Nanometer vesicle for targeted brain glioma metabolic immune remodeling as well as preparation method and application of nanometer vesicle

Abstract

The invention relates to the technical field of biological medicine, in particular to a nano-vesicle for targeted brain glioma metabolic immune remodeling and a preparation method and application thereof. According to the nano-vesicle for targeted brain glioma metabolism immune remodeling, double-target metabolism and cascade responsive cleavage site modified biological vesicles are integrated, and effective penetration of a drug blood brain barrier and precise targeting of brain glioma can be achieved. The ROS responsive nanoparticles II are prepared by selecting BSA as a drug carrier, applying a nanoprecipitation method and utilizing SLC1A5, LDHA antagonists and responsive linkers, so that the metabolic double-target inhibitor only responds to specific ROS of tumor cells, and specific release in the tumor cells is realized. The cascade response polypeptide provides a simple and convenient delivery scheme with low invasiveness and application prospects for treatment of brain glioma, and is expected to realize precise and individualized treatment in the field of treatment of malignant glioma.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a nanovesicle for targeting metabolic and immune remodeling in gliomas, its preparation method, and its application. Background Technology

[0002] Glioblastoma multiforme (GBM) is the most malignant and deadliest primary brain tumor in the central nervous system, classified as a grade IV astrocytoma in the World Health Organization's classification of central nervous system tumors. The high heterogeneity of GBM, the restriction of the blood-brain barrier, and the immunosuppressive microenvironment collectively contribute to the complexity and difficulty of its treatment.

[0003] Malignant tumor cells construct unique energy supply networks through metabolic reprogramming. Glutamine-addictive metabolism and Warburg effect-mediated hyperlactic acid synthesis form a dynamically complementary metabolic buffer system, becoming the core mechanism for maintaining tumor survival. Existing research is mostly limited to single-pathway metabolic inhibition protocols for tumor treatment. However, tumor cells can compensate for single-target metabolic limitations through metabolic reprogramming, thereby reducing the efficacy of metabolic therapy.

[0004] SLC1A5 is an important glutamine transporter, highly expressed in various tumors, and plays a crucial role in the metabolism and proliferation of tumor cells. Lactate dehydrogenase A (LDHA) is a key enzyme in the glycolysis process of tumor cells, converting pyruvate to lactate. In recent years, specific small-molecule inhibitors targeting both have become a novel strategy for tumor treatment and have attracted widespread attention. Among them, the SLC1A5 antagonist V-9302 has shown good anti-tumor activity in gastric cancer, glioblastoma, and pancreatic cancer models, but it increases glucose uptake by all tumor cell populations in the tumor microenvironment (TME). In a melanoma model, the LDHA inhibitor FX-11 can inhibit intratumoral lactate production, thereby enhancing the infiltration of CD8+ within the tumor. + and CD4 + The activity and proportion of T lymphocytes. Simultaneous inhibition of lactate production and glutamine transport can disrupt the compensatory mechanisms between the above metabolic pathways. This multi-dimensional approach can overcome the compensatory limitations of traditional single-target therapy, significantly reduce the energy supply of tumor cells, and thus affect the normal function and survival of tumor cells.

[0005] Abnormal metabolism in glioma cells (glutamine addiction, Warburg effect) is not only fundamental to their survival but also a key mechanism mediating immune escape. Tumor cells remodel their metabolic pathways to maximize nitrogen / carbon source utilization, leading to nutrient depletion infiltrating immune cells within the tumor microenvironment. Although small-molecule metabolic regulators have made some progress in targeting the lactate metabolism pathway, the inhibitory effect of single metabolic pathways on a large number of immunosuppressive metabolites is limited and lacks broad-spectrum efficacy. Therefore, targeting and regulating a wide range of metabolic pathways to inhibit tumor cell metabolism can provide sufficient interstitial nutrition for tumor-infiltrating immune cells while reducing the production of immunosuppressive metabolites, thereby improving the efficacy of immunotherapy. By targeting the metabolic network to disrupt the tumor-immune interaction balance, a paradigm shift from "palliative care" to "radical treatment" is expected.

[0006] The blood-brain barrier restricts drug entry into the brain, resulting in only about 2% of small-molecule drugs being able to effectively enter the central nervous system (for example, the concentration of temozolomide in cerebrospinal fluid is only 20% of its plasma concentration), thus creating a trade-off between the delivery efficiency and safety of glioma treatment drugs. Although chemical drugs or physical stimulation methods, such as mannitol, angiotensin, opioids, or ultrasound stimulation, can increase the permeability of the blood-brain barrier, these methods may cause adverse reactions such as infection, inflammation, increased blood pressure, and seizures. Currently, China has achieved tumor homology targeting using cell membrane biomimetic technology; however, integrating multiple responsive elements on the cell membrane to achieve precise targeting of gliomas still faces technological barriers. Summary of the Invention

[0007] This invention aims to at least solve one of the technical problems existing in the related art. Therefore, the first objective of this invention is to provide a method for preparing nanovesicles for targeting metabolic and immune remodeling in gliomas; the second objective of this invention is to provide nanovesicles for targeting metabolic and immune remodeling in gliomas; and the third objective of this invention is to provide an application of nanovesicles for targeting metabolic and immune remodeling in gliomas.

[0008] To achieve the first objective, the technical solution adopted by this invention is as follows: A method for preparing nanovesicles for targeting metabolic and immune remodeling in gliomas includes the following steps: S100. Add organic solution I containing SLC1A5 antagonist and LDHA antagonist to an aqueous solution containing bovine serum albumin and mix well to obtain an aqueous phase containing nanoparticles I. Among them, SLC1A5 is member 5 of solute carrier group 1, and LDHA is lactate dehydrogenase A; S200. Add organic solution II containing responsive linkers to an aqueous phase containing nanoparticles I to prepare ROS responsive nanoparticles II. The responsive linker is selected from N-hydroxysuccinimide-ketothiol-N-hydroxysuccinimide (NHS-TK-NHS). S300. By encapsulating the ROS-responsive nanoparticles II with an APT-containing cell membrane, nanovesicles for targeting metabolic and immune remodeling in gliomas are obtained. APT is a cascade-response polypeptide, and its structural composition is shown below: IgK signal peptide-ANG-responsive cleavage site-tLYP1-PDGFR transmembrane domain; Among them, ANG is a blood-brain barrier permeable peptide with a responsive cleavage site that is a matrix metalloproteinase-specific cleavage site, and tLYP1 is a glioma homing peptide.

[0009] Preferably, the SLC1A5 antagonist is selected from V-9302, and the LDHA antagonist is selected from FX-11.

[0010] Preferably, in step S100, the organic solution I contains equal masses of SLC1A5 antagonist and LDHA antagonist.

[0011] Preferably, the solvents for both organic solution I and organic solution II are selected from dimethyl sulfoxide.

[0012] Preferably, in step S300, the preparation of the APT-containing cell membrane includes the following steps: S310. Three lentiviral vector plasmids were transfected into HEK293T cells to obtain mouse glioma cells GL261. Among them, the three lentiviral vector plasmids are psPAX2, pMD2.G and pCDH-GFP-APT; S320. Mouse glioma cells GL261 were screened using puromycin, and surviving cells were collected to obtain mouse glioma cells GL261 carrying APT, which were named GL261-APT. S330. The cell membrane in GL261-APT was separated using a multi-step membrane purification method to obtain a cell membrane containing APT.

[0013] Preferably, in step S310, the mass ratio of the three lentiviral vector plasmids psPAX2, pMD2.G and pCDH-GFP-APT is 1:1.5:2.

[0014] Preferably, in step S300, the ROS-responsive nanoparticles II are encapsulated in a cell membrane containing APT using a polycarbonate membrane extrusion method.

[0015] Preferably, in step S300, the amino acid sequence of the IgK signal peptide is shown in SEQ ID NO.1, the amino acid sequence of ANG is shown in SEQ ID NO.2, the amino acid sequence of the responsive cleavage site is shown in SEQ ID NO.3, the amino acid sequence of tLYP1 is shown in SEQ ID NO.4, and the amino acid sequence of the PDGFR transmembrane domain is shown in SEQ ID NO.5.

[0016] To achieve the second objective, the technical solution adopted by this invention is as follows: A nanovesicle for targeting metabolic and immune remodeling in gliomas is prepared using any of the above-described methods for preparing nanovesicles for targeting metabolic and immune remodeling in gliomas.

[0017] Peptides, with their advantages of good biocompatibility, diverse functions, high in vivo responsiveness, and simple and easy synthetic modification methods, have been widely used in constructing targeted drug delivery systems. Drug delivery systems based on peptides with targeting and stimulus-responsive properties can deliver drugs specifically to tumor regions. Once the drug delivery system reaches the tumor tissue, precise drug release can be achieved under the influence of the specific tumor microenvironment or exogenous stimuli. This type of peptide carrier with specific tumor-targeting and stimulus-responsive properties can maximize the anti-tumor efficacy of drugs and reduce their toxic side effects.

[0018] The nanovesicles provided by this invention for targeting metabolic and immune remodeling in gliomas integrate dual-target metabolism and cascade-responsive cleavage site modification into biological vesicles, enabling effective drug penetration across the blood-brain barrier and precise targeting of gliomas. In this technical solution, bovine serum albumin (BSA) is selected as the drug carrier. Using a nanoprecipitation method, ROS-responsive nanoparticles II are prepared with an SLC1A5 antagonist, an LDHA antagonist, and a reactive oxygen species (ROS)-responsive linker NHS-TK-NHS. This aims to ensure that the dual-target metabolic inhibitor responds only to tumor cell-specific ROS, achieving tumor-specific release. Furthermore, the technical solution provided by this invention includes a cascade-responsive polypeptide (APT), the structural composition of which, from the N-terminus to the C-terminus, is shown below: IgK signal peptide-ANG-responsive cleavage site-tLYP1-PDGFR transmembrane domain; This structure links the blood-brain barrier permeability peptide angiopoietin (ANG) to the glioma homing peptide tLyp-1 via a responsive cleavage site (which serves as a matrix metalloproteinase-specific cleavage site). It also fuses the C-terminus of the cascade-responsive peptide with the transmembrane domain of the platelet-derived growth factor receptor (PDGFR). Utilizing the properties of this transmembrane domain, cell membrane anchoring is achieved, allowing the peptide to function on the exosome surface. The nanovesicles provided by this invention can efficiently penetrate the blood-brain barrier using ANG peptide nanoparticles. Subsequently, in the tumor microenvironment with high MMP expression, the transmembrane peptide is detached, and the tumor-targeting peptide is exposed, further promoting the targeted internalization of the nanomaterial into tumor cells and penetrating deep into the tumor. This invention constructs a synergistic therapeutic system of metabolic intervention and immune activation. During the inhibition of tumor cell metabolism, metabolic competition is weakened, promoting CD4 activation in the tumor immune microenvironment. + and CD8 + T cell differentiation. Simultaneously, the targeted responsive cascade peptide provided by this invention offers a simple, low-invasive, and promising delivery method for the treatment of gliomas. The technical solution of this invention is based on multidimensional regulation of glioma metabolism-immunity-delivery; therefore, it holds promise for achieving precision and personalized treatment in the field of malignant glioma therapy.

[0019] To achieve the third objective, the technical solution adopted by this invention is as follows: An application of nanovesicles for targeting metabolic and immune remodeling in gliomas, comprising preparing a pharmaceutical formulation using any of the nanovesicles described above for targeting metabolic and immune remodeling in gliomas, the pharmaceutical formulation being used for the treatment of glioblastoma.

[0020] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: This invention provides a nanovesicle for targeting metabolic and immune remodeling in gliomas, which can dually block lactate and glutamine metabolism within the tumor while inhibiting tumor metabolic competition, thereby remodeling the immune microenvironment. The nanovesicle contains an APT-containing cell membrane, which has been verified to have a targeting effect on gliomas (compared to the control group, the drug concentration at the tumor site increased threefold after application of this cell membrane). The nanovesicle for targeting metabolic and immune remodeling in gliomas provided by this invention establishes a synergistic therapeutic system of metabolic intervention and immune activation, weakening the metabolic competition generated during the "starvation" of tumor cells and promoting CD4+ in the tumor immune microenvironment. + and CD8 + This study also provides a simple, low-invasive, and promising drug formulation for the treatment of gliomas.

[0021] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0022] Figure 1 This describes the cleavage of VTF and VCF provided in Example 1 of the present invention in 10mM H2O2 aqueous solution and PBS buffer.

[0023] Figure 2 This is a schematic diagram of the cascade response polypeptide (APT) structure provided in Embodiment 2 of the present invention.

[0024] Figure 3 This is the APT expression status in GL261-APT cells provided in Example 2 of the present invention.

[0025] Figure 4 This is the case where the membrane peptide provided in Example 2 of the present invention expresses APT on the cell membrane.

[0026] Figure 5 This is a particle size distribution and morphology feature diagram of VTF@APT provided in Embodiment 2 of the present invention.

[0027] Figure 6 This invention examines the effects of VTF@APT provided in Example 1 on the oxidative stress level and cell morphology changes of tumor cells.

[0028] Figure 7 This is the detection result of the effect of VTF@APT on inflammation and pyroptosis-related molecules provided in Example 1 of this invention.

[0029] Figure 8 This is an example of the effect of VTF@APT provided in Example 1 of this invention on pyroptosis and death of tumor cells.

[0030] Figure 9 This invention examines the toxicity and targeted delivery effect of VTF@APT provided in Example 1.

[0031] Figure 10 This is a statistical chart showing the results of T cell subsets and their biomarkers after different nanoparticle treatments provided in Example 2 of this invention.

[0032] Figure 11 This is the result of the in vivo brain targeting ability test of different nanoparticles provided in Test Example 3 of the present invention.

[0033] Figure 12 These are in vivo near-infrared (NIR) images of different treatment groups at 8, 11, 14, 17, 20 and 23 days of treatment provided in Example 4 of this invention.

[0034] Figure 13This is a graph showing the inhibitory effect of different treatment groups on tumor growth provided in Example 4 of this invention.

[0035] Figure 14 The results are the pathological examination results of mouse brain tissue in different treatment groups at 23 days of treatment, as provided in Example 4 of this invention.

[0036] Figure 15 These are the survival curves of mice in different treatment groups provided in Example 4 of this invention. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention.

[0038] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0039] Example 1 The process for preparing ROS-responsive nanoparticles is as follows: The small molecule inhibitors V-9302 (1 mg) and FX-11 (1 mg) were dissolved in the organic solvent dimethyl sulfoxide (DMSO) (100 μL) to form an organic phase. Then, the organic phase was slowly added dropwise to an aqueous solution (1 mL) containing 10 wt% BSA and mixed thoroughly to obtain an aqueous solution containing BSA-encapsulated V-9302 and FX-11 nanoparticles I. Preparation of NHS-TK-NHS organic solution: Dissolve 1 mg of ROS-responsive NHS-TK-NHS in 100 μL of DMSO to obtain NHS-TK-NHS organic solution. The NHS-TK-NHS organic solution was slowly added dropwise to an aqueous solution (1 mL) containing BSA-encapsulated V-9302 and FX-11 nanoparticles I. After the addition was complete, the reaction was stirred for 30 min. The reaction solution was then dialyzed using a 10 kDa membrane to remove free molecules and residual DMSO solvent. The dialyzed solution was transferred to an ultrafiltration tube, centrifuged at 3000 g for 10 min, collected, and concentrated to obtain ROS-responsive nanoparticles, denoted as VTF. Meanwhile, for comparison, the ROS-responsive fragmentation NHS-TK-NHS (1mg) in the above steps was replaced with the ROS-non-responsive fragmentation NHS-C7-NHS (1mg), and the rest was the same as the above preparation process, to obtain ROS-non-responsive nanoparticles, denoted as VCF.

[0040] The lysis of VTF and VCF in 10mM H2O2 aqueous solution and PBS buffer (control) is as follows: Figure 1 As shown; Figure A shows TEM images of VTF and VCF (scale bar is 100 nm). As can be seen from the figure, VTF has a complete spherical structure in PBS buffer, but it breaks down and aggregates in H2O2 aqueous solution, indicating that H2O2 triggered its structural disintegration; VCF has a relatively complete structure in both PBS buffer and H2O2 aqueous solution, and no lysis occurred. Figure B is a histogram of VTF particle size distribution, and Figure C is a bar chart of VTF particle size. From these two figures, we can see that in the presence of 10mM H2O2, the particle size of VTF decreased from 109.1 nm to 8.85 nm, indicating that VTF can be degraded in the tumor microenvironment. Figure D shows the cumulative drug release curve of VTF, and Figure E shows the drug release bar chart of VTF. From these two figures, we can see that after 48 hours in 10 mM H2O2, VTF can achieve a drug release efficiency of 92.69%, which is 3.4 times that of the non-responsive group in PBS buffer. This indicates that VTF can release the dual-target small molecule inhibitors V-9302 and FX-11 in response to the ROS concentration in the tumor microenvironment.

[0041] Example 2 Design and synthesis of cell membrane carriers modified with cascade-responsive peptides (APTs), the structural composition of which is as follows: Figure 2 As shown in the figure, the IgK signal peptide, Flag, ANG, PLGVR, tLYP1 and PDGFR transmembrane domains are connected sequentially.

[0042] The amino acid sequence of the IgK signal peptide is shown in SEQ ID NO.1, which is as follows: MASLLVLAALASALGQARC; Flag tags are a widely used short peptide epitope tag (amino acid sequence DYKDDDDK) used as protein tags for protein detection and localization; ANG is a blood-brain barrier permeation peptide, and its amino acid sequence is shown in SEQ ID NO.2. SEQ ID NO.2 is as follows: TFFYGGSRGKRNNFKTEEY; PLGVR is a responsive cleavage site, which is the amino acid sequence of SEQ ID NO.3; tLyp1 is a glioma homing peptide, and its amino acid sequence is shown in SEQ ID NO.4, which is as follows: CGNKRTRGC; The amino acid sequence of the transmembrane domain of PDGFR is shown in SEQ ID NO.5, which is as follows: AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR.

[0043] Based on the above design, a lentiviral expression vector plasmid encoding this responsive polypeptide was synthesized and named pCDH-GFP-APT. The preparation process is as follows: I. A stable transfection system of three plasmid lentiviruses was used to construct mouse glioma cells GL261.

[0044] The lentiviral packaging vector plasmids psPAX2 and pMD2.G and the lentiviral expression vector plasmid pCDH-GFP-APT were transfected into HEK293T at a mass ratio of 5:7.5:10, as detailed below: After thoroughly mixing PEI transfection reagent (40 μL, HY-K2014) with psPAX2 (5 μg), pMD2.G (7.5 μg), and pCDH-GFP-APT (10 μg), and allowing it to stand for 5 min, serum-free DMEM medium (1 mL) was added. After mixing and standing for 15 min, the mixture was directly added to HEK293T cells that had been starved for 1 h. Viral supernatants were collected at 48 h and 72 h post-transfection. The collected viral supernatants were then added to the well-growing mouse glioma cell line GL261. Two days after viral transfection, a two-week selection process using puromycin (1 μg / ml) was initiated, and a single clone of cells was isolated and named GL261-APT. The APT expression of this cell line is as follows: Figure 3 As shown in the figure, this figure verifies the efficient and stable expression of APT in cells from three dimensions: fluorescence phenotype, protein level, and cell population ratio. Figure A shows a fluorescence microscope image (scale bar is 20 μm), Figure B shows a Western blot image of the protein, and Figure C shows the results of flow cytometry analysis.

[0045] The expression of APT on the cell membrane by transmembrane peptides, such as... Figure 4As shown in the figure; Figure A is a Western blot image, which shows that compared with non-expressing cells, the monoclonal cells screened by this invention can detect APT tags on the membrane surface, proving that APT is effectively expressed on the membrane surface; Figure B is a flow cytometry analysis result, which shows that APT can be cleaved by matrix metalloproteinase 2 / 9 (MMP2 / 9).

[0046] APT-containing cell membranes were isolated using a multi-step membrane purification method, as follows: GL261-APT cells were resuspended in hypotonic lysis buffer at 4°C and incubated overnight. Subsequently, the cell suspension underwent three cycles of liquid nitrogen freezing and thawing, followed by sonication at 4°C for 10 min (100W, 3 seconds per sonication followed by a 5-second pause). After centrifugation at 3200 g for 5 min, the supernatant was collected. This supernatant was then centrifuged at 14500 g for 20 min, and the supernatant was collected again to obtain APT-containing cell membranes. The APT-containing cell membranes (5 mg) were thoroughly mixed with VTF (1 mg each of V-9302 and FX-11). The mixture was then extruded 10–20 times through a porous polycarbonate membrane (100 nm) using a micro-extruder (to ensure uniform coating of the cell membrane onto the nanoparticle surface), yielding APT-containing cell membrane-coated VTF nanoparticles, denoted as VTF@APT. The particle size distribution and morphological characteristics are as follows: Figure 5 As shown in the figure, the results indicate that APT successfully coated VTF while maintaining the particle size at around 123 nm. Figure A is a TEM image (scale bar 100 nm), and Figure B is a particle size analysis diagram of dynamic light scattering (DLS).

[0047] To facilitate subsequent nanoparticle fluorescence bioimaging, fluorescently labeled nanoparticles were also prepared in this embodiment. Deep red fluorescent dye (DiD) (1 mg) and distearate-phosphatidylethanolamine-polyethylene glycol (DSPE-PEG) (4 mg) were added to DMSO (100 μL) and co-extruded with APT-unexpressed GL261 cell membranes (containing 5 mg). Nanoparticles, denoted as VIF@261, were then prepared according to the aforementioned method.

[0048] Test Example 1: Test of the ability of VTF@APT to kill tumor cells in vitro.

[0049] I. The effects of VTF@APT on oxidative stress levels and cell morphological changes in tumor cells were investigated using two techniques: reactive oxygen species fluorescence probe detection (DCFH-DA staining) and bright-field microscopy. The results are as follows: Figure 6As shown in the figure; Figure A is a DCFH-DA fluorescence staining image (scale bar is 20 μm), which shows that VTF@APT significantly upregulates ROS in tumor cells and induces an imbalance in ROS metabolic homeostasis in tumor cells; Figure B is a bright-field microscopy image (scale bar is 20 μm), which shows that the cells in the PBS group adhere well and have normal morphology; the tumor cells in the VTF@APT treatment group swell, become rounded, and form "bubble-like" protrusions, which shows that VTF@APT has a significant killing effect on tumor cells.

[0050] II. Detection results of the effects of VTF@APT on molecules related to inflammation and pyroptosis, such as... Figure 7 As shown in the figure, the results indicate that VTF@APT can significantly induce pyroptosis in tumor cells and upregulate pyroptosis-related markers (ATP, Caspase-1, GSDMD-N and IL-1β). Figure A shows a bar chart of ATP release levels, Figure B shows a bar chart of IL-1β secretion levels, and Figure C shows a Western blot of proteins.

[0051] III. Analysis results of the effects of VTF@APT on tumor cell pyroptosis and death, such as... Figure 8 As shown, VTF can effectively induce pyroptosis in tumor cells.

[0052] IV. Toxicity and Targeted Delivery Efficacy of VTF@APT, such as Figure 9 As shown in the figure, the results indicate that VTF@APT NPs can induce tumor cell death more effectively than unencapsulated VTF. Figure A shows the cell viability curve; Figure B shows the bright-field cell morphology (scale bar is 20 μm); Figure C shows the fluorescence staining of live / dead cells (scale bar is 20 μm).

[0053] Test Example 2: Test for the in vitro induced T cell immune activity of VTF@APT.

[0054] Splenic T cells were isolated from C57BL / 6 mice and co-cultured with GL261 cells treated with different nanoparticles (PBS, V-9302, FX-11, VTF, and VTF@APT) for 48 h. Flow cytometry was used to analyze the T cell subsets and their biomarkers after different nanoparticle treatments. The statistical results are as follows: Figure 10 As shown in the figure, the results indicate that VTF@APT can amplify effector T cells (CD4+). + CD8 + IFNγ +It reduces immunosuppressive Tregs and alleviates T cell exhaustion (low PD-1 expression), thereby enhancing the body's cellular immune response; compared with other treatments, VTF@APT significantly enhances the in vitro induced T cell immune activity.

[0055] Figure A shows the CD3 after treatment with different nanoparticles. + CD4 + The bar chart shows the percentage of T cells, and Figure B shows the CD3 concentration after different nanoparticle treatments. + CD8 + The bar chart shows the percentage of T cells, and Figure C shows the active CD8+ cells after different nanoparticle treatments. + CD3 + IFNγ in cells + A bar chart showing the percentage of cells, with Figure D representing the active CD4 cells after treatment with different nanoparticles. + CD3 + FOXP3 in cells + CD25 + A bar chart showing the percentage of Treg cells; Figure E shows the CD3 percentage after different nanoparticle treatments. + CD8 + PD-1 in cells + A bar chart showing the percentage of cells.

[0056] Test Example 3: Testing the in vivo brain targeting ability of VTF@APT.

[0057] Using the GL261 glioma orthotopic model, different DiR-labeled nanoparticles (VTF, VIF@261, and VTF@APT) were injected via tail vein. The in vivo brain-targeting ability of VTF@APT was examined using real-time near-infrared (NIR) fluorescence imaging. The results are as follows: Figure 11 As shown in the figure, the results indicate that compared with the control group (VTF and VTF@261 nanoparticles), VTF@APT nanoparticles significantly increased in accumulation in the brain. VTF@APT has excellent in vivo brain targeting ability and can be efficiently and specifically enriched in brain tumor sites. Figure A shows a live near-infrared (NIR) imaging image. It can be seen from the image that the fluorescence intensity in the brain of the VTF@APT group continued to increase over time and was significantly higher than that of the VTF and VTF@261 groups, indicating that it has a stronger ability to stay and accumulate in the brain. Figure B shows the fluorescence intensity quantitative analysis curve. From this figure, it can be seen that the fluorescence intensity of the VTF@APT group continuously increases over time and is significantly higher than that of the VTF and VTF@261 groups at each time point. This result indicates that VTF@APT has better brain targeting efficiency and retention.

[0058] Figure C shows the quantitative analysis curve of tissue distribution. From this figure, it can be seen that the fluorescence intensity of VTF@APT in the brain tumor site is significantly higher than that in other tissues and other treatment groups. This result indicates that it has brain-targeting specificity, is highly enriched in the brain, and is less distributed in other tissues, which can reduce systemic side effects. Figure D shows an immunofluorescence imaging image of tissue sections. As can be seen from the figure, the brain sections of mice treated with VTF@APT showed strong red fluorescence, mainly concentrated in the tumor area, with almost no off-target distribution. In addition, there was almost no colocalization between the nanoparticle-derived red fluorescence and the CD31-stained green blood vessels, indicating that the VTF@APT nanoparticles successfully infiltrated from the blood vessels and penetrated into the glioma parenchyma. In contrast, the fluorescence signal detected in mice receiving the control group (VTF or VTF@261) was significantly weaker, which is consistent with their poor targeting ability.

[0059] Test Example 4: Detection of the in vivo glioma-killing ability of VTF@APT.

[0060] GL261 cells labeled with biotin were in situ seeded into the brains of mice. On day 8 post-GL261-Luc cell engraftment, mice were randomly assigned to five treatment groups: PBS, V9302, VTF, VTF@261, and VTF@APT. Treatment was administered via tail vein injection every 3 days. Tumor progression was monitored every 3 days using bioluminescence imaging. In vivo near-infrared (NIR) imaging and statistical curves of the results are shown below. Figure 12 and Figure 13 As shown in these two figures, tumor progression was rapid in the PBS group, while V9302 monotherapy had limited efficacy (only 1 out of 5 mice showed tumor regression). The VTF group showed significant tumor suppression compared to the V9302 monotherapy group, confirming the advantage of dual-targeted metabolic anabolic lethality in overcoming compensatory mechanisms. The tumor growth inhibition effects of VTF and VTF@261 were comparable, indicating that although tumor cell membrane coating has targeting potential, the effect of VTF@261 in enhancing therapeutic efficacy is limited due to the lack of APT expression. However, VTF@APT showed excellent anti-tumor activity, with 3 out of 5 mice showing near-complete inhibition of bioluminescent signaling and the lowest tumor burden among all treatment groups.

[0061] On day 23 of treatment, brain tissue was collected from the treated mice for pathological examination, and the results were as follows: Figure 14As shown in the figure; Figure A is a pathological section of tumor tissue after H&E staining. It can be seen from the figure that the tumor area in the VTF@APT group is significantly smaller and more regular in shape, indicating that VTF@APT has the most significant inhibitory effect on tumor growth; Figure B is a Ki67 immunohistochemical staining result (scale bar is 20μm). It can be seen from the figure that there are very few Ki67 positive cells in the VTF@APT group, indicating that VTF@APT can significantly inhibit the proliferation of tumor cells.

[0062] Survival curves of mice in different treatment groups, such as Figure 15 As shown in the figure, compared with the PBS control group, the median survival of mice treated with V9302 was 27–35 days, the median survival of the VTF group was 38 days, the median survival of the VTF@261 group was 42 days, and 60% of the mice in the VTF@APT group survived for more than 60 days, which was significantly higher than all other groups.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing nanovesicles for targeting metabolic and immune remodeling in gliomas, characterized in that, Includes the following steps: S100. Add organic solution I containing SLC1A5 antagonist and LDHA antagonist to an aqueous solution containing bovine serum albumin and mix well to obtain an aqueous phase containing nanoparticles I. Among them, SLC1A5 is member 5 of solute carrier group 1, and LDHA is lactate dehydrogenase A; S200. Add organic solution II containing responsive linkers to an aqueous phase containing nanoparticles I to prepare ROS responsive nanoparticles II. The responsive linker is selected from N-hydroxysuccinimide-ketothiol-N-hydroxysuccinimide; S300. By encapsulating the ROS-responsive nanoparticles II with an APT-containing cell membrane, nanovesicles for targeting metabolic and immune remodeling in gliomas are obtained. APT is a cascade-response polypeptide, and its structural composition is shown below: IgK signal peptide-ANG-responsive cleavage site-tLYP1-PDGFR transmembrane domain; Among them, ANG is a blood-brain barrier permeable peptide with a responsive cleavage site that is a matrix metalloproteinase-specific cleavage site, and tLYP1 is a glioma homing peptide.

2. The method for preparing nanovesicles for targeting metabolic and immune remodeling in gliomas as described in claim 1, characterized in that, The SLC1A5 antagonist is selected from V-9302, and the LDHA antagonist is selected from FX-11.

3. The method for preparing nanovesicles for targeting metabolic and immune remodeling in gliomas as described in claim 1, characterized in that, In step S100, the organic solution I contains equal masses of SLC1A5 antagonist and LDHA antagonist.

4. The method for preparing nanovesicles for targeting metabolic and immune remodeling in gliomas as described in claim 1, characterized in that, The solvents for both organic solution I and organic solution II are selected from dimethyl sulfoxide.

5. The method for preparing nanovesicles for targeting metabolic and immune remodeling in gliomas as described in claim 1, characterized in that, In step S300, the preparation of the APT-containing cell membrane includes the following steps: S310. Three lentiviral vector plasmids were transfected into HEK293T cells to obtain mouse glioma cells GL261. Among them, the three lentiviral vector plasmids are psPAX2, pMD2.G and pCDH-GFP-APT; S320. Mouse glioma cells GL261 were screened using puromycin, and surviving cells were collected to obtain mouse glioma cells GL261 carrying APT, which were named GL261-APT. S330. The cell membrane in GL261-APT was separated using a multi-step membrane purification method to obtain a cell membrane containing APT.

6. The method for preparing nanovesicles for targeting metabolic and immune remodeling in gliomas as described in claim 5, characterized in that, In step S310, the mass ratio of the three lentiviral vector plasmids psPAX2, pMD2.G and pCDH-GFP-APT is 1:1.5:

2.

7. The method for preparing nanovesicles for targeting metabolic and immune remodeling in gliomas as described in claim 1, characterized in that, In step S300, the ROS-responsive nanoparticles II are encapsulated in a cell membrane containing APT using a polycarbonate membrane extrusion method.

8. The method for preparing nanovesicles for targeting metabolic and immune remodeling in gliomas as described in claim 1, characterized in that, In step S300, the amino acid sequence of the IgK signal peptide is shown in SEQ ID NO.1, the amino acid sequence of ANG is shown in SEQ ID NO.2, the amino acid sequence of the responsive cleavage site is shown in SEQ ID NO.3, the amino acid sequence of tLYP1 is shown in SEQ ID NO.4, and the amino acid sequence of the PDGFR transmembrane domain is shown in SEQ ID NO.

5.

9. A nanovesicle for targeting metabolic and immune remodeling in gliomas, characterized in that, It is prepared using the method for preparing nanovesicles for targeting metabolic and immune remodeling in gliomas as described in any one of claims 1 to 8.

10. An application of nanovesicles for targeting metabolic and immune remodeling in gliomas, characterized in that, A pharmaceutical formulation is prepared using the nanovesicles for targeting metabolic and immune remodeling in gliomas as described in claim 9, the pharmaceutical formulation being used for the treatment of glioblastoma.

Images