*3.2. Self-Assembly of Dendrimersomes*

Dendritic amphiphile molecules are efficiently self-organised into supramolecular associates in water medium. The CMC determination plots in PB and PBS are given in the Figure 2. Comparing two plots, a clear difference is observed. In a low-salt solution (PB), there is only one association stage, quite broad in terms of concentration range, with CMC of 9.5 ± 1.2 μM. In contrast, at physiological salt concentration (PBS), there are two association stages with CMC of 2.1 ± 1.0 μM and 21 ± 1.1 μM. Apparently, the high salt concentration fosters the association of dendron molecules at low concentrations, with these aggregates further rearranging into vesicle-like dendrimersomes at higher concentrations.

**Figure 2.** I373/I383 plots corresponding to the amphiphilic dendron association in PB and PBS.

Vesicle-like nanoconstructions, dendrimersomes, are readily assembled upon quite simple treatment (short sonication and relaxation). High uniformity of nanoconstructions' topology and size, likely defined by the molecular structure of the dendron precursor, is achieved without specific post-treatment (e.g., extrusion). Having been formed at neutral pH, dendrimersomes have mean diameter of 20–30 nm; however, when put into acidic medium (pH below 6.5), they reorganize into larger particles 100–150 nm diameter (Figure 3). TEM shows that the vesicle-like structure of dendrimersomes is maintained upon reorganization.

Based on the findings above, we suggest a provisional scheme of the bilayer behaviour (Figure 3D). The main driving force of self-assembly is the interaction of hydrophobic tails of amphiphilic dendron molecules additionally stabilized by stacking interactions of triazine moieties [34]. On the other hand, the bilayer structure is destabilized by the steric and electrostatic repulsion of dendron fragments. Such a balance of factors favouring and unfavouring the bilayer stability likely gives the supramolecular flexibility to the whole dendrimersome. In acidic medium, triazine moieties get protonated, and the stacking is distorted. Thus, the stabilizing factor turns into the destabilizing one; this causes loosening of the bilayer followed by fusion with other vesicles.

It is worth noting that the effects of pH-sensitivity are observed at slightly acidic pH (below 6.5), which corresponds to early stages of endosome maturation. Thus, once delivered into a cell by endocytosis, an encapsulated biologically active cargo can be released into the cytosol in the most favourable moment to escape endosomes. Fusing dendrimersomes can contribute to the endosomal escape release by assisting the endosome membrane disruption.

**Figure 3.** Self-assembly of dendritic amphiphiles. DLS profiles for supramolecular associates of dendron (100 μM) exposed to different pH in 10 mM Na-phosphate buffer (**A**); representative TEM images of dendrons' supramolecular associates at pH 7.0 (**B**) and pH 5.0 (**C**); tentative scheme of the pH-dependent behaviour of a dendron bilayer (**D**).
