Carbohydrate Polymers 200 (2018) 93–99

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Hyaluronic acid functionalized green reduced graphene oxide for targeted T

cancer photothermal therapy
Rita Lima-Sousaa, Duarte de Melo-Diogoa, Cátia G. Alvesa, Elisabete C. Costaa, Paula Ferreirab,
⁎
Ricardo O. Louroc, Ilídio J. Correiaa,b,
a
CICS-UBI – Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6200-506 Covilhã, Portugal
b
CIEPQPF – Departamento de Engenharia Química, Universidade de Coimbra, Rua Silvio Lima, 3030-790 Coimbra, Portugal
c
ITQB – Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal

A R T I C LE I N FO A B S T R A C T

Keywords: Reduced graphene oxide (rGO) nanomaterials display promising properties for application in cancer photo-
2D materials thermal therapy (PTT). rGO is usually obtained by treating graphene oxide (GO) with hydrazine hydrate.
Green chemistry However, this reducing agent contributes for the low cytocompatibility exhibited by rGO. Furthermore, rGO has
Hyaluronan a low water stability and does not show selectivity towards cancer cells. Herein, rGO attained using an en-
Photothermal effect
vironmentally-friendly method was functionalized with a novel hyaluronic acid (HA)-based amphiphilic polymer
Targeted therapy
to be used in targeted cancer PTT. Initially, the green-reduction of GO with L-Ascorbic acid was optimized
considering the near infrared absorption and the size distribution of the nanomaterials. Then, rGO was func-
tionalized with the HA-based amphiphile. The functionalization of rGO improved its stability, cytocompatibility
and internalization by CD44 overexpressing cells, which indicates the targeting capacity of this nanoformulation.
Furthermore, the on-demand PTT mediated by HA-functionalized rGO induced cancer cells’ ablation, thereby
confirming its potential for targeted cancer therapy.

1. Introduction
Photothermal therapy (PTT) mediated by nanomaterials has been
showing promising results in cancer treatment (de Melo-Diogo, Pais-
Silva, Costa, Louro, & Correia, 2017; de Melo-Diogo, Pais-Silva, Dias,
Moreira, & Correia, 2017). This type of therapy benefits from the ability
of nanostructures to accumulate in the tumor through the so called
enhanced permeability and retention effect (EPR effect) (Fang,
Nakamura, & Maeda, 2011; Maeda, 2012). Afterwards, the tumor zone
is irradiated with laser light, and the nanomaterials used for this type of
therapy absorb laser light energy and convert it into heat (Frazier &
Ghandehari, 2015). For photothermal applications, the use of near in-
frared light (NIR; 750–1000 nm) is crucial due to its low interaction
with biological components, thus achieving a high penetration depth
(Strangman, Boas, & Sutton, 2018). In this way, nanostructures with a
high NIR absorption are the most appealing for application in cancer
PTT, since they may produce an on-demand therapeutic effect upon
interaction with NIR light (Akhavan, Ghaderi, Aghayee, Fereydoonia, &
Talebia, 2012; Akhavan, Ghaderi, & Emamy, 2012; de Melo-Diogo,
Pais-Silva, Costa et al., 2017).
Among the different NIR-responsive nanomaterials, nanosized gra-
phene oxide (GO) has sparked a great interest for cancer PTT (Chung
et al., 2013; Li, Tang, Yuan, Sun, & Wang, 2013). This 2D carbon ma-
terial has a graphitic lattice with several types of oxygen functional
groups (e.g. epoxy, hydroxyl or carboxyl groups) and presents NIR ab-
sorption (Chen, Feng, & Li, 2012; Liu, Cui, & Losic, 2013). The reduc-
tion of GO can further improve its NIR absorption, by restoring the
aromatic lattice of this material, leading to an improved photothermal
capacity (Zhang, Yang et al., 2010). Hydrazine hydrate (HH) is the most
commonly used agent to produce reduced GO (rGO) (Hu, Sun, Ding,

Abbreviations: ANOVA, analysis of variance; CLSM, confocal laser scanning microscopy; DLS, dynamic light scattering; DMEM-F12, Dulbecco’s modified Eagle’s
medium-F-12; DMSO, dimethyl sulfoxide; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; EPR, enhanced permeability and retention; FBS, fetal bovine
serum; FTIR, fourier transform infrared spectroscopy; GO, graphene oxide; HA, hyaluronic acid; HA-g-PMAO, HA-grafted PMAO; HA-rGO, HA-g-PMAO functionalized
rGO; HH, hydrazine hydrate; IC50, half maximal inhibitory concentration; LAA, L-ascorbic acid; MCF-7, Michigan cancer foundation-7; NIR, near infrared; NHDF,
normal human dermal fibroblasts; NHS, N-hydroxysuccinimide; PBS, phosphate buffered saline; PEG, poly(ethylene glycol); PMAO, poly(maleic anhydride-alt-1-
octadecene); PTT, photothermal therapy; rGO, reduced graphene oxide; Rhod B, Rhodamine B; SD, standard deviation; TEM, transmission electron microscopy
⁎
Corresponding author at: CICS-UBI – Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6200-506 Covilhã, Portugal.
E-mail address: icorreia@ubi.pt (I.J. Correia).

https://doi.org/10.1016/j.carbpol.2018.07.066
Received 25 May 2018; Received in revised form 23 July 2018; Accepted 23 July 2018
Available online 24 July 2018
0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
R. Lima-Sousa et al.
Chen, & Chen, 2016; Robinson et al., 2011). However, the rGO obtained
by using HH is highly cytotoxic, in part due to the inherent toxicity of
this hazardous reducing agent (Robinson et al., 2011). Therefore, it is
imperative to develop and optimize alternative methods for the re-
duction of GO.
L-Ascorbic acid (LAA), or Vitamin C, is a water soluble natural
compound with reducing capacities (De Silva, Huang, Joshi, &
Yoshimura, 2017; Du, Cullen, & Buettner, 2012). This molecule has
revealed promising results in the reduction of GO (Huang et al., 2015;
Liu et al., 2012). However, the conditions for the LAA-mediated re-
duction of GO are yet to be optimized considering the NIR absorption
and the size distribution of the obtained materials. This is of paramount
importance, since the potential of rGO-based nanomaterials in cancer
therapy is highly dependent on these properties.
Furthermore, the direct use of rGO in cancer therapy is also im-
paired by its poor water solubility (Hashemi et al., 2017; Liu et al.,
2011; Yang et al., 2012) and lack of selectivity towards cancer cells
(Robinson et al., 2011). To surpass the solubility problems, rGO can be
functionalized with amphiphilic polymers through non-covalent inter-
actions (Li et al., 2014). For instance, the functionalization of rGO with
poly(ethylene glycol) (PEG)-based amphiphilic polymers can greatly
improve their water solubility (Robinson et al., 2011). Moreover, this
type of functionalization can also improve the biocompatibility of rGO
materials (Li et al., 2014; Miao et al., 2013; Robinson et al., 2011).
However, the majority of the amphiphilic polymers that have been used
to functionalize rGO do not possess targeting capacity. Therefore, in-
vestigating amphiphilic polymers with cancer cell targeting properties
is of upmost interest in order to produce rGO materials capable of
performing a selective PTT.
In this work, we hypothesized that the concentration of LAA and the
reaction time used in the environmentally-friendly reduction of GO
could be fine-tuned in order to yield rGO with a suitable size and NIR
absorption for cancer-related applications. Furthermore, it was also
hypothesized that the functionalization of rGO with a novel hyaluronic
acid (HA)-based amphiphilic polymer, produced by grafting HA onto
poly(maleic anhydride-alt-1-octadecene) (PMAO), could render nanos-
tructures with an improved stability and cytocompatibility, capable of
performing a targeted cancer PTT. In this regard, HA was selected due
to its hydrophilic character and targeting capacity to the CD44 re-
ceptors, which are overexpressed on cancer cells’ membrane (Calcagno
et al., 2010; Olsson et al., 2011). Additionally, CD44 receptors are in a
quiescent state on normal cells, i.e. they do not display binding capacity
to HA (Yang et al., 2015), which further highlights the therapeutic
potential of targeting this receptor. To produce the amphiphilic
polymer, HA will be deacetylated in order to be grafted onto PMAO,
due to the capacity of the later to adsorb on rGO surface (Shi, Chen,
Ehlerding, & Cai, 2014; Yang et al., 2012).
2. Experimental section
2.1. Materials
Dimethyl sulfoxide (DMSO) and LAA were purchased from Fisher
Scientific (Oeiras, Portugal). Dulbecco’s modified Eagle’s medium-F-12
(DMEM-F12), GO nanocolloids, N-hydroxysuccinimide (NHS), paraf-
ormaldehyde, PMAO (30000–50000 Da), penicillin/streptomycin and
resazurin were acquired from Sigma–Aldrich (Sintra, Portugal). HA
sodium salt (9.27 kDa) was obtained from Carbosynth (Berkshire, UK).
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was purchased
from Merck (Darmstadt, Germany). Michigan cancer foundation-7
(MCF-7) cell line was acquired from ATCC (Middlesex, UK). Normal
human dermal fibroblasts (NHDF) were obtained from Promo-Cell
(Heidelberg, Germany). Fetal bovine serum (FBS) was purchased from
Biochrom AG (Berlin, Germany). Water used in all experiments was
double deionized (0.22 μm filtered; 18.2 MΩ cm).
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Carbohydrate Polymers 200 (2018) 93–99
2.2. Methods
2.2.1. Optimization of the green reduction of GO
The optimization of the environmentally-friendly reduction of GO
was performed by adapting previously described methods (Fernández-
Merino et al., 2010; Zhang, Yang et al., 2010). GO was sonicated for 6 h
before its use. In brief, 1 mL solutions containing GO (200 μg/mL) and
LAA (3 mM or 1.5 mM) were reacted at 80 °C during 30, 45, 60, 90 or
120 min. Afterwards, the reduction process was stopped by cooling the
samples in an ice-water bath.
2.2.2. Functionalization of rGO with HA-g-PMAO
For the functionalization of rGO, this material (200 μg/mL, 1 mL)
was mixed with HA-g-PMAO (500 μg; the synthesis of HA-g-PMAO is
described in the supplementary information) and sonicated for 60 min
(Branson 5800, Branson Ultrasonics, CT, USA). Afterward, this solution
was dialyzed against water (14 kDa cut-off dialysis membrane) for
90 min to remove LAA and centrifuged to remove any aggregates. The
supernatant was then recovered, yielding HA-g-PMAO functionalized
rGO (HA-rGO).
2.2.3. Characterization of rGO and HA-rGO-based materials
The reduction efficiency of GO was monitored by UV–vis absorption
spectroscopy on an Evolution 201 spectrophotometer (Thermo
Scientific Inc.), over the wavelength range from 200 to 1000 nm, and by
dynamic light scattering (DLS) in a Zetasizer Nano ZS (Malvern
Instruments, Worcestershire, UK), at a scattering angle of 173°. The
successful functionalization of rGO with HA-g-PMAO was confirmed by
DLS, UV–vis absorption spectroscopy and Fourier transform infrared
spectroscopy (FTIR), using a Nicolet iS10 spectrometer (Thermo
Scientific Inc., MA, USA). Furthermore, HA-rGO nanosized dimensions
were determined by transmission electron microscopy (TEM), using a
HT7700 microscope (Hitachi, Japan), operated at an accelerating vol-
tage of 100 kV. For this purpose, samples were stained with phospho-
tungstic acid (2% (w/v)). The photothermal capacity of HA-rGO was
assessed by irradiating the samples with NIR radiation (808 nm, 1.7 W/
cm2) over a period of 5 min, and the temperature changes were mon-
itored with a thermocouple thermometer.
2.2.4. Evaluation of the cytocompatibility of rGO and HA-rGO
The cytotoxic profile of rGO and HA-rGO was determined in MCF-7
cells and NHDF as we previously reported (Gaspar et al., 2015). In brief,
96-well plates were seeded with 1 × 104 cells/well in DMEM-F12
medium supplemented with 10% FBS and 1% of penicillin/strepto-
mycin, and were incubated in a humified incubator at 37 °C, 5 % CO2.
After 24 h, the medium was replaced by fresh cell culture medium
containing rGO or HA-rGO at different concentrations, and cells were
incubated for 24 and/or 48 h. After this period, the medium was re-
placed with fresh cell culture medium containing resazurin (10% (v/v))
and cells were incubated for 4 h in the dark (37 °C, 5% CO2). Cells’
viability was then assessed by analyzing the fluorescence of resorufin
(λex = 560 nm; λem = 590 nm) in a Spectramax Gemini EM spectro-
fluorometer (Molecular Devices LLC, CA, USA). Cells solely incubated
with culture medium (without rGO derivatives) were used as negative
(K-) control, whereas those treated with ethanol (70% (v/v)) represent
the positive (K+) control.
2.2.5. Investigation of the targeting capacity of HA-rGO
To evaluate HA-rGO cellular uptake, this nanomaterial was labelled
with rhodamine B (Rhod B) as previously described elsewhere (Zhang,
Xia, Zhao, Liu, and Zhang, 2010). In brief, HA-rGO (200 μg/mL, 1 mL)
was sonicated with Rhod B for 30 min. Afterwards, the solution was
dialyzed against water for 90 min (1000 Da cut-off dialysis membrane)
to remove the non-bounded Rhod B and centrifuged to remove any
bigger aggregates.
To confirm the targeting capacity of HA-rGO to CD44
R. Lima-Sousa et al.
overexpressing cancer cells, the uptake of Rhod B labelled HA-rGO by
MCF-7 cells (overexpressing CD44) (Calcagno et al., 2010; Olsson et al.,
2011) and NHDF (that do not overexpress CD44) (Miyatake et al.,
2013) was analyzed by confocal laser scanning microscopy (CLSM).
Briefly, μ-slide 8-well imaging plates (Ibidi GmbH, Munich, Germany)
were seeded with 1.5 × 104 cells/well. After 48 h, cells were incubated
with cell culture medium containing Rhod B labelled HA-rGO (68.7 μg/
mL of Rhod B equivalents) or free Rhod B (68.7 μg/mL) during 4 h.
Subsequently, cells were fixed with paraformaldehyde 4% for 15 min,
and then rinsed with a phosphate buffered saline (PBS) solution. Zeiss
LSM 710 confocal microscope (Carl Zeiss AG, Oberkochen, Germany)
was used for imaging experiments (λex = 514 nm; λem = 513–703 nm).
Non-treated cells and cells incubated with free-Rhod B were used as
controls.
2.2.6. In vitro evaluation of the phototherapeutic effect mediated by HA-
rGO
The photothermal effect mediated by HA-rGO was evaluated by
using a previously described protocol (de Melo-Diogo, Pais-Silva, Costa
et al., 2017). In brief, 96-well plates were seeded with MCF-7 cells at a
density of 1 × 104 cells/well. After 24 h, the medium was replaced by
fresh medium containing different concentrations of HA-rGO (25, 50
and 75 μg/mL of rGO equivalents). After 4 h, cells were irradiated with
NIR light (808 nm, 1.7 W/cm2, 5 min). After 24 h of incubation, cells’
viability was determined through the resazurin assay as described
above.
2.2.7. Statistical analysis
One-way analysis of variance (ANOVA) with the
Student–Newman–Keuls test was used for multiple groups comparison.
A p-value lower than 0.05 (p < 0.05) was considered statistically
significant. For data analysis, GraphPad Prism v6.0 (Trial version,
GraphPad Software, CA, USA) was used.
3. Results and discussion
3.1. Optimization of the environmentally-friendly reduction of GO
The application of reduced forms of GO in cancer therapy is ap-
pealing since these materials have a higher NIR absorption than GO,
resulting in an improved photothermal capacity (Robinson et al., 2011).
However, rGO is usually obtained by treating GO with HH, which is a
highly toxic hazardous reducing agent (Hussain & Frazier, 2002).
In order to avoid the problems associated with the use of HH, an
environmentally-friendly method was performed to accomplish the
reduction of GO (Fernández-Merino et al., 2010; Huang et al., 2015; Liu
et al., 2012; Zhang, Yang et al., 2010). For such, LAA was investigated
in the reduction of GO at two different concentrations (1.5 and 3 mM)
and various incubation times at 80 °C (Fig. 1A). Overall, by increasing
the LAA concentration and the reaction time, a darkening of the GO
solution and an increase on materials’ NIR absorption were attained
(Fig. S1 A and B). Nevertheless, the size distribution of the materials
remained un-affected for the conditions tested (Fig. S1 C and D). This is
of uttermost importance, since the reduction of GO greatly reduces its
solubility and could have increased the nanomaterials’ size through
agglomeration. Therefore, rGO obtained by using 3 mM of LAA and
60 min of reaction time was selected for the subsequent assays since it
presented a good balance between NIR absorption (mass extinction
coefficient of 12.67 L/(g.cm), at 808 nm) and preparation time.
In comparison to a previous study where LAA was used to reduce
GO (Zhang, Yang et al., 2010), we used a lower concentration of LAA (3
vs. 5.7 mM) and a shorter reaction time (1 vs. 12–48 h). Such results
attest the convenience of the optimized green reduction method.
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Carbohydrate Polymers 200 (2018) 93–99
3.2. Functionalization of rGO with HA-g-PMAO
A simple sonication method was used to functionalize rGO with HA-
g-PMAO (the successful synthesis of HA-g-PMAO was confirmed by FTIR
(Fig. S2 and S3), 1H NMR (Fig. S4 and S5) and TGA analysis (Fig. S6) -
reported in the supplementary information). In this process, the hy-
drophobic alkyl tail of HA-g-PMAO binds to the aromatic lattice of rGO
through non-covalent interactions (hydrophobic-hydrophobic interac-
tions). Moreover, the HA segments form a hydrophilic corona, which
may confer a cancer cell targeting capacity to this nanoformulation
(Fig. 1A).
The successful functionalization of rGO with HA-g-PMAO (HA-rGO)
was confirmed through FTIR analysis (Fig. 1B). Before functionaliza-
tion, rGO displays a peak at 1614 cm−1 (C]C stretch) and one with a
low intensity at 1737 cm−1 (C]O stretch), which also confirm the
restoration of the graphitic matrix of this material upon reduction.
Furthermore, the peak at 3258 cm−1 (OeH stretch) suggests that some
LAA traces are still present in rGO. The FTIR spectrum of HA-rGO
presents peaks at 2919 cm−1 (CeH stretch) and 1730 cm−1 (C]O
stretch), which belong to the alkyl and carbonyl groups of HA-g-PMAO,
respectively, thus confirming the successful preparation of HA-rGO.
The DLS analysis demonstrated that the functionalization of rGO
with HA-g-PMAO did not impact on its size distribution (Fig. 1C). In
fact, when compared to rGO, HA-rGO presents a monomodal size dis-
tribution, which suggests that the HA-based coating can improve the
stability of the nanostructures. The nanosized dimensions of HA-rGO
were then confirmed by TEM (Fig. S7), revealing that these materials
have an average lateral size of 108 ± 51 nm. These values are within
the optimal range for passive tumor accumulation through the EPR
effect (Hobbs et al., 1998). The zeta potential of HA-rGO was de-
termined to be slightly more negative than that of rGO (LAA stabilized)
(HA-rGO: −28.6 ± 1.0 mV; rGO: −26.9 ± 0.3 mV). Nevertheless, the
surface charge of HA-rGO is in line with that of other HA-based mate-
rials reported in the literature (He, Zhao, Yin, Tang, & Yin, 2009; Huang
et al., 2014; Li et al., 2012). Furthermore, rGO and HA-rGO exhibited a
similar NIR absorption, thus confirming that the functionalized rGO
preserved its photothermal potential (Fig. 2A).
The direct functionalization of as-prepared rGO with HA-g-PMAO
led to the production of nanomaterials with suitable physicochemical
properties for application in cancer therapy. In contrast, the functio-
nalization of GO reduced by HH usually requires an additional pur-
ification step to remove this toxic reductant (Mungse & Khatri, 2014;
Yang et al., 2012). This fact highlights the convenience of our approach.
3.3. Photothermal capacity of HA-rGO
The temperature variations induced, under NIR laser irradiation, by
HA-rGO were then studied to confirm the photothermal capacity of this
nanomaterial (Fig. 2B). In general, HA-rGO produced a time and con-
centration-dependent temperature increase when it was irradiated.
After 5 min of irradiation, HA-rGO could induce a temperature increase
of about 22 °C at 25 μg/mL (of rGO equivalents) (Fig. 2B). In turn, a
photoinduced heat of 33 °C was achieved for the highest concentration
tested (75 μg/mL of rGO equivalents) (Fig. 2B). Attaining such tem-
perature increase is of paramount importance since it can induce the
death of cancer cells (Chu & Dupuy, 2014; de Melo-Diogo, Pais-Silva,
Costa et al., 2017). Furthermore, the response of water (control) to NIR
light was meaningless (< 2 °C) (Fig. 2B). Together, these results attest
the photothermal efficiency of HA-rGO.
In a previous work, our laboratory developed a hydrothermal
treatment (80 °C for 24 h) to reduce GO and functionalize it with a PEG
derivative (de Melo-Diogo, Pais-Silva, Costa et al., 2017). When com-
pared to the previous protocol, the HA-rGO preparation method is more
convenient since it requires a lower reduction time (1 vs. 24 h) (de
Melo-Diogo, Pais-Silva, Costa et al., 2017). Moreover, HA-rGO also
produced a higher photoinduced heat (33 vs. 27 °C, at 75 μg/mL of rGO
R. Lima-Sousa et al.
Fig. 1. Preparation and characterization of HA-rGO. Schematic representation of the reduction and functionalization of rGO with HA-g-PMAO and its application in
cancer photothermal therapy (A). FTIR spectra of rGO, HA-g-PMAO and HA-rGO (B). DLS size distribution of GO, rGO (LAA stabilized) and HA-rGO (C).
equivalents) (de Melo-Diogo, Pais-Silva, Costa et al., 2017), thus dis-
playing an enhanced photothermal capacity.
3.4. Cytocompatibility of HA-rGO
Before assessing the phototherapeutic capacity of HA-rGO, first the
cytocompatibility of this nanomaterial when not irradiated was in-
vestigated in MCF-7 cells (breast cancer cell model) and NHDF (healthy
cell model). HA-rGO did not induce meaningful effects on the viability
of both cell lines, in the tested concentration range (1–75 μg/mL of rGO
equivalents) and incubation times (24 or 48 h) (Fig. 3). In contrast, rGO
(non-functionalized) was showed to induce some cytotoxicity to both
cells (Fig. S8). In this way, the functionalization of rGO with HA-g-
PMAO improved its cytocompatibility, which is fundamental for the
application of HA-rGO in cancer on-demand therapy.
HA-rGO displayed an enhanced cytocompatibility in comparison to
other functionalized rGO materials attained using HH reported in the
literature (Miao et al., 2013; Robinson et al., 2011). For instance, PE-
Gylated rGO displayed an IC50 towards MCF-7 cells of about 80 μg/mL
96
Carbohydrate Polymers 200 (2018) 93–99
(Robinson et al., 2011), while HA-rGO did not affect meaningfully the
viability of these cells up to 75 μg/mL (of rGO equivalents). In another
work, HH-reduced GO functionalized with HA decreased the viability of
epidermal carcinoma cells (KB cells) to about 80% at 40 μg/mL (of rGO
equivalents) (Miao et al., 2013). Therefore, the environmentally-
friendly approach used to reduce GO produces materials with improved
cytocompatibility upon functionalization with HA-g-PMAO, that may be
able to perform a spatial-temporal controlled therapy.
3.5. Targeting capacity of HA-rGO
To confirm the targeting capacity of HA-rGO, the internalization of
this nanostructure by MCF-7 cells (CD44 overexpressing cell line)
(Calcagno et al., 2010; Olsson et al., 2011) and NHDF (low CD44 ex-
pression) (Miyatake et al., 2013) was analyzed by CLSM. Prior to this
assay, HA-rGO was labelled with Rhod B for its cellular uptake be vi-
sualized (Zhang, Xia et al., 2010). The Rhod B labelling was confirmed
by analyzing the UV–vis spectrum of Rhod B labelled HA-rGO (Fig. 4A
and S9A), which displays an absorption peak at 575 nm that is
Fig. 2. Characterization of the photothermal
capacity of HA-rGO. UV-Vis absorption spectra
of GO (25 μg/mL), rGO (LAA stabilized; 25 μg/
mL of rGO equivalents), and HA-rGO (25 μg/
mL of rGO equivalents) (A). Temperature var-
iation curves of HA-rGO at different con-
centrations (of rGO equivalents) during 5 min
of NIR irradiation (808 nm, 1.7 W/cm2) (B).
R. Lima-Sousa et al. Carbohydrate Polymers 200 (2018) 93–99

Fig. 3. Evaluation of the cytocompatibility profile of HA-rGO. Cytocompatibility of HA-rGO towards MCF-7 cells (A) and NHDF (B) at different concentrations (of
rGO equivalents) for 24 and 48 h. Data represent mean ± SD, n = 5. K- and K + represent negative and positive controls, respectively.

attributed to the fluorescent probe (Zhang, Xia et al., 2010). Moreover,
DLS analysis confirmed that the Rhod B labelling did not affect the size
distribution of the nanomaterials (Fig. 4B). Taking into account that
rGO-based materials have a high fluorescence quenching capacity, we
then studied the fluorescence emitted by Rhod B labelled HA-rGO (Fig.
S9B). Compared to free Rhod B, the Rhod B labelled HA-rGO emitted
fluorescence with a lower intensity (Fig. S9B). Despite of the quenching
effect, the fluorescence signals emitted by Rhod B labelled HA-rGO
should still enable its use in CLSM studies (Zhong et al., 2016).
The uptake of Rhod B labelled HA-rGO by MCF-7 cells and NHDF
was then studied by CLSM (Fig. 4C). The results revealed that the in-
ternalization profile of the nanostructures by MCF-7 cells and NHDF
was different (Fig. 4C). Fluorescence signals with a higher intensity
were observed in the cytoplasm of MCF-7 cells incubated with Rhod B

Fig. 4. Targeting capacity of HA-rGO. UV-Vis absorption spectra of Rhod B labelled HA-rGO (A). DLS size distribution of HA-rGO and Rhod B labelled HA-rGO (B).
Representative CLSM images of Rhod B labelled HA-rGO and free Rhod B uptake by MCF-7 and NHDF cells (λex = 514 nm; λem = 513–703 nm) (C).

97
R. Lima-Sousa et al.
labelled HA-rGO when compared to those found on NHDF (Fig. 4C). As
expected, the internalization of free Rhod B appeared to be similar on
both types of cells (Fig. 4C). In other reports, nanomaterials functio-
nalized with HA also demonstrated a higher uptake by MCF-7 cells (Gao
et al., 2017; Wang et al., 2016). Taken together these results suggest
that HA-rGO has a preferential internalization by MCF-7 cells, thus
confirming the nanostructures’ CD44 targeting ability.
3.6. Phototherapeutic effect mediated by HA-rGO
After assessing the HA-rGO cytocompatibility and preferential up-
take by MCF-7 cells, the phototherapeutic effect mediated by this na-
nomaterial was investigated (Fig. 5A). HA-rGO produced, under NIR
laser irradiation, a dose-dependent reduction on MCF-7 cells’ viability
(Fig. 5B). At the lowest concentration tested (25 μg/mL of rGO
equivalents), HA-rGO did not induce meaningful cytotoxicity towards
cancer cells (viability of ≈ 96%). In stark contrast, the photothermal
effect mediated by HA-rGO at 75 μg/mL (of rGO equivalents) induced a
reduction of cancer cells’ viability to ≈ 6%. Furthermore, the use of
NIR light alone did not induce any toxicity on cancer cells (Fig. 5B).
Additionally, non-irradiated HA-rGO did not induce any deleterious
effect on cancer cells (Fig. 5B). Taken together, these results confirm
that HA-rGO can produce an on-demand therapeutic effect upon NIR
laser irradiation.
In another report, rGO produced using dopamine as a reducing
agent and functionalized with the antiarrhythmic peptide 10 (AAP10)
produced a phototherapeutic effect towards MCF-7 cells similar to that
of HA-rGO, although it required a higher power density (2–2.2 vs.
1.7 W/cm2) and a higher dose (120 vs. 75 μg/mL) (Yu et al., 2017). In
other work, the PTT mediated by arginine-glycine-aspartic acid (RGD)-
functionalized PEGylated rGO nanoribbons induced cancer cells abla-
tion at a low dose (10 μg/mL) but demanded a higher intensity (7.5 W/
cm2) and a longer period of irradiation (8 min) (Akhavan, Ghaderi, &
Emamy, 2012). In this way, HA-rGO is a promising nanomaterial for
CD44 targeted cancer PTT.
4. Conclusion
The two-major hypotheses of this research work were confirmed.
The environmentally-friendly reduction of GO was optimized con-
sidering nanomaterials’ NIR absorption and size distribution. On the
other hand, the HA-g-PMAO was successfully synthesized (confirmed by
FTIR, 1H NMR and TGA analysis) and explored in the coating of rGO.
The functionalization of rGO with HA-g-PMAO improved its stability
and cytocompatibility. Moreover, HA-rGO appeared to have a pre-
ferential internalization by CD44 overexpressing cells, which indicates
the targeting capacity of this nanoformulation. Furthermore, the on-
demand PTT mediated by HA-rGO induced cancer cells’ ablation.
Overall, HA-rGO is a promising nanomaterial for CD44 targeted cancer
98
Carbohydrate Polymers 200 (2018) 93–99
Fig. 5. Evaluation of the photothermal capa-
city of HA-rGO. Schematic representation of
the PTT mediated by HA-rGO (A). Effect of HA-
rGO in MCF-7 cells without NIR (w/o NIR) and
with NIR (w/ NIR) laser irradiation (808 nm,
1.7 W/cm2, 5 min) (B). K- w/o NIR represents
the negative control. K- w/ NIR represents cells
solely treated with NIR light. Data represents
mean ± SD, n = 5 (*p < 0.01; **p <
0.0001), n.s.= non significant.
PTT.
Declarations of interest
None.
Acknowledgements
This work was supported by FEDER funds through the POCI –
COMPETE 2020 – Operational Programme Competitiveness and
Internationalisation in Axis I – Strengthening research, technological
development and innovation (Project POCI-01-0145-FEDER-007491)
and National Funds by FCT – Foundation for Science and Technology
(Project UID/Multi/00709/2013). The funding from CENTRO-01-0145-
FEDER-028989 is also acknowledged. Duarte de Melo-Diogo and
Elisabete C. Costa acknowledge individual PhD fellowships from FCT
(SFRH/BD/103506/2014 and SFRH/BD/103507/2014).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.carbpol.2018.07.066.
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