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 [1,2,3,4,5,6]. 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 [7, 8]. Surfactant replacement therapy (SRT) substantially reduces mortality and disability in premature neonates suffering from RDS [9,10,11,12]. The standard therapeutic approach for exogenous surfactant involves the intratracheal liquid instillation of 100–200 mg PL/kg [13]. 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 [8, 14,15,16,17,18].

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 [8, 19,20,21,22]. 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 disturbances in cerebral blood flow and neurological impairment [23,24,25,26,27,28,29,30]. 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 [12, 14, 31,32,33].

Nebulized liquid surfactant aerosol has been investigated as a potential treatment for RDS. Surfactant aerosol has several proposed benefits over standard intratracheal liquid SRT, including the potential for more uniform pulmonary drug distribution, fewer per-dosing complications, better clinical responses, and lower duration for invasive mechanical ventilation [27, 34,35,36,37]. Combining surfactant aerosol in intubated patients could allow more rapid weaning from the ventilator with CPAP and prevent the need for ventilation. Findings from small animal models of RDS have demonstrated marginal improvements in oxygenation responses despite low pulmonary deposition of nebulized surfactant aerosols (~ 2–10% lung deposition) [27, 37,38,39,40,41].

Clinical trials in preterm infants receiving surfactant aerosol have failed to significantly improve mortality, morbidity, and other important outcomes [36, 42,43,44,45,46]. 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 [47], airway interfaces [48], and conducting airways [34, 37, 49,50,51]. 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 time-consuming, 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 [52, 53]. 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 [34, 49, 54]. 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 [55].

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 [56, 57]. 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 [53].

Motivated by the drawbacks and technical limitations of existing pulmonary delivery modalities for liquid SRT, we show in Part I of this two-part translational study that in vitro administration of a new synthetic lung surfactant (SLS) excipient enhanced growth (EEG) dry powder aerosol produced using the Buchi Nano Spray Dryer (Nano SD) and aerosolized with an infant dry powder aerosol delivery system (iDP-ADS) could hold a clinical advantage over other surfactant delivery options. The Nano SD SLS-EEG formulation in combination with iDP-ADS is capable of high emitted aerosol dose (> 80% of loaded dose) with a mass median aerodynamic diameter (MMAD) of < 2 µm. Prior to testing in vivo, Part I of this two-part study implemented an in vitro animal model to estimate the lung dose delivered using realistic aerosolization. Testing in an intubated small rabbit airway and lung chamber model indicated that over half of the loaded dose could penetrate the lower lung regions. Using these studies, it was 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. Part I also developed a dose delivery protocol for the administration of the total 60 mg loaded powder dose and achieved the resulting 15 mg PL/kg to the lower lung with the iDP-ADS using an improved version of the aerosol generation device (aerosolization engine), referred to as the Infant Design 2 (D2) air-jet DPI.

This proof-of-concept animal study (Part II) aims to determine 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 SpO2 was monitored (Rad 7, Massimo, Italy) with a tail probe, and body temperature was monitored continuously with a rectal temperature probe and maintained normothermic (38-39ºC) 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 ºC 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 cmH2O. The lungs were depleted of surfactant and injured by cyclic alveolar collapse (PEEP of 0 cmH2O) with repeated lavage using 25 mL/kg of warmed (39ºC) 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 [58]. 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 PaCO2 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 [59]. 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 [8]. 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 ½ dose was followed by ventilation for several minutes before rotating the position to the opposite side and instilling the remaining ½ 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 (Part 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 Part I, two 30 mg formulation doses were loaded (from capsules) into the D2 DPI aerosolization chamber and delivered 15 min apart. Also determined in Part I 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, our 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 cmH2O, PEEP of 5 cmH2O, VT of 7 mL/kg, flow of 3 L/min, and actuation time of ~ 0.20–0.22 s (see Fig. 1). 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 in the accompanying Part I study. 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 (Fig. 1). 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.

Fig. 1
figure 1

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 cmH2O), PEEP of 5 cmH2O, 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.

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Physiologic Measurements

Figure 2 shows a diagram of the animal procedures and study time points during the experiments, which considers the dose delivery protocol developed in Part I. 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 Fig. 2).

Fig. 2
figure 2

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 Part I of this two-part study.

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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/cmH2O/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 [11, 60, 61]. 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 cmH2O (PEEP 0 cmH2O). 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 cmH2O. 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 μm thickness. Hematoxylin and eosin (H&E) slide staining was examined under light microscopy (× 8.5) to inspect the extent of lung injury and uniformity of lung inflation [62]. 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/cmH2O/kg at baseline (P < 0.001) with increased PaCO2 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 I).

Table I Baseline Data
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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 cmH2O) by the ventilator during dosing. The SLS-EEG 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 = 0.15) 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 [63]. 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 the Part I study, the total aerosol delivery time, including ventilator reconnection to enable device reloading and stabilization, was completed in < 5 min.

Gas Exchange

Gas exchange data for the different groups is shown in Fig. 3. 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 baseline 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.

Fig. 3
figure 3

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 PaO2 values fully recovered to the pre-lavage basal levels (96–100%) at 3.5 h. 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. Both surfactant treatment groups had higher PaO2 than the sham controls from 60 min until the 3.5-h endpoint. The PaCO2 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.

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The mean PaCO2 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 Fig. 2).

Lung Compliance and Disease Severity

Lung compliance and indices of disease severity data for the different groups is shown in Fig. 4. 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.

Fig. 4
figure 4

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 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.

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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 120-min 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 Fig. 5. The gross macroscopic appearance of the anterior lungs is shown while being ventilated ex vivo with peak inspiratory pressures of 20 cmH2O (5a-c). The control lungs are underinflated with large areas of discoloration suggestive of pulmonary collapse, edema, and hemorrhage (5a). The liquid Curosurf-treated lungs (5b) 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 (5c). The gross appearance of the excised tracheas following medial incision (5d-f), shows the control animal with a well hydrated and patent trachea (5d). The Curosurf-treated animal (5e) shows abundant foamy liquid assumed to be surfactant obstructing the trachea with overflow from the airway. The aerosol SLS-EEG-treated animal (5f) 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. 5g-i. In the control subject (5 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 (5 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 (5i) 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. 5j). In contrast, lung tissue from the liquid surfactant group (5 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 (5 l).

Fig. 5
figure 5

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 in Fig. 5. The gross macroscopic appearance of the anterior lungs is shown while being ventilated ex vivo with peak inspiratory pressures of 20 cmH2O (5a-c). The control lungs are underinflated with large areas of discoloration suggestive of pulmonary collapse, edema, and hemorrhage (5a). The liquid Curosurf-treated lungs (5b) 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 (5c). The gross appearance of the excised tracheas following medial incision (5d-f), shows the control animal with a well hydrated and patent trachea (5d). The Curosurf-treated animal (5e) shows abundant foamy liquid assumed to be surfactant obstructing the trachea with overflow out of the airway. The aerosol SLS-EEG-treated animal (5f) 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 (g-i). In the control subject (g), 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 (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 SLS-EEG aerosol (5i) sample shows a homogenous layer of less foamy surfactant evenly distributed across the internal surfaces of the transected lung lobe.

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Discussion

Using an established small animal model of surfactant deficiency and severe RDS, we demonstrated 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. Our findings indicate greater delivery efficiency to the acinar lung regions (Fig. 4), 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 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.

Introducing an effective aerosolized surfactant globally could substantially reduce neonatal mortality due to respiratory distress syndrome, especially if combined with noninvasive respiratory support [53]. Unfortunately, pulmonary deposition of medical aerosols in spontaneously breathing preterm infants is negligible, and with standard liquid nebulizers producing aerosols with MMAD of 3.2 and 5.0 µm resulted in minimal lung deposition of 0.89% and 0.46% of the nominal nebulizer dose, respectively [64]. High dose surfactant aerosol (375 mg) generated by a jet and ultrasonic nebulizer applied in conjunction with mechanical ventilation is no better (< 1%) with only minimal effects on PaO2 [59]. Vibrating mesh nebulizers have demonstrated improved pulmonary aerosol deposition (~ 14–16%) in neonatal animals supported by mechanical ventilation and CPAP [51, 65].

Despite improvements in nebulizer performance, animal clinical and model assessments of nebulized liquid surfactant formulations have required surfactant doses of 210–800 [11, 34, 37, 51, 66,67,68,69,70] and 100–600 mg PL/kg [35, 36, 44, 45, 55, 71], respectively, and often do not achieve a high efficacy response. Multiple clinical trials in preterm infants have shown only minimal or no improvements in oxygenation and failed to demonstrate substantial reductions in mortality and morbidity by employing several different methods to administer surfactant aerosols [36, 44,45,46, 71]. Surfactant aerosol therapy typically requires long delivery times and results in high impaction drug losses and congestion in the conducting airways, reflux, congestion, alveolar flooding, reduced lung compliance, and impaired gas exchange (retained CO2), producing many of the same obstructive side effects as liquid bolus instillation [34, 37, 44, 45, 49,50,51, 62, 72].

We attribute the high efficacy of Nano SD SLS-EEG dry powder formulation in animals 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%. The aerosol size characteristics of the SLS-EEG aerosol with the Infant D2 air-jet DPI are relatively smaller (< 2 µm) than other attempts to administer liquid and dry powder aerosols [70, 73]. We can infer from the studies in vitro, 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.

A recent study in ventilated lung-lavaged rabbits with mild-moderate RDS, investigating the efficacy of a breath-synchronized vibrating mesh nebulizer (Aerogen Pharma Corp., San Mateo, CA), showed similar improvement (~ 75% recovery in PaO2) at equivalent doses of animal-derived surfactant (Alveofact, 108 mg/kg and 97 PL/kg, Lyomark Pharma, Germany) for both directly instilled and aerosolized surfactant delivery methods [74]. Interestingly, the lung compliance values remained unchanged (0.55 vs. 0.58 mL/cmH2O) three hours following aerosol administration. Applying the same surfactant aerosol product in a large pediatric porcine model of ARDS (~ 35 kg), a total surfactant dose of 30 mg/kg (27 mg PL/kg) showed improvements in oxygenation, but compliance was reduced from baseline (13.5 vs.10.2 mL/cmH2O) [62]. After therapy, a two-fold increase in airway resistance resulted from accumulating residual surfactant foamy liquids and alveolar flooding. Failure to show rapid improvement in lung compliance several hours following SRT is also common with liquid instillation, which could take 12 to 24 h to show substantial improvements [75, 76]. Nonetheless, these studies highlight the importance of inspiratory aerosol delivery with a high efficiency nebulizer and the potential need to consider lower doses with aerosol delivery than those traditionally used for standard SRT (~ 100–200 mg/kg).

The Curosurf-treated animals from the current study 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 [17, 20, 72]. The persistently low compliance values (0.6 to 0.75 ml/cmH2O) 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 [77]. 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 [29, 30].

The animals in the current study had a more severe form of RDS (P/F < 100, OI > 15) than previous liquid aerosol studies [74], 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 [75]. 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.

Compared with previous dry powder studies, the highly dispersible small particle SLS-EEG dry powder aerosol achieved improved efficacy at ~ 1/10 the PL dose compared with Walther et al. [67] for their evaluation of ARCUS® dry powder surfactant aerosol. A best-case dose of 240 mg/kg (168 mg PL/kg) [78] 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 [70].

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. These agglomeration phenomena were associated with the formation of powder aggregates blocking the trachea of animals, as observed in other studies [69, 79, 80]. A study in vitro with ARCUS® dry powder surfactant aerosol reported MMAD of ~ 3.7 µm, and there was a noteworthy reduction in lung delivery efficiency due to accumulation of the dry powder surfactant in the airway interfaces and nasal airway occlusion when realistic humidity conditions (RH = 99%) were applied with CPAP in a premature infant nasal and lung model [73].

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.

Limitations

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 (1 s) 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. Attempts could be made to 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). 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 µm could still be delivered effectively in line with a ventilator system delivery and provide a similar benefit. Our Part I 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 VILI. 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.

We also did not directly measure aerosol deposition in the lungs of these animals. A common limitation in most in vivo aerosol studies is the need for more data regarding lung deposition and pulmonary distribution of surfactants. We described the distribution of surfactant and lung injury based on images from a single animal from each group. We did not directly quantify surfactant content in the lung or assess histology in all animals and thus could not perform lung injury scores. Studies are underway to objectively quantify inflammatory markers, lung injury, and PL levels in the lung tissue.

Conclusion

Our findings 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. At a ~ 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. Further studies will address the development of an inhaler capable of delivering a 30 mg powder bolus over a series of positive pressure actuations and focus on the transition from intratracheal administration to delivery through the neonatal nasal airways to the lungs, as will be required for future clinical applications. Transnasal dosing strategies that focus on applying this therapy in spontaneously breathing infants receiving nasal CPAP and other noninvasive support are needed. 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. However, work is still needed to develop this new dry powder surfactant aerosol product for clinical applications to realize these benefits.