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Compound 3: According to the literature procedure,[13] dry b-cyclo-
dextrin (25.0 g, 22.0 mmol) was dissolved in 0.4m aqueous NaOH
(250 mL) and cooled down to 08C, followed by the addition of p-
toluenesulfonyl chloride (17.5.0 g, 92 mmol) in small portions
under vigorous stirring over 10 min into the solution. Then the re-
sulting suspension was stirred for 30 min below 58C, and filtered
quickly. Hydrochloric acid was used to neutralize the filtrate to
pH 8.5 and stirred for another 1 h. The resultant precipitate was fil-
tered off, rinsed several times with water and dried at 608C for
added to each well. And after 4 h incubation, the supernatant was
removed, followed by the addition of 200 mL DMSO to dissolve the
obtained blue formazan crystals. PerkinElmer 1420 Multi-label
counter was used to measure the absorbance with an excitation
wavelength of 490 nm.
The flow cytometry analysis[17] was also conducted to assess the
cell internalization of FITC-b-CD/Fc-RB and the ability to detect the
H2O2 in HeLa and L929 cells correspondently. Because of their own
fluorescence, the supramolecular fluorescent nanoparticles were
added to the cells directly and incubated for indicated time that is
5, 15, 30 and 60 min. The cellular uptake experiments were per-
formed on flow cytometry. Firstly, cells were seeded in six-well in-
cubated for another day. Then FITC-b-CD/Fc-RB were dissolved in
DMEM culture medium with a final concentration of 5 mm and
added to each well. After that the cells were cultured at 378C for
5, 15, 30 and 60 min. Finally, the cells were collected by removing
the supernatant, washing with cold PBS buffer several times, and
treating with Trypsin. 1.0104 cells were counted and analysis was
conducted with BD FACSCalibur flow cytometer and CELLQuest
software.
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48 h. Yield: 8.58 g, 6.85 mmol, 30%). H NMR (400 MHz, [D6]DMSO,
208C): d =7.73 (d, J=8.3 Hz, 2H), 7.41 (d, J=8.2 Hz, 2H), 5.70 (s,
12H), 4.80 (dd, J=15.3, 11.7 Hz, 7H), 4.58–4.05 (m, 7H), 3.81–3.41
(m, 28H), 3.27 (dq, J=18.8, 9.3 Hz, 14H), 2.40 ppm (d, J=6.9 Hz,
3H).
Compound 4:[13] Compound 3 (5.0 g) was reacted with excess
amount of EDA (30 mL) at 758C for 4 h. Then the mixture was
cooled to room temperature and cold acetone (30 mL) was added.
The precipitate was repeatedly dissolved in water (30 mL) and then
poured into cold acetone (50 mL) three times to remove the un-
reacted EDA. The sample obtained was dried at 508C for 3 d in
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a vacuum oven, and b-CD-EDA was obtained (2.3 g, 49%). H NMR
Moreover, the confocal laser scanning microscopy (CLSM) measure-
ments[17] were performed to evaluate the imaging efficiency and
FRET effect of FITC-b-CD/Fc-RB nanoparticles in HeLa cells and
L929 cells. Cells were seeded in six-well plates with a density of 2
105 cells per well in 2 mL of complete DMEM. After incubation for
another day, culture medium was removed and 2 mL DMEM
medium containing 5 mm of FITC-b-CD/Fc-RB nanoparticles was
added. The cells were cultured at 378C for desired time, followed
by being rinsed with cold PBS buffer, fixed with 4% paraformalde-
hyde for 30 min at 258C. Then the slides were washed with cold
PBS buffer for several times and HOECHST 33342 was used to stain
the nuclei for 15 min. After being washed with cold PBS buffer, the
slides were mounted and observed with a fluorescence microscope
(Leica DMI6000 B) and a confocal laser scanning microscope
(Nikon A1Si). What’s more, in order to make sure if the hydrogen
peroxide in the HeLa cells worked, L929 cells incubated with H2O2
of 50 mm were also used in this experiment as control.
(400 MHz, D2O, 208C): d =4.91 (d, J=19.0 Hz, 7H), 3.77 (ddd, J=
27.5, 16.4, 6.8 Hz, 28H), 3.57–3.39 (m, 14H), 2.92 (d, J=11.0 Hz,
1H), 2.70 (s, 3H), 2.59 ppm (d, J=6.1 Hz, 2H).
Compound 5: Compound 4 (213 mg, 0.2 mmol) was dissolved in
15 mL DMF, to which 0.22 mmol FITC in DMF was added slowly at
08C. Then the mixture was heated to at 258C and reacted for an-
other 4 h with vigorous stirring. The reaction solution was evapo-
rated to 1 mL and precipitated in acetone (15 mL), and the precipi-
tate was filtered. After repeating the operation above for three
times, the collected sample was purified by silica gel column chro-
matography using n-propyl alcohol/H2O/ammonia water mixture
and dried in vacuum oven at 608C for 3 d. 1H NMR (400 MHz,
[D6]DMSO, 208C): d =8.4 (s, 1H), 7.79 (s, 1H), 7.14 (d, J=8.6 Hz,
1H), 6.67 (s, 2H), 6,57 (q, J=8.7 Hz, 4H), 5.71 (s, 7H), 4.80 (s, 7H),
3.62 (s, 37H), 2.87 ppm (s, 1H).
Self-assembly of FITC-b-CD/Fc-RB in mixed solvents: Compound 2
(3.32 mg, 0.004 mmol) and compound 5 (6.26 mg, 0.004 mmol)
were dissolved in DMF (1 mL), respectively, mixed together and
stirred for 6 h. Later the mixture was added dropwise into 8 mL of
ultrapure water with stirring, followed by dialysis in a 1000 Da dial-
ysis bag against deionized water to remove DMF. After 24 h, the
solution was diluted to 20 mL by adding water to obtain an aggre-
gate solution with a concentration of 200 mm for further experi-
ments.
NMR spectroscopy: Varian Mercury Plus 400 MHz spectrometer
was used to record NMR spectra with [D6]DMSO, and D2O as sol-
vents at 208C.
FTIR spectroscopy: Paragon 1000 instrument was used to record
the FTIR spectra through the KBr sample holder method.
Transmission electron microscopy (TEM): JEOL JEM-100CX-II in-
strument was used to investigate the morphology of the nanopar-
ticles at a voltage of 200 kV. The nanoparticle solution was drop-
ped onto the carbon-coated copper grids and then air-dried
before measurement.
All of the above reactions were done in the dark, and all com-
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pounds were characterized by H and 13C nuclear magnetic reso-
Dynamic light scattering (DLS): Malvern Zetasizer Nano S appara-
tus (Malvern Instruments Ltd) equipped with a 4.0 mW He/Ne laser
operating at l=633 nm was used to do the DLS measurements.
nance.
Cell experiments:[17] All cells were cultured at 378C, 5% CO2 in
a humid incubator. And the HeLa cancer cells and L929 normal
cells were incubated in DMEM (Dulbecco’s Modified Eagle’s
Medium) with 10% FBS (Fetal Bovine Serum), and 1% antibiotics.
And the confluent cells were sub-cultured every 3 days following
standard procedure.
UV/Vis spectrophotometry: Thermo Evolution 300 UV/Vis spectro-
photometer was used to record the UV/Vis spectra in the range of
200–650 nm.
MTT: PerkinElmer 1420 Multi-label counter was used to record the
absorbance with an excitation wavelength of 490 nm.
The MTT method[17] was performed in HeLa cells to evaluate the
cytotoxicity of FITC-b-CD/Fc-RB. Cells were seeded into 96-well
plates with a density of 5103 cells/well in 200 mL medium and
cultured until 70–80% confluence. After incubation for another
day, the culture medium was carefully removed and 200 mL of
medium with 50 mL serial concentrations of FITC-b-CD/Fc-RB nano-
particles was added. After grown for another day, 20 mL MTT assays
stock solution with a concentration of 5 mgmLÀ1 in PBS was
Acknowledgements
This work was financially supported by the National Basic Re-
search Program (2015CB931801), National Natural Science
Chem. Eur. J. 2015, 21, 11427 – 11434
11433
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim