Choline biomimetic amphiphilic polymer nanodiscs, preparation method and application thereof

Abstract

This invention provides a choline-inspired amphiphilic polymer nanodisc, the raw material for which includes a polymethacrylate copolymer; the polymethacrylate copolymer is prepared by polymerization of 2-methacryloyloxyethylphosphorylcholine and hexyl methacrylate. This choline-inspired amphiphilic polymer nanodisc possesses efficient BBB-crossing and brain-targeting capabilities. It can efficiently cross the BBB via choline transport receptors in both in vitro BBB models and in vivo in mice. The disc-shaped morphology and nanoscale size of the choline-inspired amphiphilic polymer nanodisc promote effective penetration of blood vessels, matrix, and cell barriers, exhibiting excellent solid tumor infiltration capabilities. This invention also provides a method for preparing the above-mentioned choline-inspired amphiphilic polymer nanodisc and its application in the preparation of brain tumor detection reagents or drugs for the prevention and treatment of brain tumors.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a choline-inspired amphiphilic polymer nanodisc, its preparation method, and its application. Background Technology

[0002] The blood-brain barrier is composed of endothelial cells of the capillary wall, astrocyte terminale surrounding the capillaries, and pericytes embedded in the capillary basement membrane. Its tight junctions restrict the entry of over 98% of small molecule drugs and almost all large molecule biological drugs (such as antibodies and nucleic acids) into brain tissue. Nanoparticle platforms, including liposomes (Lp), polymer nanoparticles, and micelles, have been used for brain delivery. However, nonspecific clearance, nonspecific distribution, and excessively large particle size often hinder their brain delivery efficiency.

[0003] With the rising incidence of Alzheimer's disease (AD), Parkinson's disease (PD), and gliomas, the development of delivery systems that can efficiently penetrate the body's biological barrier (BBB) ​​and possess high biocompatibility has become an urgent need in the biomedical field. Nanodisks, as highly uniform, disc-shaped phospholipid bilayer structures (typically 10-30 nm in diameter), possess excellent penetration capabilities in microcirculation due to their extremely small particle size and flat geometry. The MSP nanodisks developed by Professor Sligar et al. mimic the structure of high-density lipoprotein (HDL). While achieving great success in structural biology, large-scale industrial production faces significant challenges. Traditional membrane scaffold proteins (MSPs) are derived from human proteins, posing immunogenicity risks and high purification costs in large-scale production.

[0004] The surface of mainstream SMA polymer nanodisks carries a large number of negative charges, which react with Ca... 2+ / Mg 2+ When subjected to divalent cations, cross-linking and precipitation occur, severely limiting their stability in vivo. Although the small hydrodynamic diameter allows nanodiscs to cross the blood-brain barrier more easily, the tight junctions of the blood-brain barrier make brain entry efficiency via physical diffusion (passive transport) extremely low.

[0005] Therefore, it is essential to develop a safe and efficient nanodelivery platform with the ability to cross the BBB and target tumors. Summary of the Invention

[0006] Synthetic amphiphilic polymers (such as SMA, DIBMA, or novel synthetic polymers) not only eliminate the potential risk of immune responses from proteins, but also provide polymer scaffolds with enhanced stability at different temperatures. The diameter of nanodisks can be precisely controlled by adjusting the monomer ratio, molecular weight, and polymer / lipid ratio. Research on amphiphilic polymer-based nanodisks holds promise for developing safe and efficient nanodelivery platforms with BBB-crossing and tumor-targeting capabilities for the treatment of various brain tumors and other central nervous system diseases.

[0007] The present invention aims to at least partially solve one of the technical problems existing in the prior art. Therefore, in a first aspect, the present invention provides a choline-inspired amphiphilic polymer nanodisk, wherein the raw material for preparing the choline-inspired amphiphilic polymer nanodisk includes a polymethyl methacrylate copolymer; the polymethyl methacrylate copolymer is prepared by polymerization reaction of 2-methacryloyloxyethyl phosphorylcholine and hexyl methacrylate.

[0008] This invention synthesizes a polymethyl methacrylate copolymer (zPMA) via photocontrolled free radical polymerization. The zPMA is polymerized from hydrophobic hexyl methacrylate (HMA) and 2-methacryloyloxyethyl phosphorylcholine (MPC) in a specific molar ratio. It possesses both hydrophobic alkyl side chains and hydrophilic choline MPC head groups. Without the need for proteins or surfactants, it can self-assemble with phospholipids (such as DOPE and DOPS) to form disc-shaped choline-inspired amphiphilic polymer nanodisks (zNDs). After drug loading, these nanodisks exhibit efficient BBB-crossing capabilities and brain-targeting abilities, demonstrating clinical translational value. The choline-inspired amphiphilic polymer nanodisks (zNDs) demonstrate efficient BBB-crossing via choline transport receptors in both in vitro BBB models and in vivo in mice.

[0009] A schematic diagram illustrating the self-assembly of liposomes composed of polymethyl methacrylate copolymer (zPMA) and lipids to form disc-shaped choline-inspired amphiphilic polymer nanodiscs (zND), which then penetrate the BBB and infiltrate glioblastoma, is shown below. Figure 1 As shown.

[0010] Preferably, the molar ratio of 2-methacryloyloxyethyl phosphorylcholine to hexyl methacrylate is 1:(2-4).

[0011] Preferably, the preparation method of the polymethacrylate copolymer includes the following steps: Step 1): Add 2-methacryloyloxyethyl phosphorylcholine and hexyl methacrylate to the organic solvent; Step 2): Add chain transfer agent and photoinitiator; Step 3): React under 350-380 nm ultraviolet light for 0.5-3 h, purify, and obtain the polymethacrylate copolymer.

[0012] Preferably, the organic solvent in step 1) is isopropanol.

[0013] Preferably, the chain transfer agent in step 2) is methyl 3-mercaptopropionate, and the photoinitiator is benzoin dimethyl ether.

[0014] Preferably, in step 3), the purification includes the following steps: a: Remove the solvent, dissolve in methanol, and precipitate with diethyl ether; b: After removing the ether, dissolve in water, remove impurities using a 3.5 kDa molecular weight cutoff regenerated cellulose dialysis bag, freeze dry, and obtain the polymethacrylate copolymer.

[0015] Preferably, the Mn of the polymethacrylate copolymer (zPMA) is 23-26 kg mol. -1 .

[0016] Preferably, the raw materials for preparing the choline-inspired amphiphilic polymer nanodiscs further include liposomes, wherein the raw materials for preparing the liposomes include lipids, and the lipids include DOPS, DOPE and cholesterol in a molar ratio of (50-60):(25-45):5.

[0017] Preferably, the lipids are prepared into liposomes by thin-film dispersion.

[0018] Specifically, liposomes are prepared by thin-film dispersion, comprising: dissolving the lipids in an organic solvent, removing the organic solvent, forming a film, dispersing the film in a buffer solution, hydrating, dispersing again, and homogenizing the particle size to obtain the liposomes. Preferably, the organic solvent is chloroform and the buffer solution is PBS buffer.

[0019] Preferably, the mass ratio of the polymethacrylate copolymer to the liposome is (1.5-2.5):1.

[0020] Secondly, the present invention provides a method for preparing the above-mentioned choline-inspired amphiphilic polymer nanodisc, comprising: dispersing the polymethacrylate copolymer and liposomes in a solvent, incubating, and self-assembling to obtain the choline-inspired amphiphilic polymer nanodisc.

[0021] Preferably, the solvent is PBS buffer.

[0022] Preferably, the incubation is performed by shaking incubation at 20-50°C for 8-20 hours.

[0023] Thirdly, the present invention provides the application of the above-mentioned choline biomimetic amphiphilic polymer nanodisk or the choline biomimetic amphiphilic polymer nanodisk prepared by the above-mentioned preparation method in the preparation of brain tumor detection reagents or brain tumor prevention and treatment drugs.

[0024] Preferably, the brain tumor is a glioblastoma.

[0025] Fourthly, the present invention provides a detection reagent comprising the above-mentioned choline biomimetic amphiphilic polymer nanodisc or the choline biomimetic amphiphilic polymer nanodisc prepared by the above-mentioned preparation method.

[0026] Preferably, the detection reagent further includes a fluorescent dye, wherein the choline-inspired amphiphilic polymer nanodiscs encapsulate the fluorescent dye, and the fluorescent dye is selected from at least one of Cy5, Cy5.5, Cy7, IR820, ICG, Dir, Did, and Dil.

[0027] In the constructed in vitro BBB model, Dil-labeled choline-inspired amphiphilic polymer nanodisks (Dil-zND), Dil-labeled liposomes (Dil-Lp), and Dil-zND combined with hemicholinium-3 (HC-3, 1 μM, pre-incubated in the upper chamber for 30 minutes, an inhibitor of choline transporters) were added to the upper chamber. The basolateral transport efficiency of zND was significantly higher than that of Lp. After pretreatment of the model with HC-3, a selective inhibitor of choline transporters (CHTs), the transmembrane transport of zND was significantly inhibited, confirming that this process is a CHT-dependent active transport mechanism.

[0028] Specifically, the preparation method of the detection reagent includes: Step 1): Fluorescent dye-liposomes are prepared by dispersing lipids and fluorescent dyes in a thin film. Step 2): The polymethacrylate copolymer and fluorescent dye-liposome are dispersed in a solvent, incubated, and self-assembled to obtain the detection reagent.

[0029] Preferably, in step 1), the lipids in the raw materials for preparing the fluorescent dye-liposomes include DOPS, DOPE, and cholesterol in a molar ratio of (50-60):(25-45):5. The molar ratio of DOPS, DOPE, fluorescent dye, and cholesterol is (50-60):(25-45):(5-15):5.

[0030] Preferably, the molar ratio of DOPS, DOPE, fluorescent dye, and cholesterol is 55:30:10:5.

[0031] Specifically, step 1) involves preparing fluorescent dye-liposomes from lipids and fluorescent dyes via a thin-film dispersion method, which includes: dissolving lipids and fluorescent dyes in an organic solvent, removing the organic solvent, forming a film, dispersing the film in a buffer solution, hydrating, dispersing again, and homogenizing the particle size to obtain the fluorescent dye-liposomes. Preferably, the organic solvent is chloroform, and the buffer solution is PBS buffer.

[0032] Preferably, in step 2), the solvent is PBS buffer.

[0033] Preferably, in step 2), the mass ratio of the polymethacrylate copolymer to the fluorescent dye-liposome is (1.5-2.5):1.

[0034] Preferably, in step 2), the incubation is performed by shaking incubation at 20-50°C for 8-20 hours.

[0035] Fifthly, the present invention provides the application of the above-described detection reagent in the preparation of diagnostic reagents for brain tumors.

[0036] Preferably, the brain tumor is a glioblastoma.

[0037] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the above-described choline-inspired amphiphilic polymer nanodisc or the choline-inspired amphiphilic polymer nanodisc prepared by the above-described preparation method.

[0038] Preferably, the pharmaceutical composition further includes an active substance that has therapeutic effects on brain tumors, wherein the choline-inspired amphiphilic polymer nanodisc encapsulates the active substance that has therapeutic effects on brain tumors.

[0039] Preferably, the active substance with therapeutic effects on brain tumors is magnolol.

[0040] In a seventh aspect, the present invention provides the use of the above-described pharmaceutical composition in the preparation of a medicament for the prevention and treatment of brain tumors.

[0041] Preferably, the brain tumor is a glioblastoma.

[0042] Eighthly, the present invention provides the application of polymethacrylate copolymer in the preparation of choline-inspired amphiphilic polymer nanodisks, wherein the polymethacrylate copolymer is prepared by polymerization of monomer 2-methacryloyloxyethylphosphorylcholine and monomer hexyl methacrylate.

[0043] Preferably, the preparation method of the polymethacrylate copolymer includes the following steps: Step 1): Add 2-methacryloyloxyethyl phosphorylcholine and hexyl methacrylate to the organic solvent; Step 2): Add chain transfer agent and photoinitiator; Step 3): React under 350-380 nm ultraviolet light for 0.5-3 h, purify, and obtain the polymethacrylate copolymer.

[0044] Preferably, the organic solvent in step 1) is isopropanol.

[0045] Preferably, the chain transfer agent in step 2) is methyl 3-mercaptopropionate, and the photoinitiator is benzoin dimethyl ether.

[0046] Preferably, in step 3), the purification includes the following steps: a: Remove the solvent, dissolve in methanol, and precipitate with diethyl ether; b: After removing the ether, dissolve in water, remove impurities using a 3.5 kDa molecular weight cutoff regenerated cellulose dialysis bag, freeze dry, and obtain the polymethacrylate copolymer.

[0047] Preferably, the Mn of the polymethacrylate copolymer (zPMA) is 23-26 kg mol. -1 .

[0048] The beneficial effects of this invention are as follows: 1. This invention provides a choline-inspired amphiphilic polymer nanodisc, prepared from polymethyl methacrylate copolymer (zPMA). The zPMA, synthesized via photocontrolled free radical polymerization, is composed of hydrophobic hexyl methacrylate (HMA) and 2-methacryloyloxyethyl phosphorylcholine (MPC) in a specific molar ratio. It possesses both hydrophobic alkyl side chains and hydrophilic choline MPC head groups. Without the need for proteins or surfactants, it can self-assemble with phospholipids (such as DOPE, DOPS, etc.) to form disc-shaped choline-inspired amphiphilic polymer nanodiscs (zND). After drug loading, these choline-inspired amphiphilic polymer nanodiscs exhibit efficient BBB-crossing and brain-targeting capabilities, efficiently crossing the BBB via choline transport receptors in both in vitro BBB models and in vivo in mice. The disc-shaped morphology and nanoscale size of this choline-inspired amphiphilic polymer nanodisc promote efficient penetration into blood vessels, matrix, and cell barriers. Its modular phospholipid-polymer combination enables the co-delivery and multi-site modification of drugs such as chemotherapeutic agents, immunomodulators, or gene-editing agents, fully expanding the platform's therapeutic versatility. The absence of protein or ligand components minimizes immunogenicity, simplifying clinical translation. Furthermore, this choline-inspired amphiphilic polymer nanodisc exhibits superior solid tumor infiltration ability, significantly exceeding that of traditional liposomes.

[0049] 2. This invention provides a method for preparing the above-mentioned choline-inspired amphiphilic polymer nanodiscs. This method is simple, cost-controllable, and has strong clinical translational value.

[0050] 3. This invention provides the application of the above-mentioned choline biomimetic amphiphilic polymer nanodisk or the choline biomimetic amphiphilic polymer nanodisk prepared by the above-mentioned preparation method in the preparation of brain tumor detection reagents or brain tumor prevention and treatment drugs. Attached Figure Description

[0051] Figure 1A schematic diagram illustrating the self-assembly of liposomes composed of polymethyl methacrylate copolymer (zPMA) and lipids to form disc-shaped choline-inspired amphiphilic polymer nanodiscs (zND), which penetrate glioblastoma through the BBB. Figure 2 This is a synthetic pathway diagram for polymethyl methacrylate copolymer (zPMA); Figure 3 The image shows the 1H NMR spectrum of poly(zPMA) copolymer. Figure 4 Characterized by gel permeation chromatography for polymethyl methacrylate copolymer (zPMA); Figure 5 The results show the physicochemical characterization of Lp and zND; Where a is the transmission electron microscope (TEM) image and particle size distribution of LP, b is the transmission electron microscope (TEM) image and particle size distribution of zND, c is the hydrated particle size of Lp and zND, and d is the dynamic light scattering (DLS) detection result of the zeta potential of Lp and zND (n=3). Figure 6 The results of quantitative analysis of the penetration efficiency of the zND group, Lp group, and zND+HC-3 group across the in vitro blood-brain barrier model (n=3). Figure 7 The results of in vivo blood-brain barrier penetration ability tests for the zND group, Lp group, and zND+HC-3 group; Among them, a represents the in vivo continuous fluorescence imaging results 1, 2, 4, 24, 48, 72, and 96 h after injection (n=3), and b represents the corresponding quantitative analysis of the change in fluorescence intensity of brain tissue over time 1, 2, 4, 24, 48, 72, and 96 h after injection (n=3). Figure 8 Representative fluorescence imaging images of infiltration in tumor spheres in the zND and Lp groups, and radial fluorescence intensity distribution curves of the tumor sphere core; Figure 9 The results of in vivo tumor enrichment capacity detection for the zND group and Lp group; Figure 10 Representative fluorescence imaging images of tumor infiltration in the zND and Lp groups. Detailed Implementation

[0052] The present invention will be further described below with reference to specific embodiments. However, the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, the following embodiments were carried out under conventional conditions or conditions recommended by the manufacturer. All starting materials used are commercially available products or prepared using methods disclosed in the art. Unless otherwise specified, the methods used are conventional methods known in the art, and the consumables and reagents used are commercially available. Unless otherwise stated, the technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be applied to the present invention.

[0053] Example 1 The reaction formula for preparing polymer PMA (zPMA) from monomers MPC and HMA is as follows: Figure 2 As shown.

[0054] The preparation method of polymethyl methacrylate copolymer (zPMA) includes the following steps: Step 1): Add 2-methacryloyloxyethylphosphorylcholine (MPC, 1.25 mmol, 369.1 mg) and hexyl methacrylate (HMA, 3.75 mmol, 638.4 mg) to 5 ml of isopropanol at a specific monomer molar ratio of 1:3 (MPC:HMA). Step 2): Add chain transfer agent methyl 3-mercaptopropionate (MMP, 0.05 mmol, 6.0 mg) and photoinitiator benzoin dimethyl ether (DMPA, 0.05 mmol, 18.5 mg). Step 3): Nitrogen gas is bubbled to remove some dissolved oxygen, and the reaction is carried out at room temperature under 365 nm ultraviolet light for 1 hour; Step 4): Remove the solvent by rotary evaporation at 40 °C, dissolve the product in 1-2 ml of methanol, and precipitate it with 20 times its volume of diethyl ether; Step 5): After centrifugation at 3000 rpm for 5 min, discard the ether and treat in a vacuum drying oven for 2 h to completely remove the ether. Then add 10 ml of deionized water to dissolve it, use a 3.5 kDa molecular weight cutoff regenerated cellulose dialysis bag to remove impurities, freeze dry, and obtain polymethyl methacrylate copolymer (zPMA).

[0055] Figure 3 The image shows the proton NMR spectrum of zPMA. In the spectrum, the characteristic peak at 4.00 ppm corresponds to the proton signal of –COOCH2 in hexyl methacrylate (HMA), the characteristic peak at 4.23 ppm corresponds to the proton signal of –COOCH2 in 2-methacryloyloxyethylphosphorylcholine (MPC), and the characteristic peak at 3.30 ppm is attributed to the proton signal of –N(CH3)3 in MPC.

[0056] Figure 4 The polymethyl methacrylate copolymer (zPMA) was characterized by gel permeation chromatography (GPC, Agilent 1260 Infinity II, USA) using hexafluoroisopropanol as a diluent. GPC results showed that the synthesized zPMA had a Mn content of 24.9 kg mol. -1 Mw is 48.7 kg mol -1 The polydispersity was 1.93. This result further indicates that the polymerization reaction was successful.

[0057] Example 2 The preparation methods of Lp and zND include the following steps: Step 1): Total lipids were obtained by thoroughly mixing DOPS, DOPE, and cholesterol in a molar ratio of 55:40:5. The total lipids were dissolved in 1 mL of chloroform, and the concentration of total lipids in the system was controlled at 3 mM. Then, the mixture was blown under a nitrogen stream for 20 min to slowly evaporate the organic solvent, so that the total lipids formed a uniform film on the container wall. Finally, it was placed in a vacuum drying oven for 12 h to completely remove residual organic solvents.

[0058] Step 2): At 37 °C, add 1 mL of phosphate-buffered saline (PBS) to the obtained lipid film for hydration, and vortex for 30 min to fully disperse the lipids and form a lipid suspension.

[0059] Step 3): The obtained lipid suspension was subjected to 5 freeze-thaw cycles. Then, using a small lipid extruder, it was extruded through 400 nm and 200 nm polycarbonate membranes, 30 times each, to finally obtain Lp with uniform particle size distribution.

[0060] Step 4): Dissolve the polymethyl methacrylate copolymer (zPMA) in PBS beforehand to prepare a stock solution with a concentration of 10 mg / mL of polymethyl methacrylate copolymer (zPMA).

[0061] Step 5): Mix the prepared Lp with the above stock solution, control the mass ratio of Lp to polymethyl methacrylate copolymer (zPMA) in the system to be 1:2, vortex thoroughly, and incubate at 37 °C for 16 h to form a disc-shaped choline-inspired amphiphilic polymer nanodisk zND through self-assembly.

[0062] Figure 5The physicochemical properties of Lp and choline-inspired amphiphilic polymer nanodisks zND are characterized. Among them, a is the transmission electron microscopy (TEM) image and particle size distribution of LP, b is the transmission electron microscopy (TEM) image and particle size distribution of choline-inspired amphiphilic polymer nanodisks zND, c is the hydrated particle size of Lp and choline-inspired amphiphilic polymer nanodisks zND, and d is the dynamic light scattering (DLS) detection result of the zeta potential of Lp and choline-inspired amphiphilic polymer nanodisks zND (n=3).

[0063] The preparation methods of Dil-Lp and Dil-zND include the following steps: Step 1): Total lipids and fluorescent dye were prepared by thoroughly mixing DOPS, DOPE, Dil, and cholesterol in a molar ratio of 55:30:10:5. The total lipids and fluorescent dye were then dissolved in 1 mL of chloroform, maintaining a total concentration of 3 mM. The mixture was then blown under a nitrogen stream for 20 min to slowly evaporate the organic solvent, allowing the total lipids and fluorescent dye to form a uniform film on the container wall. Finally, it was placed in a vacuum drying oven for 12 h to completely remove any residual organic solvent.

[0064] Step 2): At 37 °C, add 1 mL of phosphate-buffered saline (PBS) to the obtained lipid film for hydration, and vortex for 30 min to fully disperse the lipids and form a lipid suspension.

[0065] Step 3): The obtained lipid suspension was subjected to five freeze-thaw cycles. Then, using a small lipid extruder, it was extruded through 400 nm and 200 nm polycarbonate filters, 30 times each, to finally obtain Dil-Lp with uniform particle size distribution.

[0066] Step 4): Dissolve the polymethyl methacrylate copolymer (zPMA) in PBS buffer beforehand to prepare a stock solution with a concentration of 10 mg / mL.

[0067] Step 5): Mix the prepared Dil-Lp with the stock solution obtained in Step 4), control the mass ratio of Dil-Lp to polymethyl methacrylate copolymer (zPMA) in the system to be 1:2, vortex thoroughly, and incubate at 37 °C for 16 h to form Dil-zND through self-assembly.

[0068] The preparation methods of Dir-Lp and Dir-zND are the same as those of Dil-Lp and Dil-zND, respectively. The only difference is that the equimolar amount of Dil is replaced with Dir.

[0069] Example 3 The in vitro blood-brain barrier penetration experiments of Lp and zND include the following steps: Step 1): The bEnd.3 cells were loaded at a rate of 1 × 10⁻⁶. 5 Cells were seeded at a density of cells / well in the upper chamber of a disposable cell embedding dish (Guangzhou Jetech Biofiltration Co., Ltd.). The upper and lower chambers of the disposable cell embedding dish were separated by a polycarbonate membrane. 500 μL of DMEM culture medium was added to the upper chamber and 1500 μL of DMEM culture medium was added to the lower chamber. Transendothelial cell resistance (TEER) was monitored using a cell resistance meter.

[0070] Step 2): When TEER ≥ 150 Ω When the concentration reaches 1 cm², the in vitro blood-brain barrier (BBB) ​​model is considered successfully constructed. Parallel setups were established for the zND group, Lp group, and zND+HC-3 group. 100 μL of a 0.05 mM Dil-labeled choline-inspired amphiphilic polymer nanodisc zND (Dil-zND) solution (PBS solvent) was added to the upper chamber as the zND group; 100 μL of a 0.05 mM Dil-labeled liposome (Dil-Lp) solution (PBS solvent) was added to the upper chamber as the Lp group; and HC-3 was added to the upper chamber to a final concentration of 1 μM, incubated for 30 min, and then 100 μL of a 0.05 mM Dil-labeled choline-inspired amphiphilic polymer nanodisc zND (Dil-zND) solution (PBS solvent) was added as the zND+HC-3 group. The dosing time for this group started from the addition of Dil-zND.

[0071] Step 3): At 2 h and 6 h after drug administration, 100 μL of culture medium was taken from the lower chamber for fluorescence intensity detection, and the blood-brain barrier penetration efficiency of the sample was calculated.

[0072] Step 4): Record the TEER value again 6 h after drug administration to verify the integrity of the bEnd.3 cell barrier.

[0073] Figure 6 The results of quantitative analysis of the penetration efficiency of zND, Lp, and zND+HC-3 groups across the in vitro blood-brain barrier model (n=3) showed that the penetration amount of zND into the lower ventricle was significantly higher than that of Lp. Pretreatment with HC-3 significantly reduced the penetration of zND, confirming that its transport depends on an active transport mechanism mediated by cholinergic transporters.

[0074] Example 4 The in vivo blood-brain barrier penetration experiments of Lp and zND include the following steps: Step 1): Female C57BL / 6 mice were used in this study, weighing 18±1 g and aged 6-8 weeks. All experimental animals were housed in a specific pathogen-free (SPF) environment with strictly controlled conditions: temperature maintained at 22-25 ℃, relative humidity controlled at 50%-70%, and a light / dark cycle of 12 hours of light / 12 hours of darkness. Animals had free access to standard rodent feed and water.

[0075] Step 2): Mice were randomly divided into three groups. The zND group was injected with 100 μL of 0.15 mM Dir-labeled choline-inspired amphiphilic polymer nanodisk zND (Dir-zND) solution (solvent: PBS) via tail vein. The Lp group was injected with 100 μL of 0.15 mM Dir-labeled liposome (Dir-Lp) solution (solvent: PBS) via tail vein. The zND+HC-3 group was injected intraperitoneally 20 min in advance, followed by a tail vein injection of 100 μL of 0.15 mM Dir-labeled choline-inspired amphiphilic polymer nanodisk zND (Dir-zND) solution (solvent: PBS). The administration time for this group was started from the injection of Dir-zND.

[0076] Step 3): At 1, 2, 4, 24, 48, 72, and 96 h after injection, in vivo fluorescence imaging was performed using a small animal in vivo imaging system, and the fluorescence signals at each time point were quantitatively analyzed.

[0077] Figure 7 The results show the in vivo blood-brain barrier penetration ability of the zND group, Lp group, and zND+HC-3 group. Specifically, a represents in vivo continuous fluorescence imaging results at 1, 2, 4, 24, 48, 72, and 96 hours after injection (n=3), and b represents the corresponding quantitative analysis of brain tissue fluorescence intensity changes over time at 1, 2, 4, 24, 48, 72, and 96 hours after injection (n=3). The results indicate that after intravenous injection of Dir-labeled zND into GL261 glioma-bearing mice, zND accumulation can be detected in brain tissue within 1 hour, reaching a peak at 2 hours, and gradually decreasing after 96 hours. HC-3 pretreatment significantly reduced zND uptake in the brain, further confirming the choline transporter-mediated transport mechanism.

[0078] Example 5 The in vitro tumor penetration assay for zND includes the following steps: Step 1): Take U251 cells in logarithmic growth phase and use 5 × 10⁻⁶ cells. 3Cells / well were seeded at a density of 96-well ultra-low adsorption medium and cultured in DMEM medium at 37 °C in a 5% CO2 incubator for 48 hours to form dense 3D tumor spheres.

[0079] Step 2): After incubation, carefully remove the culture medium from the wells and set up the zND and Lp groups in parallel. Add 100 μL of DMEM medium containing 0.003 mM Dil-zND solution (solvent: PBS) to the zND group; add 100 μL of DMEM medium containing 0.003 mM Dil-Lp solution (solvent: PBS) to the Lp group. Continue incubation for 12 hours.

[0080] Step 3): Gently wash the tumor spheres twice with phosphate-buffered saline (PBS) to remove residual culture medium. Transfer the tumor spheres to a glass-bottom culture dish and use laser confocal microscopy z-stack technology for imaging analysis to evaluate the infiltration depth and spatial distribution characteristics of the nano-formulations Dil-zND and Dil-Lp within the tumor spheres.

[0081] Figure 8 The figures show representative fluorescence imaging images of the infiltration of the zND and Lp groups into the tumor spheres, as well as the radial fluorescence intensity distribution curves of the tumor sphere core. As can be seen from the figures, in the 3D glioblastoma sphere model, zND exhibits significantly enhanced infiltration ability compared to Lp. Confocal Z-axis tomography and radial fluorescence quantification results show that zND can be uniformly distributed in the tumor sphere core region, while Lp is mainly confined to the periphery of the tumor sphere and cannot penetrate deep into the interior.

[0082] Example 6 In vivo tumor detection experiments include the following steps: Step 1): Construct a C57BL / 6 mouse orthotopic glioblastoma (GBM) model using GL261-Luc cells. First, collect GL261-Luc cells in logarithmic growth phase, resuspend them in sterile PBS solution, and adjust the cell concentration.

[0083] Step 2): Before the experiment, mice were anesthetized with isoflurane and fixed in a stereotaxic apparatus to ensure precise injection location. A microsyringe was used to inject 2 μL of a solution containing 5 × 10⁻⁶ ppm of isoflurane. 4 One GL261-Luc cell was slowly injected into the right striatum of a mouse with PBS solution. The injection coordinates were set as follows: 1 mm anterior to the anterior fontanelle, 2 mm lateral to the midline, and 3 mm below the surface of the skull.

[0084] Step 3): 7-10 days after cell implantation, verify the tumor colonization status in mice: inject 200 μL of D-fluorescein potassium salt with a concentration of 15 mg / mL into the peritoneum. After the fluorescein is fully distributed, bioluminescence imaging (BLI) is performed using an in vivo imaging system under continuous isoflurane anesthesia.

[0085] Step 4): The established mouse orthotopic glioblastoma model was injected via tail vein with 100 μL of 0.15 mM Dir-zND solution (solvent: PBS) and 100 μL of 0.15 mM Dir-Lp solution (solvent: PBS), designated as the zND group and Lp group, respectively. Bioluminescence imaging was performed 2 h after injection, followed by fluorescence imaging.

[0086] Step 5): The mouse brain was then dissected and immediately subjected to in vitro bioluminescence and fluorescence imaging. GL261-LUC was used to indicate the tumor location. The co-localization of the Dir fluorescence and GL261-LUC luminescence sites was observed to determine the tumor enrichment capacity of the sample.

[0087] Figure 9 The results show the in vivo tumor accumulation capacity of the zND and Lp groups. The results indicate that, compared to Lp, fluorescently labeled zND showed stronger accumulation at brain tumor sites. This selective accumulation may be due to the passive targeting effect caused by tumor vascular leakage, as well as the selective uptake of zND by glioblastoma cells.

[0088] Example 7 The in vivo tumor penetration assay for zND includes the following steps: Step 1): Establish an in situ glioblastoma animal model according to Example 6 (Step 1 and Step 2 in Example 6). Step 2): The participants were randomly divided into two groups. One group received 100 μL of Dil-zND solution (0.15 mM Dil, solvent: PBS) and the other group received 100 μL of Dil-Lp solution (0.15 mM Dil, solvent: PBS) via tail vein injection. The two groups were designated as the zND group and the Lp group, respectively.

[0089] Step 3): After 3 hours, the mice were sacrificed and perfused to obtain brain tissue. The tissue was dehydrated in gradients of 10% and 30% sucrose solutions, then frozen and sectioned. The tissue was stained with DAPI and mounted for observation and imaging under a confocal microscope.

[0090] Figure 10Representative fluorescence imaging images of tumor sections from the zND and Lp groups show infiltration. As can be seen, the fluorescence signal of zND within the tumor tissue is significantly stronger than that of Lp, and zND is widely distributed within the tumor parenchyma, rather than being concentrated only around blood vessels or in the stromal region. This efficient and widespread intratumoral distribution indicates that zND can effectively cross the glioma vascular barrier and overcome the transport resistance of the tumor stroma, achieving uniform and efficient biodistribution within the glioma microenvironment.

[0091] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the invention.

Claims

1. A choline-inspired amphiphilic polymer nanodisc, characterized in that, The raw materials for preparing the choline-inspired amphiphilic polymer nanodisc include polymethyl methacrylate copolymer; the polymethyl methacrylate copolymer is prepared by polymerization reaction of 2-methacryloyloxyethyl phosphorylcholine and hexyl methacrylate.

2. The choline-inspired amphiphilic polymer nanodisc according to claim 1, characterized in that, The molar ratio of 2-methacryloyloxyethyl phosphorylcholine to hexyl methacrylate is 1:(2-4). The preparation method of the polymethacrylate copolymer includes the following steps: Step 1): Add 2-methacryloyloxyethyl phosphorylcholine and hexyl methacrylate to the organic solvent; Step 2): Add chain transfer agent and photoinitiator; Step 3): React under 350-380 nm ultraviolet light for 0.5-3 h, purify, and obtain the polymethacrylate copolymer.

3. The choline-inspired amphiphilic polymer nanodisc according to claim 1, characterized in that, The raw materials for preparing the choline-inspired amphiphilic polymer nanodisc also include liposomes, and the raw materials for preparing the liposomes include lipids, and the lipids include DOPS, DOPE and cholesterol in a molar ratio of (50-60):(25-45):

5.

4. The choline-inspired amphiphilic polymer nanodisc according to claim 3, characterized in that, The lipids were prepared into liposomes by thin-film dispersion.

5. A method for preparing the choline-inspired amphiphilic polymer nanodisc according to any one of claims 1-4, characterized in that, include: The polymethacrylate copolymer and liposomes were dispersed in a solvent, incubated, and self-assembled to obtain the choline-inspired amphiphilic polymer nanodisc.

6. The use of a choline-inspired amphiphilic polymer nanodisk according to any one of claims 1-4 or a choline-inspired amphiphilic polymer nanodisk prepared by the preparation method according to claim 5 in the preparation of a brain tumor detection reagent or a brain tumor prevention or treatment drug.

7. A detection reagent, characterized in that, Includes the choline-inspired amphiphilic polymer nanodisk as described in any one of claims 1-4 or the choline-inspired amphiphilic polymer nanodisk prepared by the preparation method described in claim 5.

8. The use of the detection reagent according to claim 7 in the preparation of diagnostic reagents for brain tumors.

9. A pharmaceutical composition, characterized in that, Includes the choline-inspired amphiphilic polymer nanodisk as described in any one of claims 1-4 or the choline-inspired amphiphilic polymer nanodisk prepared by the preparation method described in claim 5.

10. The use of the pharmaceutical composition of claim 9 in the preparation of a medicament for the prevention and treatment of brain tumors.

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