Lipid@polymer hybrid nanoparticles for efficient siRNA transport across the lung barriers: Mechanistic insights into the role of Ionizable lipids

Abstract

Building on growing evidence that ionizable lipids improve RNA delivery, in this work, we developed ionizable lipid/poly(lactic-co-glycolic acid) hybrid nanoparticles (iLipid@PLGA hNPs), consisting in a PLGA core modified at surface with either 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA), 1,2-dioleoyl-3-trimethylammonium-propane (DODAP), or the branched-tail proprietary amino lipid ALC-0315. iLipid@PLGA hNPs were engineered to meet key requirements for inhalation. Thorough physicochemical characterization revealed how the choice of ionizable lipid influences pH responsiveness, surface composition, and architecture of iLipid@PLGA hNPs. In vitro studies demonstrated effective siRNA encapsulation, adjustable release kinetics, and poor interactions with mucus components, as assessed by combined UV–Vis, Dynamic Light Scattering, and Small Angle X-ray Scattering analyses. Confocal microscopy analysis of A549 cells transfected with iLipid@PLGA hNPs showed reduced colocalization of AlexaFluor647-labeled siRNA with lysosomes over time, suggesting enhanced endosomal escape in the case of DODMA@PLGA hNPs. Functional validation using GAPDH-targeting siRNA (siGAPDH) confirmed cellular uptake and gene silencing in normal human bronchial epithelial (NHBEs) cells, confirming the superior performance of DODMA@PLGA hNPs. Finally, representative fluorescently labeled DODMA@PLGA hNPs successfully diffused across a 3D air–liquid interface (ALI) cell model, simulating the human bronchial epithelial barrier. These findings highlight the successful integration of ionizable lipids into polymeric nanoparticles, establishing iLipid@PLGA hNPs as versatile and efficient carriers for siRNA therapeutics. This breakthrough supports their continued development in respiratory nanomedicine and in the local treatment of lung diseases.

Introduction

A variety of non-viral vectors, either lipid- or polymer-based, are being developed for efficient delivery of RNA therapeutics to the lungs [1]. Among them, polymer nanoparticles, particularly those made from biodegradable materials such as poly(lactic-co-glycolic acid) (PLGA), offer several advantages, including protection and controlled release of the RNA cargo, as well as evasion of natural lung clearance mechanisms, both in vivo and in vitro. Nevertheless, the behavior of inhalable nanoparticles at the interface with the biological environment is critical for overcoming lung barriers. Therefore, surface engineering plays a vital role in the design of effective PLGA nanoparticles for inhalation.

Recent advancements have led to the development of muco-inert, cell-penetrating core-shell hybrid nanoparticles (hNPs) by surface-engineering PLGA with lipids, thereby integrating the advantages of polymeric and lipid nanoplatforms. Various lipids and fabrication methods have been explored to fine-tune the technological features of lipid@PLGA hNPs and enhance their in vitro/in vivo performance [2], [3], [4]. With respect to siRNA delivery to the lungs, our research has focused on dipalmitoyl phosphatidylcholine (DPPC), the primary component of lung surfactant, and 2-distearoyl-sn-glycero-3-phosphoethanolamine–poly(ethylene glycol) 2000 (DSPE-PEG), a lipid that could enhance mucus penetration through PEGylation of lipid@PLGA hNPs [5], [6]. Nonetheless, to go beyond mucus penetration and to improve the transfection efficiency of lipid@PLGA hNPs, a systematic investigation of different lipids is a pressing need.

Among lipid components, ionizable lipids have emerged as uniquely powerful carriers for enhancing RNA delivery efficiency in vitro/in vivo, and even in humans [7]. These lipids, which undergo protonation and deprotonation in response to environmental pH changes, significantly enhance the ability of lipid nanoparticles (LNPs) to interact with and penetrate cell membranes, particularly in acidic endosomal compartments [8]. Ionizable lipids destabilize the endosomal membrane under acidic conditions, facilitating the release of RNA into the cytoplasm and preventing endo-lysosomal degradation [9]. This distinct feature makes ionizable lipids very intriguing materials for tackling cell barriers to inhaled RNAs.

While the role of ionizable lipids in LNPs has been the subject of extensive studies, their potential function within polymeric nanoparticles, such as lipid@PLGA hNPs, remains largely unexplored. A recent study on intramuscular mRNA vaccination suggests that mRNA lipid/polymer nanoparticles engineered with C12–200, a model ionizable lipid, outperform conventional mRNA LNPs, eliciting stronger antigen-specific IgG responses and more effectively reducing viral load in the nasal cavity [10].

In this work, we exploit the potential role of ionizable lipids in the design of mucus- and cell-penetrating ionizable lipid@PLGA hNPs (iLipid@PLGA hNPs) for pulmonary delivery of siRNA. The hNPs have been engineered with three model lipids selected based on their structural properties. Since it is well-known that the saturation of the ionizable lipids can strongly affect the fluidity and the delivery efficiency of the ionizable lipids, in the unsaturated family 1,2-dioleyloxy-3-dimethylaminopropane (DODMA) and 1,2-dioleoyl-3-dimethylammonium-propane (DODAP) were selected. Among them, DODAP is particularly similar in structure to the linoleyl tail of the ionizable lipid Dlin-MC3-DMA (MC3), which is the key lipid used in the first FDA-approved siRNA therapy, patisiran (Onpattro®, Alnylam Pharmaceuticals, Inc.), showing robust hepatic gene silencing. The third lipid instead is 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminium (ALC-0315), a multi-tail ionizable lipid, structurally distinguished from two-tail ionizable lipids ones by having three or more tails and are expected to produce a more cone-shaped structure with enhanced endosome-disrupting ability due to increased cross-section of the tail region and that has shown great success in the Pfizer-BioNTech mRNA-based COVID-19 vaccines [11]. All the iLipid@PLGA hNPs produced were thoroughly characterized for size, surface charge, siRNA entrapment efficiency, and in vitro release rate. Small-angle X-ray scattering (SAXS) analysis at physiologically relevant pHs provided structural insights into the developed iLipid@PLGA hNPs. Further insight into the ability of the iLipid@PLGA hNPs to escape endo-lysosomal were achieved through in vitro trafficking studies in A549 adenocarcinoma lung epithelial cells. The behavior of iLipid@PLGA hNPs in physiologically relevant conditions was predicted through a combination of in vitro assays, including mucin-particle interaction, diffusion studies and SAXS analysis in an artificial mucus model. The diffusion of fluorescently labeled hNPs was tested in a 3D airway epithelial model, revealing insights into transport, permeability, and retention relevant to in vivo performance. Finally, the functional delivery of siRNA from the developed iLipid@PLGA hNPs was confirmed through in vitro gene silencing studies in normal human bronchial epithelial (NHBE) cells using a reporter siRNA against GAPDH.

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Materials

Resomer RG 502H (uncapped PLGA 50:50, inherent viscosity 0.16 0.24 dL/g) was purchased from Evonik Industries AG (Germany). 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine–poly (ethylene glycol)2000 (DSPE-PEG) were kindly gifted from Lipoid GmbH (Switzerland). 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA), 1,2-dioleoyl-3-trimethylammonium-propane (DODAP), and ((4-hydroxybutyl)azanediyl)bis(hexano-6,1-diyl)bis(2-hexyldecanoate) (ALC0315) were purchased from Avanti® Polar Lipids (USA). The Silencer® GAPDH siRNA, Silencer® Negative control siRNA (13,000 g/mol, 21 bp), the Quant-IT™ RiboGreen® kit and UltraPure™ distilled water DNase/RNase free (UPWDNase/RNase free) were purchased from Life Technologies (Carlsbad, CA, USA). Citric acid, sodium citrate, sodium hydroxide, Egg Yolk Emulsion, deoxyribonucleic acid (DNA), diethylenetriaminepentaacetic acid (DTPA), RPMI 1640 amino acid solution, bovine mucin type II, dichloromethane (DCM), and 96% (v/v) ethanol were purchased from Merck KGaA (Darmstadt, Germany). The permeable Transwell® supports were obtained from Corning (NY, USA). Fluorescent labeled poly(lactide-co-glycolide)-rhodamine B (PolySciTech™ PLGA-Rhod, LG 50:50, Mn 10,000–30,000 Da) was purchased from Microtech Srl (Italy). All chemicals and reagents were of analytical grade and used as received without further purification. Ultrapure water (UPW, Type I) was obtained using a Purelab® Option-Q system (Elga Labwater, Italy) and used throughout the study.

Susy Brusco, Ersilia Villano, Teresa Silvestri, Amar J. Azad, Muge Molbay, Ivana d’Angelo, Agnese Miro, Paola Brocca, Olivia M. Merkel, Sarah Hedtrich, Gabriella Costabile, Francesca Ungaro, Lipid@polymer hybrid nanoparticles for efficient siRNA transport across the lung barriers: Mechanistic insights into the role of Ionizable lipids, Journal of Colloid and Interface Science, 2026, 140683, ISSN 0021-9797, https://doi.org/10.1016/j.jcis.2026.140683.

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