Modeling the Interaction of Aerosol Particles With Lung Epithelial Cells Using the Lung-On-A-Chip Platform

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Evaluation of the inhalation toxicity of natural and artificial aerosols, as well as the efficacy of aerosolized drugs, is an important practical task. Inhalation administration also offers a promising alternative method for systemic drug delivery, especially for drugs with poor oral bioavailability, such as peptides and proteins, as well as with compounds that are sensitive to first-pass metabolism in the liver and intestines [1]. The main value in understanding risk factors and mechanisms of action lies in data regarding the impact of aerosol substances on humans. However, such studies that involve human subjects are typically expensive, time-consuming, and require special permissions, especially in cases where the substances under investigation are presumed to be toxic [2]. Consequently, in vivo studies examining the biological effects of aerosols are primarily conducted on animals. Nevertheless, in addition to scientific and ethical concerns, in vivo experiments often allow for the observation of only the mediated effects of the complex interactions of aerosols on the organism. The development of organ-on-a-chip systems has recently emerged as a reliable alternative to animal testing. These micro-engineered cell systems provide cells with an environment that mimics their natural surroundings in vivo [3]. Lately, a variety of approaches for in vitro testing of inhalation toxicity have been developed [4]. Until quite recently, drug testing in vitro was performed using lung tissue cells that were completely submerged in culture medium [5]. There are also microfluidic "lung-on-chip" systems based on stem cells that model lung tissue [6]. For inhalation therapy, this scenario represents a significant deviation from physiological conditions, as drugs are deposited in the form of aerosols onto the air-facing bronchial and alveolar epithelium of the lungs. Although several cellular models for the upper respiratory tract are currently available, modeling the distal alveolar region presents several constraints that make the standardization of reliable in vitro alveolar models rather difficult. One of the new versions of those platforms for testing aerosols on cells at the air-liquid interface is the ALICE-CLOUD aerosol delivery system. In this technology, cells modeling lung epithelium (the adenocarcinoma cell line A549) are placed on Transwell plastic inserts and cultured for 24 hours at the air-liquid interface. Then, an aerosol cloud generated by a nebulizer is deposited onto the cells. The authors demonstrated that the therapeutic activity of the aerosolized drug (Bortezomib) upon deposition on the cell surface was comparable to the efficacy of the drug in solution form, but with a faster absorption kinetics. However, this model lacks the capability to cyclically alter the liquid level above the cells, which would mimic the breathing cycle. The Cloud α AX12 platform, utilizing an ultrathin porous AX12 membrane, represents a system that is capable of simulating the breathing cycle through membrane deformation. This system provides key physiological conditions for cultivating lung epithelial cells, including the air-liquid interface and three-dimensional cyclic stretch. Another comparable model is based on a stretchable and biodegradable membrane made of collagen and elastin on a gold mesh, which mimics a network of tiny alveoli with sizes close to those found in vivo [7].

 

Aim

Aim - the purpose of the study is to develop a platform that mimics the internal surface of pulmonary alveoli, consisting of a layer of cells located at the air-liquid interface. This platform aims to replicate the breathing cycle and enhance the delivery of charged aerosol particles to the cell surface through the application of a directed electric field.

Materials and methods

Cell cultures

The A549 cell line was obtained from the vertebrate cell culture collection at the Institute of Cytology, Russian Academy of Sciences.

Reagents

Doxorubicin, glucose, 96% ethanol, bisbenzimid, propidium iodide, and calcein (Sigma-Aldrich, USA); DMEM/F12 culture medium (Paneco, Russia) supplemented with 10% fetal bovine serum (Biosera, France); Molykote High Vacuum Grease (Dow Corning, USA).

Materials for device fabrication

AnyCubic Basic Clear polymer resin for 3D printing (China); track membrane made of polyethylene terephthalate with a pore size of 0.4 µm (LLC "Reatrak-Filter," Obninsk, Russia); laser module with a wavelength of 650 nm TXL-04 (China); photodiode FDK-155 (Russia).

Experimental setup for depositing charged aerosol onto the cell layer

The schematic of the setup is presented in Figure 1 (A - exploded view, B - longitudinal section of the chamber). The cell layer (1) is pre-cultured on a porous membrane (2). Beneath the porous membrane, there is a reservoir containing the culture medium (3), connected via a pipe (4) and a flexible tube to a pressure control system (not shown in the diagram), allowing for controlled and cyclic changes in the pressure applied to the liquid beneath the membrane. The pressure control system consists of a 50 ml plastic container connected to the reservoir (3). The container is mounted on a special holder connected to a movable stage (a stepper motor (Parts Hub Store, China) is connected to the stage via a worm gear). The motor is controlled through an open-source Arduino UNO microcontroller board. Pressure is regulated by creating a height difference between the liquid levels in the movable container and the chamber. The aerosol delivery system over the cell layer includes a quadrangular chamber, the lower wall of which is made of a metal plate (5), while the upper wall is made of glass with a conductive coating (6). All plastic components of the chamber are fabricated from dielectric material using an Anycubic photopolymer 3D printer (China). An opening with an area of 1 cm² is created in the lower wall of the chamber, where the porous membrane with cells is placed. At opposite ends of the chamber, there are pipes for aerosol circulation (7).

The aerosol flow rate through the chamber was maintained at 2 L/min for all experiments. The conductive walls of the chamber were connected to opposite poles of a high-voltage power source. The reservoir containing the culture medium was connected to the metallic part of the lower wall of the chamber via a detachable contact. An optical system for monitoring cell hydration was positioned on the upper wall of the chamber, consisting of a pair of lasers (8) and photodiodes (9) aligned opposite each other through a layer of insulating material and directed at equal angles toward the surface of the membrane. The laser was connected to a power supply, while the photodiode was connected to a voltage meter. The electric field within the chamber was generated using a high-voltage source VIDN-30 (OST, Russia), applying a voltage of 10 kV, with the negative pole of the source connected to the membrane containing the cells.

Experiments with the cell layer exposed to air

The cells were cultured on the surface of a polyethylene terephthalate track membrane with a pore size of 0.4 µm. Prior to cell application, the membrane was treated with low-pressure air plasma for 5 seconds to enhance wettability and improve adhesion properties. Subsequently, using an automatic pipette and a specialized stencil with an area of 1 cm², a suspension of cell culture at a density of 10^5 cells/cm² ± 1.6% in DMEM/F12 culture medium supplemented with 10% fetal bovine serum was applied. After deposition, the membrane with cells was placed in a CO2 incubator for two days at a temperature of 37.1 °C to allow for the formation of a monolayer adhered to the membrane surface. The resulting cell layer samples were positioned on the liquid surface in the chamber reservoir filled with culture medium (the cell layer was oriented toward the air-facing side of the membrane). The membrane was then secured using a clamping plate, and by adjusting the liquid level in the vessel containing the medium, which was flexibly connected to the reservoir in the chamber, the required “negative” pressure was generated beneath the membrane. Finally, the upper and lower parts of the chamber were connected, and readings from the optical sensor were recorded.

Analysis of Cell Viability

After 2 days of cultivation at 37 °C in an atmosphere containing 5% CO2, cells were stained with 2 µM calcein AM, 1 µM propidium iodide, and 1 µM bisbenzimide for 15 minutes at 37 °C. Cell viability was assessed by analyzing microphotographs obtained using a fluorescent microscope (Axiovert 200M, Zeiss, Germany). The cell count was performed using the open-source software ImageJ (NIH ImageJ). A minimum of five fields of view were analyzed for image assessment. The cell survival parameter was calculated as the ratio of the number of viable cells in the sample to the number of viable cells in the control, expressed as a percentage. All experiments were conducted in triplicate (n = 3). Data are presented as mean ± standard deviation. A two-sample t-test was employed to evaluate statistically significant differences. A p-value of <0.05 was considered statistically significant for all statistical assessments. Statistical analysis was conducted using Origin 2022 software (OriginLab Corporation, USA).

Preparation and Characterization of Charged Nanoaerosols

Charged nanoaerosols with positively charged aerosol particles were generated using a charged nanoaerosol generator based on the principle of electro-spraying followed by gas-phase electron neutralization [8, 9]. A solution of the solid material forming the aerosol was atomized from a capillary connected to a positive high voltage source. A capillary connected to a negative source was filled with 96% ethanol. The electric current through the positive and negative capillaries was measured at 100 nA and 40 nA, respectively. The aerosol generation rate was 2 L/min. The size distribution of aerosol particles was measured using an aerosol spectrometer (DAS-2702, AeronanoTech, Russia). The mass concentration of aerosols was determined using a dust meter (KANOMAX 3521, Japan).

Measurement of the Amount of Substance Deposited on the Membrane

To assess the quantity of nanoparticles deposited per unit area of the membrane, a porous membrane was replaced with foil of equivalent area. Aerosol deposition was conducted in a chamber at a voltage of 10 kV for 30 minutes. Subsequently, the deposited aerosol particles were washed off the surface of the foil using 10 µL of phosphate buffer. The concentration of doxorubicin in the obtained solution was determined using a spectrophotometer (Cary 100 Scan, USA).

Results

Evaluation of exposure parameters for cells in the aerosol deposition chamber that do not result in cell death

A549 lung carcinoma cells were cultured on the membrane for 48 hours to form a monolayer. Subsequently, sterile air was pumped through the chamber above the cell layer at a flow rate of 2 L/min with varying exposure times and different values of negative pressure applied to the liquid beneath the membrane. Following exposure, the membrane with the cells was fully immersed in culture medium. Cell viability was assessed 24 hours later.

The relationship between cell viability and the value of negative pressure applied to the liquid beneath the membrane during a 10-minute exposure in the aerosol deposition chamber is illustrated in Figure 2. Exposure of the cells at negative pressure magnitudes values ranging from 0 to 3 cm did not result in the death of a significant proportion of cells (~90% viable cells). The relationship between cell viability and exposure time at a negative pressure of 2 cm of water column applied to the liquid beneath the membrane is presented in Figure 3. Increasing the exposure time to 30 minutes resulted in a gradual decrease in the proportion of surviving cells to approximately 70%.

 

Verification of Optical Sensor Efficiency

Figure 4 presents a typical dependency of the sensor readings on the negative pressure applied to the liquid beneath the membrane. The pressure is generated by creating a height differential between the liquid in the reservoir beneath the membrane and that in the communicating movable vessel. Therefore, the pressure on the graph is expressed in units of water column height. As the pressure decreases, the cells protrude more from the liquid surface, resulting in less reflected laser light detected by the photodiode. At a negative pressure of 2 cm of water column and below, cells can be visually observed protruding above the liquid layer on the membrane surface. For a clean membrane surface, the relationship between light scattering and pressure variation is virtually absent. All subsequent experiments were conducted at a negative pressure of 2 cm of water column applied to the liquid beneath the membrane unless otherwise specified.

Assessment of Cytotoxicity of Electro-Sprayed Water and Ethanol Products, as well as Non-Toxic Nanoaerosol (Glucose) Deposited on A549 Cell Monolayers

Control experiments were conducted in which glucose nanoaerosol was deposited on the cell monolayer for 10 minutes, along with the exposure to the products of electro-spraying from the generator using pure water and ethanol for the same duration. A 10 mg/mL aqueous solution of glucose was aerosolized in the generator to produce the glucose aerosol. The particle size distribution of the resulting nanoaerosol is shown in Figure 5A. The mass concentration of the aerosol was 0.58 ± 0.04 µg/L, with an average aerosol particle diameter of 55 nm and a particle concentration of 79 ± 3 x 10³/cm³. Due to the fact that the current flowing through the positive capillary of the generator was 2.5 times higher than that through the negative capillary, the aerosol produced contained a significant fraction of positively charged aerosol particles.

After exposure, the membrane with the cells was fully immersed in culture medium. Cell viability was assessed 24 hours later. The results of the cell viability analysis from the control experiments are presented in Figure 6.

Analysis of the Cytostatic Effect of Doxorubicin Hydrochloride in Nanoaerosol Form on A549 Cell Culture

The nanoaerosol form of doxorubicin hydrochloride was obtained by pulverizing a solution of doxorubicin at a concentration of 3 mg/mL in 30% aqueous ethanol. The particle size distribution of the resulting nanoaerosol is presented in Figure 5B. The mass concentration of the aerosol was 0.45 ± 0.03 µg/L, with an average particle diameter of 90 nm and a particle concentration of 197 ± 27 x 10³/cm³.

Following the deposition of the aerosol, the membranes with the cells were incubated in culture medium for 72 hours, after which cell viability was assessed. The cell survival parameter was calculated as the ratio of the number of viable cells in the sample to the number of viable cells in the control, expressed as a percentage. The relationship between cell survival and the duration of aerosol deposition is shown in Figure 7. A characteristic inverse relationship between the proportion of surviving cells and the duration of aerosol deposition is observed. The LD50 for doxorubicin aerosol particles is reached with an exposure duration of 2 minutes.

The efficacy of the drug was also evaluated at a higher level of cell hydration (the negative pressure applied to the liquid beneath the membrane was changed from 2 to 0.5 mm of water column) and in the absence of voltage applied to the chamber. Both modifications to the aerosol deposition conditions resulted in a significant increase in cell survival (Figures 7A and 7B).

Assessment of the Quantity of Aerosol Particles Deposited Per Unit Area of the Membrane Over Time

According to measurements, 0.10 mg of doxorubicin settles on 1 cm² of membrane per minute, corresponding to the deposition of 1.0E+12 particles with a diameter of 90 nm. Additionally, the mass and quantity of aerosol particles deposited per cell were estimated, based on that the average diameter of A549 cells is 13 µm [10]. It was estimated that 1.7E+06 nanoparticles of doxorubicin settle on each cell per minute, corresponding to a total mass of deposited particles equal to 0.17 ng.

Assessment of Cell Viability During Operation of the System in the Mode of Cyclic Changes in Cell Layer Hydration

Experiments were conducted to evaluate the pressure control system under the simulation of breathing and cyclic exposure conditions for a duration of 60 minutes. The range of negative pressure values applied to the liquid beneath the membrane varied from 0.3 to 2 cm of water column. An illustration of the laser sensor operation for monitoring cell hydration in cyclic mode is presented in Figure 8. The peaks of maximum light scattering by the cell layer coincided temporally with the points of application of the largest value of negative pressure and vice versa. For comparison, another experiment was conducted with static exposure of the cells to air for the same duration at a pressure of -2 cm of water column. After exposure, the membrane with the cells was fully submerged in culture medium. Cell viability was assessed 24 hours later. The pressure change modes and results of the viability analysis for A549 cells are presented in Figure 9 (A – Breathing simulation mode; B - Cyclic exposure mode; C – Static exposure; D – Viability of the cells). It was demonstrated that cyclic modes with periodic increases in the hydration level of the cell layer allow for maintaining cell viability at over 80% for the duration of 60 minutes. In contrast, after exposure for the same duration under static conditions, cell viability was significantly lower, averaging around 60%.

Discussion

In this study, tests were conducted on an experimental "lung-on-chip" platform that simulates the inner surface of pulmonary alveoli. This platform consists of a cell layer positioned at the air-liquid interface and is capable of mimicking the respiratory cycle. To control the degree of cellular immersion in the liquid, the cell layer is cultured on a porous membrane fixed to the surface of a reservoir containing the culture medium. The placement of cells on a porous membrane for modeling the distal regions of the lungs has been previously described in the literature [7, 11]. One of the primary distinctions of the proposed design compared to existing analogs is the method for altering the liquid level above the cells, which involves the pressurization of the culture medium through the pores of a stretch-resistant membrane based on the pressure applied to the liquid beneath the membrane. The membrane is firmly secured to the reservoir surface, preventing deformation. The fixed position of the membrane enables the operation of an optical sensor, which represents the second significant difference of the proposed design from existing models. The software of the setup allows for the management of the pressure level applied to the liquid beneath the membrane via a PC, recording and retrieving real-time readings from a laser sensor measuring humidity, as well as implementing a cyclic pressure variation mode with specified frequency and amplitude. The connection of conductive walls within the chamber to a high-voltage source facilitates the creation of a directed electric field perpendicular to the aerosol flow passing over the cell layer, which is connected to one of the electrodes. The presence of an electric field can significantly enhance the delivery rate of charged aerosol particles to the cell surface. The charge on the aerosol particles can be imparted by interaction with charged air ions generated by a commercial source or through electro-spraying in a generator that produces a charged nano-aerosol via electro-spraying [9, 12]. The overall schematic of the main components of the experimental setup is illustrated in Figure 10.

The human adenocarcinoma alveolar basal epithelial cell line A549 was utilized as a model culture to model pulmonary epithelium. This cell line is widely employed as a model for pulmonary epithelial cells in drug metabolism studies [13] and in "lung-on-a-chip" models [14]. The cells were cultured on a plasma-activated porous membrane surface for 48 hours to form a monolayer. Exposure of the cells on the membrane surface within an aerosol deposition chamber, while sterile air was pumped at a flow rate of 2 L/min and negative pressure values ranged from 0 to -3 cm H₂O for 10 minutes, did not result in significant cell death, as over 90% of the cells maintained viability 24 hours post-experiment. According to measurements from the laser sensor, light scattering was distinctly observed at a pressure of -2 cm H₂O due to the protrusion of the cellular monolayer membranes from the liquid.

For the static operation mode of the system, an exposure time of 10 minutes and a maximum applied negative pressure of 2 cm H₂O were selected. Additionally, operational modes with periodic increases in the hydration level of the cell layer were tested. Two patterns of periodic pressure variation were chosen: first one involving continuous pressure modulation to simulate the expansion and contraction of pulmonary alveoli during respiration (Figure 9A). As indicated by the laser sensor readings, the periodic pressure changes were actually associated with fluctuations in the hydration levels of the cells. The second pattern is the cyclic exposure method, in which cells are predominantly exposed to air but periodically immersed in culture medium (Figure 9B). This approach aims to maximize the deposition of aerosol particles onto the cell surfaces. It was demonstrated that in both operational modes, periodic immersion of the cells in liquid significantly extended the duration of exposure of the cell layer at the liquid-air interface (from 10 minutes to 1 hour) while maintaining a cell viability level above 80%. The incorporation of this system into the experimental setup allows for a substantial increase in the potential duration of aerosol deposition and facilitates the study of interaction patterns between the model epithelium and aerosol in a simulated breathing scenario.

It was shown that the aerosolized products of water and ethanol from the generator, as well as a non-toxic glucose aerosol, did not exhibit cytotoxic effects upon deposition. This data is consistent with previous studies by the authors, which demonstrated that under standard operating conditions of the generator, there is minimal formation of reactive oxygen species [15]. This also indicates that the particles, when deposited in an electric field, do not possess sufficient kinetic energy to disrupt the cell membrane upon contact with the cell surface.

The specific activity of the aerosol form of the antitumor cytostatic drug doxorubicin hydrochloride was evaluated in relation to the A549 lung carcinoma cell culture. The LD50 for doxorubicin aerosol particles is reached with an exposure duration of 2 minutes. At the dosage delivered over 10 minutes, nearly complete cell death is observed (Figure 7). Based on estimates of the number of deposited particles, approximately 10¹³ doxorubicin particles with an average diameter of 90 nm, totaling a mass of about 2 ng, settle on each cell within 10 minutes. Direct comparison of the aerosol dosage with the LD50 for the liquid phase is challenging; however, as shown in Figure 7A, an increase in cell hydration corresponding to changes in the pressure applied to the liquid beneath the membrane results in more than a threefold increase in cell survival at the same deposition time. This is likely due to the fact that some aerosol particles dissolve in the liquid instead of contacting the cell membrane. According to literature, direct contact of aerosol particles with cells can enhance the absorption rate of certain drugs by up to 12 times compared to solutions in water [14]. Thus, the preservation of the specific activity of the drug upon interaction with cells in nanoaerosol form has been demonstrated. The model aerosol of a non-toxic substance (glucose) does not exhibit toxic effects under similar conditions.

According to data presented in Figure 5B, the aerosol of doxorubicin has an average particle size of 90 nm. Particles of this size, when inhaled, are capable of penetrating deep into the lungs and reaching the alveoli [16]. The deposition of nanoparticles within the body is expected to occur predominantly in the acinar region of the lungs via a diffusion mechanism [17]. The difference in deposition rates between doxorubicin particles in the chamber under the influence of an electric field and the deposition of aerosol with equivalent particle diameters in the lungs can be estimated using the Mathematical Particle-Phase Deposition (MPPD) model, which predicts aerosol deposition efficiency in the lungs of mice based on particle diameter [18, 19]. For particles with a diameter of 90 nm, the MPPD model predicts a deposition efficiency of 39% in the lungs of mice. The surface area of the lungs in mice is approximately 600 cm² [20], and the volume of air inhaled per minute is about 32 mL. Therefore, based on the volumetric concentration of nanoparticles in the doxorubicin aerosol, which is 197 ± 27 x 10³/cm³, and the deposition efficiency data, approximately 3600 aerosol particles will settle on 1 cm² of lung surface per minute when inhaling an uncharged aerosol of 90 nm diameter. In contrast, in the chamber of the setup, the deposition rate on the same area would be 10^12 particles/min. Consequently, the presence of a charge on the aerosol particles allows for the use of an electric field to increase the deposition rate by approximately 8 orders of magnitude. This enables the use of a small area cell layer sample for the rapid deposition of a large number of aerosol particles, significantly accelerating the acquisition of data regarding the interaction of aerosol particles with cells, cytotoxicity, drug efficacy, and other related parameters. It should be noted that charged aerosol particles will also deposit more effectively in the lungs compared to neutral particles, even in the absence of an external electric field, due to the electrostatic interactions between the particle and the induced mirror charge that occurs when a charged particle approaches a conductive surface in the airways [21].

Conclusion

In this study, we conducted tests using an experimental "lung-on-a-chip" platform that simulates the interaction between inhaled aerosol particles and the pulmonary epithelium on the inner surface of the pulmonary alveoli. The model pulmonary epithelial cell culture is cultivated on a porous polymer membrane positioned on the surface of a reservoir containing the culture medium. The membrane with the cells is placed inside a chamber through which the aerosol under investigation is pumped. For the first time, a method for assessing the degree of scattering of a laser beam reflected from the surface of the cell layer was employed to evaluate the extent of cell immersion in the liquid. The setup allows for the control of the pressure level applied to the liquid beneath the membrane, real-time recording of the laser sensor readings for hydration, and the implementation of a periodic pressure variation mode with specified frequency and amplitude, simulating the breathing cycle of pulmonary alveoli while maintaining a high level of cell viability for at least one hour. The preservation of the specific activity of the drug (doxorubicin hydrochloride) upon interaction with adenocarcinoma lung cells in nanoaerosol form was demonstrated. The model aerosol of a non-toxic substance (glucose) did not exhibit toxic effects under similar conditions. It was shown that the presence of a charge on the aerosol particles enables the use of an electric field within the chamber to increase the deposition rate by approximately 8 orders of magnitude compared to the diffusion deposition of neutral particles of similar size. This allows for the use of a small area cell layer sample for the rapid deposition of a large number of aerosol particles, significantly accelerating the acquisition of data regarding the interaction of aerosol particles with cells, cytotoxicity, and the efficacy of the deposited substance. As a result, the developed setup represents a comprehensive platform for studying the inhalation toxicity of natural and artificial aerosols, as well as for assessing the safety and efficacy of aerosolized forms of drugs in situ.

Источник финансирования. Научное исследование проведено при поддержке Российского научного фонда (грант РНФ №23-25-00478).

Funding source. This work was supported by the Russian Science Foundation (Grant No. 23-25-00478).

Конфликт интересов. Авторы декларируют отсутствие явных и потенциальных конфликтов интересов, связанных с публикацией настоящей статьи.

Competing interests. The authors declare that they have no competing interests.

Вклад авторов. Все авторы подтверждают соответствие своего авторства международным критериям ICMJE (все авторы внесли существенный вклад в разработку концепции, проведение исследования и подготовку статьи, прочли и одобрили финальную версию перед публикацией). Наибольший вклад распределён следующим образом: И.Л. Канев — концепция работы, дизайн и испытания экспериментальной установки, проведение экспериментов, обзор литературы написание текста и редактирование статьи; М. В. Верхолашин — дизайн и испытания экспериментальной установки, проведение экспериментов; М.Е. Тайлаков — проведение экспериментов, анализ данных, анализ литературных источников, редактирование статьи; О.Ю. Антонова — дизайн и проведение экспериментов, анализ данных, написание текста и редактирование статьи.

Author contribution. All authors confirm that their authorship meets the international ICMJE criteria (all authors have made a significant contribution to the development of the concept, research and preparation of the article, read and approved the final version before publication). I.L. Kanev — concept of the work, design and testing of the experimental setup, experimental procedures, literature review, writing the text and editing the article; M.V. Verkholashin — design and testing of the experimental setup, experimental procedures; M.E. Tailakov — experimental procedures, data analysis, analysis of literary sources, editing the article; O.Y. Antonova — experimental design and procedures, data analysis, writing the text and editing the article.

Благодарности. Авторы выражают свою признательность: научному сотруднику ИТЭБ РАН Кочетковой Ольге Юрьевне за консультации в работе с клеточными культурами и плодотворные дискуссии.

Acknowledgments. We acknowledge the help of O.Y. Kochetkova (ITEB RAS) in our work with cell cultures and fruitful discussions.

Figure 1. Schematic representation of a chamber for the deposition of aerosol onto cells. A - exploded view, B - longitudinal section of the chamber. The description of the specified elements is given in the main text.

Figure 2. Dependence of the viability of A549 cells on the magnitude of negative pressure applied to the liquid under the membrane during 10-minute exposure in an aerosol deposition chamber.

Figure 3. Dependence of the viability of A549 cells on the exposure time in the chamber at a negative pressure value applied to the liquid under the membrane equal to 2 cm Н₂O.

Figure 4. Dependence of liquid level sensor readings on the negative pressure applied to the liquid under the membrane.

Figure 5. Size distribution of aerosol nanoparticles generated by spraying solution in generator: A - 10 mg/ml glucose in water, B - 3 mg/ml doxorubicin hydrochloride in 30% ethanol.

Figure 6. Viability of A549 cells after deposition of glucose nanoaerosol and water electrospray products for 10 min.

Figure 7. A549 cell survivability as a function of doxorubicin nanoaerosol deposition time. A – doxorubicin aerosol deposition for 5 minutes with increased cell hydration (negative pressure equal to 5 mm Н₂O is applied to the liquid under the membrane). B – doxorubicin deposition for 5 minutes in the absence of an electric field inside the chamber.

Figure 8. Dependence of liquid level sensor readings on the pressure applied to the liquid under the membrane in cyclic mode.

Figure 9. Testing the operating modes of the chamber with periodic pressure changes. A–C – different pressure changes patterns. D – Viability of the A549 cell culture after 60 minutes of exposure in the chamber.

Figure 10. A schematic illustration of the setup for studying the effects of aerosol deposition on a cell layer in a lung-on-a-chip model.

About the authors

Igor Leonidovich Kanev

Federal state budget institution of science Institute of theoretical and experimental biophysics RAS

Email: 4kanev@gmail.com
ORCID iD: 0000-0002-6860-0630
Scopus Author ID: 55253110400
ResearcherId: JRY-6005-2023

PhD

Russian Federation, 142290, Moscow region, t. Pushchino, Institutskaja str., b.3

Mikhail Viktorovich Verkholashin

Federal State Budgetary Educational Institution of Higher Education "Tula State University", Tula

Email: www.mike2016@mail.ru
Russian Federation, 300012, Tula Oblast, Tula, Lenin Prospect, b. 92

Maxim Evgenievich Taylakov

Federal state budget institution of science Institute of theoretical and experimental biophysics RAS

Email: max29111999@gmail.com
Russian Federation, 142290, Moscow region, t. Pushchino, Institutskaja str., b.3

Olga Yurievna Antonova

Institute of theoretical and experimental biophysics

Author for correspondence.
Email: ol_antonova@mail.ru
ORCID iD: 0000-0003-3311-8745

Ph.D., senior researcher at genome research laboratory

Russian Federation

References

Supplementary files