Fig. 2: Experimental design and evaluation of anti-PEG IgM production in rat.

a Schematic illustration of the experimental protocols. Wistar rats were injected intramuscularly with 0.009 (L-LNP group), 0.342 (M-LNP group), or 2.358 (H-LNP group) mg phospholipids/kg LNP on Day 0 and Day 21, respectively. Rats in the Control group were injected with PBS. Serum samples were collected at the indicated time points (Day 0, 3, 5, 7, 14, 21, 24, 26, 28, 35, 42 and 49) for further evaluation of the presence and level of anti-PEG antibodies with ELISA. b Quantitative analysis of anti-PEG IgM (Log₁₀ CONC) (log₁₀-transformed concentration of anti-PEG IgM) induced by LNP in rat serum. Data were presented as “mean ± standard deviation”, with n = 8 for Control group and n = 15 for all LNP-treated groups. Differences in anti-PEG IgM (Log₁₀ CONC) among various groups were analyzed using Mann–Whitney U test, with P values adjusted for FDR (false discovery rate). Peak levels of anti-PEG IgM (Log₁₀ CONC) induced during the initial and second injection cycle were as follows: L-LNP, 1.996 on Day 5 and 2.374 on Day 28; M-LNP, 2.704 on Day 5 and 3.692 on Day 26; H-LNP, 2.492 on Day 5 and 4.262 on Day 26. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. c Representative quality control data of ELISA for detecting anti-PEG IgM. The left image shows the intra-assay precision/Coefficient of Variation (CV%) of anti-PEG IgM standards (CV% = 3.884 ± 3.046%) and serum samples (CV% = 5.237 ± 6.192%), and the right image shows the goodness of fit/coefficient of determination (R² = 0.992 ± 0.004) of the standard curve (see “Methods” for acceptance criteria). Data were presented as “mean ± standard deviation”, with n = 56 for intra-assay precision (CV%) of anti-PEG IgM standards, n = 636 for intra-assay precision (CV%) of serum samples and n = 8 for R². d Time-course of anti-PEG IgM induced by PEGylated LNP. The changing curves of mean anti-PEG IgM (Log₁₀ CONC) levels over time were fitted by the R package called “ggalt”.