Induction of Spontaneous Liposome Adsorption by Exogenous Surface Modification with Cell-Penetrating Peptide-Conjugated Lipids.

The use of amphiphilic molecules such as poly(ethylene glycol)-conjugated phospholipid (PEG-lipid) enables incorporation into liposome surfaces by exogenous addition as a result of the self-assembly with lipids. This technique can be applicable for manipulation of both liposomes and cells. In this study, we aimed to characterize Tat peptide (YGRKKRRQRRR)-conjugated PEG-lipids when used to exogenously surface modify liposomes (size: ca. 100 nm). We earlier reported that cells, which were surface modified with Tat peptides conjugated to PEG-lipids could attach spontaneously to material surfaces without any chemical modification. Here, we synthesized different types of Tat-PEG-lipids by combining PEG of different molecular weights (5 and 40 kDa) with different lipids with three acyl chains (myristoyl, palmitoyl, and stearoyl, respectively) and then studied the spontaneous adsorption of modified liposomes onto a substrate surface induced by the different Tat-PEG-lipids. The amount of adsorbed liposomes strongly depended on the number of incorporated Tat-PEG-lipid moieties: a decrease in both the PEG and the acyl chain lengths led to adsorption of higher amounts of liposomes. Furthermore, when a collagenase-cleavable amino acid sequence was inserted between the Tat sequence and the PEG segment, adsorbed liposomes could be harvested from the substrate by collagenase treatment with no difference in desorption efficiency between the different Tat-PEG-lipids. Thus, Tat-PEG-lipid can be a suitable tool for the manipulation of liposomes and cells.

Pluronic Micelle-Mediated Tissue Factor Silencing Enhances Hemocompatibility, Stemness, Differentiation Potential, and Paracrine Signaling of Mesenchymal Stem Cells.

Mesenchymal stem/stromal cells (MSCs) evoke great excitement for treating different human diseases due to their ability to home inflamed tissues, suppress inflammation, and promote tissue regeneration. Despite great promises, clinical trial results are disappointing as allotransplantation of MSCs trigger thrombotic activity and are damaged by the complement system, compromising their survival and function. To overcome this, a new strategy is presented by the silencing of tissue factor (TF), a transmembrane protein that mediates procoagulant activity. Novel Pluronic-based micelles are designed with the pendant pyridyl disulfide group, which are used to conjugate TF-targeting siRNA by the thiol-exchange reaction. This nanocarrier design effectively delivered the payload to MSCs resulting in ∼72% TF knockdown (KD) without significant cytotoxicity. Hematological evaluation of MSCs and TF-KD MSCs in an ex vivo human whole blood model revealed a significant reduction in an instant-blood-mediated-inflammatory reaction as evidenced by reduced platelet aggregation (93% of free platelets in the TF-KD group, compared to 22% in untreated bone marrow-derived MSCs) and thrombin-antithrombin complex formation. Effective TF silencing induced higher MSC differentiation in osteogenic and adipogenic media and showed stronger paracrine suppression of proinflammatory cytokines in macrophages and higher stimulation in the presence of endotoxins. Thus, TF silencing can produce functional cells with higher fidelity, efficacy, and functions.

Complement inhibition in biomaterial- and biosurface-induced thromboinflammation.

Therapeutic medicine today includes a vast number of procedures involving the use of biomaterials, transplantation of therapeutic cells or cell clusters, as well as of solid organs. These treatment modalities are obviously of great benefit to the patient, but also present a great challenge to the innate immune system, since they involve direct exposure of non-biological materials, cells of non-hematological origin as well as endothelial cells, damaged by ischemia-perfusion in solid organs to proteins and cells in the blood. The result of such an exposure may be an inappropriate activation of the complement and contact/kallikrein systems, which produce mediators capable of triggering the platelets and PMNs and monocytes, which can ultimately result in thrombotic and inflammatory (i.e., a thrombo-inflammatory) response to the treatment modality. In this concept review, we give an overview of the mechanisms of recognition within the innate immunity system, with the aim to identify suitable points for intervention. Finally, we discuss emerging and promising techniques for surface modification of biomaterials and cells with specific inhibitors in order to diminish thromboinflammation and improve clinical outcome.

Cell Surface Engineering for Regulation of Immune Reactions in Cell Therapy.

Transplantation of the pancreatic islets of Langerhans (islets) is a promising cell therapy for treating insulin-dependent type 1 diabetes mellitus. Islet transplantation is a minimally-invasive technique involving relatively simple surgery. However, after intraportal transplantation, the transplanted islets are attacked by the recipient's immune system, because they activate a number of systems, including coagulation, complement response, inflammation, immune rejection, and recurrence of autoimmune disease. We have developed a surface modification and microencapsulation technique that protects cells and islets with biomaterials and bioactive substances, which may be useful in clinical settings. This approach employs amphiphilic polymers, which can interact with lipid bilayer membranes, without increasing cell volume. Molecules attached to these polymers can protect transplanted cells and islets from attack by the host immune system. We expect that this surface modification technique will improve graft survival in clinical islet transplantation.

Thromboinflammation in Therapeutic Medicine.

Thromboinflammation is primarily triggered by the humoral innate immune system, which mainly consists of the cascade systems of the blood, i.e., the complement, contact/coagulation and fibrinolytic systems. Activation of these systems subsequently induces activation of endothelial cells, leukocytes and platelets, finally resulting in thrombotic and inflammatory reactions. Such reactions are triggered by a number of medical procedures, e.g., treatment with biomaterials or drug delivery devices as well as in transplantation with cells, cell clusters or whole vascularized organs. Here, we (1) describe basic mechanisms for thromboinflammation; (2) review thromboinflammatory reactions in therapeutic medicine; and (3) discuss emerging strategies to dampen thromboinflammation.

Stent coating containing a charged silane coupling agent that regulates protein adsorption to confer antithrombotic and cell-adhesion properties.

The evolution of endovascular therapies, particularly in the field of intracranial aneurysm treatment, has been truly remarkable and is characterized by the development of various stents. However, ischemic complications related to thrombosis or downstream emboli pose a challenge for the broader clinical application of such stents. Despite advancements in surface modification technologies, an ideal coating that fulfills all the desired requirements, including anti-thrombogenicity and swift endothelialization, has not been available. To address these issues, we investigated a new coating comprising 3-aminopropyltriethoxysilane (APTES) with both anti-thrombogenic and cell-adhesion properties. We assessed the anti-thrombogenic property of the coating using an in vitro blood loop model by evaluating the platelet count and the level of the thrombin-antithrombin (TAT) complex, and investigating thrombus formation on the surface using scanning electron microscopy (SEM). We then assessed endothelial cell adhesion on the metal surfaces. In vitro blood tests revealed that, compared to a bare stent, the coating significantly inhibited platelet reduction and thrombus formation; more human serum albumin spontaneously adhered to the coated surface to block thrombogenic activation in the blood. Cell adhesion tests also indicated a significant increase in the number of cells adhering to the APTES-coated surfaces compared to the numbers adhering to either the bare stent or the stent coated with an anti-fouling phospholipid polymer. Finally, we performed an in vivo safety test by implanting coated stents into the internal thoracic arteries and ascending pharyngeal arteries of minipigs, and subsequently assessing the health status and vessel patency of the arteries by angiography over the course of 1 week. We found that there were no adverse effects on the pigs and the vascular lumens of their vessels were well maintained in the group with APTES-coated stents. Therefore, our new coating exhibited both high anti-thrombogenicity and cell-adhesion properties, which fulfill the requirements of an implantable stent.

Regulation of the innate immune system by fragmented heparin-conjugated lipids on lipid bilayered membranes in vitro.

Surface modification with heparin is a powerful biomaterial coating strategy that protects against innate immunity activation since heparin is a part of the proteoglycan heparan sulfate on cell surfaces in the body. We studied the heparinization of cellular and material surfaces via lipid conjugation to a heparin-binding peptide. In the present study, we synthesized fragmented heparin (fHep)-conjugated phospholipids and studied their regulation of the innate immune system on a lipid bilayered surface using liposomes. Liposomes have versatile applications, such as drug-delivery systems, due to their ability to carry a wide range of molecules. Owing to their morphological similarity to cell membranes, they can also be used to mimic a simple cell-membrane to study protein-lipid interactions. We investigated the interaction of complement-regulators, factor H and C4b-binding protein (C4BP), as well as the coagulation inhibitor antithrombin (AT), with fHep-lipids on the liposomal surface. Herein, we studied the ability of fHep-lipids to recruit factor H, C4BP, and AT using a quartz crystal microbalance with dissipation monitoring. With dynamic light scattering, we demonstrated that liposomes could be modified with fHep-lipids and were stable up to 60 days at 4 °C. Using a capillary western blot-based method (Wes), we showed that fHep-liposomes could recruit factor H in a model system using purified proteins and assist in the degradation of the active complement protein C3b to iC3b. Furthermore, we found that fHep-liposomes could recruit factor H and AT from human plasma. Therefore, the use of fHep-lipids could be a potential coating for liposomes and cell surfaces to regulate the immune system on the lipid surface.

Effect of liposome surface modification with water-soluble phospholipid polymer chain-conjugated lipids on interaction with human plasma proteins.

Alternative liposome surface coatings for PEGylation to evade the immune system, particularly the complement system, have garnered significant interest. We previously reported poly(2-methacryloyloxyethyl phosphorylcholine) (MPC)-based lipids (PMPC-lipids) and investigated the surface modification of liposomes. In this study, we synthesize PMPC-lipids with polymerization degrees of 10 (MPC10-lipid), 20 (MPC20-lipid), 50 (MPC50-lipid), and 100 (MPC100-lipid), and coated liposomes with 1, 5, or 10 mol% PMPC-lipids (PMPC-liposomes). Non-modified and PEGylated liposomes are used as controls. We investigate the liposome size, surface charge, polydispersity index, and adsorption of plasma proteins to the liposomes post incubation in human plasma containing N,N,N',N'-ethylenediamine tetraacetic acid (EDTA) or lepirudin by some methods such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and automated capillary western blot, with emphasis on the binding of complement protein C3. It is shown that the coating of liposome PMPC-lipids can suppress protein adsorption more effectively with an increase in the molecular weight and molar ratio (1-10 mol%). Apolipoprotein A-I is detected on PMPC-liposomes with a higher molecular weight and higher molar ratio of PMPC-lipids, whereas α2-macroglobulin is detected on non-modified, PEGylated, and PMPC-liposomes with a shorter polymer chain. In addition, a correlation is shown among the PMPC molecular weight, molar ratio, and C3 binding. The MPC10-lipid cannot inhibit C3 binding efficiently, whereas surface modifications with 10 mol% MPC20-lipid and 5 mol% and 10 mol% MPC50-lipid suppress both total protein and C3 binding. Hence, liposome modification with PMPC-lipids can be a possible strategy for avoiding complement activation.

Synthesis of poly(2-methacryloyloxyethyl phosphorylcholine)-conjugated lipids and their characterization and surface properties of modified liposomes for protein interactions.

Poly(ethylene glycol) (PEG) is frequently used for liposomal surface modification. However, as PEGylated liposomes are cleared rapidly from circulation upon repeated injections, substitutes of PEG are being sought. We focused on a water-soluble polymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units, and synthesized poly(MPC) (PMPC)-conjugated lipid (PMPC-lipid) with degrees of MPC polymerization ranging from 10 to 100 (calculated molecular weight: 3 to 30 kDa). In addition, lipids with three different alkyl chains, myristoyl, palmitoyl, and stearoyl, were applied for liposomal surface coating. We studied the interactions of PMPC-lipids with plasma albumin, human complement protein C3 and fibrinogen using a quartz crystal microbalance with energy dissipation, and found that adsorption of albumin, C3 and fibrinogen could be suppressed by coating with PMPC-lipids. In particular, the effect was more pronounced for PMPC chains with higher molecular weight. We evaluated the size, polydispersity index, surface charge, and membrane fluidity of the PMPC-lipid-modified liposomes. We found that the effect of the coating on the dispersion stability was maintained over a long period (98 days). Furthermore, we also demonstrated that the anti-PEG antibody did not interact with PMPC-lipids. Thus, our findings suggest that PMPC-lipids can be used for liposomal coating.

Promotion of cell membrane fusion by cell-cell attachment through cell surface modification with functional peptide-PEG-lipids.

Cell fusion is a fundamental event in various biological processes and has been applied to a number of biotechnologies. However, cell fusion efficiency is still low and strongly depends on cell lines and skills, though some improvements have been made. Our hypothesis is that two distinct cell membranes need to be brought together for cell membrane fusion, which is important for mimicking cell fusion in vitro. Here, we aimed to improve the homogeneous and heterogeneous cell fusion efficiency using a cell-cell attachment technique. We modified cellular membranes with two distinctive poly(ethylene glycol)-lipids (PEG-lipids) carrying oligopeptide, three repeated units of the EIAALEK and KIAALKE sequences (fuE3 and fuK3, respectively), which induce cell-cell attachment. The ratio and area of cell-cell attachment can be controlled through surface modification with fuE3-and fuK3-PEG-lipids by changing the number of each incorporated peptide. By combining this technique with the PEG-induced method, the cell fusion efficiency was significantly improved for homogeneous and heterogeneous cell fusion compared to conventional PEG-induced methods. For homogeneous CCRF-CEM cell fusion, the efficiency increased up to 64% from the 8.4% with the PEG-induced method. In addition, for heterogeneous cell fusion of myeloma cells and splenocytes, the efficiency increased up to 18% from almost zero. Thus, cell membrane fusion could be promoted effectively between closely contacted cell membranes induced by the cell-cell attachment technique.

Identification of Metal-Binding Peptides and Their Conjugation onto Nanoparticles of Superparamagnetic Iron Oxides and Liposomes.

Metallic materials are used for clinical medical devices such as vascular stents and coils to treat both ischemic and hemorrhagic vascular diseases. An antiplatelet drug is required to avoid thromboembolic complication until metallic surface is covered with a neo-endothelial cell layer. It is important to identify endothelial cell coverage on the metallic surface. However, it is difficult since there are no selective ligands. Here, we used the phage display method to identify peptide ligands that had high affinity for the metallic surface of Ni-Ti stents, Pt-W coils, and Co-Cr stents. The binding assay using fluorescence labeling revealed that several synthetic peptides could bind onto those surfaces. We also chose some oligopeptides for the conjugation onto superparamagnetic iron oxide (SPIO) nanoparticles and liposome-encapsulating SPIO nanoparticles and studied their ability to bind to the stent and coils. By SEM and fluorophotometry, we found that those modified SPIOs and liposomes were selectively bound onto those surfaces. In addition, both treated stents and coils could be detected by magnetic resonance imaging due to the magnetic artifact through the SPIOs and liposomes that were immobilized onto the surface. Thus, we identified metal-binding peptides which may enable to stop antiplatelet therapy after vascular stenting or coiling.

Modification of human MSC surface with oligopeptide-PEG-lipids for selective binding to activated endothelium.

Promising cell therapies using mesenchymal stem cells (MSCs) is proposed for stroke patients. Therefore, we aimed to efficiently accumulate human MSC (hMSC) to damaged brain area to improve the therapeutic effect using poly(ethylene glycol) (PEG)-conjugated phospholipid (PEG-lipid) carrying an oligopeptide as a ligand, specific for E-selectin which is upregulated on activated endothelial cells under hypoxia-like stroke. Here we synthesized E-selectin-binding oligopeptide (ES-bp) conjugated with PEG spacer having different molecular weights from 1 to 40 kDa. We found that ES-bp can be immobilized onto the hMSC surface through PEG-lipid without influence on cell growth and differentiation into adipocytes and osteocytes, respectively. It is also possible to control the immobilization of ES-bp on hMSC surface (<108 ES-bp per cell). Immobilized ES-bp can be continuously immobilized at the outside of cell membrane when PEG-lipids with PEG 5 and 40 kDa were used. In addition, the modified hMSC can specifically attach onto E-selectin-immobilized surface as a model surface of activated endothelium in human blood, indicating the sufficient number of immobilized ES-bp onto hMSC. Thus, this technique is one of the candidates for hMSC accumulation to cerebral infarction area. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1779-1792, 2019.

Validation of an MPC Polymer Coating to Attenuate Surface-Induced Crosstalk between the Complement and Coagulation Systems in Whole Blood in In Vitro and In Vivo Models.

Artificial surfaces that come into contact with blood induce an immediate activation of the cascade systems of the blood, leading to a thrombotic and/or inflammatory response that can eventually cause damage to the biomaterial or the patient, or to both. Heparin coating has been used to improve hemocompatibility, and another approach is 2-methacryloyloxyethyl phosphorylcholine (MPC)-based polymer coatings. Here, the aim is to evaluate the hemocompatibility of MPC polymer coating by studying the interactions with coagulation and complement systems using human blood in vitro model and pig in vivo model. The stability of the coatings is investigated in vitro and MPC polymer-coated catheters are tested in vivo by insertion into the external jugular vein of pigs to monitor the catheters' antithrombotic properties. There is no significant activation of platelets or of the coagulation and complement systems in the MPC polymer-coated one, which was superior in hemocompatibility to non-coated matrix surfaces. The protective effect of the MPC polymer coat does not decline after incubation in human plasma for up to 2 weeks. With MPC polymer-coated catheters, it is possible to easily draw blood from pig for 4 days in contrast to the case for non-coated catheters, in which substantial clotting is seen.

Development and characterization of an innovative heparin coating to stabilize and protect liposomes against adverse immune reactions.

Liposomes have been recognized as excellent drug delivery systems, but when they come in direct contact with different blood components they may trigger an immediate activation of the innate immune system. The aim of the present study was to produce long-circulating, blood-compatible liposomes by developing a construct of liposomes covered by a novel unique heparin complex (CHC; 70 heparin molecules per complex) to avoid recognition by the innate immune system. Unilamellar, cationic liposomes were produced by hand extrusion through a 100-nm polycarbonate membrane. Coating of liposomes with the macromolecular CHC was accomplished by electrostatic interactions. Dynamic light scattering as well as QCM-D measurements were used to verify the electrostatic deposition of the negatively charged CHC to cationic liposomes. The CHC-coated liposomes did not aggregate when in contact with lepirudin anti-coagulated plasma. Unlike previous attempts to coat liposomes with heparin, this technique produced freely moveable heparin strands sticking out from the liposome surface, which exposed AT binding sites reflecting the anticoagulant potentials of the liposomes. In experiments using lepirudin-anticoagulated plasma, CHC-coated liposomes, in contrast to non-coated control liposomes, did not activate the complement system, as evidenced by low C3a and sC5b-9 generation and reduced leakage from the liposomes. In conclusion, we show that liposomes can be successfully coated with the biopolymer CHC, resulting in biocompatible and stable liposomes that have significant application potential.

Reciprocal relationship between contact and complement system activation on artificial polymers exposed to whole human blood.

BACKGROUND: Inappropriate and uncontrolled activation of the cascade systems in the blood is a driving force in adverse inflammatory and thrombotic reactions elicited by biomaterials, but limited data are available on the activation of the contact system by polymers and the present study was undertaken to investigate these mechanisms in established models. METHODS: Polymer particles were incubated in (1) EDTA-plasma (10 mM) to monitor the adsorption of 20 selected proteins; (2) lepirudin-anticoagulated plasma to evaluate contact system activation, monitored by the formation of complexes between the generated proteases factor[F]XIIa, FXIa and kallikrein and the serpins C1-inhibitor [C1INH] and antithrombin [AT]; (3) lepirudin-anticoagulated whole blood to determine cytokine release. RESULTS: Strong negative correlations were found between 10 cytokines and the ratio of deposited FXII/C1INH, generated FXIIa-C1INH complexes, and kallikrein-C1INH complexes. Formation of FXIIa-C1INH complexes correlated negatively with the amount of C3a and positively with deposited IgG. CONCLUSIONS: A reciprocal relationship was found between activation of the contact system and the complement system induced by the polymers studied here. The ratios of FXII/C1INH or C4/C4BP, adsorbed from EDTA-plasma are useful surrogate markers for cytokine release and inflammatory response to materials intended for blood contact.

Heparinization of cell surfaces with short peptide-conjugated PEG-lipid regulates thromboinflammation in transplantation of human MSCs and hepatocytes.

Infusion of therapeutic cells into humans is associated with immune responses, including thromboinflammation, which result in a large loss of transplanted cells. To address these problems, heparinization of the cell surfaces was achieved by a cell-surface modification technique using polyethylene glycol-conjugated phospholipid (PEG-lipid) derivatives. A short heparin-binding peptide was conjugated to the PEG-lipid for immobilization of heparin conjugates on the surface of human mesenchymal stem cells (hMSCs) and human hepatocytes. Here three kinds of heparin-binding peptides were used for immobilizing heparin conjugates and examined for the antithrombogenic effects on the cell surface. The heparinized cells were incubated in human whole blood to evaluate their hemocompatibility by measuring blood parameters such as platelet count, coagulation markers, complement markers, and Factor Xa activity. We found that one of the heparin-binding peptides did not show cytotoxicity after the immobilization with heparin conjugates. The degree of binding of the heparin conjugates on the cell surface (analyzed by flow cytometer) depended on the ratio of the active peptide to control peptide. For both human MSCs and hepatocytes in whole-blood experiments, no platelet aggregation was seen in the heparin conjugate-immobilized cell group vs. the controls (non-coated cells or control peptide). Also, the levels of thrombin-antithrombin complex (TAT), C3a, and sC5b-9 were significantly lower than those of the controls, indicating a lower activation of coagulation and complement. Factor Xa analysis indicated that the heparin conjugate was still active on the cell surface at 24h post-coating. It is possible to immobilize heparin conjugates onto hMSC and human hepatocyte surfaces and thereby protect the cell surfaces from damaging thromboinflammation. STATEMENT OF SIGNIGFICANCE: We present a promising approach to enhance the biocompatibility of therapeutic cells. Here we used short peptide-conjugated PEG-lipid for cell surface modification and heparin conjugates for the coating of human hepatocytes and MSCs. We screened the short peptides to find higher affinity for heparinization of cell surface and performed hemocompatibility assay of heparinized human hepatocytes and human MSCs in human whole blood. Using heparin-binding peptide with higher affinity, not only coagulation activation but also complement activation was significantly suppressed. Thus, it was possible to protect human hepatocytes and human MSCs from the attack of thromboinflammatory activation, which can contribute to the improvement graft survival.

Heparin molecularly imprinted surfaces for the attenuation of complement activation in blood.

Heparin-imprinted synthetic polymer surfaces with the ability to attenuate activation of both the complement and the coagulation system in whole blood were successfully produced. Imprinting was achieved using a template coated with heparin, a highly sulfated glycosaminoglycan known for its anticoagulant properties. The N,N'-diacryloylpiperazine-methacrylic acid copolymers were characterized using goniometry, AFM and XPS. The influence of the molecular imprinting process on morphology and template rebinding was demonstrated by radioligand binding assays. Surface hemocompatibility was evaluated using human whole blood without anticoagulants followed by measurement of complement activation markers C3a and sC5b-9 and platelet consumption as a surrogate coagulation activation marker. The observed low thrombogenicity of this copolymer combined with the attenuation of complement activation induced by the molecular imprint offer potential for the development of self-regulating surfaces with important potential clinical applications. We propose a mechanism for the observed phenomena based upon the recruitment of endogenous sulfated glycosaminoglycans with heparin-like activities.

Prediction of inflammatory responses induced by biomaterials in contact with human blood using protein fingerprint from plasma.

Inappropriate complement activation is often responsible for incompatibility reactions that occur when biomaterials are used. Complement activation is therefore a criterion included in legislation regarding biomaterials testing. However, no consensus is yet available regarding appropriate complement-activation-related test parameters. We examined protein adsorption in plasma and complement activation/cytokine release in whole blood incubated with well-characterized polymers. Strong correlations were found between the ratio of C4 to its inhibitor C4BP and generation of 10 (mainly pro-inflammatory) cytokines, including IL-17, IFN-γ, and IL-6. The levels of complement activation products correlated weakly (C3a) or not at all (C5a, sC5b-9), confirming their poor predictive values. We have demonstrated a direct correlation between downstream biological effects and the proteins initially adhering to an artificial surface after contact with blood. Consequently, we propose the C4/C4BP ratio as a robust, predictor of biocompatibility with superior specificity and sensitivity over the current gold standard.

Mediation of a non-proteolytic activation of complement component C3 by phospholipid vesicles.

Liposomes are becoming increasingly important as drug delivery systems, to target a drug to specific cells and tissues and thereby protecting the recipient from toxic effects of the contained drug. Liposome preparations have been described to activate complement. In this study, we have investigated complement activation triggered by neutral dimyristoyl-phosphocholine (DMPC) liposomes in human plasma and whole-blood systems. Incubation in plasma led to the generation of complement activation products (C3a and sC5b-9). Unexpectedly, investigations of surface-bound C3 revealed contact activated, conformationally changed C3 molecules on the liposomes. These changes were characterized by Western blotting with C3 monoclonal antibodies, and by incubating liposomes with purified native C3 and factors I and H. Quartz crystal microbalance analysis confirmed binding of C3 to planar DMPC surfaces. In addition, we demonstrated that DMPC liposomes bound to or were phagocytized by granulocytes in a complement-dependent manner, as evidenced by the use of complement inhibitors. In summary, we have shown that C3 is activated both by convertase-dependent cleavage, preferentially in the fluid phase, by mechanisms which are not well elucidated, and also by contact activation into C3(H2O) on the DMPC surface. In particular, this contact activation has implications for the therapeutic regulation of complement activation during liposome treatment.

Autoregulation of thromboinflammation on biomaterial surfaces by a multicomponent therapeutic coating.

Activation of the thrombotic and complement systems is the main recognition and effector mechanisms in the multiple adverse biological responses triggered when biomaterials or therapeutic cells come into blood contact. We have created a surface which is auto-protective to human innate immunity by combining three fundamentally different strategies, all developed by us previously, which have been shown to induce substantial, but incomplete hemocompatibility when used separately. In summary, we have conjugated a factor H-binding peptide; and an ADP-degrading enzyme; using a PEG linker on both material and cellular surfaces. When exposed to human whole blood, factor H was specifically recruited to the modified surfaces and inhibited complement attack. In addition, activation of platelets and coagulation was efficiently attenuated, by degrading ADP. Thus, by inhibiting thromboinflammation using a multicomponent approach, we have created a hybrid surface with the potential to greatly reduce incompatibility reactions involving biomaterials and transplantation.