Adsorptive-mediated Transcytosis Research Articles

Structure and function of the blood-brain barrier

Structure and function of the blood-brain barrier

CNS delivery via adsorptive transcytosis.

Adsorptive-mediated transcytosis (AMT) provides a means for brain delivery of medicines across the blood-brain barrier (BBB). The BBB is readily equipped for the AMT process: it provides both the potential for binding and uptake of cationic molecules to the luminal surface of endothelial cells, and then for exocytosis at the abluminal surface. The transcytotic pathways present at the BBB and its morphological and enzymatic properties provide the means for movement of the molecules through the endothelial cytoplasm. AMT-based drug delivery to the brain was performed using cationic proteins and cell-penetrating peptides (CPPs). Protein cationization using either synthetic or natural polyamines is discussed and some examples of diamine/polyamine modified proteins that cross BBB are described. Two main families of CPPs belonging to the Tat-derived peptides and Syn-B vectors have been extensively used in CPP vector-mediated strategies allowing delivery of a large variety of small molecules as well as proteins across cell membranes in vitro and the BBB in vivo. CPP strategy suffers from several limitations such as toxicity and immunogenicity--like the cationization strategy--as well as the instability of peptide vectors in biological media. The review concludes by stressing the need to improve the understanding of AMT mechanisms at BBB and the effectiveness of cationized proteins and CPP-vectorized proteins as neurotherapeutics.

Blood-brain barrier transport of 125I-labeled basic fibroblast growth factor.

This study was carried out to examine the blood-brain barrier (BBB) transport of human basic fibroblast growth factor (bFGF) and investigate its mechanism. The BBB transport of 125I-bFGF was measured by several in vivo methods including intravenous administration, in situ internal carotid artery perfusion, and intracerebral microinjection. The in vitro binding of 125I-bFGF was characterized using freshly prepared bovine brain capillaries. The distribution volume of 125I-bFGF in the postvascular supernatant increased with the perfusion time, and exceeded the space occupied by the brain microvasculature and its trichloroacetic acid (TCA) precipitability was more than 90%. 125I-bFGF avidly bound to isolated bovine brain capillaries with a Bmax of 206 +/- 48 pmol/mg protein, and a Kd of 36.5 +/- 15.7 nM. This binding was significantly inhibited by unlabeled bFGF and heparin in a concentration-dependent manner. The cationic peptides, protamine and poly-L-lysine (each 300 microM), produced over 85% inhibition of 125I-bFGF binding to brain capillaries. Furthermore, glycosaminoglycans with a sulfate residue, chondroitin sulfate B and C (each 10 microg/mL) also inhibited the binding of 125I-bFGF The in vivo transcytosis of 125I-bFGF from the luminal side to the brain was also inhibited by the presence of heparin (10 microg/mL) and poly-L-lysine (300 microM), whereas neither hyaruronic acid (10 microg/mL) nor insulin (10 microM) had any effect. In addition to these results, the brain efflux index method was used to confirm that the transcytosis of 125I-bFGF from brain to blood across the BBB was negligible. These results suggest that 125I-bFGF is transported across the BBB, possibly by an adsorptive-mediated transcytosis mechanism that is triggered by binding to negatively charged species on the luminal membrane surface of the brain microvasculature, such as heparan sulfate proteoglycans.

Transport of Cationized Anti‐Tetanus Fab'2 Fragments Across an In Vitro Blood‐Brain Barrier Model: Involvement of the Transcytosis Pathway

Tetanus neurotoxin reaches the CNS by axonal retrograde transport and thus becomes inaccessible to current treatments. A possible strategy to improve current therapy for tetanus disease would be the vectorization of Fab'2 fragments, allowing their delivery into the CNS. The purpose of this study was to investigate whether after cationization anti-tetanus Fab'2 fragments are able to cross the blood-brain barrier, the first obstacle to CNS delivery. We used primary cocultures of bovine brain capillary endothelial cells and newborn rat astrocytes as an in vitro model to study the binding and transport of cationized Fab'2 (cFab'2) fragments across the brain endothelium. We first show that cationization does not alter Fab'2 affinity for tetanus toxin. Then we demonstrate that after cationization Fab'2 fragments are able to bind to the negative charges on the surface of endothelial cells and subsequently to be transported across the endothelial cell monolayer without any modification of affinity. Finally, using fluorescence microscopy, we show that cFab'2 fragments are transported through endocytotic vesicles. The present study demonstrates that cationization allows Fab'2 directed against tetanus toxin to be transported through brain endothelium by adsorptive-mediated transcytosis. We suggest that this vectorization way could be a promising delivery strategy for carrying anti-tetanic immunoglobulin fragments across the blood-brain barrier to improve tetanus treatment.

Intestinal absorption of fluorescence-derivatized cationic peptide 001-C8-NBD via adsorptive-mediated transcytosis

The intestinal absorption of an intact oligopeptide was investigated in rats using a synthetic cationic peptide, 001-C8 (H-MeTyr-Arg-MeArg- d-Leu-NH(CH 2) 8NH 2). The peptide was coupled with 4-nitrobenzo-2-oxa-1,3-diazole (NBD) to prepare a fluorescence-labeled derivative 001-C8-NBD (H-MeTyr-Arg-MeArg- d-Leu-NH(CH 2) 8NH-NBD) for the purpose of quantification. The degradation half-life of 001-C8-NBD in jejunal homogenate (1 mg/mL) was 99.5 min, which was significantly longer than that of natural leucine enkephalin (1.14 min). The absorption of 001-C8-NBD was evaluated by the vascular-perfusion method. Intact 001-C8-NBD appeared in the blood time-dependently and the absorption volume at 30 min (2.75 ± 0.14 μL/cm intestine) was significantly larger than that of [ 3H]PEG 900 (0.88 ± 0.13 μL/cm intestine), of which membrane permeability is very low. The absorption of 001-C8-NBD was greatly reduced by an adsorptive-mediated endocytosis inhibitor, protamine (10 mM). No inhibition of the absorption of [ 3H]PEG 900 by protamine was observed. The intestinal absorption was also measured by an in vivo loop method. The absorption clearance of 001-C8-NBD measured by this method (0.083 ± 0.008 μL/min/cm intestine) was comparable to that obtained by the vascular perfusion method (0.092 ± 0.005 μL/min/cm intestine). All of these data suggested that 001-C8-NBD was absorbed as the intact oligopeptide in the intestine in vivo. Adsorptive-mediated transcytosis is suggested to have enormous potential as an oral delivery system for peptide and/or protein drugs.

Adsorptive-mediated transcytosis of a synthetic basic peptide, 001-C8 in Caco-2 cells.

Adsorptive-mediated transcytosis of a synthetic basic peptide, 001-C8 in Caco-2 cells.

Macromolecular permeability across the blood-nerve and blood-brain barriers.

The permeability of insulin (Ins), nerve growth factor (NGF), albumin (Alb), transferrin (Trf), and IgG across the blood-nerve barrier (BNB) and blood-brain barrier (BBB) in normal adult rats was quantified by measuring the (permeability coefficient x surface area) product (PS) with the i.v. bolus-injection technique in the cannulated brachial vein and artery using radioiodinated proteins. The PS values of the BNB for IgG and Alb were low: 0.079 +/- 0.029 x 10(-6) and 0.101 +/- 0.088 x 10(-6) ml.g-1.s-1, (mean +/- SD, respectively). The PS values for NGF and Trf were 16.1-fold and 25.5-fold higher than for Alb. The PS for Ins across the BNB was 33.190 +/- 2.053 x 10(-6) ml.g-1.s-1--a remarkable 329-fold increase compared with Alb. The PS values of the BBB for IgG and Alb in different brain regions were all low, from 0.028 +/- 0.017 to 0.151 +/- 0.035 x 10(-6) ml.g-1.s-1 (mean +/- SD). NGF and Trf had comparable PS values from 13- to 32-fold higher than for Alb, except for the brain stem, where the PS for Trf was 66-fold higher than for Alb. The mean PS for Ins across the BBB ranged from 15.78 +/- 5.45 x 10(-6) ml.g-1.s-1 for the cortex to 22.62 +/- 7.50 x 10(-6) ml.g-1.s-1 for the brain stem--again a remarkable 105- to 390-fold increase relative to Alb. Because reliable PS measurements were obtained for all proteins tested, the BBB and BNB cannot be considered impermeable to proteins--a concept that has plagued brain- and nerve-barrier research. The low PS values for IgG and Alb indicate low rates of transfer; however, Alb, in particular, is the major protein of endoneurial and ventricular fluid, which suggests that these PS values may be significant. Ins had the highest PS values, which likely reflect the mechanism of transport across the barriers--that is, receptor-mediated transport. Because NGF and Trf had PS values 13- to 66-fold higher than for Alb, whether this reflects receptor-mediated uptake, adsorptive-mediated transcytosis, or some other mechanism is unclear. That the PS values for NGF and Trf differ from Alb and IgG clearly suggests, however, a different uptake mechanism. Finally, the remarkably high PS values for Ins across the BBB and BNB identify this protein and its putative receptor on capillary endothelial cells as a potential target for drug delivery into the central and peripheral nervous systems.