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The blood-brain barrier (BBB) remains a significant obstacle to the delivery of therapeutic agents into the central nervous system (CNS). Primary cell cultures of brain capillary endothelial cells represent the closest possible phenotype to the in vivo BBB cell providing a convenient model for the study of transport systems and events that mediate solute delivery to the CNS. In this investigation we have characterized an in vitro primary BBB model from porcine brain microvascular endothelial capillary (PBMVEC) cells after recovery from cryopreservation of upto 12 months and studied their modulation by astrocytes. Co-cultures of PBMVECs with astrocytes (C6 astroglioma) resulted in trans-endothelial electrical resistance of up to approximately 900Omega cm2 and marked discrimination between the para- and trans- cellular markers sucrose and propranolol. Micrographs of confluent monolayers of PBMVECs showed the presence of tight junction complexes and vesicles with the morphological characteristics of either caveolae or clathrin coated pits. Extensive RT-PCR evaluation highlighted the expression of tight junction transcripts, ABC transporters, leptin receptor and select nutrient transporters. Functional studies examined the kinetics of transport of glucose, large neutral amino acids and p-glycoprotein (P-gp). Our findings indicate primary PBMVECs retain many barrier characteristics and transport pathways of the in vivo BBB. Further, primary cells can be stored as frozen stocks which can be thawed and cultured without phenotypic drift many months after isolation. Frozen PBMVECs therefore serve as a robust and convenient in vitro cell culture tool for research programs involving CNS drug delivery and targeting and in studies addressing blood-brain barrier transport mechanisms.

The blood-brain barrier (BBB) protects the central nervous system (CNS) from potentially harmful xenobiotics and endogenous molecules. Anatomically, it comprises the brain microvasculature whose functionality is nevertheless influenced by associated astrocyte, pericyte and neuronal cells. The highly restrictive paracellular pathway within brain microvasculature restricts significant CNS penetration to only those drugs whose physicochemical properties afford ready penetration into hydrophobic cell membranes or are capable of exploiting endogenous active transport processes such as solute carriers or endocytosis pathways. Endocytosis at the BBB is an essential pathway by which the brain obtains its nutrients and affords communication with the periphery. The development of strategies to exploit these endocytic pathways for the purposes of drug delivery to the CNS is still an immature field although some impressive results have been documented with the targeting of particular receptors. This current article initially provides an overview of general endocytosis processes and pathways showing evidence of their functional existence within the BBB. Subsequent sections provide, in an entity-specific manner, comprehensive reviews on BBB transport investigations of endocytosis involving: transferrin and the targeting of the transferrin receptor; hormones; cytokines; cell penetrating peptides; microorganisms and toxins, and nanoparticles aimed at more effectively delivering drugs to the CNS.

Multidrug resistance mediated by overexpression of P-glycoprotein (P-gp) is a major obstacle in the chemotherapeutic management of cancer. The objectives of the current work were to examine if fatty acids affect the intracellular transport and dynamics of doxorubicin in drug-resistant cancer cell lines, and to assess if such effects were mediated through modulation of P-gp efflux pump activity. Among the range of fatty acids tested in this study, eicosapentaenoic acid diester (EPADI) increased doxorubicin accumulation [A] to 137% and retention [R] to 212% in doxorubicin-resistant MCF-7/ADR breast carcinoma cells, and [A] to 147% and [R] to 163% in vinblastine-resistant KBVI nasopharyngeal carcinoma cells. Consistent with EPADI-induced increases in intracellular doxorubicin concentrations, EPADI (10 microg/ml) sensitized MCF-7/ADR cells to the cytotoxic effects of doxorubicin (1 microg/ml) as assessed by MTT assay (viability < 50% of control), while EPADI itself displayed no cytotoxicity. The combination of EPADI (10 microg/ml) with verapamil (1 microM) resulted in a considerable increase in the [A] and [R] of the model P-gp substrate rhodamine-123 within drug-resistant cells compared to when either agent were used alone. KBV1 cells treated with combination of EPADI (10 microg/ml) and verapamil (1 microM) achieved 160% and 1120% greater [A] and [R] of rhodamine-123, respectively, compared to untreated cells. The P-gp modulatory effects of EPADI either alone, or as part of a combination with more potent inhibitors, should be further investigated.