Biomacromolecules
Article
1H NMR (DMSO-d6, 300 MHz, δ in ppm): 1.36 (s, 9H), 1.8−2.3
(m, 4H), 2.96−3.41 (m, 8H), 3.46 (s, 2H), 3.52−3.56 (m, 2H), 4.19−
4.38 (m, 1H), 4.47 (d, 2H, 3J = 4.5 Hz), 6.64 (d, 2H, 3J = 8.5 Hz), 6.76
(m, 1H), 6.92 (m, 1H), 7.08 (s, 1H), 7.64 (m, 2 H), 7.89 (m, 1H),
7.95 (s, 1H), 8.03 (m, 1H), 8.63 (s, 1H), 10.95 (s, 1H).
days in total (for two consecutive passages) to stimulate the expression
of membrane-anchored FRα. Three days before cell seeding, the
standard DMEM medium was substituted by a folic acid free custom-
made DMEM medium (Invitrogen, Germany), which included all
other ingredients as in the standard DMEM medium. One day after
cell seeding, the HES nanocapsules were added to the cell culture
(described above). A total of 1 h before the nanocapsules were added
to the cell culture, cells were preincubated with folic acid containing
DMEM medium in different concentrations (0.1, 0.5, and 1.0 mM) in
a humidified incubator. The expression of FRα (on HeLa cells) was
confirmed by flow cytometric measurements with a fluorescent
allophycocyanin (APC)-conjugated mouse monoclonal antihuman
antibody (FOLR1-APC, R&D Systems, U.K.). For CLSM, the cells
were analyzed on the ibiTreat μ-slides and washed twice with
phosphate-buffered saline without calcium and magnesium (PBS,
Invitrogen, Germany) before membrane staining and analysis. For flow
cytometry analysis six-well plates were used, the cells were washed
with PBS, trypsinized, centrifuged (3 min, 3000 rpm = 956g (m·s−2)),
and finally resuspended in PBS. The cell viability was confirmed by a
MTS assay (CellTiter 96 Aqueous One Solution Cell Proliferation
Assay, Promega, U.S.A.), performed according to the product insert in
96-well assay plates (Corning Incorporated costar 3603, Corning,
Germany). Each well was populated with 10000 cells the day before
the experiment. Absorbance (490 nm) of this assay was measured with
a microplate reader (Infinite M1000, Tecan, Switzerland).
Synthesis of (N-(2-[2-(2-aminoethoxy)ethoxy]ethyl) folic acid (2).
In a round-bottom flask, 1.5 g (2.2 mmol, 1.0 equiv) of 1 were mixed
with 10 mL of TFA (trifluoracetic acid) and stirred for 2 h at 40 °C in
a water bath. TFA was removed under reduced pressure and the
remaining brownish gel was dissolved in 150 mL of water. The
solution was poured into 800 mL of acetonitrile and a brownish
precipitate was collected via filtration. The solid was dried under
reduced pressure for 18 h at room temperature. Yield: 610 mg (50%).
The reaction scheme is shown in Figure 2. The 1H NMR spectrum of
(N-(2-[2-(2-aminoethoxy)ethoxy]ethyl) folic acid is given in the
Supporting Information, see Figure S2.
1H NMR (DMSO-d6, 300 MHz, δ in ppm): 1.88−2.07 (m, 2H),
3
2.19−2.31 (m, 2H), 2.95 (t, 1H, J = 4.7 Hz), 3.17−3.42 (m, 6H),
3
3.48−3.61 (m, 5H), 4.29−4.36 (m, 1H), 4.48 (d, 2H, J = 5.8 Hz),
6.64 (d, 8.7 Hz), 6.95 (m, 2H), 7.64 (m, 2H), 7.87−8.13 (m, 2H),
8.64 (s, 1H), 11.26 (s, 1H). FD-MS: 572 (M + H)+ (100), 594 (M +
Na)+ (91), 610 (M + K)+ (32).
Synthesis of Folic Acid-Conjugated HES Nanocapsules. Folic
acid has two COOH groups positioned at the end of the molecule.
The reactivity of γ-COOH is higher, and therefore, this group will
react faster with NH2 groups of the other molecules.39 A 3.5 g aliquot
of the carboxymethylated HES nanocapsules dispersion (solid content
1.0 wt %, 0.0045 mmol COOH groups) was mixed with 70 mg (0.45
mmol) EDC and 20 mg (0.09 mmol) sulfo-NHS to activate the
carboxyl groups. After stirring for 30 min, the nanocapsules were
centrifuged at 4000 rpm for 30 min (Sigma 3k-30, RCF 1467) to
remove residuals of EDC and sulfo-NHS. The supernatant was
removed and the pellet was resuspended in demineralized water. Then
30 mg (0.06 mmol) of NH2-terminated folic acid conjugate were
added and the mixture was stirred for another 12 h at ambient
conditions. After the coupling procedure the nanocapsules were
centrifuged at 4000 rpm for 20 min and dialyzed (MWCO: 12000
g·mol−1) in order to remove residues of nonreacted NH2-terminated
folic acid conjugate.
Characterization of Folic Acid-Conjugated HES Nanocap-
sules. The amount of folic acid conjugate coupled to the HES
nanocapsules was studied by measuring the fluorescence intensity
using a plate reader (Infinite M1000, Tecan, Switzerland). A 3D-scan
was performed to determine the absorption and emission signals of the
folic acid conjugate. After the coupling procedure, the HES capsules
were centrifuged (30 min at 4000 rpm) and the amount of folic acid
conjugate present in the supernatant was calculated from the
fluorescence intensity data. For the calculations, the calibration curve
obtained with different amounts of dissolved folic acid conjugate was
used.
Methods of Characterization. The average size and the size
distribution of nanocapsules were analyzed by means of dynamic light
scattering (DLS) at 25 °C using a Nicomp 380 submicrometer particle
sizer equipped with a detector at a 90° angle to the incident beam
(Nicomp Particle Sizing Systems, U.S.A.) at 20 °C. The zeta potential
of nanocapsules was measured in 10−3 M potassium chloride solution
with a Nicomp zeta sizer (Nicomp Particle Sizing Systems, U.S.A.) at
20 °C.
The amount of surface-charged groups was calculated from the
results of the titration experiments performed on a particle charge
detector (Mutek GmbH, Germany) in combination with a Titrino
̈
Automatic Titrator (Metrohm AG, Switzerland). The carboxylic
groups were titrated against the positively charged polycation
poly(diallyl dimethyl ammonium chloride) (poly-DADMAC). The
titration was performed on 10 mL of the nanocapsules dispersion with
a solid content of 1 g·L−1. The amount of groups per gram of polymer
was calculated from the consumed volume of the polyelectrolyte
solution. Morphological studies were performed with scanning
electron microscopy (SEM). The images were recorded by using a
field emission microscope (LEO (Zeiss) 1530 Gemini, Oberkochen,
Germany) operated at an accelerating voltage of 170 V. Generally, the
samples were prepared by diluting the capsule dispersion in
cyclohexane or demineralized water (for redispersed samples) to
about 0.01% solid content. A droplet of dispersion was placed onto
silica wafers and dried under ambient conditions.
Cell Culture. The cellular uptake of the HES nanocapsules before
and after folic acid coupling was confirmed by confocal laser scanning
microscopy (CLSM) and flow cytometry. Human cervix carcinoma
cells (HeLa cells) and adenocarcinoma human alveolar basal epithelial
cells (A549 cells) were kept in Dulbecco's Modified Eagle Medium
(DMEM, Invitrogen, Germany). All were supplemented with 10%
fetal calf serum (FCS), 100 units of penicillin, and 100 mg·mL−1
streptomycin, (all from Invitrogen, Germany). Cells were grown in a
humidified incubator at 37 °C and 5% CO2. On the day prior to the
experiments, the adherent cells were detached by trypsin (Gibco,
Germany) and seeded in a FCS-supplemented medium at a density of
50000 cells·cm−2 in six-well plates (Greiner, Germany) for flow
cytometry and 10000 cells·cm−2 in ibiTreat μ-slides (IBIDI, Germany)
for CLSM analysis. On the following day, HES nanocapsules (2.76 ×
1013 nanocapsules per mL, labeled with SR101) were added to the
medium in the presence of 10% FCS without using a transfection
agent. Incubation periods had been carried out in a humidified
incubator (37 °C, 5% CO2). In the case of cellular uptake experiments
carried out with and without additional folic acid in cell culture
medium, cells were kept in folic acid free medium for a period of five
The analysis of the nanocapsules before and after coupling of the
folic acid conjugate was performed by FTIR measurements. A total of
3 mg of freeze-dried sample was pressed with KBr to form a pellet and
a spectrum between 4000 and 400 cm−1 was recorded using the BX
spectrometer from Perkin-Elmer. 13C NMR spectra were recorded on
a Bruker Avance 300 spectrometer operating with 75 MHz frequency.
The confocal laser scanning microscopy (CLSM) experiments were
performed to determine the intracellular localization of the HES
nanocapsules. The images were taken using a commercial setup (LSM
SP5 STED Leica Laser Scanning Confocal Microscope, Leica,
Germany) consisting of an inverse fluorescence microscope DMI
6000 CS equipped with a multilaser combination and five detectors
operating in the range of 400−800 nm. A HCX PL APO CS 63×/1.4−
0.6 oil-immersion objective was used in these studies. For the
excitation of the nanocapsules, a DPSS λ = 561 nm (∼20 mW) laser
was used. The emission was detected at 580−620 nm. The
nanocapsules are pseudocolored in red in the obtained images. The
cell membrane was stained with CellMask Deep Red plasma
membrane stain (2.5 μg·mL−1, Invitrogen, Germany). In the images,
the cell membrane is shown pseudocolored in green (excited with
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dx.doi.org/10.1021/bm300653v | Biomacromolecules 2012, 13, 2704−2715