G. Saravanakumar, et al.
Journal of Controlled Release 327 (2020) 627–640
chemical structure of the hydrophobic block segment [28,29]. A wide
range of macromolecular amphiphiles with diverse architectures in-
cluding diblock, triblock, graft and dendritic copolymers have been
explored for the preparation of polymersomes [30,31]. Recent advances
in polymer syntheses and various click reactions such as azide-alkyne
cycloaddition [32], thiol-ene [33], thiol-acrylate [34], and Diels-Alder
reactions [35] have emerged as a powerful tool and greatly expanded
our ability to construct such functional polymer structures. More re-
cently, Tang et al. reported a new type of spontaneous click poly-
merization between terminal alkynoates and secondary (2°) amines to
synthesize poly(β-aminoacrylate) (PBAC) homopolymers [36]. It should
be emphasized that this click polymerization could produce regio- and/
or stereoregular polymers of high molecular weight under mild reaction
condition without any catalyst. Interestingly, the β-aminoacrylate
Industries (Osaka, Japan). All other chemicals were of analytical grade
and used as received unless otherwise specifically mentioned.
2.2. Instrumental methods
1
H NMR spectra were recorded on a FT-300 and 500 MHz Bruker
Aspect Spectrometer using the residual proton resonance of the solvent
as the internal standard. The chemical shifts are reported in parts per
million (ppm). The dynamic light scattering (DLS) measurements were
conducted using a Zetasizer Nano S90 system (Malvern Instruments,
Worcestershire, UK). All the measurements were carried out at 25 °C
with a 90° detection angle. UV–visible spectra were recorded on a UV
2550 spectrophotometer (Shimadzu), and fluorescence spectra were
recorded on a RF-5301 PC spectrofluorophotometer (Shimadzu). The
morphology of self-assembled triblock copolymers was examined using
transmission electron microscope (TEM, JEM-1011, JEOL, Tokyo,
Japan). The samples were prepared by placing a drop of colloidal sus-
pension on a carbon coated copper grid, followed by blotting excess of
water, air-dried and negatively stained with uranyl acetate.
linkage that generated during the polymerization can be cleaved by
1
reactive oxygen species (ROS) singlet oxygen ( O
2
) [37,38]. Despite
these favorable characteristics, no significant efforts were made to ex-
ploit PBAC as building blocks to construct functional nanomaterials for
1
biomedical applications [39,40]. In order to design O
2
-responsive drug
delivery systems using the β-aminoacrylate linkage, it is also highly
indispensable to synthesis of well-defined amphiphilic PBAC-based
block copolymers rather than simple homopolymers.
2.3. Cell lines
Towards this aim, in this study, by exploiting amino-alkynoate click
polymerization, we have synthesized two pairs of ABA-type amphi-
philic copolymers composed of poly(ethylene glycol) (PEG) as the hy-
drophilic segment and PBAC with either aromatic or aliphatic mole-
cular backbone structure unit as the discrete hydrophobic segment. The
as-synthesized copolymers were self-assembled into nanosized poly-
4 T1 cells (murine mammary carcinoma cells) and CT26 cells
(murine colon carcinoma cells) were cultured in Dulbecco's modified
Eagle's medium (DMEM, Hyclone) supplemented with 10% fetal bovine
−
1
−1
serum (FBS, Hyclone), 100 U mL
penicillin, and 100 g mL
strep-
tomycin. The cells were incubated at 37 °C in a 5% CO
incubator.
2
humidified
mersome structures in an aqueous condition. To explore, the potential
1
of the polymersomes as a
O
2
-responsive drug carrier, a hydrophobic
2.4. Synthesis of dialkynoate monomers
near infrared (NIR) photosensitizer IR-780 and a hydrophobic antic-
ancer drug doxorubicin (DOX) were physically co-encapsulated into
their membrane structure. The rationale behind choosing the IR-780
photosensitizer is that it exhibits a strong fluorescence intensity and
2.4.1. Bisphenol A dipropiolate monomer (M2a) [41]
Bisphenol A (1.63 g, 7.14 mmol), DCC (4.42 g, 21.42 mmol), DMAP
(0.349 g, 2.86 mmol), and p-TsOH (0.485 g, 2.55 mmol) were dissolved
in 120 mL of dry DCM/THF (3:1 v/v). Then, the solution was cooled to
0 °C using an ice-water bath, into which propiolic acid (1.1 g,
15.71 mmol) dissolved in 10 mL of dichloromethane/tetrahydrofuran
(DCM/THF; 3:1 v/v) was added under stirring via a dropping funnel.
The reaction mixture was stirred overnight. After filtering out the solid,
the solution was concentrated by a rotary evaporator. The crude pro-
better stability compared to that of other clinically approved NIR-ab-
1
sorbing dye indocyanine green. In addition to the generation of O
2
, IR-
7
80 dye also converts the NIR photon energy into heat, thus it enables
to realize both photodynamic therapy (PDT) and photothermal therapy
(
PTT). More importantly, unlike ultraviolet radiation, NIR light has a
much deeper penetrating depth for biological tissues with minimum
photodamage to the living cells. We further envisioned that the PEG
shell of the polymersomes provides a long circulation half-life and
duct was purified by a silica gel column using CH
2
Cl /hexane (1:2 v/v)
2
1
as eluent. H NMR (300 MHz, CDCl
3
) δ, ppm: 1.66 (s, 6H), 3.06 (s, 2H),
1
3
improves their accumulation at the tumor site, after systemic admin-
7.03(d, 4H), 7.22 (d, 4H). C NMR (75 MHz, CDCl ) δ, ppm: 151.2,
3
1
istration. The
O
2
-generated upon NIR light irradiation of IR-780 and
148.7, 147.9, 128.2, 120.9, 76.9, 74.5, 42.8, 31.1.
DOX co-loaded polymersomes not only exert PDT effect, but also induce
cleavage of β-aminoacrylate linkages and dissociation of the polymer-
2.4.2. Hexane-1,6-diyl dipropiolate monomer (M2b)
some structure, triggering the release of DOX to show tumor-specific
1,6-Hexanediol (1.18 g; 10 mmol), propiolic acid (2.10 g; 30 mmol)
and p-TsOH (0.19 g; 1 mmol) were taken in a round-bottom flask
equipped with a Dean-Stark apparatus, and 100 mL dry toluene was
added as a solvent. The resulting mixture was allowed to reflux for 36 h
with continuous removal of the yielded water. After completion of the
reaction, the solution was allowed to cool to room temperature, and
concentrated using a rotary evaporator. The residue was dissolved in
1
chemotherapy (Scheme 1). Thus, the designed
O -responsive poly-
2
mersome nanoplatform could overall enhance antitumor efficacy by
combining the chemo-phototherapies.
2
. Materials and methods
2.1. Materials
CH
2
Cl
2
, and washed with 5% aqueous NaHCO and water, and then
3
dried over MgSO . After filtration and solvent evaporation, the crude
4
Monomethoxy poly(ethylene glycol) (mPEG-OH, Mn = 2000 g/
product was purified by a silica gel column using n-hexane/ethyl
1
mol), propiolic acid (95%), 1,6-hexanediol (99%), bisphenol
A
acetate to afford M2b. (Yield: 2.09 g, 91%). H NMR (300 MHz, CDCl
3
)
(
≥99%), 4,4′-trimethylenedipiperidine (TMDP, 97%), N,N′-dicyclo-
hexycarbodiimide (DCC, 99.0%), p-toluenesulfonic acid monohydrate
p-TsOH, 98.5%), N,N-dimethyl-4-nitrosoaniline (RNO, 97%), and IR-
80 iodide (2-[2-[2-chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-
indol-2-ylidene)ethylidene]-1-cyclohexen-1-yl]ethenyl]-3,3-dimethyl-
δ, ppm: 4.17 (t, J = 6.0 Hz, 2H), 2.86 (s, 1H), 1.68(m, 2H), 1.39 (m,
13
2H). C NMR (75 MHz, CDCl ) δ, ppm: 152.9, 74.9, 74.8, 66.3, 28.3,
3
(
25.6.
7
2.5. Synthesis of mPEG-alkynoate
1
-propylindolium iodide, IR-780, 98%) were purchased from Sigma-
Aldrich Co. (St. Louis, MO). 4-Dimethylaminopyridine (DMAP, 99.0%),
was obtained from Samchun Chemicals Co. (Korea). Doxorubicin hy-
drochloride (DOX HCl) were supplied from Wako Pure Chemical
mPEG-OH (10 g; 5 mmol), propiolic acid (0.525 g; 7.5 mmol) and p-
TsOH (0.19 g; 1 mmol) were taken in a round-bottom flask equipped
with a Dean-Stark apparatus, and 100 mL dry toluene was added as a
628