Evaluation of supramolecule conjugated magnetic nanoparticles as a simultaneous carrier for methotrexate and tamoxifen

Article abstract of DOI:10.1016/j.jddst.2018.07.006

Supramolecular host–guest complexes have the ability to prolong the circulation time, and promote the efficiency of hydrophobic drugs. Therefore, in this project we immobilized cyclodextrin (CD), as a host molecule for tamoxifen (TMX), to mitoxantrone (MTX) conjugated magnetic nanoparticles for simultaneous delivery of anti-cancer drugs toward folate-positive cancer cells. FESEM photography of the magnetic multi-drugs carrier confirmed a hollow circular structure for self-assembled layers of cyclodextrin conjugated to MNPs with average size about 95 nm. Drugs release from supramolecular magnetic nanoparticles (with 86.08% of TMX encapsulation and 60.3% of MTX loading) revealed that protease enzymes can enhance the MTX liberation at pH 5, but don't have any noticeable effect on liberation of TMX at both pH conditions. Also, the simultaneous presence of two drugs onto MNPs induced cytotoxicity in two cancer cell lines, MCF-7 and A549, which is different from the impact of each one of drugs alone. Therefore, we believe that this simultaneous carrier could enhance the efficiency of TMX and MTX anti-cancer drugs and be useful for the intended applications, due to hydrophilicity of supramolecular host–guest complexes and its inherent ability to target folate receptors, simultaneously.

Full text of DOI:10.1016/j.jddst.2018.07.006

JournalofDrugDeliveryScienceandTechnology47(2018)115–122
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology
journal homepage: www.elsevier.com/locate/jddst
Evaluation of supramolecule conjugated magnetic nanoparticles as a
simultaneous carrier for methotrexate and tamoxifen
Azam Sargazia, Mina Azhoogha,b, Sajedeh Allahdada,b, Mostafa Heidari Majda,∗
a
Department of Medicinal Chemistry, Faculty of Pharmacy, Zabol University of Medical Sciences, Zabol, Iran
Student Research Committee, Zabol University of Medical Sciences, Zabol, Iran
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Supramolecular
Simultaneous Carrier
Magnetic Nanoparticles
Methotrexate
Supramolecular host–guest complexes have the ability to prolong the circulation time, and promote the effi-
ciency of hydrophobic drugs. Therefore, in this project we immobilized cyclodextrin (CD), as a host molecule for
tamoxifen (TMX), to mitoxantrone (MTX) conjugated magnetic nanoparticles for simultaneous delivery of anti-
cancer drugs toward folate-positive cancer cells. FESEM photography of the magnetic multi-drugs carrier con-
firmed a hollow circular structure for self-assembled layers of cyclodextrin conjugated to MNPs with average size
about 95 nm. Drugs release from supramolecular magnetic nanoparticles (with 86.08% of TMX encapsulation
and 60.3% of MTX loading) revealed that protease enzymes can enhance the MTX liberation at pH 5, but don't
have any noticeable effect on liberation of TMX at both pH conditions. Also, the simultaneous presence of two
drugs onto MNPs induced cytotoxicity in two cancer cell lines, MCF-7 and A549, which is different from the
impact of each one of drugs alone. Therefore, we believe that this simultaneous carrier could enhance the
efficiency of TMX and MTX anti-cancer drugs and be useful for the intended applications, due to hydrophilicity
of supramolecular host–guest complexes and its inherent ability to target folate receptors, simultaneously.
Tamoxifen
Host-Guest inclusion complexes
1. Introduction
In recent decades, magnetic nanoparticles (MNPs) due to their po-
tential applications in biotechnology have captured much attention
Supramolecules or host-guest inclusion complexes demonstrate the
combined advantages of high entrapping capacity for hydrophobic
molecules and reducing undesirable release of them in non-target sites
by various weak intramolecular noncovalent bonding such as H-
bonding, π … π, hydrophobic effects etc [1,2]. Tamoxifen (TMX) is one
of the best clinical choice for the antiestrogen treatment of all stages of
breast cancer. Also it has been approved as a primary treatment for
early stage breast cancer or as a prevention approach in high-risk
women [3]. Nevertheless, TMX can motivate dose-dependent side ef-
fects include increased blood clotting, liver cancer and etc [4]. So, use
of lower doses with colloidal delivery systems can be a benefit key to
provide maximal pharmacological effects with minimal inadvertent
side effects [5]. The inclusion complex with CDs especially β-cyclo-
dextrin (β-CD), with a hydrophilic outer surface and a hydrophobic
inner surface, can have a big effect on aqueous solubility of hydro-
phobic drugs such as TMX and prolong the circulation time of them
[6,7]. Therefore, nanocapsules may be prepared by immobilizing host-
guest inclusion complexes onto polymer-coated magnetic nanoparticles
which ensures the combined advantages of high drug loading, aqueous
solubility and magnetic separation [3,8].
[9–11]. There is no doubt that the surface of MNPs should be modified
by using polymeric or other layers in order to minimizing agglomera-
tion and decreasing elimination by the immune system. Polyethylene
glycol (PEG) self-assembled monolayer is one of the best choice for
modifying the surface structure of MNPs, because PEGylated MNPs
have proven to be non-immunogenic and non-antigenic as well as
passively accumulate in the tumors via leaky vasculature through the
enhanced permeability and retention effect (EPR). The PEG moiety as
an active functional group also could endow MNPs to be coupled with
tumor targeting ligands to be useful for specific targeting and drug
delivery [12,13].
Folate receptors (FRs), which are of best pathways for endocytosis
of macromolecules, are overexpressed on many cancer cells including
breast, ovarian, lung etc., and show high affinity for the folic acid (FA)
vitamin and its analogues [14–16]. Methotrexate (MTX) as an analogue
of FA has pivotal roles in targeting and treatment of several cancers
simultaneously which overexpress FRs on their surfaces [14]. There-
fore, coupling MTX with water-soluble polymers as macromolecular
carriers can lead to adjusting drug doses, improving drug distribution
and reducing drug disadvantages [17].
∗
Corresponding author.
E-mail address: mostafamajd@live.com (M. Heidari Majd).
https://doi.org/10.1016/j.jddst.2018.07.006
Received 25 February 2017; Received in revised form 5 July 2018; Accepted 8 July 2018
Availableonline10July2018
1773-2247/©2018ElsevierB.V.Allrightsreserved.

A. Sargazi et al.
JournalofDrugDeliveryScienceandTechnology47(2018)115–122
To test this possibility, we began by conjugation of MTX to
temperature, products were precipitated by excess ethanol, then col-
lected via centrifugation. Fig. 1 shows the two-step of synthesis pro-
cedures. Finally, FTIR analysis of products was done. The yield was
89.3%.
PEGylated-MNPs and immobilizing β-CD onto this carrier, and con-
tinued by loading of non-steroidal anti-oestrogen tamoxifen into hy-
drophobic cavity of β-cyclodextrin, to make a multifunctional system.
As a consequence, we hypothesize that this multifunctional system will
demonstrate the combined advantages of selective targeting of cancer
cells by using MTX, high drug loading capacity by using β-CD, aqueous
solubility as well as passive accumulation in tumor tissues by using
PEG, magnetic separation by using MNPs, and also treatment of cancer
cells by using two anticancer drugs TMX and MTX, simultaneously.
2.4. Conjugation of methotrexate to the terminal primary amino groups
(-NH2) of magnetic nanoparticles
The synthesis of modified Fe3O4 NPs (Fe3O4-DPA-PEG-NH2 NPs),
and also conjugation of methotrexate (MTX) to them were reported in
our previous manuscripts [7,15,19]. Briefly, MTX (25 mg, 0.055 mmol)
was activated by 2.2 M excess of DCC (25 mg, 0.121 mmol) at 0 °C and
then, 176.5 mg of Fe3O4-DPA-PEG-NH2 NPs (0.055 mmol equal to the
number of moles of the terminal primary amino groups) were reacted
with them at room temperature (RT) to produce Fe3O4-DPA-PEG-NH2-
MTX NPs. Drug loading efficiency of MTX conjugated to the MNPs was
measured by UV spectrophotometer (CECIL, CE1021) at 302 nm, where
the line equation of standard curve using five concentrations of meth-
otrexate (0.076, 0.038, 0.019, 0.0095, 0.00475 mg/mL) was
y = 32.677x + 0.0157 (R2 = 1). Also, FTIR spectroscopy was em-
ployed to confirm the validity of synthesis procedures.
2. Material and methods
2.1. Materials
β-cyclodextrin (β-CD) was purchased from Acros (New jersey, USA).
Protease was purchased from Sigma Aldrich (Schnelldof, Germany),
methotrexate (MTX) was purchased from Sigma-Aldrich (Steinheim,
Germany) and dialysis bag (MWCO = 12 KD) was purchased from
Sigma-Aldrich (St. Louis, MO, USA). Polyethylene glycol (PEG, Mw,
2000), 4-Toluenesulfonyl chloride (tosyl chloride or TsCl), N-
Hydroxysuccinimide (NHS), Dimethylformamide (DMF), 3-(4, 5-di-
methyl-2-thiazolyl)-2, 5- diphenyl-2H-tetrazolium bromide (MTT),
2.5. Conjugation of β-CD-en to Fe3O4-DPA-PEG-NH2-MTX NPs
Ethylenediamine
(EDA),
Triethylamine
(TEA)
and
1,1′-
Carbonyldiimidazole (CDI) were obtained from Merck (Schuchardt,
Germany). N,N′-Dicyclohexylcarbodiimide (DCC) was purchased from
Alfa Aesar (Karlsruhe, Germany). A549 and MCF-7 cell lines were ob-
tained from the Pasteur Institute Cell Bank (Tehran, Iran). RPMI 1640,
penicillin G (10000 units/ml)-streptomycin (10000 μg/mL), trypsin
0.25%, Phosphate-buffered saline (PBS) were purchased from Caisson
labs (North Logan, UT, USA). Fetal bovin serum (FBS) was purchased
from Gibco (Carlsbad, CA, USA).
The MTX-conjugated modified MNPs (0.055 mmol) were dispersed
in 5 mL of DMF and 6-deoxy-6-aminoethylamino-β-cyclodextrin
(64.7 mg, 0.055 mmol) was added to the reaction mixture. Then, 100 μL
of triethylamine (TEA) was added dropwise to the mixture over 2 h and
the solution was stirred overnight at RT. The Fe3O4-DPA-PEG-NH2-
MTX-CD NPs were precipitated by excess amount of ether and were
separated via centrifugation. Finally, the product was characterized by
FTIR and also field emission scanning electron microscopy (FESEM)
(Mira 3-XMU). Fig. 2 shows the two-steps of synthesis procedures.
2.2. Synthesis of mono-6-tosylated-β-CD (β-CD-OTs)
2.6. Encapsulation of TMX in CD-conjugated Fe3O4-DPA-PEG-NH2-MTX
NPs
β-CD (2 g, 1.76 mmol) was dissolved in 10 mL of pyridine which was
previously dried with sodium sulfate. The solution was stirred and
cooled to 4 °C in an ice-water bath under an argon blanket for 10 min.
Subsequently, p-toluenesulfonyl chloride (TsCl) (0.44 g, 2.288 mmol)
was added and the reaction mixture was stirred for 1 h at 4 °C, there-
upon the reaction was continued overnight at room temperature under
the argon blanket. For purification of products, 300 μL of distilled water
was added and the white solid products were precipitated by acetone
(3 × ). The residual acetone was dried with a stream of argon gas.
TMX (100 mg) was dissolved in 2 mL of DMSO and was stirred very
vigorously to obtain a uniform solution. Separately, CD-conjugated
magnetic nanoparticles containing MTX anticancer drug (0.055 mmol)
were dispersed in phosphate buffered saline (16 mL), and 2 mL of NaCl
20 mM was added to the reaction mixture. Subsequently, two solutions
were mixed to find overall solution and took place for 48 h under
shaking conditions (RT, 150 rpm). The Fe3O4-DPA-PEG-NH2-MTX-CD-
TMX NPs were finally collected by an external magnetic field (Dynamag
TM-50 system).
2.3. Synthesis of 6-deoxy-6-aminoethylamino-β-cyclodextrin (β-CD-en)
The entrapped drug quantity (mg/mL), entrapment efficiency (mg
TMX/mg CD) and percentage of drug encapsulated (%) related to TMX
loaded in cavity of CD-conjugated MNPs were evaluated by UV spec-
trophotometer (CECIL, CE1021) at 278 nm where the line equation of
All tosylated-β-CDs produced in the previous step were dissolved in
5 mL (ethylenediamine) EDA and were stirred at 65 °C for 15 h under an
argon blanket [18]. After cooling the reaction mixture to room
Fig. 1. Schematic representation for synthesis of β-CD-en.
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JournalofDrugDeliveryScienceandTechnology47(2018)115–122
Fig. 2. The preparation steps of self-assembled Fe3O4-DPA-PEG-NH2-MTX-CD NPs.
standard curve using five concentrations of methotrexate (0.24, 0.12,
then were submerged in 40 mL of sodium acetate buffer (pH 5) for 72 h.
Thereupon, proper amounts of buffer medium were withdrawn and
liberation of MTX and TMX from MNPs were measured at pre-
determined time intervals using UV spectrophotometer. Also, for the
control, release of free MTX and free TMX were done in phosphate-
buffered saline.
0.06, 0.03, 0.015 mg/mL) was y = 5.7384x + 0.2061 (R2 = 0.9991).
2.7. Drugs release assay
The in vitro drug release studies for MTX and TMX were done at pH
7.4 and also pH 5 in the presence of protease enzyme at 37 °C. For this
purpose, two dialysis bag were prepared according to following pro-
cedures: I) 10.0 mg modified MNPs in 5 mL PBS were put at 37 °C into a
dialysis bag, which were sealed and then placed in 40 mL of PBS for
72 h. II) 10.0 mg modified MNPs in 5 mL sodium acetate buffer were
sealed at 37 °C in a dialysis bag containing 1 mg/mL of protease and
2.8. Cytotoxicity analysis of the Fe3O4-DPA-PEG-NH2-MTX-CD-TMX NPs
In order to investigate the FRs-targeted drug delivery of the Fe3O4-
DPA-PEG-NH2-MTX-CD-TMX NPs, folate-positive MCF-7 breast cancer
cell line and folate-negative A549 lung cell line were chosen. The cells
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were cultured in a pellet culture, with control medium consisted of
RPMI 1640 media, 10% FBS and 100 μL of Penicillin G/Streptomycin
[20,21]. Then, the cells at a density of 1 × 104 cells/well were trans-
ferred onto 96-well culture plates and incubated for 24 h at 37 °C in a
humidified CO2 incubator. Five different concentrations of Fe3O4-DPA-
PEG-NH2-MTX-CD-TMX NPs, free TMX and free MTX were prepared,
and cell lines were treated with them for 48 and 72 h. According to
loading efficiency of drugs, definite amount of prepared MNPs (1.3 mg/
mL) contain different concentrations of MTX (200 μM) and TMX
(975 μM). Therefore, adjusting the dosage of free MTX and Free TMX
were done according to loading amount of them onto MNPs. At ap-
propriate times, 200 μL of MTT solutions composed of 150 μL of fresh
media and 50 μL MTT (2 mg in 1 mL PBS) were added to wells and
incubated for 4 h. MTT was aspirated off and the formazan crystals of
each well were solubilized in 200 μL of DMSO and 25 μL Sorenson's
buffer followed by reading the absorbance at 570 nm using a spectro-
photometer (BioTek Instruments, Inc., Bad Friedrichshall, Germany).
Fe3O4 NPs, and strong peak at 1100 cm−1 is related to repetitive C-O-C
bonds of PEG. Also, characteristic peaks at 1390 cm−1 (C–N stretch),
1450 cm−1 (C–H bend) and 1630 cm−1 (amide bond) indicated the
existence of amide bond between MTX and MNPs.
Finally, in the last step, other active carboxyl group (probably γ)
was reacted with amino group of β-CD-en to gain Fe3O4-DPA-PEG-NH2-
MTX-CD NPs. In the FTIR spectra of final product (inset of Fig. 4B), in
addition to absorption peaks related to Fe3O4, CD, PEG, and MTX, an
extra peak is found at 1730 cm−1 which is related to amide bond be-
tween MTX and β-CD-en.
So, it is concluded that two amide bond are created I) between
carboxylate group of MTX and terminal amine of MNPs, and II) between
another carboxylate of conjugated MTX to MNPs and amine group of
modified CD.
The FESEM micrographs reveal the morphology and size of pre-
pared MNPs (Fig. 5). It is conceivable that by adding more layers onto
MNPs, their size increase; so that size of Fe3O4-DPA-PEG-NH2 NPs from
14 nm has been changed to 47 nm for Fe3O4-DPA-PEG-NH2-MTX NPs
and 96 nm for Fe3O4-DPA-PEG-NH2-MTX-CD NPs (Fig. 5, inset A-C). On
the other hand, in the inset picture of Fig. 5D, a number of hollow
circular structures are appeared at low magnification, which seem to be
related to self-assembled layers of cyclodextrin conjugated MNPs. These
structures that may have arisen due to hydrogen bonds, are schemati-
cally depicted in Fig. 2. Several findings about host–guest inclusion
complex or cyclodextrin nanogels were published in which a two-di-
mensional self-assembly structure similar with our report were fabri-
cated [26,27].
3. Result and discussion
3.1. Characterization of synthesized MNPs
Despite the therapeutic advantages of the anticancer drugs, they
possess critical side effects that could be harmful for patients. These
side effects are attributed to low solubility, repetitive high dosages, low
blood circulation, and accumulation in non-target tissues [22].
Common procedures for minimize drug side effects, are nanoparticle
drug delivery systems, so the β-cyclodextrin conjugated MNPs were
synthesized and characterized for smart delivery of tamoxifen and
methotrexate toward folate receptor-positive tumor cells.
3.2. Drug loading and sustained release efficiency of anticancer drugs
As the starting step, one hydroxyl group of cyclodextrin was acti-
vated by tosyl chloride (TsCl), and then β-CD-OTs was transferred to β-
CD-en through the nucleophilic substitution reaction. Analytical tools
such as FTIR are required to confirm that the synthesis procedures are
performed correctly. As illustrated in Fig. 3, the main characteristic
peaks in the FTIR spectra of β-CD-en are appeared at 1030 and
1070 cm−1, which are related to the coupled C-C/C-O of β-CD. Also, the
antisymmetric stretching band of the C-O-C glycosidic bridge and vi-
bration band of α-pyranyl are appeared at 1180 and 945 cm−1, re-
spectively. On the other hand, absorption peaks at 1320 and 1590 cm−1
well confirmed the primary amino group of EDA [23,24].
In a separate step of the synthesis, both α and γ carboxyl groups of
methotrexate were activated by DCC according to the method of
Sargazi et al. with some modifications [15]. Then, an amide bond was
constructed between one of them (probably α [25]) and primary amino
group (-NH2) of Fe3O4-DPA-PEG-NH2 NPs (Fig. 2). As demonstrated in
Fig. 4A, absorption peaks at 422, 588 and 630 cm−1 are related to the
Cyclodextrin-based host–guest supramolecular magnetic nano-
particles (CD-MNPs) which were made in this work can affect solubility,
passive targeting and circulation time of tamoxifen. Nevertheless, pas-
sive targeting procedures lack selective targeting of tumor cells.
Therefore, methotrexate was conjugated to the MNPs with a maximum
loading efficiency about 60.3% because of its inherent ability to target
FR receptors. Then, tamoxifen was accumulated into the hydrophobic
cavity of immobilized β-CD to create a water-soluble CD drug complex.
The entrapment efficiency into β-CD was measured by calculating of
the entrapped drug quantity (4.3 mg/mL), the entrapment efficiency
(1.33 mg TMX/mg CD) and percentage of drug encapsulated (86.08%)
[3] using a UV spectrophotometer.
Fig. 6 illustrates the in vitro release profiles of free drugs (MTX and
TMX) and Fe3O4-DPA-PEG-NH2-MTX-CD NPs in two different condi-
tions (phosphate-buffered saline and sodium acetate buffer containing
protease). It is obvious that free TMX and MTX rapidly passed through
the pores of the dialysis membrane. On the contrary, the release of MTX
Fig. 3. The FTIR spectra of β-CD-en. The main bonds are appeared at 1030 and 1590 cm−1
.
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JournalofDrugDeliveryScienceandTechnology47(2018)115–122
Fig. 4. The FTIR spectra of A) Fe3O4-DPA-PEG-NH2-MTX NPs and B) Fe3O4-DPA-PEG-NH2-MTX-CD NPs.
Fig. 5. The FESEM micrographs of A) Fe3O4-DPA-PEG-NH2 NPs, B) Fe3O4-DPA-PEG-NH2-MTX NPs and C) Fe3O4-DPA-PEG-NH2-MTX-CD NPs. Inset D represents
hollow circular structures related to self-assembled layers of β-CD conjugated MNPs at low magnification.
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On the other hand, TMX encapsulation in cavity of β-CD is a phy-
sical loading, therefore presence or absence of protease don't have any
noticeable impact on sustained release of TMX. So, the liberation of
TMX in both pH conditions after 72 h was almost similar (∼70%).
3.3. In vitro cytotoxicity MTT assay
Fe3O4-DPA-PEG-NH2-MTX-CD-TMX NPs have the ability to inter-
nalize actively and passively into cancer cells via folate receptor-
mediated endocytosis, and also via abnormal blood vessels through the
enhanced permeability and retention effect (EPR) [33–35]. This ability
might be due to the hydrophilic properties of PEG and β-CD, and also
inherent ability of MTX to target folate receptors [7,15]. To test this
capability, FRs-positive MCF-7 breast cancer cell line and FRs-negative
A549 lung cancer cell line were chosen in order to investigate the cy-
totoxicity of the Fe3O4-DPA-PEG-NH2-MTX-CD-TMX NPs. Fig. 7 illus-
trates cytotoxicity results for two cancer cells incubated with five dif-
ferent concentrations of prepared MNPs. As
a consequence, the
designed MNPs containing MTX and TMX did not show any appreciable
cytotoxicity in A549 cell lines after 48 h exposure (cell viability 56%),
but after 72 h incubation, the cell viability of A549 cell lines decreased
to 30% of the control.
Fig. 6. Release profile of TMX and MTX from CD-conjugated MNPs at two
different conditions. Nearly 65.12% of MTX was released after 72 h in the
presence of protease. However, 99.4% of free MTX passed through the pores of
the dialysis bag only in 7 h.
These results are different with our previous observations in which
MTX conjugated MNPs didn't show any impact on cytotoxicity of
A549 cell lines in the entire times of treatments (24, 48 and 72 h) [15].
These different results are observed due to simultaneous presence of
both TMX encapsulated into β-CD cavities and MTX conjugated on
MNPs. On the contrary, the Fe3O4-DPA-PEG-NH2-MTX-CD-TMX NPs
showed significant increased cellular cytotoxic effects on cell viability
in the MCF-7 cell lines at both 48 h and 72 h, because of inherent ability
of MTX to target folate receptors (20% cell viability after 72 h ex-
posure). Therefore, the sustained release of TMX from the molecular
host–guest system which we have designed, has a big effect on the cell
viability of both A549 and MCF-7 cell lines. The IC50 values of Fe3O4-
DPA-PEG-NH2-MTX-CD-TMX NPs in A549 and MCF-7 cell lines after
72 h were evaluated 0.98 μM and 0.66 μM, respectively. Also, the re-
lative IC50 values of 56.23 and 39.8 μM were calculated by logarithmic
graphs for prepared MNPs in A549 and MCF-7 cell lines, respectively.
Fig. 8 gives an overview of cytotoxic effects of TMX (non-steroidal
anti-oestrogen anticancer) in oestrogen receptor-negative A549 cell
lines and estrogen receptor-positive MCF-7 cell lines.
from prepared MNPs under physiological condition (pH = 7.4) was
largely inhibited by amide bond between MTX and primary amino
group (-NH2) of Fe3O4-DPA-PEG-NH2 NPs. The rate of liberation of
MTX in the above condition and after 72 h was 18.94%, but release
amount at lysosome-like condition (pH 5.4, 37 °C) was greater than this
amount (65.12%), probably due to the presence of protease enzyme in
the dialysis bag. Lysosomal enzymes such as glycosidases, proteases and
sulfatases are active at the acidic pH (about 4–5) [28], therefore we
hypothesize that the release of the chemically conjugated MTX from
MNPs be an enzyme-dependent process. The first enzymatic hydrolysis
of active substances bound to the polymer carrier was done by Jakubke
and Busch [29]. Since then, a lot of work has been done with various
enzymes to study the cleavage of the amide bond; for example the
enzymatic degradation of γ-PGA nanoparticles with Pronase (non-spe-
cific protease) was studied by Akagi et al. [30]. Or, Mallik et al. con-
firmed that liposomes containing MMP, a matrix metalloproteases
which are over-expressed in diseased tissue, e.g., cancerous tissue, can
induce liposome destabilization and drug release [31]. As a new and
same work, Craig and co-workers used of amide bonds between amino
groups of PLL and free carboxyl groups of PLGA for stabilizing the
core–shell microspheres [32]. They confirmed that protease readily
catalyzed degradation of the amide bonds of microspheres, thus re-
leasing the drug when triggered.
It is obvious that TMX has the ability to reduce cell viability of both
cancer cell lines throughout the entire range of concentrations fol-
lowing 48 h and 72 h exposure. Similar with our work, Croxtall et al.
confirmed that TMX has an inhibitory effect on growth of A549 cell
lines [36]. On the other hand, MTX display a trend of time and con-
centration dependent inhibitory effects only on MCF-7 cell lines
(Fig. 9).
Fig. 7. Cytotoxicity results for cancer cells (MCF-7 and A549) incubated with five different concentration of Fe3O4-DPA-PEG-NH2-MTX-CD-TMX NPs (n = 3) +SD.
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Fig. 8. Cytotoxic study of free TMX in oestrogen receptor-negative A549 cell lines and estrogen receptor-positive MCF-7 cell lines.
Fig. 9. Cytotoxic study of free MTX in FRs-negative A549 cell lines and FRs-positive MCF-7 cell lines. The cell viability remained at more than 60% in all cell lines
following 72 h exposure.
4. Conclusion
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Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
Acknowledgements
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