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Calibrated flux measurements reveal a nanostructure-stimulated transcytotic pathway.

ABSTRACT: Transport of therapeutic agents across epithelial barriers is an important element in drug delivery. Transepithelial flux is widely used as a measure of transit across an epithelium, however it is most typically employed as a relative as opposed to absolute measure of molecular movement. Here, we have used the calcium switch approach to measure the maximum rate of paracellular flux through unencumbered intercellular junctions as a method to calibrate the flux rates for a series of tracers ranging in 0.6-900kDa in size across barriers composed of human colon epithelial (Caco-2) cells. We then examined the effects of nanostructured films (NSFs) on transepithelial transport. Two different NSF patterns were used, Defined Nanostructure (DN) 2 imprinted on polypropylene (PP) and DN3 imprinted on polyether ether ketone (PEEK). NSFs made direct contact with cells and decreased their barrier function, as measured by transepithelial resistance (TER), however cell viability was not affected. When NSF-induced transepithelial transport of Fab fragment (55kDa) and IgG (160kDa) was measured, it was unexpectedly found to be significantly greater than the maximum paracellular rate as predicted using cells cultured in low calcium. These data suggested that NSFs stimulate an active transport pathway, most likely transcytosis, in addition to increasing paracellular flux. Transport of IgG via transcytosis was confirmed by immunofluorescence confocal microscopy, since NSFs induced a significant level of IgG endocytosis by Caco-2 cells. Thus, NSF-induced IgG flux was attributable to both transcytosis and the paracellular route. These data provide the first demonstration that transcytosis can be stimulated by NSFs and that this was concurrent with increased paracellular permeability. Moreover, NSFs with distinct architecture paired with specific substrates have the potential to provide an effective means to regulate transepithelial transport in order to optimize drug delivery.

SUBMITTER: Stewart T

PROVIDER: S-EPMC5501187 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Calibrated flux measurements reveal a nanostructure-stimulated transcytotic pathway.

Stewart Tarianna T Koval William T WT Molina Samuel A SA Bock Suzanne M SM Lillard James W JW Ross Russell F RF Desai Tejal A TA Koval Michael M

Experimental cell research 20170405 2

Transport of therapeutic agents across epithelial barriers is an important element in drug delivery. Transepithelial flux is widely used as a measure of transit across an epithelium, however it is most typically employed as a relative as opposed to absolute measure of molecular movement. Here, we have used the calcium switch approach to measure the maximum rate of paracellular flux through unencumbered intercellular junctions as a method to calibrate the flux rates for a series of tracers rangin ...[more]

PMID: 28390677

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Project description:Rationale and objectivesMammographic density is an important risk factor for breast cancer, but translation to the clinic requires assurance that prior work based on mammography is applicable to current technologies. The purpose of this work is to evaluate whether a calibration methodology developed previously produces breast density metrics predictive of breast cancer risk when applied to a case-control study.Materials and methodsA matched case control study (n = 319 pairs) was used to evaluate two calibrated measures of breast density. Two-dimensional mammograms were acquired from six Hologic mammography units: three conventional Selenia two-dimensional full-field digital mammography systems and three Dimensions digital breast tomosynthesis systems. We evaluated the capability of two calibrated breast density measures to quantify breast cancer risk: the mean (PGm) and standard deviation (PGsd) of the calibrated pixels. Matching variables included age, hormone replacement therapy usage/duration, screening history, and mammography unit. Calibrated measures were compared to the percentage of breast density (PD) determined with the operator-assisted Cumulus method. Conditional logistic regression was used to generate odds ratios (ORs) from continuous and quartile (Q) models with 95% confidence intervals. The area under the receiver operating characteristic curve (Az) was also used as a comparison metric. Both univariate models and models adjusted for body mass index and ethnicity were evaluated.ResultsIn adjusted models, both PGsd and PD were statistically significantly associated with breast cancer with similar Az of 0.61-0.62. The corresponding ORs and confidence intervals were also similar. For PGsd, the OR was 1.34 (1.09, 1.66) for the continuous measure and 1.83 (1.11, 3.02), 2.19 (1.28, 3.73), and 2.20 (1.26, 3.85) for Q2-Q4. For PD, the OR was 1.43 (1.16, 1.76) for the continuous measure and 0.84 (0.52, 1.38), 1.96 (1.19, 3.23), and 2.27 (1.29, 4.00) for Q2-Q4. The results for PGm were slightly attenuated and not statistically significant. The OR was 1.22 (0.99, 1.51) with Az = 0.60 for the continuous measure and 1.24 (0.78, 1.97), 0.98 (0.60, 1.61), and 1.26, (0.77, 2.07) for Q2-Q4 with Az = 0.60.ConclusionThe calibrated PGsd measure provided significant associations with breast cancer comparable to those given by PD. The calibrated PGm performed slightly worse. These findings indicate that the calibration approach developed previously replicates under more general conditions.

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Dataset Information

Calibrated flux measurements reveal a nanostructure-stimulated transcytotic pathway.

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Calibrated flux measurements reveal a nanostructure-stimulated transcytotic pathway.

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