Drug-loaded microalgae motor as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of micro-nano, and particularly relates to a drug-loaded microalgae motor and a preparation method and application thereof, and the drug-loaded microalgae motor comprises a polydopamine / ferroferric oxide / chlamydomonas reinhardtii micromotor and a drug loaded on the polydopamine / ferroferric oxide / chlamydomonas reinhardtii micromotor, the drug is combined with catechol groups on the surface of polydopamine in the polydopamine / ferroferric oxide / chlamydomonas reinhardtii micromotor through covalent bonds; the medicine is an anti-cancer medicine or an anti-inflammatory medicine. The microalgae motor has the characteristics of simple preparation method, good material biocompatibility, high biodegradability and the like. The polydopamine / ferroferric oxide / chlamydomonas reinhardtii drug-loaded micromotor prepared by the invention has the characteristic of self-driving in intestinal juice, so that the polydopamine / ferroferric oxide / chlamydomonas reinhardtii drug-loaded micromotor can target an intestinal disease part under the guidance of an external magnetic field to improve the treatment effect and reduce the side effect of a drug, thereby expanding the application space of a micro-nano motor in the field of biomedicine.

Description

Technical Field

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

[0002] Micro-nanomotors are miniaturized devices that can convert chemical or other forms of energy into mechanical force to propel themselves. Like macroscopic intelligent robots, micro-nanomotors can perform delicate operations in complex environments. Due to their small size and autonomous motion, they have shown broad prospects in areas 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. Most micro-nanomotors have problems such as complex preparation processes, high production costs, difficulty in surface modification, and poor biocompatibility and biodegradability. Therefore, exploring new materials to build micro-nanomotors with better performance is a research focus at this stage.

[0003] To meet these growing demands, people have begun to focus on using living cells as carriers to build active biohybrid micro-nanomotors. After millions of years of evolution, microorganisms have evolved complex motor systems with self-propulsion capabilities and strategic movement capabilities in response to different stimuli. Natural dynamic systems enable microorganisms to adapt to diverse environments and extreme conditions. In order to mimic the movement strategies of microorganisms, various methods have been designed to synthesize micro-nanomotors based on advanced synthetic materials, such as artificial flagella made of magnetized spirals 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 to achieve biomimetic behaviors. Many biomaterials, including bacteria, algae, and sperm, have been selected as the basic driving elements of biohybrid micro-nanomotors. They can generate strong long-term thrust, self-propulsion at high speed, without external drive, and can sense and respond to changes or stimuli in the surrounding environment. However, existing micro-nanomotors 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 found in oceans or freshwater lakes, are easy to obtain and cultivate, and are now used in the production of food, health products and fuels. In recent years, researchers have gradually realized that microalgae as biomaterials also have great application potential 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. It has fluorescence imaging and photoacoustic imaging capabilities and can be used in 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 targeted drug delivery. Therefore, Chlamydomonas reinhardtii shows great advantages in bioimaging, drug delivery, hypoxic tumor treatment, wound healing and other aspects. However, due to the limitations of the surface properties of Chlamydomonas reinhardtii, its types and capacity for loading drugs are limited, and the drugs directly interact with Chlamydomonas reinhardtii, which may have a significant impact on its activity and lifespan. In addition, the types of drugs currently used for drug-loaded microalgae motors are single, the types of drugs loaded are limited, the drug loading amount is small, and the drugs have a significant impact on the activity and lifespan of the 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 shortcomings of the prior art. The present invention aims to combine polydopamine, ferroferric oxide nanoparticles and microalgae to construct a new drug-loaded microalgae motor with strong controllability, good biocompatibility and long life, increase its drug-loaded types and drug-loaded amounts, so as to expand the application field of drug-loaded microalgae motors in targeted drug transport.

[0006] In order to achieve the above object, the technical solution of the present invention is: In a first aspect, the present invention provides a drug-loaded microalgae motor, comprising a polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor and a drug loaded thereon, wherein the drug is bound to the catechol group on the surface of polydopamine in the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor through intermolecular interactions; the drug is an anticancer drug or an anti-inflammatory drug.

[0007] In some other embodiments, the anti-cancer drug is doxorubicin, and the anti-inflammatory drug is curcumin.

[0008] In some other embodiments, 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.

[0009] In some other embodiments, the mass ratio of the polydopamine to the ferrosoferric oxide nanoparticles is (0.05-0.5): (1-2); 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.

[0010] The overall inventive concept adopted by the present invention is: The present invention makes full use of the great advantages of Chlamydomonas reinhardtii in bioimaging, drug delivery, hypoxic tumor treatment, wound healing, etc. Polydopamine is a mussel biomimetic material obtained by polymerization of dopamine. Since the catechol group and primary amine group on polydopamine make it have adhesion and metal coordination, it can be adsorbed on the surface of almost all solid materials (such as precious metals, carbon materials, organic polymers, etc.) to form a layer of polydopamine film. In addition, the catechol group can form a covalent bond with an amino or thiol reagent through different chemical reactions, which is conducive to the grafting of various drugs to the surface of polydopamine, and can be slowly released under appropriate conditions. Due to its good tissue adhesion, it can adhere to the intestinal mucosal layer, further prolonging the retention time of the drug in the intestine, which is conducive to improving the utilization rate of the drug. Therefore, polydopamine is a good carrier material for drugs.

[0011] At the same time, due to the advantages of low cytotoxicity and high biocompatibility, polydopamine has broad application prospects in biomedicine and environmental governance. In direct drug delivery research, polydopamine nanoparticles are mostly used as drug carriers because of their large specific surface area and high drug loading rate. However, in solutions, especially in biological systems, polydopamine nanoparticles are unstable and easy to agglomerate, which limits their further application in drug delivery. Ferroferric oxide nanoparticles have the advantages of good biocompatibility, strong magnetism, and easy preparation. Using polydopamine nanoparticles and ferroferric oxide nanoparticles as surface modifiers for Chlamydomonas reinhardtii can improve the drug loading amount and drug types of Chlamydomonas reinhardtii through the synergistic effect of the three, and reduce the stimulation of drugs to Chlamydomonas reinhardtii.

[0012] In a second aspect, the present invention provides a method for preparing the drug-loaded microalgae motor according to the first aspect, comprising 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 to obtain a polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor; Drugs were added to the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotors for loading, and the drug-loaded microalgae motors were obtained after incubation in the dark. In some other embodiments, the pretreatment method of the Chlamydomonas reinhardtii algae liquid is to centrifuge the Chlamydomonas reinhardtii algae liquid, remove the supernatant and add water for later use.

[0013] In some other embodiments, the rotation speed of the centrifugal treatment is 500-1500 rpm, and the centrifugation time is 1-10 min.

[0014] In some other embodiments, 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 pH of the Tris-HCl buffer solution is 7.0-9.0, and the concentration is 0.005-0.02 mol / L.

[0015] In some other embodiments, 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.

[0016] In a third aspect, the present invention provides use of the drug-loaded microalgae motor described in the first aspect in the preparation of disease-targeted therapeutic drugs, wherein the disease-targeted therapeutic drugs are particularly used for the treatment of diseases in the gastrointestinal tract.

[0017] In some other embodiments, the disease targeted therapeutic drug is a capsule containing polydopamine / ferroferric oxide / Chlamydomonas reinhardtii drug-loaded micromotors; 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.

[0018] In some other embodiments, the hydrophobic inner layer is trimethoxy(octadecyl)silane; and the pH-sensitive polymer is Eudragit L100-55.

[0019] In a fourth aspect, the present invention provides an enteric-coated capsule, comprising a capsule shell and a drug contained in the capsule shell, wherein the capsule shell comprises 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.

[0020] Beneficial effects of the present invention: (1) The present invention uses Chlamydomonas reinhardtii, which is green, biocompatible, highly biodegradable, and has antioxidant, anti-inflammatory and intestinal flora regulating functions, as the base material of the micro-nanomotor, and adopts a simple in situ synthesis method to prepare the microalgae motor without producing any toxic substances.

[0021] (2) In the present invention, the catechol groups on the surface of polydopamine can form covalent bonds with amino or thiol reagents through different chemical reactions, so that the motor surface can be connected to a variety of drugs, thereby achieving the loading and release of a variety of drugs.

[0022] (3) The motor prepared by the present invention can achieve sustained autonomous movement in intestinal fluid. This long-term movement can significantly improve the intestinal distribution of the motor, thereby enhancing the drug retention range. In addition, the motor can be controlled to move to the part that needs treatment through an external magnetic field 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] FIG1 is a scanning electron microscope image of the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor and Chlamydomonas reinhardtii prepared in Example 1 of the present invention, wherein a is Chlamydomonas reinhardtii, b is a partial enlarged view of a, c is the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor prepared in Example 1, and d is a partial enlarged view of c; FIG2 is a self-driven motion diagram of the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor prepared in Example 1 of the present invention moving in a directional manner toward a magnet in a simulated intestinal fluid, wherein 1, 2, 3, and 4 are randomly selected motion trajectories of the dopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor; FIG3 is a graph showing the change in the loading amount of different concentrations of doxorubicin on the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor prepared in Example 1 of the present invention over time; Figure 4 This is a graph showing the relationship between the drug loading fluorescence intensity of the anti-inflammatory drug curcumin and the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor prepared in Example 2 of the present invention; Figure 5 The cytotoxicity of Chlamydomonas reinhardtii and the polydopamine / ferric oxide / Chlamydomonas reinhardtii micromotor prepared in Example 1 of the present invention and the killing effect of the motor loaded with doxorubicin on colon cancer cell Caco-2 were tested, wherein CR is Chlamydomonas reinhardtii, the PDA / Fe3O4 / CR motor is the polydopamine / ferric oxide / Chlamydomonas reinhardtii microalgae motor, and the DOX / PDA / Fe3O4 / CR motor is the doxorubicin / polydopamine / ferric oxide / Chlamydomonas reinhardtii microalgae motor. DETAILED DESCRIPTION

[0025] As described in the background technology, existing micro-nano motors have the disadvantages of high toxicity of the basic materials used, poor biodegradability, and few surface functional groups. Therefore, the present invention proposes a preparation method of polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor and its application in intestinal diseases. The present invention is further described in conjunction with the examples. If the specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. The components used without indicating the manufacturer are all conventional products that can be obtained commercially.

[0026] The Chlamydomonas reinhardtii in the embodiment was purchased from Guangyu Biotechnology Co., Ltd., ferroferric 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 administration) were purchased from Torpac; the simulated gastric fluid composition was: 0.2% (w / w) NaCl and 0.7% (w / w) HCl, and the simulated intestinal fluid composition was: 0.68% (w / w) NaH2PO4 and 0.4% (w / w) NaOH.

[0027] The fluorescence intensity was determined by using a fluorescence spectrometer (FLS-920, Edinburgh Instruments Ltd, UK) at an excitation wavelength of 467 nm and an emission wavelength of 590 nm to measure the fluorescence intensity of doxorubicin in the supernatant.

[0028] Since the drug-loaded micromotor formed after polydopamine / ferroferric oxide / Chlamydomonas reinhardtii is loaded with drugs, it converts chemical energy into mechanical kinetic energy based on chemical reactions to drive the motor movement, but once it contacts the fluid in the gastrointestinal tract (GI), the retention time is shortened and the interaction with the gastrointestinal mucosa is weakened, which will lead to a decrease in the bioavailability of the drug. Therefore, in order to prolong the retention time of the drug in the intestinal mucosa, improve the delivery efficiency of the drug, and achieve efficient and controllable driving of the micromotor, the present invention transforms the commercial capsule, and encapsulates the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor into the modified capsule and delivers it to the mouse intestine to achieve controllable release of the drug.

[0029] The specific process of commercial capsule transformation is as follows: (1) Cover the inside of the commercial capsule with a hydrophobic inner coating so that the motor solution can be stored in the capsule. The specific method is: stir 2-20 wt% trimethoxy(octadecyl)silane solution and ethanol at room temperature for 1-5 hours, fill 2-10 μL into the capsule each time, cure at 80-200 °C for 1-5 hours to completely evaporate the solvent, repeat 10-20 times, and obtain a capsule with a hydrophobic inner coating.

[0030] (2) Coating the outside of the capsule with polymer Eudragit L100-55 to prevent it from being dissolved by gastric acid. The specific method is: dissolving Eudragit L100-55 in ethanol to prepare a 1-10% enteric coating solution, and then immersing the capsule covered with a hydrophobic inner coating into the enteric coating solution by dip coating, then taking out the capsule and evaporating the solvent, repeating 10-20 times to obtain the modified capsule.

[0031] (3) The liquid drug-loaded microalgae motor was loaded into the modified capsule through a flat-tip microsyringe (50 μL, Hamilton). The capsule loaded with the drug-loaded microalgae motor was fed into the mouth of the mouse, thereby achieving drug release in the intestine. If controlled by an external magnetic field, targeted drug release at the disease site can also be achieved.

[0032] Example 1 1. A method for preparing a polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor, comprising the following steps: First, the algae solution of Chlamydomonas reinhardtii was centrifuged at 1000 rpm for 5 min, the supernatant was removed, and deionized water was added for later use. The density of Chlamydomonas reinhardtii was determined by counting with a hemocytometer to obtain the treated algae solution. Dopamine hydrochloride, ferric oxide nanoparticle dispersion (10-50 nm), Tris-HCl buffer solution (pH=7.5) and deionized water were added to the treated algae solution to obtain 10 mL algae solution (containing 0.05 mg / mL dopamine hydrochloride, 1 mg / mL ferric oxide nanoparticles, and 0.005 mol / L Tris-HCl buffer solution).

[0033] During the research process, the inventors found that if the amount of dopamine hydrochloride is too much, Chlamydomonas reinhardtii may stop moving due to the thick coating; if the amount of nano-ferroferric oxide dispersion and Tris-HCl buffer solution is too much, the irritation to Chlamydomonas reinhardtii will be too strong, causing the algae to stop moving or even die; if the amount of dopamine hydrochloride, nano-ferroferric oxide dispersion, and Tris-HCl buffer solution added is too small, a longer reaction time is required to obtain a motor, and the surface coating falls off quickly, and the motor maintains its magnetism for a short time.

[0034] By measuring the density of the algae solution and controlling the amount of algae solution, the final density of Chlamydomonas reinhardtii in each reaction was 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.

[0035] 2. A drug delivery method for polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor, comprising the following steps: (1) Polydopamine / Fe3O4 / Chlamydomonas reinhardtii micromotors loading the anticancer drug doxorubicin 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.

[0036] (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.

[0037] Example 2 1. A method for preparing a polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor, comprising the following steps: First, the algae solution of Chlamydomonas reinhardtii was centrifuged at 1000 rpm for 5 min, the supernatant was removed, and deionized water was added for later use. The density of Chlamydomonas reinhardtii was determined by counting with a hemocytometer to obtain the treated algae solution. Dopamine hydrochloride, nano-ferric oxide dispersion (10-50 nm), Tris-HCl buffer solution (pH=8.0), and a certain amount of deionized water were added to the treated algae solution to obtain 10 mL of algae solution (containing 0.02 mg / mL dopamine hydrochloride, 1.5 mg / mL ferric oxide nanoparticles, and 0.01 mol / L Tris-HCl buffer solution).

[0038] By measuring the density of the algae solution and controlling the amount of algae solution, the final density of Chlamydomonas reinhardtii in each reaction was controlled to 3×10 6 About 1000 micrograms / mL. The reaction was carried out for 15 hours under the growth conditions suitable for Chlamydomonas reinhardtii. After 15 hours of reaction, the microalgae motor and some magnetic nanoparticles with larger particle size were centrifuged to the bottom by 1000 rpm centrifugation for 5 minutes. The supernatant was removed, deionized water was added, and the magnet was placed under the centrifuge tube for 15 minutes. The magnetic nanoparticles and some motors that were not removed by centrifugation were sucked to the bottom of the centrifuge tube. 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 polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotors.

[0039] 2. A drug delivery method for polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotors, comprising the following steps: (1) Polydopamine / FeO4 / Chlamydomonas reinhardtii micromotors loading the anti-inflammatory drug curcumin 0.5-3 μg / mL of curcumin was added to the polydopamine / ferric oxide / Chlamydomonas reinhardtii micromotors, incubated in the dark, and centrifuged to obtain motors loaded with curcumin. The fluorescence intensity of the motor surface after loading with different concentrations of curcumin was observed in the dark field of an inverted fluorescence microscope. At the same time, the changes in motor activity after loading with different concentrations of curcumin were observed to select the most suitable loading drug concentration.

[0040] (2) Drug release of curcumin / polydopamine / ferric oxide / Chlamydomonas reinhardtii micromotors in different environments In simulated gastric fluid (pH=1.2) and simulated intestinal fluid (pH=6.8), the polydopamine / ferric oxide / Chlamydomonas reinhardtii micromotors with a loading concentration of 1.0 μg / mL curcumin (1.5×10 6 / mL) were loaded into the modified capsules and 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 under different environments and time.

[0041] Comparative Example 1 The difference from Example 1 is that polydopamine / ferroferric oxide is not added. The other preparation methods are the same as those in Example 1 to obtain doxorubicin / Chlamydomonas reinhardtii micromotors.

[0042] Comparative Example 2 The difference from Example 1 is that ferrosoferric oxide is not added. The other preparation methods are the same as those in Example 1 to obtain doxorubicin / polydopamine / Chlamydomonas reinhardtii micromotors.

[0043] Comparative Example 3 The difference from Example 1 is that Spirulina (Guangyu Biotechnology Co., Ltd.) is used instead of Chlamydomonas reinhardtii. The other preparation methods are the same as those in Example 1 to obtain doxorubicin / polydopamine / ferroferric oxide / Spirulina micromotors.

[0044] Performance Testing: 1. Microstructure observation The polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotors prepared in Examples 1 and 2 were observed using a scanning electron microscope. Figure 1 As shown, a is Chlamydomonas reinhardtii, b is a partial enlarged view of a, c is the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor prepared in Example 1, and d is a partial enlarged view of c. Figure 1 It can be seen that the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor prepared in Example 1 has a surface morphology and structure similar to that of Chlamydomonas reinhardtii, but the surface of the sample in Example 1 is covered with a layer of polydopamine film. From the local image, it can be observed that the surface of the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor contains polydopamine nanoparticles with a particle size of 100 nm and ferroferric oxide nanoparticles with a particle size of 20 nm, thereby confirming that the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor is successfully prepared in the present invention.

[0045] 2. Self-driving performance test of polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotors The movement of the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor prepared in Example 1 was observed. The movement trajectory of the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor when a magnet was attracted to the right side of the droplet was as follows: Figure 2 As shown, 1, 2, 3, and 4 are randomly selected trajectories of dopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotors. Figure 2 It can be seen that the movement of the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotors prepared in Example 1 is similar, all of which move in a directional manner toward the magnet, with a spiral trajectory and a movement speed of about 85 μm / s. It has self-driving ability and can achieve sustained movement in simulated intestinal fluid.

[0046] 3. Test of drug loading capacity of microalgae motor The loading capacity of the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotors prepared in Example 1 for different concentrations of doxorubicin (1.5-3.5 μg / mL) changes over time (0-40 min), as shown in FIG. Figure 3 As shown. Figure 3 It can be seen that the polydopamine / ferric oxide / Chlamydomonas micromotor can quickly bind to about 70% of doxorubicin within 2.5 minutes. As the concentration of doxorubicin in the solution increases, the loading amount of doxorubicin by the polydopamine / ferric oxide / Chlamydomonas micromotor increases. Considering the effect of doxorubicin on the activity of Chlamydomonas, 2.5 μg / mL was selected for loading.

[0047] Example 2 The relationship between the fluorescence intensity of the anti-inflammatory drug curcumin and the survival rate of the prepared polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor, as shown in Figure 4 As shown. Figure 4 It can be seen that when the volume ratio of solvent is ethanol: water is 1:19 and the concentration of curcumin is 1.0 μg / mL, the effect of Chlamydomonas reinhardtii loading curcumin is better.

[0048] In vitro anti-tumor effect test The thiazolyl blue method (MTT method) was used to test the cytotoxicity of Chlamydomonas reinhardtii and the polydopamine / ferric oxide / Chlamydomonas reinhardtii micromotors prepared in Example 1, as well as the killing effect of the motor loaded with doxorubicin on colon cancer cells Caco-2 (purchased from the cell bank of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences). Figure 5 As shown, CR is Chlamydomonas reinhardtii, PDA / Fe3O4 / CR motor is polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor, and PDA / Fe3O4 / CR motor is doxorubicin / polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor. Figure 5 It can be seen that even if the density of Chlamydomonas reinhardtii and polydopamine / FeO4 / Chlamydomonas reinhardtii micromotors is as high as 6×10 6 / mL, colon cancer cells can still maintain a survival rate of about 95% in simulated intestinal fluid, indicating that Chlamydomonas reinhardtii and polydopamine / ferric oxide / Chlamydomonas reinhardtii micromotors have good biocompatibility and have no effect on cell activity. After the polydopamine / ferric oxide / Chlamydomonas reinhardtii micromotors loaded with doxorubicin (drug-loaded microalgae motors) released drugs in simulated intestinal fluid, the cell survival rate dropped sharply as the number of drug-loaded microalgae motors increased. The density of drug-loaded microalgae motors was 6×10 6 When the cell viability was only 36.68% at 400 μg / mL, it indicated that the drug-loaded microalgae motors had good anti-tumor effects in vitro.

[0049] In Comparative Example 1, the activity of the prepared doxorubicin / Chlamydomonas reinhardtii micromotors was tested. Chlamydomonas reinhardtii could load doxorubicin, but due to the stimulation of doxorubicin, the mortality rate of Chlamydomonas reinhardtii was still very high (>60%) even at a lower concentration (2 μg / mL).

[0050] The polydopamine / Chlamydomonas reinhardtii micromotor prepared in Comparative Example 2 has no magnetism and cannot achieve targeted drug delivery. This microalgae motor can still load doxorubicin (2 μg / mL), but the loading amount is relatively small (<50%). This is because the ferrosoferric oxide nanoparticles are negatively charged and can also load a certain amount of doxorubicin.

[0051] The polydopamine / ferric oxide / spirulina micromotor prepared in comparative example 3 was successfully constructed after testing, indicating that our method can be used not only for Chlamydomonas reinhardtii, but also for drug delivery systems of other algae. However, since Spirulina itself is very easy to break and die under external forces (such as high-speed centrifugation) and loses its autonomous movement ability, it is difficult to apply the living Spirulina micromotor to the targeted drug delivery system. The existing work is to fix Spirulina in paraformaldehyde and then use the dead Spirulina skeleton as a carrier to construct an external field driven micromotor.

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A drug-loaded microalgae motor, characterized in that: The invention 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 the polydopamine in the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotor through intermolecular interaction; and the drug is an anticancer drug or an anti-inflammatory drug.

2. The drug-loaded microalgae motor according to claim 1, characterized in that: The anticancer drug is doxorubicin, and the anti-inflammatory drug is curcumin.

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

4. The drug-loaded microalgae motor according to claim 3, characterized in that: The mass ratio of the polydopamine to the ferroferric oxide nanoparticles is (0.05-0.5): (1-2); the particle size of the ferroferric 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.

5. A method for preparing the drug-loaded microalgae motor according to any one of claims 1 to 4, 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; Drugs were added to the polydopamine / ferroferric oxide / Chlamydomonas reinhardtii micromotors for loading, and the drug-loaded microalgae motors were obtained after incubation in the dark.

6. The method for preparing the drug-loaded microalgae motor according to claim 5, 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.

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

8. The method for preparing the drug-loaded microalgae motor according to claim 5, characterized in that: 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 pH of the Tris-HCl buffer solution is 7.0-9.0, and the concentration is 0.005-0.02 mol / L.

9. The method for preparing the drug-loaded microalgae motor according to claim 5, characterized in that: 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.

10. Use of the drug-loaded microalgae motor according to any one of claims 1 to 4 in the preparation of a disease-targeted therapeutic drug, wherein the disease-targeted therapeutic drug is used for the treatment of diseases in the gastrointestinal tract.

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