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

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — A The 1:32 mAB-protamine conjugate mixture was depleted of excess free SMCC-protamine (free P) by protein G-affinity chromatography. Fractions without excess free SMCC-protamine were compared to fractions still containing excess free SMCC-protamine and the unconjugated SMCC-protamine (without αEGFR-mAB-P) in dynamic light scattering spectroscopy (DLS). Only fractions containing αEGFR-mAB-P, excess unconjugated SMCC-protamine and siRNA exhibited the ability to form larger nanostructures overnight confirmed by dynamic light scattering spectroscopy (black curves, particle size 427 ± 12 nm), but not SMCC-protamine-depleted (red curves, 3.2 nm), or preparations only consisting of free SMCC-protamine and control (scramble, scr) siRNA (blue curves, 5.7 nm) after 2 h of self-assembly. B–D Non-purified αEGFR-mAB-P conjugate preparations in complex with Alexa488-siRNA were mounted on glass slides and subjected to fluorescence microscopy. The particles detected in DLS analysis could be verified in microscopy in fluorescence (B and C) as well as bright field microscopy (D, same frame as in C). E αEGFR-mAB-P conjugate depleted from free SMCC-protamine in complex with Alexa488-siRNA were mounted on glass slides and subjected to fluorescence microscopy. No nanoparticles could be observed here. Also, formulations lacking αEGFR-mAB-P, consisting only of SMCC-protamine formed no such particles visible in microscopy (not shown). F αEGFR-mAB-P/P-scrm siRNA nanoparticles were left to form for 2 h and subjected to electron microscopy on copper grids by phospho-Wolfram negative staining. G–I αEGFR-mAB-P/P-Alexa488-siRNA nanocarriers formed for 2 h (G, green), were immobilised o/n on treated glass surface, were stained with Alexa647-anti-human-IgG (αhuman-IgG-Alexa647) (H, red). Nanocarrier structures show prominent staining of αhIgG-Alexa647 of the targeting cetuximab antibodies only on surface regions and siRNA within the vesicles (I, overlay).