Fig. 4.

Scattering signature and pH-induced transitions in CIL bulk vs. LNP core phase. (A) SAXS scattering at pH 7 (red curves) of MC3/chol/water $L_{\mathit{II}}$ bulk phase, MC3/chol/water/polyA $L_{\mathit{II}}^C$ bulk phase, MC3/chol/water/mRNA $L_{\mathit{II}}^C$ bulk phase, and mRNA LNPs and corresponding SAXS data at pH 5 (black curves). The MC3/chol/water/polyA preparation at pH 7 showed time dependence with the $H_{\mathit{II}}^C$ peak dissolving over weeks. We denote the dashed line as micellar isotropic $L_{\mathit{II}}^C$ phase. The mRNA filled bulk phases and LNPs exhibit less long-range order due to mRNA secondary structure. (B) Schematic drawing of the polyA condensed $H_{\mathit{II}}^C$ phase at pH 5, the polyA condensed $L_{\mathit{II}}^C$ (polyA) phase at pH 7, and an mRNA condensed $L_{\mathit{II}}^C$ (mRNA) phase. mRNA is assumed to retain secondary structure in a disordered $L_{\mathit{II}}$ —micellar-like lipidic phase. (C) Next nearest neighbor distances as a function of pH showing pH-induced transitions in CIL bulk phases and (D) in polyA containing complexed phases. The gray area indicates the transition region with cubic phases. The reddish area indicates metastability of $H_{\mathit{II}}^c$ . (E) pH-dependent correlation distance in LNPs derived from SAXS data (F) Fluorescence anisotropy of a hydrophobic probe (DPH) as a function of pH for MC3-, KC2-, and DD-LNPs, respectively. Increasing anisotropy indicates decreasing mobility. The critical pH values of half-maximum are 6.0, 6.1, and 6.0 for MC3-, KC2-, and DD, respectively. Data shown in (C and D) are measured at room temperature, (E and F) at 37 °C.