Fig. 4: In-depth analysis of lead siRNA-aNP physicochemical properties, in vivo behaviour and therapeutic application.
a, Lead aNP-siRNA (aNP18) composition (wt%). b, siRNA recovery, entrapment and retention, data represent mean ± s.d. of one experiment (n = 12 formulation batches). c, Apolipoprotein A1 (apoA1) content. Data represent mean ± s.d. of one experiment (n = 9 formulation batches). d, Hydrodynamic diameter represented as the number mean and dispersity. Data represent mean ± s.d. of one experiment (n = 12 formulation batches). e, Zeta potential. Data represent mean ± s.d. of one experiment (n = 6 formulation batches). f, Cryo-EM analysis of six individual aNP18-siRNA batches. g, Quantitative biodistribution of aNP18-89Zr-siRNA 15 min, 1 h, 4 h and 24 h after intravenous administration in mice (8 µCi per mouse) determined by ex vivo gamma counting. Radioactivity is expressed as %ID per gram. Data represent mean ± s.d. of one experiment, and each data point represents one animal (n = 3 mice). h, Schematic showing therapeutic effect of CCR2 silencing by inhibiting immunosuppressive monocyte migration to the tumour microenvironment. i, Schematic treatment regimen involving MC38-tumour-bearing mice (n = 5 mice per group) receiving repeated intravenous administrations of aNP18 containing scrambled siRNA (aNP18-siCtrl) or CCR2 siRNA (aNP18-siCCR2) (0.5 mg kg–1 per administration, cumulative dose of 3.5 mg kg–1). CCR2 expression was determined 14 days after the first administration by flow cytometry. j–l, CCR2 levels indicated by mean fluorescence intensity (MFI) in ly6Clo monocytes in the bone marrow (j), spleen (k) and blood (l). Data represent mean ± s.d. of one experiment (n = 5 mice) and were analysed using Student's t-test. Statistically significant differences between aNP18-siCCR2 versus aNP18-siCtrl formulations are indicated by: **P ≤ 0.01, ***P ≤ 0.001. m, Number of CCR2+ macrophages per gram of tumour tissue as determined by flow cytometry. Data represent mean ± s.d. of one experiment (n = 5 mice) n, Fraction of CCR2+ macrophages within tumour tissue expressed as percentage (%) of myeloid cells. Data represent mean ± s.d. of one experiment (n = 5 mice) and analysed using Student's t-test. Statistically significant differences between aNP18-siCCR2 versus aNP18-siCtrl formulations are indicated by: **P ≤ 0.01. o, Biocompatibility of siRNA-aNP18-siCCR2 as determined by serum levels of alanine aminotransferase (ALAT, left), aspartate aminotransferase (ASAT, middle left), creatinine (middle right) and urea (right) two days following treatment (cumulative dose of 3.5 mg kg–1). Data represent mean ± s.d. of one experiment (n = 3 or 4 mice). LLOQ, lower limit of quantification. p, Representative liver sections stained with haematoxylin and eosin from mice treated with aNP18-siCtrl (left) and aNP18-siCCR2 (right).
