Curcumin, bisdemethoxycurcumin and dimethoxycurcumin complexed with cyclodextrins have structure specific effect on the paracellular integrity of lung epithelia in vitro.

The phytochemical curcumin may improve translocation of the cystic fibrosis transmembrane regulatory (CFTR) protein in lung epithelium and therefore be helpful in the treatment of cystic fibrosis (CF) symptoms. However, previous studies often use commercial curcumin that is a combination of curcumin, demethoxycurcumin and bisdemethoxycurcumin which could affect the investigated cells differently. In the present study, we investigated the potential difference between curcumin, bisdemethoxycurcumin and dimethoxycurcumin on the epithelial tight junction complex, in the bronchial epithelial cell line VA10, by measuring transepithelial electrical resistance (TER), immunofluorescence and western blotting of tight junction proteins. The curcuminoids were complexed with hydroxypropyl-γ-cyclodextrin for increased solubility and stability. Curcumin (10 µg/ml) increased the TER significantly after 24 h of treatment while four times higher concentration of bisdemethoxycurcumin was required to obtain similar increase in TER as curcumin. Interestingly, dimethoxycurcumin did not increase TER. Curcumin clearly affected the F-actin structures both apically and basolaterally. These results begin to define possible effects of curcuminoids on healthy bronchial epithelia and shows that difference in the phenyl moiety structure of the curcuminoids influences the paracellular epithelial integrity.

Challenges in evaluation of chitosan and trimethylated chitosan (TMC) as mucosal permeation enhancers: From synthesis to in vitro application.

The polysaccharide chitosan and the water soluble chitosan derivative N,N,N-trimethyl chitosan (TMC) have been widely investigated as permeation enhancers of mucosal surfaces with numerous papers published over the last two decades. Although both chitosan and TMC increase permeation of markers through mucosal membranes, such as the intestinal and airway epithelium as well as in in vivo models, these investigations have not led to their use in marketed drug formulations. In this review, the reported extent of the permeation enhancement and cell viability after chitosan or TMC treatment in intestinal and airway models is critically evaluated and concluded that the apparent discrepancies can be explained by differences in polymer structure, experimental conditions and in vitro models. Additionally, aspects regarding the synthesis of TMC and its structural characterization are described, focusing on new synthetic strategies implemented to reduce O-methylation. Finally recommendations are provided on how studies can be conducted to improve understanding of the structure­activity relationship and elucidate possible mechanism of action.

deltaNp63 has a role in maintaining epithelial integrity in airway epithelium.

The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its ΔN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the ΔN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that ΔNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium.

N-alkylation of highly quaternized chitosan derivatives affects the paracellular permeation enhancement in bronchial epithelia in vitro.

This study describes the structure-activity relationship for carefully characterized N-alkyl-N-quaternary chitosan derivatives as permeation enhancers for drugs that are mainly absorbed through the paracellular pathway, such as macromolecular drugs and hydrophilic drugs, in a well defined bronchial epithelial cell line. The O-methyl free derivatives used in the study were fully trimethylated (100%) N,N,N-trimethyl chitosan (TMC) and N-propyl-(QuatPropyl), N-butyl-(QuatButyl) and N-hexyl (QuatHexyl)-N,N-dimethyl chitosan, with 85-91% degree of quaternization. The fully trimethylated TMC, from 0.25mg/ml, decreased transepithelial electrical resistance (TER) in a reversible manner and enhanced the permeation of the macromolecule FITC-dextran 4kDa (FD4) 2-5 fold. TMC did not cause any alterations in the tight junction (TJ) protein claudin-4 or in F-actin architecture. QuatHexyl was the most effective polymer to produce enhanced permeation and decreased TER from 0.016mg/ml. Nevertheless, this enhanced permeation was accompanied by reduced viability and dissociation of F-actin and claudin-4 proteins. The structure-activity relationship suggests that more lipophilic derivatives show more permeation enhancement, TJ disassembly, and less viability in the order of hexyl≈butyl>propyl>methyl and demonstrates that the permeation effect is not only mediated by permanent positive charge but also by the extent of N-alkylation. These results are relevant to elucidate the structural factors contributing to the permeation enhancement of chitosan derivatives and for potential use in pulmonary applications.

Drug delivery characteristics of the progenitor bronchial epithelial cell line VA10.

PURPOSE: To determine the integrity and permeability properties of the immortalized human VA10 bronchial epithelial cell line for its suitability as an in vitro drug permeation model. METHODS: Cells were grown under liquid-covered culture (LCC) or air-liquid interface (ALI) culture, characterized using electron microscopy and immunostaining. Integrity was measured using transepithelial electrical resistance (TER) and permeability of fluorescein sodium (Flu-Na). General permeability was established with dextrans and model drugs and P-glycoprotein (P-gp) function determined with bidirectional flux of rhodamine-123. RESULTS: ALI culture resulted in 2-3 cell layers with differentiation towards ciliated cells but LCC showed undifferentiated morphology. VA10 cells formed TJ, with higher TER in LCC than ALI (∼2500 vs. ∼1200 Ω*cm(2)) and Flu-Na permeability ∼1-2 × 10(-7) cm/s. ALI cultured cells expressed P-gp and distinguished between compounds depending on lipophilicity and size, consistent with previous data from Calu-3 and 16HBE14o-cell lines. CONCLUSIONS: ALI cultured cell layers capture the in vivo-like phenotype of bronchial epithelium and form functional cell barrier capable of discriminating between compounds depending on physiochemical properties. The VA10 cell line is an important alternative to previously published cell lines and a relevant model to study airway drug delivery in vitro.