Endothelial targeting of nanocarriers loaded with antioxidant enzymes for protection against vascular oxidative stress and inflammation

Abstract

Endothelial-targeted delivery of antioxidant enzymes, catalase and superoxide dismutase (SOD), is a promising strategy for protecting organs and tissues from inflammation and oxidative stress. Here we describe Protective Antioxidant Carriers for Endothelial Targeting (PACkET), the first carriers capable of targeted endothelial delivery of both catalase and SOD. PACkET formed through controlled precipitation loaded ~30% enzyme and protected it from proteolytic degradation, whereas attachment of PECAM monoclonal antibodies to surface of the enzyme-loaded carriers, achieved without adversely affecting their stability and functionality, provided targeting. Isotope tracing and microscopy showed that PACkET exhibited specific endothelial binding and internalization in vitro. Endothelial targeting of PACkET was validated in vivo by specific (vs IgG-control) accumulation in the pulmonary vasculature after intravenous injection achieving 33% of injected dose at 30 min. Catalase loaded PACkET protects endothelial cells from killing by H2O2 and alleviated the pulmonary edema and leukocyte infiltration in mouse model of endotoxin-induced lung injury, whereas SOD-loaded PACkET mitigated cytokine-induced endothelial pro-inflammatory activation and endotoxin-induced lung inflammation. These studies indicate that PACkET offers a modular approach for vascular targeting of therapeutic enzymes.

Keywords: Antioxidant enzymes; Inflammation; In vivo vascular targeting; Nanoparticles; Platelet endothelial cellular adhesion molecules.

Fig. 1

Endothelial targeted antioxidant nanoparticles formation scheme by controlled precipitation. Legend symbols signify as follows: grey dotted line represents oleate anion, and beneath that it surrounds a small black sphere indicating the oleate coated magnetite. Calcium cations that bind to free and magnetite-bound oleate anions are shown as blue spheres, and antioxidant enzymes (AOE; either catalase or SOD) are large black spheres. The amphiphilic block copolymer Pluronic F127 is indicated by a blue (for hydrophilic polyoxyethylene (POE) moieties) and red (for hydrophobic polyoxypropylene (PPE) line and then below that the dual biotinylation of the molecule is indicated with green spheres on terminal POE groups. The green symbol labeled mAb-SA indicates monoclonal antibody functionalized with streptavidin. Throughout the text “Ab” represents either human or mouse anti-PECAM; Ab62 or Mec13.3 respectively. (A) combination of aqueous suspension of oleate coated magnetite, AOE and Pluronic F127+/− biotinylation with CaCl₂ drives the controlled precipitation and forms 300–400 nm PACs (B) with surface exposed biotinylated PEO chains. Streptavidin modified Ab/IgG are added, binding to biotinylated PACs (C). Unbound fractions are separated by magnetic decantation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Inclusion of biotinylated Pluronic F127 enables mAB-SA binding to pre-formed PACs. (A) Binding of mAb-SA+/− inclusion of biotinylated Pluronic F127. Ab-SA to PAC binding measured through magnetic decantation separated unbound fraction mAbs from PAC-bound mAbs; radioactivity of fractions measured by gamma counter. (B) Stability of Ab-PAC in serum versus 5% glucose. Data shown as percentage of total released over 60 min at 37 °C over time measured as in (A).

Fig. 3

Binding of Ab-PAC to cultured endothelial cells.(A) ¹²⁵I labeled PACs incubated with cells at 37 °C, rinsed, lysed and measured for radioactivity. Binding quantified by tracing PACs labeled with a 5% fraction of ¹²⁵I-IgG relative to targeting Ab. Low biotin represents 2% biotinylated fraction included in the Pluronic PF127 and high biotin represents 5%. PAC-bound/cell calculated based on PAC concentration 2 × 10¹¹ and cell density of 5 × 10⁴/cm². Throughout the manuscript, data are shown as mean ± st dev; n ≥3 unless otherwise stated. (B) Ab-PACs added 5000 PAC/cell incubated for 60 min at 37 °C, rinsed, permeabilized, fixed, and stained with Alexa 488 to particles (green) and DAPI to cell nuclei (blue). (C) Non-specific control IgG-PAC treated as in (B). (D–E) Endocytosis of PACS containing fluorescently labeled catalase with secondary red fluorescent staining to surface PAC Abs in non-permeabilized ECs. Yellow indicates surface-bound PACS whereas green shows endocytosed PACs. (D) Incubation 2500#/cell for 15 min; (E) incubation 2500#/cell for 30 min. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

In vivo endothelial targeting Ab-PAC. (A) Tissue distribution of intravenous injected PACs into mice after 30 min circulation time shown as injected dose per gram radioactivity relative to organ mass. Radioactivity of PAC traced by inclusion of 10% ¹²⁵I-labeled rIgG-SA per targeting Ab or IgG-control bound to PAC. N = 5. (B) Organ to blood ratio of % ID/g in selected organs. (C) Lung to liver ratio of Ab-PAC vs non-specific IgG PACs. Throughout this manuscript significance is shown as *p < 0.05, **p < 0.001, ***p < 0.005.

Fig. 5

Protection by endothelial-targeted catalase PACs from oxidative stress in vitro and in vivo. (A) Chromium 51-labeled endothelial cells incubated with catalase-loaded Ab or IgG PACs and exposed to H₂O₂ (10 mm). Protection is determined by reduction in ⁵¹Cr release as an indicator of cell death as compared to untreated cells exposed to H₂O₂ relative to naïve cells. (B) Brochoalveolar lavage (BAL) protein in lungs of mice exposed to LPS via intratracheal injection testing barrier function 24 h prior to intravenous injection of catalase loaded Ab vs IgG PACs. Lung lavaged 1 h after PAC administration. Protein measured by modified Lowry assay. (C) Reduction of white blood cell infiltration (same experiment as B). Cells counted 3× by light microscopy on a hemocytometer. Significance throughout manuscript shown as *p <0.05, **p <0.001, ***p <0.005.

Fig. 6

Reduction of inflammatory markers by Ab SOD PACs. (A) PECAM-targeted PACs loaded with SOD, but not catalase, reduce VCAM expression in activated endothelial cells. Western blot measurement of cell lysate for inflammatory-marker VCAM normalized to actin. ECs incubated 1 h with Ab/IgG PACs loaded with catalase or SOD, rinsed, and exposed to 10 ng/ml TNF at 37 °C for 4 h. Quantification of western blots measured by densitometry. Significance of Ab-PAC SOD demonstrated against each group. Inset: Mouse lung homogenate expression of VCAM measured by western blot from animals exposed to LPS for 24 h (as described in Fig. 5 B and C) injected with Ab/IgG PACs laden with SOD for 1 h. Red dotted lines in all graphs represent marker expression in treated (top) and naive (bottom) controls. (B) PECAM-targeted PAC loaded with SOD reduce cytokine protein expression LPS exposed mice. Lung homogenate measured in animals injected with Ab or IgG PACs laden with SOD and exposed to LPS for 24 h for either MIP2 (left axis) or TNF (right axis) by ELISA. Significance measured against +LPS. Both concentration of MIP2/TNF determined relative to a standard curve where R² = 0.99. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)