Targeting endothelium and its dynamic caveolae for tissue-specific transcytosis in vivo: a pathway to overcome cell barriers to drug and gene delivery

Abstract

Site-directed pharmacodelivery is a desirable but elusive goal. Endothelium and epithelium create formidable barriers to endogenous molecules as well as targeted therapies in vivo. Caveolae provide a possible, yet unproven, transcellular pathway to overcome such barriers. By using an antibody- and subfractionation-based strategy, we generated a monoclonal antibody specific for lung caveolae (TX3.833) that targets rat lungs after i.v. injection (up to 89% of dose in 30 min). Unlike control antibodies (nonbinding or to lipid rafts), TX3.833 targets lung caveolae that bud to form free vesicles for selective and quantal transendothelial transport to underlying tissue cells in vivo. Rapid sequential transcytosis can occur to the alveolar air space via epithelial caveolae. Conjugation to TX3.833 increases drug delivery to the lung up to 172-fold and achieves rapid, localized bioefficacy. We conclude that: (i) molecular heterogeneity of the endothelium and its caveolae permits vascular targeting to achieve theoretical expectations of tissue-specific delivery and bioefficacy; (ii) caveolae can mediate selective transcytosis in vivo; and (iii) targeting caveolae may provide a tissue-specific pathway for overcoming key cell barriers to many drug and gene therapies in vivo.

Figure 1

Characterization of TX3.833. (A) Proteins (10 μg each lane) from the indicated H and P were subjected to Western analysis by using antibodies to the indicated proteins as in our past work (22, 27). (B) Proteins (5 μg) from rat lung H, P, V, and silica-coated plasma membranes stripped of caveolae (P-V) were immunoblotted by using TX3.833 and monoclonal antibodies to 5′NT and caveolin-1. (C) Ultra-thin cryo-sections of rat lung tissue were labeled with TX3.833 followed by reporter antibody conjugated to gold particles and were examined by EM. Arrowheads indicate endothelial caveolae. (Bar = 90 nm.)

Figure 2

In vivo biodistribution of TX3.833. (A) Blood levels and tissue uptake of ¹²⁵I-labeled TX3.833 and control IgG₁ (10 μg) were determined 30 min after i.v. injection. The mean value in μg IgG/g of tissue or blood is plotted with SD bars (n ≥ 3). (B) Rats were injected with 1 mg of unlabeled TX3.833 or control IgG 30 min before ¹²⁵I-TX3.833 (10 μg) and assessed for radioactivity in the lung and blood, which is expressed as a percentage of signal without unlabeled antibody.

Figure 3

Caveolar targeting and trafficking of TX3.833-Au in situ. The rat lung was perfused in situ with TX3.833-Au for 2.5 min (A), 5 min (B), 10 min (C–E), and 15 min (F and G), and processed for EM. Int, interstitial space; cp, coated pits; bvl, blood vessel lumen; pvs, perivascular space; mvb, multivesicular bodies; endo, endothelium; epi, epithelium; alv, alveolar space. [Bar = 90 nm (A), 128 nm (B), 116 nm (C and D), 124 nm (E), 90 nm (F), 85 nm (G).]

Figure 4

Analysis of the binding and transport of TX3.833-Au vs. IgG-Au in rat lung. Comparative morphometric analysis was performed on the electron micrographs of rat lungs perfused with TX3.833-Au or mouse IgG-Au (see Methods) to quantify over time the number of gold particles (A) in lung microvascular endothelial caveolae per unit membrane and (B) within the interstitial space per unit area (μm²).

Figure 5

TX3.833 targets dynamic caveolae in vivo. (A) Rat lungs were perfused for 5 min with 1 mg of biotinylated TX3.833 before flushing and silica perfusion for isolation of P that was subjected to the caveolae budding assay with or without GTP (20, 22). The budded caveolae (V_b), isolated by flotation away from the repelleted membrane (P-V_b), were immunoblotted with either streptavidin or the indicated antibodies. (B) After tail vein injection (15 min), TX3.833-Au is seen selectively in caveolae throughout the endothelial cell, consistent with transcytosis by the caveolae trafficking system. Arrowheads indicate a caveola or group of caveolae with gold particles inside. Arrows show gold particles exiting abluminal caveolae. Bvl, blood vessel lumen; int, interstitial space. (Bar = 91 nm.)

Figure 6

Drug bio-efficacy studies in vivo. Hematoxylin and eosin staining of rat lungs 24 h after treatment with (A) control antibody-dgRA or (B) TX3.833-dgRA. Note the evident tissue damage with gross change in tissue morphology, infiltration of blood cells into tissue, edema, and septal thickening. (Bar = 1.3 μM.)