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. 2020 Apr 9;11:266. doi: 10.3389/fphys.2020.00266

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Copyright © 2020 Das, Curstedt, Agarwal, Prahaladan, Ramirez, Bhandari, Syed, Salomone, Casiraghi, Pelizzi and Bhandari.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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CS can be used as a successful delivery vehicle, in vitro and in vivo. Adult epithelial MLE12 cells (A,B) and fetal type II alveolar epithelial cells (C–E) were transfected with CHOP siRNA, Ang2 siRNA, and miR34a Inh, respectively, following which the expression of CHOP (A), Ang2 (B), Sirt 1 (D), and Ang1 (E) (Sirt1 and Ang2 are targets of miR34a) were quantified by Western Blotting (A,B,D,E) with respective densitometric analysis (extreme right) and qPCR (C). Lipofectamine (LF) was used as a positive control. The cells that were treated with Curosurf^® (CS), showed equal efficiency of transfection as that of LF, as is evident from decreased expression of CHOP and Ang2. miR34a is increased during hyperoxia, but after treatment with CS, the expression of miR34a is suppressed which leads to an increase in Sirt1 (C,D) or Ang1 (E) expression. Neonatal mouse pups were intranasally delivered with CS + CHOP siRNA in each nostril on PN2 and PN4 of hyperoxia treatment. Western blotting shows decreased expression of CHOP (F), along with densitometric quantification (on the right). Ang2 siRNA was administered intratracheally along with CS in preterm rabbit kits. Western blotting shows decreased expression of Ang2 (G), along with densitometric quantification (on the right). Q-PCR showing relative expression of Ang1 after treatment with miR34a inh (H). On treatment with miR34a Inh alone, we saw a significant decrease in the expression of Ang1 (target of miR34a) in the ventilated group as compared to non-ventilated controls. However, adjuvant therapy with a combination of CS + miR34a Inh restored the level of Ang1 above the basal level, which was otherwise compromised after ventilation (H). β-actin is the loading control for all the western blotting while GAPDH was used as a normalizing gene for qPCR. ^∗p ≤ 0.05; ^∗∗p ≤ 0.001; ^∗∗∗p ≤ 0.0001; (N = 2–4). CS: Curosurf^® (surfactant); CHOP: CCAAT enhancer-binding protein, i.e., C/EBP homologous protein, also known as growth arrest and DNA damage-inducible gene 153/GADD153 or Ddit3; si: silencing; Ang2: Angiopoietin 2; miR: microRNA; Inh: inhibitor; Sirt: Sirtuin; LF: Lipofectamine; RA: Room Air. Hyp: Hyperoxia.

Overall, the data convincingly prove that CS can be used as an effective vehicle for administration of test agents; CS is stable when used in combination with the small molecule inhibitors, and the latter are capable of successfully suppressing the expression of the proteins of interest, following injury.