Nanoparticles for targeted regulation of meningeal lymphatic system and preparation method and application thereof

Abstract

The present invention provides a nanoparticle for targeted regulation of the meningeal lymphatic system, and a preparation method and application thereof. By injecting phospholipid nanoprobe α-PLNPs into the cerebellomedullary cisterna magna, the meningeal lymphatic pathway from the meningeal lymphatic vessels to the peripheral draining cervical lymph nodes can be effectively labeled and tracked over a long distance; by subcutaneous injection of α-PLNPs in the neck, the reverse pathway from CLNs to MLVs is also successfully labeled, thereby promoting the non-invasive delivery of immunomodulators to the meningeal lymphatic system, and then using α-M-PLNPs carrying melittin to induce the regulation of the reverse pathway of the meningeal lymphatic system, effectively preventing the occurrence of BCBM. The present invention highlights the potential of the meningeal lymphatic reverse pathway in BCBM immunotherapy, which not only provides a new method and strategy for the treatment of tumor brain metastasis, but also brings great hope for the treatment of central nervous system diseases without the need for administration through the BBB.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomedicine, and relates to a nanoparticle for targeted regulation of the meningeal lymphatic system, and a preparation method and application thereof. Background Art

[0002] Tumor brain metastasis is a common central nervous system disease, and approximately 20% of cancer patients will eventually develop brain metastasis. Metastatic breast cancer is the second most common cancer associated with brain metastasis after lung cancer. In the past few decades, with the development of cancer research, the survival rate of patients with advanced breast cancer has been greatly improved, but the incidence of brain metastasis has also increased accordingly. Breast cancer patients, especially those with human epidermal growth factor receptor 2-positive and triple-negative breast cancer, are more likely to develop brain metastasis, and their incidence of brain metastasis is 2-5 times higher than that of hormone receptor-positive breast cancer patients. Although the current advanced treatment of BC has significantly prolonged the survival of patients, the prognosis of brain metastasis of breast cancer is still not ideal.

[0003] In recent years, thanks to the rapid development of nanotechnology, drug delivery systems based on nanomaterials have become potential effective methods for treating brain tumors in non-invasive or invasive ways. Non-invasive methods mainly include different nano drug delivery systems carrying ligands that specifically target brain endothelial cell receptors, such as transferrin receptors, low-density lipoprotein receptors, insulin receptors, or glutathione receptors, so as to improve the ability of drugs to penetrate the BBB / BTB and enhance the therapeutic effect of brain metastasis of breast cancer. Unfortunately, the accumulation of nanoparticles delivered to the brain and brain tumors is still very low, which may be attributed to the heterogeneity of the BBB / BTB and the characteristics of the tumor cells themselves. Invasive methods mainly include cannula-mediated drug administration (i.e., intrathecal, intraventricular or intratumoral injection, and convection-enhanced drug delivery, etc.) or using transient ultrasound to open the blood-brain barrier to enhance drug delivery to the brain. Although these strategies can improve drug uptake in brain tumors, however, these technologies are difficult to control spatially and may be problematic for long-term use, which limits the applicability of these methods. In the past decade, targeting immune checkpoints has become an effective treatment strategy for various metastatic solid malignancies. Although immune checkpoint therapy has made great clinical progress in the treatment of metastatic breast cancer, only a small proportion of patients benefit from it, and it is accompanied by serious side effects. According to subgroup analyses of multiple clinical trials, breast cancer patients with liver cancer or brain metastases have a lower response rate to anti-PD-1 / L1 immune checkpoint therapy compared with patients with metastases in other sites. Therefore, the treatment of brain tumors remains largely challenging, and there is an urgent need to develop more effective immunotherapy strategies.

[0004] The central nervous system lacks a classic lymphatic drainage system, and the traditional view is that the brain is an immune-privileged organ. However, recent studies have found that the meningeal lymphatic vessels (MLV) exist on the transverse sinus and superior sagittal sinus of the dura mater and are connected to the deep and superficial cervical lymph nodes. As a bridge for the exchange of fluid and immune cells between cerebrospinal fluid and cervical lymph nodes, the meningeal lymphatic vessels not only play a drainage role in clearing metabolic waste from the brain, but also play a very important role in immune surveillance and participate in immune regulation in the occurrence and development of a variety of neurological diseases.Based on the above findings, some studies have reported that the production of MLVs can be regulated by delivering vascular endothelial growth factor C (VEGF-C) via intraventricular injection of adeno-associated virus (AAV) vectors to improve or enhance the therapeutic effects of Alzheimer's disease and brain gliomas (Da Mesquita S, Louveau A, Vaccari A, Smirnov I, Cornelison RC, Kingsmore KM, Contarino C, Onengut-Gumuscu S, Farber E, Raper D, Viar KE, Powell RD, Baker W, Dabhi N, Bai R, Cao R, Hu S, Rich SS, Munson JM, LopesMB, Overall CC, Acton ST, Kipnis J. Functional aspects of meningeal lymphatics inageing and Alzheimer's disease.Nature.2018;560(7717):185-191.,Hu vesselsregulate brain tumor drainage and immunity.Cell Res.2020;30(3):229-243.,SongE,Mao T,Dong H,Boisserand LSB,Antila S,Bosenberg M,Alitalo K,Thomas JL,Iwasaki A.VEGF-C-driven lymphatic drainage enables immunosurveillance of brain tumours.Nature.2020;577(7792):689-694.).However, the method of regulating meningeal lymphatic vessels based on AAV viral vectors has many shortcomings: first, AAV has strong immunogenicity, which may lead to poor effects of continuous administration to the same host; second, AAV viral vectors lack cell targeting to the lymphatic system; in addition, the method of intraventricular drug injection is invasive and technically difficult, making it difficult to be widely used in clinical treatment research. The other is to use laser transcranial irradiation to regulate MLVs proliferation, which can effectively improve the symptoms of Alzheimer's disease and intraventricular hemorrhage (Wang M, Yan C, Li X, Yang T, Wu S, Liu Q, Luo Q, Zhou F. Non-invasive modulation of meningeal lymphaticsameliorates ageing and Alzheimer's disease-associated pathology and cognitionin mice. Nat Commun. 2024; 16; 15(1): 1453., Li D, Liu S, Yu T, Liu Z, Sun S, Bragin D, Shirokov A, Navolokin N, Bragina O, Hu Z, Kurths J, Fedosov I, Blokhina I, DubrovskiA, Khorovodov A, Terskov A, Tzoy M, Semyachkina-Glushkovskaya O, Zhu D. Photostimulation of brain lymphatics in male newborn and adult rodents for therapy of intraventricular hemorrhage. Nat Commun. 2023 29; 14(1): 6104.). However, the specific molecular mechanism of transcranial light stimulation for the treatment of brain diseases remains to be further explored. In addition, nanomedicines can also be delivered to the brain through the meningeal lymphatic system to effectively control the growth of Parkinson's disease and in situ brain gliomas, but the delivery efficiency still needs to be improved. Summary of the invention

[0005] The present invention aims to provide a nanoparticle for targeted regulation of the meningeal lymphatic system and a preparation method and application thereof, highlighting the potential of the meningeal lymphatic reverse pathway in BCBM immunotherapy. It not only provides a new method and strategy for the treatment of tumor brain metastasis, but also brings great hope for the treatment of central nervous system diseases without the need for administration through the BBB.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The present invention provides a method for preparing nanoparticles for targeted regulation of the meningeal lymphatic system, comprising:

[0008] (1) Dissolve different phospholipids DMPC, DMPE, MHPC and DSPE-PEG2000 in a chloroform:methanol mixture. After all components are mixed, perform water bath sonication to ensure that each component is fully dissolved.

[0009] (2) nitrogen is introduced to remove the solvent, and then the mixture is placed in a desiccant and vacuum filtered to remove the residual solvent;

[0010] (3) adding 50-60°C ddH2O, flushing with nitrogen, sealing, and shaking to completely hydrate the nanoparticles;

[0011] (4) subjecting the hydrated nanoparticles to water bath ultrasonic treatment to make the nanoparticles uniform and transparent, with the water bath temperature being 48 to 50° C.;

[0012] (5) Add the α-helical peptide R4F that mimics apolipoprotein A-I, slowly dropwise, shake while adding, fill with nitrogen, then seal and incubate at 4°C overnight;

[0013] (6) The nanoparticle solution prepared in step (5) is centrifuged through a 30 kDa concentrator centrifuge tube to remove free polypeptides in the nanoparticles and replace the solution with PBS buffer to synthesize nanoparticles α-PLNP containing the R4F polypeptide.

[0014] Preferably, the volume ratio of chloroform to methanol in the chloroform:methanol mixture in step (1) is 3:1.

[0015] Preferably, in step (2), the nitrogen ventilation duration is 0.5 to 1 h, and the vacuum filtration time is 1 to 2 h.

[0016] Preferably, the weight ratio of DMPC, DMPE, MHPC, DSPE-PEG2000 and R4F is 6:2:2:2.5:2.

[0017] Preferably, the components dissolved in the chloroform:methanol mixture in step (1) also include water-soluble or fat-soluble near-infrared fluorescent dye and iodized oil.

[0018] The present invention also provides a nanoparticle for targeted regulation of the meningeal lymphatic system, which is prepared by the above preparation method.

[0019] The present invention also provides the use of the nanoparticles in marking and tracking a meningeal-cervical lymph node system.

[0020] The present invention also provides a method for preparing melittin nanoparticles for targeted regulation of the meningeal lymphatic system, comprising the above-mentioned preparation method, and adding melittin to the α-PLNP synthesized in step (6), incubating at 4° C. overnight, and then removing free unbound melittin by concentration and centrifugation to obtain nanoparticles α-M-PLNP.

[0021] Preferably, the weight ratio of melittin to R4F is 1:1.

[0022] The present invention also provides a melittin nanoparticle for targeted regulation of the meningeal lymphatic system, which is prepared by the above-mentioned preparation method.

[0023] The present invention further provides the use of the above-mentioned melittin nanoparticles in the preparation of immunotherapeutic drugs for brain tumors or brain metastases of tumors.

[0024] The melittin in this application is directly combined with the nanoparticle α-PLNP through the electrostatic effect, and no additional chemical reaction modification is required. The melittin alone can exert the tumor immunosuppressive function. The melittin nanoparticles can not only be used for immunotherapy of breast cancer brain metastasis, but also for melanoma brain metastasis and brain metastasis of other types of tumors. In addition, in addition to subcutaneous injection of nanoparticles in the neck, immunotherapy of brain tumors or tumor brain metastasis can also be achieved by directly injecting nanoparticles into the cervical lymph nodes.

[0025] The beneficial effects of the present invention are:

[0026] The present invention proposes a potential strategy for immunotherapy of breast cancer brain metastases (BCBM) by targeting and regulating the reverse meningeal lymphatic pathway through peripheral administration, thereby bypassing the restriction of the blood-brain barrier (BBB). CT / fluorescence dual-modality imaging showed that phospholipid nanoprobes (α-PLNPs) injected into the cerebellomedullary cisterna magna can effectively label and remotely track the meningeal lymphatic pathway from meningeal lymphatic vessels (MLVs) to peripheral draining cervical lymph nodes (CLNs). And through subcutaneous injection in the neck, α-PLNPs also successfully labeled the reverse pathway from CLNs to MLVs, thereby facilitating the non-invasive delivery of immunomodulators to the meningeal lymphatic system. In view of this, we used α-M-PLNPs carrying melittin to induce the regulation of the reverse pathway of the meningeal lymphatic system, promoting CD8 + T cells migrate to metastatic brain tumors, thus effectively preventing the occurrence of BCBM. The present invention highlights the potential of the meningeal lymphatic reverse pathway in BCBM immunotherapy, which not only provides a new method and strategy for the treatment of tumor brain metastasis, but also brings great hope for the treatment of central nervous system diseases without the need for drug administration through the BBB. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the principle of the bee venom peptide nanoparticles of the present invention in targeted regulation of the meningeal lymphatic system to inhibit brain metastasis of breast cancer tumors.

[0028] Figure 2 Schematic diagram of the results, particle size and charge of the nanoparticles prepared in the examples of the present invention.

[0029] Figure 3 CT imaging showing the three-dimensional structure of the meningeal lymphatic system.

[0030] Figure 4 The ability of the neck-injected nanoparticles to migrate into the brain was examined for in vivo fluorescence imaging.

[0031] Figure 5 Whole mount immunofluorescence imaging of meninges.

[0032] Figure 6 Fluorescence imaging was used to verify that the nanoparticles entered the brain via the cervical lymph node-meningeal lymphatic pathway.

[0033] Figure 7 It was shown that bee venom peptide nanoparticles α-M-PLNP significantly inhibited brain metastasis of breast cancer.

[0034] Figure 8 The immunological mechanism of melittin nanoparticles α-M-PLNP in inhibiting brain metastasis of breast cancer was shown. DETAILED DESCRIPTION

[0035] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0036] Example

[0037] 1. Synthesis and Characterization of Nanoparticles α-PLNP and α-M-PLNP

[0038] (a) Preparation of nanoparticle components: Dissolve different phospholipids DMPC, DMPE, MHPC, DSPE-PEG2000, near-infrared dye DiR-BOA, iodized oil Lipiodol and other components in 4 mL of chloroform: methanol mixture (volume ratio 3:1). After all the components are mixed, transfer the mixture to a round-bottom flask with a magnetic stirrer and ultrasonicate in a water bath for 20 seconds to ensure that all components are fully dissolved. The nanoparticle components and ratios are shown in Table 1:

[0039] Table 1 Components and ratios of nanoparticles

[0040]

[0041] (b) Solvent removal: The solvent was removed by passing nitrogen gas for about 1 h.

[0042] (c) Removal of residual solvent: In order to ensure that the residual organic solvent in the flask is fully removed, place it in a desiccant and continue vacuum filtration for 2 h.

[0043] (d) Hydration treatment: prepare 50-60°C ddH2O, take 4 mL and add it to a round-bottom flask. Immediately shake the flask gently, then flush nitrogen into the flask, seal the flask with a sealing film, and place the round-bottom flask on a vortex shaker to fully oscillate for 5 min to completely hydrate the nanoparticles.

[0044] (e) Ultrasound: In order to obtain nanoparticles with uniform particle size and stable structure, the hydrated nanoparticles are subjected to water bath ultrasonic treatment. The nanoparticles become uniform and transparent through the action of ultrasonic shear force. The ultrasonic time is 30 min and the temperature is 48-50°C.

[0045] (f) Adding polypeptide: Add α-helical polypeptide R4F that mimics apolipoprotein A-I, slowly dropwise, shaking the glass test tube while adding, fill with nitrogen, then seal with sealing film, and incubate at 4°C overnight.

[0046] (g) Concentration and dialysis: The prepared nanoparticle solution is centrifuged through a 30 kDa concentrating centrifuge tube to remove the free polypeptide in the nanoparticles and replace the solution with PBS buffer, which is stored in a refrigerator at 4°C for later use. The synthesized nanoparticles containing the R4F polypeptide are named α-PLNP. α-PLNP can be loaded with the near-infrared dye DiR-BOA and iodized oil Lipiodol, or it can be not loaded with the near-infrared dye DiR-BOA and iodized oil Lipiodol.

[0047] (h) 6 mg of melittin was added to α-PLNP and incubated at 4°C overnight. Free unbound melittin was removed by concentration and centrifugation, and the resulting nanoparticles were named α-M-PLNP. α-M-PLNP was not loaded with near-infrared dye DiR-BOA and lipiodol.

[0048] The multifunctional nanoparticles targeting the meningeal lymphatic system were synthesized by the above method. The schematic diagram of the particle structure is shown in Figure 2 As shown in A. The hydrated particle size of the nanoparticles was detected, and the particle size of the α-PLNP containing near-infrared dye and α-PLNP without DiR-BOA dye and iodized oil was 52.76±0.19nm and 21.68±0.14nm ( Figure 2B). The particle size of α-M-PLNP nanoparticles without DiR-BOA dye and iodized oil is 22.44±0.17nm. The surface charge results of the nanoparticles show that the surface charges of α-PLNP and α-M-PLNP are -8.15±2.0mV and -0.82±0.63mV respectively ( Figure 2 C).

[0049] 2. CT 3D Imaging and Fluorescence Imaging of the Meningeal Lymphatic System

[0050] 1. CT imaging:

[0051] First, we injected 10 μL of α-PLNP (containing 0.015 mg DiR-BOA and 0.72 mg iodized oil) into the cerebellum and medullary cisterna of C57BL / 6 mice, and performed CT imaging of the mouse brain 1 hour after the injection. From the results of the 3D reconstruction of the CT signal, we can see that the CT signal of α-PLNP is mainly distributed in the transverse sinus, superior sagittal sinus and base, and is close to the skull. At the same time, we also see that the CT signals of these parts are continuous ( Figure 3 ), and can also display cervical lymph nodes and lymphatic vessels. The above results show that α-PLNP combined with CT imaging has a good ability to display the three-dimensional structure of the meningeal lymphatic system.

[0052] 2. Fluorescence imaging:

[0053] First, 100 μL of α-PLNP nanoparticles were subcutaneously injected into the front neck of C57BL / 6 mice, and then fluorescence imaging of the mouse brain was performed at 1h, 3h, 6h, 12h, 24h, 48h and 72h after the injection of the nanoparticles. The fluorescence imaging results and quantitative analysis results are shown in Figure 4 As shown in the figure, the fluorescence imaging results show that only after 3 hours of subcutaneous injection of α-PLNP in the neck, a relatively obvious fluorescence signal can be shown in the mouse brain. At 24 hours, the fluorescence signal in the mouse brain reaches the strongest, and the fluorescence signal can last until 72 hours.

[0054] After imaging, we peeled the meninges from the skull for whole-mount immunofluorescence imaging of the meninges. First, the meninges were labeled with lymphatic vessels Lyve-1 antibody, and then confocal imaging was used to detect whether the signal of the nanoparticles on the meninges co-localized with the Lyve-1 signal. The immunofluorescence imaging results showed that the DiR-BOA fluorescence signal of α-PLNP co-localized with the signal of Lyve-1 in the meningeal lymphatic vessels ( Figure 5 ), but the α-PLNP fluorescence signal did not co-localize with the vascular CD31 antibody fluorescence signal. Based on the above results, we speculate that α-PLNP can effectively label the meningeal-cervical lymph node system and may migrate to the brain through this pathway rather than draining into the brain through blood vessels.

[0055] In order to further verify that the nanoparticles injected subcutaneously in the neck enter the brain through the cervical lymph node-meningeal lymphatic vessel pathway, we first removed the deep cervical lymph nodes (dcLNs) and superficial cervical lymph nodes (scLNs). After the mice recovered for a week, we injected nanoparticles subcutaneously in the neck again, and compared the fluorescence signal intensity of the nanoparticles in the brains of normal mice and mice with cervical lymph node removal by fluorescence imaging. The fluorescence imaging results showed that compared with normal mice, the fluorescence signal intensity in the brains of mice with cervical lymph node removal was significantly weaker than that in the brains of normal mice ( Figure 6 ). At 12h and 24h after nanoparticle injection, the fluorescence signal intensity values ​​were 2.6 times and 2.5 times lower than those of the cervical lymph nodes of normal mice, respectively ( Figure 6 ). The above results show that the removal of cervical lymph nodes can indeed reduce the ability of nanoparticles to enter the brain, which reflects that nanoparticles can indeed enter the brain through cervical lymph nodes.

[0056] The above results indicate that after subcutaneous injection of nanoparticles in the neck, α-PLNP can bypass the BBB and enter the brain parenchyma, and can effectively mark the meningeal lymphatic system, thus laying the foundation for effectively regulating the function of the meningeal lymphatic system.

[0057] 3. In vivo treatment of breast cancer brain metastasis model with α-M-PLNP nanoparticles carrying melittin and its mechanism

[0058] Combination Figure 1 First, a tumor brain metastasis model was established by intracardiac injection of fluorescent protein-labeled mCherry-EO771 breast cancer cells. One week later, 20 nmol of bee venom peptide nanoparticles were injected subcutaneously in the neck. The drug was administered three times in total, with one day between each administration. Then, fluorescence imaging was used to dynamically monitor the fluorescence signals of tumor cells metastasized in the brains of mice in different treatment groups on different days of treatment. The results of in vivo fluorescence imaging are shown in Figure 2. Figure 7 As shown in A, a relatively weak fluorescence signal can only be detected in the brain on the 14th day after tumor inoculation, and the range of the tumor signal area gradually increases over time. However, compared with other treatment groups, the signal in the brain of the α-M-PLNP treatment group was the weakest and the tumor area was the smallest. After 28 days of treatment, the mice were killed and the brains of the mice in each treatment group were taken out for in vitro tumor fluorescence imaging. The fluorescence imaging results showed that many tumor foci were metastatic to the dorsal and bottom sides of the brains of mice in the PBS and α-PLNP treatment groups, but the number of tumor foci metastatic to the brains of the α-PLNP (also without DiR-BOA and Lipiodol) and α-M-PLNP treatment groups was relatively small, and no tumor foci were detected at all in some mouse brains ( Figure 7B). The quantitative results of tumor foci showed that the average number of tumor metastasis foci in the brain of the PBS, PLNP and melittin groups was 2.6, 2.2 and 1.4, respectively, while that of the α-M-PLNP treatment group was only 0.4 ( Figure 7 C). The statistical results of tumor survival rate showed that α-M-PLNP significantly prolonged the survival time of mice compared with other control groups ( Figure 7 D).

[0059] Finally, we further explored the immunological mechanism of α-M-PLNP in inhibiting tumor brain metastasis. We detected the number of T cells and cytokines in the brain and cervical lymph nodes and the changes in APC by flow cytometry. Flow cytometry showed that compared with the PBS group, α-M-PLNP treatment could significantly increase the total CD8 + T cells and CD8 + Ki67 + The number of T cells (14.8 times) Figure 8 A) At the same time, Ki67 + CD8 + T cells and CD4 + Foxp3 + At the same time, we used flow cytometry to detect the expression of T cell cytokines and APC activation in the cervical lymph nodes. Compared with the PBS group, α-M-PLNP could significantly increase IFN-γ + CD8 + (4.4-fold) and IFN-γ + CD4 + (3.6-fold) ratio of T cells, and TNF-α + CD8 + (4.7-fold) and TNF-α + CD4 + (2.3-fold)T cells ratio ( Figure 8 B). Finally, we further evaluated the effect of α-M-PLNP stimulation on APC activation in cervical lymph nodes, and detected the expression levels of DC and macrophage co-stimulatory molecules by flow cytometry. Compared with the PBS control group, α-M-PLNP significantly increased the expression of DC and Macrophage CD80 and CD86. + CD86 + The proportion of DC and Macrophage increased from 15.6% to 28.5%, and from 6.4% to 20.2%, respectively. Figure 8C), but α-PLNP and melittin had no significant effect on the expression of CD80 and CD86 of DC and Macrophage. The above results indicate that α-M-PLNP can induce the activation of APC in cervical lymph nodes, promote T cell triggering in cervical lymph nodes and CD8+T cell infiltration into brain tumors, thereby enhancing the anti-tumor immune response of metastatic brain tumors.

[0060] The present invention develops a method for targeted regulation of the meningeal lymphatic system reverse pathway immunotherapy for breast cancer brain metastasis, providing a new perspective for further analyzing the three-dimensional structure and function of the meningeal lymphatic system and developing diagnostic techniques and treatment methods for central nervous system-related diseases.

[0061] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. Use of nanoparticles for targeting and regulating the meningeal lymphatic system in the preparation of drugs for marking and tracking the meningeal-cervical lymph node system, the preparation method of the nanoparticles comprising: (1) Dissolving different phospholipids DMPC, DMPE, MHPC, DSPE-PEG2000, water-soluble or fat-soluble near-infrared fluorescent dyes and iodized oil in a chloroform:methanol mixture. After all components are mixed, water bath sonication is performed to ensure that each component is fully dissolved. The volume ratio of chloroform to methanol in the chloroform:methanol mixture is 3:1; (2) nitrogen is introduced to remove the solvent, and then the mixture is placed in a desiccant and vacuum filtered to remove the residual solvent; (3) adding 50-60°C ddH2O, flushing with nitrogen, sealing, and shaking to completely hydrate the nanoparticles; (4) subjecting the hydrated nanoparticles to water bath ultrasonic treatment to make the nanoparticles uniform and transparent, with the water bath temperature being 48 to 50° C.; (5) adding the α-helical peptide R4F that mimics apolipoprotein A-I, slowly dropping it, shaking it while dropping it, filling it with nitrogen, and then sealing it, incubating it at 4°C overnight, and the weight ratio of DMPC, DMPE, MHPC, DSPE-PEG2000 and R4F was 6:2:2:2.5:2; (6) The nanoparticle solution prepared in step (5) is centrifuged through a 30 kDa concentrator centrifuge tube to remove free polypeptides in the nanoparticles and replace the solution with PBS buffer to synthesize nanoparticles α-PLNP containing the R4F polypeptide.

2. Use of melittin nanoparticles for targeted regulation of the meningeal lymphatic system in the preparation of immunotherapy drugs for brain tumors or brain tumor metastases, the preparation method of the melittin nanoparticles comprising: (1) Dissolving different phospholipids DMPC, DMPE, MHPC, DSPE-PEG2000, water-soluble or fat-soluble near-infrared fluorescent dyes and iodized oil in a chloroform:methanol mixture. After all components are mixed, water bath sonication is performed to ensure that each component is fully dissolved. The volume ratio of chloroform to methanol in the chloroform:methanol mixture is 3:1; (2) nitrogen is introduced to remove the solvent, and then the mixture is placed in a desiccant and vacuum filtered to remove the residual solvent; (3) adding 50-60°C ddH2O, flushing with nitrogen, sealing, and shaking to completely hydrate the nanoparticles; (4) subjecting the hydrated nanoparticles to water bath ultrasonic treatment to make the nanoparticles uniform and transparent, with the water bath temperature being 48 to 50° C.; (5) adding the α-helical peptide R4F that mimics apolipoprotein A-I, slowly dropping it, shaking it while dropping it, filling it with nitrogen, and then sealing it, incubating it at 4°C overnight, and the weight ratio of DMPC, DMPE, MHPC, DSPE-PEG2000 and R4F was 6:2:2:2.5:2; (6) centrifuging the nanoparticle solution prepared in step (5) through a 30 kDa concentrator centrifuge tube to remove free polypeptides in the nanoparticles and replacing the solution with PBS buffer to synthesize nanoparticles α-PLNP containing the R4F polypeptide; (7) Add melittin to the α-PLNP synthesized in step (6), incubate at 4° C. overnight, and then remove free unbound melittin by concentration and centrifugation to obtain nanoparticles α-M-PLNP, wherein the weight ratio of melittin to R4F is 1:1.

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