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. 2022 Feb 26;41(15):2210–2224. doi: 10.1038/s41388-022-02241-w

Fig. 4. Deciphering preconditions for effective nanoparticle formation between anti-EGFR-mAB-SMCC-protamine conjugate, free SMCC-protamine and siRNA.

Fig. 4

A–F αEGFR-mAB was conjugated with rising excess of free SMCC-protamine ranging from 1:1 molar ratio to 1:100 excess of SMCC-protamine in chamber slides (see Fig. 1A for reaction details). Resulting conjugates were used to bind siRNA in a cell-free standardised assay. The 1:32 ratio mAB to SMCC-protamine formed a homogeneous population of stable particles in the range up to 0.5–2 µm (D), see detail in G, whereas the other conjugates were incompetent to form stable particles. Stable particles subjected to confocal laser scan (CLS) microscopy optical sections showed a homogeneous distribution of fluorescent Alexa488 signals within the particle (G). H–K αEGFR-mAB-P depleted of free SMCC-protamine can form nanoparticles when at 32x free SMCC-protamine (J) is re-added to the conjugate with Alexa488-siRNA. L αEGFR-mAB-P depleted of free SMCC-protamine is effective in the inhibition of A549 cell colony formation when 32x free SMCC-protamine is re-added to the conjugate with anti-KRAS-siRNA in contrast to unspecific control (scrambled, scr) siRNA. Mean ± SD of three independent experiments. Two-sided t-test, *p < 0.05 (two-sided t-test). M–S Vesicle formation with 60 nM αEGFR-mAB-P in presence of 32x SMCC-protamine and rising (1:0.6–1:40) molar ratios of Alexa488-control-siRNAs compared to the antibody concentration. Vesicle formation can be observed at 5 to 10x molar excess of siRNA (P, Q). Upper panels: Fluorescence microscopy of Alexa488-siRNA positive vesicle. Lower panels: Phase contrast of the same preparations as in upper panels.