Preparation method and application of diABZI-loaded targeted nanoparticle preparation

Abstract

The present invention discloses a preparation method and application of a diABZI-loaded targeted nanoformulation, and relates to the field of biomedicine technology. The present invention combines a stem cell membrane and a targeting peptide pHLIP, which helps to enhance the targeting of the nanoformulation to colorectal cancer cells; stem cells are used as the coating material of nanoparticles, and the stem cell membrane has excellent biocompatibility and bioactivity, which enhances the stability and bioactivity of the nanoparticles in the body. diABZI is an effective drug for treating colorectal cancer. By loading it into nanoparticles and preferentially delivering it to the lesion site, effective enrichment of diABZI in colorectal cancer can be achieved, thereby exerting its therapeutic effect and reducing the body's inflammatory response. The stem cell membrane bionic targeted nanoformulation loaded with diABZI prepared by the present invention provides a new method for the precise treatment of colorectal cancer through targeted delivery and effective enrichment strategies, improves the therapeutic effect and biosafety, and marks an important technological progress in this field.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a preparation method and application of a diABZI-loaded targeted nano preparation. Background Art

[0002] The liver is the main target organ for hematogenous metastasis of colorectal cancer (CRC) cells. About 15% to 25% of patients develop liver metastasis after surgery for the primary CRC lesion, and the vast majority (80% to 90%) of liver metastases cannot be radically resected. The 2023 Chinese Society of Clinical Oncology (CSCO) diagnosis and treatment guidelines point out that the addition of immune checkpoint inhibitors (PD-1 / PD-L1) to the treatment of grade III CRC liver metastasis (CRLM) marks the entry of the clinical treatment of CRLM into the era of immunotherapy. PD-1 / PD-L1 can enhance the immune capacity of immune cells and exert anti-tumor effects. However, most patients have too little immune cell infiltration in the tumor microenvironment and respond poorly to PD-1 / PD-L1 treatment. Therefore, it is of great clinical value to find alternative immunotherapy methods in the treatment of CRC, expand the scope of application of immunotherapy, and thus improve the immunotherapy effect of CRC.

[0003] Diamidobenzimidazole (diABZI) is a new type of STING agonist that can be injected intravenously. It exerts a strong anti-tumor effect by remodeling the tumor microenvironment. diABZI also has a good anti-tumor effect on "cold tumors" by promoting the initiation and recruitment of specific CD8+ T cells in CRC tissues, thereby making up for the insufficient clinical therapeutic effect of PD-1 / PD-L1. Therefore, non-nucleic acid STING agonists may be a blockbuster in the field of CRC immunotherapy after PD-1 / PD-L1. However, STING protein is widely expressed in immune cells and non-immune cells. In addition to producing the tumor-killing factor interferon β (IFN-β), systemic administration of STING agonists or other administration methods can easily induce increased inflammatory cytokines, leading to inflammatory diseases and autoimmune diseases, and even triggering a "cytokine storm". Therefore, exploring more effective diABZI targeted delivery strategies is of great significance for CRC immunotherapy.

[0004] Poly(lactic-co-glycolic acid) is a degradable high molecular organic compound with good biocompatibility, low immunogenicity, good encapsulation and film-forming properties. It has been widely used in the field of drug delivery and tumor immunotherapy. Biomimetic nanoparticles based on mesenchymal stem cell (MSC) membranes show the ability to enhance blood circulation, targeting and reduce immune escape of nanoparticles. The cumbersome process of modifying nanoparticles by traditional chemical means is avoided; in addition, the biomimetic nanomaterials of stem cell membranes enter the blood and can effectively avoid the adsorption of a large amount of plasma proteins on the surface. The above advantages are due to the camouflage function of the biological membrane, which becomes a "nano protective clothing". Therefore, biomimetic nanoparticles based on stem cell membranes are expected to enhance the effective accumulation of drugs in CRC cells. At the same time, the uniqueness of the inserted targeting peptide pHLIP is that it can use the acidic environment of the tumor microenvironment (tumor cells are usually more acidic than normal cells) to deliver drugs, imaging agents or other therapeutic molecules to tumor cells in a targeted manner. Summary of the invention

[0005] The technical solution of the present invention to solve the above technical problems is to provide a method for preparing a diABZI-loaded targeted nanoformulation, comprising the following steps:

[0006] Step 1: preparing polylactic acid nanoparticles of diABZI: using a nano-sedimentation method, an organic solvent containing PLGA and diABZI is added dropwise to ultrapure water containing a surfactant, and an emulsion is prepared by magnetic stirring and ultrasonic treatment under ice bath conditions; the solvent is rotatably volatilized to obtain polylactic acid nanoparticles loaded with diABZI;

[0007] Step 2: Encapsulating stem cells: extracting stem cell membranes, and repeatedly extruding them through polycarbonate membranes with different pore sizes using a liposome extruder, so as to encapsulate the cell membranes on the surface of polylactic acid nanoparticles loaded with diABZI, thereby forming stem cell membrane-encapsulated polylactic acid biomimetic nanoparticles loaded with diABZI;

[0008] Step 3: Preparation of bionic targeted nanoformulations: vertically suspend the solution containing the targeting polypeptide pHLIP and the diABZI-loaded polylactic acid bionic nanoformulation coated with the stem cell membrane, and insert the targeting polypeptide pHLIP into the lipid bilayer of the cell membrane by the post-insertion method to form a targeting polypeptide pHLIP-modified diABZI-loaded polylactic acid bionic targeted nanoformulation.

[0009] Furthermore, the preparation of diABZI polylactic acid nanoparticles comprises the following steps:

[0010] The surfactant polyvinyl alcohol (PVA) or sodium cholate or the emulsifier Tween-20 is dissolved in ultrapure water.

[0011] PLGA and diABZI were dissolved in an organic solvent.

[0012] PLGA and diABZI organic solvent were added dropwise into ultrapure water containing surfactant, magnetically stirred for 6 h, and ultrasonically prepared emulsion under ice bath conditions;

[0013] The emulsion is subjected to rotary volatilization to obtain polylactic acid nanoparticles loaded with diABZI;

[0014] Wherein, the organic solvent includes dichloromethane and / or acetone; and the concentration of the surfactant is 1%.

[0015] Furthermore, the ultrasound intensity is 50%, the ultrasound time is 30 s, and the ultrasound mode is pulsed.

[0016] Furthermore, the method for rotating and volatilizing the solvent is a solvent volatilization method or a solvent diffusion method.

[0017] Furthermore, in step 2, the pore sizes of the polycarbonate membrane are 200 nm, 400 nm and 800 nm; and the number of extrusions is 10 times.

[0018] Furthermore, in step 3, the treatment suspension time is 6 hours.

[0019] In order to solve the above technical problems, the present invention also proposes the use of diABZI-loaded targeted nanoformulation in the treatment of colorectal cancer. The diABZI-loaded targeted nanoformulation is prepared by the preparation method of the diABZI-loaded targeted nanoformulation as described above.

[0020] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0021] 1. This application combines stem cell membrane and targeting peptide pHLIP, which helps to enhance the targeting of nanoformulations to colorectal cancer cells, thereby improving the therapeutic effect.

[0022] 2. This application uses stem cells as the coating material of nanoparticles. The stem cell membrane has excellent biocompatibility and bioactivity, which enhances the stability and bioactivity of nanoparticles in the body. In addition, the stem cell membrane also helps to achieve specific targeting of diseased tissues and reduce the impact on normal tissues.

[0023] 3. diABZI is an effective drug for the treatment of colorectal cancer. By loading it into nanoparticles and preferentially delivering it to the lesion site, diABZI can be effectively enriched in colorectal cancer, thereby exerting its therapeutic effect and reducing the body's inflammatory response.

[0024] In summary, stem cell membrane biomimetic targeted nanoformulations loaded with diABZI provide a new approach for the precise treatment of colorectal cancer through targeted delivery and effective enrichment strategies, improve therapeutic efficacy and biosafety, and mark an important technological advance in this field. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0026] Figure 1 This is a flow chart of the steps of the preparation method of the diABZI-loaded targeted nanoformulation of the present invention;

[0027] Figure 2 TEM images of diABZI@NP, stem cell membrane (MSCM) and diABZI@NP-M-pHLIP nanoparticles of the present invention;

[0028] Figure 3 : is the particle size distribution diagram of diABZI@NP and diABZI@NP-M-pHLIP nanoparticles of the present invention;

[0029] Figure 4 This is a diagram of the target head pHLI P of the present invention being linked to the outer layer of nanoparticles wrapped with stem cells;

[0030] Figure 5 For this purpose, it was determined that the stem cell membrane was wrapped in the outer layer of PLGA;

[0031] Figure 6 This is a laser confocal image showing the cellular uptake of diABZI@NP and diABZI@NP-M-pHLIP nanoparticles of the present invention;

[0032] Figure 7 This is a flow cytometry diagram showing the cellular uptake of diABZI@NP-M-pHLIP nanoparticles of the present invention;

[0033] Figure 8 Cell apoptosis diagram (%) of CT26 cells treated with diABZI@NP and diABZI@NP-M-pHLIP preparations of the present invention;

[0034] Fig. 9 Colony formation images and scratch images of CT26 cells after treatment with diABZI@NP and diABZI@NP-M-pHLIP preparations of the present invention (n=3);

[0035] Fig.10 is a schematic diagram of the acute toxicity and sample collection scheme of the present invention;

[0036] Fig.11 This is a graph showing changes in the body weight of mice of the present invention;

[0037] Fig.12 ALT, HYP and AST levels were measured for the present invention;

[0038] Fig.13 The present invention evaluates BUN, UA and serum CRE level graphs;

[0039] Fig.14 Figure E shows the in vivo optical analysis of the biodistribution of Cy5.5-labeled NPs in orthotopic CRC mice after 12 hours; Figure F shows the assessment of CRC tumor growth in mice.

[0040] Fig.15 This is a graph showing the survival rate of mice after tail vein injection of different preparations of the present invention;

[0041] Fig.16 Figure 2 shows the tumor weight of mice after tail vein injection of different preparations of the present invention (n=6 in each group);

[0042] Fig.17 This is a diagram showing the expression of IRF3 and TBK1 in mouse tumors of the present invention. DETAILED DESCRIPTION

[0043] A preparation method and application of a diABZI-loaded targeted nanoformulation is intended to design a preparation method of a diABZI-loaded targeted nanoformulation capable of improving therapeutic effects.

[0044] The preparation method of the diABZI-loaded targeted nanoformulation proposed by the present invention will be described in the following specific examples:

[0045] In the technical solution of this embodiment, Figure 1 As shown, a method for preparing a diABZI-loaded targeted nanoformulation comprises the following steps:

[0046] Step 1: preparing polylactic acid nanoparticles of diABZI: using a nano-sedimentation method, an organic solvent containing PLGA and diABZI is added dropwise to ultrapure water containing a surfactant, and an emulsion is prepared by magnetic stirring and ultrasonic treatment under ice bath conditions; the solvent is rotatably volatilized to obtain polylactic acid nanoparticles loaded with diABZI;

[0047] Step 2: Encapsulating stem cells: extracting stem cell membranes, and repeatedly extruding them through polycarbonate membranes with different pore sizes using a liposome extruder, so as to encapsulate the cell membranes on the surface of polylactic acid nanoparticles loaded with diABZI, thereby forming stem cell membrane-encapsulated polylactic acid biomimetic nanoparticles loaded with diABZI;

[0048] Step 3: Preparation of bionic targeted nanoformulations: vertically suspend the solution containing the targeting polypeptide pHLIP and the diABZI-loaded polylactic acid bionic nanoformulation coated with the stem cell membrane, and insert the targeting polypeptide pHLIP into the lipid bilayer of the cell membrane by the post-insertion method to form a targeting polypeptide pHLIP-modified diABZI-loaded polylactic acid bionic targeted nanoformulation.

[0049] Specifically, polylactic acid-polyglycolic acid (PLGA) is a widely used biodegradable polymer, often used in the production of drug carriers and biomedical engineering materials. The addition of PEG can effectively increase the blood circulation time of PLGA and avoid being cleared by the mononuclear phagocytic system (RES). The amphiphilicity of PLGA-PEG enables it to self-assemble, with the hydrophobic PLGA located inside the micelle and PEG located outside, forming a stable polymer micelle structure.

[0050] The particle size dispersion coefficient of diABZI-loaded targeted nanoparticles ranges from 0.1 to 0.2. In most applications, a smaller PDI (usually less than 0.2) is considered ideal because it indicates a narrow size distribution of the nanoparticles, which means that the size and shape of the particles are relatively consistent. The biomimetic modification of diABZI-loaded targeted nanoparticles is a stem cell membrane, such as mesenchymal stem cells, hematopoietic stem cells, bone marrow stem cells, and tumor stem cells. The diABZI-loaded targeted nanoparticles can target colorectal cancer cells in an acidic environment, including but not limited to colorectal cancer, such as liver cancer, lung cancer, and breast cancer.

[0051] Targeted delivery strategy: The targeting of pHLIP depends on the acidity of the tumor microenvironment, which enables it to target all acidic tumors, not just colorectal cancer. Studies have shown that pHLIP can target a variety of tumors such as breast cancer, lung cancer, and prostate cancer. This study used colorectal cancer as the subject to verify the therapeutic effect of the targeted nanoformulation loaded with diABZI. This method combines stem cell membranes and the targeting peptide pHLIP, which helps to enhance the targeting of the nanoformulation to colorectal cancer cells, thereby improving the therapeutic effect.

[0052] Stem cell membrane coating: As a coating material for nanoparticles, stem cell membrane has excellent biocompatibility and bioactivity, enhancing the stability and bioactivity of nanoparticles in the body. In addition, stem cell membrane can also help to achieve specific targeting of diseased tissues and reduce the impact on normal tissues.

[0053] Reduce toxic side effects: diABZI is an effective drug for the treatment of colorectal cancer. By loading it into nanoparticles and preferentially delivering it to the lesion site, diABZI can be effectively enriched in colorectal cancer, thereby exerting its therapeutic effect and reducing the body's inflammatory response.

[0054] In summary, stem cell membrane biomimetic targeted nanoformulations loaded with diABZI provide a new approach for the precise treatment of colorectal cancer through targeted delivery and effective enrichment strategies, improve therapeutic efficacy and biosafety, and mark an important technological advance in this field.

[0055] Furthermore, the preparation of diABZI polylactic acid nanoparticles comprises the following steps:

[0056] The surfactant polyvinyl alcohol (PVA) or sodium cholate or the emulsifier Tween-20 is dissolved in ultrapure water.

[0057] PLGA and diABZI were dissolved in an organic solvent.

[0058] PLGA and diABZI organic solvent were added dropwise into ultrapure water containing surfactant, magnetically stirred for 6 h, and ultrasonically prepared emulsion under ice bath conditions;

[0059] The solvent is evaporated by rotation to obtain polylactic acid nanoparticles loaded with diABZI;

[0060] Wherein, the organic solvent includes dichloromethane and / or acetone; and the concentration of the surfactant is 1%.

[0061] Furthermore, the ultrasound intensity is 50%, the ultrasound time is 30 s, and the ultrasound mode is pulsed.

[0062] Furthermore, the method for rotating and volatilizing the solvent is a solvent volatilization method or a solvent diffusion method.

[0063] Furthermore, in step 2, the pore sizes of the polycarbonate membrane are 200 nm, 400 nm and 800 nm; and the number of extrusions is 10 times.

[0064] Furthermore, in step 3, the treatment suspension time is 6 hours.

[0065] The present invention also proposes the use of a diABZI-loaded targeted nanoformulation in the treatment of colorectal cancer. The diABZI-loaded targeted nanoformulation is prepared by the preparation method of the diABZI-loaded targeted nanoformulation as described above.

[0066] Example 1: A method for preparing a diABZI-loaded targeted nanoformulation has achieved some positive results during the research and development or use process, and does have great advantages over the prior art. The following content is described in conjunction with the data, charts, etc. of the experimental process.

[0067] Step 1: Prepare a certain amount of diABZI and polylactic acid-polyethanol dissolved in 1 mL of dichloromethane as the oil phase (O), add to 100 ml of ultrapure water containing 0.5% sodium cholate as the water phase (W), ultrasonicate in an ice water bath for 120 s, with an ultrasonic intensity of 50% to prepare a small and uniform emulsion, and then place it in a fume hood to stir and solidify overnight. The solidified nanoformulation is washed 3-5 times by centrifugation with ultrapure water to obtain a polylactic acid-polyglycolic acid copolymer (PLGA) nanoformulation loaded with diABZI;

[0068] like Figure 3 As shown, the PLGA nanoparticles loaded with diABZI have good dispersibility, PDI 0.241, particle size 112nm, encapsulation efficiency 43%, and diABZI loading rate 3.1μg / mg.

[0069] Step 2: The PLGA nanoformulation loaded with diABZI in step 1 is used as the core, and the stem cell membrane is used as the shell to cover its surface. Accurately weigh 5 mg of the PLGA nanoformulation loaded with diABZI, take 1 mg of the stem cell membrane solution, mix the two in a ratio of 1:1, and then extrude them step by step through 800nm, 400nm, and 200nm polycarbonate membranes to coat the cell membrane on the surface of the nanoformulation.

[0070] Step 3: Then, the solution containing 10 μL of the targeting peptide was mixed with 1 mL of the bionic nanoformulation coated with the cell membrane and suspended vertically at 4°C for 4 h to insert the targeting peptide into the lipid bilayer of the cell membrane to form a targeting peptide-modified polylactic acid bionic targeting nanoformulation loaded with diABZI. The results are shown in FIG. Figure 2 , Figure 4 , Figure 5 shown.

[0071] Nanoparticle morphology investigation:

[0072] The morphology of NPs was observed using TEM (Jeol JEM1230, Jeol, Tokyo, Japan). Briefly, before TEM analysis, diABZI@NP-M-pHLIP and diABZI@NP were dropped onto a carbon-coated 400-mesh copper grid and then allowed to evaporate naturally. They were stained with 0.1% phosphotungstic acid solution (PAS) for 20 min, and after removing the excess PAS with filter paper, they were photographed on a transmission electron microscope.

[0073] Cellular uptake investigation:

[0074] CT26 cells at a density of 2×104 cells / mL were seeded in a six-well plate and cultured overnight at 5% (volume fraction) CO2 and 37°C. 1.0 mL of diABZI@NP-M-pHLIP preparation was added to the corresponding wells and incubated for 12 h. The wells were then rinsed 3 times with 1.0 mL of PBS each time, and then 1.0 mL of PBS and 2 μL of 1 mg / mL DAPI dye were added to stain the nucleus. The wells were allowed to stand for 15 min and observed and photographed using a laser confocal microscope or detected by flow cytometry.

[0075] like Figure 6 , Figure 7 As shown, the uptake of the targeted diABZI@NP-M-pHLIP formulation was significantly better than that of the non-targeted diABZI@NP-M formulation.

[0076] Apoptosis and colony formation assays:

[0077] CT26 cells were treated with 0.25% trypsin at 37°C. Digestion was terminated by adding DMEM and then washing CT26 cells three times with PBS and collecting the cells by centrifugation at 1500 rpm / min and discarding the supernatant. Annexin V-FITC apoptosis detection kit was used to measure the targeted diABZI@NP-M-pHLIP formulation or non-targeted diABZI@NP-M formulation. Photos were taken under a microscope to record the colony formation. In addition, another set of stained cells were incubated for 10-15 minutes in the dark at 25°C for detection by flow cytometry.

[0078] The results are as follows Figure 8 , Fig. 9 As shown, the polylactic acid biomimetic targeted nanoformulation loaded with diABZI (diABZI@NP-M-pHLIP) significantly promoted the apoptosis of CT26 cells and inhibited the cloning formation of colorectal cancer cells, indicating that the polylactic acid biomimetic targeted nanoformulation loaded with diABZI can target colorectal cancer cells and inhibit the growth of colorectal cancer cells.

[0079] 200 μL of PBS and Cy3-labeled diABZI@NP-M and diABZI@NP-M-pHLIP were injected into the tail vein. Three mice were in each group. The fluorescence distribution in the two groups of mice was observed by the small animal in vivo imaging system for 12 hours. The main organs (heart, liver, spleen, lung, kidney and tumor tissue) of each group of mice were dissected to observe their fluorescence distribution. The results are shown in Figures 10 to 14 As shown, diABZI@NP-M-pHLIP is more concentrated in the tumor site, proving that diABZI@NP-M-pHLIP has good tumor targeting ability.

[0080] Study on the in vivo inhibition of the development of in situ colorectal cancer induced by diABZI biomimetic targeted nanoformulations. C57 / J mice (6-8 weeks) were used for in vivo experiments, and the cell line CT26-LUC was used to construct homologous transplanted orthotopic tumor-bearing mice; the development of the tumor was monitored by a live imaging device. When the luciferase intensity value of the tumor reached 5×109p / sec / cm2 / sr, the nanoformulation was intravenously injected according to a certain dosing procedure, and the luciferase intensity of the tumor was recorded every two days. The end point was the luciferase intensity of the tumor of about 5×1011p / sec / cm2 / sr. The diABZI biomimetic targeted nanoformulation (diABZI@NP-M-pHLIP) effectively delayed the occurrence and development of in situ colorectal cancer and significantly prolonged the survival of mice with in situ colorectal cancer. Fig.15 After one week of administration, 6 mice were taken from each group and euthanized. Tumor tissues were taken to observe the tumor inhibition effect of the nanoparticle preparation:

[0081] like Fig.16 As shown, the nanoformulation reduced the weight of colorectal cancer tissue in mice and significantly inhibited the formation and development of colorectal cancer tissue.

[0082] Real-time PCR experiment, 1. RNA extraction:

[0083] To prevent RNA degradation and contamination, RNase-free pipette tips and EP tubes were used in the following experimental operations.

[0084] 2. Extraction of RNA from Tissues:

[0085] (1) Take 50 mg of mouse colorectal cancer tissue and place it in an EP tube. Add 500 μL of Trizol reagent and homogenize it three times using a tissue homogenizer. Centrifuge at 4°C and 14,000 rpm for 15 min and carefully pipette the supernatant into a new EP tube.

[0086] (2) Add 1 / 5 volume of Trizol in chloroform, vortex and shake, mix thoroughly, and let stand at room temperature for 5 minutes. Centrifuge at 4°C and 12,000 rpm for 15 minutes. The solution in the EP tube is separated into layers, and the total RNA is dissolved in the upper aqueous solution. Carefully pipette the upper liquid into a new EP tube.

[0087] (3) Add an equal volume of isopropanol to the liquid aspirated in (2) to precipitate the RNA. Slowly invert 6-8 times to mix. Let stand at room temperature for 10 min, and centrifuge at 12,000 rpm for 15 min. Observe the white precipitate at the bottom of the EP tube and discard the supernatant.

[0088] (4) After precooling 75% ethanol prepared with DEPC-treated water, add 500 μL to the EP tube and centrifuge at 12,000 rpm for 15 min. Aspirate the supernatant and open the lid to evaporate the ethanol for 3 min. Add an appropriate amount of precooled DEPC-treated water and gently pipette to dissolve the RNA precipitate.

[0089] 3. Cell RNA extraction:

[0090] (1) After harvesting the plates, aspirate the culture medium in the six-well plates and add Trizol reagent (500 μL / well). After lysing at room temperature for 5 min, repeatedly blow and vortex until the cells are completely detached and transfer to a 1.5 mL EP tube.

[0091] (2) Add chloroform that is 1 / 5 the volume of Trizol. The subsequent steps are the same as those for extracting tissue RNA.

[0092] 4. Determination of total RNA concentration:

[0093] The absorbance at 260 nm was measured using an ELISA reader to determine the RNA concentration of the sample, with DEPC-treated water as the Blank group. The concentration of each sample was calculated and adjusted with DEPC-treated water to make the RNA concentration of each sample consistent.

[0094] (1) Prepare the genomic DNA removal system according to the table below and react at 42°C for 2 min.

[0095] Table 1 Reverse transcription reaction configuration system:

[0096] (2) Add 10 μL of 2×Hifair® Ⅱ SuperMix plus reagent to the reaction solution in the above step and perform reverse transcription according to the parameters in the following table:

[0097] Table 2 Reverse transcription reaction parameters:

[0098] (3) Real-time PCR detection:

[0099] The experimental steps were referred to the instructions of the FastStart Universal SYBR Green Master (ROS) Kit.

[0100] 1) Reaction system:

[0101] Table 3 Real-time PCR reaction system:

[0102] 2) Reaction parameters:

[0103] Table 4 Real-time PCR reaction parameters:

[0104]

[0105] 3) The relative quantitative method 2-△△Ct was used to analyze the data and calculate the expression fold of the target gene relative to the internal reference gene GAPDH.

[0106] The experimental steps were referred to the instructions of the FastStart Universal SYBR Green Master (ROS) Kit.

[0107] The results are as follows Fig.17 As shown, biomimetic targeted nanoformulations produce the tumor-killing factor IFN beta to promote tumor killing

[0108] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing a diABZI-loaded targeted nanoformulation, characterized in that: The following steps are involved: Step 1: preparing polylactic acid nanoparticles of diABZI: using a nano-sedimentation method, an organic solvent containing PLGA and diABZI is added dropwise to ultrapure water containing a surfactant, and an emulsion is prepared by magnetic stirring and ultrasonic treatment under ice bath conditions; the solvent is rotatably volatilized to obtain polylactic acid nanoparticles loaded with diABZI; Step 2: Encapsulating stem cells: extracting stem cell membranes, and repeatedly extruding them through polycarbonate membranes with different pore sizes using a liposome extruder, so as to encapsulate the cell membranes on the surface of polylactic acid nanoparticles loaded with diABZI, thereby forming stem cell membrane-encapsulated polylactic acid biomimetic nanoparticles loaded with diABZI; Step 3: Preparation of bionic targeted nanoformulations: vertically suspend the solution containing the targeting polypeptide pHLIP and the diABZI-loaded polylactic acid bionic nanoformulation coated with the stem cell membrane, and insert the targeting polypeptide pHLIP into the lipid bilayer of the cell membrane by the post-insertion method to form a targeting polypeptide pHLIP-modified diABZI-loaded polylactic acid bionic targeted nanoformulation.

2. The method for preparing the diABZI-loaded targeted nanoformulation according to claim 1, characterized in that: The preparation of polylactic acid nanoparticles of diABZI comprises the following steps: Dissolve the surfactant in ultrapure water. PLGA and diABZI were dissolved in an organic solvent. The organic solvent containing PLGA and diABZI was added dropwise into ultrapure water containing a surfactant, and the mixture was stirred magnetically for 6 h and ultrasonically prepared under ice bath conditions; The emulsion is subjected to rotary volatilization to obtain polylactic acid nanoparticles loaded with diABZI; Wherein, the organic solvent includes dichloromethane and / or acetone; the surfactant is polyvinyl alcohol or sodium cholate or emulsifier Tween-20, and the concentration of the surfactant is 1%.

3. The method for preparing the diABZI-loaded targeted nanoformulation according to claim 2, characterized in that: In the step of preparing diABZI polylactic acid nanoparticles, the ultrasonic intensity is 50%, the ultrasonic time is 30s, and the ultrasonic method is pulsed.

4. The method for preparing the diABZI-loaded targeted nanoformulation according to claim 2, characterized in that: In the step of preparing diABZI polylactic acid nanoparticles, the method of rotating and volatilizing the emulsion is a solvent volatilization method or a solvent diffusion method.

5. The method for preparing the diABZI-loaded targeted nanoformulation according to claim 1, characterized in that: In step 2, the pore sizes of the polycarbonate membrane are 200 nm, 400 nm and 800 nm; and the number of extrusions is 10 times.

6. The method for preparing the diABZI-loaded targeted nanoformulation according to claim 1, characterized in that: In step 3, the treatment suspension time is 6 hours.

7. Use of a diABZI-loaded targeted nanoformulation in the preparation of a preparation for treating colorectal cancer, wherein the diABZI-loaded targeted nanoformulation is prepared by the method for preparing a diABZI-loaded targeted nanoformulation according to any one of claims 1 to 6.

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