1. Introduction
The airway epithelium is directly exposed to the external environment and, consequently, pollutants, such as cigarette smoke, dust, silica, and other biological threats that can result in infection and inflammation. This, in combination with individual genetic and lifestyle factors, can lead to susceptibility to developing chronic bronchopulmonary diseases such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis and acute respiratory distress syndrome. Biochemical signalling for inflammation and oxidative stress are the drivers for a number of these lung diseases [
1,
2,
3,
4]. Different research studies present in the literature are focused on the prevention of chronic bronchopulmonary diseases by the modulation and reduction of inflammation and oxidative stress. While in the present research work, we verify whether the presence of one mechanical barrier layer on the cells could reduce the possibility of starting the inflammatory and oxidating mechanism caused by the direct contact with external environmental hazards, such as UD, which is produced in urbanised areas because of environmental pollution.
Specifically, hyaluronic acid, a major component of the extracellular matrix and is composed of glucuronic acid and N-acetyl glucosamine units, has recently become the focus of respiratory-related studies. The central role of hyaluronic acid in the respiratory system has been associated with tissue homeostasis through different factors such as hydric balance and biomechanical integrity [
5,
6,
7,
8]. Characteristically, hyaluronic acid or sodium hyaluronate is known to be an ingredient that can form a physical barrier (hydrogel) when exposed to aqueous solutions that might play an important role in the biomechanical integrity of the tissues [
9,
10]. Sittek et al. showed when dissolved in artificial saliva, the presence of sodium hyaluronate increased the viscosity of the saliva. The polymers absorb the saliva forming three-dimensional solution structures stabilised by hydrogen or ionic bonds, thus creating a physical barrier [
11]. Hyaluronic acid is very often used in combination with other compounds such as mannitol. Mannitol is a naturally occurring sugar alcohol found in the sap of the manna tree and many vegetables and is generally regarded as safe. It can be used as an excipient in dry powder inhaler (DPI) formulations, where it can act as a carrier molecule to enhance the flow ability and inhalation performance of the product [
12,
13]. Moreover, mannitol has been theorised to help preserve the viscoelastic properties of hyaluronic acid [
14].
PolmonYDEFENCE/DYFESATM is a brand name of a medical device product that consists of a DPI formulation based on sodium hyaluronate and mannitol as principal ingredient and excipient, respectively, delivered with the disposable monodose PillHaler® DPI device. The associated formulation was developed as a preventative therapeutic, which can act as a defensive barrier to protect the upper respiratory tract from environmental hazards.
With the present study, we hypothesised that the formation of the barrier layer by PolmonYDEFENCE/DYFESATM blend on the epithelium will exhibit greater protective effects against environmental hazards in Calu-3 epithelia in vitro, resulting in a reduction of inflammation and oxidative stress in the epithelium through an indirect protective mechanism based on the barrier layer. This is because the presence of the barrier layer prevents the environmental hazard from being in direct contact with the epithelium.
To test this hypothesis, our study aimed to conduct a comprehensive assessment (
Supplementary Figure S1) of the protective effects in the upper respiratory tract and the primary bronchi of the barrier-forming product PolmonYDEFENCE/DYFESA
TM on the lung epithelium in vitro. This will be achieved by, firstly, determining its protective abilities against environmental hazards such as exposure to environmental pollutants and wounds. Secondly, by establishing the deposition of sodium hyaluronate delivered using the PillHaler
® DPI device into a pharmacopoeia-approved impactor (Anderson cascade impactor).
2. Materials and Methods
2.1. Materials
Hyaluronic acid (Altergon Italia Srl, Morra De Sanctis, Italy), mannitol (Giusto Faravelli S.p.A., Milan, Italy), the PolmonYDEFENCE/DYFESATM (medical device product formulated and patented by SOFAR S.p.A., Trezzano Rosa, Milan, Italy) and the PillHaler® DPI Device patented by Hollycon Italy (Gaggiano, Milan, Italy) and used as supplied. Urban dust (UD) was purchased from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA). Calu-3 cells were supplied by American Type Cell Culture Collection (ATTC, Rockville, MD, USA). Transwell® polyester cell inserts (0.33 cm2 surface area, 0.4 μm pore size) and Snapwell™ polyester cell inserts (1.12 cm2 surface area, 0.4 μm pore size) were obtained from Corning by Sigma-Aldrich (Sydney, Australia). Triton® X-100, L-ascorbic acid, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), Menadione and 200 mM L-glutamine solution were purchased from Sigma-Aldrich (Milan, Italy, and Sydney, Australia). Other cell culture reagents, including Dulbecco’s Modified Eagle’s medium/F-12, phosphate buffer saline (PBS) and foetal bovine serum (FBS), were obtained from Gibco by ThermoFisher Scientific (Sydney, Australia). Methyl tetrazolium salt (MTS) reagents were purchased from Promega (Sydney, Australia). Enzyme-linked immunoassay (ELISA) kits for the determination of the inflammation markers interleukin-6 (IL-6) and interleukin-8 (IL-8) were obtained from BD Bioscience (Sydney, Australia). Water was purified by Milli-Q reverse Osmosis (Molsheim, France).
2.2. Cell Culture
The lung epithelial cancer-derived Calu-3 cell line (ATCC HTB-55) was chosen as in vitro respiratory epithelium model. Cells were cultured between passages 26 to 36 in 75 cm
2 flasks containing Dulbecco’s Modified Eagle’s medium/F-12 enriched with 10% (
v/
v) foetal bovine serum (FBS), 1% (
v/
v) non-essential amino acids solution and 1% (
v/
v) L-glutamine solution. Cells were maintained in a humidified 95% air, 5% CO
2 atmosphere at 37 °C until confluency was reached. The medium was replaced three times a week and cells were passaged according to American Type Culture Collection Recommendations—ATCC guidelines. This cell line has been well optimised to reflect the main characteristics and secretory activity of the airway epithelium when grown at an air-liquid interface (ALI) [
15]. For the ALI model, cells were seeded at a density of 7.92 × 10
4 cells/insert on a Transwell polyester insert (0.33 cm
2 growth area) containing 100 µL in the apical chamber and 600 µL in the basolateral chamber. The medium from the apical chamber was removed after 24 h from seeding and every day afterwards until an ALI was achieved, while the medium from the basolateral chamber was replaced every second day up to 14 days of culture. Snapwell polyester inserts (1.12 cm
2 growth area) were used for impaction studies with a cell density of 1.58 × 10
5 cell/insert and ALI culture was established using 2 mL of media in the basolateral chamber.
2.3. Cytotoxicity Assay
MTS assay was performed on Calu-3 cells to measure cytotoxicity on cellular metabolic activity. MTS assay is a colorimetric test based on tetrazolium reduction into formazan. This reaction occurs only in active metabolic cells [
16]. Briefly, 100 μL of 5 × 10
4 cells/well were seeded into a 96 well-plate. After 48h, cells were exposed to pre-warmed media containing a series of 2-fold dilutions of urban dust, the blend, and equivalent concentrations of Na-Hya and mannitol. Background controls (medium) and untreated controls (untreated cells) were included in the experiment, as well as a positive control containing 20% DMSO. A blank plate (cell-free) with equal concentrations of urban dust was used to correct for absorbance reading from the urban dust alone. After 2 h or 24 h of incubation with treatments at 37 °C, in a humidified atmosphere at 5% CO
2, Calu-3 cells were incubated for another 2 h, in the same conditions, with 20 μL of MTS solution (20%
v/
v). Finally, the 96 well-plate was read at 490 nm using a SpectraMax M2 plate reader. The absorbance values were directly proportional to cell viability (%). Experiments were performed in triplicate. Data were expressed as % cell viability relative to untreated control and plotted against compound concentrations (mg/mL).
2.4. Analysis of Intracellular Reactive Oxygen Species (ROS)
Oxidative stress was evaluated by quantifying intracellular ROS produced by Calu-3 cells treated with 0–1 mg/mL % Na-Hya and equivalent concentration of the PolmonYDEFENCE/DYFESA
TM, with and without UD induction. ROS levels were determined by converting the non-fluorescent DCFH-DA into the fluorescent dichlorofluorescein (DCF). Briefly, 100 μL of Calu-3 cells were seeded with a density of 5 × 10
4 cells/well into a 96 well-plate (black, clear-bottom) and incubated overnight at 37 °C in a humidified atmosphere at 5% CO
2. Afterwards, Calu-3 were incubated in the dark for 30 min (37 °C, 5% CO
2) with 100 μL of 5 μM DCFH-DA [
17]. Then, the DCFH-DA-containing medium was removed and 100 μL of treatments were added to DCFH-DA-loaded cells, protected from light. Background controls (medium), untreated and unlabelled controls (untreated and unlabelled cells), untreated controls (untreated cells), negative controls (cells treated with 5 mM N-Acetyl Cysteine) and positive controls (cells treated with 100 µM menadione) were included in the experiment. Plates were read immediately at time 0 min and after incubation (37 °C, 5% CO
2) at the annotated times by a SpectraMax microplate reader with an excitation filter set at 485 nm and an emission filter set at 520 nm. Experiments were performed in triplicate and results were expressed as fold change of ROS production over time 0min. The antioxidant activity of the samples was also investigated by their ability to reduce oxidative stress in Calu-3 cells after induction of ROS production by UD.
2.5. Transepithelial Electrical Resistance (TEER)
Transepithelial electrical resistance (TEER) of Calu-3 cells in ALI culture was measured as described previously [
18]. Briefly, pre-warmed Hanks’ Balanced Salt Solution (HBSS) was added to the apical chamber and allowed to equilibrate for 30 min at 37 °C under 5% CO
2. TEER was measured using EVOM2
® epithelial voltohmmeter (World Precision Instruments, Sarasota, FL, USA) connected to STX-2 chopstick electrodes at the annotated conditions. Blank controls (cell-free inserts containing HBSS) and untreated controls (inserts of cells in medium) were included in the study. Experiments were performed in triplicate. TEER (Ω cm
2) was calculated from the measured potential resistance difference (Ω) between the apical and basolateral sides, normalised by subtracting the blank insert and multiplying by the surface area of the Transwell or Snapwell inserts, according to the following equation:
2.6. Sodium Fluorescein Paracellular Permeability
The functionality of tight junctions and paracellular permeability of the cell layer was investigated using the sodium fluorescein permeability assay. Briefly, sodium fluorescein (2.5 mg/mL) (Sigma Aldrich) was added to the apical chamber and pre-warmed HBSS was added to the basolateral chamber. Transwells or Snapwells were incubated for 4 h at 37 °C with 5% CO
2, with basolateral samples (100 µL) collected at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3 and 4 h to measure the rate of transport (flux) of the sodium fluorescein from the apical chamber to the basolateral chamber. For analysis, the collected basolateral sample fluorescence was measured using the SpectraMax M2 plate reader (excitation: 485 nm; emission: 538 nm). The permeation coefficient (Papp) was calculated according to equation 2, where
is the volume in the basolateral chamber,
is the surface area of the Transwell membrane,
is the initial concentration in the apical chamber, and
is flux (cumulative) of Na-Flu through the membrane.
2.7. Pro-Inflammatory Marker Expression on ALI Modelled Calu-3 Epithelial Layer
The inflammatory response to exposure to urban dust with and without the PolmonYDEFENCE/DYFESA
TM barrier layer was evaluated in the ALI-generated Calu-3 epithelial layers by the detection of the pro-inflammatory cytokines IL-6 and IL-8 using ELISA kits according to the manufacturer’s instructions. The inflammatory trigger UD and test compounds were deposited onto the epithelial layers as a suspension in HPFH (2H,3H-decafluoropentane, used as model propellant), as previously reported [
19,
20]. HPFH is a hydrophobic and highly volatile propellant, which can be handled as a liquid at ambient pressure and evaporates rapidly after exposure to air, thus leaving the epithelial layer exposed to the dry powders only. After 24 h of exposure, the media from the basolateral chamber was collected for interleukin quantification.
2.8. Wound Healing Study with ALI Culture
The wound-healing assay, also known as the scratch assay, was performed on the ALI model of Calu-3 cells. Experiments were performed after 14 days of ALI culture. On the apical side of the cell layer, a scratch was made with a pipette tip (P200 μL) along the diameter of the Transwell membrane. Na-HYA and the blend were deposited on top of the wound using HPFP and control cells were treated with an equal volume of HPFP only. Transwells were kept in a humidified chamber at 37 °C in a 5% CO
2 atmosphere and 95% humidity, and the wound was observed using a Nikon Eclipse Ti microscope (Nikon, Tokyo, Japan) with Coolsnap ES2 camera. Pictures were taken every 20 min for the first 2 h, followed by every 30 min for 18 h using NIS-Elements (version 3.22.01, Nikon Instruments Inc., New York, NY, USA) after formulation deposition. The images were analysed using Fiji ImageJ, measuring the wound closure area using an in-house macro. The percentage of wound closure was calculated using the following Equation (3), where
At is the wound area at a given time and
A0 is the initial wound area.
2.9. Impaction Studies Using the Andersen Cascade Impactor (ACI)
The deposition profile of the product PolmonYDEFENCE/DYFESATM across the ACI stages was investigated. The eight-stage ACI, including a USP induction port, was connected to a rotary vein pump (Westech Scientific Instruments, Essex, UK) and the flow rate was adjusted to 60 L/min using a calibrated flow meter (TSI Model Instruments, Shoreview, MN, USA). To minimise particle bouncing, 50 µL of Brij 35: glycerol: ethanol (10:50:40 v/v/v) solution was used to coat ACI plates. After a single shot (dose 30 mg/shot) deposition at 60 L/min for 4 s, the ACI was disassembled, and every stage was washed separately using HPLC grade water to collect the blend from each section. Emitted dose (ED) was defined as the amount of DPI that leaves the device (mouthpiece to the ACI). Experiments were performed in triplicate and Na-Hya was quantified using high-performance liquid chromatography (HPLC).
2.10. Sodium Hyaluronate Chemical Quantification by HPLC
Na-Hya detection and quantification were conducted using high-performance liquid chromatography (HPLC) system equipped with SPD-20A UV–Vis detector, an LC-20AT liquid chromatography, a SIL-20A HT autosampler (Shimadzu, Kyoto, Japan) and a BioSep SEC-S2000 column (300 × 7.8 mm, 5 µm, 145A, Phenomenex, Torrance, CA, USA). The mobile phase was 0.05M KH2PO4, pH 7.0. Samples were analysed at 205 nm, a flow rate of 1 mL/minute and an injection volume of 10 µL. Linearity was obtained between 2.5 and 500 µg/mL (R2 = 0.99) with a retention time of 5.0 min.
2.11. Scanning Electron Microscopy (SEM)
Powder samples were placed on adhesive black carbon tabs and mounted onto aluminium stubs. The samples were gold coated with a sputter coater (BAL-TEC SCD 005, Tokyo, Japan) and particles were examined under a scanning electron microscope (JEOL-JCM 6000 NeoScope Benchtop SEM, Tokyo, Japan) at 100× magnifications using 15 keV accelerating voltage.
2.12. Statistical Analysis
The data are presented as the mean ± standard deviation of three independent experiments. Statistical analysis was performed using Prism software version 8.0 (GraphPad, San Diego, CA, USA). Means were compared by one-way analysis of variance (ANOVA) followed by the annotated tests for multiple comparisons.
4. Conclusions
This study showed a comprehensive assessment of the potential protective effect of PolmonYDEFENCE/DYFESATM product, based on an inhaled formulation of sodium hyaluronate (key ingredient) and mannitol (excipient), delivered as DPI using the PillHaler® DPI device. It was observed that most of the blend was deposited in the ACI throat up to the primary stages (S0 and S1) without deposition in the lower stages of the ACI, confirming that the target of the product is the upper respiratory tract up to the primary bronchi without any deposition in the lower stages of the impactor. This aligns with its intended purpose since it could be used as a localised physical barrier for the upper respiratory tract to protect against external harmful pollutants, including potentially viruses and bacteria. Furthermore, it was demonstrated that the presence of the barrier prevents the direct contact of UD with the cells, reducing in this way the inflammatory response caused by UD. It was also observed to significantly be more effective in wound healing compared to the main ingredient alone.
While in the solubilised form, the formulation could give indirect protection against the UD and reduce levels of oxidative stress in the short term (2 h) without exhibiting any cytotoxic effects. This protection can be attributed to the viscous solution formed by sodium hyaluronate present in the product can bring to reduce the deposition of the UD on the cells and consequently guarantee its protection.
Collectively, our data demonstrate the exciting prospects of using the PolmonYDEFENCE/DYFESATM blend as a physical protective barrier against environmental pollutants. The formulation demonstrates significant potential to be used as a protective and preventative mechanism to protect the upper respiratory system in highly polluted environments or instances when an extra layer of protection for the respiratory system is desired by the user.