Synthesis of 2-deoxy-d-glucose coated Fe3O4nanoparticles for application in targeted delivery of the Pt(iv) prodrug of cisplatin-a novel approach in chemotherapy

Article abstract of DOI:10.1039/c9nj05989j

A water soluble Pt(iv) prodrug of cisplatin was synthesized by oxidation of cisplatin followed by treatment with succinic anhydride to achieve easily reducible ester linkage at axial positions which was evidenced from cyclic voltammetric analyses. Because of this modification the Pt(iv) prodrug achieved better physicochemical and pharmacological properties like water solubility and reduced toxicity for normal (non-cancerous) CHO cells respectively, as compared to cisplatin. Later, this Pt(iv) prodrug was loaded on 2-deoxy-d-glucose (2DG) functionalized over silica coated Fe3O4 magnetic nanoparticles (MNPs) to achieve the desired formulation. It exhibited potency as evidenced from the cytotoxicity evaluation against MCF-7 human breast cancer cell lines (IC50 ～ 14 μM). This encouraged us to further study the percentage viability, apoptosis and cell death evaluations on MCF-7, Colo-205 and CHO cells by flow cytometry. The cytotoxic potency of the formulation towards cancer cells, Colo-205 and MCF-7 (22-30% apoptosis), was revealed while the parent formulation was non-toxic to non-cancerous, CHO cell lines (3% apoptosis) as compared to cisplatin. It revealed that the formulation is comparable to cisplatin in its cell killing efficiency. Additionally the FITC labeled MNPs coated with 2DG exhibited efficient cell uptake and fast internalization (within 3 h) accumulating mainly in the cytoplasm and at the cell surface. Besides this, the formulation exhibited heating efficacy suggesting its possible application for hyperthermia treatment also. These results indicate the possible utility of the formulation for site specific delivery of the Pt(iv) prodrug of cisplatin. This journal is

Full text of DOI:10.1039/c9nj05989j

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DOI: 10.1039/C9NJ05989J.
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Synthesis of 2-deoxy-D-glucose coated Fe O nanoparticles for ap
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in targeted delivery of Pt(IV) prodrug of cisplatin - a novel approach in
chemotherapy
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K. Shitaljit Sharma , Akhil K. Dubey , Arunkumar S. Koijam , Chandan Kumar , Anand
Ballal , Sudip Mukherjee , Prasad P. Phadnisa, f*, Rajesh K. Vatsaa, f*
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Chemistry Division, Bhabha Atomic Research Centre, Mumbai-400 085, India, Email:
phadnisp@barc.gov.in, Tel.: +91 22 2559 2229; rkvatsa@barc.gov.in; Tel.: +91 22 2559
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438, Fax: +91 22 2550 5151
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Bio-Organic Division, Bhabha Atomic Research Centre, Mumbai-400 085, India
Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai-400 085, India
Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai-400 085, India
UGC-DAE Consortium for Scientific Research, Mumbai Centre, Mumbai-400 085, India
Homi Bhabha National Institute, Anushaktinagar, Mumbai-400 094, India
Abstract
Water soluble Pt(IV) prodrug of cisplatin was synthesized by oxidation of cisplatin
followed by treatment with succinic anhydride to achieve easily reducible ester linkage at
axial positions which was evidenced from cyclic voltammetric analyses. Because of this
modification Pt(IV) prodrug has achieved better physicochemical and pharmacological
properties like water solubility and reduced toxicity for normal (non-cancerous) CHO cells
respectively, as compared to cisplatin. Later, this Pt(IV) prodrug was loaded on 2-deoxy-D-
glucose (2DG) functionalized over silica coated Fe O magnetic nanoparticles (MNPs) to
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achieve a desired formulation. It exhibited the potency as evidenced from the cytotoxicity
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evaluation against MCF-7 human breast cancer cell lines (IC ~ 14 µM). This encouraged to
DOI: 10.1039/C9NJ05989J
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further study for the percentage viability, apoptosis and cell death evaluations on MCF-7,
Colo-205 and CHO cells by flow cytometry. It displayed the cytotoxic potency of
formulation to cancer cells, Colo-205 and MCF-7 (22-30% apoptosis) while the parent
formulation is non-toxic to non-cancerous, CHO cell lines (3% apoptosis) as compared to
cisplatin. It revealed that, the formulation is worthy to increase the efficacy of killing cancer
cells equivalent to cisplatin. Additionally the FITC labeled MNPs coated with 2DG exhibited
efficient cell uptake and fast internalization (within 3 h) accumulating mainly in cytoplasm
and at cell surface. Besides this, the formulation exhibited heating efficacy anticipating its
possible application for hyperthermia also. These results are indicating possible utility of
formulation for site specific delivery of Pt(IV) prodrug of cisplatin.
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Keywords: Fe O MNPs, 2-deoxy-D-glucose, Pt(IV) prodrug of cisplatin, Warburg effect
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1
.
Introduction
Globally, platinum based anticancer drugs, especially cisplatin, are widely used for
treatment of cancers like carcinomas, lymphomas, sarcomas and malignant tumors etc. [1-3].
However, they exhibit inherent undesired dose-limiting toxic side effects because of non-
selective side reactions with methallothionein and thiols like glutathione which leads to their
deactivation, lesser bioavailability, short retention time in bloodstream and acquiring of
resistance [4, 5]. Many approaches are being carried out to improve their therapeutic index
such as introduction of stable ligands or changing the oxidation state of Pt metal ion. As a
result of these studies, it has been recognized that the octahedral shaped Pt(IV) oxidation
state which can be reduced to the active cytotoxic Pt(II) form by cellular reductants, is a
promising option for cancer treatment and referred as the Pt(IV) prodrug approach [6, 7].
Generally, Pt(IV) prodrug consists of two axial hydroxyl groups and through these groups,
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variety of groups like lipophilic, fluorescence moieties [8] or drugs which act synergistically
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adjunct) can be conjugated [9]. Moreover, the choice of axial ligands plays a crucial role to
enhance the efficacy of a prodrug by allowing to reduce [10, 11] easily at cancer site or by
introducing moieties which enhance cellular accumulation and uptake [12, 13]. The Pt(IV)
based prodrugs of cisplatin, carboplatin, and oxaliplatin are being studied for potent
anticancer activity [14]. The transfer of Pt(IV) prodrug from research to clinical modalities
however needs to understand the cellular pathway and perform on-demand release of drug
specifically to the tumor site using delivery vehicles. To achieve this goal, the researchers
have designed and evaluated various drug delivery vehicles on basis of rationale like
biocompatibility, non-toxicity to normal cells, water dispersibility, high drug loading
capacity, usefulness in treatment evaluation by luminescence and imaging [15]. Eventually
diverse drug delivery systems [16] like inorganic nanoparticles [17], metal-organic
frameworks (MOFs) [18] and biodegradable polymer [19], protein, peptide [20], lipid-based
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21] and dendrimers based [22] delivery systems were explored. Among these, inorganic
nanoparticles have attracted considerable attention because of their excellent effects, which
have been accomplished by use of nanoparticles like gold, iron oxide (Fe O ), carbon
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nanotubes and mesoporous silica for delivery of Pt(IV) prodrug [23]. Out of these inorganic
nanoparticles, iron oxide (Fe O ) nanoparticles have been explored for their biocompatibility
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up to certain concentrations and superparamagnetic nature. Above these, Fe O based nano-
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formulations are preferably employed as drug delivery vehicles as they can be localized at
desired site with the help of an external magnet [24] or to kill cancer cells by using thermal
energy released under an AC magnetic field (hyperthermia treatment) [25]. For better
targeting, disperibility and internalization, Fe O magnetic nanoparticles are usually coated
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by various biologically active molecules like antibody, antigen, proteins and peptides etc.
26-28].
[
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As cancer cells solely depend on glycolysis for energy and nutrient to survive and
proliferate, use of 2DG which lacks a hydroxyl group at 2-position (C-2) as compared to D-
glucose can inhibit glycolysis by blocking the conversion of 2-deoxy-D-glucose-6-phosphate
to fructose-6-phosphate [29, 30].
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In our present study, Fe O magnetic nanoparticles were synthesized by thermolysis
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method using oleic acid as the capping agent and 1-octadecene as the solvent. As the
synthesized Fe O magnetic nanoparticles are easily dispersible in non-polar solvent but not
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in polar solvent like water, the Fe O nanoparticles were coated with silica homogenously to
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achieve water dispersibility [31-33] followed by functionalizing with amine functional
groups. Later the amine functional groups were conjugated with 2-DG through thiourea
linkage giving hydroxyl functional groups on the surface of the nanoparticles. Using these
hydroxyl groups, Pt(IV) prodrug of cisplatin was loaded through the ester bond. To the best
of our knowledge, the present formulation has been reported for the first time as it involved
the linkage of 2DG by chemical bond. Here we are reporting synthesis of monodispersed
Fe O based nanoparticles coated with silica (SiO ) and functionalized with
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poly(amidoamine) and 2DG for delivering Pt(IV) prodrug of cisplatin and evaluation of its
cytotoxic activity against the CHO, Colo-205 and MCF-7 breast cancer cell lines, This result
encouraged us to extend the studies to flow cytometric evaluation for percentage viability,
apoptotic cells and dead cells. The results are reported herein.
2.
Experimental
2
.1.
Materials and Methods
The chemicals and some solvents required for this study, viz., ferric chloride, oleic
acid, 1-octadecene, tetraethyl orthosilicate (TEOS), tetra-n-butylammonium bromide
TBAB), 3-aminopropyl trimethoxysilane (APTMS), methyl acryalate, ethylenediamine,
(
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benzoyl chloride, zinc metal, Igepal-500, lithium bromide, hydrogen bromide (33%) in acetic
acid, DOWEX-500 resins, 2-deoxy-D-glucose (2DG), D-glucose (DG), ammonium chloride,
acetic acid, hydrogen peroxide, succinic anhydride, potassium thiocyanate, N, N′-
dicyclohexylcarbodiimide (DCC), hydrazine hydrochloride, Concanavalin A (ConA),
Fluorescein isothiocyanate (FITC), 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium
bromide (MTT), pyridine, dimethyl formamide (DMF), dimethyl sulfoxide (DMSO) etc.
were purchased from commercial sources (Sigma-Aldrich). Guava ViaCount reagent was
purchased from Luminex Corporation, USA. The 4′, 6-diamidino-2-phenylindole (DAPI) was
purchased from Molecular Probes Inc., USA. All solvents were dried following the standard
procedure [34].
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The Pt(IV) compound was purified by recrystallization in ethanol-water mixture at
room temperature. NMR spectra were recorded on the Agilent / Varian 600 MHz NMR
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spectrometer with a Unity Inova console operating at 599.88 ( H), 150.84 ( C{ H}) and
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28.95 MHz ( Pt{ H}). The H and C{ H} NMR chemical shifts were relative to internal
DMSO peak. The 195Pt{ H} NMR chemical shifts were relative to external Na PtCl in D O
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(
δ 0 ppm). The powder X-ray diffraction (XRD) patterns were recorded on a Pan analytical
X′pert PRO X-ray diffractometer using Cu-Kα radiation in the 2θ range of 10-70° with step
size and time of 0.02° and 1.20 s, respectively. XRD patterns were analyzed by comparing
with reported data for the end member compositions (JCPDF) files. Magnetization
measurements were carried out on a vibrating sample magnetometer (VSM) coupled to a 9
Tesla physical property measurement system (PPMS) (Model: 6000, Quantum Design,
USA).The transmission electron microscopic (TEM) image was recorded on Libra-120 plus
TEM (Carl Zeiss, Germany) operated at 120 kV as the accelerating voltage. TEM images
were analyzed by using Image J software taking 100 nanoparticles statistically. The cyclic
voltammetric studies were performed potentiostatic control using Eco Chemie Potentiostat,
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AUTOLAB-100 with the VA663 stand. Electrochemical impedance measurements were
carried out using the frequency response analyzer (FRA) module attached with Autolab-100
potentiostat.
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Cell culture experiments
CHO, Colo-205 and MCF-7 cell lines were obtained from the National Center for Cell
Sciences (NCCS) Pune, India and cultured in DMEM supplemented with 10 % serum
(
Invitrogen CA, USA) and antibiotic/antimycotic solution. Cells were grown in humidified 5
%
CO atmosphere in incubator at 37 °C and passaged for every alternate day. The
2
cytotoxicity of formulation was evaluated against human breast adenocarcinoma cell line
(MCF-7) by MTT assay. The cell viability, apoptosis and cell death of different cancer and
normal cell lines were evaluated by Guava flow cytometer (Luminex Corp. USA). The cell
lines were treated with test formulations followed by Guava ViaCount reagent followed by
evaluation with flow cytometer. The data was analyzed using guavaSoft 3.1.1 software.
Internalization of MNPs-2DG-FITC with MCF-7 cells stained with DAPI was studied by
visualizing image at 40x magnification using a fluorescence microscope (Axioplan, Carl-
Zeiss, Germany).
2
.2.
Preparation of formulation
The formulation MNPs-Pt(IV) prodrug was synthesized in following steps.
2
.2.1. Synthesis of Fe O magnetic nanoparticles (MNPs) [35]
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In a beaker containing ferric chloride (5 g, 30 mmol) dissolved in methanol (40 ml)
and water (90 ml), oleic acid (31.13 ml, 27.86 g, 98.6 mmol) dissolved in petroleum ether (50
ml) was added and stirred vigorously. Later a solution of sodium hydroxide (3.94 g, 98.5
mmol) was added slowly to it. After stirring the reaction mixture for about 1 h, the formation
of two phases was observed with clear and transparent lower aqueous phase. The upper
organic layer was collected using a separating funnel. It was refluxed for 1 h and
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concentrated in vacuo. The slurry of ferric oleate was obtained. Further the ferric oleate (2 g)
and oleic acid (2 ml) were dissolved in 1-octadecene (15 ml) placed in a 150 ml three necked
round bottom flask. After purging with argon gas for 10 min., the reaction mixture was
heated at a rate of 4 °C per min until it reached to 320 °C. Heating was maintained at this
temperature for 30 min and then cooled to room temperature. Oleic acid coated Fe O MNPs
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were precipitated out by acetone and washed by dispersion and precipitation using hexane
and acetone. The Fe O MNPs were characterized by X-ray diffraction (XRD) patterns; 2θ in
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deg (°): 20, 35, 40, 55, 60 (Figure 1 A) which matched with the JCPDF: 771545 reported data
files. Further the MNPs were characterized with vibrating sample magnetometer (VSM) and
transmission electron microscopy (TEM).
2
.2.2. Coating of MNPs by silica
[36]
To oleic acid coated Fe O MNPs (200 mg) dispersed in cyclohexane (400 ml), the
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Igepal-500 (2 g) was added and stirred for 20 min followed by addition of ammonia solution
500 µl) and TEOS (1.5 ml) over a period of 16 h. The reaction mixture was stirred further
(
for 72 h and then (3-aminopropyl)trimethoxysilane (APTMS) (3 ml) was added to it. After
stirring the reaction mixture further for 24 h, methanol (20 ml) was added and stirred for next
2
0 min. The methanol layer was separated and MNPs@SiO -NH (800 mg) was collected by
2 2
centrifugation. It was characterized by FT-IR spectroscopy (Figure 2 C). Further these MNPs
were characterized with transmission electron microscopy (TEM).
2.2.3. Functionalization of the silica coated MNPs by PAMAM (MNPs-PAMAM)
To the MNPs@SiO -NH (100 mg) dispersed in ethanol (10 ml), methyl acrylate (2
2
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ml) was added at 0 °C and then stirred at 45 °C for 24 h. Then the reaction mixture was
centrifuged and the nanoparticles were obtained. They were washed with ethanol for several
times till there was no smell of methyl acrylate and then dispersed in ethanol (10 ml). Then
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ethylenediamine (2 ml) was added to the dispersion at 0 °C and stirred at 45 °C for 24 h. The
nanoparticles were collected by centrifugation. The whole procedure was repeated two times.
2.2.4. Functionalization of MNPs-PAMAM by 2-DG (MNPs-2DG)
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It was synthesized by reaction of MNPS-PAMAM loaded with 3, 4, 5-tri-O-benzoyl-
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-deoxyglycopyranosyl isothiocyanate (Suppl. Info.: Synthesis Procedures S 1: Glucose
precursor & Figures S 1 - S 10).
The MNPs-PAMAM (50 mg) were dispersed into dimethyl formamide (5 ml), to
which 3, 4, 5-tri-O-benzoyl-2-deoxyglycopyranosyl isothiocyanate (100 mg) was added and
stirred at 60 °C for 12 h. The nanoparticles were separated by centrifugation and washed with
acetone several times. Nanoparticles were again dispersed in methanol (5 ml) and cooled to 0
°
C, followed by addition of hydrazine solution (20 ml, 30 % in water) and stirring for
overnight at room temperature. The particles were washed with water and followed by
methanol. The obtained nanoparticles were dried under the IR lamp. They are hence further
referred to MNPs-2DG.
2
.2.5. Quantification of 2DG on the surface of the MNPs-2DG
The amount of 2DG on the surface of MNPs-2DG was confirmed by monitoring the
amount of sulphur generated in elemental analyses. Along with this, presence of 2DG
moieties on the surface of the nanoparticles was confirmed by treating the nanoparticles with
FITC labeled Con A and measuring the emission of the supernatant obtained after separating
the nanoparticles using an external magnetic field.
2
.2.6. Loading of Pt(IV) prodrug of cisplatin over MNPs-2DG
It was performed in following steps.
(i)
Synthesis of Pt(IV) prodrug of cisplatin
[37]
The cisplatin, [PtCl (NH ) ] (0.135 g, 0.449 mmol) was dissolved in water (10 ml)
2
3 2
followed by treatment with aqueous solution of hydrogen peroxide (30 %). The reaction
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mixture was warmed to 60 °C for 1 h and cooled to room temperature and stirred further for
overnight. The reaction mixture was concentrated in vacuo, filtered and washed with ethanol
for three times giving almost quantitative yields (0.146 g, 97%) of Pt(IV) prodrug of
cisplatin. The Pt(IV) prodrug was dissolved in dimethyl sulfoxide (5 ml) and succinic
anhydride (0.095 g, 0.077 ml, 0.945 mmol) was added and stirred for overnight at room
temperature. The solvent was evaporated by lyophilizer yielding a creamy compound which
was washed with acetone three times and then dried in vacuo to yield a solid compound
1
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(
0.207 g, 86%). At this stage the purity of prodrug was evaluated by HPLC system on a
reverse phase column using methanol-water (10:90 v/v) as mobile phase. It revealed
acceptable purity as evidenced from a single peak. It was characterized by NMR
1
spectroscopy. H NMR in dmso-d δ: 2.36 (t, 8 H), 2.49 (t, 9 H), 6.44 (br, S, 13 H) ppm;
6
1
3
1
195
1
C{ H} NMR in dmso-d δ: 30.2, 30.8, 174.2, 180.0 ppm; Pt{ H} NMR in dmso-d δ:
6
6
1220 ppm (Suppl. Info. Figures S 12-14).
(ii)
Cyclic voltammetric studies for evaluation of reduction of Pt(IV) prodrug of
cisplatin to cisplatin at pH 6.5 and 7.4 with various scan rates
The Pt(IV) prodrug of cisplatin was dissolved in PBS buffer (10 mM) and adjusted
pH at 6.5 and 7.4 respectively. The 1 ml stock solution was diluted in 20 ml of distilled water
and then the electrolytic measurements were performed. The cyclic voltammetric analyses of
1
.0 mM Pt(IV) prodrug of cisplatin in PBS buffer solution at pH 6.5 and 7.4 were performed
-1
using Ag/AgCl electrode. The different scan rates viz., 10, 20, 30, 40, 50 mV.s were
employed for these electrolytic studies.
(
iii) Loading of Pt(IV) prodrug of cisplatin on MNPs-2DG (MNPs-Pt(IV) prodrug)
To the Pt(IV) prodrug of cisplatin (3 mg) dissolved in DMF (500 µl) at 0 °C, the N,
N′-dicyclohexylcarbodiimide (1 mg per 10 ml in DMF; 1 equivalent) was added and stirred at
room temperature for overnight. Then the MNPs-2DG (3 mg) dissolved in DMF (2 ml) was
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added to it and stirred further for overnight. The mixture was centrifuged and the MNPs were
separated and then washed with methanol (2 times) and acetone (2 times). Loading of Pt(IV)
prodrug of cisplatin on the surface of the MNPs was confirmed by ICP-OES analysis.
Prior to loading, the Pt(IV) prodrug on nanoparticles the electrolytic studies were
performed to assess the reduction of Pt(IV) prodrug of cisplatin to Pt(II) i.e., cisplatin.
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.2.7. Evaluation of cytotoxicity of MNPs-Pt(IV) prodrug formulation against MCF-7
cell lines in vitro by MTT assay [38]
The MTT assay was performed in cell culture media. The cytotoxicity of cisplatin,
MNPs-2DG and Pt(IV) prodrug loaded NPs i.e., MNPs-Pt(IV) prodrug was evaluated in vitro
against MCF-7 human breast cancer cell lines using MTT assay method. 10000 cells were
plated in a 96 well plate and incubated for overnight in humidified incubator with 5 % CO2.
Stock solution of the test compound (10 mM) was prepared either in PBS or in 0.9 M saline
solutions. The cells were exposed to various concentrations of the compounds (5, 10, 20 and
4
0 µM) for 48 h. After the exposure, MTT was added (10 µl from 5 mg / ml) and incubated
for 4 h. The formazan crystals formed were dissolved in DMSO and absorbance was recorded
at 590 nm in a microplate reader. Toxicity expressed as percentage cytotoxicity, was
calculated by the following formula:
Controlabs - Testabs
Percentage cytotoxicity =
x 100
Testabs
2
.2.8. Estimation of percentage viable, apoptotic and dead cells by flow cytometer [39]
The evaluations were performed with CHO, Colo-205 and MCF-7 cell lines. In a
5
typical experiment 1 x 10 cells were plated in 12 well plates for overnight and 10 μM of
cisplatin, Pt(IV) prodrug and corresponding amounts of MNPs-2DG, MNPs-Pt(IV) prodrug
were added in each well and incubated for 48 h. All experiments were carried out in
triplicate. After completion of incubation, cells were washed, trypsinized and Guava
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ViaCount (Luminex Corp. USA) reagent was added in each well. The cells were analyzed by
Guava Flow cytometer (Luminex Corp. USA). Percentage viable, apoptotic and dead cells
were estimated for all the three cell lines. (Please see the details in Suppl. Info. Table S 1,
Figures S 15-17).
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.2.9. Labeling of the nanoparticles with FITC [40]
Labeling of the MNPs with FITC was done by reacting amine groups present on the
surface of the nanoparticles. To study internalization of the MNPs-2DG labeling with FITC
was performed prior to the conjugation with 2DG. After labeling, the MNPs were washed
with water two times by centrifugation and dialyzed using dialysis tubing in 0.1 M saline
solution for two days with frequent change of medium.
2
.2.10. Internalization study on MCF-7 cell lines [41]
For studying the intracellular uptake, MNPs-2DG were labeled with FITC, followed
by testing its internalization efficacy in MCF-7 cells (protocol for FITC labeling is given in
6
Section 2.2.9,). Briefly, MCF-7 cells (1 x 10 ) were seeded on glass cover slips for overnight
at culture conditions, followed by treatment with MNP-2DG-FITC for 3 h. The cells were
then washed with PBS, followed by fixing in 4% paraformaldehyde for 20 min. at room
temperature (RT). The cells were further washed with PBS and mounted on slide using
Prolong Gold mounting media containing DAPI (Molecular Probes, USA). The cells were
visualized by fluorescence microscopy under 40 X magnification.
2.2.11 Hyperthermia studies
[35, 42]
Induction heating of MNPs-2DG coated with 2-DG, was performed in plastic micro-
centrifuge tube (1.5 ml) using AC magnetic field (Easy Heat 8310, Ambrell, UK) with 6 cm
diameter (4 turns) coil. Particles (0.5-2 mg) suspended in 1 ml of distilled water were placed
at the centre of coil and frequency was applied at 265 kHz.
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3
.
Results and discussion
In order to obtain the nano carrier for drug delivery, synthesis of Fe O (magnetite)
3
4
magnetic nanoparticles (MNPs) was initiated through thermal decomposition of ferric oleate.
The reaction of FeCl .6H O in methanolic aqueous solution and oleic acid (C H COOH)
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17 33
solution in petroleum ether in basic medium at room temperature followed by reflux yielded
ferric oleate. Its thermal decomposition in 1-octadecene at 320 °C yielded the Fe O
3
4
nanoparticles [43]. They were characterized by powder X-ray diffraction (XRD) which
revealed the cubic spinel phase of Fe O (JCPDS PDF No. 771545) MNPs (Figure 1A) [44].
3
4
The broadening of a peak in a diffraction pattern reveals the small size of NPs. Hence the
MNPs were characterized with more sensitive and superior tool of TEM which gives the
direct (real space) visualization of NPs of very small size as well demonstrates morphological
pattern [45]. The morphology of Fe O MNPs was studied with TEM (Figure 1B) and
3
4
observed to be monodispersed (uniform shape and size). The size of the nanoparticles was
determined by statistical evaluation of 100 nanoparticles and was found to have 16 ± 1 nm
cuboid nanoparticles. The magnetic properties of the Fe O nanoparticles were studied by
3
4
vibrating sample magnetometer (VSM) [46]. There is no coercivity (Hc) of the Fe O
3
4
nanoparticles at room temperature but coercivity (Hc) is observed when measured at 5 K
Figure 1C). This reveals that the synthesized nanoparticles are superparamagnetic at room
temperature which is desired in theranostic [47], drug delivery [47] as well as hyperthermia
49]. The saturation magnetization of the nanoparticles was calculated by plotting M Vs 1/H.
(
[
The blocking temperature Tb (temperature at which superparamagnetism to ferrimagnetism
transition takes place) was found out to be 190 K (Figure 1D).
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Size of nanoparticles (nm)
2
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40
 (degree)
60
80
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1
1
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8
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2.0
1.5
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FC
-4
-
8
-3
-2 -1
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1
0
.0
.5
-5
10
15
-
-
310 K
5 K
0.0
ZFC
-
100-80 -60 -40 -20
0 20 40 60 80 100
H(kOe)
0
100
200
T (K)
300
Figure 1. Characterization of the Fe O MNPs by powder XRD (1A), TEM (1B) and
3
4
vibrating sample magnetometer (VSM) (1C & 1D) respectively
The presence of oleic acid on these NPs renders them hydrophobic in nature.
However, for better biocompatibility in health related applications [50]; hydrophilic MNPs
[
51] are preferred which is possible by surface modifications [52]. Hence the MNPs were
coated with biocompatible silica by modified Stöber et al., [53] method which produced
Fe O @SiO MNPs. They were characterized by powder X-ray diffraction (XRD) which
3
4
2
revealed the presence of cubic spinel phase of Fe O (JCPDF PDF no. 771545) and SiO
2
3
4
(
JCPDF PDF no. 821576) in XRD pattern (Figure 2A) confirmed the formation of Fe O and
3 4
SiO phases. Formation of the core shell Fe O @SiO was further confirmed by observing
2
3
4
2
TEM image (Figure 2B) due to the difference in refractive index of the shell (SiO ) and the
2
core (Fe O ). Determination of thickness of the shell and overall size of the nanoparticles was
3
4
done by statistical evaluation of 100 NPs. The thickness of the shell was found to be in the
range of 35 ± 5 nm and overall size of the nanoparticles was in the range of 55 ± 10 nm. The
functional groups present on the Fe O @SiO -NH were characterized by FT-IR
3
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spectroscopy (Figure 2C). The bands at 564 cm , 796 cm and 1097 cm are assigned
respectively for Fe-O stretching [54], Si-O-Si symmetrical and asymmetrical stretching [54].
-1
-1
The bands at 670 cm (N-H wag), 1617 cm (N-H bending) and the broad band centered at
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300 cm presence of amine groups on the surface of Fe O @SiO -NH [55]. Such surface
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2
2
functionalized Fe O MNPs have also exhibited potent applications in theranostic as well as
3
4
drug delivery applications [56].
-
--- Fe
3
O
4
MNPs (JCPDF: 771545)
(JCPDF: 821576)
------ SiO
2
3 4 2 2
Fe O @SiO -NH
MNPs-2DG
MNPs-2DG(Bz)
Figure 2. Characterization of Fe O @SiO MNPs by XRD (2A), TEM (2B) and
3
4
2
Fe O @SiO -NH by FT-IR (2C); MNPs-2DG and MNPs-2DG-(Bz) (2D)
3
4
2
2
To increase the loading capacity of prodrug quantity of amine groups on the surface
of the nanoparticles was increased by treating them with methyl acrylate followed by
ethylenediamine [57]. The whole process was repeated three times and the amount of the
amine groups was found to be enhanced ten times of the Fe O @SiO -NH (Please see
3
4
2
2
2
.2.2.). The resultant species MNPs-PAMAM (Scheme 1) were characterized with FT-IR
spectroscopy.
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Scheme 1. Synthesis of Fe O MNPS and MNPs-PAMAM
3
4
(
1: aq. MeOH solution and petroleum ether / aq. NaOH, oleic acid at RT (1 h),
reflux (1 h); 2: in 1-octadecene at 320 °C (30 min) ; 3: in cyclohesane, aq. NH3,
Igepal-500, TEOS (74 h) and APTES (24 h); 4: methyl acrylate, ethylenediamine
(at 0 °C), then at 45 °C (24 h).
Further the loading of 2DG [58] was done by thiourea bond that was formed by the
reacting amine groups [59] on the surface of the nanoparticles and isothiocyanate group of 3,
4
, 5-tri-O-benzoyl-2-deoxyglycopyranosyl isothiocyanate. The synthesis of 3, 4, 5-tri-O-
benzoyl-2-deoxyglycopyranosyl isothiocyanate was done by following the synthesis
procedure as given in the procedures (Suppl. Info. Synthesis Procedures S 1: Glucose
precursor). The conjugation of 3, 4, 5-tri-O-benzoyl-2-deoxyglycopyranosyl isothiocyanate
with the amine functional groups was done by treating it with the nanoparticles dispersed in
dried dimethylformamide (DMF) and heating at 60 °C for 6 h. The benzoyl protecting groups
were removed by reacting with hydrazine hydrochloride in ethanol at 0 °C [60] (Scheme 2).
The nanoparticles were characterized with FT-IR spectroscopy (Figure 2 D).
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OH
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DOI: 10.1039/C9NJ05989J
HN
S
OH
BzO
O
O
HN
O
NCS
BzO
NH2
N
H
O
1
.
BzO
OH
O
N
H
OH
OH
Si
N
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O
O
H
N
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Si
N
O
O
H
N
NH
H
2.
NH NH2
2
NH2
N
O
S
O
O
Scheme 2. Conjugation of MNPs-PAMAM with 2DG by reacting 3, 4, 5-tri-O-benzoyl-2-
deoxyglycopyranosyl isothiocyanate (1) with the amine groups of MNPs-
PAMAM and deprotection of benzoyl protecting groups by hydrazine
hydrochloride (2).
-1
The peak observed at 1652 cm correspond to C=O stretching of amide of PAMAM.
-1
The peaks at 696, 967, 1396, 1448 and 1535 cm are attributable to C=S stretching and
bending of thiourea linkage formed between 2-deoxy-D-glucose isothiocyanate. The broad
-1
peak observed at 3289 cm corresponds to the amine group of amide and O-H stretching
-1
vibrations of MNPs-2DG (Figure 2D). Band in region 3500-3200cm corresponds to the
hydrogen bonds associated between neighbouring hydroxyl groups. The pyranose ring
-
1
skeletal vibrations bands are represented by 990-840 cm region. The intensive bands in
-
1
1384-1250 cm region correspond to deformation vibration of hydroxyl group. The presence
of these peaks on FT-IR spectra of MNPs-(benzoyl)-2DG and MNPs-2DG (Figure 2D)
confirms the presence of 2DG moieties on the surface of MNPs. Further, the presence of 2-
DG moiety on the surface of the MNPs-2DG was confirmed by treating ConA labeled with
FITC [61]. With the increase in the amount of MNPs-2DG added to the fixed volume of the
solution of Con A (1 mM) followed by measuring fluorescent intensity of the supernatant. A
decrease in fluorescent intensity in accordance with the amount of MNPs-2DG was observed.
(
Suppl. Info. Figure S 11) This reveals that Con A was adsorbed on surface of MNPs-2DG
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due to the interaction of Con A moiety with 2DG on the surface of the nanoparticles. Once
the MNPs-2DG formulation was ready, the kinetically inert Pt(IV) prodrug of cisplatin [62]
was prepared by oxidation of cisplatin with hydrogen peroxide by following the literature
procedure (Scheme 3). The Pt(II) metal centre of cisplatin was oxidized to Pt(IV) by using
H O , generating two axial hydroxyl groups which was further made to react with succinic
1
1
1
1
1
1
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anhydride and formed two additional carboxyl groups [63]. The synthesized Pt(IV) prodrug
1
13
1
195
1
of cisplatin was characterized by NMR ( H, C{ H}, Pt{ H}) spectroscopy (Suppl. Info.
Figures S 12-14).
OH
O
O
O
Cl
Cl
NH3
Cl
Cl
NH3
1. H O
2
2
Pt
O
Pt
NH3
NH3
O
2.
O
O
O
OH
O
Scheme 3. Synthesis of Pt(IV) prodrug of cisplatin by oxidizing cisplatin by H O and further
2
2
reaction of the axial hydroxyl groups with succinic anhydride
After achieving the pure Pt(IV) prodrug, the labiality of axial groups i.e., reduction
was evaluated at an acidic pH (6.5) as well as a physiological pH (7.4) by cyclic voltammetry
since it is well established that the cancerous cells have acidic environment due to formation
of lactic acid as the end product during glycolysis. Hence it was anticipated that the axial
ester groups should preferentially reduce in acidic pH conditions. The cyclic voltammetric
studies show that maximum reduction of axial ester groups occur under acidic conditions as
depicted in Figure 3 [64].
The redox potential for reduction of the Pt(IV) prodrug of cisplatin at pH 6.5 and 7.4
unit values was evaluated. These electrochemical analyses revealed that the prodrug exhibited
an irreversible cyclic voltammetric response for the Pt(IV) / Pt(II) couple near -1.00 V vs.
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DOI: 10.1039/C9NJ05989J
Ag/AgCl in PBS buffer 1:10 mixture of compound solution in PBS buffer at pH 6.5 while
relatively very less reduction at -1.20 V at pH 7.4 respectively at scanning rate of 30 mV.s-1
(Figure 3). These reduction potentials are indicative that the prodrug is amply stable towards
reduction in the bloodstream throughout delivery to the target cancer cells by the
nanoparticles.
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Figure 3. Cyclic voltammetry of 1.0 mM Pt(IV) prodrug of cisplatin in PBS buffer solution
-1
using Ag/AgCl with different scan rates of 10, 20, 30, 40, 50 mV s indicating
variation of peak current of the first oxidation peak with the square root of the
scan rates.
Loading of prodrug of cisplatin on MNPs-2DG was performed by well-known
process of DCC coupling [65]. The activation of carboxylic groups of Pt(IV) prodrug was
performed by N, N'-Dicyclohexylcarbodiimide (DCC) followed by its treatment with MNPs-
2
DG in DMF solvent at room temperature (Scheme 4) leading to ester bond formation
between hydroxyl groups on the surface of MNPs-2DG and carboxyl groups of Pt(IV)
prodrug. It is the desired formulation. The loading efficiency of Pt(IV) prodrug of cisplatin
loaded on the surface of the nanoparticles was found to be 90 % which was quantified by
ICP-OES analysis.
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OH
OH
OH
O
OH
O
O
HN
S
OR
O
O
O
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O
Cl
Cl
NH3
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N
H
Cl
Cl
NH3
NH3
Compound-5
DCC, DMAP
OH
Pt
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R =
OH
Si
N
H
NH3
Pt
O
O
N
NH
S
H
O
O
N
OR
O
O
OH
O
R = Cis Pt (IV)
Cis Pt (IV)
Scheme 4: Loading of Pt(IV) prodrug over surface functionalized Fe O MNPs
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This MNPs-Pt(IV) prodrug formulation was evaluated for its cytotoxicity in vitro
against MCF-7 cell lines by MTT assay. The cytotoxicity of Pt(IV) prodrug of cisplatin,
MNPs-2DG and MNPs-Pt(IV) prodrug prodrug was compared with the cisplatin.
Cytotoxicity was evaluated against MCF-7 human breast cancer cell lines by treating with
various concentrations of all test compounds and formulation at 5, 10, 20 and 40 µM
concentrations (Figure 4). The IC value of cisplatin has been observed to be 10 µM while
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the Pt(IV) prodrug exhibited IC value of ~ 30 µM which is indicative of kinetic inertness of
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Pt(IV) derivative as anticipated. This also demonstrates that the carrier MNPs-2DG exhibited
negligible cytotoxicity. The 2DG itself has IC of 10 milli-molar (mM). Since the loading of
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DG molecules over MNPs-2DG is in small amount, it does not contribute much to
cytotoxicity. The formulation MNPs-Pt(IV) prodrug exhibited IC of ~ 14 µM. Although the
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IC value of prodrug is approximately triple than that of cisplatin, it is anticipated that the
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prodrug will be less toxic due to its kinetic inertness as a result of the octahedral structure.
Hence, the present formulation will help to enhance the activity of prodrug of the cisplatin
with reduced side effects. Due to the presence of 2DG, the above-mentioned formulation is
supposed to be target specific, an aspect which needs to be checked in a subsequent work. It
revealed the potency of formulation as observed IC ~ 14 µM.
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Figure 4. MTT assay for treatment of MCF-7 cell lines with cisplatin, its prodrug and
formulation
After initial evaluation of percentage cytotoxicity by MTT, we extended the
cytotoxicity studies further to estimate the percentage of viable, apoptotic and dead cells by
flow cytometric studies. Accordingly the evaluations on CHO, Colo-205 and MCF-7 cell
lines treated with 10 µM solutions of cisplatin, Pt(IV) prodrug and corresponding
concentrations of MNPs-2DG and MNPs-Pt(IV) prodrug were performed by using flow
cytometer as shown in Table 1. Percentage viability, control, 10 µM Pt(IV) prodrug treated
and 10 µM MNPs-2DG treated CHO cell lines were respectively 86.93 ± 0.87, 89.52 ± 1.03
and 86.21 ± 0.22. These values suggest the non-toxicity of Pt(IV) prodrug and our
formulation, MNPs-2-DG. The values of percentage viability in case of cisplatin and MNPs-
Pt(IV) prodrug were respectively 59.35 ± 3.28 and 50.67 ± 4.22 which show their similar
activity. Values of percentage viability for Colo-205 cell lines treated with 10 µM solutions
of cisplatin, Pt(IV) prodrug and corresponding concentrations of MNPs-2DG and MNPs-
Pt(IV) prodrug are 76.1 ± 1.41, 66.6 ± 3.54, 64.87 ± 3.7, 74.2 ± 0.14 and 63.73 ± 2.29.
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Similarly values of MCF-7 cell lines are respectively 75.235 ± 2.58, 66.98 ± 3.07, 66.1 ±
2
.84, 69.77 ± 6.93 and 45.37 ± 4.0. Overall, the cytotoxic effect desired to kill cancer cells
were observed with the current formulation as seen in treatment of MCF-7 and COLO 205
cell lines. The cytotoxic effect was however also observed in the normal CHO cell line. A
possible explanation to this is the fusion of internalisation-efficient MNPs-Pt(IV) prodrug
with lysosomes within cells. The acidic condition within lysosomes might have activated the
MNPs-Pt(IV) prodrug which eventually exert its cytotoxic effect in subsequent cell cycles.
Another possible explanation include the lesser DNA or chromosome content per cell. CHO
cells typically contain 22 chromosomes compared to COLO 205 or MCF-7 which typically
contain 46 chromosomes. The intercalation frequency of internalisation-efficient MNPs-
Pt(IV) prodrug to DNA within CHO cells must likely be higher compared to COLO 205 and
MCF-7 cells. Lower percentage viability of Cisplatin treated CHO cells compared to
Cisplatin treated COLO 205 and MCF-7 cells (Please see the details in Suppl. Info. Table S
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, Figures S 15-19) support this assumption. The reduction of cytotoxicity to normal cells
without compromising efficiency to kill cancer cells need to be studied deeper in future.
Table1. Cell toxicity studies on CHO, Colo-205 and MCF-7 cell lines in the presence of 10
μM of respective drugs and corresponding concentrations of MNPs-2DG and MNPs-
Pt(IV) prodrug
Percentage
viability of
Cell lines
CHO
Colo-205
MCF-7
Control
Prodrug
Cisplatin
MNPs-2DG MNPs-Pt(IV)
prodrug
86.93 ± 0.87
76.1 ± 1.41
75.235 ± 2.58
89.52 ± 1.03 59.35 ± 3.28 86.21 ± 0.22 50.67 ± 4.22
66.6 ± 3.54 64.87 ± 3.7 74.2 ± 0.14 63.73 ± 2.29
66.98 ± 3.07 66.1 ± 2.84 69.77 ± 6.93 45.37 ± 4.0
After cytotoxicity studies it was worthwhile to assess the cellular uptake of the
formulation. To study the higher intracellular uptake of MNPs-2DG in MCF-7 cells, MNPs-
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DG were labeled with a fluorescent dye (fluorescein iso-thiocyanate, FITC), followed by
DOI: 10.1039/C9NJ05989J
treatment of MCF-7 cells with 400 µl of MNPs-2DG-FITC. The cells were visualized by
fluorescence microscopy where green fluorescence is observed in the cytoplasm and near the
surface of the cells. The nucleus stained with DAPI didn’t show co-localization with MNPs-
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DG-FITC as observed in the merged image (Figure 5) suggesting that the MNPs are
localized mainly in the cytoplasm and cell surface within 3 h. It exhibited efficient cellular
uptake of test MNPs-2DG. In this case, the organic moiety attached at axial position of Pt(IV)
prodrug of cisplatin tunes the lipophilicity of the prodrug, consequently improving the ability
of the compound to accumulate into cancer cells
Figure 5. Fluorescence microscopy images of MCF-7 cells after treatment with MNPs-2DG-
FITC. Nucleus is stained with DAPI (blue) and green color indicates MNPs-2DG
labeled with FITC. Scale bar: 10 µm.
The cellular uptake leads to internalization and eventually cell killing which are in
correlation. As discussed earlier the flow cytometric evaluations have exhibited substantial
percentage of apoptotic cells. It's well established that appearance of apoptosis has been
recognized as an outcome of DNA damage. Literature reports have clearly stated about
cisplatin, Pt(IV) prodrugs internalize in cell through Pt-DNA binding through covalent bond
which induce the DNA damage leading to cancer cell death by triggering apoptotic pathway.
Hence, the DNA biding ability of Pt compounds correlates with its ability to induce apoptosis
[
66]. In our case, from cyclic voltammetric studies, it's clear that Pt(IV) prodrug of cisplatin
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was reduced to active form Pt(II) i.e., cisplatin in acidic pH similar in cancer cells. Hence it
reveals that that in cell the cisplatin was formed which binds with DNA leading to DNA
damage which induced apoptosis and eventually cell death. This phenomenon of Pt
compound-induced cell death should helpful in the development of more effective therapeutic
strategies for the treatment of cancer.
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Additionally the formulation was evaluated for heating effect under AC magnetic
field [35]. For typical demonstration of heating efficacy, Fe O particles coated with SiO are
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dispersed in water; and the dispersed particles (5 or 10 mg per 1 ml) are kept in induction coil
frequency = 265 kHz, current = 400 A). 0.5 mg/ml of Fe O particles produce 31-33 °C
(
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within 600 seconds at 400 A (335 Oe) (Figure 6) but could not reach hyperthermia
temperature (HT = 42 °C). Similarly, 1 mg/ml particles at 400 A could not reach HT. But, 2
mg/ml at 400 A could reach HT in 1200 s.
Figure 6: The evaluation of heat released with respect to time under AC magnetic field.
Specific absorption rates (SAR) of particles with concentrations of 0.5 mg / ml, 1
-1
mg/ml, 2 mg/ml and 5 mg/ml are found to be 2.09, 3.26 and 4.96 W.g , respectively and
here, SAR is expressed in term of Watts per 1 g of Fe O . The heat generation from magnetic
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fluid under AC magnetic field comes from relaxation phenomena and hysteresis loop.
Relaxation phenomena include Brownian motion of particles with liquid medium and single
domain relaxation (10 -10 s-1) and hysteresis loss arises during AC frequency (265 kHz)
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and applied magnetic field. Relaxation phenomena are dominating factor in case of smaller
particles sizes, whereas hysteresis loss is dominating factor in case of bigger particles
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Conclusions
In the present study, monodispersed, water dispersible Fe O MNPs coated with silica
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SiO ) were synthesized. The SiO surface was functionalized with poly(amidoamines)
2 2
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PAMAM) which in turn conjugated with 2DG through thiourea bond formation. Further the
DG was linked with Pt(IV) prodrug of cisplatin through the ester bond to afford the
2
formulation. Here the silica coating and 2DG linking render the water dispersibility and
biocompatibility to formulation. This formulation exhibited cytotoxicity against MCF-7
human breast cancer cell lines (IC value ~ 14 µM). Even though it is comparable to cell
50
killing potency of cisplatin, the formulation is anticipated to exhibit reduced severe side
effects of cisplatin. It was evidenced from the flow cytometric evaluations performed on
MCF-7, Colo-205 cancer cells and CHO non-cancer cell. The MNPs-Pt(IV) prodrug is
responsible to induce apoptosis in cancer cells evidenced from ~ 30% and ~ 22% of apoptotic
cells in case of MCF-7 and COLO-205 cell lines respectively while only 3% for CHO cell
lines for parental formulation. Further, the MNPs-2DG-FITC exhibited their efficient cell
internalization in cytoplasm of MCF-7 cell lines within 3 h. It reveals the efficient and fast
internalization of proposed formulation. It leads to conclusion that after the internalization,
the Pt moiety from formulation interacts with DNA leading binding which leads to cell death
through apoptotic pathway. Additionally the heating effect studies indicated that the
formulation has the potential for hyperthermia applications also. The results divulge that the
formulation is biocompatible and it is anticipated to be a competent pharmacological agent
leading to further research for its clinical applications.
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5
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Supplementary information
† Electronic supplementary information (ESI) available: The characterization data of
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Pt(IV) prodrug of cisplatin viz., NMR ( H. C{ H} and Pt{ H}) and other experimental
details like synthesis of 2-deoxy-D-glucose derivatives etc. are given in supplementary
materials.
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Acknowledgements
Authors are thankful to Dr. S. Kapoor Associate Director (D), Chemistry Group and
Dr. A. K. Tyagi, Head, Chemistry Division, BARC, Trombay, Mumbai for the
encouragement and support for this research work. Authors also acknowledge the fruitful
discussions with Dr. R. S. Ningthoujam, Chemistry Division, BARC as well as Dr. Dibakar
Goswami, Bio-Organic Division, BARC. KSS is grateful to University Grants Commission
(
UGC), New Delhi for the award of fellowship. The authors gratefully acknowledge help
from Analytical Chemistry Division, BARC, for providing the cyclic voltammetry and ICP-
OES analyses.
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Author information
Affiliations
Chemistry Division, Bhabha Atomic Research Centre, Mumbai, 400 085, India
K. Shitaljit Sharma
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Chemistry Division, Bhabha Atomic Research Centre, Mumbai, 400 085, India &
Homi Bhabha National Institute, Anushaktinagar, Mumbai-400 094, India
Prasad P. Phadnis and Rajesh K. Vatsa
Bio-organic Division, Bhabha Atomic Research Centre, Mumbai, 400 085, India
Akhil K. Dubey
Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai-400 085, India
Arunkumar S. Koijam and Chandan Kumar
Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai-400 085, India
Anand Ballal
UGC-DAE Consortium for Scientific Research, Mumbai Centre, Mumbai-400 085, India
Sudip Mukherjee
Corresponding authors
Correspondence to: Dr. Prasad P. Phadnis; Dr. Rajesh K. Vatsa
Email: phadnisp@barc.gov.in; rkvatsa@barc.gov.in
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Author contributions
KSS and PPP designed the work and performed synthesis of silica coated Fe O
3
4
nanoparticles and Pt(IV) prodrug. KSS, AKD and PPP conducted the synthesis of glucose
derivative and its functionalization with nanoparticles. SM performed magnetic
measurements. AB performed TEM experiments. KSS evaluated the cell internalization of
formulation as well as cytotoxicity against MCF-7 cancer cell lines by MTT assays. ASK and
CK performed assessment of percentage viable, apoptotic and dead cells by flow cytometer.
RKV supervised the experiments. KSS, PPP and RKV wrote the manuscript. All authors
discussed the results and worked on the manuscript and approved the final version of
manuscript.
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Ethics declarations
Conflicts of interest
“There are no conflicts to declare”.
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References
[1]
B. Rosenberg, L. Van Camp, J. E. Trosko and V. H. Mansour, Nature, 1969, 222,
85-386.
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