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Photothermally Controlled Methotrexate Release System Using β-

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 nanomaterials

Article
Photothermally Controlled Methotrexate Release
System Using β-Cyclodextrin and Gold Nanoparticles
Nataly Silva 1,2,3 , Ana Riveros 3,4 , Nicolás Yutronic 1 , Erika Lang 5 , Boris Chornik 6 ,
Simón Guerrero 3,4 , Josep Samitier 7,8,9 , Paul Jara 1, * and Marcelo J. Kogan 3,4, *
1 Department of Chemistry, University of Chile, Las Palmeras 3425, 7800003 Santiago, Chile;
n.silvag@ug.uchile.cl (N.S.); nyutroni@uchile.cl (N.Y.)
2 Department of Chemistry of Materials, University of Santiago de Chile, Av. Libertador Bernardo O’Higgins
3363, 9170022 Santiago, Chile
3 Department of Pharmacological and Toxicological Chemistry, University of Chile, Sergio Livingston 1007,
8380492 Santiago, Chile; ariveros@postqyf.uchile.cl (A.R.); simon.daiblogt@gmail.com (S.G.)
4 Advanced Center for Chronic Diseases (ACCDiS), University of Chile, Sergio Livingstone 1007,
8380000 Independencia Santiago, Chile
5 Department of Biology, CEM, University of Chile, Las Palmeras 3425, 7800003 Santiago, Chile;
Elang@uchile.cl
6 Department of Physics, University of Chile, Beauchef 850, 8370448 Santiago, Chile; bchornik@ing.uchile.cl
7 Nanobioengineering Laboratory, Institute for Bioengineering of Catalonia (IBEC), Barcelona Institute of
Science and Technology (BIST) Baldiri Reixac, 10–12, 08028 Barcelona, Spain; jsamitier@ibecbarcelona.eu
8 Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN),
Monforte de Lemos 3–5, Pabellón 11, 28029 Madrid, Spain
9 Department of Electronics and Biomedical Engineering, University of Barcelona (UB), Martí I Franques, 1,
08028 Barcelona, Spain
* Correspondence: pjara@uchile.cl (P.J.); mkogan@ciq.uchile.cl (M.J.K.); Tel.: +56-22-978-7396 (P.J.);
+56-22-978-2897 (M.J.K.); Fax: +56-22-271-3888 (P.J.); +56-22-978-2890 (M.J.K.)




Received: 2 October 2018; Accepted: 22 November 2018; Published: 28 November 2018


Abstract: The inclusion compound (IC) of cyclodextrin (CD) containing the antitumor drug Methotrexate
(MTX) as a guest molecule was obtained to increase the solubility of MTX and decrease its inherent
toxic effects in nonspecific cells. The IC was conjugated with gold nanoparticles (AuNPs), obtained by
a chemical method, creating a ternary intelligent delivery system for MTX molecules, based on the
plasmonic properties of the AuNPs. Irradiation of the ternary system, with a laser wavelength tunable
with the corresponding surface plasmon of AuNPs, causes local energy dissipation, producing the
controlled release of the guest from CD cavities. Finally, cell viability was evaluated using MTS assays
for β-CD/MTX and AuNPs + β-CD/MTX samples, with and without irradiation, against HeLa tumor
cells. The irradiated sample of the ternary system AuNPs + β-CD/MTX produced a diminution in
cell viability attributed to the photothermal release of MTX.

Keywords: inclusion compound; cyclodextrin; Methotrexate; delivery system; gold nanoparticles;

photothermal release; laser; irradiation

1. Introduction
Studies on the usage of nanoparticles (NPs) in the field of medicine have increased considerably
in recent years mainly because NPs can act as vectors for the diagnosis and treatment of several
diseases. These studies highlight the role of gold nanoparticles (AuNPs) in drug delivery, targeting
and imaging applications as alternative therapies for cancer. AuNPs improve drug delivery to tumors
through passive and active vectorization [1,2]. Passive vectorization involves the accumulation of

Nanomaterials 2018, 8, 985; doi:10.3390/nano8120985 www.mdpi.com/journal/nanomaterials

Nanomaterials 2018, 8, 985 2 of 15

NPs in tumor tissue due to their enhanced permeability and retention through the pores of the blood
vessels, whereas active vectorization involves conjugation to the NPs of molecules that are capable
of specifically recognizing the therapeutic target. A nanocarrier based on NPs, which combines both
the vectorization mechanisms, is considered to be an ideal carrier. Furthermore, the optical properties
of AuNPs allow external activation with an appropriate electromagnetic source [3–5]. This process is
advantageous to perform cancer treatment using the following two possible mechanisms: (a) local
hyperthermic effects of AuNPs and (b) release of a potential antitumor drug conjugated to AuNPs,
via irradiation. Therefore, through the use of AuNPs, it is possible to improve the traditional cancer
treatments that exhibit severe limitations mainly related to the low specificity of the chemotherapeutic
agents to the tumor cells. AuNPs are useful as a drug delivery system that allows to spatially
and temporally control the release of drugs in the tumor to avoid unspecific effects. AuNPs can be
conjugated to antitumor drugs and after external localized stimuli with a laser, it is possible to trigger
the release of the drugs in the affected tissue due to the local photothermal effect [6–9].
Methotrexate (MTX) is formed from a pteridine ring, a p-aminobenzoic acid residue, and a
glutamic acid residue. Substitution of 4-hydroxyl with an amino group creates a folic acid analog,
which is an antimetabolite. MTX competitively inhibits the enzyme of dihydrofolate reductase (DHFR)
and impedes the formation of tetrahydrofolate, which is required to synthesize essential purines and
pyrimidines, which are involved in both DNA and RNA synthesis. Therefore, MTX is considered to be
an antifolate, which can be recognized by its pteridine ring by folate receptors that are overexpressed
on the surfaces of various cells [10]. MTX was considered to be an effective antineoplastic to treat cell
proliferation disorders such as lymphocytic leukemia; breast carcinoma; tongue, pharynx, bladder,
and brain tumors; and skin neoplasms [10–12]. However, it also exhibits several undesirable side effects
such as stomach irritation, nausea, and dizziness caused by the administration of high dosages of MTX,
due to the tumor cells possibly being drug resistant, increasing the risk of toxicity [13]. Such effects
are related to the poor selectivity in MTX distribution. Therefore, developing intelligent systems for
drug delivery is essential. One strategy is to encapsulate the MTX within a matrix, such as CD, to form
IC with the objective to increase the drug solubility and avoid interaction with the folate receptors in
non-tumor cells [14–16]. Ensuring the release of MTX in the tumor will allow for a considerably high
selectivity of action. Another strategy is to conjugate the MTX on the surface of AuNPs to obtain a
nanotransporter that can be actively and passively vectorized [17–21].
In previous studies, we obtained ternary systems that comprised 1:2 α-cyclodextrin-octylamine
(α-CD-OA) [22,23] and 1:1 β-cyclodextrin–phenylethylamine (β-CD-PhEA) [24]. The ICs were
conjugated to AuNPs that were obtained by chemical or physical methods, respectively. We reported
the effects of green-laser irradiation of AuNPs on the IC structure and demonstrated that the
heat generated in the irradiated nanostructure was transferred to the supramolecular structure,
which caused the disassembly of the AuNP-IC system that further caused the guest release from
the CD cavity by topotactic transition.
In this study, we obtained and characterized a ternary system formed from MTX, β-CD,
and AuNPs (Figure 1) that were obtained by Turkevich synthesis. We irradiated the complex and
evaluated, for the first time, the biological activity in the HeLa cells; thus, we observed that the effects
of MTX on cell viability are dependent on the irradiation of the ternary complex.
Nanomaterials 2018, 8, 985 3 of 15
Nanomaterials 2018, 8, x FOR PEER REVIEW 3 of 15

Figure1.1.Schematic
Figure Schematicrepresentation
representationof ofthe
theprecursors
precursorsMTX
MTXand β-CDto
andβ-CD toform
formthe
theinclusion
inclusioncompound
compound
(IC)
(IC) and subsequently of the ternary system, where the IC was conjugated to the AuNP. Laserlight
and subsequently of the ternary system, where the IC was conjugated to the AuNP. Laser lightisis
absorbed by the AuNP and is transformed into local heat, which is dissipated into the
absorbed by the AuNP and is transformed into local heat, which is dissipated into the environment environment
and
and induces
induces the
the release
release of
ofMTX
MTX from
fromthe
theCD
CDcavity.
cavity. Further,
Further,the
thefree
freedrug
druginteracts
interactswith
withthe
thefolate
folate
receptors in the HeLa cells.
receptors in the HeLa cells.
2. Experimental
2. Experimental
2.1. Purification of MTX
2.1. Purification of MTX
A lyophilized vial of MTX (500 mg) with mannitol and NaOH was donated by the Kampar
A lyophilized
Oncology Laboratory vial of MTX Chile).
(Santiago, (500 mg) with mannitol
Therefore, it was and NaOHtowas
necessary donated
separate the by thefrom
drug Kamparthe
Oncology Laboratory (Santiago, Chile). Therefore, it was necessary
excipients before usage. The lyophilized compounds were dissolved in nanopure water (Merck,to separate the drug from the
excipients before
Darmstadt, Germany) usage.(20 The
mL).lyophilized
The solution compounds
was furtherwere dissolved
acidified usingin 0.1nanopure water (Merck,
M HCl (Sigma-Aldrich,
Darmstadt, Germany) (20 mL). The solution was further acidified using 0.1 M HCl
St. Louis, MO, USA) to precipitate the drug because it was insoluble at a pH value of 4. The solution was (Sigma-Aldrich,
St. Louis,
further MO, USA)
centrifuged at to precipitate
1485 ×g and was the washed
drug because
thrice itusing
was water,
insoluble at a pH
ensuring thevalue of 4. The
complete solution
elimination
was
of further Proton
mannitol. centrifuged
nuclear atmagnetic
1485×g and was washed
resonance 1
( H-NMR) thrice
wasusing
used to water,
verifyensuring the complete
the elimination of the
elimination of mannitol. Proton nuclear magnetic resonance ( 1H-NMR) was used to verify
excipients from the sample and to characterize the structure of MTX (characterization by H-NMR, 1 the
elimination of the excipients from the sample and to characterize
see Figure S1 and Table S1). No signals corresponding to mannitol [25] were observed after the the structure of MTX
(characterization
MTX purification.by H-NMR, see Figure S1 and Table S1). No signals corresponding to mannitol [25]
1

were observed after the MTX purification.

2.2. Preparation and Characterization of the β-CD/MTX Inclusion Compound
2.2. Preparation and Characterization of the β-CD/MTX Inclusion Compound
Equimolar amounts of MTX crystals and β-CD (Sigma-Aldrich, Saint Louis, MO, USA) (1 g of
β-CDEquimolar
and 0.4 g of amounts
MTX) were of MTX crystals
mixed in 10 and
mL β-CD
water,(Sigma-Aldrich,
with continuousSaint and Louis,
vigorousMO, USA) (1
stirring, at groom
of β-
CD and 0.4 gfor
temperature of48
MTX)
h. The were mixedwas
solution in 10 mL
left to water, withfor
crystallize continuous
7 days. The and vigorous
obtained stirring,
solid at room
was washed
temperature
using water and for 48 h. The
dried at 60 ◦ C (yields:
solution was left to crystallize
β-CD/MTX for 7 days.
64.25%). The The obtained
inclusion solid was(IC)
compound washed
was
using water and
characterized usingdried
NMRatspectroscopy.
60 °C (yields:All β-CD/MTX 64.25%).
the spectra were The inclusion
recorded at 400 MHz compound (IC) was
and a temperature
of 27 ◦ C on a Bruker
characterized using NMR
Avance spectroscopy. All the spectra were
400 MHz superconducting NMRrecorded
spectrometerat 400inMHz and asulfoxide-d
dimethyl temperature 6
of 27 °C on
(DMSO-d 6 ) a(Sigma-Aldrich,
Bruker Avance 400 SaintMHz superconducting
Louis, MO, USA). To NMR spectrometer
conduct in dimethyl
the 2D-ROESY sulfoxide-d6
(Rotating-frame
(DMSO-d6) (Sigma-Aldrich,
Overhauser Spectroscopy) NMR, Saint the
Louis, MO, USA).
WG-ROESY To conduct the 2D-ROESY
(Watergate-ROESY) pulse sequence (Rotating-frame
was used,
Overhauser
and 2D-ROESY Spectroscopy)
measurements NMR,
werethe WG-ROESY
performed under(Watergate-ROESY) pulse sequence
the following experimental was used,
conditions: and
64 scans;
2D-ROESY measurements were performed under the following
an acquisition time of 0.150 s; a pulse delay of 8 s; and 1024 data points. experimental conditions: 64 scans;
an acquisition time of 0.150 s; a pulse delay of 8 s; and 1024 data points.
2.3. Synthesis and Characterization of Colloidal AuNPs and Their Conjugation with the β-CD/MTX
2.3. Synthesis and Characterization
The following of Colloidal
chemical reagents wereAuNPs and Theiravailable
commercially Conjugation with
and the β-CD/MTX
were used as received:
tetrachloroauric (III) chemical
The following acid (Sigma-Aldrich,
reagents wereSaint Louis, MO, available
commercially USA); sodium citrateused
and were (Sigma-Aldrich,
as received:
Saint Louis, MO,(III)
tetrachloroauric USA);acidsodium hydroxideSaint
(Sigma-Aldrich, (Sigma-Aldrich, Saint Louis,
Louis, MO, USA); sodiumMO, USA);
citrate and sodium
(Sigma-Aldrich,
phosphate buffer
Saint Louis, MO,(PBS)
USA);(Sigma-Aldrich, Saint Louis,
sodium hydroxide MO, USA).Saint Louis, MO, USA); and sodium
(Sigma-Aldrich,
The AuNPs
phosphate bufferwere
(PBS)synthesized in accordance
(Sigma-Aldrich, withMO,
Saint Louis, the Turkevich
USA). method [26]. In a round-bottom
flask equipped with a condenser, 1.0 mM aqueous HAuCl (100 mL)
The AuNPs were synthesized in accordance with the4Turkevich method was brought
[26].to
Inrolling boil with
a round-bottom
flask equipped with a condenser, 1.0 mM aqueous HAuCl4 (100 mL) was brought to rolling boil with
vigorous stirring; further, 38.8 mM aqueous Na3C6H5O7 (10 mL) was rapidly added to the solution
Nanomaterials 2018, 8, 985 4 of 15

vigorous stirring; further, 38.8 mM aqueous Na3 C6 H5 O7 (10 mL) was rapidly added to the solution
while stirring continuously. The solution was heated for an additional period of 30 min and was then
maintained at room temperature. It was further filtered through a 0.45-µm cellulose acetate membrane
filter. The β-CD/MTX IC was conjugated to the AuNPs using the same protocol as that used for the
conjugation of MTX to AuNPs [17]. The AuNPs suspension was eight times concentrated in amicon
ultra centrifugal filters (500 µL) and 200 mM IC (5 µL) was added to 10 mM PBS (500 µL). The solution
was stirred for 24 h at room temperature (Schematic representation of the methodologic protocol,
Figure S2).
Further, the solution was centrifuged at 13,362× g for 20 min, and the precipitate was separated
from the supernatant and the pellet was resuspended in 10 mM PBS (1 mL).
The AuNPs and the conjugated AuNPs + MTX and AuNPs + β-CD/MTX were characterized
by UV-Vis spectrophotometry. This confirmed the conjugation of both MTX and IC on the surface of
AuNPs by displacing the characteristic surface plasmon band of the AuNPs. A Shimadzu UV-3101PC
spectrophotometer (Kyoto, Japan) was used. The spectra were recorded between 280 and 800 nm.
The morphologies and sizes of the AuNPs and conjugates were determined by transmission
electron microscopy (TEM). TEM was performed using a JEOL JEM-1010 transmission electron
microscope (120 keV, Peabody, MA, USA). The sample was dropped on a copper mesh that was
coated with carbon and Formvar.
The size was also determined by dynamic light scattering (DLS). The DLS spectra were obtained
in triplicate on Malvern Instruments Zetasizer Nano S90 (Worcestershire, England, UK) using 1-cm
plastic cuvettes.
X-ray photoelectron spectroscopy (XPS) was used to determine the energy that was associated
with the interactions between the IC and AuNPs. The XPS spectra were recorded on a Perkin Elmer
1257 photoelectron spectrometer (Eden Prairie, MN, USA) fitted with an ultra-high vacuum main
chamber (Eden Prairie, MN, USA), a hemispherical electron analyzer (Eden Prairie, MN, USA), and an
X-ray source providing unfiltered Al Kα radiation. Energy calibration was performed by assigning a
binding energy of 284.8 eV to the C-C component at the 1 s peak.
Electrophoresis was used to qualitatively determine the charges on the compounds by comparing
the mobilities of various compounds in an electric field. Twenty-five-fold concentrated samples
(AuNPs, AuNPs + MTX, and AuNPs + IC, 20 µL) were further mixed with 10 µL of the loading buffer
(70% glycerol with 30% 1 × Tris-acetate–EDTA (TAE)). The samples were subjected to 1% agarose gel
electrophoresis at 130 V for 10 min in 1 × TAE.
The AuNPs + β-CD/MTX sample was characterized by thermal analysis with the aim to determine
the stability and thermal events, and to provide information about the physical properties of the
samples. Thermogravimetric analysis (TGA) was performed using a thermobalance (TGA-SDTA
851e/SF/1100, Mettler Toledo, Greifensee, Switzerland). The sample was placed in an aluminum
capsule. Nitrogen was used as the purge gas, and the temperature range was 25–200 ◦ C, with a heating
rate of 10 ◦ C min−1 . Differential scanning calorimetry (DSC) was performed using a DSC-822e/400
instrument, with an aluminum capsule as the holder and reference. Nitrogen was used as the purge
gas, and the temperature was cycled between 25 and 200 ◦ C, with a heating rate of 10 ◦ C min−1 .

2.4. Evaluation of the Effect of MTX and Its Derivatives on Cell Viability

2.4.1. Cell Culture and Cells

The HeLa cell line (Sigma, St. Louis, MO, USA) was cultured in low-glucose Dulbecco’s Modified
Eagle’s medium (DMEM), supplemented with 5% fetal bovine serum (FBS) and 100 U/mL penicillin;
100 µg/mL streptomycin was obtained from Invitrogen. The cell line was maintained in a 95% air and
5% CO2 atmosphere at 37 ◦ C.
Nanomaterials 2018, 8, 985 5 of 15

2.4.2. Cell Viability Determination by MTS Assay

HeLa cells were exposed to experimental conditions in low-glucose DMEM supplemented with
5% FBS. Cells were exposed to MTX and β-CD/MTX for 1 h, then the culture medium was removed
and cells were washed three times with phosphate-buffered saline (PBS). The cells were maintained
in a 95% air, 5% CO2 atmosphere at 37 ◦ C in the cell-culture medium for 72 h and their viability was
determined using MTS assay (Promega, Madison, WI, USA). The MTX was dissolved with DMSO at a
final concentration of 1% v/v. The untreated control cells were evaluated with and without 1% DMSO,
to confirm that it did not affect HeLa cell viability. MTS, a tetrazolium compound, is bioreduced by
cells to the formazan product. The absorbance of the formazan was determined with a colorimetric
end-point kit according to the manufacturer’s instructions. A cell calibration curve was produced to
ensure linearity during the study duration. The background was subtracted and data were expressed as
percentages of surviving cells (mean ± SEM) relative to the control (cellular medium without DMSO).

2.4.3. Effect of the Irradiated AuNPs + β-CD/MTX on Cell Viability

First, the AuNPs + β-CD/MTX system was irradiated with a 532-nm and 45-mW continuous
laser for 15 min under sterile conditions. The HeLa cells were further incubated with an irradiated
and nonirradiated (control) sample for 1 h to a final concentration of 0.1 mM, and cell viability was
determined 71 h later (in triplicate) by MTS assay according to the manufacturer’s instructions.

2.4.4. Data Analysis

All the results are presented as the mean and SEM, obtained from three or more independent
experiments. Statistical analysis of the data was performed using GraphPad Prism 5 Software,
Inc., (San Diego, CA, USA) ANOVA that was followed by the Bonferroni or Dunn’s post hoc tests,
which were considered to be significant when p was less than 0.05.

3. Results and Discussion

3.1. Characterization of β-CD/MTX

The 2D-ROESY NMR method was used to confirm the IC formation and to determine the
intermolecular interactions of the guest (MTX) with the matrix (β-CD) during inclusion. The segmented
2D-ROESY spectrum, depicted in Figure 2 and Figure S3 (Supplementary Materials), illustrated the
aforementioned interactions. The interactions of the external -OH2 and -OH3 protons of β-CD with the
HL of the drug were observed; additionally, the strong interaction between the H3 and H5 protons of
the internal cavity of the matrix with HD , the amino proton of the pteridine ring of the MTX, and the
weaker interaction of H3 with HE , another proton of the amino group near HD , that also belonged to
MTX, were observed.
The interaction of the HG proton of MTX with the group of external protons of the secondary
hydroxyl group, OH6 of CD, was also observed. These protons are located in the narrowest rim of the
cone, indicating that another functional amino group of the pteridine ring of MTX undergoes a dipolar
interaction with the secondary hydroxyl of the matrix. These results indicate that the pteridine ring of
MTX is located in the cavity of β-CD.
The absence of interactions between β-CD and the remainder of the MTX molecules confirms that
p-aminobenzoic acid and glutamic acid waste were eliminated from the β-CD cavity. Therefore, it can
be unequivocally confirmed that the IC presents a 1:1 matrix:guest stoichiometry.
The information obtained using the NMR spectroscopy is consistent with that obtained from
a previous study that investigated the molecular modeling of the inclusion of MTX in the β-CD
cavity [14].
Nanomaterials 2018, 8, 985 6 of 15

3.2. Obtaining and Characterizing the Ternary System AuNPs+β-CD/MTX

Figure 3 depicts the UV-Vis spectra of AuNPs that are stabilized with citrate, MTX, and β-CD/MTX
IC. An absorption band at 520 nm corresponds to the characteristic surface plasmon band of spherical
AuNPs with
Nanomaterials a diameter
2018, 8, x FOR PEER of 12REVIEW
nm, stabilized with citrate ions [27]. When the citrate ions are exchanged
6 of 15
with MTX or β-CD/MTX IC, a bathochromic shift of the plasmon band was observed from 520 to
from 520due
525 nm to 525 nmenvironmental
to the due to the environmental changes
changes around the around the gold nanoparticle.
gold nanoparticle. Furthermore,Furthermore,
two bands
Nanomaterials 2018, 8, x FOR PEER REVIEW 6 of 15
two bands were observed at 311 and 370 nm that corresponded with the
were observed at 311 and 370 nm that corresponded with the MTX absorption bands, which MTX absorption bands,
were
which were
conjugated conjugated
to the AuNPs to the
[21].AuNPs [21].
from 520 to 525 nm due to the environmental changes around the gold nanoparticle. Furthermore,
two bands were observed at 311 and 370 nm that corresponded with the MTX absorption bands,
which were conjugated to the AuNPs [21].

Figure 2. Segmented 2D-ROESY spectrum of β-CD/MTX in DMSO-d6 solvent and the schematic
Figure 2.
Figure Segmented 2D-ROESY
2. Segmented 2D-ROESY spectrum
spectrum of
of β-CD/MTX
β-CD/MTXininDMSO-d
DMSO-d6 solvent
solventand
and the
the schematic
schematic
representation and proton assignment with numbers and letters for β-CD 6and MTX, respectively.
representationand
representation andproton
proton assignment
assignment with
with numbers
numbers and
and letters
letters for
for β-CD and MTX,
β-CD and MTX, respectively.
respectively.

1.6 AuNPs
1.6 AuNPs + MTX
AuNPs
1.4 AuNPs + β -CD/MTX
AuNPs + MTX
MTX
1.4 1.2 AuNPs + β -CD/MTX
β-CD/MTX
MTX
Absorbance (a.u.)

1.2 1.0 520 β-CD/MTX

Absorbance (a.u.)

1.0 0.8 520

0.8 0.6 525

0.4
0.6 525
0.2
0.4
0.0
0.2
200 300 400 500 600 700 800
0.0 λ (nm)

3. UV-Vis
FigureFigure 200
absorption 600 300
700(IC) and
spectra
of of MTX, inclusion compound 400
MTX, 500
800 AuNPs stabilized with
inclusion
3. UV-Vis absorption spectra compound (IC) and AuNPs stabilized with
citratecitrate
ions, ions,
MTXMTX
molecules, andand
molecules, β-CD/MTXIC.
β-CD/MTX IC. λ (nm)

FigureTEM3. UV-Vis absorption

provides spectra
information of MTX,
about inclusion compound
the morphologies and sizes (IC) and
of the AuNPs
AuNPs stabilized
and with
can, therefore,
citrate ions, MTX
statistically molecules,
determine the and
sizeβ-CD/MTX IC.
of their population in different areas. Figure 4A depicts a TEM
micrograph and histogram of AuNPs. A homogeneous distribution is observed with respect to both
TEM provides information about the morphologies and sizes of the AuNPs and can, therefore,
statistically determine the size of their population in different areas. Figure 4A depicts a TEM
Nanomaterials 2018, 8, 985 7 of 15

TEM provides information about the morphologies and sizes of the AuNPs and can, therefore,
statistically determine
Nanomaterials 2018, the REVIEW
8, x FOR PEER size of their population in different areas. Figure 4A depicts a 7TEM of 15
micrograph and histogram of AuNPs. A homogeneous distribution is observed with respect to both the
shape
the and and
shape size. size.
The histogram
The histogram that isthat
obtained from afrom
is obtained population of 100 particles
a population depicts an
of 100 particles average
depicts an
diameter of 12 ± 2 nm, which is consistent with that exhibited by the 520
average diameter of 12 ± 2 nm, which is consistent with that exhibited by the 520 nm plasmon. nm plasmon.
indicatedaahydrodynamic
DLS indicated hydrodynamicdiameter diameterofof 1919
nmnm withwith a polydispersity
a polydispersity indexindex
(PDI)(PDI) of The
of 0.2. 0.2.
The
valuevalue obtained
obtained by technique
by this this technique was different
was different fromfrom
thosethose obtained
obtained by TEM,
by TEM, which which is because
is because DLS
DLS provides
provides information
information aboutabout
the the
sizessizes of the
of the particles
particles that
that areare presentininthe
present thedispersion
dispersion with
with their
corresponding hydration
corresponding hydration sphere,
sphere, i.e.,
i.e., the metal particle and its surroundings appear as citrate and
associated ions.
Figure 4B4Band
Figures and Figure S4 depict
S4 depict a TEMa TEM micrograph
micrograph of of
thethe AuNPsthat
AuNPs thatare
arecoated
coated with
with β-CD/MTX.
β-CD/MTX.
Homogeneously distributed
Homogeneously distributedspherical
sphericalparticles
particles ± 312
of 12 of nm±diameter
3 nm were observed.
diameter wereThe modification
observed. The
of the surface of the AuNPs with the IC maintains the shape and size unaltered.
modification of the surface of the AuNPs with the IC maintains the shape and size unaltered. TEM TEM micrograph
shows an organic
micrograph showsstained layer stained
an organic present layer
around several
present gold spheres,
around several which could correspond
gold spheres, which could to
β-CD/MTX.toThe
correspond histogramThe
β-CD/MTX. exhibits an average
histogram diameter
exhibits of 12 ±
an average 3 nm forofa 12
diameter population
± 3 nm for ofa100 particles.
population
The hydrodynamic
of 100 particles. Thediameter obtaineddiameter
hydrodynamic using DLS was 17.2
obtained nm (PDI
using DLS was0.2), which
17.2 nm is (PDI
consistent with the
0.2), which is
diameter obtained
consistent with theby TEM. The
diameter hydrodynamic
obtained by TEM. diameter is reduced diameter
The hydrodynamic because the water molecules
is reduced because theare
reorganized
water due to
molecules arethereorganized
presence of duethe IC. It was
to the reported
presence of that CD It
the IC. molecules exhibitthat
was reported a high
CD capacity
molecules to
bind water
exhibit molecules
a high capacityvia tohydrogen
bind water bonds to the hydroxyl
molecules groups
via hydrogen at thetoedges
bonds of their structures
the hydroxyl groups at[23],the
decreasing
edges thestructures
of their hydration[23], sphere around the
decreasing the nanoparticle.
hydration sphere around the nanoparticle.

Figure 4. TEM micrographs and size histograms

histograms of
of AuNPs
AuNPs stabilized
stabilized with
withcitrate
citrate(A)
(A)and
andβ-CD/MTX
β-CD/MTX
(B). The red arrows indicate the presence of an organic stained layer present around several gold
spheres, which could correspond to the IC.

Qualitative analysis of the surface was performed (see Figure S5). The general XPS spectra
between 0 and 1200 eV are observed to exhibit C, O, and N 1 s peaks. Peaks of 4p, 4d, and 4f Au
orbitals are also observed. Further, we record the high-resolution spectra of the oxygen 1 s region in
MTX without and after conjugation with the nanoparticles.
Figure 5A depicts the high-resolution spectra of oxygen 1s in the β-CD/MTX IC. Two bands were
obtained by curve fitting at 531.43 and 532.50 eV. The MTX molecule contains two carboxylic acid
Nanomaterials 2018, 8, 985 8 of 15

Qualitative analysis of the surface was performed (see Figure S5). The general XPS spectra between
0 and 1200 eV are observed to exhibit C, O, and N 1 s peaks. Peaks of 4p, 4d, and 4f Au orbitals are
also observed. Further, we record the high-resolution spectra of the oxygen 1 s region in MTX without
and after conjugation with the nanoparticles.
Figure 5A depicts the high-resolution spectra of oxygen 1s in the β-CD/MTX IC. Two bands were
obtained by curve fitting at 531.43 and 532.50 eV. The MTX molecule contains two carboxylic acid
groups with pKa values of 4.8 and 5.5. Both acids are deprotonated under working conditions at a pH
value of 7.4. The band having a high intensity at 532.61 eV corresponds to the oxygen atoms of the
carboxylate groups (–COO− ). The band at 531.43 eV corresponds to the oxygen of the
Nanomaterials 2018, 8, x FOR PEER REVIEW
carbonyl group
8 of 15
(>C=O) [17,28].
groups with pKa values of 4.8 and 5.5. Both acids are deprotonated under working conditions at a
Figure 5B depicts
pH valueaofhigh-resolution
7.4. The band havingspectrum of the
a high intensity IC that
at 532.61 is conjugated
eV corresponds to theatoms
to the oxygen AuNPs. of Two bands
are observed at the
532.15 andgroups
carboxylate 532.77 eV.−).The
(–COO band
The band at at 532.15
531.43 eV corresponds
eV corresponds to the oxygento of the oxygen atom of the
the carbonyl
group (>C=O) [17,28].
carboxylate groups that interact with the AuNPs. The decrease in the binding energy of the 1s orbital
Figure 5B depicts a high-resolution spectrum of the IC that is conjugated to the AuNPs. Two
electron from 532.61
bands toare 532.15
observed eV is due
at 532.15 and to theeV.
532.77 increased
The band atshielding by AuNPs,
532.15 eV corresponds to thewhich leads to increased
oxygen atom
electron densityofin
the the
carboxylate
oxygen. groupsFinally,
that interact with
the the AuNPs.
band The decrease
at 532.77 in the binding energy
eV corresponds to ofthe
the carbonyl
1s group.
orbital electron from 532.61 to 532.15 eV is due to the increased shielding by AuNPs, which leads to
According to the studies
increased thatdensity
electron are related to the
in the oxygen. interaction
Finally, the band at of carboxylic
532.77 acids
eV corresponds with
to the AuNPs, both the
carbonyl
group. of
carboxylate groups According
MTXtoare the studies
assumed that aretorelated
presentto the interaction of carboxylic acids
weak interactions onwiththeAuNPs,
AuNP surfaces as
both the carboxylate groups of MTX are assumed to present weak interactions on the AuNP surfaces
compared with asthe strength
compared withof thethe covalent
strength interactions
of the covalent interactions[29].
[29].

Figure 5. High-resolution XPS spectra

Figure 5. High-resolution of the
XPS spectra OO11 ss region
of the region of MTX
of MTX in β-CD/MTX
in β-CD/MTX (A) and AuNPs(A)
+ β- and AuNPs +
CD/MTX (B). The measured spectrum (black line) and the fitting curves (blue light and blue dark
β-CD/MTX (B).lines)
Thearemeasured spectrum
also depicted.
(black line) and the fitting curves (blue light and blue dark
lines) are also depicted.
The mobility of supramolecular species was evaluated in the presence of an electric field using
agarose gel electrophoresis. The migration of species was determined by their charge and particle
The mobility of supramolecular species was evaluated in the presence of an electric field using
size. Figure 6 depicts the agarose gel image and the respective migration of AuNPs + β-CD/MTX IC,
agarose gel electrophoresis.
AuNPs + MTX, andThe AuNPs migration of that
species. AuNPs species was
are coated determined
using by their
β-CD/MTX (Sample charge
A) exhibit less and particle
size. Figure 6 depicts the agarose gel image and the respective migration of AuNPs + β-CD/MTX
IC, AuNPs + MTX, and AuNPs species. AuNPs that are coated using β-CD/MTX (Sample A) exhibit
less electrophoretic mobility to the anode compared to AuNPs that are coated using MTX (Sample B)
Nanomaterials 2018, 8, 985 9 of 15

(Figure 6).Nanomaterials
This is probably
2018, 8, x FORcaused by the two negative charges on the MTX carboxylate
PEER REVIEW 9 ofions
15 that are
partially masked by CD.
electrophoretic mobility to the anode compared to AuNPs that are coated using MTX (Sample B)
In the C lane, where the AuNPs were charged, a bluish coloration characteristic of colloid
(Figure 6). This is probably caused by the two negative charges on the MTX carboxylate ions that are
aggregation was masked
partially observed. by CD.This is probably because the citrate ion fails to stabilize them under the
observed workingIn the conditions
C lane, where(pH the 9–10
AuNPsdue weretocharged,
the TAE buffer).
a bluish This instability
coloration characteristicofofAuNPs
colloid in buffer
aggregation was observed. This is probably because the citrate ion fails
prevents migration, which is contrary to the expectations based on the size of the AuNPs and to stabilize them under the the triple
observed working conditions (pH 9–10 due to the TAE buffer). This instability of AuNPs in buffer
negative charge afforded by the citrate ion.
prevents migration, which is contrary to the expectations based on the size of the AuNPs and the
The aforementioned
triple negative charge results confirm
afforded by the that both
citrate ion. samples (AuNPs + MTX and AuNPs + β-CD/MTX)
exhibit a negative charge that results
The aforementioned induces their
confirm migration
that both samplestoward
(AuNPsthe + MTXanode. Furthermore,
and AuNPs + β-CD/MTX) the AuNPs
exhibit a negative
exhibited stability charge that
and integrity induces
while their
being migration toward
conjugated to thetheICanode. Furthermore,
and while being the AuNPs
coated by the MTX
exhibited stability and integrity while being conjugated to the IC and while being coated by the MTX
molecules under these working conditions.
molecules under these working conditions.

Figure 6. Electropherograms
Figure 6. Electropherograms of AuNPs
of AuNPs + β-CD/MTX (A),
+ β-CD/MTX (A),AuNPs
AuNPs+ MTX (B), and
+ MTX AuNPs
(B), and (C) on 1.0%
AuNPs (C) on 1.0%
agarose gel.
agarose gel.
TGA and DSC were performed to determine the thermal stability and to elucidate the thermal
TGA transitions
and DSCofwere performed
precursors of the IC to
anddetermine the thermal
the ternary system. Figure 7stability
depicts theand
DSCto elucidate
analysis of MTX,the thermal
transitionsandof three
precursors
endothermicof the IC and
peaks have the
beenternary
observed. system.
The firstFigure 7 depicts
broad peak, between the65.3
DSCandanalysis
138 °C, of MTX,
and three corresponded
endothermic to peaks
the thermal
haveinstability of the MTX
been observed. Thetrihydrate and therefore
first broad to the mass
peak, between and 138 ◦ C,
65.3loss
associated with these water molecules. The second endothermic peak, at 142°C, related to a possible
corresponded to the thermal instability of the MTX trihydrate and therefore to the mass loss associated
pseudo melting or dissolution [30] and the third peak, at 178 °C, corresponded to its intrinsic melting
with thesepoint
water(174molecules.
°C) [15]. The second endothermic peak, at 142◦ C, related to a possible pseudo
melting or dissolution [30] and the third peak, ◦ C, corresponded to its intrinsic melting point
at 178a single
The DSC analysis of the β-CD matrix presented broad peak with a maximum at 109 °C.
(174 ◦ C) [15].
The thermal event was associated with a continuous mass loss from 30 °C to 129.4 °C, corresponding
to a 12.8% mass of evaporated water molecules. After 130 °C, the matrix remained stable throughout
The DSC analysis of the β-CD matrix presented a single broad peak with a maximum at 109 ◦ C.
the temperature range evaluated. ◦ ◦ C, corresponding
The thermal event was associated
The β-CD/MTX IC thermicwith a continuous
analysis massendothermic
showed a broad loss from 30 peakCatto 129.4
141.8 °C associated
to a 12.8%with
mass ◦
anof evaporated
overlap of thermal water molecules.
events related to aAfter
loss of130
waterC,molecules
the matrixand remained
pure MTX peak. stableThethroughout
sample showed
the temperature range evaluated. a continuous mass loss (6.3%) from 30 °C to 140.6 °C. The absence of free β-CD
confirms the IC formation. The IC presented a possible amorphization of MTX with an increase
The β-CD/MTX IC thermic analysis showed a broad endothermic peak at 141.8 ◦ Cinassociated
thermal stability [31]. The second peak at 164.2 °C was associated with a temperature-induced
with an overlap of thermal
rearrangement of theevents related
IC. Similar resultstowere
a loss of water
observed molecules
for other and pure MTX peak. The sample
ICs [22,23].
showed a continuous mass of
The DSC analysis loss
the(6.3%)
IC conjugated 30 ◦the
from with C to 140.6
AuNPs
◦ C. The absence of free β-CD confirms
presents an endothermic peak at 163 °C
with a shoulder
the IC formation. The at IC180 °C. The mean
presented peak was associated
a possible with a temperature-induced
amorphization of MTX with an rearrangement
increase in thermal
of the IC. This is considered to be◦ pharmacologically advantageous because the plasmonic heat
stability [31]. The second peak at 164.2 C was associated with a temperature-induced rearrangement
generated by laser irradiation will produce irreversible leakage of the MTX guest from the β-CD
of the IC. Similar
cavity. The results
shoulder were observed
at 180 for other
°C corresponds to the ICs [22,23].
melting temperature of MTX which acquires a high
The DSC analysis of CD
thecavity
IC conjugated with the AuNPs presents an endothermic peak ◦
stability inside the [15]. The DSC of AuNPs + β-CD/MTX IC differs significantly from the at 163 C
◦
with a shoulder at 180 C. The mean peak was associated with a temperature-induced rearrangement
of the IC. This is considered to be pharmacologically advantageous because the plasmonic heat
generated by laser irradiation will produce irreversible leakage of the MTX guest from the β-CD
cavity. The shoulder at 180 ◦ C corresponds to the melting temperature of MTX which acquires a high
stability inside the CD cavity [15]. The DSC of AuNPs + β-CD/MTX IC differs significantly from the
IC. This difference may be explained based on the geometry of the inclusion. Proton-NMR studies
depicted that only the pteridine ring is contained in the cavity of the β-CD molecule and that the
Nanomaterials 2018, 8, x FOR PEER REVIEW 10 of 15
Nanomaterials 2018, 8, x FOR PEER REVIEW 10 of 15
Nanomaterials 2018, 8, 985may
IC. This difference be explained based on the geometry of the inclusion. Proton-NMR studies 10 of 15
IC. This difference may be explained based on the geometry of the inclusion. Proton-NMR studies
depicted that only the pteridine ring is contained in the cavity of the β-CD molecule and that the
depicted that only the pteridine ring is contained in the cavity of the β-CD molecule and that the
remainder of
remainder of the
the host
host structure,
structure, which
which interacts
interacts with
with the
the AuNPs,
AuNPs, is excluded.
excluded. Conjugation with the
AuNPs, is is excluded. Conjugation with the
AuNPs
AuNPs occurs with the loss of electron density of the guest molecule, which favors the inclusion of
AuNPs occurs
occurswith
withthetheloss
lossofofelectron
electrondensity
densityofof
thethe
guest molecule,
guest molecule, which favors
which the the
favors inclusion of the
inclusion of
the pteridine
pteridine ring,
ring,ring, providing
providing increased
increased stability
stability to the system.
to the system. Itobserved
It is alsoIt is also observed that
that there that there
is nothere is no
significant
the pteridine providing increased stability to the system. is also observed is no
significant
mass loss ormass loss or
decomposition decomposition by
by the sample the sample between
◦ 30 °C and
◦ 250 °C. The sample decreases
significant mass loss or decomposition by thebetween 30 C and
sample between 30250
°C and C. 250
The°C.sample decreases
The sample 8.93%
decreases
8.93% which
which is is attributable
attributable to the to the
loss of loss
waterofmolecules
water molecules
[32]. [32].
8.93% which is attributable to the loss of water molecules [32].

Figure 7. TGA and DSC analysis of MTX, IC, and AuNPs + IC.
Figure 7. TGA and DSC analysis of MTX, IC, and AuNPs + IC.

3.3. Evaluation
3.3. Evaluation ofof the
the Effect
Effect of
of MTX
MTX and Its Its Derivatives onon Cell Viability
Viability
3.3. Evaluation of the Effect of MTX and and Its Derivatives
Derivatives on Cell
Cell Viability
To confirm the
To the stability ofof the MTX
MTX samples in in a saline phosphate
phosphate buffer (10 (10 mM PBS),
PBS), stability
To confirm
confirm the stability
stability of thethe MTX samples
samples in aa saline
saline phosphate buffer
buffer (10 mM mM PBS), stability
stability
analysis
analysis was conducted by UV-Vis spectrophotometry. Figure 8 depicts a plot of the wavelengths of
analysis was conducted by UV-Vis spectrophotometry. Figure 8 depicts a plot of the wavelengths of
was conducted by UV-Vis spectrophotometry. Figure 8 depicts a plot of the wavelengths of
the characteristic
the characteristic absorption
absorption bands of of MTX (370
(370 nm) andand AuNPs
AuNPs (520(520 nm)
nm) that
that are
are obtained
obtained from the the
the characteristic absorption bands
bands of MTXMTX (370 nm)nm) and AuNPs (520 nm) that are obtained from
from the
absorption
absorption spectra of MTX, IC, AuNPs + MTX, AuNPs + β-CD/MTX, and AuNPs at different times
absorption spectra
spectra ofof MTX,
MTX, IC,IC, AuNPs
AuNPs ++ MTX,
MTX, AuNPs
AuNPs ++ β-CD/MTX,
β-CD/MTX, and and AuNPs
AuNPs at at different
different times
times
(details Figure
(details FigureS6 S6ininSupplementary
Supporting Materials). These samples were stable ininthe PBS buffer, with the
(details Figure S6 in Supporting Materials). These samples were stable in the PBS buffer, with the
Materials). These samples were stable the PBS buffer, with the
exception of AuNPs.
exception AuNPs. This
This difference
difference in in the
the superficial
superficial plasmon
plasmon band wavelength,
wavelength, whichwhich begins
begins after
exception ofof AuNPs. This difference in the superficial plasmon band
band wavelength, which begins after
after
48
48 h and
h and increases
and increases after
increases after 72
after 72 h,
72 h, is
h, is introduced
is introduced due
introduced due to
due to the
to the colloidal
thecolloidal suspension
colloidal suspension
suspension of of AuNPs
of AuNPs
AuNPs in in the
inthe PBS
thePBS buffer.
PBSbuffer.
buffer.
48 h
However, when the
However, the AuNPs werewere conjugated with with either MTXMTX or IC,IC, they remained
remained stable during
during the
However, whenwhen the AuNPs
AuNPs were conjugated
conjugated with either
either MTX or or IC, they
they remained stablestable during thethe
evaluation time.Based
evaluation Based onthese
these results,a aproliferation
proliferation study was performed through MTS assay in
evaluation time.
time. Basedon on theseresults,
results, a proliferation study
studywaswasperformed
performed through
throughMTS assay
MTS in the
assay in
the
HeLa HeLa cells.
cells.
the HeLa cells.

8. Schematic of the
the wavelengths of the
the characteristic absorption bands of MTX, IC, AuNPs +
Figure 8. Schematic
Figure 8. Schematic of the wavelengths
wavelengths of the characteristic
characteristic absorption bands of MTX, IC, AuNPs +
MTX, AuNPs + + IC, and AuNPs at time zero and at 2, 5, 24, 48, and 72
72 h.
h.
MTX, AuNPs + IC, and AuNPs at time zero and at 2, 5, 24, 48, and 72 h.
Nanomaterials 2018, 8, 985 11 of 15

Nanomaterials 2018, 8, x FOR PEER REVIEW

MTX is an analog of folic acid, binding to the folate receptors through its pteridine 11ring; of 15

this inhibits the action of the DHFR enzyme, which further interrupts the cell cycle [17,33,34].
MTX is an analog of folic acid, binding to the folate receptors through its pteridine ring; this
To evaluate whether the inherent cytotoxicity of MTX was reduced when it was encapsulated by CD,
inhibits the action of the DHFR enzyme, which further interrupts the cell cycle [17,33,34]. To evaluate
the effects of MTX and IC on the viability of the HeLa cells at equivalent concentrations were compared.
whether the inherent cytotoxicity of MTX was reduced when it was encapsulated by CD, the effects
Figure 9 depicts a significant reduction in cell viability at high concentrations of free MTX (tested at
of MTX and IC on the viability of the HeLa cells at equivalent concentrations were compared.
0.1 and 0.2 mM) with respect to β-CD/MTX IC. The 50% inhibitory concentrations (IC50) of MTX and
Figure 9 depicts a significant reduction in cell viability at high concentrations of free MTX (tested
the IC were calculated from the following equations obtained by nonlinear curve Fit (doseResp) in
at 0.1 and 0.2 mM) with respect to β-CD/MTX IC. The 50% inhibitory concentrations (IC50) of MTX
OriginPro software:
and the IC were calculated from the following equations obtained by nonlinear curve Fit (doseResp)
in OriginPro software: 103.41 − 0.67 8.56
y = 0.67 + ( .
0.08392 .
− x )∗− 21.60524
and y = .
− 2.62 ( x −0.20484)
, respectively.
𝑦 = 0.67 + 1 + 10 . ∗ . and 𝑦 = 1+e
. . , respectively.
The
The IC50
IC50 obtained forMTX
obtained for MTX(0.0839
(0.0839mM)mM) waswas smaller
smaller thanthan
thatthat obtained
obtained for β-CD/MTX
for β-CD/MTX IC
IC (0.239
(0.239 mM) because this required a concentration that was 2.8 times higher than
mM) because this required a concentration that was 2.8 times higher than that of MTX to significantly that of MTX to
significantly reduce cell
reduce cell viability. viability.
This This effect
protective protective
of theeffect
IC isofprobably
the IC is due
probably
to thedue to the inclusion
inclusion of the
of the pteridine
pteridine ring of
ring of MTX MTXthe
inside inside
CD the CD cavities,
cavities, which which
preventsprevents its interaction
its interaction with with the folate
the folate receptors
receptors and
and reduces its effect on cell proliferation. In order to evaluate the effects of β-CD/MTX
reduces its effect on cell proliferation. In order to evaluate the effects of β-CD/MTX IC and MTX on IC and MTX
on healthy
healthy cells,
cells, wewe usedendothelial
used endothelialcells
cellsBEND3
BEND3and andembryonic
embryonic kidneykidney cells HEK293T
HEK293T as as aamodel
model
brain.
brain. The treatment did not exert any effect on cell viability after 72 h of treatment, which isvery
The treatment did not exert any effect on cell viability after 72 h of treatment, which is very
relevant
relevantconsidering
consideringpotential
potentialapplications
applicationsininterms
termsofofselectivity
selectivity(Figure
(FigureS7).
S7).
On
Onthetheother
otherhand, we tested
hand, the effects
we tested of AuNPs
the effects of on cell viability
AuNPs on cellbyviability
MTS assay.byThe
MTS nanoparticles
assay. The
did not affect cell viability in the assayed doses (Figure S8).
nanoparticles did not affect cell viability in the assayed doses (Figure S8).

Figure9.9.Comparison
Figure Comparisonofofcell cellviability
viabilityofofthe
theHeLa
HeLacells
cellstreated
treatedwith
withMTX
MTXororβ-CD/MTX
β-CD/MTXatatdifferent
different
MTX
MTX concentrations (0.002–0.2 mM). The data are expressed as percentages of thelive
concentrations (0.002–0.2 mM). The data are expressed as percentages of the livecells
cellsrelative
relativetoto
the
theuntreated
untreatedcells
cells(controls).
(controls).The
Thevalues
values represent thethe
represent mean
mean and SEM
and SEMof three separate
of three experiments
separate experimentsin
triplicate (* p(*
in triplicate < p0.05).
< 0.05).

3.4. Evaluation of Cell Viability by MTS Assays of AuNPs + β-CD/MTX Samples with and without Irradiation
3.4. Evaluation of Cell Viability by MTS Assays of AuNPs + β-CD/MTX Samples with and without
Irradiation
The AuNPs were conjugated with the IC to induce drug release from an intelligent delivery
system using the photothermal effect of the AuNPs. To achieve this objective, an AuNPs + β-CD/MTX
The AuNPs were conjugated with the IC to induce drug release from an intelligent delivery
sample was irradiated with a continuous laser at 532 nm for 15 min. The HeLa cells were then
system using the photothermal effect of the AuNPs. To achieve this objective, an AuNPs + β-CD/MTX
incubated with these samples (irradiated and not irradiated) at a final concentration of 0.1 mM.
sample was irradiated with a continuous laser at 532 nm for 15 min. The HeLa cells were then
We select this concentration because it significantly reduces cell viability (Figure 9) with MTX but
incubated with these samples (irradiated and not irradiated) at a final concentration of 0.1 mM. We
not with β-CD/MTX IC. Figure 10 depicts a significant reduction in cell viability with respect to
select this concentration because it significantly reduces cell viability (Figure 9) with MTX but not
the nonirradiated control. This illustrates that the heat generated by the plasmonic effect of AuNPs
with β-CD/MTX IC. Figure 10 depicts a significant reduction in cell viability with respect to the
is sufficient to disassemble the IC, which can cause the release of the MTX molecule. Moreover,
nonirradiated control. This illustrates that the heat generated by the plasmonic effect of AuNPs is
we determined the release profile of MTX in the presence of irradiation following a protocol described
sufficient to disassemble the IC, which can cause the release of the MTX molecule. Moreover, we
in reference 24, observing that after 15 min of irradiation the 40 ± 9% of the free drug is released
determined the release profile of MTX in the presence of irradiation following a protocol described
in reference 24, observing that after 15 min of irradiation the 40 ± 9% of the free drug is released from
the ternary system (See Figure S9A–C). Thus the free drug interacts with the folate receptors in cells,
interferes with the nucleic acid synthesis, inhibits the cell proliferation and induces cytotoxic effects
Nanomaterials 2018, 8, 985 12 of 15
Nanomaterials 2018, 8, x FOR PEER REVIEW 12 of 15

from the ternary

[17,33,34]. system (See
Furthermore, it hasFigure S9A–C).established
been widely Thus the free
thatdrug
MTXinteracts withthe
diminishes the folate receptors
antioxidant in
systems
cells, interferes with the nucleic acid synthesis, inhibits the cell proliferation and induces
of cells [35–37]; these increased intracellular reactive oxygen species (ROS) levels develop a cytotoxic
effects [17,33,34].
mitochondrial Furthermore,
dysfunction andit induce
has been widely established
apoptosis thateffect
[38,39]. This MTXwasdiminishes the antioxidant
not observed with the
systems of cells [35–37]; these increased intracellular
irradiated IC without nanoparticles (see Figure S10). reactive oxygen species (ROS) levels develop
a mitochondrial dysfunction and induce apoptosis [38,39]. This effect was not observed with the
irradiated IC without nanoparticles (see Figure S10).

Figure 10. Test of

Figure 10. of the
the viability
viability ofof the
the HeLa
HeLa cells
cellstreated
treatedwith
withAuNPs
AuNPs++β-CD/MTX,
β-CD/MTX, previously
previously
irradiated for 15 min with a 532-nm laser (50 mW). The HeLa cells were incubated
irradiated for 15 min with a 532-nm laser (50 mW). The HeLa cells were incubated for 1 h with for 1 AuNPs
h with
AuNPs + β-CD/MTX
+ β-CD/MTX (0.1 mM)(0.1 (irradiated
mM) (irradiatedand and nonirradiated)
nonirradiated) samples.
samples. After
After 71 71h,h,cell
cell viability
viability was
was
determined
determined through
through MTS
MTS assay.
assay. The
The results
results are
are expressed
expressed as
as percentages
percentages compared
compared with with untreated
untreated
cells
cells and
and represent
representthethemean
meanand andSEM
SEMof ofthe
thethree
threeindependent
independentexperiments
experimentsin triplicate(*(*pp<<0.05).
intriplicate 0.05).

4. Conclusions
4. Conclusions
Purification and inclusion of MTX in the β-CD cavity were achieved, which also resulted in the
Purification and inclusion of MTX in the β-CD cavity were achieved, which also resulted in the
formation of an IC with 1:1 stoichiometry. An MTS viability assay on the HeLa cell line treated using
formation of an IC with 1:1 stoichiometry. An MTS viability assay on the HeLa cell line treated using
MTX and β-CD/MTX IC depicted that the inclusion of the drug in CD reduced the cytotoxic effect as
MTX and β-CD/MTX IC depicted that the inclusion of the drug in CD reduced the cytotoxic effect as
compared to that observed in free MTX.
compared to that observed in free MTX.
An MTX nanocarrier system was designed to ensure controlled drug release using the photothermal
An MTX nanocarrier system was designed to ensure controlled drug release using the
effect of AuNPs. Thus, the β-CD/MTX samples were conjugated to AuNPs and were irradiated using
photothermal effect of AuNPs. Thus, the β-CD/MTX samples were conjugated to AuNPs and were
a 532-nm laser (laser of wavelength tunable with the plasmon), which caused a decrease in cell
irradiated using a 532-nm laser (laser of wavelength tunable with the plasmon), which caused a
viability relative to that observed in the case of nonirradiated control after the release of MTX from the
decrease in cell viability relative to that observed in the case of nonirradiated control after the release
β-CD cavities.
of MTX from the β-CD cavities.
These results may contribute to the treatment of cancer and promote the design of a drug delivery
These results may contribute to the treatment of cancer and promote the design of a drug
strategy to control the local and temporary photothermal release of antitumor drugs.
delivery strategy to control the local and temporary photothermal release of antitumor drugs.
Supplementary Materials:The
Supplementary Materials: Thefollowing
followingare areavailable
availableonline
onlineat at
http://www.mdpi.com/2079-4991/8/12/985/
www.mdpi.com/xxx/s1. Figure S1. 1H-NMR
s1. Figure S1. 1 H-NMR spectra of reported mannitol, MTX + mannitol, purified MTX and reported MTX,
spectra of reported mannitol, MTX + mannitol, purified MTX and reported MTX, Figure S2. Schematic
Figure S2. Schematic representation of the methodologic protocol for the synthesis of colloidal AuNPs and
representation
their conjugation of with
the methodologic
the β-CD/MTX, protocol
FigureforS3.
the2D-ROESY
synthesis of colloidalofAuNPs
spectrum β-CD/MTX and their conjugation
IC in DMSO-d6 with the
solvent,
β-CD/MTX,
Figure S4. TEM Figure S3. 2D-ROESY
micrograph spectrum
of AuNPs of β-CD/MTX
stabilized IC in DMSO-d
with β-CD/MTX 6 solvent,
IC. The Figureindicate
red arrows S4. TEMthemicrograph
presence
of
of an organic
AuNPs stainedwith
stabilized layer present around
β-CD/MTX IC. Theseveral goldindicate
red arrows spheres,the which couldofcorrespond
presence to the IC,
an organic stained Figure
layer S5.
present
XPS general spectrum of AuNPs+β-CD/MTX IC, Figure S6. Absorption spectra of (a) MTX, (b) IC, (c) AuNPs
+around
MTX, (d)several
AuNPs gold+spheres,
IC and which couldincorrespond
e) AuNPs, PBS at time to zero,
the IC,2, Figure S5.and
5, 24, 48 XPS72 general spectrum
h, Figure of AuNPs+β-
S7. Viability after
CD/MTX treatments
applying IC, Figure S6. Absorption
of 0.1mM spectra of
β-CD/MTX. (a) MTX,
Viability (b) IC, (c)using
evaluated AuNPs the+MTS
MTX, (d) AuNPs
assay over (A)+ IC and e)cells
BEND3 AuNPs,
and
(B) HEK293
in PBS at timefor zero,
24 h (n
2, =5,3),
24,Figure
48 andS8.72Effects of AuNPs
h, Figure on cellafter
S7. Viability viability of HeLa
applying cells. Viability
treatments of 0.1mM wasβ-CD/MTX.
evaluated
using the evaluated
Viability MTS assayusing over the
HeLaMTScells for over
assay 24 h (n
(A)= BEND3
3), Figure S9A.
cells andAbsorbance
(B) HEK293spectra
for 24 hof(n=3),
MTX Figure
in chloroform at
S8. Effects
different concentrations. The insert shows the calibration curve obtained measuring the absorbance intensity
of 266
at AuNPs
nm vs onthecellconcentration
viability of HeLa cells.(nViability
of MTX. was S9B.
= 2), Figure evaluated
Release using the MTSofassay
percentage MTXoverunderHeLa cells for(left)
irradiation 24 h
(n=3),
or Figure
without S9A. Absorbance
irradiation (right) forspectra
differentof cumulative
MTX in chloroform
times (n at different
= 3). concentrations.
The irradiation The insertwith
was performed shows the
a laser
of 532 nm (50
calibration mW),
curve Figure measuring
obtained S9C. Absorbance spectra of
the absorbance organicat
intensity phase different
266 nm vs theaccumulative
concentrationtimes with(n=2),
of MTX. and
Figure S9B. Release percentage of MTX under irradiation (left) or without irradiation (right) for different
cumulative times (n = 3). The irradiation was performed with a laser of 532 nm (50 mW), Figure S9C. Absorbance
Nanomaterials 2018, 8, 985 13 of 15

without irradiation with a 532nm laser (50 mW). (n = 3), Figure S10. Test of HeLa cells viability treated with
β-CD/MTX, irradiated previously during 15 min with a 532-nm laser (45 mW). Then HeLa cells were incubated
for 1 h with both β-CD/MTX (0.1mM) (irradiated and non-irradiated) samples. After 71 h it was determined,
the cell viability through MTS assays. The results are expressed as percentages compared with untreated cells,
and represent the mean and SEM of three independent experiments in triplicate (* p < 0.05). Table S1. Chemical
shifts of MTX + mannitol, purified MTX and reported MTX. Schematic representation and proton assignment
of MTX.
Author Contributions: N.S. synthesis, characterization, discussion and paper writing. A.R. effect of the irradiation
on cell viability, discussion and paper writing. N.Y. contributed reagents/materials/analysis tools. E.L., H-RMN
experiments and analysis. B.C., XPS experiments and analysis. S.G. cell viability determination by MTS Assay. J.S.,
supervised the work related with the characterization of the AuNPs and received N.S. as student in his laboratory.
P.J. and M.J.K., paper writing, discussion and contributed with reagents/materials/analysis tools.
Funding: The authors would like to express their gratitude for the support from the laboratory NMR USACH
(Bruker Avance 400 MHz). We thank the Kampar oncology laboratory for the donation of the Methotrexate
drug. Fondecyt 1170929, 1171611, and 1160114. Scholarships Conicyt N.S. Grand AT-24121127, BECAS CHILE
Convocatoria 2011. Fondecyt Postdoctoral 3150271. Fondap 15130011. MECESUP 0811. This work was partially
supported by the MIND funded by the Spanish Ministry of Science and Innovation (MICINN) under the National
Program for the internationalization of R&D, the Spanish Ministry of Economy, Industry and Competitiveness
through the Centro de Excelencia Severo Ochoa Award and by the Generalitat de Catalunya through the CERCA
program. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010,
Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the
European Regional Development Fund. The Nanobioengineering group has support from the Commission for
Universities and Research of the Department of Innovation, Universities, and Enterprise of the Generalitat de
Catalunya (2014 SGR 1442).
Conflicts of Interest: The authors declare that they have no conflicts of interest.

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