An intramolecular photoswitch can significantly promote photoactivation of Pt(iv) prodrugs

Article abstract of DOI:10.1039/d0sc06839j

Selective activation of prodrugs at diseased tissue through bioorthogonal catalysis represents an attractive strategy for precision cancer treatment. Achieving efficient prodrug photoactivation in cancer cells, however, remains challenging. Herein, we report two Pt(iv) complexes, designated as rhodaplatins {rhodaplatin 1, [Pt(CBDCA-O,O′)(NH3)2(RhB)OH]; rhodaplatin 2, [Pt(DACH)ox(RhB)(OH)], where CBDCA is cyclobutane-1,1-dicarboxylate, RhB is rhodamine B, DACH is (1R,2R)-1,2-diaminocyclohexane, and ox is oxalate}, that bear an internal photoswitch to realize efficient accumulation, significant co-localization, and subsequent effective photoactivation in cancer cells. Compared with the conventional platform of external photocatalyst plus substrate , rhodaplatins presented up to 4.8 × 104-fold increased photoconversion efficiency in converting inert Pt(iv) prodrugs to active Pt(ii) species under physiological conditions, due to the increased proximity and covalent bond between the photoswitch and Pt(iv) substrate. As a result, rhodaplatins displayed increased photocytotoxicity compared with a mixture of RhB and conventional Pt(iv) compound in cancer cells including Pt-resistant ones. Intriguingly, rhodaplatin 2 efficiently accumulated in the mitochondria and induced apoptosis without causing genomic DNA damage to overcome drug resistance. This work presents a new approach to develop highly effective prodrugs containing intramolecular photoswitches for potential medical applications. This journal is

Full text of DOI:10.1039/d0sc06839j

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An intramolecular photoswitch can significantly
promote photoactivation of Pt(IV) prodrugs†
Cite this: DOI: 10.1039/d0sc06839j
Zhiqin Deng, ab Cai Li,ab Shu Chen,ab Qiyuan Zhou,ab Zoufeng Xu,ab Zhigang Wang,c
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
a
ab
Houzong Yao,ab Hajime Hirao
and Guangyu Zhu
*
Selective activation of prodrugs at diseased tissue through bioorthogonal catalysis represents an attractive
strategy for precision cancer treatment. Achieving efficient prodrug photoactivation in cancer cells,
however, remains challenging. Herein, we report two Pt(IV) complexes, designated as rhodaplatins
{rhodaplatin 1, [Pt(CBDCA-O,O0)(NH3)2(RhB)OH]; rhodaplatin 2, [Pt(DACH)ox(RhB)(OH)], where CBDCA is
cyclobutane-1,1-dicarboxylate, RhB is rhodamine B, DACH is (1R,2R)-1,2-diaminocyclohexane, and ox is
oxalate}, that bear an internal photoswitch to realize efficient accumulation, significant co-localization,
and subsequent effective photoactivation in cancer cells. Compared with the conventional platform of
“external photocatalyst plus substrate”, rhodaplatins presented up to 4.8
ꢀ
104-fold increased
photoconversion efficiency in converting inert Pt(IV) prodrugs to active Pt(II) species under physiological
conditions, due to the increased proximity and covalent bond between the photoswitch and Pt(IV)
substrate. As a result, rhodaplatins displayed increased photocytotoxicity compared with a mixture of
RhB and conventional Pt(IV) compound in cancer cells including Pt-resistant ones. Intriguingly,
rhodaplatin 2 efficiently accumulated in the mitochondria and induced apoptosis without causing
genomic DNA damage to overcome drug resistance. This work presents a new approach to develop
highly effective prodrugs containing intramolecular photoswitches for potential medical applications.
Received 15th December 2020
Accepted 29th March 2021
DOI: 10.1039/d0sc06839j
rsc.li/chemical-science
The emerging concept of combining photocatalysis and
bioorthogonal reactions for biological and medicinal applica-
tions has drawn much attention.6 A common strategy is to use
Introduction
Conventional chemotherapeutic drugs have achieved great
success in ghting cancer.1 Their therapeutic outcome,
however, is limited by severe adverse effects induced by unde-
sired activation of the drugs at nonpathological sites.2 Various
strategies have been developed to address this limitation,
including developing kinetically inert prodrugs that can be
preferably activated only in cancer cells,3 adding external cata-
lysts to selectively activate drugs at the desired site,4 or in situ
drug synthesis.5 The improved biological potential has been
observed for these strategies. The activation of these anticancer
agents is, however, passively reliant on the intracellular envi-
ronment of cancer cells; premature or incomplete activation is
likely to occur and compromise their anticancer activities.
Therefore, drugs that can be specically and effectively acti-
vated at the pathological site are still highly desired for preci-
sion medicine.
external photocatalysts to activate Pt(IV) anticancer prodrugs in
cancer cells to improve the prodrugs' cancer selectivity. For
instance, riboavin has been found to catalytically reduce Pt(IV)
prodrugs to active Pt(II) drugs upon visible light irradiation.7
Recently, a ruthenium-based photosensitizer has also been
utilized as the photocatalyst to activate Pt(IV) prodrugs.8
However, the photocatalysts do not always effectively co-localize
with the Pt(IV) substrate in the cancer cells, limiting their pho-
tocatalytic efficiency. Another concern is the intracellular
stability of the Pt(IV) prodrugs, some of which may be photo-
activated outside of cells. Moreover, other intracellular
biomolecules may competitively react with the Pt(IV) substrate
or catalyst.9 Therefore, the current platform of “external pho-
tocatalyst plus Pt(IV) substrate” has its own limitations
regarding biological applications. Indeed, the co-treatment
strategy has achieved only limited enhancement in cytotox-
icity compared with the original Pt(II) drugs.
aDepartment of Chemistry, City University of Hong Kong, Hong Kong SAR, P. R. China.
E-mail: guangzhu@cityu.edu.hk
bCity University of Hong Kong Shenzhen Research Institute, Shenzhen 518057, P. R.
To address these limitations, we designed a new class of
photoactivatable Pt(IV) prodrugs based on clinical Pt(II) drugs.
The highly stable prodrugs contain an internal photoswitch to
realize effective photoactivation in cancer cells. The internal-
ized photoswitch that is colocalized with the Pt center ensures
the prodrugs dramatically boosted intracellular activation
China
cSchool of Pharmaceutical Sciences, Health Science Center, Shenzhen University,
Shenzhen, P. R. China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0sc06839j
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efficiency and signicantly increased photocytotoxicity
compared with the “external catalyst plus substrate” platform.
Interestingly, one of the prodrugs precisely located and
damaged the mitochondria, an unconventional target of Pt-
based complexes. Compared with nuclei, mitochondria lack
the function of nucleotide excision repair (NER) and histone
protection,10 the two main factors responsible for the resistance
of cancer cells towards Pt drugs. In addition, inducing mito-
chondrial DNA damage could initiate mitochondria-mediated
cell death pathways.11 Therefore, by targeting mitochondria,
rhodaplatins may effectively kill cancer cells and overcome Pt
resistance. We provide a novel strategy to develop highly effec-
tive photoactivatable Pt(IV) prodrugs for controllable and
selective activation in cancer cells.
Fig.
1 (A) The chemical structures of rhodamine B (RhB) and
Results and discussion
complexes 1a–3c. The percentage of (B) complexes 2a–2c (10ꢁ4 M)
and (C) complexes 3a–3c (10ꢁ4 M) that are catalytically reduced by
RhB (10ꢁ4 M) in the presence of sodium ascorbate (2 ꢀ 10ꢁ3 M) upon
irradiation (400–760 nm, 4 mW cmꢁ2).
Rhodamine B (RhB, Fig. 1A), a widely used uorescent dye,12 has
been employed as a photocatalyst for various reactions.13 We
noticed that the oxidation potential of the photo-excited RhB
(RhB+c/RhB*: ꢁ1.3 V)13a is theoretically sufficient to reduce most
of the conventional Pt(IV) prodrugs.14 Therefore, we rst inves-
tigated the ability of RhB to catalytically reduce Pt(IV) prodrugs
in the presence of a reducing agent upon visible light irradia-
tion. Cisplatin-, carboplatin-, and oxaliplatin-based Pt(IV) pro-
drugs containing various axial ligands were obtained
(complexes 1a–3c; Fig. 1A, S1–S6 and Scheme S1†). The oxali-
platin- and carboplatin-based but not the cisplatin-based Pt(IV)
prodrugs were stable in the presence of reducing agents
(Fig. S7†). Thus, the cisplatin-based Pt(IV) prodrugs were
excluded from the following studies. For the carboplatin-based
Pt(IV) complexes, complexes 2a–2c (10ꢁ4 M) were mixed with
prodrugs, respectively (Fig. 2A, S19, S20 and Scheme S2†). As
rhodaplatins were designed to be activated in cancer cells,
which have abundant reducing agents (e.g., sodium ascorbate,
RhB (10ꢁ4 M) in PBS buffer (pH 7.4) containing 2 ꢀ 10ꢁ3
M
sodium ascorbate. No reduction of the complexes was observed
in the absence of light. Upon irradiation with white light (400–
760 nm, 4 mW cmꢁ2), although the reduction of 2a was barely
observed, 4% of 2b and 9% of 2c were reduced to carboplatin
aer irradiation for 5 h, and no signicant change of RhB was
observed during irradiation (Fig. S8–S10†), indicating that RhB
could catalytically reduce complexes 2b and 2c to carboplatin
but with poor catalytic efficiency (Fig. 1B). A similar scenario
was observed for the oxaliplatin-based Pt(IV) substrates (Fig. 1C
and S11–S13†). Our further study indicated that the catalytic
efficiency correlated with the reduction potential of Pt(IV)
substrate (Fig. S14, S15 and Table S1†), and the low electron
transfer efficiency between RhB and the Pt(IV) center was the
bottleneck that limited the catalytic efficiency of such photo-
catalysis platform (Fig. S16–S18†).
To improve the photoconversion performance, we specu-
lated that shortening the distance between the photocatalyst
and the substrate to increase the electron transfer efficiency
Fig. 2 (A) The chemical structures of rhodaplatin 1 and rhodaplatin 2.
might be a promising approach. To verify this hypothesis, we The percentage of remained (B) rhodaplatin 1 (10ꢁ4 M) and (C) rho-
daplatin 2 (10ꢁ4 M) in the PBS buffer (pH 7.4) containing 2 ꢀ 10ꢁ3
M
directly conjugated RhB with carboplatin- and oxaliplatin-based
Pt(IV) complexes, such that the distance between the photo-
switch and the Pt(IV) center signicantly decreased. The
synthetic complexes were designated as rhodaplatin 1 and
sodium ascorbate with or without irradiation with white light (400–
760 nm, 4 mW cmꢁ2). (D) The calculated conversion rate of rhoda-
platin 1 and RhB towards 2a–2c at the concentration of 10ꢁ4 M. (E) The
calculated conversion rate of rhodaplatin 2 and RhB towards 3a–3c at
rhodaplatin 2, for the carboplatin- and oxaliplatin-based the concentration of 10ꢁ4 M.
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Scheme 1 The proposed photoreduction mechanism of rhodaplatins in the presence of ascorbate under physiological conditions. Curly
brackets are used to denote transient and undetected intermediates.
was 6.6 ꢀ 104 to 1.32 ꢀ 106 times lower than that of riboavin,
but rhodaplatins presented a comparable photoconversion rate
with that from riboavin. These data conrm that the enhanced
proximity and the covalent bond between the photoswitch and
the Pt(IV) center could signicantly accelerate the photo-
conversion process.
To further investigate how the distance and the covalent
bond between the photoswitch and the Pt center would affect
the conversion efficiency, the photo-reduction of rhodaplatins
Fig. 3 The IC50 value of (A) RhB, RhB + 2c, and rhodaplatin 1; (B) RhB,
was investigated in the presence of excess Pt(IV) substrate or
RhB + 3c, and rhodaplatin 2 in A2780cisR cells upon irradiation with
RhB at various concentrations. As shown in Fig. S26 and S27,†
visible light. Cells were treated with complexes for 6 h, then the culture
no increase in the photoconversion efficiency of rhodaplatins
was observed, even when high concentrations of Pt(IV) complex
or RhB were added, indicating that a distantly separated Pt(IV)
substrate and RhB can hardly affect the internal photo-
reduction of rhodaplatins, further emphasizing the impor-
tance of the distance and covalent bond between the photo-
switch and the Pt(IV) center. Next, to study the rate law of
rhodaplatin, the impacts of irradiation power density and
concentration of rhodaplatin and sodium ascorbate on the
photo-reduction of rhodaplatin were analyzed. The photore-
duction rate of rhodaplatin increased linearly with the power
density of irradiation and the concentration of rhodaplatin,
indicating a rst-order reaction (Fig. S28 and S29†).
As rhodaplatins are presented as monovalent cations in
aqueous solutions, the prodrugs may be able to form ion-pairs
with ascorbate. To investigate such a possibility, we rst
determined the ion-pair formation between rhodaplatin 2 and
ascorbate in Milli-Q water. The prodrug formed ion-pair with
ascorbate in a 1 : 1 stoichiometry (Fig. S30†). The association
constant (Ka) value of such ion-pair in Milli-Q water was deter-
mined to be 3149 Mꢁ1 by UV-Vis spectroscopic titration
(Fig. S31†).16 A similar result was obtained by uorescence
spectroscopic titration (Fig. S32†). In PBS buffer, however, the
anions including phosphates showed a much higher affinity
towards rhodaplatin cations (Fig. S33†); only very limited
medium was replaced with fresh medium. Cells were irradiated with
visible light (400–760 nm, 4 mW cmꢁ2) for 30 min, and cultured for
another 42 h. The IC50 value was determined by MTT assay.
GSH, or NADPH),15 we monitored the stability and photore-
duction of rhodaplatins in the presence of such reducing
agents. Rhodaplatins showed high dark stability even in the
presence of sodium ascorbate; more than 94% of
rhodaplatin 1 and 88% of rhodaplatin 2 remained aer incu-
bation for 12 h (Fig. S21†). Upon irradiation with low-dose
visible light (400–760 nm, 4 mW cmꢁ2), however, 95% of rho-
daplatins were converted to the corresponding Pt(II) drugs
within 5 min in PBS buffer (pH 7.4) containing sodium ascor-
bate (Fig. 2B, C, S22 and S23†). High stability and rapid
photoreduction were also observed in the presence of gluta-
thione (Fig. S24 and S25†). In the presence of ascorbate, the
conversion rate of rhodaplatin 1 at 10ꢁ4 M was calculated to be 2
ꢀ 10ꢁ5 M minꢁ1, which is 1.5 ꢀ 104-fold and 6.7 ꢀ 103-fold
higher than that of RhB towards complex 2b and 2c, respectively
(Fig. 2D). Similarly, the conversion rate of rhodaplatin 2 at
10ꢁ4 M was up to 4.8 ꢀ 104-fold higher than that of RhB towards
oxaliplatin-based Pt(IV) substrates (Fig. 2E). Notably, compared
with riboavin, an effective photocatalyst to convert Pt(IV) pro-
drugs to Pt(II) forms,7a the photocatalysis efficiency of free RhB
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Table 1 The cytotoxicity of different complexes against various cancer cell lines. Cells were treated with complex for 6 h, then the culture
medium was replaced with drug-free fresh medium, and cells were irradiated with or without (bold text) visible light (400–760 nm, 4 mW cmꢁ2
)
for 30 min. Then cells were cultured for another 42 h. The IC50 value was determined by the MTT assay
IC50 [mM]
IC50 [mM]
Cell line
A2780
Carboplatin RhB
RhB + 2cb
Rhodaplatin 1 PIc FId
Oxaliplatin
RhB + 3cb
Rhodaplatin 2 PIc FId
301 ꢂ 28
322 ꢂ 33
258 ꢂ 19
242 ꢂ 19
204 ꢂ 19
254 ꢂ 21
220 ꢂ 17
44 ꢂ 5
5.0 7.3
68 ꢂ 6
64 ꢂ 6
263 ꢂ 18
235 ꢂ 9
108 ꢂ 9
25 ꢂ 2
4.4 2.6
6.7 9.8
3.1 2.4
3.7 3.1
4.0 5.0
6.3 3.4
222 ꢂ 14
265 ꢂ 27
A2780cisR (RF)a >400
250 ꢂ 18
6.1 >9.8 187 ꢂ 19
284 ꢂ 17
136 ꢂ 13
>400 (—)
>400
>400
>400
>400
246 ꢂ 18 (1.1) 234 ꢂ 19 (1.1) 41 ꢂ 5 (0.9)
199 ꢂ 21 (3.1) 242 ꢂ 19 (1.0) 20 ꢂ 7 (0.8)
MCF-7
A549
301 ꢂ 30
255 ꢂ 15
288 ꢂ 23
247 ꢂ 17
305 ꢂ 29
325 ꢂ 22
279 ꢂ 18
291 ꢂ 16
219 ꢂ 18
284 ꢂ 19
245 ꢂ 17
77 ꢂ 9
3.2 >5.2 113 ꢂ 13
103 ꢂ 12
311 ꢂ 22
264 ꢂ 25
277 ꢂ 20
211 ꢂ 15
274 ꢂ 17
133 ꢂ 11
43 ꢂ 3
251 ꢂ 15
57 ꢂ 5
4.4 >7.0 95 ꢂ 12
87 ꢂ 6
104 ꢂ 15
29 ꢂ 5
A549cisR (RF)a >400
289 ꢂ 17
4.7 >6.5 212 ꢂ 9
142 ꢂ 10
>400 (—)
>400
265 ꢂ 19 (1.1) 228 ꢂ 19 (1.0) 61 ꢂ 6 (1.1)
218 ꢂ 10 (2.5) 232 ꢂ 16 (1.1) 33 ꢂ 5 (1.1)
HCT116
246 ꢂ 24
184 ꢂ 16
262 ꢂ 32
257 ꢂ 20
201 ꢂ 17
277 ꢂ 21
248 ꢂ 18
47 ꢂ 5
>300
5.3 >10.7 58 ꢂ 4
60 ꢂ 4
239 ꢂ 26
163 ꢂ 16
242 ꢂ 19
112 ꢂ 6
18 ꢂ 1
>400
>400
MRC-5
—
—
82 ꢂ 7
116 ꢂ 7
—
—
a
Resistance factor (RF): the IC50 in A2780cisR (A549cisR) cells under irradiation/the IC50 in A2780 (A549) cells under irradiation. b The IC50 values of
free complex 2c or 3c are >200 mM in all the tested cells. c Phototoxic index (PI): the IC50 of the dark group treated with rhodaplatin/the IC50 of the
irradiation group treated with rhodaplatin. d Fold increase (FI): the IC50 of carboplatin (or oxaliplatin) of irradiation group/the IC50 of rhodaplatin 1
(or rhodaplatin 2) of irradiation group.
rhodaplatin 2 could form ion-pair with ascorbate, determined rhodaplatin 2 and sodium ascorbate upon irradiation was
by NMR titration (Fig. S34†), indicating that rhodaplatin may conrmed by electron paramagnetic resonance (EPR) spec-
need to obtain electrons directly from free ascorbate in PBS troscopy (Fig. S39†), ascertaining that ascorbate is the electron
buffer. At low concentrations of ascorbate, the conversion rate donor for the photoreduction of rhodaplatins. Based on these
of rhodaplatin in PBS buffer increased with the ascorbate observations, we propose a possible photoreduction mecha-
concentration, but the rate became nearly constant at high nism of rhodaplatins in the presence of reducing agents (e.g.,
concentrations of ascorbate. The leveling off effect is dependent sodium ascorbate). As shown in Scheme 1, rhodaplatins were
on irradiation power intensity (Fig. S35†), indicating that when designed to be activated in physiological environments, in
there is sufficient ascorbate, the number of photoexcited rho- which rhodamine and its derivatives are present in the cationic
daplatin is the limiting factor for the photoconversion rate in form (I).20 Upon visible light irradiation, the rhodaplatin is rst
PBS buffer.
transformed to its excited state (II), then an electron from
As the reduction potential of a free rhodamine ligand ascorbate is transferred to the RhB ligand to form the Pt(IV)
signicantly decreases aer photoexcitation,17 electron transfer intermediate containing the RhB radical (III).13a Since RhB and
from the excited rhodamine ligand to the Pt center may occur to the Pt(IV) center are connected by a covalent bond, which can
reduce the Pt(IV) complex. To verify this hypothesis, we deter- facilitate the transfer of the extra electron to the Pt(IV) center to
mined the uorescence quantum yield and lifetime of RhB and yield the Pt(III) intermediate (IV) along with the release of
rhodaplatins in aqueous solutions. As shown in Table S2,† free a hydroxyl group. Aer that, the Pt(III) intermediate is promoted
RhB presented a higher quantum yield (0.34 vs. 0.18 and 0.19) to its excited state by irradiation, and steps (II) to (IV) are
and a longer uorescence lifetime (2.0 vs. 1.0 and 1.1 ns) than repeated to generate the Pt(II) drug and free RhB ligand.
rhodaplatins, indicating electron transfer from the excited
As rhodaplatins presented signicantly greater reduction
rhodamine moiety to the Pt center.18 Aer photoactivation, the efficiency than the “RhB plus Pt(IV)” platform, we subsequently
absorption and uorescence intensity of the completely pho- analyzed how this boosted photoconversion efficiency would
toactivated products were very close to those from the same affect their biological activities. We rst measured the cellular
amount of free RhB (Fig. S36 and S37†); no fragment of RhB was accumulation of rhodaplatins in A2780cisR platinum-resistant
detected in the photoreduction products (Fig. S22, S23 and ovarian cancer cells. Most rhodaplatins accumulated in the
S25†), indicating the RhB ligand remained intact during the cells within 6 h (Fig. S40†); thus 6 h was chosen as the treatment
photoactivation process. Therefore, electron transfer from time for the following cell-based assays. More importantly, aer
reducing agents to the Pt center through the excited RhB ligand incubation for 6 h, more than 90% of rhodaplatins have
may occur. For this reason, we measured the interaction remained stable in the culture medium or cancer cells; whereas
between free RhB and sodium ascorbate. In the presence of aer irradiation for 10 min with white light, more than 93% of
sodium ascorbate, the absorption of RhB rapidly changed rhodaplatins were reduced (Fig. S41†), indicating that rhoda-
(Fig. S38†), indicating the formation of the reduced counter- platins are sufficiently stable in the physiological environment
part.19 The formation of ascorbate radicals in the mixture of but can be rapidly photoactivated in cells. The high stability of
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Compared with Pt(II) drugs, rhodaplatins also displayed signif-
icantly enhanced photocytotoxicity (Table 1 and S3†). We
observed a similar effect in A549cisR platinum-resistant lung
cancer cells as well as cancer cells from other origins. For
example, photoactivated rhodaplatins exhibited not only
signicantly increased photocytotoxicities than the parent Pt(II)
drugs but also a greater ability to overcome drug resistance,
with the resistance factor (RF) values in the range of 0.8 to 1.1,
suggesting rhodaplatins may possess a distinct mechanism of
action to overcome developed drug resistance. Rhodaplatins
showed negligible dark cytotoxicity in normal cells.
As rhodaplatin 2 exhibited higher photocytotoxicity in the
tested cell lines and possessed greater potential to overcome
drug resistance in both monolayer and 3D tumor spheroid
models (Fig. S43†), we further explored its mechanism of action
to overcome drug resistance. Rhodaplatin 2 presented consid-
erable uorescence in an aqueous solution (Fig. S37†), which
enabled us to monitor its subcellular distribution. Since rho-
daplatin 2 is presented as a lipophilic cation, which may easily
cross the phospholipid bilayers and accumulate in the mito-
chondria or endoplasmic reticulum (ER),21 we treated the cells
with rhodaplatin 2 and co-stained the cells with uorescent
trackers of the mitochondria and ER. As shown in Fig. 4A and
S44,† the Pearson's colocalization coefficient (PCC) values of the
mitochondrial- and ER-trackers with rhodaplatin 2 are 0.90 and
0.73, respectively. The prodrug showed a similar subcellular
distribution tendency in MCF-7 cells (Fig. S45 and S46†), indi-
cating its strong mitochondria-targeting ability. As Pt-based
drugs are well-known DNA damaging agents,22 we measured
the interaction of rhodaplatin 2 with nuclear DNA (nDNA) and
mitochondrial DNA (mtDNA). For rhodaplatin 2- and
oxaliplatin-treated cells, the amount of Pt on nDNA is 0.084 and
0.72 ng Pt per mg DNA, respectively (Fig. S47†); no nDNA damage
response was triggered in the rhodaplatin 2-treated cells
(Fig. S48†), indicating that nDNA is not the target. In contrast,
photoactivated rhodaplatin 2 caused a much higher level of Pt–
mtDNA binding (Fig. 4B) and greater mtDNA damage (Fig. 4C
and S49†) than that of oxaliplatin. Notably, although complex 4
(Fig. S50†), another oxaliplatin-based Pt(IV) prodrug containing
triphenylphosphonium (TPP) as the mitochondria-targeting
group,23 exhibited comparable mitochondrial accumulation
efficiency to that of rhodaplatin 2 (Fig. S51†), complex 4 induced
much lower Pt–mtDNA binding amount (0.16 ng Pt per mg
mtDNA) than rhodaplatin 2 (2.7 ng Pt per mg mtDNA, Fig. 4B).
Moreover, complex 4 was found to be nontoxic towards cancer
cells (Table S4†), suggesting that it was not sufficiently activated
in cells, thus emphasizing the importance of developing tar-
geted Pt drugs with controllable activation properties. Subse-
quently, the loss of mitochondrial membrane potential, a major
event aer intense mtDNA damage,24 was also detected in the
cells treated with photoactivated rhodaplatin 2 but not oxali-
platin (Fig. S52†). Following this observation, apoptosis-
inducing factor (AIF) and endonuclease G (endo G), two
important apoptogenic factors that respond to mitochondrial
damage,25 were translocated from the mitochondria to the
nucleus (Fig. S53 and S54†), resulting in chromatin condensa-
tion (Fig. S55†).26 At the same time, the photoactivated
Fig. 4 Rhodaplatin 2 effectively accumulated in the mitochondria and
induced mitochondrial DNA damage after photoactivation. (A) The
cellular distribution of rhodaplatin 2 in A2780cisR cells. Cells were
treated with rhodaplatin 2 (10ꢁ5 M) for 6 h, then co-stained with
mitotracker. (B) The Pt–mtDNA binding amount in A2780cisR cells
after different treatments. Cells were treated with complexes (10ꢁ5 M)
for 6 h, then the culture medium was replaced, and cells were irradi-
ated with or without visible light (400–760 nm, 4 mW cmꢁ2) for 30 min
and further cultured for 1 h. (C) The relative amplification of nDNA and
mtDNA in A2780cisR cells treated with rhodaplatin 2. Cells were
treated with complex (10ꢁ5 M) for 6 h, then the culture medium was
replaced with fresh medium, and irradiated with or without visible light
(400–760 nm, 4 mW cmꢁ2) for 30 min. After irradiation, cells were
cultured for another 1, 6, and 12 h. (D) The apoptosis level of A2780cisR
cells after different treatments. Cells were treated with complex (10ꢁ5
M) for 6 h, then the medium was replaced with fresh medium, and cells
were irradiated with or without visible light (400–760 nm, 4 mW cmꢁ2
)
for 30 min and cultured for another 12 h. As a positive control, cells
were treated with oxaliplatin (10ꢁ4 M) for 6 h, then the medium was
replaced with fresh medium, and cells were cultured for another 12 h.
rhodaplatins in cells also ensures the colocalization of RhB and
the Pt center.
Next, we compared the photocytotoxicity of free RhB,
a mixture of RhB and Pt(IV), and rhodaplatins in A2780cisR
platinum-resistant ovarian cancer cells. No signicant change
in cell viability was observed aer irradiation (Fig. S42†). RhB
presented poor photocytotoxicity with an IC50 value of 2.46 ꢀ
10ꢁ4 M (Fig. 3). Compared with free RhB, a mixture of RhB and
Pt(IV) did not result in increased photocytotoxicity, suggesting
that the Pt(IV) substrates cannot be efficiently photocatalyzed to
their Pt(II) counterparts in cancer cells. In contrast, signicantly
enhanced photocytotoxicity was achieved for rhodaplatins, and
the IC50 values of rhodaplatin 1 and rhodaplatin 2 were 5.7- and
12-fold lower than that of the “RhB plus Pt(IV)” platform, indi-
cating the effective intracellular activation of rhodaplatins.
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rhodaplatin 2 triggered the release of cytochrome c (Fig. S56†),
an essential mitochondrial factor for intrinsic apoptosis,27 and
activated caspase-3 and -7 (Fig. S57†), the key mediators
responsible for mitochondria-mediated apoptosis,28 indicating
the initiation of apoptosis. As expected, photoactivated rhoda-
platin 2 induced a remarkably higher level of apoptosis than
oxaliplatin in A2780cisR cells (Fig. 4D). Both activation of
caspase-3/7 and nuclear fragmentation could be diminished by
co-treatment with the apoptosis inhibitor Z-VAD-FMK (Fig. S57
and S58†). These data conrmed that rhodaplatin 2 could
induce mtDNA damage and activate the nDNA-damage-
independent intrinsic apoptosis to overcome drug resistance.
Acknowledgements
We thank the Hong Kong Research Grants Council (Grant Nos.
CityU 11307419, 11304318, and 11303320) and the National
Natural Science Foundation of China (Grant Nos. 21877092 and
22077108) for funding support.
Notes and references
1 (a) B. A. Chabner and T. G. Roberts, Nat. Rev. Cancer, 2005, 5,
65–72; (b) L. Kelland, Nat. Rev. Cancer, 2007, 7, 573–584; (c)
´
E. Pasquier, M. Kavallaris and N. Andre, Nat. Rev. Clin.
Oncol., 2010, 7, 455–465.
2 D. C. Liebler and F. P. Guengerich, Nat. Rev. Drug Discovery,
2005, 4, 410–420.
Conclusion
In summary, we developed a couple of photoactivable Pt(IV)
prodrugs that could be effectively converted from their inert
Pt(IV) state to clinically active Pt(II) drugs via internal photo-
switch in physiological conditions. Compared with the
conventional “photocatalyst plus Pt(IV) substrate” platform, our
rhodaplatins possess signicantly closer proximity between the
photoswitch ligand and Pt(IV) center by a covalent bond,
enabling up to 4.8 ꢀ 104-fold increased photoconversion effi-
ciency and signicantly increased photocytotoxicity in cancer
cells. Aer the cellular entrance, rhodaplatin 2 effectively
accumulated in the mitochondria of cancer cells, and induced
mtDNA but not nDNA damage aer visible light irradiation.
Subsequently, the intense mtDNA damage led to the loss of
mitochondrial membrane potential and the release of pro-
apoptotic factor endoG, AIF, and cytochrome c in a nDNA-
damage-independent manner. These translocated factors
further triggered the condensation of chromatin and activation
of caspase-3/7 to initiate apoptosis and overcome drug resis-
tance (Fig. S59†). Taken together, our results suggest that the
distance between photocatalyst and Pt(IV) substrate is the
bottleneck for conventional photocatalysis platforms; short-
ening the distance by integration in the same molecule can
signicantly improve the photoconversion efficiency. In addi-
tion, this study may pave the way to design highly effective
platinum-based prodrugs with built-in photoswitches,29 and
explore their potential applications for cancer treatment, espe-
cially against drug-resistant cancers by activating nDNA-
damage-independent pathways.
3 (a) S. Zhang, H. Yuan, Y. Guo, K. Wang, X. Wang and Z. Guo,
Chem. Commun., 2018, 54, 11717–11720; (b) M. V. Babak,
Y. Zhi, B. Czarny, T. B. Toh, L. Hooi, E. K. H. Chow,
W. H. Ang, D. Gibson and G. Pastorin, Angew. Chem., Int.
Ed., 2019, 58, 8109–8114; (c) E. Petruzzella, J. P. Braude,
J. R. Aldrich-Wright, V. Gandin and D. Gibson, Angew.
Chem., Int. Ed., 2017, 56, 11539–11544; (d) S. G. Awuah,
Y.-R. Zheng, P. M. Bruno, M. T. Hemann and S. J. Lippard,
J. Am. Chem. Soc., 2015, 137, 14854–14857; (e)
D. Y. Q. Wong, C. H. F. Yeo and W. H. Ang, Angew. Chem.,
Int. Ed., 2014, 53, 6752–6756; (f) A. Sharma, M.-G. Lee,
M. Won, S. Koo, J. F. Arambula, J. L. Sessler, S.-G. Chi and
J. S. Kim, J. Am. Chem. Soc., 2019, 141, 15611–15618; (g)
G. Thiabaud, G. He, S. Sen, K. A. Shelton, W. B. Baze,
L. Segura, J. Alaniz, R. M. Macias, G. Lyness, A. B. Watts,
H. M. Kim, H. Lee, M. Y. Cho, K. S. Hong, R. Finch,
Z. H. Siddik, J. F. Arambula and J. L. Sessler, Proc. Natl.
Acad. Sci. U. S. A., 2020, 117, 7021–7029.
´
4 (a) I. Romero-Canelon, M. Mos and P. J. Sadler, J. Med.
Chem., 2015, 58, 7874–7880; (b) J. T. Weiss, J. C. Dawson,
´
K. G. Macleod, W. Rybski, C. Fraser, C. Torres-Sanchez,
E. E. Patton, M. Bradley, N. O. Carragher and A. Unciti-
¨
Broceta, Nat. Commun., 2014, 5, 3277; (c) T. Volker,
F. Dempwolff, P. L. Graumann and E. Meggers, Angew.
Chem., Int. Ed., 2014, 53, 10536–10540.
5 (a) H. Wang, R. Wang, K. Cai, H. He, Y. Liu, J. Yen, Z. Wang,
M. Xu, Y. Sun and X. Zhou, Nat. Chem. Biol., 2017, 13, 415–
424; (b) P. Thirumurugan, D. Matosiuk and K. Jozwiak,
Chem. Rev., 2013, 113, 4905–4979; (c) F. Wang, Y. Zhang,
Z. Liu, Z. Du, L. Zhang, J. Ren and X. Qu, Angew. Chem.,
Int. Ed., 2019, 58, 6987–6992.
6 (a) A. Unciti-Broceta, Nat. Chem., 2015, 7, 538–539; (b) J. Li,
H. Kong, C. Zhu and Y. Zhang, Chem. Sci., 2020, 11, 3390–
3396; (c) H. Huang, S. Banerjee, K. Qiu, P. Zhang,
O. Blacque, T. Malcomson, M. J. Paterson, G. J. Clarkson,
M. Staniforth, V. G. Stavros, G. Gasser, H. Chao and
P. J. Sadler, Nat. Chem., 2019, 11, 1041–1048.
Author contributions
Z. D. and G. Z. designed the study. Z. X., H. Y., Q. Z., and Z. D.
synthesized these complexes. Z. D. performed the experiments.
S. C. and Z. D. detected the photo properties of rhodaplatin.
C. L. and Z. D. completed cell-based experiments. H. H. carried
out the DFT calculation. Z. W. carried out the EPR experiments.
Z. D. and G. Z. analyzed the data. Z. D. and G. Z. wrote the paper.
All authors edited and approved the nal manuscript.
7 (a) S. Alonso-de Castro, E. Ruggiero, A. Ruiz-de-Angulo,
´
E. Rezabal, J. C. Mareque-Rivas, X. Lopez, F. Lopez-Gallego
Conflicts of interest
and L. Salassa, Chem. Sci., 2017, 8, 4619–4625; (b)
´
´
There are no conicts to declare.
J. Gurruchaga-Pereda, V. Martınez-Martınez, E. Rezabal,
Chem. Sci.
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X. Lopez, C. Garino, F. Mancin, A. L. Cortajarena and 17 Y. Kim, H.-B. Kim and D.-J. Jang, J. Mater. Chem. A, 2014, 2,
L. Salassa, ACS Catal., 2019, 10, 187–196. 5791–5799.
8 D. Norman, A. Gambardella, A. Mount, A. Murray and 18 Y. Koide, Y. Urano, K. Hanaoka, T. Terai and T. Nagano, ACS
M. Bradley, Angew. Chem., Int. Ed., 2019, 58, 14189–14192.
Chem. Biol., 2011, 6, 600–608.
´
´
9 (a) S. Alonso-de Castro, A. L. Cortajarena, F. Lopez-Gallego 19 J. Piella, F. Merkoçi, A. Genç, J. Arbiol, N. G. Bastus and
and L. Salassa, Angew. Chem., Int. Ed., 2018, 57, 3143–3147;
(b) M. D. Hall and T. W. Hambley, Coord. Chem. Rev., 2002, 20 M. Homma, Y. Takei, A. Murata, T. Inoue and S. Takeoka,
232, 49–67; (c) O. Pinato, C. Musetti and C. Sissi, Chem. Commun., 2015, 51, 6194–6197.
Metallomics, 2014, 6, 380–395; (d) A. Lasorsa, 21 (a) S. Louzoun-Zada, Q. Z. Jaber and M. Fridman, Angew.
V. Puntes, J. Mater. Chem. A, 2017, 5, 11917–11929.
´
´
O. Stuchlıkova, V. Brabec, G. Natile and F. Arnesano, Mol.
Chem., Int. Ed., 2019, 58, 15584–15594; (b) M. P. Murphy,
Biochim. Biophys. Acta, Bioenerg., 2008, 1777, 1028–1031.
Pharm., 2016, 13, 3216–3223.
10 L. Kazak, A. Reyes and I. J. Holt, Nat. Rev. Mol. Cell Biol., 22 T. C. Johnstone, K. Suntharalingam and S. J. Lippard, Chem.
2012, 13, 659–671. Rev., 2016, 116, 3436–3486.
11 (a) S. Marrache, R. K. Pathak and S. Dhar, Proc. Natl. Acad. 23 S. Marrache and S. Dhar, Proc. Natl. Acad. Sci. U. S. A., 2012,
Sci. U. S. A., 2014, 111, 10444–10449; (b) S. P. Wisnovsky, 109, 16288–16293.
J. J. Wilson, R. J. Radford, M. P. Pereira, M. R. Chan, 24 J. H. Santos, L. Hunakova, Y. Chen, C. Bortner and B. Van
R. R. Laposa, S. J. Lippard and S. O. Kelley, Chem. Biol.,
2013, 20, 1323–1328.
12 M. Beija, C. A. Afonso and J. M. Martinho, Chem. Soc. Rev.,
2009, 38, 2410–2433.
Houten, J. Biol. Chem., 2003, 278, 1728–1734.
25 (a) L. Y. Li, X. Luo and X. Wang, Nature, 2001, 412, 95–99; (b)
J. M. Penninger and G. Kroemer, Nat. Cell Biol., 2003, 5, 97–
99.
13 (a) E. Yoshioka, S. Kohtani, T. Jichu, T. Fukazawa, T. Nagai, 26 S. Kilbride and J. Prehn, Oncogene, 2013, 32, 2703–2711.
A. Kawashima, Y. Takemoto and H. Miyabe, J. Org. Chem., 27 R. M. Kluck, E. Bossy-Wetzel, D. R. Green and
2016, 81, 7217–7229; (b) M. N. Hopkinson, B. Sahoo and
F. Glorius, Adv. Synth. Catal., 2014, 356, 2794–2800.
14 (a) M. D. Hall, H. L. Daly, J. Z. Zhang, M. Zhang,
R. A. Alderden, D. Pursche, G. J. Foran and T. W. Hambley,
D. D. Newmeyer, Science, 1997, 275, 1132–1136.
28 S. A. Lakhani, A. Masud, K. Kuida, G. A. Porter, C. J. Booth,
W. Z. Mehal, I. Inayat and R. A. Flavell, Science, 2006, 311,
847–851.
Metallomics, 2012, 4, 568–575; (b) H. P. Varbanov, 29 (a) V. E. Y. Lee, C. F. Chin and W. H. Ang, Dalton Trans., 2019,
S. M. Valiahdi, C. R. Kowol, M. A. Jakupec, M. Galanski
and B. K. Keppler, Dalton Trans., 2012, 41, 14404–14415; (c)
J. Z. Zhang, E. Wexselblatt, T. W. Hambley and D. Gibson,
Chem. Commun., 2012, 48, 847–849.
48, 7388–7393; (b) G. Thiabaud, J. F. Arambula, Z. H. Siddik
and J. L. Sessler, Chem.–Eur. J., 2014, 20, 8942–8947; (c)
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; (d)
15 G. Saito, J. A. Swanson and K.-D. Lee, Adv. Drug Delivery Rev.,
2003, 55, 199–215.
ˇ
´
J. Kasparkova, H. Kostrhunova, O. Novakova, R. Krikavova,
ˇ
´ ´ˇ
16 P. Thordarson, Chem. Soc. Rev., 2011, 40, 1305–1323.
J. Vanco, Z. Travnıcek and V. Brabec, Angew. Chem., Int.
Ed., 2015, 54, 14478–14482.
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