Aerosol surfactant therapy (AST) for respiratory distress syndrome (RDS)

Abstract

New formulations and techniques for aerosol therapy are superior to clinical-practice liquid bolus instillation in the critical areas of required dose (order-of-magnitude reduction), delivery time, biological response rate, and efficacy. An exemplary dry powder synthetic lung surfactant (SLS) formulation includes one or more phospholipids (PL) and one or more hygroscopic excipients and high dispersibility (e.g., particle fractions less than 5 μm greater than 88% and greater than 98%, at 0.5 and 4.0 bar respectively). An exemplary method includes emitting the formulation as an aerosol from an inhaler at a fine particle dose (FPD) <5μm equal to or greater than 5.0 mg PL / kg patient mass over one or more aerosol delivery periods forming a total therapy time.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent App. No. 63 / 639,794, filed April 29, 2024, the complete contents of which are herein incorporated by reference. FIELD OF THE INVENTION This invention is generally directed to dry powder formulations and therapies and, more particularly, synthetic surfactant powder formulations and therapies for rapid delivery and onset of action. BACKGROUND Pulmonary surfactant deficiency is associated with lung immaturity and the acute onset of atelectasis, reduced compliance, arterial hypoxemia, and development of respiratory distress syndrome (RDS), a major cause of morbidity and mortality in premature (< 34 weeks GA) infants worldwide. Surfactant is an essential biochemical compound composed primarily (80%) of phospholipids (PL) and surfactant proteins that form a lipid bilayer between the gas-liquid interface of the terminal airways and developing alveolar sacs. Surfactant contributes to lung stability by coating the internal surfaces and neutralizing the surface tension of the fluids that coat the acinar structures, preventing lung collapse, improving compliance, and maintaining pulmonary gas exchange. Figure 1A illustrates in a side-by-side comparison normal alveoli with collapsed alveoli. Surfactant replacement therapy (SRT) substantially reduces mortality and disability in premature neonates suffering from RDS. All commonly used surfactant administration techniques (i.e. endotracheal tube (ETT) delivery; INSURE (INtubate-SURfactant-Extubate), LISA (less invasive surfactant administration), MIST(minimally invasive surfactant therapy)) require intubation beyond the vocal cords with a tube or catheter and instillation of large liquid volumes (1.2-4 mL / kg) directly to the lungs. For instance, the standard therapeutic approach for exogenous surfactant involves the intratracheal liquid instillation of 100-200 mg PL / kg. This method relies heavily on natural animal-derived liquid surfactant formulations and highly skilled personnel trained to perform tracheal intubation with an endotracheal tube (ETT) and manage a mechanical ventilator. Ventilator assistance is vital for supporting infants experiencing substantial deterioration in lung mechanics during treatment and aiding in the dispersion of the liquid surfactant plug from the fluid-filled trachea, which is distributed into the peripheral lung units over several hours. Although it is lifesaving, standard SRT also poses considerable treatment risks to the patient, including acute clinical deterioration from transient clogging or complete obstruction by residual surfactant in the airways, leading to a nonuniform distribution of the liquid medication in the lung periphery, reduced pulmonary compliance, hypoxemia, hypercarbia, hemodynamic instability, pulmonary hemorrhage, and ventilator malfunction. Further side effects of the conventional invasive approach can include oxygen desaturation (frequently requiring resuscitation), bradycardia, pneumothorax, hemodynamic instability, and reduced cerebral blood flow. The ventilation duration following surfactant instillation typically spans several hours or even days, which, even in the short-term, predisposes infants to acute lung inflammation, ventilator-induced lung injury (VILI), delayed surfactant response, and neurological impairment. These treatment risks are well recognized, leading to routine use of early noninvasive respiratory support (i.e., continuous positive airway pressure, CPAP) immediately after birth, with selective surfactant administration considered in deteriorating infants. Nebulized liquid surfactant aerosol has been investigated as a potential treatment for RDS. However, clinical trials in preterm infants receiving surfactant aerosol have failed to significantly improve mortality, morbidity, and other important outcomes. Most nebulizers used for SRT generate aerosol during the entire respiratory cycle, although some are now breath synchronized. The high aerosol fraction that is not inhaled but still produced during exhalation (50-75% of the nebulized dose) ends up depositing in the ventilator or CPAP system, airway interfaces, and conducting airways. To account for these high extrapulmonary losses, it is common to apply multiples of the standard liquid surfactant doses to increase lung delivery, which still provides only minimal clinical responses with aerosol, making therapy timeconsuming, cost-prohibitive, and potentially unsafe, especially in developing countries where premature births and RDS mortality are high and access to surfactant, appropriate clinical monitoring, and ventilators is scarce. Despite the ongoing efforts over the last three decades to improve surfactant formulations and nebulizer performance, nebulized aerosol administration has yet to be established as a standard clinical practice. Recent advances in breath synchronized next generation mesh nebulizer technology developed by Aerogen Pharma (San Mateo, CA) have been applied to deliver surfactant aerosols to neonates, and when coupled with CPAP, was shown to be safe and well tolerated in a population of premature infants. However, doses of surfactant remained high, with patients receiving between one and three nebulized doses (194 mg PL / kg per nebulized dose), which were equivalent to two (194 mg PL / kg) and six (583 mg PL / kg) standard liquid doses over a prolonged treatment period (Jardine L, Lui K, Liley HG, Schindler T, Fink J, Asselin J, Durand D. Trial of aerosolised surfactant for preterm infants with respiratory distress syndrome. Arch Dis Child Fetal Neonatal Ed. 2022;107(l):51-5). As an alternative to nebulized liquid surfactants, dry powder synthetic surfactant aerosol combined with an inhaler and manual resuscitator or ventilator was reported over 40 years ago in preterm infants with RDS and has only recently been advanced. Assuming the technical challenges of dry powder surfactant could be met, this could improve survival in the millions of preterm infants that die globally each year from RDS and improve morbidity in all resource settings. Animal model studies over multiple decades have demonstrated the use of aerosol surfactant therapy (AST) for infant RDS-like conditions using both liquid nebulized and dry powder aerosols. Considering human subject testing with liquid nebulized aerosols, at least six surfactant aerosol trials have been conducted in preterm infants over the last six years with the primary outcome of non-invasive ventilation (NIV) failure and progression to invasive mechanical ventilation. Results of these clinical studies have varied between significant reduction in NIV failure rate, success only in older age groups or non-significant trends, and no improvement. Dry powder AST has not progressed to human subjects testing in recent years. Typical limitations of AST in previous animal models and human subject trials in comparison with the current clinical practice of liquid bolus instillation through an endotracheal tube or intubation catheter include (i) higher administered dose for an adequate therapeutic response, (ii) longer delivery times, (iii) equivalent or slower response times, and (iv) muted biological response at similar or increased aerosol doses. Considering the need for a higher required dose of the active phospholipid (PL) components when delivered as an aerosol, instilled liquid bolus products typically administer 100-200 mg-PL per infant kg of body weight (mg-PL / kg). In comparison, recent animal model and human subject studies with aerosol surfactants have administered 100-800 mg-PL / kg. After intubation, surfactant delivery by liquid bolus instillation requires approximately 10-15 min to complete. In comparison, liquid nebulized aerosol surfactant therapy typically requires 30 minutes to 2.5 h. The high dose ARCUS® dry powder aerosol reduced the delivery time to 15 min in an animal model, but required -400 device actuations timed with infant respiration and produced a muted biological response (Walther FJ, Waring AJ, Otieno M, and DiBlasi RM: Efficacy, dose-response, and aerosol delivery of dry powder synthetic lung surfactant treatment in surfactant-deficient rabbits and premature lambs. Respiratory Research. 2022;23(l): 1 -16). In summary, AST typically requires a substantially longer administration time and higher dose compared with liquid bolus instillation. Response rate to surfactant replacement therapy is often evaluated in animal models based on recovery of oxygenation (or blood gases) and lung biomechanics metrics over time, either after surfactant administration or at the start of AST. Consistently, response time to AST has been either slower than or equivalent with liquid bolus instillation. Often, liquid bolus instillation provides a faster initial response in PaOi (albeit at the cost of higher PaCOo and ventilatory pressure requirements than aerosol) and then blood gas levels and lung biomechanical metrics continue to recover over a period of 2-3.5 h. The response rate is also significantly impacted by the level of lung damage in the RDS model and the use of positive-pressure lung inflations, typically associated with mechanical ventilation or positive-pressure resuscitation. Considering biological response, several studies have reported similar or muted efficacy with AST compared with liquid bolus instillation. For example, in the recent nebulizer-based study of DiBlasi et al. (DiBlasi RM, Micheletti KJ, Zimmerman JD, Poli JA, Fink JB, and Kajimoto M: Physiologic effects of instilled and aerosolized surfactant using a breath-synchronized nebulizer on surfactant-deficient rabbits. Pharmaceutics. 2021; 13( 10): 1580) with a rabbit surfactant washout model of RDS, identical aerosol and instilled doses of Alveofact produced similar biological improvements in most metrics considered. Moreover, final PaO2 on 50% oxygen did not exceed 175 mmHg and lung compliance was reduced from pre-treatment baseline at the time point of 180 min after AST. Considering dry powder aerosol delivery of the Walther et al. study identified above, using ARCUS® particle technology at a powder dose of 240 mg / kg and a similar rabbit surfactant washout model, blood oxygenation and airway compliance values only recovered to 63% (approximate final arterial oxygenation = 300 mmHg on 100% oxygen) and 54% (final compliance = 0.49 mL / cm HoO) of pre-washout values, which were well below and slower than observed with liquid bolus instillation. SUMMARY While efficient aerosol delivery to preterm infants is challenging, many of the limitations of current AST can be overcome, and high-efficacy performance over liquid bolus instillation realized, with a dry powder surfactant aerosol with high activity rapidly and efficiently delivered to the deep lung regions, and preferably, all the way to the alveoli. Embodiments include a dry powder aerosol synthetic lung surfactant (SLS) product which accommodates aerosol administration through endotracheal tube (ETT), trans-nasal direct-to-infant, and non-invasive ventilation (NIV) interfaces. Significant components of an exemplary aerosol SLS product include use of the small-particle aerosol excipient enhanced growth (EEG) delivery and targeting strategy and a dry powder micrometer-sized SLS-EEG formulation. Within this disclosure, “formulation” and “composition” may be used interchangeably. A “dry powder formulation” / “dry powder composition” may be used to refer to a dry powder which is in an aerosolized form (i.e., an aerosol). Likewise, “dry powder formulation” / “dry powder composition” may be used to refer to a dry powder which is in a non-aerosolized form (e.g., a powder bed). A significant characteristic of exemplary formulations and methods of delivery is micrometer sized primary particles for high efficiency delivery to the alveolar regions of the lung. Moreover, the particles are highly dispersible particles to enable dispersion at the flow rates and volumes compatible with neonatal lung delivery. That said, though the present technology is demonstrated with models tailored to infants, exemplary formulations and methods can be used with patients of any age including adolescents and adults. Exemplary formulations are scalable. The combination of rapid delivery of a dispersible and micrometer sized dry powder synthetic surfactant formulation that is capable of targeted delivery to the alveolar region of the lungs with high efficiency (e.g., >60% of the dose delivered via intratracheal (IT) aerosolization) enables therapeutic response that is quick and achieved at lower doses compared to either liquid instillation or aerosol nebulization. An exemplary SLS-EEG formulation currently contains one or more pharmaceuticalgrade phospholipids (e.g., DPPC and POPG), hygroscopic excipients (e.g., D-mannitol (MN) and sodium chloride (NaCl)) and a dispersion enhancer (e.g., 1-leucine). In some embodiments such formulation may further include a synthetic peptide mimic of surfactant protein B (B-YL). In some alternative embodiments, however, the SLS-EEG formulation expressly excludes any surfactant proteins and synthetic peptide mimic of surfactant proteins. Some exemplary embodiments of this disclosure enable a dose of surfactant formulation of less than 50 mg phospholipid I kg to the subject, and preferably less than 30 mg phospholipid I kg using delivery flow rates less than 7 L / min in an infant and less than 45 L / min in an adult with actuation air or gas volumes of less than 11 mL / kg and preferably 6-7 mL / kg. The volume of delivered gas depends on the weight / mass of the infant or subject, and is typically in a range of 4-10 mL / kg. The composition is delivered at a rate of more than 10 mg phospholipid I second and preferably more than 20 mg phospholipid / second. An exemplary formulation (i.e., composition) may be spray dried to produce particles less than 2 pm, preferably less than 1.5 pm, and more preferably <1.3 pm, depending on the embodiment. SLS-EEG aerosol surfactant therapy offers a number of advantages including the avoidance of intubation for lung surfactant administration, elimination of side effects associated with intubation and surfactant liquid bolus instillation, improved efficacy at reduced doses (with associated reduced costs), and the ability to administer the therapy repeatedly as needed, possibly as a preventative measure, due to a low-risk and high-reward therapeutic profile. Surfactant-based formulations described in this disclosure may be used alone as a medication or co-formulated with other therapeutics to treat a range of lung conditions. Such other therapeutics may include but are not limited to anti-inflammatory medications, antibiotics, antivirals, gene therapies, and monoclonal antibodies. Some embodiments comprise bolus delivery of milligram quantities of phospholipid (PL) surfactant formulation per second, enabling treatment to be completed in less than 1-5 minutes. Some embodiments are characterized by unexpected efficacy at -20-30 mg PL / kg nominal doses and estimated lower lung doses (based on realistic in vitro model testing that extends through at least airway generation G4; bifurcation B3) of 12-15 mg PL / kg (or more). Some embodiments are characterized by unexpected in vivo efficacy at -20-30 mg PL / kg nominal doses and estimated total lung doses (based on realistic in vitro model testing that extends to the trachea) of 15-18 mg PL / kg (or more). This is unexpected because the current clinical dose is approximately 100-200 mg PL / kg administered once or multiple times to a subject through liquid bolus instillation to achieve an adequate response. As described, other aerosol surfactant therapies have typically needed to administer the same or higher doses of PL / kg to match the results of surfactant administered through liquid bolus instillation. Previous work has not reported aerosol surfactant therapy administered at lower doses as being more effective than liquid bolus instillation. Some embodiments comprise aerosolization at flow rates and volumes compatible with neonatal lung capacities. Some embodiments comprise formulations and delivery methods with strong therapeutic activity without surfactant protein or surfactant protein peptide mimic (i.e., the formulation is free of surfactant protein and surfactant protein peptide mimic). Some embodiments comprise enhancement of surfactant PL function through the inclusion of 1-leucine or similar weak surfactant amino acids. According to an embodiment, a composition that when delivered to the lungs as a small particle aerosol (0.5-2 pm) using low flow rates (2-5 L / min) and air volumes (5-15 ml) enables rapid (1-30 mins) reversal of respiratory distress syndrome at doses -10 times lower (10-30 mg Phospholipid / kg) than current therapies. According to some embodiments, compositions are micrometer sized dry powder formulations consisting of phospholipids (PL) (DPPC, POPG), leucine as a dispersion enhancer, mannitol and sodium chloride as Excipient Enhanced Growth (EEG) excipients. A surfactant protein mimic may or may not be included. According to some embodiments, an exemplary composition contains 60-70 % phospholipids, 15-25% leucine, 5-15% mannitol, 4-10% sodium chloride, and 0-5% surfactant protein peptide mimic (BYL). According to some embodiments, the phospholipid composition contains 70-100% DPPC and 0-30% POPG. According to some embodiments, a small particle aerosol is designed to minimize inhaler and upper airway losses and increasing in size using the EEG mechanism to enable targeted deposition in the alveolar airways. The composition may be prepared by spray drying a low concentration (0.05 - 0.5 %w / v) dispersion containing both hydrophilic and hydrophobic components in a water (95%) / ethanol (5%) vehicle, for example. According to some embodiments, an exemplary composition is highly dispersible with mean geometric diameter dispersed at 0.5 bar < 2.0 pm and dispersed at 4.0 bar < 1.3pm. Corresponding values for particle fractions less than 1 pm were >20% and >35%, at 0.5 and 4.0 bar respectively. Corresponding values for particle fractions less than 5 pm were >88% and >98%, at 0.5 and 4.0 bar respectively. According to some embodiments, an exemplary dry powder composition and method of delivery to an infant, child or test animal enables the efficient aerosolization of a mass of spray dried SLS powder formulation with very low flow rates (2-5 L / min) that are not traditionally used for dry powder inhaler formulations. The composition and method of delivery enables the efficient aerosolization of a mass of spray dried SLS powder formulation with very low air volumes (5-15 mL or 4-10mL / kg) that are not traditionally used for dry powder inhaler formulations. The composition and method of delivery enables the efficient aerosolization of a mass of spray dried SLS powder formulation over short durations of delivery (100-300 msec) that are not traditionally used for dry powder inhaler formulations. According to some embodiments, an exemplary dry powder composition and method of delivery enables a high delivery rate during inhalation of spray dried SLS powder formulation in the range of 5-50 mg / sec of formulation during inhalation which corresponds to 3-30 mg / sec of phospholipids per inhalation. The dry powder composition and method of delivery enables high fractional delivery of spray dried SLS powder formulation to the lower lung of an in vitro rabbit lung model with -60% of the 30 mg loaded dose delivered equivalent to 18 mg of formulation or 10.8 mg of phospholipid delivered in 5 x 200 msec actuations which is equivalent to a delivery rate during inhalation of 10.8 mg / sec of phospholipid. The dry powder composition and method of delivery enables micrometer size aerosol particles to be generated and delivered through millimeter and sub-millimeter size airway passages with high efficiency to target delivery in the lower lungs. The small particle aerosol was active at doses 10-30 mg phospholipid / kg which is approximately -10 times lower doses compared to other aerosol products and the liquid instillation (100-300 mg Phospholipid / kg). According to some embodiments, an exemplary small particle aerosol is capable of delivering > 70% of the powder formulation from the inhaler using less than 10 air actuations and preferentially less than 4 air actuations to deliver 30 mg of formulation equivalent to 18 mg Phospholipid. According to some embodiments, an exemplary dry powder composition and method of delivery enables a balanced aerosol mass per actuation delivery rate of, for example 30 mg x 4+ actuations and >70% device emitted dose (or 60 mg x 8+ actuations): i) <6.67 mg / kg of formulation delivered per actuation ii) <4 mg / kg of phospholipid delivered per actuation According to some embodiments, an exemplary dry powder composition and method of delivery enables a high rate of increase in PaO21 FiCE levels in a surfactant depletion animal model in the range of 5-15 mm Hg per min when the composition is delivered at doses of 20-30 mg phospholipid / kg. According to some embodiments, an exemplary composition and method of delivery enables an increase in dynamic lung compliance increasing by 70-90% of post lavage washout levels and recovering to 70-90 % of baseline levels within the first 15-30 minutes post administration. According to some embodiments, an exemplary dry powder composition and method of delivery limits the amount of hygroscopic (water-soluble) components introduced into the lung over any one time delivery window. A dose delivery window may encompass the rapid administration period + stabilization and monitoring period. An exemplary dry powder method and composition may limit hygroscopic components delivered to the lungs over a 10-30 minute and preferably 15-minute incremental dose delivery period. According to some embodiments, an exemplary dry powder composition contains less than 26 mg of hygroscopic components / kg, and preferably <20 and <15 mg / kg. In comparison with the current clinical practice of liquid bolus instillation through an endotracheal tube or intubation catheter, exemplary embodiments of this disclosure have one or more of the following advantages (and in some embodiments all of the following advantages): (i) lower administered dose for an adequate therapeutic response, (ii) shorter delivery times, (iii) equivalent or faster biological response times, and (iv) significant biological response. Examples 1 and 2 present a two-part study demonstrating that a combination of a D2 air-jet DPI and SLS- EEG NSD1 formulation present advantages (i) and (iv) by achieving equivalent to superior biological response compared with liquid bolus instillation at an order of magnitude reduced PL dose. Example 3 provides in vivo animal model evidence that an aerosol therapy can surpass the performance of liquid bolus instillation in all four areas (i) through (iv), and in most cases with order of magnitude improvements. Example 4 reports the efficacy of a simplified (no B-YL surfactant protein peptide mimic) synthetic lung surfactant excipient enhanced growth (SLS-EEG) dry powder aerosol formulation and delivery method that surpass the performance of liquid bolus instillation in all four areas (i) through (iv). In Example 1 below, a specific SLS-EEG formulation (produced with the Buchi Nano Spray Dryer B-90 HP; resulting in the Nano Spray-Dried or NSD1 formulation) aerosolized using a “D2” DPI device was capable of high emitted dose (> 80% of loaded) and generated an aerosol with a mass median aerodynamic diameter (MMAD) of <2 pm. The D2 device performed well with up to 20 mg loaded powder doses but was limited to < 20 mg mass loadings. Testing with a realistic in vitro rabbit model indicated that over half of the loaded dose could penetrate into the lower lung regions. Using the characterization data, a dose delivery protocol was designed in which a 60 mg total device-loaded formulation mass (24 mg-PL / kg) would be administered and deliver an approximate lung dose of 14.7-17.7 mg phospholipids / kg with a total aerosol delivery period < 5 min. A resulting 15 mg PL / kg to the lower lung was achieved. Using Example 1, it is estimated that for a 60 mg nominal dose of Nano SD SLS-EEG formulation containing 36 mg of PL, approximately 60% of the aerosol would be delivered beyond the fourth lung generation and into the deep lung. Assuming an animal body weight of 1.5 kg would translate to an approximate dose of 15 mg PL / kg directly to the lower lung region. In Example 2 below, evaluates the performance of the D2-NSD1 device and formulation combination and drug delivery profile (DDP) of Example 1 are evaluated in an intubated rabbit model of severe RDS induced by surfactant washout and compared response with liquid bolus instillation of Curosurf (200 mg-PL / kg). The total loaded dose of the dry powder SLS-EEG NSD1 formulation (60 mg containing 24 mg-PL / kg) was administered over multiple dose delivery cycles due to the 20 mg maximum limit of the D2 device (for optimal performance). Despite the order of magnitude lower PL dose of the aerosol product compared with Curosurf, the relatively high lung transmission of the aerosol beyond the upper tracheobronchial (TB) airways (see Example 1), and possibly deeper within the lungs, resulted in near-complete recoveries of arterial oxygenation (PaCE; 96-100% recovery) and oxygenation index over the 3.5 h monitoring period, with a recovery rate that was very similar to Curosurf administered with liquid bolus instillation. Importantly, the aerosol group had superior recovery of lung compliance compared with liquid-instilled Curosurf, as well as less histological evidence of lung tissue injury, greater uniformity in lung aeration and more uniform distribution of surfactant in the lung parenchyma. Total delivery time of the aerosol was ~5 mins; however, total treatment time including device reloading and animal stabilization on the ventilator was ~20 mins. In Example 3 below, the SLS excipient enhanced growth (EEG) dry powder formulation was advanced though production on a scalable nozzle-based spray dryer system and tested with an air-jet dry powder inhaler with a variable-volume aerosolization chamber. The “D3-MSD2” device and formulation combination produced a small-particle aerosol with high fine particle fraction (FPF<5gm=87.9%; FPF<2.5Mm=61.6%) and percent emitted dose (77.4% of loaded). Additional in vitro testing highlighted consistent aerosol size (DV50= 1.6pm) and emitted dose across multiple actuations. In the animal model experiments, a total device-loaded formulation mass of 60mg produced a phospholipid (PL) dose of 24mg-PL / kg and a device-emitted dose of 18mg-PL / kg, compared with the 200mg-PL / kg clinical dose of Curosurf liquid. In vivo response rate for the D3-MSD2 aerosol therapy was considerably more rapid with arterial oxygenation recovering 5-12 times faster than for liquid Curosurf. Biological response for the D3-MSD2 aerosol therapy was also superior with 2-fold improvement in final lung compliance compared to liquid Curosurf. Based on speed and efficiency of delivery, and efficacy of the formulation, an exemplary embodiment includes a method in which the subject is rapidly removed from respiratory support and the therapy is rapidly delivered, followed by a return to conventional respiratory support. For subjects receiving invasive mechanical ventilation, the ventilator is quickly removed from the endotracheal tube (ETT), the aerosol and full inhalation breaths are rapidly delivered through the ETT, and then the subject is returned to the ventilator. For subjects receiving non-invasive respiratory support, as with a face mask or nasal cannula, the conventional respiratory support interface is quickly removed, a dedicated nose-to-lung delivery interface is inserted, the formulation is rapidly administered, and then the conventional respiratory support interface is rapidly returned. Using this approach, which is enabled by the high-efficacy of the formulation and ability for rapid delivery, hardware compatibility with multiple invasive and noninvasive respiratory support systems is not required. That is to say, exemplary methods of this disclosure have the advantage of not requiring any changes or modification to any existing and future invasive and noninvasive respiratory support systems. According to some embodiments, exemplary systems, devices, and methods consistent with this disclosure may include one or more features described in related work by the inventors, namely US9433588, US2020 / 0139058A1 (US11690964B2), U.S. Patent App. No. 17 / 794,875 (US2023 / 0071308A1), U.S. Patent App. No. 17 / 794,660 (US20230173203A1), and WO / 2025 / 072946 (Application No. PCT / US2024 / 049285), all of which are herein incorporated by reference. For instance, some exemplary embodiments may entail dry powder inhaler (DPI) devices in accordance with the foregoing publications. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A is a side-by-side comparison of normal alveoli with collapsed alveoli. Figure IB is a chart providing a high level overview of three distinct but interrelated aspects of exemplary embodiments, namely formulation, delivery system, and delivery method. Figure 2 is a scanning electron micrograph of exemplary spray-dried SLS-EEG dry powder particles at 15k magnification. Figure 3 is a block diagram of an exemplary infant dry powder aerosol delivery system (iDP-ADS). Figure 4 is an exemplary PMC unit depicted in cross-section. Figure 5 is an aerosolization engine and diffusional flow pathway for a “DI” air-jet DPI. Figure 6 is an aerosolization engine and diffusional flow pathway for a “D2” air-jet DPI. Figure 7A is an aerosolization engine and diffusional flow pathway for a “D3” air-jet DPI. Figure 7B is a component of the variable aerosolization chamber of the D3 air-jet DPI including lower unit level indicators. Figure 8 is a diagram of an exemplary method of treatment. Figure 9A is an in vitro model used in Example 1 for realistic aerosol delivery testing including the upper tracheobronchial (TB) airway geometry with outlet diameters (in millimeters) labeled. Figure 9B is an in vitro model used in Example 1 for realistic aerosol delivery testing including the complete Rabbit Lung Chamber (LC) Model. Figure 10 is a scanning electron micrograph of spray-dried SLS-EEG dry powder particles of Example 1 at 15k magnification. Figure 11 is X-ray powder diffractograms of the SLS-EEG powder of Example 1. Figure 12 is dynamic vapor sorption behavior of the SLS-EEG powder of Example 1. Figure 13A is a pressure waveform from Example 1 measured within the Rabbit LC Model for the first gas delivery scenario and maximum pressure of 25.9 cm H2O. Figure 13B is a pressure waveform from Example 1 measured within the Rabbit LC Model for the second gas delivery scenario and maximum pressure of 28.6 cm H2O. Figure 13C is a pressure waveform from Example 1 measured within the Rabbit LC Model for the third gas delivery scenario and maximum pressure of 25.7 cm H2O. Figure 14 is a full dose delivery protocol developed in Example 1 based on multiple product performance factors and biological considerations intended for in vivo efficacy testing in Example 2. Figure 15 Schematic of the Surfactant Aerosol Delivery System (iDP-ADS) used in Rabbits. During aerosol delivery, the animals were removed from the ventilator and delivered aerosol under positive pressure, generated with the DPI actuator (electromechanical timer) and oxygen gas source. The D2 air-jet DPI connected directly to the rabbit ET tube, enabling precise control over the PIP (25 ± 2 cmH20), PEEP of 5 cmH20, Vt of 7 mL / kg, flow of 3 L / min, and actuation time of ~ 0.20-0.22 s. Each positive pressure actuation of the DPI was controlled by manually depressing a PIP / PEEP valve manifold attached to the outlet of the DPI. The DPI employs an adjustable pressure relief valve (> 25 cm H2O) and an analog pressure manometer, akin to a manual resuscitator, to prevent barotrauma. The aerosolization chamber was manually filled with formulation, and the DPI was actuated to deliver the aerosol and inflate the lungs, followed by an approximately 1-s breath hold, after which exhalation was allowed. The animal was promptly returned to the ventilator following dosing. Figure 16 Time schedule for animal experimental procedures. The sequence of procedures for lung lavage to establish surfactant deficiency and lung injury, treatment groups, time schedule for procedures, surfactant dosing, and physiologic measurements are shown. Additional details on the dose delivery protocol and use of the iDP-ADS are provided in Example 1. Figure 17 Gas Exchange. Gas exchange data are shown as mean ± SEM for control (black squares) and surfactant treatment groups during the 210-min study period. The grey dashed line represents the timepoint (0 min) following the completion of liquid Curosurf (black circles) administration and the initial 30 mg dose of SLS-EEG surfactant aerosol (white circles). Among the surfactant groups, arterial oxygenation was not different at any timepoint following administration, and PaO? values fully recovered to the pre-lavage basal levels (96-100%) at 3.5 h. The PaCh values at 30 min did not differ between the surfactant groups, but the liquid Curosurf group had higher PaO2 than did the controls. Both surfactant treatment groups had higher PaCE than the sham controls from 60 min until the 3.5-h endpoint. The PaCOi values in the controls were increased throughout the experiment, and they did not differ between the groups. The liquid Curosurf group had a higher pH than the controls at 180 and 210-min time points. Two-way ANOVA and post-hoc Tukey’s test compared mean differences at each time point between the groups. *p < 0.05, SLS-EEG aerosol vs. liquid CuroSurf. +p < 0.05, control vs SLS-EEG aerosol. #p < 0.05, control vs liquid Curosurf. Figure 18 Lung Compliance and Disease Severity. Gas exchange data are shown as mean ± SEM for control (black squares) and surfactant treatment groups during the 210-min study period. The grey dashed line represents the timepoint (0 min) following the completion of liquid Curosurf (black circles) administration and the initial 30 mg dose of SLS-EEG surfactant aerosol (white circles). The SLS-EEG aerosol group had greater compliance than the liquid Curosurf and control groups from the 150-min timepoint until the end of the experiments. At the 3.5-h timepoint, the compliance values in the liquid Curosurf and sham control group were not different, with only 14% improvement from baseline in the liquid group. In contrast, the surfactant SLS-EEG compliance had doubled and recovered to 60% of the pre-washout value. The OI remained low for the control cases over the 3.5-h observation period. In contrast, surfactant SLS-EEG aerosol and liquid Curosurf groups provided values below five within 90 min and near two at 3.5 h. The VEI was higher in the SLS-EEG surfactant group than the controls at 30 and 120-min time points and did not vary between the surfactant groups. Two-way ANOVA and post-hoc Tukcy’s test compared mean differences at each time point between the groups. *p < 0.05, SLS-EEG aerosol vs. liquid CuroSurf. +p < 0.05, control vs SLS-EEG aerosol. #p < 0.05, control vs liquid Curosurf. Figure 19 Postmortem Histological Evaluation of Lung Tissue. Macro and Microscopic Images are shown between the different groups. The macro and microscopic images of the lungs for the surfactant and control groups are illustrated. The gross macroscopic appearance of the anterior lungs is shown while being ventilated ex vivo with peak inspiratory pressures of 20 cmH20 (19a-c). The control lungs are underinflated with large areas of discoloration suggestive of pulmonary collapse, edema, and hemorrhage (19a). The liquid Curosurf-treated lungs (19b) show moder-ate inflation with areas of heterogeneous ventilation and petechial hemorrhage. The surfactant aerosol SLS-EEG-treated lungs are well-inflated with very little tissue damage (19c). The gross appearance of the excised tracheas following medial incision (19d-f), shows the control animal with a well hydrated and patent trachea (19d). The Curosurf-treated animal (19e) shows abundant foamy liquid assumed to be surfactant obstructing the trachea with overflow out of the airway. The aerosol SLS-EEG-treated animal (19f) reveals a patent trachea free from powder residual or agglomeration and well hydrated. Following dissection, the macroscopic appearance of the excised rabbit lungs is a cross-sectional slice of the lung lobes (19g-i). In the control subject (19g), diffuse hemorrhage with a singular surfactant pool (black triangle) or lung edema fluid on the cut surface of the lung lobe section. The liquid Curosurf (19h) lung sample shows a heterogeneous distribution of what appears to be foamy surfactant at the level of the lobar bronchi and in the lung periphery liquid (black triangles). The SLS-EEG aerosol (19i) sample shows a homogenous layer of less foamy surfactant evenly distributed across the internal surfaces of the transected lung lobe. Figure 20 Time schedule for animal experimental procedures. The sequence of procedures for lung lavage to establish surfactant deficiency and lung injury, treatment groups, time schedule for procedures, surfactant dosing, and physiologic measurements are shown. (SLS-EEG: synthetic lung surfactant-excipient enhanced growth formulation; ABG: arterial blood gas). Figure 21 In vivo testing setup illustrating aerosol delivery from the iDP-ADS using the D3 Air-Jet DPI with the endotracheal tube (ETT) interface. (iDP-ADS: infant air-jet dry powderaerosol delivery system; PIP: peak inspiratory pressure; PEEP: positive end expiratory pressure). Figure 22A Outer surface from SEM imaging of the SLS-EEG MSD2 particles indicating relatively spherical particles with wrinkled outer surfaces. Figures 22B and 22C Outer surface and inner structures from FIB / SEM imaging of the SLS-EEG MSD2 particles indicating intermediate size inner hollow voids. FIB sectioning revealed intermediate sized void structures (-100-300 nm) in most particles >1 pm upon sufficient sectioning. Location 1 in Figure 22B is the start of an addition pore that is revealed at location 2 in Figure 22C with the next FIB cut of -200 nm. Location 3 then indicates the start of a deeper void. Figure 23A Physicochemical characterization of the SLS-EEG MSD2 formulation from X-ray powder diffractograms. Figure 23B Physicochemical characterization of the SLS-EEG MSD2 formulation from dynamic vapor sorption (DVS). The particles experience little water mass uptake until >70% relatively humidity (denoted as % P / Po), which is ideal for dry powder EEG aerosol storage and lung targeting. Figure 24 Maximum intensity plume images for successive actuations of the spray (taken -50 msec after actuation when the leading plume edge has reached -100 mm from the tip of the orifice) with the Spray VIEW system indicating qualitatively consistent emitted dose over each of the four actuations (A1-A4) during a single experimental run (RI) with 30 mg mass loading: (a) Al, (b) A2, (c) A3, (d) A4. The red vertical line indicates 50 mm from the tip of the orifice (origin; where cone angle lines originate) where the cone angles and widths are taken; the red horizontal line indicates the midline as an angle bisector. Scale is 20 x 10 mm (L x H). Figure 25 Representative graphical output for plume intensity (black line, left axis; Spray VIEW), and gas flow (gray line, right axis; Flow meter) for each device actuation of the D3-MSD2 device and formulation combination when delivering a 30 mg loaded mass during a single experimental run (RI) and using one actuation per turn: (a) Al, (b) A2, (c) A3, (d) A4. Figure 26 Representative Spraytec laser diffraction particle size distributions for each device actuation of the D3-MSD2 device and formulation combination when delivering a 30 mg loaded mass during a single experimental run (RI) and using one actuation per turn: (a) Al, (b) A2, (c) A3, (d) A4. Reported Dvso values represent the mean (standard deviation; SD) of the data for each actuation number evaluated over three runs (n=3). Figure 27 Percent Emitted Dose (%ED) and Emitted Mass (mg) for each device actuation (A1-A4) of the D3-MSD2 device and formulation combination when delivering a 30 mg loaded mass and using one actuation per turn. Bars represent mean values evaluated over three runs (n=3) and error bars are standard deviations. Total represents the net emission of all four actuations. Figure 28 Gas exchange data are shown as mean ± SEM for control and surfactant treatment groups during the 210-minute study period. The vertical dashed line represents the timepoint (0 minutes) following the completion of liquid Curosurf (red circles) administration and the initial 30 mg dose of D3-MSD2 (SLS-EEG) aerosol (green circles) or sham control (blue circles). The mean PaO2 was higher in the aerosol treatment group than Curosurf and sham groups. Following the second (final) aerosol (30 mg) and sham deliveries at 15 minutes, the PaO2 values between 30 and 150 minutes were greater with aerosol than the Curosurf and control groups. The Curosurf group had higher PaO2 than the controls. Among the surfactant groups, arterial oxygenation was not different at 180 and 210 minutes, and PaO? recovered to 94% of the pre-lavage values with liquid Curosurf and 99% with aerosol at 3.5 hr. The liquid Curosurf PaCO2 values were not different from controls during the experiment. The aerosol group had statistically lower PaCO2 than the liquid Curosurf and control groups at 30- and 60-minute time points but did not differ between groups following the remainder of the experiment. The aerosol treatment group had greater pH than controls at all time points throughout the experiment. Compared with liquid Curosurf, the aerosol group had greater pH at the 5, 30, and 60-minute time points but was not different at the other time points. Two-way ANOVA and post-hoc Tukey’s test compared mean differences at each time point between the groups. *p<0.05, D3-MSD2 aerosol vs. liquid Curosurf; +p<0.05, control vs. D3-MSD2 aerosol; #p<0.05, control vs. liquid Curosurf Figure 29 Lung compliance, oxygenation index (OI), and ventilation efficiency index (VEI) data are shown as mean ± SEM for control and surfactant treatment groups during the 210-minute study period. The vertical dashed line represents the timepoint (0 minutes) following the completion of liquid Curosurf (red circles) administration and the initial 30 mg dose of D3-MSD2 (SLS-EEG) aerosol (green circles) or sham control (blue circles). The grey dashed line represents the timepoint (0 minutes) following the completion of liquid Curosurf administration and the initial 30 mg dose of D3-MSD2 surfactant aerosol. The lung compliance improved rapidly in the aerosol group 5 minutes following the initial 30 mg surfactant dose and continued to improve following the 30 mg aerosol delivery at 15 minutes. The aerosol group had greater compliance than the liquid Curosurf and control groups from the 5-minute timepoint until the end of the experiments. At the 3.5-hour time, the compliance in the liquid Curosurf and sham control groups was not improved from baseline, but surfactant D3-MSD2 compliance approximately doubled compared to washout baseline and recovered to 91% of the pre-washout value. The VEI was higher in the D3-MSD2 aerosol surfactant group than the controls at 30 minutes, and both surfactant treatment groups had higher VEI than the controls from 60 minutes until the 3.5-hour observation period. Two-way ANOVA and post-hoc Tukey’s test compared mean differences at each time point between the groups. *p<0.05, D3-MSD2 aerosol vs. liquid Curosurf; +p<0.05, control vs D3-MSD2 aerosol; #p<0.05, control vs liquid Curosurf Figure 30 is the setup of Example 4 with a chimeric CPAP in vivo animal model. Figure 31 is the procedure of Example 4. Figure 32 is In Vivo Rabbit Model Arterial Oxygenation data comparing the DPPC / POPG + B-YL formulation of Example 3 with the DPPC / POPG, No B-YL formulation of Example 4. Removal of the surfactant protein mimic did not appear to alter the efficacy of the SLS-EEG aerosol. Figure 33 is In Vivo Rabbit Model Compliance data comparing the DPPC / POPG + B-YL formulation of Example 3 with the DPPC / POPG, No B-YL formulation of Example 4. Removal of the surfactant protein mimic did not appear to alter the efficacy of the SLS-EEG aerosol. Figure 34A shows particle size distributions of the SLS-EEG formulation produced using the Mini SD and Nano SD measured at 4.0 bar dispersion pressure. Figure 34B shows particle size distributions of the SLS-EEG formulation produced using the Mini SD and Nano SD measured at 0.5 bar dispersion pressure. DETAILED DESCRIPTION Figure IB is a chart providing a high level overview of three distinct but interrelated aspects of exemplary embodiments, namely formulation, delivery system, and delivery method. Embodiments of this disclosure include a new dry powder aerosol synthetic lung surfactant (SLS) product capable of rapid and efficient aerosol delivery to the alveolar region that can be applied during invasive mechanical ventilation, non-invasive ventilation (NIV) support, or directly to an infant using the nose-to-lung route (delivery of the aerosol through the nose and to the lungs). The efficacy of aerosol surfactant therapy (AST) can be increased at reduced doses, with associated reduced cost, if highly active surfactant formulations can be rapidly and effectively delivered to the lungs, beyond the lobar bronchi and preferentially all the way to the alveolar region with a relatively homogeneous deposition pattern. A central aspect of improved AST is the dry powder formulation 101. An exemplary dry powder synthetic surfactant formulation comprises one or more phospholipids (PL) and one or more hygroscopic excipients. The formulation may be produced using spray drying techniques with a nozzle-based or mesh-based nebulizer system, for example. In its finished form suitable for packaging and distribution, an exemplary dry powder formulation is one or more of: (i) is dispersible to a mean geometric diameter of less than 2.0 pm at 0.5 bar and a mean geometric diameter of less than 1.3pm at 4.0 bar, (ii) contains particle fractions less than 1 pm greater than 20% and greater than 35%, at 0.5 and 4.0 bar, respectively, and (iii) contains particle fractions less than 5 pm greater than 88% and greater than 98%, at 0.5 and 4.0 bar respectively. Compared with nebulized liquids, potential advantages of the dry powder formulation 101 include the formation of a smaller-particle aerosol with less input energy, more rapid delivery, improved stability and the ability to better integrate delivery flow pathways with existing ventilation equipment. An exemplary but non-limiting example of formulation 101 comprises or consists of pharmaceutical-grade phospholipids (DPPC and POPG), a synthetic surfactant protein mimic (B-YL), hygroscopic excipients (MN and NaCl) and a dispersion enhancer (1-leucine). Further details of these various components are discussed below in connection with Figure 2, for example. Significant elements of delivery 103 include an excipient enhanced growth (EEG) aerosol delivery strategy of the highly dispersible micrometer-sized surfactant formulation. An exemplary EEG targeting strategy involves beginning with a micrometer-sized particle dry powder aerosol that can efficiently penetrate the delivery system, infant (or test animal) extrathoracic airways and upper lung regions. Hygroscopic growth excipients (e.g., mannitol and sodium chloride) included in the formulation cause the micrometer-sized aerosol to increase in size during transport into the lungs through condensational growth and help foster alveolar deposition as opposed to the dose exhalation that would otherwise occur. A balance is often required with an initial aerosol size that is just small enough for effective lung entry but that also has sufficient size and growth potential (in the form of excipient content) to achieve deep lung deposition. With high dose applications, it is also important to control the total mass and delivery rate of the powder and excipients to the lungs. A high efficiency dry powder aerosol delivery system 102 is important hardware for performing the delivery 103. Components of an exemplary delivery system 102 may include a positive pressure gas source, aerosolization engine, pressure monitoring and control (PMC) unit, diffusional flow pathway, and a patient interface. Exemplary air-jet dry powder inhaler (DPI) devices include an aerosolization engine (comprising at least an aerosolization chamber) and in many cases a diffusional flow pathway which enable high efficiency aerosol formation across a range of available air volumes. An exemplary air-jet DPI may include one or more small diameter inlet flow passages, an aerosolization chamber, at least one outlet flow passage (capillary), and a diffusional flow pathway. The design of these elements can be engineered to control the amount of aerosol deaggregation, emitted dose (ED), and the formulation delivery rate from the device. An additional component of the delivery strategy 103 to be accomplished with the aerosol delivery system 102 is to administer the aerosol with positive-pressure breaths. For both ETT and nose-to-lung administration of the aerosol, the delivery interface will form an airtight seal with the infant’s airways to facilitate positive-pressure breaths, and exhalation can be controlled through the PMC unit. Positive-pressure administration of the surfactant aerosol is expected to open (recruit) collapsed lung regions in the treatment of RDS, and the subsequent deposition of surfactant with rapid formation of a surface lining layer will maintain the patency of these regions, preventing the inflammation and injury that arise from cyclic opening and collapse of alveoli. Based on ventilation of preterm neonates, targeted maximum and minimum delivered pressures are 25 ± 2 to 5 ± 2 cm H2O, for example. The delivery method 103 achieves these targeted pressures during aerosol delivery with the system 102 across a range of potential lung compliances. With EEG aerosols, it is important to establish that overly rapid aerosol growth does not occur in the upper TB region thereby significantly reducing the fraction delivered to the deep lung. Based on the small number of actuations required to deliver the aerosol with the air-jet DPI (e.g., 3-5 actuations expected), the gas actuation volume (GAV) for some embodiments may be either dry or at room humidity and temperature conditions with minimal impact on the lungs. Above 60% RH, the moisture uptake increased significantly, indicating the hygroscopic nature of the formulation once the 1-leucine barrier layer (if present, depending on the embodiment) is overcome at elevated RH. This biphasic behavior is well suited for the EEG dry powder aerosol application. Specifically, the water vapor sorption behavior indicates low moisture uptake during storage under ambient conditions and elevated uptake at higher humidity similar to the airway environment when hygroscopic growth in the humid lung airways is required for EEG particle size growth to significantly enhance particle deposition and retention. Figure 2 is a scanning electron micrograph of exemplary spray-dried SLS-EEG dry powder particles at 15k magnification. Analysis metrics for embodiments of this disclosure include emitted dose (ED), mass median aerodynamic diameter (MMAD), fine particle fraction (FPF) and geometric standard deviation (GSD). Emitted dose is calculatable as the mass in the loaded dose minus the mass remaining in the device divided by the initial loaded mass. Aerosol size calculations were based on the mass recovered in the NGI (including pre-separator) or using the Malvern Spraytec. Based on an airflow rate of 45 L / min, the NGI stage cut-off diameters were determined using the formula specified in United States Pharmacopeia (USP) 35 (Chapter 601, Apparatus 5). The mass median aerodynamic diameter (MMAD) and fine particle fractions (FPFs < 5 pm or < 2.5 pm or < 1 pm) were calculated using Copley Inhaler Data Analysis Software (CITDAS). A number of the metrics used in this disclosure are characterized on a basis of per kg of patient mass ( / kg). It should be understood that such metrics may be multiplied by the mass of any patient or patient group to cause the unit of measurement to drop the per kg basis. Table I shows representative weight / mass data per age demographic according to the US Center for Disease Control and Prevention (CDC). Infant, toddler, early childhood, and late childhood may be grouped together as “pediatric”. Table I. Representative Weight ranges as 50th percentile per age demographic Term Age Weight 50th percentile in kg Preterm neonate Extremely preterm to term 0.5 kg - <3.3 kg Neonate Up to the 27th day of life 3.3-<4.5 Infant 30 days - 12 months 4.5- <9.9 Toddler 13 months - 2 years 9.9-< 15 Early childhood 3 years - 5 years 15- <21 Late childhood 6 years - 11 years 21 -< 41 Adolescent 12 years - 17 years 41-<64 Adult Older than 17 years >64 Expected adult range 27 kg to 127 kg An exemplary dry powder synthetic lung surfactant (SLS) formulation comprises one or more phospholipids (PL) and one or more hygroscopic excipients. Moreover, the dry powder SLS formulation is characterized by one or more of the following: (i) is dispersible to a mean geometric diameter of less than 2.0 pm at 0.5 bar and a mean geometric diameter of less than 5    1.3pm at 4.0 bar, (ii) contains particle fractions less than 1 pm greater than 20% and greater than 35%, at 0.5 and 4.0 bar, respectively, and (iii) contains particle fractions less than 5 pm greater than 88% and greater than 98%, at 0.5 and 4.0 bar respectively. Such an exemplary formulation may be free of (i.e., without any) surfactant protein and surfactant protein peptide mimic. Alternatively, an exemplary formulation may include a surfactant protein peptide mimic such as 10 but not limited to B-YL. An exemplary formulation may furthermore include at least one dispersion enhancer such as but not limited to 1-leucine. The phospholipid(s) of an exemplary formulation may be but are not necessarily limited to DPPC and / or POPG. Of total PL content of a formulation, the breakdown among types of PL may be, for example, 70-100% DPPC and 030% POPG. The hygroscopic excipient(s) of an exemplary formulation may include but are not 15 necessarily limited to mannitol and / or sodium chloride. The foregoing listed components may be combined together in relative amounts of, for example, 60-70% phospholipids, 15-25% 1-leucine, 5-15% mannitol, 4-10% sodium chloride, and 0-5% surfactant protein peptide mimic, with such percentages being measured by mass (e.g., %w / w). Some exemplary powder formulations have a dispersion of at least Dv50 of 1.32 pm at 0.5 bar and at least Dv50 of 0.82 pm at 4 bar based on laser diffraction powder testing. This small primary particle size together with the inclusion of the hygroscopic excipient enhances dissolution of the surfactant particles during aerosol transport and upon deposition, which is required for rapid and effective surfactant spreading and function. Furthermore, micrometersized primary particles deliver higher particle number concentrations to better coat the large alveolar surface area. That is, higher numbers of particles provide more deposited surface area to coat the alveolar region with a thin layer of surfactant, without increasing the mass of the delivered PL. For example, a primary particle diameter of 0.82 pm provides 6.1-fold more deposited particles than a 1.5 pm diameter particle, and 14.5-fold more deposited particles than a 2 pm diameter particle, all delivering the same total particle mass. In some exemplary formulations, hygroscopic components constitute less than 40% (e.g., no more than 15%) of the dry powder formulation by mass. This feature is significant because hygroscopic components can induce osmotic stress if not fully solubilized during aerosol transport. An exemplary aerosol has an MMAD of 2.3 pm or less. Another exemplary aerosol has an MMAD of 2.0 pm or less. A target EEG initial aerosol size range is expected to be approximately 0.9 to 2.0 pm with growth at current excipient levels to approximately 2.0 to 4.4 pm within the lungs. In some embodiments, the SLS-EEG formulation does not include a liquid diluent (i.e., is free of any liquid diluents). The SLS-EEG formulation does not require a liquid diluent, which minimizes the risks for liquid accumulation, foaming, airway congestion, and obstruction previously observed with standard liquid bolus and nebulized liquid surfactant aerosol administration. Some exemplary SLS-EEG dry powder formulations include a dispersion enhancer to promote deaggregation to micrometer-sized primary particles and hygroscopic growth excipients (e.g., mannitol and sodium chloride) that enable the micrometer-sized aerosol to increase in size by condensational growth during transport through the lungs to promote deep lung deposition. An exemplary dispersion enhancer is L-leucine. Some exemplary formulations include one or more protein mimics. Considering synthetic lung surfactants, third-generation synthetic surfactant protein mimics, such as Mini-B, Super Mini-B and the SP-B protein mimic B-YL, have shown high potential for surface tension reduction and efficacy when combined with pharmaceutical-grade PLs. An inhaler may be used to form an aerosol from any formulations described in or based on this disclosure. An exemplary aerosol according to this disclosure may have an MMAD of, for example, less than 2.5 pm, less than 2.4 pm, less than 2.3 pm, less than 2.2 pm, less than 2.1 pm, less than 2.0 pm, less than 1.9 pm, less than 1.8 pm, less than 1.7 pm, less than 1.6 pm, less than 1.5 pm, less than 1.4 pm, less than 1.3 pm, less than 1.4 pm, less than 1.3 pm, less than 1.2 pm, less than 1.1 pm, less than 1.0 pm, less than 0.9 pm, less than 0.8 pm, less than 0.7 pm, or less than 0.6 pm. In various embodiments, it is desirable that the MMAD be at least 0.5 pm, at least 0.6 pm, at least 0.7 pm, at least 0.8 pm, at least 0.9 pm, at least 1.0 pm, at least 1.1 pm, at least 1.2 pm, at least 1.3 pm, at least 1.4 pm, at least 1.5 pm, or at least 1.6 pm, for example. An exemplary aerosol according to this disclosure may have a fine particle fraction (FPF) <5 pm expressed as a percentage of emitted dose (ED) from an inhaler of, e.g., at least 27% ED, at least 30%ED, at least 40% ED, at least 50% ED, at least 60% ED, at least 70% ED, at least 80%, or at least 88% ED. An exemplary aerosol according to this disclosure may have a fine particle fraction (FPF) <2.5 pm expressed as a percentage of emitted dose (ED) from an inhaler of, e.g., at least 21% ED, at least 30% ED, at least 40% ED, at least 50% ED, at least 60% ED, or at least 62% ED. An exemplary aerosol according to this disclosure may have a fine particle fraction (FPF) <1 pm expressed as a percentage of emitted dose (ED) from an inhaler of at least 9% ED, at least 10% ED, at least 20% ED, or at least 30% ED, for example. Figure 3 is a schematic overview of an exemplary infant dry powder air-jet aerosol delivery system (iDP-ADS) 300. The delivery system 300 is configured for, e.g., the administration of the dry powder aerosol to the lungs of intubated infants or similarly sized test animals. The delivery system 300 is also exemplary for administration of a dry powder aerosol to patients of any age (e.g., all demographics in Table I). In this disclosure, “inhaler” may be used to generally refer to one or more pails of a system 300 excepting the patient interface 304. That is to say, an “inhaler” may comprise one or more (up to all of) air source 301, dry powder inhaler (DPI) 302, and PMC 303. However, the “inhaler” excludes the patient interface 304. Patient interface 304 may be, for example, an endotracheal tube, nasal cannula, or face mask. Elements of an exemplary delivery system 300 may include an electromechanically controlled timer air source (EM Timer) 301, DPI 302(which includes, c.g., an acrosolization engine and a diffusional flow pathway and connector for making a connection with a patient interface), pressure monitoring and control (PMC) unit 303, and patient interface 304, e.g., endotracheal tubes (ETTs), nasal cannula, or face mask. For infant subjects, exemplary ETT internal diameters are 2.5 to 3.5 mm. An exemplary EM Timer includes a digital pressure regulator, micro-solenoid valves, and a timer control unit. The PMC unit may be connected in the diffusional flow pathway very near the ETT to minimize additional rebreathing dead space (e.g., < 0.5 mL). An exemplary system 300 is configured for delivering a powder bolus (of, e.g., 30 mg or less) over a series of positive pressure actuations. Intratracheal administration is but one exemplary form of delivery. Alternatives include delivery through neonatal nasal airways to the lungs, for example. Some delivery systems may fully automate and synchronize inspiratory delivery of dry powder surfactant aerosol with intrinsic breathing efforts so infants can receive more effective dosing at lower inspiratory times (< 1 s). Some embodiments comprise transnasal dosing strategies that focus on applying AST in spontaneously breathing infants receiving nasal CPAP and other noninvasive support. Figure 4 is a cross-sectional view of an exemplary PMC unit. The PMC unit is configured to serve the multiple purposes of applying a resistance during animal exhalation to help maintain positive end expiratory pressure (PEEP), monitoring pressure near the ETT inlet, and providing pop-off valve pressure relief if ETT pressure exceeded a predetermined threshold. For a set amount of delivered gas volume (represented as GAV or more commonly as tidal volume, Vt; mL) and inlet flow rate (Qin; L / s) as provided by the iDP-ADS 300, the maximum pressure sensed by the device (Pd) at the PMC unit 303 is the sum of the airway elastic pressure contribution (APe), resistive pressure contribution (APr), and PEEP present at the end of the previous breath, where Pd can be calculated as Po = A / ’£ +    + PEEP ~ ~ • Vt + R •   + PEEP. (1) In Eq. (1), C is the lung average compliance (mL / cm H2O) and R is the interface and airway total resistance (cm H2O / L / s). The resistive pressure term is largely dissipated (by viscous shear stress) in the interface and conducting airways and represents the pressure loss required to deliver the gas through these conducting pathways to the alveolar region at the specified flow rate. Pressure within the lungs becomes a transient volume filling problem that reaches a steady state equilibrium within 3-4 time constants (t), where r = R C. For the gas delivery conditions (i.e., constant Qin and Vt), the maximum airway pressure (PAW) in the alveolar region, approximated with the LC pressure, can therefore be represented as: Pw « AP£ 4- PEEP = 1 • K + PEEP.        (2) A significant component of any aerosol delivery system is aerosol generation using an air-jet aerosolization engine. Air-jet DPIs are designed with geometrically simple elements (small diameter flow passages and smoothly curving aerosolization chamber) and engineered to enable the efficient generation of small-particle aerosols with limited volumes of air. Following are three exemplary air-jet DPIs by way of example. Figures 5 and 6 are two alternative air-jet DPIs, referred to respectively as “DI” and “D2” in this disclosure, with fixed volume aerosolization chambers. In the DI device, the outlet 551 of the aerosolization chamber 501 is flush with the chamber walls. There is no overhang of the capillary 652 into the aerosolization chamber 501. A gradual expansion (GE) flow diffuser (i.e., diffusional flow pathway) 553 dissipates the high velocity aerosol flow prior to entry into the patient interface (e.g., ETT) which may be connected to the DI DPI via connector 550. With the D2 device, the outlet flow passage (capillary) 652 protrudes into the aerosolization chamber 601 (e.g., approximately 2 mm) and is smoothly connected to the remainder of the aerosolization chamber using continuously curving walls (see protrusion 670). The intent of this configuration is to slow the release of the aerosolized powder and reduce the size of the particles that could exit the chamber 601 while not increasing velocity in the outlet flow passage 652. Instead of the GE flow diffuser of DI, the D2 device has a rapid expansion (RE) geometry diffusional flow pathway 653. This difference may reduce the occurrence of aggregate buildup and reentrainment that may be expected to occur in the GE of DI. Both DI and D2 include a connection port 654 for connection of a pressure monitoring control (PMC) unit. Figure 7A is an air-jet DPI, referred to as “D3” in this disclosure, with a variable-volume aerosolization chamber 701 with a movable lower unit 308 which controls the floor height of the chamber. The lower unit 308 is configured for raising the floor of the chamber 701. Air-jet devices like the D3 device capable of accepting significantly higher dose loadings and controlling the amount of powder delivered per actuation. For loading of high powder masses, the floor of the aerosolization chamber 701 is positioned at the lowest level, maximizing the available chamber volume. With a first actuation, the geometry is designed such that secondary air currents aerosolize a defined mass of powder on the top of the powder bed, while leaving the remaining powder relatively undisturbed. The floor of the variable-volume chamber 701 is then raised by a defined distance using a small turn of the lower unit 308, exposing the next dose of powder for aerosolization with the next actuation. By following an actuate-and-tum sequence, the dose per actuation can be substantially unified while enabling high powder mass loading and maintaining high aerosol quality. The D3 device includes downstream of the aerosolization chamber 701 an outlet flow capillary 757 and a RE diffusional flow pathway 753. An exemplary size and material for the outlet flow capillary 757 is 0.89 mm and metal (e.g., stainless steel). As illustrated in Figure 7B, alignment of the positioning tab 781 on the lower unit 308 with the indicia 403 (e.g., numbers) indicating levels of the aerosolization chamber floor is usable to guide rotation of the lower unit 308 through four defined levels. Additional details of exemplary variable aerosolization chamber size DPIs are given in WO / 2025 / 072946 (Application No. PCT / US2024 / 049285), which is incorporated herein by reference. Figure 8 is a diagram of an exemplary aerosol delivery method. A single AST therapy administration may be described as having a total therapy time. A total therapy time can be calculated as the sum of the one or more aerosol delivery periods (e.g., 1, 2, 3, or more than 3) and, if included, any stabilization and monitoring periods which may be include between successive aerosol delivery periods. Figure 8 shows, simply as one non-limiting example, a total therapy time as including two aerosol delivery periods with a stabilization and monitoring period in between. One aerosol delivery period following by one stabilization and monitoring period may be referred to together as a dose delivery cycle. A dose delivery cycle may or may not include a stabilization monitoring period. Within any one aerosol delivery period, the dose to be administered during that aerosol delivery period may be administered in one or more pails. This is advantageous in some embodiments to accommodate limits of some DPIs as to how much dry powder can be loaded at a time. If the acceptable load limit of a DPI is below the dose amount for an aerosol delivery period, the dose may be divided into parts. In such case the first part is loaded into the DPI and administered to the patient, then the DPI is loaded with the second part which is administered to the patient. This subroutine may be repeated as many times as required until the full dose for the aerosol delivery period has been administered. Between administration of dose parts, a patient may be returned to a ventilator if the delivery method in question entails removing the patient from a ventilator to administer the dry powder SLS aerosol. As discussed elsewhere in this disclosure, a variety of patient interfaces may be used for administration of aerosols to patients. Not all patients will necessarily be on a ventilator or, in the case a patient is on a ventilator, not all methods necessarily require taking the patient off the ventilator to administer the aerosol. In some embodiments an aerosol may be administered through or parallel with the ventilator interface. A single loaded dose or part of a loaded dose may be administered to a patient in one or more DPI actuations. That is to say, multiple actuations of the DPI may be used to clear the loaded dry powder from the DPI. For example, for each aerosol delivery period the aerosol may be emitted with 10 or fewer air actuations, 4-10 actuations, or less than 4 actuations. In many exemplary embodiments, it is desirable to time actuations with patient inhalations. It is also advantageous in many embodiments for aerosol emission from the inhaler to the patient interface for administration to the patient be performed with positive pressure. One advantage of exemplary methods like those summarized by Figure 8 is relatively fast aerosol delivery times and total therapy time. As non-limiting but good examples, Figure 8 indicates a total delivery time may be, for example, less than 5 minutes, and a total therapy time may be, for example, less than 20 minutes. A patient may of course continue to be monitoring after total therapy time is complete. Infants being treated for RDS are hospitalized patients and are typically monitored continuously during their stay at the hospital. As one example of an exemplary method in accordance with Figure 8, exemplary dry powder compositions of this disclosure enable a rapid administration period followed by a 5-30 minute, and preferably 15-minute stabilization and monitoring period, before addition administration (if necessary). As exemplary but non-limiting illustration, dose delivery over the rapid administration period may occur within the following time range: a) Typical infant RDS inhalation'. 0.3 sec b) Typical breath hold period to enhance deposition of the aerosol: 1 to 5 sec c) Typical infant RDS exhalation'. 0.7 sec d) Number of required actuations: 4 to 10 i) Total rapid administration period minimum: 4 actuations x (0.3 sec + 1 sec + 0.7 sec) = 8 see (so > 8 sec) ii) Total rapid administration period maximum: 10 actuations x (0.3 sec + 5 sec +0.7 sec) = 60 sec (so < 60 sec) iii) overall range of 8-60 sec for min and max An exemplary method of using a dry powder synthetic lung surfactant (SLS) formulation includes forming an aerosol, with an inhaler, containing a dry powder synthetic lung surfactant (SLS) formulation comprising one or more phospholipids (PL) and one or more hygroscopic excipients; and emitting the aerosol from the inhaler at a fine particle dose (FPD) <5pm equal to or greater than 5.0 mg PL / kg patient mass over one or more aerosol delivery periods forming a total therapy time. For the total therapy time the aerosol may be emitted from the inhaler at a nominal dose of 30 mg or less PL / kg patient mass. The aerosol may be emitted at an FPD <2.5pm equal to or greater than 4.0 mg PL / kg patient mass over the one or more aerosol delivery periods. In some embodiments, the FPD may instead be higher than these thresholds. For instance, the aerosol may be emitted at an FPD <5pm equal to or greater than 16.0 mg PL / kg patient mass over the one or more aerosol delivery periods, and / or the aerosol may be emitted at an FPD <2.5pm equal to or greater than 11.0 mg PL / kg patient mass over the one or more aerosol delivery periods. In general, an aerosol may be emitted from an inhaler at a fine particle dose (FPD) <5pm of > 5.0 mg PL / kg (patient mass), > 6.0 mg PL / kg, > 7.0 mg PL / kg, > 8.0 mg PL / kg, > 9.0 mg PL / kg, > 10.0 mg PL / kg, > 11.0 mg PL / kg, > 12.0 mg PL / kg, > 13.0 mg PL / kg, > 14.0 mg PL / kg, > 15.0 mg PL / kg, or > 16.0 mg PL / kg. In general, an aerosol may be emitted from an inhaler at a fine particle dose (FPD) <2.5pm of > 4.0 mg PL / kg (patient mass), > 5.0 mg PL / kg, > 6.0 mg PL / kg, > 7.0 mg PL / kg, > 8.0 mg PL / kg, > 9.0 mg PL / kg, > 10.0 mg PL / kg, or > 11.0 mg PL / kg. Depending on the delivery system employed (e.g., particulars of the DPI used), individual loaded doses may be delivered over multiple actuations of equal or non-equal amounts. For instance, in reference to the protocol employed in Example 1 below, the rationale for administering two 30 mg aerosol doses 15 min apart (with each 30 mg aerosol dose divided into sequential 20 and 10 mg device loaded doses, delivered approximately 2 min apart) is to prevent excessive delivery and not overwhelm the lungs from the high output powder formulation, allowing the airways to receive heated and humidified gases from the ventilator between doses. In Example 3 below, which employs the D3 device, 30 mg of powder formulation is loaded at once and delivery doses are substantially the same across multiple actuations. In either case, exemplary methods make possible delivery of highly surface-active aerosol into the distal lung regions in < 5 min total aerosol delivery time. According to some embodiments, an exemplary delivery strategy includes (i) administration through an ETT interface, and (ii) delivery with positive-pressure ventilation. ETT delivery provided a majority of the aerosol to the lower lung region (in the absence of extreme humidity) and < 5% aerosol loss in the 3 mm (internal diameter) ETT. Administration of the surfactant aerosol with positive-pressure breaths is intended as a way to recruit collapsed lung regions, better distributing the aerosol to the alveoli and throughout the lungs, and then the deposited surfactant serves to hold the recruited lung regions open through dissolution and reduction of the airway surface liquid surface tension. Exemplary actuation flow volumes are as low as 100 - 600 pL for veiy small test animals (e.g., mice), 1-5 or 1-20 mL for intermediate size test animals (e.g., rats and rabbits), 3-100 mL for human infants, 100 mL to 2 L or 3 L for human pediatric subjects, and 300 mL - 4.5 L for adults. However, if bypass channels are employed, or if the aerosol is injected into an inhalation gas stream, then the lower end actuation flow volumes (1-100 mL) may be used with any age group or subject. For devices actuated by a positive pressure gas source typical actuation pressures across system may range from 1-60 kPa, or higher, depending on desired flow rate and device resistance. For passive devices operated only by a patient’s inhalation effort, pressure drops across system may be 0.5 to 6 kPa or higher. According to some embodiments, an exemplary delivery strategy involves a delivered tidal volume limited to 10 mL / kg or less and preferably 4-7 mL / kg administered with the air-jet DPI for only 4 to 10 breaths per aerosol delivery cycle. An exemplary delivery strategy may entail administration of controlled lung pressure of approximately APE < 20 cm H2O and LC pressure < 25 ± 2 cm H2O. A high efficacy of Nano SD SLS-EEG dry powder formulation in animals in Example 2 below is attributable to rapid bolus delivery combined with positive pressure and high lower-lung deposition of a micrometer-sized aerosol, which was also demonstrated in studies in vitro, showing high-efficiency lung delivery of approximately 61 to 74% (see Example 1). In Example 3 below, the total device-loaded dose of active PL components (combination of DPPC and POPG) was 24 mg / kg. Two dose delivery cycles were employed, each requiring an aerosol delivery time of ~20 s. Total aerosol delivery time with the D3-MSD2 combination was therefore <1 min. Based on NGI testing, the previous D2-NSD1 combination produced FPFs (as a % of ED) with cutoffs of <5, <2.5 and <1 pm of 27, 21, and 9 %, respectively. With the new D3-MSD2 combination, where the D3 device enabled increased dose loading and better regulated %ED / actuation, corresponding FPFs were 88, 62, and 10 %, respectively. Implementing an FPF<2.5pni as the best available particle size cutoff metric, previous D2-NSD1 combination administered a total fine particle dose (FPD) <2.5 pm of 4 mg-PL / kg; and the current D3-MSD2 therapy administered an FPD<2.5gm of 11 mg-PL / kg. Exemplary components of a dose delivery protocol may include one or more of the following features: • -15 mg-PL / kg delivered to the lungs, as predicted by in vitro LC model testing • FPD with <1 and <5 pm cutoffs of >2 and >5 mg-PL / kg based on NGI testing. • FPD<2.5^m > 5-10 mg-PL / kg, preferably administered with positive pressure inflations with a maximum lung pressure of approximately 20-25 cm H2O. • DDP metric for severe RDS of FPD<2.5gm >5-10 mg-PL / kg, • Use of the EEG aerosol delivery strategy, • Frontloading the aerosol delivery into the first portion of the inhalation waveform, • Small primary particles (e.g., - 1 pm) containing some (e.g., 15% w / w) hygroscopic material, and • Administration of the aerosol with positive pressure breaths. Table 11 and Table 111 summarize data from Examples below and recommended ranges from exemplary formulations and delivery procedures support by the data in the Examples. An example of a 60 mg total dose of the SLS-EEG formulation (administered over successive delivery cycles) to a test animal weight of 1.5 kg (consistent with both preterm infants and rabbit test-animals), the following PL dose estimates were calculated for the Nano Spray Dried PL60%w / w +BYL5%w / w Formulation of Example 1 (nominal subject weight 1.5 kg): • Nominal Loaded PL Dose: 60 mg x 0.6 (60% PL content) = 36 mg PL • Nominal Loaded PL / kg Dose: 36 mg PL / 1.5 kg (typical weight) = 24 mg PL / kg • Emitted PL / kg Dose: 24 mg PL / kg x 0.8 (-80% emitted dose) = 19.2 mg PL / kg Initial fine particle dose (FPD) was also estimated based on aerosol size fractions. An FPDclpm can be based on the measured fine particle fraction <lpm of 9.3%. An FPD<2.5pm can be based on the measured fine particle fraction <2.5pm of 21%. Similar, an FPD<5pm can be based on fine particle fraction <5pm of 27.1%. Using these first order assumptions: • FPD<lpm PL I kg Dose: 19.2 mg PL / kg x 0.093 = 1.8 mg PL I kg • FPD<2.5pm PL I kg Dose: 19.2 mg PL / kg x 0.21 = 4.0 mg PL / kg • FPD<5pm PL I kg dose: 19.2 mg PL / kg x 0.271 = 5.2 mg PL I kg The following PL dose estimates were calculated For the Mini Spray Dried PL62.5%w / w + BYL2.5%w / w Formulation of Example 3 (nominal subject weight 1.5 kg): • Nominal Loaded PL Dose: 60 mg x 0.6 (62.5% PL content) = 37.5 mg PL • Nominal Loaded PL / kg Dose: 37.5 mg PL / 1.5 kg (typical weight) = 25 mg PL I kg • Emitted PL / kg Dose: 25 mg PL / kg x 0.75 (-75% emitted dose) = 18.8 mg PL / kg Initial fine particle dose (FPD) was also estimated based on aerosol size fractions. An FPDclpm can be based on the measured fine particle fraction clpm of 10.0%. An FPD<2.5pm can be based on the measured fine particle fraction <2.5pm of 62%. Similar, an FPD<5pm can be based on fine particle fraction <5pm of 87.9%. Using these first order assumptions: • FPD<lpm PL I kg Dose: 18.8 mg PL / kg x 0.10 = 1.9 mg PL / kg • FPD<2.5pm PL I kg Dose: 18.8 mg PL / kg x 0.62 = 11.6 mg PL / kg • FPD<5pm PL I kg dose: 18.8 mg PL / kg x 0.879 = 16.5mg PL I kg The compositions containing 15% hygroscopic components were successful in reversing surfactant washout induced respiratory distress syndrome in a rabbit animal model. The delivered dose of the composition contained 6 mg / kg of hygroscopic components (Using a 60 mg dose of the dry powder composition: 60 mg x 0.15 (MN and NaCl) = 9 mg / 1.5kg = 6 mg / kg hygroscopic components over a 15-minute). The range of hygroscopic components administered would encompass 3 mg / kg (based on current data) to 6 mg / kg (observed with nose-to-lung) to 10 mg / kg (reasonable upper limit) over a 15-minute window. Table IL Instillation D2 with Nano SD SLS EEG PL60 D3 with Mini SD SLS-EEG PL62.5 Recommended Range Emitted Dose (% Loaded) 80 75 Lower Lung Dose (% Loaded) 61.1 48.8 Total Lung Dose (% Loaded) 73.8 61.1 FPFcl um (% Emitted) 9.3 10 >9, >10 FPF<5 um (% Emitted) 27.1 87.9 >27, >88 FPF<2.5 um (% Emitted) 21 62 >21, >62 Average Rabbit weight per study (kg) 1.5 1.5 Percent PL in formulation 60 62.5 Percent hygroscopic material in formulation 15 15 Total Nominal Loaded Powder Mass (mg) 60 60 Table III. Dose Summary Instillation D2 D3 Recommended Range Nominal Loaded PL Dose (mg PL) 36.0 37.5 Nominal Loaded PL / kg Dose (mg PL / kg) 200.0 24.0 25.0 Emitted PL / kg (mg PL / kg) 19.2 18.8 PL Estimates Based on Rabbit Chamber Testing Estimated PL / kg Lower Lung Dose (mg PL / kg) 14.7 12.2 10-15 or higher Estimated PL / kg Total Lung Dose (mg PL / kg) 17.7 15.3 12-20 or higher PL Estimates Based on NGI Aerosol Size FPD<lum Emitted from Device (mg PL / kg) 1.8 1.9 21.8,21.9 FPD<5 urn Emitted from Device (mg PL / kg) 5.2 16.5 25.2, 216.5 FPD<2.5 um Emitted from Device (mg PL / kg) 4.0 11.6 24.0,211.6 Hygroscopic Material in the Lungs Estimated mg / kg Lower Lung Dose (mg / kg) 2.9 2.2 <6-10 Estimated mg / kg Total Lung Dose (mg / kg) 3.5 2.7 <6-10 Timing Rapid aerosol delivery period 5 mins 20 secs Total therapy administration time ~20 mins ~15 mins Fine Particle Dose < 5um of PL / kg FPD at 60 mg loaded (mg PL / kg) 5.2 16.5 5-17 or higher Emitted PL / actuation for 60 mg Loaded 8x actuations (4x for each 30 mg) (mg PL / actuation) 3.6 3.5 1-4 or higher 16x actuations (8x for each 30 mg) (mg PL / actuation) 1.8 1.8 1-4 or higher Rates of PL delivery for a 0.2 sec actuation; 10 mL at 3 L / min PL rate with 8x actuations (4x for each 30 mg) (mg PL / sec) 18 17.6 5-20 or higher PL rate with 16x actuations (8x for each 30 mg) (mg PL / sec) 9 8.8 5-20 or higher Rate of PL delivery with a 2 sec breath cycle (0.3 sec inhale; 1 sec hold; 0.7 sec exhale) PL rate with 8x actuations (4x for each 30 mg) (mg PL / sec) 1.8 1.8 0.5-2 or higher PL rate with 16x actuations (8x for each 30 mg) (mg PL / sec) 0.9 0.9 Abbreviations A#       device actuation number A J         air-jet AST      aerosol surfactant therapy C        lung compliance CI        confidence interval CT      computed tomography D#       design number of air-jet DPI aerosolization engine DDP     dose delivery profile DPPC dipalmitoyl phosphatidylcholine DPI      dry powder inhaler Dvso     volumetric median diameter DVS     dynamic vapor sorption ED      emitted dose EM      electromechanical ETT     endotracheal tube EEG     excipient enhanced growth FIB      focused ion beam FiOi      fraction of inspired oxygen FPD      fine particle dose FPF       fine particle fraction G#       airway generation number with the trachea denoted as one GAV    gas actuation volume GE       gradual expansion GSD     geometric standard deviation HPLC    high performance liquid chromatography ID        internal diameter iDP-ADS infant dry powder aerosol delivery system LC      lung chamber MMAD mass median aerodynamic diameter MN     mannitol MSD2    mini spray dryer formulation #2 NaCl     sodium chloride NGI      next generation impactor NIV      non-invasive ventilation NSD1    nano spray dryer formulation #1 OI       oxygenation index PaO2     partial pressure of oxygen (arterial oxygenation) Paw      airway pressure Pd        pressure at the device (PMC unit measured) PEEP    positive end expiratory pressure PIP       peak inspiratory pressure PL       phospholipid PMC     pressure monitoring and control POPG     l-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (sodium salt) PSD      particle size distribution Qin        inhalation flow rate R         lung resistance R#       run or replicate number RE       rapid expansion RDS      respiratory distress syndrome RH       relative humidity SEM     scanning electron microscope or standard error or mean SD       spray dried or standard deviation SLS       synthetic lung surfactant SpO2     oxygen saturation TB       tracheobronchial UV       ultraviolent VEI      ventilation efficiency index Vt        tidal volume XRPD   X-ray powder diffraction 3D       three dimensional APe      Change in elastic pressure APr      Change in resistive pressure EXAMPLES Example 1. A New Dry Powder Aerosol Synthetic Lung Surfactant Product for Neonatal Respiratory Distress Syndrome (RDS) - In Vitro Testing and Characterization This Example summarizes an experimental study presenting an embodiment of a new aerosol SLS product, including details about the initial aerosol delivery strategy and formulation. This Example characterizes performance based on aerosol formation and realistic in vitro airway testing leading to aerosol delivery recommendations (i.e., a complete dose delivery protocol) for a subsequent in vivo animal model testing (described in Example 2). An exemplary formulation and delivery protocol is capable of generating a micrometer-sized surfactant powder aerosol using ~ 10 mL of dispersion air delivered at a flow of < 3 L / min over a time interval of < 300 ms. Formulation Materials Phospholipids (dipalmitoyl phosphatidylcholine (DPPC) and l-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (sodium salt) (POPG)) and surfactant protein mimic peptide (B-YL) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and CSBio (Menlo Park, CA), respectively. L-leucine was purchased from Sigma-Aldrich Chemical Co. (St Louis, MO). Sodium chloride and D-mannitol together with other chemicals were purchased from Fisher Scientific Co. (Hanover Park, IL). Throughout the study, freshly collected deionized water was used. Formulation Production Multiple batches of Nano spray-dried (SD) SLS-EEG powder formulation were prepared by spray drying of feed dispersions containing phospholipids (DPPC and POPG), surfactant protein mimic (B-YL), hygroscopic excipients (mannitol and sodium chloride) and dispersion enhancer (l-leucine) using the Buchi Nano Spray Dryer B-90 HP (Biichi Labortechnik AG, Flawil, Switzerland) following the same procedure as previously reported

[45] . Briefly, the feed dispersions were prepared with a solids composition of DPPC 52.5%, POPG 7.5%, B-YL 5%, mannitol 9%, sodium chloride 6% and l-leucine 20% w / w in an ethanol-water (5:95% v / v) cosolvent system, which was sonicated for 40 min in a heated water bath (45-55°C) with intermittent manual shaking every 10 min. The total solids content of the feed dispersion was 0.125% w / v. The feed dispersion was spray-dried using the small nozzle option at a spray rate of ~ 0.40 mL / min. Throughout the spray drying process, the inlet temperature was set to 70 °C, resulting in an outlet temperature of 35-38 °C. During spray drying, the feed dispersion was continuously stirred using a magnetic stirrer as excess feed dispersion was recycled into the stock until completion of the batch processing. The Nano SD SLS-EEG powders were collected on an electrostatic precipitator and transferred into glass vials and stored in a desiccator (0% RH) at -20 °C. It is noted that while we are targeting ~ 1 pm primary particle aerodynamic diameters, the formulation nomenclature of Nano is used to denote the spray drying system (Buchi Nano Spray Dryer B-90 HP) implemented for production. Analytical Methods: DPPC and B-YL HPLC Methods Validated HPLC methods were used to measure the content uniformity of DPPC and B-YL in the SLS-EEG formulations together with quantifying the aerosol performance by measuring DPPC deposition. For DPPC, the system consisted of an Alliance 2695 Separation Module with a photodiode array UV detector and Empower 3 data acquisition software (Waters Corporation, Milford, MA). The sample injection volume was 100 pL and column temperature was 45°C. The chromatographic separation was achieved using the Waters XSelect CSH C18 XP, (2.5 pm, 4.6 x 100 mm; Waters Corporation, Milford, MA). A gradient elution system employed: Solvent A: 5 mM tetrabutylammonium bisulfate in water, pH 8.52: Solvent B acetonitrile: isopropanol: ammonium hydroxide: perchloric acid (40:60:0.05:0.0005 v / v), and Solvent C: acetonitrile, pumped at a flow rate of 0.8 mL / min. Following chromatographic separation UV absorbance was measured at 204 nm. The retention time for DPPC was ~ 9 min. For B-YL, the system was modified to employ a gradient elution with, Solvent A: water:trifluoroacetic acid (100:0.075 v / v), Solvent B: acetonitrile:trifluroacetic acid: polysorbate 80 (100:0.075:0.002 v / v), an injection volume of 40 pL and a detection wavelength of 214 nm. All other conditions were unchanged. A DPPC stock solution (2 mg / mL) was prepared by dissolving a sufficient amount of DPPC in 2.5 mM TBA in water: ethanol: polysorbate 80 (15:85:0.005 v / v (diluent)). Diluted standard solutions of DPPC in the concentration range of 50 - 400 pg / mL were prepared by further dilution of the stock standard solution in diluent. A B-YL stock solution (100 pg / mL) was prepared by dissolving a sufficient amount of B-YL in diluent. Diluted standard solutions of B-YL in the concentration range of 7 - 25 pg / mL were prepared by further dilution of the stock standard solution in diluent. Formulation Characterization: DPPC Content Uniformity The DPPC content of the powders was evaluated by quantitative analysis of the DPPC using the HPLC-UV method. Approximately 1 mg of Nano SD SLS-EEG formulation was dissolved in 3 mL diluent and quantitatively analyzed for the DPPC content. Triplicate samples were prepared and analyzed for each of the powder samples. Formulation Characterization: B-YL Content Uniformity The B-YL content of the powders was evaluated by quantitative analysis of the B-YL using the HPLC-UV method. Approximately 1 mg of Nano SD SLS-EEG formulation was dissolved in 3 mL diluent and quantitatively analyzed for the BY-L content. Triplicate samples were prepared and analyzed for each of the powder samples. Formulation Characterization: Laser Diffraction Particle Sizing Powder dispersibility and primary particle size of the Nano SD SLS-EEG formulation was determined using laser diffraction at multiple dispersion pressures. Specifically, components employed included the Sympatec HELOS (Sympa-tec GmbH, Germany) equipped with a RODOS / M disperser and an ASPIROS sample feeder. A small glass vial was filled with approximately 3 mg of spray-dried powder, placed in the sample feeder and dispersed in the laser beam using the RODOS / M disperser with an RI lens at dispersion pressures of 0.5 and 4.0 bar. The size distribution results (DvlO, Dv50 and Dv90) were obtained using Sympatec WINDOX 5.10 software (Sympatec GmbH, Germany). Size distribution results determined at 4 bar are assumed to be close to fully deaggregated primary particles, and the difference between the size distributions at 0.5 and 4 bar provides an indication of the powder dispersibility. Formulation Characterization: Scanning Electron Microscopy The particle morphology of the Nano SD SLS-EEG formulation was evaluated using a Zeiss EVO-50XVP scanning electron microscope (SEM) (Carl Zeiss, Oberkochen, Germany) at an accelerating voltage of 5.0 kV. A small amount of powder sample was mounted on a metal SEM stub using double-sided adhesive tape and loose powders were removed using compressed air. The samples were sputter coated with gold grain using an EMS550X sputter coater (Electron Microscopy Sciences, Hatfield, PA) before taking the images. Formulation Characterization: X-ray Powder Diffraction (XRPD) The Nano SD SLS-EEG formulation was characterized using the Rigaku Miniflex 6G (Rigaku, Japan) X-ray powder diffraction technique. Briefly, a small amount of powder was placed on the center of the XRPD sample holder and then the powder surface was gently smoothed using a glass slide. The samples were scanned using a continuous mode in the range of 3-50° 29 at a scan speed of 1.5° / min. Formulation Characterization: Moisture Sorption To understand the water uptake of the Nano SD SLS-EEG formulation as a function of relative humidity (RH), sampleswere exposed using the dynamic vapor sorption system (DVS Adventure, Surface Measurement Systems Ltd., UK). Briefly, a small amount of powder sample (about 5 mg) was equilibrated at 0% RH and then exposed to increasing RH from 0 to 95% (sorption cycle) and decreasing RH from 95 to 0% (desorption cycle). At each RH, once the defined equilibrium condition (the rate of mass change (dm / dt) less than 0.002%) was achieved, the RH was changed to the next level. Setup of the iDP-ADS An overview of the iDP-ADS used in this Example is provided in Figure 3. The outlet flow rate of compressed air (in vitro testing) or pure oxygen (in vivo aerosol delivery) was set to approximately 3 L / min and the timer was set toprovide either 10 mL (in vitro testing) or approximately 7 mL / kg (in vivo aerosol delivery) of gas actuation volume (GAV). The air-jet DPI was characterized by three inlet flow passages arranged in a triangular pattern (0.5 mm inlet diameters), a 0.68 mL aerosolization chamber in an approximate capsule shape, and a single 0.89 mm stainless steel capillary outlet (see Figure 5). The capillary outlet tube had an outlet length of 6.7 cm and was connected to a diffusional flow pathway, as with the gradual expansion illustrated in Figure 5, which then led to an ETT interface connection. The PMC unit corresponded with the depiction in Figure 4. The pop-off valve was configured to provide pressure relief if ETT pressure exceeded approximately 25 + 2 cm H2O. Connections from the PMC central region led to an analog pressure gauge (AG Industries, St. Louis, MO) and a pop-off valve (Ambu 22 mm PEEP valve, Ballerup, Denmark). The top of the PMC was a flow resistor cap with a 0.5 mm diameter central aperture, which served as an exhalation port. For the in vivo studies, the operator covered this exhalation port with a thumb during device actuation and a brief breath-hold period (~ 1 s) and then released the port to allow exhalation. By observing the pressure gauge, the exhalation port was covered near the end of exhalation to maintain ~ 5 cm H2O of PEEP in the lungs prior to the next device actuation. In this Example, two alternative air-jet DPIs are employed. Namely, this Example employed DI of Figure 5 and D2 of Figure 6. Preliminary testing of DI indicated that best aerosolization performance occurred with maximum 10 mg powder loadings and the aerosol emptied very quickly (within three actuations and mostly on the first actuation). Development of Rabbit Lung Chamber (LC) Model To test lung delivery of the dry powder AST product when administered through an ETT to an infant-size test animal, an in vitro model of an approximate rabbit upper tracheobronchial (TBupper) region surrounded by a cylindrical lung chamber (TBupPer + LC) was generated, as shown in Figures 9A and 9B. This model was used to predict aerosol deposition in the upper TB region (starting with the trachea and extending through approximately 4-6 bronchial generations, or G4-G6), delivery beyond the upper TB region, and to provide approximate airway resistance and compliance during aerosol testing. To form the upper TB animal airway, a cast or scan of rabbit airways was not directly available. As an alternative, we selected a pre-existing cast of a similar size small mammal (ferret airway described in Oldham MJ, Phalen RF, Huxtable RF. Growth of the ferret tracheobronchial tree. Lab Anim Sei. 1990;40:186-91) that had airway dimensions very similar to young mature New Zealand White Rabbits. Specifically, Hack and Gearhart (Hack CE, Gearhail JM. A rabbit lung morphology model for aerosol deposition and clearance. J Aerosol Sci. 2016;99:144-56) reported the mean diameters for the first three generations of mature rabbit airways beginning with the trachea to be 0.53, 0.42 and 0.26 cm, respectively. For this Example, the young adult Fe#4 model of Oldham et al. was selected with corresponding airway diameters of 0.52, 0.38, and 0.29 cm, respectively. Airway lengths were also similar, with the exception of the trachea, which was conservatively longer (i.e., producing higher deposition) in the ferret model (9.4 cm ferret vs. 5.6 cm rabbit). An original young adult ferret TB silicone cast from the study of Oldham et al. was translated to a digital object using a Bruker SkyScan 1173 microCT scanner (Bruker Corporation, Billerica, MA) and Slicer 4.10.2 software. Based on available scan resolution, the final upper TB airway model contained pathways with between 3 to 5 bifurcations, or 4 to 6 generations, where the trachea was counted as generation Gl. Final outlet diameters ranged between 2.34 mm and 0.76 mm and are illustrated in Figure 9A. A majority of the trachea (~ 5 cm) was constructed in cast silicone to provide realistic insertion conditions for the ETT interface. The remainder of the upper TB airway was constructed as a hollow body and 3D printed by Quickparts (Seattle, Washington) using SLA technology with Accura ClearVue resin. Considering that a majority of airway resistance resides in the upper TB region, this upper airway section was used to approximate the expected airway resistance during ETT aerosol delivery. The complete Rabbit LC Model is illustrated in Figure 9B with the LC surrounding the TB geometry. The main body of the LC was constructed using a clear acrylic cylinder with a volume of ~ 400 mL to approximate the expected in vivo post-washout rabbit lung compliance of approximately 0.4-0.6 mL / cm H2O. Volume of the clear cylinder, outlet pathway and filter housing produced a total chamber compressible volume of ~ 500 mL, and resulted in a measured compliance of 0.45 mL I cm H2O. The outlet pulmonary filter (Pulmoguard II™, Queset Medical, North Easton, MA) was used to capture aerosol that did not deposit in the upstream sections. During aerosol testing, a final three-way valve was used to release the chamber pressure after each delivery cycle while capturing the aerosol on the filter, and for connection to a downstream vacuum pump for a final purge of the chamber after the last actuation, if needed. A pressure measurement port was also included on the chamber side wall for measurement of the LC pressure conditions. Air-Jet DPI Recommended Settings and Pressure Testing in the Rabbit LC Model During aerosol delivery, the rabbit surfactant washout in vivo model was ventilated by the air-jet DPI (with aerosol contained in each breath). As a result, delivered gas conditions were critical to maintain oxygenation of the animal and to avoid additional lung damage, either through pressure trauma, volume trauma, or lung collapse (atelectotrauma). As an initial guide consistent with the animal model ventilator settings, the air-jet DPI delivered approximately 7 mL / kg GAV with each inhalation. A challenge with using a fixed tidal volume (per kg) in all cases was that as lung compliance decreases with surfactant washout, the administered pressure required to achieve 7 mL / kg increases, often exceeding safe levels. In reference to Equation 1 above, for the monopodial small-mammal airway structure and ETT delivery, measured R was relatively small (i.e., 4 cm H2O I L / s). As a result, the administration time of - 0.2 s represents > 10 time-constants, indicating little delay between device and lung pressures. Similarly, there is negligible difference between device and LC pressure conditions, which was also verified experimentally (< 0.3 cm H2O). During aerosol delivery, pressure targets were set as an elastic pressure rise (APe) of < 20 cm H2O and a PEEP value of ~ 5 cm H2O. Maximum and minimum device and LC pressures should then be in a range of approximately 25 ± 2 to 5 ± 2 cm H2O. Given the very close similarity in LC and device pressures, only LC pressures were recorded and reported. To enable rapid setting of the gas delivery conditions and achieve the targeted pressure range, for variable test animal weights and lung compliances, Table IV was generated. The pressure regulator was preset and calibrated to provide a flow rate of 3 L / min (0.05 L / s). For a selected rabbit weight, Table IV provided the baseline timer settings for entry into the EM Timer device to provide approximately 7 mL / kg tidal volumes. Grey shading was used in Table IV to indicate the change in pressure at different airway compliances based on the elastic effect alone (APe). The actual APe was also available in Table IV. For APe values > 20 cm H2O (light and dark grey), it was recommended that the timer setting be reduced to the first row of that compliance column with a value that would produce a APe < 20 cm H2O (no shading). For example, for a 1.6 kg rabbit with a lung compliance of 0.45 mL I cm H2O, the baseline tidal volume of 7 mL / kg would generate a APe of 25.0 cm H2O. At this compliance, it is recommended that the aerosol delivery time from the DPI be reduced to 0.18 s generating a APe of 20 cm H2O and an acceptable tidal volume of 5.7 mL / kg. Table IV. EM Timer Settings (t), Predicted Change in Elastic Pressure (APe) and Administered Tidal Volume Across a Range of Lung Compliances for an Air-let DPI Delivered Flow Rate of 3 L / min and Targeted Tidal Volume of 7 mL / kg Compliance (mL / cm IPO) 0,4 0.45 0,50 0,55 0,69 0.65 Weight t(sec) APi: (cm HjO) V( (mt) t (sec) APe (esa HjO) Vs < ml.( t (sec) APe (cm HjO) V; ( ail) t (see) AP» (on HjO) V, unf ) 1(sec) APe (cm HjO) Vi (iwL) 1 (see) APe (cm HjO) V» f mL) 1 kg 0.14 IS 7.0 0.14 16 7.0 0.14 14 7,0 0.14 13 7.9 044 12 7.0 9,14 I! 7.9 LI kg 0.15 19 7 / 7 0.15 J ‘ 0.15 15 7 . / 0.15 14 7 7 0.15 13 7 0.15 * L2 0.17 19 S.4 0.17 n 8.4 0.17 15 8.4 0,17 .14 8.4 0.17 8.4 1.3 kg titlillfiifl 0.18 20 9.1 0,17 18 9.1 0.)8 17 9.1 0.18 15 9.1 0.18 14 9.1 1.4 kg iBIilltilti liillililli 9.20 20 9.8 0.20 0.20 18 9.8 0.20 IS 9.8 1- 5 kg (Lit. W Biiiiliilii iiiiillilliii liiiiiiiifs: 0.21 19 10.5 0,2 i IS 19.5 0.21 16 ■0.5 L6 kg oilllllllll slisiiiiiiiji lifilllfllii 0.2.2 20 11.2 0.22 19 1).2 {>,22 H.2 l? kg saM iiiiiiiiiiii 0.24 20 11.9 0.24 IS 11.9 1.X kg ES 8,25 lililllllll: iiiifiiiiii 0.25 :9 12.6 I V kg TO? w HT 17,27 "TO? ITT iiiiiiilliil iiiiiiiiiiiii 0.27 2.0 kg oi TO- TO 38 TO lllllllllllil iliflllllllll Light Grey Cells: Predicted to create a APe of > 20 cm H2O; total administered pressure may exceed 25 cm H2O; operator should consider reducing actuation time by moving up to the first clear cell in the same column 5 Dark Grey Cells: Predicted to create a APe of > 25 cm H2O; total administered pressure may exceed 30 cm H2O; operator should consider reducing actuation time by moving up to the first clear cell in the same column To test administered lung pressures, the delivery device was connected to the Rabbit LC Model, with a measured lung compliance of 0.45 mL / cm H2O, and operated in a similar manner 10 with the planned in vivo experiments. The LC pressure relief valve was maintained in the closed position and the PMC exhalation port was used to release each delivered breath, after the brief breath hold. Pressure profiles were recorded at the lung chamber pressure port (LC pressure). The dry powder formulation was not delivered during LC pressure testing experiments. The respiration rate for controlling actuation of the EM Timer depended on the GAV time, an approximate 1-s breath hold, plus the exhalation time required for the chamber to reach the desired PEEP pressure of 5 ± 2 cm H2O. Pressure testing conditions were: • Scenario 1: 1.3 kg rabbit; 0.45 mL I cm H2O; 0.18 s timer setting (7 mL / kg Vt) • Scenario 2: 1.6 kg rabbit; 0.45 mL I cm H2O; 0.22 s timer setting (7 mL / kg Vt) • Scenario 3: 1.6 kg rabbit; 0.45 mL / cm H2O; 0.18 s timer setting (5.7 mL / kg Vt) Based on estimated conditions, Scenario 1 should satisfy the desired APe and LC pressure requirements; Scenario 2 represents selection of 7 mL / kg for a 1.6 kg rabbit without adjusting for lung compliance; and Scenario 3 provides the correct settings for a 1.6 kg rabbit considering both tidal volume and lung compliance that satisfies the pressure requirements. Aerosol Size Characterization using Cascade Impaction The air-jet DPI aerosol characterization experiments utilized a Next Generation Impactor (NGI; MSP, TSI Incorporated, Shoreview, MN) for aerosol particle size analysis. To assess the aerosol size distribution, the air-jet DPI (with the ETT attached to the outlet to assess size of the lung delivered dose) was connected to the pre-separator inlet of the NGI using a custom adapter, as previously described by Howe et al. (Howe C, Momin MA, Aladwani G, Strickler S, Hindle M, Longest W. Advancement of a High-Dose Infant Air-Jet Dry Powder Inhaler (DPI) with Passive Cyclic Loading: Performance Tuning for Different Formulations. Int J Pharm. 2023;643:123199). The adapter positioned the ETT exit approximately 1 cm away from the center of the pre-separator inlet with open space allowing for co-flow room air to enter the NGI, which was operated at a constant flow rate of 45 L / min using a downstream vacuum pump; while the device was operated with the intended 3 LPM flow rate. The NGI was positioned 90° off horizontal using an angle guide to allow the device to remain level during use (as intended for a horizontal infant or test animal) and maintained an inline flow path from the device outlet to the NGI inlet. Each stage of the NGI was coated with MOLYKOTE® 316 silicone spray (Dow Corning, Midland, MI) to minimize particle bounce and re-entrainment. The NGI flow rate of 45 L / min was chosen to ensure collection of the aerosol, minimize any effects of settling and provide appropriate stage cutoff diameters for evaluating small aerosol sizes. Before each set of experimental runs, the flow rate was confirmed using a flow sensor (Sensirion SFM3000, Scnsirion AG, Stafa, Switzerland) connected to the NGI inlet. The air-jet DPI was loaded with 10, 20 or 30 mg of the Nano SD SLS-EEG formulation and actuated into the NGI via the EM Timer air source using multiple (three or more) 10 mL GAVs. The flow rate and actuation time were 3 L / min and 0.2 s, respectively. For each device and loaded formulation mass, the device was actuated until exiting aerosol was no longer visible, followed by one additional actuation. Three replicate runs for each experiment were performed and quantitative analysis of DPPC deposition using the HPLC-UV assay was used to assess the aerosol performance characteristics of the SLS-EEG powder formulation. Analysis metrics included emitted dose (ED), mass median aerodynamic diameter (MMAD), fine particle fraction (FPF) and geometric standard deviation (GSD). Emitted dose was calculated as the mass of DPPC in the loaded dose minus the mass of DPPC remaining in the device divided by the initial loaded mass of DPPC. Aerosol size calculations were based on the mass of DPPC recovered in the NGI (including pre-separator). Based on an airflow rate of 45 L / min, the NGI stage cut-off diameters were determined using the formula specified in United States Pharmacopeia (USP) 35 (Chapter 601, Apparatus 5). The mass median aerodynamic diameter (MMAD) and fine particle fractions (FPFs < 5 pm or < 1 pm) were calculated using Copley Inhaler Data Analysis Software (CITDAS). In Vitro Testing of Lung Delivery Efficiency with Rabbit LC Model The Rabbit LC Model in vitro system was used to conduct realistic aerosol delivery testing and training for use of the air-jet DPI. To evaluate regional aerosol deposition in the LC Model, the inner walls of the airway model were coated with MOLYKOTE® 316 silicone spray (Dow Corning, Midland, MI) to minimize particle bounce and simulate an airway fluid coating. The air-jet DPI was loaded with 10 or 20 mg of the SLS-EEG formulation and connected to the ETT, which was inserted into the flexible silicone trachea. The LC Model was positioned horizontally on a flat surface, similar to the orientation of the rabbits in the in vivo experiments. The air-jet DPI was actuated for multiple cycles (typically three times for DI and five times for D2) with the LC pressure monitored and released after each actuation. After the air-jet DPI ETT connection was removed, the final pressure chamber evacuation was performed to capture any remaining suspended aerosol on the filter using a down-stream vacuum pump. Deposited powder formulation was recovered from the device, ETT, upper TB model, chamber and filter using appropriate wash volumes of methanol and those solutions were analyzed hy HPLC-UV to determine the DPPC content. The chamber and filter deposition fractions were grouped together as a lower lung deposition fraction. The upper TB, chamber and filter deposition fractions were grouped together as a total lung deposition fraction. All deposition fractions were expressed as a percentage of device nominal loaded dose based on the HPLC quantification of DPPC and were calculated from loaded powder mass and previously determined DPPC content. With EEG aerosols, it is important to establish that overly rapid aerosol growth does not occur in the upper TB region thereby significantly reducing the fraction delivered to the deep lung. Based on the small number of actuations required to deliver the aerosol with the air-jet DPI [3-5 actuations expected], we anticipate that the GAV can be either dry or at room humidity and temperature conditions with minimal impact on the lungs. To capture the impact of lung humidity conditions near 100% RH, experiments (using 20 mg device loaded mass) were conducted in which the walls of the LC Model were pre-wetted. Briefly, realistic humidity conditions (37 ± 2°C and 98 ± 2% RH) were created in an environmental chamber (ESPEC Corp., Grand Rapids, Michigan) and the Rabbit LC Model equilibrated in the chamber. Heated and humidified air was passed through the ETT to upper TB and LC regions until the walls of the system were saturated, with visibly wet surfaces. Large condensed water droplets were visible in the ETT, upper TB model, and LC walls. The SLS-EEG formulation delivery was repeated as previously described into the humified Rabbit LC Model. Statistical Analysis Statistical analysis for comparing aerosol delivery performance and estimated lung delivery efficiencies were performed using JMP Pro 17 (SAS Institute Inc., Cary, NC). The NGI particle size distribution and regional deposition data between DI and D2 device were compared using Student’s t-Test while comparison of multiple cases (effect of different loaded mass on NGI deposition fractions) utilized one-way ANOVA followed by post hoc Tukey. Statistical tests used a significance limit of p - 0.05. Results Formulation Characterization: DPPC and B-YL Content Uniformity Formulation content uniformity in terms of DPPC and B-YL content were assessed using validated HPLC-UV methods. Over the concentration ranges (50-400 pg / mL for DPPC and 725 pg / mL for B-YL), for both DPPC and B-YL, the calibration curves were linear with correlation coefficients (R2) > 0.998. The accuracy (%relative error) in the linear concentration range was < 9.6% for DPPC and < 4.6% for B-YL. The precision (%CV) in the linear concentration range was < 7.0% for DPPC and <7.7% for B-YL. The limit of quantitation (LOQ) for DPPC and B-YL was 6.8 pg / mL and 4.0 pg / mL, respectively. Over the course of the study, multiple batches of the Nano SD SLS-EEG formulation were produced. The mean (SD) DPPC content of seven batches of spray-dried synthetic lung surfactant powder formulation was 50.5 (1.8) %w / w, which was 96.6% of the nominal content (52.5%). The coefficient of variation for DPPC content was 3.5%, indicating good reproducibility in DPPC content across batches. The mean (SD) B-YL content was 2.8 (0.0) which was 55.9% of the nominal content (5%). The lower measured content of B-YL in the formulation may have been due to peptide degradation during the mesh-nebulizer-based spray drying process. Analysis of the feed solution indicated that the nominal amount of B-YL was present prior to and after completion of the spray drying. Interestingly, an identical formulation spray-dried using the nozzle based Buchi S300 spray dryer did not have any change in the B-YL content (5.1 (0.1) %w / w) following spray drying, indicating the loss of B-YL was likely specific to the Buchi B-90 HP spray dryer. Formulation Characterization: Laser Diffraction Particle Sizing Evaluation of the particle size of the SLS-EEG spray-dried formulation is shown Table V with the volumetric particle size determined at dispersion pressures of 0.5 and 4.0 bar for seven different batches. The mean primary particle size (Dv50) of the SLS-EEG formulation batches measured at 4 bar dispersion pressure was 0.82 ± 0.02 pm with 100% of particles < 5 pm. Significantly, there is a large submicrometer fraction with 57.8% of particles being less than 1 pm. The coefficient of variation for the Dv50 was 2% reflecting the consistent size of the spray dried SLS-EEG formulation for the seven batches considered and reproducibility of the powder preparation process. The mean Dv50 of the batches measured at 0.5 bar dispersion pressure was 1.32 ± 0.22 pm, suggesting good dispersibility of the powders. The spray drying process produces micrometer-sized particles suitable for deep lung delivery, which were nearly fully dispersed to primary particles using the high dispersion pressure. Some small degree of aggregation was observed when the lower dispersion pressure was employed and this dispersion was associated with greater variability; however, the mean (Dv50) powder formulation size of 1.32 pm remained suitable for the EEG approach and deep lung delivery. Table V Mean ± SD Particle Size Parameters of the Nano SD Micrometer-Sized SLS-EEG Formulation at 4.0 and 0.5 Bar Dispersion Pressures (data arc mean ± standard deviation of 7 different batches). Values in Parenthesis Represent the Coefficient of Variation Dispersion Particle size pressure DvlO (pm) Dv50 (pm) Dv90 (pm) 4.0 bar     0.49 ±0.01 (2%) 0.82 ±0.02 (2%) 1.50±0.08 (5%) 4.0 bar    0.60 ±0,03 (5%) 1,32 ±0.22 (16%) 3.57 ±1,18 (33%) Formulation Characterization: Scanning Electron Microscopy Figure 10 shows the scanning electron micrographs of the spray-dried SLS-EEG powder formulation and indicates good agreement with the laser diffraction measurements. In general, the powder particles were spherical with wrinkled surfaces, which is clearly visible at high magnification (15K). Formulation Characterization: XRPD and Moisture Sorption The solid-state properties of the SLS-EEG powder formulations were characterized using XRPD and water vapor moisture sorption. Figure 11 shows the X-ray powder diffractograms of three different batches of the SLS-EEG formulation. X-ray powder diffraction showed evidence of a partially crystalline structure with peaks corresponding to mannitol and 1-leucine. There were no obvious differences in the peak diffractograms indicating reproducible particle production between the batches. The water vapor sorption behavior of the SLS-EEG powder measured with DVS is shown in Figure 12. Following drying of the powder formulation during an equilibration period at 0% RH, water uptake by the powder under ambient-like conditions (10-60% RH; red line) was less than 9%, indicating relatively low uptake for an unprotected spray dried powder (Figure 12). Above 60% RH, the moisture uptake increased significantly, indicating the hygroscopic nature of the formulation once the 1-leucine barrier layer is overcome at elevated RH. This biphasic behavior is well suited for the EEG dry powder aerosol application. Specifically, the water vapor sorption behavior indicates low moisture uptake during storage under ambient conditions and elevated uptake at higher humidity similar to the airway environment when hygroscopic growth in the humid lung airways is required for EEG particle size growth to significantly enhance particle deposition and retention. Air-Jet DPI Pressure Testing in the Rabbit LC Model Pressure testing in the Rabbit LC Model was conducted based on three aerosol delivery scenarios described in the Methods with the use of the EM Timer settings provided in Tabic IV. For Scenario 1 delivery (1.3 kg rabbit; 0.45 mL / cm H2O; 0.18 timer setting), Table IV predicts that the APe will meet the < 20 cm H2O requirement at a tidal volume of 7 mL / kg. The measured LC pressure (Figure 13 A) illustrates the first administered breath (starting from zero-gauge pressure in the lung chamber) followed by three additional breath cycles. In each case, APe falls within the < 20 cm H2O range, together with 5 cm H2O PEEP, providing a total LC pressure < 25 ± 2 cm H2O, as targeted. The operator was also able to capture the 5 cm H2O PEEP with each cycle and apply the approximately 1-s recommended breath hold. For Scenario 2 (1.6 kg rabbit; 0.45 mL I cm H2O; 0.22 s timer setting), as indicated in Table IV, the measured APe was approximately 25 cm H2O, and when combined with 5 cm H2O PEEP, produced a total LC pressure that was out of the desired range (i.e., >25 + 2 cm H2O Fig. 13B). In some cases, the administering clinician may wish to prioritize maintaining an administered tidal volume of 7 mL / kg and accept the higher administered pressure for a limited number of breaths, for example to prioritize lung recruitment. In cases where the clinician wishes to prioritize maintaining the lung pressure < 25 ± 2 cm H2O, Scenario 3 adjustments should be applied. In this case, the clinician moves up the 0.45 mL / cm H2O compliance column in Table IV to the first clear cell, which administers a APe of 20 cm H2O based on reducing the EM Timer setting to 0.18 s. The resulting total tidal volume is 9.1 mL, which for the 1.6 kg subject results in a weight-based tidal volume of 5.7 mL / kg. As indicted in Fig. 13C, when applied to the Rabbit LC Model the Scenario 3 measured LC pressure was then <25 + 2 cm H2O, meeting the specified lung pressure recommendation. Aerosol Size Characterization: Comparison of DI and D2 Designs Table VI provides the aerosol size distribution of the Nano SD SLS-EEG formulation determined by using the Next Generation Impactor (NGI) and Copley Inhaler Testing Data Analysis Software (CITDAS) after delivering 10 mg loaded mass from the DI and D2 devices. The powder formulation device retention (as measured by DPPC assay) for the D2 device (18.8%) was significantly higher than the DI device (5.6%) (p < 0.05). However, the preseparator deposition, the fraction of the aerosol greater than -13 pm, for the D2 device was about 20% (absolute difference) lower than the DI device (38.4% vs 60.8%) (p < 0.05), suggesting more efficient aerosolization using the D2 device. This agreed well with the measured MM AD, which revealed that the D2 device produced a significantly smaller MMAD than the DI device (1.7 pm vs 2.3 pm). Correspondingly, the FPF<5 pm for the D2 device was also higher than the DI device (37% vs 21%) (p < 0.05). The observed differences between the micrometersize primary particles measured by Sympatec and the measured MMADs reflect incomplete deaggregation of the primary particles at the lower dispersion pressures used in the air-jet DPI. The implemented inhaler design modifications improved the aerosol size characteristics of the emitted aerosol with only a small impact on the overall emitted dose. Given the smaller aerosol size (MMAD) and acceptable emitted dose (80%) properties observed with NGI testing, the D2 device was selected for the subsequent dose planning study, described below. Table VI Aerosol Size Distribution of Nano SD SLS-EEG Formulation Determined by Using Next Generation Impactor (NGI) and Copley Inhaler Testing Data Analysis Software (CITDAS) After Delivering 10 mg Loaded mass from the DI and D2 Devices (data are means ± standard deviations, n = 3; MMAD: mass median aerodynamic diameter, FPF: fine particle fraction) Sections and terms               DI               D2 Device retention (%) 5.6 + 0,5 18.8+1.9* Pre-separator fraction (%) 60.8 ±1.3 38.4+ 1..+ M M A D (pm) 2.3 + 0,2 L7±0.3$ FPF < 1 pm (%) 8.1+2.3 12.0 + 4.3 PPP <5 pm (%) 20.9 ±2.5 37.4±L5* Emitted Dose (%) 94.4 ± 0,5 8 L2±L9* Total recovery (%) 96.4+1.4 92.7 ±0.6 * Significant difference in comparison to DI; Student’s t-test, p < 0.05 Dose Planning: Impact of Higher Loaded Doses Two additional SLS-EEG formulation doses (20 and 30 mg) were used to evaluate the impact of loaded dose on aerosol performance from the D2 device prior to in vivo testing (Table VII). Loaded dose mass had a significant effect on device retention and NGI sizing. At 30 mg loaded mass, the emitted dose (87.2%) was higher than with 10 mg (ED = 81.2%) and 20 mg (ED = 78.4%) loaded masses (Table VII). However, no differences were found in the emitted doses of between 10 and 20 mg loaded mass (p > 0.05). With increase in loaded mass, the preseparator fraction also increased (p < 0.05), which contributed to the decrease in fine particle fractions less than 5pm and 1 pm (Table VII). Considering a need to maintain an ED of ~ 80% or higher, an MMAD preferably < 2 pm and maximum FPFs, loaded dose masses of 10 and 20 mg with the D2 device were selected for additional testing in the Rabbit LC Model. Table VII Deposited Mass Fraction of Nano SD SLS-EEG Formulation after Delivering Different Loaded Masses to Next Generation Impactor (NGI) Using the D2 Device (data are means ± standard deviations, n = 3) Sections and terms Loaded mass 30 mg 10 me 20 mg Device remnticm Av 18,8 ±1.9 21,6 ± 1.7 123±13* Pre-separator fraction MMAD (pm)* 38,4 ± 1.3 L7 ±03 44,4 ±L5A L9 ±0.1 54,4 ±0,7* 2,9 ±0.5* FPF < 1 gm (%)* 12.0 ±4.3 9.3 ±0.5 6,4 ±0.7* IW<5 gm (%)* 37.4 ± 1.5 27.1 ± 1.5* 193 ±0,4* Emitted Dose 81.2 ±1-9 78.4 ±L7 87,2 ±13* Total recovery (%) 92.7 ±0.6 95.5 ± 1,7 943 ±2,1 *p < 0.05 significant effect of loaded mass on deposition fraction (one-way ANOVA) < 0.05 significant difference compared to 10 mg loaded mass (post-hoc Tukey) In Vitro Rabbit LC Model: Performance of the D2 Design The in vitro regional deposition fractions and estimated rabbit lung delivery efficiencies of SLS-EEG formulation were assessed using the in vitro Rabbit LC Model when delivered from the D2 device and the results are summarized in Table VIII. Considering the D2 device loaded with 10 mg of SLS-EEG formulation, the emitted dose was 80.4%, which was similar to the value that had been observed previously in the NGI studies. Aerosol deposition in the ETT and TB region was low, with only 3% of the aerosol being deposited in the 3 mm ETT (Table VIII). High fractions of the loaded dose were delivered to the simulated rabbit lungs, in terms of both the lower lung dose (filter + lung chamber) and total lung dose (TB + filter + lung chamber) delivery with values of 57.3% and 72.7%, respectively, using the D2 device and 10 mg loaded dose. Comparing 10 and 20 mg loaded doses delivered to the Rabbit LC Model with the D2 device resulted in only minor differences between the evaluated metrics. The only significant difference was in the drug mass deposited on the walls of the lung chamber, which increased from 49.0 to 54.5% of loaded dose with 20 mg mass loading. Key metrics of lower lung dose (LC + Filter) and total lung dose (TB + LC + Filter) with 20 mg loading remained high at 61.1 and 73.8%, respectively, with no significant difference from 10 mg loading. As a result, both 10 and 20 mg mass loadings with the Nano SD SLS-EEG powder appear to be good options in developing a drug delivery protocol for the in vivo animal model studies. The effect of system humidification on in vitro rabbit lung delivery was also evaluated using a 20 mg loaded SLS-EEG formulation dose (Table VIII). Both lung doses were significantly different when used in the humidified system for 20 mg loaded mass delivery (p < 0.05) (Table VIII), although no differences were observed in the device retention as a result of humidification conditions. In comparison to un-humidified condition, the deposition fractions in the ETT and TB were higher, but the deposition fraction was lower in the lung chamber for humidified condition (p < 0.05) (Table VIII). Specifically, with humified conditions and a 20 mg loaded dose of the Nano SD SLS-EEG formulation, lower lung and total lung doses were reduced to 30.3 and 66.0%, respectively. While these reductions are not ideal, they do appear to be acceptable considering the degree to which large water droplets were observed to obstruct the ETT and TB flow pathways. Overall, based on acceptable lung delivery efficiency results evaluated with realistic in vitro model testing, the developed formulation and device combination appeared capable of dry powder surfactant aerosol delivery via intratracheal administration for subsequent proof of concept in vivo testing, as described in Example 2 below. Specifically, a reasonable balance between loaded dose and aerosol performance with good lung delivery was observed when using the D2 device and f0 or 20 mg powder mass loadings of the Nano SD SLS-EEG formulation. Table VIII Regional Deposition Fractions (%) and Lung Delivery Efficiencies of Different Loaded Masses of Nano SD SLS-EEG Formulations Delivered from the D2 Device to the in vitro Rabbit LC Model (data are means ± standard deviations, n = 3) Sections and terms Deposited mass (%) 10 mg 20 mg 20 mg (humidified) Air-Jet Device 19.6 ±0.1 18.5 ±0.3 18.9 ±1.7 Endotracheal lube (ETT) 3.2 ±0.9 2.1 ± 0.6 13.5 + 0.4* Tracheobronchial region (TB) 15,4 ±1.9 12.7 ±1.9 35.7 ±4.7* Lung chamber (LC) 49.0 + 2.5 54.5 ± 3.3A 23.7±0.3* Filter 8,3 4? 1 ,4 6.6 ±0.4 6.6+0.6 Lower lung dose (LC4-Filter) 57.3 ±2.7 6 L1 i 3,0 30.3 ±0.7* Total lung dose (TB+LC+Filter) 72.7 ±1.4 73.8 ±1.2 66.0 ±4.2* Emitted Dose 80.4 ±0.1 81.5 ±0.3 81.0+1.7 Total Recovery 95.6± 1.5 94.4 + 2.0 98.4 ±3.7 ASignificant difference in comparison to 10 mg loaded mass; Student’s t-test, p < 0.05 * Significant difference in comparison to un-humidified condition; Student’s t-test, p < 0.05 Discussion This Example (i) presents a new dry powder SLS aerosol product that may be capable of high efficacy at relatively low doses and (ii) characterizes the aerosol delivery and performance of this product based on realistic in vitro testing in order to guide a best-case dose delivery protocol for subsequent animal model experiments (see Example 2 below). As described, the SLS aerosol product investigated by this Example included a newly developed formulation (called “Nano SD SLS-EEG”) administered with iDP-ADS hardware and a particular aerosol delivery strategy. Primary observations related to each product element arc reviewed below, followed by recommendations for a dose delivery protocol to test surfactant efficacy at low PL doses in surfactant-deficient animals. Considering first the new Nano SD SLS-EEG formulation, high PL content (60%) was achieved with good powder dispersion. The new SLS formulation had notably higher PL content and lower hygroscopic excipient content than the Survanta-EEG formulation summarized in the Background section above. For the administration of a 30 or 60 mg total dose to an infant or test animal, it was important to reduce the water-soluble hygroscopic components, which can induce osmotic stress if not fully solubilized during aerosol transport. In this Example’s tested formulation, water-soluble hygroscopic components were only 15% of the total dry powder formulation by mass. L-leucine concentration was maintained at 20% w / w for particle dispersion, which should have minimal biological impact considering that it has very low water solubility and also functions as a weak surfactant. Based on laser diffraction powder testing (without the air-jet DPI), high dispersion was observed with a Dv50 of 1.32 pm at 0.5 bar. The Dv50 at 4 bar better captures the expected primary particle size of 0.82 pm, as also observed in the SEM imaging. This small primary particle size together with the inclusion of the hygroscopic excipient are expected to enhance dissolution of the surfactant particles during aerosol transport and upon deposition, which is required for rapid and effective surfactant spreading and function. Furthermore, micrometer-sized primary particles deliver higher particle number concentrations to better coat the large alveolar surface area. That is, higher numbers of particles provide more deposited surface area to coat the alveolar region with a thin layer of surfactant, without increasing the mass of the delivered PL. For example, a primary particle diameter of 0.82 pm provides 6.1-fold more deposited particles than a 1.5 pm diameter particle, and 14.5-fold more deposited particles than a 2 pm diameter particle, all delivering the same total particle mass. Considering the iDP-ADS and design of the aerosolization engine, changes from DI to D2 devices were observed to improve the aerosol quality by doubling the FPF<5pm and reducing the MMAD from approximately 2.3 to 1.7 pm. The inward protrusion of the aerosolization chamber outlet with D2 reduced the rate of aerosol release and may have retained some undispersed aggregates within the chamber; however, emitted dose was acceptable at ~ 80%. Similarly, the metal RE reduced the aerosol size increase that was previously observed to occur with the use of the GE flow diffuser. A significantly larger aerosol size was emitted with the 30 mg powder mass loading. Setting the maximum MMAD at 2 pm for effective EEG aerosol delivery to an infant, both 10 and 20 mg mass loadings of the D2 device were acceptable options (i.e., had MMAD values < 2 pm). Similarly, 20 mg mass loading of the D2 design produced increased lower lung dose (LC + filter) and total lung dose (TB + LC + filter) values of ~ 61.1 and 73.8%, respectively. Delivering approximately 61% of the loaded dose beyond the upper TB model outlets and well into the lobar lung regions was impressive, especially considering the very small outlets of the upper TB model. While this lung delivery efficiency appears high relative to the large fraction of aerosol that was > 5 pm, it is noted that the iDP-ADS limited the aerosol delivery flow rate to 3 L / min, which maintained a low impaction parameter. Extreme humidification of the system with the formation of large occlusive water droplets in the TB region did significantly reduce the lower lung dose; however, 30% of the loaded dose was still delivered to this lung region. Components of the delivery strategy that we evaluated in this Example included (i) administration through an ETT interface, and (ii) delivery with positive-pressure ventilation. As described above, ETT delivery provided a majority of the aerosol to the lower lung region (in the absence of extreme humidity) and <5% aerosol loss in the 3 mm (internal diameter) ETT. Administration of the surfactant aerosol with positive-pressure breaths is intended as a way to recruit collapsed lung regions, better distributing the aerosol to the alveoli and throughout the lungs, and then the deposited surfactant serves to hold the recruited lung regions open through dissolution and reduction of the airway surface liquid surface tension. This Example limited the delivered tidal volume to the higher end of currently acceptable ventilator settings, i.e., 7 mL / kg, which is intended to be administered with the air-jet DPI for only ~ 4 to 10 breaths per aerosol delivery cycle. With this delivery strategy, this Example illustrates the administration of controlled lung pressure of approximately APE < 20 cm HoO and LC pressure <25 + 2 cm H2O. This Example also provides a method for maintaining these lung pressure conditions when compliance is extremely low and the tidal volume, therefore, needs to be reduced at the discretion of the clinician administering the therapy. An important component of the aerosol delivery strategy implements an enhanced excipient growth (EEG) approach. In the current formulation, mannitol and sodium chloride are included as hygroscopic excipients at dry powder mass fractions of 9% and 6% w / w, respectively. While this combination of hygroscopic excipients accounts for only 15% of the dry particle mass, the combination provides significant potential size increase. Based on previously developed and experimentally validated growth correlations (Hindle M, Longest PW. Condensational growth of combination drug-excipient submicrometer particles for targeted high efficiency pulmonary delivery: Evaluation of formulation and delivery device. I Pharm Pharmacol. 2012;64:1254-63 ; Longest PW, Hindle M. Numerical model to characterize the size increase of combination drug and hygroscopic excipient nanoparticle aerosols. Aerosol Sci Technol. 2011;45:884-99) the SLS-EEG particles have a growth coefficient (GC2) value of approximately 3.0, which predicts a final to initial diameter ratio increase of approximately 2.2 within the lungs at 99.5% relative humidity. As a result, particles falling within the 0.9 pm and 1.9 pm size bins will grow to 2.0 and 4.2 pm within the lungs when fully hydrated. Based on predictions of regional aerosol deposition within the lungs of infants, this size increase is expected to significantly improve the alveolar retention and reduce the exhaled dose of the aerosol. A primary outcome of this study is the application of the aerosol delivery characteristics to predict a dose delivery protocol for testing the efficacy of low dose AST in the rabbit surfactant washout model. Approximately 1.5 to 10 mg PL / kg delivered mostly to the lower lung region should be sufficient for high surfactant efficacy. Beginning with a 60 mg total dose of the SLS-EEG formulation (administered over successive delivery cycles) and using the experimental in vitro delivery predictions for 20 mg individual loadings and an average expected test animal weight of 1.5 kg (consistent with both preterm infants and rabbit test-animals), the following PL dose estimates were calculated: • Nominal Loaded PL Dose: 60 mg x 0.6 (60% PL content)= 36 mg PL • Nominal Loaded PL / kg Dose: 36 mg PL / 1.5 kg (typical weight) = 24 mg PL / kg • Emitted PL / kg Dose: 24 mg PL / kg x 0.8 (~ 80% emitted dose) = 19.2 mg PL I kg Based on Rabbit LC Model testing conditions with multiple 20 mg dose loadings (and the same 60 mg total device loaded dose): • Estimated PL / kg Lower Lung Dose: 24 mg PL / kg x 0.61 = 14.6 mg PL / kg • Estimated PL I kg Total Lung Dose: 24 mg PL / kg x 0.74 = 17.8 mg PL I kg Fine particle doses (FPD) of PL entering the lungs were also calculated, based on the production of the emitted PL dose and fine particle fractions, as follows: • Fine Particle Dose < 1 pm (PL I kg): 19.2 mg PL / kg x 0.093 = 1.8 mg PL / kg • Fine Particle Dose < 5 pm (PL I kg): 19.2 mg PL / kg x 0.271 = 5.2 mg PL / kg As a result, with a SLS-EEG formulation dose (administered over multiple deliveries) of 60 mg, the in vitro lung model predicted range for lung-delivered PL I kg dose is 14.6 to 17.8 mg PL / kg. The PL FPD values that fall within the aerosol size distributions of < 5 and < 1 pm are 1.8 and 5.2 mg PL / kg, respectively. After initial deposition, some dissolution and spreading of the surfactant is expected. Some of the upper lung delivered dose will likely spread to the alveolar region over time, increasing this fraction. However, there is a substantial coating cost associated with moving surfactant from the tracheobronchial to the alveolar airways, which is proportional to the size of the treated subject. Furthermore, some of the aerosol may be exhaled, even with significant EEG growth. Taking all of these factors into consideration, an initial SLS-EEG formulation dose of 60 mg appears to provide a good starting point for achieving and testing the efficacy of low dose AST and approaches an initial desired range of 1.5 to 10 mg PL I kg- In addition to the total SLS-EEG formulation dose to be administered, a primary outcome of this Example is the recommended dose delivery protocol timing and use of the device, as described below. Factors to be considered in determining the dose delivery protocol included: i. Limitation of the D2 device to 10 or 20 mg powder mass loadings, partly necessitating multiple dose delivery cycles; ii. A dose delivery cycle can be divided into an aerosol delivery period and a stabilization and monitoring period; the final dose delivery cycle does not include a stabilization and monitoring period; iii. Each dose delivery cycle requires the test animal (or ventilated infant) to be removed from the ventilator for connection with the current D2 device at least once, causing a brief lack of PEEP support and potential risk of partial lung collapse and oxygen desaturation; iv. The maximum amount of the SLS-EEG formulation that can be safely delivered over an aerosol delivery period is currently unknown; and v. The epithelial layer is expected to be capable of absorbing and / or dissolving a majority of the hygroscopic material in the formulation within a 15-45 min window. In order to limit the amount of dry powder formulation delivered as a bolus during an aerosol delivery period (based on a lack of current knowledge about the SLS-EEG formulation), and to minimize the number of ventilator disconnects (which will be detrimental for oxygenation), one option is delivering the 60 mg total dose over two equally divided 30 mg dose delivery cycles. Considering that only 3 mg / kg of hygroscopic material is contained within each 30 mg dose (and only 2.2 mg / kg of this reaches the lungs), it is anticipated that a 15-min stabilization and monitoring period is more than sufficient before delivery of the second dose. The resulting full dose delivery protocol is illustrated in Fig. 14. Based on the limitation that the D2 device can only be loaded with a maximum of 20 mg (for acceptable performance), and that aerosolization performance was moderately improved with a 10 mg loading, it is recommended that each 30 mg dose be divided into subsequent 20 and 10 mg device loadings. Each of these administrations can be delivered quickly, with five actuations requiring ~ 10 s. Between these sequential 20 and 10 mg doses, the animal is required to go back on the ventilator for ~ 2 min of respiratory support, while a second device is connected to the EM Timer. As illustrated in Fig. 14, each aerosol delivery period therefore requires a total of ~ 2 min and 20 s (i.e., 2.3 min) based on current maximum dose loading limitations of the device. As shown in Fig. 14, a total therapy time can be calculated as the sum of the two aerosol delivery periods and the stabilization and monitoring period. After the second and final aerosol delivery period, a subsequent stabilization and monitoring period is not required as part of the therapeutic delivery. Furthermore, each of the aerosol delivery periods can be assumed to require ~ 2.3 min, on average. Therefore, a total therapy time can be estimated to require < 20 min. Considering that a majority of this time is used for stabilization and monitoring in these early experiments, a total aerosol delivery time should also be considered, which can be calculated as the sum of the aerosol delivery periods, which is < 5 min. Considering limitations of the device used in this Example, increasing the capability for single dose loadings to 30 or 60 mg is desirable and is discussed in Example 3 below. While the administered flow rate is low and we observed good penetration through the upper TB geometry was observed, increasing the FPF of the aerosol is also desirable. Resistance in the Rabbit LC Model was lower than will be experienced in the animal model experiments and future nose-to-lung studies; however, compliance was accurately matched, and even with the in vivo studies, compliance will largely control pressure at the device and in the lungs. Furthermore, increasing the airway resistance will only increase the dissipation of pressure energy in the conducting airways and reduce pressure exposure in the alveolar region, which is where the danger of alveolar rupture and pneumothorax occur. We therefore view the recommended guidelines as conservatively safe for administering the targeted pressure ranges of 25 ± 2 and 5 ± 2 cm H2O. Considering limitations of the delivery strategy, these initial animal model efficacy experiments are intended to benchmark potential efficacy. In conclusion, a new SLS powder aerosol product was developed and refined in this Example. Realistic in vitro testing indicated excellent aerosol delivery efficiency as a percentage of loaded dose to the lower lung (61.1%) and total lung (73.8%) regions. Safe positive-pressure ventilation by the device during aerosol delivery over a range of test animal weights and lung compliances was also demonstrated. Realistic in vitro testing in a Rabbit LC Model proved to be highly useful in developing a product that could safely deliver a majority of the aerosol to the lower lung region. With the aim of testing the efficacy of the SLS aerosol product at low surfactant doses, a dose delivery protocol was recommended that would rapidly administer two 30 mg powder masses, interspersed by a 15-min monitoring and stabilization period, equating to an approximate 20-min total therapy administration time. The resulting total loaded 60 mg SLS-EEG dose contains 24 mg PL / kg, which is approximately an order of magnitude below what has previously been reported in most all other animal surfactant studies and what is administered with the current clinical practice of high volume surfactant liquid bolus instillation. Example 2. A New Dry Powder Aerosol Synthetic Lung Surfactant Product for Neonatal Respiratory Distress Syndrome (RDS) -In vivo Efficacy Testing in a Rabbit Surfactant Washout Model This Example is a proof-of-concept animal study assessing the preclinical biological efficacy of the Nano SD SLS-EEG dry powder formulation aerosolized with the iDP-ADS and D2 air-jet DPI using a preterm-infant-sized animal model (e.g., 1500 g) of surfactant washout and acute lung injury (i.e., RDS). Experiments were designed to compare the pulmonary gas exchange and other lung parameters in ventilated juvenile rabbits with RDS between the standard liquid dose of intratracheally instilled liquid surfactant (200 mg / kg) and the Nano SD SLS-EEG dry powder aerosol (60 mg device loaded dose) delivered with the iDP-ADS. Materials and Methods Surgical Preparation / Instrumentation All experimental animal procedures were conducted according to the National Institute of Health’s Guide for the Care and Use of Laboratory Animals and approved by the Seattle Children’s Institutional Animal Care and Use Committee. Juvenile female New Zealand White rabbits (1.56 + 0.1 kg, Western Oregon Rabbit Company, Philomath, OR) were tranquilized with 1 mg / kg acepromazine and anesthetized with 33 mg / kg ketamine and 6.6 mg / kg intramuscular xylazine. The SpOz was monitored (Rad 7, Massimo, Italy) with a tail probe, and body temperature was monitored continuously with a rectal temperature probe and maintained normothermic (38-39°C) using a warming pad. The neck and upper chest were shaved. Cetacaine spray (0.1%) was applied topically to the oral pharynx and glottis to reduce gag reflex and laryngospasm during oral intubation. Local anesthesia was provided around the trachea with lidocaine. The trachea was dissected and isolated, and animals were intubated with a 3.0 mm ID ETT. Following intubation, a strand of umbilical tape was used to anchor the trachea and ETT to prevent displacement, gas leakage, and saline leakage during lavage. Animals were ventilated (Draeger, Babylog VN500, Lubeck Germany) with volume guarantee in the assist / control mode with FiO2 of 1.0, minimum respiratory rate of 40 breaths / min, a tidal volume (Vt) of 6 mL / kg, inspiratory time of 0.30 s, and a positive end-expiratory pressure (PEEP) of 5 cm H2O. Medical gases were conditioned to 37- 40 °C with a heated humidifier (MR 850, Fisher & Paykel Healthcare, Auckland, New Zealand) and heated-wire ventilator circuits. A calibrated proximal hot-wire flow sensor placed between the ventilator circuit patient wye, and ETT provided real-time respiratory rate, airway pressures, flow, Vt, and compliance measurements. The respiratory rate was initially adjusted to maintain pH between 7.35-7.45 and CO2 34-45 torr. Animals were administered rocuronium bromide to induce neuromuscular blockade during the lung lavage and surfactant administration. Subsequent rocuronium doses were withheld to promote spontaneous inspiratory efforts to assist mandatory ventilator breaths, consistent with the clinical management of preterm infants. A 20-gauge angiocatheter was placed in the right jugular vein for the administration of fluids and medications. Sedation and analgesia were maintained to minimize pain and promote spontaneous breathing efforts using continuous IV infusions of ketamine and xylazine (3 and 0.18 mg’kg-1’hr-1, respectively) and titrated based on blood pressure, heart rate, and maintenance of an anesthetic plane. Maintenance IV fluids were provided with a continuous infusion at 3 mL’kg-1’hr-1 of 0.9% saline containing 5% dextrose. A 22-gauge angiocatheter was placed in the right carotid artery for heart rate and blood pressure monitoring and sampling for arterial blood-gas analyses (ABGs). ABGs (pH, PaCO2, PaO2) were measured at regular intervals using a Radiometer ABL 800 (Radiometer America, Brea, CA). Before lung lavage for baseline RDS condition, ABGs, lung mechanics, ventilation parameters, and vital signs (i.e., heart rate, blood pressure, temperature) were acquired. Induction of Surfactant Deficiency and Lung Injury The FiO2 was increased to 1.0, and PEEP was reduced to 0 CIT1H2O. The lungs were depleted of surfactant and injured by cyclic alveolar collapse (PEEP of 0 cmH20) with repeated lavage using 25 mL / kg of warmed (39°C) normal saline (0.9%), with 5-min recoveries between lavages until the static lung compliance was 50% of the pre-lavaged value, SpO2 of 90-92% on 1.0 FiO2 and a PEEP of 5 cm H2O. The animals were deemed surfactant deficient when PaO2 <  100 mmHg was confirmed by two consecutive ABGs 30 min apart following lavage. Additional lavages were administered to establish these surfactant depletion and lung injury goals. Following the establishment of surfactant deficiency, the ventilator rate was initially titrated based on PaCOr values of 55-60 mmHg and pH 7.25-7.30, and animals remained in these settings throughout the rest of the experiment. If pH < 7.25 at any point, the respiratory rate was increased as needed. Experimental Protocol The sample size for the experimental animal group (Nano SD SLS-EEG) was calculated with a 95% confidence interval (CI), a power of 80%, and a standard deviation (SD) of 0.20; the total sample size was five (n = 5) based on the mean and standard deviation for PaO2 from a previous animal study that compared surfactant aerosol to liquid (Fok TF, al-Essa M, Dolovich M, Rasid F, Kirpalani H. Nebulisation of surfactants in an animal model of neonatal respiratory distress. Arch Dis Child Fetal Neonatal Ed. 1998;78(l):F3-9). The negative control (sham) and positive control (liquid Curosurf) groups included four animals in each group (n = 4). Following surfactant washout, animals in the treatment group received dry powder Nano SD SLS-EEG formulation from the iDP-ADS using the D2 device (n - 5), a clinical dose of intratracheally instilled liquid surfactant (Curosurf at 200 mg / kg) (n = 4), or the aerosol delivery process without powder (n = 4) as a (negative sham) control. All subjects were supported for 3.5 h following the initial intervention. Intratracheal Liquid Surfactant Administration (Curosurf) The animals in the liquid Curosurf group received standard intratracheal administration of porcine-derived Curosurf surfactant (poractant alfa, Chiesi, Parma, Italy). Curosurf comprises approximately 96% phospholipids (80 mg / mL) and 1% surfactant proteins B and C and is only approved for administration by tracheal liquid instillation. The vials of liquid Curosurf (200 mg / kg; 2.5 ml / kg) were drawn up into a syringe and administered based on a standard protocol with the animal supine and head in a midline position during mechanical ventilation (Walsh BK, Daigle B, DiBlasi RM, Restrepo RD, American Association for Respiratory C. AARC Clinical practice guideline surfactant replacement therapy: 2013. Respir Care. 2013;58(2):367 75). Using a 5 FR Multi Access Catheter (Kimberly-Clark, Dallas, TX) advanced to the distal end of the ETT; liquid surfactant was divided into two separate aliquots with each delivered rapidly (< 5 s) with gentle rotation with either the right or left side down. Instillation of dose was followed by ventilation for several minutes before rotating the position to the opposite side and instilling the remaining Yz dose. Additional time was given, as needed, between aliquots for the animal to stabilize throughout dosing. Dry Powder Surfactant Aerosol Delivery System Animals in the aerosol group received 60 mg Nano SD SLS-EEG (device loaded dose) dry powder formulation delivered directly to the ETT. A separate in vitro assessment of lung delivery efficiency (Example 1) was used to derive these values. A series of positive pressure actuations of oxygen were used to manually ventilate the animal and aerosolize the formulation from the iDP-ADS for delivery into the lungs. As described in the dose delivery protocol established in Example 1, two 30 mg formulation doses were loaded (from capsules) into the D2 DPI aerosolization chamber and delivered 15 min apart. Also determined in Example 1 in vitro studies, the D2 device was limited to a 20 mg powder mass loading (for acceptable performance), and aerosolization performance was moderately improved with a 10 mg loading; therefore, the 30 mg doses were divided into sequential 20 and 10 mg device loaded doses, delivered approximately 2 min apart. The animals needed to be disconnected and reconnected to the ventilator for each dosing. With only limited knowledge of the impact of the osmotic formulation components on the lungs, the rationale for administering two 30 mg aerosol doses 15 min apart was to prevent excessive delivery and not overwhelm the lungs from the high output powder formulation, allowing the airways to receive heated and humidified gases from the ventilator between doses. During aerosol delivery, the D2 air-jet DPI connected directly to the rabbit ET tube, enabling precise control over the peak inspiratory pressure (PIP) of 25 ± 2 cmH20, PEEP of 5 cmH20, Vt of 7 mL / kg, flow of 3 L / min, and actuation time of ~ 0.20-0.22 s (see 15). The actuator (electromechanical timer) settings for the DPI were determined based on the post-lavage compliance values and weight (kg) using a reference chart, which is described in greater detail above in Example 1. Each positive pressure actuation of the DPI (10 per each 30 mg dose) was controlled by a single operator (RMD) by manually depressing a PIP / PEEP valve manifold attached to the outlet of the DPI (Figure 15). A total of 10 actuations were employed to deliver each 30 mg dose. The iDP-ADS employs an adjustable pressure relief valve (> 25 cm H2O) and an analog pressure manometer, akin to a manual resuscitator, to measure pressure delivery and prevent barotrauma. The powder chamber was manually filled with formulation, and the DPI was actuated to deliver the aerosol and inflate the lungs, followed by an approximately 1 -s breath hold, after which exhalation was allowed. The animal was promptly returned to the ventilator following dosing. Physiologic Measurements Figure 16 shows a diagram of the animal procedures and study time points during the experiments, which considers the dose delivery protocol developed in Example 1. Gas exchange (ABGs), ventilation parameters, and hemodynamic measurements were obtained at pre-lavage (-60-min timepoint) and post-lavage (‘baseline’ RDS, pre-treatment condition, -5-min timepoint). Between -5 and 0 min, the initial Nano SD SLS-EEG (30 mg) dose or sham, was delivered, and liquid instillation of the total dose of Curosurf was completed. The one-minute time point measurements were used to assess the short-term gas exchange effects immediately following therapy due to lung volume derecruitment from ventilator disconnections with SLS-EEG or sham delivery and airway obstruction from liquid Curosurf or powder agglomeration. Measurements were obtained at 30-min intervals thereafter for 3.5 h. (See Figure 16). Respiratory rate, peak inspiratory pressure, PEEP, and MAP were acquired from the ventilator. The pressure-volume relationship was evaluated by performing a brief inspiratory hold and recording the static compliance. The compliance was normalized based on the individual rabbit weights (mL / cmlLO / kg). The oxygenation index (OI) and ventilation efficiency index (VEI) were calculated using ventilator parameters and ABG values to assess disease severity at each timepoint. Animals were euthanized with 100 mg / kg Euthasol following the 3.5-h post-surfactant measurements and remained ventilated for necropsy. Postmortem Histologic Assessment of Lung Samples After median sternotomy, the lungs were removed en bloc while ventilated at peak inspiratory pressure of 20 cml EO (PEEP 0 cmH20). After taking gross morphological photos to assess the anterior lung inflation, the left lung was ligated and excised, a medial slice was taken of the left lower lobe, and the tissue was evaluated for the presence of surfactant on the lung surface. The right lung remained ventilated, and the ET tube was advanced into the right mainstem bronchus; 10% neutral formaldehyde fixative was infused at a constant pressure of 25 cmHiO. The right lung was then immersed in a container of formaldehyde for 72 h and placed in 70% ethanol. Right lower lobe samples were embedded in paraffin, and the tissue was sectioned at 5 pm thickness. Hematoxylin and eosin (H&E) slide staining was examined under light microscopy (x 8.5) to inspect the extent of lung injury and uniformity of lung inflation. Images were captured with a digital camera mounted on a Nikon Eclipse 80i microscope using NIS-Elements Advanced Research Software v4.13 (Nikon Instruments Inc.) and processed with ImageScope software (version 12.4.6.5003, Leica Biosystem). Statistical Analysis Data were calculated as mean ± SEM for all continuous physiologic variables at each timepoint for surfactant-treated and control groups. An ordinal one-way ANOVA was used to compare differences in lavage numbers, body weight, gas exchange, and disease severity data among groups following the completion of lung lavage. Two-way ANOVA was used to assess the treatment effects of each group and time and their interaction. Tukey’s multiple comparisons test compared mean post hoc differences in physiologic outcomes at baseline and at each time point following treatment between the different groups. The criterion for significance was established a priori P < 0.05 for all comparisons. Descriptive findings were the histological assessment of macroscopic and microscopic lung tissue on inflation, injury, and surfactant distribution. Results Animal Descriptions and Dosing Following lung lavage, the mean PaO2 was reduced from ~ 450 to 65 mmHg and compliance from - 1.1 to 0.4 mL / cmHiO / kg at baseline (P < 0.001) with increased PaCOi and disease severity (OI >15 and VEI ~ 0.04) values, all indicative of severely depleted surfactant pools, elevated alveolar surface tension and severe RDS. Baseline data following induction of lung lavage for surfactant deficiency were not different between groups for gas exchange, number of lung lavages, disease severity, and lung compliance (Table IX). All the animals in the liquid Curosurf group experienced some degree of acute airway obstruction from the liquid, resulting in hypoxemia (SpO2 < 85%), hemodynamic instability (e.g., bradycardia or hypotension) or increased PIP (> 25 cmH20) by the ventilator during dosing. The SLS-EEG aerosol and control groups experienced acute hypoxemia (SpCh < 85%) upon ventilator disconnection that quickly resolved when positive pressure actuations were applied for therapy with the iDP-ADS. Following the treatment, the heart rate (P = 0.15) and blood pressure (P = 0.9) did not vary between the groups. Based on an average rabbit weight of ~ 1.5 kg, the nominal dose of 60 mg of the Nano SD SLS-EEG powder formulation contained 24 mg PL / kg compared to the approximate 200 mg PL / kg with Curosurf. The total treatment duration, which included stabilization, was - 10 min to administer Curosurf. The aerosol delivery required a total of 20 actuations for 60 mg of Nano SD SLS-EEG powder formulation. As described in Example 1, the total aerosol delivery time, including ventilator reconnection to enable device reloading and stabilization, was completed in < 5 min. Table IX Baseline Data. Post-lavage baseline variables are shown following the induction of surfactant deficiency and lung injury. BW body weight; PaCO2 partial pressure of carbon dioxide in the arterial blood; PaO2 partial pressure of oxygen in the arterial blood; OI oxygenation index; VEI ventilation efficiency index; p values were compared between groups using one-way ANOVA. Values are shown as mean ± SD Control          Direct tn sti.lLtii.srs       SLSAiLG           Value 0^4)       (^4)            Aerosol^ ^5) BW (kg) PaCOs (mmHg) 1.57 ±0.07 52 ± 2 ! .57 ±0.03 63 ±9 1.54 ± 0,03 59 ±3 028 0 45 PaO? OnmHg) 59 + 18 62 + 11 82 + 6 0 / 11 Compliance tmVcmHiXMg) O ri ±0.06 0.38 ±0.09 0.43 * 0,06 0.27 Ol 17.3 + 6.12 14.6+125 0.41 VEI (mL&g / mmHg) 0 03 ±0.05 0.04 + 0.02 0.04 ±0.02 0.45 Lavages (n) 13.5 ± 7.6 128 + 6.4 114+5 0.98 Gas Exchange Gas exchange data for the different groups is shown in Figure 17. Following surfactant depletion, all groups had mean PaO2 values below 75 mm Hg, on FiO2 of 1.0 at baseline. As expected, PaO2 values increased from base-line in the surfactant groups immediately following therapy and remained <100 mm Hg in the control group throughout the experiment. The mean PaO2 and compliance at the 1-min timepoint were not different among the treatment groups, indicating similar short-term dosing effects between aerosol and liquid surfactant deliveries. Following the second and final aerosol and sham deliveries at 15 min, the PaO2 values at 30 min did not differ between the surfactant groups, but the liquid Curosurf group had higher PaO2 than did the controls. Among the surfactant groups, arterial oxygenation was not different at any timepoint following administration and PaO2 values fully recovered to the pre-lavage basal levels (96-100%) at 3.5 h. Both surfactant treatment groups had higher PaO2 than the sham controls from 60 min until the 3.5-h study endpoint. The mean PaCCE increased, and pH decreased among groups due to severe RDS induced by lung lavage. The PaCO2 values in controls remained increased throughout the experiment and trended downward in both surfactant treatment groups, although they did not differ. The pH values remained between 7.25 and 7.30 in the controls and remained low, and the liquid Curosurf group had higher pH than the controls at 3 and 3.5 h (see Figure 16). Lung Compliance and Disease Severity Lung compliance and indices of disease severity data for the different groups is shown in Figure 18. The lung compliance improved following surfactant delivery in both groups and began trending downward with liquid Curosurf following the 30-min timepoint. Following the 60-min timepoint, the compliance values in the SLS-EEG aerosol group began further increasing and compliance decreased in both the liquid Curosurf and control groups. The SLS-EEG aerosol group had greater compliance than the liquid Curosurf and control groups from the 150-min timepoint until the end of the experiments. At the 3.5-h time point, the compliance in the liquid Curosurf and sham control groups was not improved (0.4 vs 0.3 mL / cm H2O / kg, respectively) from baseline. In contrast, the surfactant SLS-EEG compliance had doubled (0.84 mL / cm H2O / kg) and recovered to 77% of the pre-washout value. Surfactant washout produced OI > 15 for all cases. As expected, improvement was not observed for the control cases over the 3.5-h observation period. In contrast, surfactant SLS-EEG aerosol and liquid Curosurf groups provided values below 5 within 90 min and near 2 at 3.5 h. The VEI was higher in the SLS-EEG aerosol surfactant group than the controls at 30 and 120min time points and did not vary between the surfactant groups. Postmortem Histological Evaluation of Lung Tissue The macro and microscopic images of the lungs for the surfactant and control groups are illustrated in Figure 19. The gross macroscopic appearance of the anterior lungs is shown while being ventilated ex vivo with peak inspiratory pressures of 20 cmH20 (19a-c). The control lungs are underinflated with large areas of discoloration suggestive of pulmonary collapse, edema, and hemorrhage (19a). The liquid Curosurf-treated lungs (19b) show moderate inflation with areas of heterogeneous ventilation and petechial hemorrhage. The surfactant aerosol SLS-EEG-treated lungs are well-inflated with very little tissue damage (19c). The gross appearance of the excised tracheas following medial incision (19d-f), shows the control animal with a well hydrated and patent trachea (19d). The Curosurf-trcatcd animal (19c) shows abundant foamy liquid assumed to be surfactant obstructing the trachea with overflow from the airway. The aerosol SLS-EEG-treated animal (19f) reveals a patent trachea free from powder residual agglomerates and well hydrated. The macroscopic appearance of the excised rabbit lungs following dissection is shown in a cross-sectional slice of the lung lobes in Figs. 19g-i. In the control subject (19 g), there is diffuse hemorrhage with a singular surfactant pool (black triangle) or lung edema fluid on the cut surface of the lung lobe section. The liquid Curosurf (19 h) lung sample shows a heterogeneous distribution of what appears to be foamy surfactant at the level of the lobar bronchi and in the lung periphery liquid (black triangles). The surfactant Aerosol SLS-EEG (19i) sample shows a homogeneous layer of less foamy surfactant that is evenly distributed across the internal surfaces of the transected lung lobe, implying improved therapeutic distribution across the alveolar surfaces. The microscopy image in the control group had obvious pathological changes, including neutrophilic inflammation change, capillary congestion, alveolar hemorrhage, lung edema, hyaline membrane formation, and alveolar wall thickening (Fig. 19j). In contrast, lung tissue from the liquid surfactant group (19 k) had almost normal structures in some areas with mild hyperinflation, and adjacent areas showed greater alveolar collapse and injury (arrows). The SLS-EEG aerosol treated subject showed uniform ventilation with well inflated airspaces and mild septal thickening (191). Discussion Using an established small animal model of surfactant deficiency and severe RDS, this Example demonstrates that the biological efficacy of surfactant aerosol administration could be increased at a proportionally lower PL dose for the combination Nano SD SLS-EEG powder formulation administered using the iDP-ADS, resulting in a similar recovery in oxygenation and reduction in disease severity as standard liquid surfactant delivery. The findings of this Example indicate greater delivery efficiency to the acinar lung regions (Figure 18), resulting in a two-fold greater improvement in lung compliance than standard therapy. Remarkably, this was achieved with SLS-EEG aerosol using nominally ~ 10% (24 mg PL / kg) of the total instilled dose with liquid Curosurf (200 mg PL / kg). The potency of the aerosol delivery may be even greater when considering the actual dose delivered to the airways. In vitro studies (see Example 1) indicated that with the D2 air-jet DPI and Nano SD SLS-EEG formulation, approximately 74% of the dose reaches the lungs, and 61% reaches the targeted lower airways. This would indicate that following Nano-SD SLS-EEG dry powder aerosol administration, recovery equivalent to and surpassing the liquid instillation was achieved with a lower airway dose of approximately 15 mg PL / kg. These findings show an effective delivery of highly surface-active aerosol into the distal lung regions in < 5-min total aerosol delivery time that could avoid many of the treatment risks of liquid boluses and improve the overall efficacy of surfactant aerosol administration. The high efficacy of Nano SD SLS-EEG dry powder formulation in animals in this Example is attributable to rapid bolus delivery combined with positive pressure and high lower-lung deposition of a micrometer-sized aerosol, which was also demonstrated in studies in vitro, showing high-efficiency lung delivery of approximately 61 to 74% (see Example 1). The aerosol size characteristics of the SLS-EEG aerosol with the Infant D2 air-jet DPI are relatively smaller (< 2 pm) than other attempts in the field to administer liquid and dry powder aerosols. It can be inferred from the studies in vitro (see Example 1), which showed minimal deposition in the tracheobronchial airways (down to the 6th generation on some pathways), that most of the formulation bypassed the upper airways and deposited in the peripheral lungs. In conjunction with histology findings from the current study in vivo, there was no evidence of central airway deposition or obstruction, resulting in the high efficiency delivery of surfactant aerosols in the acinar lung regions. The Nano SD SLS-EEG formulation was manually timed to deliver aerosol by actuating the iDP-ADS, preventing high expiratory drug losses, and minimizing impaction losses in the airways, which may have limited the efficacy in previous animal and clinical trials that applied surfactant liquid aerosols with continuous output nebulizers. The Curosurf-treated animals from the current Example showed a high residual accumulation of surfactant liquids and foaming in the trachea and lobar bronchi with poor distribution to the alveoli. Liquid bolus instillation is associated with a highly heterogeneous surfactant distribution, frequent alveolar flooding, structural damage to the alveolar network, and increased inflammation of the lungs. The persistently low compliance values (0.6 to 0.75 ml / cmthO) from baseline can be related to the ventilation heterogeneity resulting from regional differences in VT due to alveolar collapse from liquid obstruction in the airways following SRT. Conversely, the well inflated regions are more prone to regional hyperinflation and gas trapping. These differences in regional VT delivery are a major factor contributing to the development of VILI. The liquid Curosurf-treated animals showed patchy hemorrhage and alveolar collapse due to VILI, which also may have contributed to low pulmonary compliance values. These findings on VILI are consistent with other histological findings among deceased infants who were treated with concurrent liquid SRT and mechanical ventilation. The animals in the current study had a more severe form of RDS (P / F < 100, OI > 15) than previous liquid aerosol studies (DiBlasi RM, Micheletti KJ, Zimmerman JD, Poli JA, Fink JB, Kajimoto M. Physiologic effects of instilled and aerosolized surfactant using a breath-synchronized nebulizer on surfactant-deficient rabbits. Pharmaceutics. 2021; 13(10): 1580), yet the mean PaO2 was recovered to 96% of pre-washout values with only two 30 mg doses of powder surfactant aerosol (24 mg PL / kg) at the end of the 3.5-h observation period. The Nano SD SLS-EEG aerosol animals had approximately doubled lung compliance (0.41 to 0.84 mL / cm H2O / kg) with 77% recovery to pre-lavage baseline, and the surfactant was homogenously distributed within the acinar regions, resulting in more uniform lung inflation than liquid Curosurf. This rapid increase in compliance after SLS-EEG aerosol indicates substantial improvements in the end-expiratory lung volumes (i.e., FRC) associated with stabilizing small airways and alveolar units by surfactant. The SLS-EEG formulation does not require a liquid diluent, which minimizes the risks for liquid accumulation, foaming, airway congestion, and obstruction previously observed with standard liquid bolus and nebulized liquid surfactant aerosol administration. The highly dispersible small particle SLS-EEG dry powder aerosol achieved improved efficacy at ~ 1 / 10 the PL dose compared with Walther et al. (Walther FJ, Gupta M, Lipp MM, Chan H, Krzewick J, Gordon LM, Waring AJ. Aerosol delivery of dry powder synthetic lung surfactant to surfactant-deficient rabbits and preterm lambs on noninvasive respiratory support. Gates Open Res. 2019;3:6) for their evaluation of ARCUS® dry powder surfactant aerosol. A best-case dose of 240 mg / kg (168 mg PL / kg) resulted in a final PaO2 of - 300 mmHg on FiO2 1.0, 63% of pre-washout values, and final compliance did not rise above 0.5 mL / cm H2O. The ARCUS dry powder aerosol product also required an order of magnitude more dose and more device actuations (e.g., 400) and aerosol delivery time compared with the SLS-EEG aerosol product. While effective, the biological response with this dry powder aerosol formulation (240 mg / kg) in preterm lambs supported on B-CPAP was somewhat muted with blood oxygenation and airway compliance values that only recovered to 63% and 54% (final compliance = 0.49 mL / cm H2O), respectively (Walther FJ, Waring AJ, Otieno M, DiBlasi RM. Efficacy, dose-response, and aerosol delivery of dry powder synthetic lung surfactant treatment in surfactant-deficient rabbits and premature lambs. Respir Res. 2022;23(l):l-16). A challenge for dry powder surfactant formulations is ensuring complete deaggregation and prevention of agglomeration upon delivery, which could explain the reduced compliance and muted effects with previous attempts to deliver dry powder surfactant aerosols. The Nano SD SLS-EEG dry powder formulation includes L-leucine as a dispersion enhancer to promote deaggregation to micrometer-sized primary particles and hygroscopic growth excipients (mannitol and sodium chloride) that enable the micrometer-sized aerosol to increase in size by condensational growth during transport through the lungs to promote deep lung deposition. Remarkably, no evidence of powder agglomeration in the airway was observed with SLS-EEG formulation for studies in vitro or when the aerosol was combined with the in vivo high humidity (RH of 100%) in the animals, which showed no macroscopic or microscopic evidence of airway obstruction from aerosol aggregation. While the in vivo response to the D2-NSD1 combination was excellent at an order of magnitude less PL dose compared with current clinical practice, some limitations related to the device and SLS-EEG formulation were identified. Considering performance of the device, acceptable aerosolization limited the powder mass loading to 20 mg per dose cycle, which extended the total treatment time and required several otherwise unnecessary cycles of disconnecting and reconnecting the mechanical ventilator. With each 20 mg loaded dose, the D2 device was only actuated -4 times; however, inconsistent emptying of the device was observed across these four actuations, with a majority of the powder mass emptying on the first actuation. Overly rapid emptying of the powder resulted in an excessively dense aerosol cloud, potentially leading to an aerosol fine particle fraction (FPF) <5 pm that was relatively low at -30% of the ETT emitted dose. Finally, the response rate to the administered aerosol appeared more consistent with mid-level TB deposition of the aerosol followed by subsequent slow spreading of the surfactant to the alveoli, whereas true alveolar deposition of a majority of the dose may theoretically provide a very rapid recovery of lung function. In this initial in vivo study employing a preterm-infant-size test animal model, administering surfactant aerosol with short-term positive pressure actuations (~ 0.2 s) and applying a small breath hold (Is) with the DPI could represent a best-case scenario for aerosol deposition in the acinar lung regions. This could be achieved because the animals were heavily sedated and under neuromuscular blockade with rocuronium. It remains to be seen how the efficacy of this direct-to-lung powder aerosol delivery would translate into spontaneously breathing infants with RDS, who commonly breathe rapidly (60-100 / min) and have extremely short inspiratory times of 0.2 to 0.3 s. The surfactant aerosol delivery efficiency may be influenced by the operator’s ability to observe chest rise and appropriately time the positive pressure actuations and aerosol delivery during spontaneous breathing. Applying aerosol directly to the ETT minimizes aerosol humidity exposure and aerosol agglomeration from the ventilator system gases, which are usually heated and humidified. However, it is still likely that such a small dry powder aerosol size of 1-2 pm could still be delivered effectively in line with a ventilator system delivery and provide a similar benefit. Example 1’s in vitro study findings indicate similar delivery efficiency within a high-humidity setting with minimal agglomeration due to the brief bolus delivery process. It is not typical to remove patients from a ventilator to provide aerosol in the clinical setting due to the risks of alveolar collapse, deterioration, and VILE However, these risks are likely much lower than standard SRT for the bolus administration of the Nano SD SLS-EEG powder formulation due to its very short administration time (< 5 min total aerosol delivery time), which is also delivered with positive pressure. Nonetheless, we did not show an acute reduction in PaO2 from derecruitment at the one-minute timepoint, and the lungs in the aerosol group did not appear injured due to the ventilator disconnections. Considering the formulation, the NSD1 dry powder was spray-dried using the Buchi Nano system, which employs a unique mesh nebulizer design and electrostatic precipitator to collect the powder. These unique elements of the Buchi Nano system are advantageous for forming very small primary particles; however, represent a laboratory scale approach, which is inconsistent with the two-fluid nozzle system used in most industrial scale spray dryers. Furthermore, spray drying with the Buchi Nano was found to degrade -50% of the B-YL peptide, possibly in the electrostatic precipitator, which is currently the most expensive component of the formulation despite its low concentration. The findings of this Example demonstrate the high biological efficacy of aerosol surfactant administration at a pointedly lower dose requirement for the dry powder aerosol formulation and device combination tested in this study compared to the current standard of care. Ata - tenfold lower dose, there were similar improvements in gas exchange and disease severity as standard liquid surfactant delivery, believed to be due to higher distribution of surfactant to the alveoli and associated reduced surface tension via aerosol delivery. In addition, the dry powder aerosol delivery improved lung compliance considerably compared to liquid instillation. The Nano SD SLS EEG formulation and powder aerosol delivery have the potential to be a safe, effective, and economical alternative to bolus liquid surfactant instillation and other investigational nebulized liquid aerosol surfactant products. This study showed that dry powder surfactant and EEG technologies can dramatically reduce the dose of surfactant and the required total aerosol delivery time to < 5 min. Example 3. Preclinical Testing of a New Dry Powder Aerosol Synthetic Lung Surfactant Formulation and Device Combination for the Treatment of Neonatal Respiratory Distress Syndrome This Example builds on the work of Examples 1 and 2 towards the demonstration of exemplary dry powder AST embodiments including formulations for administration to, e.g., preterm infants via an ETT interface. Specifically, the objectives of this Example include demonstration of physicochemical characterization of an exemplary scalable powder formulation together with aerosol formation and in vitro testing, and then evaluation of biological efficacy of the resulting AST in a preterm infant-size animal model of RDS. Formulations are tested in this Example with a variable-volume chamber air-jet DPI referred to as “D3” for simplicity of discussion in combination with the iDP-ADS, both of which are explained above in this disclosure. In brief review, the aerosolization engine, termed the D3 Air-Jet DPI, employed a variable-volume aerosolization chamber design, which was intended to enable higher drug mass loadings and better unify dose emission per actuation, potentially improving aerosol quality at high powder mass loadings. The new SLS-EEG formulation was produced on a scalable nozzle-based spray drying system (Buchi S-300 Mini Spray Dryer) and termed the SLS-EEG MSD2 formulation. Quality of the formulation was first evaluated based on imaging and physicochemical analysis. Aerosolization performance of the new device and formulation combination was then determined using in vitro assessment, including cascade impaction, laser diffraction sizing and testing in a realistic airway model. Initial goals for aerosol quality included maximizing FPFs with cutoffs <5, <2.5 and <1 pm to the extent possible compared with the previous D2-NSD1 combination (with FPF<5gm -30% of emitted dose) and achieving MMAD and median volumetric diameter (DV5o) values in the range of approximately 1.5 - 2.0 pm. Once aerosol quality and emitted dose metrics were achieved, in vivo performance of the new D3-MSD2 aerosol therapy was established in an intubated rabbit model of severe RDS with a body weight consistent with preterm infants at a gestational age (GA) of 31 weeks (-1.5 kg). Finally, in vitro data and in vivo responses were compared to determine DDP metrics leading to a high-efficacy response, in terms of both required active (PL) dose and recovery response rate. Materials and Methods Formulation Materials Phospholipids (dipalmitoyl phosphatidylcholine (DPPC) and l-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (sodium salt) (POPG)) and surfactant protein mimic peptide (B-YL) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and CSBio (Menlo Park, CA), respectively. L-leucine was purchased from Sigma-Aldrich Chemical Co. (St Louis, MO). Growth excipients NaCl and MN together with other chemicals were purchased from Fisher Scientific Co. (Hanover Park, IL). Throughout the study, freshly collected deionized water was used. Formulation Production and Analytical Methods Multiple batches of the micrometer-sized SLS-EEG MSD2 formulation were produced using the two-fluid spray nozzle based Buchi S-300 Mini Spray Dryer system (Biichi Labortechnik AG, Flawil, Switzerland). Briefly, the feed dispersions were prepared with a solids composition of DPPC 55%, POPG 7.5%, surfactant protein B peptide mimic B-YL 2.5%, mannitol 9%, sodium chloride 6% and l-leucine 20% w / w in an ethanol-water (5:95% v / v) cosolvent system, which was sonicated for 40 min in a heated water bath (45-55°C) with intermittent manual shaking every 10 min. The resulting phospholipid (DPPC and POPG) and B-YL nominal content of the MSD2 formulation were 62.5 and 2.5%, respectively; compared to the previous NSD1 formulation with corresponding theoretical values of 65 and 5%, respectively. The total solids content of the MSD2 feed dispersion was 0.125% w / v. During spray drying, the feed dispersion was continuously stirred using a magnetic stirrer. Spray drying conditions included use of a 0.5 mm spray nozzle, drying gas and spray gas flow rates of 500 and 20 L / min, drying temperature of ~80 °C, and an outlet temperature of -45-46 °C. Dried powder formulations were collected in a high-efficiency cyclone with a backup bag filter. Immediately post-collcction, powder formulations were equilibrated in a glass vial (-17% RH at -24 °C for 30 min), then stored in a sealed desiccator at 0%RH / -20 °C until needed for use. Validated HPLC methods were used to quantify DPPC mass in determining aerosol size distribution from cascade impaction and lung delivery efficiency from the in vitro Lung Chamber (LC) model. The HPLC system consisted of an Alliance 2695 Separation Module with a photodiode array UV detector and Empower 3 data acquisition software (Waters Corporation, Milford, MA). The sample injection volume was 100 pL and column temperature was 45°C. The chromatographic separation was achieved using the Waters XSelect CSH Cl8 XP, (2.5 pm, 4.6 x 100 mm; Waters Corporation, Milford, MA). Further details including descriptions of the gradient elution system and UV absorbance, as well as a separate method using a similar approach for the quantification of B-YL used in content uniformity testing, are reported above in Example 1. Laser Diffraction Particle Sizing Powder dispersibility and primary particle size of the SLS-EEG MSD2 formulation were determined using laser diffraction at multiple dispersion pressures. Specifically, components employed included the Sympatec HELOS (Sympatec GmbH, Germany) equipped with a RODOS / M disperser and an ASPIROS sample feeder. A small glass vial was filled with approximately 3 mg of spray-dried powder, placed in the sample feeder and dispersed in the laser beam using the RODOS / M disperser with an RI lens at dispersion pressures of 0.5 and 4.0 bar. The particle size distribution results (Dvio, Dvso and DV90) were obtained using Sympatec WINDOX 5.10 software (Sympatec GmbH, Germany). Powder Imaging The particle surface morphology of the SLS-EEG MSD2 formulation was evaluated using a Hitachi SU-70 scanning electron microscope (SEM) (Hitachi High-Tech, Tokyo, Japan) at an accelerating voltage of 5.0 kV. A small amount of powder sample was mounted on a metal SEM stub using double-sided carbon tape and loose particles were removed using compressed air. The samples were sputter coated with platinum grain using a Denton Desk V sputter coater (Denton Vacuum, Moorestown, NJ) before imaging. Aperture, astigmatism (X&Y), and beam alignment were adjusted for optimal imaging and then the image was captured at 5 kV with a 5 mm working distance. To capture internal particle structure, a combination Focused-Ion Bean (FIB) cutting and SEM imaging approach was employed through the use of a Scios 2 DualBcam system (ThermoFisher Scientific, Waltham, MA). Once the sample preparation was complete as described above, the specimen was fixed in the sample holder and then inserted into the FIB chamber. Contrast and brightness, astigmatism (X&Y), and focus were adjusted to identify an area of interest using the SEM mode. Then, in FIB mode, the sample was positioned at a 7 mm working distance from the electron beam and tilted at 52° with horizontal. The FIB column was set to 30 kV with ion beam current of 10 pA to mill the particles while minimizing damage to the internal structure, and then the SEM mode was activated to capture the image at 5 kV with a T2 detector setting. X-ray Powder Diffraction (XRPD) and Moisture Sorption Testing The Rigaku Miniflex 6G (Rigaku, lapan) X-ray powder diffraction technique was used to characterize the SLS-EEG MSD2 formulation. Briefly, a small amount of powder was placed on the center of the XRPD sample holder and then the powder surface was gently smoothed using a glass slide. The samples were scanned using a continuous mode in the range of 3-50° 20 at a scan speed of 1.5° / min. To understand the water uptake of the SLS-EEG MSD2 formulation across a wide range of relative humidity (RH) values, samples were exposed using the dynamic vapor sorption system (DVS Adventure, Surface Measurement Systems Ltd., UK). Briefly, ~5mg of powder sample was equilibrated at 0% RH and then exposed to increasing RH from 0 to 95% (sorption cycle) and decreasing RH from 95 to 0% (desorption cycle). At each RH, once the defined equilibrium condition (the rate of mass change (dm / dt) < 0.002%) was achieved, the RH was changed to the next level. Aerosol Delivery System and D3 Air-Jet DPI The infant air-jet dry powder aerosol delivery system (iDP-ADS) used in this study consisted of an electromechanically controlled timer air source (EM Timer), aerosolization engine, pressure monitoring and control (PMC) unit, and endotracheal tube (ETT) interface connector (see Figure 3). Considering the EM Timer, the inlet pressure regulator was set to provide a 3 L / min flow rate to the device of compressed air (in vitro testing) or pure oxygen (in vivo testing). The timer setting was adjusted to deliver a gas actuation volume (GAV) of 10 mL during in vitro testing and approximately 7 mL / kg for the in vivo experiments. In the animal model experiments, a table was used to ensure that for the measured lung compliance of individual animals, the delivery of 7 mL / kg would result in an clastic lung pressure rise (APe = 1 / C*GAV; where C is the lung compliance in mL / cmH20) < 20 cmFhO (see Example 1 above). If the APe was greater than the 20 cmH20 limit, then the GAV was reduced to meet the pressure limit, at the discretion of the administering clinician. Further details of this procedure, and the corresponding lookup table, are provided above in Example 1. The PMC unit enabled the control of respiration, pressure monitoring and pressure protection during the in vivo experiments and in vitro LC testing. A flow control orifice (0.5 mm diameter) was used in the exhalation port to slow exhalation and better enable the administrator to maintain a positive end expiratory pressure (PEEP) of ~5 cm H2O (Figure 7A). Additional ports on the PMC unit led to an analog pressure gauge (AG Industries, St. Paul, MO) and pressure relief valve, adjusted to ~25 cm H2O. For device actuation, at a lung pressure of ~5 cm H2O, the administering clinician covered the exhalation port, creating an airtight seal with the lungs, and actuated the device. The exhalation port remained covered for ~1 s. The administering clinician then uncovered the exhalation port and monitored the lung pressure during exhalation until ~5 cm H2O was achieved, at which point the process was repeated. Approximately 4 device actuations were used, accompanied by a defined rotation of the variable-volume chamber lower unit after each actuation, for each 30 mg device-loaded dose. A primary component of the iDP-ADS was the Infant Air-Jet (AJ) DPI (see Figure 7A). Air-jet DPIs are designed with geometrically simple elements (small diameter flow passages and smoothly curving aerosolization chamber) and engineered to enable the efficient generation of small-particle aerosols with limited volumes of air. In Examples 1 and 2 above, the aerosolization chamber was of fixed dimensions and volume, which for the SLS-EEG and other surfactant formulations limited device loading to <20 mg to maintain high quality aerosolization, in the absence of a cyclic loading mechanism. Higher mass loadings either emitted the aerosol too quickly, degrading aerosol quality, or had reduced emitted dose. This Example used a variable-volume aerosolization chamber with a movable lower unit which controls the floor height of the chamber (see Figure 7A). During in vitro testing, one actuation per level was employed. During in vivo delivery, one actuation per level was also used; however, the operator could employ additional actuations at each level with minimal powder emissions if needed to maintain animal ventilation. Aerosol Size Characterization using Cascade Impaction The aerosol emitted from the D3 Air-Jet DPI with the ETT adaptor attached was characterized using cascade impaction with a Next Generation Impactor (NGI; MSP, TSI Incorporated, Shoreview, MN). The D3 device was connected to the pre-separator inlet of the NGI, as previous illustrated by Howe et al.,52 using a custom adapter. The NGI was oriented at 90° to horizontal to enable the device to remain in the intended level orientation for use, and the custom adaptor positioned the ETT adapter of the D3 device in the center of and ~1 cm away from the pre-separator inlet. Side openings in the custom adapter enabled the entrainment of room air around the aerosol plume, allowing the NGI to be operated at a constant flow rate of 45 L / min, generated from a downstream vacuum source. The NGI flow rate of 45 L / min was chosen to ensure collection of the aerosol, minimize any effects of settling and provide appropriate stage cutoff diameters for evaluating small aerosol sizes. Each stage of the NGI was coated with MOLYKOTE® 316 silicone spray (Dow Coming, Midland, MI) to minimize particle bounce and re-entrainment. Before each set of experimental runs, the flow rate was confirmed using a flow sensor (Sensirion SFM3000, Sensirion AG, Stafa, Switzerland) connected to the NGI inlet. For aerosol size determination, the D3 device was loaded with 30 mg of the MSD2 formulation. Consistent with aerosol delivery to a 1.5 kg infant, 10 mL GA Vs were employed at 3 L / min, which required an actuation time of -0.21 s. One actuation was used for each height of the variable-volume chamber device across four levels (three marked as shown in Figure 7B with a final top level at the maximum floor height). Preliminary development experiments were conducted to determine the correct chamber volume and rate of floor rise to efficiently deliver a high-quality aerosol and maintain a high emitted dose for a 30 mg device-loaded formulation mass and four device actuations, resulting in a target powder mass per actuation of -5-10 mg. Three replicate runs for each experiment were performed and quantitative analysis of DPPC deposition using the HPLC-UV assay was used to assess the aerosol performance characteristics of the SLS-EEG powder formulation. Analysis metrics included emitted dose (ED), MMAD, FPF and geometric standard deviation (GSD). Percent emitted dose was calculated as the mass of DPPC in the loaded dose minus the mass of DPPC remaining in the device divided by the initial loaded mass of DPPC (xlOO for %ED). Aerosol size calculations were based on the mass of DPPC recovered in the NGI (including pre-separator). Based on an airflow rate of 45 L / min, the NGI stage cut-off diameters were determined using the formula specified in USP 35 (Chapter 601, Apparatus 5). The MMAD and FPFs (<5 pm, <2.5 pm and <1 pm) were calculated using Copley Inhaler Data Analysis Software (CITDAS). Characterization by Actuation using SprayVIEW, Spraytec and Emitted Mass A combination of light sheet microscopy (Proveris Scientific SprayVIEW®), laser diffraction particle sizing (Malvern Spraytec®) and gravimetric analysis was used to characterize aerosolization performance on the basis of individual actuations. For each experiment, 30 mg of the MSD2 formulation was loaded into the D3 device, and the device was actuated one time at each level of the variable-volume chamber, denoted as actuations A1-A4, using 10 mL GAVs delivered at 3 L / min from the EM Timer. All experiments were completed in triplicate, denoted as runs R1-R3. With the SprayVIEW system, the device outlet capillary was placed at the furthest position within the camera field of view, and the laser housing was adjusted to the center of the orifice in the vertical and horizontal directions. To simultaneously record gas flow rate, an inline flow meter (Sensirion SFM3400-AW and RS485-USB / 2028) was connected between the EM Timer outlet and D3 device inlet. Image acquisition was set at a frame rate of 500 Hz over a 1 s time acquisition window. As with all in vitro cases, the EM Timer was actuated for 0.21 s, which generated the 10 mL GAV at a flow rate of 3 L / min. Analysis of the SprayVIEW data was used to report plume geometry, velocity and intensity. While a quantitative correlation is not available between SprayVIEW reported plume intensity and aerosol mass emission, intensity over actuation time was used as a qualitative metric of aerosol emission relative to device actuation flow. To determine aerosol size distribution for each of the four actuations, the Malvern Spraytec laser diffraction system was employed in the default (open bench) configuration with 1kHz data acquisition rate and employed a 300 mm lens (0.1-900.0 pm nominal size range). Refractive indices were selected as standard opaque particles (1.50+0.50i) sprayed into air (l.OO+O.OOi). Considering the density of the aerosol cloud, necessary system modifications included only considering detector ranges from 16 to ‘Last’ and removal of plastic flanges to prevent cloud recirculation into dead zones. All other parameters remained under manufacturer default values. Concentrated-weighted, time-averaged aerosol polydisperse size distributions (PSDs) and median volumetric diameters (DV5o) values were determined using the Malvern Spraytec 4.00 software for each of the four device actuations (A1-A4). The emitted formulation mass for each actuation was determined in separate experiments in which the unloaded and loaded D3 device was weighed, using an Ohaus Analytical Plus (Model: AP250D) microbalance (Ohaus Corporation, NJ, USA), and then the loaded device was reweighed after each actuation. Following each actuation, the emitted mass for that actuation was determined as the difference between the previous loaded device mass and current device mass (in mg). Percent emitted dose per actuation (%ED) was calculated as the percentage of the emitted mass relative to the original loaded mass (loaded device mass - unloaded device mass). Total emitted mass and %ED were determined as the sum of the emitted masses over the four actuations and the percentage of this sum relative to the original loaded mass, respectively. In method validation experiments, gravimetric analyses of percent emptying matched HPLC quantification (to within 10% relative difference), further validating gravimetrically recorded mass losses for calculating %ED and emitted mass. In Vitro Testing of Lung Delivery Efficiency with the LC Model To approximate the lung delivery efficiency achieved in the in vivo animal model experiments, a realistic rabbit lung chamber (LC) model was employed, as described above in Example 1. Briefly, upper airway aerosol transport and deposition was captured using a 3D-printed model of the upper TB airways extending from the trachea to outlet generations from approximately G4 to G6, which was extracted from a CT scan of a small mammal with body mass, airway structure and dimensions very similar to young mature New Zealand White Rabbits. Outlet diameters of the upper TB region ranged from 0.8 to 2.3 mm. A majority of the trachea was constructed in flexible silicone material to enable realistic insertion of the ETT with airway sealing. The upper TB model was surrounded by a cylindrical lung chamber, with a total sealed system volume of -500 mL, resulting in a realistic measured compliance of 0.45 mL / cmHiO, consistent with preterm infants experiencing RDS. A filter (Pulmoguard II™, Queset Medical, North Easton, MA) and pressure release valve were used to capture aerosol that did not deposit in the upstream components. To evaluate regional aerosol deposition in the LC model, the inner walls of the airway model were coated with MOLYKOTE® 316 silicone spray (Dow Corning, Midland, MI) to minimize particle bounce and simulate an airway fluid coating. The D3 device was loaded with 30 mg of the SLS-EEG formulation and connected to the ETT, which was inserted into the flexible silicone trachea. The LC model was positioned horizontally on a flat surface, similar to the orientation of the rabbits in the in vivo experiments. The air-jet DPI was actuated at 3 L / min with 10 mL GA Vs one time at each of the four variable-volume chamber levels, with the LC pressure monitored and released after each actuation. After removing the air-jet DPI ETT connection, a final pressure chamber evacuation was performed to capture any remaining suspended aerosol on the filter using a downstream vacuum pump. Deposited powder formulation was recovered from the device, ETT, upper TB model, chamber and filter using appropriate wash volumes of methanol, and those solutions were analyzed by HPLC-UV to determine the DPPC content. The chamber and filter deposition fractions were grouped together as a lower lung deposition fraction. The upper TB, chamber and filter deposition fractions were grouped together as a total lung deposition fraction. All deposition fractions were expressed as a percentage of device nominal loaded dose based on the HPLC quantification of DPPC and were calculated from loaded powder mass and previously determined DPPC content. In Vivo Surgical Preparation and Instrumentation All experimental animal procedures were conducted according to the National Institute of Health’s Guide for the Care and Use of Laboratory Animals and approved by the Seattle Children’s Institutional Animal Care and Use Committee. Juvenile female New Zealand White rabbits (Western Oregon Rabbit Company, Philomath, OR) were tranquilized, sedated, instrumented, monitored, and ventilated according to identical methods established in previously published animal studies (e.g., see Example 2 above). At the time of in vivo testing, body weight of the D3-MSD2 aerosol group was 1.59 ± 0.23 kg, and body weight of the entire cohort including sham and Curosurf liquid historical controls was 1.56 ± 0.07 kg. Animals were administered rocuronium bromide to induce neuromuscular blockade during the lung lavage and surfactant administration. Subsequent rocuronium doses were withheld to promote spontaneous inspiratory efforts to assist mandatory ventilator breaths, consistent with the clinical management of preterm infants. Before lung lavage for baseline RDS condition, arterial blood gases (ABGs), lung mechanics, ventilation parameters, and vital signs (i.e., heart rate, blood pressure, temperature) were acquired at a timepoint of -60 min, as summarized in Figure 20. In Vivo Induction of Surfactant Deficiency and Lung Injury Severe lung injury was induced by a multiple lung insult (hit) model that included hyperoxia, atelectotrauma, extensive surfactant washout (typically 6 to 10 cycles) and mechanical ventilation. For injury induction, the FiO2 was increased to 1.0, and PEEP was reduced to 0 cmH20. The lungs were depleted of surfactant and injured by cyclic alveolar collapse (PEEP of 0 cmH20) with repeated lavage using 25 mL / kg of warmed (39°C) normal saline (0.9%), with 5-min recoveries between lavages until the static lung compliance was 50% of the pre-lavaged value, SpO2 of 90-92% on 1.0 FiCE and a PEEP of 5 cm H2O. The animals were deemed surfactant deficient when PaCh <100 mmHg was confirmed by two consecutive ABGs 30 min apart following lavage. Additional lavages were administered as needed to establish these surfactant depletion and lung injury goals. Following the establishment of surfactant deficiency and severe lung injury, the ventilator rate was initially titrated based on PaCO2 values of 55-65 mmHg and pH 7.25-7.35, and animals remained on these settings throughout the rest of the experiment. If pH<7.25 at any point, the respiratory rate was increased as needed. Animals with PaCO2 <40, pH> 7.40, and evidence of alveolar overdistension (reduced compliance for the terminal 20% of inspiration, C20 / C ratios < 0.80) had the respiratory rate reduced to prevent pneumothorax. In Vivo Experimental Protocol The sample size for the experimental animal group (SLS-EEG) was calculated with a 95% confidence interval (CI), a power of 90%, and a standard deviation (SD) of 0.20; the total recommended sample size was seven (n=7) for the experimental group based on the mean and standard deviation for PaO2 from a previous animal study that compared between two different surfactant aerosol delivery systems (Fok TF, al-Essa M, Dolovich M, Rasid F, and Kirpalani H: Nebulisation of surfactants in an animal model of neonatal respiratory distress. Arch Dis Child Fetal Neonatal Ed. 1998;78( 1 ):F3-9). The negative control (sham) and positive control (liquid Curosurf) groups included four and five animals in each group, respectively. Figure 20 shows a diagram of the animal procedures, study time points during the experiments, and the dose delivery protocol. Following surfactant washout, animals in the aerosol treatment group received MSD2 aerosol formulation from the iDP-ADS using the D3 device (n=7). Animals receiving the D3-MSD2 combination aerosol therapy were compared with historical controls reported above in Example 2 that received either a clinical dose of intratracheally instilled liquid surfactant (Curosurf at 200 mg / kg) (n=5), or the aerosol delivery process without powder (n=4) as a (negative sham) control at time point 0. All subjects were supported for 3.5 h following the initial intervention. Intratracheal Liquid Surfactant Administration (Curosurf) As reported in Example 2 above, the animals in the liquid Curosurf control group received standard intratracheal administration of porcine-derived Curosurf surfactant (poractant alfa, Chiesi, Parma, Italy). Curosurf comprises approximately 96% phospholipids (80 mg / mL) and 1 % surfactant proteins B and C and is only approved for administration by tracheal liquid instillation. The vials of liquid Curosurf (200 mg / kg; 2.5 ml / kg) were drawn up into a syringe and administered based on a standard protocol, described in greater detail elsewhere (Walsh BK, Daigle B, DiBlasi RM, Restrepo RD, and American Association for Respiratory C: AARC Clinical Practice Guideline. Surfactant replacement therapy: 2013. Respir Care. 2013;58(2):367-375). Dry Powder Surfactant Aerosol Delivery Animals in the D3-MSD2 aerosol group received 60 mg (device-loaded dose) dry powder formulation delivered directly to the ETT. The 60 mg total device-loaded formulation mass was divided into two dose cycles, each delivering 30 mg device-loaded formulation masses and administered 15 min apart (t=0 and 15 mins). This dose magnitude and delivery timing was selected for consistency with the study of Example 2 which used the D2-NSD1 combination, in order to enable direct comparison of the in vivo responses and best isolate the in vivo impact of D3-MSD2 improvements in aerosol quality. With the updated MSD2 formulation, containing less B-YL, and a 1.59 kg average rabbit weight for the aerosol group, the initial total deviceloaded PL dose was 24 mg-PL / kg, which is consistent with the 24 mg-PL / kg from the previous NSD1 formulation (see Examples 1 and 2). It is noted that each 30 mg device loaded formulation mass of MSD2 contains only 12 mg-PL / kg. Furthermore, the experimentally-determined device percent emitted dose (%ED) of approximately 77.4% (NGI) from the ETT adaptor results in a device total emitted mass of 18 mg-PL / kg entering the ETT. Considering the Curosurf dose of 200 mg-PL / kg is also delivered directly to the ETT, the AST here may be considered to be delivering approximately one order of magnitude less active therapeutic (PL dose) to the animal. During aerosol delivery, the D3 air-jet DPI was connected directly to the rabbit ETT, enabling precise control over the peak inspiratory pressure (PIP) of 25+2 cmHoO, PEEP of 5 cmkhO, Vt of 7 mL / kg, flow of 3 L / min, and actuation time of ~0.20-0.22 s with the iDP-ADS (see Figure 21). The EM Timer settings for the DPI were determined based on the post-lavage compliance values and weight (kg) using a reference calculation chart, which is described in greater detail in the study of Example 1 above. A total of ~4 actuations were employed to deliver each 30 mg dcvicc-loadcd formulation mass. The iDP-ADS employed an adjustable pressure relief valve (~25 cm H2O) and an analog pressure manometer, akin to a manual resuscitator, to measure pressure delivery and prevent barotrauma. The powder chamber was manually filled with formulation from previously packaged capsules (Qualicaps Inc, Whitsett, NC) which were stored in Al-Al blister packs, and the DPI was actuated to deliver the aerosol and inflate the lungs, followed by an approximately 1-s breath hold, after which exhalation was allowed through the exhalation port. The animal was promptly returned to the ventilator following each of the two dosing cycles (with 30 mg device-loaded formulation mass each) conducted at t= 0 and 15 mins. Physiologic Measurements Figure 20 shows a diagram of the animal procedures, study time points during the experiments, and the DDP. Following lung lavage, gas exchange (ABGs), ventilation parameters, and hemodynamic measurements were obtained at the pre-treatment (RDS baseline) condition at -5 min. Between -5 and 0 min, the initial D3-MSD2 aerosol (30 mg) dose or sham, was delivered, or liquid instillation of the total dose of Curosurf was completed. Five-minute time point measurements were then taken to assess the short-term gas exchange effects immediately following therapy due to lung volume de-recruitment from the initial ventilator disconnection with the D3-MSD2 aerosol therapy or sham delivery, potential airway obstruction from liquid Curosurf or powder agglomeration, as well as the potential for very rapid response to the AST. The D3-MSD2 aerosol group had two additional measurements at 10 and 15 min, the latter of which occurred just before the second 30 mg device-loaded dose. Standard post-treatment measurements were obtained at 30-min intervals thereafter for 3.5 h. (See Figure 20). Respiratory rate, PIP, PEEP, and mean airway pressure (MAP) were acquired from the ventilator. The pressure-volume relationship was evaluated by performing a brief inspiratory hold and recording the static compliance. The compliance was normalized based on the individual rabbit weights (mL / cmH20 / kg). The oxygenation index (OI) and ventilation efficiency index (VEI) were calculated using ventilator parameters and ABG values to assess disease severity at each timepoint. Animals were euthanized with 100 mg / kg Euthasol following the 3.5-h post-surfactant measurements and remained ventilated for necropsy. Statistical Analysis Statistical analysis of the in vitro data for comparing aerosol delivery performance and simulated lung delivery efficiencies were performed using JMP Pro 17 (SAS Institute Inc., Cary, NC). For in vivo data, results were calculated as mean ± SEM for all continuous physiologic variables at each timepoint for surfactant-treated and control groups. Two-way ANOVA was used to assess the treatment effects of each group and time point, as well as their interactions. Tukey’s multiple comparisons test compared mean post hoc differences in physiologic outcomes at pre- and post-lavage baseline and at each time point following treatment between the different groups. The criterion for significance was established a priori as P< 0.05 for all comparisons. The two additional measurements at 10 and 15 min in the D3-MSD2 aerosol animals were descriptive and not included in the statistical analysis, based on a lack of data in the sham and Curosurf liquid historical controls (see Example 2). Results Spray Drying and Powder Formulation Particle Sizing Multiple batches of the SLS-EEG MSD2 formulation were produced over the course of the study using the Buchi S-300 Mini Spray Dryer system. The spray rate of five batches of MSD2 powder formulation was -2.1 mL / min. The DPPC content (mean±SD) of these 5 batches assessed using the previously validated HPLC-UV method was 55.5+2.9 %w / w, which was 101.1% of the nominal content (55.0%). The B-YL content (mean+SD) assessed over the same five batches using a validated HPLC-UV method was 2.5±0.1 %w / w, which was 100.7% of the nominal content (2.5%). The coefficient of variation for DPPC and B-YL content was -5%, indicating good reproducibility in key components across batches. Table X provides the volumetric particle size of the MSD2 formulation determined using the Sympatec laser diffraction technique at dispersion pressures of 0.5 and 4.0 bar, tested across five different batches. The primary particle size (Dvso) of the MSD2 formulation batches measured at 4.0 bar dispersion pressure was 1.2 pm with coefficient of variation 2.1%, reflecting the consistent size of the formulation for the five batches considered and reproducibility of the powder preparation process. The Dvso (mean±SD) of the batches measured at 0.5 bar dispersion pressure was 1.8+0.0 pm, suggesting good dispersibility of the powders. Overall, the spray drying process produces micrometer-sized particles with good dispersibility, which are suitable for the EEG approach and deep lung delivery. Table X. Dispersion properties of the SLS-EEG MSD2 formulation at 4.0 and 0.5 bar dispersion pressures from Sympatcc testing (data arc mean ± standard deviation of 5 different batches). Values in parenthesis represent the coefficient of variation. Particle size Dv90 (pm) Dispersion pressure Dvio (pm) Dvso (pm) 4.0 bar 0.6±0.0 (3.7%) 1.2+0.0 (2.1%) 2.2+0.1 (3.1%) 0.5 bar 0.7+0.0(1.4%) 1.8+0.0 (2.5%) 4.3+0.1 (3.0%) Powder Formulation Imaging Scanning electron micrograph (SEM) images of particles composing the MSD2 formulation are displayed in Figures Fig. 22A, Fig. 22B, and Fig. 22C, at magnifications of either 20k or 50k. Imaging of the external particle structure indicated wrinkled surfaces with relatively spherical particle shapes and geometrical diameters consistent with Sympatec volumetric diameter results at 4.0 bar testing (Figure Fig. 22A). Focused ion beam (FIB) sectioning of particles indicated relatively large voids were present in most particles with particle diameters approximately >1 pm. As illustrated in Figure Fig. 22B, internal voids had diameters of -100-300 nm with between-void spacings of -100 nm or greater. Smaller openings, as shown at location 1 in Figure 22B progressed to larger voids as with location 2 in Figure Fig. 22C during successive FIB sectioning. X-ray Powder Diffraction (XRPD) and Moisture Sorption Testing Figures 23A and 23B show the solid-state properties of the MSD2 powder formulations characterized using XRPD and dynamic vapor sorption (DVS). The X-ray powder diffractograms of three different batches of the MSD2 formulations (Figure 23A) showed evidence of a partially crystalline structure with no differences in the peak diffractograms indicating reproducible production of different batches of powders. Characteristic peaks were observed for L-leucine and mannitol as being in the crystalline state. Figure 23B shows that following an equilibration period at 0% RH, water uptake by the powder at 10-60% RH (black line) was less than 9%, indicating relatively low uptake for an unprotected (i.e., unpackaged) spray-dried powder. Above 70% RH, the water uptake increased significantly, indicating the hygroscopic nature of the formulation at elevated RH, which is well suited for the EEG dry powder aerosol application. Low water uptake during storage under ambient conditions and elevated water uptake at higher humidity conditions are important for powder handling and when hygroscopic growth in the humid lung airways is required for EEG particle size increase. Aerosol Size Characterization Using Cascade Impaction Table XI provides the aerosol particle sizing of the MSD2 formulation delivered with the D3 device (one actuation per level) and a 30 mg device-loaded formulation mass based on cascade impaction with the NGI. The device retention (as measured by DPPC assay) was 22.6% with a pre-separator deposition fraction of 5%. The low pre-separator fraction led to a fine particle fraction <5 pm (FPF<5Mm) of 87.9%, which was more than 4 times higher than the previously reported FPF<5Mm (19.3%) for the D2-NSD1 combination (see Example 1). The mean FPF<2.5pm was 61.6% after 4 actuations, suggesting the potential to deliver a substantial amount of powder to the alveolar region. The MMAD of the MSD2 formulation delivered from the D3 device was 2.0 pm, which is lower than the previously reported D2-NSD1 combination of 2.9 pm (see Example 1), suggesting more efficient aerosolization and better aerosol quality using the new D3-MSD2 combination. Table XL Aerosol size distribution of the SLS-EEG MSD2 formulation determined by using Next Generation Impactor (NGI) after delivering 30 mg loaded dose from the D3 device with one actuation per level (data are means ± standard deviations, n=3; MMAD: mass median aerodynamic diameter, FPF: fine particle fraction). Sections and terms Values Device retention (%) 22.6+2.1 Pre-separator fraction (%) 5.0+2.0 MMAD (pm) 2.0+0.0 FPF<lMm (%) 10.0+0.6 FPF<2.5Mm (%) 61.6+0.2 FPF<5pm (%) 87.9+3.3 Emitted Dose (%) 77.4+2.1 Total recovery (%) 98.7+3.8 Characterization by Actuation from SprayVIEW, Spraytec, and Emitted Mass Figure 24 shows a snapshot of the plume geometry of powder aerosol emitted from the D3 device over 4 actuations. Mean cone angles of plumes during their maximum signal intensity (-50 ms from start of actuation and -100 mm from tip of the device orifice) for actuations Al-A4 were 26.6°, 29.2°, 30.7° and 27.1°, respectively, with a 1.9° standard deviation across actuations; corresponding mean cone widths for actuations A1-A4 were 23.7, 26.2, 27.5 and 24.1 mm, respectively, with a 1.8 mm standard deviation across actuations. The Plume Front Velocity (PFV) for the leading edge of the emitted aerosol cloud up to 100 mm from the orifice for each actuation A1-A4 were 3.0, 1.5, 1.9 and 2.0 mm / ms, respectively, with a 0.6 mm / ms standard deviation across actuations. Plume durations (PD) measured with the Spraytec analysis for actuations A1-A4 were 319, 315, 313, and 270 ms, respectively, with a standard deviation of 22.8 ms. For comparison, the PD observed in Spray VIEW were 10-20% longer than with Spray tec. Spray tec PD measurements were expected to be more accurate, as the plume enters and exits the measurement zone with minimum recirculation when the detector tower flanges are removed. A comparison of simultaneous aerosol emission vs. device actuation flow rate over time based on Spray VIEW testing for actuations A1-A4 is provided in Figure 25. Spray VIEW intensity measurements were used to approximate aerosol emission over time and selected as a conservatively longer option compared with the Spraytec measurement of obscuration (100-%Transmission). Flow profiles indicated an airflow actuation duration of -0.21 s, as expected, as well as an asymptotically decreasing flow through -0.3 s, likely due to compressible flow effects within the D3 device. Interestingly, for all actuations, a majority of the aerosol plume intensity, approximating powder emission, occurred during the first half of the actuation (-100 ms). This relationship provides evidence that the D3 device is capable of controlling the amount of emitted mass such that a majority of the aerosol enters the first half of the inhalation volume. Spraytec evaluation of aerosol size distributions for individual actuations A1-A4 over a single run are illustrated in Figure 26. In each case, a majority of the aerosol volume occurs in particles with geometric diameters <2 pm, and very little volume (-10%) occurred in particles with geometric diameters >10 pm. The volumetric PSD also appears consistent across the four actuations. Mean±SD Dvso values evaluated for each actuation A1-A4 resulted in a narrow range of 1.5+0.2 pm to 1.6+0.3 pm. Furthermore, Spraytec laser diffraction FPF values less than 2.5 pm based on volumetric diameter for actuations 1-4 were 64%, 68%, 65% and 71%, respectively. In summary, both the mean geometric aerosol size and the particle size distribution was very consistent across the four actuations of the D3 device loaded with 30mg of the MSD2 formulation, and similar to cascade impaction aerodynamic sizing (Tabic XI). Percent emitted dose (%ED) and emitted mass are provided in Figure 27 for actuations A1-A4, and as the sum of all actuations (‘Total’). Relatively high and consistent emitted masses within the targeted range of 5-10 mg / actuation were observed over actuations A1-A3. Emitted mass with A4 was just below the target range (4.1 mg); however, this was viewed as acceptable as the final A4 actuation may be considered a final chamber emptying step. Variability and low emission in actuations A1-A3 was typically compensated for with higher emissions in the subsequent actuations, resulting in low overall variability on total emitted mass. The Total mean+SD %ED was 76.7+4.6%, which agreed well with the NGI findings (77.4%+2.1; Table XI). In Vitro Testing of Lung Delivery Efficiency with the LC Model Table XII provides the in vitro regional deposition fractions and estimated rabbit lung delivery efficiencies of the D3-MSD2 combination assessed using the in vitro LC model when administering a device-loaded formulation mass of 30 mg. The emitted dose from the D3 device was 70.0%, with lower lung (filter+lung chamber) and total lung (TB+filter+lung chamber) delivery efficiencies of 40.1% and 56.8%, respectively, indicating a high fraction of the loaded dose was delivered to the simulated rabbit lungs. Although the lung delivery efficiency from D3 device was slightly lower than the D2-NSD2 combination (see Example 1), higher FPF<5|jm in the NGI study suggests significant improvements in aerosol quality, which will be critical for delivering the aerosol directly to the alveolar region with the D3-MSD2 combination in the in vivo experiments. Table XII. Regional deposition fractions (%) and lung delivery efficiencies of the SLS-EEG MSD2 formulation (30 mg loaded mass) delivered from the D3 device to the in vitro LC model (data are means + standard deviations, n=3). Sections and terms PL Mass (%) Air-Jet Device 30.0+2.7 Endotracheal tube (ETT) 13.4+2.5 Tracheobronchial region (TB) 16.7±0.7 Lung chamber (LC) 30.3±3.2 Filter 9.8±0.4 Lower lung dose (LC+Filter) 40.1±3.3 Total lung dose (TB+LC+Filter) 56.8±2.8 Emitted Dose 70.0±2.7 Total Recovery 100.2±2.3 In Vivo Animal Descriptions and Dosing Following lung lavage, the mean PaO2 was reduced from -460 to 65 mmHg and weight normalized compliance from -1.1 to 0.4 mL / cmH2O / kg at baseline (P<0.001) with increased PaCO2 and disease severity (OI>15 and VEI-0.03) values, all indicative of severely depleted surfactant pools, elevated alveolar surface tension and severe RDS. Baseline data following induction of lung lavage for surfactant deficiency were not different between groups for body weight, gas exchange, disease severity, and lung compliance. All the animals in the liquid Curosurf group (see historical controls in Example 2) experienced some degree of acute airway obstruction from the liquid, resulting in hypoxemia (SpO2<85%), hemodynamic instability (e.g., bradycardia or hypotension) or increased PIP (>25 cndEO) by the ventilator during dosing. The D3-MSD2 aerosol and control groups experienced acute hypoxemia (SpO2<85%) upon ventilator disconnection that quickly resolved when positive pressure actuations were applied for therapy with the iDP-ADS. Following the treatment, the heart rate (P=0.94) and blood pressure (P=0.93) did not vary between the groups. As described, based on an experimental mean rabbit weight of 1.59 kg, the device-loaded dose of 60 mg of the SLS-EEG MSD2 powder formulation contained 24 mg-PL / kg compared to the approximate 200 mg-PL / kg with Curosurf. The total treatment duration, which included stabilization, was -10 min to administer Curosurf. The aerosol delivery required eight actuations for 60 mg delivery of the D3-MSD2 formulation with the D3 device and could be completed in <1 min. The total therapy delivery time, including ventilator reconnection to enable device reloading and stabilization (i.e., two 30 mg device-loaded dose delivery cycles), was completed in <5 min. In Vivo Gas Exchange Gas exchange data for the different in vivo groups is shown in Figure 28. Following surfactant depletion, all groups had mean PaO2 values below 70 mm Hg, on FiO2 of 1.0 at baseline, indicative of severe RDS. As expected, PaCh values increased from baseline in the surfactant groups immediately following therapy and remained <100 mm Hg in the sham control group throughout the experiment. The mean PaO2 at the 5-min timepoint was not different between the Curosurf and control groups. In contrast, at the 5-min timepoint (which included only the first 30 mg device-loaded dose), the D3-MSD2 aerosol group was significantly greater than both the sham control and Curosurf liquid groups, with a PaO2 of -300 mmHg. Following the second (final) device-loaded dose of 30 mg and delivery at 15 min, the PaO2 values between 30 and 150 min were significantly greater with aerosol than the Curosurf and sham control groups. As expected, the Curosurf group had higher PaO2 than the sham control throughout the experiment. Among the surfactant groups, arterial oxygenation was not different at 180 and 210 min, and PaO2 values recovered to 94% of the pre-lavage with Curosurf liquid. For the D3-MSD2 combination, PaO2 values exceeded the basal levels at 180 mins, and ended with a final PaO2 level that was 99% of the pre-washout value at 210 mins. As expected, the mean PaCO2 increased, and pH decreased among groups due to severe RDS induced by lung lavage (Figure 28). The PaCO2 values in controls remained increased throughout the experiment, and although PaCO2 trended downward in both surfactant groups, the liquid Curosurf values were not different from sham controls during the experiment. The PaCCh values with the D3-MSD2 aerosol were statistically lower than liquid Curosurf and controls at 30- and 60-min time points but did not differ from the Curosurf or sham control groups for the remainder of the experiment. The mean pH values remained between 7.25 and 7.30 in the controls and remained low but were no different from liquid Curosurf, except for the 180 and 210-min timepoints, where Curosurf pH was higher than control. The aerosol treatment group had greater pH than controls at all time points throughout the experiment, beginning with the 5min blood gas. Compared with liquid Curosurf, the aerosol group had greater pH at the 5, 30, and 60-min time points, but was not statistically different at the remaining time points (see Figure 28), despite having a higher trend at all time points. In Vivo Lung Compliance and Disease Severity Lung compliance and indices of disease severity data for the different groups are shown in Figure 29. The lung compliance improved rapidly in the aerosol group at 5-min following the initial 30 mg device-loaded dose and continued to improve following the second 30 mg aerosol delivery at 15 min. The D3-MSD2 aerosol group had greater compliance than the liquid Curosurf and control groups from the 5-min timepoint until the end of the experiments. Following a small initial increase between 5 and 30 min, the compliance values stabilized and then slightly decreased with liquid Curosurf and were not statistically different from sham controls at any of the time points over the 3.5-h observation period. At the 3.5-h final time, the compliance in the liquid Curosurf and sham control groups was not improved (0.4 vs. 0.3 mL / cm HiO / kg, respectively) from baseline. In contrast, the D3-MSD2 aerosol group compliance nearly doubled from baseline (0.46 to 0.91 mL / cm FLO / kg) and recovered to 91% of the pre-washout value. Furthermore, the D3-MSD2 aerosol group demonstrated an increasing trend in compliance through the final hour of the experiment that continued to the 3.5-h final time point. Surfactant washout produced OI >15 for all cases, consistent with severe respiratory disease. As expected, improvement was not observed for the sham control case over the 3.5-h observation period, and OI values were higher than the surfactant-treated groups at all time points. In contrast, the surfactant aerosol group showed a rapid reduction in OI (<2) with 80-91% recovery from baseline (pre-lavage) that was maintained from the 30-min timepoint until the end of the 3.5h period. The liquid Curosurf group showed a favorable reduction in OI following treatment with a more gradual recovery, ranging from 28-65% between 30 and 180 min and 75% at 3.5h. Considering VEI, liquid Curosurf demonstrated an improved trend compared with the sham control throughout the observation period that did not rise to the level of statistical significance. In contrast, the D3-MSD2 aerosol group demonstrated a statically significant improvement vs. the sham control at 30 mins, which continued through the 3.5-h experiment. Furthermore, the D3-MSD2 aerosol group demonstrated statistically significant improvement vs. liquid Curosurf at 60 min, which also continued through the remainder of the 3.5-h observation period. Discussion Considering the administered dose of the aerosol lung surfactant product, the total device-loaded dose of active PL components (combination of DPPC and POPG) was 24 mg / kg. Based on in vitro testing in the NGI, device %ED was 77.4%, resulting in a device emitted dose of 18 mg-PL / kg, which was administered directly to the ETT. In comparison, Curosurf was administered at a clinically recommended first dose of 200 mg / kg and consisted almost entirely of PL components. Furthermore, based on high PL and surfactant protein content, Curosurf is often the preferred clinical option for surfactant replacement therapy in RDS. Despite the order of magnitude lower administered PL dose associated with the aerosol, the AST met or exceeded response to Curosurf for every metric tested. Furthermore, the AST exceeded Curosurf performance at every time point, often significantly, with the exception of pH for the final two time points where performance largely overlapped. Superior performance of the aerosol at order-of-magnitude reduced doses was possible due, at least in part, to improved delivery of a small particle formulation directly to the alveolar region. Liquid bolus instillation is expected to be associated with a significant ‘coating cost’ in the TB airways, such that alveolar delivery is a low percentage of the instilled dose. Furthermore, liquid bolus instillation is often associated with blockage of the ETT, significant surfactant reflux, clogging of the trachea, oxygen desaturation and additional alveolar injury. In contrast, the aerosol therapy of this Example showed no evidence of airway blockage and is expected to have improved delivery of the active components directly to the alveolar region. Considering the time required for therapeutic aerosol delivery, previous animal and human subject AST studies reported aerosol administration times of 15 min to 2.5 h, with multiple delivery cycles often required. For administration of the D3-MSD2 combination in this study, two dose delivery cycles were employed, each requiring an aerosol delivery time of ~20 s. Total aerosol delivery time with the D3-MSD2 combination was therefore <1 min. In practice, animal care together with disconnection and reconnection with the ventilator required a total therapy delivery time of <5 min, which could also be reduced to <1 min by better integration of the iDP-ADS with the ventilator. One of the most striking advantages of the AST in this study was the rapid and almost instantaneous response achieved in the animal model of severe RDS. For example, at the 5-min blood gas reading when only the first 30 mg device-loaded dose had been administered (12 mg-PL / kg), the mean PaOi was already at approximately 300 mmHg. This 5-min recovery value is already far in excess of the -50-80 mmHg value achieved by a recent nebulization study after 15 min and -200 mmHg at 180 min following administration of 100 mg of surfactant (DiBlasi RM, Micheletti KJ, Zimmerman JD, Poli JA, Fink JB, and Kajimoto M: Physiologic effects of instilled and aerosolized surfactant using a breath-synchronized nebulizer on surfactant-deficient rabbits. Pharmaceutics. 2021; 13(10): 1580) and equal to the final 180-min value achieved with a previous dry powder therapy (Walther FJ, Waring AJ, Otieno M, and DiBlasi RM: Efficacy, dose-response, and aerosol delivery of dry powder synthetic lung surfactant treatment in surfactant-dcficicnt rabbits and premature lambs. Respiratory Research. 2022;23(l):l-16). Similarly, almost instantaneous response to the AST with significant differences at the 5-min time point (vs. sham control and vs. liquid Curosurf) were observed for the in vivo metrics of pH and compliance. PaCO? and VEI with AST then reached the level of statistical difference (vs. sham control and vs. liquid Curosurf) at the 30- and 60-min time points, respectively. The times required for the D3-MSD2 aerosol therapy and instilled Curosurf liquid to restore oxygenation to 300 mmHg were -5 min and ~60 min, respectively; resulting in an ~12-fold faster response with the aerosol therapy. Similar times to reach approximately 400 mmHg were 30 mins and 150 mins, respectively; resulting in an ~5-fold faster response with the aerosol. Considering a 500mmHg metric, the D3-MSD2 aerosol therapy required 60 min, whereas the Curosurf liquid did not reach this level. Based on these observations, response rate to the D3-MSD2 therapy appears to be increased by approximately an order of magnitude, compared with both previous aerosol therapies considered and with the current clinical standard of liquid Curosurf administration in the same animal model. While previous aerosol therapies have reported relatively muted biological response compared with liquid bolus instillation, the response to the D3-MSD2 formulation may be considered dramatic. As described above, PaO2 levels responded 5- to 12-fold times faster with aerosol, were significantly higher than with liquid Curosurf for a majority of the experiment (i.e., through 150 mins), and achieved values >500 mmHg, also not achieved with the liquid. One of the largest differences in therapeutic response was observed in the lung compliance where the D3-MSD2 therapy produce statistically higher values than the sham control and liquid Curosurf at the 5-min time point and at all subsequent time points (for which comparison data was available). Moreover, compliance of the sham control animals averaged -0.35 mL / cmH2O-kg and liquid Curosurf produced an approximate 43% recovery, compared with the pre-lavage compliance, to an average value of 0.48 mL / cm H2O / kg. The D3-MSD2 aerosol therapy resulted in an average value of -0.78 mL / cm H2O / kg and a final value of 0.90 mL / cm H2O / kg. The final compliance recovery with the D3-MSD2 therapy was 91% of the original pre-lavage value of 0.99 mL / cm H2O / kg. Similarly, the VEI with the D3-MSD2 therapy remained significantly higher than both the control and liquid Curosurf beginning at 60 min and continuing through the remainder of the experiment. In summary, the D3-MSD2 therapy appeared biologically superior to liquid Curosurf instillation with strong statistical significance in the rate of oxygenation, PaCO2 and pH recovery, and in the rate and final magnitude of compliance and VEI. The significant improvements in therapeutic response associated with the D3-MSD2 aerosol therapy compared with the previous D2-NSD1 combination (see preceding Examples above) are most likely associated with improvements in the aerosol quality and delivery protocol, leading to higher mass of active surfactant components delivered deeper within the lungs and possibly all the way to the alveolar region using positive pressure actuations. Based on NGI testing, the previous D2-NSD1 combination produced FPFs (as a % of ED) with cutoffs of <5, <2.5 and <1 pm of 27, 21, and 9 %, respectively. With the new D3-MSD2 combination, where the D3 device enabled increased dose loading and better regulated %ED / actuation, corresponding FPFs were 88, 62, and 10 %, respectively. Previously conducted numerical simulations in term infants with an ETT interface estimated that an aerosol size without EEG growth of 2.5 pm provided an optimal delivery to the alveolar region, taking into account upstream deposition and the potential for aerosol exhalation. Implementing an FPF^yum as the best available particle size cutoff metric, as well as the NGI testing data from Table XI, the previous D2-NSD1 combination administered a total fine particle dose (FPD) <2.5 pm of 4 mg-PL / kg; and the current D3-MSD2 therapy administered an FPD<2.5uni of 11 mg-PL / kg. In terms of aerosol quality, evaluated by FPF, the new D3 device increased the FPF<2.5gm by a factor >3-fold. While the D3 device had a slightly reduced total %ED, the D3-MSD2 therapy also improved FPD<2.5Mm by approximately 3-fold. Additional aerosol delivery advantages provided by the D3-MSD2 combination included the ability to load 30 mg of powder formulation at once, near-elimination of a significant fraction of particle aggregates (captured in the NGI pre-separator and likely depositing in the upper TB region), front-loading of the aerosol into the first —1 / 2 of the inhalation waveform, and unifying the delivery across multiple actuations. Based on the in vitro testing described above in Example 1 and theoretical considerations of surfactant pool size and the previous results of Kamga et al. (Kamga Gninzeko FJ, Valentine MS, Tho CK, Chindal SR, Boc S, Dhapare S, Momin MAM, Hassan A, Hindle M, and Farkas DR: Excipient Enhanced Growth Aerosol Surfactant Replacement Therapy in an In Vivo Rat Lung Injury Model. Journal of Aerosol Medicine and Pulmonary Drug Delivery. 2020;33(6):314-322), a dose delivery profile (DDP) was constructed for dry powder aerosol lung surfactant therapy, which resulted in a high-efficacy response in the subsequent study described above in Example 2. Components of this DDP included: • ~15 mg-PL / kg delivered to the lungs, as predicted by in vitro LC model testing • FPD with <1 and <5 pm cutoffs of >2 and >5 mg-PL / kg based on NGI testing. In the current study, the D3-MSD2 combination approximates or exceeds these requirements, as described above. Moreover, to better refine the DDP with the goal of a high-efficacy and rapid response, we recommend the addition of an FPD<2.5pm metric. As described above, the previous study of Momin et al.48 had an FPD<2.5gm of 4 mg-PL / kg. The current study provided an FPD<2.5Mm of 11 mg-PL / kg with a full 60 mg total device loaded formulation mass. However, a dramatic response and recovery rate were observed after the first 30 mg deviceloaded dose, containing an FPD<2.5pm of only 5.5 mg-PL / kg. Therefore, as an additional DDP metric in a rabbit washout model of severe RDS, what is recommended for exemplary embodiments of this disclosure is: • FPD<2.5pm > 5-10 mg-PL / kg, preferably administered with positive pressure inflations with a maximum lung pressure of approximately 20-25 cm H2O. While this Example suggests improvement in aerosol quality played a predominate role in the improved performance of the D3-MSD2 aerosol therapy observed in this study, there were multiple other aspects of aerosol delivery that are also expected to be important for achieving a rapid high-efficacy response and should also be considered pail of the target DDP. Firstly, excipient enhanced growth of the aerosol likely minimized the exhaled dose fraction, despite the small aerosol size. During in vivo testing, very little aerosol was observed in the exhaled breath. Even with EEG growth, it is possible to make the initial aerosol size too small. A target EEG initial aerosol size range is expected to be approximately 0.9 to 2.0 pm with growth at current excipient levels to approximately 2.0 to 4.4 pm within the lungs. Front-loading of the aerosol into the first half of inhalation was also previously shown to be important by Longest et al. (Longest PW, Azimi M, and Hindle M: Optimal delivery of aerosols to infants during mechanical ventilation. Journal of Aerosol Medicine and Pulmonary Drug Delivery. 2014;27(5):371-385), and was demonstrated with a dry powder aerosol in the current study. Rapid delivery of the aerosol, to minimize the time that the recipient is exposed to ventilation pressures without surfactant, likely resulted in less lung damage, as illustrated in Example 2. The small primary particles of the aerosol likely helped to form an initial thin surface coating of surfactant over the large surface area of the alveolar region. The presence of hygroscopic excipients may have also helped to dissipate and spread the surfactant components upon deposition at the micrometer level. Finally, administration with positive-pressure inflations is expected to better open collapsed lung regions, which is a common goal in ventilation therapy, and these lung regions are then held open by the locally deposited and quickly spreading surfactant droplets. A primary limitation of the current study is aerosol delivery through an ETT, which does not realize the full non-invasive potential of AST. Despite CPAP and NIV being the favored clinical approaches for all infants, a significant proportion (often >40%) of these infants will fail NIV and / or CPAP support and require invasive ventilation. The exemplary iDP-ADS with the ETT adaptor can be used directly in these scenarios. The surfactant washout rabbit model provided multiple benefits including consistent body weight with a preterm infant and a multiple-hit injury approach from which the control animals did not recover over the time course of the experiments. One limitation of this model was that the alveolar structure of juvenile rabbits is largely mature. In contrast, depending on the gestational age of a preterm infant, the alveolar structure is still forming and may lack full septation and volume-filling. In addition, the amount of surfactant remaining in the lungs after washout was not fully quantified. A small amount of surfactant likely remains, potentially including trace amounts of surfactant proteins B and C. A further limitation of the in vivo model may be the use of FiCb of 1.0 throughout the experiment. As described, this non-clinical level of hyperoxia is intended to standardize oxygen therapy and ensure adequate oxygenation and prevent deterioration despite potential complications that could arise during therapy, cause additional lung injury, and prevent the formation of endogenous surfactant. However, in the current study, the animals recovered so quickly in the aerosol group that it was challenging to reduce the ventilation rate and ventilation support fast enough to avoid the deleterious effects of hyperinflation and increased alveolar dead space due to the rapid improvements in lung compliance. Based on the rapid recovery of the animals, there is the potential to extubate and discontinue mechanical ventilation shortly after surfactant administration. This Example has demonstrated that a high efficiency AST approach can significantly exceed the performance of the current clinical standard of high-volume liquid bolus surfactant instillation in an animal model of severe RDS across the critical areas of (i) dose administered, (ii) delivery time, (iii) response rate and (iv) response magnitude. The D3-MSD2 aerosol therapy enabled an order of magnitude reduction in administration of the active component (PL) dose compared with liquid bolus instillation of the clinically-favored animal-derived Curosurf product while achieving an equivalent or, for some metrics, superior response. At the end of the experimental period, the AST produced significant improvements in comparison with liquid Curosurf for the in vivo metrics of compliance and VEI. The response rate to the AST was also dramatic, as exemplified by a 5- to 12-fold faster recovery of oxygenation compared with Curosurf. These recovery rates and compliance values were also superior to the SLS-EEG surfactant aerosol studies of Example 1 and 2 which employed the D2-NSD1 combination. It is expected that the high-efficacy response and rapid recovery rate arc largely due to further improvements in the aerosol quality, better enabling the targeting of small-particle aerosol directly to the alveolar region. Comparisons between the in vitro testing and in vivo response lead to an additional DDP metric for severe RDS of FPD<2.5|im >5-10 mg-PL / kg, determined with NGI testing. Additional advantages of the D3-MSD2 aerosol therapy administered with the iDP-ADS that may have contributed to a high-efficacy rapid response and should be considered in the recommended DDP include: • Use of the EEG aerosol delivery strategy, • Frontloading the aerosol delivery into the first portion of the inhalation waveform, • Small primary particles (e.g., ~ 1 pm) containing some (e.g., 15% w / w) hygroscopic material, and • Administration of the aerosol with positive pressure breaths. Example 4. (SLS-EEG) dry powder aerosol formulation without B-YL surfactant protein peptide mimic This Example reports the efficacy of a simplified (no B-YL surfactant protein peptide mimic) synthetic lung surfactant excipient enhanced growth (SLS-EEG) dry powder aerosol formulation with the purpose of reducing formulation cost and complexity. The SLS-EEG formulation containing DPPC, POPG, L-leucine, D-mannitol and sodium chloride was produced using a scalable two-fluid spray nozzle-based Buchi S-300 Mini Spray Dryer (Buchi Labortechnik AG, Flawil, Switzerland). The formulation was characterized for in vitro physicochemical properties along with the aerosol aerodynamic properties testing with the high-dose air-jet dry powder inhaler (D3) device. In preparation for in vivo testing, the aerosolization performance and lung delivery efficiency of the SLS-EEG powder were evaluated by delivering 30 mg of powder formulation from the D3 device to the 3D-printed rabbit upper tracheobronchial (TB) airway model at 3 LPM flow rate using a 10 mL air actuation volume. The D3 device and simplified SLS-EEG formulation combination was then evaluated for in vivo efficacy in a rabbit surfactant depletion model with PaOi and lung compliance changes measured after intra-tracheal administration of 60 mg SLS-EEG formulation and compared to the current standard of care liquid instillation of Curosurf. The experimental setup and procedure are depicted in Figure 30 and Figure 31, respectively. The primary particle size (Dv50) measured for the spray dried powder formulations at 4 bar dispersion pressure was ~1.2 pm with %CV of 2.2 suggesting the reproducibility of the spray drying process. The Dv50 of the particles measured at 0.5 bar dispersion pressure was -1.7 pm suggesting a good dispersibility of the powder formulations. The mean DPPC and POPG content of the formulations were 99.4+2.9% and 98.2+1.0%, respectively, of the theoretical content. X-ray powder diffraction showed evidence of crystalline structure in the synthetic lung surfactant powder formulations. The in vitro rabbit model study showed 79.2+2.3% dose emitted from the D3 device with 67.3+0.8% total lung delivery efficiency, based on loaded dose. The aerosol aerodynamic properties measured using NGI showed 1.9 pm mass median aerodynamic diameter (MMAD) of SLS-EEG powder delivered from the D3 device with a fine particle fraction < 5 pm of 90.8%. In the in vivo rabbit experiments, a total device-loaded formulation mass of 60 mg produced a phospholipid (PL) dose of 26 mg-PL / kg and a device emitted dose of 20.2 mg-PL / kg (with a fine particle dose (FPD) of 18.3 mg PL / kg) compared with the 200 mg-PL / kg clinical dose of Curosurf liquid. The in-vivo rabbit study showed 5-12 times faster recovery of PaO2 and two-fold increase in pulmonary compliance for the simplified (B-YL free) powder aerosol formulation than the liquid instillation of Curosurf suggesting the superior performance of the simplified powder aerosol therapy at -10-fold lower doses than the current standard of care liquid therapy. Figure 32 shows In Vivo Rabbit Model Arterial Oxygenation data comparing the DPPC / POPG + B-YL formulation of Example 3 with the DPPC / POPG, No B-YL formulation of Example 4. Removal of the surfactant protein mimic did not appear to alter the efficacy of the SLS-EEG aerosol. Figure 33 is In Vivo Rabbit Model Compliance data comparing the DPPC / POPG + B-YL formulation of Example 3 with the DPPC / POPG, No B-YL formulation of Example 4. Removal of the surfactant protein mimic did not appear to alter the efficacy of the SLS-EEG aerosol. This simplified formulation / device combination may be suitable for treating premature neonates and could overcome the challenges of liquid bolus instillation of surfactant replacement therapy. Example 5. Comparison of In Vitro Aerosol Performance and Powder Characteristics of a Synthetic Lung Surfactant Powder Aerosol Formulation Produced using Two Spray Drying Methods The main objective of this Example is to evaluate physicochemical properties and aerosol performance of a synthetic lung surfactant (SLS) powder EEG formulation when prepared with two alternative spray dryer configurations (vibrating mesh vs. two-fluid nozzle) of the Buchi S-300 Mini Spray Dryer (Mini SD). The Buchi S-300 Mini Spray Dryer (Mini SD) utilizes a two-fluid nozzle and pressurized gas that are easily varied to control parameters such as outlet temperature, humidity, and gas flow to optimize powder characteristics and performance. The Buchi B-90 HP Nano Spray Dryer (Nano SD) employs a vibrating mesh for droplet generation. Methods The SLS-EEG formulation contained phospholipids (DPPC and POPG), surfactant mimic peptide (B-YL), hygroscopic excipients (mannitol and sodium chloride), and a dispersion enhancer (Lleucine). Feed dispersions were prepared following an optimized ratio and procedure previously reported (Boe S, Momin MA, Farkas DR, Longest W, Hindle M: Development and characterization of excipient enhanced growth (EEG) surfactant powder formulations for treating neonatal respiratory distress syndrome. AAPS PharmSciTech 2021, 22: 1-12), then spray-dried on either the Nano SD (vibrating mesh) or Mini SD (two-fluid nozzle) (Biichi Labortechnik AG, Flawil, Switzerland; n = 3 batches each) using the operating parameters detailed in Table XIII. Immediately post-collection, powder formulations were equilibrated in a glass vial (~ 17% RH at ~ 24 °C for 30 min), then stored in a sealed desiccator at 0% RH / -20 °C until further analysis. Table XIII. Spray drying process parameters. |        i SotW Con®      1 Spray Noxtla reF" T ,CC| | w* '    1     I | Nano SD ;       0.128        j ’smaff        moth 128 I N / A t          TFi 35-37 J | MW SO !                    G.5 mm swo-fM nozzte ......................1................... i        80 45-46    1 Fdrying: drying gas flow rate. Fspray: spray gas flow rate. Tin: inlet temperature. Tout: outlet temperature. The Nano and Mini spray-dried powders were characterized by DPPC and B-YL recovery, spray rate, bulk density, particle size distribution and aerosol performance. Primary particle size was assessed using a Sympatec HELOS equipped with a RODOS / M disperser (Sympatec GmbH, Clausthal-Zellerfeld, Germany) at dispersion pressures 4.0 and 0.5 bar. DPPC and B-YL content in the formulation was determined using validated high performance liquid chromatography (HPLC) methods. In vitro aerodynamic properties were determined using a Next Generation Impactor (NGI; MSP, TSI Inc., Shoreview, USA) following methods previously detailed [6]. Briefly, formulations (30 mg) were aerosolized using the VCU D3 low-volume air-jet inhaler designed for surfactant powder delivery that was actuated with ~ 10 mL volume of air at a 3 L / min flow rate. Aerosol performance was characterized by mass median aerodynamic diameter (MMAD), fine particle fraction (FPF< 5 pm / ED), fine particle dose (FPD) and emitted dose based on quantitative analysis of DPPC. All data values represent the mean ± standard deviation of three batches, unless otherwise stated. Results and Discussion Table XIV shows the effects of the spray dryer on DPPC and B-YL content in the SLS-EEG formulations. The DPPC content was in close agreement with the nominal concentration for both spray dried formulations; however, the B-YL content was lower than nominal for the Nano SD formulation, possibly due to denaturation during the mesh nebulization process. The SLS-EEG formulation produced using the Mini SD had a B-YL content close nominal value with a measured value of 4.9% w / w (97.8 ± 1.6% of nominal). The Mini SD had a faster spray rate of 2.1 mL / min compared to 0.4 mL / min for the Nano SD (0.4 mL / min). Lower yields were observed, however, with the Mini SD due low collection efficiency from the cyclone compared to the electrostatic precipitator used in the Nano SD (data not shown). Table XIV. Mean (SD) DPPC and B-YL content, spray rate, and bulk density for the SLS-EEG formulations produced using Mini SD and Nano SD methods. [         DPPC B-YL Spray Kate (mL / min} Buik Density {g / snt) j Mirts SD | 53.3*0 6 | 191.511.1 4.9 *5 r j 97.8*1,8* 0.08*0.01      | | Nim SD | 51,7*0 8   |   98.7*1.7 28*9,9’ |   58,1 *G.4* 0 HO 02    j *Significant difference (student t-test, p < 0.05). The primary particle size distribution for the SLS-EEG formulations are shown in Figures 34A and 34B. Figure 34A shows particle size distributions of the SLS-EEG formulation produced using the Mini SD and Nano SD measured at 4.0 bar dispersion pressure. Figure 34B shows particle size distributions of the SLS-EEG formulation produced using the Mini SD and Nano SD measured at 0.5 bar dispersion pressure. The Mini SD formulation had a mean Dv50 of 1.1 ± 0.0 pm with 100% of the particles < 5 pm and 42.7% < 1 pm measured at 4 bar. The Nano SD formulation had a slightly smaller primary particle size (Dv50 = 0.9 ± 0.03 pm) and a higher sub-micrometer fraction (60.5 ± 2.1%) at 4 bar. However, measurements at 0.5 bar revealed that Nano SD formulation did not disperse as well as the Mini SD formulation with a mean Dv50 of 1.7 pm and 1.5 pm, respectively. Aerosol performance and emitted dose of the SLS-EEG formulations are shown in Table XV. Both formulations showed high emitted doses and small MMADs (Table XV). However, while the Nano SD produced formulations displayed a higher emitted dose (85.0 ± 2.8%), nearly 40.3 ± 5.0% of this dose was deposited on the pre-separator. From this evaluation, it appears that the Mini SD formulations produced the superior aerosol; 78.9 ± 2.3% of the loaded dose was emitted, depositing only 3.5 ± 1.4% on the pre-separator. The Mini SD formulation was observed to have an FPF< 5 pm of 91.1 ±2.1% with a DPPC FPD of 11.9 ± 1.0 mg compared to the Nano SD formulation (42.7 ± 6.5% FPF < 5 pm; 6.6 ± 0.7 mg FPD). The improved performance of the Mini SD formulation could be associated with the method of collection and associated secondary drying in the high efficiency cyclone compared to the lower temperatures experienced on the electrostatic precipitator with the Nano SD formulation. Table XV. In vitro aerosol performance of the SLS-EEG formulation produced using the Mini SD and Nano SD aerosolized using a novel low-volume air-jet inhaler and quantified based on DPPC deposition. j Device {%} SWsspatatsw (%) :     £0(¾) FPU jmgj FPF <5       i swao (fsmj Mirs SO |   21,h23* i 78.9 $2.3* Sf.hzr | ■ijsQ.i Nano SD |   15.012.7» 40.3*5.0» 42.710.5*   |     1.510.1 *Significant difference (student t-test, p < 0.05). Formulations suitable for lung delivery were produced by both Mini (nozzle based) and Nano (vibrational mesh based) spray dryers. Both aerosol formulations had high emitted doses and respirable fractions > 40% suitable for infant delivery using the low-volume air-jet inhaler. There arc a number of advantages to using the Mini SD system and associated formulations (compared with the Nano SD system and formulations) including increased production rate, B-YL stability, scalability of the nozzle-based process and improved aerosol de-aggregation characteristics that may translate to more efficient pulmonary delivery of the Mini SD SLS formulation. It is noted that, as used herein and in the appended claims, the singular’ forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely”, “only”, and the like in connection with the recitation of claim elements, or use of a “negative” limitation. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are described. It is to be understood that the terminology and explanations used herein are for the purpose of describing exemplary embodiments only, and arc not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Claims

1. A dry powder synthetic lung surfactant (SLS) formulation, comprising one or more phospholipids (PL); and one or more hygroscopic excipients, wherein the dry powder SLS formulation(i) is dispersible to a mean geometric diameter of less than 2.0 pm at 0.5 bar and a mean geometric diameter of less than 1.3pm at 4.0 bar, and / or(ii) contains particle fractions less than 1 pm greater than 20% and greater than 35%, at 0.5 and 4.0 bar, respectively, and / or(iii) contains particle fractions less than 5 pm greater than 88% and greater than 98%, at 0.5 and 4.0 bar respectively.

2. The dry powder SLS formulation of claim 1, wherein the formulation is free of surfactant protein and surfactant protein peptide mimic.

3. The dry powder SLS formulation of claim 1, further comprising at least one dispersion enhancer.

4. The dry powder SLS formulation of claim 3, wherein the at least one dispersion enhancer comprises 1-leucine.

5. The dry powder SLS formulation of claim 1, wherein the one or more PL comprise DPPC and / or POPG, and wherein the one or more hygroscopic excipients comprise mannitol and / or sodium chloride.

6. The dry powder SLS formulation of claim 5, wherein the one or more PL is 70-100% DPPC and 0-30% POPG.

7. The dry powder SLS formulation of claim 5, further comprising a surfactant protein peptide mimic.

8. The dry powder SLS formulation of claim 7, wherein the surfactant protein peptide mimic is B-YL.

9. The dry powder SLS formulation of claim 5, wherein the dry powder SLS formulation contains 60-70% phospholipids, 15-25% 1-leucine, 5-15% mannitol, 4-10% sodium chloride, and 0-5% surfactant protein peptide mimic.

10. The dry powder SLS formulation of claim 5, wherein the one or more hygroscopic excipients are no more than 15% of the dry powder SLS formulation.

11. The dry powder SLS formulation of claim 1, wherein the dry powder SLS formulation is an aerosol.

12. An aerosol of a dry powder synthetic lung surfactant (SLS) formulation comprising one or more phospholipids (PL) and one or more hygroscopic excipients, wherein the aerosol has a mass median aerodynamic diameter (MMAD) of 0.5-2 pm,wherein the dry powder SLS formulation(i) is dispersible to a mean geometric diameter of less than 2.0 pm at 0.5 bar and a mean geometric diameter of less than 1.3pm at 4.0 bar, and / or(ii) contains particle fractions less than 1 pm greater than 20% and greater than35%, at 0.5 and 4.0 bar, respectively, and / or(iii) contains particle fractions less than 5 pm greater than 88% and greater than 98%, at 0.5 and 4.0 bar respectively.

13. The aerosol of claim 12, wherein the aerosol is free of surfactant protein and surfactant protein peptide mimic.

14. The aerosol of claim 12, further comprising at least one dispersion enhancer.

15. The aerosol of claim 14, wherein the at least one dispersion enhancer comprises 1-lcucinc.

16. The aerosol of claim 12, wherein the one or more PL comprise DPPC and / or POPG, andwherein the one or more hygroscopic excipients comprise mannitol and / or sodium chloride.

17. The aerosol of claim 16, wherein the one or more PL is 70-100% DPPC and 0-30% POPG.

18. The aerosol of claim 16, further comprising a surfactant protein peptide mimic.

19. The aerosol of claim 18, wherein the surfactant protein peptide mimic is B-YL.

20. The aerosol of claim 16, wherein the formulation contains 60-70% phospholipids, 15-25% 1-leucine, 5-15% mannitol, 4-10% sodium chloride, and 0-5% surfactant protein peptide mimic.

21. The aerosol of claim 16, wherein the one or more hygroscopic excipients are no more than 15% of the formulation.

22. The aerosol of claim 12, wherein(i) the aerosol has a fine particle fraction (FPF) <5 pm expressed as a percentage ofemitted dose (ED) from an inhaler of at least 27% ED, and / or(ii) the aerosol has a fine particle fraction (FPF) <2.5 pm expressed as a percentage ofemitted dose (ED) from an inhaler of at least 21% ED, and / or(iii) the aerosol has a fine particle fraction (FPF) <1 pm expressed as a percentage of emitted dose (ED) from an inhaler of at least 9% ED.

23. The aerosol of claim 22, wherein(i) the aerosol has a fine particle fraction (FPF) <5 pm expressed as a percentage ofemitted dose (ED) from an inhaler of at least 88% ED, and / or(ii) the aerosol has a fine particle fraction (FPF) <2.5 pm expressed as a percentage ofemitted dose (ED) from an inhaler of at least 62% ED, and / or(iii) the aerosol has a fine particle fraction (FPF) <1 pm expressed as a percentage of emitted dose (ED) from an inhaler of at least 10% ED.

24. The aerosol of claim 12, wherein the MMAD is less than 2.0 pm.

25. The aerosol of claim 12, wherein the MMAD is less than 1.7 pm.

26. An aerosol of a dry powder synthetic lung surfactant (SLS) formulation comprising one or more phospholipids (PL) and one or more hygroscopic excipients, wherein(i) the aerosol has a mass median aerodynamic diameter (MMAD) of less than 2.3 pm, and / or(ii) the aerosol has a fine particle fraction (FPF) <5 pm expressed as a percentage of emitted dose (ED) from an inhaler of at least 27% ED, and / or(iii) the aerosol has a fine particle fraction (FPF) <2.5 pm expressed as a percentage of emitted dose (ED) from an inhaler of at least 21% ED, and / or(iv) the aerosol has a fine particle fraction (FPF) <1 pm expressed as a percentage of emitted dose (ED) from an inhaler of at least 9% ED.

27. The aerosol of claim 26, wherein(i) the aerosol has a fine particle fraction (FPF) <5 pm expressed as a percentage of emitted dose (ED) from an inhaler of at least 88% ED, and / or(ii) the aerosol has a fine particle fraction (FPF) <2.5 pm expressed as a percentage of emitted dose (ED) from an inhaler of at least 62% ED, and / or(iii) the aerosol has a fine particle fraction (FPF) <1 pm expressed as a percentage of emitted dose (ED) from an inhaler of at least 10% ED.

28. The aerosol of claim 26, wherein the MMAD is less than 2.0 pm.

29. The aerosol of claim 26, wherein the MMAD is less than 1.7 pm.

30. The aerosol of claim 26, wherein the dry powder SLS formulation(i) is dispersible to a mean geometric diameter of less than 2.0 pm at 0.5 bar and a mean geometric diameter of less than 1.3pm at 4.0 bar, and / or(ii) contains particle fractions less than 1 pm greater than 20% and greater than 35%, at 0.5 and 4.0 bar, respectively, and / or(iii) contains particle fractions less than 5 pm greater than 88% and greater than 98%, at 0.5 and 4.0 bar respectively.

31. The aerosol of claim 26, wherein the aerosol is free of surfactant protein and surfactant protein peptide mimic.

32. The aerosol of claim 26, further comprising at least one dispersion enhancer.

33. The aerosol of claim 32, wherein the at least one dispersion enhancer comprises 1-leucine.

34. The aerosol of claim 26, wherein the one or more PL comprise DPPC and / or POPG, and wherein the one or more hygroscopic excipients comprise mannitol and / or sodium chloride.

35. The aerosol of claim 34, wherein the one or more PL is 70-100% DPPC and 0-30% POPG.

36. The aerosol of claim 34, further comprising a surfactant protein peptide mimic.

37. The aerosol of claim 36, wherein the surfactant protein peptide mimic is B-YL.

38. The aerosol of claim 34, wherein the dry powder SLS formulation contains 60-70% phospholipids, 15-25% 1-leucine, 5-15% mannitol, 4-10% sodium chloride, and 0-5% surfactant protein peptide mimic.

39. The aerosol of claim 34, wherein the one or more hygroscopic excipients are no more than 15% of the dry powder SLS formulation.

40. A method of using a dry powder synthetic lung surfactant (SLS) formulation, comprisingforming an aerosol, with an inhaler, containing a dry powder synthetic lung surfactant (SLS) formulation comprising one or more phospholipids (PL) and one or more hygroscopic excipients; andemitting the aerosol from the inhaler at a fine particle dose (FPD) <5pm equal to or greater than 5.0 mg PL / kg patient mass over one or more aerosol delivery periods forming a total therapy time,wherein for the total therapy time the aerosol is emitted from the inhaler at a nominal dose of 30 mg or less PL / kg patient mass.

41. The method of claim 40, wherein the aerosol is emitted at an FPD <2.5 p m equal to or greater than 4.0 mg PL / kg patient mass over the one or more aerosol delivery periods.

42. The method of claim 40, whereinthe aerosol is emitted at an FPD <5pm equal to or greater than 16.0 mg PL / kg patient mass over the one or more aerosol delivery periods, and / orthe aerosol is emitted at an FPD <2.5pm equal to or greater than 11.0 mg PL / kg patient mass over the one or more aerosol delivery periods.

43. The method of claim 40, wherein(i) the aerosol has a fine particle fraction (FPF) <5 pm expressed as a percentage of emitted dose (ED) from an inhaler of at least 27% ED, and / or(ii) the aerosol has a fine particle fraction (FPF) <2.5 pm expressed as a percentage of emitted dose (ED) from an inhaler of at least 21% ED, and / or(iii) the aerosol has a fine particle fraction (FPF) <1 pm expressed as a percentage of emitted dose (ED) from an inhaler of at least 9% ED.

44. The method of claim 43, wherein(i) the aerosol has a fine particle fraction (FPF) <5 pm expressed as a percentage of emitted dose (ED) from an inhaler of at least 88% ED, and / or(ii) the aerosol has a fine particle fraction (FPF) <2.5 pm expressed as a percentage of emitted dose (ED) from an inhaler of at least 62% ED, and / or(iii) the aerosol has a fine particle fraction (FPF) <1 pm expressed as a percentage of emitted dose (ED) from an inhaler of at least 10% ED.

45. The method of claim 40, wherein the dry powder SLS formulation (i) is dispersible to a mean geometric diameter of less than 2.0 pm at 0.5 bar and a mean geometric diameter of less than 1.3pm at 4.0 bar, and / or (ii) contains particle fractions less than 1 pm greater than 20% and greater than 35%, at 0.5 and 4.0 bar, respectively, and / or (iii) contains particle fractions less than 5 pm greater than 88% and greater than 98%, at 0.5 and 4.0 bar respectively.

46. The method of claim 40, wherein each aerosol delivery period is less than 5 minutes.

47. The method of claim 46, wherein the total therapy time is less than 20 minutes.

48. The method of claim 40, wherein the nominal dose is 10-30 mg PL / kg.

49. The method of claim 40, wherein the nominal dose is 20-30 mg PL / kg.

50. The method of claim 40, wherein for each aerosol delivery period the aerosol is emitted with 10 or fewer air actuations, each actuation being timed to coincide with an inhalation.

51. The method of claim 40, wherein for each aerosol delivery period the aerosol is emitted with 4-10 air actuations, each actuation being timed to coincide with an inhalation.

52. The method of claim 40, wherein for each aerosol delivery period the aerosol is emitted with less than 4 air actuations, each actuation being timed to coincide with an inhalation.

53. The method of claim 40, wherein the emitting step is performed with positive pressure.

54. The method of claim 40, wherein the patient interface is an endotracheal tube, nasal cannula, or face mask.

55. The method of claim 40, wherein the patient is an infant.

56. The method of claim 40, wherein the aerosol has an MMAD of less than 2.3 pm.

57. The method of claim 56, wherein the MMAD is less than 2.0 pm.

58. The method of claim 56, wherein the MMAD is less than 1.7 pm.

59. The method of claim 40, wherein the dry powder SLS formulation is free of surfactantprotein and surfactant protein peptide mimic.

60. The method of claim 40, wherein the dry powder SLS formulation further comprises at least one dispersion enhancer.

61. The dry powder SLS formulation of claim 60, wherein the at least one dispersion enhancer comprises 1-leucine.

62. The method of claim 40, wherein the one or more PL comprise DPPC and / or POPG, and wherein the one or more hygroscopic excipients comprise mannitol and / or sodium chloride.

63. The method of claim 62, wherein the one or more PL is 70-100% DPPC and 0-30% POPG.

64. The method of claim 62, further comprising a surfactant protein peptide mimic.

65. The method of claim 64, wherein the surfactant protein peptide mimic is B-YL.

66. The method of claim 62, wherein the dry powder SLS formulation contains 60-70% phospholipids, 15-25% 1-leucine, 5-15% mannitol, 4-10% sodium chloride, and 0-5% surfactant protein peptide mimic.

67. The method of claim 62, wherein the one or more hygroscopic excipients are no more than 15% of the dry powder SLS formulation.

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