CN114425088B - Yeast bionic immune micro-nano biological robot and preparation method and application thereof - Google Patents
Yeast bionic immune micro-nano biological robot and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a yeast bionic immune micro-nano biological robot and a preparation method and application thereof. The multi-layer assembled and expanded biotinylase layer and the streptavidin layer greatly increase the content of biological enzyme, thereby improving the driving force of the yeast bionic immune micro-nano biological robot, and simultaneously, the chassis yeast cell wall microcapsule can stimulate immune cells to produce anti-inflammatory factors and play an anti-inflammatory role.
Description
Technical Field
The invention belongs to the technical field of drug carriers, and particularly relates to a yeast bionic immune micro-nano biological robot and a preparation method and application thereof.
Background
Inflammation-related diseases including tumor, obesity, atherosclerosis, osteoporosis, osteosclerotic disease, hepatitis, pneumonia, neurodegenerative diseases, arthritis, cystitis, colitis, diabetes, preeclampsia, etc. seriously affect human health, and due to physiological barrier effects in the body, such as mucus barrier, blood brain barrier, placenta barrier, qi-blood barrier, etc., whether free drugs or conventional nano-delivery systems hardly break through the physiological barrier to reach focus sites, it has been found in recent researches that only 0.7% of nanoparticles are successfully delivered into solid tumors, and this low value seriously affects clinical transformation of nano-drugs.
The emerging micro-nano robot is expected to bring subversion change to the field of biological medicine due to the advantages of active transportation, barrier penetration and the like. The enzyme-driven micro-nano biological robot is a micro-nano device which converts chemical energy into mechanical energy by utilizing the catalytic reaction between biological enzyme and a corresponding substrate. Because it does not need a complex external device, it is more suitable for application in an in vivo environment. However, the application of enzyme-driven micro-nano robots in vivo faces several challenges: (1) Most of the materials are substrates of micro-nano biological robots, and the materials have no biological effect and poor biocompatibility; (2) At present, the micro-nano robot is prepared by common spraying, electrochemical deposition and other methods, only focuses on the movement function, and has no medicine carrying part and low medicine carrying quantity; (3) the driving force of the enzyme-driven micro-nano robot is weak at present. Cannot move under high salt and high viscosity conditions.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a yeast bionic immune micro-nano biological robot and a preparation method and application thereof. The yeast bionic immune micro-nano biological robot is expanded through multi-layer assembly, so that the content of biological enzyme is greatly increased, the driving force of the yeast bionic immune micro-nano biological robot is improved, and meanwhile, the chassis yeast cell wall microcapsule can stimulate immune cells to produce anti-inflammatory factors, so that the yeast bionic immune micro-nano biological robot provided by the invention is particularly suitable for treating and detecting inflammation related diseases.
The method is realized by the following technical scheme:
the yeast bionic immune micro-nano biological robot comprises a yeast cell wall microcapsule, drug-loaded nanoparticles, a biotinylase layer and a streptavidin layer, wherein the biotinylase layer and the streptavidin layer are assembled and expanded in a plurality of layers and are modified on part of the outer surface of the yeast cell wall microcapsule, and the drug-loaded nanoparticles are wrapped inside the yeast cell wall microcapsule.
Further, the yeast cell wall microcapsule is taken from one or more of Saccharomyces cerevisiae, hansenula polymorpha, issatchenkia orientalis, kluyveromyces pichia, pichia membranaceus, meinachia Meinaki, rhodosporidium and Candida.
Further, the nanoparticle in the drug-loaded nanoparticle is one or more of charged lipid polymer nanoparticle, liposome, polymer nanoparticle and albumin nanoparticle.
Further, the drug carried by the drug-carrying nano-particles is one or more of small molecular drugs, polypeptides, macromolecular protein drugs, gene drugs, heavy metal capturing agents, virus capturing agents or bacteria capturing agents.
Further, the enzyme in the biotinylase layer is one or more of glucose oxidase, catalase, urease, lipase, trypsin, acetylcholinesterase, deoxyribonuclease (dnase) or triphosphatase (atpase).
The invention also provides a preparation method of the yeast bionic immune micro-nano biological robot, which comprises the following steps:
(1) Embedding the drug-carrying nano-particles into yeast cell wall microcapsules by an electrostatic deposition mode to obtain drug-carrying yeast microcapsules;
(2) Adding a masking solution into the drug-loaded yeast microcapsule prepared in the step (1), binding the drug-loaded yeast microcapsule on a plate by the masking solution, masking part of the drug-loaded yeast microcapsule, adding an activating agent, floating above the masking solution by the activating agent, and carrying out surface activation on the unmasked part of the drug-loaded yeast microcapsule;
(3) Incubating the biotinylated enzyme and the surface part activated yeast microcapsule prepared in the step (2) to obtain a yeast bionic immune micro-nano robot with a surface part coated with a single-layer biotinylated enzyme layer;
(4) Incubating the yeast bionic immune micro-nano biological robot with the surface partially coated with the single-layer biotinylase layer prepared in the step (3) with streptavidin, centrifuging and washing, and collecting precipitate to obtain the yeast bionic immune robot with the surface partially coated with the single-layer biotinylase layer and the single-layer streptavidin layer;
(5) The yeast bionic immune robot with the surface partially coated with the single-layer biotinylase layer and the single-layer streptavidin layer prepared in the step (4) is incubated with biotinylase to obtain the yeast bionic micro-nano biological robot modified by double-layer biotinylase;
(6) And (5) repeating the steps (4) to (5) to obtain the multilayer biotinylase modified yeast bionic micro-nano biological robot.
Preferably, the mass ratio of the drug-loaded yeast microcapsule to the masking solution is 1: (0.01-0.25), the mass ratio can ensure that the drug-loaded yeast microcapsule is partially masked, and the condition that the biotinylation enzyme cannot be modified because the drug-loaded yeast microcapsule is completely masked is avoided.
Preferably, in the step (2), the mass ratio of the drug-loaded yeast microcapsule to the activator is 1: (40-80).
Further, the masking solution includes, but is not limited to, at least one of ethylene glycol, glycerol, propylene glycol, polyethylene glycol, maltitol, xylitol, and sorbitol.
Further, the activator is a hydroxyl activator including, but not limited to, tosyl chloride, cyanogen bromide, disuccinimidyl carbonate, N-hydroxysuccinimidyl chloroformate, carbonyldiimidazole, sodium periodate, N-acetyl-D-galactosamine, galactose oxidase, chloroacetic acid, or isothiocyanate.
Further, in the step (3), the biotinylated enzyme is excessively added relative to the surface-partially-activated yeast microcapsule, so that on one hand, successful modification of the biotinylated enzyme can be ensured, and on the other hand, the excessive enzyme can be recycled.
The invention also provides application of the yeast bionic immune micro-nano biological robot in drug transportation, in particular to preparation of inflammatory disease drugs.
The inflammatory diseases include, but are not limited to, one or more of tumors, obesity, atherosclerosis, osteoporosis, osteosclerotic diseases, hepatitis, pneumonia, neurodegenerative diseases, arthritis, cystitis, gastric ulcers, colitis, diabetes, preeclampsia.
The beneficial effects of the invention include the following aspects:
1. the yeast bionic immune micro-nano biological robot provided by the invention takes the yeast cell wall microcapsule as a chassis cell, and the yeast cell wall microcapsule is derived from edible yeast cells, so that the yeast bionic immune micro-nano biological robot has excellent biocompatibility;
2. the yeast cell wall has an immunological effect, can target macrophages and stimulate the macrophages to produce anti-inflammatory factors such as interleukin 10 (IL 10) and the like;
3. the yeast bionic immune micro-nano biological robot provided by the invention greatly increases the content of biological enzymes through multi-layer assembly expansion, thereby improving the driving force of the yeast bionic immune micro-nano biological robot.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a preparation process of a yeast bionic immune micro-nano biological robot provided by the invention;
FIG. 2 is a scanning electron microscope image comparison diagram of a yeast cell wall microcapsule and multilayer urease modified yeast bionic immune micro-nano biological robot;
FIG. 3 is a graph showing the comparison of motion trajectories of single-layer urease and multi-layer urease modified yeast bionic immune micro-nano biological robots in urea solution;
FIG. 4 is a graph comparing diffusion results of single-layer urease and multi-layer urease modified yeast bionic immune micro-nano biological robots in mucus;
FIG. 5 is a diagram of the motion trace of a multi-layered glucose oxidase modified yeast biomimetic immunomicrobial robot in 0.9% physiological saline and 3% high salt solution;
fig. 6 is a graph comparing the therapeutic effect of gastric ulcer in mice, wherein fig. 6 (1) is a graph showing the therapeutic effect of oral administration of free curcumin to mice, fig. 6 (2) is a graph showing the therapeutic effect of oral administration of single-layer urease modified curcumin-carrying yeast immune micro-nano robot to mice, and fig. 6 (3) is a graph showing the therapeutic effect of oral administration of multi-layer urease modified curcumin-carrying yeast immune micro-nano robot to mice.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides a yeast bionic immune micro-nano biological robot and a preparation method thereof.
In some embodiments, the yeast cell wall microcapsule is taken from one or more of Saccharomyces cerevisiae, hansenula polymorpha, issatchenkia orientalis, kluyveromyces pichia pastoris, pichia mozukuri, mei Qiqi, rhodosporidium and Candida.
In some embodiments, the nanoparticle in the drug-loaded nanoparticle may be selected from one or more of a charged lipid polymer nanoparticle, a liposome, a polymer nanoparticle, and an albumin nanoparticle.
In some embodiments, the drug loaded nanoparticle is one or more of a small molecule drug, a polypeptide, a macromolecular protein drug, a genetic drug, a heavy metal capture agent, a viral capture agent, or a bacterial capture agent.
The enzyme in the biotinylated enzyme layer may be selected from one or more of glucose oxidase, catalase, urease, lipase, trypsin, acetylcholinesterase, deoxyribonuclease (dnase), and atpase (atpase).
The yeast bionic immune micro-nano biological robot is expanded through multi-layer assembly, so that the content of biological enzyme is greatly increased, the driving force of the yeast bionic immune micro-nano biological robot is improved, meanwhile, the main component of the chassis yeast cell wall microcapsule is beta-1, 3-glucan which can be specifically endocytosed by macrophages, immune cells can be stimulated to produce anti-inflammatory factors such as interleukin 10 (IL 10), and the like, so that the anti-inflammatory effect is exerted. Therefore, the yeast bionic immune micro-nano biological robot provided by the invention is particularly suitable for preparing medicines for treating and detecting inflammatory diseases. Inflammation-related disorders include, but are not limited to, one or more of tumors, obesity, atherosclerosis, osteoporosis, osteosclerotic disorders, hepatitis, pneumonia, neurodegenerative disorders, arthritis, cystitis, gastric ulcers, colitis, diabetes, preeclampsia.
The invention also provides a preparation method of the yeast bionic immune micro-nano biological robot, which comprises the following steps:
(1) Embedding the drug-carrying nano-particles into yeast cell wall microcapsules by an electrostatic deposition mode to obtain drug-carrying yeast microcapsules;
(2) Adding a masking solution into the drug-loaded yeast microcapsule prepared in the step (1), wherein the masking solution binds the drug-loaded yeast microcapsule on a plate, and partially masking the drug-loaded yeast microcapsule, wherein the masking solution comprises at least one of ethylene glycol, glycerol, propylene glycol aqueous solution, polyethylene glycol, maltitol, xylitol and sorbitol; the mass ratio of the drug-loaded yeast microcapsule to the masking solution is preferably 1: (0.01-0.25).
Then adding an activating agent, floating above the masking solution by the activating agent, and carrying out surface activation on the unmasked part of the drug-carrying yeast microcapsule; the activator is a hydroxyl activator including, but not limited to, tosyl chloride, cyanogen bromide, disuccinimidyl carbonate, N-hydroxysuccinimidyl chloroformate, carbonyldiimidazole, sodium periodate, N-acetyl-D-galactosamine, galactose oxidase, chloroacetic acid, isothiocyanate; the mass ratio of the drug-loaded yeast microcapsule to the activator is preferably 1: (40-80).
(3) Incubating the biotinylated enzyme and the surface part activated yeast microcapsule prepared in the step (2) to obtain a yeast bionic immune micro-nano robot with a surface part coated with a single-layer biotinylated enzyme layer;
(4) Incubating the yeast bionic immune micro-nano biological robot with the surface partially coated with the single-layer biotinylase layer prepared in the step (3) with streptavidin, centrifuging and washing, and collecting precipitate to obtain the yeast bionic immune robot with the surface partially coated with the single-layer biotinylase layer and the single-layer streptavidin layer;
(5) The yeast bionic immune robot with the surface partially coated with the single-layer biotinylase layer and the single-layer streptavidin layer prepared in the step (4) is incubated with biotinylase to obtain the yeast bionic micro-nano biological robot modified by double-layer biotinylase;
(6) And (5) repeating the steps (4) to (5) to obtain the multilayer biotinylase modified yeast bionic micro-nano biological robot.
Example 1
A preparation method of a yeast bionic immune micro-nano biological robot comprises the following steps:
(1) Mixing 1mg of drug-loaded cationic nanoparticles with 10mg of yeast cell walls, incubating at 37 ℃ for 24 hours, embedding cations into the negatively charged yeast cell walls due to electrostatic effect, washing with ultrapure water for 4 times, and collecting precipitate to obtain the drug-loaded yeast microcapsule;
(2) 10mg of drug-loaded yeast microcapsule is dissolved in 0.1mg of glycerin aqueous solution, uniformly mixed, poured into a plate, glycerin can bind the drug-loaded yeast microcapsule on the plate, dried for 2 hours at 50 ℃, and then washed with ultrapure water for 3 times to wash out excessive glycerin. 400mg of carbonyldiimidazole activator was added, and the carbonyldiimidazole activator was allowed to float above the glycerin masking solution, was activated at room temperature of 25℃for 2 hours, and was then washed with ultrapure water 3 times to remove carbonyldiimidazole.
(3) Adding 1mg of biotinylated urease, incubating for 6 hours at room temperature of 25 ℃, and then washing with ultrapure water for 3 times to remove unreacted urease, thus obtaining the single-layer urease modified yeast bionic immune microbial robot;
(4) 0.25mg of streptavidin is added, and the mixture is incubated for 30min at the room temperature of 25 ℃; then washing 3 times with ultrapure water;
(5) Adding 1mg of biotinylated urease, incubating for 6 hours at room temperature of 25 ℃, and then washing with ultrapure water for 3 times to remove unreacted urease, thus obtaining the double-layer urease modified yeast bionic immune micro-nano biological robot;
(6) And (5) repeating the step (4) and the step (5) to obtain the multilayer urease modified yeast bionic immune micro-nano biological robot.
Fig. 1 is a schematic diagram of a preparation of a yeast bionic immune micro-nano biological robot, wherein A is a yeast cell wall microcapsule, B is a drug-loaded nanoparticle, and the drug-loaded nanoparticle is introduced into the yeast cell wall microcapsule to obtain a drug-loaded yeast microcapsule C. Adding a masking agent D for half-face masking, adding an activating agent R for half-face activation, and then adding a biotinylase E and a streptavidin F to enable the surface of the drug-loaded yeast microcapsule C to be modified with multiple layers of biotinylase, and then dissolving the masking agent to obtain the yeast bionic immune micro-nano biological robot G.
In fig. 2, fig. 2 (1) is a scanning electron microscope image (bar=1 μm) of a yeast cell wall microcapsule, and fig. 2 (2) is a scanning electron microscope image (bar=1 μm) of a multi-layer urease-modified yeast biomimetic immunomicrobial robot, and it can be seen that the urease is modified on a part of the outer surface of the yeast microcapsule.
FIG. 3 is a graph showing the comparison of motion trajectories of a single-layer urease-modified and a multi-layer urease-modified yeast bionic immune micro-nano biological robot in a urea solution, wherein FIG. 3 (1) is a motion trajectory of the single-layer urease-modified yeast bionic immune micro-nano biological robot 10S in a 10mM urea solution, the length is 15.32 μm, and the average speed is 1.53 μm/S; FIG. 3 (2) is a trace of movement of the multilayered urease modified yeast biomimetic immunomicrobial robot 10s in 10mM urea solution, with a length of 40.35 μm and an average speed of 4.03 μm/s. It can be seen that the movement length of the multilayer urease is 25.03 μm greater than that of the single layer urease and the average speed is 1.25 μm/s greater in the same time. (bar=5μm) it is shown that the multilayer enzyme modified yeast bionic immune micro-nano biological robot greatly improves the movement speed of the existing monolayer enzyme modification.
FIG. 4 is a graph showing the comparison of diffusion results of drug-loaded yeast microcapsules and multi-layer urease modified yeast bionic immune micro-nano biological robots in 1% bionic mucus with 10mM urea as a substrate for 3 min. Wherein, the figure 4 (1) is the diffusion result of the drug-loaded yeast microcapsule, and the figure 4 (2) is the diffusion result of the multilayer urease modified yeast bionic immune micro-nano biological robot. It can be seen that the drug-loaded yeast microcapsules still aggregated together within 3 min. The multilayer urease modified yeast bionic immune micro-nano biological robot still has activity in 1% of bionic mucus, and rapidly and uniformly diffuses to the periphery (bar=100 μm). Description: the multilayer urease modified yeast bionic immune micro-nano biological robot can move in mucus autonomously, and the penetrability of a mucus barrier is greatly improved.
Example 2
A preparation method of a yeast bionic immune micro-nano biological robot comprises the following steps:
(1) Mixing 1mg of drug-loaded nanoparticles with 10mg of yeast cell walls, incubating for 24 hours at 37 ℃, washing with ultrapure water for 4 times, and collecting precipitate to obtain the drug-loaded yeast microcapsule.
(2) 5mg of drug-loaded yeast microcapsules were dissolved in 1.25mg of glycerol, mixed uniformly, then poured into a dish, glycerol was able to bind the drug-loaded yeast microcapsules to the dish, dried at 50 ℃ for 2 hours, and then washed 3 times with ultrapure water to wash out excess glycerol. 250mg of carbonyldiimidazole activator was added, and the carbonyldiimidazole activator was allowed to float above the glycerin masking solution, was activated at room temperature of 25℃for 2 hours, and was then washed with ultrapure water 3 times to remove carbonyldiimidazole.
(3) Adding 1mg of biotinylated glucose oxidase, incubating for 6 hours at room temperature of 25 ℃, and then washing with ultrapure water for 3 times to remove unreacted glucose oxidase, thus obtaining the single-layer glucose oxidase modified yeast bionic immune micro-nano biological robot.
(4) 0.25mg of streptavidin is added, and the mixture is incubated for 30min at the room temperature of 25 ℃; after that, the substrate was washed 3 times with ultrapure water.
(5) Adding 1mg of biotinylated glucose oxidase, incubating for 6 hours at room temperature of 25 ℃, and then washing with ultrapure water for 3 times to remove unreacted glucose oxidase, thus obtaining the double-layer glucose oxidase modified yeast bionic immune micro-nano biological robot.
(6) And (5) repeating the step (4) and the step (5) to obtain the multi-layer glucose oxidase modified yeast bionic immune micro-nano biological robot.
Fig. 5 is a graph of motion profile of a multi-layered glucose oxidase modified yeast biomimetic immunomicrobial robot in 0.9% physiological saline and 3% high salt solution for 10s (bar=5 μm). Wherein, FIG. 5 (1) is a diagram of the motion trace of the multilayer glucose oxidase modified yeast bionic immune microbial robot in a 3% high-salt solution, and the motion speed of the multilayer glucose oxidase modified yeast bionic immune microbial robot is 2.30 μm/s after the 3% high-salt solution is added, wherein, 0.9% physiological saline is added into the 20mM glucose solution. Therefore, in physiological conditions and high-salt solution, the multilayer glucose oxidase modified yeast bionic immune micro-nano biological robot still keeps high autonomous movement, and the salt solution hardly affects the movement of the robot, which is caused by the following reasons: glucose oxidase breaks down glucose, thereby forming a glucose concentration difference on the surface of the yeast robot, thereby causing self-diffusion electrophoresis of the yeast bionic immune micro-nano biological robot, regardless of the particle concentration.
Experimental example
Helicobacter pylori in the stomach can decompose urea through urease generated on the surface to neutralize gastric acid, so that gastric mucus layer is penetrated, and based on the bionic principle, the multilayer enzyme modified yeast immune micro-nano robot can be used for treating stomach diseases. In the drinking water of mice, 10% ethanol solution was added daily, thereby causing gastric ulcers. From day three, three drugs, free curcumin (Free Cur), single layer urease modified curcumin-loaded yeast immune micro-nano robot (cur@monoy-robot) and multi-layer urease modified curcumin-loaded yeast immune micro-nano robot (cur@multy y-robot), were orally administered to mice every two days with a curcumin equivalent of 10 μg/g. On day 8, an active oxygen fluorescent probe (L-012) was injected intraperitoneally to confirm the effect of treatment of gastric ulcer based on the intensity of fluorescence.
As can be seen from fig. 6, the mice were imaged on day 8 after oral administration of 10% ethanol, and the control group had strong fluorescence, indicating that gastric ulcers caused strong inflammatory responses, and that model modeling was successful. When Free Cur was taken orally (see fig. 6 (1)), the fluorescence intensity did not decrease significantly, indicating that Free Cur hardly reached the stomach wall after oral administration. After the Cur@MonoY-robot is orally taken (see (2) of fig. 6), the fluorescence intensity is slightly reduced, which proves that the immune yeast micro-nano robot modified by the single-layer urease has a certain treatment effect on gastric ulcer, and after the Cur@multY-robot is orally taken (see (3) of fig. 6), the fluorescence intensity is weakest, which proves that the immune yeast micro-nano robot modified by the multi-layer urease has an obvious treatment effect on gastric ulcer. Overall results indicate that the multilayer urease modified yeast immune micro-nano robot is superior to the single-layer urease modified therapeutic effect.
In conclusion, the yeast bionic immune micro-nano biological robot provided by the invention takes the yeast cell wall microcapsule as a chassis cell, and the yeast cell wall is derived from edible yeast cells, so that the yeast bionic immune micro-nano biological robot has excellent biocompatibility; secondly, the yeast cell wall has an immunological effect and can target macrophages to stimulate the macrophages to produce anti-inflammatory factors; in addition, the yeast bionic immune micro-nano biological robot provided by the invention greatly increases the content of biological enzymes through multi-layer assembly expansion, so that the driving force of the yeast bionic immune micro-nano biological robot is improved.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (6)
1. The yeast bionic immune micro-nano biological robot is characterized by comprising a yeast cell wall microcapsule, a drug-loaded nanoparticle, a biotinylase layer and a streptavidin layer, wherein the biotinylase layer and the streptavidin layer are assembled, expanded and modified on part of the outer surface of the yeast cell wall microcapsule in a multilayer manner, and the drug-loaded nanoparticle is wrapped in the yeast cell wall microcapsule;
the preparation method of the yeast bionic immune micro-nano biological robot comprises the following steps:
(1) Embedding the drug-carrying nano-particles into yeast cell wall microcapsules by an electrostatic deposition mode to obtain drug-carrying yeast microcapsules;
(2) Adding a masking solution into the drug-loaded yeast microcapsule prepared in the step (1), binding the drug-loaded yeast microcapsule on a plate by the masking solution, masking part of the drug-loaded yeast microcapsule, adding an activating agent, floating above the masking solution by the activating agent, and carrying out surface activation on the unmasked part of the drug-loaded yeast microcapsule;
(3) Incubating the biotinylated enzyme and the surface part activated yeast microcapsule prepared in the step (2) to obtain a yeast bionic immune micro-nano robot with a surface part coated with a single-layer biotinylated enzyme layer;
(4) Incubating the yeast bionic immune micro-nano biological robot with the surface partially coated with the single-layer biotinylase layer prepared in the step (3) with streptavidin, centrifuging and washing, and collecting precipitate to obtain the yeast bionic immune robot with the surface partially coated with the single-layer biotinylase layer and the single-layer streptavidin layer;
(5) The yeast bionic immune robot with the surface partially coated with the single-layer biotinylase layer and the single-layer streptavidin layer prepared in the step (4) is incubated with biotinylase to obtain the yeast bionic micro-nano biological robot modified by double-layer biotinylase;
(6) Repeating the steps (4) to (5) to obtain the multi-layer biotinylation enzyme modified yeast bionic micro-nano biological robot;
in the step (2), the mass ratio of the drug-loaded yeast microcapsule to the masking solution is 1: (0.01-0.25);
the enzyme in the biotinylation enzyme layer is one or more of glucose oxidase, catalase, urease, lipase, trypsin, acetylcholinesterase, deoxyribonuclease or triphosadenidase;
the masking solution includes, but is not limited to, at least one of ethylene glycol, glycerol, propylene glycol, polyethylene glycol, maltitol, xylitol, or sorbitol;
the activator is a hydroxyl activator.
2. The yeast bionic immune micro-nano biological robot of claim 1, wherein the drug loaded nanoparticle is one or more of a small molecule drug, a polypeptide, a macromolecular protein drug, a gene drug, a heavy metal capturing agent, a virus capturing agent or a bacteria capturing agent.
3. A method for preparing the yeast bionic immune micro-nano biological robot according to claim 1 or 2, which is characterized by comprising the following steps:
(1) Embedding the drug-carrying nano-particles into yeast cell wall microcapsules by an electrostatic deposition mode to obtain drug-carrying yeast microcapsules;
(2) Adding a masking solution into the drug-loaded yeast microcapsule prepared in the step (1), binding the drug-loaded yeast microcapsule on a plate by the masking solution, masking part of the drug-loaded yeast microcapsule, adding an activating agent, floating above the masking solution by the activating agent, and carrying out surface activation on the unmasked part of the drug-loaded yeast microcapsule;
(3) Incubating the biotinylated enzyme and the surface part activated yeast microcapsule prepared in the step (2) to obtain a yeast bionic immune micro-nano robot with a surface part coated with a single-layer biotinylated enzyme layer;
(4) Incubating the yeast bionic immune micro-nano biological robot with the surface partially coated with the single-layer biotinylase layer prepared in the step (3) with streptavidin, centrifuging and washing, and collecting precipitate to obtain the yeast bionic immune robot with the surface partially coated with the single-layer biotinylase layer and the single-layer streptavidin layer;
(5) The yeast bionic immune robot with the surface partially coated with the single-layer biotinylase layer and the single-layer streptavidin layer prepared in the step (4) is incubated with biotinylase to obtain the yeast bionic micro-nano biological robot modified by double-layer biotinylase;
(6) And (5) repeating the steps (4) to (5) to obtain the multilayer biotinylase modified yeast bionic micro-nano biological robot.
4. The method for preparing a yeast bionic immune micro-nano biological robot according to claim 3, wherein in the step (2), the mass ratio of the drug-loaded yeast microcapsule to the activator is 1: (40-80).
5. The method for producing a yeast biomimetic immunomicrobial robot according to claim 3, wherein in the step (3), the biotinylated enzyme is excessively added with respect to the surface part-activated yeast microcapsule.
6. Use of a yeast biomimetic immunomicrobial robot according to claim 1 or 2 or a yeast biomimetic immunomicrobial robot prepared according to any one of the preparation methods of claims 3-5 for drug delivery.
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