A kind of conjugated GO-203 and load DMAMCL lipid nano-carrier, preparation method and application thereof

Abstract

The application relates to a kind of lipid nanocarriers coupled with GO-203 and loaded with DMAMCL and its preparation method and application, belong to the field of biological medicine and nano-preparation.The lipid nanocarriers include water phase core, lipid bilayer and surface modification layer from inside to outside, wherein water-soluble dimethylamine methyl chloride (DMAMCL) is loaded in the water phase core;The lipid bilayer is wrapped in the water phase core, and coumarin-6 is embedded as fluorescent marker for tracing and detection;Functional modification layer is arranged on the outer surface of the lipid bilayer, including DSPE-PEG2000-COOH and DSPE-PEG2000-GO-203, wherein GO-203 is cell membrane penetrating peptide.The lipid nanocarrier provided by the application can realize the synergistic delivery of GO-203 and DMAMCL, be used for regulating tumor immune microenvironment and be used for the treatment of triple-negative breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanoparticle formulations, specifically relating to a lipid nanocarrier, its preparation method, and its application. More particularly, it relates to a lipid nanocarrier with surface-coupled GO-203 and loaded with DMAMCL, its preparation method, and its application in regulating the tumor immune microenvironment and treating triple-negative breast cancer. Background Technology

[0002] Triple-negative breast cancer (TNBC) is a highly heterogeneous subtype of breast cancer that lacks estrogen receptor, progesterone receptor, and HER2 expression. It is characterized by high invasiveness, high metastasis rate, high recurrence risk, and poor clinical prognosis. Currently, there are no clearly effective molecular targeted therapies for TNBC, and its treatment efficacy and patient survival remain limited.

[0003] Studies have shown that the formation of a tumor immunosuppressive microenvironment (TME) is one of the core reasons for the limited efficacy of TNBC immunotherapy. Tumor cells can evade immune surveillance by constructing an immunosuppressive "cold tumor microenvironment" (cTME), and the immunosuppressive microenvironment presented by TNBC is characterized by "multi-target synergistic effects and stronger inhibitory effects." Its formation mechanism is mainly reflected in the following aspects. First, tumor cells themselves and their secreted exosomes highly express programmed death ligand 1 (PD-L1), inhibiting T cell activation through the PD-1 / PD-L1 signaling axis, which is one of the key barriers to TNBC immune escape. Second, tumor-associated macrophages (TAMs) in the TNBC microenvironment tend to polarize towards the immunosuppressive M2 type, which is an important regulatory node for enhancing the immunosuppressive state. M2 TAMs can not only secrete a variety of immunosuppressive factors, but also directly inhibit CD8. + The proliferation and activation of T cells weaken their killing effect on tumor cells. Furthermore, the TNBC microenvironment is characterized by increased infiltration of regulatory T cells (Tregs) and aggregation of myeloid-derived suppressor cells (MDSCs). Simultaneously, tumor cells consume large amounts of glucose and produce lactate through the "Warburg effect," causing local metabolic immunosuppression. This, combined with the physical barrier formed by cancer-associated fibroblasts (CAFs) hindering immune cell infiltration, ultimately constitutes a highly stable and irreversible immunosuppressive network. Therefore, fundamentally reshaping the immunosuppressive microenvironment of TNBC and improving the response rate to immunotherapy are crucial technical challenges that urgently need to be addressed.

[0004] Currently, various lipid nanocarriers have been used for drug delivery and treatment of triple-negative breast cancer (TNBC). However, existing technologies mainly focus on enhancing drug accumulation in tumor tissue or improving cytotoxicity, with few reports on strategies to improve immunotherapy response rates by modulating the tumor immunosuppressive microenvironment in TNBC. Therefore, how to construct a lipid nanocarrier system that can effectively modulate the tumor immune microenvironment for TNBC treatment remains a pressing technical problem to be solved in this field. This invention proposes a lipid nanocarrier based on reshaping the tumor immunosuppressive microenvironment for the treatment of triple-negative breast cancer.

[0005] Building upon existing research, GO-203, a cell-penetrating polypeptide molecule, can target the mucin 1 cytoplasmic domain (MUC1-C), blocking the formation of MUC1-C homodimers and inhibiting the activation of related signaling pathways. Previous studies have shown that MUC1-C can promote the expression of programmed death ligand 1 (PD-L1) in tumor cells by activating the NF-κB p65 signaling pathway. Inhibiting MUC1-C-related signaling pathways with GO-203 can, to some extent, reduce the expression level of PD-L1 in tumor cells and their exosomes, and help improve the tumor immune microenvironment, such as promoting CD8+ expression. + T cells and other immune effector cells infiltrate tumor tissues, thereby enhancing the body's anti-tumor immune response. In addition, the decrease in PD-L1 expression may also affect the polarization state of tumor-associated macrophages (TAMs), thereby alleviating the tumor immunosuppressive environment to some extent.

[0006] On the other hand, the parthenolide derivative ACT001 significantly reduces PD-L1 expression levels in tumor cells by inhibiting the phosphorylation of signal transduction and transcription activator 3 (STAT3). ACT001 is the fumarate form of dimethylaminomethyl chloride (DMAMCL), which can slowly and continuously release MCL with antitumor activity in vivo. MCL can reduce tumor cell viability and induce apoptosis in a dose-dependent manner. Since the STAT3 signaling pathway is generally highly activated in TNBC, ACT001 shows good PD-L1 inhibitory potential in this tumor type.

[0007] Therefore, GO-203 and ACT001 can influence tumor cell-related immune regulation mechanisms through different signaling pathways, thereby downregulating PD-L1 expression levels to some extent. Decreased PD-L1 expression helps regulate the tumor immune microenvironment, such as affecting tumor-associated macrophage (TAM) polarization and promoting CD8+ expression. +T cells and other immune effector cells exert anti-tumor effects. Therefore, the combined application of GO-203 and ACT001 is expected to regulate the immunosuppressive microenvironment of triple-negative breast cancer (TNBC) at multiple levels, thereby providing a new technical approach to improve the treatment efficacy of TNBC.

[0008] Building upon this foundation, this technology further introduces lipid nanocarriers as a co-delivery system to achieve the synergistic delivery of GO-203 and DMAMCL. Due to their excellent biocompatibility and biodegradability, lipid nanocarriers have become one of the most widely studied nano-drug delivery systems. Their structure typically consists of bilayered spherical vesicles spontaneously formed by phospholipid molecules in an aqueous medium, possessing both hydrophilic and lipophilic characteristics, enabling them to efficiently encapsulate various types of active drugs.

[0009] During in vivo administration, via tail vein injection, the lipid nanocarrier passively accumulates at the tumor site due to the high permeability and retention effect (EPR effect) of tumor tissue. Simultaneously, GO-203 possesses cell-penetrating capabilities, promoting the entry of the nanocarrier and its loaded drug into tumor cells, thereby improving drug uptake efficiency. In this delivery system, the synergistic mechanism of GO-203 targeting and inhibiting the MUC1-C signaling pathway and ACT001 inhibiting STAT3 phosphorylation synergistically affects PD-L1 expression in tumor cells at different signaling pathway levels. Changes in PD-L1 expression levels may further influence the polarization state of tumor-associated macrophages (TAMs) and regulate CD8+. + The anti-tumor activity of immune effector cells such as T cells can improve the immunosuppressive microenvironment of triple-negative breast cancer (TNBC) to some extent.

[0010] Therefore, developing a lipid nanocarrier drug delivery system with surface-conjugated GO-203 and loaded with DMAMCL can achieve synergistic delivery of the above-mentioned active ingredients, providing a new technical solution for regulating the tumor immune microenvironment of TNBC and for the treatment of triple-negative breast cancer. Summary of the Invention

[0011] To address the shortcomings of existing technologies in the treatment of triple-negative breast cancer, this invention provides a lipid nanocarrier coupled with GO-203 and loaded with DMAMCL, along with its preparation method and applications. This lipid nanocarrier, through the synergistic delivery of GO-203 and DMAMCL, achieves regulation of the tumor immune microenvironment, thus providing a novel technical solution for the treatment of triple-negative breast cancer.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A lipid nanocarrier coupled with a cell membrane-penetrating peptide (GO-203) and loaded with dimethylaminomethyl chloride (DMAMCL) is a lipid nanocarrier for treating triple-negative breast cancer from the perspective of reshaping the tumor immunosuppressive microenvironment. The lipid nanocarrier mainly consists of three parts from the inside out: a core (aqueous phase) of DMAMCL, a lipid bilayer membrane, and a surface-modified layer with GO-203. Specifically, the aqueous phase contains water-soluble DMAMCL, which is encapsulated within the innermost layer of the lipid nanocarrier, effectively preventing degradation by the external environment. Encapsulating the core (aqueous phase) is a lipid bilayer structure with embedded coumarin-6 as a fluorescent label for tracking and detection. Two key components are embedded on the outer surface of the lipid bilayer: DSPE-PEG... 2000 -COOH provides long-cycle and stealth properties, while the carboxyl group (-COOH) can be used for further functionalization; the second is DSPE-PEG. 2000 GO-203, a transmembrane peptide, is modified on the surface of lipid nanocarriers, endowing them with the ability to target triple-negative breast cancer cells. After intravenous injection of this lipid nanocarrier suspension, the lipid nanocarriers accumulate at the tumor site through the EPR effect. Combined with the transmembrane effect of GO-203, the lipid nanocarriers cross the cell membrane to reach the tumor cells, releasing DMAMCL in the cytoplasm. GO-203 and DMAMCL synergistically inhibit the expression of PD-L1 in tumor cells and their exosomes, further inhibiting the differentiation of TAMs into M2-type cells, while simultaneously restoring CD8+ expression. + T cells kill tumor cells, thereby reshaping the immunosuppressive microenvironment and achieving good results.

[0013] A method for preparing a lipid nanocarrier coupled with GO-203 and loaded with DMAMCL includes the following steps: The first step is to combine GO-203 with DSPE-PEG. 2000 Coupling with -COOH yields DSPE-PEG. 2000 -GO-203 powder; Step 1.1) Dissolve NHS and EDC separately in DMSO, mix well to obtain EDC solution and NHS solution. Take DSPE-PEG 2000 -COOH is dissolved in fresh DMSO. First, EDC solution is added dropwise, followed by NHS solution, to obtain a mixed solution. The pH is then adjusted to between 4.0 and 6.5, and the solution is stirred in the dark for 30-40 minutes to obtain a solution containing DSPE-PEG. 2000 A solution of -NHS.

[0014] Furthermore, the concentration of the EDC solution is 1 mg / mL, and the concentration of the NHS solution is 1 mg / mL.

[0015] Furthermore, 23.94 mg of DSPE-PEG was added to each 1 mL of new DMSO. 2000 -COOH, 0.5-1.5 mL of EDC solution, and 0.5-1.5 mL of NHS solution.

[0016] Furthermore, the pH of the mixed solution was adjusted to between 4.0 and 6.5 using the remaining EDC and NHS solutions.

[0017] Furthermore, DMSO solvent can be replaced with DMF solvent.

[0018] Step 1.2) Dissolve GO-203 in DMSO and mix well to obtain a GO-203 solution. Then, take the DSPE-PEG obtained in step 1.1). 2000 -NHS solution was slowly added dropwise to the GO-203 solution, and the pH was adjusted to 7.2-8.5 with triethylamine. The solution was then stirred at room temperature in the dark for 36-60 hours to obtain DSPE-PEG. 2000 -GO-203 solution. Concentration was determined using a BCA kit. In this step, DSPE-PEG... 2000 The -NHS group reacts with the primary amino group (-NH2) of the GO-203 peptide to form a stable amide bond, thereby coupling GO-203 to DSPE-PEG. 2000 -NHS, to obtain DSPE-PEG 2000 A solution of -GO-203.

[0019] Furthermore, 10 mg of GO-203 and 1 mL of DSPE-PEG were added to each 1 mL of DMSO. 2000 -NHS solution.

[0020] Step 1.3) Dialyze the DSPE-PEG2000-GO-203 solution obtained in Step 1.2) with purified water for 36-60 hours, then freeze-dry. This yields DSPE-PEG. 2000 -GO-203 powder, and DSPE-PEG 2000 -GO-203 powder was characterized.

[0021] Furthermore, the characterization in step 1.3) includes detecting its coupling rate using a BCA kit and characterizing its characteristic peaks using Fourier transform infrared spectroscopy (FITR).

[0022] The second step is to prepare lipid nanocarriers. Step 2.1) Add egg yolk lecithin, cholesterol, and DSPE-PEG. 2000 -COOH,DSPE-PEG 2000-GO-203 powder was added to an organic solvent, followed by the addition of coumarin-6. The mixture was then transferred to a round-bottom flask and evaporated using a rotary evaporator until a homogeneous film was formed. In this step, egg yolk lecithin, cholesterol, and DSPE-PEG were also added. 2000 -COOH,DSPE-PEG 2000 -GO-203 powder and coumarin-6 undergo physical dissolution in an organic solvent. The solvent is evaporated by rotary evaporation, and the solute is physically deposited on the inner wall of the flask to form a uniform thin film. The entire process involves no covalent chemical reaction and relies solely on intermolecular forces to maintain the stability of the film structure.

[0023] Furthermore, organic solvents such as methanol and dichloromethane can be selected.

[0024] Furthermore, the egg yolk lecithin, cholesterol, and DSPE-PEG... 2000 -COOH,DSPE-PEG 2000 The mass ratio of GO-203 powder is 20-40:8-12:0.8-1.2:1.

[0025] Furthermore, the amount of coumarin-6 added is 0.5-1.5% of egg yolk lecithin.

[0026] Furthermore, the rotary evaporation temperature is 40~60℃.

[0027] Step 2.2) Dissolve DMAMCL in PBS and add it to the flask from Step 2.1). Shake continuously at 100-200 rpm on a 37°C shaker for 30 min-1 h until the membrane is completely detached from the flask wall.

[0028] Furthermore, each 10 mg of DMAMCL is added to 10-15 mL of PBS.

[0029] Step 2.3) At room temperature, the membrane detached in step 2.2) is subjected to sonication using a cell disruptor to obtain a relatively clear lipid suspension until the Tyndall effect is observed, and finally a crude lipid nanocarrier solution is obtained.

[0030] Furthermore, the ultrasonic power is 100-150W, and the duration is 10-15min.

[0031] Furthermore, the method for determining the Tyndall effect is as follows: a visible laser beam (such as a laser pointer) is shone into the system to be tested. If a bright "path" formed by the laser beam can be observed, it indicates that the system has the Tyndall effect; if no bright path is observed, then there is no Tyndall effect.

[0032] Furthermore, a lipid nanocarrier extruder can be used to extrude the relatively clear lipid suspension obtained by ultrasonic treatment multiple times to make its particle size more uniform.

[0033] Step 2.4) The crude lipid nanocarrier solution obtained in Step 2.3 was purified using a dextran gel G-50 chromatography column to obtain the lipid nanocarrier LP(D)-G, which was then characterized.

[0034] Furthermore, the characterization in step 2.4) includes: characterizing the morphology of the lipid nanocarrier using scanning electron microscopy (SEM) (Nova NanoSEM450), characterizing its particle size and potential using dynamic light scattering (DLS), and characterizing the drug release behavior of LP(D)-G DMAMCL using dialysis.

[0035] The application of a lipid nanocarrier coupled with cell membrane-penetrating peptide (GO-203) and loaded with dimethylaminomethyl chloride (DMAMCL) to treat triple-negative breast cancer from the perspective of reshaping the tumor immunosuppressive microenvironment.

[0036] The beneficial effects of this invention are as follows: (1) This invention provides a lipid nanocarrier based on remodeling the tumor immunosuppressive microenvironment for the treatment of triple-negative breast cancer. The lipid nanocarrier is aqueously loaded with DMAMCL and conjugated to the surface with the membrane-penetrating peptide GO-203. DSPE-PEG is also present. 2000 -COOH endows the lipid nanocarriers with "stealth" and "long-circulation" properties. Following intravenous injection, the nanoparticles accumulate at the tumor site via the EPR effect and enter tumor cells through the membrane-penetrating action of GO-203. Subsequently, DMAMCL is released into the cytoplasm. GO-203 and DMAMCL synergistically inhibit PD-L1 expression in tumor cells and their exosomes, reduce TAM differentiation into M2-type cells, and restore CD8+ expression. + The tumor-killing function of T cells is enhanced, thereby reshaping the immunosuppressive microenvironment and achieving significant effects.

[0037] (2) Preparation method of the lipid nanocarrier of the present invention: ① The coupling reaction is highly efficient and controllable, and the product is highly stable: GO-203 and DSPE-PEG2000-COOH are coupled by EDC / NHS activation method. Specific binding is achieved by adjusting the reaction parameters. After purification, lyophilization and FTIR characterization, the product has high purity, stable structure, and the coupling rate can be accurately detected with good repeatability.

[0038] ② The lipid membrane preparation process is simple and mild, with no side reactions: The rotary evaporation method for preparing lipid membranes involves only physical changes without side reactions; the key process parameters are clear, the operation is simple and highly controllable, and it is suitable for large-scale preparation.

[0039] ③ Synergistic optimization of drug loading and nanoforming, resulting in excellent carrier performance: DMAMCL loading and ultrasonic nanoforming are synergistically optimized, the Tyndall effect ensures dispersibility, an optional extrusion step optimizes particle size, and chromatographic purification can remove impurities, resulting in excellent carrier performance.

[0040] ④ A comprehensive characterization system can fully verify the quality of the vector: A complete characterization system has been established, with corresponding characterization methods from the coupling product to the final vector, which can fully verify the quality of the vector and provide data support for subsequent applications.

[0041] ⑤ The materials are reasonably selected and suitable for biomedical applications: the materials have good biocompatibility and low toxicity, DSPE-PEG2000 modification improves stability, and GO-203 gives them targeting potential, making them suitable for biomedical applications and promising. Attached Figure Description

[0042] Figure 1 The coupling rate of GO-203 was detected using the BCA kit.

[0043] Figure 2 This is a qualitative diagram of GO-203 coupling detected by infrared spectroscopy.

[0044] Figure 3 The image shows the morphology of lipid nanocarriers as observed using scanning electron microscopy. Figure 3 (a) in the figure is a morphological diagram of the lipid nanocarrier LP; Figure 3 (b) in the figure is a morphological diagram of the lipid nanocarrier LP-G; Figure 3 (c) in the figure is a morphological diagram of the lipid nanocarrier LP(D); Figure 3 (d) in the figure is a morphology diagram of the lipid nanocarrier LP(D)-G.

[0045] Figure 4 This is a particle size distribution obtained using dynamic light scattering. Figure 4 (a) in the figure is the particle size distribution of the lipid nanocarrier LP; Figure 4 (b) in the figure is the particle size diagram of the lipid nanocarrier LP-G; Figure 4 (c) in the figure is the particle size diagram of the lipid nanocarrier LP(D); Figure 4 (d) in the figure represents the particle size distribution of the lipid nanocarrier LP(D)-G.

[0046] Figure 5 The 7-day particle size distribution, obtained using dynamic light scattering, is used to demonstrate its stability.

[0047] Figure 6 The potential of the lipid nanocarrier was detected using dynamic light scattering.

[0048] Figure 7 The drug release curve of DMAMCL was detected by dialysis.

[0049] Figure 8 CCK-8 results for DMAMCL and its lipid nanocarriers.

[0050] Figure 9 The results for CCK-8 assays of GO-203 and its lipid nanocarriers are shown.

[0051] Figure 10 The cellular uptake results of lipid nanocarriers are shown. The horizontal distributions of LP, LP-G, LP(D), and LP(D)-G represent the groups of lipid nanocarriers, while the vertical distributions of DAPI, Cou-6, and Merge represent the staining labels and fusion images: DAPI: blue fluorescence, used to label cell nuclei, indicating cell location and number. Cou-6: green fluorescence, used to label lipid nanocarriers, indicating the distribution and uptake of the carriers within cells. Merge: a fusion image of DAPI and Cou-6, which visually shows the intracellular localization of the nanocarriers. For example, the first image in the upper left corner is the DAPI staining image of the LP group, showing only the blue fluorescence of the cell nuclei in this group.

[0052] Figure 11 The effects of lipid nanoparticle carrier treatment on the expression of proteins such as MUC1, NF-κB p65, PD-L1, STAT3, and p-STAT3 were investigated. Figure 11(a) shows the Western blot results of MUC1 and dimer (MUC1 (Dimers)) protein expression, with GAPDH and β-Tubulin as internal controls, used to evaluate the effects of different treatment groups (Con, GO-203, LP-G, LP(D), LP(D)-G) on MUC1 protein expression and dimerization; Figure 11(b) shows the Western blot results of NF-κB p65 and PD-L1 protein expression, with GAPDH as the internal control, used to analyze the regulatory effects of different treatment groups on the NF-κB pathway and the expression of the immune checkpoint molecule PD-L1; Figure 11(c) shows the Western blot results of p-STAT3 (phosphorylated STAT3), total STAT3 and PD-L1 protein expression, with GAPDH as the internal control, used to evaluate the effects of different treatment groups on JAK / STAT3 signaling pathway activation and PD-L1 expression.

[0053] Figure 12 Scanning electron microscope image of exosomes derived from 4T1.

[0054] Figure 13 This is a particle size distribution of exosomes derived from 4T1.

[0055] Figure 14 The protein expression status of exosomes derived from 4T1.

[0056] Figure 15 The protein expression of RAW264.7 after co-culturing 4T1 and RAW264.7.

[0057] Figure 16 This is a tumor quality graph for each group after treatment.

[0058] Figure 17 The tumor volume diagrams for each group are shown.

[0059] Figure 18 This image shows the Ki67 expression detected by immunofluorescence. The horizontal distribution of PBS, GO-203, DMAMCL, LP-G, LP(D), and LP(D)-G represents different treatment groups, while the vertical distribution of DAPI, Ki67, and Merge represents the staining markers and fusion images. Specifically, DAPI (blue fluorescence) labels the cell nucleus, indicating cell location and number; Ki67 (green fluorescence) labels the proliferation-related protein Ki67, indicating cell proliferation activity; and Merge is a fusion image of DAPI and Ki67, visually showing the distribution and proportion of proliferating cells in the overall cell population. The first image in the upper left corner shows the DAPI staining image of the PBS group, used to label the cell nucleus and indicate cell location and number.

[0060] Figure 19 shows the PD-L1 expression as detected by immunofluorescence. The horizontal distribution of PBS, GO-203, DMAMCL, LP-G, LP(D), and LP(D)-G represents different treatment groups, while the vertical distribution of DAPI, PD-L1, and Merge represents the staining markers and fusion images. DAPI: blue fluorescence, marking cell nuclei to indicate cell location and number. PD-L1: green fluorescence, marking the immune checkpoint protein PD-L1 to indicate its expression level and distribution in cells. Merge: a fusion image of DAPI and PD-L1, visually showing the location and expression intensity of PD-L1 in cells. The first image in the upper left corner shows the DAPI staining image of the PBS group, used to mark cell nuclei and indicate cell location and number.

[0061] Figure 20 To detect M1 macrophages and CD8+ in tumor tissues of each group by flow cytometry + The proportion of T cells; Figure 20 (a) represents the proportion of M1 macrophages in each group of tumor tissues; Figure 20 (b) in the figure represents the CD8 concentration in tumor tissues of each group. +The proportion of T cells. The horizontal axis represents different treatment groups: PBS, GO-203, DMAMCL, LP-G, LP(D), and LP(D)-G. The vertical axis represents: (a) Figure: Analysis of co-stimulatory molecule expression in dendritic cells or macrophages, with CD86 as the vertical axis and CD80 as the horizontal axis. (b) Figure: Analysis of T lymphocyte subset proportions, with CD8+ as the vertical axis and CD4+ as the horizontal axis.

[0062] Figure 21 This image shows iNOS expression as detected by immunofluorescence, used to assess the effects of different treatment groups on macrophage activation and nitric oxide production: Horizontal axis: PBS, GO-203, DMAMCL, LP-G, LP(D), LP(D)-G represent different treatment groups; Vertical axis: DAPI: blue fluorescence, marking cell nuclei and indicating cell location and number; iNOS: green fluorescence, marking inducible nitric oxide synthase, indicating the degree of macrophage M1 activation and NO production potential; Merge: fusion image of DAPI and iNOS, visually showing the location and expression intensity of iNOS in cells. The first image in the upper left corner is a DAPI staining image of the PBS group, used to mark cell nuclei and indicate cell location and number.

[0063] Figure 22 This image shows CD8 expression as detected by immunofluorescence, used to assess the effects of different treatment groups on the infiltration and activation of cytotoxic T cells (CD8+ T cells). Horizontally: PBS, GO-203, DMAMCL, LP-G, LP(D), and LP(D)-G represent different treatment groups. Vertically: DAPI: blue fluorescence, marking cell nuclei and indicating cell location and number; CD8: green fluorescence, marking CD8 molecules and indicating the distribution and number of cytotoxic T cells; Merge: fusion image of DAPI and CD8, visually showing the infiltration of CD8+ T cells in tissues. The first image in the upper left corner shows the DAPI staining image of the PBS group, used to mark cell nuclei and indicate cell location and number.

[0064] Figure 23 This is a bar chart showing the detection of cytokines and cytotoxic molecules, used to evaluate the effects of different treatment groups (PBS, GO-203, DMAMCL, LP-G, LP(D), LP(D)-G) on immune activation and antitumor immune responses. Figure 23 (a) in the text represents TNF-α (tumor necrosis factor-α). Figure 23 (b) in the text refers to IL-6 (interleukin-6). Figure 23 (c) in the text refers to IFN-γ (interferon-γ). Figure 23(d) in the text represents Gzm B (granulase B).

[0065] Figure 24 This is a bar chart showing the results of complete blood count and liver and kidney function tests, used to evaluate the effects of different treatment groups (PBS, GO-203, DMAMCL, LP-G, LP(D), LP(D)-G) on the body's hematopoietic system and liver and kidney function: Figure 24 (a) in the text represents WBC (white blood cells); Figure 24 (b) in the text represents RBCs (red blood cells); Figure 24 (c) in the text refers to PLT (platelets); Figure 24 (d) in the text represents HGB (hemoglobin); Figure 24 (e) in the text represents ALT (alanine aminotransferase). Figure 24 (f) in the text represents AST (aspartate aminotransferase). Figure 24 (g) in the text refers to Urea (urea); Figure 24 (h) in the text stands for CREA (creatinine). Detailed Implementation

[0066] The present invention will be further described below with reference to specific embodiments.

[0067] Example 1 The first step was to synthesize LP(D)-G lipid nanocarriers. Step 1.1) Activate DSPE-PEG 2000 -COOH Dissolve 10 mg each of NHS and EDC in 1 mL of DMSO, and simmer until fully dissolved. Prepare a 10 mg / mL solution. Take DSPE-PEG... 2000 Dissolve 23.940 mg (0.023940 g) of COOH in 650 μL of DMSO, and vortex until fully dissolved. Add DSPE-PEG. 2000 Add -COOH to a 10mL round-bottom flask, add 750μL of EDC, then add 600μL of NHS. Measure the pH of the solution using a pH meter to ensure it is between 4.0 and 6.5 (ideally 5.0-6.0). If not, slowly add small amounts of EDC (acidic) and NHS (weakly alkaline) under stirring to adjust the pH. Once the pH is satisfactory, stir in the dark and activate for 30 minutes to obtain DSPE-PEG. 2000 -NHS.

[0068] Step 1.2) DSPE-PEG 2000 -NHS and GO-203 combined Take another 10mL round-bottom flask, dissolve 10mg of GO-203 in 1mL of DMSO, and simmer until fully dissolved. Add the DSPE-PEG prepared in step 1.1). 2000 -NHS was slowly added dropwise to GO-203 under light-protected stirring. After the addition was complete, the pH value was measured to ensure it was near 8. If not, triethylamine was used to adjust the pH. After pH adjustment, the reaction was carried out under light-protected stirring for 48 hours. Approximately every 8 hours, 10 μL of the sample was taken, diluted 10-fold with 90 μL of DMSO, and 20 μL was taken for BCA method to determine the concentration of free GO-203 in the solution (two additional parallel experiments were conducted, totaling 180 μL). The reaction endpoint is reached when the GO-203 concentration stops decreasing. The concentration of GO-203 can be determined from the standard curve. The results are as follows... Figure 1 As shown: In the first 0-6 hours: the coupling rate rises rapidly from 0% to about 70%, indicating a fast initial reaction rate; in the second 6-36 hours: it continues to increase slowly, reaching about 80% around 36 hours; in the third 36-48 hours: the curve flattens out, and the coupling rate stabilizes at around 80%, indicating that the reaction has basically reached equilibrium or saturation.

[0069] BCA method for measuring GO-203 concentration: Prepare a suitable working solution (A:B = 50:1) by mixing 1000 μL of BCA reagent A with 20 μL of BCA reagent B, and then pipette to mix thoroughly. Add 20 μL of diluted test sample to the sample wells (when adding reagents to the wells, touch the bottom of the plate; add slowly to avoid generating air bubbles). For the blank control group, add 20 μL of DMSO reagent, followed by vertically suspending 200 μL of BCA working solution. On the microplate reader, vortex to mix for 20 seconds. Cover with a membrane and incubate at 37°C for 30 minutes. Measure the OD value at 562 nm using the microplate reader. Compare with the standard curve and calculate the concentration.

[0070] Step 1.3) Dialysis purification of DSPE-PEG 2000 -GO-203, and freeze-dried Cut the dialysis bag to a suitable length using scissors, then place it in distilled water and boil in a water bath for 30 minutes to activate the bag. During boiling, continuously stir the distilled water to prevent the bottom temperature from becoming too high and damaging the bag. Remove the dialysis bag from the distilled water, select a suitable dialysis bag clamp, fold it, and clamp the bottom of the bag. Next, open the top of the dialysis bag and use a dropper to add the solution to be dialyzed into the bag. Remove any air from the bag, fold the dialysis clamp to secure the top of the bag, leaving about 1 / 3 of the volume above the liquid level to prevent the bag from bursting due to increased solution volume during dialysis. After loading the sample into the dialysis bag, place it in 1L of distilled water for dialysis. During dialysis, add a stir bar to the beaker and place it on a magnetic stirrer. Change the distilled water every 1, 2, 4, 8, 12, and 24 hours. After dialysis is complete, remove the sample and place it in a beaker, ensuring the thickness is no more than 0.7 cm. Seal the beaker with sealing film and freeze at -20°C overnight.

[0071] After it has completely frozen into a solid, freeze dry it using a freeze dryer.

[0072] Step 1.4) Infrared spectroscopy characterization and identification Power on the instrument and wait 20 minutes for it to warm up. Set the wavenumber range to 3900 to 400 cm⁻¹. -1 Between [times], 16 scans were performed at a resolution of 4. 1-2 mg of sample was finely ground and mixed with 100 mg of pure KBr, then pressed into transparent tablets on a tablet press (pressure increased to 18 MPa, waited 2 min) for determination. Simultaneously, the raw material DSPE-PEG was used... 2000 -NHS, SPDE-PEG 2000 The infrared spectrum of -COOH is used as a reference, and the result is shown in the figure below. Figure 2 As shown: DSPE-PEG 2000 -GO-203 at 1630–1650 cm -1 A new strong absorption peak appeared nearby, which is a characteristic signal of the amide bond (-CONH-). The presence of the amide bond directly proves DSPE-PEG. 2000 -COOH and GO-203 were successfully coupled via an amidation reaction.

[0073] Step 1.5) Prepare lipid nanocarriers using thin-film hydration method Accurately weigh 30mg egg yolk lecithin, 10mg cholesterol, and 10mg DSPE-PEG. 2000 -COOH, 10mg DSPE-PEG 2000 -GO-203 and 10μg of coumarin-6 were dissolved together in 10mL of chloroform and added to a 100mL round-bottom flask.

[0074] Assemble the flask into a rotary evaporator and use a water bath at 50°C, a vacuum of approximately -0.08 MPa, and a rotation speed of approximately 80-120 rpm for rotary evaporation. Continue evaporating until the solvent evaporates completely and a uniform lipid film forms on the inner wall of the flask.

[0075] Hydration: DMAMCL was dissolved in PBS solution and preheated to 50°C. This solution was then added to a round-bottom flask and incubated in a 50°C water bath until a uniform lipid film detached from the flask wall. This resulted in the synthesis of lipid nanocarriers with relatively large particle sizes.

[0076] Homogenization: Add the hydrated mixed solution to a 15mL centrifuge tube and sonicate in an ice-water bath using a cell disruptor. Parameter settings: power 100W, turn on for one second and off for three seconds, run for a total of 15 minutes until the particle size is around 100nm, then stop sonicating.

[0077] Characterization of lipid nanocarriers: (1) The morphological characteristics of the lipid nanocarriers were observed using scanning electron microscopy, such as... Figure 3 As shown: LP is prone to aggregation and has poor dispersibility; GO-203 surface-modified LP-G and LP(D)-G), GO-203 improves dispersibility and reduces aggregation, but makes the particle surface rough; LP(D) loaded with DMAMCL maintains both good dispersibility and surface smoothness; in the final group LP(D)-G, the morphology and dispersibility of the nanocarrier were not significantly damaged after drug loading, proving that lipid nanocarriers have good drug loading stability.

[0078] (2) Dynamic light scattering (DLS) was used to detect the particle size of the lipid nanocarriers, such as... Figure 4 As shown. Particle size variation: From LP to LP-G, LP(D), and then to LP(D)-G, the particle size increases to a certain extent, which is consistent with the structural changes caused by GO-203 modification and drug DMAMCL loading; Distribution uniformity: LP(D)-G has the narrowest particle size distribution, indicating that GO-203 modification improves dispersibility.

[0079] (3) The particle size of the lipid nanocarrier was continuously measured for 7 days using dynamic light scattering (DLS) to obtain its stability, such as... Figure 5 As shown in the figure, the stability from strongest to weakest is: LP(D)-G, LP-G, LP(D), LP. This indicates that the introduction of GO-203 significantly improves the particle size stability of the lipid nanocarrier and effectively inhibits particle size growth during storage. Regarding drug loading: drug loading alone (LP(D)) slightly increases the particle size, but the GO-203-modified drug carrier LP(D)-G exhibits the best stability, indicating that this lipid nanocarrier has good application potential.

[0080] (4) Dynamic light scattering (DLS) was used to detect the potential of the lipid nanocarrier, such as... Figure 6 As shown in the figure, the potentials of each lipid nanocarrier are negative, indicating a certain degree of stability.

[0081] (5) The in vitro drug release behavior of DMAMCL in lipid nanocarrier LP(D)-G was detected by dialysis. Specifically, this lipid nanocarrier has the following characteristics: ① pH responsiveness: The DMAMCL carrier exhibits obvious pH-dependent release behavior, with a faster release rate in stronger acidity. This characteristic makes it very suitable as a carrier for tumor-targeted drugs; ② Tumor-targeting advantage: The drug can be effectively released in the tumor microenvironment (pH 6.5) and intracellular lysosomes (pH 5.3), while remaining stable in normal tissues (pH 7.4), achieving the goal of "targeted release and reduced systemic toxicity"; ③ Application potential: This pH-responsive release characteristic provides important experimental evidence for the application of DMAMCL in precision tumor treatment.

[0082] The sample preparation method for scanning electron microscopy is as follows: Lipid nanocarrier samples were diluted with pure water at three gradients: 10, 100, and 1000 times. Glutaraldehyde fixative was added at a ratio of sample to glutaraldehyde fixative of 1:1, mixed thoroughly, and allowed to stand for 5 minutes. A single-sided polished silicon wafer was taken, with the "mirror side" facing upwards. 10 μL of the fixed sample was added to the mirror side of the wafer and placed at room temperature until dry. Then, the wafer was immersed in a 50% methanol solution for 15 minutes; followed by immersion in a 60% methanol solution for 15 minutes; then in a 70% methanol solution for 15 minutes; then in an 80% methanol solution for 15 minutes; then in a 90% methanol solution for 15 minutes; then in a 100% methanol solution for 15 minutes; and finally in a 100% methanol solution for 15 minutes. The 100% methanol solution was used twice. The methanol solution was prepared by mixing anhydrous methanol and pure water in a volume ratio.

[0083] The method for detecting drug release by dialysis is as follows: Three pH PBS solutions (7.4, 6.5, and 5.3) are prepared in advance. PBS at pH 7.4 simulates the normal tissue environment; PBS at pH 6.5 simulates the tumor tissue environment; and PBS at pH 5.3 simulates the lysosomal environment of tumor cells. Dialysis bags with a molecular weight cutoff of 8000-14000 are used and activated by soaking in pure water. Equal amounts of lipid nanocarriers are placed into the corresponding dialysis bags and then placed in PBS at the specific pH values. The solution is continuously shaken at 37°C and 100 rpm, and the drug content in the dialysate is measured at specific time points of 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 hours. The percentage of drug release is then calculated.

[0084] The second step is to study the in vitro antitumor effects of lipid nanocarriers. Step 2.1) Efficacy of in vitro combined therapy with lipid nanocarriers quantified using DMAMCL Four T1 cells were seeded into 96-well plates, with 8000-10000 cells per well, and a cell-free control group was set up. The cells were incubated overnight at 37°C in a CO2 incubator until they adhered. The next day, the culture medium was discarded, and different concentrations of DMAMCL, LP(D), and LP(D)-G culture medium suspensions (0 μM, 0.78 μM, 1.56 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM) were added to the wells. After incubation for 24 hours, the old culture medium was removed, and fresh culture medium containing 10% CCK-8 was added. The cells were cultured for another 1 hour. The absorbance (OD) value was measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = [(OD)] 样品 -OD 空白 ) / (OD 对照 -OD 空白 )】×100%; The results are as follows Figure 8 As shown.

[0085] from Figure 8 It can be seen that DMAMCL has a concentration-dependent effect on 4T1 tumor cells: low doses promote cell growth, while high doses inhibit proliferation. In contrast, LP(D) only showed slightly better cytotoxicity than DMAMCL. LP(D)-G showed the strongest tumor-killing effect, and when the concentration reached 100 μM, it almost completely inhibited the survival of tumor cells, demonstrating the best anti-tumor performance.

[0086] Step 2.2) Quantification of the in vitro combined therapeutic effect of lipid nanocarriers using GO-203 Four T1 cells were seeded into 96-well plates, with 8000-10000 cells per well, and a cell-free control group was set up. The cells were incubated overnight at 37°C in a CO2 incubator until adhesion. The next day, the culture medium was discarded, and different concentrations of GO-203, LP-G, and LP(D)-G culture medium suspensions (0 μM, 1 μM, 3 μM, 5 μM, 7 μM, 9 μM) were added to the wells. After incubation for 24 hours, the old culture medium was removed, and fresh culture medium containing 10% CCK-8 was added. The cells were cultured for another 1 hour. The absorbance (OD value) was measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = [(OD)] 样品 -OD 空白 ) / (OD 对照 -OD 空白)】×100%; The results are as follows Figure 9 As shown.

[0087] from Figure 9 It can be seen that: GO-203 exhibited some cytotoxicity in 4T1 tumor cells; however, its killing effect tended to stabilize when the drug concentration exceeded 5 μM, possibly due to the lack of a drug delivery system. In stark contrast, both LP-G and LP(D)-G formulations showed significant dose-dependent inhibitory effects, with LP(D)-G performing particularly well. At a dosage concentration of 9 μM, LP(D)-G almost completely blocked tumor cell proliferation, demonstrating superior anticancer activity.

[0088] Step 2.3) Specific targeting ability of lipid nanocarrier LP(D)-G: 4T1 cells were incubated overnight in 24-well plates. The next day, the culture medium was removed, and different concentrations of LP, LP-G, LP(D), and LP(D)-G suspensions (5 μg / mL, quantified as coumarin-6) were added to the wells. After incubation for another 4 hours, the original culture medium was discarded, and 4% paraformaldehyde fixative was added to fix the cell morphology. The cell nuclei were then stained with DAPI solution, followed by the addition of an anti-fluorescence quencher, and the cells were mounted. Uptake was analyzed using laser confocal microscopy to infer the specific targeting ability.

[0089] The results are as follows Figure 10 As shown.

[0090] from Figure 10 It can be seen that the unmodified lipid nanocarriers already possess cellular uptake capabilities; however, after surface modification with GO-203, their uptake efficiency is significantly enhanced. This phenomenon indicates that lipid nanocarriers can not only enhance drug accumulation within cells, but also further amplify this effect through the transmembrane properties of GO-203, thereby significantly improving drug cytotoxicity.

[0091] The third step is to study the in vitro immunogenicity of lipid nanocarriers. Step 3.1) Investigation into the mechanism by which lipid nanocarriers inhibit PD-L1 in cells Four T1 cells were seeded into 6-well plates, with 500,000 cells per well. The plates were incubated overnight at 37°C in a CO2 incubator until the cells reached 50% cell growth. The next day, the culture medium was discarded, and PBS, GO-203, LP-G, LP(D), and LP(D)-G culture medium suspensions were added to the wells, respectively. The concentration of GO-203 was 5 μM, and the concentration of DMAMCL was 50 μM. After incubation for 24 hours, the old culture medium was removed, protein extraction was performed, and Western blotting was conducted.

[0092] The results are as follows Figure 11 As shown.

[0093] from Figure 11 It can be seen that GO-203 can effectively block the dimerization process of MUC1-C; in comparison, the LP-G treatment group showed a slightly better inhibitory effect; while LP(D)-G showed the most significant inhibitory effect on MUC1-C dimerization. Meanwhile, when GO-203 blocks the dimerization process of MUC1-C, it can effectively reduce the level of NF-κB p65, thereby significantly downregulating PD-L1 expression. In comparison, the LP-G combination showed a slightly better inhibitory effect, while LP(D)-G showed the strongest PD-L1 inhibitory ability. Furthermore, LP(D) can effectively weaken the phosphorylation of STAT3, thereby reducing the production of PD-L1; coincidentally, GO-203 and LP-G also showed similar inhibitory effects; it is particularly noteworthy that LP(D)-G showed the most outstanding performance in blocking STAT3 phosphorylation and downregulating PD-L1 expression.

[0094] Step 3.2) Lipid nanocarriers inhibit exosomal PD-L1 Four T1 cells were cultured in 10 cm dishes using complete culture medium prepared with exosome-free serum until they reached the logarithmic growth phase. Exosomes were then extracted using differential centrifugation. The specific steps are as follows: Take the original cell culture medium, centrifuge at 300g and 4℃ for 10min, and collect the supernatant; Take the supernatant from the previous step, centrifuge at 2000g and 4℃ for 10 minutes, and take the supernatant. Take the supernatant from the previous step, centrifuge at 10,000g and 4℃ for 30 minutes, and take the supernatant. Take the supernatant from the previous step, centrifuge at 100,000g and 4℃ for 70 minutes, and collect the precipitate, which is the exosome.

[0095] The precipitate was resuspended in 100 μL of PBS and stored at -80°C.

[0096] The morphology was characterized by scanning electron microscopy (SEM); the particle size was detected by dynamic light scattering (DLS); and the expression of its marker protein and PD-L1 was detected by Western blotting.

[0097] Scanning electron microscopy results as follows Figure 12 As shown; dynamic light scattering results are as follows Figure 13 Indicates; Western blotting experiment as Figure 14 express.

[0098] The results show that scanning electron microscopy reveals a typical cup-shaped structure, and dynamic light scattering analysis indicates an average diameter of approximately 140 nm. Furthermore, Western blotting experiments detected the expression of exosome-specific marker proteins ALIX, TSG101, and CD63, confirming successful exosome isolation. We then further examined the expression level of PD-L1 protein on the cell surface. Experimental data showed that, compared to the control group, all other experimental groups exhibited varying degrees of downregulation in PD-L1 expression.

[0099] Step 3.3) Investigation of the in vitro immunomodulatory effects of lipid nanocarriers 4T1 cells were cultured in 10cm dishes, and PBS, GO-203, LP-G, LP(D), and LP(D)-G culture medium suspensions were added, respectively. The concentration of GO-203 was 5 μM, and the concentration of DMAMCL was 50 μM. After incubation for 24 h, the original culture medium was mixed with fresh culture medium at a 1:1 ratio and added to RAW264.7 macrophage cells, and incubated for another 24 h. After 24 h, proteins were extracted from RAW264.7 cells and Western blot experiments were performed.

[0100] Western blotting results are as follows: Figure 15 express.

[0101] The results show that, compared to the M0 group, the expression level of Arg1, a marker of M2 polarization, was significantly increased in RAW264.7 cells of the control group, while the expression level of iNOS, a marker of M1 polarization, did not show a significant change. Notably, after intervention with the drug and lipid nanocarrier in each group, the expression level of iNOS increased in all samples, with the LP(D)-G treatment group showing particularly significant improvement; simultaneously, the expression of Arg1 gradually decreased. These in vitro results confirm that the lipid nanocarrier LP(D)-G has the ability to convert M2 macrophages into M1 macrophages, thereby exerting an immunomodulatory function.

[0102] Step 4: In vivo experimental results of lipid nanocarriers Step 4.1) In vivo antitumor effect of lipid nanocarriers 4T1 cells (5×10) 5 100 μL of PBS suspension was subcutaneously injected into the left hind limb of each mouse to induce tumor formation. Tumors were induced when the tumor volume reached 50 mm². 3Mice were randomly divided into 6 groups of 7 mice each. (1) PBS; (2) GO-203; (3) DMAMCL; (4) LP-G; (5) LP(D); (6) LP(D)-G. The drugs were administered on days 1, 3, 5, 7, and 9. Tumor volume was recorded every two days. After the experiment, all mice were euthanized, and the tumors were removed and weighed. Tumor mass was as follows: Figure 16 As shown. Figure 17 The graphs showing the changes in tumor volume in each group of mice show that, compared with the PBS control group, the other experimental groups all showed varying degrees of tumor growth inhibition, with the LP(D)-G group exhibiting the most significant tumor-inhibiting effect.

[0103] Then, tumor tissue was taken for paraffin sectioning, and the expression of PD-L1 and Ki67 proteins in the tumor tissue was detected using immunofluorescence technology. Figure 18 and 19 As shown, the LP(D)-G treatment group was able to significantly reduce both PD-L1 expression and Ki67 positivity rate, suggesting that this therapy may achieve synergistic anti-tumor effects through a dual mechanism of "blocking proliferation + relieving immune escape".

[0104] Step 4.2) In vivo immunotherapy effect of lipid nanocarriers This patent uses flow cytometry and tissue immunofluorescence techniques to conduct in-depth analysis of the effects of immunotherapy.

[0105] like Figure 20 , 21 As shown in Figure 22, compared with the PBS group, the expression ratios of CD80 and CD86 in all treatment groups were significantly increased. Among them, the expression ratios of CD80 and CD86 in the LP(D)-G treatment group reached 65.4%, significantly enhancing its tumor cell killing effect. Immunofluorescence results also verified this finding. Compared with the PBS group, the expression ratios of CD80 and CD86 in all treatment groups were significantly increased. + The proportion of T cells increased in all groups. Specifically, the CD8+ group in the LP(D)-G treatment group showed an increase. + The proportion of T cells was the highest, reaching 43.5%. Immunofluorescence results further confirmed this finding. CD8 + T cells are the core effector cells in the immune system responsible for specific cell killing. They can directly kill tumor cells through the perforin and granzyme B pathways, and can also indirectly inhibit tumor cell growth by secreting cytokines such as IFN-γ and TFN-α.

[0106] To further verify M1 type TAMs and CD8 + This patent demonstrates the killing effect of immune cells such as T cells on tumor cells using ELISA experiments on tumor tissue, thereby proving the immune-mediated cytokine-mediated promotion of immune function. Figure 23As shown in Figure EFGH, TNF-α (tumor necrosis factor α) is mainly secreted by macrophages and T cells. It is a pro-inflammatory cytokine involved in anti-infection and anti-tumor immunity, and also mediates inflammatory responses. IL-6 (interleukin-6) is a multifunctional cytokine that can promote inflammatory responses, activate immune cells, and participate in the proliferation and differentiation of immune cells, serving as a key hub for inflammation and immune regulation. IFN-γ (interferon-γ) is mainly secreted by activated T cells and NK cells. It is an important antiviral and anti-tumor cytokine that can enhance the phagocytic and killing ability of macrophages and regulate adaptive immunity. Gzm B (granulase B) is mainly found in the cytotoxic granules of cytotoxic T cells (CTLs) and NK cells. It is a core effector molecule mediating target cell apoptosis and directly participates in the specific killing of tumor or virus-infected cells. Figure EFGH shows that, compared with the PBS group, other treatment groups significantly promoted the secretion of TNF-α, IL-6, IFN-γ, and Gzm B. These treatment groups significantly enhanced the body's innate and adaptive immune responses by upregulating pro-inflammatory cytokines (TNF-α, IL-6), antiviral / antitumor cytokines (IFN-γ), and cytotoxic effector molecules (Gzm B). Among them, LP(D)-G showed the best immune activation effect in the treatment groups.

[0107] Step 4.3) Biosafety assessment of lipid nanocarriers A good treatment regimen should be both safe and effective. Based on the excellent results achieved in previous research regarding tumor treatment efficacy and the remodeling of the tumor immunosuppressive microenvironment, this patent further explores its safety, such as… Figure 24 As shown.

[0108] Blood routine tests showed no significant changes in any indicators except for a slight decrease in white blood cell (WBC) count. The slight decrease in WBC count may suggest that rapid tumor growth caused some damage to the mice, and this damage was alleviated after treatment, indirectly reflecting a good in vivo anti-tumor effect. Liver function tests showed that, compared with the PBS control group, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in each treatment group were not significantly increased, indicating that the treatment did not cause significant liver damage. Kidney function tests showed that the levels of creatinine (CREA) and urea (UREA) in each treatment group were not significantly different from the control group, indicating that the treatment did not cause significant kidney damage.

[0109] Example 2 LP lipid nanocarriers were prepared using a thin-film hydration method. Step 1: Accurately weigh 30mg egg yolk lecithin, 10mg cholesterol, and 10mg DSPE-PEG. 2000-COOH, 10μg coumarin-6, dissolved together in 10mL chloroform, and added to a 100mL round-bottom flask.

[0110] The second step involves assembling the flask into a rotary evaporator and using a water bath at 40°C, a vacuum of approximately -0.08 MPa, and a rotation speed of approximately 80-120 rpm for rotary evaporation. This continues until the solvent evaporates completely and a uniform lipid film adheres to the inner wall of the flask.

[0111] The third step is hydration: preheating to 40°C. The solution is then added to a globular flask and placed in a 40°C water bath until a uniform lipid film detaches from the flask wall. This completes the synthesis of lipid nanocarriers with relatively large particle sizes.

[0112] Step 4, homogenization: Add the hydrated mixed solution to a 15mL centrifuge tube and sonicate in an ice-water bath using a cell disruptor. Parameter settings: power 150W, turn on for one second and off for three seconds, run for a total of 10 minutes, until the particle size is around 100nm, then stop sonicating.

[0113] The fifth step involves observing the morphology of the lipid nanocarriers using scanning electron microscopy; detecting the particle size of the lipid nanocarriers using dynamic light scattering (DLS); continuously detecting the particle size of the lipid nanocarriers using dynamic light scattering (DLS) for 7 days to obtain their stability; and detecting the potential of the lipid nanocarriers using dynamic light scattering (DLS).

[0114] Example 3 The first step was to synthesize LP-G lipid nanocarriers. Step 1.1) Activate DSPE-PEG 2000 -COOH Dissolve 10 mg each of NHS and EDC in 1 mL of DMSO, and simmer until fully dissolved. Prepare a 10 mg / mL solution. Take DSPE-PEG... 2000 Dissolve 23.940 mg (0.023940 g) of COOH in 650 μL of DMSO, and vortex until fully dissolved. Add DSPE-PEG. 2000 Add -COOH to a 10mL round-bottom flask, add 750μL of EDC, then add 600μL of NHS. Measure the pH of the solution using a pH meter to ensure it is between 4.0 and 6.5 (ideally 5.0-6.0). If not, slowly add small amounts of EDC (acidic) and NHS (weakly alkaline) under stirring to adjust the pH. Once the pH is satisfactory, stir in the dark and activate for 40 minutes to obtain DSPE-PEG. 2000 -NHS.

[0115] Step 1.2) DSPE-PEG 2000 -NHS and GO-203 combined Take another 10mL round-bottom flask, dissolve 10mg of GO-203 in 1mL of DMSO, and simmer until fully dissolved. Add the DSPE-PEG prepared in step 1.1). 2000 -NHS was slowly added dropwise to GO-203 under light-protected stirring. After the addition was complete, the pH value was measured to ensure it was near 8. If not, triethylamine was used to adjust the pH. After pH adjustment, the reaction was stirred under light for 36 hours. Approximately every 8 hours, 10 μL of the sample was taken, diluted 10-fold with 90 μL of DMSO, and 20 μL was taken for the BCA method to determine the concentration of free GO-203 in the solution (two additional parallel experiments were conducted, totaling 180 μL). The reaction reached its endpoint when the GO-203 concentration no longer decreased. The concentration of GO-203 can be determined from the standard curve.

[0116] Step 1.3) Dialysis purification of DSPE-PEG 2000 -GO-203, and freeze-dried Cut the dialysis bag to a suitable length using scissors, then place it in distilled water and boil in a water bath for 30 minutes to activate the bag. During boiling, continuously stir the distilled water to prevent the bottom temperature from becoming too high and damaging the bag. Remove the dialysis bag from the distilled water, select a suitable dialysis bag clamp, fold it, and clamp the bottom of the bag. Next, open the top of the dialysis bag and use a dropper to add the solution to be dialyzed into the bag. Remove any air from the bag, fold the dialysis clamp to secure the top of the bag, leaving about 1 / 3 of the volume above the liquid level to prevent the bag from bursting due to increased solution volume during dialysis. After loading the sample into the dialysis bag, place it in 1L of distilled water for dialysis. During dialysis, add a stir bar to the beaker and place it on a magnetic stirrer. Change the distilled water every 1, 2, 4, 8, 12, and 24 hours. After dialysis is complete, remove the sample and place it in a beaker, ensuring the thickness is no more than 0.7 cm. Seal the beaker with sealing film and freeze at -20°C overnight.

[0117] After it has completely frozen into a solid, freeze dry it using a freeze dryer.

[0118] Step 1.4) Infrared spectroscopy characterization and identification Power on the instrument and wait 30 minutes for it to warm up. Set the wavenumber range to 3900 to 400 cm⁻¹. -1 Between [times], 16 scans were performed at a resolution of 4. 1-2 mg of sample was finely ground and mixed with 100 mg of pure KBr, then pressed into transparent tablets on a tablet press (pressure increased to 18 MPa, waited 2 min) for determination. Simultaneously, the raw material DSPE-PEG was used... 2000 -NHS, SPDE-PEG 2000 The infrared spectrum of -COOH is used as a reference, and the result is shown in the figure below. Figure 2 As shown.

[0119] Step 1.5) Prepare lipid nanocarriers using thin-film hydration method Accurately weigh 20mg egg yolk lecithin, 8mg cholesterol, and 8mg DSPE-PEG. 2000 -COOH, 1mg DSPE-PEG 2000 -GO-203 and 10μg of coumarin-6 were dissolved together in 10mL of chloroform and added to a 100mL round-bottom flask.

[0120] Assemble the flask into a rotary evaporator and use a water bath at 60°C, a vacuum of approximately -0.08 MPa, and a rotation speed of approximately 80-120 rpm for rotary evaporation. Continue evaporating until the solvent evaporates completely and a uniform lipid film forms on the inner wall of the flask.

[0121] Hydration: PBS preheated to 60°C was used for hydration. The PBS was added to a gaiwan flask and incubated in a 60°C water bath until a uniform lipid film detached from the flask wall. This resulted in the synthesis of lipid nanocarriers with relatively large particle sizes.

[0122] Homogenization: Add the hydrated mixed solution to a 15mL centrifuge tube and sonicate in an ice-water bath using a cell disruptor. Parameter settings: power 120W, turn on for one second and off for three seconds, run for a total of 13 minutes until the particle size is around 100nm, then stop sonicating.

[0123] The morphology of lipid nanocarriers was observed using scanning electron microscopy; the particle size of lipid nanocarriers was detected using dynamic light scattering (DLS); the stability of lipid nanocarriers was obtained by continuously detecting the particle size of lipid nanocarriers using dynamic light scattering (DLS) for 7 days; and the potential of lipid nanocarriers was detected using dynamic light scattering (DLS).

[0124] Example 4 The first step was to synthesize LP(D) lipid nanocarriers. Step 1.1) Prepare lipid nanocarriers using thin-film hydration method Accurately weigh 40mg egg yolk lecithin, 12mg cholesterol, and 12mg DSPE-PEG. 2000 -COOH, 10μg coumarin-6, dissolved together in 10mL chloroform, and added to a 100mL round-bottom flask.

[0125] Assemble the flask into a rotary evaporator and use a water bath at 60°C, a vacuum of approximately -0.08 MPa, and a rotation speed of approximately 80-120 rpm for rotary evaporation. Continue evaporating until the solvent evaporates completely and a uniform lipid film forms on the inner wall of the flask.

[0126] Hydration: PBS containing DMAMCL was preheated to 60°C and used for hydration. The solution was added to a globular flask and incubated in a 60°C water bath until a uniform lipid film detached from the flask wall. This synthesized a lipid nanocarrier with a relatively large particle size.

[0127] Homogenization: Add the hydrated mixed solution to a 15mL centrifuge tube and sonicate in an ice-water bath using a cell disruptor. Parameter settings: power 150W, turn on for one second and off for three seconds, run for a total of 10 minutes until the particle size is around 100nm, then stop sonicating.

[0128] The morphology of lipid nanocarriers was observed using scanning electron microscopy; the particle size of lipid nanocarriers was detected using dynamic light scattering (DLS); the stability of lipid nanocarriers was obtained by continuously detecting the particle size of lipid nanocarriers using dynamic light scattering (DLS) for 7 days; and the potential of lipid nanocarriers was detected using dynamic light scattering (DLS).

[0129] The above embodiments are merely illustrative of the implementation methods of the present invention, but should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the protection scope of the present invention.

Claims

1. A method for preparing a lipid nanocarrier coupled with GO-203 and loaded with DMAMCL, characterized in that, The preparation method includes the following steps: The first step is to combine GO-203 with DSPE-PEG. 2000 -COOH is coupled to obtain DSPE-PEG. 2000 -GO-203 powder; Step 1.1) Dissolve NHS and EDC separately in DMSO, mix well to obtain EDC solution and NHS solution; take DSPE-PEG 2000 -COOH is dissolved in fresh DMSO. EDC solution is added dropwise first, followed by NHS solution, to obtain a mixed solution. The pH is adjusted to acidic, and the solution is stirred in the dark to obtain a solution containing DSPE-PEG. 2000 -NHS solution; Step 1.2) Dissolve GO-203 in DMSO and mix well to obtain a GO-203 solution; then dissolve the DSPE-PEG obtained in step 1.1). 2000 -NHS solution was slowly added dropwise to GO-203 to obtain a mixed solution, and the pH of the mixed solution was adjusted to alkaline; the solution was stirred in the dark to obtain a solution containing DSPE-PEG. 2000 -GO-203 solution; Step 1.3) Dialyze the DSPE-PEG2000-GO-203 solution obtained in Step 1.2) with purified water, and then freeze-dry to obtain DSPE-PEG. 2000 -GO-203 powder; The second step is to prepare lipid nanocarriers. Step 2.1) Add egg yolk lecithin, cholesterol, and DSPE-PEG. 2000 -COOH,DSPE-PEG 2000 -GO-203 powder was added to an organic solvent, and coumarin-6 was added. The mixture was then rotary evaporated in a flask until a uniform film was formed. Step 2.2) Dissolve DMAMCL in PBS and add it to the flask from Step 2.1), and shake until the membrane is completely detached; Step 2.3) The membrane detached in step 2.2) is subjected to ultrasonic treatment to obtain a relatively clear lipid suspension until the Tyndall effect is observed, thus obtaining a crude lipid nanocarrier solution. Step 2.4) The crude lipid nanocarrier solution obtained in step 2.3 is purified to obtain lipid nanocarrier LP(D)-G.

2. The method for preparing a lipid nanocarrier coupled with GO-203 and loaded with DMAMCL according to claim 1, characterized in that, In step 1.1): The concentration of the EDC solution is 1 mg / mL, and the concentration of the NHS solution is 1 mg / mL; Add 23.94 mg of DSPE-PEG to each 1 mL of new DMSO. 2000 -COOH, 0.5-1.5 mL of EDC solution, 0.5-1.5 mL of NHS solution; Adjust the pH of the mixed solution to between 4.0 and 6.5 using the remaining EDC and NHS solutions; stir for 30-40 minutes in the dark. DMSO solvent can also be replaced with DMF solvent.

3. The method for preparing a lipid nanocarrier coupled with GO-203 and loaded with DMAMCL according to claim 1, characterized in that, In step 1.2): Add 10 mg of GO-203 and 1 mL of DSPE-PEG to each 1 mL of DMSO. 2000 -NHS solution; The pH of the mixed solution was adjusted to 7.2-8.5 using triethylamine; Stir at room temperature in the dark for 36-60 hours.

4. The method for preparing a lipid nanocarrier coupled with GO-203 and loaded with DMAMCL according to claim 1, characterized in that, In step 1.3), the dialysis time is 36-60 hours.

5. The method for preparing a lipid nanocarrier coupled with GO-203 and loaded with DMAMCL according to claim 1, characterized in that, In step 2.1): The organic solvent is selected from methanol or dichloromethane; The egg yolk lecithin, cholesterol, and DSPE-PEG 2000 -COOH,DSPE-PEG 2000 The mass ratio of GO-203 powder is 20-40:8-12:0.8-1.2:1; The amount of coumarin-6 added is 0.5-1.5% of the egg yolk lecithin content; The rotary evaporation temperature is 40~60℃.

6. The method for preparing a lipid nanocarrier coupled with GO-203 and loaded with DMAMCL according to claim 1, characterized in that, In step 2.2): Each 10 mg of DMAMCL should be added to 10-15 mL of PBS; Use a 37℃ shaking incubator to continuously oscillate at a speed of 100-200 rpm for 30 minutes to 1 hour.

7. The method for preparing a lipid nanocarrier coupled with GO-203 and loaded with DMAMCL according to claim 1, characterized in that, In step 2.3): At room temperature, the membrane detached in step 2.2) was subjected to ultrasonic treatment using a cell disruptor with an ultrasonic power of 100-150W for 10-15 minutes. The lipid nanocarrier extruder can also be used to extrude the relatively clear lipid suspension obtained by ultrasonic treatment multiple times to make its particle size more uniform.

8. The method for preparing a lipid nanocarrier coupled with GO-203 and loaded with DMAMCL according to claim 1, characterized in that, In step 2.4), the crude lipid nanocarrier solution is purified using a dextran gel G-50 chromatography column.

9. A lipid nanocarrier coupled with GO-203 and loaded with DMAMCL, prepared by any one of the preparation methods described in claims 1-8, characterized in that, The lipid nanocarrier is mainly divided into three parts from the inside out: a core, which is an aqueous DMAMCL; a lipid bilayer membrane; and a surface-modified layer with GO-203. The aqueous phase is loaded with water-soluble dimethylamine methyl chloride (DMAMCL), which is encapsulated in the innermost layer of the lipid nanocarrier. The lipid bilayer membrane encapsulates the core, which has a lipid bilayer structure and embeds coumarin-6 as a fluorescent label for tracking and detection. Two key components are embedded on the outer surface of the lipid bilayer: one is DSPE-PEG. 2000 -COOH; secondly, DSPE-PEG 2000 -GO-203.

10. The application of the lipid nanocarrier conjugated with GO-203 and loaded with DMAMCL as described in claim 9, characterized in that, It is applied to the treatment of triple-negative breast cancer from the perspective of reshaping the tumor immunosuppressive microenvironment.

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