Antioxidant protection by PECAM-targeted delivery of a novel NADPH-oxidase inhibitor to the endothelium in vitro and in vivo

Abstract

Oxidant stress caused by pathological elevation of reactive oxygen species (ROS) production in the endothelial cells lining the vascular lumen is an important component of many vascular and pulmonary disease conditions. NADPH oxidase (NOX) activated by pathological mediators including angiotensin and cytokines is a major source of endothelial ROS. In order to intercept this pathological pathway, we have encapsulated an indirect NOX inhibitor, MJ33, into immunoliposomes (Ab-MJ33/IL) targeted to endothelial marker platelet endothelial cell adhesion molecule (PECAM-1). Ab-MJ33/IL, but not control IgG-MJ33/IL are specifically bound to endothelium and attenuated angiotensin-induced ROS production in vitro and in vivo. Additionally, Ab-MJ33/IL inhibited endothelial expression of the inflammatory marker vascular cell adhesion molecule (VCAM) in cells and animals challenged with the cytokine TNF. Furthermore, Ab-MJ33/IL alleviated pathological disruption of endothelial permeability barrier function in cells exposed to vascular endothelial growth factor (VEGF) and in the lungs of mice challenged with lipopolysaccharide (LPS). Of note, the latter beneficial effect has been achieved both by prophylactic and therapeutic injection of Ab-MJ33/IL in animals. Therefore, specific suppression of ROS production by NOX in endothelium, attainable by Ab-MJ33/IL targeting, may help deciphering mechanisms of vascular oxidative stress and inflammation, and potentially improve treatment of these conditions.

Fig. 1. MJ33 inhibits NOX mediated ROS

(a) Ang II actives PLA₂ thereby releasing lysoPC from the plasma membrane resulting in NOX activation and release of ROS. (b) MJ33 inhibition of PLA2 activity prevents the release of lysoPC whereupon NOX2 does not translocate to the plasma membrane or release ROS. (c) PECAM targeted lipid nanoparticle structure with MJ33 cargo d) MJ33 (1-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol, Li) MW 498.5 g/mol.

Fig. 2. MJ33 immunoliposome characterization

(a) DLS mean hydrodynamic diameter of liposomes measured at 1:40 dilution in ultrapurified DI water. Data shows stability of particle size of the IgG-SA coated liposomes size initially and at two months stored at 4°C. (b) Varying concentrations of radiolabeled IgG-SA were incubated with biotinylated liposomes for 1 hr at RT with unbound IgG-SA separated from ILs by ultracentrifugation. Binding was calculated by comparison of cpm in purified samples relative to cpm added. (c) MJ33 PLA₂ activity retained in antibody conjugated liposome formulation. Activity represents cleavage of FFA. Error is standard deviation, n>=3, for all data. Significance p<0.001.

Fig. 3. Anti-PECAM conjugated liposomes bind specifically to endothelial cell targets in vitro

(a) Binding of Ab-MJ33/IL fluorescently labeled with the inclusion of CF-DSPE compared to (b) IgG-ILs. (c) Binding of anti-PECAM (Ab62) and IgG liposomes to cultured HUVECs quantified by radiotracing ¹²⁵I labeled IgG dosed with unlabeled mAbs at 10% by mass. Expressed data was calculated by comparing radioactivity measures per well of lysed cells post incubation normalized to cell number relative to the activity of known activity and concentration of the liposome aliquot added. n=4, Error bars represent standard deviation.

Fig. 4. Biodistribution of anti-PECAM MJ33 liposomes vs IgG control liposomes

(a) Tissue localization of anti-PECAM targeted liposomes versus IgG controls 60 mins post IV administration. Individual organs were weighed and gamma counted. Biodistribution data shown are calculated as cpm in the organ divided by the injected dose and normalized by mass of organ (%ID/g) (b) Tissue level of MJ33 found in lung homogenate measured by mass spectroscopy.

Fig. 5. Inhibition of ROS production with NOX2 agonist Ang II in vitro and in vivo

(a) Fluorescence from DCFDA dye representing ROS generation in Ang II treated MS1 cells. Representative fluorescent images in each treatment group taken 10 mins after adminstration of Ang II to ensure equivalance of potential ROS generation. Images left to right: (1) Untreated; (2) Ang II treatment, no MJ33; (3) Ang II + untargeted MJ33 liposomes;(4) Ang II + PECAM targeted MJ33 liposomes (b) Corresponding quantifications of fluorescence. Normalized relative to the untreated control. Quantification based on no less than 6 images. Error bars represent standard deviation. Significance between targeted and untargeted *p < 0.001. (c) Inhibition of ROS production in Ang II (50 mM) treated isolated perfused lung model. Reduction of ROS 2.5 fold by targeted MJ33 liposomes over untargeted from lung perfusate. Fluorescence generated from reaction of Amplex Red with ROS. Values shown represent slopes of the linear regression of the time vs FL units relative to untreated controls. Error bars represent standard deviation. n=3.

Fig. 6. Inhibition of pro-inflammatory endothelial activation by targeted MJ33 liposomes in vitro and in vivo

(a) Quantification of VCAM expression normalized to actin from cells treated +/- anti-PECAM (Ab62) ILs challenged by 10 ng/ml TNF for 4 hr. Resulting cell lysates were measured for VCAM by Western blot. Error bars represent standard deviation. **p < 0.005. (b) Quantification of VCAM expression normalized to actin in mice lung homogenates +/- anti-PECAM (Mec13) ILs challenged by intratracheally administered LPS (I mg/kg) 24 hr prior to lung harvest. Resulting lung homogenates were measured for VCAM by Western blot. Error bars represent standard deviation, n=3 for targeted and untargeted ILs. **p<0.001

Fig. 7. Attenuation of permeability by targeted delivery of MJ33 in vitro and in vivo

(a) HUVECs incubated with anti-PECAM (Ab62) Ils IgG ILs +/- VEGF challenge were evaluated for permeability by measuring leakage of FITC-dextran across the endothelial monolayer in transwell culture dishes. Fluorescence in top and bottom wells ere measured. Values shown are normalized percentages of the total FITC-dextran crossing the cells relative to that of the untreated controls. Error bars represent st dev. (n=4) ** p<0.001 vs VEGF controls. B. BAL protein with MJ33 treatment 15 mins before and 60 mins post LPS treatment (1 mg/kg intratracheally). The bottom dotted line represents BAL protein/ g weight in unchallenged animals that received sham PBS injections. The top solid line represents BAL protein/g weight LPS challenged animals untreated by ILs. Error bars represent st dev. n>= 4 for all groups. **p<0.001 vs LPS treated controls.