Transition Temperature-Guided Design of Lipid Nanoparticles for Effective mRNA Delivery

Abstract

Lipid nanoparticles (LNPs) are promising mRNA delivery vehicles due to their biocompatibility and tunable characteristics. While current rational design approaches focus on ionizable lipids' pK_a and zeta potential to optimize mRNA encapsulation and endosomal escape, the selection of helper lipids remains largely empirical. We propose that the lipid transition temperature (T_m), marking the shift from the gel to the liquid crystalline phase, can guide rational helper lipid selection. Through screening 54 ionizable lipids, we identified H7T4, which displayed favorable physicochemical properties when combined with its tail variants but exhibited poor transfection efficiency. Using nano differential scanning calorimetry (nDSC) and biological small-angle X-ray scattering (BioSAXS), we found that lowering the system's T_m by combining H7T4 (high transition temperature) with a low-transition-temperature helper lipid such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) significantly enhanced mRNA cellular uptake both in vitro and in vivo. These findings establish T_m as a crucial parameter for a rational LNP design.

Keywords: SAXS; ionizable lipid; lipid nanoparticle; mRNA; nDSC.

Figure 1

Synthesis, characterization, and spectroscopic analysis of H7T4: MS and NMR studies of raw lipid reaction products. (A) Reaction scheme and chemical structure of H7T4. (B) Chemical structures of H7T4 variants (left: H7T4–1; right: H7T4–4). (C) Mass spectra of H7T4 raw mixture. (D) NMR data of pure H7T4–4.

Figure 2

Characterization of LNPs with H7T4 variants. (A) Sizes of LNPs using H7T4 variants with different tail numbers. (B) PDIs of LNPs using H7T4 variants with different tail numbers. (C) Encapsulation efficiencies of LNPs with H7T4 variants. (D) Unencapsulated mRNA concentrations post-LNP formulation with H7T4 variant. (E) Cryo-TEM for H7T4–1 only LNPs H7T4–1/DSPC/cholesterol/PEG-lipid (50/10/38.5/1.5 mol %) and H7T4–1 + H7T4–4 LNPs H7T4–1/H7T4–4/DSPC/cholesterol/PEG-lipid (25/25/10/38.5/1.5 mol %). Cryo-TEM analysis followed dialysis against PBS to remove ethanol (pH 4.5) and neutralize the LNPs (pH 7.4). Scale bar = 100 nm. (F) In vitro firefly luciferase assays (n = 3, biologically independent samples). LNPs were formulated with H7T4 variant(s)/DSPC/cholesterol/PEG-lipid (50/10/38.5/1.5 mol %), using either single H7T4 variants or H7T4 variant combinations in equal proportions (25:25 mol %). Two muscle cell lines (C2C12 and SkMC) were treated with firefly luciferase mRNA-loaded H7T4 LNPs at a 10 ng/well mRNA dose for 24 h. Data are represented as mean ± SD.

Figure 3

nDSC thermograms of H7T4 variants. Nano differential scanning calorimetry (nDSC) thermograms of (A) buffer (ethanol 100%) and H7T4 variants (B: H7T4–1 and C: H7T4–4) were obtained to characterize the phase transition of lipids as a function of temperature. The transition temperature of H7T4 variants is indicated as T_m, and enthalpy changes during phase transition, as ΔH.

Figure 4

Characterization of H7T4–4 LNPs with other helper lipids. (A) Encapsulation efficiency of H7T4–4 LNPs with different helper lipids (H7T4–4/helper lipid/cholesterol/PEG-lipid at 50/10/38.5/1.5 mol %). (B) In vitro mRNA delivery of H7T4–4 LNPs with different helper lipids in C2C12 cells. (C) Size distribution data for H7T4–4 LNPs with different helper lipids. (D) SAXS data for H7T4–4 LNPs with different helper lipids SAXS data for H7T4 lipid variants. To accommodate the SAXS data, each set was given an offset (empty: 0.2 and POPC-TO: 1 unit).

Figure 5

Optimization of H7T4–4 LNP formulation for mRNA delivery in vitro and in vivo. (A) In vitro firefly luciferase expression (n = 3, biologically independent samples). The pancreatic cancer cell line (AsPC-1) was treated with firefly luciferase mRNA-loaded H7T4 LNPs at a 10 ng/well mRNA dose for 24 h. (B) In vivo firefly luciferase expression (n = 4, biologically independent samples). Mice were injected intratumorally (i.t.) with firefly luciferase-loaded H7T4 LNPs at various mRNA doses (1 or 5 μg per mouse). IVIS luminescence imaging was performed at 24 h post-treatment, and total flux was quantified. (C) In vivo firefly luciferase expression (n = 3, biologically independent samples). Mice were administered intranasally (i.n.) with firefly luciferase-loaded H7T4 LNPs at various mRNA doses (5, 25 μg per mouse). IVIS luminescence imaging was performed at 6 and 24 h post-treatment, and total flux was quantified. Data are presented as mean ± SD. Statistical significance was evaluated using a one-way ANOVA with Tukey’s post-hoc test for multiple comparisons.