Drug-loaded biomimetic magnetic micro-robot for anti-tuberculosis and preparation method thereof

Abstract

The application discloses a drug-loaded biomimetic magnetic micro-robot for resisting tuberculosis and a preparation method thereof, and belongs to the technical field of drug carriers. The application takes magnetotactic bacteria AMB-1 as a core carrier, and in view of different properties of active pharmaceutical ingredients (such as anti-tuberculosis drugs, mRNA, protein particles), the active pharmaceutical ingredients are loaded on the surface of the bacteria by adopting electrostatic adsorption, hydrophobic insertion, covalent coupling or cationic polymer-mediated electrostatic binding mode. The micro-robot can actively move under the mediation of an external magnetic field, penetrates physiological barriers such as lung dense granuloma, targets and delivers drugs to the core area of a lesion, prolongs the in-vivo residence time of the drugs, and improves the treatment efficiency. The application has good biological safety, and provides a new strategy for the targeted treatment of tuberculosis and other diseases.

Description

Technical Field

[0001] This invention belongs to the field of drug carrier technology, specifically relating to a drug-carrying biomimetic magnetic microrobot for anti-tuberculosis and its preparation method. Background Technology

[0002] The World Health Organization's "Global Tuberculosis Report 2025" shows that tuberculosis remains one of the world's deadliest infectious diseases, causing more than 1.2 million deaths in 2024. Traditional treatments for tuberculosis often involve chemotherapy with various drugs, but these methods have drawbacks, such as short drug retention time in the body, easy clearance, lack of targeting, and even toxic side effects on systemic tissues. Furthermore, Mycobacterium tuberculosis forms dense granulomas in the lungs, leading to dense fibrosis of lung tissue and creating a physiological barrier that prevents drugs from penetrating the barrier to reach the lesion area, resulting in insufficient drug concentration and inadequate bactericidal effect. Granulations can also hinder the aggregation of immune cells, thus impairing immune clearance. Therefore, tuberculosis treatment requires a new approach that can penetrate dense granulomas to reach deep into the lesion, precisely targeting lung lesions and prolonging the retention time in the body. This is of great significance for the treatment of tuberculosis.

[0003] In recent years, microrobots have been widely used in biomedicine and have shown promising medical prospects. Among them, magnetotactic bacterial microrobots are an ideal drug delivery carrier for the treatment of tuberculosis. Magnetotactic bacteria, with the help of magnetosomes in their endosomes, can actively move in the body according to a designed route under the control of an external magnetic field, passing through the lung mucus layer, penetrating granulomas and reaching the central lesion area. Furthermore, with the support of the magnetic field, the residence time in the body can be prolonged, while also exhibiting good biocompatibility. Summary of the Invention

[0004] To address the issues of non-targeting and insufficient drug concentration caused by traditional chemotherapy drugs' difficulty in crossing the lung barrier, this invention utilizes magnetotactic bacteria as drug delivery carriers, building upon traditional drug therapy to achieve targeted treatment for tuberculosis. On one hand, mediated by a magnetic field, magnetotactic bacteria, through their magnetosomes, can actively transport drugs within the lungs over time, penetrating dense granulomas and delivering drugs to the core of the lesion. On the other hand, the magnetic field also prolongs the drug's time in the body, thereby improving therapeutic efficiency. Furthermore, magnetotactic bacteria exhibit good biocompatibility, without causing serious toxic side effects on body tissues, increasing its feasibility in clinical treatment.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] A drug-carrying biomimetic magnetic microrobot includes magnetotactic bacteria and a drug active ingredient loaded on the surface of the magnetotactic bacteria;

[0007] The active pharmaceutical ingredient is selected from anti-tuberculosis drugs, anti-tuberculosis mRNA, or anti-tuberculosis protein particles;

[0008] The active pharmaceutical ingredient is loaded onto the surface of the magnetotactic bacteria in any of the following ways:

[0009] (a) Electrostatic adsorption;

[0010] (b) Hydrophobic insertion;

[0011] (c) Covalent coupling; or

[0012] (d) Electrostatic bonding mediated by cationic polymers.

[0013] Furthermore, the magnetotactic bacteria is strain AMB-1. Magnetotactic bacteria AMB-1 itself carries a negative charge.

[0014] Furthermore, the anti-tuberculosis drug is streptomycin, amikacin, kanamycin, isoniazid, rifampin, ethambutol, para-aminosalicylic acid, bedaquiline, or delamani;

[0015] Furthermore, the anti-tuberculosis mRNA is mRNA-Ag85B, mRNA-ESAT-6, or mRNA-M72;

[0016] Furthermore, the anti-tuberculosis protein particles are LL37, BSA nanoparticles, Ag85B antigen protein particles, or HSA / BSA drug-loaded protein nanoparticles.

[0017] Furthermore, the electrostatic adsorption is suitable for positively charged active pharmaceutical ingredients;

[0018] Furthermore, the hydrophobic insertion is suitable for lipid-soluble pharmaceutical active ingredients;

[0019] Furthermore, the covalent coupling is applicable to pharmaceutical active ingredients containing carboxyl or amino groups, and is connected to the surface of magnetotactic bacteria via amide or imine bonds;

[0020] Furthermore, the cationic polymer-mediated electrostatic binding is suitable for loading negatively charged pharmaceutical active ingredients onto magnetotactic bacteria coated with polyethyleneimine (PEI) or poly-L-lysine (PLL).

[0021] A method for preparing the aforementioned drug-loaded biomimetic magnetic microrobot includes the following steps:

[0022] Step 1: Cultivate magnetotactic bacteria to the logarithmic growth phase;

[0023] Step 2: Collect and wash magnetotactic bacteria;

[0024] Step 3: Based on the properties of the active pharmaceutical ingredient, select any one of the following methods: electrostatic adsorption, hydrophobic insertion, covalent coupling, or cationic polymer-mediated electrostatic binding, to load the active pharmaceutical ingredient onto the surface of magnetotactic bacteria.

[0025] Step 4: Purify to obtain drug-loaded biomimetic magnetic microrobots.

[0026] Furthermore, in step three:

[0027] When the active pharmaceutical ingredient is a positively charged anti-tuberculosis drug, it is loaded by electrostatic adsorption;

[0028] When the active pharmaceutical ingredient is a lipid-soluble anti-tuberculosis drug, it is loaded by inserting it into the hydrophobic region of the cell membrane of magnetotactic bacteria;

[0029] When the active pharmaceutical ingredient is an anti-tuberculosis drug containing carboxyl or amino groups, it is loaded by covalent coupling using an EDC / NHS crosslinking agent;

[0030] When the active pharmaceutical ingredient is mRNA or anionic protein particles, the magnetotactic bacteria are first coated with a cationic polymer and then loaded by electrostatic adsorption.

[0031] When the active pharmaceutical ingredient is any type of protein, the carboxyl groups on the surface of magnetotactic bacteria are activated using EDC / NHS and loaded via covalent coupling.

[0032] Further, in step three, when rifampin is loaded, the specific steps include: dissolving rifampin in dimethyl sulfoxide to obtain a mother liquor, diluting it to the working concentration, mixing it with the pretreated magnetotactic bacterial suspension, and incubating it in the dark at 25°C or 4°C for 2-6 hours.

[0033] Furthermore, in step three, when loading mRNA-Ag85B, the specific steps include: modifying the surface of magnetotactic bacteria with poly-L-lysine to make it positively charged, then adding the mRNA-Ag85B solution dropwise, and incubating on ice for 30 minutes at an N / P ratio of 5:1 to 10:1, so that the mRNA can cover the bacterial surface by electrostatic attraction.

[0034] Furthermore, in step three, when loading LL-37 protein particles, the specific steps include: activating the carboxyl groups on the surface of magnetotactic bacteria using EDC and NHS, then adding LL-37 self-assembled protein, and incubating at 4°C for 2-4 hours or at room temperature for 1 hour, so that the protein is covalently coupled to the bacterial surface via amide bonds.

[0035] Furthermore, the drug-loaded biomimetic magnetic microrobot is used to prepare drug formulations for treating tuberculosis.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] This invention utilizes the excellent biocompatibility of magnetotactic bacteria, which do not cause serious damage to the body. The resulting drug-carrying biomimetic magnetic microrobot can actively move under the guidance of an external magnetic field, possessing the ability to target lesions and increase drug concentration in the lesion area. It can penetrate physiological barriers such as pulmonary granulomas, reaching the center of the lesion directly. Detailed Implementation

[0038] To gain a deeper understanding of this invention, we will provide a comprehensive and detailed description. However, this invention has various implementations and is not limited to the specific examples listed herein. These examples are presented to enhance a full understanding of the disclosure of this invention.

[0039] Example 1:

[0040] This embodiment provides a specific method for preparing rifampicin-loaded magnetotactic bacteria, the steps of which are as follows:

[0041] 1. Cultivation of magnetotactic bacteria:

[0042] (1) Prepare Revised Magnetic Spirulina Growth Medium (MSGM) culture medium with the following formula:

[0043] 10.0 mL Wolfe's Vitamin Solution (see below), 5.0mL Wolfe's MineralSolution (see below), 2.0mL 0.01M Ferric Quinate (see below), 0.68g KH2PO4, 0.12gNaNO3, 0.035g Ascorbic acid, 0.37g Tartaric acid, 0.37g Succinic acid, 0.05gSodium acetate, 1000.0 mL Distilled water

[0044] (2) Take 50 mL of culture medium and add 5 mL of AMB-1 bacterial solution. Place it in a 30℃ incubator for anaerobic constant temperature culture until AMB-1 reaches the logarithmic phase (at this time, the magnetosomes are most complete and the magnetism is strongest).

[0045] 2. Preparation of rifampicin solution:

[0046] Powdered rifampicin was dissolved in dimethyl sulfoxide (DMSO) to prepare a high-concentration rifampicin stock solution (1 mg / mL).

[0047] 3. Pretreatment of magnetotactic bacteria:

[0048] (1) Collect bacterial cells: Take the cultured bacterial solution and centrifuge at 8000 rpm for 10 minutes at 4℃.

[0049] (2) Washing: Discard the supernatant of the culture medium and resuspend the cells in sterile PBS buffer.

[0050] (3) Magnetic purification: Use a magnetic separator to collect bacteria with strong magnetic induction and remove non-magnetic or weakly magnetic fragments. Repeat washing 2-3 times, and finally adjust the bacterial concentration to a fixed OD600 value.

[0051] 4. Preparation of working concentration dilution for rifampicin

[0052] Dilute rifampicin to the desired concentration (e.g., 50 μg / mL). Note that the final concentration of DMSO should be kept below 1% to avoid killing bacteria.

[0053] 5. Mixed incubation

[0054] (1) Mix the pretreated AMB-1 bacterial suspension with rifampicin solution in a certain proportion.

[0055] (2) Incubate at 25℃ or 4℃ (low temperature helps maintain bacterial motility) in the dark on a low-speed shaker (about 50 rpm) for 2 to 6 hours.

[0056] (3) Centrifugation: After incubation, centrifuge at 4°C to remove unbound drugs.

[0057] (4) Magnetic washing: Resuspend the precipitate in PBS and place it on the side of the magnetic separator. After the bacteria have completely adhered to the wall, discard the supernatant. Repeat this step 3 times until the supernatant shows no rifampicin signal under UV detection.

[0058] (5) Final resuspension: The drug-loaded AMB-1 was resuspended in sterile PBS for later use.

[0059] Example 2:

[0060] This example provides a specific method for preparing mRNA-Ag85B loaded with magnetotactic bacteria, the steps of which are as follows:

[0061] 1. mRNA preparation: Obtain the mRNA encoding Ag85B through in vitro transcription. Ensure it has a 5' guanylic acid cap and a 3' poly(A) tail.

[0062] 2. Cationic modification of magnetotactic bacteria (AMB-1):

[0063] (1) Bacterial washing: Magnetotactic bacteria AMB-1 in the logarithmic growth phase were collected by magnetic separation and washed three times with RNase-free PBS.

[0064] (2) Surface modification: The bacteria were resuspended in PBS containing 0.1% poly-L-lysine (PLL) and incubated with gentle shaking at 4°C for 30 minutes. PLL will attach to the lipopolysaccharide / protein of the bacterial cell wall through electrostatic interaction.

[0065] (3) Removal of excess polymers: Collect bacteria by centrifugation or magnetic separation, and wash twice with PBS to remove unbound PLLs. At this point, the surface charge of the bacteria should change from negative to positive by a potentiometer.

[0066] 3. Loaded with mRNA-Ag85B

[0067] (1) Ratio optimization: Set different N / P ratios (the ratio of nitrogen atoms in the polymer to phosphate groups in the mRNA), usually between 5:1 and 10:1.

[0068] (2) Electrostatic assembly: Dilute mRNA-Ag85B in RNase-free PBS. Add the mRNA solution dropwise to the PLL-modified AMB-1 suspension while gently pipetting. Incubate on ice for 30 minutes to allow the mRNA to completely cover the bacterial surface through electrostatic attraction.

[0069] (3) Magnetic purification: Use an external magnet to attract the mRNA-loaded bacteria to the tube wall. Discard the supernatant containing unloaded mRNA. Gently wash once with cold PBS.

[0070] Example 3

[0071] This embodiment provides a specific method for preparing protein particles LL-37 loaded onto the surface of magnetotactic bacteria AMB-1, the steps of which are as follows:

[0072] 1. Surface activation of AMB-1 bacteria

[0073] (1) Take 10 mL of AMB-1 culture medium, collect the bacteria using a magnetic separator, and discard the culture medium.

[0074] (2) Wash twice with sterile PBS, and then wash once with MES buffer.

[0075] (3) Resuspend the bacteria in 2 mL of MES buffer and add EDC (20-50 mM) and NHS (50-100 mM).

[0076] (4) Shake gently for 30 minutes at room temperature away from light.

[0077] 2. Remove excess crosslinking agent

[0078] (1) Place the activated bacteria on a magnetic separator and let it stand until the liquid becomes clear.

[0079] (2) Discard the supernatant and wash twice quickly with PBS (pH 7.4) to remove unreacted EDC / NHS.

[0080] 3. Protein loading (coupling)

[0081] (1) Add the prepared LL-37 self-assembled protein (concentration of about 100~500 μg / mL) to a PBS suspension containing activated AMB-1.

[0082] (2) Incubate at low speed at 4℃ for 2-4 hours (or at room temperature for 1 hour).

[0083] 4. Washing and purification

[0084] (1) A magnetic separator was used to attract AMB-1 loaded with protein.

[0085] (2) Collect the supernatant (for subsequent detection of the content of unbound protein).

[0086] (3) Wash the bacteria gently with PBS three times until no protein is detected in the eluent.

[0087] Finally, LL-37@AMB-1 was resuspended in PBS or sterile saline.

[0088] Contents not described in detail in this specification are prior art known to those skilled in the art. Although illustrative specific embodiments of the invention have been described above to facilitate understanding by those skilled in the art, it should be understood that the invention is not limited to the scope of the specific embodiments. Various modifications are readily apparent to those skilled in the art as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims, and all inventions utilizing the concept of this invention are protected.

Claims

1. A drug-carrying biomimetic magnetic microrobot, characterized in that, Includes magnetotactic bacteria and pharmaceutically active ingredients loaded on the surface of the magnetotactic bacteria; The active pharmaceutical ingredient is selected from anti-tuberculosis drugs, anti-tuberculosis mRNA, or anti-tuberculosis protein particles; The active pharmaceutical ingredient is loaded onto the surface of the magnetotactic bacteria in any of the following ways: (a) Electrostatic adsorption; (b) Hydrophobic insertion; (c) Covalent coupling; or (d) Electrostatic bonding mediated by cationic polymers.

2. The drug-carrying biomimetic magnetic microrobot according to claim 1, characterized in that, The magnetotactic bacteria is strain AMB-1.

3. The drug-carrying biomimetic magnetic microrobot according to claim 1, characterized in that, The anti-tuberculosis drugs are streptomycin, amikacin, kanamycin, isoniazid, rifampin, ethambutol, para-aminosalicylic acid, bedaquiline, or delamani; The anti-tuberculosis mRNA is mRNA-Ag85B, mRNA-ESAT-6, or mRNA-M72; The anti-tuberculosis protein particles are LL37, BSA nanoparticles, Ag85B antigen protein particles, or HSA / BSA drug-loaded protein nanoparticles.

4. The drug-carrying biomimetic magnetic microrobot according to claim 1, characterized in that: The electrostatic adsorption is suitable for positively charged active pharmaceutical ingredients; The hydrophobic insertion is suitable for lipid-soluble pharmaceutical active ingredients; The covalent coupling is applicable to pharmaceutical active ingredients containing carboxyl or amino groups, and is connected to the surface of magnetotactic bacteria via amide or imine bonds; The cationic polymer-mediated electrostatic binding is suitable for negatively charged pharmaceutical active ingredients, which are loaded by magnetotactic bacteria coated with polyethyleneimine or poly-L-lysine.

5. A method for preparing the drug-loaded biomimetic magnetic microrobot according to any one of claims 1 to 4, characterized in that, Includes the following steps: Step 1: Cultivate magnetotactic bacteria to the logarithmic growth phase; Step 2: Collect and wash magnetotactic bacteria; Step 3: Based on the properties of the active pharmaceutical ingredient, select any one of the following methods: electrostatic adsorption, hydrophobic insertion, covalent coupling, or cationic polymer-mediated electrostatic binding, to load the active pharmaceutical ingredient onto the surface of magnetotactic bacteria. Step 4: Purify to obtain drug-loaded biomimetic magnetic microrobots.

6. The method according to claim 5, characterized in that, In step three: When the active pharmaceutical ingredient is a positively charged anti-tuberculosis drug, it is loaded by electrostatic adsorption; When the active pharmaceutical ingredient is a lipid-soluble anti-tuberculosis drug, it is loaded by inserting it into the hydrophobic region of the cell membrane of magnetotactic bacteria; When the active pharmaceutical ingredient is an anti-tuberculosis drug containing carboxyl or amino groups, it is loaded by covalent coupling using an EDC / NHS crosslinking agent; When the active pharmaceutical ingredient is mRNA or anionic protein particles, the magnetotactic bacteria are first coated with a cationic polymer and then loaded by electrostatic adsorption. When the active pharmaceutical ingredient is any type of protein, the carboxyl groups on the surface of magnetotactic bacteria are activated using EDC / NHS and loaded via covalent coupling.

7. The method according to claim 5, characterized in that, In step three, when rifampin is loaded, the specific steps include: dissolving rifampin in dimethyl sulfoxide to obtain a mother liquor, diluting it to the working concentration, mixing it with the pretreated magnetotactic bacterial suspension, and incubating it in the dark at 25°C or 4°C for 2-6 hours.

8. The method according to claim 5, characterized in that, In step three, when loading mRNA-Ag85B, the specific steps include: modifying the surface of magnetotactic bacteria with poly-L-lysine to make it positively charged, then adding the mRNA-Ag85B solution dropwise, and incubating on ice for 30 minutes at an N / P ratio of 5:1 to 10:1, so that the mRNA can cover the bacterial surface by electrostatic attraction.

9. The method according to claim 5, characterized in that, In step three, when loading LL-37 protein particles, the specific steps include: using EDC and NHS to activate the carboxyl groups on the surface of magnetotactic bacteria, then adding LL-37 self-assembled protein, and incubating at 4°C for 2-4 hours or at room temperature for 1 hour, so that the protein is covalently coupled to the bacterial surface through amide bonds.

10. The drug-carrying biomimetic magnetic microrobot according to claim 1, characterized in that, The drug-loaded biomimetic magnetic microrobot is used to prepare drug formulations for the treatment of tuberculosis.