A reactive oxygen species (ROS)-responsive polymer for safe, efficient, and targeted gene delivery in cancer cells

Article abstract of DOI:10.1002/anie.201209633

Stimuli-responsive release: The high levels of reactive oxygen species (ROS) in prostate cancer cells can be exploited to trigger cancer-targeted gene delivery. A ROS-responsive thioketal-based cationic polymer was synthesized and functionalization with a cancer-targeting peptide led to selective and enhanced gene transfection in prostate cancer cells (see scheme). Copyright

Full text of DOI:10.1002/anie.201209633

Angewandte
Chemie
Communications
Targeted Delivery
Stimuli-responsive release: The high
levels of reactive oxygen species (ROS) in
prostate cancer cells can be exploited to
trigger cancer-targeted gene delivery. A
ROS-responsive thioketal-based cationic
polymer was synthesized and functional-
ization with a cancer-targeting peptide
led to selective and enhanced gene
transfection in prostate cancer cells (see
scheme).
M. S. Shim, Y. Xia*
&&&& — &&&&
A Reactive Oxygen Species (ROS)-
Responsive Polymer for Safe, Efficient,
and Targeted Gene Delivery in Cancer
Cells
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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DOI: 10.1002/anie.201209633
Targeted Delivery
A Reactive Oxygen Species (ROS)-Responsive Polymer for Safe,
Efficient, and Targeted Gene Delivery in Cancer Cells**
Min Suk Shim and Younan Xia*
Over the past decade, cancer gene therapy has been one of the
most rapidly evolving areas in the clinic as a result of
improved understanding of cancer at the molecular level.
cient and safe gene delivery in cancer cells by developing
a novel ROS-responsive, cationic, water-soluble polymer
composed of biodegradable thioketal linkages that are readily
[1]
[
8]
The clinical success of cancer gene therapy critically depends
on the development of safe, efficient, and targeted gene
cleavable in ROS-abundant conditions. It was hypothesized
that the ROS-cleavable thioketal-based polymeric carrier
could enhance the gene delivery efficiency in cancer cells by
facilitating intracellular release of the encapsulated nucleic
acids in response to high levels of intracellular ROS (Fig-
ure 1a). We further functionalized this polymer with a cancer-
targeting peptide to achieve cancer-targeted gene delivery.
This modification could enhance the gene delivery efficiency
and thus reduce the potential systemic toxicity by avoiding
nonspecific accumulation in normal cells.
[
2]
carriers. In recent years, synthetic nonviral vectors have
attracted great attention as a promising alternative to
conventional viral vectors owing to their lower risk of
[3]
immunogenicity and larger gene delivery capacity. How-
ever, nonviral vectors often suffer from low transfection
efficiency owing to their poor extra- and intracellular
[
4]
processes as compared to viral vectors. In addition, the
lack of target-specificity for nonviral vectors is another critical
obstacle that needs to be overcome before their maximized
The ROS-cleavable, cationic polymer was synthesized by
polymerization of oligoamines with acrylamide thioketal
cross-linkers (Figure 1b). Briefly, trifluoroacetate (TFA)-
[
4]
therapeutic efficacy can be achieved at the target site.
Recently, various types of smart polymers capable of
responding to the stimuli intrinsic to a tumor environment
have emerged as efficient nonviral gene carriers for the
[
5]
treatment of cancer. Most of the stimuli-responsive nonviral
vectors rely on the use of extracellular environments of
tumors, such as low extracellular tumor pH value and matrix
metalloproteinases (MMPs), for cancer-targeted gene deliv-
[
6]
ery. Few of them have been developed with responses to the
unique intracellular stimuli of cancer cells, which could be
used to greatly improve their intracellular trafficking and
gene transfection.
Recent studies reported that, owing to oncogenic trans-
formation, cancer cells constantly generate high levels of
intracellular reactive oxygen species (ROS), including H O ,
2
2
hydroxyl radical, and superoxide, in comparison with normal
[
7]
cells. Therefore, in this study, the intracellular ROS in
cancer cells was utilized as a unique cancer-related stimulus to
mediate intracellular gene delivery. We demonstrated effi-
[+]
[
*] Dr. M. S. Shim, Prof. Y. Xia
The Wallace H. Coulter Department of Biomedical Engineering
School of Chemistry & Biochemistry and
School of Chemical & Biomolecular Engineering
Georgia Institute of Technology
Atlanta, GA 30332 (USA)
E-mail: younan.xia@bme.gatech.edu
+
[
] Current address: Division of Bioengineering, Incheon National
University (Republic of Korea)
[
**] This work was supported in part by a 2006 NIH Director’s Pioneer
Award (DP1 OD000798) and start-up funds from Georgia Institute
of Technology. The authors thank Dennis Oakley of the Bakewell
Neuroimaging Core at Washington University School of Medicine
for assistance with some of the confocal microscopy imaging
studies.
Figure 1. a) Illustration of intracellular delivery of plasmid DNA to the
nucleus of a cancer cell using ROS-cleavable poly(amino thioketal)
(PATK). After the DNA/PATK polyplexes have been internalized by the
cancer cell and escaped from the endosome, ROS-triggered degrada-
tion of the PATK facilitates the intracellular release of DNA from the
particle, leading to enhanced gene transfection. b) Synthetic scheme of
ROS-responsive PATK.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201209633.
2
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Chemie
protected N,N’-bis(2-aminoethyl)-1,3-propanediamine was
polymerized with the thioketal cross-linkers by Michael
addition conjugation, followed by deprotection of the TFA
groups, resulting in poly(amino thioketal) (PATK). Fig-
ure S1a in the Supporting Information shows the detailed
synthesis. Non-degradable poly(amine) without thioketal
linkages was also synthesized as a ROS-insensitive counter-
part to investigate the effects of ROS sensitivity on the
intracellular release of nucleic acids and consequent gene
delivery efficiency (see Figure S1b for detailed synthesis).
1
The formation of PATK was confirmed by the H NMR
spectroscopy (Figure S2a). The molecular weights of PATK
were M = 5.7 kDa and M = 9.4 kDa with a polydispersity
n
w
index of 1.65, as determined by gel permeation chromatog-
raphy (GPC).
The degradation profile of PATK under simulated ROS
1
conditions was quantified by H NMR spectroscopy. In
a typical experiment, PATK was dissolved in D O containing
2
H O and trace amounts of transition-metal ions (e.g., 1.6 mm
2
2
CuCl ) to trigger its degradation by ROS. 3-(trimethylsilyl)-
2
propionic acid sodium salt (TSP) was also added into the D O
2
solvent as a stable reference. When the PATK was incubated
in the solvent at 378C for different times, the disappearance of
the thioketal linkage peak (d = 1.62 ppm) was monitored and
1
quantified using H NMR spectroscopy in comparison with
1
the TSP peak. The H NMR spectrum confirmed that the
thioketal linkages were efficiently cleaved by ROS, generat-
ing acetone (d = 2.16 ppm) as a by-product during the
cleavage process (data not shown). This finding was in
agreement with a previous study regarding the dethioacetal-
[
9]
ization using H O . The degradation rate of thioketal
2
2
linkages was proportional to the concentration of H O as
2
2
shown in Figure 2a. For example, the half-lives of thioketal
linkages in PATK incubated with 100 mm and 200 mm H O2
solution were approximately 20 h and 11 h, respectively
2
Figure 2. a) Degradation profiles of thioketal linkages in PATK after
exposure to H O at various concentrations for different times, as
2
2
(
Figure 2a). The decrease in molecular weight of PATK
1
determined by H NMR spectroscopy. b) Transmission electron micro-
graphs of the DNA/PATK polyplexes (N/P=100) before and after
incubation with H O for 12 h at 378C. Scale bars indicate 100 nm.
after incubation with 100 mm H O and 1.6 mm CuCl at 378C
2
2
2
was also confirmed by GPC (Figure S2b).
2
2
c) Generation of intracellular ROS in PC3 cells confirmed by fluores-
cence microscopy. Strong green fluorescence throughout the cells
demonstrates the high levels of intracellular ROS in PC3 cells. Cellular
nuclei were counter-stained with DAPI in blue color. d) Confocal
micrographs illustrating the intracellular localization of DNA/PATK and
DNA/non-degradable poly(amine) polyplexes in PC3 cells. Cellular
nuclei were counter-stained with DRAQ5 (blue); DNA (red), poly-
mer (green), yellow dots represent co-localized polymer and DNA.
Scale bars: 10 mm.
Efficient DNA complexation by cationic PATK was
confirmed by measuring the particle size and surface charge
of DNA/PATK polyplexes. As shown in Figure S3a, PATK
efficiently complexed with DNA, forming stable polyplexes
with diameters ranging from 130 to 200 nm at amine to
phosphate (N/P) ratios of 50 and higher. The zeta potentials
of DNA/PATK polyplexes were in the range of + 15 to
+
25 mVat N/P ratios of 50 and higher (Figure S3b). Efficient
ROS-triggered dissociation of DNA from DNA/PATK poly-
plexes was confirmed by an agarose gel electrophoresis assay.
Before incubation with a H O solution, DNAwas completely
hydroxyl radical, H O , and superoxide, was also examined
2
2
2
2
retained by PATK at N/P ratios of 20 and higher (Figure S4).
However, DNA was efficiently liberated from the PATK
polyplexes when the samples were exposed to ROS (Fig-
ure S4). TEM images also demonstrated ROS-triggered
destabilization of the DNA/PATK polyplexes (Figure 2b).
In addition, destabilization of the DNA/PATK polyplexes in
response to biologically relevant levels of H O (e.g., 100 mm
using the EtBr exclusion assay (Figure S5b). Among the
tested ROS, thioketal linkages showed the most efficient
degradation in response to the hydroxyl radical.
To confirm whether cancer cells spontaneously generate
ROS, we visualized intracellular ROS generation using 2’,7-
dichlorofluoresceindiacetate (DCFDA), a cell permeable
fluorescent dye that is rapidly oxidized to a fluorescent
2
2
[10]
[11]
and 1 mm)
was confirmed by ethidium bromide (EtBr)
molecule by intracellular ROS.
The PC3 cell, a human
exclusion assay (Figure S5a). The degradation selectivity of
the DNA/PATK polyplexes under various ROS, including
prostate cancer cell line, was used as a model system due to its
inherently high levels of ROS. Fluorescent micrographs of
[
7a]
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DCFDA-stained PC3 cells clearly demonstrated the gener-
ation of intracellular ROS throughout the cell (Figure 2c). To
investigate if ROS in PC3 cells trigger efficient intracellular
disassembly of DNA/PATK polyplexes, their intracellular
disassembly in PC3 cells was examined by confocal laser
scanning microscopy (Figure 2d). For comparison, non-
degradable poly(amine) and 25 kDa branched PEI (B-PEI)
were also complexed with DNA and incubated with PC3 cells.
As shown in Figure 2d, a significant amount of free DNA
DNA from the gene carrier is crucial for efficient gene
[
12]
transfection. The confocal micrographs in Figure 2d clearly
shows that DNA was efficiently released from PATK poly-
plexes in PC3 cells. Taken together, enhanced gene trans-
fection by the DNA/PATK polyplexes in comparison with
DNA/non-degradable poly(amine) and DNA/B-PEI poly-
plexes can be attributed to their efficient intracellular
disassembly in ROS-generating PC3 cells. In addition to
higher gene transfection, ROS-sensitive DNA/PATK poly-
plexes exhibited lower cytotoxicity than DNA/B-PEI poly-
plexes (Figure 3b). In general, the cytotoxicity of cationic
polymers increases with their charge density and molecular
(
labeled with Alexa Fluor 568 dye, red) was disassembled
from the Alexa Fluor 488-labeled PATK (green), demon-
strating the efficient intracellular disassembly of DNA/PATK
polyplexes in the ROS-generating PC3 cells. In contrast,
limited intracellular release of DNAwas observed with DNA/
non-degradable poly(amine) polyplexes and DNA/B-PEI
polyplexes as represented by many yellow dots (Figure 2d
and Figure S6).
[
13]
weights. Therefore, the reduced cytotoxicity of PATK can
be attributed to its lower molecular weight and lower cationic
surface charge (DNA/PATK: ca. 20 mV, DNA/B-PEI: ca.
30 mV; Figure S3b).
To investigate if the enhanced gene transfection by PATK
is selectively triggered in response to high levels of ROS in
cancer cells, its transfection efficiency in PC3 cells was
compared with the Chinese hamster ovary (CHO) cells that
produce significantly lower levels of ROS (Figure S7a). PATK
showed significantly higher transfection efficiency in PC3
cells than in non-cancerous CHO cells (Figure 3a and Fig-
ure S7b), whereas non-degradable B-PEI showed similar
transfection efficiencies for both PC3 and CHO cells. Con-
sidering that the total cellular uptake of both DNA/PATK and
DNA/B-PEI polyplexes was similar for both PC3 and CHO
cells (Figure S7c), the noticeably enhanced gene transfection
by PATK in PC3 cells can be attributed to more efficient
disassembly of the DNA/PATK polyplexes in PC3 cells that
are known to have higher levels of ROS. The significantly
enhanced gene transfection by PATK in response to high
levels of ROS in prostate cancer cells was also consistently
observed in LNCaP cells, another prostate cancer cell line
showing higher levels of intracellular ROS than those of non-
cancerous CHO cells (Figure S7).
Enhanced gene transfection by ROS-responsive PATK
was investigated by comparing its transfection efficiency with
reference to non-degradable poly(amine). As shown in
Figure 3a, eGFP expression (transfection) of the PC3 cells
incubated with PATK polyplexes complexing eGFP-encoding
plasmid DNA was significantly higher than that of the cells
incubated with the DNA/non-degradable poly(amine) poly-
plexes. The transfection efficiency of PATK was also com-
pared with B-PEI, a commercially available superior trans-
fecting agent. Clearly, DNA/PATK polyplexes transfected
PC3 cells more efficiently than DNA/B-PEI polyplexes
(
Figure 3a). It is established that intracellular release of
Cancer-targeted gene delivery is a key requirement for
[
14]
future cancer gene therapy in the clinic. To demonstrate the
feasibility of achieving cancer-targeted gene delivery using
the ROS-responsive PATK, we further conjugated it with
GRP78-binding peptide (GRP78P, peptide sequence =
WIFPWIQL) (Figure S8), which selectively binds to GRP78
proteins over-expressed by many types of tumor cells includ-
[
15]
ing the prostate cancer cells. We then complexed fluores-
cently labeled DNA with the GRP78P-conjugated PATK
(GRP78P-PATK) or GRP78P-free PATK to quantify their
cellular uptake. Notably, the conjugation of GRP78P signifi-
cantly increased (ca. three-fold higher) cellular uptake of the
PATK polyplexes in PC3 cells (Figure 4). Consequently,
DNA/GRP78P-PATK polyplexes yielded a two-fold higher
gene transfection efficiency in PC3 cells in comparison with
DNA/GRP78P-free PATK polyplexes. To confirm that the
increased transfection efficiency by DNA/GRP78P-PATK
polyplexes was mainly caused by the receptor-mediated
cellular uptake of GRP78P, a competitive assay where free
GRP78P was pre-incubated with PC3 cells prior to the
transfection was conducted. When 200 mm of free GRP78P
was pre-incubated with the cells prior to the transfection, the
DNA/GRP78P-PATK polyplexes showed significantly
Figure 3. a) Transfection efficiency and b) cell viability for DNA/PATK
N/P=100), DNA/non-degradable poly(amine) (N/P=120), and
DNA/B-PEI polyplexes (N/P=9) in PC3 cells. The N/P ratios of the
polyplexes were determined by optimized transfection efficiency and
cytotoxicity. * indicates p<0.05. ** indicates p<0.01.
(
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In conclusion, ROS-responsive thioketal-based PATK
was synthesized for safe, efficient, and targeted gene delivery
in prostate cancer cells. Degradation of thioketal linkages in
PATK under ROS conditions led to efficient intracellular
release of the complexed DNA in prostate cancer cells. As
a result, the DNA/PATK polyplexes exhibited efficient gene
transfection in prostate cancer cells. Incorporation of GRP78-
binding peptide to the PATK achieved cancer-targeted gene
transfection. Most importantly, this study demonstrates that
the high levels of intracellular ROS in cancer cells are unique
biological stimuli that can be utilized for efficient and
targeted gene delivery in cancer cells.
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Received: December 1, 2012
Revised: April 17, 2013
Published online: && &&, &&&&
[
[
[
Keywords: cancer · gene expression · nanoparticles ·
.
reactive oxygen species
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