Sticky nanoparticles: A platform for siRNA delivery by a bis(zinc(II) dipicolylamine)-functionalized, self-assembled nanoconjugate

Article abstract of DOI:10.1002/anie.201105565

Delivering the goods: Multifunctional, self-assembled, polymeric nanoparticles for the simultaneous delivery of small-molecule drugs and siRNA have been synthesized. The nanoparticles are composed of biodegradable hyaluronic acid, for tumor targeting and

Full text of DOI:10.1002/anie.201105565

Angewandte
Chemie
DOI: 10.1002/anie.201105565
Drug Delivery
Sticky Nanoparticles: A Platform for siRNA Delivery by a Bis(zinc(II)
dipicolylamine)-Functionalized, Self-Assembled Nanoconjugate**
Gang Liu, Ki Young Choi, Ashwinkumar Bhirde, Magdalena Swierczewska, Juan Yin,
Sang Wook Lee, Jae Hyung Park, Jong In Hong, Jin Xie, Gang Niu, Dale O. Kiesewetter,
Seulki Lee,* and Xiaoyuan Chen*
Small interfering RNA (siRNA) has specific and effective
gene silencing effects and has rapidly emerged as a potent
strategy for cancer therapy.[1] Naked siRNA cannot readily
cross cellular membranes, and the delivery of siRNA to its
target remains the biggest hurdle to clinical translation.[2]
Various types of synthetic nanovectors have been investigated
for siRNA delivery.[3] Although many promising systems for
the delivery of siRNA have been reported, clinical translation
of siRNA therapeutics remains a challenge. Herein, we report
a nanoparticle-based system for delivering siRNA into cells
that uses a biodegradable conjugate with a high cell perme-
ability for targeting tumors, and a universal phosphate-
binding analogue for high siRNA binding affinity.
formulate and cost-effective to manufacture.[4,5] Similar to
other nanoparticle (NP) formulations, HA-NPs deliver
encapsulated drugs into tumors by the enhanced permeability
retention (EPR) effect. Furthermore, as HA is the main
ligand for CD44,[6] which is overexpressed on the surfaces of a
variety of tumor cells, HA-NPs can actively accumulate in
tumor cells. Once HA-NPs accumulate in tumor cells by
passive and active targeting mechanisms, the encapsulated
drugs are released efficiently into the tumor cells through
enzyme-triggered degradation of the NPs by the HA-degrad-
ing intracellular enzyme hyaluronidase-1 (Hyal-1).[7]
Although HA-NPs are an ideal platform for new nano-
formulations with small-molecule anticancer drugs, they are
not adequate for siRNA delivery. This is because HA-NPs are
composed of highly anionic polymer that cannot form nano-
sized polyelectrolyte complexes with anionic siRNA for facile
delivery into cells (Scheme 1).
Recently, we developed a hyaluronic acid (HA) based
nanoformulation for tumor therapy that is nontoxic, highly
specific for targeting tumor cells, drug-loadable, and biode-
gradable. Importantly, this HA-based system is easy to
Previously, we reported a series of chemosensors based on
ZnII–dipicolylamine complexes (Zn–DPA) that are highly
selective for phosphate-containing molecules in aqueous
solution. The selectivity is achieved through specific inter-
actions between the coordinated zinc ions of the DPA and the
anionic phosphate moieties.[8] Zn–DPA analogues have also
been used as probes for the anionic membrane surfaces of
dead or dying cells by targeting phosphatidylserine exposed at
the cell membrane.[9] Although various types of Zn–DPA
analogues have been developed, to our knowledge none have
been applied for the delivery of therapeutic molecules that
contain phosphate groups, such as siRNA. By using the
principles of supramolecular chemistry, we hypothesized that
synthetic receptor molecules, such as Zn–DPA, may have the
ability to associate selectively with the phosphodiester groups
that constitute the backbone of siRNA.
[*] Dr. G. Liu,[+] Dr. K. Y. Choi,[+] Dr. A. Bhirde, M. Swierczewska,
Dr. G. Niu, Dr. D. O. Kiesewetter, Dr. S. Lee, Dr. X. Chen
Laboratory of Molecular Imaging and Nanomedicine (LOMIN)
National Institute of Biomedical Imaging and
Bioengineering (NIBIB), National Institutes of Health (USA)
E-mail: seulki.lee@nih.gov
shawn.chen@nih.gov
Dr. G. Liu[+]
Sichuan Key Laboratory of Medical Imaging
North Sichuan Medical College (China)
Dr. J. Yin
National Cancer Institute (NCI)
National Institutes of Health (USA)
S. W. Lee, Prof. J. I. Hong
Department of Chemistry, Seoul National University (Korea)
Prof. J. H. Park
Herein, we demonstrate a functional, simple, and cost-
effective delivery system for siRNA that works by utilizing
Zn–DPA to bind siRNA, and HA-NPs as a carrier ligand to
target tumor cells. Unlike many other conventional siRNA
delivery systems that involve chemical or physical conjuga-
tion of the siRNA to polycations, HA-NPs that are con-
jugated with Zn–DPA (HADPA-Zn-NPs, Scheme 1) have a high
affinity for siRNA. This affinity improves the targeting of the
system for gene silencing and maintains the advantages of
HA-NPs for cellular delivery of the siRNA. As siRNA and
hydrophobic anticancer drugs can be simultaneously loaded
onto the surface and the inner core of HADPA-Zn-NPs, this
system can be used as a co-delivery carrier to maximize and
synergize therapeutic effects. Polymeric NPs that are func-
Department of Polymer Science and Engineering
Sungkyunkwan University (Korea)
Prof. J. Xie
Department of Chemistry and Bio-Imaging Research Center
University of Georgia (USA)
[+] These authors contributed equally to this work.
[**] This work was supported by the Intramural Research Program of the
NIBIB, NIH and partially supported by an NIH Pathway to
Independence (K99/R00) Award, the National Natural Science
Foundation of China (NSFC, grant nos. 81101101 and
2011JQ0032), the International Cooperative Program of the NSFC
(grant no. 81028009), and NRF grant 2009-0080734 from the MEST
(Korea).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201105565.
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siLuc and analyzed in a retar-
dation assay by agarose gel
electrophoresis. As shown in
Figure 1a, the HADPA-Zn-NPs
were able to bind siLuc but
HA and the HADPA-NPs alone
did not bind to siLuc at any
concentration. To confirm that
this complex structure was
based
between
on
the
coordination
phosphate
groups of the siRNA and Zn–
DPA, excess sodium phos-
phate was added to induce
decomplexation of the siLuc
from the HADPA-Zn-NPs (Fig-
ure 1b). After the addition of
sodium phosphate, siLuc was
released from HADPA-Zn-NPs,
whereas no significant change
was observed after the addi-
tion of other salts. This result
indicates that siLuc was bound
on the HADPA-Zn-NPs by coor-
dination with Zn–DPA. SiLuc/
HADPA-Zn-NPs had a negative
zeta potential of (À15.7 Æ
4.0) mVand an average hydro-
dynamic diameter of (227.6 Æ
Scheme 1. Synthesis and chemical structure of HADPA-Zn-NPs. Boc=tert-butyloxycarbonyl, MC=CH2Cl2,
TFA=trifluoroacetic acid.
tionalized with Zn–DPA provide highly targeted small-
molecule delivery and efficient intraceullular transfer of
25.4) nm (see Figure S7 in the Supporting Information). In
terms of biocompatibility, in vitro cellular toxicity studies
showed that the HADPA-Zn-NPs were nontoxic (Figure 1c).
Prior to functional biological studies, we evaluated the
cellular uptake of siRNA/HADPA-Zn-NPs in cells that were
strongly positive for CD44 (4T1-fluc) and cells that were less-
strongly positive for CD44 (293T). CD44 expression was
confirmed by flow cytometry (see Figure S8 in the Supporting
Information). As shown in Figure 1d, the red fluorescence
from Cy3-labeled siRNA was more intense in 4T1-fluc cells
that were treated with siRNA/HADPA-Zn-NPs than in the cells
that were treated with Lipofectamine 2000 (Lipo2K)/siRNA.
In contrast, no fluorescence was observed in cells that were
treated with HA, HADPA-NPs, and Zn–DPA (see Figure S9 in
the Supporting Information). Furthermore, the uptake of
siRNA/HADPA-Zn-NPs into 4T1-fluc cells was (3.7 Æ 0.5)-fold
higher than the uptake into 293T cells (Figure 1e). To
determine whether CD44 receptors are responsible for the
efficient cellular uptake of siRNA/HADPA-Zn-NPs, the CD44
receptors were blocked with excess HA or the anti-CD44
antibody HERM-1. Additionally, to investigate whether the
cellular uptake of NPs is energy-dependent and proceeds by
endocytosis, the active transport processes were strongly
inhibited by using a low temperature or by introducing
metabolic inhibitors. Each of the two cell lines were treated
with siRNA/HADPA-Zn-NPs and incubated at 48C without a
metabolic inhibitor or at 378C with the metabolic inhibitor
sodium azide. All of the treatments clearly reduced the
efficiency of siRNA/HADPA-Zn-NPs uptake into 4T1-fluc cells
((36.0 Æ 6.2)%, (28.7 Æ 1.5)%, (28.3 Æ 3.5)%, and (26.3 Æ
siRNA with low toxicity. This combination makes HADPA-Zn
-
NPs promising as safe and efficient vectors for targeted gene/
anticancer drug therapy.
The HADPA-Zn-NP conjugates were prepared by covalent
conjugation of the aminated bis(DPA) analogue 3 to the
carboxyl groups of HA-NP by using a standard ethyl-
(dimethylaminopropyl) carbodiimide/N-hydroxysuccinamide
(EDC/NHS) activation reaction in a buffer solution. HA-NP
and 3 were synthesized as previously reported (Scheme 1; see
also Figure S1–S4 in the Supporting Information).[4b,8c] After
dialysis of the mixture against distilled water, zinc ions were
incorporated into the complex by the addition of excess
Zn(ClO4)2·6H2O and sonication. Zn–DPA has an apparent
Kd value of 70 nm.[10] The HADPA-Zn-NP conjugate was isolated
1
as a white powder by lyophilization. Analysis by H NMR
spectroscopy showed that HADPA-Zn-NPs were conjugated to
about 32 molecules of 3 per 100 repeating units of HA (see
Figure S5 in the Supporting Information). As a result of its
amphiphilic structure, the HADPA-Zn-NPs self-assembled in
aqueous solution and remained stable in a solution of buffer
for at least two months at 48C. TEM images show that the
conjugation of 3 does not affect the structure of the NPs (see
Figure S6 in the Supporting Information). The ability of
HADPA-Zn-NPs to form Zn–DPA-mediated complexes with
siRNA was investigated by using a siRNA (siLuc) that targets
the firefly luciferase (fluc) gene as a model compound.
Different amounts of HA and NPs with (HADPA-Zn-NPs) and
without zinc ions (HADPA-NPs) were mixed with 15 pmol of
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Figure 1. In vitro characterization of siRNA/HADPA-Zn-NPs complexes. a) Electrophorectic retardation analysis of siRNA (15 pmol) binding with
different carriers (0–100 mg). b) Release of siRNA from siRNA/HADPA-Zn-NPs after the addition of the salts indicated. c) Cytotoxicity of HA-NPs and
HADPA-Zn-NPs in 4T1-fluc cells. d) Confocal microscopy images of 4T1-fluc cells treated with Cy3-siRNA complexed with HADPA-Zn-NPs or
Lipofectamine 2000 (L2K); blue: nuclei (DAPI), red: siRNA. e) Intracellular uptake of Cy3-siRNA/HADPA-Zn-NPs (NP) by 4T1-fluc cells and 293T
cells at low temperature (48C) or in the presence of various metabolic inhibitors at 378C; *p<0.005. f) Suppression of fluc gene expression by
HADPA-Zn-NPs (NP) or Lipo2K (L2K) complexed with control nontargeting siRNA or siLuc; B=buffer only *p<0.005 versus control, **p<0.005
versus Lipo2K/siLuc.
3.1)% for HA, HERM-1, 48C, and sodium azide, respec-
tively), which demonstrated that HADPA-Zn-NP/siRNA com-
plexes enter the cells to some extent through CD44 receptor
mediated endocytosis, and also in an energy-dependent
manner (Figure 1e). The ability of HADPA-Zn-NPs to deliver
siRNA was confirmed by treating 4T1-fluc cells with siLuc/
HADPA-Zn-NPs and measuring the expression of fluc (Fig-
ure 1 f). The bioluminescence imaging (BLI) signal intensity
of untreated 4T1-fluc cells was set to 100% fluc expression. In
agremment with the uptake of Cy3-siRNA shown in Fig-
ure 1d, siLuc/HADPA-Zn-NPs showed a remarkably high gene
silencing efficacy in a dose-dependent manner (10 mg, 20 mg,
and 40 mg of siLuc/HADPA-Zn-NPs reduced the expression of
fluc to (54.9 Æ 5.6)%, (33.4 Æ 3.5)%, and (10.5 Æ 1.2)%,
respectively). The HADPA-Zn-NP/siLuc complex was (2.2 Æ
0.4)-fold more effective in silencing the expression of fluc
than the Lipo2K formulation. To rule out the possibility that
the decreased expression of fluc was caused by a reduction in
cell viability because of nonspecific cytotoxicity, we per-
formed an MTT assay to assess cell viability after the BLI
experiment. No significant cytotoxicity was detected in the
4T1-fluc cells after BLI, which confirmed that the gene
silencing effect was a consequence of the treatment with
HADPA-Zn-NP/siLuc and was not caused by a reduced number
of viable cells (see Figure S10 in the Supporting Information).
The HA-NPs were designed with the objective of creating
tumor-targeting NPs for anticancer therapy. These NPs are
intended to carry hydrophobic drugs in their inner cores and,
once the NPs are delivered into tumor cells, release the
encapsulated drug by Hyal-1-mediated degradation of the
NPs. To demonstrate that HADPA-Zn-NPs can be used as a co-
delivery carrier for both an anticancer drug and for siRNA,
the hydrophobic and fluorescent carbocyanine 3,3’-dioctade-
cyloxacarbocyanine perchlorate (DiO) was used as a model
compound (see Figure S11 in the Supporting Information).
As DiO is a lipophilic molecule, it was readily encapsulated in
HADPA-Zn-NPs in its fluorescently quenched state. The release
of DiO from the NPs can be easily monitored by measuring
changes in fluorescence. The encapsulation of DiO along with
siRNA in the HADPA-Zn-NPs slightly increased the mean
diameter of the HADPA-Zn-NPs to (290.6 Æ 31.7) nm (see
Figure S7 in the Supporting Information). After the siRNA/
DiO/HADPA-Zn-NPs complex was exposed to a solution of
buffer that contained Hyal-1 (120 unitsmLÀ1), the fluores-
cence intensity increased significantly and was saturated
within 30 min as a result of the release of DiO from the
HADPA-Zn-NPs (Figure 2a). To monitor the release of DiO in
cells, 4T1-fluc and 293T cells were treated with siRNA/DiO/
HADPA-Zn-NPs, and the fluorescence intensity was recorded by
a fluorescence microplate reader. As shown in Figure 2b, a
gradual increase in the fluorescence occurred as early as
10 min after the cells were treated with siRNA/DiO/HADPA-
Zn-NPs. The maximum fluorescence signal was detected after
30 min in 4T1-fluc cells. This implies that 4T1-fluc cells are
highly permeable to the HADPA-Zn-NPs and that the HADPA-Zn
-
NPs are highly susceptible to intracellular Hyal-1, which
readily destroys the structural integrity of the HADPA-Zn-NPs
and leads to rapid release of the payload. To verify if the
intracellular release of DiO is Hyal-1-dependent, the release
of DiO was also tested in NIH3T3 cells, which have a lower
level of intracellular Hyal-1. The results demonstrate that the
release of DiO was significantly lower in NIH3T3 cells
relative to 4T1-fluc cells (see Figure S12 in the Supporting
Information). The finding that HADPA-Zn-NPs enhance cellu-
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implies that the downregulation
of the fluc gene was selectively
induced by the gene silencing
mechanism.
The development of biocom-
patible, efficient, and clinically
relevant
delivery
systems
remains an important challenge
for the clinical applications of
siRNA-based therapeutics. In
this study we demonstrated a
siRNA delivery platform that is
based on DPA coordinated to
ZnII, which is an artificial recep-
tor for phosphate anions. A com-
bination
of
self-assembled,
tumor-targeting NPs and a Zn–
DPA analogue conferred high
siRNA binding affinity on the
complex, which is based on the
multivalent interactions between
the NPs and the siRNAs. This
binding affinity enables specific,
intracellular siRNA delivery and
maintains the enhanced cell-pen-
etrating and tumor-targeting
properties of HA-based NPs.
Figure 2. In vitro enzyme-triggered release of DiO from DiO/HADPA-Zn-NPs. Fluorescence quantification
of DiO/HADPA-Zn-NPs after incubation a) with or without Hyal-1 enzyme (120 unitsmLÀ1) in acetate
buffer at 378C and b) with 4T1-fluc and 293T cells; *p<0.005 versus 293T cells. c) Intracellular uptake
of siRNA/DiO/HADPA-Zn-NPs in 4T1-fluc cells; blue: nuclei (DAPI), green: DiO, and red: siRNA.
lar uptake of both encapsulated drugs and siRNAs that are
coordinated to Zn–DPA is supported by confocal microscopy
images of 4T1-fluc cells after treatment with siRNA/DiO/
HADPA-Zn-NPs (Figure 2c).
To demonstrate the use of the siLuc/HADPA-Zn-NPs
in vivo, we investigated the gene silencing effect of siLuc/
HADPA-Zn-NPs in a 4T1-fluc xenograft mouse model. Four
groups of Balb/C mice (n = 5 per group) with subcutaneous
4T1-fluc tumors were treated with an intratumor injection of
80 mL of a buffer solution that contained siLuc/HADPA-Zn-NPs
(100 pmol/200 mg), HADPA-Zn-NPs (200 mg), naked siLuc
(100 pmol), or phosphate-buffered saline (PBS). The silenc-
ing of the fluc gene was measured by in vivo BLI. Figure 3a
shows a series of typical optical images of fluc-expressing
tumors after the administration of each formulation. After
72 hours, the level of fluc expression was significantly reduced
in the tumor that was treated with siLuc/HADPA-Zn-NPs;
however, the expression of fluc did not decrease in the other
control groups. Quantitative analysis showed that the relative
level of fluc expression was suppressed at 48 hours after the
injection of siLuc/HADPA-Zn-NPs and was significantly inhib-
ited to (À6.2 Æ 3.7)% at 72 hours. In contrast the control
groups had increased levels of fluc expression ((30.0 Æ 4.1)%,
(38.2 Æ 3.9)%, and (39.2 Æ 5.3)%, for HADPA-Zn-NPs, naked
siRNA, and PBS, respectively, Figure 3b). The tumors from
each group were harvested and weighed after BLI to
determine whether the apparent gene silencing effect was a
result of a reduction in the volume of the tumors as a
consequence of the toxicity of the formulation (see Figure S13
Figure 3. a) In vivo optical images and b) quantitative analysis of fluc-
expressing tumor after intratumor injection of siLuc/HADPA-Zn-NPs(NP),
HADPA-Zn-NPs, naked siLuc, or PBS (n=5 per group). *p<0.005 versus
control groups.
in the Supporting Information). As shown by the in vitro
testing, there was no significant difference in the neoplastic
weights of the tumors between the various groups. This result
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Keywords: dipicolylamines · drug delivery · hyaluronic acid ·
nanoparticles · siRNA
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