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iChol15-C4A2
Formulation & Performance
Formulation & Performance
Study-specific compositions, preparation conditions and experimental outcomes for iChol15-C4A2.
Newly checked against the author-hosted complete article and official ACS Supporting Information. The initial screen and optimized formulation, intramuscular and intravenous studies, and luciferase expression and Cy5 distribution are kept separate.
1. Formulations and component amounts
Reported composition and calculated mass are separate. Molecular-weight inputs use catalog values or an explicitly identified reference average; DMG-PEG2000 is fixed initially at 2507. Edit inputs only when using a verified alternative material.
Initial three-component screening formulation
Molar parts (sum 101.5), not percentages; no separately added free cholesterolSupporting Information p. 4 and Table S3, p. 60
| Component | Molecular weight | Molar parts | Normalized mol% | Amount (µmol) | Weight (mg) |
|---|---|---|---|---|---|
| iChol15-C4A2 | 49.261 | 51.0165 | 55.8524 | ||
| DSPC | 49.261 | 51.0165 | 40.3107 | ||
| DMG-PEG2000 | 1.478 | 1.5305 | 3.8370 |
Calculated total: 100.0000 mg lipid. RNA, solvent and process loss are excluded.
Optimized Formulation 8
Molar parts (sum 111.5), not percentages; selected by intramuscular screening and used in the subsequent intravenous studiesMain Fig. 5c–e; Supporting Information pp. 4–5 and Fig. S49
| Component | Molecular weight | Molar parts | Normalized mol% | Amount (µmol) | Weight (mg) |
|---|---|---|---|---|---|
| iChol15-C4A2 | 53.812 | 55.0684 | 60.2883 | ||
| DSPC | 44.843 | 45.8903 | 36.2602 | ||
| DMG-PEG2000 | 1.345 | 1.3767 | 3.4514 |
Calculated total: 100.0000 mg lipid. RNA, solvent and process loss are excluded.
DMG-PEG2000 calculations use the catalog nominal molecular weight 2507.
How component weights are calculated
For total lipid mass M (mg), molar parts rᵢ and molecular weight MWᵢ: component mass = M × rᵢ × MWᵢ / Σ(r × MW). Component amount (µmol) = component mass (mg) × 1000 / MWᵢ. Molar parts are normalized only for the displayed percentage. The calculation does not infer N/P or RNA dose.
2. Preparation and experimental setup
| Parameter | Reported procedure / limitation | Source |
|---|---|---|
| Aqueous phase | mRNA in 25 mM citrate buffer, pH 4.4. | Supporting Information p. 4 |
| Organic phase | The lipid mixture was dissolved in ethanol at 10 mg/mL. | Supporting Information p. 4 |
| Mixing | Microfluidic mixing at aqueous:ethanol 3:1 by volume; total flow rate not reported. The formulation-optimization subsection also describes ethanol dilution. | Supporting Information pp. 4–5 |
| Lipid:mRNA loading | iChol lipid:mRNA 25:1 by mass; this is the ionizable lipid mass, not the combined lipid mass and not N/P. | Supporting Information pp. 4–5 |
| Buffer exchange | Overnight dialysis against PBS, pH 7.4, with a 20 kDa dialysis cassette. | Supporting Information p. 4 |
| Analytical methods | DLS for hydrodynamic size and zeta potential; a modified RiboGreen assay for RNA encapsulation. | Supporting Information p. 4 |
| Unreported parameters | No experimentally established N/P or target-specific apparent particle pKa was identified. | Full article and Supporting Information review |
3. Particle characterization
| Measurement / batch | Reported result | Evidence context |
|---|---|---|
| Initial formulation: size | 90.76 ± 4.8 nm | Table S3, p. 60; 50:50:1.5 molar parts |
| Initial formulation: PDI | 0.088 | Table S3, p. 60; 50:50:1.5 molar parts |
| Initial formulation: encapsulation | 93.56 ± 1.06% | Table S3, p. 60; 50:50:1.5 molar parts |
| Optimized Formulation 8: size | 106 nm | Main Results accompanying Fig. 5c–e and SI Fig. S49; 60:50:1.5 molar parts |
| Optimized Formulation 8: PDI / encapsulation | 0.09 / 94.7% | Main Results accompanying Fig. 5c–e and SI Fig. S49 |
4. Performance by experiment
| Experiment and dose | Readout and comparator | Result and source |
|---|---|---|
| Initial intramuscular reporter screen BALB/c mice; luciferase mRNA 5 µg/mouse; initial 50:50:1.5 formulation; n = 3 | Readout: 6 h Comparator: Experimental MC3 and ALC-0315 LNPs | iChol15-C4A2 was among the leading local-expression candidates. This is injection-site expression, not an intravenous organ-targeting measurement.Main Fig. 5a–b; SI p. 4 |
| Intramuscular formulation optimization BALB/c mice; luciferase mRNA 1 µg/mouse; ten compositions; n = 3 | Readout: 6 h Comparator: Ten formulations spanning iChol 40–60, DSPC 30–60 and DMG-PEG2000 1.5–5 molar parts | Formulation 8 (60:50:1.5) was selected for higher local reporter expression and carried forward.Main Fig. 5c–e; SI pp. 4–5 and Fig. S49 |
| Intravenous functional mRNA expression Mice; luciferase mRNA 0.1 mg/kg; optimized 60:50:1.5 formulation; n = 3 | Readout: 6 h Comparator: Experimental MC3 and ALC-0315 LNPs | Spleen:liver luminescence ratio 4.78, versus 0.24 for MC3 and 0.12 for ALC-0315. The ratio is approximately 39.8 times the ALC-0315 ratio; this is not a 39.8-fold increase in absolute splenic expression.Main Fig. 5f–i and accompanying Results; SI p. 5 |
| Intravenous fluorescent RNA distribution BALB/c mice; Cy5-labelled mRNA 0.3 mg/kg; optimized formulation; n = 3 | Readout: 6 h Comparator: ALC-0315 LNPs | The article describes an approximately 1.0 spleen:liver fluorescence ratio for Tc-LNPs versus approximately 0.2 for ALC-0315. Fluorescence tracks labelled material and does not establish translated protein or percentage of injected dose.Main Fig. 5j–l and accompanying Results; SI p. 5 |
| ApoE-dependent cellular uptake and expression HepG2 and HEK293 cells; LNPs preincubated with ApoE at 0–1 g ApoE/g total lipid for 15 min at 37 °C; 1 µg reporter mRNA | Readout: Cy5 uptake assessed after 2 h; expression assessed in the parallel reporter assay Comparator: ALC-0315 LNPs; with and without ApoE preincubation | ApoE increased ALC-0315 uptake and expression in LDLR-expressing HepG2 cells, while the tested Tc-LNPs changed minimally. The comparison supports reduced dependence on ApoE-mediated uptake.Main Fig. 6a–f; SI pp. 5–6 and Figs. S51–S52 |
| Protein-corona analysis LNPs mixed 1:1 by volume with mouse plasma at 37 °C, then isolated and analysed by LC–MS | Readout: 1 h plasma incubation Comparator: ALC-0315 LNPs | The tested Tc-LNP coronas contained less ApoE than the ALC-0315 comparator. Main-text and SI quantitative summaries are not identical, so no single exact ApoE percentage is assigned here.Main Fig. 6g–j; SI p. 6 and Tables S5–S7 |
| pH-dependent membrane disruption Mouse erythrocytes exposed to Tc-LNPs; physiological versus acidic buffer | Readout: 1 h at 37 °C Comparator: Physiological pH 7.4 and assay controls | The tested Tc-LNPs showed greater membrane-disruptive activity under acidic conditions. The methods specify pH 5.5, whereas one Results passage uses pH 5.0; no exact acidic-pH percentage or pKa is assigned here.Main Fig. 7b; SI p. 6 |
| Storage of formulated RNA-loaded particles Optimized 60:50:1.5 luciferase-mRNA LNPs stored at 4 °C for 0, 1, 3, 7, 14 and 30 days; particle assays and cell reporter assays; IM test 5 µg/mouse, n = 3 | Readout: Up to 30 days; mouse reporter readout 6 h after injection Comparator: Experimental four-component MC3 and ALC-0315 LNPs | The authors report retained physical properties, encapsulation and reporter activity over the tested storage period. This does not establish storage stability of neat lipid or empty LNPs.SI p. 7 and Fig. S57; main Results |
| Short-term in vivo tolerability BALB/c mice; intravenous RNA-loaded Tc-LNPs, 0.5 mg mRNA/kg | Readout: 24 h Comparator: PBS control | No significant changes in ALT, AST, BUN or creatinine, or obvious major-organ histopathology, were reported under the tested conditions. This is a short-term mouse observation.Main Fig. 7d–e; SI p. 7 |
Mechanistic evidence
The ionizable lipid incorporates a cholesteryl group, enabling a three-component iChol/DSPC/DMG-PEG2000 particle without separately added free cholesterol. Protein-corona and ApoE/LDLR experiments support reduced ApoE-dependent hepatocyte uptake. Acid-dependent membrane activity and cellular imaging support an endosomal-delivery rationale; they do not measure a target-specific apparent pKa.
5. Interpretation and unresolved details
- Use 50:50:1.5 only for the initial screen and Table S3 values; use 60:50:1.5 for optimized Formulation 8 and subsequent intravenous studies. Normalize these molar parts before calculating molar percentages.
- The 25:1 loading ratio refers to iChol lipid:mRNA mass. It is neither total lipid:mRNA nor N/P.
- Keep the 0.1 mg/kg luciferase study separate from the 0.3 mg/kg Cy5-labelled RNA study. A spleen:liver ratio is not an absolute expression fold improvement.
- The study demonstrates reporter delivery in cells and mice. It does not establish T-cell-specific transfection, in vivo CAR-T activity, T-cell-engager efficacy, non-human-primate efficacy or clinical performance.
- The acidic hemolysis condition is described as pH 5.5 in SI methods but pH 5.0 in one main-text passage. The SI toxicity subsection also contains an isolated DEG-PEG2000 spelling, inconsistent with DMG-PEG2000 throughout the formulation record; this is not treated as a separate PEG component.
- Published particle measurements are study-specific and do not represent lot-specific COA results. Thirty-day stability applies to the formulated RNA-loaded particles tested.
Results apply to the stated LNP composition, cargo, process, dose, route and model. LNP experimental measurements are not batch-release specifications for the isolated lipid.
6. References and data access
Teng Y, et al. Computationally Aided Design of Ionizable Cholesteryl Lipids for Lipid Nanoparticles to Modulate Hepatic mRNA Accumulation. J Am Chem Soc. 2025;147:38865–38878. doi:10.1021/jacs.5c14870.
Download experiment table (CSV) · Open printable Formulation & Performance guide
Revision FP-20261024-b2 · Source review: 24 October 2026 · DC Chemicals