Activity and biophysical inhibition resistance of a novel synthetic lung surfactant containing Super-Mini-B DATK peptide

Abstract

Background/objectives. This study examines the surface activity, resistance to biophysical inhibition, and pulmonary efficacy of a synthetic lung surfactant containing glycerophospholipids combined with Super Mini-B (S-MB) DATK, a novel and stable molecular mimic of lung surfactant protein (SP)-B. The objective of the work is to test whether S-MB DATK synthetic surfactant has favorable biophysical and physiological activity for future use in treating surfactant deficiency or dysfunction in lung disease or injury. Methods. The structure of S-MB DATK peptide was analyzed by homology modeling and by FTIR spectroscopy. The in vitro surface activity and inhibition resistance of synthetic S-MB DATK surfactant was assessed in the presence and absence of albumin, lysophosphatidylcholine (lyso-PC), and free fatty acids (palmitoleic and oleic acid). Adsorption and dynamic surface tension lowering were measured with a stirred subphase dish apparatus and a pulsating bubble surfactometer (20 cycles/min, 50% area compression, 37 °C). In vivo pulmonary activity of S-MB DATK surfactant was measured in ventilated rabbits with surfactant deficiency/dysfunction induced by repeated lung lavages that resulted in arterial PO2 values <100 mmHg. Results. S-MB DATK surfactant had very high surface activity in all assessments. The preparation adsorbed rapidly to surface pressures of 46-48 mN/m at 37 °C (low equilibrium surface tensions of 22-24 mN/m), and reduced surface tension to <1 mN/m under dynamic compression on the pulsating bubble surfactometer. S-MB DATK surfactant showed a significant ability to resist inhibition by serum albumin, C16:0 lyso-PC, and free fatty acids, but surfactant inhibition was mitigated by increasing surfactant concentration. S-MB DATK synthetic surfactant quickly improved arterial oxygenation and lung compliance after intratracheal instillation to ventilated rabbits with severe surfactant deficiency. Conclusions. S-MB DATK is an active mimic of native SP-B. Synthetic surfactants containing S-MB DATK (or related peptides) combined with lipids appear to have significant future potential for treating clinical states of surfactant deficiency or dysfunction, such as neonatal and acute respiratory distress syndromes.

Keywords: Acute lung injury (ALI); Acute respiratory distress syndrome (ARDS); Neonatal respiratory distress syndrome (NRDS); SP-B peptide mimics; Super Mini-B; Super Mini-B DATK; Synthetic lung surfactant; Synthetic surfactant peptides.

Figure 1. Homology-modeled secondary structure of reduced S-MB DATK peptide.

The predicted homology-modeled structure from the amino acid sequence of S-MB DATK peptide was calculated via I-TASSER 3.0 (Zhang, 2008; Roy, Kucukural & Zhang, 2010) (http://zhanglab.ccmb.med.umich.edu/I-TASSER) and rendered using PyMOL (TM). Helical residues are highlighted in green ribbon, disordered and loop-turn segments are represented as green tubes, and the ion-pair that stabilizes the turn is rendered in red (Asp⁻-23) and blue (Lys⁺-26). The characteristic “saposin fold” of the reduced S-MB DATK encompasses the N-terminal α-helix (residues 8–21), the loop-turn (residues 22–29) and C-terminal α-helix (residues 30–37), while the additional N-terminal insertion sequence includes residues 1–7 (see text).

Figure 2. Representative FTIR spectrum of reduced S-MB DATK in surfactant lipids.

FTIR spectra were measured for reduced S-MB DATK in multilayer films with synthetic surfactant lipids (5:3:2 DPPC:POPC:POPG) (Methods). The peptide to lipid mole ratio in the mixed film was 1:10. The representative FTIR spectrum shown is consistent with the primary overall helix-turn-helix motif of homology-modeled S-MB DATK in Fig. 1. Proportions of specific secondary structure determined for reduced S-MB DATK by Fourier self-deconvolution from the Amide I band of the spectra are given in Table 1. See text for details.

Figure 3. Inhibitory effect of bovine serum albumin (BSA) on the adsorption (A) and dynamic surface tension lowering (B) of synthetic surfactant mixtures.

(A) Surface pressure (amount by which surface tension is lowered below the pure subphase value of 70 mN/m at 37 °C) is plotted as a function of time during adsorption. Synthetic surfactant mixtures with or without BSA were injected at time zero beneath the surface of a well-stirred buffered subphase. Final adsorption subphase concentration of synthetic surfactant mixtures was 0.0625 mg/mL (doubled in high concentration surfactant case). Data are mean ± SEM, n = 3–5. All adsorption curves with 50–83% albumin present are significantly different from SSM alone, and curve for 2x SSM concentration with 83% albumin is significantly improved compared to the equivalent low surfactant concentration curve at the same albumin level (P < 0.05 or less by one-way ANOVA). (B) Data are minimum surface tension as a function of time for dispersions of surfactant plus inhibitors in a pulsating bubble surfactometer (20 cycles/min at 37 °C). Surfactant phospholipid concentration was 5.0 mg/mL (doubled in high concentration surfactant case). Data are mean ± SEM, n = 3–5. All dynamic surface tension lowering curves with albumin present at 50–83% are significantly different from SSM alone, and curve for 2x SSM concentration with 83% albumin is significantly improved compared to the equivalent low surfactant concentration curve at the same albumin level (P < 0.05 or less by one-way ANOVA). See Methods and text for details.

Figure 4. Inhibitory effect of Lyso-PC (LPC) on the adsorption (A) and dynamic surface tension lowering (B) of synthetic surfactant mixtures.

(A) Surface pressure (amount by which surface tension is lowered below the pure subphase value of 70 mN/m at 37 °C) is plotted as a function of time during adsorption. Synthetic surfactant mixtures with or without LPC were injected at time zero beneath the surface of a well-stirred buffered subphase. Final adsorption subphase concentration of synthetic surfactant mixtures was 0.0625 mg/mL (doubled in high concentration surfactant case). Data are mean ± SEM, n = 3–5. All adsorption curves with 20–40% LPC present are significantly different from SSM alone, and curve for 2x SSM concentration with 40% LPC is significantly improved compared to the equivalent low surfactant concentration curve at the same LPC level (P < 0.05 or less by one-way ANOVA). (B) Data are minimum surface tension as a function of time for dispersions of surfactant plus inhibitors in a pulsating bubble surfactometer (20 cycles/min at 37 °C). Surfactant phospholipid concentration was 5.0 mg/mL (doubled in high concentration surfactant case). Data are mean ± SEM, n = 3–5. All dynamic surface tension lowering curves with LPC present at 20–40% are significantly different from SSM alone, and curve for 2x SSM concentration with 40% LPC is significantly improved compared to the equivalent low surfactant concentration curve at the same LPC level (P < 0.05 or less by one-way ANOVA). See Methods and text for details.

Figure 5. Inhibitory effect of palmitoleic acid (PA) on the adsorption (A) and dynamic surface tension lowering (B) of synthetic surfactant mixtures.

(A) Surface pressure (amount by which surface tension is lowered below the pure subphase value of 70 mN/m at 37 °C) is plotted as a function of time during adsorption. Synthetic surfactant mixtures with or without PA were injected at time zero beneath the surface of a well-stirred buffered subphase. Final adsorption subphase concentration of synthetic surfactant mixtures was 0.0625 mg/mL (doubled in high concentration surfactant case). Data are mean ± SEM, n = 3–5. All adsorption curves with 10–35% PA present are significantly different from SSM alone, and curve for 2x SSM concentration with 35% PA is significantly improved compared to the equivalent low surfactant concentration curve at the same PA level (P < 0.05 or less by one-way ANOVA). (B) Data are minimum surface tension as a function of time for dispersions of surfactant plus inhibitors in a pulsating bubble surfactometer (20 cycles/min at 37 °C). Surfactant phospholipid concentration was 5.0 mg/mL (doubled in high concentration surfactant case). Data are mean ± SEM, n = 3–5. All dynamic surface tension lowering curves with PA present at 20–35% are significantly different from SSM alone, and curve for 2x SSM concentration with 35% PA is significantly improved compared to the equivalent low surfactant concentration curve at the same PA level (P < 0.05 or less by one-way ANOVA). See Methods and text for details.

Figure 6. Inhibitory effect of oleic acid (OA) on the adsorption (A) and dynamic surface tension lowering (B) of synthetic surfactant mixtures.

(A) Surface pressure (amount by which surface tension is lowered below the pure subphase value of 70 mN/m at 37 °C) is plotted as a function of time during adsorption. Synthetic surfactant mixtures with or without OA were injected at time zero beneath the surface of a well-stirred buffered subphase. Final adsorption subphase concentration of synthetic surfactant mixtures was 0.0625 mg/mL (doubled in high concentration surfactant case). Data are mean ± SEM, n = 3–5. All adsorption curves with 10–35% OA present are significantly different from SSM alone, and curve for 2x SSM concentration with 35% OA is significantly improved compared to the equivalent low surfactant concentration curve at the same OA level (P < 0.05 or less by one-way ANOVA). (B) Data are minimum surface tension as a function of time for dispersions of surfactant plus inhibitors in a pulsating bubble surfactometer (20 cycles/min at 37 °C). Surfactant phospholipid concentration was 5.0 mg/mL (doubled in high concentration surfactant case). Data are mean ± SEM, n = 3–5. All dynamic surface tension lowering curves with OA present at 20–35% are significantly different from SSM alone, and curve for 2x SSM concentration with 35% OA is significantly improved compared to the equivalent low surfactant concentration curve at the same OA level (P < 0.05 or less by one-way ANOVA). See Methods and text for details.

Figure 7. Effect of S-MB DATK synthetic surfactant on oxygenation and lung compliance in rats with surfactant-deficient ARDS.

Synthetic surfactant or lipid-only control was instilled intratracheally at time zero into ventilated rats following in vivo lavage to induce clinical oxygenation criteria for ARDS. Arterial oxygenation (A) and dynamic lung compliance (B) are shown as a function of time following tracheal instillation of S-MB DATH surfactant or lipid-only control. Data are mean ± SEM for n = 3–5. Values for both oxygenation and dynamic compliance at all times greater than zero are significantly improved for surfactant-treated animals compared to controls (P < 0.001 by one-way ANOVA). See Methods and text for details.