Human Nasal Epithelial Cell Monolayers Research Articles

Intranasal delivery of insulin by self-emulsified nanoemulsion system: In vitro and in vivo studies

The main objective of this research was to develop a self-emulsified nanoemulsion (SEN) dosage form of insulin where insulin is loaded into the lipid phase of the nanoemulsion for enhanced absorption through intranasal delivery. When loaded into the lipid droplets (oil phase), insulin can be protected from enzymatic degradation, can penetrate through the mucus gel barrier in a comparatively effective manner and can be absorbed through transcellular permeation along with paracellular route. To incorporate lipophilicity to insulin molecule, Ins-SPC (Soy-L-α-phosphatidylcholine) complex was prepared by solid dispersion method to load insulin into the oil phase. The cytotoxicity of SPC and the developed nanoemulsions was tested on the human nasal epithelial cells in vitro. An optimized formulation with high loading of insulin and low in vitro cytotoxicity was developed and characterized. To predict the absorption of insulin through nasal mucosa in vivo by the nanoemulsion system, the insulin-loaded SEN along with controls was tested for the transport through human nasal epithelial cell monolayer in vitro. The insulin-loaded SEN significantly (p < 0.01) enhanced the permeation of insulin by three times as compared to the insulin solution. The in vivo absorption of insulin after intranasal delivery of the insulin-loaded SEN was evaluated in anesthetized rats. The results show that the Cmax (maximum plasma concentration) and the bioavailability (relative to the subcutaneous delivery) of the insulin-loaded SEN was 255.9 µU/ml and 68 %, respectively, while the intranasal delivery of the insulin solution resulted in only 5.8 µU/ml of Cmax and 5% of relative bioavailability. Intranasal delivery of 3.6 IU/kg insulin-loaded SEN decreased the plasma glucose level remarkably, achieving a maximum reduction of 70%, and the glucose reduction activity lasted for the whole experimental period of 4 h. Histological examination of the nasal mucosa showed no apparent signs of toxicity at the site of administration after single dose of the insulin-loaded SEN. These results demonstrate that the insulin-loaded SEN significantly enhanced insulin absorption through intranasal delivery, indicating that the developed nanoemulsion system offers a favorable approach for intranasal delivery of insulin.

Comparing ex vivo and in vitro translocation of silver nanoparticles and ions through human nasal epithelium

Silver nanomaterials are widely used in clinically approved devices, consumer goods, and over-the-counter nutraceutical products. Despite the increase in silver nanomaterial research, few investigations have specifically distinguished the biological effects resulting from silver nanoparticles (AgNPs) versus silver ions released from AgNPs. This is in part, due to the complex analytical methods required to characterize silver ion release from AgNPs in biological media. This study sought to analyze silver ion release from AgNPs in biological media, compare silver transport from soluble AgNO3 and AgNPs through ex vivo full thickness sinus human tissue explants and human nasal epithelium and determine fractional AgNP internalization by human nasal epithelial cells. Rapid silver ion release is observed from AgNPs in human nasal epithelial cell medium over 3 h (9.6% of total silver mass). Significantly lower translocation of AgNPs is observed through human nasal epithelial cell monolayers and ex vivo human sinus tissue explants compared to silver ion (AgNO3). AgNP internalization is directly observed in AgNP-exposed human nasal epithelial cell monolayers by live cell scanning transmission electron microscopy (STEM), providing one potential mechanism for AgNP transcytosis. However, in vitro AgNP dissolution experiments suggest that silver in human nasal epithelium is primarily silver ion. Ionic AgNO3 produces significantly higher silver translocation, supporting previous results claiming silver ion as primarily responsible for biological effects of AgNPs.

In vitro safety evaluation of human nasal epithelial cell monolayers exposed to carrageenan sinus wash.

Carrageenans have shown to reduce the viral load in nasal secretions and lower the incidence of secondary infections in children with common cold. Despite the widespread use of carrageenans in topical applications, the effect of carrageenans on the sinonasal epithelial barrier has not been elucidated. We investigate the effect of different carrageenans on the sinonasal epithelial barrier and inflammatory response in vitro. Iota and Kappa carrageenan delivered in saline irrigation solutions applied to air-liquid interface (ALI) cultures of primary human nasal epithelial cells from chronic rhinosinusitis patients and controls. Epithelial barrier structure was assessed by measuring the transepithelial electrical resistance (TEER) and immunolocalization of F actin. Ciliary beat frequency (CBF), toxicity, and inflammatory response was studied. Kappa or Iota carrageenan in the different solutions was not toxic, did not have detrimental effects on epithelial barrier structure and CBF. Rather, application of Kappa carrageenan significantly increased TEER and suppressed interleukin 6 (IL-6) secretion in ALI cultures from CRS patients. Kappa or Iota carrageenan solution was safe and did not negatively affect epithelial barrier function. Kappa carrageenan increased TEER and decreased IL-6 production in CRS patients, indicating positive effects on epithelial barrier function in vitro.

Enhancing effect of borneol and muscone on geniposide transport across the human nasal epithelial cell monolayer.

Geniposide is widely used in the treatment of cerebral ischemic stroke and cerebrovascular diseases for its anti-thrombotic and anti-inflammatory effects. Recent studies demonstrated that geniposide could be absorbed promptly and thoroughly by intranasal administration in mice and basically transported into the brain. Here, we explored its transport mechanism and the effect of borneol and muscone on its transport by human nasal epithelial cell (HNEC) monolayer. The cytotoxicity of geniposide, borneol, muscone and their combinations on HNECs was evaluated by the MTT assay. Transcellular transport of geniposide and the influence of borneol and muscone were studied using the HNEC monolayer. Immunostaining and transepithelial electrical resistance were measured to assess the integrity of the monolayer. The membrane fluidity of HNEC was evaluated by fluorescence recovery after photobleaching. Geniposide showed relatively poor absorption in the HNEC monolayer and it was not a P-gp substrate. Geniposide transport in both directions significantly increased when co-administrated with increasing concentrations of borneol and muscone. The enhancing effect of borneol and muscone on geniposide transport across the HNEC may be attributed to the significant enhancement on cell membrane fluidity, disassembly effect on tight junction integrity and the process was reversible. These results indicated that intranasal administration has good potential to treat cerebrovascular diseases.

Expression and functional activity of P-glycoprotein in passaged primary human nasal epithelial cell monolayers cultured by the air–liquid interface method for nasal drug transport study

P-glycoprotein (P-gp) is an efflux transporter encoded by the multidrug resistance gene (MDR1), which is also known as the human ABCB1 gene (ATP-binding cassette, subfamily-B). The objectives of this study were to investigate the expression of P-gp in passaged primary human nasal epithelial (HNE) cell monolayer, cultured by the air-liquid interface (ALI) method, and to evaluate its feasibility as an in-vitro model for cellular uptake and transport studies of P-gp substrates. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to verify the expression of the MDR1 gene. Transport and cellular uptake studies with P-gp substrate (rhodamine123) and P-gp inhibitors (verapamil and cyclosporin A) were conducted to assess the functional activity of P-gp in HNE cell monolayers cultured by the ALI method. MDR1 gene expression in primary HNE cell monolayers cultured by ALI method was confirmed by RT-PCR. The apparent permeability coefficient (P(app) ) of the P-gp substrate (rhodamine123) in the basolateral to apical (B to A) direction was 6.9 times higher than that in the apical to basolateral (A to B) direction. B to A transport was saturated at high rhodamine123 concentration, and the treatment of P-gp inhibitors increased cellular uptake of rhodamine123 in a time- and concentration-dependent manner. These results support the MDR1 gene expression and the functional activity of P-gp in primary HNE cell monolayers cultured by the ALI method.

Poloxamer/Cyclodextrin/Chitosan-Based Thermoreversible Gel for Intranasal Delivery of Fexofenadine Hydrochloride

To enhance permeation and solubility of an intranasal delivery system of fexofenadine hydrochloride (FXD HCl), a new formulation using poloxamer 407 (P407)/hydroxypropyl-β-cyclodextrin (HP-β-CD)-based thermoreversible gels with chitosan, was developed. Prepared gels were characterized by gelation temperature, viscosity, viscoelasticity, and drug release profile. The in vitro permeation study was performed in primary human nasal epithelial cell monolayers cultured by air-liquid interface method. The addition of chitosan caused the slight elevation of gelation temperature and viscosity-enhancing effect. Viscosity enhancement by the incorporation of chitosan caused the retardation of drug release from P407 gels in in vitro release test. The in vitro permeation profile showed that the increase in chitosan content (0.1% and 0.3%, w/v) significantly enhanced the permeation of FXD HCl. After intranasal administration of P407/HP-β-CD-based thermoreversible gels containing 0.1% and 0.3% of chitosan in rabbits at 0.5 mg/kg dose, plasma concentrations of FXD HCl were significantly higher than those of nasal solutions (p < 0.05). In particular, the bioavailability of the optimized thermoreversible gel containing 0.3% chitosan was about 18-fold higher than that of the solution type. These results suggested the feasibility that thermosensitive gels could be used as an effective dosage form to enhance the nasal absorption of FXD HCl.

In vitro Nasal Cell Culture Systems for Drug Transport Studies

Growing interest in the nasal route as a drug delivery system calls for a reliable in vitro model which is crucial for efficiently evaluating drug transport through the nasal cells. Various in vitro cell culture systems has thus been developed to displace the ex vivo excised nasal tissue and in vivo animal models. Due to species difference, results from animal studies are not sufficient for estimating the drug absorption kinetics in humans. However, the difficulty in obtaining reliable human tissue source limits the use of primary culture of human nasal epithelial cells. This shortage of human nasal tissue has therefore prompted studies on the passage culture of nasal epithelial cells. A serially passaged primary human nasal epithelial cell monolayer system developed by the air-liquid interface (ALI) culture is known to promote the differentiation of cilia and mucin gene and maintain high TEER values. Recent studies on the in vitro nasal cell culture systems for drug transport studies are reviewed in this article.

Enhancing effect of surfactants on fexofenadine·HCl transport across the human nasal epithelial cell monolayer

The effect of various surfactants (sodium cholate, sodium taurocholate, Tween 80 and Poloxamer F68) on enhancing the transepithelial permeability of fexofenadine·HCl was evaluated in a human nasal epithelial cell monolayer model. The cytotoxicity of the surfactants on the human nasal epithelial cells was evaluated by the MTT assay. A dose-dependent reduction of cell viability was observed at higher than critical micelle concentration (CMC) of the surfactants, and the IC 50 of non-ionic surfactants (Tween 80 and Poloxamer F68) was higher than that of ionic surfactants (sodium cholate and sodium taurocholate). The TEER values significantly decreased after 2 h incubation with the ionic surfactants, but were recovered after 24 h in the fresh culture media. Ionic surfactants significantly increased the transepithelial permeability ( P app) of fexofenadine·HCl compared to the non-ionic surfactants. The reduction of TEER values upon exposing the cell monolayer to the surfactants for 2 h correlated well with the P app of fexofenadine·HCl, which suggests that the permeation-enhancing mechanism of the ionic surfactants is by altering the tight junction property of the paracellular pathway. F-actin staining showed that the effect of ionic surfactants on the tight junction is temporary and reversible, which is consistent with the TEER value recovery within 24 h. These results imply that ionic surfactants are potentially useful permeation enhancers for nasal delivery of hydrophilic compounds, such as fexofenadine·HCl. This study also indicated the usefulness of the human nasal epithelial cell monolayer model not only for evaluating the in vitro nasal drug transport but also for studying the mechanism and toxicity of enhancers.

The identification of P-gp expression in Air-Liquid Interface(ALI) culture of human nasal epithelial cell monolayers

The identification of P-gp expression in Air-Liquid Interface(ALI) culture of human nasal epithelial cell monolayers

Transport of anti-allergic drugs across the passage cultured human nasal epithelial cell monolayer

The purpose of this study was to investigate the nasal absorption characteristics of a series of anti-allergic drugs across the human nasal epithelial cell monolayer, which was passage cultured by the liquid-covered culture (LCC) method on Transwell ®. Characterization of this cell culture model was achieved by bioelectric measurements and morphological studies. The passages 2–4 of cell monolayers exhibited the TEER value of 1731 ± 635 Ω cm 2 after 2 days of seeding and maintained high TEER value for 4–6 days. Morphological study by TEM and SEM showed the existence of the tight junctions, and the cuboidal shaped epithelial cells monolayer. A series of anti-allergic drugs, albuterol hemisulfate, albuterol, fexofenadine HCl, dexamethasone, triamcinolon acetonide, and budesonide were selected as model compounds for transport studies. All the drugs were assayed using reversed-phase HPLC under isocratic conditions. Results indicated that within the log P (apparent 1-octanol/water partition coefficient) range from −1.58 (albuterol) to 3.21 (budesonide), there existed 100-fold difference in the apparent permeability coefficients ( P app). A log-linear relationship was shown between the drug log P and the P app across passaged human nasal epithelial monolayers. The amount of fexofenadine HCl and dexamethasone across passaged human nasal cell monolayers was concentration-dependent in the direction of apical to basolateral. The direction dependent transport studies were investigated among all these drugs and no significant difference in the two directions was observed. In conclusion, this LCC passaged human nasal epithelial culture model may be a useful in vitro model for studying the passive transport processes in nasal drug delivery.

Air-Liquid Interface Culture of Serially Passaged Human Nasal Epithelial Cell Monolayer for In Vitro Drug Transport Studies

The objective of this study was to establish a drug transport study using human nasal epithelial (HNE) cell monolayers cultured by the air-liquid interface (ALI) method using serum-free medium (BEGM:DME/F12, 50:50). The cells were developed and characterized in comparison to those that have been previously cultured by the liquid-covered culture (LCC) method. The epithelial cell monolayer cultured by the ALI method resulted in a significantly higher transepithelial electrical resistance value (3,453 ± 302 ohm × cm2) that was maintained (>1,000 ohm × cm2) for up to 20 days compared with that cultured by the LCC method. Observation by scanning electron microscopy revealed mature cilia after 2 weeks in the ALI culture, while flatten unhealthy ciliated cells were observed in the LCC method. After 21 days, higher level of MUC5AC and 8 mRNA were expressed in ALI culture which confirmed the secretory differentiation of HNE monolayers in vitro. No significant difference in the permeability coefficients of a model hydrophilic marker (14C-mannitol) and a lipophilic drug (budesonide) was observed between the two conditions on day 7. The passage 2–3 of the HNE monolayer using ALI condition retained the morphology and differentiated features of normal epithelium. Thus it would be a suitable model for in vitro nasal drug delivery studies.

약물의 in vitro 투과 실험을 위한 사람의 비강점막상피세포 단층막의 일차배양

The primary culture of human nasal epithelial cell monolayer was performed on a Transwell. The effect of various factors on the tight junction formation was observed in order to develop an in vitro experimental system for nasal transport studies. Human nasal epithelial cells, collected from human normal inferior turbinates, were plated onto diverse inserts. After 4 days, media of the apical surface was removed for air-liquid interface (ALI) culture. Morphological characteristics was observed by transmission electron microscopy (TEM). A polyester membrane of pore size was determined as the most effective insert based on the change in the transepithelial electric resistance (TEER) value as well as the transport study. The ALI method was effective in developing the tight junction as observed in the further increase in the TEER value and reduction in the permeability coefficient of transport. Results of the transport study of a model drug, budesonide, showed that the primary culture system developed in this study could be further developed and applied for in vitro nasal transport studies.

ATP-activated basolateral Na+/H+ exchange in human normal and cystic fibrosis airway epithelium.

This study investigated the distribution (apical/basolateral membrane) of Na+/H+ exchange in human nasal epithelial cell monolayers from cystic fibrosis (CF) and non-CF individuals. Monolayers of non-CF and CF preparations were loaded with the pH probe 2',7'-bis(carboxyethyl)-5(6)- carboxyfluorescein, and intracellular pH (pH1) was measured with a microfluorimeter. In nominally HCO3(-)-free NaCl Ringer solution, basal pHi values in non-CF and CF monolayers were essentially identical (approximately 7.1). When cells were acid loaded (NH4+ prepulse), subsequent alkalinization of non-CF and CF cell monolayers required serosal Na+ and was blocked by serosal, but not mucosal, amiloride (500 microM). In the presence of extracellular Na+, initial rates (delta pHi/min) of recovery from an acid load in non-CF and CF preparations were statistically identical (approximately 0.08). Pretreatment of monolayers with ATP (100 microM) or phorbol 12-myristate 13-acetate (PMA; 100 nM) caused initial rates of recovery to increase by twofold over control values in both preparations. These data provide evidence in support for an ATP- and PMA-activated Na+/H+ exchanger located at the basolateral surface of airway epithelia.