A biomimetic nanomedicine for reversing hepatic stellate cell activation and its application

Abstract

The present application belongs to the technical field of preparation of bionic nanomedicines, and specifically relates to a patent application for a bionic nanomedicine for reversing the activation of hepatic stellate cells and its application. The bionic nanomedicine is prepared by the following steps: preparing bionic nanocarriers HNPs with vascular endothelial cell HUVEC membranes, modifying and transforming B7‑33, loading and constructing polypeptide bionic nanomedicines, etc. In the present application, the inventors selected and constructed bionic nanocarriers with targeting and modified the B7‑33 polypeptide, based on the principle of receptor ligand affinity technology, and achieved a better effect of drug delivery to the liver on the basis of achieving efficient reprinting of drug molecules. Preliminary experimental results show that after the polypeptide B7‑33 is delivered to the liver in a targeted manner, the fibrosis mediated by the activation of HSCs is reversed, and then the microenvironment regulation effect before pancreatic cancer liver metastasis is reversed, which can lay a technical foundation for inhibiting pancreatic cancer liver metastasis and thus improving the prevention and treatment effect of pancreatic cancer.

Description

Technical Field

[0001] The present application belongs to the technical field of bionic nanomedicine preparation, and specifically relates to a patent application for a bionic nanomedicine for reversing hepatic stellate cell activation and its application. Background Art

[0002] In cancer prevention and treatment, most existing treatment options target only the tumor cells themselves, while the metastasis of tumor cells (cancer cells) is a crucial factor influencing the effectiveness of tumor prevention and treatment. Research suggests that a key step in tumor metastasis is the successful colonization of circulating tumor cells in secondary or distant organs, a process influenced by the local microenvironment of the distant organ. To this end, tumor cells can alter the local microenvironment of distant organs (i.e., the pre-metastatic microenvironment) through the secretion of cytokines and extracellular vesicles, paving the way for tumor cell colonization and growth in these distant organs. Given this characteristic, in-depth research on the pre-metastatic microenvironment is a key breakthrough in improving the prevention and treatment of these tumors (cancers).

[0003] Research has shown that liver metastasis is one of the leading causes of death in pancreatic cancer patients. During this process, the activation of hepatic stellate cells (HSCs) plays a crucial role in the initial formation of the premetastatic niche. By secreting large amounts of extracellular matrix (ECM), including collagen I and fibronectin, HSCs mediate liver fibrosis and regulate the premetastatic niche to promote tumor cell adhesion and colonization. Therefore, liver fibrosis mediated by activated HSCs is crucial for the development of pancreatic cancer liver metastasis. Based on this characteristic, targeted inhibition of HSC activation may inhibit the occurrence of pancreatic cancer liver metastasis, thereby laying a foundation for the prevention and treatment of related tumors.

[0004] Relaxin, a peptide hormone, is currently used clinically to treat acute heart failure. Studies on its mechanism of action suggest that it possesses potent anti-fibrotic effects. The primary mechanism is that relaxin binds to relaxin receptor 1, thereby inhibiting Smad2 / 3 phosphorylation induced by pro-fibrotic factors (such as TGF-β), ultimately inhibiting collagen deposition and related matrix degradation. Because relaxin receptor 1 is expressed on the surface of hepatic stellate cells, some studies have shown that relaxin can inhibit the fibrotic process mediated by activated hepatic stellate cells. However, due to the abundance of disulfide bonds in its molecular structure, relaxin's synthetic preparation process is complex and expensive, which to some extent limits its practical clinical application.

[0005] It should be noted that peptide B7-33, a relaxin analog with only a B-chain structure, exhibits anti-fibrotic effects similar to relaxin. Given its ease of synthesis, ease of modification, and low immunogenicity, it is expected to replace relaxin in clinical applications. However, since the half-life of peptide B7-33 after intravenous administration is only a few minutes, increasing its in vivo half-life while maintaining its maximum bioavailability is one of the greatest technical challenges in improving the efficacy of peptide B7-33. Furthermore, since the target site delivery efficiency of systemic B7-33 is extremely low, improving the targeting of B7-33 during its application is also key to improving its efficacy.

[0006] In recent years, the rapid development of biomimetic nanocarrier technologies, exemplified by biomembranes, has provided a new approach to improving the delivery of relevant drug molecules due to their inherently long in vivo circulation and excellent biocompatibility. Furthermore, with the maturation of biopeptide modification to improve their half-life and bioavailability, combining relevant therapeutic peptide drug molecules with nano-bionic carrier technology to design an in vivo delivery system with strong targeting capabilities and high delivery efficiency has positive technical significance for improving the prevention and treatment of relevant diseases. Summary of the Invention

[0007] Based on the preparation of biomimetic nanocarriers from vascular endothelial cells (HUVECs) and the modification of B7-33, the purpose of this application is to provide a biomimetic nanomedicine that can reverse the activation of hepatic stellate cells, thereby laying a certain technical foundation for the prevention and treatment of pancreatic cancer liver metastasis, and further laying a foundation for improving and enhancing the prevention and treatment effects of pancreatic cancer. It can also provide certain reference and reference for the prevention and treatment of other tumors (cancers).

[0008] The technical solutions adopted in this application are described in detail as follows.

[0009] A biomimetic nanomedicine for reversing hepatic stellate cell activation is prepared by the following steps:

[0010] (1) Preparation of biomimetic nanocarriers

[0011] The biomimetic nanocarrier HNPs was prepared using HUVEC membrane.

[0012] The vascular endothelial cell membrane can be prepared by a classic cell membrane extraction method. The specific operation is as follows:

[0013] (1) Gradient centrifugation to obtain cell membrane

[0014] Pour off the culture medium of vascular endothelial cells that have been cultured and grown all over the culture dish, wash gently with PBS three times (retain 1 ml of PBS for the last wash), scrape the cells, and centrifuge at 1000 r for 5 min;

[0015] Discard the supernatant, add IB-1 (2 ml) to the cell pellet to resuspend, and transfer to a glass homogenizer and grind for about 40 times (operate on ice);

[0016] The ground cell solution was centrifuged at 3000 g for 5 min at 4 °C and the supernatant was retained;

[0017] The supernatant was centrifuged again at 4°C and 10,000 g for 10 min, and the supernatant was retained;

[0018] Add the supernatant to the ultracentrifuge tube, fill up with IB-1, centrifuge at 10,000 g for 2 h at 4°C, and save the precipitate (for subsequent steps or store at -80°C for later use).

[0019] IB-1 (isolation buffer, needs to be prepared immediately) is: SB + 0.5% BSA + 0.5mmol EGTA. To prepare, first dissolve 0.5g BSA in 100ml SB solution, then add 500ul 100mmol EGTA (pH 7.4) and 1% protease inhibitors.

[0020] The protein inhibitors were prepared in DMSO, with the following concentrations: 200 mM AEBSF, 30 μM Aprotinin, 13 mM Bestatin, 1.4 mM E64, and 1 mM Leupeptin;

[0021] The SB (prepared one day in advance) is: 225 mmol mannitol + 75 mmol sucrose + 30 mmol Tris-HCl (pH 7.4); during preparation, 20.5 g mannitol and 13 g sucrose were first dissolved in 400 ml ddH2O, then 15 ml Tris-HCl was added, and the mixture was incubated at 4°C for 30 min to adjust the pH to 7.4. Finally, the volume was adjusted to 500 ml with ddH2O.

[0022] (2) Using an extruder to extrude HNPs into cell membrane biomimetic nanocarriers

[0023] The vascular endothelial cells obtained by centrifugation in step (1) were placed in a cell ultrasonic disruptor and ultrasonicated at an ultrasonic power of 100 W for 3 min (ultrasonication for 5 s, pause for 5 s). Subsequently, the cells were extruded using an Avanti Mini Extruder (Avanti Polar Lipids):

[0024] First, the ultrasonically treated vascular endothelial cell membrane was extruded up and down 10 times using a 0.8 μm polycarbonate (PC) membrane;

[0025] Subsequently, 0.4 μm and 0.1 μm PC films were used to perform up and down extrusion treatments 10 times respectively;

[0026] Finally, uniform nanoparticles (bionic nanocarriers HNPs) with an average particle size of about 100 nm were prepared for use;

[0027] (2) Modification of B7-33

[0028] The RGD sequence was modified by coupling with B7-33 (RGD-B7-33);

[0029] The polypeptide B7-33 has an amino acid sequence as shown in SEQ ID No. 1, specifically: VIKLSGRELVRAQIAISGMSTWSKRSL;

[0030] The amino acid sequence of the modified B7-33 (RGD-B7-33) is: VIKLSGRELVRAQIAISGMSTWSKRSL-GG-c(RGDfK);

[0031] The RGDfK is: Arg-Gly-Asp-Phe-Lys;

[0032] (III) Loading and constructing peptide biomimetic nanomedicines

[0033] Based on the affinity between the receptor (Integrin αv (β3) on the HUVEC cell membrane surface) and the ligand (RGD), the therapeutic peptide B7-33 (RGD-B7-33) was efficiently loaded onto the biomimetic nanocarrier to construct the peptide biomimetic nanoparticles (B7-33-HNPs). Specifically, the following steps were performed:

[0034] The B7-33 (RGD-B7-33) prepared in step (2) and the biomimetic nanocarrier HNPs prepared in step (1) were incubated at 4°C for 12 h to prepare the biomimetic nanomedicine B7-33-HNPs;

[0035] During incubation, the reference ratio of RGD-B7-33 to HNPs was 1.5% (i.e., 1.5 g RGD, 98.5 g HNPs).

[0036] The application of the bionic nanomedicine for reversing hepatic stellate cell activation in the preparation of a pancreatic cancer prevention and treatment agent is used to prevent or reverse the metastasis of pancreatic cancer (tumor) cells to liver tissue in an anti-fibrotic manner.

[0037] In the prior art, although it is known that fibrosis mediated by activated hepatic stellate cells (HSCs) plays an important role in regulating the microenvironment before pancreatic cancer liver metastasis, it is still unclear how to deliver relevant therapeutic drugs (such as B7-33) to the liver and whether they can reverse the fibrotic process mediated by activated HSCs. To this end, in this application, the inventors selected and constructed a targeted biomimetic nanocarrier and modified the B7-33 polypeptide. Based on the principle of receptor ligand affinity technology, they achieved a better effect of targeted drug delivery to the liver while achieving efficient drug transfer.

[0038] Preliminary experimental results show that after the peptide B7-33 is delivered to the liver in a targeted manner, it reverses the fibrosis mediated by HSCs activation, and thus reverses the regulatory effect of the microenvironment before pancreatic cancer liver metastasis, which can lay a good technical foundation for inhibiting pancreatic cancer liver metastasis and thus improving the prevention and treatment of pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The characterization results of the prepared B7-33-HNPs nanomedicine are shown in Figure 2.

[0040] A is Western blot analysis of the protein integrin αv(β3) in purified HUVEC membranes, HNPs, and RNPs; erythrocyte membranes were used as negative controls;

[0041] B is the size distribution results of RNPs, B7-33-RNPs, HNPs, and B7-33-HNPs (n = 3);

[0042] C shows the zeta potential results of RNPs, B7-33-RNPs, HNPs, and B7-33-HNPs (n = 3);

[0043] D is the TEM image of erythrocyte membrane RNPs, B7-33-RNPs, HNPs, and B7-33-HNPs; scale bar: 100 nm;

[0044] E is a schematic diagram of the complex of magnetic beads binding FITC-labeled B7-33-RGD and RNPs or HNPs;

[0045] F is the flow cytometry results of magnetic bead-bound FITC-labeled B7-33-RGD and RNPs or HNPs complexes (n = 6), used to detect and evaluate the binding of B7-33-RGD to HNPs or RNPs; in the figure, RNPs and HNPs refer to the bead-bound filtered RNPs or HNPs retentate, used as negative control; B7-33-RNPs and B7-33-HNPs refer to the bead-bound filtered FITC-labeled B7-33-RGD and RNPs or HNPs complex retentate;

[0046] Figure 2 The molecular weights of RGD-B7-33 and RGD-B7-33-FITC were determined using electrospray ionization mass spectrometry (ESI-MS).

[0047] A is the detection result of RGD-B7-33. It can be seen that the peak size is consistent with the predicted molecular weight of RGD-B7-33 (theoretical value: 3,687.30; measured value: 3,686.50);

[0048] B is the detection result of RGD-B7-33-FITC, which shows that the peak size is consistent with the predicted molecular weight of RGD-B7-33-FITC (theoretical value: 4,204.48; measured value: 4,203.60);

[0049] Figure 3 Figure 3 shows the results of an in vitro cell experiment with B7-33-HNPs, used to evaluate the binding of B7-33-HNPs to the cell membrane of LX-2 cells (HSCs). In the experiment, RGD-B7-33, RNPs, HNPs, B7-33-RNPs, or B7-33-HNPs were incubated with cells for 4 hours. HNPs were labeled with Cy5.5, and RGD-B7-33 was labeled with FITC. Scale bar: 10 μm.

[0050] Figure 4 The effect of B7-33-HNPs on TGF-β-mediated HSCs activation;

[0051] A is the immunofluorescence (IF) staining results of α-SMA, collagen I, and fibronectin in LX-2 after treatment with the indicated preparations for 48 h, scale bar: 10 μm;

[0052] B is the Western blot result showing the inhibition of protein expression of α-SMA, collagen I, and fibronectin in LX-2 cells after 48 h of TGF-β activation; protein levels were normalized to GAPDH;

[0053] C is the result of phosphorylated protein detection, which is used to show the inhibitory effect on Smad2 or Smad3 phosphorylation in LX-2 after 1 hour of TGF-β activation; protein levels are normalized to SMAD2 and SMAD3;

[0054] D is the quantitative analysis result of the normalized α-SMA, type I collagen, and fibronectin expression using Image J software;

[0055] E is the quantitative analysis result of normalized pSMAD2 and pSMAD3 protein expression using Image J software;

[0056] Figure 5 The in vivo pharmacokinetics and biodistribution of nanomedicine B7-33-HNPs are as follows:

[0057] A is the fluorescence images of mouse blood at the indicated time points after intravenous injection of free B7-33-RGD and B7-33-HNPs (FITC-labeled B7-33-RGD);

[0058] B is the quantification of fluorescence intensity at several time points after administration; it can be seen that the clearance time of RGD-B7-33 peptide was significantly prolonged by decoration on HNPs;

[0059] C is the in vitro fluorescence image of the main organs of pancreatic cancer liver metastasis model mice 24 hours after injection of B7-33-HNPs-cy5.5, C7-33-HNPs-cy5.5 and HNPs-cy5.5;

[0060] D is the semi-quantitative analysis of organ fluorescence intensity;

[0061] E shows the distribution of B7-33-HNPs-cy5.5, C7-33-HNPs-cy5.5, and HNPs-cy5.5 in the liver metastatic niche 24 hours after injection. In color view, hepatic stellate cells were stained with α-SMA (green) and cell nuclei were stained with DAPI (blue); scale bar, 50 μm;

[0062] Figure 6 H&E-stained pathological sections of the main organs in each treatment group were used for evaluation. Scale bar: 100 μm.

[0063] Figure 7 Biochemical indicators of liver and kidney function; among them:

[0064] A is the biochemical test result of ALT (alanine aminotransferase) in serum liver function;

[0065] B is the biochemical test result of AST (aspartate aminotransferase) in serum liver function;

[0066] C is the biochemical test result of TBIL (total bilirubin) in serum renal function;

[0067] D is the biochemical test result of CR (urine creatinine) index in serum renal function;

[0068] E is the biochemical test result of BUN (blood urea nitrogen) index in serum renal function;

[0069] Figure 8 The immune response of C57BL / 6 mice to B7-33-HNPs after 24 hours of treatment;

[0070] A is the concentration of IL-2 in serum;

[0071] B is the concentration of IFN-γ in serum;

[0072] C is the concentration of IL-6 in serum;

[0073] Figure 9 The formation of a fibrotic premetastatic niche in a mouse model of liver metastasis;

[0074] A shows the H&E and immunofluorescence staining results of α-SMA and fibronectin expression in mice with liver metastatic and non-metastatic tumors at different days (d0-d7) after inoculation of Pan02 cells in the pre-metastatic stage. In the color view, white circles are used to outline the metastatic niche. Scale bar: 100 μm.

[0075] B, quantitative analysis of the normalized level of α-SMA protein;

[0076] C is the quantitative analysis of the normalized level of fibronectin;

[0077] Figure 10 The B7-33 formulation prevents liver metastasis in mice; wherein:

[0078] A is a schematic diagram of the PC (pancreatic cancer) liver metastasis model and the time points of drug administration;

[0079] B is the in vivo luminescence images of mice inoculated with PC cells at the beginning (day 3) and end point (day 17) of treatment with PBS, HNPs, C7-33-HNPs, RGD-B7-33, RGD-B7-33+RNPs, and B7-33-HNPs;

[0080] C shows H&E-stained liver sections in each group on day 17 after treatment. In the color view, the dark purple area outlined by white dashed lines represents metastatic nodules. Scale bar, 500 μm.

[0081] D is the growth curve of total luminescence of each treatment group (n = 6);

[0082] E is the area of ​​metastatic nodules in the liver of each treatment group;

[0083] F is the liver weight of mice in each treatment group after treatment;

[0084] Figure 11 The results show the reversal of HSC activation and reduction of fibrosis in a pancreatic cancer liver metastasis model;

[0085] A, Histological study of liver metastatic niches in vivo using collagen trichrome staining and immunohistochemical staining of α-SMA, fibronectin, and collagen I; scale bar, 50 μm;

[0086] B is the quantitative analysis results of the normalized α-SMA, collagen, type I collagen, and fibronectin levels using Image J software;

[0087] In the above figures, when data processing and statistics are involved, the data are mean ± standard deviation (SD) results. At the same time, *P<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0088] The present application will be further explained below with reference to the following examples. Before introducing the specific examples, a brief description of the experimental background of some of the following examples is given below.

[0089] Biomaterials:

[0090] Blood was collected and provided by the Physical Examination Department of the First Affiliated Hospital of Zhengzhou University (all volunteers provided signed informed consent and were approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University, approval number: #KY-398).

[0091] Hepatic stellate cells (LX-2), a commonly used experimental cell type in biomedical research, were obtained from Xiangya Medical College (Changsha, China);

[0092] C57BL / 6 mice (6-8 weeks, male, approximately 20 g), a commonly used experimental mouse strain in biomedical research, are publicly available.

[0093] Pancreatic cancer Pan02 cells, a commonly used experimental cell line in biomedical research, were purchased from the American Type Culture Collection (Manassas, VA, USA) for use in the experiments related to this application.

[0094] Some experimental reagents and equipment:

[0095] EDTA, Hepes-NaOH, and protease inhibitor cocktail were products of Sigma-Aldrich (St. Louis, MO, USA);

[0096] Matrigel matrix, a product of Corning (Chicago, IL, USA);

[0097] Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were products of HyClone (Logan City, UT, USA);

[0098] Cell Counting Kit-8 (CCK-8), a product of Dojindo Molecular Technologies (Tokyo, Japan);

[0099] SMAD2 (D43B4) XP Rabbit mAb (Catalog #5339S), SMAD3 (C67H9) Rabbit mAb (Catalog #9513), Phospho-SMAD2 (Ser465 / Ser467) (E8F3R) Rabbit mAb (Catalog #18338), and Phospho-SMAD3 (Ser423 / 425) (C25A9) Rabbit mAb (Catalog #9520S) are products of CST (Boston, MA, USA);

[0100] Rabbit polyclonal antibodies against GAPDH (catalog #10494-1-AP), fibronectin (catalog #15613-1-AP), and type I collagen (catalog #14695-1-AP) were products of Proteintech (Chicago, IL, USA);

[0101] Recombinant mouse TGF-β protein (catalog #7666-MB-005) and mouse relaxin R1 antibody (catalog #MAB8898) were products of R&D Systems (Minneapolis, MN, USA);

[0102] Antibodies against Na+-K+ ATPase (catalog #ab76020), α-SMA (catalog #ab5694), integrin alphaV (catalog #ab179475), and integrin 3 (catalog #ab119992) are all products of Abcam (Cambridge, UK).

[0103] Secondary antibody: Alexa Fluor® 488-conjugated goat anti-rabbit IgG (H + L) (Cat. #A-11034), a product of Thermo Fisher Scientific (Waltham, MA, USA);

[0104] 4 μm diameter aldehyde / sulfate-containing latex beads (lot #2198595), product of Invitrogen (Waltham, MA, USA);

[0105] 100 kDa diafiltration tubes (UFC510096) were manufactured by Millipore (Burlington, MA, USA).

[0106] Example 1

[0107] In previous research, the inventors believed that platelet-derived biomimetic nanomedicines could deliver drugs to the tumor microenvironment through the EPR effect (enhanced permeability and retention effect of solid tumors). Furthermore, through receptor-ligand-mediated interactions, the drugs could be targeted to tumor cells, thereby enhancing the drug's anti-tumor efficacy. Similarly, by exploiting the differences between the pre-metastatic microenvironment of tumors and normal tissues, it is possible to design and synthesize nanomedicines that can achieve targeted delivery of anti-tumor drugs, thereby laying the foundation for preventing (or even reversing) tumor metastasis.

[0108] Based on the above technical concepts, combined with the inventors' long-term experience in peptide drug modification, and innovatively utilizing receptor and ligand principles, the inventors conducted experimental drug loading experiments and prepared a new biomimetic nanopharmaceutical, B7-33-HNPs. The preparation process of this nanopharmaceutical is briefly described below.

[0109] (1) Preparation of biomimetic nanocarriers

[0110] The biomimetic nanocarrier HNPs was prepared using the cell membrane of vascular endothelial cells (HUVEC). The specific operation is as follows.

[0111] First, the classic cell membrane extraction method is used to extract the vascular endothelial cell membrane. Specifically, the following steps are performed:

[0112] (1) Gradient centrifugation to obtain cell membrane

[0113] Pour off the culture medium of vascular endothelial cells that have been cultured and grown all over the culture dish, wash gently with PBS three times (retain 1 ml of PBS for the last wash), scrape the cells, and centrifuge at 1000 r for 5 min;

[0114] Discard the supernatant, add IB-1 (2 ml) to the cell pellet to resuspend, and transfer to a glass homogenizer and grind for about 40 times (operate on ice);

[0115] The ground cell solution was centrifuged at 3000 g for 5 min at 4 °C and the supernatant was retained;

[0116] The supernatant was centrifuged again at 4°C and 10,000 g for 10 min, and the supernatant was retained;

[0117] Add the supernatant to the ultracentrifuge tube, fill up with IB-1, centrifuge at 10,000 g for 2 h at 4°C, and save the precipitate (for subsequent steps or store at -80°C for later use).

[0118] IB-1 (isolation buffer, needs to be prepared immediately) is: SB + 0.5% BSA + 0.5mmol EGTA. To prepare, first dissolve 0.5g BSA in 100ml SB solution, then add 500ul 100mmol EGTA (pH 7.4) and 1% protease inhibitors.

[0119] The SB (prepared one day in advance) is: 225 mmol mannitol + 75 mmol sucrose + 30 mmol Tris-HCl (pH 7.4); during preparation, 20.5 g mannitol and 13 g sucrose were first dissolved in 400 ml ddH2O, then 15 ml Tris-HCl was added, and the mixture was incubated at 4°C for 30 min to adjust the pH to 7.4. Finally, the volume was adjusted to 500 ml with ddH2O.

[0120] (2) Using an extruder to extrude HNPs into cell membrane biomimetic nanocarriers

[0121] The vascular endothelial cell membrane obtained by centrifugation in step (1) was placed in a cell ultrasonic disruptor and ultrasonicated for 3 min at an ultrasonic power of 100 W (ultrasonication for 5 s, pause for 5 s). Subsequently, the membrane was extruded using an Avanti Mini Extruder (Avanti Polar Lipids):

[0122] First, the ultrasonically treated vascular endothelial cell membrane was extruded up and down 10 times using a 0.8 μm polycarbonate (PC) membrane;

[0123] Subsequently, 0.4 μm and 0.1 μm PC films were used to perform up and down extrusion treatments 10 times respectively;

[0124] Finally, uniform nanoparticles (bionic nanocarriers HNPs) with an average particle size of about 100 nm are prepared for use.

[0125] As a control, the inventors also prepared biomimetic nanocarrier RNPs using erythrocyte membranes derived from erythrocytes. The specific operation is as follows.

[0126] First, the red blood cell membrane is obtained using the hypotonic method. Specifically, the following steps are performed:

[0127] Freshly collected blood samples (whole blood) were centrifuged at 1000 × g for 5 min at 4°C to remove serum and other blood cells, and the pellet (erythrocytes) was retained and washed with PBS (three times);

[0128] The above precipitate was resuspended in ice-cold hypotonic solution (0.25× PBS) for 1 hour, centrifuged at 20,000×g for 30 minutes at 4°C, and the precipitate (white pellet) was retained;

[0129] Resuspend in PBS and incubate at 37°C for 1 hour.

[0130] Subsequently, referring to the above operation, an Avanti Mini Extruder (Avanti Polar Lipids) was used to extrude through 0.4 μm and 0.22 μm PC membranes in sequence to obtain biomimetic nanocarrier RNPs.

[0131] The inventors used Western blotting to examine the expression of integrin αv (β3) in the prepared biomimetic nanocarriers (using red blood cell membrane-derived nanoparticles (RNPs) as a negative control). Integrin αv and β3 are typically expressed during angiogenesis in various cancers and are therefore used as tumor or tumor cell metastasis signaling molecules. They also serve as receptor molecules for subsequent drug loading in this application. Transmission electron microscopy and a Malvern particle size analyzer were also used to characterize the nanocarriers' structural morphology, size, surface potential, and other performance parameters.

[0132] Transmission electron microscopy (TEM) characterization results of HNPs and RNPs are shown in Figure 2. Figure 1 ( Figure 1D), it can be seen that they are all spherical structures. The size distribution of RNPs and HNPs shows the characteristics of good dispersion and have almost the same hydrodynamic size, with an average particle size of about 120 nm ( Figure 1 B). At the same time, the further zeta potential situation is also basically the same ( Figure 1 C). However, further Western Blot results showed that integrin αv (β3) was only expressed in endothelial cells ( Figure 1 A).

[0133] (2) Modification of B7-33

[0134] The polypeptide B7-33 has an amino acid sequence as shown in SEQ ID No. 1, specifically: VIKLSGRELVRAQIAISGMSTWSKRSL;

[0135] The amino acid sequence of the modified RGD-B7-33 is: VIKLSGRELVRAQIAISGMSTWSKRSL-GG-c (RGDfK);

[0136] The sequence of RGD-B7-33-FITC after FITC labeling is:

[0137] VIKLSGRELVRAQIAISGMSTWSKRSL-K(FITC)-GG- c(RGDfK);

[0138] As a control, the sequence of RGD-C7-33 after FITC labeling is:

[0139] RLRSRVASGKSVILGLKSEQMSTAIWI-K(FITC)-GG-c(RGDfK).

[0140] The above-mentioned related sequences were commissioned to Anhui Guoping Pharmaceutical Co., Ltd. (Anhui, China) for synthesis using solid-phase peptide synthesis (SPPS).

[0141] Taking the preparation of RGD-B7-33 as an example, the specific synthetic operation process can be referred to as follows:

[0142] (1) First, weigh 0.5 g of 2-CTC resin and add it to the peptide synthesis reaction tube. Add 5 ml of DCM to the reaction tube and let it swell for 10 minutes. After swelling, add 5 ml of DMF and wash three times, each time for 30 seconds.

[0143] (2) Add 0.15 mmol Fmoc-ASP(OALL)-resin and 0.5 mmol DIEA, and condense by bubbling nitrogen for 2 h;

[0144] After the condensation was completed, the reaction solution was removed and washed with 5 ml of DMF four times, each time for 30 s. After the fifth wash, the solution was dried.

[0145] (3) Add methanol: DIEA: DCM (1:1:2) to the above-mentioned drained solid phase reaction tube, bubble nitrogen for 30 minutes and then seal the tube. After the reaction is completed, remove the reaction solution and add 5 ml DMF to wash 5 times, each time for 30 seconds;

[0146] (4) Add 5 ml of 20% piperidine / DMF solution to the reaction tube and allow nitrogen to bubble for 20 min to remove Fmoc.

[0147] 0.5 mmol Fmoc-GLY-OH and 0.5 mmol HOBT were added to a solid phase reaction tube, followed by 0.5 mmol DIC, and nitrogen was bubbled for 1 h for condensation.

[0148] (5) Repeat the above washing and condensation steps, add Fmoc-ARG(PBF)-OH, Fmoc-LYS(DDE)-OH, and Fmoc-D-PHE-OH for synthesis to obtain the uncyclized peptide chain RGDfK-resin;

[0149] Subsequently, 0.03 mmol of tetrakistriphenylphosphine palladium catalyst was dissolved in DCM and placed in the reactor to remove the oall protecting group for 3 h;

[0150] (6) Using DIC and HOBT as condensation reagents, cyclization was performed to obtain c(RGDfK(dde))-resin;

[0151] Subsequently, dde was removed with 2% hydrazine hydrate DMF solution to obtain c(RGDfK)-resin;

[0152] Finally, washing and condensation were repeated, and the remaining amino acids were coupled in sequence to prepare RGD-B7-33.

[0153] Electrospray ionization mass spectrometry (ESI-MS) was used to identify the prepared compounds. Figure 2The results showed that the molecular weight of the prepared RGD-B7-33 was 3687.50 (consistent with the theoretical value: 3,687.30); the molecular weight of the prepared RGD-B7-33-FITC was 4203.60 (consistent with the theoretical value: 4204.48). This result indicates that the relevant compounds were successfully synthesized.

[0154] (III) Loading and constructing peptide biomimetic nanomedicines

[0155] Based on the affinity between the receptor (Integrin αv (β3) on the surface of HUVEC cell membrane) and the ligand (RGD), after mixing the nanocarrier HNPs prepared in step (1) with the RGD-B7-33 prepared in step (2), the therapeutic peptide B7-33 (RGD-B7-33) can be efficiently loaded on the biomimetic nanocarrier, thereby finally constructing the peptide biomimetic nanomedicine (B7-33-HNPs).

[0156] The biomimetic nanodrug B7-33-HNPs (or negative control B7-33-RNPs) and the control biomimetic nanodrug C7-33-HNPs were prepared by incubating RGD-B7-33 (or RGD-C7-33) with endothelial cell membrane biomimetic nanocarriers HNPs (or red blood cell membrane biomimetic nanocarriers RNPs) at 4°C for 12 h.

[0157] Taking the loading of RGD-B7-33-FITC on HNPs as an example, the loading efficiency of RGD-B7-33-FITC on nanoparticles HNPs at different ratios was measured using a fluorescence spectrophotometer. The results are shown in Table 1 below.

[0158] Table 1. Loading efficiency of RGD-B7-33-FITC in nanoparticles

[0159] .

[0160] The data in the table above demonstrates that when the RGD-B7-33 to HNP ratio is 1.5% (i.e., 1.5g RGD-B7-33 to 98.5g HNPs), RGD exhibits high drug loading efficiency and entrapment efficiency, reaching 1.26% and 73.55%, respectively. Further calculations indicate that the average number of B7-33 molecules loaded per nanovesicle is approximately 13.

[0161] The morphological characteristics of each material after loading the drug component were further characterized, and the specific results are briefly described as follows.

[0162] (1) Morphological characteristics and potential

[0163] As mentioned above, the morphology of RNPs and HNPs before loading with drugs was spherical, but after loading with drugs, the diameter and properties of the nanoparticles did not change significantly ( Figure 1 B. Figure 1 D), indicating that drug loading did not affect the hydrodynamic properties of the nanoparticles. However, zeta potential results showed that after drug loading, the zeta potential of B7-33-HNPs increased from -21.9 ± 3.1 mV (unloaded HNPs) to -6.4 ± 2.4 mV. This is presumably due to the fact that the drug molecule RGD-B7-33 is a positively charged peptide with additional highly polar residues. In contrast, the zeta potential of the RNPs in the control group changed only slightly before and after modification with RGD-B7-33 loading. This directly reflects the significant differences in the physicochemical properties of RNPs and HNPs.

[0164] (2) Drug loading "firmness"

[0165] Referring to the above operation, HNPs were incubated with RGD-B7-33-FITC, and unbound RGD-B7-33 was removed by continuous diafiltration; then, the retentate (i.e., B7-33-HNPs after loading with FITC-labeled drug) was bound to aldehyde / sulfate-containing latex beads and detected by flow cytometry (the same operation was performed, but RNPs or HNPs were used as negative controls).

[0166] The results are as follows Figure 1 ( Figure 1 E. Figure 1 Figure 5 (F). It can be seen that the RGD-B7-33-loaded HNP complex exhibits a higher fluorescence signal. Analysis suggests that this is due to the efficient loading of RGD-B7-33 onto HNPs through receptor-ligand interaction, thus ensuring better detection results. In contrast, RNPs only interact nonspecifically with RGD-B7-33 through electrostatic adsorption, resulting in a slightly brighter fluorescence signal for the RGD-B7-33-loaded RNPs after filtration. In other words, the receptor-ligand interaction effectively ensures the loading of the drug molecule RGD-B7-33 onto HNPs.

[0167] Example 2

[0168] Based on Example 1, the inventors conducted further in vitro cell experiments on the prepared nanomedicine B7-33-HNPs. The specific experimental process and results are briefly described as follows.

[0169] (I) In vitro localization of nanomedicines on hepatic stellate cells

[0170] To investigate whether B7-33-HNPs bind to HSCs (hepatic stellate cells), we incubated B7-33-HNPs with LX-2 cells in vitro and then performed the assay. The specific procedures are as follows.

[0171] Hepatic stellate cells LX-2 in the logarithmic growth phase were seeded into confocal culture dishes and cultured overnight. The pro-fibrotic factor TGF-β 5 ng / ml was added to stimulate the LX-2 cells for 48 hours to activate them.

[0172] Then, equal volumes of RGD-B7-33, HNPs, RNPs, B7-33-RNPs, and B7-33-HNPs (FITC-labeled RGD-B7-33, Cy5.5-labeled HNPs) were added to the culture dish so that the concentration of Cy5.5 was 5 ng / ml.

[0173] After different exposure times, laser confocal microscopy was used to take pictures (to visually and qualitatively observe B7-33-HNPs bound to the cell surface).

[0174] The results are as follows Figure 3 As shown in Figure 2, it can be seen that FITC-labeled RGD-B7-33 effectively bound to the LX-2 cell membrane surface after 4 hours of incubation.

[0175] Due to cellular endocytosis, the Cy5.5 fluorescence intensity inside the cells of the HNPs or RNPs treatment group was much higher than that of the B7-33-HNPs treatment group.

[0176] On the other hand, the overlap between RGD-B7-33-FITC and HNPs-Cy5.5 on the cell membrane was higher in the B7-33-HNPs-treated group than in the B7-33-RNPs-treated group. This difference is believed to be primarily due to the fact that, although RGD-B7-33 loaded by RNPs was also adsorbed to the cell membrane in the B7-33-RNPs-treated group, as previously described, the RNPs were unable to effectively load RGD-B7-33, resulting in a difference in efficacy during endocytosis.

[0177] Overall, the intuitive observation results of laser confocal microscopy showed that B7-33-HNPs can be stably adsorbed on the cell membrane through B7-33 binding to RXFP1 (Relaxin family peptide receptor 1) on the surface of hepatic stellate cells, which is a prerequisite for the nanomedicine to reverse the activation function of HSCs.

[0178] (II) Inhibition of hepatic stellate cell activation-mediated fibrosis

[0179] To clarify the effect of B7-33-HNPs on HSC activation, LX-2 cells were stimulated with TGF-β and the effects of the nanoparticles were observed. The specific procedures are as follows.

[0180] Hepatic stellate cells LX-2 in the logarithmic growth phase were seeded into 6-well plates. Equal volumes of PBS, HNPs, RNPs, B7-33, RGD-B7-33, B7-33-RNPs, C7-33-HNPs, or B7-33-HNPs were added to the plates (the concentration of B7-33 was 20 nmol / L). One hour later, 5 ng / ml TGF-β was added to stimulate the cells. After 48 hours, the expression of proteins such as a-SMA, Fibronectin, and Collagen I in hepatic stellate cells was observed by cell immunofluorescence and Western Blot.

[0181] In addition, the same method was used to study the effect of B7-33-HNPs on the fibrosis-related signaling pathways of hepatic stellate cells. Specifically, after TGF-β was added to stimulate the cells for 1 h, Western Blot was used to detect the expression of Smad2 / 3 phosphorylation and NOS proteins in hepatic stellate cells.

[0182] Specific test results such as Figure 4 As shown. From the immunofluorescence (IF) staining images ( Figure 4 A) It can be seen that compared with the untreated control, the B7-33-HNPs-treated group significantly reduced the levels of α-SMA (one of the markers of activated HSCs) and other proteins.

[0183] The detection of microenvironment matrix-related components is mainly used to evaluate the regulatory ability of B7-33-HNPs on ECM (extracellular matrix), and then to evaluate whether nanomedicines can be used to metastasize tumor (cancer) cells. Specific results:

[0184] Compared with the control group, the expression (content) of collagen and fibronectin in the B7-33-HNPs treatment group was significantly reduced (p<0.05); and the related reduction results were consistent with those of B7-33 ( Figure 4 B. Figure 4 D). This result indicates that B7-33 loaded onto HNPs does not affect the function of the drug molecule itself. Based on this result, it can be concluded that B7-33-HNPs regulate matrix composition by inhibiting the expression of α-SMA, fibronectin, and collagen I, thereby suppressing fibrosis.

[0185] To further evaluate whether B7-33-HNPs exerts its regulatory effects by inhibiting TGF-β downstream signaling (such as phosphorylation of Smad2 and Smad3), the inventors further detected the expression of related pSmad2 (Smad2) and pSmad3 (Smad3). The results showed that B7-33-HNPs or B7-33 significantly inhibited and reduced the TGF-β-induced pSmad2 / Smad2 and pSmad3 / Smad3 ratios ( Figure 4 C. Figure 4 E). This result suggests that B7-33-HNPs (or B7-33) inhibits hepatic stellate cell fibrosis by reducing the phosphorylation pathways of Smad2 and Smad3 (i.e., B7-33-HNPs inhibit TGF-β-induced HSC activation by inhibiting the pSmad2 and pSmad3 pathways).

[0186] Example 3

[0187] Based on Example 2, the inventors conducted further animal experiments to further accurately evaluate the actual preventive and therapeutic effects of B7-33-HNPs. The specific experimental results are briefly described below.

[0188] (I) Pharmacokinetic analysis

[0189] C57BL / 6 mice were intravenously injected with RGD-B7-33-FITC or B7-33-HNPs.

[0190] (FITC-conjugated RGD-B7-33) (Dose: 5 mg / kg) The fluorescence intensity of FITC in blood collected from the tail vein at different times was analyzed using an IVIS spectral imaging system.

[0191] The results showed that ( Figure 5 A. Figure 5 B): The fluorescence intensity of the RGD-B7-33-treated group rapidly decreased to 25% within 10 minutes, indicating that the circulating half-life of RGD-B7-33-FITC is only approximately 5 minutes. In contrast, the fluorescence intensity of B7-33-HNPs remained around 50% after 1 hour (i.e., a half-life of approximately 1 hour).

[0192] This result demonstrates that the newly designed B7-33-HNPs indeed enhance the in vivo stability of the nanomedicine and prolong the half-life of the peptide drug B7-33 in circulation. In other words, this result indicates that the B7-33-HNPs provided in this application have good blood circulation stability and low clearance rate.

[0193] (2) Safety evaluation

[0194] In order to study whether the B7-33-HNPs designed in this project meet the requirements of biosafety, the inventors used normal mice to evaluate their immunogenicity and safety.

[0195] First, PBS, HNPs, B7-33, B7-33-RNPs, and B7-33-HNPs were injected intravenously. 24 hours after the single intravenous injection, blood was collected for the detection of inflammatory cytokines (IFNγ, IL-6, and IL-2) to study the effect of B7-33-HNPs on the immune response of mice.

[0196] At the same time, according to the above grouping, the mice were given the drug every other day. After 5 doses, the body weight, blood routine and blood biochemical levels (liver function: aspartate aminotransferase, AST; alanine aminotransferase, ALT; renal function: total bilirubin, TBIL; urine creatinine, CR; blood urea nitrogen, BUN) of the mice were monitored. At the same time, H&E staining was used to observe the toxicity of B7-33-HNPs to major organs such as the heart, liver, spleen, lungs and kidneys.

[0197] Some experimental results are as follows Figure 6 、 Figure 7 、 Figure 8 The analysis showed that there were no significant differences in the physiological indicators among the treatment groups, indicating that the provided B7-33-HNPs had good safety.

[0198] (III) In vivo targeting

[0199] Specifically, tumor-bearing mice were intravenously injected with HNPs-cy5.5, C7-33-HNPs (cy5.5 labeled), or B7-33-HNPs (cy5.5 labeled), and ex vivo imaging of major organs and tumors was performed 24 hours after injection.

[0200] IVIS spectral imaging system (Figure 5C, Figure 5 D) Results showed that the HNPs-delivered group was primarily concentrated in the liver, demonstrating excellent liver targeting. Furthermore, the total fluorescence intensity in the liver of the B7-33-HNPs-treated group was 1.4-fold and 1.6-fold higher than that of the C7-33-HNPs-treated and HNPs-treated groups (B7-33-HNPs: 1511.35 ± 141.7, C7-33-HNPs: 1079.00 ± 81.79, HNPs: 942.40 ± 193.38).

[0201] This result suggests that the combination of B7-33 and HNPs exhibits a better liver-delayed effect (i.e., better liver targeting) when confronted with actual tumor (cancer) cells.

[0202] Immunohistofluorescence analysis of liver tissue was further performed to investigate the co-localization of B7-33-HNPs with hepatic stellate cells in the pre-metastatic microenvironment ( Figure 5 E, FITC was used to stain α-SMA in tissue sections): The results showed dense accumulation of B7-33-HNPs in tumor tissue sections, with red fluorescence (Cy5.5-labeled HNPs) overlapping and closer to green fluorescence (α-SMA; elevated α-SMA expression is characteristic of the liver metastatic niche), indicating that the liver metastasis-targeting properties of B7-33-HNPs benefit not only from their excellent blood circulation properties but also from the targeting ability of the surface drug molecule B7-33, allowing for modulation of stromal components and reversal of HSC activation.

[0203] Example 4

[0204] Based on the above experimental results, the inventors further studied and clarified the preventive and therapeutic effects of the prepared B7-33-HNPs by establishing a mouse pancreatic cancer liver metastasis model. The specific experimental process and results are briefly described below.

[0205] (I) Construction of a mouse pancreatic cancer liver metastasis model

[0206] Each C57BL / 6N mouse was injected intrasplenicly (near the hepatosplenic vein) with 1×10 pancreatic cancer cells. 6 A pancreatic cancer liver metastasis model was established using pan02-luci cells.

[0207] During the experiment, the microenvironment before liver metastasis in mice was detected and analyzed. Specifically:

[0208] Liver tissues were removed on days 0, 2, 4, 7, and 10 of modeling, and the expression of proteins such as a-SMA, Fibronectin, and Collagen I in hepatic stellate cells was observed by immunofluorescence and H&E staining. The time required for the formation of macroscopically visible metastatic lesions was also determined to evaluate whether the mouse pancreatic cancer liver metastasis model was successfully established and the specific conditions of the pre-metastatic fibrotic microenvironment.

[0209] Related staining and protein expression results are as follows Figure 9 ( Figure 9 A. Figure 9 B. Figure 9 C), combined with actual observation results, it can be evaluated and determined that visible metastatic lesions can be formed one week after tumor inoculation, at which point it can be determined that the mouse pancreatic cancer liver metastasis model has been successfully established (subsequent models will be constructed with reference to this time).

[0210] (II) Inhibition of tumor (cancer) cells during pancreatic cancer liver metastasis by nanomedicine

[0211] The liver is one of the most common metastatic sites for pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer with high metastatic potential. To evaluate the inhibitory effect of B7-33-HNPs on the formation of liver metastases in the Pan02 mouse model, the inventors conducted further experiments based on the previously described pancreatic cancer liver metastasis model. The specific procedures are as follows.

[0212] Referring to the above operation, pan02-luci was injected to establish a pancreatic cancer liver metastasis model. On the second day of the establishment of the pancreatic cancer liver metastasis model, PBS, HNPs, RGD-B7-33, RGD-B7-33+RNPs, C7-33-HNPs, and B7-33-HNPs were intravenously injected, with 6 mice in each group, and the drug was administered every other day for 6 times (refer to Figure 10 A time point for relevant sampling or operation).

[0213] Related test results such as Figure 10 As shown. Specifically:

[0214] From the IVIS spectral imaging results, we can see that ( Figure 10 B. Figure 10 D): The RGD-B7-33, C7-33-HNPs, HNPs, and RGD-B7-33+RNPs treatment groups all showed some inhibitory effects on the metastatic growth of tumor (cancer) cells. The poor inhibitory effect of RGD-B7-33 on tumor (cancer) cell metastasis is speculated to be related to its extremely short half-life and low accumulation in the metastatic site (liver). In contrast, the B7-33-HNPs treatment group significantly reduced the formation of tumor (cancer) cell liver metastases.

[0215] In addition, the H&E staining and statistical results of liver organs showed that ( Figure 10 C. Figure 10 E): The metastatic area of ​​tumor (cancer) cells in the B7-33-HNPs-treated group was significantly smaller than that in the other treatment groups. The quantitative results of the specific percentage of liver metastatic niches showed that ( Figure 10 E): PBS, 41.10 ± 8.51; HNPs, 33.74 ± 4.32; C7-33-HNPs, 31.97 ± 9.01; RGD-B7-33, 22.54 ± 7.02; RGD-B7-33 + RNPs, 24.51 ± 10.36; B7-33-HNPs, 5.94 ± 4.07; the number of metastatic areas in the B7-33-HNPs-treated group was significantly lowest.

[0216] Furthermore, the liver weight statistics showed that ( Figure 10 F): Liver weight was reduced by 54.5% in the B7-33-HNPs-treated group compared with the PBS group (at the same time, liver weight in the B7-33-HNPs-treated group was comparable to that in healthy, age-matched, tumor-free mice). This result also indicates that B7-33-HNPs treatment is associated with minimal metastatic tumors (i.e., there is no significant increase in liver weight due to the inhibitory effect on tumor (cancer) cell metastasis).

[0217] In summary, the above results preliminarily indicate that B7-33-HNPs has the strongest inhibitory activity on tumor cell colonization and metastasis formation among all preparations by regulating the pre-metastatic microenvironment of tumor (cancer) cells.

[0218] (III) In vivo mechanism of inhibition of pancreatic cancer liver metastasis

[0219] Since the formation of pancreatic cancer cell liver metastasis is closely related to the fibrosis process of cells, the inventors further explored the mechanism of anti-fibrosis in vivo. The specific experimental process is briefly described below.

[0220] During the experiment, the drug was administered every other day according to the experimental group. After 6 doses, the mouse liver tissue was collected at the end of the experiment for frozen sectioning. After acetone fixation, a-SMA, Fibronectin and Collagen I staining were performed to study the mechanism of action of B7-33-HNPs in inhibiting the fibrotic microenvironment before liver metastasis.

[0221] Furthermore, B7-33-HNPs can significantly reverse the activation of HSCs (activated HSCs can regulate the liver microenvironment to support the colonization and growth of metastatic tumor cells), and significantly reduce the expression of a-SMA, Fibronectin and Collagen I proteins in activated HSCs. That is, B7-33-HNPs nanoparticles can significantly inhibit the activation of hepatic stellate cells, thereby significantly inhibiting fibrosis in liver metastases.

[0222] Immunohistochemistry (IHC) staining results in liver metastatic niches Figure 11 ( Figure 11 As shown in A). Analysis showed that the B7-33-HNPs-treated group significantly reduced the levels of collagen, α-SMA, and fibronectin in metastatic lesions, which are major ECM (extracellular matrix) proteins (ECM proteins are directly related to the degree of fibrosis);

[0223] Combined with the relevant statistical results ( Figure 11B) Analysis showed that compared with the RGD-B7-33-treated group, B7-33-HNPs treatment led to a significant decrease in ECM scaffold protein expression, which further demonstrated that our designed B7-33-HNPs indeed played a better role in blood (body fluid) circulation by prolonging its half-life and enhancing its stability in vivo.

[0224] Similarly, the expression of α-SMA and collagen was similarly decreased in the RGD-B7-33 + RNPs-treated group, while fibronectin was detected in the RGD-B7-33-treated group; HNPs or C7-33-HNPs alone did not affect the expression of ECM proteins in liver metastases.

[0225] Based on the above results, it can be concluded that compared with the non-RGD-B7-33 treatment group (e.g., C7-33-HNPs, HNPs) or the control group, the application of B7-33-HNPs can significantly reduce the degree of fibrosis in the liver metastasis microenvironment, that is, it can reversibly inhibit the liver metastasis process of pancreatic cancer tumor (cancer) cells through anti-fibrosis means.

[0226] In summary, the inventors of this application developed and prepared targeted nanoparticles (B7-33-HNPs) that can be used to modify the liver metastatic microenvironment and inhibit metastasis formation and progression. During nanoparticle preparation, HNPs that overexpress integrin αv (β3) specifically carry the anti-fibrotic peptide B7-33 modified with an RGD peptide. This modulates the fibrotic microenvironment within the metastatic microenvironment and inhibits the colonization and progression of disseminated tumor cells in the liver. The RGD-B7-33 surface modification of the HNPs enables them to significantly target activated hematopoietic stem cells (HSCs), resulting in long circulation and excellent targeting of metastatic niches. Experimental results demonstrated effective inhibition of fibrosis in the liver fibrotic niche both in vitro and in vivo, leading to the inhibition of pancreatic cancer liver metastasis progression in a mouse pancreatic cancer (high liver metastasis) model with high efficacy and excellent biocompatibility. Furthermore, B7-33-HNPs exhibited low or no cytotoxicity in mammalian systems. In summary, this treatment strategy has great potential for systemic treatment of the formation and progression of pancreatic cancer liver metastasis and can better support clinical application in the future.

Claims

1. A biomimetic nanomedicine for reversing hepatic stellate cell activation, characterized in that: The bionic nanomedicine is prepared by the following steps: (1) Preparation of biomimetic nanocarriers The biomimetic nanocarrier HNPs was prepared using HUVEC membrane. (2) Modification of B7-33 The RGD sequence was coupled and modified with the peptide B7-33; The polypeptide B7-33 has an amino acid sequence as shown in SEQ ID No. 1, specifically: VIKLSGRELVRAQIAISGMSTWSKRSL; The amino acid sequence of the modified B7-33 is: VIKLSGRELVRAQIAISGMSTWSKRSL-GG-c(RGDfK); (III) Loading and constructing peptide biomimetic nanomedicines Based on the affinity of receptor and ligand, peptide B7-33 was loaded on biomimetic nanocarrier HNPs to construct peptide biomimetic nanomedicine B7-33-HNPs. The specific operation is as follows: The modified B7-33 in step (ii) is mixed with the biomimetic nanocarrier HNPs prepared in step (i) and incubated to prepare the biomimetic nanomedicine B7-33-HNPs.

2. The biomimetic nanomedicine for reversing hepatic stellate cell activation according to claim 1, characterized in that: In step (1), the vascular endothelial cell membrane is prepared by a cell membrane extraction method, and the specific operation is as follows: (1) Gradient centrifugation to obtain cell membrane The cultured vascular endothelial cells were collected, resuspended with IB-1, and then homogenized and ground; The ground cell suspension was centrifuged at 3000 g for 5 min at 4°C and the supernatant was retained. The supernatant was centrifuged again at 4°C and 10,000 g for 10 min, and the supernatant was retained; The supernatant was added to an ultracentrifuge tube, supplemented with IB-1, and centrifuged at 10,000 g for 2 h at 4°C to retain the precipitate; The IB-1 is: SB + 0.5% BSA + 0.5mmol EGTA; The SB is: 225 mmol mannitol + 75 mmol sucrose + 30 mmol Tris-HCl with pH = 7.4; (2) Using an extruder to extrude HNPs into cell membrane biomimetic nanocarriers The precipitate obtained by centrifugation in step (1) was placed in a cell ultrasonic disruptor and ultrasonicated at an ultrasonic power of 100W for 3 minutes. Subsequently, the precipitate was extruded using an extruder: First, the ultrasonically treated vascular endothelial cells were extruded up and down 10 times using a 0.8 μm polycarbonate (PC) membrane. Subsequently, 0.4 μm and 0.1 μm polycarbonate PC films were used to perform up and down extrusion 10 times respectively; Finally, uniform biomimetic nanocarrier HNPs nanoparticles with an average particle size of about 100 nm are prepared for use.

3. The biomimetic nanomedicine for reversing hepatic stellate cell activation according to claim 1, wherein: In step (iii), during incubation, the proportion of modified B7-33 was 1.5% by mass.

4. The biomimetic nanomedicine for reversing hepatic stellate cell activation according to claim 1, wherein: In step (3), incubate at 4°C overnight.

5. Use of the bionic nanomedicine for reversing hepatic stellate cell activation according to any one of claims 1 to 4 in the preparation of a medicament for preventing and treating pancreatic cancer, characterized in that: Used to prevent or reverse the metastasis of pancreatic cancer cells to liver tissue through anti-fibrosis means.

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