The key role of the scaffold on the efficiency of dendrimer nanodrugs

Abstract

Dendrimers are well-defined macromolecules whose highly branched structure is reminiscent of many natural structures, such as trees, dendritic cells, neurons or the networks of kidneys and lungs. Nature has privileged such branched structures for increasing the efficiency of exchanges with the external medium; thus, the whole structure is of pivotal importance for these natural networks. On the contrary, it is generally believed that the properties of dendrimers are essentially related to their terminal groups, and that the internal structure plays the minor role of an 'innocent' scaffold. Here we show that such an assertion is misleading, using convergent information from biological data (human monocytes activation) and all-atom molecular dynamics simulations on seven families of dendrimers (13 compounds) that we have synthesized, possessing identical terminal groups, but different internal structures. This work demonstrates that the scaffold of nanodrugs strongly influences their properties, somewhat reminiscent of the backbone of proteins.

Figure 1. Chemical structure of dendrimers 1-G₁–4-G₁.

The azabisphosphonic salts are in red, the linkers in blue and variable internal structures in black.

Figure 2. Chemical structure of dendrimers 5a,b-G₁–8a-G₁.

The azabisphosphonic salts are in red, the linkers in blue and variable internal structures in black.

Figure 3. The different methods of synthesis of the dendrimers.

Dendrimer 3-G₁ is synthesized as 3-G₂; dendrimers 6a-G₁ and 6b-G₁ as 6b-G₂; and dendrimers 7a-G₁ as 7b-G₂ (HOBt: hydroxybenzotriazole, DCC: N,N′-dicyclohexylcarbodiimide, BrTMS: bromotrimethylsilane).

Figure 4. Activation of human monocytes by the series of dendrimers 1-G₁, 2-G₂, 3-G₁, 3-G₂, 4-G₁, 5a-G₁ and 5b-G₁.

The bioactivity of the dendrimers is analysed by flow cytometry. Each dot in the plots is indicative of morphological change (size—the Forward Scatter (FSC) parameter on the x axis—and granularity—the Side Scatter (SSC) parameter on the y axis) undergone by purified monocytes in the presence of the different dendrimers at 20, 2, and 0.2 μM (left, middle, and right graphs respectively). Red points are monocytes (gated in the polygon), green points are remaining lymphocytes after purification, black points are died or dying cells. For each dendrimer, the number of terminal functions is indicated in parentheses. The score attributed to each dendrimer appears in red on the left, from 0 (no activation) to +++ (the highest activity, attributed to 1-G₁). Data are from one representative experiment out of six.

Figure 5. Activation of human monocytes by the series of dendrimers 6a-G₁, 6b-G₁, 6b-G₂, 7a-G₁, 7b-G₂ and 8a-G₂.

The bioactivity of the dendrimers is analysed by flow cytometry. Each dot in the plots is indicative of morphological change (size—the forward scatter (FSC) parameter on the x axis—and granularity—the side scatter (SSC) parameter on the y axis) undergone by purified monocytes in the presence of the different dendrimers at 20, 2, and 0.2 μM (left, middle and right graphs, respectively). Red points are monocytes (gated in the polygon), green points are remaining lymphocytes after purification, black points are died or dying cells. For each dendrimer, the number of terminal functions is indicated in parentheses. The score attributed to each dendrimer appears in red on the left, 0 means no activation. The negative control, without any dendrimer, is given first. Data are from one representative experiment out of six.

Figure 6. Equilibrated configurations of the 13 dendrimers and their size, obtained from the MD simulations.

(a) Radius of gyration (R_g) of the different dendrimers extracted from the equilibrated phase of the MD simulations in solution, number of azabisphosphonic surface groups (END) and biological efficiency score. Shape analysis: (b) MD equilibrated snapshots of the thirteen dendrimers displaying their shape. Dotted circles are added around the dendrimers to emphasize differences in the displacement of the azabisphosphonic functions around the dendrimers surface (symmetrical or directional molecules).

Figure 7. Radial distribution functions of the dendrimer terminal groups.

(a) Radial distribution functions—g(r)—of the terminal groups (END) with respect to the dendrimer core unit (CEN); (b) normalized peaks of the g(r) curves (only the topmost 10%) revealing the average (most probable) END to CEN distances in the dendrimers; (c) radial distribution functions, g(r), of the terminal groups with respect to each other; (d) normalized peaks of the g(r) curves of c (only the topmost 10%) revealing the average (most probable) END to END distances. In all graphs, the distance (x axis) is expressed in R_g units to allow comparison between different size dendrimers, and the data corresponding to dendrimer 1-G₁ is given in dotted black lines.

Figure 8. Comparison of biological activity and data from MD simulations for 10 dendrimers.

(a) The number of terminal groups of dendrimers versus the biological efficiency. (b) Quantification of relative granularity (arbitrary units, means from three donors) of the monocytes treated with the dendrimers and of negative control monocytes. (c) G_sol values from MD (relative hydrophilicity). (d) Calculated distance between terminal groups (in R_g units), as an indication of molecular directionality. (e,f) Comparison between biological data and MD data: granularity versus hydrophilicity (e) and granularity versus directionality (f). The black dots correspond in all cases to the lead compound 1-G₁.