6Ac1-C12
Formulation & Performance

1. Verified product identity

Formula: C106H208N6O13. Molecular weight: 1774.86. Values refer to the neutral parent lipid. The corrected structure has six tertiary amines and six ester groups.

SMILES and the structure image have been reconciled with SI Fig.S2. Average MW is calculated from the verified structure using standard average atomic weights (RDKit result 1774.859 g/mol, rounded to 1774.86). Open high-resolution structure.

2. Formulation composition

mol% = individual molar parts / sum of parts × 100. Calculations use unrounded values; displayed percentages may sum to 99.9999% or 100.0001%. DOTAP is the chloride salt; DDAB is dimethyldioctadecylammonium bromide. The five-component formulation includes 62 parts DOTAP, which is 50 mol% of the final mixture. The three-component lung system removes both DOPE and cholesterol.

3. Preparation and RNA loading

These mass ratios use the ionizable lipid alone, not total lipid mass. The 20:1 ratio is stated alongside the three-component lung recipes; it is not separately restated for every four-/five-component comparator. Their RNA mass is therefore not automatically calculated. A microfluidic device, flow rates, dialysis MWCO and mixing lipid/RNA concentrations are not specified in the checked method. An analytical-assay lipid concentration of 1 mM in SI pp.2–3 is not a universal preparation concentration.

4. Particle properties

Mean ± sample SD calculated from the author Source Data, n = 3. Liver: Figure 3, M4:M6 / M8:M10 and Supplementary Fig.9, D13:F13 / C41:E41. Lung: Supplementary Fig.17, N4:S7, O14:Q16 and B20:E22. The authors call the RNA endpoint “binding efficacy”; it is not silently relabeled as encapsulation efficiency. Different source panels used different preparations; keep their values and conditions separate.

Apparent LNP pKa: 5.97 for the four-component 6Ac1-C12 formulation (main Fig.3j, p.5; TNS method in SI p.3). This value is not established for all lung formulations.

5. Cell transfection and organ accumulation

Ai9 mice; IV Cre mRNA 0.25 mg/kg; analysis at 48 h; n = 3. Percentages are within the indicated cell subset, not a fraction of all organ cells. Means ± sample SD calculated from Source Data Figure 5: C16:K16 and C47:K47. Conditions: main p.10; SI Figs.24 and 29, pp.25,30.

C57BL/6 mice; IV Cy5-mRNA 0.5 mg/kg; 2 h; n = 3. Means ± sample SD calculated from Source Data Supplementary Fig.25, F4:K6; see SI p.25 and main p.9. These are relative organ fluorescence values, not percentages of injected dose. The three-component formulation reduces hepatic accumulation but does not eliminate it. Cy5 accumulation and Cre-dependent reporter expression measure different endpoints.

In the matched lung comparison, three-component DOTAP LNPs produced higher pulmonary luciferase signals than four-/five-component counterparts (IV Fluc mRNA 0.25 mg/kg, 6 h; SI Fig.22, p.21; main p.9). No fold changes are transferred across different assays or administration routes.

6. Stability, tolerability and interpretation

The study tracked size during 30-day storage; the liver formulation was stored at 4 °C (main p.5). In the three-component DOTAP stability series, size was 113.17 ± 0.75 nm on day 0 and 115.93 ± 0.45 nm on day 30 (mean ± sample SD, n = 3; Source Data Supplementary Fig.31, C4:E4 / C8:E8; SI p.31). Size stability alone does not establish preservation of RNA integrity or expression potency.

At IV mRNA 0.5 mg/kg, liver/kidney markers and hematology were comparable to PBS in the reported 24-hour mouse evaluation; histology was also examined (main p.10; SI Figs.32–34, pp.32–33). These findings do not establish chronic or clinical safety. The lipid hydrolysis experiment used 6 M HCl under reflux for 24 h (SI p.3), so it must not be described as a measured physiological degradation half-life.

7. Calculated stock preparations

Uses MW 1774.86 g/mol. V (mL) = mass (mg) × 1000 / [MW × concentration (mM)]. Values are final solution volumes, not a measured solubility limit or the volume of solvent to add. 10 mM = 17.7486 mg/mL; confirm complete dissolution.

9. Product handling

Supplied as pure lipid. For small quantities, dissolve the complete vial in a compatible solvent to minimize weighing loss. Ethanol is used in the published lipid phase. Follow vial instructions: pure lipid at −20 °C for 1 year; stock solutions at −80 °C for 6 months or −20 °C for 1 month, tightly sealed and protected from light. These supplier handling periods are separate from the paper’s formulated-LNP stability study. Research use only.

Sources

[1] Su K, Shi L, Sheng T, et al. Reformulating lipid nanoparticles for organ-targeted mRNA accumulation and translation. Nature Communications 15, 5659 (2024). DOI: 10.1038/s41467-024-50093-7. Original article. Main PDF: 12 pages. [2] Supplied Supplementary Information: 33 pages; all three uploaded filenames are byte-identical copies. Page references count from the first PDF page. [3] Author Source Data, 41467_2024_50093_MOESM4_ESM.xlsx. Numeric summaries use the cited sheets and cells, with arithmetic means and sample SD (n−1).

Paper and associated data: Su et al., 2024, CC BY 4.0. Content has been summarized; structure graphics have been redrawn and mol%, component weights and data summaries calculated by DC Chemicals. Helper-lipid MW inputs come from existing catalog records and the previously confirmed DMG-PEG2000 average MW.