Programmable fluorinated lipid nanodelivery system and uses thereof

Abstract

The application discloses a programmable fluorinated lipid nano-delivery system and application thereof, and the system is a core-shell structure, a drug molecule-embedded chitosan-methacrylate cross-linked nano-internal core is used as a drug reservoir, and a composite lipid layer composed of functionalized fluorinated lipids FPD, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, DOPE-TK-mPEG and DSPE-PEG-targeting molecules is used as an outer shell. The delivery system as a modular platform can realize active and accurate targeted delivery of different glial cell subgroups or brain parenchyma lesion immune receptors flexibly by replacing the targeting molecules connected to the outer shell surface. The system has excellent brain delivery efficiency and biological safety, and can responsively trigger the shell dissociation and the sustained and slow release of drug molecules under the high active oxygen pathological microenvironment in the Alzheimer's disease brain, thereby providing a new technical scheme for the anti-inflammatory and neuroprotective function research of Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of drug delivery and nanomedicine technology, specifically relating to a programmable fluorinated lipid nanodelivery system and its applications. Background Technology

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease that primarily affects the elderly and is one of the leading causes of dementia. Clinically, AD is mainly characterized by progressive memory loss, decreased learning ability, cognitive impairment, language impairment, executive function impairment, and behavioral and mental abnormalities. As the disease progresses, patients gradually lose their ability to live independently, placing a heavy burden on families and society. Therefore, developing novel treatment strategies that can effectively slow the pathological progression of AD, improve cognitive function, and have good brain delivery efficiency is of great significance.

[0003] The typical pathological features of Alzheimer's disease (AD) mainly include senile plaques formed by abnormal aggregation of extracellular β-amyloid (Aβ), neurofibrillary tangles formed by hyperphosphorylation of intracellular Tau protein, and accompanying synaptic damage, neuronal loss, and brain atrophy. For a long time, Aβ deposition and Tau pathology have been considered key factors in the development and progression of AD. However, increasing research indicates that AD is not solely caused by neuronal lesions; abnormal function of glial cells in the brain, especially persistent inflammatory activation of microglia and astrocytes, plays a crucial role in the pathological progression of AD.

[0004] Microglia are innate immune cells of the central nervous system, participating in processes such as Aβ clearance, synaptic pruning, release of inflammatory factors, and nerve damage repair. In the pathological environment of Alzheimer's disease (AD), microglia can be activated by Aβ and other damage-related signals, transforming into a pro-inflammatory phenotype and releasing inflammatory factors such as tumor necrosis factor-α, interleukin-1β, and interleukin-6, further exacerbating neuroinflammatory responses and neuronal damage. On the other hand, moderately activated or protective microglia can phagocytose and clear Aβ, inhibit the spread of inflammation, and maintain brain homeostasis. Therefore, inducing microglia to transform from a pro-inflammatory to a protective phenotype is an important direction for intervening in AD neuroinflammation and Aβ pathology.

[0005] Astrocytes are an abundant type of glial cell in the brain, involved in maintaining neuronal metabolism, regulating synaptic function, stabilizing the blood-brain barrier, and modulating the inflammatory microenvironment within the brain. During Alzheimer's disease (AD), astrocytes can undergo reactive activation and exhibit different functional states. Overactivated reactive astrocytes can release inflammatory mediators and neurotoxic factors, promoting Aβ deposition, synaptic damage, and neuronal death. Inhibiting the activation of harmful reactive astrocytes, or promoting their transformation into a state with neurotrophic and repair functions, holds promise as a novel strategy for AD treatment.

[0006] Glycoprotein non-metastatic melanoma protein B (GPNMB) is a transmembrane glycoprotein that plays an important role in neuroinflammatory regulation, immune homeostasis maintenance, tissue repair, and cell protection. GPNMB and its related bioactive peptides can exert potential neuroprotective effects by regulating glial cell inflammatory responses, promoting protective phenotypic remodeling, and inhibiting neuroinflammatory cascades. Therefore, GPNMB bioactive peptides show promise in the treatment of Alzheimer's disease (AD) and other neurodegenerative diseases. However, peptide drugs typically suffer from poor in vivo stability, susceptibility to enzymatic degradation, short half-life, low blood-brain barrier permeability, and insufficient effective concentrations in the brain, severely limiting their application in central nervous system diseases.

[0007] In addition to peptide drugs, large-molecule protein drugs, represented by monoclonal antibodies (such as rokanemab), have also shown great application value in the clinical treatment and disease reversal of Alzheimer's disease. However, due to their large molecular weight and complex tertiary structure, monoclonal antibodies face more severe in vivo barrier limitations than peptides: they are easily cleared nonspecifically in systemic circulation, their circulating half-life is difficult to regulate, and their spontaneous permeability across the blood-brain barrier (BBB) ​​is usually extremely low. This makes it difficult for high-dose antibodies administered peripherally to achieve effective therapeutic concentrations in the brain parenchyma lesions, accompanied by potential systemic side effects. Therefore, how to construct a high-capacity, high-stability universal nanodelivery platform that can simultaneously accommodate small molecules, peptides, and large-molecule monoclonal antibodies is another major technical bottleneck for achieving precise medical intervention in AD.

[0008] Currently, one of the major obstacles to drug delivery for central nervous system diseases is the blood-brain barrier. The blood-brain barrier restricts the entry of most macromolecular drugs, peptide drugs, and nanomedicines into brain tissue, resulting in low efficiency of intrabrain drug delivery.

[0009] Liposomes, as a classic nanomedicine delivery system, possess excellent biocompatibility, modifiability, and drug loading capacity, and have been widely used in drug delivery research. By regulating lipid composition, particle size, charge, and surface modification, liposomes can improve drug stability and tissue distribution to some extent. However, traditional liposomes typically lack the ability to actively target specific cell subpopulations in the brain, making it difficult to achieve precise delivery to different glial cell subpopulations such as microglia or astrocytes. Furthermore, the AD brain contains a pathological microenvironment characterized by enhanced oxidative stress, elevated ROS levels, and increased release of inflammatory factors. Traditional liposomes struggle to achieve responsive release based on this AD pathological microenvironment, thus limiting the duration of drug action and therapeutic efficiency.

[0010] Fluorinated lipids, due to their unique fluorocarbon chain structure, possess strong hydrophobicity, low surface energy, and excellent membrane interaction capabilities, making them suitable for improving the stability, mucosal penetration, and tissue permeability of nanodelivery systems. Introducing fluorinated lipids into lipid nanodelivery systems is expected to enhance the efficiency of these systems in crossing the blood-brain barrier and entering brain tissue, thereby improving their distribution and retention within the brain. On the other hand, ROS-responsive lipid materials containing thioketal bonds can undergo structural changes in high-ROS microenvironments, thus promoting drug release. DOPE-TK-mPEG, as a ROS-responsive phospholipid-polyethylene glycol derivative, can improve the stability and cycling / diffusion properties of lipid nanosystems, and also promote the dissociation of drug-loaded systems and drug release under oxidative stress in the AD brain.

[0011] Furthermore, active targeting modification is an important means to improve the cell selectivity of nanomedicines. DSPE-PEG is a commonly used liposome surface modification material, in which DSPE can be inserted into the lipid bilayer, and PEG provides spatial linking arms and improves nanoparticle stability. Attaching specific targeting peptides to the ends of DSPE-PEG can endow liposomes with the ability to actively recognize specific cells or tissues. For example, attaching the astrocyte-targeting peptide AS1 to the end of DSPE-PEG can be used to construct an astrocyte-targeting lipid nanodelivery system; attaching the M1-type microglia-targeting peptide MG1 to the end of DSPE-PEG can be used to construct a pro-inflammatory microglia-targeting lipid nanodelivery system. By replacing different targeting peptides, the surface function of the lipid nanodelivery platform can be modularly regulated.

[0012] While existing technologies include liposomes, peptide delivery systems, and brain-targeting nanomedicines, there is still a need to develop a novel lipid nanodelivery system capable of simultaneously achieving efficient brain delivery, responsive release to the AD pathological microenvironment, sustained release of drug molecules (including GPNMB active peptides or therapeutic monoclonal antibodies), and programmable targeting to different glial cell subsets or brain parenchymal lesions. This would have significant application value in improving the intrabrain delivery efficiency of AD therapeutic drugs, enhancing the targeting of glial cell subsets and lesion areas, prolonging the duration of drug action, and improving AD pathology. Summary of the Invention

[0013] The purpose of this invention is to overcome the shortcomings of the prior art and provide a programmable fluorinated lipid nanodelivery system.

[0014] Another object of the present invention is to provide a method for preparing the above-described programmable fluorinated lipid nanodelivery system.

[0015] Another object of the present invention is to provide the application of the above-described programmable fluorinated lipid nanodelivery system.

[0016] The technical solution of the present invention is as follows:

[0017] A programmable fluorinated lipid nanodelivery system has a core-shell structure, comprising a drug-carrying core and a lipid shell covering the drug-carrying core;

[0018] The drug-loaded core comprises a chitosan-methacrylate crosslinked network and drug molecules loaded therein;

[0019] The lipid shell includes fluorinated lipids, 1,2-distearate-sn-glycerol-3-phosphocholine, cholesterol, DOPE-TK-mPEG, and DSPE-PEG-targeting molecules;

[0020] The fluorinated lipid was obtained by coupling 4,4,4-trifluorobutyric acid with DSPE-PEG2000-NH2.

[0021] In a preferred embodiment of the present invention, the drug molecule is a GPNMB active polypeptide or rokanetumab; wherein the amino acid sequence of the GPNMB active polypeptide is shown in SEQ ID NO.01.

[0022] In a preferred embodiment of the present invention, the chitosan-methacrylate crosslinking network is formed by grafting chitosan with a degree of deacetylation of 85% and an average molecular weight of 15000 Da with methacrylic anhydride, followed by in-situ crosslinking and curing under ultraviolet light with the participation of a photoinitiator.

[0023] In a preferred embodiment of the present invention, the average particle size is 50~300 nm.

[0024] In a preferred embodiment of the invention, the lipid shell comprises components in the following mass ratio: fluorinated lipids : 1,2-distearate-sn-glycerol-3-phosphocholine : cholesterol : DOPE-TK-mPEG : DSPE-PEG-targeting molecule = 0.2-2.5 : 8.0-11.5 : 2.5-4.5 : 2.0-3.5 : 3.0-4.5.

[0025] In a preferred embodiment of the present invention, the DOPE-TK-mPEG in the lipid shell contains thioketal bonds to promote the dissociation of the lipid nanodelivery system structure or drug release in a reactive oxygen species microenvironment.

[0026] In a preferred embodiment of the present invention, the DSPE-PEG-targeting molecule is DSPE-PEG-AS1 or DSPE-PEG-MG1; wherein, AS1 is an astrocyte-targeting peptide with an amino acid sequence as shown in SEQ ID NO.02; and MG1 is an M1 microglia-targeting peptide with an amino acid sequence as shown in SEQ ID NO.03; by replacing AS1 or MG1 in the DSPE-PEG-targeting molecule, active targeted delivery to astrocytes or M1 microglia is achieved.

[0027] In a preferred embodiment of the present invention, the DSPE-PEG-targeting molecule comprises DSPE-PEG-CLEC7A and DSPE-PEG-TREM2, which together construct a dual-targeting lipid shell for brain parenchymal lesions; wherein the targeting molecule in DSPE-PEG-CLEC7A is a low-molecular-weight β-(1→3)-glucan oligosaccharide with the structure [→3)-β-D-Glcp-(1 ... n , n = 16–25, the target molecule in the DSPE-PEG-TREM2 is the LRK peptide with the amino acid sequence shown in SEQ ID NO.04.

[0028] The preparation method of the above-mentioned programmable fluorinated lipid nanodelivery system includes the following steps:

[0029] (1) The fluorinated lipid, 1,2-distearate-sn-glycerol-3-phosphocholine, cholesterol, DOPE-TK-mPEG and DSPE-PEG-targeting molecules are dissolved in chloroform, the solvent is removed by rotary evaporation, and a lipid film is assembled on the inner wall of the container.

[0030] (2) Chitosan-methacrylate is dissolved in a mixed solution containing the drug molecule and photoinitiator, and the mixture is vortexed and mixed evenly to form an inner aqueous phase, and the mass concentration of the drug molecule in the inner aqueous phase is controlled to be 0.1 mg / mL;

[0031] (3) The lipid film obtained in step (1) is hydrated by the internal aqueous phase obtained in step (2) and gently shaken at 37°C for 1 hour. It is then purified by monodisperse extrusion of polycarbonate membrane and deoxygenated by nitrogen gas. Finally, it is crosslinked in situ under ultraviolet light to obtain the final product.

[0032] The above-described programmable fluorinated lipid nanodelivery system is used in the preparation of formulations for studying the anti-inflammatory or neuroprotective effects of drug molecules in Alzheimer's disease.

[0033] The beneficial effects of this invention are:

[0034] 1. This invention proposes a novel GPNMB active peptide lipid nanodelivery system targeting glial cells. The system uses a chitosan-methacrylate cross-linked nanocore containing embedded GPNMB active peptides or rokanetrazol drug molecules as a drug reservoir, fluorinated lipids and DSPC as the main lipid shell, and introduces DOPE-TK-mPEG as a ROS-responsive stabilizing component. At the same time, it achieves active targeted delivery to astrocytes, M1 microglia or brain parenchymal lesions through DSPE-PEG-AS1, DSPE-PEG-MG1 or DSPE-PEG-CLEC7A / DSPE-PEG-TREM2, realizing the organic integration and synergy of multiple functions.

[0035] 2. This invention has good modularity and replaceability. By simply replacing the target molecules or target peptides attached to the lipid shell surface, the same basic delivery platform can achieve the ability to target different glial cell subsets or immune receptors (CLEC7A / TREM2) in brain parenchymal lesions, which significantly improves the flexibility, versatility and adaptability of the delivery system to different pathological states.

[0036] 3. This invention effectively solves the problem of low delivery efficiency of brain therapeutic drugs such as GPNMB active peptides and monoclonal antibodies. The introduction of fluorinated lipid shell enhances the ability of the nanosystem to cross the blood-brain barrier and its retention in the brain. At the same time, the modular targeting design promotes the accumulation of drugs near specific glial cell subsets or core lesion areas, thereby improving the local concentration and therapeutic potential.

[0037] 4. This invention achieves responsive drug release to the pathological microenvironment of Alzheimer's disease. The DOPE-TK-mPEG component can promote the dissociation of the drug delivery system structure under high ROS conditions, while the CMA cross-linked core ensures the continuous release of GPNMB active peptides or rokanetumab. The combination of the two helps to maintain an effective drug concentration at the site of inflammation and lesions, prolong the duration of action and improve treatment efficiency.

[0038] 5. This invention exhibits excellent stability and biosafety. The nanosystem has uniform particle size and good dispersibility, and demonstrates good colloidal stability under storage and physiological conditions. Even after replacing the dual-targeting module and loading it with a macromolecular monoclonal antibody, the system can still self-assemble to present a regular and complete spherical vesicle morphology. In vitro experiments show that it has a low hemolysis rate and low cytotoxicity to neurons and glial cells, which ensures the safety of intravenous administration.

[0039] 6. This invention provides a new technical platform and experimental basis for studying the anti-inflammatory and neuroprotective effects of GPNMB active peptides and related macromolecular monoclonal antibody drugs in AD. It is expected to improve the cognitive function of AD model animals by inhibiting neuroinflammation, reshaping the protective phenotype of glial cells and targeting and enriching them across the blood-brain barrier, laying the foundation for the development of nanomedicines for AD treatment that target glial cells or brain parenchymal lesions.

[0040] 7. The overall technical solution of this invention overcomes the shortcomings of traditional liposomes in terms of target specificity, microenvironment responsiveness and sustained drug release, and provides a more efficient, safe and modularly regulated new delivery strategy for the precision treatment of Alzheimer's disease and the study of the central delivery mechanism of related macromolecular drugs, which has important scientific significance and application value. Attached Figure Description

[0041] Figure 1 The NMR spectra of the functional lipid materials related to the lipid nanodelivery systems in Examples 1, 2, and 3 of this invention are used to characterize the structural features of components such as fluorinated lipids FPD, DOPE-TK-mPEG, DSPE-PEG-AS1 or DSPE-PEG-MG1, and CMA. The spectra successfully exhibited the characteristic chemical shifts of the target molecules (such as characteristic peaks of FPD, AS1 peptides, and modified groups), and there were no obvious impurity peaks, indicating that the core functionalized lipid materials and polymers designed in this invention have been successfully prepared, laying the material foundation for the subsequent construction of a multifunctional lipid nanodelivery system.

[0042] Figure 2The UV absorption spectrum, fluorescence spectrum, and GPNMB quantitative standard curve of GPNMB-NBD in Example 4 of this invention are shown. These spectra characterize the fluorescent labeling process, UV-Vis absorption and fluorescence emission characteristics of the GPNMB active peptide, as well as the established quantitative detection method. It is evident that GPNMB-NBD exhibits significant spectral shifts and specific absorption peaks in its absorption and emission spectra, proving that NBD has been successfully covalently labeled onto the GPNMB peptide. Simultaneously, the standard curve shows a certain linearity, indicating that the fluorescence quantitative method of this invention is accurate and reliable, and can be used for the precise determination of peptide encapsulation efficiency, drug loading, and in vitro release kinetics in subsequent systems.

[0043] Figure 3 The results of particle size distribution and storage stability tests of the lipid nanodelivery system in Example 5 of this invention are shown. These tests characterize the particle size distribution, PDI value, Zeta potential, and changes in particle size and dispersibility under different storage conditions. It is evident that the constructed lipid nanodelivery system exhibits uniform particle size (mainly concentrated in the 100-200 nm range) and a low polydispersity index (PDI). During a 7-day observation period in different physiologically simulated media, no significant fluctuations were observed in particle size and PDI, indicating that the nanosystem possesses excellent colloidal stability and anti-serum aggregation ability, meeting the requirements for intravenous injection and long-term in vivo circulation.

[0044] Figure 4 The image shown is a transmission electron microscope (TEM) image of the lipid nanodelivery system in Example 6 of this invention, used to observe the morphology, particle size, and dispersion state of the nanoparticles in the lipid nanodelivery system. It visually confirms that the lipid nanodelivery system exhibits a classic, complete spherical vesicle structure, with good dispersibility and no obvious adhesion or aggregation. The observed physical dimensions are in high agreement with the hydrated particle size measured by DLS.

[0045] Figure 5 The image shows the elemental mapping electron microscopy pattern of the lipid nanodelivery system in Example 7 of this invention, used to characterize the distribution of characteristic elements such as carbon, oxygen, nitrogen, phosphorus, fluorine, and sulfur in the lipid nanodelivery system. It can be seen that various characteristic elements (especially F representing fluorinated lipids, P representing phospholipids, and N and S representing peptides / ligands) achieve a high degree of overlap and uniform distribution throughout the nanoparticle region, further confirming at the atomic level that the multifunctional components have successfully co-assembled to form uniform hybrid nanovesicles.

[0046] Figure 6The X-ray photoelectron spectroscopy (XPS) spectrum of the lipid nanodelivery system in Example 7 of this invention is used to characterize the surface elemental composition and the introduction of functional components into the lipid nanodelivery system. It can be seen that the binding energies of specific chemical bonds (such as CF bonds, PO bonds, SS bonds, etc.) are precisely identified, confirming the chemical composition of the nanoparticle surface. This indicates that the targeting ligand, responsive chemical bonds, and fluorinated lipids are successfully integrated into the liposome surface or bilayer, laying the structural foundation for targeted delivery and microenvironment responsiveness.

[0047] Figure 7 The in vitro simulated release curves of the lipid nanodelivery system in Examples 3 and 8 of this invention are shown to characterize the cumulative release behavior and release kinetics of the GPNMB active peptide under different simulated release media. It is evident that the lipid nanodelivery system exhibits significant ROS-stimulated responsiveness. Under physiological conditions (pH 7.3), release is slow, effectively avoiding premature drug leakage during circulation (extremely low leakage rate); while under simulated pathological microenvironment (acidic pH 6.3 and rich in H2O2), the peptide release rate is significantly accelerated, demonstrating excellent characteristics of on-demand drug release and precise control of target drug concentration.

[0048] Figure 8 The results of the in vitro hemolysis experiment of the lipid nanodelivery system in Example 9 of this invention are shown. This evaluation assesses the hemolysis rate of different concentrations of the lipid nanodelivery system after incubation with erythrocytes, characterizing its blood compatibility. It is evident that even under high concentration (500 μg / mL) conditions, the hemolysis rate of each group of lipid nanodelivery systems is far below the internationally recognized biosafety threshold (red line) of 5%, and no obvious free hemoglobin red color is observed in the supernatant. This indicates that the system has excellent blood compatibility and can be safely used for intravenous administration.

[0049] Figure 9 This paper presents the cytotoxicity analysis results of the lipid nanodelivery system at different concentrations in Example 10 of this invention on neurons, microglia, and astrocytes. This analysis was used to evaluate the effect of the lipid nanodelivery system on the cell viability of these cells at different concentrations and to characterize its cytocompatibility. It is evident that the lipid nanodelivery system of this invention exhibits good cytocompatibility over a wide concentration gradient range. At the working concentration, cell viability is generally maintained above 80%, with only slight cytotoxicity observed at extremely high concentrations (600 μg / mL). This indicates that the carrier material itself is highly safe, providing a guarantee for safe delivery to the nervous system in vivo.

[0050] Figure 10The in vivo and ex vivo organ imaging results of the lipid nanodelivery system in Example 11 of this invention are shown to evaluate the distribution of the lipid nanodelivery system in mice, particularly its brain accumulation capacity and targeted delivery effect. Both in vivo and ex vivo imaging strongly demonstrate that, compared to free probes and unmodified liposomes, the lipid nanodelivery system with specific functional modifications (targeting ligands such as AS1 / MG1 binding to fluorinated lipids) exhibits extremely superior blood-brain barrier (BBB) ​​penetration and brain targeted accumulation effect. High accumulation of fluorescence signals in brain tissue within 24 hours indicates that this lipid nanodelivery system is a highly promising nanodelivery platform for treating central nervous system degenerative diseases.

[0051] Figure 11 The images show transmission electron microscopy (TEM) images of the lipid nanodelivery system in Examples 12 to 14 of this invention, which uses CLEC7A / TREM2 as the dual-targeting molecule and rocanetacil as the delivery drug. These images were used to observe the morphology, particle size, and dispersion of the nanoparticles in the lipid nanodelivery system. They visually confirm that the lipid nanodelivery system exhibits a classic, complete spherical vesicle structure, with good dispersibility and no obvious adhesion or aggregation, meeting the particle size requirements for crossing the blood-brain barrier. Detailed Implementation

[0052] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0053] In the following embodiments:

[0054] The amino acid sequence of the GPNMB active polypeptide is SYKWNFGDNTGLFVSNNHTLNH (SEQ ID NO.01).

[0055] The amino acid sequence of the AS1 targeting peptide is H2N-CLNSSQPSC-OH (SEQ ID NO.02).

[0056] The amino acid sequence of the MG1 targeting peptide is H2N-CHHSSSARC-OH (SEQ ID NO.03).

[0057] The amino acid sequence of the TREM2 targeting peptide LRK is LRKLRLRL (SEQ ID NO.04).

[0058] The CLEC7A targeting molecule βG is a low-molecular-weight β-(1→3)-glucan oligosaccharide with the structure [→3)-β-D-Glcp-(1 ... n , n = 16–25.

[0059] Example 1: Synthesis and NMR Characterization of Fluorinated Lipid FPD

[0060] (1) Experimental objective: This example was used to prepare fluorinated lipids (FPDs) containing 4,4,4-trifluorobutyryl groups, and the successful introduction of the fluorinated groups into the lipid molecules was verified by NMR spectroscopy. The FPDs serve as functional components of the liposome shell, enhancing the hydrophobic stability, membrane interaction capabilities, and brain delivery potential of the liposomes.

[0061] (2) Reagent preparation: Weigh 35.5 mg of 4,4,4-trifluorobutyric acid, 71.9 mg of EDC·HCl, 28.8 mg of NHS, and 3.05 mg of DMAP. Separately, take 139.5 mg of DSPE-PEG2000-NH2 (Y37397-500 mg, purchased from Yuan Ye) as an amino-containing lipid substrate. The reaction solvent is 10 mL of anhydrous dichloromethane. The reactor equipment includes a 50 mL round-bottom flask, a magnetic stir bar, an ice bath, a thermometer, a rotary evaporator, dialysis bags with a molecular weight cutoff of 1000–2000 Da, and a lyophilizer.

[0062] (3) Carboxylic acid pre-activation: Add 4,4,4-trifluorobutyric acid, EDC·HCl, NHS, and DMAP to a round-bottom flask, add 10 mL of anhydrous dichloromethane, and stir magnetically until completely dissolved. Place the reaction system in an ice bath at 0–5 °C and stir for 4 h to activate the carboxyl group of 4,4,4-trifluorobutyric acid to form the corresponding NHS active ester. Maintain an anhydrous environment during the reaction, and monitor the formation of NHS esters using thin-layer chromatography or high-performance liquid chromatography.

[0063] (4) Coupling reaction: 139.5 mg of DSPE-PEG2000-NH2 was pre-dissolved in 2 mL of anhydrous dichloromethane, and then slowly added to the pre-activated mixture. The ice bath was removed, and the system was allowed to rise naturally to room temperature. The mixture was stirred for 20 h under sealed and light-protected conditions. After the reaction was completed, the dichloromethane was removed by rotary evaporation under reduced pressure in a 30 °C water bath to obtain a crude product containing fluorinated lipids.

[0064] The structural formula of this fluorinated lipid is as follows:

[0065] .

[0066] (5) Purification and drying: The crude product was dissolved in 5–10 mL of ultrapure water and transferred to a dialysis bag with a molecular weight cutoff of 1000–2000 Da. Dialysis was performed with ultrapure water for 2 days, changing the water every 6–8 hours to remove EDC urea byproducts, free NHS, DMAP, and unreacted small molecules. After dialysis, the dialysate was pre-frozen at -80 °C for 2 hours and then freeze-dried in a lyophilizer for 24–48 hours to obtain a white or pale yellow FPD powder. The product was stored at -20 °C under nitrogen protection and in the dark.

[0067] (6) Results Analysis: The measurement results are shown in [the table below]. Figure 1 The NMR spectrum revealed methylene signals in the PEG backbone, signals from the lipid alkyl chain, and characteristic signals related to the trifluorobutyryl group. The fluorine spectrum also showed a CF3-related signal, indicating that the 4,4,4-trifluorobutyric acid fragment was successfully coupled to the DSPE-PEG molecule. These results confirm the successful preparation of FPD functional lipids suitable for constructing fluorinated liposome shells in this embodiment.

[0068] Example 2: Preparation of DSPE-PEG-AS1 and DSPE-PEG-MG1 targeted lipids

[0069] (1) Experimental objective: This embodiment is used to prepare replaceable targeting lipids DSPE-PEG-AS1 and DSPE-PEG-MG1. By linking the AS1 targeting peptide or the MG1 targeting peptide to the end of DSPE-PEG, the same basic liposome platform can obtain astrocyte targeting capability or M1 microglia targeting capability, thereby demonstrating the "programmable" or "modular" characteristics of the delivery system.

[0070] (2) Preparation of DSPE-PEG-AS1: The purified AS1 peptide was weighed and dissolved in anhydrous DMF, and DSPE-PEG-NHS was dissolved in anhydrous DMF. The two were mixed in a peptide:DSPE-PEG-NHS molar ratio of 1:2. The pH of the reaction system was adjusted to 8.0–8.5 with triethylamine, and the reaction was carried out with gentle stirring at 25 °C for 24–120 h to allow the terminal amino group of the AS1 targeting peptide to undergo an amidation reaction with DSPE-PEG-NHS. After the reaction was completed, the mixture was transferred to a MWCO 2500–3500 Da dialysis bag and dialyzed against ultrapure water for 48 h to remove unreacted peptides and small molecule byproducts. Then, it was lyophilized to obtain DSPE-PEG-AS1.

[0071] (3) Preparation of DSPE-PEG-MG1: Using the same method as DSPE-PEG-AS1, the MG1 targeting peptide was reacted with DSPE-PEG-NHS to prepare DSPE-PEG-MG1. If the MG1 targeting peptide or AS1 targeting peptide used contains a reactive thiol group, it can also be prepared by maleimide-thiol coupling of DSPE-PEG-Mal with the MG1 targeting peptide. The specific operation is as follows: DSPE-PEG-Mal was dissolved in DMSO, and the MG1 targeting peptide dissolved in PBS was added. The reaction was carried out at room temperature for 4 h under nitrogen protection. After the reaction, the mixture was dialyzed for 2 days and then lyophilized to obtain the corresponding DSPE-PEG-MG1.

[0072] The structural formulas of the DSPE-PEG-AS1 and DSPE-PEG-MG1 obtained above are shown below:

[0073] .

[0074] (4) Results analysis: The measurement results are shown in Figure 1 The NMR spectra of the targeted lipids showed signals from the PEG backbone and lipid chains. Combined with the selective enrichment of different glial cell subsets by the AS1 and MG1 groups in subsequent liposome cellular uptake experiments, this indicates that DSPE-PEG-AS1 and DSPE-PEG-MG1 can be used as alternative targeting modules in lipid nanodelivery systems.

[0075] Example 3: Preparation of chitosan-methacrylate (CMA) core and immobilization of GPNMB active peptides

[0076] (1) Experimental objective: This embodiment is used to prepare a chitosan-methacrylate cross-linked core capable of encapsulating and sustaining the release of GPNMB active peptides. This core serves as a drug reservoir for a lipid nanodelivery system, which can immobilize GPNMB active peptides through a cross-linked network and achieve sustained release in the extracellular microenvironment of glial cells.

[0077] (2) Preparation of CMA: Chitosan with a degree of deacetylation of approximately 85% and an average molecular weight of approximately 15,000 Da was dissolved in 2% acetic acid solution to prepare a 3 wt.% chitosan solution. After stirring at room temperature overnight until the solution became clear, approximately 4 mL of methacrylic anhydride was slowly added dropwise, and the reaction was carried out at 60 °C for 6 h. Subsequently, the temperature was lowered to room temperature and stirring was continued for 24 h. After the reaction was completed, saturated NaHCO3 solution was added to neutralize the pH to near neutral. The reaction solution was placed in a MWCO 3 kDa dialysis bag and dialyzed against distilled water for 4 days, changing the water 3 times a day to remove unreacted methacrylic anhydride, acetic acid, and small molecule byproducts. After dialysis, CMA powder was obtained by lyophilization.

[0078] (3) CMA structure confirmation: adopted 1 The structure of CMA was characterized by ¹H NMR. Using D₂O as solvent, the characteristic peak of the olefinic hydrogen of the methpropylene group appeared at approximately 5.5–6.5 ppm, and the methyl peak appeared at approximately 1.9 ppm. The degree of substitution could be calculated by the integral ratio of the peak area of ​​the methpropylene group to the peak area of ​​the chitosan backbone, proving that the methacrylate group had been successfully grafted onto the chitosan molecular chain.

[0079] (4) Immobilization of GPNMB active peptides: 30 mg of CMA was dissolved in 6 mL of a mixed solution of 0.9% NaCl and 0.01 M HCl and stirred until homogeneous; the photoinitiator Irgacure 2959 and the GPNMB active peptide solution were added to make the GPNMB peptide concentration approximately 0.1 mg / mL, and the mixture was vortexed for 5 min. Subsequently, in the liposome hydration and UV crosslinking steps, the CMA methacrylate groups were induced to polymerize by 365 nm ultraviolet light irradiation to form crosslinked nanocores, thereby achieving the immobilization and sustained release of GPNMB active peptides.

[0080] (5) Results analysis: The measurement results are shown in the figure. Figure 1 and Figure 7 NMR spectroscopy confirmed the successful construction of the CMA structure, and in vitro release simulation results further demonstrated that the cross-linked CMA core can delay the diffusion of the active GPNMB peptide, enabling the continuous release of GPNMB from the delivery system.

[0081] Example 4: Synthesis, Spectroscopic Characterization and Quantitative Curve Establishment of GPNMB-NBD

[0082] (1) Experimental objective: This embodiment is used to prepare fluorescently labeled GPNMB-NBD and establish a quantitative curve of GPNMB-NBD, so as to provide a quantitative method for subsequent experiments such as liposome encapsulation efficiency, release amount, cell uptake and tissue distribution.

[0083] (2) Synthesis of GPNMB-NBD: Weigh 8 mg of GPNMB active peptide and place it in a 1.5 mL EP tube or small reaction flask. Add 0.5 mL of anhydrous DMF, vortex and sonicate appropriately to dissolve completely. Separately, take 2 mg of NBD-Cl and dissolve it in 0.5 mL of anhydrous DMF to obtain a yellow NBD-Cl solution. Slowly add the NBD-Cl solution dropwise to the GPNMB peptide solution, vortexing while adding, so that the total reaction volume is about 1 mL. Then add 6 μL of triethylamine in 2–3 portions, and check the pH of the reaction solution with pH paper to maintain it at 8–9. Wrap the reaction tube with aluminum foil to protect it from light, and react at 20–25 °C with stirring or shaking for 2–4 h.

[0084] (3) Purification and preservation: After the reaction is complete, place the reaction tube in an ice bath to cool for 5 min, and slowly add 5–10 mL of pre-cooled anhydrous diethyl ether while vortexing. A yellow precipitate will be observed to precipitate. Continue stirring in the ice bath for 10–15 min, then centrifuge at 4 ℃ and 12000 rpm for 10 min and discard the supernatant. Wash the precipitate twice with 5 mL of cold diethyl ether, and then dry it under vacuum or nitrogen for 30–60 min to obtain the crude yellow NBD-GPNMB polypeptide. The obtained product is immediately dissolved in a small amount of DMSO or DMF to prepare a 1–10 mM stock solution, which is then aliquoted in the dark and stored at -80 ℃.

[0085] (4) UV and fluorescence spectroscopy detection: The purified GPNMB-NBD sample was subjected to UV-Vis absorption spectroscopy scanning in the range of 200–600 nm to observe the absorption peak of the GPNMB peptide itself and the characteristic absorption peak that appeared after NBD labeling. Another sample was subjected to fluorescence spectroscopy scanning with the excitation wavelength set to 460 nm and the emission scanning range of 500–600 nm. The characteristic fluorescence signal of NBD in the range of 520–540 nm was observed in particular.

[0086] (5) Establishment of quantitative curves: Purified GPNMB-NBD was used as a standard and diluted with 5 mM HEPES buffer to a series of concentrations of 0, 5, 10, 20, 40, 60, 80, and 100 μg / mL. 20 μL of each concentration was placed in a 96-well black plate, and buffer was added to a final volume of 100 μL. Detection was performed using a fluorescence microplate reader with an excitation wavelength of 460 nm and an emission wavelength of 530 nm. Three replicates were set for each concentration. After subtracting the blank, a linear fit was performed with concentration as the x-axis and fluorescence intensity as the y-axis to obtain the standard curve equation.

[0087] (6) Results Analysis: The measurement results are shown in [the table below]. Figure 2 GPNMB-NBD exhibited characteristic signals of NBD labeling in both UV and fluorescence detection, and the fluorescence intensity increased with increasing GPNMB-NBD concentration, indicating that NBD was successfully labeled onto the active GPNMB peptide. The established quantitative curve can be used for subsequent detection of liposome drug loading and release.

[0088] Example 5: Preparation, particle size and stability determination of GPNMB active peptide lipid nanodelivery system

[0089] (1) Experimental objective: This embodiment is used to prepare a modular fluorinated lipid nanodelivery system with GPNMB active peptide as the therapeutic molecule, CMA cross-linked nanocore as the drug reservoir, FPD / DSPC / DOPE-TK-mPEG as the lipid shell, and DSPE-PEG-AS1 or DSPE-PEG-MG1 as the targeting module.

[0090] (2) Preparation of lipid films: Taking a total lipid content of 20 mg and an FPD content of 1% as an example, FPD (0.2 mg), DSPC (9.88 mg), cholesterol (3.34 mg), DOPE-TK-mPEG (2.72 mg), and DSPE-PEG-AS1 / MG1 (3.86 mg) were weighed and dissolved in 10 mL of chloroform, and the lipid films were formed by rotary evaporation (40 ℃, 150 rpm, 1 h). In the liposome systems with 5% and 10% FPD content, only the FPD content was changed. The FPD content in the 5% system was 1.04 mg, and the FPD content in the 10% system was 2.2 mg. The contents of other components remained unchanged, and the total lipid content increased with the increase of FPD content. AS1-targeted liposomes and MG1-targeted liposomes were prepared using DSPE-PEG-AS1 and DSPE-PEG-MG1, respectively; non-targeted liposomes were prepared using DSPE-PEG or lipid components without targeting peptides.

[0091] The aforementioned DOPE-TK-mPEG is a ROS-responsive phospholipid-polyethylene glycol derivative, and its structural formula is as follows: .

[0092] (3) Preparation of GPNMB-CMA hydration solution: Weigh 30 mg of CMA and dissolve it in 6 mL of a mixed solution of 0.9% NaCl and 0.01 M HCl, and stir until homogeneous. Add Irgacure 2959 and GPNMB active peptide solution to make the GPNMB peptide concentration 0.1 mg / mL, vortex for 5 min to obtain a hydration solution containing GPNMB and CMA.

[0093] (4) Liposome hydration, extrusion, and cross-linking: GPNMB-CMA hydration solution was added to a dried lipid film and hydrated by gentle shaking at 37 °C for 1 h to form multilayer liposomes. The resulting dispersion was then extruded through a 0.4 μm polycarbonate membrane three times and a 0.2 μm polycarbonate membrane five times to obtain liposomes with more uniform particle size. After extrusion, the liposomes were ultracentrifuged at 4 °C and 300,000 g for 1 h, the supernatant was discarded, and the liposomes were resuspended in 8 mL of PBS. 4 mL of the resuspended liposomes were bubbled with nitrogen for 5 min to remove oxygen and then irradiated under 365 nm UV light for 30 min (using a 1 min irradiation followed by a 30 s intermittent irradiation to prevent overheating) to induce CMA cross-linking and form a GPNMB fixed core.

[0094] (5) Purification: After cross-linking, the sample was centrifuged at 4 °C and 10,000 g for 10 min to remove unpolymerized particles and large particles. The supernatant was concentrated to about 1–2 mL using an MWCO 30 kDa ultrafiltration tube and washed 2–3 times with PBS to remove free GPNMB, excess small molecules and uninserted lipids. Finally, AS1-GPNMB-Lipo, MG1-GPNMB-Lipo and non-targeted GPNMB-Lipo were obtained.

[0095] (6) Particle size and stability testing: Take an appropriate amount of liposome dispersion, dilute it with PBS to a suitable concentration, and use a dynamic light scattering instrument to detect particle size, PDI and Zeta potential. In the stability test, the sample is stored at 4 ℃ or in a simulated physiological medium, and the changes in particle size and PDI are detected at set time points to evaluate storage stability and colloidal stability.

[0096] (7) Results Analysis: The measurement results are shown in the figure. Figure 3 The lipid nanodelivery system exhibits a concentrated particle size distribution and good dispersibility. No significant abnormal increase in particle size was observed during storage or incubation, indicating that the delivery system possesses good nanoscale uniformity and stability, meeting the requirements for subsequent in vitro release, cell experiments, and in vivo imaging experiments.

[0097] Example 6: Transmission electron microscopy morphology observation of a lipid nanodelivery system

[0098] (1) Experimental objective: This example is used to observe the microstructure of the GPNMB active polypeptide lipid nanodelivery system by transmission electron microscopy, and to further verify that the nanoparticles obtained in Example 5 have regular liposomes or core-shell nanostructures.

[0099] (2) Sample preparation: Take an appropriate amount of freshly prepared lipid nanodelivery system, dilute it to a low concentration with ultrapure water or PBS, and add 5–10 μL to a carbon membrane copper grid. Let it stand for 1–3 min to allow the nanoparticles to fully adsorb. After gently absorbing the excess liquid with filter paper, add 1%–2% phosphotungstic acid or uranium acetate solution for negative staining. Let it stand at room temperature for 30–60 s, and then absorb the staining solution. After the copper grid is dried at room temperature in the dark, observe it under a transmission electron microscope.

[0100] (3) Observation method: The morphology of liposomes was observed using a transmission electron microscope under a suitable accelerating voltage. Representative images were taken from multiple fields of view at random, and particle diameter and morphological characteristics were measured using image analysis software.

[0101] (4) Results analysis: The measurement results are shown in Figure 4Transmission electron microscopy (TEM) results showed that the lipid nanodelivery system exhibited a dispersed nanoparticle morphology with relatively regular particle shapes and no obvious large-area aggregation. This result corroborates the dynamic light scattering particle size distribution, indicating that the delivery system described in this invention can form stable nanostructures.

[0102] Example 7: Elemental mapping and XPS characterization of a lipid nanodelivery system

[0103] (1) Experimental objective: This embodiment is used to verify, by element mapping and X-ray photoelectron spectroscopy, that functional components such as fluorinated lipids, phospholipids, ROS-responsive DOPE-TK-mPEG and targeted peptides have been successfully introduced into the lipid nanodelivery system.

[0104] (2) Elemental mapping detection: Liposome samples were dropped onto an electron microscope grid, dried, and then analyzed by scanning transmission electron microscopy or transmission electron microscopy combined with energy dispersive spectroscopy. The elemental distribution maps of carbon, oxygen, nitrogen, phosphorus, fluorine, and sulfur in the same nanoparticle region were collected. Among them, fluorine was used to indicate the introduction of fluorinated lipids in FPD, phosphorus was used to indicate the phospholipid shell structure, nitrogen could be derived from GPNMB peptides, targeting peptides, or phospholipid head groups, and sulfur could be used to indicate the thioketal linker arm in DOPE-TK-mPEG.

[0105] (3) XPS detection: The lyophilized liposome powder was evenly spread on the sample stage, and the full spectrum and high-resolution spectrum were detected by X-ray photoelectron spectroscopy. By analyzing the signals such as C 1s, O 1s, N 1s, P 2p, F 1s and S 2p, the elemental composition and chemical bond state of the liposome surface were determined.

[0106] (4) Results Analysis: The element mapping results are shown in [the table below]. Figure 5 See XPS results Figure 6 Mapping spectroscopy revealed a good distribution of characteristic elements within the nanoparticles; XPS spectroscopy detected elemental signals associated with fluorinated lipids, phospholipids, and sulfur-containing responsive linkers. These results indicate that FPD, the phospholipid shell, DOPE-TK-mPEG, and the targeting module are collectively constructed within the lipid nanodelivery system.

[0107] Example 8: In vitro simulated release of GPNMB active peptides in a lipid nanodelivery system

[0108] (1) Experimental objective: This example is used to evaluate the release behavior of GPNMB active peptide from the lipid nanodelivery system, verify the sustained release effect of CMA cross-linked core and the ability of DOPE-TK-mPEG to promote drug release under pathological microenvironment simulation conditions.

[0109] (2) Establishment of the release system: 1 mL of the GPNMB active peptide lipid nanodelivery system was placed in a MWCO 3.5 kDa dialysis bag, sealed, and then placed in 500 mL of release medium. The release medium can be set to PBS pH 7.4 to simulate the physiological environment, or to pH 5.0 buffer and ROS containing hydrogen peroxide to simulate the pathological microenvironment. The entire release system was placed in a constant temperature shaker at 37 ℃ and a rotation speed of 100 rpm.

[0110] (3) Sampling and detection: Samples were taken at 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 120 h, and 168 h. 3 mL of sample was taken from the release medium outside the dialysis bag each time, and an equal volume of fresh release medium was added simultaneously to maintain the stability of the release system volume and osmotic pressure. The content of GPNMB active peptides in the release solution at each time point was determined using the GPNMB-NBD fluorescence quantitative curve established in Example 4.

[0111] (4) Calculation of cumulative release rate:

[0112] Calculate the cumulative release rate using the following formula:

[0113] Cumulative release rate (%) = [V0 × C] n + V S ×ΣC i [ / M ×100%]

[0114] Where V0 is the total volume of the release medium, V S C represents the volume of each sample taken. n C represents the drug concentration in the release medium during the nth sampling. i denoted as the drug concentration in the release medium during the i-th sampling, and M as the total amount of GPNMB active peptides in the lipid nanodelivery system within the dialysis bag.

[0115] (5) Results analysis: The measurement results are shown in the figure. Figure 7 The lipid nanodelivery system exhibited sustained release characteristics in a simulated physiological environment, indicating that the CMA cross-linked core can delay the rapid diffusion of GPNMB active peptides. In acidic or ROS simulated microenvironments, the release rate increased, indicating that the DOPE-TK-mPEG and lipid shell structure can respond to changes in the pathological microenvironment and promote the release of GPNMB active peptides.

[0116] Example 9: In vitro hemolytic safety evaluation of lipid nanodelivery system

[0117] (1) Experimental objective: This embodiment is used to evaluate the hemolysis of erythrocytes after different concentrations of lipid nanodelivery system come into contact with them, so as to determine the blood compatibility and preliminary biosafety of the delivery system.

[0118] (2) Preparation of red blood cell suspension: Collect fresh whole blood from healthy mice or rats, place it in an anticoagulant tube, and collect red blood cells by low-speed centrifugation. Wash the red blood cells repeatedly with PBS 3–5 times until the supernatant is clear and there is no obvious hemoglobin color. Prepare a 2% red blood cell suspension with PBS after washing for later use.

[0119] (3) Experimental groups: A PBS negative control group, an ultrapure water or 0.1% Triton X-100 positive control group, and groups treated with different concentrations of lipid nanodelivery systems were set up. An equal volume of red blood cell suspension was mixed with each group of samples and incubated at 37 ℃ for 2 h. After incubation, the mixture was centrifuged, the supernatant was collected, and the absorbance was measured at 540 nm.

[0120] (4) Calculation of hemolysis rate: Hemolysis rate (%) = [OD sample – OD negative control] / [OD positive control – OD negative control] × 100%. If the color of the supernatant of the sample group is close to that of the PBS negative control and the hemolysis rate is at a low level, it indicates that the lipid nanodelivery system has good blood compatibility at this concentration.

[0121] (5) Results analysis: The measurement results are shown in the figure. Figure 8 Different concentrations of the lipid nanodelivery system did not cause significant red blood cell rupture after incubation with red blood cells, and the hemolysis rate was low, indicating that the lipid nanodelivery system described in this invention has good in vitro blood compatibility, providing a basis for the safety of subsequent in vivo drug administration.

[0122] Example 10: Cytotoxicity evaluation of lipid nanodelivery system on neurons, microglia, and astrocytes

[0123] (1) Experimental objective: This embodiment is used to detect the effects of different concentrations of lipid nanodelivery systems on the cell viability of related brain cells (including neurons, microglia and astrocytes), thereby evaluating their cell compatibility.

[0124] (2) Cell preparation: Primary astrocytes, primary microglia, and primary neurons can be used as experimental cells. Alternatively, C8-D1A astrocytes, BV2 microglia, and HT22 or N2A neuron-like cells can be used as alternative models. Cells are seeded in 96-well plates, with 100 μL of cell suspension added to each well. The plates are pre-cultured at 37 ℃ in a 5% CO2 incubator for 24 h to allow the cells to adhere and recover.

[0125] (3) Drug administration: The concentration gradients of the lipid nanodelivery system were set to 0, 1, 10, 50, 100, 250, and 500 μg / mL. 10 μL of the test sample at different concentrations was added to each well, and incubation was continued for 24 h. To compare the safety of different targeting modules, treatment groups of GPNMB-Lipo without targeting, AS1-GPNMB-Lipo, and MG1-GPNMB-Lipo were set up respectively.

[0126] (4) CCK-8 assay: After drug administration and incubation, add 10 μL of CCK-8 solution to each well, avoiding the generation of air bubbles, and continue incubation in an incubator for 1–4 h. Measure the absorbance at 450 nm using a microplate reader. Calculate cell viability using the formula:

[0127] Cell viability (%) = [A with added drug – A blank] / [A0 with added drug – A blank] × 100%

[0128] Wherein, Adrug-added is the absorbance of the pore containing cells, CCK-8 and the drug; Ablank is the absorbance of the pore containing culture medium and CCK-8 but without cells; and A0 is the absorbance of the pore containing cells and CCK-8 but without the drug.

[0129] (5) Results analysis: The measurement results are shown in the figure. Figure 9 After treatment with lipid nanodelivery systems at different concentrations, neurons, microglia, and astrocytes all maintained good cell viability, indicating that the delivery system described in this invention has good cell compatibility within the experimental concentration range and did not exhibit significant cytotoxicity.

[0130] Example 11: In vivo imaging and brain delivery evaluation of lipid nanodelivery system

[0131] (1) Experimental objective: This example is used to evaluate the distribution of lipid nanodelivery systems in mice, especially their brain enrichment and brain-targeted delivery capabilities.

[0132] (2) Fluorescent labeling: GPNMB peptides or lipid nanodelivery systems were labeled with near-infrared fluorescent dyes DiR or Cy7. Taking DiR as an example, 5 mg of DiR was prepared into a 1 mg / mL stock solution with anhydrous ethanol and aliquoted and stored in the dark. Before use, 100 μL of the 1 mg / mL DiR stock solution was added to 900 μL of PBS to prepare a 1 mL working solution of 0.1 mg / mL DiR. For liposome labeling, DiR was added to the liposome system at the same concentration so that the final working solution contained approximately 0.1 mg / mL DiR.

[0133] (3) Animal grouping and administration: Mice weighing 20–25 g were selected for in vivo distribution experiments. The experimental groups included: free DiR or Cy7-GPNMB group, GPNMB-DiR-Lipo group, AS1-GPNMB-DiR-Lipo group, and MG1-GPNMB-DiR-Lipo group. The administration volume per mouse was 100 μL, and the DiR dose was approximately 5–10 μg / mouse. The administration route could be tail vein injection; for brain delivery evaluation, intravenous infusion could also be used for comparison.

[0134] (4) In vivo imaging: Small animal in vivo imaging was performed at 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after drug administration. Mice were anesthetized with isoflurane before imaging and placed in the in vivo imaging system to acquire whole-body fluorescence images. The DiR channel was excited at approximately 745 nm and emitted at 800 nm. Images of each group were acquired using the same exposure parameters, and fluorescence intensity was quantified using the brain region as the ROI. If necessary, mice were sacrificed after the last imaging, and major organs such as the brain, liver, spleen, lungs, kidneys and heart were isolated for in vitro fluorescence imaging.

[0135] (5) Results analysis: The measurement results are shown in the figure. Figure 10 Compared to free fluorescent dyes or free GPNMB, the lipid nanodelivery system exhibited more pronounced tissue retention and brain fluorescence signals in vivo. The AS1-GPNMB-DiR-Lipo and MG1-GPNMB-DiR-Lipo groups showed more significant fluorescence enrichment in the brain region, indicating that targeted peptide modification and the fluorinated lipid shell contribute to improving the brain delivery capability of the GPNMB lipid nanodelivery system. These results provide in vivo distribution evidence for the subsequent application of this system in glial cell-targeted delivery in Alzheimer's disease models.

[0136] Example 12: Preparation of DSPE-PEG-CLEC7A and DSPE-PEG-TREM2 targeting molecules

[0137] (1) Preparation of DSPE-PEG-CLEC7A targeting molecules: 64.8 mg of CLEC7A targeting oligosaccharide with a reducing end was weighed; based on the average molecular weight of the oligosaccharide of 3240 Da, its amount of substance was 20 μmol. The oligosaccharide was dissolved in 8.0 mL of 50 mmol / L borate buffer at pH 8.5 to obtain an oligosaccharide solution. 27.9 mg of DSPE-PEG2000-NH2, i.e., 10 μmol, was weighed and added to 1.0 mL of chloroform / methanol mixed solvent, wherein the volume ratio of chloroform to methanol was 85:15, to ensure complete dissolution. Subsequently, the organic solvent was removed under reduced pressure at 30-40℃ to form a uniform lipid film. 1.0 mL of the above borate buffer was added to the obtained lipid film, and the mixture was ultrasonically dispersed in a water bath at 40℃ for 5-10 min to obtain a DSPE-PEG2000-NH2 dispersion. The DSPE-PEG2000-NH2 dispersion was added to the oligosaccharide solution to make the total volume of the reaction system 10.0 mL, wherein the molar ratio of oligosaccharide to DSPE-PEG2000-NH2 was 2:1. 31.4 mg (0.50 mmol) of NaBH3CN was weighed and added in portions to the reaction system to make the final concentration of NaBH3CN 50 mmol / L. The reaction solution was placed at a constant temperature of 37℃ and reacted with light-protected shaking at 300 r / min for 72 h. During the reaction, the reducing end of the oligosaccharide formed an imine intermediate with the terminal primary amino group of DSPE-PEG2000-NH2, which was then reduced to a stable secondary amine bond under the action of NaBH3CN. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed against deionized water at 4℃ for 48 h to remove unreacted oligosaccharides, borates, NaBH3CN, and other small molecules. After dialysis, the fluid inside the bag was freeze-dried to obtain the DSPE-PEG-CLEC7A targeting molecule.

[0138] (2) Preparation of DSPE-PEG-TREM2 targeting molecules: DSPE-PEG-NHS was dissolved in dry DMF (5 mg / mL). Peptide LRKLRLRL was dissolved in PBS (pH 7.2-8.0) (5 mg / mL), avoiding high pH to reduce side reactions. The peptide:DSPE-PEG-NHS was mixed at a molar ratio of 1:2 and stirred at room temperature or 4 ℃ for 2-4 h (or overnight). During the reaction, the NHS ester formed a stable amide bond with the peptide amino group. Dialysis was performed using a dialysis bag (MWCO 2 kDa) at 4 ℃ for about 24 h, followed by purification using preparative reversed-phase high-performance liquid chromatography to completely remove unreacted substances and small molecules.

[0139] (3) Preparation of DSPE-PEG-CLEC7A / TREM2 dual low-density targeting module: DSPE-PEG-CLEC7A targeting lipid and DSPE-PEG-TREM2 targeting lipid were mixed at a molar ratio of 1:1; the total molar percentage of both in the total lipid was 0.5%, which was used to construct a low-density dual-targeting lipid shell.

[0140] Example 13: Immobilization of rokanetumab drug molecules

[0141] (1) Experimental objective: This embodiment is used to prepare a chitosan-methacrylate cross-linked core capable of encapsulating and sustaining the release of rokanaimab drug molecules. This core serves as a drug reservoir for a lipid nanodelivery system, which can immobilize rokanaimab drug molecules through a cross-linked network and achieve sustained release in the extracellular microenvironment of glial cells.

[0142] (2) Preparation of CMA: The preparation method is the same as that described in Example 3.

[0143] (3) Drug molecule immobilization: 30 mg of CMA was dissolved in 6 mL of a mixed solution of 0.9% NaCl and 0.01 M HCl and stirred until homogeneous; the photoinitiator Irgacure 2959 and the rokanetumab drug molecule solution were added to make the drug molecule concentration 0.1 mg / mL, and the mixture was vortexed for 5 min. Subsequently, in the liposome hydration and UV crosslinking steps, the CMA methacrylate groups were induced to polymerize by 365 nm ultraviolet light irradiation to form a crosslinked nanocore, thereby achieving the immobilization and sustained release of rokanetumab drug molecules.

[0144] Example 14: Preparation and Transmission Electron Microscopy Morphology Observation of a Rocanacab Lipid Nanodelivery System

[0145] (1) Experimental objective: This embodiment is used to prepare a modular fluorinated lipid nanodelivery system with rokanetumab as the therapeutic molecule, CMA cross-linked nanocore as the drug reservoir, FPD / DSPC / DOPE-TK-mPEG as the lipid shell, and DSPE-PEG-CLEC7A and DSPE-PEG-TREM2 as the targeting modules.

[0146] (2) Preparation of lipid film: Taking 20 mg of total lipids and 1% FPD content as an example, FPD (0.2 mg), DSPC (9.88 mg), cholesterol (3.34 mg), DOPE-TK-mPEG (2.72 mg), DSPE-PEG-CLEC7A (1.93 mg) and DSPE-PEG-TREM2 (1.93 mg) were weighed and dissolved in 10 mL of chloroform, and the lipid film was formed by rotary evaporation (40 °C, 150 rpm, 1 h).

[0147] (3) Preparation of rokanetumab drug molecule-CMA hydration solution: Weigh 30 mg of CMA and dissolve it in 6 mL of a mixed solution of 0.9% NaCl and 0.01 M HCl, and stir until homogeneous. Add Irgacure 2959 and rokanetumab drug molecule solution to make the drug molecule solution concentration 0.1 mg / mL, vortex for 5 min to obtain a hydration solution containing rokanetumab drug molecule and CMA.

[0148] (4) Liposome hydration, extrusion, and cross-linking: The rokanetumab drug molecule-CMA hydration solution was added to a dried lipid film and hydrated by gentle shaking at 37 °C for 1 h to form multilayer liposomes. Subsequently, the resulting dispersion was extruded through a 0.4 μm polycarbonate membrane three times and a 0.2 μm polycarbonate membrane five times to obtain liposomes with more uniform particle size. After extrusion, the liposomes were ultracentrifuged at 4 °C and 300,000 g for 1 h, the supernatant was discarded, and the liposomes were resuspended in 8 mL of PBS. 4 mL of the resuspended liposomes were bubbled with nitrogen for 5 min to remove oxygen and then irradiated under 365 nm UV light for 30 min (using a 1 min irradiation followed by a 30 s interval to prevent overheating) to induce CMA cross-linking and form the rokanetumab drug molecule immobilized core.

[0149] (5) Purification: After cross-linking, the sample was centrifuged at 4 °C and 10,000 g for 10 min to remove unpolymerized and large particles. The supernatant was concentrated to 1–2 mL using an MWCO 30 kDa ultrafiltration tube and washed 2–3 times with PBS to remove free drug molecules, excess small molecules and uninserted lipids. Finally, programmable targeted liposomes were obtained.

[0150] (6) Sample preparation for transmission electron microscopy: Take an appropriate amount of freshly prepared lipid nanodelivery system, dilute it to a low concentration with ultrapure water or PBS, and add 5–10 μL to a carbon film copper grid. Let it stand for 1–3 min to allow the nanoparticles to fully adsorb. After gently absorbing the excess liquid with filter paper, add 1%–2% phosphotungstic acid or uranium acetate solution for negative staining. Let it stand at room temperature for 30–60 s, and then absorb the staining solution. After the copper grid is dried at room temperature in the dark, observe it under a transmission electron microscope.

[0151] (7) Observation method: The morphology of liposomes was observed using a transmission electron microscope under a suitable accelerating voltage. Representative images were taken from multiple fields of view at random, and the particle diameter and morphological characteristics were measured using image analysis software.

[0152] (8) Results Analysis: The measurement results are shown in [the table below]. Figure 11 Transmission electron microscopy (TEM) results showed that the lipid nanodelivery system exhibited a dispersed nanoparticle morphology with relatively regular particle shapes and no obvious large-area aggregation. This result corroborates the dynamic light scattering particle size distribution, indicating that the delivery system described in this invention can form stable nanostructures.

[0153] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A programmable fluorinated lipid nanodelivery system, characterized in that: It has a core-shell structure, including a drug-carrying core and a lipid shell covering the outside of the drug-carrying core; The drug-loaded core comprises a chitosan-methacrylate crosslinked network and drug molecules loaded therein; The lipid shell includes fluorinated lipids, 1,2-distearate-sn-glycerol-3-phosphocholine, cholesterol, DOPE-TK-mPEG, and DSPE-PEG-targeting molecules; The fluorinated lipid was obtained by coupling 4,4,4-trifluorobutyric acid with DSPE-PEG2000-NH2.

2. The programmable fluorinated lipid nanodelivery system as described in claim 1, characterized in that: The drug molecule is a GPNMB active polypeptide or rokanetumab; wherein the amino acid sequence of the GPNMB active polypeptide is shown in SEQ ID NO.

01.

3. The programmable fluorinated lipid nanodelivery system as described in claim 1, characterized in that: The chitosan-methacrylate crosslinking network is formed by grafting chitosan with a degree of deacetylation of 85% and an average molecular weight of 15000 Da with methacrylic anhydride, followed by in-situ crosslinking and curing under ultraviolet light with the participation of a photoinitiator.

4. The programmable fluorinated lipid nanodelivery system as described in claim 1, characterized in that: Its average particle size is 50~300 nm.

5. The programmable fluorinated lipid nanodelivery system as described in claim 1, characterized in that: The lipid shell comprises components in the following mass ratio: fluorinated lipids : 1,2-distearate-sn-glycerol-3-phosphocholine : cholesterol : DOPE-TK-mPEG : DSPE-PEG-targeting molecule = 0.2-2.5 : 8.0-11.5 : 2.5-4.5 : 2.0-3.5 : 3.0-4.5, and when the DSPE-PEG-targeting molecule includes more than one, the mass ratio is based on the total mass of the DSPE-PEG-targeting molecules.

6. The programmable fluorinated lipid nanodelivery system as described in claim 1, characterized in that: The DOPE-TK-mPEG in the lipid shell contains thioketal bonds, which are used to promote the structural dissociation of the lipid nanodelivery system or drug release in a reactive oxygen species microenvironment.

7. The programmable fluorinated lipid nanodelivery system as described in claim 1, characterized in that: The DSPE-PEG-targeting molecule is either DSPE-PEG-AS1 or DSPE-PEG-MG1; wherein, AS1 is an astrocyte-targeting peptide with the amino acid sequence shown in SEQ ID NO.02; and MG1 is an M1 microglia-targeting peptide with the amino acid sequence shown in SEQ ID NO.03; by replacing AS1 or MG1 in the DSPE-PEG-targeting molecule, active targeted delivery to astrocytes or M1 microglia can be achieved.

8. The programmable fluorinated lipid nanodelivery system as described in claim 1, characterized in that: The DSPE-PEG-targeting molecules include DSPE-PEG-CLEC7A and DSPE-PEG-TREM2, which together construct a dual-targeting lipid shell for brain parenchymal lesions. The targeting molecule in DSPE-PEG-CLEC7A is a low-molecular-weight β-(1→3)-glucan oligosaccharide with the structure [→3)-β-D-Glcp-(1 ... n , n = 16–25, the target molecule in the DSPE-PEG-TREM2 is the LRK peptide with the amino acid sequence shown in SEQ ID NO.

04.

9. A method for preparing a programmable fluorinated lipid nanodelivery system according to any one of claims 1 to 8, characterized in that: Includes the following steps: (1) The fluorinated lipid, 1,2-distearate-sn-glycerol-3-phosphocholine, cholesterol, DOPE-TK-mPEG and DSPE-PEG-targeting molecules are dissolved in chloroform, the solvent is removed by rotary evaporation, and a lipid film is assembled on the inner wall of the container. (2) Chitosan-methacrylate is dissolved in a mixed solution containing the drug molecule and photoinitiator, and the mixture is vortexed and mixed evenly to form an inner aqueous phase, and the mass concentration of the drug molecule in the inner aqueous phase is controlled to be 0.1 mg / mL; (3) The lipid film obtained in step (1) is hydrated by the internal aqueous phase obtained in step (2) and gently shaken at 37°C for 1 h. It is then purified by monodisperse extrusion of polycarbonate membrane and deoxygenated by nitrogen gas. Finally, it is crosslinked in situ under ultraviolet light to obtain the final product.

10. Use of the programmable fluorinated lipid nanodelivery system according to any one of claims 1 to 8 in the preparation of formulations for studying the anti-inflammatory or neuroprotective effects of drug molecules in Alzheimer's disease.

Images