MC3/SAINT-O-Somes, a novel liposomal delivery system for efficient and safe delivery of siRNA into endothelial cells

Increased understanding of chronic inflammatory diseases and the role of endothelial cell (EC) activation herein, have urged interest in sophisticated strategies to therapeutically intervene in activated EC to treat these diseases. Liposome-mediated delivery of therapeutic siRNA in inflammation-activated EC is such a strategy. In this study, we describe the design and characterisation of two liposomal siRNA delivery systems formulated with the cationic MC3 lipid or MC3/SAINT mixed lipids, referred to as MC3-O-Somes (MOS) and MC3/SAINT-O-Somes (MSS). The two formulations showed comparable physicochemical properties, except for better siRNA encapsulation efficiency in the MSS formulation. Antibody-mediated VCAM-1 targeting (AbVCAM-1) increased the association of the targeted MOS and MSS with activated EC, although the targeted MOS showed a significantly higher VCAM-1 specific association than the targeted MSS. AbVCAM-1 MSS containing RelA siRNA achieved significant downregulation of RelA expression, while AbVCAM-1 MOS containing RelA siRNA did not downregulate RelA expression in activated EC. Additionally, AbVCAM-1 MSS containing RelA siRNA showed low cytotoxicity in EC and at the same time prohibited endothelial inflammatory activation by reducing expression of cell adhesion molecules. The AbVCAM-1 MSS formulation is a novel siRNA delivery system based on a combination of the cationic lipids MC3 and SAINT, that shows good physicochemical characteristics, enhanced endothelial cell association, improved transfection activity, low toxicity and significant anti-inflammatory effect, thereby complying with the requirements for future in vivo investigations.</p

MC3/SAINT-O-Somes, a novel liposomal delivery system for efficient and safe delivery of siRNA into endothelial cells

Increased understanding of chronic inflammatory diseases and the role of endothelial cell (EC) activation herein, have urged interest in sophisticated strategies to therapeutically intervene in activated EC to treat these diseases. Liposome-mediated delivery of therapeutic siRNA in inflammation-activated EC is such a strategy. In this study, we describe the design and characterisation of two liposomal siRNA delivery systems formulated with the cationic MC3 lipid or MC3/SAINT mixed lipids, referred to as MC3-O-Somes (MOS) and MC3/SAINT-O-Somes (MSS). The two formulations showed comparable physicochemical properties, except for better siRNA encapsulation efficiency in the MSS formulation. Antibody-mediated VCAM-1 targeting (AbVCAM-1) increased the association of the targeted MOS and MSS with activated EC, although the targeted MOS showed a significantly higher VCAM-1 specific association than the targeted MSS. AbVCAM-1 MSS containing RelA siRNA achieved significant downregulation of RelA expression, while AbVCAM-1 MOS containing RelA siRNA did not downregulate RelA expression in activated EC. Additionally, AbVCAM-1 MSS containing RelA siRNA showed low cytotoxicity in EC and at the same time prohibited endothelial inflammatory activation by reducing expression of cell adhesion molecules. The AbVCAM-1 MSS formulation is a novel siRNA delivery system based on a combination of the cationic lipids MC3 and SAINT, that shows good physicochemical characteristics, enhanced endothelial cell association, improved transfection activity, low toxicity and significant anti-inflammatory effect, thereby complying with the requirements for future in vivo investigations.</p

Neovascularization and vascular markers in a foreign body reaction to subcutaneously implanted degradable biomaterial in mice

To study the spatiotemporal processes of angiogenesis during a foreign body reaction (FBR), biodegradable bovine collagen type-1 (COL-I) discs were implanted in mice for a period up to 28 days. The cellular infiltration (consisting mainly of macrophages, giant cells and fibroblasts), and the extent of neovascularization into the discs were determined. Also the expression levels and/or distribution of the endothelial cell markers von Willebrand factor (vWF), platelet endothelial cell adhesion molecule-1 (PECAM-1)/CD31, MECA-32 antigens and endomucin, and of the basal lamina marker collagen type IV (Coll IV) were analysed. In time, a strong neovascularization of the discs was observed, with frequently occurring vascular sprouting, and intussusceptive growth of vessels. In this model, vWF, MECA-32 and endomucin antibodies often failed to stain neovessels in the COL-I discs. In contrast, staining for collagen IV basal lamina component in combination with CD31 covered the complete range of neo-vessels. We conclude that the model described in this study is a useful model to study FBR induced angiogenesis because of the active neovascularization taking place during prolonged periods of time.</p

Development of a Combined Lipid-Based Nanoparticle Formulation for Enhanced siRNA Delivery to Vascular Endothelial Cells

Low transfection efficiency in endothelial cells (EC) is still a bottleneck for the majority of siRNA-based vascular delivery approaches. In this work, we developed a lipid-based nanoparticle (LNP) formulation based on a combination of a permanently charged cationic lipid-DOTAP and a conditionally ionized cationic lipid-MC3 (DOTAP/MC3) for the enhanced delivery of siRNA into EC. Compared with a single DOTAP or MC3-based benchmark LNP, we demonstrated that the DOTAP/MC3 LNP formulation shows the best transfection efficiency both in primary EC in vitro and in endothelium in zebrafish. The high transfection activity of the DOTAP/MC3 LNP formulation is achieved by a combination of improved endothelial association mediated by DOTAP and MC3-triggered efficient siRNA intracellular release in EC. Furthermore, AbVCAM-1-coupled DOTAP/MC3 LNP-mediated siRNARelA transfection showed pronounced anti-inflammatory effects in inflammatory-activated primary EC by effectively blocking the NF-κB pathway. In conclusion, the combination of permanent and ionizable cationic lipids in LNP formulation provides an effective endothelial cell delivery of siRNA

Differential effects of oleate on vascular endothelial and liver sinusoidal endothelial cells reveal its toxic features in vitro

Several fatty acids, in particular saturated fatty acids like palmitic acid, cause lipotoxicity in the context of non-alcoholic fatty liver disease . Unsaturated fatty acids (e.g. oleic acid) protect against lipotoxicity in hepatocytes. However, the effect of oleic acid on other liver cell types, in particular liver sinusoidal endothelial cells (LSECs), is unknown. Human umbilical vein endothelial cells (HUVECs) are often used as a substitute for LSECs, however, because of the unique phenotype of LSECs, HUVECs cannot represent the same biological features as LSECs. In this study, we investigate the effects of oleate and palmitate (the sodium salts of oleic acid and palmitic acid) on primary rat LSECs in comparison to their effects on HUVECs. Oleate induces necrotic cell death in LSECs, but not in HUVECs. Necrotic cell death of LSECs can be prevented by supplementation of 2-stearoylglycerol, which promotes cellular triglyceride (TG) synthesis. Repressing TG synthesis, by knocking down DGAT1 renders HUVECs sensitive to oleate-induced necrotic death. Mechanistically, oleate causes a sharp drop of intracellular ATP level and impairs mitochondrial respiration in LSECs. The combination of oleate and palmitate reverses the toxic effect of oleate in both LSECs and HUVECs. These results indicate that oleate is toxic and its toxicity can be attenuated by stimulating TG synthesis. The toxicity of oleate is characterized by mitochondrial dysfunction and necrotic cell death. Moreover, HUVECs are not suitable as a substitute model for LSECs.</p

Identification of LPS-Activated Endothelial Subpopulations With Distinct Inflammatory Phenotypes and Regulatory Signaling Mechanisms

Sepsis is a life-threatening condition caused by a dysregulated host response to infection. Endothelial cells (EC) are actively involved in sepsis-associated (micro)vascular disturbances and subsequent organ dysfunction. Lipopolysaccharide (LPS), a Gram-negative bacterial product, can activate EC leading to the expression of pro-inflammatory molecules. This process is molecularly regulated by specific receptors and distinct, yet poorly understood intracellular signaling pathways. LPS-induced expression of endothelial adhesion molecules E-selectin and VCAM-1 in mice was previously shown to be organ- and microvascular-specific. Here we report that also within renal microvascular beds the endothelium expresses different extents of E-selectin and VCAM-1. This heterogeneity was recapitulated in vitro in LPS-activated human umbilical vein EC (HUVEC). Within 2 h after LPS exposure, four distinct HUVEC subpopulations were visible by flow cytometric analysis detecting E-selectin and VCAM-1 protein. These encompassed E-selectin−/VCAM-1− (–/–), E-selectin+/VCAM-1− (E-sel+), E-selectin+/VCAM-1+ (+/+), and E-selectin−/VCAM-1+ (VCAM-1+) subpopulations. The formation of subpopulations was a common response of endothelial cells to LPS challenge. Using fluorescence-activated cell sorting (FACS) we demonstrated that the +/+ subpopulation also expressed the highest levels of inflammatory cytokines and chemokines. The differences in responsiveness of EC subpopulations could not be explained by differential expression of LPS receptors TLR4 and RIG-I. Functional studies, however, demonstrated that the formation of the E-sel+ subpopulation was mainly TLR4-mediated, while the formation of the +/+ subpopulation was mediated by both TLR4 and RIG-I. Pharmacological blockade of NF-κB and p38 MAPK furthermore revealed a prominent role of their signaling cascades in E-sel+ and +/+ subpopulation formation. In contrast, the VCAM-1+ subpopulation was not controlled by any of these signaling pathways. Noteworthy is the existence of a “quiescent” subpopulation that was devoid of the two adhesion molecules and did not express cytokines or chemokines despite LPS exposure. Summarizing, our findings suggest that LPS activates different signaling mechanisms in EC that drive heterogeneous expression of EC inflammatory molecules. Further characterization of the signaling pathways involved will enhance our understanding of endothelial heterogeneous responses to sepsis related stimuli and enable the future design of effective therapeutic strategies to interfere in these processes to counteract sepsis-associated organ dysfunction

Leukocyte-Released Mediators in Response to Both Bacterial and Fungal Infections Trigger IFN Pathways, Independent of IL-1 and TNF-alpha, in Endothelial Cells

In sepsis, dysregulated immune responses to infections cause damage to the host. Previous studies have attempted to capture pathogen-induced leukocyte responses. However, the impact of mediators released after pathogen-leukocyte interaction on endothelial cells, and how endothelial cell responses vary depending on the pathogen-type is lacking. Here, we comprehensively characterized the transcriptomic responses of human leukocytes and endothelial cells to Gram negative-bacteria, Gram positive-bacteria, and fungi. We showed that whole pathogen lysates induced strong activation of leukocytes but not endothelial cells. Interestingly, the common response of leukocytes to various pathogens converges on endothelial activation. By exposing endothelial cells to leukocyte-released mediators, we observed a strong activation of endothelial cells at both transcription and protein levels. By adding IL-1RA and TNF-α antibody in leukocyte-released mediators before exposing to endothelial cells, we identified specific roles for IL-1 and TNF-α in driving the most, but not all, endothelial activation. We also showed for the first time, activation of interferon response by endothelial cells in response to leukocyte-released mediators, independently from IL-1 and TNF-α pathways. Our study therefore, not only provides pathogen-dependent transcriptional changes in leukocytes and endothelial cells during infections, but also reveals a role for IFN, together with IL1 and TNFα signaling, in mediating leukocyte-endothelial interaction in infections

MC3/SAINT-O-Somes, a novel liposomal delivery system for efficient and safe delivery of siRNA into endothelial cells

Increased understanding of chronic inflammatory diseases and the role of endothelial cell (EC) activation herein, have urged interest in sophisticated strategies to therapeutically intervene in activated EC to treat these diseases. Liposome-mediated delivery of therapeutic siRNA in inflammation-activated EC is such a strategy. In this study, we describe the design and characterisation of two liposomal siRNA delivery systems formulated with the cationic MC3 lipid or MC3/SAINT mixed lipids, referred to as MC3-O-Somes (MOS) and MC3/SAINT-O-Somes (MSS). The two formulations showed comparable physicochemical properties, except for better siRNA encapsulation efficiency in the MSS formulation. Antibody-mediated VCAM-1 targeting (AbVCAM-1) increased the association of the targeted MOS and MSS with activated EC, although the targeted MOS showed a significantly higher VCAM-1 specific association than the targeted MSS. AbVCAM-1 MSS containing RelA siRNA achieved significant downregulation of RelA expression, while AbVCAM-1 MOS containing RelA siRNA did not downregulate RelA expression in activated EC. Additionally, AbVCAM-1 MSS containing RelA siRNA showed low cytotoxicity in EC and at the same time prohibited endothelial inflammatory activation by reducing expression of cell adhesion molecules. The AbVCAM-1 MSS formulation is a novel siRNA delivery system based on a combination of the cationic lipids MC3 and SAINT, that shows good physicochemical characteristics, enhanced endothelial cell association, improved transfection activity, low toxicity and significant anti-inflammatory effect, thereby complying with the requirements for future in vivo investigations.</p

MC3/SAINT-O-Somes, a novel liposomal delivery system for efficient and safe delivery of siRNA into endothelial cells

Increased understanding of chronic inflammatory diseases and the role of endothelial cell (EC) activation herein, have urged interest in sophisticated strategies to therapeutically intervene in activated EC to treat these diseases. Liposome-mediated delivery of therapeutic siRNA in inflammation-activated EC is such a strategy. In this study, we describe the design and characterisation of two liposomal siRNA delivery systems formulated with the cationic MC3 lipid or MC3/SAINT mixed lipids, referred to as MC3-O-Somes (MOS) and MC3/SAINT-O-Somes (MSS). The two formulations showed comparable physicochemical properties, except for better siRNA encapsulation efficiency in the MSS formulation. Antibody-mediated VCAM-1 targeting (AbVCAM-1) increased the association of the targeted MOS and MSS with activated EC, although the targeted MOS showed a significantly higher VCAM-1 specific association than the targeted MSS. AbVCAM-1 MSS containing RelA siRNA achieved significant downregulation of RelA expression, while AbVCAM-1 MOS containing RelA siRNA did not downregulate RelA expression in activated EC. Additionally, AbVCAM-1 MSS containing RelA siRNA showed low cytotoxicity in EC and at the same time prohibited endothelial inflammatory activation by reducing expression of cell adhesion molecules. The AbVCAM-1 MSS formulation is a novel siRNA delivery system based on a combination of the cationic lipids MC3 and SAINT, that shows good physicochemical characteristics, enhanced endothelial cell association, improved transfection activity, low toxicity and significant anti-inflammatory effect, thereby complying with the requirements for future in vivo investigations.</p

Trimethylene Carbonate and -Caprolactone Based (co)Polymer Networks: Mechanical Properties and Enzymatic Degradation

High molecular weight trimethylene carbonate (TMC) and -caprolactone (CL) (co)polymers were synthesized. Melt pressed (co)polymer films were cross-linked by gamma irradiation (25 kGy or 50 kGy) in vacuum, yielding gel fractions of up to 70%. The effects of copolymer composition and irradiation dose on the cytotoxicity, surface properties, degradation behavior, and mechanical and thermal properties of these (co)polymers and networks were investigated. Upon incubation with cell culture medium containing extracts of (co)polymers and networks, human foreskin fibroblasts remained viable. For all (co)polymers and networks, cell viabilities were determined to be higher than 94%. The formed networks were flexible, with elastic moduli ranging from 2.7 to 5.8 MPa. Moreover, these form-stable networks were creep resistant under dynamic conditions. The permanent deformation after 2 h relaxation was as low as 1% after elongating to 50% strain for 20 times. The in vitro enzymatic erosion behavior of these hydrophobic (co)polymers and networks was investigated using aqueous lipase solutions. The erosion rates in lipase solution could be tuned linearly from 0.8 to 45 mg/(cm2 × day) by varying the TMC to CL ratio and the irradiation dose. The copolymers and networks degraded essentially by a surface erosion mechanism