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Lipid 6
Formulation & Performance
Formulation & Performance
Study-specific compositions, preparation conditions and experimental outcomes for Lipid 6.
Main article and complete supplementary PDF reviewed; screening and optimized recipes are kept separate.
1. Formulations and component amounts
Reported composition and calculated mass are separate. Molecular-weight inputs use the current catalog values; DMG-PEG2000 is fixed initially at 2507. Edit inputs only when using a verified alternative material.
Initial screen
mol%Main Methods; SI Table S1
| Component | Molecular weight | Molar parts | Normalized mol% | Amount (µmol) | Weight (mg) |
|---|---|---|---|---|---|
| Lipid 6 | 35.000 | 37.0682 | 61.7126 | ||
| DOPE | 16.000 | 16.9455 | 12.6079 | ||
| Cholesterol | 46.500 | 49.2477 | 19.0416 | ||
| DMG-PEG2000 | 2.500 | 2.6477 | 6.6379 |
Calculated total: 100.0000 mg lipid. RNA, solvent and process loss are excluded.
B11
mol%SI Table S2
| Component | Molecular weight | Molar parts | Normalized mol% | Amount (µmol) | Weight (mg) |
|---|---|---|---|---|---|
| Lipid 6 | 30.000 | 34.9303 | 58.1533 | ||
| DOPE | 10.000 | 11.6434 | 8.6631 | ||
| Cholesterol | 57.500 | 66.9497 | 25.8861 | ||
| DMG-PEG2000 | 2.500 | 2.9109 | 7.2975 |
Calculated total: 100.0000 mg lipid. RNA, solvent and process loss are excluded.
B13
rounded mol% (sum 100.01)SI Table S2
| Component | Molecular weight | Molar parts | Normalized mol% | Amount (µmol) | Weight (mg) |
|---|---|---|---|---|---|
| Lipid 6 | 29.997 | 34.7377 | 57.8327 | ||
| DOPE | 14.999 | 17.3688 | 12.9229 | ||
| Cholesterol | 53.125 | 61.5205 | 23.7869 | ||
| DMG-PEG2000 | 1.880 | 2.1769 | 5.4575 |
Calculated total: 100.0000 mg lipid. RNA, solvent and process loss are excluded.
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 |
|---|---|---|
| RNA complexation | N/P 10 selected; positivity plateaued near N/P 5, whereas fluorescence intensity was highest at N/P 10. | Main Fig. 3 and optimization text |
| Phases / mixing | Ethanol lipid phase and RNA in 10 mM citrate, pH 4; ethanol:aqueous 1:3 by microfluidic mixing. Total flow rate not located. | STAR Methods |
| Purification | 14 kDa dialysis against approximately 50 volumes DPBS overnight at 4 °C; 0.22 µm filtration. | STAR Methods |
| T-cell preparation | Activate for 48 h; IL-2 100 U/mL and 2% FBS in the reported culture workflow. LNP exposure 6 h, three washes, then expression readout. | STAR Methods |
| Optimized T-cell condition | 150 ng reporter mRNA per 5 × 10⁵ cells with ApoE4 1 µg/mL in the optimization text. | Main Fig. 3; do not transfer this dose to the inconsistent CAR passage |
3. Particle characterization
| Measurement / batch | Reported result | Evidence context |
|---|---|---|
| Initial-screen Lipid 6 LNP | 79.6 nm; PDI 0.134; encapsulation 99.40%; zeta potential 1.13 mV | SI Table S1; not a B13-specific release specification |
| Screened formulations | 26 DOE entries, A1–A13 and B1–B13 | SI Table S2 |
| High-lipid-fraction formulations | Some formulations containing 50% ionizable lipid precipitated on storage | Main optimization discussion |
4. Performance by experiment
| Experiment and dose | Readout and comparator | Result and source |
|---|---|---|
| Mouse reporter delivery Luciferase mRNA; intravenous or intramuscular; 0.5 mg/kg | Readout: 6 h; IM time-course also follows expression through 72 h Comparator: SM-102 and ALC-0315 study formulations | Both routes were tested. The 6 h comparison and the IM persistence experiment are separate endpoints; no cross-route fold ratio is inferred.Fig. 2; n=3, mean ± SD; ANOVA as specified in the figure |
| Formulation optimization Activated human T cells; eGFP reporter; optimization conditions above | Readout: 24 h expression assessment Comparator: Initial recipe and DOE formulations | B11 and B13 yielded the strongest fluorescence intensity; B-series formulations produced >90% positive cells under the reported assay.Fig. 3; SI Table S2 |
| Cell-type breadth Reporter mRNA in primary immune-cell preparations | Readout: Study expression endpoint Comparator: Cell-type-specific untreated / assay controls | Reported positivity: DC/NK >90%, TIL >97%, activated T cells >95%, unactivated T cells >80%. These are separate cell populations, not pooled efficacy.Fig. 4; n=3 donors |
| CAR expression and phenotype Primary human T cells; CAR mRNA | Readout: 24 h CAR expression Comparator: Mock-treated cells | Approximately 90% CAR-positive cells; viability, proliferation, CD4/CD8 distribution and tested exhaustion markers were not significantly altered in the reported comparison. CAR dose wording is inconsistent (see interpretation).Fig. 5; n=3 |
| Antigen-dependent killing MUC1-positive SKOV3 / MDA-MB-468 targets; 10⁴ target cells seeded 24 h before effectors; varied E:T ratios | Readout: Real-time cytotoxicity assay Comparator: Mock T cells and assay controls | CAR T-cell activity depends on target line and E:T ratio. No single killing percentage is assigned across these conditions.Fig. 6; assay Methods |
| Storage of RNA-loaded LNP 4 °C formulation storage; eGFP delivery assay | Readout: Up to 110 days Comparator: 25 °C and 37 °C storage conditions | The SI reports near-complete eGFP positivity and >80% viability after 4 °C storage. Warmer conditions lose activity; this is not a neat-lipid shelf-life claim.SI Fig. S3 |
Mechanistic evidence
ApoE supplementation and formulation optimization were evaluated experimentally. The study supports context-specific transfection of immune cells; it does not establish the same performance in every donor or activation state.
5. Interpretation and unresolved details
- B13 totals 100.01 because of reported rounding. Mass calculations normalize the displayed values explicitly.
- The CAR text reports 150 ng/µL whereas the optimization text uses 150 ng per 5 × 10⁵ cells; these are not interchangeable. A figure legend also differs in ApoE units. Do not treat the CAR paragraph as a ready-to-use dosing instruction.
- N/P is a charge ratio, not an ionizable-lipid:RNA mass ratio. No automatic conversion is made.
- B11/B13 performance is not assigned the initial-screen particle size and PDI.
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
Gao H, et al. Lipid-based mRNA chimeric antigen receptor T cells for enhanced in vitro antitumor activity. iScience. 2026;29:117390. doi:10.1016/j.isci.2026.117390
DOI: 10.1016/j.isci.2026.117390
Download experiment table (CSV) · Open printable Formulation & Performance guide
Revision FP-20261022 · Source review: 22 October 2026 · DC Chemicals