pharmaceutics Article Nanocarrier Lipid Composition Modulates the Impact of Pulmonary Surfactant Protein B (SP-B) on Cellular Delivery of siRNA Roberta Guagliardo 1, Pieterjan Merckx 1 , Agata Zamborlin 1, Lynn De Backer 1, Mercedes Echaide 2, Jesus Pérez-Gil 2, Stefaan C. De Smedt 1,* and Koen Raemdonck 1,* 1 Ghent Research Group on Nanomedicines, Laboratory of General Biochemistry and Physical Pharmacy, Department of Pharmaceutics, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium 2 Departamento de Bioquímica y Biología Molecular, Facultad de Biologia, and Research Institute Hospital 12 de Octubre, Universidad Complutense, José Antonio Novais 12, 28040 Madrid, Spain * Correspondence: [email protected] (S.C.D.S.); [email protected] (K.R.); Received: 30 June 2019; Accepted: 13 August 2019; Published: 23 August 2019 Abstract: Two decades since the discovery of the RNA interference (RNAi) pathway, we are now witnessing the approval of the first RNAi-based treatments with small interfering RNA (siRNA) drugs. Nevertheless, the widespread use of siRNA is limited by various extra- and intracellular barriers, requiring its encapsulation in a suitable (nanosized) delivery system. On the intracellular level, the endosomal membrane is a major barrier following endocytosis of siRNA-loaded nanoparticles in target cells and innovative materials to promote cytosolic siRNA delivery are highly sought after. We previously identified the endogenous lung surfactant protein B (SP-B) as siRNA delivery enhancer when reconstituted in (proteo) lipid-coated nanogels. It is known that the surface-active function of SP-B in the lung is influenced by the lipid composition of the lung surfactant. Here, we investigated the role of the lipid component on the siRNA delivery-promoting activity of SP-B proteolipid-coated nanogels in more detail. Our results clearly indicate that SP-B prefers fluid membranes with cholesterol not exceeding physiological levels. In addition, SP-B retains its activity in the presence of different classes of anionic lipids. In contrast, comparable fractions of SP-B did not promote the siRNA delivery potential of DOTAP:DOPE cationic liposomes. Finally, we demonstrate that the beneficial effect of lung surfactant on siRNA delivery is not limited to lung-related cell types, providing broader therapeutic opportunities in other tissues as well. Keywords: siRNA delivery; nanoparticles; pulmonary surfactant 1. Introduction Over the last two decades, research in the field of RNAi therapeutics has gained attention as it allows to address diseases at the transcriptome level [1]. Once they reached the cytosol, small interfering RNAs (siRNAs) activate the RNAi machinery, leading to post-transcriptional gene silencing through sequence-specific degradation of mRNA [2,3]. High target specificity and versatility of this emerging class of therapeutics represent some of the main advantages compared to conventional small molecule drugs and monoclonal antibodies, providing a wide range of biomedical uses [1,3]. However, their application in the clinic is limited by many extra- and intracellular delivery barriers. Most importantly, negatively charged hydrophilic macromolecules like siRNAs cannot cross biological membranes, making cellular delivery challenging [1,4]. Viral vectors are often applied carriers to guide cellular delivery of nucleic acids. However, labor-intensive large-scale production and safety issues remain important drawbacks, hence encouraging Pharmaceutics 2019, 11, 431; doi:10.3390/pharmaceutics11090431 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2019, 11, x 2 of 17 Pharmaceutics 2019, 11, 431 2 of 16 Viral vectors are often applied carriers to guide cellular delivery of nucleic acids. However, labor-intensive large-scale production and safety issues remain important drawbacks, hence encouragingresearch for non-viralresearch alternativesfor non-viral [4– 6alternatives]. Encapsulation [4–6]. of Encapsulation siRNA into synthetic of siRNA nanoparticles into synthetic (NPs) nanoparticlesallows its internalization (NPs) allows by its cells internalization through endocytosis by cells through followed endocytosis by release followed of the encapsulated by release of RNA the encapsulatedinto the cytosol RNA (i.e., into endosomal the cytosol escape). (i.e., Amongendosomal the vastescape). number Among of NPs the under vast number investigation, of NPs cationic under investigation,lipid nanoparticles cationic (LNPs) lipid currently nanoparticles are the (LNPs) preferred currently material are for the RNA preferred delivery material [7]. To date, for manyRNA deliverycationic lipid[7]. To materials date, many have cationic been synthetized lipid material fors LNPhave productionbeen synthetized [8–13]. for However, LNP production the endosomal [8–13]. However,escape effi ciencythe endosomal often remains escape poor efficiency [4,14–16 often]. Moreover, remains poor concerns [4,14–16]. remain Moreover, regarding concerns their safety remain and regardingimmunogenicity their safety [12,17 and]. As immunoge such, to expeditenicity [12,17]. clinical As translationsuch, to expedite of this highlyclinical promisingtranslation classof this of highlytherapeutics, promising lipid-based class of siRNA therapeutics, formulations lipid-based are needed siRNA to mergeformulations efficient are cellular needed delivery to merge with efficientacceptable cellular toxicity. delivery with acceptable toxicity. As synthetic polymer-and lipid-based lipid-based NPs NPs often often fail fail to to combine combine biocompatibility biocompatibility and and efficacy, efficacy, there isis a a growing growing interest interest in using in using bio-inspired bio-inspired materials materials [18]. We [18]. recently We reportedrecently on reported a bio-inspired on a bio-inspirednanocomposite, nanocomposite, composed of composed a siRNA-loaded of a siRN polymericA-loaded matrix polymeric core surrounded matrix core by surrounded a shell of clinical by a α ® shellpulmonary of clinical surfactant, pulmonary i.e., poractantsurfactant, i.e.,(Curosurf poractant) (Figure α (Curosurf1)[19].®) (Figure 1) [19]. Figure 1. Visual representation of thethe core-shellcore-shell surfactant-coatedsurfactant-coated nanogelnanogel structure.structure. SiRNA-loaded dextran nanogelsnanogels (siNGs) (siNGs) were we coatedre coated with Curosurfwith Curosurf® (poractant® (poractantα; porcine α; derivedporcine clinical derived pulmonary clinical pulmonarysurfactant (PS)) surfactant or with (PS)) a PS-inspired or with a lipidPS-inspired coating lipid containing coating the containing surfactant the protein surfactant B (SP-B) protein andan B (SP-B)anionic and lipid an mixture. anionic lipid PC = mixture.phosphatidylcholine, PC = phosphatidylcholine, PG = phosphatidylglycerol. PG = phosphatidylglycerol. Pulmonary surfactant (PS) is a surface-active material that is produced and secreted into the alveolar space by specialized alveolar type II epithelialepithelial cells. PS PS covers the entire alveolar surface and itsits mainmain physiological physiological role role is tois maintainto maintain low surfacelow surface tension tension upon expirationupon expiration to prevent to alveolarprevent alveolarcollapse [20collapse]. Natural [20]. human Natural PS hashuman a complex PS has composition a complex of lipidscomposition (~90 wt%) of andlipids proteins (~90 wt%) (~10 wt%). and proteinsThe lipid (~10 fraction wt%). mainly The containslipid fraction zwitterionic mainly phosphatidylcholine contains zwitterionic (PC) phosphatidylcholine (~60–70 wt%) as well (PC) as (~60–70anionic phosphatidylglycerolwt%) as well as anionic (PG) phosphatidylglycerol (~10 wt%) species and(PG) neutral (~10 wt%) lipids, species of which and cholesterolneutral lipids, is the of whichmost abundant cholesterol (~8–10 is the wt%). most The abundant protein fraction (~8–10 consistswt%). The of two protein major fraction classes of consists specialized of two surfactant major classesproteins of (SPs), specialized i.e., the largersurfactant and hydrophilicproteins (SPs), SP-A i.e., and the SP-D, larger as welland ashydrophilic the smaller SP-A hydrophobic and SP-D, SP-B as welland SP-Cas the [21 smaller,22]. PS hydrophobic has been extensively SP-B and studied SP-C mainly[21,22]. because PS has of been its functional extensively role studied in mammalian mainly becausebreathing of [ 22its, 23functional]. In the contextrole in ofmammalian inhalation breathing therapy with [22,23]. nanomedicines, In the context PS of is primarilyinhalation regarded therapy withas one nanomedicines, of the extracellular PS is primarily barriers in regarded the deep as lung one thatof the needs extracellular to be overcome barriers toin gainthe deep access lung to thatunderlying needs to target be overcome cells upon to gain inhalation access therapyto underl [21ying]. Itstarget current cells therapeuticupon inhalation use istherapy limited [21]. to theIts currenttreatment therapeutic of respiratory use distressis limited syndrome to the treatmen in prematuret of infants,respiratory where distress modified syndrome PS from in animal premature origin infants,(e.g., Curosurf where® modified) is approved PS from for so-calledanimal origin surfactant (e.g., Curosurf replacement®) is therapyapproved [23 for]. so-called surfactant replacementUnexpectedly, therapy we [23]. observed that the PS outer layer in the above mentioned nanocomposites significantlyUnexpectedly, enhanced we intracellularobserved that siRNA the PS delivery outer