Dendronized Semiconducting Polymer as Photothermal Nanocarrier for Remote Activation of Gene Expression

Article abstract of DOI:10.1002/anie.201705543

Regulation of transgene systems is needed to develop innovative medicines. However, noninvasive remote control of gene expression has been rarely developed and remains challenging. We herein synthesize a near-infrared (NIR) absorbing dendronized semicondu

Full text of DOI:10.1002/anie.201705543

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Accepted Article
Title: Dendronized semiconducting polymer as photothermal
nanocarrier for remote activation of gene expression
Authors: Yan Lyu, Dong Cui, He Sun, Yansong Miao, Hongwei Duan,
and Kanyi Pu
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COMMUNICATION
Dendronized Semiconducting Polymer as Photothermal
Nanocarrier for Remote Activation of Gene Expression
Yan Lyu, Dong Cui, He Sun, Yansong Miao, Hongwei Duan, and Kanyi Pu*
[
+]
[+]
[
*] Y. Lyu, D. Cui, Prof. Y. Miao, Prof. H. Duan, Prof. K. Pu
School of Chemical and Biomedical Engineering
Nanyang Technological University
Singapore, 637457
E-mail: kypu@ntu.edu.sg
H. Sun, Prof. Y. Miao
School of Biological Science
Nanyang Technological University
Singapore, 637551
[
+] These authors contributed equally to this work.
Abstract: Regulation of transgene systems is imperative to develop
innovative medicines. However, noninvasively remote control of gene
expression has been rarely developed and remains to be challenging.
We herein synthesize a near-infrared (NIR) absorbing dendronized
semiconducting polymer (DSP) and utilize it as a photothermal
nanocarrier not only to efficiently deliver genes but also to
spatiotemporally control gene expression in conjunction with heat-
Semiconducting polymer nanoparticles (SPNs) have evolved
[8]
as versatile optical agents for molecular imaging. As SPNs
comprise completely benign organic components, they bypass
the issue of metal ion induced toxicity but possess optical
advantages equal or sometimes superior to inorganic
[9]
semiconductor nanoparticles. SPNs have also been revealed to
efficiently convert photon energy into heat, making them
[
10]
applicable for photoacoustic imaging and photothermal therapy.
Particularly, they often exhibit higher absorption and photothermal
conversion efficiencies as compared with other nanoparticles
such as carbon nanotubes and gold nanorods, enabling faster
inducible promoter. DSP has
a high photothermal conversion
efficiency (44.2%) at 808 nm, permitting fast transduction of NIR light
into thermal signals for intracellular activation of transcription. Such a
DSP-mediated remote activation can rapidly and safely result in 25-
and 4.5-fold increments in the expression levels of proteins in living
cells and mice, respectively. This study thus provides a promising
approach to optically regulate transgene systems, which has the
potential to be translated into nanomedicines for on-demand
therapeutic transgene dosing.
[11]
heating. Based on such excellent photothermal properties, we
recently developed SPN-based bioconjugates that specifically
targeted and rapidly activated the protein thermosensitive ion
[
11a]
channels in neurons.
These studies imply that SPNs could
serve as photothermal nanomodulators to optically regulate
[
12]
cellular behaviors.
We herein report the development of a near-infrared (NIR)
absorbing dendronized semiconducting polymer (DSP) and
demonstrate its proof-of-concept application as a photothermal
nanocarrier and intracellular nanotransducer for remote activation
of gene expression. DSP comprises three key components
(Figure 1a): the hydrophobic semiconducting backbone, the
cationic third-generation polyamidoamine (PAMAM3) side chains,
and the neural poly(ethylene glycol) (PEG) blocks, which serve as
the NIR photothermal nanotransducer, the gene vector and the
water-solubility enhancer, respectively. The conversion of
photothermal signal generated from DSP into the cue for gene
expression is realized by simple incorporation of plasmids with the
gene of interest at the downstream of heat shock promoter
(HSP70). Thus, gene expression can be regulated by HSP70 and
activated by photothermal signals. The working mechanism starts
with the electrostatic attraction induced formation of
nanocomplexes between DSP and HSP70-regulated gene
(Figure 1b). After cellular internalization, the gene is released from
the complex and enters into cellular nucleus, while DSP retains in
cytoplasm. Upon laser irradiation at 808 nm, DPS generates heat,
stimulating the transformation of heat shock factor (HSF) from
monomers to trimers; HSF trimers then translocate to the cellular
nucleus, bind to heat shock element (HSE) in the HSP70 and
Regulation of cellular behaviors and transgene systems can
provide useful tools to understand their physiological roles and
potentially lead to innovative therapies. In this regard, gene
therapy that utilizes viral or non-viral vectors to deliver exogenous
nucleic acids into specific cells has become
a promising
therapeutic approach to treat inherited genetic diseases and
[
1]
cancers. However, it is challenging to precisely regulate gene
expression at designated location and time so as to minimize off-
[
2]
target expression.
therapeutic nucleic acids is one feasible solution,
however is less likely to be generalized for various diseases.
Encoding tissue-specific promoters in
[3]
which
[
4]
[5]
[6]
Recently, gas molecules, radio waves and light have been
used to remotely switch on gene expression. Among them, light
is the most ideal inducer due to its low toxicity, easy tunability and
high spatiotemporal resolution. However, current methods are
[
7]
[6a,b]
generally limited to ultraviolet and visible light
with shallow
tissue penetration, preventing them from in vivo applications;
moreover, optical responsive components are knocked in through
[
6b,c]
synthetic biology to trigger the complex cascade response,
partially complicating their implementation.
[
13]
activate transcription.
As such, DSP sequentially acts as the
1
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COMMUNICATION
gene
nanocarrier and
the
intracellular photothermal
nanotransducer to efficiently deliver and remotely control
transgene expression. Note that although carbon nanotubes have
been used to activate HSP70 in the previous studies, they are
simply used as the heater and gene delivery need to be
[
14]
conducted in a separate step.
a
DSP
Self-
Assembly
Backbone:
PAMAM:
PEG:
Photothermal
Gene Water-Solubility
Nanotransducer Vector
Enhancer
b
HSF
Monomer
HSF
Trimer
P P P
4
8
08 nm
5
3
Binding
Entry
Heat
P
P
P
P P P
RNA
Polymerase
2
Release
HSP-
DSP
Regulated
Gene
HSE
1
Internalization
6
Expression
Figure 2. In vitro characterization of DSP and its transfection capability. (a)
Absorption and fluorescence spectra of DSP in 1×PBS at pH 7.4. (b) Solution
temperatures of DSP (132 µg/mL) as a function of laser irradiating time. The
Proteins
Nanocomplex
-2
laser wavelength was 808 nm and its power intensity was 0.67 W cm . Inset:
thermal images of DSP and PBS solutions at their respective maximum
temperatures. (c) Agarose gel electrophoresis of DNA, DSP and DSP/DNA
nanocomplexes at various N/P ratios. (d) DLS profiles of DSP and DSP/DNA
nanocomplex with the N/P ratio of 5:1. Inset: representative TEM images of DSP
and DSP/DNA complex; scale bars indicate 100 nm. (e) Hydrodynamic
diameters and zeta potential of DNA, DSP and DSP/DNA nanocomplexes at
various N/P ratios. (f) Transfection efficacy of DSP/DNA nanocomplexes at
various N/P ratios. Error bars represented standard deviations of three separate
measurements.
Figure 1. Design DSP as photothermal nanocarrier and nanotransducer for
remote activation of gene expression. (a) Chemical structure and self-assembly
of DSP. (b) Illustration of DSP-mediated gene delivery and remote photothermal
activation of gene expression under NIR laser irradiation at 808 nm.
DSP was synthesized via a graft-on approach (Schemes S1
and S2, Supporting Information). Pd-catalyzed Suzuki
polymerization of PEG-functionalized endcapping agent (4), 4,4’-
bis-(5-bromopentyl)-4H-cyclopenta[2,1-b:3,4-b’]dithiophene (5),
and 2,1,3- benzothiadiazole-4,7-bis (boronic acid pinacol ester)
To evaluate the complexation of DSP with genes, the pSV40-
Luc plasmid (luciferase as the reporter protein) was used as an
example, and the gel retardation, diameters, zeta potentials of the
DSP/plasmid nanocomplexes were studied at different N/P ratios.
Agar gel electrophoresis revealed that plasmids were fully loaded
by DSP at a low N/P ratio of 5:1, because nearly all the plasmids
were retarded in the slots and no migration band was observed
(
6) led to the PEG-endcapped triblock copolymer (P1). The
bromide groups of P1 were converted into the azide groups,
allowing it to undergo the copper(I)-catalyzed alkyne-azide
cycloaddition (CAAC) reaction to graft PAMAM2.5 onto it. At last,
the resulted grafted SP (P3) reacted with ethylenediamine via
aza-Michael addition reaction to convert PAMAM2.5 into the third-
generation form (PAMAM3), affording the final polymer (DSP).
The intermediate polymers and DSP were characterized by NMR
(
Figure 2c). Dynamic light scattering (DLS) and transmission
electron microscopy (TEM) were conducted to analyze the
condensation between DSP and plasmids. DSP itself had a
spherical morphology with the average hydrodynamic diameter of
(
Figures S1-S3, Supporting Information). DSP could be directly
∼
15 nm (Figure 2d and Figure S5, Supporting Information),
dissolved in aqueous solution and buffer, making it feasible for
gene delivery.
remaining nearly unchanged after storage for 42 days (Figure S6,
Supporting Information). After complexation with plasmids, the
morphology remained spherical. However, the diameters
dramatically decreased from 396 to 38 nm with increasing the N/P
DSP had a broad absorption peak in the NIR region ranging
from 500 to 900 nm with a maximum at 635 nm, and its
fluorescence maximum was located at 791 nm (Figure 2a). Such
a NIR optical feature is beneficial for in vivo applications, enabling
utilization of NIR light excitation to generate both fluorescence
and photothermal signals. Upon continuous laser irradiation at
ratio from 0.54:1 to 7.5:1 (Figure 2e). This was well observed for
[15]
non-viral vectors,
and could be contributed to enhanced
DSP/plasmid condensation with increased N/P ratios. The
diameters slightly changed when the N/P ratio was larger than
8
08 nm, the temperature of DSP solution gradually increased and
7
.5:1, indicating the formation of compact and stable
reached plateau of 58.8ºC at t= 480 s (Figure 2b). The
photothermal conversion efficacy of DSP was calculated to be
nanocomplexes. The zeta potential of the nanocomplexes
increased from 3 to 29 mV with increasing the N/P ratio and
reached maximum at the N/P ratio of 5:1 (Figure 2e). These data
proved that DSP was an ideal carrier with the efficient gene
loading and condensation capabilities.
[
11a]
4
4.2±2.8%, which was higher than gold nanorods (~17%).
Moreover, the maximum temperature of DSP could be adjusted
using different laser powers and it remained nearly unchanged
after 6 reversible heating and natural cooling cycles, proving its
excellent photothermal stability (Figure S4, Supporting
Information).
The ability of DSP for in vitro gene transfection was studies in
Hela cells using luciferase genes (pSV40-Luc) as the reporter
gene. The transfection efficacy was evaluated by the expression
2
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level of luciferase, which can be quantified with bioluminescence
monitored along with the laser irradiation (Figure 3c). The highest
expression appeared after laser irradiation for only 5 min. At this
(
BL). With increased N/P ratio, the BL intensity gradually
increased, and reached maximum at the N/P ratio of 7.5:1 (Figure
f). This N/P ratio was consistent well with the gene loading and
time point, ∆BL/BL
0
was 25-fold higher than that without
2
irradiation, indicating the significantly enhanced gene expression
level triggered by DSP in a rapid manner. Note that during the
laser irradiation, the solution temperature was increased by ∼2.4
ºC at most. Such a temperature increase did not cause obvious
cytotoxicity (Figure S9, Supporting Information). These cell data
verified that DSP served as the cytocompatible photothermal
nanocarrier and nanotransducer to remotely control gene
expression. Note that light irradiation of cellular medium alone
could trigger transgene expression due to the heat generation
condensation data, wherein the compact and stable DSP/plasmid
nanocomplexes formed. When the N/P ratios were above 7.5:1,
the BL intensity slightly decreased, probably caused by the over
condensation that blocked plasmid release. In addition to the
excellent performance in transfection, DSP showed good
cytocompatibility even at the high concentration (Figure S7,
Supporting Information).
[
17]
from water absorption. However, due to the weak photothermal
effect of water, it requires high power and long irradiation time,
which may cause cytotoxicity issue.
Figure 3. In vitro DSP-mediated photothermal activation of gene expression. (a)
Fluorescence images of HeLa cells transfected with DSP/pHSP70-EGFP
nanocomplexes with the N/P ratio of 7.5:1 with and without laser irradiation at
-
2
8
(
08 nm (0.75 W cm ) for 30 min. Quantification of changes in the BL intensities
b) and temperature (c) of HeLa cells transfected with DSP/pHSP70-Luc
nanocomplexes with the N/P ratio of 7.5:1 with and without laser irradiation at
08 nm (0.75 W cm ) for 30 min. The excitation and emission wavelengths were
40/655-710 nm for DSP, 488/500-550 nm for EGFP and 405/410-470 nm for
Figure 4. In vivo DSP-mediated photothermal activation of gene expression. (a)
Illustration of laser irradiation in living nude mice. (b) whole-animal BL images
at representative time-points with (left) and without (right) laser irradiation at 808
-
2
-2
8
6
nm (0.42 W cm ). The red/white dashed circles indicated the respective regions
subcutaneously implanted with the HeLa cells pellets (2, 520,000 cells)
transfected with DSP/pHSP70-Luc nanocomplexes and treated with/without
laser irradiation. Quantification of changes in the BL intensities (c) and
temperature (d) as a function of time with (left) and without (right) laser
irradiation. Error bars represented standard deviations of three separate
measurements.
Hoechst. Error bars represented standard deviations of three separate
measurements.
To test the photothermal ability of DSP to remotely control
transgene expression, enhanced green florescence proteins
(
EGFPs) and luciferases were employed as the reporter proteins
To validate the potential of DSP for in vivo activation of gene
expression, the HeLa cells pellets (∼2, 520,000 cells) transfected
with DSP/pHSP70-Luc nanocomplexes were subcutaneously
injected into the back of living mice (Figure 4a). The BL images
were longitudinally recorded and quantified for the areas with or
without laser irradiation. To minimize the temperature increase,
the laser treatment was set in a discontinuous manner (Figure
S10, Supporting Information). BL intensity gradually increased
along with laser irradiation and reached maximum at 19 min
at the downstream of HSP70, and activation was conducted on
HeLa cells. The sequences were designed according to the
previous literature.
[
16]
Hela cells were incubated with the
DSP/pHSP70-EGFP plasmid nanocomplexes at the optimal N/P
ratio of 7.5:1, recovered with fresh media and treated with or
without laser irradiation at 808 nm for 30 min. With the NIR
fluorescence of DSP, confocal fluorescence microscopy could be
applied to track the location of DSP and the expression of EGFP
in cells. After incubation, the NIR fluorescence from DSP was
observed in the cytoplasm for both laser treated or non-treated
cells, indicating the efficient cellular internalization of
DSP/pHSP70-EGFP plasmid nanocomplexes (Figure 3a). The
location of DSP was also ideal to trigger HSP70-regulated gene,
as associated HSF monomers located in cytoplasm. Only upon
laser irradiation, green fluorescence was observed, showing the
remotely controlled expression of EGFP. Such a laser-triggered
expression was not observed in the absence of HSP70 (Figure
S8, Supporting Information), confirming that the activation was
triggered by DSP-mediated photothermal transduction.
(
Figures 4b&4c), while it slightly changed without laser irradiation.
Such a phenomenon was not observed for the cells transfected
with luciferase gene at the downstream of the constitutive
promoter (SV40) (Figure S11, Supporting Information). This
proved that the expression of luciferase was selectively controlled
by the photothermal generation of DSP under high tissue
[
11]
penetrating NIR light irradiation at 808 nm. At t =19 min, ∆BL/BL
0
was 4.5-fold higher than that without laser irradiation. Note that
the temperature increased only by 5.2 ºC at most during laser
irradiation (Figure 4d). The highest temperature was 39.5 ºC
below the threshold temperature to induce apoptosis (43 ºC)
(Figure S12, Supporting Information). These in vivo data clearly
validate the feasibility of using DSP for remotely controlled gene
expression at desired location and time in living animals.
To quantify the DSP-mediated photothermal activation of
gene expression, similar cell experiments were conducted on
HeLa cells using the DSP/pHSP70-Luc plasmid nanocomplexes
at the optimal N/P ratio of 7.5:1. The luciferase expression level
was longitudinally monitored for ∼70, 000 cells and quantitatively
described as the incremental percentage of BL intensities
In conclusion, we have synthesized a multifunctional water-
soluble polymer (DSP) that can not only efficiently deliver genes
into cells but also convert NIR light into heat for photothermal
applications. In association with heat inducible promoter
(
0
∆BL/BL ) (Figure 3b). Meanwhile, the solution temperature was
3
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regulated genes, DSP acts as the intracellular photothermal
nanotransducer to remotely activate gene expression upon NIR
light irradiation at 808 nm. Real-time monitoring of luminescence
indicates that gene expression can be rapidly triggered and
significantly enhanced by 25- and 4.5-fold within minutes in living
cells and mice, respectively. Thus, this study provides an
innovative polymeric approach to noninvasively control gene
expression at designated location and time. To the best of our
knowledge, this is the first polymer system that integrates the
functionalities of gene delivery and photothermal activation. In
view of its completely benign organic components and NIR
absorption with high-tissue penetration, such kind of polymers
holds promise to be developed into nanomedicines for controlled
therapeutic transgene dosing.
[5] S. A. Stanley, J. E. Gagner, S. Damanpour, M. Yoshida, J. S. Dordick, J. M.
Friedman, Science 2012, 336, 604-608.
[6] a) X. Wang, X. Chen, Y. Yang, Nat. Methods 2012, 9, 266-269; b) H. F. Ye,
M. Daoud-El Baba, R. W. Peng, M. Fussenegger, Science 2011, 332,
1565-1568; c) M. Folcher, S. Oesterle, K. Zwicky, T. Thekkottil, J. Heymoz,
M. Hohmann, M. Christen, M. D. El-Baba, P. Buchmann, M. Fussenegger,
Nat. Commun. 2014, 5.
[
7] B. C. McKay, L. J. Stubbert, C. C. Fowler, J. M. Smith, R. A. Cardamore, J.
C. Spronck, Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 6582-6586.
[
8] a) C. Zhu, L. Liu, Q. Yang, F. Lv, S. Wang, Chem. Rev. 2012, 112, 4687-
4
735; b) C. Wu, D. T. Chiu Angew. Chem., Int. Ed. 2013, 52, 3086-3109;
Angew. Chem. 2013, 125, 3164-3190; c) Q. Miao, K. Pu, Bioconjugate
Chem. 2016, 27, 2808-2823.
[
[
9] a) K. Pu, N. Chattopadhyay, J. Rao, J. J. Controlled Release 2016, 240, 312-
3
22; b) Zhen, C. Zhang, C. Xie, Q. Miao, K. L. Lim, K. Pu, ACS Nano 2016,
0, 6400-6409.
1
10] a) K. Pu, A. J. Shuhendler, J. V. Jokerst, J. Mei, S. S. Gambhir, Z. Bao, J.
Rao, Nat. Nanotechnol. 2014, 9, 233-239; b) Q. Miao, Y. Lyu, D. Ding, K.
Pu, Adv. Mater. 2016, 28, 3662-3668; c) Y. Lyu, Y. Fang, Q. Miao, X. Zhen,
D. Ding, K. Pu, ACS Nano 2016, 10, 4472-4481; d) L. Xu, L. Cheng, C.
Wang, R. Peng, Z. Liu, Polym. Chem. 2014, 5, 1573-1580.
Acknowledgements
This work was supported by Nanyang Technological University
start-up grants (NTU-SUG: M4081627.120&M4081533.080),
Academic Research Fund Tier 1 (RG133/15: M4011559.120),
[11] a) Y. Lyu, C. Xie, S. A. Chechetka, E. Miyako, K. Pu, J. Am. Chem. Soc.
2016, 138, 9049-9052; b) C. Xie, P. K. Upputuri, X. Zhen, M. Pramanik, K.
Pu, Biomaterials 2017, 119, 1-8.
Academic Research Fund Tier
2
(MOE2016-T2-1-098,
MOE2015-T2-1-112&MOE-2016-T2-1-005S), and Academic
Research Fund Tier 3 (MOE2013-T3-1-004) from Singapore
Ministry of Education.
[
12] C. Tortiglione, M. R. Antognazza, A. Tino, C. Bossio, V. Marchesano, A.
Bauduin, M. Zangoli, S. V. Morata, G. Lanzani, Sci. Adv. 2017, 3, No.1.
13] R. I. Morimoto, Science 1993, 259, 1409-1410.
[
[
14] E. Miyako, T. Deguchi, Y. Nakajima, M. Yudasaka, Y. Hagihara, M. Horie,
M. Shichiri, Y. Higuchi, F. Yamashita, M. Hashida, Y. Shigeri, Y. Yoshida,
S. Iijima, Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 7523-7528.
[
15] a) L. C. Yin, Z. Y. Song, K. H. Kim, N. Zheng, H. Y. Tang, H. Lu, N.
Keywords: dendronized semiconducting polymer • non-viral
gene carrier • photothermal effect • light activation •
nanoparticles
Gabrielson, J. J. Cheng, Biomaterials 2013, 34, 2340-2349;
b) L. Yin,
H. Tang, K. Kim, N. Zheng, Z. Song, N. P. Gabrielson, H. Lu, J. Cheng,
Angew. Chem., Int. Ed. 2013, 52, 9182-9186; Angew. Chem. 2013, 125,
9
352-9356.
[
[
[
[
1] L. Naldini, Nature 2015, 526, 351-360.
[16] B. J. Wu, R. E. Kingston, R. I. Morimoto, Proc. Natl. Acad. Sci. U. S. A.
1986, 83, 629-633.
2] M. A. Kay, Nat. Rev. Genet. 2011, 12, 316-328.
3] D. M. Nettelbeck, V. Jerome, R. Muller, Trends Genet. 2000, 16, 174-181.
4] W. Weber, M. Rimann, M. Spielmann, B. Keller, M. Daoud-El Baba, D. Aubel,
C. C. Weber, M. Fussenegger, Nat. Biotechnol. 2004, 22, 1440-1444.
[17] Y. Kamei, M. Suzuki, K. Watanabe, K. Fujimori, T. Kawasaki, T. Deguchi,
Y. Yoneda, T. Todo, S. Takagi, T. Funatsu, S. Yuba, Nat. methods 2009,
6, 79-81.
4
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COMMUNICATION
COMMUNICATION
Yan Lyu, Dong Cui, He Sun, Yansong
Miao, Hongwei Duan, and Kanyi Pu*
Page No.1 – Page No.4
Title: Dendronized Semiconducting
Polymer as Photothermal Nanocarrier
for Remote Activation of Gene
Expression
Text for Table of Contents
TOC Description
Remote Control: A near-infrared (NIR)
absorbing dendronized semiconducting
polymer (DSP) is synthesized and utilized
as a photothermal nanocarrier not only to
efficiently deliver gene but also to
photothermally control gene expression
at designated location and time.
5
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