Supporting Information

Hyaluronic acid functionalized green reduced graphene

oxide for targeted cancer photothermal therapy

Rita Lima-Sousa, Duarte de Melo-Diogo, Cátia G. Alves, Elisabete C. Costa, Paula

Ferreira, Ricardo O. Louro, Ilídio J. Correia*

1. Methods

1.1. Deacetylation of hyaluronic acid

Deacetylated hyaluronic acid (dHA) was prepared following a method described

elsewhere with slight modifications [​1​]. Briefly, 250 mg of hyaluronic acid (HA) were
dissolved in 12.5 mL of NaOH 13.7N. This solution was left to stir for 2 h at 60 ºC.
Afterward, the solution’s pH was adjusted to 7 with HCl 5N. The resulting solution
was dialyzed against water (3.5 kDa cut-off dialysis membrane) for 3 days and it was
then freeze dried (ScanVac CoolSafe, LaboGene Aps, Lynge, Denmark), yielding
dHA.

1.2. Opening of the anhydride ring in poly(maleic anhydride-​alt​-1-octadecene)

Hydrolyzed poly(maleic anhydride-​alt​-1-octadecene) (oPMAO) was produced by

adapting a method previously described in the literature [​2​]. In brief, 4 mL of a
solution of NaOH 2N was added to 200 mg of PMAO and allowed to stir for 5 h at
room temperature (RT). After adjusting the pH to 7, the opaque solution was dialyzed
against water (14kDa cut-off dialysis membrane) for 1 day and then was freeze dried,
yielding oPMAO.

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1.3. Preparation of HA-grafted PMAO

To obtain HA-grafted PMAO (HA-​g​-PMAO), oPMAO was conjugated with dHA

through EDC chemistry [​3​]. In brief, 50 mg of oPMAO was activated with EDC (27.3
mg) and NHS (16.4 mg) for 1 h (RT) in 25 mL of DMSO. Afterward, 50 mg of dHA
dissolved in water (25 mL) was added dropwise to the former solution. This solution
was allowed to stir for another 5 h at RT. Then, the solution was dialyzed against
water (14 kDa cut-off dialysis membrane) and it was freeze dried, yielding
HA-​g-​ PMAO (801.28 kDa).

​ MAO
1.4. Characterization of HA, dHA, PMAO, oPMAO and HA-​g-P

The average molecular weight of HA and HA-​g​-PMAO was determined in a Zetasizer

Nano ZS (Malvern Instruments, Worcestershire, UK). The deacetylation of HA, the
hydrolysis of PMAO and the grafting of dHA onto oPMAO were confirmed by Fourier
transform infrared spectroscopy (FTIR) using a Nicolet iS10 spectrometer (Thermo
Scientific Inc., MA, USA). Proton nuclear magnetic resonance (​1​H NMR) analysis was
also performed to confirm the deacetylation of HA and the synthesis of HA-​g​-PMAO
in a Brüker Avance III 400MHz spectrometer (Brüker Scientific Inc., NY, USA). For
such, HA (9:1 (v/v) H​2​O/D​2​O), dHA (9:1 (v/v) H​2​O/D​2​O), oPMAO (CDCl​3​) and
HA-​g-​ PMAO (1:1 (v/v) DMSO-d​6​/(9:1 (v/v) H​2​O/D​2​O)) were analyzed at 298 K. The
acquired spectra were processed in MNova software (Mestrelab Research, SL,
Santiago de Compostela, Spain). The samples were also analyzed by TGA by using
a SDT Q500 from Thermal Analysis (TA) Instruments. For the analysis, samples
were heated from 25 ºC to 600 ºC at a heating rate of 5 ºC/min under nitrogen purge.
Universal Analysis 2000 software was used for data analysis.

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2. Results

2.1. Optimization of the environmentally-friendly reduction of GO

Fig. S1. ​Vis-NIR absorption spectra of GO (25 µg/mL) and rGO (25 µg/mL) at
different times of reduction (A-B). DLS size distribution of GO (25 µg/mL) and rGO
(25 µg/mL) at different times of reduction (C-D). (A) and (C) refer to GO reduced with
1.5 mM of LAA. (B) and (D) refer to GO reduced with 3 mM of LAA.

2.2. FTIR characterization

FTIR analysis was performed to confirm the deacetylation of HA, the hydrolysis of
PMAO and the grafting of dHA onto oPMAO. The FTIR spectrum of HA exhibited a
peak at 1570 cm​-1 (NH deformation), that could be attributed to the secondary amides
of HA (Fig. S2A). The intensity of this peak decreased after deacetylation, thus
suggesting the successful of production of dHA (Fig. S2A).

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Previous to the hydrolysis, the FTIR spectrum of PMAO displayed peaks at 1856 cm​-1
and 1776 cm​-1​ (C=O stretches), which are characteristic of the anhydride ring
(Fig. S2B). After the base-catalyzed hydrolysis of PMAO, the previous peaks
disappeared (Fig. S2B). Furthermore, the FTIR spectrum of oPMAO displayed new
peaks at 1698 cm​-1 and 1558 cm​-1 (C=O stretches), which can be attributed to
carboxylic acid groups, thereby confirming the opening of the PMAO anhydride rings
(Fig. S2B).

Fig. S2.​ FTIR spectra of HA and dHA (A). FTIR spectra of PMAO and oPMAO (B).

HA-​g-​ PMAO FTIR spectrum (Fig. S3) displayed peaks at 2919 and 2850 cm​-1 (C-H
stretch), which are also present in the spectrum of oPMAO (Fig. S3). Furthermore, a
peak at 1043 cm​-1 (primary alcohol C-O stretch) is also present on the FTIR spectra
of dHA and HA-​g​-PMAO (Fig. S3). The spectrum of HA-​g-​ PMAO displays a peak at
1639 cm​-1 (secondary amide stretch), which confirm the conjugation of the primary
amines of dHA with the carboxylic acid groups of oPMAO (Fig. S3). Taken together
these results confirm the successful preparation of HA-​g-​ PMAO.

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Fig. S3.​ FTIR spectra of oPMAO, dHA and HA-​g​-PMAO.

2.3. NMR characterization of dHA and HA-​g-​ PMAO

1​
H NMR analysis confirmed the deacetylation of HA, since the base-catalyzed
treatment led to a reduction of the area of the peak at δ ≈ 2.0 ppm (-C​H​3​; acetyl
group protons) (Fig. S4). Based on this analysis, the deacetylation degree of dHA
was determined to be ≈ 21 %.

Fig. S4.​ 1​H NMR spectroscopy analysis of HA (A) and dHA (B).

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1​
H NMR was also performed to confirm the synthesis of HA-​g​-PMAO (Fig. S5). The
spectrum of oPMAO displays peaks at δ ≈ 1.2 ppm and δ ≈ 0.8 ppm (Fig. S5C),
belonging to the methylene (-C​H​2​) and methyl (-C​H​3​) protons of the alkyl chain of
PMAO, respectively [​4​]. In turn, the dHA spectrum presents several characteristic
peaks of HA, namely at δ ≈ 4.6-4.4 (-C​H​; anomeric protons) and 2.0 (-C​H​3​; acetyl
group protons) ppm (Fig. S5B) [​5​]. Furthermore, the peaks at δ ≈ 4.0-3.2 ppm
(remaining -C​H​2 and -C​H protons of HA sugar ring) present on dHA spectrum are
also characteristic of HA (Fig. S5B) [​5​]. In turn, the spectrum of HA-​g-​ PMAO (Fig.
S5A) displays peaks at δ ≈ 4.0-3.2 and 1.2-0.9 ppm, which are also present on dHA
(Fig. S5B) and oPMAO (Fig. S5C) spectra, respectively, thereby confirming the
grafting of dHA onto oPMAO.

S5. 1​​ H NMR spectroscopy analysis of HA-​g​-PMAO (A), dHA (B) and
 Fig.
oPMAO (C).
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2.4. TGA characterization of HA-​g-​ PMAO

TGA analysis also confirmed the successful synthesis of HA-​g​-PMAO (Fig. S6A).
The results demonstrated that the temperature of maximum degradation of dHA and
oPMAO occurred at 218 ºC and 425 ºC, respectively (Fig. S6B). In contrast, the first
maximum degradation temperature of HA-​g​-PMAO was determined to be at 230 ºC.
Such result suggests that the dHA portion on HA-​g​-PMAO display an enhanced
thermal stability. Moreover, the TGA thermogram of HA-​g​-PMAO presents a second
stage of degradation at 395 ºC that overlaps the degradation profile of oPMAO. Both
facts corroborate the grafting of dHA onto oPMAO.

Fig. S6. ​Thermogravimetric curves of dHA, oPMAO and HA-​g-​ PMAO (A) and
respective derivative thermogravimetric thermograms (B).

2.5. TEM characterization of HA-rGO

Fig. S7. TEM image of HA-rGO (A). Lateral size distribution of HA-rGO determined
by measuring the size of HA-rGO based on TEM images (B).
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2.6. Cytocompatibility profile of rGO

Fig. S8. Cytocompatibility of rGO at different concentrations towards MCF-7 cells

​ 0.05; **​p <
and NHDF after 48 h of incubation. Data represents mean ± SD (*​p <
0.01), n=5. K+ and K- represent positive and negative controls, respectively. n.s. =
non significant.

2.7. Characterization of Rhod B labelled HA-rGO

Fig. S9. UV-Vis absorption spectrum of free Rhod B (dissolved in water) (A).
Fluorescence spectra of free Rhod B, Rhod B labelled HA-rGO and water (excitation
at 560 nm) (B).

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References

[1] T. Wada, S. Chirachanchai, N. Izawa, Y. Inaki, K. Takemoto, Synthesis and

Properties of Hyaluronic Acid Conjugated Nucleic Acid Analogs—1: Synthesis of
Deacetylhyaluronan and Introduction of Nucleic Acid Bases, Journal of Bioactive and
Compatible Polymers, 9 (1994) 429-447.
[2] S.M. Henry, M.E.H. El-Sayed, C.M. Pirie, A.S. Hoffman, P.S. Stayton,
pH-Responsive Poly(styrene-alt-maleic anhydride) Alkylamide Copolymers for
Intracellular Drug Delivery, Biomacromolecules, 7 (2006) 2407-2414.
[3] J. Qiu, R. Cheng, J. Zhang, H. Sun, C. Deng, F. Meng, Z. Zhong,
Glutathione-Sensitive Hyaluronic Acid-Mercaptopurine Prodrug Linked via Carbonyl
Vinyl Sulfide: A Robust and CD44-Targeted Nanomedicine for Leukemia,
Biomacromolecules, 18 (2017) 3207-3214.
[4] E. Peng, E.S. Choo, C.S. Tan, X. Tang, Y. Sheng, J. Xue, Multifunctional
PEGylated nanoclusters for biomedical applications, Nanoscale, 5 (2013) 5994-6005.
[5] W. Zhang, H. Mu, D. Dong, D. Wang, A. Zhang, J. Duan, Alteration in immune
responses toward N-deacetylation of hyaluronic acid, Glycobiology, 24 (2014)
1334-1342.