Magnetic composite probiotics capable of being remotely magnetically controlled as well as preparation method and application of magnetic composite probiotics

Abstract

The invention relates to the technical field of composite material preparation, in particular to magnetic composite probiotics capable of being remotely magnetically controlled and a preparation method and application thereof.Positively charged amino modified ferroferric oxide nano-particles are attached to the surface of negatively charged polydopamine coated lactobacillus rhamnosus by means of electrostatic interaction force, so that the magnetic composite probiotics capable of being remotely magnetically controlled are obtained; and the magnetic lactobacillus rhamnosus composite probiotics capable of being remotely magnetically controlled are successfully prepared. In order to solve the problems of poor delivery effect, too short residence time and the like of the current probiotics in the gastrointestinal field, the invention provides a scheme which is easy to manufacture and feasible.

Description

Technical Field

[0001] The present invention relates to the technical field of composite material preparation, and in particular to a magnetic composite probiotic capable of remote magnetic manipulation, a preparation method and application thereof. Background Art

[0002] As a kind of active microorganisms that are very beneficial to human health, probiotics play multiple key roles in maintaining human health. They can effectively promote the digestion and absorption of nutrients, help the human body to fully absorb various nutrients; at the same time, they can also significantly enhance the body's immunity, comprehensively regulate the systemic immune function, actively maintain the balance of intestinal flora, and ensure the stability and health of the intestinal microecological environment. These excellent health-promoting properties allow probiotics to show extremely broad application prospects in the prevention and treatment of various gastrointestinal diseases, and have become a highly watched research hotspot in the fields of medicine and health. However, in the actual application process, probiotics still face many severe challenges. First, the gastrointestinal environment is complex and harsh, especially gastric acid is highly corrosive. Probiotics must have excellent tolerance to avoid being inactivated by gastric acid in large quantities when passing through the stomach, and then smoothly reach the intestine to play a role. Secondly, the gastrointestinal tract is in a state of continuous peristalsis, and this normal physiological process greatly limits the residence time of probiotics in the intestine. Once the probiotics are quickly cleared from the gastrointestinal tract, their bioavailability will drop significantly, and the therapeutic efficacy will be greatly reduced. In summary, enhancing the retention rate of probiotics in the intestine has become the key to improving its therapeutic effect.

[0003] A variety of delivery systems have been developed to improve the residence time of probiotics in the intestine. Among them, the sustained-release system based on microdevices can release probiotics at a slow and steady rate, effectively prolonging its action time in the intestine; the adhesion system uses a special adhesion mechanism to make probiotics closely adhere to the intestinal wall to avoid being quickly discharged; the targeted modification technology accurately guides probiotics to specific parts of the intestine to enhance the targeted effect. These strategies have successfully extended the residence time of probiotics in the intestine to a certain extent. However, it cannot be ignored that these advanced delivery systems also face significant limitations. The manufacturing process is complicated and cumbersome, often involving high-precision equipment and complex operating procedures, which not only puts extremely high demands on production technology, but also greatly increases the difficulty and time cost of production. At the same time, the high manufacturing cost has severely restricted large-scale production and wide application, making it difficult to meet the large-scale needs of clinical and market. Therefore, developing a probiotic delivery strategy that is both simple, low-cost, and highly adaptable is of great significance for promoting the widespread application of probiotics in clinical treatment.

[0004] As a physical field that is easy to generate and precisely controllable in three-dimensional space, magnetic field has significant technical advantages: it is highly controllable and can achieve precise control of target objects by adjusting parameters such as field strength and gradient; it is highly safe and its non-contact mechanism enables it to work effectively in complex biological environments, avoiding tissue damage and infection risks that may be caused by traditional contact operations; it has a fast response speed and can achieve real-time control at the millisecond level. Based on these characteristics, magnetic control systems have shown broad application prospects in biomedical applications such as targeted drug delivery, cell manipulation, and micro-robot navigation, and have been widely and deeply studied. For example, magnetically controlled ferromagnetic robots can achieve precise navigation and positioning in narrow, tortuous and complex vascular networks with the guidance of external magnetic fields, providing innovative solutions for vascular interventional treatment; magnetic clusters, driven by external magnetic fields, can achieve active intravascular delivery, opening up new avenues for drug transportation and disease treatment.

[0005] Based on the successful application of magnetic control systems in the biomedical field, the introduction of magnetic control technology into the probiotic delivery strategy is expected to achieve controlled retention and efficient aggregation of probiotics in the intestine. This can not only significantly enhance the effect of probiotics in the intestine, but also further enhance its therapeutic efficacy in the treatment of gastrointestinal diseases, providing a new strategy for the treatment of gastrointestinal diseases.

[0006] In view of the above-mentioned defects, the inventors of the present invention finally obtained the present invention after a long period of research and practice. Summary of the invention

[0007] The purpose of the present invention is to solve the problems of poor delivery effect and short retention time of probiotics in the gastrointestinal field, and to provide a magnetic composite probiotic that can be remotely magnetically controlled, a preparation method and an application thereof.

[0008] In order to achieve the above object, the present invention discloses a method for preparing a magnetic composite probiotic that can be remotely magnetically controlled, comprising the following steps:

[0009] S1, preparing ferroferric oxide nanoparticles by a solvothermal method, and modifying the ferroferric oxide nanoparticles with (3-aminopropyl)triethoxysilane to obtain amino-modified ferroferric oxide nanoparticles;

[0010] S2, gently stirring Lactobacillus rhamnosus and dopamine in a Tris-HCl solution with a magnetic stirrer to obtain polydopamine-coated Lactobacillus rhamnosus;

[0011] S3, adding the solution of the aminated ferroferric oxide nanoparticles obtained in step S1 dropwise to the solution of the polydopamine-coated Lactobacillus rhamnosus, fully mixing by vortex oscillation to obtain a mixed solution, and centrifuging to obtain the magnetic composite probiotics. The binding force between the aminated magnetic ferroferric oxide nanoparticles and the polydopamine-coated Lactobacillus rhamnosus is an electrostatic interaction force.

[0012] The specific preparation method of the aminated ferroferric oxide nanoparticles in step S1 comprises the following steps:

[0013] S11, adding ferric chloride hexahydrate and polyacrylic acid to a mixed solution of ethylene glycol and diethylene glycol, stirring thoroughly, adding sodium acetate to the mixed solution, stirring evenly and transferring to a polytetrafluoroethylene reactor, and collecting the precipitate by centrifugation and washing after the reaction is completed;

[0014] S12, adding the ferrosoferric oxide nanoparticles obtained in step S11 dropwise to a mixed solution of ethanol, distilled water and ammonia water, then adding tetraethyl silicate and (3-aminopropyl) triethoxysilane to the mixed solution, stirring rapidly to mix the solution evenly, then adding (3-aminopropyl) triethoxysilane to the mixed solution, stirring overnight, centrifuging and washing after the reaction is completed, and dispersing the collected amination ferrosoferric oxide nanoparticles in the aqueous solution for later use.

[0015] In the step S11, the reaction temperature is 180° C. and the reaction time is 12 h.

[0016] In the step S2, the polydopamine coating on the surface of the polydopamine-coated Lactobacillus rhamnosus is prepared by self-polymerization, and the specific preparation method is as follows: under room temperature, Lactobacillus rhamnosus and dopamine are added to a Tris-hydrochloric acid buffer solution, and magnetic stirring is performed for 30 minutes to prepare the polydopamine-coated Lactobacillus rhamnosus. After the reaction is completed, the mixture is centrifuged and washed three times with a phosphate buffer solution, and then dispersed in a phosphate buffer solution and stored at 4°C.

[0017] In step S3, the concentration of the aminated ferroferric oxide nanoparticles in the mixed solution is 500 μg / mL, and the total number of viable cells of polydopamine-coated Lactobacillus rhamnosus is not less than 1×10 8 CFU / mL.

[0018] In step S3, the obtained magnetic composite probiotics are washed in a phosphate buffer solution with a pH of 7.4 and then stored at 4°C.

[0019] The invention also discloses remotely magnetically controlled magnetic composite probiotics prepared by the preparation method.

[0020] The present invention also discloses the use of the magnetic composite probiotics capable of remote magnetic manipulation in the preparation of medicines for treating gastrointestinal diseases.

[0021] The magnetic composite probiotics in the present invention use electrostatic forces to combine the positively charged aminoferric oxide with the negatively charged polydopamine-wrapped probiotics. Under the harsh conditions of simulating the gastrointestinal environment, the magnetic composite probiotics can not only maintain the stability of their own structure, but also ensure that the biological activity of the probiotics is not affected. In addition, the magnetic composite probiotics can be controlled to move and target aggregation under the drive of an external magnetic field. This probiotic delivery strategy based on magnetic control technology provides an easy-to-scale, efficient and controllable method to improve the effectiveness of probiotics in the treatment of gastrointestinal diseases. Based on the above advantages, the magnetically controlled probiotic delivery strategy shows great potential for application in clinical transformation.

[0022] Compared with the prior art, the beneficial effect of the present invention is that the present invention combines magnetic nanomaterials and probiotics based on the electrostatic force assembly method, giving the probiotics excellent magnetic response performance. Under the action of the external magnetic field, the magnetic composite probiotics show excellent controllability, can achieve precise directional movement and efficient aggregation, breaking through the bottleneck of random distribution and short residence time of traditional probiotics in the intestine. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Transmission electron microscope images and scanning electron microscope images of ferroferric oxide nanoparticles;

[0024] Figure 2 Transmission electron microscopy and scanning electron microscopy images of amination-modified ferrosoferric oxide nanoparticles;

[0025] Figure 3 is a particle size distribution diagram of ferroferric oxide nanoparticles and amination ferroferric oxide nanoparticles;

[0026] Figure 4 The zeta potential diagram of ferroferric oxide nanoparticles and amination ferroferric oxide nanoparticles;

[0027] Figure 5 It is the infrared absorption spectrum of ferroferric oxide nanoparticles and amination ferroferric oxide nanoparticles;

[0028] Figure 6 The X-ray diffraction patterns of ferroferric oxide nanoparticles and amination ferroferric oxide nanoparticles;

[0029] Figure 7 Optical photos and transmission electron microscope images of probiotics;

[0030] Figure 8Optical photos and transmission electron microscopy images of polydopamine-coated probiotics;

[0031] Figure 9 Transmission electron microscope images of magnetic composite probiotics at different concentrations of amino-ferric oxide nanoparticles;

[0032] Figure 10 The optical photos and transmission electron microscope images of magnetic composite probiotics;

[0033] Figure 11 Zeta potential diagrams of amino-ferric oxide nanoparticles, polydopamine-coated probiotics and magnetic composite probiotics;

[0034] Figure 12 This is an optical photograph of the magnetic composite probiotics being attracted by a magnet;

[0035] Figure 13 This is the Zeta potential diagram of magnetic composite probiotics after being placed at different pH values ​​for 2 hours;

[0036] Figure 14 Optical photos of the magnetic composite probiotics placed in a simulated gastric fluid environment at different time periods for 2 hours and transmission electron microscope images after 24 hours;

[0037] Figure 15 Optical photos of the magnetic composite probiotics placed in a simulated intestinal fluid environment at different time periods for 2 hours and transmission electron microscope images after 24 hours;

[0038] Figure 16 The growth curves of probiotics, polydopamine-coated probiotics and magnetic composite probiotics are shown;

[0039] Figure 17 It is a bar graph showing the survival number of probiotics, polydopamine-coated probiotics and magnetic composite probiotics in a simulated gastric fluid environment;

[0040] Figure 18 It is a bar graph showing the survival number of probiotics, polydopamine-coated probiotics and magnetic composite probiotics in bile salt solution;

[0041] Figure 19 The dead / alive bacteria fluorescence staining microscope images of probiotics, polydopamine-coated probiotics and magnetic composite probiotics, the scale bar is 25 μm;

[0042] Figure 20 This is a microscope image of the magnetic composite probiotics during the magnetic drive process, with a scale of 100 μm;

[0043] Figure 21 This is an optical photograph of the magnetic composite probiotic suspension under magnetic manipulation aggregation, the scale is 2 cm;

[0044] Figure 22 This is the optical density bar graph of magnetic composite probiotics under circulating flow after different treatments (untreated, without magnetic field, with magnetic field);

[0045] Figure 23 This is a microscope image of the magnetic aggregation process of magnetic composite probiotics under the action of a magnetic field, with a scale of 200 μm;

[0046] Figure 24 This is an optical photograph of the controlled aggregation and retention of magnetic composite probiotics in the simulated intestine, with a scale of 2 cm;

[0047] Figure 25 This is a fluorescence image of fluorescently labeled magnetic composite probiotics in the mouse intestine 24 hours after administration. DETAILED DESCRIPTION

[0048] The above and other technical features and advantages of the present invention are described in more detail below in conjunction with the accompanying drawings.

[0049] Example 1

[0050] Preparation method of ferroferric oxide nanoparticles: dissolve ferric chloride hexahydrate (1.08 g), polyacrylic acid (0.1 g) and sodium acetate (4.0 g) in the above mixture. After fully dissolved, transfer the solution to a polytetrafluoroethylene reactor and react at 200°C for 10 hours. After the reaction is completed, centrifuge and collect the resulting precipitate, which is ferroferric oxide nanoparticles.

[0051] Figure 1 The transmission electron microscope image and scanning electron microscope image of ferroferric oxide nanoparticles are shown in Figure 1. It can be seen from the figure that the ferroferric oxide nanoparticles are spherical, have a relatively uniform size and a rough surface.

[0052] Example 2

[0053] The preparation steps of amination of ferroferric oxide nanoparticles are as follows:

[0054] Ferroferric oxide nanoparticles (0.3 g) were added to a mixed solution of ethanol (140 mL), distilled water (30 mL) and ammonia (3 mL) and fully dispersed under ultrasound. After tetraethyl silicate (0.1 mL) and (3-aminopropyl) triethoxysilane (0.5 mL) were dissolved in ethanol (10 mL), they were added dropwise to the above solution and stirred for 2 hours. Subsequently, (3-aminopropyl) triethoxysilane (0.5 mL) was dissolved in ethanol (10 mL), added to the above mixture at the same rate, and continued to stir for 6 hours. After being fully washed with ethanol and water, the amination of the obtained ferroferric oxide nanoparticles was stored in water.

[0055] Figure 2 The transmission electron microscope image and scanning electron microscope image of the aminated ferroferric oxide nanoparticles are shown in Figure 1. From the figure, it can be seen that the aminated ferroferric oxide nanoparticles have the same morphology as the unmodified ferroferric oxide nanoparticles, which are spherical and have a relatively uniform size. However, due to the modification of the particles with the silane coupling agent, the surface of the aminated ferroferric oxide nanoparticles is smoother;

[0056] Figure 3 The particle size distribution diagram of ferroferric oxide nanoparticles and amination ferroferric oxide nanoparticles. It can be found from the figure that the particle size of the modified nanoparticles is slightly increased;

[0057] Figure 4 The Zeta potential diagram of ferroferric oxide nanoparticles and aminated ferroferric oxide nanoparticles. Compared with ferroferric oxide nanoparticles (~-35 mV), the Zeta potential of aminated ferroferric oxide nanoparticles is ~17 mV, which proves the successful modification of amino groups.

[0058] Figure 5 The infrared absorption spectra of Fe3O4 nanoparticles and amination Fe3O4 nanoparticles confirm the successful modification of amino groups.

[0059] Figure 6 The X-ray diffraction patterns of ferroferric oxide nanoparticles and amino-modified ferroferric oxide nanoparticles prove that the successful modification of the amino group does not affect the structure of ferroferric oxide.

[0060] Example 3

[0061] The preparation of probiotics with polydopamine coating is as follows:

[0062] 1×10 8 CFU / mL of Lactobacillus rhamnosus and 0.8 mg / mL of dopamine were added to a Tris-hydrochloride buffer solution (pH=8.5), and gently stirred with a magnetic stirrer at room temperature for 30 minutes to generate polydopamine-coated probiotics. The mixed solution was centrifuged and washed three times with a phosphate buffer solution (pH=7.4), and the obtained probiotics were stored in a phosphate buffer solution at 4°C.

[0063] Figure 7 These are optical photographs and transmission electron microscope images of probiotics. It can be seen from the figures that the surface of Lactobacillus rhamnosus is smooth and the overall solution is white.

[0064] Figure 8 These are optical photographs and transmission electron microscope images of polydopamine-coated probiotics. It can be seen from the figures that after the in-situ polymerization of polydopamine, a layer of polydopamine shell grows on the surface of Lactobacillus rhamnosus and the color of the solution turns light black.

[0065] Example 4

[0066] A preparation method of a magnetic composite probiotic that can be remotely magnetically controlled, comprising the following steps:

[0067] The concentrations of 250 μg / mL, 500 μg / mL, and 700 μg / mL of aminated ferroferric oxide nanoparticles were added to three groups of 1×10 8 CFU / mL of polydopamine-coated probiotic suspension. Subsequently, the mixed solution was oscillated and mixed evenly under a vortexer. The mixed solution was centrifuged and washed three times with a phosphate buffer solution, and the obtained magnetic composite probiotics were stored in a phosphate buffer solution at 4°C.

[0068] Figure 9 The transmission electron microscope images of the magnetic composite probiotics when different concentrations of aminated ferroferric oxide nanoparticles are added, from which it can be seen that when the concentration of aminated ferroferric oxide nanoparticles is 250 μg / mL, the aminated ferroferric oxide nanoparticles adsorbed on the surface of Lactobacillus rhamnosus are less; when the concentration of aminated ferroferric oxide nanoparticles increases to 500 μg / mL, the aminated ferroferric oxide nanoparticles adsorbed on the surface of Lactobacillus rhamnosus increase accordingly; when the concentration of aminated ferroferric oxide nanoparticles increases to 750 μg / mL, unadsorbed nanoparticles appear around Lactobacillus rhamnosus. The subsequent study of the present invention selects a concentration of aminated ferroferric oxide nanoparticles of 500 μg / mL.

[0069] Figure 10 The optical photograph and transmission electron microscope image of the magnetic composite probiotics prepared when the concentration of the aminated ferroferric oxide nanoparticles is 500 μg / mL, from which it can be seen that the surface of Lactobacillus rhamnosus has obvious magnetic particles attached and the color of the solution turns into dark brown;

[0070] Figure 11 Zeta potential diagram of amino-iron tetroxide nanoparticles, polydopamine-coated probiotics and magnetic composite probiotics. After assembly with positively charged magnetic particles, the Zeta potential of the magnetic composite probiotics is significantly reduced compared with the Zeta potential of the polydopamine-coated probiotics.

[0071] The above data prove that electrostatic force assembly is introduced during the preparation process, and the positively charged amino-ferric oxide nanoparticles are adsorbed on the surface of the negatively charged polydopamine-coated probiotics to prepare magnetic composite probiotics.

[0072] Example 5

[0073] The magnetic manipulation performance test of magnetic composite probiotics is as follows:

[0074] Add 1.5 mL of magnetic composite probiotics into a plastic dish, place a magnet next to the plastic dish, and observe the changes in the magnetic composite probiotic solution.

[0075] Figure 12 The optical photos of the magnetic composite probiotics before and after being attracted by a magnet show that the color of the magnetic composite probiotic suspension changes from brown to colorless, indicating that the magnetic composite probiotics have excellent magnetic manipulation properties.

[0076] Example 6

[0077] The structural stability of magnetic composite probiotics is characterized by the following steps:

[0078] 1) The magnetic composite probiotics were mixed with solutions of different pH values, and the Zeta potential of the solutions was tested after 2 hours.

[0079] 2) The magnetic composite probiotics were mixed with simulated gastric fluid and simulated intestinal fluid, adsorbed using magnets at different time points, and the color of the solution was observed.

[0080] 3) The magnetic composite probiotics were mixed with simulated gastric fluid and simulated intestinal fluid, and the structure of the magnetic composite probiotics was observed by transmission electron microscopy after 24 hours.

[0081] Figure 13 The Zeta potential diagram of the magnetic composite probiotics after being placed at different pH values ​​for 2 hours shows that the magnetic composite probiotics can maintain charge stability in an acidic environment (pH = 4 and pH = 2);

[0082] Figure 14 The optical photos of the magnetic composite probiotics at different time periods during 2 hours of being placed in a simulated gastric fluid environment and the transmission electron microscope images after being placed for 24 hours show that the magnetic composite probiotics are attracted after the magnetic field is applied, and the color of the adsorbed solution does not change within 2 hours, indicating that the magnetic composite probiotics are not corroded and degraded. The transmission electron microscope image shows that the structure of the magnetic composite probiotics remains stable within 24 hours.

[0083] Figure 15 These are optical photographs of the magnetic composite probiotics at different time periods during a 2-hour period in a simulated intestinal fluid environment and a transmission electron microscope image after 24 hours of placement. The figures are consistent with the phenomena in a simulated gastric fluid environment, indicating that the magnetic composite probiotics can maintain their structural stability in a simulated intestinal fluid environment.

[0084] The above data prove that the magnetic composite probiotics have excellent structural stability and can withstand harsh gastrointestinal conditions while maintaining structural stability.

[0085] Example 7

[0086] The reproductive activity of magnetic composite probiotics and their survival rate test under simulated gastrointestinal conditions are as follows:

[0087] 1) Add 1 mL of probiotics, polydopamine-coated probiotics and magnetic composite probiotics to MRS medium, and then culture at 37° C. and 140 rpm. Measure the optical density (OD) of the culture at a wavelength of 600 nm every 2 hours.

[0088] 2) Each containing 1×10 8 Probiotics, polydopamine-coated probiotics, and magnetic composite probiotic strains with CFU / mL were placed in 1mL simulated gastric fluid (pH 2.0) or 0.3mg / mL bile solution. Then they were cultured at 37°C and 140rpm under shaking conditions. Every 1h and 2h, 100μL of samples were extracted from each group and dropped on solid MRS agar, and colonies were counted after overnight culture at 37°C. In addition, live and dead bacteria staining kits were used for staining and observed by inverted fluorescence images.

[0089] Figure 16 The growth curves of probiotics, polydopamine-coated probiotics and magnetic composite probiotics are shown in the figure. As can be seen from the figure, within the same culture time, the growth curves of the three are similar, and there is no significant difference in OD values. The results show that the adsorption of polydopamine coating and amino-ferric oxide has almost no effect on the activity of probiotics.

[0090] Figure 17 The figure shows the number of surviving probiotics in the simulated gastric fluid environment of probiotics, polydopamine-coated probiotics and magnetic composite probiotics. It can be seen from the figure that the number of surviving probiotics in the polydopamine-coated probiotics and magnetic composite probiotics groups is much higher than that in the simple probiotics group, proving that the polydopamine-modified probiotics and magnetic composite probiotics can resist the killing of probiotics by gastric acid and maintain a high survival rate.

[0091] Figure 18 The survival rate of probiotics, polydopamine-coated probiotics and magnetic composite probiotics in bile salt solution was tested. The results were consistent with those in simulated gastric fluid. Even after 2 hours of exposure, polydopamine-coated probiotics and magnetic composite probiotics still showed high activity.

[0092] Figure 19 The dead / alive bacterial fluorescence staining microscope images of probiotics, polydopamine-coated probiotics, and magnetic composite probiotics show that the probiotics showed strong red and weak green fluorescence signals in the simulated gastric fluid group, indicating that most of the probiotics were inactivated. The bile salt solution treatment group also showed strong green and weak red fluorescence signals, indicating a high survival rate. It is worth noting that strong green fluorescence signals were observed in all different treatment groups of polydopamine-coated probiotics and magnetic composite probiotics.

[0093] Figures 16 - 19 It was proved that the magnetic composite probiotics prepared by electrostatic force assembly have good protective properties provided by the polydopamine coating, so that the magnetic probiotics can maintain a high survival rate in an acidic environment and improve their survival rate after entering the intestine.

[0094] Example 8

[0095] The in vitro magnetic driving performance of magnetic composite probiotics is as follows:

[0096] 1) Disperse 1 μL of magnetic composite probiotics in 100 μL of phosphate buffer solution, and pipette and drop it onto a glass slide.

[0097] 2) The magnetic actuation performance was evaluated by observation using an inverted fluorescence microscope at magnetic field strengths of 10 and 15 mT, respectively.

[0098] Figure 20 This is a microscope image of the magnetic composite probiotics during the magnetic drive process (scale is 100μm). When the magnetic field strength is 10mT, it takes 30s for the magnetic composite probiotics to move to the other end. When the magnetic field strength is 15mT, it takes 15s. The results show that the magnetic composite probiotics can respond to the external magnetic field, and its magnetic drive behavior is related to the magnetic field strength.

[0099] Figure 21 This is an optical photo of the magnetic composite probiotic suspension aggregated under magnetic manipulation. It can be seen from the figure that when the microfluidic device is used to simulate the intestine, in the absence of a magnetic field, no aggregation of magnetic composite probiotics is observed on the pipeline after 30 minutes of circulation. Under the action of the magnetic field, the magnetic composite probiotics aggregate in the pipeline, showing an obvious retention effect;

[0100] Figure 22 This is a bar graph of the optical density of magnetic composite probiotics under circulation flow after different treatments (untreated, without magnetic field, with magnetic field). It can be seen from the figure that in the absence of a magnetic field, the difference in optical density values ​​before and after circulation flow is small; in the presence of a magnetic field, the optical density value of the magnetic composite probiotic solution is significantly reduced;

[0101] Figure 23 This is a microscope image of the magnetic aggregation process of magnetic composite probiotics under the action of a magnetic field. It can be seen from the figure that in the original state, the magnetic composite probiotics are randomly dispersed in the solution. When an external magnetic field is applied, the magnetic composite probiotics move along the direction of the magnetic field and gradually form large aggregates;

[0102] Figure 24This is an optical photograph of the controllable aggregation and retention of magnetic composite probiotics in the simulated intestine. Even after washing for 30 minutes, the magnetic composite probiotics still remain in an aggregated state. After removing the magnetic field, the aggregated magnetic composite probiotics are washed away by the phosphate buffer solution. These results show that the magnetic composite probiotics have excellent magnetic drive performance and can be controlled to aggregate and retain.

[0103] Figures 20 - 24 It was proved that the magnetic composite probiotics have excellent magnetic driving performance based on electrostatic force assembly. Under the action of the external magnetic field, the magnetic composite probiotics achieved controllable movement and demonstrated controllable aggregation and retention functions.

[0104] Example 9

[0105] The in vivo magnetically controlled retention performance of magnetic composite probiotics is as follows:

[0106] 1) Add the fluorescent material FITC in the step of modifying the probiotics with polydopamine, so that the prepared probiotics have a fluorescent effect. Add the amino-containing ferroferric oxide nanoparticles (500 μg) to 1×10 8 The mixture was then mixed with a fluorescently labeled probiotic suspension of 100 CFU / mL. The mixed solution was then vortexed and mixed evenly. The mixed solution was centrifuged and washed three times with a phosphate buffer solution, and then dissolved in 3 mL of a phosphate buffer solution.

[0107] 2) The material prepared in step 1) (~1×10 8 CFU, 300 μL / mouse) was intragastrically administered into the stomach of mice. 24 hours after administration, the mice were euthanized and dissected to remove the intestines. Subsequently, the fluorescent signal in the intestine was detected using an IVIS spectrometer (Perkin Elmer).

[0108] Figure 25 This is a fluorescence image of the fluorescently labeled magnetic composite probiotics in the intestines of mice 24 hours after administration. As can be seen from the figure, the fluorescence signal in the intestines of the mice group without magnets is weak. In contrast, the fluorescence signal in the intestines of the mice group with magnets is very high.

[0109] The results show that in the real intestinal environment, the magnetic composite probiotics still exhibit excellent magnetic manipulation properties, achieving controllable aggregation and retention.

[0110] The above description is only a preferred embodiment of the present invention, which is only illustrative and not restrictive of the present invention. Those skilled in the art understand that many changes, modifications, and even equivalences may be made to the present invention within the spirit and scope defined by the claims of the present invention, but all of them will fall within the scope of protection of the present invention.

Claims

1. A method for preparing a magnetic composite probiotic that can be remotely magnetically controlled, characterized in that: The following steps are involved: S1, preparing ferroferric oxide nanoparticles by a solvothermal method, and modifying the ferroferric oxide nanoparticles with (3-aminopropyl)triethoxysilane to obtain amino-modified ferroferric oxide nanoparticles; S2, gently stirring Lactobacillus rhamnosus and dopamine in a Tris-HCl solution with a magnetic stirrer to obtain polydopamine-coated Lactobacillus rhamnosus; S3, adding the solution of the aminated ferroferric oxide nanoparticles obtained in step S1 dropwise to the solution of the polydopamine-coated Lactobacillus rhamnosus, fully mixing by vortex oscillation to obtain a mixed solution, and obtaining the magnetic composite probiotics after centrifugal separation.

2. The method for preparing a magnetic composite probiotic capable of remote magnetic manipulation according to claim 1, characterized in that: The specific preparation method of the aminated ferroferric oxide nanoparticles in step S1 comprises the following steps: S11, adding ferric chloride hexahydrate and polyacrylic acid to a mixed solution of ethylene glycol and diethylene glycol, stirring thoroughly, adding sodium acetate to the mixed solution, stirring evenly and transferring to a polytetrafluoroethylene reactor, and collecting the precipitate by centrifugation and washing after the reaction is completed; S12, adding the ferrosoferric oxide nanoparticles obtained in step S11 dropwise to a mixed solution of ethanol, distilled water and ammonia water, then adding tetraethyl silicate and (3-aminopropyl) triethoxysilane to the mixed solution, stirring rapidly to mix the solution evenly, then adding (3-aminopropyl) triethoxysilane to the mixed solution, stirring overnight, centrifuging and washing after the reaction is completed, and dispersing the collected amination ferrosoferric oxide nanoparticles in the aqueous solution for later use.

3. The method for preparing a magnetic composite probiotic capable of remote magnetic manipulation according to claim 2, characterized in that: In the step S11, the reaction temperature is 180° C. and the reaction time is 12 h.

4. The method for preparing a magnetic composite probiotic capable of remote magnetic manipulation according to claim 1, characterized in that: In step S2, the preparation method of polydopamine-coated Lactobacillus rhamnosus is as follows: under room temperature, Lactobacillus rhamnosus and dopamine are added to a Tris-hydrochloric acid buffer solution, and magnetically stirred for 30 minutes to prepare Lactobacillus rhamnosus coated with a polydopamine coating. After the reaction is completed, the mixture is centrifuged and washed three times with a phosphate buffer solution, and then dispersed in a phosphate buffer solution and stored at 4°C.

5. The method for preparing a magnetic composite probiotic capable of remote magnetic manipulation according to claim 1, characterized in that: In step S3, the concentration of the aminated ferroferric oxide nanoparticles in the mixed solution is 500 μg / mL, and the total number of viable cells of polydopamine-coated Lactobacillus rhamnosus is not less than 1×10 8 CFU / mL.

6. The method for preparing a magnetic composite probiotic capable of remote magnetic manipulation according to claim 1, characterized in that: In step S3, the obtained magnetic composite probiotics are washed in a phosphate buffer solution with a pH of 7.4 and then stored at 4°C.

7. A magnetic composite probiotic capable of remote magnetic manipulation, prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the remotely magnetically controllable magnetic composite probiotics as claimed in claim 7 in the preparation of medicines for treating gastrointestinal diseases.

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