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Lipid S4
Formulation & Performance
Initial S4
Main pp. 3–4; original molar parts retained. OS4T uses a different PEG lipid and is excluded from these mass calculations.
| Component | Molecular weight | Molar parts | Normalized mol% | Amount (µmol) | Weight (mg) |
|---|---|---|---|---|---|
| Lipid S4 | 45.889 | 65.6698 | 56.5844 | ||
| DOPE | 22.945 | 32.8349 | 24.4302 | ||
| Cholesterol | 30.593 | 43.7799 | 16.9275 | ||
| DMG-PEG2000 | 0.574 | 0.8209 | 2.0579 |
Calculated total: 100.0000 mg lipid.
Calculated catalog planning quantities; RNA, solvent and process loss are excluded. DMG-PEG2000 uses 2507. Component mass = total lipid mass × molar parts × molecular weight / sum(parts × molecular weight). This conversion does not infer N/P.
Optimized OS4
Main pp. 3–4; original molar parts retained. OS4T uses a different PEG lipid and is excluded from these mass calculations.
| Component | Molecular weight | Molar parts | Normalized mol% | Amount (µmol) | Weight (mg) |
|---|---|---|---|---|---|
| Lipid S4 | 28.419 | 44.7896 | 38.5930 | ||
| DOPE | 28.419 | 44.7896 | 33.3248 | ||
| Cholesterol | 42.629 | 67.1844 | 25.9768 | ||
| DMG-PEG2000 | 0.533 | 0.8398 | 2.1054 |
Calculated total: 100.0000 mg lipid.
Calculated catalog planning quantities; RNA, solvent and process loss are excluded. DMG-PEG2000 uses 2507. Component mass = total lipid mass × molar parts × molecular weight / sum(parts × molecular weight). This conversion does not infer N/P.
Formulation & In Vivo Data
Formulation identity and evidence scope
Lipid S4 is an SR-57227-derived ionizable lipid studied for systemic mRNA delivery to the mouse brain. Initial S4, optimized OS4 and Tat-conjugated OS4T are distinct research formulations. The highest reported brain expression and glioblastoma treatment results used OS4T. These are formulation-specific preclinical results.
1. Lipid composition
| Component | Initial S4 mol% | OS4 mol% | MW used (g/mol) |
|---|---|---|---|
| Lipid S4 | 45.8891 | 28.4192 | 861.65 |
| DOPE | 22.9446 | 28.4192 | 744.03 |
| Cholesterol | 30.5927 | 42.6288 | 386.65 |
| DMG-PEG2k | 0.5736 | 0.5329 | 2507 |
Original molar parts: initial S4 = 60:30:40:0.75 (sum 130.75); OS4 = 40:40:60:0.75 (sum 140.75). mol% = parts / sum of parts × 100; rounding can produce a 0.0001% display difference. Calculations use unrounded parts. OS4 corresponds to formulation 24 in Fig. 2A. [1: pp. 3–4]
2. OS4T requires a different PEG lipid and Tat coupling
Replace DMG-PEG2k with DSPE-PEG2000-maleimide, then attach the cysteine-containing Tat peptide shown in Fig. 3A: GRKKRRQRRRPPQC. The Methods describe PEG replacement in the OS4 formulation; they do not list a separate numerical OS4T ratio. Do not substitute unconjugated OS4 for OS4T.
- Dissolve lipids in ethanol at the selected molar ratio and mix with aqueous mRNA. The paper states a lipid:mRNA mass ratio of 10:1.
- For peptide conjugation, dialyze the LNPs in PBS.
- Mix cysteine-containing peptide and maleimide-functionalized LNPs at cysteine:maleimide = 2:1 mol/mol; allow Michael addition for at least 1 hour at room temperature.
The paper does not explicitly define whether “lipid” in the 10:1 mass ratio means S4 alone or all lipids. N/P, mixing device, flow rates, aqueous/ethanol volume ratio, formulation-buffer pH, post-coupling purification and measured Tat coupling yield are not specified in the supplied documents. Derived component weights and stock volumes are provided below; the RNA mass basis remains unresolved. [1: pp. 5, 10]
3. Particle properties
| Formulation | Size | PDI | Encapsulation | Zeta potential | Source |
|---|---|---|---|---|---|
| Initial S4 | ~120 nm | ~0.10 | ~83% | ~−7 mV | SI p. 7, Figs. S2–S3 |
| OS4 | ~100 nm | ~0.03 | Not individually stated | ~−7 mV | Main p. 5, Fig. 3B; SI p. 8, Fig. S5 |
| OS4T | ~130 nm | ~0.20 | Not individually stated | ~+17 mV | Main p. 5, Fig. 3B; SI p. 8, Fig. S5 |
All approximate numbers in this table are visual estimates of plotted means, not author-tabulated values or release specifications. n = 3 in the cited characterization plots. Error bars were not digitized. Exact means/SDs and apparent pKa are not provided in the supplied text. Initial S4 encapsulation must not be assigned to OS4 or OS4T.
4. Delivery, efficacy and tolerability
| Formulation / assay | Experimental conditions | Reported result | Source |
|---|---|---|---|
| OS4 · brain FLuc | C57BL/6J mice; IV 0.6 mg mRNA/kg; 6 h; n = 3 | 13.3-fold MC3, 12.5-fold SM-102, 9.6-fold ALC-0315, and 2-fold initial S4. | Main pp. 3–4, Fig. 2D–E |
| OS4T · brain FLuc | Same dose and endpoint; separate comparator panels; n = 3 | 12.7-fold OS4 in Fig. 3C. In Fig. 3D–E, 59.1-fold MC3 and 17.2-fold MC3T. | Main pp. 3, 5, Fig. 3C–E |
| OS4 / OS4T · brain-cell GFP | C57BL/6J mice; IV 1 mg mRNA/kg; 12 h; n = 3 | OS4 improved GFP-positive fractions versus MC3 by about 8× in neurons, 4× in astrocytes, 6× in microglia and 3× in BCECs. OS4T further improved neurons, microglia and BCECs. | Main p. 6, Fig. 4A |
| OS4T · Cre reporter activation | Ai14 mice; one IV dose, 1 mg Cre mRNA/kg; Results specify day 7 | tdTomato-positive neurons 13.1 ± 1.2%; astrocytes 9.9 ± 1.2%; microglia 10.2 ± 0.4%; BCECs 11.6 ± 1.6%. MC3: <2%. | Main pp. 6–7, Fig. 4B–C |
| OS4T–eIL-12 · orthotopic GBM | CT-2A-Luc tumors in C57BL/6J mice; IV 1 mg mRNA/kg on days 3, 8 and 13; survival n = 7 | Median survival 37 days versus 17 days for PBS. This result requires Tat-conjugated OS4T and engineered IL-12 mRNA. | Main pp. 7–8, Fig. 5D–F |
| OS4 / OS4T · acute tolerability | IV FLuc mRNA 1 mg/kg; AST/ALT/BUN at 24 and 48 h; n = 3; histology at 48 h | Biomarkers were comparable to PBS; no notable tissue pathology in the reported short-term mouse experiment. | Main pp. 5–6, 10; SI p. 12, Fig. S11 |
Fold changes compare complete LNP formulations in their own experimental panels. Do not multiply fold changes across panels. MC3 and SM-102 controls used DSPC and 50:10:38.5:1.5; ALC-0315 used DSPC/ALC-0159 and 45.8:10.4:42.2:1.6. MC3T denotes the peptide-modified comparator. BCECs = brain capillary endothelial cells. [1: p. 10]
5. Interpretation and unresolved source details
- Cre timing: Results and Fig. 4C indicate a day-7 single-dose readout and doses on days 0, 7 and 14 with analysis on day 21. The Methods instead state collection 5 days after the final Cre dose. These descriptions are not fully consistent; retain the figure/Results schedule and confirm the protocol before replication. [1: pp. 6–7, 10]
- BBB mechanism: Tat improved transport in the bEnd.3/N2a Transwell model, but did not further improve uptake or endosomal escape versus OS4 in the reported N2a assays. This does not establish exclusive brain delivery; Tat also increased lung delivery versus unmodified counterparts. [1: pp. 5–6, 9; 2: Figs. S7–S9]
- Payload contribution: GBM survival and reduced off-target cytokine activity concern engineered IL-12, not S4 alone. Short-term mouse tolerability does not establish human safety or chronic tolerability. [1: pp. 7–8; 2: Figs. S13–S14]
Preparation molecular weight: 861.65 g/mol. The catalog formula field omits chlorine present in the supplied paper structure and catalog SMILES; formula verification remains open. All S4 mass-to-mole calculations in this guide use 861.65 g/mol.
6. Product handling
Follow the vial storage conditions. Keep pure lipid tightly sealed and protected from light; the product-page storage period is 1 year at −20 °C. Prepare fresh solutions where possible; if stored, use tightly sealed aliquots and avoid repeated freeze–thaw cycles. The product page lists solutions at −80 °C for 6 months or −20 °C for 1 month. These are supplier handling statements, not stability data for OS4/OS4T LNPs. Use 861.65 g/mol for S4 molar stocks and confirm complete dissolution.
Calculated S4 stock preparations
S4 molecular weight = 861.65 g/mol. 1 mg = 1.160564 μmol; 1 mM = 0.86165 mg/mL; 5 mM = 4.30825 mg/mL; 10 mM = 8.6165 mg/mL.
| S4 mass | Amount (μmol) | Final volume: 1 mM | Final volume: 5 mM | Final volume: 10 mM |
|---|---|---|---|---|
| 10 mg | 11.6056 | 11.6056 mL | 2.3211 mL | 1.1606 mL |
| 25 mg | 29.0141 | 29.0141 mL | 5.8028 mL | 2.9014 mL |
| 50 mg | 58.0282 | 58.0282 mL | 11.6056 mL | 5.8028 mL |
| 100 mg | 116.0564 | 116.0564 mL | 23.2113 mL | 11.6056 mL |
| 250 mg | 290.1410 | 290.1410 mL | 58.0282 mL | 29.0141 mL |
V (mL) = mass (mg) × 1000 / [861.65 × concentration (mM)]. These are final solution volumes, not measured solubility limits. Dissolve in compatible solvent and make up to the stated final volume. Ethanol is the lipid-phase solvent in the paper; verify complete dissolution.
OS4T: PEG replacement and Tat feed calculation
At 25 mg S4, retaining OS4 molar parts 40:40:60:0.75 while replacing DMG-PEG2k with DSPE-PEG2000-maleimide gives 0.544014 μmol maleimide PEG lipid. The reported cysteine:maleimide feed ratio 2:1 corresponds to 1.088029 μmol Tat peptide, assuming one cysteine per peptide and one maleimide per PEG lipid. The sequence shown is GRKKRRQRRRPPQC.
This is a stoichiometric planning calculation based on the Methods description of PEG replacement; a separate numerical OS4T ratio is not tabulated. DSPE-PEG2000-maleimide mass (mg) = PEG-lipid amount (μmol) × its supplier average MW / 1000. Peptide mass uses the exact supplied peptide/salt MW in the same way. Do not use DMG-PEG2k MW 2507 for DSPE-PEG2000-maleimide. Feed stoichiometry does not establish coupling yield, accessible maleimide or final peptide loading.
RNA mass and N/P: calculation limits
The paper reports lipid:mRNA = 10:1 by mass without explicitly defining whether lipid means S4 alone or total lipid. For the 10 mg total-lipid OS4 example above, assuming total lipid:mRNA = 10:1 gives 1.0000 mg RNA; assuming S4:mRNA = 10:1 gives 0.3859 mg RNA. These are alternative calculations, not a resolved literature recipe.
N/P, pKa, particle size, encapsulation and delivery performance cannot be determined from S4 molecular weight. Literature results remain in their separate experimental sections.
Sources
[1] Cao D et al. Lipid nanoparticles for mRNA delivery in brain via systemic administration. Science Advances 11, eadw0730 (2025). DOI: 10.1126/sciadv.adw0730. Main article PDF: 14 supplied pages, with article content on pp. 1–13.
[2] User-supplied Supplementary Materials, 18 PDF pages. Page references count from the first PDF page. Particle properties: p. 7; CPP zeta potential: p. 8; safety: Fig. S11. Values labeled calculated or estimated are distinguished from directly reported results.