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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., Title in the list of papers of Anna Adler's thesis: Regulation of innate immune system by fragmented heparin-conjugated lipids on lipid bilayer membranes

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., Title in the list of papers of Anna Adler's thesis: Regulation of innate immune system by fragmented heparin-conjugated lipids on lipid bilayer membranes

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., Title in the list of papers of Anna Adler's thesis: Regulation of innate immune system by fragmented heparin-conjugated lipids on lipid bilayer membranes

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 degree celsius. 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.

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 degree celsius. 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.

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 degree celsius. 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.

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 degree celsius. 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.

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., Title in the list of papers of Anna Adler's thesis: Regulation of innate immune system by fragmented heparin-conjugated lipids on lipid bilayer membranes

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 degree celsius. 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.

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., Title in the list of papers of Anna Adler's thesis: Regulation of innate immune system by fragmented heparin-conjugated lipids on lipid bilayer membranes

Asymmetrical lipid nanoparticles are interesting nanocarriers for charged molecules, like nucleic acids. They promise control over inner and outer charge. High charge density on the inside is favorable for efficient condensation and charge neutralization of highly charged biopharmaceuticals, while a neutral or slightly negative outer layer promotes biocompatibility. The main goal of this work was the development and characterization of asymmetric liposomes, prepared using water in oil (w/o) nanoemulsions of phospholipids and squalene in a centrifugal field. This method enables the control over the lipid composition of each monolayer. Liposomes were prepared by passing phospholipid w/o nanoemulsions through an oil-water interface previously saturated with phospholipids. We used NBD-PE orNBD-PC as a fluorescent marker for either the inner or outer lipid layer and plasmid DNA as nucleic acid payload. The final liposomes had sizes below 200 nm and polydispersity indexes of 0.3 and had a bilayer asymmetry of 70%, thus shielding the charge of positive phospholipids in the inner bilayer leaflet. Final formulations were examined using negative staining transmission electron microscopy (TEM). Plasmid encapsulation efficiency of the method was 10-15%. Our results indicate that the w/o nanoemulsion-centrifugation method allows the successful production of liposomes with tailored features for encapsulation of nucleic acid therapeutics.

Lysolipid-containing thermosensitive liposomes (LTSL) are clinically-relevant drug nanocarriers which have been used to deliver small molecule cytostatics to tumors in combination with local hyperthermia (42 °C) to trigger local drug release. The objective of this study was to investigate the feasibility of LTSL for encapsulation and triggered release of macromolecular drugs such as plant-derived cytotoxins. As therapeutic protein we used Mistletoe lectin-1 (ML1) - a ribosome-inactivating protein with potent cytotoxic activity in tumor cells. Model macromolecules (dextrans, albumin) and ML1 were encapsulated in small unilamellar LTSL with varying lipid compositions by the thin film hydration method and extrusion. LTSLs showed molecular weight dependent heat-triggered release of the loaded cargo. The most promising composition, ML1 formulated in LTSL composed of 86:10:4 %mol DPPC:MSPC:DSPE-PEG2000, was further studied for bioactivity against murine CT26 colon carcinoma cells. Confocal live-cell imaging showed uptake of released ML1 after mild hyperthermia at 42 °C, subsequently leading to potent cytotoxicity by LTSL-ML1. Our study shows that LTSL in combination with localized hyperthermia hold promise as local tumor delivery strategy for macromolecular cytotoxins.

Many food preparations, pharmaceuticals, and cosmetics use water-in-oil (W/O) emulsions stabilized by phospholipids. Moreover, recent technological developments try to produce liposomes or lipid coated capsules from W/O emulsions, but are faced with colloidal instabilities. To explore these instability mechanisms, emulsification by sonication was applied in three cycles, and the sample stability was studied for 3 h after each cycle. Clearly identifiable temporal structures of instability provide evidence about the emulsion morphology: an initial regime of about 10 min is shown to be governed by coalescence after which Ostwald ripening dominates. Transport via molecular diffusion in Ostwald ripening is commonly based on the mutual solubility of the two phases and is therefore prohibited in emulsions composed of immiscible phases. However, in the case of water in oil emulsified by phospholipids, these form water-loaded reverse micelles in oil, which enable Ostwald ripening despite the low solubility of water in oil, as is shown for squalene. As is proved for the phospholipid dipalmitoylphosphatidylcholine (DPPC), concentrations below the critical aggregation concentration (CAC) form monolayers at the interfaces and smaller droplet sizes. In contrast, phospholipid concentrations above the CAC create complex multilayers at the interface with larger droplet sizes. The key factors for stable W/O emulsions in classical or innovative applications are first, the minimization of the phospholipids' capacity to form reversed micelles, and second, the adaption of the initial phospholipid concentration to the water content to enable an optimized coverage of phospholipids at the interfaces for the intended drop size.

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.

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. (C) 2016 Elsevier B.V. All rights reserved.

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. (C) 2013 Elsevier Ltd. All rights reserved.

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. (C) 2013 Elsevier Ltd. All rights reserved.