[Paper Review] Pulmonary surfactant inhibition of nanoparticle uptake by alveolar epithelial cells

Milad Radiom, Matthieu Sarkis+4 more|arXiv (Cornell University)|Oct 20, 2020

Inhalation and Respiratory Drug DeliveryMedicine55 references64 citations

TL;DR

This study demonstrates that pulmonary surfactant significantly reduces uptake of positively charged silica nanoparticles by alveolar epithelial cells (A549 and NCI-H441), inhibiting internalization by up to two orders of magnitude. Using in vitro models with glass coverslips and air-liquid interface transwells, the researchers show that surfactant forms protective aggregates with nanoparticles, reducing cellular interaction and uptake, as confirmed by fluorescence and confocal microscopy, with colocalization of nanoparticles and lipid vesicles inside cells.

ABSTRACT

Pulmonary surfactant forms a sub-micrometer thick fluid layer that covers the surface of alveolar lumen and inhaled nanoparticles therefore come in to contact with surfactant prior to any interaction with epithelial cells. We investigate the role of the surfactant as a protective physical barrier by modeling the interactions using silica-Curosurf-alveolar epithelial cell system in vitro. Electron microscopy displays that the vesicles are preserved in the presence of nanoparticles while nanoparticle-lipid interaction leads to the formation of mixed aggregates. Fluorescence microscopy reveals that the surfactant decreases the uptake of nanoparticles by up to two orders of magnitude in two models of alveolar epithelial cells, A549 and NCI-H441, irrespective of immersed culture on glass or air-liquid interface culture on transwell. Confocal microscopy corroborates the results by showing nanoparticle-lipid colocalization interacting with the cells. Our work thus supports the idea that pulmonary surfactant plays a protective role against inhaled nanoparticles. The effect of surfactant should therefore be considered in predictive assessment of nanoparticle toxicity or drug nanocarrier uptake. Models based on the one presented in this work may be used for preclinical tests with engineered nanoparticles.

Motivation & Objective

• To investigate the role of pulmonary surfactant as a physical barrier against inhaled nanoparticles in the alveolar region.
• To assess how surfactant affects nanoparticle uptake in two alveolar epithelial cell models: A549 and NCI-H441.
• To compare nanoparticle uptake under standard culture conditions (glass coverslip) versus air-liquid interface (transwell) to evaluate physiological relevance.
• To determine whether surfactant-lipid interactions alter nanoparticle sedimentation and effective cellular dose.
• To evaluate the impact of surfactant concentration on nanoparticle-cell interactions and uptake efficiency.

Proposed method

• Used a model system combining Curosurf® (clinical surfactant substitute) and positively charged aminated silica nanoparticles.
• Performed exposure experiments on A549 and NCI-H441 cells cultured on glass coverslips and on transwells under air-liquid interface conditions.
• Employed fluorescence microscopy to quantify nanoparticle uptake by measuring fluorescence intensity over time.
• Conducted confocal microscopy to analyze colocalization of fluorescent nanoparticles and lipid-labeled surfactant vesicles within cells.
• Used transmission electron microscopy (TEM) to visualize nanoparticle-surfactant interactions and internalization into vesicles.
• Measured dynamic light scattering (DLS) to assess nanoparticle aggregation and hydrodynamic size changes in the presence of surfactant.

Experimental results

Research questions

• RQ1 How does pulmonary surfactant affect the uptake of positively charged silica nanoparticles by alveolar epithelial cells?
• RQ2 Does the presence of surfactant alter the sedimentation profile or effective dose of nanoparticles delivered to epithelial cells?
• RQ3 How do different culture models (glass vs. transwell) influence nanoparticle uptake in the presence of surfactant?
• RQ4 To what extent do surfactant-lipid aggregates reduce nanoparticle internalization, and is this effect dose-dependent?
• RQ5 Is there a difference in nanoparticle uptake between A549 and NCI-H441 cells when surfactant is present?

Key findings

• Pulmonary surfactant reduced nanoparticle uptake by up to two orders of magnitude in both A549 and NCI-H441 cells, regardless of culture substrate or interface condition.
• At a Curosurf® to nanoparticle mixing ratio of X = 2, uptake was reduced to 1.5–2.1% of the control (neat nanoparticles), with no further reduction at X = 20.
• Confocal microscopy confirmed colocalization of nanoparticles and surfactant lipids within cells, indicating direct involvement of surfactant in modulating uptake.
• TEM imaging revealed that nanoparticles form mixed aggregates with surfactant vesicles and are internalized within lipid-bound structures.
• NCI-H441 cells showed lower uptake than A549 cells—only 2% of particles internalized in transwell conditions—suggesting better intrinsic protection due to tight junctions.
• The surfactant-lipid complexation altered nanoparticle sedimentation, likely due to density matching with vesicles, reducing effective cellular exposure.