Polymer Lung Surfactants

Abstract

Animal-derived lung surfactants annually save 40 000 infants with neonatal respiratory distress syndrome (NRDS) in the United States. Lung surfactants have further potential for treating about 190 000 adult patients with acute respiratory distress syndrome (ARDS) each year. To this end, the properties of current therapeutics need to be modified. Although the limitations of current therapeutics have been recognized since the 1990s, there has been little improvement. To address this gap, our laboratory has been exploring a radically different approach in which, instead of lipids, proteins, or peptides, synthetic polymers are used as the active ingredient. This endeavor has led to an identification of a promising polymer-based lung surfactant candidate, poly(styrene-b-ethylene glycol) (PS-PEG) polymer nanomicelles. PS-PEG micelles produce extremely low surface tension under high compression because PS-PEG micelles have a strong affinity to the air-water interface. NMR measurements support that PS-PEG micelles are less hydrated than ordinary polymer micelles. Studies using mouse models of acid aspiration confirm that PS-PEG lung surfactant is safe and efficacious.

Keywords: acute respiratory distress syndrome; block copolymer micelle; lung surfactant; neonatal respiratory distress syndrome; pulmonary surfactant.

Figure 1.

(a) Contour plot of the Laplace pressure (ΔP) of a spherical alveolus calculated as a function of surface tension (γ) and radius (R). (b) Surface pressure vs relative area isotherms of Infasurf and Survanta obtained during repeated continuous compression−expansion cycles. The data displayed represent the last 10 compression−expansion cycles of total 50 continuous cycles. The subphase solution used contained 150 mM NaCl, 2 mM CaCl₂, and 0.2 mM NaHCO₃ (pH 7.0−7.4, 25 °C). The monolayer was compressed and expanded at a rate of 50 mm/min; one compression−expansion cycle took 7.18 min. At the “100% relative area”, 10 mg of Infasurf and Survanta was spread on water in a Langmuir trough with a 780 cm² surface area and a 1.4 L subphase volume; “100% relative areas” corresponded to 0.972 square angstroms per molecule for both Infasurf and Survanta.

Figure 2.

Molecular characteristics of polymer LS candidate materials investigated in this study. The dagger indicates a ratio of lactic acid to glycolic acid of 47:53 by mole.

Figure 3.

Constant-compression surface pressure−area isotherms of chloroform-spread and water-spread PLGA(4030)−PEG(5000) monolayers on the surface of Milli-Q-purified water (18 MΩ·cm resistivity) at 25 °C. Surface pressure was measured during compression at a rate of 3 mm/min. In the water spread experiments, the mean hydrodynamic diameter of the PLGA−PEG micelles was 75.1 ± 3 nm (measured by DLS). For each solvent group, measurements were performed in four different area ranges because of a small (finite) compression distance of the Langmuir trough to construct an isotherm curve over a full range of area per monomer values; the curves from right to left were obtained, respectively, by initially spreading 4, 16, 64, and 256 μL of 5 mg/mL PLGA(4030)− PEG(5000) solution (in chloroform or water) on water in a Langmuir trough with a 780 cm² surface area and a 1.4 L subphase volume. Note that the water-spread isotherms obtained in different area ranges do not overlap with each other; this indicates that the water spreading process involves significant loss of polymer to the aqueous subphase.

Figure 4.

(a) 1D ¹H NMR Spectra for PS(5610)−PEG(5000) and PLGA(4030)−PEG(5000) in D₂O at 25 °C. (b) 2D ¹H−¹³C heteronuclear multiple bond correlation (HMBC) NMR spectra for PS(5610)−PEG(5000) in D₂O at 25 °C. (c) Longitudinal relaxation decay curves for PEG protons at 25 °C. Solid curves are fits to a mono-exponential decay function (G(t) = exp(−t/T₁)). (d) Transverse relaxation decay curves for PEG protons at 25 °C. As demonstrated in panel a, spectra from PS(5610)−PEG(5000) and PS(13832)−PEG(5000) micelles exhibited two PEG peaks (a sharp peak at ~3.61 ppm and a broad peak at ~3.56 ppm). The decay curves of these peaks were separately fitted with a mono-exponential decay function. Open symbols represent broad PEG peaks, and filled symbols represent sharp PEG peaks. Spectra from PEG(5000) and PLGA(4030)−PEG(5000) exhibited single PEG peaks (also demonstrated in panel a). The decay curve of PEG(5000) was fit with the monoexponential function, and that of PLGA(4030)−PEG(5000) was fit with a bi-exponential function (G(t) = a × exp(−t/T₂₁) + (1 − a) × exp(−t/ T₂₂)). (e) Best-fit T₁ and T₂ values. For all regressions the coefficient of determination (R²) was greater than 0.99. Daggers indicate fractions of PEG segments contributing to the sharp and broad PEG peaks out of the total number of PEG segments available in the system, estimated based on pyridine internal reference. Double daggers indicate the coefficient of the first term of the biexponential decay function, a. Also shown for comparison is a predicted T₂ value for PEG(5000) melt at 100 °C (see the text for details).

Figure 5.

(a) TEM images of PS−PEG micelles formed in bulk water solutions. The dried micelle samples were negatively stained with uranyl acetate. Summarized in the table at the bottom are diameters of PS−PEG micelles as determined by TEM or DLS. Daggers indicate that they exclude elongated micelles. Molecular-packing properties of PS−PEG micelles (i.e., the micelle aggregation number, the interfacial area per chain, the dimensionless PEG grafting density, and the degree of PS chain stretching) estimated from the TEM data are summarized in Table S1. Constant-compression surface pressure−area isotherms of (b) chloroform-spread and (c) water-spread monolayers of 4 different PS−PEG materials at 25 °C. Milli-Q-purified water (18 MΩ·cm resistivity) was used as the subphase. In each panel, the isotherm curves from right to left were obtained, respectively, by initially spreading 4, 16, and 64 μL of 5 mg/mL PS−PEG solution in (b) chloroform or (c) water on the 780 cm² subphase surface. The subphase volume was 1.4 L. The monolayer compression rate was 3 mm/min.

Figure 6.

Surface pressure−area isotherms for (a) Infasurf (10 mg; 7.14 micrograms per milliliter of subphase) with and without the addition of BSA (30 mg; 21.4 micrograms per milliliter of subphase), and (b) water-spread PS(4418)−PEG(5000) (10 mg; 7.14 micrograms per milliliter of subphase) with and without the addition of BSA (30 mg; 21.4 micrograms per milliliter of subphase) during repeated compression−expansion cycles. A typical experiment was performed as follows: (1) Infasurf or PS(4418)−PEG(5000) was water-spread on water; (2) 30 min were waited for equilibration; (3) BSA was injected into the subphase without perturbing the Infasurf or PS(4418)−PEG(5000) interface; (4) compression−expansion cycles were initiated following a 10 min waiting period. The data displayed represent the last 10 compression−expansion cycles of a total 50 continuous cycles performed after spreading Infasurf or PS−PEG micelles. The subphase solution used contained 150 mM NaCl, 2 mM CaCl₂, and 0.2 mM NaHCO₃ (pH 7.0−7.4, 25 °C). The monolayer was compressed and expanded at a rate of 50 mm/min; one compression−expansion cycle took 7.18 min. At the “100% relative area”, 10 mg of Infasurf or PS(4810)−PEG(5000) was spread on water in a Langmuir trough with 780 cm² surface area and 1.4 L subphase volume; “100% relative areas” corresponded to 0.972 and 12.2 square angstroms per molecule for Infasurf and PS(4418)− PEG(5000), respectively.

Figure 7.

(a) Mouse body weights recorded as a function of time following intratracheal instillation of different doses of PS(4418)−PEG(5000) micelles at day 0 (N = 1). (b) A representative H&E-stained histological section of the lungs taken at 7 days after intratracheal injection of 240 mg PS(4418)−PEG(5000) micelles per kilogram of body weight in mice (N = 1). (c) Levels of albumin and 4 different cytokines in BAL fluids collected from mice at 7 days after intratracheal injection of 240 mg/kg PS(4418)−PEG(5000) micelles. BAL fluids from untreated mice were used as control. Measurements were performed in quadruplicate (N = 4). Error bars represent standard deviations. The assessment of statistical significance is summarized in Table S2.

Figure 8.

(a) Diagrammatic description of the procedures used in LS efficacy tests using acid aspiration lung injury ARDS mouse models. (b) Closed-chest pressure−volume (PV) curves of acid-injured mouse lungs following intratracheal instillation of PS(4418)−PEG(5000) micelles at four different polymer doses. (c) Closed-chest PV curves of acid-injured mouse lungs following intratracheal instillation of PS(4418)−PEG(5000) micelles (0.600 mg/mL × 4.0 mL/kg), PLGA(4030)−PEG(5000) micelles (0.714 mg/mL × 4.0 mL/kg) or PEG(5000) homopolymers (0.3185 mg/mL × 4.0 mL/kg). (d) Closed-chest PV curves of acid-injured mouse lungs following intratracheal instillation of PS(4418)−PEG(5000) (0.600 mg/mL × 4.0 mL/kg), Infasurf (35.0 mg/mL × 3.0 mL/kg), or saline (4.0 mL/kg). Also included is the curve from noninjured mice (“No Injury”). Error bars represent standard deviations (N values shown in legends). The assessment of statistical significance is summarized in Table S3.