A liposome material for real-time in situ fluorescence imaging of primary central nervous system lymphoma, its preparation method and application.

Abstract

This invention discloses a liposome material for real-time in situ fluorescence imaging of primary central nervous system lymphoma, its preparation method, and its application, belonging to the field of fluorescence imaging technology. The liposome material provided by this invention comprises a liposome carrier composed of soybean lecithin and cholesterol, a brain-targeting peptide, an anti-CD20 monoclonal antibody, and a near-infrared fluorescent dye; the brain-targeting peptide and the anti-CD20 monoclonal antibody are modified on the surface of the liposome carrier; the brain-targeting peptide is a transferrin receptor-targeting peptide; and the near-infrared fluorescent dye is DiR. This material avoids non-specific binding to normal brain tissue and non-CD20-positive lesions, significantly improving the diagnostic specificity for primary central nervous system lymphoma, effectively reducing the risk of imaging confusion with gliomas, metastatic tumors, and inflammatory demyelinating diseases of the central nervous system, while avoiding invasive biopsy procedures, achieving non-invasive or minimally invasive real-time in situ diagnosis.

Description

Technical Field

[0001] This invention relates to the field of fluorescence imaging technology, and in particular to a liposome for real-time in situ fluorescence imaging of primary central nervous system lymphoma, its preparation method, and its application. Background Technology

[0002] The clinical manifestations of primary central nervous system lymphoma (PCNSL) lack specificity and highly overlap with the imaging features of gliomas, metastatic tumors, and inflammatory demyelinating diseases, leading to a high rate of misdiagnosis. Among existing diagnostic methods, stereotactic brain biopsy is the gold standard, but it is an invasive procedure with risks of bleeding, infection, and neurological deficits; some patients cannot undergo this procedure due to the deep location of the lesion or poor general condition. Lumbar puncture for cerebrospinal fluid cytology has a low positive rate and is prone to false negatives. Conventional neuroimaging techniques such as MRI and PET / CT are insufficient for specific molecular-level differentiation, and atypical manifestations are often confused with various intracranial lesions, resulting in insufficient specificity. Although some fluorescent probes have been developed for intraoperative identification, most rely on local administration or target other brain tumor types; there is a lack of diagnostic tools that can be administered systemically, actively cross the blood-brain barrier, and target PCNSL lesions for real-time in vivo fluorescence imaging. Current technologies still have significant limitations in terms of diagnostic accuracy, minimal invasiveness, and real-time performance. Summary of the Invention

[0003] In view of this, the present invention provides a liposomal material for real-time in situ fluorescence imaging of primary central nervous system lymphoma, its preparation method and application, so as to achieve real-time, in situ, and highly specific fluorescence imaging diagnosis of primary central nervous system lymphoma.

[0004] In a first aspect, the present invention provides a liposome material for real-time in situ fluorescence imaging of primary central nervous system lymphoma, comprising a liposome carrier composed of soybean lecithin and cholesterol, a brain-targeting peptide, an anti-CD20 monoclonal antibody, and a near-infrared fluorescent dye. The brain-targeting peptide and the anti-CD20 monoclonal antibody are modified on the surface of the liposome carrier; The brain-targeting peptide is a transferrin receptor-targeting peptide; the near-infrared fluorescent dye is DiR.

[0005] Preferably, the transferrin receptor targeting peptide is Transferrin 7, and its amino acid sequence is HAIYPRH.

[0006] Furthermore, Transferrin 7 is connected to DSPE-PEG2000 via a thioether bond to form DSPE-PEG2000-Transferrin 7, which is anchored to the surface of the liposome carrier.

[0007] Furthermore, the mass ratio of soybean lecithin, cholesterol, DSPE-PEG2000-Transferrin 7 and near-infrared fluorescent dye is (19.5~20.5): (4.5~5.5): (3.5~4.5): (0.8~1.2).

[0008] Preferably, the anti-CD20 monoclonal antibody is inserted into the surface of the liposome carrier via phospholipid modification.

[0009] Preferably, the average particle size of the liposome material is 100~150 nm.

[0010] Secondly, the present invention provides a method for preparing the above-mentioned liposome material, comprising the following steps: Soybean lecithin, cholesterol, brain-targeting peptides and near-infrared fluorescent dyes were dissolved in an organic solvent, and a liposome solution loaded with near-infrared fluorescent dyes and modified with brain-targeting peptides on the surface was obtained by thin-film hydration method. The liposome solution was mixed with the phospholipid-modified anti-CD20 monoclonal antibody solution, incubated, centrifuged, and resuspended to obtain the final product.

[0011] Preferably, the organic solvent is at least one of methanol and chloroform; the concentration of the liposome solution is 0.8~1.5 mg / mL; and the concentration of the anti-CD20 monoclonal antibody solution is 0.8~1.5 mg / mL.

[0012] Further, the step of mixing and incubating the liposome solution with the phospholipid-modified anti-CD20 monoclonal antibody is as follows: the liposome solution and the phospholipid-modified anti-CD20 monoclonal antibody solution are mixed and incubated at a volume ratio of (4.8~5.2): (1.8~2.2) at a temperature of 20~35℃ for 4~8 h; after incubation, the mixture is sonicated at 20~35℃ for 20~40 min, and then centrifuged.

[0013] Preferably, the centrifugation speed is 15000~18000 rpm, the temperature is 2~5℃, the centrifugation time is 60~120 min, and the number of centrifugations is 1~4.

[0014] Thirdly, the present invention provides the application of the above-mentioned liposome material or the liposome material prepared by the above-mentioned preparation method in the preparation of a real-time in situ fluorescence imaging diagnostic reagent for primary central nervous system lymphoma.

[0015] Compared with the prior art, the present invention has achieved the following beneficial effects: The liposome material of this invention consists of soybean lecithin and cholesterol as liposome carriers, and is modified with transferrin receptor-targeting peptides and anti-CD20 monoclonal antibodies on its surface. The transferrin receptor-targeting peptides mediate the crossing of the blood-brain barrier, enabling the liposome material to be actively delivered to the central nervous system. The anti-CD20 monoclonal antibody specifically recognizes the CD20 antigen on the surface of primary central nervous system lymphoma cells, achieving active targeted enrichment of the lesion site. The near-infrared fluorescent dye DiR loaded in the liposomes provides a fluorescence signal with low tissue autofluorescence interference and high tissue penetration depth in in vivo imaging, thereby displaying the tumor location and boundary in real time in vivo. This material combines brain-targeted delivery, lymphoma-specific molecular recognition, and near-infrared fluorescence imaging, avoiding non-specific binding to normal brain tissue and non-CD20-positive lesions. It significantly improves the diagnostic specificity for primary central nervous system lymphomas, effectively reduces the risk of imaging confusion with central nervous system lesions such as gliomas, metastatic tumors, and inflammatory demyelinating diseases, and avoids invasive biopsy procedures, achieving non-invasive or minimally invasive real-time in situ diagnosis. Attached Figure Description

[0016] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0017] Figure 1 These are structural characterizations of the liposome materials of Examples 1 and Comparative Examples 1-2 of the present invention; wherein, a is a transmission electron microscope (TEM) image of the liposome material of Example 1; b is a comparison diagram of the hydration kinetics particle size of the liposome materials of Examples 1 and Comparative Examples 1-2; c is a Zeta potential diagram of the liposome materials of Examples 1 and Comparative Examples 1-2; d is a comparison diagram of the hydration kinetics particle size of the liposome materials of Example 1 (sample 1 in the figure) and Comparative Example 3 (sample 2 in the figure); e is an image of the liposome materials in the static state of two parallel experiments of Comparative Example 4; f is a comparison diagram of the hydration kinetics particle size of the liposome materials of Example 1 (sample 1 in the figure) and Comparative Example 4 (sample 2 in the figure). Figure 2 These are the cytotoxicity test results of the liposome material of Example 1 of the present invention on Raji cells and Karpas-299 cells; where a is Raji cells; b is Karpas-299 cells; Figure 3 These are the results of the live-cell specific targeting ability test of the liposome materials in Embodiment 1 and Comparative Example 2 of the present invention; wherein, a is the result of flow cytometry analysis; b is the average fluorescence intensity data output result of a; and c is the result of live-cell laser confocal imaging. Figure 4 These are the live-cell laser confocal imaging results of the liposome materials in Embodiment 1 and Comparative Example 5 of the present invention; Figure 5 These are the live-cell laser confocal imaging results of the liposome materials in Comparative Example 6, Example 1, and Comparative Example 7 of this invention; Figure 6 The results of the specific recognition test of the liposome material prepared in Example 1 of this invention on different cell lines are shown; where a is the result of flow cytometry analysis; b is the average fluorescence intensity data output result of a. Figure 7 These are the results of the targeting ability verification of the liposome materials of Example 1 and Comparative Example 2 of the present invention against central nervous system lymphoma in mice; where a is a mouse with a brain tumor of similar size; b is the fluorescence signal at the location of the brain tumor in the mouse with central nervous system lymphoma after injection of the material of Example 1 (left) and the material of Comparative Example 2 (right); Figure 8 The results show the imaging diagnostic application of the liposome materials of Example 1 and Comparative Example 8 of this invention on central nervous system lymphoma in mice; a) is the fluorescence signal of the brain tumor location in mice with central nervous system lymphoma injected in Example 1 and healthy control mice, respectively; b) is the fluorescence signal of the brain tumor location in mice with central nervous system lymphoma injected in Comparative Example 8 and healthy control mice, respectively. Figure 9 The results show the imaging differential diagnosis of central nervous system lymphoma in mice using the liposome material of Example 1 of this invention; fluorescence signals in mouse models of central B-cell lymphoma, central T-cell lymphoma, and glioma after injection of the material of Example 1. Detailed Implementation

[0018] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0019] Primary central nervous system lymphoma (PCNSL) is a rare but highly aggressive type of non-Hodgkin's lymphoma, with over 95% of cases being diffuse large B-cell lymphoma. Due to its nonspecific clinical presentation and the fact that its imaging features often highly overlap with gliomas, metastases, and inflammatory demyelinating diseases, the rate of misdiagnosis is high. Among existing diagnostic methods, stereotactic brain biopsy is the gold standard, but it is an invasive procedure with risks of bleeding, infection, and neurological deficits, and is not feasible for some patients due to deep lesion locations or poor general condition. Lumbar puncture cerebrospinal fluid cytology has a low positive rate and is prone to false negatives. Conventional neuroimaging examinations lack specificity, making it difficult to achieve molecular-level differential diagnosis. To address these problems, this invention provides the following technical solution.

[0020] This invention provides a liposome material for real-time in situ fluorescence imaging of primary central nervous system lymphoma, comprising a liposome carrier composed of soybean lecithin and cholesterol, a brain-targeting peptide, an anti-CD20 monoclonal antibody, and a near-infrared fluorescent dye; wherein the brain-targeting peptide and the anti-CD20 monoclonal antibody are modified on the surface of the liposome carrier; the brain-targeting peptide is a transferrin receptor-targeting peptide; and the near-infrared fluorescent dye is DiR.

[0021] In this invention, the liposome carrier is composed of soybean lecithin and cholesterol. Soybean lecithin and cholesterol are the basic membrane materials for forming the liposome bilayer, exhibiting good biocompatibility, biodegradability, and low immunogenicity. Soybean lecithin, as the main phospholipid component, provides the bilayer framework, while cholesterol, embedded in the phospholipid bilayer, regulates membrane fluidity and stability, reduces liposome permeability, and minimizes leakage of the load.

[0022] In this invention, the brain-targeting peptide is a transferrin receptor-targeting peptide. The transferrin receptor is highly expressed on the endothelial cell membrane of the blood-brain barrier and is an important target for mediating the transport of macromolecules across the blood-brain barrier. The transferrin receptor-targeting peptide specifically binds to the transferrin receptor on the surface of blood-brain barrier endothelial cells, is internalized through receptor-mediated endocytosis, and then transported across the cell to the brain parenchyma, thereby achieving the active delivery of liposome materials across the blood-brain barrier.

[0023] Preferably, the transferrin receptor targeting peptide is Transferrin 7, with the amino acid sequence HAIYPRH. Transferrin 7 is a short transferrin receptor targeting peptide obtained through phage display technology. Compared to full-length transferrin, it has a smaller molecular weight, lower immunogenicity, lower production cost, and maintains a high affinity for the transferrin receptor.

[0024] Furthermore, the brain-targeting peptide Transferrin 7 is linked to DSPE-PEG2000 via a thioether bond to form DSPE-PEG2000-Transferrin 7, which is anchored to the surface of the liposome carrier. The lipid tail of DSPE (distearate phosphatidylethanolamine) can be inserted into the liposome bilayer membrane, and PEG2000 (polyethylene glycol, molecular weight 2000) acts as a flexible spacer arm to modify the Transferrin 7 onto the liposome surface. At the same time, the PEG chain can reduce the recognition and clearance of liposomes by the reticuloendothelial system in the blood circulation, thus prolonging the systemic circulation time.

[0025] Furthermore, the mass ratio of soybean lecithin, cholesterol, DSPE-PEG2000-Transferrin 7, and near-infrared fluorescent dye is (19.5~20.5): (4.5~5.5): (3.5~4.5): (0.8~1.2). This ratio range can balance the stability of the liposome membrane, the surface density of the targeting ligand, and the encapsulation efficiency of the dye. Exceeding this range may lead to problems such as uneven liposome particle size, decreased stability, insufficient targeting ligand density affecting brain targeting efficiency, or insufficient imaging signal due to excessively low dye encapsulation rate.

[0026] In this invention, the anti-CD20 monoclonal antibody is modified on the surface of a liposome carrier. CD20 is a specific marker on the surface of B lymphocytes, highly expressed in over 95% of primary central nervous system lymphoma cells, but not expressed or expressed at low levels in normal brain tissue and other central nervous system lesions (such as gliomas, metastases, and inflammatory demyelinating diseases). The anti-CD20 monoclonal antibody achieves active targeting of primary central nervous system lymphoma lesions by specifically recognizing and binding to the CD20 antigen through the liposome material.

[0027] Preferably, the anti-CD20 monoclonal antibody is inserted into the surface of the liposome carrier via phospholipid modification. Phospholipid modification refers to covalently linking hydrophobic phospholipid molecules to the antibody molecule. The modified antibody inserts into the liposome bilayer membrane through the hydrophobic interaction of its phospholipid tail, achieving stable anchoring of the antibody on the liposome surface. This method avoids potential interference with the antibody's active site caused by chemical cross-linking, thus preserving the antibody's antigen-binding ability.

[0028] In this invention, the near-infrared fluorescent dye is DiR. DiR is a lipophilic near-infrared fluorescent film dye with excitation and emission wavelengths both located in the near-infrared region (approximately 750 nm and 780 nm). It exhibits minimal scattering and absorption in biological tissues, allowing for tissue penetration depths of several centimeters, making it suitable for imaging deep tissues in living animals. Furthermore, DiR possesses good photostability and is not easily quenched rapidly, making it suitable for long-term dynamic observation of fluorescence signal changes at lesion sites. DiR is loaded into the lipid bilayer of a liposome carrier, utilizing its lipophilicity to stably locate within the lipid membrane.

[0029] Preferably, the average particle size of the liposome material is 100-150 nm. This particle size range is beneficial for the stable existence of liposomes in the blood circulation, and at the same time, it can be enriched at the lesion site through the passive targeting effect (EPR effect) of areas with impaired blood-brain barrier (such as tumor sites). Preferably, the average particle size is 120-140 nm; for example, it can be 110 nm, 125 nm, 130 nm, 135 nm or 145 nm; more preferably, it is 130-150 nm. If the particle size is too small, it may be cleared too quickly in the blood circulation, while if the particle size is too large, it may affect the ability to cross the blood-brain barrier.

[0030] The present invention also provides a method for preparing the above-mentioned liposome material, comprising the following steps: dissolving soybean lecithin, cholesterol, brain-targeting peptide and near-infrared fluorescent dye in an organic solvent, and obtaining a liposome solution loaded with near-infrared fluorescent dye and modified with brain-targeting peptide on the surface by thin-film hydration method; mixing the liposome solution with phospholipid-modified anti-CD20 monoclonal antibody, centrifuging and resuspending to obtain the final product.

[0031] Preferably, the organic solvent is at least one of methanol and chloroform. Both solvents can effectively dissolve soybean lecithin, cholesterol, and DSPE-PEG2000-Transferrin 7, and have low boiling points, making them easy to remove by rotary evaporation.

[0032] In a preferred embodiment of the present invention, the preparation method of DSPE-PEG2000-Transferrin 7 includes: dissolving DSPE-PEG2000-MAL and Transferrin 7 in phosphate buffer (PBS) at a molar ratio of 1:(1~2), reacting at room temperature for 12~24 hours, and forming a thioether bond between the terminal thiol group and the maleimide group of Transferrin 7 through a Michael addition reaction, and purifying to obtain DSPE-PEG2000-Transferrin 7. DSPE-PEG2000-MAL is a bifunctional lipid derivative with a structure consisting of three parts: DSPE (distearate phosphatidylethanolamine) as a lipid anchoring part that can be inserted into the liposome bilayer membrane; PEG2000 (polyethylene glycol, molecular weight 2000) as a flexible hydrophilic spacer arm; and the terminal maleimide (MAL) group for specific Michael addition reactions with thiol-containing molecules (such as Transferrin 7) to form a stable thioether bond.

[0033] Specifically, the membrane hydration method includes: dissolving soybean lecithin, cholesterol, DSPE-PEG2000-Transferrin 7, and DiR dye in an organic solvent at a mass ratio of (19.5~20.5):(4.5~5.5):(3.5~4.5):(0.8~1.2), and sonicating for 10~15 min to ensure complete dissolution; removing the organic solvent by rotary evaporation under reduced pressure (temperature 25~35℃, speed 40~60 rpm) to form a uniform phospholipid bilayer film on the bottle wall, and vacuum drying overnight to completely remove residual solvent; adding deionized water for hydration, shaking by hand until the film layer completely detaches, and then sonicating at 25~35℃ for 10~15 min; extruding through 0.45 μm and 0.22 μm aqueous phase filter membranes 2~4 times each to initially remove large particles and unstable aggregates; and then using a liposome extruder to pass through a 100 A polycarbonate film of nm is extruded back and forth 10-15 times to obtain a liposome solution with uniform particle size, which is then refrigerated for later use. Preferably, the concentration of the liposome solution is 0.8-1.5 mg / mL.

[0034] In a preferred embodiment of the present invention, the preparation method of the phospholipid-modified anti-CD20 monoclonal antibody is as follows: Activated phospholipids with terminal NHS groups are mixed with anti-CD20 monoclonal antibody at a mass ratio of (1.5~2.5):(0.8~1.2) and allowed to stand at room temperature for 1.5~3 hours; the activated phospholipids with terminal NHS groups are covalently coupled to the primary amine groups on the anti-CD20 monoclonal antibody through their NHS groups; after the reaction is completed, the mixture is centrifuged 2~4 times using high-speed centrifugation (12000~15000 rpm, 2~6℃, 30~60 min) to remove unreacted phospholipids.

[0035] Preferably, the step of mixing and incubating the liposome solution with the phospholipid-modified anti-CD20 monoclonal antibody solution specifically involves: mixing the liposome solution and the phospholipid-modified anti-CD20 monoclonal antibody solution at a volume ratio of (4.8~5.2):(1.8~2.2) and incubating at a temperature of 20~35℃ for 4~8 h; after incubation, sonicating at 20~35℃ for 20~40 min, followed by centrifugation. The preferred incubation temperature is 25~30℃; the preferred incubation time is 4~6 h. The concentration of the phospholipid-modified anti-CD20 monoclonal antibody solution is 0.8~1.5 mg / mL. In this step, the phospholipid-modified anti-CD20 monoclonal antibody inserts into the liposome bilayer membrane through its phospholipid tail; sonication helps accelerate the insertion process and improve insertion efficiency.

[0036] Preferably, the centrifugation speed is 15000~18000 rpm, the temperature is 3~5℃, the centrifugation time is 60~120 min, and the number of centrifugations is 1~4. Preferably, the speed is 17000 rpm, the temperature is 4℃, the centrifugation time is 90 min, and the number of centrifugations is 2. High-speed centrifugation can effectively remove free phospholipid-modified antibodies and DiR dye that may leak from the liposomes. After centrifugation, the supernatant is discarded, and the precipitate is resuspended in phosphate-buffered saline (PBS) to obtain the final target liposome material with a concentration of 0.8~1.5 mg / mL, which is then refrigerated (2~5℃) for later use.

[0037] This invention also provides the application of the above-mentioned liposome material or the liposome material prepared by the above-mentioned preparation method in the preparation of a real-time in situ fluorescence imaging diagnostic reagent for primary central nervous system lymphoma.

[0038] In use, the liposome material is administered to mice via intraperitoneal injection or other methods, and the distribution of fluorescence signals in the brain is observed in real-time using a small animal in vivo imaging system. The liposome material reaches the brain via blood circulation, and Transferrin 7 mediates its transport across the blood-brain barrier into the brain parenchyma. An anti-CD20 monoclonal antibody specifically anchors the liposomes to the surface of primary central nervous system lymphoma cells. DiR dye emits fluorescence under near-infrared excitation light, thus displaying the location, size, and boundaries of the tumor in real-time, in situ, and with high contrast at the in vivo level. This diagnostic reagent can be used for early detection, intraoperative navigation, efficacy evaluation, and recurrence monitoring of primary central nervous system lymphoma.

[0039] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention does not impose any special restrictions on the source of reagents used in the following embodiments; commercially available products well known to those skilled in the art can be used.

[0040] In the following examples, DSPE-PEG2000-Transferrin 7 was prepared as follows: DSPE-PEG2000-MAL and Transferrin 7 peptide (amino acid sequence HAIYPRH, with a free thiol group introduced at the H residue terminus) were dissolved in PBS buffer (pH 7.4) at a molar ratio of 1:1.2 to achieve a DSPE-PEG2000-MAL concentration of 5 mg / mL. The reaction system was stirred and reacted at room temperature under nitrogen protection for 24 hours in the dark. During the reaction, the thiol group at the end of Transferrin 7 and the maleimide group at the end of DSPE-PEG2000-MAL underwent a Michael addition reaction to form a stable thioether bond, generating DSPE-PEG2000-Transferrin 7. After the reaction was completed, the reaction solution was dialyzed to remove unreacted Transferrin 7 peptide and small molecule byproducts. The dialysis product was freeze-dried to obtain a white flocculent solid, which was the purified DSPE-PEG2000-Transferrin 7, and stored at -20℃ for later use.

[0041] Example 1 This embodiment provides the preparation of liposome materials for real-time in situ fluorescence imaging of primary central nervous system lymphoma.

[0042] (1) Dissolve 2 mg of soybean lecithin, 0.5 mg of cholesterol, 0.4 mg of DSPE-PEG2000-Transferrin 7, and 0.1 mg of near-infrared fluorescent dye DiR in 20 mL of methanol, place in a 250 mL round-bottom flask, and sonicate for 10 min to ensure complete dissolution. Remove the organic solvent by rotary evaporation under reduced pressure (30 °C, 50 rpm) to form a uniform phospholipid bilayer liposome film on the flask wall, and vacuum dry overnight to completely remove residual solvent.

[0043] (2) Add 3 mL of deionized water to the above liposome membrane for hydration, shake by hand until the membrane layer is completely detached, and then sonicate at 30℃ for 10 min. Extrude three times each through 0.45 μm and 0.22 μm aqueous phase filter membranes, and then extrude 12 times each through a 100 nm polycarbonate membrane using a liposome extruder to obtain a liposome solution with a concentration of 1.0 mg / mL, and store it in a refrigerator at 4℃ for later use.

[0044] (3) Phospholipid-conjugated anti-CD20 monoclonal antibody was prepared using a commercially available phospholipid-conjugated antibody-protein kit (Ruixiong Biotechnology, Xi'an). Following the instructions, a phospholipid-conjugated anti-CD20 monoclonal antibody solution with a concentration of 1.0 mg / mL was obtained. The phospholipids in the kit contain NHS groups, which can covalently couple with the primary amine groups on the anti-CD20 monoclonal antibody. The reaction conditions were as follows: 2 mg of phospholipid was mixed with 1 mg of anti-CD20 monoclonal antibody and allowed to stand at room temperature for 2 h without stirring or shaking; then, the mixture was centrifuged three times at high speed (13000 rpm, 4℃, 40 min) to remove unreacted phospholipids. The resulting phospholipid-conjugated anti-CD20 monoclonal antibody solution was stored at -20℃.

[0045] (4) The liposome solution obtained in step (2) was mixed with the phospholipid-modified anti-CD20 monoclonal antibody obtained in step (3) at a volume ratio of 5:2. The mixture was incubated at room temperature for 5 h and then sonicated at 30 °C for 30 min to allow the phospholipid-modified antibody to insert into the liposome surface. The mixture was centrifuged twice at high speed (17000 rpm, 4 °C, 90 min) to remove free phospholipid-modified antibody and any dye that may have leaked out. The precipitate was resuspended in PBS (pH 7.4) to obtain liposome material with a concentration of 1 mg / mL, denoted as PCNSL-Lipo-CD20 mAb, and stored at 4 °C for later use.

[0046] Comparative Example 1 The difference between this comparative example and Example 1 is that in step (3) of this comparative example, the phospholipid-modified anti-CD20 monoclonal antibody is replaced with an equal amount of phospholipid, while the other steps and conditions remain unchanged. This example is denoted as Lipo.

[0047] Comparative Example 2 The difference between this comparative example and Example 1 is that the phospholipid-based anti-CD20 monoclonal antibody was replaced with a phospholipid-based anti-IgG monoclonal antibody, while the other steps and conditions remained unchanged. The final liposome material was designated PCNSL-Lipo-IgG mAb.

[0048] Comparative Example 3 The difference between this comparative example and Example 1 is that the ratio of soybean lecithin to cholesterol in the preparation of the liposome solution was changed from soybean lecithin:cholesterol:DSPE-PEG2000-Transferrin 7:DiR = 20:5:4:1 to 21:7:4:1, while the other steps and conditions remained the same. The final liposome material was designated PCNSL-Lipo-CD20 mAb (21:7:4:1).

[0049] Comparative Example 4 The difference between this comparative example and Example 1 is that the ratio of dye DiR was changed during the preparation of the liposome solution. The ratio was changed from soybean lecithin:cholesterol:DSPE-PEG2000-Transferrin 7:DiR = 20:5:4:1 to 20:5:4:3, while the other steps and conditions remained the same. The final liposome material was designated PCNSL-Lipo-CD20 mAb (20:5:4:3).

[0050] Comparative Example 5 The difference between this comparative example and Example 1 is that the ratio of dye DiR was changed during the preparation of the liposome solution. The ratio was changed from soybean lecithin:cholesterol:DSPE-PEG2000-Transferrin 7:DiR = 20:5:4:1 to 20:5:4:0.3, while the other steps and conditions remained the same. The final liposome material was designated PCNSL-Lipo-CD20 mAb (20:5:4:0.3).

[0051] Comparative Example 6 The difference between this comparative example and Example 1 is that the volume ratio of the liposome solution to the phospholipid-modified anti-CD20 monoclonal antibody is different, changed from 5:2 to 5:1, while the other steps and conditions remain unchanged. The final liposome material is designated PCNSL-Lipo-CD20 mAb (5:1).

[0052] Comparative Example 7 The difference between this comparative example and Example 1 is that the volume ratio of the liposome solution to the phospholipid-modified anti-CD20 monoclonal antibody is different, changed from 5:2 to 5:4, while the other steps and conditions remain unchanged. The final liposome material is designated PCNSL-Lipo-CD20 mAb (5:4).

[0053] Comparative Example 8 This comparative example differs from Example 1 in that the DiR dye was replaced with Cy7 dye, while the other steps and conditions remained unchanged. The final liposome material was designated PCNSL-Lipo-CD20 mAb-Cy7. Experimental Example 1. Structural characterization Figure 1 In Figure 'a', the image is a transmission electron microscope (TEM) image of PCNSL-Lipo-CD20 mAb prepared in Example 1. It can be seen that the material consists of uniformly dispersed spherical nanoparticles with a particle size of approximately 130 nm.

[0054] Figure 1 b and c in the figure are the hydration kinetic particle size diagram and Zeta potential diagram of the liposome materials of Example 1 and Comparative Examples 1-2, respectively. It can be seen that the hydration kinetic particle size of the liposome material of Example 1 is about 130 nm (PDI is 0.142), which is basically consistent with the electron microscopy results and is between Comparative Example 1 and Comparative Example 2. The Zeta potential is about -17 mV.

[0055] Figure 1 In the diagram, 'd' represents the hydration kinetics particle size distribution of Example 1 and Comparative Example 3. It can be seen that changing the ratio of soybean lecithin to cholesterol increases the particle size of the liposome material, making it prone to aggregation, precipitation, and other unstable conditions.

[0056] Figure 1 The two samples in 'e' are images of the static state from two parallel experiments in Comparative Example 4. Figure 1 In the figures, f represents the hydration kinetics particle size distribution for Example 1 and Comparative Example 4, respectively. It can be seen that increasing the mass of the encapsulated dye leads to larger liposome particle sizes, resulting in aggregation, precipitation, and material instability.

[0057] 2. Dye encapsulation efficiency and drug loading rate The dye DiR was prepared into solutions of different concentrations using 80% methanol, and absorbance standard curves were obtained. Then, the liposome material prepared in Example 1 was diluted in 80% methanol to obtain a liposome solution of 100 μg / mL. After sonication at room temperature for 5 min, the absorbance of dye DiR at a wavelength of 750 nm was measured using a UV-Vis spectrophotometer. The concentration of dye DiR was then calculated based on the measured standard curves, and the content of dye DiR in the liposome material prepared in Example 1 was then calculated.

[0058] The encapsulation efficiency and drug loading of the liposome material prepared in Example 1 for the dye DiR were calculated using the following formula: Encapsulation efficiency = (Mass of liposome-loaded dye DiR / Initial total mass of added dye DiR) × 100% Drug loading rate = (mass of DiR dye loaded on liposomes / total mass of liposomes) × 100% According to the test results, the encapsulation efficiency of the dye DiR in the liposome material prepared in Example 1 was about 30%, and the drug loading rate was about 1%.

[0059] 3. Cytotoxicity The cytotoxicity of the liposome material in Example 1 was assessed using the CCK-8 assay. Raji cells (human Burkitt lymphoma cells, B-cell lymphoma cell line) and Karpas-299 cells (human degenerative large cell lymphoma cells, T-cell lymphoma cell line) in logarithmic growth phase were respectively cultured at 1 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of 100 μg / mL in 96-well plates and cultured overnight to allow cell adhesion. PCNSL-Lipo-CD20 mAb material prepared in Example 1 was added at concentrations ranging from 0 to 200 μg / mL, and after 24 h of incubation, 10 μL of CCK-8 reagent was added to each well, followed by another 2 h of incubation. Cell viability was calculated by measuring absorbance at 450 nm using a microplate reader.

[0060] Figure 2 The results showed that, within the concentration range of 0~200 μg / mL, the survival rate of the two cell types did not decrease significantly compared with the control group, indicating that the material of Example 1 of the present invention has good biocompatibility and no obvious cytotoxicity.

[0061] 3. Specific targeting capability test at the live cell level The PCNSL-Lipo-CD20 mAb prepared in Example 1 and the PCNSL-Lipo-IgG mAb prepared in Comparative Example 2 were diluted to 10 μg / mL with cell culture medium 1640 and added to the U2932 cell line (B-cell lymphoma cell line). After incubation in an incubator for 6 h, the cells were washed 5 times with PBS and then analyzed by flow cytometry and confocal imaging.

[0062] like Figure 3 Flow cytometry analysis of examples a and b showed that after adding PCNSL-Lipo-CD20 mAb material from Example 1, the cell fluorescence intensity was significantly higher than that of PCNSL-Lipo-IgG mAb material from Comparative Example 2, demonstrating that the material PCNSL-Lipo-CD20 mAb can specifically target B-cell lymphoma cell lines.

[0063] Figure 3 The confocal imaging analysis results showed that the fluorescence intensity of U2932 cells with the material of Example 1 was significantly higher than that of cells with the material of Comparative Example 2, further indicating that the anti-CD20 monoclonal antibody used in this invention has excellent targeting ability for B-cell lymphoma cell lines.

[0064] The PCNSL-Lipo-CD20 mAb prepared in Example 1 and the PCNSL-Lipo-CD20 mAb (20:5:4:0.3) prepared in Comparative Example 5 were diluted to 10 μg / mL with cell culture medium 1640 and added to the U2932 cell line (B-cell lymphoma cell line), respectively. After incubation in an incubator for 6 h, the cells were washed 5 times with PBS and then subjected to confocal imaging analysis. The imaging results are as follows: Figure 4 As shown, the fluorescence intensity of U2932 cells with the material of Example 1 was significantly higher than that of cells with the material of Comparative Example 5, indicating that the dye encapsulation amount was too low in Comparative Example 5, resulting in insufficient imaging signal.

[0065] The PCNSL-Lipo-CD20 mAb prepared in Example 1, the PCNSL-Lipo-CD20 mAb (5:1) prepared in Comparative Example 6, and the PCNSL-Lipo-CD20 mAb (5:4) prepared in Comparative Example 7 were diluted to 10 μg / mL with cell culture medium 1640 and added to the U2932 cell line (B-cell lymphoma cell line). After incubation for 6 h, the cells were washed 5 times with PBS and then subjected to confocal imaging analysis. The imaging results are as follows: Figure 5 As shown, the cellular fluorescence intensity was stronger when the volume ratio of liposomes to phospholipid-modified anti-CD20 monoclonal antibody was 5:2 than that when it was 5:1, indicating that introducing more phospholipid-modified antibody onto the liposome surface enhanced the material's targeting and imaging capabilities. Furthermore, the imaging effects of the liposomes to phospholipid-modified anti-CD20 monoclonal antibody volume ratios of 5:2 and 5:4 were essentially the same, indicating that the amount of antibody inserted into the liposome material surface reached saturation at a volume ratio of 5:2. Therefore, a volume ratio of liposomes to phospholipid-modified anti-CD20 monoclonal antibody of 5:2 is the optimal ratio.

[0066] 4. Specific recognition test for different cell lines The PCNSL-Lipo-CD20 mAb prepared in Example 1 was diluted to 10 μg / mL with cell culture medium 1640 and added to B-cell lymphoma cell lines (U2932 cell line, Raji cell line), T-cell lymphoma cell lines (Karpas-299 cell line, Jurkat cell line), myeloma cell lines (H929 cell line, RPMI-8226 cell line), and glioma cell line (U251 cell line), respectively. After incubation for 6 h, the cells were washed 5 times with PBS and analyzed by flow cytometry.

[0067] Figure 6The results showed that strong fluorescence signals were detected only in B-cell lymphoma cell lines (U2932, Raji), while no obvious fluorescence signals were detected in other cell lines, indicating that the material of the present invention has high specificity for CD20-positive B-cell lymphoma.

[0068] 5. Specific targeting ability test of mouse model of central nervous system lymphoma A mouse model of central nervous system lymphoma was established by injecting U2932-Luc cells into the brain parenchyma of nude mice using a stereotaxic instrument. One week after modeling, tumor formation was assessed using a small animal in vivo imaging system, and mice with similar brain tumor sizes were selected. Figure 7 (a) in the middle.

[0069] Mice were injected intraperitoneally with the materials from Example 1 (PCNSL-Lipo-CD20 mAb) and Comparative Example 2 (PCNSL-Lipo-IgG mAb) at a dose of 0.75 mg / kg. In vivo imaging of the small animals was performed 1 h after injection.

[0070] Imaging results showed that the mouse brain injected with the material of Example 1 exhibited the highest fluorescence intensity, with clearly discernible tumor boundaries; the mouse brain injected with the material of Comparative Example 2 showed significantly lower fluorescence intensity than that of Example 1. These results indicate that the material of Example 1 of this invention can specifically target and visualize central nervous system lymphomas in the mouse brain. Figure 7 (b).

[0071] 6. In vivo imaging diagnosis of a mouse model of central nervous system lymphoma The materials from Example 1 (PCNSL-Lipo-CD20 mAb) and Comparative Example 8 (PCNSL-Lipo-CD20 mAb-Cy7) were injected intraperitoneally into mice with central nervous system lymphoma and healthy control mice, respectively, at a dose of 0.75 mg / kg. In vivo imaging of the small animals was performed 1 h after injection.

[0072] Figure 8 Imaging results showed that the brains of mice with central nervous system lymphoma injected with the material of Example 1 exhibited high fluorescence intensity and clearly distinguishable tumor boundaries, while the brains of healthy control mice showed virtually no fluorescence. In contrast, the fluorescence intensity in the brains of mice with central nervous system lymphoma injected with the material of Comparative Example 8 was only slightly higher than that in healthy control mice. These results demonstrate that the material of Example 1 can specifically diagnose central nervous system lymphoma in the brain, exhibiting superior imaging diagnostic performance compared to the material of Comparative Example 8, highlighting the importance of encapsulating with DiR dye.

[0073] 7. Differential diagnostic test for central nervous system lymphoma and other brain tumors A mouse model of central nervous system B-cell lymphoma, a mouse model of central nervous system T-cell lymphoma, and a mouse model of glioma were constructed. PCNSL-Lipo-CD20 mAb prepared in Example 1 was injected intraperitoneally into both groups of mice at a dose of 0.75 mg / kg. In vivo imaging of the small animals was performed 1 h after injection.

[0074] like Figure 9 As shown in the imaging results, the fluorescence intensity in the brain of the mouse model of central nervous system B-cell lymphoma was significantly higher than that in the brains of the mouse models of central nervous system T-cell lymphoma and glioma, with a difference in fluorescence intensity greater than 3 times. This result demonstrates that the material of this invention can effectively distinguish central nervous system lymphoma from other brain tumors, exhibiting excellent differential diagnostic capabilities and significantly reducing the clinical misdiagnosis rate.

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A liposome material for real-time in situ fluorescence imaging of primary central nervous system lymphoma, characterized in that, It includes liposome carriers composed of soybean lecithin and cholesterol, brain-targeting peptides, anti-CD20 monoclonal antibodies, and near-infrared fluorescent dyes; The brain-targeting peptide and the anti-CD20 monoclonal antibody are modified on the surface of the liposome carrier; The brain-targeting peptide is a transferrin receptor-targeting peptide; the near-infrared fluorescent dye is DiR.

2. The liposome material as described in claim 1, characterized in that, The transferrin receptor targeting peptide is Transferrin 7, and its amino acid sequence is HAIYPRH.

3. The liposome material as described in claim 2, characterized in that, The Transferrin 7 and DSPE-PEG2000 are linked by a thioether bond to form DSPE-PEG2000-Transferrin 7, which is anchored on the surface of the liposome carrier.

4. The liposome material as described in claim 3, characterized in that, The mass ratio of soybean lecithin, cholesterol, DSPE-PEG2000-Transferrin 7 and near-infrared fluorescent dye is (19.5~20.5): (4.5~5.5): (3.5~4.5): (0.8~1.2).

5. The liposome material as described in claim 1, characterized in that, The anti-CD20 monoclonal antibody is inserted into the surface of the liposome carrier through phospholipid modification; the average particle size of the liposome material is 100~150 nm.

6. The method for preparing the liposome material according to any one of claims 1 to 5, characterized in that, Includes the following steps: Soybean lecithin, cholesterol, brain-targeting peptides and near-infrared fluorescent dyes were dissolved in an organic solvent, and a liposome solution loaded with near-infrared fluorescent dyes and modified with brain-targeting peptides on the surface was obtained by thin-film hydration method. The liposome solution was mixed with the phospholipid-modified anti-CD20 monoclonal antibody solution, incubated, centrifuged, and resuspended to obtain the final product.

7. The preparation method according to claim 6, characterized in that, The organic solvent is at least one of methanol and chloroform; the concentration of the liposome solution is 0.8~1.5 mg / mL; and the concentration of the anti-CD20 monoclonal antibody solution is 0.8~1.5 mg / mL.

8. The preparation method according to claim 7, characterized in that, The specific steps for mixing and incubating the liposome solution with the phospholipid-modified anti-CD20 monoclonal antibody are as follows: the liposome solution and the phospholipid-modified anti-CD20 monoclonal antibody solution are mixed and incubated at a volume ratio of (4.8~5.2): (1.8~2.2) at a temperature of 20~35℃ for 4~8 h; after incubation, the mixture is sonicated at 20~35℃ for 20~40 min, and then centrifuged.

9. The preparation method according to claim 6, characterized in that, The centrifugation speed is 15000~18000 rpm, the temperature is 2~5℃, the centrifugation time is 60~120 min, and the number of centrifugations is 1~4.

10. The application of the liposome material according to any one of claims 1 to 5 or the liposome material prepared by the preparation method according to any one of claims 6 to 9 in the preparation of a real-time in situ fluorescence imaging diagnostic reagent for primary central nervous system lymphoma.

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