Biokinetics of Aerosolized Liposomal Ciclosporin A in Human Lung Cells In Vitro Using an Air-Liquid Cell Interface Exposure System.

BACKGROUND: Inhalation of aerosolized drugs is a promising route for noninvasive targeted drug delivery to the lung. Nanocarrier systems such as liposomes have been explored for inhalation therapy opening new avenues, including stabilization of nonsoluble drugs (e.g., Ciclosporin A [CsA]) and controlled release. METHODS: The biokinetic behavior of the immunosuppressive drug CsA encapsulated in liposomes (L-CsA) at the lung epithelial barrier was studied in vitro. Human lung epithelial cells (alveolar A549 and bronchial 16HBE14o- epithelial cells) were exposed to aerosolized L-CsA at the air-liquid interface (ALI) using a dose-controlled air-liquid interface cell exposure (ALICE) system and the temporal profile of the L-CsA dose in the apical, basal, and cell compartment was monitored up to 24 hours. RESULTS: Aerosolization of different volumes of L-CsA solution with the ALICE resulted in dose-controlled, spatially uniform, and reproducible L-CsA delivery. Cell viability at 24 hours postexposure was not impaired and immunofluorescence staining revealed the typical epithelial cell morphology in control as well as in L-CsA-exposed cells. The (pro-)inflammatory interleukin-8 levels were not elevated under any condition. The biokinetic analysis revealed that both cell types formed a tight, but imperfect, barrier for L-CsA resulting in initially high transbarrier L-CsA transport rates, which ceased after about 4 hours. Although substantial transbarrier L-CsA transport was observed for both cell types, respectively, a 150-fold higher L-CsA concentration was established in the apical and cell compared to the basal compartment. Most importantly, for pulmonary drug targeting, a high cellular L-CsA dose level (20%-25% of the delivered dose) was obtained rapidly (<1 hour) and maintained for at least 24 hours. CONCLUSIONS: The ALICE system combined with lung epithelial cells cultured at the ALI offers a reliable and relevant in vitro platform technology to study the effects of inhalable substances such as L-CsA under biomimetic conditions.

Ultrathin Ceramic Membranes as Scaffolds for Functional Cell Coculture Models on a Biomimetic Scale.

Epithelial tissue serves as an interface between biological compartments. Many in vitro epithelial cell models have been developed as an alternative to animal experiments to answer a range of research questions. These in vitro models are grown on permeable two-chamber systems; however, commercially available, polymer-based cell culture inserts are around 10 µm thick. Since the basement membrane found in biological systems is usually less than 1 µm thick, the 10-fold thickness of cell culture inserts is a major limitation in the establishment of realistic models. In this work, an alternative insert, accommodating an ultrathin ceramic membrane with a thickness of only 500 nm (i.e., the Silicon nitride Microporous Permeable Insert [SIMPLI]-well), was produced and used to refine an established human alveolar barrier coculture model by both replacing the conventional inserts with the SIMPLI-well and completing it with endothelial cells. The structural-functional relationship of the model was evaluated, including the translocation of gold nanoparticles across the barrier, revealing a higher translocation if compared to corresponding polyethylene terephthalate (PET) membranes. This study demonstrates the power of the SIMPLI-well system as a scaffold for epithelial tissue cell models on a truly biomimetic scale, allowing construction of more functionally accurate models of human biological barriers.

Translocation of gold nanoparticles across the lung epithelial tissue barrier: Combining in vitro and in silico methods to substitute in vivo experiments.

BACKGROUND: The lung epithelial tissue barrier represents the main portal for entry of inhaled nanoparticles (NPs) into the systemic circulation. Thus great efforts are currently being made to determine adverse health effects associated with inhalation of NPs. However, to date very little is known about factors that determine the pulmonary translocation of NPs and their subsequent distribution to secondary organs. METHODS: A novel two-step approach to assess the biokinetics of inhaled NPs is presented. In a first step, alveolar epithelial cellular monolayers (CMLs) at the air-liquid interface (ALI) were exposed to aerosolized NPs to determine their translocation kinetics across the epithelial tissue barrier. Then, in a second step, the distribution to secondary organs was predicted with a physiologically based pharmacokinetic (PBPK) model. Monodisperse, spherical, well-characterized, negatively charged gold nanoparticles (AuNP) were used as model NPs. Furthermore, to obtain a comprehensive picture of the translocation kinetics in different species, human (A549) and mouse (MLE-12) alveolar epithelial CMLs were exposed to ionic gold and to various doses (i.e., 25, 50, 100, 150, 200 ng/cm(2)) and sizes (i.e., 2, 7, 18, 46, 80 nm) of AuNP, and incubated post-exposure for different time periods (i.e., 0, 2, 8, 24, 48, 72 h). RESULTS: The translocation kinetics of the AuNP across A549 and MLE-12 CMLs was similar. The translocated fraction was (1) inversely proportional to the particle size, and (2) independent of the applied dose (up to 100 ng/cm(2)). Furthermore, supplementing the A549 CML with two immune cells, i.e., macrophages and dendritic cells, did not significantly change the amount of translocated AuNP. Comparison of the measured translocation kinetics and modeled biodistribution with in vivo data from literature showed that the combination of in vitro and in silico methods can accurately predict the in vivo biokinetics of inhaled/instilled AuNP. CONCLUSION: Our approach to combine in vitro and in silico methods for assessing the pulmonary translocation and biodistribution of NPs has the potential to replace short-term animal studies which aim to assess the pulmonary absorption and biodistribution of NPs, and to serve as a screening tool to identify NPs of special concern.

Engineering an in vitro air-blood barrier by 3D bioprinting.

Intensive efforts in recent years to develop and commercialize in vitro alternatives in the field of risk assessment have yielded new promising two- and three dimensional (3D) cell culture models. Nevertheless, a realistic 3D in vitro alveolar model is not available yet. Here we report on the biofabrication of the human air-blood tissue barrier analogue composed of an endothelial cell, basement membrane and epithelial cell layer by using a bioprinting technology. In contrary to the manual method, we demonstrate that this technique enables automatized and reproducible creation of thinner and more homogeneous cell layers, which is required for an optimal air-blood tissue barrier. This bioprinting platform will offer an excellent tool to engineer an advanced 3D lung model for high-throughput screening for safety assessment and drug efficacy testing.