Y. Dong, et al.
InternationalJournalofPharmaceutics588(2020)119723
2.2. Synthesis methods
a dark red powder after freeze-drying.
2.2.1. Compound 1
2.4. Analysis and characterization
Compound 1 was synthesized by refluxing 4-bromo-1, 8-naphthalic
anhydride (5.52 g, 20 mmol) and ethanolamine (2.60 g, 43 mmol) in
temperature, the precipitate was collected by filtration and then wa-
shed with ethanol three times, and compound 1 was obtained after
drying with a yield of 87%. 1H NMR (400 MHz, DMSO‑d6, Fig. S1) δ
8.49 (dd, J = 15.0, 7.8 Hz, 2H), 8.32 – 8.22 (m, 1H), 8.16 (t,
J = 5.8 Hz, 1H), 8.01 – 7.86 (m, 1H), 4.83 (s, 1H), 4.13 (t, J = 6.4 Hz,
2H), 3.74 – 3.52 (m, 2H).
1H NMR spectra (proton nuclear magnetic resonance) were obtained
on a JEOL ECS 400 MHz NMR spectrometer.
The morphology of the prodrug nanoparticles was characterized on
a JEM1200 EX/S transmission electron microscope (TEM).
The hydrodynamic diameter and distribution of the prodrug nano-
particles were analyzed on a BI-200SM device (Brook Haven Instrument
Company, USA) using dynamic light scattering (DLS) technology.
The fluorescence spectra were recorded on a Hitachi F-7000 fluor-
escence spectrometer.
2.2.2. Compound 2
Compound 2 was synthesized by heating compound 1 (2.32 g,
7.2 mmol), p-hydroxybenzaldehyde (1.06 g, 8.7 mmol) and anhydrous
potassium carbonate (2.0 g, 14.4 mmol) in 50 mL of anhydrous DMF at
mixture of ethyl acetate (50 mL) and H2O (50 mL). The aqueous phase
was washed three times with ethyl acetate (50 mL × 3) and the organic
phases were combined. Then, the organic layer was washed with sa-
turated brine and water three times each and dried with anhydrous
MgSO4. The crude product of compound 2 was obtained after the sol-
vent was removed under reduced pressure and used directly for the next
step without further purification.
2.5. In vitro DOX release
pH-triggered DOX release was performed at 37 °C as follows:
10.0 mL of a dispersion of PPEGMA20-PNAP8-DOX prodrug nano-
particles (5.0 mg) in a buffered solution (PBS (pH 7.4) or ABS (pH 5.0))
was transferred into a dialysis bag (MWCO: 1000 Da). Then, the dis-
persion was dialyzed against the corresponding buffered solution
(100.0 mL). Five millilitres of each dialysate was removed to measure
the DOX concentration with a TU-1901 UV–vis spectrophotometer at
480 nm, and 5.0 mL of the fresh buffered solution was added to keep
the dialysate volume constant.
2.2.3. Compound 3
2.6. In vitro cytotoxicity assays
Compound 2 (1.00 g, 1.7 mmol) and triethylamine (0.91 g, 9 mmol)
were dissolved in CH2Cl2 (20 mL) and the solution was cooled to 0 °C.
Then, 5.0 mL of a methylacryloyl chloride (0.94 g, 9 mmol) solution in
dichloromethane was added dropwise over 1 h, and the reaction was
Afterwards, the resultant solution was poured into water and the or-
ganic layer was separated, washed with saturated aqueous Na2CO3 and
water three times each and dried with anhydrous MgSO4 overnight. The
organic layer was concentrated to obtain the crude product as a yellow
oil. Compound 3 was purified by column chromatography using silica
gel as the stationary phase and a mixture of ethyl acetate/hexane (1:4)
as the eluent. The pure product was obtained as a plain yellow powder
with a yield of 74.6%. 1H NMR (400 MHz, CDCl3, Fig. S2) δ 10.02 (s,
1H), 8.68 (dd, J = 7.3, 1.1 Hz, 1H), 8.62–8.44 (m, 2H), 8.03–7.92 (m,
2H), 7.80 (dd, J = 8.4, 7.4 Hz, 1H), 7.31–7.26 (m, 2H), 7.13 (d,
J = 8.2 Hz, 1H), 6.06 (s, 1H), 5.54–5.48 (m, 1H), 4.57 (t, J = 5.0 Hz,
2H), 4.50 (t, J = 5.0 Hz, 2H), 1.88 (s, 3H).
Cytotoxicity was evaluated in vitro via MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide) assay in the HepG2 cell line.
After the cells had been cultivated in 96-well plates (1.0 × 105 cells per
well) and incubated for 24 h, they were coincubated with the
PPEGMA20-PNAP8-DOX prodrug nanoparticles, free DOX or blank
PPEGMA20-PNAP8 nanoparticles at various concentrations for different
times. Then, 20.0 μL of an MTT solution (5.0 mg/mL) was added to
each well, followed by incubation for additional 4 h. Finally, the MTT
solution was removed, and 150 μL of DMSO was added to dissolve the
insoluble formazan crystals. Cell viability was detected by measuring
absorbance at 490 nm on a microplate absorbance reader (Bio-Rad
Laboratories, iMark).
2.7. Cellular uptake and intracellular distribution
Cellular uptake of the PPEGMA20-PNAP8-DOX prodrug nano-
particles into HepG2 cells was assessed via CLSM. After coincubation of
HepG2 cells with 15 μg/mL PPEGMA20-PNAP8-DOX nanoparticles for
24 h, the culture medium was discarded, and the cells were washed
with culture solution three times to remove the prodrug residue.
Finally, the cells were fixed with 4% paraformaldehyde, and the nuclei
were stained with DAPI. Fluorescence images were obtained using an
inverted fluorescence microscope (IX-71, Olympus).
2.2.4. PPEGMA20-PNAP8 copolymer
S-1-Dodecyl-S′-(α,α′-dimethyl-α″-acetic
acid)
trithiocarbonate
(DDMAT) was synthesized as a macro-chain transfer agent for re-
versible addition–fragmentation chain transfer (RAFT) polymerization
Then, the PPEGMA20-PNAP8 copolymer was synthesized via RAFT
polymerization, as follows: PEGMA (380 mg, 0.8 mmol), DDMAT
(44 mg, 0.12 mmol), AIBN (3.28 mg, 0.02 mmol), and compound 3
(172 mg, 0.4 mmol) were dissolved into 5.0 mL of DMF in a glass tube.
After three freeze–pumpthaw cycles, the tube was sealed, and the
polymerization was performed in an oil bath at 70 °C for 24 h. The
product was collected as a yellow sticky oil, by precipitation in cold
ether three times and dried under vacuum overnight.
3. Results and discussion
3.1. Synthesis and characterization of the polymer prodrug PPEGMA20
PNAP8-DOX
-
As shown in Scheme 1, the fluorescent copolymer PPEGMA20
-
PNAP8 containing naphthalimide side groups as the FRET energy donor
was synthesized via the RAFT polymerization of PEGMA and compound
3 as comonomers, with AIBN and DDMAT as the initiator and chain
2.3. PPEGMA20-PNAP8-DOX prodrug nanoparticles
DOX·HCl (50 mg) and triethylamine (100 μL) were added to the
PPEGMA20-PNAP8 (50 mg) solution in 10 mL of DMSO in the dark.
After the mixture had been stirred for 24 h at room temperature, it was
dialyzed against DMSO to remove free DOX and then PBS (pH 7.4) for
24 h (MWCO = 1000 Da). The prodrug nanoparticles were obtained as
transfer agent, respectively. In the 1H NMR spectrum of the PPEGMA20
-
PNAP8 copolymer (Fig. 1a), the polymerization degrees of PEGMA and
compound 3 were 20 and 8 respectively, as determined by calculating
the ratio of the integral area of the characteristic methyl proton signals
of PEGMA (δ = 3.34 ppm) and the benzaldehyde proton signal of
3