Anticancer drug encapsulated in inorganic lattice can overcome drug resistance

Article abstract of DOI:10.1039/b925831k

A methotrexate (MTX)-layered double hydroxide (LDH) hybrid have been developed as a drug delivery system, in which an anticancer drug, MTX, was intercalated into a 2-dimensional LDH nanovehicle to form a nanohybrid. According to the comparative cell viability studies between MTX only and its LDH nanohybrid on MTX sensitive and resistant cell culture lines, it was found that the MTX-LDH nanohybrid could bypass the MTX resistance and eventually inhibit cancer cell proliferation very effectively compared to free MTX, due to an enhanced permeability and retention effect of MTX-LDH nanoparticles even in dihydrofolate reductase-overexpressing MTX-resistant cells. This is definitely associated with the uptake mechanism via a clathrin-mediated endocytic pathway for the MTX-LDH nanohybrid particles, the same as for the LDH nanocarrier only, which is completely different from the cellular uptake mechanism for MTX only, the reduced folate carrier (RFC) and/or the folate receptor entries.

Full text of DOI:10.1039/b925831k

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PAPER
www.rsc.org/materials | Journal of Materials Chemistry
Anticancer drug encapsulated in inorganic lattice can overcome drug
resistance†‡
a
b
c
b
b
b
Soo-Jin Choi, Go Eun Choi, Jae-Min Oh, Yeon-Ji Oh, Myung-Chul Park and Jin-Ho Choy*
Received 7th December 2009, Accepted 22nd March 2010
DOI: 10.1039/b925831k
A methotrexate (MTX)-layered double hydroxide (LDH) hybrid have been developed as a drug
delivery system, in which an anticancer drug, MTX, was intercalated into a 2-dimensional LDH
nanovehicle to form a nanohybrid. According to the comparative cell viability studies between MTX
only and its LDH nanohybrid on MTX sensitive and resistant cell culture lines, it was found that the
MTX-LDH nanohybrid could bypass the MTX resistance and eventually inhibit cancer cell
proliferation very effectively compared to free MTX, due to an enhanced permeability and retention
effect of MTX-LDH nanoparticles even in dihydrofolate reductase-overexpressing MTX-resistant
cells. This is definitely associated with the uptake mechanism via a clathrin-mediated endocytic
pathway for the MTX-LDH nanohybrid particles, the same as for the LDH nanocarrier only, which is
completely different from the cellular uptake mechanism for MTX only, the reduced folate carrier
(
RFC) and/or the folate receptor entries.
Introduction
cellular uptake mechanism of LDH nanoparticles, namely, the
clathrin-mediated endocytosis, the most common energy-
6,8
Chemotherapy with cytotoxic drugs is one of the most widely
prescribed methods for treating various cancers. However, one
serious problem associated with chemotherapy is the frequent
development of tumor cell resistance to these agents during
dependant endocytic pathway in all mammalian cells.
Methotrexate (MTX), a folate analogue, is used to treat a variety
of cancers including osteogenic sarcoma, acute lymphoblastic
leukemia, breastcancer, and head and neck cancers byinhibiting the
1–4
clinical treatment. For successful cancer therapy, high efficacy
and low side effect drugs are highly needed, which might possibly
be achieved by drug-nanoparticle hybrid systems. Recently,
various inorganic nanomaterials have emerged as an important
class of drug delivery systems in nanomedicine fields. Among
them, layered double hydroxides (LDHs) have attracted a great
deal of attention due to their ability to release drugs in
a controlled manner, to facilitate cellular interaction, and
enhance cellular uptake and eventually lead to a high efficacy of
drugs. In our previous reports, we found that anticancer drugs-
LDH nanohybrid systems could considerably enhance the
cellular uptake of drugs and consequently suppress the cancer
9
metabolism of folate. In mammalian cells, MTX molecules can
enter the cell membranes via two major transport systems, the
10,11
reduced
folate
carrier
(RFC)
and
the
12,13
glyco-
sylphosphatidylinositol-anchored folate receptor,
the same
uptake route to that of folate. The MTX molecules can then serve as
a substrate for folylpolyglutamyl synthetase (FPGS) and convert it
14
into MTX polyglutamates. This process leads to the irreversible
inhibition of the enzyme dihydrofolate reductase (DHFR) and
eventually induces blocking of the intracellular pathway whereby de
novo biosynthesis of thymidine and purine occurs for DNA
synthesis in a later process, and finally contributes to anti-prolifer-
ation. However, a low therapeutic response of MTX is often found
to be associated with the clinical resistance of certain cancers. In
general, acquirement of MTX resistance is known to be related to
the following: 1) decreased MTX transport attributed by the
5–7
cell proliferation effectively as well. Furthermore we were able
to clearly demonstrate that such a positive effect of an inorganic
drug delivery vehicle is directly associated with the specific
15,16
absence or defects of RFC;
2) loss of MTX polyglutamation
17,18
activity through decreased FPGS expression;
a
Department of Food Science and Technology, Seoul Women’s University,
Seoul, 139-774, Korea. E-mail: sjchoi@swu.ac.kr; Fax: +82 2 970 5977;
Tel: +82 2 970 5634
3) increased levels
b
Center for Intelligent NanoBio Materials, Division of Nano Science BK21,
Department of Bioinspired Science and Department of Chemistry and Nano
Science, Ewha Womans University, Seoul, 120-750, Korea. E-mail:
jhchoy@ewha.ac.kr; Fax: + 82 2 3277 4340; Tel: +82 2 3277 4135
c
Department of Chemistry and Medical Chemistry, College of Science and
Technology, Yonsei University, Wonju, Gangwondo, 220-720, Korea.
E-mail: jaemin.oh@yonsei.ac.kr; Fax: +82 33 760 2182; Tel: +82 33 760
2
368
This paper is part of a Journal of Materials Chemistry themed issue on
†
Advanced Hybrid Materials, inspired by the symposium on Advanced
Hybrid Materials: Stakes and Concepts, E-MRS 2010 meeting in
Strasbourg. Guest editors: Pierre Rabu and Andreas Taubert.
‡
Electronic supplementary information (ESI) available: X-Ray
diffraction pattern of pristine LDH and MTX-LDH. See DOI:
0.1039/b925831k
Scheme 1 The mechanism of action of MTX-LDH nanohybrid for
1
bypassing drug resistance (DR).
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15,19,20
of DHFR resulting from gene amplification
21–23
mutation resulting in reduced affinity for MTX.
or DHFR
cells were washed twice with phosphate-buffered saline (PBS),
and harvested by trypsin-EDTA treatment, and the cell pellets
were collected by centrifugation. After washing twice with PBS,
cell lysis radioimmunoprecipitation assay (RIPA) buffer (25 mM
Tris$HCl pH 7.6, 150 mM NaCl, 1% Nonidet-P40 (NP-4), 0.1%
sodium deoxycholate, 0.1% SDS, protease inhibitors), was
In this study, we employed LDH intercalatively encapsulated
with MTX (MTX-LDH nanohybrid) to overcome MTX resis-
tance and to enhance drug efficacy in DHFR-overexpressing
human osteosarcoma (HOS) cells (Scheme 1). The mechanism of
action as to whether the hybrid system could bypass the drug
resistance and enhance the anticancer activity has been system-
atically investigated.
ꢂ
added, and the pellets were incubated for 20 min at 4 C followed
by ultrasonication for 20 s. (Sonics & Materials Inc.). The crude
cell lysates were then centrifuged at 10,000 ꢃ g for 5 min. DHFR
and b-actin (42 kDa) were detected by incubating the samples
with a monoclonal anti-DHFR antibody (Abnova, 1 : 1000
dilution) and anti-actin antibody (Sigma, 1 : 1000 dilution), fol-
lowed by secondary antibody IgG (Pierce, 1 : 5000 dilution)
conjugated to alkaline phosphatase. Proteins were visualized
using a 1-step NBT-BCIP (nitro blue tetrazolium chloride and 5-
bromo- 4-chloro-3-indolylphosphate-p-toluidine salt) according
to the manufacturer’s instructions (Pierce Chemical). Protein
concentration of the cell lysate was determined with a Bradford
Protein assay (Bio-Rad).
Materials and methods
Synthesis of the MTX-LDH nanohybrid
The MTX-LDH nanohybrid was prepared by a conventional co-
precipitation route. Powdered MTX (0.80 g) was first dispersed in
decarbonated water (53 mL), and titrated with NaOH solution
(
0.5 M) to produce Na-salt type MTX solution at pH 7.0. 53 mL of
mixed metal solution containing MgCl $6H O (0.72 g) and
AlCl $6H O (0.43 g) with a molar ratio of Mg/Al ¼ 2/1 was then
2
2
3
2
added to the MTX solution, and it was then titrated with a NaOH
solution (0.5 M) until pH ꢀ9.5 to produce yellowish precipitates.
The prepared suspension was stirred at room temperature for 48 h
under nitrogen purging, and finally the resulting yellowish
precipitate was isolated by centrifugation, washed thoroughly
with decarbonated water, and freeze-dried. The above procedure
was also applied without the MTX solution in order to prepare
LDH carriers only (denoted as LDHs). All the samples were
characterized by powder X-ray diffraction (Rigaku X-ray
diffractometer with the monochromated beam of Cu-Ka radia-
tion (l ¼ 1.5418)) and Fourier transform infrared (FT-IR) spec-
troscopy (JASCO FT/IR-6100) analyses using the standard KBr
disk method. The surface charges of powdered MTX, LDHs and
the MTX-LDH nanohybrid were measured by zeta potentiometer
DHFR activity assay
Confluent cell monolayers were washed with PBS, harvested by
scraper, resuspended in a solution containing 20 mM Tris pH 7.5,
and 1 mM EDTA, and then sonicated twice with an ultra-
sonicator (Sonics & Materials Inc.) for 20 s. The supernatant
product was used in the DHRF activity assay; the cell extracts
2
were incubated in the presence of 66 mM dihydrofolate (FH )
and 0.1 mM NADPH in 50 mM potassium phosphate (pH 7.0)/
ꢂ
0
.1 M KCl buffer at 25 C. The decrease of the absorbance was
monitored at 340 nm according to the oxidation of NADPH to
+
NADP . One unit of activity is defined as the amount of enzyme
which reduces 1 mM of FH to FH per min. Protein concen-
2
4
tration of the cell lysates was determined by Bradford assay (Bio-
Rad).
(
Malvern Nano ZS), and the particle sizes of LDHs and MTX-
LDH were measured by transmission electron microscopy (TEM)
and dynamic light scattering method (zeta sizer: Malvern Nano
ZS). The chemical formulae of LDHs and MTX-LDHs were
determined by inductively coupled plasma-atomic emission
spectroscopy (ICP-AES) and elemental analyses.
In vitro cytotoxicity study
Cytotoxicity of free MTX and MTX-LDH for parental HOS and
MTX-resistant HOS/Mtx cells was evaluated by MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay;
3
cells were seeded onto 96-well plates at a density of 2 ꢃ 10 cells/
Cell cultures
ꢂ
well and incubated overnight at 37 C under 5% CO atmo-
2
Human osteosarcoma (MNNG/HOS) cells were purchased from
American Type Culture Collection (ATCC, Rockville, MD) and
grown in minimum essential medium (MEM) supplemented with
sphere. The medium in the wells was then replaced with fresh
ꢁ
1
medium containing drugs in the range of 0.001 to 10 mg ml and
incubated under standard conditions. After 72 h, 10 ml of MTT
solution (Roche) was added to each well and further incubated
for 4 h, and the reaction was terminated by adding 100 ml of 10%
SDS in 0.01 M HCl solution (Roche). The solution was further
incubated overnight, and the supernatant product was trans-
ferred onto a new microtiter plate after centrifugation to remove
nanoparticles which may disturb the test, and finally absorbance
was measured with a plate reader at 570 nm.
1
0% heat inactivated fetal bovine serum (Welgene. Ltd), 100 units/
ꢁ
1
ml penicillin, and 100 mg ml streptomycin under a humidified
ꢂ
atmosphere (5% CO plus 95% air) at 37 C. MTX-resistant HOS
2
cells (HOS/Mtx) were obtained by serial passage of parental HOS
ꢁ
9
cells in stepwise increasing concentration of MTX from 1 ꢃ 10
ꢁ6
to 1 ꢃ 10 M over a period of 10 months. HOS/Mtx cells were
subsequently maintained in a medium containing 1 mM MTX
until their use in the growth studies. For enzyme assays, they were
grown for 2 weeks in the medium without MTX.
Cellular uptake and retention of MTX
For the quantitative analysis of MTX uptake in both parental
6
Western blot analysis
HOS and MTX-resistant HOS/Mtx cells, the cells (1 ꢃ 10 )
ꢁ1
Western blot analysis was performed to compare the DHFR
expression levels of the two cell lines used. HOS and HOS/Mtx
grown on a 60 mm culture dish were treated with 50 mg mL of
ꢁ1
MTX or 112 mg mL of MTX-LDH, where the concentration of
9
464 | J. Mater. Chem., 2010, 20, 9463–9469
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ꢁ1
ꢁ1
MTX in MTX-LDH (50 mg mL of MTX and 62 mg mL of
LDH) was adjusted to maintain equivalent amounts of free
MTX. After 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 h, the cells were washed
twice with PBS, harvested by scraper, and the cell pellets were
collected by centrifugation. To measure the MTX retention in
cells, the cells were treated with MTX or MTX-LDH for 1 h,
washed twice with PBS, and further incubated in fresh medium
without drugs. After incubation for a given time, the pellets were
washed twice with PBS and lysed with buffer (20 mM Tris-HCl,
pH 7.5, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, and
protease inhibitors) followed by incubating in an ice bath for
2
0 min and ultrasonicating for 20 s. (Sonics & Materials Inc.).
The crude cell lysates were then centrifuged at 10000 ꢃ g for
min. Two volumes of acetonitrile were added to the cell
5
supernatants and mixed vigorously to precipitate proteins. After
centrifugation at 10000 ꢃ g for 3 min, four volumes of chloro-
form were added to the supernatant to remove lipids. The
supernatants were then collected by centrifugation at 2000 ꢃ g
for 10 min and analyzed by high performance liquid chroma-
tography (HPLC). The MTX content in the cell was quantified
by HPLC with an Agilent 1100 series HPLC system on a C18
column (Nucleosil, 250 ꢃ 4.6 mm, Alltech). The mobile phase
was 0.05 M KH PO , 10% acetonitrile, pH 6.6, and the flow rate
2
4
ꢁ1
ꢂ
was 1 mL min with the column temperature of 40 C. And
MTX was detected by UV absorption band at 304 nm.
Endocytic pathway inhibition
Fig. 1 FT-IR spectra for (a) LDHs (b) MTX (c) Na-MTX and (d)
MTX-LDH nanohybrid. The triangles and the arrows in the top image
stand for the characteristic peaks in MTX and LDHs, respectively. The
dotted and dash-dot lines in the bottom image represent the stretching
The effects of endocytosis inhibitors on cellular MTX uptake
ꢁ1
were evaluated. Endocytosis inhibitors, 10 mg ml chlorproma-
ꢁ1
zine (CPZ), 1 mg ml filipin, and 100 mM amiloride hydrochlo-
ride, were used to examine the cellular uptake modulations, by
ꢁ
vibration of COOH and COO , respectively.
ꢂ
pre-incubating the cells with the inhibitor for 1 h at 37 C, fol-
ꢁ
1
ꢁ1
lowed by incubation with 50 mg mL of MTX or 112 mg mL of
MTX-LDH for 1 h. The cells were washed twice with PBS,
harvested by scraping, suspended in lysis buffer, and then
intracellular MTX content was analyzed by HPLC as described
above.
observed in the spectra for Na-MTX and MTX-LDH. It is worth
noting here that the peaks associated with carboxylic acid and
carboxylate are distinguishable as follows; in powdered MTX
(
Fig. 1(b)), the carboxylic acid shows three characteristic finger-
ꢁ1
prints at 1636, 1300 and 1410 cm corresponding to the C]O
and C–O stretching bands, and the O–H bending one in COOH
group (dotted vertical lines). On the other hand, the O–H
bending mode disappeared in both the Na-MTX and MTX-
LDH nanohybrid (Fig. 1(c) and (d)). Furthermore, the
Results and discussion
Characterization of the MTX-LDH nanohybrid
ꢁ1
The crystal structures of the LDHs and the MTX-LDH nano-
hybrid were determined by powder X-ray diffractometer,
showing well developed 2-dimensional structures for both LDHs
carboxylate group shows a strong peak at 1625 cm and a broad
ꢁ1
one at around 1400 cm , which are assigned to asymmetric and
ꢁ
symmetric stretching vibrations of the COO group (dash-dot
24
and MTX-LDHs (ESI Fig. S1‡) as reported previously. Upon
vertical lines). The splitting of the COOH stretching mode to
ꢁ
intercalation of MTX, the interlayer distance expanded from
ꢁ
symmetric and asymmetric COO in MTX-LDH nanohybrid
0
.78 (Cl intercalated LDHs) to 1.99 nm.
indicates the electrostatic stabilization of MTX molecules in the
LDH nanocarriers.
The FT-IR spectra clearly showed the stabilization of MTX
molecules in LDHs through electrostatic interaction (Fig. 1). The
The size and morphology of the LDHs and the MTX-LDH
nanohybrid were observed with transmission electron micros-
copy (Fig. 2). Both nanoparticles showed plate-like shape with
average primary particle size of 40 nm. The electron diffraction
patterns of both the LDHs and MTX-LDHs showed a well
developed 2-dimensional, hexagonal lattice type structure.
Since the MTX-LDH hybrid systems were tested with a form
of aqueous suspension for the cell culture test, we had to
measured the colloidal particle sizes of the LDHs and MTX-
LDH nanohybrids. As shown in Table 1, the particle sizes
ꢁ1
broad band at the region 2500–3500 cm found in every spec-
trum is attributed to the O–H stretching absorption, and the
ꢁ1
peaks at around 700 and 450 cm found in LDHs and MTX-
LDHs can be ascribed to the M–O and O–M–O stretching ones
(
arrows) in the LDH framework. The spectrum of powdered
MTX (Fig. 1(b)) showed characteristic stretching vibration
ꢁ1
modes such as the C–H stretching at around 2900 cm , the C]C
ꢁ1
ones in the backbone of aromatic rings at 1453 cm , and the
ꢁ1
C–N one at 1100 cm (triangles). These peaks are commonly
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Fig. 2 TEM images of LDHs (a and b) and MTX-LDH nanohybrid (c
and d). The insets in (c) and (d) represents the selected area electron
diffraction (SAED) patterns for LDHs and MTX-LDH nanohybrid,
respectively.
determined by the light scattering method are about ꢀ100 nm for
both the LDHs and MTX-LDHs. In colloids the nanoparticles
are often solvated and aggregated in solution, and therefore
determined to be larger in size than that measured by micros-
copy. In the case of the MTX only, it tends to aggregate in
aqueous solution, resulting in a larger particle size of around
Fig. 3 A western blot analysis of DHFR (ꢀ21 kDa) in HOS and HOS/
Mtx cells, respectively, showing increased DHFR expression level in
HOS/Mtx cells (a). Increased DHFR activity in HOS/Mtx cells compared
with wild-type HOS cells (b).
1
000 nm, due to its limited solubility. It is therefore worth noting
here that the particle size of MTX-LDH hybrids should be
optimized to ꢀ100 nm, which is suitable for efficient delivery via
8
clathrin-mediated endocytosis. The surface charge (x-potential)
HOS cells with a stepwise increasing concentration of MTX over
a period of 10 months. Abnormal overexpression of the DHFR
level in HOS/Mtx cells compared with wild-type HOS cells was
confirmed by western blot analysis (Fig. 3(a)). On the other hand,
an abnormal increased level of P-gp expression could not be
observed in HOS/Mtx cells (data not shown). This result indi-
cated that an MTX-resistant cell line was well established by
increasing the DHFR expression level. Higher DHFR activity in
HOS/Mtx cells than in wild-type HOS cells was also verified by
measuring the DHFR enzyme activity (Fig. 3(b)).
measurement data also indicate that the MTX molecules are
stabilized via electrostatic interaction with LDHs, since the LDH
particles are determined to be highly positive with a surface
charge of ꢀ+45 mV, whereas the MTX-LDH hybrids exhibit
slightly negative x potential (ꢀꢁ8 mV). Such a result is not that
surprising because it originates from the charge neutralization
and surface adsorption of the MTX molecules. The small nega-
tive x potential of the MTX (ꢀ10 mV) is due to the low ionization
efficiency of weak carboxylic acids in MTX.
Preparation of HOS/Mtx cell line
Effect of the MTX-LDH nanohybrid on cell proliferation of
HOS/Mtx cells
In terms of multi-drug resistance (MDR), P-glycoprotein (P-gp),
an efflux pump with broad substrate specificity, protects cells by
decreasing the intracellular accumulation of many cytotoxic
drugs and is responsible for a clinically important phenotype of
Effect of free MTX or the MTX-LDH nanohybrid on the inhi-
bition of cancer cell proliferation was evaluated in wild-type
HOS cells and HOS/Mtx cells, respectively (Fig. 4). Cell prolif-
eration was measured with the MTT assay, based on the fact that
the yellow tetrazolium salt MTT is reduced by metabolically
active cells, resulting in purple formazan crystals. When free
MTX was treated, inhibition of cell proliferation remarkably
2,25
MDR in tumor cells.
However, P-gp-overexpressing multi-
drug resistant cell lines do not always show remarkable
26,27
cross-resistance to MTX.
Therefore, we have developed
MTX-resistant HOS/Mtx, for the first time, by treating parental
Table 1 Physico-chemical properties of MTX, LDHs and the MTX-LDH nanohybrid
a
Particle size /nm
b
Particle size /nm
Chemical formulae
x-potential/mV
MTX
LDHs
MTX-LDH
C
20
H
22
N
8
O
5
ꢁ10
+45
ꢁ8
N. D.
40
40
1000
100
110
Mg0.67Al0.33(OH)
Mg0.67Al0.33(OH)
2
Cl0.33$mH
(MTX)0.17$mH
2
O
2
2
O
a
b
Particle size measured by TEM. Particle size measured by light scattering method in suspension.
9
466 | J. Mater. Chem., 2010, 20, 9463–9469
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Fig. 4 Comparative growth inhibition profile of LDH, free MTX, and
the MTX-LDH nanohybrid in wild-type HOS and HOS/Mtx cell lines.
decreased in HOS/Mtx cells compared to wild-type HOS cells,
suggesting that MTX resistance resulted in decreased drug effi-
cacy. Whereas, MTX-LDH inhibited both HOS cells and HOS/
Mtx cells with the same degree. It is important to note that MTX
acts as a DHFR inhibitor and the DHFR level in HOS/Mtx cells
is abnormally overexpressed. This result indicates that MTX-
LDH can play a role as an effective anticancer drug even in
DHFR-overexpressing HOS cells.
Fig. 5 Time profiles of the cellular uptake of free MTX (a) and MTX
delivered by MTX-LDH nanohybrid (b) in wild-type HOS cells HOS/
Mtx cells, respectively.
Cellular uptake of the MTX-LDH nanohybrid in HOS/Mtx cells
Drug efficacy of the MTX-LDH nanohybrid is strongly associ-
ated with the intracellular concentration of MTX delivered by
LDH nanocarriers. The time profile of MTX uptake by HOS
nanohybrid was well retained with high concentration level
under a given experimental condition without extracellular
drugs. This result suggests that the uptake amount of MTX
ꢁ1
cells or HOS/Mtx cells, treated with 50 mg mL of MTX or an
equivalent molar amount of MTX-LDH, is shown in Fig. 5.
One thing to note here is that intracellular MTX levels in both
the HOS cells and HOS/Mtx cells treated with free MTX were
very low, showing no significant difference between wild-type
HOS cells and HOS/Mtx cells. As expected, MTX uptake
delivered by MTX-LDH in both cell lines was remarkably high
compared to free MTX. Moreover, no significant difference in
the cellular uptake amount of MTX between HOS cells and
HOS/Mtx cells could be seen, when the MTX-LDH nanohybrid
was treated. Therefore, this result clearly indicates that a large
amount of MTX-LDH can be effectively delivered into MTX-
resistant HOS/Mtx cells, which is enough to block high DHFR
activity as demonstrated in Fig. 4.
Cellular retention of the MTX-LDH nanohybrid in HOS/Mtx
cells
Once an anticancer drug is delivered into cells, the intracellular
drug concentration should be highly retained and accumulated.
The retention of the drug in cells may be related to the P-gp efflux
pump whose function is the exclusion of cytotoxic compounds
into the extracellular environment, or to insufficient initial drug
uptake that leads to rapid degradation in cellular organelle.
Thus, the retention of MTX in both wild-type HOS cells and
HOS/Mtx cells was investigated by incubating the cells in the
absence of drugs after pre-incubation with free MTX or MTX-
LDH for 1 h. Fig. 6 demonstrates that only a negligible amount
of MTX was retained in both cell lines when treated with free
MTX. On the other hand, MTX delivered by the MTX-LDH
Fig. 6 Time profiles of cellular retention of free MTX (A) and MTX
delivered by the MTX-LDH nanohybrid (B) in wild-type HOS cells HOS/
Mtx cells, respectively.
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delivered by LDH nanocarrier for the first 1 h is large enough to
maintain the high intracellular MTX concentration even after
MTX conversion into MTX polyglutamate, and is also well
retained and accumulated in the MTX-resistant cell line.
responsible for an enhanced cellular internalization of MTX,
allowing the drug resistance to be bypassed.
Conclusions
We present here the first example of a drug-LDH nanohybrid
system to overcome drug resistance. The MTX-LDH hybrid
system can be highly effective in both wild-type HOS cells and
MTX-resistant HOS/Mtx cells in terms of inhibition of cancer
cell proliferation, cellular uptake, and intracellular retention. We
also demonstrated that the uptake mechanism of MTX-LDH in
HOS/Mtx cells was mediated by the clathrin-mediated endocytic
pathway, the same entry for the LDH nanocarrier only. It is,
therefore, concluded that the clathrin-coated pit for the inter-
nalization of MTX-LDH, which is completely different from that
for free MTX, is responsible for accumulating high drug
concentrations, even in HOS/Mtx cells and bypassing the MTX
resistance.
Uptake mechanism of the MTX-LDH nanohybrid in HOS/Mtx
cells
To further investigate the possible mechanism responsible for the
MTX-LDH uptake, the uptake modulation after the inhibition
of different endocytic pathways was evaluated. Either wild-type
HOS or HOS/Mtx cells were pre-incubated with endocytosis
ꢂ
inhibitors for 1 h at 37 C followed by treatment with MTX or
MTX-LDH for 1 h. CPZ, filipin, and amiloride were used to
inhibit the clathrin-mediated endocytosis, caveolae-mediated
one, and macropinocytosis, respectively. Since MTX acts as
a folate antagonist, both cell lines were also pre-incubated with
excess folate to induce competitive inhibition and thereby satu-
rate the folate transporter systems. The intracellular MTX
amount was then quantified by HPLC analysis. As shown in
Fig. 7, the uptake amount of MTX was modulated in a different
manner in the HOS/Mtx cells. When free MTX was treated in the
cells, the cellular uptake of MTX could be decreased by filipin,
amiloride, and folate pre-incubation, as expected. As is well
Acknowledgements
This work was supported by the National Research Foundation
of Korea Grant funded by the Korean Government (2010-
0
001487 and 2010-0001488), and partly by the National
Research Foundation of Korea Grant funded by the Korean
Government (2010-0001487) and grants from the Ministry of
Knowledge Economy (10030036).
28
known, the folate-receptors enter the cells via the caveolae site,
the decrease in MTX uptake in the cell is not surprising after pre-
treatment with filipin. In particular, MTX uptake was found to
be highly suppressed by folate pre-incubation, proving the same
entry of MTX to folate. On the contrary, intracellular amounts
of MTX delivered by the MTX-LDH nanohybrid was consid-
erably decreased only when the clathrin-mediated endocytosis
was blocked by pre-treatment with CPZ. The same result was
obtained in wild-type HOS cells (data not shown). This result
suggests the important role of a clathrin-coated pit for the entry
of MTX-LDH, which is completely different from that of free
MTX. Our previous reports demonstrated that LDH nano-
carriers are internalized into HOS cells via clathrin-mediated
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