Dual-drug loaded Janus graphene oxide-based thermoresponsive nanoparticles for targeted therapy

Article abstract of DOI:10.1016/j.polymer.2018.03.022

Graphene oxide (GO) is extensively recognized as an effective material in a variety of fields and specifically for various biomedical applications. However, the modification of each side of a GO sheet, needed for preparation of heterogeneous nanoparticles, is yet hypothetical and unclear. Here, we have developed a new strategy for the synthesis of two-faced GOs by simultaneous anchoring of different polymers with different hydro-affinity via Pickering emulsion (two-phase media). Alkynyl-modified GO was prepared for unsymmetrically attaching azidated-poly(?-caprolactone) (PCL) and (N-isopropylacrylamide-co-acrylamide-co-allylamine) terpolymer to the surface of GO through click reaction. During this reaction, a thermoresponsive nanoparticles with specific morphology, known as “Janus” nanostructure, will be produced. PCL/terpolymer homostructure was also fabricated via solution (one-phase) reaction, where two types of polymers were grafted on the both surfaces of GO disorderly, named “mixed” nanoparticles. In the following, drug loaded nanocarriers were synthesized by simultaneously using quercetin and 5-FU as hydrophobic and hydrophilic drugs. The bioapplicability of these nanoparticles were evaluated by comparison of their in vitro release profiles and cell cytotoxicity at two temperatures of 37 and 40 °C.

Full text of DOI:10.1016/j.polymer.2018.03.022

Accepted Manuscript
Dual-drug loaded Janus graphene oxide-based thermoresponsive nanoparticles for
targeted therapy
Sepideh Khoee, Mohammad Reza Karimi
PII:
S0032-3861(18)30227-1
10.1016/j.polymer.2018.03.022
JPOL 20435
DOI:
Reference:
To appear in: Polymer
Received Date: 18 September 2017
Revised Date: 5 March 2018
Accepted Date: 12 March 2018
Please cite this article as: Khoee S, Karimi MR, Dual-drug loaded Janus graphene oxide-
based thermoresponsive nanoparticles for targeted therapy, Polymer (2018), doi: 10.1016/
j.polymer.2018.03.022.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Dual-drug loaded Janus graphene oxide-based thermoresponsive
nanoparticles for targeted therapy
Sepideh Khoee*, Mohammad Reza Karimi
Polymer Laboratory, School of Chemistry, College of Science, University of Tehran, PO Box
1
4155 6455, Tehran, Iran
*
Corresponding author. Email: khoee@khayam.ut.ac.ir Tel: (098) 21 61113301
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Abstract
Graphene oxide (GO) is extensively recognized as an effective material in a variety of fields
and specifically for various biomedical applications. However, the modification of each side
of a GO sheet, needed for preparation of heterogeneous nanoparticles, is yet hypothetical and
unclear. Here, we have developed a new strategy for the synthesis of two-faced GOs by
simultaneous anchoring of different polymers with different hydro-affinity via Pickering
emulsion (two-phase media). Alkynyl-modified GO was prepared for unsymmetrically
attaching azidated- poly(ɛ-caprolactone) (PCL) and (N-isopropylacrylamide-co-acrylamide-
co-allylamine) terpolymer to the surface of GO through click reaction. During this reaction, a
thermoresponsive nanoparticles with specific morphology, known as “Janus” nanostructure,
will be produced. PCL/terpolymer homostructure was also fabricated via solution (one-phase)
reaction, where two types of polymers were grafted on the both surfaces of GO disorderly,
named “mixed” nanoparticles. In the following, drug loaded nanocarriers were synthesized by
simultaneously using quercetin and 5-FU as hydrophobic and hydrophilic drugs. The
bioapplicability of these nanoparticles were evaluated by comparison of their in vitro release
profiles and cell cytotoxicity at two temperatures of 37 and 40 °C.
Keywords: Graphene oxide; Janus nanoparticles; Microemulsion; Dual-drug delivery;
Thermoresponsive polymer
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1
. Introduction
Through utilizing the unique capabilities of nanotechnology, smart nanocarriers have been
introduced which can overcome many drawbacks of traditional drug delivery systems
through improvements in drug release properties in targeted delivery sites to reduce side
effects in other regions [1]. Such side effects can be fatal in the case of cancer chemotherapy.
In addition, while chemotherapeutic agents can effectively treat cancerous cells, repeating the
single drug treatment may lead to chemotherapeutic drug resistance [2]. To address such
issues, combination therapy has been used in many clinical trials as a promising approach for
cancer treatment. Through combination therapy, two or more therapeutic components are
simultaneously introduced into the body. Co-delivery of different chemotherapeutics through
dual drug delivery systems, as an example, aim to maximize the efficacy of therapy [1-4].
Due to the synergistic effects of different therapeutics and lowered levels for the
administration of total dosage of these cytotoxic agents, such combinatory methods have
become the most favored therapeutic modality to overcome side effects caused by high doses
of single drugs [5]. Combination of chemotherapeutics and anti-oxidants have been reported
to be effective for different cancer therapies [6], 5-fluorouracil and quercetin have shown to
exhibit synergistic effects in colorectal and pancreatic cancers [1, 7]. Finding carriers with
proper load, and release properties are then of major concern. Janus particles, named after the
mythological Roman god of gates, have been recently shown to be applicable in biomedical
and drug delivery systems [8-11]. Such nanoparticles are generally made up of two different
hemispheres with different chemistry and physical properties where they can play at least two
different simultaneous roles of targeting and stimuli-based release for different payloads with
different hydro-affinities [12].
Among the currently available synthetic approaches, Pickering emulsion strategy has proven
to be successful in the synthesis of Janus particles. Pickering emulsion provides suitable
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conditions for the conjugation of hydrophobic and hydrophilic components solved in water or
oil phases through the stabilizing effect by bringing solid nanoparticles at a vast interface
between two liquid phases. On the other hand, graphene oxide (GO) is considered as an
appropriate platform for biodevice because of its highly efficient properties like
biocompatibility, biodegradablility, biosensing, bio-imaging and photo-thermal therapy [13,
1
4]. Having multiple oxygen containing functional groups both at the surface and the edges
can provide opportunity for graphene layers to be dispersed in both aqueous and polar
organic phases [4]. Existence of such functional groups can also be used for chemical
reactions to achieve permanent stabilization for long-term usage.
Previous attempts toward preparation of Janus GO mainly involved electrostatic assembly
[
15] and functionalization of large scale materials with different hydro-affinity functional
groups in different sides and Pickering emulsion [16, 17]. As it has unique amphiphilic
structure, solid stabilization of foams and emulsions is considered as one of the most
outstanding duties of Janus graphene oxide [18, 19]. Yang et al. fabricated Janus GO
nanosheets by using cationic polystyrene microspheres as micro-template and electrostatic
assembling of GO nanosheets on their surface. Anchoring hydrophilic poly(2-
(dimethylamino)ethyl methacrylate) chains on the other side can make GO-based Janus
nanoparticles [15]. To prepare Janus GO with different hydro-affinity properties, millimeter-
scale reduced GO sponges were synthesized by freeze drying, and their wettability was
controlled by fluorine (hydrophobic in air) or oxygen (hydrophilic in air) functionalizations
[
16]. Wu et al. prepared janus GO nanosheet through protecting one side of GO via wax-in-
water Pickering emulsion method and grafting the free surface of the GO with epoxy groups.
Janus GO, compared with symmetrically modified GO, shows potent stabilization in
Pickering emulsion method [17].
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Of the various significant research activities on GO chemistry that have been conducted,
nearly no work to date has been focused on the simultaneous functionalization of graphene
oxide using two hydrophobic and hydrophilic polymers via Pickering emulsion method. And
between some few research articles about preparation of Janus GO, no one deals with
practical applications of dual drugs delivery systems. In the present study, we demonstrate
the synthesis of a new Janus graphene oxide nanoparticle, via covalently attaching different
types of polymers on its two faces simultaneously, as an amphiphilic nanovehicle for
combinatorial drug delivery approach. Our strategy in fabrication of this nanocarrier is based
on the utilization of poly(N-isopropylacrylamide-co-acrylamide-co-allylamine) terpolymer as
the thermo-sensitive hydrophilic component and poly(ɛ-caprolactone) as the hydrophobic
one. Graphene oxide was symmetrically decorated with alkynylated groups and the prepared
pre-polymers were covalently attached to the surface of nano graphene oxide (NGO) through
click reaction via two methods. Janus nanoparticles were synthesized via oil in water
Pickering emulsion and “mixed” nanoparticle was obtained through a homogeneous solution.
The procedure was followed by simultaneously or individually loading of quercetin and 5-FU
as respective hydrophobic and hydrophilic drugs to investigate the possibility of using these
particles in cancer combination therapy. In-vitro release profiles of 5-FU and quercetin were
observed in phosphate buffer solutions at two temperatures, 37 and 40 °C (pH=7.4), to
investigate the stimuli-responsiveness of the particles.
2
. Experimental
.1 Materials
Graphite flakes, potassium permanganate (KMnO ), phosphoric acid (H PO ), N,N-
2
4
3
4
dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), sodium azide (NaN3),
copper bromide (CuBr), azobisisobutyronitrile (AIBN), copper(II) sulfate pentahydrate and
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propargyl amine were obtained from Sigma-Aldrich. ɛ-Caprolactone, N-isopropylacrylamide
(
NIPAAm), acrylamide (AAm), allylamine (AA), pentamethyldiethylene triamine
PMDETA), ascorbic acid, anhydrous magnesium sulfate, epichlorohydrin, sodium
(
bicarbonate, tetra-n-butylammonium bromide (TBAB), dichloromethane (DCM), diethyl
ether, N,N-dimethylformamide (DMF), sulfuric acid (H SO ), hydrochloric acid (HCl),
2
4
hydrogen peroxide (H O ), 1,4-dioxane, and stannous octoate (Sn(Oct) ) were all purchased
2
2
2
from Merck Chemical Co. Triethylamine (TEA) was obtained from Fluka. All reagents and
solvents were of analytical grade and used without any purification.
2
2
.2 Preparation of nanoparticles
.2.1 Azidation of epichlorohydrin
Azidated epichlorohydrin (AE) was synthesized according to the procedure
described as previously reported [20]. Briefly, 5 g sodium azide (77 mmol) was
dissolved in 15 mL deionized water, containing tetrabutylammonium bromide (0.16
g, 0.5 mmol) as a phase transfer catalyst, in a round-bottom flask followed by the
dropwise addition of 7.11 g epichlorohydrin (6 mL, 77 mmol). Since the reaction
products are highly photosensitive, reaction vessels were covered by aluminum foil
and the mixture was allowed to proceed at room temperature for about 16 h. 20 mL
dichloromethane was then added to the reaction mixture and non-aqueous layer was
separated using a separating funnel. The organic layer was dried over anhydrous
magnesium sulfate and its solvent was evaporated under reduced pressure by
rotatory evaporator to yield the azidated epichlorohydrin with 72% yield.
2
.2.2 Synthesis of poly(ɛ-caprolactone) with AE initiator
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5
mL ɛ-caprolactone (45 mmol), 5 mL DMF and 675 mg AE (5 mmol) were poured into a
three-necked round-bottom flask and purged with dried nitrogen. The mixture was heated to
0 °C and a catalytic amount of Sn(Oct) was added to the reaction medium. The reaction
8
2
temperature was raised up to 120 °C and kept constant at this temperature for 24 h. The crude
product was precipitated in cold water and dried in a vacuum oven at 40 °C for 24 h to
produce azide-terminated poly(ɛ-caprolactone) 73.2% yield.
2
.2.3 Synthesis of Poly(N-isopropylacrylamide-co-acrylamide-co-allylamine) Poly(NIPAAm-
co-AAm-co-AA), a thermo-sensitive polymer:
Poly(NIPAAm-co-AAm-co-AA)
(terpolymer) was prepared via random radical
polymerization [21]. Briefly‚ 0.518 g (5 mmol) NIPAAm‚ 0.016 g (0.22 mmol) AAm‚ 0.022
mL (0.29 mmol) AA and 5 mL of dioxane were added into a three-necked round-bottom flask
and let to stir for 30 min, under continuous nitrogen purging. 0.01 g AIBN was added to the
mixture and the reaction temperature was adjusted to 60 °C using an oil bath and the system
was stirred at this temperature for 24 h under nitrogen atmosphere. The resulting polymers
were re-precipitated into cold diethyl ether and then dried under vacuum to obtain terpolymer
with 77% yield.
2
.2.4 Functionalized Poly(NIPAAm-co-AAm-co-AA) by azide groups
Terpolymer (1.5 g) was dissolved in 1,4-dioxane, in a three-necked round-bottom flask, at
room temperature and purged with nitrogen gas. 2 mL triethylamine (2.75 g) was added
slowly to the above mixture and followed by dropwise addition of freshly-prepared azidated
epichlorohydrin (3.5 g). The reaction solution was then stirred at 50 °C for 48 h. The reaction
mixture was poured into cold diethyl ether, filtered and then dried under vacuum to yield
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crude azidated terpolymer. In order to further purify, the product was re-dissolved in 1,4-
dioxane and re-precipitated in cold diethyl ether to yield purified product with 78% yield.
2
.2.5 Synthesis of graphene oxide
Graphene oxide was synthesized from graphite powder using improved hummers
method [22]. In brief, 1 g graphite was mixed with 200 mL acid mixture containing
H SO and H PO with a 9:1 vol/vol ratio, respectively, under constant stirring.
2
4
3
4
After steady addition of 6 g KMnO , the mixture was let to stir for 3 days in 50 °C.
4
The solution was poured into ice water and then H O solution was added dropwise
2
2
till the color of the suspension changed to bright yellow. The obtained graphite
oxide solution was centrifuged and washed twice with 1 M of HCl aqueous solution
and then three times with deionized water to set the pH value of the solution to 4–5.
Graphene oxide sheets were separated by centrifugation and freeze-dried.
2
.2.6 Synthesis of Alkynyl-GO
In order to utilize the click reaction in the next steps of this work, we had to modify
the surface of the GO with alkynyl groups. Alkynyl-GO was prepared using
propargyl amine. In a typical procedure [23], GO (0.1 g) was dispersed in 5 mL
DMF using Bandelin probe sonicator (20 min, 50%, 5 second on/3 second off).
Propargyl amine (0.268 g, 2.73 mmol) was added into the GO suspension and
dissolved using stirrer. DCC (4 g, 20.87 mmol) and DMAP (0.3 g, 2.46 mmol) were
gradually poured into the mixture during 20 min. The reaction solution was let to
stir for 16 h at room temperature. After the completion of reaction, the suspension
was diluted in 30 mL of DMF. The diluted suspension was centrifuged and washed
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thoroughly using DMF and then dried under vacuum overnight to produce 0.207 g
alkynyl-decorated GO.
2
.2.7 Synthesis of Janus poly(ɛ-caprolactone)-graphene oxide-Poly(NIPAAm-co-AAm-co-AA)
(J-(PCL-NGO-Terpolymer))
J-(PCL-NGO-Terpolymer) was prepared using Pickering oil in water (O/W)
emulsion/solvent evaporation method. Briefly, azide-terminated PCL (20 mg) was dissolved
in DCM (2 mL) to form the oil phase. On the other hand, alkynated-GO (10 mg) was
dispersed in 10 mL phosphate buffer saline (PBS) (pH adjusted to 7.4) and sonicated for 30
min (5 second on/3 second off) to obtain a homogeneous colloidal suspension of GO
nanosheets (NGO) as water phase. Afterwards, a 2 mL orange colored aqueous solution
containing ascorbic acid (21.13 mg, 0.12 mmol), sodium bicarbonate (30.24 mg, 0.12 mmol)
and 10 mg of copper(II) sulfate pentahydrate was added to the aqueous phase. Oil phase was
then added dropwise to an emulsifier-free aqueous solution containing NGO. Oil-in-water
(O/W) micro-emulsion was formed and was let to stir for 16 h at room temperature to
produce one side PCL coated graphene oxide J-(PCL-NGO). Terpolymer (50 mg) was added
to the reaction solution and stirred for a day at room temperature. After the completion of
reaction, the mixture was kept at room temperature for 24 h to evaporate the organic solvent
and form J-(PCL-NGO-Terpolymer). The product was washed once with DCM and twice
with distilled water. The resultant material was freeze-dried to remove the trapped water
without destroying the nanoparticle structures.
2
.2.8 Synthesis of mixed poly(ɛ-caprolactone)-graphene oxide-Poly(NIPAAm-co-AAm-co-AA)
(m-(PCl-NGO-Terpolymer))
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GO (10 mg) was dispersed in 10 mL DMF, using Bandelin probe sonicator (20 min, 50%).
CuBr (15 mg) and PMDETA (22 µL) were successively added into the GO suspension and
dissolved by stirring. Azide containing PCL and terpolymer were added simultaneously to the
mixture and the reaction solution was let to stir for 48 h at room temperature. After the
reaction was completed, the obtained m-(PCL-NGO-Terpolymer) was centrifuged and
washed thoroughly using DMF and water, then dried overnight under vacuum.
m-(PCL-NGO) and J-(PCL-NGO) samples were synthesized in the absence of terpolymer
through this method separately for TEM analysis.
2
.3 Characterization and Equipment
Polymers were characterized using Fourier transform-infrared spectroscopy (Bruker-
1
1
Equinox55 FT-IR) and H-NMR (Bruker, 500 MHz). H-NMR spectrum of azide-terminated
PCL and terpolymer were recorded in CDCl and D O, respectively. The number averaged
3
2
1
molecular weight (Mn) of azide-terminated PCL was calculated with H-NMR data. Number
average molecular weight (Mn), weight average molecular weight (Mw) and polydispersity
index (PDI) of poly (NIPAAm-co-AAm-co-AA) were determined in tetrahydrofuran
containing 0.25% of TBAB at 25 °C and in the flow rate of 0.7 mL/min by gel permeation
chromatography (GPC) using a PL-EMD 950 instrument. Calibration was carried out with
monodisperse polystyrene standards. GPC was used to determine Mn, Mw and PDI of PCL,
too. PCL was dissolved in tetrahydrofuran and GPC was run in the flow rate of 1 mL/min at
2
5 °C. The morphology of GO and functionalized GO were investigated by transmission
electron microscopy (TEM) (Zeiss LEO 906 TEM instrument) and scanning electron
microscopy (SEM) (FESEM; Zeiss SuprATM 55, Germany). The materials were dispersed in
distilled water and one drop of the dispersed mixture was placed on a carbon coated grid. The
grid was completely dried and used for TEM/SEM analysis (150 kV). Thermal gravimetric
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analysis (TGA) was performed to display the content of polymer on graphene oxide surface
(TGA Q50 V6.3 Build 189). Samples were heated at a rate of 20 °C/min under nitrogen
blanket and the data was recorded from 20 to 600 °C. The X-ray diffraction (XRD) patterns
were obtained on an X-ray diffractometer (X’Pert PRO MPD, PANalytical Company,
Netherlands) with Cu Kα radiation. The topography of the GO nanosheets was characterized
by atomic force microscopy (AFM, Auto Probe CP Research- Veeco Instruments Inc., USA)
on a freshly cleaved mica substrate. The drug release was studied using ultraviolet–visible
light (UV–Vis) spectrophotometer spectra (Perkin-Elmer Lambda 25 spectrophotometer).
Cloud point temperature (Tcp) was determined using an UV–Vis spectrophotometer coupled
with a temperature controller. The polymer solutions were prepared using standard phosphate
buffer (PBS, pH=7.4). The heating rate was 2 °C/10 min and 0.2 °C/10 min near the cloud
point (CP). From the calibration curve based on normalized absorbance vs. temperature, CP
was determined as the temperature in which the absorbance is 10%. The volume phase
transition temperature, VPTT, of the “Janus” and “mixed” nanoparticles were investigated
using a micro-differential scanning calorimeter (VP-DSC, Micro Cal Inc.).The micro-DSC
were measured from 20 °C to 80 °C at a heating rate of 1 °C/min under a nitrogen atmosphere
with 0.1% (W/V) of each nanoparticle in aqueous solution. The average hydrodynamic
diameter, size distribution and surface charge (ζ zeta potential) of aqueous dispersion of all
nanoparticles were determined by dynamic light scattering (DLS) (Nano ZS, malven
instrument, UK) at 25 °C. Morphological assessment of nanoparticles after loading
fluorescein as a hydrophobic dye was performed with a Zeiss light microscope. 10 mg of
mixed and Janus nanoparticles were dispersed in 10 mL acetone and then 1 mg of
fluorescein, as a fluorescent tracer, was added to the above suspension. After one hour, a few
drops of suspension were diluted, transferred to a lamella and allowed to dry.
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2
.4 Drug loading and encapsulation efficiency
0 mg of each nanocarrier (m-(PCL-NGO-Terpolymer) and J-(PCL-NGO-Terpolymer)) was
1
separately dispersed in a mixture of deionized water (5 mL) and DMF (5 mL). 1 mg 5-FU as
hydrophilic anti-cancer drug and 1 mg quercetin as hydrophobic one were added into the
dispersions. The mixture was incubated for 48 h to prepare drug loaded nanocarriers. Drug
loaded nanocarriers were separated from the solution by centrifuging the mixture and were
washed twice with solvent to remove unloaded drug molecules. The product was freeze-dried
for 36 h in order to obtain dried drug loaded nanocarriers without any agglomeration. Single
drug loading, i.e. 5-FU or quercetin, was carried out according to the procedure described
above and was used to yield 5-FU or quercetin loaded J-(PCL-NGO-Terpolymer)
nanoparticles. Dried solid nanoparticle samples were obtained after lyophilization.
Drug loading and encapsulation efficiencies of these nanoparticles were determined by UV-
Vis spectrophotometer. A pre-determined amount of dried nanocarriers were dissolved in
DMF/H O (1:1) to determine the amount of encapsulated drug. The DMF/H O mixture could
2
2
instantaneously dissolve both polymers and consequently, the entrapped quercetin and 5-FU
immigrate into solvents media suddenly, and the residue of nanoparticles were separated by
high speed centrifuge. The absorbance of the supernatant was determined by UV-Vis
spectrophotometer (287 nm for 5-FU and 376 nm for quercetin) in accordance with the
standard calibration curve of drug in solvent (1:1 DMF/H O). Encapsulation efficiency (EE)
2
and drug loading (DL) were calculated according to the following equations (Eqs 1 and 2):
ꢀ
ꢁꢂꢃꢄꢅꢆꢇꢈꢆꢉꢊꢋꢃꢆꢁꢌꢅꢊꢍꢎꢎꢁꢉꢆꢏꢂꢅꢄꢂꢌꢆꢌꢍꢌꢇꢎꢍꢊꢅꢂꢐꢑꢁꢒꢆ
ꢓꢇꢅꢍꢑꢆꢏꢁꢂꢃꢄꢅꢆꢇꢈꢆꢌꢍꢌꢇꢎꢍꢊꢅꢂꢐꢑꢁꢒ
DL(%) =
×100 (Eq. 1)
ꢀ
ꢁꢂꢃꢄꢅꢆꢇꢈꢆꢉꢊꢋꢃꢆꢁꢌꢅꢊꢍꢎꢎꢁꢉꢆꢏꢂꢅꢄꢂꢌꢆꢌꢍꢌꢇꢎꢍꢊꢅꢂꢐꢑꢁꢒ
ꢓꢇꢅꢍꢑꢆꢉꢊꢋꢃꢆꢍꢉꢉꢁꢉꢆ
EE% =
×100 (Eq. 2)
2
.5 In-vitro release of drugs from nanocarrier:
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In-vitro release profiles of 5-FU and quercetin from mixed and Janus nanoparticles were
performed in phosphate buffer solutions of 7.4 at 37 and 40 °C. The same amount for every
sample (1.5 mg) was dispersed in 2 mL PBS and transferred to a dialysis bag (molecular
weight cut off 12 KD). The bag was then immersed into the flask containing 60 mL PBS
(pH=7.4), and was stirred at 100 rpm at 37 and 40 °C. At determined time intervals, 2 mL of
the sample was taken out from the release media and replaced with fresh buffer (2 mL). The
amount of released quercetin and 5-FU in PBS media were determined by UV-Vis
spectroscopy, at wavelengths of 202 nm and 260 nm, respectively. The drug release
percentage can be determined according to Equation 3:
Drug release (%) = M /M × 100
Eq (3)
t
0
2
.6 cell cytotoxicity assay
The relative cell viability was evaluated in the presence of quercetin, 5-FU, quercetin/5-FU,
J-(PCL-NGO-Terpolymer), quercetin-loaded J-(PCL-NGO-Terpolymer), 5-FU-loaded J-
(PCL-NGO-Terpolymer), (quercetin/5-FU)-loaded J-(PCL-NGO-Terpolymer), m-(PCL-
NGO-Terpolymer) and mixed (quercetin/5-FU)-loaded m-(PCL-NGO-Terpolymer) rat C6
glioblastoma and OLN-93 cells (rat brain non-tumor cell line) via conventional 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The cells were seeded
3
at the density of 5×10 cells per well in 96-well plates and incubated at 37 °C in a humidified
atmosphere containing 5% CO for 24 h. After pre-incubation, cell culture was removed and
2
cells were treated with 100 µL fresh medium, containing either above-mentioned samples at
various concentrations for 24 h and at two temperatures, 37 and 40 °C. The control and
treated cells were observed and photographed using an inverted phase contrast microscope
(BEL, Italy) equipped a camera. 1 mM freshly-prepared MTT was added to each well and the
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samples were incubated for 4 hours at desired temperatures. Then, the culture media were
removed and 100 µL DMSO was added to each well. Prepared samples were incubated for 10
minutes at 37 °C. The absorption of obtained solution was measured at 540 nm.
3
. Results and discussion
poly(ɛ-caprolactone) and (N-isopropylacrylamide-co-acrylamide-co-allylamine) terpolymer
were synthesized by ring opening polymerization (ROP) and free radical polymerization. GO
was synthesized by improved Hummers method and was functionalized with alkynyl groups.
Then, alkynyl-GO was modified with PCL and terpolymer via two methods. First method
was oil-in-water microemulsion method in which dichloromethane and PBS were used as
organic and aqueous layers, respectively. In this technique, alkynyl-GO works as an
emulsifier. PCL in the oil phase was reacted with the internal sides of alkynyl-GO and the
external sides of alkynyl-GO were functionalized with terpolymer. In this reaction, “Janus”
nanoparticles were produced as a specific kind of nanoparticles. In the second method for
functionalization of alkynyl-GO, DMF was used as a sole organic solvent to disperse alkynyl-
GO and dissolve both polymers. Under this condition, two different types of polymers will be
grafted on both surfaces of graphene oxide disorderly named “mixed” nanoparticles (Figure
1
).
1
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Figure 1. Synthesis of GO, Alkynyl-GO, Mixed and Janus nanoparticles
3
.1 Preparation and characterization of hydrophobic and hydrophilic domains of
nanoparticles
The initiator, containing azide group, was initially prepared for poly(ɛ-caprolactone)
synthesis. 1-Chloro-2-hydroxy-3-azido propane (azidated epichlorohydrin) was synthesized
using epichlorohydrin and sodium azide in the presence of tetrabutylammonium bromide as a
phase-transfer catalyst (Figure 2, Step 1).
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Figure 2. Synthesis of azidated epichlorohydrin (Step 1), azide-terminated PCL (Step 2),
Terpolymer (Step 3) and Azidated Terpolymer (Step 4)
Figure 3a shows FT-IR spectrum of azidated epichlorohydrin. The intense absorption at
-1
-1
2
088 cm and a characteristic peak at 1267 cm are attributed to azide and CH -Cl groups,
2
-1
respectively. The wide absorption at 3413 cm is attributed to OH stretching bond.
1
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1
Figure 3. FT-IR spectra of azidated epichlorohydrin (a), azide-terminated PCL (b) and H-
NMR spectrum of azide-terminated PCL (c)
N -PCL was synthesized via ring opening polymerization of ɛ-caprolactone in the presence of
3
azidated epichlorohydrin as initiator and Sn(Oct) as catalyst at 120 °C (Figure 2, Step2).
2
FT-IR spectrum of azide-terminated PCL is illustrated in Figure 3b. The PCL characteristic
-1
peaks were located at 1721 and 1172 cm , related to C=O and C-O stretching bonds of
-1
esteric groups, respectively. The absorption band at 2943 cm corresponds to C-H stretching
-1
bond and that at 2103 cm belongs to the azide end groups. Presence of these peaks confirms
the formation of azide-terminated PCL chain. As well as, four major peaks related to the
repeating units of PCL (d, e, f and g), three small but discernible peaks (a, b and c) are
1
observable in the H-NMR spectrum of azide-terminated PCL in CDCl (Figure 3c) which is
3
related to azidated epichlorohydrin (AE). The methylenic proton resonance peaks of PCL
chains appear as multiple peaks at 1.3 (f), 1.6 (e), 2.3 (d) and 4 (g) ppm. The peaks related to
the protons of the used initiator can be detected at 4.1 (c), 3.7 (b) and 3.2 (a) ppm. The
number average molecular weight of azide-terminated PCL was calculated from peak area
ratio at 1.3 ppm (peak “f” of PCL) and 3.7 ppm (peak “b” of AE). According to the integral
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values of these two peaks, number average molecular weight of PCL is about 2350 g/mol.
GPC results of PCL showed that Mn and PDI were 2030 g/mol and 1.13, respectively.
Poly(N-isopropylacrylamide) and its copolymers are the most relevant thermo-responsive
polymers, because it possesses a cloud point temperature (Tcp) that makes it an appropriate
nanovector for smart delivery of the drug only at the target site. Tcp of pure PNIPAAm is
around 32 °C in aqueous solutions and to have a polymer with a Tcp value closely similar to
the cancer cell temperature, some extra monomers should be applied to increase this
temperature. Here and similar to the previously reported procedure [24], acrylamide and
allylamine were used as co-monomers for adjusting the Tcp value of PNIPAAm, and thus,
the theoretical molar ratio of NIPAAm:AAm:AA was selected to be 15:0.75:1 (sample T2).
Consequently, poly(NIPAAm-co-AAm-co-AA) was synthesized through free radical
polymerization using AIBN as initiator according to Figure 2, Step3. FT-IR spectrum of
terpolymer is shown in Figure 4a. C=O stretching vibration of amide group can be seen as a
-1
-1
peak at 1635 cm and the peaks at 1453 and 3293 cm are ascribed to N-H bending and
stretching vibration frequency, respectively. C-H stretching bond in terpolymer appeared as a
-1
-1
tiny sharp peak at 2969 cm . Peaks at 1455 and 1387 cm indicate the methylene groups of
the main polymer chain and isopropyl group of NIPAAm, respectively.
1
H-NMR spectrum of terpolymer is shown in Figure 4c. Characteristic peaks of the
backbone protons are 1.43 (c) and 1.87 (d) ppm. The broad peaks at 7.68 (g) and 7.93 (h)
ppm, respectively indicate primary and secondary amide groups in acrylamide and N-
isopropylacrylamide. Furthermore, the proton resonance peaks related to the amine groups in
allylamine appeared at 3.42 (e) and 3.50 (f) ppm. These peaks affirm the existence of all three
monomers in the terpolymer structure. The molecular weight and polydispersity index of the
terpolymer were measured by gel permeation chromatography (GPC). The results showed
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that the number average and the weight average molecular weights were 9983 and 14176
g/mol, respectively. The PDI of terpolymer was 1.42 which is a relatively modest value.
1
Figure 4. FT-IR spectrum of terpolymer (a), azidated terpolymer (b) and H-NMR spectra of
terpolymer in D O (c)
2
Terpolymer was azidated in the presence of AE and triethylamine at 50 °C (Figure 2, Step
4
). Coupling of azidated epichlorohydrin and terpolymer has been confirmed using FT-IR
(Figure 4b). Stretching vibration of azide groups attached to terpolymer can be seen at 2102
-
1
cm .
To evaluate the relationship between the Tcp values of the azidated terpolymer solutions at
two different media (water and buffer) and the monomer ratios of the corresponding
terpolymers, UV-Vis spectrophotometer coupled with a temperature controller was used. As
shown in Figure 5, variation in the investigated media exhibits a significant change in the
Tcp transition, which sensitively depends on the type and concentration of salts used in
buffer. In PBS media a sharp phase separation was observed at 35.90 °C (Figure 5), which is
lower than the desired value. As monomer content in terpolymer has direct relationship to
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Tcp and in order to increase the Tcp value, two more formulations were prepared similar to
the above polymerization method, except that the weight percentages of three monomers
were changed according to Table 1. Strikingly, the exact same trend is also observed in the
Tcp values of azidated terpolymer in water and PBS media and suggests that the solvent
must play a determining role in Tcp transitions.
Figure 5. Tcp profile of azidated terpolymer in PBS at different ratio of monomers (table 1)
Table 1. Influence of feed monomer ratio on the Tcp
a
%
mole ratio in the feed
Tcp (°C )
Sample
NIPAAm
Aam
1
0.75
1
AA
1
1
pH=7.4
35.90
37.50
41.02
H O
2
T1
T2
T3
17
15
10
39.10
43.00
46.30
1
a
The concentration of terpolymer solution always being 1%, w/v
3
.2 Preparation and characterization of alkynylated GO
The focus of this paper is to synthesize Janus nanoparticles through the reaction of each
surface of nano-graphene oxide (NGO) with two different polymers. So, [2+3] click reaction
has been selected to provide this purpose and accordingly, the alkynated graphene oxide,
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azide-terminated poly(ε-caprolactone) and azidated poly(NIPAAm-co-AAm-co-AA) were
designed. Indeed, it was found that GO can locate between aqueous and oil phases and can
act as an emulsifier to create stable submillimeter-sized oil droplets in water [25]. However,
freshly prepared GO revealed little surface-active properties in water, and GO sheets started
to appear as a colloidal surfactant just after a few hours, which is due to their large size and
high molecular mass. To overcome this drawback in this work, we designed a mass reducing
process using probe sonicator to accelerate the migration of GO sheets to the surface as well
as convert millimetric droplets to nanometric ones.
As shown in Figure 1, graphene oxide was synthesized by oxidation of graphite via
improved hummer’s method, introduced by Marcano et al. [22]. They showed that increasing
the amount of KMnO and using a mixture of H SO /H PO can improve the oxidation
4
2
4
3
4
process.
FT-IR analysis was done to characterize the nature of the GO surface (Figure 6a). The broad
-1
band at approximately 3300-3500 cm is attributed to the stretching vibration of OH group.
-1
The appearance of the absorption bands at 1725, 1626 and 1223 cm is assigned to C=O,
C=C and C-O stretching vibrations of the carboxylic, aromatic hydrocarbon, and epoxy
groups, respectively.
Alkynylated GO was then prepared after amidation of the GO carboxylic acids with
propargyl amine (Figure 1). Several new peaks appeared in the FT-IR spectrum of alkynyl-
GO. The characteristic peaks of amide (–C(O)NH–) stretching vibration appeared at 1560
−
1
cm and indicates the presence of the amide bond which was formed through the reaction
between GO and propargyl amine. Moreover, methylene (–CH –) stretching vibration can be
2
−
1
observed at 2926 and 2853 cm (Figure 6b).
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Figure 6. FT-IR of GO (a), alkynyl-GO (b), J-(PCL-NGO) (c), J-(PCL-NGO-Terpolymer)
(d) and m-(PCL-NGO-Terpolymer) (e)
3
.3 Preparation of Janus (PCL-NGO-Terpolymer) and Mixed (PCL-NGO-Terpolymer)
nanoparticles
As illustrated in Figure 7 (step 1), J-(PCL-NGO-Terpolymer) nanoparticles were prepared
via solvent evaporation of spontaneously formed oil-in-water microemulsion. In this system,
oil phase contained azide-terminated PCL, water phase contained azidated terpolymer and
graphene oxide was used as the emulsifier. Oil phase was dispersed in water through stirring
to make oil droplets. Graphene oxide acts as a surfactant and forms a monolayer at the
interface between oil and water to stabilize the emulsion. Alkyne groups in both sides of the
graphene oxide would face either oil or water phase [26]. NGO alkyne groups that face to oil
phase were activated using sodium ascorbate (sodium bicarbonate+ ascorbic acid) and copper
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(
II) sulfate pentahydrate to react with oil-solvated azide-terminated PCL. Alkyne groups
facing water phase would react with water-solvated azidated terpolymer via click reaction in
the presence of copper and ligand to form triazole ring and graft azidated poly(NIPAAm-co-
AAm-co-AA) on the graphene oxide surface.
Figure 7. Illustrated method for formation of “Janus” nanoparticle (Step 1) and “mixed”
nanoparticle (Step 2)
To confirm the conjugation of azid-terminated PCL on the surface of graphene oxide, the
reaction was stopped at the first step before adding azidated terpolymer and the sample was
evaluated using FT-IR (Figure 6c). Compared with alkynyl-graphene spectrum, the strong
-1
vibration at 1721 cm , relating to ester carbonyl stretching (C=O) as well as the appearance
of PCL pattern in the FT-IR spectrum of one-side polymer attached GO would confirm the
grafting of PCL on the surface of graphene oxide.
Janus nanoparticles (J-(PCL-NGO-Terpolymer)) have been characterized using FT-IR
(Figure 6d). Compared with J-(PCL-NGO) spectrum (Figure 6c), the peaks at 3251, 1624
-1
and 1550 cm , respectively relating to N-H stretching vibration frequency, C=O stretching
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vibration of amide group and N-H bending vibration frequency, have become stronger. The
-1
weak peak at 2101 cm ascribes to the unreacted azide groups on terpolymer chains.
Presence of peaks relating to these groups confirms the conjugation of azidated terpolymer on
-1
the surface of graphene oxide. The peak at 2173 cm for alkyne group of propargyl amine
has been disappeared, showing that the conjugation has been performed via click reaction.
A mixture of azide-terminated PCL and azidated terpolymer can be randomly grafted on the
surfaces of graphene oxide in a common solvent to make mixed nanoparticles. To perform
the click reaction, copper bromide and PMDETA were used in DMF. Both polymers were
linked on the surface of GO through their azide heads in a one-step reaction to produce mixed
nanoparticles (Figure 7, Step 2).
Compared with FT-IR spectrum of Janus nanoparticle (Figure 6d), the spectrum of mixed
nanoparticle (Figure 6e) shows all important absorptions of various functional groups present
in the structure of nanoparticle, but with different intensities. In Janus morphology, there is a
-1
distinct band at 1721 and 1624 cm (corresponding to the PCL and terpolymer carbonyl
groups), while for the mixed one there is a broad band in this region. Due to the interactions
between the functional groups of PCL and terpolymer units in the mixed structure, these
bands become wide, while these interactions are not so important in the separated structure of
Janus form. This difference between two morphologies is similar to the pattern observed in
block and random copolymers [27].
Thermogravimetric analysis is a good complementary technique which can determine the
changes in thermal stability of the Janus and mixed structure of (PCL-NGO-Terpolymer).
TGA curves of graphene oxide (black), J-(PCL-NGO-Terpolymer) (red) and m-(PCL-NGO-
Terpolymer) (blue) are shown in Figure 8A. In the pure GO sample CO, CO and vapor from
2
the most labile functional groups are released during a single weight loss event between 200
and 220 °C relating to pyrolysis of unstable oxygen-containing functional groups (Figure
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8
a). However, a small mass loss below 100 °C relates to water evaporation. The major weight
loss was started at 120 °C and at temperatures below 600 °C a total weight loss of about 67%
was observed. In contrast, both modified NGO with either Janus or mixed structures showed
higher thermal stability than pure graphene oxide. Actually, the weight loss curve of modified
NGO shows the amount of grafted polymer onto the surface of NGO after each step of
functionalization.
Figure 8. (A): TGA thermograms of GO (a), J-(PCL-NGO-Terpolymer) (b) and m-(PCL-
NGO-Terpolymer) (c); (B): XRD diffractograms of graphite (d), GO (e), J-(PCL-NGO) (f),
J-(PCL-NGO- Terpolymer) (g), m-(PCL-NGO-Terpolymer) (h)
Functionalizing NGO with azide-terminated PCL and azidated terpolymer would
impressively enhance thermal stability of NGO sheets, which is due to the role of PCL and
terpolymer as a barrier against heat. m-(PCL-NGO-Terpolymer) lost 51 % of its weight from
2
30 °C to 600 °C which is related to the decomposition of conjugated polymers, while J-
(PCL-NGO-Terpolymer) lost only 43% of its weight in this region (Figure 8b and c).
Around 360 °C, the plots for both Janus and mixed samples have notable drops, which
indicates degradation of the conjugated polymers from the surface of both nanoparticles. This
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slump is more significant for the Janus particles, which is due to the difference in particle
structure.
Usually homopolymer of polyallylamine shows three distinct regions of weight loss. The first
one, which is below 150 °C, is related to the release of bound water in the polymer matrix.
The second between 150 and 370 °C corresponds to the scission of the amine side-chains.
The last weight loss around 370 to 550 °C is attributed to the polymer chain scission [28].
Similar to polyallylamine, polyacrylamide also showed an explicit weight loss as soon as
heating was started. During this process, the adsorbed water was isolated and this event was
completed below 100 °C. Separation of carbon dioxide from pendant groups of
polyacrylamide was also detected below 200 °C and as the temperature raised more, the
degradation of the polymers began [24]. TGA measurements provide valuable information
that can be used to prove the formation of Janus nanoparticle. The overall TGA curves
indicate that thermal stability of the mixed nanoparticles is higher than Janus ones. The
proximity of polyacrylamide chains in Janus nanoparticles promotes higher polymer-polymer
interactions, which naturally results in lower chain mobility. By heating the nanoparticle, the
retained energy will be released and accordingly, lower temperature is needed for degradation
of Janus nanoparticle. TGA of mixed nanoparticles demonstrates that an additional amount of
energy is needed for further degradation which is due to the inaccessibility of reactive sites of
polymer chains to each other and consequent lower amount of hydrogen bonding.
Micro DSC heating curves of Janus and mixed nanoparticles in aqueous solution shows the
switch-ability of the polymeric shell and the temperature sensitivity of the whole
nanoparticles. As we described above, volume phase transition temperature (VPTT) of
azidated terpolymer in buffer solution was about 37.5 °C (T sample). This VPTT will be
2
changed by incorporation of hydrophobic PCL and hydrophilic NGO in nanoparticle
structure. Mixed nanoparticle shows a similar temperature-sensitive behavior in buffer media
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as Janus nanoparticles (i.e. 40 °C), but with slightly higher critical temperature of about 42
°
C, higher than Tcp of azidated terpolymer. The higher VPTT of nanoparticles in contrast to
Tcp of azidated terpolymer arises from the hydrophilic nature of graphene oxide.
Crystallographic structure of the nanoparticles series was determined by X-ray diffraction
(Figure 8B). In X-ray diffraction pattern (XRD), graphite shows a characteristic peak at 2θ =
2
6° (Figure 8d). XRD of graphene oxide showed a distinct peak at 2θ =10.28°, which is
related to the interlayer spacing of GO layers that is about 8.60 Å (Figure 8e). XRD pattern
of the as-prepared J-(PCL-NGO) showed a sharp peak at 2θ of 7.76°, indicating the interlayer
distance of 11.38 Å. The shift of XRD peak of NGO from 10.28° to 7.76° in one-side coated
NGO suggested that the polymeric NGO has been separated well. However, some new peaks
at 21.52° and 23.87° relating to PCL chains were observed in the XRD pattern of J-(PCL-
NGO) structure (Figure 8f). In the XRD patterns of Janus and mixed nanoparticles, multi-
peaks of terpolymer in several 2θ shifts appeared in addition to the PCL chains peaks. Weak
peak of GO at 5.82° revealed that the interlayer spacing was approximately 15.17 Å (Figure
8
g and h). 2θ shift and spacing increase for GO interlayer demonstrate the placement of
polymer chains between GO layers and exhibit higher extent of modification in comparison
with alkynated NGO.
The functionalized NGO morphology was investigated using scanning electron microscopy
(SEM) and transmission electron microscopy (TEM). SEM ascertained different
morphologies of nanoparticles obtained from different synthetic methods. SEM image of
graphite shows a dense morphology (Figure 9a), while, the presence of extensive peel-like
feature in the graphene oxide sample proves the oxidation of graphite (Figure 9b). The image
of as-prepared Janus nanoparticles (without washing and drying after the reaction) exhibits
clearly semi-individual particles with bright and dark contrast (Figure 9c), whereas a sheet
morphology was observed for the mixed nanoparticle. After drying the nanoparticles, the
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mixed one displayed a continuous layered morphology (Figure 9g and h), while, several
partitioned fluffy domains could be identified in the Janus nanoparticles (Figure 9d and e).
Figure 9. Scanning electron microscopy pictures of graphite (a), graphene oxide (b), Janus
nanoparticles before drying (c), Janus nanoparticles after drying with two magnifications (d
and e) mixed nanoparticles before drying (f), mixed nanoparticles after drying with two
magnifications (g and h)
We believe this behavior in Janus nanoparticle is based on two important features: (i) the
hydrophobic interaction between the PCL chains, which results in the self-assembly of
nanoparticles to form micrometer-sized core, and (ii) the hydrophilic chains on the periphery
of the macrocluster, which are designed from the interaction between azidated terpolymer
faces of several Janus nanoparticles to optimize the repulsive interactions. In this case, the
implementation of two different hydroaffinities in two dissimilar sides of the nanoparticle
2
8