Introduction The success of a therapeutic intervention ultimately depends on the ability of the drug molecule to reach its predetermined target inside the body at relevant concentrations. Clearance and active metabolism limit the population and circulation life times of the drug molecules in the blood stream while the physical barrier of the endothelium limits their transport to the organ cells or tissue. In the case of high toxicity drug constructs, specificity in binding to the predetermined target is an additional constraint. A strategy to overcome these limitations would be to conjugate the drug molecule to a chaperone that has high circulation life times on account of slow clearance and metabolism, has predetermined transport pathways for crossing the endothelium out of and back into the circulation and has some specificity in terms of access to distinct compartments in the body. An obvious choice, and with some precedents, would be the use of physiological transport proteins. Multicellular organisms routinely use proteins to transport molecular cargo between cells, and eventually across the vasculature, via a well regulated, highly vectored process of transcytosis. We focused attention on three such proteins, previously implicated in transcytosis, to study their characteristics as chaperones for carrying drug molecules in circulation: human serum albumin (HSA), transferrin (Tf) and immunoglobulin complex type G (IgG). Initial studies were geared towards understanding the molecular transport of these proteins across the vascular endothelium.

Methods The endothelium was modeled in an in vitro system as a monolayer of primary human aortic endothelial cells (HAECs). Cells were obtained from the aorta of healthy individuals and were cultured through 2 passages post receipt. 100,000 cells were seeded in 250 µL of endothelial growth media on 6.5 mm diameter TranswellŽ filter supports. Integrity of monolayers was continuously monitored by transendothelial electrical resistance (TEER) measurements. Monolayers were considered to be tightly formed and fully functional at TEER numbers of 40-50 -cm2. The cell monolayers were incubated with the test protein and fluxes were measured in both: apical to basolateral (AB) and basolateral to apical (BA) directions. Flux (mg/s) was converted into permeability (cm/s) by normalizing with concentration of the test protein (mg/ml) and the area of diffusion (cm2). For each protein, permeability was measured over a range of concentrations, in media and buffer, and at 37 °C and 4 °C.

Results At 4 °C, a drop in the HSA permeability numbers from those measured at 37 °C points toward a receptor mediated transport process consistent with transcytosis of HSA. The permeability of HSA across HAEC monolayers was concentration dependent at low concentrations. Permeability decreased with increasing HSA concentration and eventually reached a plateau (1E-6 cm/s) at physiologically relevant concentrations (35-50 mg/ml) in agreement with previous reports. BA transport of HSA showed a similar profile but was 2-3 folds lower in magnitude as compared to AB transport indicating specificity in the direction of HSA transport. Permeability numbers measured in buffer were seen to be higher as compared to those measured in media at all concentrations. IgG transport in the AB direction showed weak concentration dependence. Permeability increased gradually with concentration and reached a plateau (5E-7 cm/s) at physiologically relevant concentrations (15-20 mg/ml). BA permeability of IgG on the other hand showed very strong concentration dependence and was 2-10 times higher as compared to AB permeability. BA permeability rapidly decreased with increasing concentration and approached permeability numbers in the AB direction at physiologically relevant concentrations. Tf permeability was measured in the AB direction and showed weak concentration dependence. Permeability increased marginally with increasing concentration before reaching a plateau (4E-7 cm/s) at physiologically relevant concentrations (8-10 mg/ml).

Conclusion The different proteins studied exhibit different transport characteristics across the endothelium depending upon their physiological function and bio-chemical attributes. Quantitative transport measurements such as those described above will be useful in constructing a theoretical framework to explain transport of model proteins across cells and membranes. Such a theoretical framework will not only provide insights into transcytosis as an active transport process but will also help in designing drug conjugates using these proteins as chaperones. Future work will focus on experimental elucidation of the various steps in the transcytosis process starting from receptor binding at the cell surface, cytoplasmic transport inside the cell to final exocytosis at the opposite membrane. In addition, effect of drug conjugation on transport properties of proteins will be assessed via similar transport measurements.

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