A biomimetic glycocalyx layer material, immunosuppressive compositions and their use in endothelial cell protection and inhibition of immune response to organ transplantation

Abstract

The application belongs to the technical field of biological medicine and glycopeptide preparation, and particularly relates to a biomimetic glycocalyx layer material, an immunosuppressive composition and application thereof in endothelial cell protection and inhibition of organ transplantation immune response. The biomimetic glycocalyx layer material (self-assembled glycopeptide) has the effects of maintaining cell integrity and preventing immune cell infiltration, maintaining the integrity of endothelial cells, and thus achieving the effect of protecting endothelial cells. On the basis of the above scheme, the biomimetic glycocalyx layer material and an immunosuppressive agent are combined for use. In the process of use, the biomimetic glycocalyx layer material can encapsulate the immunosuppressive agent, significantly improve the solubility of the immunosuppressive agent, reduce systemic toxicity, prolong the circulation time of the immunosuppressive agent, enhance the accumulation of the immunosuppressive agent at a disease site, and thus prolong the survival time of a graft, reduce the dose of the immunosuppressive agent, and achieve the effect of better inhibition of organ transplantation immune response.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and glycopeptide preparation technology, specifically relating to a biomimetic glycocalyx material, an immunosuppressive composition, and its application in endothelial cell protection and inhibition of immune responses in organ transplantation. Background Technology

[0002] Endothelial cells (ECs) are a thin layer of epithelial cells, composed of a single layer of flattened, polygonal cells with serrated edges, interlocking with each other. Endothelial cells form the inner wall of blood vessels, serving as the interface between the blood within the vessel lumen and the other vessel walls (a single layer of squamous epithelium). Endothelial cells extend throughout the entire circulatory system, from the heart down to the smallest capillaries.

[0003] Endothelial cells play a crucial role in heart transplantation. They serve not only as the physical barrier between the transplanted heart and the recipient but also in several key physiological processes, such as vascular regulation, inflammation, immune response, coagulation, and fibrinolysis. During heart transplantation, cryopreservation and reperfusion processes can damage fragile endothelial cells. Damaged endothelial cells release damage-associated molecular patterns (DAMPs), such as HMGB1, which in turn activate innate immune cells. These immune cells further signal with T (Treg) cells and B (Breg) cells, leading to adaptive immune activation. Furthermore, endothelial cells themselves act as hapten-presenting cells, participating in the activation of adaptive immunity. Conversely, endothelial cells are among the first cells to come into contact with the recipient's immune system, becoming primary targets of immune attack and further exacerbating endothelial damage.

[0004] While early and adequate use of broad-spectrum immunosuppressants can suppress immune activation, it not only increases susceptibility to infection, raises cancer risk, and causes liver and kidney toxicity, but also damages endothelial cells. Studies have shown that immunosuppressants such as tacrolimus, mycophenolate mofetil, and cyclosporine reduce endothelial cell migration at damaged sites and inhibit endothelial cell proliferation, leading to endothelial cell dysfunction and inhibiting angiogenesis, thus causing toxicity to the vascular system. Damaged endothelium leads to a series of vascular homeostasis imbalances, including inflammation, complement activation, and coagulation activation. Therefore, maintaining endothelial cell integrity is crucial for the function of transplanted hearts.

[0005] Alternatives to traditional immunosuppressants include the introduction of immune modulation using cell therapies, such as regulatory T cells or regulatory B cells. Engineered T and B cells have induced immune tolerance in allogeneic grafts. Conversely, combined bone marrow transplantation utilizes a transient hybrid chimerism approach, enabling the recipient to possess the immune systems of both the donor and recipient, and has achieved some success in achieving sustained chimerism. However, these approaches are currently suboptimal because they rely on combination therapies, inconsistencies in donor-specific tolerance induction, and potential challenges in adapting to diverse patients. Therefore, alternatives to immunosuppressants also currently face significant challenges.

[0006] However, there are currently no good technologies or methods that can simultaneously suppress the immune response to organ transplantation and maintain the integrity of endothelial cells. Summary of the Invention

[0007] The purpose of this invention is to provide a biomimetic glycocalyx material, an immunosuppressive composition, and their application in endothelial cell protection and suppression of immune responses during organ transplantation. The biomimetic glycocalyx material can effectively suppress immune responses during organ transplantation, thereby protecting endothelial cells, reducing damage to endothelial cells from immune responses, and maintaining the integrity of endothelial cells.

[0008] This invention provides a biomimetic saccharide calyx material. The raw materials for preparing the biomimetic saccharide calyx material include polypeptide SBP1, self-assembled peptide FFVDF, and sialyl lactose. The sialyl lactose is coupled to peptide bonds formed by the dehydration condensation of polypeptide SBP1 and self-assembled peptide FFVDF through an azide group. The amino acid sequences of polypeptide SBP1 and self-assembled peptide FFVDF are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0009] Preferably, the structural formula of the biomimetic sugar calyx material is shown in Formula I:

[0010]

[0011] The present invention also provides a method for preparing the biomimetic calyx material described in the above technical solution, comprising: preparing the biomimetic calyx material by solid-phase synthesis using polypeptide SBP1, self-assembled peptide FFVDF and sialyl glycosylose as raw materials.

[0012] Preferably, the molar ratio of the polypeptide SBP1, the self-assembled peptide FFVDF, and the sialyl lactose is 1:1:2.

[0013] The present invention also provides an immunosuppressive composition comprising individually packaged biomimetic calyx material and an immunosuppressant; wherein the biomimetic calyx material is the biomimetic calyx material described in the above technical solution or the biomimetic calyx material prepared by the preparation method described in the above technical solution.

[0014] Preferably, the immunosuppressant includes tacrolimus.

[0015] The present invention also provides the application of the biomimetic saccharide calyx material described in the above technical solution, or the biomimetic saccharide calyx material prepared by the preparation method described in the above technical solution, or the immunosuppressive composition described in the above technical solution, in the preparation of endothelial cell protection products.

[0016] The present invention also provides the application of the biomimetic calyx material described in the above technical solution, or the biomimetic calyx material prepared by the preparation method described in the above technical solution, or the immunosuppressive composition described in the above technical solution, in the preparation of products that inhibit immune responses in organ transplantation.

[0017] Preferably, the organ transplant includes a heart transplant.

[0018] Preferably, the product includes a drug.

[0019] Beneficial effects:

[0020] This invention provides a biomimetic glycocalyx material. The raw materials for preparing the biomimetic glycocalyx material include polypeptide SBP1, self-assembled peptide FFVDF, and sialyl lactose. The sialyl lactose is coupled to polypeptide SBP1 and self-assembled peptide FFVDF via an azide group, respectively. The amino acid sequences of polypeptide SBP1 and self-assembled peptide FFVDF are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. The biomimetic glycocalyx material (self-assembled glycopeptide) of this invention maintains cell integrity and prevents immune cell infiltration, thus preserving the integrity of endothelial cells and achieving the effect of protecting endothelial cells.

[0021] Based on the above-mentioned solutions, the present invention also provides an immunosuppressive composition comprising an independently packaged biomimetic glycocalyx material and an immunosuppressant. Specifically, the biomimetic glycocalyx material described in the above-mentioned technical solution is used in combination with the immunosuppressant. During use, the biomimetic glycocalyx material can encapsulate the immunosuppressant, reducing systemic toxicity, prolonging the circulation time of the immunosuppressant, enhancing its accumulation at the disease site, thereby prolonging the survival time of the transplant, reducing the dosage of the immunosuppressant, and achieving the effect of suppressing the immune response in organ transplantation. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0023] Figure 1 This is the mass spectrum of the SBP peptide in Example 1;

[0024] Figure 2 The graph shows the molecular structure of EPG (A) and the results of electrospray ionization mass spectrometry detection (B) in Example 2.

[0025] Figure 3 This is a graph showing the high-performance liquid chromatography (HPLC) detection results of EPG in Example 2;

[0026] Figure 4 The graph shows the molecular structure of EP in Example 2 (A) and the results of electrospray ionization mass spectrometry detection (B).

[0027] Figure 5 This is a graph showing the high-performance liquid chromatography (HPLC) detection results of EP in Example 2;

[0028] Figure 6 The TEM images of EPG and EP in Example 3 are shown, with a scale bar of 200 nm.

[0029] Figure 7 The images show DLS detection data of EPG+SLC9A3R2 and EPG at different time points in Example 3.

[0030] Figure 8 The retention time curve of EPG on the surface of HUVEC cells in Example 4;

[0031] Figure 9 The intensity curve of the FITC-labeled BSA in Example 4;

[0032] Figure 10 This is a CLSM diagram of the interaction between EPG and HUVEC cells in Example 5, with a scale bar of 12 μm;

[0033] Figure 11 This is a SEM image of the interaction between EPG and HUVEC cells in Example 5;

[0034] Figure 12 This is a flowchart of the mouse heart transplantation process involving EPG intervention in Example 6;

[0035] Figure 13 The survival curves after transplantation for the control group and EPG group in Example 6 are shown. "**" indicates a significant difference (P < 0.01).

[0036] Figure 14The survival curves after transplantation in the FK506 group and the FK506+EPG group in Example 6 are shown. "**" indicates a significant difference (P < 0.01).

[0037] Figure 15 The images show the H&E and immune cell infiltration patterns of myocardial tissue in the control group and EPG group in Example 6. "**" and "***" indicate significant differences (P < 0.01 and P < 0.001, respectively).

[0038] Figure 16 The images show the hematologic and immune cell infiltration patterns of myocardial tissue in the FK506 group and the FK506+EPG group in Example 6. "*", "**", and "***" indicate significant differences, P < 0.05, P < 0.01, and P < 0.001, respectively.

[0039] Figure 17 This is the synthesis reaction formula for sialyl lactose in this invention. Detailed Implementation

[0040] This invention provides a biomimetic glycocalyx material (EPG), the raw materials for which the biomimetic glycocalyx material is prepared include polypeptide SBP1, self-assembled peptide FFVDF, and sialyl lactose (Sia3Lac). The sialyl lactose is coupled to peptide bonds formed by the dehydration condensation of polypeptide SBP1 and self-assembled peptide FFVDF through an azide group. The amino acid sequences of polypeptide SBP1 and self-assembled peptide FFVDF are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0041] In this invention, as one embodiment, the sialyl lactose has the structural formula shown in Formula II:

[0042]

[0043] As one embodiment, the method for synthesizing sialyl glycosyl ... Figure 17 As shown in the figure), a polysaccharide as shown in Formula II is generated.

[0044]

[0045] In one embodiment, the reaction system for the glycosyltransferase reaction of the present invention includes CMP sialic acid (CAS: 3063-71-6), 4-hydroxyethylpiperazine ethanesulfonic acid (Hepes), and MgCl2, wherein the concentrations of CMP sialic acid, 4-hydroxyethylpiperazine ethanesulfonic acid (Hepes), and MgCl2 in the reaction system are 50 mM, 50 mM, and 20 mM, respectively. In another embodiment, the glycosyltransferase reaction is carried out at a temperature of 37°C for 4 hours, with a pH of 7.5.

[0046] In this invention, the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2 are as follows:

[0047] SEQ ID NO:1: KQVFKRH; SEQ ID NO:2: RRRFDVFF. The polypeptide SBP1 described in this invention has a high affinity for SLC9A3R2, a characteristic molecule on the surface of endothelial cells, and can specifically bind to SLC9A3R2, targeting endothelial cells. The self-assembling peptide FFVDF described in this invention serves as an assembly unit, enabling EPG molecules to form nanostructures.

[0048] As one embodiment, the structural formula of the biomimetic calyx layer material of the present invention is shown in Formula I:

[0049]

[0050] This invention also provides a method for preparing the biomimetic calyx material described in the above technical solution, comprising: preparing the biomimetic calyx material by solid-phase synthesis using polypeptide SBP1, self-assembled peptide FFVDF, and sialyl glycosides as raw materials. As one embodiment, the molar ratio of polypeptide SBP1, self-assembled peptide FFVDF, and sialyl glycosides in this invention is 1:1:2. This invention does not specifically limit the specific steps of the solid-phase synthesis; conventional solid-phase synthesis steps in the art can be used.

[0051] This invention also provides an immunosuppressive composition comprising individually packaged biomimetic glycocalyx material and an immunosuppressant; the biomimetic glycocalyx material is the biomimetic glycocalyx material described in the above-described technical solution or a biomimetic glycocalyx material prepared using the preparation method described in the above-described technical solution. As one embodiment, the immunosuppressant of this invention may be tacrolimus (FK506).

[0052] The present invention also provides the application of the biomimetic saccharide calyx material described in the above technical solution, or the biomimetic saccharide calyx material prepared by the preparation method described in the above technical solution, or the immunosuppressive composition described in the above technical solution, in the preparation of endothelial cell protection products.

[0053] The present invention also provides the application of the biomimetic calyx material described in the above technical solution, or the biomimetic calyx material prepared by the preparation method described in the above technical solution, or the immunosuppressive composition described in the above technical solution, in the preparation of products that inhibit immune responses in organ transplantation.

[0054] In one implementation, the organ transplant includes a heart transplant. In another implementation, the product is a drug.

[0055] This invention leverages the inherent advantages of self-assembled glycopeptide nanotechnology by encapsulating traditional immunosuppressants with biomimetic glycocalyx materials (self-assembled glycopeptides). This reduces the systemic toxicity of immunosuppressants, prolongs circulation time, and enhances their accumulation at disease sites. This method can be applied in the early stages of acute immune rejection to enhance graft protection, thereby preventing transplant rejection and ultimately protecting endothelial cells from damage and maintaining their integrity.

[0056] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0057] Example 1

[0058] This invention identifies a characteristic of vulnerable endothelial cells after heart transplantation—high expression of SLC9A3R2, a cell membrane expression molecule—through single-cell RNA sequencing analysis of porcine allogeneic heart transplants. Validation using human, porcine, and mouse specimens revealed that endothelial cell damage due to high SLC9A3R2 expression is consistent across different species.

[0059] In this embodiment, a library was constructed using all standard amino acids except cysteine ​​for standard solid-phase peptide synthesis. SLC9A3R2, covalently labeled with the fluorescent dye fluorescein isothiocyanate (FITC, green), was used as the screening target. 100,000 beads from the 7-mer linear OBOC peptide library were incubated with FITC-labeled albumin (100 μg / mL) in phosphate-buffered saline (PBS) for 12 h. Then, the strongly fluorescent beads were picked up at 488 nm under a fluorescence microscope for sequencing, and the results are as follows. Figure 1 As shown, in Figure 1 In the diagram, A2, A3, and A4 represent [M-4H] respectively. 4- [M-3H] 3- [M-2H] 2- .

[0060] Depend on Figure 1It can be concluded that the obtained sequences were identified as SBPs, including KQVFKRH (SBP1, SEQ ID NO:1) and WHIQQIE (SBP2, SEQ ID NO:3). Among them, SBP1 has a high specific binding ability to SLC9A3R2, with a maximum binding affinity of 1.3 × 10⁻⁶. -9 (Table 1)

[0061] Table 1. Amino acid sequence and related information of SLC9A3R2 targeting peptide.

[0062]

[0063]

[0064] Table 1 shows the peptide sequences, GRAVY values, and isoelectric points of peptides with high affinity for SLC9A3R2 obtained from OBOC screening. Through specific binding to SLC9A3R2, these peptides can target cardiac endothelial cells, effectively improving the efficiency of endothelial cell engineering.

[0065] Example 2

[0066] This embodiment successfully prepared a biomimetic sugar calyx material (EPG). The specific steps are as follows: using solid-phase synthesis technology, the peptide KQVFRHG-Sia3Lac-RRRFDVFF (EPG) and the negative control molecule KQVFRHG-RRRFDVFF (EP) were synthesized using the Fmoc coupling chemical method.

[0067] The compounds with the structure shown in Formula III and the compounds with the structure shown in Formula IV undergo a glycosyltransferase reaction under the action of α-2,3-sialyltransferase (reaction formula as shown in Figure 1). Figure 17 As shown), Sia3Lac is generated; the reaction system consists of 50mM CMP sialic acid, 50mM 4-hydroxyethylpiperazine ethanesulfonic acid (Hepes) and 20mM MgCl2. The glycosyltransfer reaction is carried out at a temperature of 37℃ for 4 hours and at a pH of 7.5.

[0068] EPG and EP were characterized by high performance liquid chromatography (HPLC) and electrospray ionization (ESI) mass spectrometry, and the results are as follows: Figures 2-5 As shown, where Figure 2 The graph shows the molecular structure of EPG (A) and the results of electrospray ionization mass spectrometry detection (B). Figure 3 The image shows the high-performance liquid chromatography (HPLC) detection results for EPG. Figure 4 The graph shows the molecular structure of EP (A) and the results of electrospray ionization mass spectrometry detection (B). Figure 5 This is a graph showing the high-performance liquid chromatography (HPLC) detection results of EP.

[0069] Depend on Figures 2-5 It can be concluded that EPG and EP were successfully prepared.

[0070] Example 3

[0071] To evaluate the potential of EPG as a biomimetic calyx layer, this embodiment presents a detailed analysis of the self-assembled nanostructures of EPG.

[0072] By adding the SLC9A3R2 extracellular receptor protein (i.e., SLC9A3R2 protein), expression was achieved in human umbilical vein endothelial cells (HUVEC, purchased from the Cell Culture Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), transforming nanoparticles into nanofibers, and then culturing them at 37°C for several hours. Specifically, HUVEC cells in EPG, SLC9A3R2 protein, and DMEM medium were co-incubated at room temperature. The amount of SLC9A3R2 protein added was 1 / 10 of the amount of EPG. Samples were prepared at 0, 0.5, 2, and 4 hours. The co-incubation solution was used for size measurement (DLS, NanoZS, Malvern) and transmission electron microscopy (Philips CM-120TEM, USA). TEM samples were stained with uranium acetate.

[0073] TEM images show that the diameters of EPG and EP were approximately 10 nm at 0 h, gradually increasing to 20 nm. Nanofiber structures with a diameter of approximately 9 nm were observed; the number of nanofibers increased within 2 h, and the nanofiber network was clearly visible after 4 h, exhibiting a wide size distribution. Figure 6 DLS measurements revealed the size distribution of EPG and EP aggregates, confirming the structural transition from nanoparticles to nanofibers. These results indicate that both EPG and EP possess stable nanostructures. Figure 7 ).

[0074] Example 4

[0075] In this embodiment, Cy7-labeled EPG was perfused into mouse donor hearts at a flow rate of 2 mL for transplantation experiments. Small animal fluorescence imaging was performed at different time points, and the half-life was subsequently calculated from the fluorescence signals. The results are as follows: Figure 8 As shown;

[0076] The steps for labeling Cy7 with EPG were as follows: EPG was dissolved in 3 mL of PBS buffer, then 1 mL of 1 / 10 molar Cy7 was added, along with an appropriate amount of triethylamine. The pH was adjusted to 9.4°C and stirred overnight in an ice bath. The reaction solution turned fluorescent blue. Dialysis was then performed using a dialysis bag with a molecular weight of 1000 Diatoms, changing the water every 1 hour, for 2 days. The resulting residue was lyophilized for later use.

[0077] Depend on Figure 8 It can be concluded that EPG can prolong its half-life and enhance resistance to immune rejection. The half-life of EPG on the cell surface is as long as 21 hours.

[0078] HUVEC cells with 60-80% confluence were treated with trypsin at a concentration of 100,000 cells / cm³. 2 The inoculation rates were respectively inoculated into β-cell immunoglobulins containing tumor necrosis factor TNF-α (TNF-α, purchased from Abcam, CAS: 94948-59-1, 2000 U / mL), EPG (EPG, 50 μM), and EFG+TNF (EPG, 50 μM, TNF-α 2000 U / mL) (3 μm pore size, 0.33 cm). 2 The tissue culture inserts (3415, Corning) were incubated for 3 hours at 37°C and 5% CO2, with a blank control group included. The tissue culture inserts were placed in 24-well plates. HUVEC cells were allowed to grow in the tissue culture inserts for 1 week before performing transendothelial protein channel experiments, with the cell culture medium changed every 2 days.

[0079] The passage of FITC-labeled BSA (A92771, Sigma) in a monolayer was tracked for 3 hours. First, the medium in both compartments was replaced with serum-free medium for 1 hour. Then, 0.5 mg / mL FITC-BSA medium was added to the upper compartment, and 0.5 mg / mL unlabeled BSA (A-7888, Sigma) medium was added to the lower compartment. Over 3 hours, 100 μL aliquots were collected hourly from the lower compartment and replaced with medium containing 0.5 mg / mL unlabeled BSA. The fluorescence of the collected medium was recorded, and the concentration was calculated based on the standard curve of FITC-labeled BSA. The results are shown below. Figure 9 As shown.

[0080] Depend on Figure 9 It can be concluded that EPG does not affect the passage of BSA through endothelial cells.

[0081] Example 5

[0082] CLSM and SEM were used to verify the structural transformation of EPG on the cell surface, and the steps were as follows:

[0083] HUVEC cells were cultured in glass dishes for 12 hours. EPG (50 μM) was incubated with cells in DMEM at 37°C for 6 or 12 hours, respectively. For confocal laser scanning microscopy (CLSM, LSM800, ZEISS), samples were fixed with glutaraldehyde (4%) for 10 minutes, washed three times with PBS, and examined using a 405 nm laser and a 40× immersion objective. The results are as follows: Figure 10 As shown.

[0084] To further verify the binding of EPG to SLC9A3R2, this invention used a rabbit anti-SLC9A3R2 (Abcam, USA) monoclonal antibody (Sigma Aldrich, USA) to detect the extracellular domain of SLC9A3R2 on the surface of HUVECs. Specifically, the rabbit anti-SLC9A3R2 monoclonal antibody was co-incubated with the aforementioned EPG-bound HUVEC cells in DMEM medium at 5% CO2 and 37°C. For scanning electron microscopy (SEM, Philips XL30 TMP, FEI, Hillsborough), the cells were fixed overnight with glutaraldehyde (4%) and then plated with gold for 2 minutes. Figure 11 As shown in the figure, the first three icons have a ruler of 20μm, and the last icon has a ruler of 5μm.

[0085] Depend on Figure 10 and Figure 11 It can be concluded that EPG has a good binding ability with SLC9A3R2, and that EPG on the cell surface can achieve structural transformation.

[0086] Example 6

[0087] To further evaluate the protective effect of EPG on endothelial cells (ECs) in vivo, this embodiment constructed four mouse heart transplantation (HTx) models: control group, EPG group, FK506 group, and EPG+FK506 group.

[0088] Control group: 2 mL of HTK solution was infused into the donor heart via the inferior vena cava;

[0089] EPG group: 2 mL of HTK solution containing EPG was perfused into the donor heart via the inferior vena cava, and the final concentration of EPG in the HTK solution was 1 mg / mL;

[0090] FK506 group: 2 mL of HTK solution was infused into the donor heart via the inferior vena cava;

[0091] EPG+FK506 group: 2 mL of HTK solution containing EPG was perfused into the donor heart via the inferior vena cava. The final concentration of EPG in the HTK solution was 1 mg / mL.

[0092] The mouse heart transplantation protocol was based on a cervical heterotopic transplantation model (J Vis Exp. 2014 Oct12; (92):e50753). In short, a mouse heterotopic heart transplantation model was constructed using BALB / c (H-2d) and C57BL / 6 (H-2b) mice (6–8 weeks old). C57BL / 6 mice were anesthetized and placed supine. After preparing the skin, layers were incised to expose and release the left external jugular vein and common carotid artery. The vessels were then cut, slid onto specially designed cannulas, and secured with sutures. Under deep anesthesia, the thoracic cavity of the BALB / c mice was opened, and the ascending aorta and pulmonary trunk of sufficient length were cut. The remaining vessels were ligated and cut sequentially, and the heart was removed. The donor heart was placed in the recipient's neck, and the aorta was connected to the common carotid artery, and the pulmonary trunk to the external jugular vein, all with sutures. The skin was then sutured, and the mice were placed in a warm environment for recovery. All model mice were housed in an SPF environment with ample food and water. The FK506 and EPG+FK506 groups received FK506 at 0.5 mg / kg daily, as shown in the flowchart. Figure 12 As shown.

[0093] Survival was assessed and monitored in all groups by visual inspection and palpation. Kaplan-Meier analysis was performed to compare the protective effect of EPG on heart grafts. Results showed that the median survival time in the control group and the EPG group was 11 days and 7 days, respectively, with a slight increase in graft survival time in the EPG group compared to the control group. Figure 13 ,exist Figure 13 and the following Figures 14-16 In the text, PBS represents the control group. As a classic immunosuppressant, the survival time in the FK506 treatment group (0.5 mg / kg / day) was 12 days. Notably, the EPG+FK506 group achieved a survival time of over 30 days. Figure 14 ).

[0094] To further evaluate the protective effect of EPG on the endothelium, mouse heart grafts were obtained from the control and EPG groups 3 days post-surgery. The first 3 days are the period of most severe endothelial injury after heart transplantation, and early endothelial protection is crucial for heart graft survival. Routine HE pathological examination was performed, and staining for T cell and macrophage surface markers CD3 (ZA-0503, zsbio) and CD68 (ZM-0060, zsbio) was performed to assess endothelial integrity and the degree of myocardial inflammatory infiltration. In the control group, significant endothelial swelling and detachment were observed due to IRI and immune damage, while the EPG endothelium remained intact and tightly attached to the vascular lumen. Simultaneously, the EPG group showed less immune cell infiltration than the control group. Figure 15 ).

[0095] To further evaluate the protective effect of EPG on long-term survival of heart grafts, mouse heart grafts were obtained 7 days post-surgery from the FK506 group and the FK506+EPG group. Heart grafts that did not receive any treatment were rejected within 7 days; therefore, 7 days is the standard observation period for the therapeutic effect after heart transplantation. Routine HE pathological examination was also performed in this embodiment, and staining for T cell and macrophage surface markers CD3 (ZA-0503, zsbio) and CD68 (ZM-0060, zsbio) was performed to assess endothelial integrity and the degree of myocardial inflammatory infiltration. The results at 7 days showed that the FK506+EPG group had minimal T cell and macrophage infiltration (…). Figure 16 ).

[0096] As can be seen from the above embodiments, the biomimetic calyx material of the present invention can effectively inhibit the immune response during organ transplantation, thereby protecting endothelial cells, reducing the damage of the immune response to endothelial cells, and maintaining the integrity of endothelial cells.

[0097] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A biomimetic sugar calyx layer material, characterized in that, The raw materials for preparing the biomimetic calyx material include polypeptide SBP1, self-assembled peptide FFVDF, and sialyl lactose. The sialyl lactose is coupled to peptide bonds formed by the dehydration condensation of polypeptide SBP1 and self-assembled peptide FFVDF through an azide group. The amino acid sequences of polypeptide SBP1 and self-assembled peptide FFVDF are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. The structure of the biomimetic sugar calyx material is shown in Formula I: Formula I.

2. The method for preparing the biomimetic sugar calyx material according to claim 1, characterized in that, include: The biomimetic calyx material was prepared by solid-phase synthesis using polypeptide SBP1, self-assembled peptide FFVDF, and sialyl lactose as raw materials.

3. The preparation method according to claim 2, characterized in that, The molar ratio of the polypeptide SBP1, the self-assembled peptide FFVDF, and the sialyl lactose is 1:1:

2.

4. An immunosuppressive composition, characterized in that, The immunosuppressive composition comprises individually packaged biomimetic calyx material and an immunosuppressant; the biomimetic calyx material is the biomimetic calyx material of claim 1 or the biomimetic calyx material prepared by the preparation method of claim 2 or 3.

5. The immunosuppressive composition according to claim 4, characterized in that, The immunosuppressants include tacrolimus.

6. The use of the biomimetic calyx material of claim 1, or the biomimetic calyx material prepared by the preparation method of claim 2 or 3, or the immunosuppressive composition of claim 4 or 5, in the preparation of a medicament for inhibiting immune responses in organ transplantation.

7. The application according to claim 6, characterized in that, The organ transplantation mentioned includes heart transplantation.

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