*3.3. In Vivo siRNA Delivery Performance of the Double-Tail Self-Assembling Dendron AD*

The final steps to assess the therapeutic potential of siRNA delivery based on the double-tail self-assembled amphiphilic dendron **AD** were constituted by in vivo experiments using prostate cancer (PC-3) xenograft mouse models and Hsp27 as the target gene for knockdown. Figure 12a,b reveal that, following a 5-week animal treatment by intraperitoneal injections (2/week) of siRNA/**AD** complexes (3 mg/kg Hsp27 siRNA, **AD** at N/P = 5), there was a remarkable downregulation of Hsp27 at both mRNA (62%) and protein (73%) levels. This was paralleled by an effective inhibition of tumor growth, as shown in Figure 12c. Further, as for the case of the single-tail amphiphilic analogue **4**, no major organ injury or histopathological changes were evidenced in the animal subjected to **AD**/siRNA treatments (Figures 12d and A2).

**Figure 12.** In vivo effective gene silencing of Hsp27 at the mRNA (**a**) and protein (**b**) levels observed in prostate cancer (PC-3) xenograft mice after treatment with Hsp27 siRNA delivered by **AD** (3 mg/kg siRNA and **AD** at N/P = 5, injection twice a week for 5 weeks). Hsp27 mRNA and protein levels were quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting, respectively. Non-treated mice were used for the control. **AD** alone, siRNA alone and a scrambled (non-silencing) siRNA sequence delivered by **AD** were used as negative controls. (**c**) Tumor volume during treatment of PC-3 mice xenografts with siRNA/**AD** complexes (red symbols), compared with non-treated animals (green), and animals treated by AD alone (light blue), siRNA alone (dark blue), and a scrambled (non-silencing) siRNA sequence delivered by **AD** (gray symbol) used as negative controls. (**d**) Hepatic enzyme levels (alanine transferase ALT (light green bars), gamma-glutamine transferase γ-GLT (teal bars), (kidney functions (creatinine CRE (mmol/L, sky blue bars), blood urea nitrogen BUN (μmol/L, dark blue bars), and cholesterol level in the blood (gray bars, values on the right axis) measured in male C57BL/6 mice treated with intravenous administration of siRNA/AD (3 mg/kg siRNA and **AD** at N/P = 5). Untreated mice were used for control. Adapted from [6] with permission of John Wiley and Sons, 2014.

In summary, the nanosized micelles generated upon self-assembly of the double-tail amphiphilic PAMAM-based dendron **AD** were not able to deliver siRNA to a different panel of cancer cell lines with efficiency in vitro and in vivo comparable to those formed by its single-tail precursor **4**. However, they were also capable of eliciting gene silencing effects in the highly challenging and treatment-refectory human primary cells and stem cells. Accordingly, these versatile and safe delivery properties of **AD**, coupled with easy formulation, render AD a promising nanocarrier for highly efficient, cell-type-independent RNAi therapeutics.
