Piotr S. Kowalski

Anti-VCAM-1 and anti-E-selectin SAINT-O-Somes for selective delivery of siRNA into inflammation-activated primary endothelial cells.

Activated endothelial cells play a pivotal role in the pathology of inflammatory diseases and present a rational target for therapeutic intervention by endothelial specific delivery of short interfering RNAs (siRNA). This study demonstrates the potential of the recently developed new generation of liposomes based on cationic amphiphile SAINT-C18 (1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride) for functional and selective delivery of siRNA into inflamed primary endothelial cells. To create specificity for inflamed endothelial cells, these so-called SAINT-O-Somes were harnessed with antibodies against vascular cell adhesion protein 1 (VCAM-1) or respectively E-selectin and tested in TNF-α activated primary endothelial cells from venous and aortic vascular beds. Both targeted SAINT-O-Somes carrying siRNA against the endothelial gene VE-cadherin specifically downregulated its target mRNA and protein without exerting cellular toxicity. SAINT-O-Somes formulated with siRNA formed small particles (106 nm) with a 71% siRNA encapsulation efficiency. SAINT-O-Somes were stable in the presence of serum at 37 °C, protected siRNA from degradation by serum RNases, and after i.v. injection displayed pharmacokinetic comparable to conventional long circulating liposomes. These anti-VCAM-1 and anti-E-selectin SAINT-O-Somes are thus a novel drug delivery system that can achieve specific and effective delivery of siRNA into inflamed primary endothelial cells and have physicochemical features that comply with in vivo application demands.

Targeted siRNA delivery to diseased microvascular endothelial cells—Cellular and molecular concepts

Increased insight in the role of endothelial cells in the pathophysiology of cancer, inflammatory and cardiovascular diseases, has drawn great interest in pharmacological interventions aiming at the endothelium in diseased sites. Their location in the body makes them suitable targets for therapeutic approaches based on targeted drug delivery. Functional heterogeneity of the microvascular bed in normal organ homeostasis has been appreciated for a long time, and more recent studies have revealed heterogeneity in endothelial reactivity to inflammatory stimuli as well. Upon stimulation, each organ displays a vascular bed specific pattern of cell adhesion molecules providing challenging opportunities to deliver drugs or small RNAs to organ specific (micro)vascular endothelial subsets. In this review we introduce general concepts of endothelial heterogeneity in relation to disease state and its consequences for targeted therapeutic interventions. Furthermore, we will describe novel approaches to interfere with endothelial cell engagement in disease with a main focus on siRNA therapeutics and currently used nonviral lipid and polymer‐based siRNA delivery systems. The last part of this review addresses some technical issues that are essential in proving the concept of target mRNA knock down in a vascular bed specific manner, and the further development of effective endothelial cell specific drug delivery devices.

Anti-VCAM-1 SAINT-O-Somes enable endothelial-specific delivery of siRNA and downregulation of inflammatory genes in activated endothelium in vivo.

The pivotal role of endothelial cells in the pathology of inflammatory diseases raised interest in the development of short interfering RNA (siRNA) delivery devices for selective pharmacological intervention in the inflamed endothelium. The current study demonstrates endothelial specific delivery of siRNAs and downregulation of inflammatory genes in activated endothelium in vivo by applying a novel type of targeted liposomes based on the cationic amphiphile SAINT-C18 (1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride). To create specificity for inflamed endothelial cells, these so-called SAINT-O-Somes were harnessed with antibodies against vascular cell adhesion protein 1 (VCAM-1). In TNFα challenged mice, intravenously administered anti-VCAM-1 SAINT-O-Somes exerted long circulation times and homed to VCAM-1 expressing endothelial cells in inflamed organs. The formulations were devoid of liver and kidney toxicity. Using anti-VCAM-1 SAINT-O-Somes we successfully delivered siRNA to knock down VE-cadherin mRNA in inflamed renal microvasculature, as demonstrated by using laser microdissection of different microvascular beds prior to analysis of gene expression. Using the same strategy, we demonstrated local attenuation of endothelial inflammatory response towards lipopolysaccharide in kidneys of mice treated with anti-VCAM-1 SAINT-O-Somes containing NFκB p65 specific siRNA. This study is the first demonstration of a novel, endothelial specific carrier that is suitable for selective in vivo delivery of siRNAs into inflamed microvascular segments and interference with disease associated endothelial activation.

Effective siRNA delivery to inflamed primary vascular endothelial cells by anti-E-selectin and anti-VCAM-1 PEGylated SAINT-based lipoplexes

The endothelium represents an attractive therapeutic target due to its pivotal role in many diseases including chronic inflammation and cancer. Small interfering RNAs (siRNAs) specifically interfere with the expression of target genes and are considered an important new class of therapeutics. However, due to their size and charge, siRNAs do not spontaneously enter unperturbed endothelial cells (EC). To overcome this problem, we developed novel lipoplexes for siRNA delivery that are based on the cationic amphiphilic lipid SAINT-C18. Antibodies recognizing disease induced cell adhesion molecules were employed to create cell specificity resulting in so-called antibody-SAINTargs. To improve particle stability, antibody-SAINTargs were further optimized for EC-specific siRNA-mediated gene silencing by addition of polyethylene glycol (PEG). Although PEGylated antibody-SAINTargs maintained specificity, they lost their siRNA delivery capacity. Coupling of antibodies to the distal end of PEG (so-called antibody-SAINTPEGargs), resulted in anti-E-selectin- and anti-vascular cell adhesion molecule (VCAM)-1-SAINTPEGarg that preserved their antigen recognition and their capability to specifically deliver siRNA into inflammation-activated primary endothelial cells. The enhanced uptake of siRNA by antibody-SAINTPEGargs was followed by improved silencing of the target gene VE-cadherin, demonstrating that antibody-SAINTPEGargs were capable of functionally delivering siRNA into primary endothelial cells originating from different vascular beds. In conclusion, the newly developed, physicochemically stable, and EC-specific siRNA carrying antibody-SAINTPEGargs selectively down-regulate target genes in primary endothelial cells that are generally difficult to transfect.

A critical role for Egr-1 during vascular remodelling in pulmonary arterial hypertension

AIMS Pulmonary arterial hypertension (PAH) is characterized by the development of unique neointimal lesions in the small pulmonary arteries, leading to increased right ventricular (RV) afterload and failure. Novel therapeutic strategies are needed that target these neointimal lesions. Recently, the transcription factor Egr-1 (early growth response protein 1) was demonstrated to be up-regulated early in experimental neointimal PAH. Its effect on disease development, however, is unknown. We aimed to uncover a novel role for Egr-1 as a molecular inductor for disease development in PAH. METHODS AND RESULTS In experimental flow-associated PAH in rats, we investigated the effects of Egr-1 down-regulation on pulmonary vascular remodelling, including neointimal development, and disease progression. Intravenous administration of catalytic oligodeoxynucleotides (DNA enzymes, DNAzymes) resulted in down-regulation of pulmonary vascular Egr-1 expression. Compared with vehicle or scrambled DNAzymes, DNAzymes attenuated pulmonary vascular remodelling, including the development of occlusive neointimal lesions. Selective down-regulation of Egr-1 in vivo led to reduced expression of vascular PDGF-B, TGF-β, IL-6, and p53, resulting in a reduction of vascular proliferation and increased apoptosis. DNAzyme treatment further attenuated pulmonary vascular resistance, RV systolic pressure, and RV hypertrophy. In contrast, in non-neointimal PH rodents, DNAzyme treatment had no effect on pulmonary vascular and RV remodelling. Finally, pharmacological inhibition of Egr-1 with pioglitazone, a peroxisome proliferator activated receptor-γ ligand, attenuated vascular remodelling including the development of neointimal lesions. CONCLUSIONS These results indicate that Egr-1 governs pulmonary vascular remodelling and the development of characteristic vascular neointimal lesions in flow-associated PAH. Egr-1 is therefore a potential target for future PAH treatment.

VCAM-1 specific PEGylated SAINT-based lipoplexes deliver siRNA to activated endothelium in vivo but do not attenuate target gene expression

In recent years much research in RNA nanotechnology has been directed to develop an efficient and clinically suitable delivery system for short interfering RNA (siRNA). The current study describes the in vivo siRNA delivery using PEGylated antibody-targeted SAINT-based-lipoplexes (referred to as antibody-SAINTPEGarg/PEG2%), which showed superior siRNA delivery capacity and effective down-regulation of VE-cadherin gene expression in vitro in inflammation-activated primary endothelial cells of different vascular origins. PEGylation of antibody-SAINTPEGarg resulted in more desirable pharmacokinetic behavior than that of non-PEGylated antibody-SAINTPEGarg. To create specificity for inflammation-activated endothelial cells, antibodies against vascular cell adhesion molecule-1 (VCAM-1) were employed. In TNFα-challenged mice, these intravenously administered anti-VCAM-1-SAINTPEGarg/PEG2% homed to VCAM-1 protein expressing vasculature. Confocal laser scanning microscopy revealed that anti-VCAM-1-SAINTPEGarg/PEG2% co-localized with endothelial cells in lung postcapillary venules. Furthermore, they did not exert any liver and kidney toxicity. Yet, lack of in vivo gene silencing as assessed in whole lung and in laser microdissected lung microvascular segments indicates that in vivo internalization and/or intracellular trafficking of the delivery system and its cargo in the target cells are not sufficient, and needs further attention, emphasizing the essence of evaluating siRNA delivery systems in an appropriate in vivo animal model at an early stage in their development.

SAINT-liposome-polycation particles, a new carrier for improved delivery of siRNAs to inflamed endothelial cells

Interference with acute and chronic inflammatory processes by means of delivery of siRNAs into microvascular endothelial cells at a site of inflammation demands specific, non-toxic and effective siRNA delivery system. In the current work we describe the design and characterization of siRNA carriers based on cationic pyridinium-derived lipid 1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride) (SAINT-C18) and the transfection enhancer protamine, complexed with siRNA/carrier DNA or siRNA only. These carriers, called SAINT-liposome-polycation-DNA (S-LPD) and SAINT-liposome-polycation (S-LP), have a high efficiency of siRNA encapsulation, low cellular toxicity, and superior efficacy of gene downregulation in endothelial cells in vitro as compared to DOTAP-LPD. Incorporation of 10 mol% PEG and anti-E-selectin antibody in these formulations resulted in selective siRNA delivery into activated endothelial cells. Furthermore, we showed that the physicochemical characteristics of S-LPD and S-LP, including size-stability and maintenance of the siRNA integrity in the presence of serum at 37 °C, comply with requirements for in vivo application.

Endothelium-targeted delivery of dexamethasone by anti-VCAM-1 SAINT-O-Somes in mouse endotoxemia

Microvascular endothelial cells play a pivotal role in the pathogenesis of sepsis-induced inflammatory responses and multiple organ failure. Therefore, they represent an important target for pharmacological intervention in the treatment of sepsis. Glucocorticosteroids were widely used in the treatment of sepsis but vast evidence to support their systemic use is lacking. The limited effects of glucocorticoids in the treatment of sepsis may be explained by differential effects of drug initiated NF-κB inhibition in different cell types and insufficient drug delivery in target cells. The current study aimed therefore to investigate the effects of an endothelial targeted delivery of dexamethasone in a mouse model of endotoxemia induced by two consecutive i.p. injections of lipopolysaccharide (LPS). To achieve endothelial cell specific delivery of dexamethasone, we modified SAINT-O-Somes, a new generation of liposomes that contain the cationic amphiphile SAINT-C18 (1-methyl-4-(cis-9-dioleyl) methyl-pyridinium chloride, with antibodies against vascular cell adhesion molecule-1 (VCAM-1). In LPS challenged mice, the systemic administration of free dexamethasone had negligible effects on the microvascular inflammatory endothelial responses. Dexamethasone-loaded anti-VCAM-1 SAINT-O-Somes specifically localized at VCAM-1 expressing endothelial cells in the microvasculature of inflamed organs. This was associated with a marginal attenuation of the expression of a few pro-inflammatory genes in kidney and liver, while no effects in the lung were observed. This study reveals that, although local accumulation of the targeted drug was achieved, endothelial targeted dexamethasone containing anti-VCAM-1 SAINT-O-Somes exhibited marginal effects on inflammatory endothelial cell activation in a model of endotoxemia. Studies with more potent drugs encapsulated into anti-VCAM-1 SAINT-O-Somes will in the future reveal whether this delivery system can be further developed for efficacious endothelial directed delivery of drugs in the treatment of sepsis.