Adaptive amphiphilic dendrimer-based nanoassemblies as robust and versatile siRNA delivery systems

Article abstract of DOI:10.1002/anie.201406764

siRNA delivery remains a major challenge in RNAi-based therapy. Here, we report for the first time that an amphiphilic dendrimer is able to self-assemble into adaptive supramolecular assemblies upon interaction with siRNA, and effectively delivers siRNAs to various cell lines, including human primary and stem cells, thereby outperforming the currently available nonviral vectors. In addition, this amphi-philic dendrimer is able to harness the advantageous features of both polymer and lipid vectors and hence promotes effective siRNA delivery. Our study demonstrates for the first time that dendrimer-based adaptive supramolecular assemblies represent novel and versatile means for functional siRNA delivery, heralding a new age of dendrimer-based self-assembled drug delivery in biomedical applications.

Full text of DOI:10.1002/anie.201406764

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Angewandte
Communications
DOI: 10.1002/anie.201406764
Adaptive Supramolecular Assemblies
Adaptive Amphiphilic Dendrimer-Based Nanoassemblies as Robust
and Versatile siRNA Delivery Systems**
Xiaoxuan Liu, Jiehua Zhou, Tianzhu Yu, Chao Chen, Qiang Cheng, Kheya Sengupta,
Yuanyu Huang, Haitang Li, Cheng Liu, Yang Wang, Paola Posocco, Menghua Wang, Qi Cui,
Suzanne Giorgio, Maurizio Fermeglia, Fanqi Qu, Sabrina Pricl, Yanhong Shi, Zicai Liang,
Palma Rocchi, John J. Rossi, and Ling Peng*
Abstract: siRNA delivery remains a major challenge in RNAi-
based therapy. Here, we report for the first time that an
amphiphilic dendrimer is able to self-assemble into adaptive
supramolecular assemblies upon interaction with siRNA, and
effectively delivers siRNAs to various cell lines, including
human primary and stem cells, thereby outperforming the
currently available nonviral vectors. In addition, this amphi-
philic dendrimer is able to harness the advantageous features of
both polymer and lipid vectors and hence promotes effective
siRNA delivery. Our study demonstrates for the first time that
dendrimer-based adaptive supramolecular assemblies repre-
sent novel and versatile means for functional siRNA delivery,
heralding a new age of dendrimer-based self-assembled drug
delivery in biomedical applications.
size, shape, structure, chemistry, and mode of action have
[
4–8]
been developed for siRNA delivery.
These delivery
systems can be generally classified into two major groups:
lipid and polymer vectors. For both, the efficiency is often
dependent on the cell type, with siRNA delivery to primary
and stem cells being particularly difficult. Here, we report for
the first time an adaptive amphiphilic dendrimer-based
nanoassembly acting as versatile vector for effective siRNA
delivery and gene silencing, and demonstrate its efficacy for
primary and stem cells as well as in vivo. This amphiphilic
dendrimer AD (Scheme 1A) self-assembles into vesicle-like
[
9]
dendrimersome nanostructures, which, in the presence of
siRNA, spontaneously undergo structural rearrangement into
smaller, spherical micelles, thereby maximizing the interac-
tions between the siRNA and AD (Scheme 1B). The peculiar
self-assembly of AD into adaptive supramolecular structures
alongside its advantageous combination of lipid and dendri-
mer vector features is evocative of virus-like delivery. Our
study hence opens new perspectives in nonviral vector design
for siRNA-based gene therapy.
S
mall interfering RNA (siRNA) can efficiently silence
disease-related genes in a sequence-specific manner, creating
[1,2]
an entirely new siRNA-based gene therapy.
and effective siRNA delivery remains a major challenge.
However, safe
[
2,3]
Although viral vectors are very effective, increasing concerns
over their safety and immunogenicity substantiate the need to
develop alternative nonviral vectors. During the past years,
myriads of natural and synthetic nonviral vectors differing in
The amphiphilic dendrimer AD (Scheme 1A) was readily
[10]
synthesized by click chemistry (Scheme S1–S3). It sponta-
neously self-assembled in water into spherical nanostructures
[*] X. Liu, T. Yu, C. Chen, K. Sengupta, C. Liu, Y. Wang, M. Wang,
Q. Cui, Y. Shi
S. Giorgio, L. Peng
Department of Neurosciences
Beckman Research Institute of City of Hope (USA)
Aix-Marseille Universitꢀ, CNRS, Centre Interdisciplinaire de Nano-
science de Marseille, UMR 7325 (France)
E-mail: ling.peng@univ-amu.fr
P. Posocco, M. Fermeglia, S. Pricl
Molecular Simulation Engineering (MOSE), DEA
University of Trieste (Italy)
X. Liu, P. Rocchi
Centre de Recherche en Cancꢀrologie de Marseille, INSERM,
UMR1068 (France)
Q. Cui, Y. Shi, J. J. Rossi
Irell and Manella Graduate School of Biological Sciences
Beckman Research Institute of City of Hope (USA)
X. Liu, P. Rocchi
Institut Paoli-Calmettes
Marseille (France)
[
**] Financial support from the international ERA-Net EURONA-
NOMED European Research project DENANORNA, the Ministry of
Science and Technology of China (No.2012AA022501), Agence
National de la Recherche (projects DENANORNA and SANAM),
Association FranÅaise contre les Myopathies (X.L.), Association
pour la Recherche sur les Tumeurs de la Prostate (X.L.), La bourse
Eiffel (T.Y., M.W.), China Scholarship Council (Y.W., C.C., C.L.),
INCa, Canceropꢁle PACA, Wuhan University, CNRS, INSERM, and
the European COST Action TD0802 “Dendrimers in Biomedical
Applications” is acknowledged.
X. Liu, P. Rocchi
Aix-Marseille Universitꢀ (France)
X. Liu, P. Rocchi
CNRS, UMR7258, Marseille (France)
J. Zhou, H. Li, J. J. Rossi
Department of Molecular and Cellular Biology
Beckman Research Institute of City of Hope (USA)
T. Yu, C. Liu, M. Wang, F. Qu
College of Chemistry and Molecular Sciences
Wuhan University (China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201406764.
Q. Cheng, Y. Huang, Z. Liang
Institute of Molecular Medicine, Peking University (China)
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Scheme 1. A) The dendrimer AD and B) representation of its adaptive self-assembly upon interaction with siRNA for siRNA delivery.
of ca. 200 nm in diameter, as
shown by dynamic light scattering
(
DLS) and transmission electron
microscopy (TEM) (Figure 1A
and B). The TEM images showed
a corona structure (Figure 1C),
which we inferred to be a bilayer,
from the fact that its thickness was
7
nm—approximately twice the
theoretical length of AD
(
ꢀ 3.5 nm). Accordingly, AD
readily self-assembled to form
unilamellar vesicles aptly termed
dendrimersomes.
[
9]
In addition,
AD formed a Langmuir mono-
layer film at the air/water inter-
face and could be swelled to form
stable giant vesicles (Figure S1),
providing further indication of its
lipid-like behavior and vesicle-
forming properties.
Most interestingly, upon inter-
action with siRNA, the AD-den-
drimersomes underwent struc-
tural rearrangement to form nano-
sized siRNA/AD complexes (Fig-
ure 1D) composed of many highly
ordered smaller spherical sub-
structures of 6–8 nm diameter
(
Figure 1E and F). From this
size, which is twice the size of
AD, we conclude that these sub-
structures are micelles. This vesic-
ular to micellar structural transi- Figure 1. Dendrimer AD self-assembles into vesicle-like dendrimersomes, which undergo structural
rearrangement into smaller, spherical micelles to interact with siRNA. A) DLS analysis and B,C) TEM
imaging of dendrimersomes formed by AD in water. D) DLS analysis and E,F) TEM imaging of the
siRNA/AD complexes. Computer modeling results showing nanostructures formed by AD alone (H),
a section plane (G), and in the presence of siRNA (I). The hydrophilic units are portrayed in light
tion is expected to expose more of
the positively charged dendrimer
surface area, thereby providing
stronger stabilizing electrostatic
green, the hydrophobic units in dark green, and siRNAs as orange sticks (see the Supporting
interactions with the negatively
Information for more details). Light grey spheres are used to portray some representative water
charged siRNA. This implies that molecules.
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Figure 3. AD-mediated siRNA delivery in human prostate cancer PC-3
cells, human CCRF-CEM T-cells, human primary cells (PBMC-CD4+),
and hematopoitic stem cells (CD34+). Gene silencing of Hsp27 at
A) mRNA and B) protein levels in PC-3 cells treated with 20 nm siRNA
and AD at N/P ratio 10. Down-regulation of CD4 at C) mRNA and
D) protein expression on CEM cells treated with 50 nm dsiRNA and
AD at N/P ratio 5. Knockdown of Tat/Rev mRNA expression on
E) PBMC-CD4+ cells and F) CD34+ stem cells with 50 nm dsiRNA
and AD at N/P ratio of 5. Oligofectamine (oligo) and Trans IT-TKO
(TKO) were used as controls. *, **, ***, and **** differ from control
(pꢁ0.05, pꢁ0.01, pꢁ0.001, and pꢁ0.0001, respectively) by Student’s
t-test.
Figure 2. The siRNA/AD nanoparticles are able to A) protect siRNA
from RNase digestion and B) promote cell uptake by macropinocytosis
(
C, D). C) Uptake of Alexa488-labeled siRNA/AD complexes in PC-3
cells in the presence of specific inhibitors and D) confocal imaging of
fluorescent markers of different endocytosis pathways. The inhibitors
and biomarkers are: cytochalasin D and dextran for macropinocytosis,
genistein and CTX-B for caveolae-mediated endocytosis, chlorproma-
zine and transferrin for clathrin-mediated endocytosis. * differ from
control (pꢁ0.05) by Student’s t-test.
micelles (Figure 1I) indeed provides more exposed positively
charged surfaces, thus allowing for more efficient electrostatic
interactions with the negatively charged siRNA. This in turn
results in the formation of stable and compact siRNA/AD
nanoparticles in silico.
AD is able to dynamically self-assemble into responsive and
adaptive supramolecular assemblies (Scheme 1B) in the
presence of external stimuli.
Indeed, the siRNA/AD complexes are stable colloidal
nanoparticles (Figure S2) with a z-potential of + 25 mV.
These nanoparticles could effectively protect siRNA from
degradation (Figure 2A) and promote rapid and efficient cell
uptake, with almost 100% internalization being attained
within 30 min (Figure 2B). Neither the clathrin-mediated
endocytosis inhibitor chlorpromazine nor the caveolae-medi-
ated endocytosis inhibitor genistein was able to influence the
cellular uptake of the siRNA/AD complexes; conversely, the
macropinocytosis inhibitor cytochalasin D significantly
decreased their internalization (Figure 2C). Confocal imag-
ing revealed significant co-localization of the siRNA/AD
complexes with the macropinocytosis biomarker dextran,
whereas little co-localization was observed for either the
clathrin-mediated endocytosis biomarker transferrin or the
caveolae-mediated endocytosis biomarker CTX-B (Fig-
ure 2D). Furthermore, after treatment with siRNA/AD
To verify this hypothesis and gain insight into the adaptive
supramolecular assemblies formed by AD, we performed
[
11]
mesoscale computational studies using dissipative particle
[12]
dynamics (DPD). Our simulations show that AD assembles
into large supramolecular structures (Figure 1G), whose
planar section indeed shows a double-layered dendrimersome
(
Figure 1H). The formation of the dendrimersome is con-
trolled by the balance of two opposing forces: hydrophobic
interactions among alkyl chains that favor cooperative con-
struction of the supramolecular assemblies, and repulsive
interactions/steric effects among the positively charged head
groups within the dendrons that constrain the assemblies to
remain finite sized. Such balance is altered in the presence of
siRNA. The conversion of the larger vesicles into smaller
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complexes, actin depolymerization, the hallmark of macro-
pinocytosis, was also observed (Figure S4). Collectively, these
findings indicate that macropinocytosis, an advantageous
mechanism for drug delivery, is the dominant uptake pathway.
Encouraged by the rapid and favorable cellular uptake
results of the siRNA/AD complexes, we went on to assess
AD-mediated siRNA delivery in adherent human cancer cells
such as prostate cancer PC-3 cells (Figure 3C and D) and
breast cancer MCF-7 and MDA-MB231 cells (Figure S5A and
unable to effectively deliver siRNA to human primary and
stem cells (Figure S10), whereas Trans IT-TKO (TKO)
[
17]
exhibited notable toxicity on these two cell types.
Our
results thus pave the way for the use of AD as a promising
vector for future stem cell research and cellular therapy.
To understand the unique and outstanding delivery
capability of AD, it was important to explore the delivery
mechanism. We hypothesized that, in addition to being able to
form supramolecular nanostructures (Scheme 1 and
Figure 1), AD might also benefit from the combined advan-
tages of lipid and dendrimer vectors, since AD is in fact
a lipid/dendrimer hybrid bearing a hydrophobic alkyl chain
[13]
B), taking the delivery of Hsp27 (heat shock protein 27)
siRNA as an example. Indeed, a significant attenuation of
Hsp27 expression at both mRNA and protein levels (Fig-
ure 3A and B) as well as the related anticancer activity
[10,18]
entity and a hydrophilic PAMAM dendron.
Indeed, the
(
Figure S8) were achieved. Such RNAi efficacy was compa-
effective delivery ability of AD can be attributed to its
peculiar amphiphilic architecture, since neither the hydro-
phobic alkyl chain 1 nor the hydrophilic dendron 2 alone led
to any notable gene silencing (Figure 4A). Importantly,
whereas AD could form stable complexes with siRNA and
completely retard the migration of siRNA in gel at an N/P
ratio of 2.5, dendron 2 alone was not able to do so even at N/
P = 10 (Figure 4B). This indicates that the long alkyl chains
favor the self-assembly of AD into supramolecular structures,
which, in turn, increase the stability of the siRNA/AD
nanocomplexes. Interestingly, gene silencing could be signifi-
cantly enhanced in the presence of the fusogenic lipid
dioleoylphosphatidylethanolamine (DOPE) which is often
employed as a helper lipid to promote the delivery ability of
lipid vectors (Figure 4C), underlining the lipid-vector-like
character of AD.
rable to or even better than that obtained with the gold
reference of oligofectamine (oligo; Figure 3A and B).
Remarkably, the silencing effect could be maintained over
one week (Figure S7A), even in serum-containing medium in
the presence of chloroquine (Figure S7B).
We next demonstrated successful AD-mediated siRNA
delivery to suspended human T (CCRF-CEM) and B (Jeko-1)
cells (Figure 3C and D, Figure S5C), with dicer-substrate
[
14,15]
siRNAs (hereafter denoted as dsiRNAs)
targeting CD4,
[
16]
the primary receptor for HIV-1, as an example. A consid-
erable decrease of CD4 expression at both mRNA and
protein level was achieved (Figure 3C and D). No inhibition
was observed with either AD alone or a mutant dsiRNA/AD
complex. Again, AD was more effective than the relevant
commercial reference Trans IT-TKO (TKO; Figure 3D).
The next important chal-
lenge was the delivery to
primary cells and stem cells.
AD was indeed able to exer-
cise functional delivery of
RNAi
therapeutics
to
human primary peripheral
blood mononuclear cells
(
PBMC-CD4 +)
(Fig-
ure 3E),
hematopoietic
stem cells (HSC-CD34 +)
Figure 3F), and glioblas-
(
toma stem cells (GSCs) (Fig-
ure S5D). Following treat-
ment with the anti-tat/rev
dsiRNA delivered by AD,
substantial reductions in tat/
rev mRNA were observed in
both the PBMC-CD4 + cells
and the HSC-CD34 + cells
(
Figure 3E and F), leading
to more than 50% reduction
of viral infection (Figure S9).
To our knowledge, this is the
first report on the successful
and safe delivery of siRNA
into primary and stem cells.
In fact, the commercially
available vector Lipofecta-
Figure 4. AD-mediated siRNA delivery benefits the advantages of both lipid and dendrimer vectors.
A) Compared to AD, neither the alkyl chain entity 1 nor the dendron 2 led to any gene silencing with 20 nm
siRNA at N/P ratio 10. B) Gel retardation of siRNA in agarose gel with AD compared to 2 at varying N/P
ratios. C) Dioleoylphosphatidylethanolamine (DOPE) enhanced the gene silencing of Hsp27 with 10 nm
siRNA at N/P ratio 10. D) Bafilomycin A1 decreased the AD-mediated gene silencing of Hsp27 observed with
2
0 nm siRNA at N/P ratio 10. PC-3 cells were used. ** differ from in absence of DOPE or Bafilomycin A1
mine RNAiMAX (lipo) was (pꢁ0.01) and *** differ from control (pꢁ0.001) by Student’s t-test.
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We further verified whether the so-called “proton sponge
As a final, decisive step to assess the effective therapeutic
potential of AD in siRNA delivery, we conducted in vivo
delivery of Hsp27 siRNA using a prostate cancer PC-3
xenograft mouse model. Remarkable down-regulation of
Hsp27 at both mRNA and protein levels was achieved
following treatment with Hsp27 siRNA/AD (Figure 5A and
B), leading to an effective inhibition of tumor growth
(Figure 5C), which is in line with the functional role of
[
19]
effect”, often invoked to explain the nucleic acid release
from the dendrimer complexes, also contributed to AD-
mediated siRNA delivery. It is believed that inside the acidic
endosomal environment, the tertiary amines in the dendrimer
interior are protonated, and thus promote endosome dis-
ruption and subsequent cargo release in the cytoplasm. Our
finding that the gene silencing effect was significantly reduced
in the presence of bafilomycin A1 (Figure 4D), a proton
pump inhibitor that prevents the acidification of endosomes,
implies that AD-mediated siRNA delivery was indeed
dependent on the endosomal acidification process and that
the proton sponge effect played a role in the delivery
[
21]
Hsp27.
Importantly, there was no discernible toxicity
observed since neither weight alteration (Figure S11) nor
organ injury nor histopathological changes (Figure S12) were
noted.
In summary, here we have presented the novel amphi-
philic dendrimer AD and its self-assembled adaptive nano-
structures as versatile nanocarrier for functional siRNA
delivery. AD, composed of a positively charged dendron
and two lipid-like alkyl chains, combines the advantages of
dendrimer and lipid vectors. It readily self-assembles into
dendrimersome nanovesicles which, upon interaction with
siRNA, rearrange spontaneously into small spherical
micelles. These micelles, by exposing more efficiently the
positively charged dendrimer surface, effectively interact,
entrap, and condense the negatively charged siRNAs into
nanoparticles, which protect the siRNA from degradation.
These nanoparticles successfully deliver siRNA into a wide
range of cell types including the highly challenging human
primary cells and stem cells. They are also efficacious for
in vivo delivery. This robust, versatile and nontoxic delivery
activity of AD, coupled with its easy formulation, yields
a promise for a new, highly effective, and cell-type-independ-
ent nanovector for efficient and safe siRNA delivery.
[
20]
process.
Received: July 8, 2014
Revised: August 3, 2014
Published online: September 12, 2014
Keywords: dendrimers · gene silencing · nanocarriers ·
.
nonviral vectors · self-assembly
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