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Lipid nanoparticles (LNPs) are the main platform for delivering RNA drugs. Optimizing the transfection efficiency of these systems has drawn a lot of attention. The standard preparation process involves dissolving lipids in ethanol and mixing them with a pH 4 buffer containing mRNA, followed by dialysis with pH 7.4 physiological saline buffer to remove ethanol and raise the pH. The dialysis process causes the mRNA-lipid complexes and small vesicles formed at pH 4 to fuse, driven by two steps: first, the ionizable lipids transform from positively charged bilayer structures to neutral oil-like structures; second, high ionic strength triggers fusion. This article explores a two-step dialysis process: first, forming the complexes in 25 mM NaOAc at pH 4, and then dialyzing with a high ionic strength buffer at pH 4 to induce fusion while keeping the ethanol concentration at 25% (v/v). Finally, dialysis with PBS buffer is done to raise the pH. We show that, compared with the standard process, the two-step method increases the mRNA load per LNP, reduces the proportion of “empty” particles, and boosts transfection efficiency both in vitro and in vivo compared to LNP mRNA formed with the traditional one-step buffer exchange process.
Summary
In the preparation of lipid nanoparticles, traditional dialysis can cause abrupt pH changes that lead to mRNA-lipid complexes fusing with empty vesicles, reducing drug loading efficiency. We explored a two-step dialysis process: first, we prepare the complexes in a low-concentration acetate solution under acidic conditions, then dialyze with a high ionic strength buffer at the same pH to induce fusion while maintaining the ethanol concentration, and finally raise the pH using a phosphate buffer. This process increases the mRNA load per particle, reduces the proportion of empty particles, and boosts transfection efficiency both in vitro and in vivo.
This work reveals the key effects of pH and ionic strength timing during the dialysis process on particle fusion and drug loading, providing practical parameters for fine-tuning the LNP preparation process. In the future, buffer composition could be optimized for different RNA types or ionizable lipid structures. The two-step method doesn’t rely on extra equipment and can be easily adjusted on existing production lines. Reducing the proportion of empty particles helps lower dosage and toxicity. This strategy could also be applied to other nucleic acid delivery systems, like the assembly and purification of polymer or inorganic nanoparticles. Long-term stability and consistency in large-scale production still need to be verified.
References:
DOI: 10.1002/adma.75174
