D. Zhang et al.
Journal of Drug Delivery Science and Technology 45 (2018) 281–286
receiver fluid was PBS (pH 7.4, 0.15 M) with 5% SDS (w/w) and the
donor phase was micelle suspension (2 mL) in PBS (pH 7.4, 0.15 M).
The donor and receiver compartments were separated by a regenerated
cellulose membrane (MWCO: 3500 Da). The potential membrane
binding of LU was investigated by incubating LU (15 μg/mL) with the
cold PBS (pH 7.4, 0.15 M) solution. The obtained cells were suspended
in of binding buffer (100 μL), followed by adding Annexin V-FITC (5 μL)
and PI (10 μL) staining solutions. After 15 min's incubation, the binding
buffer (400 μL) was added with gentle mixing on ice. Samples were then
analyzed by flow cytometry employing a FACSCalibur flow cytometer
(Becton Dickinson, USA) coupled with Cell Quest Pro Software (Becton
Dickinson). The cell cycle analysis was performed subsequently uti-
lizing FlowJo software (Tree Star Inc. USA).
2
membrane (2 cm ) in the mixture of water and methanol at 37 °C; after
24 h, drug recovery was calculated (n = 3). The donor compartment
was filled with RM or CM micellar solution that contained ca. 330 μg
lutein. At pre-determined time points, the receiver fluid (0.5 mL) was
withdrawn for drug quantification, followed by rapid supplementation
of the same volume of fresh receiver fluid to maintain the total volume
constant. The drug release at acidic environment utilized the citric acid-
2.8. Statistical analysis
The data were expressed as the mean ± standard deviation (SD). A
statistically significant difference was determined at a minimal level of
significance of 0.05 via Student's t-test or analysis.
2 4
Na HPO buffer (pH 5.0, 0.15 M) as the receiver fluid that also con-
tained 5% (w/w) SDS to maintain the sink conditions. The release
curves were presented as the cumulative amount of released drug
against time.
3. Results and discussion
2.5. Cytotoxicity analysis
3.1. Micelle preparation and characterization
Cervical cancer cells (HeLa cells) were obtained from the State Key
Laboratory of Medicinal Chemical Biology, Nankai University. The cells
were seeded in RPMI 1640 medium (Gibco, NY, USA), which containing
Both types of polymers could successfully self-assemble into mi-
cellar nanocarriers with hydrophobic lutein being physically en-
capsulated. The drug loading was 3.5 ± 0.1% (w/w, RM) and
0.9 ± 0.1% (w/w, CM), respectively (Fig. 1A). The drug recovery from
micelles, dialysis medium and precipitate demonstrated a LU recovery
over 95% for all samples, which validated the removal of non-en-
capsulated drug and micelle purification. The high loading in the re-
sponsive micelles was presumed due to the potential hydrogen bonding
between imidazole and lutein; the nitrogen in imidazole moiety can act
as the hydrogen donor, whereas the hydroxyl group of lutein can
function as the hydrogen acceptor. Such hydrogen bonding-enhanced
cargo loading has also been observed in previous work [19]. The iron
level in placebo RM and drug-loaded RM (RM/LU) was similar at
0.20 ± 0.01% (w/w) (Fig. 1B). This indicated that the loading process
did not affect the coordination between imidazole and iron, which
concurred well with previous report [17]. However, the presence of
lutein slightly increased the hydrodynamic size of micelles irrespective
of the micelle type and medium acidity (p < 0.05) (Fig. 1C and D).
Polymeric micelles often display a hydrophobic core-hydrophilic shell
structure with the hydrophobic cargos locating in the core. Upon drug
loading, the drug would occupy some space to enable core expansion,
leading to an enlarged micelle [27], which was the case in the current
study. Regarding the responsive micelles, the hydrodynamic diameters
increased clearly with decreasing pH. As the micelle self-assembly is
1
% penicillin/streptomycin and 10% fetal bovine serum. HeLa cells
were culturing at 37 °C with CO /air (5:95) humidified atmosphere.
2
Cytotoxicity of free lutein (LU), placebo CM, drug-loaded micelles (CM/
LU), placebo RM, drug-loaded micelles (RM/LU) was evaluated by the
standard MTT assay. The LU dose ranged from 0 to 80 μM (n = 6). HeLa
3
cells were seeded in 96-well plates at a density of 4 × 10 cells/well.
After 24 h incubation, five samples were added to the 96-well plates
after medium removal, followed by incubating for another 48 h. The
medium contained LU was removed and 100 μL of the PBS (pH 7.4,
0
.01 M) was added into each well. Subsequently the cells were treated
with MTT solution (100 μL, 0.5 mg/mL) for 4 h in the dark. The medium
was slowly removed and 100 μL of the DMSO was added into each well
to dissolve formazan. After 20 min, the absorbance of formazan is de-
termined at 490 nm by a microplate reader (BioTek Instruments Inc.,
VT, USA). The percentage of viable cells and the half maximal in-
hibitory concentrations (IC50) values were calculated accordingly.
2
.6. Cell cycle analysis
Cell cycle analyses were explored by the same cell type and culture
condition as cytotoxicity experiment. The cells were incubated for 24 h
5
on 6-well plates at a seeding density of 5 × 10 cells/well (n = 3). After
24 h, the medium was removed and cells were washed with 1 mL PBS
(
pH 7.4, 0.01 M) twice, followed by incubation for 48 h with the
medium. Afterwards, the cell supernatant was removed from 6-well
plate and the cells were digested with 1 mL trypsin solution for 1 min
followed by adding 2 mL medium. Then the cells were washed with
1
4
mL ice-cold PBS (pH 7.4, 0.01 M) and suspended in 75% ethanol at
°C overnight. After fixation, the cell pellets were washed with ice-cold
PBS in triplicate, followed by incubation with PBS solution containing
5
2
0 μg/mL PI, 100 μg/mL RNase A, and 0.2% Triton-X-100 at 4 °C for
0 min. Afterwards, the cells were analyzed by flow cytometry em-
ploying a FACSCalibur flow cytometer (Becton Dickinson, USA).
2.7. Apoptosis analysis
Detection of cell apoptosis were used the same cell type and the
culture condition as cell cycle evaluation. The cells were incubated for
5
2
4 h on 6-well plates at a seeding density of 5 × 10 cells/well (n = 3).
After 24 h, the medium was removed and cells were washed with 1 mL
PBS (pH 7.4, 0.01 M) twice, followed by sample supplementation (LU,
RM/LU, CM/LU). After 48 h, the cells were centrifuged at 300 g for
Fig. 1. Pharmaceutical assessment of lutein (LU)-loaded crosslinked responsive micelles
(RM) and control micelles (CM) (n = 3). (A) Drug loading; (B) Iron loading; (C–D)
5
min. The collected cells were digested with pancreatin and cen-
Hydrodynamic size.
trifuged at 300 g for 5 min. After that, cells were washed with 1 mL ice-
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