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Fig. 2 Design and physico-chemical characterization of targeted LNPs. A) Schematic representation of the targeted LNP production: a lipid phase in ethanol containing MC3, Cholesterol, DSPC, DSPE-PEG2000, DSPE-PEG2000 folate (FA), DSPE-PEG2000 maleimide in different molar ratios were mixed with an aqueous phase containing plasmid DNA using a microfluidic mixer (N/P ratio = 6). Subsequently, Herceptin (Her) was coupled to maleimide moieties via Michael addition. Purified samples were analyzed using dynamic light scattering to determine the hydrodynamic diameter (DH, B), the polydispersity index (PDI, C), and ?-potentials at pH 7.4 (D). Results are shown for Her- (turquoise), FA- (pink), and Dual- (purple) LNPs. Values are means ± SD, n ? 3. E) Representative cryogenic transmission electron microscopy (cryo-TEM) images of Her-, FA-, and Dual-LNPs. Black arrows show bleb structures and turquoise arrows show conjugated Herceptin on the surface of the LNPs. Scale bar = 100 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)