Formulation & Performance — Lipid-392

DCL0020 · Updated 20 October 2026

Lead selection and study scope

Lipid-392 stLNP is the author-selected lead for intravenous lung RNA delivery. The work optimizes formulations of Lipid-392 rather than ranking a library of different lipid molecules. The final system removes cholesterol, selects POPE, adjusts N/P to 10 and uses 20 mM acetate at pH 4.0. Evidence includes mouse reporter expression, acute tolerability, Cre recombination, base editing, siRNA knockdown and an IL-10 treatment model.

Reported literature outcomes are specific to the tested formulation and experimental conditions; they are not batch release specifications or guaranteed performance.

Final formulation: cholesterol-free Lipid-392 stLNP

Component	Catalog number	mol%	Role
Lipid-392	DCL0020	90	Ionizable carrier
POPE	DC65745	8.5	Helper phospholipid
DMG-PEG2000	DC40169	1.5	PEG lipid

Source: Fig.2E; Results p.15351; Fig.3A–C; Methods p.15357.

Component molecular weights and mass-ratio tables

Calculated lipid-only quantities: mass fraction = (mol% × molecular weight) / Σ(mol% × molecular weight). Input molecular weights are shown as numbers; DMG-PEG2000 uses the catalog convention 2507. POPE uses 718.00, consistent with the catalog formula C39H76NO8P. These calculations do not include RNA, water, counterions, solvent, purification loss or overage.

Final stLNP — 90:8.5:1.5 mol%

Component	Molecular weight	mol%	Mass per 100 mg total lipid	Mass per 100 mg Lipid-392
Lipid-392	1275.05	90	92.085 mg	100.000 mg
POPE	718.0	8.5	4.897 mg	5.318 mg
DMG-PEG2000	2507	1.5	3.018 mg	3.277 mg

Initial G4 comparator — 50:10:38.5:1.5 mol%

Component	Molecular weight	mol%	Mass per 100 mg total lipid	Mass per 100 mg Lipid-392
Lipid-392	1275.05	50	70.600 mg	100.000 mg
DSPC	790.15	10	8.750 mg	12.394 mg
Cholesterol	386.65	38.5	16.485 mg	23.350 mg
DMG-PEG2000	2507	1.5	4.164 mg	5.899 mg

N/P 10 is a molar nitrogen-to-phosphate ratio, not a 10:1 lipid-to-RNA mass ratio. The source does not unambiguously define which nitrogens enter its N/P calculation. Lipid-392 contains three tertiary amines and six hydrazide nitrogens; they must not automatically be treated as nine equivalent protonatable amines. An RNA-to-total-lipid mass calculator is therefore not supplied.

Preparation and characterization

Parameter	Reported information
Buffer	20 mM acetate, pH 4.0 (B6)
N/P	10, selected in the N/P screen
Mixing	Rapid mixing of RNA-containing aqueous buffer with the ethanol lipid phase; aqueous:ethanol = 3:1 v/v. No microfluidic flow-rate setting is reported.
Incubation	10 minutes at room temperature after mixing
Buffer exchange	Dialysis against 1×PBS, pH 7.4, for 4 h using a 100 kDa MWCO membrane
Size / morphology	Cryo-TEM: well-dispersed spherical particles below 100 nm. DLS, PDI and EE are plotted in the SI; exact numerical means are not tabulated.
Apparent pKa	7.67 for the formulated stLNP, measured by TNS fluorescence with half-maximal fitted signal; pH 2.5–11, n=3.
Encapsulation	RiboGreen; EE = (total RNA − free RNA) / total RNA ×100. No exact final value transcribed from a graph.
Physical stability	SI S9 monitors size/PDI/EE through day 9; main text describes one week. Storage temperature and validated shelf life are not established by this result.

Source: Methods p.15357; Fig.3D–E; SI S9.

Sequential optimization: keep each comparison separate

Step	Selected condition	Matched comparison	Reported change
Ionizable lipid fraction	G8, 90 mol% Lipid-392	G4,50 mol%	~12-fold lung expression
Cholesterol removal	G8-5,90:8.5:0:1.5	G8-2,90:2:6.5:1.5	~1.9-fold lung expression
Phospholipid	POPE,8.5 mol%	DSPC,8.5 mol%	~1.7-fold lung expression
N/P	10	5/10/15/20	Author-selected condition; no exact fold assigned
Buffer	B6,20 mM acetate,pH4	B1,10 mM citrate,pH3	~8-fold lung expression

Source: Figs.1–3; SI S2–S8.

The authors describe an overall improvement exceeding 100-fold versus initial G4 after sequential optimization. This is not a comparison between two chemical identities, and stage-specific fold changes are not multiplied here. The N/P plot does not establish a statistically unique global optimum; 10 is the condition adopted by the authors.

Initial G1–G9 composition screen

Group	Lipid-392	DSPC	Cholesterol	DMG-PEG2000	Sum mol%
G1	0	14.1	54.4	1.5	70.0
G2	30	14.1	54.4	1.5	100.0
G3	40	12.1	46.4	1.5	100.0
G4	50	10	38.5	1.5	100.0
G5	60	7.9	30.6	1.5	100.0
G6	70	5.9	22.6	1.5	100.0
G7	80	3.8	14.7	1.5	100.0
G8	90	1.8	6.7	1.5	100.0
G9	100	0	0	0	100

Source: Fig.1B.

G1 sums to 70 mol% as printed; its missing fraction is not inferred. G9 has 0% PEG in the figure despite the general fixed-PEG statement. These source entries are retained for traceability and are not used as calculated preparation recipes.

G8 helper-lipid screen

Group	Lipid-392	DSPC	Cholesterol	DMG-PEG2000	N/P
G8-1	90	0	8.5	1.5	12
G8-2	90	2	6.5	1.5	12
G8-3	90	4	4.5	1.5	12
G8-4	90	6	2.5	1.5	12
G8-5	90	8.5	0	1.5	12

Source: SI Fig.S4A.

Buffer screen

Group	Buffer	Concentration	pH
B1	Citrate	10 mM	3.0
B2	Citrate	20 mM	3.0
B3	Citrate	10 mM	4.0
B4	Citrate	20 mM	4.0
B5	Acetate	10 mM	4.0
B6	Acetate	20 mM	4.0

Source: Fig.3A; buffer reporter comparison n=2.

Experiment-specific performance

Endpoint	Finding	Experimental context / comparator	Source
Initial formulation screen	G8 (90 mol% Lipid-392) gave approximately 12-fold higher lung expression than G4 (50 mol%).	Luc mRNA 0.1 mg/kg IV; C57BL/6 mice; 6 h; n=3; N/P 12; comparator: Matched G1–G9 formulation series	Fig. 1B–F; SI S2
Cholesterol removal	G8-5 (90:8.5:0:1.5) gave approximately 1.9-fold higher expression than G8-2 (90:2:6.5:1.5).	Mouse IV luciferase screen; dose/time not separately restated in Fig.2 caption; n=3; Lipid-392/DSPC/cholesterol/DMG-PEG2000; N/P 12; comparator: G8-1 through G8-5	Fig. 2A–B; SI S4
Phospholipid selection	POPE gave the greatest reported expression, approximately 1.7-fold versus DSPC.	Mouse IV luciferase screen; dose/time not separately restated in Fig.2 caption; n=3; Lipid-392/helper/DMG-PEG2000 90:8.5:1.5; N/P 12; comparator: DSPC, DOPC, 4ME, DOPE, POPE	Fig. 2C–D; SI S5
N/P selection	Authors selected N/P 10 for the POPE-containing three-component formulation.	Mouse IV luciferase screen; dose/time not separately restated in Fig.2 caption; n=3; comparator: N/P 5, 10, 15, 20	Fig. 2E–F; SI S6
Buffer selection	B6 (20 mM acetate pH 4.0) produced approximately 8-fold higher lung expression than B1 (10 mM citrate pH 3.0).	Lipid-392 stLNP; IV luciferase screen; n=2; exact screen dose not restated in Fig. 3A–C caption; comparator: Six buffer conditions B1–B6	Fig. 3A–C; SI S8
Matched lung benchmark	4.93-fold higher lung luciferase signal; approximately 99% of measured lung/liver/spleen signal localized to lung.	Luc mRNA 0.25 mg/kg IV; 6 h; n=4; comparator: DOTAP-5-LNP	Fig. 3F–G; Results p.15353
RNA-loaded acute tolerability	Displayed ALT/AST and inflammatory markers were comparable to PBS; DOTAP-5-LNP elevated multiple markers.	Luc mRNA 1 mg/kg IV; 6, 24, 48 h; n=5; comparator: PBS and DOTAP-5-LNP	Fig. 3H; SI S12
High-dose empty particles	Transient liver-enzyme/cytokine elevations; highest-dose markers remained mildly elevated at 48 h. No overt distress or organ damage reported over the observation period.	EMPTY stLNP; lipid exposure equivalent to RNA doses 5, 8, 10, 12 mg/kg; IV; 6, 24, 48 h; n=3; comparator: PBS	Fig. 2G–H; SI S7; Results p.15351
Lung-cell uptake	Cy5-positive: approximately 82% endothelial, 78% epithelial and 50% total lung cells. This is uptake, not editing.	Cy5-labeled mRNA; IV; 6 h; n=3; dose CONFLICT: 0.3 mg/kg in Fig. 4A caption, 0.5 mg/kg in Results/Methods; comparator: PBS	Fig. 4A–D; SI S13; Methods p.15358
Cre reporter activation	tdTomato-positive: approximately 68% endothelial, 70% epithelial, 32% immune and 37% total lung cells.	Ai14 mice; Cre mRNA IV; 48 h; n=3; dose CONFLICT: 0.5 mg/kg in Fig. 4E caption, 0.3 mg/kg in Results/Methods; comparator: PBS	Fig. 4E–H; Methods p.15358
Single-dose kinetics	Luciferase signal detectable from 0.5 h to 48 h; the 48 h image uses a different color scale.	Luc mRNA 0.75 mg/kg IV; 0.5, 2, 6, 12, 24, 48 h; n=4; comparator: Within-formulation time course	Fig. 5A–C
Repeat dosing	Similar expression peaks were obtained after dosing every 48 h over three administrations.	Luc mRNA 0.75 mg/kg IV at 0, 48, 96 h; follow-up through 144 h; n=4; comparator: Repeated stLNP administration	Fig. 5A/D–E
Adenine base editing	3.24% editing in lung tissue by NGS; separate from the much larger Cre reporter percentages.	ABE reporter mice; ABE mRNA:sgRNA 2:1 mass ratio; total RNA 1 mg/kg IV; day 5; n=3; comparator: PBS	Fig. 5F–H; Methods p.15358
Acute lung injury treatment	IL-10 delivery reduced TNF-alpha and IL-6 in lung tissue and BALF and improved histology. Empty LNP did not produce the same effect.	LPS-induced mouse ALI; IL-10 mRNA 1 mg/kg IV 2 h after induction; n=4; LPS dose and endpoint timing conflict between Results and Methods; comparator: Healthy, LPS/PBS, LPS/empty LNP	Fig. 5I–K; SI S15–S16; Methods p.15358
siTie2 knockdown	Lung Tie2 mRNA ED50 = 0.056 mg/kg; protein suppression also evaluated.	Single IV siTie2 dose-response; lung tissue at 72 h; n=3; exact dose range differs between plotted series and Methods list; comparator: PBS / dose-response	Fig. 6A–C; Methods p.15359
siEGFP knockdown	Approximately 50% lung EGFP mRNA knockdown with reduced reporter fluorescence.	EGFP reporter mice; IV; day 3; n=3; dose CONFLICT: 1 mg/kg in Fig. 6D caption, 2 mg/kg in Methods; comparator: LNP-only control	Fig. 6D–F; Methods p.15359
Particle characterization	Apparent LNP pKa 7.67 by TNS; spherical particles below 100 nm by cryo-TEM. No exact DLS mean assigned from a plotted bar.	Final stLNP; TNS half-maximal fitted fluorescence, pH 2.5–11; n=3; comparator: No isolated-lipid pKa measurement	Fig. 3D–E
Short-term physical stability	Main text describes one-week stability; SI S9 plots observations through day 9. No validated shelf life or storage temperature assigned.	Size, PDI and encapsulation monitored; n=3; comparator: Within-formulation time course	SI S9; Results p.15353

Benchmark and dose interpretation

DOTAP-5-LNP was MC3/DSPC/cholesterol/DMG-PEG2000/DOTAP = 25:5:19.3:0.8:50 as printed (sum 100.1 from rounding). It is a separate five-component comparator. The 4.93-fold result is a matched 0.25 mg/kg reporter experiment; acute safety was compared at 1 mg/kg RNA. The 12 mg/kg result uses empty particles at an RNA-equivalent lipid exposure, not demonstrated delivery of 12 mg/kg RNA without toxicity.

Approximately 99% refers to the relative reporter signal among the measured organs, not 99% of injected dose, 99% of all lung cells, or 99% editing. Uptake, Cre reporter activation, ABE editing and siRNA knockdown are distinct endpoints.

Structure and analytical identity

Item	Evidence
Structure mapping	SI Fig.S1 labels the final structure NNCC14; the figure caption and synthesis identify it as Lipid-392. NNCC14 is retained as a source-scheme alias.
Architecture	N-methylbis(3-aminopropyl)amine-derived scaffold, three tertiary amines, three palmitoyl diacylhydrazide branches and one N-linked 2-hydroxydecyl arm. No ester linker is present.
Formula	C74H147N9O7; explicitly reported and independently recovered from the depicted neutral structure.
Average molecular weight	1275.05; calculated from the verified neutral structure.
Salt / stereochemistry	No isolated counterion or solvate assigned. The OH-bearing stereocenter is unspecified; no enantiopurity claim.
HRMS discrepancy	SI reports [M+H]+ calculated 1275.15 and found 1275.1793. Toolkit theoretical value is 1275.1496; the approximately 23 ppm offset is unresolved without the raw spectrum.
Analytical record	SI p.3 supplies 1H NMR shifts and ELSD-HPLC 96.30% for the research batch. This is not a certificate for a DC Chemicals lot.
Derived SMILES	`CCCCCCCCCCCCCCCC(=O)NNC(=O)CCN(CCCN(C)CCCN(CCC(=O)NNC(=O)CCCCCCCCCCCCCCC)CC(O)CCCCCCCC)CCC(=O)NNC(=O)CCCCCCCCCCCCCCC`

Source: SI Fig.S1 pp.2–3; main Fig.1A.

Synthesis and structural interpretation

The reported route prepares palmitoyl hydrazide from methyl palmitate, installs an acryloyl group to form M-2, and opens 1,2-epoxydecane with N-methylbis(3-aminopropyl)amine to form M-3. Three aza-Michael additions of M-2 onto M-3 give Lipid-392; the final step reports 37.8% isolated yield. The M-3 text name contains a diol wording inconsistency; the scheme and formula show one alcohol.

Observed formulation relationship: raising Lipid-392 content, removing cholesterol, selecting POPE and optimizing the aqueous buffer improved lung reporter expression in the tested series. This work does not isolate headgroup, chain-length or linker SAR using matched molecular analogues.

Design hypothesis: the hydrazide linkers may influence adsorbed protein corona and pulmonary distribution; this is proposed rather than directly established. Priorities for future matched studies include hydrazide-linker substitution, acyl-chain length and the hydroxyl-bearing arm, while keeping formulation and dose fixed.

Source discrepancies and limits

Issue	How it is handled
1	Published HRMS [M+H]+ calculated 1275.15, found 1275.1793; toolkit theoretical 1275.1496. The approximately 23 ppm difference remains unresolved without the raw spectrum. Depicted connectivity, route and reported neutral formula agree.
2	Fig. 4 Cy5-mRNA dose 0.3 mg/kg versus Results/Methods 0.5 mg/kg; Cre dose 0.5 mg/kg versus Results/Methods 0.3 mg/kg. Do not silently swap these.
3	ALI LPS induction: Results 2 mg/kg versus Methods 10 micrograms/mouse; endpoint 24 h after treatment versus 24 h after induction. Not presented as a fully resolved reproduction protocol.
4	siEGFP dose: Fig. 6D 1 mg/kg versus Methods 2 mg/kg. siTie2 Methods dose list omits lower plotted doses.
5	Initial G1 recipe sums to 70 mol% as printed. G9 has zero PEG despite the general fixed-PEG description. Neither is corrected by inference.
6	DOTAP-5-LNP printed ratio sums to 100.1 because of rounding. No silently normalized mass recipe supplied.
7	N/P definition does not unambiguously specify which lipid nitrogens are counted. No RNA mass-to-lipid conversion is supplied.
8	No CAS, assigned stereochemistry, isolated counterion, commercial lipid pack prices, batch COA or raw spectral/source-data files supplied.
9	S9 does not establish a neat-material or stock-solution shelf life; main text one week versus plot through day 9.
10	Lipid-392 is the investigated lipid; G1–G9 and B1–B6 are formulations of that lipid, not different chemical compounds.

References

Fei Y, Lin Y, Liu P, Zhong K, Gao Y, Ma Q, Zhou Z, Luo S, Hu R, Cheng Q. Optimization of Lipid Nanoparticles for Safe and Versatile Lung-Targeted RNA Delivery and Disease Therapy. ACS Nano. 2026;20:15347–15362. doi:10.1021/acsnano.6c03013. Original article and Supporting Information.

Main article: Figs.1–6 and Methods pp.15357–15359. Supporting Information: Fig.S1 and synthesis pp.2–3, Figs.S2–S16 and Tables S1–S4. The supplied full article and SI were inspected; separate raw source data and raw analytical spectra were not supplied.