Shell-detachable nanoparticles based on a light-responsive amphiphile for enhanced siRNA delivery

Article abstract of DOI:10.1039/c3ra44866e

Novel light-responsive nanoparticles based on an amphiphile with a single photolabile linker between its hydrophilic head and hydrophobic tail was developed for small interfering RNA (siRNA) delivery. Upon UV exposure, cleavage of the linkage resulted in rapid shell detachment of the nanoparticles, which facilitated siRNA release and enhanced gene silencing efficiency.

Full text of DOI:10.1039/c3ra44866e

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Shell-detachable nanoparticles based on a
light-responsive amphiphile for enhanced
Cite this: RSC Adv., 2014, 4, 1961
siRNA delivery†
Received 3rd September 2013
Accepted 21st November 2013
Hong-Jun Li, Hong-Xia Wang, Chun-Yang Sun, Jin-Zhi Du* and Jun Wang*
DOI: 10.1039/c3ra44866e
www.rsc.org/advances
Novel light-responsive nanoparticles based on an amphiphile with a aspect of intracellular siRNA release, versatile smart vectors
single photolabile linker between its hydrophilic head and hydro- with stimuli-triggered sensitivities have been developed. For
phobic tail was developed for small interfering RNA (siRNA) delivery. example, various pH-, light- or redox-responsive delivery vectors
Upon UV exposure, cleavage of the linkage resulted in rapid shell have been constructed and demonstrated to be capable of
detachment of the nanoparticles, which facilitated siRNA release and enhancing gene silencing efficiency by facilitating siRNA release
11–16
enhanced gene silencing efficiency.
in the cytoplasm.
Despite these impressive achievements, either hydro-
phobization or stimuli-triggered siRNA release only meets one
aspect of the design dilemma. Studies that simultaneously
solved the two aspects are rarely reported. The design of siRNA
delivery systems can still be advanced and optimized. In the
present study, we propose a light-responsive amphiphile as a
proof-of-concept model in an attempt to address such a design
dilemma. Light-responsive linkage was used because it can be
remotely applied with high spatial and temporal precision and
Small interfering RNA (siRNA) is one of the most powerful
technologies for sequence-specic suppression of genes, and is
considered highly promising to treat a variety of human
1
disease. However, its application is markedly hampered by
inherent drawbacks, including susceptibility to degradation by
2
serum nucleases and poor cellular uptake. Successful applica-
tions of siRNA-based therapy rely on the development of safe
and efficient delivery vectors. Up to now, various promising
has been widely used as an external stimulus to control cargo
3
delivery systems, including cationic polymers, lipid-based
17–19
release from delivery systems.
The amphiphile in this study
4
5
vectors, and inorganic nanoparticles have been developed for
siRNA delivery.
has a long hydrophobic tail and a cationic head with a single
photolabile 2-nitrobenzyl (NB) ester bond as a bridge. In
aqueous solution, the amphiphile self-assembles into UV-
sensitive nanoparticles (USNPs) with a hydrophobic core and a
cationic shell. The hydrophobic core maintains the stability of
the nanoparticles, while the cationic shell provides complex
sites for siRNA condensation. When exposed to UV light irra-
diation, cleavage of the NB bond causes detachment of the
hydrophilic shell and subsequently facilitates siRNA release
from the complex to eventually enhance gene silencing effi-
ciency Scheme 1.
One great challenge for successful gene silencing is to
address an inherent dilemma in designing ideal vectors; that is,
the vectors should on the one hand form a tight and stable
complex with siRNA during circulation, and on the other hand
6
they should be able to facilitate siRNA release in the cytoplasm.
To address the former aspect, hydrophobic modication of
cationic vectors has been considered a useful solution. Several
studies have conrmed that hydrophobic modication of water
soluble polycations signicantly improves the binding ability,
stability and gene silencing efficacy of siRNAs as compared to
The cationic amphiphile 1 was synthesized through a
3,7
the parent vectors. Furthermore, the incorporation of hydro-
phobic segments in polycations could produce siRNA
complexes with enhanced stability, improved blood circulation
multiple reaction procedure, as illustrated in the ESI
1
(
Scheme S1†). Its chemical structure was veried by H NMR
and ESI-MS data (Fig. S1†). USNPs of amphiphile 1 were
prepared by a dialysis method. As shown in Fig. 1a, the trans-
mission electron microscopy (TEM) image indicates that USNPs
were well-dispersed with an average diameter approximately
8
–10
and biodistribution for in vivo studies.
To solve the latter
School of Life Sciences, University of Science and Technology of China, Hefei, Anhui
2
+
†
30027, P.R. China. E-mail: djzhi@mail.ustc.edu.cn; jwang699@ustc.edu.cn; Fax: 230 nm, which is in good agreement with the dynamic light
86 551 63600402; Tel: +86 551 63600335
scattering (DLS) results shown in Fig. 1b.
Electronic supplementary information (ESI) available: Experimental details and
raw data. See DOI: 10.1039/c3ra44866e
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Fig. 2 (a) Size and (b) zeta potential measurements of USNP/siRNA
Scheme
1
Illustration of the preparation of UV-sensitive nano- complexes at different N/P ratios. (c) Agarose gel analysis of siRNA
particles/siRNA complexes (USNP/siRNA). The complexes can be migration of the USNP/siRNA complexes at N/P ratios varying from 1/1
taken up by cancer cells and effectively release siRNA by the trigger of to 20/1. (d) Effect of UV irradiation time on siRNA migration from the
UV light.
USNP/siRNA complexes at N/P ¼ 25.
345 nm against irradiation time, one can see that the absor-
bance decreased rapidly in the initial 200 s and attened
gradually aer 300 s (Fig. S2†), demonstrating the rapid
degradation of the NB ester bond upon UV irradiation.
Due to the presence of amino groups on the surface of
USNPs, the zeta potential of the nanoparticles was highly
positive (52.2 ꢀ 0.5 mV). As siRNA is negatively charged, the
USNPs were expected to bind siRNA through electrostatic
interactions. Aer mixing USNPs with siRNA at various
nitrogen/phosphate (N/P) ratios, the size and zeta potential of
the complexes were determined. As shown in Fig. 2a and b, with
an increase in the N/P ratio, the size of the complexes changed
dramatically, transforming from 234.3 nm at N/P ¼ 5 to
2
3
687.2 nm at N/P ¼ 10, then decreased and remained less than
00 nm when the N/P ratio was greater than 25. Meanwhile, the
Fig. 1 Size measurement of USNPs by (a) TEM and (b) DLS. (c) Partial
H NMR spectra of amphiphile 1 before UV irradiation (bottom), after
UV irradiation for 300 s (middle) and pure palmitic acid (top). (d) UV-Vis
spectra of USNPs after exposure to UV light at times varying from 0 to
zeta potential of the complexes increased consistently with an
increase in the N/P ratio. It should be noted that the maximum
particle size at N/P ¼ 10 is caused by neutral surface charge
1
21
9
00 s. The USNPs solution was subjected to UV irradiation at the induced particle aggregation. In accordance with these results,
ꢁ2
intensity of 10 mW cm (lmax ¼ 365 nm).
the gel retardation assay in Fig. 2c also shows that the migration
of siRNA could be completely halted by the USNPs when the N/P
ratio was greater than 10, demonstrating successful siRNA
The UV-light triggered degradation of USNPs was studied by binding by USNPs. In comparison, the pure water soluble
H NMR and UV-Vis absorption methods. Upon UV irradiation, cationic molecule diethylenetriamine hydrochloride was also
1
the NB ester bond was cleaved, which causes characteristic used; this molecule has an identical structure to the cationic
1
changes in both the H NMR spectrum and UV-Vis absorption head of amphiphile 1 but could not bind siRNA, even at an N/P
curve. As shown in Fig. 1c, aer UV irradiation for 300 s, the ratio up to 50 (Fig. S3†). This result implies that the formation
chemical shis of peak a and b shied from 1.55 and 2.38 ppm of nanoparticles by introducing a hydrophobic tail indeed
to 1.50 (a*) and 2.18 ppm (b*), respectively. By comparing the improves siRNA binding capacity.
1
ratio of peak b and b* of the middle H NMR spectrum, the
siRNA release from the complex is a crucial step for effective
degradation efficiency was calculated to be 72% aer 300 s of gene silencing. One key design of our USNPs was the UV-
UV irradiation. Meanwhile, the UV-Vis absorption spectra also induced degradation of the carrier to facilitate siRNA release. To
showed a rapid decrease at 345 nm along with a concomitant test this, USNP/siRNA complexes at an N/P ratio of 25 were
increase at 265 nm upon UV irradiation (Fig. 1d). This spectral subjected to UV irradiation for a predetermined period of time
change was attributed to cleavage of the linker and the forma- and analyzed by agarose gel electrophoresis. As shown in
20
tion of nitrosobenzaldehydes. By plotting the absorbance at Fig. 2d, with an increase in irradiation time, more free siRNAs
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were observed to migrate from the loading position, indicating Fig. 3c, compared with the PBS control, neither free siLuc nor
that UV-induced nanoparticle degradation facilitated siRNA the UNSP complex with negative control siRNA (USNP/siN.C.)
release.
generated a gene silencing effect. Furthermore, the gene
Next, cellular uptake and intracellular siRNA release of the silencing efficiencies did not change considerably, even aer UV
USNP/siRNA complexes were studied by ow cytometry (FACS) irradiation for 300 s, implying that UV irradiation did not
and confocal laser scanning microscopy (CLSM) in MDA-MB- induce cell cytotoxicity under these experimental conditions. In
231 cells. As depicted in Fig. 3a, compared to free FAM-labeled contrast, the USNP/siLuc complexes showed better performance
siRNA (FAM-siRNA), USNP/FAM-siRNA complexes showed aer UV irradiation. Without UV irradiation, USNP/siLuc only
much stronger uorescent signals, whether the incubation time exhibited a slight gene silencing effect (ꢂ15%). However, aer
was 3 or 5 h, demonstrating that USNPs were effective in UV irradiation for 300 s, the gene silencing effect was signi-
delivering siRNA into cancer cells. CLSM images also conrmed cantly enhanced up to 50%, which could be attributed to the
this result. As shown in Fig. 3b, with free siRNA, no detectable enhanced intracellular siRNA release induced by UV-triggered
FAM-siRNA uorescence could be seen in the cells. In contrast, nanoparticle degradation. Additionally, an MTT assay revealed
clear green uorescence could be observed when cells were that the USNPs did not induce obvious cell cytotoxicity at the
treated with USNP/FAM-siRNA complexes. Furthermore, by test concentrations (Fig. S4†).
careful observation, it was found that the green uorescence of
In summary, we have successfully developed a novel light-
the complexes without UV irradiation was mainly in an aggre- responsive shell-detachable nanoparticle based on an amphi-
gated form, whereas that upon UV exposure was much more phile with a photolabile linker for enhanced siRNA delivery. The
dispersed throughout the entire cytoplasm and even in the formation of nanoparticles enhanced siRNA binding when
nuclei. These results conrm our initial expectation that USNPs compared to the water-soluble cationic counterpart at identical
could promote intracellular siRNA release in response to UV N/P ratios. More importantly, when exposed to UV light,
light irradiation.
cleavage of the linker caused rapid shell detachment of the
Based on these observations, we further studied whether the nanoparticle and accordingly facilitated intracellular siRNA
enhanced cellular uptake and intracellular siRNA release would release and enhanced gene silencing efficiency. Our design
result in enhanced in vitro gene silencing. The siRNA sequence provides a new strategy to manipulate delivery systems for
targeting the luciferase gene (siLuc) was chosen to form a advanced performance. Just recently, near-infrared (NIR) light
complex with USNPs, and the silencing efficiency was tested in with deeper tissue penetration and lower toxicity has attracted
stable luciferase-expressing MDA-MB-231 cells. As shown in more and more attentions in biomedical applications. By
combining UV-sensitive leaving groups with upconversion
nanoparticles, a couple of materials have been reported to be
22
capable of responding to NIR light to promote cargo release.
Based on these progresses, our newly designed siRNA vector can
extend to NIR responsiveness and further expands its applica-
tions in the future.
Acknowledgements
This work was supported by the National Basic Research
Program of China (973 Programs, 2012CB932500,
2013CB933900) and the National Natural Science Foundation of
China (51125012, 51390482) and SRFDP (20133402110019).
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