Formulation & Performance — 9C-SCC-10

Product identity and study scope

DC67451 is uncomplexed 9C-SCC-10. It is not preformed Zn-9C-SCC-10. The lung-selective results below require the zinc-coordinated system. Gd-12C-SCC-10, used elsewhere in the paper for imaging, is a different compound.

Values shown are literature-reported results for the cited formulation and study conditions. They do not guarantee performance of the supplied product in other formulations or experiments.

Formulations kept separate

Source: SI S29 §4; S9 Fig. S9; S11 Figs. S15–S16; S12 Fig. S18.

Component molecular weights and molar-to-mass conversion

The published ratios total 63 or 78 molar parts. The mol% and amounts below are calculated normalizations, not the source notation. Amounts use a 10 µmol total-component basis. A definitive molecular weight for the isolated zinc complex, including counterions or hydration, is not established by this SI. Its mass and total mixture mass fractions are therefore left unresolved.

Original zinc-coordinated formulation

Optimized zinc-coordinated formulation

Uncomplexed 9C-SCC-10 MW: 1481.40. This value must not be substituted for the zinc complex. For a free-lipid stock, 1 mg corresponds to 0.6750 µmol; a 1 mM solution requires 1.4814 mg/mL. Helper-lipid MW values are catalog calculation inputs; DMG-PEG2000 uses 2507.

Source: Raw ratios: SI S29 §4 and S11 Fig. S15. Normalized mol%, helper masses and parent stock conversions: calculated.

Reported zinc coordination and LNP preparation

The coordination experiment combines 33 mg (0.022 mmol) 9C-SCC-10 with 6.5 mg (0.022 mmol) zinc nitrate hexahydrate in 220 µL methanol at 40 °C for 24 h, followed by evaporation. This is a 1:1 ligand-to-zinc feed. The isolated counterion and hydration stoichiometry are not analytically established. The procedure contains a final “Zn-9C-SC-10” label although the stated starting lipid and target are 9C-SCC-10 / Zn-9C-SCC-10.

Source: SI S23, synthesis of Zn-9C-SCC-10.

The 21.4 µL lipid phase is rapidly pipette-mixed with mRNA in 64.2 µL of 25 mM sodium acetate, pH 5.2 (aqueous:organic = 3:1 v/v). Incubate for 20 min at room temperature, dialyze against 1× PBS for 2 h using a 3.5 kDa cutoff, and dilute to 200 µL for the reported intravenous experiment. The stock quantities are reproduced as a source example; they are not a validated mass reconstruction of the declared molar ratio. RNA loading, N/P and a total lipid:RNA mass ratio are not resolved.

The general method also states a 0.5 ng/µL working RNA concentration. That concentration at 200 µL gives 0.1 µg and does not reconcile with 0.2 mg/kg in an approximately 20 g mouse. Use the experiment-specific dose as reported; the dilution discrepancy remains unresolved.

Source: SI S29 §4 and S30–S31 §§11–12.

Uncomplexed lipid versus zinc-coordinated lipid

Source: SI S8 Fig. S7; S9 Fig. S9; S11 Fig. S15; S29 §5; S30 §11.

Zinc formulation optimization and delivery results

The original-to-optimized comparison showed increased Fluc expression, a smaller number-mean particle size and increased zeta potential while retaining lung-biased expression. Exact particle size, zeta potential, PDI and encapsulation efficiency are not entered as tabulated values because the supplied figures do not provide a complete numerical dataset.

Source: SI S11 Figs. S15–S16; S12 Fig. S18 (TEM).

Source: SI S12 Fig. S17. The caption uses a “Cy5” label in panel b despite the Fluc experiment context; organ signal proportions are interpreted within the stated Fluc experiment.

Functional Cre reporter delivery — zinc complex only

The readout is Cre-dependent tdTomato reporter activation with cell-population flow cytometry. These percentages are not bulk DNA-sequencing editing rates and are not results for uncomplexed DC67451.

Source: SI S16 Table S1; S31 §12; gating panels S14–S15.

Additional experiment context

Cy5-RNA uptake was examined in MLE-12 and A549 cells after 4 h using 125 ng RNA per treatment; fluorescence uptake is distinct from functional protein expression. Cy5-RNA biodistribution experiments used intravenous 0.2 mg/kg and a 6 h endpoint. For the optimized 30:15:30:3 composition, the Fig. S13 pie chart assigns 22% of major-organ Cy5 fluorescence to lung, which must not be substituted for the approximately 97% lung share of Fluc expression. The paper also examines metal-coordinated particles in serum-containing medium and short-term histology; those assays do not establish long-term or clinical safety.

Source: SI S10 Fig. S13; S13 Figs. S19–S20; S17 Fig. S23; S30 §§8–9.

Structure and analytical support

The neutral parent has a 1,4,7-triazonane core, three disulfide-containing arms, six ester groups and three decylthio tails. No stereochemistry is assigned to the three branched centers. The supplied SI includes labeled ¹H and ¹³C NMR spectra and HRMS for 9C-SCC-10. HRMS [M+H]⁺: calculated 1480.7135, found 1480.7164; the ion formula is C69H130N3O12S9⁺. Removing the added proton gives the neutral formula C69H129N3O12S9. The displayed average MW, 1481.40, is calculated from the verified neutral structure and is distinct from the HRMS m/z.

Source: SI S3 Fig. S1; S4–S6 Figs. S2–S4; S16 Table S1; S22–S23 synthesis.

Evidence coverage and remaining gaps

This analysis uses the complete supplied 32-page Supporting Information and the official article abstract. The full main article was not available for a complete figure-by-figure review. Unresolved details include the uncomplexed screening formulation ratio, N/P, the isolated zinc complex formula and MW, the dilution inconsistency and some figure-specific dose assignments. Gd-12C-SCC-10 MRI findings are not assigned to DC67451.

References

Chen, Z.; Yang, Y.; Qiu, X.; Zhou, H.; Wang, R.; Xiong, H. Crown-like Biodegradable Lipids Enable Lung-Selective mRNA Delivery and Dual-Modal Tumor Imaging In Vivo. J. Am. Chem. Soc. 2024, 146 (49), 34209–34220. DOI: 10.1021/jacs.4c14500.

Supporting Information: ja4c14500_si_001.pdf, S1–S32.