Cellular Uptake But Low Permeation of Human Calcitonin-Derived Cell Penetrating Peptides and Tat(47-57) Through Well-Differentiated Epithelial Models

PURPOSE: To investigate whether cell penetrating peptides (CPP) derived from human calcitonin (hCT) possess, in addition to cellular uptake, the capacity to deliver their cargo through epithelial barriers. METHODS: Cellular uptake of hCT(9-32) and permeation of six hCT-derived peptides, namely, hCT(9-32), hCT(12-32), hCT(15-32), hCT(18-32), hCT(21-32), and a random sequence of hCT(9-32) were evaluated in fully organized confluent Madin-Darby canine kidney (MDCK), Calu-3, and TR146 cell culture models. For comparison, Tat(47-57) and penetratin(43-58) were investigated. The peptides were N-terminally labeled with carboxyfluorescein (CF). Uptake in the well-differentiated epithelial models was observed by confocal laser scanning microscopy (CLSM), whereas permeation through the models was analyzed by reversed-phase (RP)-HPLC. RESULTS: In MDCK epithelium hCT(9-32), Tat(47-57) and penetratin(43-58) demonstrated punctuated cytoplasmic distribution. In Calu-3, Tat(47-57) and penetratin(43-58) were simultaneously localized in a punctuated cytoplasmic and paracellular distribution, whereas hCT(9-32) showed strict paracellular distribution. By contrast, in TR146 cells, Tat(47-57) was located strictly paracellularily, whereas penetratin(43-58) showed a punctuated cytoplasmic pattern and hCT(9-32) both. The transepithelial permeability of all tested peptides and their cargo was lower than that of paracellular markers. CONCLUSIONS: The CPP uptake pattern depends on both the type of peptide and the cell culture model. In general, the investigated CPP have no apparent potential for systemic drug delivery across epithelia. Nevertheless, distinct patterns of cellular distribution may offer a potential for localized epithelial delivery.     

Electroporation-mediated delivery of molecules to model intestinal epithelia

This study was conducted to determine if electroporation can deliver membrane-impermeant molecules intracellularly to intact, physiologically competent monolayers that mimic the intestinal epithelium. In addition, the long-term effects of electroporation on these monolayers were studied to determine the kinetics with which monolayers recover barrier function. Caco-2 and T84 cells were electroporated as monolayers using calcein and fluorescein-labeled bovine serum albumin as marker molecules for measuring delivery into cells. Confocal microscopy and flow cytometry were used, respectively, to visualize and quantify uptake of these molecules. Transepithelial resistance was used as a measure of physiologic barrier function. We found that intracellular uptake of calcein and bovine serum albumin occurred uniformly throughout both types of model epithelia and increased as a function of voltage, pulse length, and pulse number. There was no significant difference in uptake resulting from single and multiple pulses of the same total exposure time. We also observed that monolayers exposed to electroporation that induced uptake of up to 10⁶ molecules/cell were able to recover normal barrier function within one day. These findings suggest that electroporation may be useful for intracellular delivery into monolayers to study epithelial biology and, possibly, for drug delivery to intestinal epithelium.     

Differentiation Restricted Endocytosis of Cell Penetrating Peptides in MDCK Cells Corresponds with Activities of Rho-GTPases

Purpose Cellular entry of biomacromolecules is restricted by the barrier function of cell membranes. Tethering such molecules to cell penetrating peptides (CPPs) that can translocate cell membranes has opened new horizons in biomedical research. Here, we investigate the cellular internalization of hCT(9-32)-br, a human calcitonin derived branched CPP, and SAP, a γ-zein related sequence. Methods Internalization of fluorescence labelled CPPs was performed with both proliferating and confluent MDCK cells by means of confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS) using appropriate controls. Internalization was further elaborated in an inflammatory, IFN-γ/TNF-αa induced confluent MDCK model mimicking inflammatory epithelial pathologies. Activities of active form Rho-GTPases (Rho-A and Rac-1) in proliferating and confluent MDCK cells were monitored by pull-down assay and Western blot analysis. Results We observed marked endocytic uptake of the peptides into proliferating MDCK by a process suggesting both lipid rafts and clathrin-coated pits. In confluent MDCK, however, we noted a massive but compound-unspecific slow-down of endocytosis. This corresponded with a down-regulation of endocytosis by Rho-GTPases, previously identified to be intimately involved in endocytic traffic. In fact, we found endocytic internalization to relate with active Rho-A; vice versa, MDCK cell density, degree of cellular differentiation and endocytic slow-down were found to relate with active Rac-1. To our knowledge, this is the first study to cast light on the previously observed differentiation restricted internalization of CPPs into epithelial cell models. In the inflammatory IFN-γ/TNF-αa induced confluent MDCK model mimicking inflammatory epithelial pathologies, CPP internalization was enhanced in a cytokine concentration-dependent way resulting in maximum enhancement rates of up to 90%. We suggest a cytokine induced redistribution of lipid rafts in confluent MDCK to cause this enhancement. Conclusion Our findings emphasize the significance of differentiated cell models in the study of CPP internalization and point towards inflammatory epithelial pathologies as potential niche for the application of CPPs for cellular delivery. This work was supported by the Commission of the European Union (EU project on Quality of Life and Management of Living Resources, Project No. QLK2-CT-2001-01451.     

Cellular Internalization of Human Calcitonin Derived Peptides in MDCK Monolayers: A Comparative Study with Tat(47-57) and Penetratin(43-58)

PURPOSE: The objective of this study was to evaluate key motif requirements of human calcitonin (hCT)-derived peptides for the permeation through the plasma membrane of MDCK monolayers, as epithelial model. METHODS: Truncated and sequence-modified fluorescent-labeled hCT-derived peptides were synthesized through Fmoc chemistry. Peptide uptake by confluent MDCK was observed by confocal laser scanning microscopy. The cytotoxic effect of the peptides on cellular integrity was followed by LDH release. For direct comparison we covered the cellular uptake of established cell penetrating peptides, Tat(47-57) and penetratin(43-58). RESULTS: Truncated sequences of hCT, from hCT(9-32) to hCT(18-32), penetrated the plasma membrane and demonstrated a sectoral, punctuated cytoplasmic distribution. The uptake process appeared to be temperature-, time- and concentration-dependent. Amino acid modifications of hCT(18-32) indicated that both the proline in position 23 and the positive charge of lysine in position 18 are crucial for peptide uptake. The reverse sequence hCT(32-18) did not penetrate the membrane, indicating the importance of sequence orientation. Tat(47-57) and penetratin(43-58) showed a similar punctuated cytoplasmic distribution in MDCK and HeLa cell lines. No relevant toxicity was observed. CONCLUSIONS: Selected hCT-derived peptides have cell penetrating properties. The uptake mechanism seems to involve an endocytic pathway.     

In vitro effect of the Phoneutria nigriventer spider venom on cell viability, paracellular barrier function and transcellular transport in cultured cell lines.

Phoneutria nigriventer spider venom (PNV) induces, in rats, local edema as result of an increased vascular permeability, as well as causes blood-brain barrier (BBB) breakdown by altering transendothelial transport routes in hippocampal microvessels. In this work we investigated the in vitro effects of PNV on cell viability and cellular transport routes using three cell lines, the ECV304 endothelial-, the C6 glioma- and the MDCK epithelial cells. We showed that PNV (14.6 and 292 microg crude venom/ml culture medium) had no direct cytotoxic effect on both the ECV304 and the MDCK cell lines but slightly reduced the viability of C6 glioma cells (P<0.05) at the highest concentration, as revealed by the cellular neutral red uptake assay. The PNV effects on cell transport were evaluated in MDCK cell line. PNV seems do not cause any disturbance in the paracellular barrier function of the cultured MDCK cells, as shown by the lack of a significant change in the distribution and expression of the junctional proteins, ZO-1, occludin, E-cadherin and the cytoskeletal F-actin. In contrast, PNV-treated MDCK monolayers showed an enhancement in the transepithelial electrical resistance and a tendency towards an increased occludin expression. In addition, the PNV significantly increased the apical endocytosis of HRP, which was not followed by an equivalent exocytosis at the basal side, as revealed by biochemical and ultrastructural methods. We conclude that the venom of P. nigriventer displays a relatively low cytotoxicity in vitro as well as activates directly the endocytic transport pathway in MDCK cells without disrupting the paracellular route.     

Lack of cross-tolerance following heat and cadmium exposure in functional MDCK monolayers

Exposure of monolayers of Madin-Darby canine kidney epithelial (MDCK) cells to a mild heat stimulus induces a state of physiological thermotolerance in which epithelial barrier function is maintained following a second more severe heat stress. We have previously shown that expression of exogenous HSP70 fully mimics the effects of the conditioning heat stress. Exposure of MDCK cells to elevated temperatures or medium containing CdCl2 caused a robust increase in cellular levels of HSP70. Pretreatment of MDCK monolayers with cadmium but not heat caused a small protection of epithelial barrier function against a second challenge with cadmium. In addition, a prior exposure of monolayers to cadmium at levels sufficient to induce HSP70 expression and increased cellular chaperone activity did not afford protection against a subsequent thermal challenge. Therefore multiple stress-specific cellular pathways impinge on the ability of heat shock proteins to induce physiological thermotolerance. Occludin, a component of tight junctions, is induced in MDCK cells engineered to express high levels of exogenous HSP70, potentially accounting for an elevation in baseline resistance. However neither basal levels of occludin, nor alterations in occludin expression, were correlated with epithelial barrier function in MDCK cells either exposed to elevated temperatures or challenged with cadmium.     

Comparison of Bidirectional Cephalexin Transport Across MDCK and Caco-2 Cell Monolayers: Interactions with Peptide Transporters

Purpose. Bidirectional transport studies were conducted to determine whether Madin-Darby canine kidney (MDCK) cell monolayers could be used as an alternative to the traditional Caco-2 assay as a fast-growingin vitro model of peptide transport. Methods. Transport of cephalexin and glycylsarcosine across MDCK and Caco-2 cell monolayers was quantified using LC-LC/MS. Glycylsarcosine, p-aminohippuric acid (PAH), and tetraethylammonium chloride (TEA) were tested as inhibitors of cephalexin transport. Results. The ratio of apparent cephalexin permeabilities (apical to basolateral/basolateral to apical) obtained from MDCK monolayers was almost 5-fold greater than that obtained from Caco-2 monolayers. The opposite trend was observed for glycylsarcosine. When MDCK monolayers were used, glycylsarcosine reduced the cephalexin/apparent permeability ratio almost 90%. PAH and TEA did not inhibit cephalexin transport across MDCK or Caco-2 cell monolayers. Conclusion. MDCK cell monolayers may be a promising, fast-growing alternative to Caco-2 cells for identifying peptide transporter substrates. However, differences in the apical-to-basolateral transport of cephalexin and glycylsarcosine suggest that the basolateral transport mechanisms for these compounds are different in the two cell lines. Additionally, because the activity of the peptide transporter in MDCK cells was low, scaling factors may be required when using this cell line to predict in vivo drug absorption.     

The role of anion exchange in the uptake of Pb by human erythrocytes and Madin-Darby canine kidney cells

Anion exchange (AE) plays a critical role in regulating intracellular pH in erythrocytes and epithelial cells and has been suggested to facilitate the transport of lead (Pb) across the erythrocyte cell membrane. In this study we examined the role of AE in the uptake of Pb by human erythrocytes and by Madin-Darby canine kidney (MDCK) cells, the kidney epithelial cell line. Functional AE in MDCK cells was evidenced by: increased uptake of SO(4)(2-) at pH 6.0 over pH 7.0, and inhibition of SO(4)(2-) uptake by the AE inhibitor 4, 4'-diisothiocyanostilbene-2, 2'- disulfonic acid (DIDS) as well as by non-halide anions. Accumulation of Pb into MDCK cells was time and temperature dependent. DIDS inhibited uptake of Pb into human erythrocytes but not MDCK cells. In conclusion, uptake of Pb into erythrocytes but not kidney epithelial cells occurs through AE.     

A Drug Absorption Model Based on the Mucus Layer Producing Human Intestinal Goblet Cell Line HT29-H

A new drug absorption model based on monolayers of the human intestinal goblet cell line HT29-H grown on permeable filters has been characterized. HT29-H cells have been shown (a) to form monolayers of mature goblet cells under standard cell culture conditions, (b) to secrete mucin molecules, (c) to produce a mucus layer that covers the apical cell surface, and (d) that this mucus layer is a significant barrier to the absorption of the lipophilic drug testosterone. This is the first demonstration of an intact human mucus layer with functional barrier properties produced in cell culture. The results indicate that monolayers of HT29-H cells provide a valuable complement to mucus-free drug absorption models based on absorptive cell lines such as Caco-2 cells.     

A novel cell compatible impingement system to study in vitro drug absorption from dry powder aerosol formulations

A modified Astra type multistage liquid impinger (MSLI) with integrated bronchial cell monolayers was used to study deposition and subsequent drug absorption on in vitro models of the human airway epithelial barrier. Inverted cell culture of Calu-3 cells on the bottom side of cell culture filter inserts was integrated into a compendial MSLI. Upside down cultivation did not impair the barrier function, morphology and viability of Calu-3 cells. Size selective deposition with subsequent absorption was studied for three different commercially available dry powder formulations of salbutamol sulphate and budesonide. After deposition without size separation the absorption rates from the aerosol formulations differed but correlated with the size of the carrier lactose particles. However, after deposition in the MSLI, simulating relevant impaction and causing the separation of small drug crystals from the carrier lactose, the absorption rates of the three formulations were identical, confirming the bioequivalence of the three formulations.     

Effect of thrombin on permeability of human epithelial cell monolayers

Epithelial cells play an important role in maintaining the airway barrier, which is impaired in inflammatory conditions. Recently, thrombin was reported to be increased in the airway of patients with asthma, and thrombin has been shown to increase the permeability of endothelial cell monolayers. Therefore, we suspected that thrombin affects airway permeability. Calu-3 cell monolayers were established on microporous membranes of tissue culture cell inserts. We examined the effects of topically applied thrombin or thrombin receptor-activating peptide (TRAP) on: (1) transepithelial permeability (luminal to serosal transfer) of radiolabeled mannitol and albumin, (2) changes in electrical resistance, and (3) actin fiber content as assessed by fluorescence microscopy. Compared with untreated control cultures, treatment of the monolayers for 24 h with thrombin or TRAP significantly decreased the electrical resistance and increased the permeability to mannitol and albumin. In addition, these treatments enhanced the appearance of actin stress fibers, and small gaps became visible at areas of cell-cell contact. Thrombin appears to increase epithelial permeability by receptor-mediated reorganization of the actin network in airway epithelial cells. This is likely to contribute to the impairment of the airway barrier function.     

Functional characterization of monocarboxylic acid,large neutral amino acid,bile acid and peptide transporters,and P-glycoprotein in MDCK and Caco-2 cells

Bidirectional transport studies were conducted to determine whether substrates of five intestinal transporters showed carrier-mediated asymmetric transport across MDCK (Madin-Darby canine kidney) cell monolayers grown under standard conditions. Drug concentrations were quantitated using liquid scintillation counting, liquid chromatography/mass spectrometry/mass spectrometry, or liquid chromatography/mass spectrometry. In the presence of a pH gradient, benzoic acid exhibited net apical-to-basolateral transport, with apparent permeability ratios (apical-to-basolateral permeability/basolateral-to-apical permeability) ranging from 14 to 25. The addition of valproic acid reduced the permeability ratio by 70-90%. Cephalexin transport also exhibited net absorption in the presence of a pH gradient, with apparent permeability ratios ranging from 14 to 71, depending on growth conditions. Radiolabeled phenylalanine exhibited a low level of carrier-mediated absorption with an apparent permeability ratio of 1.8 that was reduced to 1.0 in the presence of unlabeled L-phenylalanine. Taurocholic acid did not exhibit carrier-mediated absorption. Cyclosporine and fexofenadine exhibited P-glycoprotein-mediated efflux from both MDCK and Caco-2 cells, which was more sensitive to inhibition in MDCK cells. These results suggest that although MDCK cell monolayers may be a useful model for evaluating transport by the absorptive monocarboxylic acid and peptide transporters and the efflux transporter, P-glycoprotein, they are not useful for predicting large neutral amino acid or bile acid transport in the intestine.     

A modified Tat peptide for selective intracellular delivery of macromolecules

Objectives The Tat peptide has been widely used for the intracellular delivery of macromolecules. The aim of this study was to modify the peptide to enable regulation of cellular uptake through a dependency on activation by proteases present in the local environment. Methods The native Tat peptide sequence was altered to inhibit the initial interaction of the peptide with the cell membrane through the addition of the consensus sequence for urokinase plasminogen activator (uPA). uPA expression was characterised and semi‐quantitatively rated in three cell lines (U251mg, MDA‐MB‐231 and HeLa). The modified peptide was incubated with both recombinant enzyme and with cells varying in uPA activity. Cellular uptake of the modified Tat peptide line was compared with that of the native peptide and rated according to uPA activity measured in each cell line. Key findings uPA activity was observed to be high in U251mg and MDA‐MB‐231 and low in HeLa. In MDA‐MB‐231 and HeLa, uptake of the modified peptide correlated with the level of uPA expression detected (93 and 52%, respectively). In U251mg, however, the uptake of the modified peptide was much less (19% observed reduction) than the native peptide despite a high level of uPA activity detected. Conclusions Proteolytic activation represents an interesting strategy for the targeted delivery of macromolecules using peptide‐based carriers and holds significant potential for further exploitation.     

A novel hPepT1 stably transfected cell line: establishing a correlation between expression and function

Stably transfected MDCK/hPepT1-V5&His clonal cell lines expressing varying levels of epitope-tagged hPepT1 protein were established to quantify the relationship between transgene hPepT1 expression levels and its functional kinetics in facilitating peptide and peptide-like drug uptake and transport in vitro. The hPepT1 sequence was amplified from Caco-2 cell mRNA, inserted into the pcDNA3.1 -V5&His TOPO plasmid, and transfected into MDCK cells. Transgene protein levels were quantified by Western Blot analysis utilizing a standard curve generated with a positive control protein containing a V5&His epitope. Three clones expressing different levels of the hPepT1 fusion protein (low, medium, and high) were selected for the functional characterization with [14C]Gly-Sar and [3H]carnosine. The MDCK/hPepT1 cells expressed a novel hPepT1/epitope tag protein with an apparent molecular mass of 110 kDa. The [14C]Gly-Sar uptake in the transfected cells was sodium-independent and pH-dependent, demonstrating enhanced uptake, the rate of which increased significantly from the weakly to strongly expressing hPepT1 MDCK/hPepT1 -V5&His clones as compared to the mock cell line at pH 6.0. The uptake and permeability of [14C]Gly-Sar and [3H]carnosine demonstrated a direct correlation between the hPepT1 level of expression, uptake, and transport capabilities. Molecular and functional characterization of the MDCK/hPepT1-V5&His cell line confirmed a directly proportional relationship between Vmax and Papp versus the molar levels of hPepT1 transgene expression. This stably transfected hPepT1 cell line may serve as a useful in vitro model for screening and quantifying peptide and peptide-like drug transport as a function of hPepT1 expression in drug discovery.     

Passive Transepithelial Absorption of Thyrotropin-Releasing Hormone (TRH) via a Paracellular Route in Cultured Intestinal and Renal Epithelial Cell Lines

Transport studies using intestinal brush-border membrane vesicles isolated from rats and rabbits have failed to demonstrate proton- or Na⁺-dependent carrier-mediated transport of thyrotropin-releasing hormone (TRH), despite a pharmacologically relevant oral bioavailability. To examine the hypothesis that reported levels of oral bioavailability reflect predominately a paracellular rather than transcellular route for transepithelial transport of TRH, we have studied TRH transport in cultured epithelial cell types of intestinal (Caco-2 and T₈₄) and renal (MDCK I, MDCK II, and LLC-PK₁ origin, whose paracellular pathways span the range of permeability values observed in natural epithelia. Transport of TRH across monolayers of intestinal Caco-2 cells was similar to the flux of mannitol (1–4% per 4 hr), and unlike other putative substrates for the di-/tripeptide carrier, apical-to-basolateral transport was not increased by the presence of an acidic pH in the apical chamber. TRH transport did not show saturation, being uneffected in the presence of 20 mM cold TRH. In each cell type studied TRH and mannitol transport were similar and positively correlated with the conductance of the cell layers, consistent with a passive mechanism of absorption. This evidence suggests that, providing that a peptide is resistant to luminal hydrolysis, small but pharmacologically significant amounts of peptide absorption may be achieved by passive absorption across a paracellular route.     

Parathyroid hormone-stimulated cadmium accumulation in Madin-Darby canine kidney cells.

Although most renal cadmium transport occurs in proximal tubules indirect evidence suggests that distal tubules may also transport this heavy metal. Since the distal nephron is the site at which parathyroid hormone (PTH) regulates calcium absorption, we evaluated the effects of PTH on Cd2+ accumulation in Madin-Darby canine kidney (MDCK) cells. MDCK cells express a distal-like phenotype including PTH-sensitive adenylyl cyclase and stimulation of calcium transport. MDCK cells were grown to confluence in phenol red-free Dulbecco's modified Eagle's medium. PTH increased 109CdCl2 accumulation in a concentration-dependent manner over the range of 10(-11)-10(-9) M bPTH[1-34]. At 10(-9) M, PTH increased Cd2+ accumulation maximally by 205%. The PTH antagonist, bPTH[3-34], failed to augment 109Cd2+ accumulation. The dihydropyridine agonist, Bay k 8644, in the presence of PTH, increased 109Cd2+ uptake by 200% over vehicle-treated controls and by approximately 100% over PTH or Bay k 8644 alone. The apparent Km for Bay k 8644 activation was 1.3 microM. Bay k 8644-augmented 109Cd2+ uptake was competitively inhibited by the calcium channel antagonist nifedipine. No voltage dependence of Bay k 8644-amplified 109Cd2+ uptake could be detected. Based on these observations we conclude: (1) MDCK cells accumulate Cd2+; (2) PTH increases Cd2+ uptake into MDCK cells; and (3) Cd2+ entry in kidney epithelial cells is mediated, at least in part, by dihydropyridine-sensitive calcium channels.     

Effects of Deoxycholate on the Transepithelial Transport of Sucrose and Horseradish Peroxidase in Filter-Grown Madin–Darby Canine Kidney (MDCK) Cells

Madin–Darby canine kidney (MDCK) epithelial cells grown on microporous polycarbonate filters were used as a model system to investigate the mechanisms of enhancement by deoxycholate in the transepithelial transport of horseradish peroxidase (HRP) and ¹⁴C-sucrose. Deoxycholate at 0.025% had no effect on the transepithelial electrical resistance (TEER); a fivefold enhancement on the transepithelial transport of HRP, but not on that of ¹⁴C-sucrose, was observed. Deoxycholate at 0.05% induced a reversible decrease of TEER; a 2- and 50-fold enhancement on the transepithelial transport of ¹⁴C-sucrose and HRP, respectively, was observed. At 0.1%, deoxycholate induced an irreversible decrease in TEER and the epithelial barrier in the cell monolayer was completely eliminated. A 3.3-fold increase in cellular uptake in HRP, but not in ¹⁴C-sucrose, was also observed in the presence of 0.025% deoxycholate. The increase in cellular uptake was abolished when HRP was conjugated to polylysine. These results suggest that deoxycholate can increase the transepithelial transport by at least two different mechanisms, i.e., a transcellular pathway, possibly due to the enhancement of cellular uptake of selective molecules, and a nonselective paracellular pathway, due to the loosening of tight junctions by deoxycholate at higher concentrations.     

The fd phage and a peptide derived from its p8 coat protein interact with the HIV-1 Tat-NLS and inhibit its biological functions

Filamentous fd bacteriophages are used to construct phage-display peptide libraries, which have been instrumental in selecting peptides that interact with specific domains within target molecules. Here we demonstrate that the fd bacteriophage itself, as well as NTP8 - a synthetic peptide derived from it and bearing amino acids 1-20 of the phage p8 protein - interact with the nuclear localization signal (NLS) of the HIV-1 Tat protein. Accordingly, fd bacteriophage and the NTP8 peptide inhibit binding mediated by the Tat-NLS to the nuclear-import receptor importin beta and Tat-NLS-mediated translocation into cell nuclei. The NTP8 peptide, at 100 microM concentration, also caused about 50% inhibition of HIV-1 propagation in cultured cells. The fd bacteriophage prevents heparan sulfate proteoglycans-mediated uptake of extracellular Tat by target cells and consequently transactivation of a chloramphenicol acetyltransferase (CAT) reporter gene. A BSA-NTP8 conjugate inhibits Tat-NLS-mediated binding to heparin immobilized on a BIAcore surface. BLAST analysis of the NTP8 amino-acid sequence revealed similarity to sequences in several human proteins, including ADA2 and CD53.     

绿原酸跨细胞转运机制研究

目的利用Caco-2和MDCK细胞单层模型研究绿原酸(chlorogenic acid,CGA)的跨细胞转运过程及其机制。方法①Caco-2和MDCK细胞单层模型建立:Caco-2、MDCK细胞分别按密度1×105、5×104个细胞/cm2接种到Milli-cell-CM culture plate inserts上培养,待细胞单层达到一定致密程度后进行透过实验。②透过实验:用M2e酶标仪测定CGA在不同方向、不同浓度下的跨细胞转运情况并计算累积透过量。结果在两种细胞模型上CGA均有不同程度的双向跨细胞转运(吸收和分泌),P-gp抑制剂维拉帕米能明显减少CGA的分泌。结论CGA跨细胞转运同时存在吸收和分泌的动力学过程。P-gp部分参与CGA的分泌机制。     

Reduction of doxorubicin resistance in P-glycoprotein overexpressing cells by hybrid cell-penetrating and drug-binding peptide

Drug efflux by the membrane transporter P-glycoprotein (P-gp) plays a key role in multidrug resistance (MDR). In order to bypass P-gp, thus overcoming MDR, a hybrid peptide comprising a cell penetrating peptide (Tat) and a drug binding motif (DBM) has been developed to noncovalently bind and deliver doxorubicin (Dox) into MDR cells. The uptake of Dox into the leukemia cell line K562 and its P-gp overexpressing subline KD30 increased in the presence of DBM-Tat peptide. Confocal microscopy indicated that DBM-Tat associated Dox was directed to a perinuclear area of KD30 cells, while this was not observed in parent K562 cells. When KD30 cells were pretreated with the endosomotropic agent chloroquine (CLQ), peptide associated Dox redistributed into the cytosol, indicating that endocytosis was the predominant uptake route. Altered drug uptake kinetics observed by cellular accumulation assay also supported an endocytic uptake. In the presence of CLQ, DBM-Tat was able to enhance the cytotoxicity of Dox by 68.4% at 5?µM peptide concentration in KD30 cells but there were only minor effects on Dox cytotoxicity in K562 cells even in the presence of CLQ. Thus, combining Dox with DBM-Tat reduces P-gp mediated drug efflux, without a requirement for drug modification or inhibiting P-gp function.