RU2808054C2 - Treatment of ex vivo organs with peg-phospholipide molecules - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: following is described: a method of ex vivo providing coverage of at least a portion of the endothelial lining of the vascular system of an organ or part of an organ. The method includes the steps of ex vivo infusion of a solution containing a PEG-phospholipid molecules into the vascular system of an organ or a part of an organ; and ex vivo incubation of a solution containing PEG-phospholipid molecules in the vascular system.

EFFECT: method makes it possible to form protection against thrombus inflammation caused by perfusion of an organ or a part of an organ.

15 cl, 58 dwg, 4 ex

Description

TECHNICAL FIELD

The present invention generally relates to ex vivo organ treatments and, in particular, to those ex vivo organ treatments that are capable of inhibiting or preventing thromboinflammation and hypotension caused by organ reperfusion.

BACKGROUND OF THE ART

Ischemia-reperfusion (I/R) injury (IRI) is a major contributor to the pathology of several conditions, including transplantation, thrombotic disorders, sepsis, and cardiopulmonary bypass, all of which cause thromboinflammation.

Today, kidney transplantation is the most effective treatment for patients with end-stage renal failure. Despite impressive improvements in treatment outcomes since introduction in 1960, graft loss still occurs in 25% of patients after 5 years and in almost 50% after 10 years. A major target for improving kidney transplant outcomes is IRI, as IRI causes acute renal failure, delayed graft function, and distant organ failure. Immunosuppression does not significantly affect the humoral innate immune system and therefore does not reduce IRI. The field of transplantation also faces the challenge of organ shortages. One option to increase the donor pool is to use organs of marginal suitability more frequently, such as organs from cardiac deceased donors. However, these organs are also severely affected by IRI.

During ischemia, metabolism switches to an anaerobic state, which ultimately leads to an inflammatory state that is triggered by the up- and down-regulation of multiple genes, including nuclear factor kappa light chain enhancer of activated B cells (NF-κB), and through it cytokines , chemokines and tissue factor (TF), but also genes that protect against ischemia, such as heme oxygenase 1 (HO-1), hypoxia-inducible factors (HIF) 1 and 2, and nitrotyrosine. Under normal oxygen conditions, the surface of endothelial cells is covered with a negatively charged “hairy” network called the glycocalyx (GCX), which contains a complex network of proteoglycans, glycosaminoglycans and glycoproteins that contains anti-inflammatory and anticoagulant proteins to ensure microvascular homeostasis and prevent thrombinflammation. Moreover, thick GCX protects the cell surface and adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1), vascular endothelial adhesion molecule 1 (VCAM-1) and E-selectin, thereby preventing the recruitment of leukocytes and platelets to the endothelial surface. GCX is very sensitive to oxidative stress and ischemia-induced structural damage, which often results in rapid loss of GCX coating. It is caused by the expression of heparinase and metalloproteinase in the lining of endothelial cells, which leads to the cleavage and destruction of GCX, which protects endothelial cells from attack by the innate immune system. GCX contains many growth factors, such as hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), which are released during the breakdown of GCX. This will also cause complement, coagulation and contact regulators to be released from the cell surface. Such regulators are, for example, antithrombin (AT), protein C, tissue factor pathway inhibitor (TFPI), C4b-binding protein (C4BP), factor H and C1-inhibitor (C1-INH), resulting in the endothelial cell surface being not protected from attack by the complement, coagulation and contact systems. Finally, if ischemia is prolonged, adenosine triphosphate (ATP) stores are depleted, leading to apoptosis and necrosis.

Reperfusion of an ischemic organ induces a repair process, but initially it triggers detrimental damage to the organ due to pathological changes already caused by ischemia. Rapid oxygenation creates damage, including the formation of reactive oxygen species (ROS), and the unprotected endothelial surface triggers destructive activation of the complement and junction systems. During transplantation, expressed collectin-11/12 (CL-11/12) in the kidney graft recognizes ischemic cells and promotes complement activation through the lectin pathway (LP). Also other recognition molecules such as mannose binding lectin (MBL) and ficolin-2 LP pathways have been associated with complement activation. Evidence of complement activation is the deposition of C4d on endothelial cells in combination with the deposition of membrane attack complex (MAC) in the perivascular parenchyma, indicating cell damage in these areas. Triggered by increased TF expression and activation of the junction system, fibrin deposition indicates that the coagulation system is involved. In combination with complement activation, increased expression and exposure to von Willebrand factor (vWF) with subsequent binding and activation of platelets and polymorphonuclear neutrophils (PMN) to the endothelial surface, thromboinflammation is caused. Much of this thromboinflammation is triggered by locally produced complement factors, as was clearly demonstrated in a mouse model of kidney transplantation in which C3 knockout protected the transplanted organ from rejection.

Reperfusion of a transplanted organ is also associated with hemodynamic instability after reperfusion. This phenomenon, previously described as post-reperfusion syndrome (PRS), was first described in liver transplant recipients who experienced severe hemodynamic instability after liver graft reperfusion. However, this phenomenon has also been observed in kidney and pancreas transplant recipients, who often suffer from immediate hemodynamic instability after reperfusion. As a result, a rapid decrease in systemic blood pressure leads to impaired graft perfusion and prolongation of warm ischemia of the graft. These patients may suffer from delayed graft function, increased risk of rejection, and premature graft loss.

The amphiphilic polymer polyethylene glycol-phospholipid (conjugated to PEG-phospholipid, or simply PEG-phospholipid) spontaneously inserts into the lipid bilayer of the cell membrane through hydrophobic interactions [1, 2]. Moreover, the creation of an artificial cell surface layer by PEG-phospholipid can attenuate the immediate blood-mediated inflammatory response (IBMIR) and prolong both islet survival and function after portal vein infusion in diabetic mice [3, 6].

PEG phospholipid conjugated to innate immune regulators such as apyrase and factor H has previously been shown to protect cells and material surfaces from attack by the innate immune system in vitro [4, 5].

However, there is a need for improvements in organ transplantation.

BRIEF DESCRIPTION OF THE INVENTION

The main goal is to create improvements in organ transplantation.

This and other objectives are satisfied by the embodiments described in this application.

The present invention is defined by independent claims. Additional embodiments of the present invention are defined by the dependent claims.

A method of ex vivo treatment of an organ or part of an organ includes ex vivo infusion of a solution containing PEG-phospholipid molecules into the vascular system of the organ or part of an organ. The method also includes ex vivo incubation of a solution containing PEG-phospholipid molecules in the vascular system to ensure that at least a portion of the endothelial lining of the vasculature is coated with PEG-phospholipid molecules while maintaining the organ or part of an organ immersed in the organ preservation solution containing PEG phospholipid molecules.

The present invention also provides a PEG phospholipid for use in inhibiting or preventing reperfusion-associated hypotension that develops following organ transplant reperfusion in a recipient subject.

In an organ transplantex vivo, processed PEG phospholipid molecules observed significantly less thromboinflammation compared with untreated control organ transplants, as assessed by plasma levels of coagulation markers such as TAT and complement markers such as C3a and sC5b-9. Also, the expression of cytokines, particularly IL-6 and IL-1β, was significantly suppressed in PEG-phospholipid-treated organ transplants. Organ function was improved in PEG-phospholipid-treated organ transplants. PEG-phospholipid-treated organ transplants also showed reduced deposition of complement markers such as C3b and MAC, and expression of C5aR. Quite unexpected, but significant from a medical point of view, the effect of treatmentex vivo organ transplant with PEG-phospholipid molecules was such that such PEG-phospholipid treatment was able to reduce the detrimental drop in recipient blood pressure that is often associated with organ transplant reperfusion. Moreover, after 4 days, treatment with PEG-phospholipid was able to reduce the increase in T-cell-dependent cytokines IL-4 and IL-12, which are associated with activation of the acquired immune response.

BRIEF DESCRIPTION OF THE FIGURES

The embodiments, together with their additional objects and advantages, will be best understood by reference to the following description in conjunction with the accompanying figures, in which:

In fig. Figure 1 schematically shows the mechanism of hydrophobic interaction of a PEG-lipid molecule with lipid bilayers of cell membranes.

In fig. 2A - 2C illustrate the effects of different PEG phospholipid functional groups on blood coagulation (Figure 2A) and complement (Figures 2B and 2C) when coated mesenchymal stem cells (MSCs) were assayed in an in vitro Chandler loop model compared to controls (uncoated cells).

In fig. Figure 3 shows the concentration of the liquid phase over 60 minutes during perfusion of 25 ml of 2 mg/ml FITC-PEG phospholipid. Circles indicate without a connected kidney and squares indicate with a connected kidney. The calculated absorption of PEG phospholipid was 76%.

In fig. 4A - 4F illustrate biopsies extracted from control and treated kidneys at time points of 5 minutes (Figure 4A: control, Figure 4B: treated), 60 minutes (Figure 4C: control, Figure 4D: treated) and 360 minutes ( Fig. 4E: control, Fig. 4F: treated) after reperfusion. The kidneys were perfused with a biotin-PEG-phospholipid solution, and confirmation of coverage of the renal endothelium with PEG-phospholipid was achieved by counterstaining with streptavidin-488. Sections were analyzed using confocal microscopy.

In fig. 5A - 5E illustrate complement activation in a short-term porcine allogeneic kidney transplantation model. Deposition of Fig. 5A C3a, fig. 5B sC5b-9, fig. 5C C4d, fig. 5D C3b and FIG. 5E MAC from plasma in kidney biopsies. Squares and bars with arrows indicate PEG-phospholipid-treated buds and circles and bars without arrows indicate untreated control. * p<0.05; **p<0.01; *** p<0.001; ****p<0.0001.

In fig. 6A - 6D illustrate coagulation activation in a short-term allogeneic kidney transplantation model. Fig. 6A TAT, fig. 6B TF, fig. 6C FXII/AT and FIG. 6D FXII/C1INH in plasma. Squares indicate PEG-phospholipid-treated buds and circles indicate untreated control. * p<0.05; **p<0.01; *** p<0.001.

In fig. 7A - 7C illustrate the concentration of pro-inflammatory cytokines in a short-term allogeneic kidney transplantation model. Fig. 7A IL-1β, FIG. 7B IL-6 and FIG. 7C TNF in plasma. Squares indicate PEG-phospholipid-treated buds and circles indicate untreated control. * p<0.05; **p<0.01.

In fig. 8A - 8C illustrate complement activation and coagulation in a long-term allogeneic kidney transplantation model. Fig. 8A C3a, fig. 8B sC5b-9 and FIG. 8C TAT in plasma. Squares indicate PEG-phospholipid-treated buds and circles indicate untreated control. * p<0.05; **p<0.01.

In fig. 9A - 9L illustrate the concentration of cytokines/chemokines in a long-term allogeneic kidney transplantation model. Fig. 9A INFγ, FIG. 9B IL-1β, FIG. 9C IL-1α, FIG. 9D IL-1R, FIG. 9E IL-2, FIG. 9F IL-4, FIG. 9G IL-6, FIG. 9H, IL-10, Fig. 9I IL-8, FIG. 9J IL-12, Fig. 9K IL-18 and Fig. 9L TNF in plasma. Squares indicate PEG-phospholipid-treated buds and circles indicate untreated control.

In fig. 10A and 10B illustrate functional evaluation of a PEG-phospholipid-treated kidney in a short-term (FIG. 10A) and long-term (FIG. 10B) allogeneic kidney transplantation model. In fig. 10A assessed diuresis and FIG. 10B measured creatinine levels. Squares indicate PEG-phospholipid-treated buds and circles indicate untreated control. *p<0.05.

In fig. 11 shows a functional assessment of the compensatory increase in heart rate immediately after reperfusion in pigs transplanted with PEG-phospholipid-treated kidneys compared with animals transplanted with control kidneys in long-term allogeneic kidney transplantation models. *p<0.05.

In fig. Figure 12 shows gross pathological examination of porcine kidneys after (1) collection, (2) static hypothermic perfusion, and (3) PEG-phospholipid perfusion.

In fig. 13 shows the retention of PEG phospholipid molecules with different PEG subunit lengths (1 kDa circles, 5 kDa squares and 40 kDa triangles) using CCRF-CEM cells.

In fig. 14 shows the synthesis of [ ⁶⁸ Ga]NO2A-propyl azide.

In fig. 15 shows the synthesis of ⁶⁸ Ga-NO2A-PEG lipid.

In fig. 16A and 16B illustrate TAT values and plasma creatinine values after incubating porcine kidneys for 24 hours in PEG-phospholipid-supplemented HTK solution (treated) or HTK solution alone (control) at 4°C.

In fig. 17A - 17D illustrate complement and coagulation activation and creatinine levels in a long-term allogeneic kidney transplant model: TAT (FIG. 17A), C3a (FIG. 17B), sC5b-9 (FIG. 17C) and creatinine (FIG. 17D) in plasma. Lines with squares indicate PEG-phospholipid-treated buds and lines with circles indicate untreated control. *p<0.05.

In fig. 18A - 18G illustrate the concentration of cytokines/chemokines in a long-term allogeneic kidney transplant model: IL-1β (Fig. 18A), IL-2 (Fig. 18B), IL-4 (Fig. 18C), IL-6 (Fig. 18D) , IL-8 (Fig. 18E), IL-10 (Fig. 18F) and TNF (Fig. 18G) in plasma. Squares indicate PEG-phospholipid-treated buds and circles indicate untreated control. *p<0.05.

In fig. 19 shows the uptake of 5 kDa PEG-lipid and 40 kDa PEG-lipid after infusion of PEG-lipid molecules into kidneys from four different pigs, one kidney being used for 5 kDa PEG-lipid and the other for 40 kDa PEG-lipid. Kidneys were perfused with FITC-PEG-phospholipid solution, and confirmation of PEG-phospholipid coverage of the renal endothelium and parenchyma was visualized. Sections were analyzed using confocal microscopy.

In fig. 20 shows the ability to mix PEG-lipid molecules with three commonly used perfusion fluids (HTK, IGL-1 and UW). No precipitation was observed in any of these mixtures at a PEG-lipid concentration of 2 mg/ml.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally provides ex vivo organ treatments and, in particular, ex vivo organ treatments that are capable of treating, inhibiting or preventing thromboinflammation that precedes activation of the adaptive immune system and hypotension caused by organ reperfusion.

The ex vivo method of the present invention results in significant improvements in organ transplantation and organ processing by providing protective shielding or coating of the endothelial lining or endothelium of the vascular system and the parenchyma of such organs or portions of such organs. This protective coating provides effective protection against thrombus inflammation and the harmful effects of ischemia-reperfusion (I/R) injury (IRI).

Glycocalyx is necessary to ensure microvascular hemostasis and prevent thromboinflammation. This glycocalyx is, however, very sensitive to oxidative stress and ischemia-induced damage. Consequently, the protective coating of the glycocalyx is rapidly lost during ischemia that occurs in association with organ harvesting and in vitro organ preservation. Once an organ graft has been transplanted and reperfused, a repair process is initiated that begins detrimental damage to the organ graft due to missing or damaged glycocalyx. Consequently, the complement and contact systems are activated. The platelets are taken up and the white blood cells become activated and infiltrate the organ graft. Due to activation of these systems, cytokines are produced, ultimately leading to cell death and graft loss or delayed graft function. This detrimental injury to an organ transplant is thromboinflammation, which is primarily triggered by the humoral innate immune system, which primarily consists of the blood cascade systems, i.e., the complement, contact/coagulation, and fibrinolytic systems. Activation of these systems subsequently causes activation of endothelial cells, leukocytes and platelets, ultimately leading to thrombotic and inflammatory reactions.

As shown herein, by ex vivo treatment of an organ or part of an organ with polyethylene glycol phospholipid (PEG-phospholipid) molecules, the organ or part of an organ can be protected from thromboinflammation. Consequently, PEG phospholipid molecules are able to bind to the membranes of the cells of the endothelial lining of the vascular system of an organ or part of an organ and thereby compensate for the loss of the glycocalyx. Consequently, the PEG phospholipid molecules form a protective coating on the endothelium that inhibits or at least significantly reduces the deleterious effects of thromboinflammation, including lower levels of coagulation and complement activation, suppression of cytokine expression, and overall improvement in graft function.

In particular, ex vivo treatment of the organ with PEG-phospholipid molecules resulted in a significant reduction in cytokines, such as T cell-specific cytokines including interleukin 4 (IL-4) and IL-12. Untreated control organs exhibited high levels of such cytokines for several days after transplantation, which had a negative impact on continued immunization and future immune suppression. Therefore, coating the endothelium with PEG-phospholipid molecules inhibits cytokine-induced effects and damage to an organ or part of an organ after transplantation, and also improves the outcome of immune suppression after transplantation.

Another very unexpected effect achieved by ex vivo treatment of organs or parts of organs with PEG phospholipid molecules was the treatment, inhibition or prevention of hemodynamic instability with increased heart rate and reperfusion-associated hypotension that developed after organ transplant reperfusion. This transplant-associated hypotension can lead to poor perfusion, organ failure, and even lead to patient death. Hypotension is caused when an organ graft is connected to the patient's vascular system and thereby subjected to reperfusion. Treatment of such hypotension is currently an important task in organ transplantation, since if hypotension cannot be treated, then there is a significant risk of death of the patient during transplantation. However, by ex vivo treatment of the organ graft prior to transplantation with PEG phospholipid molecules, hypotension can be prevented or at least significantly inhibited or reduced. Therefore, the risk of hypotension-induced injury associated with organ transplantation is reduced with the use of the PEG phospholipid molecules of the present invention. The PEG phospholipid molecules of the present invention are therefore useful for the treatment, prevention and/or inhibition of post-reperfusion syndrome (PRS).

In a particular aspect of the present invention, therefore, a method is provided for treating an organ or part of an organ ex vivo . The method involves ex vivo infusion of a solution containing PEG phospholipid molecules into the vascular system, and optionally into the parenchyma, organ or part of an organ. The method also includes incubating an ex vivo solution containing a PEG phospholipid molecule in the vascular system, and optionally in the parenchyma, to ensure that at least a portion of the endothelial lining of the vascular system, and preferably the parenchyma, is coated with the PEG phospholipid molecules while exposing the organ or part of an organ immersed in an organ preservation solution containing PEG-phospholipid molecules.

Thus, the ex vivo method involves introducing PEG phospholipid molecules into the vascular system of an organ or part of an organ and allowing the PEG phospholipid molecules there to interact and bind to the membranes of endothelial and parenchyma cells. In fig. 1 schematically illustrates this principle with PEG phospholipid molecules hydrophobically interacting with the membrane of the endothelial lipid bilayer to thereby anchor or attach the PEG phospholipid molecules to the cell membrane through the phospholipid group.

The interaction between the PEG-phospholipid molecules and the lipid bilayer of the membrane of the endothelium and optionally the parenchyma, such as the renal parenchyma in the case of the kidney, occurs ex vivo while the organ or part of an organ is immersed or bathed in an organ preservation solution containing the PEG-phospholipid molecules. The experimental data presented in this application indicate that keeping an organ immersed in an organ preservation solution containing PEG-phospholipid molecules leads to significantly less activation of the cascade system and cytokine expression compared to keeping the organ or part of an organ immersed in the solution alone for organ preservation in which there are no PEG-phospholipid molecules, even if the vascular system, and optionally the parenchyma, of an organ or part of an organ is coated with PEG-phospholipid molecules.

Therefore, in order to achieve efficient processingex vivo organs or parts of organs, the vascular system, and optionally the parenchyma of an organ or part of an organ must be brought into contact with PEG phospholipid molecules and the organ or part of an organ must be immersed in an organ preservation solution containing PEG phospholipid molecules. Antithromboinflammatory effect of such treatmentex vivo significantly improved compared to infusionex vivo only a solution containing PEG-phospholipid molecules into the vascular system, and optionally into the parenchyma, organ or part of an organ. In fact, the antithromboinflammatory effects of the treatmentex vivo according to the present invention have already been induced during a period of cold ischemia when the organ or organ part is kept immersed in an organ preservation solution containing PEG phospholipid molecules, i.e., even before reperfusion of the organ or organ part.

In a specific embodiment, a solution containing PEG phospholipid molecules is first infused ex vivo into an organ or organ portion into the vascular system, and optionally into the parenchyma, organ, or organ portion. Such ex vivo infusion preferably occurs as soon as possible after explantation and removal of the organ or part of an organ from the donor's body. The perfused organ or portion of an organ is then immersed in and maintained in an organ preservation solution containing PEG phospholipid, preferably at a reduced temperature, such as approximately 4° C., as discussed later in this application.

In another specific embodiment, the organ or part of an organ is first immersed in an organ preservation solution containing PEG-phospholipid molecules, and then the solution containing PEG-phospholipid molecules is infused ex vivo into the vascular system, and optionally into the parenchyma, organ or part organ. Such ex vivo infusion can be accomplished by keeping the organ or part of an organ immersed in an organ preservation solution containing PEG phospholipid molecules. Alternatively, the organ or part of an organ is temporarily removed from the organ preservation solution for ex vivo infusion and then placed back into the organ preservation solution containing the PEG phospholipid molecules.

In one embodiment, the method also includes infusing an ex vivo organ preservation solution into the vascular system to flush unbound PEG phospholipid molecules from the vascular system. Therefore, unbound PEG phospholipid molecules are preferably washed away in one or multiple, ie at least two, washing steps using the organ preservation solution.

Such an organ preservation solution may also be used to flush the vasculature of an organ or part of an organ prior to infusion of a solution containing PEG phospholipid molecules in one or multiple flushing steps. In such an embodiment, the method also includes infusion of an ex vivo organ preservation solution into the vascular system prior to infusion of an ex vivo solution containing PEG phospholipid molecules into the vascular system. Pre-infusion of an organ preservation solution prior to the addition of PEG-phospholipid molecules will flush the vasculature of the organ or part of an organ to thereby facilitate effective coating of the endothelium with PEG-phospholipid molecules.

In one embodiment, the infusionex vivo solution containing PEG-phospholipid molecules includes the installationex vivo clamp on one of the arteries and veins of the vascular system. This embodiment also includes infusionex vivo solution containing PEG-phospholipid molecules into the other from the artery and vein and installing a clampex vivoon the other from an artery and a vein.

Therefore, in one embodiment, a clamp is first placed on a vein (or artery) of the vascular system of an organ or part of an organ to prevent the solution to be added from flowing out of the vascular system. A solution containing PEG phospholipid molecules is added to an artery (or vein) of the vascular system and a clamp is then placed on the artery (or vein) to prevent the solution to be added from leaking out of the vascular system. Consequently, the incoming and outgoing blood vessels of the vascular system of an organ or part of an organ are closed by installing a clamp to thereby retain the solution containing the PEG phospholipid molecules within the vascular system and thereby ensure the communication of the PEG phospholipid molecules with the endothelium of the vascular system.

In another embodiment, a solution containing PEG phospholipid molecules is infused into an artery (or vein) of the vascular system of an organ or part of an organ until the solution appears in a vein (or artery) of the organ or part of an organ. This confirms that the solution with PEG-phospholipid molecules filled the vascular system. At this point, a clamp is placed on the artery and vein.

A solution containing PEG phospholipid molecules can be added either through a vein or an artery. In a specific embodiment, the solution is administered by infusion into an artery. In such a particular embodiment, the optional initial installation of the clamp onto the vein of the vascular system is then preferably performed.

The clamp embodiments described above are particularly useful when the solution to be infused ex vivo into the vascular system is different from the organ preservation solution in which the organ or part of an organ and/or the PEG phospholipid molecule is immersed. contained in the solution are different from the PEG-phospholipid molecules contained in the organ preservation solution. In other cases, the clamp will not be required because the same PEG-phospholipid containing organ preservation solution will then be infused ex vivo as the solution in which the organ or part of the organ is immersed.

The solution containing the PEG phospholipid molecules is preferably incubated ex vivo in the vascular system for a period of time from 10 minutes to 48 hours to ensure hydrophobic interaction of the PEG phospholipid molecules with the endothelial cell membranes and thereby coat at least a portion of the vascular system organ or part of an organ. Ex vivo incubation is preferably carried out for from 20 minutes to 36 hours and more preferably from 30 minutes to 24 hours, for example from 30 minutes to 12 hours, up to 8 hours, up to 4 hours or up to 1 hour.

The amount of solution containing PEG-phospholipid molecules that is administered by infusion into the vascular system depends on the type of organ and the size of the organ (adult/child). As a rule, the volume of solution should be sufficient to fill the vascular system of the organ. In most practical applications, 5 ml to 250 ml of a solution containing PEG phospholipid molecules is administered into the vascular system by ex vivo infusion. In a preferred embodiment, from 5 ml to 100 ml and preferably from 5 ml to 50 ml of the solution containing the PEG phospholipid molecules is administered by ex vivo infusion into the vascular system.

In one embodiment, the solution contains from 0.25 mg/ml to 25 mg/ml PEG phospholipid molecules. In a preferred embodiment, the solution contains from 0.25 mg/ml to 10 mg/ml, preferably from 0.25 mg/ml to 5 mg/ml, for example 2 mg/ml PEG phospholipid molecules.

The concentrations of PEG phospholipid molecules described above can also be used for an organ preservation solution containing PEG phospholipid molecules.

According to the present invention, a solution containing PEG-phospholipid molecules is incubated ex vivo in the vascular system while keeping the organ or part of an organ submerged or flooded with an organ preservation solution containing PEG-phospholipid molecules. In addition, the organ or part of an organ is also preferably kept 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.

In this embodiment, the organ or part of an organ is immersed in an organ preservation solution containing PEG phospholipid molecules during incubation where the PEG phospholipid molecules are allowed to interact and bind to the membranes of endothelial cells in the vascular system. The organ or part of the organ is preferably also kept cold, i.e. at a temperature close to but above 0°C.

According to the present invention, an organ or part of an organ is kept immersed in an organ preservation solution containing PEG phospholipid molecules.

The theoretical ideal temperature for organ preservation has been shown to be 4°C - 8°C. While higher temperatures lead to hypoxic organ damage due to insufficient metabolic reduction, temperatures lower than 4°C increase the risk of cold injury with protein denaturation.

Today, the gold standard for preserving donor organs during organ transplantation in the clinic uses three plastic bags and a refrigerator. The first plastic bag contains the organ itself, immersed in an organ preservation solution. This first plastic bag is placed in a second plastic bag filled with saline solution, and then these two plastic bags are placed in a third plastic bag filled with saline solution, which is then placed in a refrigerator. More advanced organ preservation devices are available and can be used to store organs in a temperature-controlled environment, such as Sherpa Pak™ transport systems from Paragonix Technologies, Inc., Waves from Waters Medical Systems, LifePort transport systems from Organ Recovery Systems, and etc.

The solution containing the PEG phospholipid molecules may be a saline solution, an aqueous buffer solution, or an organ preservation solution.

Typical but non-limiting examples of aqueous buffer solutions that can be used include phosphate buffered saline (PBS) and citrate solution.

An organ preservation solution that can be used to infuse PEG-phospholipid molecules and/or flush the vascular system of an organ or part of an organ before or after ex vivo infusion of PEG-phospholipid molecules and/or into which an organ or part of an organ can be immersed can be selected from known solutions for organ preservation. Typical 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 Institute George Lopez-1 (IGL) solution. -1).

Experimental data presented in this application showed that PEG-phospholipid molecules are easily mixed with organ preservation solutions without any precipitation (Fig. 20).

In one embodiment, the PEG phospholipid molecules are non-functionalized PEG phospholipid molecules composed of PEG phospholipids. Therefore, in this embodiment, the PEG phospholipid molecules do not contain any functional groups. This corresponds to the presence of R corresponding to CH ₂ or H in FIG. 1.

In another embodiment, the PEG phospholipid molecules are functionalized PEG phospholipid molecules comprising a corresponding functionalized molecule attached to a PEG of the PEG phospholipid molecules.

In a further embodiment, the solution contains a mixture of non-functionalized PEG phospholipid molecules consisting of PEG phospholipids and functionalized PEG phospholipid molecules containing a corresponding functionalized molecule attached to the PEG of the PEG phospholipid molecules.

In particular embodiments comprising only functionalized PEG phospholipid molecules or a mixture of functionalized and non-functionalized PEG phospholipid molecules, the functionalized molecule is preferably selected from the group consisting of a complement inhibitor, a blood clotting inhibitor, a platelet inhibitor, a molecule capable of binding a complement inhibitor, a molecule, a molecule capable of binding a blood clotting inhibitor, a molecule capable of binding a platelet inhibitor, and mixtures thereof.

Exemplary but non-limiting examples of complement inhibitors include Factor H; C4b-binding protein (C4BP); 4 to 6 N-terminal short consensus repeats (SCRs) of complement receptor 1 (CR1), also known as C3b/C4b receptor or cluster of differentiation 35 (CD35); complement regulatory protein CD46 (CD46), also known as membrane cofactor protein (MCP); and complement decay accelerating factor (DAF), also known as CD55.

A typical but non-limiting example of blood clotting inhibitors includes heparin.

A typical but non-limiting example of a platelet inhibitor is an adenosine diphosphate (ADP) cleaving enzyme such as apyrase and ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase 1), also known as CD39.

Representative but non-limiting examples of a molecule capable of binding a complement inhibitor are Factor H binding peptide such as 5C6 described in [4]; and a C4BP binding peptide such as the Streptococcus M protein-derived peptide M2-N, M4-N or M22-N described in [5]

A typical but non-limiting example of a molecule capable of binding a coagulation inhibitor is a heparin binding peptide such as described in [7].

Therefore, in one embodiment, the functionalized molecule is selected from the group consisting of a heparin binding peptide, 4 to 6 N-terminal SCRs CR1, CD46, DAF, a Factor H binding molecule, an ADP-cleaving enzyme, and mixtures thereof.

In a specific embodiment, the functionalized molecule is selected from the group consisting of 5C6 peptide, apyrase, CD39, C4B, and a mixture thereof.

In one embodiment, the PEG phospholipid molecules have formula (I) or formula (II):

In one embodiment, n, m are integers independently selected from 10 to 16. The parameters n, m are preferably independently selected as 10, 12, 14, or 16, and more preferably n = m = 14.

In one embodiment, p is selected such that the PEG chain has an average molecular weight selected in the range of 1,000 Da to 40,000 Da. The parameter p is preferably chosen such that the PEG chain has an average molecular weight of from 3,000 to 10,000 Da, and more preferably 5,000 Da.

Average molecular weight, as defined herein, indicates that individual PEG phospholipid molecules may have a molecular weight different from this average molecular weight, but that the average molecular weight represents the average of the molecular weights of the PEG phospholipid molecules. This further implies that there will be a natural distribution of molecular weights around a given average molecular weight of the PEG phospholipid sample.

The terminal group R is, in a particular embodiment, H, CH ₂ , NH ₂ , a maleimide group, a biotin group, a streptavidin group, an avidin group, or a functionalized molecule. In the case of a functionalized molecule, it is preferably selected from the group consisting of a complement inhibitor, a blood clotting inhibitor, a platelet inhibitor, a molecule capable of binding a complement inhibitor, a molecule capable of binding a blood clotting inhibitor, a molecule capable of binding a platelet inhibitor, and mixtures thereof.

The fatty acid chains of the PEG phospholipid molecule can be saturated. At least one or both fatty acid chains may alternatively be unsaturated, ie, contain at least one -CH=CH- group, at least one -C≡C- group, or a combination thereof. Each fatty acid chain can be straight or branched.

The PEG phospholipid molecules that are administered ex vivo by infusion into the vascular system, and optionally the parenchyma, organ or part of an organ, may be of the same type as the PEG phospholipid molecules contained in the organ preservation solution, immersed in which the organ is held or part of an organ. In another embodiment, different types of PEG phospholipid molecules can be used in a solution that is administered ex vivo by infusion into the vascular system, and optionally into the parenchyma, organ or organ part, as compared to an organ preservation solution immersed in which the organ or part is kept organ. For example, the PEG chain size may vary among PEG phospholipid molecules, with the PEG chain size of the PEG phospholipid molecules being selected to achieve the target size for the vascular system, and optionally parenchyma, organ, or portion of an organ. In fig. 19 shows that the size of the PEG chain limits the portions of the vasculature and parenchyma that PEG phospholipid molecules can reach. In particular, 5 kDa PEG phospholipid molecules can reach larger parts of the vasculature and parenchyma compared to larger 40 kDa PEG phospholipid molecules.

Therefore, in a particular embodiment, the PEG phospholipid molecules contained in a solution that is administered by ex vivo infusion into the vascular system, and optionally the parenchyma, organ, or portion of an organ, may have a different average PEG chain size compared to the PEG phospholipid molecules contained in organ preservation solution in which an organ or part of an organ is immersed.

Alternatively or in addition, the PEG phospholipid molecules in the ex vivo infusion solution and the PEG phospholipid molecules in the organ preservation solution may contain different functional groups, ie, R terminal groups in formula (I) and (II).

The ex vivo method of the present invention can be applied to any organ, or part of an organ, containing a vascular system and which can be used as a transplantable organ, ie, transplanted into a recipient. Typical but non-limiting examples of such organs include the kidney, liver, pancreas, heart, lung, uterus, bladder, thymus and intestines.

In a specific embodiment, the organ is a kidney. In this particular embodiment, ex vivo incubation of a solution containing PEG-phospholipid molecules in the vasculature allows coating of not only the renal endothelium of the renal vasculature, but preferably also the tubules of the renal parenchyma with PEG-phospholipid molecules. Thus, the experimental data presented in this application show that PEG-phospholipid molecules were evenly distributed in the peritubular vessels, glomeruli and tubules of the kidney upon ex vivo injection of a solution containing PEG-phospholipid molecules through the aortic remnant.

Experimental data presented herein showed that PEG-phospholipid-treated organ transplants had significantly less thromboinflammation compared with untreated control organ transplants, as measured by plasma levels of coagulation markers such as TAT and complement markers such as C3a and sC5b-9. Also, the expression of cytokines, particularly IL-6 and IL-1β, was significantly suppressed in PEG-phospholipid-treated organ transplants. Organ function was improved in PEG-phospholipid-treated organ transplants. PEG-phospholipid-treated organ transplants also showed reduced deposition of complement markers, such as C3b and membrane attack complexes, and expression of C5aR.

In summary, ex vivo coating of organ transplants with PEG phospholipid molecules protects against I/R-induced thromboinflammation, suggesting that it is effective against I/R injury during transplantation in the clinic.

In a further aspect of the present invention, a PEG phospholipid is provided for use in treating, inhibiting or preventing reperfusion-associated hypotension following organ transplant reperfusion.

In another aspect of the present invention, a PEG phospholipid is provided for use in treating, inhibiting or preventing PRS.

A complication during and immediately, usually up to 24 hours, after organ transplantation is a significant drop in blood pressure after reperfusion of the organ graft into the recipient. This hemodynamic change in the recipient can, in severe cases, lead to ischemia in the recipient's organs and in the transplanted graft, causing damage to any of these organs and even death of the patient if treatment is not successful.

The observed protection by PEG-phospholipid against vascular instability may be a consequence of inhibition of the contact/kallikrein system. Unexpectedly, it was found that the contact/kallikrein system was activated after reperfusion. The contact/kallikrein system generated bradykinin, a highly vasoactive peptide that causes vasodilation and increased permeability leading to hypotension, which causes compensatory tachycardia. During surgery, lowering blood pressure is treated with pharmaceutical substances and fluids, however, a life-threatening situation may occur. Thus, the ability to prevent hypotension is of great clinical importance.

Thus, preventing, treating, or at least reducing such hypotension and the fall in blood pressure associated with organ transplant reperfusion is a significant improvement in organ transplantation. The experimental data presented herein demonstrated that ex vivo treatment of an organ graft prior to transplantation with a solution containing PEG-phospholipid molecules protected the recipient from such hypotension or a drop in blood pressure when the organ graft was reperfused.

In one embodiment, the PEG phospholipid is in the form of a solution containing PEG phospholipid molecules and is intended for ex vivo infusion into the vasculature of an organ graft to provide coverage of the endothelial lining of the vasculature and parenchyma with PEG phospholipid molecules.

In a similar aspect, the present invention provides the use of a PEG phospholipid for the manufacture of a medicament for treating, inhibiting or preventing reperfusion-associated hypotension occurring after organ transplant reperfusion. The aspect also provides a method for treating, inhibiting, or preventing reperfusion-associated hypotension that develops following organ transplant reperfusion. This method includes the previously described method steps of ex vivo infusion of a solution containing PEG-phospholipid molecules into the vascular system of an organ transplant and incubation of an ex vivo solution containing PEG-phospholipid molecules in the vascular system to provide coverage of at least a portion of the endothelial lining of the vascular system. system with PEG-phospholipid molecules, optionally, but preferably, while keeping the organ transplant immersed in an organ preservation solution containing PEG-phospholipid molecules.

experiments

EXPERIMENT 1

MATERIALS, METHODS AND ANIMAL EXPERIMENTS

Synthesis of PEG-phospholipid derivatives

a. FITC-PEG-phospholipid

The synthesis of PEG phospholipids was carried out using methods described previously [1]. Briefly, 1,2-dipalmitoyl- sn -glycero-3-phosphatidylethanolamine (NOF Corporation, Tokyo, Japan, DPPE, 21 mg) was dissolved in 3 mL dichloromethane (Sigma-Aldrich Chemical Co. (St. Louis, MO, USA)) . α- N -Hydroxysuccinimidyl-ω-tert-butoxycarbonylpolyethylene glycol (NHS-PEG-Boc, Mw: 5000, from NOF, 185 mg) and triethylamine (3 μL, Nacalai Tesque (Kyoto, Japan)) were added to the reaction solution, and then stirred for 4 days at room temperature (RT ~20°C). The NHS group NHS-PEG-Boc readily reacted with the amino group of phospholipids. The Boc amino group was deprotected by adding 2 mL of 99% trifluoroacetic acid (Wako Pure Chemical (Osaka, Japan)) and further incubation at 4°C for 30 min. The crude product was purified by reprecipitation in diethyl ether (Nacalai Tesque). After extraction with chloroform (Nacalai Tesque) and evaporation, PEG phospholipids were obtained as a white solid. The yield was 85%. For fluorescent labeling with fluorescein isothiocyanate (FITC) (Dojindo Laboratories (Kumamoto, Japan)), PEG phospholipids (200 mg) were allowed to react with FITC (31 mg) in acetone for 12 h. FITC PEG phospholipid was purified by gel permeation chromatography (Sephadex G-25, GE Healthcare (Buckinghamshire, UK)).

The synthesis of FITC-PEG phospholipid (40 kDa) was carried out according to the method described below. α-N-Hydroxysuccinimidyl-ω-maleimidylpolyethylene glycol (NHS-PEG(40k)-Mal, Mw: 40000) and DPPE (9.6 mg) were dissolved in 3 ml of dichloromethane and triethylamine (3 μl, Nacalai Tesque) was added to the reaction solution. and then stirred for 4 days at room temperature (RT, ~20°C). The crude product was purified by reprecipitation in diethyl ether. After extraction with chloroform and evaporation, Mal-PEG phospholipids (40 kDa) were obtained as a white solid. The yield was 80%. For fluorescent labeling with fluorescein isothiocyanate (FITC), Mal-PEG phospholipids (40 kDa) (1200 mg) were dissolved in DMSO (Nacalai Tesque, 20 ml), and then FITC-glycine cysteine (FITC-GC, BEX Co., Ltd.) was added. , Tokyo, Japan, 12 mg) and incubated for 24 h at 37°C. The crude product was purified by reprecipitation in diethyl ether. After extraction with chloroform and evaporation, FITC-PEG phospholipids (40 kDa) were obtained as a yellow solid.

b. Biotin-PEG-phospholipid

Biotin-PEG phospholipid was synthesized by combining polyethylene glycol ether ( N -hydroxysuccinimide-5-pentanoate)-2-(biotinylamino)ethane (biotin-PEG-NHS, Mw: 5000 from NOF, 180 mg) and 1,2-dipalmitoyl- sn -glycero-3-phosphatidylethanolamine (DPPE) (20 mg) with triethylamine (50 µl) and dichloromethane (4 ml) and stirring for 48 h at RT. Precipitation with diethyl ether produced biotin-PEG phospholipid as a white powder (165 mg; 80% yield). Biotin-PEG phospholipid was used for imaging without further purification.

c. PEG phospholipid

PEG phospholipid was synthesized by combining α-succinimidyloxysuccinyl-ω-methoxypolyoxyethylene (MeO-PEG-NHS, Mw: 5000 from NOF, 171 mg) and DPPE (22 mg) with triethylamine (25 μl) and dichloromethane (5 ml) and stirring in within 72 hours with CT. Precipitation with diethyl ether produced PEG phospholipid as a white powder (160 mg; 80% yield). PEG phospholipid was used to treat the kidney without further purification.

Functional evaluation of different types of PEG phospholipids

In order to evaluate whether the functional group had any effect on the protective properties, a series of experiments were carried out on primary human mesenchymal stem cells (hMSC). hMSCs were coated with PEG-phospholipid, FITC-PEG-phospholipid, and biotin-PEG-phospholipid. After surface modification, the viability of the coated cells was analyzed using the Alamar Blue assay (Life technologies, Stockholm; Sweden), according to the manufacturer's instructions. Briefly, coated cells were cultured in MSC culture medium in a 96-well microcell culture plate. After 6 hours of culture, 10 μl of alamar blue reagent was added to 100 μl of culture medium, and then incubated for 4 hours at 37°C. Then fluorescent emission was measured at 580 - 610 nm.

To determine the hemocompatibility of hMSCs, surface-modified MSCs were contacted with whole blood in a loop model described previously [7]. Briefly, whole blood was collected from healthy volunteers into heparin-coated tubes (Corline AB, Uppsala; Sweden). Heparinized peripheral intravenous catheter tubing (30 cm) was used. Each tube was filled with 3 ml of blood, to which 10,000 MSC/ml was added. The tubes were rotated at 30 rpm at 37°C for 60 min. After incubation, 1.2 ml of blood was collected into 2 ml tubes (Eppendorf, Germany) containing EDTA at a final concentration of 10 mM. The blood was then centrifuged at 4200× g for 15 min at 4°C to obtain plasma. Plasma was aliquoted and stored at -80°C until further analysis.

Conservation of PEG phospholipid with different PEG subunit lengths

Cells (CCRF-CEM, suspension cell line; n=3) were incubated with FITC-PEG phospholipid (2 mg/ml in PBS) for 30 min at RT and then washed with culture medium. In this case, FITC-PEG phospholipids with different molecular weights of the PEG chain were used - 1 kDa, 5 kDa and 40 kDa. Treated cells were cultured in culture medium at 37°C and collected for flow cytometry analysis at 0, 1, 3, 6, 24, and 48 h.

Allogeneic transplantation model in pigs

The pig was chosen as a large animal model because of its anatomy and physiology, which is very similar to that of humans. In particular, the structure and function of the kidney are very similar to those of the said primate organ. All pig studies used common breeds with a high standard of health. In long-term survival experiments, animals were allowed to acclimatize over a two-week period and undergo a training and socialization program. Clinical investigations, including urinary tract ultrasound, hematology, and clinical pathology, were performed frequently, and SLA typing to identify donor-recipient mismatches was performed before transplantation. Recipient pigs recovered quickly from surgery but were not treated with immunosuppressive drugs. Administration of such drugs disrupts the immune response and was excluded from the experiments. Pigs were therefore euthanized after 96 h to avoid acute graft rejection. A complete necropsy was performed after killing. All studies were approved by the Animal Experimentation Ethics Committee, Uppsala, Sweden.

A. Short-term model (kidney perfused with PEG-phospholipid alone for 40 min)

Ischemia-reperfusion injury (I/R injury) is a major contributor to the pathology of several conditions, including transplantation, thrombotic disorders, sepsis, and cardiopulmonary bypass. The present experiment examined whether ex vivo coating of ischemic cell surfaces of porcine kidney grafts with PEG phospholipid protects against I/R injury in a model of en bloc allogeneic kidney transplantation.

Animals. The study was carried out at the Hedenstierna Experimental Laboratory, Uppsala University Hospital, Sweden. The study included six donor pigs and six recipient pigs of both sexes. The body weight and age of the pigs ranged from 30 - 35 kg and 10 - 12 weeks, respectively. Non-survival procedures were used and animals were sacrificed at the end of an 8-hour post-transplant observation period.

Anesthesia. Upon arrival at the sedation laboratory, an intramuscular injection of 2.2 mg/kg xylazine (Rompun; Bayer, Leverkusen, Germany) in combination with 6 mg/kg zolazepam (Zoletil; Virbac, Carreau, France) was administered. The animals were then fixed in a prone position. Peripheral intravenous catheters were inserted into both ear veins to induce and maintain anesthesia and to administer fluids. An epitracheal small incision was made to expose the trachea and transtracheal intubation for mechanical ventilation using a mechanical ventilator Servo-I, MAQUET, Medical Systems, USA. The animals were mechanically ventilated with 30% oxygen in the air reaching 5.0 - 5.5 kPa CO ₂ . The goal was a mean arterial pressure (MAP) >60 mmHg to ensure adequate organ perfusion.

Transplantation model . We have developed a porcine transplant model that allows double en bloc retrieval and implantation of both kidneys from a donor animal into a recipient animal. By isolated ex vivo incubation with PEG phospholipid of just one randomly selected kidney from a single block set, this technique allowed us to cotransplant the treated organ with perfectly matched control tissue into a single recipient pig, which not only reduced the number of experimental animals, but also minimized experimental confounding. variables.

The trunk of the supra- and infrarenal aorta, as well as the inferior vena cava, were mobilized, so that a single block set was created, consisting of two kidneys, ureters and the entire renal vasculature. The single block set was immediately removed and washed with cold HTK solution (Custodiol; Chemie GmbH, Germany). The single block set was then cold stored in HTK solution at 4°C for 24 hours.

After storage, one kidney from each single block set was randomly selected for incubation with PEG phospholipid. A clamp was placed on the vein and artery of the contralateral kidney (control), as well as on the vein of the selected kidney. A total of 10 - 15 ml of PEG-phospholipid solution at a concentration of 2 mg/ml was slowly infused into the selected kidney. A clamp was then placed on the artery of the treated kidney and the single block set was cold stored for an additional 40 min at 4°C in HTK. After incubation and before implantation, excess PEG phospholipid was washed out from the treated kidney using HTK without removing the clamps from the control.

Recipient pigs were handled and anesthetized as previously described. The single block set was placed horizontally inside the peritoneum. The distal end of the vena cava and aorta of the graft was connected by a terminolateral anastomosis to the vena cava and aorta of the recipient, respectively. After removal of the clamp, the treated and control kidneys were sequentially reperfused with delayed release of the clamp from the control kidney at intervals of several seconds, during which time the eluate was washed out from the treated kidney. This was done to prevent contamination of the control kidney with remaining PEG phospholipid solution within the aortic conduit. Both transplanted ureters were catheterized separately to document the amount of urine produced.

B. Long-term model (kidney perfused with PEG-phospholipid alone for 40 min)

Animals. Based on the SLA typing results, crossbred donor (n=12) and recipient (n=21) pigs were selected for incompatible transplantation. Animals were housed in separate pens measuring 3 ^m2 within sight and hearing of each other. Straw and sawdust were used as bedding. A 14:10 h light/dark cycle was used (lights on at 06:00 h) and an infrared lamp was provided in each pen (24 h). The temperature was 16 - 18°C. Animals were given commercially available finishing feed without growth stimulants (SOLO 330, Sweden) twice a day, the amount was dependent on body weight and was consistent with the Swedish University of Agricultural Sciences swine diet. Water was provided without restriction. Donor pigs were anesthetized with a combination of medetomidine (Domitor® vet, 1 mg/ml; Orion Pharma Animal Health, Sollentuna, Sweden) at a dose of 0.1 mg/kg body weight, butorphanol (Butomidor vet, 10 mg/ml; Salfarm Scandinavia, Helsingborg, Sweden) at a dose of 0.2 mg/kg body weight and ketamine (Ketaminol® vet, 100 mg/ml; Intervet International BV, Boxmeer, the Netherlands) at a dose of 5 mg/kg body weight intramuscularly (IM) and the kidneys were studied with ultrasound (Logiq e R6, GE Healthcare, Wawatosa, USA) using linear (10 MHz) and curved (4 MHz) probes to exclude pigs with renal cysts. Length, echogenicity, and corticomedullary differentiation were assessed, the peritoneal abdominal region was assessed for signs of pyelectasia, and color Doppler was used to assess the presence of renal blood flow. During the two-week acclimation period, pigs were cared for daily and allowed to become accustomed to the various examinations to be performed without restraint after transplantation.

Transplantation model. Anesthesia was induced with a combination of medetomidine (Domitor® vet, 1 mg/ml; Orion Pharma Animal Health, Sollentuna, Sweden) at a dose of 0.05 mg/kg body weight and tiletamine and zolazepam (Zoletil®, 50 mg + 50 mg/ml; Virbac , Reading, Carreau, France) at a dose of 5 mg/kg body weight IM. Buprenorphine (Vetergesic® vet, 0.3 mg/ml; Orion Pharma Animal Health, Sollentuna, Sweden) at a dose of 0.01 mg/kg body weight intramuscularly and epidural morphine (Morfin Epidural Meda, 2 mg/ml; Meda , Solna, Sweden) at a dose of 0.1 mg/kg was given preoperatively for additional analgesia. Procaine-benzylpenicillin (Penovet® vet, 300 mg/ml; Boehringer Ingelheim Vetmedica, Ingelheim am Rhein, Germany) was given preoperatively at a dose of 20 mg/kg body weight IM. After induction of anesthesia, long-term venous catheters (Careflow™, 3Fr, 200 mm, BD, Franklin Lakes, NJ, USA) were inserted into the ear vein. (vena auricularis) according to the Seldinger method. After surgery, these catheters made it easier to draw blood without stress. Catheters were flushed once daily with 100 IU/mL heparinized saline (LEO Heparin, 5000 IU/mL; Leo Pharma, Ballerup, Denmark). Anesthesia was maintained with isoflurane (IsoFlo® vet; Orion Pharma Animal Health, Sollentuna, Sweden) vaporized in oxygen. Pigs were continuously treated with crystalloid fluids (Ringer acetate; Fresenius-Kabi, Bad Homburg, Germany). Colloids (Gelofusin® 40 mg/ml; Braun, Melsungen, Germany) and dobutamine (Dobutamin Carino, 250 mg/ml; Carinopharm, Elze, Germany) were given as needed to maintain blood pressure within reference values. Respiratory and cardiovascular parameters were monitored during anesthesia.

Donation procedure. In the first set of experiments (3 + 3 donors and 6 + 5 recipients), both kidneys were removed, similarly perfused with HTK (n=11), kept on ice overnight, and then one kidney was randomly treated with PEG-phospholipid 60 min before implantation, while the other kidney was left untreated. An alternative application of PEG-phospholipid was performed with another ten kidneys (5 donors and 10 recipients) that were treated with either HTK perfusion containing PEG-phospholipid or conventional HTK perfusion solution. After the procedure, donor pigs were euthanized by intravenous injection of sodium pentobarbital (Allfatal vet, 100 mg/ml; Omnidea AB, Stockholm, Sweden) under general anesthesia.

Transplant operation. Recipient pigs underwent allogeneic transplantation of one kidney. All procedures were performed intraperitoneally through a 15 to 20 cm midline incision, and the iliac vessels in the right iliac fossa were identified and carefully mobilized. The distal fragment of the vena cava, as well as the iliac artery (from its exit from the aorta) was mobilized by ligating the surrounding lymphoid tissue. The kidney graft was then placed in the right iliac fossa near the iliac vessels. The renal vein and artery were trimmed and subsequently anastomosed in a terminolateral manner to the right iliac artery and distal vena cava of the recipient using a running suture using 7/0 polypropylene suture (PROLENE®, Ethicon, USA). The ureters were implanted by extravesical ureterocystoneostomy at the bladder apex using 6-0 polydioxanone suture (PDS®, Ethicon, USA). After this, bilateral nephrectomy of the patient's own kidneys was performed. The midline incision was closed using a continuous fascial suture using 2/0 polyglactin suture (Vicryl® Ethicon, USA) and skin staples. The average duration of surgical intervention was 3 hours (2.5 - 5.5 hours).

At the end of surgery, isoflurane was stopped, the fraction of inspired oxygen was increased (FiO ₂ 1.0) and the pigs were removed from the mechanical ventilator, and spontaneous breathing was restored. The pigs were placed in an intensive care cage where staff could easily monitor their condition continuously. The pigs were given supplemental oxygen and pulse rate, respiratory rate, oxygen saturation, and body temperature were monitored until the pigs regained consciousness. During the first 24 hours, the pigs were hand-fed fruit and helped to drink water when needed. They were also assisted in standing and walking and were given several massages of the gluteus medius, biceps femoris and gluteus maximus muscles. For postoperative pain relief, buprenorphine was administered intravenously for two days.

Killing and necropsy. Recipients were euthanized with an overdose of sodium pentobarbital (Allfatal vet, 100 mg/ml; Omnidea AB, Stockholm, Sweden) after 96 hours. Kidney biopsies for histological examination were collected immediately after death and post-mortem examination of all pigs was carried out at the Department of Biomedical Sciences and of Veterinary Public Health, in the Department of Pathology, SLU.

Pre-treatment with HTK solution. In another series of experiments, four pigs were transplanted with PEG-phospholipid-treated kidneys and five pigs with control kidneys. In this series of experiments, after removal, the kidneys were perfused with a solution of HTK (Custodiol; Chemie GmbH, Germany) with 2 mg/ml PEG phospholipid (treated kidneys) or without PEG phospholipid (control kidneys). Kidneys were also stored immersed in HTK solution with 2 mg/ml PEG-phospholipid (treated kidneys) or in HTK solution without added PEG-phospholipid (control kidneys) at 4°C for 24 hours.

C. Long-term model to assess PEG-phospholipid shedding from renal endothelium.

Kidneys were removed from donor pigs and transplanted into recipient pigs after two weeks of acclimation and preparation as described above. The kidneys were processed using the same procedure as for the grafts, which were injected with PEG phospholipid 60 min before implantation. To measure release from the endothelium, PEG phospholipid was fluorescently labeled. The same anesthetic and surgical protocol were used, but the recipients retained their own kidneys (n = 4). Pigs were euthanized after 12, 24, 48, and 72 h, respectively. All pigs underwent necropsy and biopsies were collected for histological analysis and fluorescence monitoring.

Taking blood samples

In the short-term model, blood samples were obtained from the veins of the left and right kidney grafts at 5, 15, 30, 60, 120, 240, and 360 minutes after the start of reperfusion. In the long-term model, blood was obtained at 1, 24, 48, 72, and 96 hours. Samples were collected in EDTA Vacutainer tubes (Becton Dickinson AB, Sweden) and immediately centrifuged at RT for 15 minutes. Collected plasma was stored at -80°C for further analysis.

ELISA TAT, C3a and sC5b-9

Plasma TAT, C3a, and sC5b-9 were measured by sandwich ELISA as previously described [7]. Briefly, to assess TAT, plasma samples were diluted 1/100 in normal plasma with citrate-phosphate-dextrose (CPD). Monoclonal anti-human thrombin antibody was used for capture and HRP-conjugated anti-human antithrombin (AT) antibody was used for detection (Enzyme Research Laboratories, South Bend, IN, USA). The standard was prepared by diluting pooled human serum into normal CPD plasma. Values were expressed in units of μg/L. To assess C3a, plasma was diluted 1/1000 in running buffer (PBS containing 0.05% Tween 20, 10 mg/ml BSA, and 10 mM EDTA). Anti-human C3a mAb 4SD17.1 was used to capture C3a and a biotinylated rabbit polyclonal anti-C3a antibody followed by HRP-conjugated streptavidin to detect C3a in plasma. Zymosan-activated serum, calibrated against purified C3a, served as a standard. Values were expressed in units of ng/ml. For sC5b-9, plasma was diluted 1/50 in running buffer (as described above). sC5b-9 was captured by the anti-human C5b-9 mAb (a9e) and detected with rabbit polyclonal anti-human C5 antibody (Dako) and HRP-conjugated anti-rabbit IgG (Dako). Zymosan-activated serum containing 6x104 u/ml was used as a standard. Values were expressed in units of a.u./ml.

Histological analysis

Kidney tissue biopsies were taken from control and biotin-PEG-phospholipid-treated kidneys at 5, 60, and 360 minutes after reperfusion. To detect PEG phospholipid, biopsies were immediately frozen in liquid nitrogen and stored at -80°C until further analysis. Frozen kidney sections (4 μm thick) treated with biotin-PEG phospholipid were incubated in Alexa488-streptavidin (GE Healthcare, 1:500) at RT for 10 min. Sections were analyzed using laser scanning confocal microscopy (LSM510, META, Carl Zeiss, Germany). Both biotin-PEG-phospholipid and FITC-PEG-phospholipid (5 kDa, 40 kDa) were evaluated.

Tissue samples were also fixed in 4% paraformaldehyde overnight and stored in 70% ethanol until embedded in paraffin. Paraffin sections (5 μm thick) were deparaffinized with xylene and rehydrated in ethanol. Heat-induced antigen retrieval was performed by boiling sections twice in sodium citrate buffer. Blocked with goat serum before incubation with primary antibody. The resulting sections were stained with hematoxylin and eosin (HE stain) and antibodies to complement markers such as C5aR (Acris Antibodies, Herford, Germany), C3b (Bioss Antibodies, Woburn, MA, USA), C4d (Abcam, Cambridge, UK) and C5b -9 (MAC; Abcam). Rabbit IgG (Dako, Glostrup, Denmark) and mouse IgG (Abcam) were used as controls. Primary antibody detection was performed using a Dako real alkaline phosphatase/red detection system (Dako) followed by hematoxylin counterstaining. Slides were imaged using a Zeiss Axio Imager A1 microscope. The objective was a 10X objective and the areas assessed were 800,000 μm ² in area. Quantitative analysis of square areas was performed to determine the intensity and area of immunoreactive areas using Axio Visio software (ed. 4.8) (Zeiss, Jena, Germany). The results are presented as mean densitometric sum readings. Histological analysis was performed by a pathologist under double anonymity of the samples to detect tubular dilation and necrosis, glomerular damage and vacuolization.

Statistical analysis

Results are presented as means ± standard error of the mean. Data plotting and statistical analysis were performed using Prism software, version 6, for Macintosh (Graphpad, San Diego, CA, USA). Differences between means for two groups were assessed statistically using the paired Wilcoxon test, whereas statistical significance between more than two groups was analyzed using two-way repeated measures ANOVA followed by Bonferroni post hoc estimation. Comparisons between groups are considered significant when p < 0.05, while values above are considered not significant (n/s).

results

Comparison of the functional properties of various PEG-phospholipid preparations

hMSCs were treated with different types of PEG-phospholipid preparations. The viability of hMSCs was not affected by surface modification with PEG phospholipids alone (97.3%), FITC-labeled PEG phospholipids (98.3%), and biotin-labeled PEG phospholipids (97%), indicating that PEG- phospholipids do not affect viability.

PEG phospholipid retention with different PEG subunit lengths was examined using CCRF-CEM suspension cells. Cells were coated with PEG phospholipid with 1 kDa, 5 kDa, and 40 kDa PEG subunits conjugated to FITC. Cells were incubated at 37°C and binding was monitored by flow cytometry. They showed that the binding of all drugs was lost by 90% after 24 hours, but the 5 kDa drug persisted for the longest time (Fig. 13).

TAT levels were significantly reduced when PEG-phospholipid-coated cells were brought into contact with whole blood compared with uncoated cells. This reduction in TAT formation was similar for cells coated with either PEG phospholipid alone (61.5 ± 2.9 versus 110 ± 7.6), FITC-labeled PEG phospholipid (60.1 ± 2.7), or biotin-labeled PEG-phospholipid (61.4 ± 5.1), compared with uncoated PEG-phospholipid controls (110 ± 7.6) (Figure 2A).

A similar trend was observed for markers of complement activation when coated and uncoated cells were brought into contact with whole blood. At 60 min post-blood exposure, plasma levels of C3a and sC5b-9 were significantly reduced when cells were coated with FITC-labeled PEG phospholipid (50.8 ± 3.2, 37.7 ± 1.5) (51 .3 ± 3.8, 35.0 ± 2.1) and biotin-labeled PEG-phospholipid (49.8 ± 4.1, 38.3 ± 2.0) compared to uncoated cells (104 ± 7.7, 104 ± 6.2) (Figs. 2A and 2B).

Distribution of FITC-labeled phospholipid in porcine kidneys treated with PEG phospholipids

Allogeneic kidneys were transplanted en bloc into recipients (n = 6) as described in Materials and Methods. The distribution of PEG phospholipid in the kidney tissue after reperfusion in the recipient was analyzed in biopsies taken after 5, 60 and 360 min. In fig. Figure 4 shows that pre-reperfusion fluorescence from FITC-labeled PEG-phospholipid was uniformly distributed in the peritubular vessels, glomeruli, and tubules of the entire kidney, reflecting that the kidney tissue was uniformly modified with PEG-phospholipid by injecting the PEG-phospholipid solution through the aortic remnant. In contrast, no fluorescence was observed in the tissues of the control kidneys, indicating that there is no significant flow between the test and control kidneys.

When kidney tissues were compared at 5, 60, and 360 min, fluorescence, particularly in the glomeruli, but also in the peritubular vessels and tubules, in FITC-PEG-phospholipid-treated kidney tissues gradually decreased over time, indicating that PEG- The phospholipid was released from the cells, although some PEG phospholipid still remained associated with the tubules after 360 min.

In the long-term study to assess detachment (C. Long-term model to assess the detachment of PEG-phospholipid from the kidney endothelium), only minor fluorescence was visible after 24 hours, and no fluorescence was observed after 48 hours, indicating that all PEG-phospholipid had detached. Ex vivo, FITC-labeled PEG phospholipid also adhered to cardiac vascular endothelium.

A. Short-term allogeneic transplantation model

Immunohistochemical analysis of transplanted kidneys

Transplanted kidneys were analyzed by immunohistochemistry for albumin leakage, complement deposition, and receptor expression (C3a, sC5b-9, C4d, C3b; Figures 5A to 5D). Histological evaluation revealed lower levels of complement activation markers such as C3b/iC3b and C5b-9 and complement receptor C5aR expression in PEG-phospholipid-treated kidney grafts compared with control kidneys. There was a significant decrease in C3b/iC3b deposition after 5 min in the PEG-phospholipid-treated kidney after perfusion. For albumin or the oxidative stress markers HO-1, iNOS, and nitrotyrosine, there was a trend (n/d) that these markers were lower in PEG-phospholipid-treated kidneys compared to control kidneys. Thus, the results of histological ranking confirm that treatment of the kidney graft surface with PEG-phospholipid protects against complement activation and that there is a tendency to suppress oxidative stress.

Activation of blood coagulation and complement

To evaluate the effect of PEG-phospholipid treatment on the vascular innate immune system after renal reperfusion in a short-term allogeneic en bloc transplantation model in pigs, markers of coagulation activation (TAT, Fig. 6A) and complement activation (C3a, sC5b-9, Fig. 6A) were analyzed. 5A and 5B) in the blood. TAT levels were lower in the blood of recipients with PEG-phospholipid-treated kidneys compared with control recipients. There was a significant decrease in TAT levels in PEG-phospholipid-treated kidneys compared to uncoated controls at 5 min (46.1 ± 5.2 vs. 73 ± 12.3, p: 0.01), 15 min (48 ± 8. 4 vs. 73 ± 9, p: 0.02), 30 min (41 ± 9.1 vs. 57 ± 4.9, n/s), 60 min (32 ± 4.8 vs. 58 ± 3.7, p: 0.02), and 120 min (28.6 ± 5.1 vs. 53 ± 4, p: 0.02), but not at 240 min (32 ± 4 vs. 49 ± 6.1, n/s) and 360 min (29 ± 9 compared with 41 ± 14, n/s) after reperfusion. These results indicated that the activation of blood coagulation was significantly suppressed by surface modification after kidney transplantation.

Similar results were observed for complement markers (C3a and sC5b-9). There was a significant decrease in blood C3a levels in recipients receiving PEG-phospholipid-treated kidneys compared to control recipients at 5 min (40 ± 2.8 versus 63.6 ± 5.4, p: 0.03), 15 min (39 ± 4 vs. 61 ± 7.2, p: 0.04), 60 min (41 ± 5.2 vs. 68 ± 5.1, p: 0.01), 120 min (44 ± 6.1 vs. 67 ± 3.1, p: 0.03), 240 min (45 ± 12 vs. 93 ± 3, p < 0.0001) and 360 min (45 ± 12 vs. 80 ± 16, p<0.001), but not 30 min (39 ± 5.8 vs. 54 ± 5.9, n/s) after reperfusion. In addition, a significant decrease in sC5b-9 levels was also observed compared to control recipients. The formation of sC5b-9 in samples from treated kidneys was reduced at all time points, except at 5 min, compared with controls. Plasma sC5b-9 levels 5 min after reperfusion of treated and control kidneys were (16 ± 6 vs. 31 ± 5, p: 0.2), after 15 min (15 ± 3 vs. 45 ± 11, p: 0.01), after 30 min (16 ± 4 vs. 45 ± 3, p: 0.01), after 60 min (25 ± 7 vs. 54 ± 4, p: 0.01), after 120 min (36 ± 12 vs. 64 ± 9, p: 0.01), at 240 min (45 ± 11 vs. 75 ± 11, p: 0.01) and at 360 min (44 ± 14 vs. 79 ± 11, p=0.008). Thus, these results showed that complement activation was significantly suppressed when endothelium was coated with PEG phospholipid.

Kidney function

Renal function was analyzed by assessing urine output after kidney transplantation with and without PEG-phospholipid treatment (Fig. 10A). There was a significant increase in the total amount of urine produced in recipients with PEG-phospholipid-treated kidneys compared with control recipients 360 min after reperfusion (48.3 ± 22.2 ml versus 33.5 ± 18.2 ml, p < 0. 05). However, no significant differences were observed between groups regarding urine output time or serum creatinine levels because the recipients' kidneys were not removed.

B. Model of long-term allogeneic transplantation

Activation of blood coagulation and complement

To evaluate the effect of PEG-phospholipid treatment on the innate immune system after renal reperfusion in a porcine model of long-term allogeneic transplantation, markers of complement activation (C3a, sC5b-9, Fig. 8A and 8B) and activation of coagulation (TAT, Fig. 8C) were analyzed. ) in blood. TAT levels were lower in the blood of recipients with PEG-phospholipid-treated kidneys compared with control recipients. Significant reductions in TAT levels in PEG-phospholipid-treated kidneys compared to uncoated controls were observed at 24 hours (372 ± 123 vs. 137 ± 21, p < 0.05), 48 hours (365 ± 82 vs. 152 ± 17, p<0.05), 72 hours (467 ± 96 versus 135 ± 18, p<0.001) and 96 hours (428.6 ± 111 versus 168 ± 23, p<0.01), but not after 1 hour (378 ± 82 vs. 176 ± 24, n/s) after reperfusion. These results indicated that the activation of blood coagulation was significantly suppressed by surface modification after kidney transplantation.

Similar results were observed for complement markers (C3a and sC5b-9). There was a significant decrease in C3a levels in the blood of recipients transplanted with PEG-phospholipid-treated kidneys compared to control recipients at 48 hours (119 ± 40 versus 67 ± 9, p < 0.001), 72 hours (95 ± 13 versus 63 ± 10, p<0.01), but not after 1 hour (68 ± 11 compared with 54 ± 4, n/s), 24 h min (128 ± 54 compared with 63 ± 18, n/s) or 96 hours (103 ± 21 vs. 74 ± 8, n/s) after reperfusion. In addition, a significant decrease in sC5b-9 levels was also observed compared to control recipients. The formation of sC5b-9 in samples from treated kidneys was reduced at all time points except 5 min compared with controls. Plasma sC5b-9 levels 5 min after reperfusion of treated and control kidneys were 24 h min (73 ± 29 vs. 33 ± 16, p < 0.05), 48 h min (94 ± 33 vs. 33 ± 11 , p<0.001), 72 hours (126 ± 46 vs. 41 ± 18, p<0.0001) and 96 (108 ± 23 vs. 51 ± 11, p<0.01), but not after 1 hour (63 ± 11 vs. 33 ± 16, n/c). Thus, these results showed that complement activation was significantly suppressed when endothelium was coated with PEG-phospholipid.

Pro-inflammatory cytokines

Cytokine/chemokine concentrations were assessed during long-term allogeneic kidney transplantation. Plasma concentrations of INFγ, IL-1β, IL-2, IL-1α, IL-1R, IL-4, IL-6, IL-10, IL-18, IL-8, IL-12 and TNF were assessed using a multiplex assay. , see fig. 9A - 9L. Levels of IL-1β, IL-1R, IL-4, IL-6, IL-12, IL-18, and TNF were significantly reduced in animals transplanted with PEG-lipid-treated kidneys after 4 days.

Kidney function

All animals recovered well from transplantation and were eating within 24 hours. Urine production was confirmed by ultrasound and urine was detected in the bladder within 72 hours in all pigs. Kidney function was analyzed by assessing creatine levels after kidney transplantation with and without PEG-phospholipid treatment (Fig. 10B). Significantly lower levels were observed in recipients with PEG-phospholipid-treated kidneys compared to control recipients over the entire four-day period.

Necropsy

No signs of disease were observed, with the exception of changes associated with kidney transplantation. The pathological examination was carried out at the Department of Pathology, SLU, Uppsala.

Postperfusion syndrome

Our long-term survival studies in pigs showed that animals transplanted with PEG-phospholipid-treated kidneys had minimal or no changes in heart rate and blood pressure after reperfusion. Thus, the PEG-phospholipid product also prevented activation of the contact system and ultimately the ORS, see FIG. eleven.

Pre-treatment with HTK solution

In fig. 16A and 16B show the results of an evaluation of porcine kidneys incubated for 24 hours in a PEG-phospholipid-supplemented HTK solution at 4°C. The TAT values are presented in Fig. 16A and plasma creatinine values are presented in FIG. 16B. Significant improvements were observed when porcine kidneys were incubated in HTK solution supplemented with PEG-phospholipid compared to the control with HTK solution alone and without PEG-phospholipid treatment. The low plasma creatinine levels observed in treated porcine kidneys were achieved even without any additional immunosuppression.

Organ storage

When PEG-lipid was dissolved in the perfusion fluid in combination with long-term graft storage at +4°C, significantly less activation of the cascade system (FIGS. 17A - 17D) and cytokine expression levels (FIGS. 18A - 18G) were observed in PEG-lipid-treated kidneys after reperfusion.

Histological analysis of FITC-PEG-phospholipid

In fig. 19 shows confocal microscopy images of kidney biopsies treated with FITC-PEG phospholipid (5 kDa) and FITC-PEG phospholipid (40 kDa) along with controls. These images therefore show the uptake of 5 kDa PEG-lipid and 40 kDa PEG-lipid after ex vivo infusion of PEG-lipids into kidneys from four different pigs, where one kidney was used for 5 kDa PEG-lipid and the other for 40 kDa. PEG lipid. In fig. 19 shows that the 5 kDa PEG lipid penetrated the kidney parenchyma more effectively compared to the 40 kDa PEG lipid. Accordingly, 40 kDa PEG lipid molecules were less efficiently filtered through the nephron glomeruli than 5 kDa PEG lipid molecules.

Discussion

In many diseases and pathological conditions, ischemia causes pathological changes. Basically, four types of mechanisms cause ischemia in tissue, namely thrombosis, transplantation, clamping of the artery leading to the organ, and sepsis. IRI is an inevitable event in organ transplantation, but it also occurs in a wide range of other clinically significant conditions, such as brain death, sepsis, cardiac surgery, and after reperfusion of the ischemic region for impending myocardial infarction or stroke. In this experiment, we demonstrate that coating the endothelial lining and tubular system with a PEG-phospholipid construct prior to transplantation in both short-term and long-term porcine allogeneic transplantation models functionally replaces GXC and strongly attenuates the primary I/R response to injury in vivo .

The transplanted kidney is subject to ischemic injury at various stages, ranging from the period of time in the donor to the actual transplantation event. Since cadaveric donors serve as the main sources of donor organs, hemodynamic instability of blood flow in cadaveric donors is one of the main causes of ischemia. Warm ischemia occurs during organ harvesting from cadaveric donors and cold ischemic injury occurs during organ preservation at lower temperatures. Reperfusion of the organ in the recipient completes the I/R injury.

To overcome these problems, we proposed a new method for local protection against innate immune system attack in kidney transplantation. Kidney endothelium and tubules were coated with PEG phospholipids through hydrophobic interactions with the lipid bilayer of the cell membrane, thereby replacing the GXC layer. PEG chains are inert and highly biocompatible. Therefore, PEG phospholipid alone protects against I/R injury by protecting endothelial and tubular cell membranes and preventing proteins from reaching and activating them on the surfaces of renal endothelial cells.

PEG-phospholipid-treated kidneys showed less immune system activation in recipients, as reflected by attenuated plasma levels of TAT, FXIIa, TF, C3a, and sC5b-9 and deposition of C3b on the endothelial surface of kidney grafts. This showed that PEG chains protruding from the lipid bilayer of the membrane can inhibit complement activation and protect cell membranes from the deposition of complement components after reperfusion with fresh blood from recipients. This also led to suppression of coagulation activation. Activation of complement and coagulation is a hallmark of I/R, and decreased levels of complement markers support the concept of a protective effect against I/R injury leading to rejection.

In a short-term study, the graft was observed for up to 6 hours to assess renal function and local hemocompatibility. When the PEG phospholipid suspension was injected through a common aortic fragment for two kidney grafts, the tissue of the entire test kidney was uniformly coated with PEG phospholipid, including the glomeruli (Figures 4B, 4D, 4F). Macroscopic pathological examination of the kidney grafts was unchanged, indicating that treatment of the kidney graft with PEG-phospholipid did not have an undesirable effect on cell viability. Also, when the total urine output was compared between the control kidney and the PEG-phospholipid-treated kidney, significant improvements in urine output were observed in the test kidney. These results indicate not only that the renal endothelium was protected from attack by the innate immune system, but also that renal function was well preserved despite the glomeruli being coated with PEG phospholipid. Given the established relationship between morphological changes after ischemic injury and renal function as a tool to distinguish between viable and nonviable kidneys ex-vivo , our short-term follow-up study demonstrated the effectiveness of PEG-phospholipid coating in attenuating I/R attack without any adverse effects on function kidney

The effect of PEG phospholipid is limited to the cell surface since binding is mediated by hydrophobic interaction. By monitoring the fluorescence of PEG phospholipid bound in the kidneys in both the short-term and long-term models, it was shown that there was a decrease in intensity during the observation period, indicating that the PEG phospholipid was detached from the endothelium. Fluorescence appeared to migrate from the glomeruli into the tubular system, eventually reaching the base of the tubular cells 6 hours after transplantation. Fluorescence was detected in urine during the observation period, confirming that PEG phospholipid molecules were metabolized by the kidney. In a long-term study, significant fluorescence was detected up to 12 to 24 hours after reperfusion and completely disappeared after 48 hours. It is possible that there is an initial oversaturation of PEG phospholipid, which is easily washed away, and that the actual protection of the endothelial cells is carried out by molecules that produce only weak fluorescence. This is supported by the relatively low fluorescence of PEG phospholipid in a short-term study at 6 hours post-transplant in combination with a protective effect against innate immune system attack, as evidenced by significant differences in TAT, C3a and sC5b-9 levels in the blood and in C3b deposition in kidney grafts . This was also supported by the fact that efficacy was maintained in a long-term study of up to 72 hours, although a lower density of PEG phospholipid embedded in the endothelial surface was observed. Therefore, the long-term stability of PEG phospholipids is sufficient.

This short-term study provides proof of concept for the effectiveness of PEG-phospholipid-only coating to attenuate I/R injury events by directly inhibiting thromboinflammation. PEG phospholipid was designed to act as a binder and spacer for cell-associated innate immune regulators. Functionalized PEG phospholipid molecules are very effective at attenuating various types of innate immune and thrombotic responses such as complement activation, junction system activation and platelet activation. For example, a peptide with affinity for Factor H inhibits complement activation, and apyrase is able to reduce thrombotic reactions associated with platelet activation. Together, these two regulators can completely inhibit the thromboinflammation triggered in the xenogeneic system when porcine aortic endothelial cells are brought into contact with human blood in an in vitro whole blood system. When considering the immersion effect of PEG-phospholipid alone, the addition of regulators to PEG-phospholipid further improves the design with respect to inhibition of I/R injury in allogeneic kidney grafts.

Activation of the contact system, which is part of the innate immune system, can cause serious complications during surgery, such as cardiovascular disorders and hemodynamic instability after reperfusion. This phenomenon, which was previously described as post-reperfusion syndrome (PRS), was first observed in liver transplant recipients who experienced severe hemodynamic instability after liver graft reperfusion. However, this phenomenon has also been observed in kidney and pancreas transplant recipients, who often suffer from immediate hemodynamic instability after reperfusion. During PRS, the plasma protein factor XII, pre-kallikrein and high molecular weight kininogen are activated, leading to activation of the coagulation cascade and production of bradykinin, which is a very effective vasodilator causing hemodynamic instability. Consequently, a rapid decrease in systemic blood pressure leads to impaired graft perfusion and prolongation of warm graft ischemia. These patients may suffer from delayed graft function, increased risk of rejection, and premature graft loss. In healthy individuals, an increased heart rate may partially compensate for a drop in blood pressure.

Our long-term survival studies in pigs showed that animals transplanted with PEG-phospholipid-treated kidneys had minimal or no changes in heart rate and blood pressure after reperfusion. Thus, the PEG-phospholipid product also prevented the activation of the contact system and, ultimately, the ORS.

The difference between the various long-term experiments was that the graft in the earlier experiments was stored in a refrigerator at +4°C for 24 hours without immersion in liquid for perfusion and that the PEG phospholipid was injected by perfusion into the vascular system 40 minutes before transplantation kidneys In later experiments, the graft was perfused with PEG-phospholipid immediately after harvest and then immersed in PEG-phospholipid dissolved in the perfusion fluid for 24 hours at +4°C until transplantation and reperfusion. Already in the first study, significantly less activation of the cascade system (Figs. 8A - 8C) and cytokine expression (Figs. 9A - 9L) was observed in animals treated with PEG-phospholipid. However, in the second study, activation of the cascade system (Figs. 17A - 17D) and cytokine expression (Figs. 18A - 18G) were much lower compared to the first study and, in addition, treatment with PEG-phospholipid resulted in a significant decrease in activation of the system cascade (Fig. 17A - 17D) and cytokine expression (Fig. 18A - 18G). Therefore, the present experiment clearly demonstrated that the combination of perfusion fluid and PEG-phospholipid was the best combination. This experiment also showed that cytokine expression was clearly lower immediately after reperfusion of PEG-phospholipid-treated grafts (Figs. 18A - 18G), indicating that the anti-inflammatory effect was already induced during the period of cold ischemia before reperfusion, possibly due to an inhibitory effect on CL-11 during ischemia, as mentioned above.

EXPERIMENT 2

The aim of the experiment was to evaluate the safety of PEG conjugated to phospholipid (1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine, DPPE) in rats. A single dose of the compound was administered intravenously, followed by a recovery period of 14 days. In a clinical setting, 2 g of PEG (MW: 5.0 kDa)/phospholipid compound will be diluted in 1000 ml of perfusion solution, which is typically injected into ex vivo organ grafts prior to implantation into the recipient. Rats in the low dose group were given the same concentration, and rats in the high dose group were given 10 times the concentration, respectively.

Study design

Thirty Sprague Dawley rats of both sexes were purchased from Charles River and transported to Uppsala. The experiment lasted a total of four weeks, starting with an acclimation period of two weeks, after which the animals were randomly injected with either NaCl, a low dose, or a high dose of PEG phospholipid (day 1). Injections were given into the lateral tail vein. The rats were then observed repeatedly in their cages over a 6-hour period using the Irwin observation test, which involves extensive clinical examination of neurological and motor function to detect signs of acute toxicity. Treatment was blinded to the observing staff. Clinical studies five days a week then continued for two weeks. At the end of the study, blood was collected by cardiac puncture under terminal anesthesia. After sacrifice, all animals were immediately subjected to necropsy (day 15).

Group 1: 10 control animals (5 females, 5 males) received NaCl, 1 ml

Group 2: 10 rats (5 females, 5 males) received 2 mg/ml PEG phospholipid, 1 ml

Group 3: 10 rats (5 females, 5 males) received 20 mg/ml PEG phospholipid, 1 ml

Clinical observations

Throughout the study, all animals were examined and manipulated 5 days a week. They were removed from the cage and placed on a piece of synthetic fleece, one or two at a time. Three times a week, all rats were released into a large play area, 5 rats at a time, to climb, bathe, and explore. Positive reinforcement in the form of corn flakes was used immediately after the manipulations. They underwent a full clinical examination upon arrival at the vivarium and before injection of the substance. Immediately after injection, rats were monitored for signs of toxicity for six hours at specified intervals. Thereafter, they were examined regularly by a qualified veterinarian. No clinical signs of disease or toxicity were observed in any animal during the acclimation and experimental periods.

Body mass

The body weights of all animals were recorded before and after intravenous injection twice a week for the remainder of the study. Animal weight gain was similar in all three groups.

Ophthalmological examination

All animals underwent an ophthalmological examination with biomicroscopy (KOWA-SL17) and ophthalmoscopy (Heine Omega 500) one week before and one week after administration by PEG-phospholipid injection. Rats received Mydriacyl® (tropicamide) eye drops, 0.5%, 5 mg/ml before examination. They were held in place by gentle manual fixation throughout the procedure.

No signs of toxicity were observed on ophthalmologic examination. Before administration of the substance, opacity of the cornea of the right eye was observed in one female rat, and nuclear opacity of the lens was observed in two female rats: in one in both eyes and in the other in only the right eye. No ophthalmological changes were observed after administration of the substance, with the exception of mild irritation of the epithelium in one of the females. In two male rats, corneal degeneration was observed before injection of the substance: in one, bilateral, and in the other, only the right cornea. Nuclear lens defects were observed in one male, and cataracts were observed in another. Intravitreal hemorrhage in the left eye could be observed in one male rat. After administration of the substance, no changes were observed in male rats, and the hemorrhage resolved.

Clinical pathology

At the end of the study, blood was taken from all animals by cardiac puncture; one ml of blood was divided into tubes with EDTA and Li-heparin. After centrifugation at 3000 rpm for 10 min, plasma was collected and stored at -20°C until clinical chemistry analysis. EDTA blood was immediately analyzed for hematology using a Siemens Advia 2120 medical device and plasma using an Architect c4000, Abbott Laboratories, according to the user manual and standard operating procedures, at the Department of Clinical Pathology, SLU, Uppsala. These assays are validated for rodents. The following hematological studies were performed: blood (B)-platelet (B-TPK), B-erythrocyte (B-EPK), B-hemoglobin (B-Hb), B-mean erythrocyte volume (B-MCV), B-mean concentration erythrocyte hemoglobin (B-MCHC), erythrocyte-reticulocyte, erythrocyte volume distribution width (RDW), mean erythrocyte hemoglobin (MCH), mean platelet volume (MPV), erythrocyte-morphology, B-leukocyte (B-LPK) , B-neutrophils, B-eosinophils, B-basophils, B-lymphocytes, B-monocytes, - and the following measurements were performed as clinical biochemistry: sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein and albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and bile acids. When possible, the following urine tests were performed: pH, protein, glucose, and blood.

All blood and plasma test results were within the normal range for Sprague Dawley rats 8 to 16 weeks of age. Blood was found in five urine samples.

Pathology

All animals were euthanized as part of a scheduled necropsy on day 15 by exsanguination under isoflurane and oxygen anesthesia. Body weights were recorded after sacrifice and gross pathology was performed. No macroscopic abnormalities were found in any of the animals.

conclusions

Clinical studies, including ophthalmic studies, did not reveal any signs of toxicity in any of the low dose, high dose and control groups of animals. All hematological and biochemical tests were within the normal range and no macroscopic abnormalities were detected at necropsy in any of the animals. It can be concluded that intravenous administration of PEG phospholipid is well tolerated in rats at concentrations up to 20 mg/ml.

EXPERIMENT 3

Gallium-68 ( ⁶⁸ Ga) is a positron emitter with a half-life of 68 min that is widely used as a diagnostic radionuclide in positron emission tomography (PET)/computed tomography (CT). The radiolabeled PEG phospholipid molecule ⁶⁸ Ga-NO2A-PEG phospholipid was prepared by a two-step synthesis.

Preparation of buffers and stock solutions.

Hydrochloric acid (HCl) 0.1 M for elution ⁶⁸ Ga. Concentrated HCl (9.00 ml, d=1.17 g×ml ^-1 , 11.23 M, 32-35% Ultrapure NORMATOM for trace metal analysis) was added to water (1000 ml, Thermo Fisher, Ultra Trace Analytical Grade) and mixed and stored at 4°C in the dark directly in the water vial. An aliquot was kept at RT in a plastic vial (Nalgene, low-metal high-density polyethylene resin, Thermo Fischer) before elution from the generator.

Water without metals. Obtain deionized water in a glass vial from a water purifier (MillieQ). The aliquot was transferred to a plastic container and stored over Chelex-100 to remove metal ions.

NO2A-azide, 1.0 M solution in DMSO and water. 1,4,7-triazacyclononane-1,4-bis(acetic acid)-7-(3-azidopropylacetamide)trihydrochloride (NO2A-azide, Macrocyclics, molecular weight 494.8 g/mol) (1.0 mg , 2.0 µmol) was added to an Eppendorf tube (2 ml). The solid was first dissolved in dimethyl sulfoxide (DMSO, 50 μL) and diluted very slowly dropwise with metal-free water (1950 μL) to obtain a homogeneous 1.0 M solution.

Sodium acetate, 1.0 M, buffer pH 3.5. Metal-free water was used at all stages of buffer preparation. Sodium acetate (8.203 g, 0.100 mol, 99.995% by weight metals, TraceSELECT) was dissolved in water (100 ml) in a plastic bottle (Nalgene, HDPE, low metal resin) and pH adjusted to 4.5, 4.0 or 3.5 by adding concentrated acetic acid (≥99%, TraceSELECT for trace element analysis) using a plastic pipette. The final volume was greater than 100 mL due to the addition of salt and acid, hence the molarity of the final solution was less than 1.0 M. Approximately 5 mL aliquots of the buffer were removed sequentially and the pH was measured using a calibrated pH meter (Mettler Toledo). Aliquots were discarded after pH measurements to avoid any metal contamination from the pH probe. The final buffer was stored over Chelex 100 and filtered through a 0.5 μm syringe filter before further use.

Sodium hydroxide, 1.0 M solution. Sodium hydroxide solution (100 μl, 10 M, biological purity above standard, Sigma Aldrich) was diluted with metal-free water (900 μl) in an Eppendorf tube.

Elution from 68Ga generator

⁶⁸Ga was eluted from generator⁶⁸Ga/⁶⁸Ge (50 mCi generator, Obninsk, Russia) with 0.1 M HCl (5.0 ml, approx. 0.8 ml/min), using a 10 ml plastic syringe and a motorized syringe infusion pump (Univentor 864, Malta). The resulting solution was fractionated in Eppendorf tubes to obtain a low volume and a high concentration of radioactivity.

Fraction 1: time 0 - 45 s, 600 µl, 0 MBq - discarded

Fraction 2: time 45 - 1 min, 15 s, 400 µl, 212 MBq - saved for labeling

Fraction 3: time 1 min, 15 s - 6 min, 15 s, 4000 µl, 281 MBq - discarded

HPLC method

A LaPrep Sigma HPLC system (Hitachi) equipped with a manual injector (Rehodyne 7225i, 20 µL loop size), a UV detector (4D LWL, Knauer) with an external analytical UV flow cell (2 µL) connected by optical cables, and a radioactivity detector ( Flow Count, Bioscan) equipped with an FC 6106 extended range module (Eckert and Ziegler) and a PMT/NaI FC-3300 well detector (Eckert and Ziegler), which was connected in series after the UV detector. A stainless steel loop was preferred over a plastic loop inside the radio detector due to the tendency of the radioactive materials being analyzed to stick. The HPLC apparatus was controlled by OpenLAB software (Agilent) and the results were analyzed using the same software.

Ionization chamber (VDC 202, Veenstra Instruments, calibration factor used for 68Ga was 751).

Method A: reverse phase, column C18 YMC-Pack ODS-AL, AL12S05-1046WT, AL-301, (YMC Europe GmbH) 100x4.6 mm (id), 5 µm, 120A. Solvent A: water and 0.1% TFA, solvent B: acetonitrile and 0.1% TFA. Solvent gradient 5 - 70% B over 5 min, then 70% B over 3 min. Flow rate 1.0 ml∙min ^-1 .

Method B: Size Exclusion Chromatography, Column: Superdex™ Peptide 10/300 GL, GE Healthcare. Isocratic eluent 30% B. Flow rate 0.80 ml∙min ^-1 .

Synthesis of [68Ga]NO2A-azide

Sodium hydroxide 1.0 M (40 µL, 40 µmol) and sodium acetate buffer 0.10 M (25 µL, pH 3.5) were mixed in an Eppendorf tube (2 ml) and added to ⁶⁸ Ga (179 MBq) at 0. 1 M HCl (400 µl). NO2A-azide solution (10 µl, 1.0 M in DMSO/pure water (1/39) v/v) was then added (total reaction volume 250 µl) and the tube was vortexed, centrifuged and heated to 80°C in a heating device for 15 minutes to obtain ⁶⁸ Ga labeled NO2A azide, see FIG. 14 The crude reaction mixture was analyzed using radio-HPLC Method A, wavelength λ = 215 nm, t _R : no signal (UV), 3.7 min (radio), and used for subsequent reactions without further purification. Radio-HPLC Method B was used to obtain unreacted Click Reaction Standard 1, t _R : no signal (UV), t _R : 25.7 min (radio).

Synthesis of dibenzocyclooctine (DBCO)-PEG-lipid

Mal-PEG lipid (20 mg) was reacted with cysteine (2 mg) in PBS (1 ml) for 1 h at RT and purified using a spin column (Protein Desalting Spin Column, Thermo Fisher Scientific) to obtain Cys- PEG lipid after lyophilization. Cys-PEG lipid (10 mg in PBS) was then reacted with DBCO-C6-NHS ester (6 mg, Click Chemistry Tools LLC) in DMSO (1 mL) for 1 h at RT. After adding 10 ml of pure water, the precipitate was removed by filtration (pore size: 0.2 μm, Millex®-GV, Merck Millipore Ltd.), followed by drying under vacuum to obtain DBCO-PEG lipid (5 mg). The labeling efficiency of DBCO was 80%, which was calculated from the UV absorbance of DBCO.

Synthesis ⁶⁸ Ga-NO2A-PEG-lipid

DBCO-PEG-lipid (2.0 mg, 333 nmol lipid, 75% DBCO functionalization, 250 nmol DBCO) and PBS (50 μl) were added to an Eppendorf tube (2 ml). A centrifuge was used to sediment all the material to the bottom of the tube. Then 450 μl of the previously obtained [ ⁶⁸ Ga]NO2A-azide solution (116 MBq, 9.0 nmol) was added. The final DBCO/azide ratio in the reaction mixture was 1/28 molar. The reaction mixture was left at RT for 10 min and then heated to 60°C in a heating device for 10 min.

450 µl of the reaction mixture was transferred to a NAP-5 column (Sephadex ® G-25 column, DNA grade, pre-equilibrated with 5 ml PBS, 5 kDa threshold, GE Healthcare), which was eluted with PBS in 200 µl fractions. Desired product ⁶⁸ Ga-NO2A-PEG-lipid, see FIG. 15, eluted in fractions 2 - 4 (35.0 MBq, decay-corrected radiochemical yield of 32% in the click reaction based on the amount of [ ⁶⁸ Ga]NO2A-azide added, decay-corrected radiochemical yield of 7% based on the total amount eluted ⁶⁸ Ga) and analyzed on a radio-HPLC equipped with a size exclusion chromatography column (Superdex™ Peptide, 10/300 GL, GE), wavelength λ=254 nm, t _R : 21.5 min, radiochemical purity >92%. The main impurity was unreacted [ ⁶⁸ Ga]NO2A-azide (5%), t _R : 25.7 min.

EXPERIMENT 4

PET/magnetic resonance imaging (MRI) studies were performed based on in vitro studies to monitor the distribution of the PEG phospholipid construct in porcine kidneys and to estimate the amount of PEG phospholipid that was distributed into the kidneys.

⁶⁸ Ga-NO2A-PEG-lipid (in PBS) was mixed with unlabeled MeO-PEG-lipid (in HTK solution) immediately before use ( ⁶⁸ Ga-NO2A-PEG-lipid/MeO-PEG-lipid = 1/4 molar ratio , [PEG-lipid] = 2.0 mg/ml).

Blood was washed out of the pig kidney using cold PBS, and then the kidney was washed with HTK solution at 4°C and placed in a plastic bag filled with HTK solution overnight. A solution of ⁶⁸ Ga-NO2A-PEG lipid (2 mg/ml, in HTK, 20 ml, at least 10 MBq) was carefully injected into the porcine renal artery using a syringe and incubated for 30 min on ice. After flushing the kidney with cold HTK solution (50 mL), the treated kidney was placed on the PET/MRI device platform (nanoScan®, Mediso Medical Imaging Systems, Budapest, Hungary). The platform was designed for a large rat from a PET/3T MRI scanner for preclinical studies of a small animal. An MRI exploratory scan (GRE Multi-FOV) was used to establish bed position for whole kidney PET scanning. A static PET study, multiple bed scans, was performed over 40 minutes. MRI measurements were performed at 3 T using a transceiver coil to examine the whole body of the porcine kidney. A gradient echo sequence (GRE MultiFOV) was acquired with the following parameters: number of excitations (NEX): 2; repetition time (TR): 396 ms; echo time (TE): 4.15 ms; coup: 45; slices: 180; cut thickness: 1 mm; field of view (FOV): 64×64 mm ² ; spatial resolution: 0.5×0.5 mm ² ; capture time: 14 min. PET data in list mode were reconstructed using the Tera tomo 3D algorithm (4 iterations, voxel size: 0.40 mm, matrix: 212 × 212 × 582).

PET and MRI images were analyzed using PMOD (version 3.510; PMOD Technologies Ltd.). Volumes of interest were manually delineated on overlaid PET-MRI images to contain the entire kidney and the area around the medulla. The distribution of the isotopic marker in the rest of the kidney was estimated by subtracting the value for the area around the medulla from the value for the whole kidney.

Uptake into each tissue was expressed in MBq, and decay was corrected for the time of administration. The amount of administered radioactivity and venous eluates from all washing steps were measured on a dose calibrator (VDC-405, Veenstra Instrumenten, the Netherlands) and decay was corrected for the time of administration. All measurements were performed using CT. After the first PET/MRI and gamma counter measurement, the kidney was again flushed with cold HTK solution (50 mL), and then the kidney was placed on the PET/MRI device platform for the second measurement.

results

The porcine kidney was treated with ⁶⁸ Ga-NO2A-PEG-lipid in the same manner as for kidney transplantation into recipient pigs, and then PET/MRI imaging was performed. After treating the kidney with ⁶⁸ Ga-NO2A-PEG-lipid by injection and arterial washout, imaging was performed to evaluate the distribution of ⁶⁸ Ga-NO2A-PEG-lipid. The ⁶⁸ Ga-NO2A-PEG-lipid signal was distributed throughout the kidney, and also a higher signal was particularly found in the medulla region (81%) compared to the cortex region (19%). When the same kidney was reimaged 90 min after additional HTK wash, the imaging result was almost the same. The ⁶⁸ Ga-NO2A-PEG-lipid signal was still distributed throughout the kidney, and higher signal was found in the medulla region (93%) compared to the cortex region (7%). These results showed that all the tissues and vessels in the kidney were coated with ⁶⁸ Ga-NO2A-PEG lipid due to the injection, and the tissues and vessels in the medulla region were well modified with ⁶⁸ Ga-NO2A-PEG lipid.

The embodiments described above are to be understood as several illustrative examples of the present invention. It will be apparent to 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 parts of the solutions in different embodiments can be combined in other configurations when technically possible. The scope of the present invention is, however, defined by the appended claims.

reference materials

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[2] Teramura et al., Behavior of Synthetic Polymers Immobilized on Cell Membrane. Biomaterials , 2008, 29:1345-1355.

[3] Teramura and Iwata, Improvement of graft survival by surface modification with poly(ethylene glycol)-lipid and urokinase in intraportal islet transplantation. Transplantation . 2011, 91(3):271-278.

[4] Nilsson et al., Autoregulation of thromboinflammation on biomaterial surfaces by a multicomponent therapeutic coating. Biomaterials . 2013, 34(4):985-994.

[5] Engberg et al., Inhibition of complement activation on a model biomaterial surface by streptococcal M protein-derived peptides. Biomaterials . 2009, 30(13):2653-2659.

[6] Teramura and Iwata, Surface Modification of Islets With PEG-Lipid for Improvement of Graft Survival in Intraportal Transplantation. Transplantation . 2009, 88(5):624-630.

[7] Asif et al., Heparinization of cell surfaces with short peptide-conjugated PEG-lipid regulates thromobinflammation in transplantation of human MSCs and hepatocytes. Acta Biomateriala. 2016, 35:194-205.

Claims (34)

1. An ex vivo method of providing coverage of at least a portion of the endothelial lining of the vascular system of an organ or part of an organ, the method comprising: ex vivo infusion of a solution containing polyethylene glycol phospholipid (PEG phospholipid) molecules into said vascular system of an organ or part of an organ; And incubating ex vivo a solution containing PEG-phospholipid molecules in the vascular system to ensure that at least a portion of the endothelial lining of the vascular system is coated with PEG-phospholipid molecules, while maintaining the organ or part of an organ immersed in the organ preservation solution containing the PEG-phospholipid molecules, wherein said coating forms protection against thrombus inflammation caused by perfusion of said organ or part of an organ. 2. The method according to claim 1, additionally including: infusion of an ex vivo organ preservation solution into the vascular system to flush out unbound PEG phospholipid molecules from the vascular system. 3. Method according to claim 1 or 2, additionally including: infusion of an ex vivo organ preservation solution into the vascular system prior to infusion of an ex vivo solution containing PEG phospholipid molecules into the vascular system. 4. Method according to any one of paragraphs. 1-3, characterized in that the ex vivo infusion of the solution includes: placing an ex vivo clamp on one vessel selected from an artery and a vein of the vascular system; ex vivo infusion of a solution containing PEG phospholipid molecules into another vessel selected from an artery and a vein; And placing an ex vivo clamp on another vessel selected from an artery and a vein. 5. Method according to any one of paragraphs. 1-4, characterized in that the infusion of the ex vivo solution includes the infusion of an ex vivo solution containing from 0.25 mg/ml to 25 mg/ml, preferably from 0.25 mg/ml to 10 mg/ml and more preferably from 0 .25 mg/ml to 5 mg/ml, for example 2 mg/ml, PEG phospholipid molecules into the vascular system. 6. Method according to any one of paragraphs. 1-5, characterized in that the ex vivo infusion of the solution includes the ex vivo infusion of from 5 ml to 250 ml, preferably from 5 ml to 100 ml, and more preferably from 5 ml to 50 ml of a solution containing PEG-phospholipid molecules into the vascular system . 7. Method according to any one of paragraphs. 1-6, characterized in that ex vivo incubation includes incubation of an ex vivo solution containing PEG-phospholipid molecules in the vascular system, while keeping the organ or part of an organ immersed in an organ preservation solution containing PEG-phospholipid molecules and at a temperature above 0 °C but below 8°C, preferably above 0°C but below 6°C, and more preferably above 0°C but equal to or below 4°C. 8. Method according to any one of paragraphs. 1-7, characterized in that the organ preservation solution is selected from the group consisting of histidine tryptophan ketoglutarate (HTK) solution, citrate solution, University of Wisconsin (UW) solution, Collins solution, Celsior solution, Kyoto University solution and Institute solution George Lopez-1 (IGL-1). 9. Method according to any one of paragraphs. 1-8, characterized in that the ex vivo incubation includes incubation of an ex vivo solution containing PEG phospholipid molecules in the vascular system for a period of time from 10 minutes to 48 hours, preferably from 20 minutes to 36 hours, and more preferably from 30 minutes up to 24 hours. 10. Method according to any one of paragraphs. 1-9, characterized in that the PEG phospholipid molecules are selected from the group consisting of non-functionalized PEG phospholipid molecules consisting of PEG phospholipids, functionalized PEG phospholipid molecules containing a corresponding functionalized molecule attached to the PEG of the PEG phospholipid molecules, and mixtures thereof; And said functionalized molecule is selected from the group consisting of a complement inhibitor, a blood clotting inhibitor, a platelet inhibitor, a molecule capable of binding a complement inhibitor, a molecule capable of binding a blood clotting inhibitor, a molecule capable of binding a platelet inhibitor, and mixtures thereof. 11. Method according to any one of paragraphs. 1-10, characterized in that the PEG phospholipid molecules have formula (I) or formula (II): , (I) , (II) Where n, m are integers independently selected from 10 to 16, preferably n, m are independently 10, 12, 14 or 16 and more preferably n=m=14; p is selected such that the PEG chain has an average molecular weight selected in the range of 1000 Da to 40,000 Da, preferably 3,000 to 10,000 Da, and more preferably 5,000 Da; And R is H, CH ₃ , NH ₂ , a maleimide group, a biotin group, a streptavidin group, an avidin group, or a functionalized molecule selected from the group consisting of a complement inhibitor, a blood clotting inhibitor, a platelet inhibitor, a molecule capable of binding a complement inhibitor, a molecule molecule capable of binding a blood clotting inhibitor, a molecule capable of binding a platelet inhibitor, and mixtures thereof. 12. The method of claim 10 or 11, wherein the functionalized molecule is selected from the group consisting of a heparin binding peptide, 4 to 6 N-terminal short consensus repeats (SCRs) of complement receptor 1 (CR1), complement regulatory protein CD46 (CD46), complement degrading accelerating factor (DAF), Factor H binding molecule, adenosine diphosphate (ADP) degrading enzyme, and mixtures thereof, preferably selected from the group consisting of 5C6 peptide, apyrase, cluster of differentiation 39 (CD39), C4b binding protein (C4BP) and mixtures thereof. 13. Method according to any one of paragraphs. 1-12, characterized in that the organ is selected from the group consisting of kidney, liver, pancreas, heart, lung, uterus, bladder, thymus and intestines. 14. The method according to claim 13, characterized in that the organ is a kidney; And ex vivo incubation involves incubating an ex vivo solution containing PEG-phospholipid molecules in the vasculature to provide coverage of the endothelium of the renal vasculature and coating of renal parenchyma tubules with PEG-phospholipid molecules while maintaining the kidney immersed in an organ preservation solution containing the PEG-phospholipid molecules. 15. The method according to claim 1, characterized in that the PEG phospholipid molecules can be obtained by combining α-succinimidyloxysuccinyl-ω-methoxypolyoxyethylene having an average molecular weight of 5000 Da and 1,2-dipalmitoyl -sn -glycero-3-phosphatidylethanolamine with triethylamine and dichloromethane and stirring for 72 hours at room temperature, followed by precipitation with diethyl ether.