Toward supramolecular nanozymes for the photocatalytic activation of Pt(iv) anticancer prodrugs

Article abstract of DOI:10.1039/d0cc03450a

A supramolecular nanozyme for the photocatalytic conversion of a Pt(iv) anticancer complex to cisplatin is described herein. We employed 1.9 nm Au nanoparticles decorated with thiol ligands bearing a TACN (1,4,7-triazacyclononane) headgroup to encapsulate FMN (riboflavin-5′-phosphate). In the presence of an electron donor, flavin-loaded nanoparticles photocatalyzed the reductive activation of the prodrug cis,cis,trans-[Pt(NH3)2(Cl2)(O2CCH2CH2COOH)2] to cisplatin, achieving turnover frequency values of 7.4 min-1.

Full text of DOI:10.1039/d0cc03450a

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Toward supramolecular nanozymes for the
photocatalytic activation of Pt(IV) anticancer
Cite this:DOI: 10.1039/d0cc03450a
prodrugs†
Laura F. Mazzei,abc Alvaro Martınez,* Lucia Trevisan,
Received 14th May 2020,
Accepted 28th July 2020
a
c
c
´
Daniele Rosa-Gastaldo,
´
bd
ade
Aitziber L. Cortajarena,
Fabrizio Mancin *c and Luca Salassa
*
DOI: 10.1039/d0cc03450a
rsc.li/chemcomm
A supramolecular nanozyme for the photocatalytic conversion of
We recently reported a novel type of bioorthogonal photo-
a Pt(IV) anticancer complex to cisplatin is described herein. We catalytic reactions in which flavins (e.g. riboflavin, FMN and
employed 1.9 nm Au nanoparticles decorated with thiol ligands FAD) and certain flavoproteins transform Pt(IV) precursors into
bearing a TACN (1,4,7-triazacyclononane) headgroup to encapsulate the clinically approved anticancer drugs cisplatin and carbo-
FMN (riboflavin-50-phosphate). In the presence of an electron donor, platin in biological media (Fig. 1A).11–13 As rarely observed in
flavin-loaded nanoparticles photocatalyzed the reductive activation of catalysis,14,15 metal complexes act as substrates rather than
the prodrug cis,cis,trans-[Pt(NH3)2(Cl2)(O2CCH2CH2COOH)2] to cispla- catalysts in these unconventional processes. Mechanistic stu-
tin, achieving turnover frequency values of 7.4 minꢀ1
.
dies highlighted that light excitation of flavins in the presence
Bioorthogonal nanozymes are nm-sized catalysts capable of
performing artificial reactions in biological media.1 To date,
nanozymes have been fabricated by entrapping metallic nano-
particles (e.g. Pd, Au, Cu) into polymer matrixes,2–4 by decorat-
ing nanometric Au scaffolds with catalytic units5 or by
encapsulating organometallic catalysts (e.g. Ru, Pd complexes)
in the coating ligand shell.6 Especially in the latter case, the
nanostructure is fundamental to improve the solubility of the
embedded catalyst and to protect it from poisoning reactions
that easily occur in biological environments.7 Besides, proper
design of the nanozyme enables controlling the localization of
the molecular catalyst in specific cellular compartments3,8 or
tissues9 and triggering their catalytic activity upon external
stimuli.6 To the best of our knowledge, bioorthogonal nano-
zymes developed so far catalyze the deprotection of caged
organic imaging and therapeutic agents or promote the in situ
synthesis of drug derivatives.1,4,10
a Donostia International Physics Center, Paseo Manuel de Lardizabal 4, Donostia,
20018, Spain. E-mail: lsalassa@dipc.org
b
´
CIC biomaGUNE, Basque Research Technology Alliance, BRTA, Parque Tecnologico
´
´
´
de San Sebastian, Paseo Miramon 194, 20014 Donostia/San Sebastian, Spain
c
`
Dipartimento di Scienze Chimiche, Universita di Padova, via Marzolo 1, Padova,
35131, Italy. E-mail: fabrizio.mancin@unipd.it
d Ikerbasque, Basque Foundation for Science, Bilbao, 48011, Spain
Fig. 1 (A) Photocatalytic activation scheme of the cisplatin prodrug cis,cis,-
trans-[Pt(NH3)2(Cl2)(O2CCH2CH2COOH)2] (1); (B) photocatalyst (FMN) and Au
nanoparticles decorated with a C11-thiol bearing a 1,4,7-triazacyclononane
headgroup (TACN AuNPs); (C) schematic representation of the nanozyme
developed in this work.
e Kimika Fakultatea, Euskal Herriko Unibertsitatea, UPV/EHU, Donostia, 20080,
Spain
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0cc03450a
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of an electron donor (e.g. zwitterionic buffers or NADH) affords
the doubly-reduced flavin form (Fig. 1A) which is the active
catalyst prompting the bioorthogonal substrate conversion.11,13
In vitro experiments also demonstrated that this activation
strategy hold promise for its capacity to deliver lethal concen-
trations of Pt drugs with minimal light doses.11,16
The relevance of Pt(IV) complexes in photochemotherapy
research is proved by the numerous photoactivatable prodrugs
and activation approaches developed in recent years.17–20
Motivated by these results and the progress in the design of
nanomaterials with enzyme-like features, we report herein our
first steps in the implementation of photocatalysis towards
Pt substrates by nanozymes. To such aim, we selected FMN,
1.9 nm Au nanoparticles decorated with a C11-thiol bearing a
1,4,7-triazacyclononane headgroup (TACN AuNPs), and the
complex cis,cis,trans-[Pt(NH3)2(Cl2)(O2CCH2CH2COOH)2] (1) as
photocatalyst, nanozyme scaffold, and substrate, respectively
(Fig. 1A and B; Fig. S1 and S2, ESI†).
Fig. 2 DOSY spectra of 1 in the absence (A) or presence (B) of TACN
AuNPs in D2O at 28 1C. [1] = 500 mM, [TACN]AuNPs = 2 mM.
We opted for spherical nanoparticles with a Au core smaller to be in the 50–60 mM range for both types of TACN AuNPs.
than 2 nm to have a nanoplatform size in the biomolecular Furthermore, DOSY and NOE-pumping NMR experiments con-
scale and to minimize light absorption and scattering by the firmed the anticipated interaction between TACN AuNPs and 1
nanoparticles due to the surface plasmon band, whose inten- (Fig. 2 and Fig. S4, ESI†), including under the condition
sity depends on the nanoparticle size.21 Previously, Rotello and employed in catalysis tests (vide infra). As expected for such a
co-workers used similar AuNPs for the controlled photodelivery scenario, signals of 1 undergo a relevant broadening in the
of anticancer drugs using UV-A light.22 NMR sensing studies on presence of TACN AuNPs (Fig. S4, ESI†) and the diffusion rate
TACN AuNPs closely related to the ones used in this work of 1 determined by DOSY decreased up to ca. 4 times in the
showed that FMN strongly binds to the thiol monolayer via presence of the nanoparticles with respect to the free complex
electrostatic and hydrophobic interactions.23 When in the (1.26 ꢁ 10ꢀ10 vs. 4.15 ꢁ 10ꢀ10 m2 sꢀ1). NOE pumping unequi-
protonated form (TACNNH pKa 10.4, 6.8, 2.4)24 or bound to vocally demonstrated the fast exchange of nanoparticle-bound
Zn2+, the positively charged TACN group can interact with the 1 in the NMR time scale. In fact, magnetization transfer from
negatively charged phosphate of the FMN ribityl sidechain, the AuNPs to the metal complex can only be observed in the
while the hydrophobic environment provided by the C11 alkyl case of close (o4 Å) and sufficiently prolonged contacts.
spacer of the ligand can accommodate the aromatic isoallox-
For testing the catalytic performance of the nanozyme, we
azine ring system of FMN. Likewise, 1 is also capable of hence loaded the nanoparticles with 25 mM FMN, a concen-
electrostatically binding the TACN headgroups, since the suc- tration considerably below the saturation limit to avoid the
cinato ligands of the complex are deprotonated at pH above 6.25 presence of free catalyst in solution during the reaction. The
Therefore, TACN AuNPs are valuable candidates to operate as concentration of substrate 1 was fixed at 500 mM to allow
1
supramolecular nanoscaffolds that favor co-localization of FMN monitoring the photocatalysis progression by H-NMR. Impor-
and 1, ultimately affording a nanozyme for the photocatalytic tantly, fluorescence control experiments showed that FMN is
conversion of a Pt(IV) prodrug into cisplatin (Fig. 1C).
not significantly displaced by 1 at such concentrations (Fig. S5,
To test this hypothesis and assemble the nanozyme, we ESI†), confirming the higher affinity of the TACN AuNPs for
firstly investigated the loading of FMN onto TACN AuNPs amphiphilic guests as FMN than for hydrophilic guests as 1.28,29
(100 mM of TACN ligand) in MES buffer (5 mM, pH 6) by
Under these conditions, we evaluated the capacity of the
fluorescence quenching measurements. These experiments flavonanozymes to photocatalytically activate 1 upon irradiation
were performed in the absence and presence of 1 equivalent with blue light (460 nm, 5.30 mW cmꢀ2) and compared their
of Zn(NO3)2 (100 mM), that is with the amino groups of TACN efficiency with 25 mM of free FMN (Fig. 3 and Fig. S6, ESI†).
positively charged due to protonation (FMN@TACN AuNPs) or
Substrate conversion was monitored by quantifying the
coordination of Zn2+ (FMN@Zn-TACN AuNPs) respectively. release of succinato ligands by 1H-NMR as a function of the
MES was selected since it acts as electron donor in the irradiation time. Previous work demonstrated that this process
Pt(IV) - Pt(II) catalytic conversion and protects flavins from corresponded to the generation of cisplatin.16
photodecomposition.11,26 Upon interaction with TACN AuNPs,
FMN@TACN and FMN@Zn-TACN AuNPs displayed an
FMN fluorescence emission (lem = 527 nm) was quenched by almost identical behavior in terms of catalytic activity, reaching
the Au core of the nanoparticles, enabling determination of the 80% conversion already within 7 min and a plateau at 90%
FMN concentration at which the nanoscaffold binding sites are conversion after 30 min of light irradiation. These data corre-
saturated. After correcting FMN emission intensity for inner sponded to a turnover number (TON) of 18 and turnover
filter effect27 (Fig. S3, ESI†), we found saturation concentrations frequency (TOF) of 7.4 minꢀ1 (calculated in the range 15–45%
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consistent with the plateau observed in the substrate conversion
curves of Fig. 3.
As previously reported, FMN alone cannot catalytically
reduce 1 in the absence of light, nor under light irradiation
without electron donor.13 Controls showed that TACN AuNPs
only prompted a 15% substrate conversion in the dark, both
with and without FMN loaded (Fig. S7 and S8, ESI†). Conver-
sely, the nanoscaffold (TACN AuNPs) displayed certain catalytic
activity upon light irradiation, reaching 50–70% conversion in
30 min. Importantly, the reaction efficiency was significantly
lower in terms of TOF value (o0.9 minꢀ1) compared to
FMN@TACN AuNPs and free FMN (Fig. S9 and S10, ESI†). High
TOF values under light irradiation are valuable for application
in photochemotherapy since they permit rapid conversion of
Pt(IV) prodrugs with low light doses.
Although further investigation is required, the background
catalytic activity of TACN AuNPs towards the photoreduction of
1 could be reasonably ascribed to an excited-state electron
transfer process promoted by the nanomaterial, considering
that its absorption tail extends over 800 nm (Fig. S2, ESI†).
Undoubtedly, the roadmap to bioorthogonal nanozymes for
the catalytic activation of Pt drugs in complex biological envir-
onments still demands a more refined design than the one
described in this work. Use of these systems in vitro and in vivo
requires that their robustness increases without reducing the
catalytic activity. For instance, inclusion of PEGylated ligands
in the nanoparticle monolayer will likely improve the colloidal
stability and biocompatibility of FMN@TACN and FMN@Zn-
TACN,30 while providing enhanced protection of the FMN
catalyst from the chemical and biological components present
in cells and tissues. While these key aspects are worth exploring
in the future, the results described here highlight that TACN
AuNPs are suitable components to develop supramolecular
nanomaterials capable to carry out unconventional flavin-
mediated catalytic reactions using Pt(IV) prodrugs as substrates.
Besides extending the repertoire of reactions catalyzed by
TACN-based nanoparticles,31–33 this work holds promise as
strategy to control spatio-temporally the effects of Pt anticancer
drugs via light activation and catalytic amplification.
Fig. 3 Photocatalytic activation of 1 by FMN, FMN@TACN AuNPs and
FMN@Zn-TACN AuNPs. Experimental conditions: [FMN] = 25 mM, [1] = 500 mM,
[MES]pH6 = 5 mM, [TACN]AuNPs = 0 mM or 100 mM, [Zn(NO3)2] = 0 mM or
100 mM, hn 460 nm (5.30 mW cmꢀ2). Each point corresponds to an
average of a triplicate of triplicate independent measurements.
conversion). Similar TON and TOF values were obtained for free
FMN under the same conditions. Furthermore, we investigated
the stability of the nanozymes under light irradiation to discard
leaking of FMN from the AuNPs during catalysis (Fig. 4). In the
presence of TACN AuNPs, the fluorescence emission of FMN
remained quenched throughout the whole light-irradiation
time window, thus demonstrating that the catalyst was associated
to the nanozyme scaffold during such period. In addition, the
emission intensity of FMN@TACN AuNPs underwent a further
slight decrease over time due to FMN photodegradation,11 with
almost complete depletion after 10 min. However, the process
occurred at a lower rate than for free FMN (Fig. 4, inset plot),
suggesting a moderate protecting effect of the AuNPs towards
FMN photobleaching. We speculate that such increase in stability
positively contributed to the catalytic activity of the nanozymes.
Inactivation of the catalyst after 10 min of light irradiation was
We acknowledge financial support from the Spanish State
Research Agency for the grants CTQ2016-80844-R, BIO2016-
77367-R, PID2019-111649RB-I00 and PCI2018-092984. This work
was performed under the Severo Ochoa Centres of Excellence and
Maria de Maeztu Units of Excellence Program from the Spanish
State Research Agency – Grant No. CEX2018-000867-S (DIPC) and
MDM-2017-0720 (CIC biomaGUNE). We also thank Fondazione
´
Cariparo (Ricerca Scientifica di Eccellenza Grant ‘‘SELECT’’). A. M.
has received funding from the European Union’s Horizon 2020
Research and Innovation Programme under Marie Sklodowska-
Curie Actions grant no. 793702. We thank the Spanish Multi-
MetDrugs network (RED2018-102471-T) for fruitful discussion.
Fig. 4 Photobleaching of FMN@TACN AuNPs and free FMN. Experimental
Conflicts of interest
conditions: [FMN] = 25 mM, [1] = 500 mM, [MES]pH6 = 5 mM, [TACN]AuNPs
0 mM or 100 mM, hn 460 nm (5.30 mW cmꢀ2).
=
There are no conflicts to declare.
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´
´
17 J. Gurruchaga-Pereda, A. Martınez, A. Terenzi and L. Salassa, Inorg.
Chim. Acta, 2019, 495, 118981.
Notes and references
1 X. Zhang, R. Huang, S. Gopalakrishnan, R. Cao-Milan and V. M. Rotello,
Trends Chem., 2019, 1, 90–98.
2 R. M. Yusop, A. Unciti-Broceta, E. M. V. Johansson, R. M. Sanchez-
´
18 N. J. Farrer, J. A. Woods, L. Salassa, Y. Zhao, K. S. Robinson,
G. Clarkson, F. S. Mackay and P. J. Sadler, Angew. Chem., Int. Ed.,
2010, 49, 8905–8908.
19 D. J. Norman, A. Gambardella, A. R. Mount, A. F. Murray and
M. Bradley, Angew. Chem., Int. Ed., 2019, 58, 14189–14192.
20 Z. Wang, N. Wang, S.-C. Cheng, K. Xu, Z. Deng, S. Chen, Z. Xu,
K. Xie, M.-K. Tse, P. Shi, H. Hirao, C.-C. Ko and G. Zhu, Chemistry,
2019, 5, 3151–3165.
´
´
Martın and M. Bradley, Nat. Chem., 2011, 3, 239–243.
´
´
´
3 A. M. Perez-Lopez, B. Rubio-Ruiz, V. Sebastian, L. Hamilton,
C. Adam, T. L. Bray, S. Irusta, P. M. Brennan, G. C. Lloyd-Jones,
´
D. Sieger, J. Santamarıa and A. Unciti-Broceta, Angew. Chem., Int.
Ed., 2017, 56, 12548–12552.
4 J. Clavadetscher, S. Hoffmann, A. Lilienkampf, L. Mackay,
R. M. Yusop, S. A. Rider, J. J. Mullins and M. Bradley, Angew. Chem.,
Int. Ed., 2016, 55, 15662–15666.
5 F. Mancin, L. J. Prins, P. Pengo, L. Pasquato, P. Tecilla and
P. Scrimin, Molecules, 2016, 21, 1014.
6 G. Y. Tonga, Y. Jeong, B. Duncan, T. Mizuhara, R. Mout, R. Das,
S. T. Kim, Y. C. Yeh, B. Yan, S. Hou and V. M. Rotello, Nat. Chem.,
2015, 7, 597–603.
21 X. Liu, M. Atwater, J. Wang and Q. Huo, Colloids Surf., B, 2007, 58,
3–7.
22 S. S. Agasti, A. Chompoosor, C. You, P. Ghosh, C. K. Kim and
V. M. Rotello, J. Am. Chem. Soc., 2009, 131, 5728–5729.
23 M. Diez-Castellnou, M. V. Salvia, S. Springhetti, F. Rastrelli and
F. Mancin, Chem. – Eur. J., 2016, 22, 16957–16963.
24 X. Yu and J. Zhang, Macrocyclic Polyamines, Wiley-VCH Verlag GmbH
& Co. KGaA, Weinheim, Germany, 2017, pp. 1–5.
7 H. Wei and E. Wang, Chem. Soc. Rev., 2013, 42, 6060–6093.
8 A. Gupta, R. Das, G. Yesilbag Tonga, T. Mizuhara and V. M. Rotello,
ACS Nano, 2018, 12, 89–94.
´
25 A. Garaikoetxea Arguinzoniz, N. Gomez Blanco, P. Ansorena Legarra
and J. C. Mareque-Rivas, Dalton Trans., 2015, 44, 7135–7138.
26 L. C. P. Gonçalves, H. R. Mansouri, E. L. Bastos, M. Abdellah,
9 J. T. Weiss, J. C. Dawson, K. G. Macleod, W. Rybski, C. Fraser,
´
B. S. Fadiga, J. Sa, F. Rudroff and M. D. Mihovilovic, Catal. Sci.
´
C. Torres-Sanchez, E. E. Patton, M. Bradley, N. O. Carragher and
Technol., 2019, 9, 1365–1371.
A. Unciti-Broceta, Nat. Commun., 2014, 5, 3277.
27 B. Birdsall, R. W. King, M. R. Wheeler, C. A. Lewis, S. R. Goode,
R. B. Dunlap and G. C. K. Roberts, Anal. Biochem., 1983, 132,
353–361.
28 M.-V. Salvia, F. Ramadori, S. Springhetti, M. Diez-Castellnou,
B. Perrone, F. Rastrelli and F. Mancin, J. Am. Chem. Soc., 2015,
137, 886–892.
29 L. Gabrielli, D. Rosa-Gastaldo, M.-V. Salvia, S. Springhetti,
F. Rastrelli and F. Mancin, Chem. Sci., 2018, 9, 4777–4784.
30 R. Tavano, D. Segat, E. Reddi, J. Kos, M. Rojnik, P. Kocbek, S. Iratni,
D. Scheglmann, M. Colucci, I. M. R. Echevarria, F. Selvestrel,
F. Mancin and E. Papini, Nanomedicine, 2010, 5, 881–896.
31 M. Diez-Castellnou, F. Mancin and P. Scrimin, J. Am. Chem. Soc.,
2014, 136, 1158–1161.
10 S. Alonso-de Castro, A. Terenzi, J. Gurruchaga-Pereda and L. Salassa,
Chem. – Eur. J., 2019, 25, 6651–6660.
11 S. Alonso-de Castro, E. Ruggiero, A. Ruiz-de-Angulo, E. Rezabal,
´
J. C. Mareque-Rivas, X. Lopez, F. Lopez-Gallego and L. Salassa,
Chem. Sci., 2017, 8, 4619–4625.
12 S. Alonso-de Castro, A. L. Cortajarena, F. Lopez-Gallego and
´
L. Salassa, Angew. Chem., Int. Ed., 2018, 57, 3143–3147.
´
´
13 J. Gurruchaga-Pereda, V. Martınez-Martınez, E. Rezabal, X. Lopez,
C. Garino, F. Mancin, A. L. Cortajarena and L. Salassa, ACS Catal.,
2020, 10, 187–196.
14 L. Gong, Z. Lin, K. Harms and E. Meggers, Angew. Chem., Int. Ed.,
2010, 7955–7957.
´
15 A. Ruffoni, F. Julia, T. D. Svejstrup, A. J. McMillan, J. J. Douglas and
32 S. Neri, S. Garcia Martin, C. Pezzato and L. J. Prins, J. Am. Chem. Soc.,
2017, 139, 1794–1797.
33 F. della Sala, J. L.-Y. Chen, S. Ranallo, D. Badocco, P. Pastore,
F. Ricci and L. J. Prins, Angew. Chem., Int. Ed., 2016, 55,
10737–10740.
D. Leonori, Nat. Chem., 2019, 11, 426–433.
16 S. Alonso-de Castro, A. Terenzi, S. Hager, B. Englinger, A. Faraone,
´
J. C. Martınez, M. Galanski, B. K. Keppler, W. Berger and L. Salassa,
Sci. Rep., 2018, 8, 17198.
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