Surfactant Protein B Promotes Cytosolic SiRNA Delivery by Adopting a Virus-like Mechanism of Action

Abstract

RNA therapeutics are poised to revolutionize medicine. To unlock the full potential of RNA drugs, safe and efficient (nano)formulations to deliver them inside target cells are required. Endosomal sequestration of nanocarriers represents a major bottleneck in nucleic acid delivery. Gaining more detailed information on the intracellular behavior of RNA nanocarriers is crucial to rationally develop delivery systems with improved therapeutic efficiency. Surfactant protein B (SP-B) is a key component of pulmonary surfactant (PS), essential for mammalian breathing. In contrast to the general belief that PS should be regarded as a barrier for inhaled nanomedicines, we recently discovered the ability of SP-B to promote gene silencing by siRNA-loaded and lipid-coated nanogels. However, the mechanisms governing this process are poorly understood. The major objective of this work was to obtain mechanistic insights in the SP-B mediated cellular delivery of siRNA. To this end, we combined siRNA knockdown experiments, confocal microscopy and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) imaging in an in vitro non-small cell lung carcinoma model with lipid mixing assays on vesicles that mimic the composition of (intra)cellular membranes. Our work highlights a strong correlation between SP-B mediated fusion with anionic endosomal membranes and cytosolic siRNA delivery, a mode-of-action resembling that of certain viruses and virus-derived cell-penetrating peptides. Building on these gained insights, we optimized the SP-B proteolipid composition, which dramatically improved delivery efficiency. Altogether, our work provides a mechanistic understanding of SP-B induced perturbation of intracellular membranes, offering opportunities to fuel rational design of SP-B inspired RNA nanoformulations for inhalation therapy.

Gold Nanoparticles as Carriers for Functional RNA Nanostructures

“…They are capable of entering cells and may be released in cytoplasm, which is visible in the TEM images. This is interesting in view of recent findings about cytosolic delivery of RNA under a virus-like mechanism . It is also clear that free structural RNA can be processed in cells by Dicer endonuclease, which leads to gene regulation via RNAi.…”

Perfluorocarbon Nanoemulsions Enhance Therapeutic siRNA Delivery in the Treatment of Pulmonary Fibrosis

“…The cellular uptake and endosome escape results showed that most siRNA could escape from the lysosomes and be transported to the cytosol, which suggested that the mechanism may involve the perturbation of intracellular phospholipid membranes. [ 30 ] Further studies need to be performed to gain better understanding.…”

Pulmonary surfactant as a versatile biomaterial to fight COVID-19

“…In addition, the latter enabled to investigate the impact of the lipid components on the delivery-promoting activity of SP-B in more detail. In vitro studies demonstrated the importance of a fluid lipid bilayer to support SP-B, with cholesterol not exceeding physiological levels, while the type of anionic lipid was less critical [ 50 ].…”

“…Unfortunately, current state-of-the-art siRNA nanomedicines have difficulties in overcoming the many extra- and intracellular barriers upon topical administration to the lung, leading to inefficient cytosolic delivery [ 43 , 44 ]. In this regard, it was recently disclosed by Raemdonck and co -workers that exogenous PS has the unexpected property of promoting cytosolic siRNA delivery by polymeric nanomedicines [ [45] , [46] , [47] , [48] , [49] , [50] ]. As such, exogenous PS can be regarded as a multifaceted biomaterial, enabling both direct treatment of COVID-19-related lung dysfunction as well as improved pulmonary delivery of small molecular- and macromolecular drugs.…”