Influence of the Internalization Pathway on the Efficacy of siRNA Delivery by Cationic Fluorescent Nanodiamonds in the Ewing Sarcoma Cell Model

Small interfering RNAs (siRNAs) are powerful tools commonly used for the specific inhibition of gene expression. However, vectorization is required to facilitate cell penetration and to prevent siRNA degradation by nucleases. We have shown that diamond nanocrystals coated with cationic polymer can be used to carry siRNAs into Ewing sarcoma cells, in which they remain traceable over long periods, due to their intrinsic stable fluorescence. We tested two cationic polymers, polyallylamine and polyethylenimine. The release of siRNA, accompanied by Ewing sarcoma EWS-Fli1 oncogene silencing, was observed only with polyethylenimine. We investigated cell penetration and found that the underlying mechanisms accounted for these differences in behavior. Using drugs selectively inhibiting particular pathways and a combination of fluorescence and electronic microscopy, we showed that siRNA gene silencing occurred only if the siRNA:cationic nanodiamond complex followed the macropinocytosis route. These results have potential implications for the design of efficient drug-delivery vectors.

The expression of EWS-Fli1 oncogene in cell is described as an early event in cancerous cell transformation. This oncogene modulates many regulatory pathways such as IGF1 growth factor pathway [1] and inhibition of apoptosis involving p21 protein [2]. In this context, we may expect that inhibition of EWS-Fli1 restores a functional apoptosis into cell and then cell sensitivity to chemotherapy.
The apoptotic status of NIH/3T3 EWS-Fli1 was tested after siRNA treatment by the annexin V methods. The cells were treated for 72 h with 50 nM of free siRNA (control) or siRNA:polycationic ND as described in Materials and Methods. After annexin V (ApopNexin Annexin V FITC Apoptosis Kit, EMD Millipore Corp., USA) and propidium iodine (PI) coloration, the cells are analysed by flow cytometry (Accuri C6, BD Biosciences, USA). Apoptosis cells fixed the annexin V-fluorescein (green fluorescence) but have intact membrane and are not permeant to PI (red fluorescence). Apoptotic cells therefore correspond to anexin V + /PI − population, which is displayed in Figure S1 taking cells not treated with siRNA:polycationic ND as the reference.
We observe that free siRNA and the two types of cationic ND alone do not induce a modification  It is well known that cationic polymer such as PEI are efficient vectors for nucleic acids (including siRNA) delivery to cells [4]. Thus, the siRNA inhibitory effect we observed could be the result of the polycations detaching from the ND, then encapsulating the siRNA, and finally triggering its internalization. To check if such a scenario takes place, we have compared in the same experiment, the ability of free and ND-bound PEI and PAH to vectorise efficiently the siRNA and then promote EWS-Fli1 silencing in cells.
In the case of siRNA associated to ND-PAH or ND-PEI, we used the optimal siRNA:ND-polycation  Figure S2 shows that ND-PEI vectorized siRNA promote 40% inhibition of EWS-Fli1 mRNA expression, while no significant inhibition is observed for siRNA vectorised by ND-PAH as previously reported [3], neither for siRNA vectorised by free PEI or PAH. These results are confirmed at the protein level by western blotting analysis (data not shown). The inefficiency of free PEI to vectorise siRNA in cell may be due to the use of low molecular weight polymer (800 Da) as reported by Pereira et al. [5] who observed that siRNA:PEI (1300 Da) polyplex is not stable in physiological conditions contrary to siRNA:PEI (10,000 Da).

Distribution of 200 nm ND-PAH nanoparticles as observed by TEM
In order to evaluate the role of the particle size in the internalization pathway followed, we prepared ND-PAH with size 200 nm diamond nanocrystlals similar to the size of the aggregates of 50 nm 3 ND (Fig. 3C). After 4 h of incubation of these cationic nanodiamonds with NIH/3T3 EWS-Fli1 cells, we detect only a few nanocrystals in lysosomal and endosomal compartments ( Figure S3), while the majority are still in the culture medium and a few are located at the cell surface. Therefore, the use of diamond nanocrystals larger than 50 nm resulted in a smaller internalization yield, and cannot account for the triggering of the macropynocytosis pathway.