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The medical utility of proteins, e.g. therapeutic enzymes, is greatly restricted by their labile nature and inadequate delivery. Most therapeutic enzymes do not accumulate in their targets and are inactivated by proteases. Targeting of enzymes encapsulated into substrate-permeable polymer nano-carriers (PNC) impermeable for proteases might overcome these limitations. To test this hypothesis, we designed endothelial targeted PNC loaded with catalase, an H(2)O(2)-detoxifying enzyme, and tested if this approach protects against vascular oxidative stress, a pathological process implicated in ischemia-reperfusion and other disease conditions. Encapsulation of catalase (MW 247 kD), peroxidase (MW 42 kD) and xanthine oxidase (XO, MW 300 kD) into approximately 300 nm diameter PNC composed of co-polymers of polyethylene glycol and poly-lactic/poly-glycolic acid (PEG-PLGA) was in the range approximately 10% for all enzymes. PNC/catalase and PNC/peroxidase were protected from external proteolysis and exerted enzymatic activity on their PNC diffusible substrates, H(2)O(2) and ortho-phenylendiamine, whereas activity of encapsulated XO was negligible due to polymer impermeability to the substrate. PNC targeted to platelet-endothelial cell (EC) adhesion molecule-1 delivered active encapsulated catalase to ECs and protected the endothelium against oxidative stress in cell culture and animal studies. Vascular targeting of PNC-loaded detoxifying enzymes may find wide medical applications including management of oxidative stress and other toxicities.

Vascular immunotargeting to Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM) facilitates drug delivery to endothelium. We used human PECAM-transfected REN cells (REN/PECAM) as a model to compare targeting of antioxidant enzyme catalase conjugated with PECAM antibody (anti-PECAM/catalase) with adenoviral catalase delivery. Anti-PECAM/(125)I-catalase bound to REN/PECAM, but not to REN cells (70 vs. 1 ng/well vs. < 2 ng/well of unmodified catalase). At a virus-to-cell ratio of 1, elevated levels of catalase protein were detected by immunoblotting after adenoviral transfection of REN/PECAM and REN cells alike; H(2)O(2)-degrading activity of cell lysates was elevated at ratios of 10 and higher. REN/PECAM cells internalize 66% of cell-bound anti-PECAM/(125)I-catalase. Confocal microscopy localized anti-PECAM/catalase to intracellular vesicles, while catalase expressed by adenovirus was distributed in vesicles and throughout the cytosol. Within 15 min of delivery, anti-PECAM/catalase augmented H(2)O(2)-degrading activity and survival of H(2)O(2)-exposed REN/PECAM cells. The effects of conjugate delivery reached a plateau within 1 h and declined to the basal level within 12 h. In contrast, adenoviral delivery required several hours for transduction and development of the effects, but permitted much longer duration of protection (at least 48 h). Simultaneous exposure of REN/PECAM cells to anti-PECAM/catalase and catalase-encoding adenovirus afforded protection against H(2)O(2) with a rapid onset and a prolonged duration. Therefore, PECAM-directed immunotargeting provides a specific, antigen-directed intracellular delivery of catalase that affords a rapid but transient protection against H(2)O(2) and may complement gene delivery strategies for antioxidant protection.

In mouse 3T3-L1 preadipocytes, the glucocorticoid dexamethasone has been shown to promote a switch in beta-adrenoceptor subtype expression from beta 1 to beta 2 and to increase the total number of beta-adrenoceptors. The present study demonstrates that sodium butyrate also modulates beta-adrenoceptor expression in these cells. Incubation of preadipocytes with 2-10 mM butyrate for 24-48 h promoted a dose- and time-dependent switch in beta-adrenoceptor subtype from a near equal mixture of beta 1 and beta 2 to greater than 85% beta 2 and caused an approximate doubling of the receptor number. beta-Adrenoceptors were assayed in membranes prepared from 3T3-L1 cells using the radiolabeled antagonist [125I]iodocyanopindolol and the beta 2-selective antagonist ICI 118.551. Other short chain acids were not as effective as butyrate in promoting changes in beta-adrenoceptor expression. Cycloheximide (1.0 microgram/ml) inhibited the effects of butyrate on both beta-adrenoceptor subtype and number. Alterations in beta-adrenoceptor phenotype promoted by either butyrate or dexamethasone were functionally correlated with cAMP accumulation in these cells. Comparison of the effects of butyrate and dexamethasone on beta-adrenoceptor expression suggests that these two agents regulate beta-adrenoceptors by different mechanisms.