(M1230-07-41) Effects of Lipidoid-Polymer Hybrid Nanoparticles on the Biophysical Function and Structure of Pulmonary Surfactant

Purpose: Inhaled nanoparticles (NPs) must first interact with the pulmonary surfactant (PS) film that covers the entire internal surface of the respiratory tract and plays an important role in surface tension reduction and as the first line of host defense. The interaction between NPs and PS determines the potential therapeutic or toxicological effect of inhaled NPs. Lipidoids, which contain one or several amine centers and multiple hydrophobic tails, and depending on their degree of alkylation, they are subdivided into different groups, e.g., L₅N₁₂ ^[1]. Lipidoid-polymer hybrid nanoparticles (LPNs) display an unprecedented ability to deliver nucleic acids to cells in vitro and in vivo ^[1,2]. They are composed of L₅N₁₂ and the polymer poly(lactic-co-glycolic acid). Recently, we formulated dry powder inhaler formulations containing tumor necrosis factor (TNF)-α small interfering RNA (siRNA)-loaded LPNs with optimized aerosolization properties for inhalation therapy in cases of lung inflammation ^[3]. We hypothesize that understanding the interactions between LPNs and PS is crucial for predicting the safety profile and fate of inhaled LPNs. However, little is known about how LPNs interact with PS, and whether LPNs display adverse effects on the biophysical function and lateral structure of PS. The aim of this study was to investigate the effect of LPNs on the biophysical function and lateral structure of PS for the rational design of inhaled nanoparticles.

Methods: Two surfactant models were used: (i) an organic extract (hydrophobic fraction) of porcine PS, which contains all the surfactant lipids and the hydrophobic surfactant proteins B (SP-B) and SP-C, and (ii) a simplified model composed of dipalmitoylphosphatidylcholine (DPPC). Monolayer experiments were performed at 37 °C in a thermostated Langmuir–Blodgett trough. LPNs were subsequently deposited onto preformed PS films. The effect of the LPNs on the structure of PS was analyzed by using epifluorescence microscopy and cryogenic transmission electron microscopy (cryo-TEM).

Results: The interaction between bulk lipidoid and PS improved apparently the biophysical properties of the latter, preventing surfactant material loss during cycling and facilitating maximal reduction of the surface tension at the air/liquid interface (Figure 1A and B). Lipidoid coating of PLGA nanoparticles (LPNs) facilitated the incorporation of the surfactant film up to a surface pressure of 50 mN/m, at which the nanomaterial was excluded from the film without significantly influencing the surface behavior of PS under physiologically relevant conditions (Figure 1C). The interaction between LPNs and PS seems to be mediated by the miscibility between the lipidoid and DPPC, the main phospholipid component and the main surface-active molecule in PS. Epifluorescence micrographs of mixed DPPC/LPN films showed that the nanomaterial (i) affected both the nucleation shape and growth of compression-driven segregated tilted condensed (TC) domains, and (ii) facilitated the intermixing of TC and liquid expanded (LE) domains (Figure 2). This indicates a preferential location of the nanomaterial at defect structures of the LE/TC domain boundaries, as previously observed for other polymeric nanoparticles ^[4]. Finally, a strong interaction between LPNs and PS bilayers generated negatively curved structures in surfactant liposomes (Figure 3). In addition, although the lipidoid component of LPNs facilitated incorporation of LPNs into the interfacial PS layer, the LPNs did neither affect the surface tension-lowering capabilities of PS nor it’s re-spreading, because the nanomaterial is ultimately translocated towards the aqueous subphase.

Conclusion: : Our results demonstrate that (i) L₅N₁₂ is a safe material to use for pulmonary delivery, and (ii) the interaction between LPNs and PS facilitates the translocation of the LPNs towards the aqueous subphase without disrupting the lateral structure and biophysical function of PS. Hence, understanding LPN-PS interactions may not only provide new insights into the safety of nanocarriers, but also shed light on the feasibility of using LPNs for pulmonary drug delivery strategies.

References: [1] Thanki, K.; Zeng, X.; Justesen, S., et al, Eur. J. Pharm. Biopharm. 2017, 120, 22-33.
[2] Jansen, M. A. A.; Klausen, L. H.; Thanki, K., et al., Eur. J. Pharm. Biopharm. 2019, 142, 38-48.
[3] Xu, Y., Harinck, L., Lokras, A. G., et al, Int. J. Pharm. 2022, 121758.
[4] Cañadas, O.; García-García, A.; Prieto, M.A.; Pérez-Gil, Nanomaterials. 2021, 11, 1482.

Acknowledgements: We gratefully acknowledge the financial support from the Novo Nordisk Foundation, Denmark (grant no. NNF17OC0026526), Independent Research Fund, Denmark (grant no. DFF-4184-00422), the regional Government of Madrid (grant P2018/NMT­4389) and the Spanish Ministry of Science and Innovation (grant RTI2018-094564-B-I00). We acknowledge the China Scholarship Council (CSC) for the Scholarship to You Xu (201906210064).


Figure 1. Surface activity of porcine PS (A), lipidoid (B) and LPNs + PS (C) along 5 consecutive compression-expansion cycles: 1st (red), 2nd (green), 3rd (blue), 4th (purple) and 5th (orange).


Figure 2. Representative epifluorescence microscopy images of interfacial films taken at different surface pressures.


Figure 3. Representative cryo-TEM images of PS and LPNs/PS interaction.