1 Pennington Biomedical Research Center, Louisiana State University System, 6400 Perkins Road, Baton Rouge, LA 70808, USA
2 BioMarin Pharmaceutical Incorporated, 371 Bel Marin Keys Boulevard, Suite 210, Novato, CA 94949, USA
3 Department of Pediatrics, and Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA
4 Department of Molecular Biopharmacy and Genetics, Tohoku University, 1-1 katahira 2-chome, Aoba-ku, Sendai, 980-8577, Japan
* Author for correspondence (e-mail: weihong.pan{at}pbrc.edu)
Accepted 24 June 2004
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Summary |
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Key words: Receptor-associated protein, Blood-brain barrier, Drug delivery, Megalin, LRP, Receptor-mediated transport
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Introduction |
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Megalin is expressed primarily in a subset of epithelial cell layers including those lining the renal proximal tubule, thyroid colloid, epididymis, alveolae, brain vasculature, and the ciliary body of the eye (Orlando and Farquhar, 1993; Zheng et al., 1994
). Of particular interest are the cerebral microvessel endothelial cells (specialized squamous epithelial cells) composing the blood-brain barrier (BBB). In at least three cases, megalin has been shown to transcytose ligands in the apical-to-basolateral direction across some of these epithelial cell layers. Marinò and colleagues have shown efficient megalin-dependent transcytosis of thyroglobulin across the thyroid epithelium (Marinò et al., 2003
) and megalin-dependent transcytosis of retinol binding protein across the renal proximal tubule epithelium (Marinò et al., 2001
). Transport of apoJ across the brain capillary endothelium in situ also was shown to involve megalin-dependent transcytosis (Zlokovic et al., 1996
; Shayo et al., 1997
). Another study has shown that the complex between RAP and megalin remains stable as far as the late endosome (Czekay et al., 1997
), an indication of pH stability that often signifies transcytotic competence.
Exogenously applied RAP can be endocytosed efficiently by all members of the LDL receptor family (Bu et al., 1994; Savonen et al., 1999
; Li et al., 2000
; Li et al., 2001b
). RAP has also been shown to be functional in both N- and C-terminal fusions with other proteins (personal communication, BioMarin Pharmaceutical). Thus, RAP has potential as a vehicle to bring other proteins into cells by receptor-mediated endocytosis. If RAP undergoes megalin-dependent transcytosis across the brain capillary endothelium as do thyroglobulin and retinol binding protein across other epithelial cells (Marinò et al., 2003
; Marinò et al., 2001
), and if the RAP fusion proteins maintain domain conformation and the ability to be transported across the BBB, RAP could serve as a vehicle for receptor-mediated transcytotic delivery of other proteins into the brain in vivo.
The BBB dynamically regulates the availability of proteins from blood to brain and spinal cord. In general, peptides and proteins in the periphery are excluded from substantial entry into the central nervous system. The ability to treat neurological disorders with powerful protein and peptide drugs is largely hindered by this effect. Therefore, vehicle-mediated delivery targeting brain endothelial antigens as transporters has been widely explored as a means of protein-based drug delivery. The transferrin receptor is highly expressed in brain microvessel endothelial cells, and transferrin may be transcytosed by this receptor or recycled back to the apical surface of the endothelial cells (Morris et al., 1992; Ueda et al., 1993
; Moos and Morgan, 2001
). Moreover, high concentrations of transferrin in the blood block efficient transcytosis of exogenously administered transferrin in vivo. The murine OX26 monoclonal antibody to the rat transferrin receptor has been used as a vehicle for the delivery of attached ligands to the brain (Skarlatos et al., 1995
; Broadwell et al., 1996
). The related ligand, human melanotransferrin, which shares 37% sequence homology with transferrin, has been shown to have a high rate of brain uptake and a high permeability constant in cultured bovine brain capillary endothelial cells (Demeule et al., 2002
). In this report, we describe studies to determine whether RAP crosses the BBB and how the rate at which this occurs compares with that of transferrin and melanotransferrin.
There are two main implications for the study of RAP transport across the BBB. First, the transport system could be enhanced to facilitate the transport of ligands such as RAP and its homologous proteins. An alternative approach is the use of a transport system with a ligand as a carrier protein. Therefore, it may be possible to attach the target drug to RAP to provide efficient transport into the brain.
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Materials and Methods |
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BBB permeability studies
Male CD1 mice, weighing 25-35 g (Charles River Laboratories), were anesthetized immediately before the study. For multiple time regression analysis (Kastin et al., 2001), the mouse received a bolus mixture of [125I]-RAP and [131I]-albumin (0.5-1 µCi/mouse in 100 µl of lactated Ringer's with 1% albumin, except when otherwise specified) through the left jugular vein. At designated times (1-30 minutes after injection), blood was collected from a cut in the right common carotid artery and the mouse was decapitated immediately. Brain and peripheral tissue samples were obtained, weighed, and assayed for radioactivity. Radioactivity in 50 µl of serum was also measured for calculation of the volume of distribution.
To correct for the decay of serum radioactivity over time, `exposure time' was calculated as the integral of serum radioactivity from time zero to time t divided by the radioactivity at time t. The unidirectional influx constant, Ki, expressed in µl/g-minute, and the apparent volume of distribution, Vi, in µl/g, were determined from the linear portion of the following equation:
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where Am is the amount of radioactivity in a tissue sample per unit mass (cpm/g), Cpt is the amount of radioactivity in 1 µl serum at time t (cpm/µl), and exposure time is measured by the term (Blasberg et al., 1983
; Patlak et al., 1983
).
The Ki values were compared by analysis of variance (ANOVA) to determine whether there were differences within groups. This was followed by a Newman-Keuls post-hoc test to determine which values within a group differed. The standard deviation of the mean for the slope was taken as the standard error of the mean and, because two means (the slope and the intercept) were calculated from the data, n-1 was used as the n value in the ANOVA and range tests. Statistically significant differences among groups were determined with the aid of the GraphPad Prism statistical program (Banks et al., 2003).
For in situ brain perfusion, the descending aorta was clamped and bilateral jugular veins were severed. After a minute of perfusion with oxygenated, modified Zlokovic's buffer (Zlokovic et al., 1990; Pan et al., 1998
), the mouse received [125I]-RAP and [99mTc]-albumin (0.5-1 µCi/ml each) at a perfusion rate of 2 ml/minute for designated times between 1 and 10 minutes. Mice were then perfused with buffer alone for another minute before decapitation. In some experiments, [125I]transferrin and [125I]-melanotransferrin were studied under conditions identical to [125I]-RAP.
Degradation assays
Acid precipitation was performed with the stock solution, perfusion buffer, serum and tissue homogenates. Brain and peripheral tissue were homogenized in 1 ml phosphate-buffered saline containing a protease inhibitor cocktail. The supernatant was precipitated with an equal volume of 30% trichloroacetic acid. Control samples to assess ex vivo degradation of radioactively labeled compounds during processing were prepared simultaneously by direct addition of [125I]RAP to the test tubes.
In vitro transport assays
Stably transfected MDCK cells and their control were originally obtained from Maria Paz Marzolo (Catholic University, Chile). The LRP1 mini-receptor constructs contain the fourth ligand-binding domain of human LRP1, followed by the transmembrane domain and either the complete cytosolic tail of LRP1 (mLRP/LRPTmT=LRPt) or the complete cytoplasmic tail of megalin (mLRP/LRPTmMegT= MEGt). The LRPt is distributed basolaterally as shown by indirect immunofluorescence with an anti-HA antibody, and MEGt localizes to the apical surface of the transfected MDCK cells (Marzolo et al., 2003). These cells were plated on the surface of polyacetate membrane inserts of the Transwell system (Costar, Cambridge, MA) with a uniform pore size of 0.4 µm. Cells were seeded at a density of 2x105 cells/ml and cultured in DMEM supplemented with 10% FBS. Medium was exchanged every three days. The cells were kept in a 5% CO2 incubator at 37°C. Transcytosis studies were performed in triplicate for either apical-to-basolateral or basolateral-to-apical transport, with or without inclusion of 2 µg/ml of excess unlabeled RAP.
Twenty minutes before the transport assay, the Transwell insert and its supporting endothelial cell monolayer were equilibrated in the transport buffer (Hank's balanced salt solution with 25 mM HEPES and 0.1% albumin) at 37°C. Transport was initiated by addition of [125I]-RAP (1 µCi/ml) and [99mTc]-albumin (2 µCi/ml) to the donor (upper or lower) chamber at time zero. The plate was kept at 37°C with gentle mixing at about 130 rpm during the entire procedure. At 5, 10, 15, 20, 30, 40, 50 and 60 minutes, a 10 µl aliquot of sample was collected from the acceptor (lower or upper) chamber of each well. At 60 minutes, solutions in both chambers were transferred to separate test tubes at 4°C. The radioactivity of [125I]-RAP and [99mTc]-albumin was measured simultaneously in a -counter with a dual-channel program. The amount of intact [125I]-RAP and [99mTc]-albumin after transport was measured by acid precipitation. HPLC analysis was performed on selected samples, with a linear gradient of 10-90% acetonitrile in 0.1% trifluoroacetic acid over 40 minutes with 1 ml fractions collected.
To determine the flux transfer constant, a linear regression analysis was performed for the acceptor/donor ratio of radioactivity (mean±s.e.m.) and time of transport by use of the GraphPad Prism program. The slope of the regression line represents the permeability-surface area product (PS, in cm3/second). The permeability coefficient P was obtained by dividing the PS product by the filter surface area (1 cm2 in the 12-well insert) and expressed in cm/second. The permeability coefficients among groups were compared.
All in vitro experiments were performed in triplicate. Group designs of the experiments are described in detail in the Results section. For experiments with repeated acquisition of data, statistical analysis was performed by use of SPSS with one-way ANOVA followed by Tukey's post-hoc test. For comparison of linear regression lines, analysis of covariance was performed with the aid of GraphPad Prism software.
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Results |
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To determine the influx transfer constant of [125I]-RAP after an iv bolus injection, four groups of mice were studied simultaneously. The amount of [125I]-RAP differed in the groups: 0.3, 0.6, 1.2, and 2.4 µCi/mouse, respectively. In each group, blood, brain, liver, kidney and muscle samples from eight mice were obtained at 1, 2, 5, 7, 10, 15, 20 and 30 minutes after injection (each mouse represented one time point). Disappearance of serum radioactivity fitted a one-phase exponential decay model and was not significantly different among the four groups. The mean serum half-life of RAP was 1.5±0.1 minutes (Fig. 1). In the kidney and liver, uptake of [125I]-RAP had a rapid distribution phase peaking at about 12 and 17 minutes, respectively, followed by a slower distribution phase. In the gluteus major muscle, the linear transfer constant was similar among groups with a mean of 3.1±0.2 µl/g-minute. In the brain, the linear influx transfer constant was not significantly different among groups and had a mean of 0.6±0.1 µl/g-minute (Fig. 2). The amount of [125I]-RAP in the brain at 30 minutes was 1%/g brain of the total [125I]-RAP delivered intravenously at 0.3 µCi/mouse. This, however, was reduced to 0.5% in groups receiving 0.6, 1.2, and 2.4 µCi/mouse of [125I]RAP without additional unlabeled RAP, suggesting possible saturability of the blood-brain transfer. In all groups, the vascular marker [99mTc]-albumin did not have significant entry. The results indicate that despite the first-pass effect and metabolism in the liver and kidneys, intact RAP had linear influx to a third peripheral organ (muscle) and also across the BBB to the brain.
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Interactions of RAP with peripheral blood before reaching the BBB
Excess non-radioactively labeled RAP, when injected intravenously with [125I]-RAP, did not significantly decrease the influx transfer constant of [125I]-RAP but rather enhanced it. However, 25 minutes after intravenous injection, the percentage of brain uptake of the injected dose of [125I]-RAP was decreased from 0.9%/g brain to 0.4% and 0.2% in mice receiving 5 µg/mouse and 200 µg/mouse of excess unlabeled RAP, respectively. This again suggested the possible presence of a saturable transport system at the BBB. The lack of a significant decrease of the influx transfer constant in the groups receiving excess RAP may be explained by peripheral binding, although preliminary studies with capillary electrophoresis did not show high-affinity binding of RAP with serum proteins. Nonetheless, RAP does have potential binding to blood cells and undergoes clearance by the liver and kidney (Warshawsky et al., 1993). Regardless, the degradation and kinetic transfer studies indicate that most [125I]-RAP in the circulation is available to interact with the BBB before being degraded in the periphery.
Influx of [125I]-RAP after in situ brain perfusion
To determine the direct interactions of RAP with the BBB, we used a blood-free perfusion system. The influx transfer constant of [125I]-RAP, determined from mice studied during 1-10 minutes of in situ brain perfusion was 6.2±1.3 µl/g-minute. By contrast, there was no significant influx of [125I]-melanotransferrin or [99mTc]-albumin in the same study (Fig. 3).
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The influx of [125I]-RAP into the right hyoglossus muscle was measured simultaneously during in situ brain perfusion as a positive control. The value was 10.8±1.8 µl/g-minute. By contrast, the influx rate of 99mTc-albumin to the same muscle was 2.7±1.5 µl/g-minute, whereas [125I]-melanotransferrin had no significant influx (Fig. 4).
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In addition to the comparison of RAP with melanotransferrin, transferrin was also studied in separate experiments. During a perfusion period of 1-5 minutes, [125I]-RAP entered both cortical and subcortical areas significantly faster than [125I]-transferrin (Fig. 5).
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The compartmental distribution of [125I]-RAP after in situ brain perfusion was determined by capillary depletion studies. After 5 minutes of in situ perfusion, the amount of radioactivity in the brain parenchyma was significantly higher than in the vasculature. As shown in Fig. 6, more than 70% of [125I]-RAP that had reached the brain compartment was present in the parenchyma. Moreover, addition of 5 µg/mouse of excess unlabeled RAP decreased the uptake of [125I]-RAP in parenchyma significantly [F(1,9)=5.7, P<0.05] without affecting that in the capillary fraction. This indicates the presence of a saturable transport system for RAP at the BBB. The uptake of [99mTc]-albumin was significantly lower in brain parenchyma than that of [125I]-RAP and was not decreased in the presence of excess non-radioactively labeled RAP. Collectively, these results indicate that the uptake of [125I]-RAP into brain parenchyma was a saturable process.
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Degradation of [125I]-RAP after intravenous injection and in situ brain perfusion
Acid precipitation of the supernatant of the brain homogenate, corrected for ex vivo degradation, showed that intact [125I]-RAP accounted for 72%, 73%, and 61% of the radioactivity in the brain at 2, 10 and 30 minutes after intravenous injection, respectively. This is consistent with HPLC results. Similarly, intact [125I]-RAP accounted for 89% and 88% of total radioactivity at 1 and 10 minutes after in situ brain perfusion, respectively. Because of the lack of substantial degradation of [125I]-RAP in either blood or perfusion buffer during the multiple-time regression study, the influx transfer constants measured probably reflect entry of [125I]-RAP rather than free iodine or RAP degradation products.
Involvement of megalin in the transcytosis of [125I]-RAP
To identify the receptor system responsible for delivering RAP across the BBB, apical-to-basolateral and basolateral-to-apical transport of [125I]-RAP was studied in three types of MDCK cells: (a) non-transfected MDCK cells; (b) MDCK cells stably transfected with an LRP domain IV mini-receptor (LRPt); and (c) MDCK cells stably transfected with a chimeric mini-receptor consisting of LRP1 extracellular domain IV and the megalin tail (MEGt). Our previous studies have shown that LRPt mimics the endogenous LRP and distributes basolaterally. MEGt, however, resembles endogenous megalin and localizes to the apical membrane (Marzolo et al., 2003). The transepithelial electrical resistance (TEER) of the confluent monolayer, an indicator of the tightness of the barrier, was 757±35
cm2 (mean±s.e.m.) for native MDCK, 364±22
cm2 for LRPt transfected MDCK, and 370±29
cm2 for MEGt transfected MDCK.
The transcytosis assays were initiated by simultaneous addition of [125I]-RAP and the paracellular permeability marker [99mTc]-albumin at time zero. At the end of the study (60 minutes), intact [125I]-RAP accounted for 99% of the acid precipitable radioactivity in the donor chamber and 91% of that in the acceptor chamber. This indicates that the majority of radioactivity measured represented intact [125I]-RAP. Extension of the study period to 120 minutes did not change the percentage of intact [125I]-RAP or the flux rate. For apical-to-basolateral flux in non-transfected MDCK cells, the permeability coefficient of [125I]-RAP after transport for 60 minutes was 5.1±0.8x10-6 cm/second. This is probably explained by low-level expression of native megalin by MDCK. By contrast, in MDCK cells transfected with MEGt, the permeability coefficient of [125I]-RAP was increased to 18.1±1.2x10-6 cm/second, significantly higher than the control [F(1,11)=77.9, P<0.001]. No significant flux was observed in MDCK cells transfected with LRPt, consistent with the basolateral localization pattern of this receptor (Fig. 7). In all groups, [99mTc]-albumin had no significant flux.
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Addition of excess non-radioactively labeled RAP at 2 µg/ml significantly decreased the permeability coefficient of [125I]-RAP in MEGt-transfected cells (6.3±0.4x10-6 cm/second) [F(1,12)=86.1, P<0.0001]. Whereas the non-transfected cells had no significant flux after addition of excess RAP, the difference between the groups with and without excess RAP was also statistically significant [F(1,11)=24, P<0.0005]. Thus, the results support the presence of a saturable transport system for RAP at the apical surface and the essential role of megalin in the transport process.
For basolateral-to-apical flux, the transport of [125I]RAP in all three groups was not significantly higher than that of [99mTc]-albumin, the marker of paracellular permeability. For MDCK cells stably transfected with MEGt, the apical-to-basolateral permeability coefficient of [125I]-RAP was 460 times higher than the basolateralto-apical permeability coefficient (Fig. 8). Taken together, these results support megalin-mediated transcytosis of RAP.
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LRPt-transfected cells had lower flux transfer constants in both apical-to-basolateral and basolateral-to-apical directions than the non-transfected MDCK cells. It is possible that whereas LRP1 is universally expressed in the native cells, overexpression of LRPt interferes with RAP endocytosis. Therefore, our finding of megalin-mediated transcytosis does not exclude the possibility of co-existing LRP1-mediated transport in either direction.
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Discussion |
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In order to assess the feasibility of RAP delivery from the periphery to the brain, we first determined its fate after intravenous injection. The relative stability of [125I]-RAP within the study period indicates that the brain/blood ratios of radioactivity reflect intact RAP in tissue samples, and that there is a sufficient window of time for RAP to interact with the BBB.
At 30 minutes after intravenous bolus delivery, about 0.1% of [125I]-RAP was able to enter a gram of brain. Moreover, this uptake was decreased to half its value when excess RAP was added to the injection. This suggests the presence of a saturable transport system at the BBB. To further test the possibility of saturable transport, we co-administered excess nonradioactively RAP with [125I]-RAP in a blood-free perfusion system. In situ brain perfusion, although less physiological than intravenous injection, avoids confounding factors like peripheral binding of RAP to heparin sulfate proteoglycans (Berryman and Bensadoun, 1995; Melman et al., 2001
), LRP1, and megalin, especially in the liver and kidney (Warshawsky et al., 1993
). Although we did not detect a decrease of the [125I]-RAP influx transfer constant by excess unlabeled RAP in the intravenous studies, the self-inhibition characteristic of a specific saturable transport system was evident during in situ perfusion. This is similar to the finding with insulin-like growth factor 1 (IGF-1) (Pan and Kastin, 2000
), where a saturable transport system occurs during in situ brain perfusion despite the observation that excess unlabeled IGF-1 paradoxically increases the influx of [125I]-IGF-1 after intravenous administration. Furthermore, entry of [125I]-RAP into brain parenchyma was confirmed by capillary depletion analyses, indicating that RAP, unlike transforming growth factor ß, which also has a high apparent influx transfer constant (Pan et al., 1999
), does not merely associate with the cerebral vasculature but crosses the BBB completely.
Transferrin receptors are highly expressed on endothelial cells, and molecules targeted at the transferrin receptor (such as OX-26 antibody) have been designed as delivery vehicles to bring peptide and protein ligands into the brain. Therefore, we used transferrin and its related ligand melanotransferrin as positive controls for blood-to-brain transfer (Skarlatos et al., 1995; Demeule et al., 2002
). In both in vivo and in vitro situations, BBB permeability appeared higher for RAP than for either transferrin or melanotransferrin. This indicates that RAP is a promising vehicle for efficient delivery across the BBB.
One of the RAP receptors, LRP1, is abundantly expressed in brain microvessels in young mice (Shibata et al., 2000). Although megalin has a more restricted distribution (Kounnas et al., 1994
; Zheng et al., 1994
), it is expressed in brain microvessels and the choroid plexus (Chun et al., 1999
). Megalin plays an important role in forebrain development (Wilnow et al., 1996
) and is probably the receptor that mediates the transport of apoJ/amyloid ß protein across the BBB and blood-cerebrospinal fluid barrier (Zlokovic et al., 1996
). The important role of LRP1 in the efflux transport of amyloid ß protein (Shibata et al., 2000
) supports its basolateral localization and therefore LRP1 is probably not essential for blood-to-brain transport of RAP, whereas megalin is apically expressed and might be directly responsible for RAP transport at the BBB.
To test this possibility, we studied the kinetics of transcytosis in MEGt transfected MDCK cells. MDCK cells polarize and form tight junctions when cultured in Transwell inserts and have been used as an in vitro model of the BBB for drug screening (Irvine et al., 1999; Vilhardt et al., 1999
; Zhang et al., 2002
). As members of the RAP-binding LDL receptor family are large transmembrane proteins (Li et al., 2001a
), functional analysis has been accomplished by domain reconstruction and studies of mini-receptors. In the transfected MDCK cells used for this study, the LRP mini-receptor (LRPt) is sorted basolaterally because of the NPTY motif in its cytoplasmic domain (Marzolo et al., 2003
). Our previous studies have shown that the megalin cytoplasmic tail (MEGt) directs apical sorting. Thus, the polarized localization of the two mini-receptors in MDCK cells in vitro is identical to that occurring in vivo. Our results here show that megalin is mainly responsible for the apical-to-basolateral transport of RAP.
Basolateral-to-apical flux in vitro represents brain-to-blood efflux out of the brain in vivo. The basolateral-to-apical flux of [125I]-RAP, even in the presence of LRPt, was significantly lower than that in control non-transfected cells. This indicates that neither overexpressed megalin nor LRPt mini-receptors is involved in basolateral-to-apical transport. It is possible that the transfected MDCK cells had reduced expression of endogenous LRP1 that was responsible for significant efflux of RAP in the control cells.
Published results with intracellular trafficking of RAP show its dissociation from megalin (Czekay et al., 1997) or LRP1 (Bu et al., 1995
) at low pH accompanied by receptor recycling. However, the RAP/megalin complex is more stable than other LRP complexes. It remains together as far as the late endosome whereas the lipoprotein lipase/megalin complex only lasts as far as the early endosome. Thus, a significant amount of RAP may escape intracellular degradation and undergo complete transcytosis in polarized epithelial/endothelial cells. The lack of significant degradation in the donor chambers in all our study groups indicates that the radioactivity measured represents actual transfer of RAP across the cell barrier rather than endocytosis and degradation. This is supported by the high TEER and the minimal transfer of the paracellular permeation marker albumin. Therefore, RAP has saturable, receptor-mediated apical-to-basolateral transport in MEGt-transfected MDCK cells.
In summary, RAP crosses the BBB by an efficient, saturable transport system probably mediated by megalin. In this process, RAP enters the brain parenchyma intact. Identification of a specific transport system for RAP at the BBB indicates the potential for RAP-mediated delivery of therapeutic peptides and proteins from blood to brain.
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Acknowledgments |
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