Multifunctional bionic nano-preparation, preparation method and application

Abstract

This application belongs to the field of pharmaceutical formulation technology. This application discloses a multifunctional biomimetic nanoparticle formulation, its preparation method, and its application. The multifunctional biomimetic nanoparticle formulation of this application utilizes the dual functions of ginsenoside Rg3 as both a drug and a membrane stabilizer, as well as the natural targeting ability of platelet membranes to circulating tumor cells and metastatic lesions. Simultaneously, it utilizes cationic liposomes to adsorb negatively charged NETs nucleic acid protein complexes, achieving potential reversal and active targeting. Furthermore, by co-loading paclitaxel (PTX) and a photothermal agent (PCP), it integrates photothermal-chemotherapy-immunogenic death (ICD) induction functions, effectively inhibiting tumor-associated fibroblast activation, efficiently capturing circulating tumor cells, reshaping the tumor immune microenvironment, effectively killing tumor cells, and inhibiting distant tumor metastasis.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical formulation technology, and specifically relates to a multifunctional biomimetic nano-formulation, its preparation method, and its application. Background Technology

[0002] Tumor metastasis is the leading cause of clinical treatment failure and patient death. While current liposome-based nanodelivery systems have improved drug pharmacokinetic behavior to some extent, their targeting relies heavily on passive EPR effects or single active targeting modifications, resulting in limited targeting efficiency for highly heterogeneous metastatic lesions. Furthermore, existing delivery systems struggle to simultaneously achieve precise controlled drug release, local microenvironment remodeling, and systemic immune activation, severely limiting their anti-metastatic efficacy. In summary, current technologies for tumor treatment suffer from the following shortcomings: firstly, a lack of synergistic targeting capabilities across multiple stages of the metastasis chain (primary tumor, central tumor cells (CTCs), and metastatic lesions); secondly, an inability to effectively regulate the tumor microenvironment and immunosuppressive state; and thirdly, poor formulation stability, short in vivo circulation time, and easy clearance, impacting efficacy and safety. Summary of the Invention

[0003] Purpose of the Invention: To address the problems of existing technologies, this application provides a multifunctional biomimetic nanoparticle formulation, its preparation method, and its application. The multifunctional biomimetic nanoparticle formulation of this application comprises a first liposome and a second liposome encapsulated on a platelet membrane and co-loaded with berberine (BBR) and ginsenoside Rg3 (G-RG3). The structure of the second liposome comprises a cationic liposome encapsulated in an extracellular trap of neutrophils and co-loaded with paclitaxel (PTX) and a photothermal agent (PCP). The multifunctional biomimetic nanoparticle formulation of this application can achieve multi-target synergy and multi-mechanism linkage, especially in inhibiting tumor metastasis, exhibiting multiple functions such as highly efficient targeting, intelligent drug release, immune regulation, and microenvironment remodeling.

[0004] Technical Solution: This application provides a multifunctional biomimetic nanoparticle formulation, comprising a first liposome and a second liposome; the first liposome comprises a core structure and a platelet membrane coating the core structure; the core structure comprises lecithin, cholesterol, and ginsenoside Rg3 (G-RG3) in a mass ratio of (20~30):(5~10):(1~1.5); the first liposome further comprises berberine, and the berberine loading is 2%~4% based on the total mass of the first liposome; the amount of platelet membrane is: M1=(M2×M0) / 120, where M1 is the mass of the platelet membrane in mg, M2 is the mass of lecithin in mg, and M0 is the mass of platelet membrane extracted from 1 mL of whole blood in mg;

[0005] The second liposome comprises an inner layer structure and a shell covering the outer layer of the inner layer structure; the inner layer structure comprises (2,3-dioleoxypropyl)trimethylammonium chloride and cholesterol in a mass ratio of (20~30):(5~10); the second liposome further comprises paclitaxel and a photothermal agent; based on the total mass of the second liposome, the drug loading of paclitaxel is 2%~5% and the drug loading of the photothermal agent is 1%~3%; the shell comprises a neutrophil extracellular trap, and the mass ratio of the neutrophil extracellular trap to the (2,3-dioleoxypropyl)trimethylammonium chloride is 1:100.

[0006] This application's multifunctional biomimetic nanoparticle formulation utilizes G-RG3, which possesses both drug-like and membrane-stabilizing functions, to prepare a first liposome. The first liposome is coated with a platelet membrane, which endows it with natural targeting capabilities against circulating tumor cells and metastatic lesions. The first liposome is also loaded with berberine, combining the therapeutic effects of berberine on tumors. Simultaneously, the second liposome structure comprises a cationic liposome co-loaded with paclitaxel (PTX) and a photothermal agent (PCP) encapsulated within a neutrophil extracellular trap (NETs nucleoprotein complex). This cationic liposome utilizes the adsorption of negatively charged... NETs enable potential reversal and active targeting, and integrate photothermal-chemotherapy-immunogenic death (ICD) induction functions, giving the multifunctional biomimetic nanoparticles in this application embodiments a dual active targeting function. They can simultaneously identify primary tumors and metastatic lesions. Furthermore, these nanoparticles can be triggered by exogenous near-infrared light to stimulate drug release, achieving synergistic treatment through multiple modalities including fusion chemotherapy, photothermal therapy, and immunomodulation. This inhibits tumor-associated fibroblast (CAF) activation, promotes immune cell infiltration, and activates anti-tumor immune responses, achieving microenvironment remodeling and effectively inhibiting tumor metastasis. In other words, the multifunctional biomimetic nanoparticles of this application can effectively inhibit tumor-associated fibroblast activation, efficiently capture circulating tumor cells, remodel the tumor immune microenvironment, effectively kill tumor cells, and inhibit distant tumor metastasis.

[0007] In some embodiments, the mass ratio of lecithin, cholesterol, and ginsenoside Rg3 is 24:8:1.

[0008] In some embodiments, the mass ratio of (2,3-dioleoxypropyl)trimethylammonium chloride to cholesterol is 3:1.

[0009] In some embodiments, the particle size of the first liposome is 91 nm to 105 nm; the particle size of the second liposome is 147 nm to 160 nm.

[0010] In some embodiments, the neutrophil extracellular trap is prepared by stimulating neutrophils with phorbol 12-tetradecanoate 13-acetate to induce the neutrophil extracellular trap.

[0011] In some specific embodiments, the neutrophil extracellular trap (NETs nucleic acid-protein complex) of this application is prepared by the following method:

[0012] Mouse peripheral blood was collected, neutrophils were extracted, and NETs nucleic acid-protein complexes were induced to form NETs nucleic acid-protein complexes by phorbol 12-tetradecanoate 13-acetate (PMA). The complexes were then extracted using the TRIzol method. TRIzol reagent was added, and after incubation, chloroform was added, followed by centrifugation and collection of the aqueous phase. Isopropanol was then added to precipitate the NETs nucleic acid-protein complexes, and the mixture was washed to obtain the NETs nucleic acid-protein complexes. This application also provides a method for preparing multifunctional biomimetic nanoparticles, including the following steps:

[0013] Preparation of the first liposome: Lecithin, cholesterol, berberine, and ginsenoside Rg3 were dissolved in anhydrous ethanol to obtain the first solution. PBS solution was used as the second solution. The second solution was heated to 50℃~60℃. The first solution was then added dropwise to the second solution. Anhydrous ethanol was removed by rotary evaporation. The resulting solution was sonicated in an ice bath, mixed with platelet membrane, and extruded to obtain the first liposome.

[0014] Preparation of the second liposome: (2,3-dioleoxypropyl)trimethylammonium chloride, cholesterol, paclitaxel, and photothermal agent were dissolved in chloroform, the chloroform was removed by rotary evaporation, PBS solution was added for hydration, the resulting solution was sonicated in an ice bath, and co-incubated with neutrophil extracellular traps (NETs nucleic acid protein complexes), filtered, and the second liposome was obtained.

[0015] In some specific embodiments, after incubation, the resulting second liposomes are filtered through filter membranes with 220 nm and 480 nm pore sizes, respectively.

[0016] This application also provides the application of the above-mentioned multifunctional biomimetic nanoparticles or the preparation method of the above-mentioned multifunctional biomimetic nanoparticles in the preparation of drugs for treating tumors and drugs for inhibiting tumor metastasis.

[0017] In some embodiments, the multifunctional biomimetic nanoparticle formulation of this application, combined with photothermal therapy, can better enhance the therapeutic effect.

[0018] In some embodiments, the tumor is oral squamous cell carcinoma.

[0019] Beneficial Effects: This application provides a multifunctional biomimetic nanoparticle formulation, its preparation method, and its application. The multifunctional biomimetic nanoparticle formulation utilizes the dual functions of ginsenoside Rg3 as both a drug and a membrane stabilizer, along with the natural targeting ability of platelet membranes to circulating tumor cells and metastatic lesions. Simultaneously, cationic liposomes co-loaded with paclitaxel (PTX) and photothermal agent (PCP) are encapsulated in a NETs nucleic acid-protein complex. The cationic liposomes adsorb negatively charged NETs, ​​achieving potential reversal and active targeting, and integrating photothermal-chemotherapy-immunogenic death (ICD) induction function. The nanoparticle formulation of this application can effectively inhibit tumor-associated fibroblast activation, efficiently capture circulating tumor cells, remodel the tumor immune microenvironment, effectively kill tumor cells, and inhibit distant tumor metastasis. Attached Figure Description

[0020] Figure 1 The embodiments of this application present cytotoxicity experiments with different ratios of BBR and RG3, PTX and PCP. Figure A shows the results of cytotoxicity experiments with different ratios of BBR and G-RG3. The blue area represents the treatment group with a BBR to G-RG3 mass ratio of 3:1, the red area represents the treatment group with a BBR to G-RG3 mass ratio of 1:1, and the green area represents the treatment group with a BBR to G-RG3 mass ratio of 1:3. Figure B shows the results of cytotoxicity experiments with different ratios of PTX and PCP. The blue area represents the PTX to PCP mass ratio of 3:1, the red area represents the PTX to PCP mass ratio of 2:1, the green area represents the PTX to PCP mass ratio of 1:1, the purple area represents the PTX to PCP mass ratio of 1:2, and the orange area represents the PTX to PCP mass ratio of 1:3. The analyzed data are expressed as mean ± standard deviation, n=3, ns: no significant difference, *P<0.05;

[0021] Figure 2 Figure A shows the particle size distribution of different liposomes prepared in the embodiments of this application. Figure B shows the particle size of liposomes BBR&RG3 / LIP prepared by BBR and G-RG3 in a mass ratio of 1:3 without platelet membrane coating; Figure B shows the particle size of liposomes BBR&RG3 / LIP prepared by BBR and G-RG3 in a mass ratio of 3:1 without platelet membrane coating; Figure C shows the particle size of liposomes BBR&RG3 / LIP prepared by BBR and G-RG3 in a mass ratio of 1:1 without platelet membrane coating; and Figure D shows the particle size of liposomes BBR&RG3 / PL prepared by BBR and G-RG3 in a mass ratio of 1:1 with platelet membrane coating.

[0022] Figure 3Electron microscopy and electrophoresis results of different liposomes prepared for the embodiments of this application are shown. Figure A is an electron micrograph of BBR&RG3 / LIP, Figure B is an electron micrograph of BBR&RG3 / PL, and Figure C is a CD47 Western Blot and Coomassie Brilliant Blue assay result of platelet membrane (PLTm), BBR&RG3 / LIP and BBR&RG3 / PL.

[0023] Figure 4 The following are particle size distribution diagrams of different liposomes prepared in the embodiments of this application: Figure A is the particle size distribution diagram of PTX&PCP / LIP liposomes, and Figure B is the particle size distribution diagram of PTX&PCP / NL liposomes.

[0024] Figure 5 The electron microscopy and electrophoresis results of different liposomes are shown in Figure A, which is an electron microscopy image of PTX&PCP / LIP liposomes, Figure B is an electron microscopy image of PTX&PCP / NL liposomes, and Figure C is a Coomassie Brilliant Blue result image of NETs and PTX&PCP / NL.

[0025] Figure 6 The particle size change of PTX&PCP / NL and BBR&RG3 / PL liposomes prepared for the embodiments of this application after 14 days is presented. The data are expressed as mean ± standard deviation, n=3.

[0026] Figure 7 Drug release curves of BBR&RG3 / LIP and BBR&RG3 / PL liposomes prepared for the embodiments of this application within 48 hours. The data analyzed are expressed as mean ± standard deviation, n=3.

[0027] Figure 8 Drug release curves of PTX&PCP / LIP and PTX&PCP / NL liposomes prepared for the embodiments of this application within 48 hours were analyzed. The data are expressed as mean ± standard deviation, n=3.

[0028] Figure 9 The variation curves of PTX&PCP / NL prepared for the embodiments of this application under different concentrations and temperature conditions;

[0029] Figure 10 Hemolysis test of NLIP (PTX&PCP / NL) and PLIP (BBR&RG3 / PL) vectors prepared for embodiments of this application;

[0030] Figure 11 Verification test results were generated for the different liposomes prepared in the embodiments of this application to generate ROS.

[0031] Figure 12The figures show the quantitative results of ATP release from cells after treatment with different drug groups in the embodiments of this application. In the figure, 1 represents the CAL27+MRC-5 treatment group, 2 represents the PTX treatment group, 3 represents the PCP treatment group, 4 represents the PCP+L treatment group, 5 represents the PTX+PCP+L treatment group, 6 represents the BBR+G-RG3 treatment group, 7 represents the PTX+PCP+BBR+G-RG3+L treatment group, 8 represents the PTX&PCP / LIP+L treatment group, 9 represents the PTX&PCP / NL+L treatment group, and 10 represents the PTX&PCP / NL+BBR&RG3 / PL+L treatment group. The analyzed data are expressed as mean ± standard deviation, n=3, ns: no significant difference, ***P<0.001;

[0032] Figure 13 The images show confocal images and flow cytometry quantitative results of the uptake of C6&RG3 / PL and C6&PCP / NL prepared in MRC-5 and CAL27 cells, respectively, in the embodiments of this application. Figures A and B show the uptake of C6, C6&RG3 / LIP, and C6&RG3 / PL in MRC-5 (CAFs) cells induced by tumor supernatant, and their structures analyzed by flow cytometry. Figures C and D show the uptake effect of C6, C6&PCP / LIP, and C6&PCP / NL in tumor cells, and their quantitative results by flow cytometry. The analyzed data are expressed as mean ± standard deviation, n=3, **P<0.01, ***P<0.001;

[0033] Figure 14 The MTT test results for each drug group in the embodiments of this application are presented as mean ± standard deviation, n=3, *P<0.05, ***P<0.001;

[0034] Figure 15 Figure A shows the cell scratch assay results of different treatment groups in the embodiments of this application. Figure B shows the quantitative results of the cell scratch assay of different treatment groups on the CAL27 single-cell model, Figure C shows the cell scratch assay of different treatment groups on the CAL27 single-cell model, and Figure D shows the quantitative results of the cell scratch assay of different treatment groups on the CAL27+MRC-5 co-cultured cell model. The analyzed data are expressed as mean ± standard deviation, n=3, ns: no significant difference, **P<0.01;

[0035] Figure 16 The Transwell experimental results for different treatment groups in the embodiments of this application are shown. Figure A is an inverted microscope image, and Figure B is the quantitative result. The analyzed data are expressed as mean ± standard deviation, n=3, **P<0.01;

[0036] Figure 17 The figures show the biodistribution of NLIP and PLIP prepared in this application in vivo. Figure A shows the biodistribution of different formulations in vivo at different time points (2, 4, 8, 12, 24, 32 and 48 hours). Figure B shows the in vitro fluorescence imaging. Figures C and D show the results of fluorescence quantitative analysis of the two formulations in major organs and tumors. The analyzed data are expressed as mean ± standard deviation, n=3, *P<0.05, **P<0.01;

[0037] Figure 18 In the embodiments of this application, near-infrared light irradiation (1.5W / cm²) is used. 2 Temperature change curves at the tumor sites of mice in each group after [following the previous steps].

[0038] Figure 19 Images of tumors after treatment with the formulations prepared in the embodiments of this application;

[0039] Figure 20 The data for changes in mouse body weight during treatment with the formulation prepared in the embodiments of this application are expressed as mean ± standard deviation, n=3, **P<0.01, ***P<0.001;

[0040] Figure 21 The results of tumor analysis after treatment with the formulation prepared in the embodiments of this application are shown in Figure A, which is a graph of tumor volume change, and Figure B is a quantitative result of tumor inhibition rate of each treatment group. The analyzed data are expressed as mean ± standard deviation, n=3, **P<0.01, ***P<0.001;

[0041] Figure 22 These are staining images of tumors after treatment with the formulations prepared in the embodiments of this application, wherein image A is an H&E staining image and image B is a Masson staining image;

[0042] Figure 23 The images show the H&E staining results of major organs (heart, liver, spleen, lungs, and kidneys) of mice after treatment in each group of the embodiments of this application.

[0043] Figure 24 Figure A shows the results of anti-inflammatory factor expression detection in tumor tissue in the embodiments of this application. Figure B shows the results of TNF-α detection, Figure C shows the results of IFN-γ detection, Figure D shows the results of IL-6 detection, and Figure D shows the results of IL-10 detection. The analyzed data are expressed as mean ± standard deviation, n=3, *P<0.05, **P<0.01, ***P<0.001;

[0044] Figure 25 The figures shown are the results of the tumor lung metastasis inhibition experiment in the embodiments of this application. Figure A shows the quantitative results of lung nodules, Figure B shows a visual diagram of lung nodules, and Figure C shows the results of H&E staining of the lungs. Detailed Implementation

[0045] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0046] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. In addition, in the description of this application, the term "comprising" means "including but not limited to". The terms first, second, third, etc. are used only as illustrative purposes and do not impose numerical requirements or establish an order. Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and conciseness and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range. Additionally, whenever a numerical range is specified in this document, it means that any referenced number (fraction or integer) within the range is included.

[0047] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art described herein. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this application. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail. Unless otherwise stated, "%" is a percentage based on mass, and the solvent ethanol used refers to an ethanol solution with a mass percentage of ≥95%.

[0048] I. Materials and Methods

[0049] 1. Materials

[0050] Lecithin (egg yolk lecithin, PC), (2,3-dioleoxypropyl)trimethylammonium chloride (DOTAP), and cholesterol were purchased from AVT Pharmaceuticals Ltd. (Shanghai, China). Paclitaxel (PTX), berberine (BBR), and ginsenoside Rg3 (G-RG3) were purchased from Solarbio Science & Technology Ltd. (Beijing, China). Poly(bisthiophene-benzothiadiazole) alternating copolymer (PCPDTBT, PCP) was purchased from MedChemExpress Ltd. (Shanghai, China). Antibodies against E-cadherin, vimentin, and CD31 were purchased from Abcam Pharmaceuticals Ltd. (Massachusetts, USA). All other chemical reagents were of analytical or chromatographic grade.

[0051] 2. Cells and animals

[0052] Human tongue squamous cell carcinoma cell line (CAL27) and human embryonic lung fibroblasts (MRC-5) were purchased from Beijing Institute of Biotechnology. BALB / c-nu mice (female, 5 weeks old, 14-16g) were purchased from Shandong Pengyue Experimental Animal Center. All animal experiments were approved by the Animal Experiment Ethics Committee of Shandong Second Medical University (SDSMU, 2019-045), and the care and use of experimental animals strictly complied with the "Animal Management Regulations" (No. 55, 001) issued by the National Health Commission of the People's Republic of China.

[0053] II. Testing Methods

[0054] 1. Physicochemical characterization of BBR&RG3 / PL and PTX&PCP / NL

[0055] The particle size distribution and zeta potential of BBR&RG3 / PL and PTX&PCP / NL liposomes were measured using a Malvern particle size potentiometer. BBR&RG3 / PL and PTX&PCP / NL liposomes were diluted 10-fold in DMEM medium containing 10% fetal bovine serum and stored at 4°C. Particle size changes in DMEM medium containing 10% fetal bovine serum were measured every other day. The liposomes were diluted 20-fold with PBS, negatively stained with phosphotungstic acid, and their morphology was observed using transmission electron microscopy (TEM) after drying. The content of G-RG3 in BBR&RG3 / PL and the content of PTX (227 nm) in PTX&PCP / NL were determined by high-performance liquid chromatography (HPLC). The contents of BBR (357 nm) in BBR&RG3 / PL and PCP (690 nm) in PTX&PCP / NL were measured using a UV spectrophotometer. The encapsulation efficiency (EE) and drug loading (LE) were calculated using the following formulas:

[0056]

[0057] 2. In vitro drug release of BBR&RG3 / PL and PTX&PCP / NL

[0058] The in vitro drug release of BBR from BBR&RG3 / PL was evaluated in PBS buffer at different pH values ​​(pH=7.4, 6.5, or 5.5). 1 mL of BBR&RG3 / PL was added to a dialysis bag (MWCO=3500), sealed, and placed in a 50 mL centrifuge tube containing 30 mL of PBS buffer at different pH values ​​(containing 0.5% Tween-80). The release system was placed in a constant temperature shaking incubator at 37℃ and 100 rpm. At predetermined time points (0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h), 2 mL of release medium was collected, and 2 mL of fresh release medium was added simultaneously. The amount of BBR released was determined by high-performance liquid chromatography (HPLC) at 357 nm.

[0059] PTX & PCP / NL are affected by near-infrared (NIR) light irradiation. Therefore, the drug release of PTX under NIR irradiation was detected in PBS buffer at pH 7.4 using the same method as described above. High-performance liquid chromatography (HPLC) was used to detect PTX drug release.

[0060] 3. In vitro photothermal performance evaluation

[0061] To investigate suitable PTX&PCP / NL concentrations and laser irradiation power, a series of PTX&PCP / NL concentration-temperature and irradiation power-temperature curves were constructed. First, 1 mL of PBS solution and a series of PTX&PCP / NL concentrations (0, 30 μg / mL, 60 μg / mL, 125 μg / mL, 250 μg / mL) were placed in centrifuge tubes and irradiated with an 808 nm near-infrared laser for 5 min at a power of 1.5 W / cm². 2 Temperatures were recorded at predetermined times. Then, a concentration of 125 μg / mL PTX & PCP / NL was subjected to irradiation with an 808 nm laser at different irradiation powers (0.5 W / cm²). 2 1W / cm 2 1.5W / cm 2 2W / cm 2 Irradiation was performed for 5 minutes under the specified conditions, with temperature recorded at predetermined time points. Additionally, PTX & PCP / NL (75 μg / mL) were irradiated for 5 minutes using an 808 nm laser at a power of 1.5 W / cm². 2Temperature changes were recorded and photographs were taken to evaluate the photothermal conversion performance of the formulation. PTX & PCP / NL (125 μg / mL) was irradiated with an 808 nm laser for three on / off cycles at a power of 1.5 W / cm². 2 Temperature changes are recorded at predetermined times to assess the photothermal stability of the formulation.

[0062] 4. Evaluation of hemolytic activity

[0063] Blood was collected from healthy mice using the enucleation method. 1 mL of blood was placed in a centrifuge tube and centrifuged at 3500 rpm for 10 min. The supernatant was discarded, and the red blood cells were washed with physiological saline. The cells were resuspended, and the centrifugation process was repeated until the supernatant was colorless. The red blood cells were diluted with physiological saline to obtain a 2% red blood cell suspension. Liposomes were mixed with the red blood cell suspension to obtain samples with different carrier concentrations (0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 5 mg / mL). Triton X-100 and physiological saline were used as positive and negative controls, respectively. All samples were incubated in a constant temperature shaking incubator for 2 h, and then centrifuged (3500 rpm, 10 min). The samples were observed and photographed, and the absorbance (OD value) of the supernatant was measured at 545 nm using a microplate reader. The hemolysis rate was calculated using the following formula:

[0064]

[0065] A, A1, and A2 represent the absorbance values ​​of the positive control group, the experimental group, and the negative control group, respectively.

[0066] 5. Generation of reactive oxygen species

[0067] To evaluate the effect of PTX combined with photothermal therapy on reactive oxygen species (ROS) production, CAL27 cells were collected and divided into two wells at a ratio of 2 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates and cultured for 24 hours. Different drug groups (PBS, PTX+PCP, PTX+PCP+BBR+G-RG3, PTX&PCP / LIP, PTX&PCP / LIP+BBR&RG3 / LIP, PTX&PCP / NL+BBR&RG3 / PL) were then added for treatment. The light group received 1.5 W / cm² treatment after 6 hours. 2 Irradiate with (808nm) near-infrared laser for 5 min, continue culturing for 24 h, add reactive oxygen species fluorescent probe (DCFH-DA) probe and stain at 37℃ in the dark for 30 min, wash off excess dye with PBS, and observe with an inverted fluorescence microscope.

[0068] The control group received PBS solution; the PTX+PCP treatment group consisted of PTX at 0.5 μg / mL and PCP at 0.2 μg / mL; the PTX+PCP+BBR+G-RG3 treatment group consisted of PTX at 0.5 μg / mL, PCP at 0.2 μg / mL, BBR at 0.5 μg / mL, and G-RG3 at 0.5 μg / mL; the PTX&PCP / LIP treatment group consisted of PTX at 0.5 μg / mL and PCP at 0.2 μg / mL. mL; the PTX&PCP / LIP+BBR&RG3 / LIP treatment group had PTX at 0.5 μg / mL, PCP at 0.2 μg / mL, BBR at 0.5 μg / mL, and G-RG3 at 0.5 μg / mL; the PTX&PCP / NL+BBR&RG3 / PL treatment group had the same dosage as the PTX&PCP / LIP+BBR&RG3 / LIP group, the difference being that they were loaded with platelet membrane and NETs nucleic acid protein complex, respectively.

[0069] 6. Cellular immunogenic death

[0070] To verify whether photothermal therapy can induce immunogenic death of tumor cells, the expression level of adenosine triphosphate (ATP) in cells was measured. CAL27+ MRC-5 cells (5 × 10⁻⁶) were used for evaluation. 4 Cells (CAL27 and MRC-5 cells in a 5:1 ratio) were seeded in eight-well chambers and cultured for 24 h. Control groups (CAL27 + MRC-5), PTX, PCP, PCP+L, PTX + PCP+L, BBR + G-RG3, PTX + PCP + BBR + G-RG3+L, PTX & PCP / LIP+L, PTX & PCP / NL+L, and PTX & PCP / NL + BBR & RG3 / PL+L were then incubated with the cells for 6 h. The cells were then treated with 1.5 W / cm² water. 2 Irradiate with (808nm) near-infrared laser for 5 min and incubate overnight. Then, collect the cell supernatant, and use an ATP assay kit to detect the ATP content in the collected cell supernatant. Quantitative analysis is performed using a microplate reader.

[0071] The control group was the CAL27+MRC-5 culture group, without any drug treatment; PTX treatment groups: PTX concentration 0.5 μg / mL; PCP treatment groups: PCP concentration 0.2 μg / mL; PCP+L treatment groups: PCP concentration 0.2 μg / mL, combined with a 3-min light exposure; PTX+PCP+L treatment groups: PTX concentration 0.5 μg / mL, PCP concentration 0.2 μg / mL, combined with a 3-min light exposure; BBR+G-RG3 treatment groups: BBR concentration 0.5 μg / mL, G-RG3 concentration 0.5 μg / mL; PTX+PCP+BBR+G-RG3+L treatment groups: PTX concentration 0.5 μg / mL, PCP concentration 0.2 μg / mL. / mL, BBR treatment concentration was 0.5μg / mL, G-RG3 treatment concentration was 0.5μg / mL, and the light exposure time was 3min; PTX&PCP / LIP+L treatment group: liposomes without NETs nucleic acid protein complex encapsulation, the drug dosage in the treatment group was the same as the PTX+PCP+L treatment group, and the light exposure time was 3min; PTC&PCP / NL+L treatment group: the second liposome treatment group, the drug dosage in the treatment group was the same as the PTX+PCP+L treatment group, and the light exposure time was 3min; PTC&PCP / NL+BBR&RG3 / PL+L treatment group: the first and second liposome treatment groups, the drug dosage in the treatment group was the same as the PTX+PCP+BBR+G-RG3+L treatment group, and the light exposure time was 3min.

[0072] 7. Cell uptake assay

[0073] To investigate the uptake of liposomes by tumor cells, coumarin 6 (C6) was used as a fluorescent agent instead of BBR and PTX to prepare fluorescent liposomes C6&RG3 / PL and C6&PCP / NL. Because the two formulations have different sites of action, the uptake of C6&RG3 / PL was detected by MRC-5 cells induced into tumor-associated fibroblasts (CAFs) using tumor supernatant, and the uptake of C6&PCP / NL was detected by CAL27 cells. The induced MRC-5 and CAL27 cells were seeded in cell culture dishes (5 × 10⁻⁶ cells / year). 4After cells adhered and grew in culture dishes, free C6, liposomes loaded with C6 and G-RG3 (C6&RG3 / LIP), and platelet membrane-encapsulated pH-sensitive liposomes loaded with C6 and G-RG3 (C6&RG3 / PL) (C6 concentration 10 μg / mL) were added to the MRC-5 cell model. In the CAL27 cell model, free C6, liposomes loaded with C6 and PCP (C6&PCP / LIP), and NETs-encapsulated liposomes co-loaded with C6 and PCP (C6&PCP / NL) (C6 concentration 10 μg / mL) were added. Treatment with these drugs lasted 30 min. After washing three times with PBS, cells were fixed in tissue fixative for 10 min and stained with 4,6-diamidinyl-2-phenylindole (DAPI, concentration 1 μg / mL) nuclear dye for 10 min. After washing three times with PBS, images were taken using a laser confocal microscope, and quantitative analysis was performed using an Accuri C6 Plus flow cytometer.

[0074] 8. In vitro cytotoxicity test

[0075] The MTT assay was used to detect the toxicity of different formulations to tumor cells. To better evaluate the role of drugs in the tumor microenvironment, we established two cell models: (1) CAL27 cells; (2) a "CAL27+MRC-5" co-culture cell model (the ratio of CAL27 to MRC-5 cells was 5:1). First, both cell models were seeded into 96-well plates, with 5 × 10⁶ cells added to each well. 3 Cells were cultured overnight at 37°C in a 5% CO2 incubator. PTX, PCP, PTX+PCP, BBR+G-RG3, PTX+PCP+BBR+G-RG3, PTX&PCP / NL, and PTX&PCP / NL+BBR&RG3 / PL were added, and the incubator was operated at 1.5 W / cm². 2 Cells were irradiated with near-infrared laser at 808 nm for 5 min, with PTX concentrations ranging from 0.01 μg / mL to 10 μg / mL, for 48 h. MTT solution (5 mg / mL) was added to each well, and the cells were incubated for 4 h. The culture medium was removed, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. The OD values ​​at 570 nm were measured using a microplate reader (ELX800, BioTek, USA), and cell viability was calculated for each group.

[0076] In different treatment groups, the dosage of BBR and G-RG3 was the same as the treatment concentration of PTX, and the concentration ratio of PCP to PTX was 1:2.5.

[0077] 9. Cell migration and invasion analysis

[0078] The in vitro anti-migration and anti-invasive capabilities of the control group, PTX, PCP, PCP+L, PTX+PCP+L, BBR+G-RG3, PTX+PCP+BBR+G-RG3+L, PTC&PCP / LIP+L, PTC&PCP / NL+L, and PTC&PCP / NL+BBR&RG3 / PL+L were evaluated using scratch healing and Transwell assays.

[0079] (1) Scratch assay: CAL27 and MRC-5 cells (5:1 ratio) were seeded in six-well plates. When the cell confluence reached 80%, the cell layer was scratched with the tip of a 200µL sterile pipette. After washing with PBS, the width of the scratch was observed under an inverted microscope at 0h. The cells were then treated with different preparations and treated with 1.5W / cm² of PBS. 2 After irradiating the wound with an 808nm near-infrared laser for 5 min and incubating for 24 h, the wound was washed three times with PBS. The wound width was observed again after 24 h, and the migration rate was quantified using ImageJ software. The calculation is shown in the following formula:

[0080]

[0081] In the formula, d 0h The width of the 0h wound is in μm, d 24h The width of the wound over 24 hours is expressed in μm.

[0082] (2) Transwell Assay: The Transwell invasion assay requires a layer of Matrigel at the bottom of the upper chamber of the Transwell. First, dilute the Matrigel (1:5) with serum-free medium on ice. Add the prepared Matrigel to the Transwell chamber and incubate at 37°C for 1 hour. Pretreat cells with different formulations, collect cells after 24 hours, and resuspend them in serum-free medium (cell density, 5 × 10⁻⁶). 5 200 µL of cell suspension was added to the chamber, and 600 µL of culture medium containing 10% fetal bovine serum was added to the 24-well plate below the chamber as an inducing agent. After incubation at 37°C for 16 h, the upper chamber medium was discarded, and the upper matrix gel of the chamber was gently wiped off with a sterile cotton swab. The cells were washed three times with PBS, fixed with tissue cell fixation solution for 20 min, washed three times with PBS, stained with crystal violet for 10 min, rinsed three times with PBS, and observed under a microscope. Finally, ImageJ software was used to quantitatively analyze the cells that migrated to the lower surface of the chamber.

[0083] CAL27+MRC-5 culture group, no drug treatment; PTX treatment group: PTX concentration 0.5 μg / mL; PCP treatment group: PCP concentration 0.2 μg / mL; PCP+L treatment group: PCP concentration 0.2 μg / mL, combined with 3 min of light exposure; PTX+PCP+L treatment group: PTX concentration 0.5 μg / mL, PCP concentration 0.2 μg / mL, combined with 3 min of light exposure; BBR+G-RG3 treatment group: BBR concentration 0.5 μg / mL, G-RG3 concentration 0.5 μg / mL; PTX+PCP+BBR+G-RG3+L treatment group: PTX concentration 0.5 μg / mL, PCP concentration 0.2 μg / mL. L, BBR treatment concentration was 0.5 μg / mL, G-RG3 treatment concentration was 0.5 μg / mL, and the illumination time was 3 min; PTX&PCP / LIP+L treatment group: liposomes without NETs nucleic acid protein complex encapsulation, the drug dosage in the treatment group was the same as the PTX+PCP+L treatment group, and the illumination time was 3 min; PTC&PCP / NL+L treatment group: the second liposome treatment group, the drug dosage in the treatment group was the same as the PTX+PCP+L treatment group, and the illumination time was 3 min; PTC&PCP / NL+BBR&RG3 / PL+L treatment group: the first and second liposome treatment groups, the drug dosage in the treatment group was the same as the PTX+PCP+BBR+G-RG3+L treatment group, and the illumination time was 3 min.

[0084] 10. In vivo imaging analysis

[0085] Two fluorescent substances, DID and DIR, were used instead of drugs to detect the biodistribution of PTX&PCP / NL and BBR&RG3 / PL in "CAL27+MRC-5" tumor-bearing mice. A "CAL27+MRC-5" tumor-bearing model was established by subcutaneous injection of the mixed cell suspension into BALB / c-nu mice. When the tumor volume was approximately 120 mm², the tumor was considered to be present. 3 Mice were divided into three groups: DID+DIR+L, DID&PCP / LIP+DIR&RG3 / LIP+L, and DID&PCP / NL+DIR&RG3 / PL+L. The biodistribution of DID and DIR in mice was monitored at different time points using a near-infrared fluorescence imaging system (NIRF). Organs and tumors were harvested 48 hours later for in vitro fluorescence intensity analysis.

[0086] Among them, the DID+DIR+L treatment group: DID was 3 mg / kg, DIR was 3 mg / kg, and the light exposure time was 3 min (L).

[0087] DID&PCP / LIP+DIR&RG3 / LIP+L treatment group: DID is 3 mg / kg, PCP is 2 mg / kg, DIR is 3 mg / kg, G-RG3 is 5 mg / kg, combined with a photoperiod of 3 min (L).

[0088] The treatment groups were: DID & PCP / NL + DIR & RG3 / PL + L, with DID at 3 mg / kg, PCP at 2 mg / kg, DIR at 3 mg / kg, and G-RG3 at 5 mg / kg, combined with a photoperiod of 3 min (L).

[0089] 11. In vivo photothermal performance assessment

[0090] A subcutaneous CAL27 tumor-bearing mouse model was established. When the tumor grew to a suitable size, PTX+PCP, PTX&PCP / LIP, or PTX&PCP / NL were injected via the tail vein. Eight hours later, the mice were anesthetized and irradiated with an 808nm laser for 5 minutes at a power of 1.5W / cm². 2 Temperature changes are recorded every 30 seconds, and images are captured using a near-infrared thermal imager.

[0091] In the PTX+PCP, PTX&PCP / LIP, or PTX&PCP / NL treatment groups, the dosage of PTX was 5 mg / kg and the dosage of PCP was 2 mg / kg.

[0092] 12. Evaluation of tumor suppression in subcutaneous xenografts

[0093] To evaluate the efficacy of PTX&PCP / NL and BBR&RG3 / PL in treating oral squamous cell carcinoma, a mouse model of subcutaneous co-implantation of CAL27+MRC-5 was established, with a tumor volume of approximately 120 mmHg. 3 The drug was divided into 6 groups (n=3): (1) control group; (2) PTX+PCP+L; (3) PTX+PCP+L+BBR+G-RG3+L; (4) PTX&PCP / LIP+L; (5) PTX&PCP / LIP+L+BBR&RG3 / LIP; (6) PTX&PCP / NL+L+BBR&RG3 / PL. The drug was administered every other day, and an 8-hour interval was observed after administration of 1.5 W / cm² for 3 minutes. 2 Near-infrared light irradiation was performed seven times. Tumor size and body weight were monitored every other day after drug treatment. Two weeks later, the mice were euthanized, and major organs (heart, liver, spleen, lungs, and kidneys) and tumors were harvested and fixed in 4% paraformaldehyde. Tumor sections were then stained with H&E and Masson staining.

[0094] The control group received PBS solution; the PTX+PCP+L treatment group consisted of 5 mg / kg PTX and 2 mg / kg PCP, with a photoperiod of 3 min (L); the PTX+PCP+L+BBR+G-RG3+L treatment group consisted of 5 mg / kg PTX, 2 mg / kg PCP, 5 mg / kg BBR, and 5 mg / kg G-RG3, with a photoperiod of 3 min (L); and the PTX&PCP / LIP+L treatment group consisted of 5 mg / kg PTX and 2 mg / kg PCP. The phototherapy time was 3 min (L). The PTX&PCP / LIP+L+BBR&RG3 / LIP treatment group had PTX 5 mg / kg, PCP 2 mg / kg, BBR 5 mg / kg, and G-RG3 5 mg / kg, with a phototherapy time of 3 min (L). The PTX&PCP / NL+L+BBR&RG3 / PL treatment group had the same dosage as PTX&PCP / LIP+L+BBR&RG3 / LIP, but was loaded with platelet membrane and NETs nucleic acid protein complex, respectively.

[0095] 13. Immunological research

[0096] To investigate the function of liposomes in activating anti-tumor immunity in mice, the expression levels of cellular immune factors (TNF-α, IFN-γ, IL-6, and IL-10) in tumor tissues of tumor-bearing mouse models were detected by ELISA. The specific steps were as follows: fresh mouse tumor tissues were collected, minced, added to pre-cooled PBS buffer, and thoroughly ground. The tissues were centrifuged at 4°C, and the supernatant was collected. Following the ELISA kit instructions, the levels of cellular immune factors such as TNF-α, IFN-γ, IL-6, and IL-10 in the supernatant were detected. Finally, quantitative analysis was performed using a microplate reader.

[0097] G1 was the control group: PBS solution; G2 was the PTX+PCP+L treatment group: PTX 5 mg / kg, PCP 2 mg / kg, with a photoperiod of 3 min (L); G3 was the PTX+PCP+L+BBR+G-RG3+L treatment group: PTX 5 mg / kg, PCP 2 mg / kg, BBR 5 mg / kg, G-RG3 5 mg / kg, with a photoperiod of 3 min (L); G4 was the PTX&PCP / LIP+L treatment group: PTX 5 mg / kg, PCP 2 mg / kg, with a photoperiod of 3 min (L). The combined light exposure time was 3 min (L); G5 was the PTX&PCP / LIP+L+BBR&RG3 / LIP treatment group: PTX 5 mg / kg, PCP 2 mg / kg, BBR 5 mg / kg, G-RG3 5 mg / kg, combined with a light exposure time of 3 min (L); G6 was the PTX&PCP / NL+L+BBR&RG3 / PL treatment group, which had the same dosage as PTX&PCP / LIP+L+BBR&RG3 / LIP, but was different in that it was loaded with platelet membrane and NETs nucleic acid protein complex respectively.

[0098] 14. Experiment on inhibiting lung metastasis

[0099] 1×10 via tail vein injection 6 A lung metastasis model of advanced cancer was constructed using CAL27 cells. Mice were then randomly divided into 6 groups (n=3): (1) control group; (2) PTX+PCP+L; (3) PTX+PCP+L+BBR+G-RG3+L; (4) PTX&PCP / LIP+L; (5) PTX&PCP / LIP+L+BBR&RG3 / LIP; (6) PTX&PCP / NL+L+BBR&RG3 / PL. Treatment was administered every 2 days, with a 3-minute pulse at 1.5 W / cm² every 8 hours after administration. 2 Near-infrared light irradiation was performed seven times. Mice were sacrificed 14 days after the end of treatment. The lungs were dissected and fixed with 4% paraformaldehyde. The number of metastatic nodules was measured, and lung tissue sections were stained with H&E.

[0100] In the lung metastasis inhibition experiment, the dosage in the treatment group was the same as that in the tumor suppression evaluation treatment group for subcutaneous xenografts.

[0101] 15. Statistical Analysis

[0102] Data are expressed as mean ± standard deviation. Prism 8.0 (GraphPad) was used for data visualization and statistical analysis. Differences between two groups were assessed using Student's t test or ANOVA, with p < 0.05 considered statistically significant.

[0103] III. Sample Preparation and Experimental Results

[0104] Example 1: Preparation of the first liposome (BBR&RG3 / PL)

[0105] In the embodiments of this application, the first liposome is a platelet membrane-encapsulated macroglobule co-loaded with BBR and G-RG3 (BBR&RG3 / PL), prepared by the following method:

[0106] (1) Preparation of platelet membrane: 1 mL of whole blood from BALB / c mice was collected by ocular sampling into a centrifuge tube containing 50 μL of heparin sodium anticoagulant. The tube was centrifuged at 300 × g for 10 min at 4°C, and the supernatant and the white cell layer at the liquid surface were collected. The supernatant was centrifuged at 2000 × g for 10 min at 4°C, and the white platelet precipitate was collected and washed twice with PBS containing anticoagulant. After repeated freeze-thaw cycles, the platelet membrane was obtained.

[0107] (2) Optimization of BBR&RG3 / PL preparation process: Since G-RG3 can bind with phospholipids and cholesterol to form liposomes, it not only acts as a drug but also participates in the formation of liposomes, which will change the liposome particle size and other properties. Therefore, the effect of G-RG3 dosage on the formulation particle size, drug loading and encapsulation efficiency needs to be investigated in the preparation of BBR&RG3 / LIP.

[0108] Different mass ratios of BBR and G-RG3 (1:1, 3:1, 1:3) were selected to prepare co-loaded liposomes (BBR&RG3 / LIP) using the ethanol injection method. The core structure of the liposomes included lecithin, cholesterol, and ginsenoside Rg3 in a mass ratio of 24:8:1. The specific steps included: dissolving 120 mg of lecithin, 40 mg of cholesterol, 5 mg of berberine, and 5 mg of ginsenoside Rg3 in 2 mL of anhydrous ethanol to obtain a first solution; using 8 mL of PBS solution as a second solution, the second solution was placed on a constant temperature magnetic stirrer (50°C~60°C, 20 rpm~30 rpm) and stirred. Then, the first solution was added dropwise to the second solution, and the anhydrous ethanol was removed by rotary evaporation to obtain BBR&RG3 / LIP.

[0109] (3) Then determine the ratio of platelet membrane to liposome. According to the ratio of platelets taken from 1 mL of whole blood to liposomes prepared from 120 mg of lecithin, the solution obtained in step (2) was broken by sonication in an ice bath, and the platelet membrane prepared in step (1) was added and mixed. The platelet membrane was then wrapped on the surface of the liposome by extrusion to obtain BBR&RG3 / PL. The morphology and the platelet membrane wrapping were observed by electron microscopy.

[0110] Results of BBR&RG3 / PL preparation process optimization: BBR&RG3 / LIP prepared with different mass ratios of BBR and G-RG3 in step (2) were tested for particle size, potential, drug loading, and encapsulation efficiency; subsequently, platelet membrane and BBR&RG3 / LIP were co-extruded by extrusion and passed through 440nm and 220nm polyethersulfone filters to obtain BBR&RG3 / PL, and its particle size, potential, drug loading, encapsulation efficiency, and drug synergy were tested. The results are shown in Table 1 and Figure 2 As shown, Figure 2 Figure A shows the particle size of unencapsulated platelet membrane liposomes BBR&RG3 / LIP prepared with BBR and G-RG3 at a mass ratio of 1:3; Figure B shows the particle size of unencapsulated platelet membrane liposomes BBR&RG3 / LIP prepared with BBR and G-RG3 at a mass ratio of 3:1; Figure C shows the particle size of unencapsulated platelet membrane liposomes BBR&RG3 / LIP prepared with BBR and G-RG3 at a mass ratio of 1:1; and Figure D shows the particle size of encapsulated platelet membrane liposomes BBR&RG3 / PL prepared with BBR and G-RG3 at a mass ratio of 1:1. (From Table 1 and...) Figure 2 The results showed that as G-RG3 increased, the liposome particle size decreased and the drug loading of BBR decreased. This may be because G-RG3 has a steroidal structure similar to cholesterol, which is embedded in the lipid bilayer, making its structure more compact, resulting in smaller particle size and reduced encapsulation efficiency. However, after being encapsulated in the platelet membrane, the particle size increased slightly.

[0111] Table 1. Physicochemical properties of BBR&RG3 liposomes

[0112]

[0113] Based on the above experimental results, a synergistic test of BBR and G-RG3 was conducted: using the CAL27 cell model, the toxicity of BBR and G-RG3 to tumor cells was detected by the MTT assay. The results are as follows: Figure 1 As shown in Figure A, the results indicate that the two drugs did not exhibit significant synergy. Considering all indicators, a 1:1 mass ratio of BBR to G-RG3, which yielded the highest drug utilization, was ultimately selected as the formulation for the first liposome.

[0114] BBR&RG3 / LIP and BBR&RG3 / PL structural assays: Comparison of BBR&RG3 / LIP and BBR&RG3 / PL liposomes, results are as follows Figure 3 As shown, from Figure 3 Figure A and Figure 3 As can be seen from Figure B, BBR&RG3 / PL has a more obvious shell-membrane structure compared to BBR&RG3 / LIP, and from... Figure 3As shown in Figure C, the Coomassie Brilliant Blue and WB results indicate that the formulation encapsulating the platelet membrane has the same protein expression as the platelet membrane, and the CD44 protein band is clear.

[0115] Example 2: Preparation of the second liposome (PTX & PCP / NL)

[0116] In this embodiment, the second liposome NETs nucleic acid-protein complex encapsulating a photothermal targeting liposome (PTX&PCP / NL) co-loaded with PTX and PCP is prepared by the following method:

[0117] (1) Preparation of neutrophil extracellular traps (NETs nucleic acid-protein complexes): Neutrophils were stimulated with phorbol 12-tetradecanoate 13-acetate (PMA) to induce neutrophil extracellular traps (NETs nucleic acid-protein complexes). The specific steps are as follows: Neutrophils extracted from mouse peripheral blood were resuspended in RPMI 1640, stimulated with 50 nM PMA for 8 h, the culture medium was removed, and the cells were washed with RPMI 1640 medium. At this concentration, PMA did not promote apoptosis or necrosis, but caused the characteristic formation of NETs nucleic acid-protein complexes. After removing the supernatant, 2 mL of cold PBS was added to wash the NETs adhering to the bottom of the six-well plate, and the plate was centrifuged at 4°C, 1000×g for 10 min. The precipitate was collected and resuspended with PBS, and centrifuged at 20×g for 5 min to collect the NETs nucleic acid-protein complexes (DNA-protein complexes).

[0118] (2) Photothermal liposomes co-loaded with PTX and PCP were prepared by thin-film dispersion method. According to different ratios of positive phospholipid (2,3-dioleoxypropyl)trimethylammonium chloride (DOTAP) to cholesterol (2:1, 3:1, 4:1, 5:1), DOTAP, cholesterol, PTX and PCP were dissolved in 2 mL of chloroform. The chloroform was removed by rotary evaporation under the conditions of 50°C~60°C water bath and 10rpm~30rpm. 8 mL of PBS solution was added for hydration for 45min~60min. The resulting solution was then ultrasonically broken up in an ice bath to obtain PTX&PCP / LIP. By testing the potential, encapsulation efficiency and particle size of PTX&PCP / LIP, the mass ratio of DOTAP to cholesterol was finally selected as 3:1 to prepare PTX&PCP / LIP.

[0119] (3) Add NETs nucleic acid protein complex and co-incubate for 1 h to adsorb NETs nucleic acid protein complex onto PTX&PCP / LIP to reverse its positive potential. Then pass through 0.45 μm and 0.22 μm polyether sulfone filter membranes in sequence to obtain liposomes PTX&PCP / NL encapsulated by NETs nucleic acid protein complex.

[0120] Screening of the amount of NETs nucleic acid protein complex in PTX&PCP / NL: PTX&PCP / LIP was prepared by thin film dispersion method. The pre-extracted NETs nucleic acid protein complex was added and co-incubated at 37℃ for 10 min. The NETs nucleic acid protein complex was encapsulated on the surface of the formulation by electrostatic adsorption of the positive and negative charges of positive phospholipids (DOTAP). The mixture was then filtered through 440 nm and 220 nm polyethersulfone membranes to obtain PTX&PCP / NL. Its particle size, potential, drug loading, encapsulation efficiency and other characterization characteristics were detected.

[0121] Table 2. Screening results of the amount of NETs nucleic acid protein complex in PTX&PCP / NL.

[0122]

[0123] Due to the addition of positive phospholipids, the PTX&PCP / LIP potential differed from that of traditional liposomes, being 34.03 mV (Table 2). However, after incubation with the NETs nucleic acid protein complex, when the ratio of DOTAP to the NETs nucleic acid protein complex was 100:1, the potential reversed from positive to negative. Ultimately, the optimal addition ratio of the NETs nucleic acid protein complex was determined to be 100:1. The particle size of the second liposome with the added NETs nucleic acid protein complex was slightly larger. Figure 4 (Figures A and B) and the potential reverses to -5.22mV, which is more conducive to its circulation in vivo. Compared with PTX&PCP / LIP, the electron micrographs of PTX&PCP / NL ( Figure 5 The presence of a clear shell-membrane structure in Figures A and B confirms that the NETs nucleic acid-protein complex was successfully encapsulated on the formulation surface. To determine whether the NETs nucleic acid-protein complex still contains the characteristic protein before and after encapsulation, we used Coomassie Brilliant Blue staining (…). Figure 5 (Figure C) Comparing the protein bands of the NETs nucleic acid-protein complex and PTX&PCP / NL, it was found that PTX&PCP / NL still contained the characteristic protein bands of the NETs nucleic acid-protein complex, with no significant protein loss. The final preparation method of PTX&PCP / NL was determined by comprehensively considering the PCP concentration, its photothermal conversion efficiency, drug loading, and encapsulation efficiency.

[0124] Table 3 Physicochemical properties of PTX & PCP liposomes

[0125]

[0126] (4) Coordination test of PTX and PCP

[0127] The CAL27 cell model was used, and the toxicity of PTX and PCP to tumor cells was detected by the MTT assay. The results are as follows: Figure 1As shown in Figure B, the results indicate that PTX and PCP exhibit significant synergy at a mass ratio of 2:1. Considering the particle size, encapsulation efficiency, and stability of the formulation, a mass ratio of 2.5:1 for PTX and PCP was ultimately selected as the formulation for the second liposome.

[0128] The formulation of the second liposome PTX&PCP / NL is as follows: 120 mg of positive phospholipid (DOTAP), 40 mg of cholesterol, 5 mg of PTX, 2 mg of PCP, and 1.2 mg of NETs nucleic acid protein complex.

[0129] Example 3: Performance Testing

[0130] (1) In vitro stability testing of PTX & PCP / NL and BBR & RG3 / PL

[0131] To investigate the in vitro stability of the formulations, the prepared PTX&PCP / NL and BBR&RG3 / PL were diluted tenfold in DMEM culture medium containing 10% FBS, and their particle size was measured every other day for a total of seven times. The results showed that ( Figure 6 PTX&PCP / NL and BBR&RG3 / PL showed no significant change in particle size within 14 days, indicating good stability.

[0132] (2) In vitro drug release performance testing of PTX&PCP / NL and BBR&RG3 / PL

[0133] To investigate whether platelet membrane encapsulation affects the drug release performance of liposomes, the in vitro release effects of BBR&RG3 / PL and BBR&RG3 / LIP were compared. Figure 7 As shown, the formulation exhibits better sustained-release efficacy after being encapsulated by the platelet membrane.

[0134] To investigate whether light irradiation affects the drug release of PTX & PCP / NL, near-infrared light irradiation at a power of 1.5 W / cm² was applied 8 hours after drug release. 2 Time: 5 minutes; Results: Figure 8 As shown, after light irradiation (PTX&PCP / NL+L), the release rate of PTX in PTX&PCP / NL is significantly accelerated, indicating that the drug release of PTX&PCP / NL has near-infrared light responsiveness, which is expected to realize photoresponsive drug controlled release.

[0135] (3) In vitro photothermal performance of PTX & PCP / NL

[0136] To investigate the photothermal conversion efficiency of the formulation in vitro, PTX&PCP / NL concentrations were diluted to 20 μg / mL, 40 μg / mL, 60 μg / mL, 125 μg / mL, and 250 μg / mL, with PBS as a control, at 1.5 W / cm².2 Near-infrared light was irradiated at a power of [value], and its temperature change curve was plotted. The results are as follows: Figure 9 As shown, within 5 minutes, the concentration was positively correlated with the rate of temperature rise and the maximum temperature. 20 μg / mL reached 42℃ in 5 minutes, while there was no significant difference between 125 μg / mL and 250 μg / mL.

[0137] Mild photothermal therapy (41℃~45℃) aims to induce immunogenic cell death and assist chemotherapy drugs in killing tumor cells. The drug loading capacity of PTX&PCP / NL can meet the needs of photothermal therapy.

[0138] (4) Evaluation of hemolytic activity

[0139] The results of the hemolysis test are as follows Figure 10 As shown, both NLIP (PTX&PCP / NL) and PLIP (BBR&RG3 / PL) exhibited hemolysis rates of less than 5% (maximum 4.94%) within 5 mg / mL, indicating that both vectors have good biocompatibility.

[0140] (5) Generation of reactive oxygen species

[0141] Reactive oxygen species (ROS) are a key marker of photothermal therapy-induced immunogenic cell death. This study investigated ROS generation to assess whether drug combination therapy affects ROS production. Results are as follows: Figure 11 As shown, the combined drug regimen significantly increased the production of reactive oxygen species (ROS), with the final formulation group exhibiting the highest ROS production. This indicates that the photothermal-chemotherapy-immune three-dimensional regulatory formulation can effectively enhance ROS generation.

[0142] (6) Validation of immunogenicity-induced death

[0143] To verify the photothermal effect of PCP inducing ICD (immunogenic cell death), supernatants were extracted after treatment with each drug formulation, and changes in adenosine triphosphate (ATP), a hallmark of ICD, were detected. The results showed ( Figure 12 After photothermal therapy, the release of ATP was effectively increased, while the introduction of BBR and G-RG3 did not cause a significant change in the release of APT. The final formulation group had the highest release.

[0144] (7) In vitro drug uptake and retention experiments of PTX&PCP / NL and BBR&RG3 / PL

[0145] To investigate the uptake of PTX & PCP / NL and BBR & RG3 / PL in CAL27 and MRC-5 cells, coumarin 6 was used instead of PTX and BBR. DAPI was used as the green fluorescence tracking agent to locate the cell nucleus. The two dyes were co-localized to the cells, and the staining effect was observed. The fluorescence intensity was further quantified by flow cytometry (FCM).

[0146] In MRC-5 ( Figure 13 Figures A and B) and CAL27 ( Figure 13 In the confocal images (Figures C and D) and flow cytometry experiments, after encapsulation with the biomimetic membrane, the uptake effect of both C6&PCP / NL and C6&RG3 / PL was stronger than that of traditional liposomes, and there was no significant difference compared with free drugs. This indicates that encapsulation with the biomimetic membrane is beneficial for the uptake of the formulation by cells.

[0147] (8) In vitro drug toxicity detection of PTX&PCP / NL and BBR&RG3 / PL

[0148] The toxicity of each drug group to CAL27+MRC-5 co-cultured cells was detected by the MTT assay. The results showed that ( Figure 14 PCP alone exhibits low cytotoxicity in mixed cell processes, but shows strong cytotoxicity after light exposure, and shows good synergistic effects when used in combination with PTX. Furthermore, BBR and G-RG3 alone have no significant cytotoxicity, but show great anti-tumor potential when used in combination with PTX and PCP. This may be because BBR and G-RG3 themselves have no significant killing effect on tumor cells, but can inhibit the promoting effect of MRC-5 on CAL27, causing tumor cells to lose the protection of tumor-associated fibroblasts, indirectly enhancing the killing effect of chemotherapy drugs and photothermal therapy on tumor cells.

[0149] (9) In vitro anti-migration experiments of PTX&PCP / NL and BBR&RG3 / PL

[0150] To evaluate the anti-migration effects of each formulation on tumor cells, we separately tested the CAL27 single-cell model (…). Figure 15 (Figures A and B) and the CAL27+MRC-5 co-culture cell model ( Figure 15Scratch assays were performed (Figures C and D). Results showed that the co-culture model had a higher migration rate (58.21%) compared to the traditional single-cell model (33.95%). In the single-cell culture model, BBR+G-RG3 had negligible inhibitory effect on cell migration (10.54%), while in the co-culture model, it effectively inhibited tumor cell migration (24.19%). This may be because BBR+G-RG3 interrupted the pro-migration effect of CAFs on tumor cells, but had no significant inhibitory effect on tumor cells alone. Mild photothermal therapy alone had almost no inhibitory effect on tumor migration, but when combined with the chemotherapy drug PTX, it effectively inhibited tumor cell migration (43.05%). Furthermore, the drug combination effectively inhibited tumor cell migration, with the best inhibitory effect observed in the final formulation group (PTX&PCP / NL+BBR&RG3 / PL+L) (76.55%).

[0151] Subsequently, a Transwell assay was performed to further evaluate the anti-invasive efficacy of the formulation. Figure 16 The results were similar to those of the scratch assay, showing that the final formulation effectively inhibited tumor cell invasion.

[0152] (10) Biodistribution of PTX&PCP / NL and BBR&RG3 / PL in vivo

[0153] To verify the tumor-targeting properties of the formulation, DID was used instead of PTX, and DIR was used instead of BBR. The distribution of the formulation in mice was tracked using a small animal in vivo imaging system. The results showed that the free drug group did not accumulate significantly at the tumor site, while after encapsulation with the biomimetic membrane, fluorescence accumulated significantly at the tumor site at 8 hours and remained strong at 48 hours, indicating good tumor targeting and long-term circulating effect in vivo. Figure 17 As shown in Figure A). Results of fluorescence imaging and quantitative fluorescence quantification of major organs (…). Figure 17 In Figures B, C, and D, the fluorescence in the liver of the free drug group was the strongest, indicating that most of the drug is metabolized in the liver. After being encapsulated by the biomimetic membrane, the accumulation of fluorescence at the tumor site was effectively improved, demonstrating good tumor targeting.

[0154] (11) Detection of in vivo photothermal conversion performance of PTX & PCP / NL

[0155] To investigate the photothermal conversion effect of the formulations at animal tumor sites, free PTX+PCP, PTX&PCP / LIP, and PTX&PCP / NL were injected into mice (at the same dosage as in the tumor inhibition evaluation test of subcutaneous xenografts), at a concentration of 1.5 W / cm². 2 Near-infrared light was used to irradiate tumor sites in mice, and the temperature change over time was observed. The results showed that ( Figure 18PTX & PCP / NL exhibit better photothermal conversion efficiency at the tumor site, reaching 48.3°C at 3 minutes, meeting the requirements for mild photothermal therapy. In contrast, free drugs and traditional drug-loaded liposomes still have lower temperatures at 5 minutes, failing to meet the needs of photothermal therapy. This may be due to the highly efficient tumor-targeting properties of liposomes encapsulated by NETs nucleic acid-protein complexes, leading to greater accumulation of PCP at the tumor site.

[0156] (12) In vivo antitumor and biocompatibility analysis of PTX&PCP / NL and BBR&RG3 / PL

[0157] To evaluate the in vivo antitumor effect of the formulation, a subcutaneous tumor-bearing model of CAL27 was constructed. Results showed no significant change in mouse body weight during treatment. Figure 20 The final formulation group had the smallest tumor volume ( Figure 19 , Figure 21 Figure A shows that it has the strongest tumor-suppressing efficiency. Figure 21 Figure B). In each group of tumors, H&E (… Figure 22 (Figure A) and Masson ( Figure 22 The staining results (Figure B) show that the combined drug significantly promoted tumor apoptosis and reduced collagen fiber deposition. Compared with traditional liposomes, biomimetic membrane encapsulation has a stronger anti-tumor effect. Furthermore, the H&E staining results of major organs in mice after treatment (Figure B) Figure 23 In the study, no significant tissue damage was found in any of the groups, indicating that the final formulation has good biosafety.

[0158] (13) Histoimmunological testing

[0159] The expression of pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL-6 (high levels inhibit tumor development) and the immunosuppressive factor IL-10 (inhibits tumor immune responses and promotes tumor progression) in tumor tissue was detected to assess the immune changes induced by drug treatment. The results showed ( Figure 24 (Figures A, B, and C) The combined drug therapy significantly increased the expression of TNF-α, IFN-γ, and IL-6, and decreased the expression of the immunosuppressive factor IL-10 (…). Figure 24 (See Figure D). This effect was particularly pronounced in the final formulation group. This may be because photothermal chemotherapy and ginsenoside Rg3 jointly regulate the immune system, thereby enhancing the immune system's anti-tumor function.

[0160] (14) PTX&PCP / NL and BBR&RG3 / PL in vivo anti-lung metastasis

[0161] Advanced tumor metastasis remains a major challenge in clinical cancer treatment. To evaluate the anti-metastatic effect of the formulation, an advanced lung metastasis model was established by tail vein injection of CAL27. Figure 25 As shown ( Figure 25 (Figures A and B) After drug combination, the number of metastatic lung nodules decreased, with the final formulation group having the fewest nodules. This was observed in the lung H&E staining results ( Figure 25 As shown in Figure C, the biomimetic membrane significantly reduced lung tumor metastasis compared to traditional liposomes. This may be because the platelet membrane can target tumor cells via P-selectin, and the naked nucleic acid proteins of the NETs nucleic acid protein complex can capture circulating tumor cells, targeting and killing them to inhibit tumor metastasis.

[0162] In the above embodiments, the descriptions of each embodiment have different focuses. For parts not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0163] The foregoing has provided a detailed description of a multifunctional biomimetic nano-formulation, its preparation method, and its application. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. At the same time, those skilled in the art will recognize that there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A multifunctional biomimetic nano-formulation, characterized in that, The multifunctional biomimetic nanoparticle formulation includes a first liposome and a second liposome; the first liposome is a liposome co-loaded with berberine and ginsenoside Rg3 encapsulated in a platelet membrane; the structure of the second liposome includes a cationic liposome co-loaded with paclitaxel and a photothermal agent encapsulated in an extracellular trap of neutrophils; the first liposome includes lecithin, cholesterol and ginsenoside Rg3 in a mass ratio of (20~30):(5~10):(1~1.5); the berberine loading is 2%~4% based on the total mass of the first liposome; the amount of platelet membrane used is: M1=(M2×M0) / 120, where M1 is the mass of the platelet membrane in mg, M2 is the mass of lecithin in mg, and M0 is the mass of the platelet membrane extracted from 1 mL of whole blood in mg; The second liposome comprises (2,3-dioleoxypropyl)trimethylammonium chloride and cholesterol in a mass ratio of (20-30):(5-10); the drug loading of paclitaxel is 2%-5% based on the total mass of the second liposome, and the drug loading of the photothermal agent is 1%-3%; the mass ratio of the neutrophil extracellular trap to the (2,3-dioleoxypropyl)trimethylammonium chloride is 1:

100.

2. The multifunctional biomimetic nano-formulation according to claim 1, characterized in that, The mass ratio of lecithin, cholesterol, and ginsenoside Rg3 is 24:8:

1.

3. The multifunctional biomimetic nano-formulation according to claim 1, characterized in that, The mass ratio of (2,3-dioleoxypropyl)trimethylammonium chloride to cholesterol is 3:

1.

4. The multifunctional biomimetic nano-formulation according to claim 1, characterized in that, The average particle size of the first liposome ranges from 91 nm to 105 nm; the average particle size of the second liposome ranges from 147 nm to 160 nm.

5. The multifunctional biomimetic nano-formulation according to claim 1, characterized in that, The neutrophil extracellular traps were prepared by stimulating neutrophils with phorbol 12-tetradecanoate 13-acetate to induce the neutrophil extracellular traps.

6. The method for preparing multifunctional biomimetic nanoparticles according to claim 1, characterized in that, Includes the following steps: Preparation of the first liposome: Lecithin, cholesterol, berberine, and ginsenoside Rg3 were dissolved in anhydrous ethanol to obtain the first solution. PBS solution was used as the second solution. The second solution was heated to 50℃~60℃. The first solution was then added dropwise to the second solution. Anhydrous ethanol was removed by rotary evaporation. The resulting solution was sonicated in an ice bath, mixed with platelet membrane, and extruded to obtain the first liposome. Preparation of the second liposome: (2,3-dioleoxypropyl)trimethylammonium chloride, cholesterol, paclitaxel and photothermal agent were dissolved in chloroform, the chloroform was removed by rotary evaporation, PBS solution was added for hydration, the resulting solution was sonicated in an ice bath, and co-incubated with extracellular traps of neutrophils. After filtration, the second liposome was obtained.

7. The use of a multifunctional biomimetic nanoformulation prepared by any one of claims 1 to 5 or by the preparation method of the multifunctional biomimetic nanoformulation as described in claim 6 in the preparation of drugs for treating tumors and drugs for inhibiting tumor metastasis.

8. The application according to claim 7, characterized in that, The tumor in question is oral squamous cell carcinoma.

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