Dual Stimuli-Responsive Block Copolymers with Adjacent Redox- And Photo-Cleavable Linkages for Smart Drug Delivery

Article abstract of DOI:10.1021/acs.biomac.0c00773

A novel dual-stimuli cleavable linker containing adjacent UV light-sensitive o-nitrobenzyl ester and GSH-responsive disulfide bonds was first designed and synthesized to increase the responsivity to external stimuli. The functionalized linker was then uti

Full text of DOI:10.1021/acs.biomac.0c00773

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Article
Dual Stimuli-responsive Block Copolymers with Adjacent
Redox- and Photo-cleavable Linkages for Smart Drug Delivery
Yu-Lun Lo, Ming-Fong Tsai, Yugendhar Soorni, Chin Hsu, Zi-Xian Liao, and Li-Fang Wang
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.0c00773 • Publication Date (Web): 14 Jul 2020
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Dual Stimuli-responsive Block Copolymers with Adjacent Redox- and
Photo-cleavable Linkages for Smart Drug Delivery
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Yu-Lun Lo,a,b# Ming-Fong Tsai,a# Yugendhar Soorni,a Chin Hsu,b Zi-Xian Liaoc, and Li-
Fang Wang*a,c,d
aDepartment of Medicinal and Applied Chemistry, College of Life Sciences, Kaohsiung
Medical University, Kaohsiung 807, Taiwan
bDepartment of Physiology, Faculty of Medicine, College of Medicine, Kaohsiung
Medical University, Kaohsiung 807, Taiwan
cInstitute of Medical Science and Technology, National Sun Yat-Sen University,
Kaohsiung 804, Taiwan
dDepartment of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung
807, Taiwan
Correspondence to: Li-Fang Wang, Professor of Medicinal & Applied Chemistry
College of Life Sciences
Kaohsiung Medical University
100, Shih-Chuan 1st Rd, Kaohsiung City 807, Taiwan
Tel: 011-886-7-3121101-2217
Fax: 011-886-7-3125339
E-mail: lfwang@kmu.edu.tw
Manuscript submitted to Biomacromolecules on May 18, 2020
1st revision on June, 20, 2020
2nd revision on July 9, 2020
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ABSTRACT
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A novel dual-stimuli cleavable linker containing adjacent UV light-sensitive o-
nitrobenzyl ester (ONB) and GSH-responsive disulfide bonds was first designed and
synthesized to increase the responsivity to external stimuli. The functionalized linker was
then utilized to prepare a dual-responsive amphiphilic block copolymer using ring
opening polymerization and atom transfer radical polymerization. The copolymer formed
a micelle in an aqueous solution and showed dual-stimuli responses including photo-
mediated cleavage under UV light irradiation at 365 nm as well as reduction-responsive
degradation in the presence of a reducing agent. The micelle was nontoxic against three
cell lines and majorly internalized via clathrin-mediated endocytosis. Doxorubicin (Dox)
was loaded in the hydrophobic core of the micelle. Enhancement of a cell-killing effect
against A549 cells was clearly observed in the Dox-encapsulated micelle when exposed
to UV light.
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Keywords: micelle, dual-stimuli response, ring opening polymerization, atom transfer
radical polymerization, clathrin-mediated endocytosis, doxorubicin
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INTRODUCTION
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Stimuli-responsive amphiphilic block copolymers have been extensively studied
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with respect to biomedical applications because of their tremendous responsiveness to
external triggers such as light, pH, redox, ultrasound, and ionic forces.1 Integration of
different kinds of the stimuli-reactive linkers into a block copolymer can be achieved by
designing dual or multi stimuli-responsive compartments either as pendent groups or in
the backbone chain of parent block copolymers.2 The main goal of all designs is to induce
polymeric micelles to undergo efficient and controllable degradation upon external
stimuli and consequently, encapsulated agents in the hydrophobic domain of polymeric
micelles can be efficiently released at a target tissue on-demand.3
Among external physical stimuli, a photo-mediated therapy strategy is attractive
because light can direct active molecules into targeted site in highly spatiotemporal
precision under light irradiation at a specific wavelength. In addition, it is noninvasive
and shows minimal toxicity to normal tissue.4 Light-cleavable moieties include organic
coumarin, 3′, 5′-di(carboxymethoxy) benzoin acetate and o-nitrobenzyl (ONB)
derivatives and inorganic (Ru, Pt, and Mn complexes) compounds, undergoing an
irreversible reaction.5 Although an organic ONB family is widely developed for drug
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delivery applications,6-8 nevertheless, it has some drawbacks. For example, the release
rates of the ONB family are slower than those of coumarin derivatives.9 After irradiation,
the produced fragment (i.e. nitrosoaldehyde) can react with amines to form imine bonds,
which can especially be a problem for abundance of surrounding proteins in living
tissues.10
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Glutathione (GSH) is a thiol-containing tripeptide, showing different concentrations
in intracellular (1 - 10 mM) and extracellular areas (2 - 20 μM) in living cells.11 The
cytosolic GSH concentrations in cancer cells are several times higher than those in
normal cells.12 Thus, the use of GSH concentration gradient is a promising approach to
respond to the tumor microenvironment (TME) and to control therapeutics released at the
tumor site. The combination of a redox-cleavable disulfide linkage and a photo-cleavable
ONB linkage has been widely reported in several polymers13-15 and hydrogel16 to
demonstrate the dual-responsive behavior; however, the disulfide and ONB linkages are
positioned at different places of the polymeric structures. We speculate such designs
might need a higher concentration of reducing agents or longer time exposed to light than
those placing two responsive linkages together.
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Herein, a novel dual-stimuli cleavable linker containing adjacent UV light-sensitive
ONB ester and GSH-responsive disulfide bonds, 2-(2-(2-bromo-2-methylpropanoyl) oxy
ethyl disulfanyl) ethyl 4-(hydroxymethyl)-3-nitrobenzoate (OH-ONB-SS-Br) is first
designed and synthesized to increase the responsivity to the external stimuli. This
functionalized linker is utilized to prepare a dual-responsive amphiphilic block
copolymer using ring opening polymerization (ROP) and atom transfer radical
polymerization (ATRP). The synthesized dual-stimuli cleavable linker is located between
the hydrophobic and hydrophilic junctions of the amphiphilic copolymer in which the
ONB ester moiety undergoes photolysis under UV light irradiation and the disulfide
linkage can be dissipated by reducing agents like dithiothreitol (DTT) or GSH.13, 17-19
Similar degradation fragments will be formed either with the presence of a reducing
agent or under light irradiation. The dual-stimuli responsive cleavage may decrease the
required amount of ONB and simultaneously solve the aforementioned disadvantage of
ONB; additionally, the GSH-responsible behavior has the merit of increasing the drug
release at the tumour site. Here, the artificially-made amphiphilic block copolymer will
be well-characterized and its responsiveness to the external stimuli will be thoroughly
assayed to demonstrate the success of the design.
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EXPERIMENTAL SECTION
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Materials. 4-Bromomethyl-3-nitrobenzoic acid, sodium carbonate (Na2CO3), p-
toluenesulfonic acid (pTSA), 3,4-dihydro-2H-pyran (dihydropyran), 2-bromo-2-
methylpropionyl bromide, triethylamine (TEA), N,N’-dicyclohexylcarbodiimide
(DCC), 4-dimethylaminopyridine (DMAP), tin(II) 2-ethylhexanoate (Sn(Oct)2)
copper(I) bromide (CuBr), and trifluoroacetic acid (TFA) were purchased from
Alfa Aesar. 4-(Bromomethyl)-3-nitrobenzoic acid, ε-caprolactone (CL), tertbutyl
methacrylate (tBMA), N,N,N’,N”,N”-pentamethyldiethylenetriamine (PMDETA),
(1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride) (EDC), and N-
hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was from MP
Biomedicals. Phosphate buffer saline (PBS), Dulbecco's Modified Eagle Medium
(DMEM), Roswell Park Memorial Institute (RPMI) 1640 Medium, trypsin–EDTA
and fetal bovine serum (FBS) were purchased from Invitrogen. Doxorubicin (Dox)
was purchased from Combi-Blocks.
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Synthesis of OH-ONB-SS-Br Macroinitiator (6)
Synthesis of 4-(Hydroxymethyl)-3-nitrobenzoic Acid (2). The reaction procedure of OH-
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ONB-SS-Br macroinitiator is shown in Scheme 1. To synthesize compound 2, the solution
containing 4-(bromomethyl)-3-nitrobenzoic acid (5.00 g, 19.23 mmol) and Na2CO3 (7.13
g, 67.30 mmol) in water: acetone (1:1 v/v, 150 mL) was refluxed at room temperature (RT)
for 5 h and purified according to the literature.22 The compound 2 is a pale brown solid
(3.5 g, 94.6% yield). 1H-NMR (200 MHz, DMSO-d6, ppm) 8.58 (s, 1H, –CH–Phenyl.–),
8.37 (d, 1H, –CH–Phenyl.–), 8.03 (d, 1H, –CH–Phenyl.–), 5.75 (t, 1H, OH–CH2–Phenyl.–),
4.97 (d, 2H, OH–CH2–Phenyl.–). MS (ESI) m/z calcd for C8H7NO5: 197.0; found: 195.97
[M-1]+.
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Synthesis of 2-(2-Hydroxyethyl-disulfanyl) ethyl-2-bromo-2-methylpropanoate (HO-SS-
Br) (3a). The HO-SS-Br was synthesized according to the literature.20 Briefly, the solution
consisting of bis(2-hydroxyethyl) disulfide (5.00 g, 32.4 mmol) and TEA (12.0 mL, 86.1
mmol) in THF was stirred at RT for 20 min under an argon atmosphere. The compound 3a
is a pale yellowish liquid (4.0 g, 40.8% yield). 1H-NMR (200 MHz, CDCl3, ppm) 4.45 (t,
2H, –CH2OC (O)–), 3.90 (t, 2H, HO–CH2–), 3.00 (t, 2H, –SS–CH2––), 2.90 (t, 2H, (–CH2–
SS–), 1.95 (s, 6H, –C(CH3)2Br). MS (ESI) m/z calcd for C8H15BrO3S2: 302.0; found:
303.93 [M+1]+.
Synthesis of 3-Nitro-4-((tetrahydro-2H-pyran-2-yl) oxy methyl) Benzoic Acid (THP-
ONB-COOH) (4). 4-(Hydroxymethyl)-3-nitrobenzoic acid (5.00 g, 25.4 mmol) and pTSA
(0.021 g, 0.12 mmol) were dissolved in THF (50 mL) and the mixture was stirred at RT for
15 min. Under an argon condition, 4.05 mL of 3, 4-dihydro-2H-pyran was dropwise added
to the mixture and refluxed for 10 h. After the solvent was removed by rotary evaporator,
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the residue was purified by column chromatography (silica gel 75-200 mesh) using 10%
EA: petroleum ether mobile phase to yield a pale yellow solid of 4 (7.0 g, 98.6% yield).
1H-NMR (200 MHz, DMSO-d6, ppm) 8.80 (s, 1H, –CH-Phenyl.–), 8.37 (d, 1H, –CH-
Phenyl.–), 8.00 (d, 1H, –CH-Phenyl.–), 5.30 – 4.95 (m, 2H, –O–CH2–Phenyl.–), 4.80 (s,
1H, –O–CH–O–) 3.95 – 3.80 (m, 2H, –C–CH2–C–), 3.70 – 3.50 (m, 2H, –C–CH2–C–),
2.00 – 1.77 (m, 2H, –C–CH2–C–), 1.70 – 1.50 (m, 2H, –C–CH2–C–). MS (ESI) m/z calcd
for C13H15NO6: 281.1; found: 279.90 [M-1]+.
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Synthesis of 2-(2-(2-Bromo-2-methylpropanoyl) oxy ethyl disulfanyl ethyl-3-nitro-4-
(tetrahydro-2H-pyran-2-yl) oxy methyl) Benzoate (THP-ONB-SS-Br) (5). 3-Nitro-4-
((tetrahydro-2H-pyran-2-yl)oxy methyl) benzoic acid (5.00 g, 17.8 mmol) and 2-(2-
hydroxyethyl-disulfanyl) ethyl-2-bromo-2-methylpropanoate (6.40 g, 21.3 mmol) were
mixed in DCM (100 mL). Under an argon atmosphere, DMAP (0.43 g, 3.55 mmol) and
DCC (1.80 g, 8.88 mmol) were added, and the mixture was stirred at RT for 16 h. After
complete reaction, the residue was purified by column chromatography using 5% EA:
petroleum ether mobile phase to yield a yellowish green liquid of 5 (6.5 g, 65.0% yield).
1H-NMR (200 MHz, CDCl3, ppm) 8.70 (s, 1H, –CH-Phenyl.–), 8.30 (d, 1H, –CH-Phenyl.–),
7.95 (d, 1H, –CH-Phenyl.–), 5.30 – 4.90 (m, 2H, –O–CH2–Phenyl.–), 4.80 (s, 1H, –O–CH–
O–), 4.70 (t, 2H, –CH2OC (O)–), 4.50 (t, 2H, –CH2OC (O)–), 3.97 – 3.80 (m, 2H, –C–
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CH2–C–), 3.65 – 3.50 (m, 2H, –C–CH2–C–), 3.95 – 2.95 (m, 4H, –SS–CH2–CH2OC (O)–),
2.00 (s, 6H, –O=C–(CH3)2–Br–), 1.90 – 1.70 (m, 2H, –C–CH2–C–), 1.65 – 1.50 (m, 2H, –
C–CH2–C–).
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Synthesis of 2-(2-(2-Bromo-2-methylpropanoyl) oxy ethyl disulfanyl) ethyl 4-
(hydroxymethyl)-3-nitrobenzoate (OH-ONB-SS-Br) (6). Compound 5 (5.00 g, 8.82 mmol)
was put into a clean round bottom flask and dissolved in THF (40 mL). Under an argon
atmosphere, pTSA (0.021 g, 0.12 mmol) was added to the mixture and stirred at RT for 18
h. The column chromatography was utilized to purify the residue with 10% EA: petroleum
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ether mobile phase to yield a pale green liquid of 6 (2.5 g, 59.5% yield). H-NMR (200
MHz, CDCl3, ppm) 8.75 (s, 1H, –CH-Phenyl.–), 8.35 (d, 1H, –CH-Phenyl.–), 7.95 (d, 1H,
–CH-Phenyl.–), 5.15 (d, 2H, HO–CH2–Phenyl.–), 4.65 (t, 2H, –CH2OC (O)–), 4.45 (t,
2H, –CH2OC (O)–), 3.95 – 2.95 (m, 4H, –SS–CH2–CH2OC (O)–), 2.00 (s, 6H, –O=C–
(CH3)2–Br–). MS (ESI) m/z calcd for C8H15BrO3S2: 481.0; found: 481.80 [M]+.
Synthesis of PCL-ONB-SS-PMAA Diblock Copolymer
Synthesis of Poly(ε-caprolactone)-ONB-b-bromide with a Disulfide Linkage (PCL-
ONB-SS-Br). The PCL-ONB-SS-Br was synthesized under an argon atmosphere using
HO-ONB-SS-Br (96 mg, 0.31 mmol), CL (2.19 mL, 19.5 mmol), Sn(Oct)2 (15.9 mg, 0.049
mmol) and toluene (2.1 mL). The ROP was carried out at 120 °C for 6 h in a glove box,
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and the PCL-ONB-SS-Br was purified according to previous publication.21
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Synthesis of PCL-ONB-SS-PMAA. The asymmetric diblock copolymer of PCL-ONB-
SS-PtBMA was synthesized by ATRP using PCL-ONB-SS-Br (200 mg, 0.028 mmol),
CuBr (12.3 mg, 0.085 mmol), PMDETA (21.0 mg, 0.091 mmol), tBMA (617 mg, 4.34
mmol) and toluene (1.5 mL) in a round-bottom flask. The flask was degassed by
consecutive standard freeze–pump–thaw cycles for three times. The ATRP was carried out
at 50 °C for 6 h in the glove box. The tert-butyl ester groups of PCL-ONB-SS-PtBMA
were removed in an acid condition of TFA to yield PCL-ONB-SS-PMAA according to
previous publication.21
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Characterization of Block Copolymer. H-nuclear magnetic resonance (1H-NMR)
spectra were recorded on a Varian Gemini (200 MHz) NMR spectrometer using CDCl3 or
DMSO-d6 as a solvent to determine chemical structures. Gel permeation chromatography
(GPC) was acquired to determine molecular weights (MW) using an Agilent 1200 series
equipped with a Shodex-KF804L and KF-803 connected column. FT-IR spectrum was
acquired from Bruker ALPHA-T spectrometer. Mass data was acquired from
TRACEGC-POLARISQ (Thermo Finnigan) and Waters ZQ 4000 mass
spectrometer.
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Preparation and Characterization of Micelles
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Micelle Formation. A dialysis method was acquired for preparation of PCL-ONB-SS-
PMAA micelle (POSPm). The morphologies of POSPm were observed by transmission
electron microscope (TEM HT7700, Hitachi), and the size distribution of POSPm was
measured by dynamic light scattering (DLS) zeta-sizer (ELSZ-2000, Otsuka Electronics).
The critical aggregation concentrations (CAC) of POSPm were determined using a Cary
Eclipse fluorescence spectrophotometer (Varian) with pyrene as a probe according to
previous publication.21
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Reductive- and Photo-triggered Cleavage of POSPm. Redox-driven cleavage of the
disulfide linkage of POSPm was carried out with 10 mM GSH for 20 min. POSPm was
dispersed in DD water containing GSH at 0.5 mg/mL. For photo-triggered cleavage of
POSPm, two millimeters of POSPm aqueous solution were placed into a cuvette (Hellma)
and stirred continuously. The cuvette was kept in the dark and irradiated by continuous
wave (CW) UV laser diode (365 nm, max. 430 mW/cm2, TANYU) horizontally under
continuous stirring for 20 min. Following reductive- and photo-triggered treatments, the
change in size and morphology of POSPm was observed by DLS and TEM respectively.
Dox-encapsulated POSPm (POSPm@Dox) and Dox Release. The Dox∙HCl powder was
dissolved in DMSO at a concentration of 2 mg/mL and an equimolar amount of TEA was
added for desalting. One mL of the Dox solution was added into the solution containing 20
mg of the PCL-ONB-SS-PMAA copolymer in 4 mL of DMSO.29 A dried sample of
POSPm@Dox was dissolved in DMSO at a concentration of 1 mg/mL, and the Dox content
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was calculated against a calibration curve using a UV-Vis spectrometer (Agilent 8453) at
the wavelength of 485 nm. The loading efficiency (LE) and encapsulation efficiency (EE)
were reported as follows.
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LE (%) = (amount of drug in micelle / total amount of POSPm@Dox) x 100
EE (%) = (amount of drug in micelle / amount of drug in feed) x 100
(1)
(2)
The redox- and photo-trigger-released behavior of Dox from POSPm@Dox was
determined in PBS at pH 7.4 and 37 °C for simulating a physiological condition. The 800
μL of POSPm@Dox solution at 3 mg/mL was put in a DiaEasy™ Dialyzer (MWCO
3.5kDa, BioVision) and immersed in 10 mL of PBS (1) for whole time (control), (2) for 6
h, followed by treatment with 10 mM GSH, (3) for 6 h, followed by treatment with UV
irradiation for 20 min, (4) for 6 h, followed by treatment with 10 mM GSH and UV
irradiation for 20 min. At a certain time interval, one mL of PBS solution was carefully
removed from the tube and replaced with 1 mL of fresh PBS to maintain a sink condition.
Besides being irradiated at the time point of 6 h, groups 3 and 4 were also treated with light
irradiation for 20 min at another two time points of 12 and 24 h. The Dox concentration
was determined using an UV−vis spectrophotometer as aforementioned.
Cell Experiments
Cell Culture. Non-small cell lung carcinoma A549 cells were cultured in RPMI, and
human normal lung fibroblasts HEL-299 cells and non-small cell lung carcinoma CRL-
5802 cells were cultured in DMEM, respectively containing 10% fetal bovine serum (FBS)
and 100 μg/mL penicillin/streptomycin in a 37°C incubator with a humidified atmosphere
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Relative Cell Viability of POSPm Micelle and POSPm@Dox Micelle. The cell viability
of POSPm was determined with an MTT assay against A549 cells, CRL-5802 cells and
HEL-299 cells according to previous publication.28 The cytotoxic efficacy of POSPm@Dox
was done in A549 cells alone. To test the photo-triggered cytotoxicity of POSPm@Dox,
A549 cells were seeded into 12 well plates (1 × 105 cells/well) for 24 h and added with an
equivalent concentration of free Dox or POSPm@Dox at 2 μg/mL. Following 4 h
incubation, the medium was replaced with 1 mL of fresh complete medium, and the cells
were exposed to UV light (365 nm, 430 mW/cm2) for 20 min and postincubated for another
48 h. The cytotoxic efficacy of POSPm@Dox and free Dox was determined with the MTT
assay.
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Endocytosis Pathway of Micelle. Rhodamine 123 (R123) was conjugated with POSPm
by EDC/NHS coupling reaction to examine the internalization pathway of POSPm into
A549 cells.21 A549 cells (2 × 105 cells/well) were seeded in 6-well plates and incubated
for 24 h. The cells were pretreated with inhibitors: chlorpromazine (10 μg/mL),
wortamannin (50 nM), genistein (200 μM), separately for 30 min and analyzed by flow
cytometry.28
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scanning microscope (CLSM, LSM 700 Zeiss Confocal Microscopy). A549 cells (1.0 ×
105 cells/well) were seeded in 12-well plates with one sterilized glass coverslip/well for 24
h, and the cells were treated as same as aforementioned in the MTT assay. The fluorescence
intensity was analyzed by MetaVue software. The Annexin-V/PI (propidium iodide) dual
staining assay was performed to estimate apoptosis-inducing efficacy of free Dox,
POSPm@Dox before and after UV light irradiation as well.29
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Statistical Analysis. Experiments were repeated at least three times, and data are expressed
as the mean ± standard deviation. Student’s t-test was performed to determine the statistical
significance of the respective group. *p < 0.05 or **p < 0.01 indicated a significant
difference.

RESULTS AND DISCUSSION
Copolymer Synthesis and Characterization. The synthetic approach of PCL-ONB-
SS-PMAA was associated with eight steps (Scheme 1). Initially, compound 2 with
the ONB moiety was synthesized according to the procedure reported22 and the 1H-
NMR and LC/MS spectra of compound 2 are in good consistency (Supporting
Information Figs. 1Sa & 1Sb). Secondly, the benzyl hydroxyl group was protected
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with 3,6-dihydro-2H-pyran to yield compound 4 (Supporting Information Figs. S1c
& S1d) and proceeded for further esterification process with compound 3a
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1
(Supporting Information Figs. S1e & S1f) to yield compound 5. The H-NMR
spectrum of compound 5 is shown in Figure 1a. Compound 5 was de-protected with
p-TSA to obtain compound 6 (macroinitiator) containing hydroxyl (-OH) group on
one end and tertiary bromide on the other end. Compound 6 was applicable for ROP
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and ATRP respectively. The H-NMR and LC/MS spectra of the compound 6 are
shown in Figures 1b & 1c. Thirdly, the ε-caprolactone monomer was used to
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synthesize compound 8 using Sn(Oct)2 as a catalyst through ROP. The H-NMR
spectrum of PCL-ONB-SS-Br (compound 8) is shown in Figure 2a. In the further
step, compound 10 was synthesized using the compound 8, CuBr and PMDETA via
ATRP. Following the reaction, the polymer solution was dissolved in THF and
1
passed through the neutral alumina to remove the residual Cu+ ion. The H-NMR
spectrum of PCL-ONB-SS-PtBMA (compound 10) is shown in Figure 2b and the
increase in MW before and after ATRP was measured by GPC as shown in Figure
2c. The tert-butyl moieties of the ester groups were removed into carboxylic acid
groups upon treating the copolymer solution with TFA to obtain compound 11,
1
where the carboxylic acid proton peak appears ~12.4 ppm in H-NMR spectrum
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(Figure 2d).
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In light of the literature review,23-26 the ONB ether linkage is commonly used to make
the copolymers containing ONB moiety instead of ONB ester linkage. Thus, we initially
designed and synthesized an ONB ether linkage between the junction of PMAA and PCL
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(Supporting Information Scheme S1). However, a shoulder peak was observed at ~15
min elution time in a GPC profile (Supporting Information Fig. S2a). This shoulder peak
always existed in GPC profiles even though the reaction time was prolonged to 12 h
(Supporting Information Fig. S2b). We speculated that the ONB ether linkage was
o
unstable as the ROP reaction was carried out at a high temperature of 90 C. The ONB
ether linkage degraded and formed a fragmented PCL-ONB-OH structure as indicated in
Fig. S2a. The positive inductive effect of methyl groups on aromatic ether position of the
polymer containing ONB ether linkage accelerated the disintegration of the ether linkage27
and thus converted it into hydrophobic polymer fragments bearing phenolic -OH group
(PCL-ONB-OH), which was not applicable for further ATRP reaction. Hence, we utilized
the ONB ester as the macroinitiator for further diblock copolymer synthesis and intended
to make the copolymer more stable towards temperature and mild acidic conditions.
Micellar Formulation and Drug Encapsulation. A dialysis method was adopted to
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prepare a PCL-ONB-SS-PMAA micelle (POSPm). The CAC values of POSPm were
determined using a fluorescence pyrene as the probe, i.e. 1.18 x 10-2 mg/mL at pH 5.5 and
8.25 x 10-3 mg/mL at pH 7.4 respectively (Supporting Information Fig. S3). The low
CAC value implied good stability in the physiological condition of pH 7.4.
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Scheme 2 depicts photo- and redox-stimuli responsive segments of POSPm. The
nitro group present on the benzene ring at ortho position undergoes homolysis, and
causes the degradation of the ester bond when it absorbs the UV light as a proposed
mechanism shown in Scheme 2. In this process, two fragments were formed after UV
irradiation, leading to the formation of an o-nitrosobenzaldehyde group attached on
PMAA segment and a carboxylic acid group attached on the end of PCL segment. An
aldehyde signal peak clearly appeared at ~10.2 ppm in the NMR spectrum of the PCL-
ONB-SS-PMAA block copolymer in d6-DMSO after UV irradiation at 365 nm (430
mW/cm2) for 20 min (Supporting Information Fig. S4). Additional evidence for photo-
and redox-responsive characteristics of the block copolymer was confirmed by GPC
(Figure 3). PCL-ONB-SS-PtBMA was utilized for testing because of the insolubility of
PCL-ONB-SS-PMAA in THF. A mono MW distribution of the block copolymer was
observed at initiation; nevertheless, a bimodal distribution profile appeared when the
copolymer was treated with DTT (50 mM in THF) at a regular time interval. An increase
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in MW distribution appearing at longer elution time went hand-in-hand with a decrease in
MW distribution appearing at a shorter elution time, indicating the gradual cleavage of
the disulfide linkage of the block copolymer (Figure 3a). A similar trend of the MW
change of PCL-ONB-SS-PtBMA was observed in GPC profiles under UV light
irradiation (Figure 3b). These results reconfirmed that the block copolymer underwent a
characteristic o-nitrobenzyl cleavage when the polymer was irradiated by UV light.
The morphological change of POSPm was acquired by TEM. The particle size of
POSPm is ~ 80 nm estimated from the TEM image (Figure 4a). Irregular shapes were
observed when the micelle was treated with 10 mM GSH for 20 min, UV light irradiation
for 20 min, and both combined (Supporting Information Fig. S5a-c), indicating the
micelle disintegrated because of the cleavage of the disulfide linkage as well as the ONB
moiety.
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In parallel, the hydrodynamic size of the micelle was determined to be ~120 nm and
the size distribution became broader and broader with time when POSPm was treated with
GSH (Figure 4b). A bimodal size distribution appeared under UV light irradiation and
shifted to the large size distribution when the micelle was treated with both stimuli. From
GPC, TEM and DLS results, it seemed that the response of POSPm degradation under UV
light irradiation was superior to GSH stimulus. A similar finding has been reported in a
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dual-stimuli-responsive ABA-type triblock copolymer micelle, where o-nitrobenzyl and
disulfide groups were positioned separately in the hydrophobic middle block.15 A burst
release of a hydrophobic drug from disintegrated micelles was traced by UV light but a
slow release was followed in the presence of a reducing agent.
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In Vitro Cytotoxicity and Cellular Uptake Pathway. The human lung carcinoma
cell lines CRL-5802, HEL-299 and A549 cells were cultured in a DMEM or RPMI
medium. The cells containing different micellar concentrations within 0–200 µg/mL
were added to each well and further incubated for another 24 h under 5% CO2 at 37
oC.28 No obvious cytotoxicity of the cells exposed to the micelle was found at the
test concentrations, and their viabilities were still >95% at the highest concentration
of 200 μg/mL. The cytotoxicity of the micelle was independent on the cell lines used
(Figure 5a).
The cellular uptake of the micelle was tested in CRL-5802 and A549 cells using a
flow cytometer, where the internalization of the micelle was higher in A549 cells than
in CRL-5802 cells because of the larger right shift in fluorescence intensity (Figure
5b). Three commonly-used chemical inhibitors were adopted for testing the
internalization pathway of the micelle into the cells using wortmannin for
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micropinocytosis, chlorpromazine for clathrin-mediated endocytosis (CME) and genistein
for caveolae-mediated endocytosis.29 Flow cytometric histograms showed a left shift when
A549 cells and CRL-5802 cells were pretreated with chlorpromazine, indicating an
inhibition effect and no shifts when the cells were pretreated with genistein and
wortmannin. This fact implied that CME is the major internalization pathway. The
endocytic pathway highly determined the intracellular destination of nanoparticles (NPs).
CME seemed the most promising pathway for cellular entry in drug delivery systems
because the drug could be released rapidly from drug-encapsulated NPs.30-34
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Encapsulation of DOX into POSPm (POSPm@Dox) and its Cytotoxicity. A micelle
has the capability to encapsulate lipophilic agents inside the hydrophobic core for
smart drug delivery. The desalt form of doxorubicin (Dox) was used as a hydrophobic
drug model, and the Dox calibration curve was established to calculate LE and EE of the
drug, i.e. ~7.0% and 75.6% respectively. The in vitro Dox released from the micelle was
performed in PBS of pH 7.4 to simulate the physiological condition. Initially, four groups
of the test samples were immersed in PBS of pH 7.4 for 6 h. About 17% of Dox was
gradually released as shown in Figure 6a. After that, the samples in DiaEasy™ Dialyzer
containing POSPm@Dox were continually dialyzed against 10 mL of PBS at pH 7.4 and
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separately treated with (1) nothing (control), (2) 10 mM GSH, (3) UV irradiation for 20
min, and (4) both 10 mM GSH and UV irradiation for 20 min. After the time point of 6 h,
the drug release rate was in the order of Group (4) > Group (3) ≈ Group (2) > Group (1).
Because the difference of drug release profiles among groups was insignificant, another
light irradiation was applied at 12 h and 24 h. In Group 4, the drug release percentage
clearly increased at 12 h and was larger than those in Groups 2 and 3, and then significantly
increased at 24 h, ~20% increase in Dox release. Groups 2 and 3 showed similar release
profiles. As depicted in Scheme 2, the micelle might preserve the hydrophobic core and
the encapsulated drug gradually becomes released with a single stimulus. In contrast, the
micelle disintegrated upon two stimuli and the drug was rapidly released. The
combination of redox-responsive disulfide along with the UV-sensitive linkage in
the copolymer clearly enhanced disintegration of the micelle in the physiological
condition, where the concentration of GSH (3 - 10 mM) was found to be higher than
extracellular areas (~ 2.8 μM) in living cells.35, 36 This fact benefits an effective
cellular-triggered drug release because of the cleavage of disulfide linkages in the
micelle via disulfide-thiol exchange reactions.22
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The cytotoxicity of POSPm@Dox was tested against A549 cells (Figure 6b). For 24 h
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20 μg/mL. For 48 h postincubation, the IC50 values (the concentration of Dox to
inhibit 50% of cell viability) were 1.65 μg/mL for POSPm@Dox and 1.50 μg/mL for
free Dox. The potency of POSPm@Dox in induced apoptosis was similar to that of
free Dox against A549 cells. Dox is an anthracycline antibiotic, and its anticancer
activity is widely known towards solid tumors like lung, breast, ovarian, thyroid
and gastric cancers.37
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For inhibition of cell growth testing, we first checked whether A549 cells alone or
exposed to POSPm could be damaged under UV light irradiation at 365 nm with 430
mW/cm2 for 20 min. Indeed, no significant difference in cell viability was found under
UV light irradiation, and the cells maintained an intact morphology as compared with the
control group (Supporting Information Figs. S6a & S6b). Subsequently, the cells were
exposed to POSPm@Dox at a relevant Dox concentration of 2 μg/mL, slightly larger than
its IC50 value. Figure 7a shows 63.40 ± 7.36% and 47.33 ± 4.95% cell viabilities for the
cells treated with free Dox and POSPm@Dox respectively. The cells died significantly as
exposed to POSPm@Dox under UV light irradiation (p < 0.05, compared with free Dox
or POSPm@Dox). The relative cell viability was 16.75 ± 1.61%. The cellular uptake of
Dox into the cells was traced by CLSM as well. Figure 7b shows the highest fluorescence
intensity of Dox was observed in POSPm@Dox with UV light irradiation, indicating that
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the cleavage of ONB linkage indeed enhanced the drug release. The higher concentration
of Dox released from POSPm@Dox+UV also resulted in more serious damage of the
cells. A distorted morphology of the cells is clearly seen in the DIC image.
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Annexin-V/PI dual-staining assay was acquired for apoptosis testing. Consistent
results were found as those observed by MTT assay (Figure 8a). The percentages of
Annexin-V/PI staining positive cells (except lower left quadrant) are summed and plotted
in Figure 8b, being 36.01 ± 4.66% for the cells treated with DOX, 43.90 ± 4.14% for the
cells under UV irradiation, and 62.56 ± 3.18% for the cells with the combined treatment.
Compared with free Dox and POSPm@Dox, the significant increase in amount of death
cells was found for POSPm@Dox+UV.

CONCLUSION
A novel PCL-ONB-SS-PMAA diblock copolymer with photo- and redox-dual
stimuli responsive sites adjacently located within the hydrophobic-hydrophilic
core-shell junctions was successfully and firstly synthesized by –OH protection,
de-protection method, ROP, and ATRP reactions. The stimuli-responsive
degradable ability of the micelle was confirmed from TEM images, GPC profiles
and UV-visible spectra. In in vitro studies, the micelle showed low cytotoxicity
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against CRL-5802, A549 and HEL-299 cells. The intracellular uptake of the
micelle was higher in A549 cells than in CRL5802 cells, and the internalization
mechanism was mainly mediated by CME. The model drug Dox was efficiently
encapsulated inside the core compartment of the micelle, and a synergistic Dox-
released profile was found in the group with the treatment of GSH and UV light
irradiation. This combination of redox- and photo-responsive cleavage behavior
could enhance disintegration of the micelle in the tumor microenvironment. The
highest released amount of Dox from POSPm@Dox+UV showed the highest cell
killing effect. Thus, this artificially-made micelle is promising for smart drug
delivery.
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
CONFLICTS OF INTEREST
The authors declare that they have no conflict of interest.

ASSOCIATED CONTENT
The Supporting Information is available free of charge at https://pubs.acs.org
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Synthetic procedures of PCL-ONB-ether-SS-PMAA, NMR spectra of synthesized
compounds, GPC profiles, Critical aggregation concentration, NMR spectrum and TEM
image after UV light irradiation, and Cell viability by MTT assay.
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
ACKNOWLEDGEMENTS
We are grateful for financial support from the Ministry of Science and Technology of
Taiwan (grant nos. MOST106-2320-B-037-004-MY3 and MOST108-2221-B-037-002-
MY3. This study was also supported by Kaohsiung Medical University under grant no.
KMU-DK(個)109001, and by the NSYSU-KMU Joint Research Project under grant no.
NSYSUKMU 109-P009. We appreciate the experimental support of a confocal laser
scanning microscope and a transmission electron microscope provided by the Center for
Research Resources and Development of Kaohsiung Medical University.

REFERENCES
(1) Fleige, E.; Quadir, M. A.; Haag, R., Stimuli-responsive polymeric nanocarriers for
the controlled transport of active compounds: concepts and applications. Adv. Drug
Deliv. Rev. 2012, 64, 866-884.
(2) Cheng, R.; Meng, F.; Deng, C.; Klok, H. A.; Zhong, Z., Dual and multi-stimuli
responsive polymeric nanoparticles for programmed site-specific drug delivery.
Biomaterials 2013, 34, 3647-3657.
(3) Zhang, Q.; Aleksanian, S.; Nohbc, S. M.; Oh, J. K., Thiol-responsive block
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copolymer nanocarriers exhibiting tunable release with morphology changes. Polym.
Chem.-Uk 2013, 4, 351-359.
(4) Yu, M.; Zheng, J., Clearance pathways and tumor targeting of imaging
nanoparticles. ACS Nano 2015, 9, 6655-6674.
(5) Liu, G.; Liu, W.; Dong, C. M., UV- and NIR-responsive polymeric nanomedicines
for on-demand drug delivery. Polym. Chem.-Uk 2013, 4, 3431-3443.
(6) Aujard, I.; Benbrahim, C.; Gouget, M.; Ruel, O.; Baudin, J. B.; Neveu, P.; Jullien,
L., o-Nitrobenzyl photolabile protecting groups with red-shifted absorption: syntheses
and uncaging cross-sections for one- and two-photon excitation. Chemistry (Easton)
2006, 12, 6865-6879.
10
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(7) Marinzi, C.; Offer, J.; Longhi, R.; Dawson, P. E., An o-nitrobenzyl scaffold for
peptide ligation: synthesis and applications. Bioorg. Med. Chem. 2004, 12 (10), 2749-
2757.
(8) Piggott, A. M.; Karuso, P., Synthesis of a new hydrophilic o-nitrobenzyl
photocleavable linker suitable for use in chemical proteomics. Tetrahedron Lett. 2005,
46, 8241-8244.
(9) Schade, B.; Hagen, V.; Schmidt, R.; Herbrich, R.; Krause, E.; Eckardt, T.; Bendig,
J., Deactivation behavior and excited-state properties of (coumarin-4-yl)methyl
derivatives. 1. Photocleavage of (7-methoxycoumarin-4-yl)methyl-caged acids with
fluorescence enhancement. J. Org. Chem. 1999, 64, 9109-9117.
(10) Beaute, L.; McClenaghan, N.; Lecommandoux, S., Photo-triggered polymer
nanomedicines: From molecular mechanisms to therapeutic applications. Adv. Drug
Deliv. Rev. 2019, 138, 148-166.
(11) Cheng, R.; Feng, F.; Meng, F.; Deng, C.; Feijen, J.; Zhong, Z., Glutathione-
responsive nano-vehicles as a promising platform for targeted intracellular drug and gene
delivery. J. Control. Release 2011, 152, 2-12.
(12) Huang, L.; Chaurasiya, B.; Wu, D.; Wang, H.; Du, Y.; Tu, J.; Webster, T. J.; Sun,
C., Versatile redox-sensitive pullulan nanoparticles for enhanced liver targeting and
efficient cancer therapy. Nanomedicine 2018, 14, 1005-1017.
(13) Xuan, J.; Han, D.; Xia, H.; Zhao, Y., Dual-stimuli-responsive micelle of an ABC
triblock copolymer bearing a redox-cleavable unit and a photocleavable unit at two block
junctions. Langmuir 2014, 30, 410-417.
(14) Huang, Q.; Liu, T.; Bao, C.; Lin, Q.; Ma, M.; Zhu, L., Light and reductive dual
stimuli-responsive PEI nanoparticles: “AND” logic response and controllable release. J.
Mater. Chem. B 2014, 2, 3333-3339.
26
ACS Paragon Plus Environment

Page 27 of 39
Biomacromolecules
1
2
3
4
5
6
7
8
9
(15) Han, D.; Tong, X.; Zhao, Y., Block copolymer micelles with a dual-stimuli-
responsive core for fast or slow degradation. Langmuir 2012, 28 (5), 2327-2331.
(16) Cao, Z.; Zhou, X.; Wang, G., Selective release of hydrophobic and hydrophilic
cargos from multi-stimuli-responsive nanogels. ACS Appl. Mater. Inter. 2016, 8, 28888-
28896.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(17) Han, D.; Tong, X.; Zhao, Y., Block copolymer micelles with a dual-stimuli-
responsive core for fast or slow degradation. Langmuir 2012, 28, 2327-2331.
(18) Zhang, P.; Zhang, H.; He, W.; Zhao, D.; Song, A.; Luan, Y., Disulfide-linked
amphiphilic polymer-docetaxel conjugates assembled redox-sensitive micelles for
efficient antitumor drug delivery. Biomacromolecules 2016, 17, 1621-1632.
(19) Chen, J.; Qiu, X.; Ouyang, J.; Kong, J.; Zhong, W.; Xing, M. M., pH and reduction
dual-sensitive copolymeric micelles for intracellular doxorubicin delivery.
Biomacromolecules 2011, 12, 3601-3611.
(20) Chan, N.; An, S. Y.; Oh, J. K., Dual location disulfide degradable interlayer-
crosslinked micelles with extended sheddable coronas exhibiting enhanced colloidal
stability and rapid release. Polym. Chem.-Uk 2014, 5, 1637-1649.
(21) Liu, Y. S.; Huang, S. J.; Huang, X. S.; Wu, Y. T.; Chen, H. Y.; Lo, Y. L.; Wang,
L. F., The synthesis and comparison of poly(methacrylic acid)–poly(ε-caprolactone)
block copolymers with and without symmetrical disulfide linkages in the center for
enhanced cellular uptake. RSC Adv. 2016, 6, 75092-75103.
(22) Li, W.; Wang, J.; Ren, J.; Qu, X., Near-infrared upconversion controls photocaged
cell adhesion. J. Am. Chem. Soc. 2014, 136, 2248-2251.
(23) Bertrand, O.; Poggi, E.; Gohy, J. F.; Fustin, C. A., Functionalized stimuli-
responsive nanocages from photocleavable block copolymers. Macromolecules 2014, 47,
183-190.
(24) Lee, J. E.; Ahn, E.; Bak, J. M.; Jung, S. H.; Park, J. M.; Kim, B. S.; Lee, H. I.,
Polymeric micelles based on photocleavable linkers tethered with a model drug. Polymer
2014, 55, 1436-1442.
(25) Lee, R. S.; Li, Y. C.; Wang, S. W., Synthesis and characterization of amphiphilic
photocleavable polymers based on dextran and substituted-ɛ-caprolactone. Carbohydr.
Polym. 2015, 117, 201-210.
(26) Jalani, G.; Naccache, R.; Rosenzweig, D. H.; Haglund, L.; Vetrone, F.; Cerruti,
M., Photocleavable hydrogel-coated upconverting nanoparticles: A multifunctional
theranostic platform for NIR imaging and on-demand macromolecular delivery. J. Am.
Chem. Soc. 2016, 138, 1078-1083.
27
ACS Paragon Plus Environment

Biomacromolecules
Page 28 of 39
1
2
3
4
5
6
(27) Zhao, H.; Sterner, E. S.; Coughlin, E. B.; Theato, P., o-Nitrobenzyl alcohol
derivatives: opportunities in polymer and materials science. Macromolecules 2012, 45,
1723-1736.
7
8
9
(28) Lo, Y. L.; Chou, H. L.; Liao, Z. X.; Huang, S. J.; Ke, J. H.; Liu, Y. S.; Chiu, C. C.;
Wang, L. F., Chondroitin sulfate-polyethylenimine copolymer-coated superparamagnetic
iron oxide nanoparticles as an efficient magneto-gene carrier for microRNA-encoding
plasmid DNA delivery. Nanoscale 2015, 7, 8554-8565.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(29) Liu, Y. S.; Chiu, C. C.; Chen, H. Y.; Chen, S. H.; Wang, L. F., Preparation of
chondroitin sulfate-g-poly(epsilon-caprolactone) copolymers as a CD44-targeted vehicle
for enhanced intracellular uptake. Mol. Pharm. 2014, 11, 1164-1175.
(30) Harush-Frenkel, O.; Debotton, N.; Benita, S.; Altschuler, Y., Targeting of
nanoparticles to the clathrin-mediated endocytic pathway. Biochem. Biophys. Res.
Commun. 2007, 353, 26-32.
(31) Li, W.; Chen, C.; Ye, C.; Wei, T.; Zhao, Y.; Lao, F.; Chen, Z.; Meng, H.; Gao, Y.;
Yuan, H.; Xing, G.; Zhao, F.; Chai, Z.; Zhang, X.; Yang, F.; Han, D.; Tang, X.; Zhang,
Y., The translocation of fullerenic nanoparticles into lysosome via the pathway of
clathrin-mediated endocytosis. Nanotechnology 2008, 19, 145102.
(32) Rappoport, J. Z., Focusing on clathrin-mediated endocytosis. Biochem. J. 2008,
412, 415-423.
(33) Smith, P. J.; Giroud, M.; Wiggins, H. L.; Gower, F.; Thorley, J. A.; Stolpe, B.;
Mazzolini, J.; Dyson, R. J.; Rappoport, J. Z., Cellular entry of nanoparticles via serum
sensitive clathrin-mediated endocytosis, and plasma membrane permeabilization. Int J
Nanomedicine 2012, 7, 2045-2055.
(34) Chen, G. J.; Hsu, C.; Ke, J. H.; Wang, L. F., Imaging and chemotherapeutic
comparisons of iron oxide nanoparticles chemically and physically coated with
poly(ethylene glycol)-b-poly(ε-caprolactone)-g-poly(acrylic acid). J. Biomed. Nanotech.
2015, 11, 951-963.
(35) Bae, Y. H.; Park, K., Targeted drug delivery to tumors: myths, reality and
possibility. J. Control. Release 2011, 153, 198-205.
(36) Spain, S. G.; Cameron, N. R., A spoonful of sugar: the application of
glycopolymers in therapeutics. Polym Chem-Uk 2011, 2, 60-68.
(37) Tacar, O.; Sriamornsak, P.; Dass, C. R., Doxorubicin: an update on anticancer
molecular action, toxicity and novel drug delivery systems. J. Pharm. Pharmacol. 2013,
65, 157-170.
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Scheme 1. Synthetic procedures of amphiphilic block copolymer PCL-ONB-SS-PMAA (POSP)
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