Detection method of bionic nano-carrier system based on GPC3 engineering macrophage membrane in liver cancer treatment

Abstract

The invention relates to the technical field of biological detection, and particularly discloses a detection method of a bionic nano-carrier system based on a GPC3 engineering macrophage membrane in liver cancer treatment, and the detection method comprises the steps of S1, nanoparticle preparation, S2, drug encapsulation, S3, macrophage membrane MM preparation, S4, nanoparticle and macrophage membrane combination, S5, GPC3 targeted modification, and S6, in-vitro cell experimental verification. Through modification of the GPC3 targeting peptide, the nanoparticles can specifically target liver cancer cells, so that the targeting property of liver cancer treatment is greatly improved, and damage to normal tissues is reduced; the use of the macrophage membrane effectively avoids the phagocytosis of an immune system, enhances the circulation time and stability of the drug in vivo, improves the immunotherapy effect, and can significantly induce ferroptosis of liver cancer cells in combination with sorafenib (Sor) and astragaloside IV (As), thereby providing a novel treatment strategy and improving the treatment effect of liver cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to a detection method for a biomimetic nanocarrier system based on GPC3 engineered macrophage membrane in the treatment of liver cancer. Background Art

[0002] With the development of nanotechnology, cell membrane-camouflaged nanoparticles (NPs) have shown great potential as a new drug delivery platform in cancer treatment, immunotherapy, and other fields. This technology utilizes the natural properties of the cell membrane to effectively prolong the circulation time of drugs in the body, improve drug targeting, and enhance drug permeability and accumulation in tissues, especially in targeted tumor therapy.

[0003] Compared to traditional liposomes and extracellular vesicles, cell membrane-camouflaged nanoparticles can not only carry drugs but also precisely target tumor tissues through ligand-receptor interactions on the cell membrane, reducing the drug's impact on non-target tissues and thus minimizing off-target effects. Furthermore, macrophage membranes, as innate immune cell membranes, can avoid phagocytosis by the immune system and enhance anti-tumor immune responses, thus providing a new strategy for tumor immunotherapy.

[0004] 1. Application of Cell Membrane Camouflage Nanoparticles

[0005] Cell membrane-camouflaged nanoparticles are typically derived from the following cell membranes: red blood cells, macrophages, and platelets. Macrophage membranes, due to their strong immune response, have been widely used in tumor immunotherapy. For example, macrophage-camouflaged nanoparticles can enhance tumor-specific immune responses and increase the activation of tumor immune cells in anti-tumor immunotherapy, further enhancing the therapeutic effect.

[0006] 2. Application of GPC3 in liver cancer

[0007] Hepatocellular carcinoma (HCC) is a common malignant tumor with a generally poor prognosis. GPC3 (glycosylphosphatidylinositol-anchored glycoprotein 3), a molecular marker for HCC, is highly expressed in over 70% of liver cancer cases. High GPC3 expression in HCC is associated with a poor prognosis, and numerous studies have investigated GPC3-targeted therapies, particularly the use of GPC3-targeted peptides, which have become an important area of ​​HCC treatment.

[0008] 3. Ferroptosis and liver cancer treatment

[0009] Ferroptosis is a novel type of programmed cell death characterized by the accumulation of lipid peroxides (LPO), which increases intracellular iron concentrations and induces oxidative stress. Research on ferroptosis has attracted widespread attention in recent years, particularly in the treatment of hepatocellular carcinoma (HCC). As a novel therapeutic mechanism, ferroptosis offers new avenues for the treatment of HCC. Sorafenib (Sor), a chemotherapy drug used to treat HCC, has been shown to induce ferroptosis. However, due to its limited properties and therapeutic efficacy, it has not yet achieved the desired therapeutic effect.

[0010] 4. Limitations of PD-1 Inhibitors

[0011] Immune checkpoint inhibitors, particularly PD-1 / PD-L1 inhibitors, have demonstrated remarkable efficacy in the treatment of liver cancer. However, the efficacy of PD-1 inhibitors alone remains unsatisfactory, with objective response rates in liver cancer patients ranging from only 15% to 20%. This is primarily due to immune escape mechanisms within the liver cancer microenvironment, particularly low T cell infiltration, which severely impact the therapeutic efficacy of PD-1 inhibitors. Therefore, improving the efficacy of PD-1 inhibitors has become a research hotspot in liver cancer immunotherapy.

[0012] Problems and shortcomings of existing technologies

[0013] 1. Insufficient targeting: Although cell membrane-camouflaged nanoparticles have good targeting capabilities, current technology cannot completely avoid the off-target effects of nanoparticles, resulting in some drugs failing to effectively concentrate in tumor tissues.

[0014] 2. Low drug delivery efficiency: Existing drug delivery systems often face the problems of low bioavailability and low targeted accumulation of drugs in the body, especially for traditional chemotherapy drugs (such as sorafenib), which have insufficient accumulation in tumor tissues and limited efficacy.

[0015] 3. Immune escape mechanism: Although macrophage membranes can activate immune responses, immune escape remains a key issue in the liver cancer microenvironment due to insufficient T cell infiltration and the influence of immunosuppressive factors.

[0016] By combining the macrophage membrane-targeted delivery system, the application of GPC3-targeted peptides, and the combined strategy of ferroptosis and immunotherapy, the present invention proposes a new treatment plan that aims to overcome the shortcomings of the existing technology and provide a more effective liver cancer treatment plan.

[0017] In response to this, the inventors proposed a detection method for a biomimetic nanocarrier system based on GPC3 engineered macrophage membrane in the treatment of liver cancer to solve the above problems. Summary of the Invention

[0018] The purpose of the present invention is to provide a detection method for a biomimetic nanocarrier system based on GPC3 engineered macrophage membrane in the treatment of liver cancer, so as to solve the problems raised in the above background technology.

[0019] To achieve the above object, the present invention provides the following technical solutions:

[0020] A method for detecting a biomimetic nanocarrier system based on GPC3 engineered macrophage membrane in the treatment of liver cancer comprises the following steps:

[0021] S1. Nanoparticle preparation: SiO2 nanoparticles were modified with PDA and their surface was chemically modified to enable them to bind iron ions to form MPDA / Fe nanospheres.

[0022] S2. Drug encapsulation: The therapeutic drugs sorafenib (Sor) and astragaloside IV (As) were encapsulated in MPDA / Fe nanoparticles using a solvent evaporation method to ensure that the drugs were stably encapsulated in the nanoparticles.

[0023] S3. Preparation of macrophage membrane MM: Cell membranes were extracted from mouse macrophage RAW 264.7 cells and macrophages were obtained by ultrasonic disruption.

[0024] S4. Binding of nanoparticles to macrophage membranes: MPDA / Fe nanoparticles encapsulated with sorafenib Sor and astragaloside IV As were fused with the cell membrane of recipient cells to obtain MM@Sor-AS@MPDA / Fe complexes;

[0025] S5. GPC3 targeted modification: DSPE-PEG-GPC3 targeting peptide was used to modify the surface of MM@Sor-AS@MPDA / Fe, enabling it to specifically target GPC3-expressing liver cancer cells.

[0026] S6. In vitro cell experiment verification steps: The prepared MM@Sor-AS@MPDA / Fe complex is co-cultured with GPC3-positive liver cancer cells, and flow cytometry or fluorescence microscopy is used to observe whether the complex is successfully targeted and bound to the surface of liver cancer cells, and cell activity experiments are used to evaluate the killing effect of the carrier system on liver cancer cells.

[0027] Preferably, the reaction temperature in the PDA-modified SiO2 nanoparticles in step S1 is room temperature, and the reaction time is 4 hours.

[0028] Preferably, the concentration of sorafenib in step S2 is 5 mg / mL, and the concentration of astragaloside IV is 1 mg / mL;

[0029] In the solvent evaporation method, the solvent evaporation temperature is 40° C. and the evaporation time is 8 hours to ensure that the drug is completely encapsulated.

[0030] Preferably, in the cell culture medium in step S3, the concentration of cell membrane extract is 1×10^6 cells / mL;

[0031] The ultrasonic power was 250 W and the ultrasonic time was 5 minutes.

[0032] Preferably, the ratio of the receptor cell membrane to the nanoparticles in step S4 is 1:10;

[0033] The reaction temperature was 37°C and the reaction time was 2 hours.

[0034] Preferably, the concentration of the GPC3 targeting peptide in step S5 is 50 μg / mL;

[0035] The reaction time was 1 hour and the reaction temperature was room temperature.

[0036] Preferably, the receptor cell membrane is at least one of a macrophage membrane, a T cell membrane and a dendritic cell membrane.

[0037] Preferably, step S6 further includes evaluating the drug release characteristics: collecting the culture supernatant at different time points, and using high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS) to detect the concentrations of sorafenib Sor and astragaloside IV As released into the culture medium to evaluate the release rate and release pattern of the drug from the carrier, thereby optimizing the treatment strategy.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) The present invention modifies the GPC3 targeting peptide, so that the nanoparticles can specifically target liver cancer cells, greatly improving the targeting of liver cancer treatment and reducing damage to normal tissues; the use of macrophage membranes effectively avoids the phagocytic effect of the immune system, enhances the circulation time and stability of the drug in the body, and improves the immunotherapy effect.

[0040] (2) The present invention combines sorafenib (Sor) and astragaloside IV (As) to significantly induce ferroptosis in liver cancer cells, thereby providing a new treatment strategy to improve the therapeutic effect of liver cancer; astragaloside IV can activate tumor-infiltrating lymphocytes (TILs) by regulating the tumor microenvironment, thereby improving the effect of immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a flow chart of a detection method for a biomimetic nanocarrier system based on GPC3 engineered macrophage membrane in the treatment of liver cancer according to the present invention;

[0042] Figure 2 is a transmission electron microscope (TEM) image of the nanocarrier of the present invention;

[0043] Figure 3 This is a phalloidin staining image of the nanocarrier of the present invention;

[0044] Figure 4 This is a flow cytometry analysis diagram of GPC3-MM@Sor-AS@MPDAFe of the present invention;

[0045] Figure 5 This is a flow cytometry analysis diagram of Sor-AS@MPDAFe of the present invention;

[0046] Figure 6 This is a graph analyzing the toxicity of the CCK8 detection material of the present invention to liver cancer cells;

[0047] Figure 7 This is the cell live-death of the present invention: GPC3-MM@Sor-AS@MPDAFe Figure 1;

[0048] Figure 8 Cell live-death of the present invention: GPC3-MM@Sor-AS@MPDAFe Figure 2;

[0049] Figure 9 Cell live-death of the present invention: GPC3-MM@Sor-AS@MPDAFe Figure 3;

[0050] Figure 10 Cell live-death of the present invention: Sor-AS@MPDAFe G figure;

[0051] Figure 11 This is the cell live-death of the present invention: Figure 1 of Sor-AS@MPDAFe;

[0052] Figure 12 The cell live-death of the present invention: Sor-AS@MPDAFe Figure 2;

[0053] Figure 13 is one of the ROS diagrams of the present invention;

[0054] Figure 14 This is the second ROS diagram of the present invention;

[0055] Figure 15 This is one of the lipid ROS: GPC3-MM@Sor-AS@MPDAFe diagrams of the present invention;

[0056] Figure 16 This is the lipid ROS of the present invention: GPC3-MM@Sor-AS@MPDAFe Figure 2;

[0057] Figure 17 This is the lipid ROS of the present invention: GPC3-MM@Sor-AS@MPDAFe Figure 3;

[0058] Figure 18 This is one of the lipid ROS: Sor-AS@MPDAFe diagrams of the present invention;

[0059] Figure 19 This is the lipid ROS of the present invention: Sor-AS@MPDAFe Figure 2;

[0060] Figure 20 This is the third figure of lipid ROS: Sor-AS@MPDAFe of the present invention. DETAILED DESCRIPTION

[0061] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0062] Example 1:

[0063] See also Figure 1 As shown, a method for detecting a biomimetic nanocarrier system based on GPC3 engineered macrophage membrane in the treatment of liver cancer comprises:

[0064] S1. Nanoparticle preparation: SiO2 nanoparticles were modified with PDA and their surface was chemically modified to enable them to bind iron ions to form MPDA / Fe nanospheres.

[0065] The reaction temperature in the PDA-modified SiO2 nanoparticles is room temperature and the reaction time is 4 hours;

[0066] The iron ion concentration was controlled at 1 mM to form MPDA / Fe nanoparticles;

[0067] S2. Drug encapsulation: The therapeutic drugs sorafenib (Sor) and astragaloside IV (As) were encapsulated in MPDA / Fe nanoparticles using a solvent evaporation method to ensure that the drugs were stably encapsulated in the nanoparticles.

[0068] The sorafenib concentration is 5 mg / mL, and the astragaloside IV concentration is 1 mg / mL

[0069] S3. Preparation of macrophage membrane MM: Cell membranes were extracted from mouse macrophage RAW 264.7 cells and macrophages were obtained by ultrasonic disruption.

[0070] In the cell culture medium, the concentration of cell membrane extract is 1×10^6 cells / mL;

[0071] The ultrasonic power was 250 W and the ultrasonic time was 5 min;

[0072] S4. Binding of nanoparticles to macrophage membranes: MPDA / Fe nanoparticles encapsulated with sorafenib Sor and astragaloside IV As were fused with macrophage membranes to obtain MM@Sor-AS@MPDA / Fe complexes;

[0073] The ratio of macrophage membrane to nanoparticles was 1:10;

[0074] The reaction temperature was 37°C and the reaction time was 2 hours;

[0075] S5. GPC3 targeted modification: DSPE-PEG-GPC3 targeting peptide was used to modify the surface of MM@Sor-AS@MPDA / Fe, enabling it to specifically target GPC3-expressing liver cancer cells.

[0076] The concentration of GPC3 targeting peptide was 50 μg / mL;

[0077] The reaction time was 1 hour and the reaction temperature was room temperature;

[0078] S6. In vitro cell experiment verification step: The prepared MM@Sor-AS@MPDA / Fe complex was co-cultured with GPC3-positive liver cancer cells. Flow cytometry or fluorescence microscopy was used to observe whether the complex successfully targeted and bound to the liver cancer cell surface. Cell viability experiments were also performed to evaluate the killing effect of the carrier system on liver cancer cells.

[0079] It also includes drug release characteristics evaluation: the culture supernatant is collected at different time points (2 hours, 4 hours, 8 hours, 24 hours, and 48 hours), and the concentrations of sorafenib Sor and astragaloside IV As released into the culture medium are detected by high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS) to evaluate the release rate and release pattern of the drug from the carrier, thereby optimizing the treatment strategy.

[0080] As can be seen from the above, in the present invention, through modification of the GPC3 targeting peptide, the nanoparticles can specifically target liver cancer cells, greatly improving the targeting of liver cancer treatment and reducing damage to normal tissues;

[0081] The use of macrophage membranes effectively avoids the phagocytic effect of the immune system, enhances the circulation time and stability of drugs in the body, and improves the effect of immunotherapy;

[0082] Combining sorafenib (Sor) and astragaloside IV (As) can significantly induce ferroptosis in liver cancer cells, thus providing a new therapeutic strategy to improve the treatment effect of liver cancer;

[0083] Astragaloside IV can improve the effect of immunotherapy by regulating the tumor microenvironment and activating tumor-infiltrating lymphocytes (TILs).

[0084] Example 2:

[0085] The nanocarrier preparation process used in the present invention is as follows: First, iron ions are bound to the surface of PDA-modified SiO2 nanoparticles (MPDA / Fe) to form MPDA / Fe nanospheres. Next, Sor and astragaloside IV (As) are encapsulated in the MPDA / Fe particles to obtain Sor-AS@MPDA / Fe. Subsequently, Sor-AS@MPDA / Fe is encapsulated using macrophage membranes (MM) to prepare MM@Sor-AS@MPDA / Fe particles. Finally, MM@Sor-AS@MPDA / Fe is modified with a GPC3 targeting peptide (DSPE-PEG-GPC3) to obtain a targeted GPC3-MM@Sor-AS@MPDA / Fe material.

[0086] Example 3:

[0087] refer to Figure 2 As shown, the transmission electron microscopy (TEM) image of the nanocarrier

[0088] This image, a transmission electron microscopy (TEM) image, shows the morphology and structure of the GPC3-MM@Sor-AS@MPDA / Fe nanocarriers. The nanocarriers clearly exhibit uniform particle shape, a smooth surface, and a diameter of approximately 100 nanometers, meeting the size requirements for nanoparticle drug delivery. The nanoparticles shown in the image contain uniformly distributed iron ions (Fe) and drug carriers.

[0089] Attachment Figure 3 :Photo of phalloidin staining of nanocarriers

[0090] It can be observed from the figure that the fluorescence is mainly distributed around the cells or aggregated into cluster structures, indicating that the phalloidin-labeled nanomaterials can specifically target cells.

[0091] Example 4:

[0092] refer to Figure 4 and Figure 5 Figure 2 shows flow cytometry analysis of macrophage membrane-camouflaged nanocarriers.

[0093] This figure shows the flow cytometry analysis, comparing the endocytosis efficiency of unmodified and GPC3-modified nanocarriers in RAW macrophages. Figure 4 ) and unmodified Sor-AS@MPDA / Fe were labeled (ref. Figure 5 ) and analyzed its endocytosis in cells. The figure shows that the endocytosis efficiency of the GPC3-MM@Sor-AS@MPDA / Fe group is significantly higher than that of the Sor-AS@MPDA / Fe group, demonstrating the advantage of targeted modification of GPC3 in improving cellular uptake.

[0094] Embodiment 5:

[0095] refer to Figure 6 As shown, the in vitro anti-hepatocellular carcinoma effect evaluation of GPC3-MM@Sor-AS@MPDA / Fe

[0096] Figure 6 The following is a graph showing the toxicity of the CCK8 assay material on liver cancer cells, the CCK-8 cell viability assay results, and the cell survival using live-dead staining. The results show the toxic effects of GPC3-MM@Sor-AS@MPDA / Fe on liver cancer cell lines at different concentrations. CCK-8 staining showed that the viability of liver cancer cells treated with GPC3-MM@Sor-AS@MPDA / Fe was significantly lower than that of the control group; live-dead staining showed that ( Figures 7 to 12 )GPC3-MM@Sor-AS@MPDA / Fe significantly promoted cell death compared with Sor-AS@MPDAFe, demonstrating that the nanocarrier has a strong ability to inhibit the proliferation of liver cancer cells.

[0097] Example 6:

[0098] Targeting and drug release experiments of biomimetic nanocarrier system based on GPC3 engineered macrophage membrane in liver cancer cells:

[0099] Used to verify the targeting properties and drug release effect of GPC3-MM@Sor-AS@MPDA / Fe complex on GPC3-positive liver cancer cells.

[0100] 1. Materials and Methods

[0101] 1.1 Material preparation

[0102] GPC3-MM@Sor-AS@MPDA / Fe complex (prepared by steps S1 to S5 above).

[0103] GPC3-positive hepatocellular carcinoma cells (HepG2 cell line).

[0104] FITC labeling reagent, used to label nanocarriers.

[0105] High performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS).

[0106] 1.2 Experimental equipment

[0107] Flow cytometer.

[0108] Fluorescence microscopy.

[0109] Cell culture incubator (5% CO2, 37°C).

[0110] 2. Experimental Procedure

[0111] 2.1 Complex labeling and cell co-culture

[0112] The GPC3-MM@Sor-AS@MPDA / Fe complex was labeled with FITC to emit green fluorescence;

[0113] The labeled complex was co-cultured with HepG2 cells for 4 hours.

[0114] 2.2 Flow cytometry analysis

[0115] Flow cytometry was used to detect the binding efficiency of GPC3-MM@Sor-AS@MPDA / Fe complex with HepG2 cells;

[0116] The binding rates of the unmodified Sor-AS@MPDA / Fe complex (control group) and the GPC3-MM@Sor-AS@MPDA / Fe complex (experimental group) were compared and analyzed, as shown in Table 1 below;

[0117] Table 1: Flow cytometry analysis data - Binding efficiency of nanocarriers in HepG2 cells:

[0118]

[0119] As shown in Table 1, the fluorescence intensity of the GPC3-modified nanocarriers was significantly higher than that of the control group, proving that targeted modification improved the binding efficiency and cellular endocytosis ability.

[0120] 2.3 Fluorescence microscopy observation

[0121] Fluorescence microscopy was used to observe whether the nanocarriers were successfully targeted and bound to the surface of HepG2 cells;

[0122] Comparing the cell fluorescence intensity of the experimental group and the control group, the experimental data are shown in Table 2 below:

[0123] Table 2: Fluorescence microscopy data - Fluorescence intensity of nanocarriers targeting HepG2 cells

[0124]

[0125] As shown in Table 2, the fluorescence intensity on the cell surface and inside the cells of the experimental group were much higher than those of the control group, indicating that the targeted modification improved the targeting ability of the complex.

[0126] 2.4 Evaluation of drug release characteristics

[0127] The culture supernatant was collected at different time points (e.g., 1 hour, 4 hours, 12 hours, 24 hours);

[0128] The concentrations of sorafenib (Sor) and astragaloside IV (As) released from the culture medium were detected by HPLC or LC-MS;

[0129] The drug release curve was recorded to evaluate its release rate and release pattern.

[0130] 3. Experimental Results

[0131] 3.1 Target binding verification (flow cytometry analysis)

[0132] Figure 4 It was shown that the fluorescence intensity of the GPC3-MM@Sor-AS@MPDA / Fe complex in HepG2 cells was significantly higher than that of the Sor-AS@MPDA / Fe complex, indicating that GPC3 modification significantly improved the targeting binding efficiency of the complex.

[0133] Data analysis showed that the endocytosis efficiency of the experimental group was more than twice that of the control group.

[0134] 3.2 Target binding verification (fluorescence microscopy)

[0135] In the fluorescence microscopy images, the distribution of the experimental group on the cell surface and intracellularly was significantly higher than that in the control group, proving the targeting of GPC3 targeted modification.

[0136] 3.3 Drug release characteristics

[0137] HPLC / LC-MS test results showed that Sor and As exhibited good controlled-release properties in the complex: the release rate was low in the first 4 hours, and the release rate increased significantly from 12 to 24 hours.

[0138] The drug release curve showed a biphasic release pattern (initial slow release and later accelerated release) that was consistent with the characteristics of nano drug delivery systems;

[0139] Table 3: Drug release characteristics data - Release curve data of Sor and As:

[0140] Time (hours) Sor concentration (μg / mL) As concentration (μg / mL) Cumulative release rate (%) 1 1.2±0.1 0.8±0.1 10±2 4 3.5±0.2 2.2±0.1 28±3 12 9.8±0.5 6.4±0.3 67±5 24 14.2±0.6 10.1±0.5 98±2

[0141] As shown in Table 3, the drug release curve presents a biphasic pattern of initial slow release (first 4 hours) and later accelerated release (12-24 hours), which meets the controlled release requirements for tumor treatment.

[0142] 4. Experimental Conclusion

[0143] The GPC3-MM@Sor-AS@MPDA / Fe complex exhibited excellent liver cancer cell targeting ability and drug release performance;

[0144] GPC3 modification enhanced the targeting efficiency of the nanocarrier, and the drug release properties supported its use as an effective therapeutic strategy for liver cancer.

[0145] Embodiment seven:

[0146] refer to Figures 13 to 20 As shown, the effect of GPC3-MM@Sor-AS@MPDA / Fe on ferroptosis of liver cancer cells

[0147] This figure shows the assessment of ferroptosis by lipid ROS staining and ROS staining ( Figures 13 to 20 ), the ROS content in the liver cancer cells of the GPC3-MM@Sor-AS@MPDA / Fe group was significantly increased (P<0.01), and the lipid peroxidation level was significantly increased (P<0.01), indicating that the nanocarrier effectively induced ferroptosis of liver cancer cells.

[0148] As shown above, the GPC3 targeting peptide modification enables nanoparticles to effectively target liver cancer cells, significantly improving the accuracy of drug delivery. Compared with traditional non-targeted treatments, the nanocarriers of this invention have significantly enhanced their in vivo targeting effect, particularly in terms of targeting liver cancer cells and accumulation in tumor tissue.

[0149] Experimental data: In in vitro experiments, immunofluorescence technology was used to analyze the cellular endocytosis efficiency of nanoparticles. The results showed that the endocytosis efficiency of the GPC3-MM@Sor-AS@MPDA / Fe group was three times that of the unmodified nanoparticles (Sor-AS@MPDA / Fe) (P<0.05), demonstrating the significant role of targeted modification in improving the targeting of liver cancer cells.

[0150] In the description of this specification, the reference terms "one embodiment", "some embodiments", "examples", "specific examples" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0151] In the drawings of the embodiments disclosed in the present invention, only the structures related to the embodiments disclosed in the present invention are involved. Other structures can refer to the general design. In the absence of conflict, the same embodiment and different embodiments of the present invention can be combined with each other.

[0152] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting a biomimetic nanocarrier system based on GPC3 engineered macrophage membrane in the treatment of liver cancer, characterized in that: The following steps are involved: S1. Nanoparticle preparation: SiO2 nanoparticles were modified with PDA and their surface was chemically modified to enable them to bind iron ions to form MPDA / Fe nanospheres. S2. Drug encapsulation: The therapeutic drugs sorafenib (Sor) and astragaloside IV (As) were encapsulated in MPDA / Fe nanoparticles using a solvent evaporation method to encapsulate the two drugs in the nanoparticles. S3. Preparation of macrophage membrane MM: Cell membranes were extracted from mouse macrophage RAW 264.7 cells and macrophages were obtained by ultrasonic disruption. S4. Binding of nanoparticles to macrophage membranes: MPDA / Fe nanoparticles encapsulated with sorafenib Sor and astragaloside IV As were fused with the cell membrane of recipient cells to obtain MM@Sor-AS@MPDA / Fe complexes; S5. GPC3 targeted modification: DSPE-PEG-GPC3 targeting peptide was used to modify the surface of MM@Sor-AS@MPDA / Fe, enabling it to specifically target GPC3-expressing liver cancer cells. S6. In vitro cell experiment verification steps: The prepared MM@Sor-AS@MPDA / Fe complex is co-cultured with GPC3-positive liver cancer cells, and flow cytometry or fluorescence microscopy is used to observe whether the complex is successfully targeted and bound to the surface of liver cancer cells, and cell activity experiments are used to evaluate the killing effect of the carrier system on liver cancer cells.

2. The method for detecting a biomimetic nanocarrier system based on GPC3 engineered macrophage membrane in the treatment of liver cancer according to claim 1, characterized in that: The reaction temperature for the PDA-modified SiO2 nanoparticles in step S1 is room temperature and the reaction time is 4 hours; The iron ion concentration is controlled at 1 mM to form MPDA / Fe nanoparticles.

3. The method for detecting a biomimetic nanocarrier system based on GPC3 engineered macrophage membrane in the treatment of liver cancer according to claim 1, characterized in that: In step S2, the concentration of sorafenib is 5 mg / mL, and the concentration of astragaloside IV is 1 mg / mL; In the solvent evaporation method, the solvent evaporation temperature is 40° C. and the evaporation time is 8 hours to ensure that the drug is completely encapsulated.

4. The method for detecting a biomimetic nanocarrier system based on GPC3 engineered macrophage membrane in the treatment of liver cancer according to claim 1, characterized in that: In the cell culture medium in step S3, the concentration of cell membrane extract is 1×10^6 cells / mL; The ultrasonic power was 250 W and the ultrasonic time was 5 minutes.

5. The method for detecting a biomimetic nanocarrier system based on GPC3 engineered macrophage membrane in the treatment of liver cancer according to claim 1, characterized in that: The ratio of the receptor cell membrane to the nanoparticles in step S4 is 1:10; The reaction temperature was 37°C and the reaction time was 2 hours.

6. The method for detecting a biomimetic nanocarrier system based on GPC3 engineered macrophage membrane in the treatment of liver cancer according to claim 1, characterized in that: The concentration of the GPC3 targeting peptide in step S5 is 50 μg / mL; The reaction time was 1 hour and the reaction temperature was room temperature.

7. The method for detecting a biomimetic nanocarrier system based on GPC3 engineered macrophage membrane in the treatment of liver cancer according to claim 1, characterized in that: The receptor cell membrane is at least one of a macrophage membrane, a T cell membrane and a dendritic cell membrane.

8. The method for detecting a biomimetic nanocarrier system based on GPC3 engineered macrophage membrane in the treatment of liver cancer according to claim 1, characterized in that: Step S6 also includes evaluating the drug release characteristics: the culture supernatant is collected at different time points, and the concentrations of sorafenib Sor and astragaloside IV As released into the culture medium are detected by high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS) to evaluate the release rate and release pattern of the drug from the carrier.

Images