Bionic glycocalyx layer material and application thereof

Abstract

The invention belongs to the technical field of biological medicine and glycopeptide preparation, and particularly relates to a bionic glycocalyx layer material and application thereof. The bionic glycocalyx layer material is prepared from the following raw materials: polypeptide HSDVHK, self-assembled peptide FFLDF and saliva lactoyl sugar, the polypeptide HSDVHK and the self-assembled peptide FFLDF are subjected to dehydration condensation to form a peptide bond, and the saliva lactoyl sugar is coupled with the peptide bond through an azide group. In the organ transplantation process, the bionic glycocalyx layer material (self-assembled glycopeptide and EPG) can prevent immune cell infiltration, the integrity of endothelial cells is kept while immune response is effectively inhibited, and then the effect of protecting the endothelial cells is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and glycopeptide preparation, and particularly relates to a biomimetic glycocalyx layer material and its application. Background Art

[0002] Endothelial cells (ECs) are a single-cell structure that covers the inner walls of all blood vessels such as arteries, capillaries, and veins. Endothelial cells form a barrier between blood vessels and tissues and control the flow of substances and fluids in and out of tissues. In all blood vessels in the body, endothelial cells form a semipermeable barrier between blood and surrounding tissues. As an important endocrine organ, this single layer of cells has been shown to play an important role in regulating blood transmission, fibrinolysis and macromolecular transfer, immune response, and participating in inflammatory response.

[0003] Endothelial cells are a physical barrier between the transplanted heart and the recipient, and thus play an important role in the process of heart transplantation. During heart transplantation, the cold storage and reperfusion processes can cause damage to the fragile endothelial cells. The damaged ECs will activate innate immune cells, leading to the activation of adaptive immunity. At the same time, ECs will also act as hapten-presenting cells to activate adaptive immunity, and then cause ECs to become the primary target of immune attack, exacerbating endothelial damage.

[0004] Xenogeneic heart transplantation has broad application prospects due to its biogenicity and modifiability, but the complex immune rejection reaction is an important obstacle to the clinical application of xenogeneic heart transplantation. In the early stage, sufficient broad-spectrum immunosuppressants in heart transplantation, especially xenogeneic heart transplantation, can inhibit immune activation, but the immunosuppressants themselves will damage endothelial cells. Although the use of cell therapy to introduce immune regulation to replace traditional immunosuppressants can reduce the damage to endothelial cells after xenogeneic heart transplantation, there are potential challenges in the inconsistent induction of donor-specific tolerance and adaptation to different patients. Therefore, there is currently a lack of materials or technologies for protecting endothelial cells during heart transplantation, especially xenogeneic heart transplantation. Summary of the Invention

[0005] The purpose of the present invention is to provide a biomimetic glycocalyx layer material and its application. The biomimetic glycocalyx layer material can inhibit the immune reaction during organ transplantation, reduce the damage of the immune reaction to endothelial cells, and maintain the integrity of endothelial cells.

[0006] The present invention provides a biomimetic glycocalyx layer material. The preparation raw materials of the biomimetic glycocalyx layer material include polypeptide HSDVHK, self-assembling peptide FFVDF, and sialyllactose. The polypeptide HSDVHK and the self-assembling peptide FFVDF are dehydrated and condensed to form peptide bonds, and the sialyllactose is coupled with the peptide bonds through azide groups.

[0007] Preferably, the structural formula of the biomimetic glycocalyx layer material is as shown in Formula I:

[0008]

[0009] The present invention also provides a method for preparing the biomimetic glycocalyx layer material described in the above technical solution, including the following steps:

[0010] The biomimetic glycocalyx layer material is obtained by solid-phase synthesis technology using polypeptide HSDVHK, self-assembling peptide FFVDF, and sialyllactose.

[0011] Preferably, the molar ratio of the polypeptide HSDVHK, self-assembling peptide FFVDF, and sialyllactose is 1:1:2.

[0012] The present invention also provides the use of the biomimetic glycocalyx layer material described in the above technical solution or the biomimetic glycocalyx layer material prepared by the preparation method described in the above technical solution in the preparation of a product for protecting endothelial cells.

[0013] The present invention also provides the use of the biomimetic glycocalyx layer material described in the above technical solution or the biomimetic glycocalyx layer material prepared by the preparation method described in the above technical solution in the preparation of a product for inhibiting the immune response of organ transplantation.

[0014] Preferably, the organ transplantation includes heart transplantation.

[0015] Preferably, the heart transplantation includes xenogeneic heart transplantation.

[0016] Preferably, the product includes drugs.

[0017] The present invention also provides a drug for protecting endothelial cells and / or inhibiting the immune response of organ transplantation, and the active ingredient of the drug includes the biomimetic glycocalyx layer material described in the above technical solution or the biomimetic glycocalyx layer material prepared by the preparation method described in the above technical solution.

[0018] Beneficial effects:

[0019] The present invention provides a biomimetic glycocalyx layer material. The preparation raw materials of the biomimetic glycocalyx layer material include polypeptide HSDVHK, self-assembling peptide FFVDF, and sialyllactose. The polypeptide HSDVHK and the self-assembling peptide FFVDF are dehydrated and condensed to form peptide bonds, and the sialyllactose is coupled with the peptide bonds through azide groups. The biomimetic glycocalyx layer material (self-assembled glycopeptide, EPG) of the present invention can prevent the infiltration of immune cells during organ transplantation, effectively inhibit the immune response while maintaining the integrity of endothelial cells, and thus achieve the effect of protecting endothelial cells. Description of the Drawings

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings required for the embodiments will be briefly introduced below.

[0021] Figure 1 It is a diagram showing the molecular structural formula (A) of EPG and the results of electrospray ionization mass spectrometry detection (B) in Example 1;

[0022] Figure 2 It is a diagram showing the results of high performance liquid chromatography detection of EPG in Example 1;

[0023] Figure 3 It is a diagram showing the molecular structural formula (A) of EPP and the results of electrospray ionization mass spectrometry detection (B) in Example 1;

[0024] Figure 4 It is a diagram showing the results of high performance liquid chromatography detection of EPP in Example 1;

[0025] Figure 5 It is a TEM detection diagram of EPG and EPP in Example 2; wherein, the scale bar is 200 nm;

[0026] Figure 6 It is a DLS detection diagram of EPG and EPP in Example 2;

[0027] Figure 7 It is the retention time curve of EPG on the surface of HUVEC cells in Example 3;

[0028] Figure 8 It is the intensity curve of FITC-labeled BSA in Example 3;

[0029] Figure 9 It is a CLSM diagram of the interaction between EPG and HUVEC cells or MCF-7 cells in Example 4; wherein, the scale bar is 10 μm;

[0030] Figure 10 It is a SEM diagram of the interaction between EPG and HUVEC cells in Example 4;

[0031] Figure 11 It is the flow chart of mouse heart transplantation intervened by EPG in Example 5;

[0032] Figure 12 It is the survival curve after transplantation of the control group EPP group and EPG group in Example 5;

[0033] Figure 13 It is the H&E and immune cell infiltration diagram of the myocardial tissue of the control group EPP group and EPG group in Example 5;

[0034] Figure 14 It is the synthesis reaction formula of salivary lactose. Detailed implementation mode

[0035] The present invention provides a biomimetic glycocalyx layer material. The raw materials for preparing the biomimetic glycocalyx layer material include polypeptide HSDVHK (SEQ ID NO: 1), self-assembling peptide FFVDF (SEQ ID NO: 2), and sialyllactose (Sia3Lac). The polypeptide HSDVHK and the self-assembling peptide FFVDF undergo dehydration condensation to form peptide bonds, and the sialyllactose is coupled to the peptide bonds through azide groups.

[0036] As an implementation mode, the polypeptide HSDVHK and the self-assembling peptide FFVDF in the present invention undergo dehydration condensation through terminal amino acids to form peptide bonds; as another implementation mode, the structural formula of the biomimetic glycocalyx layer material is preferably as shown in Formula I:

[0037]

[0038] In the present invention, the structural formula of the sialyllactose in the present invention is preferably as shown in Formula II:

[0039]

[0040] The structural formula in Formula II can also be as follows:

[0041]

[0042] The synthesis method of the sialyllactose in the present invention preferably includes the following steps:

[0043] The compound with the structure shown in Formula III and the compound with the structure shown in Formula IV undergo a glycosyltransferase reaction under the action of α-2,3-sialyltransferase (the reaction formula is as Figure 14 shown), to generate sialyllactose as shown in Formula II.

[0044]

[0045] The reaction system of the glycosyltransferase reaction in the present invention preferably includes CMP-sialic acid (CAS: 3063-71-6), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), and MgCl 2 ; the concentrations of CMP-sialic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), and MgCl 2 in the reaction system are preferably 50 mM, 50 mM, and 20 mM respectively. The temperature of the glycosyltransferase reaction in the present invention is preferably 37 °C, the time is preferably 4 h, and the pH value is preferably 7.5.

[0046] The polypeptide HSDVHK of the present invention has a high affinity for the characteristic molecule SLC9A3R2 on the surface of endothelial cells, can specifically bind to SLC9A3R2, and targets endothelial cells. The self-assembling peptide FFVDF of the present invention functions as an assembly unit to enable EPG molecules to form nanostructures. The structural formula of the biomimetic glycocalyx layer material of the present invention is shown in Formula I.

[0047] The present invention also provides a preparation method of the biomimetic glycocalyx layer material described in the above technical solution, including the following steps:

[0048] The biomimetic glycocalyx layer material is obtained by solid-phase synthesis technology using the polypeptide HSDVHK, the self-assembling peptide FFVDF, and lactosyl lactose.

[0049] The molar ratio of the polypeptide HSDVHK, the self-assembling peptide FFVDF, and lactosyl lactose of the present invention is preferably 1:1:2. The present invention has no special limitation on the specific process of the solid-phase synthesis, and the steps of conventional solid-phase synthesis in the art can be adopted.

[0050] The present invention also provides the application of the biomimetic glycocalyx layer material described in the above technical solution or the biomimetic glycocalyx layer material prepared by the preparation method described in the above technical solution in the preparation of endothelial cell protection products. The product of the present invention is preferably a drug.

[0051] The present invention also provides the application of the biomimetic glycocalyx layer material described in the above technical solution or the biomimetic glycocalyx layer material prepared by the preparation method described in the above technical solution in the preparation of organ transplantation immune response inhibition products. The organ transplantation of the present invention is preferably heart transplantation; the heart transplantation is preferably xenogeneic heart transplantation. The product of the present invention is preferably a drug.

[0052] The present invention also provides a drug for endothelial cell protection and / or inhibition of organ transplantation immune response. The active ingredient of the endothelial cell protection drug includes the biomimetic glycocalyx layer material described in the above technical solution or the biomimetic glycocalyx layer material prepared by the preparation method described in the above technical solution.

[0053] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the drawings and examples, but they cannot be construed as limiting the protection scope of the present invention.

[0054] Example 1

[0055] A preparation method of a biomimetic glycocalyx layer material (EPG) is as follows:

[0056] Synthesis method of sialyllactose: A compound with the structure shown in Formula III and a compound with the structure shown in Formula IV undergo a glycosyltransferase reaction under the action of α-2,3-sialyltransferase (the reaction formula is as shown in Figure 14 ) to produce sialyllactose as shown in Formula II; the glycosyltransferase reaction system is 50 mM CMP-sialic acid, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 20 mM MgCl 2 2, and the reaction conditions are a pH value of 7.5 and a reaction at 37 °C for 4 h.

[0057] Using solid-phase synthesis technology, the polypeptide FFVDF-(Sia3Lac)-HSDVHK is synthesized by the Fmoc coupling chemical method, denoted as EPG;

[0058] The negative control molecule FFVDF-HSDVHK is prepared by the same method, denoted as EPP.

[0059] EPG and EPP are characterized by high-performance liquid chromatography (HPLC) and electrospray ionization (ESI) mass spectrometry, and the results are as shown in Figures 1 to 4 , where Figure 1 are the molecular structural formula (A) and electrospray ionization mass spectrometry detection (B) results of EPG, Figure 2 is the high-performance liquid chromatography detection result of EPG; Figure 3 are the molecular structural formula (A) and electrospray ionization mass spectrometry detection (B) results of EPP, Figure 4 is the high-performance liquid chromatography detection result of EPP.

[0060] From Figures 1 to 4 it can be concluded that EPG and EPP are successfully prepared.

[0061] Example 2

[0062] The potential of EPG as a biomimetic glycocalyx layer in Example 1 is analyzed and evaluated by analyzing the self-assembled nanostructure of EPG, and the steps are as follows:

[0063] By adding CaCl 2 , EPG is expressed in human umbilical vein endothelial cells (HUVEC, purchased from the Cell Culture Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences), the morphology of the nanoparticles is transformed into nanofibers, and they are cultured at 37 °C for several hours.

[0064] Specifically: EPG, CaCl 2 and HUVEC cells in DMEM medium are co-incubated at room temperature, and CaCl 2The addition amount is 1 / 10 of the amount of substance of EPG. Samples were prepared at 0, 2, and 4 h. The co-incubation solution was used to measure the size (DLS, Nano ZS, Malvern) and transmission electron microscopy (Philips CM-120 TEM, USA). TEM samples were stained with uranyl acetate. The experiment without adding CaCl 2 was used as the control group.

[0065] Similarly, EPP was replaced with EPG, and the experimental group and the control group were set under the same experimental conditions.

[0066] TEM images showed that: at 0 h, the diameters of EPG and EPP were about 10 nm, and then gradually increased to 20 nm. At 2 h, nanofiber structures with a diameter of about 9 nm were observed. The number of nanofibers increased within 2 h. After 4 h, the nanofiber network was clearly visible and the size distribution was wide ( Figure 5 ); while the group without adding CaCl 2 did not form obvious nanofiber structures.

[0067] DLS measurements showed that: the distribution sizes of EPG and EPP aggregates confirmed the structural transformation from nanoparticles to nanofibers ( Figure 6 ).

[0068] These results indicate that both EPG and EPP have stable nanostructures.

[0069] Example 3

[0070] In this example, FITC-labeled EPG was used to perform flow cytometry experiments on HUVEC cells at different time points, and then the half-life was calculated through fluorescence signals. The results are as Figure 7 shown;

[0071] The steps for labeling EPG with FITC were as follows: EPG was dissolved in 3 mL of PBS buffer, then 1 mL of FITC with a molar amount of 1 / 10 was added thereto, an appropriate amount of triethylamine was added, the pH was adjusted to 9, and it was stirred overnight in an ice bath at 4 °C. Then it was dialyzed with a dialysis bag with a molecular weight of 1000 Diatoms, the water was changed once an hour, and dialysis was carried out for 2 days. The obtained residue was freeze-dried and reserved for use.

[0072] It can be concluded from Figure 7 that the half-life of EPG on the cell surface is as long as 12.5 h.

[0073] HUVEC cells with 60 - 80% confluence were treated with trypsin and seeded on a polycarbonate support (3 μM pore size, 0.33 cm 2In a tissue culture insert (3415, Corning) with a surface area of [surface area value not provided], 10,000 cells were seeded per square centimeter. The tissue culture insert was placed in a 24-well plate and incubated at 37 °C and 5% CO 2 for 3 h.

[0074] Different experimental groups were set up by adding different substances to the tissue culture insert as follows:

[0075] TNF-α: Containing tumor necrosis factor TNF-α (TNF-α purchased from Abcam, CAS: 94948-59-1, 2000 U / mL); EPG (EPG, 50 μM); EFG+TNF (EPG, 50 μM, TNF-α 2000 U / mL); EPP (EPP, 50 μM); and EPP+TNF (EPP, 50 μM, TNF-α 2000 U / mL); and a blank control group, namely the PBS group, was set up.

[0076] Before performing the transendothelial protein passage experiment, HUVEC cells were allowed to grow in the tissue culture insert for 1 week, and the cell culture medium was changed every 2 days.

[0077] FITC-labeled BSA (A92771, Sigma) was traced through the monolayer channel within 3 h. First, the medium in both compartments was replaced with serum-free medium for 1 h. Then, 0.5 mg / mL of FITC-BSA medium was added to the upper compartment, and 0.5 mg / mL of unlabeled BSA (A-7888, Sigma) medium was added to the lower compartment. Within 3 h, 100 μL aliquots were collected from the lower compartment every hour and replaced with medium containing 0.5 mg / mL of unlabeled BSA. The fluorescence of the collected medium was recorded, and the concentration was calculated according to the standard curve of FITC-labeled BSA. The results are as Figure 8 shown.

[0078] It can be concluded from Figure 8 that EPG does not affect the passage of BSA through endothelial cells.

[0079] Example 4

[0080] CLSM and SEM were used to verify the structural transformation of EPG on the cell surface as follows:

[0081] 1) HUVEC cells were cultured in a glass-bottom dish for 12 h. FITC-fluorescently labeled EPG (50 μM) was incubated with HUVEC cells in DMEM at 37 °C for 12 h. Images were taken with a confocal laser scanning microscope (CLSM, LSM 800, ZEISS), washed 3 times with PBS, and examined using a 405 nm laser and a 40× immersion objective. The results are as Figure 9as shown in the first row of pictures.

[0082] 2) Replace HUVEC cells with MCF-7 cells and perform the same experiment as in step 1). The results are as Figure 9 shown in the second row of pictures.

[0083] 3) To further verify the ability of EPG to form fibrous structures on the surface of endothelial cells. Specifically, EPG and HUVEC cells are co-incubated in DMEM medium in a 5% CO 2 , 37 °C incubator. For scanning electron microscopy (SEM, Philips XL30 TMP, FEI Company, Hillsboro), the cells are fixed overnight with glutaraldehyde (4%), and then gold-plated for 2 min, as Figure 10 shown.

[0084] It can be concluded from Figure 9 and Figure 10 that EPG has a good binding ability with HUVECs cells, and EPG on the cell surface can achieve structural transformation.

[0085] Example 5

[0086] To further evaluate the protective effect of EPG on endothelial cells (ECs) in vivo, three groups of xenogeneic mouse heart transplantation models are constructed in this example: a control group, an EPG group, and an EPP group.

[0087] Control group: Perfuse 2 mL of PBS solution into the donor heart from the inferior vena cava;

[0088] EPG group: Perfuse 2 mL of PBS solution containing EPG into the donor heart from the inferior vena cava. The final concentration of EPG in the PBS solution is 1 mg / mL;

[0089] EPP group: Perfuse 2 mL of PBS solution containing EPP into the donor heart from the inferior vena cava. The final concentration of EPP in the PBS solution is 1 mg / mL;

[0090] The mouse heart transplantation protocol was based on the construction of a heterotopic transplantation model in the neck (J Vis Exp. 2014 Oct 12; (92): e50753). Briefly, an ectopic mouse heart transplantation model was constructed using Sprague-Dawley (SD) rats (1 week old) and BALB / c mice (6 - 8 weeks old). The SD rats were anesthetized and placed supine. After preparing the skin, incisions were made layer by layer to expose and release the left external jugular vein and the common carotid artery. Then, the blood vessels were transected, slipped onto specially designed cannulas, and fixed with sutures. Under deep anesthesia, the chest of the SD rat was opened, and the ascending aorta and the pulmonary artery trunk were cut after releasing a sufficient length. The remaining blood vessels were ligated and transected in sequence, and the heart was removed. The donor heart was placed in the neck of the recipient BALB / c mouse, and the aorta was connected to the common carotid artery and the pulmonary artery trunk was connected to the external jugular vein with sutures. Then the skin was sutured, and the mice were placed in a warm environment to recover. All model mice were housed in a specific pathogen-free (SPF) environment with sufficient food and water. The flow chart is as Figure 11 shown.

[0091] The survival of all groups was monitored by visual inspection and palpation. To compare the protective effect of EPG on heart grafts, Kaplan-Meier analysis was performed on the survival time. The results showed that the median survival time of the EPG group was 8.2 days, and the graft survival time in the EPG group increased by 1.62 times compared with the control group (at Figure 12 , PBS represents the control group).

[0092] To further evaluate the protective effect of EPG on endothelial cells, in this example, mouse heart grafts of the control group (transplant group), EPG group, EPP group, and normal group without any treatment were obtained 3 days after surgery. The 3-day period is the time when endothelial injury is most severe after heart transplantation, and early endothelial cell protection is crucial for heart grafts. In this example, routine HE pathological examination was performed to evaluate endothelial integrity. In the control group, due to immune injury, obvious endothelial swelling and detachment were observed, while the EPG endothelium remained intact and tightly adhered to the vascular lumen ( Figure 13 ).

[0093] From the above examples, it can be concluded that the biomimetic glycocalyx layer material of the present invention can inhibit the immune response during organ transplantation, reduce the damage of the immune response to endothelial cells, and maintain the integrity of endothelial cells.

[0094] Although the above examples have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments according to this example without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A bionic glycocalyx layer material, characterized in that: The raw materials for preparing the biomimetic glycocalyx layer material include polypeptide HSDVHK, self-assembling peptide FFVDF and sialyl lactose. The polypeptide HSDVHK and the self-assembling peptide FFVDF are dehydrated and condensed to form a peptide bond, and the sialyl lactose is coupled to the peptide bond through an azide group.

2. The bionic glycocalyx layer material according to claim 1, characterized in that: The structural formula of the bionic glycocalyx layer material is shown in Formula I:

3. The method for preparing the bionic glycocalyx layer material according to claim 1 or 2, characterized in that: The steps include: The bionic glycocalyx layer material is obtained by synthesizing polypeptide HSDVHK, self-assembling peptide FFVDF and sialyllactyl sugar through solid phase synthesis technology.

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

2.

5. Use of the bionic glycocalyx layer material according to claim 1 or 2 or the bionic glycocalyx layer material prepared by the preparation method according to claim 3 or 4 in preparing products for protecting endothelial cells.

6. Use of the bionic glycocalyx layer material according to claim 1 or 2 or the bionic glycocalyx layer material prepared by the preparation method according to claim 3 or 4 in preparing products that inhibit immune response in organ transplantation.

7. The use according to claim 6, characterized in that The organ transplant includes a heart transplant.

8. The use according to claim 7, characterized in that The heart transplant includes xenogeneic heart transplant.

9. The use according to any one of claims 5 to 8, characterized in that: The products include pharmaceutical products.

10. A drug for protecting endothelial cells and / or inhibiting immune response in organ transplantation, characterized in that: The active ingredient of the medicine includes the bionic glycocalyx layer material according to claim 1 or 2 or the bionic glycocalyx layer material prepared by the preparation method according to claim 3 or 4.

Images