Colloids and Surfaces B: Biointerfaces 110 (2013) 74–80
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Colloids and Surfaces B: Biointerfaces
journal homepage: www.elsevier.com/locate/colsurfb
Drug resistance reversal activity of anticancer drug loaded solid lipid
nanoparticles in multi-drug resistant cancer cells
Jing Miaoa
, Yong-Zhong Dub
, Hong Yuanb
, Xing-guo Zhanga
, Fu-Qiang Hub,∗
a
Department of Pharmacy, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, PR China
b
College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, PR China
a r t i c l e i n f o
Article history:
Received 16 November 2012
Received in revised form 11 March 2013
Accepted 19 March 2013
Available online 17 April 2013
Keywords:
Solid lipid nanoparticles
Multi-drug resistance
Paclitaxel
Doxorubicin
Cytotoxicity
a b s t r a c t
The aim of our study was to enhance the cytotoxicity of anticancer drugs by reversing the resistance of
multi-drug resistant cancer cells. The cytotoxicities of paclitaxel (PTX) and doxorubicin (DOX), either as
single agents or loaded in solid lipid nanoparticles (SLN) by a solvent diffusion method, were examined
using drug sensitive cancer cells and drug resistant cells by measuring the drug concentration required
for 50% growth inhibition (IC50). Compared to Taxol and DOX·HCl solution, both PTX and DOX loaded
in SLN exhibited higher cytotoxicities in human breast tumor drug sensitive MCF-7 and drug resistant
MCF-7/ADR cells. The ability of PTX loaded SLN and DOX loaded SLN to reverse the drug resistance of
MCF-7 cells compared to MCF-7/ADR cells was 31.0 and 4.3 fold, respectively. Both PTX and DOX loaded
SLN showed the same trends of enhanced cytotoxicity against a second wild type/drug resistant human
ovarian cancer cell pair SKOV3 and SKOV3-TR30 cells. The reversal powers were 3.8 and 1.9 fold for PTX
loaded SLN and DOX loaded SLN, respectively.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
Paclitaxel (PTX) and doxorubicin (DOX) are typical and commonly
used drugs against a wide spectrum of solid tumors in the
clinic. However, similar to other anticancer drugs, even when they
are located in the tumor interstitium they can have limited efficacy
against numerous solid tumor types, because cancer cells
are able to develop mechanisms of resistance to drugs and evade
chemotherapy [1]. The multi-drug resistance (MDR) phenotype,
mainly due to expression of the MDR gene family encoding for the
P-glycoprotein (P-gp) membrane proteins [2], represents an important
problem in chemotherapy. The P-gp proteins are capable of
extruding various generally positively charged xenobiotics, including
some anticancer drugs, out of the cell via an ATP-dependent
mechanism leading to intracellular reduction in the concentration
of drug [3].
Nanoparticle (NP) delivery systems are known to carry the
incorporated PTX or DOX into cells and improve the intracellular
concentration of the drug [4]. Studies have shown that intracellular
entry of drug loaded NPs delivery systems can be via endocytosis,
followed by release of entrapped agent in cytoplasm. This is an
alternative route of drug entry that enables bypassing or inhibiting
the P-gp-mediated efflux [5,6].
∗ Corresponding author. Tel.: +86 571 88208441; fax: +86 571 88208439.
E-mail address: hufq@zju.edu.cn (F.-Q. Hu).
One type of NP delivery system is solid lipid nanoparticles (SLN)
developed as a colloidal carrier based on solid lipid materials. The
SLN incorporate drugs into the lipid matrix resulting in the advantages
of controlled release [7], high bioavailability by nonparenteral
administration [8] and better tolerability [9]. In our previous studies
[10–12], hydrophobic drugs were encapsulated in SLN with a
controlled in vitro release rate. Moreover, higher cellular uptake
of incorporated drugs in SLN was also observed. Therefore, SLN
loaded anticancer drugs could enter the cells by an endocytotic
pathway, thus bypassing the P-gp-dependent efflux, leading to an
increased intracellular drug concentration and drug cytotoxicity,
and reversing MDR activity in MDR cancer cells.
In this study, PTX and DOX were chosen as hydrophobic cytotoxic
drugs. The cytotoxicities of drug loaded SLN prepared by a
solvent-dispersion method were investigated in two human cancer
cells (human breast cancer MCR-7 cells and human ovarian cancer
SKOV3 cells) and their multi-drug resistant variants. Our aim was
to evaluate and analyze the drug resistance reversal activity of drug
loaded SLN in MDR cells (PTX and DOX resistant cells) compared to
free drug solutions.
2. Materials and methods
2.1. Materials
Doxorubicin hydrochloride (DOX·HCl) was a gift from
Hisun Pharm. (Zhejiang, China). Paclitaxel was purchased from
0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.colsurfb.2013.03.037
J. Miao et al. / Colloids and Surfaces B: Biointerfaces 110 (2013) 74–80 75
Zhanwang Biochemical (Huzhou, China). Glyceryl monostearate
(Monostearin, C21H42O4) (Shanghai Chemical Reagent Co., China)
was used as a solid lipid material for nanoparticles and ␣-
hydro-␻-hydroxypoly(oxyethylene)80poly(oxypropylene)27poly
(oxyethylene)80 block copolymer (poloaxmer 188, HO(C2H4O)80
(C3H6O)27(C2H4O)80H) (Shenyang Pharmaceutical university Jiqi
Co. Ltd., China) was used as the surfactant. 3-(4,5-Dimethylthiazol-
2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased
from Sigma (St. Louis, MO, USA). Trypsin and RPMI 1640 Medium
were purchased from Gibco BRL (Gaithersberg, MD, USA). Fetal
bovine serum (FBS) was purchased from Sijiqing Biologic (Hanghzou,
China). All other chemicals were analytical or chromatographic
grade.
2.2. Preparation of SLN
Before loading into the SLN, doxorubicin hydrochloride
(DOX·HCl) was stirred with twice the number of mole of triethylamine
(TEA) in DMSO overnight to obtain the DOX base [13].
The preparation method of drug loaded SLN was developed
in our previous studies [10–12]. Briefly, 3 mg hydrophobic drug
(PTX or DOX) and 60 mg monostearin were dissolved in 6 ml warm
ethanol. The resultant organic solution was quickly dispersed into
distilled water (with volume ration 1:10) under mechanical stirring
(DC-40, Hangzhou Electrical Engineering Instruments, China) at
400 rpm in water bath of 70 ◦C for 5 min. The obtained pre-emulsion
(melted lipid droplets) was then cooled to room temperature till
drug loaded SLN formation was obtained. Blank SLN were also prepared
as described above but omitting drug in the organic solution.
The pH value of the obtained SLN dispersion above was adjusted
to 1.20, to allow SLN aggregation, by adding 0.1 M of hydrochloric
acid. Then the SLN precipitate was harvested by centrifugation at
46,282 × g for 15 min (3K30, Sigma, Germany). The precipitate of
SLN or NLC was collected for drug entrapment efficiency and drug
loading determination.
The SLN precipitate was re-dispersed in the aqueous solution
containing 0.1% poloxamer 188 (w/v) by probe-type ultrasonic
treatment with 20 sonic bursts (200 W, active every 2 s for a 3 s
duration) (JY92-II, Scientz Biotechnology Co. Ltd., China).
Then the resultant dispersion was fast frozen under −64 ◦C in
a deep-freezer (Sanyo Ultra Low Temperature Freezer MDF-192,
Japan) for 5 h and then the sample was moved to the freeze-drier
(Freezone 2.5L, LABCONCO, USA). The drying time was controlled
in 72 h and then the SLN powders were collected for in vitro release
study.
2.3. Particle size and zeta potential measurement
The blank or drug loaded SLN in dispersion after sonication were
diluted 20 times with distilled water, of which the volume average
diameter and zeta potential were determined with Zetasizer
(3000HS, Malvern Instruments, UK).
2.4. Drug entrapment efficiency and drug loading determination
The collected SLN precipitate was re-dispersed in phosphate
buffer solution (PBS, pH 7.2, = 0.1 M) medium and subjected to
vortex mixing (XW-80A, Instruments factory of Shanghai Medical
University, China) at 2800 rpm for 3 min to dissolve the surface
attached drugs, and treated with centrifugation at 46,282 × g for
15 min. Drug content in the supernatant was measured as follows.
For PTX, the supernatents were diluted in PBS medium containing
2 M sodium salicylate, and quantified by HPLC (Agilent 1100
series, USA), using C18 column (DiamohsilTM 250 mm × 4.6 mm,
5 ␮m) in 35 ◦C, a UV detector (Agilent, USA) at a set wavelength
of 227 nm. The mobile phase was a mixture of acetonitrile and
water (50:50, v/v) with flow rate of 1.0 ml/min. Injected volume
of the sample was 20 ␮l. The calibration curve of peak area against
concentration of paclitaxel was y = 32.4x − 16.3 under the concentration
of paclitaxel 0.5–120 ␮g/ml (R2 = 0.9994, where y = peak
area and x = paclitaxel concentration), the limit of detection was
0.01 ␮g/ml.
For DOX, the PBS medium contained 0.1% (w/v) sodium
lauryl sulfate, and DOX concentration was determined by use
of a fluorescence spectrophotometer (F-2500, Hitachi, Japan),
excitation at 505 nm and emittion at 560 nm. The calibration
curve of fluorescent intensity against concentration of
doxorubicin was y = 244x + 11.399 under the concentration of doxorubicin
0.2–10 ␮g/ml (R2 = 0.9993, where y = fluorescent intensity
and x = doxorubicin concentration), the limit of detection was
0.05 ␮g/ml.
The drug entrapment efficiency (EE) and drug loading (DL) of
SLN were calculated from Eqs. (1) and (2):
EE (%) =
Wa − Ws1
− Ws2
Wa
× 100 (1)
DL (%) =
Wa − Ws1
− Ws2
Wa − Ws1
− Ws2
+ WL
× 100 (2)
where Wa was the amount of drug added in system, Ws1
was the
analyzed amount of drug in supernatant after the first centrifugation,
Ws2
was the analyzed amount of drug in supernatant after the
second centrifugation. WL was the weight of lipid added in system.
2.5. In vitro release study
To investigate the release kinetics of drugs from SLN, separated
SLN precipitate containing PTX or DOX was re-dispersed in respective
release medium (sodium salicylate and sodium lauryl sulfate
for PTX and DOX particles respectively as described above in Section
2.4) and mixed by vortexing at 2800 rpm for 3 min, and then
shaken horizontally (Shellab1227-2E, Shellab, USA) at 37 ◦C and
60 strokes/min for 24 h. One ml of the dispersion was withdrawn
from the system at various time intervals and centrifuged. The drug
concentration in release medium was measured as described in
Section 2.4. The amount of the released drug at each time was then
calculated. Each formulation was investigated independently three
times.
2.6. Cell culture
MCF-7 (human breast cancer cells) and MCF-7/ADR (multi-drug
resistant variant) were donated from the first Affiliated Hospital,
College of Medicine, Zhejiang University (Hangzhou, China). SKOV3
(human ovarian cancer cells) and SKOV3-TR30 (multi-drug resistant
variant) were obtained from Women’s Hospital, College of
Medicine, Zhejiang University (Hangzhou, China). Cells were grown
at 37 ◦C in a humidified atmosphere containing 5% CO2 in RPMI
1640 medium supplemented with 10% FBS, 100 units/ml penicillin,
and 100 units/ml streptomycin.
2.7. Cytotoxicity assay and reversal power calculation
Using PTX solution (TaxolTM, a 50:50 mixture of Cremophor EL
and ethanol) and DOX·HCl solution as control, the cytotoxicities
of PTX loaded SLN and DOX loaded SLN were performed against
MCF-7, MCF-7/Adr, SKOV3 and SKOV3-TR30 cells. Briefly, cells were
seeded in a 96-well plate at a seeding density of 10,000 cells per
well in 0.2 ml of growth medium consisting of RPMI 1640 with
10% FBS and antibiotics. After cells were cultured at 37 ◦C for 24 h,
the growth medium was removed and growth medium containing
the different amount of drug, in solution or loaded in SLN,
76 J. Miao et al. / Colloids and Surfaces B: Biointerfaces 110 (2013) 74–80
were added respectively. Blank SLN at different concentrations in
growth medium were also added to cells. All the experiments were
performed in triplicate.
The cells were further incubated for 48 h at 37 ◦C. Then 100 ␮l
of fresh growth medium containing 50 mg MTT was added to each
well and cells were incubated for 4 h at 37 ◦C. After removing the
unreduced MTT and medium, each well was washed with 100 ␮l of
PBS and 180 ␮l of DMSO were then added to each well to dissolve
the MTT formazan crystals at room temperature. Finally, the plates
were shaken for 20 min at room temperature and the absorbance of
formazan product was measured at 570 nm in a microplate reader
(BioRad, Model 680, USA). Survival percentage was calculated as
compared to mock-treated cells (100% survival).
The reversal power was calculated from Eq. (3):
Reversal power =
Rf/RN
Sf/SN
(3)
where Rf was the IC50 value of drug solution against drug resistant
cells, RN was the IC50 value of drug loaded SLN against drug resistant
cells; Sf was the IC50 value of drug solution against drug sensitive
cells, SN was the IC50 value of drug loaded SLN against drug sensitive
cells.
2.8. Analysis of reversal activity
2.8.1. Cellular uptakes of fluorescent SLN
The conjugate of octadecylamine-fluorescein isothiocyanate
(ODA-FITC) was synthesized according to our previous researches
[12], and encapsulated into SLN as a fluorescence probe to
investigate the cellular uptakes. MCF-7, MCF-7/ADR, SKOV3 and
SKOV3-TR30 cells were seeded in a 24-well plate at a seeding density
of 10,000 cells per well in 1 ml of growth medium and allowed
to attach for 24 h. Cells were incubated with FITC-ODA loaded SLN
suspension (the concentration were 100 ␮g/ml) in growth medium
for different times. After the incubation, cells were washed thrice
with PBS (pH 7.4) to remove the fluorescent SLN adsorbed on the
cell membrane, and then directly viewed under a fluorescence
microscope (Leica DM4000 B, Leica, Germany).
2.8.2. Cellular uptake of drug
Cells were seeded in a 24-well plate at a seeding density of
10,000 cells per well in 1 ml of growth medium and allowed to
attach for 24 h. Then cells were incubated with free drug solution,
drug loaded SLN (drug concentration: 2 ␮g/ml) in growth
medium for 1, 2, 4, 12, 24 h. After the cells were washed with
PBS thrice, 100 ␮l trypsin PBS solution (2.5 ␮g/ml) was added. The
cells were further incubated for 5 min. According to the method
reported by us previously [12], the cells were then harvested by
adding 400 ␮l methanol, and were subjected to probe-type ultrasonic
treatment (400 W, 10 cycles with 2 s active-3 s duration,
JY92-II, Scientz Biotechnology Co. Ltd., China) in an ice bath. The
obtained cell lysate was centrifuged at 22,360 × g for 10 min. The
PTX content in the supernatant after centrifugation was measured
by a HPLC method as described in Section 2.4, while the DOX content
in the supernatant after centrifugation was determined using
a fluorescence spectrophotometer as described in Section 2.4.
The protein content in the cell lysate was measured using the
Micro BCA protein assay kit. The drug uptake percentages were
calculated from Eq. (4):
Drug uptake percentage (%) =
C/M
C0/M0
× 100 (4)
where C was intracellular drug concentration at a particular time,
M was unit weight (milligram) of cellular protein at a particular
time, C0 was initial drug concentration, M0 was initial unit weight
(mg) of cellular protein.
Fig. 1. In vitro release profiles of paclitaxel or doxorubicin loaded SLN. Mean ± SD,
n = 3.
3. Results
3.1. Characteristic of SLN
Blank and drug loaded SLN were prepared by a solvent diffusion
method. The volume average diameters, zeta potential and
the polydispersity indexes of the resulting SLN are listed in Table 1.
As SLN used in our study were the redispersions in 0.1% poloxamer
188 solution, the results were the data of SLN redispersions.
From Table 1, it was also found that the sizes of drug loaded SLN
were higher than that of blank SLN due to the incorporation of drug
in the SLN matrix which increased the amount of solid phase. All
of the absolute values for zeta potential of the SLN were above
30 mV, which demonstrated that the nanoparticles redispersion
was a physically stable system. Table 1 also reports the encapsulation
efficiency (EE) and drug loading (DL) of drug loaded SLN. About
60 wt% EE and 3 wt% DL could be reached by the present preparation
method.
3.2. In vitro release study
Fig. 1 shows the PTX and DOX release profiles from drug loaded
SLN formulations within 24 h. The cumulative amount of PTX
released over 24 h was 41.6%, while the cumulative amount of DOX
released over 24 h was 67.3%. It can be seen from the results that
the PTX presented sustained release profile from PTX loaded SLN. As
PTX is a more lipophilic drug with better affinity for lipid material
compared to DOX, the PTX loaded SLN shown a relatively slower
in vitro release profile than DOX loaded SLN.
3.3. The anti-cancer and reversal MDR activities of drug loaded
SLN
Blank SLN formulations showed very high value of IC50 in both
the breast and ovarian cell lines and their multi-drug resistant variants
(Table 2), suggesting these lipid materials were safe to prepare
SLN as drug carriers.
Table 2 reports IC50 values for drug solution and drug loaded SLN
against drug sensitive cells and drug resistant cells. Fig. 2 shows cell
survival curves after exposuring to drug solution and drug loaded
SLN. From Table 2 and Fig. 2, it was found that SLN apparently
improved the cytotoxicity of PTX comparing to Taxol in sensitive
human breast cancer cells (MCF-7). In the MDR cells (MCF-7/ADR),
the IC50 value of Taxol was nearly 30-fold higher than sensitive
cells, however, PTX loaded in SLN had an even lower IC50 value than
the sensitive cells, which meant the PTX loaded SLN could totally
J. Miao et al. / Colloids and Surfaces B: Biointerfaces 110 (2013) 74–80 77
Table 1
Properties of blank and drug loaded SLN (n = 3).
Formulation dv (nm) PI (–) (−mV) EE (%) DL (%)
Blank SLN 273 ± 44 0.101 ± 0.015 36.4 ± 2.1 – –
PTX loaded SLN 437 ± 68 0.436 ± 0.068 41.2 ± 5.2 61.8 ± 1.7 2.92 ± 0.15
DOX loaded SLN 481 ± 31 0.300 ± 0.038 46.6 ± 4.3 58.2 ± 2.8 2.83 ± 0.13
dv, PI, , EE and DL indicate the volume average diameter, polydispersity index, zeta potential, drug entrapment efficiency and drug loading, respectively.
Table 2
Cytotoxicities of blank SLN, drug solution and drug loaded SLN against drug sensitive cells and drug resistant cells (n = 3).
Formulation IC50 (␮g/ml) Reversal power IC50 (␮g/ml) Reversal power
MCF-7 MCF-7/ADR SKOV3 SKOV3-TR30
Blank SLN 549 ± 29 564 ± 22 – 544 ± 24 558 ± 24 –
Taxol 0.290 ± 0.011 8.61 ± 0.28 – 0.160 ± 0.003 9.35 ± 0.25 –
PTX loaded SLN 0.092 ± 0.002 0.088 ± 0.003 31.0 0.089 ± 0.008 1.36 ± 0.05 3.8
DOX·HCl solution 0.176 ± 0.005 6.20 ± 0.22 – 0.52 ± 0.08 1.83 ± 0.11 –
DOX loaded SLN 0.120 ± 0.005 0.99 ± 0.09 4.3 0.48 ± 0.07 0.91 ± 0.07 1.9
Fig. 2. Cell survival curves against drug concentration after MCF-7 (A), MCF-7/ADR (B), SKOV3 (C) and SKOV3-TR30 (D) were incubation with drug solution and drug loaded
SLN. Mean ± SD, n = 3.
78 J. Miao et al. / Colloids and Surfaces B: Biointerfaces 110 (2013) 74–80
Fig. 3. Fluorescence images of the MCF-7, SKOV3 cells and their multi-drug resistant variants when incubated with fluorescent SLN (the concentrations of the SLN were
100 ␮g/ml). Original magnification 40×.
reverse the PTX-resistant activity in MCF-7/ADR cells. As a result,
the reversal fold of PTX in SLN against MCR-7/ADR was 31.0, which
could be caused by the enhanced endocytosis of SLN delivered drug
bypassing or “inhibiting” the pump efflux by P-gp.
In MCF-7 cells, SLN could not apparently improve the cytotoxicity
of DOX over DOX·HCl solution much. This result may be due
to the burst drug release behavior of DOX loaded SLN at the initial
stage, which led to a lower DOX concentration internalized into
cells. However, in MCF-7/ADR cells, after loading into SLN, the cytotoxicity
of DOX was improved about 6-fold, and the reversal power
of SLN was 4.3.
In SKOV3 cells, PTX loaded SLN did show an apparent cytotoxicity
improvement in both sensitive and resistant cells, with reversal
power of 3.8 (Table 2). Also in SKOV3 cells, DOX loaded SLN showed
a poorer improvement of cytotoxicity in both sensitive and resistant
cells, as a result, the reversal power of DOX loaded SLN was
below 2.
3.4. Analysis of reversal activity
3.4.1. Cellular uptakes of fluorescent SLN
Fig. 3 shows the fluorescence images when MCF-7, MCF-7/ADR,
SKOV3 and SKOV3-TR30 cells were incubated with fluorescent SLN
for different incubation time.
It was found that the uptake of SLN were without obvious differences
between drug sensitive cells and drug resistant cells in
both MCF-7 and SKOV3 cells. On the other hand, the uptake of fluorescent
SLN increased with incubation time in all four cell lines.
The SLN may enter the cells via endocytosis, which is not a P-gp
dependent pathway.
3.4.2. Cellular uptake of drug
Fig. 4A shows the cellular uptake percentages of PTX at different
incubation time when the MCF-7 and MCF-7/ADR cells were
incubated with PTX in different formulations. The cellular uptake
percentages of PTX loaded in SLN obviously increased more compared
with that of Taxol. The cellular uptake percentages of PTX
delivered by SLN in MCF-7/ADR cells were similar or even higher
than that in MCF-7 cells at the same incubation time. This result
was consistent with the total reversal of PTX-resistant activity in
MCF-7/ADR cells, in which the cytotoxicity of PTX loaded SLN in
MCF-7/ADR cells was higher than that in MCF-7 cells.
Fig. 4B shows the cellular uptake percentages of DOX at different
incubation times when the MCF-7 and MCF-7/ADR cells were
incubated with DOX in different formulations. The cellular uptake
percentages of DOX loaded in SLN obviously increased compared
with that of DOX·HCl solution. However, the cellular uptake percentages
of DOX delivered by SLN in MCF-7/ADR cells were still
lower than that in MCF-7 cells at the same incubation time. This
result was consistent with the partial reversal of DOX-resistant
activity in MCF-7/ADR cells, in which the cytotoxicity of DOX loaded
SLN in MCF-7/ADR cells was still lower than that in MCF-7 cells.
Fig. 5A shows the cellular uptake percentages of PTX at different
incubation time when the SKOV3 and SKOV3-TR30 cells were
incubated with PTX of different formulations. The cellular uptake
percentages of PTX loaded in SLN obviously increased compared
with that of Taxol. However, the cellular uptake percentages of PTX
delivered by SLN in SKOV3-TR30 cells were still lower than that in
SKOV3 cells at the same incubation time. This result was consistent
with the partial reversal of PTX-resistant activity in SKOV3-TR30
cells, in which the cytotoxicity of PTX loaded SLN in SKOV3-TR30
cells was still lower than that in SKOV3 cells.
Fig. 5B shows the cellular uptake percentages of DOX at different
incubation time when the SKOV3 and SKOV3-TR30 cells were
incubated with DOX of different formulations. The cellular uptake
percentages of DOX loaded in SLN obviously increased compared
with that of DOX·HCl solution. However, the cellular uptake percentages
of DOX delivered by SLN in SKOV3-TR30 cells were still
lower than that in SKOV3 cells at the same incubation time. This
result was consistent with the partial reversal of DOX-resistant
activity in SKOV3-TR30 cells, in which the cytotoxicity of DOX
loaded SLN in SKOV3-TR30 cells was still lower than that in SKOV3
cells.
4. Discussion
In this study, we prepared SLN by the method of solventdispersion.
The EE and DL of obtained SLN was around 60 wt% and
J. Miao et al. / Colloids and Surfaces B: Biointerfaces 110 (2013) 74–80 79
Fig. 4. PTX (A) or DOX (B) uptake percentage against incubation time after MCF-7
and MCF-7/ADR cells were incubated with Taxol and PTX loaded SLN. Mean ± SD,
n = 3.
3 wt%, respectively. The low EE and DL might due to the shortage of
SLN including limited drug loading capacity, some drug loss into the
aqueous system during the formulation, and drug expulsion during
storage [14,15]. In addition, the lower EE and DL of DOX loaded in
SLN than that of PTX was mainly due to the lower solubility of DOX
in water.
The profiles of in vitro release showed the release plateau at
40% for PTX and 65% for DOX over 24 h. After 24 h, the drug was
still in SLN, and could be released still PTX or DOX from SLN.
These results indicated that PTX and DOX could be released slowly
from SLN and be kept at a constant concentration for a relatively
long period. Therefore, the frequency of administration might be
reduced, which is beneficial for clinical application.
The difference between the release properties of PTX and DOX
was attributed to the prolonged release function of SLN. Lipophilic
drug was held by the hydrophobic carrier, and the drug was
released primarily through dissolution and diffusion. As PTX is a
more lipophilic drug with better affinity for lipid material compared
to DOX, the PTX loaded SLN shown a relatively slower in vitro
release profile than DOX loaded SLN.
In MDR cancer cells, P-gp is an organic cation pump that is capable
of transporting a variety of structurally and functionally diverse
Fig. 5. PTX (A) or DOX (B) uptake percentage against incubation time after SKOV3
and SKOV3-TR30 cells were incubated with DOX·HCl solution and DOX loaded SLN.
Mean ± SD, n = 3.
chemotherapeutic drugs [16]. Owing to the overexpression of P-gp,
cancer cells actively efflux the drug, leading to reduced intracellular
drug accumulation and decreased therapeutic efficacy.
Based on our previous study, we concluded SLN could be taken
up by target cells efficiently due to the membrane affinity of lipid
material and nano-scale size of SLN. Herein, SLN loading drugs with
controlled release profiles within 24 h, accumulated in cytoplasm,
served as a drug-reservoir releasing the drug continuously to the
cytoplasm to overcome the drug reduction from P-gp mediated
drug efflux. On the other hand, it was reported that the cellular
uptake of drug loaded NPs was an ATP-mediated action [3]. Thus,
lipid NPs were presumed to act as a competitive inhibitor to P-gp’s
action, which would be responsible for the drug uptake increase
and MDR reversal in multi-drug resistant cells. In this study, compared
with free drug solution, the drug loaded in SLN not only
improve cytotoxicity against sensitive cancer cells, which were also
seen in a previous study in vitro [17] and in vivo [18], but also
improve cytotoxicity against resistant cancer cells. As a result, SLN
showed MDR reversal activity in both two multi-drug resistant cell
lines (MCF-7/ADR and SKOV3-TR30).
In our work, we made drug loaded SLN with considerable anticancer
and MDR reversal activity. However, whether and to what
80 J. Miao et al. / Colloids and Surfaces B: Biointerfaces 110 (2013) 74–80
extent SLN could overcome the P-gp’s efflux and deliver drug
into MDR cells was still uncertain. Thus the cellular uptake of
PTX or DOX delivered by SLN was measured to establish a clear
relationship between the reversal activity and the drug concentration
in cells. Lipid matrix-based SLN can increase the drug
transport into cancer cells by efficiently cellular uptake both in
sensitive and resistant cancer cells. As low intracellular drug concentration
is the universal character in multi-drug resistant cells
when the anticancer agent was administrated, the drug concentration
increase in the target cells is the key point for reversing
the MDR. Thus the cellular drug uptake result of anticancer
drug loaded SLN was consistent with its drug resistance reversal
activity.
5. Conclusion
Intracellular drug concentration deficiency caused by P-gpmediated
efflux in cancer cells is a key mechanism for the
occurrence of MDR. Herein, solid lipid nanoparticles drug delivery
system can increase the transport of standard anticancer drugs
(PTX or DOX) into cancer cells and enhance the cytotoxicity against
sensitive cancer cells and their multi-drug resistant variant cells,
compared with free drug solutions. That is to say, lipid matrix based
SLN can protect the encapsulated drug from the P-gp efflux mechanism
of the cell and overcome multi-drug resistance, and revealing
a potential application of this drug resistance reversal mechanism
in drug resistant human cancer cells.
Acknowledgments
We appreciate the financial support from the National Basic
Research Program of China (973 Program) under Contract
2009CB930300, and Zhejiang Provincial Program for the Cultivation
of High-level Innovative Health Talents.
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