Angewandte
Chemie
inside the cell, which should lead to a better silencing
effect.[7e,10] In order to exploit this possibility, we next
functionalized the siRNA duplexes of the dendrimers with
a glucose molecule at the 3’ ends.[11] Glucose forms H-bonds
with the RNA duplex and increases the water solubility. The
introduction of the 3’ modification was achieved by hybrid-
izing the passenger-strand-containing dendrimers with the
corresponding glucose-modified guide strands. Indeed, the
glucose-end-capped dendrimers showed
a significantly
enhanced efficacy. It is likely that the large siRNA dendrimes
suffer from serious degradation in the endosomes after
internalization.[10a,12] This may be retarded by glucose cap-
ping.[13]
The advantage of capping of the trimeric siRNA dendri-
mer is quite large, as shown in Figure 3B. The threefold
glycosylated structure finally gave a silencing effect of
approximately 80%. Thus, in comparison to the monomeric
duplex, the silencing efficiency was increased by a factor of
2.5, despite the reduced total concentration.
We next investigated the possibility of utilizing the
dendritic siRNA structure to induce silencing in difficult to
transfect cells such as neurons (Figure 4). Consequently, we
tested the uptake of the nanoparticles into neural stem cells.
We prepared siRNA dendrimers in which we replaced the
glucose units at the 3’ end of the guide strand by Alexa Flour
647. Indeed, when we added the fluorophore-modified siRNA
trimer to the neural stem cells, efficient uptake was detected
by confocal microscopy (Figure 4A).
In order to prove the ability of the siRNA dendrimers to
silence an endogenous gene, we next transfected the neural
stem cells with a trimeric siRNA dendrimer targeting Tet1.
This enzyme was recently shown to oxidize 5-methylcytosine
to 5-hydroxymethylcytosine and 5-carboxycytosine, which is
essential for the differentiation process.[14] Using qPCR, we
monitored the expression level of Tet1 (Figure 4B). Indeed,
the siRNA dendrimer led to a significant reduction of the
level of endogenous Tet1 expression, proving the effect of the
siRNA despite it being incorporated into a dendritic struc-
ture. Again, we observed the strongest silencing effect with
the trimeric siRNA structure.
Finally we were interested in the ability of our dendritic
siRNA structures to exhibit a medicinally relevant function in
primary neurons that are hard to transfect by normal
methods.[3f,15] As a target, we chose the neurotropic rabies
virus (RABV), which causes over 55000 deaths per year and
is not treatable after the onset of clinical symptoms.[16] For this
study, we infected mouse E14 cortical neurons with the
RABV and subsequently treated the cells with two siRNA
dendrimers (AEA-[3siRNA]-Glc) against the mRNAs of the
viral nucleoprotein (N protein) and the phosphoprotein
(P protein). The siRNA sequences were designed in silico
using the program siDESIGN. Both proteins are essential for
viral transcription and replication and are therefore consid-
ered promising targets to efficiently counteract RABV
infection.[17] In addition, the P protein of RABV is an
important antagonist of the innate immune system, com-
pletely suppressing both IFNb induction and signaling path-
ways.[18] Down-regulation of P protein expression should
consequently promote the innate immune response, thereby
Figure 4. A) Anandamide-mediated delivery of dendritic siRNA nano-
structures to neural stem cells. Left: trimeric siRNA modified with
Alexa Fluor647 was incubated with neural stem cells. Right: As
a negative control the stem cells were incubated with siRNA lacking
the ligand modification (red: siRNA, green: cell membranes. The
contrast settings for siRNA signals were equal for both images).
B) Successful delivery of nanostructures to stem cells was additionally
demonstrated by regulation of Tet1 monitored by real-time PCR.
a: AEA-[1siRNA-Tet1], b: AEA-[3siRNA-Tet1]-Glc. C) Down-regulation of
RABV titers in E14 cortical neurons by treatment with different AEA-
modified siRNA structures targeting the P protein of rabies virus.
c: AEA-[1siRNA-P-protein], d: AEA-[3siRNA-P-protein]-Glc.
further restricting RABV infection. The data of the experi-
ment clearly show that treatment of the infected neurons with
either one of the dendrimers AEA-[3siRNA]-Glc (anti-P and
anti-N) lead to a strongly reduced viral titer (Figure 4C; for
data of targeting N protein Figures S2 and S3). The titer was
reduced by two orders of magnitude in relation to the control
experiment performed with a nontargeting anandamide-
modified siRNA. The trimeric dendrimer was able to
reduce the viral titer to a level close to the detection limit
of the experiment. Important are also the observations that
the reduction of the viral titer is dose-dependent and that with
the standard lipofectamin2000-based transfection, only a ten-
fold reduction of the viral titer was observed. These results
show the improved efficacy of the siRNA dendrimers.
In summary, we have presented novel dendritic siRNA
nanostructures that allow the transfection of difficult-to-
transfect cells, such as stem cells and primary neurons.[19]
A
click-chemistry-based approach, in which the passenger
strands of the siRNAs are covalently bound to the dendritic
structure, allowed us to exactly control the monodispersity of
the particles, which enabled the control of the number of
Angew. Chem. Int. Ed. 2014, 53, 1 – 5
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers!
Brunner, Korbinian
