Preparation of ROS-responsive core crosslinked polycarbonate micelles with thioketal linkage

Article abstract of DOI:10.1016/j.colsurfb.2020.111276

Herein, we prepared novel reactive oxygen species (ROS) responsive core crosslinked (CCL/TK) polycarbonate micelles conveniently by click reaction between amphiphilic diblock copolymer poly(ethylene glycol)-poly(5-methyl-5-propargylxycar-bonyl-1,3-dioxane-2-one) (PEG-PMPC) with pendant alkynyl group and thioketal containing azide derivative bis (2-azidoethyl) 3, 3′- (propane-2, 2-diylbis (sulfanediyl)) dipropanoate (TK-N₃). The CCL/TK micelles were obtained with small size of 146.4 nm, showing excellent stability against dilution and high doxorubicin (DOX) loading. In vitro toxicity tests demonstrated that the obtained CCL/TK micelles have good biocompatibility and low toxicity with cell viability above 95 %. Furthermore, DOX-loaded CCL/TK micelles showed significantly superior toxicity with IC₅₀ values for HeLa and MCF-7 cells about 3.74 μg/mL and 3.91 μg/mL, respectively. Confocal laser scanning microscope (CLSM) and flow cytometry showed excellent internalization efficiency and intracellular drug release of DOX-loaded CCL/TK micelles. The obtained ROS-responsive CCL/TK micelles showed great potential for anticancer drug delivery.

Full text of DOI:10.1016/j.colsurfb.2020.111276

Journal Pre-proof
Preparation of ROS-responsive Core Crosslinked Polycarbonate Micelles
with thioketal linkage
Deqi Wang (Methodology) (Investigation) (Formal analysis) (Writing
- original draft), Song Wang (Investigation) (Formal analysis),
Yingchun Xia (Methodology) (Writing - review and editing), Simeng
Liu (Validation), Ruixin Jia (Investigation) (Formal analysis), Gege
Xu (Formal analysis), Junjie Zhan (Investigation) (Validation),
Yanbing Lu (Conceptualization) (Methodology) (Supervision)
(Writing - review and editing)
PII:
S0927-7765(20)30632-9
DOI:
https://doi.org/10.1016/j.colsurfb.2020.111276
COLSUB 111276
Reference:
To appear in:
Colloids and Surfaces B: Biointerfaces
Received Date:
Revised Date:
Accepted Date:
17 March 2020
22 July 2020
23 July 2020
Please cite this article as: Wang D, Wang S, Xia Y, Liu S, Jia R, Xu G, Zhan J, Lu Y,
Preparation of ROS-responsive Core Crosslinked Polycarbonate Micelles with thioketal
linkage, Colloids and Surfaces B: Biointerfaces (2020),
doi: https://doi.org/10.1016/j.colsurfb.2020.111276

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Preparation of ROS-responsive Core Crosslinked
Polycarbonate Micelles with thioketal linkage
Deqi Wang a, #, Song Wang b, #, Yingchun Xia a, Simeng Liu a, Ruixin Jia a, Gege Xu a, Junjie
Zhan a, Yanbing Lu*a, b
a College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082,
P. R. China.
b State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry
and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular
Chemical Biology, Hunan University, Changsha 410082, P. R. China
# Contributed equally.
* To whom correspondence should be addressed. E-mail: yanbinglu@hnu.edu.cn.
Graphic abstract
DOX
1

Highlights



ROS-responsive core crosslinked micelles were conveniently prepared.
The obtained CCL/TK micelles exhibited good biocompatibility and low toxicity.
DOX-loaded CCL/TK micelles had higher toxicity effects for HeLa and MCF-7
cells.
Abstract:
Herein, we prepared novel reactive oxygen species (ROS) responsive core
crosslinked (CCL/TK) polycarbonate micelles conveniently by click reaction between
amphiphilic diblock copolymer poly(ethylene glycol)-poly(5-methyl-5-propargylxycar-
bonyl-1,3-dioxane-2-one) (PEG-PMPC) with pendant alkynyl group and thioketal
containing azide derivative bis (2-azidoethyl) 3, 3'- (propane-2, 2-diylbis (sulfanediyl))
dipropanoate (TK-N3). The CCL/TK micelles were obtained with small size of 146.4 nm,
showing excellent stability against dilution and high doxorubicin (DOX) loading. In vitro
toxicity tests demonstrated that the obtained CCL/TK micelles have good
biocompatibility and low toxicity with cell viability above 95%. Furthermore, DOX-
loaded CCL/TK micelles showed significantly superior toxicity with IC50 values for HeLa
and MCF-7 cells about 3.74 μg/mL and 3.91 μg/mL, respectively. Confocal laser
scanning microscope (CLSM) and flow cytometry showed excellent internalization
efficiency and intracellular drug release of DOX-loaded CCL/TK micelles. The obtained
ROS-responsive CCL/TK micelles showed great potential for anticancer drug delivery.
Keywords: polycarbonate; core crosslinked micelles; drug delivery; ROS responsive
2

materials; thioketal
1. Introduction
Nanocarriers have wide application prospects in the field of biomedicine, and have
unique advantages in drug delivery, biosensors and bioimaging [1-3]. Great progress
has been made in targeted delivery of different types of active agents such as
anticancer drugs, genetic agents, and proteins, which are based on nanocarriers
represented by liposomes, polymer nanoparticles, amphiphilic polymer micelles and
dendrimers [4, 5]. Nanocarriers self-assembled by amphiphilic polymer micelles have
several distinct merits in the delivery of anticancer drugs. For example, it improves the
stability of the drug during transportation, increases the bioavailability of the drug,
and extends the circulation time [6-8]. In addition, nano-sized polymer micelles can
improve tumor accumulation through the enhanced permeability and retention (EPR)
effect [9]. Polymer micelles are promising as a platform for specific targeted delivery
of anticancer drugs. In the past decades, various types of nanocarriers had emerged
[10]. However, many nanocarriers have the problem of premature drug leakage during
transportation, which severely limits their clinical applications [11]. Undesired
disintegration occurs prematurely because nanocarriers are diluted by blood when
injected into a blood vessel. This not only reduces the therapeutic effect, but also
causes a large number of aggregates in normal cells or tissues which cause serious side
effects [12, 13]. Therefore, addressing the stability of nanocarriers during
transportation and achieving drug release at diseased sites play an extremely
3

important role in cancer treatment.
Stimuli-responsive core crosslinked (CCL) micelles have been extensively studied as
a strategy to improve the stability of nanocarriers [14-16]. Drugs encapsulated by
stimulus-responsive CCL micelles can be released by the triggers such as pH [17], redox
[18], reactive oxygen species (ROS) and enzymes [19], in appropriate cellular
microenvironment. Compared with uncrosslinked (UCL) micelles, CCL micelles have
the obvious superiority to improved drug loading and prevented the disintegration of
the drug carrier by the extreme dilution of biological fluids [20]. However, while the
crosslinked micelles prevent dilution, they also restrain the drug from being released
in the hydrophobic inner core, which resulting in insufficient therapeutic effects [21].
In comparison with permanent CCL micelles, decrosslinked micelles can solve this
limitation and achieve a strategy for enhance the effect of cancer treatment [22, 23].
ROS is a substance that contains oxygen and has high reactivity, including singlet
oxygen, superoxide anion radicals, hydroxyl radicals and hydrogen peroxide [24].
Compared with normal tissues, the level of ROS in cancer cells is higher than that in
normal tissues, the difference in ROS concentration provides a physiological and
chemical basis for developing a novel stimulus-responsive nanocarrier which can be
applied to cancer therapy [25, 26]. Currently, ROS-responsive functional groups are
often used to construct and design anticancer drug carriers such as Se [27], Te [28],
arylboronic acid [29], thioketal [30], and thioether [31]. The thioketal groups can be
broken under the stimulation of a suitable tumor microenvironment, and have been
widely explored to build stimuli-responsive drug carrier to achieve the triggered
4

release of drugs at the target site [25, 32-37]. Li [38] synthesized a diblock copolymer
(PEG-TK-PLA) by using thioketal as a connecting bridge between PEG and PLA, which
was used to encapsulate the anti-cancer drug PTX to achieve on-demand drug delivery.
What’s more, Xu [39] designed a ROS-responsive prodrug micelle based on PEG and
anticancer drug DOX in combination with ROS-cleavable thioketal linkage between
PEG and DOX, and Li [40] synthesized polyphosphoester-based, core crosslinked ROS
responsive micelles for delivery of anticancer drugs.
Aliphatic polyesters and polycarbonates have the advantages of low toxicity,
biodegradability and excellent biocompatibility, and have been widely studied for used
in medical devices, genes, and drug delivery [41]. Based on biodegradability and
biocompatibility, polycarbonates that used to design amphiphilic polymer micelles
with different stimuli responsiveness has been continuously explored for the delivery
of anticancer drugs [42]. Polycarbonates as the hydrophobic core can not only
improved the solubility of hydrophobic drugs but also enhanced the biocompatibility
of polymer micelles. In addition, polycarbonates had the adjustable side chain
structure, which showed attractive advantages in the construction and design of CCL
micelles [43]. Wu [44] prepared pH-responsive CCL PEG-b-poly(mono-2, 4, 6-
trimethoxy pentaerythritol benzylidene-carbonate) (PEG-b-P(TMBPEC-co-AC)) based
micelles for intracellular release of paclitaxel (PTX). Li [45] prepared of pH and redox
dual-sensitive CCL micelles though the reaction of bis-(azidoethyl) disulfide with the
alkynyl group on the side chain of a pH responsive PEG-p (TMBPEC-co-MPMC) for DOX
delivery. Our group [46] also synthesized redox sensitive CCL micelles based on
5

amphiphilic diblock copolymer PEG-b-polycarbonate (p(MPC)), in which a-lipoic acid
derivative (LA) was introduced to form crosslinked network structure under the
catalysis of DTT, enabling release of encapsulated drugs in the existence of 10 mM GSH.
Based on self-assembly of amphiphilic diblock polyethylene glycol-polycarbonate
micelles (PEG-PMPC), ROS responsive core crosslinked polymer micelle was
synthesized by the azide-alkyne click method for delivery of anticancer drug DOX
(Scheme 1). The shell composing of PEG could prolong the circulation time in the blood,
polycarbonate block as the inner core of micelles could improve the biocompatibility
of CCL/TK micelles and reduce biotoxicity. The thioketal containing crosslinker TK-N3,
not only imparted ROS responsiveness to the amphiphilic micelles, but also improved
the stability and drug loading of the micelles. The cytotoxicity of micelles were
investigated in HeLa and MCF-7 cells. The anticancer effect and internalization
efficiency of DOX-loaded CCL/TK micelles were also investigated. Results showed that
the crosslinked micelles have low biotoxicity, and the DOX-loaded crosslinked micelles
have excellent anti-cancer effects and internalization efficiency.
Scheme 1 Illustration of ROS responsive CCL/TK micelles for DOX delivery in vitro. DOX loaded
CCL/TK micelles with high DOX loading efficiently accumulate and trigger encapsulated DOX release.
2. Experimental
6

2.1 Materials
2-Bromoethanol, 2,2-dimethylolpropionic acid, 3-mercaptopropionic acid, 3-
bromopropyne, 4-dimethylaminopyridine (DMAP), 1,8-diazabic-yclo[5.4.0]undec-7-
ene (DBU), N,N’-Dicyclohexylcarbodiimide (DCC), 1,6-dibromohexane and doxorubicin
hydrochloride (DOX·HCl) were purchased from Energy Chemical. Monomethoxy
poly(ethylene glycol) (PEG5k) was purchased from Sigma-Aldrich and L-sodium
ascorbate was purchased from Shanghai Titan Scientific Co., Ltd. Ethyl chloroformate
was purchased from Beijing Xiya Chemistry Co., Ltd. Cyclic carbonate monomer 5-
Methyl-5-propargylxycarbonyl-1,3-dioxane-2-one (MPC) was prepared as reported
before [47]. HeLa and MCF-7 cells were obtained from the cell bank of the central
laboratory at Hunan Xiangya hospital. DAPI was purchased from USA Solarbio.
RPMI1640, DMEM medium was obtained from Beijing neuronbc. Fetal bovine serum
(FBS) was purchased from Biological Industries, USA. Penicillin-Streptomycin, 0.25%
Trypsin-EDTA was obtained from Suzhou NCM Biotech. All other reagents and solvents
were purchased from Sinopharm Chemical Reagent Co. Ltd.
2.2 Characterization
1H NMR and 13C NMR spectra were tested by a 400 MHz spectrometer (Bruker, USA).
The number molecular weight (Mn) and polydispersity index (Ð) of the obtained
copolymer were performed on a Waters 1515 gel permeation chromatography (GPC)
instrument (Waters, USA). The sizes of micelles were measured by Nano ZS90 zeta-
potential and particle analyzer (Malvern, UK). Transmission electron microscopy (TEM,
7

JEM-2100Plus) was used to observe the morphology of micelles. Fluorescent spectra
were tested using a Hitachi F-4600 fluorescence spectrophotometer (Hitachi, Japan).
Cell viability assay was measured on a SynergyTM Mx multi-mode microplate reader
(BioTek, USA). Confocal fluorescence imaging was obtained on a confocal laser
scanning microscope (CLSM, C1-Si, Nikon, Japan). Flow cytometric analysis was
measured on a Gallios flow cytometer (Beckman-Coulter, USA).
2.3 Synthesis of bis (2-azidoethyl) 3, 3'-(propane-2, 2-diylbis (sulfanediyl))
Dipropanoate (TK-N3)
As shown in Scheme S1, the thioketal containing crosslinker TK-N3 was prepared in
three steps. 3, 3'-(propane-2, 2-diylbis (sulfanediyl)) dipropionic acid (TK) was firstly
synthesized according to previous reports [39]. 3-Mercaptopropionic acid (10 g, 94.2
mmol) and anhydrous acetone (10.9 g, 188.4 mmol) were mixed in a flask and dry
hydrogen chloride atmosphere (HCl (g)) was continuously introduced for 4 h. Then, the
reaction solution was crystallized in an ice-salt bath. The obtained product was washed
three times with hexane (20 mL) and cold water (20 mL) respectively. TK was collected
as a white solid. Yield: 13.1 g (55%).
TK (2.00 g, 7.94 mmol) and DCC (3.60 g, 17.46 mmol) were dissolved in anhydrous
DCM (60 mL), and the mixture was stirred at 0oC for 30 min. Then, 2-bromoethanol
(1.89 g, 15.12 mmol) and DMAP (0.20 g, 1.59 mmol) were added. After keeping
incubated at 0oC for 1 h, the reaction was stirred at room temperature for 24 h. The
mixture was filtered, and the filtrate was washed with saturated NaHCO3 and saturated
NaCl aqueous solution respectively. The organic phase was then dried over anhydrous
8

MgSO4, filtered, and evaporated to remove the solvent. The residue was further
purified by silica column chromatography with petroleum ether/ethyl acetate (4/1, v/v)
as eluent. TK-Br was obtained as colorless oil. Yield: 1.79 g (51%).
TK-Br (1.14 g, 2.46 mmol) and NaN3 (0.48 g, 7.42 mmol) were dissolved in DMF (25
mL), and the mixture was stirred at 80oC for 24 h. The reaction was quenched with
deionized water (50 mL) and extracted with ethyl ether (3×30 mL). The organic phase
was then washed with DI water (3×50 mL), dried over anhydrous MgSO4, filtered, the
solvent was removed by evaporation. TK-N3 was obtained as yellow oil. Yield: 0.62 g
(65%).
The non-responsive cross-linker, 1,6-diazidohexane was prepared according to the
method reported previously[45].
2.4 Synthesis of diblock copolymer PEG-PMPC
The diblock copolymer PEG-PMPC was synthesized via ring-opening polymerization
(ROP) of cyclic carbonate monomer MPC using PEG5k as a macro-initiator and DBU as
the catalyst. MPC (0.2 g, 1.01 mmol), PEG5k (0.202 g, 0.0404 mmol), DBU (0.77 mg,
1.01×10-2 mmol) and anhydrous DCM (2 mL) were put into a dried schlenk flask, and
degassed by three freeze–pump–thaw cycles. Subsequently, the mixture was keep
stirring at room temperature for 7 h. After that, the solution was precipitated by cold
ether and centrifugation. The product was dried in vacuum and a white powder was
obtained. Yield: 288 mg (71.3%).
2.5 Preparation and characterization of UCL micelles
9

UCL micelles were prepared via a dialysis method. PEG-PMPC (5 mg) was dissolved
in DMF (1 mL). Then, 10 mL of DI water was slowly dropwise added under vigorous
stirring. After keeping stirring at room temperature for 2 h, the micelles were prepared
via further dialysis against deionized water for 24 h (MWCO=3.5 kDa).
Nile Red (NR) was used as a fluorescent probe to measure the critical micelle
concentration (CMC) of PEG-PMPC. NR dissolved in THF (0.12 mg/L, 30 μL) was put in
the vials. After THF was evaporated, a series of micelles of different concentrations
(2×10-4-0.2 mg/mL) were added to the vials. Subsequently, the solution was kept
stirring overnight. The fluorescence intensity value of micelles were measured by a
fluorescence spectrometer (λex=550 nm, λem=560-720 nm).
2.6 Synthesis of CCL micelles
Taking CCL/TK micelles as an example. PEG-PMPC (5.0 mg, 0.5×10-3 mmol, 1 eq. of
alkyne groups) and TK-N3 (3.9 mg, 10.24×10-3 mmol, 0.8 eq. of alkyne groups) were
dissolved in 1 mL DMF and stirred for 1h. Then, 10 mL of DI water was added dropwise
under vigorous stirring. After stirred for 2 h, CuSO4·5H2O (0.78 mg, 3.125×10-3 mmol,
0.4 eq. of alkyne groups) and sodium ascorbate (0.62 mg, 3.125×10-3 mmol, 0.4 eq. of
alkyne groups) were added into the mixed solution. After stirred at room temperature
for 24 h, the CCL/TK micelles were prepared by dialysis against deionized water for 24
h (MWCO=3.5 kDa). Non-responsive cross-linked (CCL/CC) micelles were synthesized
using 1,6-diazidohexane as a crosslinker according to the same method.
2.7 DOX-loaded and ROS responsive release of DOX
10

DOX-loaded CCL micelles were prepared by the similar step as for blank CCL micelles.
Taken 20% of theoretical drug loading contents as an example, PEG-PMPC (10 mg/mL
in DMF, 0.2 mL), TK-N3 (1.56 mg) and DOX (5 mg/mL in DMSO, 100 μL) were mixed
together. Then, DI water (3 mL) was added dropwise into the mixture under vigorous
stirring. After 2 h, CuSO4·5H2O (0.312 mg, 1.25×10-3 mmol, 0.4 eq of alkyne groups)
and sodium ascorbate (0.248 mg, 1.25×10-3 mmol, 0.4 eq of alkyne groups) were
added into the mixed solution. After stirred at room temperature for 24 h, the DOX-
loaded CCL/TK micelles were obtained by dialysis against deionized water for 24 h. The
whole process was maintained in dark. DOX-loaded CCL/CC micelles were synthesized
by replacing TK-N3 crosslinker with 1,6-diazidehexane according to the same method.
In the absence of crosslinker, the DOX loaded UCL micelles were prepared via the same
procedure.
The drug loading capacity (DLC) and drug loading efficiency (DLE) of the micelles
were determined via calculating the fluorescence intensity of DOX in the medium
collected outside the dialysis bag. The content of loaded drug was measured by
spectrofluorimetry (DOX: λex=485 nm, λem=592 nm). The DLC and DLE were calculated
via the following formulation.
DLC (%) = (weight of loaded drug/total weight of polymer and loaded drug) × 100%
DLE (%) = (weight of loaded drug/weight of feeding drug) × 100%
The in vitro drug release profiles from DOX-loaded CCL/TK micelles were studied in
different media (PBS (10 mM, pH=7.4), 5 mg/mL and 10 mg/mL H2O2). 1 mL of DOX-
loaded CCL/TK micelles (CCL/TK micelles: 0.4 mg/mL, DOX: 88 μg) were placed in a
11

dialysis bag (MWCO=8.0-14.0 kDa), which was immersed in 25 mL of various media
and followed by shaking at 37°C. At certain time intervals, 3 mL of release medium was
removed to determine the DOX concentration by fluorescence, and replenished with
an equal volume of same solution. The release results were performed in three parallel
experiments.
2.8 Cytotoxicity assay in vitro
In vitro cytoxicities of micelles were evaluated in HeLa and MCF-7 cells. Briefly, cells
were seeded on 96-well plates (6000 cells per well) and incubated in fresh RPMI-1640
medium containing 10% FBS for 24 h. Then, micelle solutions with different
concentrations were added into the wells, which had been replaced with fresh culture
media. In absent of micelle solution, cells were treated as control. After 24 h, MTT (5
mg/mL) was added and further incubated for another 4 h. After that, the unreacted
MTT was removed carefully and replaced with DMSO to dissolve the crystals formed
by the living cells. The absorbance of the each well at the wavelength of 570 nm was
measured using a microplate reader to determine cell relative viability. The
cytotoxicities of DOX-loaded CCL/TK, CCL/CC, UCL micelles and free DOX were tested
in the same way. In addition, the cytoxicities of DOX-loaded CCL/TK micelles and free
DOX after being cultured for 48 h were also investigated. The cytoxicities of CCL/TK
micelles with exogenous H2O2 was also investigated in HeLa cells for 24 h, according to
the method reported previously [48].
2.9 Cellular uptake in vitro
12

HeLa cells were planted on 24-well plates (2×104 cells per well) and incubated in
RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin for 24 h (37°C,
5% CO2) and then cultured with DOX-loaded CCL/TK, CCL/CC, UCL micelles and free
DOX for 4 h and 8 h respectively (37°C, 5% CO2) at a final DOX concentration of 2 μg/mL.
The cells were fixed with 4% paraformaldehyde, and the nuclei were stained with DAPI.
Fluorescent images were observed in a confocal laser scanning microscope (C1-si,
Nikon, Japan). The cell uptake was further detected using a flow cytometer. HeLa cells
were seeded on 24-well plates (2×104 cells per well) containing fresh cell culture
medium for 24 h and then cultured with DOX-loaded CCL/TK, CCL/CC, UCL micelles and
free DOX (DOX dose: 5 μg/mL) for 4 h and 8 h, respectively. The cells were then
digested by trypsin and collected by centrifugation. Subsequently, the sample was
analysis by flow cytometry.
3. Results and discussion
3.1 Preparation and characterization of ROS responsive core crosslinked micelles
Amphiphilic diblock copolymer PEG-PMPC was obtained by ring-opening
polymerization of cyclic MPC using PEG as initiator and DBU as catalyst (Scheme S1)
according to the method described previously [47]. PEG-PMPC was successful
synthesized and confirmed by 1H NMR analysis (Fig. 1a). The theoretical degree of PEG-
PMPC of the polymer was set at 25 and calculated to be 23, comparing the integrals
of peaks at δ = 3.58 (methylene protons of PEG) and δ = 2.48 (the alkynyl proton of
PMPC). GPC curve of MPC-PMPC showed that the number average molecular weights
13

and polydispersity index (Ð) of the obtained PEG-PMPC was 8.9 kDa and 1.11 (Table
S1, Fig. S1). The thioketal containing crosslinker TK-N3 and non-responsive crosslinker
1,6-diazidohexane were synthesized according the route shown in Scheme S1. The
1
successful synthesis of the TK-N3 and 1,6-diazidohexane were confirmed via H NMR
spectra (Fig. S2-S4), 13C NMR spectra (Fig. S5) and FT-IR spectra (Fig. S6).
(a)
c
a
b
d
b
e
f
CDCl3
d
e
c
f
a
(b)
(c)
Cross-linked polymer
H2O
PEG
DMSO
e
b
d
a
c
e
CDCl3
c
d
a
b
8
7
6
5
4
3
2
1
0
δ (ppm)
Fig.1. 1H NMR spectra of PEG-PMPC (a), crosslinked polymer (b) and crosslinker TK-N3 (c).
ROS-responsive core crosslinked micelles were prepared via click reaction between
thioketal containing crosslinker TK-N3 and pendant alkynyl groups of hydrophobic
polycarbonates block of PEG-PMPC. As shown in Fig. 1b, the proton peaks of the
hydrophobic inner core of the core crosslinked polymer and TK-N3 were not observed.
It was probably because the crosslinked structure hindered the dissolution of the inner
core and shielded the proton peak of the hydrophobic inner core [18].
The critical micelle concentration (CMC) of PEG-PMPC was determined to be
approximately 0.029 mg/mL using NR as a fluorescent probe (Fig. 2a). The size and
morphology of CCL/TK, CCL/CC and UCL micelles were determined with DLS and TEM.
The particle size of CCL/TK, CCL/CC and UCL micelles were 146.4 nm, 144.1 nm and
14

98.8 nm measured by DLS (Fig. 2b). The increased size of micelles after crosslinking
may due to the crosslinked network structure formed by the hydrophobic core, which
made the core of the micelles swell [18, 49]. TEM images of CCL/TK, CCL/CC and UCL
micelles showed a spherical morphology formed in the aqueous solution with a size of
35.6 nm, 33.1 nm and 28.7 nm (Fig. 2e). The smaller particle size obtained by TEM than
DLS was due to the shrinkage of the hydrophilic shell after drying [17, 20]. Consistent
with the results obtained by DLS, TEM images also showed that the particle size of
CCL/TK micelles was larger than those of CCL/CC and UCL micelles.
In order to evaluate the stability of CCL/TK, CCL/CC and UCL micelles, micelles
against extensive dilution were investigated and their changes in diameters were
determined by DLS. When diluted with 10-fold volume of DMF, the CCL/TK and CCL/CC
micelles still maintained their structures while the sizes only increased slightly, but the
sizes of UCL micelles became chaotic and smaller sizes were observed (Fig. 2c).
Moreover, after 100-fold dilution with water to let the micelle concentration below
the CMC of PEG-PMPC, the sizes of the CCL/TK and CCL/CC micelles increased slightly.
It may be due to the presence of crosslinked network structure of the CCL micelles that
only increased the sizes because of swelling. In contrast, the UCL micelles disintegrated
under 100-fold dilution of water (Fig. 2d). Additionally, the stability of the micelles in
PBS (10 mM, pH 7.4) and plasma were also evaluated. Compared with UCL micelles,
the particle sizes of CCL/TK and CCL/CC micelles were not significantly different in PBS
and plasma, while the particle sizes of UCL micelles increased slightly (Fig. S6). These
results indicated that CCL/TK and CCL/CC micelles had excellent stability due to the
15

core cross-linked network.
(a)1500
(b)
(d)
UCL: 98.8 nm (PDI: 0.16)
CCL/TK: 146.4 nm (PDI: 0.23)
CCL/CC: 144.1 nm (PDI: 0.19)
40
30
20
10
0
1200
900
PEG-PMPC
600
CMC=0.029 mg/mL
300
0
1
10
100
1000
10000
-4.0
-3.5
-3.0
-2.5
-2.0
-1.5
-1.0
-0.5
Diameter (nm)
LogC (C= mg/mL)
(c)
(e)
UCL-DMF: 575.0 nm (PDI: 0.87)
CCL/TK-DMF: 319.4 nm (PDI: 0.17)
CCL/CC-DMF: 213.3 nm (PDI: 0.21)
UCL-water: 591.1 nm (PDI: 0.83)
CCL/TK-water: 167.6 nm (PDI: 0.30)
CCL/CC-water: 166.5 nm (PDI: 0.31)
40
30
20
10
0
40
30
20
10
0
1
10
100
1000
10000
1
10
100
1000
10000
Diameter (nm)
Diameter (nm)
CCL/TK
UCL
CCL/CC
CCL/CC
200 nm
CCL/TK+H2O2
200 nm
CCL/CC+H2O2
200 nm
UCL+H2O2
200 nm
200 nm
200 nm
Fig. 2. The CMC value of UCL micelles (a). The size distributions of UCL, CCL/TK and CCL/CC micelles
in water (b), after 10-fold dilution with DMF (c), after 100-fold dilution with water (d). TEM images
of CCL/TK, CCL/CC and UCL micelles before and after treatment with 10 mM H2O2 (e).
The stability of CCL/TK micelles against reactive oxygen species H2O2 was also
evaluated. After 48 h of incubation with 5 mM, 10 mM and 20 mM H2O2, the sizes of
CCL/TK micelles decreased gradually to 137.0 nm, 127.3 nm and 120.9 nm respectively
16

(Fig. S7). However, the sizes of CCL/CC and UCL micelles remained unchanged. It was
possibly due to decrosslinking of CCL/TK micelles caused by thioketal cleavage. It can
also be obtained from the TEM images that the diameters of CCL/TK micelles became
smaller after cultured with H2O2, while CCL/CC and UCL micelles maintained
unchanged morphology (Fig. 2e).
3.2 DOX loading and the vitro drug release of CCL micelles
DOX-loaded micelles were formed by hydrophobic interactions. The theoretical DOX
loading contents were set at 10, 20 and 30 wt.%. The DLC and DLE values of DOX-
loaded CCL/TK and CCL/CC micelles were remarkably higher than those of DOX-loaded
UCL micelles under the same theoretical drug loading (Table 1). It demonstrated that
the crosslinking network of the CCL/TK and CCL/CC micelles could prevent the leakage
of the loaded DOX, thereby extending the circulation time in the blood. Compared with
unloaded micelles, the sizes of DOX-loaded CCL/TK, CCL/CC and UCL micelles increased
significantly. It was due to the encapsulated DOX expanding the core of the micelles.
Table 1. Characterization of DOX-loaded micelles
UCL Micelles
CCL/CC Micelles
CCL/TK Micelles
DOX feed ratio
(wt%)
DLC
(%)
DLE
Size
DLC
(%)
DLE
(%)
Size
DLC
(%)
DLE
(%)
Size
PDI
PDI
PDI
(%)
(nm)
144.1
155.1
227.1
(nm)
151.7
171.7
192.6
(nm)
156.8
191.9
241.4
10
20
30
4.8
50.1
62.5
54.0
0.12 7.7
0.20 14.7
0.20 20.9
84.7
88.2
90.6
0.27 7.6
0.14 16.9
0.18 25.8
75.6
84.9
86.1
0.11
0.38
0.34
10.4
12.5
The in vitro ROS responsiveness of DOX-loaded CCL/TK micelles was performed at
different concentrations of H2O2 (0, 5, 10 mM). In the absence of H2O2 (PBS, pH 7.4),
DOX-loaded CCL/TK micelles had a cumulative release of only 20% in 48 h. When the
concentration of H2O2 increased to 5 mM and 10 mM, the cumulative release of DOX
17

reached 51% and 65%, respectively (Fig. 3). At the same time, DOX cumulatively
released 16% from DOX-loaded UCL micelles at pH 7.4, and 18% from DOX-loaded
CCL/CC in the presence of 10 mM H2O2. It demonstrated that the DOX-loaded CCL/TK
micelles showed ROS-responsive behavior and could undergo decrosslinking to release
encapsulated DOX.
UCL-pH 7.4
100
CCL/CC-10 mM H2O2
CCL/TK-pH 7.4
80
CCL/TK-5 mM H2O2
CCL/TK-10 mM H2O2
60
40
20
0
0
10
20
30
40
50
Time (h)
Fig. 3. In vitro DOX release profiles of DOX-loaded micelles in the presence and absence of H2O2.
3.3 In vitro cytotoxicity assay
The in vitro cytotoxicities of CCL/TK, CCL/CC, UCL micelles against HeLa and MCF-7
cells were evaluated by MTT assay. The cells were incubated for 48 h at varying
concentrations of micelles from 0 to 500 μg/mL. Results showed that CCL/TK, CCL/CC
and UCL micelles had excellent biocompatibility and low toxicity. HeLa and MCF-7 cells
had a high viability more than 95% at all tested concentrations (Fig. 4a-4b).
The in vitro cytotoxicities of DOX loaded micelles also were evaluated in HeLa and
MCF-7 cells using MTT assay (Fig. 4c-4d) with free DOX as control. The results showed
that cell survival rate decreases with increasing DOX concentration. The half-maximal
inhibitory concentration (IC50) of free DOX (2.52 μg/mL for HeLa, 2.67 μg/mL for MCF-
18

7) was significantly lower than those of DOX-loaded CCL/TK (3.74 μg/mL for HeLa, 3.91
μg/mL for MCF-7), DOX-loaded CCL/CC (5.15 μg/mL for HeLa, 4.67 μg/mL for MCF-7)
and DOX-loaded UCL (7.18 μg/mL for HeLa, 6.74 μg/mL for MCF-7) micelles. Compared
with DOX-loaded CCL/CC and UCL micelles, DOX-loaded CCL/TK micelles had higher
cytoxicity to HeLa and MCF-7 cells. DOX-loaded CCL/TK micelles exhibited similar
cytotoxicity as free DOX, and could release loaded DOX to kill cancer cells. Compared
with free DOX, DOX-loaded CCL/TK micelles had lower cytotoxicities. The possible
reason was that the process of DOX release from the ROS-responsive DOX-loaded
CCL/TK micelles was delayed after CCL micelles endocytosed into the cell [45]. When
the incubation time extended to 48 h, DOX-loaded CCL/TK micelles (1.68 μg/mL for
HeLa, 0.97 μg/mL for MCF-7) showed higher cytoxicity than free DOX (2.37 μg/mL for
HeLa, 2.04 μg/mL for MCF-7) (Fig. S8). Because the H2O2 concentration of in vitro
cancer cells was much lower than that of tumor tissue and the cell culture medium
could consume H2O2 [48], exogenous H2O2 was added to explore whether the addition
of exogenous H2O2 could enhance the cytoxicity of DOX-loaded CCL/TK in HeLa cells.
The cytoxicity of H2O2 was evaluated by the MTT assay (Fig. S9) and the appropriate
concentration of H2O2 was set to be 30 μM. As shown in Fig. S10, trace amount of
exogenous H2O2 had significant effect on the toxicity of DOX-loaded micelles. With the
addition of 30 μM exogenous H2O2, the IC50 of DOX loaded CCL/TK, CCL/CC and UCL
micelles were 2.39, 3.37 and 3.79 μg/mL respectively. The addition of exogenous H2O2
could not only enhance the toxicity of DOX loaded CCL/TK micelles but also enhanced
the toxicity of DOX loaded CCL/CC and UCL micelles. It should further be noted that
19

the cytotoxicity of DOX-loaded CCL/TK micelles might be enhanced via some ways to
further increase ROS levels in cancer cells such as loaded photosensitizer [38, 40].
140
120
100
80
140
120
100
80
(a)
(b)
CCL/TK
CCL/CC
UCL
CCL/TK
CCL/CC
UCL
60
60
40
40
20
20
0
0
0
50
100
200
300
400
500
0
50
100
200
300
400
500
Micellce Concentration (μg/mL)
Micellce Concentration (μg/mL)
120
100
80
60
40
20
0
120
100
80
60
40
20
0
(c)
(d)
Free DOX
Free DOX
CCL/TK-DOX
CCL/CC-DOX
UCL-DOX
CCL/TK-DOX
CCL/CC-DOX
UCL-DOX
0
2
4
6
8
10
0
2
4
6
8
10
DOX Concentration (μg/mL)
DOX Concentration (μg/mL)
Fig. 4. Cytotoxicity of micelles in HeLa cells (a) and MCF-7 cells (b), free DOX and DOX loaded
micelles in HeLa (c) and MCF-7 cells (d). The cells were incubated for 24 h. Data were expressed as
mean ± standard deviation (n=3).
3.4 In vitro cellular uptake
The cellular internalization and intracellular distribution of DOX-loaded micelles
were investigated in HeLa cells by CLSM and flow cytometry. From the CLSM image
(Fig. 5 and Fig. S11), the brighter red DOX fluorescence appeared surrounding the
nucleus after DOX-loaded CCL/TK micelles incubated for 4 h. When extending the
incubation time to 8 h, stronger DOX fluorescence was observed around the nucleus.
However, the fluorescence intensity of free DOX was relatively weak under same
culture time (4 h, 8 h), which was possibly due to the fluorescence quenching of free
20

DOX after intercalation into nuclear DNA [50]. Another possible reason was the
different ways to enter the cell. Free DOX entered the cell by diffusion, and it was easily
effluxed from the cell [51]. DOX loaded micelles entered the cells through endocytosis,
while the DOX released from loaded CCL/TK micelles may not be easily effluxed for a
relatively long period [52]. Compared with DOX loaded CCL/CC and UCL micelles, DOX
loaded CCL/TK micelles were observed to have stronger DOX fluorescence. This may
be attributed to the fact that DOX loaded CCL/TK micelles could release more DOX in
cancer cells, which were demonstrated by in vitro DOX release expriments. According
to previous reports, the fluorescence of DOX was quenched when DOX was
encapsulated in the core of micelles [53]. When DOX was released from the micelles,
its fluorescence became stronger. Furthermore, flow cytometry results (Fig. 6 and Fig.
S12) were consistent with those of CLSM. The fluorescence intensity of DOX-loaded
CCL/TK micelles in HeLa cells was markedly higher than those of DOX loaded CCL/CC,
DOX loaded UCL and free DOX after being cultured for 4 h or 8 h. The results proved
that the DOX-loaded CCL/TK micelles had excellent internalization efficiency, which
could effectively release the encapsulated DOX in cancer cells.
21

DAPI
DOX
Merge
Free DOX-4 h
100 μm
100 μm
100 μm
100 μm
100 μm
100 μm
100 μm
CCL/TK-4 h
CCL/CC-4 h
100 μm
100 μm
UCL/DOX-4 h
100 μm
100 μm
100 μm
Fig. 5. CLSM images of HeLa cells after incubation with the free DOX, DOX loaded CCL/TK, CCL/CC
and UCL micelles for 4 h (DOX dosage: 2 μg/mL).
UCL-DOX-4 h
CCL/CC-DOX-4 h
CCL/TK-DOX-4 h
Free DOX-4 h
Control
102
103
104
Intensity
105
106
Fig. 6. Flow cytometry analysis of the HeLa cells incubated with free DOX and DOX loaded CCL/TK,
CCL/CC and UCL micelles for 4h (DOX dosage: 5 μg/mL).
4. Conclusions
In conclusion, novel ROS-responsive core crosslinked polycarbonate micelles were
22

conveniently synthesized. The introduction of thioketal containing crosslinker TK-N3
not only enhanced the stability of the micelles and improved the drug loading, but also
endowed ROS-responsiveness for triggered intracellular drug release. The obtained
CCL/TK micelles exhibited good biocompatibility and low toxicity for HeLa and MCF-7
cells. DOX-loaded CCL/TK micelles had significantly higher toxicity effects for HeLa and
MCF-7 cells, and exhibited excellent internalization efficiency and drug release inside
the cancer cells. As a result, polycarbonate based CCL/TK micelles are promising
potential as a nanocarrier for drug delivery.
Credit Author Statement:
Deqi Wang: Methodology, Investigation, Formal analysis, Writing-Original draft
preparation Song Wang: Investigation, Formal analysis Yingchun Xia: Methodology,
Writing-Reviewing and Editing Simeng Liu: Validation, Ruixin Jia: Investigation, Formal
analysis Gege Xu: Formal Analysis Junjie Zhan: Investigation, Validation Yanbing Lu:
Conceptualization, Methodology, Supervision, Writing-Reviewing and Editing
Declaration of interests
The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this
paper.
Author Contributions
23

Yanbing Lu* contributed to design and guide of the research.
Deqi Wang# and Song Wang# contributed equally to this work.
Deqi Wang performed the synthesis and characterization of the CCL micelles and
data analyses, and wrote the manuscript.
Song Wang contributed to the in vitro experiments.
Yingchun Xia, Simeng Liu, Ruixin Jia, Gege Xu and Junjie Zhan contributed to the
conception of the study and discussion.
Yanbing Lu contributed to design of the research, data investigation, the conception
of the study and discussion, and wrote the manuscript
Disclosure statement
The authors declare no competing financial interests.
Fund
This research did not receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors.
Appendix A. Supplementary data
Supplementary material in online version
24

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