Carver for writing assistance. targeting caveolae may provide a tissue-specific pathway for overcoming key cell barriers to many drug and gene therapiesin vivo. Molecular medicine has discovered many new therapeutic modalities by using state-of-the-art techniques in molecular biology. High through-put,in vitroassays that screen for pharmacological actions on the desired cell type are frequently used to design new drugs. Although such agents are certainly justified by their successin vitro, they frequently perform much less effectivelyin vivowhere the agent must reach its target cells in a tissue in sufficient quantities to be potent while sparing bystander organs (1). Depending on the route of administration, the endothelium and/or epithelium form significant barriers that greatly limit thein vivoaccessibility of many drugs, antibodies, and gene vectors to their intended target sites of pharmacological action, namely, the cells inside the tissue (14). For example, poor tissue penetration has hindered many monoclonal antibodies from reaching their cell-specific antigens to achieve effective tissue- or cell-directed pharmaco-deliveryin vivo(1,36). Moving the target from the tissue cell surface to the surface of vascular endothelium has theoretical advantages in tissue-specific delivery (710). This vascular targeting strategy still requires the identification of a tissue-specific target on endothelium, validation of expected deliveryin vivoand, to be useful for Glyburide many therapies, a means by which to enter and even cross the vascular wall for access to underlying tissue cells (7). The microvascular endothelium in most organs acts as a significant barrier to the free passage of bloodborne molecules and cells to the underlying interstitium and tissue cells (8,11). Specific transport mechanisms are expected to exist for the transendothelial transport of essential circulating blood macromolecules to the subendothelial space to meet the metabolic needs of the surrounding tissue cells (8). Continuous endothelium contains distinct flask-shaped invaginations in the plasma membrane called caveolae that are open to the luminal blood vessel space where circulating molecules may enter them (1214). These caveolae may provide a PKCC trafficking pathway for macromolecules into and possibly across cells (7,8,1114). Based on morphological studies showing few plasmalemmal vesicles existing free and unattached to other membranes inside the cell, some investigators have concluded that caveolae are not dynamic but rather static structures (1518). Yet, caveolae can bud from the plasma membrane via a dynamin-mediated, GTP-dependent fission process (19,20), and they contain key functional docking and fusion proteins (2024). Glyburide Whether caveolae can traffic their cargo across cells (transcytosis) remains unproven, primarily because comparative analysis has not been possible by using probes capable of targeting caveolae with high affinity and specificityin vivovs. physically identical, nontargeting control probes. The utility of caveolae in overcoming cell barriers to facilitate efficient pharmacodeliveryin vivois unknown. The molecular composition of caveolae, including possible tissue-specific differences, is unknown. == Methods == == Antibody Production. == Monoclonal antibodies were generated by standard somatic cell hybridization using 100 g of silica-coated luminal endothelial cell plasma membranes (P) as an immunogen and were screened by ELISA with P adsorbed onto 96-well trays. == In VivoBiodistribution Studies. == IgG was purified by Protein G chromatography (Pierce) and radiolabeled with125I using Iodogen (25). The rat tail vein was injected with 10 g of125I-IgG in 500 l of rat serum albumin (10 mg/ml). After 30 min, the rats were anesthetized for thoracotomy, blood sampling by cardiac puncture, and organ removal. Tissues were weighed before counting radioactivity to determine antibody per g of tissue. In initial studies,51Cr-labeled red blood cells were injected to determine Glyburide actual tissue uptake by subtracting tissue blood volumes. For TX3.833, this correction was negligible, and the practice was discontinued. == Antibody-Au Conjugates. == A monodispersed solution of colloidal gold (average diameter = 6 nm) (EM Science) was adjusted to pH 9.2 with K2CO3before adding purified IgG and stirring rapidly for 30 min. Polyethylene glycol (Mr20,000) was added to a concentration of 0.5 mg/ml for the last 5 min. After centrifugation at 105,000 gfor 1 h at 4C, the loose pellets were collected and resuspended in 5 mM phosphate before dialysis against 50 mM Tris during which NaCl was added slowly to a concentration of 150 mM. The conjugates were used within 48 h of preparation. == Tracking Antibody-Au Complexes Perfusedin Situandin Vivo. == For thein situexperiments, the cranial lobe of the.
