Han et al
The morphology of rLips before and after incubation
Cell Uptake
with BSA and FA-BSA were observed under TEM (FEI
Company, Hillsboro, OR, USA) with negative stain
method. In short, NLC, B-rLips and FB-rLips were
dropped onto a copper grid, then negatively stained with
1% uranyl acetate and naturally evaporated at room tem-
perature. Particle diameter distribution and zeta potential
were performed by using a NICOMP 380/ZLS zeta poten-
tial/particle size analyzer (PSS.NICOMP Particle Systems,
Santa Barbara, CA, USA). Prior to each measurement,
samples were dissolved with PBS (0.1 M, pH 7.4).
To demonstrate the cellular uptake behavior in vitro, cou-
marin-6 (C6) was selected instead of PTX as the fluorescent
dye for labeling the carriers according to the same drug
encapsulation process. HepG2 and LO2 cells were seeded
in 24-well plates at a density of 2.5×104 cells/well and grown
for 24 h until 90% confluence was attained. The cells were
treated with C6-loaded NLC, C6-loaded B-rLips and C6-
loaded FB-rLips nanosuspensions (the final concentration
in the medium was 100 μg/mL) and co-incubated for two
hours. After washing three times with normal saline, the cells
were fixed in 4% paraformaldehyde for 10 min. The cellular
uptake was qualitatively observed by inverted fluorescence
microscope (Olympus Corporation, Tokyo, Japan) and quan-
In vitro Release Behavior
Release behavior in vitro was performed using the dialysis
method. 3 mL NLC, B-rLips and FB-rLips suspension
loaded with PTX was transferred to dialysis bags (MW
CO:8000–14,000), then placed in a beaker with 100 mL
PBS pH7.4 containing 1 M sodium salicylate, and gently
shaken at 37°C and 100 rpm in the thermostatic oscillator
(Jintan District Baita Instrument Factory, Changzhou,
China). In comparison, 3 mL PTX solution in cremophor
EL and ethanol (1:1, v/v) was carried out in the same way.
Release medium (1.0 mL) was collected at set intervals and
fresh buffer with equal volume was replenished at the same
time. Samples were filtered through 0.22 μm pore-size mem-
brane filter and the continuous filtrate was served for deter-
mination by HPLC as described above.
titatively measured (λex:466nm,
λem:504 nm) with
a multimode microplate reader.
To clarify the mechanism of the cellular internalization
of FB-rLips, HepG2 cells were seeded in 24-well plates
(2.5×104 cells/well) and incubated for 24 h before use.
Then various endocytosis inhibitors including chlorproma-
zine hydrochloride (CPZ, 10 μg/mL), sodium azide (1 mg/
mL), colchicine (50 μg/mL), bacteriocin (6 μg/mL) and
free FA (50 μg/mL, 100 μg/mL, 200 μg/mL) were intro-
duced and co-incubated with HepG2 cells for 0.5 h, fol-
lowed by addition of 100 μg/mL C6-loaded FB-rLips and
incubation for another two hours. After that, the cells were
washed three times by normal saline and determined the
fluorescence intensity of C6 as the above method.
Hemolysis Tests
Cytotoxicity Evaluation in vitro
Two millilters of PFB-rLips, normal saline (as negative
control) and distilled water (as positive control) were
mixed with 2 mL fresh rabbit red blood cells suspensions
diluted with normal saline (2.5%, v:v) in different formu-
lation concentrations of 500 μg/mL and 1000 μg/mL. After
incubation at 37°C for one hour, the hemoglobin release of
different groups was observed and photographed. The
suspension was centrifuged at 955×g for 10 min, and the
absorbance of the supernatant was measured at 541 nm.
The hemolysis rate (HR) was calculated according to the
following formula:
The killing effects of P-NLC, PB-rLips and PFB-rLips on
HepG2 cells were estimated through CCK-8 assay. HepG2
cells were seeded in 96-well plates at a density of 1×104
cells/well for 24 h. Then the cells were co-incubated with
different PTX-loaded formulations at the final PTX con-
centration from 0.5 to 10 µg/mL for another 24 h. Taxol®
(paclitaxel) formulation was used as a control and under-
went the same procedure. At given time intervals, the
medium was withdrawn and 100 μL CCK8 working solu-
tion was added for one-
hour incubation, and the UV absorbance was measured by
a multimode microplate reader at a set wavelength of 450
nm. Besides, the toxicities of blank NLC, B-rLips and FB-
rLips were also evaluated in HepG2 cells and LO2 cells at
the final suspension concentration of 10 μg/mL, 500 μg/
A1 ꢀ A2
HRð%Þ ¼
ꢁ 100%
(3)
A3 ꢀ A2
Formula (3): where HR (%) is the hemoglobin release of
different formulation, A1, A2 and A3 are the absorbance of
the experimental group, negative control and positive con- mL, and 1000 μg/mL, respectively. Cell viability was
trol, respectively.
expressed by the following formula (4):
International Journal of Nanomedicine 2020:15
3437
Han, Mengmeng
