(M1030-09-61) Lyotropic Liquid Crystalline Nanoparticles for Gene Delivery: Influence of Composition on Physicochemical Properties, Cellular Uptake, and DNA Encapsulation

Purpose: Lyotropic liquid crystalline nanoparticles (LLCNPs) have emerged as promising next-generation nanocarriers for the delivery of various sensitive biomolecules, such as nucleic acids and proteins [1, 2]. LLCNPs are mesophase self-assembled systems composed of amphiphilic lipids and surfactants dispersed in an aqueous environment. Their tunable internal structure, including lamellar, hexagonal, and cubic phases, offers several advantages for nucleic acid delivery, including enhanced colloidal stability, protection from enzymatic degradation, and improved cellular uptake [3]. Following the successful development of SARS-COV-2 vaccines using lipid nanoparticle (LNP) technology, increasing attention has been directed toward LLCNPs as a versatile and structurally ordered alternative, with the potential to enhance drug loading and stability under physiological conditions. In this study, LLCNPs were developed using glyceryl monooleate (GMO) and Pluronic P407, combined with either the cationic lipid DOTAP or the ionizable lipid ALC-0315, to investigate the impact of lipid composition on nanoparticle physicochemical behavior, cytotoxicity, cellular uptake, and DNA encapsulation.

Methods: Lyotropic liquid crystalline nanoparticles encapsulating DNA were developed using glyceryl monooleate (GMO) and Pluronic P407 combined with either the cationic lipid DOTAP or the ionizable lipid ALC-0315. First, lipids were heated to 80°C, and the melted mixture was hydrated with an aqueous dispersion of Pluronic P407 containing DNA at specific ratios. The resulting dispersion was extruded through polycarbonate membranes to achieve uniform small size. The physicochemical properties of the LLCNPs, including hydrodynamic diameter (D_h) and polydispersity index (PDI), were characterized by dynamic light scattering (DLS), while zeta potential was measured via electrophoretic light scattering (ELS). Colloidal stability was assessed over 90 days of storage. Moreover, LLCNs were incubated with serum isolated from mice to assess protein corona formation and the potential of Pluronic P407 to provide stealth properties. The encapsulation efficiency of DNA was quantified using fluorescence spectroscopy. The cytotoxicity of the nanoparticles was assessed in HeLa cells using MTT assay at a wide range of concentrations (1- 100μg/mL) after 24 h of treatment. Subsequently, confocal microscopy was utilized to evaluate nanoparticle cellular uptake and intracellular distribution in HeLa cells at predetermined time points (2h, 4h, 8h). To visualize subcellular localization, nuclei were stained with Hoechst and F-actin cytoskeleton was labeled with FITC-phalloidin.

Results: The prepared LLCNs were stable in terms of particle size and size distribution over 90 days, indicating their potential for long-term storage at room temperature. Specifically, LLCNPs formulated with the permanently charged cationic lipid DOTAP exhibited a mean hydrodynamic diameter approximately 150nm, while those containing the ionizable lipid ALC-0315 showed larger particle size (~200nm), as measured by DLS. The polydispersity index (PDI) of all the prepared nanoparticles was approximately 0.2, indicating great homogeneity. The z-potential values were close to neutral for the LLCNPs containing the ionizable lipid, indicating effective interaction between the positively charged amine groups of the lipid and the negatively charged phosphate groups of DNA. However, DOTAP-based nanoparticles showed slightly positive z-potential values (~ 10 mV), indicating sufficient amine groups to interact with DNA at higher concentrations. Following incubation with serum isolated from mice, the nanoparticles retained their physicochemical properties, providing an indication that Pluronic P407 not only acts as a stabilizer in the formulations but also prevents protein corona formation, which is known to restrict nanoparticle biodistribution, half-life and efficacy in vivo. All formulations exhibited high encapsulation efficiency, with DOTAP-based nanoparticles reaching up to 80% and ALC-0315 nanoparticles up to 65%. Regarding cytotoxicity, MTT assay demonstrated that all the prepared LLCNs were well tolerated at the examined concentration range (5- 100 μg/mL), with cell viability consistently above 70%. Confocal microscopy revealed that the nanoparticles were efficiently internalized into HeLa cells, with DOTAP-based LLCNPs exhibiting notably higher cellular uptake and interestingly without showing signs of cytotoxicity. The cellular uptake was time-dependent with higher internalization achieved at 8h of treatment for DOTAP-based formulations. In contrast, the formulations containing the ionizable lipid ALC-0315 showed low cellular uptake even at 8h of treatment, demonstrating that nanoparticle lipid composition plays a crucial role in cellular uptake. These findings can be attributed to the distinct surface charge of the nanoparticles, as it is demonstrated in previous studies that positively charged nanoparticles can enhance cellular uptake, although with increased cytotoxicity due to membrane disruption. In our case, the positively charged DOTAP-based nanoparticles can interact more efficiently with cellular membranes, resulting in a notably higher internalization at 8h compared to LLCNPs containing the ionizable lipid ALC-0315. However, the presence of Pluronic P407 can compromise cytotoxicity at all formulations and play a crucial role in preventing protein corona formation.

Conclusion: This study presents a comprehensive evaluation of LLCNPs encapsulating DNA. The results indicate that lipid composition significantly affects encapsulation efficiency, in vitro cytotoxicity and cellular uptake. Specifically, DOTAP-based LLCNPs showed promising results compared to LLCNPs composed of the ionizable lipid ALC-0315, suggesting their potential as a safe and effective non-viral gene delivery platform.

References: 1. Zhai J, Fong C, Tran N, Drummond CJ (2019) Non-Lamellar Lyotropic Liquid Crystalline Lipid Nanoparticles for the Next Generation of Nanomedicine. ACS Nano 13:6178–6206
2. Kim H, Leal C (2015) Cuboplexes: Topologically Active siRNA Delivery. ACS Nano 9:10214–10226
3. Yap SL, Dyett B, Hobro AJ, Nguyen H, Smith NI, Drummond CJ, Conn CE, Tran N The Internal Nanostructure of Lipid Nanoparticles Influences Their Diverse Cellular Uptake Pathways. Small n/a:2500903

Figure 1. The hydrodynamic diameter (Dh) of the prepared GMO:P407:ALC-0315 and GMO:P407:DOTAP lyotropic liquid crystalline nanoparticles (LLCNPs) encapsulating DNA over 90 days of storage at 25 °C.

Figure 2. Confocal laser scanning microscopy (CLSM) images of HeLa cells stained with Hoechst (blue) and FITC-phalloidin (green) at 2h, 4h and 8h of treatment with GMO:P407:DOTAP lyotropic liquid crystalline nanoparticles encapsulating DNA (red) (50 μg/mL).