Mycoderm drug loading system as well as preparation method and application thereof

Abstract

The invention provides a pellicle drug-loading system and a preparation method and application thereof, the pellicle drug-loading system is of a structure that an outer membrane wraps a core, and the outer membrane is a bacterial outer membrane; the core is formed by loading near-infrared two-region AIE photo-thermal molecules on a tetrahedral DNA (deoxyribonucleic acid) nano structure. The invention has the advantages that: the mycoderm drug delivery system can realize spatial dual targeting and time visual regulation and control on pathogens, and constructs a positive feedback cycle of drug delivery-immune activation through triple function synergy, thereby providing a drug basis for treatment of bacterial infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug preparation, in particular to the technical field of mycomembrane drug delivery system. BACKGROUND

[0002] In the field of tuberculosis treatment, the existing technology mainly includes: traditional nanocarrier systems such as liposomes and polymer nanoparticles to deliver rifampicin, a first-line drug. These systems improve drug enrichment at the lesion site by passive targeting or surface modification of targeting molecules; at the same time, some studies attempt to combine immunomodulators such as cytokines or checkpoint inhibitors to enhance therapeutic effect. And photothermal therapy, the conventional approach is to use inorganic materials such as gold nanorods or carbon nanotubes as photothermal agents, combined with chemotherapy drugs for treatment. The most advanced in the existing technology is the macrophage membrane-wrapped nanocarrier, which uses the homologous targeting of biological membranes to improve drug delivery efficiency.

[0003] These existing technologies have improved drug targeting and reduced systemic toxicity to some extent, but still have obvious limitations: the drug loading efficiency of traditional nanocarriers is generally low, and it is difficult to break through the dense barrier of tuberculosis granuloma; the traditional photothermal agents used in photothermal therapy are prone to aggregation and inactivation in the high-viscosity tuberculosis lesion microenvironment, greatly reducing the treatment effect, and photothermal damage to normal tissues; immunomodulation strategies are mostly systemic administration, lack of lesion specificity, and are prone to side effects such as excessive immune activation; the macrophage membrane-wrapped system improves biocompatibility, but cannot provide specific immune activation against Mycobacterium tuberculosis, such as patent CN118141782A discloses the application of a biomimetic nanoparticle in tuberculosis diagnosis and photothermal therapy, the nanoparticle is a core-wrapped membrane structure, the outer membrane is the cell membrane of macrophages pre-activated by Mycobacterium tuberculosis, and the core is an aggregation-induced luminescent material TPE-BT-BBTD encapsulated in a degradable polymer to form an aggregation-induced luminescent composite material.

[0004] These technical defects directly lead to the existing treatment scheme having long treatment cycle (current tuberculosis treatment requires more than 6 months), easy recurrence, poor effect on drug-resistant tuberculosis, etc. In particular, the existing technology cannot simultaneously achieve efficient enrichment of drugs in granulomas and lymph nodes, and it is difficult to coordinate the timing relationship between chemotherapy and immune activation, so it is of great significance to provide a comprehensive treatment scheme that integrates efficient sterilization, precise targeting of granulomas and lymph nodes, and immune regulation, effectively solving the key problems of low drug delivery efficiency, poor lesion penetration, and inaccurate immune activation in the existing technology. SUMMARY

[0005] The present application provides a mycomembrane drug delivery system, its preparation method and application, to solve the problems of long treatment cycle, easy recurrence and poor antibacterial effect of existing antibacterial treatment drugs.

[0006] To achieve the above object, the present application adopts the following technical scheme:

[0007] The present application provides a bacterial membrane drug delivery system, which is a structure of an outer membrane wrapping a core, wherein the outer membrane is a bacterial outer membrane; and the core is composed of a tetrahedral DNA nanostructure loaded with near-infrared two-region AIE photothermal molecules.

[0008] Further, the tetrahedral DNA nanostructure is self-assembled from four single-stranded DNAs, and the sequences of the four single-stranded DNAs are shown in SEQ ID NO. 1-4.

[0009] Further, the bacterial outer membrane is a Mycobacterium marinum membrane.

[0010] Further, the bacterial outer membrane is a Mycobacterium marinum membrane containing key membrane proteins.

[0011] Further, the bacterial outer membrane is a Mycobacterium marinum membrane containing Ag85 complex.

[0012] The present application also provides a preparation method of the bacterial membrane drug delivery system, which comprises the following steps:

[0013] (1) synthesizing the four single-stranded DNAs;

[0014] (2) self-assembling the four single-stranded DNAs into a tetrahedral DNA nanostructure;

[0015] (3) loading AIE photothermal molecules onto the tetrahedral DNA nanostructure to obtain an intermediate product TDN+AIE;

[0016] (4) mixing the Mycobacterium marinum membrane and the TDN+AIE and co-extruding to wrap the TDN+AIE with the Mycobacterium marinum membrane, thereby obtaining the bacterial membrane drug delivery system TDN+AIE@MM.

[0017] Further, the detailed process of step (2) is as follows: dissolve the four single-stranded DNAs in TM buffer respectively, and the concentration of each single-stranded DNA solution is 50 µM; then take 2 µl of each single-stranded DNA solution into an eight-tube containing 92 µl of TM buffer, thereby obtaining a self-assembly reaction system; after denaturation at 95 ℃ for 10 min and slow annealing to room temperature for 20 min, the tetrahedral DNA nanostructure is obtained and stored at 4 ℃.

[0018] Further, the detailed process of step (3) is as follows: 200 µg / ml AIE photothermal molecules and 100 nM tetrahedral DNA nanostructures are mixed at 4 ℃ for 24 h. The AIE photothermal molecules are loaded onto the tetrahedral DNA nanostructures through intermolecular forces to obtain the intermediate product TDN+AIE.

[0019] Further, in step (4), the Mycobacterium marineis membrane with a protein concentration of 2 mg / ml and TDN+AIE are mixed at a volume ratio of 1:1 and co-extruded to coat TDN+AIE with the Mycobacterium marineis membrane, thus obtaining the membrane-loaded drug system TDN+AIE@MM.

[0020] The present invention also provides the application of the bacterial film drug delivery system in the preparation of drugs for treating bacterial infections.

[0021] The present invention also provides the application of the bacterial film drug delivery system in the preparation of drugs for treating tuberculosis.

[0022] The advantages of this invention are:

[0023] It provides an innovative synergistic treatment system for tuberculosis by encapsulating Mycobacterium marinum membranes around tetrahedral DNA nanostructures (TDN) loaded with AIE (aggregation-induced emission) photothermal molecules, thus constructing a multifunctional biomimetic drug delivery platform to achieve dual synergistic treatment of "photothermal clearance + immune activation," providing a brand-new solution for tuberculosis treatment.

[0024] The core components and functional advantages of this system are as follows: First, using TDN as a carrier, leveraging its high loading capacity, programmability, and excellent biocompatibility, it not only provides efficient drug delivery space but also effectively protects drug stability and precisely controls release, exhibiting stability far exceeding that of traditional carriers such as liposomes and polymers. Second, the AIE photothermal molecule, through its unique molecular design, restricts intramolecular rotation during aggregation, directing excited-state energy towards fluorescence emission or heat generation. This overcomes the challenges of fluorescence quenching during aggregation in traditional photosensitizers, achieving synergy between imaging and treatment. Furthermore, it generates localized high heat under near-infrared light excitation without significant inactivation, enabling spatiotemporally controllable targeted sterilization and avoiding damage to normal tissues. Thirdly, the encapsulation of Mycobacterium marineum membrane endows the system with natural characteristics. On the one hand, it achieves dual spatiotemporal targeting through membrane surface specific antigens, mimicking the natural infection pathway of pathogens and simultaneously enriching in tuberculous granulomas (lesions) and draining lymph nodes (immune regulation centers), breaking through physical barriers and immunosuppressive microenvironments, and solving the pain point of insufficient drug penetration in existing therapies. On the other hand, the bacterial membrane protein can activate the TLR pathway, downregulate inhibitory cells such as Treg and MDSC, reverse the immunosuppressive state in granulomas, promote the transformation of granulomas from a "closed state" to an "open state", and at the same time target lymph nodes to enhance systemic immune activation and form long-term immune memory.

[0025] This system ultimately achieves dual spatial targeting, temporal visualization and control, and triple functional synergy, constructing a positive feedback loop of "drug delivery-immune activation." Its photothermal-immune synergistic effect can reduce antibiotic dosage and lower the risk of drug resistance. Immune microenvironment remodeling can shorten the treatment course, exhibiting a particularly strong clearance ability against persistent and drug-resistant bacteria, significantly improving treatment efficacy and reducing tuberculosis recurrence rates. It is expected to promote the transformation of tuberculosis from "chronic control" to "radical treatment and prevention of recurrence." This invention is the first to apply bacterial outer membrane encapsulation technology to tuberculosis treatment drug carriers, combining the carrier advantages of TDN, opening a new path for collaborative tuberculosis treatment. Attached Figure Description

[0026] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, do not constitute an undue limitation of the invention. In the drawings:

[0027] Figure 1 The figure shows the characterization results of TDN.

[0028] Figures 2-3 The figure shows the characterization results of TDN+AIE@MM.

[0029] Figure 4 The image shows the verification results of the TDN+AIE@MM targeting effect.

[0030] Figures 5-6 This is a diagram showing the in vitro sterilization effect of TDN+AIE@MM.

[0031] Figure 7 Image showing the CFU results of lung tissue after TDN+AIE@MM treatment.

[0032] Figure 8 HE-stained section of lung tissue after TDN+AIE@MM treatment.

[0033] Figure 9 TNF-α slices and statistical graphs of lung tissue after TDN+AIE@MM treatment.

[0034] Figure 10 This is a graph showing the changes in inflammatory factors after TDN+AIE@MM treatment. Detailed Implementation

[0035] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0036] 1. Preparation of a bacterial film-loaded drug delivery system

[0037] The preparation process of the bacterial film drug delivery system includes:

[0038] (1) Four types of single-stranded DNA (ssDNA) were synthesized, and their base sequences are shown in Table 1:

[0039] Table 1

[0040]

[0041] (2) Prepare a self-assembly reaction system containing 4 types of ssDNA. Dissolve the 4 types of ssDNA in TM buffer. The concentration of each ssDNA solution is 50 µM. Then take 2 µl of each solution into an eight-tube containing 92 µl of TM buffer.

[0042] (3) After denaturation at 95 °C for 10 min and slow annealing to room temperature for 20 min, a tetrahedral DNA nanostructure (TDN) was obtained from the self-assembly reaction system and stored at 4 °C. The successful synthesis of TDN was confirmed by agarose gel electrophoresis and transmission electron microscopy, as shown in the results below. Figure 1 .

[0043] (4) AIE photothermal molecules and TDN were incubated together at 4 °C for 24 h to make the final concentrations of the mixed system as follows: AIE photothermal molecules 200 µg / mL, TDN 100 nM. AIE photothermal molecules were loaded onto TDN through intermolecular forces to obtain the intermediate product TDN+AIE.

[0044] (5) Culture *Mycobacterium marineum* in liquid culture medium until the logarithmic growth phase, then centrifuge to collect approximately [amount missing]. One bacterial cell was treated with lysozyme (60 μg / mL) at room temperature for 30 minutes, followed by the addition of sodium carbonate to adjust the pH to 11–13 to terminate the enzyme reaction. The bacterial suspension was ultrasonically disrupted, centrifuged at 4 °C to remove the precipitate, and the supernatant was subjected to sucrose density gradient ultracentrifugation (100,000 g, 2 h) to obtain Mycobacterium marineis membranes containing key membrane proteins such as the Ag85 complex.

[0045] (6) The Mycobacterium marineis membrane (2 mg / ml protein concentration) and TDN+AIE were mixed at a volume ratio of 1:1 and co-extruded to coat TDN+AIE with the Mycobacterium marineis membrane, thus obtaining the membrane-loaded drug system TDN+AIE@MM. The successful preparation of the membrane-loaded drug system TDN+AIE@MM was verified by agarose gel electrophoresis, potential, transmission electron microscopy, particle size analysis, etc. Figure 2 and Figure 3 As shown.

[0046] 2. Mechanism of action of the TDN+AIE@MM biofilm drug delivery system as a treatment for pulmonary tuberculosis

[0047] The specific process is as follows:

[0048] (1) Targeted delivery stage: The specific antigens on the surface of the bacterial film mediate the active uptake by macrophages, which then target and accumulate in tuberculous granulomas and lymph nodes through the lymphatic circulation. AIE fluorescence tracing enables real-time monitoring of the delivery process, which improves the targeting efficiency compared with rifampicin alone.

[0049] (2) Therapeutic stage: In the lesion, near-infrared light excites AIE photothermal molecules to generate heat, and the local temperature can reach 60 ℃. The bacterial membrane protein activates the TLR pathway to reverse immunosuppression, integrating photothermal / immune dual therapy.

[0050] 3. Treatment efficacy

[0051] Experiments using flow cytometry and confocal microscopy demonstrated the in vitro targeting effect of TDN+AIE@MM on granulomas and lymph nodes. Figure 4 As shown, transmission electron microscopy (TEM) images clearly show the directional aggregation of TDN+AIE@MM around H37Ra. Subsequent colony-forming unit (CFU) measurements yielded the following results: Figure 5 As shown, without 808 nm laser irradiation, TDN+AIE and TDN+AIE@MM had no killing effect on H37Ra. The combination of TDN+AIE@MM and 808 nm laser irradiation (1.5 W / cm², 5 min) resulted in a bacterial mortality rate exceeding 99%. Regardless of the presence or absence of 808 nm laser irradiation, the Rif group showed a certain killing effect on the bacteria. Subsequently, the effects of different treatments on the structure of H37Ra were observed using scanning electron microscopy (SEM), such as... Figure 6 As shown, after being treated with an 808 nm laser, the complete morphology of H37Ra was significantly damaged, and obvious wrinkling and collapse appeared on the surface. This morphological change directly confirms the highly efficient bactericidal effect of TDN+AIE@MM.

[0052] m-cherry BCG was injected via the tail vein. A mouse model of Mycobacterium tuberculosis infection was constructed using a concentration of [number] cells / mL (200 μL). Modeling was completed after 21 days. Mice were then divided into different treatment groups as follows: control, Rif, TDN+AIE, and TDN+AIE@MM. Each group included both 808 nm light irradiation and non-light irradiation treatments. The AIE photothermal molecule-mediated photothermal effect can directly and physically kill H37Ra through localized hyperthermia, achieving rapid clearance of the pathogen. CFU results are shown below. Figure 7As shown, the number of H37Ra bacteria in the granulomatous lesions of the treatment group showed a significant decreasing trend. This result directly confirms that TDN+AIE@MM combined with light treatment can exhibit a strong clearance ability against M.tb in the in vivo environment. Simultaneously, HE staining of lung tissue in each mouse disease model group yielded the following results: Figure 8 As shown, the alveolar structural integrity of mouse lung tissue treated with TDN+AIE@MM combined with light irradiation was the best among all groups, specifically manifested as a significant reduction in alveolar wall thickness, a marked decrease in the area of ​​fibrotic lesions, and a substantial reduction in the number of hemorrhagic lesions. Tissue samples from the lesions were subjected to TNF-α immunohistochemistry, and the results are as follows... Figure 9 As shown, after TDN+AIE@MM combined with phototherapy, the positive expression of TNF-α in lung tissue decreased sharply, and its expression level was similar to that of normal mouse lung tissue. Furthermore, the alveolar structure was clear, inflammatory infiltration significantly subsided, and the overall morphological appearance was indistinguishable from normal tissue, suggesting that lung inflammation had been largely controlled and well-repaired. Simultaneously, the expression levels of inflammatory factors in the samples were detected, and the results were as follows... Figure 10 As shown, compared with other groups, the TDN+AIE@MM group had the lowest expression levels of IL-6, IL-1β, TNF-α, and IFN-γ after light treatment.

[0053] This invention has significant advantages over existing technologies:

[0054] In terms of therapeutic efficacy, the TDN+AIE drug delivery system, through the Mycobacterium marineis membrane encapsulation technology, possesses both the ability to evade immune clearance and the ability to actively target the target.

[0055] In terms of treatment mechanism, the innovative AIE photothermal agent overcomes the problem of aggregation and quenching of traditional photosensitizers in high-viscosity lesions, greatly improving photothermal conversion efficiency and achieving synergistic bactericidal effect when combined with chemotherapy; the bacterial film protein can specifically activate the TLR pathway and effectively reverse the immunosuppressive microenvironment.

[0056] In terms of fabrication technology, the TDN self-assembly process simplifies the carrier preparation process. In clinical applications, the fluorescence properties of AIE enable real-time monitoring of the treatment process, avoiding the blindness of traditional therapies; the synergistic mechanism shortens the treatment cycle and significantly reduces treatment costs.

[0057] In terms of environmental friendliness, the system is biodegradable and does not have the metal residue problem of traditional photothermal agents.

[0058] In summary, this invention is significantly superior to existing technologies in terms of therapeutic effect, preparation process, and clinical application, providing a more efficient, precise, and safe solution for the treatment of tuberculosis.

[0059] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A bacterial film-based drug delivery system, characterized in that, The bacterial membrane drug delivery system is a structure in which the outer membrane encapsulates the core, and the outer membrane is the bacterial outer membrane; the core is composed of tetrahedral DNA nanostructures loaded with near-infrared II AIE photothermal molecules.

2. The bacterial film drug delivery system according to claim 1, characterized in that, The tetrahedral DNA nanostructure is self-assembled from four single-stranded DNA molecules, the sequences of which are shown in SEQ ID NO. 1~4.

3. The bacterial film drug delivery system according to claim 1, characterized in that, The outer membrane of the bacteria is a Mycobacterium marineum membrane.

4. The bacterial film drug delivery system according to claim 3, characterized in that, The bacterial outer membrane is a Mycobacterium marineis membrane containing key membrane proteins.

5. The bacterial film drug delivery system according to claim 3, characterized in that, The bacterial outer membrane is a Mycobacterium marineis membrane containing the Ag85 complex.

6. A method for preparing a bacterial film-loaded drug delivery system as described in any one of claims 1-5, characterized in that the process... include: (1) Four single-stranded DNAs were synthesized, and the sequences of the four single-stranded DNAs are shown in SEQ ID NO.1~4; (2) Four types of single-stranded DNA were self-assembled into tetrahedral DNA nanostructures; (3) AIE photothermal molecules were loaded onto tetrahedral DNA nanostructures to obtain the intermediate product TDN+AIE; (4) Mix the Mycobacterium marineis film and TDN+AIE and co-extrude them to encapsulate TDN+AIE with the Mycobacterium marineis film, and obtain the film-loaded drug system TDN+AIE@MM.

7. The method for preparing a bacterial film-loaded drug delivery system according to claim 6, characterized in that, The detailed process of step (2) is as follows: four single-stranded DNAs are dissolved in TM buffer, each with a concentration of 50 µM. Then, 2 µl of each solution is taken into an eight-tube containing 92 µl of TM buffer to obtain a self-assembly reaction system. The self-assembly reaction system is denatured at 95 °C for 10 min and then slowly annealed to room temperature for 20 min to obtain a tetrahedral DNA nanostructure, which is stored at 4 °C.

8. The method for preparing a bacterial film-loaded drug delivery system according to claim 6, characterized in that, The detailed process of step (3) is as follows: 200 µg / ml AIE photothermal molecules and 100 nM tetrahedral DNA nanostructures are mixed at 4 °C for 24 h. The AIE photothermal molecules are loaded onto the tetrahedral DNA nanostructures through intermolecular forces to obtain the intermediate product TDN+AIE.

9. The method for preparing a bacterial film-loaded drug delivery system according to claim 5, characterized in that, In step (4), the Mycobacterium marineis membrane with a protein concentration of 2 mg / ml and TDN+AIE are mixed at a volume ratio of 1:1 and co-extruded to coat TDN+AIE with the Mycobacterium marineis membrane, thus obtaining the membrane-loaded drug system TDN+AIE@MM.

10. The use of the bacterial film drug delivery system according to any one of claims 1-5 in the preparation of a medicament for treating bacterial infections.

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