Brain delivery nanocarriers of a transcellular transport pathway and methods of making the same

Abstract

This invention discloses a brain delivery nanocarrier via a transcellular transport pathway. It uses a lecithin-modified inorganic nanomaterial as its core and a membrane-fused liposome-cell membrane as its shell. The membrane-fused liposome-cell membrane coats the surface of the lecithin-modified inorganic nanomaterial, forming a core-shell structure. The membrane-fused liposome-cell membrane is formed by the hybridization and fusion of membrane-fused liposomes with the cell membranes of metastatic brain cancer cells. This brain delivery nanocarrier can efficiently cross the blood-brain barrier via a membrane fusion pathway, bypassing the classic endocytosis-lysosome pathway to enter brain microvascular endothelial cells, thus solving the problems of low delivery efficiency and easy retention and degradation in lysosomes of existing nanomaterials.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a brain delivery nanocarrier via a transcellular transport pathway that can bypass the classic endocytosis-lysosome pathway to enter brain microvascular endothelial cells, effectively reducing their retention and degradation in cellular lysosomes. Background Technology

[0002] With the increasing incidence of neurological diseases worldwide, treatment methods for brain diseases are constantly being innovated. However, the treatment of brain diseases continues to face a significant obstacle: the blood-brain barrier. The blood-brain barrier, mainly composed of brain microvascular endothelial cells, astrocytes, pericytes, and the basement membrane, is a highly selective semi-permeable membrane barrier between the brain and the circulatory system. It protects the brain from harmful substances in the blood and regulates and maintains the stability of the brain's internal environment. However, it also prevents most drugs from entering the brain parenchyma, affecting the therapeutic effect. Therefore, developing a strategy that can efficiently cross the blood-brain barrier is crucial for brain drug delivery and the diagnosis and treatment of brain diseases.

[0003] Various strategies have been developed to overcome the blood-brain barrier and improve drug delivery efficiency to the brain, among which focused ultrasound, functional ligand modification, and biomembrane encapsulation are widely used. Biomembrane encapsulation, in particular, demonstrates significant advantages due to its good biocompatibility, low immunogenicity, and inherent biomimetic properties. However, current biomimetic nanocarrier design strategies primarily focus on enhancing targeting ability against the blood-brain barrier through ligand-receptor interactions, thus still heavily relying on the classic vesicle transport pathway during blood-brain barrier transport. This pathway has significant limitations: nanomedicines are easily captured and retained within lysosomes during transmembrane transport, subsequently suffering from the acidic microenvironment or enzymatic degradation, leading to a substantial loss of bioactivity before reaching the brain parenchyma. Therefore, overcoming the dependence of existing biomimetic carriers on the endocytosis pathway and developing novel biomimetic strategies that can efficiently circumvent lysosomal degradation and achieve highly active brain-targeted drug delivery remains a key technological bottleneck that urgently needs to be overcome in the field of brain drug delivery. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a brain delivery nanocarrier via a transcellular transport pathway to address the shortcomings of the prior art. This nanocarrier efficiently crosses the blood-brain barrier through membrane fusion, bypassing the classic endocytosis-lysosome pathway to enter brain microvascular endothelial cells, effectively reducing its retention and degradation in lysosomes, thereby solving the problems of low delivery efficiency and easy retention and degradation in lysosomes of existing nanomaterials.

[0005] The technical solution adopted by the present invention to solve the above-mentioned problems is as follows: A brain delivery nanocarrier via a transcellular transport pathway comprises an inorganic nanomaterial modified with lecithin as its core and a membrane-fused liposome-cell membrane as its outer shell. The membrane-fused liposome-cell membrane is coated on the surface of the inorganic nanomaterial modified with lecithin. The membrane-fused liposome-cell membrane is formed by the fusion of the membrane-fused liposome with the cell membrane of metastatic brain cancer cells.

[0006] Furthermore, the membrane-fusion liposomes are nanoscale lipid vesicles with strong membrane fusion capabilities, prepared by thin-film hydration method from 70%-80% neutral phospholipids, 15%-25% cationic lipids, and 4%-6% PEG-modified phospholipids by molar percentage. Neutral phospholipids constitute the largest proportion, serving as the structural framework of the lipid bilayer, which helps to construct stable vesicles, regulate membrane flexibility and fluidity, reduce the membrane fusion energy barrier, and improve biocompatibility. Cationic lipids provide the core driving force for membrane fusion, adsorbing negatively charged cell membranes through electrostatic interactions and inducing liposome fusion with cell membranes. PEG-modified phospholipids can form a protective layer on the surface of the liposomes, playing a role in long-term circulation and stabilization, prolonging in vivo circulation time, and preventing particle aggregation. Specifically, neutral phospholipids are preferably DMPC (1,2-dimyristic-sn-glycerol-3-phosphocholine), cationic lipids are preferably DOTAP (1,2-dioleoyl-3-trimethylammonium propane), and PEG-modified phospholipids are preferably DSPE-PEG2000 (distearate-phosphatidylethanolamine-polyethylene glycol 2000).

[0007] Furthermore, the mass ratio of cell membrane to membrane-fused liposomes in brain metastatic cancer cells was (1~4):1.

[0008] Furthermore, the mass ratio of membrane-fused liposomes-cell membranes to inorganic nanomaterials is (1~3):1.

[0009] Furthermore, the lecithin surface-modified inorganic nanomaterial is an inorganic nanomaterial with surface-modified amphiphilic ligand lecithin, and is water-soluble.

[0010] This invention also provides a method for preparing a brain delivery nanocarrier via the aforementioned transcellular transport pathway, comprising the following steps: (1) The cell membrane of brain metastatic cancer cells and membrane fusion liposomes were mixed in a buffer solution by low-temperature intermittent sonication for 5-10 min to obtain membrane fusion liposome-cell membrane; (2) The membrane-fused liposome-cell membrane and the lecithin-modified inorganic nanomaterials were mixed in a buffer solution and ultrasonically treated at a temperature of 0~4℃ to obtain the brain delivery nanocarrier of the transcellular transport pathway.

[0011] Further, in step (1), the concentrations of the cell membrane and membrane fusion liposomes of brain metastatic cancer cells in the buffer solution are 5~10 mg / mL and 2~5 mg / mL, respectively; more preferably, the concentrations of the cell membrane and membrane fusion liposomes of brain metastatic cancer cells in the buffer solution are 5~8 mg / mL and 2~4 mg / mL, respectively.

[0012] Further, in step (1), the low-temperature intermittent ultrasound power is 60~70 W, the temperature is 0~4℃, and the intermittent ultrasound is paused for 1~2 min after every 3~5 min of ultrasound, for a total of 2~4 cycles. The cell membrane of brain metastatic cancer cells and membrane fusion liposomes are difficult to form a stable and functionally complete coating layer structure through simple mixing. The low-temperature intermittent ultrasound used in this invention is the key to achieving effective integration of the two: the low temperature condition and intermittent ultrasound can avoid continuous heat generation by ultrasound, which can not only protect the natural membrane proteins from denaturation, but also prevent the liposomes from structural damage due to excessive temperature; at the same time, by alternating ultrasound and resting, the liposomes can be gradually inserted into the cancer cell membrane and restore their conformation, and finally obtain a membrane fusion liposome-cell membrane with preserved membrane protein activity and stable liposome function as a composite coating layer.

[0013] Furthermore, in step (1), the mass ratio of the cell membrane of the brain metastatic cancer cells to the membrane fusion liposomes is (1~4):1.

[0014] Furthermore, in step (2), the ultrasonic power and ultrasonic time are preferably 60~70 W and 10~20 min, respectively.

[0015] Further, in step (2), the concentrations of the membrane-fused liposome-cell membrane and the lecithin-modified inorganic nanomaterials in the buffer solution are 2~12 mg / mL and 1~6 mg / mL, respectively. More preferably, the concentrations of the membrane-fused liposome-cell membrane and the lecithin-modified inorganic nanomaterials in the buffer solution are 4~8 mg / mL and 2~4 mg / mL, respectively.

[0016] Furthermore, in step (2), the mass ratio of membrane-fused liposome-cell membrane to lecithin-modified inorganic nanomaterial is (1~3):1.

[0017] Furthermore, in steps (1) and (2), the concentration range of the buffer solution is 5~20 mM, and the pH range is 7.0~8.0. The buffer solution should be a non-toxic and stable buffer that is suitable for cells, preferably Tris-HCl buffer, HEPES buffer, TES buffer, MOPS buffer, etc.

[0018] The present invention also provides the above-mentioned brain delivery nanocarrier that can enter brain microvascular endothelial cells via membrane fusion pathway for the delivery of drugs and other functional components to target brain regions.

[0019] The main technical concept of this invention is as follows: On the one hand, this invention utilizes various functional proteins (such as transferrin receptor, ApoE, integrin, and CD44) retained on the surface of brain metastatic cancer cell membranes to specifically recognize corresponding receptors overexpressed on brain microvascular endothelial cells, guiding the accumulation of nanocarriers at the blood-brain barrier, enhancing the adhesion and retention of the carriers at the target site, and providing high-concentration contact conditions for subsequent transmembrane processes. On the other hand, this invention utilizes membrane-fusion-promoting components (such as DOTAP) contained in membrane-fusion liposomes, which, after close contact between the carrier and the cell membrane, trigger the reconstruction and merging of their lipid structures. This process does not rely on clathrin or caveolin-mediated endocytosis, thereby avoiding the carrier being carried into the lysosomal pathway, reducing the retention and degradation of drugs and other functional components in lysosomes, and improving delivery efficiency. This invention combines brain metastatic cancer cell membranes with membrane-fusion liposomes, achieving efficient integration of the two through a special preparation process of low-temperature intermittent ultrasound, exerting a synergistic effect of active targeting and membrane fusion delivery, achieving a dual synergistic effect of blood-brain barrier targeted enrichment and efficient direct intracellular delivery, which can significantly improve delivery efficiency. Therefore, the brain delivery nanocarrier described in this invention can efficiently and directly fuse with the cell membrane of brain microvascular endothelial cells, thereby delivering the loaded drugs and other functional components into the cells.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The brain delivery nanocarrier described in this invention innovatively adopts a membrane fusion mechanism to directly fuse with the cell membrane of brain microvascular endothelial cells, thereby achieving a dual synergistic effect of targeted enrichment of the blood-brain barrier and efficient direct intracellular delivery. This solves the problem that existing biomimetic nanocarriers are subject to large-scale capture and degradation by lysosomes due to reliance on endocytosis when crossing the blood-brain barrier via transcellular transport pathways, resulting in low delivery efficiency.

[0021] (2) This brain delivery nanocarrier integrates the advantages of membrane-fused liposomes with the membrane of brain metastatic cancer cells, which not only achieves efficient brain enrichment based on natural targeting molecules, but also facilitates the direct release of functional components into the cytoplasm through an efficient membrane fusion process, significantly improving its delivery efficiency as a carrier and enhancing the intracellular bioavailability of functional components.

[0022] In summary, this invention bypasses the classic endocytosis-lysosome pathway through an enhanced membrane fusion strategy, ensuring the efficient and complete delivery of functional components such as drugs into the cytoplasm, thereby significantly improving the brain delivery efficiency of the biomimetic membrane carrier for brain metastatic cancer cells via the transcellular transport pathway. Attached Figure Description

[0023] Figure 1 Transmission electron microscopy (TEM) images of the nanocarriers prepared in Example 1 and the nanocarriers prepared in Comparative Examples 1-2.

[0024] Figure 2 Confocal images showing the co-localization of nanocarriers NPs in Comparative Example 1 and nanocarriers Lipo-CMNPs in Example 1 with lysosomes during the uptake process by brain microvascular endothelial cells; Lyso-Tracker Red is a commercially available lysosomal fluorescent dye. Scale bar: 10 μm.

[0025] Figure 3 In Application Example 1, the process by which nanocarriers NPs, 95D-CMNPs, and Lipo-CMNPs incubated with and entered brain microvascular endothelial cells is described; where scale bar: 50 μm.

[0026] Figure 4 In Application Example 2, the fluorescence recovery of R18 after co-incubation with brain microvascular endothelial cells on different nanocarriers loaded with R18 dye is shown.

[0027] Figure 5 The image shows flow cytometry fluorescence images of the uptake of different nanocarriers by brain microvascular endothelial cells before and after treatment with the membrane fusion inhibitor Z-FFF in Application Example 3.

[0028] Figure 6 The image shows a flow cytometry fluorescence quantitative analysis of different nanocarriers taken up by brain microvascular endothelial cells before and after treatment with the membrane fusion inhibitor Z-FFF, as shown in Application Example 3.

[0029] Figure 7 This is an in vivo imaging image showing the enrichment of different nanocarriers in the brain of a healthy nude mouse, as shown in Application Example 4.

[0030] Figure 8 This is a fluorescence quantitative analysis diagram showing the enrichment of different nanocarriers in the brains of healthy nude mice in Application Example 4. Detailed Implementation

[0031] To better understand the present invention, detailed descriptions will be provided below in conjunction with embodiments and illustrations, but the present invention is not limited to the embodiments described below.

[0032] In this invention, the lecithin-modified inorganic nanomaterial is obtained by surface modification of oleic acid-modified inorganic nanomaterials with lecithin. The preparation method can be as follows: the oleic acid-modified inorganic nanomaterials and lecithin are ultrasonically dispersed evenly in a mixed solvent of chloroform and n-hexane. After removing the organic solvent by rotary evaporation, the solid product is washed and collected, which is the lecithin-modified inorganic nanomaterial. The mass ratio of oleic acid-modified inorganic nanomaterials to lecithin is 1:(4~6); the volume ratio of chloroform to n-hexane is 1:(1~3); and the concentration range of the oleic acid ligand inorganic nanomaterials in the mixed solvent (i.e., chloroform and n-hexane) is 2~3 mg / mL.

[0033] In this invention, the inorganic nanomaterial can be one or a mixture of several of rare earth nanomaterials, Fe3O4 nanomaterials, and SiO2 nanomaterials in any proportion. Modifying the surface of the inorganic nanomaterial with oleic acid ligands is suitable for this invention, followed by lecithin surface modification to serve as the core of the brain delivery nanocarrier described in this invention. Preferably, the particle size of the inorganic nanomaterial is in the range of 40-100 nm.

[0034] In the following embodiments, the inorganic nanomaterials are rare-earth nanomaterials directly using oleic acid ligands. Taking NaYF4:Yb / Er@NaYF4 nanomaterial as an example, its surface is modified with oleic acid ligands, labeled OA-RENPs. In some embodiments, NaYF4:Yb / Er and NaYF4 are composited in a molar ratio of 1:0.45, wherein Y in NaYF4:Yb / Er... 3+ Yb 3+ Er 3+ The molar ratio is 68:30:2. Specifically, this invention provides a method for preparing rare earth nanoparticles (OA-RENPs) with oleic acid ligands, as follows: using rare earth chlorides (such as YCl3, YbCl3, ErCl3) as raw materials, the rare earth elements are prepared according to the molar ratio of each element in the chemical formula (such as NaYF4:Yb / Er), and oleic acid / octadecene is used as a composite solvent system. The rare earth nanoparticles (such as NaYF4:Yb / Er) are prepared by a solvothermal method. Then, using the rare earth nanoparticles as the core, rare earth chlorides are added again (prepared according to the shell chemical formula, such as the shell being NaYF4), and the reaction is continued by a solvothermal method using oleic acid / octadecene as the composite solvent system. Core-shell structured rare earth nanomaterials (such as NaYF4:Yb / Er@NaYF4) can be prepared.

[0035] In the following examples, the membrane-fusion liposomes are nanoscale lipid vesicles with strong membrane fusion capabilities, prepared by thin-film hydration method from commercially available DMPC, DOTAP, and DSPE-PEG2000 in a molar percentage of 75%:20%:5%. The specific synthesis steps are as follows: 20 mg of the three components were completely dissolved in 5-10 mL of chloroform and transferred to a round-bottom flask. After removing the organic solvent by rotary evaporation, a relatively transparent film was visible at the bottom of the flask. Ultrapure water was added and hydrated in a 50°C water bath to uniformly disperse the film in the ultrapure water. After sonication, the solution was transferred to a centrifuge tube, yielding the membrane-fusion liposome solution. The concentration of the membrane-fusion liposome solution was 10 mg / mL, and it was stored at 4°C for later use.

[0036] In the following examples, the cell membranes of brain metastatic cancer cells were taken from human highly metastatic lung cancer cells 95D. Cell membrane fragments were dispersed in HEPES buffer (pH=7.4, 10 mM) at a concentration typically of 5–15 mg / mL and stored at -80°C for later use. This invention can also utilize cell membranes from brain metastatic cancer cells such as human triple-negative breast cancer cells MDA-MB-231 and mouse melanoma cells B16-F10.

[0037] Example 1 A brain delivery nanocarrier via a transcellular transport pathway is prepared using the following steps: (1) 72 mg of OA-RENPs, 360 mg of lecithin, 10 mL of chloroform and 20 mL of n-hexane were ultrasonically dispersed in a round-bottom flask until uniformly dispersed. After removing the organic solvent by rotary evaporation, a relatively transparent film was visible at the bottom of the flask. 25 mL of ultrapure water was added and ultrasonically treated to make the film uniformly dispersed in the ultrapure water. After centrifugation (12000 rpm, 30 min), the precipitate obtained was the lecithin-modified rare earth nanomaterial (lecithin-RENPs). The precipitate was added to 10 mL of ultrapure water again, ultrasonicated and centrifuged (2000 rpm, 3 min). The supernatant was taken, which was the aqueous solution of water-soluble lecithin-RENPs.

[0038] (2) Mix the membrane fusion liposome solution (concentration of 10 mg / mL, volume of 30 μL) and the 95D cell membrane solution (concentration of 13 mg / mL, volume of 70 μL). At this time, the concentration of the membrane fusion liposome in the mixed system is 3 mg / mL and the concentration of the 95D cell membrane in the mixed system is 9 mg / mL. Then, perform low-temperature intermittent sonication (sonication power of 60 W, sonication temperature of 4℃, pause for 2 min after every 3 min of sonication, and repeat 4 times in total) to obtain the membrane fusion liposome-cell membrane solution, abbreviated as Lipo-CM solution.

[0039] (3) The Lipo-CM solution (concentration of 12 mg / mL, volume of 100 μL) and the aqueous solution of lecithin-RENPs (concentration of 6 mg / mL, volume of 100 μL) were mixed in HEPES buffer (pH=7.4, 10 mM). The total volume of the mixture was about 0.6 mL. The mixture was then sonicated in an ice-water bath for 15 min to obtain brain delivery nanocarriers dispersed in the buffer, abbreviated as Lipo-CMNPs.

[0040] like Figure 1 As shown, the brain delivery nanocarrier prepared in this embodiment has a particle size of 45~65 nm, good uniformity, and a clear cell membrane coating layer with a thickness of 5~15 nm is visible on its surface.

[0041] To facilitate subsequent characterization, the nanocarriers (Lipo-CMNPs) prepared in Example 1 were fluorescently labeled. The specific method was as follows: the component DSPE-PEG2000 of the synthetic membrane fusion liposome was replaced with DSPE-PEG2000-FITC or DSPE-PEG2000-Cy5.5 to achieve fluorescent labeling of the liposome coating layer; cell membrane dye DiO or DiD (1 mM, 5 μL) was mixed with lecithin-RENPs (1 mg) in HEPES buffer, vortexed and centrifuged to achieve fluorescent labeling of the nanocarrier core. The prepared material can be named DiO-NPs or DiD-NPs. In step (3), Lipo-CM (1.5 mg) and DiO-NPs (1 mg) were mixed in HEPES buffer and sonicated in an ice-water bath to obtain dye-labeled Lipo-CMNPs.

[0042] Example 2 A brain delivery nanocarrier via a transcellular transport pathway is prepared using the following steps: (1) The operation is basically the same as step (1) of Example 1, except that the mass of lecithin is 300 mg and the mass of n-hexane is 15 mL; (2) Mix the membrane fusion liposome solution (concentration of 10 mg / mL, volume of 50 μL) and the 95D cell membrane solution (concentration of 10 mg / mL, volume of 100 μL). At this time, the concentration of the membrane fusion liposome in the mixed system is 3.3 mg / mL and the concentration of the 95D cell membrane in the mixed system is 6.7 mg / mL. Then, perform low-temperature intermittent sonication (power of 60 W, temperature of 4℃, pause for 2 min after every 5 min of sonication, for a total of 2 cycles) to obtain the membrane fusion liposome-cell membrane solution, abbreviated as Lipo-CM solution.

[0043] (3) The Lipo-CM solution (concentration of 10 mg / mL, volume of 150 μL) and the aqueous solution of lecithin-RENPs (concentration of 6 mg / mL, volume of 200 μL) were mixed in HEPES buffer (pH=7.4, 10 mM). The total volume of the mixture was 0.6 mL. The mixture was then sonicated in an ice-water bath for 20 min to obtain brain delivery nanocarriers dispersed in the buffer, abbreviated as Lipo-CMNPs.

[0044] Example 3 A brain delivery nanocarrier via a transcellular transport pathway is prepared using the following steps: (1) The operation is basically the same as step (1) of Example 1, except that: the mass of lecithin is 288 mg and the mass of n-hexane is 15 mL; (2) Mix the membrane fusion liposome solution (concentration of 10 mg / mL, volume of 75 μL) and the 95D cell membrane solution (concentration of 10 mg / mL, volume of 75 μL). At this time, the concentration of the membrane fusion liposome in the mixed system is 5 mg / mL, and the concentration of the 95D cell membrane in the mixed system is 5 mg / mL. Then, perform low-temperature intermittent sonication (power of 65 W, temperature of 4℃, pause for 1 min after every 4 min of sonication, for a total of 3 cycles) to obtain the membrane fusion liposome-cell membrane solution, abbreviated as Lipo-CM solution.

[0045] (3) Take Lipo-CM solution (concentration of 10 mg / mL, volume of 150 μL), aqueous solution of lecithin-RENPs (concentration of 5 mg / mL, volume of 200 μL) and HEPES buffer (pH=7.4, 10 mM) and mix them. The total volume of the mixture is 0.5 mL. Sonicate in an ice water bath for 20 min to obtain brain delivery nanocarriers dispersed in buffer, abbreviated as Lipo-CMNPs.

[0046] Comparative Example 1 A nanocarrier, the specific preparation method of which includes the following steps: (1) Prepare water-soluble lecithin-RENPs according to step (1) of Example 1.

[0047] (2) An aqueous solution of lecithin-RENPs (concentration 5 mg / mL, volume 200 μL) was mixed with HEPES buffer (pH=7.4, 10 mM) to a total volume of 0.3 mL. The mixture was then sonicated in an ice-water bath for 15 min. The product obtained was a HEPES solution of lecithin-RENPs, labeled as nanocarrier NPs.

[0048] The transmission electron microscopy characterization results show that the nanocarrier prepared in Comparative Example 1 has a particle size distribution of 40-50 nm and exhibits a uniform spherical morphology. Figure 1 ).

[0049] In the lysosomal colocalization experiment, brain microvascular endothelial cells HCMEC / D3 were co-incubated with DiO-labeled comparative example 1 nanocarriers NPs and example 1 nanocarriers Lipo-CMNPs (the carrier concentration in the cell culture medium was 150 μg / mL during co-incubation). After incubation for different times, lysosomal staining was performed (dye Lyso-Tracker Red, staining concentration 50 nM, staining time 15 min). Subsequently, the colocalization of different carriers with lysosomes was observed using confocal microscopy. Figure 2 As shown, the nanocarriers NPs prepared in Comparative Example 1 exhibited significant co-localization with lysosomes during the uptake process by brain microvascular endothelial cells, indicating that uncoated NPs enter cells via the classical endocytosis pathway and are easily retained and degraded in lysosomes. In contrast, the brain delivery nanocarriers Lipo-CMNPs prepared in Example 1 adhered to the cell membrane surface before entering the cell interior during the uptake process by brain microvascular endothelial cells, and showed no significant co-localization with lysosomes throughout the process. This indicates that the brain delivery nanocarriers prepared in Example 1 can effectively avoid retention and degradation caused by lysosome capture.

[0050] Comparative Example 2 A nanocarrier, the specific preparation method of which includes the following steps: (1) Prepare water-soluble lecithin-RENPs according to step (1) of Example 1.

[0051] (2) Disperse 95D cell membrane solution (concentration of 10 mg / mL, volume of 150 μL) in HEPES buffer (pH=7.4, 10 mM). After mixing the two, the total volume is 1 mL. Vortex for 5 min and then centrifuge (13000 rpm, 40 min) to obtain cell membrane CM. (3) Mix CM (centrifuged product 1.5 mg) with an aqueous solution of lecithin-RENPs (concentration 5 mg / mL, volume 200 μL) in HEPES buffer (pH=7.4, 10 mM). The total volume of the mixture is 0.5 mL. Sonicate in an ice-water bath for 15 min. The product obtained is cell membrane-coated lecithin-RENPs, labeled as nanocarrier 95D-CMNPs.

[0052] Transmission electron microscopy characterization results show that the nanocarriers prepared in Comparative Example 2 have a particle size of 45-65 nm, good uniformity, and a distinct cell membrane coating layer with a thickness of 5-15 nm visible on their surface. Figure 1 ).

[0053] For ease of subsequent comparison, the nanocarriers (95D-CMNPs) prepared in Comparative Example 2 were labeled with fluorescent dyes, and the operation method was the same as the dye labeling process in Example 1.

[0054] Application Example 1 demonstrates that nanocarriers can enter brain microvascular endothelial cells via membrane fusion. Human brain microvascular endothelial cells (HCMEC / D3) in good growth condition were prepared into a cell suspension (0.5~1×10⁻⁶). 6 HCMEC / D3 cells were seeded in confocal microscopy dishes at a concentration of 0.15 mg / mL (1.5 mL). Once the cell density reached approximately 80%, the liquid culture medium was discarded, and new liquid culture medium containing different nanocarriers (0.15 mg / mL carrier concentration, 1.2 mL) was added to the dishes. The different nanocarriers were: NPs prepared in Comparative Example 1, 95D-CMNPs prepared in Comparative Example 2, and Lipo-CMNPs prepared in Example 1. These nanocarriers were co-incubated with the cells for different times (1 h, 2 h, 4 h, 6 h). The original culture medium was then discarded, and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde, and the nuclei of HCMEC / D3 cells were stained with DAPI dye (5 μg / mL, 10 min). After washing three times with PBS, the cells were preserved by soaking in 1 mL of PBS. Laser confocal microscopy was used to image the cells to analyze the pathways by which different nanocarriers entered HCMEC / D3 cells.

[0055] The results are as follows Figure 3 As shown, the nanocarriers Lipo-CMNPs prepared in Example 1 and the nanocarriers 95D-CMNPs prepared in Comparative Example 2 both exhibited significant membrane fusion after being incubated with brain microvascular endothelial cells for different times, and the carrier distribution sites showed obvious co-localization with the cell membrane. However, the NPs prepared in Comparative Example 1 did not exhibit membrane fusion, and their fluorescence signals did not show obvious co-localization with the cell membrane. With the extension of incubation time, the cell uptake of Lipo-CMNPs gradually increased and was higher than that of the 95D-CMNPs treatment group, indicating that Lipo-CMNPs have a stronger uptake potential by brain microvascular endothelial cells.

[0056] The results above demonstrate that the brain delivery nanocarrier prepared in Example 1 can enter brain microvascular endothelial cells through membrane fusion and be taken up by brain microvascular endothelial cells more efficiently, thereby crossing the blood-brain barrier; bypassing the classic endocytosis-lysosome pathway, it can efficiently and completely deliver functional components into the cytoplasm, thus improving its delivery efficiency as a nanocarrier.

[0057] Application Example 2: R18 dye fluorescence self-quenching recovery experiment to evaluate the membrane fusion capability of nanocarriers. R18 dye (R18 concentration of 5 mg / mL) was loaded onto 0.2 mg of CM prepared in Comparative Example 2 or Lipo-CM prepared in Example 1. After vortexing for 5 min, the mixture was centrifuged (12600 rpm, 40 min) to obtain cell membrane precipitates or membrane-fused liposome-cell membrane precipitates loaded with R18 dye. 0.1 mg of water-soluble lecithin-RENPs prepared in Example 1 (final concentration of 1 mg / mL, total volume of 100 μL) was added, and the mixture was sonicated in an ice-water bath for 15 min to obtain two nanocarriers loaded with R18 dye, named R18-95D-CMNPs or R18-Lipo-CMNPs.

[0058] Three flasks (T25 culture flasks) of HCMEC / D3 cells in logarithmic growth phase were prepared. After digestion and centrifugation, 400 μL of PBS was added to prepare a single-cell suspension for subsequent experiments. In the control group, only 40 μL of R18-95D-CMNPs or R18-Lipo-CMNPs were added; in the experimental group, 40 μL of R18-95D-CMNPs or R18-Lipo-CMNPs and 100 μL of the prepared cell suspension were added. The samples from the experimental and control groups were balanced with PBS (keeping the total volume of each group at 400 μL) and incubated in a shaker at 37°C for 40 min. The fluorescence of the control group was measured (F0), and the fluorescence of the experimental group was measured (F).

[0059] The results are as follows Figure 4 As shown, after incubation with brain microvascular endothelial cells for 40 min, the fluorescence intensity of R18-95D-CMNPs increased by 2.34-fold; the fluorescence intensity of R18-Lipo-CMNPs increased by 4.38-fold after incubation. The specific mechanism is that when the R18-labeled lipid membrane fuses with the unlabeled lipid membrane, the R18 concentration is locally diluted, the self-quenching phenomenon is weakened, leading to an increase in fluorescence signal. These results indicate that the brain delivery nanocarrier prepared in this invention can effectively fuse with cell membranes in vitro via membrane fusion, thereby restoring the fluorescence signal of the self-quenched R18 dye at higher concentrations. Furthermore, the membrane fusion efficiency of R18-Lipo-CMNPs is higher than that of R18-95D-CMNPs coated with pure cell membranes.

[0060] Application Example 3 confirms that the cellular uptake process of the nanocarrier is inhibited by the membrane fusion inhibitor Z-FFF. HCMEC / D3 cells in the logarithmic growth phase were prepared into single-cell suspensions, at a density of 2 mL per well and a cell density of 0.5–1 × 10⁻⁶ cells / well. 6 Cells were seeded at 150 μg / mL in six-well plates. Once the cell confluence reached approximately 80%, the original liquid culture medium was discarded, and 2 mL of liquid culture medium containing the membrane fusion inhibitor Z-FFF was added. The plates were incubated at 37°C for 1 h (Z-FFF concentration was 150 μg / mL). After incubation, the original liquid culture medium was discarded, and liquid culture medium containing different nanocarriers was added according to the group (carrier concentration was 0.15 mg / mL, 2 mL; different nanocarriers were: NPs prepared in Comparative Example 1, 95D-CMNPs prepared in Comparative Example 2, and Lipo-CMNPs prepared in Example 1). The cells were then co-incubated with the brain microvascular endothelial cells at 37°C for another 1 h. After incubation, the original culture medium was discarded, and the cells were washed twice with PBS. The cells in each well were digested and centrifuged (1000 rpm, 5 min). The cell pellet was resuspended in 200 μL of PBS to prepare a cell suspension, which was then analyzed by flow cytometry.

[0061] The results are as follows Figure 5 As shown, after treatment with the membrane fusion inhibitor Z-FFF, the uptake of Lipo-CMNPs prepared in Example 1 by cells decreased significantly, while the uptake of 95D-CMNPs prepared in Comparative Example 2 decreased slightly. This indicates that the membrane fusion inhibitor Z-FFF has a significant impact on the cellular uptake of the nanocarrier Lipo-CMNPs. In contrast, after Z-FFF treatment, there was no significant change in the uptake of NPs prepared in Comparative Example 1 by cells, indicating that the membrane fusion inhibitor has little effect on the cellular uptake process of NPs, and that NPs are not taken up by brain microvascular endothelial cells through the membrane fusion pathway.

[0062] like Figure 6 As shown, quantitative fluorescence analysis of the flow cytometry results revealed that after treatment with the membrane fusion inhibitor Z-FFF, the fluorescence signal of the NPs prepared in Comparative Example 1 showed no significant change compared to the untreated group in brain microvascular endothelial cells. However, the fluorescence signals of 95D-CMNPs prepared in Comparative Example 2 and Lipo-CMNPs prepared in Example 1 decreased by 52% and 78%, respectively. This indicates that the membrane fusion inhibitor Z-FFF significantly inhibits the uptake of 95D-CMNPs and Lipo-CMNPs by brain microvascular endothelial cells. These results demonstrate that the cellular uptake of the brain-delivered nanocarriers prepared in this invention is significantly affected by the membrane fusion inhibitor Z-FFF, indirectly proving that it primarily enters brain microvascular endothelial cells via the membrane fusion pathway.

[0063] Application Example 4 demonstrates that nanocarriers can efficiently cross the blood-brain barrier. BALB / c nude mice were randomly divided into two groups, and different dye-labeled nanocarriers (95D-CMNPs prepared in Comparative Example 2 and Lipo-CMNPs prepared in Example 1 were both dispersed in 0.9% NaCl solution at a concentration of 0.2 mg / mL, with an injection volume of 500 μL) were injected via tail vein. In vivo imaging of the small animals was performed at multiple time points after injection (1 h, 3 h, 5 h, 7 h, 9 h, 11 h, and 24 h).

[0064] The results are as follows Figure 7 As shown, the brain enrichment of Lipo-CMNPs prepared in Example 1 was much greater than that of 95D-CMNPs prepared in Comparative Example 2. Figure 8 Quantitative fluorescence analysis of the brain showed that the fluorescence enrichment of Lipo-CMNPs in the brain was approximately twice that of 95D-CMNPs, exhibiting a significant difference. Furthermore, the fluorescence changes in the nude mouse brain at various time points showed a trend of first increasing and then decreasing, reaching a maximum fluorescence enrichment at 11 h and decreasing at 24 h. This indicates that the nanocarrier in nude mice follows a process of initial enrichment followed by metabolism. These results demonstrate that the brain-delivery nanocarrier prepared in Example 1 significantly improved the enrichment of the material in the mouse brain.

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A brain delivery nanocarrier across a cell transport pathway, characterized by, Using lecithin-modified inorganic nanomaterials as the core and membrane-fused liposome-cell membranes as the shell, the membrane-fused liposome-cell membranes coat the surface of the lecithin-modified inorganic nanomaterials to form a core-shell structure; wherein, the membrane-fused liposome-cell membranes are formed by the hybridization and fusion of membrane-fused liposomes and the cell membranes of metastatic brain cancer cells.

2. The brain delivery nanocarrier via a transcellular transport pathway according to claim 1, characterized in that, The membrane-fusion liposomes are nanoscale lipid vesicles with membrane fusion capabilities.

3. The brain delivery nanocarrier via a transcellular transport pathway according to claim 1, characterized in that, The membrane-fused liposomes are prepared by membrane hydration method from 70% to 80% neutral phospholipids, 15% to 25% cationic lipids, and 4% to 6% PEG-modified phospholipids in molar percentage.

4. The brain delivery nanocarrier via a transcellular transport pathway according to claim 1, characterized in that, The mass ratio of cell membrane to membrane-fused liposomes in brain metastatic cancer cells was (1~4):1; the mass ratio of membrane-fused liposomes-cell membrane coating layer to inorganic nanomaterials was (1~3):

1.

5. The method for preparing a brain delivery nanocarrier via a transcellular transport pathway according to claim 1, characterized in that, The lecithin-modified inorganic nanomaterial is an inorganic nanomaterial with surface-modified amphiphilic ligand lecithin, obtained by changing the surface of oleic acid-modified inorganic nanomaterial from oily to watery through lecithin modification.

6. The method for preparing a brain delivery nanocarrier via a transcellular transport pathway according to claim 1, characterized in that, Includes the following steps: (1) The cell membrane of brain metastatic cancer cells and membrane fusion liposomes were mixed in a buffer solution by low-temperature intermittent sonication to obtain membrane fusion liposome-cell membrane; (2) The membrane-fused liposome-cell membrane and lecithin-modified inorganic nanomaterials are mixed in a buffer solution and ultrasonically treated at a temperature of 0~4℃ to obtain the brain delivery nanocarrier of the transcellular transport pathway.

7. The method for preparing a brain delivery nanocarrier via a transcellular transport pathway according to claim 6, characterized in that, In step (1), the mass ratio of the cell membrane of the brain metastatic cancer cells to the membrane fusion liposome is (1~4):1; in step (2), the mass ratio of the membrane fusion liposome to the cell membrane and the inorganic nanomaterial modified with lecithin surface is (1~3):

1.

8. The method for preparing a brain delivery nanocarrier via a transcellular transport pathway according to claim 6, characterized in that, In step (1), the concentrations of the cell membrane and membrane fusion liposomes of brain metastatic cancer cells in the buffer solution are 5~10 mg / mL and 2~5 mg / mL, respectively; the low-temperature intermittent ultrasound power is 60~70 W, the temperature is 0~4℃, and the intermittent ultrasound is achieved by pausing for 1~2 minutes after every 3~5 minutes of ultrasound, and the cycle is repeated 2~4 times. In step (2), the concentrations of membrane-fused liposome-cell membrane and lecithin-modified inorganic nanomaterials in the buffer solution are 2~12 mg / mL and 1~6 mg / mL, respectively, the ultrasonic power is 60~70 W, and the ultrasonic time is 10~20 min.

9. The method for preparing a brain delivery nanocarrier via a transcellular transport pathway according to claim 6, characterized in that, In steps (1) and (2), the pH of the buffer solution is 7.0~8.0 and the concentration range is 5~20 mM; the buffer solution system includes: Tris-HCl buffer, HEPES buffer, TES buffer, and MOPS buffer.

10. The application of the brain delivery nanocarrier according to claim 1 in delivering functional components of brain microvascular endothelial cells.

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