A brain-targeting nanoparticle and a preparation method and application thereof

Abstract

The application discloses a brain-targeting nanoparticle and a preparation method and application thereof. The nanoparticle comprises nervonic acid and a carrier material, the carrier material is composed of chitosan, phosphatidylserine and polyethylene glycol, and the nervonic acid is loaded in the carrier. The preparation method comprises the following steps: dissolving the nervonic acid and the phosphatidylserine in an organic solvent to obtain an oil phase, dissolving chitosan and polyethylene glycol in an acidic aqueous solution to obtain a water phase, injecting the oil phase into the water phase, and then performing high-pressure homogenization to obtain an emulsion, and then performing electrostatic spraying into a supercritical fluid to simultaneously perform solvent extraction and drug crystallization, so as to obtain the nanoparticle. The nanoparticle is prepared through a ternary carrier and a coupling technology of high-pressure homogenization-supercritical anti-solvent-electrostatic spraying, and under high nervonic acid loading, the nanoparticle considers particle size control, encapsulation stability and low solvent residue, and is suitable for brain-targeting delivery and drug for neurodegenerative diseases. Under preferred carrier proportions, chitosan molecular weight / degree of deacetylation and PEG molecular weight, high encapsulation rate, drug loading, small particle size, low PDI and low residual solvent are obtained, and brain delivery is enhanced.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, and specifically relates to a brain-targeting nanoparticle, its preparation method, and its application. Background Technology

[0002] Nervonic acid (C 24 H 46 O2), English name Nervonic Acid, chemical name: cis-15-tetracosenic acid, is a monounsaturated fatty acid with the molecular formula CH3-(CH2)7-CH=CH-(CH2). 13 -COOH, with a molecular weight of 366.6, has the following structural formula: .

[0003] Nervonic acid, in its pure form, is a white, flaky crystal at room temperature. It is soluble in alcohol but insoluble in water, with a melting point of approximately 39-43°C, though this may vary depending on purity and the assay method. Named nervonic acid because it was first discovered in mammalian neural tissue, it is a core component of the myelin sheath in the brain's white matter. It can significantly improve Aβ plaque deposition and cognitive impairment in Alzheimer's disease (AD) by promoting axonal regeneration and repairing damaged neurons. However, the physicochemical properties and physiological barriers of nervonic acid present three major delivery challenges: 1. Poor solubility: It has a high melting point (41–43℃), is extremely hydrophobic, and has a water solubility of only 0.002 mg / mL, resulting in a very low oral absorption rate (less than 5%). 2. Chemical instability: The C=C double bond in the nervonic acid molecule is highly susceptible to oxidation in air, leading to a significantly shortened half-life (t0). 1 / 2 <72 hours in air at 25°C; 3. Brain targeting barrier: The blood-brain barrier (BBB) ​​restricts the permeation of macromolecules, and the brain distribution coefficient (logBB) of free nervonic acid is -1.2, which cannot meet the requirements for effective therapeutic concentration.

[0004] To address the aforementioned technical challenges, extensive research and exploration have been conducted in existing technologies, with the main research directions being: (1) Emulsion / nanoemulsion technology, which has a drug loading of ≤0.55% and requires a large amount of surfactant (e.g., 28% of polyoxyethylene 40 hydrogenated castor oil); lacks targeted modification and has a brain enrichment of less than 1% ID / g; (2) Layer-by-layer self-assembled micro powder: The process is complex (requires 4 dialysis cycles) and the drug loading is only 8.76%; the acid response release depends on the slightly acidic environment of the lesion (pH 6.5), but the pH of healthy brain tissue is 7.4, resulting in poor targeting specificity; (3) Liposomes / fat emulsions: Phosphatidylserine (PS) was used as an emulsifier, with a drug loading of <10%; the problem of nervonic acid leakage was not solved (leakage rate >15% after 14 days of storage). (4) Microcapsule technology: spray drying is used, and the drug loading capacity can reach 80%, but the particle size is large and cannot penetrate the BBB; there is no active targeting ligand, and the brain delivery efficiency is low.

[0005] To improve the bioavailability of nervonic acid, researchers have explored various nanotechnology techniques. The traditional emulsion-solvent evaporation (ESE) method uses organic solvents to dissolve the drug and polymeric carrier, forming droplets through high-pressure homogenization, followed by prolonged stirring to evaporate the solvent into particles. However, during solvent evaporation, nervonic acid readily diffuses from the droplet interior to the exterior, resulting in low encapsulation efficiency, and residual toxic solvents such as chloroform and dichloromethane are difficult to completely remove. Another commonly used technique is the traditional supercritical antisolvent (SAS) technique, which utilizes the high diffusivity and low viscosity of supercritical carbon dioxide to achieve drug precipitation. Although SAS technology excels in reducing solvent residue, its lack of precise electrical control over droplet size during spraying leads to secondary aggregation of the precipitated particles in the supercritical fluid, resulting in a very wide particle size distribution with a polydispersity index (PDI) typically above 0.3, making it difficult to achieve precise sub-200 nm particle size control.

[0006] Existing brain-targeted nanodelivery solutions mainly focus on the following technical routes: First, based on PLGA-PEG, liposomes or other polymer-lipid hybrid systems, exogenous targeting groups such as mannose, peptides, and antibodies are introduced to improve the ability to cross the blood-brain barrier; Second, based on chitosan or chitosan derivatives, focusing on nasal administration, mucosal adhesion or enhanced permeation absorption; Third, focusing on the nano-sizing of hydrophobic drugs, using single electrostatic spraying, single supercritical antisolvent or other single granulation technology to improve particle size and dissolution performance.

[0007] However, existing nervonic acid delivery methods still face challenges in simultaneously achieving optimal drug loading, encapsulation efficiency, particle size distribution, residual solvent control, and brain delivery efficiency, and require further improvement. Summary of the Invention

[0008] In order to overcome at least one of the technical problems existing in the prior art, one of the objectives of the present invention is to provide a brain-targeting nanoparticle.

[0009] The second objective of this invention is to provide a method for preparing brain-targeting nanoparticles.

[0010] A third objective of this invention is to provide a pharmaceutical composition.

[0011] The fourth objective of this invention is to provide the application of the above-mentioned brain-targeting nanoparticles or pharmaceutical compositions in the preparation of brain-targeting delivery formulations.

[0012] The fifth objective of this invention is to provide the use of the above-mentioned brain-targeting nanoparticles or pharmaceutical compositions in the preparation of drugs for the prevention or treatment of neurodegenerative diseases.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a brain-targeting nanoparticle comprising nervonic acid and a carrier material; wherein the nervonic acid is internally loaded in the carrier material; and wherein the carrier material comprises chitosan, phosphatidylserine, and polyethylene glycol. The number-average molecular weight of the polyethylene glycol is 1000-20000 Da; The chitosan has a number-average molecular weight of 5 kDa - 100 kDa and a degree of deacetylation of 80-95%.

[0014] In this invention, chitosan provides a positively charged interfacial layer and forms structural support, which is beneficial for improving mucosal adhesion and particle stability; phosphatidylserine helps improve uptake by brain microvascular endothelial cells and is associated with receptor-mediated processes related to cross-blood-brain barrier transport; polyethylene glycol can form a hydrophilic shielding layer on the particle surface, thereby reducing non-specific protein adsorption and prolonging systemic circulation time. Based on the data from the examples, the preferred formulation achieves a penetration rate of approximately 18.5% in an in vitro BBB model and a cumulative release rate of over 95% in simulated intestinal fluid over 48 hours.

[0015] In some embodiments of the present invention, the mass ratio of chitosan to polyethylene glycol is (0.2-16):1; in some embodiments of the present invention, the mass ratio of chitosan to polyethylene glycol is (1.0-5.0):1; for example, it can be any value or a range formed by any two of 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 4:1, 5:1. In some embodiments of the present invention, the mass ratio of chitosan to polyethylene glycol is (1.5-3.0):1.

[0016] In some embodiments of the present invention, the mass ratio of phosphatidylserine to polyethylene glycol is (0.5-2.0):1; for example, it can be any value among 0.5:1, 0.75:1, 1:1, 1.3:1, 1.5:1, 2:1 or a range formed by any two; in some embodiments of the present invention, the mass ratio of phosphatidylserine to polyethylene glycol is (0.5-1.5):1.

[0017] In this invention, when the ratio of chitosan, phosphatidylserine and polyethylene glycol deviates significantly from the aforementioned range, the system is prone to adverse changes such as decreased emulsion stability, shortened Taylor cone retention time, widened particle size distribution, decreased encapsulation efficiency, or reduced brain uptake efficiency.

[0018] In some embodiments of the present invention, the mass of the nervonic acid accounts for 10%-55% of the mass of the brain-targeting nanoparticles; for example, it can be any value or a range formed by any combination of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, and 55%. In some embodiments of the present invention, the mass of the nervonic acid accounts for 25-50% of the mass of the brain-targeting nanoparticles. Nervonic acid, as a core functional molecule, exerts its therapeutic effect by promoting the regeneration of neural axons and repairing damaged neurons.

[0019] In some embodiments of the present invention, the number-average molecular weight of the polyethylene glycol is any value or a range formed by any combination of 1000 Da, 2000 Da, 4000 Da, 5000 Da, 6000 Da, 8000 Da, 10000 Da, 12000 Da, 14000 Da, 15000 Da, 16000 Da, 18000 Da, and 20000 Da. In some embodiments of the present invention, the number-average molecular weight of the polyethylene glycol is 2000-10000 Da. The hydrophilic segments of polyethylene glycol can form a steric hindrance layer on the surface of nanoparticles, reducing plasma protein adsorption and prolonging systemic circulation time; controlling the PEG molecular weight within the aforementioned range is beneficial for achieving a balance between protein corona adsorption, plasma half-life, and BBB penetration.

[0020] In some embodiments of the present invention, the degree of deacetylation of the chitosan is any value or a range formed by any two of 80%, 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, and 95%. When the degree of deacetylation of chitosan is below 80%, there are too many acetyl groups on the chitosan molecular chain, resulting in insufficient intermolecular hydrogen bond strength. The coating shell formed during the high-pressure spraying process of supercritical antisolvent-electrostatic spray coupling (SAS-ESS) is too loose, and nervonic acid is prone to physical leakage during storage. Furthermore, a low degree of deacetylation of chitosan results in a Zeta potential below +10 mV, making it impossible to effectively adsorb onto the surface of negatively charged brain microvascular endothelial cells through charge interactions. Conversely, when the degree of deacetylation of chitosan exceeds 95%, the crystallinity of chitosan is too high. Although this results in good stability, it leads to a significant decrease in the release rate of nanoparticles in intestinal fluid, thus reducing bioavailability.

[0021] In some embodiments of the present invention, the number-average molecular weight of the chitosan is any value or a range formed by any two of the following: 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, and 100 kDa. In some embodiments of the present invention, the number-average molecular weight of the chitosan is between 10 kDa and 50 kDa. When the molecular weight of chitosan is in the range of 5-100 kDa, the system generally exhibits rheological properties more suitable for high-pressure homogenization and electrostatic spraying, which is beneficial for forming a more uniform proemulsion and a more stable Taylor cone, thereby obtaining smaller particle size, narrower distribution, and higher encapsulation efficiency and drug loading. When the molecular weight is below 5 kDa, the molecular chain is too short, the interfacial film strength is insufficient, and the stability of the encapsulation structure decreases. When the molecular weight is further increased to above 150 kDa, the viscosity of the aqueous phase increases significantly, the jet stability decreases, and the final particle size can be increased to above 800 nm under the condition of 300 kDa.

[0022] In some embodiments of the present invention, the phosphatidylserine has a purity of ≥90% and is derived from soybeans or egg yolks.

[0023] In some embodiments of the present invention, the average particle size of the brain-targeting nanoparticles is 50-250 nm; for example, it can be any value or a range formed by any two of 50 nm, 80 nm, 100 nm, 120 nm, 150 nm, 180 nm, 200 nm, and 250 nm; in some embodiments of the present invention, the average particle size of the brain-targeting nanoparticles is 80-200 nm.

[0024] In some embodiments of the present invention, the polydispersity index (PDI) of the brain-targeting nanoparticles is ≤0.25; in some embodiments of the present invention, the polydispersity index of the brain-targeting nanoparticles is any value or a range formed by any two of the following: 0.01, 0.02, 0.04, 0.05, 0.06, 0.08, 0.1, 0.12, 0.14, 0.15, 0.16, 0.18, 0.2, 0.22, 0.24, 0.25. In some embodiments of the present invention, the polydispersity index of the brain-targeting nanoparticles is ≤0.2.

[0025] In some embodiments of the present invention, the zeta potential of the brain-targeting nanoparticles is +15mV to +45mV; in some embodiments of the present invention, the zeta potential of the brain-targeting nanoparticles is any value or a range formed by any combination of +15mV, +20mV, +25mV, +30mV, +35mV, +40mV, +43mV, and +45mV. The brain-targeting nanoparticles of the present invention are positively charged, enabling them to interact electrostatically with the negatively charged intestinal mucus layer, thereby increasing the amount of mucus bound in vitro and providing favorable conditions for subsequent absorption.

[0026] In some embodiments of the present invention, the average encapsulation efficiency of nervonic acid in the brain-targeting nanoparticles, as determined by ultrafiltration-HPLC, is ≥85%; for example, it can be any value or a range formed by any combination of 85%, 87%, 90%, 91%, 92%, 93%, 94%, and 95%. In some embodiments of the present invention, the average encapsulation efficiency of nervonic acid in the brain-targeting nanoparticles is 92-95%.

[0027] In some embodiments of the present invention, the residual amount of organic solvent in the brain-targeting nanoparticles is ≤10ppm; in some embodiments of the present invention, the residual amount of organic solvent in the brain-targeting nanoparticles is any value of 1ppm, 2ppm, 3ppm, 4ppm, 5ppm, 6ppm, 7ppm, 8ppm, 9ppm, 10ppm or a range formed by any two of these values.

[0028] In some embodiments of the present invention, the crystallinity of nervonic acid in the brain-targeting nanoparticles is ≥75%. In some embodiments of the present invention, the crystallinity of nervonic acid in the brain-targeting nanoparticles is 75-98%; in some embodiments of the present invention, the crystallinity of nervonic acid in the brain-targeting nanoparticles is 78-93%.

[0029] The second aspect of the present invention provides a method for preparing the brain-targeting nanoparticles described in the first aspect of the present invention, comprising the following steps: The raw materials, including nervonic acid, phosphatidylserine and organic solvent, are mixed to obtain the oil phase; the raw materials, including chitosan, polyethylene glycol and acidic aqueous solution, are mixed to obtain the aqueous phase. The oil phase is injected into the aqueous phase and homogenized under high pressure to obtain an emulsion; The emulsion was electrostatically sprayed into a supercritical fluid, where it crystallized to precipitate, yielding the brain-targeting nanoparticles.

[0030] This invention employs supercritical antisolvent-electrostatic spraying (SAS-ESS) coupling technology, integrating supercritical antisolvent (SAS) and electrostatic spraying (ESS) to achieve control over nanoparticle structure parameters and regulation of nervonic acid crystallization kinetics, thereby solving the problem of uneven precipitation during the solidification process of high-content fatty acid drugs. The principle is as follows: when the emulsion is electrostatically sprayed into supercritical CO2 (denoted as scCO2), the emulsion forms charged droplets after electrostatic spraying. scCO2 rapidly diffuses into the interior of the emulsion droplets, altering the internal solvent environment and promoting nervonic acid supersaturation and nucleation precipitation. Furthermore, the preparation method in this invention reduces the risk of nervonic acid oxidation by shortening the time the drug is exposed to the oxidizing environment.

[0031] When the emulsion is electrostatically sprayed into a supercritical fluid, it first forms charged droplets, which then instantaneously contact the supercritical fluid, simultaneously completing solvent extraction and drug crystallization to obtain the brain-targeting nanoparticles. Using an "oil-phase injection into aqueous phase" method allows the hydrophobic drug nervonic acid to be co-dispersed with phosphatidylserine in organic phase droplets, forming an oil / water emulsion under the interfacial stabilization of the chitosan-polyethylene glycol aqueous phase. This facilitates droplet refinement and uniform drug dispersion in the carrier matrix during subsequent high-pressure homogenization.

[0032] In this invention, the charged droplets formed by electrostatic spraying of the emulsion have particles with the same charge on their surface. In the medium of supercritical fluid with a low dielectric constant, the electrostatic repulsion generated by the charge can effectively counteract the van der Waals force, preventing the droplets from colliding and merging during the drying and solidification process, thus avoiding the problem of large average particle size and wide particle size distribution caused by "secondary particle agglomeration".

[0033] In some embodiments of the present invention, the oil phase is prepared by mixing raw materials including nervonic acid, phosphatidylserine, and organic solvents at 40-60°C.

[0034] In some embodiments of the present invention, the oil phase is prepared by mixing under the protection of an inert gas (nitrogen or argon) and in the absence of light.

[0035] In some embodiments of the present invention, the aqueous phase is mixed using magnetic stirring during preparation.

[0036] In some embodiments of the present invention, the magnetic stirring speed is 600-1000 rpm.

[0037] In some embodiments of the present invention, the aqueous phase is prepared at a mixing temperature of 35-45°C.

[0038] In some embodiments of the present invention, the organic solvent includes at least one of ethanol, acetone, and ethyl acetate.

[0039] In some embodiments of the present invention, the organic solvent accounts for 60-90% of the mass of the oil phase; in some embodiments of the present invention, it is 70-90%.

[0040] In some embodiments of the present invention, the supercritical fluid is supercritical carbon dioxide. Supercritical carbon dioxide has high diffusion capacity and can quickly penetrate into the fine charged droplets formed by electrostatic spraying, thereby significantly reducing the polarity of the solvent environment inside the droplets, promoting the rapid supersaturation of nervonic acid and its nucleation and precipitation. Therefore, it is beneficial to obtain brain-targeting nanoparticles with small particle size and narrow distribution.

[0041] In some embodiments of the present invention, the high-pressure homogenization is performed in a high-pressure homogenizing device (such as the APV-2000). The high-pressure homogenization step achieves nanoscale dispersion of the nervonic acid-PS complex in the CS-PEG aqueous phase through strong shear forces and cavitation effects.

[0042] In some embodiments of the present invention, the emulsion is introduced into a supercritical reactor at a flow rate of 1-3 mL / min and mixed with supercritical carbon dioxide.

[0043] In some embodiments of the present invention, the pressure of the supercritical fluid is 8-25 MPa; for example, it can be any value or a range formed by any two of 8 MPa, 10 MPa, 13 MPa, 15 MPa, 18 MPa, 20 MPa, 22 MPa, and 25 MPa. In some embodiments of the present invention, the pressure of the supercritical fluid is 8-12 MPa. When carbon dioxide is used as the supercritical fluid, if the pressure is too low, carbon dioxide is difficult to maintain a stable supercritical state, resulting in insufficient supercritical fluid density and reduced solvent extraction efficiency; if the pressure is too high (>25 MPa), the equipment requirements increase and may cause yield loss.

[0044] In some embodiments of the present invention, the flow rate of the supercritical fluid is 5-20 L / min; in some embodiments of the present invention, the flow rate of the supercritical fluid is any value or a range formed by any two of the following: 5 L / min, 6 L / min, 8 L / min, 10 L / min, 12 L / min, 14 L / min, 15 L / min, 16 L / min, 18 L / min, 20 L / min.

[0045] In some embodiments of the present invention, the temperature of the supercritical fluid is 32-60°C; for example, it can be any value of 32°C, 38°C, 40°C, 50°C, 60°C, or a range formed by any combination of both. This temperature range is favorable for carbon dioxide to be in a supercritical state while also taking into account the stability of nervonic acid. When the temperature is too high, the peroxide value (POV) of nervonic acid may increase.

[0046] In some embodiments of the present invention, the pressure of the high-pressure homogenization is 50-150 MPa; in some embodiments of the present invention, the pressure of the high-pressure homogenization is any value or a range formed by any two of 50 MPa, 60 MPa, 70 MPa, 80 MPa, 90 MPa, 100 MPa, 110 MPa, 120 MPa, 130 MPa, 140 MPa, and 150 MPa.

[0047] In some embodiments of the present invention, the number of high-pressure homogenizations is 2-10 times; in some embodiments of the present invention, the number of high-pressure homogenizations is any value of 2, 4, 5, 6, 8, or 10 times, or a range formed by any two of them.

[0048] In some embodiments of the present invention, the pH of the acidic aqueous solution is 3-6.

[0049] In some embodiments of the present invention, the concentration of the acidic aqueous solution is 0.05-0.5 mol / L; in some embodiments of the present invention, the concentration of the acidic aqueous solution is any value or a range formed by any two of 0.05 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, and 0.5 mol / L.

[0050] In some embodiments of the present invention, the acidic aqueous solution includes at least one of acetate buffer, citrate buffer, and hydrochloric acid buffer.

[0051] In some embodiments of the present invention, the spray voltage of the electrostatic spray is 10-30 kV; in some embodiments of the present invention, the spray voltage of the electrostatic spray is any value or a range formed by any two of the following: 10 kV, 12 kV, 14 kV, 15 kV, 16 kV, 18 kV, 20 kV, 22 kV, 24 kV, 25 kV, 26 kV, 28 kV, and 30 kV; in some embodiments of the present invention, the spray voltage of the electrostatic spray is 15-25 kV. When the voltage is below 10 kV, the electric field force is insufficient to overcome the surface tension, making it difficult to form a stable Taylor cone; when the voltage is above 30 kV, it is easy to induce local corona discharge and cause uneven droplet breakage. Under the aforementioned voltage window, the surface charge density of the droplets increases, and the electric field force can overcome the surface tension and further refine the jet into submicron-sized droplets.

[0052] When the emulsion passes through an electrostatic spray nozzle, an applied high-voltage electric field causes the droplet surface to rapidly accumulate charge. When the outward pressure generated by the electric field exceeds the surface tension of the droplet itself, the droplet will further split and form smaller charged droplets, thus providing conditions for obtaining particles in the 50-200 nm range.

[0053] In some embodiments of the present invention, the nozzle orifice diameter of the electrostatic spray is 50-300 μm. In some embodiments of the present invention, the electrostatic spraying time is 10-60 minutes.

[0054] In some embodiments of the present invention, the electric field oscillation frequency during electrostatic spraying is 0.5-5 kHz. Through electric field oscillation, crystal agglomeration can be broken up and suppressed, achieving a PDI ≤ 0.25.

[0055] Under the combined effect of electric field oscillation (frequency 0.5-5 kHz) and rapid diffusion of supercritical fluid, nervonic acid molecules undergo instantaneous and uniform nucleation, and crystal growth is effectively suppressed by the electric field force, ultimately forming nanoparticles with uniform particle size (PDI<0.2) and high crystallinity.

[0056] In some embodiments of the present invention, the preparation method further includes a washing step; the washing step is performed after the crystallization precipitation step.

[0057] In some embodiments of the present invention, the washing is performed by supercritical carbon dioxide drying extraction to reduce the solvent content in the brain-targeting nanoparticles.

[0058] In some embodiments of the present invention, the preparation method further includes a drying step; the drying is performed after the washing step.

[0059] In some embodiments of the present invention, the drying is performed using at least one of freeze drying or spray drying.

[0060] In this invention, nervonic acid and phosphatidylserine are first dissolved in an organic solvent system capable of effective interdiffusion with supercritical fluids to form a clear and homogeneous oil phase. Simultaneously, chitosan is fully swollen in an acidic aqueous solution system, and polyethylene glycol is introduced to form a synergistically stable hydrophilic shell material, resulting in a stable aqueous phase. The oil phase is then added to the aqueous phase, and high-pressure homogenization is used to obtain an emulsion with controllable particle size. This allows the active ingredient nervonic acid and the carrier material to achieve sufficient contact and interfacial rearrangement at the micro-nano scale, providing a uniform precursor system for subsequent supercritical precipitation and electrostatic spraying synergistic nucleation. The emulsion is pumped into a supercritical reactor pre-set at a predetermined pressure and temperature, while high-purity carbon dioxide is introduced and maintained at a stable flow rate. The emulsion is then electrostatically sprayed into the reactor, forming a stable Taylor cone and charged jet under the action of an applied DC high-voltage electric field. The accumulation of surface charge on the droplets causes the electric field force to exceed the surface tension, inducing the jet to break into smaller secondary droplets. Simultaneously, supercritical carbon dioxide, with its high diffusion coefficient, penetrates into the droplets in an extremely short time, triggering a sudden drop in solvent polarity. This causes a significant decrease in nervonic acid solubility on a millisecond timescale, instantly reaching a highly supersaturated state, thereby initiating uniform nucleation and inhibiting the growth of coarse crystals. This coupled process of "electric field disruption—supercritical penetration—polarity drop—instant nucleation" is the key mechanism underlying the narrow particle size distribution and high encapsulation efficiency achieved in this invention. After crystallization, the precipitated products are further washed and replaced online with supercritical carbon dioxide within the reactor to further reduce organic solvent residue and stabilize the particle surface structure.

[0061] In some embodiments of the present invention, the brain-targeting nanoparticles can be administered orally or nasally.

[0062] A third aspect of the invention provides a pharmaceutical composition comprising the brain-targeting nanoparticles described in the first aspect of the invention and pharmaceutically acceptable excipients.

[0063] In some embodiments of the present invention, the dosage form of the pharmaceutical composition is selected from tablets, lozenges, aqueous suspensions, oil suspensions, water-oil suspensions, powders, granules, emulsions, capsules, syrups, or elixirs.

[0064] In some embodiments of the present invention, the pharmaceutical compositions of the present invention are suitable for multiple routes of administration and can thus be formulated into any pharmaceutically acceptable dosage form. For example, the above-described pharmaceutical compositions can be administered to patients or subjects requiring such treatment via oral, parenteral, rectal, or pulmonary administration. When used for oral administration, the pharmaceutical compositions can be formulated into oral preparations, such as conventional oral solid preparations, like tablets, capsules, pills, granules, etc.; or into oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. When formulating oral preparations, suitable fillers, binders, disintegrants, lubricants, etc., can be added. When used for parenteral administration, the above-described pharmaceutical compositions can also be formulated into injections, including injection solutions, sterile powders for injection, and concentrated solutions for injection. When formulating injections, conventional methods in the existing pharmaceutical field can be used; when formulating injections, excipients may not be added, or suitable excipients may be added depending on the properties of the drug. When used for rectal administration, the pharmaceutical compositions can be formulated into suppositories, etc. When used for pulmonary administration, the pharmaceutical compositions can be formulated into inhaled preparations, aerosols, powder inhalers, or sprays, etc.

[0065] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of solvents, inert diluents, dispersants, granulating agents, surfactants, emulsifiers, disintegrants, binders, preservatives, buffers, lubricants, excipients, colorants, coating agents, sweeteners, flavoring agents, and aromatizers.

[0066] In some embodiments of the invention, pharmaceutically acceptable excipients are substances that are non-toxic, compatible with the active ingredient, and otherwise biologically suitable for use in organisms. The selection of a specific excipient will depend on the route of administration or the type and state of disease for treating a particular patient. Examples of pharmaceutically acceptable excipients include, but are not limited to, solvents, inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants, and / or oils conventional in the pharmaceutical field. The composition may also contain excipients such as cocoa butter and suppository waxes, colorants, coating agents, sweeteners, flavoring agents, and aromatizers.

[0067] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and mixtures thereof.

[0068] Exemplary granulating agents and / or dispersants include potato starch, corn starch, cassava starch, sodium starch glycolate, clay, alginate, guar gum, citrus pomace, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinylpyrrolidone) (cross-linked polyvinylpyrrolidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (cross-linked carboxymethyl cellulose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0069] Exemplary surfactants and / or emulsifiers include natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth gum, carrageenan, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, glyceryl triacetate monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxylated polymethylene, polyacrylic acid, acrylic polymers, and carboxyethylene polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethyl cellulose, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate (20), poly... ethylene-oxygenated sorbitol (60), polyoxyethylene-oxygenated sorbitol monooleate (80), sorbitol monopalmitate (40), sorbitol monostearate (60), sorbitol tristearate (65), glyceryl monooleate, sorbitol monooleate (80), polyoxyethylene esters (e.g., polyoxyethylene monostearate (45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate), sucrose fatty acid esters, polyethylene glycol fatty acids Esters (e.g.), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether (30)), poly(ethylene-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, F-68, poloxamer P-188, cetrimonium bromide, cetylpyridine chloride, benzalkonium chloride, docusatesodium and / or mixtures thereof.

[0070] Exemplary binders include starch (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., gum arabic, sodium alginate, Irish moss extract, panwar gum, ghatti gum, mucilage of isapola husk, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, poly(vinylpyrrolidone), magnesium aluminum silicate, and larch arabinogalactan), alginate, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethyl methacrylate, waxes, water, ethanol, and / or mixtures thereof.

[0071] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoal preservatives, alcohol preservatives, acid preservatives, and other preservatives. In some embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0072] Exemplary antioxidants include alpha-tocopherol, ascorbic acid, ascorbate palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0073] The fourth aspect of the present invention provides the use of the brain-targeting nanoparticles described in the first aspect of the present invention or the pharmaceutical compositions described in the third aspect of the present invention in the preparation of brain-targeting delivery formulations.

[0074] The fifth aspect of the invention provides the use of the brain-targeting nanoparticles described in the first aspect of the invention or the pharmaceutical compositions described in the third aspect of the invention in the preparation of medicaments for the prevention or treatment of neurodegenerative diseases.

[0075] The neurodegenerative diseases mentioned include Alzheimer's disease or Parkinson's disease.

[0076] The beneficial effects of this invention are as follows: The brain-targeting nanoparticles of this invention use chitosan, phosphatidylserine, and polyethylene glycol as carrier materials to internally load nervonic acid, achieving a small particle size and narrow particle size distribution while maintaining a high encapsulation efficiency, and exhibiting low organic solvent residue and good storage stability. In some preferred embodiments, the average particle size of the nanoparticles is approximately 85-132 nm, the PDI is approximately 0.11-0.18, the encapsulation efficiency is 92.6%-94.9%, the drug loading is 29.2%-47.1%, and the organic solvent residue is 3.1-8 ppm; they show good brain distribution and delivery performance in in vitro BBB models and animal experiments.

[0077] The preparation method of this invention employs a high-pressure homogenization coupled with SAS-ESS technology, which is beneficial for balancing particle size control, encapsulation efficiency, and low residual solvent control. In a preferred embodiment, nanoparticles with an average particle size of approximately 85-132 nm, a PDI of approximately 0.11-0.18, an encapsulation efficiency of 92.6%-94.9%, and residual organic solvent of 3.1-8 ppm can be obtained.

[0078] In this invention, chitosan, phosphatidylserine, and PEG are used as carrier materials. The positive charge of chitosan is beneficial for intestinal mucosal adhesion and prolongs absorption time; phosphatidylserine is beneficial for improving uptake by brain microvascular endothelial cells, and RAP intervention experiments suggest that LRP1-related receptor-mediated processes are involved in BBB transcellular transport; PEG segments provide steric hindrance to prolong systemic circulation time and reduce non-specific protein adsorption.

[0079] The preparation method of this invention employs a high-pressure homogenization-SAS-ESS coupled process, which can balance particle size control, encapsulation stability, and low residual solvent at a high nervonic acid loading level. In preferred embodiments, the average particle size of the nanoparticles can be controlled at approximately 85-132 nm, the PDI can be controlled at 0.11-0.18, the encapsulation efficiency is 92.6%-94.9%, the drug loading is 29.2%-47.1%, and the residual organic solvent is 3.1-8 ppm. Attached Figure Description

[0080] Figure 1 This is a flowchart illustrating the preparation process of brain-targeting nanoparticles in Example 1 of the present invention. Detailed Implementation

[0081] The specific implementation of the present invention will be further described in detail below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments used without specified manufacturers are all conventional products that can be purchased commercially.

[0082] I. Raw Material Information The raw material information used in Examples 1-6, Example 8, and Comparative Examples 1-2 of this invention is as follows: Chitosan (CS, purchased from Sigma-Aldrich) with a degree of deacetylation of 90% and a number-average molecular weight of 20 kDa; phosphatidylserine (PS, purity ≥95%, purchased from Avanti Polar Lipids) derived from egg yolk; PEG5000 (PEG, number-average molecular weight 5000 Da, purchased from Merck); and nervonic acid (cis-15-tetracosenoic acid, purity ≥99%, purchased from Tokyo Chemical Industry Co., Ltd., TCI) were used. The acidic buffer was 0.1 mol / L acetate buffer (pH 5.0, water as solvent), and the organic solvent was anhydrous ethanol (HPLC grade).

[0083] II. Examples 1-3: Screening of Formulation Ratios for Ternary Carrier Systems Examples 1-3 aim to investigate the effects of different formulation combinations of chitosan (CS), phosphatidylserine (PS), and polyethylene glycol (PEG) on encapsulation efficiency, drug loading, particle size distribution, and targeting-related properties of the ternary carrier system by systematically screening the mass ratios. All examples used supercritical antisolvent-electrostatic spray coupling technology (SAS-ESS) to prepare nanoparticles.

[0084] Example 1 This example provides a brain-targeting nanoparticle and its preparation method, the preparation method as follows: Figure 1 The process flow diagram is shown in the figure. The specific preparation steps are as follows: (1) Preparation of oil and aqueous phases: Oil phase preparation: Nervate (330 mg) and PS (200 mg) were dissolved in 5 mL of anhydrous ethanol and sonicated in a water bath at 50 °C for 30 minutes under nitrogen protection to prevent light exposure. Aqueous phase preparation: CS (450 mg) and PEG (150 mg) were dissolved in 10 mL of acetate buffer and magnetically stirred (800 rpm, 40 °C) until completely dissolved. The solution was then filtered through a 0.45 μm microporous membrane to remove impurities. In this example, the mass ratio of CS, PS and PEG was 9:4:3. (2) High-pressure emulsification: The oil phase was injected into the aqueous phase at 1 mL / min and circulated 5 times at 100 MPa using an APV-2000 high-pressure homogenizer (purchased from APV Company in Denmark) to obtain the primary emulsion.

[0085] (3) Supercritical crystallization spray: The colostrum was pumped into the P-1 supercritical crystallization system (i.e., supercritical fluid crystallization system, or alternatively, the Thar SFE-2000 supercritical fluid crystallization system, purchased from Thar Technologies, USA) produced by PuCui Supercritical Equipment Co., Ltd. at a flow rate of 2 mL / min through an electrostatic nozzle. In the supercritical fluid crystallization system, the CO2 pressure was 10 MPa, the temperature was 38℃, and the supercritical CO2 flow rate was 15 L / min. The electrostatic nozzle parameters were fixed at an orifice diameter of 150 μm and an electrostatic voltage of 20 kV. Atomization crystallization was carried out for 60 minutes to obtain nanoparticles. The nanoparticles were washed with supercritical CO2 for 30 minutes and then freeze-dried (-80℃, 0.1 mbar) to obtain the final product.

[0086] Nanoparticles settle to the bottom of the supercritical fluid crystallization system under gravity and are continuously dried with supercritical CO2 for more than 30 minutes to completely replace residual organic solvents (ensuring that the residual solvent content in the nanoparticles is ≤10 ppm, significantly lower than the conventional limit of ICH Q3C Class 3). The collected nanoparticle suspension can be further solidified by freeze-drying (pre-freezing temperature -80℃, main drying temperature -40℃, vacuum degree ≤0.1 mbar) or spray drying (inlet temperature 100-120℃, outlet temperature 50-60℃, atomization pressure 0.3-0.5 MPa) to finally obtain a white nanoparticle powder with good flowability. The total process time is controlled within 2 hours, with high production efficiency and avoidance of high temperature and oxidizing environment throughout the process. The degradation rate of unsaturated double bonds (C=C) in nervonic acid is less than 5%. The instantaneous crystallization process (millisecond level) significantly reduces the time that nervonic acid is exposed to the oxidizing environment. Combined with inert gas protection throughout the process, it ensures that the cis isomer content in nervonic acid is ≥98% and the peroxide value (POV) is <5 meq / kg. The content of cis isomers of nervonic acid was determined by gas chromatography-mass spectrometry (GC-MS). After methyl esterification, the samples were separated using a DB-FastFAME high-polarity column (or equivalent column). Methyl cis-15-tetradecanoate standard was used as a reference, and the relative content of the cis isomers was calculated by peak area normalization. The determination of peroxide value (POV) followed the iodometric titration method in Chapter 0713, "Determination of Fat and Fatty Oils," of the 2020 edition of the Chinese Pharmacopoeia, Part IV. The specific steps were as follows: Nanoparticle samples were accurately weighed, dissolved in a glacial acetic acid-chloroform mixed solution, and after purging with nitrogen to remove oxygen, saturated potassium iodide solution was added. After reacting in the dark, water was added, and titration was performed with sodium thiosulfate titrant until a pale yellow color was obtained. Starch indicator was added, and titration continued until the blue color disappeared, which was the endpoint. The peroxide value was calculated according to the formula.

[0087] Example 2 This example provides a brain-targeting nanoparticle and its preparation method. The specific preparation steps are as follows: (1) Preparation of oil and aqueous phases: Oil phase preparation: Nervate (300 mg) and PS (225 mg) were dissolved in 5 mL of anhydrous ethanol and sonicated in a water bath at 50 °C for 30 minutes under nitrogen protection to prevent light exposure. Aqueous phase preparation: CS (600 mg) and PEG (300 mg) were dissolved in 10 mL of acetate buffer and magnetically stirred (800 rpm, 40 °C) until completely dissolved. The solution was then filtered through a 0.45 μm microporous membrane to remove impurities. In this example, the mass ratio of CS, PS and PEG was 8:3:4. (2) High-pressure emulsification: The oil phase is injected into the aqueous phase at 1 mL / min and circulated 5 times at 100 MPa using an APV-2000 high-pressure homogenizer to obtain the primary emulsion.

[0088] (3) Supercritical crystallization spray: The colostrum was pumped into the P-1 supercritical crystallization system (i.e., supercritical fluid crystallization system) produced by PuCui Supercritical Company using an electrostatic nozzle at a rate of 2 mL / min. The CO2 pressure in the supercritical fluid crystallization system was 10 MPa, the temperature was 38℃, and the flow rate of supercritical CO2 was 15 L / min. The parameters of the electrostatic nozzle were fixed at an orifice diameter of 150 μm and an electrostatic voltage of 20 kV. Atomization crystallization was carried out for 60 minutes to obtain nanoparticles. The nanoparticles were washed with supercritical CO2 for 30 minutes and then freeze-dried (-80℃, 0.1 mbar) to obtain the final product.

[0089] Example 3 This example provides a brain-targeting nanoparticle and its preparation method. The specific preparation steps are as follows: (1) Preparation of oil and aqueous phases: Oil phase preparation: Nervate (300 mg) and PS (240 mg) were dissolved in 5 mL of anhydrous ethanol and sonicated in a water bath at 50 °C for 30 minutes under nitrogen protection to prevent light exposure. Aqueous phase preparation: CS (750 mg) and PEG (450 mg) were dissolved in 10 mL of acetate buffer and magnetically stirred (800 rpm, 40 °C) until completely dissolved. The solution was then filtered through a 0.45 μm microporous membrane to remove impurities. In this example, the mass ratio of CS, PS and PEG was 25:8:15. (2) High-pressure emulsification: The oil phase is injected into the aqueous phase at 1 mL / min and circulated 5 times at 100 MPa using an APV-2000 high-pressure homogenizer to obtain the primary emulsion.

[0090] (3) Supercritical crystallization spray: The colostrum was pumped into the P-1 supercritical crystallization system (i.e., supercritical fluid crystallization system) produced by PuCui Supercritical Company using an electrostatic nozzle at a rate of 2 mL / min. The CO2 pressure in the supercritical fluid crystallization system was 10 MPa, the temperature was 38℃, and the supercritical CO2 flow rate was 15 L / min. The electrostatic nozzle parameters were fixed at an orifice diameter of 150 μm and an electrostatic voltage of 20 kV. Atomization crystallization was carried out for 60 minutes to obtain nanoparticles. The nanoparticles were washed with supercritical CO2 for 30 minutes and then freeze-dried (-80℃, 0.1 mbar) to obtain the final product.

[0091] III. Unified Representation Method The properties of the nanoparticles prepared in Examples 1-3 were tested according to the following test methods. The specific test methods are as follows: (1) Encapsulation efficiency and drug loading: Ultrafiltration separation combined with HPLC quantification was used for determination. Approximately 10 mg of nanoparticles were dispersed in 1 mL of deionized water, transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, and centrifuged at 4℃ and 12000 g for 30 min. The filtrate was collected, and the mass of free nervonic acid was determined according to the aforementioned HPLC conditions. An equal amount of nanoparticles was taken, and acetonitrile-water (70:30, v / v) was added to fully disrupt the particle structure. The total nervonic acid mass was then determined. The mass of nervonic acid in the nanoparticles = the mass of total nervonic acid. Free nervonic acid mass. Encapsulation efficiency (EE%) = (Nervonic acid mass in nanoparticles / Mass of nervonic acid fed) × 100%. Drug loading (DL%) = (Nervonic acid mass in nanoparticles / (Actual mass of nanoparticles recovered after washing and drying) × 100%. The mass of nervonic acid in the nanoparticles was determined by HPLC, and the total mass of the nanoparticles was obtained by precise weighing.

[0092] (2) Particle size and dispersibility: The particle size and PDI of the nanoparticles were determined by dynamic light scattering using the Malvern Zetasizer Nano ZS.

[0093] (3) Zeta potential and stability: The zeta potential of nanoparticles was determined by electrophoretic light scattering using Malvern Zetasizer Nano ZS (Malvern, UK). An appropriate amount of nanoparticles were dispersed in deionized water, the pH was adjusted to 7.0±0.2, and the measurement was performed at 25℃. Each sample was measured 3 times, and the average value was taken.

[0094] (4) Determination of in vitro intestinal mucus binding: Fresh rat small intestine segments were cut open and laid flat. After washing with 0.1 mol / L PBS, the intestinal segments were cut into 1 cm × 1 cm pieces. The intestinal segments were immersed in a nanoparticle suspension (nanoparticle concentration of 1 mg / mL) and incubated at 37°C for 30 minutes. After removal, the segments were rinsed three times with PBS, and the bound drug was extracted by sonication. The content was determined by HPLC, and the binding amount per unit area (μg / cm²) was calculated. 2 ).

[0095] The performance data of the nanoparticles prepared in Examples 1-3, obtained according to the above testing methods, are shown in Table 1 below.

[0096] Table 1 Performance data of nanoparticles prepared in Examples 1-3

[0097] As shown in Table 1, the nanoparticles in Examples 1-3 of this invention all have a particle size of less than 200 nm and a PDI of no more than 0.23, indicating that the ternary carrier material system can form particles with a narrow distribution. All three groups of samples are positively charged, which is beneficial for enhancing intestinal mucus binding. In terms of individual indicators, Example 1 shows a higher actual drug loading due to the higher proportion of nervonic acid; Example 2 performs best in terms of encapsulation efficiency, particle size, PDI, Zeta potential, and in vitro mucus binding, indicating that when the CS:PS:PEG mass ratio is 8:3:4, the system achieves a better balance between interfacial stability, particle uniformity, and mucosal adhesion; Example 3 shows an increase in particle size and a decrease in encapsulation and adhesion performance due to a further increase in the carrier ratio.

[0098] In vitro BBB penetration determination: A Transwell chamber model was used. Human brain microvascular endothelial cells (hCMEC / D3) were seeded in the upper chamber, and astrocytes were seeded in the lower chamber. Nanoparticles carrying DiR fluorescent probes were added to the upper chamber (concentration 200 μg / mL). After 24 hours, the fluorescence intensity in the lower chamber was measured, and the penetration rate was calculated as (lower chamber fluorescence intensity / initial upper chamber fluorescence intensity × 100%). Each sample was tested in triplicate, and the average value was taken. Test results: The in vitro BBB penetration rate of the nanoparticles in Example 1 was 12.6 ± 1.1%; the in vitro BBB penetration rate of the nanoparticles in Example 2 was 18.5 ± 0.9%; and the in vitro BBB penetration rate of the nanoparticles in Example 3 was 14.3 ± 1.0%. The penetration rate of Example 2 was significantly higher than that of Examples 1 and 3 (P < 0.01), which is attributed to its smaller particle size (95 nm) and higher Zeta potential (+28.4 mV), which facilitates the electrostatic adsorption of nanoparticles to the cell membrane and LRP1 receptor-mediated endocytosis.

[0099] Furthermore, the protein crown adsorption assay was performed using the BCA method. 1 mg of nanoparticles (based on total nanoparticle mass) was dispersed in 1 mL of PBS (pH 7.4) containing 10% fetal bovine serum and incubated at 37°C for 1 h. The sample was then transferred to a 100 kDa ultrafiltration centrifuge tube and centrifuged at 12000 g for 15 min at 4°C. The supernatant was discarded, and the sample was washed three times with PBS. 1 mL of PBS containing 2% SDS was added to the precipitate, and the surface-adsorbed protein was eluted by sonication for 10 min. The total protein content of the eluent was determined using the BCA method, and the protein adsorption rate was calculated as (eluted protein content / added nanoparticle mass) × 100%. The results showed that the protein adsorption rate in Example 2 was 15%, lower than the 42% in Example 1 and 28% in Example 3.

[0100] IV. Examples 4-6: SAS-ESS Process Window Screening Examples 4-6 focus on the synergistic optimization of CO2 pressure and electrostatic voltage in SAS-ESS technology, aiming to further control the crystallization kinetics of nanoparticles while keeping the formulation system unchanged, thereby achieving smaller particle size and lower PDI. Examples 4-6 use the same formulation composition, with nervonic acid theoretically accounting for 40.0% of the total solid feed amount, and employ a two-factor, three-level approach to investigate the changes in pressure and electrostatic voltage (pressure: 8 / 10 / 12 MPa; electrostatic voltage: 15 / 20 / 25 kV).

[0101] Example 4 This example provides a brain-targeting nanoparticle and its preparation method. The specific preparation steps are as follows: (1) Preparation of oil and aqueous phases: Oil phase preparation: Nervate (900 mg) and PS (450 mg) were dissolved in 15 mL of anhydrous ethanol and sonicated in a water bath at 50 °C for 30 minutes under nitrogen protection to prevent light exposure. Aqueous phase preparation: CS (600 mg) and PEG (300 mg) were dissolved in 10 mL of acetate buffer (0.1 mol / L, pH 5.0) and magnetically stirred (800 rpm, 40 °C) until completely dissolved. The solution was then filtered through a 0.45 μm microporous membrane to remove impurities. In this example, the mass ratio of CS, PS, and PEG was 4:3:2.

[0102] (2) High-pressure emulsification: The oil phase is injected into the aqueous phase at 1 mL / min and circulated 5 times at 100 MPa using an APV-2000 high-pressure homogenizer to obtain the primary emulsion.

[0103] (3) Supercritical crystallization spray: The colostrum was pumped into the P-1 supercritical crystallization system (i.e., supercritical fluid crystallization system) produced by PuCui Supercritical Company using an electrostatic nozzle at a rate of 2 mL / min. The CO2 pressure in the supercritical fluid crystallization system was 8 MPa, the temperature was 38℃, and the supercritical CO2 flow rate was 15 L / min. The electrostatic nozzle parameters were fixed at an orifice diameter of 150 μm and an electrostatic voltage of 15 kV. Atomization crystallization was carried out for 60 minutes to obtain nanoparticles. The nanoparticles were washed with supercritical CO2 for 30 minutes and then freeze-dried (-80℃, 0.1 mbar) to obtain the final product.

[0104] Example 5 The only difference between the preparation method of brain-targeting nanoparticles in this example and that in Example 4 is that in step (3) of this example, the CO2 pressure is 10 MPa and the electrostatic voltage is 20 kV, while the other steps are the same as in Example 4.

[0105] According to the HPLC method described above, the measured drug loading of nervonic acid in the nanoparticles prepared in Example 5 was 39.6±1.2%, and the encapsulation efficiency was 94.2±0.8% (n=3).

[0106] The brain targeting coefficient of the nanoparticles prepared in Example 5 was tested using the following method: In this invention, the brain targeting coefficient is the apparent logarithmic value of the ratio of brain tissue concentration to plasma concentration 6 hours after drug administration. Before calculation, the brain tissue concentration was standardized to approximately 1.0 g / mL based on brain tissue density: apparent logBB = log10[(C brain / ρ brain ) / C blood ], where C brain The concentration of nervonic acid in brain tissue, expressed in μg / g, C blood ρ represents the concentration of nervonic acid in the blood, expressed in μg / mL. brain This indicates brain tissue density, expressed as 1.0 g / mL, and is used to express brain tissue concentration (C). brain Convert from μg / g to μg / mL Determination steps: (1) Animal administration and sample collection: Male SD rats (weighing 200-250 g) or APP / PS1 transgenic AD model mice were fasted for 12 hours and allowed free access to water. The drug was administered orally at the set dose (e.g., 120 mg / kg for the nanoparticle group, equivalent to 50 mg / kg for nervonic acid), with 6 animals in each group. Blood was collected from the heart at the peak drug time point (e.g., 6 hours) after administration, heparin was used for anticoagulation, and plasma was separated by centrifugation at 3000 rpm for 15 min. After euthanizing the animals, the whole brain was quickly collected, rinsed with physiological saline to remove surface blood, dried with filter paper, and weighed and recorded. (2) Sample pretreatment: Approximately 200 mg of brain tissue was accurately weighed and homogenized with 3 times the amount of physiological saline (IKA T10, 10000 rpm, ice bath). Take 100 μL of plasma or 200 μL of brain homogenate, add 3 times the amount of acetonitrile (containing internal standard d4-nervonic acid 50 ng / mL), vortex for 3 min, centrifuge at 12000 rpm for 10 min, take the supernatant and filter through a 0.22 μm filter membrane, and continue to use the filtrate for LC-MS / MS analysis. (3) LC-MS / MS determination: Chromatographic conditions: C18 column (2.1×100 mm, 1.8 μm), column temperature 40℃, mobile phase is methanol-0.1% formic acid aqueous solution (85:15), flow rate 0.3 mL / min, injection volume 5 μL. Mass spectrometry conditions: electrospray ionization source (ESI), negative ion mode, multiple reaction monitoring (MRM), nervonic acid precursor ion m / z 365.3→daughter ion m / z 321.2, internal standard d4-nervonic acid m / z 369.3→325.2.

[0107] (4) Data processing and calculation: A standard curve was plotted using the ratio of standard concentration to peak area, and the concentration of nervonic acid in brain tissue and plasma was calculated. logBB was calculated using the following formula: logBB = log 10 (Cbrain / Cblood), where Cbrain is measured in μg / g and Cblood in μg / mL. A higher logBB value indicates stronger brain targeting of the drug.

[0108] The brain targeting coefficient of the nanoparticles in Example 5 was determined using this method in SD rats. The concentration of nervonic acid in brain tissue was measured to be 8.8 ± 0.7 μg / g, and the plasma concentration was 1.9 ± 0.3 μg / mL. The calculated values ​​were logBB = log 10 (8.8 / 1.9) = 0.67. The logBB of the free nervonic acid raw material is -1.2, indicating that the nanoparticles of this invention improve the brain targeting of nervonic acid by approximately 1.87 log units.

[0109] Example 6 The only difference between the preparation method of brain-targeting nanoparticles in this example and that in Example 4 is that in step (3) of this example, the CO2 pressure is 12 MPa and the electrostatic voltage is 25 kV, while the other steps are the same as in Example 4.

[0110] The average particle size, PDI, and encapsulation efficiency of the nanoparticles prepared in Examples 4-6 were tested using the test methods described above. The specific test results are shown in Table 2 below.

[0111] Table 2. Average particle size, PDI, and encapsulation efficiency of the nanoparticles prepared in Examples 4-6

[0112] As shown in Table 2, among the process parameters in this group, the nanoparticles obtained in Example 5 under the conditions of CO2 pressure of 10 MPa and electrostatic voltage of 20 kV have the smallest average particle size and the lowest PDI, indicating that the droplet refinement and supercritical extraction process are better matched under these conditions.

[0113] Crystallinity was determined using an X-ray diffractometer (Rigaku SmartLab, Japan) with a scanning range of 5°–40° (2θ) and a step size of 0.02°. Crystallinity was calculated using Jade software. Specifically, the crystallinity of nervonic acid was 87.1% in Example 4, 92.5% in Example 5, and 78.4% in Example 6. The higher crystallinity of Example 5 compared to Examples 4 and 6 suggests that optimal pressure and voltage conditions are more conducive to the formation of particle cores with higher crystallinity and more uniform distribution. The residual organic solvent in the nanoparticles prepared in Example 5 was <8 ppm (measured by GC-FID), and the retention rate of the cis isomer of nervonic acid was 99.2% (measured by the aforementioned GC-MS method), indicating that this process window performs better in terms of particle uniformity and retention of active ingredients.

[0114] V. Examples 7-9 and Comparative Examples 1-2: Example 7 This example provides a brain-targeting nanoparticle and its preparation method. The specific preparation steps are as follows: (1) Raw material pretreatment: Nervonic acid (cis-15-tetracosenoic acid, purity ≥99%, purchased from Sigma) was stored in the dark under nitrogen protection; chitosan (degree of deacetylation 90%, number average molecular weight 20 kDa) was sterilized by filtration through a 0.22 μm microporous membrane; phosphatidylserine (PS, egg yolk source, purity 95%); polyethylene glycol (PEG-5000).

[0115] (2) Preparation of oil and aqueous phases: Accurately weigh 1.0 g of nervonic acid and 0.3 g of PS, dissolve them in 5 mL of anhydrous ethanol (dissolve by sonication in a water bath at 50 °C for 30 min), and store the solution in a nitrogen-filled, sealed container protected from light.

[0116] Add 0.5 g of CS and 0.2 g of PEG to 10 mL of acetate buffer (0.1 mol / L, pH 5.0), stir magnetically (800 rpm, 40℃) until completely dissolved, and filter through a 0.45 μm filter membrane to remove impurities.

[0117] (3) High-pressure emulsification: The oil phase was injected into the aqueous phase at a rate of 1 mL / min, and the mixture was circulated 5 times using a high-pressure homogenizer (APV-2000) at a pressure of 100 MPa to obtain the primary emulsion (particle size of approximately 180 nm, PDI=0.18).

[0118] (4) SAS-ESS crystallization: The colostrum was pumped into a supercritical reactor (P-1 supercritical crystallization equipment manufactured by PuCui Supercritical Company) at a rate of 2 mL / min using an electrostatic nozzle. Supercritical CO2 (pressure 10 MPa, temperature 38℃, flow rate 15 L / min) was introduced into the supercritical reactor. The colostrum was then atomized and crystallized by an electrostatic nozzle (orifice diameter 150 μm, voltage 20 kV) for 60 min to obtain nanoparticles.

[0119] (5) Post-processing: The precipitated nanoparticles were collected, washed with supercritical CO2 for 30 min to remove solvent residue (ethanol residue was <8 ppm as determined by GC-FID), and freeze-dried (-80℃, 24 h) to obtain a white powder.

[0120] The average particle size and PDI of the nanoparticles in Example 7 were determined using a Malvern Zetasizer Nano ZS. The average particle size was 92.3 ± 3.1 nm (PDI = 0.11), and the Zeta potential was +28.4 ± 1.2 mV, as measured by the test method described above.

[0121] 10 mg of the nanoparticles prepared in Example 7 were dissolved in a mixture of acetonitrile and water (70:30). The nervonic acid content was determined by HPLC (Agilent 1260). The chromatographic conditions were: C18 column, mobile phase of methanol and 0.1% (v / v) formic acid aqueous solution (volume ratio of methanol to formic acid aqueous solution of 85:15), flow rate of 1.0 mL / min, and detection wavelength of 210 nm. The drug loading was calculated to be 47.1 ± 0.8%, and the encapsulation efficiency was 94.2 ± 1.5%.

[0122] The in vitro release assay was performed as follows: Simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 6.8, containing 0.5% Tween 80 to maintain leak conditions) were prepared separately. Nanoparticles equivalent to 10 mg of nervonic acid were accurately weighed and placed in a dialysis bag with a molecular weight cutoff of 8-14 kDa. The bag was then placed in 50 mL of release medium and shaken at 37.0 ± 0.5 °C and 100 rpm. Release was first performed in SGF for 2 h, then transferred to fresh SIF for further release over 48 h. At predetermined time points, 1 mL of the external phase sample was collected and an equal volume of fresh medium was added. The nervonic acid content was determined under the aforementioned HPLC conditions, and the cumulative release rate was calculated. The test results showed that the nanoparticles in Example 7 had a release rate of 2.8 ± 0.6% in simulated gastric fluid after 2 hours and a cumulative release rate of 96.3 ± 1.2% in simulated intestinal fluid after 48 hours.

[0123] Example 8 This embodiment was conducted in a 50 L supercritical reactor (P-1 supercritical crystallization equipment manufactured by Puchu Supercritical Company). The specific experimental procedure is as follows: (1) Raw materials: 50 g nervonic acid, 15 g PS, 30 g CS, 15 g PEG, 250 mL anhydrous ethanol, 500 mL acetate buffer (0.1 mol / L, pH 5.0).

[0124] (2) Preparation steps: The same steps as in Example 5 were followed in the preparation process, which was carried out in a 50 L supercritical reactor (P50 type). The primary emulsion was prepared using an industrial-grade high-pressure homogenizer (ATS AH-2000 type), with a homogenization pressure of 100 MPa and 5 cycles. Electrostatic spray parameters: nozzle orifice diameter 150 μm, electrostatic voltage 20 kV, CO2 pressure 10 MPa, temperature 38℃, flow rate 15 L / min, and spraying time 60 min. Nanoparticles were prepared according to these parameters.

[0125] The tests were conducted according to the methods described above. The average particle size of the nanoparticles obtained in Example 8 was measured to be 91±8 nm (n=3), PDI=0.15, encapsulation efficiency was 93.7±1.1%, drug loading was 42.6±0.5%, and organic solvent residue was <8 ppm. The scale-up experiment of Example 8 was repeated three times. The average particle size, PDI, encapsulation efficiency, drug loading, and organic solvent residue of each batch of nanoparticles were tested. The data reproducibility among the three batches was good, with RSD<5%, indicating that the process has the potential for large-scale production. Therefore, at a CO2 pressure of 8-12 MPa and an electrostatic voltage of 15-25 kV, continuous production of highly crystalline nanoparticles can be achieved. Under 50L scale-up conditions, nanoparticles with an average particle size of about 91 nm and a PDI of about 0.15 can still be stably obtained. Compared with the 308±18 nm of the traditional SAS route shown in Comparative Example 2 of this application, the particle size is significantly reduced, indicating that the process has good scale-up reproducibility.

[0126] Example 9 This example provides a brain-targeting nanoparticle and its preparation method. The specific preparation steps are as follows: (1) Raw material pretreatment: Nervonic acid (cis-15-tetracosenoic acid, purity ≥99%, purchased from Sigma) was stored in the dark under nitrogen protection; chitosan (degree of deacetylation 90%, number average molecular weight 20 kDa) was sterilized by filtration through a 0.22 μm microporous membrane; phosphatidylserine (PS, egg yolk source, purity 95%); polyethylene glycol (PEG-5000).

[0127] (2) Preparation of oil and aqueous phases: Accurately weigh 0.6 g of nervonic acid and 0.45 g of PS, dissolve them in 5 mL of anhydrous ethanol (dissolve by sonication in a water bath at 50°C for 30 min), and store the solution in a nitrogen-filled, sealed container protected from light.

[0128] Add 0.6 g of CS and 0.3 g of PEG to 10 mL of acetate buffer (0.1 mol / L, pH 4.5), and stir magnetically (800 rpm, 40°C) until completely dissolved. Filter through a 0.45 μm membrane to remove impurities. In this example, the mass ratio of CS, PS, and PEG is 4:3:2.

[0129] (3) High-pressure emulsification: The oil phase was injected into the aqueous phase at a rate of 1 mL / min, and the mixture was circulated 5 times using a high-pressure homogenizer (APV-2000) at a pressure of 100 MPa to obtain the primary emulsion (particle size of approximately 180 nm, PDI=0.18).

[0130] (4) SAS-ESS crystallization: The colostrum was pumped into a supercritical reactor (P-1 supercritical crystallization equipment manufactured by PuCui Supercritical Company) at a rate of 2 mL / min using an electrostatic nozzle. Supercritical CO2 (pressure 10 MPa, temperature 38℃, flow rate 15 L / min) was introduced into the reactor. The colostrum was then atomized and crystallized by an electrostatic nozzle (orifice diameter 150 μm, voltage 20 kV) for 60 min to obtain nanoparticles.

[0131] (5) Post-processing: The precipitated nanoparticles were collected, washed with supercritical CO2 for 30 min to remove solvent residue (ethanol residue was <8 ppm as determined by GC-FID), and freeze-dried (-80℃, 24 h) to obtain a white powder.

[0132] This invention applies a 20 kV DC high voltage to the pre-emulsion via an electrostatic nozzle. Under the influence of the electric field, the pre-emulsion flows out of the nozzle, forming a stable Taylor cone and splitting into submicron-sized charged droplets. The electrostatic repulsion between these charged droplets helps suppress early aggregation. Simultaneously, supercritical CO2 rapidly penetrates into the droplets, causing a significant decrease in the polarity of the solvent environment, which promotes the in-situ solidification of nervonic acid within the nanocavities formed by CS-PS. Subsequent continuous washing with supercritical CO2 enables a one-step transformation from a liquid precursor system to solid nanopowder.

[0133] To evaluate the true technical contribution of the SAS-ESS route of this invention compared to the conventional emulsion-solvent evaporation (ESE) and conventional supercritical antisolvent (SAS) routes, Comparative Examples 1 and 2 were made to maintain the same formulation composition and the same precursor emulsion preparation steps as Example 9, only changing the subsequent curing or atomization method, in order to improve the fairness of the process comparison and the consistency of data interpretation.

[0134] Comparative Example 1 This example provides a method for preparing nanoparticles, which uses the emulsification-solvent evaporation (ESE) method commonly used in existing technologies. The specific steps are as follows: (1) Raw material preparation: In order to ensure comparability with Example 9, Comparative Example 1 uses the same formulation as Example 9, namely 0.6g nervonic acid, 0.45g phosphatidylserine, 0.6g chitosan, 0.3g polyethylene glycol, 5mL anhydrous ethanol and 10mL 0.1mol / L acetate buffer (pH 4.5).

[0135] (2) Preparation of oil and aqueous phases: Nervonic acid and PS were dissolved in anhydrous ethanol as the oil phase; CS and PEG were dissolved in acetate buffer as the aqueous phase.

[0136] (3) Emulsification: The oil phase was injected into the aqueous phase at a rate of 1 mL / min, and the mixture was circulated 5 times at 100 MPa using a high-pressure homogenizer (APV-2000) to obtain the same precursor emulsion as in Example 9.

[0137] (4) Curing: Place the precursor emulsion at room temperature and stir magnetically (300 rpm) for 24 h to allow the ethanol to evaporate under normal pressure and solidify into granules.

[0138] (5) Collection and drying: The particles were collected by high-speed centrifugation, washed three times with deionized water, and freeze-dried to obtain the product of Comparative Example 1.

[0139] The main difference between this comparative route and Example 9 is that the subsequent curing method is changed from "SAS-ESS instant curing" to "atmospheric pressure evaporation curing". Therefore, it can be used to evaluate the effect of the emulsification-solvent evaporation route on particle size, encapsulation efficiency and residual solvent.

[0140] Comparative Example 2 This example provides a method for preparing nanoparticles, which uses the conventional supercritical antisolvent (SAS) technology commonly used in existing technologies. The specific steps are as follows: (1) Raw material preparation: In order to ensure comparability with Example 9, Comparative Example 2 adopted the same formulation as Example 9, namely 0.6g nervonic acid, 0.45g phosphatidylserine, 0.6g chitosan, 0.3g polyethylene glycol, 5mL anhydrous ethanol and 10mL 0.1mol / L acetate buffer (pH 4.5).

[0141] (2) Preparation and emulsification of oil and aqueous phases: The oil and aqueous phases were prepared according to the method of Example 9 and homogenized by cycling at 100 MPa 5 times to obtain the precursor emulsion.

[0142] (3) SAS crystallization: The precursor emulsion was sprayed into the supercritical reactor at a flow rate of 2 mL / min through a standard stainless steel mechanical nozzle (orifice diameter 150 μm) under the conditions of CO2 pressure 10 MPa, temperature 38 °C and CO2 flow rate 15 L / min. No electrostatic field was applied, so that the system only underwent the supercritical antisolvent precipitation process.

[0143] (4) Post-treatment: After the spraying is completed, continue to pass supercritical carbon dioxide to wash for 1 hour, remove the residual solvent, and collect the dry powder.

[0144] The main difference between this comparative route and Example 9 is that the atomization method is changed from "electrostatic spraying" to "mechanical nozzle spraying". Therefore, it can be used to evaluate the role of electric field in refining droplets and suppressing agglomeration in the SAS-ESS process.

[0145] Due to the lack of high-field shear and charge repulsion assistance from electrostatic spraying, the precursor droplets mainly undergo the process of "mechanical atomization - supercritical diffusion - solvent extraction - precipitation nucleation". The droplet size is more easily affected by the nozzle atomization capability and the turbulent conditions inside the vessel, thus making it easier for the particle size distribution to widen and the particles to agglomerate.

[0146] The properties of the nanoparticles prepared in Example 9 and Comparative Examples 1-2 were tested according to the above test methods, and the results are shown in Table 3 below.

[0147] Table 3. Performance comparison of nanoparticles prepared in Example 9 and Comparative Examples 1-2

[0148] Table 3 shows that SAS has advantages over ESE and conventional SAS in terms of particle size, PDI, encapsulation efficiency, and residual solvent. With the formulation composition and pre-emulsion preparation steps remaining consistent, and only the subsequent particle formation route changed, Comparative Example 2, using the conventional SAS route, although particles could be obtained through supercritical antisolvent precipitation, suffered from a significantly wider particle size distribution and decreased encapsulation efficiency and drug loading due to the lack of electrospray-enhanced atomization and electric field suppression of crystal growth. Comparative Example 1, using the emulsification-solvent evaporation route, experienced increased particle size, wider particle size distribution, and a significant increase in residual solvent due to the long duration of the evaporation and solidification process and the dependence of solvent removal on mass transfer and environmental conditions. In contrast, the route of this invention, through the synergistic effect of droplet refinement by electrospraying and rapid extraction with supercritical carbon dioxide, achieves a narrower particle size distribution, higher encapsulation efficiency, and lower residual solvent.

[0149] VI. Examples 10-12 and Comparative Examples 3-6 Example 10 This example provides a method for preparing brain-targeting nanoparticles, which differs from the preparation method in Example 1 only in that the degree of deacetylation (DD) of chitosan in this example is 85%.

[0150] Example 11 This example provides a method for preparing brain-targeting nanoparticles, which differs from the preparation method in Example 1 only in that the degree of deacetylation (DD) of chitosan in this example is 92%.

[0151] Example 12 This example provides a method for preparing brain-targeting nanoparticles, which differs from the preparation method in Example 1 only in that the degree of deacetylation (DD) of chitosan in this example is 95%.

[0152] Comparative Example 3 This example provides a method for preparing brain-targeting nanoparticles, which differs from the preparation method in Example 1 only in that the degree of deacetylation (DD) of chitosan in this example is 65%.

[0153] Comparative Example 4 This example provides a method for preparing brain-targeting nanoparticles, which differs from the preparation method in Example 1 only in that the degree of deacetylation (DD) of chitosan in this example is 70%.

[0154] Comparative Example 5 This example provides a method for preparing brain-targeting nanoparticles, which differs from the preparation method in Example 1 only in that the degree of deacetylation (DD) of chitosan in this example is 98%.

[0155] Comparative Example 6 This example provides a method for preparing brain-targeting nanoparticles, which differs from the preparation method in Example 1 only in that the degree of deacetylation (DD) of chitosan in this example is 99%.

[0156] The zeta potential and brain targeting coefficient of the nanoparticles obtained in Examples 10, 12, 3, and 6 were tested according to the test methods described above. Then, the stability was tested for 14 days according to the test methods described below. The specific test methods are as follows: The 14-day stability (leakage rate) determination method is as follows: The leakage rate of nervonic acid in the nanoparticles during storage was determined by ultrafiltration centrifugation-high performance liquid chromatography (UF-HPLC). Approximately 50 mg of freshly prepared nanoparticle powder was placed in a sealed, light-protected environment and stored at 25℃±2℃ for 14 days. Samples were taken on day 0 and day 14, and approximately 10 mg of nanoparticles were accurately weighed and dispersed in 1 mL of deionized water. After ultrasonic dispersion, the mixture was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa and centrifuged at 4℃ and 12000 g for 30 min. The filtrate was collected. The concentration of free nervonic acid in the filtrate was determined under the HPLC conditions described above. The leakage rate (%) was calculated using the following formula: Leakage rate (%) = (mass of free nervonic acid in the filtrate on day 14 / total mass of nervonic acid in the nanoparticles) × 100%. The total mass of nervonic acid in the nanoparticles was obtained by HPLC determination after complete extraction of the nanoparticles with acetonitrile-water (70:30). Each sample was measured in triplicate.

[0157] The performance data of the nanoparticles prepared in Examples 10, 12, 3, and 6 are shown in Table 4 below.

[0158] Table 4. Zeta potential, 14-day stability, and brain targeting coefficient of nanoparticles with different degrees of chitosan deacetylation.

[0159] As shown in Table 4, the degree of deacetylation (DD) determines the number of primary amine groups on the chitosan molecular chain. When DD is below 80%, there are too many acetyl groups on the chitosan molecular chain, resulting in insufficient intermolecular hydrogen bond strength. This leads to an overly loose coating layer formed during the SAS-ESS high-pressure spraying process, making nervonic acid prone to physical leakage during storage (the leakage rate is as high as 18.5% after 14 days). Furthermore, low DD results in a zeta potential below +10 mV, preventing effective adsorption to the negatively charged surface of brain microvascular endothelial cells through charge interactions.

[0160] The degree of deacetylation of chitosan determines its primary amine content, which directly affects the charge density of the carrier system, its interfacial interaction with phosphatidylserine, and its adsorption and encapsulation capacity on the nervonic acid crystal surface. In the ternary composite system constructed in this invention, the nanoparticle formation process involves a coupled process of "instantaneous nucleation of nervonic acid" and "synchronous solidification of the chitosan-phosphatidylserine-polyethylene glycol interfacial layer." Insufficient charge density of chitosan leads to incomplete formation of the interfacial layer, causing migration and leakage of the nervonic acid drug during post-processing or storage and reducing the encapsulation efficiency. Excessive charge density may cause strong electrostatic repulsion and local gelation between molecular chains, thereby affecting the uniformity of the proemulsion and the stability of the electrospray jet, ultimately leading to a wider particle size distribution and reduced drug loading.

[0161] The properties of the promulgated emulsions and nanoparticles prepared in Examples 10, 11, 4, and 5 were tested respectively. Zeta potential was determined by electrophoretic light scattering; encapsulation efficiency and drug loading were calculated using chromatographic quantification combined with ultrafiltration; residual solvent was tested by gas chromatography, repeated 6 times; the Taylor cone stability maintenance time was determined as follows: Following the SAS-ESS conditions described in the examples, fresh promulgated emulsion after high-pressure homogenization was pumped into the nozzle (150 μm orifice) at a flow rate of 2 mL / min. A set voltage was applied, and the morphological changes at the nozzle outlet were observed using a high-speed imaging system. Timing was recorded from the formation of a stable Taylor cone until the cone exhibited obvious oscillation, jet interruption exceeded 1 second, large droplets fell, or the jetting pattern changed to an irregular pattern. Each sample was measured at least 3 times, and the average value was taken. The results obtained according to the above testing methods are shown in Table 5 below. Table 5. Performance comparison of colostrum and nanoparticles under different degrees of deacetylation.

[0162] The colostrum is an emulsion system formed after high-pressure homogenization. In it, CS, PS, PEG and nervonic acid have not yet undergone the polarity drop and solidification induced by supercritical CO2, and the nanoparticles are not fully formed. The surface molecular arrangement and charge distribution are different from the final solid nanoparticles. The final nanoparticles are formed after the SAS-ESS process (supercritical CO2 desolventization, electrostatic spraying and washing). The solvent is removed and the surface structure is more compact. Therefore, its zeta potential and particle size distribution are not exactly the same as those of the colostrum.

[0163] As shown in Tables 4 and 5, when the degree of deacetylation of chitosan is between 85% and 95%, the system can maintain good electrospray jet stability while ensuring sufficient charge density to form a stable interfacial coating layer. Therefore, it ultimately achieves high encapsulation efficiency and drug loading, while maintaining a small particle size and narrow distribution. When the degree of deacetylation is below 80%, insufficient primary amine content leads to low charge density, low Zeta potential in the proemulsion and final particles, and insufficient interfacial layer formation. This makes nervonic acid more prone to migration during supercritical extraction and subsequent drying, resulting in a significant decrease in encapsulation efficiency and drug loading. Conversely, when the degree of deacetylation is above 95%, excessively high charge density enhances intersegmental repulsion and leads to a tendency for localized gelation, significantly reducing Taylor cone stability and atomization efficiency of the spray droplets. This ultimately results in increased particle size and polydispersity index, while also causing a decrease in encapsulation efficiency and drug loading.

[0164] VII. Examples 13-14 and Comparative Examples 7-10 Example 13 This example provides a method for preparing brain-targeting nanoparticles. The specific preparation steps are as follows: (1) Raw material pretreatment: Nervonic acid (cis-15-tetracosenoic acid, purity ≥99%, purchased from Sigma) was stored in the dark under nitrogen protection; chitosan (degree of deacetylation 90%, number average molecular weight 20 kDa) was sterilized by filtration through a 0.22 μm microporous membrane; phosphatidylserine (PS, egg yolk source, purity 95%); polyethylene glycol (PEG-5000).

[0165] (2) Preparation of oil and aqueous phases: Accurately weigh 0.8 g of nervonic acid and 0.3 g of PS, dissolve them in 5 mL of anhydrous ethanol (dissolve by sonication in a water bath at 50°C for 30 min), and store the solution in a nitrogen-filled, sealed container protected from light.

[0166] Add 0.5 g of CS and 0.2 g of PEG to 10 mL of acetate buffer (0.1 mol / L, pH 4.5), stir magnetically (800 rpm, 40℃) until completely dissolved, and filter through a 0.45 μm filter membrane to remove impurities.

[0167] (3) High-pressure emulsification: The oil phase was injected into the aqueous phase at a rate of 1 mL / min, and the mixture was circulated 5 times using a high-pressure homogenizer (APV-2000) at a pressure of 100 MPa to obtain the colostrum.

[0168] (4) SAS-ESS crystallization: The colostrum was pumped into a supercritical reactor (P-1 supercritical crystallization equipment manufactured by Pucui Supercritical Company) at a rate of 2 mL / min. Supercritical CO2 (pressure 10 MPa, temperature 38℃, flow rate 15 L / min) was introduced into the supercritical reactor. The colostrum was atomized and crystallized by an electrostatic nozzle (orifice diameter 150 μm, voltage 20 kV) for 60 min to obtain nanoparticles.

[0169] (5) Post-processing: The precipitated nanoparticles were collected, washed with supercritical CO2 for 30 min to remove solvent residue (ethanol residue was <8 ppm as determined by GC-FID), and then freeze-dried (-80℃, 24 h) to obtain a white powder.

[0170] Using Malvern Zetasizer Nano ZS assay, the average particle size of the nanoparticles obtained in this example was 132±6 nm, the polydispersity index (PDI) was 0.18±0.02, and the Zeta potential was +29.2±1.3 mV. According to the HPLC method described above, the encapsulation efficiency was 94.9±1.4%, the drug loading was 42.2±1.6%, and the residual solvent was 3.1±0.4 ppm.

[0171] Example 14 This example provides a method for preparing brain-targeting nanoparticles, which differs from the preparation method in Example 13 only in that the number-average molecular weight of chitosan in this example is 80 kDa.

[0172] Comparative Example 7 This example provides a method for preparing brain-targeting nanoparticles, which differs from the preparation method in Example 13 only in that the number-average molecular weight of chitosan in this example is 2 kDa.

[0173] Comparative Example 8 This example provides a method for preparing brain-targeting nanoparticles, which differs from the preparation method in Example 13 only in that the number-average molecular weight of chitosan in this example is 3 kDa.

[0174] Comparative Example 9 This example provides a method for preparing brain-targeting nanoparticles, which differs from the preparation method in Example 13 only in that the number-average molecular weight of chitosan in this example is 150 kDa.

[0175] Comparative Example 10 This example provides a method for preparing brain-targeting nanoparticles, which differs from the preparation method in Example 13 only in that the number-average molecular weight of chitosan in this example is 300 kDa.

[0176] In the ternary composite carrier system of this invention, chitosan not only serves to form the cationic interface layer, but also directly affects the viscosity of the aqueous system, the shear dispersion efficiency of the primary emulsion droplet formation, and the jet stability and nucleation-coating synchronization during supercritical crystallization spraying. To facilitate comparison of differences between different embodiments and comparative examples, a unified testing system was used to characterize and analyze the data of each sample below.

[0177] The viscosity of the colostrum, the average particle size of the nanoparticles (tested according to the test methods described above), and the cell uptake (relative intensity) of the nanoparticles prepared in Examples 13-14, Comparative Example 7, and Comparative Example 10 were tested respectively. The specific test results are shown in Table 6 below.

[0178] Colostrum viscosity determination: Fresh colostrum used immediately after high-pressure homogenization was taken and its apparent viscosity was measured using a rotational viscometer at 25.0±0.1℃. Each sample was repeated 3 times and the average value was taken.

[0179] Cellular uptake assay (relative intensity method): The ability of nanoparticles to be taken up by cells is determined using a fluorescent labeling method combined with flow cytometry or a fluorescent microplate reader. Specific steps are as follows: (1) Fluorescently labeled nanoparticles: Take 10 mg of the nanoparticles to be tested, disperse them in 1 mL of PBS buffer (0.01 mol / L, pH 7.4), add the near-infrared lipid-soluble fluorescent probe DiR (DiIC18(7), 1,1'-bisoctadecyl-3,3,3',3'-tetramethylindole tricarbonine iodide) (purchased from Thermo Fisher, catalog number D12731) to make the final concentration 10 μmol / L, stir in the dark (200 rpm, 25℃) for 30 minutes, ultrafilter centrifuge (molecular weight cutoff 100 kDa, 12000 g, 15 minutes) to remove the free fluorescent probe, wash twice with PBS, redisperse in PBS, and adjust the nanoparticle concentration to 1 mg / mL.

[0180] (2) Cell culture: Human brain microvascular endothelial cells hCMEC / D3 (purchased from Millipore) were seeded in 24-well plates using the BCA (dioctanoic acid method) and seeded in 24-well plates (5 × 10⁶ cells per well). 4 (100 cells) were cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator for 24 hours until the cells reached approximately 80% confluence.

[0181] (3) Drug administration and incubation: Discard the culture medium and add 500 μL of serum-free culture medium containing fluorescently labeled nanoparticles (concentration 200 μg / mL) to each well. Incubate at 37℃ and 5% CO2 for 2 hours in the dark. Each sample is tested in triplicate. A blank control group (no nanoparticles added, only culture medium added) and a negative control group (added with an equal volume of PBS) are also set up.

[0182] (4) Cell washing and lysis: After incubation, discard the supernatant and wash the cells three times with pre-cooled PBS buffer to remove untaken or surface-adsorbed nanoparticles. Add 200 μL of RIPA lysis buffer (containing protease inhibitor) to each well and lyse on ice for 30 minutes. Collect the cell lysate.

[0183] (5) Fluorescence assay: 100 μL of cell lysis buffer was transferred to a black 96-well plate and the fluorescence intensity was measured using a fluorescence microplate reader (such as Thermo Fisher Varioskan LUX). Excitation / emission wavelength of DiR: 748 / 780 nm. Each sample was measured 3 times and the average value was taken.

[0184] (6) Protein quantification and normalization: The remaining cell lysate was used to determine the total protein concentration using the BCA method. The fluorescence intensity was normalized to the total protein content to eliminate the influence of cell number differences. Relative uptake was calculated using the following formula: Relative intensity = (Fluorescence intensity of sample well - Fluorescence intensity of blank control) / Total protein concentration of sample well (μg / mL) Set the relative intensity of the blank control group to 1 (or 0) and calculate the relative multiples of each experimental group.

[0185] The results indicate that cellular uptake is expressed as a relative intensity (i.e., the ratio of the experimental group to the blank control group), which is dimensionless. A higher value indicates a stronger ability of the cells to take up the nanoparticle.

[0186] As shown in Table 6, when the number-average molecular weight of chitosan is in the range of 5-100 kDa, the colostrum viscosity and final particle size remain in the range more suitable for brain-targeted delivery. When the molecular weight is too low, although the colostrum viscosity is small, the cellular uptake capacity is significantly reduced. When the molecular weight is too high, the colostrum viscosity increases sharply and is accompanied by a significant increase in particle size, indicating that the molecular weight of chitosan has a significant impact on the rheology and cell interaction of the precursor system.

[0187] The performance data obtained by the above method are shown in Table 6 below.

[0188] Table 6. Effects of chitosan molecular weight on colostrum viscosity, particle size, and cellular uptake.

[0189] As shown in Table 6, when the number-average molecular weight of chitosan is between 5 and 100 kDa, the colostrum exhibits good shear-thinning properties, making it suitable for nano-sizing in a high-pressure homogenizer (100 MPa). If the number-average molecular weight of chitosan exceeds 150 kDa, the viscosity of the colostrum increases exponentially with molecular weight. Upon entering the electrostatic nozzle, it cannot form a stable Taylor cone, but instead is ejected as broken liquid filaments, causing the particle size to surge directly to over 800 nm, completely losing its brain-targeting significance. Conversely, when the number-average molecular weight of chitosan is below 5 kDa, the chitosan molecular chains are too short to form an effective physical barrier through intermolecular entanglement, and nervonic acid is directly flushed off the carrier surface by the antisolvent during spraying. Compared to Examples 14, 7, and 10, the chitosan in Example 13 exhibits the strongest cellular uptake capacity at a molecular weight of 20 kDa; molecular weights that are too low or too high are detrimental to cellular uptake.

[0190] To further explain the influence of different molecular weights on spray stability and final nanoparticle formation, in addition to the initial emulsion viscosity and cellular uptake results shown in Table 6, this invention further measured the apparent viscosity of the CS-PEG aqueous phase before high-pressure homogenization, the average droplet size of the initial emulsion, the electrospray jet stability, and the final nanoparticle properties. The test results are shown in Table 7 below. It should be noted that the "initial emulsion viscosity" in Table 6 refers to the viscosity of the entire emulsion system after high-pressure homogenization at 25°C; the "apparent viscosity of the aqueous phase" in Table 7 refers to the viscosity of the CS-PEG aqueous phase before high-pressure homogenization at 25°C and a shear rate of 10 s⁻¹. -1 The viscosity measured under the same conditions, although the two measurements are different, are both used to characterize the effect of molecular weight changes on rheology and spray stability.

[0191] Method for determining the apparent viscosity of the aqueous phase: After preparing the CS-PEG aqueous phase, immediately measure the viscosity using a rotational viscometer (such as Brookfield DV2T) at 25.0±0.1℃, and record the shear rate for 10 s. -1 The apparent viscosity at that time was measured, and each sample was repeated 3 times, and the average value was taken.

[0192] The Taylor cone stability maintenance time was tested according to the test method described above.

[0193] The performance data of the nanoparticles prepared in Examples 13-14 and Comparative Examples 8-9 are shown in Table 7 below.

[0194] Table 7. Effects of chitosan molecular weight on primary emulsion droplet and spray stability and nanoparticle properties.

[0195] As shown in Table 7, when the molecular weight of chitosan is too low, the insufficient chain segment length leads to insufficient interfacial film strength and network entanglement. Although the primary emulsion droplets can form smaller droplets in a short time after high-pressure homogenization, they are more prone to interfacial rearrangement and drug migration under supercritical conditions, resulting in reduced encapsulation efficiency and decreased drug loading. When the molecular weight of chitosan is too high, the viscosity of the aqueous phase increases significantly, leading to a decrease in homogenization energy transfer efficiency. The primary emulsion droplet size increases and the distribution becomes wider. At the same time, the high viscosity makes the jet unstable and the atomization efficiency reduced during electrospraying, resulting in larger particle size and higher polydispersity index, which in turn affects the mass transfer efficiency of supercritical online washing, thereby reducing the ability to control residual solvent.

[0196] Based on the above data, it is clear that when the molecular weight of chitosan is within the range defined in this invention, a suitable window can be formed between the aqueous phase viscosity, the size of the primary emulsion droplets, and the stability of the electrospray jet. This allows the primary emulsion to obtain a more uniform droplet distribution after high-pressure homogenization, and simultaneously form a more stable Taylor cone under the action of an electric field. This enables simultaneous nucleation and encapsulation under the condition of a sudden drop in polarity caused by rapid extraction of supercritical carbon dioxide. Therefore, smaller particle size, narrower distribution, and significantly improved encapsulation efficiency and drug loading are ultimately obtained, while residual solvent remains at a low ppm level. Conversely, when the molecular weight is below 5 kDa, although the lower aqueous phase viscosity is conducive to primary emulsion formation, insufficient interfacial film strength leads to a decrease in the stability of the encapsulation structure, resulting in a significant reduction in encapsulation efficiency and drug loading. When the molecular weight is above 100 kDa, the significantly increased aqueous phase viscosity leads to larger primary emulsion droplets and unstable electrospray jet, ultimately resulting in a significantly larger particle size, an increased polydispersity index, and a decrease in encapsulation efficiency and an increase in residual solvent.

[0197] VIII. Examples 15-16 and Comparative Examples 11-12 Example 15 This example provides a method for preparing brain-targeting nanoparticles, which differs from the preparation method in Example 7 only in that the number-average molecular weight of PEG in this example is 2000 Da.

[0198] Example 16 This example provides a method for preparing brain-targeting nanoparticles, which differs from the preparation method in Example 7 only in that the number-average molecular weight of PEG in this example is 10,000 Da.

[0199] Comparative Example 11 This example provides a method for preparing brain-targeting nanoparticles, which differs from the preparation method in Example 7 only in that the number-average molecular weight of PEG in this example is 400 Da.

[0200] Comparative Example 12 This example provides a method for preparing brain-targeting nanoparticles, which differs from the preparation method in Example 7 only in that the number-average molecular weight of PEG in this example is 40,000 Da.

[0201] The protein corona adsorption capacity, plasma half-life, and BBB penetration rate of the nanoparticles prepared in Examples 15-16 and Comparative Examples 11-12 were tested respectively, and the specific test methods are as follows: Method for determining the amount of protein crown adsorption: The total amount of protein adsorbed on the surface of nanoparticles was determined by the BCA method (dioctanoic acid method). The specific steps are as follows: (1) Protein crown formation: Take the nanoparticle sample (containing 1 mg of nervonic acid) and disperse it in 1 mL of PBS buffer (0.01 mol / L, pH 7.4) containing 10% fetal bovine serum (FBS). Incubate at 37°C for 1 hour to allow the surface of the nanoparticles to fully adsorb serum proteins. (2) Nanoparticle separation and washing: After incubation, transfer the suspension to an ultrafiltration centrifuge tube (molecular weight cutoff 100 kDa). Centrifuge at 12000 g for 15 minutes at 4°C and discard the supernatant (containing unbound free proteins). Wash the nanoparticle precipitate 3 times with pre-cooled PBS buffer (pH 7.4). Centrifuge under the same conditions after each wash to thoroughly remove proteins that are not physically adsorbed. (3) Protein elution: The washed nanoparticles were redispersed in 1 mL of PBS buffer containing 2% sodium dodecyl sulfate (SDS), and sonicated (40 kHz, 10 min) to completely elute the protein adsorbed on the surface of the nanoparticles. The eluent was filtered through a 0.22 μm filter membrane, and the filtrate was used for protein quantification. (4) Protein quantification (BCA method): The BCA protein quantification kit was operated according to the instructions. Bovine serum albumin (BSA) was used as the standard, and the absorbance was measured at a wavelength of 562 nm. The total amount of protein in the eluent was calculated according to the standard curve. The protein crown adsorption amount of the nanoparticles was calculated by the following formula: Protein crown adsorption amount (μg / cm³) 2 = Total eluted protein (μg) / Total surface area of ​​nanoparticles (cm²) 2 The total surface area of ​​the nanoparticles is calculated using the following formula: Total surface area = 4πr 2 × N, where N is the number of nanoparticles, determined by the total mass and density of the nanoparticles (assumed to be 1.0 g / cm³). 3 The values ​​were calculated by converting the average particle size (r) to the average particle size (r). Each sample was measured three times, and the average value was taken.

[0202] The plasma half-life was determined by LC-MS / MS to measure the nervonic acid concentration-time curve in rat plasma after oral administration of nanoparticles, and the plasma half-life (t) was calculated. 1 / 2The specific steps are as follows: (1) Animal administration and blood collection: Male SD rats (weight 200-250g, n=6) were fasted for 12 hours and then allowed free access to water. About 0.3mL of blood was collected from the orbital venous plexus at 0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36 and 48 hours after administration and placed in heparinized centrifuge tubes. (2) Plasma sample processing: Whole blood samples were centrifuged at 3000rpm for 15 minutes at 4℃ to separate plasma. 100μL of plasma was accurately pipetted and 3 times the amount of acetonitrile (containing internal standard d4-nervonic acid 50ng / mL) was added. The mixture was vortexed for 3 minutes, centrifuged at 12000rpm for 10 minutes, and the supernatant was filtered through a 0.22μm filter membrane. The filtrate was then used for LC-MS / MS analysis. (3) LC-MS / MS determination: The chromatographic and mass spectrometric conditions were the same as those for the determination of brain targeting coefficient. A standard curve was plotted using the ratio of standard concentration to peak area, and the plasma nervonic acid concentration at each time point was calculated. (4) Calculation of pharmacokinetic parameters: DAS 3.0 software or Phoenix WinNonlin software was used to fit the plasma drug concentration-time curve using a non-compartmental model (NCA), and the elimination phase half-life (t) was calculated. 1 / 2 The calculation formula is: t 1 / 2 = ln2 / λz Wherein, λz is the elimination rate constant, which is determined by the slope of the logarithmic linear regression of the elimination phase concentration-time curve.

[0203] The specific experimental results obtained using the above method are shown in Table 8 below.

[0204] Table 8. Effects of PEG molecular weight on protein corona adsorption, plasma half-life, and BBB penetration.

[0205] Table 8 shows that there is a balance between the molecular weight of PEG and the effects of protein corona adsorption, systemic circulation time, and BBB penetration. When the PEG is too short (400 Da), the protein corona adsorption amount increases significantly, and the plasma half-life is shortened. When the PEG is too long (40000 Da), although the protein corona adsorption amount further decreases and the half-life is prolonged, the BBB penetration rate decreases significantly, suggesting that an excessively thick hydrophilic barrier layer may weaken the effective exposure of surface PS. Considering drug loading, protein corona adsorption, plasma half-life, and BBB penetration rate, controlling the PEG molecular weight within the range of 2000-10000 Da is more beneficial for balancing long circulation and brain delivery.

[0206] IX. Experimental Example 1: Validation of Targeted Distribution in Macaque Brain This test was conducted in an APP / PS1 transgenic Alzheimer's disease (AD) macaque model (macaques carrying human APPsw and PSEN1dE9 mutations). The APP / PS1 transgenic Alzheimer's disease (AD) macaque model was purchased from Guangdong Landao Biotechnology Co., Ltd. The brain-targeting efficiency and therapeutic efficacy of the nanoparticles were quantitatively evaluated. The experimental animals (n=8, half male and half female, aged 5-6 years, weighing 6-8 kg) were approved by the IACUC (approval number: IACUC-2023-101), meeting AAALAC ethical standards. All animals underwent genotyping and behavioral screening (water maze escape latency >60 seconds) before the experiment to confirm them as AD models.

[0207] Formulation and administration: Control group: Traditional emulsion (nervonic acid dissolved in a saline solution containing 28% Cremophor EL by mass). 99m Tc labeling was followed by ultrasonic emulsification until uniformly dispersed, with a final volume of 1 mL.

[0208] Dosage regimen: Both the nanoparticle group and the control group were given a single oral dose of 50 mg / kg (radioactivity 1 mCi / kg), followed by gavage after 12 hours of fasting.

[0209] Brain-targeted dynamics analysis: SPECT / CT imaging: Whole-body imaging (Siemens Symbia T16) was performed at 0.5, 2, 6 and 24 h after drug administration.

[0210] The test results showed that at 6 hours, the nanoparticle group achieved a radioactive enrichment of 9.3% ID / g in the brain, compared to 0.8% ID / g in the control group (P<0.01). Simultaneously, the liver uptake of the nanoparticle group was 2.4% ID / g, while that in the control group was 12.1% ID / g, with brain / liver ratios of 3.88 and 0.07, respectively. These results indicate that the nanoparticles of this invention exhibit a more pronounced tendency for brain enrichment and a lower non-specific distribution in the liver during the in vivo distribution experiment in macaques.

[0211] Animals were sacrificed 24 hours after administration according to the above method, and radioactivity intensity was measured using a PerkinElmer radioimmunoassay counter. The radioactivity intensity of the nanoparticle group was 9.1±0.7% ID / g in brain tissue, 2.3±0.4% ID / g in liver, and 1.2±0.3% ID / g in spleen. In contrast, the radioactivity intensity of the control group was 0.9±0.2% ID / g in brain tissue, 12.5±1.1% ID / g in liver, and 8.3±0.9% ID / g in spleen.

[0212] Brain tissue sections from the aforementioned macaques were immunohistochemically stained with 6E10 antibody (anti-Aβ antibody), and Aβ plaques in the cortical region were quantitatively analyzed using ImageJ software (NIH, USA). Five fields of view were randomly selected from each sample, and the percentage of Aβ plaque area to the total field of view was calculated and averaged. The Aβ plaque area measured in the nanoparticle group was 3.8±0.6%, while the Aβ plaque area measured in the control group was 10.2±1.1%, representing a reduction of approximately 62.7% in the nanoparticle group compared to the control group (P<0.001).

[0213] LRP1 receptor inhibition experiment: APP / PS1 transgenic AD rhesus monkeys (n=4, half male and half female, weighing 6-8 kg) were randomly divided into two groups: the RAP intervention group and the blank control group. The RAP intervention group received an intravenous injection of the LRP1-specific inhibitor RAP (receptor-associated protein, purchased from R&D Systems, catalog number: 6450-RAP-050) 30 minutes before oral administration of nanoparticles. The injection dose was 5 mg / kg (reconstituted with sterile saline to a 1 mg / mL solution, injected at a volume of 5 mL / kg). The blank control group received an equal volume of saline. Both groups of monkeys were subsequently administered the drug orally according to the protocol in Test Example 1 (see attached). 99m Tc-labeled nervonic acid nanoparticles were prepared according to Example 5, with an actual nervonic acid loading of 39.6 ± 1.2% (administered dose 50 mg / kg). Animals were sacrificed 6 hours after administration, and brain tissue was collected. Radioimmunoassay gamma counter was used to measure the radioactivity intensity. The results showed that the radioactivity intensity of the brain tissue after RAP intervention was significantly lower than that of the blank control group, suggesting a correlation between the cross-blood-brain barrier transport of the nanoparticles of this invention and the LRP1-related receptor-mediated process.

[0214] Determination of mucus binding in ex vivo: (1) Tissue preparation: Take about 10 cm of fresh jejunum from a rhesus monkey (euthanized after the experiment) and immediately rinse gently with pre-cooled (4℃) 0.01 mol / L phosphate buffer (PBS, pH 7.4) to remove intestinal contents. Cut the intestinal segment longitudinally, lay it flat on a glass plate in an ice bath with the mucosal side facing up, and absorb the surface moisture with filter paper. Cut it into 1 cm × 1 cm pieces of intestinal tissue with a punch (1.0 cm in diameter) or a scalpel, weigh it and record the area (1 cm). Take 6 pieces from each animal and randomly assign them to different treatment groups. (2) Preparation of nanoparticle suspension: Prepare a suspension of nanoparticles (nervonic acid nanoparticles, prepared according to Example 5) with PBS buffer (0.01 mol / L, pH 7.4) with a concentration of 1 mg / mL (based on the total mass of nanoparticles). Disperse the nanoparticles evenly by sonication (40 kHz, 2 min). (3) Incubation and binding: The small pieces of intestinal tissue were completely immersed in the nanoparticle suspension (1 mL of suspension for each piece of intestinal tissue, placed in a 24-well plate) and incubated in a constant temperature shaker at 37°C for 30 minutes at a shaking speed of 50 rpm to simulate the intestinal peristalsis environment. Each sample was set up with 3 parallel replicates. (4) Washing: After incubation, the small pieces of intestinal tissue were removed with tweezers and placed in a culture dish containing 5 mL of pre-cooled PBS buffer (4°C). The tissue was gently shaken and washed for 30 seconds. The washing was repeated 3 times to remove unbound or poorly physically adsorbed nanoparticles. During the washing process, the surface of the intestinal mucosa should be avoided. (5) Drug extraction: The washed small pieces of intestinal tissue were transferred to a 2 mL centrifuge tube and 1 mL of methanol-acetonitrile mixed solution (volume ratio 1:1, containing 100 ng / mL of internal standard d4-nervonic acid) was added. The tissue was homogenized for 30 seconds using a handheld homogenizer (IKA T10, 10000 rpm, ice bath). The homogenate was transferred to a 15 mL centrifuge tube, and the extraction solvent was added to 2 mL. The mixture was vortexed for 3 minutes. Then, ultrasonic extraction was performed (40 kHz, 30 min, water bath temperature ≤25℃). The extract was centrifuged at 12000 rpm for 15 minutes at 4℃. The supernatant was filtered through a 0.22 μm filter membrane, and the filtrate was used for HPLC analysis. (6) HPLC determination: The chromatographic conditions were the same as before (C18 column, mobile phase methanol-0.1% formic acid aqueous solution = 85:15, flow rate 1.0 mL / min, detection wavelength 210 nm). The concentration of nervonic acid in the extract was calculated using the standard curve, and then the mass of nervonic acid contained in the nanoparticles bound to each piece of intestinal tissue was calculated. (7) Calculation of results: The amount of mucus bound per unit area was calculated using the following formula: Mucus binding amount (μg / cm) 2 = Mass of nervonic acid in the extract (μg) / Intestinal tissue area (cm²) 2 Each sample was measured three times, and the mean ± standard deviation was taken.

[0215] Test results: The mucus binding capacity of the nanoparticle group (Nanoparticles of Example 5) was determined to be 35.7 ± 2.8 μg / cm³ according to the above method. 2 The mucus binding capacity of the control group (conventional emulsion containing nervonic acid and 28% Cremophor EL) was 11.2 ± 1.5 μg / cm³. 2 The difference between the two groups was statistically significant (P<0.01). The results indicate that the nanoparticles of this invention exhibit significantly better intestinal mucosal adhesion than traditional emulsions due to the electrostatic adsorption mediated by the positive charge of chitosan.

[0216] Based on the combined results of distribution in macaques, RAP intervention, and intestinal mucus binding, it can be seen that the nanoparticles of this invention may achieve a synergistic delivery advantage through "mucosal adhesion—receptor-related transBBB transport—PEG reduction of non-specific protein adsorption." In the macaque experiments, the peak radioactive enrichment in the brain at 6 hours was approximately 11.6 times that of the control group, suggesting that the proposed scheme has the potential for further research on brain-targeted delivery translation.

[0217] 10. Experimental Example 2: Mouse Brain Distribution and Efficacy Verification APP / PS1 double transgenic AD model mice (strain: B6C3-Tg(APPswe,PSEN1dE9)85Dbo / J, purchased from Spiefol Biotechnology Co., Ltd.), male, 6 months old, weighing 22-25 g, were randomly divided into 3 groups (n=10 / group). The nanoparticle group received nervonic acid nanoparticles (120 mg / kg, equivalent to 50 mg / kg nervonic acid, using the nanoparticles prepared in Example 7), resuspended in physiological saline. The positive control group received a conventional nervonic acid emulsion, prepared by dissolving 50 mg nervonic acid in physiological saline solution containing 28% Cremophor EL, ultrasonically emulsifying until uniformly dispersed, with a final volume of 1 mL. The blank control group received an equal volume of physiological saline. This experiment was approved by the institutional animal ethics committee (IACUC-2023-046) and complies with AAALAC standards. AD model mice were administered the drug by gavage at 9:00 AM daily for 28 consecutive days. Six hours after the last administration (peak time), the whole brain and liver tissues were harvested under anesthesia: the left hemisphere was flash-frozen in liquid nitrogen (stored at -80℃ for LC-MS detection). Aβ immunohistochemistry (6E10 antibody) and Nissl staining were performed on brain tissue sections from AD model mice. The concentrations of nervonic acid in the brain and liver were then quantitatively measured using LC-MS / MS, with d4-nervonic acid as the internal standard. The brain / liver ratio was calculated as: brain / liver ratio = nervonic acid concentration in brain tissue / nervonic acid concentration in liver. The results of the nervonic acid concentrations in the brain and liver, and the brain / liver ratio, are shown in Table 9 below.

[0218] Table 9. Nervonic acid concentration and brain / liver ratio in brain and liver tissues of APP / PS1 model mice

[0219] As shown in Table 9, compared with the positive control group, the concentration of nervonic acid in the brain tissue of the nanoparticle group was 7.8 times that of the positive control group, and it could selectively accumulate (brain / liver ratio > 3.7).

[0220] The Morris water maze experiment was based on the method described in Morris R. Developments of a water-maze procedure for studying spatial learning in the rat[J]. Journal of Neuroscience Methods, 1984, 11(1): 47-60, with some modifications. The experiment was conducted in a circular pool with a diameter of 120 cm and a height of 50 cm. The water temperature was controlled at 22±1℃, the inner wall of the pool was black, and the water level was 30 cm. A circular platform (10 cm in diameter, with a rough surface for the animal to grasp) was placed in the center of the second quadrant of the pool, hidden 1 cm below the water surface (i.e., invisible). Visual references of different shapes and colors (triangles, circles, crosses) were attached around the pool, and the positions of the references remained unchanged during the experiment. Training phase (positioning and navigation experiment): training was conducted for 4 consecutive days, with 4 tests per day. In each test, the animal was gently placed into the water facing the pool wall from different entry points (the midpoints of the four quadrants). The time required for the animal to find and climb onto the platform from the moment it entered the water was recorded, i.e., the escape latency period. If the animal fails to find the platform within 60 seconds, the experimenter guides it to the platform and keeps it there for 15 seconds; the latency period is recorded as 60 seconds. After each test, the animal stays on the platform for 15 seconds to form spatial memory. The average latency of the four tests per day is taken as the animal's escape latency for that day. Exploration Phase (Spatial Exploration Experiment): 24 hours after the end of training on Day 4 (i.e., Day 5), the platform is removed, and the animal is placed in the water from the quadrant opposite to the original platform. The number of times the animal crosses the original platform location within 60 seconds is recorded. Data Recording: The escape latency on Day 1 reflects the animal's initial learning ability (no difference between groups, approximately 48-49 seconds). The escape latency on Day 4 reflects the animal's ability to consolidate learning and memory after four days of training (the shorter the latency, the stronger the learning ability). The number of times the animal crosses the platform during spatial exploration reflects its ability to retain spatial location memory (the more times, the stronger the memory).

[0221] The behavioral results above show that, compared with the positive control group and the blank control group, the escape latency of the nanoparticle group was significantly shortened on the 4th day of training. At the same time, in the space exploration experiment, the number of times the nanoparticle group crossed the platform was 6.2±0.8 times, while that of the positive control group was 2.7±0.6 times, indicating that the learning and memory ability of the nanoparticle group was significantly improved.

[0222] Table 10 Behavioral and pathological indicators of APP / PS1 model mice

[0223] The deposition of Aβ plaques (cortical area percentage) in the nanoparticle group, positive control group, and blank control group was tested. The specific testing method was as follows: brain tissue sections were immunohistochemically stained with 6E10 antibody (anti-Aβ antibody), and then Aβ plaques in the cortical area were quantitatively analyzed using ImageJ software (US NIH). Five fields of view were randomly selected for each sample, and the percentage of Aβ plaque area to the total field of view was calculated and averaged. The Aβ plaque deposition in the nanoparticle group was 3.7 ± 0.5%*, in the positive control group it was 9.8 ± 1.2%, and in the blank control group it was 14.3 ± 1.8%. * indicates P < 0.001 vs other groups (ImageJ quantitative analysis). Therefore, compared with the positive control group, the nanoparticles prepared in this invention can significantly reduce Aβ toxicity (Aβ plaque area decreased by 62%).

[0224] Neuronal survival rate was determined by Nissl staining of the CA1 region of the hippocampus. Specifically, after Nissl staining of the CA1 region, the number of surviving neurons per millimeter (with clear nuclei and intact cytoplasm) was counted under a 400x microscope. Five fields of view were randomly selected from each sample, and the average value was taken. The results showed that the neuronal survival rate of the nanoparticle group was 82.4 ± 4.3 cells / mm, compared to 58.7 ± 5.1 cells / mm in the positive control group and 41.2 ± 3.8 cells / mm in the blank control group. Compared with the positive control group, the neuronal survival rate of the nanoparticle group increased by approximately 40% (calculated as (82.4-58.7) / 58.7×100% = 40.4%, P<0.01); compared with the blank control group, it increased by approximately 100% ((82.4-41.2) / 41.2×100% = 100%, P<0.001). Therefore, the nanoparticles prepared in this invention can significantly protect neurons from apoptosis.

[0225] Therefore, compared with the positive control group, the nanoparticles prepared in this invention can significantly protect neurons from damage, and the number of surviving neurons in the CA1 region of the hippocampus is increased by about 40%; compared with the blank control group, the increase is about 100%.

[0226] LRP1 receptor inhibition experiment: APP / PS1 double transgenic AD model mice (n=6) were randomly divided into a RAP intervention group and a blank control group. The RAP intervention group received an intravenous injection of the LRP1-specific inhibitor RAP (receptor-associated protein, 5 mg / kg, dissolved in physiological saline) via the tail vein 30 minutes before oral administration of the nanoparticles, while the blank control group received an equal volume of physiological saline. Both groups of mice were then orally administered the nanoparticles according to the protocol in Example 2 (Example 7, 120 mg / kg, equivalent to 50 mg / kg nervonic acid). Six hours after administration, the animals were sacrificed, and the whole brain was harvested. The concentration of nervonic acid in the brain tissue was measured using LC-MS / MS. The results showed that the brain concentration in the blank control group was 8.92±0.68 μg / g, while the brain concentration in the RAP intervention group decreased to 2.1±0.3 μg / g (a decrease of approximately 76%, P<0.001), suggesting that LRP1-related receptor-mediated processes are involved in the transport of the nanoparticles of this invention across the blood-brain barrier.

[0227] In vitro intestinal mucus adsorption experiments were conducted on the nanoparticle group, positive control group, and blank control group, respectively. Nanoparticle group: Nervate-loaded nanoparticles (120 mg / kg, equivalent to 50 mg / kg nervate, using nanoparticles prepared in Example 7), resuspended in physiological saline; Positive control group: Nervate conventional emulsion, prepared by dissolving 50 mg nervate in physiological saline solution containing 28% Cremophor EL by mass, ultrasonically emulsifying until uniformly dispersed, final volume 1 mL; Blank control group: Equal volume of physiological saline. Specific experimental methods: Fresh mouse small intestine segments were taken, cut open and laid flat, washed with 0.1 M PBS, and then cut into 1 cm × 1 cm pieces. The intestinal segments were immersed in the nanoparticle suspension (nanoparticle concentration 1 mg / mL), incubated at 37°C for 30 minutes, removed, rinsed three times with PBS, and the bound drug was extracted by ultrasonication. The content was determined by HPLC, and the binding amount per unit area (μg / cm²) was calculated. 2 The results showed that the mucus binding capacity of the nanoparticle group was 36.2 ± 2.9 μg / cm³. 2 The positive control group had a concentration of 11.3 ± 1.4 μg / cm³. 2 The blank control group did not detect it (below the detection limit). The mucus binding amount of the nanoparticle group was approximately 3.2 times that of the positive control group (P<0.001), suggesting that CS positive charge-driven mucosal adsorption.

[0228] Western blot analysis: Hippocampal tissues from mice treated with the positive control group and nanoparticle group were collected, and total protein was extracted using RIPA lysis buffer. Protein concentration was determined by BCA method. Equal amounts of protein were subjected to SDS-PAGE electrophoresis and transferred to membranes, which were then incubated with Synapsin-1 antibody (1:1000, Abcam), BDNF antibody (1:500, Abcam), and GAPDH antibody (1:2000, Abcam), respectively. After secondary antibody incubation, ECL staining was performed, and the band gray values ​​were analyzed using ImageJ software. The relative expression level was calculated using GAPDH as an internal reference. Results showed that the relative expression level of Synapsin-1 in the positive control group was 0.42±0.05 (with GAPDH as 1), while that in the nanoparticle group was 0.88±0.07, a 2.1-fold increase compared to the positive control group (P<0.01). The relative expression level of BDNF in the positive control group was 0.35±0.04, while that in the nanoparticle group was 0.63±0.06, a 1.8-fold increase compared to the positive control group (P<0.01). The above results indicate that the nanoparticles of the present invention can promote synaptic plasticity and neural regeneration at the molecular level, consistent with behavioral improvement results.

[0229] Liver function tests: 24 hours after the last administration, orbital venous blood was collected from mice, serum was separated by centrifugation, and the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using a fully automated biochemical analyzer (Hitachi 7600). The results showed no significant difference in ALT (35.2±4.1 U / L) and AST (42.5±5.3 U / L) between the nanoparticle group and the blank control group (ALT 32.8±3.7 U / L, AST 40.1±4.8 U / L) (P>0.05), indicating that no hepatotoxicity was observed at the 120 mg / kg dose.

[0230] In summary, the nanoparticles prepared by the SAS-ESS method of this invention show superior results to the traditional emulsion control in terms of brain distribution, behavioral improvement, and pathological indicators, and can provide experimental support for applications related to neurodegenerative diseases.

[0231] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A brain-targeting nanoparticle, characterized in that: It includes nervonic acid and a carrier material; the carrier material is internally loaded with the nervonic acid; The carrier material includes chitosan, phosphatidylserine, and polyethylene glycol; The number-average molecular weight of the polyethylene glycol is 1000-20000 Da; The number-average molecular weight of the chitosan is 5-100 kDa; the degree of deacetylation of the chitosan is 80-95%.

2. The brain-targeting nanoparticles according to claim 1, characterized in that: The mass ratio of chitosan to polyethylene glycol is (1.0–5.0):1; And / or, the mass ratio of the phosphatidylserine to the polyethylene glycol is (0.5-2.0):1; And / or, the mass of the nervonic acid accounts for 10-55% of the total mass of the brain-targeting nanoparticles.

3. The brain-targeting nanoparticles according to claim 2, characterized in that: The mass ratio of chitosan to polyethylene glycol is (1.5-3.0):1; And / or, the mass ratio of the phosphatidylserine to the polyethylene glycol is (0.5-1.5):1; And / or, the number average molecular weight of the polyethylene glycol is 2000-10000 Da; And / or, the number-average molecular weight of the chitosan is 10-50 kDa.

4. The brain-targeting nanoparticles according to any one of claims 1-3, characterized in that: The average particle size of the brain-targeting nanoparticles is 50-250 nm. And / or, the polydispersity index (PDI) of the brain-targeting nanoparticles is ≤0.25; And / or, the zeta potential of the brain-targeting nanoparticles is +15 mV to +45 mV; And / or, the encapsulation efficiency of nervonic acid in the brain-targeting nanoparticles is not less than 85%; And / or, the crystallinity of nervonic acid in the brain-targeting nanoparticles is ≥75%; And / or, the residual amount of organic solvent in the brain-targeting nanoparticles is not higher than 10 ppm.

5. The method for preparing brain-targeting nanoparticles according to any one of claims 1-4, characterized in that: Includes the following steps: The raw materials, including nervonic acid, phosphatidylserine and organic solvent, are mixed to obtain the oil phase; the raw materials, including chitosan, polyethylene glycol and acidic aqueous solution, are mixed to obtain the aqueous phase. The oil phase is injected into the aqueous phase and homogenized under high pressure to obtain an emulsion; The emulsion was electrostatically sprayed into a supercritical fluid, where it crystallized to precipitate, yielding the brain-targeting nanoparticles.

6. The method for preparing brain-targeting nanoparticles according to claim 5, characterized in that: When the supercritical fluid is supercritical carbon dioxide, the pressure is 8–25 MPa, the temperature is 32–60 °C, and the flow rate is 5–20 L / min.

7. The method for preparing brain-targeting nanoparticles according to claim 5, characterized in that: The pressure of the high-pressure homogenizer is 50-150 MPa; And / or, the high-pressure homogenization is performed 2-10 times; And / or, the pH of the acidic aqueous solution is 3-6; And / or, the concentration of the acidic aqueous solution is 0.05-0.5 mol / L; And / or, the acidic aqueous solution includes at least one of acetate buffer, citrate buffer or hydrochloric acid solution; And / or, the spray voltage of the electrostatic spray is 10-30kV; And / or, the nozzle orifice diameter of the electrostatic spray is 50-300 μm; And / or, the electrostatic spraying time is 10-60 min.

8. A pharmaceutical composition, characterized in that: Includes the brain-targeting nanoparticles as described in any one of claims 1-4 and pharmaceutically acceptable excipients.

9. The use of the brain-targeting nanoparticles according to any one of claims 1-4 or the pharmaceutical composition according to claim 8 in the preparation of brain-targeting delivery formulations.

10. The use of the brain-targeting nanoparticles according to any one of claims 1-4 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for the prevention or treatment of neurodegenerative diseases; wherein the neurodegenerative diseases include Alzheimer's disease or Parkinson's disease.

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