Self-immolative nanoparticles triggered by hydrogen peroxide and pH

Article abstract of DOI:10.1002/pola.27203

The azomethine-based oligomers bearing boronate groups and imine moieties in the main chain were synthesized from a dialdehyde monomer and an aromatic (oligomer 4) diamine or an aliphatic diamine (oligomer 5). Based on the oligomers, the nanoparticles with hydrogen peroxide (H₂O₂) and pH dual-responsive properties were constructed and encapsulated nile red inside. The nanoparticles disassembled either by the trigger of H ₂O₂ or by the attack of H⁺, thus leading to the release of loaded species. Compared to oligomer 4, oligomer 5 showed a faster degradation rate in the presence of H₂O₂, especially in a weak acidic environment. No significant cytotoxicity was observed as HeLa cells incubated in the nanoparticles with the concentration up to 200 μg/mL evidenced by cytotoxicity assay in vitro. Such a system capable of dual response of H₂O₂ and H⁺ may have potential application as a carrier for drug delivery.

Full text of DOI:10.1002/pola.27203

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Self-Immolative Nanoparticles Triggered by Hydrogen Peroxide and pH
Zhijian Wang, Jianbo Sun, Xinru Jia
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Polymer Chemistry and Physics of
the Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
Correspondence to: X. Jia (E-mail: xrjia@pku.edu.cn)
Received 28 September 2013; accepted 31 March 2014; published online 00 Month 2014
DOI: 10.1002/pola.27203
ABSTRACT: The azomethine-based oligomers bearing boronate
groups and imine moieties in the main chain were synthesized
from a dialdehyde monomer and an aromatic (oligomer 4) dia-
mine or an aliphatic diamine (oligomer 5). Based on the
oligomers, the nanoparticles with hydrogen peroxide (H2O2)
and pH dual-responsive properties were constructed and
encapsulated nile red inside. The nanoparticles disassembled
either by the trigger of H2O2 or by the attack of H1, thus lead-
ing to the release of loaded species. Compared to oligomer 4,
oligomer 5 showed a faster degradation rate in the presence of
H2O2, especially in a weak acidic environment. No significant
cytotoxicity was observed as HeLa cells incubated in the nano-
particles with the concentration up to 200 lg/mL evidenced by
cytotoxicity assay in vitro. Such a system capable of dual
response of H2O2 and H1 may have potential application as a
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carrier for drug delivery.
2014 Wiley Periodicals, Inc. J.
Polym. Sci., Part A: Polym. Chem. 2014, 00, 000–000
KEYWORDS: nanoparticles; oligomers; step-growth polymeriza-
tion; stimuli-sensitive polymers
lower in the lysosome.19,20 Based on such behavior, many
drug-delivery systems with pH sensitivity were reported to
show the advantages of controlled and targeted drug
release.21–24 For example, Lahann and coworkers reported
the synthesis of acetal-modified dextran that could be
cleaved into hydrophilic dextran species in the presence of
protons.25 Besides, the polymers with tertiary amines or
imidazole rings in the structure were also widely used in the
design of carriers. Such polymers could transfer from hydro-
phobic to hydrophilic by the protonation, resulting in the
release of payloads.26
INTRODUCTION It is known that the excessive level of hydrogen
peroxide (H2O2) (up to 1 mM)1 will break the reactive oxygen
species (ROS) balance and cause various human diseases includ-
ing cancers, tissue damage, atherosclerosis, diabetes, and
chronic hepatitis because of the occurrence of oxidation stress
and damage to DNA.2 However, H2O2, as a major source of ROS
in living cells, is highlighted currently for its functions of regulat-
ing the intracellular signaling pathways and acting as a mediator
in cell proliferation, differentiation, and migration.3 In recent
years, a number of drug carriers were developed with H2O2 as a
trigger, such as H2O2-induced hydrophobic–hydrophilic transi-
tion,4,5 oxidation of selenium-containing polymers,6,7 cleavage
of diselenide bond,8 degradation of peroxalate ester bonds,9 and
disruption of boronate groups.10–14 A typical example was
Due to the sophisticated environments in the human body, it
is imperative to create drug-delivery systems with multiple
stimuli-responsive properties for precise control on both
degradation kinetics and release of guest species. Up to now,
many drug carriers with two triggers, such as reduction and
pH, have been developed.20,26,27 However, a drug-delivery
system with the sensitivity of oxidation and pH is still rare,
although some phagosomes are of oxidizability and acidic.15
An example reported by Almutairi and coworkers described
that the polythioether ketal that was cleaved in an acidic
aqueous solution changed the hydrophobicity in the presence
of H2O2.5
ꢀ
reported by Frechet and coworkers, who integrated the self-
immolative structures into the dextran-based drug carrier.15 In
their case, the increased water-solubility of the polymer was
ascribed to the disruption of hydrophilic–hydrophobic balance
by a H2O2-induced oxidation reaction, leading to dissociation of
the aggregates and liberation of the payloads. Recently, a self-
immolative polymer responding to H2O2 has been reported.16
Such main chain degradation polymer showed unique proper-
ties for the achievement of structure degradation and the
release of loaded species.17,18
As reported, pHs in the intracellular compartments are dif-
ferent with near pH 5 in the late endosome and pH even
Herein, we report a new type of self-immolative nanopar-
ticles from azomethine-based oligomers with the guest
Additional Supporting Information may be found in the online version of this article.
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molecules inside. Our aim is to construct a dual responsive
model system with H2O2 responsive moieties and pH sensi-
tive groups in the main chain for exploring its potential
application as a drug carrier. The nanoparticles disassembled
either by breaking the boronate bonds with the trigger of
H2O2, or by rupturing the C5N bonds at lower pHs.
obtained which could be further converted into the hydrody-
namic radius Rh by using the Stokes–Einstein equation:
D5kBT=6pgRh
where D, kB, T, g are the translational diffusive coefficient,
the Boltzmann constant, the absolute temperature, and the
viscosity of the solvent, respectively.
EXPERIMENTAL
Materials
Measurements
2,6-Dimethyl phenyl boronic acid (J&K Chemical Company),
pinacol (Alfa Aesar), N-bromosuccinimide (NBS)(Alfa Aesar),
and 4-hydroxy benzaldehyde (Alfa Aesar) were used as
received. Azodiisobutyonitrile (AIBN) and p-phenylenedi-
amine were recrystallized from methanol and ethanol,
respectively. THF and chloroform was distilled following the
standard procedures. Other solvents and reagents, unless
stated specifically, were purchased from Beijing Chemical
Reagent Co. and used as received.
1H NMR spectra were recorded on 300 MHz (Varian Mer-
cury) and 400 MHz (Bruker) spectrometers operated at
room temperature with CDCl3 or D2O/acetone-d6 as the sol-
vents and tetramethylsilane (TMS) as the internal standard.
The fluorescence spectra were recorded on a Hitachi F-4500
fluorescence spectrophotometer. Transmission electronic
microscopy (TEM) measurement was operated by a JEM-100
CXII microscope with an acceleration voltage of 100 kV
(Hitachi). The samples for TEM measurements were pre-
pared as follows: a drop of the suspension was placed on a
carbon-coated Formvar copper grid. After 2 min, the grid
was tapped with filter paper to remove the aqueous solution
on the surface and air-dried. Then, a drop of 1 wt % solution
of uranyl acetate was added to the copper grid. The size of
the nanoparticles was determined by dynamic light scatter-
ing (DLS) measurement at an angle of 90ꢀ (the ZetaPlus
instrument, Brookhaven Instruments, NY). Gel permeation
chromatography (GPC) measurement was performed on an
Synthesis of Compound 1
About 10.0 g (67.0 mmol) 2,6-dimethyl phenylboronic acid
and 9.50 g (80.4 mmol) pinacol were dissolved in 150 mL
toluene in a 250-mL round bottom flask that was attached
to a Dean-Stark apparatus. The mixture was heated to vigor-
ous reflux and stirred for 6 h. Then, the solution was cooled
to room temperature and toluene was evaporated in vacuo.
The crude product was purified through a silica column
using dichloromethane as the eluent. Compound 1 was
obtained as a white solid. Yield: 92%.
Agilent 1200 series system, equipped with
a VARIAN
PolarGel-M column (300 3 7.5mm), an Iso Pump (G1310A),
a UV detector at 254 nm, and a differential refractive index
detector (RI). Except specially mentioned, the number aver-
age molecular weight (Mn) and the polydispersity (PDI)
reported here were from the RI detector. N,N-Dimethylforma-
mide (DMF) was used as the eluent at 50 ꢀC with a flow
rate of 1 mL/min. Narrowly distributed Poly(methyl methac-
rylate (MMA)) samples (molecular weight range of 690–
1,944,000 g/mol, from Polymer Laboratories) were used as
the calibration standard.
Compound 1: 1H NMR (400 MHz, CDCl3, TMS, T 5 298 K):
7.12 (1 H, s), 6.93 (2 H, d, J 5 7.6 Hz), 2.39 (6 H, s), 1.38
(12 H, s). 13C NMR (100 MHz, CDCl3, TMS, T 5 298 K):
141.74, 129.14, 127.46, 126.40, 83.62, 24.95, 22.19. High
Resolution-Electron Spray Ionization-Mass Spectroscopy (HR-
ESI-MS): C14H22BO2; Mass: calculated 233.17099, measured
233.17036.
Synthesis of Compound 2
About 3.51 g (15.1 mmol) compound 1, 5.93 g (33.3 mmol)
NBS, and 0.50 g (3.0 mmol) AIBN were dissolved in 25 mL tet-
rachloromethane in a Schlenk tube. After thoroughly deoxyge-
nation, the tube was sealed and heated to 90 ꢀC for 4 h. Then,
the mixture was cooled to room temperature and filtrated.
The filtrate was collected and purified through a silica column
with petroleum ether/ethyl acetate (20/1) as the eluent. Com-
pound 2 was obtained as a white solid. Yield: 95%.
Both DLS and static light scattering (SLS) measurement of
nanoparticles were performed on a Brookhaven goniometer
(BI-200SM) equipped with a BI-TurboCorr digital correlator
and a thermostatic bath with temperature accuracy of 0.01
ꢀC. A vertically polarized solid-state laser operating at 532
nm was used as the light source (100 mW, CNI Changchun
GXC-III, China). For a dilute solution, the root mean-square
radius of gyration (Rg) can be obtained from SLS data on the
basis of the following equation:
Compound 2: 1H NMR (400 MHz, CDCl3, TMS, T 5 298 K):
7.33–7.20 (3 H, m), 4.89–4.75 (4 H, m), 1.52–1.39 (12 H, m).
13C NMR (100 MHz, CDCl3, TMS, T 5 298 K): 147.80, 144.08,
h
i
HC=Rvv ðhÞ5ð1=MwÞ 11ð1=3ÞR2gq2 12A2C
130.35,
14H23BBr2NO2; Mass: calculated 406.01836, measured:
408.01670.
129.83,
84.21,
33.90,
25.07.
HR-ESI-MS:
C
where H 5 4p2n2(dn/dC)2/(NAk4), and q 5 4pn/k sin(h/2),
NA, n, dn/dC, and k are the Avogadro’s number, the solvent
refractive index, the specific refractive index increment, and
the wavelength of light in a vacuum, respectively. In DLS, by
using a Laplace inversion program, CONTIN, the normalized
distribution function of the characteristic line width was
Synthesis of Compound 3
About 1.00 g (2.56 mmol) compound 2 and 0.78 g (6.39
mmol) 4-hydroxybenzaldehyde were dissolved in 50 mL ace-
tone followed by addition of 4 Å molecular sieves. Then,
2
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3.54 g (2.56 mmol) anhydrous potassium carbonate was
added into the mixture, stirred, and heated. After 8 h, the
mixture was cooled to room temperature and filtrated. The
filtrate was collected and evaporated in vacuo. The crude
product was purified via a silica column with dichlorome-
thane as the eluent. Compound 3 was obtained as a white
solid. Yield: 41%.
The degradation of oligomer 5 was conducted in the mixed solu-
tion of CDCl3 and 0.1 M NaHCO3. The solution containing 1 mg
oligomer 5 in 500 lL CDCl3 was added into 200 lL 0.1 M
NaHCO3 solution. The 1H NMR spectrum of mixture was
recorded via scanning in the 300 MHz NMR spectrometer for 12
h. Then, 0.5 lL 30% H2O2 was added into the system. After
being incubated at room temperature for 24 h, the 1H NMR spec-
trum was recorded again. The spectra were just of CDCl3 phase.
Compound 3: 1H NMR (400 MHz, CDCl3, TMS, T 5 298 K):
9.89 (1 H, s), 7.84 (2 H, d, J 5 8.7 Hz), 7.08 (2 H, d, J 5 8.7
Hz), 5.34 (2 H, s), 1.16 (6 H, s). 13C NMR (100 MHz, CDCl3,
TMS, T 5 298 K): 190.76, 163.85, 141.59, 131.95, 130.29,
130.02, 128.66, 115.03, 83.93, 70.49, 24.93. HR-ESI-MS:
The Release of Nile Red from Nanoparticles
The release of nile red was measured by the F4500 fluores-
cence spectrometer. The excitation and emission slit width of
the fluorometer were set at 10 and 10 nm, respectively. The
excitation wavelength was set up at 550 nm, and the emis-
sion spectra were recorded from 570 nm to 700 nm at a
scanning rate of 60 nm/min. For the measurement, 30%
H2O2 solution was added into the nanoparticle suspension
for affording the resulting solution in the presence of H2O2
at different concentrations. HCl solution was added for
adjusting a weak acidic environment. The fluorescence spec-
tra were recorded at different time point. The excitation
intensity value at 610 nm was used to record the release of
nile red from the nanoparticles.
C28H30BO6;
Mass:
calculated
473.21348,
measured
473.21267.
Synthesis of Oligomer 4
About 94.4 mg (0.20 mmol) compound 3 and 21.7 mg (0.20
mmol) p-phenylenediamine were dissolved in 2 mL anhy-
drous chloroform in a Schlenk tube. After deoxygenation, the
ꢀ
tube was sealed and heated to 90 C for 24 h. After the solu-
tion was cooled to room temperature, the solvent was evapo-
rated and a yellow solid was obtained. 1H NMR (400 MHz,
CDCl3, TMS, T 5 298 K): 9.90–9.87, 8.52–8.35, 7.92–7.76,
7.51–7.38, 7.17–6.98, 6.77–6.65, 5.39–5.23, 1.12–1.10.
Cell Culture
HeLa cells were routinely grown in Dulbecco’s Modified
Eagle’s Medium (DMEM) medium supplemented by 10%
heated-inactivated fetal-bovine serum (FBS), 100 U/mL peni-
cilliꢀn, and 100 lg/mL streptomycin. Cells were maintained at
37 C with 5% CO2.
Synthesis of Oligomer 5
About 198.2 mg (0.42 mmol) compound 3 and 24.0 mg
(0.40 mmol) ethylene diamine were dissolved in 2 mL anhy-
drous chloroform in a Schlenk tube. After deoxygenation, the
ꢀ
tube was sealed and heated to 90 C for 24 h. After the solu-
tion was cooled to room temperature, the solvent was evapo-
rated and a yellow solid was obtained. 1H NMR (400 MHz,
CDCl3, TMS, T 5 298 K): 9.90–9.87, 8.41–8.00, 7.72–7.56,
7.56–7.49, 7.49–7.32, 7.11–6.76, 5.39–5.05, 4.04–3.75, 1.34–
0.87.
In Vitro Cytotoxicity Assay
HeLa cells were seeded into 96-well culture plates at a den-
sity of 5 3 103 cells/well and grown for 24 h. Then, the
nanoparticles prepared from the oligomers 4 and 5 were
added into 96-well culture plates, respectively. The final con-
centrations of oligomers were controlled in the range of 0.1–
200 lg/mL. After incubation for 48 h and staining the SRB
assay, the cell viability was measured by a microplate reader
at 549 nm. The following formula was used to calculate the
cell survival:
Preparation of the Nanoparticles
The nanoparticles were prepared by using a common emul-
sion method. About 1.56 mg oligomer 4 and 0.15 mg nile red
was dissolved in 1 mL dichloromethane. The resulting red
solution was added to a 0.1 M NaHCO3 solution containing 1%
Polyvinyl alcohol (PVA). The mixture was oscillated under
ultrasound for 2 min and then the emulsion was stirred at
room temperature. The suspension was purified by dialysis
using a dialysis membrane (Mw 5 5000) to remove PVA, then
filtered using a 0.45-lm PES filter (Millipore) and followed by
a 0.22-lm PES filter (Millipore). The size of nanoparticles was
measured by particle sizing instrument and TEM.
Survival %5ðA540nm for the treated cells
=A540nm for the control cells Þ3100%
where the A540nm is the absorbance value. The cytotoxicity
assay was repeated for three times.
RESULTS AND DISCUSSIONS
H2O2-Triggered Degradation of the Oligomers
Generally, the imines from aromatic amines and aldehydes
are more stable than those from aliphatic amines.28 In order
to understand the dependence of responsive property on the
structure, oligomers 4 and 5 were designed with either aro-
matic or aliphatic Schiff base groups in the main chains. The
The degradation of oligomer 4 was measured by 1H NMR
measurement. The solution of 1 mg oligomer 4 in 200 lL
acetone-d6 was added into 300 lL 0.1 M NaHCO3 solution in
D2O. The 1H NMR spectrum of mixture was recorded via
scanning in the 300 MHz NMR spectrometer for 12 h. Then,
0.5 lL 30% H2O2 was added into the NMR tube. After being
incubated at room temperature for 24 h, the 1H NMR spec-
trum was recorded again.
oligomers were synthesized through
a
condensation
polymerization from a dialdehyde monomer 3 containing a
boronate group and an aromatic diamine (oligomer 4) or an
aliphatic (oligomer 5) diamine. Scheme
1 depicts the
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SCHEME 1 Synthesis and the structures of azomethine-based oligomers 4 and 5.
structures of oligomers 4 and 5. The detailed synthetic pro-
cedures and characterizations are described in the Experi-
mental section.
hydroxyl groups in the oligomer structure. Then the
oligomer chain undergoes an 1,4-quinone methide rearrange-
ment, thus to cleave the ether bonds and generate the degra-
dation
products
of
2,6-bis(hydroxymethyl)
phenol,
Monomer 3 was prepared by the following reactions. Pinacol
reacted with 2,6-dimethyl phenylboronic acid to generate
boronic ester 1. The bromination of methyl groups of 1 by
NBS in vacuo afforded compound 2. Monomer 3 was
obtained via a typical etherification of 4-hydrophenyl alde-
hyde with benzyl bromide groups of 2. Finally, the target
oligomers 4 and 5 were simply synthesized by a well known
necleophilic addition reaction between aldehyde groups of 3
and p-phenylenediamine or ethylene diamine, respectively.
azomethine derivatives, and 4-hydroxy benzaldehyde. While
in an acidic environment, N atom of Schiff base group is pro-
tonated, then by the attack of H2O, the C5N bonds are rup-
tured to form the corresponding diamine and dialdehyde
monomers.
The degradation of oligomers was confirmed by 1H NMR
measurement (Figure 1). Oligomer 4 was incubated for 24 h
in the acetone-d6/NaHCO3/D2O buffer solution containing 10
mM H2O2. As a result, the signals at 5.46 ppm corresponding
to methylene protons of benzyl ether disappeared, indicating
the breakage of benzyl ether structure. The proton signal of
formyl groups shifted to high field at 9.5 ppm, and two dou-
blet peaks near 7.78 and 6.75 ppm emerged obviously, con-
firming the formation of 4-hydrophenyl aldehyde
(Supporting Information Fig. S3), one of the degradation
products of oligomer 4. However, the signals of the diimine
were not observed because of its poor solubility in the
mixed solvent.
The structures and molecular weight of 4 and 5 were char-
acterized by 1H NMR and GPC measurements (Supporting
Information Figs. S1 and S2). It was found that the signal at
8.45 ppm related to the protons of azomethine appeared,
indicating the formation of azomethine structure. From the
integral ratio of aldehyde at 9.90–9.87 ppm and azomethine
protons at 8.52 and 8.35 ppm, the repeating unit was calcu-
lated to be about 5 for both of the oligomers 4 and 5 in
average, corresponding to the molecular weight of 2900 and
3500, respectively. The polydispersity of the obtained
oligomers was also measured by GPC with the results of
2.28 for oligomer 4 and 2.07 for oligomer 5 (Supporting
Information Fig. S2).
Figure 2 shows the 1H NMR spectra of oligomer 5. It was
incubated in a buffer of CDCl3/NaHCO3/D2O containing 10
mM H2O2 or at pH 3 separately at room temperature for 24
h. We used a mixed solvent of CDCl3/NaHCO3/D2O instead of
acetone-d6/NaHCO3/D2O buffer solution for 1H NMR mea-
surement because of the poor solubility of oligomer 5 in the
later one. The spectra were just of the CDCl3 phase. The
Scheme 2 depicts a proposed cleavage process of the
azomethine-based oligomers. In the presence of H2O2, the
boronates are oxidized and eliminated, which affords the
SCHEME 2 The proposed cleavage process of azomethine-based oligomers triggered by H2O2 and pH.
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The size of nanoparticles was measured by TEM and DLS at
an angle of 90ꢀ by the ZetaPlus instrument. The size of par-
ticles from oligomer 5 was about 180 nm (TEM), and 230
nm (DLS) in average. While the nanoparticles from oligomer
4 were smaller with the size of about 50 nm (TEM) and 150
nm (DLS), which might be due to the stronger p–p interac-
tions of aromatic groups in the structure of oligomer 4.
The H2O2 responsive behaviour of nanoparticles was exam-
ined by the fluorescence spectroscopy using nile red as a
probe whose emission intensity would decrease when trans-
ferred from hydrophobic environment into an aqueous
medium. Figure 3 shows the plots of normalized fluorescent
intensity of nile red vs. time when the nanoparticles were
treated with H2O2 at different concentrations. It shows that
the emission intensity remains constant without H2O2, but
reduces over time upon treated with H2O2, indicating the
disassembly of nanoparticles and the release of payloads
into the aqueous solution. We noticed that the nanoparticles
from oligomers 4 and 5 displayed different degradation
rates, particularly by increasing the concentration of H2O2.
At the concentration of 10 mM, the emission intensity of
nanoparticles from oligomer 5 reduced to 60%, while for the
nanoparticles from oligomer 4, it was 85%. With increasing
the concentration of H2O2 to 100 mM, the emission intensity
of nanoparticles from oligomer 5 decreased rapidly to 40%
within a short time (60% for the particles from oligomer 4
under the same condition), indicating the nanoparticles from
oligomer 4 degraded slower. That might be because its dense
structure gave difficulty for the attack of H2O2.
FIGURE 1 1H NMR spectra of (a) oligomer 4 in 0.1 M NaHCO3
buffer (D2O): acetone-d6 (3:2); (b) oligomer 4 after incubated
for 24 h in 0.1 M NaHCO3 buffer containing 10 mM H2O2 (D2O):
acetone-d6 (3:2).
To understand the disassembly of nanoparticles, we
observed the morphologies of nanoparticles before and after
treating with H2O2 by TEM. The images are shown in Figure
peak at 3.91 ppm corresponding to the signal of ethyl dia-
mine was not detected because the product transferred into
the aqueous phase. Moreover, when the sample was incu-
bated with 10 mM H2O2 for 24 h, the signals assigned to the
methylene protons of 2,6-bis(hydroxymethyl) phenol
appeared at 4.77 ppm, demonstrating the cleavage of ether
bonds [Fig. 2(b)]. At pH 3.0, the signal at 9.98 ppm corre-
sponding to the aldehyde protons increased obviously, and
the signal at 8.21 ppm disappeared, approving the cleavage
of C5N bonds [Fig. 2(c)].
The GPC traces also confirmed the degradation of oligomers
by the oxidization of H2O2 or at an acidic environment (Sup-
porting Information Fig. S4). As a result, the oligomers
degraded into fragments with different length. The number-
average molecular weight (Mn) of oligomers
4 and 5
decreased to 1900 and 1200 after being incubated in 100
mM H2O2 for 24 h. Moreover, the Mn of both oligomers 4
and 5 decreased to 600 at pH 3.
FIGURE 2 1H NMR spectra of oligomer 5 incubated in a mixed
solvent of buffer (D2O) containing 0.1 M NaHCO3 and CDCl3
(1:5) (a) without H2O2, (b) with 10 mM H2O2, and (c) at pH 3 for
24 h at room temperature. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
To investigate the release of guest species, we used a simple
emulsion method29–32 to prepare the nanoparticles with nile
reds encapsulated inside for its good stability and as an
excellent fluorescence probe in the presence of H2O2.33–35
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oligomer 4 (Supporting Information Fig. S5), suggesting the
superior sensitivity of oligomer 5 to H2O2. The results were
in agreement with the degradation kinetics measured by flu-
orescence spectroscopy. We also tried DLS measurement to
observe how the size of nanoparticles from oligomer 4 and
oligomer 5 changed according to the time of adding H2O2.
However, Rh,app did not show any significant change after the
addition of H2O2 because the particle size distribution is too
broad to give the useful information (Supporting Information
Fig. S6).
Moreover, we noticed that the H2O2-triggered cleavage reac-
tion was speeded up at a weak acidic environment. The fluo-
rescent intensity was 60% in a buffer solution with 10 mM
H2O2 at pH 8.5, it dropped to nearly 40% at pH 5.0, while it
was 85% at pH 5.0 without H2O2. The results demonstrated
that the enhanced degradation and release efficiency were
achieved in a weak acidic medium and low concentration of
H2O2. Such conditions are more close to the physiological
environment.
However, the situation was different at pH 3.0. The intensity
decreased to 40% either at pH 3.0 or at pH 3.0 with 10 mM
H2O2 in the system, which might be due to the rupture of
C5N groups dominating the degradation because of the
strong protonation of imine groups at pH 3.0 as shown in
Figure 5(a,b).
FIGURE 3 The plots of time vs. emission intensity (at 610 nm)
of nile red encapsulated in nanoparticles treated with different
concentrations of H2O2 (a) from oligomer
4 and (b) from
oligomer 5. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
4 and Supporting Information Figure S5, respectively. The
nanoparticles are sphere-like in original [Fig. 4(a)]. The loose
particles with larger size [Fig. 4(b)] were observed when
incubated the particles in a solution containing 100 mM
H2O2 for 24 h, which evidences the disassembly process.
Such phenomenon is in accordance with the report by Almu-
tairi and coworkers who found that the polymeric particles
expanded and ripped when exposed to H2O2.16 Interestingly,
we found that the nanoparticles from oligomer 5 (Fig. 4)
showed a more serious ruptured morphology than that from
FIGURE 5 The plots of time vs. emission intensity of nile red
(at 610 nm) encapsulated in nanoparticles (oligomer 5) at (a)
pH 5 and (b) pH 3. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
FIGURE 4 TEM images of nanoparticles prepared from 5 incu-
bated (a) without H2O2 and (b) with 100 mM H2O2 for 24 h.
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based on azomethine oligomers with boronates and imine
groups in the main chain. The nanoparticles from oligomers
4 and 5 degraded with the trigger of H2O2 via a 1,4-quinone
methide rearrangement. In addition, the nanoparticles could
also break into monomers and release the loaded species
under acidic condition. Compared to oligomer 4, oligomer 5
showed a faster degradation rate in the presence of H2O2,
especially in a weak acidic environment. Such a system capa-
ble of dual response of H2O2 and H1 may have potential
application as a carrier for drug delivery.
ACKNOWLEDGMENTS
This work is financially supported by the National Natural Sci-
ence Foundation of China (21174005 and 21274004) to X.R.J.
The authors thank Dehai Liang for the help in the characteriza-
tion of nanoparticles.
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