Yonsei Med J.  2017 Jul;58(4):823-828. 10.3349/ymj.2017.58.4.823.

A Combination of Short and Simple Surfactant Protein B and C Analogues as a New Synthetic Surfactant: In Vitro and Animal Experiments

• Choi YS
• Chung SH
• Bae CW

• KMID: 2419090
• DOI: http://doi.org/10.3349/ymj.2017.58.4.823

Abstract

PURPOSE
Pulmonary surfactants for preterm infants contain mostly animal-derived surfactant proteins (SPs), which are essential for lowering surface tension. We prepared artificial pulmonary surfactants using synthetic human SP analogs and performed in vitro and in vivo experiments.
MATERIALS AND METHODS
We synthesized peptide analogues that resemble human SP-B (RMLPQLVCRLVLRCSMD) and SP-C (CPVHLKRLLLLLLLLLLLLLLLL). Dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol (PG), and palmitic acid (PA) were added and mixed in lyophilized to render powdered surfactant. Synsurf-1 was composed of DPPC:PG:PA:SP-B (75:25:10:3, w/w); Synsurf-2 was composed of DPPC:PG:PA:SP-C (75:25:10:3, w/w); and Synsurf-3 was composed of DPPC:PG:PA:SP-B:SP-C (75:25:10:3:3, w/w). We performed in vitro study to compare the physical characteristics using pulsating bubble surfactometer and modified Wilhelmy balance test. Surface spreading and adsorption test of the surfactant preparations were measured. In vivo test was performed using term and preterm rabbit pups. Pressure-volume curves were generated during the deflation phase. Histologic findings were examined.
RESULTS
Pulsating bubble surfactometer readings revealed following minimum and maximum surface tension (mN/m) at 5 minutes: Surfacten® (5.5±0.4, 32.8±1.6), Synsurf-1 (16.7±0.6, 28.7±1.5), Synsurf-2 (7.9±1.0, 33.1±1.6), and Synsurf-3 (7.1±0.8, 34.5±1.0). Surface spreading rates were as follows: Surfacten® (27 mN/m), Synsurf-1 (43 mN/m), Synsurf-2 (27 mN/m), and Synsurf-3 (27 mN/m). Surface adsorption rate results were as follows: Surfacten® (28 mN/m), Synsurf-1 (35 mN/m), Synsurf-2 (29 mN/m), and Synsurf-3 (27 mN/m). The deflation curves were best for Synsurf-3; those for Synsurf-2 were better than those for Surfacten®. Synsurf-1 was the worst surfactant preparation. Microscopic examination showed the largest aerated area of the alveoli in the Synsurf-3 group, followed by Synsurf-1 and Surfacten®; Synsurf-2 was the smallest.
CONCLUSION
Synsurf-3 containing both SP-B and SP-C synthetic analogs showed comparable and better efficacy than commercially used Surfacten® in lowering surface tension, pressure-volume curves, and tissue aerated area of the alveoli.

Keyword

Pulmonary surfactants; pulmonary surfactant-associated proteins; respiratory distress syndrome; newborn; synthetic surfactant

MeSH Terms

Animals
Animals, Newborn
Birth Weight
Emulsions
Humans
Lung/drug effects
Pressure
Pulmonary Surfactant-Associated Protein B/*pharmacology
Pulmonary Surfactant-Associated Protein C/*pharmacology
Rabbits
Surface Properties
Emulsions
Pulmonary Surfactant-Associated Protein B
Pulmonary Surfactant-Associated Protein C

Figure

• 

  Fig. 1 Diagrams generated by modified Wilhelmy balance test. (A) Surface spreading rate test. Synsurf-1 was slow. Synsurf-2 and -3 and Surfacten® were similarly fast in regards to surface spreading rate test. (B) Surface adsorption was best for Synsurf-3, followed by Surfacten®, Synsurf-2, and Synsurf-1 in that order. (C) In surface tension area diagram, all four preparations showed hysteresis curves. Among these, Surfacten® showed the lowest minimum-ST, which was the best. Synsurf-2 and -3 showed similarly in the middle and Synsurf-1 was the worst. ST, surface tension.

• 

  Fig. 2 Pressure volume curves. (A) shows the difference between term (n=9) and preterm controls (n=13, p<0.001). (B) shows deflation curves for the four different surfactant preparations. Synsurf-2 (n=7, p=0.0068), Synsurf-3 (n=6, p=0.0031), and Surfacten® (n=18, p<0.001) showed higher pressure volume curves in the term group than the preterm group; Synsurf-1 (n=6, p=0.4811) showed no difference. Mann Whitney U test was used to compare volumes at 15 cm H2O pressure.

• 

  Fig. 3 Histologic differences among six groups (hematoxylin and eosin stain, ×100). Four surfactant preparations-treated lung tissues demonstrated better aerated features than preterm controls. For statistical analysis, alveolar areas were compared which and revealed significant differences, as shown in Fig. 4.

• 

  Fig. 4 Aerated area percent comparison. Tissues treated with Sufacten® (n=34), Synsurf-1 (n=24), Synsurf-2 (n=31), and Synsurf-3 (n=30), demonstrated significantly higher aerated area rate than controls (n=49, p<0.001). Synsurf-3 was significantly higher than Surfactant® (p=0.020), while Synsurf-1 and Synsurf-2 were not different from Surfactant ’ (p=0.729 and 0.722). Unpaired t-test was used.

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