A drug-loaded microalgae motor and its preparation method and application

Abstract

The present invention belongs to the field of micro-nano technology, and particularly relates to a drug-loaded microalgae motor and its preparation method and application, including a polydopamine / magnetite / chlamydomonas reinhardtii micro-motor and a drug loaded thereon. The drug is covalently bonded to the catechol group on the surface of polydopamine in the polydopamine / magnetite / chlamydomonas reinhardtii micro-motor; the drug is an anti-cancer drug or an anti-inflammatory drug. This microalgae motor has the characteristics of simple preparation method, good biocompatibility of materials, high biodegradability, etc. The prepared polydopamine / magnetite / chlamydomonas reinhardtii drug-loaded micro-motor of the present invention has the characteristic of self-driving in intestinal fluid. Therefore, under the guidance of an external magnetic field, it can target the intestinal disease site to improve the treatment effect and reduce the side effects of the drug, thereby expanding the application space of micro-nano motors in the biomedical field.

Description

Technical Field

[0001] The present invention belongs to the field of micro-nano technology, and particularly relates to a drug-loaded microalgae motor and its preparation method and application. Background Art

[0002] Micro-nano motors are miniaturized devices that can convert chemical or other forms of energy into mechanical forces to drive their own movement. Similar to macroscopic intelligent robots, micro-nano motors can perform fine operations in complex environments. Due to their small size and self-propelled movement characteristics, they have shown broad prospects in fields such as drug delivery, minimally invasive surgery, biochemical sensing, and environmental remediation. However, the medical applications of these micro-devices are still in their infancy, and most micro-nano motors have problems such as complex preparation processes, high manufacturing costs, difficulty in surface modification, poor biocompatibility, and biodegradability. Therefore, exploring new materials to construct micro-nano motors with better performance is a research focus at the present stage.

[0003] To meet these growing demands, people have begun to focus on using living cells as carriers to construct active biohybrid micro-nano motors. After millions of years of evolution, microorganisms have evolved complex motility systems, with self-propulsion capabilities and strategic motility capabilities to respond to different stimuli. Natural dynamic systems enable microorganisms to adapt to diverse environments and extreme conditions. To mimic the motility strategies of microorganisms, various synthesis methods of micro-nano motors based on advanced synthetic materials have been designed, such as artificial flagella made of magnetized helices or flexible structures, to achieve simulated movement under external magnetic fields. However, the design of synthetic motors cannot fully mimic the attractive intrinsic characteristics of biological systems, including driving power, energy conversion efficiency, flexibility, complex control, as well as tropism and sensing capabilities. Biohybrid motors provide an attractive strategy for achieving biomimetic behavior. Many biological materials, including bacteria, algae, and sperm, have been selected as the basic driving elements of biohybrid micro-nano motors. They can generate strong long-term thrust, have a fast self-propulsion speed, do not require external drive, and can sense and respond to changes or stimuli in the surrounding environment. However, existing micro-nano motors have disadvantages such as high toxicity of the basic materials used, poor biodegradability, and few surface functional groups.

[0004] Microalgae are a common type of single-celled photosynthetic organisms, including prokaryotic algae (such as cyanobacteria) and eukaryotic algae (such as diatoms, green algae, etc.). They are widely distributed in the ocean or freshwater lakes, are easy to obtain and cultivate, and have now been applied in the production of food, health products, fuels, etc. In recent years, researchers have gradually recognized that microalgae also have great application potential as biological materials in the medical field. Among them, Chlamydomonas reinhardtii is rich in photosynthetic pigments such as natural fluorescein and chlorophyll, which can be used as natural photosensitizers, have fluorescence imaging and photoacoustic imaging capabilities, and can be applied to medical imaging. It can achieve self-driving through the beating of flagella, has a unique surface structure that makes its surface easy to modify, has good biocompatibility and biosafety, and can be used for drug targeted delivery. Therefore, Chlamydomonas reinhardtii shows great advantages in biological imaging, drug delivery, hypoxic tumor treatment, wound healing, etc. However, due to the limitations of the surface properties of Chlamydomonas reinhardtii, the types and capabilities of drugs it can load are limited, and the direct interaction between drugs and Chlamydomonas reinhardtii may have a greater impact on its activity and lifespan. Currently, the varieties of drug-loaded microalgae motors are single, the types of drugs loaded are limited, the drug loading capacity is small, and the drugs have a greater impact on the activity and lifespan of microalgae. Summary of the Invention

[0005] The purpose of the present invention is to provide a drug-loaded microalgae motor and its preparation method and application, so as to overcome the deficiencies of the prior art. The present invention aims to combine polydopamine, iron oxide nanoparticles with microalgae to construct a new type of drug-loaded microalgae motor with strong controllability, good biocompatibility and long lifespan, increase its drug loading types and drug loading capacity, and expand the application field of drug-loaded microalgae motors in targeted drug transportation.

[0006] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0007] In the first aspect, the present invention provides a drug-loaded microalgae motor, including a polydopamine / iron oxide / Chlamydomonas reinhardtii micromotor and the drug loaded thereon. The drug is combined with the catechol groups on the surface of polydopamine in the polydopamine / iron oxide / Chlamydomonas reinhardtii micromotor through intermolecular interactions; the drug is an anti-cancer drug or an anti-inflammatory drug.

[0008] In some other embodiments, the anti-cancer drug is doxorubicin, and the anti-inflammatory drug is curcumin.

[0009] In some other embodiments, the polydopamine / iron oxide / Chlamydomonas reinhardtii micromotor includes Chlamydomonas reinhardtii and a coating wrapped on the surface of Chlamydomonas reinhardtii. The coating is composed of polydopamine and iron oxide nanoparticles.

[0010] In some other embodiments, the mass ratio of the polydopamine to the iron oxide nanoparticles is (0.05 - 0.5):(1 - 2); the particle size of the iron oxide nanoparticles is 10 - 50 nm, and the particle size of the polydopamine is 80 - 100 nm;

[0011] The density of the Chlamydomonas reinhardtii is 1×10 6 -1×10 7 cells / mL.

[0012] The overall inventive concept adopted in the present invention is:

[0013] The present invention makes full use of the great advantages of Chlamydomonas reinhardtii in aspects such as bioimaging, drug delivery, hypoxic tumor treatment, wound healing, etc. Polydopamine is a mussel - biomimetic material obtained by the polymerization of dopamine. Due to the catechol groups and primary amine groups on the polydopamine, it has adhesiveness and metal - coordination properties, and can adsorb on the surface of almost all solid materials (such as noble metals, carbon materials, organic polymers, etc.) to form a layer of polydopamine film. In addition, the catechol groups can form covalent bonds with amino or mercapto reagents through different chemical reactions, which is conducive to grafting various drugs onto the surface of polydopamine and can be slowly released under appropriate conditions. Due to its good tissue adhesiveness, it can adhere to the intestinal mucosal layer, further prolonging the retention time of the drug in the intestine and being beneficial to improving the utilization rate of the drug. Therefore, polydopamine is a good carrier material for drugs.

[0014] Meanwhile, due to the advantages of low cytotoxicity and high biocompatibility of polydopamine, it has broad application prospects in biomedicine and environmental governance and other aspects. In direct drug - loading research, because the specific surface area of polydopamine nanoparticles is relatively large and the drug - loading rate is high, they are mostly used as drug carriers. However, in solution, especially in biological systems, polydopamine nanoparticles are unstable and prone to aggregation, which limits the further expansion of their application in drug loading. Iron oxide nanoparticles have the advantages of good biocompatibility, strong magnetism, and easy preparation. Using polydopamine nanoparticles and iron oxide nanoparticles as surface modifiers of Chlamydomonas reinhardtii can, through the synergistic cooperation of the three, improve the drug - loading capacity and types of drugs of Chlamydomonas reinhardtii and reduce the stimulation of drugs to Chlamydomonas reinhardtii.

[0015] In a second aspect, the present invention provides a method for preparing the drug - loaded microalgae motor described in the first aspect, including the following steps: adding a hydrochloric acid dopamine solution, an iron oxide nanoparticle dispersion, a Tris - HCl buffer solution, and deionized water to the pretreated algal solution of Chlamydomonas reinhardtii for reaction to obtain a polydopamine / iron oxide / Chlamydomonas reinhardtii micro - motor;

[0016] Adding a drug to the polydopamine / iron oxide / Chlamydomonas reinhardtii micro - motor for loading, and obtaining the drug - loaded microalgae motor after dark incubation.

[0017] In some other embodiments, the pretreatment method of the Chlamydomonas reinhardtii algal solution is to centrifuge the Chlamydomonas reinhardtii algal solution, remove the supernatant, and add water for standby.

[0018] In some other embodiments, the rotation speed of the centrifugation treatment is 500 - 1500 rpm, and the centrifugation time is 1 - 10 min.

[0019] In some other embodiments, the concentration of the dopamine hydrochloride solution is 0.05 - 0.5 mg / mL; the concentration of the iron oxide nanoparticles dispersion is 1 - 2 mg / mL; the pH of the Tris-HCl buffer solution is 7.0 - 9.0, and the concentration is 0.005 - 0.02 mol / L.

[0020] In some other embodiments, the reaction time is 10 - 24 h;

[0021] The concentration of the drug is 0.5 - 50 μg / mL, and the loading time is 2.5 - 60 min.

[0022] In a third aspect, the present invention provides an application of the drug-loaded microalgae motor described in the first aspect in the preparation of a disease-targeted therapeutic drug, and the disease-targeted therapeutic drug is particularly used for the treatment of diseases in the gastrointestinal tract.

[0023] In some other embodiments, the disease-targeted therapeutic drug is a capsule containing a polydopamine / iron oxide / Chlamydomonas reinhardtii drug-loaded micromotor; the inner wall of the capsule is provided with a hydrophobic coating, and the outer wall of the capsule is provided with a pH-sensitive polymer.

[0024] In some other embodiments, the hydrophobic inner layer is trimethoxy(octadecyl)silane; the pH-sensitive polymer is Eudragit L100-55.

[0025] In a fourth aspect, the present invention provides an enteric-coated capsule, which includes a capsule shell and a drug contained in the capsule shell. The capsule shell includes a capsule body, a hydrophobic coating provided on the inner wall of the capsule body, and an enteric coating provided on the outer wall of the capsule body; the drug contained in the capsule shell is the drug-loaded microalgae motor described in the first aspect.

[0026] Advantages of the present invention:

[0027] (1) The present invention uses Chlamydomonas reinhardtii, which is green, has good biocompatibility, high biodegradability, and has functions of antioxidation, anti-inflammation, and regulating intestinal flora, as the matrix material of the micro-nano motor, and prepares the microalgae motor by a simple in-situ synthesis method without generating any toxic substances.

[0028] (2) In the present invention, the catechol groups on the surface of polydopamine can form covalent bonds with amino or mercapto reagents through different chemical reactions, enabling various drugs to be connected to the surface of the motor, thereby achieving the loading and release of multiple drugs.

[0029] (3) The motor prepared in the present invention can achieve persistent autonomous movement in intestinal fluid. This long-term movement can significantly improve the intestinal distribution of the motor, thereby enhancing the drug residence range. Moreover, the movement of the motor can be controlled by an external magnetic field to reach the site that needs treatment, so as to improve the treatment effect and reduce the side effects of the drug, thereby expanding the application space of micro-nano motors in the field of biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The attached drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0031] FIG. 1 is a scanning electron micrograph of polydopamine / iron oxide / Chlamydomonas reinhardtii micro-motors prepared in Example 1 of the present invention and Chlamydomonas reinhardtii. Among them, a is Chlamydomonas reinhardtii, b is a partial enlarged view of a, c is the polydopamine / iron oxide / Chlamydomonas reinhardtii micro-motor prepared in Example 1, and d is a partial enlarged view of c;

[0032] FIG. 2 is a self-driven movement diagram of the polydopamine / iron oxide / Chlamydomonas reinhardtii micro-motor prepared in Example 1 of the present invention moving directionally towards the magnet in simulated intestinal fluid. Among them, 1, 2, 3, and 4 are the movement trajectories of randomly selected dopamine / iron oxide / Chlamydomonas reinhardtii micro-motors;

[0033] FIG. 3 is a graph showing the change of the loading amount of different concentrations of doxorubicin by the polydopamine / iron oxide / Chlamydomonas reinhardtii micro-motor prepared in Example 1 of the present invention over time;

[0034] Figure 4 It is a graph showing the relationship between the drug-loading fluorescence intensity of the polydopamine / iron oxide / Chlamydomonas reinhardtii micro-motor prepared in Example 2 of the present invention and the anti-inflammatory drug curcumin;

[0035] Figure 5 The cytotoxicity of Chlamydomonas reinhardtii and the polydopamine / iron oxide / Chlamydomonas reinhardtii micro-motor prepared in Example 1 of the present invention, as well as the killing effect of the doxorubicin-loaded motor on colon cancer cells Caco-2, were tested. Among them, CR is Chlamydomonas reinhardtii, the PDA / Fe3O4 / CR motor is the polydopamine / iron oxide / Chlamydomonas reinhardtii micro-algae motor, and the DOX / PDA / Fe3O4 / CR motor is the doxorubicin / polydopamine / iron oxide / Chlamydomonas reinhardtii micro-algae motor. DETAILED DESCRIPTION OF THE INVENTION

[0036] As described in the background art, existing micro-nano motors have disadvantages such as high toxicity of the base materials used, poor biodegradability, and few surface functional groups. Therefore, the present invention proposes a preparation method of poly-dopamine / iron oxide / Chlamydomonas reinhardtii micro-motor and its application in intestinal diseases. The present invention will be further described below in conjunction with examples. Unless otherwise specified in the examples, the procedures are carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all components used are conventional products commercially available.

[0037] Chlamydomonas reinhardtii in the examples was purchased from Guangyu Biotechnology Co., Ltd., iron oxide nanoparticles were purchased from Aladdin Reagent Co., Ltd., a cell counting chamber was purchased from Fuzhou Taimei Biotechnology Co., Ltd., and commercial capsules (size M for small animal drug administration) were purchased from Torpac; the simulated gastric juice composition was: 0.2% (w / w) NaCl and 0.7% (w / w) HCl, and the simulated intestinal juice composition was: 0.68% (w / w) NaH2PO4 and 0.4% (w / w) NaOH.

[0038] The method for measuring the fluorescence intensity was as follows: using a fluorescence spectrometer (FLS-920, Edinburgh Instruments Ltd, UK), the fluorescence intensity of doxorubicin in the supernatant was measured under the conditions of an excitation wavelength of 467 nm and an emission wavelength of 590 nm.

[0039] Since the drug-loaded micro-motor formed by poly-dopamine / iron oxide / Chlamydomonas reinhardtii after loading drugs converts chemical energy into mechanical kinetic energy based on chemical reactions to drive the motor, once it comes into contact with the fluid in the gastrointestinal (GI) tract, the residence time is shortened and the interaction with the gastrointestinal mucosa is weakened, resulting in a decrease in the bioavailability of the drug. Therefore, in order to prolong the residence time of the drug in the intestinal mucosa, improve the drug delivery efficiency, and achieve efficient and controllable driving of the micro-motor, the present invention modifies commercial capsules, and encapsulates poly-dopamine / iron oxide / Chlamydomonas reinhardtii micro-motor into the modified capsules and transports them into the mouse intestine to achieve controllable release of the drug.

[0040] The specific process of modifying commercial capsules is as follows:

[0041] (1) Coat a hydrophobic inner coating inside the commercial capsule so that the motor solution can be stored in the capsule. The specific method is: stir a 2-20 wt% solution of trimethoxy(octadecyl)silane and ethanol at room temperature for 1-5 hours, fill 2-10 μL into the capsule each time, and cure at 80-200 °C for 1-5 hours to completely evaporate the solvent. Repeat 10-20 times to obtain a capsule with a hydrophobic inner coating.

[0042] (2) Coating the outside of the capsule with the polymer Eudragit L100-55 to prevent it from being dissolved by gastric acid. The specific method is as follows: Dissolve Eudragit L100-55 in ethanol to prepare a 1-10% enteric coating solution, then immerse the capsule with a hydrophobic inner coating inside into the enteric coating solution by dip coating method. Then take out the capsule and evaporate the solvent, repeating 10-20 times to obtain the modified capsule.

[0043] (3) Load the liquid drug-loaded microalgae motor into the modified capsule through a flat-tip micro syringe (50 μL, Hamilton). Feed the capsule loaded with the drug-loaded microalgae motor into the mouse's mouth, thereby enabling the drug to be released in the intestine. If under the control of an external magnetic field, targeted release of the drug at the disease site can also be achieved.

[0044] Example 1

[0045] 1. A preparation method of a polydopamine / magnetite / chlamydomonas reinhardtii micro motor, comprising the following steps:

[0046] First, centrifuge the chlamydomonas reinhardtii algal solution at a speed of 1000 rpm for 5 min, remove the supernatant, and add deionized water for standby. Determine the density of chlamydomonas reinhardtii through a hemocytometer to obtain the treated algal solution. Add dopamine hydrochloride, magnetite nanoparticle dispersion (10-50 nm), Tris-HCl buffer solution (pH = 7.5) and deionized water to the treated algal solution to obtain 10 mL of algal solution (containing 0.05 mg / mL dopamine hydrochloride, 1 mg / mL magnetite nanoparticles, 0.005 mol / L Tris-HCl buffer solution).

[0047] The inventor found during the research process that if the amount of dopamine hydrochloride is too much, chlamydomonas reinhardtii may stop moving due to too thick a coating; if the amount of nano-magnetite dispersion and Tris-HCl buffer solution is too much, the irritation to chlamydomonas reinhardtii is too strong, which will cause the algae to stop moving or even die; if the amount of dopamine hydrochloride, nano-magnetite dispersion, and Tris-HCl buffer solution added is too small, it will take a longer reaction time to obtain the motor, and the surface coating will fall off quickly, and the motor will maintain magnetism for a short time.

[0048] By measuring the density of the algal solution and controlling the amount of the algal solution used, the final density of chlamydomonas reinhardtii in each reaction is controlled to be 1×10 6The reaction was carried out for 10 hours under the conditions of 12 / 12 hours of light / darkness, 19-25℃, and light intensity of about 4000 lux. After 10 hours of reaction, the microalgae motors and some magnetic nanoparticles with larger particle sizes were centrifuged to the bottom after being centrifuged at 1000 rpm for 5 minutes. The supernatant was removed, deionized water was added, and the magnet was placed under the centrifuge tube for 10 minutes. The magnetic nanoparticles and some motors that were not removed by centrifugation were sucked to the bottom of the centrifuge tube. When the magnet was removed, the motors would swim back to the supernatant, while the magnetic nanoparticles were still gathered at the bottom of the centrifuge tube. The supernatant was taken out to obtain the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotors.

[0049] 2. A drug delivery method for polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor, comprising the following steps:

[0050] (1) Polydopamine / Fe3O4 / Chlamydomonas reinhardtii micromotors loading the anticancer drug doxorubicin

[0051] Add 0.5 μg / mL of the anticancer drug doxorubicin to the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotors, incubate in the dark, centrifuge and take the supernatant to measure its fluorescence intensity, and bring it into the fluorescence standard curve of doxorubicin F=8352.85x+324.5, where F is the fluorescence intensity and x is the concentration of doxorubicin (μg / mL) to obtain the loading amount of the anticancer drug doxorubicin by the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotors. At the same time, observe the changes in motor activity after loading different concentrations of doxorubicin in order to select the most suitable loading drug concentration.

[0052] (2) Drug release of doxorubicin / polydopamine / ferric oxide / Chlamydomonas reinhardtii micromotors in different environments Drug release was performed in simulated gastric fluid (pH = 1.2) and simulated intestinal fluid (pH = 6.8). The polydopamine / ferric oxide / Chlamydomonas reinhardtii micromotors (1.5×10 6 / mL) were loaded into the modified capsules, and then placed in simulated gastric fluid and simulated intestinal fluid respectively. The supernatant was taken at 2.5 min, 5 min, 10 min, 15 min, 20 min, and 30 min and detected by fluorescence spectrometer to determine the release rate of the drug in different environments and time.

[0053] Example 2

[0054] 1. A method for preparing a polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor, comprising the following steps:

[0055] First, centrifuge the Chlamydomonas reinhardtii algal solution at 1000 rpm for 5 min, discard the supernatant, and add deionized water for standby. Determine the density of Chlamydomonas reinhardtii by counting with a hemocytometer to obtain the processed algal solution. Add dopamine hydrochloride, nano-ferroferric oxide dispersion (10 - 50 nm), Tris-HCl buffer solution (pH = 8.0), and a certain amount of deionized water to the processed algal solution to obtain 10 mL of algal solution (containing 0.02 mg / mL dopamine hydrochloride, 1.5 mg / mL ferroferric oxide nanoparticles, and 0.01 mol / L Tris-HCl buffer solution).

[0056] By measuring the density of the algal solution and controlling the amount of the algal solution used, the final density of Chlamydomonas reinhardtii in each reaction is controlled to be about 3×10 6 cells / mL. React for 15 hours under the suitable growth conditions of Chlamydomonas reinhardtii. After reacting for 15 hours, centrifuge at 1000 rpm for 5 minutes. The microalgae motors and some magnetic nanoparticles with larger particle sizes are centrifuged to the bottom. Discard the supernatant, add deionized water, place a magnet under the centrifuge tube for 15 minutes. The magnetic nanoparticles that are not completely removed by centrifugation and some motors are attracted to the bottom of the centrifuge tube. Remove the magnet, and the motors will swim back to the supernatant again, while the magnetic nanoparticles still aggregate at the bottom of the centrifuge tube. Take out the supernatant to obtain the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micro-motors.

[0057] 2. A method for loading drugs onto polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micro-motors, comprising the following steps:

[0058] (1) Loading of anti-inflammatory drug curcumin onto polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micro-motors

[0059] Add 0.5 - 3 μg / mL of curcumin to the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micro-motors, incubate in the dark, and centrifuge to obtain the motors loaded with curcumin. Observe the fluorescence intensity on the surface of the motors after loading different concentrations of curcumin under the dark field of a fluorescence inverted microscope. At the same time, observe the changes in the activity of the motors after loading different concentrations of curcumin to select the most suitable drug loading concentration.

[0060] (2) Drug release of curcumin / polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micro-motors in different environments. In simulated gastric fluid (pH = 1.2) and simulated intestinal fluid (pH = 6.8). Select the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micro-motors loaded with 1.0 μg / mL of curcumin (1.5×10 6Loaded into the modified capsules at (number / mL), and placed in simulated gastric juice and simulated intestinal juice respectively. Take the supernatant at 2.5 min, 5 min, 10 min, 15 min, 20 min, and 30 min, and detect it with a fluorescence spectrometer to determine the release rate of the drug under different environments and times.

[0061] Comparative Example 1

[0062] Different from Example 1, without adding polydopamine / iron oxide, and other preparation methods are the same as those in Example 1, to obtain doxorubicin / Chlamydomonas reinhardtii micromotors.

[0063] Comparative Example 2

[0064] Different from Example 1, without adding iron oxide, and other preparation methods are the same as those in Example 1, to obtain doxorubicin / polydopamine / Chlamydomonas reinhardtii micromotors.

[0065] Comparative Example 3

[0066] Different from Example 1, using Spirulina (Guangyu Biotechnology Co., Ltd.) instead of Chlamydomonas reinhardtii, and other preparation methods are the same as those in Example 1, to obtain doxorubicin / polydopamine / iron oxide / Spirulina micromotors.

[0067] Performance test:

[0068] 1. Microscopic tissue observation

[0069] The polydopamine / iron oxide / Chlamydomonas reinhardtii micromotors prepared in Examples 1 and 2 were observed by scanning electron microscopy. The results are as Figure 1 shown, where a is Chlamydomonas reinhardtii, b is a partial enlarged view of a, c is the polydopamine / iron oxide / Chlamydomonas reinhardtii micromotor prepared in Example 1, and d is a partial enlarged view of c. From Figure 1 it can be seen that the surface morphology and structure of the polydopamine / iron oxide / Chlamydomonas reinhardtii micromotor prepared in Example 1 are similar to those of Chlamydomonas reinhardtii, but the surface of the sample in Example 1 is covered with a polydopamine film. It can be observed from the local view that the surface of the polydopamine / iron oxide / Chlamydomonas reinhardtii micromotor contains polydopamine nanoparticles with a particle size of 100 nm and iron oxide nanoparticles with a particle size of 20 nm, thus confirming that the polydopamine / iron oxide / Chlamydomonas reinhardtii micromotor was successfully prepared in the present invention.

[0070] 2. Self-propelled performance test of polydopamine / iron oxide / Chlamydomonas reinhardtii micromotors

[0071] Observe the movement of the polydopamine / iron oxide / Chlamydomonas reinhardtii micromotor prepared in Example 1. The movement trajectory diagram of the polydopamine / iron oxide / Chlamydomonas reinhardtii micromotor when attracted by a magnet on the right side of the droplet. The results are asFigure 2 as shown, where 1, 2, 3, and 4 are the movement trajectories of randomly selected dopamine / iron oxide / Chlamydomonas reinhardtii micromotors. From Figure 2 it can be seen that the movement of the polydopamine / iron oxide / Chlamydomonas reinhardtii micromotors prepared in Example 1 is similar. They all move directionally towards the magnet, with a helical movement trajectory and a movement speed of about 85 μm / s. They have self-driving ability and can achieve persistent movement in simulated intestinal fluid.

[0072] 3. Test on the drug-loading ability of microalgae motors

[0073] The variation of the loading amount of the polydopamine / iron oxide / Chlamydomonas reinhardtii micromotors prepared in Example 1 with respect to different concentrations of doxorubicin (1.5 - 3.5 μg / mL) over time (0 - 40 min) is as Figure 3 shown. From Figure 3 it can be seen that the polydopamine / iron oxide / Chlamydomonas reinhardtii micromotors can rapidly bind approximately 70% of doxorubicin within 2.5 minutes. As the concentration of doxorubicin in the solution increases, the loading amount of doxorubicin by the polydopamine / iron oxide / Chlamydomonas reinhardtii micromotors increases. Considering the influence of doxorubicin on the activity of Chlamydomonas reinhardtii, 2.5 μg / mL is selected for loading.

[0074] The relationship between the drug-loading fluorescence intensity and the algal survival rate of the polydopamine / iron oxide / Chlamydomonas reinhardtii micromotors prepared in Example 2 for the anti-inflammatory drug curcumin is as Figure 4 shown. From Figure 4 it can be seen that when the solvent is ethanol:water with a volume ratio of 1:19 and the curcumin concentration is 1.0 μg / mL, the effect of Chlamydomonas reinhardtii in loading curcumin is relatively good.

[0075] The in vitro anti-tumor effect was tested by the thiazolyl blue method (MTT method) for the cytotoxicity of Chlamydomonas reinhardtii and the polydopamine / iron oxide / Chlamydomonas reinhardtii micromotors prepared in Example 1, as well as the killing effect of the doxorubicin-loaded motors on colon cancer cells Caco-2 (purchased from the cell bank of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences). As Figure 5 shown, where CR is Chlamydomonas reinhardtii, PDA / Fe3O4 / CR motor is the polydopamine / iron oxide / Chlamydomonas reinhardtii micromotor, and PDA / Fe3O4 / CR motor is the doxorubicin / polydopamine / iron oxide / Chlamydomonas reinhardtii microalgae motor. From Figure 5 it can be seen that even when the density of Chlamydomonas reinhardtii and the polydopamine / iron oxide / Chlamydomonas reinhardtii micromotors is as high as 6×10 6cells / mL. The colon cancer cells can still maintain a survival rate of about 95% in simulated intestinal fluid, indicating that Chlamydomonas reinhardtii and polydopamine / magnetite / Chlamydomonas reinhardtii micromotors have good biocompatibility and have no effect on cell viability. After the polydopamine / magnetite / Chlamydomonas reinhardtii micromotors loaded with doxorubicin (drug-loaded microalgae motors) release drugs in simulated intestinal fluid, as the number of drug-loaded microalgae motors increases, the cell survival rate drops sharply. When the density of the drug-loaded microalgae motors is 6×10 6 cells / mL, the cell survival rate is only 36.68%, indicating that the drug-loaded microalgae motors have good in vitro anti-tumor effects.

[0076] In Comparative Example 1, the activity of the prepared doxorubicin / Chlamydomonas reinhardtii micromotors was tested. Chlamydomonas reinhardtii can load doxorubicin, but due to the stimulation of doxorubicin, even at a low concentration (2 μg / mL), the mortality rate of Chlamydomonas reinhardtii is still very high (>60%).

[0077] The polydopamine / Chlamydomonas reinhardtii micromotors prepared in Comparative Example 2 have no magnetism and cannot achieve targeted drug loading. These microalgae motors can still load doxorubicin (2 μg / mL), but the loading amount is relatively small (<50%), because magnetite nanoparticles are negatively charged and can also load a certain amount of doxorubicin.

[0078] The polydopamine / magnetite / spirulina micromotors prepared in Comparative Example 3 can also be successfully constructed after testing, indicating that our method can not only be used for Chlamydomonas reinhardtii, but also be extended to the drug delivery systems of other algae. However, due to the fact that spirulina is extremely easy to break and die under external forces (such as high-speed centrifugation) and loses its self-propelling ability, it is difficult to apply living spirulina micromotors to the targeted drug delivery system. Existing work has fixed spirulina in paraformaldehyde and then used the dead spirulina skeleton as a carrier to construct externally driven micromotors.

[0079] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. 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 drug-loaded microalgae motor, characterized in that: It comprises a polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor and a drug loaded thereon, wherein the drug is combined with the catechol group on the surface of polydopamine in the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor through intermolecular interaction; the drug is an anticancer drug or an anti-inflammatory drug; The anticancer drug is doxorubicin, and the anti-inflammatory drug is curcumin; The polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor comprises Chlamydomonas reinhardtii and a coating coated on the surface of Chlamydomonas reinhardtii, wherein the coating is composed of polydopamine and ferroferric oxide nanoparticles; The mass ratio of polydopamine to ferrosoferric oxide nanoparticles is (0.05-0.5): (1-2); The preparation method of the drug-loaded microalgae motor comprises the following steps: adding a dopamine hydrochloride solution, a ferroferric oxide nanoparticle dispersion, a Tris-HCl buffer solution and deionized water to the pretreated algae liquid of Chlamydomonas reinhardtii to react and obtain a polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor; Add drugs to polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotors for loading, and prepare drug-loaded microalgae motors after incubation in the dark; The concentration of the dopamine hydrochloride solution is 0.05-0.5 mg / mL; the concentration of the ferroferric oxide nanoparticle dispersion is 1-2 mg / mL; The reaction time is 10-24 h; The concentration of the drug is 0.5-50 μg / mL, and the loading time is 2.5-60 min.

2. The drug-loaded microalgae motor according to claim 1, characterized in that: The particle size of the ferrosoferric oxide nanoparticles is 10-50 nm, and the particle size of the polydopamine is 80-100 nm; The density of Chlamydomonas reinhardtii is 1×10 6 -1×10 7 Pieces / mL.

3. The drug-loaded microalgae motor according to claim 1, characterized in that: The drug is doxorubicin.

4. A method for preparing the drug-loaded microalgae motor according to any one of claims 1 to 3, characterized in that: The following steps are involved: Adding a dopamine hydrochloride solution, a ferroferric oxide nanoparticle dispersion, a Tris-HCl buffer solution and deionized water to the pretreated Chlamydomonas reinhardtii algae liquid to react and obtain polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotors; Add drugs to polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotors for loading, and prepare drug-loaded microalgae motors after incubation in the dark; The concentration of the dopamine hydrochloride solution is 0.05-0.5 mg / mL; the concentration of the ferrosoferric oxide nanoparticle dispersion is 1-2 mg / mL; The reaction time is 10-24 h; The concentration of the drug is 0.5-50 μg / mL, and the loading time is 2.5-60 min.

5. The method for preparing the drug-loaded microalgae motor according to claim 4, characterized in that: The pretreatment method of the Chlamydomonas reinhardtii algae liquid is to centrifuge the Chlamydomonas reinhardtii algae liquid, remove the supernatant and add water for standby use.

6. The method for preparing the drug-loaded microalgae motor according to claim 5, characterized in that: The rotation speed of the centrifugal treatment is 500-1500 rpm, and the centrifugal time is 1-10 min.

7. The method for preparing the drug-loaded microalgae motor according to claim 4, characterized in that: The pH of the Tris-HCl buffer solution is 7.0-9.0, and the concentration is 0.005-0.02 mol / L.

8. Use of the drug-loaded microalgae motor according to claim 3 in the preparation of a disease-targeted therapeutic drug, wherein the disease-targeted therapeutic drug is used to treat a disease in the gastrointestinal tract; the disease is colon cancer.

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