C. Sun, et al.
Biomaterials225(2019)119514
hydrochloric acid (HCl) (30 mL). Tin (II) chloride (1.428 g) was added,
while stirring, and the mixture was then heated to 65 °C for 4 h after
reaction, the mixture was poured into ice water (100 mL) and further
neutralized with ammonium hydroxide until pH = 8. The mixed solu-
tion was then extracted with DCM until colorless. The organic layer was
concentrated under vacuum and further purified by column chroma-
tography isolation using DCM: ethyl acetate (EA) = 4:1 as elution to
give 100 mg purple mixture of 5, 10(15)-bis (4-aminophenyl)-15,20-
diphenylporphyrin.
sulfoxide, and then added into 10 mL of deionized water dropwise
under ultrasonication. Subsequently, the polymeric nanoparticles were
purified through 48 h dialysis (molecular cut off = 3.5 k), and finally
diluted to pre-determined concentration.
2.6. Singlet oxygen generation
Singlet oxygen sensor green (SOSG) was employed to detect the
generation of 1O2, which could be oxidized by 1O2, followed by en-
hancement of fluorescence, with excitation peaks at 504 nm and
emission peaks at 525 nm. Solution of PP3–Se with SOSG (~10 μM) was
irradiated with a 660 nm laser at a power density 1.0 W/cm2 for 2 or
4 min. The fluorescence intensity was recorded by RF-5301PC SPECT-
ROFLUOROPHOTOMETER (SHIMADZU).
1H NMR (400 MHz, DMSO‑d6) δ (ppm) 8.95 (d, J = 6.1 Hz, 4H),
8.78 (d, J = 7.2 Hz, 4H), 8.22 (dt, J = 7.6, 2.4 Hz, 4H), 7.86 (dtd,
J = 10.0, 5.4, 4.7, 2.2 Hz, 10H), 7.01 (dd, J = 8.4, 3.0 Hz, 4H), 5.60 (d,
J = 4.2 Hz, 4H), −2.80 (d, J = 4.3 Hz, 2H). 13C NMR (400 MHz,
DMSO‑d6) δ (ppm): 149.12, 141.99, 141.91, 136.03, 135.98, 134.67,
131.82, 128.97,128.86, 128.41, 127.43, 122.16, 121.66, 119.93,
119.48, 113.04, 113.02, 101.59. HR-MS (ESI+), calcd. 644.26885,
Found:645.27667 (M + H+).
1O2 levels were detected by the EPR trapping technique using
2,2,6,6-tetramethyl-4-piperidone (TEMP) as a spin trap. After irradiated
with a 660 nm laser at a power density 1.0 W/cm2 for 2 or 10 min, the
EPR spectra was recorded by JES-FA200 ESR Spectrometer.
2.3. Synthesis of bis(6-hydroxyhexyl) 3,3′-selenodipropionate and bis(6-
hydroxyhexyl) 6,6′-selenodihexanoate
2.7. Cell culture
3-Bromopropionyl Chloride (3.428 g, 20 mmol) was added dropwise
with stirring to a solution of 1,6-hexanol (2.3636 g, 20 mmol) in an-
hydrous tetrahydrofuran (THF) (20 mL) in ice water bath. The resulting
solution was stirred at room temperature for 12 h. After evaporation of
THF, the colourless oil was purified on a plug of silica gel, eluted with
EA:PE (from 1:3 to 1:2). After evaporation of the solvent, the residue
was dissolved into THF (20 mL) and then added into an aqueous so-
lution of selenium (0.5 equiv.) and sodium borohydride (4 equiv.). The
mixture solution was stirred at 50 °C overnight, and then extracted by
DCM (3*50 mL). After evaporation of solvent, the faint yellow oil was
purified through column chromatography isolation, eluting with EA:PE
(from 1:1 to 2:1).
Cell lines MDA-MB-231 (human breast cancer cells), A549 (human
lung cancer cells) and L-02 (human hepatocyte cells) were cultured in
DMEM supplied with 10% (v/v) FBS, streptomycin (100 μg/mL) and
penicillin (100 U/mL). Cells were incubated at 37 °C in a humidified 5%
CO2 atmosphere.
2.7.1. In vitro cytotoxicity and apoptosis
CCK-kit 8: MDA-MB-231, A549 and L-02 cells were seeded on 96-
well plates (7000 cells per well). After seeding for overnight, the cells
were cultured with various drug formulations for pre-determined time
(e.g. 48 h or 72 h). The cell viability was evaluated by CCK-8 kit assay
according to the manufacturer's instructions.
1H NMR (400 MHz, Chloroform-d) δ 4.09 (t, J = 6.6 Hz, 4H), 3.62
(t, J = 6.6 Hz, 4H), 2.81 (dd, J = 7.7, 5.8 Hz, 4H), 2.71 (dd, J = 7.7,
5.8 Hz, 4H), 1.61–1.51 (m, 8H), 1.39 (p, J = 3.5 Hz, 8H). 13C NMR
(400 MHz, CDCl3) δ (ppm): 172.30, 64.79, 62.70, 35.82, 32.51, 28.63,
25.80, 25.44, 18.01. HR-MS(ESI+), calcd. 426.15206, Found:
449.14122 (M + Na+).
Apoptosis: MDA-MB-231 cells were seeded on 6-well plates (for
HHA) or 24-well plates (for polymeric nanoparticles) at a density of 200
k or 80 k cells per well. After seeding 12 h, the cells were then co-
incubation with various drug formulations for 24 or 48 h (for HHA) or
for 4 h for PP3–Se or PP6–Se nanoparticles. To polymeric nanoparticles
group, we then irradiated the well for a certain time (660 nm laser,
0.75 W/cm2), and further cultured for 48 h. Cells were then stained
with Annexin V-FITC/PI Apoptosis Detection Kit, and further analysed
by FCM.
Bis(6-hydroxyhexyl) 6,6′-selenodihexanoate was prepared and pur-
ified in the similar way.
1H NMR (400 MHz, Chloroform-d) δ 4.07 (t, J = 6.7 Hz, 4H), 3.64
(t, J = 6.6 Hz, 4H), 2.55 (t, J = 7.4 Hz, 4H), 2.31 (t, J = 7.4 Hz, 4H),
1.74–1.56 (m, 12H), 1.53–1.32 (m, 16H). 13C NMR (400 MHz, CDCl3) δ
(ppm): 173.80, 64.40, 62.80, 34.32, 32.70, 30.40, 29.55, 28.80, 25.88,
25.49, 24.66, 23.81. HR-MS(ESI+), calcd. 510.24596, Found:
533.23519 (M + Na+).
2.8. Cell uptake and intracellular distribution
Cell uptake: MDA-MB-231 cells were seeded at a density of 100 k
per well onto 12-well plates, and then co-incubation with PP3–Se na-
noparticles (100 μg/mL) for 4, 8 or 24 h. The medium was then washed
by phosphate buffer saline (PBS) for three times and then replaced with
fresh medium. The cells were incubated with 1 mL trypsin for 1 min,
and then centrifugated to collect cells and then resuspended into 2 mL
PBS. Owing to the fluorescence imaging ability of Por, FCM was em-
ployed to analyse the cell uptake ability.
Intracellular distribution: MDA-MB-231 cells were seeded at a den-
sity of 100 k cells per well onto a glass-bottom dish with 2 mL medium
and then cultured overnight. The cells was then incubated with PP3–Se
nanoparticles (100 μg/mL) for 4 or 24 h at the dose of 19 μg/mL Por at
37 °C. And then the cells were washed by PBS for three times, and re-
placed with 1.0 mL fresh medium, which was contained with Hoechst
33343 (1.0 μg/mL) and LysTracker Green DND-26 (100 nM). After
20 min incubation, the cells were washed by PBS for three times before
CLSM observation (Leica SP8).
2.4. Synthesis of PP3–Se, PP6–Se, and poly-C6-C3(6)-Se-PEG2k
100 mg 5, 10(15)-bis (4-aminophenyl)-15,20-diphenylporphyrin
and 330.1 mg bis(6-hydroxyhexyl) 3,3′-selenodipropionate was dis-
solved in 5 mL of anhydrous DMF in a 100 mL flask and sealed with a
rubber plug. A solution of TDI (200 μL) in 10 mL anhydrous DMF was
injected to the flask under the atmosphere of nitrogen. After stirring for
6 h at 50 °C, 376 mg PEG dissolved in 10 mL anhydrous DMF was added
into this flask and the reaction was carried out for another 24 h. The
resulting products were washed by water and ethyl alcohol for three
times, respectively, followed by drying under vacuum to give a purple
sticky-solid.
PP6–Se, and Poly-C6-C3(6)-Se-PEG2k were prepared and purified in
the similar way.
2.5. Preparation of polymeric nanoparticles
2.9. ROS detection
20 mg of Se-polymers were first dissolved into 4 mL dimethyl
MDA-MB-231 cells were seeded at a density of 80 k cells per well
3