Hyperbranched Self-Immolative Polymers (hSIPs) for Programmed Payload Delivery and Ultrasensitive Detection

Article abstract of DOI:10.1021/jacs.5b05060

Upon stimuli-triggered single cleavage of capping moieties at the focal point and chain terminal, self-immolative dendrimers (SIDs) and linear self-immolative polymers (l-SIPs) undergo spontaneous domino-like radial fragmentation and cascade head-to-tail depolymerization, respectively. The nature of response selectivity and signal amplification has rendered them a unique type of stimuli-responsive materials. Moreover, novel design principles are required for further advancement in the field of self-immolative polymers (SIPs). Herein, we report the facile fabrication of water-dispersible SIPs with a new chain topology, hyperbranched self-immolative polymers (hSIPs), by utilizing one-pot AB₂ polycondensation methodology and sequential postfunctionalization. The modular engineering of three categories of branching scaffolds, three types of stimuli-cleavable capping moieties at the focal point, and seven different types of peripheral functional groups and polymeric building blocks affords both structurally and functionally diverse hSIPs with chemically tunable amplified-release features. On the basis of the hSIP platform, we explored myriad functions including visible light-triggered intracellular release of peripheral conjugated drugs in a targeted and spatiotemporally controlled fashion, intracellular delivery and cytoplasmic reductive milieu-triggered plasmid DNA release via on/off multivalency switching, mitochondria-targeted fluorescent sensing of H₂O₂ with a detection limit down to ～20 nM, and colorimetric H₂O₂ assay via triggered dispersion of gold nanoparticle aggregates. To further demonstrate the potency and generality of the hSIP platform, we further configure it into biosensor design for the ultrasensitive detection of pathologically relevant antigens (e.g., human carcinoembryonic antigen) by integrating with enzyme-mediated cycle amplification with positive feedback and enzyme-linked immunosorbent assay (ELISA).

Full text of DOI:10.1021/jacs.5b05060

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Article
Hyperbranched Self-Immolative Polymers (hSIPs) for
Programmed Payload Delivery and Ultrasensitive Detection
Guhuan Liu, Guofeng Zhang, Jinming Hu, Xiaorui Wang, Mingqiang Zhu, and Shiyong Liu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b05060 • Publication Date (Web): 01 Sep 2015
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Hyperbranched Self-Immolative Polymers (hSIPs) for Pro-
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Guhuan Liu,† Guofeng Zhang,†,‡ Jinming Hu,† Xiaorui Wang,† Mingqiang Zhu,‡ and Shiyong Liu*,†
† CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Sciences at the Microscale,
iChem (Collaborative Innovation Center of Chemistry for Energy Materials), Department of Polymer Science and
Engineering, University of Science and Technology of China, Hefei, Anhui 230026, China. ‡ Wuhan National Labora-
tory for Optoelectronics, College of Chemistry and Chemical Engineering, Huazhong University of Science and
Technology, Wuhan, Hubei 430074, China
ABSTRACT: Upon stimuli-triggered single cleavage of capping moieties at the focal point and chain terminal, self-immo-
lative dendrimers (SIDs) and linear self-immolative polymers (l-SIPs) undergo spontaneous domino-like radial fragmenta-
tion and cascade head-to-tail depolymerization, respectively. The nature of response selectivity and signal amplification
has rendered them an unique type of stimuli-responsive materials. Moreover, novel design principles are required for fur-
ther advancement in the field of self-immolative polymers (SIPs). Herein, we report the facile fabrication of water-dispersi-
ble SIPs with a new chain topology, hyperbranched self-immolative polymers (hSIPs), by utilizing one-pot AB2 polyconden-
sation methodology and sequential post-functionalization. The modular engineering of three categories of branching scaf-
folds, three types of stimuli-cleavable capping moieties at the focal point, and seven different types of peripheral functional
groups and polymeric building blocks affords both structurally and functionally diverse hSIPs with chemically tunable am-
plified-release features. Based on the hSIP platform, we explored myriad functions including visible light-triggered intra-
cellular release of peripheral conjugated drugs in a targeted and spatiotemporally controlled fashion, intracellular delivery
and cytoplasmic reductive milieu-triggered plasmid DNA release via on/off multivalency switching, mitochondria-targeted
fluorescent sensing of H2O2 with a detection limit down to ~20 nM, and colorimetric H2O2 assay via triggered dispersion of
gold nanoparticle aggregates. To further demonstrate the potency and generality of hSIP platform, we further configure it
into biosensor design for the ultrasensitive detection of pathologically relevant antigens (e.g. human carcinoembryonic
antigen) by integrating with enzyme-mediated cycle amplification with positive feedback and enzyme-linked immuno-
sorbent assay (ELISA).
unimolecular level are non-conventional, with self-
immolative dendrimers (SIDs)6 and linear self-immolative
polymers (l-SIPs)7 being rare examples of this type.
■ INTRODUCTION
Biological systems can sense and respond to exogenous
and endogenous signals through well-ordered multiple
molecular events to achieve signal transduction and
amplification.1 The long-standing pursuits of mimicking
intricate processes and functions intrinsic of living
organisms2 have spurred the development of stimuli-
responsive polymeric materials.3 Current challenges in this
emerging field include achieving sophisticated structural
and sequence control, establishing advanced biomimetic
functions, and fabricating more sensitive and selective
systems.4 The latter relies on the integration with chemical
and biochemical amplification strategies. In this context,
enzyme-mediated catalytic reactions, which are closely
relevant to amplification detection techniques such as
polymerase chain reaction (PCR) and enzyme-linked
immunosorbent assay (ELISA), have led to responsive
polymers with improved sensitivity, selectivity, and
specificity.5 However, the multi-molecular nature of these
enzyme-involved systems renders largely compromised
signal amplification performance in highly complex
biological milieu.1 On the other hand, responsive polymers
Originally proposed in 2003,6a-c SIDs can undergo
spontaneous domino-like radial fragmentation upon
triggered cleavage of a single capping moiety at the focal
point. The release of peripheral functional groups allows
for exponential signal amplification with the extent
depending on the generation of SIDs. This feature as well
as advantages including programmed fragmentation and
tunable multivalency8 have promoted the construction of
advanced drug/ gene delivery and diagnostic functions,
mostly in aqueous media.6 However, SIDs suffer from
challenging multistep synthesis and tedious purification
procedures (Scheme 1).9
exhibiting input signal amplification features at
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Previous Works
Multistep
Synthesis
The invention of the concept of l-SIPs later in 2008 by
Polymerization &
Post-Modification
Shabat et al.7a can partially solve the above issues as the
synthesis of l-SIPs can be conducted in one-pot via either
polycondensation10 or chain growth approaches.11 Note
that functional reporters in l-SIPs can be either installed at
the distal end or conjugated as side moieties. In the former
case, no signal amplification can be achieved; whereas in
the latter case, triggered cleavage of capping terminal
moiety will release multiple functional moieties with the
extent of amplification dictated by the chain length
(Scheme 1). However, l-SIPs equipped with side chain
release motifs are also synthetically challenging,7a,12 and
thus most of reported l-SIPs have not utilized this feature
to achieve functional amplification.7 As compared to those
of SIDs,6 core functions of l-SIPs in aqueous media (e.g.,
amplified drug delivery and sensing) have been far less
explored.7 Existing few examples are based on l-SIPs in the
form of molecularly dissolved state7a,12 or colloidal
dispersions.10a-c,11d,11e,13
Dendritic Self-Immolative
Polymers (SIDs)
Linear Self-Immolative
Polymers (l-SIPs)
This Work
One-Pot
Polymerization &
Post-Modification
Trigger Removal &
Self-Immolative
Depolymerization
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Hyperbranched Self-
Immolative Polymers (hSIPs)
Scheme 1. Schematic illustration of hyperbranched self-im-
molative polymers (hSIPs), which represent a new chain to-
pology in the field of self-immolative polymers exhibiting trig-
gered cascade depolymerization characteristics. For clarity,
schematic structures of conventional SIDs and l-SIPs are also
shown. Upon stimuli-triggered cleavage of capping moiety at
the focal point, self-immolative depolymerization of hSIPs re-
leases multiple bioactive agents initially conjugated at the pe-
riphery, as well as small molecule branching units.
Functional
Groups (FG)
PEG or
PDMAEMA
Targeting
Moieties
i) CDI
ii) FG-NH2 or FG-OH
i) N3-PEG-Mal
ii) N3-PEG or
N3-PDMAEMA
iii) 2-Propynylamine
Triggers:
Functional
Groups (FG):
HS-cRGD
(Integrin Targeting)
or HS-CGKRK
(Mitochondrion Targeting)
Scheme 2. Reaction schemes employed for the synthesis and functionalization of hSIPs possessing three categories of branching
scaffolds (M1-M3) and three types of capping moieties at the focal point which are responsive to visible light (perylen-3-yl, per),
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H2O2 (phenylboronic ester, PB), and intracellular reductive milieu (disulfide, SS), with inert benzyl (Bn) capping functionality
serving as a control. A variety of bioactive and structural agents are conjugated at the periphery of hSIPs for specific functions
including chemotherapy (doxorubicin, DOX), fluorescent sensing (caged coumarin, AMC), enzymatic reaction substrate for signal
amplification (choline, CHO), triggered elimination of multivalent interactions with negatively charged species such as plasmid
DNA and gold nanoparticles (PDMAEMA block, CHO), cell membrane or mitochondrion targeting (HS-cRGD or HS-CGKRK),
and dispersibility of hSIPs in aqueous media (PEG).
9
The current status of both SIDs and l-SIPs revealed that
it has remained a considerable challenge to fabricate self-
immolative polymers (SIPs) with facile synthesis, high
chemical amplification potency, modular design, and
multifunctional integration. Further advancement in the
field of SIPs thus requires the introduction of novel design
criteria. It is well-known that besides linear, cyclic, star,
and dendritic polymers, hyperbranched polymers (HBPs)
represent an unique type of chain topology possessing
highly branched and three-dimensional globular
architecture. HBPs can be readily synthesized via one-pot
further expanded through rich choices of functionalization
at the periphery. On the basis of hSIP platform, we
construct myriad functions including visible light-
triggered spatiotemporal and intracellular delivery of
peripheral conjugated drugs, intracellular delivery and
cytoplasmic reductive milieu-triggered plasmid DNA
release via on/off multivalency modulation, mitochondria-
targeted fluorescent sensing of H2O2 with enhanced
detection limit, and colorimetric H2O2 assay via triggered
dispersion of gold nanoparticle aggregates. We also
configure hSIPs into biosensor design for the ultrasensitive
detection of pathologically indicative antigens by
integrating with enzyme-mediated cycle amplification and
ELISA technique.
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polymerization
of
ABX
monomers14
or
inimers/comonomers.15 Despite being less structurally
uniform than dendrimers, HBPs are distinguished by a
large number of peripheral terminal moieties, which can
be facilely functionalized (e.g., drugs and fluorophores) for
drug/gene delivery, optical sensing, and theranostic
applications.16 Our previous interest in responsive
polymers with self-immolative motifs (building blocks13
and side linkages17) and hyperbranched polymers with self-
immolative drug comonomers18 has prompted us to
speculate the possibility of fabricating hyperbranched self-
immolative polymers (hSIPs). Theoretically, hSIPs are
expected to not only inherit advantages of conventional
HBPs such as facile synthesis, modular design, and multi-
■ RESULTS AND DISCUSSION
Modular Synthesis of hSIPs. Inspired by the
pioneering work of Shabat et al.7a concerning l-SIPs and
relevant literature reports,12a,19 our initial design of hSIPs
involves the use of trifunctional benzene derivatives with
one caged isocyanate and two hydroxyl-containing
moieties at ortho and para positions by learning the
principle developed by Frechet et al.14d for the synthesis of
conventional
derivative is prone to intramolecular cyclization with 6-
membered ring formation during branching
HBPs.
2,4-Bis(hydroxymethyl)aniline
functionalization,
but
possess
potent
chemical
amplification and programmed release characteristics
(Scheme 1).
polycondensation. On the other hand, choosing 2-(3-
hydroxy-1-propenyl)-4-hydroxymethylaniline derivative as
latent precursor involves multistep synthesis with
unsatisfactory overall yield, and the final hSIP scaffold is
poorly water-dispersible.20 The limited success of these
initial trials prompted us to explore alternate design
approaches for hSIPs. We recently came across with the
idea that bridging the caged isocyanate moiety with two
reactive hydroxyl functionalities through self-immolative
linkages might endow AB2 polycondensation precursors
with improved structural stability and better synthetic
access (Schemes 1 and 2).
Herein, we report on the facile fabrication of SIPs with a
hyperbranched chain topology. The general design
principle of hSIPs is shown in Scheme 2. Starting from
three categories of AB2-type branching precursors (M1, M2,
and M3), one-pot polycondensation reaction and
subsequent caging reactions with three types of capping
moieties (responsive to visible light, H2O2, and intracellu-
lar reductive milieu, respectively) at the focal point afford
a
series of hSIP scaffolds with chemically tunable
depolymerization rates and varying responsive modes. The
structural and functional diversity of hSIPs could be
Table 1. Structural parameters of hyperbranched self-immolative polymers (hSIPs) synthesized in this work.
Branch-
ing
Units
Total No.
Terminal
Units a,b
Functional
Polymer
(FP)
Target-
ing Moi-
eties
Capping
Moieties
Functional
Groups (FG)
NFP
Mn,MALS Mw/
a
Entry
NFG
a
c
(kDa) c Mn
hSIP1
hSIP2
hSIP3
M1
M2
M3
per
per
per
19
14
16
DOX
DOX
DOX
10
6
PEG45
PEG45
PEG45
6.5
5.5
6
cRGD
cRGD
cRGD
30.5
25.4
28.6
1.66
1.82
1.73
8
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hSIP4
hSIP5
M1
M1
SS
SS
16
16
DOX
/
8
/
PEG45
5.5
13
/
/
26.3
29.3
1.89
PDMAEMA8
1.76
5
6
Bn
control
hSIP6
M1
17
/
/
PDMAEMA8
11
/
27.8
1.69
7
8
9
hSIP7
hSIP8
hSIP9
hSIP10
M1
M1
M1
M1
PB
PB
PB
PB
18
18
18
18
AMC
AMC
9
9
PEG45
PEG45
/
5
5
CGKRK
27.3
27.3
/
1.88
1.88
/
/
/
/
CHO
16
/
10
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CHO/AMC
8/2.5
PEG45
4.5
/
/
a Calculated from 1H NMR results. b Total number of peripheral functional moieties (including modified and unmodified hydroxymethyl residues) conjugated
at the periphery of hSIP scaffolds. Molecular weights, Mn, and molecular weight distributions, Mw/Mn, were evaluated by GPC/MALS using DMF as the
eluent (1.0 mL/min).
c
M1-M3
branching
precursors
with
varying
addition, just as conventional hyperbranched polymers, as-
prepared hSIPs are less branched and possess higher MWs
compared to dendrimers with a similar number of
peripheral functional groups.
Next, peripheral hydroxymethyl moieties of as-
synthesized hyperbranched scaffolds with caging moieties
at the focal point were activated by N,N'-
carbonyldiimidazole (CDI) at first and a variety of
functional, bioactive, or structural agents including
doxorubicin (DOX), 7-amino-4-methylcoumarin (AMC),
choline (CHO), and propargylamine were conjugated.
Finally, structural or functional hydrophilic segments such
hydroxymethyl substituting positions and activatable
linkages (O or S) were then designed to achieve chemically
tunable depolymerization rates for the hSIP scaffolds
(Scheme S1, Figures S1-S3). The hyperbranched scaffolds of
hSIPs (hSIP1-hSIP10) were synthesized through
polycondensation of M1, M2, or M3 AB2-type precursors
(Scheme 2) at 110 C.7a,14d The polycondensation reactions
were quenched by reacting with three types of capping
agents
o
including
perylen-3-yl
methanol
(per),
hydroxymethyl phenylboronic ester (PB), and diethanol
disulfide (SS); this process introduced responsiveness of
capping moieties at the hSIP focal point towards visible
light, H2O2, and cytoplasmic reductive milieu, respectively.
Benzyl alcohol (Bn) was also utilized as an inert capping
agent to serve as a control. The total number of terminal
hydroxyl groups at the periphery of as-synthesized hSIP
scaffolds were determined to be in the range of 14-19 by 1H
NMR analysis (Table 1, Figures S4-S6). When fully
conjugated with releasable reporter molecules at the
periphery, these hSIPs will exhibit signal amplification
capability comparable to SIDs of the fourth generation, the
synthesis of which has not been accomplished yet.6 In
as
poly(ethylene
oxide)
(PEO),
poly(2-
(dimethylamino)ethyl methacrylate) (PDMAEMA), and
targeting peptides (intergrin-targeting cRGD peptide and
mitochondrion-targeting CGKRK peptide) were covalently
attached to the periphery through copper-catalyzed azide-
alkyne cycloaddition (CuAAC) or thiol-maleimide click
reactions, affording target hSIPs (hSIP1-hSIP10). All
intermediate precursors and hSIPs were well characterized
1
by H NMR, FT-IR, and GPC (Figures S4-S7). Structural
parameters of all hSIPs are summarized in Table 1.
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a)
3
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460 nm light
7
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hSIP1,
hSIP2, hSIP3
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cRGD (For Integrin Targeting)
100
b)
c)
d)
DOX
ABA
4BHP
Benzyl Alcohol (Stardand)
w/o Light
0.5 h Irradiation
1 h after Light
2 h after Light
4 h after Light
6 h after Light
PEG45
80
60
40
20
0
DOX
4BHP
ABA
0
2
4
6
8
10
0
60 120 180 240 300 360
10
12
14
16
Elution time / min
Time / min
Elution time / min
e)
f)
g) 100
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
Increasing Time
80
60
40
20
0
w/o Irradiation
60 120 180 240 300
0.0
hSIP1
hSIP2
hSIP3
650 700 750 800
Wavelength/ nm
0
Time / min
0
60
120
180
240
300
0
60
120
Time /1m80in
240
300
0
60 120 180 240 300 360
Time / min
Time / min
Figure 1. (a) Schematic illustration of self-immolative cascade depolymerization of hSIP1, hSIP2, and hSIP3 triggered by 460 nm
blue light irradiation. (b) HPLC traces (MeOH/H2O 7/3 v/v; 260 nm absorbance; benzyl alcohol as an external standard) and (c)
time-dependent evolution of the extent of spontaneous release of ABA, 4BHP, and DOX recorded for the aqueous solution of
hSIP1 (0.1 g/L, 25 oC) upon blue light irradiation for 30 min (blue region), followed by extended incubation under dark condition.
(d) DMF GPC traces recorded for hSIP1 without irradiation (black line) and upon incubating for 0 h (red line), 1 h (yellow line), 2
h (cyan line), 4 h (green line), and 6 h (blue line) after 30 min blue light irradiation. (e) Normalized scattering light intensities
recorded for aqueous hSIP1 solution upon 30 min blue light irradiation followed by extended incubation. (f) Nile red (NR) release
profile from the hydrophobic core domains of hSIP1; the inset shows the evolution of fluorescence emission spectra (ex = 550 nm)
of NR. (g) In vitro release profiles of ABA from hSIP1, hSIP2, and hSIP3 (0.1 g/L, 25 oC); the extent of spontaneous release of ABA
was monitored by HPLC during 30 min blue light irradiation (blue region) and upon extended incubation under dark condition.
Visible
Light-Triggered
Spatiotemporal
molecule ABA, 4BHP, and DOX drug (Figure 1b). A total of
~6 h was required for complete depolymerization of hSIP1
(>95% DOX release, Figure 1c). The degradation process
can be also monitored by the evolution of GPC traces,
revealing a gradual decrease in molecular weights (MW)
upon extending incubation duration (Figure 1d). Notably,
the final MW of residual fragments was consistent with
that of peripheral PEO segments conjugated at the
periphery of hSIP1 (Mn ~ 2 kDa) (Figure 1d). This confirmed
complete disintegration of hSIP1 upon removal of capping
moieties by photo irradiation.
Intracellular Drug Delivery. With these functionalized
hSIPs in hand, we first examined visible light
responsiveness of hSIP1-hSIP3 with the same visible light-
responsive capping moiety but different hyperbranched
scaffolds (Figure 1a). Due to the presence of hydrophilic
PEO segments (~6 per hSIP molecule), hSIP1 can be
directly dispersed into water and exists as relatively
monodisperse spherical nanoparticles as evidenced by
TEM and dynamic light scattering (DLS) measurements,
the latter revealed an intensity-average hydrodynamic
diameter, <Dh>, of ~18 nm (Figure S8). After 30 min blue
light irradiation at 460 nm to fully remove per capping
moieties, the subsequent spontaneous self-immolative
radial fragmentation of hSIP1 was traced by GPC and
HPLC via monitoring the concentrations of released small
In addition, DLS measurements revealed >90% decrease
in scattering light intensities within 6 h after 30 min light
irradiation (Figure 1e). In sharp contrast, without light
irradiation, almost no change in scattering intensities was
discernible within the same time range (inset in Figure 1e),
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confirming that hSIP1 was intrinsically inert to
hSIP1 as well (Figure 1g), possibly due to the slow
spontaneous hydrolysis. Further, using a polarity-sensitive
probe, Nile red, encapsulated within the hydrophobic core
domain of hSIP1, photo-triggered degradation of hSIP1
generation of thiophenol. The chemical tuning of cascade
fragmentation rates through structural modulation of hSIP
scaffolds augurs the nature of programmed drug release.
Most importantly, self-immolative radial fragmentation
feature of hSIP1 can be retained when the capping moiety
at the focal point was replaced with reductive milieu-
responsive disulfide-containing one (SS). For hSIP4, GSH-
triggered cascade depolymerization was verified by
triggered DOX release in a programmed manner, time-
dependent decrease of scattering light intensity, and
release of encapsulated Nile red probe into the aqueous
milieu (Figure S9).
was manifested by
a pronounced decrease in the
fluorescence emission of Nile red following 30 min light
irradiation (Figure 1f). This indicated that Nile red was
located in highly hydrophilic milieu upon triggered hSIP1
degradation.
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a)
b)
Original
0 min
20 min
40 min
50 m
50 m
c)
Next, blue light-induced intracellular release of DOX
from hSIP1 was in situ examined with HeLa cells by
confocal laser scanning microscopy (CLSM). Upon co-
incubation without light irradiation, punctuated red-
emitting dots of hSIP1-DOX were discernible inside cells
and most of them did not colocalize with LysoTracker
green, which selectively stains late endosomes and
lysosomes (Figure S10). Upon extending the incubation
duration, the colocalization ratio between hSIP1 red
channel and LysoTracker green channel steadily decreased
(Figure S10b) and the red channel emission intensity
gradually increased (Figure S10c). This indicated that
hSIP1 were increasingly taken up into cells and mainly
located in the cytosol rather than in acidic organelles at
extended incubation, possibly benefiting from the small
size (~18 nm) and presence of cRGD targeting peptide
segments at the periphery.22 In addition, DOX red emission
was not discernible in the cell nuclei even after 4 h co-
incubation without light irradiation, possibly due to the
covalent binding nature of DOX (Figure S10).
60 min
90 min
120 min
180 min
50 m
50 m
d) 100
80
60
40
20
0
w/o Light
with Light
free DOX
10-2
10-1
100
101
Red Channel: DOX / Green Channel: Acridine Orange
CDOX / g/mL
Figure 2. (a) Representative confocal laser scanning micros-
copy (CLSM, scar bar: 20 m) images recorded for HeLa cells
upon incubating for 0-180 min after one scan of 458 nm laser
irradiation; top row: red channel images (DOX, 560 ± 20 nm);
bottom row: overlay of red channel and green channel (acri-
dine orange, 500 ± 20 nm) images. (b,c) CLSM images (scar
bar: 50 m) were recorded for HeLa cells subjected to further
incubation for (b) 0 min and (c) 120 min after one scan of laser
irradiation (458 nm) at the blue square box area; left row: red
channel images (DOX, 560 ± 20 nm); right row: overlay of red
channel and green channel (acridine orange, 500 ± 20 nm) im-
ages. (d) In vitro cytotoxicity determined by MTT assay
against HeLa cells for aqueous solution of cRGD-decorated
hSIP1 without or with 30 min blue LED light irradiation (460
nm). In all cases, the cells were incubated with aqueous hSIP1
solution for 4 h at first before blue light irradiation. Error bars
represent mean ±SD, n = 4.
To monitor intracellular light-triggered DOX release, a
single HeLa cell after co-incubation with hSIP1 for 4 h was
arbitrarily chosen and subjected to one scan of 458 nm
Argon laser irradiation. Upon extending the incubation
time, punctuated red emissive dots from DOX gradually
weakened and eventually disappeared, whereas
continuously enhanced red emission in the cell nucleus
appeared and well co-localized with acridine orange (AO),
which selectively stains cell nucleus (Figure 2a). This
suggested that initially covalently conjugated DOX was
cleaved from periphery of hSIP1 upon 458 nm laser
irradiation and entered into cell nucleus. Visible light-
induced DOX release and subsequent nuclear entry were
further studied on a larger scale under CLSM (Figures 2b-
2c), in which three cells in the square box were selectively
irradiated by 458 nm argon laser for one scan. After further
incubation for 120 min, continuous red emission of
released DOX well co-localized with AO-stained cell nuclei
within the irradiation square area (Figure 2c). In contrast,
for those cells outside the irradiation zone, discrete green
and red emissions were clearly located in the cell nuclei
and cytoplasm, respectively (Figure 2c). These results
The rate of triggered fragmentation of SIPs plays a vital
role considering their biomedical functions such as drug
delivery. Presumably, the depolymerization rate could be
modulated by the chemical structure, chain sequence, and
topology of SIPs.11b,11c,21 To achieve adjustable release rates,
two extra branching precursors, M2 and M3, were utilized
to fabricate photo-responsive hSIP2 and hSIP3, possessing
distinct core scaffolds but the same coronas as compared
to hSIP1. After 30 min blue light irradiation, the release
rate of ABA from hSIP1 was faster than that of hSIP2
(Figure 1g). This is in agreement with the stepwise
depolymerization mechanism that the release of para
substituent via 1,6-elimination is more preferred than that
of ortho substituent via 1,4-elimination5d. Also, the
depolymerization rate of hSIP3 was slower than that of
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indicated that
light irradiation
triggered the
depolymerization of hSIP1, and this is followed by DOX
release at later stages and further diffusion into the cell
nuclei; whereas intact hSIP1 cannot efficiently enter into
the nuclei. We can also conclude that intracellular drug
release from hSIPs can be achieved in a spatiotemporal and
programmed manner.
intermediates and products from the hyperbranched
scaffold might also contribute to the observed cytotoxicity.
We then conducted additional cell viability assays
concerning the cytotoxicity of hSIP4-hSIP7 possessing
inert or reductive/H2O2-cleavable capping moieties (Figure
S11). It was found that nondegradable hSIP6, serving as a
control, exhibited negligible cytotoxicity. Although DOX-
loaded and disulfide-capped hSIP4 exhibited potent
cytotoxicity, hSIP5 and hSIP7 (capped with disulfide and
boronic ester moieties, respectively) without peripherally
conjugated drugs also exhibited cytotoxicity to some
extent. The latter should be ascribed to self-immolative
degradation intermediates (e.g., quinone methide) and
products.23 This poses a challenge towards their future in
vivo drug delivery applications; however, upon appropriate
chemical design, synergistic chemotherapy might also be
achieved.23
In vitro cytotoxicity of hSIP1 was then examined via
MTT assay against HeLa cells (Figure 2d). The cell viability
of hSIP1 without blue light irradiation exhibited negligible
cytotoxicity up to a DOX equivalent concentration of 10
g/mL. However, cell viability dramatically decreased to
~18% at a DOX equivalent concentration of 10 g/mL when
subjected to 30 min blue light irradiation. Note that
control experiments using blue light irradiation alone did
not apparently affect the cell viability. Except light-
triggered release of DOX from hSIP1, degradation
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a)
GSH
PDMAEMA8
hSIP5
b)
Blue + Red
1h
Green
Blue + Red + Green
c)
4h
d)
e)
1.0
0.8
0.6
0.4
0.2
Blue channel / Green Channel
Red channel / Blue Channel
8h
0
4
8
12
16
Time / h
106
105
104
hSIP5
hSIP6
16h
0
4
8
12 16 20 24 28 32
N/P ratio
Blue Channel: hSIP5; Red Channel: Cy5-pDNA;
Green channel: LysoTracker
Figure 3. (a) Schematic illustration of the formation of electrostatic polyplexes between pDNA and hSIP5 bearing multiple cationic
PDMAEMA segments at the periphery; the subsequent hSIP5 depolymerization and pDNA release was triggered by intracellular
reductive milieu (e.g., GSH). (b) Gel electrophoresis analysis of pDNA mobility upon formation of electrostatic complexes with
hSIP5 at varying N/P ratios (left) before and (right) after treating with GSH. (c) Representative CLSM images (scar bar: 20 m) of
red (Cy5-pDNA, 670 ± 20 nm), green (LysoTracker, 520 ± 20 nm), and blue (cou-hSIP5, 450 ± 20 nm) channels recorded for HeLa
cells upon incubating with hSIP5/pDNA complex (N/P = 8) for varying periods. (d) Colocalization ratio analysis of blue channel
to green channel and red channel to blue channel. (e) In vitro transfection efficiencies determined for polyplexes formed between
luciferase-expressing pDNA and hSIP5 or hSIP6 at varying N/P ratios. Error bars represent mean ±SD, n = 4.
Intracellular Reductive Milieu-Triggered pDNA
Delivery by hSIPs Functionalized with Peripheral
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Cationic Segments. Apart from intracellular DOX
These results strongly suggested that cou-hSIP5/Cy5-
pDNA polyplexes underwent endosomal escape into the
cytosol after incubation and the complexes
delivery, we expect that hSIPs could be readily engineered
into nonviral gene delivery vectors by replacing peripheral
chemotherapeutic DOX drug with cationic PDMAEMA
segments. Intracellular pDNA release can be achieved by
the elimination of multivalent interactions triggered by
self-immolative degradation of cationic hSIP vectors
(Figure 3a).24 Installed with disulfide-containing capping
agent (SS) at the focal point, hSIP5 with multiple cationic
PDMAEMA in the periphery can effectively condense
negatively charged pDNA via the formation of electrostatic
polyplexes due to synergistic multivalent interactions,
despite the fact that PDMAEMA8 homopolymer was
deemed infeasible for efficient pDNA binding.24b,24c
8
h
disassembled in the cytosol after being subjected to GSH-
triggered hSIP5 depolymerization and pDNA release. A
quantitative assay of the transfection efficiency was
performed using luciferase-expressing pDNA as the
reporter gene (Figure 3e), and non-degradable
hSIP6/pDNA complexes were employed as a control since
cytoplasmic reductive milieu cannot cleave Bn
functionality at the focal point. In the N/P range of 4-32, in
vitro gene transfection of hSIP5/pDNA polyplexes
systematically presented higher efficiency than
hSIP6/pDNA polyplexes, presumably due to the poor
unpackaging efficacy for the latter. This conclusion was
further verified by utilizing green fluorescent protein
(GFP)-expressing pDNA as the reporter gene (Figure S14).
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The formation of hSIP5/pDNA polyplexes was
confirmed by TEM, zeta-potential, and DLS analysis,
displaying increased size dimension and reversal of Zeta
potentials (Figure S12). In addition, complete gel
retardation can be achieved at N/P ratios higher than 4
(Figure 3b). After treating with 10 mM GSH, hSIP5
underwent depolymerization due to cleavage of disulfide
bond and subsequent cascade radial fragmentation.
Comparable to photo-triggered hSIP1 depolymerization
(Figures 1d-1e), GSH-actuated hSIP5 degradation was also
evidenced by a steady decrease of MW (Figure S13a). We
surmised that GSH-triggered depolymerization of hSIP5
should significantly attenuate pDNA binding capability,
thereby unpacking pDNA payload. As expected, after GSH
addition, pDNA cannot be efficiently bound even at an N/P
ratio as high as 16, as confirmed by gel electrophoresis
analysis (Figure 3b). The disassociation of hSIP5/pDNA
polyplexes upon treating with 10 mM GSH was also
evidenced by the substantially decreased scattering light
intensity and <Dh> upon extending incubation duration
(Figure S13b).
Mitochondria-Targeted Fluorescent Sensing of
H2O2. In addition to spatiotemporal intracellular drug and
gene delivery, we further explored the application scope of
hSIPs and utilized them for intracellular fluorescent
imaging and sensing. The focal point of hSIP7 was caged
with H2O2-responsive phenylboronic ester (PB); the
periphery of hSIP7 was dually functionalized with
mitochondrion-targeting CGKRK peptide and AMC as
caged
fluorescent
reporter.
H2O2-triggered
depolymerization of hSIP7 was then conducted and
monitored by GPC analysis (Figure S15). This process was
accompanied with an evident bathochromic shift from 326
nm to 346 nm in the UV-vis spectra (Figure 4b) and a
cumulative ~25.5 fold fluorescence emission increase
within 240 min (Figure 4c). Compared with the small
molecule control BAC (Figure S16), hSIP7-based H2O2
probe exhibited significant fluorescence emission increase
and enhanced detection sensitivity (Figure 4d). Specifically,
the detection limits (defined as 10% emission increase
relative to the blank sample) towards H2O2 were
determined to be ∼20 nM for hSIP7 and ∼150 nM for BAC,
which was in general agreement with the chemical
amplification factor (~9) of hSIP7 (Figure 4d and Table 1).
Moreover, the selectivity of hSIP7-based H2O2 probe over
other reactive oxygen species (ROS) was well retained due
to the high specificity of phenylboronic ester towards H2O2
(Figure 4e). Thus, hSIP7 can serve as a highly selective and
sensitive fluorescent H2O2 probe. Another concern of
hSIP7-based fluorescent H2O2 probe comes from batch-
to-batch variation, leading to discrepancy in chemical
amplification factors and signal intensities. In preliminary
experiments, we synthesized three additional batches of
hSIP7 in parallel. In the presence of excess H2O2, the
overall fluorescence emission intensities remained almost
constant (Figure S17). These results implied that batch-to-
batch variations can be neglected to some extent.
Considering high cytoplasmic GSH concentration and
reductive milieu in the cytosol, intracellular transfection
efficiency of hSIP5/ pDNA polyplexes (N/P = 8) was
evaluated by taking advantage of cytoplasmic GSH-
triggered pDNA release. By incubating polyplexes of
coumarin-decorated hSIP5 (cou-hSIP5; Scheme S2) and
Cy5-labeled pDNA (Cy5-pDNA) with HeLa cells for 1 h,
most of cou-hSIP5 blue emission overlapped with the red
emission of Cy5-pDNA and LysoTracker Green emission,
indicating that cou-hSIP5/Cy5-pDNA polyplexes retained
and mainly located within endolysosomes. Upon
extending co-incubation duration, the colocalization ratio
between cou-hSIP5 blue emission and LysoTracker Green
emission exhibited a pronounced decrease from 88% (1 h)
to 34% (8 h) and 24% (16 h). Meanwhile, the colocalization
ratio between cou-hSIP5 blue emission and Cy5-pDNA red
emission remained to be ~80% at 8 h co-incubation,
whereas it drastically decreased to ~43% after 16 h co-
incubation (Figures 3c-3d).
Upon co-incubating HeLa cells with CGKRK-decorated
hSIP7 for 12 h, strong punctuated AMC blue emissive dots
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were discerned inside cells and most of them co-localized
integrating with specific enzymatic reactions, in which
well with MitoTracker green (Figure 4f); whereas blue
emission intensities originating from decaged AMC
moieties of label-free hSIP8 and BAC small molecules were
only 57% and 7% relative to that of hSIP7 (Figures 4f-4g).
These results confirmed that hSIP7 decorated with CGKRK
targeting peptide can efficiently find mitochondrion
organelle, where phenylboronic ester capping agent was
cleaved by endogenous H2O2 and hSIP7 was subjected to
depolymerization, thereby releasing AMC fluorophores
and switching on the blue emission.
released fragments could serve as enzymatic substrate and
generate more substance capable of triggering
depolymerization of hSIPs. Previously, Shabat et al.25
originated the “dendritic chain reaction” principle to
design a series of colorimetric and fluorescent probes
solely based on SIDs of the first generation. We expected
that hSIPs, possessing higher chemical amplification
potency, installed with this cycle amplification module will
enable ultrasensitive detection of specific substance. As a
proof of concept, we fabricated an ultrasensitive
colorimetric H2O2 detection system consisting of three
components: negatively charged citric acid-stabilized gold
nanoparticles (AuNPs), CHO-labeled positively charged
hSIP9 capable of triggered fragmentation and CHO release
upon H2O2 treating, and choline oxidase (COx) (Figure 5a).
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Colorimetric Detection of H2O2 by Integrating
Enzyme-Directed Cycle Amplification with Au
Nanoparticle Aggregates. Besides the intrinsic chemical
amplification feature of hSIPs, the signal amplification
performance could be further enhanced via rationally
a)
H2O2
H2O2
BAC
CGKRK (Mitochondria Targeting Moieties)
hSIP7
0.4
0.3
0.2
0.1
0.0
30
25
20
15
10
5
2500
2000
1500
1000
500
10
8
b)
e)
c)
d)
30
20
10
0
hSIP7
BAC
Increasing Time
6
0.02 M
0
60 120 180 240
Time / min
4
0.15 M
2
10 % increase
0
0
0
250
300
350
400
450
500
400 450 500 550 600
700
0
2
4
6
8
10 0.0
0.5
1.0
Wavelength / nm650
Wavelength / nm
H2O2 / M
f)
CGKRK
Decorated hSIP7
Label-Free
hSIP8
g)
CGKRK-decorated hSIP7
label-free hSIP8
BAC
BAC
25
20
15
10
5
1.0
0.8
0.6
0.4
0.2
1.0
0.8
0.6
0.4
0.2
0.0
0
-
H2O2 ·OH OCl- ·OtBu 1O2 O2
Blue: Coumarin / Green: MitoTracker
Figure 4. (a) Schematic illustration of H2O2-triggered self-immolative depolymerization of hSIP7 possessing caged nonfluorescent
AMC at the periphery; as a control, H2O2-triggered cleavage of small molecule BAC is also shown. Time-dependent (b) UV-vis
absorbance spectra and (c) fluorescence emission spectra (ex = 350 nm) recorded for the aqueous hSIP7 solution (10 M) upon
incubating with H2O2 (100 M) for varying time intervals (0-240 min); the inset in (c) shows the evolution of emission intensity at
450 nm. (d) H2O2 concentration dependence of normalized emission intensities at 450 nm after incubating the aqueous solution
of hSIP7 (1 M, containing 9 M coumarin moieties) or BAC (9 M) with H2O2 for 12 h. (e) Fluorescence response (ex = 350 nm,
em = 450 nm) of hSIP7 towards various ROS species. (f) Representative green channel (MitoTracker, 520 ± 20 nm) and blue
channel (coumarin, 450 ± 20 nm) CLSM images (scar bar: 20 m) recorded for HeLa cells upon incubating with CGKRK-decorated
hSIP7, label-free hSIP8, and BAC for 12 h; the bottom row represents an overlay of blue and green channel images. (g) Colocali-
zation ratio analysis between blue channel and green channel fluorescence, and normalized blue channel emission intensities
quantified from CLSM images (f).
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H2O2-triggered generation of CHO from depolymerized
added, the less time was needed to reach a plateau (i.e.,
hSIP9 can be further oxidized by COx to generate more
H2O2, which can in turn trigger the degradation of other
unreacted hSIP9 and release more CHO (Figure 5a). Upon
addition of aqueous hSIP9 solution (4-60 mg/L) into
citrate-stabilized AuNP dispersion (~8 nm in average
diameter, 80 mg/L), the initial wine red color gradually
turned purple blue and the UV-vis absorption peak red-
shifted from ~520 nm to ~570 nm (Figure S18), indicating
the formation of AuNP aggregates due to electrostatic
interactions with hSIP9. However, when 9 equiv. H2O2
(relative to the phenylboronic ester caging moieties) was
added to hSIP9/AuNP hybrid aggregates in the presence
of COx, reversible color change from purple blue to wine
red was achieved within ~330 min (Figure 5b), suggesting
the disintegration of hybrid aggregates and formation of
re-dispersed AuNPs. In addition, H2O2-triggered
dispersion of AuNP aggregates can also be confirmed by
TEM observations (Figures S19).
complete depolymerization). Notably, when only ~0.1
equiv. H2O2 (1 μM) was introduced (relative to phenyl-
boronic ester moieties), the time-dependent evolution of
A520/A570 revealed a sigmoidal shape, which was in accord
with an autocatalytic process (Figure 5c). In contrast, in the
absence of H2O2, hSIP9/AuNP hybrid aggregates were
relatively stable and there was no appreciable change in
A520/A570 ratios within ~6 h co-incubation, although a slight
increase can be still discerned after 9 h co-incubation
(Figure S20). Spontaneous hydrolysis of carbonate bonds
to release CHO, which can generate H2O2 under COx
enzymatic reaction, should account for the slight change
in absorbance intensity ratio for the blank control. The
H2O2 detection limit (defined as 10% A520/A570 increase
relative to the control) was determined to be ~0.13 μM after
360 min incubation (Figure 5d), which is more sensitive
than that of previously reported colorimetric H2O2 sensing
system involving AuNPs.17c We also conducted
supplementary experiments in the absence of COx (Figure
5e). The lack of positive feedback cycle amplification aided
by enzymatic H2O2 generation led to considerably
decreased shift in absorbance intensity ratios, and this
trend was more prominent at lower H2O2 levels (e.g., ~7
times difference at 1 M H2O2).
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These promising preliminary results spurred us to
further assess H2O2-triggered signal amplification system
in the presence of lower H2O2 concentrations. Upon H2O2
addition (0-100 μΜ), time-dependent evolution of
absorbance intensity ratios (A520/A570
) were highly
dependent on H2O2 levels (Figure 5c). The more H2O2 was
a)
b)
1.2
H2O2
Increasing Time
1.0
H2O2
0.8
AuNPs
Aggregated AuNPs
0.6
0.4
0.2
H2O2
H2O2
Choline
Oxidase
(COx)
0.0
450 500 550 600 650 700 750 800
hSIP9
Wavelength / nm
c)
d)
e)
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
1.2
1.0
0.8
0.6
0.4
0.2
0.0
2.2
H2O2 (M)
w/ COx
w/o COx
x1
2.0
1.8
1.6
1.4
1.2
1.0
100
50
10
5
1
0
x2
x7
0.13 M
Increase by 10 %
20 30 40 50
0.8
0
120
240
360
480
0
10
60
1
10
H2O25/ M
Time / min
Concentration / M
Figure 5. (a) Schematic illustration of the construction of colorimetric H2O2 sensing system by integrating negatively charged Au
NPs with cationic hSIP9 exhibiting H2O2-triggered cascade fragmentation and enzyme-mediated cycle amplification features.
Time-dependent evolution of (b) UV-vis absorption spectra and (c) absorbance intensity ratios (A520/A570) recorded for the aque-
ous mixture of hSIP9 (80 mg/L, 11 M), Au NPs (80 mg/L), and COx (choline oxidase; 10 ng/mL) upon addition of H2O2. (d) H2O2
concentration dependence of absorbance intensity ratios (A520/A570) recorded for the aqueous mixture (80 mg/L hSIP9, 80 mg/mL
Au NPs, 10 ng/mL COx) upon 6 h co-incubation. (e) Absorbance intensity ratio shift, (A520/A570), recorded for the aqueous mixture
(80 mg/L hSIP9, 80 mg/mL Au NPs) upon 6 h co-incubation in the presence or absence of COx.
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Fluorescent H2O2 and Antigen Detection by
Integrating with Enzyme-Directed Cycle
(Figure 6b). Specifically, hSIP10 was fully fragmented
within ~300 min in the presence of 30 ng/mL COx (Figure
6b). When the incubation time was set at 240 min, the COx
detection limit (defined as 10% AMC fluorescence emission
increase relative to the blank control) was determined to
be ~0.95 ng/mL (Figure 6c), which is quite comparable to
the antigen detection range of commercial ELISA kit.
Amplification and ELISA. Considering that fluorescent
H2O2 detection system integrated with enzyme-directed
cycle amplification will possess more improved detection
sensitivity, we further designed hSIP10 bearing caged
fluorescent AMC moieties at the periphery (Figure 6a). The
time-depende nt emission intensity changes at 450 nm
recorded hSIP10-COx system in the presence of varying
amount of H2O2 exhibited a sigmoidal trend. Importantly,
0.02 equiv. H2O2 (relative to phenylboronic ester moieties
at the focal point) addition was capable of completely
depolymerizing hSIP10 if longer incubation duration was
given (Figure S21). Further numerical fitting of reaction
kinetics revealed that the process complied with the
positive feedback cycle amplification model developed by
Shabat research group (Figure S21).25b These results verified
that the coupling of intrinsic self-immolative chemical
amplification with enzyme-mediated cycle amplification
could significantly boost detection sensitivity.
Finally, we attempted to further explore functional
applications of hSIPs in clinical diagnosis. As a proof-of-
example, we fabricated an ELISA kit to detect human
carcinoembryonic antigen (HCEA) by taking advantage of
the cycle amplification capability of hSIP10. Towards this
goal, COx and HCEA monoclonal antibody (ACEA) were
covalently conjugated onto silica nanoparticles (SiNP-
COx-ACEA) in a sequential manner (Scheme S3). All
intermediate products and final SiNP-COx-ACEA hybrid
nanoparticles were characterized by CLSM, UV-vis and
fluorescence spectroscopy, and TEM measurements
(Figures S22-S25). Based on the hydrolysis rate of BAC
catalyzed by SiNP-COx-ACEA, the exact number of COx
enzymes per SiNP was calculated to be ~5600 (Figure S22).
Using the newly developed SiNP-COx-ACEA in
combination with hSIP10, and following standard ELISA
protocols, prominently increased fluorescence emission at
450 nm was discerned upon gradually increasing spiked
CEA concentrations (Figure 6d). The CEA detection limit
using hSIP10/SiNP-COx-ACEA system was determined to
be 1.6 ng/mL, ~7 times better than that of BAC/SiNP-COx-
ACEA or commercially available horseradish peroxidase
(HRP)-3,3',5,5'-tetramethyl-benzidine (TMB) assay (Figure
6e).26
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Note that COx concentrations also affected the
amplification efficacy of hSIP10-COx system; therefore, it
can be used for the detection of COx as well in the presence
of trace amount of H2O2 (e.g., 0.1 equiv. relative to phenyl-
boronic ester moieties). In this approach, the background
signal could be minimized due to the fact that hSIP10-COx
system cannot amplify fluorescence signals without COx
even if inadvertent hydrolysis will release CHO substrate.
Upon gradually increasing COx levels (0-30 ng/mL),
emission intensities at 450 nm of decaged AMC moieties
from depolymerized hSIP10 increased more abruptly
a)
b)
2000
Choline Oxidase:
30 ng/mL
15 ng/mL
1500
7 ng/mL
3 ng/mL
0 ng/mL
hSIP10
Human CEA
Capture Antibody
1000
500
0
Human CEA
Detection Antibody
H2O2
SiO2 Nanoparticles
Choline Oxidase (COx)
Human CEA Antigen
0
60 120 180 240 300 360 420
Time / min
c)
d)
e)
250
200
150
100
50
20
15
10
5
2000
1500
1000
500
0
HRP-TMB (Standard Protocol)
hSIP10
BAC
40 ng / mL
20 ng / mL
10 ng / mL
5 ng / mL
2 ng / mL
1 ng / mL
0 ng / mL
0.95 ng/mL
1.6 ng/mL
~10 ng/mL
10% Increase
15 20
0
0
380 400 420 440 460 480 500 520 540
0
5
10
25
30
1
10
100
Wavelength / nm
Choline Oxidase / ng/mL
Human CEA / ng/mL
Figure 6. (a) Schematics of the construction of cycle signal amplification system from COx enzyme and hSIP10 possessing caged
nonfluorescent AMC and choline moieties at the periphery, which considerably improves the detection sensitivity of sandwich
ELISA format towards human CEA. (b) Time-dependent emission intensity at 450 nm (ex = 350 nm) recorded for the aqueous
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mixture of hSIP10 (50 M) and COx at varying concentrations upon addition of H2O2 (5 M). (c) COx concentration dependence
of emission intensities at 450 nm upon incubating the aqueous mixture for 4 h. (d) Fluorescence spectra and (e) human CEA
concentration-dependent changes in emission intensities at 450 nm (ex = 350 nm) obtained via the ELISA kit; surface captured
SiNP-COx-ACEA (95/5 PBS/DMSO, pH 7.4, 37 C) was co-incubated with choline (2 M) and hSIP10 (10 M, containing 25 M
coumarin moieties) or BAC (25 M). As a control, the optical absorption at 450 nm was also recorded by following standard ELISA
o
protocols using TMB (3,3',5,5'-tetramethylbenzidine) as the HRP substrate. Error bars represent mean ±SD, n = 4.
9
The authors declare no competing financial interest.
■ CONCLUSIONS
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A library of water-dispersible and multi-functionalized
ACKNOWLEDGMENT
self-immolative polymers with
a
new type of
The financial support from the National Natural Scientific
Foundation of China (NNSFC) Project (21274137, 51273190,
5147315 and 51033005) and Specialized Research Fund for the
Doctoral Program of Higher Education (SRFDP,
20123402130010) is gratefully acknowledged.
hyperbranched chain topology, hSIPs, were synthesized by
integrating one-pot polycondensation of three types of AB2
precursors with sequential post-functionalization. By
rationally screening branching scaffolds, capping agents at
the focal point, and peripheral functional groups and
polymer segments, hSIPs with advanced drug/gene
delivery and ultrasensitive detection functions were
successfully constructed, exhibiting an intrinsic chemical
amplification factor of up to 20 per hSIP molecule. The
spontaneous radial depolymerization of hSIPs upon
stimuli-triggered single cleavage of the capping moiety
allowed for the construction of myriad functions including
visible light-actuated intracellular release of conjugated
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ASSOCIATED CONTENT
Supporting Information. Detailed experimental procedures,
NMR, UV-vis, FT-IR, MS, and fluorescence spectra, HPLC,
GPC, and DLS data, TEM and CLSM images. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
sliu@ustc.edu.cn
Notes
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Previous Works
Multistep
Synthesis
Polymerization &
Post-Modification
Dendritic Self-Immolative
Polymers (SIDs)
Linear Self-Immolative
Polymers (l-SIPs)
This Work
One-Pot
Polymerization &
Post-Modification
Trigger Removal &
Self-Immolative
Depolymerization
Hyperbranched Self-
Immolative Polymers (hSIPs)
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