BBI608-entrapped nanoparticles as well as preparation method and application of BBI608-entrapped nanoparticles

Abstract

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to BBI608-coated nanoparticles as well as a preparation method and application thereof. According to the invention, a hydrophobic material polylactic acid-glycolic acid copolymer (PLGA) with good biocompatibility and biodegradability is adopted as a carrier and is prepared into a nano preparation, so that the solubility of the medicine is improved, a better hepatocellular carcinoma treatment effect is realized through passive targeting after administration, and the nano preparation has good safety and can be used for preparing a medicine for treating hepatocellular carcinoma. The system provided by the invention evaluates the in-vivo and in-vitro anti-HCC effect of the NapatPLGA NPs, and provides a new theoretical and experimental basis for further development and clinical application of the NapatPLGA NPs, so that the NapatPLGA NPs has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and molecular biology, and specifically relates to a nanoparticle loaded with BBI608 and a preparation method and application thereof. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. As a malignant tumor of the digestive system, HCC is hidden in the early stage. Most patients are in the late stage when they develop the disease. It is difficult to treat and has a high recurrence rate. For advanced HCC, sorafenib has been the first-line drug for systemic treatment for more than ten years. However, its clinical efficacy is not ideal and can only extend the patient's survival by three months. Therefore, it is crucial to find new mechanisms for the occurrence and development of liver cancer and discover potential intervention targets.

[0004] As the convergence point of many oncogenic signaling pathways, signal transducer and activator of transcription 3 (STAT3) plays a core role in regulating anti-tumor immune responses. A large number of studies have shown that overexpression and constitutive activation of STAT3 are often found in HCC, inhibiting the expression of multiple immune activating factors or promoting the production of immunosuppressive factors, and are associated with poor prognosis of patients. IL-6 and IL-22 are the main stimuli that activate STAT3 signals in HCC cells. Activated STAT3 promotes the proliferation, anti-apoptosis, migration, invasion, angiogenesis and stem cell characteristics of HCC cells through transcriptional regulation of target genes, cooperation with NF-κB and epigenetic regulation of miRNA. Therefore, targeting the STAT3 signaling pathway has become a therapeutic strategy for many cancers.

[0005] At present, direct inhibitors of STAT3 can be divided into three categories: peptides, small molecule compounds and oligonucleotides. BBI608 (Napabucasin, CAS No.: 83280-65-3) is a small molecule inhibitor that selectively binds to the DBD domain of STAT3. It is the only inhibitor that directly acts on STAT3 that has entered Phase III trials so far. The results of its monotherapy trial showed that BBI608 has a potential effect on advanced colorectal cancer, and the FDA has approved BBI608 as an orphan drug for the treatment of gastric cancer and pancreatic cancer, but no studies have reported its clinical effects on HCC patients. Based on previous laboratory studies, the inventors found that Napabucasin exhibited excellent anti-HCC effects in vitro, but due to its low water solubility and low cell permeability, its solubility is not high, and its therapeutic index is low and its toxicity is relatively high, which is not conducive to its subsequent actual clinical application. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention aims to provide a nanoparticle loaded with BBI608 and a preparation method and application thereof. The present invention uses polylactic acid-co-glycolic acid (PLGA) as a carrier, which is a hydrophobic material with good biocompatibility and biodegradability, to prepare it into a nanoformulation, improve drug solubility, and achieve better hepatocellular carcinoma treatment effects through passive targeting.

[0007] In order to achieve the above technical objectives, the technical solution provided by the present invention is as follows:

[0008] The first aspect of the present invention provides nanoparticles encapsulating BBI608, wherein the nanoparticles use polylactic acid-co-glycolic acid as a carrier and encapsulate BBI608; wherein the average particle size of the nanoparticles is 300 nm, and the drug encapsulation rate is above 90%.

[0009] In the present invention, the nanoparticles loaded with BBI608 are referred to as Nap@PLGA NPs.

[0010] The second aspect of the present invention provides a method for preparing the above-mentioned nanoparticles, which comprises preparing the above-mentioned nanoparticles by a solvent volatilization method.

[0011] The third aspect of the present invention provides the use of the above-mentioned nanoparticles in any one or more of the following:

[0012] a1) Ability to inhibit tumor cell proliferation or to prepare products capable of inhibiting tumor cell proliferation;

[0013] a2) Inducing apoptosis of tumor cells or preparing products that induce apoptosis of tumor cells;

[0014] a3) Inhibiting tumor cell stemness or preparing products that inhibit tumor cell stemness;

[0015] a4) Inhibition of p-STAT3 in tumor cells Try705 Inhibition of p-STAT3 in tumor cells Try705 horizontal products;

[0016] a5) Inhibiting the ability of tumor cell colony formation or preparing products that inhibit the ability of tumor cell colony formation;

[0017] a7) Inhibiting the ability of tumor cells to form spheres or preparing products that inhibit the ability of tumor cells to form spheres;

[0018] a8) Inhibiting tumor growth or preparing products that inhibit tumor growth;

[0019] a9) Reducing the toxic and side effects of BBI608, alleviating drug damage, or preparing products that reduce the toxic and side effects of BBI608, alleviating drug damage;

[0020] a10) Activating anti-tumor immune response or preparing products that activate anti-tumor immune response;

[0021] a11) Reversing the function of HCC-TAMs, enhancing T cell response, or preparing products that reverse the function of HCC-TAMs and enhance T cell response.

[0022] A fourth aspect of the present invention provides a drug, which comprises at least the above-mentioned nanoparticles.

[0023] A fifth aspect of the present invention provides a method for treating tumors, comprising administering a therapeutically effective dose of the above-mentioned nanoparticles or drugs to a subject.

[0024] Beneficial technical effects of one or more of the above technical solutions:

[0025] 1. The Nap@PLGA NPs prepared by the above technical solution using the solvent volatilization method improve the solubility of the drug, reduce the drug toxicity, and enhance the anti-HCC effect of the drug in vivo and in vitro while ensuring the efficacy.

[0026] 2. The above technical solution research found that Nap@PLGA NPs can not only directly inhibit the tumor biological characteristics of HCC cells, but also promote the conversion of M2 macrophages to M1 by inhibiting STAT3 signals in HCC-TAMs, thereby improving the immune microenvironment and enhancing the anti-tumor function of T cells.

[0027] 3. The above technical scheme systematically evaluated the anti-HCC effects of Nap@PLGA NPs in vitro and in vivo, providing new theoretical and experimental basis for its further development and clinical application, and has good potential practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0029] Figure 1 Figure 1 is the characterization of Napabucasin-PLGA NPs in the embodiment of the present invention; A shows that Nap@PLGANPs have obvious Tyndall effect; B is the measurement result of nanoparticle size; C and D are UV spectra of Napabucasin and Nap@PLGA NPs, respectively; E is the encapsulation efficiency (EE%), drug loading (DL%) and drug release rate of the nanoformulation; F is the standard curve of Nap; G shows that the nanoformulation is pH responsive.

[0030] Figure 2 This is the dose-response curve of Nap@PLGANPs on HCC cell lines in the example of the present invention; HCC cells were treated with different concentrations of Napabucasin (A) or Nap@PLGANPs (B) and the cell viability was detected after 48 hours; the data show the mean±SD values ​​of three independent experiments.

[0031] Figure 3 The ability of Nap@PLGANPs to inhibit HCC cell proliferation in vitro in the present invention; HCC cells were treated with different concentrations of Napabucasin (A) or Nap@PLGA NPs (B) for 24 hours, 48 ​​hours and 72 hours, and their cell viability was detected. The data show the mean±SD values ​​of three independent experiments. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.ns, no significance).

[0032] Figure 4 The in vitro induction of apoptosis of HCC cells by Nap@PLGANPs in the present invention; Huh7 (A), Hep3B (B) and Hepa1-6 (C) cells were treated with different concentrations of Napabucasin or Nap@PLGANPs for 12 hours, and their apoptosis ratios were detected by flow cytometry. The data show the mean±SD values ​​of three independent experiments. (*p<0.05, **p<0.01, ***p<0.001).

[0033] Figure 5 Nap@PLGA NPs down-regulate p-STAT3 in HCC cells in the present invention Try705 Levels; Western Blotting detected the effects of Nap@PLGA NPs treatment on STAT3 and p-STAT3Try705 The influence of protein levels.

[0034] Figure 6 Nap@PLGANPs inhibit HCC colony formation in the examples of the present invention; Huh7 (A), Hep3B (B) and Hepa1-6 cells (C) were treated with different concentrations of Napabucasin or Nap@PLGANPs to detect their clone formation ability.

[0035] Figure 7 The results show that Nap@PLGA NPs inhibit the in vitro spheroidization of HCC cells in the present invention. Huh7 (A) and Hepa1-6 (B) cells were treated with different concentrations of Napabucasin or Nap@PLGA NPs to detect their in vitro spheroidization ability. The data show the mean±SD values ​​of three independent experiments. (**p<0.01, ***p<0.001).

[0036] Figure 8 The Nap@PLGA NPs in the present invention inhibited the growth of subcutaneous tumors in mice; A shows the treatment plan of the in vivo experiment; B shows the tumor size of each group of mice after treatment; C shows the changes in the tumor volume of each group of mice during the treatment; D shows the tumor mass of each group of mice. (*p<0.05, **p<0.01).

[0037] Fig. 9 In the example of the present invention, Napabucasin prepared into PLGA NPs can reduce the toxic and side effects of the drug; A shows the change curve of the body weight of each group of mice; B and C show the HE staining results of the main organs of each group of mice after treatment.

[0038] Fig.10 Nap@PLGA NPs enhance anti-tumor immune response in the present invention; flow cytometry was used to detect CD8 + T cell ratio (A) and IFN-γ + (B), TNF-α + (C)TIGIT + CD8 + T (D) cell ratio; flow cytometry detection of NK cell ratio (E) and IFN-γ in tumor tissue after treatment + (F),TIGIT + (G),TIM3 + (H) NK cell ratio; flow cytometry detection of CD4 + T cell ratio (I) and TNF-α + CD4 +The proportion of T (J) cells. (n=6. *p<0.05, **p<0.01, ***p<0.001. ns, no significance).

[0039] Fig.11 Nap@PLGA NPs promote TAMs infiltration and M1 polarization in the present invention; flow cytometry was used to detect CD11b in tumor tissue after treatment + F4 / 80 + Macrophage ratio (A) and CD206 + (B), iNOS + (C), MHCⅡ + (D),CD86 + (E), PD-L1 + (F) Macrophage ratio. (n=6. *p<0.05, **p<0.01, ***p<0.001. ns, no significance).

[0040] Fig.12 The results show that Nap@PLGA NPs inhibit the level of p-STAT3 in HCC-TAMs in the present invention. Western Blotting was used to detect the expression of STAT3 and p-STAT3 in HCC-TAMs after 12 hours of treatment with Napabucasin or Nap@PLGA NPs. Try705 Protein levels.

[0041] Fig.13 The results show that Nap@PLGANPs inhibited M2 polarization of RAW264.7 cells in the present invention; A shows the effect of Napabucasin or Nap@PLGA NPs on HCC-TAMs cell viability; B shows the mRNA levels of M2 and M1 markers; C and D detected the ratio of CD206 / iNOS and IL-I2 by flow cytometry. The data show the mean±SD values ​​of three independent experiments. (*p<0.05, **p<0.01, ***p<0.001.ns, no significance).

[0042] Fig.14 The example of the present invention shows that Nap@PLGA NPs inhibit the tumor-promoting effect of HCC-TAMs; HCC-TAMs were treated with Nap@PLGANPs for 24 hours, and the culture medium was changed and continued for 24 hours, and the supernatant was collected (A). The effect of the culture supernatant on the proliferation ability (B) and migration ability (C) of Hepa1-6 cells was detected. The data show the mean±SD values ​​of three independent experiments. (**p<0.01, ***p<0.001).

[0043] Fig.15 In the present invention, Nap@PLGANPs relieve HCC-TAMs from CD8 + T cell inhibitory effect; (A) CD8 + T cells were labeled with CFSE and co-cultured with HCC-TAMs treated with Nap@PLGA NPs for 72 hours and detected by flow cytometry; (B) CD8 + T cells were co-cultured with HCC-TAMs treated with Nap@PLGA NPs for 72 hours, and their activation was detected by flow cytometry; (C) CD8 + T cells, labeled with CFSE, were co-cultured with HCC-TAMs treated with Nap@PLGA NPs by Transwell for 72 hours and detected by flow cytometry. Data show mean±SD values ​​of three independent experiments. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.001.ns, no significance). DETAILED DESCRIPTION

[0044] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0045] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0046] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out according to conventional conditions or the conditions recommended by the reagent company; the reagents and consumables used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0047] As mentioned above, BBI608, as a new small molecule STAT3 inhibitor, showed excellent anti-HCC effects in vitro. However, its low solubility and high cytotoxicity limit its clinical application.

[0048] In view of this, the present invention adopts polylactic acid-glycolic acid copolymer (PLGA), a hydrophobic material with good biocompatibility and biodegradability, as a carrier, prepares it into a nanoformulation, improves drug solubility, and achieves a better treatment effect of hepatocellular carcinoma through passive targeting.

[0049] Specifically, in a typical embodiment of the present invention, a nanoparticle encapsulating BBI608 is provided, wherein the nanoparticle uses polylactic acid-glycolic acid copolymer as a carrier and encapsulates BBI608; wherein the average particle size of the nanoparticle is 300 nm, and the drug encapsulation rate is above 90%.

[0050] In the present invention, the nanoparticles loaded with BBI608 are referred to as Nap@PLGA NPs.

[0051] In another specific embodiment of the present invention, a method for preparing the above-mentioned nanoparticles is provided, and the preparation method comprises preparing the above-mentioned nanoparticles by a solvent volatilization method.

[0052] In another specific embodiment of the present invention, the preparation method comprises:

[0053] S1, dissolving BBI608 and poly(lactic acid-co-glycolic acid) copolymer in an organic solvent until they are completely dissolved, and then adding them into water and stirring to obtain a mixture;

[0054] S2, shearing the mixture obtained in step S1 at low temperature until the solution becomes a uniform white emulsion;

[0055] S3, stirring the white emulsion obtained in step S2 to volatilize the organic solvent.

[0056] Wherein, in the step S1, the mass ratio of BBI608 to polylactic acid-glycolic acid copolymer is 1:5-15, preferably 1:10;

[0057] The organic solvent can be dichloromethane; using this organic solvent is conducive to obtaining a better encapsulation rate and particle size.

[0058] The mass volume ratio of BBI608, organic solvent and water is 1 mg: 0.2-1 mL: 3-8 mL, preferably 1 mg: 0.4 mL: 5 mL.

[0059] In step S2, the low-temperature shearing treatment time is 20-60 minutes, preferably 30 minutes.

[0060] In step S3, the stirring treatment time is at least 12 hours.

[0061] In another specific embodiment of the present invention, the use of the above-mentioned nanoparticles in any one or more of the following is provided:

[0062] a1) Ability to inhibit tumor cell proliferation or to prepare products capable of inhibiting tumor cell proliferation;

[0063] a2) Inducing apoptosis of tumor cells or preparing products that induce apoptosis of tumor cells;

[0064] a3) Inhibiting tumor cell stemness or preparing products that inhibit tumor cell stemness;

[0065] a4) Inhibition of p-STAT3 in tumor cells Try705 Inhibition of p-STAT3 in tumor cells Try705 horizontal products;

[0066] a5) Inhibiting the ability of tumor cell colony formation or preparing products that inhibit the ability of tumor cell colony formation;

[0067] a7) Inhibiting the ability of tumor cells to form spheres or preparing products that inhibit the ability of tumor cells to form spheres;

[0068] a8) Inhibiting tumor growth or preparing products that inhibit tumor growth;

[0069] a9) Reducing the toxic and side effects of BBI608, alleviating drug damage, or preparing products that reduce the toxic and side effects of BBI608, alleviating drug damage;

[0070] a10) Activating anti-tumor immune response or preparing products that activate anti-tumor immune response;

[0071] a11) Reversing the function of HCC-TAMs, enhancing T cell response, or preparing products that reverse the function of HCC-TAMs and enhance T cell response.

[0072] Specifically, in the above application, the tumor cell may be an HCC cell;

[0073] The tumor is liver cancer.

[0074] In a10), the activation of anti-tumor immune response is specifically manifested as:

[0075] a10.1) Promotes tumor-infiltrating T cells and NK cell activation;

[0076] a10.2) Promote the infiltration and M1 polarization of TAMs;

[0077] a10.3) inhibiting p-STAT3 levels in HCC-TAMs;

[0078] a10.4) inhibiting the M2 polarization of HCC-TAMs;

[0079] a10.5) Inhibit the tumor-promoting effect of HCC-TAMs.

[0080] a11) is manifested by the release of TAMs from CD8 + T cell inhibitory effect.

[0081] The product may be a drug or an experimental reagent, and the experimental reagent may be used for basic research, such as basic research on liver cancer-related tumors using tumor (HCC)-related cells or animal models.

[0082] In another specific embodiment of the present invention, a drug is provided, wherein the drug at least comprises the above-mentioned nanoparticles.

[0083] The medicament can be used for the treatment of HCC.

[0084] According to the present invention, the drug may further include at least one other inactive pharmaceutical ingredient.

[0085] The inactive ingredients of the drug can be carriers, excipients and diluents commonly used in pharmacy, etc. Moreover, according to the usual method, it can be made into oral preparations, external preparations, suppositories and sterile injection solutions in the form of powders, granules, suspensions, emulsions, syrups, sprays, etc.

[0086] The non-drug active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards.

[0087] In another specific embodiment of the present invention, the carrier, excipient and diluent include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0088] In another specific embodiment of the present invention, the medicine of the present invention can be administered to the body by known means. For example, it is delivered to the tissue of interest by intravenous systemic delivery or local injection (such as intratumoral injection). Such administration can be carried out via a single dose or multiple doses. It is understood by those skilled in the art that the actual dose to be administered in the present invention can vary depending on a variety of factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0089] In another specific embodiment of the present invention, the subjects of drug administration can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc., among which humans are preferred.

[0090] In another specific embodiment of the present invention, a method for treating tumors is provided, the method comprising administering a therapeutically effective dose of the above-mentioned nanoparticles or drugs to a subject.

[0091] The nanoparticles or drugs of the present invention can be used to treat benign tumors and malignant tumors. Examples of malignant tumors that can be treated with the drugs of the present invention include solid tumors and blood tumors, solid tumors are preferred, such as liver cancer, especially hepatocellular carcinoma.

[0092] The subject refers to an animal that has been the object of treatment, observation or experiment, preferably a mammal, and most preferably a human. The "therapeutically effective amount" refers to the amount of active compounds or agents, including the compounds of the present invention, which can cause the biological or medical response of the tissue system, animal or human sought by the researcher, veterinarian, doctor or other medical personnel, including the alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated. It must be recognized that the optimal dosage and interval of the active ingredients of the present invention are determined by their properties and external conditions such as the form, route and site of administration and the specific mammal being treated, and this optimal dosage can be determined using conventional techniques. It must also be recognized that the optimal course of treatment, that is, the daily dosage of the compound over a specified period of time, can be determined by methods known in the art.

[0093] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods for the specific conditions in the following examples are usually carried out under normal conditions.

[0094] Example

[0095] 1. Materials and Methods

[0096] 1.1 Main materials and reagents

[0097] 1.1.1 Reagents related to the preparation and characterization of Nap@PLGANPs

[0098] Napabucasin was purchased from Selleck; PLGA carrier was purchased from Hangzhou Xinqiao Biotechnology Co., Ltd.; dichloromethane, Tween 80 and other reagents were purchased from Aladdin.

[0099] 1.1.2 Experimental cell lines

[0100] Human hepatocellular carcinoma cell line Huh-7, human hepatocellular carcinoma cell line Hep3B, mouse hepatocellular carcinoma cell line Hepa1-6, mouse mononuclear macrophage leukemia cell line RAW264.7, and human embryonic kidney cell line HEK293T were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0101] 1.1.3 Experimental animals

[0102] C57BL / 6J mice, male, 5 weeks old, were purchased from Beijing Huafukang Company; all mice were housed in the SPF animal room of Shandong University as required. All animal experiments were in accordance with the ethical requirements of Shandong University experimental animals.

[0103] 1.1.4 Cell culture related reagents

[0104] DMEM, MEM, and RPMI1640 culture media were purchased from HyClone; fetal bovine serum was purchased from Hangzhou Sijiqing Biological Products Co., Ltd.; trypsin was purchased from Solarbio; penicillin G sodium and streptomycin sulfate were purchased from Lukang Pharmaceutical Co., Ltd.; cell freezing solution was purchased from Dalian Meilun Biotechnology Co., Ltd.; EGF and bFGF were purchased from Peprotech, USA; B-27 was purchased from Invitrogen, USA; CCK-8 was purchased from Biosharp; Annexin V / PI apoptosis kit was purchased from Yisheng Biotechnology Co., Ltd.; anti-mouse CD3e, anti-mouse CD28, MojoSort TM Mouse CD8T Cell Isolation Kit was purchased from Biolegend; flow cytometry antibodies were purchased from Biolegend; protein extraction and Western blotting related reagents were purchased from Solebow Technology Co., Ltd. and Beyotime Biotechnology Co., Ltd.

[0105] 1.1.5 Reagents related to vector construction

[0106] Escherichia coli cloning strain DH5α and PCDH vector were stored in our laboratory; restriction endonucleases and other enzymes were purchased from Thermo Scientific; PCR Using High-Fidelity DNA Polymerase was purchased from New England Biolabs; DNA ligation kit was purchased from Acre Bio; agar was purchased from BBI Life Sciences Co., Ltd.; and plasmid extraction kit was purchased from OMEGA.

[0107] 1.1.6 Animal experiment related reagents

[0108] Collagenase IV, hyaluronic acid, and heparin sodium were purchased from Solebao Technology Co., Ltd.; DNase I was purchased from Roche; ionomycin and PMA (HY-18739) were purchased from Med Chem Express (MCE); In vivo MAb anti-mouseCD4 and In vivo MAb anti-mouse CD8α were purchased from Bio X Cell; protein transport inhibitor BFA was purchased from Biolegend; and Percoll was purchased from GE Healthcare, Sweden.

[0109] 1.2 Preparation of Nap@PLGA NPs

[0110] (1) 1 mg Napabucasin and 10 mg PLGA were dissolved in 400 μL dichloromethane, pipetted until completely dissolved, and added dropwise into 5 mL triple distilled water. The mixture was stirred at room temperature to obtain Nap@PLGA NPs. Separately, 10 mg PLGA was dissolved in 400 μL dichloromethane and added dropwise into an equal volume of triple distilled water to prepare blank PLGA NPs for the control group.

[0111] (2) The above mixtures were placed in an ultrasonic shearing machine and sheared at low temperature for 30 min until the solution became a uniform white emulsion.

[0112] (3) Place the white emulsion on a magnetic stirrer and continue stirring for more than 12 hours to evaporate and remove dichloromethane.

[0113] (4) The white emulsion was centrifuged at 10,000 rpm for 10 min, and the supernatant was taken and the encapsulation efficiency and drug loading of BBI608 in Nap@PLGA NPs were detected by UV spectrophotometer.

[0114] (5) The lower layer Nap@PLGANPs and control PLGA NPs were dispersed in sterile triple-distilled water, respectively, and the final concentration was adjusted to 1 mM. The Tyndall effect was verified with a laser pen, and the NPs were stored in a refrigerator at 4 °C for later use.

[0115] 1.3 Characterization of Nap@PLGA NPs

[0116] 1.3.1 Particle size measurement

[0117] Dynamic light scattering was used to measure the particle size of lipid nanoparticles Nap@PLGA NPs.

[0118] 1.3.2 Investigation of in vitro drug release behavior

[0119] The drug release behavior of Nap@PLGANPs was determined by dynamic membrane dialysis. The specific experimental operation was as follows: 2 mL of 1.5 mg mL-1 1 mL of Nap@PLGANPs was taken and added into two dialysis bags (3500Da) respectively. The bags were divided into two groups. One group was placed in a 10 mL PBS centrifuge tube with pH = 7.4 and 2% Tween 80, and the other group was placed in a 10 mL PBS centrifuge tube with pH = 5.0 and 2% Tween 80. The bags were placed in a 37°C constant temperature shaker for shaking. 1 mL of sample was taken out at each specific time point (0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h), and an equal amount of release medium was supplemented accordingly. Three parallel groups were set up for each group at each time point. The Napabucasin content at different time points was determined by UV spectrophotometry, the cumulative release percentage was calculated, and the in vitro cumulative release curve was drawn.

[0120] 1.4 Main experimental methods

[0121] 1.4.1 Cell culture

[0122] 1.4.1.1 Tumor cell passaging

[0123] Discard the original culture medium of the cells that have grown on the wall, add a small amount of PBS to rinse, and then add an appropriate amount of trypsin to digest for 1 to 2 minutes. When the intercellular gap increases and the shape changes, add culture medium to terminate digestion, and use a pipette to gently blow until all cells fall off. Transfer the cell suspension to a new centrifuge tube and centrifuge at 100g for 5 minutes. After centrifugation, discard the supernatant and add fresh culture medium to resuspend. According to the experimental needs, divide the cell suspension into new culture bottles and place them in a constant temperature incubator with 5% CO2 and 37℃ for culture.

[0124] 1.4.1.2 RAW264.7 cell passaging

[0125] Use a cell scraper to gently scrape off the cells that have grown on the wall, transfer them to a new centrifuge tube, and centrifuge at 100g for 5 minutes. After centrifugation, discard the supernatant and add fresh culture medium to resuspend. According to the experimental needs, divide the cell suspension into new culture bottles and place them in a 5% CO2, 37℃ constant temperature incubator for culture.

[0126] 1.4.2 Cell apoptosis experiment

[0127] The cells were seeded in 12-well plates at 1.5 × 10 5cells. After the cells adhered to the wall, Napabucasin and Nap@PLGA NPs were added for treatment for 12 hours. After 12 hours, 500 μL of EDTA-free trypsin was added to each well for digestion, and PBS was added to terminate the digestion. After the cells were resuspended, they were transferred to the flow tube and centrifuged at 100g for 5 minutes. After centrifugation, the supernatant was discarded, and 500 μL of 1×BindingBuffer was added to each well to resuspend the cells, and centrifuged at 100g for 5 minutes. After centrifugation, the supernatant was discarded, and a small amount of solution was retained. 2.5 μL Annexin V and 5 μL PI dye were added, and placed at room temperature away from light for 15 minutes. Detected using a flow cytometer as required.

[0128] 1.4.3 CCK-8 assay for cell proliferation

[0129] (1) Tumor cells were inoculated into 96-well plates, with 5000 cells per well.

[0130] (2) After the cells adhere to the wall, add 100 μL of drug-containing culture medium.

[0131] (3) After drug treatment for 24 h, 48 h, and 72 h, 10 μL of CCK8 solution was added to each well in the dark and the cells were placed in a 5% CO2, 37°C constant temperature cell culture incubator for further culturing for 2 h.

[0132] (4) After 2 h, the absorbance at 450 nm was measured using a multifunctional microplate reader.

[0133] 1.4.4 Cell spheroidization assay

[0134] (1) Tumor cells were seeded in low-adhesion six-well plates, with 5,000 cells per well.

[0135] (2) Add 40 μL of B-27 supplement (50×), 20 μL of N-2, 40 ng of EGF, and 40 ng / mL of bFGF to 2 mL of DMEM / F12 medium, and replenish the medium every 3 days.

[0136] (3) After 10 days of culture, photographs were taken under an inverted microscope, and cell spheres with a diameter > 75 μm were counted.

[0137] 1.4.5 Fluorescence quantitative PCR

[0138] (1) The cDNA obtained by reverse transcription was diluted 50 times with triple distilled water for fluorescence quantitative PCR detection.

[0139] (2) Add the reagents to the eight-row series as shown in the table below:

[0140] Table 1 RT-qPCR system

[0141]

[0142] (3) Open the computer software, use the two-step reaction program, and set the number of cycles to 49.

[0143] (4) Data analysis

[0144] 1.4.6 Protein extraction and Western blotting experiments

[0145] 1.4.6.1 Protein extraction

[0146] (1) Take the cells cultured in the six-well plate, discard the supernatant, wash once with PBS, add 100 μL of RIPA lysis buffer (PIPA:PMSF=100:1), and lyse on ice for 15-20 minutes, pipetting every 10 minutes.

[0147] (2) Centrifuge the lysed cells at 13,000 g for 15 min at 4°C and collect the supernatant.

[0148] (3) Take 5 μL of supernatant and perform quantification with the BCA kit. Add an equal volume of 2× loading buffer to the remaining supernatant, mix well, denature in a metal bath at 98°C for 15 min, and store in a -20°C refrigerator.

[0149] 1.4.6.2 Western blotting experiment

[0150] (1) Wash the glass plate and place it on a gel rack. Add distilled water to check for leaks.

[0151] (2) Prepare 10% lower separation gel according to the following table:

[0152] Table 2 10% separation gel configuration system

[0153]

[0154]

[0155] (3) Add separation gel to the gap between the glass plates and add single distilled water to flatten the gel surface to avoid the formation of bubbles. Leave the gel at room temperature.

[0156] (4) After the lower layer of gel solidifies, prepare 5% concentrated gel according to the table below.

[0157] Table 3 5% concentrated gel preparation system

[0158]

[0159] (5) Pour out the distilled water from the glass plate and dry it with absorbent paper. Then quickly add the prepared concentrated glue to the glass plate and insert a tooth comb.

[0160] (6) After the upper layer of gel solidifies, remove the comb, install the glass plate on the electrophoresis device, add electrophoresis fluid, and prepare for sample loading.

[0161] (7) When loading the sample, add 20 μg of protein sample to each well and add different volumes of protein marker on both sides.

[0162] (8) During electrophoresis, first use 80V for 30 minutes to compress the protein sample to the plane of the separation gel; then use 120V for 120 minutes to separate proteins of different molecular weights.

[0163] (9) After electrophoresis, a transfer device was constructed according to the sandwich method, and transfer solution was added. The membrane was transferred at a constant voltage of 80 V for 1 h.

[0164] (10) The PVDF membrane with protein blot was placed in 5% skim milk and blocked at room temperature for 1 h.

[0165] (11) After blocking, place the PVDF membrane in the primary antibody dilution solution and incubate on a shaker at 4°C overnight.

[0166] (12) Remove the PVDF membrane and add 1× TBST to rinse three times, each time for 5 min; then place the PVDF membrane in the secondary antibody dilution solution and incubate at room temperature for 2 h.

[0167] (13) Remove the PVDF membrane and rinse it three times with 1× TBST, each time for 5 min; then it can be photographed and developed.

[0168] 1.4.7 Incubation of RAW264.7 cells with Hepa1-6 supernatant

[0169] (1) Collect the supernatant of Hepa1-6 cells in good condition, centrifuge at 3000g for 10 min to remove cell debris, and store in aliquots at -80℃.

[0170] (2) Take RAW264.7 cells in good condition, scrape them with a cell scraper, add culture medium to resuspend them, count and plate them, and 1.5×10 cells per well were used. 5 cells.

[0171] (3) After the cells adhered, Hepa1-6 cell supernatant and culture medium were added in a 1:1 ratio and incubated for 48 h.

[0172] 1.4.8 Scratch test

[0173] (1) RAW264.7 cells were incubated with Hepa1-6 supernatant for 48 h, and then treated with Napabucasin, Nap@PLGA NPs, and PLGA NPs for 24 h. After changing the medium, the cells were cultured for another 24 h, and the supernatant was collected for the following scratch experiment.

[0174] (3) 50,000 well-growing Hepa1-6 cells were seeded into each well of a 48-well plate.

[0175] (4) When the cells have adhered to the wall and spread out, use the pipette tip to draw a horizontal line vertically in the well plate.

[0176] (5) Wash three times with PBS to remove detached cells, add 50% serum-free DMEM and 50% supernatant of macrophages treated differently in (1), and take pictures under a microscope at 0 h and 24 h, and store them.

[0177] 1.4.9 Co-incubation experiment

[0178] 1.4.9.1 Acquisition of HCC-TAMs

[0179] (1) Hepa1-6 supernatant was added to the well-growing RAW264.7 cells for induction for 48 hours to obtain HCC-TAMs; Nap@PLGA NPs and PLGA NPs were then added to the HCC-TAMs for treatment for 24 hours to obtain drug-treated HCC-TAMs.

[0180] 1.4.9.2 Obtaining Mouse Splenic Mononuclear Cells

[0181] (1) Kill the mice, separate the spleen, grind it with a 200-mesh sieve, filter it into a 50-mL centrifuge tube, and add 1× PBS to 40 mL.

[0182] (2) Centrifuge at 400 g for 5 min.

[0183] (3) Discard the upper layer of liquid, resuspend the cells with 1 mL of MojoSort 1× Buffer, and filter into a 2 mL EP tube using a 200-mesh filter.

[0184] (4) Count and draw out 5×10 7 For each cell, add 1× Buffer to 500 μL.

[0185] 1.4.9.3CD8 + T cell magnetic bead sorting

[0186] (1) Add 50 μL of antibody, mix well, and incubate at 4°C for 15 min.

[0187] (2) Add 50 μL beads, mix well, and incubate at 4°C for 15 min.

[0188] (3) Add 3 mL of 1× Buffer and centrifuge at 400 g for 5 min.

[0189] (4) Discard the upper layer of liquid, add 2.5 mL of 1× Buffer, and mix well.

[0190] (5) Separate for 5 minutes and pour into a new flow tube.

[0191] (6) Add 2 mL of 1× Buffer and mix well.

[0192] (7) Sorting for 5 minutes, pouring into a flow tube.

[0193] (8) Centrifuge at 400 g for 5 min.

[0194] (9) Discard the supernatant liquid and retain the cell pellet.

[0195] 1.4.9.4 Cell plating

[0196] (1) Culture medium: RPMI1640 complete culture medium + anti-mouse CD3 (2 g / mL) + anti-mouse CD28 (2 g / mL) + IL-2 (1000 IU / mL).

[0197] (2) Differently treated RAW264.7 cells were compared with sorted CD8 + T cells were divided into 1:4 (5.0×10 4 :2.0×10 5 ) ratio and inoculated in a 24-well plate (CFSE was added before T cell inoculation and incubated at 37°C in the dark for 15 min).

[0198] (3) Culture the cells in a 37°C, 5% CO2 cell culture incubator in the dark for 72 h, supplementing the culture medium once during the process.

[0199] 1.4.10 Establishment and treatment of mouse transplanted tumor model

[0200] (1) Hepa1-6 cells in good condition were collected, digested with trypsin, centrifuged at 100 g for 5 min, discarded the supernatant, and washed twice with 1× PBS.

[0201] (2) Resuspend the cells in 1× PBS and adjust the cell concentration to 1×10 8 / mL, keep on ice until use.

[0202] (3) Take 5-week-old healthy C57BL / 6J mice and inject 100 μL of the cell suspension into the left armpit of the mouse using a syringe.

[0203] (4) After tumor loading, the size of the mouse tumor was measured using a vernier caliper every 2 days to calculate the tumor volume and monitor the changes in mouse body weight.

[0204] (5) Seven days after tumor loading, mice were given Nap@PLGA NPs or API by gavage once every two days for 8 consecutive times.

[0205] (6) After administration, the mice were euthanized, the tumor tissues were removed, the tumor weight was measured, and photos were taken for preservation.

[0206] 1.4.11 Flow cytometry to detect cell molecule expression

[0207] 1.4.11.1 Detection of cell surface molecules

[0208] (1) Collect the cell suspension, pass it through a 200-mesh sieve, and transfer it to a flow cytometer.

[0209] (2) Centrifuge at 100 g for 5 min, discard the supernatant, add a small amount of 1× PBS to resuspend the cells, and add 10 μL of rat serum to each tube for blocking at room temperature for 30 min.

[0210] (3) Add fluorescein-labeled flow cytometry antibody and incubate at 4°C in the dark for 40 min.

[0211] (4) Add 3 mL of 1× PBS to each tube to wash away unbound antibodies and centrifuge at 400 g for 5 min.

[0212] (5) Discard the supernatant, add 100 μL of 1× PBS to resuspend the cells, and detect using a flow cytometer.

[0213] 1.4.11.2 Detection of cytokines and intracellular molecules

[0214] (1) Collect the cell suspension, pass it through a 200-mesh sieve, and transfer it to a flow cytometer.

[0215] (2) Centrifuge at 100 g for 5 min, discard the supernatant, add a small amount of 1× PBS to resuspend the cells, and add 10 μL of rat serum to each tube for blocking at room temperature for 30 min.

[0216] (3) Label the cell surface molecules according to the above experimental steps, wash away the unbound antibodies with 1× PBS, add 100 μL of 1% paraformaldehyde solution to each tube, and fix at 4°C for 30 min.

[0217] (4) Add 3 mL of 1× PBS to each tube to wash away the paraformaldehyde, centrifuge at 400 g for 5 min, add 100 μL of transmembrane solution, and incubate at room temperature for 30 min.

[0218] (5) Add fluorescein-labeled flow cytometry antibody and incubate at 4°C in the dark for 40 min.

[0219] (6) Add 3 mL of 1× PBS to each tube to wash away unbound antibodies and centrifuge at 400 g for 5 min.

[0220] (7) Discard the supernatant, add 100 μL of 1× PBS to resuspend the cells, and detect using a flow cytometer.

[0221] 1.4.12 HE staining

[0222] (1) Fixation: The heart, liver, spleen, lung and kidney of mice were separated, immersed in 4% polymethanol and stored in the dark for more than one week.

[0223] (2) Dehydration: Place the fixed tissue in an embedding frame, wash it with water overnight, and then place it in ethanol solutions of different concentrations for dehydration.

[0224] Heart: 50% ethanol for 1 hour, 70% ethanol for 1 hour, 80% ethanol for 1 hour, 95% ethanol for 1 hour, anhydrous ethanol for 1 hour;

[0225] Liver: 50% ethanol for 1 hour, 70% ethanol I for 1 hour, 70% ethanol II for 1 hour, 80% ethanol for 1 hour, 90% ethanol for 1 hour, anhydrous ethanol for 1 hour;

[0226] Spleen: 50% ethanol for 20 min, 70% ethanol for 20 min, 75% ethanol for 20 min, 80% ethanol for 20 min, 90% ethanol for 20 min, 95% ethanol I for 20 min, 95% ethanol II for 20 min, anhydrous ethanol I for 20 min, anhydrous ethanol II for 20 min;

[0227] Lung: 70% ethanol for 1 hour, 75% ethanol for 1 hour, 80% ethanol for 1 hour, 90% ethanol for 1 hour, 95% ethanol for 1 hour, anhydrous ethanol for 1 hour;

[0228] Kidney: 75% ethanol for 30 min, 80% ethanol for 30 min, 90% ethanol for 30 min, 95% ethanol I for 30 min, 95% ethanol II for 30 min, anhydrous ethanol I for 30 min, anhydrous ethanol II for 30 min.

[0229] (3) Transparency: Xylene I 40 min, Xylene II 40 min.

[0230] (4) Wax immersion: paraffin I for 60 min, paraffin II for 60 min, 60°C.

[0231] (5) Sectioning: Cut the tissue wax block into 5 μm sections, place in 30% ethanol for 30 seconds, and then place in a 45°C water bath for expansion. Place the tissue sections adsorbed on the slide in a 37°C oven to dry overnight.

[0232] (6) Dewaxing: xylene I for 15 min, xylene II for 15 min.

[0233] (7) Hydration: anhydrous ethanol for 3 min, 95% ethanol for 3 min, 80% ethanol for 3 min, 70% ethanol for 3 min, and distilled water for 5 min.

[0234] (8) Staining: Stain with hematoxylin for 5 min, rinse with running water, add 1% hydrochloric acid ethanol for differentiation for 10 s, reverse blue with tap water, add eosin for staining for 5 min, and rinse with running water.

[0235] (9) Dehydration and transparency: 80% ethanol for 30 s, 95% ethanol for 60 s, 100% ethanol for 60 s, xylene I for 15 min, xylene II for 15 min.

[0236] (10) Sealing: After the slides are naturally dried in a fume hood, add a drop of neutral resin to seal the slides, observe under a microscope, and take photos.

[0237] 1.4.13 Data Analysis

[0238] The experimental results were plotted and statistically analyzed using GraphPad Prism 8.0, and Student's or ANOVA variance test was used for statistical analysis. Each experiment was repeated at least three times, and the data were presented as mean ± standard deviation (mean ± SD). When p < 0.05, it was considered that there was a significant difference between the two groups. The correlation was determined by Pearson analysis, and when p < 0.05, it was considered that there was a significant correlation between the two groups. The closer the absolute value of R is to 1, the stronger the correlation.

[0239] 2. Experimental results

[0240] 2.1 Preparation of Nap@PLGA NPs by solvent evaporation method

[0241] Napabucasin, as a new STAT3 inhibitor, has a good anti-tumor effect on both early and late-stage liver cancer in mouse models and reduces the stem cell characteristics of liver cancer cells. In order to improve the water solubility of the drug and reduce toxic side effects, we prepared Nap@PLGA NPs using a solvent evaporation method. Next, the physicochemical properties of Nap@PLGA NPs were characterized. The results show that Nap@PLGA NPs have a significant Tyndall effect ( Figure 1 A), the particle size measurement results show that the nanoparticle size is about 300nm ( Figure 1 B), Napabucasin ( Figure 1 C) and Nap@PLGA NPs (D) UV spectra. The encapsulation efficiency (EE%), drug loading (DL%) and drug release rate of the nanoformulations at different pH were determined by UV spectrophotometry. The results showed that the drug encapsulation efficiency of the two formulations was above 90%, and the drug loading was around 10% ( Figure 1 E). And the standard curve of Nap was determined ( Figure 1 F). The drug release at pH = 5.0 is greater than that at pH = 7.4. At 48 hours, the Nap release is close to 80%, which is pH responsive ( Figure 1 G). Dissolve in sterile water and store at 4°C.

[0242] 2.2 Dose-response curve of Nap@PLGA NPs on HCC cells

[0243] In order to verify whether Napabucasin can effectively exert its anti-HCC effect after being prepared into nanoformulations, we first evaluated its dose-response curve in human HCC cell lines Huh7 and Hepa3B and mouse HCC cell line Hepa1-6, and calculated the IC50 value (Table 4). The experimental results showed that compared with the raw material, the IC50 value of Nap@PLGA NPs increased slightly, but still showed a good anti-HCC effect ( Figure 2 ).

[0244] Table 4 IC50 values ​​of Napabucasin and Nap@PLGA NPs in HCC cell lines

[0245]

[0246] 2.3 Inhibitory effect of Nap@PLGA NPs on HCC cell proliferation

[0247] According to the IC50 values ​​obtained in the above experiments, we selected three drug concentrations to treat Huh7, Hep3B and Hepa1-6 cells, and observed the effect of Nap@PLGA NPs on the proliferation of HCC cells. The experimental results showed that, similar to the raw material drug, Nap@PLGA NPs inhibited the proliferation of HCC cells in vitro in a concentration- and time-dependent manner ( Figure 3 ).

[0248] 2.4 Nap@PLGANPs induce apoptosis of HCC cells

[0249] STAT3 regulates cell proliferation and apoptosis. Inhibiting STAT3 phosphorylation in HCC cells can induce cell apoptosis. As the concentration of the inhibitor increases, the level of cell apoptosis also gradually increases. We selected three concentrations based on the IC50 value to treat Huh7( Figure 4 A), Hepa3B (Figure 4B) and Hepa1-6 cells ( Figure 4 C) Treated for 12 hours, Annexin-V-FITC and PI dual-color labeling method, flow cytometry analysis of the effect of Nap@PLGA NPs on the apoptosis of HCC cell lines. The experimental results showed that, similar to the raw material drug, Nap@PLGA NPs induced HCC cell apoptosis in vitro in a dose-dependent manner ( Figure 4 ).

[0250] 2.5 Nap@PLGA NPs inhibit HCC cell stemness

[0251] STAT3 is overactivated in many tumors and has been shown to be an important pathway for tumor stem cells to mediate tumor proliferation. Napabucasin, as a tumor stem cell inhibitor, can inhibit STAT3 phosphorylation by downregulating the expression of tumor-related stem molecules such as Nanog, SOX2, and CD163, inhibiting tumor stemness and reducing the ability of HCC cells to form spheres in vitro. Based on previous studies, we next evaluated the ability of Nap@PLGA NPs to inhibit HCC cell stemness in vitro.

[0252] 2.5.1 Nap@PLGANPs inhibit p-STAT3 in HCC cells Try705 level

[0253] First, we treated Huh7 cells with different concentrations of Nap@PLGANPs for 12 h and detected STAT3 and p-STAT3 in the cells by Western Blot. Try705 The experimental results showed that Nap@PLGA NPs could significantly inhibit the expression of p-STAT3 in HCC cells. Try705 levels, but did not affect the total STAT3 protein level ( Figure 5 ).

[0254] 2.5.2 Nap@PLGA NPs inhibit HCC cell colony formation

[0255] Cell colonies are formed by cells with stem cell characteristics through unlimited reproduction in vitro, and are an important experiment for evaluating the proliferation and stemness characteristics of tumor cells in vitro. The experimental results showed that, similar to the raw materials, Nap@PLGA NPs can significantly inhibit the colony formation ability of HCC cells ( Figure 6 ).

[0256] 2.5.3 Nap@PLGA NPs inhibit HCC cell spheroidization

[0257] The cell sphere formation experiment is also an important method for evaluating the stemness characteristics of tumor cells in vitro. Single cells are inoculated in a low-adhesion culture environment, and a variety of growth factors are added to the suspension culture medium. Tumor cells with stem cell characteristics can form regular cell spheres in vitro through continuous self-replication. We evaluate the sphere formation ability by observing the number and size of tumor cell spheres. The experimental results show that similar to the raw material drug, in Huh7( Figure 7 A), Hepa1-6 cells ( Figure 7B) Adding low concentrations of Nap@PLGA NPs (1.0 μM, 5.0 μM) during the spheroidization process can significantly reduce the number of stem cell spheres, promote the lysis of stem cell spheres, and inhibit their spheroidization ability.

[0258] 2.6 Anti-HCC effect of Nap@PLGA NPs in vivo

[0259] 2.6.1 Nap@PLGANPs inhibit tumor growth in mice with Hepa1-6 subcutaneous tumors

[0260] Given that Nap@PLGA NPs exhibited good anti-HCC effects in vitro, we then used a mouse hepatoma cell subcutaneous transplant model to verify the in vivo therapeutic effect of Nap@PLGA NPs. 7 Hepa1-6 cells were inoculated in the left armpit of C57BL / 6J mice, and the mice were treated with drugs on the 7th day of tumor bearing. The drugs were given by intraperitoneal injection once every 2 days for 8 consecutive times to evaluate the therapeutic effect of Nap@PLGA NPs ( Figure 8 A). Experimental results show that Nap@PLGA NPs can significantly inhibit tumor growth, and the therapeutic effect is comparable to that of the raw material drug ( Figure 8 BD).

[0261] 2.6.2 Preparation of Napabucasin into Nap@PLGA NPs can reduce drug damage

[0262] During the treatment, we found that high doses of Napabucasin (20 mg / kg) significantly reduced the body weight of tumor-bearing mice ( Fig. 9 A), indicating that Napabucasin may have greater toxicity. HE staining sections of mouse heart, liver, kidney, spleen and lung were used to observe the damage of the drug to major organs. The experimental results showed that when treated with a high dose of Napabucasin (20 mg / kg), the intercellular space in the mouse kidneys increased significantly, and a large number of vacuoles appeared in the liver tissue ( Fig. 9 B), while no obvious organ damage was observed in the Nap@PLGA NPs treatment group ( Fig. 9 C) These results indicate that Napabucasin has severe hepatotoxicity and renal toxicity, and preparation into PLGANPs can reduce the toxic side effects of Napabucasin while maintaining the therapeutic effect.

[0263] 2.7 Nap@PLGANPs activate anti-tumor immune response

[0264] Through the mouse Hepa1-6 cell subcutaneous transplant tumor model, we found that Nap@PLGA NPs can significantly inhibit tumor growth. Tumor growth is closely related to the immune microenvironment, and as the tumor develops, the function of effector T cells and NK cells in the tumor microenvironment is impaired and gradually exhausted; while suppressive immune cells (M2 TAMs, Tregs, MDSCs, etc.) proliferate in large numbers and secrete a variety of cytokines to promote tumor invasion, metastasis and angiogenesis. In order to further explore the mechanism by which Nap@PLGANPs exert anti-HCC effects in vivo, we once again used the Hepa1-6 cell subcutaneous transplant tumor mouse model and gave 7 Nap@PLGA NPs treatments (10 mg / kg, ip) to evaluate the activation of the mouse immune system.

[0265] 2.7.1 Nap@PLGA NPs promote tumor-infiltrating T cells and NK cell activation

[0266] As important effector cells of anti-tumor immunity, T cells and NK cells play a direct role in anti-tumor immunity. However, as the tumor continues to develop, the deteriorating immunosuppressive microenvironment induces the inactivation of T cell and NK cell effector functions and promotes the expression of multiple inhibitory receptors (PD-1, TIGIT, TIM3, LAG3, etc.), causing T cells and NK cells to gradually become exhausted.

[0267] In order to observe the effect of Nap@PLGA NPs on the immune microenvironment, we used flow cytometry to detect the activation of tumor-infiltrating T cells and NK cells. The experimental results showed that Nap@PLGANPs treatment increased CD8 + T cells, NK cells ( Fig.10 A and E) infiltration levels, promoting CD8 + The secretion level of IFN-γ in T cells and NK cells ( Fig.10 B&F), while down-regulating the expression levels of inhibitory molecules TIGIT and TIM3 ( Fig.10 D and H). In addition, Nap@PLGA NPs also promoted CD8 + T, CD4 + T cells secrete TNF-α ( Fig.10 C and J). These results indicate that Nap@PLGA NPs can enhance the expression of CD8 + The effector functions of T cells and NK cells improve the tumor microenvironment and enhance the killing effect on tumor cells.

[0268] 2.7.2 Nap@PLGANPs promote TAMs infiltration and M1 polarization

[0269] Tumor-associated macrophages (TAMs) play a powerful role in anti-tumor immunity. It is generally believed that macrophages in tumor tissues are mostly of M2 phenotype and play an immunosuppressive role. Therefore, targeting TAMs has also become an effective strategy for treating cancer. In order to observe the effect of Nap@PLGA NPs on TAMs in vivo, we isolated tumor-infiltrating mononuclear cells and detected the level of macrophage infiltration and activation in tumor tissues by flow cytometry. The experimental results showed that compared with the untreated or PLGA NPs-treated group, Nap@PLGANPs treatment increased the infiltration of macrophages in tumor tissues ( Fig.11 A), while reducing the expression level of M2 marker CD206 ( Fig.11 B), upregulation of iNOS, a marker of M1 macrophages ( Fig.11 C) CD86( Fig.11 D) and MHCⅡ( Fig.11 E) expression level and inhibited the activation of PD-L1 (Figure 112F). This result also suggests that Nap@PLGA NPs may inhibit M2 TAMs, promote the expression of co-stimulatory molecule CD86 and antigen presenting molecule MHCⅡ, enhance anti-tumor immunity, and better exert anti-HCC effect.

[0270] 2.7.3 Nap@PLGA NPs inhibit p-STAT3 levels in HCC-TAMs

[0271] More and more studies have shown that the STAT3 signaling pathway plays an important role in the polarization of M2 macrophages. To further explore the mechanism by which HCC supernatant induces M2 polarization of macrophages, we used Western Blot to detect p-STAT3 in HCC-TAMs. Try705 The experimental results showed that compared with untreated RAW264.7 cells (M), HCC supernatant-treated RAW264.7 cells (Untreated) had a higher p-STAT3 Try705 Subsequently, we used Nap@PLGA NPs and API to treat HCC-TAMs for 12 h and found that both Nap@PLGA NPs and API could significantly inhibit the phosphorylation level of STAT3 in HCC-TAMs ( Fig.12 ).

[0272] 2.7.4 Nap@PLGA NPs inhibit M2 polarization of HCC-TAMs

[0273] Compared with normal macrophages, TAMs often show higher Ki67 activity. In order to explore the effect of Nap@PLGANPs on macrophage polarization, we first tested the effect of Nap@PLGA NPs on HCC-TAMs cell viability. The experimental results showed that compared with untreated HCC-TAMs (untreated), HCC-TAMs cell viability still reached more than 80% after 24h treatment with Nap@PLGANPs ( Fig.13 A). Next, we used RT-qPCR to detect the effect of Nap@PLGANPs on HCC-TAMs cell polarization. The experimental results showed that compared with untreated and PLGANPs-treated HCC-TAMs, Nap@PLGANPs could reduce the mRNA expression levels of M2 markers CD206, IL-10, and TGF-, and increase the mRNA expression levels of M1 marker molecules iNOS and IL-12 in HCC-TAMs ( Fig.13 B). Flow cytometry assay further confirmed that the ratio of CD206 / iNOS in HCC-TAMs was significantly upregulated compared with untreated RAW264.7 cells (M), while Nap@PLGANPs could reduce the ratio of CD206 / iNOS in HCC-TAMs ( Fig.13 C) and increased the expression level of IL-12 ( Fig.13 D). The above results demonstrate that Nap@PLGANPs can inhibit the M2 polarization of macrophages.

[0274] 2.7.5 Nap@PLGANPs inhibit the tumor-promoting effect of HCC-TAMs

[0275] Next, we collected the culture supernatant of HCC-TAMs treated with Nap@PLGA NPs, incubated tumor cells for 24h and 48h, and observed the effects on tumor cell proliferation and migration. The experimental results showed that compared with the supernatant of HCC-TAMs treated with untreated and PLGA NPs, the supernatant of HCC-TAMs treated with Nap@PLGANPs could inhibit the proliferation of Hepa1-6 cells ( Fig.14 B) and migration ability ( Fig.14 C) and exhibits time dependence.

[0276] 2.8 Nap@PLGANPs reverse HCC-TAMs function and enhance T cell response

[0277] 2.8.1 Nap@PLGANPs relieve TAMs from CD8 +Inhibitory effect of T cells Macrophages, as important antigen presenting cells, can not only secrete a variety of cytokines in response to natural immune responses, but also promote anti-tumor immunity by activating T. However, TAMs exert immunosuppressive effects by enhancing the expression of immune checkpoints PD-1, TIGIT, and TIM3 on the surface of T cells, inhibiting the secretion of IFN-γ and IL-2 in T cells. We isolated CD8 + T cells were activated in vitro by adding anti-CD3 / CD28 antibodies, and then HCC-TAMs treated with Nap@PLGA NPs were combined with CD8 + T cells were co-incubated for 72 h to observe their effects on CD8 + The experimental results showed that compared with the T cells in the group without HCC-TAMs, HCC-TAMs could inhibit the proliferation of CD8+T cells, while Nap@PLGA NPs treatment could relieve the effect of HCC-TAMs on CD8 + T cell inhibitory effect ( Fig.15 A) and increase CD8 + The expression levels of IFN-γ, Perforin and Granzyme B in T cells ( Fig.15 B) Furthermore, HCC-TAMs were co-cultured with T cells using the Transwell method, and it was found that Nap@PLGA NPs-treated macrophages had a significant effect on CD8 + There was no significant effect on T cell proliferation ( Fig.15 C) These results suggest that HCC-TAMs express CD8 + The effect of T cells may be achieved through direct cell-cell contact. Nap@PLGA NPs can weaken the effect of HCC-TAMs on CD8 + T cell inhibitory effect.

[0278] The research results of this embodiment are as follows:

[0279] 1. Nap@PLGA NPs can significantly inhibit p-STAT3 in HCC cells Try705 It inhibited the proliferation of HCC cells in a concentration- and time-dependent manner in vitro, and induced the apoptosis of HCC cells in a dose-dependent manner, without affecting the total STAT3 protein level.

[0280] 2. Nap@PLGA NPs can reduce the mRNA levels of tumor stemness-related molecules such as Nanog, SOX2, OCT4, and KIF4 in HCC cells, and significantly inhibit the colony formation and in vitro spheroidization ability of HCC cells.

[0281] 3. Nap@PLGA NPs reduce p-STAT3 in HCC-TAMs Try705 levels, inhibiting the FAO process in HCC-TAMs, reducing the consumption of endogenous lipids, and decreasing the levels of ECAR and OCR, thereby altering macrophage M2 polarization and inhibiting the tumor-promoting effects of HCC-TAMs.

[0282] 4. Co-incubation experiments showed that Nap@PLGA NPs treatment could relieve HCC-TAMs from CD8 + T cell suppression and increase in CD8 + Expression levels of IFN-γ, Perforin and Granzyme B in T cells.

[0283] 5. Nap@PLGA NPs inhibited the growth of Hepa1-6 subcutaneous tumors and reduced drug damage. At the same time, Nap@PLGANPs increased macrophage infiltration in tumor tissues, promoted the conversion of TAMs to M1 phenotype, and then enhanced CD8 + T cell function and improved anti-tumor immunity. The anti-tumor effect of Nap@PLGA NPs in vivo depends on the enhanced T cell response.

[0284] In summary, the present invention demonstrates that Nap@PLGA NPs prepared by solvent evaporation method have excellent anti-HCC effect and good safety.

[0285] Matters not covered by the present invention are known technologies.

[0286] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A nanoparticle loaded with BBI608, characterized in that: The nanoparticles use polylactic acid-glycolic acid copolymer as a carrier and encapsulate BBI608; wherein the average particle size of the nanoparticles is 300nm, and the drug encapsulation rate is above 90%.

2. The method for preparing nanoparticles according to claim 1, characterized in that: The preparation method comprises the step of preparing by solvent volatilization.

3. The preparation method according to claim 2, characterized in that: The preparation method comprises: S1, dissolving BBI608 and poly(lactic acid-co-glycolic acid) copolymer in an organic solvent until they are completely dissolved, and then adding them into water and stirring to obtain a mixture; S2, shearing the mixture obtained in step S1 at low temperature until the solution becomes a uniform white emulsion; S3, stirring the white emulsion obtained in step S2 to volatilize the organic solvent.

4. The preparation method according to claim 3, characterized in that: In the step S1, the mass ratio of BBI608 to polylactic acid-co-glycolic acid is 1:5-15, further 1:10; The organic solvent is dichloromethane; The mass volume ratio of BBI608, organic solvent and water is 1 mg: 0.2-1 mL: 3-8 mL, further 1 mg: 0.4 mL: 5 mL.

5. The preparation method according to claim 3, characterized in that: In step S2, the low-temperature shearing treatment time is 20-60 minutes, and further 30 minutes.

6. The preparation method according to claim 3, characterized in that: In step S3, the stirring treatment time is at least 12 hours.

7. Use of the nanoparticles according to claim 1 or the nanoparticles prepared by the preparation method according to any one of claims 2 to 6 in any one or more of the following: a1) Ability to inhibit tumor cell proliferation or to prepare products capable of inhibiting tumor cell proliferation; a2) Inducing apoptosis of tumor cells or preparing products that induce apoptosis of tumor cells; a3) Inhibiting tumor cell stemness or preparing products that inhibit tumor cell stemness; a4) Inhibition of p-STAT3 in tumor cells Try705 Inhibition of p-STAT3 in tumor cells Try705 horizontal products; a5) Inhibiting the ability of tumor cell colony formation or preparing products that inhibit the ability of tumor cell colony formation; a7) Inhibiting the ability of tumor cells to form spheres or preparing products that inhibit the ability of tumor cells to form spheres; a8) Inhibiting tumor growth or preparing products that inhibit tumor growth; a9) Reducing the toxic and side effects of BBI608, alleviating drug damage, or preparing products that reduce the toxic and side effects of BBI608, alleviating drug damage; a10) Activating anti-tumor immune response or preparing products that activate anti-tumor immune response; a11) Reversing the function of HCC-TAMs, enhancing T cell response, or preparing products that reverse the function of HCC-TAMs and enhance T cell response.

8. The use according to claim 7, characterized in that The tumor cells are HCC cells; The tumor is liver cancer.

9. The use according to claim 7, characterized in that: In a10), the activation of anti-tumor immune response is specifically manifested as: a10.1) Promotes tumor-infiltrating T cells and NK cell activation; a10.2) Promote the infiltration and M1 polarization of TAMs; a10.3) inhibiting p-STAT3 levels in HCC-TAMs; a10.4) inhibiting the M2 polarization of HCC-TAMs; a10.5) inhibiting the tumor-promoting effect of HCC-TAMs; a11) is manifested by the release of TAMs from CD8 + The inhibitory effect of T cells; The product is a drug or an experimental reagent, and the experimental reagent is used for basic research.

10. A drug, characterized in that The drug at least comprises the nanoparticles according to claim 1 or the nanoparticles prepared by the preparation method according to any one of claims 2-6.

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