The Asymmetry of the Unstirred Water Layer in Permeability Experiments

Korjamo, Timo; Heikkinen, Aki T.; Waltari, P.; Mönkkönen, Jukka

doi:10.1007/s11095-008-9573-8

Cited by 46 publications

(48 citation statements)

References 20 publications

(39 reference statements)

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“…Several in vitro studies indicate that mechanical agitation, such as magnetic stirring, shaking, or air bubbling, reduces the thickness of the UWL. [21][22][23][24] Naruhashi et al have also shown that the results of drug permeability obtained from in vitro experiments under agitation are well correlated with those from in vivo experiments. 22) Based on these reports, we performed mechanical agitation using a shaker during in vitro transfection of pDNA via cationic liposomes to evaluate the effect of the UWL on cationic liposome permeability in a Caco-2 cell monolayer.…”

Section: Discussionmentioning

confidence: 89%

“…It is well known that the transepithelial transport of lipophilic compounds is greatly affected by a UWL. [21][22][23][24] Yano et al showed that the transport of heptyl paraben across a Caco-2 cell monolayer is the most affected by stirring compared with other shorter chain alkylparabens (butyl, amyl, and hexyl parabens). 24) Because cationic liposomes are composed of a cationic lipid (DOTAP) and cholesterol, it is conceivable that cationic liposomes are also poorly diffusable across a UWL.…”

Section: Discussionmentioning

confidence: 99%

“…[21][22][23][24] Based on these reports, we first evaluated the efficiency of pDNA transfection by cationic liposomes into a Caco-2 cell monolayer under mechanical agitation using a shaker. In this study, we prepared cationic liposomes consisting of DOTAP and cholesterol.…”

Section: Effect Of Mechanical Agitation On Pdna Transfectionmentioning

confidence: 99%

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Effect of Mechanical Agitation on Cationic Liposome Transport across an Unstirred Water Layer in Caco-2 Cells

Kono

Iwasaki

Matsuoka

et al. 2016

Biological & Pharmaceutical Bulletin

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To develop an effective oral delivery system for plasmid DNA (pDNA) using cationic liposomes, it is necessary to clarify the characteristics of uptake and transport of cationic liposome/pDNA complexes into the intestinal epithelium. In particular, evaluation of the involvement of an unstirred water layer (UWL), which is a considerable permeability barrier, in cationic liposome transport is very important. Here, we investigated the effects of a UWL on the transfection efficiency of cationic liposome/pDNA complexes into a Caco-2 cell monolayer. When Caco-2 cells were transfected with cationic liposome/pDNA complexes in shaking cultures to reduce the thickness of the UWL, gene expression was significantly higher in Caco-2 cells compared with static cultures. We also found that this enhancement of gene expression by shaking was not attributable to activation of transcription factors such as activator protein-1 and nuclear factor-kappaB (NF-κB). In addition, the increase in gene expression by mechanical agitation was observed at all charge ratios (1.5, 2.3, 3.1, 4.5) of cationic liposome/pDNA complexes. Transport experiments using Transwells demonstrated that mechanical agitation increased the uptake of cationic liposome/pDNA complexes by Caco-2 cells, whereas transport of the complexes across a Caco-2 cell monolayer did not occurr. Moreover, the augmentation of the gene expression of cationic liposome/pDNA complexes by shaking was observed in Madin-Darby canine kidney cells. These results indicate that a UWL greatly affects the uptake and transfection efficiency of cationic liposome/pDNA complexes into an epithelial monolayer in vitro.

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Section: Discussionmentioning

confidence: 89%

Section: Discussionmentioning

confidence: 99%

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Effect of Mechanical Agitation on Cationic Liposome Transport across an Unstirred Water Layer in Caco-2 Cells

Kono

Iwasaki

Matsuoka

et al. 2016

Biological & Pharmaceutical Bulletin

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“…One basic, acidic, and neutral compound was selected for the experiments. All the model compounds are known to be transported passively through Caco-2 monolayers when no pH gradient is present and are not significantly metabolized during Caco-2 permeation experiments (Engman et al, 2001;Korjamo et al, 2008).…”

Section: Chemicalsmentioning

confidence: 99%

“…Thus, accurate universal correction terms for poor recovery in permeation experiments cannot be applied. However, in some reported studies, the poor recovery has been taken into account by correcting the calculations with experimental recovery at a single time point, practically either at the end of the experiment (Youdim et al, 2003) or after the initial "cell loading" period in the beginning of the experiment (Tran et al, 2004;Korjamo et al, 2008).…”

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confidence: 99%

Kinetics of Cellular Retention during Caco-2 Permeation Experiments: Role of Lysosomal Sequestration and Impact on Permeability Estimates

Heikkinen

Mönkkönen²,

Korjamo³

2008

J Pharmacol Exp Ther

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The permeability estimation from cell monolayer permeation data is usually based on 100% recovery assumption. However, poor recovery is often seen in such experiments in practice but often neglected in data interpretation. In the present study, the cellular retention kinetics during Caco-2 permeation experiments of three passively transported compounds, weakly basic propranolol [(Ϯ)-1-isopropylamino-3-(1-naphthyloxy)-2-propanol], weakly acidic ibuprofen [␣-methyl-4-(isobutyl)phenylacetic acid], and neutral testosterone (17␤-hydroxy-4-androsten-3-one), were determined. Furthermore, the effects of cellular retention kinetics on apparent permeability were evaluated, and the role of lysosomal sequestration in cellular retention of propranolol was explored. The cellular retention profiles were observed to be direction and concentration dependent, which may cause erroneous directionality and concentration dependence in permeability estimates. Furthermore, the lysosomal sequestration was demonstrated to contribute to the extent and kinetics of the cellular retention of propranolol.Cell monolayer permeation experiments are commonly used to predict intestinal absorption potential of drug molecules and to screen compounds for their interactions with active transporters (Polli et al., 2001). In addition, cell monolayer permeation experiments are often used to study the mechanisms of drug transfer .In in vitro permeation experiments, typically, the permeability estimates are based on the appearance kinetics of the test compound to the receiver compartment. In addition, samples from both donor and receiver sides are often collected at the final time point of the experiment to determine the recovery of the test compound. The analysis and interpretation of permeation data are often based on the assumption that the cell monolayer behaves as a single barrier for the solute transfer and that the whole mass of the studied compound is in donor and receiver compartments, i.e., the recovery is 100%. However, physiologically, the permeation barrier in cell monolayer experiments consists of various serial and parallel barriers ; furthermore, reduced recovery is often observed in permeability experiments (Polli et al., 2001). Incomplete recovery is attributed to metabolism, drug binding to the plastic surfaces, and/or cellular retention of drug (Fisher et al., 1999;Ö sth et al., 2002;Palmgrén et al., 2006). The possible bias in permeability estimates caused by poor recovery is generally acknowledged but still often neglected in the data interpretation.The mechanisms causing poor recovery and, consequently, the kinetics of apparent loss of test compound in permeation experiments may be specific to compound, experimental apparatus, and permeation barrier (Ö sth et al., 2002;Tran et al., 2004). Thus, accurate universal correction terms for poor This work was supported by the National Technology Agency of Finland (TEKES).Portions of the data presented here appeared in abstract form as follows: Heikkinen AT, Korjamo T, and Mönkkönen...

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Permeability: Caco‐2/MDCK

2012

Absorption and Drug Development

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The Asymmetry of the Unstirred Water Layer in Permeability Experiments

Abstract: An orbital shaker does not produce symmetric hydrodynamics in both chambers of Transwell apparatus. The asymmetric unstirred water layer may complicate the exact analysis of polarized transport.

Cited by 46 publications

References 20 publications

Effect of Mechanical Agitation on Cationic Liposome Transport across an Unstirred Water Layer in Caco-2 Cells

Effect of Mechanical Agitation on Cationic Liposome Transport across an Unstirred Water Layer in Caco-2 Cells

Kinetics of Cellular Retention during Caco-2 Permeation Experiments: Role of Lysosomal Sequestration and Impact on Permeability Estimates

Permeability: Caco‐2/MDCK

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