Needle-shaped amphoteric calix[4]arene as a magnetic nanocarrier for simultaneous delivery of anticancer drugs to the breast cancer cells

Article abstract of DOI:10.2147/IJN.S194596

Background: Chemotherapy as an important tool for cancer treatment faces many obstacles such as multidrug resistance and adverse toxic effects on healthy tissues. Drug delivery systems has opened a new window to overcome these problems. There has been a strong interest development of new platform and system for delivof chemotherapeutic agents. Purpose: In the present study, a green synthesis method was chosen and performed for preparation of a novel amphoteric calix[4]arene (Calix) macrocycle with low toxicity to the human body. Materials and methods: The amphoteric Calix was coated on the surface of Fe3O4 magnetic nanoparticles and used as a magnetic nanocarrier for simultaneous delivery of two anticancer agents, doxorubicin and methotrexate, against MCF7 cancer cells. Several chemical characterizations were done for validation of prepared nanocarrier, and in vitro loading and release studies of drugs were performed with good encapsulation efficiency. Results: In vitro biological studies including hemolysis assay, erythrocytes sedimentation rate, red blood cells aggregation, cyto cellular internalization, and apoptosis evaluations were performed. Based on results, the developed nanocarrier has many advantages and capability for an efficient codelivery of DOX and MTX, which has a highly potent ability to kill cancer cells. Conclusion: All these results persuade us, this nanocarrier could be effectively used for cancer therapy of MCF7 breast cancer cells and is suitable for use in further animal studies in future investigations.

Full text of DOI:10.2147/IJN.S194596

International Journal of Nanomedicine
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Open Access FullText Article
O r I g I N a l r e s e a r c h
Needle-shaped amphoteric calix[4]arene as a
magnetic nanocarrier for simultaneous delivery of
anticancer drugs to the breast cancer cells
This article was published in the following Dove Medical Press journal:
International Journal of Nanomedicine
Mahdi rahimi,1 ramin
Background: Chemotherapy as an important tool for cancer treatment faces many obstacles
Karimian,2 ehsan Bahojb
Noruzi,3 Khudaverdi
such as multidrug resistance and adverse toxic effects on healthy tissues. Drug delivery sys-
tems has opened a new window to overcome these problems. There has been a strong interest
development of new platform and system for delivof chemotherapeutic agents.
ganbarov,4 Mojtaba Zarei,1
Fadhil s Kamounah,5 Bahman
Yousefi,1 Milad Bastami,6
Mehdi Yousefi,7 hossein
samadi Kafil1
Purpose: In the present study, a green synthesis method was chosen and performed for prepara-
tion of a novel amphoteric calix[4]arene (Calix) macrocycle with low toxicity to the human body.
Materials and methods: The amphoteric Calix was coated on the surface of Fe3O4 magnetic
nanoparticles and used as a magnetic nanocarrier for simultaneous delivery of two anticancer
agents, doxorubicin and methotrexate, against MCF7 cancer cells. Several chemical charac-
terizations were done for validation of prepared nanocarrier, and in vitro loading and release
studies of drugs were performed with good encapsulation efficiency.
1Drug applied research center,
Faculty of Medicine,Tabriz University
of Medical sciences,Tabriz, Iran;
2chemical Injuries research center,
systems biology and poisonings
institute, Baqiyatallah University
of Medical sciences,Tehran, Iran;
3Faculty of chemistry, Department
of Inorganic chemistry, University of
Tabriz,Tabriz, Iran; 4Department of
Microbiology, Baku state University,
Baku, republic of azerbaijan;
Results: In vitro biological studies including hemolysis assay, erythrocytes sedimentation
rate, red blood cells aggregation, cyto cellular internalization, and apoptosis evaluations were
performed. Based on results, the developed nanocarrier has many advantages and capability for
an efficient codelivery of DOX and MTX, which has a highly potent ability to kill cancer cells.
Conclusion:All these results persuade us, this nanocarrier could be effectively used for cancer therapy
of MCF7 breast cancer cells and is suitable for use in further animal studies in future investigations.
Keywords:green synthesis, amphoteric calix[4]arene, drug delivery, combination therapy, MCF7 cells
5Department of chemistry, University
of copenhagen, copenhagen.
Denmark; 6Biotechnology research
center, Faculty of Medicine,Tabriz
University of Medical sciences,Tabriz,
Iran; 7stem cell research center,
Faculty of Medicine,Tabriz University
of Medical sciences,Tabriz, Iran
Introduction
Macrocycle molecules are the significant class of drug-delivery vehicle like other
classes of the delivery vehicle such as carbon nanotubes, nanoparticles, micelles, and
dendrimers.1–4 There are a large number of macrocycles that are potentially useful
for drug delivery; only five types have been studied extensively such as calixarenes,
cyclodextrins, cucurbiturils, cryptands, crown ethers, and their derivatives.5–11 Construc-
tion of drug carrier from these macrocycles is of fundamental interest for applications
in biotechnology and medicine. Calixarenes are a class of organic macrocyclic host
molecules and have been widely studied and used in various fields since their discovery
by David Gutsche around 1970 by the condensation of substituted phenols and formal-
dehyde in the presence of inorganic bases and more rarely acids as the catalyst.12 Since
calixarenes can be modified by different functionalization at the lower and/or upper
rims of the molecule, the calixarene derivatives provide useful platforms for anchoring
a variety of binding units for ions and neutral molecules, and incorporating functional
groups, such as ester, ether, ketone, amide, and carboxylic acid groups, exhibit excellent
binding abilities.13–16 Their structures have unique three-dimensional surface, which
correspondence: ramin Karimian
chemical Injuries research center,
systems Biology and Poisonings Institute,
Baqiyatallah University of Medical
sciences, Mollasadra street, sheykh Baha’i
street, Tehran 1435915371, Iran
Tel +98 912 610 1850
email karimian.r@gmail.com
Hossein Samadi Kafil
Drug applied research center, Faculty
of Medicine, Tabriz University of Medical
sciences, Daneshgah street, Tabriz
51656-65811, Iran
Tel +98 912 718 4735
email kafilhs@tbzmed.ac.ir
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hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission
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made them as one of the best-known host molecules and used (IL-oxirane) were synthesized according to the recent lit-
in the biomedical applications of supramolecular systems erature reports.3,6 Ferric chloride hexahydrate (FeCl3.6H2O),
based on host–guest interactions.17,18 A particular interest was ferrous chloride tetrahydrate (FeCl2⋅4H2O), ammonia solu-
generated by the possibility of using calixarenes as vehicles tion (30%), 1-methylimidazole, epichlorohydrin, different
in drug delivery and other pharmacological contexts.
reagents, and solvents of analytical grade were purchased
Nano-sized magnetic particles (Fe3O4) are recently devel- from Merck Co. and used as received. Anticancer drugs were
oped and their superparamagnetic properties have opened obtained from Zahravi Pharmaceutical Co. (Tabriz, Iran) and
promising new perspectives for their application in several Sobhan Pharmaceuticals Co. (Tehran, Iran). RBCs stabilized
fields such as drug-delivery systems, MRI, bioseparation, with EDTA were obtained from the Iranian Blood Transfusion
etc.19,20 For drug targeting, they can accumulate in desired Institute and used with institutional bioethics approval. A puri-
locations within the body by using a permanent external fied Milli-Q deionized water (DI) was used for all experiments
magnet.21 Besides, using magnetic particles can significantly in this study. Furthermore, all used materials for biological
improve hyperthermia cancer treatment. This kind of therapy protocols, including trypsin, FBS, and Roswell Park Memorial
could raise the temperature of the target tissue to 43°C–46°C, Institute 1640 growth medium (RPMI) were purchased from
which makes cell more sensitive to chemotherapy and may Gibco BRL Life Technologies. MCF7 cells as human breast
show better results. Therefore, applying magnetic nanopar- cancer cells were provided by Cell Bank, Pasteur Institute of
ticles as nano-object in drug-delivery systems give several Tehran, Tehran, Iran. This study has been conducted in full
advantages to final drug carriers.
accordance with the World Medical Association Declaration
Developing drug carrier with either multidrug loading or of Helsinki and was done after confirmation from the eth-
conjugating for codelivery of the chemotherapeutic agents ics committee of Tabriz University of Medical Sciences,
is a necessary approach to overcome the multiple drug Tabriz, Iran (as local institutional bioethics committee).
resistance.22–24 The results and evidence revealed that anti-
cancer drugs effects, cytotoxicity, antiproliferative activity, characterizations
tumor cell apoptosis, and the low dose of anticancer drugs Fourier-transform infrared (FTIR) spectroscopy was used to
were increased by using drug carrier with combinational for- monitor the reaction process and to determine sample’s chem-
mulations.25 Improving the therapeutic index and overcoming ical structure. KBr method (sample to KBr ratio is 1:50) was
the undesirable side effects of anticancer drugs with similar used for recording the FTIR spectra on a Bruker (Shimadzu,
and different mechanisms of action is a promising strategy Japan). The prepared KBr disk scanned against a blank KBr
to reduce unwanted toxicity.26,27
disk at wavenumbers ranging from 500 to 4,000 cm−1. The
In recent years, there has been considerable interest in using nuclear magnetic resonance (NMR) spectroscopy (Bruker
amphiphilic materials as the drug- and gene-delivery system Co., Billerica, MA, USA) was performed to validate the
for cancer therapy.28–31 The aim of present study is to prepare chemical structure of prepared samples. D2O was used as a
an amphoteric calix[4]arene (Calix) with cationic and anionic solvent for calixarene derivatives. The morphology, size, and
rim sides by a green approach. Also, the capability of this elemental percentage analysis of samples were investigated
amphoteric carrier was evaluated for MCF7 cells as the human using a scanning electron microscopy (SEM; FESEM-EDX;
breast adenocarcinoma cell line with targeted delivery of DOX TESCAN 5001). Before the examination, samples were
and MTX as the model anticancer drugs. The novel amphoteric mounted onto a metal stub using a carbon double-sided
nanocarrier showed negligible cytotoxicity and hemolytic adhesive tape and covered with a thin layer of gold, with
effects on human red blood cells (RBCs) with good in vitro the aid of a direct current sputter technique (Emitechk450X;
loading efficacy. Finally, to show the capability of drug-loaded Quorum Technologies Ltd., Lewes, UK).
nanocarrier in killing MCF cancer cells, MTT assay, cellular
Furthermore, to accurately assess the modification pro-
uptake, and Annexin V-FITC, and PI methods were used to cess, elemental analysis was performed by using energy dis-
investigate the apoptotic effect in comparison to free drugs.
persive X-ray (EDX) spectroscopy. The transmission electron
microscopy (TEM) analysis was used to show the size and
shape of the samples (Leo 906, Zeiss, Freiburg, Germany)
with an operating voltage of 100 kV. Before analysis, the sus-
Materials and methods
Materials
Some starting materials such as Calix and 1-methyl- pension was ultrasonically dispersed using a probe-type ultra-
3-(oxirane-2-ylmethyl)–1H-imidazole-3-ium chloride sonic generator (400 W) for 6 seconds, and the suspension was
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rahimi et al
placed on a carbon-coated copper grid. Contact angle (CA) Preparation of PIl-calix-coated magnetic
was used for the evaluation of hydrophilicity of the samples nanoparticles (PIl-calix/MNPs)
by sessile drop method. Two-microliter droplets of deion- Briefly, PIL-Calix (1.0 g) was dissolved in 50 mL deionized
ized water were released slowly on the coverslips containing water, and subsequently, 1.25 g of FeCl3⋅6H2O and 0.5 g of
samples pressed layer. ImageJ, an image analyzer software, FeCl2⋅4H2O were added, and the solution was stirred at room
was used for measuring the droplet of water on the surface temperature for 30 minutes to obtain a homogenous solution.
of the sample. For CA analysis, the powder was pressed on By adding ammonia solution gradually to the above solution,
a glass slide to make a uniform and smooth thin layer. The Fe3O4 magnetic nanoparticles were formed, which modified
magnetic properties of samples were characterized with a with PIL-Calix at nanoparticles surface. The reaction mixture
vibrating sample magnetometer (VSM) (AGFM, Kashan, was stirred for 30 minutes, and the obtained PIL-Calix-coated
Iran) for determination of sample’s magnetization response vs magnetic nanoparticles (PIL-Calix/MNPs) were isolated by
applied field at room temperature. The particle size distribu- an external magnet and washed several times with ethanol
tion of samples was assessed using a dynamic laser-scattering and water. Finally, the formed PIL-Calix/MNP was dried in
technique (DLS; Zetasizer Nano ZS90; Malvern Instruments, vacuum for further use.
Malvern, UK). Before analysis, a diluted suspension of the
Blood compatibility tests
sample was prepared by PBS solution (pH 7.4) to achieve the
Human whole blood was obtained from Iranian Blood Trans-
fusion Institute stabilized with EDTA and spun at 4,000 rpm
for 10 minutes at room temperature. The plasma (upper layer)
was discarded, and the pellets (RBCs) were isolated. The
isolated RBCs were washed three times with PBS (pH 7.4)
until a clear supernatant was obtained. For the blood com-
patibility of PIL-Calix/MNPs, the following three tests were
performed according to the procedure described below.34,35
Test A – hemolysis assay: 0.5 mL suspension of PIL-
Calix/MNPs (25, 50, 100, 200, 400, and 800 µg/mL) and
0.5 mL of diluted RBCs were mixed in a microtube and
incubated at 37°C for 1 hour. The samples were centrifuged
at 5,000 rpm for 5 minutes and the supernatant was trans-
ferred to the 96-well plates. ELISA plate reader was used
to measure the released hemoglobin (at a wavelength of
540 nm) and calculate the hemolysis percentage using the
following formula:
required sample density for analysis.
green synthesis of magnetic nanocarrier
based on amphoteric calixarene
synthesis of p-sulfonatocalix[4]arene (s-calix)
This compound was synthesized using a previously reported
procedure.32 Calix (1.0 g, 2.4 mmol) was treated with concen-
trated H2SO4 (10 mL), and the mixture was stirred vigorously
at 70°C for 3 hours. An aliquot was taken out from the solution
and poured into water. The reaction progress was monitored
by adding an aliquot of reaction mixture gradually into the
deionized water. After completion of the reaction, no pre-
cipitate materials were detected in the mixture. The reaction
mixture was allowed to cool and poured gradually into 10 mL
deionized water. The solution was neutralized with aqueous
Na2CO3 solution and evaporated to dryness. The residue was
reprecipitated several times from water into absolute ethanol.
Finally, p-sulfonatocalix[4]arene (s-Calix) was dried at 50°C
for 24 hours, and a white crystalline solid was obtained.
Abssample − AbsPBS
Hemolysis rate (%) =
×100
Abswater − AbsPBS
synthesis of needle-shaped poly ionic liquid-grafted
p-sulfonated calix[4]arene (PIl-calix)
Test B – erythrocytes aggregation test: after incubation
Ring-opening polymerization of IL-oxirane on the surface of of test number A, aggregation of RBCs was observed by
s-Calix was performed by modification of a previously reported phase contrast microscope (Nikon, YS2-T; Tokyo, Japan)
method.33 s-Calix (1.0 g, 1.2 mmol) was dissolved with an with 40× magnification. Diluted RBCs treated with water
excess amount of IL-oxirane (100 mmol) in deionized water, and PBS were used as positive and negative controls with
and the pH of the solution was adjusted to 1.0 by adding HCl 100% and 0% hemolytic effects, respectively.
solution. The mixture was stirred at 60°C under a nitrogen
Test C – erythrocytes sedimentation rate: 2 mL of human
atmosphere for 24 hours. The resulting poly ionic liquid-grafted blood was drawn into the Westergren tube (a narrow vertical
p-sulfonated calix[4]arene (PIL-Calix) was reprecipitated tube) containing 0.5 mL sodium citrate, 2.0 mg of PIL-Calix/
several times in ethanol to remove unreacted IL-oxirane. The MNPs was then added, and the tube was placed in a rack in
obtained light brown solid (PIL-Calix) was dried in vacuum.
an upright position for 1 hour at room temperature. The rate
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of sedimentation is measured as the number of millimeters for cellular uptake by fluorescent microscopy and flow
of clear plasma present at the top of the column after 1 hour cytometry assays, respectively.2 After incubation of cells for
(mm/hour).
24 hours, the culture media of 6-well plates were replaced
with free media. Then, the cells were treated with different
sample suspensions including DOX-labeled nanocarrier (at
a concentration of 10 µg/mL in PBS), and the cells with no
treatment considered as a control. In the end, two following
regular procedures were performed.
In vitro DOX and MTX loading and
release studies
In vitro drug loading and release studies were performed
according to the two following procedures:
Cellular uptake by fluorescent microscopy: after incuba-
tion for 1, 2, and 3 hours, the cells were washed three times
with PBS and fixed by formaldehyde (4% w/v). The coverslip
was taken onto a glass microscope slide, and DOX/nanocarrier
uptake was observed by using a fluorescence microscope
(Olympus Microscope Bh2-RFCA, Tokyo, Japan).
Cellular uptake by flow cytometry: after incubation for
1, 2, and 3 hours, the cells were trypsinized and collected
for determination of DOX/nanocarrier uptake inside the
cells via fluorescence intensity using a FACSCalibur flow
cytometer (Becton Dickinson Immunocytometry Systems,
San Jose, CA, USA).
Loading step: simultaneous loading of DOX and MTX
was performed as follows: PIL-Calix/MNPs (100 mg) as a
nanocarrier was dispersed in PBS solution (100 mg) by water
bath sonication, and then MTX (10 mg) was added to the
suspension and mixed moderately with a magnetic stirrer for
24 hours. After isolation of MTX-loaded nanocarrier (MTX/
nanocarrier) and washing with PBS to remove unloaded MTX
and DOX, loading was done as follows: MTX/nanocarrier
was dispersed in PBS, and DOX (10 mg) was added to the
suspension. The suspension was stirred at room temperature
under the dark condition for 24 hours. Finally, DOX-MTX-
loaded magnetic nanocarrier (DOX-MTX/nanocarrier) was
obtained and used in the releasing step.
cytotoxicity assay
Releasing step: DOX-MTX/nanocarrier (10 mg) was
added to microtube, which was categorized as follows:
category 1 for pH 7.4 and category 2 for pH 5.0 in triplicate.
Two millilters of PBS solution was added to each microtube
and incubated at 37°C. At a certain time, the microtubes were
centrifuged, and the supernatant was taken for determination
of released MTX and DOX by UV–Vis spectrophotometer.
The MCF7 cells at the concentration of 10×103 cells per
well were seeded in 96-well plates and incubated under
5% CO2 atmosphere for overnight at 37°C attached at the
bottom of each well. For in vitro cytotoxicity evolutions,
following procedure was performed. Briefly, the seeded
cells were treated with different samples including (blank
nanocarrier, DOX-MTX/nanocarrier, and DOX-MTX) at
various concentrations in RPMI medium and incubated for
48 hours. After washing several times with PBS, MTT assays
were performed to determine the cell viability according to
the reported procedure. The absorbance of each well was
measured at 570 nm with an ELISA plate reader.
cell culture
MCF7 cells as the human breast adenocarcinoma cell line
were used in this study which was obtained from the National
Cell Bank of Iran (Pasteur Institute of Iran, Tehran, Iran).
The cells were cultured with RPMI 1640 medium (supple-
mented with 10% (v/v) FBS, 1% antibiotics including peni-
cillin and streptomycin with a concentration of 50 IU/mL
and 50 µg/mL, respectively). After cell culturing, the flask
containing cells were incubated under a fully humidified
atmosphere at 37°C with 5% CO2, and growth medium must
be renewed two to three times per week until the cells reached
90% confluency. After sufficient PBS washing, the cells were
detached with 0.25% trypsin in PBS (pH 7.4) and centrifuged
at 1,500 rpm for 5 minutes.36 Subsequently, the cells were
dispersed in growth medium and used for seeding in further
in vitro assays.
DaPI staining
The apoptotic effects of samples on MCF7 cells were exam-
ined by DAPI staining as a routine microscopic analysis.37
Briefly, in 6-well plates containing sterile coverslips, 5×104
MCF7 cells were seeded and allowed attaching at the bottom
of each well at 37°C for overnight. The cells were treated
with blank nanocarrier, DOX-MTX/nanocarrier, and DOX-
MTX at the concentration of IC50. The cells with no treatment
were considered as a negative control. The 6-well plates were
kept in an incubator and incubated for 48 hours. The wells
were washed three times with PBS and fixed with 4% form-
aldehyde at ambient temperature for 15 minutes. Then cells
cellular uptake
MCF7 cells were seeded in 6-well plates for quantification were permeabilized by adding Triton X-100 (0.1% w/v) and
of uptake at a density of 5×104 and 5×105 cells per well incubated for 5 minutes. Ultimately, cells were stained by
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rahimi et al
using DAPI solution (200 ng/mL), and DNA condensation statistical analysis
and fragmentation in apoptotic cells were evaluated under All data were reported as mean SD of three replicates.
a fluorescence microscope (Olympus Microscope Bh2- The ANOVA was used for statistical analysis. The
RFCA, Japan).
P-values 0.05 were considered significant.
Results and discussion
Apoptotic effects of different formulations were measured synthesis
Apoptosis assay by flow cytometry
by ApoFlowEx® FITC Kit (EXBIO Diagnostics, Vastec, As shown in Figure 1A, Calix is a cavity-shaped cyclic mol-
Czech Republic). MCF7 cancer cells at the density of 5×105 ecule with four phenol units linked with methylene bridges
cells per well were seeded in 6-well plates and incubated at ortho positions. Calix has four tertiary butyl (t-butyl)
for 24 hours attached at the bottom of the wells. Then, the functional groups at the para position of hydroxyl groups,
cells were treated with blank nanocarrier, free DOX-MTX, which render them very hydrophobic. It has a crystalline
and DOX-MTX/nanocarrier. Cells receiving only fresh structure under the microscope with a very light yellow
medium were considered as control group. After incubation color. After treating with concentrated sulfuric acid, the
for 48 hours, cells were trypsinized, washed twice with cold t-butyl groups at para position were substituted with sulfo-
PBS, resuspended in 1× binding buffer (500 µL), added 5 µL nate (−SO3H) hydrophilic groups, and the physicochemical
of Annexin V-FITC and 5 µL of propidium iodide, and incu- characteristics of the resulting compound were changed. The
bated for further 15 minutes in dark. Then flow cytometry crystalline form of sulfonate calixarene under the micro-
analysis was performed by flow cytometer (FACSCalibur; scope showed the same appearance as Calix (Figure 1B).
BD Biosciences, San Jose, CA, USA).
The sulfonate groups act as anionic active sites for loading
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Notes: calix (A), s-calix (B), PIl-calix molecules (C).
Abbreviations: calix, calix[4]arene; PIl-calix, poly ionic liquid-grafted p-sulfonated calix[4]arene; s-calix, p-sulfonatocalix[4]arene.
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rahimi et al
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Figure 2 (A) Preparation of p-sulfonatocalix[4]arene (s-calix) from 4-tert-butylcalix[4]arene (calix). (B) Preparation of needle-shaped poly ionic liquid-grafted p-sulfonated
calix[4]arene (PIl-calix) via ring-opening polymerization of Il oxirane (step 1) and preparation of PIl-calix-coated magnetic nanoparticles (PIl-calix/MNPs) as a magnetic
nanocarrier (step 2).
doxorubicin drug. On the other hand, to create cationic were taken. The FTIR characteristic absorbance and 1H NMR
active sites on these macrocycles for loading MTX drug, chemical shifts of the samples are given below.
ionic liquid polymeric chains were attached to the other
Calix: the characteristic absorption peaks at 670,
side of macrocycles causing amphoteric Calix. The result- 1,242, 1,481, 2,956, 3,049, and 3,201 cm−1 are attributed to
ing calixarene has different appearance without crystalline
structure (Figure 1C). All reaction processes were per-
formed in green approach with no toxic solvents, making
the designed nanocarrier very safe for use in the biomedical
applications (Figure 2).
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Before performing biological assays to show the safety of
nanocarrier and its capability in combinational delivery of
anticancer drugs in drug-delivery systems, all materials were
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differential scanning calorimetry (DSC), SEM, EDX, TEM,
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All samples were characterized by FTIR and H NMR
Figure 3 FTIr spectra of calix (A), s-calix (B), PIl-calix (C), and PIl-calix/MNPs
(D) at wavenumber range of 500–4,000 cm−1.
Abbreviations: calix, calix[4]arene; PIl-calix, poly ionic liquid-grafted p-sulfonated
calix[4]arene; PIl-calix/MNPs, PIl-calix-coated magnetic nanoparticles; s-calix,
p-sulfonatocalix[4]arene.
spectroscopies to determine the functional groups and chemi-
cal structure of the samples. As shown in Figure 3, the FTIR
spectra of Calix, s-Calix, PIL-Calix, and PIL-Calix/MNPs
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the =C-hour bending, C−O stretching, C=C stretching, ali-
phatic C−H stretching, aromatic C−H stretching, and O−H
stretching bonds, respectively. Due to the presence of the
t-butyl group substituted at the para position of phenol, the
intensity of the peaks at around 2,956 is high.
1H-NMR (400 MHz, CDCl3, δ ppm): 10.3 (OH, s, 4 H),
7.05 (Ar-H, s, 8 H), 4.25 (CH2, d, 4 H), 3.49 (CH2, d, 4 H),
and 1.21 (t-Bu, s, 36 H).
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s-Calix: the characteristic peaks at 669, 1,045, 1,186,
1,213, 1,471, 2,954, 3,190, and 3,446 cm−1 are assigned to
the =C-H bending, asymmetric stretching vibration of S−O,
C−O stretching, splitting of the S=O group into symmetric
and asymmetric stretching vibrations, C=C stretching, ali-
phatic C–H stretching, aromatic C−H stretching, and O−H
stretching bonds, respectively.
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Figure 4 Dsc thermographs of calix (A), s-calix (B), and PIl-calix (C) from 30°c
to 250°c at a heating rate of 5°C/min under a flowing nitrogen atmosphere.
Abbreviations: calix, calix[4]arene; Dsc, differential scanning calorimetry;
PIl-calix, poly ionic liquid-grafted p-sulfonated calix[4]arene; s-calix,
p-sulfonatocalix[4]arene.
1H-NMR (400 MHz, D2O, δ ppm): 7.60 (ArH, s, 8 H)
and 3.84 (CH2, S, 8 H).
PIL-Calix: as the PIL-Calix has polymeric chains on its peak in the temperature range of 50°C–100°C, which is
structure with several methylene groups (−CH2−), −C−N=, attributed to the recrystallization of molecules with high
−C=N−, and −C−OH, it was expected that some new peaks heat releasing rate. At this point, the fragments may obtain
or shifting absorption peaks were observed. Regarding the enough freedom of motion to arrange themselves into a
formation of many methylene groups in the polymeric chains, crystalline form spontaneously. Due to the presence of
the absorption peaks at 2,961 cm−1 were enhanced in compari- polymeric branches on the PIL-Calix, it was expected that
son to the s-Calix. The characteristic peak of −C=N− sections the recrystallization temperature shifted to the higher tem-
was appeared at 1,680 cm−1, whereas the peak of −C−N= was perature because the polymeric chains prevent rearrangement
not appeared clearly because of its low intensity. Identical of the molecules in lower temperature. By observing the
peaks in both s-Calix and PIL-Calix appeared at the same DSC graph of PIL-Calix, the height of the curve was more
range of wave number with slight shifting.
depressed than unmodified s-Calix and can be concluded that
1H-NMR (400 MHz, D2O, δ ppm): 8.58 (N=CH, s), 7.60 low exothermic heat was released. All results confirmed that
(ArH, s, 8 H), 7.32–6.94 (NCH, m), 4.18 (NCH3, s), 3.84 the modification in each step was successful.
(CH2, S, 8 H), and 3.5–3.22 (OCH2CHCH2N, m).
The water CA is a measure of the wettability of the solid
PIL-Calix/MNPs: after surface modification of magnetic surface. If the solid surface is highly hydrophilic, water
nanoparticles (Fe3O4) with PIL-Calix, the characteristic peaks droplet is strongly attracted, and then the water drop will
of both Fe3O4 and PIL-Calix were observed. Fe3O4 has a bulk completely spread out with CA (θ) of 0°. In general, water CA
network with two clear peaks at 573 and 3,458 cm−1 for Fe−O lower and more substantial than 90° is considered hydrophilic
and O−H. By following the PIL-Calix/MNPs spectrum, the and hydrophobic solid surfaces, respectively. Regarding
characteristic peaks of PIL-Calix were also found with low this, an appropriate amount of each sample was placed on a
intensity. All observations and results validated our prepara- glass slide, and another glass slide was fixed on the sample.
tion process.
The glass slides were placed in the pressure instrument, and
DSC is a technique for analyzing the thermal behavior the sample was pressed slowly. The obtained disk on glass
of the sample according to the amount of heat required to slide had a homogenous surface. Given that the conditions are
increase the temperature. The DSC graph is depicted based the same, the results could be compared to each other. Since
on heat flow vs temperature. The DSC thermographs of Calix contains four t-butyl groups, which cause it highly
Calix, s-Calix, and PIL-Calix are shown in Figure 4. Calix hydrophobic, its water CA value could not be measured by
showed a uniform curve with no exothermic and endothermic instrument due to the high hydrophobicity and escaping from
peaks in the analysis temperature range, and it has no phase the water. Figure 5 showed that the droplet could not separate
transition with a melting point above this range (melting and stand on the surface. (This picture was snapshotted from
point 300°C). However, s-Calix has a sharp exothermic the video of the measurement process.) After exchanging
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the t-butyl groups with sulfonate (−SO3H) group to yield
s-Calix, it was expected that the hydrophilicity enhanced
after substitution of t-butyl with sulfonate and the amount
of θ decrease to lower degree (34.01). The hydrophilicity
of PIL-Calix was also evaluated and results showed that
water droplet could spread out easily on the surface of film
with θ value of 28.83. All samples were tested in triplicate
at three different areas, and obtained midpoint results were
reported. Overall, the results data confirmed that the present
modification process was done.
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The surface morphology of Calix, s-Calix, PIL-Calix, and
PIL-Calix/MNPs was studied by SEM analysis and illustrated
in Figure 6. Calix has a solid structure with finger-like beads
and smooth surface. The s-Calix also has finger-like beads.
However, its morphology was changed to rough form with
several fractions on it. Further evaluation revealed that each
bead was changed to multilayer structure. Surface morphol-
ogy of PIL-Calix revealed that the structure of s-Calix was
Figure 5 Water drop contact angle for hydrophobic calix (A) and hydrophilic
s-calix (B), and PIl-calix (C) samples.
Abbreviations: calix, calix[4]arene; PIl-calix, poly ionic liquid-grafted p-sulfonated
calix[4]arene; s-calix, p-sulfonatocalix[4]arene.
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Figure 6 surface morphology and size of calix (A), s-calix (B), PIl-calix (C), and PIl-calix/MNPs (D) by seM technique at the scale of 1 µm, 1 µm, 1 µm, and 300 nm,
respectively.
Abbreviations: calix, calix[4]arene; PIl-calix, poly ionic liquid-grafted p-sulfonated calix[4]arene; PIl-calix/MNPs, PIl-calix-coated magnetic nanoparticles; s-calix,
p-sulfonatocalix[4]arene; seM, scanning electron microscopy.
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Figure 7 The eDs spectra of Fe3O4 (A) and PIl-calix/MNPs (B) for the estimation of the elemental analysis.
Abbreviations: eDs, energy dispersive X-ray spectroscopy; PIl-calix/MNPs, PIl-calix-coated magnetic nanoparticles.
completely altered to various and specific shapes, which their peaks were overlapped with each other. By appearance
will facilitate the attachment of drug molecules onto the of these elements, the intensity of Fe and O elements was
surface and PIL-Calix cavities. After surface modification decreased. All results validated the successful preparation
of PIL-Calix/MNPs. The elemental analysis data were sum-
marized in Table 1.
of magnetic nanoparticle with PIL-Calix, spherical particles
with nanosize around 40–50 nm were formed. The SEM
image demonstrated that prepared PIL-Calix/MNPs tended
to aggregate in aqueous solution because of the small size
effect caused by hydrophilic units of the compound on the
Fe3O4 nanoparticles. All observations confirmed that surface
modification processes were performed successfully.
To receive more direct information on the particle size
and morphology, TEM micrographs of PIL-Calix/MNPs
were investigated. As shown in Figure 8, TEM image shows
a difference in morphology. Some needle- and rod-shaped
calixarene-doped magnetic nanoparticles were also observed,
where magnetic nanoparticles formed on the surface of PIL-
Calix needles and rods. Many dense aggregations of pure Fe3O4
magnetic nanoparticles were observed, due to the lack of repul-
sive force. After PIL-Calix immobilization, the dispersion of
particles was improved as well, which can easily be explained
by the electrostatic repulsion force and steric hindrance between
the Calix and the surface of Fe3O4 nanoparticles.
EDX spectroscopy (EDS) or EDX is an analytical tech-
nique for chemical characterization and determination of the
elemental analysis. EDS can be used to estimate chemical
elements relative abundance. Several factors (such as over-
lapping X-ray emission peaks, the nature of the sample, etc)
affected the accuracy of the sample compositions. Fe3O4
NPs were synthesized and used as a control. As seen in
the EDS spectrum (Figure 7), the atomic percentage of the
iron atom (Fe) is 40.17% 6.65% with two characteristic
peaks at 0.705 and 6.398 keV. Also, the oxygen atom (O)
has 59.83% 5.98% atomic percentage with a characteristic
peak at 0.525 keV. After preparation of PIL-Calix/MNPs
by in situ synthesis of Fe3O4 NPs in PIL-Calix solution,
the EDS analysis was taken, and the atomic percentages
were reported. As seen in the spectrum, two new peaks
of carbon (C) and nitrogen (N) were appeared at around
0.277 and 0.392 keV, respectively, with atomic percentage
of 22.28% 6.23% and 3.64% 0.87%. As X-ray emission
peaks of N and O elements appears close to each other,
To measure magnetic properties of magnetic materi-
als, VSM was used, and the magnetization value (emu/g)
vs applied field (Oe) was determined at room temperature
Table 1 elemental analysis data based on weight percentage
(W%) and atomic percentage (a%)
Element
Fe3O4
PIL-Calix/MNPs
Fe
70.09 8.26
40.17 6.65
23.79 3.21
58.62 9.01
14.77 2.09
2.80 0.88
100.00
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59.83 5.98
66.36 9.74
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100.00
Abbreviation: PIl-calix/MNPs, PIl-calix-coated magnetic nanoparticles.
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could carry chemotherapeutic agents to the target cells by
using a permanent external magnet. After using an external
magnet, the separation time for collecting magnetic nano-
carrier beside the container is about 20 seconds. All results
confirmed that the nanocarrier is suitable for use in the
pharmaceutical field.
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Despite the size of the magnetic nanoparticles was
observed and calculated by SEM analysis. Particle size
can be determined by measuring the random changes in
the intensity of light scattered from a suspension. DLS is a
proper technique that is most commonly used for analyz-
ing nanoparticle size distribution. However, this technique
was performed in an aqueous solution to enable particles
to interact with each other freely, which caused aggrega-
tion. As seen from the DLS graph of prepared nanocarrier
(Figure 10), the size distribution is around 210 nm.
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Figure 8 TeM micrograph of PIl-calix/MNPs with a needle-shaped structure.
Abbreviations: PIl-calix/MNPs, PIl-calix-coated magnetic nanoparticles; TeM,
transmission electron microscopy.
stability of nanocarrier in the
physiological condition
(Figure 9). The prepared nanocarrier displayed saturation
magnetization equal to about 62 emu/g, which has lower
amount than Fe3O4 NPs as a control (with magnetization
value of 74 emu/g), and it is attributed to the presence of a
polymeric branch of PIL-Calix on the magnetic core. The
S shape of the magnetic nanocarrier diagram exhibited zero
coercivity and permanence, thus indicating its superpara-
magnetic feature. Therefore, this property of nanocarrier
The colloidal behavior of PIL-Calix/MNPs as a magnetic
nanocarrier was investigated, and the results discussed as
follows. The colloidal stability of magnetic nanoparticles
(Fe3O4 NPs) in an aqueous solution depends on the environ-
mental parameters and their intrinsic magnetic properties.
However, the surface modification of nanoparticles could
enhance its stability, bioavailability, and toxicity. Several
parameters (such as pH, ionic strength, and plasma protein
concentration) were affected by the properties of magnetic
nanoparticles in physiological condition. Therefore, we used
a common culture medium (RPMI 1640), which contained
mono and divalent ions (Na+, Mg2+, and Ca2+), and FBS was
also added to the culture medium for cell culturing and it
provided the simulated condition of the physiological body.
As the stability could be affected by altering nanoparticles
concentration, two different concentrations with the low and
high amount of magnetic nanocarriers (0.1% and 1%) were
evaluated. The results showed that magnetic nanocarrier sta-
bility in both concentrations has not changed after incubation
in culture medium for 10 days and no significant macroscopic
changes were observed. This demonstrates that the stability
of magnetic nanocarrier in physiological conditions is a
complex function of nanoparticles surface characteristics
and components of the surrounding medium. The results
confirmed that the suspension has good stability with low
rate sedimentation, which breaks down the aggregation by
sonication or stirring. To show the diffusing capability of
nanocarrier, a droplet of nanocarrier suspension was added to
the simulated physiological condition medium with PBS, and
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nanocarrier at room temperature.
Abbreviation: VsM, vibrating sample magnetometer.
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Abbreviation: Dls, dynamic laser scattering.
a series of pictures were snapshotted. As shown in Figure 11, presence of cationic and anionic functional groups on drugs
the nanocarrier could easily disperse in the container.
and drug carrier at the loading condition (PBS pH 7.4). The
interactions are hydrogen bonding and electrostatic interac-
tion. It is assumed that the drugs could release by changing
Drugs loading and release studies
High loading drugs on the drug carrier for increasing the medium condition and causing repulsion between drugs
therapeutic efficiency is an important role in the field of and drug carrier. In this regard, drug release profile was simu-
nanoparticulate drug-delivery systems. Regarding this issue, lated in both physiological and cancerous tissue conditions.
high-capacity drug-delivery system for loading combination DOX-MTX/nanocarrier was dispersed in PBS solution in
anticancer drugs was designed based on amphoteric Calix. both pH 7.4 and 5.0, and release profiles were evaluated for
DOX and MTX were used as the model drugs with two dif- 1 week (168 hours) (Figure 12). Both DOX and MTX release
ferent moieties (amine and carboxylic acid functional groups, profiles have burst release in first and then sustain release
respectively). Measurement of two important parameters after 1 day. The burst release of both drugs is attributed to the
is essential in drug-delivery systems including loading physical interaction between drug-carrier and drugs, so the
and encapsulation efficiencies; their calculation formula drugs could easily separate from nanocarrier surface. After
is shown below. After calculation of both parameters for release study for 168 hours, DOX and MTX showed about
both DOX and MTX, the obtained values of loading and 83.26% and 87.93% of cumulative releases, respectively.
encapsulation efficiencies for DOX were 9.02% 2.47%
and 90.22% 5.08%, respectively. However, the values Biocompatibility of nanocarrier
of these parameters for MTX were 5.86% 1.79% and In biomedical applications, nanomaterials enter the body
58.62% 6.53%, respectively. These values are related to the and contact with tissues and cells directly. It is essential
to analyze their biocompatibility. RBCs, also called
erythrocytes, represent the most abundant cellular con-
stituent of the blood (99%), which plays an essential
role in human health. Nanomaterials in drug-delivery
systems are directly contacted with RBCs. Thus they
are examined for their blood-compatible behaviors.38
Several experiments including hemolysis assay, coagula-
tion, erythrocytes aggregations, and sedimentation rate
have been carried for evaluation of biocompatibility of
biomaterials before using in biomedical applications.34
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tation rate experiments as reliable and straightforward
measures were chosen for estimating blood compatibility
Figure 11 Diffusing capability of nanocarrier in simulated physiological condition
after intravenous injection (ph 7.4 and 37°c).
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Figure 12 The cumulative release of MTX and DOX from DOX-MTX/nanocarrier at two ph values (5.0 and 7.4) at 37°c.
Abbreviation: DOX-MTX/nanocarrier, DOX-MTX-loaded magnetic nanocarrier.
of materials. By treating RBCs (erythrocytes) with various The results of the aggregation test on RBCs showed that
concentrations of nanocarrier (25, 50, 100, 200, 400, and nanocarrier has no effect on RBCs membrane charge
800 µg/mL), hemolytic effects were calculated using the and no aggregation occurred (Figure 13B). To evaluate
hemolysis rate formula. As hemolysis rate has a direct the impact of nanocarrier on RBCs membrane charge,
relationship with the concentration of nanocarrier, a erythrocyte sedimentation rate (ESR) was calculated.
negligible hemolytic effects on RBCs was observed with The ESR is the rate of RBCs sedimentation in 1 hour
2.77% hemolysis percentage even at high treated dosage. (the ESR test is a nonspecific measure of inflammation
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Abbreviations: PBs, phosphate buffered saline; rBcs, red blood cells.
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and hematology test). After treating RBCs with nanocar- performed by fluorescent microscopy and flow cytometry
rier at the dosage of 1,000 µg/mL in a Westergren tube analysis (two complementary methods). First, the MCF7
(upright tube) (containing citrate as an anticoagulant cells were treated with DOX-labeled nanocarrier in a time-
agent), the amount of sedimentation in both treated and dependent manner (1, 2, and 3 hours). As shown Figure 14,
untreated RBCs has no significant changes after 1 hour all cells could uptake the DOX-labeled nanocarrier showing
(Figure 13C). To make sure that our nanocarrier has no fluorescent light. In the next step, flow cytometry analysis was
bad effects on the RBCs surface charge, more incubation used to measure the uptake percentage and amount of fluo-
time for interacting RBCs with nanocarrier was also done rescent intensity (Figure 15). The results were confirmed by
and results showed no significant difference. Ultimately, fluorescent microscopy observations and also showed that by
based on the results of three common blood experiments, increasing treating time, fluorescent intensity was enhanced.
it was expected that the developed nanocarrier is safe for Ultimately, it could be concluded that our nanocarrier has
using in blood-contacting biomedical applications.
good internalization capability into the cells, which is a cru-
cial role for the delivery of anticancer agents into the cells.
cellular internalization
The interactions of nanoparticles with the soft surfaces of cytotoxic assay by MTT assay
biological systems such as cells play vital roles in execut- For assessing cell metabolic activity and cytotoxicity effects
ing their biomedical functions and in toxicity. Cells have a of samples DOX-MTX, DOX-MTX/nanocarrier, and blank
permeable membrane through which small molecules could nanocarrier on MCF7 breast cancer cells, the MTT assay
easily enter into them by several pathways (concentration was performed for 48 hours (Figure 16). Before comparing
gradients, active transport system such as protein pumps and the cytotoxic effects of anticancer drugs in free and nanofor-
ion channels, and endocytosis).39 Endocytosis is a pathway mulation form, the MCF7 cells (considered as model cells)
that internalizes nanomaterials in transport vesicles derived were treated with blank nanocarrier to be sure for nanocar-
from the plasma membrane.40 As our magnetic nanocarrier rier biocompatibility. The results revealed that nanocarrier
has a size of 100 nm, it could be internalized into the cells has low cytotoxic effects on cells in the concentration range
by energy-dependent clathrin-mediated endocytosis in which of 2–128 µg/mL equivalent to 99.76%–92.71% cell viability
nanocarrier is deposited in small endocytic vesicles and in a dose-dependent manner. By increasing the nanocar-
fuse with early endosomes.41 To understand the nanocarrier rier concentration, the cell viability was decreased, which
capability in the cellular uptake process, cellular uptake was attributed to the sedimentation of nanocarrier on attached
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Figure 14 cellular uptake of DOX-labeled nanocarrier by McF7 cells after treatment for 1 (A), 2 (B), and 3 hours (C) by fluorescent microscopy.
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Figure 15 Cellular uptake percentage by flow cytometry after treatment for 1 (A), 2 (B), and 3 hours (C).
cells in the wells and causing hypoxia (deficiency in the into the cells and releasing the drugs with control man-
amount of oxygen reaching the tissues). The cytotoxic ner. Free drugs can be quickly pumped out by drug efflux
effects of DOX and MTX on MCF7 cells were evaluated, transporters, which cause limitations for successful cancer
and their inhibition concentration (IC50) was measured and chemotherapy (Figure 17). However, using drugs in nano-
reported (the IC50 of both drugs are 7.22 and 9.52 µg/mL, formulation form overcomes the transporter-mediated
respectively). The cell treated with DOX-MTX/nanocarrier multidrug resistance due to the passing through of nano-
has low viability in comparison to DOX-MTX, and it could carrier from the cellular membrane and releasing drugs in
be related to the capability of nanocarrier internalization a controlled manner.
100
95
90
85
80
2
4
8
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32
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Nanocarrier concentration (µg/mL)
100
80
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40
20
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DOX-MTX
DOX-MTX/nanocarrier
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0.625
1.25
2.5
5
10
Drug concentration (µg/mL)
Figure 16 MTT assay for evaluation of cytotoxic effects of DOX-MTX, DOX-MTX/nanocarrier and blank nanocarrier on McF7 breast cancer cells for 48 hours.
Abbreviation: DOX-MTX/nanocarrier, DOX-MTX-loaded magnetic nanocarrier.
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Figure 17 Drug-loaded nanocarrier internalization into the cells and releasing the drugs with control manner (Pathway A). Easily pumping out of free drugs by drug efflux
transporters with low cytotoxic effects (Pathway B).
Based on ANOVA test results, there are significant dif- DNA. According to the DAPI characteristic, the nucleus of
ferences between and within groups (DOX-MTX/nanocarrier MCF7 cells treated with blank nanocarrier, DOX-MTX, and
and free DOX-MTX groups) with a P-value 0.001. The post DOX-MTX/nanocarrier was stained with DAPI and observed
hoc Tukey’s analysis was also performed and results showed under fluorescent microscopy. To compare the nuclear
that there are significant differences with P-value 0.001, morphological changes of cells, the cells with no treatment
except some dosages that showed P-value 0.05 (1 with 7), considered as a control. The normal nucleus of cells has a
(3 and 8), (4 and 9), (5 and 10), and (6 and 11) (Table 2).
size within a defined range, while changes in nuclear size
and shape are associated with a variety of reasons. As shown
in Figure 18A and B, the cells of control and treated groups
with blank nanocarrier have an entire nuclear structure with
apoptotic effect evaluation by DaPI
staining and flow cytometry
DAPI is a common nuclear and chromosome counterstain, normal morphology and different size of the nucleus. After
which emits blue fluorescence upon binding to AT regions of treating cells with blank nanocarrier, the population of cells is
Table 2 MTT test results of McF7 cells treated with DOX-MTX/nanocarrier with P-value 0.001
DOX-MTX group
DOX-MTX/nanocarrier group
No
Drug treating
Cell
viability SD
No
Drug treating
dose
Cell
viability SD
dose
0.312
0.625
1.250
2.500
5.000
10.000
1
2
3
4
5
6
69.270 1.549
60.299 1.016
50.779 1.762
41.609 1.537
27.733 2.758
21.606 1.113
7
0.312
0.625
1.250
2.500
5.000
10.000
68.936 2.679
51.299 2.227
46.779 3.609
32.609 2.620
20.399 2.208
12.458 2.115
8
9
10
11
12
Abbreviation: DOX-MTX/nanocarrier, DOX-MTX-loaded magnetic nanocarrier.
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Figure 18 Fluorescent microscopic images of DaPI-stained cells following 48-hour treatment with fresh culture medium as a control (A), blank nanocarrier (B), free DOX-
MTX (C), and DOX-MTX/nanocarrier (D).
Abbreviation: DOX-MTX/nanocarrier, DOX-MTX-loaded magnetic nanocarrier.
A
B
104
103
102
101
104
103
102
101
0.07%
0.01%
0.00%
2.46%
2.05%
0.62%
99.92%
21.97%
94.87%
30.27%
100
100
100
101
102
103
104
100
101
102
103
104
FITC
FITC
C
D
104
103
102
101
104
103
102
101
40.68%
44.49%
35.79%
2.56%
25.72%
0.52%
100
100
100
101
102
103
104
100
101
102
103
104
FITC
FITC
Figure 19 The apoptotic effects of MCF7 cells, determined by flow cytometry, for untreated cells as a control (A), blank nanocarrier (B), free DOX-MTX (C), and DOX-
MTX/nanocarrier (D).
Abbreviation: DOX-MTX/nanocarrier, DOX-MTX-loaded magnetic nanocarrier.
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the same as control. Whereas the cells treated with free drugs Disclosure
and drug-loaded nanocarrier have shown significant morpho- The authors report no conflicts of interest in this work.
logical changes with nuclear fragmentation, condensation, and
nucleolus pyknosis (Figure 18C and D). The results showed References
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