Polypeptide derivative for pancreas islet surface modification and inhibition of pancreas islet transplantation IBMIR reaction

Abstract

The invention relates to a polypeptide derivative for pancreas islet surface modification and inhibition of pancreas islet transplantation IBMIR reaction, and belongs to the technical field of biological medicine. The peptide derivative disclosed by the invention is obtained by carrying out an amide reaction on KF7 peptide and levodopa, and the amino acid sequence of the KF7 peptide is shown as SEQ ID NO: 1. The polypeptide derivative has good biocompatibility, the surface modification method is simple, the modification process has no influence on cell life activity, activation, aggregation, expansion and adhesion of platelets can be inhibited, the bleeding risk is reduced, a blood coagulation system is not influenced, pancreas islet intrahepatic colonization and pancreas islet functions after portal transplantation are enhanced, and the application prospect is wide. The composition can protect pancreas islet, prevent infiltration of platelets and CD11b + cells, inhibit pancreas islet transplantation IBMIR reaction, control blood sugar level, and prevent and treat diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a polypeptide derivative for islet surface modification and inhibition of the IBMIR reaction in islet transplantation. Background Art

[0002] Islet transplantation is a β-cell replacement technology that holds promise for curing type 1 diabetes. However, in clinical practice, sometimes a single recipient requires multiple islet transplants from multiple donors to achieve insulin independence, which greatly increases the shortage of pancreatic organ supply. A major reason for the overall inferior long-term effect of islet transplantation compared to pancreatic transplantation is the loss of islets caused by the instant blood-mediated inflammatory reaction (IBMIR). When islets come into direct contact with the recipient's blood after being injected into the portal vein, platelets are activated and attach to the islet surface. This attachment triggers the coagulation cascade reaction, complement attachment, and infiltration of inflammatory cells, resulting in the destruction of islets. Therefore, IBMIR exacerbates the early loss of transplanted islets. If IBMIR can be effectively controlled, the prognosis of islet transplantation will be significantly improved.

[0003] Islet surface encapsulation technology is a promising method for inhibiting IBMIR. Existing technologies include the following:

[0004] Alginate (ALG) is a widely studied material for microcapsule modification. Alginate has excellent properties such as stability, solubility, and adhesiveness, making it easy to form microcapsules on the islet surface and carry other drugs. In recent years, ALG and its polymers have shown good effects in encapsulating islets.

[0005] Polyethylene glycol (PEG) is also one of the preferred polymers for nanoparticle surface modification. PEG modification is also known as PEGylation of molecules. It can increase the stability of the modified substance and reduce its immunogenicity. Park et al. (Park H, Haque MR, Park JB, et al. Polymeric nano-shielded islets with heparin-polyethylene glycol in a non-human primate model. Biomaterials. 2018; 171: 164-77.) coated heparin on the islet surface with PEG, and the effect was better than that of systemic application of heparin alone.

[0006] The binding of streptavidin to biotin is highly specific and stable, and the binding force is the strongest non-covalent interaction in nature known so far. The interaction between biotin and avidin can be used to label or modify proteins. Cabric et al. (Cabric S, Sanchez J, Lundgren T, et al. Islet surface heparinization prevents the instant blood-mediated inflammatory reaction in islet transplantation. Diabetes. 2007;56(8):2008-15.) used the streptavidin-biotin ligation method to attach heparin to the surface of islets. The method of coating heparin with biotin is relatively simple, reducing the processing time and the risk of islet damage. Heparinization of the islet surface reduces portal vein thrombosis while avoiding the risk of bleeding caused by systemic heparin use, achieving good results.

[0007] At present, the clinical application of these research results on islet surface modification is not yet feasible, and the long-term effectiveness and safety of biomaterials need further observation and demonstration. Which biomaterials and drugs should be combined to establish a simple and effective islet transplantation method remains to be further studied in depth.

[0008] Moreover, since IBMIR can cause thrombosis, in order to inhibit IBMIR, anticoagulant drugs such as heparin are often systemically administered during islet transplantation. Although this method has certain effects, it also causes the risk of liver bleeding. Especially when islet transplantation requires percutaneous transhepatic puncture of the portal vein, after withdrawing the catheter for infusing islets, there is a channel between the portal vein and the abdominal cavity, and systemic heparinization at this time will bring a bleeding risk. In clinical islet transplantation, the bleeding risk is as high as 13.6% to 17.1%. In addition, even when heparin is used, portal vein thrombosis may still form, indicating that IBMIR still exists. Summary of the Invention

[0009] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a polypeptide derivative for islet surface modification and inhibition of the IBMIR reaction in islet transplantation.

[0010] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0011] In the first aspect, the present invention provides a polypeptide derivative, which is obtained by an amide reaction of KF7 peptide and levodopa, and the amino acid sequence of the KF7 peptide is as shown in SEQ ID NO: 1.

[0012] One of the main initiating factors for the occurrence of IBMIR is platelet activation. KEATSTF, a small peptide fragment KF7, is a newly discovered platelet inhibitor and has great potential for inhibiting IBMIR. During platelet activation, a 14-3-3ζ-c-Src-integrin-β3 complex is formed to complete the outside-in signal transduction and activate platelets. KF7 is the KEATSTF fragment on the intracellular tail of integrin β3, which can regulate and interfere with the formation of the 14-3-3ζ-c-Src-integrin-β3 complex and further inhibit platelet activation. This inhibition is specific and does not affect the binding of integrin β3 to fibrinogen, that is, fibrinogen can still play a role in hemostasis, which is also the reason why KF7 can specifically inhibit thrombus formation without causing obvious bleeding. Platelet activation plays a key role in inducing IBMIR. Therefore, the present invention enhances the engraftment of islets by inhibiting platelet activation with KF7.

[0013] L-DOPA (3,4-Dihydroxy-L-phenylalanine) is a new type of surface modification material, which contains catechol and amino groups and can adhere to any substrate through multiple covalent and non-covalent interactions. The dopamine derivative layer produced by L-DOPA surface modification has good hydrophilicity and biocompatibility, which can promote the interaction between the material and the organism and reduce the toxic and side effects of the material on the human body.

[0014] The present invention uses cell surface modification technology to perform a cell surface nano-coating on islets with the bioadhesive material L-DOPA. Utilizing the adhesion property of L-DOPA, simply suspending the islets in a culture medium containing L-DOPA enables L-DOPA to bind to the islet extracellular matrix. This technology is simple and easy to perform and does not affect the survival and function of islets. Therefore, modifying islets with L-DOPA loaded with KF7 can achieve the inhibition of IBMIR in islet transplantation while avoiding the complications caused by systemic drug administration.

[0015] Furthermore, the chemical structural formula of the polypeptide derivative is shown in Formula I:

[0016]

[0017] In a second aspect, the present invention provides the use of the polypeptide derivative in the preparation of a drug for inhibiting the immediate blood-mediated inflammatory reaction in islet transplantation.

[0018] The invention uses the platelet-specific drug KF7 peptide to modify islets, which can efficiently inhibit thrombus aggregation on the surface of islets and reduce IBMIR immune damage while avoiding the risk of systemic bleeding in the body, thereby achieving the effect of precisely protecting islets.

[0019] Furthermore, the drug also contains pharmaceutically acceptable additives and / or excipients and / or carriers.

[0020] In a third aspect, the present invention provides a drug for inhibiting the immediate blood-mediated inflammatory reaction in islet transplantation, and the drug contains the polypeptide derivative.

[0021] In a fourth aspect, the present invention provides a surface-modified islet cell, and the surface of the islet cell is modified with the polypeptide derivative.

[0022] Furthermore, the surface of the islet cell is modified with collagen, and the polypeptide derivative is modified on the surface of the islet cell through collagen.

[0023] In a fifth aspect, the present invention provides the use of the islet cell in the preparation of a product for preventing and treating diabetes.

[0024] In a sixth aspect, the present invention provides a method for surface-modifying the islet cell, including the following steps:

[0025] S1: Mix a collagen solution with islet cells and incubate for 30 - 40 min to obtain islet cells modified with collagen;

[0026] S2: Mix the islet cells modified with collagen in step S1 with the polypeptide derivative according to claim 1 or 2 and incubate for 30 - 40 min to obtain islet cells modified with collagen and the polypeptide derivative.

[0027] Furthermore, the incubation time in steps S1 and S2 is 30 min.

[0028] Furthermore, steps S1 and S2 are incubated under the conditions of 37°C and 5% v / v CO2.

[0029] Furthermore, the concentration of the collagen solution is 2 - 3 mg / mL, and the concentration of the polypeptide derivative is 2 - 3 mg / mL.

[0030] Furthermore, the concentration of the collagen solution is 2 mg / mL, and the concentration of the polypeptide derivative is 2 mg / mL.

[0031] Furthermore, after incubation in steps S1 and S2, wash with HBSS solution.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] (1) It has good biocompatibility: Levodopa itself is a neurotransmitter and has no additional drug toxicity and side effects.

[0034] (2) Simple surface modification method: The dopamine coating process is simple and mild. The entire surface modification process can be completed by simply co-incubating with islets at room temperature.

[0035] (3) The modification process has no impact on cell vital activities: Levodopa has the catecholamine functional group in the mussel-secreted adhesive protein structure. Under weakly alkaline conditions, it can be deposited on the surface of various materials through oxidative self-polymerization reaction to form a dopamine surface modification layer. It can further react with functional molecules containing amino, mercapto or carboxyl groups in the extracellular matrix through Michael addition or Schiff base reaction to achieve adsorption or fixation on the cell surface. It has no negative impact on the activity and function of islet cells. The coating thickness reaches the nanometer level, which does not affect the exchange of oxygen and nutrients, and at the same time can isolate the attack of immune factors.

[0036] (4) The polypeptide derivative of the present invention can inhibit the interaction of the 14-3-3ζ-c-Src-integrin-β3 complex, thereby inhibiting platelet activation, aggregation, spreading and adhesion, reducing the bleeding risk; the polypeptide derivative of the present invention does not affect the coagulation system, enhances the intrahepatic colonization of islets and islet function after portal transplantation, protects islets and prevents platelet and CD11b+ cell infiltration, inhibits the IBMIR reaction of islet transplantation, controls blood glucose levels, and prevents and treats diabetes. Brief Description of the Drawings

[0037] Figure 1 It is the technical flow chart of the present invention.

[0038] Figure 2 It is the surface modification mechanism of L-DOPA loaded with KF7. Among them, A is the chemical structural formula of KF7 and L-DOPA; B is the reaction of KF7 and L-DOPA to generate L-DOPA-KF7 and its chemical structural formula; C is the oxidation of the catechol functional group of L-DOPA to the quinone functional group under weak base; D is the repair of the damaged extracellular matrix by collagen; E is the covalent binding of the quinone functional group of L-DOPA to the amino group of collagen through Michael addition reaction; F is the overall process of surface modification of L-DOPA loaded with KF7 on islet cells.

[0039] Figure 3 It is the identification of L-DOPA-KF7 modified on the islet surface. Among them, A is the fluorescence confocal microscope showing that the islets are successfully modified with collagen and L-DOPA-KF7, scale bar: 50μm; B is the fluorescence density curve of the islets; C is the 3D fluorescence map of the islets, scale bar: 50μm.

[0040] Figure 4Characteristics of L-DOPA-KF7 surface modification on islets. Among them, A is the transmission electron microscopy image of L-DOPA-KF7 surface modification. The white arrow represents the collagen surface coating, and the black arrow represents the DOPA-KF7 surface coating. Scale bar: 500 nm; B is the AFM image of L-DOPA-KF7 surface modification. The coating thickness of the Collagen group is 13 nm, and the coating thickness of the Collagen-KF7 group is 53 nm.

[0041] Figure 5 Effect of surface modification with L-DOPA-KF7 on islet activity. Among them, A is the FDA / PI activity staining after islet surface modification. Scale bar: 100 μm (n = 5); B is the CCK8 experiment and detection of islet ATP content to measure cell activity after islet surface modification (n = 5); C is the TUNEL apoptosis experiment after islet surface modification. Scale bar: 50 μm (n = 5 sections / group); D is the expression of apoptosis genes Bax-1 and Caspase-3 after islet surface modification (n = 5).

[0042] Figure 6 Effect of surface modification with L-DOPA-KF7 on islet function. Among them, A is the glucose-stimulated insulin secretion experiment (n = 5); B is the survival rate of islets after surface modification and culturing for 12 h, 24 h, and 48 h respectively (n = 5); C is the co-culture of islets after surface modification with splenocytes. Scale bar: 100 μm (n = 5); D is the TF expression level after islet surface modification. There is no difference in the expression levels among groups (n = 5). *p < 0.05.

[0043] Figure 7 L-DOPA-KF7 inhibits platelet spreading and aggregation. Among them, A is the fluorescence image of L-DOPA-KF7 inhibiting platelet spreading. Scale bar: 50 μm (n = 5); B is the specific value of L-DOPA-KF7 inhibiting platelet spreading (n = 5); C is the inhibition of platelet aggregation by different concentrations of L-DOPA-KF7 (n = 5). Note: Platelets are stained red by phalloidin. ***p < 0.001.

[0044] Figure 8 L-DOPA-KF7 surface modification on islets inhibits platelet activation. Among them, A is the fluorescence image of L-DOPA-KF7 surface modification on islets inhibiting platelet adhesion to the islet surface. Scale bar: 50 μm (n = 5); B is the specific value of significantly reduced platelet adhesion to the L-DOPA-KF7-modified islet surface (n = 5). Note: Platelets are stained red by anti-CD62P antibody. ***p < 0.001.

[0045] Figure 9L-DOPA-KF7 modification on the surface of islets inhibits thrombosis. Among them, A shows that L-DOPA-KF7 modification on the surface of islets inhibits thrombosis on the surface of islets in the Tubing Loops model. Red (white arrow) represents islets, and gray represents thrombus. Scale bar: 500 μm (n = 5); B shows that L-DOPA-KF7 modification on the surface of islets significantly reduces platelet consumption in the Tubing Loops model (n = 5). ***p < 0.001.

[0046] Figure 10 DOPA-KF7 modification on the surface of islets reduces the bleeding risk without affecting the in vivo coagulation system. Among them, A is the mouse tail bleeding experiment (n = 5); B is the liver bleeding experiment (n = 5); C is the femoral artery thrombosis experiment (n = 5); D is the tail vein thrombosis experiment (n = 5). **p < 0.01.

[0047] Figure 11 L-DOPA-KF7 modification on the surface of islets inhibits the IBMIR reaction in portal transplantation. Among them, A is the liver of mice with portal islet transplantation. The white arrow indicates the site of thrombus occlusion. Scale bar: 1 cm (n = 5); B is the TAT level in the liver of mice 2 h after portal islet transplantation (n = 5); C is the C-peptide level in the peripheral blood of mice 2 h after portal islet transplantation (n = 5); D is the TNF-α expression level in the liver of mice 2 h after portal islet transplantation; E is the IL-1β expression level in the liver of mice 2 h after portal islet transplantation (n = 5); F is the IL-10 expression level in the liver of mice 2 h after portal islet transplantation; G is the IFN-γ expression level in the liver of mice 2 h after portal islet transplantation. There is no significant statistical difference between groups (n = 5). **p < 0.01, *p < 0.05.

[0048] Figure 12 L-DOPA-KF7 surface modification of islets enhances islet function after portal transplantation and prolongs graft survival. Among them, A is the blood glucose change of mice with portal islet transplantation in the Normal group at 60 days (n = 5); B is the blood glucose change of mice with portal islet transplantation in the Diabetic group at 60 days (n = 5); C is the blood glucose change of mice with portal islet transplantation in the Control group at 60 days (n = 5); D is the blood glucose change of mice with portal islet transplantation in the Collagen group at 60 days (n = 5); E is the blood glucose change of mice with portal islet transplantation in the Collagen-KF7 group at 60 days (n = 5); F is the body weight change of mice with portal islet transplantation in each group at 60 days (n = 8), p < 0.05; G is the survival curve analysis of mice with portal islet transplantation in each group at 60 days (n = 8), p < 0.05.

[0049] Figure 13This was the intraperitoneal glucose tolerance test (IPGTT) for mice with portal islet transplantation. Among them, A was the curve of blood glucose change over time after intraperitoneal injection of high glucose in mice with portal islet transplantation (n = 8); B was the comparison of the area under the blood glucose change curve after intraperitoneal injection of glucose in mice with portal islet transplantation (n = 8). **p < 0.01, *p < 0.05.

[0050] Figure 14 L-DOPA-KF7 surface modification protected islets and reduced platelet adhesion and infiltration. n = 5 sections / group, scale bar: 50 μm, ***p < 0.001.

[0051] Figure 15 L-DOPA-KF7 surface modification of islets inhibited platelet activation induced by the 14-3-3ζ-c-Src-integrin β3 complex, **p < 0.01, ***p < 0.001.

[0052] Figure 16 DOPA-KF7 surface modification of islets reduced CD11b+ cell infiltration after portal transplantation. n = 5 sections / group, scale bar: 50 μm, ***p < 0.001. Detailed implementation mode

[0053] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Other materials, reagents, etc. used in the embodiments can be obtained from commercial channels without special instructions.

[0054] Example 1 Surface modification process of L-DOPA loaded with KF7

[0055] I. Preparation of L-DOPA-KF7

[0056] Figure 1 This is the technical flow chart of the present invention. KF7 is a small molecule polypeptide with the amino acid sequence KEATSTF (SEQ ID NO: 1) and a carboxyl group at the end. The chemical structure of levodopa is C9H 11 NO4, which is composed of a carboxyl group connected to catecholamine (a benzene ring with two hydroxyl groups) via an amino group ( Figure 2 A). The amino group of levodopa and the carboxyl group of phenylalanine of KF7 were subjected to an amide reaction to prepare L-DOPA-KEATSTF, that is, L-DOPA-KF7 ( Figure 2 B), which was prepared using a twelve-channel semi-automatic polypeptide synthesizer. The method is as follows:

[0057] 1. Resin swelling: Put 2-Chlorotrityl Chloride Resin into a reaction tube, add 15 ml / g of N,N-dimethylformamide (DMF), and shake for 60 min. Add 0.5 - 1 mL of 15 ml / g of N,N-dimethylformamide per 0.1 g of 2-chlorotrityl chloride resin, which can ensure that there is enough solvent environment for the resin to swell fully and for subsequent reactions during the swelling process.

[0058] 2. Coupling the first amino acid: Filter out the solvent DMF in step 1 through a sintered glass filter, add 3-fold molar excess of Fmoc-protected amino acid (the first amino acid at the C-terminus, Fmoc-Lys(Boc)-OH, and the molar amount of Fmoc is 3 times the molar amount of the Lys it protects), then add 10-fold molar excess of N,N-diisopropylethylamine (DIEA, and the molar amount of DIEA is 10 times the molar amount of Lys). Add DMF to dissolve (the ratio of DMF to 2-chlorotrityl chloride resin is 5 - 10 mL:1 g), and shake for 30 min. Cap with methanol for 30 min to obtain the resin with the first amino acid coupled.

[0059] 3. Deprotection: Remove the DMF in step 2, add the deprotection reagent and react for 5 min, then remove the deprotection reagent, and add the deprotection reagent again and react for 15 min; the deprotection reagent is 20% (v / v) piperidine in DMF solution (DMF concentration is 15 ml / g) to obtain the resin with the first amino acid coupled after removing the Fmoc group.

[0060] 4. Detection: Remove the deprotection reagent in step 3, wash the resin with the first amino acid coupled after removing the Fmoc group three times with ethanol, add 2 - 3 drops of Kaiser reagent, and heat at 105 °C - 110 °C for 5 min. A deep blue color change indicates a positive reaction.

[0061] 5. Washing: Wash the resin with the first amino acid coupled after removing the Fmoc group that passed the detection in step 4 twice with DMF (10 ml / g), twice with methanol (10 ml / g), and twice with DMF (10 ml / g) to obtain the washed resin with the first amino acid coupled after removing the Fmoc group.

[0062] 6. Condensation: Add 3-fold molar excess of Fmoc-protected amino acid (Fmoc-Glu(Otbu)-OH), 3-fold molar excess of peptide coupling reagent (HBTU), then add 10-fold molar excess of DIEA, and finally add DMF to dissolve, and shake and react for 45 min to obtain the resin with the protected dipeptide. Then, perform detection and washing according to the methods in steps 4 and 5 to obtain the washed resin with the deprotected dipeptide.

[0063] 7. Repeat the operations in 3 - 6, replace the types of corresponding protected amino acid raw materials according to the amino acid sequence of the following L-DOPA-KF7 polypeptide sequence, and synthesize from the C-terminus to the N-terminus of the polypeptide in turn until the synthesis is completed. Until the last amino acid Fmoc protecting group is removed to obtain peptide resin; the protected amino acid raw materials used include Fmoc-Ala-OH, Fmoc-Thr(tbu)-OH, Fmoc-Ser(tbu)-OH, Fmoc-Thr(tbu)-OH, Fmoc-Phe-OH and Fmoc-DOPA(acetonide)-OH.

[0064] 8. Wash the peptide resin in step 7 twice with DMF (10 ml / g), three times with dichloromethane (DCM, 10 ml / g), and four times with methanol (10 ml / g), and drain for 10 min to obtain the washed peptide resin.

[0065] 9. Cleavage: Cleave the washed peptide resin in step 8 with the cleavage solution for 180 min to obtain cleaved L-DOPA-KF7; the cleavage solution consists of 95% (v / v) trifluoroacetic acid (TFA), 2% (v / v) water, 2% (v / v) 1,2-ethanedithiol (EDT) and 1% (v / v) triisopropylsilane (TIS).

[0066] 10. Blow-dry and wash: Blow-dry the cleaved L-DOPA-KF7 with nitrogen, precipitate with ether, centrifuge to remove the supernatant, wash the precipitate six times with ether, and then volatilize at room temperature to obtain the crude L-DOPA-KF7.

[0067] 11. Purification and preparation: For the crude product, dissolve the crude L-DOPA-KF7 in step 10 with an aqueous acetonitrile solution (H2O / ACN), and analyze on an HPLC analyzer to determine the elution time corresponding to the target peak;

[0068] Use a C18 reversed-phase chromatography preparation system, wavelength 220 nm, flow rate 15 ml / min, injection volume 20 mL, column temperature 25°C; mobile phase A is 0.1% (v / v) aqueous TFA solution, and mobile phase B is 0.1% (v / v) TFA acetonitrile solution; collect the target peak solution, and take the target peak solution for mass spectrometry confirmation and purity detection.

[0069] 12. Lyophilize the qualified target peak solution to obtain purified L-DOPA-KF7. After performing molecular weight identification by MS and purity identification by HPLC analysis, seal and package it, and store it at -20°C.

[0070] II. The catechol functional group of levodopa can be oxidized to a quinone functional group under weak base conditions ( Figure 2C). The collagen in the extracellular matrix is rich in amino groups, and levodopa can be covalently bound to the quinone functional group through a Michael addition reaction, so as to realize the loading of dopamine materials with drugs on the surface of islet cells. Since the extracellular matrix is damaged by collagenase oscillation digestion during the islet isolation process, in the present invention, collagen is used to repair the extracellular matrix, and exogenous collagen can provide more abundant amino groups for surface modification of islet cells ( Figure 2 D - F).

[0071] The method for modifying collagen and L - DOPA - KF7 is as follows:

[0072] 1. Washing: Wash the isolated islets 2 times with HBSS solution (Hank's Balanced Salt Solution) to obtain clean islets.

[0073] 2. Modifying collagen: Add 2 mg / mL collagen solution to the washed - clean islets, incubate at 37 °C and 5% (v / v) CO₂ for 30 min to make the collagen adhere to the surface of the islets, and obtain the islets incubated with collagen; wash the islets incubated with collagen 3 times with HBSS solution to elute the unbound collagen, and obtain the islets modified with collagen.

[0074] 3. Modifying L - DOPA - KF7: Add 2 mg / mL L - DOPA - KF7 solution to the islets modified with collagen, incubate at 37 °C and 5% (v / v) CO₂ for 30 min to fix collagen and L - DOPA - KF7 on the surface of islet cells, and obtain the islets incubated with collagen and L - DOPA - KF7; wash the islets incubated with collagen and L - DOPA - KF7 3 times with HBSS solution to remove the unbound L - DOPA - KF7, and obtain the islets modified with collagen and L - DOPA - KF7.

[0075] Example 2 Identification of L - DOPA - KF7 Modified on the Surface of Islets

[0076] In order to identify whether L - DOPA - KF7 is successfully modified on the surface of islets, in the present invention, fluorescein isothiocyanate (FITC) is used to fluorescently label collagen, and rhodamine is used to fluorescently label L - DOPA - KF7. Then, according to the method of Example 1, the islets modified with collagen and the islets modified with collagen and L - DOPA - KF7 are obtained, and the fluorescence intensity on the surface of the islets is evaluated with a fluorescence confocal laser scanning microscope.

[0077] As Figure 3 shown in A, the islets are successfully modified with a layer of FITC - labeled collagen (red) and a layer of rhodamine - labeled L - DOPA - KF7 (yellow).

[0078] The fluorescence density curve shows that the fluorescence signal is strong at both ends and weak in the middle, indicating that the modified material is mainly concentrated on the surface of the islets and less penetrates into the interior of the islets( Figure 3 B).

[0079] The 3D fluorescence image of the islets shows that collagen and L-DOPA-KF7 are evenly distributed and the entire surface of the islets is completely covered( Figure 3 C).

[0080] This indicates that L-DOPA loaded with KF7 has been successfully modified on the surface of islet cells.

[0081] Example 3 Characteristics of L-DOPA-KF7 Modified on the Surface of Islets

[0082] 1. Examine unmodified islets, the Collagen group (islets modified with collagen), and the Collagen-KF7 group (islets modified with collagen and L-DOPA-KF7) using a transmission electron microscope.

[0083] Compared with unmodified islets, there is a layer of coating on the surface of islets in the Collagen group, and there are two layers of coating on the surface of islets in the Collagen-KF7 group. The white arrow indicates the collagen coating, and the black arrow indicates the L-DOPA-KF7 coating( Figure 4 A).

[0084] 2. To further evaluate the physical characteristics of the surface modification, coat collagen on a glass plate to simulate the extracellular matrix of islets, then modify L-DOPA-KF7 on the collagen matrix, and evaluate the surface characteristics using an atomic force microscope AFM.

[0085] The results show that the surface coating thickness of the collagen-coated glass plate is about 10 ± 15 nm, while the average surface coating of the glass plate with collagen and L-DOPA-KF7 is 50 ± 18 nm( Figure 4 B). The surface modification does not significantly change the size and volume of the islets. Therefore, in islets modified with collagen and L-DOPA-KF7, the diffusion and transport of nutrients and metabolites will not be disturbed because the average range of the free diffusion distance of particles can be within 200 μm.

[0086] Example 4 Effects of Surface Modification with L-DOPA-KF7 on Islet Activity

[0087] Considering that the L-DOPA-KF7 modification on the cell surface may cause chemical or physical damage to the islets, the present invention evaluates the potential effects of the coating process on islet viability through FDA / PI live-dead cell staining, CCK8 assay, TUNEL apoptosis staining, and QPCR assay.

[0088] The islets coated with L-DOPA-KF7 showed no obvious difference in macroscopic appearance from the unmodified islets.

[0089] The results of FDA / PI live-dead cell staining showed that most of the islet cells in the Collagen group and the Collagen-KF7 group had high activity and showed strong green fluorescence staining; on the contrary, almost no cells stained red fluorescence (dead cells) were observed ( Figure 5 A).

[0090] The cell survival rates measured by the CCK8 assay were 100±19.1%, 92.9±12.2% and 100±28.0% in the Control group, the Collagen group and the Collagen-KF7 group respectively ( Figure 5 B), and there was no significant difference among the groups (p>0.05). In addition, the difference in the content of ATP in these three groups of islets (p>0.05) was also not significant ( Figure 5 B).

[0091] Similarly, few TUNEL-positive apoptotic stained cells were observed in all groups, and the difference was not statistically significant (p>0.05) ( Figure 5 C).

[0092] In this invention, QPCR verification was also carried out. The expression levels of apoptosis-related genes Bax-1 and Caspase-3 showed no significant difference in the above three groups of islets, indicating that surface modification would not cause apoptosis of islets ( Figure 5 D).

[0093] Therefore, these results indicate that surface modification with collagen and L-DOPA-KF7 has no toxic effect on islets and does not affect the viability of islet cells.

[0094] Example 5 Effect of Surface Modification with L-DOPA-KF7 on Islet Function

[0095] 1. To further explore the effect of surface modification on islet function, this invention conducted a glucose-stimulated insulin secretion (GSIS) experiment to monitor the ability of islets to stimulate the release of insulin in response to changes in glucose levels.

[0096] (1) Pre-incubate the islets of each group in a serum solution containing 1.67 mM glucose. The serum solution is a Krebs-Ringer solution with 0.5% (w / v) bovine serum albumin (BSA), and incubate at room temperature for 1 h for rewarming. There are 50 islets in each group to obtain pre-incubated islets.

[0097] (2) Incubate the pre-incubated islets in a serum solution with low-concentration (1.67 mM) glucose for 1 h, and then continue to incubate in a serum solution with high-concentration (16.7 mM) glucose for 1 h.

[0098] (3) Collect the supernatant after incubation in step (2) and store it in an environment at -80 °C, and detect the insulin concentration by ELISA.

[0099] (4) Collect the islets in each group in step (2), detect the DNA content, and quantify the secreted insulin by normalization.

[0100] (5) The sugar stimulation index (SI) value is obtained by dividing the amount of insulin secreted at high glucose levels by the amount of insulin at low glucose levels.

[0101] The results showed that there were no significant changes in the insulin levels released by the modified or unmodified islets under the stimulation of low glucose (1.67 mM) and high glucose (16.7 mM) respectively ( Figure 6 A). The SI values of the Control group, Collagen group, and Collagen-KF7 group were 1.95 ± 0.07, 1.96 ± 0.25, and 1.91 ± 0.10 respectively, and there was no statistical difference among the groups (p > 0.05). Therefore, surface modification with L-DOPA-KF7 will not have a harmful effect on the insulin-secreting function of islets.

[0102] 2. To evaluate the effect of L-DOPA-KF7-coated islets on the viability of islets during in vitro culture, after surface-modifying L-DOPA-KF7 on the islets modified with collagen according to the method of Example 1, continue to incubate for 12 h, 24 h, and 48 h, and observe the survival of the islets. The results showed that the islets in each group still had good survival rates ( Figure 6 B), and the loss rates of the islets in each group were small and there was no significant difference (p > 0.05).

[0103] 3. To study the immune isolation and protection effect of the surface-modified nanolayer, the present invention carried out an in vitro co-culture experiment of the surface-modified islets with splenocytes of syngeneic mice.

[0104] (1) Decapitate the mice and take out the spleens. Place the spleens in ice-cold physiological saline for washing to ensure the freshness of the tissues.

[0105] (2) Place a 70-μm cell sieve in a 50-mL centrifuge tube, place the spleen on the cell sieve, cut the spleen into pieces with scissors, grind it with the piston of a 10-mL syringe, and add RPMI-1640 medium for rinsing while grinding, and process 10 mL to obtain the sieved product.

[0106] (3) Take the sieved product of step (2) and centrifuge it at 300g for 5 min, discard the supernatant, add 2-3 mL of freshly prepared 1× red blood cell lysis buffer, gently pipette to mix, lyse at room temperature for 2 min, quickly add 10 mL of phosphate buffered saline (PBS) to terminate the lysis, centrifuge at 300g for 5 min, discard the supernatant, obtain spleen cells, resuspend the spleen cells in complete culture medium, count and retain them.

[0107] (4) 1.0×10 5 The spleen cells were co-cultured with the pancreatic islets in each group at 37°C and 5% (v / v) CO2 for 24 h, with 50 pancreatic islets in each group. After the co-culture, the activity of the pancreatic islet cells was detected.

[0108] After 24 hours of co-culture, live / dead staining showed that the number of pancreatic islet death cells in the Collagen-KF7 group was significantly less than that in the Collagen group and the Control group (p<0.05), and the surface-modified islets still maintained a good survival state. Therefore, surface modification of islets with L-DOPA-KF7 can effectively inhibit the destruction of islets by spleen cells during co-culture ( Figure 6 C).

[0109] 4. QPCR was used to evaluate the expression level of tissue factor (TF) in each group of islets. Modification of L-DOPA-KF7 on the surface of islets did not significantly increase the expression level of tissue factor in islets (p>0.05)( Figure 6 D) On the contrary, it can isolate TF from the contact with blood, and TF is also one of the main factors initiating IBMIR. Isolating TF plays a role in inhibiting the activation of the coagulation system.

[0110] Example 6: Islet surface modification with L-DOPA-KF7 inhibits platelet activation

[0111] KF7 can inhibit the interaction of 14-3-3ζ-c-Src-integrin-β3 complexes to inhibit platelet activation, but it is unclear whether KF7 modified with L-DOPA can maintain its function. Platelet activation includes platelet aggregation and expansion, so the present invention detects the effect of L-DOPA-KF7 on platelet aggregation and expansion.

[0112] 1. Platelet aggregation test

[0113] (1) Blood collection: Select 7-8 week old C57BL / 6 male mice, remove the eyeballs to collect blood, slowly drip the blood into a blood collection tube containing sodium citrate, and mix thoroughly to obtain a blood sodium citrate mixture. Then add an equal volume of benchtop buffer to the blood sodium citrate mixture, mix thoroughly, add prostaglandin E1 (PGE1) solution to make the final concentration of PGE1 to 50 ng / mL, and mix gently.

[0114] (2) Primary centrifugation: Centrifuge the blood processed in step (1) at 200×g for 16 min at 4°C, aspirate the upper pale white liquid, add PGE1 solution, and the final concentration of PGE1 is 50 ng / mL, and gently mix well.

[0115] (3) Obtain platelet-rich plasma: Centrifuge the sample processed in step (2) at 800×g for 10 min at 4°C, discard the supernatant, add 2 mL of table buffer, 8 μL of ethylenediaminetetraacetic acid (EDTA) solution and 2 μL of PGE1 solution, so that the final concentration of EDTA is 1 mM and the final concentration of PGE1 is 50 ng / mL, and gently mix well.

[0116] (4) Centrifuge the platelet-rich plasma obtained in step (3) at 750×g for 10 min at room temperature, aspirate and discard the supernatant, retain the precipitate, add table buffer, and gently mix well to resuspend the platelets to obtain washed platelets.

[0117] (5) Counting: Count the washed platelets using a hemocytometer, and adjust the platelet concentration to 3×10 8 cells / mL with table buffer to obtain a platelet diluent. Wait for the platelets to rewarm at room temperature for 30 min, and start the platelet adhesion experiment.

[0118] (6) Incubate the platelet diluent at 37°C for 5 min, add 0.03 U / mL thrombin as an agonist to induce platelet aggregation, and measure it in a platelet aggregometer, and perform professional data analysis using Aggrolink8 software.

[0119] 2. Platelet spreading experiment

[0120] (1) Coating with fibrinogen: Take a confocal glass dish, add 250 μL of fibrinogen solution pre-cooled at 4°C and incubate overnight. Aspirate and discard the fibrinogen solution, and wash it 3 times with 4°C PBS to wash away the residual unadhered fibrinogen to obtain a confocal glass dish coated with fibrinogen.

[0121] (2) Blocking: Add 250 μL of 1% (w / v) BSA to the confocal glass dish coated with fibrinogen, block it at room temperature for 1 h, and aspirate it after completion.

[0122] (3) Incubating platelets: Add 250 μL of platelet diluent with a density of 2×10 7 cells / mL to each well, add 2 mg / mL of L-DOPA-KF7, KF7 or the control reagent dimethyl sulfoxide (DMSO), and incubate at 37°C for 1 h. After the incubation, aspirate and discard the supernatant, and wash it 3 times with PBS.

[0123] (4) Fixation: Add 250 μL of 4% (w / v) paraformaldehyde solution to the platelets processed in step (3) and fix for 15 min. After the incubation ends, aspirate and wash 3 times with PBS.

[0124] (5) Permeabilization: Add 250 μL of 0.1% Triton X-100 to the platelets processed in step (4) to permeabilize the membrane for 5 min. After the incubation ends, aspirate and wash 3 times with PBS.

[0125] (6) Staining: Add FITC-labeled phalloidin (final concentration of 1 μg / mL) to the platelets processed in step (5), and incubate at 37 °C in the dark for 45 min. After the incubation ends, aspirate and wash 3 times with PBS.

[0126] (7) Observation: Observe the spreading morphology of the platelets after the treatment in step (6) using a confocal fluorescence microscope, save the pictures, and analyze the experimental results using Image J.

[0127] As Figure 7 shown in A, compared with the control group, L-DOPA-KF7 significantly inhibited the spreading of platelets on the fixed fibrinogen glass slide. The spreading area of platelets in the L-DOPA-KF7 group was significantly reduced (p < 0.001), while there was no significant difference compared with the KF7 group of unmodified L-DOPA ( Figure 7 B).

[0128] Meanwhile, L-DOPA-KF7 significantly inhibited the platelet aggregation induced by 0.03 U / mL thrombin, and the inhibitory effect became more obvious with the increase in concentration ( Figure 7 C).

[0129] These results indicate that the L-DOPA modification does not affect the biological activity of KF7. This may be because only the carboxyl group in KF7 undergoes an amide reaction with the amino group of L-DOPA, and its main active groups are not affected.

[0130] 3. To simulate the platelet adhesion process occurring on the extracellular matrix collagen fiber capsule outside the islets in vitro, the islets of each group were co-cultured with platelets at 37 °C and 5% (v / v) CO2 for 60 min. After the co-culture ended, observe the number of platelets adhering to the surface of the islets ( Figure 8 A). Compared with the Collagen group and the Control group, the number of platelets adhering to the surface of the islets in the Collagen-KF7 group was significantly reduced (p < 0.001). The surface modification of L-DOPA-KF7 could effectively inhibit the adhesion of platelets to the surface of the islets ( Figure 8 B).

[0131] 4. Currently, the technical methods for clinically studying the actual situation of IBMIR reaction after portal vein transplanted islets enter the blood are limited. Therefore, the present invention adopts an improved in vitro Tubing Loops model. In a flowing in vitro circulation model, based on the interaction between islets and blood, the IBMIR phenomenon observed after islets enter the blood in vivo is simulated.

[0132] The islets of each group were co-incubated with platelet diluent (3×10 8 cells / mL) at 37°C and 5% (v / v) CO2 for 1 h to allow platelets and islets to fully contact and interact, obtaining islets co-incubated with platelets. The islets co-incubated with platelets were gently washed with PBS to remove non-adherent platelets and other impurities.

[0133] The results showed that the islets in the Control group and the Collagen group were surrounded by thrombi, while the islets with L-DOPA-KF7 coating were relatively intact and had no thrombus infiltration ( Figure 9 A). In addition, the platelet counts in the Control group and the Collagen group decreased significantly, while the decrease in platelet count in the Collagen-KF7 group was significantly reduced (p<0.001) ( Figure 9 B).

[0134] The above results indicate that L-DOPA-KF7 inhibits platelet activation and consumption, protecting islets from IBMIR-mediated damage.

[0135] Example 7 Modification of islet surface with L-DOPA-KF7 reduces the bleeding risk and does not affect the coagulation system

[0136] KF7 is an antiplatelet drug, and previous studies have confirmed its excellent antithrombotic ability and no bleeding risk. To study the potential bleeding risk and antithrombotic ability of L-DOPA-KF7 in vivo, the present invention divided diabetic mice into a Control group, a systemic administration group (System administration, SA-KF7 group), and a surface modification group (Surfacemodification, SM-KF7 group). 2 h before portal vein islet transplantation, the mice in the SA-KF7 group were orally administered with L-DOPA-KF7 solution, the Control group was orally administered with DMSO, while the SM-KF7 group only received portal vein infusion of islets surface-modified with L-DOPA-KF7. Each mouse was transplanted or orally administered with 300 islets.

[0137] Diabetic mice were fasted for 6 - 8 hours before portal islet transplantation, and then anesthetized with isoflurane. The mice were fixed and the abdominal hair was shaved. On the sterilized surgical field, the abdominal skin was incised along the linea alba with scissors. Under a stereomicroscope, forceps and sterile cotton swabs were used in cooperation to expose the cecum and display the cecal vein. The intestinal tract was moistened with normal saline. The islets in each group were resuspended again. The needle was inserted into the cecal vein along the direction of blood flow, and the islet suspension was slowly injected, allowing it to slowly return to the portal vein along with the cecal vein. The infusion time was about 15 seconds, and the liquid volume was 0.2 mL. After injection, the needle was withdrawn, and the puncture site was compressed with gelatin sponge for 2 minutes. After confirming no bleeding, the intestinal tract was pushed back into the abdominal cavity, and 0.5 mL of normal saline was injected for fluid replacement. The abdominal cavity was then closed.

[0138] There was no significant difference in the tail vein bleeding time of the SM-KF7 group (453.7 ± 43.8 s) compared with the Control group (463.6 ± 40.4 s) and the SA-KF7 group (473.0 ± 73.9 s) (p > 0.05)( Figure 10 A).

[0139] The present invention further explored the effect of L-DOPA-KF7 on liver bleeding. There was no statistical difference in the liver bleeding time of the SM-KF7 group (177.5 ± 26.0 s) compared with the Control group (169.0 ± 16.1 s) and the SA-KF7 group (168.0 ± 27.8 s) (p > 0.05)( Figure 10 B).

[0140] The above results indicate that neither local modification nor systemic administration of L-DOPA-KF7 will increase the bleeding risk of the body.

[0141] The femoral artery thrombosis formation experiment showed that systemic administration of L-DOPA-KF7 (100 mg / kg) significantly restored FeCl3-induced femoral artery blood flow occlusion, while the SM-KF7 group (69.8 ± 5.6%) was not affected (p < 0.01)( Figure 10 C). In the tail vein thrombosis formation experiment, systemic administration of L-DOPA-KF7 significantly reduced the tail vein thrombosis formation (1.8 ± 0.2 cm) caused by intraperitoneal injection of carrageenan. However, compared with the Control group (3.0 ± 0.4 cm), the thrombus length of the SM-KF7 group (2.7 ± 0.3 cm) was not significantly reduced (p < 0.01)( Figure 10D). These results indicate that systemic administration of the L-DOPA-KF7 system can significantly inhibit in vivo thrombus formation. However, the drug with an L-DOPA-KF7 coating on the surface of the islets only acts locally on the surface of the islets and has no effect on the coagulation system of the whole body of the mice. That is, if it is applied in clinical islet transplantation, thrombi can still form in the portal puncture channel to prevent bleeding. Therefore, compared with systemic administration, this new targeted delivery system has incomparable advantages because it can directly act on the islets with a therapeutic dose of the drug, improve the drug delivery effect, and avoid the disadvantages of systemic administration.

[0142] Example 8 Modification of the surface of islets with L-DOPA-KF7 inhibits the IBMIR response in portal vein transplantation

[0143] 1. The L-DOPA-KF7 surface-modified islets were subjected to portal vein transplantation to evaluate their potential to inhibit the IBMIR response. After 2 h, visible white spots, namely embolism ischemic foci, were found at the edges of the livers in the Control group and the Collagen group, while fewer thrombus embolism foci were present in the livers receiving L-DOPA-KF7 islet transplantation ( Figure 11 A). Corresponding to this result, the TAT levels in the Control group and the Collagen group were significantly increased (p < 0.01), indicating activation of the coagulation system ( Figure 11 B), with thrombus formation. At the same time, the C-peptide levels in the Control group and the Collagen group were also significantly increased (p < 0.05). Considering that the increase in C-peptide 2 h after the operation was the result of leakage from damaged islets, it is reasonable to believe that the IBMIR response damaged the islets. In contrast, the TAT and C-peptide levels in the Collagen-KF7 group were significantly decreased, indicating inhibition of the IBMIR response ( Figure 11 C), with less islet damage.

[0144] 2. The present invention further randomly detected the levels of pro-inflammatory factors in the recipient livers 2 h after islet transplantation. Compared with the Collagen-KF7 group, the levels of TNF-α (p < 0.05) and IL-1β (p < 0.05) were significantly increased in the Control group and the Collagen group after portal vein islet transplantation, and the increase in these pro-inflammatory factors was related to early islet loss ( Figure 11 D and Figure 11 E). The present study also detected the release of cytokines IFN-γ and IL-10, but no significant differences were observed among the groups (p > 0.05) ( Figure 11 F and Figure 11 G). These results indicate that L-DOPA-KF7 surface-modified islets reduce IBMIR by reducing the production of inflammatory factors, thereby enhancing islet engraftment and maintaining function.

[0145] Example 9: L-DOPA-KF7 Engineered Islets Enhance Intrahepatic Engraftment and Islet Function after Portal Transplantation

[0146] To evaluate the effect of surface modification with L-DOPA-KF7 as an IBMIR inhibitor on the long-term function of islet transplantation, the body weights and NBG (2-NBD-glucose, a fluorescent-labeled deoxyglucose analog) levels of the mice in each group treated in Example 7 were monitored.

[0147] Figure 12 A-E show the changes in NBG levels of recipient mice. During the observation period, the blood glucose levels of the mice in the normal group were well controlled, and the blood glucose levels of all the mice (100%) in the Collagen-KF7 group also returned to normal within a few days after transplantation. Only 3 out of 8 mice (37.5%) in the control group had normal blood glucose. As a isotype control, 2 out of 8 mice (25%) in the Collagen group had normal blood glucose. Therefore, the diabetes reversal rate in the Collagen-KF7 group was significantly higher than that in the Control group and the Collagen group (p>0.05). The blood glucose control levels of all the mice with islet transplantation were better than those of the diabetic mice, indicating that islet transplantation has the effect of improving blood glucose.

[0148] In addition, the average body weight of the mice with islet transplantation in the Collagen-KF7 group continuously increased after transplantation and finally became comparable to that of the mice in the Normal group. However, the average body weights of the mice in the Diabetic group, the Control group, and the Collagen group gradually decreased, which was the result of poor blood glucose control ( Figure 12 F). The mice in the Normal group and the Collagen-KF7 group survived healthily during the experiment. 2 out of 8 mice (25%) in the Control group died, and 3 out of 8 mice (37.5%) in the Collagen group died. All the mice in the Diabetic group failed to survive at the end of the experiment. The survival status corresponded to the blood glucose control level, showing the harm of diabetes to the body. The survival times of the mice in the Normal group and the Collagen-KF7 group were significantly higher than those in the Control group, the Collagen group, and the Diabetic group (p<0.05)( Figure 12 G).

[0149] To evaluate the reactivity of the recipients of L-DOPA-KF7 islet transplantation to hyperglycemia, an intraperitoneal glucose tolerance test was performed on the mice in each group 20 days after transplantation. The mice were fasted overnight, and a glucose solution (2.0 g / kg) was injected into the peritoneal cavities of the mice in each group. Blood glucose levels were measured by collecting blood from the tail vein at 0, 30, 60, 90, and 120 min.

[0150] For the mice in the Normal group and the Collagen-KF7 group, the blood glucose level increased 15 min after glucose injection and then rapidly decreased to the normal level within 120 min, indicating a complete response to the increase in blood glucose. There was no significant difference in the area under the glucose-stimulated curve between the two groups (p>0.05). Only a partial response to IPGTT was observed in the Control group and the Collagen group. The mice in the Diabetic group showed no response to IPGTT ( Figure 13 A), and the area under the glucose-stimulated curve in the Collagen-KF7 group was significantly lower than that in the Control group and the Collagen group (p<0.05) ( Figure 13 B). These results indicate that the islets surface-modified with L-DOPA-KF7 can be protected from IBMIR damage and can successfully colonize in the liver to play a role in regulating blood glucose.

[0151] Example 10 L-DOPA-KF7 protects islets and prevents platelet and CD11b+ cell infiltration

[0152] To confirm the successful colonization of the islets in the Collagen-KF7 group in the mouse liver, the liver containing the islets was harvested 1 day after transplantation for histological analysis. The morphology of the transplanted islets in the Collagen-KF7 group was relatively intact, and the fluorescence intensity of Insulin staining was high. In contrast, the morphology of the islets in the Control group and the Collagen group was less intact, and a large number of platelets infiltrated near the damaged islets ( Figure 14 ). In contrast, fewer CD62P-positive platelet infiltrations were detected near the transplantation site of the islets surface-modified with L-DOPA-KF7 (p<0.001).

[0153] To explore the mechanism by which L-DOPA-KF7 surface modification reduces platelet infiltration, the present invention detected the complex 14-3-3ζ-c-Src-β3 that regulates platelet outside-in signaling. The expression of this complex can cause platelet activation and thus trigger the IBMIR reaction. Compared with the Control group and the Collagen group, the overall expression levels of 14-3-3ζ (p<0.001), c-Src (p<0.001), and integrin β3 (p<0.01) in the Collagen-KF7 group were decreased ( Figure 15 ). These results confirm that L-DOPA-KF7 acts as an inhibitor of the 14-3-3ζ-c-Src-β3 complex, and the surface modification with L-DOPA-KF7 plays an anti-IBMIR reaction role by inhibiting platelet activation.

[0154] CD11b+ cells are one of the earliest inflammatory cells that infiltrate and damage pancreatic islets in islet transplantation. Therefore, in this invention, CD11b antibody is used to evaluate the infiltration of inflammatory cells around islet grafts. A large number of CD11b+ cells infiltrated in the Control group and the Collagen group, while surface modification in the Collagen-KF7 group could significantly reduce the infiltration of CD11b+ cells( Figure 16 ). These results indicate that L-DOPA-KF7 not only inhibits platelet activation, but also can significantly reduce the infiltration of inflammatory cells around pancreatic islets (p<0.001). Due to these protective factors, islets modified with L-DOPA-KF7 were well engrafted and survived in the liver after transplantation.

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A polypeptide derivative, characterized in that, The polypeptide derivative is obtained by an amide reaction of KF7 peptide and levodopa, and the amino acid sequence of the KF7 peptide is as shown in SEQ ID NO:

1.

2. The polypeptide derivative according to claim 1, wherein The chemical structural formula of the polypeptide derivative is as shown in Formula I:

3. Use of the polypeptide derivative according to claim 1 or 2 in the preparation of a drug for inhibiting the blood-mediated inflammatory reaction immediately after islet transplantation.

4. The application according to claim 3, wherein The drug further contains a pharmaceutically acceptable additive or / and excipient or / and carrier.

5. A drug for suppressing the immediate blood-mediated inflammatory reaction in islet transplantation, characterized in that, The drug contains the polypeptide derivative according to claim 1 or 2.

6. A surface-modified islet cell, characterized in that, The surface of the islet cells is modified with the polypeptide derivative according to claim 1 or 2.

7. The pancreatic islet cells according to claim 6, characterized in that, The surface of the islet cells is modified with collagen, and the polypeptide derivative is modified on the surface of the islet cells through collagen.

8. Use of the islet cells according to claim 6 or 7 in the preparation of a product for preventing and treating diabetes.

9. The method for surface modification of pancreatic islet cells according to claim 6 or 7, characterized in that, Comprising the following steps: S1: Mix the collagen solution with islet cells and incubate for 30-40 min to obtain islet cells modified with collagen; S2: Mix the islet cells modified with collagen in step S1 with the polypeptide derivative according to claim 1 or 2 and incubate for 30-40 min to obtain islet cells modified with collagen and the polypeptide derivative.

10. The method according to claim 9, wherein The concentration of the collagen solution is 2-3 mg / mL, and the concentration of the polypeptide derivative is 2-3 mg / mL.

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