A Collaborative Assembly Strategy for Tumor-Targeted siRNA Delivery

Article abstract of DOI:10.1021/jacs.5b01435

A novel "collaborative assembly" approach was reported for the synthesis of an siRNA delivery system via a combination of an electrostatically driven physical assembly and a facile click reaction-mediated chemical assembly, which showed various advantages

Full text of DOI:10.1021/jacs.5b01435

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Article
A Collaborative Assembly Strategy for Tumor-Targeted siRNA Delivery
Qiong Sun, Zisheng Kang, Lingjing Xue, Yunkai Shang, Zhigui Su, Qineng Ping, Ran Mo, and Can Zhang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b01435 • Publication Date (Web): 14 Apr 2015
Downloaded from http://pubs.acs.org on May 3, 2015
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A Collaborative Assembly Strategy for Tumor-Targeted siRNA
Delivery
Qiong Sun, Zisheng Kang, Lingjing Xue, Yunkai Shang, Zhigui Su, Qineng Ping, Ran Mo*
and Can Zhang*
State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Discovery
for Metabolic Diseases, Center of Drug Discovery, China Pharmaceutical University, Nanjing
2
10009, China
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ABSTRACT
A novel “collaborative assembly” approach was reported for the synthesis of an siRNA
delivery system via a combination of an electrostatically-driven physical assembly and a
facile click reaction-mediated chemical assembly, which showed various advantages of more
safety, efficiency and flexibility over the conventional approach that is only based on the
physical assembly. This strategy remained a high cationic property of lipid-based complex for
high siRNA loading capacity. The direct chemical modification of a model polyanion,
hyaluronic acid (HA) on the cationic complex via click chemistry shielded the positive charge
of complex without affecting the siRNA binding, which reduced the toxicity and enhanced the
blood stability of the complex. In addition, the incorporated polyanion might be
pre-functionalized, which endued the carrier with better biological characteristics such as long
circulating or tumor targeting. We demonstrated that the obtained lipid-polymer hybrid
nanoparticle (RSC-HA) using collaborative assembly presented greater in vivo stability in the
blood for efficient tumor targeting than the physically-assembled RSC/HA in which HA was
physically adsorbed on the complex. After endocytosis into the cells, the protection of
RSC-HA on siRNA turned off, while the release of siRNA induced by the intracellular signals
for enhanced gene-silencing capacity. This combination of physical and chemical assemblies
provides an efficient strategy for the exploitation of safe, stable and functionalized siRNA
delivery systems.
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Keywords：click chemistry, collaborative assembly, siRNA delivery, tumor targeting, gene
silencing
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INTRODUCTION
Small interfering RNA (siRNA) has emerged as a promising therapeutic agent for targeted
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cancer therapy due to its potent gene-silencing activity. Naked siRNA have been proven to
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be unfavorable for systemic delivery due to its inherent limitations such as labile degradation
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by blood nucleases and rapid elimination by renal excretion. Up to now, although arduous
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efforts have been made, no commercial siRNA product is on the market for intravenous
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cancer treatment. The development of a safe and efficient siRNA delivery system still
remains to be a critical challenge.
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Viral and non-viral carriers have been widely reported for siRNA delivery.
Although
the viral vectors show high transfection capability, their applications are enormously limited
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by serious immunotoxicity and adverse effects.
Cationic lipids and polymers as the most
common non-viral vectors used for siRNA delivery can efficiently assemble with siRNA via
electrostatic adsorption to form a condensed complex, which provides enhanced cellular
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uptake and higher siRNA-mediated gene-silencing effect.
The development of cationic
carrier-based siRNA delivery systems is highly dependent upon the management of the
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relationship among cationic property, toxicity, siRNA binding efficiency and stability.
Higher cationic carriers result in stronger siRNA binding rates with higher stabilities.
However, such complex with high positive surface charge often induces complement
activation and inflammatory response after intravenous administration, thereby yielding
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severe toxicities in vivo.
Reducing positive charge may alleviate the toxicities in part, but
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affects the stability of the complex yet.
Moreover, not only can negatively charged blood
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proteins easily bind onto the cationic complex, which interferes with the complex stability in
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the circulation, but also competitively displace negatively charged siRNA in the complex,
which impedes the therapeutic efficacy of siRNA.
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To solve this issue, the most practical strategy is PEGylation of the cationic
lipids/polymers or noncovalent coating with polyanions to shield the high positive charge of
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the complex.
Nevertheless, the steric hindrance of the incorporated PEG chain decreases
the siRNA binding with the cationic carrier and also hampers the tumor cell uptake of the
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carrier.
Additionally, the PEGylated nanoparticles suffer from the accelerated blood
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clearance and lose their long-circulating properties after consecutive intravenous injections.
For the polyanionic coating as a physical layer-by-layer (LbL) assembly approach, the
incorporation of polyanions with higher negative charge inevitably disturbs the electrostatic
interaction between cationic constituents and anionic siRNA by competitive binding to the
complex, which causes the leakage of preloaded siRNA and the reduction in the siRNA
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loading capacity.
Furthermore, the exterior polyanions are inclined to be displaced by the
negatively charged proteins in the blood, leading to aggregation and elimination by the
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reticular epithelial system.
Accordingly, this physical assembly strategy that is mainly
depends upon the electrostatic force cannot realize an effective siRNA delivery as expected.
To address the dilemma in the cationic property-associated toxicities, siRNA binding
efficiency and stability, a new assembly strategy for siRNA packing and delivery is highly
desirable.
Herein, we report a novel “collaborative assembly” approach for synthesis of an siRNA
delivery system consisting of a lipid-polymer hybrid nanocarrier via a combination of an
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electrostatic force-based physical assembly and a facile click reaction-mediated chemical
assembly, which has a variety of advantages over the above-mentioned physical assembly
method: 1) the high cationic property of the lipid is remained without chemical modification
such as PEGylation, which renders the high siRNA binding and loading capacity; 2) the direct
chemical decoration of polyanion on the cationic vector shows no influence on the binding
between siRNA and the carrier, which can shield the cationic property, reduce the toxicity and
improve the blood stability of the complex; 3) the incorporated polyanion may be
pre-functionalized with PEG or ligands, which endues carriers with better biological
behaviors such as long circulating or tumor targeting.
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The chemical assembly is based on the azide-alkyne cycloaddition click chemistry.
Cholesterol (Chol) as a commonly used component for liposome preparation and hyaluronic
acid (HA) as a model polyanion with good biocompatibility and biodegradability are
functionalized with azide and alkyne groups as clickable modules, respectively. The
azide-modified cholesterol (Chol-N ) with a natural soy phosphatidylcholine (SPC) and a
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reduction-cleavable cationic lipid (LHSSG2C14
)
is firstly used to fabricate
a
dual-functionalized cationic liposome with redox-responsiveness and clickability, which is
further mixed with siRNA to form a complex (designated as RSC) via the
electrostatically-driven assembly (Figure 1A). The anionic alkyne-modified HA (alk-HA) is
readily coated on RSC, followed by the rapid click reaction-mediated coupling to achieve a
stable HA-conjugated RSC (RSC-HA) without the leakage of siRNA. HA also serves as an
active tumor-targeting ligand, which can bind to the cell surface receptors such as CD44 that
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is overexpressed in many tumors. Moreover, HA can be degraded by hyaluronidase (HAase)
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in the tumor intracellular compartments to “turn off” the protective function of HA,
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contributing to the exposure and release of the payloads.
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As illustrated in Figure 1B, RSC-HA after intravenous administration is expected to
have a good colloidal stability and preferentially accumulate at the tumor site by the
combined passive and active targeting effects. After internalization by the tumor cells into the
endosomes, the HA shell of RSC is degraded by HAase and sheds from RSC. Meanwhile, the
LHSSG2C14 component promotes endosomal escape of RSC due to the proton sponge effect
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of the imidazole ring of histidine, which allows migration of RSC into the cytoplasm. The
high cytoplasmic concentration of GSH further cleaves the disulfide bond in LHSSG2C via
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the thiol-disulfide exchange, which leads to the liposomal destabilization along with the
RSC disassembly and therefore triggers the release of the encapsulated siRNA in the
cytoplasm for gene silencing. This combination of physical and chemical assemblies provides
an efficient strategy for the exploitation of safe, stable and functionalized siRNA delivery
systems.
RESULTS AND DISCUSSION
Collaborative Assembly of RSC-HA.
The reduction sensitive cationic lipid, LHSSG2C14 (Scheme S1 in the Supporting
Information), the non-reduction sensitive cationic lipid, LHG2C14 (Scheme S2) and two
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clickable modules, Chol-N (Scheme S3) and alk-HA (Scheme S4) were first synthesized and
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characterized by H nuclear magnetic resonance spectroscopy. The azide-decorated
redox-sensitive cationic liposome (designated as RSN
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-L) consisting of SPC, LHSSG2C14
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and Chol-N
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(5:5:3, w:w:w) was prepared using the thin-film hydration method. RSN
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-L was
-L to
mixed with siRNA at the optimal N:P ratio of 4:1 (the molar ratio of nitrogen in RSN
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phosphate in siRNA) via electrostatic adsorption to form RSC, which had an average particle
size of 117 nm and a zeta potential of +18 mV (Figure 2A). The encapsulation of siRNA by
RSC was confirmed by the electrophoretic mobility shift assay (EMSA) (Figure 2B). No free
siRNA was observed on the siRNA band, indicating that the siRNA molecules were
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completely complexed with RSN
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-L. Subsequently, RSC was added into the alk-HA solution
of 2:1 to obtain a negatively charged alk-HA-coated RSC,
at a molar ratio of alk-HA:Chol-N
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followed by click reaction catalyzed by the in situ generation of active copper(I) species from
reduction of copper sulfate by sodium ascorbate (Figure 2C). The collaboratively-assembled
RSC-HA (N:P = 4:1, mol:mol) was finally achieved after removal of the catalysts by
centrifugal filtration. No undesirable release and destruction of the encapsulated siRNA were
determined after the click reaction-mediated assembly (Figure 2D). The obtained RSC-HA
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(alk-HA:Chol-N = 2:1, mol:mol) had a hydrodynamic diameter of about 130 nm and a zeta
potential of –26 mV. The transmission electron microscope (TEM) examination showed a
spheroid structure of RSC-HA (Figure 2E). The chemical assembly of HA on the RSC
surface was further demonstrated by both the disappearance of the characteristic infrared
-1
absorption peak of azide group at 2101 cm (Figure S1A) and the increased ultraviolet
absorbance resulting from generation of triazole group after click reaction (Figure S1B). To
evaluate the protection of siRNA by RSC-HA from nuclease degradation, RSC-HA was
incubated with ribonuclease A (RNase A), which is commonly present in the blood, for 30
o
min at 37 C, followed by the EMSA analysis (Figure 2F). Free siRNA was rapidly digested
by RNase A, while RSC-HA showed a high capability of protecting siRNA from the RNase A
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degradation.
To validate the higher stability of the collaboratively-assembled RSC-HA than the
physically-assembled complex, we took the unmodified HA-coated RSC complex with
siRNA (designated as RSC/HA) as a control, which had a resulting diameter of about 240 nm
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and a zeta potential of –27 mV at the N:P ratio of 4:1 (mol:mol) and the HA:Chol-N
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ratio of
2:1 (mol:mol) (Figure S2A). RSC/HA could fully condense siRNA at the N:P ratio higher
than 3:1 (Figure S2B). The variation in the particle size of RSC-HA and RSC/HA was
monitored after incubation under different conditions over time (Figure 3A). The particle size
of RSC-HA maintained unchanged in the presence of serum or at pH 7.4 (physiological pH)
and pH 6.5 (a typical tumor extracellular pH). By comparison, RSC/HA showed a large
fluctuation in the particle size under the same conditions, which was mainly attributed to the
unstable HA outer layer. Furthermore, the change of the HA amount on the surface of
RSC-HA and RSC/HA under a different set of circumstances was quantified using the
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cetyltrimethylammonium bromide (CTAB) turbidimetric method (Figure 3B). The free HA
shedding from the complex was separated using a size exclusion chromatography technique.
Slight change in the HA amount on RSC-HA was determined after incubation, while RSC/HA
presented a significantly decreased amount of HA. It was indicated that the
chemically-conjugated HA of RSC-HA was firmly anchored on the RSC surface for enhanced
stability, compared with the physically-bound HA of RSC/HA. Taken together, RSC-HA,
possessing a superior stability than RSC/HA, can reduce the unspecific interactions with
blood proteins and prolonged retention time in the circulation for elevated tumor targeting.
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Enzyme and Redox Combined-Triggered siRNA Release.
To explore the release of siRNA from RSC-HA under intracellular conditions, the EMSA
analysis was applied after incubating RSC-HA in the presence and absence of HAase and
GSH, respectively. The HAase-mediated HA degradation was confirmed by the surface
charge conversion of RSC-HA after treated with HAase (0.5 mg/mL) under acid condition as
time extended (Figure S3A), which was due to the degraded small molecular HA pieces
shedding from RSC. This result was in agreement with the quantitative assay (Figure S3B).
However, since HAase degraded the HA shell but the RSC core remained stable, no free
siRNA was visualized in the siRNA band after RSC-HA (N:P = 4:1, mol:mol) was incubated
with HAase (0.5 mg/mL) alone (Figure 4A and Figure S4A). On the other hand, only
treatment with a high concentration of GSH (10 mM, a typical concentration within the cells)
also showed no release of siRNA from RSC-HA (Figure 4B), which implied that the HA
shell held the inner RSC via the triazole linkages and hindered the siRNA release, although
RSC was destabilized by the GSH-mediated LHSSG2C14 cleavage. The TEM image
confirmed the structural integrality of RSC-HA after the GSH treatment (Figure 4C). In sharp
contrast, the released siRNA bands were clearly observed after incubation of RSC-HA with a
variety of concentrations of HAase in the presence of GSH (10 mM) (Figure 4D).
Additionally, the treatment with HAase and GSH at the concentration of 1 or 10 mM resulted
in the siRNA release from RSC-HA, while no released siRNA band was detected after
incubation of RSC-HA with HAase in the presence of GSH (10 µM) (a typical concentration
outside the cells) (Figure S4B). The disruption of RSC-HA was further substantiated by the
TEM imaging (Figure 4E). No spherical particles were visible after the combined treatment
with HAase (0.5 mg/mL) and GSH (10 mM). It was suggested that the rapid disassociation of
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RSC-HA accompanied by the efficient release of siRNA was synergistically triggered by a
combination of HAase and GSH inside the cells.
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To further demonstrate the specificity of the siRNA release, RSC-HA was treated with
both bovine serum albumin (BSA) and GSH, followed by the EMSA analysis. As expected,
no free siRNA band was detected (Figure S5), which indicated that the negatively charged
BSA was unable to competitively displace the HA shell conjugated on RSC and hardly induce
the release of siRNA even with the help of GSH. In addition, a non-redox-sensitive complex
(designated as nRSC-HA) was taken as a reference, which was prepared similarly as RSC-HA
except that a control cationic lipid without disulfide linkage, LHG2C14 was in place of
LHSSG2C14. nRSC-HA had an average particle size of 139 nm and zeta potential of –29 mV
at the N:P ratio of 4:1 (Figure S6A), which was capable of completely complexing siRNA
(Figure S6B). After the HAase/GSH-combined treatment, nRSC-HA did not show any
siRNA release (Figure S6C).
Next, we used a fluorescence resonance energy transfer (FRET) technique to estimate the
integrity of RSC-HA exposed to the HAase/GSH environment. NBD-PE
(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolami
ne) and RhB-PE (N-(lissamine rhodamine B sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-
phosphoethanolamine) were simultaneously pre-incorporated into the liposomal components
to obtain the FRET RSC-HA. The change in the FRET signals allowed the detection of the
integrity variation of RSC-HA. As shown in Figure 4F, the FRET RSC-HA showed an
apparent emission of RhB at 585 nm after the NBD excitation at 470 nm, suggesting that the
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integrity of RSC-HA supported the energy transfer from the NBD donor to the RhB acceptor.
Of note, treatment with either HAase or GSH had no remarkable impact on the stability of
RSC-HA, while the dissociation of RSC-HA after the HAase/GSH-combined treatment was
evidenced by the noticeably increased NBD signal together with the decreased RhB signal.
Collectively, the highly stable RSC-HA impeded the siRNA leakage under physiological
condition, while collapsed to efficiently release the encapsulated siRNA in response to
combined stimuli, HAase and GSH, both of which were highly expressed inside the tumor
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cells.
Cellular Uptake, Subcellular Distribution and Intracellular siRNA Realease.
To verify the HA-mediated active targeting of RSC-HA (N:P = 4:1, mol:mol), the human lung
adenocarcinoma epithelial (A549) cells, which overexpress the HA receptor, CD44 on the cell
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membrane, were incubated with different formulations containing FAM-labeled siRNA
(FAM-siRNA) for 6 h, followed by detecting the fluorescence intensity of FAM-siRNA
within the cells using flow cytometry (Figure 5A). RSC noticeably facilitated the cellular
uptake of siRNA upon the electrostatic attraction-based internalization compared with the
naked siRNA. However, RSC-HA showed significantly enhanced uptake of siRNA than RSC
without the HA shell, which indicated that the CD44-mediated endocytosis played an
important role in facilitating the intracellular accumulation of siRNA. Additionally, after the
cells were pre-treated with the excessive amount of free HA to block the CD44 receptor, the
cellular uptake of RSC-HA drastically reduced, which further confirmed that RSC-HA could
bind onto and enter into the cancer cells primarily via the CD44-mediated endocytosis.
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The endocytotic pathway of RSC-HA was then determined by monitoring the change in
the fluorescence intensity of siRNA after incubating the cells in the presence of different
inhibitors for specific pathways (Figure 5B). Treatment with sodium azide, an energy
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inhibitor , extremely reduced the cellular uptake of RSC-HA, indicating that RSC-HA
permeated into the cells via a typical energy-dependent endocytotic pathway. The presence of
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both nystatin, the inhibitor of caveolae-mediated endocytotic pathway and amiloride, the
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inhibitor of macropinocytosis , exhibited a pronounced effect on inhibiting the cellular
uptake of RSC-HA, suggesting that RSC-HA was localized in the caveosomes and
macropinosomes after internalization and then transported into the endosomes.
The subcellular distribution of RSC-HA in A549 cells was examined using the confocal
laser scanning microscope (CLSM) (Figure 5C). In the cells incubated with RSC-HA for 3 h,
the signal of FAM-siRNA (green) encapsulated in RSC-HA was mainly co-localized with the
signal of LysoTracker (red), a late endosome and lysosome marker, indicating that RSC-HA
was effectively taken up by the cells and entrapped into the endosomes. After an additional 3
h of incubation, a great dissociation in the signals of FAM-siRNA and LysoTracker was
observed, confirming the efficient endosomal escape of RSC-HA. This enhanced endosomal
escape was associated with the proton sponge effect of the cationic histidine-containing lipid
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in RSC-HA,
which could gradually absorb protons, produce an increased osmotic pressure
within the endosomes, and therefore cause the endosomal disruption, as confirmed by a
buffering capacity assay (Figure S7).
To evaluate the intracellular release of siRNA, the fluorescence change was also detected
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using CLSM after the cells were incubated with the double-labeled RSC-HA containing
FAM-siRNA and RhB-PE over time (Figure 5D). After 3 h of incubation, the signal of
FAM-siRNA (green) was mostly overlaid with the signal of RhB-PE (red) that was used to
label the nanocarrier, implying that siRNA was embedded in RSC-HA. Note that the FAM
and RhB signals showed an obvious separation after incubation for additional 9 h, suggesting
an efficient intracellular release and cytoplasmic distribution of siRNA due to the dissociation
of RSC-HA in response to the intracellular HAase and GSH.
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To further demonstrate the important role of HAase in the disassembly of RSC-HA in the
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cells, we applied a small-molecule inhibitor, thioguanine (TG) to inhibit the effect of HAase
on degrading HA and evaluated its inhibition effect on the HAase-mediated HA degradation.
RSC-HA was incubated with HAase in the presence of different concentrations of TG over
time, followed by determination of the HA degradation from RSC using the CTAB
turbidimetric method (Figure S8). At the concentration of 4.5 µM that has been proposed as
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the half maximal inhibitory concentration in the previous report, TG showed an efficient
capability of inhibiting HAase to degrade HA. When the concentration of TG was increased
to higher than 9 µM, the HA degradation by HAase was nearly completed suppressed.
Afterwards, the FRET technique was applied to estimate the role of HAase in the
destabilization of RSC-HA in cells. The cells were incubated with the FRET RSC-HA
containing NBD-PE and Rho-PE in the absence and presence of TG (9 µM) for different time,
followed by monitoring the FRET change (Figure S9). After the cells were incubated with the
FRET RSC-HA for 3 h, no significant change in the fluorescence spectra was found, while a
noticeable increased NBD signal along with the decreased RhB signal was observed after
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incubation with the FRET RSC-HA for additional 9 h, which suggested the disassembly of
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RSC-HA inside the cells (Figure S9A). The FRET ratio [IRhB/(IRhB + INBD) × 100%] of
RSC-HA in the cells reduced from 61.67% at 3 h to 50.06% at 3 h plus 9 h, where IRhB and
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INBD are the fluorescence intensity of RhB at 585 nm and NBD at 530 nm, respectively
(Figure S9B). In sharp contrast, the TG-treated cells presented no remarkable change in the
fluorescence spectra between 3 h and 3 h plus 9 h, suggesting that RSC-HA maintained the
structural integrality inside the cells since TG inhibited the HAase-mediated HA degradation.
The FRET ratio of RSC-HA showed no significant change as the incubation time increased
after treatment with TG. Furthermore, the intracellular siRNA release accompanied by the
disassembly of RSC-HA containing FAM-siRNA and RhB-PE was monitored in the presence
of TG using CLSM (Figure S9C). No apparent separation between the FAM and RhB signals
was observed after the TG-treated cells were incubated with RSC-HA for additional 9 h. As a
result, HAase had a significant effect on the intracellular disintegration of RSC-HA.
Gene Silencing and Apoptosis Inducing Capability.
To evaluate the siRNA-mediated gene-silencing efficiency of RSC-HA, the level of
messenger RNA (mRNA) transcription and the related protein expression were determined by
the quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis,
respectively (Figure 6 and Figure S10). Survivin, an inhibitor of apoptosis protein, functions
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associated with chemotherapy resistance. A therapeutic siRNA targeting survivin mRNA
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designed as cpusiRNA2 ) was encapsulated in RSC-HA. At the mRNA level, compared
with other formulations, the RSC-HA loaded with cpusiRNA2 showed the highest
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sequence-specific silencing efficiency on the survivin mRNA expression with 55.09%
down-regulation in A549 cells (Figure 6A) and 65.02% in the murine Lewis lung cancer
(LLC) cells that also overexpress CD44 receptor (Figure S10A), which validated that the
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enhanced cellular uptake and efficient intracellular delivery of siRNA by RSC-HA resulted in
the elevated gene-silencing effectiveness. In line with this, at the protein expression level,
cpusiRNA2 delivered by RSC-HA remarkably decreased the expression of survivin protein in
both of the lung cancer cells (Figure 6B and Figure S10B).
Next, we estimated the apoptosis-inducing effect of RSC-HA loaded with cpusiRNA2
using the Annexin V-FITC/PI apoptosis detection. As expected, the cells after treatment with
RSC-HA loaded with cpusiRNA2 showed the strongest apoptosis. The total apoptotic ratio, a
sum of the early and late apoptotic ratios, was 23.48% in A549 cells (Figure 6C) and 60.7%
in LLC cells (Figure S10C), much higher than that of other formulations. It is confirmed that
RSC-HA with the high gene-silencing efficiency exhibited a superior apoptosis-inducing
capacity.
Pharmacokinetics, Biodistribution, Therapeutic Efficacy and Safety Evaluation.
The FRET technique was first utilized to compare the stability of the conjugated HA on the
collaboratively-assembled RSC-HA with that of the adsorbed HA on the
physically-assembled RSC/HA during the systemic circulation (Figure 7A and Figure S11).
Both RSC-HA and RSC/HA were doubly labeled with NBD and RhB as a FRET pair, in
which NBD-PE was anchored into the liposome to track the complex core and RhB was
covalently conjugated to alk-HA (RhB-alk-HA) (Scheme S5) or HA (RhB-HA) (Scheme S6)
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to track the outer shell. When the HA shell was firmly coated on the complex core, the
proximity between NBD and RhB was in the FRET range, which supported the efficient
energy transfer from NBD to RhB at the excitation of NBD at 470 nm (Figure S11A). The
FRET ratio of RSC-HA and RSC/HA was calculated to be 63.62% and 61.70%, respectively.
After intravenous injection of the FRET nanocarriers into the rats for different time, the
change in the fluorescence signals in the plasma was monitored. The FRET signal maintained
up to 8 h in RSC-HA (Figure S11B), while the attenuation in the FRET effect as the
increased NBD signal with the decreased RhB signal was apparently observed in RSC/HA
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(Figure S11C). The FRET ratio of RSC/HA rapidly reduced to 50.91% within 1 h
post-injection, indicating that the adsorbed HA shell was unstable and underwent shedding
from RSC/HA in the circulation (Figure 7A). In contrast, the FRET ratio of RSC-HA retained
stable within the studied time period, suggesting that the chemical conjugation significantly
enhanced the in vivo stability of the HA shell.
The pharmacokinetics was investigated after intravenous injection of different
formulations containing FAM-siRNA (Figure 7B and Table S1). Compared with the
encapsulated siRNA in the complex, the naked siRNA showed an extremely rapid degradation
and clearance in the body, which had a half-time (t1/2) of only 0.26 h. The HA outer shell of
RSC/HA, nRSC-HA and RSC-HA increased the in vivo stability of RSC and rendered the
long circulating behavior. More importantly, RSC-HA displayed a conspicuously enhanced
blood persistence than RSC/HA, which had a higher area under the plasma concentration
versus the time curves (AUC), longer t1/2 and mean residence time (MRT), and a lower
clearance (Cl). Nevertheless, the pharmacokinetic parameters of RSC-HA were similar to that
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of nRSC-HA. It is speculated that the chemically-conjugated HA shell hold the complex core
more firmly than the physically-adsorbed one, thereby increasing the bioavailability of siRNA
in the blood for potential reinforcement of the tumor targeting and antitumor effect.
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To assess the tumor targeting property of RSC-HA, we applied an in vivo fluorescence
imaging technique to detect the biodistribution of RSC-HA loaded with Cy5-conjugated
siRNA (Cy5-siRNA) after intravenous injection into the LLC tumor-bearing mice (Figure
7C). RSC-HA showed greater tumor targetability than both RSC and RSC/HA, while no
significant difference was found in the effect between RSC-HA and nRSC-HA. The Cy5
signal of RSC-HA was clearly observed at the tumor site at 6 h post-injection and maintained
for up to 24 h. It is suggested that the enhanced HA shell via the chemical conjugation
realized the efficient and tumor-targeted intravenous siRNA delivery. To further demonstrate
the HA-mediated tumor targetability of RSC-HA, a high dose of the free HA was
intravenously pre-administrated into the mice followed by the injection of RSC-HA. The Cy5
signal was monitored to be much weaker in the tumor region after pre-injection of HA as
expected; this is because the free HA competitively blocked the binding between RSC-HA
and the CD44 receptors, which resulted in the decreased tumor accumulation of RSC-HA. At
24 post-injection, the tumor and different organs were collected for ex vivo imaging (Figure
7D). The Cy5 signal of RSC-HA at the tumor site was noticeably higher than that of RSC,
RSC/HA and the HA pre-injected RSC-HA, but comparable to that of nRSC-HA. The
quantitative region-of-interest (ROI) analysis further substantiated the superior tumor
targeting ability of RCS-HA (Figure 7E), which showed the Cy5 intensity with 2.7-, 1.7- and
2.2-fold of RSC, RSC/HA and the HA pre-injected RSC-HA at the tumor site, respectively.
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Additionally, the Cy5 intensity of RSC-HA at the tumor site was 392.4-, 1.2-, 10.2-, 58.9- and
3.3-fold of that at heart, liver, spleen, lung and kidney, respectively. There was no significant
difference in the Cy5 intensity between RSC-HA and nRSC-HA. The liver accumulation of
RSC-HA containing FAM-siRNA and RhB-alk-HA was monitored after injection for
different time (Figure S12). The liver sinusoidal endothelial cells (LSECs) specifically
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expressing CD146 receptors in the liver section were stained with the APC-conjugated
CD146 antibody. At 1 h post-injection, RSC-HA showed a clear liver accumulation, a large
part of which was rapidly taken up by the LSECs, as substantiated by the co-localization of
the FAM-siRNA (green), RhB-labeled HA (red) and CD146 (purple) expressing LSECs. As
time extended, both of FAM and RhB signals gradually attenuated and almost disappeared at
24 h post-injection, indicating the metabolism and excretion of RSC-HA by the liver.
Furthermore, the intratumoral distribution of RSC-HA containing RhB-PE and Cy5-siRNA
was also investigated at 24 h post-injection (Figure S13). The CD44 receptors in the tumor
section were stained with the FITC-conjugated CD44 antibody. Cy5-siRNA (red) and
RhB-labeled carrier (purple) of RSC-HA distributed throughout the tumor and closely
associated with CD44 (green) overexpressing cells, which implied that RSC-HA could
effectively deliver the encapsulated siRNA into the tumor region.
The therapeutic potential of RSC-HA loaded with cpusiRNA2 was evaluated on the LLC
tumor-bearing mice (Figure 8). Nanoparticle-based formulations loaded with cpusiRNA2
exhibited stronger effects on inhibiting the tumor growth, compared with the control groups,
such as saline, the naked cpusiRNA2 and RSC-HA/siN.C. (Figure 8A and Figure S14). Note
that a significant difference in the tumor growth inhibition was determined between RSC-HA
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and RSC/HA, suggesting that the in vivo stability of the HA shell on the complex played an
essential part in the tumor-targeted delivery of siRNA and the improved therapeutic efficacy.
Moreover, RSC-HA showed higher tumor growth inhibiting effect than nRSC-HA, indicating
that the efficient intracellular release of siRNA contributed to the enhanced antitumor activity.
The body weight of the mice did not show obvious change during the period of treatment with
RSC-HA (Figure S15). At the end of treatment, the tumors were harvested and weighed. The
mice treated with RSC-HA presented much lower mass ratio of the tumor to the body,
compared with other formulations (Figure 8B). Treatment with the RSC-HA loaded with
cpusiRNA2 presented the highest sequence-specific silencing efficiency on the survivin
mRNA expression with 63.70% down-regulation (Figure 8C), and remarkably decreased the
expression of survivin protein in the tumor (Figure 8D). In addition, the hematoxylin and
eosin (HE) stained tumor section for histological analysis showed a large area of cancer cells
remission after the mice received the treatment of RSC-HA (Figure S16A). The apoptosis
detection using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
assay confirmed that treatment with the RSC-HA carrying survivin-targeted siRNA produced
greater apoptosis for efficiently suppressing the tumor growth (Figure S16B).
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For the safety evaluation, the quantities of alkaline phosphatase (AKP), alanine
transaminase (ALT), aspartate transaminase (AST) for liver toxicity and blood urea nitrogen
(BUN) for kidney toxicity in the plasma were determined after RSC-HA treatment in
comparison with that after treatment with saline, respectively (Figure S17). No significant
difference in the amount of AKP, ALT, AST and BUN between RSC-HA and saline was
observed. Meanwhile, the main normal organs were harvested and weighed, such as heart,
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liver, spleen, lung, kidney and thymus. The mice treated with RSC-HA did not show any
noticeable variation in the mass ratio of the organ to the body (Figure S18A). Furthermore,
the histological analysis on the liver, spleen and kidney using the HE staining evidenced that
treatment with RSC-HA displayed no pathological change compared with treatment with
saline (Figure S18B). In addition, no gene silencing caused by RSC-HA was determined in
the liver, spleen and kidney compared with that in the tumor (Figure S19). These results
suggested that RSC-HA was a safe and potential nanocarrier for the efficient siRNA delivery
within the studied time period.
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CONCLUSIONS
In summary, we have developed a novel “collaborative assembly” strategy for a
tumor-targeted siRNA delivery system based on a combination of physical and chemical
assembly. RSC-HA was successfully fabricated by directly conjugating the polyanionic HA
polymer on the surface of the cationic lipid-based complex with siRNA via click chemistry,
which showed greater in vivo stability in the blood for efficient tumor targeting, compared
with RSC/HA in which HA was physically adsorbed on the complex. The protection of
RSC-HA on siRNA turned off inside the cells and the release of siRNA activated by the
intracellular signals for enhanced therapeutic efficacy. This assembly approach resolved the
conflict among toxicities, siRNA binding efficiency and stability related to the cationic
property of the complex. We anticipate this strategy will provide a promising platform for the
easy-to-fabrication of effective siRNA delivery systems and potentially promote the clinical
application of siRNA therapy.
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ASSOCIATED CONTENT
Supporting Information
Experimental procedures, in vitro and in vivo characterization of the nanocarrier. This material
is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
rmo@cpu.edu.cn; zhangcan@cpu.edu.cn
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (81473153,
81273468), National Basic Research Program of China (2015CB755500), State Key
Laboratory of Natural Medicines at China Pharmaceutical University (SKLNMZZCX201401),
and 111 Project from the Ministry of Education of China and the State Administration of
Foreign Expert Affairs of China (No. 111-2-07).
REFERENCES
(1) Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. C. Nature
1998, 391, 806.
(2) Whitehead, K. A.; Langer, R.; Anderson, D. G. Nat. Rev. Drug Discovery 2009, 8,
516.
(3) Salcher, E. E.; Wagner, E. Top. Curr. Chem. 2010, 296, 227.
(4) Whitehead, K. A.; Dorkin, J. R.; Vegas, A. J.; Chang, P. H.; Veiseh, O.; Matthews, J.;
21
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4
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5
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6
Fenton, O. S.; Zhang, Y. L.; Olejnik, K. T.; Yesilyurt, V.; Chen, D. L.; Barros, S.; Klebanov,
B.; Novobrantseva, T.; Langer, R.; Anderson, D. G. Nat. Commun. 2014, 5. 4277.
(
5) Nomoto, T.; Fukushima, S.; Kumagai, M.; Machitani, K.; Arnida; Matsumoto, Y.;
Oba, M.; Miyata, K.; Osada, K.; Nishiyama, N.; Kataoka, K. Nat. Commun. 2014, 5, 3545.
6) Guo, X.; Huang, L. Acc. Chem. Res. 2012, 45, 971.
0
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4
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7
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3
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(
(7) Cui, L.; Cohen, J. L.; Chu, C. K.; Wich, P. R.; Kierstead, P. H.; Frechet, J. M. J. Am.
Chem. Soc. 2012, 134, 15840.
(8) Pecot, C. V.; Calin, G. A.; Coleman, R. L.; Lopez-Berestein, G.; Sood, A. K. Nat. Rev.
Cancer 2011, 11, 59.
(9) Thompson, J. D. Drug Discovery Today: Ther. Strat. 2013, 10, e133.
(
(
(
(
10) Thomas, C. E.; Ehrhardt, A.; Kay, M. A. Nat. Rev. Genet. 2003, 4, 346.
11) Niidome, T.; Huang, L. Gene Ther. 2002, 9, 1647.
12) Descamps D, B. K. Curr. Gene Ther. 2009, 9, 115.
13) Duerner, L. J.; Schwantes, A.; Schneider, I. C.; Cichutek, K.; Buchholz, C. J. Gene
Ther. 2008, 15, 1500.
14) Schroeder, A.; Levins, C. G.; Cortez, C.; Langer, R.; Anderson, D. G. J. Intern. Med.
010, 267, 9.
(
2
(
(
(
15) Howard, K. A. Adv. Drug Delivery Rev. 2009, 61, 710.
16) Yang, X.; Du, J.; Dou, S.; Mao, C.; Long, H.; Wang, J. ACS Nano 2011, 6, 771.
17) Ornelas-Megiatto, C.; Wich, P. R.; Frechet, J. M. J. Am. Chem. Soc. 2012, 134,
1902.
(18) Yu, T.; Liu, X.; Bolcato-Bellemin, A. L.; Wang, Y.; Liu, C.; Erbacher, P.; Qu, F.;
Rocchi, P.; Behr, J. P.; Peng, L. Angew. Chem. Int. Ed. 2012, 51, 8478.
22
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Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
(
19) Zhang, S.; Zhao, B.; Jiang, H.; Wang, B.; Ma, B. J. Controlled Release 2007, 123, 1.
(20) Ozpolat, B.; Sood, A. K.; Lopez-Berestein, G. Adv. Drug Delivery Rev. 2014, 66,
1
10.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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45
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
(21) Ballarín-González, B.; Howard, K. A. Adv. Drug Delivery Rev. 2012, 64, 1717.
(
22) Wang, Y.; Xu, Z.; Zhang, R.; Li, W.; Yang, L.; Hu, Q. Colloids Surf. B:
Biointerfaces 2011, 84, 259.
23) Al-Qadi, S.; Alatorre-Meda, M.; Zaghloul, E. M.; Taboada, P.; Remunán-López, C.
Colloids Surf. B: Biointerfaces 2013, 103, 615.
24) Ballarin-Gonzalez, B.; Howard, K. A. Adv. Drug Delivery Rev. 2012, 64, 1717.
(
(
(25) Hashimoto, Y.; Abu Lila, A. S.; Shimizu, T.; Ishida, T.; Kiwada, H. J. Controlled
Release 2014, 184, 1.
(
26) Huang, Y.; Lin, D.; Jiang, Q.; Zhang, W.; Guo, S.; Xiao, P.; Zheng, S.; Wang, X.;
Chen, H.; Zhang, H.-Y.; Deng, L.; Xing, J.; Du, Q.; Dong, A.; Liang, Z. Biomaterials 2012,
3, 4653.
27) Ran, R.; Liu, Y.; Gao, H.; Kuang, Q.; Zhang, Q.; Tang, J.; Huang, K.; Chen, X.;
Zhang, Z.; He, Q. Int. J. Pharmaceutics 2014, 477, 590.
28) Buyens, K.; De Smedt, S. C.; Braeckmans, K.; Demeester, J.; Peeters, L.; van
3
(
(
Grunsven, L. A.; de Mollerat du Jeu, X.; Sawant, R.; Torchilin, V.; Farkasova, K.; Ogris, M.;
Sanders, N. N. J. Controlled Release 2012, 158, 362.
(29) Hatakeyama, H.; Akita, H.; Ito, E.; Hayashi, Y.; Oishi, M.; Nagasaki, Y.; Danev, R.;
Nagayama, K.; Kaji, N.; Kikuchi, H.; Baba, Y.; Harashima, H. Biomaterials 2011, 32, 4306.
(
30) Abu Lila, A. S.; Kiwada, H.; Ishida, T. J. Controlled Release 2013, 172, 38.
31) De Koker, S.; Hoogenboom, R.; De Geest, B. G. Chem. Soc. Rev. 2012, 41, 2867.
(
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5
5
6
(
32) de Villiers, M. M.; Otto, D. P.; Strydom, S. J.; Lvov, Y. M. Adv. Drug Delivery Rev.
2011, 63, 701.
(33) Kluger, R. J. Am. Chem. Soc. 2010, 132, 6611.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
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9
0
(34) Jiang, T.; Sun, W.; Zhu, Q.; Burns, N. A.; Khan, S. A.; Mo, R.; Gu, Z. Adv. Mater.
015, 27, 1021.
2
(35) Mo, R.; Jiang, T.; DiSanto, R.; Tai, W.; Gu, Z. Nat. Commun. 2014, 5, 3364.
(
36) Jiang, T.; Mo, R.; Bellotti, A.; Zhou, J.; Gu, Z. Adv. Funct. Mater. 2014, 24, 2295.
(37) Mo, R.; Sun, Q.; Xue, J.; Li, N.; Li, W.; Zhang, C.; Ping, Q. Adv. Mater. 2012, 24,
705.
(38) Wu, W.; Yao, W.; Wang, X.; Xie, C.; Zhang, J.; Jiang, X. Biomaterials 2015, 39,
60.
3
2
(
(
(
(
39) Qhattal, H. S.; Liu, X. Mol. Pharmaceutics 2011, 8, 1233.
40) Stern, R.; Jedrzejas, M. J. Chem. Rev. 2006, 106, 818.
41) Balendiran, G. K.; Dabur, R.; Fraser, D. Cell Biochem. Funct. 2004, 22, 343.
42) Kim, E.; Yang, J.; Park, J.; Kim, S.; Kim, N. H.; Yook, J. I.; Suh, J. S.; Haam, S.;
Huh, Y. M. ACS Nano 2012, 6, 8525.
43) Fazlollahi, F.; Angelow, S.; Yacobi, N. R.; Marchelletta, R.; Yu, A. S.;
Hamm-Alvarez, S. F.; Borok, Z.; Kim, K. J.; Crandall, E. D. Nanomedicine: NBM 2011, 7,
88.
(
5
(
44) Dudleenamjil, E.; Lin, C. Y.; Dredge, D.; Murray, B. K.; Robison, R. A.; Johnson, F.
B. J. Gen. Virol. 2010, 91, 3032.
45) Koivusalo, M.; Welch, C.; Hayashi, H.; Scott, C. C.; Kim, M.; Alexander, T.; Touret,
(
N.; Hahn, K. M.; Grinstein, S. J. Cell Biol. 2010, 189, 547.
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9
(
46) Shigeta, K.; Kawakami, S.; Higuchi, Y.; Okuda, T.; Yagi, H.; Yamashita, F.;
Hashida, M. J. Controlled Release 2007, 118, 262.
47) Shen, M.; Chao, C.; Wu, Y.; Wu, Y.; Huang, C.; Li, Y. Sens. Actuators, B 2013, 181,
(
10
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605.
(
48) Li, Y.; Xiao, W.; Xiao, K.; Berti, L.; Luo, J.; Tseng, H. P.; Fung, G.; Lam, K. S.
Angew. Chem. Int. Ed. 2012, 51, 2864.
49) Altieri, D. C. Oncogene 2003, 22, 8581.
(50) Kanwar, R. K.; Cheung, C. H.; Chang, J. Y.; Kanwar, J. R. Curr. Med. Chem. 2010,
7, 1509.
(51) Lu, Y.; Wang, K.; He, R.; Xi, T. Cancer Biother. Radiopharm. 2008, 23, 727.
52) Mandili, G.; Alchera, E.; Merlin, S.; Imarisio, C.; Chandrashekar, B. R.; Riganti, C.;
(
1
(
Bianchi, A.; Novelli, F.; Follenzi, A.; Carini, R. J. Hepatol. 2015, 62, 573.
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Figure 1. (A) Schematic illustration of the “collaborative assembly” strategy for construction
of an siRNA delivery system consisting of a lipid-polymer hybrid nanocarrier via a
combination of an electrostatically-driven physical assembly and a click reaction-mediated
chemical assembly. (B) Schematic illustration of tumor-targeted siRNA delivery by RSC-HA.
i: accumulation of RSC-HA at the tumor site; ii: endocytosis of RSC-HA into the tumor cells;
iii: HAase-mediated degradation of the HA shell of RSC-HA and endosomal escape into the
cytoplasm; iv: GSH-triggered disassembly of RSC-HA and release of the complexed siRNA
in the cytoplasm; v: gene silencing induced by the released siRNA.
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Figure 2. (A) Particle size and zeta potential of RSC at different N:P ratios. (B) Agarose gel
electrophoresis assay of RSC at different N:P ratios. I: the complexed siRNA band; II: the
free siRNA band. (C) Particle size and zeta potential of RSC-HA with siRNA at different
ratios of alk-HA:Chol-N . (D) Agarose gel electrophoresis assay of RSC-HA
3
(alk-HA:Chol-N
3
= 2:1, mol:mol) at different N:P ratios. (E) Size distribution histogram of
= 2:1, mol:mol) using the DLS measurement.
RSC-HA (N:P = 4:1, mol:mol; alk-HA:Chol-N
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Inset: TEM image of RSC-HA. Scale bar: 200 nm. (F) Agarose gel electrophoresis assay of
RSC-HA at different N:P ratios in the absence or presence of RNase A or SDS.
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Figure 3. (A) Change in the particle size of RSC-HA and RSC/HA after incubation at pH 7.4
and pH 6.5 or in the presence of serum over time. RSC-HA was constructed by the
collaborative assembly strategy, while RSC/HA was fabricated by the physical assembly
approach. (B) Quantification of the HA detached from RSC-HA and RSC/HA under different
**
conditions. P < 0.01.
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Figure 4. (A) Agarose gel electrophoresis assay of RSC-HA at different N:P ratios after 2 h
of incubation with 0.5 mg/mL HAase for 2 h. (B) Agarose gel electrophoresis assay of
RSC-HA at different N:P ratios after 2 h of incubation with 10 mM GSH. (C) Size
distribution histogram of RSC-HA after 2 h of incubation with 10 mM GSH using the DLS
measurement. Inset: TEM image of the treated RSC-HA. Scale bar: 200 nm. (D) Agarose gel
electrophoresis assay of RSC-HA after 2 h of incubation with different concentrations of
HAase and another 2 h of incubation with 10 mM GSH. (E) Size distribution histogram of
RSC-HA after 2 h of incubation with 0.5 mg/mL HAase and 10 mM GSH using the DLS
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