PEG-lipids

Abstract

PEG-lipids are produced by mixing a cationic PEG-lipid containing at least one amino group with a sulfated glycosaminoglycan containing at least one carbonyl group to form a Schiff base intermediate. A reducing agent is added to the Schiff base intermediate to form a sulfated glycosaminoglycan-PEG-lipid. The sulfated glycosaminoglycan-PEG-lipid can be used in biological tissues to combat thrombotic inflammation. Coating biological tissues with the sulfated glycosaminoglycan-PEG-lipid can be completed in a single step and does not induce any significant cell aggregation.

Description

Technical Field

[0001] This invention generally relates to poly(ethylene glycol) (PEG) lipids, particularly such PEG-lipids comprising sulfated glycosaminoglycans, and their production and medical uses. Background Technology

[0002] Although no serious side effects have been reported after cell transplantation of, for example, pancreatic islets, mesenchymal stem cells (MSCs), or hepatocytes, the biocompatibility of these therapeutic cells remains unresolved. Infusion of therapeutic cells into the human body is associated with significant loss of transplanted cells due to an immune response known as thrombotic inflammation, or immediate blood-mediated inflammation (IBMIR). Thrombotic inflammation, or IBMIR, is an innate immune attack triggered by the complement and coagulation systems, followed by rapid platelet binding and leukocyte infiltration into the clot, leading to early loss of transplanted cells. Additionally, thrombotic inflammation occurs in ischemia-reperfusion injury (IRI) in solid organ transplants such as kidney and heart transplants. This devastating response damages post-transplant tissues and organs, thereby reducing graft survival.

[0003] Therefore, in order to achieve successful treatment and high-level implantation of therapeutic cells and solid organs, it is crucial to protect the cell surface from this thrombotic inflammatory attack.

[0004] Some studies have shown that thrombotic inflammation can be modulated by systemic administration of anticoagulants (such as thrombin inhibitors, melagatum, low molecular weight dextran sulfate, and / or complement inhibitors) to prevent early adverse reactions. However, some of these techniques are difficult to apply in clinical settings due to the associated increased risk of bleeding.

[0005] Heparan sulfate is expressed on the surface of endothelial cells and plays an important role in regulating coagulation, complement, and platelet activation. Therefore, surface modification with heparin and heparin conjugates to mimic the endothelial surface has been suggested as a method for regulating thrombotic inflammation occurring in cell and organ transplantation [1-3]. However, surface modification with heparin and heparin conjugates requires several process steps: chemical modification of the cell surface and reaction with heparin, each step followed by a washing process. Another problem associated with surface modification with heparin and heparin conjugates is cell aggregation following the reaction with heparin. Heparin molecules also crosslink between cells, leading to cell clumping.

[0006] Therefore, there is a need for compounds that can be used to protect biological tissues against thrombotic inflammation without the drawbacks associated with existing technological solutions. Summary of the Invention

[0007] The overall goal is to provide molecules that can protect biological tissues against thrombotic inflammation without at least some of the drawbacks associated with existing technological solutions.

[0008] This and other objectives are achieved through the invention as defined herein.

[0009] This invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims.

[0010] One aspect of the present invention relates to a method for producing poly(ethylene glycol) lipids (PEG-lipids). The method includes mixing a cationic PEG-lipid containing at least one amino group with a sulfated glycosaminoglycan containing at least one carbonyl group, preferably at least one aldehyde group, to form a Schiff base intermediate. The method further includes adding a reducing agent to the Schiff base intermediate to form sulfated glycosaminoglycan-PEG-lipids.

[0011] Another aspect of the invention relates to a PEG-lipid comprising at least one sulfated glycosaminoglycan, the sulfated glycosaminoglycan being linked to the PEG-lipid via a bond formed between an amino group of a cationic PEG-lipid comprising at least one amino group and a carbonyl group of at least one sulfated glycosaminoglycan to form a Schiff base intermediate reduced by a reducing agent.

[0012] Other aspects of the invention relate to a biological tissue comprising at least one such PEG-lipid anchored in the cell membrane of the biological tissue; and a liposome comprising at least one such PEG-lipid anchored in the lipid bilayer of the liposome.

[0013] The invention also defines PEG-lipids according to the invention for use as medicines, for treating thromboinflammatory diseases, for treating immediate blood-mediated response (IBMIR), for treating ischemia-reperfusion injury (IRI), for treating stroke, and for treating myocardial infarction.

[0014] Another aspect of the invention relates to an in vitro method for providing a sulfated glycosaminoglycan coating to biological tissues. The in vitro method includes adding an in vitro PEG-lipid according to the invention to the biological tissue to anchor the PEG-lipid in the cell membrane of the biological tissue.

[0015] Other aspects of the invention define an ex vivo method for processing an organ or a portion of an organ. The method includes ex vivo infusion of a solution containing a PEG-lipid according to the invention into a vascular system of the organ or a portion of an organ. The method further includes ex vivo incubation of the solution containing the PEG-lipid according to the invention within the vascular system to achieve coating at least a portion of the endothelial lining of the vascular system with the PEG-lipid according to the invention.

[0016] The PEG-lipids of this invention can be used to coat lipid membrane structures, such as cells and liposomes, via a single-step procedure. Furthermore, this coating of lipid membrane structures does not cause any significant aggregation or aggregation of cells or liposomes. Thus, the PEG-lipids of this invention can be used to protect biological tissues against thrombotic inflammation without the disadvantages associated with prior art solutions. Attached Figure Description

[0017] The best understanding of the embodiments, as well as its other objects and advantages, can be obtained by referring to the following description in conjunction with the accompanying drawings:

[0018] Figure 1 This is a schematic diagram of heparin-conjugated PEG-lipids (fHep-lipids). fHep-lipids include: fHep-C-lipids, fHep-K1C-lipids, fHep-K2C-lipids, fHep-K4C-lipids, and fHep-K8C-lipids.

[0019] Figure 2 schematically illustrates the synthesis of fHep-lipids. (A) Mal-PEG-lipids react with C, K1C, K2C, K4C, or K8C, followed by conjugation with fragmented heparin (fHep). (B) Fragmentation of unfractionated heparin (UFH) into fragmented heparin (fHep). (C) fHep-KnC-lipids (n = 0, 1, 2, 4, 8).

[0020] Figure 3 This is a graph showing the absorbance of fHep and heparin at 260 nm (N=3).

[0021] Figure 4 The graphs show (A) molecular weight analysis by gel permeation chromatography (GPC) (N=9 for fragmented heparin (fHep) and N=8 for unfractionated heparin) and (B) anti-factor Xa activity of fHep and unfractionated heparin (N=4).

[0022] Figure 5 This is a graph showing the (A) size and (B) ζ-potential of fHep-lipid (N=3).

[0023] Figure 6 The analysis of the antithrombin (AT) binding activity of fHep-lipids using a quartz crystal microbalance (QCM-D) with dissipation monitoring is shown.

[0024] Figure 7 This is a graph (N=3) showing the quantitative analysis of the binding amounts of AT with fHep-lipids and cationic-PEG-lipids.

[0025] Figure 8A QCM-D-based analysis of the AT-binding activity of fHep(-)-lipids is presented.

[0026] Figure 9 This is a graph (N=3) showing the quantitative analysis of the binding amounts of AT and bovine serum albumin (BSA) to fHep(-)-lipids.

[0027] Figure 10 QCM-D-based analysis of factor H binding activity of fHep(-)- lipids is presented.

[0028] Figure 11 The graph shows the quantitative analysis of the binding amounts of factors H and AT with fHep(-)-lipids and Mal-PEG-lipids (N=3) and the calculated molar ratios of immobilized factor H with fHep(-)-lipids and Mal-PEG-lipids (N=3).

[0029] Figure 12 This is a graph showing the anti-factor Xa activity of fHep-lipid modified liposomes (N=3).

[0030] Figure 13 This is a diagram showing the size of fHep-lipid modified liposomes (N=3).

[0031] Figure 14 This is a graph showing the polydispersity index (PDI) of fHep-lipid modified liposomes (N=3).

[0032] Figure 15 This is a graph showing the zeta potential of fHep-lipid modified liposomes (N=3).

[0033] Figure 16 Fluorescent images of AT (Alexa488 labeled) on the surface of human erythrocytes treated with fHep(-)-lipid, K1C-PEG-lipid, and fHep are shown.

[0034] Figure 17 This figure shows the quantitative analysis of the binding amount of AT (Alexa488 labeled) on the surface of erythrocytes treated with fHep(-)-lipids, K1C-PEG-lipids and fHep by flow cytometry.

[0035] Figure 18 This is a graph showing the anti-factor Xa activity of fHep-lipid modified CCRF-CEM cells (*: p<0.05, N=3).

[0036] Figure 19 shows the effect of fHep-lipid modification of hMSCs on blood compatibility. (A) Confocal image of hMSCs treated with fHep-lipids and Alexa488-labeled AT. Here, fHep-lipids are fHep-K1C(-)-lipids and fHep-K8C(-)-lipids. Scale bar: 40 μm. (B) Quantitative analysis of AT binding on modified hMSCs by flow cytometry. Error bars indicate standard deviation (N=5). (C) Viability assay of modified hMSCs by trypan blue exclusion method. Error bars indicate standard deviation (N=5). (E)-(G) Loop model assay of modified hMSCs in human whole blood. At 37 °C, at 1.0 × 10⁻⁶... 5 Modified hMSCs were incubated in human whole blood (0.5 IU / mL UFH) for 2 hours at a rate of 1 cell / mL. Here, hMSCs were modified with fHep-KnC(-)-lipids (n = 1 and 8) and K1C-PEG-lipids. Whole blood supplemented with PBS and untreated hMSCs were used as controls. The figures illustrate (D) relative platelet counts and the production of (E) TAT, (F) C3a, and (G) sC5b-9. Error bars represent standard deviations (N = 6).

[0037] Figure 20 shows the ring model determination of modified hMSCs in human whole blood. At 37°C, at a concentration of 1.0 × 10⁻⁶... 4 Modified hMSCs were incubated in human whole blood (0.5 IU / mL UFH) for 2 hours at a rate of 1 cell / mL. Here, hMSCs were modified with fHep-KnC(-)-lipids (n = 1 and 8) and K1C-PEG-lipids. Whole blood supplemented with PBS and untreated hMSCs were used as controls. The figures show (A) relative platelet counts and (B) the production of TAT, (C) C3a, and (D) sC5b-9. Error bars represent standard deviations (N = 6). Detailed Implementation

[0038] This invention generally relates to poly(ethylene glycol) (PEG) lipids, particularly such PEG-lipids comprising sulfated glycosaminoglycans, and their production and medical uses.

[0039] The PEG-lipids of this invention can be used for surface modification of cell and organ grafts to mimic endothelial surfaces, thereby protecting such cell and organ grafts against thrombotic inflammation. PEG-lipids offer several advantages over prior art methods using heparin and heparin conjugates. First, surface modification with the PEG-lipids of this invention can be performed in a single step without any chemical modification of the cell surface. This means that the surface modification process of cells or organ grafts with PEG-lipids is much easier to perform than prior art, which requires several process steps, including chemical modification of the cell surface, which can adversely affect the cells. Second, the PEG-lipids of this invention do not crosslink when connected to cells. Therefore, the PEG-lipids are not compromised by the disadvantages of prior art, which can lead to cell aggregation and aggregation upon reaction with heparin or heparin conjugates.

[0040] Therefore, the PEG-lipids of the present invention can be used to protect biological tissues, including cells and organ grafts, against thrombotic inflammation.

[0041] One aspect of the present invention relates to a method for producing PEG-lipids. The method includes mixing a cationic PEG-lipid containing at least one amino group with a sulfated glycosaminoglycan containing at least one carbonyl group, preferably at least one aldehyde group, to form a Schiff base intermediate. The method further includes adding a reducing agent to the Schiff base intermediate to form sulfated glycosaminoglycan-PEG-lipids.

[0042] Glycosaminoglycan-PEG-lipids are formed by Schiff base chemistry, including nucleophilic addition to form a hemiacetal amine, followed by dehydration to generate a Schiff base intermediate. The starting material in this reaction is a cationic PEG-lipid containing at least one amino group. This at least one amino group reacts with at least one carbonyl group, preferably at least one aldehyde group, of a sulfated glycosaminoglycan to form a Schiff base intermediate (the C=N bond between the sulfated glycosaminoglycan and the cationic PEG-lipid), which is then reduced by adding a reducing agent to form sulfated glycosaminoglycan-PEG-lipids, wherein the sulfated glycosaminoglycan is linked to the PEG-lipid via a C=N bond.

[0043] Therefore, sulfated glycosaminoglycans are linked to cationic PEG-lipids via covalent bonds, more specifically, covalent bonds (i.e., CN bonds) between the carbonyl (preferably aldehyde) group of the sulfated glycosaminoglycan and the amino group of the cationic PEG-lipid.

[0044] The cationic PEG-lipid containing at least one amino group can be any PEG-lipid containing at least one amino group, including PEG-phospholipids.

[0045] PEG-lipids can have the general structure of formula (II), and the corresponding PEG-phospholipids have the general structure of formula (III), where R1 and R2 represent the lipid portion of the molecule.

[0046]

[0047] In one embodiment, Y in formulas (II) and (III) is selected from H, CH3, maleimide, and N-hydroxysuccinimide.

[0048] As used herein, PEG-lipids include any conjugate between PEG and at least one lipid (including fatty acids, phospholipids, glycerides, glycerophospholipids, sphingolipids, sterols, isoprenols, glycolipids, and polyketides). In a preferred embodiment, PEG-lipids capable of anchoring in lipid layers, such as in the cell membranes of biological materials, are selected. Currently, PEG-lipids are preferred.

[0049] Preferably, at least one amino group is introduced into the PEG-lipid to form a cationic PEG-lipid, which is formed by reacting a maleimide-conjugated PEG-lipid with a cysteine ​​peptide.

[0050] Therefore, in one embodiment, the method includes the additional step of mixing the maleimide-conjugated PEG-lipid with at least one cysteine ​​peptide to form a cationic-PEG-lipid comprising at least one amino group. In one embodiment, the at least one cysteine ​​peptide may be at least one K n C-peptide, at least one CK n A peptide or combination thereof, wherein C is cysteine, K is lysine, and n is zero or equal to or less than 20, preferably equal to or less than 15, more preferably equal to or less than 10, such as a positive integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0051] If in K n C or CK n If n=0 in a peptide, then the cationic PEG-lipid will contain a single amino group. n C or CK n Each lysine in the peptide adds an amino group to the cationic PEG-lipid, thus containing n+1 amino groups.

[0052] In one embodiment, the maleimide-conjugated PEG-lipid is formed by mixing α-N-hydroxysuccinimide-ω-maleimide PEG (NHS-PEG-Mal), triethylamine, and 1,2-dispalmitoyl-sn-glycerol-3-phosphatidylethanolamine (DPPE) in dichloromethane. The maleimide-conjugated PEG-lipid is then precipitated by adding diethyl ether to the mixture of NHS-PEG-Mal, triethylamine, and DPPE in dichloromethane.

[0053] Sulfated glycosaminoglycans contain at least one carbonyl group. Currently, the preferred carbonyl group is an aldehyde group (-CHO). However, the invention is not limited to this, but also includes sulfated glycosaminoglycans containing at least one aldehyde group, at least one ketone (-C(=O)-), at least one carboxyl group (-C(=O)OH), at least one carboxyl ester group (-C(=O)O-), and / or at least one amide group (-C(=O)NR- or –C(=O)NH-). Sulfated glycosaminoglycans may contain a single carbonyl group, such as a single aldehyde group, or multiple carbonyl groups, i.e., at least two carbonyl groups, such as multiple aldehyde groups.

[0054] Glycosaminoglycans (GAGs) are long linear polysaccharides containing repeating disaccharide units, i.e., multiple disaccharide units. The most common repeating units include amino sugars, such as N-acetylglucosamine or N-acetylglucosamine, and uronic acid sugars, such as glucuronic acid or iduronic acid, or galactose. In one embodiment, the sulfated glycosaminoglycan is selected from heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratin sulfate, and hyaluronic acid.

[0055] Currently, the preferred sulfated glycosaminoglycan is heparin containing at least one carbonyl group, and more preferably heparin containing at least one aldehyde group. In a specific embodiment, the sulfated glycosaminoglycan is fragmented heparin (fHep) containing at least one carbonyl group, and more preferably fragmented heparin containing at least one aldehyde group.

[0056] This fragmentation of heparin introduces a carbonyl group, preferably an aldehyde group, into the heparin molecule. Furthermore, compared to unfractionated heparin (UFH), fragmentation reduces the length of the heparin chain, thereby lowering the molecular weight.

[0057] In one embodiment, the fragmentation reaction includes mixing an acidic solution and an aqueous solution of sodium nitrite (NaNO2) to form a mixed solution. The pH of the mixed solution is adjusted to be in the range of 2 to 6, preferably 3 to 5, more preferably 4. Heparin, preferably in the form of sodium heparin, is added to the mixed solution to form a heparin solution. The pH of the heparin solution is adjusted to be in the range of 6 to 8, preferably 6.5 to 7.5, more preferably 7, to form fragmented heparin containing at least one carbonyl group, preferably at least one aldehyde group. The fragmentation reaction may optionally include dialyzing the fragmented heparin containing at least one carbonyl group, preferably at least one aldehyde group, with water and lyophilizing the fragmented heparin containing at least one carbonyl group, preferably at least one aldehyde group.

[0058] The acidic solution is preferably selected from sulfuric acid (H2SO4) solution or acetic acid (CH3COOH) solution, with sulfuric acid (H2SO4) solution being more preferred.

[0059] In one embodiment, adding a reducing agent comprises adding sodium cyanoborohydride (NaBH3CN) to a Schiff base intermediate to form sulfated glycosaminoglycan-PEG-lipid. Therefore, in a preferred embodiment, the reducing agent is sodium cyanoborohydride. However, the embodiments are not limited thereto. Other reducing agents besides sodium cyanoborohydride may be used alternatively or additionally, including, for example, sodium triacetoxyborohydride and sodium borohydride.

[0060] Figure 2A An example of fHep-lipid synthesis is illustrated. Mal-PEG-lipids are reacted with C-peptide (n=0), K1C-peptide (n=1), K2C-peptide (n=2), K4C-peptide (n=4), or K8C-peptide (n=8), and then conjugated with fHep containing an aldehyde group to synthesize sulfated glycosaminoglycan-PEG-lipids fHep-KnC-lipids. Figure 2B This demonstrates the fragmentation of ungraded heparin (UHF) into fragmented heparin (fHep), and Figure 2C An embodiment of sulfated glycosaminoglycan-PEG-lipid is shown.

[0061] Figure 1 It schematically shows the following based on Figures 2A to 2C The synthesized sulfated glycosaminoglycan-PEG-lipid (fHep-KnC-lipid) is anchored in a lipid bilayer membrane. Figure 1 It also points out the maximum number of fHep molecules in each fHep-KnC-lipid, namely n+1 fHep molecules.

[0062] In one embodiment, any unreacted amino groups in the sulfated glycosaminoglycan-PEG-lipid are converted to carboxyl groups.

[0063] Carboxyl groups are generally less reactive than amino groups. Therefore, converting unreacted amino groups in sulfated glycosaminoglycan-PEG-lipids to carboxyl groups results in less cytotoxicity and thus less harm to cells. Furthermore, the negative charge introduced by the carboxyl group inhibits the binding of nonspecific proteins to the surface, where sulfated glycosaminoglycan-PEG-lipids are anchored. See [link to relevant documentation]. Figure 9 .

[0064] In a particular embodiment, any such unreacted amino groups are converted into carboxyl groups by adding an anhydride to the sulfated glycosaminoglycan-PEG-lipid to convert any unreacted amino groups in the sulfated glycosaminoglycan-PEG-lipid to carboxyl groups.

[0065] Any acid anhydride can be used to convert unreacted amino groups to carboxyl groups. Non-limiting but illustrative examples include succinic anhydride (SA), glutaric anhydride, diethylene glycol anhydride, and combinations thereof, with SA being preferred.

[0066] Another aspect of the present invention relates to a PEG-lipid comprising at least one sulfated glycosaminoglycan.

[0067] The at least one sulfated glycosaminoglycan is linked to the PEG-lipid via a bond formed between the amino group of the cationic PEG-lipid containing at least one amino group and the carbonyl group of the at least one sulfated glycosaminoglycan containing at least one carbonyl group to form a Schiff base intermediate reduced by a reducing agent.

[0068] Therefore, according to the present invention, sulfated glycosaminoglycans are linked to PEG-lipids via covalent bonds, particularly a covalent bond between the carbonyl group (preferably the aldehyde group) of the sulfated glycosaminoglycan and the nitrogen group (amino group) of the cationic PEG-lipid. This covalent bond between carbon and nitrogen is a CN bond.

[0069] In one embodiment, the PEG-lipid comprises K+ linking at least one sulfated glycosaminoglycan and the PEG-lipid together. n C and / or CK n Connector. In this embodiment, C is cysteine, K is lysine, and n is a positive integer of zero or less than 20. In one embodiment, n is selected in the range of 0 to 15, preferably in the range of 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0070] In one embodiment, sulfated glycosaminoglycans are transported via K... n C and / or CK n The amino or K amino group of any lysine residue in the linker n C and / or CK nThe N-terminal amine and the carbonyl group of at least one sulfated glycosaminoglycan containing at least one carbonyl group, preferably at least one aldehyde group, in the linker are linked to the PEG-lipid.

[0071] In one embodiment, the PEG-lipid moiety of the sulfated glycosaminoglycan-PEG-lipid has formula (I).

[0072]

[0073] In formula (I), p and q are integers independently chosen within the range of 10 to 16, preferably p and q are independently 10, 12, 14 or 16, and more preferably p = q = 14. m is selected such that the average molecular weight of the PEG chain is selected in the range of 1 kDa to 40 kDa, preferably 3 kDa to 10 kDa, and more preferably 5 kDa. The sulfated glycosaminoglycan molecule can then be linked to the PEG-lipid according to formula (I) at the N-terminal amine or at the amino group of one or more lysine residues.

[0074] As defined in this paper, the average molecular weight indicates that the molecular weight of individual PEG chains may differ from this average molecular weight, but the average molecular weight represents the average molecular weight of the PEG chains. This further implies that the PEG chains will have a natural molecular weight distribution around this average molecular weight.

[0075] In one embodiment, the sulfated glycosaminoglycan is fragmented heparin.

[0076] In one implementation, the weight-average molecular weight (M) of fragmented heparin is... w The range is 2.5kDa to 15kDa, preferably 4kDa to 10kDa, such as 5kDa to 10kDa, more preferably 5kDa to 8kDa or 7kDa to 9kDa.

[0077] In one embodiment, the sulfated glycosaminoglycan-PEG-lipid does not contain any unreacted or free amino groups. In a particular embodiment, any unreacted or free amino groups in the sulfated glycosaminoglycan-PEG-lipid are converted to carboxyl groups.

[0078] As mentioned herein, unreacted or free amino groups refer to any N-terminal amine and optionally any lysine residue in the PEG-lipid, as shown in formula (I), which are not bound to any sulfated glycosaminoglycan molecules.

[0079] In one embodiment, sulfated glycosaminoglycan-PEG-lipids can be obtained or have been obtained by methods as disclosed herein.

[0080] The sulfated glycosaminoglycan-PEG-lipid antithrombin (AT) of the present invention (see) Figure 6-10 , 11A) and factor H (see Figure 10 , 11 A and 11B) have affinity.

[0081] AT is a protein molecule that inactivates several enzymes in the coagulation system. Its activity is increased many times over by the anticoagulant heparin, which enhances the binding of AT to factor IIa (thrombin) and factor Xa (FXa). This means that the sulfated glycosaminoglycan-PEG-lipid of the present invention possesses anti-FXa activity by being able to bind to AT, thereby exhibiting coagulation inhibition.

[0082] Factor H is a member of the complement activation family of regulatory factors and a complement control protein. Its primary function is to regulate the alternative pathway of the complement system, ensuring that the complement system targets pathogens or other hazardous substances without harming host tissues. Factor H regulates complement activation on its own cells and surfaces through its cofactor activity against factor I-mediated C3b cleavage and its decay-accelerating activity against the alternative pathway C3-convertase C3bBb. Factor H exerts its protective effect on its own cells and surfaces, but not on bacterial or viral surfaces. This is thought to be a result of Factor H's ability to adopt either a lower or higher active conformation as a cofactor for C3 cleavage or decay-accelerating activity. The lower active conformation is the dominant form in solution and is sufficient to control fluid phase amplification. When Factor H binds to glycosaminoglycans and / or sialic acid, which are normally present on host cells but not on pathogen surfaces, it is thought to induce a more active conformation, thus ensuring protection of the self-surface while complement increases on foreign surfaces.

[0083] Therefore, cell surfaces containing the anchored sulfated glycosaminoglycan-PEG-lipids of the present invention possess the ability to attract and bind AT and factor H, thereby protecting the cell surface from thrombotic inflammation. See also Figure 12 , 17 18. The sulfated glycosaminoglycan-PEG-lipids of the present invention also exhibit this biological effect even when linked to lipid bilayer membranes (such as cell surfaces or liposomes).

[0084] As the experimental data provided herein further demonstrate, modification of lipid bilayers with the sulfated glycosaminoglycan-PEG-lipids of this invention does not induce any aggregation or cell condensation (which is common when modifying cell surfaces with heparin according to the prior art). See [link to previous document]. Figure 13 .

[0085] The present invention also relates to a lipid layer, preferably a lipid bilayer, comprising at least one sulfated glycosaminoglycan-PEG-lipid of the present invention. In this case, as Figure 1As shown, sulfated glycosaminoglycan-PEG-lipids are linked to or anchored to a lipid layer via PEG-lipid groups. For example, the present invention relates to a liposome comprising at least one PEG-lipid according to the invention anchored in a lipid bilayer of the liposome.

[0086] Other aspects of the invention relate to a biological tissue comprising at least one PEG-lipid according to the invention anchored in the cell membrane of the biological tissue.

[0087] Biological tissues can be single cells or multiple cells, such as stem cells, including mesenchymal stem cells (MSCs) and embryonic stem cells (ESCs); hepatocytes; endothelial cells; β cells (insulin-producing cells); and erythrocytes as illustrative but not limiting examples. Alternatively, biological tissues can be cell populations, such as pancreatic islets. Biological tissues can also be in the form of tissues or organs or portions thereof, such as kidneys, hearts, pancreas, livers, lungs, uteruses, bladders, thymus, intestines, and spleens. In certain embodiments, at least a portion of the vascular system and optionally the solids of the tissue or organ or a portion thereof may be coated with at least one PEG lipid according to the invention.

[0088] Another aspect of the invention relates to a PEG-lipid according to the invention for use as a medicament.

[0089] Other aspects of the invention relate to a PEG-lipid according to the invention for treating thromboinflammatory disease, for treating immediate blood-mediated response (IBMIR), for treating ischemia-reperfusion injury (IRI), for treating stroke, and / or for treating myocardial infarction.

[0090] The relevant aspects of this invention define the use of the PEG-lipids according to the invention in the manufacture of medicaments for treating thrombotic inflammation, IBMIR, IRI, stroke and / or myocardial infarction.

[0091] The PEG-lipids of the present invention can be administered to subjects in need via systemic or local administration. Non-limiting examples of systemic administration include intravenous and subcutaneous administration. Local administration includes locally injecting the PEG-lipids of the present invention into a target organ or tissue of a subject.

[0092] The PEG-lipids of the present invention are preferably administered in the form of a PEG-lipid solution. The solution containing PEG-lipid molecules can be, for example, saline, a buffered aqueous solution, or an organ preservation solution. Exemplary but non-limiting examples of buffered aqueous solutions that can be used include phosphate-buffered saline (PBS) and citrate solutions.

[0093] Another aspect of the invention relates to an in vitro method for providing a sulfated glycosaminoglycan coating to biological tissues. The in vitro method includes adding an in vitro PEG-lipid according to the invention to the biological tissue to anchor the PEG-lipid in the cell membrane of the biological tissue.

[0094] One aspect of the invention relates to an ex vivo method for processing an organ or a portion of an organ. The method includes ex vivo infusion of a solution comprising a PEG-lipid according to the invention into the vascular system and, optionally, the parenchyma of the organ or a portion of the organ. The method further includes ex vivo incubation of the solution comprising the PEG-lipid according to the invention in the vascular system and, optionally, the parenchyma, to achieve coating of at least a portion of the endothelial lining of the vascular system, and preferably the parenchyma, with the PEG-lipid according to the invention.

[0095] In one embodiment, the in vitro incubation step includes in vitro incubating a solution containing the PEG-lipid according to the invention in the vascular system and optionally in the parenchyma to achieve coating of at least a portion of the endothelial lining of the vascular system and preferably the parenchyma with the PEG-lipid according to the invention, while keeping the organ or a portion of the organ immersed in an organ preservation solution, preferably an organ preservation solution containing the PEG-lipid according to the invention.

[0096] Therefore, in vitro methods involve introducing PEG-lipids into the vascular system of an organ or a part of an organ, and allowing PEG-lipid molecules to interact with and bind to the cell membranes of the endothelium and parenchyma. Figure 1 This principle is illustrated schematically, in which PEG-lipid molecules interact hydrophobically with the lipid bilayer membrane, thereby anchoring or attaching the PEG-lipid molecules to the cell membrane via phospholipid groups.

[0097] When an organ or a portion thereof is immersed or submerged in an organ preservation solution (preferably an organ preservation solution containing PEG-lipid molecules), the interaction between the PEG-lipid molecules and the lipid bilayer membrane of the endothelium and optionally the parenchyma (such as the renal parenchyma in the case of the kidney) preferably occurs in vitro.

[0098] In a particular embodiment, a solution containing PEG-lipid molecules is first infused ex vivo into the vascular system and optionally the parenchyma of an organ or a portion of an organ. This ex vivo infusion is advantageously performed as early as possible after the organ or a portion of an organ has been removed from the donor body. The perfused organ or a portion of an organ is then immersed in and held therein, preferably at a reduced temperature, such as about 4°C.

[0099] In another specific embodiment, the organ or a portion of an organ is first immersed in an organ preservation solution, preferably containing PEG-lipid molecules, and then the PEG-lipid-containing solution is ex vivo infused into the vascular system and optionally the parenchyma of the organ or a portion of the organ. This ex vivo infusion can be performed simultaneously with the immersion of the organ or a portion of the organ in the organ preservation solution, preferably containing PEG-lipid molecules. Alternatively, the organ or a portion of the organ may be temporarily removed from the organ preservation solution for ex vivo infusion and then returned to the organ preservation solution, preferably containing PEG-lipid molecules.

[0100] In one embodiment, the method further includes infusing an organ preservation solution ex vivo into the vascular system to flush away unbound PEG-lipid molecules from the vascular system. Therefore, it is preferable to use the organ preservation solution to wash away unbound PEG-lipid molecules with one or more (i.e., at least two) washing steps.

[0101] In one embodiment, the ex vivo infusion of a solution containing PEG-lipid molecules includes ex vivo clamping of one of the arteries and veins in a vascular system. This embodiment also includes ex vivo infusion of the solution containing PEG-lipid molecules into the other of the arteries and veins, and ex vivo clamping of the other of the arteries and veins.

[0102] In another embodiment, a solution containing PEG-lipid molecules is infused into an artery (or vein) of the vascular system of an organ or part of an organ until the solution appears at the vein (or artery) of the organ or part of an organ. This confirms that the solution containing PEG-lipid molecules has filled the vascular system. At this point, the artery and vein are clamped.

[0103] The solution containing PEG-lipid molecules can be added via vein or artery. In a particular embodiment, the solution is infused into an artery. In this particular embodiment, the vein in the vascular system is then optionally initially clamped.

[0104] The solution containing PEG-lipid molecules is preferably incubated in vitro in the vascular system for a period of 10 minutes to 48 hours to allow the PEG-lipid molecules to interact hydrophobically with the endothelial cell membrane, thereby coating at least a portion of the vascular system of the organ or part of the organ. In vitro incubation is preferably performed for 20 minutes to 36 hours, and more preferably 30 minutes to 24 hours, such as 30 minutes to 12 hours, to 8 hours, to 4 hours, or to 1 hour.

[0105] The amount of PEG-lipid-containing solution infused into the vascular system depends on the type and size of the organ (adults vs. children). Generally, the volume of solution should be sufficient to fill the organ's vascular system. In most practical applications, 5 mL to 250 mL of PEG-lipid-containing solution is infused ex vivo into the vascular system. In a preferred embodiment, 5 mL to 100 mL, and preferably 5 mL to 50 mL, of PEG-lipid-containing solution is infused ex vivo into the vascular system.

[0106] In one embodiment, the solution contains 0.25 mg / mL to 25 mg / mL of PEG-lipid molecules. In a preferred embodiment, the solution contains 0.25 mg / mL to 10 mg / mL, preferably 0.25 mg / mL to 5 mg / mL, such as 2 mg / mL of PEG-lipid molecules.

[0107] The concentration of PEG-lipid molecules mentioned above can also be used in organ preservation solutions containing PEG-lipid molecules.

[0108] According to the present invention, a solution containing PEG-lipid molecules is incubated in vitro in a vascular system while the organ or a portion thereof is immersed or submerged in an organ preservation solution preferably containing PEG-lipid molecules. Furthermore, the organ or a portion thereof is preferably maintained at a temperature above 0°C but below 8°C, preferably above 0°C but equal to or below 6°C, and more preferably above 0°C but equal to or below 4°C.

[0109] In this embodiment, during the incubation period, the organ or a portion of an organ is immersed in an organ preservation solution, preferably containing PEG-lipid molecules, while allowing the PEG-lipid molecules to interact and bind with the cell membranes of the endothelium in the vascular system. The organ or a portion of an organ is also preferably kept cold, i.e., at a temperature close to but above 0°C. It has been shown that the theoretically ideal temperature for organ preservation is 4°C–8°C. Higher temperatures can lead to hypoxic damage to the organ due to ineffective reduction of metabolism, while temperatures below 4°C increase the risk of cold damage accompanied by protein denaturation.

[0110] Currently, the gold standard for donor organ preservation in clinical organ transplantation uses three plastic bags and an icebox. The first plastic bag contains the organ itself, submerged in an organ preservation solution. The first plastic bag is placed inside a second plastic bag filled with saline solution, and these two bags are then placed inside a third plastic bag filled with saline solution, which is then placed in an icebox. More advanced organ preservation devices for maintaining organs in a temperature-controlled environment are available and usable, such as the Sherpa Pak™ transport system from Paragonix Technologies, Inc., Waves from Waters Medical Systems, and the LifePort transporter from Organ Recovery Systems.

[0111] Solutions containing PEG-lipid molecules can be saline, buffered aqueous solutions, or organ preservation solutions.

[0112] Illustrative but non-limiting examples of buffered aqueous solutions that can be used include PBS and citrate solutions.

[0113] Organ preservation solutions that can be used to wash an organ or a portion of an organ's vascular system before or after infusion of PEG-lipid molecules and / or to immerse an organ or a portion of an organ may be selected from known organ preservation solutions. Illustrative but non-limiting examples of such organ preservation solutions include: histidine-tryptophan-ketoglutarate (HTK) solution, citrate solution, University of Wisconsin (UW) solution, Collins solution, Celsior solution, Kyoto University solution, and Institut Georges Lopez-1 (IGL-1) solution.

[0114] The subject is preferably a human subject. However, the invention can also be used in veterinary applications, wherein the subject is a non-human subject, such as a non-human mammal, including but not limited to cats, dogs, horses, cattle, rabbits, pigs, sheep, goats, and guinea pigs.

[0115] Other aspects of the invention relate to a method for treating, inhibiting, or preventing thrombotic inflammation, IBMIR, IRI, stroke, and / or myocardial infarction in a subject. The method includes administering the PEG-lipid according to the invention to a subject in need. In another embodiment, the method includes the previously described steps of: ex vivo infusion of a solution containing the PEG-lipid according to the invention into the vascular system of an organ graft, and ex vivo incubation of the solution containing PEG-lipid molecules in the vascular system to achieve coating at least a portion of the endothelial lining of the vascular system with PEG-lipid molecules, optionally but preferably, while keeping the organ graft immersed in an organ preservation solution preferably containing PEG-lipid molecules.

[0116] The PEG-lipids according to the present invention can provide local protection against thrombotic inflammation by mimicking the glycocalyx on the surface of normal endothelial cells. This method also avoids the risk of bleeding because, compared with systemic application, the coating of endothelial cells on the surface of target organs requires a small amount of modulator.

[0117] In one embodiment, the PEG-lipid of the present invention comprises heparin, which has similar functions to heparan sulfate proteoglycan (HS). Since heparin, like HS, can interact with many modulators, the fHep-lipid coating obtained using the PEG-lipid of the present invention can modulate complex biological responses during IRI, making it readily applicable to clinical trials.

[0118] Various methods for heparin coating have been reported in the prior art. Layer-by-layer coating of heparin with soluble complement receptor 1 (sCR1) has been applied to mouse islets[7]. However, this method cannot be applied to the endothelial coating of the kidney due to the impracticality and complexity of using recombinant sCR1. ​​In addition, cationic avidin has been used for heparin coating of islets by electrostatic interaction[8]. However, due to the strong antigenicity of avidin, this method is difficult to use in a clinical setting. Heparin-binding peptides have been used to immobilize heparin on cell surfaces using PEG-lipids[6, 9]. However, this coating procedure still requires several cumbersome processes, making it more difficult to coat the endothelial surface of solid organs with heparin.

[0119] Example

[0120] This embodiment illustrates the production and characterization of heparin-conjugated PEG-lipids (fHep-lipids), which can be coated onto lipid membrane structures such as cells and liposomes via a single-step method.

[0121] Reagents and materials

[0122] The following reagents and materials were used in the examples:

[0123] Heparin sodium (UFH, Fujifilm Wako PureChemical Corporation, Osaka, Japan)

[0124] Sulfuric acid (H2SO4, Fujifilm and Wako Pure Chemical Industries, Ltd.)

[0125] Sodium nitrite (NaNO2, Fujifilm and Wako Pure Chemical Industries, Ltd.)

[0126] 5M Sodium Hydroxide (NaOH, Fujifilm and Kojun Chemical Co., Ltd.)

[0127] Dialysis membrane (Spectra / Por, MWCO: 3.5-5kDa, Repligen Corporation, Waltham, MA, USA)

[0128] Sodium cyanoborohydride (NaCNBH3, Sigma-Aldrich Chemical Co., St. Louis, MO, USA)

[0129] D-PBS(-) (Fujifilm and Koko Pure Chemical Industries, Ltd.)

[0130] Bio-heparin (AT+) (COSMO BIO Ltd., Tokyo, Japan)

[0131] Glucan (M) w : 1080Da, 9890Da, 43500Da, 123600Da (Sigma-Aldrich Chemicals)

[0132] Sodium chloride (NaCl, produced by Fujifilm and Kojun Chemical Co., Ltd.)

[0133] Distilled water (Fujifilm and Kazuko Pure Chemicals Co., Ltd.)

[0134] Dimethyl sulfoxide (DMSO, produced by Fujifilm and Kojun Pharmaceutical Co., Ltd.)

[0135] α-N-hydroxysuccinimide-ω-maleimide poly(ethylene glycol) (NHS-PEG-Mal, M) w 5000Da (NOF Corporation, Tokyo, Japan)

[0136] Triethylamine (Sigma-Aldrich, St. Louis, Missouri)

[0137] 1,2-Dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine (DPPE, NOF Corporation)

[0138] Dichloromethane (Sigma-Aldrich Chemical Company)

[0139] 1,6-Diphenyl-1,3,5-Hextriene (DPH, Sigma-Aldrich Chemical Company)

[0140] L-cysteine ​​(C, M) w =121.16Da, Fujifilm and Wako Pure Chemical Industries, Ltd.)

[0141] Lysine-cysteine ​​(K1C, M) w =249.335Da, BEX Co., Ltd., Tokyo, Japan)

[0142] Lysine-lysine-cysteine ​​(K2C, M) w =377.51Da, BEX Limited)

[0143] Lysine-Lysine-Lysine-Lysine-Cysteine ​​(K4C, M) w =633.85 Da, GenScript Inc., Tokyo, Japan

[0144] Lysine-lysine-lysine-lysine-lysine-lysine-lysine-lysine-cysteine ​​(K8C, M) w =1146.54Da (GenScript Corporation)

[0145] Fluoresamine (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0146] Glycine (Fujifilm and Wako Pure Chemical Industries, Ltd.)

[0147] Antithrombin (AT, KENKETU NONTHRON 500 for injection, Takeda Pharmaceutical Company Limited, Osaka, Japan)

[0148] 1-Dodecanethiol (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0149] Fetal bovine serum albumin (BSA, Sigma-Aldrich Chemical Company)

[0150] Cholesterol (Fujifilm and Kazumitsu Chemical Co., Ltd.)

[0151] Dipalmitoylphosphatidylcholine (DPPC, MC-6060, NOF Company)

[0152] Poly(2-methacryloyloxyethylphosphorylcholine-co-n-butyl methacrylate) (MPC polymer, composed of 2-methacryloyloxyethylphosphorylcholine (MPC) and n-butyl methacrylate (BMA) domains in a 3:7 ratio, NOF Corporation, Tokyo, Japan)

[0153] Polyoxyethylene dehydrated sorbitan monolaurate ( 20. Tokyo Chemical Industry Co., Ltd., Tokyo, Japan

[0154] 3,3′,5,5′-Tetramethylbenzidine (TMB, ready-to-use solution, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan)

[0155] Ethanol (99.5%, Fujifilm and Koichi Pure Chemical Industries, Ltd.)

[0156] Citric acid monohydrate (CAM, Fujifilm and Koichi Pure Chemicals Co., Ltd.)

[0157] Sodium lauryl sulfate (SDS, Fujifilm and Koichi Chemical Co., Ltd.)

[0158] Cholesterol Quantitative Reagent Kit (T-Cho E, Fujifilm and Koichi Pure Chemicals Co., Ltd.)

[0159] Dioxane (dehydrated) (Kanto Chemical Co., Ltd.)

[0160] Succinic anhydride (SA, Fujifilm and Kohden Chemical Co., Ltd.)

[0161] TrypanBlue (Thermo Fisher Scientific, Waltham, Massachusetts, USA)

[0162] DMEM (Durbecoir Modified Eagle's Medium, Thermo Fisher Scientific, Waltham, Massachusetts, USA)

[0163] Trypsin-EDTA (0.25%, Thermo Fisher Scientific, Waltham, Massachusetts, USA)

[0164] CCRF CEM (American Type Culture Collection, ATCC, Manassas, VA, USA)

[0165] Human mesenchymal stem cells (hMSCs, Lonza, Morristown, NJ, USA)

[0166] Horseradish peroxidase (HRP) conjugated streptavidin (GE Healthcare, Chicago, IL, USA)

[0167] RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA)

[0168] Fetal bovine serum (FBS, Thermo Fisher Scientific)

[0169] Penicillin-Streptomycin, liquid (P / S penicillin: 5000 IU / mL, streptomycin: 5000 g / mL, in 100 mL 0.85% NaCl aqueous solution, Thermo Fisher Scientific)

[0170] Alexa Fluor TM 488 Antibody Labeling Kit (including sodium bicarbonate and Alexa Fluor in the kit) TM 488 carboxylic acid, tetrafluorophenyl (TFP) ester, Thermo Fisher Scientific

[0171] Vacuum blood collection tubes (EDTA-2Na treated, manufactured by Terum Corporation, Tokyo, Japan)

[0172] Ethylenediaminetetraacetic acid solution (EDTA, 0.5M, pH 8.0, Ingenium Corporation)

[0173] Factor H (purified from human blood)

[0174] equipment

[0175] The following devices were used in the embodiments:

[0176] pH meter (LAQUA, Horiba Co., Ltd., Kyoto, Japan)

[0177] Nanodrop-1000 (Thermo Fisher Scientific)

[0178] Nanodrop-3300 (Thermo Fisher Scientific)

[0179] Quartz crystal microbalance with energy dissipation (QCM, qsense GmbH, Biolin Scientific, Gothenburg, Sweden)

[0180] Gel permeation chromatography (GPC, LC-2000Plus series, JASCO Corporation, Tokyo, Japan)

[0181] Zetasizer Nano ZS (Malvern Instruments Co., Ltd., Worcestershire, UK)

[0182] Board reader (AD200, Beckman Coulter, Miami, FL, USA)

[0183] Cell counter (countess, Ingenium Corporation)

[0184] Extruder (Avanti Polar Lipids, Inc., Birmingham, AL, USA)

[0185] Centrifuge (MX301, TOMY SEIKO Co., Ltd., Tokyo, Japan)

[0186] Centrifuge (Force mini SBC 140-115, BM EQUIPMENT Co., Ltd., Tokyo, Japan)

[0187] Confocal laser scanning microscope (CLSM, LSM880, Carl Zeiss, Jena, Germany)

[0188] Flow cytometer (FCM, BD LSR II, BD Biosciences, San Jose, CA, USA)

[0189] Example 1 - Synthesis and Characterization of Fragmented Heparin (fHep)

[0190] Synthesis of fragmented heparin

[0191] A 1M solution of sulfuric acid (H₂SO₄) and a 7M aqueous solution of sodium nitrite (NaNO₂) were mixed, and the pH of the mixture was adjusted to 4. 3 mL of unfractionated heparin sodium solution (20 mg / mL in water) was mixed with 11 mL of the H₂SO₄ and NaNO₂ mixture at room temperature (RT, approximately 20-25°C) for 15 minutes. The pH of the solution was then adjusted to 7 by adding approximately 4 mL of 1M NaOH aqueous solution. After dialyzing the MilliQ water reaction mixture using a dialysis membrane (3.5-5 kDa, Spectra / Por) for 1 day, the solution was lyophilized to obtain fragmented heparin (fHep). The yield was 40%.

[0192] UV spectrum

[0193] The solution of fHep (10 mg / mL, in PBS) was measured using a UV-Vis spectrophotometer (Nanodrop 1000, Thermo Fisher Scientific, Waltham, Massachusetts, USA) to examine the aldehyde group of fHep.

[0194] The molecular weight of fHep was measured using GPC.

[0195] The molecular weights of UFH and fHep were measured by GPC. The column was a Shodex SB803HQ (Showa Denko Co., Ltd., Tokyo, Japan). The eluent was a 0.1M aqueous solution of NaCl. The flow rate was 0.5 mL / min, and the column oven temperature was 25°C. Dextran (M...) was used as a standard reagent. w : 1080Da, 9890Da, 43500Da, 123600Da (Sigma-Aldrich Chemical Company, St. Louis, Missouri, USA).

[0196] FXa assay to check heparin activity

[0197] The anti-factor Xa activity of the synthesized fHep was assessed using an FXa activity assay kit (Biophen heparin (AT+), COSMO BIO Ltd.). The concentration of fHep was 0.01 mg / mL (in PBS), while the concentrations of UFH as standards were 2, 1, and 0.5 IU / mL.

[0198] result

[0199] Because the aldehyde group has absorbance at 260 nm, the fHep solution also has absorbance at that wavelength. Figure 3 ).like Figure 3 As shown, the original UFH had no absorbance. This indicates that fHep contains an aldehyde group.

[0200] The number-average molecular weights (Mn) of fHep and heparin were calculated by GPC using dextran standards. n ()( Figure 4 A). fHep's M n =6.1kDa, while UFH's M n =22kDa. The results indicate that fHep is a fragmentation of heparin. The activity of fHep was measured using factor Xa activity assay. Figure 4 B). The activity of fHep is approximately 24% of the activity of the original UFH.

[0201] Example 2 - Synthesis and Evaluation of Cationic-PEG-Lipids and fHep-Lipids

[0202] Synthesis of Mal-PEG-lipids

[0203] The synthesis of Mal-PEG-lipids was carried out as previously disclosed [4]. In short, α-N-hydroxysuccinimide-ω-maleimide-poly(ethylene glycol) (NHS-PEG-Mal, M... w Mal-PEG(5k)-lipids (5000 Da, 200 mg), triethylamine (50 μL), and 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine (DPPE, 20 mg) were dissolved in dichloromethane and stirred at room temperature for 48 hours. Precipitation with diethyl ether yielded a white powder of Mal-PEG(5k)-lipids (yield: 80%).

[0204] Synthesis of cationic PEG-lipids

[0205] To introduce at least one amino group at the end of the PEG chain, we conjugated C, K1C, K2C, K4C, and K8C to Mal-PEG-lipids, wherein each lysine residue contains an amino group. C, K4C, and K8C were dissolved in PBS, and K1C and K2C were dissolved in DMSO at a concentration of 10 mg / mL (stock solutions). Each stock solution (10 mg / mL, 21 μL for C, 55 μL for K1C, 83 μL for K2C, 117 μL for K4C, or 217 μL for K8C) was mixed with Mal-PEG(5k)-lipids (10 mg / mL, 1000 μL, in PBS). The resulting solutions were rotated at room temperature for 24 hours to produce the following cationic-PEG-lipids: C-PEG-lipids, K1C-PEG-lipids, K2C-PEG-lipids, K4C-PEG-lipids, and K8C-PEG-lipids, referred to herein as KnC-PEG-lipids (n: the number of lysine residues).

[0206] Synthesis and functional assessment of fHep lipids

[0207] Each cationic PEG-lipid (1 mL, 10 mg / mL, in PBS) was mixed with fHep (15, 30, 45, 70, and 120 mg for C-, K1C-, K2C-, K4C-, and K8C-PEG-lipids, respectively), followed by the addition of NaCNBH3 solution (6, 13, 18, 30, and 49 μL, 6.4 M, in PBS, for C-, K1C-, K2C-, K4C-, and K8C-PEG-lipids, respectively). The mixtures were stirred at room temperature for 3 days (for K8C-PEG-lipids and K4C-PEG-lipids) or 7 days (for K2C-PEG-lipids, K1C-PEG-lipids, and C-PEG-lipids) to obtain the following fHep-lipids: fHep-C-lipid, fHep-K1C-lipid, fHep-K2C-lipid, fHep-K4C-lipid, and fHep-K8C-lipid.

[0208] Following the reaction, succinic anhydride (SA) was added to convert unreacted amino groups in the fHep-lipids to carboxyl groups. Each fHep-lipid (1 mL, 10 mg / mL, in PBS) was mixed with SA solutions (33, 64, 94, 151, and 252 μL in 0.5 M dioxane for fHep-C-, fHep-K1C-, fHep-K2C-, fHep-K4C-, and fHep-K8C-lipids, respectively) and stirred at room temperature for 24 hours. The resulting solutions were then lyophilized and passed through a GPC (spin column, Pierce) column. TM Purification was performed using a polyacrylamide rotary desalting column (7K MWCO, 0.7 mL, Thermo Fisher Scientific) to obtain fHep(-)-lipids: fHep-C(-)-lipids, fHep-K1C(-)-lipids, fHep-K2C(-)-lipids, fHep-K4C(-)-lipids, and fHep-K8C(-)-lipids.

[0209] Determination of fHep lipid diameter and surface charge

[0210] The diameter, polydispersity index (PDI), and zeta potential (surface charge) of each cationic-PEG-lipid (0.5 mg / mL in PBS), fHep-lipid (0.5 mg / mL in PBS), and fHep (4 mg / mL in PBS) were evaluated by dynamic light scattering using a Zetasizer Nano ZS (Malvin Instruments Ltd., Worcestershire, UK).

[0211] Determination of fHep-lipid critical micelle concentration (CMC)

[0212] DPH was used to measure the CMC of fHep-lipids. fHep-lipids, cationic-PEG-lipids, and Mal-PEG-lipids (1 mL, 1.0 × 10⁻⁶) were used. -1 -1.0×10 -7 The solution was mixed with 2 μL of DPH solution (2 mg / mL, in PBS) and 30 μM of DPH solution (in THF) and incubated at 37 °C for 1 hour. The fluorescence intensity of the resulting solution was then measured using a fluorometer (FP-6600, JASCO, Ex: 357 nm, Em: 430 nm).

[0213] The concentration of amine groups was determined using fluorescent amine.

[0214] Each cationic-PEG-lipid and fHep-lipid was diluted with PBS (0.5 mg / mL), and fluorescein was dissolved in DMSO at a concentration of 3 mg / mL. Each cationic-PEG-lipid solution (9 μL) or fHep-lipid solution (9 μL) was mixed with the fluorescein solution (3 μL) at room temperature for 15 minutes, and the absorbance of each resulting solution was measured (at 481 nm) using a Nanodrop-3300 (Thermo Fisher Scientific, Waltham, MA). The same experiments were performed using C, K1C, K2C, K4C, and K8C solutions of the same concentration. Glycine was used to calibrate the curves to determine the amino group concentration.

[0215] result

[0216] Molecular design of fHep-lipids in Figure 1 As shown in the diagram, multiple fragmented heparin molecules can be conjugated to each PEG-lipid molecule. Figure 2A Heparin is chemically modified to obtain fragmented heparin (fHep), which has an aldehyde group at the end. Figure 2B Then, fHep was conjugated with cationic NH2-PEG-lipids (cationic-PEG-lipids) via Schiff base chemistry. Figure 2A , 2C To introduce amino groups, C, K1C, K2C, K4C, and K8C were used, which were conjugated with Mal-PEG-lipids. The following cationic-PEG-lipids were produced: C-PEG-lipid (one amino group), K1C-PEG-lipid (two amino groups), K2C-PEG-lipid (three amino groups), K4C-PEG-lipid (five amino groups), and K8C-PEG-lipid (nine amino groups).

[0217] fHep was conjugated to each cationic-PEG-lipid via Schiff base chemistry between aldehyde and amino groups, followed by reduction with NaCNBH3. The number of fHep conjugated to the cationic-PEG-lipid was calculated by measuring the unreacted amino groups of the fHep-lipid and the amino groups of the cationic-PEG-lipid using fluorescein. For fHep-K8C-lipid, fHep-K4C-lipid, fHep-K2C-lipid, fHep-K1C-lipid, and fHep-C-lipid, the percentages of reacted amino groups were calculated to be 89%, 90%, 91%, 88%, and 61%, respectively, as listed in Table 1 below. Then, for fHep-K8C-lipid, fHep-K4C-lipid, fHep-K2C-lipid, fHep-K1C-lipid, and fHep-C-lipid, the number of fHep conjugated to each PEG-lipid was 8.0, 4.5, 2.7, 1.8, and 0.6, respectively.

[0218] Table 1 – Number of fHep per PEG-lipid conjugate

[0219]

[0220]

[0221] The micelle size of each fHep-lipid was determined by DLS. Figure 5 A). All fHep-lipids were observed between 15 nm and 20 nm, while fHep was observed around 2 nm. Additionally, the zeta potential of each fHep-lipid was more negative than that of each cationic-PEG-lipid. Figure 5 B). These results indicate that fHep is conjugated with PEG-lipids.

[0222] We also measured the CMC of each fHep-lipid using DPH. The CMCs of fHep-C-lipid, fHep-K1C-lipid, fHep-K2C-lipid, fHep-K4C-lipid, fHep-K8C-lipid, C-PEG-lipid, K1C-PEG-lipid, K2C-PEG-lipid, K4C-PEG-lipid, K8C-PEG-lipid, and Mal-PEG-lipid were 0.9, 1.1, 1.1, 1.0, 0.6, 1.1, 1.1, 1.0, 1.0, 0.7, and 1.1 μM, respectively, indicating that fHep-lipids are amphiphilic and can indeed form micelles.

[0223] Example 3 - Functional assessment of fHep-lipids using QCM-D

[0224] The function of fHep-lipids was assessed using a quartz crystal microbalance with energy dissipation (QCM-D, Q-sense GmbH, Gothenburg, Sweden). The binding affinity of antithrombin (AT) to each fHep-lipid and fHep(-)-PEG-lipid was quantified using the QCM-D. After cleaning the QCM gold sensor chip with oxygen plasma treatment (300 W, 100 mL / min gas flow, PR500; Yamato Scientific Co., Ltd., Tokyo, Japan), the sensor chip was immersed in 1-dodecylthiol solution (1.25 mM, in EtOH) for 24 hours to form a hydrophobic self-assembled monolayer (CH3-SAM). After thorough washing with ethanol and water, the sensor chip was placed in the QCM-D chamber. A solution of each fHep-lipid (0.1 mg / mL, in PBS) was infused into the chamber for 30 minutes, followed by a blocking treatment with BSA solution (1 mg / mL, in PBS) for 10 minutes. Finally, AT solution (0.1 mg / mL in PBS) was flowed into the chamber for 10 minutes. PBS was flowed for 2 minutes for washing before each sample solution was flowed. Adsorption of each material was calculated from the change in resonance frequency (Δf at the 7th overtone) using the Sauerbrey equation [5].

[0225] In addition, the binding of factor H to fHep(-)-lipids (fHep-K1C(-)-lipids, fHep-K4C(-)-lipids, and fHep-K8C(-)-lipids) or Mal-PEG-lipids (as a control) was investigated using QCM-D. A solution of each fHep(-)-lipid or Mal-PEG-lipid (0.1 mg / mL in PBS) was infused into the chamber for 30 min, followed by a blocking treatment with BSA solution (1 mg / mL in PBS) for 10 min. Then, factor H solution (50 μg / mL in PBS) was infused into the chamber for 15 min. PBS was run for 2 min for washing before each sample solution flow. Afterward, AT solution (0.1 mg / mL in PBS) was infused into the chamber for 10 min. PBS was run for 10 min for washing before each sample solution flow. The adsorption of each material was calculated using the Sauerbrey equation from the change in resonance frequency (Δf at the 7th overtone)[5].

[0226] result

[0227] The binding affinity of AT to fHep-lipids was assessed using QCM-D. Figure 6A representative QCM-D map of the interaction between fHep-K8C-lipids and AT is shown. After blocking with BSA, the binding of AT to fHep-K8C-lipids can be observed on the surface. Figure 7 Data on the AT binding amounts of each fHep-lipid and cationic-PEG-lipid were summarized. fHep was also added as a control. AT binding was observed for all fHep-lipids, but not for either cationic-PEG-lipids or fHep.

[0228] We also examined the AT-binding ability of fHep-lipids treated with succinic anhydride (SA) (i.e., fHep(-)-lipids). SA was used to convert these fHep-lipids into carboxyl groups, which are less cytotoxic, due to the unreacted amine groups present on them. Figure 8 A representative QCM-D map of the interaction between fHep-K8C(-)-lipids and AT is shown. When we add BSA for blocking, we can see less BSA binding and only AT binding. Figure 9 Similar results were obtained when fHep-K4C(-)-lipids were used. These results indicate that the negative charge on fHep(-)-lipids inhibits the nonspecific binding of BSA.

[0229] We also examined the binding ability of factor H to fHep(-)-lipids using QCM-D. Figure 10 A representative QCM-D map of interaction with factor H is shown. After blocking with BSA, we can see the binding of factor H on fHep(-)-lipids, but no binding was detected on the control Mal-PEG-lipids. Figure 11 A and 11B summarize the quantitative analysis of factor H binding for each fHep(-)-lipid and Mal-PEG-lipid. Factor H binding was present for all fHep(-)-lipids, but not for Mal-PEG-lipids. The number of factor H molecules per fHep(-)-lipid was highest when using fHep-K8C(-)-lipids, compared to fHep-K1C(-)-lipids and fHep-K4C(-)-lipids. This result indicates that highly stacked fHep molecules of fHep-K8C(-)-lipids have the highest affinity for factor H, which is important for regulating complement activation via factor H recruitment.

[0230] Example 4 - Functional assessment of fHep-lipids by FXa activity assay

[0231] The function of fHep-lipids was assessed using an FXa activity assay. Here, we evaluated the binding capacity of antithrombin (AT) to each fHep-lipid (which was incorporated into liposomes).

[0232] Liposomes were prepared from dipalmitoylphosphatidylcholine (DPPC) and cholesterol (molar ratio 1:1). A cholesterol solution (530 μL, 10 mg / mL in ethanol) and a DPPC solution (1 mL, 10 mg / mL in ethanol) were mixed and evaporated using a rotary evaporator to form lipid membranes, which were then dried under vacuum for 24 hours. PBS (1 mL) was then added and the mixture was vigorously stirred with a magnetic stir bar at room temperature for 1 hour. The resulting lipid suspensions were extruded into membrane filters (Φ1000, 400, 200, and 100 nm) using an extruder (Avanti Polar Lipids, Birmingham, Alabama, USA). The lipid suspensions were passed through each filter 21 times.

[0233] To incorporate fHep-lipids into the liposome surface, an fHep-lipid solution was mixed with a liposome suspension. The liposome suspension (500 μL, 1 mg / mL in the formulation, in PBS) was centrifuged (TOMY MX301, 20,000 g, 70 min, 4 °C), and then the fHep-lipid solution (50 μL, 0.5 mg / mL in PBS) was mixed with the liposome precipitate. After incubation at room temperature for 10 min, the suspension was washed once with PBS (450 μL) by centrifugation (20,000 g, 70 min, 4 °C). Finally, fHep-lipid-modified liposomes were obtained. The cholesterol concentration in the liposomes was measured using an assay kit (T-Cho E, Fujifilm and Koichi Chemicals Co., Ltd.). The FXa activity of the liposomes was evaluated using an assay kit (Biophen heparin (AT+), COSMO BIO Ltd.).

[0234] FXa activity assay

[0235] Liposome suspension (15 μL, in PBS) was mixed with human AT (15 μL) in a 96-well plate. Bovine FXa (75 μL) was added to each well and incubated at room temperature for 120 seconds. Then, after mixing with chromogenic reagent (75 μL) for 90 seconds, citric acid aqueous solution (100 μL, 20 mg / mL) was added. After collecting each supernatant by centrifugation (20,000 g, 70 min, 4 °C), absorbance was measured (at 405 nm).

[0236] Cholesterol in liposomes was measured by mixing a liposome suspension (60 μL, in PBS) with SDS (2 μL, 15 mg / mL, in PBS) at room temperature for 30 minutes to dissolve it. Cholesterol concentration was then determined according to the company's instructions.

[0237] result

[0238] The anti-FXa activity of fHep-liposomes was evaluated. Figure 12 As a control group, each cationic-PEG-liposome modified with liposomes and fHep-treated liposomes were used in the assay. Anti-FXa activity was normalized by liposome concentration. All fHep-liposome modified with liposomes showed higher anti-FXa activity than the control group. Similarly, similar results were obtained when fHep(-)-liposome modified with liposomes were used. Figure 12 These results indicate that the surface of liposomes can be modified with fHep-lipids and fHep(-)-lipids, and that such modified liposomes possess anti-FXa activity.

[0239] Example 5 - Characterization of treated liposomes

[0240] As described in Example 4, the surface of the liposomes was modified with each of the following fHep-lipids (fHep-C-lipid, fHep-K1C-lipid, fHep-K2C-lipid, fHep-K4C-lipid, and fHep-K8C-lipid) or cationic-PEG-lipids (C-PEG-lipid, K1C-PEG-lipid, K2C-PEG-lipid, K4C-PEG-lipid, and K8C-PEG-lipid). Furthermore, fHep and PBS were used as control groups.

[0241] After centrifugation (TOMY MX301, 20,000 g, 70 min, 4 °C), a solution of fHep-lipid (0.5 mg / mL in PBS) or cationic-PEG-lipid (0.5 mg / mL in PBS) was mixed with the liposome precipitate. After incubation at room temperature for 10 min, the liposomes were washed once with PBS (450 μL) by centrifugation (20,000 g, 70 min, 4 °C). Finally, fHep-lipid-modified liposomes and cationic-PEG-lipid-modified liposomes were obtained. The diameter, polydispersity index (PDI), and zeta potential (surface charge) of the treated liposomes were evaluated by dynamic light scattering using a Zetasizer Nano ZS (Malvin Instruments Ltd., Worcestershire, UK).

[0242] result

[0243] The size of liposomes modified with fHep-lipid or cationic-PEG-lipid was measured by DLS. Figure 13 As a control group, liposomes were treated with PBS or fHep. Before treatment, the liposome size was 150 nm. The average size of liposomes modified with fHep-lipid or cationic-PEG-lipid was then between 155 nm and 175 nm, while the control liposomes were approximately 250 nm in size. Furthermore, the polydispersity index (PDI) of liposomes modified with fHep-lipid or cationic-PEG-lipid showed a lower value (approximately 0.2) than that of the control liposomes (approximately 0.5). Figure 14 These results indicate that liposomes modified with fHep-lipid or cationic-PEG-lipid dispersed well, while control liposomes aggregated.

[0244] In addition, the zeta potential of all liposomes was measured. Figure 15 All samples showed a negative charge, but liposomes modified with each fHep-lipid showed a greater negative charge than those modified with each cationic-PEG-lipid. This result indicates that the liposome surface is negatively modified with fHep-lipids.

[0245] Example 6 - Cell surface functionalization using fHep(-)-lipids

[0246] Human red blood cells (RBCs) are collected from healthy donors using vacuum blood collection tubes. Alexa fluor is used. TM The 488 antibody labeling kit was used to label antithrombin (AT) according to the company's provided protocol. RBCs (10 μL, 7 × 10⁻⁶ cells / mL) were washed with 1 mL PBS. 9Cells / mL, in 10 mM EDTA / PBS) and centrifuged (Force mini SBC 140-115, BM EQUIPMENT Ltd., 1 min). At room temperature, the cell pellet was treated with fHep(-)-lipids (fHep-C(-)-lipids, fHep-K1C(-)-lipids, fHep-K2C(-)-lipids, fHep-K4C(-)-lipids and fHep-K8C(-)-lipids), K1C-PEG-lipids (0.5 mg / mL, 20 μL per sample), fHep (4 mg / mL in PBS) or PBS (20 μL) for 30 min, followed by washing twice with 1 mL PBS. The cell pellet was then treated with Alexa488-AT (4 mg / mL) at room temperature for 10 min, followed by washing twice with 1 mL PBS and centrifuged (Force mini SBC 140-115, 1 min). The resulting cell pellet was resuspended in 1 mL PBS. The treated cells were observed using a confocal microscope (CLSM, LSM880, Carl Zeiss AG, Jena, Germany) and analyzed by flow cytometry (BDLSR II, BD Biosciences, San Jose, California, USA). The experiments were approved by the Ethics Committee of the University of Tokyo.

[0247] The function of fHep-lipids was assessed using an FXa activity assay. Here, we evaluated the binding capacity of antithrombin (AT) to each fHep-lipid (which was incorporated into live cells (CCRF-CEM cells)). To modify the cell surface of CCRF-CEM cells, fHep-lipids (fHep-C-lipids) were mixed with the cells. The cell suspension was washed with PBS (2 × 10⁶ cells / mL RPMI 1640 medium) by centrifugation (120 g, 4 °C, 3 min). 6 Cells were incubated twice with fHep-C-lipid solution (100 μL, 0.5 mg / mL, in PBS containing 1 mg / mL glycine) to form a pellet. The pellet was incubated at room temperature for 30 min, gently tapping the pellet every 10 min. As a control, fHep (100 μL, 2.5 mg / mL, in PBS containing 1 mg / mL glycine) was used. The treated cells were then washed twice with PBS by centrifugation (180 g, 4 °C, 6 min). Finally, the cells were resuspended in PBS (100 μL). Cell viability and cell number were assessed using trypan blue and a cell counter.

[0248] Next, a cell suspension (15 μL) was prepared and mixed with human AT (15 μL) in a 96-well plate. Bovine FXa (75 μL) was added to each well and incubated at room temperature for 120 seconds. Then, after mixing with the chromogenic reagent (75 μL) for 90 seconds, citric acid aqueous solution (100 μL, 20 mg / mL) was added. Finally, the absorbance was measured (at 405 nm).

[0249] result

[0250] When cells were treated with fHep(-)-lipids, fluorescence was observed on the cell membrane. Figure 16 When cells were treated with K1C-PEG-lipids, fHep, and PBS, no fluorescence was observed on the cell membrane, indicating that AT is specifically immobilized on fHep(-)-lipids on the cell surface. Figure 17 Quantitative analysis of Alexa488-AT immobilized on each cell is shown, which also indicates that AT is specifically immobilized on fHep(-)-lipids on the cell surface. Furthermore, the highest number of immobilized ATs were observed when cells were treated with fHep-K4C(-)-lipids and fHep-K8C(-)-lipids, where the highly stacked fHep effectively immobilized ATs on the fHep(-)-lipids.

[0251] The anti-FXa activity of fHep-lipid modified cells (CCRF-CEM cells) was evaluated. Figure 18 As a control group, unmodified cells were used for the assay. Cell number compensated for anti-FXa activity. fHep-lipid-modified cells showed higher anti-FXa activity than unmodified cells. Figure 18 These results indicate that the cell surface can be modified with fHep-lipids, and also show that this surface modification leads to anti-FXa activity.

[0252] Example 7 – Functional assessment of fHep-lipids using a whole blood model

[0253] hMSC surface functionalization using fHep-lipids

[0254] hMSCs were cultured in DMEM (supplemented with 10% FBS, 50 IU / mL penicillin, and 50 μg / mL streptomycin) at 37°C, in 5% CO2 and 95% air. hMSCs (1 mL, 2.5 × 10⁻⁶ cells / mL) collected by trypsin digestion (3 min, 37°C, 5% CO2) were then cultured in PBS. 5Centrifuge at 10 cells / mL (Force mini SBC 140-115, BM EQUIPMENT Ltd., 1 min). Treat the cell pellet with fHep(-)-lipids (20 μL, 10 mg / mL in PBS, fHep-K1C(-)-lipids and fHep-K8C(-)-lipids), KnC-PEG-lipids (20 μL, 10 mg / mL in PBS, K1C-PEG-lipids and K8C-PEG-lipids), fHep (20 μL, 30 and 120 mg / mL in PBS) or PBS (20 μL) at room temperature for 30 min, then wash twice with cold PBS (1 mL) and centrifuge (Force mini SBC 140-115, 1 min). Samples containing fHep (fHep-K1C(-)-lipids, fHep-K8C(-)-lipids, and fHep (30 or 120 mg / mL)) were reacted with glycine (18 mg / mL in PBS) for 4 hours, followed by purification using a spin column to inactivate the cytotoxic aldehyde groups of free fHep in solution. The cell pellet was treated with Alexa 488-AT (4 mg / mL) at room temperature for 10 minutes, followed by a single wash with 1 mL of cold PBS and centrifugation (Force mini SBC 140-115, 1 min). The resulting cell pellet was resuspended in 500 μL of PBS, and cell viability was assessed using trypan blue and a cell counter (Countess II, Ingenium). Treated cells were observed using a CLSM (LSM880, Carl Zeiss) and also analyzed by flow cytometry (BD LSR II, BD Biosciences).

[0255] Blood tests using whole human blood

[0256] hMSCs were exposed to human whole blood using the Chandler ring model [6] to evaluate the antithrombotic properties of the surface of hMSCs treated with fHep-lipids. The number of passages of hMSCs used for blood tests was 6-8. hMSCs (1 mL, 1.0 × 10⁻⁶ mg / mL in PBS) were treated with fHep-K1C(-)-lipids, fHep-K8C(-)-lipids and K1C-PEG-lipids (40 μL, 10 mg / mL in PBS, for each sample). 6 Cells were collected (cells / mL) and washed twice to remove free fHep-lipids. Cell viability and concentration were assessed using trypan blue and a cell counter (Countess II, Ingenium), and the cell concentration was adjusted to 2.5 × 10⁻⁶ cells / mL. 6 Or 2.5×10 5Cells / mL. Loops made from polyurethane tubes (Φ6.3 mm, 40 cm) and polypropylene connectors (Φ6.5 mm, ISIS Ltd., Osaka, Japan) were coated with MPC polymer (2 mL, 5 mg / mL in EtOH) for 24 hours, followed by air drying for 24 hours to prevent surface-induced blood activation. Human whole blood was drawn from healthy donors who had not received any drugs for at least 14 days prior to donation into vacuum tubes (7 mL, untreated, Terumo Ltd.). Immediately after blood collection, UFH (2.5 μL / 1 mL blood, 200 IU / mL in PBS) was mixed into the blood. Then, human whole blood (2.5 mL, containing 0.5 IU / mL UFH) was added to the MPC polymer-coated loops, followed by 100 μL of hMSC suspension (2.5 × 10⁻⁶ cells / mL) in PBS. 6 Or 2.5×10 5 1 cell / mL, treated or untreated hMSCs, or PBS as a control. Tubes were incubated at 37°C and rotated at 22 rpm for 2 hours. Blood was collected from each loop (1 mL) at 1 hour and 2 hours and mixed with EDTA solution (10 mM). Platelet counts for each sample were measured using a cell counter (pocH-80i, SYSMEX, Hyogo Prefecture, Japan). Blood samples were then centrifuged (TOMY MX301, 2,600 g, 15 min, 4°C), and plasma from each sample was collected and stored at -80°C for enzyme-linked immunosorbent assay (ELISA) of TAT, C3a, and sC5b-9. The experiments were approved by the Ethics Committee of the University of Tokyo.

[0257] Measurement of TAT, C3a and sC5b-9 in plasma

[0258] TAT, C3a, and sC5b-9 in plasma were measured using a standard sandwich ELISA. In short, the concentrations were measured using a dilution buffer (containing 0.05%...). Plasma was diluted with PBS containing 20 mM EDTA and 10 mg / mL BSA. C3a in the plasma was captured by pre-coated anti-human C3a mAb 4SD17.3 on a 96-well plate and detected by biotinylated polyclonal rabbit anti-C3a antibody and horseradish peroxidase (HRP) conjugated streptavidin. TMB was reacted with immobilized HRP (15 min) and the reaction was terminated with 1 M H2SO4 aqueous solution. Finally, absorbance at 450 nm was detected using a plate reader (AD200, Beckman Coulter, Miami, FL). Yeast polysaccharide-activated serum (calibrated against purified C3a) was used as a standard. The sC5b-9 ELISA was validated in the same manner as the C3a measurement. First, the plasma was diluted with dilution buffer. Then, sC5b-9 in plasma was captured by pre-coating anti-neoC9 mAb aE11 (Diatec Monoclonals AS, Oslo, Norway) on a 96-well plate and detected using anti-human C5 polyclonal rabbit antibody (Dako) and HRP-conjugated anti-rabbit IgG (Dako). TMB was reacted with immobilized HRP (15 min), and the reaction was terminated with 1M H2SO4 aqueous solution. The absorbance at 450 nm was then measured using a plate reader. Yeast polysaccharide-activated serum was used as a standard.

[0259] According to the company's instructions, TAT was measured using an ELISA kit (Human Thrombin-Antithrombin Complex (TAT) AssayMax ELISA Kit, Assaypro, St Charles, MO, USA). In short, plasma was diluted with a diluent. TAT was then captured by a monoclonal antibody pre-coated on a 96-well plate against human antithrombin and detected with a biotinylated polyclonal antibody against human thrombin, followed by detection with HRP-conjugated streptavidin. The peroxidase chromogen substrate, tetramethylbenzidine, was allowed to react for 20 minutes, and the reaction was terminated with 0.5N hydrochloric acid solution. The absorbance was then measured at 450 nm using a plate reader. The human TAT complex was used as a standard.

[0260] result

[0261] hMSCs were modified with fHep-lipids, fHep-K1C(-)-lipids, and fHep-K8C(-)-lipids, which have high and low AT binding abilities, respectively, to compare their antithrombotic properties in human whole blood. When hMSCs were treated with fHep-K1C(-)-lipids and fHep-K8C(-)-lipids, strong fluorescence from Alexa488-AT was observed on the hMSC membrane, while no fluorescence was observed on the cell membrane when those cells were treated with KnC-PEG-lipids (n=1 and 8), fHep, and PBS. Figure 19A This indicates that fHep(-)-lipids are immobilized on the surface of hMSCs. Flow cytometry analysis showed that hMSCs treated with fHep-K1C-lipids had more Alexa488-AT binding than hMSCs treated with fHep-K8C-lipids. Figure 19B This may be because AT binding on fHep-K8C(-)-lipids on the surface of hMSCs is inhibited, due to the high accumulation of fHep on the fHep-K8C(-)-lipids on the hMSC surface, while exogenous AT cannot fully access fHep. The viability of the treated hMSCs was approximately 80%, similar to the control group (UFH-treated, PBS-treated, and untreated cells), indicating the non-cytotoxicity of fHep(-)-lipid modification. Figure 19C High fluorescence intensity was observed in cells treated with K8C-PEG-lipids. Figure 19B , 19C These cells were found to be disrupted by K8C-PEG-lipid modification due to their cationic properties, leading to the uptake of Alexa488-AT.

[0262] Next, we incubated human whole blood with hMSCs at a concentration of 1.0 × 10⁻⁶. 4 ( Figure 20A-20D ) or 1.0×10 5 cells / mL Figure 19D-19G The hMSCs were treated with fHep-K1C(-)-lipids, fHep-K8C(-)-lipids, or K1C-PEG-lipids. Untreated hMSCs and PBS were used as controls.

[0263] Figure 19DPlatelet counts in the blood at 1 hour and 2 hours are shown. When PBS was added to the blood, there was almost no decrease in platelets. Furthermore, when hMSCs were added, the platelet count decreased over time, indicating that TF from hMSCs induces platelet aggregation. The same results were observed for K1C-PEG-lipid modified hMSCs. It appears that the positively charged K1C at the ends of the PEG layer induces platelet activation, leading to actual platelet aggregation. On the other hand, in the case of hMSCs treated with fHep-K1C(-)-lipids and fHep-K8C(-)-lipids, although the platelet count decreased slightly, the remaining platelets were much higher than in the control group of unmodified hMSCs, suggesting that surface modification with fHep-lipids can attenuate platelet activation. There was no significant difference in platelet counts between fHep-K1C(-)-lipid and fHep-K8C(-)-lipid modified hMSCs. When the hMSC concentration was 1.0 × 10⁻⁶, the platelet count was significantly lower than in the control group. 4 At 100 cells / mL, there was no significant difference in platelet count, although we could see a similar trend as seen at higher cell concentrations. Figure 20A ).

[0264] We assessed the level of TAT, which was compared with the treated hMSCs ( Figure 20B [hMSC] = 1.0 × 10 4 cells / mL, and Figure 19E [hMSC] = 1.0 × 10 5 Coagulation markers were measured during 2 hours of incubation (cells / mL). For hMSCs modified with K1C-PEG-lipids and untreated hMSCs, TAT levels increased significantly over time, while for hMSCs modified with fHep-lipids and blood supplemented with PBS, TAT levels increased slightly. These results were obtained at 1.0 × 10⁻⁶ cells / mL. 5 The concentration of hMSCs per mL was very high in the blood. Significant differences were observed between the untreated hMSC group, the K1C-PEG-lipid-modified hMSCs, and each type of fHep-lipid-modified hMSC. Figure 19E No significant differences were observed between PBS-mixed blood and hMSC-mixed blood containing each fHep-lipid modified variety. These results suggest that fHep-lipids on the surface of hMSCs can inhibit coagulation activation.

[0265] In addition, we evaluated the effect of treatment on hMSCs ( Figure 20C , 20D [hMSC] = 1.0 × 10 4 cells / mL, and Figure 19F , 19G [hMSC] = 1.0 × 10 5The production of C3a and sC5b-9 (complement markers) was observed during 2 hours of incubation (cells / mL). Essentially, the levels of both markers increased over time. However, there were no differences in levels between groups. We did not observe any effect of cell surface modification with fHep-lipids on complement activation.

[0266] The embodiments described above should be understood as several illustrative examples of the present invention. Those skilled in the art should understand that various modifications, combinations, and variations can be made to the embodiments without departing from the scope of the invention. Specifically, where technically possible, different partial solutions from different embodiments can be combined into other configurations. However, the scope of the invention is defined by the appended claims.

[0267] References

[0268] 1Cabric S, Eich T, Sanchez J, Nilsson B, Korsgren O, Larsson LA new method for incorporating function al heparin onto the surface of islets of Langerhans. Tissue Engineering, Part C, 14:141-147.2008.

[0269] 2U.S. Patent No. 8,153,147

[0270] 3U.S. Patent No. 9,795,629

[0271] 4Teramura Y, Oommen OP, Olerud J, Hilborn J, Nilsson B. Microencapsulation of cells, including islets, within stable ultra-thin membranes of maleimide-conjugated PEG-lipid with multifunctional crosslinkers. Biomaterials 34: 2683-2693.2013.

[0272] 5Teramura,Y.;Kuroyama,K.;Takai,M.Influence of Molecular Weight of PegChain on Interaction between Streptavidin and Biotin-Peg-ConjugatedPhospholipids Studied with Qcm-D.Acta Biomater 2016;30,135-143.

[0273] 6Asif,S.;Ekdahl,K.N.;Fromell,K.;Gustafson,E.;Barbu,A.;Le Blanc,K.;Nilsson,B.;Teramura,Y.Heparinization of Cell Surfaces with Short Peptide-Conjugated Peg-Lipid Regulates Thromboinflammation in Transplantation ofHuman Mscs and Hepatocytes.Acta Biomater 2016;35,194-205.

[0274] 7Luan,N.M.;Teramura,Y.;Iwata,H.Layer-by-Layer Co-Immobilization ofSoluble Complement Receptor 1and Heparin on Islets.Biomaterials 2011;32,6487-6492.

[0275] 8Cabric,S.;Sanchez,J.;Lundgren,T.;Foss,A.;Felldin,M.;Kallen,R.;Salmela,K.;Tibell,A.;Tufveson,G.;Larsson,R.;Korsgren,O.;Nilsson,B.IsletSurface Heparinization Prevents the Instant Blood-Mediated InflammatoryReaction in Islet Transplantation.Diabetes 2007;56,2008-2015.

[0276] 9 Kristina N Ekdahl, Shan Huang, Bo Nilsson, Yuji Teramura, Complement inhibition in biomaterial- and biosurface-induced thromboinflammation, SeminImmunol, 2016;28(3):268-77.doi:10.1016 / j.smim.2016.04.006.

Claims

1. A method for producing poly(ethylene glycol) lipids (PEG-lipids), the method comprising: Maleimide-conjugated PEG-lipids with K n C and / or CK n Mixed to form a PEG-lipid containing at least one amino group, wherein in the maleimide-conjugated PEG-lipid, maleimide is conjugated with PEG, and wherein C is cysteine, K is lysine, and n is zero or a positive integer equal to or less than 20. The PEG-lipid containing at least one amino group is mixed with fragmented heparin containing at least one aldehyde group to form a Schiff base intermediate. as well as A reducing agent is added to the Schiff base intermediate to form heparin-PEG-lipid.

2. The method according to claim 1, wherein n is zero or a positive integer equal to or less than 15.

3. The method of claim 2, wherein n is zero or a positive integer equal to or less than 10.

4. The method according to any one of claims 1 to 3, further comprising: Mixing α- in dichloromethane N -Hydroxysuccinimide-ω-maleimide-PEG (NHS-PEG-Mal), triethylamine and 1,2-dipalmitoyl- sn glycerol-3-phosphatidylethanolamine (DPPE); and The maleimide-conjugated PEG-lipids were precipitated by adding diethyl ether to a mixture of NHS-PEG-Mal, triethylamine, and DPPE in dichloromethane.

5. The method according to any one of claims 1 to 3, wherein the heparin is a fragmented heparin molecule containing an aldehyde group at the end of the fragmented heparin molecule.

6. The method according to claim 5, further comprising: An acidic solution and an aqueous solution of sodium nitrite (NaNO2) are mixed to form a mixed solution; The pH of the mixed solution was adjusted to be within the range of 2 to 6; Heparin is added to the mixed solution to form a heparin solution; as well as The pH of the heparin solution is adjusted in the range of 6 to 8 to form fragmented heparin containing at least one aldehyde group.

7. The method according to claim 6, further comprising: The fragmented heparin containing at least one aldehyde group was dialyzed with water and then lyophilized.

8. The method of claim 7, wherein adjusting the pH of the mixed solution comprises adjusting the pH of the mixed solution within a range of 3 to 5.

9. The method of claim 8, wherein adjusting the pH of the mixed solution comprises adjusting the pH of the mixed solution to 4.

10. The method of claim 6, wherein adjusting the pH of the heparin solution comprises adjusting the pH of the heparin solution within a range of 6.5 to 7.5 to form the fragmented heparin comprising at least one aldehyde group.

11. The method of claim 10, wherein adjusting the pH of the heparin solution comprises adjusting the pH of the heparin solution to 7 to form the fragmented heparin comprising at least one aldehyde group.

12. The method of claim 6, wherein adding heparin comprises adding sodium heparin to the mixed solution to form the heparin solution.

13. The method according to any one of claims 1 to 3, wherein adding the reducing agent comprises adding sodium cyanoborohydride to the Schiff base intermediate to form the heparin-PEG-lipid.

14. The method according to any one of claims 1 to 3, the method further comprising converting any unreacted amino groups in the heparin-PEG-lipid into carboxyl groups.

15. The method of claim 14, further comprising adding an anhydride to the heparin-PEG-lipid to convert any unreacted amino groups in the heparin-PEG-lipid into carboxyl groups.

16. The method according to any one of claims 1 to 3, wherein the PEG-lipid is anchored in the lipid layer.

17. The method of claim 16, wherein the PEG-lipid is capable of anchoring in the cell membrane of a biological tissue and / or in the lipid bilayer of a liposome.

18. The method of claim 16, wherein the PEG-lipid is a phospholipid capable of anchoring in the lipid layer.

19. A poly(ethylene glycol) lipid (PEG-lipid) comprising at least one fragmented heparin, said fragmented heparin being contained via K n C and / or CK n The K of the linker and at least one amino group of PEG-lipid n C and / or CK n The amino group of any lysine residue in the linker or the K n C and / or CK n A CN bond formed between the N-terminal amine in the linker and the aldehyde group of the at least one fragmented heparin containing at least one aldehyde group is linked to the PEG-lipid to form a Schiff base intermediate that is reduced by the addition of a reducing agent, wherein C is cysteine, K is lysine, and n is a positive integer of zero or equal to or less than 20.

20. The PEG-lipid of claim 19, wherein n is selected in the range of 0 to 15.

21. The PEG-lipid of claim 20, wherein n is selected in the range of 0 to 10.

22. The PEG-lipid according to any one of claims 19 to 21, wherein the weight-average molecular weight (M) of the fragmented heparin is... w Choose within the range of 2.5 kDa to 15 kDa.

23. The PEG-lipid of claim 22, wherein the M of the fragmented heparin w Choose within the range of 5 kDa to 10 kDa.

24. The PEG-lipid according to any one of claims 19 to 21, wherein any free amino group in the heparin-PEG-lipid is converted into a carboxyl group.

25. The PEG-lipid according to any one of claims 19 to 21, wherein the PEG-lipid has an affinity for thrombin and factor H.

26. The PEG-lipid according to any one of claims 19 to 21, wherein the PEG-lipid is capable of being anchored in a lipid layer.

27. The PEG-lipid of claim 26, wherein the PEG-lipid is capable of anchoring in the cell membrane of a biological tissue and / or in the lipid bilayer of a liposome.

28. The PEG-lipid of claim 26, wherein the PEG-lipid is a phospholipid capable of anchoring in the lipid layer.

29. A biomaterial comprising at least one poly(ethylene glycol) lipid (PEG-lipid) according to any one of claims 19 to 28, wherein the at least one PEG-lipid is anchored in the cell membrane of the biomaterial.

30. The biomaterial according to claim 29, wherein the biomaterial is selected from pancreatic islets, mesenchymal stem cells (MSCs), endothelial cells, β cells, erythrocytes, hepatocytes, kidneys, hearts, pancreas, livers, lungs, uteruses, bladders, thymus, intestines, and spleens.

31. A liposome comprising at least one poly(ethylene glycol) lipid (PEG-lipid) according to any one of claims 19 to 28, wherein the at least one PEG-lipid is anchored in the lipid bilayer of the liposome.

32. Use of the poly(ethylene glycol) lipid (PEG-lipid) according to any one of claims 19 to 28 for the preparation of a medicament for treating thrombotic inflammation.

33. Use of the poly(ethylene glycol) lipid (PEG-lipid) according to any one of claims 19 to 28 for the preparation of a medicament for treating immediate blood-mediated inflammatory response (IBMIR).

34. Use of the poly(ethylene glycol) lipid (PEG-lipid) according to any one of claims 19 to 28 for the preparation of a medicament for treating ischemia-reperfusion injury (IRI).

35. Use of the poly(ethylene glycol) lipid (PEG-lipid) according to any one of claims 19 to 28 in the preparation of a medicament for treating stroke.

36. Use of the poly(ethylene glycol) lipid (PEG-lipid) according to any one of claims 19 to 28 for the preparation of a medicament for the treatment of myocardial infarction.

37. An in vitro method for providing sulfated glycosaminoglycan coatings to biological tissues, wherein... in vitro The method includes injecting the biological tissue in vitro Add poly(ethylene glycol) lipids (PEG-lipids) according to any one of claims 19 to 28 to anchor the PEG-lipids in the cell membrane of the biological tissue.

38. An ex vivo method for processing an organ or a portion of said organ, the method comprising: In vitro infusion of a solution comprising the poly(ethylene glycol) lipid (PEG-lipid) according to any one of claims 19 to 28 into the vascular system of the organ or a portion thereof; and In vitro incubation of a solution containing PEG-lipids according to any one of claims 19 to 28 in the vascular system to achieve coating of at least a portion of the endothelial lining of the vascular system with PEG-lipids according to any one of claims 19 to 28.

39. The in vitro method of claim 38, wherein the in vitro incubation comprises in vitro incubating in the vascular system a solution containing a PEG-lipid according to any one of claims 19 to 28 to achieve coating at least a portion of the endothelial lining of the vascular system with the PEG-lipid according to any one of claims 19 to 28, while keeping the organ or the portion thereof immersed in an organ preservation solution.

40. The in vitro method of claim 39, wherein the in vitro incubation comprises in vitro incubating the vascular system with a solution containing a PEG-lipid according to any one of claims 19 to 28 to achieve coating at least a portion of the endothelial lining of the vascular system with a PEG-lipid according to any one of claims 19 to 28, while keeping the organ or the portion thereof immersed in an organ preservation solution containing a PEG-lipid according to any one of claims 19 to 28.

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