Carbohydrate Polymers 181 (2018) 1–9

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

Research Paper

IR 780-loaded hyaluronic acid micelles for enhanced tumor-targeted T

photothermal therapy
Saji Uthamana,d, Ansuja Pulickal Mathewa, Hyeong Ju Parkb, Byeong-Il Leeb, Hyung-Seok Kimc,
⁎ ⁎
Kang Moo Huhd, , In-Kyu Parka,
a
Department of Biomedical Science, BK21 PLUS Centre for Creative Biomedical Scientists, Chonnam National University Medical School, 160 Baekseo-ro, Gwangju,
61469, Republic of Korea
b
Medical Photonics Research Center, Korea Photonics Technology Institute, Gwangju, 61007, Republic of Korea
c
Department of Forensic Medicine, Chonnam National University Medical School, 160 Baekseo-ro, Gwangju, 61469, Republic of Korea
d
Department of Polymer Science and Engineering, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon, 34134, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Keywords: In this study, we propose using IR 780-loaded, CD44-targeted hyaluronic acid-based micelles (HA-IR 780) for
Photothermal therapy enhanced photothermal therapy (PTT) effects in tumors. Two kinds of HA-C18 micelles were synthesized from
Laser different C18 feed ratios with degree of substitution of 3% and 13% respectively. Three different IR 780 weight
Tumor percentages were used for micelle formation with loading content of 4.6%, 7.9%, and 10.3% respectively. The
Micelles
IC50 value of HA-IR 780 in TC1 cells was 21.89 μg mL−1 (32.81 μM). Upon irradiation of the tumor site with an
Micelles
808-nm laser (2Wcm−2) for 2 min, the temperature in the tumor in the HA-IR 780-treated groups reached
Irradiation
49.9 °C which exceeds the temperature threshold to induce irreversible tissue damage. Toxicity studies showed
that HA-IR 780 does not cause any adverse effects in organs, including heart, liver, lungs, kidney and spleen,
although it selectively caused cell damage in the tumor region upon laser irradiation. Therefore, the present
study suggests that HA-IR 780 can cause selective cell death in tumor regions due to its enhanced tumor-tar-
geting and photothermal capabilities.

1. Introduction iodide has recently been reported to exhibit tumor accumulation

Cancer is a major health problem globally, with 1,688,780 new
cancer cases and 600,920 deaths projected to occur in the United States
in 2017 (Siegel, Miller, & Jemal, 2017). Currently available treatments
include surgery, radiotherapy and small molecule-based therapies such
as chemotherapy and hormone therapy. However, the utility of these
approaches is compromised by non-specific toxicity and drug resistance
mechanisms, which have prompted the development of new ther-
apeutic tactics (Pais-Silva, de Melo-Diogo, & Correia, 2017). Among the
various alternative strategies under investigation, photothermal
therapy (PTT) is an emerging treatment approach for cancer. PTT uses
photosensitizers to generate heat upon laser irradiation, leading to
cellular necrosis and apoptosis (Melamed, Edelstein, & Day, 2015). IR
780 iodide is a near-infrared (NIR) dye that is suitable for in vivo
imaging due to its excellent imaging depth, trivial background auto-
fluorescence and optical scattering (Kim, Chen et al., 2010). The
characteristic absorption peak of IR 780 is at 780 nm, which is within
the so-called biological transparency window (700–900 nm). IR 780
properties and has shown potential as an agent for cancer imaging,
photodynamic therapy (PDT) and PTT (Guo, Yu, Wang, Tan, & Li, 2015;
Jiang et al., 2015; Lin, Yuan et al., 2017; Pais-Silva et al., 2017; Qiu
et al., 2016; Wang et al., 2016; Yi et al., 2015; Yuan et al., 2015).
However, its biomedical applications have been limited due to its li-
pophilic nature, which makes it difficult to dissolve in water.
To improve the biomedical applicability of IR 780, an appropriate
formulation should be designed for efficient solubilization with phar-
maceutically acceptable solvents and enhanced accumulation in tumors
after systemic administration. Various nanostructures have been de-
veloped over the years to improve water solubility and photostability
by chemically conjugating IR 780 to polyethylene glycol (PEG) (Qiu
et al., 2016; Yuan et al., 2015) and hyaluronic acid (HA) (Lin, Yuan
et al., 2017). However, these chemical modifications affect IR 780
fluorescence (James et al., 2013) and limit the amount of IR 780 pre-
sent in the nanostructure. Conjugation of IR 780 to HA results in the
quenching of fluorescence when dissolved in water (Lin, Yuan et al.,
2017) and since the dye was chemically conjugated to the polymer, the

⁎
Corresponding authors.
E-mail addresses: khuh@cnu.ac.kr (K.M. Huh), pik96@jnu.ac.kr (I.-K. Park).

http://dx.doi.org/10.1016/j.carbpol.2017.10.033
Received 13 May 2017; Received in revised form 30 August 2017; Accepted 6 October 2017
Available online 13 October 2017
0144-8617/ © 2017 Elsevier Ltd. All rights reserved.
S. Uthaman et al.
amount of dye conjugated to polymer was dependent on the number of
functional group in the polymer. Physical encapsulation methods using
various nanomaterials have also been reported to overcome the dis-
advantages of chemically modified IR 780-based nanostructures (Jiang
et al., 2015; Pais-Silva et al., 2017; Wang et al., 2016; Yue et al., 2013).
Amphiphilic micelles have attracted much attention due to their many
advantages, such as (i) high encapsulation efficiency, (ii) suitable
physicochemical properties that benefit from an enhanced permeability
retention (EPR) effect, (iii) the inner hydrophobic core acting as a re-
servoir for non-covalent encapsulation of hydrophobic moieties, and
(iv) hydrophilic outer layer protecting the carrier from serum proteins
and cellular components (Gong, Chen, Zheng, Wang, & Wang, 2012;
Matsumura, 2008; Nesporova et al., 2016).
HA is a linear, biodegradable, and biocompatible polysaccharide
polymer that comprises alternating disaccharide units of D-glucuronic
acid and N-acetyl-D-glucosamine linked by alternate β (1 → 4) glyco-
sidic bonds (Jiang et al., 2008; Uthaman, Maya, Jayakumar,
Cho, & Park, 2014). HA has the ability to actively target tumor cells
over expressing CD44 receptor (Aruffo, Stamenkovic, Melnick,
Underhill, & Seed, 1990; Hu et al., 2016; Lin, Lee, & Shieh, 2017; Liu
et al., 2016; Yin et al., 2015). CD44 overexpression in cancer cells in-
creases the practical usage of HA as a basal material and as an ap-
pealing targeting moiety for constructing tumor-targeting nanocarrier
systems (Chen, He, Chen, Zhao, & Li, 2016; Eliaz & Szoka, 2001; Li
et al., 2016; Lin, Lee et al., 2017; Peterson, Yu, Stamenkovic, & Toole,
2000; Udabage, Brownlee, Nilsson, & Brown, 2005). In the present
study, CD44-targeted PTT was developed by non-covalently en-
capsulating IR 780 iodide in C18-conjugated HA (HA-C18) micelles and
investigated their tumor-targeting properties, fluorescent imaging and
PTT potential upon laser irradiation (Scheme 1). This study demon-
strates HA-IR 780 could preferentially accumulate in tumor tissue via
EPR effect and internalized into tumor cells via CD 44 mediated en-
docytosis. Inside tumor tissue, HA-IR 780 would be disassembled
through hyaluronidase mediated degradation of HA backbone. Finally,
the released IR 780 can generate photothermal ablation upon laser ir-
radiation. We believe that this is the first study in which IR 780 iodide
2
Carbohydrate Polymers 181 (2018) 1–9
has been non-covalently loaded into HA-C18 micelles and used for
tumor-targeted PTT.
2. Materials and methods
2.1. Materials
HA (0.48 MDa) was purchased from Bioland (Cheonan, South
Korea) and octadecyl amine (C18), formamide, pyrene, 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide
(NHS) and IR 780 iodide were purchased from Sigma-Aldrich (St Louis,
MO, USA). N, N-dimethylformamide was purchased from Merck
(Darmstadt, Germany). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carbox-
ymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) was pur-
chased from Promega (Madison, WI, USA). Dulbecco's modified Eagle's
medium (DMEM) and Roswell Park Memorial Institute medium (RPMI-
1640) were purchased from Thermo Scientific (Waltham, MA, USA). All
chemical reagents and solvents were purchased from a commercial
supplier unless specified.
2.2. Preparation of HA-C18
HA-C18 was synthesized as previously described with slight mod-
ifications (Liu et al., 2011). Briefly, 100 mg of HA was dissolved in 5 mL
of anhydrous formamide with gentle heating. After the HA was com-
pletely dissolved, a two-molar excess of EDC and NHS was added at
room temperature. After a 2-h incubation to activate the carboxylic
group of HA, C18 was added for conjugation. Briefly, C18 was dissolved
in DMF, added to carboxylic acid activated HA solution and stirred
under a nitrogen atmosphere for 5 h. The reaction temperature was
maintained at 60 °C and then stirred at room temperature for an ad-
ditional 24 h. The resultant mixture was further subjected to dialysis
(MWCO = 10,000, Spectra/Por®2) against approximately 4 L of ethanol
(100%, 70%) and distilled water for 1 day respectively. Finally, the
solution was filtered to remove impurities and then freeze-dried. The
synthesis of HA-C18 micelles was investigated by Proton Nuclear
Scheme 1. Schematic illustration of HA-IR 780 for-
mation and photothermal therapy mediated by HA-
IR 780.
S. Uthaman et al.
magnetic resonance (1H NMR) analysis (600 Hz, Bruker, Billerica, MA,
USA), with δ = 0.83 (CH2CH2CH2), δ = 1.23 (CH2CH3) and δ = 1.95
(eNHCOCH3).
2.3. Formulation of IR-780-loaded HA-C18 micelles (HA-IR 780)
HA-IR 780 micelles were prepared using a simple dialysis method.
First, 20 mg of HA-C18 was dissolved in 10 mL of distilled water and
1 mL of IR-780 solution (2 mg mL−1) in anhydrous DMSO was added
dropwise to the HA-C18 micelles solution at room temperature under
stirring condition, followed by 30 min of sonication over ice-bath using
a micro-tip probe sonicator (VCX-500, Sonics & Material Inc.,
Connecticut, USA) at 20% amplitude. After 30 min, the solution was
dialyzed against distilled water for one day to remove DMSO. The re-
sulting solution was filtered through a 0.45-μm filter and lyophilized.
The concentration of IR 780 loaded into the HA-C18 micelles (HA-IR
780) was determined by Spectroscopy (UV-2700 Shimadzu, Japan).
Briefly, lyophilized HA-IR 780 samples were dissolved in 1 mL of an-
hydrous DMSO to destabilize the micelle and release the encapsulated
IR 780. HA-C18 micelles dissolved in anhydrous DMSO were used as a
blank. The concentration of IR 780 in HA-IR 780 was analyzed by
comparing the IR 780 absorbance peak in anhydrous DMSO (801 nm)
with that of an IR 780 standard curve in DMSO. The loading efficiency
is expressed as the weight ratio of loaded IR 780 in the HA-IR780 mi-
celles to the total weight of IR 780 added initially.
2.4. Physico-chemical characterization of HA-IR 780
2.4.1. Particle size and zeta potential measurements
The average hydrodynamic diameter and zeta potential of HA-IR
780 micelles were measured via dynamic light scattering (DLS) using a
Zetasizer Nano Z (Malvern Instruments, Malvern, UK). All samples were
analyzed in triplicate (n = 3).
2.4.2. Critical micelle concentration (CMC) determination
To determine the minimum concentration of HA-C18 required for
micelle formation, the CMC was evaluated fluorescently using pyrene as
a fluorescent probe. (Liu et al., 2011) Briefly, different concentrations
of HA-C18, ranging from 1 × 10−4 to 1 mg mL−1, were added to the
pyrene so that the final pyrene concentration in each HA-C18 micelle
was 6 × 10−7M. The fluorescence spectra were recorded using a Safire
2 microplate reader (Tecan, Austria) (Excitation at 336 nm and emis-
sion scan from 360 to 450 nm).
2.4.3. NMR spectroscopy and field-emission transmission electron
microscopy (Fe-TEM)
The 1H NMR spectra of HA-C18 wererecorded by NMR spectro-
photometry at 30 °C (600 Hz, Bruker, Billerica, MA, USA). 10 mg mL−1
of HA in deuterium oxide (D2O) and 10 mg mL−1 HA-C18 was dis-
solved in deuterated dimethyl sulfoxide DMSO-d6/D2O (7:1). The
morphology of HA-IR780 was characterized by field-emission trans-
mission electron microscopy (JEM-2100F, USA).
2.5. Receptor expression analysis
Flow cytometry analysis was used to analyze CD44 expression levels
in TC-1 and NIH3T3 cells. Briefly, 1 × 106 cells were incubated with
FITC-conjugated anti-mouse/anti-human CD44 (eBioscience, cat no:
11-0441-81) for 30 min at 4 °C. After incubation, the cells were washed
three times with ice-cold PBS and re-suspended in FACS buffer. The
fluorescence intensity was analyzed using flow cytometry.
2.6. Cell viability
The in vitro cytotoxicity of HA-IR 780 was assessed in the TC-1
tumor cell line, which was derived from C57BL/6 mouse primary lung
3
Carbohydrate Polymers 181 (2018) 1–9
epithelial cells. TC-1 cells were seeded at a density of 1 × 104 cells
mL−1 and cultured overnight at 37 °C before treatment. After incuba-
tion overnight, the incubation medium was aspirated and replaced with
HA-IR 780 at various concentrations in OPTI-MEM® and incubated at
37 °C for 6 h. Afterwards, the media was removed and replaced with
100 μL of fresh media and incubated for 24 h at 37 °C. After 24 h, MTS
assay was performed according to the manufacturer’s protocol to de-
termine cell viability.
2.7. In vitro photothermal effect
To investigate the in vitro phototherapeutic effects of HA- IR 780,
cell viability and live-dead assays were performed. For cell viability
assays, the cells were seeded and samples treated as described above.
After 24 h of incubation, the samples were subjected to NIR irradiation
and MTS assay as per the manufacturer’s protocol to determine the cell
viability. To directly observe the phototherapeutic effect of HA-IR 780,
cells irradiated with the laser were washed with PBS, and fluorescence-
based live-dead assays were performed using fluorescein diacetate
(FDA) and propidium iodide (PI).
2.8. CD44-specific intracellular internalization of HA-IR 780 micelles and
CD44-specific competitive inhibition study
To determine the intracellular internalization of HA-IR780, 1 × 105
cells per well of TC-1 cells were seeded in Lab-Tek® Chamber Slides and
incubated for 24 h at 37 °C and 5% CO2. After 24 h of incubation, the
media was aspirated and HA-IR780 samples (10 μg mL−1) in OPTI-
MEM® were added and incubated for 6 h, followed by aspiration of the
media, three washes with 1X PBS and treatment with 4% paraf-
ormaldehyde. DAPI in gold anti-fade reagent was used for nuclear
staining. Confocal laser scanning microscopy was used to visualize the
fluorescence in the samples. For the CD 44-specific competitive in-
hibition assay, the medium was aspirated and supplemented with HA
polymer (8 mg mL−1) to block CD44 prior to sample treatment.
To further quantify uptake, 1 × 106 cells/well of TC −1 cells were
incubated overnight with HA-IR 780. The cells were harvested after
incubation, washed 4 times with 1X PBS, and quantified via flow cy-
tometry (FACScan).
2.9. In vivo biodistribution and tumor-targeting characteristics of HA-IR
780
All experiments involving live animals were performed in com-
pliance with the institutional guidelines of the Chonnam National
University Medical School and Chonnam National University Hospital
(CNU IACUC-H-2015-47), South Korea. TC-1 xenografts in mice models
were prepared by subcutaneously injecting 1.0 × 106 cells into the
flanks of inbred Balb/c nude mice (5 weeks, male) (Orient Bio Inc.,
Seongnam-si, South Korea). Upon reaching a tumor volume of ap-
proximately 100 mm3, the mice were randomly grouped, and HA-IR
780 samples (10 mg kg−1) were intravenously injected. The biodis-
tribution and tumor-targeting ability of the HA-IR 780 samples were
studied by fluorescence imaging at various time-points using a
Fluorescence-labeled Organism Bio-imaging Instrument (FOBI, NEO
science, Gyeonggi, Korea). All samples were examined in triplicate, and
sample accumulation was confirmed by quantifying the near-infrared
fluorescence (NIRF) intensity in various organs.
2.10. Ex vivo organ analysis and quantification
All the major organs and tumors were dissected 24 h post-injection
of the HA-IR 780 samples. NIRF images of the dissected organs and
tumors were acquired using a FOBI in vivo imaging system (exposure,
2 s) with IR excitation/emission filter sets. To quantify the extent of HA-
IR 780 accumulation in all the organs, the integrated density was
S. Uthaman et al.
measured from the NIRF intensity. To extract IR 780, the organs were
collected and homogenized in 5 mL of DMSO and centrifuged at
9000 rpm for 15 min. To quantify the extracted IR 780, the fluorescence
of IR 780 in the NIR range was quantified by UV spectroscopy.
2.11. In vivo photothermal therapy
The increase in temperature at the tumor region during NIR irra-
diation in the different experimental groups was evaluated by in-
travenously injecting PBS, IR 780 (1 mg kg−1) and HA-IR 780
(10 mg kg−1) into mice bearing TC-1 tumors. The temperature changes
in the tumor tissues during NIR irradiation (808 nm laser, 2 W cm−2 for
2 min) were measured at 24 h post-injection. The region of maximum
temperature and infrared thermographic images were obtained using
an infrared thermal imaging camera (Avio IR camera/Thermometer,
Shinagawa-ku, Tokyo).
In vivo photothermal therapy treatment was initiated when the
tumor diameter reached 100 mm3, with tumor-bearing mice randomly
divided into 6 groups: (1) PBS without laser irradiation, (2) PBS with
laser irradiation, (3) IR 780 without laser irradiation, (4) IR 780 with
laser irradiation, (5) HA-IR 780 without laser irradiation, and (6) HA-IR
780 with laser irradiation. All samples were administered intravenously
via tail-vein injection, and the tumor was exposed to 808 nm NIR laser
(2 W cm−2) for 2 min at 24 h post-injection (day 0). The tumor size and
body weight of each mouse were recorded every 3 days. The mice were
sacrificed on day 18, and heart, lung, spleen, liver, kidney and tumor
tissues were harvested. The tissues were fixed in 4% neutral formalin,
embedded in paraffin, sectioned and stained with hematoxylin and
eosin (H & E) for histological examination. After H & E staining, the
tissues samples were analyzed, and images were acquired on a light
microscope.
3. Results and discussion
3.1. Preparation and characterization of HA-C18 and HA-IR 780 micelles
HA-C18 polymers were fabricated by conjugating the HA carboxyl
group with the C18 amine group via EDC/NHS chemistry. 1H NMR
analysis was performed to determine the degree of substitution (DS) of
the octadecyl group on the HA backbone. Two kinds of HA-C18 micelles
were synthesized from different C18 feed ratios, and their micellar
properties were characterized (Table 1). Compared with the 1H NMR
spectrum of HC 3 in D2O, the HC 3 in DMSO-d6/D2O spectrum showed
new peaks at δ (ppm) 0.83 corresponding to methylene groups
(CH2CH2CH2) and δ (ppm) 1.23 corresponding to terminal methyl
groups (CH2CH3) of C18, as well as the characteristic HA peak at δ
(ppm) 1.50 for the acetamido moiety of the N-acetyl-D-glucosamine
residue (-NHCOCH3) (Fig. 1a). This is due to the fact that D2O is a good
solvent for hydrophilic HA and bad solvent for hydrophobic C18
groups. However, when HC 3 were dissolved in co-solvents of DMSO-
d6/D2O, both HA and C18 are exposed to the solvent. The DS of C18 (in
percentage, %) was calculated by integrating the peak intensity of the
HA −NHCOCH3 group and the peak intensity of the C18 CH2CH2CH2
groups. The DS for HC 1 and HC 3 were determined as 3% and 13%,
respectively. To calculate the minimum concentration of the conjugate
required for micelle formation, CMC analysis was performed by in-
cluding the hydrophobic fluorescence probe pyrene in the HA-C18
Table 1
Physicochemical properties of HA-C18 micelles.
Sample DS (%) CMC Average Hydrodynamic Average Zeta
(mg/ml) Diameter (nm) Potential (mV)
HC 1 3 0.95 501 ± 30 −20 ± 5
HC 3 13 0.65 150 ± 5 −20 ± 5
4
Carbohydrate Polymers 181 (2018) 1–9
micelles and was found to be 0.95 mg mL−1 and 0.65 mg mL−1 for HC
1 and HC 3, respectively. The average hydrodynamic diameter and zeta
potential of HC 1 and HC 3 were determined by DLS analysis. The
average hydrodynamic diameter of HC1 and HC 3 was 501 ± 30 nm
and 150 ± 5 nm, respectively. No significant differences were ob-
served between the average zeta potential values of HC 1 and HC 3. HC
3 micelles, which had a higher DS value, were chosen for the sub-
sequent experiments due to their lower CMC and smaller size.
The HA-IR 780 micelles were prepared via dialysis. Loading effi-
ciency was calculated as the amount of IR 780 in the micelle relative to
the total amount of IR 780 used in micelle formation. Three different IR
780 wt percentages (10%, 20%, and 30%) were used for micelle for-
mation, and the micelles were named HA-IR 780-10, HA-IR780-20 and
HA-IR 780-30, respectively. The loading efficiency of the HA-IR 780-10,
HA-IR780-20 and HA-IR 780-30 micelles was 4.6%, 7.9%, and 10.3%,
respectively. DLS measurements were performed to evaluate the
average hydrodynamic diameter of the HA-IR 780 micelles (Fig. 1b). As
shown in Fig. 1c, all of the formulations showed negative zeta poten-
tials, demonstrating the encapsulation of IR 780 in the hydrophobic
core of the micelles with HA exposed outside. FE-TEM images (Fig. 1d)
showed that the HA-IR 780 micelles had a spherical morphology with a
size below 200 nm. The stability of HA-IR 780 in serum solution was
investigated (Fig. S1). The results demonstrated the size of HA-IR 780
remains consistent over 24 h, indicating that HA-IR 780 were stable in
the presence of serum solution. The absorbance spectra of HA- IR 780
(Fig. 1e) were compared with that of IR 780. IR 780 showed an ab-
sorption peak at 801 nm in DMSO, whereas the IR 780-loaded HA mi-
celles showed a peak shift, likely due to the tight packing of the dye
inside the micelles.
To evaluate the degradation and release of IR 780 from HA-IR 780,
HA-IR-780 were dispersed in PBS (pH 7.4, 37 °C) with different con-
centration of hyaluronidase (0, 50, 100, 200, 500-unit mL−1). As shown
in Figure S.2, a hyaluronidase responsive release was observed. A sig-
nificant difference among the group is observed and the sustained re-
lease profile of HA-IR 780 demonstrate that the HA-IR 780 will display
good PTT effect under in vivo environmental conditions.
3.2. Cell viability
MTS assays were performed for analyze the effect of HA-IR 780
micelles on cell viability. Fig. 2a illustrates the cell viability profiles at
different HA-IR 780 micelle concentrations versus the same con-
centrations of IR 780 alone. A concentration-dependent cell viability
profile was observed for the HA-IR 780 micelles, while an equivalent
concentration of IR 780 alone showed a lower viability profile. Thus,
loading IR 780 into polymer micelles increases cell viability, which may
be due to the biocompatibility of HA (Dosio, Arpicco, Stella, & Fattal,
2016; Necas, Bartosikova, Brauner, & Kolar, 2008). The IC50 value of
HA-IR 780 in TC1 cells was 21.89 μg mL−1 (32.81 μM), and that of IR
780 was 8.399 μg mL−1 (12.59 μM) (Fig. 2b). From these results, HA-IR
780 micelles in the concentration range of 15.625 μg mL−1 to
0.977 μg mL−1 exhibit no acute or intrinsic cellular cytotoxicity in TC-1
cells.
3.3. CD44-specific intracellular internalization of HA-IR 780 micelles and
CD44-specific competitive inhibition
CD44 expression levels in NIH3T3 and TC-1 cells were evaluated
using FACS (Varghese, Liu, Sundaram, Hilborn, & Oommen, 2016).
FACS analysis revealed 4.3-fold higher CD44 expression in TC-1 cells
than in NIH3T3 cells (Fig. 2c). To investigate the CD44-specific cellular
internalization of HA-IR 780 micelles, TC-1 cells were chosen due to
their higher CD44 expression levels. Confocal microscopy was used to
evaluate the cellular uptake of HA-IR 780 micelles in TC-1 cells. As
observed in the confocal microscopy images (Fig. 2d), stronger fluor-
escence signals were observed in the cytoplasm of TC-1 cells treated
S. Uthaman et al.
Fig. 1. Physico-chemical characterization of HA-C18 micelles. (a) 1H NMR spectra of HA (D2O), HC 3 (D2O) and HC 3 (DMSO-d6/D2O, 7:1). Peak 1 − N-acetyl (eNHCOCH3) of HA; peak
2 terminal methyl groups (CH2CH3) of C18 and peak 3 methylene groups (CH2CH2CH2) of C18, concentration of all samples was 10 mg mL−1. (b), (c) Average hydrodynamic diameter
average and zeta potential of IR 780-loaded HA-C18 micelles (HA-IR 780-10, HA-IR780-20 and HA-IR 780-30). (d) FE-TEM image of HA-IR 780-30, inset higher magnification and e)
UV–vis spectroscopy of IR 780 (0.1 mg mL−1) in DMSO and HA-IR780 (1 mg mL−1) in distilled water.
with HA-IR 780 micelles, illustrating that HA-IR 780 micelles were
readily taken up by TC-1 cells. To confirm that the cellular uptake of
HA-IR 780 micelles is mediated by CD44-specific internalization,
competitive inhibition assays were performed by pretreating the cells
with free HA (Uthaman et al., 2016). Weaker fluorescence signals were
observed in HA-IR 780 micelle-treated TC-1 cells that were pretreated
with free HA compared with those in cells without any pretreatment
with free HA. In the case of IR 780-treated TC-1 cells, the presence or
absence of HA pretreatment had no effect on the fluorescent signals in
the cytosol. These results indicated that HA-IR 780 micelles specifically
bind to CD44 and are internalized via CD44-specific receptor-mediated
endocytosis.
3.4. In vivo biodistribution and ex vivo organ analysis and quantification
To evaluate the in vivo biodistribution and tumor-targeting ability of
HA-IR 780, non-invasive, real-time imaging was performed after the
intravenous injection of HA-IR 780 into TC-1 tumor-bearing mice. As a
control, IR 780 alone at the same concentration as that loaded into HA-
IR 780 was also intravenously injected. Fluorescence images were ac-
quired at different time intervals via whole-body fluorescence intensity
(Fig. 3a). The IR 780 fluorescence signals were distributed throughout
the body until 24 h post-injection; in contrast, HA-IR 780 specifically
accumulated with in the tumor by 24 h post-injection. However, in non-
tumor bearing mice at 24 h post injection doesn’t showed any sig-
nificant different in the biodistribution profile among HA-IR 780 and IR
780 (Fig. S3). The tumor-specific preferential accumulation of HA-IR
780 might be due to the improved stability of IR 780 during blood
circulation upon encapsulation inside a micellar structure, with the
nanoscale size aiding accumulation within the tumor via an EPR effect,
(Chen, Li et al., 2016) followed by CD44-based cellular uptake (Kim,
Hur et al., 2010). It has also been reported that free IR-780 possess
preferential accumulation into tumor cells (Kuang et al., 2017; Yue
et al., 2013). As evident from the biodistribution profile in non-tumor
bearing mice and tumor bearing mice, more accumulation of HA-IR 780
was found in tumor bearing mice due to tumor specific accumulation,
followed by hyaluronidase mediated release of IR 780. Ex vivo analysis
5
Carbohydrate Polymers 181 (2018) 1–9
of excised organs at 24 h post-injection was performed to evaluate the
distribution of IR 780 (Fig. 3b). The highest HA-IR 780 micelle fluor-
escence signal was observed in the tumor, which was in accordance
with whole-animal imaging. Compared with the other organs, the liver
showed the highest fluorescence, which might be due to the cellular
uptake of the HA-IR 780 micelles by liver sinusoidal endothelial cells,
which express HARE receptors and are the phagocytic cells in the re-
ticuloendothelial system (Platt & Szoka, 2008; Zhou, Weigel,
Fauss, & Weigel, 2000). To quantify the IR 780 levels in each organ,
DMSO was used as an extracting solvent due to its easy miscibility with
water, better penetration into cell membranes and frequent use as a
solvent for drug molecules and cryoprotectants (Yu & Quinn, 1998). As
shown in Fig. 3d, a negligible amount of IR 780 was recovered from the
lungs, kidney, heart and spleen. Higher amounts of IR 780 were found
in the tumor, followed by the liver, which is consistent with the ex vivo
organ biodistribution.
3.5. In vitro and in vivo photothermal therapy
The in vitro photothermal efficacy of HA-IR 780 was investigated
using MTS assays (Fig. 4a). The groups treated with HA-IR 780 and IR
780 at the same concentrations without laser irradiation were used as
controls. Upon laser irradiation, cell viability was greatly reduced,
especially in the 25, 12.5 and 6.25 μg mL−1 groups. Similar results
were observed for the IR 780-treated groups. One possible explanation
for the high viability in the absence of laser irradiation and the lower
viability in the presence of laser irradiation could be the heat-gen-
erating properties of IR 780 upon laser irradiation (Yue et al., 2013). To
visualize the effectiveness of HA-IR 780 in killing the cells, live-dead
assays were performed using FDA and PI. As shown in Fig. 4b, no cell
death was observed in the cell-only (without HA-IR 780 treatment)
group, even after laser irradiation, whereas considerable cell death was
observed in the circular region, which coincided with the laser spot.
These results suggest that HA-IR 780 is non-toxic to cells and shows
selective toxicity upon laser exposure.
To investigate the increase in temperature in the tumor tissues
during PTT, TC-1 tumor-bearing mice were injected with PBS, free IR
S. Uthaman et al. Carbohydrate Polymers 181 (2018) 1–9

Fig. 2. (a) Cell viability profile by MTS assay. (b) IC50 values of TC-1 cells treated with various concentrations of HA-IR 780 and IR 780. (c) FACS analysis to determine CD44 expression in
TC-1 and NIH3T3 cells. Untreated cells are denoted as the positive control (P.C.), whereas cells treated with Triton X-100 are denoted as the negative control (N.C.). Viability is expressed
as a percentage of the positive control. Values are expressed as the mean ± SD. n = 4. (d) Confocal microscopy analysis of the CD44-based cellular uptake of HA-IR780 in TC-1 cells
treated as follows: (i) Untreated TC-1 cells, (ii) TC-1 cells treated with IR 780 (1 μg mL−1) (iii) TC-1 cells treated with IR 780 (1 μg mL−1) after HA treatment, (iv) TC-1 cells treated with
HA-IR 780 (10 μg mL−1) after HA treatment and (v) TC-1 cells treated with HA-IR 780 (10 μg mL−1).

780 and HA-IR 780 (1 mg kg−1 of IR 780) via tail-vein injection. TC-1
tumor-bearing mice were then irradiated with 808 nm laser light
(2 W cm−2) for 2 min at 24 h post-injection. After 2 min of irradiation,
the temperature in the tumor in the HA-IR 780-treated groups reached
49.9 °C (Fig. 4C), which exceeds the temperature threshold to induce
irreversible tissue damage (Peng et al., 2011). However, the IR 780 and
PBS-treated groups attained maximum temperatures of 44.2 °C and
41.0 °C, respectively, which are insufficient to cause irreversible tissue
damage (Wang et al., 2016; Yue et al., 2013). However, in non-tumor
bearing mice no increase in temperature (Fig. S4) was observed after
irradiated with 808 nm laser light (2 W cm−2) for PBS, free IR 780 and
HA-IR 780.
To explore the photothermal ablation efficacy of HA-IR 780, TC-1
tumor-bearing mice were intravenously injected with PBS, free IR 780
and HA-IR 780 via tail-vein injection. The tumor region was then ir-
radiated with 808 nm laser light (2W cm−2) for 2 min at 24 h post-in-
jection. Groups treated with PBS, free IR 780 and HA-IR 780 without
laser irradiation served as controls. As shown in Fig. 5a, the HA-IR 780-
treated groups that received laser irradiation showed significant re-
ductions in tumor volume compared with that in the other groups,
which showed a 16–18-fold increase in tumor volume from day 0 to day
18. Tumor weight was also measured 18 days of post-irradiation, and as
shown in Fig. 5b. Tumor weight was substantially lower in the HA-
IR780 treated groups; in fact, only scar tissue remained at the tumor
site. These results show that HA-IR 780 possesses obvious
phototherapeutic abilities to suppress tumors. We also investigated the
potential toxic side effects of HA-IR780 treatment by analyzing the loss
of body weight, which is often used as an indicator for treatment-in-
duced toxicity (Yue et al., 2013). As shown in Fig. 5c, no significant
body weight loss was observed. H & E staining was also performed to
investigate the toxic effects of HA-IR 780 treatment in various organs,
as well as the anti-tumor effects of HA-IR 780. As shown in Fig. 5d,
none of the treatments caused any apparent lesions (e.g., edema, in-
flammation, hyperplasia or necrosis) in the heart, liver, lungs, kidney or
spleen. However, tumor sections from the group treated with HA-IR 780
showed significant necrosis after laser irradiation. These results suggest
that HA-IR780 does not cause adverse effects in the heart, liver, lungs,
kidney and spleen while causing selective cell damage in the tumor
region upon laser irradiation.
4. Conclusion
We have developed IR 780-loaded tumor-targeted micelles (HA-IR
780), which absorb in the NIR region. EDC/NHS chemistry was used for
the grafting of C18 to HA. HA-C18 micelles showed an CMC of
0.6 mg mL−1. The encapsulation of IR 780 in HA-C18 micelles sig-
nificantly improves the water solubility of IR 780 and have spherical
morphology with an average diameter less than 200 nm. HA-IR 780 are
non-toxic to tumor cells but promote significant toxicity upon laser ir-
radiation. HA-IR 780 also exhibited CD44- and EPR-based tumor

6
S. Uthaman et al. Carbohydrate Polymers 181 (2018) 1–9

Fig. 3. In vivo biodistribution of HA-IR 780. (a) Biodistribution of HA-IR 780 in TC-1 tumor-bearing mice with respect to different post-injection time-points observed by NIRF imaging.
(b) Ex vivo NIRF images of the tumor and main organs (lungs, heart, liver, spleen, kidney and intestine) 24 h post-injection. (c) The quantitative NIRF intensity of the tumor and main
organs 24 h after the intravenous injection of HA-IR 780 micelles (n = 3). (d) Biodistribution of HA-IR 780 within the lungs, kidney, heart, spleen, liver and tumor 24 h post-injection.

Fig. 4. (a) Cell viability profile of TC-1 cells treated with HA-IR 780 and IR 780 at various concentrations in the presence and absence of laser irradiation. (b) Live-dead assay using FDA
and PI. (c) Infrared thermal images of TC-1 tumor-bearing mice after 24 h post-injection with HA-IR780, IR 780, or PBS and exposure to 808 nm laser light (2 Wcm−2).

7
S. Uthaman et al.
Fig. 5. PTT and toxicity of HA-IR 780 in vivo. (a) Tumor growth in TC-1 tumor-bearing mice after the indicated treatments. (b) Tumor weight 18 days post laser treatment. (c) Body
weights of mice after the indicated treatments. (d) Histopathological analysis of organs and tumors harvested from TC-1 tumor-bearing mice treated with PBS, IR 780 and HA-IR 780 at
24 h post-injection. Scale bar = 200 μm.
accumulation, and tumor regions can be visualized via fluorescence.
Upon irradiation of the tumor tissue with an 808-nm laser, HA-IR 780
produced photothermic ablation without causing any toxicity to other
organs. These results clearly demonstrated that HA-IR 780 could be
potential candidate for a safe PTT agent.
Disclosures
The authors declare no competing financial interests.
Author contributions
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
Ethical approval
All animal experiments were carried out under the guidelines of the
Chonnam National University Medical School and Chonnam National
University Hospital (CNU IACUC-H-2015-47), South Korea and in ac-
cordance with the principles of laboratory animal care (NIH publication
No. 80-23).
Acknowledgements
This work was financially supported by the Basic Science Research
Program (NRF-2015R1D1A1A09056741, 2016K2A9A1A06921661,
2016R1A2B4011184 and 2017M3A9F5030940) through the National
Research Foundation of Korea (NRF) funded by the Ministry of Science,
ICT & Future Planning; the Industrial Technology Innovation Program
(10060059, Externally Actuatable Nanorobot System for Precise
8
Carbohydrate Polymers 181 (2018) 1–9
Targeting and Controlled Releasing of Drugs) funded by the Ministry of
Trade, Industry and Energy (MOTIE, Korea); and the Pioneer Research
Center Program through the National Research Foundation of Korea
funded by the Ministry of Science, ICT & Future Planning
(2014M3C1A3053035). IKP also acknowledges the financial support
grant (CRI16071-3) from the CNUH-GIST.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.carbpol.2017.10.033.
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