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 the sulfated glycosaminoglycan-PEG-lipid. The sulfated glycosaminoglycan-PEG-lipid can be used to treat biological tissues against thrombo-inflammation. Coating biological tissues with sulfated glycosaminoglycan-PEG-lipid can be performed in a single-step procedure and does not cause significant cell aggregation.

Description

[Technical field]

[0001] The present invention relates generally to poly(ethylene glycol) (PEG) lipids, and in particular to such PEG-lipids that contain sulfated glycosaminoglycans, and their production and medical uses. [Background technology]

[0002] Although no severe side effects have been reported after cell transplantation, for example, of islets of Langerhans, mesenchymal stem cells (MSCs) or hepatocytes, the biocompatibility of these therapeutic cells remains unresolved. The infusion of therapeutic cells into the human body is associated with a large loss of transplanted cells as a result of an immune response called thromboinflammation or instantaneous blood-borne inflammatory response (IBMIR). Thromboinflammation, or IBMIR, is an innate immune attack triggered by the complement and coagulation system, followed by rapid platelet binding and infiltration of leukocytes into the clot, resulting in early loss of transplanted cells. In addition, thromboinflammatory responses occur in ischemia-reperfusion injury (IRI) in solid organ transplants, such as kidney and heart transplants. This devastating response destroys tissues and organs after transplantation, which reduces graft survival.

[0003] Therefore, to achieve successful treatment and high levels of engraftment of therapeutic cells and solid organs, it is important to protect the cell surface from this thromboinflammatory attack.

[0004] Several studies have shown that thromboinflammation can be modulated via systemic administration of anticoagulants, such as thrombin inhibitors, melagatran, low molecular weight dextran sulfate, and / or complement inhibitors, to prevent early unwanted reactions, but 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 endothelial cell surface and plays an important role in regulating aggregation as well as complement and platelet activation. Therefore, mimicking endothelial surfaces by surface modification with heparin and heparin complexes has been proposed as an approach to modulate thromboinflammation occurring in cell and organ transplantation [1-3]. However, surface modification with heparin and heparin complexes requires several process steps; chemical modification of the cell surface and reaction with heparin, with washing processes required after each step. Another problem associated with surface modification with heparin and heparin complexes is cell aggregation after reaction with heparin. Heparin molecules also cross-link between cells, thereby causing cell clumping.

[0006] Therefore, there is a need for compounds that can be used to protect biological tissues against thromboinflammation and that do not have the disadvantages associated with the prior art solutions. Summary of the Invention

[0007] A general object is to provide molecules that are useful for protecting biological tissue against thromboinflammation and that do not have at least some of the disadvantages associated with the prior art solutions.

[0008] This and other objectives are met by the present invention as defined herein.

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

[0010] An embodiment 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 also includes adding a reducing agent to the Schiff base intermediate to form a sulfated glycosaminoglycan-PEG-lipid.

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

[0012] A further aspect of the invention relates to a biological tissue comprising at least one such PEG-lipid anchored to a cell membrane of the biological tissue and a liposome comprising at least one such PEG-lipid anchored to the lipid bilayer of the liposome.

[0013] Aspects of the present invention also define PEG-lipids according to the present invention for use as a medicament, for use in the treatment of thromboinflammation, for use in the treatment of instantaneous blood-mediated inflammatory response (IBMIR), for use in the treatment of ischemia-reperfusion injury (IRI), for use in the treatment of stroke and for use in the treatment of myocardial infarction.

[0014] Another aspect of the present invention relates to an in vitro method of providing a biological tissue having a sulfated glycosaminoglycan coating, the in vitro method comprising adding a PEG-lipid according to the present invention to the biological tissue in vitro to immobilize the PEG-lipid to the cell membrane of the biological tissue.

[0015] A further aspect of the present invention defines an ex vivo method of treating an organ or part of an organ. The method comprises ex vivo injection of a solution comprising PEG-lipids according to the present invention into the vasculature of the organ or part of an organ. The method also comprises ex vivo incubation of the solution comprising PEG-lipids according to the present invention with the vasculature, allowing coating of at least a portion of the intima of the vasculature with the PEG-lipids according to the present invention.

[0016] The PEG-lipids of the present invention can be used to coat cells and lipid membrane structures such as liposomes by a single step procedure.Such coating of lipid membrane structures furthermore does not cause significant aggregation or clumping of cells or liposomes.The PEG-lipids of the present invention can thereby be used to protect biological tissues against thrombo-inflammation, but without the drawbacks associated with the prior art solutions.

[0017] The embodiments, together with further objects and advantages thereof, may best be understood by reference to the following description taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0018] [Figure 1] Figure 1 is a schematic diagram of heparin-conjugated PEG-lipids (fHep-lipids): fHep-C-lipid, fHep-K1C-lipid, fHep-K2C-lipid, fHep-K4C-lipid, and fHep-K8C-lipid. [Diagram 2] Figure 2 shows a schematic of the synthesis of fHep-lipid. (A) Mal-PEG-lipid was reacted with C, K1C, K2C, K4C, or K8C and subsequently conjugated with fragmented heparin (fHep). (B) Unfractionated heparin (UFH) was fragmented into fragmented heparin (fHep). (C) fHep-KnC-lipid (n=0, 1, 2, 4, 8). [Diagram 3] FIG. 3 shows the absorbance of fHep and heparin at 260 nm (N=3). [Figure 4] FIG. 4 shows (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). [Diagram 5] FIG. 5 shows (A) the size and (B) the zeta potential (N=3) of fHep-lipids. [Figure 6]FIG. 6 shows a quartz crystal microbalance with dissipation monitoring (QCM-D) based analysis of the antithrombin (AT) binding activity of fHep-lipids. [Figure 7] FIG. 7 shows a quantitative analysis of the amount of AT bound to fHep-lipid and cationic-PEG-lipid (N=3). [Figure 8] FIG. 8 shows a QCM-D-based analysis of the AT-binding activity of fHep(-)-lipids. [Figure 9] FIG. 9 shows the quantitative analysis of the binding amounts (N=3) of AT and bovine serum albumin (BSA) to fHep(-)-lipid. [Figure 10] FIG. 10 shows a QCM-D-based analysis of the factor H binding activity of fHep(-)-lipids. [Figure 11] FIG. 11 shows (A) quantitative analysis of the amounts of factor H and AT bound to fHep(-)-lipids and Mal-PEG-lipids (N=3) and (B) the calculated molar ratios of immobilized factor H to fHep(-)-lipids and Mal-PEG-lipids (N=3). [Figure 12] FIG. 12 shows the anti-factor Xa activity (N=3) of fHep-lipid-modified liposomes. [Figure 13] FIG. 13 shows the size of fHep-lipid-modified liposomes (N=3). [Figure 14] FIG. 14 shows the polydispersity index (PDI) (N=3) of fHep-lipid-modified liposomes. [Figure 15] FIG. 15 shows the zeta potential (N=3) of fHep-lipid-modified liposomes. [Figure 16] FIG. 16 shows fluorescence images of AT (Alexa488 labeled) on the surface of human red blood cells treated with fHep(-)-lipid, K1C-PEG-lipid, and fHep. [Figure 17]FIG. 17 shows quantitative analysis by flow cytometry of the amount of AT (Alexa488-labeled) bound to the surface of red blood cells treated with fHep(-)-lipid, K1C-PEG-lipid, and fHep. [Figure 18] FIG. 18 shows the anti-factor Xa activity of fHep-lipid-modified CCRF-CEM cells (*: p<0.05, N=3). [Figure 19] Figure 19 shows the effect of hMSC modification with fHep-lipid on blood compatibility. (A) Confocal images of hMSC treated with fHep-lipid and Alexa488-labeled AT. Here, fHep-lipid is fHep-K1C(-)-lipid and fHep-K8C(-)-lipid. Scale bar: 40 μm. (B) Quantitative analysis of AT binding to modified hMSC by flow cytometry. Error bars indicate standard deviation (N=5). (C) Viability assay of modified hMSC by trypan blue dye exclusion method. Error bars indicate standard deviation (N=5). (E)-(G) Loop model assay of modified hMSC in human whole blood. Modified hMSC were incubated in human whole blood (0.5 IU / mL UFH) at 1.0 × 105 cells / mL for 2 h at 37 °C. Here, hMSCs were modified with fHep-KnC(-)-lipid (n=1 and 8) and K1C-PEG-lipid. PBS-added whole blood and untreated hMSCs were used as controls. The figure shows (D) relative platelet counts and (E) TAT, (F) C3a, and (G) sC5b-9 production. Error bars indicate standard deviation (N=6). [Figure 20] Figure 20 shows the loop model assay of modified hMSCs in human whole blood. Modified hMSCs were incubated at 1.0x104 cells / mL in human whole blood (0.5IU / mL UFH) for 2 hours at 37°C. Here, hMSCs were modified with fHep-KnC(-)-lipid (n=1 and 8) and K1C-PEG-lipid. PBS-added whole blood and untreated hMSCs were used as controls. The figure shows (A) relative platelet counts and (B) TAT, (C) C3a, and (D) sC5b-9 production. Error bars indicate standard deviation (N=6). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention relates generally to poly(ethylene glycol) (PEG) lipids, in particular such PEG-lipids containing sulfated glycosaminoglycans, and their production and medical uses.

[0020] The PEG-lipids of the present invention are useful in the surface modification of cells and organ grafts to mimic endothelial surface, thereby protecting such cells and organ grafts against thrombo-inflammation. PEG-lipids have several advantages compared to the prior art approach of using heparin and heparin complexes. First, the surface modification using the PEG-lipids of the present invention can be carried out in a single step, without the need for any chemical modification of the cell surface. This means that the surface modification process of cells or organ grafts using PEG-lipids can be carried out more easily compared to the prior art, which requires several process steps, including chemical modification of the cell surface, which may cause harmful effects on cells. Second, the PEG-lipids of the present invention do not crosslink when attached to cells. Thereby, the PEG-lipids are not impaired by the shortcomings of the prior art, which causes cell clumping and aggregation after reaction with heparin or heparin complexes.

[0021] The PEG-lipids of the present invention are therefore useful in protecting biological tissues, including cells and organ transplants, against thromboinflammation.

[0022] One aspect of the present invention relates to a method for producing a PEG-lipid, the method comprising 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 also comprises adding a reducing agent to the Schiff base intermediate to form a sulfated glycosaminoglycan-PEG-lipid.

[0023] Glycosaminoglycan-PEG-lipids are formed by Schiff base chemistry, which involves nucleophilic addition to form hemiaminal, followed by dehydration to generate a Schiff base intermediate.The starting material in this reaction is a cationic-PEG-lipid that contains 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 sulfated glycosaminoglycan to form a Schiff base intermediate (C=N bond between sulfated glycosaminoglycan and cationic-PEG-lipid), which is reduced by adding a reducing agent to form sulfated glycosaminoglycan-PEG-lipid with sulfated glycosaminoglycan attached to PEG-lipid through a C=N bond.

[0024] Thus, the sulfated glycosaminoglycan is attached to the cationic-PEG-lipid through a covalent bond, more particularly a covalent bond between a carbonyl group, preferably a C in an aldehyde group, of the sulfated glycosaminoglycan and an N in an amino group of the cationic-PEG-lipid, i.e., a C-N bond.

[0025] The cationic-PEG-lipid containing at least one amino group can be any PEG-lipid, including a PEG phospholipid, that contains at least one amino group.

[0026] The PEG-lipid may have the general structural formula (II) with the corresponding PEG-phospholipid according to the general structural formula (III), where R1 and R2 represent the lipid portion of the molecule. [ka] [ka]

[0027] In one embodiment, Y in formula (II) and formula (III) is selected from the group consisting of H, CH3, maleimide and N-hydroxysuccinimide.

[0028] PEG-lipid as used herein includes any complex between PEG and at least one lipid, including fatty acid, phospholipid, glycerolipid, glycerophospholipid, sphingolipid, sterol, prenol, saccharolipid, and polyketide.In a preferred embodiment, PEG-lipid is selected so that it can be fixed to lipid layer, such as cell membrane of biomaterial.Currently preferred PEG-lipid is PEG-phospholipid.

[0029] At least one amino group is preferably 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.

[0030] Thus, 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 that includes at least one amino group. In one embodiment, the at least one cysteine ​​peptide has at least one K n C-peptide, at least one CK n and n is 0 or a positive integer not greater than 20, preferably not greater than 15, more preferably not greater than 10, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0031] K n C or CK n If n=0 in the peptide, the cationic-PEG-lipid will contain one amino group. n C or CK n Each lysine in the peptide adds one amino group to the cationic-PEG-lipid, which therefore contains n+1 amino groups.

[0032] In one embodiment, the maleimide-conjugated PEG-lipid is formed by mixing α-N-hydroxysuccinimidyl-ω-maleimidyl PEG (NHS-PEG-Mal), triethylamine and 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine (DPPE) in dichloromethane. The maleimide-conjugated PEG-lipid is then precipitated by adding diethyl ether to a mixture of NHS-PEG-Mal, triethylamine and DPPE in dichloromethane.

[0033] Sulfated glycosaminoglycans contain at least one carbonyl group. The currently preferred carbonyl group is an aldehyde group (-CHO). However, the present invention also includes, but is not limited to, sulfated glycosaminoglycans that contain at least one aldehyde group, at least one ketone (-C(=O)-), at least one carboxyl group (-C(=O)OH), at least one carboxylic ester group (-C(=O)O-) and / or at least one amide group (-C(=O)NR- or -C(=O)NH-). Sulfated glycosaminoglycans may contain one carbonyl group, e.g., one aldehyde group, or multiple, i.e., at least two carbonyl groups, e.g., multiple aldehyde groups.

[0034] Glycosaminoglycans (GAGs) are long linear polysaccharides that contain a repeating disaccharide unit, i.e., multiple disaccharide units. In most cases, the repeating unit contains an amino sugar, such as N-acetylglucosamine or N-acetylgalactosamine, together with an uronic acid sugar, such as glucuronic acid or iduronic acid, or galactose. In one embodiment, the sulfated glycosaminoglycan is selected from the group consisting of heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratin sulfate, and hyaluronic acid.

[0035] A currently preferred sulfated glycosaminoglycan is a heparin containing at least one carbonyl group, preferably a heparin containing at least one aldehyde group. In a particular embodiment, the sulfated glycosaminoglycan is a fragmented heparin (fHep) containing at least one carbonyl group, preferably a fragmented heparin containing at least one aldehyde group.

[0036] Such fragmentation of heparin introduces carbonyl groups, preferably aldehyde groups, into the heparin molecule. Furthermore, fragmentation shortens the length of the heparin chains, thereby reducing the molecular weight compared to unfractionated heparin (UFH).

[0037] In one embodiment, the fragmentation reaction comprises 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 within the range of 2 to a maximum of 6, preferably within the range of 3 to a maximum of 5, more preferably to 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 within the range of 6 to 8, preferably within the range of 6.5 to 7.5, more preferably to 7 to form a fragmented heparin comprising at least one carbonyl group, preferably at least one aldehyde group. The fragmentation reaction optionally comprises dialyzing the fragmented heparin comprising at least one carbonyl group, preferably at least one aldehyde group, against water, and lyophilizing the fragmented heparin comprising at least one carbonyl group, preferably at least one aldehyde group.

[0038] The acidic solution is preferably selected from a sulfuric acid (H2SO4) solution or an acetic acid (CH3COOH) solution, preferably a sulfuric acid (H2SO4) solution.

[0039] In one embodiment, adding the reducing agent comprises adding sodium cyanoboronhydride (NaBH3CN) to the Schiff base intermediate to form a sulfated glycosaminoglycan-PEG-lipid. Thus, in a preferred embodiment, the reducing agent is sodium cyanoboronhydride. However, the embodiments are not so limited. Other reducing agents besides sodium cyanoboronhydride can be used alternatively or in addition, including, for example, sodium triacetoxyborohydride and sodium borohydride.

[0040] Figure 2A shows an example of the synthesis of fHep-lipids in schematic form. Mal-PEG-lipids were reacted with C-peptide (n=0), K1C-peptide (n=1), K2C-peptide (n=2), K4C-peptide (n=4), or K8C-peptide (n=8), followed by conjugation with sulfated glycosaminoglycan-PEG-lipid fHep-KnC-lipid with fHep containing aldehyde groups. Figure 2B shows the fragmentation of unfractionated heparin (UHF) to fragmented heparin (fHep), and Figure 2C shows one embodiment of sulfated glycosaminoglycan-PEG-lipid.

[0041] Figure 1 shows a schematic of sulfated glycosaminoglycan-PEG-lipid (fHep-KnC-lipid) synthesized according to Figures 2A-C and immobilized in a lipid bilayer. Figure 1 also shows the maximum number of fHep molecules per fHep-KnC-lipid, i.e., n+1 fHep molecules.

[0042] In one embodiment, any unreacted amino groups in the sulfated glycosaminoglycan-PEG-lipid are converted to a carboxylic acid group.

[0043] Carboxylic acid groups are generally less reactive than amino groups. Therefore, converting the unreacted amino groups in sulfated glycosaminoglycan-PEG-lipids to carboxylic acid groups makes the sulfated glycosaminoglycan-PEG-lipids less toxic and therefore less harmful to cells. In addition, the negative charge introduced by the carboxylic acid group inhibits non-specific protein binding to the surface on which the sulfated glycosaminoglycan-PEG-lipids are immobilized (see FIG. 9).

[0044] In certain embodiments, any such unreacted amino groups are converted to carboxylic acid groups by adding an acid anhydride to the sulfated glycosaminoglycan-PEG-lipid to convert any unreacted amino groups in the sulfated glycosaminoglycan-PEG-lipid to a carboxylic acid group.

[0045] Any acid anhydride can be used in the conversion of unreacted amino groups to carboxylic acid groups. Illustrative, but non-limiting examples include succinic anhydride (SA), glutaric anhydride, diglycolic anhydride, and combinations thereof, with SA being preferred.

[0046] Another aspect of the present invention relates to a PEG-lipid comprising at least one sulfated glycosaminoglycan.

[0047] At least one sulfated glycosaminoglycan is attached to the PEG-lipid via a bond formed between an amino group of the cationic-PEG-lipid containing at least one amino group and a carbonyl group of the at least one sulfated glycosaminoglycan containing at least one carbonyl group to form a Schiff base intermediate that is reduced by a reducing agent.

[0048] Thus, according to the present invention, the sulfated glycosaminoglycan is attached to the PEG-lipid through a covalent bond, in particular a covalent bond between the carbonyl group, preferably the C of the aldehyde group, of the sulfated glycosaminoglycan and the N of the amino group of the cationic-PEG-lipid. This covalent bond between carbon and nitrogen is a C-N bond.

[0049] In one embodiment, the PEG-lipid comprises at least one sulfated glycosaminoglycan and a K bond that interconnects the PEG-lipid. n C and / or CK n In this embodiment, C is cysteine, K is lysine, and n is 0 or a positive integer less than or equal to 20. In one embodiment, n is selected in the interval from 0 to 15, preferably in the interval from 0 to 10, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0050] In one embodiment, the sulfated glycosaminoglycan is K n C and / or CK n The amino group or K of any lysine residue in the linkage n C and / or CK n It is attached to the PEG-lipid via a bond formed between the N-terminal amine of the linkage and a carbonyl group, preferably an aldehyde group, of at least one sulfated glycosaminoglycan that contains at least one carbonyl group, preferably at least one aldehyde group.

[0051] In one embodiment, the PEG-lipid portion of the sulfated glycosaminoglycan-PEG-lipid has the formula (I): [ka]

[0052] In formula (I), p, q are integers independently selected within the interval of 10 up to 16, preferably p, q are independently 10, 12, 14 or 16, more preferably p=q=14. m is selected such that the PEG chain has an average molecular weight selected within the range of 1 kDa up to 40 kDa, preferably 3 kDa up to 10 kDa, more preferably 5 kDa. The sulfated glycosaminoglycan molecule can then be attached to the PEG-lipid according to formula (I) at the N-terminal amine or amino group of the lysine residue(s).

[0053] The average molecular weight as defined herein indicates that, although individual PEG chains may have molecular weights that differ from this average molecular weight, the average molecular weight represents the median molecular weight of the PEG chains, which further implies that there is a natural distribution of molecular weights around this average molecular weight for the PEG chains.

[0054] In one embodiment, the sulfated glycosaminoglycan is fragmented heparin.

[0055] In one embodiment, the fragmented heparin has a weight average molecular weight (M) selected within the interval of 2.5 kDa to 15 kDa, preferably within the interval of 4 kDa to 10 kDa, for example within the interval of 5 kDa to 10 kDa, more preferably within the interval of 5 kDa to 8 kDa, or within the interval of 7 kDa to 9 kDa. w ).

[0056] In one embodiment, the sulfated glycosaminoglycan-PEG-lipid does not contain any unreacted or free amino groups. In certain embodiments, all unreacted or free amino groups in the sulfated glycosaminoglycan-PEG-lipid are converted to carboxylic acid groups.

[0057] An unreacted or free amino group as referred to herein refers to any N-terminal amine in a PEG-lipid and any amino group in any lysine residue, such as shown in formula (I), that is not attached to any sulfated glycosaminoglycan molecule.

[0058] In one embodiment, the sulfated glycosaminoglycan-PEG-lipid is obtainable or obtained by the methods as disclosed herein.

[0059] The sulfated glycosaminoglycan-PEG-lipids of the present invention have affinity for antithrombin (AT) (see Figures 6-10, Figure 11A) and factor H (see Figures 10, 11A and 11B).

[0060] AT is a protein molecule that inactivates several enzymes of the coagulation system. Its activity is increased many-fold 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 has anti-FXa activity by being able to bind to AT, thereby having an anticoagulation effect.

[0061] Factor H is a member of the regulators of complement activation family and is a complement control protein. Its main function is to regulate the alternative pathway of the complement system, ensuring that it is directed towards pathogens or other dangerous materials and not damaging host tissues. Factor H regulates complement activation on self cells and surfaces by retaining both cofactor activity for factor I-mediated C3b cleavage and decay-accelerating activity for the alternative pathway C3-convertase, C3bBb. Factor H exerts a protective effect on self cells and surfaces, but not on bacterial or viral surfaces. This is thought to be the result of factor H having the ability to adopt forms with lower or higher activity as a cofactor for C3 cleavage or decay-accelerating activity. The less active form is the predominant form in solution and is sufficient to control fluid-phase amplification. The more active form is thought to be induced when factor H binds to glycosaminoglycans and / or sialic acids that are commonly present on host cells but not normally on pathogen surfaces, ensuring that self surfaces are protected while complement proceeds unabated on foreign surfaces.

[0062] Thus, cell surfaces containing immobilized sulfated glycosaminoglycan-PEG-lipids of the invention have the ability to attract and bind AT and factor H, thereby protecting the cell surface from thromboinflammation. The sulfated glycosaminoglycan-PEG-lipids of the invention have this biological effect even when attached to lipid bilayers, such as cell surfaces or liposomes (see Figures 12, 17 and 18).

[0063] The experimental data as presented herein further demonstrates that modification of lipid bilayers with the sulfated glycosaminoglycan-PEG-lipids of the present invention does not result in any aggregation or cell clumping, which is common when cell surfaces are modified with heparin according to the prior art (see FIG. 13).

[0064] 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 such a case, the sulfated glycosaminoglycan-PEG-lipid is attached or immobilized to the lipid bilayer through the PEG-lipid group as shown in Figure 1. For example, the present invention relates to a liposome comprising at least one PEG-lipid according to the present invention immobilized in the lipid bilayer of the liposome.

[0065] A further aspect of the present invention relates to a biological tissue comprising at least one PEG-lipid according to the present invention immobilized within the cell membrane of the biological tissue.

[0066] The biological tissue may be an individual cell or a plurality of cells, such as stem cells, including, as specific but non-limiting examples, mesenchymal stem cells (MSCs) and embryonic stem cells (ESCs); hepatocytes; endothelial cells; beta cells (insulin-producing cells) and red blood cells. The biological tissue may alternatively be a cluster of cells, such as islets of Langerhans. The biological tissue may also be in the form of a tissue or organ, or a part thereof, such as kidney, heart, pancreas, liver, lung, uterus, bladder, thymus, intestine and spleen. In certain embodiments, at least a part of the vasculature and optionally the parenchyma of the tissue or organ, or a part thereof, may be coated with at least one PEG-lipid according to the present invention.

[0067] Another aspect of the present invention relates to a PEG-lipid according to the present invention for use as a medicament.

[0068] Further aspects of the invention relate to PEG-lipids according to the invention for use in the treatment of thromboinflammation, for use in the treatment of instantaneous blood-mediated inflammatory response (IBMIR), for use in the treatment of ischemia-reperfusion injury (IRI), for use in the treatment of stroke and / or for use in the treatment of myocardial infarction.

[0069] A related aspect of the present invention defines the use of a PEG-lipid according to the invention for the manufacture of a medicament for the treatment of thromboinflammation, IBMIR, IRI, stroke and / or myocardial infarction.

[0070] The PEG-lipids of the present invention can be administered to a subject in need thereof by systemic or local administration. Non-limiting examples of systemic administration routes include intravenous administration and subcutaneous administration. Local administration includes injection of the PEG-lipids of the present invention locally into a target organ or tissue of a subject.

[0071] The PEG-lipid of the present invention is preferably administered in the form of a PEG-lipid solution.The solution containing PEG-lipid molecules can be, for example, saline, aqueous buffer solution or organ preservation solution.Specific but non-limiting examples of aqueous buffer solutions that can be used include phosphate buffered saline (PBS) and citrate solution.

[0072] Another aspect of the present invention relates to an in vitro method for providing a biological tissue having a sulfated glycosaminoglycan coating, the in vitro method comprising adding a PEG-lipid according to the present invention to the biological tissue in vitro to immobilize the PEG-lipid to the cell membrane of the biological tissue.

[0073] One aspect of the present invention relates to an ex vivo method of treating an organ or part of an organ, the method comprising ex vivo injection of a solution comprising PEG-lipids according to the present invention into the vasculature and optionally into the soft tissue of the organ or part of an organ. The method also comprises ex vivo incubation of a solution comprising PEG-lipids according to the present invention in the vasculature and optionally in the soft tissue, to allow coating of at least a portion of the inner lining of the vasculature and preferably the soft tissue with PEG-lipids according to the present invention.

[0074] In one embodiment, the ex vivo incubating step comprises incubating a solution comprising the PEG-lipids according to the present invention ex vivo in the vasculature, and optionally the soft tissue, to allow coating of at least a portion of the lining of the vasculature, and preferably the soft tissue, with the PEG-lipids according to the present invention, while the organ or part of the organ is immersed in an organ preservation solution, preferably an organ preservation solution comprising the PEG-lipids according to the present invention.

[0075] Thus, ex vivo methods involve introducing PEG-lipids into the vasculature of an organ or part of an organ, where they are allowed to interact with and bind to endothelial and parenchymal cell membranes. Figure 1 shows a schematic of this principle using PEG-lipid molecules that interact hydrophobically with lipid bilayers, thereby anchoring or binding the PEG-lipid molecules in the cell membranes via the phospholipid groups.

[0076] The interaction between the PEG-lipid molecules and the lipid bilayer of the parenchyma, such as the endothelium and, optionally, in the case of the kidney, the renal parenchyma, preferably occurs ex vivo, while the organ or part of the organ is placed or immersed in an organ preservation solution, preferably an organ preservation solution containing the PEG-lipid molecules.

[0077] In certain embodiments, organ or organ part is first ex vivo injected with the solution containing PEG-lipid molecules into the vascular system of organ or organ part, and optionally into its parenchyma.This ex vivo injection is advantageously carried out as soon as possible after explantation and removal of organ or organ part from donor's body.The perfused organ or organ part is then immersed in organ preservation solution, preferably containing PEG-lipid, and is maintained therein at low temperature, preferably about 4°C.

[0078] In another particular embodiment, the organ or organ part is first immersed in an organ preservation solution, preferably containing PEG-lipid molecules, and then the solution containing PEG-lipid molecules is ex vivo injected into the vascular system of the organ or organ part, and optionally into its parenchyma.This ex vivo injection can be performed while the organ or organ part is immersed in the organ preservation solution, preferably containing PEG-lipid molecules.Alternatively, the organ or organ part is temporarily removed from the organ preservation solution, ex vivo injection is performed, and then it is returned to the organ preservation solution, preferably containing PEG-lipid molecules.

[0079] In one embodiment, the method also includes ex vivo injection of the organ preservation solution into the vascular system to flush unbound PEG-lipid molecules from the vascular system. Thus, unbound PEG-lipid molecules are preferably flushed out in one or more, i.e., at least two, washing steps using the organ preservation solution.

[0080] In one embodiment, the ex vivo injection of the solution comprising the PEG-lipid molecules comprises ex vivo clamping one of the arteries and veins of the vascular system. This embodiment also comprises ex vivo injection of the solution comprising the PEG-lipid molecules into the other of the arteries and veins and ex vivo clamping the other of the arteries and veins.

[0081] In another embodiment, the solution with PEG-lipid molecules is injected into an artery (or vein) of the vascular system of an organ or part of an organ until the solution emerges in the vein (or artery) of the organ or part of an organ. This confirms that the solution with PEG-lipid molecules has filled the vascular system. At that point, the artery and vein are clamped.

[0082] The solution containing the PEG-lipid molecules can be added intravenously or intraarterially. In certain embodiments, the solution is injected into an artery. In certain such embodiments, any initial clamping is then preferably performed on a vein in the vascular system.

[0083] The solution containing the PEG-lipid molecules is preferably incubated ex vivo in the vasculature for a period of from 10 minutes up to 48 hours to allow the PEG-lipid molecules to hydrophobically interact with the endothelial cell membrane, thereby coating at least a portion of the vasculature of the organ or part of an organ. The ex vivo incubation is preferably carried out for from 20 minutes up to 36 hours, more preferably from 30 minutes up to 24 hours, such as from 30 minutes up to 12 hours, up to 8 hours, up to 4 hours, or up to 1 hour.

[0084] The amount of solution containing PEG-lipid molecules injected into the vascular system depends on the type of organ and the size of the organ (adult vs. pediatric). In general, the volume of the solution should be sufficient to fill the vascular system of the organ. In most practical applications, 5 mL to a maximum of 250 mL of solution containing PEG-lipid molecules is injected ex vivo into the vascular system. In a preferred embodiment, 5 mL to a maximum of 100 mL, preferably 5 mL to a maximum of 50 mL of solution containing PEG-lipid molecules is injected ex vivo into the vascular system.

[0085] In one embodiment, the solution contains 0.25 mg / mL to a maximum of 25 mg / mL of PEG-lipid molecules. In a preferred embodiment, the solution contains 0.25 mg / mL to a maximum of 10 mg / mL, preferably 0.25 mg / mL to a maximum of 5 mg / mL, e.g., 2 mg / mL of PEG-lipid molecules.

[0086] The above-mentioned concentrations of PEG-lipid molecules can also be used for organ preservation solutions containing PEG-lipid molecules.

[0087] According to the invention, a solution containing PEG-lipid molecules is incubated ex vivo in the vascular system while the organ or organ part remains placed or immersed in the organ preservation solution, preferably containing PEG-lipid molecules. In addition, the organ or organ part is preferably also maintained at a temperature above 0° C. but below 8° C., preferably above 0° C. but below 6° C., more preferably above 0° C. but below 4° C.

[0088] In this embodiment, the organ or organ part is immersed in an organ preservation solution, preferably containing PEG-lipid molecules, for an incubation time during which the PEG-lipid molecules interact with and bind to the cell membrane of the endothelium of the vasculature. The organ or organ part is also preferably kept cold, i.e., at a temperature close to but higher than 0°C. It has been shown that the theoretically perfect temperature for organ preservation is between 4°C and 8°C. Higher temperatures result in hypoxic damage of the organ as metabolism is not efficiently reduced, and temperatures below 4°C increase the risk of cryoinjury accompanied by protein denaturation.

[0089] 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, which is immersed in an organ preservation solution. This first plastic bag is placed in a second plastic bag filled with saline, and then these two plastic bags are placed in a third plastic bag filled with saline, which is then placed in an icebox. More advanced organ preservation devices for maintaining organs in a temperature-controlled environment are available, such as the Sherpa Pak™ transport system from Paragonix Technologies, Waves from Waters Medical Systems, and the LifePort transporter from Organ Recovery systems.

[0090] The solution containing the PEG-lipid molecules can be saline, an aqueous buffer solution, or an organ preservation solution.

[0091] Specific, but non-limiting, examples of aqueous buffer solutions that can be used include PBS and citrate solutions.

[0092] The organ preservation solution that can be used to inject the PEG-lipid molecules and / or to wash the vasculature of the organ or organ part before or after ex vivo injection of the PEG-lipid molecules and / or in which the organ or organ part can be immersed can be selected from known organ preservation solutions. Specific, but non-limiting examples of such organ preservation solutions include histidine-tryptophan-ketoglutarate (HTK) solution, citric acid solution, University of Wisconsin (UW) solution, Collins solution, Celsior solution, Kyoto University solution and Institut Georges Lopez-1 (IGL-1) solution.

[0093] The subject is preferably a human subject, however, the invention can also be used in veterinary applications where the subject is a non-human subject, such as a non-human mammal, including, but not limited to, a cat, dog, horse, cow, rabbit, pig, sheep, goat, and guinea pig.

[0094] A further aspect of the present invention relates to a method for treating, inhibiting or preventing thromboinflammation, IBMIR, IRI, stroke and / or myocardial infarction in a subject, comprising administering a PEG-lipid according to the present invention to a subject in need thereof. In another embodiment, the method optionally but preferably comprises the previously described method steps of ex vivo injection of a solution comprising the PEG-lipid molecules according to the present invention into the vasculature of an organ graft and ex vivo incubation of a solution comprising the PEG-lipid molecules in the vasculature, allowing coating of at least a portion of the intima of the vasculature with the PEG-lipid molecules, while the organ graft is preferably immersed in an organ preservation solution comprising the PEG-lipid molecules.

[0095] The PEG-lipids according to the invention mimic the polysaccharide envelope of normal endothelial cells, thereby allowing local protection against thrombo-inflammation. This approach also avoids the risk of bleeding, since coating of the endothelial cell surface in the target organ requires a smaller amount of modulator compared to systemic administration.

[0096] In one embodiment, the PEG-lipids of the present invention contain heparin, which has a similar function to heparan sulfate proteoglycan (HS). Since heparin can interact with many regulatory factors in the same way as HS, the fHep-lipid coating obtained using the PEG-lipids of the present invention can regulate complex biological responses during IRI and therefore can be easily applied in clinical trials.

[0097] Various methods of heparin coating have already been reported in the art. A layer-by-layer coating of heparin combined with soluble complement receptor 1 (sCR1) has been applied to mouse pancreatic islets [7]. However, this approach cannot be applied to endothelial coating in kidneys, as the use of recombinant sCR1 is not practical and the procedure is complicated. Also, cationic avidin has been used for heparin coating of pancreatic islets via electrostatic interactions [8]. However, due to the strong antigenicity of avidin, it is difficult to use this method in a clinical context. Heparin-binding peptides have been used for immobilization of heparin by using PEG-lipids on cell surfaces [6,9]. However, this coating procedure still requires some laborious processes, which makes it more difficult to coat the endothelial surfaces of solid organs with heparin.

[0098] Working Example This example demonstrates the production and characterization of heparin-conjugated PEG-lipids (fHep-lipids), which can coat cells and lipid membrane structures such as liposomes by a single-step process.

[0099] Reagents and materials The following reagents and materials were used in the examples. Heparin sodium (UFH, Fujifilm Wako Pure Chemical Industries, Osaka, Japan) Sulfuric acid (H2SO4, Fujifilm Wako Pure Chemical Industries, Ltd.) Sodium nitrite (NaNO2, Fujifilm Wako Pure Chemical Industries, Ltd.) 5M Sodium Hydroxide (NaOH, Fujifilm Wako Pure Chemical Industries, Ltd.) Dialysis membrane (Spectra / Por, MWCO: 3.5-5 kDa, Repligen Corporation, Waltham, MA, USA) Sodium cyanoborohydride (NaCNBH3, Sigma-Aldrich, St. Louis, MO, USA) D-PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.) Biophen heparin (AT+) (Cosmo Bio Co., Ltd., Tokyo, Japan) Dextran (M w : 1080Da, 9890Da, 43500Da, 123600Da, Sigma-Aldrich) Sodium chloride (NaCl, Fujifilm Wako Pure Chemical Industries, Ltd.) Distilled water (Fujifilm Wako Pure Chemical Industries, Ltd.) Dimethyl sulfoxide (DMSO, Fujifilm Wako Pure Chemical Industries, Ltd.) α-N-hydroxysuccinimidyl-ω-maleimidyl poly(ethylene glycol) (NHS-PEG-Mal, M w :5000Da, NOF Corporation, Tokyo, Japan) Triethylamine (Sigma-Aldrich, St. Louis, MO) 1,2-Dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine (DPPE, NOF Corporation) Dichloromethane (Sigma-Aldrich) 1,6-diphenyl-1,3,5-hexatriene (DPH, Sigma-Aldrich) L-Cysteine ​​(C,M w =121.16Da, Fujifilm Wako Pure Chemical Corporation) Lysine-cysteine ​​(K1C, M w =249.335Da, Bex Co., Ltd., Tokyo, Japan) Lysine-Lysine-Cysteine ​​(K2C, M w =377.51Da, Vex Co., Ltd.) Lysine-lysine-lysine-lysine-cysteine ​​(K4C, M w =633.85Da, GenScript, Tokyo, Japan) Lysine-lysine-lysine-lysine-lysine-lysine-lysine-lysine-cysteine ​​(K8C, M w =1146.54Da, GenScript) Fluorescamine (Fujifilm Wako Pure Chemical Industries, Ltd.) Glycine (Fujifilm Wako Pure Chemical Industries, Ltd.) Antithrombin (AT, Nonthron 500 for injection, Takeda Pharmaceutical Co., Ltd., Osaka, Japan) 1-Dodecanethiol (Fujifilm Wako Pure Chemical Industries, Ltd.) Bovine serum albumin (BSA, Sigma-Aldrich) Cholesterol (Fujifilm Wako Pure Chemical Corporation) Dipalmitoylphosphatidylcholine (DPPC, MC-6060, NOF Corporation) Poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (MPC polymer, consisting of 2-methacryloyloxyethyl phosphorylcholine (MPC) and n-butyl methacrylate (BMA) domains in a ratio of 3:7, NOF Corporation, Tokyo, Japan) Polyoxyethylene sorbitan monolaurate (TWEEN® 20, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) 3,3',5,5'-Tetramethylbenzidine (TMB, ready-to-use solution, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) Ethanol (99.5%, Fujifilm Wako Pure Chemical Industries, Ltd.) Citric acid monohydrate (CAM, Fujifilm Wako Pure Chemical Industries, Ltd.) Sodium dodecyl sulfate (SDS, Fujifilm Wako Pure Chemical Industries, Ltd.) Cholesterol quantification kit (T-Cho E, Fujifilm Wako Pure Chemical Corporation) Dioxane (dehydrated) (Kanto Chemical Co., Ltd.) Succinic anhydride (SA, Fujifilm Wako Pure Chemical Industries, Ltd.) Trypan blue (Thermo Fisher Scientific, Waltham, MA, USA) Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA). Trypsin-EDTA (0.25%, Thermo Fisher Scientific, Waltham, MA, USA) CCRF-CEM (American Type Culture Collection, ATCC, Manassas, VA, USA) Human mesenchymal stem cells (hMSCs, Lonza, Morristown, NJ, USA) Horseradish peroxidase (HRP)-conjugated streptavidin (GE Healthcare, Chicago, IL, USA) RPMI1640 medium (Invitrogen, Carlsbad, CA, USA) Fetal bovine serum (FBS, Thermo Fisher Scientific) Penicillin-Streptomycin, liquid (P / S 0.85% NaCl in 100 mL of aqueous solution, penicillin: 5000 IU / mL, streptomycin: 5000 μg / mL, Thermo Fisher Scientific) Alexa Fluor™ 488 Antibody Labeling Kit (contains sodium bicarbonate and Alexa Fluor™ 488 carboxylic acid, tetrafluorophenyl (TFP) ester in the kit, Thermo Fisher Scientific) Vacuum blood collection tube (EDTA-2Na treated, Terumo Corporation, Tokyo, Japan) Ethylenediaminetetraacetic acid solution (EDTA, 0.5M, pH 8.0, Invitrogen) Factor H (purified from human blood)

[0100] Device The following equipment was used in the examples: pH meter (LAQUA, Horiba, Kyoto, Japan) Nanodrop-1000 (Thermo Fisher Scientific) Nanodrop-3300 (Thermo Fisher Scientific) Quartz crystal microbalance with energy dissipation (QCM, qsense, Biolin Scientific, Gothenburg, Sweden) Gel permeation chromatography (GPC, LC-2000Plus series, JASCO, Tokyo, Japan) Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK) Plate reader (AD200, Beckman Coulter, Miami, FL, USA) Cell counter (Countess, Invitrogen) Extruder (Avantipolar Lipids, Inc., Avantipolar Lipids, Inc., Birmingham, AL, USA) Centrifuge (MX301, Tommy Seiko Co., Ltd., Tokyo, Japan) Centrifuge (Force mini SBC140-115, BM Kiki Co., Ltd., Tokyo, Japan) Confocal laser scanning microscope (CLSM, LSM880, Carl Zeiss, Jena, Germany) Flow cytometer (FCM, BD LSR II, BD Biosciences, San Jose, CA, USA)

[0101] Example 1 - Synthesis and characterization of fragmented heparin (fHep) Synthesis of Fragmented Heparin A sulfuric acid (H2SO4) solution (1 M) and a sodium nitrite (NaNO2) aqueous solution (7 M) were mixed, and the pH of the mixed solution was adjusted to 4. A solution of sodium heparin (unfractionated heparin (UFH), 20 mg / mL in water, 3 mL) was mixed with the mixed solution of H2SO4 and NaNO2 (11 mL) for 15 min at room temperature (RT, approximately 20-25 °C). The pH of the solution was then adjusted to 7 by adding a 1 M aqueous NaOH solution (approximately 4 mL). After the reactants were dialyzed against MilliQ water for 1 day using a dialysis membrane (3.5-5 kDa, Spectra / Por), the solution was lyophilized to obtain fragmented heparin (fHep). The yield was 40%.

[0102] UV spectrum To examine the aldehyde groups of fHep, a solution of fHep (10 mg / mL in PBS) was measured by UV-visible spectrophotometer (Nanodrop1000, Thermo Fisher Scientific, Waltham, MA, USA).

[0103] Determination of the molecular weight of fHep using GPC The molecular weights of UFH and fHep were measured by GPC. The column was Shodex SB803HQ (Showa Denko K.K., Tokyo, Japan). The eluent was 0.1 M NaCl aqueous solution. The flow rate was 0.5 mL / min, and the column oven temperature was 25°C. Dextran (M w : 1080 Da, 9890 Da, 43500 Da, 123600 Da) (Sigma-Aldrich, St. Louis, MO, USA) were used.

[0104] FXa Assay for Testing Heparin Activity The anti-factor Xa activity of synthetic fHep was evaluated using an FXa activity assay kit (Biophen Heparin (AT+), Cosmo Bio Co., Ltd.). The concentration of fHep was 0.01 mg / mL (in PBS), while the concentrations of UFH as standard were 2 IU / mL, 1 IU / mL, and 0.5 IU / mL.

[0105] result Since aldehyde groups have absorbance at 260 nm, the fHep solution has absorbance at that wavelength (Figure 3). There was no absorbance for the original UFH, as shown in Figure 3. The results showed that fHep contains aldehyde groups.

[0106] Number average molecular weight (M n ) was calculated by GPC using dextran standards (Figure 4A). n = 6.1 kDa, whereas UFH has an M n = 22 kDa. The results showed that fHep was a fragment of heparin. The activity of fHep was measured by factor Xa activity assay (Figure 4B). The activity of fHep was about 24% of that of the original UFH.

[0107] Example 2 - Synthesis and evaluation of cationic-PEG-lipids and fHep-lipids Synthesis of Mal-PEG-lipids The synthesis of Mal-PEG-lipids was carried out as previously described [4]. Briefly, α-N-hydroxysuccinimidyl-ω-maleimidyl poly(ethylene glycol) (NHS-PEG-Mal, M w Mal-PEG(5k)-lipid (5000 Da, 200 mg), triethylamine (50 μL) and 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine (DPPE, 20 mg) were dissolved in dichloromethane and stirred for 48 h at RT. Precipitation with diethyl ether gave Mal-PEG(5k)-lipid as a white powder (yield: 80%).

[0108] Synthesis of cationic-PEG-lipids To introduce at least one amine group at the end of the PEG chain, C, K1C, K2C, K4C, and K8C were conjugated to Mal-PEG-lipids, in which each lysine residue contains one 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)-lipid (10 mg / mL, 1000 μL, in PBS). Each resulting solution was rotated at RT for 24 h. The following cationic-PEG-lipids, referred to herein as KnC-PEG-lipids (n: number of lysine residues), were produced: C-PEG-lipid, K1C-PEG-lipid, K2C-PEG-lipid, K4C-PEG-lipid, and K8C-PEG-lipid.

[0109] Synthesis and functional evaluation of fHep-lipids Each cation-PEG-lipid (1 mL, 10 mg / mL in PBS) was mixed with fHep (15 mg, 30 mg, 45 mg, 70 mg, and 120 mg for C-PEG-lipid, K1C-PEG-lipid, K2C-PEG-lipid, K4C-PEG-lipid, and K8C-PEG-lipid, respectively), followed by addition of NaCNBH3 solution (6 μL, 13 μL, 18 μL, 30 μL, and 49 μL, 6.4 M in PBS for C-PEG-lipid, K1C-PEG-lipid, K2C-PEG-lipid, K4C-PEG-lipid, and K8C-PEG-lipid, respectively). The mixed solution was stirred at RT for 3 days (for K8C-PEG-lipid and K4C-PEG-lipid) or 7 days (for K2C-PEG-lipid, K1C-PEG-lipid and C-PEG-lipid) to obtain the following fHep-lipids: fHep-C-lipid, fHep-K1C-lipid, fHep-K2C-lipid, fHep-K4C-lipid, and fHep-K8C-lipid.

[0110] After the reaction, succinic anhydride (SA) was added to convert the unreacted amine groups of fHep-lipids to carboxylic acid groups. Each fHep-lipid (1 mL, 10 mg / mL in PEG) was mixed with SA solution (33 μL, 64 μL, 94 μL, 151 μL, and 252 μL for fHep-C-lipid, fHep-K1C-lipid, fHep-K2C-lipid, fHep-K4C-lipid, and fHep-K8C-lipid, respectively, 0.5 M in dioxane) and stirred at room temperature for 24 h. The resulting solution was then lyophilized and purified by GPC (spin column, Pierce™ polyacrylamide spin desalting column, 7K MWCO, 0.7 mL, Thermo Fisher Scientific) to obtain fHep(-)-lipid: fHep-C(-)-lipid, fHep-K1C(-)-lipid, fHep-K2C(-)-lipid, fHep-K4C(-)-lipid, and fHep-K8C(-)-lipid.

[0111] Determination of diameter and surface charge of fHep-lipids The diameter, polydispersity index (PDI) and zeta potential (surface charge) of each cation-PEG-lipid (0.5 mg / mL in PBS), fHep-lipid (0.5 mg / mL in PBS) and fHep (4 mg / mL in PBS) were assessed by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK).

[0112] Determination of the critical micelle concentration (CMC) for fHep-lipids DPH was used to measure the CMC of fHep-lipid. fHep-lipid, cationic-PEG-lipid and Mal-PEG-lipid (1 mL, 1.0 × 10 -1 mg / mL to 1.0×10 -7 The DPH solution (2 μL, 30 μM, in THF) and DPH solution (2 μL, 30 μM, in THF) were mixed and incubated at 37° C. for 1 h. The fluorescence intensity of the resulting solution was then measured using a fluorometer (FP-6600, JASCO, Ex: 357 nm, Em: 430 nm).

[0113] Determination of amine group concentration using fluorescamine Each cationic-PEG-lipid and fHep-lipid was diluted in PBS (0.5 mg / mL), and fluorescamine was dissolved in DMSO at a concentration of 3 mg / mL. Each cationic-PEG-lipid solution (9 μL) or fHep-lipid (9 μL) was mixed with fluorescamine solution (3 μL) at RT for 15 min, and the absorbance (at 481 nm) of each resulting solution was measured by Nanodrop-3300 (Thermo Fisher Scientific, Waltham, MA, USA). The same experiment was performed using C, K1C, K2C, K4C, and K8C solutions with the same concentrations. Glycine was used for the calibration curve to determine the amine group concentration.

[0114] result The molecular design of fHep-lipid is shown in Figure 1. Multiple fragmented heparins can be conjugated to each PEG-lipid molecule (Figure 2A). Heparin was chemically modified to obtain fragmented heparins with terminal aldehyde groups (fHep, Figure 2B). fHep was then conjugated to cationic NH2-PEG-lipids (cationic-PEG-lipids) by Schiff base chemistry (Figure 2A, Figure 2C). To introduce amine groups, C, K1C, K2C, K4C and K8C were used and conjugated to Mal-PEG-lipids. The following cationic-PEG-lipids were produced: C-PEG-lipid (one amine group), K1C-PEG-lipid (two amine groups), K2C-PEG-lipid (three amine groups), K4C-PEG-lipid (five amine groups), and K8C-PEG-lipid (nine amine groups).

[0115] fHep was conjugated to each cationic-PEG-lipid through Schiff base chemistry between aldehyde and amine groups, followed by reduction with NaCNBH3. The number of fHep conjugated to cationic-PEG-lipids was calculated by measuring both the unreacted amine groups of fHep-lipid and the amine groups of cationic-PEG-lipid by using fluorescamine. The percentage of reacted amine groups was calculated to be 89%, 90%, 91%, 88%, and 61% for fHep-K8C-lipid, fHep-K4C-lipid, fHep-K2C-lipid, fHep-K1C-lipid, and fHep-C-lipid, respectively, as listed in Table 1 below. Thereafter, the number of conjugated fHep per PEG-lipid was 8.0, 4.5, 2.7, 1.8, and 0.6 for fHep-K8C-lipid, fHep-K4C-lipid, fHep-K2C-lipid, fHep-K1C-lipid, and fHep-C-lipid, respectively.

[0116] [Table 1]

[0117] The micelle size of each fHep-lipid was determined by DLS (Figure 5A). All fHep-lipids showed 15-20 nm, while fHep showed approximately 2 nm. In addition, the zeta potential of each fHep-lipid was more negative than that of each cation-PEG-lipid (Figure 5B). These results suggested that fHep was conjugated with PEG-lipid.

[0118] We also measured the CMC of each fHep-lipid using DPH, which were 0.9 μM, 1.1 μM, 1.1 μM, 1.0 μM, 0.6 μM, 1.1 μM, 1.1 μM, 1.0 μM, 1.0 μM, 0.7 μM and 1.1 μM for 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, respectively, suggesting that fHep-lipids are amphiphilic and may indeed form micelles.

[0119] Example 3 - Functional evaluation of fHep-lipids by QCM-D The functionality of fHep-lipids was evaluated by quartz crystal microbalance with energy dissipation (QCM-D, Q-sense, Gothenburg, Sweden). The binding ability of antithrombin (AT) to each fHep-lipid and fHep(-)-PEG-lipid was quantified by QCM-D. After cleaning the QCM gold sensor chip by oxygen plasma treatment (300 W, 100 mL / min gas flow, PR500; Yamato Scientific Co., Ltd., Tokyo, Japan), the sensor chip was immersed in 1-dodecanethiol solution (1.25 mM, in EtOH) for 24 h to form a hydrophobic self-assembled monolayer (CH3-SAM). After extensive washing with ethanol and water, the sensor chip was set in the QCM-D chamber. A solution of each fHep-lipid (0.1 mg / mL in PBS) was flowed into the chamber for 30 min, followed by BSA solution (1 mg / mL, in PBS) for 10 min for blocking treatment. Finally, AT solution (0.1 mg / mL in PBS) was flowed through the chamber for 10 min. PBS was flowed for 2 min for washing before each sample solution was flowed. The adsorption of each material was calculated from the resonant frequency change (Δf at the seventh overtone) using the Sorbery equation [5].

[0120] In addition, binding of factor H to fHep(-)-lipids (fHep-K1C(-)-lipid, fHep-K4C(-)-lipid and fHep-K8C(-)-lipid) or Mal-PEG-lipid (as a control) was studied by QCM-D. Each fHep(-)-lipid or Mal-PEG-lipid solution (0.1 mg / mL in PBS) was flowed through the chamber for 30 min, and BSA solution (1 mg / mL in PBS) was flowed for 10 min for blocking treatment. Then, factor H solution (50 μg / mL in PBS) was flowed through the chamber for 15 min. PBS was flowed for 2 min for washing before each sample solution was flowed. Then, AT solution (0.1 mg / mL in PBS) was flowed through the chamber for 10 min. PBS was flowed for 10 min for washing before each sample solution was flowed. The adsorption of each material was calculated from the resonant frequency change (Δf at the seventh overtone) using the Sorbery equation [5].

[0121] result The binding ability of AT to fHep-lipid was evaluated by QCM-D. Figure 6 shows a representative QCM-D profile of the interaction between fHep-K8C-lipid and AT. After blocking with BSA, AT binding to fHep-K8C-lipid could be confirmed on the surface. Figure 7 summarizes the data of the amount of AT binding for each fHep-lipid and cationic-PEG-lipid. Here, fHep was also added as a control. AT binding could be confirmed for all fHep-lipids, but there was no AT binding to cationic-PEG-lipid and fHep.

[0122] We also investigated the AT binding ability of fHep-lipids treated with succinic anhydride (SA), i.e., fHep(-)-lipids. Since there are unreacted amine groups on the fHep-lipids, SA was used to convert them to carboxylic acid groups, which have less cytotoxicity. Figure 8 shows a representative QCM-D profile of the interaction between fHep-K8C(-)-lipids and AT. When BSA was added for blocking treatment, less BSA binding was observed, and only AT binding was observed (Figure 9). Similar results were obtained when fHep-K4C(-)-lipids were used. These results indicated that the negative charge of fHep(-)-lipids inhibited the nonspecific binding of BSA.

[0123] We also investigated the binding ability of factor H to fHep(-)-lipids by QCM-D. Figure 10 shows a representative QCM-D profile of the interaction with factor H. After blocking with BSA, binding of factor H on fHep(-)-lipids could be confirmed, whereas no binding was detected on the control Mal-PEG-lipid. Figures 11A and 11B summarize the quantitative analysis of the amount of factor H binding to each fHep(-)-lipid and Mal-PEG-lipid. There was binding of factor H to all fHep(-)-lipids, but no binding of factor H to Mal-PEG-lipids. The number of factor H per fHep(-)-lipid molecule was the highest when fHep-K8C(-)-lipid was used compared to fHep-K1C(-)-lipid and fHep-K4C(-)-lipid. This result suggests that fHep highly loaded with fHep-K8C(-)-lipid has the highest affinity for factor H, which may be important for regulating complement activation via recruitment of factor H.

[0124] Example 4 - Functional evaluation of fHep-lipids by FXa activity assay The functionality of fHep-lipids was evaluated by FXa activity assay, in which we evaluated the binding ability of antithrombin (AT) to each fHep-lipid incorporated into liposomes.

[0125] Liposomes were prepared with dipalmitoylphosphatidylcholine (DPPC) and cholesterol (1:1 molar ratio). Cholesterol solution (530 μL, 10 mg / mL in ethanol) and DPPC solution (1 mL, 10 mg / mL in ethanol) were mixed and evaporated using a rotary evaporator to form a lipid film, followed by drying in vacuum for 24 h. Then, PBS (1 mL) was added and vigorously stirred with a magnetic stir bar at RT for 1 h. The resulting lipid suspension was extruded into membrane filters (φ1000 nm, 400 nm, 200 nm and 100 nm) using an extruder (Avanti Polar Lipids, Birmingham, AL, USA). The lipid suspension was passed through each filter 21 times.

[0126] To incorporate fHep-lipids on the liposome surface, a solution of fHep-lipids was mixed with the liposome suspension. The liposome suspension (500 μL, 1 mg / mL in 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 pellet. After 10 min of incubation at RT, the suspension was washed with PBS (450 μL) by one centrifugation (20,000 g, 70 min, 4 °C). Finally, fHep-lipid-modified liposomes were obtained. The concentration of cholesterol in the liposomes was measured by an assay kit (T-Cho E, Fujifilm Wako Pure Chemical Co., Ltd.). The FXa activity of the liposomes was evaluated using an assay kit (Biophen Heparin (AT+), Cosmo Bio Co., Ltd.).

[0127] FXa activity assay The 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 RT for 120 s. The color reagent (75 μL) was then mixed for 90 s, followed by the addition of aqueous citric acid (100 μL, 20 mg / mL). Each supernatant was collected by centrifugation (20,000 g, 70 min, 4° C.), after which the absorbance (at 405 nm) was measured.

[0128] Liposomal cholesterol was measured by mixing the liposome suspension (60 μL in PBS) with SDS (2 μL, 15 mg / mL in PBS) for 30 min at RT for solubilization, after which the cholesterol concentration was determined according to the company's instructions.

[0129] result The anti-FXa activity of fHep-lipid modified liposomes was evaluated (Figure 12). As control groups, each cationic-PEG-lipid modified liposome and fHep-treated liposomes were used for the assay. The anti-FXa activity was normalized by liposome concentration. All fHep-lipid modified liposomes showed higher anti-FXa activity than the control group. In addition, similar results were obtained when fHep(-)-lipid modified liposomes were used (Figure 12). These results indicated that the surface of liposomes can be modified with fHep-lipids and fHep(-)-lipids, and that such modified liposomes have anti-FXa activity.

[0130] Example 5 - Characterization of Treated Liposomes The surface of liposome was modified with each fHep-lipid (fHep-C-lipid, fHep-K1C-lipid, fHep-K2C-lipid, fHep-K4C-lipid, and fHep-K8C-lipid) or cationic-PEG-lipid (C-PEG-lipid, K1C-PEG-lipid, K2C-PEG-lipid, K4C-PEG-lipid, and K8C-PEG-lipid) as described in Example 4. fHep and PBS were used as controls.

[0131] 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 pellet after centrifugation (TOMY MX301, 20,000 g, 70 min, 4° C.). After 10 min of incubation at RT, the liposomes were washed with PBS (450 μL) by one 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 (Malvern Instruments Ltd., Worcestershire, UK).

[0132] result The size of liposomes modified with fHep-lipids or cationic-PEG-lipids was measured by DLS (Figure 13). As a control group, liposomes were treated with either PBS or fHep. Before treatment, the size of liposomes was 150 nm. After that, the average size of liposomes modified with fHep-lipids or cationic-PEG-lipids was 155 nm to 175 nm, while the size of control liposomes was about 250 nm. Also, the polydispersity index (PDI) of liposomes modified with fHep-lipids or cationic-PEG-lipids was lower (about 0.2) than that of control liposomes (about 0.5) (Figure 14). These results showed that liposomes modified with fHep-lipids or cationic-PEG-lipids were well dispersed, while the control liposomes were aggregated.

[0133] The zeta potential of all liposomes was also measured (Figure 15). All samples showed a negative charge, but the liposomes modified with each fHep-lipid showed a more negative charge than the liposomes modified with each cationic-PEG-lipid. This result indicated that the liposome surface was modified with negative fHep-lipid.

[0134] Example 6 - Cell surface functionalization with fHep(-)-lipids Human red blood cells (RBCs) were collected from healthy donors using vacuum blood collection tubes. Antithrombin (AT) labeling was performed using the Alexa fluor™ 488 antibody labeling kit according to the protocol provided by the company. RBCs (10 μL, 7 × 10 in 10 mM EDTA / PBS) were collected from healthy donors using vacuum blood collection tubes. Antithrombin (AT) labeling was performed using the Alexa fluor™ 488 antibody labeling kit according to the protocol provided by the company. 9Cell pellets were treated with fHep(-)-lipid (fHep-C(-)-lipid, fHep-K1C(-)-lipid, fHep-K2C(-)-lipid, fHep-K4C(-)-lipid, and fHep-K8C(-)-lipid), K1C-PEG-lipid (0.5mg / mL, 20μL for each sample), fHep (4mg / mL in PBS) or PBS (20μL) for 30min at RT, followed by rinsing twice with 1mL PBS. Cell pellets were treated with Alexa488-AT (4mg / mL) for 10min at RT, followed by rinsing twice with 1mL PBS and centrifugation (Force mini SBC140-115, 1min). The resulting cell pellet was suspended in 1 mL of PBS. The treated cells were observed using a confocal microscope (CLSM, LSM880, Carl Zeiss, Jena, Germany), and the cells were analyzed by flow cytometry (BD LSR II, BD Biosciences, San Jose, CA, USA). The experiment was approved by the ethical committee of the University of Tokyo.

[0135] The functionality of fHep-lipids was evaluated by FXa activity assay. Here, we evaluated the binding ability of antithrombin (AT) to each fHep-lipid incorporated into live cells (CCRF-CEM cells). To decorate the cell surface of CCRF-CEM cells, fHep-lipids (fHep-C-lipids) were mixed with the cells. The cell suspension (2 × 10 in 2 mL of RPMI 1640 medium) was mixed with 100 μl of fHep-lipids (2 × 10 in 2 mL of RPMI 1640 medium). 6The treated cells (100 μL, 2.5 mg / mL, in PBS containing 1 mg / mL glycine) were washed with PBS by two centrifugations (120 g, 4° C., 3 min). A solution of fHep-C-lipid (100 μL, 0.5 mg / mL, in PBS containing 1 mg / mL glycine) was mixed with the cell pellet and incubated for 30 min at RT with gentle tapping 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 with PBS by two centrifugations (180 g, 4° C., 6 min). Finally, the cells were suspended in PBS (100 μL). Cell viability and cell number were assessed using trypan blue and a cell counter.

[0136] Then, a cell suspension (15 μL) was prepared and then mixed with human AT (15 μL) in a 96-well plate. Bovine FXa (75 μL) was added to each well and incubated at RT for 120 s. Then, the color reagent (75 μL) was mixed for 90 s, followed by the addition of an aqueous citric acid solution (100 μL, 20 mg / mL). Finally, the absorbance (at 405 nm) was measured.

[0137] result Fluorescence was observed on the cell membrane when cells were treated with fHep(-)-lipid (Figure 16), while fluorescence was not observed on the cell membrane when cells were treated with K1C-PEG-lipid, fHep and PBS, suggesting that AT was specifically immobilized on the fHep(-)-lipid on the cell surface. Figure 17 shows the quantitative analysis of Alexa488-AT immobilized on each cell, which also suggested that AT was specifically immobilized on the fHep(-)-lipid on the cell surface. In addition, the number of immobilized AT was the highest when cells were treated with fHep-K4C(-)-lipid and fHep-K8C(-)-lipid, indicating that highly loaded fHep could effectively immobilize AT on fHep(-)-lipid.

[0138] The anti-FXa activity of fHep-lipid-modified cells (CCRF-CEM cells) was evaluated (FIG. 18). As a control group, unmodified cells were used for the assay. The anti-FXa activity was normalized by cell number. The fHep-lipid-modified cells showed higher anti-FXa activity than the unmodified cells (FIG. 18). These results indicate that the surface of cells can be modified with fHep-lipids and also show that such surface modification results in anti-FXa activity.

[0139] Example 7 - Functional evaluation of fHep-lipids using a whole blood model hMSC surface functionalization with fHep-lipids hMSCs were cultured in DMEM (supplemented with 10% FBS, 50 IU / mL penicillin, 50 μg / mL streptomycin) at 37°C in 5% CO2 and 95% air. hMSCs (1 mL, 2.5 × 10 in PBS) were harvested by trypsinization (3 min, 37°C, 5% CO2). 5The cells were centrifuged (Force mini SBC140-115, BM Equipment Co., Ltd., 1 min). The cell pellet was treated with fHep(-)-lipid (20 μL, 10 mg / mL in PBS, fHep-K1C(-)-lipid and fHep-K8C(-)-lipid), KnC-PEG-lipid (20 μL, 10 mg / mL in PBS, K1C-PEG-lipid and K8C-PEG-lipid), fHep (20 μL, 30 mg / mL and 120 mg / mL in PBS) or PBS (20 μL) for 30 min at RT, followed by rinsing twice with cold PBS (1 mL) and centrifugation (Force mini SBC140-115, 1 min). Samples containing fHep (fHep-K1C(-)-lipid, fHep-K8C(-)-lipid and fHep (30mg / mL or 120mg / mL)) were reacted with glycine (18mg / mL in PBS) for 4 hours, followed by purification on a spin column to inactivate the cytotoxic aldehyde groups of free fHep in solution. The cell pellet was treated with Alexa488-AT (4mg / mL) for 10 minutes at RT, followed by rinsing once with 1mL cold PBS and centrifugation (Force mini SBC140-115, 1min). The resulting cell pellet was suspended in 500μL PBS and cell viability was assessed using trypan blue and a cell counter (countessII, Invitrogen). The treated cells were assessed using CLSM (LSM800, Carl Zeiss) and cells were analyzed by flow cytometry (BD LSR II, BD Biosciences).

[0140] Blood testing using human whole blood hMSCs were exposed to human whole blood using the Chandler loop model [6] to evaluate the antithrombotic properties of the surface of fHep-lipid-treated hMSCs. hMSCs used for blood testing were at passages 6–8. hMSCs (1.0 × 10 in 1 mL PBS) were 6Cells / mL) were treated with fHep-K1C(-)-lipid, fHep-K8C(-)-lipid and K1C-PEG-lipid (40 μL, 10 mg / mL in PBS for each sample) and rinsed twice to remove free fHep-lipid. Cell viability and concentration were assessed using trypan blue and a cell counter (countessII, Invitrogen), and cell concentrations were determined to be 2.5 × 10 6 cells / mL or 2.5 x 10 5 The concentration of cells / mL was adjusted. A loop made of polyurethane tubing (φ6.3 mm, 40 cm) and polypropylene connector (φ6.5 mm, Isis Co., Ltd., Osaka, Japan) was coated with MCP polymer (2 mL, 5 mg / mL in EtOH) for 24 h, followed by drying in air for 24 h to prevent surface-induced blood activation. Human whole blood was collected in vacuum tubes (7 mL, non-treated, Terumo Co., Ltd.) from healthy donors who had not undergone medication at least 14 days prior to blood donation. Immediately after blood collection, UFH (2.5 μL / 1 mL blood, 200 IU / mL in PBS) was mixed with the blood. Then, human whole blood (2.5 mL, with 0.5 IU / mL UFH) was added to the MPC polymer-coated loop, followed by the addition of 100 μL of hMSC suspension (2.5 × 10 6 cells / mL or 2.5 x 10 5 Cells / mL, treated or non-treated hMSCs) or PBS as a control were added. The tubes were rotated at 22 rpm for 2 h in a 37°C cabinet. Blood samples (1 mL) from each loop were taken at 1 h and 2 h and mixed with EDTA solution (10 mM). Platelet counts were measured for each sample using a cell counter (pocH-80i, Sysmex Corporation, Hyogo, Japan). Afterwards, the blood samples were centrifuged (TOMY MX301, 2,600 g, 15 min, 4°C) and the plasma of each sample was collected and stored in a -80°C freezer for enzyme-linked immunosorbent assay (ELISA) for TAT, C3a and sC5b-9. The experiments were approved by the Ethics Committee of the University of Tokyo.

[0141] Measurement of TAT, C3a and sC5b-9 in plasma TAT, C3a and sC5b-9 in plasma were measured by conventional sandwich ELISA. Briefly, plasma was diluted with dilution buffer (PBS containing 0.05% TWEEN® 20, 10 mM EDTA and 10 mg / mL BSA). C3a in plasma was captured by anti-human C3a mAb 4SD17.3 pre-coated on 96-well plates 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 stopped with 1 M H2SO4aq. Finally, the absorbance at 450 nm was detected using a plate reader (AD200, Beckman Coulter, Miami, FL, USA). Zymosan-activated serum calibrated against purified C3a was used as a standard. ELISA for sC5b-9 was demonstrated in the same way as C3a measurement. First, plasma was diluted with dilution buffer. Then, sC5b-9 in plasma was captured by anti-neoC9 mAb aE11 (Diatec Monoclonals AS, Oslo, Norway) precoated on 96-well plates and detected with 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 stopped with 1 M H2SO4aq., followed by measuring the absorbance at 450 nm using a plate reader. Zymosan-activated serum was used as a standard.

[0142] TAT was measured by ELISA kit (Human Thrombin-Antithrombin Complex (TAT) AssayMax ELISA Kit, Assaypro, St. Charles, MO, USA) according to the manufacturer's instructions. Briefly, plasma was diluted with diluent. TAT was then captured by a monoclonal antibody against human antithrombin precoated on a 96-well plate, and detected with a biotinylated polyclonal antibody against human thrombin, followed by HRP-conjugated streptavidin. Tetramethylbenzidine, a peroxidase chromogenic substrate, was reacted for 20 min, and the reaction was stopped with 0.5 N hydrochloric acid solution, followed by measuring the absorbance at 450 nm using a plate reader. Human TAT complex was used as a standard.

[0143] result The surface of hMSCs was modified with fHep-lipid, fHep-K1C(-)-lipid and fHep-K8C(-)-lipid with high and low AT-binding capacity to compare their antithrombotic properties in human whole blood. Strong fluorescence from Alexa488-AT on hMSC membrane was observed when hMSCs were treated with fHep-K1C(-)-lipid and fHep-K8C(-)-lipid, but no fluorescence was observed on the cell membrane when the cells were treated with KnC-PEG lipid (n=1 and 8), fHep and PBS (Figure 19A), suggesting that fHep(-)-lipid was immobilized on the hMSC surface. Flow cytometry analysis showed that fHep-K1C-lipid-treated hMSCs had more binding of Alexa488-AT than fHep-K8C-lipid-treated hMSCs (Figure 19B). This was probably because AT binding was inhibited by fHep-K8C(-)-lipids on the hMSC surface due to the highly loaded fHep on the hMSC surface, and exogenous AT could not fully access fHep. The viability of treated hMSCs was about 80%, which was similar to the control groups (UFH-treated cells, PBS-treated cells and non-treated cells), suggesting the non-cytotoxicity of fHep(-)-lipid modification (Figure 19C). High fluorescence intensity was observed in K8C-PEG-lipid-treated cells (Figure 19B, Figure 19C). It was observed that those cells were destroyed by K8C-PEG-lipid modification due to cationic properties, resulting in the uptake of Alexa488-AT.

[0144] Next, we diluted human whole blood with 1.0 × 10 4 cells / mL (Figures 20A to 20D) or 1.0 x 10 5 hMSCs were incubated with fHep-K1C(-)-lipids, fHep-K8C(-)-lipids or K1C-PEG-lipids at a concentration of 1000 cells / mL (Figures 19D-G), where non-treated hMSCs and PEG were used as controls.

[0145] FIG. 19D shows the platelet count in blood at 1 hour and 2 hours. When PBS was added to blood, there was almost no decrease in platelets. Also, the platelet count decreased over time when hMSC was added, suggesting that TF from hMSC induced platelet aggregation. The same result was observed with K1C-PEG-lipid-modified hMSC. It seems that the positive charge K1C at the end of the PEG layer induced platelet activation, so the platelets actually aggregated. On the other hand, in the case of hMSC treated with fHep-K1C(-)-lipid and fHep-K8C(-)-lipid, the platelet count decreased slightly, but the remaining platelets were much more than the control group of unmodified hMSC, suggesting that the surface modification with fHep-lipid could weaken platelet activation. There was no obvious difference in the platelet count between fHep-K1C(-)-lipid-modified hMSC and fHep-K8C(-)-lipid-modified hMSC. When the hMSC concentration was 1.0×10 4 When cells / mL were used, a similar trend was observed as seen at higher cell concentrations, but there was no clear difference in platelet counts (Figure 20A).

[0146] We used treated hMSCs ([hMSCs] = 1.0 × 10 for Figure 20B). 4 cells / mL and 1.0 x 10 for Figure 19E 5 The levels of TAT, an aggregation marker, were assessed during a 2-hour incubation with hMSCs (1.0 × 10 cells / mL). There was a large increase in TAT levels over time for K1C-PEG-lipid-modified hMSCs and untreated hMSCs, whereas there was a small increase for fHep-lipid-modified hMSCs and PBS-spiked blood. The results were consistent with a 1.0 × 10 5 The increase in the number of cells / mL of hMSC in blood was remarkable. There was a significant difference between the untreated hMSC group or K1C-PEG-lipid-modified hMSC and each fHep-lipid-modified hMSC (Figure 19E). No significant difference was observed between PBS-mixed blood and each fHep-lipid-modified hMSC-mixed blood. These results suggested that the fHep-lipid on the hMSC surface could suppress aggregation activation.

[0147] In addition, we used treated hMSCs ([hMSCs] = 1.0 × 10 for Figures 20C and 20D). 4 cells / mL and 1.0 x 10 for Figures 19F and 19G 5 We assessed the production of C3a and sC5b-9, complement markers, during 2 h of incubation with fHep-lipids (1000 x 1000 cells / mL). Essentially, the levels of both markers increased over time. However, there were no differences in levels between groups. We could not determine any effect of cell surface modification with fHep-lipids on complement activation.

[0148] The above-described embodiments should be understood as a few illustrative examples of the present invention. It is understood by those skilled in the art that various modifications, combinations and changes can be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in different embodiments can be combined in other configurations where technically possible. The scope of the present invention is, however, defined by the appended claims.

[0149] References [Table 2]

Claims

1. 1. A method for producing poly(ethylene glycol) lipids (PEG-lipids), comprising: mixing K n C and / or CK n with a maleimide-conjugated PEG-lipid to form a cationic-PEG-lipid comprising at least one amino group, where C is cysteine, K is lysine, and n is 0 or a positive integer less than or equal to 20, preferably 0 or a positive integer less than or equal to 15, and more preferably 0 or a positive integer less than or equal to 10; mixing said cationic-PEG-lipid containing at least one amino group with heparin containing at least one carbonyl group, preferably at least one aldehyde group, to form a Schiff base intermediate; and adding a reducing agent to said Schiff base intermediate to form a heparin-PEG-lipid. A method comprising:

2. mixing α-N-hydroxysuccinimidyl-ω-maleimidyl PEG (NHS-PEG-Mal), triethylamine and 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine (DPPE) in dichloromethane; and Precipitating the maleimide-conjugated PEG-lipid by adding diethyl ether to the mixture of NHS-PEG-Mal, triethylamine and DPPE in dichloromethane. The method of claim 1 further comprising:

3. 3. The method according to claim 1 or 2, wherein the heparin is a fragmented heparin containing at least one carbonyl group, preferably a fragmented heparin containing at least one aldehyde group.

4. The acid solution and sodium nitrite (NaNO) were added to form a mixed solution. ２ ) mixing the aqueous solutions; adjusting the pH of the mixed solution to within the range of 2 to a maximum of 6, preferably 3 to a maximum of 5, more preferably 4; adding heparin, preferably sodium heparin, to said mixed solution to form a heparin solution; adjusting the pH of the heparin solution to within the interval of 6 to 8, preferably 6.5 to 7.5, more preferably 7, to form the fragmented heparin containing at least one carbonyl group, preferably at least one aldehyde group; and Optionally, dialysing said fragmented heparin containing at least one carbonyl group against water and lyophilizing said fragmented heparin containing at least one carbonyl group, preferably at least one aldehyde group. The method of claim 3 further comprising:

5. 5. The method of any one of claims 1 to 4, wherein adding the reducing agent comprises adding sodium cyanoboronhydride to the Schiff base intermediate to form the heparin-PEG-lipid.

6. 6. The method of any one of claims 1 to 5, further comprising converting any unreacted amino groups in the heparin-PEG-lipid to carboxylic acid groups.

7. 7. The method of claim 6, further comprising adding an acid anhydride to the heparin-PEG-lipid to convert any unreacted amino groups in the heparin-PEG-lipid to carboxylic acid groups.

8. a poly(ethylene glycol) lipid (PEG-lipid) comprising at least one heparin attached to the PEG-lipid via a bond formed between an amino group of any lysine residue in the K n C and / or CK n linkages or an N-terminal amine in the K n C and / or CK n linkages of the cationic-PEG-lipid comprising the K n C and / or CK n linkages and a carbonyl group, preferably an aldehyde group, of at least one heparin comprising at least one carbonyl group, preferably at least one aldehyde group, to form a Schiff base intermediate which is reduced by the addition of a reducing agent; A PEG-lipid, wherein C is cysteine, K is lysine, and n is 0 or a positive integer less than or equal to 20, preferably n is selected in the interval from 0 to 15, and more preferably n is selected in the interval from 0 to 10.

9. The PEG-lipid of claim 8, wherein the heparin is a fragmented heparin.

10. The fragmented heparin has a weight average molecular weight (M) selected within the interval of 2.5 kDa to 15 kDa, preferably within the interval of 5 kDa to 10 kDa. ｗ 10. The PEG-lipid of claim 9, having the formula:

11. The PEG-lipid of any one of claims 8 to 10, wherein any free amino groups in the heparin-PEG-lipid are converted to carboxylic acid groups.

12. 12. The PEG-lipid of any one of claims 8 to 11, having affinity for antithrombin and factor H.

13. 13. A biological tissue comprising at least one poly(ethylene glycol) lipid (PEG-lipid) according to any one of claims 8 to 12, wherein said at least one PEG-lipid is fixed in a cell membrane of said biological tissue.

14. 14. The biological tissue of claim 13, selected from the group consisting of islets of Langerhans, mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), endothelial cells, beta cells, red blood cells, hepatocytes, kidney, heart, pancreas, liver, lung, uterus, bladder, thymus, intestine and spleen.

15. A liposome comprising at least one poly(ethylene glycol) lipid (PEG-lipid) according to any one of claims 8 to 12, wherein said at least one PEG-lipid is immobilized in the lipid bilayer of said liposome.

16. A poly(ethylene glycol) lipid (PEG-lipid) according to any one of claims 8 to 12 for use as a medicament.

17. A poly(ethylene glycol) lipid (PEG-lipid) according to any one of claims 8 to 12 for use in the treatment of thromboinflammation.

18. A poly(ethylene glycol) lipid (PEG-lipid) according to any one of claims 8 to 12 for use in the treatment of instantaneous blood-mediated inflammatory response (IBMIR).

19. The poly(ethylene glycol) lipid (PEG-lipid) according to any one of claims 8 to 12 for use in the treatment of ischemia-reperfusion injury (IRI).

20. The poly(ethylene glycol) lipid (PEG-lipid) according to any one of claims 8 to 12 for use in the treatment of stroke.

21. The poly(ethylene glycol) lipid (PEG-lipid) according to any one of claims 8 to 12 for use in the treatment of myocardial infarction.

22. An in vitro method for providing a biological tissue having a heparin coating, comprising adding to the biological tissue in vitro a poly(ethylene glycol) lipid (PEG-lipid) according to any one of claims 8 to 12 to fix the PEG-lipid in a cell membrane of the biological tissue.

23. 1. An ex vivo method of treating an organ or part of an organ, comprising: injecting ex vivo into the vasculature of said organ or said part of said organ a solution comprising a poly(ethylene glycol) lipid (PEG-lipid) according to any one of claims 8 to 12; and incubating ex vivo in the vasculature the solution comprising the PEG-lipid according to any one of claims 8 to 12 to allow coating of at least a portion of the intima of the vasculature with the PEG-lipid according to any one of claims 8 to 12. A method comprising:

24. 24. The ex vivo method according to claim 23, wherein ex vivo incubation preferably comprises immersing the organ or said part of the organ in an organ preservation solution comprising a PEG-lipid according to any one of claims 8 to 12, while ex vivo incubating said solution comprising a PEG-lipid according to any one of claims 8 to 12 in said vasculature to allow coating of at least a part of the intima of said vasculature with said PEG-lipid according to any one of claims 8 to 12.

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