Development and characterization of excipient enhanced growth (EEG) surfactant powder formulations for treating neonatal respiratory distress syndrome

Abstract

This study aimed to develop and characterize a spray-dried powder aerosol formulation of a commercially available surfactant formulation, Survanta^® intratracheal suspension, using the excipient enhanced growth (EEG) approach. Survanta EEG powders were prepared by spray drying of the feed dispersions containing Survanta^® (beractant) intratracheal suspension, hygroscopic excipients (mannitol and sodium chloride) and a dispersion enhancer (l-leucine or trileucine) in 5 or 20% v/v ethanol in water using the Buchi Nano Spray Dryer B-90 HP. Powders were characterized for primary particle size, morphology, phospholipid content, moisture content, thermal properties, moisture sorption and surface activity. The aerosol performance of the powders was assessed using a novel low-volume dry powder inhaler (LV-DPI) device operated with 3 mL volume of dispersion air. At both ethanol concentrations, in comparison to trileucine, l-leucine significantly reduced the primary particle size and span and increased the fraction of submicrometer particles of the Survanta EEG powders. The l-leucine containing Survanta EEG powders exhibited good aerosolization performance with ≥ 88% of the mass emitted (% nominal) after 3 actuations from the modified LV-DPI device. In addition, l-leucine containing powders had a low moisture content (< 3% w/w) with transition temperatures close to the commercial surfactant formulation and retained their surface tension reducing activity after formulation processing. A Survanta EEG powder containing l-leucine was developed which showed efficient aerosol delivery from the modified LV-DPI device using a low dispersion air volume.

Introduction

Preterm birth is one of the leading causes of global newborn deaths (1). In comparison to other developed nations, the United States has a higher preterm birth rate, which is still on the rise (2,3). Neonatal respiratory distress syndrome (NRDS) is the most common lung disease in the preterm infants and remains one of the single major causes of infant death in developed nations (1,4). NRDS develops in the preterm infants born with underdeveloped lungs (mostly infants born <32 weeks of gestational age) resulting in an insufficient production of lung surfactant that is necessary for breathing (5).

Surfactant replacement therapy (SRT) is the standard recommended therapy for infants with NRDS and has been used for more than three decades (6). This therapy involves invasive intubation and intratracheal bolus instillation of a liquid surfactant formulation. There are two treatment strategies that physicians use to administer exogenous surfactant: preventive and therapeutic. In the preventive strategy, surfactant is administered in preterm infants at high risk of developing respiratory distress syndrome; whereas in the therapeutic strategy, surfactant is administered in infants with confirmed respiratory distress and requiring ventilation support (7,8). While the preventive strategy, with proper prenatal care, appears to be the best way to avoid NRDS, the therapeutic strategy for the confirmed NRDS cases is continuously evolving for optimal therapeutic outcomes. For example, during surfactant administration, nasal continuous positive airway pressure (NCPAP) is now more commonly used for the breathing support of infants with confirmed respiratory distress instead of the traditional mechanical ventilator (9), thereby reducing or eliminating the complications and adverse effects associated with mechanical ventilation.

Although SRT remains a life-saving treatment for infants with NRDS, there are some concerns related to the safety and efficacy of SRT, which include rapid fluctuations in hemodynamics and cerebral perfusion after liquid instillation (10,11). The delivery of a liquid bolus is also associated with non-uniform distribution of the surfactant within the lung airways and requirement of a high dosage regimen (12). In addition, increased risk of bradycardia, hypoxia and hypotension are associated with the liquid bolus instillation (13,14). Moreover, surfactant delivery during mechanical ventilation may decrease the treatment success with a chance of a higher degree of surfactant inactivation compared to delivery during spontaneous breathing (12).

Surfactant delivery as aerosol formulations during ventilation support could be an alternative delivery method to reduce or eliminate the problems related to the liquid instillation used in current SRT (12). Among the aerosol generation and delivery techniques, nebulization has been broadly studied to generate and deliver surfactant aerosols to the preterm infants with NRDS (15-20). However, prolonged delivery time and low pulmonary delivery efficiency with variable clinical efficacy have limited the use of aerosolized surfactant therapy using nebulization (18,21). Dry powder formulations of surfactant could overcome the problems associated with nebulization. In comparison to liquid formulations used in nebulization, dry powder formulations are more stable. In addition, higher lung delivery efficiency can be achieved using dry powder formulations and delivery of a powder requires less time than nebulized formulations (22-24). A number of surfactant preparations have been studied as dry powder formulations for treating respiratory distress syndrome (25-29). These surfactant formulations were prepared from synthetic surfactants. In contrast, in the current study, a naturally derived surfactant in a commercial liquid surfactant replacement product has been transformed into a spray-dried dry powder formulation.

To develop dry powder formulations for inhalation, the aerodynamic size range of the generated aerosol particles need to be 1–5 μm (30). Spray drying is one of the most attractive technique to produce inhalable dry powder particles due to its particle engineering capacity and easy scalability (31,32). Micrometer-sized particles stored as powders are notoriously difficult to disperse into their primary particles due to the agglomeration and high cohesiveness. Surface modifying excipients can be used to improve the dispersibility of powder formulations. For example, l-leucine and trileucine have been used to improve the dispersibility of spray-dried powders (33-37).

Excipient enhanced growth (EEG) is a drug formulation approach for efficient aerosol generation and delivery to the lungs (38). This technique employs micrometer-sized particles which increase in size following inhalation in order to minimize upper respiratory tract deposition and maximize targeted deposition in the lungs (39). The EEG dry powder formulation contains drug, a hygroscopic excipient and a dispersion enhancer. The EEG approach has been adapted for use with both dry powder and liquid formulations (40,41).

This study aimed to develop and characterize a spray-dried EEG dry powder formulation of a commercially available natural surfactant formulation, Survanta^® intratracheal suspension that would be used for treating NRDS during non-invasive ventilation. Survanta^® is a natural bovine lung extract suspended in 0.9% sodium chloride with 25 mg/mL of phospholipids (42,43). In addition to sodium chloride present in the commercial suspension, mannitol was added as a hygroscopic excipient. Two dry powder dispersion enhancers, l-leucine or trileucine, and the effect of ethanol concentration (5 or 20% v/v ethanol in water) in the feed dispersion were studied to explore their effects on the powder formulation characteristics. Finally, the aerosol performance of the developed spray-dried Survanta EEG powders were assessed using a novel low-volume dry powder inhaler (LV-DPI) device designed to operate with a small volume (3 mL) of air, which will be required for aerosol delivery to neonates.

Materials and methods

Materials

Dipalmitoylphosphatidylcholine (DPPC), Survanta^® (beractant) intratracheal suspension and l-leucine (Leu) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL), Cardinal Health, Inc. (Greensboro, NC) and Sigma-Aldrich Chemical Co. (St Louis, MO), respectively. Sodium chloride, trileucine (Trileu) and ethanol were purchased from Fisher Scientific Co. (Hanover Park, IL). Pearlitol^® PF-mannitol was a kind donation of Roquette Pharma (Lestrem, France). Throughout the study, freshly collected deionized water was used.

Powder preparation

Feed dispersion preparation

Spray drying feed dispersions containing commercial Survanta^® (beractant) intratracheal suspension, hygroscopic excipients (mannitol and sodium chloride) and dispersion enhancer (l-leucine or trileucine) were prepared by heated bath sonication (Fisher Scientific™ CPXH, Hanover Park, IL). All formulations contained a 3:1 ratio of mannitol (30% w/w) and sodium chloride (10% w/w) with 20% w/w of l-leucine or trileucine. Approximately 27% w/w of phospholipids (including ~14% w/w DPPC) were included in each formulation based on the label claim of Survanta^® (43). Briefly, solids were weighed and dissolved in the ethanol-water (5 or 20% v/v ethanol in water) co-solvent system followed by addition of Survanta^® for a total solids concentration of 0.125% w/v in the feed dispersion. Preliminary studies revealed that an ethanol-water co-solvent system was required to ensure a homogenous dispersion of all the added components. Dispersions were sonicated for 40 min in a water bath set at 60 °C.

The stability and homogeneity of the feed dispersions were evaluated by measuring their zeta potential and size distribution using dynamic light scattering (Zetasizer Nano S, Malvern Instruments Ltd., Worcestershire, UK). Briefly, about 1 mL of the feed dispersion was filled in an appropriate cuvette then analyzed to determine the mean particle size, z-average, size distribution and polydispersity index (PDI). All measurements were performed at 25 °C with the test material set as phospholipids (refractive index: 1.450, absorption coefficient: 0.001) and the dispersant set as water (refractive index: 1.330, viscosity: 0.887 cP, dielectric constant: 78.5). Zeta potentials were calculated using the Smoluchowski approximation for samples in aqueous media. Triplicate samples were measured for each analysis.

Spray drying

Survanta EEG powders were prepared by spray drying of the feed dispersions using the Buchi Nano Spray Dryer B-90 HP (Büchi Labortechnik AG, Flawil, Switzerland) operated in an open-mode configuration. Feed dispersions were spray-dried using the medium nozzle and the optimized spray drying parameters shown in Table I. Typical spray rates were in the range of 0.23-0.29 ml/min. Spray drying conditions were optimized in a study by Boc (2018) using a DPPC EEG formulation due to the cost associated with use of Survanta (44).

Table I.

Spray drying parameters to prepare surfactant powders.

Parameter	Setting
Inlet temperature	70 °C
Outlet temperature	37 – 41 °C
Drying gas flow	120 L/min (chamber pressure 40-42 mbar)
Spray frequency	120 kHz
Pump speed	3%
Spray percentage	80%

During spray drying, feed dispersions were continuously mixed using a stir bar and stir plate and kept cool with ice packs. The spray-dried powders were collected from the electrostatic precipitator into tared glass vials, evaluated for process yield and stored sealed in a desiccator (0% RH) in the refrigerator (2–8 °C) when not in use. The process yield of the collected powders was calculated using the following equation:

$$Processyield(\%)=\frac{Powdermasscollected}{Concentrationofsolidsindispersion\ast Volumeofdispersionspraydried}\times 100.$$ (1)

Particle size determination

The primary particle size distributions of the spray-dried Survanta EEG powders were determined using a Sympatec HELOS laser diffraction apparatus (Sympatec GmbH, Clausthal-Zellerfeld, Germany) with an R1 lens. The apparatus is equipped with RODOS/M disperser and ASPIROS sample feeder. Approximately 3 mg of powder sample filled in sample vials were placed into the ASPIROS sample feeder and dispersed in the laser beam using RODOS/M disperser at dispersion pressures of 1.0 and 4.5 bar. Triplicate samples were analyzed for each measurement at each dispersion pressure. Volume-based size distribution parameters, Dv10, Dv50 and Dv90 (particle size for 10%, 50% and 90% of the cumulative volume distribution), were calculated by WINDOX 5.0 software (Sympatec GmbH, Clausthal-Zellerfeld, Germany) using the Fraunhofer theory. The span of the particle size distribution was calculated using equation 2. The percentage of particles having sizes less than 1 and 5 μm, respectively, was also calculated using the Sympatec software.

$$Span=\frac{Dv90-Dv10}{Dv50}$$ (2)

Liquid chromatography – mass spectrometry (LC-MS)

In order to approximate the surfactant content of the Survanta EEG formulations, the content of DPPC was analytically quantified and used to estimate the total phospholipids content of this complex mixture based on the label claim for the Survanta^® product. The DPPC content was quantified using a liquid chromatography–mass spectrometry (LC-MS) method adapted from Li et al. (45). The system consisted of the Quattro micro™ mass spectrometer linked to an Alliance 2695 Separations Module with data acquisition software, MassLynx software v4.1 (all from Waters Corporation, Milford, MA). The chromatographic separation was achieved using the Atlantis hydrophilic interaction liquid chromatography (HILIC) silica column (5μm, 50×1.0 mm; Waters Corporation, Milford, MA). The isocratic mobile phase consisted of acetonitrile and 5 mM ammonium formate in water with 0.1% formic acid (85:15% v/v) pumped at a flow rate of 0.5 mL/min. The injection volume was 2 μL. Following optimization of the ionization settings of the mass spectrometer, selected ion monitoring (SIM) analysis (for m/z = 735) with positive electrospray ionization mode was applied to detect and quantify DPPC (molecular weight = 734 Daltons) following chromatographic separation. The DPPC stock standard solution (10 μg/mL) was prepared by dissolving sufficient amount of DPPC (Avanti Polar Lipids, Inc., Alabaster, AL) in methanol. The diluted standard solutions of DPPC in the concentration range of 0.5 to 5 μg/mL were prepared by dilution of the stock standard solution in methanol. The prepared stock standard and diluted standard solutions were injected as calibration standards. The calibration curve for DPPC was linear over the concentration range of 0.5–10 μg/mL with correlation coefficients (r) ≥ 0.995.

Estimation of DPPC content in Survanta EEG formulations

Approximately 1 mg of spray-dried Survanta EEG powder was dissolved in 25 mL of methanol and quantitatively analyzed for the DPPC content by the LC-MS method described above. The mean amount of DPPC per mg of Survanta EEG formulations was determined. Triplicate samples were prepared and analyzed for each of the powder samples.

Morphology

The morphology of the spray-dried Survanta EEG powder particles was examined with a Zeiss EVO-50XVP scanning electron microscope (SEM) (Carl Zeiss, Oberkochen, Germany) at an accelerating voltage of 15 kV. Powder samples were mounted on a metal SEM stub using double-sided adhesive tape and loose powders were removed using compressed air. Prior to imaging, all samples were sputter coated with gold grain using an EMS550X sputter coater (Electron Microscopy Sciences, Hatfield, PA). The microscopic images of the powder samples were captured at magnifications of 19k to 21k.

Moisture content

The estimated moisture content of the powder samples was determined by thermogravimetric analysis using the Pyris 1 TGA (PerkinElmer, Covina, CA) with TAC 7/DX thermal analysis controller. About 2 mg samples of powders were loaded in aluminum pans and heated at 10 °C/min from 25 °C to 100 °C under nitrogen gas purging at 40 mL/min. The estimated moisture content (as percentage) was determined from the percent weight loss following an isothermal hold at 100 °C for 45 min.

Thermal analysis

The thermal properties (glass transition temperature) of the spray-dried Survanta EEG powder samples were determined by differential scanning calorimetry (DSC) using the DSC 7 (PerkinElmer, Covina, CA) with TAC 7/DX thermal analysis controller. About 2 mg of each sample (spray-dried powder sample and dried sample of commercial suspension) was hermetically sealed in an aluminum DSC pan and heated over a range of 25 °C to 100 °C at 5 °C/min under a nitrogen environment (purge at 20 mL/min) (46,47). An empty pan was used as a reference.

Moisture sorption

The dynamic moisture sorption behavior of the l-leucine containing Survanta EEG powder formulation was evaluated using the dynamic vapor sorption (DVS) (DVS Adventure, Surface Measurement Systems Ltd., UK) to understand the water uptake of the powder as a function of relative humidity (RH). Powder samples (about 10 mg) were subject to equilibration at 0% RH and then exposed to increasing RH from 0% to 95% and decreasing RH from 95% to 0%. At each RH, the rate of mass change (dm/dt) less than 0.002% was considered as the equilibrium condition before automatically proceeding to the next RH.

Aerosol performance

A low-volume dry powder inhaler (LV-DPI) device modified from the original design developed by Farkas et al. (48) was used for the aerosol performance of the lead l-leucine containing Survanta EEG powder formulation. The LV-DPI device was modified to operate with a small volume (3 mL) of dispersion air so that it could be used for intratracheal administration of dry powder to small animal models and eventually for very low birth weight infants. Briefly, the device has two main parts, an inlet body containing three 0.60 mm holes with a commercial luer lock connection and an outlet body containing a 0.89 mm hole extended to a 19 gauge extra-thin-walled capillary needle with 55 mm length and 0.89 mm internal diameter (Fig. 1). When assembled, the volume of the powder chamber of LV-DPI is 0.21 mL.

Fig. 1.

Axial cross-section view of the assembled modified containment unit DPI with 55 mm delivery tube length and 0.21 mL powder chamber.

Emitted mass determination

The emitted mass of the l-leucine containing Survanta EEG powders from the modified LV-DPI device was determined by gravimetric analysis. Briefly, powder samples (about 10 mg) were manually loaded into the modified LV-DPI device, the device was weighed and then actuated by manual compression of a 5 mL syringe filled with 3 mL of room air, which was attached to the device. After actuation, the syringe was disconnected from the device and the device was reweighed. The emitted mass of the powder was determined from the weight difference of the device before and after actuation. Measurements were repeated for up to three actuations. The percentage of powder emitted mass was calculated using the following equation:

$$Emittedmass(\%)=\frac{Massofpowderloadeddevice-Massofdeviceafteractuation}{Initialpowderfillmass}\times 100.$$ (3)

Aerosol characterization

The aerosol characterization of the powders dispersed from the modified LV-DPI device was performed by laser diffraction using a Malvern Spraytec^® (Malvern Instruments, Ltd., Worcestershire, UK) with RT Sizer Software. Powder samples were manually dispersed from the device into the laser beam using 3 mL dispersion air filled in a 5 mL syringe. For each measurement, a fixed distance of the device (5 cm) from the center of the laser beam and the 100 mm focal length lens (range of 0.5–200 μm) were used. Prior to each measurement, a background reading was taken. The data were reported as median volume diameter, Dv50 and % particle fraction <1 and <5 μm, respectively.

Surface activity and integrity of surfactant proteins

Surface activity

To determine whether the surfactant activity was maintained after formulation processing, the surface tension of the commercial Survanta^® and l-leucine containing Survanta EEG powders was assessed using a bubble pressure tensiometer (BP2, Kruss, Hamburg, Germany) following the sample preparation method described by Gugliotti et al. (49). Briefly, Survanta EEG powder samples, with a target DPPC concentration of 1.5 mg/mL, were dispersed in 1 mM NaCl using a microtip probe sonicator at an amplitude of 35 Hz for 15 min. Surface tension measurements of the dispersions were performed with the tensiometer connected to a recirculating water bath that allowed for temperature-controlled measurements. The temperature was monitored using Fisher Scientific Traceable Double Thermometer with Type K thermocouple (Fisher Scientific, Waltham, MA). The surface tension was measured at 50 °C as this is above the transition temperature (~41 °C) of DPPC, increasing the fluidity of DPPC lipid layers to allow the DPPC particles to migrate to the newly formed air-liquid interfaces. The original commercial Survanta^® formulation was also analyzed using the same procedure. All measurements for each sample were performed in triplicate.

Integrity of surfactant proteins

The presence of the surfactant proteins SP-B and SP-C (present in commercial surfactant formulations) after formulation processing is very crucial to ensure proper function of pulmonary surfactant. Formulation processing can affect the structures of the proteins which can result in partial or complete loss of their biological activity. Enzymatic assay was conducted for qualitative determination of the surfactant proteins in the feed dispersions and Survanta EEG powder formulations. Enzyme-linked immunosorbent assay (ELISA) kits (MBS703513 for bovine SP-B and MBS700517 for bovine SP-C (MyBiosoource.com, San Diego, CA)) were used for SP-B and SP-C analysis. Standards were prepared following manufacturer instructions and samples were prepared by diluting commercial Survanta^®, feed dispersions and l-leucine containing Survanta EEG powders in acetonitrile-water (60/40% v/v) co-solvent with theoretical protein concentrations of 100 to 500 μg/mL. The absorbance readings were measured for all samples at 450 nm using a microplate reader (Synergy™ H1 Hybrid Multi-Mode Reader, BioTek Instruments, Inc., Winooski, VT) and relative absorbance values were calculated for the feed dispersions and Survanta EEG powders compared to the commercial Survanta^® formulation. Samples were analyzed in triplicate.

Statistical analysis

Statistical analyses of the data were performed using the student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s HSD using JMP^® Pro software (version 12.0; SAS Institute Inc., Cary, NC). P values less than 0.05 were considered as statistically significant. All data are expressed as mean ± standard deviation.

Results and discussion

Effect of dispersion enhancer and ethanol concentration on feed dispersion characteristics

Table II shows the summary of the characteristics of the spray drying feed dispersions for the study formulations determined by dynamic light scattering. For the dispersions prepared in 5% v/v ethanol in water, the l-leucine feed dispersion had a significantly smaller mean particle size (z-average: 125 ± 2 nm), higher polydispersity index (PDI: 0.48 ± 0.06) and stability (zeta potential: −40 ± 1 mV) than the trileucine containing feed dispersions (z-average: 162 ± 10 nm, PDI: 0.39 ± 0.05 and zeta potential: −36 ± 4 mV) (p<0.05). However, using the 20% v/v ethanol in water vehicle, there was no significant difference observed in the mean particle size or zeta potential for the l-leucine and trileucine feed dispersions. Again, the PDI of l-leucine containing dispersion was significantly higher (0.60) than the trileucine containing dispersions (0.41) prepared in 20% v/v ethanol in water (p<0.05). For both l-leucine and trileucine, decreased dispersion stability (lower zeta potential) was observed for the dispersions prepared with higher (20% v/v) ethanol concentration (Table II). It is known that short-chain alcohols disturb the natural microstructure of the lipid membrane by residing in the headgroup region that results in increased disorder (50). Overall, a stable feed dispersion with the smallest dispersed particle size was observed in l-leucine containing feed dispersions when prepared in 5% v/v ethanol in water.

Table II.

Effect of dispersion enhancer and ethanol concentration on the particle size (z-Average), polydispersity index (PDI) and zeta potential of the spray drying dispersion (values are mean (standard deviation), n≥3).

Dispersion enhancer	Ethanol concentration (%v/v)	z-Average (nm)	PDI	Zeta potential (mV)
L-leucine	5	125 (2)*	0.48 (0.06)*	−40 (1)*
Trileucine	5	162 (10)	0.39 (0.05)	−36 (4)
L-leucine	20	176 (15)	0.60 (0.05)#	−22 (1)
Trileucine	20	188 (9)	0.41 (0.04)	−21 (1)

^*Significant difference compared to trileucine at 5% v/v ethanol in water concentration

^#Significant difference compared to trileucine at 20% v/v ethanol in water concentration; both are student’s t-test, p<0.05.

Powder preparation, process yield and DPPC content

The Survanta EEG powders were prepared by spray drying the prepared feed dispersions. The process yields of the spray-dried EEG powders were between 65 and 76% w/w recovered from the electrostatic precipitator. The DPPC content of the Survanta EEG powders determined by LC-MS analysis was between 11 and 14% w/w which is 85 to 110% of the theoretical content, equivalent to an estimated total phospholipids content of 21–27 % w/w (based on Survanta^® label claim). The RSD values for DPPC content of the powders were very low (<1.0%), suggesting high reproducibility of the powders.

Effect of dispersion enhancer and ethanol concentration on primary particle size of spray-dried powders

The particle size distribution data of the spray-dried Survanta EEG powders are shown in Fig. 2 and Table III. The results at 4.5 bar dispersion pressure are representative of the primary particle characteristics of the powders whereas the results at the lower dispersion pressure of 1.0 bar give a measure of powder deaggregation behavior. At both dispersion pressures, l-leucine containing powders prepared in 5% v/v ethanol in water had a significantly smaller Dv50 and span with higher particle fractions (both <1 and <5 μm) than the trileucine containing powders (p<0.05) (Table III). Similarly, l-leucine containing powders prepared in 20% v/v ethanol in water had a significantly smaller Dv50 and higher particle fraction (<1 μm) than trileucine containing powders (p<0.05) at both dispersion pressures. In addition, the span values were significantly smaller in l-leucine powders than the trileucine powders prepared in 20% v/v ethanol in water when characterized at 4.5 bar dispersion pressure although they were not significantly different at 1.0 bar. The Dv90 values of the l-leucine containing powders at 1.0 bar dispersion pressure are lower than the trileucine powders (Fig. 2) suggesting less aggregation of l-leucine containing powders. However, this finding is different from the previous report showing better deaggregation of trileucine than l-leucine (51). This difference could be due to the presence of other surface-active components in the formulation, which may have influenced the droplet formation and eventually the surface of powder particles during the spray drying process. Overall, smaller primary particles with smaller span and higher particle fractions (<1 and <5 μm) were observed in l-leucine containing powders. For all the powders the primary particle size, Dv50 at 4.5 bar pressure, were approximately 1 μm and about 0.3 μm smaller than the Dv50 at 1.0 bar pressure (Table III).

Fig. 2.

Primary particle size of the Survanta EEG powders determined by Sympatec HELOS with RODOS dispersion unit (Leu: l-leucine, Trileu: Trileucine, Bars represent the mean values, error bars represent standard deviations, n≥3).

Table III.

Effect of dispersion enhancer and ethanol concentration on the primary particle size (Dv50), span and particle fraction (<1 and 5 μm) of the spray dried Survanta EEG powders at 1.0 and 4.5 bars (values are mean (standard deviation), n≥3).

Dispersion enhancer	Ethanol concentration (%v/v)	Dv50 (μm)		Span		Particle fraction, <1 μm (%)		Particle fraction, <5 μm (%)
		1.0 bar	4.5 bar	1.0 bar	4.5 bar	1.0 bar	4.5 bar	1.0 bar	4.5 bar
L-leucine	5	1.26 (0.03)	1.00 (0.01)	1.57 (0.07)	1.44 (0.01)	36.2 (0.9)	50.0 (0.5)	99.7 (0.6)	100 (0.0)
Trileucine	5	1.41 (0.08)*	1.10 (0.05)*	2.23 (0.24)*	1.76 (0.28)*	31.8 (2.1)*	43.8 (2.9)*	94.0 (2.1)*	97.7 (2.5)*
L-leucine	20	1.25 (0.05)	0.96 (0.03)^	1.91 (0.07)^	1.36 (0.03)^	36.9 (1.9)	53.3 (2.5)^	96.7 (1.1)^	100 (0.0)
Trileucine	20	1.46 (0.07)#	1.07 (0.03)#	1.98 (0.09)	1.45 (0.04)#	30.5 (1.7)#	45.6 (1.9)#	96.5 (1.0)	100 (0.0)

^*Significant difference compared to l-leucine at 5% v/v ethanol in water concentration

^#Significant difference compared to l-leucine at 20% v/v ethanol in water concentration

^{^}Significant difference compared to l-leucine at 5% v/v ethanol in water concentration; All are student’s t-test, p<0.05.

The effect of ethanol concentration in l-leucine containing powders was significant. The Dv90 and span values of the l-leucine powders prepared in 20% v/v ethanol in water were significantly larger than the powders prepared in 5% v/v ethanol in water concentration (p<0.05) at 1.0 bar dispersion pressure. Also, the particle fraction <5 μm was higher in l-leucine containing powders prepared in 5% v/v ethanol in water than the powders prepared in 20% v/v ethanol in water.

Based on the dispersion feed properties and the smaller particle size characteristics of the l-leucine Survanta EEG powders, these formulations (prepared both in 5% and 20% v/v ethanol in water) were selected for further investigation and analysis.

Morphology and solid-state properties of l-leucine containing Survanta EEG powders

The morphology of the l-leucine containing powders prepared both in 5% and 20% v/v ethanol in water concentration visualized by SEM are shown in Fig. 3. For both ethanol concentrations, the particle morphology of the powders appeared to be similar. The size range of the particles was observed to be approximately 0.5 to 2 μm which is similar to the data observed in the laser diffraction analysis (Table III).

Fig. 3.

Scanning electron micrographs of l-leucine containing Survanta EEG powders prepared in (a) 5% v/v ethanol in water, and (b) 20% v/v ethanol in water feed dispersions.

The TGA analysis showed low estimated moisture content of the spray-dried l-leucine Survanta EEG powders (Fig. 4a). Although powders produced with higher ethanol concentration were expected to have lower estimated moisture content, no significant differences were observed in the estimated moisture content between the powders prepared in 5% and 20% v/v ethanol in water concentrations. The estimated moisture content (determined as percent weight loss) of the powders prepared in 5% and 20% v/v ethanol in water concentrations were 2.5 ± 0.7% w/w and 2.2 ± 0.2% w/w, respectively. The moisture sorption analysis results also support this finding of low hygroscopicity (supplement Fig. S1). The moisture uptake of the l-leucine Survanta EEG powder was very low (about 3%) up to conditions of 60% RH, indicative of a hydrophobic surface that is resistant to water uptake and would make it suitable as a dry powder inhaler formulation when appropriately packaged. Above 60% RH, the moisture uptake was observed to increase, suggesting that the l-leucine Survanta EEG powder would also be a suitable powder formulation for hygroscopic growth in the humid airways of the lungs where moisture uptake is required to enable particle growth and deposition as proposed by the EEG strategy. The higher moisture uptake at elevated RH is likely due to the presence of hygroscopic excipients in the formulation. The moisture content of the EEG powders is slightly higher than the previously reported moisture content (less than 1.5% w/w) for dry powder synthetic surfactant formulations (29). The differences could be due to the use of different feed solvents and formulation compositions between the two studies.

Fig. 4.

Characteristic TGA (a) and DSC (b) thermograms of l-leucine containing Survanta EEG powders prepared in 5% and 20% v/v ethanol in water feed dispersions; Survanta in DSC thermograms is for commercial Survanta^® formulation.

The glass transition temperature (T_g) of the commercial Survanta^® formulation was determined to be 37 °C (Fig. 4b). The T_g of the spray-dried l-leucine containing Survanta EEG powders (34 to 36 °C) was similar to the commercial Survanta^® which is likely due to the presence of mannitol and l-leucine in the formulations that tend to maintain their crystallized form during spray drying (32,34). Although in the experimental temperature range, no recrystallization or melting temperature was observed in the spray-dried Survanta EEG powders; a broad melting range between 50 and 80 °C was observed in the commercial Survanta^® formulation. This broad melting range may be due to the relaxation or loss in chain order of the acyl chains of the phospholipid structure.

Aerosol performance of l-leucine Survanta EEG powders

Emitted mass

Fig. 5 shows the cumulative mass emitted (% nominal) from the modified LV-DPI device for over multiple actuations. For the first actuation, the mass emitted (% nominal) was significantly higher for powders prepared with 5% v/v ethanol in water (63.8%) than the powders prepared with 20% v/v ethanol in water (53.8%) (p<0.05). However, for the subsequent actuations, the cumulative mass emitted between two formulations was similar. The cumulative mass emitted on the third actuation was 90.3% and 89.5% for the powders prepared with 5% and 20% v/v ethanol in water, respectively, indicating that it is possible to efficiently empty 10 mg of Survanta EEG powder using only three 3 mL actuations with the new LV-DPI device. Overall, for both l-leucine powder formulations, ≥ 88% of the nominal mass emitted after 3 actuations, indicating good aerosolization properties of the powders.

Fig. 5.

Cumulative mass emitted (% nominal) for l-leucine containing Survanta EEG powders with modified LV-DPI using 3 mL dispersion air (Markers are the mean values, error bars are standard deviations, n=3).

Aerosol characteristics

The characteristics of the aerosolized Survanta EEG powders was determined using the Malvern Spraytec. Table IV shows the aerosol characteristics of the l-leucine containing Survanta EEG powders. At each actuation, no differences were found in the emitted mass and Dv50 values between the two formulations. While Dv50 values for the powders prepared in 5% v/v ethanol in water were observed to decrease with each successive actuation, the Dv50 values decreased from the first actuation to the second actuation and then leveled off on the third actuation for the powders prepared in 20% v/v ethanol in water. Larger Dv50 values in both formulations for the first actuation could be due to the larger mass emitted at this actuation. Except for the particle fraction <5 μm on the second actuation, no significant differences were observed in the particle fractions (<1 and <5 μm) between powders prepared in 5% v/v ethanol and 20% v/v ethanol in water at each actuation. Overall, l-leucine containing Survanta EEG powders prepared with 20% v/v ethanol in water showed less variability in aerosol particle size (Dv50) and particle fractions between three actuations compared to the powders prepared with 5% v/v ethanol in water. This could be due to the solubility differences of l-leucine and phospholipids present in the formulations in the water-ethanol co-solvent system. In comparison to the lower ethanol concentration, a higher concentration of ethanol in feed dispersions might have allowed l-leucine to precipitate sooner during the rapid spray drying process resulting in a powder particle surface more enriched with l-leucine (52-55). At both ethanol concentrations, the observed particle fractions of the powders (<1 μm: ~20% and <5 μm: ~55%) suggest the efficiency of the developed device-formulation combination to achieve a respirable aerosol with a low dispersion air volume (3 mL) which is suitable for intratracheal administration of dry powder to small animal models and for low birth weight infants.

Table IV.

Aerosol characteristics of l-leucine containing Survanta EEG powders using the modified LV-DPI with 10 mg powder fill mass and 3 mL dispersion air (values are mean (standard deviation), n=3)

Actuation	Ethanol concentration (% v/v)	Mass emitted (mg)	Dv50 (μm)	Particle fraction (%)
				<1 μm	<5 μm
1st	5	6.42 (0.37)	4.5 (0.8)	19.6 (1.1)	51.7 (2.6)
	20	5.38 (0.51)	3.9 (0.4)	18.1 (1.1)	54.6 (2.2)
2nd	5	2.18 (0.14)	3.3 (0.2)	21.6 (1.3)	56.4 (0.8)*
	20	2.57 (0.30)	3.1 (0.1)	20.6 (0.2)	60.0 (0.7)
3rd	5	0.48 (0.10)	2.6 (0.5)	25.5 (2.7)	61.7 (4.4)
	20	0.96 (0.12)	3.2 (0.3)	20.6 (1.8)	57.8 (2.6)

^*Significant difference compared to 20% v/v ethanol in water concentration for the 2^nd actuation; student’s t-test, p<0.05.

To translate the in vitro characterization of the developed Survanta EEG powders into in vivo performance, the powder was subsequently used in an in vivo study using a rat lung injury model (56). The in vivo study demonstrated improved lung compliance and elastance in surfactant depleted rats at a 30-fold lower dose of phospholipids compared to liquid Survanta^® instillation. The results of the in vivo study provides evidence for Survanta EEG powder as a potential method of surfactant replacement therapy using the developed device-formulation combination. Previous studies also support the potential of dry powder surfactant delivery in surfactant replacement therapy for treating respiratory distress syndrome. A recent study by Walther et al. (29) reported improved oxygenation and lung functions in surfactant-deficient rabbits and preterm lambs for aerosol delivery of dry powder synthetic surfactant during non-invasive respiratory support with nasal continuous positive airway pressure. A dry powder synthetic surfactant with an advanced SP-B peptide mimic was administered from a capsule-based low flow inhaler device. In comparison to our EEG powder, the geometric particle size of the Walther et al. (29) studied powders was larger (3.2 to 6 μm vs ~1 μm), which might be due to the differences in the formulation composition between the two studies as well as differences in aerosolization device efficiency. Walther et al. (29) used the ARCUS^® Pulmonary Dry Powder Technology to produce the dry powder synthetic surfactant formulations which contained a combination of phospholipids, fatty acids and stabilizing excipients; our EEG approach used a combination of drug (commercial natural surfactant formulation, Survanta^®), hygroscopic excipients and a dispersion enhancer. Pohlmann et al. (27) and Ruppert et al. (28) demonstrated the successful delivery of dry powder recombinant surfactant protein-C (rSP-C) from their developed devices. Pohlmann et al. (27) used a high-concentration continuous powder aerosolization system with a subsequent humidification step for delivery of inhalable surfactant to preterm neonates where the median particle sizes of the humidified particles at the patient interface were in the range of 3 to 3.5 μm. Ruppert et al. (28) generated aerosols with a mass median aerodynamic diameter of 1.6 μm with 85% of all particles less than 5 μm from their developed dry powder aerosolizer. All previous studies conducted to deliver dry powder surfactant formulations were based on synthetic surfactant, none of which contain both SP-B and SP-C together – the surfactant proteins necessary for regulating the surface tension in the lungs by independently promoting rapid adsorption of phospholipids from the subphase to the interface (12,57). In the Survanta EEG formulations, we used a naturally derived surfactant in a commercial liquid surfactant replacement product (Survanta^®, which contains both SP-B and SP-C), that is easily dispersed and emitted with a low actuation air volume of 3 mL from a novel DPI.

Surface activity and integrity of surfactant proteins

Tables V and VI show the surface activity and integrity of surfactant proteins for the l-leucine-Survanta EEG powder formulations compared to Survanta^®. The final surface tension of the commercial Survanta^® and spray-dried Survanta EEG powders was 29.9 ± 5.5 mN/m and 33.0 ± 1.5 mN/m, respectively. The surface tension reduction rates of the two formulations were 0.73 ± 0.16 mN/m/s and 0.58 ± 0.07 mN/m/s, respectively. There were no significant differences in the surface tension values and surface tension reduction rates between the two formulations (p>0.05), suggesting that the EEG powders retained their surface tension reducing ability after formulation processing.

Table V.

Surfactant activity of commercial Survanta^® and l-leucine containing Survanta EEG powder (values are mean (standard deviation), n=3).

Formulation	Surface age (s)	Final Surface Tension (mN/m)	Surface tension reduction rate (mN/m/s)
Survanta®	59.0 (13.1)	29.9 (5.5)	0.73 (0.16)
L-leucine-Survanta EEG	65.6 (1.3)	33.0 (1.5)	0.58 (0.07)

Table VI.

Enzyme-linked immunosorbent assay (ELISA) results of the feed dispersions and spray dried l-leucine-Survanta EEG powders relative to the commercial Survanta^® formulation using ELISA kits for bovine SP-B and SP-C (values are mean (standard deviation), n=3).

Surfactant protein	Absorbance relative to Survanta®, (%)
	Feed dispersion	L-leucine-Survanta EEG powder
SP-B	94.1 (6.0)	92.0 (7.8)
SP-C	82.2 (3.3)	90.9 (4.1)

Further, enzyme-linked assay results showed that, relative to the unprocessed commercial Survanta^®, both the feed dispersions and the spray-dried Survanta EEG powders maintained the amount of surfactant proteins, SP-B and SP-C, throughout the formulation process. In comparison to the unprocessed commercial Survanta^®, spray-dried Survanta EEG formulations had relative absorbance values for SP-B and SP-C of 92% and 91%, respectively (Table VI), suggesting that Survanta EEG powders produced using the described spray drying process retained their surfactant proteins during formulation processing, which might have helped to maintain their surface activity. These findings support the potential method of delivering lung surfactant replacement therapy as a dry powder, which can reduce the problems associated with the existing liquid bolus instillation method of surfactant replacement therapy.

Conclusions

Dry powder formulations of commercial Survanta^® intratracheal suspension were successfully produced using the EEG approach by varying dispersion enhancers (l-leucine and trileucine) and ethanol concentrations (5 and 20% v/v ethanol in water) in the spray drying vehicle. At both ethanol concentrations, l-leucine containing Survanta EEG powders had smaller primary particles, smaller spans, and higher fractions of submicrometer particles than trileucine containing powders. The developed l-leucine containing Survanta EEG powders exhibited good aerosolization performance with ≥ 88% of the mass emitted (% nominal) after 3 actuations from the modified LV-DPI device using 3 mL dispersion air. The aerosol particle size (Dv50) and particle fractions between three actuations were more consistent in l-leucine containing Survanta EEG powders prepared with 20% v/v ethanol in water than the powders prepared with 5% v/v ethanol in water. L-leucine containing powders had a low moisture content (< 3% w/w) with transition temperatures close to that of the commercial surfactant formulation. The Survanta EEG powders retained their surfactant proteins and surface tension reducing ability during and after formulation processing. This study suggests the suitability of the modified LV-DPI device to deliver developed surfactant EEG powder to low birth weight infants using a low dispersion air volume, which can be helpful for the effective treatment of NRDS.