Endothelial targeting of antibody-decorated polymeric filomicelles

Abstract

The endothelial lining of the lumen of blood vessels is a key therapeutic target for many human diseases. Polymeric filomicelles that self-assemble from polyethylene oxide (PEO)-based diblock copolymers are long and flexible rather than small or rigid, can be loaded with drugs, and--most importantly--they circulate for a prolonged period of time in the bloodstream due in part to flow alignment. Filomicelles seem promising for targeted drug delivery to endothelial cells because they can in principle adhere strongly, length-wise to specific cell surface determinants. In order to achieve such a goal of vascular drug delivery, two fundamental questions needed to be addressed: (i) whether these supramolecular filomicelles retain structural integrity and dynamic flexibility after attachment of targeting molecules such as antibodies, and (ii) whether the avidity of antibody-carrying filomicelles is sufficient to anchor the carrier to the endothelial surface despite the effects of flow that oppose adhesive interactions. Here we make targeted filomicelles that bear antibodies which recognize distinct endothelial surface molecules. We characterize these antibody targeted filomicelles and prove that (i) they retain structural integrity and dynamic flexibility and (ii) they adhere to endothelium with high specificity both in vitro and in vivo. These results provide the basis for a new drug delivery approach employing antibody-targeted filomicelles that circulate for a prolonged time yet bind to endothelial cells in vascular beds expressing select markers.

Fig. 1

Vascular oxidative stress. Pro-inflammatory insults cause endothelial exposure of cell adhesion molecules (selectins, ICAM or VCAM) and cytokine production. Cell adhesion molecules facilitate white blood cell (WBC) adhesion and transmigration. Activation of Nox (for example by angiotensin II) leads to generation of superoxide that quenches NO and thus causes vasoconstriction. Activated WBCs bind to endothelium via cell adhesion molecules and produce reactive oxygen species (ROS) and other aggressive molecules that can result in oxidative damage and death of endothelial cells. ICAM, intercellular adhesion molecule; Nox, NADPH oxidase; PMN, polymorphonuclear neutrophils; TM, thrombomodulin; ICAM, intercellular cell adhesion molecule; VCAM, vascular cell adhesion molecule; TNF, tumor necrosis factor, IL, interleukin.

Fig. 2

Reactive oxygen species pathways, antioxidant enzymes and their role in vascular oxidative stress. Superoxide is produced by several cellular enzyme systems including NADPH-oxidases, xanthine oxidase, etc. It can react with NO producing aggressive peroxynitrite anion ONOO⁻ and decreasing NO pool. Superoxide spontaneously or by action of superoxide dismutase may be reduced into hydrogen peroxide H₂O₂. Hydrogen peroxide can produce extremely reactive hydrogen radical •OH in the presence of transition metals or hypochlorous acid by myeloperoxidase. Catalase and glutathione peroxidases protect cells against hydrogen peroxide. ALI/ARDS, acute lung injury/acute respiratory distress syndrome; COX, cyclooxygenase; GSHPx, glutathione peroxidases; MPO, myeloperoxidase; ROS, reactive oxygen species; SOD, superoxide dismutase; XO, xanthine oxidase.

Fig. 3

Protective effects of targeted formulations of AOEs in models of oxidative stress in vitro and in vivo. Catalase and SOD were conjugated to antibodies against endothelial target. AngII, angiotensin II; GOX, glucose oxidase; LPS, lipopolysaccharide; PQ, paraquat; SOD, superoxide dismutase.