Dynamic Infrared Thermography of Nanoheaters Embedded in Skin-Equivalent Phantoms
Journal of Nanomaterials
Dynamic Infrared Thermography of Nanoheaters Embedded in Skin-Equivalent Phantoms
K. A. López-Varela 2
N. Cayetano-Castro 1
E. S. Kolosovas-Machuca 0
F. J. González F. S. Chiwo 0
J. L. Rodríguez-López 2
Leszek A. Dobrzan´ski
0 Coordinacio ́n para la Innovacio ́n y la Aplicacio ́n de la Ciencia y la Tecnolog ́ıa, Universidad Auto ́noma de San Luis Potos ́ı , San Luis Potos ́ı, SLP , Mexico
1 Nanoscience, Micro and Nanotechnology Center, National Polytechnic Institute , Av. Luis Enrique Erro s/n, Zacatenco, 07738 Mexico City , Mexico
2 Divisio ́n de Materiales Avanzados, Instituto Potosino de Investigacio ́n Cient ́ıfica y Tecnolo ́gica , A.C., Camino Presa San Jose ́ 2055, Lomas 4 Secc., 78216 San Luis Potos ́ı, SLP , Mexico
Nanoheaters are promising tools for localized photothermal therapy (PTT) of malignant cells. The anisotropic AuNPs present tunable surface plasmon resonances (SPR) with ideal NIR optical response to be applied as theranostic agents. To this purpose, nanoparticles with branches are suitable because of the electromagnetic field concentrated at their vertices. We standardized a protocol to synthesize multibranched gold nanoparticles (MB-AuNPs) by the seed-growth method and found a size-seed dependence tunability on the hierarchy of branching. Once the optical response is evaluated, we tested the temporal stability as nanoheaters of the MB-AuNPs immersed in skin-equivalent phantoms by dynamic infrared thermography (DIRT). The most suited sample presents a concentration of 5.2 × 108 MB-AuNPs/mL showing good thermal stability with Δ = 4.5∘C, during 3 cycles of 10 min at 785 nm laser irradiation with power of 0.15 W. According to these results, the MB-AuNPs are suitable nanoheaters to be tested for PTT in more complex models.
The last twenty years’ research reports on nanostructured
materials clearly indicate their potential to develop new
technologies for different specialized areas. The extensive
investigation work in shape controlled synthesis of metal
nanoparticles (NPs) has allowed the achievement of structures with
complicated geometric forms and the use of nontoxic
chemicals for their synthesis. Gold plays a particular and special
role in this area of size and shape controlled synthesis, and
because of their properties, those NPs are studied for
technological applications in fields such as renewable energies,
catalysis, medicine, and photonics. Because of the optical and
low-reactive properties of gold nanoparticles (AuNPs), they
have been considered for medical applications as theranostic
agents, which means that they can be simultaneously used for
drug delivery [
], medical imaging [
photothermal therapy (PTT) , and biological sensing [
Nowadays, for some specific theranostic applications
AuNPs are designed with anisotropic shapes, characteristic
that tunes their surface plasmon resonance (SPR) from the
visible to the near (NIR) and middle infrared (Mid-IR)
regions of the spectra, also presenting local concentration of
electromagnetic fields on the vertices [
]. The most common
anisotropic AuNPs studied and used have been the gold
nanorods (AuNRs) and stellated or spiky AuNPs, here termed
multibranched gold nanoparticles (MB-AuNPs). The fact that
NIR absorbance of MB-AuNPs fits the so-called therapeutic
window present in the human tissue (700–1200 nm) [
spectral region where maximum penetration of light occurs
due to the minimal absorption of blood components, such
as hemoglobin and water [
], is important for these
With the goal to apply these gold nanoheaters on the PTT
of cancer (localized heating for killing cancer cells) [
have to understand their thermal response under controlled
conditions. Thermal response has been evaluated by dynamic
thermography technique, as infrared imaging tool for
realtime recording of the temperature increases (Δ ). Since the
evaluation of nanosystems in real tissue samples presents
many out-of-control factors, such as the complexity in the
formation of protein corona on the nanoparticles and their
interaction with the physiological cellular environment [
], and besides the lack of the reproducibility of results due
to the difficulty in finding identical specimens, we decided
the use of gel phantoms as skin-equivalent model [
] which emulates the dielectric properties of the human
body surface. Previous reports had evaluated the thermal
response of AuNRs [
] in agar phantoms with intralipid (fat
emulsion) as scattered agent [
] and suggested to be effective
for tumor irradiation of 10 mm depth from the illuminated
tissue surface. We use for the accurate analysis of
MBAuNPs nanoheaters an optimal agar phantom model, with
permittivity close to that of the human skin in the 60 GHz
]. The skin-equivalent phantom uses polyethylene
powder (PEP) to decrease the real and imaginary parts of the
In this report we use the organic molecule HEPES,
2-[4(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid, as shape
directing agent. HEPES is one of the good buffers used in
cell culture [
] and is also used as reducing and shape
directing agent [
] in the synthesis of nanoparticles
by wet-chemical [
] and hydrothermal methods .
The shape promoted by HEPES in nanoparticle formation is
mostly multibranched, varying from few to several peaks.
Previously reported seeded-growth synthesis protocol, in
which the HEPES act as directing agent and hydroxylamine
], is used as reducing agent [
the seeds surface; the hydroxylamine has the role of avoiding
new nucleation events during the peak growth stage. It has
been also reported that hydroxylamine influences or
promotes the growth of stellated gold nanoparticles [
we start from this method that involves two molecules, both
with capping and direct-shaping properties. The function
of the HEPES and hydroxylamine during the growing stage
of peaks has been determined by our experimental design
on concentrations of the reactants. The seed-growth method
allows obtaining good monodispersity in the final product
shape and size. We also had found a size-seed dependence
tunability on the hierarchy of branching.
In this work, we evaluated the optical response of
MBAuNPs by UV-Vis spectroscopy, and their morphology by
electronic microscopy (SEM and HRTEM). Particle density
was determined by inducted coupled plasma spectrometry
(ICP), and the temporal stability as nanoheaters was tested
in skin-equivalent phantoms by dynamic infrared
2. Materials and Methods
Deionized water was used for all experiments. The reagents
used for NPs synthesis were obtained from Sigma-Aldrich:
HAuCl4⋅3H2O, HEPES, NH2OH⋅HCl, and sodium citrate.
For the preparation of skin-equivalent-phantom,
polyethylene powder (PEP) was obtained from Baker, Agar from
Sigma-Aldrich, and TX-151 from Oil Center, and all reagents
were used as received without further treatment or
purification. All glassware was cleaned with aqua regia (HCl : HNO3,
3 : 1) and rinsed with plenty of deionized water. The samples
were characterized with UV-Vis absorption spectroscopy,
acquired on a Cary 60 UV-Vis-NIR spectrophotometer at
room temperature. ICP elemental quantification was
performed on Variant 730-ES spectrometer. The electron
micrographs were acquired in a transmission electron microscope
JEOL 2100, and scanning electron micrographs in a
FEIHelios Nanolab. The X-ray diffractograms were obtained in a
Bruker DX-8 system at CuK source of wavelength 1.54056 A˚.
The dynamic thermographs were taken at a distance of 0.3 m
from the skin-equivalent phantom [
], using a
high-resolution infrared camera with thermal sensitivity better than
40 mK and a 480×360 focal plane array of VOx
microbolometers (FLIR T600, FLIR Systems Inc., Wilsonville, OR). The
phantoms were evaluated with a 785 nm laser diode with a
spot size of 3.5 mm and optical power of 0.15 W. Temperature
measurements of the skin-like phantom were taken with
and without MB-AuNPs. Data was analyzed with FLIR-IR
2.1. Synthesis of Multibranched Gold Nanoparticles
(MBAuNPs) Using a Seed-Growth Method. The 16 and 18 gold
nanospheres (AuNSs) used as seeds were prepared by a
reverse-modified Turkevich method  by citrate reduction
of gold salt. Shortly, in a three-neck round-bottom flask
with a condenser mounted, a citrate solution was brought to
boiling by a heating mantle for 5 mins; then a gold solution
of initial concentration of 25 mM was added, and after a
light red ruby color appeared the system was left to react
for half hour. For the synthesis of 16 nm AuNSs, the total
concentration of sodium citrate and gold salt was 1.8 mM and
0.16 mM, respectively; and for 18 nm AuNSs were 0.75 mM
and 0.25 mM. After the reaction was completed, the seeds
solution was left to cool down at ambient temperature and
used without modifications.
For the MB-AuNPs synthesis we used three
concentrations of HEPES buffer [25, 50, 75 mM] as solution. Briefly,
under slowly stirring within an ice bath (∼4∘C) to 12.5 mL
of HEPES 50 L of AuNSs (16 or 18 nm) was added, as well
as 30, 60, or 120 L of hydroxylamine [0.1 M], and finally
the dropwise addition of gold salt solution with rate of
0.375 mL/min to a f inal concentration of 0.07 or 0.13 mM. For
the reaction to be complete it is necessary to leave the sample
to rest 8 h in the fridge. The samples were washed three times
by centrifugation and a drop of the concentrated colloid was
deposited on an aluminum pin for SEM and on a lacey carbon
copper grid for HRTEM. For UV-Vis measurements, the
samples were analyzed as synthesized. The samples for ICP
quantification were centrifuged and the NPs concentrated
was dissolved in aqua regia.
2.2. Fabrication of Skin-Equivalent Phantom. For the
fabrication of the control phantom a previously reported protocol
was used, and the reagents concentration was maintained
]. The procedure is as follows: a Buchner flask with
deionized water and agar was heated until ∼80∘C under slow
magnetic stirring and vacuum. Once the agar was dissolved and
the boiling point was reached, the vacuum and heating were
turned of f, the TX-151 was added and mixed gently with a
stirring rod until its complete incorporation, and then the PEP
was added to decrease the real and imaginary parts of the
permittivity. After complete PEP incorporation the vacuum was
turned on for a moment to avoid bubbles in the final
phantom. Finally, the mix was placed into a petri box (ø = 55 mm)
and left to solidify at ambient temperature. When phantoms
with nanoparticles were prepared, a 1x concentrated of
prewashed MB-AuNPs colloid was added and completely
incorporated before TX-151 addition. For comparison, other
phantoms with AuNSs were prepared, at the same NPs
concentration of MB-AuNPs.
3.1. Multibranched Gold Nanoparticles (MB-AuNPs). In order
to prepare monodisperse MB-AuNPs, presynthesized gold
single-crystalline and multitwinned nanoparticles are used
as seeds for the growth of peaks [
], which grow along
preferential crystallographic directions of the metallic core
. According to previous reports, the capping agents [
have preferential adsorption by specific crystalline faces of the
metal seeds [
]; and this promotes an accelerated growth rate
along specific crystallographic directions [
7, 32, 38–40
The AuNSs, obtained by the reverse Turkevich method,
were monodispersed in size (see Figures 1 and SI-1); by ICP
spectroscopy was determined the concentration of 2.39 × 1011
and 3.66 × 1011 NPs/mL for 16 and 18 nm AuNSs, respectively.
We will see later that the structural characteristics of the
AuNSs will influence the growth process of peaks; as can be
seen in Figure 1(a), the 16-AuNSs present more symmetrical
shape and monocrystalline structure, being different from
18AuNSs samples (Figure 1(a)) that show multitwinned
structure and nonspherical multifaceted shapes. During the stage
of peaks formation, the promotion of second-order
branching (Figure 3) is up to the seed structure [
] and the stabilizer
agent protection [
] at the nanoparticle surface, observed in
the MB-AuNPs synthesized from 18 nm nanospheres (18
MBAuNPs), with 0.13 mM of gold salt. Also, we observed that
the growth mechanism of the MB-AuNPs is influenced by the
chemical characteristics of the reagents present in the growth
solution and by its molar ratio with respect to gold ions.
Therefore, both hydroxylamine and HEPES are dual
agents, with reducing and shape capabilities. Manipulation of
the role played by each reactive is made through experimental
conditions such as temperature (e.g., to slow the HEPES
reducing action) and concentration of hydroxylamine (using
the necessary to reduce the gold ions). Within the molar rates
used in this work, the hydroxylamine governs the reaction
kinetics and the f inal shape of the peaks and the HEPES
modulate the peaks growth. A simple way to know if the
HEPES has been or not contributing as reducing agent is by
means of UV-Vis spectroscopy (see Figure SI-2), since the
nitrogen species from degraded HEPES can be detected by
its absorption at ∼346 nm [
]. It is important to mention
that, in the reaction where no hydroxylamine is used, the
color changes of the colloid take place slowly, evidencing that
HEPES is a slow reductor. Increasing the concentration of
HEPES (from 25 to 50 mM), the growth of narrow peaks is
promoted while the cores remain small. Then the addition of
hydroxylamine is necessary to modulate the kinetics without
excess of HEPES and will help to obtain better definition
on the peaks and a monodisperse size and shape sample
(see Figure SI-3). From the mechanism reduction of Au3+
by hydroxylamine proposed by Minati et al. [
], the molar
ratio of Au3+ : NH2OH (1 : 3) was used to synthesize the
sample with 16 nm nanospheres (16 MB-AuNPs), in order
to avoid the intervention of HEPES reducing action (see
Figure SI-2). We observed that, when hydroxylamine is
present and increases its molar rate, the change of colors
gets faster indicating the necessary use of a mild reducing
agent (hydroxylamine) simultaneously with a directing shape
agent (HEPES) for better anisotropic growth. We also have
determined that an excess of hydroxylamine promotes the
second-order branching in 18 MB-AuNPs, which was
synthesized with molar rate of Au3+ : NH2OH of 1 : 6 (see Figure
It had been reported that the size of the NPs used as seeds
inf luences the shape of f inal products [
]. In our results
we observed that from 16-AuNSs, MB-AuNPs of 80 nm of
diameter can be obtained, synthesized with 0.07 mM of gold
salt (16 MB-AuNPs, see Figure SI-5), and the colloid presents
a maximum absorbance at 728 nm (Figure 2). On the other
hand, dendritic growth is only perceptible in 18 MB-AuNPs
(Figure 3), even if the concentration of gold ions is increased
in the samples synthesized from 16-AuNSs (see Figure SI-6).
The increase in gold ions leads to bigger cores and smaller
length of peaks, due to an addition of gold atoms between
peaks for minimization of energy, instead of the growth of
longer peaks [
At this stage we have shown that both sets of samples
(16 and 18 MB-AuNPs) present good optical response for
being used as nanoheaters. The next criteria for choosing a
sample is the critical size for being used in an in vitro or
in vivo environment. Thus, we decided the use of 16
MBAuNPs for being tested as nanoheaters; however, the optical
response of 18 MB-AuNPs in the 830 nm (Figure 3) is suitable
for applications in nanostructured system for SERS [
3.2. Thermal Response of MB-AuNPs. Infrared (IR) imaging
is based on the fact that any object at a temperature above
absolute zero (−273∘C) will emit IR radiation, even if only
weakly. T he human body has a low thermal emittance,
radiating in a wavelength range that starts at around 3 m and
peaks in the vicinity of 10 m and trails off from this point
into the extreme IR and, negligibly, beyond it. The emissivity
of human skin has a constant value between wavelengths of
2 and 14 m of 0.98 ± 0.01 for black skin and 0.97 ± 0.02 for
white skin [
]; thus, human skin has a known and almost
invariant emissivity in this wavelength region that makes IR
imaging an ideal procedure to evaluate surface temperature of
the human body [
]. Because of the above arguments, the
skin-equivalent phantoms with 0.62±0.01 reported emittance
 is a well suited model for IR thermal evaluation.
The thermal response of 16-AuNSs and 16 MB-AuNPs was
evaluated inside the skin-equivalent phantoms by dynamic
thermography. The samples were irradiated at 0.15 W power
laser by 3 cycles of 10 mins and recorded with an IR camera,
and the thermographs shown at Figure 4 were extracted
at 555 s using the FLIR-RIR software, which allows the
analysis of single points, line profiles, and averages of
circle/rectangular areas. The plot in Figure 4(a) shows data from
a line profile analyzed at different times, and it clearly can be
observed that not only the incident laser area has been heated
but also there is a radial propagation by diffusion of the
generated heat; thus there is energy transmitted to the adjacent
regions due to the immersed NPs. This effect is also observed
in the control phantom analysis, but over a smaller area and
with more uniform temperature as can be seen in Figure SI-7.
Figure 4(b) reports the information on how fast the system
absorbs the energy, achieving constant temperature at early
irradiation times (60 s). This was also performed for evidence
the continuity of the thermal response of immersed 16
MBAuNPs in the phantom as a function of time.
The plot in Figure 5 corresponds to a dynamic thermal
analysis, and from it we can appreciate the difference in
the temperature reached by the three phantoms: the largest
Line Profile (Li1)
increase of localized temperature is measured in the sample
prepared with 16 MB-AuNPs, compared to the phantom with
16-AuNSs and the one without the addition of NPs, used as a
A red-shift of the LSPR is present for anisotropic
MBAuNPs compared with spheres (see Figure 1), and a
substantial increase in the heating efficiency with respect the sphere
is expected. In our results, the maximum temperature
increment in 16 MB-AuNPs phantom was achieved in 60 s and
maintained during all the laser irradiation. This temperature
increment is Δ = 4.5∘C and when the light source is turned
off, a fast decay is observed. The comparison between the
AuNSs and MB-AuNPs at the same concentration shows the
evident increase of thermal response due to the anisotropic
shape of MB-AuNPs and the localized SPR’s synergy, because
the MB-AuNPs absorbs closer to the wavelength of laser
irradiation. In the case of the phantom with 16-AuNSs, the
increment in temperature can be described for the excitation
of hot-spots created in the interaction among spherical
]. The fact that there were not physical changes
observed in the phantoms under laser irradiation, as leaking
or deformation, even after the three cycles evaluation is
important to mention.
We have shown that the control on the width, the length, the
number of peaks, and the degree of dendritic growth gives
rise to an easy tuning of the plasmon resonance spectra [
and their corresponding higher transduction of light into
heat, and therefore gold nanoheaters are good candidates for
IR absorption for efficient photothermal therapy of malignant
The structural characteristics of the AuNSs influenced the
degree of ramification of the nanoparticle tips, resulting in
second-order branches for MB-AuNPs synthesized from
18AuNSs, which present crystalline defects promoting directed
anisotropy. From 16-AuNSs we could obtain monodisperse
samples. After analyzing the impact of HEPES and
hydroxylamine on the final tips shape, we estimated that the optimal
molar ratio of hydroxylamine : Au ions is 3 : 1, where this
amount is enough to avoid the intervention of HEPES in the
reduction. Even if an excess of hydroxylamine may result in a
better definition of tips, it can also cause the agglomeration of
MB-AuNPs, due to the resulting high ionic force of the
solution, that could not be used for clinical applications, and the
same argument is valid for higher concentration of HEPES
(75 mM). On the other side, the increase on the molar
concentration of Au ions (and necessary hydroxylamine) leads
to bigger cores and smaller length of tips; thus it shifts the SPR
to lower wavelengths making them not practical for PTT.
Regarding the dynamic thermography results for 16
MBAuNPs, we found that not only the incident laser area is being
heated, but also there is a radial propagation of the released
heat, which means there is transmission of energy around
the adjacent region of the spot due to the immersed NPs.
Therefore, the addition of MB-AuNPs results in the
production of more efficient centers for scattering and reemission
The temperature increment achieved by the nanoheaters
in this work (Δ ∼ 5∘C) is good enough for effective and
minimally harmful gold nanoparticles based photothermal
therapy techniques [
]. This temperature increment from
multibranched gold nanoparticles immersed in a phantom gel (as a
model approach for the real environment in bioapplications)
satisfies biomedical requirement for the treatment of
superficial diseases (mycoses, fungal infections, and many different
types of skin cancer) [
A synthesis method that uses nonaggressive chemicals for
obtaining multibranched gold nanoparticles and secondary
growth branching by tuning the seed size is presented.
Analysis of the UV-Vis spectra and SEM micrographs allows us to
understand the role of each reactant in the final shape of the
nanoparticles. The HEPES molecule mainly grow tips from
seeds with a random order; and the hydroxylamine can act as
a directing shape agent, but this role only follows after its
reduction role has been f inished in the reaction. A faster
depletion of the reactants (by higher concentration of
hydroxylamine) during the grow reaction promotes the growth of
larger tips with second-order branches.
We have determined that the synthesized multibranched
gold nanoparticles increase the temperature in a localized
area irradiated with a 785 nm laser. The temperature
increment recorded was Δ = 4.5∘C. The 16 MB-AuNPs embedded
in the phantom gel is a system that rapidly absorbs the proper
incident energy, achieving a plateau of constant temperature
within 60 s which is recovered during at least three different
cycles. This evaluation indicates MB-AuNPs as an interesting
system for being tested as nanoheater in biological models.
Furthermore, the evaluation of their photothermal behavior
in cultured cells is necessary for their direct application in
medicine. We also propose to carry on the test with an
irradiance lower than the ANSI regulation. Due to the
experimental conditions reached in our phantoms, we expect that the
transduction of light into thermal energy will be more
effective, due to the limitation of MB-AuNPs reshaping.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
The authors acknowledge Ph.D. CONACYT scholarship
number 375630 and institutional support from IPICYT. The
authors acknowledge Dra. Gladys Judith Labrada-Delgado
for SEM and Dr. Hector Gabriel Silva-Pereyra for HRTEM
analysis, as well as the infrastructure of the National
Laboratory for Nanoscience and Nanotechnology (LINAN) at
IPICYT. This work has been supported by
CONACYTMexico Grants 106437 and 216315. F. J. Gonza´lez would like to
acknowledge support from Project 32 of “Centro Mexicano de
Innovacio´n en Energ´ıa Solar” and by the National Laboratory
Program from CONACYT through the Terahertz Science and
Technology National Lab (LANCYTT).
Figure SI-1. Histograms of diameters and frequency
distribution data of populations of (a) 200 AuNSs for 16-AuNSs
and (b) 100 for 18-AuNSs. Figure SI-2. The increase of the
molar concentration of HEPES promotes strong anisotropic
formation of the MB-AuNPs. Figure SI-3. Scanning electron
microscopy micrograph of 16 MB-AuNPs; the
monodispersity and nonagglomeration of the NPs can be observed.
Figure SI-4. Diagram of the concentrations of reactants
used for the samples analyzed. Figure SI-5. Histograms of
diameters and frequency distribution data of populations
of 50 16 MB-AuNPs. Figure SI-6. SEM micrographs of
MBAuNPs synthesized in a medium of HEPES [50 mM], with a
molar ratio Au3+/NH2OH of 1 : 3, with (a) 16-AuNSs and
(b)(c) 18-AuNSs. Figure SI-7. Thermography of skin-equivalent
phantom used as control. (Supplementary Materials)
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