**1. Self-Assembling PAMAM-Based Amphiphilic Dendrons: A New Paradigm for siRNA Delivery**

In its broadest sense, self-assembly describes the natural tendency of physical systems to exchange energy with their surroundings and assume patterns or structures of low free energy. Random thermal motions bring constituent particles together in various configurations, and only those with significantly favorable interaction energy forming, tend to persist, and eventually become predominant. The information on the shape and size of the ultimate self-assembled entity is embodied in the structures of the individual components. A system slowly approaching equilibrium assumes a simple repetitive structure, while a dynamic system may generate structures of great complexity. For example, molecules in a cooling glass of water self-assemble as simple ice crystals, while the same molecules in a turbulent cloud with temperature and humidity gradients self-assemble as complex snowflakes of enormous variety. In relation to chemistry, self-assembly constitutes the quintessence of nanotechnology-based techniques leading to the design of novel materials [1–4]. It relies on the cumulative effects of multiple non-covalent interactions to assemble molecular building blocks into supramolecular entities in a reversible, controllable, and specific way, yet with relatively little synthetic effort. In particular is the ability of self-assembled structures to behave as more than the sum of their individual parts, and exhibit completely new properties [5].

With these concepts in mind, the authors envisaged the idea of creating small amphiphilic poly(amidoamine) (PAMAM)-based dendrons which, upon auto-organization into nanosized micelles, could mimic the covalent, high-generation dendrimer counterparts in size, shape and function, in particular for in vitro and in vivo siRNA delivery [6–8], as set out below.

#### **2. siRNA Delivery by Single-Tail Self-Assembling Amphiphilic Dendrons**

#### *2.1. Design, Optimization and Chemico-Physical Characterization of Single-Tail Self-Assembling Amphiphilic Dendrons*

Our work in this field started with the design, optimization and synthesis of a series of amphiphilic dendrons (**1**–**6**) characterized by a single hydrophobic alkyl chain of variable length and a hydrophilic PAMAM head with 8 primary amine terminal groups [7], as shown in Scheme 1. In addition, two non-amphiphilic dendrons characterized by a hydrophilic pentaethylene glycol (PEG) chain (**7**) and by the presence of the sole PAMAM head **(8**) were also produced as negative reference compounds.

**Scheme 1.** The chemical structure of the PAMAM-based self-assembling amphiphilic dendrons bearing a single alkyl chain of variable length (**1**–**6**). The two non-amphiphilic dendrons characterized by a hydrophilic pentaethylene glycol (PEG) chain (**7**) and by the presence of the sole PAMAM head (**8**) used as negative reference compounds are also shown (see text for details). Adapted from [7] with the permission of John Wiley and Sons, 2016.

Mesoscale simulations (see Figure S1, Table S1, and text in Supporting Information for details) were initially employed to predict the self-assembly of all dendrons **1**–**8**. According to the in silico results, the amphiphilic dendrons **1**–**6** were able to auto-organize into spherical nanosized micelles (shown in Figure 1 for dendrons **4**, **3** and **2** as an example), with an average diameter D and aggregation number Nagg ranging from 5.6 nm and 5 for micelles. This was generated by dendron **1** to 8.1 nm and 13 for those originated by the self-assembling of dendron **6** (Table 1). Contextually, no stable nanostructure formation was observed for the negative controls **7** and **8**, as shown in the left panel of Figure 1d for dendron **8**.

**Figure 1.** The computer simulation of the self-assembled micelles generated by the amphiphilic dendrons **4** (**a**), **3** (**b**), and **2** (**c**). In all panels, the hydrophilic PAMAM head is portrayed as purple and lilac beads, the hydrophobic tail is represented by light gray beads while the linker between the hydrophilic and the hydrophobic portion of the molecule is shown as green beads. Water and counterions are portrayed as a gray field for clarity. The right images in each panel show only the micellar cores. (**d**, **left**) Zoomed view of the micelles formed by dendron **4**. The micellar cores are highlighted by a light gray surface. (**d**, **right**) Computer simulation of the non-amphiphilic dendron **8** selected as negative control, showing the non-self-assembling characteristics of this system.

**Table 1.** Micelle aggregation number (Nagg), surface charge density (σm, *e*/nm2), diameter (D, nm), critical micelle concentration (CMC, μM) and free energy of micellization (ΔGm, kJ/mol) for dendrons **1**–**6** as predicted by computer simulations. The corresponding experimental values of the micelle diameter (Dexp, nm) and critical micelle concentration (CMCexp, μM) are also reported. Adapted from [7] with the permission of John Wiley and Sons.


The values of the critical micelle concentration (CMC) for the amphiphilic dendrons **1**–**6** (see Supporting Information for details) were further investigated from which a clear inverse relationship between the hydrophobic tail length and the CMC was obtained (Table 1). In other words, the amphiphilic dendrons bearing a longer hydrophobic component are able to self-assemble and pack more efficiently than those characterized by shorter alkyl tails. In addition, the associated values of the free energy of micellization (ΔGm) predicted by simulation (see Supporting Information for details) were all largely negative, supporting the spontaneous and thermodynamically favored micelle formation for all dendrons **1**–**6** (Table 1). As the hydrophilic portion was the same in all amphiphilic dendrons, the differential contribution to ΔGm (and hence to CMC) is obviously related to the length of the hydrophobic chain.

Experimental confirmation of in silico predictions were first performed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Both these techniques confirmed that the amphiphilic dendrons predominantly formed nanosized spherical micelles, as shown in Figure 2 for dendrons **3**–**6**. In particular, data analysis confirmed that an increase of the hydrophobic tail length from C14 (**3**) to C22 (**6**) was paralleled by an increase of the micellar diameter Dexp from 6.6 nm to 7.8 nm, in full agreement with the computer-based predictions (Table 1). Quite interestingly, the overall dimensions of these micellar nano-objects are indeed very close to those characterizing the high-generation structurally flexible triethanolamine (TEA)-core covalent dendrimers discussed in detail in the companion paper [9] (e.g., the average molecular diameters for generation 4, 5 and 6 TEA-core dendrimers are equal to 5.2, 7.7 and 10.1 nm, respectively). Therefore, at least from the standpoint of structure and dimensions, the self-assembled nanoparticles obtained from the amphiphilic dendrons can be considered as supramolecular dendrimer mimics.

**Figure 2.** TEM images of spherical micelles formed by the amphiphilic dendrons **3**–**6**. Adapted from [7] with the permission of John Wiley and Sons, 2016.

The predicted CMC values were also confirmed by experiments using pyrene as a hydrophobic fluorescent probe (Table 1). The progressive increase in alkyl chain length resulted in a drop of CMCexp from 398 μM for dendron **1** to 8.24 μM for dendron **6**. The high values of the computational/experimental CMC for systems **1** and **2** are the likely reason why micelle formation by these two dendrons was not observed under the conditions adopted for DLS/TEM experiments. Further, in agreement with the simulation, no CMC value could be experimentally determined for **7** and **8**, confirming the non-self-assembling properties of these two negative-control systems.
