Two strains of Madin Darby canine kidney (MDCK) cells were grown on a polycarbonate membrane with 3-micron pores without any extracellular matrix treatment. The membrane, 2.45 cm in diameter, which is part of a commercially obtained presterilized culture insert, provides two chambers when placed in a regular six-well culture plate. This device was found to be convenient for investigating transport of a few selected fluid-phase markers across the MDCK cell monolayer. Both the strain from the American Type Culture Collection (ATCC) and the so-called highly resistant strain I, at a serial passage between 65 and 70, showed a seeding concentration-dependent lag phase followed by a growth phase with a 21-hr doubling time. When seeded at 5 x 10(4) cells/cm2, cell confluence was achieved in 5 days in a modified Eagle's minimum essential medium (MEM) containing 10% fetal bovine serum under a 5% CO2 atmosphere. Similarly, transepithelial electrical resistance (TEER) also reached a plateau value in 5 days. Both light and electron microscopic examinations revealed well-defined junctional structures. Transport of the fluid-phase markers, sucrose, lucifer yellow CH (LY), inulin, and dextran across the MDCK cell monolayers was studied primarily at 37 degrees C following the apical-to-basolateral as well as the basolateral-to-apical direction. Large variations in the steady-state transport rate were observed for a given marker between the cell layer preparations. Thus, the present study proposes an "internal standard" procedure for meaningful comparisons of the transport rate. When normalized to the rate of sucrose, the rate ratio was 1.00:0.80:0.67:0.15 for sucrose:LY:inulin:dextran.(ABSTRACT TRUNCATED AT 250 WORDS)

Transport of 14C-labeled acetic, propionic (PA), butyric, valeric, heptanoic (HA), and octanoic (OA) acids across the Madin Darby canine kidney (MDCK) epithelial cell monolayer grown on a porous polycarbonate membrane was studied in Hanks' balanced salt solution (HBSS) at 37 degrees C in both apical-to-basolateral and basolateral-to-apical directions. At micromolar concentrations of solutes, metabolic decomposition was significant as evidenced by [14C]CO2 production during the OA transport. The apparent permeability (Pe) indicates that as lipophilicity increases, diffusion across the "unstirred" boundary layer becomes rate limiting. In support of this notion, transport of OA and HA was enhanced by agitation, showed an activation energy of 3.7 kcal/mol for OA, and resulted in identical Pe values for both transport directions. Analysis of Pe changes with varying alkyl chain length resulted in a delta G of -0.68 +/- 0.09 kcal/mol for -CH2-group transfer from an aqueous phase to the MDCK cells. When the intercellular tight junctions were opened by the divalent chelator EGTA in Ca2+/Mg2(+)-free HBSS, transport of the fluid-phase marker Lucifer yellow greatly increased because of paracellular leakage. PA transport also showed a significant increase, but OA transport was independent of EGTA. Although albumin also undergoes paracellular transport in the presence of EGTA and OA binds strongly to albumin, OA transport in EGTA solution was unchanged by albumin. These observations indicate that transmembrane transport is the major mechanism for lipophilic substances. The present study, together with earlier work on the transport of polar substances, shows that the MDCK cell monolayer is an excellent model of the transepithelial transport barrier.

In assessing the feasibility of utilizing the phagocytic activity of polymorphonuclear leukocytes (PMNs) for a more efficient drug delivery to the cell, the uptake of the fluid-phase marker lucifer yellow CH (LY) at 37 degrees C by human PMNs from LY-containing liposomes was compared with that from solutions. In the presence of 10% autologous serum, the LY uptake at 37 degrees C via phagocytosis of a given PMN source when the concentrations of PMN, LY, and total lipid were in the range of 10(7) cells/ml, 0.5 mg/ml, and 50 mumol/ml, respectively. As expected, the LY uptake via phagocytosis was critically dependent upon the LY entrapment efficiency in the liposome preparation. Interestingly, little LY uptake was found when the serum was heat inactivated (56 degrees C x 30 min). The serum effect was upon liposome vesicles rather than upon the cells. The present study demonstrates that the use of particular drug carriers for targeted drug delivery to PMNs and possible to an extravascular site mediated by the cell infiltration is a viable approach.

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Targeted drug delivery to peripheral blood neutrophils (PMNs) should be of therapeutic potential in various disease states. In addition, substances taken up by PMNs in the circulation may be delivered to an extravascular site via the naturally occurring cell infiltration. The present study employs an in vitro chemotaxis model to test whether particulate drug carriers such as liposomes can be transported across a cellular barrier by migrating PMNs. The system contained 10(7) human PMNs/ml, 0.3-micron liposomes at a total lipid concentration of 2.5 mM, and 10% autologous human serum in the apical side of a confluent Madin Darby canine kidney (MDCK) epithelial cell monolayer of 4.71 cm2. The MDCK cells were grown on a polycarbonate membrane with 3-micron pores without any extracellular matrix, and 10(-7) M f-Met-Leu-Phe was added to the basolateral side as a trigger of chemotaxis. The aqueous phase of the reverse-phase evaporation vesicles (REVs) contained lucifer yellow CH (LY) and [14C]sucrose. The lipid bilayer of the REVs was spiked with [3H]dipalmitoylphosphatidylcholine (DPPC). Transmission electron micrographs showed that, in response to the formyl peptide, PMNs adhered to the apical surface of MDCK cells, emigrated across the MDCK cell layer, passed through the 3-micron pores in the polycarbonate membrane, and finally, appeared in the bottom well. Epifluorescence micrographs showed that most, if not all, of the migrated PMNs contained punctate fluorescence derived from LY. Transport data over a 3.5-hr period indicated that those markers that appeared in the basal side were indeed transported by phagocytosis of REVs by PMNs and that intact serum was an essential component in the process.(ABSTRACT TRUNCATED AT 250 WORDS)