Nano-drug for simultaneously inhibiting fibroblast activation and angiogenesis

Abstract

The invention belongs to the technical field of tumor prevention and treatment, and particularly relates to a nano-drug capable of simultaneously inhibiting fibroblast activation and angiogenesis. The nano-drug for simultaneously inhibiting fibroblast activation and angiogenesis is a bionic nano-carrier SNPs (Single Nucleotide Polymorphism) loaded with modified relaxin analogue polypeptide B7-33. The bifunctional bionic nano-drug B7-33-SNPs adopted by the invention can be delivered to tumor tissues in a targeted manner, and meanwhile, the fibroblast activation-mediated extracellular matrix and angiogenesis are inhibited, and the fibroblast activation-mediated angiogenesis is also inhibited. The results can lay a good technical foundation for targeted prevention and treatment of bile duct cancer, and provide a new technical thought for prevention and treatment of other diseases.

Description

Technical Field

[0001] This application belongs to the field of tumor prevention and treatment technology, specifically relating to a nanomedicine that can simultaneously inhibit fibroblast activation and angiogenesis. Background Technology

[0002] Medical statistics show that cholangiocarcinoma (CCA) accounts for approximately 3% of gastrointestinal tumors, and its global incidence and mortality rates are on a continuous upward trend. Currently, surgical resection remains the only potentially curative treatment for this disease. However, due to factors such as low public awareness and limited timely detection, over 70% of patients are diagnosed at an advanced stage, thus losing the opportunity for surgery. Clinically, chemotherapy regimens using gemcitabine / cisplatin as first-line therapy are currently the main treatment approach. However, due to factors such as rapid development of drug resistance in patients, clinical benefits are limited. Therefore, there is an urgent need to develop new treatment strategies.

[0003] Studies on the mechanisms of tumorigenesis and development have shown that the tumor microenvironment plays a crucial role in the progression of cholangiocarcinoma, particularly the angiogenesis process driven by vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANG-2), both of which are important prognostic factors promoting tumor angiogenesis and growth. Based on these mechanistic findings, targeting the VEGFA or ANG2 signaling pathways with monoclonal antibodies or multi-target tyrosine kinase inhibitors could inhibit cholangiocarcinoma angiogenesis to some extent. However, further research indicates that drug resistance and poor treatment efficacy limit the clinical application of this therapy. Furthermore, preclinical studies have found that anti-angiogenic therapy exacerbates tumor hypoxia, promotes the upregulation of extracellular matrix (ECM) expression, and thus affects tumor angiogenesis and treatment response.

[0004] Studies on cholangiocarcinoma have revealed that a significant characteristic of the cholangiocarcinoma microenvironment is the extensive fibrosis driven by cancer-associated fibroblasts (CAFs) and the massive deposition of extracellular matrix (ECM). This not only promotes tumor progression but also leads to poor clinical outcomes and treatment resistance. Research shows that cytokines such as interleukin-6 (IL-6) and C-X-C motif chemokine ligand 12 (CXCL12), as well as ECM proteins secreted by CAFs, can enhance the growth and invasion of tumor cells. High expression of α-SMA-positive CAFs is closely associated with rapid tumor growth, high migration and invasiveness, and poor survival prognosis in cholangiocarcinoma patients. Simultaneously, the ECM proteins secreted by CAFs not only provide structural support for the tumor but also form a physical barrier hindering drug penetration, leading to reduced efficacy of anti-angiogenic drugs. Furthermore, CAFs create a pro-angiogenic microenvironment by upregulating the expression of stromal cell-derived factor 1 (SDF-1 / CXCL12) and VEGFA, thus weakening the efficacy of anti-VEGFA therapy. In summary, whether a treatment strategy based on simultaneously targeting CAF activation and inhibiting angiogenesis can be applied in clinical treatment models of cholangiocarcinoma has not been fully explored and validated.

[0005] In recent years, biomimetic nanocarriers, represented by biological membranes, have attracted widespread attention due to their advantages such as long-term circulation in vivo and good biocompatibility. Research suggests that biomimetic nanocarriers possess both the physicochemical properties of nanocarriers, facilitating targeted aggregation to specific organs, and the biological properties of natural cells, enabling the loading of drugs such as nucleic acids, peptides, and small molecules through biological (genetic engineering, membrane fusion) or physicochemical (endocytosis, direct drug delivery, and chemical modification) methods, thus offering significant advantages in the diagnosis and treatment of related diseases. Furthermore, nanocellular membranes, based on the intact lipids and receptors retained on their surface, can act as "nanosponges," safely and effectively absorbing or removing pathogenic molecules. Therefore, biomimetic nanocarriers have broad prospects for medical applications. In previous research, the inventors developed related biomimetic nanocarrier-based preventative drugs and achieved some progress. Based on this, and considering the mechanisms of cholangiocarcinoma development and the challenges of prevention and treatment, the inventors attempted to develop a new nanomedicine, or utilize existing nanocarrier drugs, that can simultaneously target and inhibit tumor matrix fibrosis and angiogenesis, thereby providing a new prevention and treatment pathway for cholangiocarcinoma. Summary of the Invention

[0006] Based on previous research findings, the purpose of this application is to provide a new nanomedicine or to experimentally apply existing nanocarrier drugs to cholangiocarcinoma, with the aim of simultaneously targeting and inhibiting fibroblast activation and angiogenesis in the cholangiocarcinoma tumor microenvironment, thereby laying a certain technical foundation for the prevention and treatment of cholangiocarcinoma.

[0007] The technical solution adopted in this application is described in detail below.

[0008] A nanomedicine that simultaneously inhibits fibroblast activation and angiogenesis is prepared by loading modified B7-33 onto biomimetic nanocarriers SNPs made from biomembranes. The preparation process involves the following steps: (I) Preparation of biomimetic nanocarriers SNPs made from biomembranes Referring to existing technologies, biomimetic nanocarriers SNPs made from biomembranes are prepared, specifically, for example, biomembranes prepared using mouse vascular endothelial cells sEND.1; or they can be prepared by referring to the following preparation procedures: (1) Gradient centrifugation was used to extract cell membranes. Discard the culture medium in the pre-cultured mouse vascular endothelial cells sEND.1 that have grown to a full culture dish, wash gently with PBS 3 times (retain 1 ml of PBS in the last wash), scrape off the cells, and centrifuge at 1000 r for 5 min. Discard the supernatant, add IB-1 (2 ml) to the cell pellet to resuspend, and transfer to a glass homogenizer to grind for about 40 times (operate on ice). Centrifuge the ground cell solution at 3000g for 5 minutes at 4℃ and collect the supernatant; Centrifuge the supernatant at 4°C and 10000g for 10 minutes, and retain the supernatant. Add the supernatant to the centrifuge tube, add IB-1 to fill the tube, centrifuge at 4°C and 10000g for 2 hours, and retain the precipitate (for subsequent operations or store at -80°C for later use).

[0009] The IB-1 (isolation buffer, to be prepared fresh) is: SB + 0.5% BSA + 0.5 mmol EGTA. To prepare, first dissolve 0.5 g BSA in 100 ml of SB solution, then add 500 μL of 100 mmol EGTA (pH 7.4) and 1% protease inhibitor (specifically, 200 mM AEBSF, 30 μM Aprotinin, 13 mM Mpestatin, 1.4 mM E64, and 1 mM eupeptin, prepared in high-purity DMSO). The SB (prepared one day in advance) is: 225 mmol mannitol + 75 mmol sucrose + 30 mmol Tris-HCl (pH 7.4); to prepare it, first dissolve 20.5 g mannitol and 13 g sucrose in 400 ml ddH2O, then add 15 ml Tris-HCl, let it stand at 4°C for 30 min, adjust the pH to 7.4, and finally make up the volume to 500 ml with ddH2O. (2) Using an extruder to extrude cell membrane biomimetic nanocarriers SNPs The precipitate (i.e., mouse vascular endothelial cells) obtained by centrifugation in step (1) was placed in a cell sonicator and sonicated for 3 min (sonic power 100w, sonication for 5s, pause for 5s). Subsequently, the cells were extruded using an Avanti Mini Extruder (AvantiPolar Lipids) extruder. First, using a 0.8 μm polycarbonate (PC) membrane, the vascular endothelial cell membrane after ultrasonic treatment was squeezed up and down 10 times. Subsequently, the process involved extruding 0.4 μm and 0.1 μm PC films 10 times each. Finally, uniform nanoparticles (biomimetic nanocarriers SNPs) with an average particle size of about 100 nm were prepared for later use. (ii) Modifications and alterations to B7-33 The modification of B7-33 is achieved by using the cRGD sequence to modify B7-33 to form cRGD-B7-33; The amino acid sequence of the polypeptide B7-33 is shown in SEQ ID No. 1, specifically: VIKLSGRELVRAQIAISGMSTWSKRSL; The modified amino acid sequence of B7-33 (cRGD-B7-33) is: VIKLSGRELVRAQIAISGMSTWSKRSL-GG-c(RGDfK); (III) Loading and constructing bifunctional biomimetic nanomedicines Based on the affinity between the receptor (sEND.1 cell membrane surface Integrin αv(β3)) and the ligand (cRGD), efficient loading of cRGD-B7-33 onto a biomimetic nanocarrier was achieved, thus constructing multifunctional biomimetic nanomedicines (B7-33-SNPs); specifically: By incubating the modified B7-33 (cRGD-B7-33) from step (II) with the biomimetic nanocarrier SNPs prepared in step (I) at 4℃ for 12 h, biomimetic nanomedicine B7-33-SNPs can be obtained.

[0010] The application of the nanomedicine that simultaneously inhibits fibroblast activation and angiogenesis in the preparation of cholangiocarcinoma prevention and treatment agents, which interferes with the association between fibroblasts and blood vessels for the prevention and treatment of cholangiocarcinoma (tumor).

[0011] In existing technologies, anti-angiogenic drugs have limited efficacy in the prevention and treatment of cholangiocarcinoma (CC), and resistance often develops rapidly. Therefore, after studying the mechanisms of CC development, the inventors believe that anti-VEGFR2 therapy activates extracellular matrix (CAF), leading to excessive ECM deposition and the formation of a physical barrier, thus weakening the efficacy of anti-angiogenic therapy in CC. In other words, extracellular matrix remodeling caused by fibroblast activation is a key factor in resistance to anti-angiogenic therapy. Therefore, in the process of preventing and treating CC based on anti-angiogenesis, a drug is needed that can simultaneously target and inhibit both fibroblasts and angiogenesis, thereby avoiding drug resistance caused by fibroblast activation during the anti-angiogenic process. Based on previous research results, the inventors applied their novel B7-33-SNP nanomedicine to cholangiocarcinoma and discovered its dual function: firstly, it retains angiogenesis factor receptors, competitively adsorbing angiogenesis factors by utilizing the nanosponges function of endothelial cell membranes to weaken their biological effects; secondly, due to high expression of integrin αvβ3, the specifically loaded cRGD peptide-modified peptide B7-33 can inhibit CAF activation. Further experiments in a subcutaneous CCA xenograft model showed that B7-33-SNPs synergistically blocked the crosstalk between CAF and angiogenesis, effectively reducing microvessel density and fibrosis deposition in the tumor microenvironment. These results demonstrate that a combined strategy based on B7-33-SNPs that simultaneously targets stromal fibrosis and the vascular microenvironment can produce a synergistically enhanced tumor growth inhibition effect in the prevention and treatment of cholangiocarcinoma, thus providing a new therapeutic option for inhibiting the occurrence and development of malignant CCA.

[0012] Overall, the bifunctional biomimetic nanomedicine B7-33-SNPs used in this application can be targeted and delivered to tumor tissues, while reducing drug resistance mediated by fibroblast activation in the extracellular matrix. It also inhibits fibroblast activation mediated during angiogenesis and retains its inhibitory effect on angiogenesis. These results lay a solid technical foundation for the targeted prevention and treatment of cholangiocarcinoma and provide new technical approaches for the prevention and treatment of other diseases. Attached Figure Description

[0013] Figure 1This study presents the expression and spatial distribution patterns of α-SMA, CD31, and RXFP1 in human cholangiocarcinoma (CCA) tissues and mouse CCA cell-derived xenograft (CDX) models. A represents the double immunofluorescence staining results (n=6) of CD31 (white), α-SMA (red), and RXFP1 (green) in human CCA tissues, adjacent normal tissues, and HUCCT1 subcutaneous xenografts; cell nuclei are displayed by DAPI counterstaining (blue); scale bar: 100 µm. B shows the quantitative analysis of normalized protein expression levels of α-SMA, CD31, and RXFP1 in adjacent normal and tumor tissues using ImageJ software (n=6). C represents the Pearson correlation analysis of the number of transcripts per million (TPM) for ACTA2 (α-SMA), PECAM1 (CD31), and RXFP1 (RXFP1) genes in human CCA tissues. Reported correlation coefficients and significance levels are expressed as mean ± standard deviation; t-test showed p < 0.01. and *p<0.001; Figure 2 To illustrate the effects of anti-VEGFR2 therapy and endothelial cell membrane-derived nanovesicles (SNPs) on cancer-associated fibroblast (CAF) activation and extracellular matrix (ECM) deposition in HUCCT1 subcutaneous xenografts; where: A represents immunofluorescence analysis of matrix remodeling in HUCCT1 tumors; representative images show the staining results of CD31 and ECM deposition (α-SMA, fibronectin, and type I collagen) in mouse tumor sections treated with PBS, anti-VEGFR2 antibody, or SNPs (n=6); cell nuclei are displayed by DAPI counterstaining (blue). Scale bar: 100 µm; B represents the quantitative analysis of CD31 and ECM standardized expression levels; results are expressed as mean ± standard deviation (n=6); t-tests showed p<0.01 and *p<0.001; Figure 3Characterization of B7-33-SNPs; where: A is a transmission electron microscopy (TEM) image of B7-33-SNPs showing their uniform spherical morphology; scale bar: 200 nm; B is a stability analysis of B7-33-SNPs in 1×PBS (pH 7.4) at 37°C; hydrodynamic diameter (red) and polydispersity index (PDI, blue) were monitored over 7 days by dynamic light scattering (DLS) (n=3); C is an SDS-PAGE analysis of membrane proteins in sEND.1 cells, B7-33-SNPs, and B7-33-RNPs; D is a Western blot analysis validating the expression of endothelial cell-specific receptors VEGFR2, Tie2, and integrin αvβ3 in sEND.1 cells, B7-33-SNPs, and B7-33-RNPs; using Na⁺ / K⁺ATP Enzymes were used as loading controls; E shows immunogold-labeled transmission electron microscopy imaging of B7-33-SNPs: labeled with anti-VEGFR2 and anti-Tie2 antibodies (black dots) on 5 nm gold nanoparticles, and the samples were counterstained with 2% uranium acetate; scale bar: 100 nm; F shows the cytokine binding capacity of B7-33-SNPs and B7-33-RNPs assessed by ELISA, demonstrating dose-dependent neutralization of VEGFA and ANG2 (n=3 biological replicates); results are expressed as mean ± standard deviation. Figure 4 The in vitro anti-angiogenic activity of B7-33-SNPs is shown below; where: A is a representative wound healing assay image of VEGFA-activated sEND.1 cells after treatment with B7-33-SNPs or B7-33-RNPs; the dashed line indicates the scratch boundary; scale bar: 200 µm; B is a quantitative analysis of wound closure rate relative to 0 hours (n=3); C is a Transwell migration assay image showing the invasion of sEND.1 cells through the Matrigel-coated filter membrane; scale bar: 200 µm; D is a normalized quantitative analysis of the number of migrating cells relative to the VEGFA control group (n=3). E shows experimental images of lumen formation on Matrigel matrix after 6 hours of treatment; scale bar: 200 µm; F shows quantitative analysis of total lumen length per field of view (n=3); results are expressed as mean ± standard deviation; t-test showed **p<0.01; Figure 5The effects of B7-33-SNPs on contractile ability and CAF-mediated angiogenesis are shown below. Specifically: A shows representative images of iMEF cell contraction induced by 5 ng / mL TGF-β after 48 hours of treatment with different formulations; B shows the quantitative assessment of iMEF cell contraction rate in different treatment groups; C shows ECM deposition in iMEF cells or TGF-β-activated iMEF cells after 48 hours of treatment with different formulations, normalized with GAPDH, detected by Western blotting; D shows the quantitative analysis of ECM protein levels relative to GAPDH in different treatment groups; E shows a schematic diagram of the Transwell invasion assay setup: sEND.1 cells were seeded in Matrigel-coated Transwell chambers and allowed to invade iMEF cells or TGF-β-activated iMEF cells treated with different formulations in the lower culture chamber for 12 hours; F shows CMFDA staining images of migrating sEND.1 cells in each group; scale bar: 100µm; G shows CMFDA-based... Quantitative analysis of migrating cells in stained images; H represents the sprouting bright-field image on Matrigel after 48 hours of co-incubation of sEND.1 three-dimensional spheres with corresponding iMEF cells or TGF-β activated iMEF cells using different formulations; scale bar: 100 µm; dashed lines indicate the spherical sprouting boundary; magnified views of the areas marked by white boxes in the above image are shown below; I represents the quantitative analysis of the sprouting degree of sEND.1 three-dimensional spheres; results are expressed as mean ± standard deviation (n=6); t-test showed p<0.01 and *p<0.001; Figure 6 The distribution of B7-33-SNPs in a cholangiocarcinoma model was shown in vivo. A represents in vitro Cy5.5 fluorescence imaging of tumor tissue 24 hours after injection of B7-33-SNPs, C7-33-SNPs, or other SNPs; B represents quantitative analysis of tumor fluorescence intensity; C represents immunofluorescence localization of Cy5.5-labeled SNPs in the tumor (CD31 / α-SMA / DAPI staining, scale bar: 100µm). Results are expressed as mean ± standard deviation (n=6); t-test showed **p<0.01. Figure 7The in vivo therapeutic effect of B7-33-SNPs on cholangiocarcinoma is shown below: A is a schematic diagram of HUCCT1 subcutaneous inoculation and treatment regimen; B is the tumor growth curve during treatment; C is the individual tumor volume of the HUCCT1 cholangiocarcinoma model in the treatment study; D is a representative image of tumor-bearing mice at the endpoint, showing a reduction in tumor burden in the B7-33-SNPs group; E is H&E stained sections of tumor tissue from mice treated with different formulations; scale bar: 200 µm; F is the Western blot analysis of tumor matrix markers (including α-SMA, type I collagen, fibronectin, and CXCL12); G is the quantitative analysis of protein expression using ImageJ software, normalized to β-actin; results are expressed as mean ± standard deviation (n=6); t-test shows... p<0.05, p<0.01 and p<0.001; Figure 8 Immunofluorescence assessment of stromal modulation in HUCCT1 subcutaneous xenografts; where: A is a representative immunofluorescence image showing tumor stromal components (collagen fibers, type I collagen, α-SMA, fibronectin, PDGFR-β, and CD31); scale bar: 50 µm; B is a quantitative analysis of the normalized levels of collagen, type I collagen, α-SMA, fibronectin, PDGFR-β, and CD31; results are expressed as mean ± standard deviation (n=6); t-test showed... p<0.05, p<0.01 and p<0.001; Figure 9 Tumor penetration analysis of SNPs-Cy5.5 after cRGD-B7-33 pretreatment; where: A is a schematic diagram of the anti-fibrotic treatment regimen: mice bearing cholangiocarcinoma were intravenously injected with cRGD-B7-33 every 2 days for a total of 8 injections, followed by SNPs-Cy5.5 administration; B is in vivo fluorescence imaging 1 hour after SNPs-Cy5.5 injection; C is quantitative analysis of tumor fluorescence intensity; D is confocal microscopy images of tumor sections (DAPI: blue; SNPs-Cy5.5: red) showing spatial distribution (dashed lines delineate tumor margin / core; scale bar = 100 μm); E is quantitative analysis of the penetration depth of SNPs-Cy5.5 within the tumor in the cRGD-B7-33 treatment group; results are expressed as mean ± standard deviation (n=6); t-test showed ***p<0.001; Figure 10 Tumor volume of cholangiocarcinoma in mice after treatment with anti-VEGFR2 monoclonal antibody and SNPs; results are expressed as mean ± standard deviation (n=6); t-test showed... p<0.05, p<0.01 ; Figure 11For histopathological evaluation of major organs; H&E stained sections of major mouse organs treated with PBS, cRGD-B7-33, cRGD-B7-33+RNPs, SNPs, C7-33-SNPs, or B7-33-SNPs; Scale bar: 100 μm. Detailed Implementation

[0014] The present application will be further explained below with reference to the embodiments. Before introducing the specific embodiments, the experimental background of some embodiments is briefly described below.

[0015] Biomaterials: The immortalized mouse embryonic fibroblast (iMEF) cell line was generously provided by Professor Lin Xi of Tsinghua University in Beijing, China. Human cholangiocarcinoma (HuCCT1) and mouse endothelial cell (sEND.1) cell lines are commonly used in current tumor research and can be obtained from the open market. When culturing, use DMEM medium (Catalog No.: SH30022.01B; HyClone) supplemented with 10% fetal bovine serum (FBS, catalog No.: S829012029; Invigentech), penicillin (100 U / mL), and streptomycin (100 µg / mL, catalog No.: sv30010; HyClone), changing the medium every 3-4 days. Culture conditions are: 37°C, 5% CO2, 95% humidity. Male BALB / c nude mice (6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; all related animal experiments were approved by the Laboratory Animal Ethics Committee of the First Affiliated Hospital of Zhengzhou University (No.: 2024-KY-1113-001). The samples used for immunofluorescence (IF) staining of human CCA tissue, including CCA tissue and adjacent normal tissue, were obtained from Shanghai Chipchao Biotechnology Co., Ltd.; all experiments involving human specimens were approved by the Shanghai Chipchao Biomedical Research Ethics Committee (No.: SHYJS-BC-2310001). Some experimental reagents and equipment: A mixture of sucrose, EDTA, Hepes-NaOH, and protease inhibitors, a product of Sigma-Aldrich, USA; Matrigel matrix, type I collagen (catalog number: 354249), and Matrigel (catalog number: 354230) are products of Corning Incorporated, Inc., USA. Duchenne Modified Eagle Medium (DMEM) and Fetal Bovine Serum (FBS), products of HyClone, USA; CXCL12 (Catalog No.: A1325), recombinant mouse VEGFA / VEGF164 protein (Catalog No.: RP01060), mouse VEGFA ELISA kit (Catalog No.: RK00028), and recombinant mouse angiopoietin-2 / ANG-2 protein (Catalog No.: RP01744) are products of ABclonal, Inc. (USA). Anti-VEGFR2 antibody (catalog number: 9698S), a product of CST Biotechnology, USA; Anti-VEGFA monoclonal antibody B20.4-1.1, a product of Genentech, USA; Recombinant mouse TGF-β protein (catalog number: 7666-MB-005) and mouse relaxin R1 antibody (catalog number: MAB8898), products of R&D Systems, USA; Anti-Na+ / K+ ATPase (Catalog No.: ab76020), anti-Tie2 antibody (Catalog No.: ab21915), α-SMA (Catalog No.: ab5694), integrin αV (Catalog No.: ab179475) and integrin β3 (Catalog No.: ab119992), products of Abcam, UK; Alexa Fluor488 conjugated goat anti-rabbit IgG (H+L) secondary antibody (catalog number: A-11034), a product of ThermoFisher Scientific, USA; Rat anti-mouse CD31 antibody (Catalog No.: 550 274), a product of BD Bioscience, USA; Mouse angiopoietin-2 ELISA kit (catalog number: ZN2511), a product of Leybold Biotech Co., Ltd. 100 kDa percolation tube (UFC510096), a product of Millipore, USA.

[0016] Example 1 Based on the need for the prevention and treatment of cholangiocarcinoma, the inventors conducted preliminary research on the pathogenesis and development mechanism of cholangiocarcinoma. The relevant research is briefly introduced below.

[0017] (a) Immunofluorescence (IF) staining of human CCA tissues The patient's CCA tissue and adjacent tissue were embedded in paraffin, sectioned, dewaxed with xylene, and then hydrated with graded ethanol (refer to the existing technical routine operation, and the specific operation will not be described in detail). Subsequently, antigen retrieval was performed using sodium citrate buffer, and nonspecific binding sites were blocked with 10% goat serum. Primary antibodies targeting mouse relaxin R1, mouse CD31 monoclonal antibody, and α-SMA antibody were added to the slides and incubated overnight at 4°C. Next, add the fluorescently conjugated secondary antibody (catalog numbers: A31573 and A21428; Invitrogen and catalog number: A-11034; Thermo Fisher Scientific) and incubate at room temperature for 1 hour; Finally, the cell nuclei were counterstained with DAPI, the fluorescence signal was observed using a fluorescence microscope, and the positively stained areas were quantitatively assessed using ImageJ software.

[0018] Relevant experimental results are as follows Figure 1 As shown.

[0019] Since the tumor microenvironment is a key determinant of resistance to anti-angiogenic therapy, in order to elucidate the matrix-angiogenic interaction in cholangiocarcinoma (CCA), the inventors performed immunofluorescence staining of α-smooth muscle actin (α-SMA, a cancer-associated fibroblast CAF marker) and CD31 (an endothelial cell marker) on untreated human CCA tissue and adjacent non-tumor liver tissue.

[0020] right Figure 1 The analysis results show that: The number of α-SMA-positive stromal cells and CD31-positive microvessels in CCA tissue was significantly higher than that in normal organ parenchyma. Immunohistochemical analysis significantly showed that relaxin R1 (RXFP1) expression was upregulated in CCA tissue and co-localized with α-SMA-positive CAF and CD31-positive endothelial cells (Figure 1A). Consistent with human data, the mouse CCA model (HUCCT1 subcutaneous xenograft) showed strong expression of α-SMA, CD31, and RXFP1 in the tumor stroma. Pearson correlation analysis on the TCGA dataset showed a significant positive correlation between the expression of the gene ACTA2 (α-SMA) and the gene PECAM1 (CD31) (r=0.52, p=0.0013), and a significant positive correlation between the expression of the gene ACTA2 and the gene RXFP1 (r=0.52, p=0.0012). Figure 1 C).

[0021] The above results indicate that there is a close spatial and functional correlation between CAF, angiogenesis, and RXFP1 signaling in CCA. Based on this, it can be preliminarily inferred that targeting RXFP1 and thereby simultaneously blocking CAF-mediated fibrosis and angiogenesis may be an effective strategy for treating cholangiocarcinoma.

[0022] (ii) Anti-angiogenesis induces extracellular matrix deposition of CAF in cholangiocarcinoma. Nude mice bearing HUCCT1 xenografts were divided into the following experimental groups: (1) PBS (control group), (2) Anti-VEGFA monoclonal antibody B20.4-1.1 (Genentech; 5 mg / kg, intraperitoneal injection, twice a week). (3) SNPs (12 mg / kg, intravenous injection, every 48 hours); (see below for preparation method) The experiment lasted for 1.5 and 3 weeks (the experiment ended on day 11 of week 1.5), and 3-5 mice in each group were sacrificed at the end of the experiment; After euthanizing the mice, the tumors were removed; the removed tumors were fixed in 4% paraformaldehyde (4°C, overnight), embedded in paraffin, and sectioned (5 µm thickness). Immunohistochemical staining was performed on the sections using antibodies against CD31 (catalog number: 550274; BD Biosciences), α-SMA (catalog number: 19245S; CST), fibronectin (catalog number: 15613-1-AP; Proteintech), and type I collagen (catalog number: 14695-1-AP; Proteintech). After staining, five fields of view were randomly selected from each sample, and ImageJ software was used to perform quantitative analysis on the positive protein expression regions. All analyses used a uniform threshold setting.

[0023] The inventors believe that activated cancer-associated fibroblasts (CAFs) secrete excessive extracellular matrix (ECM) components, thereby regulating tumor angiogenesis and resistance to anti-angiogenic therapies. However, it remains unclear whether this process affects drug efficacy by specifically forming a physical barrier through ECM deposition in cholangiocarcinoma (CCA). Therefore, the above experiments primarily aimed to investigate the differences in efficacy between single nanovesicles (SNPs) and VEGFR2 neutralizing antibodies (anti-VEGFR2) in a mouse CCA model.

[0024] Experimental results are as follows Figure 2 As shown in the figure. Analysis reveals that: After 1.5 weeks of treatment, anti-VEGFR2 and SNPs significantly reduced the primary tumor volume by 37.1% (p<0.05) and 47.3% (p<0.01) compared to the control group, respectively. Figure 10(As shown in Figure 2A). However, both groups of tumors regrowed after 3 weeks of treatment, indicating acquired anti-angiogenic resistance. Furthermore, immunofluorescence analysis confirmed that both drugs significantly inhibited CD31-positive tumor angiogenesis after 1.5 weeks of treatment (Figure 2A). Figure 2 (B) This is consistent with the observed anti-angiogenic effect. Notably, the vascular remodeling in the 3-week treatment group showed rapid tumor angiogenesis, suggesting adaptive resistance to anti-angiogenic therapy. These results indicate that SNPs exert potent anti-angiogenic and anti-tumor effects in the early stages of treatment, but efficacy declines to near baseline levels during the angiogenesis phase. Overall, these results suggest that the physical barrier formed by ECM deposition does indeed affect the continuation of drug treatment.

[0025] It is also worth noting that, compared with the PBS-treated control group, tumor histology in mice treated for 3 weeks showed evidence of CAF activation, manifested as increased α-SMA expression and increased deposition of type I collagen and fibronectin (Figure 2A). Figure 2 B). These observations further confirm that ECM upregulation induced by anti-angiogenic therapy can lead to tumor drug resistance.

[0026] In summary, the results suggest that anti-VEGFR2 targeted therapy can induce CAF-mediated ECM remodeling, which may hinder drug penetration and lead to CCA treatment resistance. Therefore, a combined strategy targeting both angiogenesis and CAF activation may be an effective way to improve the efficacy of CCA treatment.

[0027] Example 2 In their previous research, the inventors believed that biomimetic nanomedicines derived from vascular endothelial cells could efficiently load peptide drugs onto their surfaces through receptor-ligand mediated interactions, thereby targeting and delivering peptides to the tumor microenvironment and enhancing the anti-tumor effects of peptide drugs.

[0028] Based on the above technical concept, combined with the inventors' long-term experience in peptide drug modification and the preparation of cell membrane-derived biomimetic nanovesicles, and utilizing receptors on mouse vascular endothelial cell membranes that highly express angiogenesis factors (VEGFA and ANG2), the inventors tentatively conducted a bifunctional detection experiment, and prepared a bifunctional biomimetic nanodrug B7-33-SNPs that simultaneously targets and inhibits fibroblast activation and angiogenesis. The preparation process of this nanodrug is briefly described below.

[0029] In this application, biomimetic nanocarriers SNPs are prepared using the cell membrane of mouse vascular endothelial cells (sEND.1). The specific operation is as follows.

[0030] First, vascular endothelial cell membranes are extracted using the classic cell membrane extraction method. Specifically: (1) Gradient centrifugation was used to extract cell membranes. Discard the culture medium from the pre-cultured vascular endothelial cells that have grown confluently in the culture dish, wash gently with PBS 3 times (retain 1 ml of PBS during the last wash), scrape off the cells, and centrifuge at 1000 r for 5 min. Discard the supernatant, add IB-1 (2 ml) to the cell pellet to resuspend, and transfer to a glass homogenizer to grind for about 40 times (operate on ice). Centrifuge the ground cell solution at 3000g for 5 minutes at 4℃ and collect the supernatant; Centrifuge the supernatant at 4°C and 10000g for 10 minutes, and retain the supernatant. Add the supernatant to the supernatant tube, add IB-1 to fill the tube, centrifuge at 4°C and 10000g for 2 hours, and retain the precipitate (for subsequent operations or store at -80°C for later use).

[0031] The IB-1 (isolation buffer, which needs to be prepared fresh) is: SB + 0.5% BSA + 0.5mmol EGTA. To prepare it, first dissolve 0.5g BSA in 100ml of SB solution, then add 500ul of 100mmol EGTA (pH 7.4) and 1% protease inhibitor. The SB (prepared one day in advance) is: 225 mmol mannitol + 75 mmol sucrose + 30 mmol Tris-HCl (pH 7.4); to prepare it, first dissolve 20.5 g mannitol and 13 g sucrose in 400 ml ddH2O, then add 15 ml Tris-HCl, let it stand at 4°C for 30 min, adjust the pH to 7.4, and finally make up the volume to 500 ml with ddH2O.

[0032] (2) Using an extruder to extrude cell membrane biomimetic nanocarriers SNPs The vascular endothelial cell membrane obtained by centrifugation in step (1) was placed in a cell ultrasonic disruptor with an ultrasonic power of 100W and ultrasonic treatment for 3 minutes (5 seconds of ultrasonic treatment followed by 5 seconds of pause). Subsequently, the membrane was extruded using an Avanti Mini Extruder (Avanti Polar Lipids) extruder. First, using a 0.8 μm polycarbonate (PC) membrane, the vascular endothelial cell membrane after ultrasonic treatment was squeezed up and down 10 times. Subsequently, the process involved extruding 0.4 μm and 0.1 μm PC films 10 times each. Finally, uniform nanoparticles (biomimetic nanocarriers SNPs) with an average particle size of about 100 nm were prepared for later use.

[0033] As a control, the inventors also prepared biomimetic nanocarriers RNPs using red blood cell membranes derived from mouse red blood cells. The specific operation is as follows.

[0034] First, red blood cell membranes are obtained using a hypotonic method. Specifically: Freshly collected blood samples (whole blood) were centrifuged at 1000 × g for 5 minutes at 4°C to remove serum and other blood cells, retaining the precipitate (red blood cells) and washing with PBS (three times). The precipitate was resuspended in ice-cold hypotonic solution (0.25 × PBS) for 1 hour, centrifuged at 4°C and 20,000 × g for 30 minutes, and the precipitate (white particles) was retained. Resuspend in PBS and incubate at 37°C for 1 hour for later use; Subsequently, following the aforementioned procedure, the Avanti Mini Extruder (Avanti Polar Lipids) was used to extrude through 0.4 μm and 0.22 μm PC films sequentially to obtain biomimetic nanocarriers RNPs.

[0035] (3) Modifications and alterations to B7-33 The amino acid sequence of the polypeptide B7-33 is shown in SEQ ID No. 1, specifically: VIKLSGRELVRAQIAISGMSTWSKRSL; The modified amino acid sequence of cRGD-B7-33 is: VIKLSGRELVRAQIAISGMSTWSKRSL-GG-c(RGDfK); As a control (a scrambled B7-33 analog), the sequence of RGD-C7-33 is: RLRSRVASGKSVILGLKSEQMSTAIWI-GG-c(RGDfK).

[0036] The aforementioned sequences were synthesized by Anhui Guoping Pharmaceutical Co., Ltd. (Anhui, China) using the solid-phase peptide synthesis method (SPPS).

[0037] Taking the preparation of RGD-B7-33 as an example, the specific synthesis procedure can be found as follows: (1) First, weigh 0.5 g of 2-CTC resin and add it to the peptide synthesis reaction tube. Add 5 ml of DCM until the reaction tube swells for 10 min. After swelling, add 5 ml of DMF and wash 3 times, 30 s each time. (2) Add 0.15 mmol Fmoc-ASP(OALL)-resin and 0.5 mmol DIEA, and condense by bubbling under nitrogen for 2 h; After the condensation is complete, the reaction solution is removed, and 5 ml of DMF is added to wash 4 times, 30 seconds each time. After the 5th wash, the solution is dried. (3) Add methanol: DIEA: DCM (1:1:2) to the above-dried solid reaction tube, bubble the reaction with nitrogen for 30 min and then seal the tube. After the reaction is completed, remove the reaction solution and add 5 ml of DMF to wash 5 times, 30 s each time. (4) Measure 5 ml of 20% piperidine / DMF solution and add it to the reaction tube. Bubble the reaction with nitrogen for 20 min to remove Fmoc. Add 0.5 mmol Fmoc-GLY-OH and 0.5 mmol HOBT to a solid-phase reaction tube, then add 0.5 mmol DIC, and condense by bubbling with nitrogen for 1 h. (5) Repeat the washing and condensation steps above, add Fmoc-ARG(PBF)-OH, Fmoc-LYS(DDE)-OH and Fmoc-D-PHE-OH to synthesize and obtain the uncyclized peptide chain RGDfK-resin; Subsequently, 0.03 mmol of tetraphenylphosphine palladium catalyst was dissolved in DCM and placed in a reactor to remove the oall protecting group for 3 h; (6) Using DIC and HOBT as condensing agents, c(RGDfK(dde))-resin was obtained by cyclization; Subsequently, dde was removed with 2% hydrazine hydrate DMF solution to obtain c(RGDfK)-resin; Finally, the washing and condensation were repeated, and the remaining amino acids were coupled sequentially to prepare cRGD-B7-33.

[0038] The prepared compounds were identified using electrospray ionization mass spectrometry (ESI-MS). The results showed that the molecular weight of the prepared cRGD-B7-33 was 3687.50 (consistent with the theoretical value of 3687.30), indicating the successful synthesis of the relevant compound.

[0039] (4) Loading and constructing peptide biomimetic nanomedicines Based on the affinity between the receptor (sEND.1 cell membrane surface Integrin αv(β3)) and the ligand (cRGD), the nanocarrier SNPs prepared in step (2) are mixed with the cRGD-B7-33 prepared in step (3) to achieve efficient loading of the therapeutic peptide B7-33 (cRGD-B7-33) onto the biomimetic nanocarrier, thereby ultimately constructing and obtaining the peptide biomimetic nanomedicine (B7-33-SNPs). In specific operation: By incubating cRGD-B7-33 (or cRGD-C7-33) with endothelial cell membrane biomimetic nanocarriers SNPs (or erythrocyte membrane biomimetic nanocarriers RNPs) at 4°C for 12 h, biomimetic nanomedicines B7-33-SNPs (or negative control B7-33-RNPs) and control biomimetic nanomedicines C7-33-SNPs can be prepared.

[0040] The prepared nanomedicine was characterized and tested, and the results are as follows: Figure 3 As shown in the figure. Specific analysis reveals that transmission electron microscopy (TEM) of B7-33-SNPs shows that the nanoparticles have a spherical morphology. Figure 3 A); Dynamic light scattering (DLS) analysis showed that its monodisperse hydrodynamic diameter was 120.3 ± 2.1 nm (PDI = 0.25 ± 0.02), zeta potential was -5.8 ± 1.9 mV, and it remained stable for 7 days. Figure 3 B).

[0041] To verify the retention of membrane proteins during nanoparticle preparation, B7-33-SNPs were further characterized by SDS-PAGE stained with Coomassie Brilliant Blue. Figure 3 C). The proteomic profile of B7-33-SNPs closely matched that of sEND.1 cells, confirming the retention of membrane-associated proteins. Subsequent Western blot analysis revealed that key receptors expressing important pro-angiogenic factors (such as VEGFR2 and Tie2) on endothelial cells were retained on the B7-33-SNP membrane. Figure 3 D). As a negative control, the expression of these pro-angiogenic receptors in erythrocyte membrane-masked nanoparticles (B7-33-RNPs) was negligible. Western blot further confirmed the expression of integrin αvβ3 in sEND.1 cells ( Figure 3 D). Immunogold staining with target-specific antibodies visually confirmed the localization of these proteins on the surface of B7-33-SNPs. Figure 3E), thereby endowing it with the ability to bind to key angiogenesis mediators (such as VEGFA and ANG2). Unlike B7-33-RNPs, B7-33-SNPs showed dose-dependent neutralization of all tested angiogenic cytokines (E). Figure 3 F).

[0042] In summary, it can be concluded that B7-33-SNPs nanomedicine was successfully constructed and exhibited strong cytokine neutralizing ability in vitro.

[0043] Example 3 Based on Example 1, the inventors conducted further in vitro cell experiments on the prepared nanomedicine B7-33-SNPs. The specific experimental process and results are briefly described below.

[0044] (a) B7-33-SNPs inhibit VEGFA-induced endothelial cell migration To evaluate the anti-angiogenic potential of B7-33-SNPs, we conducted wound healing, Transwell migration, and lumen formation experiments using sEND.1 endothelial cells (the relevant experimental procedures and operations can be referred to the existing standard procedures and will not be elaborated further).

[0045] Experimental results are as follows Figure 4 As shown in the figure. Analysis reveals that: As shown in Figure 4A, VEGFA-stimulated sEND.1 cells could completely close the scratch wound within 24 hours, while B7-33-SNPs treatment significantly inhibited cell migration, resulting in a residual wound area of ​​59.5±9.1% (p<0.01 compared with the VEGFA control group; Figure 4B).

[0046] Consistent with the wound healing assay results, Transwell migration assays showed that B7-33-SNPs treatment reduced VEGFA-driven cell invasion by 60.1 ± 8.1% (Figure 4C). In contrast, B7-33-RNPs had no significant inhibitory effect on migration (p>0.05 compared to the VEGFA control group; Figure 4D).

[0047] In addition, capillary-like lumen formation on Matrigel cells was assessed 6 hours after treatment: VEGFA-stimulated cells formed dense tubular networks, while B7-33-SNP treatment reduced branching by 59.2 ± 9.6% (p < 0.01), and the formed lumen was fragmented and shortened (Fig. 4E). B7-33-RNPs showed no inhibitory activity, confirming the specificity of the observed effect (Fig. 4F).

[0048] In summary, the results indicate that B7-33-SNPs can effectively inhibit VEGFA-mediated endothelial cell migration and lumen formation, thus demonstrating their anti-angiogenic effect.

[0049] (ii) Simultaneous targeting of CAF and angiogenesis can block matrix-vascular interaction. To evaluate the functional effects of B7-33-SNPs on mouse fibroblasts (iMEF), the inventors conducted a TGF-β-stimulated gel shrinkage experiment (the relevant experimental procedures and operations can be referred to the conventional operation of existing technology, and will not be repeated here).

[0050] Experimental results are as follows Figure 5 As shown in the figure. Analysis reveals that: Compared with the PBS-treated control group, B7-33-SNPs treatment significantly attenuated TGF-β-induced matrix shrinkage (p<0.01), with a reduction of 51.5±9.5% in shrinkage area (Figure 5A). This inhibitory effect was relatively superior to that of free B7-33 treatment (reduction of 43.9±10.3%), and also indicated that the antifibrotic activity of nanoparticles coupled with B7-33 was preserved (Figure 5B). Western blot showed that B7-33-SNPs strongly inhibited TGF-β-induced α-SMA, fibronectin, and type I collagen levels (Figure 5C, Figure 5D). Figure 5 (D), consistent with previous findings. These data indicate that B7-33-SNPs significantly inhibit fibroblast activation by blocking the TGF-β signaling pathway.

[0051] Activated CAFs promote angiogenesis by secreting specific pro-angiogenic factors and confer resistance to anti-angiogenic therapies. To assess the angiogenic potential of activated fibroblasts, endothelial cell migration was quantitatively analyzed using a Transwell assay (Figure 5E). The quantitative analysis showed that TGF-β-activated iMEF conditioned medium (PBS treatment) induced a 1.4-fold increase in the migration of CMFDA-labeled sEND.1 cells compared to the untreated iMEF-CM control group (Figure 5F). Compared to the PBS-treated control group, B7-33-SNPs-treated iMEF conditioned medium significantly inhibited sEND.1 cell migration. It is noteworthy that B7-33-SNPs reduced migration by 57.7±0.9%, and their efficacy was significantly better than that of free B7-33 (30.0±0.8%), cRGD-B7-33 (27.3±0.6%), or naked SNPs (27.8±10.6%) (Figure 5G).

[0052] To further investigate the effects of B7-33-SNPs on activated fibroblast-mediated angiogenesis, a shoot angiogenesis experiment was conducted. Compared with the PBS-treated control group, B7-33-SNPs treatment reduced shoot growth by 83.1 ± 12.9%. These findings indicate that B7-33-SNPs not only reverse iMEF activation but also inhibit iMEF-driven angiogenesis, thereby enhancing the anti-angiogenic therapeutic effect (Figure 5H). Figure 5 I).

[0053] Example 4 Based on Example 3, the inventors conducted further animal tumor-bearing experiments to more accurately evaluate the actual preventive and therapeutic effects of B7-33-SNPs in the prevention and treatment of cholangiocarcinoma. A brief summary of the specific experimental details is as follows.

[0054] (a) In vivo targeting For effective cancer treatment, nanoparticles require enhanced cyclic stability and targeted accumulation at lesion sites. Previous studies have demonstrated that B7-33-SNPs prolong the circulating half-life of the peptide cRGD-B7-33, improving its in vivo stability. To evaluate tumor-targeting efficacy, in vivo biodistribution analysis was performed on Cy5.5-labeled nanoparticles.

[0055] During the experiment, subcutaneous xenografts bearing HUCCT1 (1×10⁻⁶) were subjected to treatment. 6 BALB / c nude mice (number of cells / mouse) were intravenously injected with Cy5.5 dye and a Cy5.5-labeled B7-33-SNPs formulation. In vitro imaging was performed 24 hours post-injection. Results showed that the tumor accumulation with B7-33-SNPs-Cy5.5 was significantly higher than that with the control formulation (Figure 6A).

[0056] Further quantitative analysis showed that the tumor fluorescence intensity (0.83±0.07) in the B7-33-SNPs-treated group was 2.0 times, 2.1 times, and 2.1 times that of C7-33-SNPs (0.42±0.04), SNPs (0.38±0.06), and free Cy5.5 (0.39±0.08), respectively (Figure 6B).

[0057] Based on the above results, it can be preliminarily concluded that the tumor fluorescence intensity of B7-33-SNPs-Cy5.5 was significantly higher than that of the control group, which is attributed to the targeting effect mediated by relaxin R1 and the binding affinity to vascular endothelial CD31. This result indicates that B7-33-SNPs is a bifunctional nanosystem that meets the CCA therapeutic targeting criteria.

[0058] To further investigate the biodistribution of the nanosystems, tumors harvested 24 hours after injection of the Cy5.5-labeled formulation were sectioned and analyzed using confocal microscopy. The results showed that, consistent with in vitro data, the tumor accumulation of B7-33-SNPs-Cy5.5 was superior to other control groups. The distribution of B7-33-SNPs was consistent with imaging data, showing significant accumulation at the tumor site (Figure 6C). Notably, the Cy5.5-labeled nanoparticles (red) co-localized with CD31⁺ endothelial cells (white) and α-SMA⁺ CAF (green), indicating a dual targeting mechanism through long-circulation properties and relaxin R1 / CD31 interaction.

[0059] (II) Actual prevention and control effects The inventors further investigated and clarified the preventive and therapeutic effects of the prepared B7-33-SNPs by constructing a mouse model of cholangiocarcinoma. A brief summary of the specific experimental process and results is as follows.

[0060] (1) Dual-targeted nanotherapy synergistically inhibits the growth of cholangiocarcinoma HUCCT1 (1×10 6 BALB / c nude mice with subcutaneous xenograft tumors (cells / mouse) were divided into six groups after 7 days of tumor growth: PBS group, cRGD-B7-33 group, cRGD-B7-33+RNPs group, SNPs group, C7-33-SNPs group, and B7-33-SNPs group, according to the flowchart shown below. Figure 7 As shown in Figure A, various drugs were administered intravenously (cRGD-B7-33: 20 µg / kg; SNPs or RNPs: 12 mg / kg).

[0061] Weight monitoring showed comparable tolerability across groups with no significant differences between groups. Tumor growth curves indicated delayed tumor progression in the cRGD-B7-33 and cRGD-B7-33+RNPs groups compared to the PBS control group. Although the SNPs and C7-33-SNPs groups further inhibited tumor growth compared to the cRGD-B7-33-based treatment group, the difference was not statistically significant (p>0.05). Compared to SNPs monotherapy (p<0.01) and cRGD-B7-33 monotherapy (p<0.01), B7-33-SNPs treatment significantly inhibited tumor growth, demonstrating a synergistic effect through dual inhibition of CAF activation and angiogenesis. Figure 7 B. Figure 7 C Figure 7 D).

[0062] H&E staining of excised tumors showed that the tissue structure and degree of necrosis were comparable between the B7-33-SNPs group and the PBS group (Figure 7E). Western blot analysis confirmed that B7-33-SNPs treatment significantly reduced key ECM proteins such as type I collagen, α-SMA, and fibronectin (Figure 7F). Figure 7 G).

[0063] Mechanistic analysis suggests that B7-33-SNPs inhibit the expression of CXCL12 (SDF-1), a multifunctional mediator of activated CAF secretion in pro-fibroproliferative tumors, which in turn promotes angiogenesis by regulating vascular endothelium. Combined with quantitative analysis, compared with the PBS control group, CXCL12 was downregulated by 54.3±12.3% in tumors treated with B7-33-SNPs. These results collectively indicate that B7-33-SNPs, as a dual-targeting therapy, can simultaneously inhibit CAF activation and angiogenesis, thereby suppressing CCA progression.

[0064] (2) Study on the mechanism of in vivo inhibition of bile duct cancer To verify the dual inhibitory effect of B7-33-SNPs on CAF activation and tumor angiogenesis in vivo, immunohistochemical (IHC) analysis was performed on tumor tissues from the B7-33-SNPs-treated group and the control group. The results are as follows: Figure 8 As shown in the figure. Analysis reveals that: Compared with the control group, the B7-33-SNPs group showed a significant reduction in type I collagen, α-SMA, fibronectin, and the endothelial marker CD31. Figure 8 A, Figure 8 B). In contrast, cRGD-B7-33-based treatments (cRGD-B7-33 and cRGD-B7-33+RNPs) only resulted in a modest reduction in these proteins and had no significant anti-angiogenic activity. Monotherapy using SNPs or C7-33-SNPs (carrying VEGFR2 / Tie2 decoy receptors) inhibits angiogenesis by isolating pro-angiogenic factors such as VEGFA and ANG2.

[0065] In summary, B7-33-SNPs uniquely reversed CAF activation in the tumor stroma and inhibited angiogenesis, demonstrating superior efficacy compared to formulations based on cRGD-B7-33 or containing SNPs (such as C7-33-SNPs and cRGD-B7-33+SNPs).

[0066] (3) Distribution of anti-angiogenic SNPs in tumor tissue after cRGD-B7-33 treatment The inventors' previous research indicated that overexpression of the ECM can hinder drug delivery, and upregulation of profibrotic proteins in the CAF of anti-angiogenic cancer cells (CCAs) resistant to anti-angiogenic therapy may lead to treatment resistance. Therefore, attenuating ECM expression may enhance intratumoral perfusion of anti-angiogenic drugs.

[0067] To verify the above hypothesis, based on the aforementioned experimental procedures, after 2 weeks of pretreatment with cRGD-B7-33, Cy5.5-labeled SNPs were used to evaluate the perfusion of nanoparticles in tumors.

[0068] Relevant experimental results are as follows Figure 9 As shown. In vivo fluorescence imaging 1 hour after injection showed that, compared with the PBS control group, mice pretreated with cRGD-B7-33 showed enhanced tumor signal aggregation (…). Figure 9 A, Figure 9 B、、 Figure 9 C). Confocal microscopy confirmed that cRGD-B7-33 pretreated tumors exhibited a wider and higher Cy5.5 fluorescence spectrum. Further quantitative analysis confirmed that the nanoparticle penetration depth in cRGD-B7-33 pretreated tumors increased by 2.7-fold (9.08±2.23 μm vs. 3.39±1.32 μm; p<0.001). Figure 9 D、 Figure 9 E).

[0069] In summary, these results indicate that cRGD-B7-33-driven ECM modulation can enhance nanoparticle perfusion in CCA xenografts.

[0070] Overall, the above studies demonstrate that the bifunctional nanomedicine B7-33-SNPs used in this application overcomes the drug resistance bottleneck of existing anti-angiogenic therapies through a dual mechanism: 1. Dual-targeting effect of matrix and blood vessel: By inhibiting CAF activation and TGF-β signaling pathway through the relaxin analog peptide (cRGD-B7-33), the deposition of ECM components such as type I collagen and fibronectin is reduced, reversing tumor matrix fibrosis; at the same time, the decoy receptor (VEGFR2 / Tie2) derived from endothelial cell membrane neutralizes pro-angiogenic factors such as VEGFA and ANG2, blocking endothelial cell migration and lumen formation, thus doubly inhibiting the interaction between CAF and vascular endothelial cells; 2. High efficiency in tumor accumulation and safety: B7-33-SNPs achieve tumor-specific accumulation through a dual targeting mechanism of relaxin R1 / CD31, with a prolonged circulating half-life. The 24-hour tumor fluorescence intensity is 2.0-2.1 times that of the control group, and no significant systemic toxicity is caused (no difference in weight monitoring between groups, and normal H&E staining of major organs). Figure 11 It exhibits good biocompatibility; 3. Synergistic anti-tumor efficacy: In tumor-bearing mouse models, B7-33-SNPs significantly inhibited tumor growth, reducing tumor volume by more than 50% compared to the single-drug treatment group (SNPs or cRGD-B7-33). p <0.01), and downregulated the expression of pro-angiogenic mediators such as CXCL12 (inhibition rate 54.3%). Simultaneously, ECM remodeling significantly enhanced the penetration depth of nanomedicines (up to 2.7 times). p <0.001), overcoming the drug delivery barriers of traditional therapies.

[0071] In short, B7-33-SNPs can delay the development of anti-angiogenic resistance by targeting CAF activation. By simultaneously intervening in CAF-mediated fibrosis and angiogenesis, they can break the vicious cycle of treatment resistance. Thus, the strategy of jointly inhibiting CAF activation and angiogenesis can synergistically enhance the anti-tumor efficacy against cholangiocarcinoma (CCA), and is expected to become a novel adjuvant therapy for CCA, with significant clinical translational potential.

Claims

1. The application of nanomedicines that simultaneously inhibit fibroblast activation and angiogenesis in the preparation of agents for the prevention and treatment of cholangiocarcinoma, characterized in that, The nanomedicine that simultaneously inhibits fibroblast activation and angiogenesis is a biomimetic nanocarrier SNP loaded with modified B7-33. The modified B7-33 amino acid sequence is: VIKLSGRELVRAQIAISGMSTWSKRSL-GG-c(RGDfK).

2. The application of the nanomedicine as described in claim 1 in the preparation of cholangiocarcinoma prevention and treatment agents, characterized in that, The nanomedicine that simultaneously inhibits fibroblast activation and angiogenesis utilizes biomimetic nanocarriers (SNPs) prepared from biomembranes to load modified B7-33.

3. The application of the nanomedicine as described in claim 2 in the preparation of cholangiocarcinoma prevention and treatment agents, characterized in that, The biomembrane was prepared using mouse vascular endothelial cells sEND.

1.

4. The application of the nanomedicine as described in claim 3 in the preparation of cholangiocarcinoma prevention and treatment agents, characterized in that, The biomimetic nanocarriers SNPs prepared from biomembranes were obtained through the following steps: (1) Gradient centrifugation was used to extract cell membranes. Discard the culture medium from mouse vascular endothelial cells that have been pre-cultured and confluent in a culture dish, and collect the cells. Discard the supernatant, resuspend the cells in IB-1, and then homogenize them in a homogenizer. Centrifuge the ground cell solution and retain the supernatant; Add the supernatant to the centrifuge tube, add IB-1 to fill the tube, centrifuge, and retain the precipitate; (2) Using an extruder to extrude cell membrane biomimetic nanocarriers SNPs The precipitate obtained by centrifugation in step (1) was placed in a cell ultrasonic disruptor and ultrasonically treated. Then, it was extruded using an extruder to prepare nanoparticles with uniform particle size.

5. The application of biomimetic nanocarriers SNPs in the preparation of agents that inhibit the activation of fibroblasts in cholangiocarcinoma, characterized in that, The biomimetic nanocarriers SNPs are biomimetic nanocarriers made from biomembranes and loaded with modified B7-33. The modified B7-33 amino acid sequence is: VIKLSGRELVRAQIAISGMSTWSKRSL-GG-c(RGDfK).

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