Ternary-responsive magnetic nanocarriers for targeted delivery of thiol-containing anticancer drugs

Article abstract of DOI:10.1039/c5nj03602j

Cancer is currently the second leading cause of death in the world and one of the most serious challenges in medical therapy. Targeted nanocarrier systems potentially propose a solution to the severe side effects associated with direct drug delivery to the tumor cells (conventional chemotherapy). A novel multi (magnetic, thermo, pH and redox) responsive drug nanocarrier was successfully prepared through the grafting of poly(N-isopropylacrylamide-co-maleic anhydride-g-S-S-Py), fully characterized by various techniques and subsequently used for loading and targeted delivery of 6-mercaptopurine (6MP), a thiol containing anticancer drug. The in vitro drug release behavior of 6MP over 24 h was investigated in the presence of GSH (10 mM and 2 μM for simulating intra and extracellular conditions, respectively) under physiologic (37 °C, pH 7.4) and simulated cancerous tissue (40 °C, pH 5.3) conditions. The thermo, pH and redox responsive 6MP release ability of the developed nanocarriers was proved through the in vitro drug release study. The cell cytotoxicity study on the Raji (Burkitt lymphoma) cell line indicated efficient performance of multi-responsive 6MP-loaded nanocarriers. It was concluded that the newly developed multi-responsive nanocarriers have promising potential for targeting of thiol containing anticancer drugs to the tumor tissues.

Full text of DOI:10.1039/c5nj03602j

View Article Online
View Journal
NJC
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: M. Ghorbani, H.
Hamishehkar, H. Hajipour, N. Arsalani and A. A. Entezami, New J. Chem., 2016, DOI:
1
0.1039/C5NJ03602J.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/njc

Page 1 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ03602J
Ternary-responsive magnetic nanocarriers for targeted delivery of thiol-
containing anticancer drugs
1
2
3
1
Marjan Ghorbani , Hamed Hamishehkar *, Hamed Hajipour , Naser Arsalani and Ali
1
Akbar Entezami *
1
Laboratory of Polymer, Faculty of Chemistry, University of Tabriz, Tabriz, Iran.
2
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
3
Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
*Corresponding authors:
Hamed Hamishehkar: Drug Applied Research Center, Tabriz University of Medical Sciences,
Tabriz, Iran. Tel: +98 41 33363231; Fax: +98 41 33363231; Eꢀmail addresses:
hamishehkarh@tbzmed.ac.ir
Ali Akbar Entezami: Laboratory of Polymer, Faculty of Chemistry, University of Tabriz, Tabriz,
Iran. Tel: + 98 41 33393119; Fax: + 98 41 33340191; Eꢀmail addresses: aentezami@tabrizu.ac.ir
1

New Journal of Chemistry
Page 2 of 31
View Article Online
DOI: 10.1039/C5NJ03602J
Abstract
Cancer is currently the second leading cause of death in the worlds and one of the most serious
challenges in the medical therapy. Targeted nanocarrier systems potentially propose a solution to
the severe side effects associated to direct drug delivery to the tumor cells (conventional
chemotherapy). A novel multi (magnetic, thermo, pH and redox) responsive drug nanocarrier
was successfully prepared through the grafting of poly(Nꢀisopropylacrylamideꢀcoꢀmaleic
anhydrideꢀgꢀSꢀSꢀPy), fully characterized by various techniques and subsequently used for
loading and targeted delivery of 6ꢀmercaptopurine (6MP), a thiol containing anticancer drug. The
in-vitro drug release behavior of 6MP over 24 h was investigated in the presence of GSH (10
mM and 2 µM for simulating intra and extracellular condition, respectively) at physiologic
(37°C, pH 7.4) and simulated cancerous tissue (40°C, pH 5.3) conditions. The thermo, pH and
redox responsive 6MP release ability of the developed nanocarriers was proved through the in-
vitro drug release study. The cell cytotoxicity study on Raji (Burkitt lymphoma) cell line
indicated the efficient performance of multiꢀresponsive 6MPꢀloaded nanocarriers. It was
concluded that the newly developed multiꢀresponsive nanocarriers have a promising potential for
targeting of thiol containing anticancer drugs to the tumor tissues.
2

Page 3 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ03602J
1
. Introduction
Cancer is a major health problem in many parts of the world; it is also one of the most serious
challenges facing medical research. Cancer is currently the second leading cause of death in the
world and is estimated to surpass cardiovascular disease as the leading cause of death in the next
few years. Therefore, tremendous efforts have been made to advance the methods of cancer
therapy. Current cancer chemotherapies often impose problems because of their severe adverse
1
effects on normal tissue . Thus, targeted nanocarrier systems propose a potential solution to this
2
–4
5
problem through direct drug delivery to tumor cells . Enhanced permeability and retention
allow nanoparticles to easily accumulate in tumor tissues; this is known as passive targetedꢀdrug
6
,7
delivery . The incorporation of magnetic nanoparticles (MNPs) into biocompatible
8
nanoparticles promotes the targeting ability of nanoparticles into cancerous tissues through the
9
aid of an external magnetic resonance field while also having EPR effects . Moreover, the use of
stimuliꢀresponsive nanocarriers offers an interesting opportunity for targeted drug delivery to
1
0–14
cancer sites
. Temperature, pH, and redox potential are examples of triggers which can be
1
5–17
used with stimuliꢀresponsive nanocarriers
. The pH is lower at systemic sites of disease,
primitive tumors, and metastasized tumors than in normal tissues. Moreover, the temperature of
tumor tissue has been reported to be a bit higher than that of normal cells. Therefore, these
1
8,19
behaviors can be used for the arrangement of stimuliꢀresponsive drug delivery systems
,
2
0,21
which can operate as the biochemical properties for targeted delivery
. Furthermore, redoxꢀ
responsive vehicles are mostly intended to release drugs in the cytosol, because a high redox
potential difference (∼100–1000 fold) between the reducing intracellular space and the oxidizing
2
2–24
extracellular space enables it to be potential stimuli for drug delivery
. Glutathione (GSH) is a
thiolꢀcontaining tripeptide which controls the intracellular redox potential and is meaningfully
3

New Journal of Chemistry
Page 4 of 31
View Article Online
DOI: 10.1039/C5NJ03602J
involved in this stimuli mechanism. To obtain nanoparticles with high extracellular stability and
fast intracellular drug release, GSHꢀresponsive nanoparticles containing disulfide bonds have
been considered as a fascinating class of biomedical materials to be used for the growth of
2
5–27
delivery systems
. Drugs containing thiol groups, such as 6ꢀmercaptopurine (6MP), can be
2
8,29
conjugated to the polymeric scaffold of nanoparticles via cleavable disulfide bonds
. 6MP is a
special kind of anticancer drug widely used as a clinically important antimetabolite drug. The
aim of the present work was to design a novel multiꢀresponsive nanocarrier by grafting a thermoꢀ
,
pHꢀ, and redoxꢀresponsive copolymer [poly(Nꢀisopropylacrylamideꢀcoꢀmaleic anhydrideꢀSꢀSꢀ
6MP)] onto the surface of magnetic nanoparticles (PolyꢀSꢀSꢀ6MPꢀMNPs). The in vitro
performance of the developed nanoparticles was assessed for drug release and cell cytotoxicity in
simulated cancerous tissue conditions (pH = 5.3, temperature = 40 °C, in the presence of 10 mM
GSH) and lymphoblastoid cells derived from a Burkitt lymphoma cell line, respectively.
2
. Materials and Methods
2
.1 Materials
Ferrous chloride tetrahydrate (FeCl , 4H O), ferric chloride hexahydrate (FeCl , 6H O),
2
2
3
2
ammonia solution (25ꢀ28%), 2ꢀaminoethanthiol (AET) and maleic anhydride (MA) were
purchased from Merck Chemicals Co. (Darmstadt, Germany). Dipyridyl disulfide (DPDS),
dimethylformamide (DMF), 1,4ꢀdioxane, dimethyl sulfoxide (DMSO), glutathione (GSH) and 6ꢀ
mercaptopurine (6MP) were purchased from SigmaꢀAldrich. NꢀIsopropylacrylamide (NIPAAm,
9
9%) was purchased from Acros Organics (Geel, Belgium). DMF was totally dried and then
distilled under reduced pressure. 1,4 dioxane was dried and distilled after refluxing for 3h.
.2 Synthesis of poly(NIPAAm-co-MA)
2
4

Page 5 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ03602J
The copolymerization of NIPAAm with MA were performed in 1,4ꢀdioxane at 65 °C with AIBN
as a radical initiator with mole ratio of [NIPAAM]/[MA]/[AIBN] 85:15:100 under a nitrogen
atmosphere. NIPAAm (1.5g, 13.2 mmol) and MA (0.228 g, 2.32 mmol) were dissolved in 1,4ꢀ
dioxane and the solution was purged with nitrogen gas for 30 min. Afterward, AIBN (0.024 g,
0.145 mmol) was added to the solution and the reaction flask was heated to 65°C under nitrogen
atmosphere with continuous mechanical stirring for 24h. The NIPAAmꢀco–MA copolymer was
separated from the reaction mixture by precipitation with diethyl ether and dried under a
moderate vacuum.
2.3 Synthesis of S-(2-Pyridylthio)cysteamine Hydrochloride (PDA)
3
0
PDA was synthesized following the method similar to the literature . In a typical procedure,
Pyridyl disulfide (2.2 g, 10 mmol) was dissolved in the mixture of 0.8 mL of acetic acid and 20
mL of methanol. Afterward, AET (0.57 g, 5 mmol in 10 mL of methanol) was added dropwise
over a period of 0.5 h and the reaction mixture was stirred for an additional 48 h. Then, the
mixture was concentrated under high vacuum to obtain yellow oil. Finally, the product was
washed with 50 mL of diethyl ether and redissolved in 10 mL of methanol for three times to
receive a white powder.
2
.4 Synthesis of poly(NIPAAm-co-MA-PDA)
Poly(NIPAAmꢀcoꢀMA) (0.4 g) was dissolved in DMF (15 mL) and a solution of PDA (0.22 g,
.2 mmol in 10 mL of DMF) was added dropwise in an ice cooled bath for 30 min and the
1
mixture was warmed up to room temperature over 12 h. Afterward, the flask was immersed into
a 60 °C oilꢀbath for 18 h under nitrogen atmosphere. Finally, the solution was purified by
5

New Journal of Chemistry
Page 6 of 31
View Article Online
DOI: 10.1039/C5NJ03602J
dialysis bag (molecular weight cut off 1000 Da, SigmaꢀAldrich, USA) against deionized water at
room temperature
2.5 Preparation of Magnetic nanoparticles (MNPs)
MNPs were prepared using chemical coꢀprecipitation method according to the following
3
1
procedure . FeCl .6H O (5.46 g, 20 mmol) and FeCl .4H O (2 g, 10 mmol) were dissolved in
3
2
2
2
150 mL of deionized water. The solution was then stirred vigorously until the iron salts were
dissolved under a nitrogen atmosphere. After 10 min of vigorous stirring, 55 mL of ammonia
aqueous solution (28%) was dropped into the reacting solution. The reaction mixture was heated
to 70°C for 30 min. The obtained black Fe O nanoparticles were purified by separation with
3
4
magnet and redispersed in first deionized water, then ethanol for several times. Finally,
magnetically separated MNPs were vacuumꢀdried (VO400, Memmert, Germany) at 50°C for 12
h.
2.6 Synthesis of poly(NIPAAm-co-MA-PDA)-MNPs ferrofluid (poly-PDA-MNPs)
In order to synthesis of polyꢀPDAꢀMNP, 100 mg of polyꢀPDA was added to 10 mL of deionized
water and stirred for 10 minutes at room temperature for the preparation of polymer solution and
then 100 mg of Fe O MNPs were added to this solution. Subsequently, the solution was
3
4
vigorously stirred and incubated at room temperature for 24 h. The unreacted Fe O particles
3
4
were precipitated from ferrofluid by centrifugation at 6000 rpm for 10 min and separated from
polyꢀPDAꢀMNPs. In addition, an external magnet was used for the removal of the unreacted
polyꢀPDA from the polyꢀPDAꢀMNPs ferrofluid.
2.7 Synthesis of poly-S-S-6MP-MNPs (poly-6MP-MNPs)
6

Page 7 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ03602J
Polyꢀ6MPꢀMNPs was synthesized by the following procedure: 4 mL of the polyꢀPDAꢀMNPs
ferrofluid (5 mg/mL) was deoxygenated with nitrogen for 20 min, and a solution of 10 mg 6MP
dissolved in 2 mL DMSO was added dropwise to the reaction system in the nitrogen atmosphere.
The mixture was stirred at room temperature for 48 h. Upon completion, the product was
collected by magnetic separation followed by washing with deionized water several times in
order to separate the unreacted 6MP and remove DMSO solvent from the MNPs. The
encapsulation efficiency (EE) and loading capacity (LC) percentages were calculated according
to the following equations:
Mass of drug in nanoparticles
Encapsulation efficiency(%,w / w) =
×100
Mass of initial added drug
Massof drugin nanoparticles
Massof nanoparticles
Loading efficiency
(
%, w/w
)
=
×100
The unloaded drug concentration was measured by UVꢀvis spectroscopy at 300 nm.
.8 Stability investigation of (poly-6MP-MNPs)
The stability of synthesized system (polyꢀ6MPꢀMNPs) was studied in different pH range (3ꢀ8).
2
ꢀ1
The absorbance of the polyꢀ6MPꢀMNPs suspensions (250 mg.L ) was measured at 542 nm at
pH 3, 4, and 5 (prepared using acetic acid/sodium acetate) and pH 6 and 8 (prepared using
Na HPO /NaH PO .2H O) in sample cell.
2
4
2
4
2
2.9 In vitro drug release
The temperature, pH and redox responsive behavior of polyꢀ6MPꢀMNPs was investigated by
drug release study. 5 mL of polyꢀ6MPꢀMNPs suspension (1 mg/mL) was put into dialysis bag
and immersed into different media against 40 mL of phosphate buffered saline (PBS, 10 mM)
7

New Journal of Chemistry
Page 8 of 31
View Article Online
DOI: 10.1039/C5NJ03602J
containing different GSH concentration (10 mM and 2µM), pH (7.4 and 5.3) and temperature
37°C and 40°C) to explore the redox potential, pH and temperature sensitive ability of the
(
developed drug carrier, respectively and prove the efficient performance of carriers for
intracellular targeted drug delivery to cancer tissues. At a predetermined time intervals, 3 mL of
the outside medium of the dialysis bag was withdrawn and same volumes of fresh medium was
added back to the reservoir. All samples were measured by UVꢀvis spectroscopy at 322 nm
(maximum absorbance wave length of 6MP) to determine the amount of drug release. Due to the
presence of pyridine 2ꢀthione in the medium used for the determination of unloaded 6MP, the
corresponded study was done in 300 nm where pyridine 2ꢀthione has the least UV absorbance.
2.10 Cell cytotoxicity study
To assess the therapeutic ability of nanocarriers, MTT assay was used by measuring the cell
4
viability of Raji cells. The cells were seeded into a 96ꢀwell plate at a density of 2×10 cells/well.
After incubation for 24 h (37 °C, 5% CO ), the culture medium was removed by plate
2
centrifugation and 200 ꢀL growth medium containing various concentrations of free 6MP, polyꢀ
6MPꢀMNPs and polyꢀPDAꢀMNPs were added to each well and incubated for 48 h and 72h.
Then, the MTT solution (5mg/mL) was added and cells were incubated for further 4 h. Finally,
the optical density (OD) was measured at 570 nm by using a microplate reader (Elx808, Biotek,
USA) and the results were compared with respect to control cells.
2.11 Measurements
ꢀ1
Fourier transform infrared (FTIR) spectra was acquired in the range of 4000–400 cm on a
1
Tensor 27 spectrometer (Bruker, Germany). HꢀNMR spectra was recorded at 25˚C on a 400
MHz NMR spectrometer (Bruker, Germany). The Samples were dissolved in DMSO at room
8

Page 9 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ03602J
temperature. Size based on zꢀaverage and Zeta potential of the polyꢀMNPs were carried out by
photoꢀcorrelation spectroscopy (PCS) (ZetasizerꢀZS, Malvern, UK). Transmission electron
microscope (TEM) image of polyꢀMNPs was obtained by a CM120, Philips, Germany with
operating at 200 kV. Scanning electron microscopy (SEM) image of the MNPs was captured by
SEMꢀTESCAN MIRA3ꢀFEG. Thermogravimetric (TG) analysis was performed (TGA/SDTA
ꢀ1
8
51/Mettlear Toledo, Spain) by heating rate of 10 ˚C min from 50 to 800 ˚C under a nitrogen
flow. The lower critical solution temperature (LCST) of polyꢀMNPs was measured by UVꢀ
visible spectrophotometer (Caryꢀ100 UVꢀVis, USA). The temperature of the solution was set
from 20 to 50 ˚C at wavelength of 542 nm. The crystallinity of the MNPs were shown using an
Xꢀray diffractometer (D5000, Siemens, Germany) with Cu Kα radiation at 40 kV in the range of
ꢀ
1
2
θ from 20 to 70 ˚C and scan rate of 3˚ min . Magnetic properties of nanoparticles were
measured by using vibratingꢀsample magnetometer (VSM; AGFM, Kashan, Iran). The
magnetization hysteresis loops were measured at room temperature.
3. Results and discussion
3.1 Synthesis and characterization of poly-6MP-MNPs
The polyꢀ6MPꢀMNPs were prepared in four sequential steps as illustrated in Scheme 1. In the
first step, poly(NIPAAmꢀcoꢀMA) containing 13.7 mol% MA was synthesized by freeꢀradical
random copolymerization. Next, PDA was covalently attached to the MA units by opening the
rings under the formation of amide bonds and carboxyl functional groups.
9

New Journal of Chemistry
Page 10 of 31
View Article Online
DOI: 10.1039/C5NJ03602J
Scheme 1. Synthetic Route of drugꢀloaded nanocarrier (polyꢀ6MPꢀMNPs).
After the synthesis of poly(NIPAAmꢀcoꢀMAꢀPDA), the Fe O MNPs were synthesized by coꢀ
3
4
precipitation of ferric and ferrous salts in an alkaline medium. Aqueous magnetic fluids were
1
0

Page 11 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ03602J
stabilized using hydrophilic polymers as steric stabilizers, and the final polymerꢀMNPs were
3
2
stabilized entirely by electrostatic repulsion .
For this reason, studies have used the carboxyl groups of polymers first to cause an interaction
with iron ions and second to provide enough negative surface charge for the creation of
3
3,34
electrostatic repulsion which guarantees the polymerꢀcoated MNPs’ aqueous stability
. Thus,
in step 3, the poly(NIPAAmꢀcoꢀMAꢀPDA) was bound to the surface of Fe O nanoparticles via
3
4
carboxyl groups of MA to suspend the Fe O nanoparticles in a fine state of dispersion, and the
3
4
poly(NIPAAmꢀcoꢀMAꢀPDA) provided both electrostatic and steric repulsion against particle
aggregation. Finally, to construct the polyꢀMNPsꢀdrug, 6MP as an anticancer drug was
conjugated to the poly(NIPAAmꢀcoꢀMAꢀPDA)ꢀMNPs by a thiolꢀdisulfide exchange reaction to
form form polyꢀ6MPꢀMNPs. The successful synthesis of polymer and polyꢀ6MPꢀMNPs was
evaluated using FTIR (Fig. 1a) and HꢀNMR (Fig. 1b) techniques.
1
1

New Journal of Chemistry
Page 12 of 31
View Article Online
DOI: 10.1039/C5NJ03602J
Fig. 1. Fourier transform infrared spectroscopy (FTIR) (a) and Proton nuclear magnetic
resonance (1H NMR) (b) spectra of synthesized polymer.
The FTIR spectrum offers the chemical structure information of poly(NIPAAmꢀcoꢀMA). The
ꢀ1
ꢀ1
spectrum exhibited strong absorptions at about 1649 cm (υ co) and 1544 cm (υ CꢀN) from the
ꢀ1
NIPAAm and peaks at 1775 and 1840 cm (υ co) attributed to the C=O stretching vibration of
3
5
MA unit . Furthermore, the stretching vibration of isopropyl groups of PNIPAAm appeared at
ꢀ1
1
386 and 1370 cm , and absorption peaks of CꢀOꢀC in MA were exhibited at about 1175 and
ꢀ1
1
125 cm . The formation of the poly(NIPAAmꢀcoꢀMAꢀPDA) was confirmed by the
1
2

Page 13 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ03602J
ꢀ1 35
disappearance of anhydride unit carbonyl bands at 1850 and 1775 cm
and the appearance of a
ꢀ1
new absorption band at 2750 cm attributed to the CꢀH stretching mode of the methylene in the
2
9
PDA group . Compared with the spectrum of poly(NIPAAmꢀcoꢀMAꢀPDA), the introduction of
6MP to the polymer was confirmed by the presence of characteristic purine ring bands between
ꢀ1 29
525 and 795 cm
. For the polyꢀ6MPꢀMNPs, characteristic peaks of copolymer were
ꢀ1
ꢀ
considered at 1649, 1544, and the peak at 586 cm , confirming the polymer linkage via –COO
to the surface of Fe O NPs. Moreover, the chemical structure and the composition of the
3
4
1
copolymers containing 13.7 mol% MA in the feeds were determined by HNMR techniques (Fig.
1
1
b). H NMR spectra of poly(NIPAAmꢀcoꢀMA) (in DMSOꢀd ) were (1) 2H, CH (backbone),
6
2
1.49–1.60; (2) 1H, CH (backbone), 1.89–2.07; (3) 1H, NH (broad peak), 6.05–7.04; (4) 3.94; (5)
and (6) 6H, CH , 1.38–1.44 for NIPAAm unit; (7) and (8) 2H, CH (backbone), 4.23 for
3
3
5
anhydride unit . Additionally, the structure of poly(NIPAAmꢀcoꢀMAꢀPDA) was characterized
1
according to the following explanations: HNMR spectra (in DMSOꢀd ) (a) 2H, CH (backbone),
6
2
1
.35–1.62; (b) 1H, CH (backbone), 1.9–2.2; (c) 3.94; (d) and (e) 6H, CH , 1.15 for NIPAAm
3
unit; (f) and (g) 2H, CH (backbone), 4.23 for anhydride unit; (h), (i), (j) and (k) hydrogen in
pyridine of PDA (side chain), 7.18ꢀ8.32 ppm belong to the hydrogen in pyridine of PDA. Fig. 1b
shows the disappearance of the pyridyl group signals at 7.2ꢀ7.8 ppm and the appearance of
signals at 8.44ꢀ8.6 ppm, indicating the successful graft of 6MP onto the backbone of the
2
8
copolymer . It should be noted that in practice, 6MP was loaded on the polymer after Fe O
3
4
nanoparticles were decorated with synthesized polymer. Since nanoparticulate systems cannot be
examined with the NMR technique, the drug was incubated with only polymer before polymer
decoration on the magnetic nanoparticles to show the ability of polymer to interact with 6MP in
the HꢀNMR experiment. The size of the nanoparticles is an important factor for in-vivo
1
3

New Journal of Chemistry
Page 14 of 31
View Article Online
DOI: 10.1039/C5NJ03602J
performance and pharmacokinetics. Fig. 2 illustrates the size, shape, and morphology of
nanoparticles (polyꢀ6MPꢀMNPs) as measured by TEM, DLS and SEM analyses.
Fig. 2. Transmission electron microscopy (TEM) image of magnetic nanoparticles (MNPs) (a)
and TEM (b) and scanning electron microscope (SEM) (c), dynamic light scattering (DLS) (d)
and Zeta potential (e) profiles of drugꢀloaded nanocarrier (polyꢀ6MPꢀMNPs). The scale bars in
TEM and SEM figures represented 50 and 200 nm, respectively.
The size of the nanoparticles is well within the preferred range (<100 nm) for effective drug
3
6
delivery against cancer tissues via passive targeting . Fig. 2a and 2b presented the TEM images
of MNPs before and after decoration with polymer, respectively. Fig. 2b showed that the
nanoparticles were well dispersed without any aggregation in comparison to Fig. 2a which is
crucial parameter for drug delivery systems especially for those used in passive targeting.
1
4

Page 15 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ03602J
Generally, nanoparticualte systems based on polymers suffer from some degree of aggregation.
Therefore, the system developed in this study is ideal for drug targeting. Spherical particles with
an average particle size of 15ꢀ20 nm were also seen in the TEM image. To further study the size
distribution of the nanoparticles, the SEM image of polyꢀ6MPꢀMNPs, which showed a much
higher population of particles in a lower magnification than TEM, revealed that the particles
were uniformly distributed in almost the same size (Fig. 2c). The DLS data was presented (Fig.
2
d) to support the particle size and distribution. It indicated a narrow size distribution of around
0 nm particles. The size resulted from DLS is a bit larger than SEM which is common because
6
of particle accumulation in aqueous medium. It was reported that the zeta potential value ±30
3
7
mV enhanced the longꢀterm stability of dispersed nanoparticles . To prove this report, it was
necessary to measure the zeta potential of the nanoparticles. As shown in Fig. 2e, the
nanoparticles showed a high negative zeta potential value (ꢀ33 mV). This value may correspond
with the presence of many carboxylic acid groups of poly(NIPAAmꢀcoꢀMAꢀPDA) bonded onto
the surface of Fe O nanoparticles, which enabled both electrostatic and steric repulsion against
3
4
particle aggregation as shown in the TEM image. The characteristics of MNPs were investigated
using the XRD (Fig. 3a) and VSM (Fig. 3b) techniques before and after decoration with polymer.
1
5

New Journal of Chemistry
Page 16 of 31
View Article Online
DOI: 10.1039/C5NJ03602J
Fig. 3. XꢀRay Diffraction (XRD) (a) and vibratingꢀsample magnetometer (VSM) (b) patterns of
neat Fe3O4 and drugꢀloaded nanocarrier (polyꢀ6MPꢀMNPs).
The wellꢀdefined XRD patterns illustrated the formation of highly crystalline iron oxide
nanoparticles. The XRD pattern of pristine Fe O nanoparticles exhibited six intense peaks
3
4
between 30 °C and 70 °C and diffraction peaks at 2Ө= 28, 35.01, 43, 55, 58.1, and 65.8, which
can be attributed to the (220), (311), (400), (422), (511), and (440) plans of Fe O in cubic
3
4
1
6

Page 17 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ03602J
3
8
phase , respectively (Fig. 3a). Moreover, the characteristic peaks of MNPs were observed in
polyꢀ6MPꢀMNP, confirming that no phase change occurred. With the introduction of the
polymer on the surface of Fe O MNPs, however, the intensities of the peaks became weaker. It
3
4
is well known that Fe O nanoparticles show a superparamagnetic property; however, the
3
4
magnetic property of the ferrofluid particles, when the polymers were bonded to the
nanoparticles, had to be studied. Therefore, the magnetic properties of Fe O and polyꢀ6MPꢀ
3
4
ꢀ1
MNPs were examined. The saturated magnetization value of polyꢀ6MPꢀMNPs was 16.5 emu g ,
a lower value than that of the saturation magnetization of pure Fe O nanoparticles (60 emu/g)
3
4
(
Fig. 3b). Despite the decrease in magnetization during the derivatization of Fe O nanoparticles
3 4
with stimuliꢀresponsive polymer, the polyꢀ6MPꢀMNPs are still advantageous, because a
ꢀ1
39
magnetization range of 7–22 emu g is usually desirable in biomedical applications . Thermal
analysis was performed to approve the polymer coating on the surface of the magnetic
nanoparticles and recorded as change in mass from the decomposition, dehydration, and
oxidation of samples. Using this technique, the Fe O content was also calculated. Weight loss is
3
4
3
8,40
not revealed in uncoated and pure iron oxide nanoparticles
, whereas for the other components
a stepꢀwise weight loss is apparent as temperature increases. Therefore, the TG and derivative
TG (DTG curve, red line) curves for the polyꢀ6MPꢀMNPs are shown in Fig. 4.
1
7

New Journal of Chemistry
Page 18 of 31
View Article Online
DOI: 10.1039/C5NJ03602J
Fig. 4. Thermogravimetry (TG) (black line) and derivative TG (DTG) (red line) curves of drugꢀ
loaded nanocarrier (polyꢀ6MPꢀMNPs).
The first stage of mass loss was attributed to the removal of the absorbed physical and chemical
water between 50 and 150 °C. The massive weight loss in the second step at 180ꢀ270 °C might
be attributed to the loss of 6MP and the decomposition of acidic segments of the copolymer.
4
1,42
According to the literature
, pure 6MP shows a sharp mass reduction step at 328 °C. The
appearance of a mass reduction in 6MP at lower temperatures might be due to the grafting on
polymer. The third weight loss which constitutes the major portion of the total weight occurred
1
3
42
from 400ꢀ500 °C and corresponded to the PNIPAAm and 6MP . Finally, from the residue
weight at 520 °C for the mass of Fe O nanoparticles, it could be concluded that the Fe O
3
4
3
4
content in the final synthesized nanopraticles was 60.1%. The LCST of the aqueous solution of
polyꢀ6MPꢀMNPs was investigated by monitoring the change in optical absorbance of the
nanoparticle suspension as a function of temperature and pH. The LCST of the synthesized
ꢀ1
nanocarriers was determined in the nanoparticle concentration of 250 mgL in PBS (pH 7.4 and
1
8

Page 19 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ03602J
5.3) and measured at 542 nm. As shown in Fig. 5, the LCST is about 39 °C in pH 7.4, which is
higher than the LCST of pure PNIPAAm (32 °C).
Fig. 5. The temperature and pHꢀresponsive behavior of drugꢀloaded nanocarrier (polyꢀ6MPꢀ
MNPs) upon temperature and pH changes.
Since the PNIPAAm is considered one of the most thermally responsive polymers which exhibits
the LCST in water, the poly(NIPAAmꢀcoꢀMAꢀ6MA)ꢀcapped Fe O nanoparticles are expected
3
4
to be thermosensitive, and any hydrophobic/hydrophilic modification may affect the LCST
4
3
behavior of PNIPAAm . Therefore, in the present study, the incorporation of a hydrophilic
monomer on the copolymer backbone may provide an obvious pHꢀinduced conformational
transition. At low pH values (5.3), polymer chains with carboxyl side groups adopt a compact
structure because of hydrophobic interactions. In this work, the carboxyl groups of the
copolymer had pHꢀdependent LCST behavior, and as pH was increased, the carboxylic acid
groups of MA segments of the copolymer were easily deprotonated. This led to an increase in the
hydrophilicity of the nanoparticles, which finally resulted in an increase in the LCST of the NPs
1
9

New Journal of Chemistry
Page 20 of 31
View Article Online
DOI: 10.1039/C5NJ03602J
nanoparticles at 36°C (pH= 5) to 39°C (pH=7.4). These findings show the nanocarriers
developed in this study to be well suited for application in targeted drug delivery to cancerous
tissues.
3.2 Investigation on the stability of poly-6MP-MNPs in various pH
The Fe O MNPs must be stable in water at physiological pH and salinity to be an appropriate
3
4
4
4
candidate for applications in biology and medical diagnosis and therapy . In the present study,
the developed nanocarriers was going to be administered for the intracellular delivery of 6MP.
Therefore, the stability of synthesized nanocarriers in the range of 3ꢀ8 was evaluated. This range
covers almost the all pH values influences intracellular drug delivery (lysosomes pH 4.5ꢀ5.0,
endosomes pH 5.0ꢀ6.5, environments in cancerous tissues pH 5.3ꢀ7.2, and For this purpose,
slightly acidic environments in cancerous tissues (pH 6.5ꢀ7.2), endosomes (pH 5.0ꢀ6.5) and
4
5,46
physiological pH of 7.4)
. The results in Fig. 6 indicated that only pH 3 caused instability of
nanocarriers (the separation of polymeric shell from magnetic core). Since the pH range for
intracellular drug delivery is 4.5ꢀ7.4 (as discussed above), it is concluded that the developed
carrier was qualified for intracellular drug delivery.
2
0

Page 21 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ03602J
Fig. 6. Stability of drugꢀloaded nanocarrier (polyꢀ6MPꢀMNPs) as a function of pH.
3.3 Determination of drug content on Poly-PDA-MNPs
To estimate the drugꢀloading capacity of MNPs, the antiꢀtumor drug 6MP was conjugated to the
PolyꢀPDAꢀMNPs by thiolꢀexchange reaction and was analyzed by UVꢀvis spectrophotometry.
The content of drug on the polyꢀMNPs was determined by quantifying the unreacted drug. The
unreacted drug was separated from the polyꢀ6MPꢀMNPs using an external magnet, and the
absorbance was recorded at 300 nm with a UVꢀvis spectrophotometer. The percentages of EE
and LC of 6MP in polyꢀ6MPꢀMNPs were found to be 84.56 ± 5.32 % and 42.28 ± 2.65 %,
respectively.
3.4 Redox-responsiveness and releasing behavior of (poly-6MP-MNPs)
In the presented work, 6MP was attached to Fe O nanoparticles decorated with smart polymer
3
4
through a disulfide bond. The newly developed polymer was designed to be responsive to a
2
1

New Journal of Chemistry
Page 22 of 31
View Article Online
DOI: 10.1039/C5NJ03602J
redoxꢀreducing environment. As is wellꢀknown, the in vivo redoxꢀreducing environment is
controlled by GSH. In this study, it was found that the level of cytoplasmic GSH in some tumor
44, 45
cells is much higher than that in normal cells
. Furthermore, a question always asked for
polymeric nanocarriers is whether these cargos release their drugs after endocytosis into cancer
cells. Thus, a drug release study was carried out at the highꢀend level of intracellular
concentration (10 mM) and the lowꢀend level of extracellular concentration (2 µM) in PBS at 37
°
C. Fig. 7 shows that nanocarriers released almost fourꢀfold more 6MP in a higher GSH
concentration.
2
2

Page 23 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ03602J
Fig. 7. Cumulative in vitro release profiles of 6ꢀmercaptopurine (6MP) at different conditions
from drugꢀloaded nanocarrier (polyꢀ6MPꢀMNPs) (data was presented as mean ± standard
deviation, n=3).
Furthermore, as shown in Fig. 7, the release of 6MP was obviously thermoꢀ and pH–dependent,
which is attributable to the presence of the temperatureꢀresponsive monomer (NIPAAm). At 37
°
C (below the LCST), 51% of MP was released; an increase in temperature to 40 °C resulted in
faster drug release (∼69%), which could be attributed to the collapse of the polymeric shell. In
other words, by heating to 40 °C (above the LCST), the hydrogen bonds between the copolymer
and water were destroyed and, accordingly, the copolymer aggregates became more
hydrophobic. Therefore, this polymer shell shrinks at 40 °C, facilitating the release of loaded
2
1, 46
drug from the nanoparticles
. Interestingly, in spite of the lower ratio of the pHꢀresponsive
segment (13.7% maleic acid), a comparison of drug release patterns under various conditions
indicated that pH played a more imperative role than temperature. The pKa value of maleic acid
is 6.07; therefore, it would be nonꢀionized in simulated cancerous tissue release conditions (pH
5.3). In such case, polymers would be more hydrophobic than under physiologic conditions and,
subsequently, would collapse and probably release the entrapped drug. In addition to the
covalently attachment of 6MP to the polymer backbone, it seems to be loaded by coordination of
6MP via its nitrogen and sulfur donor sites with metallic bonding sites to form metal
41,42
complexes
. It was claimed that the pH responsive release property of 6MP was occurred due
to the ion exchange process taking place between the 6MP anions and the anions in the buffer
solutions which was accelerated by temperature induced polymer collapse. Therefore, it can be
concluded that the disulfide bond reduction (GSH mediated), pH decrease, and temperature
increase in the mentioned order impact on 6MP release from the carriers in simulated cancerous
2
3

New Journal of Chemistry
Page 24 of 31
View Article Online
DOI: 10.1039/C5NJ03602J
tissue condition. The observed drug release patterns (Fig. 7) is wellꢀmatched with these
explanations. Finally, the release study illustrated that the developed carrier responded well to
intracellulr cancerous triggers (high level of GSH, low pH, and high temperature) by releasing 8
times more 6MP than under physiologic conditions (Fig. 7).
3.5 Cell cytotoxicity study
An MTT assay was used to quantify the cell viability of Raji cells (Burkitt lymphoma cell line)
to evaluate the therapeutic effect of the developed nanocarriers against tumor cells. To assess the
cytotoxic effects of free 6MP, polyꢀPDAꢀMNPs and polyꢀ6MPꢀMNPs at concentrations ranging
from 0.25 to16 µg mLꢀ1 were treated with Raji cells for 48h and 72h. As shown in Fig. 8, the
polyꢀPDAꢀMNPs exhibited no significant toxicity on the viability of Raji cells after 48h and 72h,
indicating the biocompatibility of the newlyꢀsynthesized nanoparticles.
2
4

Page 25 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ03602J
Fig. 8. Cell viability of 6ꢀmercaptopurine (6MP), nanocarrier (polyꢀPDAꢀMNPs) and drugꢀ
loaded nanocarrier (polyꢀ6MPꢀMNPs) with different concentrations of 6MP and nanoparticles
versus Raji cell line (Burkitt lymphoma) for 48 h (a) and 72 h (b). C indicates the untreated cells
used as control group (data was presented as mean ± standard deviation, n=6).
Polyꢀ6MPꢀMNPs showed higher inhibition activity after 48h and 72h in comparison with 6MP.
It was reported that drugꢀloaded nanoparticulate systems show better efficacy than drug solution.
It has been proposed that nanoparticles may be able to escape from Pꢀglycoprotein–mediated
2
5

New Journal of Chemistry
Page 26 of 31
View Article Online
DOI: 10.1039/C5NJ03602J
resistance. One probable mechanism is that nanoparticles may avoid recognition by the Pꢀ
glycoprotein efflux pump by means of being enveloped in an endosome when arriving the cell,
resulting in high intracellular drug concentrations. This drug cell accumulation and subsequently
cell cytotoxicity would be increased by time. The greater inhibition of cell growth for polyꢀ6MPꢀ
MNPs compared with the free form of the drug would be advantageous by lowering the dose of
the anticancer drug in application. The polyꢀ6MPꢀMNPs have many favorable qualities as an
ideal targeted drug carrier, such as excellent biocompatibility, high drugꢀloading capacity,
minimal drug release in extracellular conditions, and rapid drug release in response to the
intracellular level of pH, temperature, and reducing agent. The results showed that the polyꢀ
6MPꢀMNPs offer an efficient anticancer delivery system.
4. Conclusion
Anticancer therapy is normally accompanied by unwanted side effects caused by the nonꢀspecific
distribution of drugs into normal tissue. A number of studies have shown that stimuliꢀresponsive
nanocarriers can have significant advantages for targeted delivery. A novel smart nanocarrier
with a multiꢀresponsive polymeric shell was designed and prepared for application as a vehicle
for specific targeting of 6MP to tumors. The in vitro release study of 6MP in physiologic and
simulated cancerous tissue conditions in the presence of GSH in intracellular concentrations
revealed that the developed multiꢀresponsive magnetic nanocarriers exhibited pH, thermoꢀ and
redoxꢀresponsive 6MP release characteristics. It was concluded that this new type of nanoparticle
has great potential for targeted delivery of thiol containing antiꢀcancer drugs to tumorous cells
with thermo/pH/redox/magneticꢀresponsive properties.
Acknowledgement
2
6

Page 27 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ03602J
The authors acknowledge the financial support of the University of Tabriz.
References
1
S. Zimmermann, R. Dziadziuszko and S. Peters, Cancer Treat. Rev., 2014, 40, 716–722.
2
3
S. Bauhuber, C. Hozsa, M. Breunig and A. Göpferich, Adv. Mater., 2009, 21, 3286–3306.
M. Kanapathipillai, A. Brock and D. E. Ingber, Adv. Drug Deliv. Rev., 2014, 79-80, 107–
118.
4
5
M. H. Majd, J. Barar, D. Asgari, H. Valizadeh, M. R. Rashidi, V. Kafil, J. Shahbazi and
Y. Omidi, Adv. Pharm. Bull., 2013, 3, 189–195.
U. Prabhakar, H. Maeda, R. K. Jain, E. M. SevickꢀMuraca, W. Zamboni, O. C. Farokhzad,
S. T. Barry, A. Gabizon, P. Grodzinski and D. C. Blakey, Cancer Res., 2013, 73, 2412–
2417.
6
J. Nicolas, S. Mura, D. Brambilla, N. Mackiewicz and P. Couvreur, Chem. Soc. Rev.,
013, 42, 1147–1235.
2
7
8
9
M. K. Yu, J. Park and S. Jon, Theranostics, 2012, 2, 3–44.
J. Barar and Y. Omidi, 2014, 4, 3–14.
B. Chang, X. Sha, J. Guo, Y. Jiao, C. Wang and W. Yang, J. Mater. Chem., 2011, 21,
9239–9247.
10
11
12
S. F. Medeiros, a. M. Santos, H. Fessi and a. Elaissari, Int. J. Pharm., 2011, 403, 139–
61.
1
S. Ganta, H. Devalapally, A. Shahiwala and M. Amiji, J. Control. Release, 2008, 126,
87–204.
1
R. a Siegel, J. Control. Release, 2014, 190, 337–351.
2
7

New Journal of Chemistry
Page 28 of 31
View Article Online
DOI: 10.1039/C5NJ03602J
13
M. Fathi, A. A. Entezami, S. Arami and M.ꢀR. Rashidi, Int. J. Polym. Mater. Polym.
Biomater., 2015, 64, 541–549.
1
4
5
Y. Omidi, BioImpacts, 2011, 1, 145–147.
1
R. Cheng, F. Meng, C. Deng, H.ꢀA. Klok and Z. Zhong, Biomaterials, 2013, 34, 3647–
3657.
16
17
18
19
20
S. Mura, J. Nicolas and P. Couvreur, Nat. Mater., 2013, 12, 991–1003.
H. Namazi and Y. T. Hamrahloo, Adv. Pharm. Bull., 2011, 1, 40–47.
Y.ꢀJ. Zhu and F. Chen, Chem. - An Asian J., 2015, 10, 284–305.
J. Barar and Y. Omidi, BioImpacts, 2013, 3, 149–162.
R. Salehi, H. Hamishehkar, M. Eskandani, M. Mahkam and S. Davaran, J. Drug Target.,
2014, 22, 327–342.
21
22
23
24
25
26
B. Sahoo, K. S. P. Devi, R. Banerjee, T. K. Maiti, P. Pramanik and D. Dhara, ACS Appl.
Mater. Interfaces, 2013, 5, 3884–3893.
Z. Luo, K. Cai, Y. Hu, J. Li, X. Ding, B. Zhang, D. Xu, W. Yang and P. Liu, Adv. Mater.,
2012, 24, 431–435.
H. Sun, F. Meng, R. Cheng, C. Deng and Z. Zhong, Expert Opin. Drug Deliv., 2013, 10,
109–1122.
1
R. Cheng, F. Feng, F. Meng, C. Deng, J. Feijen and Z. Zhong, J. Control. Release, 2011,
52, 2–12.
1
Q. Zhao, C. Wang, Y. Liu, J. Wang, Y. Gao, X. Zhang, T. Jiang and S. Wang, Int. J.
Pharm., 2014, 477, 613–622.
Y. Zhuang, Y. Su, Y. Peng, D. Wang, H. Deng, X. Xi, X. Zhu and Y. Lu,
Biomacromolecules, 2014, 15, 1408–1418.
2
8

Page 29 of 31
New Journal of Chemistry
View Article Online
DOI: 10.1039/C5NJ03602J
2
7
8
B. Remant, B. Thapa and P. Xu, Mol. Pharm., 2012, 9, 2719–2729.
2
X. Zhang, F. Du, J. Huang, W. Lu, S. Liu and J. Yu, Colloids Surfaces B Biointerfaces,
012, 100, 155–162.
2
2
9
0
H. Zheng, Y. Rao, Y. Yin, X. Xiong, P. Xu and B. Lu, Carbohydr. Polym., 2011, 83,
952–1958.
1
3
Y. W. Ebright, Y. Chen, P. S. Pendergrast and R. H. Ebright, Biochemistry, 1992, 31,
0664–10670.
1
31
32
33
S. Wang, Y. Zhou, H. Niu and X. Zhang, Curr. Appl. Phys., 2011, 11, 1337–1342.
C. L. Lin, C. F. Lee and W. Y. Chiu, J. Colloid Interface Sci., 2005, 291, 411–420.
C. Schneider, M. Hanisch, B. Wedel, A. Jusufi and M. Ballauff, J. Colloid Interface Sci.,
2011, 358, 62–67.
34
35
36
A. Hajdú, M. Szekeres, I. Y. Tóth, R. A. Bauer, J. Mihály, I. Zupkó and E. Tombácz,
Colloids Surfaces B Biointerfaces, 2012, 94, 242–249.
V. Koseli, Z. M. O. Rzaev and E. Piskin, J. Polym. Sci. Part A Polym. Chem., 2003, 41,
1580–1593.
K. Cho, X. Wang, S. Nie, Z. Chen and D. M. Shin, Clin. Cancer Res., 2008, 14, 1310–
316.
1
37
38
39
V. J. Mohanraj and Y. Chen, Trop. J. Pharm. Res., 2007, 5, 561–573.
Q. Zhang, C. Wang, L. Qiao, H. Yan and K. Liu, J. Mater. Chem., 2009, 19, 8393–8402.
W. Zhang, L. Deng, G. Wang, X. Guo, Q. Li, J. Zhang and N. M. Khashab, Part. Part.
Syst. Charact., 2014, 31, 985–993.
40
S. Mohapatra, S. K. Mallick, T. K. Maiti, S. K. Ghosh and P. Pramanik, Nanotechnology,
2007, 18, 1–9.
2
9