Brain-targeting and photo-thermal therapy bionic nano delivery platform, preparation method thereof and application thereof in treatment of neurodegenerative diseases

Abstract

The invention relates to a bionic nano delivery platform for brain targeting and photothermal therapy, a preparation method of the bionic nano delivery platform and application of the bionic nano delivery platform in treatment of neurodegenerative diseases. The platform is composed of a drug-loading core and a functionalized shell, wherein the drug-loading core is a cycloastragenol-loaded liposome; the functionalized shell covers the drug loading core and is composed of a microcolloid cell membrane and a near-infrared two-region photo-thermal material dispersed in the microcolloid cell membrane. According to the structure, through cell membrane bionic modification, a platform is endowed with good brain focus targeting, efficient immune escape ability and a remarkable drug enrichment effect, and collaborative delivery and controlled release of cycloastragenol and a photo-thermal material can be achieved. The platform and the system have the comprehensive advantages of being accurate in brain targeting, remarkable in treatment synergistic effect and good in biocompatibility, and have wide clinical application prospects in the field of targeted therapy of neurodegenerative diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanotechnology, specifically relating to a biomimetic nanoplatform for brain-targeted delivery and photothermal synergistic therapy, its preparation method, and the application of the platform or a system containing the platform in the preparation of drugs for treating neurodegenerative diseases. Background Technology

[0002] First, in the field of nanodelivery carriers, existing technologies generally suffer from limited functionality. Conventionally used liposomes or polymer micelles typically possess only basic drug encapsulation capabilities or limited targeting functions. When multifunctional integration of drug delivery, specific cell targeting, and external energy response (e.g., photothermal therapy) is required, multiple different carriers must often be used in combination. This approach leads to poor synergy between carriers, difficulty in controlling in vivo distribution, and complex preparation and administration processes, thus failing to achieve effective treatment with consistent temporal and spatial consistency. Furthermore, targeting modification strategies based on single receptor-ligand interactions lack sufficient precision, making it difficult to effectively distinguish activated target cells (such as microglia) from normal cells in complex organisms, easily triggering off-target effects and systemic toxicity. Simultaneously, such carriers are readily recognized and cleared by the body's mononuclear phagocytic system, preventing the achievement and maintenance of effective drug concentrations at the lesion site.

[0003] Secondly, in the field of microglia regulation, existing methods mostly rely on small molecule inhibitors or gene silencing techniques for unidirectional functional inhibition, resulting in overly simplistic regulatory strategies. Microglia exhibit dynamic phenotype changes under pathological conditions, making it difficult for current technologies to achieve precise and timely bidirectional regulation of their pro-inflammatory and anti-inflammatory / repair phenotypes. Furthermore, these regulatory molecules lack efficient delivery systems capable of crossing the blood-brain barrier and specifically delivering to target cells; their non-specific distribution may further disrupt the immune microenvironment homeostasis of the central nervous system. Emerging photothermal-based physical regulation technologies are limited by the binding method of photothermal materials and carriers; commonly used physical adsorption loading methods are prone to uneven material distribution and leakage during in vivo circulation, leading to unstable photothermal conversion efficiency and posing a potential risk of non-specific damage to normal brain tissue.

[0004] Furthermore, in the field of pathogenic protein clearance strategies, existing technologies such as specific antibodies or molecular chaperones mostly focus on directly clearing pathogenic proteins such as α-synuclein, while neglecting their intrinsic connection with the neuroinflammatory process. Therefore, they are unable to effectively block the core pathological cycle of "abnormal protein aggregation exacerbating the inflammatory response, which in turn promotes protein aggregation." In addition, these clearance strategies rely on the phagocytic function of microglia, but do not simultaneously provide auxiliary means to enhance the lysosomal degradation activity within microglia, resulting in low efficiency in clearing protein aggregates and an inability to effectively reverse existing substantial pathological damage.

[0005] In summary, existing technologies lack an integrated solution that can simultaneously meet the following key requirements: (1) efficient crossing of the blood-brain barrier and precise targeting of key cells in the lesion area; (2) integrated and synergistic delivery of therapeutic drugs and external energy response functions; and (3) simultaneous intervention in multiple interrelated pathological processes such as neuroinflammation and clearance of pathogenic proteins. Therefore, there is an urgent need in this field to develop a novel, multifunctional integrated targeted delivery platform. Summary of the Invention

[0006] The main objective of this invention is to propose a biomimetic nanodelivery platform for brain-targeted and photothermal therapy, its preparation method, and its application in the treatment of neurodegenerative diseases.

[0007] To achieve the above objectives, the first aspect of this application proposes a biomimetic nanodelivery platform for brain-targeted and photothermal therapy, the biomimetic nanodelivery platform comprising: The drug-carrying core comprises liposomes and cycloastragalool loaded in the liposomes; The functionalized shell encapsulates the drug-loaded core and includes a cell membrane and photothermal conversion materials dispersed in the cell membrane.

[0008] In some implementations, the cell membrane is derived from microglia.

[0009] In some implementations, the microglia are BV2 microglia.

[0010] In some implementations, the photothermal conversion material is a near-infrared II photothermal material.

[0011] In some implementations, the surface of the functionalized shell is coupled with a targeting antibody.

[0012] In some implementations, the targeting antibody is a microglia-targeting antibody.

[0013] In some implementations, the microglia-targeting antibody is an anti-TRPV4 antibody.

[0014] In some embodiments, the loading of cycloastragalool in the liposomes is 10-50 wt%, for example, it can be 10 wt%, 12 wt%, 14 wt%, 15 wt%, 16 wt%, 18 wt%, 20 wt%, 22 wt%, 24 wt%, 25 wt%, 26 wt%, 28 wt%, 30 wt%, 32 wt%, 34 wt%, 35 wt%, 36 wt%, 38 wt%, 40 wt%, 42 wt%, 44 wt%, 45 wt%, 46 wt%, 48 wt%, or 50 wt%.

[0015] In some embodiments, the raw materials for the liposomes include soybean lecithin, cholesterol, and phospholipid-polyethylene glycol.

[0016] In some embodiments, the content of soybean lecithin is 65-75% based on the total mass of the liposome raw materials, for example, it can be 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75%.

[0017] In some embodiments, the cholesterol content is 3% to 4% based on the total mass of the liposome raw materials, for example, it can be 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, or 4%.

[0018] In some embodiments, the phospholipid-polyethylene glycol content is 20% to 25% based on the total mass of the liposome raw materials, for example, it can be 20%, 21%, 22%, 23%, 24%, or 25%.

[0019] In some embodiments, based on the total mass of the liposome raw materials, the content of soybean lecithin is 65% to 75%, the content of cholesterol is 3% to 4%, and the content of phospholipid-polyethylene glycol is 20% to 25%.

[0020] In some embodiments, the wavelength range of the near-infrared II region is 1000~1700 nm, for example, it can be 1000 nm, 1050 nm, 1100 nm, 1150 nm, 1200 nm, 1250 nm, 1300 nm, 1350 nm, 1400 nm, 1450 nm, 1500 nm, 1550 nm, 1600 nm, 1650 nm, or 1700 nm.

[0021] Compared to conventional NIR-I lasers, this application utilizes NIR-II lasers, which offer significant advantages in achieving precise, safe, and controllable photothermal therapy in deep brain regions, as detailed below: 1. A qualitative leap in penetration depth and therapeutic accessibility NIR-I lasers (such as 808 nm) suffer from severe scattering in biological tissues, resulting in shallow effective penetration depth and making it difficult to reach and effectively treat deep brain regions such as the substantia nigra and hippocampus without damage. In contrast, the 1064 nm NIR-II laser used in this application is located within the "optical transparency window of biological tissues," enabling a several-fold increase in penetration depth. This allows for the first-ever non-invasive, deep photothermal treatment of the aforementioned key lesion areas, something that NIR-I technology cannot achieve.

[0022] 2. A leap forward in precise temperature control and biosafety To effectively activate TRPV4, heat needs to be precisely and evenly delivered to the deep target site. NIR-I lasers, due to their rapid energy decay in surface tissues, often cause overheating or even burns to the surface tissues when trying to reach the effective deep temperature, resulting in poor controllability. In contrast, NIR-II laser energy can reach deeper tissues more efficiently, thus achieving rapid (within 5 minutes) and precise (±0.5℃) temperature control (up to 55.2℃) in deep brain regions while avoiding surface damage (significantly improving biosafety). This is crucial for activating the TRPV4 channel without causing collateral damage. 3. Closed-loop imaging-guided therapy Treatment and imaging can use the same NIR-II laser source / window. This means that while performing photothermal therapy, real-time, online treatment monitoring and efficacy evaluation can be conducted using NIR-II fluorescence, forming a closed loop of "integrated diagnosis and treatment." Conventional NIR-I systems cannot achieve this kind of deep, high signal-to-noise ratio real-time monitoring, and their treatment is relatively blind.

[0023] In some embodiments, the near-infrared II photothermal material includes near-infrared II anthocyanin dyes.

[0024] In some embodiments, the near-infrared II photothermal material includes FD1080, EtFD1080, EtFD1080-Mal, EtFD1080-NHS, and EtFD1080-N3.

[0025] A second aspect of this application provides a method for preparing the aforementioned biomimetic nanodelivery platform, comprising the step of fusing liposomes loaded with cycloastragalool with a cell membrane dispersed with a photothermal conversion material. In some embodiments, the liposomes are coated onto the cell membrane through membrane fusion.

[0026] In some embodiments, the photothermal conversion material is co-extruded with the cell membrane to obtain a cell membrane in which the photothermal conversion material is dispersed.

[0027] In some embodiments, the method further includes preparing liposomes loaded with cycloastragalool by combining the raw materials of the liposomes with cycloastragalool.

[0028] In some embodiments, the preparation of cell membranes includes separating cell membranes from microglia through hypotonic lysis, freeze-thaw cycles, and differential centrifugation.

[0029] In some embodiments, the hypotonic lysis buffer used contains 5–20 mM Tris-HCl, 0.5–2 mM EDTA, and 0.05–0.2 mM benzyl sulfonyl fluoride (PMSF). In some embodiments, the hypotonic lysis buffer contains 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mM Tris-HCl. In some embodiments, the hypotonic lysis buffer contains 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mM EDTA. In some embodiments, the low-permeability lysis solution contains 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.2 mM PMSF.

[0030] In some embodiments, hypotonic pyrolysis includes incubation in a hypotonic pyrolysis solution for 5 to 30 minutes, for example, 5, 10, 15, 20, 25, or 30 minutes. In some embodiments, the incubation temperature for hypotonic pyrolysis is 0 to 4 °C.

[0031] In some implementations, the freeze-thaw cycle temperature is -60 to -20 °C, for example, it can be -60 °C, -55 °C, -50 °C, -45 °C, -40 °C, -35 °C, -30 °C, -25 °C, or -20 °C.

[0032] In some implementations, the number of freeze-thaw cycles is 3 to 10, for example, 3, 4, 5, 6, 7, 8, 9, or 10.

[0033] In some embodiments, differential centrifugation includes a first centrifugation to remove cytoplasm and a second centrifugation to collect the cell membrane. In some embodiments, the rotation speed of the first centrifugation is 2000-4000 g, for example, 2000 g, 2500 g, 3000 g, 3500 g, or 4000 g. In some embodiments, the rotation speed of the second centrifugation is 50000-200000 g, for example, 50000 g, 100000 g, 150000 g, or 200000 g. In some embodiments, differential centrifugation further includes homogenizing the collected cell membrane by extrusion.

[0034] In some embodiments, the method for preparing liposomes loaded with cycloastragalool from liposome raw materials includes at least one of thin-film hydration, reverse evaporation, ethanol injection, microfluidics, and high-pressure homogenization.

[0035] In some embodiments, the raw materials for the liposomes include soybean lecithin, cholesterol, and phospholipid-polyethylene glycol.

[0036] In some embodiments, the content of soybean lecithin is 65-75% based on the total mass of the liposome raw materials, for example, it can be 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75%.

[0037] In some embodiments, the cholesterol content is 3% to 4% based on the total mass of the liposome raw materials, for example, it can be 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, or 4%.

[0038] In some embodiments, the phospholipid-polyethylene glycol content is 20% to 25% based on the total mass of the liposome raw materials, for example, it can be 20%, 21%, 22%, 23%, 24%, or 25%.

[0039] In some embodiments, based on the total mass of the liposome raw materials, the content of soybean lecithin is 65% to 75%, the content of cholesterol is 3% to 4%, and the content of phospholipid-polyethylene glycol is 20% to 25%.

[0040] In some embodiments, the method further includes a step of coupling a targeting antibody after membrane fusion of a liposome loaded with cycloastragalool with a cell membrane in which photothermal conversion material is dispersed.

[0041] In some embodiments, the mass ratio of cholesterol to cycloastragaloyl alcohol is 1:2 to 10, for example, it can be 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0042] In some embodiments, membrane fusion is performed by co-extruding liposomes loaded with cycloastragalool with a cell membrane dispersed with photothermal conversion material.

[0043] In some embodiments, a polycarbonate film is used for co-extrusion. In some embodiments, the pore size of the co-extruded polycarbonate film is 50~400 nm, for example, 50 nm, 100 nm, 200 nm, 400 nm, etc.

[0044] Conventional physical methods such as ultrasound or microcurrent are used to process complex systems composed of CAG and vesicles, which have many drawbacks, such as leakage and damage of small molecules: high shear forces or cavitation effects can easily cause small molecular weight and structurally unstable CAG to leak out of the complex, precipitate, or undergo chemical degradation, thus losing its efficacy; another example is damage to the carrier structure: it may destroy the delicate structure of FD1080@MM, affecting its targeting function. The method of pre-preparing CAG into liposomes in this invention is a mild and controlled biomimetic membrane fusion process, and its unique advantage for small molecule CAG complexes is: 1. Achieve efficient co-encapsulation: It can spontaneously and directionally assemble hydrophobic / amphiphilic CAG small molecules with FD1080@MM into specific positions in the lipid bilayer, achieving extremely high encapsulation efficiency and drug loading.

[0045] 2. Maximum protection of activity: The gentle process ensures that the chemical structure of CAG and the functional integrity of FD1080@MM are not compromised during preparation.

[0046] 3. Formation of stable complexes: It can form nanocomposites with dense structure, low leakage and uniform particle size, ensuring their stability in in vivo circulation.

[0047] Conventional high-energy physics methods are more direct, but the mild liposome (LP) method used in this application overcomes the inherent defects of high-energy methods when dealing with small molecule complex systems and produces synergistically enhanced drug efficacy.

[0048] A third aspect of this application provides a photothermal synergistic therapy system, comprising: Delivery module, used to apply the aforementioned biomimetic nanodelivery platform; The irradiation module is used to emit laser light with a wavelength in the near-infrared II region to the target tissue.

[0049] In some embodiments, the photothermal synergistic therapy system further includes an evaluation module for evaluating the therapeutic effect based on the fluorescence generated by the near-infrared II photothermal material under laser excitation.

[0050] In some embodiments, the delivery module stores the aforementioned biomimetic nanodelivery platform and is used to apply the aforementioned biomimetic nanodelivery platform.

[0051] A fourth aspect of this application provides the application of the aforementioned biomimetic nanodelivery platform or the aforementioned photothermal synergistic therapy system in the preparation of products for treating neurodegenerative diseases.

[0052] In some implementations, neurodegenerative diseases include any one of Parkinson's disease, Lewy body dementia, Alzheimer's disease, and multiple system atrophy.

[0053] The beneficial effects of this invention are: This invention utilizes a composite liposome to construct a nanoplatform integrating "precise targeting, photothermal activation, drug delivery, and biomimetic avoidance and rejection." Through a dual-functionalization modification (microglia cell membrane coating + TRPV4 antibody conjugation), it significantly enhances the targeting and recognition ability of microglia, reduces the risk of immune clearance, and increases the drug accumulation rate in brain lesions. It achieves efficient co-loading and stable delivery of dual active ingredients (cycloastragalol + near-infrared II photothermal reagent). Precise component ratios and process control improve the loading efficiency of active ingredients, reduce drug leakage, and ensure effective concentrations when acting on target cells. A synergistic mechanism of "targeted delivery-photothermal activation-functional regulation" is established. Near-infrared II laser precisely activates the photothermal effect, synergistically inducing microglia to shift from a pro-inflammatory phenotype to an anti-inflammatory phenotype, while simultaneously upregulating lysosomal activity and enhancing α-synuclein clearance, thus addressing the two major pathological problems of neuroinflammation and pathogenic protein accumulation.

[0054] Among them, targeted recognition: the anti-TRPV4 antibody on the surface specifically binds to the TRPV4 ion channel on the surface of microglia, while the microglia cell membrane coating structure can improve cell fusion efficiency and achieve precise targeted delivery.

[0055] Photothermal activation: Irradiation with a 1064 nm near-infrared II laser produces a locally controllable photothermal effect (power density 1.5 W / cm²). 2 (When the temperature rises from 20 ℃ to 55.2 ℃ within 5 minutes), the TRPV4 channel is activated.

[0056] Synergistic regulation: The photothermal effect and cycloastragalool work synergistically to induce microglia to transform from a pro-inflammatory phenotype to an anti-inflammatory phenotype, and reduce the levels of pro-inflammatory factors such as IL-1β and TNF-α; at the same time, it upregulates lysosomal activity and phagocytic function, and enhances the degradation and clearance of α-synuclein. Attached Figure Description

[0057] Figure 1 This is a schematic diagram illustrating the preparation principle of CAG / FD1080@MM-aTRPV4 in Example 1 of this application.

[0058] Figure 2 These are cryo-transmission electron micrographs of CAG@LP and CAG / FD1080@MM prepared in Example 1 of this application.

[0059] Figure 3The figures show the characterization results of particle size and potential of CAG@LP, CAG / FD1080@MM, and CAG / FD1080@MM-aTRPV4 prepared in Example 1 of this application. In the figures, A is the number distribution map of particle size detected by dynamic light scattering, B is the intensity distribution map of particle size detected by dynamic light scattering, and C is the zeta potential detection result of the three liposomes.

[0060] Figure 4 These are the photothermal performance test results of CAG / FD1080@MM-aTRPV4 in Example 1 of this application. Wherein, A represents the temperature change of CAG / FD1080@MM-aTRPV4 over time at different power densities; B represents the temperature change of CAG / FD1080@MM-aTRPV4 over time at different concentrations; C represents the temperature change at a power density of 1.5 W / cm². 2 The temperature change of CAG / FD1080@MM-aTRPV4 over time under the cyclic conditions of 400 μg / mL concentration, 300s irradiation and 300s irradiation stop.

[0061] Figure 5 This is the detection result of BV2 cells' targeted uptake of liposomes in vitro in Example 2 of this application. In the figure, A shows the confocal microscopy observation results after co-incubating BV2 cells with Rhodamine B-labeled CAG / FD1080@MM-aTRPV4 or Rhodamine B dye alone for 30 minutes and 2 hours. B shows the flow cytometry-quantified change in red fluorescence intensity after co-incubation of BV2 cells with Rhodamine B-labeled CAG / FD1080@MM-aTRPV4 for different durations. The scale bar in the figure is 50 μm.

[0062] Figure 6 This is the result of microglia function regulation verification in Example 2 of this application. A and B are confocal microscopy observations of the control group (LP@MM) and the experimental group (CAG / FD1080@MM-aTRPV4) after co-incubation at 0, 2, 6, and 24 hours in an α-synuclein-treated microglia culture model. In A and B, microglia morphology was stained with Iba1 antibody (red), and cell nuclei were counterstained with DAPI (blue). Microglia activation status in A was stained with CD68 antibody (green), and α-synuclein aggregation in B was stained with α-synuclein antibody (green). Co-localization of merged channels was shown in A and B (yellow). C represents the CD68 levels per microglia. + Fluorescence intensity, D is the α-synuclein per microglia + Fluorescence intensity, data are expressed as mean ± standard error (n=6), ***p<0.001 (two-way ANOVA + Tukey multiple comparisons); E is a schematic diagram of the mechanism of action.

[0063] Figure 7 This is the thermographic analysis result of the ultra-high performance liquid chromatography-mass spectrometry detection of metabolites in Example 3 of this application.

[0064] Figure 8 The results of KEGG pathway enrichment of metabolites detected by ultra-high performance liquid chromatography-mass spectrometry in Example 3 of this application are shown.

[0065] Figure 9 The results are behavioral detection results from Example 3 of this application. A shows the stride length changes of the left foreleg (FL), left hindleg (RL), right foreleg (FR), and right hindleg (RR) in different groups of gait analysis; B shows the swing speed changes in different groups of gait analysis; C shows the fall time of mice in different groups under different rotational speeds; D shows images of mice in different groups undergoing the elevated cross maze test; E shows images of mice in different groups undergoing the open field test; F-H represent the number of times mice entered the open arm, the open arm dwell time, and the open arm movement distance in the elevated cross maze test for different groups of mice, respectively; and I-J represent the movement distance and central region dwell time in the open field test for different groups of mice, respectively. Detailed Implementation

[0066] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The terminology used herein is for the purpose of describing embodiments of this application only and is not intended to limit the application. Those skilled in the art will recognize that, with the evolution of technology and the emergence of new application scenarios, the technical solutions provided in the embodiments of this application are equally applicable to similar technical problems.

[0067] Example 1: Preparation and characterization of complex liposomes This embodiment provides a composite liposome, and the preparation steps are as follows: Figure 1 The details are as follows: Step 1: Preparation of FD1080@MM Take 0.2 mL of FD1080 (methanol solution, concentration 4 mg / mL) and mix it with cell membrane fragments of BV2 microglia (containing 7.5 mg of protein), and place it in a 10 mL centrifuge tube. Transfer the mixture to a mini extruder (Avanti PolarLipids) and co-extrude it through a polycarbonate membrane (pore size 200 nm) 20 times (extrusion pressure 0.3 MPa, room temperature) to promote the stable binding of FD1080 to the cell membrane. After extrusion, transfer the solution to an ultrafiltration tube (Amicon Ultra, molecular weight cutoff 100 kDa), centrifuge at 4 ℃ and 4000 rpm for 15 minutes to remove unencapsulated free FD1080. Resuspend the residue in the ultrafiltration tube with PBS buffer (pH 7.4), and make up to 2 mL to obtain the photothermal reagent-cell membrane complex FD1080@MM, which is then sealed and stored at 4 ℃ for later use.

[0068] The cell membrane fragments of BV2 microglia were obtained by isolating the cell membranes of microglia from the BV2 cell line (CVCL_0182) using hypotonic lysis, freeze-thaw cycles, and differential centrifugation: BV2 cells were gently scraped from the cell membranes with PBS buffer (pH 7.4) and centrifuged at 1000×g for 5 minutes at 4 °C to collect the precipitate. The precipitate was resuspended in hypotonic lysis buffer containing protease inhibitors (10 mM Tris-HCl, 1 mM EDTA, 0.1 mM PMSF) and incubated on ice for 20 minutes to promote membrane dissociation. The lysis buffer was subjected to five freeze-thaw cycles in liquid nitrogen at -37 °C, and then centrifuged at 3000×g for 10 minutes to remove cytoplasmic components, collecting the supernatant rich in membrane fragments. The supernatant was centrifuged at 100,000 × g for 1 hour at 4 °C using a Beckman 70 Ti rotor. The resulting membrane precipitate was homogenized by extrusion 20 times using a 400 nm polycarbonate membrane, then resuspended in PBS and stored at 4 °C or -80 °C for later use.

[0069] Step 2: Preparation of CAG@LP 31 mg of soybean lecithin, 1.6 mg of cholesterol, 14 mg of phospholipid-polyethylene glycol (DSPE-PEG2000), and 8.75 mg of cycloastragalool (CAG, purity ≥98%) were dissolved in 40 mL of chloroform and transferred to a rotary evaporator flask. The chloroform was removed by rotary evaporation at 30 °C and 100 rpm for 30 minutes, forming a uniform lipid film on the flask wall. 5 mL of PBS buffer (pH 7.4) was added to the film, and the mixture was sonicated at 37 °C (300 W) for 30 minutes to form a milky white suspension. The suspension was centrifuged at 4000 rpm for 10 minutes, and the undissolved precipitate at the bottom was discarded. The supernatant was concentrated to 3 mL with PBS to obtain cycloastragalool-loaded liposomes CAG@LP with a particle size of approximately 30.3 nm.

[0070] Step 3: Preparation of CAG / FD1080@MM Mix 1.5 mL of CAG@LP with 1.5 mL of FD1080@MM in equal volumes and place in a 5 mL centrifuge tube. Use a mini extruder (Avanti Polar Lipids) to co-extrude through a polycarbonate membrane (pore size 200 nm) 20 times (extrusion pressure 0.3 MPa, room temperature) to ensure uniform fusion of the two components. After extrusion, centrifuge the solution at 4 ℃ and 3000 rpm for 5 minutes to remove possible agglomerates. The supernatant is the dual-active liposome CAG / FD1080@MM, which should be stored at 4 ℃.

[0071] Step 4: Preparation of CAG / FD1080@MM-aTRPV4 Mix 0.1 mL of anti-TRPV4 antibody (100 μg / mL) with 0.1 mL of bifunctional cross-linking agent sulfo-SMCC (5 mg / mL) and incubate at 4 °C for 2 hours to form an antibody-cross-linking agent conjugate. Slowly add the conjugate to 1 mL of CAG / FD1080@MM and incubate at 4 °C in the dark overnight (12 hours). After incubation, dialyze with PBS (dialysis bag molecular weight cutoff 50 kDa) for 4 hours to remove unconjugated antibody and cross-linking agent, obtaining the composite liposome CAG / FD1080@MM-aTRPV4.

[0072] Among them, the anti-TRPV4 antibody is generated against an 18-amino acid peptide (RREVTDEDTRHLSRKFKD) near the center of human TRPV4, and the immunogen is located between amino acids 380 and 430 of TRPV4.

[0073] The products prepared in each of the above steps were characterized as follows: (1) Electron microscopy characterization Take 10 μL of the CAG@LP solution prepared in step 2 and the CAG / FD1080@MM solution prepared in step 3, and add them dropwise onto a copper mesh (400 mesh). After freezing in liquid nitrogen and lyophilizing, stain the mesh. The results are observed by cryo-transmission electron microscopy (TEM). Figure 2 Among them, CAG@LP is a uniformly dispersed spherical vesicle with a diameter of about 30 nm; CAG / FD1080@MM retains the spherical structure, and the surface shows a double membrane structure formed by the cell membrane (in the green dashed box), proving that the biomimetic modification was successful.

[0074] (2) Particle size and zeta potential At room temperature, the products prepared in each step were diluted to 0.1 mg / mL with PBS buffer, and the particle size and zeta potential were detected by dynamic light scattering (DLS) at a detection angle of 90°.

[0075] The results are as follows Figure 3 As shown in the figure, the average particle size of CAG@LP is 30.3±16.1 nm, that of CAG / FD1080@MM is 28.4±12.1 nm, and that of CAG / FD1080@MM-aTRPV4 is 32.5±14.3 nm. All three have a concentrated particle size distribution (PDI<0.3) and no obvious aggregation. The zeta potential of CAG@LP is -8.5 mV, that of CAG / FD1080@MM is -8.0 mV, and that of CAG / FD1080@MM-aTRPV4 after conjugation with anti-TRPV4 antibody rises to -2.0 mV. This potential change confirms successful antibody conjugation.

[0076] (3) Photothermal performance testing A PBS control group and experimental groups with different concentrations (0, 50, 100, 200, 400 μg / mL composite liposome CAG / FD1080@MM-aTRPV4) were set up, and irradiated with 1064 nm near-infrared laser (effective power density 0, 1.0, 1.2, 1.5 W / cm²). 2 It records temperature changes in real time over 5 minutes.

[0077] The results are as follows Figure 4 As shown in the figure, the temperature of the composite liposomes increases continuously with increasing power density over the same time period, indicating that the photothermal effect of the CAG / FD1080@MM-aTRPV4 composite liposomes under 1064 nm near-infrared II illumination is power-dependent. At 1.5 W / cm², the temperature of the composite liposomes increases more rapidly. 2 At a given power density, the temperature of the composite liposomes continuously increased with increasing concentration over the same time period, indicating that the photothermal effect of the composite liposomes is positively correlated with its concentration at a fixed power density. Furthermore, during thermal cycling from 30 °C to 58 °C, the maximum temperature fluctuation was less than 3 °C, demonstrating good photothermal stability of the composite liposomes.

[0078] Example 2: In vitro functional verification 1. Experimental Model and Grouping The cell model was BV2 microglia (cultured in DMEM medium containing 10 v / v% fetal bovine serum at 37°C and 5% CO2). The groups were: blank control group (culture medium only), LP@MM group (bionic carrier without active ingredients), CAG@MM group (CAG-loaded carrier only), CAG / FD1080@MM-aTRPV4 group, and CAG / FD1080@MM-aTRPV4+laser group (1064 nm laser, 1.5 W / cm²). 2Irradiate for 5 minutes.

[0079] 2. In vitro targeted uptake validation (1) Observation by confocal microscope Log-phase BV2 cells were harvested and seeded in confocal culture dishes (2 × 10⁻⁶ cells / year). 5 Cells / plate were cultured for 24 hours; Rhodamine B-labeled CAG / FD1080@MM-aTRPV4 (final concentration 0.1 mg / mL) was added, and incubation was performed for 30 minutes and 2 hours, respectively; after incubation, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 minutes, stained with Phalloidin (green) on the cytoskeleton, and Hoechst (blue) on the nuclei; the results were observed under a laser confocal microscope. Figure 5 As shown in Figure A, weak red fluorescence was visible in the cells after 30 minutes of incubation, and the red fluorescence was significantly enhanced after 2 hours, proving that the complex liposomes were actively taken up by microglia, and the amount of take-up increased over time.

[0080] (2) Quantitative flow cytometry Log-phase BV2 cells were seeded in 6-well plates (5 × 10⁻⁶ cells per well). 5 Cells / well were cultured for 24 hours; Rhodamine B-labeled CAG / FD1080@MM-aTRPV4 (final concentration 0.1 mg / mL) was added, and incubation was performed for 0, 3, 6, 9, and 12 hours, respectively; after incubation, cells were washed three times with PBS, digested with trypsin, washed twice with PBS, and the intensity of red fluorescence was detected by flow cytometry. Results are as follows. Figure 5 As shown in Figure B, the fluorescence signal peak shifts to the right with incubation time, and the fluorescence intensity at 12 hours is 8.2 times that at 0 hours, confirming that uptake is time-dependent.

[0081] 3. Validation of microglia function regulation Log-phase BV2 cells were seeded in 6-well plates (5 × 10⁻⁶ cells per well). 5 Recombinant α-synuclein (final concentration 5 μg / mL) was added to cells / wells and cultured for 12 hours to establish an accumulation model; then CAG / FD1080@MM-aTRPV4 (final concentration 0.1 mg / mL) was added and cultured for another 24 hours. At the same time, a control group LP@MM (empty liposome-encapsulated MM) was set up to detect the temporal changes in microglial cell activation and α-synuclein degradation.

[0082] The results of the confocal microscope images are as follows: Figure 6 As shown, quantitative analysis results indicate that, compared with the LP@MM control group, the CAG@MM group significantly inhibited microglial cell overactivation and significantly promoted α-synuclein degradation. The mechanism of action is as follows: Figure 6As shown in E, CAG@MM improves α-synucleosis through a dual mechanism of enhancing microglia's ability to clear α-synucleosis and inhibiting the pathological overactivation of microglia.

[0083] Example 3: Animal efficacy verification in a mouse model of Parkinson's disease 1. Experimental Model and Grouping Model construction: C57BL / 6 mice (male, 8 weeks old) were used to construct a Parkinson's disease model by injecting either AAV-empty vector (CTL, normal control group) or AAV-α-synuclein (A53T); Grouping: Normal control group (WT), PD+LP@MM-aTRPV4 group, PD+FD1080@MM-aTRPV4+ laser group, PD+CAG / FD1080@MM-aTRPV4+ laser group (n=6 / group); Administration: Tail vein injection, dose 10 mg / kg, once a week for 4 consecutive weeks; Laser treatment: 24 hours after each administration, irradiate the head with a 1064 nm laser (1.5 W / cm²). 2 (5 minutes).

[0084] 2. Metabolic reprogramming detection Twenty-four hours after the last administration, striatal tissue was taken from mice, metabolites were extracted, and detected by UPLC-Q-TOF-MS.

[0085] The results are as follows Figure 7 and Figure 8 As shown, Figure 7 The results of the metabolite heatmap analysis show that, compared with the PD model group, the expression of 23 differential metabolites (such as ceramide and arachidonic acid) in the PD+CAG / FD1080@MM-aTRPV4+ laser group was restored to near the normal group level. Figure 8 The KEGG pathway enrichment results show that the PD+CAG / FD1080@MM-aTRPV4+ laser group significantly enriched pathways related to neural function, such as "neuroactive ligand-receptor interaction" and "phosphatidylinositol signaling pathway" (Q value < 0.05), demonstrating the effect of metabolic reprogramming.

[0086] 3. Behavioral improvement assessment (1) Gait analysis Test indicators: left forepaw stride length (FL), left hindaw stride length (RL), swing speed; The results are as follows Figure 9As shown in Figures A and B, it can be seen from the figures that, compared with the PD model group, the FL step length of the PD+CAG / FD1080@MM-aTRPV4+ laser group increased by 35.2%, the RL step length increased by 42.1%, and the oscillation speed increased by 28.7% (P<0.01).

[0087] (2) Rotating rod experiment (motor coordination) Test conditions: Motor function was assessed using an accelerated rotating bar system. Mice were placed on the rotating bar, with the rotation speed starting at 8 revolutions per minute (rpm) and increasing in steps every 5 minutes to 15, 20, 24, 31, and 40 rpm. The fall latency (seconds) of the mice was recorded, and the average of three measurements per day was taken as the score at each rotation speed.

[0088] The results are as follows Figure 9 As shown in Figure C, the normal control group (CTL) mice maintained a consistently long fall latency (107±15.4) during the accelerated test, indicating normal motor function and providing a benchmark for subsequent evaluation. After injection of blank liposomes (PD+LP@MM) into PD model mice, the fall latency in the high-speed range (24–40 rpm) was significantly reduced (lowest 50.99±16.13), indicating that the carrier alone had no therapeutic effect. After injection of the targeted carrier (PD+1080@MM-aTRPV4), the fall latency in the medium-to-high-speed range was prolonged to 80.85±17.02, suggesting that the targeted design had a certain improvement effect. Furthermore, after injection of the composite liposomes of this invention combined with thermotherapy (PD+CAG / 1080@MM-aTRPV4+Heat), the fall latency was further increased to 89.5±15.03, superior to other model groups.

[0089] (3) Elevated cross maze (EPM, anxiety behavior) and open field experiment (OFT, exploration behavior) EPM results are as follows Figure 9 As shown in Figures D and F~H, it can be seen from the figure that, compared with the PD model group, the number of open arm entry times of the PD+CAG / FD1080@MM-aTRPV4+ laser group increased by 68.3%, and the dwell time increased by 82.5%.

[0090] The OFT results are as follows: Figure 9 As shown in E, I, and J, the figure shows that compared with the PD model group, the total movement distance of the PD+CAG / FD1080@MM-aTRPV4+ laser group increased by 55.2%, and the dwell time in the central area increased by 78.4% (P<0.01), which proves that anxiety behavior is reduced and exploration ability is restored.

[0091] As can be seen from the above embodiments, the composite liposomes provided in this application have significant advantages over existing technologies in terms of targeting precision, synergistic regulation capability, structural stability, and clinical translational value, as detailed below: (1) The targeting accuracy is significantly improved and the off-target effect is greatly reduced. The dual-functional nanoplatform exhibits excellent active targeting capabilities and reduced off-target effects. Through a synergistic design of "microglia cell membrane coating" and "anti-TRPV4 antibody conjugation," this platform achieves the following effects: Specific recognition and binding: The anti-TRPV4 antibody on the surface can specifically recognize and bind to the TRPV4 ion channel on the surface of microglia. In vitro experiments confirmed that the uptake of this platform in BV2 microglia increased in a time-dependent manner, demonstrating its effective targeted binding ability.

[0092] Immune escape and long circulation: The cell membrane coating structure of microglia provides good biocompatibility for complex liposomes, which can effectively reduce the risk of immune recognition and help avoid rapid clearance by the reticuloendothelial system.

[0093] Synergistic Effect: Due to the combination of the above two functions, this composite liposome can be delivered more efficiently to the brain lesion area in vivo and specifically taken up by activated microglia. This synergistic effect of immune evasion and active targeting leads to the high enrichment of the platform in the brain, with a brain enrichment rate of 15-20%, significantly higher than existing nanocarriers (generally below 10%), and correspondingly reduces non-specific distribution in peripheral tissues, thus potentially significantly reducing toxic side effects caused by off-target distribution in peripheral tissues.

[0094] (2) To achieve simultaneous interruption and synergistic treatment of the vicious cycle of pathology. This invention innovatively constructs a cascaded therapeutic mechanism of "targeted anchoring → photothermal initiation → drug enhancement." The core of this mechanism lies in utilizing the deep spatiotemporal and functional synergy between photothermal effects and drug therapy to achieve multi-level, integrated intervention in the vicious cycle of "neuroinflammation" and "α-synuclein pathology." Experiments have confirmed that the efficacy of this platform is reflected in three closely related levels: At the molecular and cellular levels, the inhibition of pathological activation of microglia (reduction of CD68 fluorescence density) and degradation of α-synuclein (decrease in fluorescence density) were achieved simultaneously.

[0095] At the level of brain region metabolism, the pathological metabolic reprogramming of the striatum in the Parkinson's disease model was successfully reversed, restoring key energy metabolism homeostasis and providing the necessary microenvironmental basis for the recovery of neural function.

[0096] At the overall behavioral level, the treatment demonstrated fundamental functional recovery in Parkinson's disease model mice. The synergistic treatment group (CAG / FD1080@MM-aTRPV4 + photothermal) showed significant and comprehensive improvements in motor coordination (significantly prolonged latency in the rotarod test), gait balance (improved stride length and body swing speed), anxiety-like behavior, and exploratory abilities (comprehensive improvement in indicators for the elevated cruciate maze and open field tests). In contrast, the single-mechanism treatment groups, including drug-loaded liposomes only (LP@MM-aTRPV4) and photothermal nanoparticles only (FD1080@MM-aTRPV4 + laser), failed to mimic the broad behavioral benefits of the synergistic treatment group at this level.

[0097] This result strongly demonstrates that the therapeutic effect produced by this invention through "mechanism coupling" and "spatiotemporal synergy" is far beyond what a single therapy or simple drug combination can achieve, thus realizing a fundamental therapeutic breakthrough from molecular pathological improvement to overall neurological function recovery.

[0098] (3) Significantly improved structural stability and active ingredient delivery efficiency By employing the precise liposome composition ratio (soybean lecithin: cholesterol: DSPE-PEG2000: CAG = 31:1.6:14:8.75) and combining it with an optimized "two-stage membrane extrusion" process, a highly uniform and stable nanoplatform was successfully constructed. This platform exhibits the following core advantages: Excellent size uniformity and structural integrity: As shown by Cryo-TEM and DLS characterization, the nanoplatform has uniform particle size (average particle size of CAG@LP is 30.3±16.1 nm, and average particle size of CAG / FD1080@MM is 28.4±12.1 nm) and low breakage rate (less than 3%), demonstrating its excellent physical stability.

[0099] Highly efficient drug loading and controlled release: This process achieves a drug loading rate of up to 13.6% cycloastragalool (CAG) while maintaining a low drug leakage rate (below 5%) during storage and delivery. This high loading and high retention characteristic ensures that the active ingredient can be efficiently delivered to the target.

[0100] Stable photothermal component function: FD1080 achieves efficient encapsulation through the aforementioned membrane extrusion process, exhibiting a low shedding rate (less than 5%) in vivo. Its photothermal performance is stable; after multiple laser irradiation cycles (5 times), the photothermal conversion efficiency remains above 95%, ensuring the reliability and repeatability of photothermal therapy effects.

[0101] In summary, this invention, through a specific combination of components and processes, synergistically endows the nanoplatform with excellent comprehensive properties such as high load capacity, low leakage, structural stability, and long-lasting functionality.

[0102] (4) It has good biocompatibility and clear clinical translation potential. A carrier was constructed using biocompatible materials (soybean lecithin and microglia cell membrane fragments). No significant toxicity was observed in in vitro cell experiments, and in vivo experiments showed no significant impact on liver and kidney function or blood parameters. The preparation process can be optimized for large-scale production and can be further developed into a nasal delivery formulation, reducing the pain of injection and improving patient compliance. Furthermore, this technological framework can be adapted to other neurodegenerative diseases such as Alzheimer's disease and multiple system atrophy by replacing the active ingredient and targeting antibody, expanding its application scenarios and providing a novel technological solution for the etiological treatment of neurodegenerative diseases.

[0103] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A biomimetic nanodelivery platform for brain-targeted and photothermal synergistic therapy, characterized in that, include: A drug-carrying core comprising liposomes and cycloastragalool loaded in the liposomes; A functionalized shell, which encloses the drug-carrying core, comprising a cell membrane and a photothermal conversion material dispersed in the cell membrane.

2. The biomimetic nanodelivery platform according to claim 1, characterized in that, The cell membrane originates from microglia.

3. The biomimetic nanodelivery platform according to claim 1 or 2, characterized in that, The photothermal conversion material is a near-infrared II photothermal material.

4. The biomimetic nanodelivery platform according to claim 1, characterized in that, The loading amount of cycloastragalool in the liposomes is 10~50 wt.

5. The biomimetic nanodelivery platform according to claim 1, characterized in that, The raw materials constituting the liposomes include soybean lecithin, cholesterol, and phospholipid-polyethylene glycol.

6. The biomimetic nanodelivery platform according to claim 5, characterized in that, Based on the total mass of the raw materials for the liposomes, the content of soybean lecithin is 65%~75%, the content of cholesterol is 3%~4%, and the content of phospholipid-polyethylene glycol is 20%~25%.

7. The method for preparing the biomimetic nanodelivery platform according to any one of claims 1 to 6, characterized in that, The method includes the step of fusing liposomes loaded with cycloastragalool with a cell membrane in which photothermal conversion material is dispersed.

8. A photothermal synergistic therapy system, characterized in that, include: The delivery module is used to apply the biomimetic nanodelivery platform according to any one of claims 1 to 6; the irradiation module is used to emit laser light with a wavelength in the near-infrared II band to the target tissue.

9. The use of the biomimetic nanodelivery platform according to any one of claims 1 to 6, or the photothermal synergistic therapy system according to claim 8, in the preparation of a medicament for treating neurodegenerative diseases.

10. The application according to claim 9, characterized in that, The neurodegenerative disease is selected from one or more of Parkinson's disease, Lewy body dementia, and Alzheimer's disease.

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