Biomacromolecules
Article
dimethylformamide (DMF) as an eluent at 30 °C with a flow rate of
1.00 mL/min. Matrix-assisted laser desorption and ionization time-of-
flight (MALDI-ToF) mass spectrometry measurements were carried
out on an Ultraflex III MALDI mass spectrometer with α-cyano-4-
hydroxycinnamic acid as the matrix. Differential scanning calorimetry
(DSC) was performed using a differential scanning calorimeter (Q200
model, TA Instruments) in the temperature range of −80−50 °C at a
heating rate of 10 K/min under nitrogen. The zeta potential was
measured using a Malvern Zetasizer Nano-ZS.
Protection of 4-Amino-1-butanol. The precursor, t-butyl N-(4-
hydroxybutyl)carbamate was synthesized according to the literature
procedure with slight modification.35 To a solution of 4-amino-1-
butanol (10 g, 112 mmol) and triethylamine (17.1 mL, 123 mmol) in
dichloromethane (CH2Cl2, 35 mL), a solution of di-tert-butyl-
dicarbonate (25.7 g, 118 mmol) in CH2Cl2 (100 mL) was added
dropwise over 30 min at room temperature. The mixture was stirred at
room temperature for 6 h and the reaction mixture was concentrated
under reduced pressure. The crude mixture was dissolved in CH2Cl2
and washed with water and then with brine. The organic phase was
dried over Na2SO4 and concentrated under reduced pressure. The
residue was purified by flash column chromatography eluting with 10%
methanol in CH2Cl2 to give a compound as colorless oil.
(number of BBAG repeating units) × 7 (number of protons of BBAG
except t-butyl)}]/5 (number of protons of the G monomer (5H)) =
56; Mn = 74.08 (molecular weight of the G monomer) × 56 + 245.32
(molecular weight of the BBAG monomer) × 2 + 134.17 (molecular
weight of TMP) = 4773.29 g/mol. Considering the error range of
NMR integration, we used 4800 g/mol as the Mn value of the polymer
1.
Synthesis of P(G56-co-BAG2). The Boc-protected P(G56-co-
BBAG2) copolymer (polymer 1) was dissolved in CH2Cl2 with
trifluoroacetic acid (TFA) (1.0 mL) and stirred at room temperature
for 1 h. The reaction mixture was concentrated under reduced pressure
and the resulting deprotected polymer was dissolved in 1.0 mL of
methanol; the homogeneous polymer solution was then precipitated in
excess diethyl ether, and the precipitate was washed twice using diethyl
ether. The resulting deprotected P(G56-co-BAG2) polymer was dried
under vacuum at 90 °C for 1 day.
13C NMR Kinetics. To generate the initiator, TMP (20 mg, 0.204
mmol), and potassium methoxide in methanol (25 wt %, 23 μL, 0.082
mmol) were reacted in a round-bottom flask under an argon
atmosphere at 50 °C. Excess methanol was removed using a rotary
evaporator, and the resulting product was dried in a vacuum oven at 90
°C for 3 h to yield a white salt of the initiator. The initiator was added
to the comonomer mixture of G (0.151 g, 2.04 mmol) and BBAG
(0.500 g, 2.04 mmol), which was placed in a 4.0 mL vial and stirred
over an ice bath. The mixture was transferred to a conventional NMR
tube under an argon atmosphere and then sealed with a septum over
an ice bath. The kinetic measurements using 13C NMR spectroscopy
were recorded on a 600 MHz VNMRS system with a 5 mm PFG
AutoXDB probe in neat solutions. A standard kinetic 13C NMR
experiment required 64 transients, which were obtained with a 13.7 μs
90° pulse, spectral width of 1894 Hz, and recycling delay of 10 s for
each kinetic run; 43 experiments were performed over a period of 12 h
with a flip angle of 45° and inverse gated decoupling.
Rhodamine B Conjugated Hyperbranched P(G113-co-BAG21)
Polymer. Rhodamine B conjugated polymer was synthesized
according to a method previously described in the literature.36 N-
Hydroxysuccinimide (2.3 mg, 0.02 mmol) and rhodamine B (9.6 mg,
0.02 mmol) were dissolved in 1.5 mL of DMF, and then N,N′-
dicyclohexylcarbodiimide (6.2 mg, 0.03 mmol) and 4-dimethylamino-
pyridine (2.4 mg, 0.02 mmol) were added to the solution. The mixture
was stirred at room temperature for 30 min, and then, 70 mg of
P(G113-co-BAG21) (polymer 7) in 1.5 mL of DMF was added to the
solution. After stirring for 48 h at room temperature, the insoluble
residue was filtered off and the solvent was removed by a rotary
evaporator. The residues were dissolved in deionized water to dialyze
against water for 7 days, followed by lyophilization to give the
rhodamine B-conjugated polymer in a quantitative yield.
Synthesis of BBAG Monomer. A solution of t-butyl N-(4-
hydroxybutyl)carbamate (14.5 g, 76.6 mmol) in t-butanol (100 mL)
was slowly added to a solution of potassium t-butoxide (8.50 g, 76.6
mmol) in t-butanol (100 mL) with stirring for 15 min at room
temperature under argon. After stirring for an additional 15 min,
excess epichlorohydrin (35.9 mL, 459 mmol) was added dropwise for
30 min. The solution was stirred at room temperature for 24 h after
which additional water (100 mL) and CH2Cl2 (200 mL) were added.
The aqueous phase was extracted with CH2Cl2 and combined organic
layers were dried over MgSO4. The organic phase was concentrated
under reduced pressure and the residue was purified by flash column
chromatography with CH2Cl2/hexane (1:100 v/v) eluent to give 11.2
g (60%) of the BBAG monomer as a colorless oil. The synthesis of the
BBAG monomer was successfully confirmed through various
1
characterizations, including H and 13C NMR, COSY, HMQC, and
DEPT spectroscopy and ESI-MS (see Figure 2 for corresponding peak
1
NMR (600 MHz, DMSO-d6): δ ppm 3.61 (dd, 1H, J = 11.6 and 2.8
Hz, e), 3.34−3.40 (m, 2H, f), 3.18 (dd, 1H, J = 11.5 and 6.3 Hz, d),
3.03−3.05 (m, 1H, c), 2.88 (q, 2H, J = 6.7 Hz, i), 2.68 (dd, 1H, J = 4.3
and 5.1 Hz, b), 2.49 (dd, 1H, J = 5.1 and 2.6 Hz, a), 1.42−1.47 (m,
2H, g), 1.36−1.39 (m, 2H, h), 1.34 (s, 9H, j). 13C NMR (150 MHz,
DMSO-d6): δ ppm 158.66, 80.38, 74.24, 73.29, 53.41, 46.46, 42.88,
31.34, 29.65, 29.34. MS (m/z + Na+, ESI+) Calcd for C12H23NO2,
268.3; found, 268.3
Cytotoxicity Assay. Murine macrophage cell line, RAW264.7, was
purchased from the Korean Cell Line Bank (Seoul, Korea).
Cytotoxicity assays were performed using the traditional MTT assay.
Cells were seeded in 24-well plates at a density of 1 × 105 cells per well
and incubated for 24 h in 5% CO2 at 37 °C. The RAW264.7 cells were
cultured with Dulbecco’s Modified Eagle’s Medium (DMEM,
Hyclone) containing 10% fetal bovine serum (FBS) and 1%
penicillin−streptomycin. After removing the culture medium, the
wells were washed with phosphate-buffered saline (PBS). Each well
was then treated with various concentrations of P(G-co-BAG)
solutions (polymers 3, 6, and 7) and incubated for an additional 18
h. For the MTT assays, each well was washed with PBS then filled with
60 μL of a thiazolyl blue tetrazolium bromide (MTT, Sigma-Aldrich)
stock solution (5.0 mg/mL) and 940 μL of fresh media. After
incubation for 3 h, 1.0 μL of DMSO was added to the polymer
solution to solubilize the MTT-formazan product, after which the
plates were gently agitated for 15 min at room temperature. After
transferring 100 μL of each sample into the 96-well plates, the
absorbance of the solution was recorded at a wavelength of 540 nm
using 620 nm as the reference.
Synthesis of P(G56-co-BBAG2) (Polymer 1). Trimethylolpropane
(TMP; 54.6 mg, 0.407 mmol) was placed in a two-neck round-bottom
flask. Potassium methoxide in methanol (25 wt %, 45 μL, 0.163 mmol)
was diluted with 0.70 mL of methanol and then added to the flask and
stirred for 30 min at room temperature under an argon atmosphere.
Excess methanol was removed using a rotary evaporator and the
resulting product was dried in a vacuum oven at 90 °C for 3 h to yield
a white salt of the initiator. The flask was then purged with argon and
heated to 90 °C. A mixture of t-butyl 4-(oxiran-2-ylmethoxy)-
butylcarbamate (BBAG; 0.30 g, 1.22 mmol) and glycidol (G; 1.54 g,
23.23 mmol) was added dropwise over 12 h using a syringe pump.
After complete addition of the monomer, the reaction was continued
for an additional 5 h. The resulting P(G56-co-BBAG2) copolymer was
dissolved in 1.0 mL of methanol; the homogeneous polymer solution
was then precipitated in excess diethyl ether, and the precipitate was
washed twice using diethyl ether. The resulting polymer was dried
under vacuum at 90 °C for 1 day. All polymers synthesized in this
study were isolated in approximately 90% yield. The Mn of the P(G56-
co-BBAG2) polymer was determined to be 4773 g/mol, as calculated
using the following equation: number of repeating units (BBAG) =
17.25 (integration value)/9 (number of protons of t-butyl of BBAG) =
2, number of repeating units (G) = [292.13 (integration value) − {(2
Cellular Uptake Imaging. Cellular uptake of the rhodamine B-
conjugated polymers was tested by using HEK-293T cells. Cells were
seeded on a coverslip in a 24-well tissue culture plate at a density of 1
C
Biomacromolecules XXXX, XXX, XXX−XXX