## 2 Materials and Methods
### Benchmark Surfactant Aerosol Deposition Studies  
According to the in vivo compatibility check, the Westmed (infant size) and Fisher & Paykel premature NCs were selected for benchmark studies. The in vitro set-up is depicted in Figure 1 and consisted of a heated, humidified flow-generating device (OptiflowTM or Fabian ventilator, as appropriate), a customized eFlow Neos nebulizer, the corresponding NCs, a 3D-printed cast of the upper airways of a 32 weeks' gestation premature infant (PrINT model) [31], and a breath simulator programmed with the sinusoidal breathing pattern of a premature infant: \(V_{\mathrm{T}}\) of 8.9 mL, RR of 70 cycles/min and an I:E of 40/60. The nebulizer was placed right before the bifurcation of the NCs. A surfactant-collecting filter (PARI filter PAD, PARI Pharma, Starnberg, Germany) was placed in the distal airway of the PrINT model to estimate the surfactant lung dose. The PrINT model resembles the upper airways of a premature infant and is based on the three-dimensional reconstruction of a magnetic resonance image obtained from a baby born at 32 weeks' gestation, who had a birth weight of 1750 g [31]. Therefore, 350 mg of undiluted _Poractant alfa_ (4.375 mL), a dose equivalent to 200 mg/kg for the PrINT neonate, was nebulized in each in vitro experiment. After the full surfactant dose was delivered, the set-up was carefully dissembled, and the surfactant was extracted from each component using a rinsing solvent containing chloroform/methanol 50:50. The combined amount of surfactant deposited in the nebulizer and within the NCs was determined. The surfactant deposited onto the face of the PrINT model was recovered with a tissue, and together with the surfactant extracted from the upper airways of the cast (i.e., before the glottis) represented the total surfactant recovered from the PrINT model. The surfactant collected in the backup trap resembles the fraction of surfactant that impacts the PrINT's airway and moves forward as a liquid film. Lastly, the surfactant collected in the inhalation filter represents the surfactant aerosol lung dose. A validated high-pressure liquid chromatography (HPLC) method with the appropriate external standard calibration  
Figure 1: Scheme of the experimental setup. PrINT, Premature Infant Nose Throat-Model. was used for quantifying PC [13], the lead compound of _Poractant alfa_. The experiments with each type of NC were repeated at least three times. In Vivo Effect of Nebulized Surfactant during HFNC with the OptiflowTM System in Surfactant-Depleted Rabbits  
Eighteen surfactant-depleted adult rabbits were allocated to one of the three experimental groups: animals in the \"**HFNC (Control)**\" group (n = 6) were extubated after the confirmation of respiratory distress and managed with HFNC ventilation. Animals in the \"**SF Instillation + HFNC**\" group (n = 6) received a clinical dose of intratracheal _Poractant alfa_ (200 mg/kg) before extubation and were then managed with HFNC. Finally, animals in the \"**HFNC + SF (400 mg/kg)**\" group (n = 6) were extubated and received a nominal dose of 400 mg/kg of nebulized _Poractant alfa_ during HFNC ventilation. In all groups, HFNC ventilation was delivered at a flow of 8 L/min via the OptiflowTM system with the Fisher & Paykel red premature NC for 180 min. Arterial blood gases were withdrawn and analyzed for arterial carbon dioxide partial pressure (PaCO2) and PaO2 (Radiometer Medical, Copenhagen, Denmark) at baseline (healthy lung status), after the confirmation of respiratory distress (right before extubation), and at regular intervals after shifting to HFNC, at 15 and 30 min after starting the HFNC, and then every 30 min until the end of the experiment. At the end of the follow-up period (180 min), all animals were re-intubated and briefly managed with positive pressure ventilation using the same ventilation settings as at baseline. This way, the dynamic compliance (Cdyn) at baseline (healthy status), after the confirmation of respiratory distress, and after treatment could be compared.