(M1130-09-61) Small-Angle X-Ray Scattering Analysis Reveals the Internal Structure of Lyotropic Liquid Crystalline Nanoparticles Encapsulating DNA

Purpose: Lyotropic liquid crystalline nanoparticles are self-assembled nanostructures composed of amphiphilic biomolecules, such as lipids and surfactants, dispersed in water and organized into various mesophases. Non-lamellar mesophases, such as inverse hexagonal (hexosomes) and bicontinuous cubic (cubosomes) offer several advantages for nucleic acid delivery, including improved colloidal stability and encapsulation efficiency, particularly for oligonucleotides, such as siRNA and miRNA [1, 2]. In this study, lyotropic liquid crystalline nanoparticles encapsulating low molecular weight DNA (50bp) were developed and characterized for their physicochemical properties and internal structure by dynamic light scattering (DLS), electrophoretic light scattering (ELS), and small-angle x-ray scattering (SAXS). The lyotropic liquid crystalline nanoparticles were formulated using glyceryl monooleate (GMO), Pluronic P407, the ionizable lipid ALC-0315, and DNA two nitrogen to phosphate ratios (N/P ratio) to evaluate structural changes under different pH conditions simulating early and late endosomes, which present the major barriers in lipid nanoparticle-mediated gene delivery, and to assess thermal effects on their physicochemical and internal structural properties.

Methods: Nanoparticles were prepared by melting the lipids at 80 ^oC, followed by hydration with Pluronic P407 and DNA at N/P ratios of 2 and 4, respectively. The resulting dispersions were extruded through polycarbonate membranes of defined pore size to achieve uniform vesicle size. Physicochemical characterization of the nanoparticles was performed via dynamic and electrophoretic light scattering (DLS, ELS) to determine particle size, polydispersity index, and z-potential. The physicochemical behavior of the nanoparticles was assessed at various temperatures (25^oC, 37^oC, 50^oC) to evaluate their thermal stability. Small-angle X-ray scattering was also conducted at 25°C, 37°C and 50°C to evaluate temperature-induced internal structural transitions. Moreover, DLS and SAXS measurements were performed at pH 6.5, and 5.5 to monitor physicochemical and structural changes that may occur in early and late endosome environments, which present the major challenge in lipid nanoparticle-mediated gene delivery during nanoparticle trafficking. Subsequently, the encapsulation efficiency of DNA in all the prepared formulations was quantified by fluorescence spectroscopy.

Results: Lyotropic liquid crystalline nanoparticles exhibited superior colloidal stability over 90 days of storage at room temperature. The hydrodynamic diameter (D_h) of the formulations was approximately 140nm, while polydispersity was relatively low with values below 0.22, indicating great homogeneity. Colloidal stability was evaluated in biorelevant pH conditions, mimicking early and late endosomes, indicating a pH-dependent increase of the hydrodynamic diameter at N/P 4 that can be attributed to the increased protonation of the ionizable lipid, which enhances interactions between the positively charged amine groups and the negatively charged phosphate groups of nucleic acid. Moreover, SAXS analysis demonstrated that the nanoparticles retained their internal structure following incubation in various pH conditions for both N/P ratios, as there was no shift in Bragg peaks. Specifically, nanoparticles at N/P 2 exhibited one sharp Bragg peak (q= 0.15 A^-1), demonstrating a less ordered structure, which according to the literature corresponds to a lamellar phase, while at N/P 4 three Bragg peaks were detected consistent with the 1:√ 3: √ 4 reflections of the inverted hexagonal phase (H_II) [1]. This result can be attributed to the increased interaction of the ionizable lipid with DNA at higher N/P ratios, leading to transitions into non-lamellar, more ordered mesophases. In contrast, SAXS measurements at different temperatures revealed that nanoparticles at N/P 2 exhibited temperature-induced transitions weakening the internal structure with increasing temperature, while the inverted hexagonal phase at N/P 4 remained stable. Despite these internal rearrangements, DLS confirmed that both formulations maintained their overall physicochemical properties at all temperatures. In this case, we propose that the nanoparticles retain their physicochemical properties as indicated by dynamic light scattering at the examined temperatures, however small-angle X-ray scattering analysis provides insights into thermally-induced changes at N/P 2 within the aqueous core, suggesting that mesophase domains become more fluid or disordered. In contrast, nanoparticles at N/P 4 maintain inverted hexagonal phase over a wide range of temperatures, indicating that this formulation is thermally stable. Subsequently, the encapsulation efficiency of the prepared formulations was measured above 60%.

Conclusion: This study demonstrates that lyotropic liquid crystalline nanoparticles encapsulating DNA exhibit distinct thermotropic and pH-dependent behavior relative to the N/P ratio. Formulations at N/P 2 remained stable under endosome-relevant pH conditions, however exhibited temperature-induced internal rearrangements, as indicated by a transition from a sharp lamellar peak to a broader SAXS signal with increasing temperature. In contrast, nanoparticles at N/P 4 showed a pH-dependent increase in hydrodynamic diameter, attributed to enhanced interactions between ionizable lipids and DNA, while maintaining a stable inverse hexagonal phase at all tested temperatures. These results demonstrate that increasing the N/P ratio promotes the formation of thermally and structurally stable non-lamellar phases, supporting their potential for efficient DNA delivery in physiologically relevant environments. Overall, these findings may provide insights into the design of next-generation nucleic acid therapeutics and vaccines.

References: 1. Pattipeiluhu R, Zeng Y, Hendrix MMRM, Voets IK, Kros A, Sharp TH (2024) Liquid crystalline inverted lipid phases encapsulating siRNA enhance lipid nanoparticle mediated transfection. Nat Commun 15:1303
2. Nele V, Campani V, Alia Moosavian S, De Rosa G (2024) Lipid nanoparticles for RNA delivery: Self-assembling vs driven-assembling strategies. Advanced Drug Delivery Reviews 208:115291

Acknowledgements: Zetasizer ZSU3105 (Malvern, UK) instrumentation was financed by Uni-Pharma Kleon Tsetis Pharmaceutical Laboratories S.A.
The SAXS measurements were performed in a (GI-)SAXS/WAXS/USAXS beamline the acquisition of which was supported by the Hellenic Foundation for Research and Innovation (Project SAXS-SOFT, Project Number: 3401).

Figure 1. The hydrodynamic diameter (Dh) of lyotropic liquid crystalline nanoparticles (LLCNPs) encapsulating DNA at N/P 2 and N/P 4 ratios a) over 90 days of storage, b) upon incubation in various pH conditions, and c) after incubation at different temperatures.

Figure 2. SAXS patterns of lyotropic liquid crystalline nanoparticles (LLCNPs) encapsulating DNA at a) N/P 2 and b) N/P 4 ratios over a pH range from 5.5 to 7.4, and a temperature range from 25 °C to 50 °C.