Tumor-targeted nano drug delivery system as well as preparation method and application thereof

Abstract

The invention provides a tumor targeted nano drug delivery system and a preparation method and application thereof, the tumor targeted nano drug delivery system comprises a PD-L1 monoclonal antibody, radix pseudostellariae cyclic peptide B and an mPEG-PLGA-NH2 nano carrier, the radix pseudostellariae cyclic peptide B is encapsulated in the mPEG-PLGA-NH2 nano carrier, and the outer surface of the mPEG-PLGA-NH2 nano carrier is modified with the PD-L1 monoclonal antibody. The tumor-targeted nano drug delivery system has good anti-cancer activity and biocompatibility, has longer circulation time and higher bioavailability, is obviously superior to free drugs in the aspects of tumor resistance and immunoregulation, and is expected to be applied to preparation of tumor-targeted drugs, especially gastric cancer-targeted drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a tumor-targeted nano drug delivery system and a preparation method and application thereof. Background Art

[0002] Gastric cancer (GC) refers to a malignant tumor originating from the epithelial cells of the gastric mucosa, the vast majority of which are adenocarcinomas, and gastric cancer accounts for more than 95% of gastric malignant tumors. The occurrence of GC is caused by a variety of risk factors, the most common of which include Helicobacter pylori infection, smoking, high salt intake, etc. Early GC has a high chance of being completely cured by surgery, but most patients are usually in the advanced stage at the time of diagnosis and often miss the best opportunity for surgery. The main treatments for advanced gastric cancer include neoadjuvant chemoradiotherapy, molecular targeted therapy, and immunotherapy. Among them, immunotherapy based on immune checkpoint inhibitors (immune checkpoint inhibitors (ICIs), ICIs) has shown promising progress in clinical trials for locally advanced or metastatic GC.

[0003] Studies have shown that ICIs such as programmed death receptor 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) can prevent tumor immune escape and enable T cells to recognize and kill tumors. Currently, a variety of ICIs such as nivolumab, pembrolizumab, durvalumab, and atezolizumab have been approved for clinical use in advanced GC. Although some cancer patients can benefit from ICIs treatment, the overall response rate of ICIs alone is only 10-20%. Therefore, it is necessary to introduce combination therapies including ICIs to maximize their efficacy. How to overcome the limitations of traditional drugs and maximize their efficacy remains a major challenge in improving the effectiveness of ICI treatment.

[0004] The Chinese patent document discloses "a tumor-targeted nano drug delivery system modified with a slightly acidic environment targeting polypeptide and its preparation method", and its publication number is CN 103705465A. The invention uses a slightly acidic environment targeting polypeptide to modify the surface of the amino-rich dendritic polymer material to encapsulate the gene white assembly to form a slightly acidic environment targeting polypeptide modified tumor-targeted nano drug delivery system, and selects a polypeptide-modified polymer carrier derived from the transmembrane helical protein C of bacterial rhodopsin, which is concentrated and attached to the cell in a pH-sensitive cell membrane insertion manner and enters the cell in an electrostatic adsorption-mediated endocytosis manner, thereby improving the tumor cell's uptake of the drug and reducing toxic side effects. However, the preparation conditions of the tumor-targeted nano drug delivery system of the invention are harsh, and there is no relevant therapeutic effect data on gastric cancer. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a tumor-targeted nano-drug delivery system and a preparation method and application thereof, so as to solve the problem of poor efficacy of immune checkpoint inhibitors in the clinical application of gastric cancer in the prior art.

[0006] To achieve the above-mentioned and other related purposes, the present invention provides a tumor-targeted nano drug delivery system, which comprises PD-L1 monoclonal antibody, Pseudomonas aeruginosa cyclopeptide B and mPEG-PLGA-NH 2 Nanocarrier, the Pseudostellariae cyclopeptide B is encapsulated in mPEG-PLGA-NH 2 In the nanocarrier, the PD-L1 monoclonal antibody is modified with mPEG-PLGA-NH 2 The outer surface of the nanocarrier.

[0007] Pseudostellariae cyclotide B (HB) is an active ingredient from the medicinal plant Pseudostellariae cyclotide, and its structure has been identified as a cyclooctapeptide by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), mass spectrometry (MS), and nuclear magnetic resonance analysis. Cyclic peptides are generally considered to be potential anticancer drugs because of their good binding affinity, target selectivity, and low toxicity. HB has an inhibitory effect on the malignant phenotype of GC and esophageal cancer and can reduce the expression of PD-L1 by inhibiting PI3K / Akt signaling in GC.

[0008] mPEG-PLGA-NH 2 The nanocarrier is amino polylactic acid glycolic acid copolymer methoxy polyethylene glycol, which consists of three main components: NH 2 (amino), PLGA (polylactic acid-polyglycolic acid), mPEG (methoxy polyethylene glycol). 2 Contains amino groups -NH 2 The part is alkaline; PLGA is a copolymer composed of polylactic acid and polyglycolic acid; PLGA is used to achieve controlled release of drugs. mPEG is a variant of polyethylene glycol (PEG) with a methoxy group (-OCH 3 PEG can improve solubility and biocompatibility. 2 Nanocarriers can be used for drug delivery.

[0009] The present invention encapsulates the compound HB having anticancer activity in mPEG-PLGA-NH 2In the nanocarrier, HB can not only effectively hinder tumor growth, but also improve the immunosuppressive microenvironment; mPEG-PLGA-NH 2 The PD-L1 modification on the outer surface of the nanocarrier not only improves the mPEG-PLGA-NH 2 The nanocarrier has the ability to target and accumulate on tumor cells, and improves the bioavailability of PD-L1 and reduces its toxicity, thus successfully obtaining a tumor-targeted nano-drug delivery system. The tumor-targeted nano-drug delivery system of the present invention not only has good anti-cancer activity and biocompatibility, but also has a longer circulation time and higher bioavailability, and is significantly superior to free drugs in terms of anti-tumor and immunomodulation.

[0010] Preferably, the PD-L1 monoclonal antibody is αPDL1.

[0011] Preferably, the tumor-targeted nano-drug delivery system is PD-L1 monoclonal antibody modified with mPEG-PLGA-NH 2 The nanocarrier encapsulates spherical nanoparticles formed by self-assembly of Pseudostellariae Cyclopeptide B.

[0012] More preferably, the average particle size of the spherical nanoparticles is 350-450 nm.

[0013] The present invention also provides a method for preparing the above tumor-targeted nano drug delivery system, comprising the following steps: (1) Pseudostellariae cyclopeptide B and mPEG-PLGA-NH 2 The nanocarrier is dispersed in a solvent, stirred for reaction, and after solid-liquid separation, the obtained precipitate and supernatant are remixed to obtain an encapsulated carrier suspension, wherein the encapsulated carrier suspension is mPEG-PLGA-NH 2 a mixture of nanocarriers and supernatant; (2) The encapsulated carrier suspension is mixed with a cross-linking agent to obtain a solution A, the PD-L1 monoclonal antibody is mixed with a reducing agent to obtain a solution B, the solution A and the solution B are mixed and stirred for reaction, and the solid-liquid separation is performed. The obtained solid is a tumor-targeted nano-drug delivery system. In this step, the reducing agent reacts with the PD-L1 monoclonal antibody to first reduce the disulfide bond of the PD-L1 monoclonal antibody to a thiol group, and then the mPEG-PLGA-NH 2 The outer surface of the nanocarrier is modified.

[0014] The present invention first adopts mPEG-PLGA-NH 2Nanocarrier encapsulated Pseudo-P ... 2 The outer surface of the nanocarrier was modified with PD-L1 monoclonal antibody, and the tumor-targeted nano-delivery system was successfully prepared using the thin film hydration method.

[0015] Preferably, in step (1), the Pseudostellariae Cyclotide B and mPEG-PLGA-NH 2 The mass ratio of the nanocarrier is 1:(15-25).

[0016] Preferably, in step (1), the solvent is dichloromethane and water or a mixture of the two.

[0017] More preferably, in step (1), Pseudostellariae cyclotide B and mPEG-PLGA-NH 2 The nanocarriers were first dispersed in dichloromethane, and then water was added.

[0018] Preferably, in step (2), the cross-linking agent is m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS).

[0019] Preferably, in step (2), the volume ratio of the encapsulation carrier suspension to the cross-linking agent is 1:(1-1.5).

[0020] Preferably, in step (2), the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride.

[0021] The present invention also provides an application of the tumor-targeted nano drug delivery system in the preparation of tumor-targeted drugs.

[0022] Preferably, the tumor targeted drug is a gastric cancer targeted drug.

[0023] As described above, the present invention has the following beneficial effects: (1) Encapsulating the anticancer compound HB in mPEG-PLGA-NH 2 In the nanocarrier, HB can not only effectively hinder tumor growth, but also improve the immunosuppressive microenvironment; (2) Modification of mPEG-PLGA-NH 2 The PD-L1 modification on the outer surface of the nanocarrier not only improves the mPEG-PLGA-NH 2 The nanocarrier has the ability to target and accumulate on tumor cells, improves the bioavailability of PD-L1 and reduces its toxicity, and successfully obtains a tumor-targeted nano-drug delivery system; (3) The tumor-targeted nano-drug delivery system of the present invention not only has good anti-cancer activity and biocompatibility, but also has a longer circulation time and higher bioavailability. It is significantly superior to free drugs in anti-tumor and immunomodulatory aspects and is expected to be applied to the preparation of tumor-targeted drugs, especially gastric cancer-targeted drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the structure of the tumor-targeted nano drug delivery system of the present invention.

[0025] Figure 2 The mPEG-PLGA-NH 2 @HB(A) and mPEG-PLGA-NH 2 TEM image of @HB-αPDL1(b).

[0026] Figure 3 The mPEG-PLGA-NH 2 @HB and mPEG-PLGA-NH 2 @Particle size distribution diagram of HB-αPDL1.

[0027] Figure 4 The mPEG-PLGA-NH 2 @HB and mPEG-PLGA-NH 2 Zeta potential of @HB-αPDL1.

[0028] Figure 5 The mPEG-PLGA-NH 2 @HB and mPEG-PLGA-NH 2 @Absorbance of HB-αPDL1.

[0029] Figure 6 The mPEG-PLGA-NH 2 @HB and mPEG-PLGA-NH 2 @HB-αPDL1 cumulative release curve of HB at different pH values.

[0030] Figure 7 The mPEG-PLGA-NH 2 @HB, mPEG-PLGA-NH 2 @In vitro biosafety evaluation of HB-αPDL1 and HB of Comparative Example 2: A is the cell viability test result; B is the hemolysis test result.

[0031] Figure 8The mPEG-PLGA-NH 2 @HB, mPEG-PLGA-NH 2 @The cell viability test results of HB-αPDL1 and HB of comparative example 2 on different cell lines.

[0032] Fig. 9 For Transwell experiment, AGS cells were detected in the control group and the mPEG-PLGA-NH 2 @HB, mPEG-PLGA-NH 2 @The migration and invasion ability of HB-αPDL1 and comparative example 2 after HB treatment.

[0033] Fig.10 Flow cytometry was used to detect the AGS cells in the control group and the mPEG-PLGA-NH 2 @HB, mPEG-PLGA-NH 2 @The cell apoptosis levels after HB-αPDL1 and HB treatment in comparison example 2.

[0034] Fig.11 The protein expressions of p-PI3K, PI3K, p-AKT, AKT, and PD-L1 in AGS cells were evaluated for WB. Fig.12 The relative expression of PD-L1 mRNA in AGS cells was detected by qRT-PCR. * indicates p < 0.05.

[0035] Fig.13 For AGS cells and free DOX, mPEG-PLGA-NH 2 @DOX、mBEG-PLGA / NH 2 CLSM image of @DOX-αPDL1 co-incubated for 4 hours: blue fluorescence indicates nuclei labeled with DAPI; red fluorescence indicates DOX.

[0036] Fig.14 The volume (A), physical image (B) and weight (C) of the tumor of mice treated with different drugs, * indicates p < 0.05 compared with the control group; # indicates p < 0.05.

[0037] Fig.15 H&E staining images of mouse tumor tissues and major organs (heart, liver, spleen, lung, and kidney).

[0038] Fig.16 The secretion levels of TNF-α, IFN-γ and MIP-1β in the blood of mice treated with different drugs.

[0039] Fig.17 CD4 in tumor tissue after treatment with different drugs + T cells and CD8 + Flow cytometry results of T.

[0040] Fig.18 for Fig.17 Quantification bar graph of flow cytometry results.

[0041] Fig.19 These are the IHC detection results of the expression of CD4, CD8 and Foxp3 in tumor tissues after treatment with different drugs. DETAILED DESCRIPTION

[0042] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0043] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0044] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0045] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0046] Example 1 like Figure 1As shown, the embodiment of the present application provides a tumor-targeted nano drug delivery system, wherein the tumor-targeted nano drug delivery system comprises PD-L1 monoclonal antibody, Pseudomonas aeruginosa cyclopeptide B and mPEG-PLGA-NH 2 Nanocarrier, the Pseudostellariae cyclopeptide B is encapsulated in mPEG-PLGA-NH 2 In the nanocarrier, the PD-L1 monoclonal antibody is modified with mPEG-PLGA-NH 2 The outer surface of the nanocarrier, the PD-L1 monoclonal antibody adopts αPDL1.

[0047] The present application also provides a method for preparing a tumor-targeted nano-drug delivery system, comprising the following steps: (1) 2 mg HB (Yuanye Biotech, China) and 40 mg mPEG-PLGA-NH 2 Nanocarriers (Sangon Biotech, China) were dissolved in 1 mL of dichloromethane (Aladdin, China) and sonicated for 5 min. Then, 3 mL of ultrapure water was slowly added to the above solution and then sonicated for 10 min. After sonication, the entire solution was placed on a magnetic stirrer at room temperature overnight and finally centrifuged at 12,000 rpm for 10 min. The precipitated material and the supernatant were collected and resuspended together with the precipitate to obtain the encapsulated carrier suspension, which was stored in a refrigerator at 4 °C. (2) 1 mL (10 mmol / L) of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution (Aladdin, China) and m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) solution (Thermo Fisher Scientific, USA) were prepared respectively; 200 μL of the encapsulated carrier suspension and 200 μL of the MBS solution were mixed with ultrasound for 5 minutes to form solution A, and then 200 μL of the TCEP solution was reacted with αPDL1 (HY-P73361 / 66248-1-Ig, MCE / Proteintech, USA) to reduce the disulfide bond to thiol to form solution B; solution B was added to solution A and stirred at room temperature for 30 minutes; after the reaction, the solution was centrifuged at 12000 rpm for 10 minutes; the solution was washed twice with pure water to remove the reducing agent TCEP, and the tumor targeting nano drug delivery system (mPEG-PLGA-NH 2 @HB-αPDL1); finally, the resuspended material was stored at -20 °C for later use.

[0048] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that step (2) is not included, that is, the mPEG-PLGA-NH 2The nanocarrier and the rest of the process are exactly the same as those in Example 1. The tumor targeting nano drug delivery system prepared in the comparative example is denoted as mPEG-PLGA-NH 2 @HB.

[0049] Comparative Example 2 Comparative Example 2 uses Pseudostellariae Cyclopeptide B, denoted as HB.

[0050] The tumor-targeted nano drug delivery systems prepared in Example 1 and Comparative Example 1 were characterized as follows: The morphology and size of the two materials before and after αPDL1 modification were characterized using a JEM-2100 transmission electron microscope (TEM, JEOL Ltd, Japan). 2 @HB and mPEG-PLGA-NH 2 The TEM images of @HB-αPDL1 are shown in Figure 2 A and Figure 2 As shown in B. Both nanomaterials exhibit a uniform spherical appearance.

[0051] Zeta potential analyzer and particle size analyzer (Brookhaven, USA) were used to characterize the potential and size of the two materials before and after αPDL1 modification. The results are shown in Figure 3 and Figure 4 As shown. Figure 3 It can be seen that the NH 2 @HB hydrated particle size is 338nm, Example 1 PEG-PLGA-NH modified by αPDL1 2 The hydrated particle size of @HB-αPDL1 is about 427 nm, which may play a role in avoiding the rapid clearance of free αPDL1. In addition, the successful modification of αPDL1 improves the targeting and accumulation ability of the tumor-targeted nano-drug delivery system of the present application on tumor cells, thereby improving the bioavailability of αPDL1 and reducing its toxicity, which was confirmed by GC cell uptake and CCK-8 experiments on gastric mucosal epithelial cells. Figure 4 It can be seen that in Example 1, due to the successful modification of αPDL1, mPEG-PLGA-NH 2 The zeta potential of @HB decreased from -10.23±1.19mV to -17.9±0.11mV.

[0052] The bicinchoninic acid (BCA) method was used to determine the αPDL1 content of the two materials before and after αPDL1 modification: 2 mL of BCA working solution (BCA working solution: Cu = 50:1) was prepared, and 20 μL of mPEG-PLGA-NH 2 @HB (three parallel groups) and mPEG-PLGA-NH 2@HB-αPDL1 (three parallel groups) were connected to a 96-well plate in sequence. Next, 200 μL of BCA working solution was introduced into each of the six wells and then incubated in an oven with tin foil for 30 minutes. The 96-well plate was placed in a microplate reader (PerkinElmer, USA) to detect the protein absorbance at 560 nm. The results are shown in Figure 5 shown.

[0053] from Figure 5 It can be seen that mPEG-PLGA-NH 2 @HB and mPEG-PLGA-NH 2 The increase in absorbance further proves that αPDL1 has been successfully modified on mPEG-PLGA-NH 2 @HB Surface.

[0054] Determination of drug loading and encapsulation efficiency: The absorbance of the material and the supernatant containing HB was measured at 203 nm using a UV-visible spectrophotometer (Shimadzu, Japan). We calculated the encapsulation efficiency (EE%) and loading efficiency (LE%) according to the following formula: EE (%) = mass of HB on the material / total mass after adding HB × 100% LE (%) = mass of HB on material / total mass of material × 100%.

[0055] In vitro drug release: The drug release of HB under different pH conditions was determined by dialysis. 2 @HB solution and 1mL PEG-PLGA-NH 2 @HB-αPDL1 was added to the dialysis bag (MWCO = 3500Da) respectively. The dialysis bag was immersed in 29mL of phosphate buffered saline (PBS) solution of pH 5.0 and pH 7.4 containing 0.1% (w / v) Tween 80 respectively. We stirred the solution at 100rpm at 37°C for 96 hours, removed 200μL of PBS solution at fixed time points, and supplemented with 200μL of fresh PBS solution. Finally, the content of HB in the PBS solution was determined by UV spectrophotometer.

[0056] The mPEG-PLGA-NH 2 @HB and mPEG-PLGA-NH 2 The encapsulation efficiency of @HB-αPDL1 was 55% and 45%, and the loading rate was 2.75% and 2.25%, respectively.

[0057] In order to study the drug release of nanomaterials in different environments in vivo, the present application simulated the release behavior of HB in physiological and tumor environments in PBS (pH=5.0 and pH 7.4) solution containing 0.1% Tween 80. The results are shown in Figure 2. Figure 6 As shown. Figure 6 It can be seen that at pH 7.4, mPEG-PLGA-NH 2 @HB and mPEG-PLGA-NH 2 The cumulative HB release of @HB-αPDL1 after 96 hours was 48.1% and 46.2%, respectively. However, below pH 5.0, the cumulative HB release after 96 hours was 65.4% and 60.1%, respectively. The release rate of HB in an acidic environment was higher than that in a weakly alkaline environment, which may be due to the fact that mPEG-PLGA-NH 2 It is more susceptible to hydrolysis under acidic conditions. At the same time, higher proton concentrations may affect the amphiphilicity of PLGA-PEG, resulting in reduced integrity of the micelle structure.

[0058] Cell experiments were performed on the tumor-targeted nano drug delivery systems prepared in Example 1 and Comparative Example 1, and the Pseudostellariae Cyclotide B in Comparative Example 2: 1. Cell Culture The human gastric mucosal cell line GES-1 (SNL-304), human GC cell line AGS (SNL-103), HGC-27 (SNL-104), MKN-74 (SNL-176) and rat GC cell line MFC (SNL-365) used in the present application were purchased from Sunnyll Biotechnology (China). GES-1, HGC-27, MKN-74 and MFC cells were inoculated in RPMI 1640 medium (Procell, China) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S). AGS cells were inoculated in RPMI 1640 medium (Procell, China) containing 10% FBS and 1% P / S. The humidified air condition was set to 37°C with 5% CO 2 2. Cytotoxicity Assay The effects of different (nano) drugs on cell viability were tested by CCK-8 assay. GES-1, AGS, HGC-27 or MKN-74 cells were seeded in 96-well plates at a density of 8000 cells / well and incubated at 37°C, 5% CO 2 In order to systematically compare the 2 @HB and mPEG-PLGA-NH 2@HB-αPDL1 cytotoxicity, different concentrations (0, 1, 5, 50, 100, 200 μM) of drug suspension were added to the above plate. After 24 hours of incubation, we introduced 10 μL of CCK-8 solution into each well and further incubated for 4 hours. Finally, we measured and recorded the absorbance of the cell samples on a microplate reader (λ = 450 nm).

[0059] 3. Hemolysis Test In order to evaluate the biocompatibility of nanomaterials, a hemolysis experiment was performed in this application example. Blood samples were collected from the mouse orbits and the whole blood was washed with a 0.9% NaCl solution. After centrifugation at 1500rpm for 5 minutes, the supernatant was discarded and then a 0.9% NaCl solution was introduced to obtain a 5% red blood cell (RBC) suspension. In this application example, deionized water and a 0.9% NaCl solution were added to the RBC suspension as positive and negative control groups. HB, mPEG-PLGA-NH 2 @HB and mPEG-PLGA-NH 2 @HB-αPDL1 was mixed with red blood cell suspension as the experimental group. All groups were incubated at 37°C for 30 minutes, and the hemolysis status was photographed and recorded. After centrifugation at 1500rpm for 10 minutes, the absorbance of the supernatant at 545nm was measured using an enzyme reader. The hemolysis rate (RHR) was calculated according to the formula. RHR (%) = (OD sample OD negative) / (OD positive OD negative) × 100%. If the RHR is lower than the international standard requirement (5%), it indicates that the sample does not show obvious hemolysis.

[0060] In order to evaluate the biosafety performance of nanomedicines, the present invention cultured different concentrations of HB, mPEG-PLGA-NH 2 @HB and mPEG-PLGA-NH 2 @HB-αPDL1, CCK-8 experimental results are as follows Figure 7 As shown in Figure A, HB significantly reduced the survival rate of GES-1 cells in the 50μM and 100μM groups compared with the control group. 2 @HB 100μM group GES-1 cell survival rate was significantly reduced. However, mPEG-PLGA-NH 2 @HB-αPDL1Due to the biocompatibility of the nanomicelles and the presence of αPDL1, αPDL1 did not reduce the survival rate of normal cells at lower concentrations (0-100 μM).

[0061] The present application example selected 50 μM of the drug mixed with the red blood cell suspension for hemolysis experiment. Figure 7As shown in B. From left to right are negative control (tube 1), HB (tube 2), mPEG-PLGA-NH 2 @HB (tube 3), mPEG-PLGA-NH 2 @HB-αPDL1 (tube 4) and positive control (tube 5). The supernatant of tube 4 was clear in color, and the hemolysis rate of tube 4 was calculated to be lower than the internationally recognized standard (5%), indicating that the mPEG-PLGA-NH 2 @HB-αPDL1 has good blood compatibility.

[0062] IV. Transwell assay Transwell chambers (Corning Costar, USA) were used to evaluate the effects of drugs on the migration and invasion of AGS cells. First, different drugs (HB, mPEG-PLA-NH 2 @HB, and mPEG-PLA-NH 2 @HB-αPDL1) was reacted with AGS cells for 24 hours, and then the cells were suspended in serum-free medium after trypsin digestion. The cells were seeded in the upper chamber, which was not paved (for migration analysis) or paved with matrix gel (for invasion analysis). The lower chamber was RPMI-1640 medium containing 10% FBS. After 24 hours, the cells that did not migrate or invade in the upper chamber were wiped with a cotton swab, then fixed with 75% methanol, stained with 0.5% crystal violet, and the results were observed under a microscope and the number was counted (MSHOT, China).

[0063] 5. Cell apoptosis assay Flow cytometry was used to detect the apoptosis of AGS cells under each drug treatment. Specifically, AGS cells were treated with the same concentration of drugs (HB, mPEG-PLGA-NH 2 @HBmPEG-PLGA-NH 2 @HB-αPDL1) for 24 hours. The cells were resuspended in PBS buffer and then stained with AnnexinV-FITC / PI double staining kit (Beyotime, China) at room temperature in the dark for 30 minutes. Flow cytometry was immediately performed using a flow cytometer equipped with Agilent (Agilent, China).

[0064] In order to evaluate the in vitro therapeutic effect of nanomedicine, the present application tested the anticancer activity of nanomedicine through a series of cell function experiments. Figure 8 As shown. 2 Compared with @HB, mPEG-PLGA-NH 2 @HB-αPDL1 hindered cell survival at lower doses.

[0065] After that, the present application selected AGS cells with good cell condition and more sensitive to HB for subsequent experiments and 50 μM dose of drug treatment, and the results are as follows Fig. 9 and Fig.10 As shown. Fig. 9 Transwell assay results and Fig.10 The apoptosis detection results of HB and mPEG-PLGA-NH 2 @HB and mPEG-PLGA-NH 2 After treatment with HB-αPDL1, the migration and invasion abilities of AGS cells were significantly inhibited, and the apoptosis rate was significantly increased. 2 The cell migration and invasion times of the @HB-αPDL1 group were the lowest, while the apoptosis rate was the highest, indicating that mPEG-PLGA-NH 2 @HB-αPDL1 may have the strongest anti-tumor activity at the same dose. HB can downregulate the expression of PD-L1 by inhibiting the PI3K / AKT signaling pathway.

[0066] 6. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted from each group of AGS cells using TRIzol (CWBIO, China). AdvanceFast First-Strand cDNA Synthesis Kit (YEASEN, China) was used to reverse transcribe RNA to obtain cDNA. We used SYBRGreen (Biorad, USA) to perform qPCR on cDNA on real-time fluorescence quantitative PCR (ABI 7500, USA). β-actin was selected as an internal control (quantified and normalized to 2 -ΔΔCt Table 1 is a primer sequence table.

[0067] Table 1. Primer sequence list 7. Western Blot (WB) Proteins were extracted using lysis buffer (CST, USA), and protein concentrations were then detected using the BCA method. The protein samples were loaded on 10% sodium dodecyl sulfate polyacrylamide gels and then subjected to electrophoresis. After electrophoresis, the proteins in the gel were transferred to a PVDF membrane (Millipore, USA). The membrane was blocked with 5% skim milk for 1 hour at room temperature to reduce nonspecific binding. The membrane was incubated with anti-PI3K antibody (A22730), anti-p-PI3K antibody, anti-AKT antibody (A17909), anti-p-AKT antibody, anti-PD-L1 antibody (A18103), or anti-β-actin antibody (AC004) at 4°C overnight. After washing, horseradish peroxidase (HRP)-labeled IgG H&L (A0208) and HRP-labeled IgG H&L (A0216) were incubated at room temperature for 2 hours. Antibodies were purchased from Abclonal and Beyotime (China). Finally, the protein bands were detected using a chemiluminescence imaging system (Clinx, China) and enhanced chemiluminescence (ECL) reagent (Biosharp, China), and the grayscale values ​​of the bands were calculated using ImageJ v1.8.0 (National Institutes of Health, USA) software.

[0068] This application further studied the effects of different drug treatments on downstream signaling, and the results were as follows Fig.11 and Fig.12 As shown, mPEG-PLGA-NH 2 @HB-αPDL1 significantly reduced the ratios of p-PI3K / PI3K and p-AKT / AKT, as well as the expression of PD-L1. 2 @HB-αPDL1 has good anticancer activity in vitro.

[0069] 8. Cellular Uptake Laser scanning fluorescence microscopy (Keyence, China) was used to observe the cellular uptake of different drugs. Since HB itself does not emit fluorescence, constructing nanomedicines with fluorescent labels is the best way to analyze the cellular uptake of different nanomedicines. We used doxorubicin (DOX) instead of HB to prepare mPEG-PLGA-NH 2 @DOX and mPEG-PLGA-NH 2 @DOX-αPDL1 and fluorescent dye. AGS cells (1×10 5 cells / well) were seeded in 6-well plates and incubated overnight. Free DOX, mPEG-PLGA-NH 2 @DOX and the introduction of mPEG-PLGA-NH 2@DOX-αPDL1 was incubated for 4 hours. Then the cells were washed 3 times with PBS. The cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma, USA) for 5 minutes. Finally, the results were observed under a fluorescence microscope and photographed.

[0070] Cellular uptake is one of the indicators for evaluating the bioavailability of nanomedicines. In order to analyze the cellular uptake of different nanomedicines, this application uses DOX instead of HB to prepare nanomaterials with fluorescent properties for tracking. Fig.13 In the presence of free DOX and mPEG-PLGA-NH 2 A small amount of red fluorescence of DOX was observed in the AGS cells of the @DOX group; 2 When co-incubated with @DOX-αPDL1, red fluorescence was visible in the cells, indicating that the presence of αPDL1 increased the uptake of nanomaterials by cancer cells. 2 HB delivery as a carrier can effectively enhance the uptake of HB by cancer cells and is expected to increase intratumoral drug concentration and therapeutic efficacy.

[0071] In vivo animal experiments: 1. Construction of allogeneic transplantation mouse model To test the therapeutic effects of different drugs in animals, the present applicant purchased 4- or 5-week-old female 615 mice (Beiyou Biotechnology, China) and injected 2×10 7 / mL MFC cells to establish an allogeneic tumor model. When the tumor volume reached about 100mm 3 The mice were randomly divided into 7 groups (n=5) and intravenously injected with normal saline (control group), HB, αPDL1, HB+αPDL1 (combination group), mPEG-PLGA-NH 2 , mPEG-PLGA-NH 2 @HB and mPEG-PLGA-NH 2 @HB-αPDL1. The tumor volume of mice was measured every week, and the mice were killed after 5 weeks. The tumor tissue was removed for photography and weighing.

[0072] 2. Enzyme-linked immunosorbent assay (ELISA) In the present application, blood samples of mice of different groups were collected and cytokines (IFN-γ, TNF-α, MIP-1β) were detected using ELISA kits (Elabscience, China). The samples and enzyme-coupled antibodies were incubated in the assay wells for 1 hour. After incubation for 30 minutes, we washed the plate 5 times. Then we added the chromogenic substrate for 10 minutes of color development. Finally, the stop solution was introduced. The OD 450 value was evaluated within 15 minutes using a microplate reader.

[0073] 3. Flow cytometry detection of immune cell populations In the present application, a single cell suspension was prepared from the crushed tumor tissue through a 100 μm cell filter, and the cells were stained with the following antibodies: APC anti-CD3 antibody (E-AB-F1013E), PerCP anti-CD8 antibody (E-ABC-F1104F), PE anti-CD4 antibody (E-ABA-F1353D). The antibodies were purchased from Elabscience (China). Finally, the data were processed in a flow cytometer.

[0074] 4. Histological Detection For immunohistochemistry (IHC) analysis, fresh tumor tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned, then dewaxed in xylene for 20 min. Sections were antigen-retrieved with EDTA antigen retrieval solution and then immersed in 3% H 2 O 2 The sections were incubated with CD4 antibody (ab237722), CD8 antibody (ab217344), or Foxp3 antibody (ab215206) at 4°C overnight, followed by incubation with secondary antibody (ab205718) for 0.5 h. 3,3'-diaminobenzidine (DAB) (Sigma, USA) staining solution was introduced for color development. Finally, we re-stained the nuclei with hematoxylin.

[0075] V. Statistical Analysis The experimental data were processed using Graphpad Prism 8.0 (La Jolla, USA) software and expressed as mean ± standard deviation. T test was used for comparison between two groups, and univariate analysis of variance (ANOVA) was used for comparison between multiple groups. P < 0.05 was considered statistically significant. The data were from at least three independent experiments.

[0076] To evaluate the mPEG-PLGA-NH 2 @HB-αPDL1's anti-tumor effect in animals: This application example used 615 mice to construct a GC allogeneic tumor model. When the tumor volume reached about 100 mm3 When HB, αPDL1, combined, and mPEG-PLGA-NH 2 、mPEG-PLGA-NH 2 @HB, mPEG-PLGA-NH 2 @HB-αPDL1 and saline were used as controls. The results are as follows Fig.14 As shown, compared with the control group, except for mPEG-PLGA-NH 2 Except for the group, all other drug treatment groups had significant inhibitory effects on mouse tumors. 2 @HB-αPDL1 showed the strongest anti-cancer effect and effectively controlled the growth of GC subcutaneous tumors. After killing the mice, the applicant performed H&E staining on fresh tumor tissues and important organs. The results are as follows Fig.15 As shown. Fig.15 It can be seen that mPEG-PLGA-NH 2 @HB-αPDL1 treatment did not cause significant damage to the heart, liver, spleen, lung, and kidney of mice, but resulted in extensive nuclear coagulation and necrosis of tumor tissue. 2 @HB-αPDL1 has good anti-tumor activity in mice and has no obvious toxic side effects on normal organs.

[0077] To further elucidate the mPEG-PLGA-NH 2 @HB-αPDL1 treatment mechanism of GC, the present application example analyzed the changes in the mouse immune system after different drug treatments by ELISA, flow cytometry and IHC. Fig.16 As shown, the use of drugs increased the secretion of immune stimulatory cytokines (TNF-α, IFN-γ, MIP-1β) in the blood of mice. 2 @HB-αPDL1 has the strongest immunostimulatory effect.

[0078] Flow cytometry was used to detect the abundance of specific immune cells. Fig.17 and Fig.18 It can be seen that after drug treatment, CD4 + T cells and CD8 + The abundance of T cells increased significantly. 2 The abundance of cells in the HB-αPDL1 group changed the most. Fig.19 The results of IHC staining shown in the figure showed that after treatment with HB or (and) αPDL1, the expression of CD4 and CD8 increased, and the expression of Foxp3 decreased, while mPEG-PLGA-NH 2@HB-αPDL1 treatment seems to have a greater effect on CD4, CD8, and Foxp3 expression. In conclusion, mPEG-PLGA-NH2@HB-αPDL1 can modulate the tumor immune microenvironment and enhance anti-tumor immunity The regulation of HB on PD-L1 means that HB may have an immunomodulatory effect in the tumor microenvironment. By constructing an allogeneic tumor model, the present application found that HB not only effectively hindered tumor growth but also improved the immunosuppressive microenvironment. This effect was observed in mPEG-PLGA-NH 2 @HB-αPDL1 treatment group, ELISA, flow cytometry and IHC assays showed that HB and αPDL1 significantly enhanced the anti-tumor immune response of GC mice under nanomicelle delivery.

[0079] The tumor targeting nano drug delivery system (mPEG-PLGA-NH 2 @HB-αPDL1) not only has good anticancer activity and biocompatibility, but also has a longer circulation time and higher bioavailability. It is significantly superior to free drugs in anti-tumor and immunomodulation, and is expected to be used in the preparation of tumor-targeted drugs, especially gastric cancer-targeted drugs. Among them, αPDL1 is used to target PD-L1 on the surface of tumor cells to reduce immunosuppression. HB exerts its anticancer activity, thereby synergistically improving the immunosuppressive microenvironment. At the same time, mPEG-PLGA-NH 2 The nanocarrier exhibited good biocompatibility and was able to safely deliver HB and αPDL1, effectively inhibiting the development of GC. The in vitro studies of this application showed that mPEG-PLGA-NH 2 @HB-αPDL1 has good anticancer activity and biocompatibility. By constructing an allogeneic tumor model, mPEG-PLGA-NH 2 @HB-αPDL1 in vivo studies have shown that mPEG-PLGA-NH 2 @HB-αPDL1 is significantly superior to free drugs in anti-tumor and immunomodulatory aspects. This application shows that mPEG-PLGA-NH 2 @HB-αPDL1 may be a promising drug for GC treatment.

[0080] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of methods and compositions in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A tumor-targeted nano drug delivery system, characterized in that: The tumor-targeted nano drug delivery system comprises PD-L1 monoclonal antibody, Pseudostellariae Radix Cyclopeptide B and mPEG-PLGA-NH2 nanocarrier, wherein the Pseudostellariae Radix Cyclopeptide B is encapsulated in the mPEG-PLGA-NH2 nanocarrier, and the PD-L1 monoclonal antibody is modified on the outer surface of the mPEG-PLGA-NH2 nanocarrier.

2. The tumor-targeted nano drug delivery system according to claim 1, characterized in that: The PD-L1 monoclonal antibody is αPDL1.

3. The tumor-targeted nano drug delivery system according to claim 1, characterized in that: The tumor-targeted nano drug delivery system is a spherical nanoparticle formed by self-assembly of PD-L1 monoclonal antibody-modified mPEG-PLGA-NH2 nanocarriers encapsulating Pseudostellariae cyclopeptide B, and the average particle size of the spherical nanoparticles is 350-450 nm.

4. A method for preparing a tumor-targeted nano drug delivery system according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) dispersing Pseudo-radix cyclotide B and mPEG-PLGA-NH2 nanocarriers in a solvent, stirring for reaction, and after solid-liquid separation, remixing the obtained precipitate and supernatant to obtain an encapsulated carrier suspension, wherein the encapsulated carrier suspension is a mixture of mPEG-PLGA-NH2 nanocarriers encapsulating Pseudo-radix cyclotide B and the supernatant; (2) The encapsulated carrier suspension is mixed with a cross-linking agent to obtain a solution A, and the PD-L1 monoclonal antibody is mixed with a reducing agent to obtain a solution B. The solution A and the solution B are mixed and stirred to react, and the solid-liquid separation is performed to obtain a solid that is a tumor-targeted nano-drug delivery system.

5. The preparation method according to claim 4, characterized in that: In step (1), the mass ratio of the Pseudostellariae cyclopeptide B to the mPEG-PLGA-NH2 nanocarrier is 1:(15-25).

6. The preparation method according to claim 4, characterized in that: In step (1), the solvent is dichloromethane and water or a mixture of the two.

7. The preparation method according to claim 4, characterized in that: In step (2), the cross-linking agent is m-maleimidobenzoyl-N-hydroxysuccinimide ester; and the volume ratio of the encapsulation carrier suspension to the cross-linking agent is 1:(1-1.5).

8. The preparation method according to claim 4, characterized in that: In step (2), the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride.

9. Use of the tumor-targeted nano drug delivery system as claimed in any one of claims 1 to 3 in the preparation of tumor-targeted drugs.

10. The use according to claim 9, characterized in that: The tumor targeted drug is a gastric cancer targeted drug.

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