Elizabeth D. Hood

Endothelial delivery of antioxidant enzymes loaded into non-polymeric magnetic nanoparticles

Antioxidant enzymes have shown promise as a therapy for pathological conditions involving increased production of reactive oxygen species (ROS). However the efficiency of their use for combating oxidative stress is dependent on the ability to achieve therapeutically adequate levels of active enzymes at the site of ROS-mediated injury. Thus, the implementation of antioxidant enzyme therapy requires a strategy enabling both guided delivery to the target site and effective protection of the protein in its active form. To address these requirements we developed magnetically responsive nanoparticles (MNP) formed by precipitation of calcium oleate in the presence of magnetite-based ferrofluid (controlled aggregation/precipitation) as a carrier for magnetically guided delivery of therapeutic proteins. We hypothesized that antioxidant enzymes, catalase and superoxide dismutase (SOD), can be protected from proteolytic inactivation by encapsulation in MNP. We also hypothesized that catalase-loaded MNP applied with a high-gradient magnetic field can rescue endothelial cells from hydrogen peroxide toxicity in culture. To test these hypotheses, a family of enzyme-loaded MNP formulations were prepared and characterized with respect to their magnetic properties, enzyme entrapment yields and protection capacity. SOD- and catalase-loaded MNP were formed with average sizes ranging from 300 to 400 nm, and a protein loading efficiency of 20-33%. MNP were strongly magnetically responsive (magnetic moment at saturation of 14.3 emu/g) in the absence of magnetic remanence, and exhibited a protracted release of their cargo protein in plasma. Catalase stably associated with MNP was protected from proteolysis and retained 20% of its initial enzymatic activity after 24h of exposure to pronase. Under magnetic guidance catalase-loaded MNP were rapidly taken up by cultured endothelial cells providing increased resistance to oxidative stress (62+/-12% cells rescued from hydrogen peroxide induced cell death vs. 10+/-4% under non-magnetic conditions). We conclude that non-polymeric MNP formed using the controlled aggregation/precipitation strategy are a promising carrier for targeted antioxidant enzyme therapy, and in combination with magnetic guidance can be applied to protect endothelial cells from oxidative stress mediated damage. This protective effect of magnetically targeted MNP impregnated with antioxidant enzymes can be highly relevant for the treatment of cardiovascular disease and should be further investigated in animal models.

Nanocarriers for vascular delivery of antioxidants.

Antioxidant enzymes (AOEs) catalase and superoxide dismutase (SOD) detoxify harmful reactive oxygen species, but the therapeutic utility of AOEs is hindered by inadequate delivery. AOE modification by poly-ethylene glycol (PEG) and encapsulation in PEG-coated liposomes increases the AOE bioavailability and enhances protective effects in animal models. Pluronic-based micelles formed with AOEs show even more potent protective effects. Furthermore, polymeric nanocarriers (PNCs) based on PEG-copolymers protect encapsulated AOEs from proteolysis and improve delivery to the target cells, such as the endothelium lining the vascular lumen. Antibodies to endothelial determinants conjugated to AOEs or AOE carriers provide targeting and intracellular delivery. Targeted liposomes, protein conjugates and magnetic nanoparticles deliver AOEs to sites of vascular oxidative stress in the cardiovascular, pulmonary and nervous systems. Further advances in nanodevices for AOE delivery will provide a basis for the translation of this approach in the clinical domain.

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

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.

Endothelial targeting of liposomes encapsulating SOD/Catalase mimetic EUK-134 alleviates acute pulmonary inflammation

Production of excessive levels of reactive oxygen species (ROS) in the vascular endothelium is a common pathogenic pathway in many dangerous conditions, including acute lung injury, ischemia-reperfusion, and inflammation. Ineffective delivery of antioxidants to the endothelium limits their utility for management of these conditions. In this study, we devised a novel translational antioxidant intervention targeted to the vascular endothelium using PEG-liposomes loaded with EUK-134 (EUK), a potent superoxide dismutase/catalase mimetic. EUK loaded into antibody-coated liposomes (size 197.8±4.5 nm diameter, PDI 0.179±0.066) exerted partial activity in the intact carrier, while full activity was recovered upon liposome disruption. For targeting we used antibodies (Abs) to platelet-endothelial cell adhesion molecule (PECAM-1). Both streptavidin-biotin and SATA/SMCC conjugation chemistries provided binding of 125-150 Ab molecules per liposome. Ab/EUK/liposomes, but not IgG/EUK/liposomes: i) bound to endothelial cells and inhibited cytokine-induced inflammatory activation in vitro; and, ii) accumulated in lungs after intravascular injection, providing >60% protection against pulmonary edema in endotoxin-challenged mice (vs <6% protection afforded by IgG/liposome/EUK counterpart). Since the design elements of this drug delivery system are already in clinical use (PEG-liposomes, antibodies, SATA/SMCC conjugation), it is an attractive candidate for translational interventions using antioxidant molecules such as EUK and other clinically acceptable drugs.

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

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.

Nanocarriers for Vascular Delivery of Anti-Inflammatory Agents

There is a need for improved treatment of acute vascular inflammation in conditions such as ischemia-reperfusion injury, acute lung injury, sepsis, and stroke. The vascular endothelium represents an important therapeutic target in these conditions. Furthermore, some anti-inflammatory agents (AIAs) (e.g., biotherapeutics) require precise delivery into subcellular compartments. In theory, optimized delivery to the desired site of action may improve the effects and enable new mechanisms of action of these AIAs. Diverse nanocarriers (NCs) and strategies for targeting them to endothelial cells have been designed and explored for this purpose. Studies in animal models suggest that delivery of AIAs using NCs may provide potent and specific molecular interventions in inflammatory pathways. However, the industrial development and clinical translation of complex NC-AIA formulations are challenging. Rigorous analysis of therapeutic/side effect and benefit/cost ratios is necessary to identify and optimize the approaches that may find clinical utility in the management of acute inflammation.

Vascular Immunotargeting to Endothelial Determinant ICAM-1 Enables Optimal Partnering of Recombinant scFv-Thrombomodulin Fusion with Endogenous Cofactor

The use of targeted therapeutics to replenish pathologically deficient proteins on the luminal endothelial membrane has the potential to revolutionize emergency and cardiovascular medicine. Untargeted recombinant proteins, like activated protein C (APC) and thrombomodulin (TM), have demonstrated beneficial effects in acute vascular disorders, but have failed to have a major impact on clinical care. We recently reported that TM fused with an scFv antibody fragment to platelet endothelial cell adhesion molecule-1 (PECAM-1) exerts therapeutic effects superior to untargeted TM. PECAM-1 is localized to cell-cell junctions, however, whereas the endothelial protein C receptor (EPCR), the key co-factor of TM/APC, is exposed in the apical membrane. Here we tested whether anchoring TM to the intercellular adhesion molecule (ICAM-1) favors scFv/TM collaboration with EPCR. Indeed: i) endothelial targeting scFv/TM to ICAM-1 provides ∼15-fold greater activation of protein C than its PECAM-targeted counterpart; ii) blocking EPCR reduces protein C activation by scFv/TM anchored to endothelial ICAM-1, but not PECAM-1; and iii) anti-ICAM scFv/TM fusion provides more profound anti-inflammatory effects than anti-PECAM scFv/TM in a mouse model of acute lung injury. These findings, obtained using new translational constructs, emphasize the importance of targeting protein therapeutics to the proper surface determinant, in order to optimize their microenvironment and beneficial effects.

Dual targeting of therapeutics to endothelial cells: collaborative enhancement of delivery and effect

Anchoring pharmacologic agents to the vascular lumen has the potential to modulate critical processes at the blood‐tissue interface, avoiding many of the off‐target effects of systemically circulating agents. We report a novel strategy for endothelial dual targeting of therapeutics, which both enhances drug delivery and enables targeted agents to partner enzymatically to generate enhanced biologic effect. Based on the recent discovery that paired antibodies directed to adjacent epitopes of platelet endothelial cell adhesion molecule (PECAM)‐1 stimulate each others binding, we fused single‐chain fragments (scFv) of paired anti‐mouse PECAM‐1 antibodies to recombinant murine thrombomodulin (TM) and endothelial protein C receptor (EPCR), endothelial membrane proteins that partner in activation of protein C (PC). scFv/TM and scFv/EPCR bound to mouse endothelial PECAM‐1 with high affinity (EC50 1.5 and 3.8 nM, respectively), and codelivery induced a 5‐fold increase in PC activation not seen when TM and EPCR are anchored to distinct cell adhesion molecules. In a mouse model of acute lung injury, dual targeting reduces both the expression of lung inflammatory markers and trans‐endothelial protein leak by as much as 40%, as compared to either agent alone. These findings provide proof of principle for endothelial dual targeting, an approach with numerous potential biomedical applications.—Greineder, C. F., Brenza, J. B., Carnemolla, R., Zaitsev, S., Hood, E.D., Pan, D.C., Ding, B.‐S., Esmon, C. T., Chacko, A. M., Muzykantov, V. R. Dual targeting of therapeutics to endothelial cells: collaborative enhancement of delivery and effect. FASEB J. 29, 3483‐3492 (2015). www.fasebj.org

Targeting to Endothelial Cells Augments the Protective Effect of Novel Dual Bioactive Antioxidant/Anti-Inflammatory Nanoparticles

Oxidative stress and inflammation are intertwined contributors to numerous acute vascular pathologies. A novel dual bioactive nanoparticle with antioxidant/anti-inflammatory properties was developed based on the interactions of tocopherol phosphate and the manganese porphyrin SOD mimetic, MnTMPyP. The size and drug incorporation efficiency were shown to be dependent on the amount of MnTMPyP added as well as the choice of surfactant. MnTMPyP was shown to retain its SOD-like activity while in intact particles and to release in a slow and controlled manner. Conjugation of anti-PECAM antibody to the nanoparticles provided endothelial targeting and potentiated nanoparticle-mediated suppression of inflammatory activation of these cells manifested by expression of VCAM, E-selectin, and IL-8. This nanoparticle technology may find applicability with drug combinations relevant for other pathologies.

Vascular Accessibility of Endothelial Targeted Ferritin Nanoparticles

Targeting nanocarriers to the endothelium, using affinity ligands to cell adhesion molecules such as ICAM-1 and PECAM-1, holds promise to improve the pharmacotherapy of many disease conditions. This approach capitalizes on the observation that antibody-targeted carriers of 100 nm and above accumulate in the pulmonary vasculature more effectively than free antibodies. Targeting of prospective nanocarriers in the 10-50 nm range, however, has not been studied. To address this intriguing issue, we conjugated monoclonal antibodies (Ab) to ICAM-1 and PECAM-1 or their single chain antigen-binding fragments (scFv) to ferritin nanoparticles (FNPs, size 12 nm), thereby producing Ab/FNPs and scFv/FNPs. Targeted FNPs retained their typical symmetric core-shell structure with sizes of 20-25 nm and ∼4-5 Ab (or ∼7-9 scFv) per particle. Ab/FNPs and scFv/FNPs, but not control IgG/FNPs, bound specifically to cells expressing target molecules and accumulated in the lungs after intravenous injection, with pulmonary targeting an order of magnitude higher than free Ab. Most intriguing, the targeting of Ab/FNPs to ICAM-1, but not PECAM-1, surpassed that of larger Ab/carriers targeted by the same ligand. These results indicate that (i) FNPs may provide a platform for targeting endothelial adhesion molecules with carriers in the 20 nm size range, which has not been previously reported; and (ii) ICAM-1 and PECAM-1 (known to localize in different domains of endothelial plasmalemma) differ in their accessibility to circulating objects of this size, common for blood components and nanocarriers.