TR146 Cell Culture Research Articles

Two types of core/shell fibers based on carboxymethyl chitosan and Sodium carboxymethyl cellulose with self-assembled liposome for buccal delivery of carvedilol across TR146 cell culture and porcine buccal mucosa.

Two novel core-shell fibers which could self-assemble liposome were fabricated based on bioadhesive polymer carboxymethyl chitosan (CMCS) and Sodium carboxymethyl cellulose (CMC-Na), separately. The shell layers were CMCS/PVA and CMC-Na/PVA, respectively, and the core layer was mixture of PVP, Phospholipids (PC) and Carvedilol (Car). The diameter distribution and core/shell structure were examined by SEM and CLSM. FTIR and XRD were also used to characterize the fiber. Self-assembled liposome was observed by TEM, and other parameters like drug encapsulation efficiency was also determined. In vitro adhesive force was conducted to evaluate bioadhesive property of fibers. Dissolution test demonstrated Car was almost completely released within 2 h and presented linear release. The permeation studies across porcine TR146 cell culture and buccal mucosa were carried out, indicating self-assembled liposome and bioadhesive polymer both promoted drug penetration. MTT test showed TR146 cells were safe after incubation with fibers' extraction medium under the concentration of 10 mg/mL. In summary, this design based on self-assembled liposome and core/shell fiber using water-soluble bioadhesive polymer was desirable for Car buccal absorption.

Buccal delivery of metformin: TR146 cell culture model evaluating the use of bioadhesive chitosan discs for drug permeability enhancement

The oral cavity is considered an attractive site of drug administration. Metformin is currently, used in oral diabetes treatment. The aim of the current study was to study the feasibility of metformin, to permeate the buccal epithelium applying a bioadhesive and permeation enhancing drug delivery system. The in vitro TR146 cell culture model was used to study the effect of drug concentration (5-100mM) and the impact of a bioadhesive chitosan formulation (discs) and chitosan in solution (0-20mg/mL) acting as a permeation enhancer. The permeation of metformin occurred by passive diffusion via the paracellular pathway driven by the concentration gradient, yet with a possibility of increasing the metformin transport by using higher, donor concentrations. When using floating baskets, as a new application of the TR146 cell culture model, it was possible to observe a time-dependent effect of the bioadhesive metformin discs and, metformin permeation may be increased due to a combination of bioadhesion and permeation enhancement induced by chitosan, although the permeation enhancing effect of chitosan was not statistically significant. The limited apparent buccal permeability of metformin observed in vitro, suggest that in vivo absorption of therapeutic doses of metformin needs to take place as a combination of buccal and intestinal absorption as metformin therapy requires the use of high doses.

In vitro, ex vivo and in vivo examination of buccal absorption of metoprolol with varying pH in TR146 cell culture, porcine buccal mucosa and Göttingen minipigs

This work studied the buccal absorption of metoprolol in vitro, ex vivo and in vivo as a function of buffered pH at 7.4, 8.5, 9.0 and 9.5. Permeability studies showed a correlation (r2=0.92) between in vitro TR146 cell culture and ex vivo porcine buccal mucosa in a modified Ussing chamber. A higher apparent permeability was observed at higher pH values, i.e. the more compound that was unionised the higher the permeability. In vivo studies were conducted in anaesthetised Göttingen mini-pigs. A clear influence of pH on the absorption was seen and a significant higher absolute bioavailability was obtained after buccal dosing (58–107%) compared to oral (3%) administration, ranging 58–107% and 3%, respectively. Macroscopically, no local toxic effects were observed by visual inspection of mini-pig cheeks. A very clear level C in vitro in vivo correlation (r2=0.98) was obtained between the observed in vitro permeabilities and the bioavailability observed in vivo, suggesting that the two in vitro models have good predictive power for drug delivery, which could be a useful tool for future formulation developments intended for buccal delivery.

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

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. 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. 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. 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.

Nicotine permeability across the buccal TR146 cell culture model and porcine buccal mucosa in vitro: effect of pH and concentration

The present study was conducted to investigate and compare the effect of pH and drug concentration on nicotine permeability across the TR146 cell culture model and porcine buccal mucosa in vitro. As a further characterization of the TR146 cell culture model, it was explored whether the results were comparable for bi-directional and uni-directional transport in the presence of a transmembrane pH gradient. Nicotine concentrations between 10 −5 and 10 −2 M were applied to the apical side of the TR146 cell culture model or the mucosal side of porcine buccal mucosa. Buffers with pH values of 5.5, 7.4 and 8.1 were used to obtain different fractions of non- and mono-ionized nicotine. The apparent permeability ( P app) of nicotine across both models increased significantly with increasing pH, and the P app values obtained with the two models could be correlated in a linear manner. With increasing concentrations of nicotine, the P app values decreased, which can partly be explained by an effect on the paracellular pathway. Similar results were also obtained when using the models for bi-directional as well as for uni-directional studies. The TR146 cell culture model may be used as model for buccal epithelium in studies with ionized drugs and a transmembrane pH gradient.

The potential of chitosan in enhancing peptide and protein absorption across the TR146 cell culture model-an in vitro model of the buccal epithelium.

To investigate the potential of chitosan (CS) to enhance buccal peptide and protein absorption, the TR146 cell culture model, a model of the buccal epithelium, was used. The sensitivity of TR146 cells to several CS solutions (different salts with different MW) was investigated by using the MTS/ PMS assay. Permeability studies were performed to determine the enhancing effect of CS glutamate (1, 20, 40, 60, and 100 microg/mL) on the permeability of 3H-mannitol and fluorescein isothiocyanate labeled dextrans (FD) with various MW (4.4-19.5 kD) across the cell culture model. Sensitivity of TR146 cells to CS solutions depended on the concentration, the pH, and the type of CS salt. CS glutamate solutions (pH 6.0) were found to be the least harmful. CS glutamate was able to increase the permeability of model substances with MW up to 9.5 kD across the cell model. An enhancing effect was found for CS concentrations of 20 microg/mL and higher, correlating with a decrease in TEER values. The 20 microg/mL CS concentration had a negligible effect on the enzyme activity of the cells as determined by the MTS/PMS assay. CS glutamate is effective in enhancing the transport of macromolecules across the buccal TR146 cell culture model. Therefore. it might be a promising vehicle for peptide and protein buccal administration.

TR146 cells grown on filters as a model of human buccal epithelium: V. Enzyme activity of the TR146 cell culture model, human buccal epithelium and porcine buccal epithelium, and permeability of leu-enkephalin

The objective of the present study was to characterise the TR146 cell culture model as an in vitro model of human buccal mucosa with respect to the enzyme activity in the tissues. For this purpose, the contents of aminopeptidase, carboxypeptidase and esterase in homogenate supernatants of the TR146 cell culture model, and human and porcine buccal epithelium were compared. The esterase activity in the intact cell culture model and in the porcine buccal mucosa was compared. Further, the TR146 cell culture model was used to study the permeability rate and metabolism of leu-enkephalin. The activity of the three enzymes in the TR146 homogenate supernatants was in the same range as the activity in homogenate supernatants of human buccal epithelium. In the TR146 cell culture model, the activity of aminopeptidase (13.70±2.10 nmol/min per mg protein) was approx. four times the activity of carboxypeptidase (3.73±0.53 nmol/min per mg protein), whereas the level of esterase activity was significantly higher (223.39±69.82 nmol/min per mg protein). In the TR146 cell culture model, the apical esterase activity was found significantly higher than the basal activity, and found comparable to the porcine buccal mucosa. However, the esterase activity on the serosal side of the porcine buccal mucosa was higher than in the TR146 cell culture model. Approx. 1.5% of leu-enkephalin permeated the TR146 cell layers within 5 h ( P app 7.38±0.83×10 −7 cm/s) and approx. 77% of intact peptide was still present in the donor phase after 5 h. The present study suggests that the TR146 cell culture model is a valuable in vitro model for permeability and metabolism studies with enzymatically labile drugs, such as leu-enkephalin, intended for buccal drug delivery.

TR146 cells grown on filters as a model of human buccal epithelium: IV. Permeability of water, mannitol, testosterone and β-adrenoceptor antagonists. Comparison to human, monkey and porcine buccal mucosa

The objective of the present study was to evaluate the TR146 cell culture model as an in vitro model of human buccal epithelium. For this purpose, the permeability of water, mannitol and testosterone across the TR146 cell culture model was compared to the permeability across human, monkey and porcine buccal mucosa. Further, the permeability rates of ten β-adrenoceptor antagonists (acebutolol, alprenolol, atenolol, labetalol, metoprolol, oxprenolol, pindolol, propranolol, timolol and tertatolol) across the TR146 cell culture model and porcine buccal mucosa were related to their lipophilicity (log D oct; 7.4) and capacity factor ( k′) and to their polar water accessible surface area (PWASA). For water, mannitol, testosterone and some of the β-adrenoceptor antagonists, the permeability enhancement across the TR146 cell culture model in the presence of sodium glycocholate (GC) was determined. The mannitol and testosterone permeability across the TR146 cell culture model could be related to the permeability across porcine and human buccal mucosa. The permeability of the β-adrenoceptor antagonists across the TR146 cell culture model varied between 2.2×10 −6 cm/s (atenolol) and 165×10 −6 cm/s (metoprolol). For propranolol the cellular permeability value ( P c) was lower than expected, probably due to accumulation in the TR146 cell layers. Limited correlation of permeability with k′ was observed both for the TR146 cell culture model and the porcine buccal mucosa, although the porcine permeability values were ≈100 times less than the values determined with the TR146 cell culture model. The permeability values were also found to decrease with increasing PWASA. The PWASA value seemed to be more predictable for permeability than k′. The presence of 12.5 mM GC increased the permeability only for the hydrophilic atenolol, which may help explain the mechanism for GC-induced enhancement. The present results indicate that the TR146 cell culture model can be used as an in vitro model for permeability studies and mechanistic studies of human buccal drug delivery of drugs with different lipophilicity.

TR146 cells grown on filters as a model of human buccal epithelium: III. Permeability enhancement by different pH values, different osmolality values, and bile salts

The aim of the present study was to evaluate the TR146 cell culture model as an in vitro model of human buccal epithelium with respect to the permeability enhancement by different pH values, different osmolality values or bile salts. For this purpose, the increase in the apparent permeability ( P app) of the hydrophilic marker mannitol due to exposure to solutions with pH values or osmolality values different from the physiological values was studied. As in studies with solutions of either taurocholate (TC), glycocholate (GC) or glycodeoxycholate (GDC) the results were compared to the increase in P app of mannitol obtained in analog studies using porcine buccal mucosa in an Ussing chamber. The effect of the exposure on the electrical resistance of the TR146 cell culture model and the porcine buccal mucosa was measured, and the degree of protein leakage due to GC exposure was investigated in the TR146 cell culture model. The porcine buccal mucosa was approximately ten times less permeable to mannitol than the TR146 cell culture model. The P app values obtained with both in vitro models were dependent on the concentration of bile salt, the degree of hydroxylation of the bile salt, and the type of conjugation to the bile salt as the enhancement effect decreased in the order GDC⪢GC>TC. Increased P app values correlated with a decrease in the electrical resistance of the TR146 cell culture model and the porcine buccal mucosa. GC was shown to induce concentration dependent protein leakage in the TR146 cell culture but only from the site of application, and the results indicate that duration of exposure further than 120 min was of minor importance. The present results indicate that the TR146 cell culture model may be a suitable in vitro model for efficacy studies and mechanistic studies of enhancers with potential use in human buccal drug delivery.

TR146 cells grown on filters as a model of human buccal epithelium: permeability of fluorescein isothiocyanate-labelled dextrans in the presence of sodium glycocholate

The aim of the present study was to characterize the TR146 cell culture model as an in vitro model of human buccal epithelium with respect to the permeability of test substances with different molecular weights (Mw). For this purpose, the apparent permeability (Papp) values for mannitol and for fluorescein isothiocyanate (FITC)-labelled dextrans (FD) with various Mw (4000–40 000) were compared to the Papp values obtained using porcine buccal mucosa as an in vitro model of the human buccal epithelium. The effect of 10 mM sodium glycocholate (GC) on the Papp values was examined. To identify the pathways by which FD of Mw 4000–40 000 were transported, the confocal laser scanning microscope (CLSM) was used.The Papp values obtained with the TR146 cell culture model in the absence of a permeability enhancer linearly decreased with increasing Mw of the test substance from 0.65±0.055×10−8 to 44±7.5×10−8 cm/s, as the Papp values obtained with porcine buccal mucosa. In the presence of the permeability enhancer, GC, the permeability of the FD across the cultured TR146 cell culture model increased in a parabolic manner, reaching maximum at Mw 10 000. In the absence of the enhancer, only paracellular localization of FD was observed while, in the presence of GC, FD also could be detected in the cytosol of some of the superficial cells. The GC-induced enhancement of FD permeation may be partially attributed to changed permeation pathways. The present results indicate that the TR146 cell culture model is a suitable in vitro model for mechanistic permeability studies of human buccal drug permeability.

Filter-grown TR146 cells as an in vitro model of human buccal epithelial permeability.

Use of a cell culture model of a specific epithelium requires documentation of its differentiation. This study reports permeability of mannitol concurrent with a profile of differentiation markers of filter-grown TR146 cells, a cell line originating from a human buccal carcinoma, cultivated submerged or at the air-liquid interface for 23 to 31 d. A multilayered squamous epithelial-like tissue was found. The maximal permeability barrier and the most distinct stratification were obtained at day 23, when cultured submerged (apparent permeability coefficient 4.08 +/- 0.15 (x 10(-6)) cm/s; transepithelial electrical resistance 102 +/- 5 omega x cm2). The profile of differentiation markers demonstrated similarities to normal human buccal epithelium by expression of K4, K10, K13, K16, and K19 keratins, plasma membrane-associated transglutaminase, involucrin, and epidermal growth factor receptor. Uniform expression of K5, K8 and K18 was consistent with the carcinogenic origin of TR146 cells. Identical profiles of differentiation markers were obtained irrespective of method or time of culture. Karyotyping proved the human origin of TR146 cells. Three different passages had near triploid (3n+-) chromosome compliments and consistent occurrence of four marker chromosomes [mar4, mar5, mar9, and add(5)(p)], while differences between them defined them as subclones. The results indicate that a submerged filter-grown TR146 cell culture at day 23 of culture has the potential to model the human buccal epithelial barrier for permeation of drugs.