iL-19 — Formulation & Performance

Literature formulation

Component order below is standardized as iL-19 / DSPC / cholesterol / DMG-PEG2000. The paper lists cholesterol before DSPC; the formulation itself is unchanged.

Total: 100 mol%. Ionizable (cationic) lipid:circRNA mass ratio: 30:1. N/P ratio: not reported. The 30:1 ratio is not a total-lipid:RNA ratio and is not N/P.

Source: Main p. 4; SI §2, p. 3; §5.1, p. 27; §6.3, p. 34 [1].

Component mass calculator

Calculate lipid amounts from total lipid quantity. PEG2000 is a chain designation; the complete conjugate molecular weight must be confirmed for the batch.

Calculated mg = total µmol × mol% ÷ 100 × MW ÷ 1000. Mass fractions use the summed component mass. No RNA amount or N/P is inferred.

Preparation details & reproducibility notes

Source: SI §2, pp. 3–5; §6.5, p. 39 [1].

Formulation optimization

The authors varied helper lipids, sterols and lipid:circRNA mass ratios. The standard iL-19 formulation remained the preferred condition in the tested IM screen; substituting DOPE or alternative sterols reduced the displayed expression.

Female BALB/c mice, IM, circLuc, figure-labeled dose 1.5 µg, 12 h; n = 2. Percentages are printed figure labels, not a statistical proof that every pair differs. No error bars are reconstructed.

Source: SI Fig. S7, p. 34 [1].

Physicochemical properties: circRNA vs linear mRNA

Reported mean ± SD, triplicate measurements. Size/PDI/zeta: DLS; encapsulation: RiboGreen; apparent LNP pKa: TNS fluorescence titration (half-maximal fluorescence). These are formulated-particle values, not intrinsic properties of neat iL-19.

Source: SI Table S6, p. 27; Table S7, p. 30; methods pp. 3–4 [1].

Reference

Cook A, Yadav PK, Helmy EW, Vlasova K, Chang HY, Wender PA. Circular RNA Delivery In Vitro and In Vivo Using Doubly Branched Aminoethoxy-Functionalized Ionizable Lipid Nanoparticles. Journal of the American Chemical Society (2026). DOI: 10.1021/jacs.6c14175. Main article (9 PDF pages) and Supporting Information (85 pages). Volume and final pagination are not assigned in the supplied version.

Why iL-19 is the overall lead

iL-19 and iL-20 were both strong C2C12 candidates: the plotted expression values are 2800% and 2890% of SM-102, respectively. iL-20 is numerically higher in that cell assay, but the authors selected iL-19 as the overall lead based on the combined structure–delivery and in vivo IM evidence. The study does not establish iL-19 as best for every route or payload.

Source: Main Figs. 3–4, pp. 4–5; SI Figs. S10–S11, pp. 37–38 [1].

In vitro circRNA expression: C2C12 myocytes

circLuc, 60 ng per well; 15,000 cells seeded per well; 12-hour expression readout. Main Fig. 3 reports n = 6 independent experiments, with SD and one-way ANOVA/Dunnett versus SM-102 (all six second-generation lipids P < 0.0001). The general SI procedure additionally describes triplicate wells; these are not substituted for the figure-specific n.

HEK293T experiments provide supporting evidence of the structure–delivery trends (SI Figs. S4–S6, n = 3). Cell luminescence is a protein-expression endpoint, not an editing rate.

Source: Main Fig. 3, p. 4; SI methods pp. 4–5 and Figs. S4–S6, pp. 32–33 [1].

Intramuscular validation: benchmark comparison

Female BALB/c mice, 6–8 weeks, approximately 20 ± 2 g; circLuc; 12 h; n = 6 per group. The experiment is labeled 1.5 µg circLuc and uses bilateral quadriceps injections (33 µL per quadriceps). The text does not unambiguously resolve whether the labeled dose is per site or across both sites, so no total-dose conversion is made here.

Original display of the rounded percentages printed in SI Fig. S11; numeric SD is not tabulated and is not reconstructed. SM-102 is normalized to 100%.

The authors report iL-19 expression approximately 2.5× SM-102, 7.8× ALC-0315, 33.4× DLin-MC3-DMA and 2.3× δO3. These reported fold values are retained rather than recalculated from rounded bar labels.

Benchmark LNPs used their respective published optimized formulations. The comparisons share the study model, cargo and readout, but do not use one identical lipid composition for every benchmark. One-way ANOVA/Dunnett tested comparisons against SM-102; iL-19 P < 0.0001. Significance is not inferred for each iL-19-versus-benchmark pair.

Source: Main p. 5 and Fig. 4; SI Fig. S11, p. 38 [1].

A separate extended IM screen used n = 4 per group (SI Fig. S10, p. 37). Its data are not pooled with the n = 6 benchmark study.

Preliminary intravenous experiment

In BALB/c mice given 3.3 µg circLuc by retro-orbital IV injection, 12-hour expression for iL-19 was plotted at 118% of SM-102 (100%), with n = 2. The authors describe efficacy as comparable, with qualitative liver localization. This small study does not establish statistically significant superiority, quantitative liver selectivity or performance in other organs.

Source: SI §6.5 and Fig. S12, p. 39 [1].

Structure–delivery analysis

Source: Main Figs. 2–3 and Results pp. 3–5; SI iL-19 p. 24 and precursor S9 p. 15 [1].

Stability & tolerability

Size, PDI and zeta potential showed negligible changes during the reported 14-day LNP storage study at 4 °C. This does not establish RNA integrity, preserved expression over that entire period, frozen-product shelf life or shipping stability.

Mice showed normal behavior, no observed acute clinical signs and no reported weight loss during the study. The authors specifically identify the positively charged lead formulations as needing further safety, immunogenicity and PK/PD evaluation. No clinical safety claim or vaccine-protection outcome is established.

Source: Main p. 5; SI Figs. S1–S3, pp. 28–29 [1].

Full reference

Cook A, Yadav PK, Helmy EW, Vlasova K, Chang HY, Wender PA. Circular RNA Delivery In Vitro and In Vivo Using Doubly Branched Aminoethoxy-Functionalized Ionizable Lipid Nanoparticles. Journal of the American Chemical Society (2026). DOI: 10.1021/jacs.6c14175. Main article (9 PDF pages) and Supporting Information (85 pages). Volume and final pagination are not assigned in the supplied version.