Novel multi-sensitive pseudo-poly(amino acid) for effective intracellular drug delivery

Article abstract of DOI:10.1039/c5ra03423j

Novel intracellular pH, glutathione (GSH) and reactive oxygen species (ROS)-responsive nanoparticles were obtained using mPEG_2k-block-redox dual sensitive chain-block-mPEG_2k (PRDSP) which was prepared by Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) click polymerization. The disulfide bond, peroxalate ester and triazole units were regularly and repeatedly arranged in the hydrophobic blocks. The disulfide bond was GSH-sensitive and the peroxalate ester structure could be disrupted by acid and hydrogen peroxide. In addition, the triazole units are capable of forming pH-responsive hydrogen bonds. Dynamic Light Scattering (DLS) and transmission electron microscopy (TEM) were used to investigate the pH, GSH and ROS sensitivity of the PRDSP nanoparticles (NPs). The results indicated that the average diameter, size distribution and morphology greatly changed upon adding GSH/H₂O₂ or modulating the pH. As the preloaded model anticancer drug, doxorubicin (DOX) was quickly released from DOX-loaded PRDSP (PRDSP@DOX) NPs by addition of 10 mM glutathione (GSH), or 10 mM H₂O₂ or under acidic conditions rather than under physiological conditions. Confocal laser scanning microscopy (CLSM) and flow cytometric analyses revealed that PRDSP@DOX could effectively deliver DOX into the cytoplasm and nucleus of cells. Therefore, PRDSP NPs may be a promising redox heterogeneity-sensitive carrier for efficient and controlled anticancer drug release.

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ARTICLE TYPE
Novel Multi-Sensitive Pseudo-poly(amino acid) For Effective
Intracellular Drug Delivery
Yanjuan Wu,a,bDongfang Zhou,aYanxin Qi,aZhigang Xie,aXuesi Chen,aXiabin Jinga and
Yubin Huang*a
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
A novel intracellular pH, glutathione (GSH) and reactive oxygen species (ROS)ꢀresponsive nanoparticles
were obtained using mPEG2kꢀblockꢀredox dual sensitive chainꢀblockꢀmPEG2k (PRDSP) which was
prepared by Cu(I)ꢀcatalyzed azideꢀalkyne cycloaddition (CuAAC) Click polymerization. The disulfide
10 bond, peroxalate ester and triazole units were regularly and repeatedly arranged in the hydrophobic
blocks. The disulfide bond was GSHꢀsensitive and peroxalate ester structure could be disrupted by acid
and hydrogen peroxide. In addition, triazole units are capable of forming pHꢀresponsive hydrogen bonds.
Dynamic Light Scattering (DLS) and Transmission electron microscopy (TEM) was used to investigate
the pH, GSH and ROS sensitivity of PRDSP nanoparticles (NPs).The results indicated that the average
15 diameter, size distribution and morphology greatly changed upon adding GSH /H2O2 or modulating pH.
As the preloaded model anticancer drug, Doxorubicin (DOX) was quickly released from DOXꢀloaded
PRDSP (PRDSP@DOX) NPs by addition of 10 mM Glutathione (GSH), 10 mM H2O2 or under acidic
condition rather than under physiological condition. Confocal laser scanning microscopy(CLSM) and
flow cytometric analyses revealed that PRDSP@DOX could effectively deliver DOX into the cytoplasm
20 and nucleus of cells. Therefore, PRDSP NPs may be a promising redox heterogeneityꢀsensitive carrier for
efficient and controlled anticancer drug release.
45 at tumor site.13,14 In addition, cancer cells may have the
millimolar level of intracellular glutathione (GSH), which is
reported to be severalꢀfold higher than that of plasma GSH.
Zhong’s groups had introduced disulfide bonds into the main
chain, crossꢀlinking unit or side chain of the block copolymer
50 micelles for rapid intracellular release of anticancer drugs.15,16,17
It should be further noted that low or intermediate levels of ROS
are endogenous class of carcinogens, causing DNA damage, cell
mutation and promoting cell proliferation, and ultimately
inducing carcinogenesis.18 The high oxidative stress in the
55 cytosol and cell nuclei of tumor cells have recently been
exploited for active intracellular release of various drugs.19,20,21,22
Despite the considerable development of various stimuliꢀ
sensitive block copolymers, few of the DDSs have achieved
optimal outcomes. The challenges still exist. First, an ideal
60 controllable DDS should be fast but not burst release. In most
reported copolymer systems, stimuliꢀsensitive bonds located
either in the crossꢀlinking unit, or on the side chain, or in the
main chain with limited number (like only one such bond in the
main chain).23,24 Therefore, under the environmental stimuli, the
65 assembly structure of the drugꢀloading system could not be
entirely destroyed, leading to a slow but incomplete drug release.
Drug efficiency is another crucial point. High drug loading
content (DLC) and drug loading efficiency (DLE) are commonly
Introduction
Nanoꢀscaled functional polymeric nanocarriers offer several
25 unique features, such as improving drug bioavailability and
solubility, decreasing side effects, and preferential accumulation
at the tumor sites via the enhanced permeability and retention
(EPR) effect. Therefore, in the past decades, functional polymeric
materials have emerged as a most viable and promising
30 controlled drug delivery system (DDS) for cancer treatment.
Multifarious stimuliꢀresponsive DDSs have been extensively
studied.1,2,3,4,5,6,7 Of these DDSs, the block copolymer micelle is
one of the most important species because of their palpable
advantages, including viable modularization, ordinary synthetic
35 procedures and characterizations, and simple selfꢀassemble
process. Consequently, enormous efforts have been made to the
development of block copolymer micelles in response to internal
stimulus (e.g. pH, reductive potential, glucose, oxidative stress,
and lysosomal enzymes) or external stimulus (e.g. light, magnetic
40 field, and ultrasound).8,9,10,11,12 For example, pH levels in late
endosomes (pH 5.0ꢀ6.0) and lysosomes (pH 5.0) are notablely
lower than normal extracellular pH (7.4). Taking advantages of
slightly acidic environments, enormous pHꢀsensitive block
copolymers have been developed to realize enhanced drug release
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necessary. However, most stimuliꢀresponsive bonds could not
provide additional interaction with the loaded drugs to improve
DLC and DLE levels. The most important challenge is the
purification.
Lꢀcystine
and
(Benzotriazolꢀ1ꢀ
yloxy)tris(dimethylamino)phosphoniumhexafluophosphate(BOP
reagent) were obtained from GL Biochem (Shanghai) Ltd.
heterogeneity of cancer cells.25 For example, different stimuli 60 N,N,N',N'',N''ꢀPentamethyldiethylenetriamine (PMDETA), 4ꢀ
5
may coexist in one cancer cell, but also may exist in different
regions of tumor site. Furthermore, one cancer cell may has
different stimuli levels at different living stages. Thus,
nanocarriers bearing single sensitivity may not sufficiently
dimethylaminopyridine and oxalyl chloride were purchased from
Aladdin (Shanghai) Ltd. Sodium azide (NaN3) and Glutathione
(GSH) were obtained from Energy Chemical (Shanghai) Ltd.
Doxorubicin hydrochloride (DOX•HCl) was purchased from
release the loaded drugs under the complex situation of internal 65 Zhejiang Hisun Pharmaceutical Co. Ltd. Hoechst 33258, amplex
10 stimuli inside tumor cells, leading to unsatisfied therapeutic
efficacy. For these reasons, design of multiꢀresponsive block
copolymers becomes rational and available.
red reagent and 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenylteꢀ
trazolium bromide (MTT) were purchased from SigmaꢀAldrich
Co. Reactive Oxygen Species Assay Kit was purchased from
Beyotime. After being immersed with CaH2 for 2 weeks,
In this work, a novel redox dual sensitive polymer was
designed and synthesized by introducing disulfide bonds and 70 Dichloromethane (DCM) and Triethylamine (TEA) were dried by
15 peroxalate ester units repeatedly into polymer backbone. To our
best knowledge, conventional polymerization methods, such as
ring opening polymerization (ROP), could also be used to
synthesize multiꢀresponsive nanocarrier. However, the stimuliꢀ
sensitive bonds were usually arranged in the copolymer randomly.
20 We noticed that Cu(I)ꢀcatalyzed azideꢀalkyne cycloaddition
(CuAAC) click polymerization was generally used to synthesize
functional polymer due to its ordinary manipulation, gentle
reaction condition, selectivity and orthogonality with various
functional groups.26,27,28,29,30 CuAAC click polymerization is a
25 very convenient tool to import two or more functions into one
system. Plenty of triazole groups formed in polymer backbone
could be served as hydrogen bond acceptors and donors. In our
design, cystine, a natural amino acid with one disulfide group,
was chosen as starting material to synthesis dialkyned monomer
30 first. Then peroxalate ester unit was imported into the diazided
monomer. After CuAAC click polymerization under the catalysis
of CuCl/PMDETA, the prepared hydrophobic block was further
reacted with azideꢀterminated mPEG2k to obtain an ABAꢀtype
triblock copolymer (PRDSP). In this PRDSP copolymer,
35 disulfide, peroxalate ester and triazole units were repeatedly
interposed into polymer backbone without any protection and
deprotection steps. Because of the sensitive characteristics, this
polymer could be degraded by enzyme and disrupted under redox
circumstance. The existence of triazole groups may form
40 hydrogen bond and also involve the electrostatic interaction with
DOX to improve the drug loading efficiency. DOX was loaded
into PRDSP NPs as a model drug. The DOX loading, endocytosis
and intracellular drug release were presented in Scheme 1. The
effective loading of DOX and triggered release in response to
45 different pH values, distinct concentrations of GSH and disparate
H2O2 level were demonstrated. The intracellular pH, GSH, ROS
multiꢀsensitive Pseudoꢀpoly(amino acid) NPs may be potential
candidates as platforms for intracellular drug delivery.
refluxing with CaH2and then distilled. Dimethylformamide
(DMF) was dried by stirring with CaH2 and then distilled under
vacuum.
Measurement
75 1H NMR spectra were recorded using a Bruker AVANCE DRX
400 spectrometer in CDCl3, D2O or DMSOꢀd6 at 25℃. Gel
permeation chromatography (GPC) was measured in DMF using
a series of liner Tskgel Super columns (AW3000 and AW5000)
and Waters 515 HPLC pump with OPTILAB DSP
80 Interferometric Refractometer (Wyatt Technology) as the detector.
DMF containing 0.01 M LiBr was used as eluent with a flow rate
of 1.0 mL/min at 50℃. Transmission electron microscopy (TEM)
images were performed on a JEOL JEMꢀ1011 transmission
electron microscope. Dynamic light scattering (DLS)
85 measurements were tested on Brookhaven 90Plus size analyzer.
UVꢀVis spectroscopy (UVꢀ2450PC, Shimadzu) was used to
measure the amount of DOX. Zeta potential measurements were
conducted on a Malvern Zetasizer Nano ZS.
90 Synthesis of dialkyned-cystine derivative:
2.2’-Dithiobis[1-(prop-2-ynylcarbamoyl)-ethyl-
carbamicacidtert-butyl ester] (monomer1)
The dialkynedꢀcystine derivative was synthesized according to a
prior published literature with some modification.31 A stirred
95 solution of BOCꢀcystine (3.960 g, 9 mmol) and TEA (7 mL, 50.4
mmol) in 80 mL anhydrous DCM was chilled in an iceꢀbath, and
propargylamine (2.5 mL, 36 mmol) was added portionwisely.
Afterwards the mixture was kept at 0℃ upon addition of BOP
reagent (9.558 g, 21.6 mmol) in 20 mL of DCM under argon for
100 0.5 h. The ice bath was removed after 2 h and the reaction was
maintained at room temperature for 24 h. Dichloromethane was
removed by reduced pressure distillation. The crude product was
dissolved in 350 mL ethyl acetate, and the mixture was washed
with saturated potassium hydrogensulphate (KHSO4), saturated
105 sodium dicarbonate (NaHCO3) solution and saturated sodium
chloride (NaCl). After drying over magnesium sulphate (MgSO4),
the solvent was removed by evaporation. Purification was
achieved by column chromatography. Yield: 74.7%.1H NMR
(CDCl3, 400 MHz) δ1.48 (s, 18H), 2.18 (s, 2H), 2.94(m, 4H),
110 4.08 (m, 4H), 4.92(br s, 2H), 5.53 (d, 2H), 8.07 (br s, 2H).
Experimental section
50 Materials
Cuprous chloride (CuCl) was purified following the prior
experimental manipulation. N,NꢀDi(tertꢀbutoxycarbonyl)ꢀLꢀ
cystine (BOCꢀcystine) was synthesized according to traditional
procedure. Poly(ethylene glycol) methyl ether with an Mn of ca.
55 2000, 3ꢀChloroꢀ1ꢀpropanol, mesyl chloride and proparygl amine
were purchased from SigmaꢀAldrich and used without further
Synthesis of bis(3-azidopropyl) oxalate (monomer2).
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Bis(3ꢀazidopropyl) oxalate was synthesized according to the
abundant water to obtain the product (hydrophobic block). In
another Schlenk tube, the hydrophobic block (0.2 g) was reacted
with mPEG2kꢀN3 (0.500 g, 0.25 mmol) under the catalysis of
60 CuCl (0.050 g, 0.5 mmol), and PMDETA (0.173 g, 1 mmol) was
used as chelating agents. The reaction was proceeded at 40 ℃for
24 h. The products were purified by dialysis against DMF,
ethylenediaminetetraacetic acid disodium salt (EDTA·Na)
solution and pure water to remove the catalyst and unreacted
65 mPEG2kꢀN3. After lyophilization, the final product (PRDSP) was
obtained.
Scheme 1.
following steps. First, 3ꢀChloroꢀ1ꢀpropanol (9.454 g, 100 mmol),
sodium azide (19.503 g, 300 mmol) and sodium hydroxide (1.000
g, 25 mmol) were added into 80 mL distilled water in a
Schlenkflask. After stirring at 80 ℃for 48 h, the product was
extracted with diethyl ether. The combined organic extracts were
washed with saturated sodium chloride (NaCl), and dried over
MgSO4. After volatiles evaporation, the colorless oil (3ꢀazidoꢀ1ꢀ
5
10 propanol) was obtained. Yield: 90%. 1H NMR (CDCl3, 400MHz):
δ(ppm) = 3.75 (t, 2H,), 3.45 (t, 2H,), 1.84 (m, 2H), 1.91 (s, 1H).
Second, bis(3ꢀazidopropyl) oxalate was prepared according to
the literature with minor modification.32,33 A solution of oxalyl
chloride (1.5 mL, 18 mmol) in 70 mL of DCM was added
15 dropwisely to a well stirred iceꢀcold solution of 3ꢀazidoꢀ1ꢀ
propanol (3.030 g, 30 mmol), TEA (12.5 mL, 90 mmol) and
DMAP (3.665 g, 3 mmol) in DCM (60 mL). The reaction was
stirred vigorously at room temperature for 24 h under argon
atmosphere. After filtration, the filtrate was washed with brine for
20 five times and dried over MgSO4. The obtained oil was purified
by column chromatography. Yield: 73%. 1H NMR (CDCl3,
400MHz): δ(ppm) = 4.4 (t, 2H,), 3.46 (t, 2H,), 2.02 (m, 2H).
Scheme 2.
Determination of critical aggregation concentration (CAC) of
PRDSP NPs
70 Critical aggregation concentration (CAC) of PRDSP NPs in
deionized water was determined using pyrene as probe. PRDSP
NPs were prepared with various concentrations (6.1 ×10−5～1.25
× 10−1 mg/mL) in deionized water, and the final concentration of
pyrene was immobilized at 6.0 × 10−7 mol/L. The fluorescence
75 spectra were monitored using a fluorescence spectrophotometer.
The excitation spectra of PRDSP@pyrene solutions were scanned
from 308 to 350 nm at room temperature. The intensity ratios of
I337.5 to I333 were plotted against the logarithm of PRDSP
concentrations.
Synthesis of Azido-mPEG2k ( mPEG2k-N3).
mPEG2kꢀN3 was synthesized as reported procedure with minor
25 modification.34 First, in a 250 mL dried Schlenk flask, TEA (13.9
mL, 100 mmol) was syringed into a solution of mPEG2k (10 g, 5
mmol) in 90 mL of DCM. Then the mixture was cooled to 0 ℃in
an ice bath before a DCM solution (90 mL) of mesyl chloride
(3.9 mL, 50 mmol) was added dropwisely. After reaction at room
30 temperature for 24 h, the solution was extracted with 20 mL of
saturated brine for fourth. After drying the solution with MgSO4,
the concentrated solution was precipitated in diethyl ether and
dried in vacuum for 24 h.
Second, the obtained methylsufonylꢀmPEG2k (6.000 g, 3 mmol)
35 and NaN3 (9.751 g, 150 mmol) were dissolved in 30 mL dried
DMF. The reaction mixture was degassed by three freezeꢀpumpꢀ
thaw cycles and the reaction proceeded at 80 °C for 24 h. After
that, the reactive mixture was cooled to room temperature, and
the excess amount of sodium azide and the side product (sodium
40 bromide) were filtered off. After reduced pressure distillation, the
mixture was dissolved in 300 mL of DCM, washed with saturated
NaCl solution and dried over MgSO4. The organic phase was
concentrated, precipitated in 250 mL of diethyl ether and the
product mPEG2kꢀN3 was dried in vacuum for 24 h. Yield:71%. 1H
45 NMR (CDCl3, 400MHz): δ(ppm) = 3.64 (t, 181H,), 3.38 (t, 4H,).
80 Preparation of the PRDSP and PRDSP@DOX NPs
Blank NPs were prepared by a modified nanoꢀprecipitation
method. PRDSP (10 mg) was dissolved in 2 mL of DMF and
stirred for 2 h. Deionized water (15 mL) was then added
lentamente. The mixture was dialyzed against deionized water for
85 24 h at room temperature by using a dialysis membrane bag
(MWCO 3500 Da).
DOX was loaded into PRDSP copolymer nanocarriers as a
model drug by a dialysis method. Briefly, DOX·HCl (10 mg,
0.017 mmol) was stirred with TEA (7 ꢁL, 0.051 mmol) in
90 dimethyl sulfoxide (0.6 mL) for 2 h in the dark. The PRDSP
copolymer (50 mg) was dissolved in DMF (2 mL). The mixture
was stirred at room temperature overnight and then added
dropwisely into 40 mL of deionized water. The solution was
transferred into the dialysis bag (MWCO 3500) and dialyzed
95 against deionized water for 24 h (the medium was changed every
3 h). The obtained DOXꢀloaded micelles (PRDSP@DOX NPs)
were lyophilized in the dark.
Characterization of the PRDSP and PRDSP@DOX NPs
DLS was used to investigate size and size distribution of the NPs
100 aggregates, and TEM was used to determine the morphological
and size of the NPs aggregates. The TEM sample was prepared as
follow: a drop of sample solution (0.5 mg/mL) was deposited
onto a copper grid coated with carbon, and the sample was
maintained at room temperature to volatilize the solvent.
Synthesis of polymer-RPDSP
Polycycloaddition reactions of the dialkyned monomer1 with
diazided monomer2 were carried out under argon in Schlenk tube.
In general, dialkyned monomer1 (0.514 g, 1 mmol), diazided
50 monomer2 (0.256 g, 1 mmol), PMDETA (0.069 g, 0.4 mmol) and
CuCl (0.020 g, 0.2 mmol) were reacted in 10 mL of dried DMF at
40℃ for 24 h after a deoxidation process. At the end of the
polymerization, the degassed solution of dialkyned monomer1
(0.103 g, 0.2 mmol) was added to the Schlenk tube to achieve
55 dialkyne end groups on polymer. After precipitated in cold
diethyl ether and filtered, the precipitation was washed with
105
Table 1.
PH and redox-triggered destabilization of PRDSP NPs
The size change of PRDSP NPs in response to pH, reduction
agent (GSH), ROS (H2O2) was followed by DLS and TEM
measurements.
110
For pH sensitivity, 20 mg of freeze dried NPs were dispersed
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Reduction of cell ROS
in 40mL of water solution (pH = 7.4 or 5.0). At predesigned time
point, the change of PRDSP NP sizes was monitored by DLS. To
determine the reductionꢀtriggered destabilization of PRDSP NPs,
0, 10 µM, 100 µM or 10 mM GSH were appended to 5 mL
solution of PRDSP NPs in MilliꢀQ (MQ). The solution was
placed in a constant temperature shaker at 37 ℃ with a rotation
speed of 100 rpm. To determine the oxidation sensitivity,
PRDSPNPs were dispersed in MQ at specific concentrations (0.5
mg/mL), then certain amounts of H2O2 were added to make the
10 final H2O2 concentrations to be 0, 0.1 and 10 mM, respectively.
Eventually, to mimic blood and tumor circumstance, GSH were
added to 5 mL of PRDSP NPs solutions in PBS (pH 7.4 or 5.0) to
make the final GSH concentration to be 10 µM or 10 mM. The
alteration of NP sizes was monitored in time by DLS, and the
15 change of morphology under different conditions were observed
using TEM.
The intracellular ROS production was measured by
dichlorofluoresceinꢀdiacetate (DCFHꢀDA) staining. HeLa cells (5
×105 cells/well) were seeded in a 6ꢀwell plates and incubated for
60 24 h. Cells were disposed with 20 ꢁg or 200 ꢁg of PRDSP NPs
for 12 h and then treated with 5 ꢁL of rosup (1 mg/mL) for 10 h.
Cells with only rosup treatment were used as the positive control,
and cells with no treatment were used as the negative control. 20
ꢁM of DCFHꢀDA was added to each dish, and 1 h later, the
65 fluorescence images were observed by confocal laser scanning
microscope (CLSM). Flow cytometry was also performed to
quantify the fluorescent cells.
5
Cell Viability Assays
In vitro cytotoxicity of PRDSP NPs against L929 and HeLa were
70 assessed by methyltetrazolium (MTT) assay. Briefly, L929 cells
were seeded onto 96ꢀwell microtiter plates at a density of 1×104
cells per well in 100 ꢁL of DMEM and incubated at 37 ℃ and
5% CO2 for 24h. Then the culture medium was withdrawn. 100
ꢁL of PRDSP solutions with different concentrations
75 (0.0312−1mg/mL) in complete DMEM were added. After being
incubated for another 24 h in cell incubator, cells were subjected
to MTT assay. The absorbance at 490 nm was measured (Bioꢀ
Rad 808 microplate reader) with control wells containing cell
culture medium only. The relative cell viability was determined
80 by comparing the absorbance at 490 nm. At the same time, cells
were placed in 96ꢀwell plates (5 × 103 cells/well) in 100 ꢁL of
complete DMEM and incubated for 24 h to adhere, 100 ꢁL of the
PRDSP NPs at various concentrations were applied to replace the
culture medium. After incubation for 48 h, MTT assay was used
85 to evaluate cell viability.
The scavenging of H2O2
3 mg of PRDSP NPs or PRDSP@DOX NPs were suspended in 2
mL of H2O2 solutions (10 µM), and the solution was placed in a
20 constant temperature shaker at 37℃ under gentle shaking. At
appropriate time intervals, the solution was centrifuged (10000
r/min) and the H2O2 concentration of the supernatant was
determined by Amplex Red assay according to the
manufacturer’s protocol.
25 In vitro drug release
To determine the DLC and DLE, DOXꢀloaded polymer micelles
were dissolved in DMF. The amount of DOX was quantified via
fluorescence measurement according to a calibration curve. DLC
and DLE of PRDSP NPs were calculated by the following
30 equations:
The cytotoxicity of PRDSP@DOX NPs against HeLa and
A549 cells were also determined by using a standard MTT assay.
Briefly, four density (10×103, 7×103, 4×103, 2×103 cells per well)
of HeLa cells were seeded in four 96ꢀwells plates and incubated
90 for 24 h. After the culture medium was discarded, cells were
treated with 0 or 10 mM GSH for 2 h, and then rinsed with fresh
DMEM. Free DOX and PRDSP@DOX at various concentrations
were added. The MTT assay was carried out after incubation for
24, 48, 72 and 96 h, respectively. A549 cells were also used to
95 evaluate the cytotoxicity of PRDSP@DOX NPs.
DLC (wt%)= (weight of DOX in nanoparticles/total weight of
DOXꢀloaded nannoparticles) ×100%
DLE (wt%) = (weight of DOX in nanoparticles/total weight of
DOX in feed) ×100%
35
In vitro release profiles of DOX from PRDSP@DOX NPs
were investigated in distinct buffer solutions (pH 7.4 phosphate
buffer, pH 5.0 acetate buffer, pH 7.4 phosphate buffer with 10µM
of GSH, pH 7.4 phosphate buffer with 10 mM of GSH, pH 5.0
acetate buffer with 10 mM of GSH, MQ with 0, 100 ꢁM or 10
40 mM of H2O2). The preꢀweighed PRDSP@DOX NPs were
suspended in 5 mL of corresponding buffer solution. The solution
was subsequently transferred into a dialysis membrane bag
(MWCO 3500 Da), and the dialysis bag was immersed into 50
mL of the homologous buffer solution. At predetermined time
45 intervals, 2 mL of release medium was withdrawn for UVꢀvis
analysis and then equal volume of fresh buffer was added. The
concentration of released DOX was quantified using fluorescence
measurement (λex= 480 nm) base on the standard curve.
Intracellular Drug Release
CLSM and flow cytometric analyses were applied to monitor the
cellular uptake and intracellular distribution of DOX and
PRDSP@DOX NPs towards HeLa and A549 cells. For example,
100 HeLa cells were seeded on the coverslips in 6ꢀwell plates (3 × 105
cells/well) and cultured in 2 mL of DMEM for 24 h, and then
treated with GSH (10 mM) for 2 h. After washed by PBS, 2 mL
of DOX or PRDSP@DOX NPs (at a final DOX concentration of
10 ꢁg·mLꢀ1) in DMEM were added, and cells were incubated for
105 additional 0.5 or 4 h. Cells without GSH treatment were used as
the control. In this study, the nuclei were stained by Hoechst
33258 (10 ꢁg/well) and transferred to a blue color by software,
accordingly, DOX was transferred to a red color. The images of
cells were detected using a CLSM.
Cell lines
50 Three cell lines, L929 (mouse fibroblasts cells), A549 (nonꢀsmall
lung carcinoma cells) and HeLa (human cervical carcinoma cells)
were selected for cell tests. L929, A549 and HeLa, which were
offered by the Medical Department of Jilin University, China,
were incubated in Dulbecco's modified Eagle's medium (DMEM,
55 Gibco).
110
For flow cytometric analyses, the experimental process was
similar to the preparation of CLSM samples with little
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modification. HeLa cells were placed into 6ꢀwell plates (3 × 105
Figure 1.
cells/well) with no coverslip. After cultured for 24 h to adhere,
preprocessed with 10 mM of GSH, and treated with 2 mL of
DOX or PRDSP@DOX NPs (at a final DOX concentration of 10
ꢁg·mLꢀ1), cells were washed with PBS three times and
trypsinized. Then, 1.0 mL of PBS was added, and the suspensions
were centrifuged for 5 min at 1000 rpm and cells were reꢀ
suspended in 0.5 mL of PBS. The analysis was performed by
flow cytometer. CLSM and flow cytometric analyses were also
CAC measurement
60 To demonstrate the formation of micelles, pyrene was used as a
hydrophobic fluorescence probe to investigate the critical
aggregate concentration (CAC) value (Fig. S5). CAC value of
PRDSP was calculated to be 1.64 mg/L, which was obtained by
poltting I337.5/I333 against the logarithm of PRDSP concentrations.
65 The low CAC value illuminated good stability of the aggregates
under highly diluted conditions, which was highly advantageous
to intravenous administration.
5
10 applied to investigate the cellular uptake and intracellular
distribution of DOX and PRDSP@DOX NPs towards A549 cells.
Aggregates formation and characterization
Results and discussion
Amphiphilic polymers are capable of selfꢀassembly into
70 aggregates in specific solvents. In this study, micelles of PRDSP
block copolymers were prepared by the modified
nanoprecipitation method. After completely dissolving the
copolymer PRDSP in DMF, the deionized water was added
lentamente. Then the mixture was dialyzed against the deionized
75 water for 1 day to form the aggregate. DOX was used as a model
anticancer drug for encapsulation. The aggregates’ size, size
distribution and the morphology were determined by DLS（Fig.
2A and Fig. S6A） and TEM (Fig. 2B and Fig. S6B). For blank
PRDSP NPs, DLS measurements showed that PRDSP formed
80 aggregates with size of 97 nm. In TEM micrographs, PRDSP NPs
had clear spherical morphology with the average diameter around
70 nm. This phenomenon is ubiquitous and should ascribe to the
shrinkage of the PEG shell during the sample preparation for
TEM. After DOX encapsulation, the average size increased and
85 the size distribution became broad. This phenomenon was
common and also reported by other literatures.35
Preparation and characterization of “clickable” monomers
and PRDSP triblock copolymer
15 In this work, dialkynedꢀcystine, diazide monomer and azideꢀ
terminated mPEG2k were applied to synthesis the linear redox
dualꢀsensitive triblock copolymer PRDSP （Scheme 2 ）by
CuAAC step polymerization with catalysis of CuCl.
Cystine was chosen as a raw material with amino groups
20 protected by BOC to introduce the disulfide groups into polymer
backbone. To avoid the racemization of Lꢀcystine, BOP was
selected as condensation agent instead of DCC or EDC. The
chemical structure of dialkynedꢀcystine was confirmed by 1H
NMR (Fig. S1A). The signal corresponding to the alkynl group
25 (2.18 ppm) could be clearly observed. The resonances at 4.08,
1.48 and 2.94 ppm were assigned to protons on CH2 (ꢀSꢀCH2ꢀ), ꢀ
BOC and CH2 (ꢀNHꢀCH2) moiety, correspondingly. Fig.S1C
shows the 1H NMR spectrum of the diazide monomer. All
chemical shifts were assigned to the protons of diazide monomer,
30 respectively. Fourier transform infrared spectroscopy (FTꢀIR)
analysis of monomers was also performed (Figure S2). The
appearance of azide peak at 2103 cm−1 further suggested the
successful synthesis of azideꢀterminated monomers.
Figure 2.
Stability of PRDSP NPs
It has been confirmed that cancer tissue has idiosyncratic
90 microenvironments including reduced extracellar and intracellar
pH, increased temperature, elevated reductive agent level and
oxidative stress. In this work, triazole, disulfide bond and oxalate
structures were regularly and repeatedly positioned in polymer
backbone, which could provide DDS with potential pH, reductive
95 and oxidative sensitivities to release drugs around or inside tumor
cells as much as possible. For this purpose, we investigated the
triggered disassemble behavior of PRDSP NPs in response to
different pH and concentration of GSH or H2O2.
For CuAAC step polymerization, CuCl/PMDETA was used as
35 the catalyst with minor excessive dialkyned monomer to produce
alkyne groups on both ends of hydrophobic chain. Due to the
hydrophobic property of the synthetic segment, abundant water
was applied to wash the polymer to eliminate the catalyst. The
acquired hydrophobic block was then reacted with azideꢀ
40 terminated mPEG2k to obtain the final amphiphilic triblock
copolymer. For hydrophobic block, chemical shifts at 1.37 and
1
2.22 ppm in H NMR were ascribed to the protons of BOC and
CH2 (adjacent to ꢀCH2ꢀC=O), respectively (Fig. S3). And the
proton signals of the triazole ring at 8.0 and 8.44 ppm were
Figure 3.
1
45 explicitly observed. The H NMR of PRDSP polymer also had
100
The size change of PRDSP NPs in distinct condition as a
function of time was followed by DLS measurement (Fig. 3).
Infinitesimal variety was found for PRDSP NPs in the absence of
GSH or in 10 ꢁM of GSH. Particle size had enormous change
while 100 ꢁM GSH was added. Notably, when GSH was
105 increased to 10 mM, fast aggregation was observed for PRDSP
NPs. The size increased from 135 nm to 328 nm in 10 h, and
reached to over 2900 nm after 14 h, showing significant reductive
sensitivity (Fig. 3A). In the presence of 100 ꢁM or 10 mM H2O2,
PRDSP NPS also showed good oxidative responsiveness with
110 immediately increased particle size (Fig. 3B). PRDSP NPs were
quite stable with no size change at pH 7.4 with 10 ꢁM of GSH to
the above chemical shifts with additional resonances at 3.52 ppm
assigning to CH2 (ꢀCH2ꢀCH2ꢀOꢀ) groups of mPEG2k (Fig.1). The
degree of polymerization (PD) was determined by calculating the
relative peak area ratio of CH2 ascribed to PEG to BOC groups
50 (Fig.S3B). PD was about 7.5, which corresponds to an average
molecular weight of 9775 g/mol. The 1H NMR spectra of PRDSP
in D2O was also measured (Fig. 1), and the resonances at 3.62
ppm were assigned to CH2(ꢀCH2ꢀCH2ꢀOꢀ) groups of mPEG2k.
However, other signals on hydrophobic block were not clearly
55 observed, implying the coreꢀshell structure of PRDSP NPs in
water. GPC was also used to investigate the Numberꢀaverage
Molecular Weight (Mn) (Fig. S4).
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mimic the extracellular circumstance (Fig. 3C). When pH was
decreased to 5.0, PRDSP NPs were found unstable with
continuously increased particle size (Fig. 3C). The abundant
A significant difference was observed for the DOX release profile
at pH 7.4 and 5.0. This phenomenon could attribute to two
factors. First, DOX had higher solubility in acidic media. Second,
triazole groups in polymer backbone would have stronger 60 there
hydrogenꢀbondꢀaccepting/ꢀdonating abilities compared with
amide. These hydrogen bonds might be more sensitive and easier
5
Figure 6.
are one primary amine, three carbonyl groups and five hydroxyl
groups in one DOX molecule. These groups would have the
possibility to form hydrogen bonds with triazole and amide
65 groups on PRDSP, which are easier to be destroyed under acidic
condition. About 49% of DOX was released from PRDSP@DOX
NPs at pH 5.0 in 75 h. While in the case of pH 7.4, the released
DOX was only 15%.
PH 7.4 and 10 ꢁM GSH were used again to mimic the
70 physiological condition. Less than 15 % of DOX was released in
75 h, suggesting the satisfying stability of PRDSP@DOX during
blood circulation. Increasing of GSH concentration (10 mM, pH
7.4) would definitely accelerate the release speed. In the presence
of 10 mM of GSH at pH 5.0 to mimic the intracellular
75 environment, DOX release had the fastest speed. More than 82%
of DOX was released in 75 h, indicating the excellent reductive
sensitivity of PRDSP@DOX NPs triggered by GSH. H2O2ꢀ
triggered DOX release from PRDSP@DOX NPs was monitored
in the absence or presence of H2O2 in MQ (Fig. 6B). The results
80 showed that PRDSP@DOX released DOX more rapidly in H2O2.
In 75 h, about 44% and 20 % of DOX were released in the
presence of 10 mM and 100 ꢁM of H2O2. The above results
indicated that drug release from PRDSP@DOX could be
controlled under the acidic and redox conditions. The well
destroyed under lower pH, leading to instability of the NPs. In the
case of treating with 10 mM of GSH at pH 5.0 to mimic the
intracellular environment, significant size diversification (Fig.
10 3C) and broader size distribution (Fig. S7) of PRDSP NPs were
confirmed.
It is assumed that when the disulfide bonds or oxalate
structures in PRDSP backbone begun to be disrupted by the
addition of GSH or H2O2, the stability of PRDSP NPs was
15 destroyed, inducing the reꢀassembling or aggregation of NPs.
While most disulfide bonds or oxalate structures in the main
chain were consequently broken, the entire NPs structure could
be dissociated, leading to smaller particle size. The diversification
of the morphology and size of PRDSP NPs under different
20 conditions was also monitored by TEM (Fig. 4). Similarly,
PRDSPNPs were stable at pH 7.4 with little alteration in size,
size distribution and morphology, but greatly changed with
lowered pH and increased concentration of GSH or H2O2.
Figure 4.
25
Another evidence for oxidative sensitivity of PRDSP NPs is
H2O2 scavenging test by Amplex Red assay. Both blank PRDSP
NPs and PRDSP@DOX could significantly reduce the
concentration of H2O2 to 49 and 65 % of its original level at 36 h,
respectively (Fig. 5). H2O2 is one of the most important oxidative 85 displayed multiꢀsensitivities endow this system with excellent
30 agents in cancer cell. It has been proved that peroxalate ester
bond can be oxidized by H2O2 instantaneously and
spontaneously, and eventually decomposed into CO2.36,37 The
repeatedly located peroxalate ester bonds in PRDSP would
therefore react with H2O2to induce the cleavage of polymer
35 backbone, and consequently reduce the H2O2ꢀmediatedoxidative
stress. All these results clearly demonstrated the redox dualꢀ
sensitive characteristics of the PRDSP NPs.
characteristics for smart intracellular drug delivery in tumor cells.
Inhibition of intracellular ROS
The impactful treatment of inflammation and other ROSꢀrelated
diseases, such as malignant tumor, requires effective suppression
90 or elimination of ROS production. In order to estimate the
potential of PRDSP NPs as ROS scavenger, we investigated their
ability to suppress ROS generationin HeLa cells stimulated by
Rosup as an endotoxin. DCFHꢀDA was used as ROS probe to test
the level of ROS generated in cells. DCFHꢀDA is nonꢀfluorescent
95 until it can be oxidized by the intracellular ROS. The production
of ROS in cells was determined by CLSM and flow cytometry.
After Rosup treatment for 12 h, a tremendous amount of ROS
was produced in cells, which was evidenced by the stronger
DCFH fluorescence in CLSM images (Fig. 7). 20 ꢁg of PRDSP
100 NPs showed slightly inhibitory effects on ROS generation.
However, when 200 ꢁg of PRDSP NPs was added, the
remarkably reduced red fluorescence in cells was observed,
suggesting the reliable potent of PRDSP as ROS scavenger. Flow
cytometry results (Fig. S8) were consistent with CLSM results.
Figure 5.
In vitro GSH, H2O2 triggered DOX release
40
DOX is one of the most potent and widely used anthracycline
anticancer drugs to treat diverse types of solid malignant tumors
by interacting with DNA or RNA to inhibit the macromolecular
biosynthesis. In the current study, to confirm the feasibility of
using the redox dualꢀsensitive PRDSP NPs for intracellular drug
45 delivery, DOX was loaded into the PRDSP NPs as a model drug.
DOXꢀloaded PRDSP NPs were prepared by dialysis method. The
theoretical DOX loading content was set at 20 wt%. The results
showed that DLC and DLE for PRDSP NPs were approximately
16% and 80%, respectively.
105
Figure 7.
50
The pH, GSH, H2O2ꢀdependent DOX release behaviors of the
PRDSP@DOX were investigated by dialysis method under
physiological condition (pH 7.4) and endosomal circumstance
(pH 5.0) at 37℃ (Fig. 6). It has to be noted that it is difficult to
introduce reductive and oxidative agents together in one system.
In vitro cellular proliferation assay
The cytotoxicity of the blank PRDSPNPs was evaluated using
L929 cells and HeLa cells (Fig.8). The viabilities of L929 and
HeLa cells treated with PRDSP NPs for 24 h and 48 h exceeded
110 80% at all test concentrations up to 1 mg·mLꢀ1, showing
55 Therefore, the drug release behaviors of PRDSP@DOX under
reductive and oxidative stimulus have to be evaluated separately.
negligible cytotoxicity and favourable compatibility and safety of
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PRDSP NPs to cells.
cells (cultured for 0.5 h).Several researchers have reported that,
compared with the DOX in NPs at the same concentration, the
free DOX have stronger fluorescence due to the selfꢀquenching
60 effect of DOX.38 The intracellular DOX release from
PRDSP@DOX towards A549 cells was also investigated by
CLSM (Fig. S11). The results also showed that the
PRDSP@DOX NPs could be internalized by A549 cells. All the
results indicated that the synthetic copolymer PRDSP was a
65 biologically responsive anticancer drug carrier and could realize
controlled drug release.
Figure 8.
In vitro cellular proliferation inhibitions of free DOX,
PRDSP@DOX against HeLa and A549 cells (Fig. 9 and Fig.
S10) were also evaluated. To confirm the intracellular
concentration of GSH uniformly, cells were first pretreated with
10 mM of GSH for 2 h and then incubated with free DOX or
PRDP@DOX NPs (gradient concentrations from 10 to 0.156
ꢁg/mL) for predetermined time. Cells with no pretreatment were
5
10 used as control. In all groups, higher drug dose would lead to
increased cytotoxicity. Unlike the free diffusion of free DOX into
cell, DOX released from PRDSP@DOX is a time consuming
procedure. PRDSP@DOX thus exhibited slightly lower cell
inhibition efficiency than free DOX. The IC50 values
15 (halfmaximal (50%) inhibitoryconcentration) of different samples
with different cultural time were listed in Table 2. The results
showed that compared with nonꢀpretreated groups,
PRDSP@DOX NPs in the presence of GSH exhibited higher
inhibition efficacy with significantly decreased IC50 values. While
20 in control groups, the pretreatment of GSH had no impact on the
cytotoxicity of HeLa/A549cells cultured with free DOX. The
results demonstrated that faster DOX release from
PRDSP@DOXNPs could be well triggered by the higher
intracellular reduction agent level.
Conclusion
A novel biologically responsive triblock copolymer PRDSP was
fabricated as a biocompatible and biodegradable drug delivery
70 carrier. The amphiphilic copolymer PRDSP was synthesized via
CuAAC click polymerization, and disulfide bond, peroxalate
ester and triazole units were repeatedly positioned in the mainꢀ
chain of hydrophobic blocks. DLS, TEM, GPC results clearly
showed responsiveness of PRDSPNPs under acidic or redox
75 conditions. DOX was loaded into the PRDSP NPs as a model
drug, and could be responsively released in the presence of
biologically relevant concentrations of GSH and H2O2. Lower pH
would also accelerate the DOX release speed. In vitro cellular
experiment confirmed that PRDSP NPs was ROSꢀsensitive.
80 MTT, CLSM and flow cytometry results indicated that the
internalization and the anticancer efficacy of PRDSP@DOX NPs
were enhanced in the presence of 10 mM GSH. The investigation
indicated that the PRDSP NPs were potent drug delivery systems
with heterogeneityꢀsensitivity for cancer therapy.
25
Figure 9.
Table 2.
Intracellular DOX release
To determine whether PRDSP NPs were effective to deliver
DOX into cells, the cellular internalization of DOX and
30 PRDSP@DOX NPs towards HeLa cells were monitored by
CLSM and flow cytometry. HeLa cells were incubated with DOX
or PRDSP@DOX in the absence or presence 10 mM of GSH for
the designed time, and detected using CLSM (Fig. 10). As
85 Acknowledgments
The authors would like to thank the financial support from
National Natural Science Foundation of China (No.
51321062and 21174143), the Ministry of Science and
Technology of China (863 Project, No. SS2012AA020603), and
90 “100 Talents Program” of the Chinese Academy of Sciences
(No.KGCX2ꢀYWꢀ802).
Figure 10.
35 expected, stronger red fluorescence in cells was clearly observed
with increased incubation time. After 0.5 or 4 h incubation with
free DOX, the DOX fluorescence was observed to be aggregated
in the nuclei in both samples. There was no difference between
nonꢀpretreated and GSH pretreated groups. In the case of
40 PRDSP@DOX groups, stronger intracellular DOX fluorescence
in GSH pretreated cells was observed, in comparison with nonꢀ
pretreated group. After incubation with PRDSP@DOX for 0.5 h,
DOX fluorescence was found to be aggregated basically around
the nuclei. With increasing culture time to 4 h, DOX fluorescence
45 mostly emerged in the nuclei. These data suggested that PRDSP
NPs was potent to deliver DOX into cells. For further
confirmation, cellular uptake of DOX and PRDSP@DOX NPs
into HeLa cells were determined by flow cytometry (Fig. S9), and
accordant results were acquired. As shown in Fig. S9, for free
50 DOX, there was no fluorescence intensity distinction between
nonꢀpretreated and 10 mM GSH pretreated group. And for
PRDSP@DOX NPs, the fluorescence intensity decreased in the
following order: PRDSP@DOX in 10 mM GSH pretreated cells
(cultured for 4 h)>PRDSP@DOX in GSH nonꢀpretreated cells
55 (cultured for 4 h)> PRDSP@DOX in 10 mM GSH pretreated
cells (cultured for 0.5 h)>PRDSP@DOX in GSH nonꢀpretreated
Notes and references
aState Key Laboratory of Polymer Physics and Chemistry, Changchun
Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun
95 130022, P. R. China E-mail: ybhuang@ciac.jl.cn; Fax: +86 431
85262769; Tel: +86 431 85262769
bGraduate University of the Chinese Academy of Sciences, Beijing
100039, P. R. China
† Electronic Supplementary Information (ESI) available: 1H NMR
100 spectra, FTꢀTR spectra, GPC spectra, a representative CAC plot of
PRDSP, DLS and TEM images, other in vitro cellular data. See
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Schematic illustration of DOX loading, endocytosis and intracellular
microenvironment triggered release from PRDSP@DOX NPs.

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Novel Redox Heterogeneity-Sensitive Pseudo-poly(aminoacid)
For Effective Intracellular Drug Delivery
Scheme 1. Schematic illustration of DOX loading, endocytosis and intracellular
microenvironment triggered release from PRDSP@DOX NPs.

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Scheme 2. Synthetic pathway for clickable monomers (A), ABAꢀtype triblock
copolymer PRSDP (B). Reagents and conditions:(a) proparygl amine, BOP, TEA,
CH2Cl2, 0℃ for 2 h, room temperature (rt) for 24 h; (b) NaN3, NaOH, H2O, 80℃ for
48 h; (c) oxalylchloride, TEA, DMAP, CH2Cl2, 0℃ to rt for 24 h; (d) mesyl chloride,
TEA, DMAP, CH2Cl2, 0℃ to rt for 24 h; (e) NaN3, DMF, 80℃ for 24 h.
Figure 1. 1H NMR characterization of synthetic copolymer PRDSP in DMSOꢀD6 and
D2O.

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Table 1. Information about the synthetic copolymer and DOXꢀloaded NPs.
Entry copolymer
Mna
Mnb
CACc
(10ꢀ3
Diametersd DLCe DLE
(g·molꢀ1) (g·molꢀ1)
1H NMR
(nm)
(wt%) (wt%)
mg·mLꢀ1)
GPC
1
PRDSP
9775
22750
1.64
97
16 80
aCalculated by 1H NMR with DMSOꢀd6 as the solvent;
bCalculated by GPC with DMF as the eluent ;
cDetacted by luminescence spectrometer using pyrene as probe;
dDetermined by DLS;
eDetermined by UVꢀvis spectroscopy with assistance of calibration curve.
Figure 2. (A)Average diameters of blank PRDSP NPs monitored by DLS, and
(B)TEM micrograph of blank PRDSP NPs (scale bars: 500 nm).
Figure 3. Changes in average diameters of blank PRSDP NPs as a function of time (A)
in MQ with different concentrations of GSH; (B) in MQ with different concentrations
of H2O2; (C)in PBS at pH 7.4, pH 5.0 and conditions mimicking plasma and cancer
cell environment monitored by DLS.

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Figure 4. The pH, GSH, ROSꢀsensitivity of PRDSP NPs monitored by TEM (scale
bars: 500 nm).
Figure 5. H2O2 scavenging activity of PRDSP NPs.
Figure 6. In vitro DOX release under different conditions.

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DOI: 10.1039/C5RA03423J
Figure 7. Reduction of intracellular ROS, representative CLSM images of
Rosupꢀstimulated cells stained with DCFHꢀDA (bar:100ꢁm).
Figure 8. In vitro cytotoxicities of PRDSP NPs to L929 and HeLa cells.

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Figure 9. In vitro cytotoxicity of PRDSP@DOX NPs and free DOX·HCl at various
DOX concentrations towards 10 mM GSHꢀpretreated or nonꢀpretreated cells with
different incubation time. (A) HeLa, 24 h; (B) HeLa, 48 h; (C) A549, 24 h; (D) A549,
48 h.
Table 2. IC50 values of DOX and PRDSP@DOX NPs towards 10 mM GSH
pretreated or nonꢀpretreated HeLa/A549 cells for different incubation time.
HeLa cells
A549 cells
IC50
(ꢁg/mL)
24 h
IC50
(ꢁg/mL)
48 h
IC50
(ꢁg/mL)
24 h
IC50
(ꢁg/mL)
48 h
DOX
1.44
0.68
3.08
0.50
DOX+10mM GSH
1.48
0.75
3.17
0.58
PRDSP@DOX NPs
PRDSP@DOX+10 mM GSH
3.58
2.41
1.67
1.02
8.92
5.32
2.17
1.34

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Figure 10. Cellular uptake of DOX and PRDSP@DOX NPs in the presence or
absence of 10 mM GSH towards HeLa cells ( blue:Hoechst, red:DOX. Bar:50 ꢁm).