Liposome drug, application and preparation for diagnosing early alzheimer's disease

Abstract

The present application relates to the biomedical field, and in particular to a liposome drug, application and preparation for diagnosing early Alzheimer's disease. The liposome drug comprises a LilrB2 extracellular domain D1D2 modified protein capable of targeted binding to beta-amyloid oligomers, and a liposome coated with the D1D2 modified protein, and the D1D2 modified protein is connected with a magnetic resonance imaging contrast agent. The method obtains the modified protein through a biological orthogonal reaction, and adopts a film hydration method to prepare the liposome drug. The drug realizes accurate targeting of the early pathological marker of Alzheimer's disease, and makes it capable of autonomously penetrating the blood-brain barrier, has biological safety and clinical application feasibility, and effectively supports the safe, sensitive and repeatable in vivo magnetic resonance diagnosis of early Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to a liposomal drug, its application, and a formulation for diagnosing early-stage Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease that has a wide-ranging impact on human health, yet effective treatments remain elusive. Early diagnosis is crucial for developing timely and targeted treatment strategies. Typical pathological features of Alzheimer's disease include amyloid plaques formed by the abnormal aggregation of amyloid-β (Aβ) protein, and neurofibrillary tangles caused by abnormal phosphorylation of tau protein in the brain. Current Alzheimer's diagnosis relies on positron emission tomography (PET) probes to identify Aβ fibers in the brain; however, due to the involvement of ionizing radiation, PET scans pose significant health risks, making them unsuitable for routine screening for early Alzheimer's disease, causing patients to miss the window of opportunity for early intervention.

[0003] LilrB2 is expressed on the surface of human nerve cells. Studies have confirmed that Aβ oligomers can specifically bind to the extracellular D1D2 domain of LilrB2, thereby triggering synaptic toxicity and promoting neuroinflammation. Based on its high physiological affinity, specific binding to Aβ oligomers, and low immunogenicity, the human protein LilrB2 D1D2 holds promise as a highly prospective targeted tool for AD diagnosis.

[0004] However, since LilrB2 D1D2 is a biological macromolecule and does not possess in vivo imaging signals, and exogenous protein molecules cannot autonomously penetrate the blood-brain barrier, the application scenarios of directly using LilrB2 D1D2 for in vivo imaging of Aβ-targeting in the brain to achieve in vivo diagnosis of AD are greatly limited. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a liposomal drug, its application, and a formulation for the diagnosis of early Alzheimer's disease.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: This invention provides a liposomal drug, characterized in that it comprises: A modified LilrB2 extracellular domain D1D2 protein that can target and bind to β-amyloid oligomers, wherein the modified D1D2 protein is linked to a magnetic resonance imaging contrast agent. Liposomes encapsulating the D1D2 modified protein.

[0007] Based on the above technical solution, the present invention can be further improved as follows.

[0008] Furthermore, the magnetic resonance imaging contrast agent includes gadolinium-based contrast agents, iron-based contrast agents, and manganese-based contrast agents.

[0009] Furthermore, the liposomes are composed of lecithin, cholesterol, DSPE-PEG2k and DSPE-PEG-MA, with a molar ratio of 60-70:25-35:1-5:1-5.

[0010] Furthermore, the liposomes have a particle size of less than 200 nm and a polydispersity index of less than 0.2.

[0011] The present invention also provides a method for preparing a liposomal drug as described above, comprising preparing a magnetic resonance imaging contrast agent precursor and a D1D2 modified protein precursor, mixing the two and performing a bioorthogonal reaction to obtain the modified protein; and encapsulating the D1D2 modified protein in the liposomes using a thin-film hydration method to obtain the liposomal drug.

[0012] Furthermore, the following steps are included: S1. The magnetic resonance imaging contrast agent precursor N3-DOTA-Gd was prepared by reacting DOTA-N3 with GdCl3·6H2O. S2. The D1D2 modified protein precursor D1D2-DBCO was prepared by reacting LilrB2 D1D2 protein with NHS-DBCO. S3. D1D2-DBCO and N3-DOTA-Gd were reacted at pH 7.4 and temperature 4℃ for 12-16 hours to obtain the D1D2 modified protein D1D2-Gd. S4. After mixing and hydrating the lipid membrane with a solution containing D1D2-Gd, the mixture is extruded using a polycarbonate membrane to obtain the liposome drug Lipo@D1D2-Gd, in which the D1D2-Gd modified protein is encapsulated in liposomes using a thin-film hydration method.

[0013] Furthermore, in step S4, the hydration method is to rotate at 4°C for 30 minutes.

[0014] Furthermore, in step S4, the polycarbonate film is 200 nm thick and the extrusion times are 9-13.

[0015] The present invention also provides the use of the liposomal drug as described above in the preparation of a formulation for diagnosing early Alzheimer's disease.

[0016] The present invention also provides an agent for diagnosing early Alzheimer's disease, comprising the liposomal drug as described above.

[0017] The liposomal drug of this invention uses the LilrB2 extracellular domain D1D2 fragment, which targets and binds to β-amyloid oligomers, as the core recognition element. It utilizes the high affinity and specific interaction between LilrB2 and Aβ oligomers to achieve precise targeting of early pathological markers of Alzheimer's disease. At the same time, a bioorthogonal reaction is used to covalently link a gadolinium-based magnetic resonance imaging contrast agent to the D1D2 protein, endowing the biomolecule with stable magnetic resonance signal enhancement capabilities, enabling non-invasive imaging detection at the in vivo level. Furthermore, the liposomal encapsulation solves the key bottleneck that exogenous proteins cannot autonomously penetrate the blood-brain barrier.

[0018] This invention employs a mild and efficient thin-film hydration method combined with polycarbonate membrane extrusion technology. Under mild conditions, it ensures the activity of D1D2 protein and the stability of gadolinium contrast agent while obtaining a high encapsulation rate and monodisperse liposome population. The method is simple to operate, the conditions are controllable, and it is easy to scale up production, ensuring the biosafety and clinical feasibility of the final product. Overall, it strongly supports safe, sensitive, and repeatable in vivo magnetic resonance imaging diagnosis of early Alzheimer's disease. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the process of modifying LilrB2 D1D2 in Embodiment 1 of the present invention; Figure 2 This is an SDS-PAGE result image of the modified protein in Example 1 of the present invention; Figure 3 The above are the experimental results for verifying the affinity of D1D2-Gd for Aβ in Example 2 of the present invention. Figure 3 In the figure, 'a' represents the ELISA experimental results. Figure 3 Figure b in the middle shows the results of the Pull-down experiment; Figure 4 This is a schematic diagram of the preparation process of the liposome-coated modified protein Lipo@D1D2-Gd in Example 3 of the present invention; Figure 5 This is a DLS result diagram of the liposome-coated modified protein Lipo@D1D2-Gd in Example 3 of the present invention; Figure 6 The experimental results are for Example 4 of the present invention. Figure 4 Image a shows an in vivo magnetic resonance imaging (MRI) image of the liposome-coated modified protein Lipo@D1D2-Gd. Figure 4 In the image, b represents the quantization result. Detailed Implementation

[0020] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0021] The liposomal drug of the present invention comprises: a modified LilrB2 extracellular domain D1D2 protein capable of targeting and binding to β-amyloid oligomers, the modified D1D2 protein being linked to a magnetic resonance imaging contrast agent; and liposomes encapsulating the modified D1D2 protein.

[0022] The liposomal drug of this invention achieves precise targeted recognition of early pathological markers of Alzheimer's disease by specifically binding with the D1D2 fragment of the LilrB2 extracellular domain to β-amyloid oligomers with high affinity. Simultaneously, it utilizes a bioorthogonal reaction to label magnetic resonance imaging contrast agents onto the D1D2 protein, enabling biomolecules that previously lacked in vivo imaging signals to acquire magnetic resonance imaging capabilities, thus overcoming the technical barrier that traditional protein molecules cannot be directly used for in vivo imaging. More importantly, by encapsulating the modified D1D2 protein with liposomes, the interaction between liposomes and blood-brain barrier endothelial cells enables efficient delivery of protein macromolecules across the blood-brain barrier, solving the key bottleneck that exogenous protein molecules cannot autonomously enter the brain. This allows the diagnostic probe to perform non-invasive detection of Aβ oligomers in the brain at the in vivo level.

[0023] Compared to existing diagnostic methods that rely on radioactive positron emission tomography (PET), the liposomal drug of this invention provides a basis for diagnosis using magnetic resonance imaging (MRI) technology, completely avoiding the health risks associated with ionizing radiation. It is more suitable for routine screening and multiple follow-up monitoring of early Alzheimer's disease, helping patients to receive timely diagnosis and treatment during the early intervention window. Furthermore, the liposomal drug has a mature preparation process, good biocompatibility, and strong targeting, and has broad prospects for clinical application.

[0024] Preferably, the magnetic resonance imaging contrast agent includes gadolinium-based contrast agents, iron-based contrast agents, and manganese-based contrast agents, with gadolinium-based contrast agents being the most preferred.

[0025] The paramagnetic properties of gadolinium effectively improve the sensitivity and spatial resolution of liposomal drugs in magnetic resonance imaging, making it possible to detect trace amounts of β-amyloid oligomers in the brains of early-stage Alzheimer's patients. Simultaneously, gadolinium-based contrast agents exhibit good biocompatibility and metabolic stability. Chelation with macrocyclic ligands such as DOTA effectively avoids the toxic release of free gadolinium ions, ensuring safety for in vivo application. Furthermore, the bioorthogonal linkage between gadolinium contrast agents and D1D2 proteins ensures high labeling efficiency and site specificity, without affecting the protein's original spatial conformation and targeting binding ability. This allows the liposomal drug to achieve both high-sensitivity imaging and excellent targeting recognition, providing a reliable technical means for the early non-invasive diagnosis of Alzheimer's disease.

[0026] Preferably, the liposomes are composed of lecithin, cholesterol, DSPE-PEG2k and DSPE-PEG-MA, with a molar ratio of 60-70:25-35:1-5:1-5, and the optimal ratio is 65:30:2.5:2.5.

[0027] Lecithin, as the main membrane structural component, provides the basic framework and biocompatibility of liposomes. Cholesterol, inserted into the phospholipid bilayer in an appropriate proportion, enhances the stability and rigidity of the membrane, effectively reducing drug leakage and premature dissociation of liposomes in vivo. The introduction of DSPE-PEG2k significantly prolongs the half-life of liposomes in blood circulation through polyethylene glycol modification, reduces the clearance efficiency of the reticuloendothelial system, and improves drug bioavailability. DSPE-PEG-MA provides additional functional sites or charge properties, which help enhance the interaction between liposomes and the blood-brain barrier and promote cross-barrier transport.

[0028] The liposome composition and ratio of the present invention enable the liposomes to have suitable particle size and surface properties, which can effectively encapsulate biomacromolecules such as D1D2 modified proteins, while maintaining good colloidal stability and uniform dispersion, ensuring stable drug delivery in vivo and targeted enrichment in the brain, thereby achieving efficient diagnosis of early Alzheimer's disease.

[0029] Preferably, the liposomes have a particle size of less than 200 nm and a polydispersity index of less than 0.2.

[0030] The liposome preparation method of the present invention involves preparing a magnetic resonance imaging contrast agent precursor and a D1D2 modified protein precursor, then mixing the two and performing a bioorthogonal reaction to obtain the modified protein; and using a thin-film hydration method to encapsulate the D1D2 modified protein in the liposomes to obtain a liposomal drug.

[0031] Bioorthogonal reactions possess high selectivity and mild reaction conditions, enabling them to proceed efficiently in aqueous physiological environments without interfering with the native structure and function of biomolecules. Furthermore, the high reaction rate and yield of bioorthogonal reactions ensure the high efficiency and uniformity of contrast agent labeling, making the amount of contrast agent carried by each protein molecule controllable and reproducible. This guarantees the stability and reliability of subsequent in vivo magnetic resonance imaging signals. This chemical modification strategy exhibits good biocompatibility and low immunogenicity.

[0032] The thin-film hydration method involves dissolving membrane materials such as phospholipids in an organic solvent and then evaporating them under reduced pressure and rotational conditions to form a uniform and dense lipid film. This allows the membrane molecules to arrange themselves in an orderly manner and achieve full hydration. The resulting lipid film can effectively encapsulate water-soluble biomolecules such as the D1D2-Gd modified protein, achieving high encapsulation efficiency and drug loading.

[0033] Preferably, the liposome preparation method of the present invention includes the following steps: S1. The magnetic resonance imaging contrast agent precursor N3-DOTA-Gd was prepared by reacting DOTA-N3 with GdCl3·6H2O. S2. The D1D2 modified protein precursor D1D2-DBCO was prepared by reacting LilrB2 D1D2 protein with NHS-DBCO. S3. React D1D2-DBCO and N3-DOTA-Gd at pH 7.4 and temperature 4℃ for 12-16 hours to obtain the D1D2 modified protein D1D2-Gd.

[0034] In the above process, the azido-alkyne cycloaddition reaction was promoted by the strain between DBCO and the azide group, which achieved site-specific covalent linkage between LilrB2 D1D2 protein and magnetic resonance imaging contrast agent. This reaction does not require metal catalyst catalysis, avoiding the potential damage to protein activity by heavy metal ions. At the same time, the reaction conditions are mild, effectively maintaining the three-dimensional conformation of D1D2 protein and its high affinity binding ability to β-amyloid oligomers.

[0035] S4. The lipid membrane is mixed with a solution containing D1D2-Gd and hydrated to allow the lipid membrane to fully swell and spontaneously curl into a closed bilayer vesicle structure. Then, it is extruded using a polycarbonate membrane to obtain liposomes Lipo@D1D2-Gd, in which the D1D2-Gd modified protein is encapsulated by a thin-film hydration method.

[0036] Preferably, the lipid membrane is prepared by dissolving lecithin, cholesterol, DSPE-PEG2k, and DSPE-PEG-MA in a chloroform / methanol (volume ratio 3:1) solution at a molar ratio of 65:30:2.5:2.5, rotating the solution at 50 r / min for 30 min under reduced pressure (0.08 MPa) at 37°C, and evaporating the organic solvent to form a uniform lipid membrane. The above process ensures complete removal of the organic solvent and avoids the destruction of protein activity by high temperature.

[0037] Preferably, the hydration method is to rotate at 4°C for 30 min; this condition is mild and preserves the targeting binding activity of the D1D2 protein and the imaging function of the gadolinium contrast agent to the greatest extent.

[0038] Preferably, the polycarbonate film is 200 nm thick and the number of extrusions is 9-13 times; this allows for precise control of the liposome particle size distribution, resulting in a monodisperse liposome population with uniform particle size and low polydispersity index, significantly improving the physical stability and batch-to-batch reproducibility of the formulation.

[0039] The liposomal drug of the present invention can be used in the preparation of formulations for the diagnosis of early Alzheimer's disease.

[0040] The formulation of the present invention for diagnosing early Alzheimer's disease includes the liposomal drug described above; preferably, the formulation also includes other pharmaceutically acceptable excipients, auxiliaries, or buffer solutions.

[0041] The effects of the present invention will be illustrated by specific embodiments below.

[0042] Example 1: Modification of LilrB2 D1D2 This embodiment modifies LilrB2 D1D2 using a biological orthogonal method, such as... Figure 1 As shown, the specific steps are as follows: (1) Under vigorous stirring, DOTA-N3 was slowly added dropwise to an equimolar amount of GdCl3·6H2O aqueous solution. Under the reaction conditions of pH 6.0 and temperature 60℃, the mixture was stirred continuously for 24 hours, and the solution was freeze-dried to obtain a white solid product N3-DOTA-Gd.

[0043] (2) Under the reaction conditions of pH 7.4 and temperature 4℃, LilrB2 D1D2 was mixed with excess NHS-DBCO and reacted overnight, and D1D2-DBCO was obtained by purification.

[0044] The amino acid sequence of LilrB2 D1D2 in this embodiment is shown in SEQ ID NO:1: SEQ ID NO:1:MGSSHHHHHHSSGGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLV LVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSK (3) Under reaction conditions of pH 7.4 and temperature 4℃, D1D2-DBCO was mixed with excess N3-DOTA-Gd and reacted overnight, and D1D2-Gd was obtained by purification.

[0045] (4) The reaction products were characterized by SDS-PAGE to verify the successful conduct of the reaction.

[0046] Figure 2 The above are the SDS-PAGE results of the modified protein in this embodiment. D1D2-DBCO* is purified D1D2-DBCO, and D1D2-Gd* is purified D1D2-Gd. Figure 2 The changes in molecular weight of D1D2-DBCO and D1D2-Gd confirm the success of the covalent modification. Therefore, this example yielded high-purity D1D2-Gd.

[0047] Example 2: Verification of the affinity of D1D2-Gd for Aβ In this embodiment, ELISA and pull-down experiments are used to verify the binding of D1D2-Gd with Aβ, in order to verify the affinity of D1D2-Gd.

[0048] (1) ELISA experiment: D1D2 and D1D2-Gd were dissolved in PBS at a concentration of 3 μg / mL, and 100 μL was added to each well of the ELISA plate. The plate was incubated overnight at 4°C. After washing the ELISA plate with PBST solution (PBS, pH 7.4, 0.05% Tween-20), 200 μL of BSA solution (PBS, pH 7.4, 0.05% Tween-20, 5% w / v BSA) was added, and the plate was blocked at 37°C for 1 h.

[0049] After washing the ELISA plate with PBST solution, add 100 μL of Aβ oligomer solutions with concentrations of 0, 0.25, 0.5, and 1 μM, and react at 37°C for 1 h. After washing the ELISA plate with PBST solution, add 100 μL of Aβ-specific antibody 6E10 dilution buffer (1:10000) and incubate at room temperature for 2 h.

[0050] After washing the ELISA plate with PBST solution, add 100 μL of secondary antibody dilution buffer (HRP crosslinked goat anti-mouse IgG1, 1:3000) and incubate at room temperature for 1 h. After washing the ELISA plate with PBST solution, add 100 μL of TMB solution and incubate at room temperature for 5-20 min. Then add 50 μL of ELISA stop solution and detect the absorbance at 450 nm.

[0051] The absorbance comparison results of the ELISA experiment are as follows: Figure 3 As shown in Figure a, in 0.25, 0.5, and 1 μM solutions of Aβ oligomers, the affinity of D1D2-Gd for Aβ was not significantly different from that of LilrB2 D1D2, indicating that D1D2-Gd is fully capable of recognizing and detecting Aβ.

[0052] (2) Pull-down experiment: After washing and equilibrating the Ni column with PBS, add 100 μL of a mixture of D1D2-Gd and Aβ oligomers, and 100 μL of binding buffer (PBS, pH 7.4, 20 mM imidazole). Incubate overnight at 4°C by rotation. Centrifuge at 1000 xg for 1 min, collect the supernatant as FT (Flow Through), and wash the column three times with 200 μL of binding buffer (PBS, pH 7.4, 20 mM imidazole). Collect the supernatant as Wash.

[0053] Add 50 μL of elution buffer (PBS, pH 7.4, 500 mM imidazole), mix for 15 min, and collect the sample as Elu. (Elution). Add non-reducing loading buffer to the sample solution in proportion, and perform non-reducing gel electrophoresis under constant voltage of 150V.

[0054] In the environment of rapid transfer buffer, the protein was transferred to a 0.2 μm PVDF membrane at a constant current of 400 mA for 30 min; the membrane was blocked with 5% skim milk solution at room temperature for 1 h; and incubated overnight at 4 °C with Aβ-specific antibody 6E10 dilution (1:2000).

[0055] After washing the membrane with TBST solution, add secondary antibody dilution buffer (HRP crosslinked goat anti-mouse IgG1, 1:3000) and incubate at room temperature for 2 h; after washing the membrane with TBST solution, add ECL chemiluminescence solution for chemiluminescence imaging.

[0056] The results of the pull-down experiment are as follows: Figure 3 As shown in Figure b, this result also demonstrates that D1D2-Gd has a good affinity for Aβ.

[0057] Example 3 Preparation of Lipo@D1D2-Gd like Figure 4 As shown, this embodiment uses a thin-film hydration method to prepare Lipo@D1D2-Gd, and the specific steps are as follows: (1) Dissolve lecithin, cholesterol, DSPE-PEG2k and DSPE-PEG-MA in a chloroform / methanol (volume ratio 3:1) solution at a mass ratio of 65:30:2.5:2.5. Rotate at 50 r / min for 30 min under reduced pressure (0.08 MPa) at 37℃ to evaporate the organic solvent and form a uniform lipid film.

[0058] (2) Add D1D2-Gd solution and rotate at 4℃ for 30 min to completely hydrate the lipid film. (3) The protein-lipid mixture was extruded 10 times using a 200 nm polycarbonate membrane to obtain homogeneous Lipo@D1D2-Gd.

[0059] (4) The liposome particle size and polydispersity index were determined by DLS, and the results are as follows: Figure 5 As shown.

[0060] DLS analysis results showed that the average particle size of the liposomes was 173.7 nm and the PDI was 0.136, proving that the preparation process can obtain a stable formulation with uniform particle size and good dispersibility.

[0061] Example 4: Verification of Lipo@D1D2-Gd in vivo magnetic resonance imaging results In this embodiment, Lipo@D1D2-Gd was injected into mice via the tail vein, and wild-type mice and AD model mice were distinguished by in vivo magnetic resonance imaging. The specific steps are as follows: (1) Wild-type and AD model mice aged 4 months were fasted for 24 h before imaging examination. (2) After completely anesthetizing the mice with 2.5% isoflurane, fix them on the scanning table and continue to anesthetize them by nasal injection of 1-2% isoflurane.

[0062] (3) After coil calibration, the brain images of mice were scanned using a T1-weighted rapid low-angle imaging sequence (T1-FLASH: repetition time = 50.0 ms, echo time = 1.9 ms, slice thickness = 1.0 mm, image size = 256 × 256, field of view = 30.0 mm × 30.0 mm, number of slices = 40).

[0063] (4) Inject Lipo@D1D2-Gd (0.064 μmol D1D2 / kg) into the tail vein of mice, and perform T1-FLASH sequence scans on the brain images of mice again 40 min after injection.

[0064] (5) Quantify the gray values ​​of mouse brain regions obtained from magnetic resonance scanning to distinguish between wild-type mice and AD model mice.

[0065] The in vivo magnetic resonance imaging image of the liposome-coated modified protein Lipo@D1D2-Gd in this embodiment is as follows: Figure 6 As shown in Figure a, the quantization results are as follows: Figure 6 As shown in b.

[0066] according to Figure 6It can be seen that 40 minutes after tail vein injection of Lipo@D1D2-Gd, there was a significant increase in T1 signal in the brain of AD model mice, while no significant change was observed in wild-type mice. Quantitative analysis confirmed the significant difference between the two groups, indicating that the liposomal drug can successfully cross the blood-brain barrier and specifically recognize Aβ oligomers in the brain, thus achieving effective differential diagnosis of early Alzheimer's disease.

[0067] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A liposomal drug, characterized in that, include: A modified LilrB2 extracellular domain D1D2 protein that can target and bind to β-amyloid oligomers, wherein the modified D1D2 protein is linked to a magnetic resonance imaging contrast agent. Liposomes encapsulating the D1D2 modified protein.

2. The liposomal drug according to claim 1, characterized in that, The magnetic resonance imaging contrast agents include gadolinium-based contrast agents, iron-based contrast agents, and manganese-based contrast agents.

3. A liposomal drug according to claim 1, characterized in that, The liposomes are composed of lecithin, cholesterol, DSPE-PEG2k and DSPE-PEG-MA, with a molar ratio of 60-70:25-35:1-5:1-5.

4. A liposomal drug according to any one of claims 1-3, characterized in that, The liposomes have a particle size of less than 200 nm and a polydispersity index of less than 0.

2.

5. A method for preparing a liposome drug as described in any one of claims 1-4, characterized in that, A magnetic resonance imaging contrast agent precursor and a D1D2 modified protein precursor were prepared separately, then the two were mixed and subjected to a bioorthogonal reaction to obtain the modified protein; the D1D2 modified protein was then encapsulated in liposomes using a thin-film hydration method to obtain the liposomal drug.

6. The method for preparing a liposomal drug according to claim 5, characterized in that, Includes the following steps: S1. The magnetic resonance imaging contrast agent precursor N3-DOTA-Gd was prepared by reacting DOTA-N3 with GdCl3·6H2O. S2. The D1D2 modified protein precursor D1D2-DBCO was prepared by reacting LilrB2 D1D2 protein with NHS-DBCO. S3. React D1D2-DBCO and N3-DOTA-Gd at pH 7.4 and temperature 4℃ for 12-16 hours to obtain the D1D2 modified protein D1D2-Gd; S4. After mixing and hydrating the lipid membrane with a solution containing D1D2-Gd, the mixture is extruded using a polycarbonate membrane to obtain the liposome drug Lipo@D1D2-Gd, in which the D1D2-Gd modified protein is encapsulated in liposomes using a thin-film hydration method.

7. A method for preparing a liposome drug according to claim 6, characterized in that, In step S4, the hydration method is to rotate at 4°C for 30 minutes.

8. A method for preparing a liposome drug according to claim 6, characterized in that, In step S4, the polycarbonate film is 200 nm thick, and the extrusion times are 9-13.

9. The use of the liposomal drug as described in any one of claims 1-4 in the preparation of a formulation for diagnosing early Alzheimer's disease.

10. A formulation for diagnosing early-stage Alzheimer's disease, characterized in that, Including the liposomal drugs as described in any one of claims 1-4.

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