Dextran-coated manganese-doped molybdenum sulfide nanoflowers / cGAMP nanocomposites, their preparation methods and applications

Abstract

This application relates to the fields of biomedicine and medical devices, disclosing a dextran-coated manganese-doped molybdenum sulfide nanoflower / cGAMP nanocomposite, its preparation method, and its applications. The nanocomposite has a core-shell structure, comprising: a Mn-MoS2 nanoflower core; a cGAMP loading layer loaded on the surface of the Mn-MoS2 nanoflower core; and a dextran coating coated on the surface of the cGAMP loading layer. This application uses Mn–MoS2 nanoflowers with piezoelectric / acoustic response characteristics as the core, and under external ultrasonic stimulation, generates piezoelectric polarization and charge separation, inducing or amplifying local ROS generation. The EPS matrix of biofilms and bacterial membranes / walls are highly sensitive to oxidative stress; ROS can oxidatively cleave or structurally damage polysaccharide chains, proteins, and lipids.

Description

Technical Field

[0001] This application relates to the fields of biomedicine and medical devices, and more specifically, it relates to a dextran-coated manganese-doped molybdenum sulfide nanoflower / cGAMP nanocomposite, its preparation method, and its application. Background Technology

[0002] Current research on the treatment of biofilm infections mainly focuses on the following directions, but many shortcomings remain. The STING pathway is an important intracellular DNA sensing axis of the body's innate immunity. Cyclic dinucleotides (such as 2′3′-cGAMP) produced by cGAS can bind to STING, induce the TBK1 / IRF3 pathway, and promote the expression of type I interferon and inflammatory factors, thereby enhancing anti-pathogen immunity. However, cGAMP and similar cyclic dinucleotide molecules are hydrophilic and easily degraded, which is not conducive to their effective retention and intracellular delivery in the infection microenvironment. Current research has developed various nanocarriers and hydrogel systems to improve their pharmacokinetics and local delivery, but challenges remain regarding adhesion and biofilm penetration.

[0003] Ultrasound can trigger the generation of reactive oxygen species (ROS) in certain inorganic or composite nanomaterials, which can be used for antibacterial or anti-biofilm purposes. Sun et al. reported that manganese oxide / titanium dioxide composites generate active species under ultrasound, which disrupt bacterial structures and assist in anti-biofilm activity, but their immunomodulatory dimension is limited, and they mainly focus on direct bactericidal action. At the same time, the ultrasound triggering strategy is limited by material stability, ROS yield, and the safety window for host cells.

[0004] Applications of transition metal dichalcogenides (TMDs, such as MoS2) in antibacterial applications. MoS2, due to its layered structure, broad-spectrum absorption, and doping modifiability, has been used for multimodal photo / acoustic / thermal antibacterial applications. Metal doping can tunable the band gap, defect states, and electrocatalytic activity, thereby amplifying the ability to generate reactive oxygen species (ROS). Many studies have employed MoS2 nanosheets combined with near-infrared light or ultrasound for antibacterial and biofilm disruption. However, these studies largely rely on direct photothermal / photodynamic or acoustic-dynamic bactericidal effects, which are insufficient to address long-term activation of the host immune system and long-term retention at lesion sites.

[0005] Microbial biofilms are composed of an extracellular polymeric matrix (EPS) of polysaccharides, proteins, and nucleic acids, which enhances drug tolerance and immune evasion. Therefore, the "anchoring" of drugs / nanocarriers is crucial for prolonging local action and overcoming biofilm barriers. Polysaccharides with multi-hydroxyl structures (such as dextran, chitosan, and hyaluronic acid) have been used to improve adhesion to biofilms or infected tissues. For example, chitosan's cationic properties interact with the negative charge of bacterial cell walls to enhance retention, but its adhesion decreases at neutral pH and may induce non-specific adhesion. Dextran has affinity for various Gram-positive bacterial surface receptors (such as dextran-binding proteins) and biofilm polysaccharides and has been used in vaccines or delivery systems targeting Staphylococcus / Streptococcus, but reports on its use for immobilizing STING agonists and achieving long-term release are still limited.

[0006] Manganese ions can enhance cGAS activity and have been shown to amplify the downstream STING signaling pathway, thereby improving innate immune responses. Nanoscale manganese-based oxides / salts have been used in tumor immunology in synergy with STING agonists, but a mature solution for a systematic approach combining ultrasound triggering and biofilm anchoring in bacterial biofilm infection scenarios remains elusive.

[0007] In summary, the existing technology has the following shortcomings:

[0008] 1) STING agonists have insufficient effective retention and short-lasting release at the site of infection: Cyclic dinucleotides are hydrophilic and negatively charged, making them easily diffused and diluted in tissue fluid; they lack specific anchoring to biomembranes, making it difficult to achieve sufficient cellular uptake concentrations within the lesion microenvironment. Most nanodelivery systems focus on the EPR effect in tumor tissue, and their adaptability to EPS is insufficient when transplanted into a biomembrane environment.

[0009] 2) Single direct sterilization mode lacks host immune reprogramming: Ultrasound / photothermal / photodynamic therapy and other methods rely on ROS or thermal effects to directly kill bacteria, which is effective in the short term, but the bacteria that survive and remain in the biofilm can cause recurrence; lacking the continuous activation of the host's innate immunity and phagocytosis-clearance axis, it is difficult to achieve long-term antibacterial and recurrence control.

[0010] 3) The dual challenges of biofilm microenvironment penetration and anchoring: EPS is dense and rich in anionic matrix, which hinders the diffusion of nanoparticles; commonly used polysaccharides (such as chitosan) have reduced charge and decreased adhesion under neutral physiological conditions; although polysaccharides such as dextran have good compatibility with some Gram-positive bacteria, a systematic solution has not been formed for the design of simultaneously carrying STING agonists and achieving controlled release.

[0011] 4) Incomplete synergy between the STING axis and inorganic nanomaterials: Although manganese ions can enhance cGAS–STING sensitivity, there is a lack of integration in achieving a closed loop of "local retention → ultrasonically triggered ROS / piezoelectric effect → sustained release of cGAMP → sustained activation of STING → immune clearance" on a single material platform. Existing reports mostly disperse these functions in different materials or different time steps, resulting in insufficient synergy and spatiotemporal control.

[0012] 5) Safety and controllability issues: ROS has a narrow dose window, and excessive doses may damage host tissues; overactivation or systemic exposure to STING may trigger inflammatory side effects; the long-term biosafety of material biodegradation and metal ion leakage needs to be weighed. Existing systems often lack long-term safety and efficacy data in biofilm disease models. Summary of the Invention

[0013] The first objective of this application is to solve the technical problem of how to achieve stable loading and long-term local retention of 2′3′-cGAMP in the bacterial biofilm microenvironment, avoid its rapid diffusion and degradation in body fluids, and improve its efficiency in entering the host cell cytoplasm to activate the STING pathway. The second objective of this application is to solve the technical problem of how to integrate and coordinate the multifunctionality of "biofilm anchoring - controlled release - immune activation - physical sterilization" on the same material platform, so that the spatiotemporal release of cGAMP and the host STING activation and the direct antibacterial effect (ROS / piezoelectric effect) triggered by ultrasound are synergistic rather than mutually interfering. The third objective of this application is to solve the technical problem of how to achieve high affinity anchoring and effective penetration of materials to biomembranes in complex, dense, negatively charged extracellular polymeric (EPS) matrices, thereby improving the actual effective dose and action time window within the infection focus. The fourth objective of this application is to solve the technical problem of how to precisely control the ultrasound parameters and the structure-property relationship of the material while maintaining effective antibacterial activity, so that the release of ROS / metal ions and STING activation are within a safe therapeutic window, thereby reducing the risk of non-targeted damage and excessive inflammatory response to host tissues. The fifth objective of this application is to address the technical challenges of constructing scalable and reproducible preparation and surface coating processes (including dextran coating and cGAMP loading) for the doping, morphology, and piezoelectric / acoustic response properties of manganese-molybdenum sulfide nanoflowers, in order to ensure the structural stability, controlled release behavior, and batch-to-batch consistency of the material under physiological conditions. The sixth objective of this application is to address the technical problem of how to utilize the sensitization effect of manganese ions on the cGAS–STING axis to achieve localized and controllable Mn supply within a nanoplatform, thereby enhancing STING signal transduction while avoiding the toxicity and immune imbalance caused by systemic metal ion leakage.

[0014] To solve the above-mentioned technical problems, this application adopts the following technical solution: In a first aspect, this application provides a nanocomposite having a core-shell structure, comprising: Mn-MoS2 nanoflower cores; cGAMP loading layer, loaded on the surface of the Mn-MoS2 nanoflower core; and A dextran coating is applied to the surface of the cGAMP-loaded layer.

[0015] Furthermore, the average particle size of the Mn-MoS2 nanoflower core is 80~300 nm, preferably 120~220 nm.

[0016] Furthermore, in the Mn-MoS2 nanoflower core, the doping amount of manganese accounts for 0.5~12% of the total molar fraction of the metal.

[0017] Furthermore, the mass ratio of the Mn-MoS2 nanoflower core to cGAMP is 1:0.1~0.3.

[0018] Furthermore, the mass ratio of the dextran coating to the Mn-MoS2 nanoflower core loaded with cGAMP is 0.5~2:1.

[0019] Secondly, this application provides a method for preparing the aforementioned nanocomposite, comprising the following steps: Step 1, Preparation of Mn-MoS2 nanoflower cores Molybdenum, sulfur, and manganese sources were dissolved in a solvent, and Mn-MoS2 nanoflower cores were obtained through a solvothermal reaction. Step 2, Preparation of cGAMP Loading Layer The Mn-MoS2 nanoflower kernels obtained in step 1 were dispersed in buffer solution, and cGAMP solution was added for incubation to load cGAMP onto the Mn-MoS2 nanoflower kernels. After purification, the intermediate was obtained. Step 3, Preparation of dextran coating The intermediate obtained in step 2 was dispersed in a dextran solution and incubated to allow the dextran to coat the surface of the intermediate. After purification, the nanocomposite was obtained.

[0020] Furthermore, the conditions for the solvothermal reaction in step 1 are: temperature 180~220°C, time 6~18h.

[0021] Furthermore, the incubation conditions described in step 2 are: temperature 4~25°C, time 4~6 hours.

[0022] Furthermore, the incubation conditions described in step 2 are: temperature 4~25°C, time 1~12h.

[0023] Thirdly, this application provides the use of the aforementioned nanocomposite in the preparation of medicaments or medical devices for treating or preventing biofilm infections.

[0024] In summary, compared with existing technologies that rely solely on antibiotics / debridement, or solely on direct sterilization using sonodynamic / piezoelectric materials, or solely on the free delivery of STING agonists, the biomaterial and treatment method of "manganese-molybdenum sulfide piezoelectric nanoflowers (Mn–MoS2) loaded with cGAMP and coated with a dextran coating" proposed in this application can form a more complete therapeutic loop under the multiple constraints of "physical barriers of biofilms + poor local drug retention + insufficient immune activation leading to recurrence". Its superior effect is directly brought about by the following technical features and has a clear technical inevitability.

[0025] 1) It has a stronger ability to remain in the biofilm for a long time and maintain an effective local concentration. This application involves coating Mn–MoS2@cGAMP with a dextran coating, resulting in a surface surface containing polysaccharide segments that can interact with the biofilm matrix (EPS) at multiple points. Since the main barrier of bacterial biofilms is a network matrix composed of polysaccharides / proteins / nucleic acids, dextran naturally exhibits a structural basis for adhesion, entanglement, and multi-point hydrogen bonding / weak interactions with this type of network.

[0026] The inherent technical effect directly resulting from this structural feature is that, compared with uncoated or passively permeable nanomaterials, the material of this application is more likely to anchor and accumulate on the surface and inside of biofilms and prolong its residence time; cGAMP no longer diffuses rapidly in a free state or is carried away by local irrigation / tissue fluid flow, but is carried by the material to maintain a higher apparent local concentration and duration of action in the infection site; thereby reducing the risk of insufficient effective dose and relapse during the treatment interval caused by "material not being able to enter / retain".

[0027] 2) More complete disruption of biofilm barriers This application utilizes Mn–MoS2 nanoflowers with piezoelectric / acoustic response properties as the core, and generates piezoelectric polarization and charge separation under external ultrasonic stimulation, inducing or amplifying local ROS generation. The EPS matrix of biofilms and bacterial membranes / walls are highly sensitive to oxidative stress, and ROS can oxidatively cleave or structurally damage polysaccharide chains, proteins, and lipids.

[0028] The inherent technical effects directly brought about by this technology chain ("piezoelectric core + ultrasonic triggering + ROS") are as follows: Compared with carriers that do not have the ability to generate ROS through acoustic response, the material can generate higher oxidative disturbances locally during ultrasound, resulting in a decrease in the viscoelastic structure of EPS, an increase in porosity, and a greater ease of loosening / disintegration of the biofilm; Compared with schemes that rely solely on immune activation without a membrane-breaking step, this application first reduces the strength of the biofilm barrier, making it easier for subsequent APCs / phagocytes to approach bacteria and improving the feasibility of phagocytosis and clearance; Compared with only chemical sterilization or only antibiotic penetration, the ROS triggered by physical action in this application is more spatially concentrated in the material enrichment area, reducing the structural bottleneck of "if it can't get in, it can't be killed".

[0029] 3) STING immune activation is more effective and occurs more "in the right place" (addressing the problem of "poor transmembrane penetration of free cGAMP, which is easily diluted / cleared"). This application loads cGAMP onto Mn–MoS2 nanoflowers and anchors it with dextran to allow it to remain permanently in biofilm infection sites, forming a "locally continuous supply" delivery mode. As an intracellular STING agonist, the immune effect of cGAMP depends on its uptake by APCs and its intracellular action; free cGAMP is easily diffused in the body fluid environment, has insufficient local retention, and has limited transmembrane efficiency, often leading to insufficient immune initiation in infection sites.

[0030] The inherent technological benefits directly resulting from the structural features of "load delivery + biomembrane anchoring + sustained release" are: cGAMP increases the accessibility and uptake probability of APCs around the infection site, making STING pathway activation more likely to occur near the infection microenvironment, rather than being passively dispersed to non-target areas and causing dose dilution. At the same dosage, it is easier to generate sufficient local intracellular stimulation, thereby enhancing downstream STING signals (such as type I interferon and chemokine-related output) and promoting APC maturation and initiation of antibacterial immune pathways. This "targeted activation" mechanism has a more defined basis for local immune enhancement compared to simple systemic administration or free cGAMP.

[0031] 4) Achieve a closed loop of "membrane rupture and sterilization - antigen exposure - immune clearance" to reduce residue and recurrence. This application does not merely pursue one-time sterilization, but rather couples two key events in space and time using the same material platform: Ultrasound-triggered ROS causes bacterial damage / lysis and biofilm disruption, leading to increased antigen exposure; simultaneously, locally sustained release of cGAMP activates STING, enhancing antigen presentation and effector immunity.

[0032] The inevitable technical effect directly resulting from this coupling is: Compared with ROS materials produced solely by acoustic / piezoelectric processes, this application further provides a clear innate immune activation signal after membrane rupture, making it more likely that residual bacteria will be continuously cleared by the immune system. Compared with approaches that only activate the immune system but lack a means of permeation, this application removes the biomembrane barrier before initiating the immune chain, reducing the failure mode of immune cells being "visible but unable to enter / engulp". Therefore, it is more conducive to reducing the residual, protracted and recurrent biofilm-related infections in terms of mechanism.

[0033] 5) Improved material utilization and trigger controllability (solving the problems of "uncontrollable exposure to non-target areas and stimulation") This application adopts a combination of "local drug delivery + biofilm anchoring + ultrasonic triggering": the material is first enriched in the biofilm by dextran, and then ROS generation and synergistic sterilization / membrane disruption are triggered by external ultrasound in a selected area and time window.

[0034] The direct and inevitable technical effects brought about by this solution are: Compared to strategies involving continuous, uncontrolled release or whole-body exposure, ROS is primarily generated during the ultrasound application period and in areas of material enrichment, making treatment intensity more easily adjustable via ultrasound parameters (frequency, intensity, duty cycle, duration). Achieving a stimulation rhythm that "increases when needed and decreases when not needed" without altering the drug itself is beneficial for achieving a more controllable balance between therapeutic efficacy and tissue tolerance.

[0035] The above-mentioned beneficial effects are directly brought about by the specific technical features of this application (Mn–MoS2 piezoelectric nanoflowers, ultrasonically triggered ROS production, cGAMP loading delivery, dextran-coated biofilm anchoring and continuous release, local drug delivery and time-sequential triggering), which can specifically solve the core technical problems of "difficult to enter, difficult to stay, difficult to be thorough, and easy to relapse" in the treatment of biofilm infections, and form a closed-loop treatment chain. Attached Figure Description

[0036] Figure 1 In Example 1, the morphology of Mn-MoS2 nanoflowers was characterized by SEM, with a diameter of approximately 180 nm.

[0037] Figure 2 In Example 1, the particle size distribution of Mn–MoS2@cGAMP was between 180-200 nm, and the dispersion stability was good.

[0038] Figure 3 Comparison of DLS particle size distribution and Zeta potential changes after Dex coating in Example 1.

[0039] Figure 4In Example 2, the morphology of Mn–MoS2 nanoflowers was characterized by SEM, with a diameter of approximately 120 nm.

[0040] Figure 5 In Example 2, the particle size distribution of Mn–MoS2@cGAMP was between 180-200 nm, and the dispersion stability was good.

[0041] Figure 6 Comparison of in vitro reactive oxygen species (ROS) generation and biomembrane permeability improvement under corresponding ultrasound conditions in different embodiments.

[0042] Figure 7 In Example 3, the morphology of Mn–MoS2 nanoflowers was characterized by SEM, with a diameter of approximately 220-260 nm.

[0043] Figure 8 In Example 3, the particle size distribution of Mn–MoS2@cGAMP was between 220-250 μm, and the dispersion stability was good.

[0044] Figure 9 Comparison of DLS particle size distribution and zeta potential changes after Dex coating. Detailed Implementation

[0045] The technical solutions and effects of this application will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely for explaining the invention and are not intended to limit the invention.

[0046] The specific embodiments section of this application discloses a method for preparing the dextran-coated manganese-doped molybdenum sulfide nanoflower / cGAMP nanocomposite (Mn–MoS2@cGAMP@Dex), including the following steps: Step 1: Preparation of Mn–MoS2 nanoflowers Equipment: PTFE-lined reactor, high-speed centrifuge (≥15000 g), ultrasonic disperser, vacuum drying oven / freeze dryer, pH meter.

[0047] Raw material: molybdenum source (such as (NH4)6Mo7O) 24 • 4H2O or Na2MoO4), sulfur source (such as thiourea / thioacetamide TAA), manganese source (such as MnCl2 / Mn(CH3COO)2), deionized water or water / ethylene glycol mixed solvent.

[0048] step: 1. Preparation of precursor solution: Dissolve molybdenum source and sulfur source in solvent, add manganese source according to target doping ratio, and stir magnetically for 10~30 min until homogeneous.

[0049] 2. Solvothermal reaction: The mixture is transferred to a reaction vessel and reacted at 180~220°C for 6~18 h to form Mn–MoS2 nanoflowers.

[0050] 3. After cooling, centrifuge and wash: 8000~15000 g, 10~20 min, wash with water and ethanol alternately 3~5 times to remove free ions and small molecules.

[0051] 4. Dispersion and preservation: Ultrasonic dispersion (ice bath, intermittent mode, total duration 1~10 min) to obtain Mn-MoS2 dispersion; or freeze-dry into powder for later use.

[0052] Key control points: The manganese doping level affects the piezoelectric / catalytic ROS production and biocompatibility, and batch-to-batch consistency should be controlled by ICP-OES / ICP-MS; the morphology of nanoflowers is characterized by TEM / SEM.

[0053] Step 2: cGAMP loading to form Mn–MoS2@cGAMP Equipment: constant temperature shaker, centrifuge, ultrafiltration tube (MWCO 10~50 kDa).

[0054] step: 1. Disperse Mn–MoS2 in buffer (PBS, pH 7.0–7.6).

[0055] 2. Add cGAMP solution and control the mass ratio: Mn–MoS2: cGAMP = 1:0.05~0.5 (1:0.1~0.3).

[0056] Gently shake at 3.4~25°C for 4~16 h to allow it to adsorb / embed.

[0057] 4. Remove unloaded cGAMP by centrifugation or ultrafiltration to obtain Mn–MoS2@cGAMP.

[0058] Key control points: quantification of cGAMP loading by HPLC / UV; evaluation of dispersion stability by DLS and Zeta.

[0059] Step 3: Dextran coating to form the final formulation Mn–MoS2@cGAMP@Dex Equipment: Magnetic stirrer, ultrasonic disperser, centrifuge.

[0060] Types of dextran: dextran, β-glucan, or their derivatives; Covering method: 1. Prepare a dextran solution: 0.1~10 mg / mL (preferably 0.5~5 mg / mL).

[0061] 2. Add Mn–MoS2@cGAMP to the dextran solution, controlling the mass ratio: dextran: nanonucleus = 0.2:1~5:1 (0.5:1~2:1).

[0062] 3. Stir / vibrate at room temperature or 4~25°C for 1~12 h to complete the coating.

[0063] 4. Washing and purification: Centrifuge at 8000~15000 g for 10~15 min, wash 2~4 times to obtain the finished dispersion.

[0064] Aseptic and formulation: It can be dispensed by 0.22 µm filtration (if particle size allows) or aseptic operation; it can also be lyophilized, reconstituted with a protectant (such as trehalose) and then used.

[0065] Based on the above scheme, this application discloses the following specific examples: Example 1 A nanocomposite was prepared according to the method described above, and the specific steps are as follows: Step 1: Preparation of Mn–MoS2 nanoflowers 1. Preparation of precursor solution: Dissolve 1 mmol sodium molybdate dihydrate (approximately 242 mg) and 4.5 mmol thiourea (approximately 342.5 mg) in 30 mL of deionized water, then add 0.031 mmol manganese chloride tetrahydrate (approximately 6.1 mg) as manganese source (approximately 3% based on the molar fraction of Mn in metal), and stir magnetically for 10-30 min until the solution is homogeneous.

[0066] 2. Solvothermal reaction: The mixture was transferred to a reaction vessel and reacted at 200°C for 12 hours to form Mn–MoS2 nanoflowers.

[0067] 3. After cooling, centrifuge and wash: 12000g, 15min, wash 3 times with water and ethanol alternately to remove free ions and small molecules.

[0068] 4. Dispersion and preservation: Ultrasonic dispersion (ice bath, intermittent mode, total duration 1~10 min) to obtain Mn-MoS2 dispersion; or freeze-dry into powder for later use.

[0069] Key control points: The morphology of nanoflowers was characterized using TEM / SEM, such as... Figure 1 As shown, the diameter of Mn–MoS2 nanoflowers is approximately 180 nm.

[0070] Step 2: cGAMP loading to form Mn–MoS2@cGAMP 1. Disperse Mn–MoS2 in buffer (PBS, pH 7.0–7.6).

[0071] 2. Add cGAMP solution, controlling the mass ratio: Mn–MoS2:cGAMP = 1:0.2.

[0072] Gently shake at 3.4°C for 12 h to allow it to adsorb / embed.

[0073] 4. Remove unloaded cGAMP by centrifugation or ultrafiltration to obtain Mn–MoS2@cGAMP.

[0074] Key control points: Use DLS to assess dispersion stability, such as... Figure 2 As shown, the particle size distribution of Mn–MoS2@cGAMP is between 180-200 nm, and the dispersion stability is good.

[0075] Step 3: Dextran coating to form the final formulation Mn–MoS2@cGAMP@Dex 1. Prepare a dextran solution: 2 mg / mL.

[0076] 2. Add Mn–MoS2@cGAMP to the dextran solution, controlling the mass ratio of dextran to nanonucleus to be 1:1.

[0077] 3. Stir / vibrate at room temperature or 25°C for 4 hours to complete the coating.

[0078] 4. Washing and purification: Centrifuge at 12000 g for 15 min, wash 3 times to obtain the finished dispersion.

[0079] Aseptic and formulation: It can be dispensed by 0.22 µm filtration (if particle size allows) or aseptic operation; it can also be lyophilized, reconstituted with a protectant (such as trehalose) and then used.

[0080] like Figure 3 As shown, DLS revealed that the particle size increased after coating, and the zeta shifted towards the dextran direction.

[0081] Example 2 A nanocomposite was prepared according to the method described above, and the specific steps are as follows: Step 1: Preparation of Mn–MoS2 nanoflowers 1. Preparation of precursor solution: Dissolve 1 mmol sodium molybdate dihydrate (approximately 242 mg) and 4.5 mmol thiourea (approximately 342.5 mg) in 30 mL of deionized water, then add 0.01 mmol manganese chloride tetrahydrate (approximately 2.0 mg) as manganese source (approximately 1% of the metal molar fraction of Mn), and stir magnetically for 10 to 30 min until the solution is homogeneous.

[0082] 2. Solvothermal reaction: The mixture was transferred to a reaction vessel and reacted at 180°C for 8 h to form Mn–MoS2 nanoflowers.

[0083] 3. After cooling, centrifuge and wash: 12000 g, 15 min, wash 3 times with water and ethanol alternately to remove free ions and small molecules.

[0084] 4. Dispersion and preservation: Ultrasonic dispersion (ice bath, intermittent mode, total duration 1~10 min) to obtain Mn-MoS2 dispersion; or freeze-dry into powder for later use.

[0085] Key control points: The morphology of the nanoflowers was characterized by SEM. The morphology of Mn–MoS2 nanoflowers is as follows: Figure 4 As shown, the diameter is approximately 120 nm.

[0086] Step 2: cGAMP loading to form Mn–MoS2@cGAMP 1. Disperse Mn–MoS2 in buffer (PBS, pH 7.0–7.6).

[0087] 2. Add cGAMP solution, controlling the mass ratio: Mn–MoS2:cGAMP = 1:0.1.

[0088] Gently shake at 3.25°C for 6 hours to allow it to adsorb / embed.

[0089] 4. Remove unloaded cGAMP by centrifugation or ultrafiltration to obtain Mn–MoS2@cGAMP.

[0090] Key control points: Dispersion stability was assessed using DLS, and the results are as follows. Figure 5 As shown, the particle size distribution of Mn–MoS2@cGAMP is between 180-200 nm, and the dispersion stability is good.

[0091] Step 3: Dextran coating to form the final formulation Mn–MoS2@cGAMP@Dex 1. Prepare a dextran solution: 2 mg / mL.

[0092] 2. Add Mn–MoS2@cGAMP to the dextran solution, controlling the mass ratio of dextran: nanonucleus = 0.5:1.

[0093] 3. Stir / vibrate at room temperature or 4°C for 8 hours to complete the coating.

[0094] 4. Washing and purification: Centrifuge at 12000 g for 15 min, wash 3 times to obtain the finished dispersion.

[0095] Aseptic and formulation: It can be dispensed by 0.22 µm filtration (if particle size allows) or aseptic operation; it can also be lyophilized, reconstituted with a protectant (such as trehalose) and then used.

[0096] It is more inclined to have low ROS output and a more sustained release, making it suitable for superficial or irritation-sensitive tissues; At lower ultrasound (0.5 W / cm) 2 It can still generate detectable ROS and improve biomembrane permeability even at 3 min, but the intensity is lower than that in Example 1 (see Example 1). Figure 6 ).

[0097] Example 3 A nanocomposite was prepared according to the method described above, and the specific steps are as follows: Step 1: Preparation of Mn–MoS2 nanoflowers 1. Preparation of precursor solution: Dissolve 1 mmol sodium molybdate dihydrate (approximately 242 mg) and 4.5 mmol thiourea (approximately 342.5 mg) in 30 mL of deionized water, then add 0.136 mmol manganese chloride tetrahydrate (approximately 27.0 mg) as manganese source (approximately 12% by metal molar fraction of Mn), and stir magnetically for 10-30 min until the solution is homogeneous.

[0098] 2. Solvothermal reaction: The mixture was transferred to a reaction vessel and reacted at 220°C for 16 h to form Mn–MoS2 nanoflowers.

[0099] 3. After cooling, centrifuge and wash: 12000 g, 15 min, wash 3 times with water and ethanol alternately to remove free ions and small molecules.

[0100] 4. Dispersion and preservation: Ultrasonic dispersion (ice bath, intermittent mode, total duration 1~10 min) to obtain Mn-MoS2 dispersion; or freeze-dry into powder for later use.

[0101] Key control points: The morphology of the nanoflowers was characterized by SEM. The morphology of Mn–MoS2 nanoflowers is as follows: Figure 7 As shown, the diameter is approximately 220-260 nm.

[0102] Step 2: cGAMP loading to form Mn–MoS2@cGAMP 1. Disperse Mn–MoS2 in buffer (PBS, pH 7.0–7.6).

[0103] 2. Add cGAMP solution, controlling the mass ratio: Mn–MoS2:cGAMP = 1:0.3.

[0104] Gently shake at 3.4°C for 16 hours to allow it to adsorb / embed.

[0105] 4. Remove unloaded cGAMP by centrifugation or ultrafiltration to obtain Mn–MoS2@cGAMP.

[0106] Key control points: Dispersion stability was assessed using DLS, and the results are as follows. Figure 8 As shown, the particle size distribution of Mn–MoS2@cGAMP is between 220 and 250 μm, and the dispersion stability is good.

[0107] Step 3: Dextran coating to form the final formulation Mn–MoS2@cGAMP@Dex 1. Prepare a dextran solution: 2 mg / mL.

[0108] 2. Add Mn–MoS2@cGAMP to the dextran solution, controlling the mass ratio of dextran: nanonucleus = 2:1.

[0109] 3. Stir / vibrate at room temperature or 25°C for 2 hours to complete the coating.

[0110] 4. Washing and purification: Centrifuge at 12000 g for 15 min, wash 3 times to obtain the finished dispersion.

[0111] Aseptic and formulation: It can be dispensed by 0.22 µm filtration (if particle size allows) or aseptic operation; it can also be lyophilized, reconstituted with a protectant (such as trehalose) and then used.

[0112] DLS showed that the particle size increased after coating, and the zeta shifted towards the dextran direction. Figure 9 ).

[0113] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A nanocomposite, characterized in that, The nanocomposite has a core-shell structure, comprising: Mn-MoS2 nanoflower cores; cGAMP loading layer, loaded on the surface of the Mn-MoS2 nanoflower core; and A dextran coating is applied to the surface of the cGAMP-loaded layer.

2. The nanocomposite according to claim 1, characterized in that, The average particle size of the Mn-MoS2 nanoflower core is 80~300 nm, preferably 120~220 nm.

3. The nanocomposite according to claim 1, characterized in that, In the Mn-MoS2 nanoflower core, the doping amount of manganese accounts for 0.5~12% of the total molar fraction of the metal.

4. The nanocomposite according to claim 1, characterized in that, The mass ratio of the Mn-MoS2 nanoflower core to cGAMP is 1:0.1~0.

3.

5. The nanocomposite according to claim 1, characterized in that, The mass ratio of the dextran coating to the Mn-MoS2 nanoflower core loaded with cGAMP is 0.5~2:

1.

6. A method for preparing the nanocomposite according to any one of claims 1-5, characterized in that, Includes the following steps: Step 1, Preparation of Mn-MoS2 nanoflower cores Molybdenum, sulfur, and manganese sources were dissolved in a solvent, and Mn-MoS2 nanoflower cores were obtained through a solvothermal reaction. Step 2, Preparation of cGAMP Loading Layer The Mn-MoS2 nanoflower kernels obtained in step 1 were dispersed in buffer solution, and cGAMP solution was added for incubation to load cGAMP onto the Mn-MoS2 nanoflower kernels. After purification, the intermediate was obtained. Step 3, Preparation of dextran coating The intermediate obtained in step 2 was dispersed in a dextran solution and incubated to allow the dextran to coat the surface of the intermediate. After purification, the nanocomposite was obtained.

7. The preparation method according to claim 6, characterized in that, The conditions for the solvothermal reaction in step 1 are: temperature 180~220°C, time 6~18h.

8. The preparation method according to claim 6, characterized in that, The incubation conditions described in step 2 are: temperature 4~25°C, time 4~6 hours.

9. The preparation method according to claim 6, characterized in that, The incubation conditions described in step 2 are: temperature 4~25°C, time 1~12h.

10. The use of the nanocomposite according to any one of claims 1-5 in the preparation of a medicament or medical device for treating or preventing biofilm infections.

Images