placental trophoblast micro-nano robot according to claim 1, characterized in that: The drug-loaded nanoparticles targeted to placental trophoblast cells include any one of lipid polymer nanoparticles, liposomes and polymer nanoparticles.

5. The step-by-step targeted placental trophoblast micro-nano robot according to claim 1, characterized in that: The drug-loaded nanoparticles targeted to placental trophoblast cells include any one or more of small molecule drugs, polypeptides, macromolecule drugs, gene drugs and mitochondria.

6. The method for preparing the micro-nano robot for step-by-step targeting of placental trophoblast cells according to any one of claims 1 to 5, characterized in that: The steps include: (1) Obtaining capsule-like yeast cell walls to obtain drug-loaded yeast microcapsules; (2) adding an activator to the drug-loaded yeast microcapsules described in step (1) to obtain drug-loaded yeast microcapsules with completely activated surfaces; (3) co-incubating one or more biological enzymes with the drug-loaded yeast microcapsules described in step (2) to obtain a yeast bionic micro-nano robot precursor with asymmetrically modified biological enzymes on its surface; (4) preparing drug-loaded nanoparticles and linking them with placental-like chondroitin sulfate A binding polypeptide; (5) embedding the drug-loaded nanoparticles obtained in step (4) into the yeast biomimetic micro-nano robot precursor described in step (3) by electrostatic deposition; (6) Purifying the yeast bionic micro-nano robot precursor prepared in step (5) to obtain the step-by-step targeted placental trophoblast micro-nano robot.

7. The preparation method according to claim 6, characterized in that: In step (2), the mass ratio of the drug-loaded yeast microcapsules to the activator is 1:(40-80).

8. The preparation method according to claim 6, characterized in that: In step (5), the mass ratio of the yeast biomimetic micro-nano robot precursor to the drug-loaded nanoparticles is 1:(10-50).

9. The preparation method according to claim 6, characterized in that: The activator in step (2) is a hydroxy activator; The hydroxyl activator is any one of tosyl chloride, cyanogen bromide, disuccinimidyl carbonate, N-hydroxysuccinimidyl chloroformate, carbonyl diimidazole, sodium periodate, N-acetyl-D-galactosamine, galactose oxidase, chloroacetic acid and isothiocyanate.

10. Use of the step-by-step targeted placental trophoblast micro-nano robot described in any one of claims 1 to 5 or the step-by-step targeted placental trophoblast micro-nano robot obtained by the preparation method described in any one of claims 6 to 9 in drug transportation.

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