Polyhedron 249 (2024) 116793

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Polyhedron
journal homepage: www.elsevier.com/locate/poly

New hyaluronan-terpyridine conjugate: Metal complexes and their

biological activity
Roberta Panebianco a, Maurizio Viale b, Graziella Vecchio a, *
a
Dipartimento di Scienze Chimiche, Università degli Studi di Catania, Viale A. Doria 6, 95125 Catania, Italy
b
IRCCS Ospedale Policlinico San Martino, U.O.C. Bioterapie, L.go R. Benzi 10, 16132 Genova, Italy

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

Keywords: Terpyridine coordination compounds have attracted broad interest concerning their biomedical applications. We
Cancer devised a terpyridine-hyaluronic acid conjugate and studied its copper(II), iron(II) and zinc(II) metal complexes.
Copper We determined the antiproliferative activities of the ligand and the metal complexes in human cell lines A2780
Hyaluronic acid
(ovary, adenocarcinoma), A549 (lung, carcinoma), MDA-MB-453 (breast, carcinoma), SKHep1 (liver, carcinoma)
Iron
Zinc
and compared them to 4′-Chloro-2,2′:6′,2′’-terpyridine systems. The terpyridine-hyaluronan conjugate showed
pharmacologically significant antiproliferative activity in all cell lines tested. The ternary metal complexes of
terpyridine-hyaluronan conjugate and 4-chloro-terpyridine showed significant IC50 values (nanomolar range)
depending on the cell line. Additionally, Cu2+ and Zn2+ ternary complexes exhibited higher antiproliferative
activity than Doxorubicin.

1. Introduction some tpy metal complexes could affect cell activity by inhibiting the
normal function of nucleic acids and enzymes [18,23–27].
Metal complexes have been investigated as potential therapeutics However, tpy metal complexes are hardly water-soluble, but their
because of their biological activity [1]. Despite the development of functionalisation can improve water solubility. It is known that the
metallodrugs based on second or third-row transition metal ions, dissolution in aqueous media allows it to reach therapeutic concentra­
remarkable results have been obtained with new metallodrugs based on tion. Various approaches have been investigated to overcome the poor
the naturally more abundant and intrinsically less toxic first-row tran­ solubility issue and increase bioavailability [28,29].
sition metal ions. Zinc(II), copper(II), cobalt(II), and iron(II) are of pri­ Amongst them, carbohydrate conjugation stands out in obtaining
mary consideration due to their biocompatibility and involvement in new hybrids with improved solubility, bioavailability, enhanced bio­
biological processes in the human body [2–5]. It has been observed that logical activity and selective targeting properties [30,31].
various coordination compounds containing these metals exhibit Hyaluronic acid or hyaluronan (HA) is an endogenous polymer of D-
outstanding anticancer efficacy [6–10]. glucuronic acid and N-acetyl-D-glucosamine [32,33]. HA is the principal
In this context, terpyridine (tpy) derivative coordination compounds component of the extracellular matrix (ECM) and is involved in different
have attracted wide interest concerning biomedical applications biological processes, such as cell proliferation and cell migration, and it
[4,9,11]. regulates the inflammatory state of the ECM. Exogenous HA fragments
Terpyridine is an N-heterocycle pincer-ligand with a high binding of low molecular weight have been shown to affect cell behaviour
affinity towards transition metal ions, such as iron(II), zinc(II), and through binding to CD44 and RHAMM (Receptor for Hyaluronan
copper(II). This characteristic has been widely used to build supramo­ Mediated Motility) receptors [34].
lecular systems, mainly soluble in organic solvents [12–14]. In particular, the CD44 receptor belongs to a family of cell adhesion
Tpy metal complexes have been used in medicinal chemistry because molecules [32,35]. It is a widely distributed transmembrane glycopro­
of their peculiar structure and range of biological activities [15–18]. tein that plays a critical role in malignant cell activities, including
Indeed, the anticancer activity of tpy and its coordination compounds adhesion, migration, invasion, and survival; it is also strongly implicated
has also been reported in recent years [19–22]. It has been proven that in the cell signalling cascades associated with cancer [36]. CD44 is a

* Corresponding author.
E-mail address: [email protected] (G. Vecchio).

https://doi.org/10.1016/j.poly.2023.116793
Received 17 September 2023; Accepted 12 December 2023
Available online 14 December 2023
0277-5387/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
R. Panebianco et al. Polyhedron 249 (2024) 116793

from the Varian library.

2.3. UV–Vis spectroscopy

UV–Vis spectra were recorded with a Cary 3500 UV-Vis spectro­

photometer equipped with a Peltier temperature control module.
UV–Vis titrations were carried out in phosphate buffer (pH = 7.4).
HAtpy was dissolved in the buffer. Freshly prepared FeSO4 solution in 2
mM H2SO4, Zn(ClO4)2 and Cu(NO3)2 were used. The titrant (metal so­
lution) was added by a Hamilton micro-syringe to the solution of the
ligand in the cuvette, under stirring at 25 ◦ C.

2.4. Dynamic light scattering

Fig. 1. Tpy conjugated with HA (n = 30, x = 7). Dynamic light scattering (DLS) measurements were performed with
Zetasizer Nano ZS (Malvern Instruments, UK) operating at 633 nm
critical component in the internalisation and metabolism of HA and is (He–Ne laser) at 25 ◦ C. The mean hydrodynamic diameter (d) of the NPs
expressed at low levels in various cell types. Tumour-derived cells, was calculated from intensity measurement after averaging ten mea­
however, express CD44 in a high-affinity state that can promote the surements. The samples (4 mg/ml) were prepared in phosphate buffer
binding and internalisation of HA [33]. Aside from this targeting ability, (pH 7.4) in ultrapure filtered water (0.2 μm filter).
HA nanosystems show several interesting features, such as very low
immunogenicity, biodegradability, non-inflammatory reactions, 2.5. Synthesis of HAtpy
bioavailability and biocompatibility [32,34]. Also, hyaluronidase de­
grades HA to low molecular weight components [34]. HA (150 mg, 0.013 mmol) was dissolved in 15 ml of MilliQ water. An
Due to these biological features, there is a prominent interest in aqueous solution of DMTMM (128 mg, 0.463 mmol) and an acetonitrile
obtaining HA-based functional biomaterials [34]. solution of TpyNH2 (142 mg, 0.40 mmol) were prepared. DMTMM and
HA-based NPs have been widely used as drug delivery systems TpyNH2 were added to the HA solution under stirring.
[37–40]. Specifically, HA-drug conjugates enhance drug circulation After about 12 h, unreacted TpyNH2 precipitated. The white solid
time, stability, solubility and accumulation in CD44 overexpressing was eliminated by centrifugation. The supernatant solution was purified
cancer cells [40,41]. through a Sephadex G-15 column eluted with MilliQ water.
1
The HA affinity towards some biometals has also been studied H NMR (500 MHz, D2O) δ(ppm): 8.86–8.66 (br. s, H6, H6″, H3, H3″
[42–44]. Although, few derivatives of HA with metal chelators have of tpy), 8.62–6.35 (br. s, H3′, H5′, H4, H4″ of tpy), 7.97–7.76 (br. s, H5,
been synthesised [45–47]. H5″ of tpy), 4.46 (br. s, H-1 of D-glucuronic acid unit), 4.39 (s, H-1 of N-
Based on the interest in HA properties, here we report a new multi­ acetyl-D-glucosamine unit), 4.34 (sh, a of propylenic linker), 3.89–3.12
metal system based on the coordination properties of tpy functionalising (m, H-2, H-3, H-4, H-5, H-6 of HA backbone and c of propylenic linker),
HA polymers (Fig. 1). Particularly, we functionalised low molecular 2.06 (m, b of propylenic linker), 1.91 (s, CH3 of HA).
weight HA (about 11 kDa) with tpy moieties to exploit the coordination
ability of tpy in aqueous solutions. We studied metal complexes of the 2.6. Synthesis of HAtpy metal complexes
new derivative with zinc(II), copper(II) and iron(II). We also determined
the antiproliferative activities of the HAtpy derivative and its metal Cu2+, Fe2+ and Zn2+ complexes were synthesised by adding
complexes in human cell lines A2780 (ovary, adenocarcinoma, respectively Cu(NO3)2, FeSO4 or Zn(ClO4)2 water solution to the ligand
HTL98008), A549 (lung, carcinoma, HTL03001), MDA-MB-453 (breast, water solution in a 1:2 M/L (L is tpy unit) molar ratio. Ternary com­
carcinoma, HTL01013), SKHep1 (liver, carcinoma) and compared them plexes were synthesised by adding Cu(NO3)2, FeSO4 or Zn(ClO4)2 water
to free tpy systems. The polysaccharide backbone confers water solu­ solution to HAtpy and TpyCl in water/DMSO 10/1 solution at a M/L/
bility to the tpy-based system and may extend the application of tpy TpyCl 1:1:1 (L is tpy unit) molar ratio.
chemistry in water solutions. HAtpy-Cu-TpyCl: UV–Vis spectrum λ nm (ε, L M− 1cm− 1): 270 (sh,
96000), 276 (97300), 286 (85300), 312 (sh, 45300), 325 (60000), 337
2. Materials and methods (54700).
HAtpy-Fe-TpyCl: UV–Vis spectrum λ nm (ε, L M− 1cm− 1): 273
2.1. Materials (111000), 282 (sh, 93300), 316 (97300), 509 (sh, 16000), 558 (25300).
HAtpy-Zn-TpyCl: UV–Vis spectrum λ nm (ε, L M− 1cm− 1): 266 (sh,
Commercially available reagents were used directly unless otherwise 86700), 274 (94700), 282 (89300), 322 (82700), 330 (68000).
noted. Hyaluronic acid (~30 units, 8–15 kDa) was purchased from
Carbosynth, 4′-Chloro-2,2′:6′,2′’-terpyridine (TpyCl) and DMTMM (4- 2.7. Evaluation of the antiproliferative activity
(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride)
were purchased from TCI (Tokyo, Japan). Human cell lines A2780 (ovary, adenocarcinoma, HTL98008), A549
3-([2,2′:6′,2′’-terpyridin]-4′-yloxy)propan-1-amine (TpyNH2) was (lung, carcinoma, HTL03001), MDA-MB-453 (breast, carcinoma,
synthesised as reported elsewhere [48]. HTL01013) (all these cell lines obtained from Interlab Cell Line
TLC (Thin layer chromatography) was carried out on silica gel plates Collection, Genova, Italy), and SKHep1 (liver, carcinoma, 52 HTB, ob­
(Merck 60-F254). Carbohydrate derivatives were detected on TLC with tained from American Type Culture Collection, Rockville, MA, USA),
UV light and the anisaldehyde test. were plated in 180 μl into flat-bottomed 96-well microtiter plates at the
opportune numbers per well in complete media (RPMI 1640 or DMEM)
2.2. NMR spectroscopy added with antibiotics and 10 % fetal bovine serum (FBS). After 6–8 h,
cells were administered with 20 μl containing five concentrations of
1
H NMR spectra were recorded with a Varian UNITY PLUS-500 HAtpy derivatives diluted in water plus 0.75 % DMSO.
spectrometer at 499.9 MHz at 300 K, using standard pulse programs Plates were then processed as described elsewhere [49]. The NP

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R. Panebianco et al. Polyhedron 249 (2024) 116793

Fig. 2. 1H NMR spectrum of HAtpy (D2O, 500 MHz).

concentrations inhibiting 50 % cell growth (IC50) were calculated based

on the analysis of the concentration–response curves. Each experiment
was repeated 4–5 times. IC50 values higher than 30 µM were considered
pharmacologically irrelevant.

2.8. Statistical analysis of data

The Student’s t-test for parametric data was used for statistical
comparisons.

3. Results and discussion

HAtpy conjugate was synthesised by coupling HA with TpyNH2 by

amidation reaction, using DMTMM as the coupling agent (Fig. S1).
1
H NMR and UV–vis spectroscopy confirmed the conjugation of tpy
with HA. The 1H NMR spectrum of HAtpy (Fig. 2 and S2) shows broad
signals, as typically found for polymers. 1H NMR spectrum of HA is re­
ported for comparison in Fig. S2.
In the 1H NMR spectrum, the broad peaks between 8.86 and 7.76
ppm are due to the tpy protons. H-1 protons of the D-glucuronic acid and
N-acetyl-D-glucosamine of HA resonate at 4.5–4.3 ppm. Other HA pro­ Fig. 3. UV–Vis spectrophotometric titration of HAtpy (6.0x10-6 M, phosphate
tons resonate from 4.0 to 3.0 ppm. The signal at 1.91 ppm was assigned buffer pH 7.4) with Fe2+. Inset: Absorbance at 560 nm upon the addition
to the methyl group in the HA backbone. The b CH2 of the propylenic of Fe2+.
chain of the tpy linker resonates at 2.06 ppm. Other CH2 resonances (a
and c) overlap with HA signals (3.5 and 4.3 ppm). From the integration 3.1. Metal complexes of HAtpy
of aromatic signals of tpy and –CH3 of HA, a degree of functionalisation
of about 25 % was estimated (about 7 of the 30 repeating disaccharide To study the complexation properties of HAtpy, we carried out
units of HA were functionalised with tpy). UV–Vis titrations of HAtpy with M2+ salt solutions (M = Fe, Cu, Zn) in
UV–vis spectrum of the HAtpy ligand showed bands at 240 and 280 phosphate buffer at pH = 7.4. The titration curves showed characteristic
nm with a shoulder at 300 nm due to π-π* transitions, as reported for tpy bands.
(Fig. S3). During the titration, a new band arose at about 313 nm (Fig. 3). This
The particle size distribution of HAtpy was investigated with DLS. band is characteristic of the conformational change of tpy from the
HAtpy forms NPs in water at pH 7.4. The DLS spectra of HAtpy showed a trans/trans conformation of the free ligand to the perfectly planarised
main population with a diameter of the hydrodynamic volume 8 nm ± 4 cis/cis conformation in the complex [50].
nm (Fig. S4, Z-average 11 nm). The functionalisation of HA with tpy did In the case of the Fe2+ system, an intense band appeared in the
not change the size of the nanoparticles but its peak size distribution (HA visible region at 560 nm due to a metal-to-ligand charge transfer
diameter is 9 nm ± 4 nm, Z-average 26 nm). (MLCT). The titration showed a clear isosbestic point at about 300 nm,
suggesting that only two species, the free ligand and the metal complex,
are present during the titration process. Spectra changed up to a Fe2+/
HAtpy 3.5 or Fe2+/L (L is tpy unit) 1:2 ratio (Fig. 3), considering the

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R. Panebianco et al. Polyhedron 249 (2024) 116793

Table 1
IC50 values (µM) of HAtpy, its binary Cu2+, Fe2+ and Zn2+ complexes (M/L 1:2) and ternary complexes with TpyCl (M/L/TpyCl 1:1:1, L is tpy unit)). [M(TpyCl)2]2+ are
reported for comparison.
Cell lines HAtpy HAtpy-Fe HAtpy- HAtpy- HAtpy-Fe- HAtpy-Cu- HAtpy-Zn- TpyCl [Fe [Cu [Zn
Cu Zn TpyCl TpyCl TpyCl (TpyCl)2]2+ (TpyCl)2]2+ (TpyCl)2]2+

A2780 4.91 ± >30 6.68 ± >30 0.17 ± 0.04 ± 0.12 ± 0.31 ± 0.22 ± 0.04 0.26 ± 0.01 0.06 ± 0.01e
0.97 2.88 0.02a 0.02b 0.04c 0.08
A549 4.45 ± >30 >30 >30 0.51 ± 0.05 ± 0.06 ± 0.01 0.42 ± 0.26 ± 0.05 0.28 ± 0.02 0.04 ± 0.01e
0.94 0.09b 0.01b 0.19
SKHep1 11.5 ± 1.74 ± >30 ND >30 >30 ND 0.64 ± 0.45 ± 0.14 0.32 ± 0.14 ND
1.0 0.66 0.26
MDA-MB- 2.68 ± >30 29.4 ± >30 13.9 ± 3.9 0.09 ± 0.12 ± 1.40 ± 1.47 ± 0.70 1.33 ± 0.33 0.06 ± 0.01e
453 0.85 8.4 0.01b 0.02d 0.58
a
p < 0.05 as compared to the treatment with [Fe(TpyCl)2]2+; bp < 0.01, as compared to the treatment with [Cu(TpyCl)2]2+; cp < 0.02, as compared to the treatment
with [Zn(TpyCl)2]2+; dp < 0.001, as compared to the treatment with [Zn(TpyCl)2]2+; ep < 0.001, as compared to the treatment with [Fe(TpyCl)2]2+ and [Cu
(TpyCl)2]2+.

functionalisation degree of HA obtained by NMR spectra. It suggests that activity on all cell lines except for SKHep1 cells (IC50s > 30 µM) and
[Fe(L)2]2+ complex is the main species. Hence, as reported for free HAtpy-Fe-TpyCl in MDA-MB-453 cells (IC50, 13.9 ± 3.9 µM).
terpyridine ligand, two tpy moieties are coordinated to Fe2+ in an ML2- It is noteworthy that the treatment with the ternary complex HAtpy-
like coordination environment [50]. Metal coordination can occur be­ Cu-TpyCl caused a significant reduction of IC50 values in MDA-MB-453,
tween tpy units intramolecularly within the same HAtpy molecule or A549 and A2780 cells, compared to the treatment with [Cu(TpyCl)2]2+.
intermolecularly in different HAtpy molecules. In MDA-MB-453, the IC50 value of HAtpy-Cu-TpyCl (0.09 ± 0.01 µM) is
As for Zn2+complexes, in the UV–Vis spectra, a well-developed lower than that we found for Doxorubicin (0.18 µM) [48].
bimodal band increases in the region between 310 and 322 nm and The ternary complex HAtpy-Fe-TpyCl showed an IC50 value slightly
the isosbestic point is evident at 300 nm (Fig. S6). The spectra are lower than [Fe(TpyCl)2]2+ only in the A2780 cell line. Regarding [Zn
characterised by a straight slope of the absorbance values at 322 nm (TpyCl)2]2+, its activity was consistently higher than that of the analo­
versus metal ion concentration and an abrupt saturation at a 1/2 M/L (L gous Cu2+ and Fe2+ complexes (Table 1). [Zn(TpyCl)2]2+ showed IC50
is tpy unit) ratio. values lower (A2780 and MDA-MB-453 cells) or similar (A549 cells)
A similar trend was found when Cu2+ was added to the HAtpy so­ than the corresponding ternary complex HAtpy-Zn-TpyCl.
lution (Fig. S7). Isosbestic point is present up to 0.5 equivalents of Cu2+.
When Cu2+/tpy unit ratio > 0.5, spectra are subjected to bathochromic 4. Conclusions
shift (Fig. S7) and the intensity of the bands between 310 and 320 nm
decreased. The titration of HAtpy with copper (II) is the only example The new hyaluronic acid functionalised with terpyridine units can
where a significant spectral change can be observed in the range of M/L form metal complexes with Fe2+, Zn2+ and Cu2+. We studied the anti­
ratio between 0.5 and 1 due to the coexistence of ML and ML2 species proliferative activity of the conjugate and its binary and ternary com­
(Fig. S8). CuL and CuL2 species have been characterised in the case of plexes with 4-chloro-terpyridine in cancer cells. The HAtpy-M-TpyCl
terpyridine ligands [11,51]. complexes showed antiproliferative activity in the nM range, in
UV–Vis spectra of ternary complexes HAtpy-M-TpyCl are reported in dependence of the cell line. In particular, HAtpy-Cu-TpyCl IC50s were
Fig. S9. The spectra are similar to the ML2 spectra. lower than [Cu(TpyCl)2]2+. Specifically, the ternary copper complex
The particle size distribution of HAtpy-M2+ (M = Fe, Cu, Zn) was showed the best improvement of the antiproliferative activity in MDA-
investigated by DLS. The addition of metal ions revealed an increase in MB-453 cells, and the IC50 value was lower than that of Doxorubicin.
the hydrodynamic volume compared to the ligand (Fig. S5, Table S1). Drawing from the antiproliferative activity data of ternary com­
This is in keeping with the formation of intermolecular complex species. plexes in comparison to [Fe(TpyCl)2]2+ and [Cu(TpyCl)2]2+, we may
HAtpy-Cu2+ showed the smallest Z-average (18 nm, Table S1), which propose that the presence of hyaluronic acid may enhance the cellular
may be due to the small amount of ML species. HAtpy-Fe2+ and HAtpy- uptake of the copper complex (in three of the four cell lines), and of the
Zn2+ NPs showed a higher hydrodynamic volume (Z-average is 25 nm iron complex (in one of the four cell lines). In the case of HAtpy-Zn-
and 30 nm respectively), which may suggest the formation of intermo­ TpyCl, the hyaluronic acid moiety did not improve the cytotoxicity
lecular ML2 species (Table S1, Fig. S5). Similar behaviour has been re­ compared to [Zn(TpyCl)2]2+.
ported for other tpy polymers [52]. The conjugation with hyaluronic acid is a fruitful approach for
improving the water-solubility of terpyridine and their metal complexes.
The coordination properties of terpyridine ligand can be extended to
3.2. Antiproliferative activity other tpy derivatives by tuning the properties of the final system.

The antiproliferative activity of HAtpy and its Fe2+, Cu2+ and Zn2+
5. Authorship contributions
binary and ternary complexes with TpyCl (Fig. S10) was studied in
MDA-MB-453, SKHep1 (except for Zn complexes), A549, and A2780 cell
R. Panebianco: Data curation, Investigation, Original draft, Writing
lines (Table 1). We have already investigated Fe2+ and Cu2+ TpyCl
– review & editing. M. Viale: Data curation, Formal analysis, Investi­
complexes [18]. The antiproliferative activity of [Zn(TpyCl)2]2+ [53]
gation, Writing – review & editing. Graziella Vecchio: Supervision,
was studied for comparison. The results are reported in Table 1.
Data curation, Investigation, Original Draft Writing – review & editing.
Data showed that HAtpy has IC50 values that are pharmacologically
significant in all cell lines tested, although higher than TpyCl. Binary
complexes of HAtpy were generally pharmacologically inactive on all Declaration of competing interest
cell lines except HAtpy-Fe2+, which showed significant IC50 of about
1.74 ± 0.66 µM in SKHep1 cells. A similar trend was found for cross- The authors declare that they have no known competing financial
linked cyclodextrin polymers functionalised with tpy [48]. interests or personal relationships that could have appeared to influence
On the contrary, ternary complexes had a high antiproliferative the work reported in this paper.

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R. Panebianco et al. Polyhedron 249 (2024) 116793

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