Formation of particulate lipid lyotropic liquid crystalline nanocarriers using a microfluidic platform

Real‐Time pH‐Dependent Self‐Assembly of Ionisable Lipids from COVID‐19 Vaccines and In Situ Nucleic Acid Complexation

“…For mRNA and pDNA, K (72.6 s −1 ) of mRNA@SM/MO is faster than that (56.6 s −1 ) of pDNA@SM/MO, which may manifest from the smaller molecular weight and single‐stranded molecular structure of mRNA. In addition, the rate constant K (56.6 s −1 ) of this direct self‐assembled pDNA@SM/MO is greater than the previously reported fastest K (31.4 s −1 ) for the complexation of pDNA with nanostructured lipid nanocarriers, [40] accelerated by the transient lipid oversaturation created during solvent mixing [29] . The millisecond kinetic of nucleic acid‐lipid complexation under a comparable flow condition with the manufacturing of COVID‐19 vaccines reveals the real rates of the formation of self‐assembled structures between various classes of nucleic acids and ionisable lipids.…”

“…Both ILNPs were negatively charged (approximately −10 mV) at higher pH over 10, crossed zero around pH 9.5, and became positively charged at pH below 9. The negative zeta potentials at high pH are most likely a result of base‐catalysed hydrolysis of the ionisable lipid ester bonds resulting in the formation of anionic fatty acid salts [29] . The zeta potential of ILNPs were approximately 12 mV at pH 9 and further increased as pH continued to decrease, reaching 40 mV for ILNP SM and 27 mV for ILNP ALC , respectively, at the acidic environment with the lowest pH 3.…”

“…In order to understand the kinetics of the pH‐driven inverse structure formation of the ionisable lipids, we performed time‐resolved SAXS at the millisecond (ms) timescale at the ID02 beamline, ESRF. Rapid microfluidic mixing has been widely used for the formation of various LNPs with advanced size control for drug delivery [29,37,38] . Combining with the in situ SAXS characterisation, the high‐brilliance synchrotron X‐ray source coupled with a stopped‐flow rapid‐mixing device at the ID02 beamline has enabled this technique to be used as a powerful tool to capture the rate of structural formation and intermediate structural evolution of biomembranes and many other self‐assembled systems [28,39–41] .…”

“…For the kinetic study at the ID02 beamline, three types of NAs were employed, including a polyA (single‐stranded, 2100—10000 nt, Mw 700–3500 kDa), a GFP mRNA (single‐stranded, 996 nt, Mw 320 kDa) and a plasmid DNA (pDNA, double‐stranded, 5487 bp, Mw 3390 kDa). As sketched in Figure 5a, the in situ rapid‐mixing apparatus mounted at the ID02 beamline has the solvent solution and aqueous solution simultaneously injected into the microfluidic channel with a fixed 1 : 3 volume ratio and a total flow rate of 6.7 mL/s, which is comparable with the manufacturing conditions (over 20 L/h) of both Pfizer and Moderna COVID‐19 mRNA vaccines using NanoAssemblr GMP System [29] . In 3D time‐resolved SAXS patterns (Figures 5b–d), the appearance and growth of the single peak were observed between 0.12 Å −1 and 0.15 Å −1 for all the complexation during the recording time of 150 ms.…”

Inverse Cubic and Hexagonal Mesophase Evolution within Ionizable Lipid Nanoparticles Correlates with mRNA Transfection in Macrophages

“…As shown in Figure a,b, MO LNPs (α = 0) displayed the Q 2 P mesophase independently of pH in a range of 3–9. The phase transition of MO LNPs observed at higher pH (over 9) is related to the hydrolysis of the MO to oleic acid in a basic environment. , In contrast, pure ionizable LNPs (α = 100) displayed a structural transition toward complex inverse mesophases at lower pH and this phenomenon has been described in detail in our earlier publication …”