Zhiqin
Deng
ab,
Cai
Li
ab,
Shu
Chen
ab,
Qiyuan
Zhou
ab,
Zoufeng
Xu
ab,
Zhigang
Wang
c,
Houzong
Yao
ab,
Hajime
Hirao
a and
Guangyu
Zhu
*ab
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. China
cSchool of Pharmaceutical Sciences, Health Science Center, Shenzhen University, Shenzhen, P. R. China
First published on 1st April 2021
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.
The emerging concept of combining photocatalysis and bioorthogonal reactions for biological and medicinal applications has drawn much attention.6 A common strategy is to use external photocatalysts to activate Pt(IV) anticancer prodrugs in cancer cells to improve the prodrugs' cancer selectivity. For instance, riboflavin 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 photocatalytic efficiency. Another concern is the intracellular stability of the Pt(IV) prodrugs, some of which may be photoactivated outside of cells. Moreover, other intracellular biomolecules may competitively react with the Pt(IV) substrate or catalyst.9 Therefore, the current platform of “external photocatalyst plus Pt(IV) substrate” has its own limitations regarding biological applications. Indeed, the co-treatment strategy has achieved only limited enhancement in cytotoxicity compared with the original Pt(II) drugs.
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 internalized photoswitch that is colocalized with the Pt center ensures the prodrugs dramatically boosted intracellular activation efficiency and significantly 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 mitochondrial 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 effective photoactivatable Pt(IV) prodrugs for controllable and selective activation in cancer cells.
To improve the photoconversion performance, we speculated that shortening the distance between the photocatalyst and the substrate to increase the electron transfer efficiency might be a promising approach. To verify this hypothesis, we directly conjugated RhB with carboplatin- and oxaliplatin-based Pt(IV) complexes, such that the distance between the photoswitch and the Pt(IV) center significantly decreased. The synthetic complexes were designated as rhodaplatin 1 and rhodaplatin 2, for the carboplatin- and oxaliplatin-based 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, GSH, or NADPH),15 we monitored the stability and photoreduction 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 after incubation for 12 h (Fig. S21†). Upon irradiation with low-dose visible light (400–760 nm, 4 mW cm−2), however, 95% of rhodaplatins were converted to the corresponding Pt(II) drugs within 5 min in PBS buffer (pH 7.4) containing sodium ascorbate (Fig. 2B, C, S22 and S23†). High stability and rapid photoreduction were also observed in the presence of glutathione (Fig. S24 and S25†). In the presence of ascorbate, the conversion rate of rhodaplatin 1 at 10−4 M was calculated to be 2 × 10−5 M min−1, 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 10−4 M was up to 4.8 × 104-fold higher than that of RhB towards oxaliplatin-based Pt(IV) substrates (Fig. 2E). Notably, compared with riboflavin, an effective photocatalyst to convert Pt(IV) prodrugs to Pt(II) forms,7a the photocatalysis efficiency of free RhB was 6.6 × 104 to 1.32 × 106 times lower than that of riboflavin, but rhodaplatins presented a comparable photoconversion rate with that from riboflavin. These data confirm that the enhanced proximity and the covalent bond between the photoswitch and the Pt(IV) center could significantly accelerate the photoconversion 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 was investigated in the presence of excess Pt(IV) substrate or RhB at various concentrations. As shown in Fig. S26 and S27,† 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 importance of the distance and covalent bond between the photoswitch 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 photoreduction rate of rhodaplatin increased linearly with the power density of irradiation and the concentration of rhodaplatin, indicating a first-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 first 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 determined to be 3149 M−1 by UV-Vis spectroscopic titration (Fig. S31†).16 A similar result was obtained by fluorescence 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 rhodaplatin 2 could form ion-pair with ascorbate, determined by NMR titration (Fig. S34†), indicating that rhodaplatin may need to obtain electrons directly from free ascorbate in PBS buffer. At low concentrations of ascorbate, the conversion rate of rhodaplatin in PBS buffer increased with the ascorbate concentration, but the rate became nearly constant at high concentrations of ascorbate. The leveling off effect is dependent on irradiation power intensity (Fig. S35†), indicating that when there is sufficient ascorbate, the number of photoexcited rhodaplatin is the limiting factor for the photoconversion rate in PBS buffer.
As the reduction potential of a free rhodamine ligand significantly decreases after photoexcitation,17 electron transfer from the excited rhodamine ligand to the Pt center may occur to reduce the Pt(IV) complex. To verify this hypothesis, we determined the fluorescence quantum yield and lifetime of RhB and rhodaplatins in aqueous solutions. As shown in Table S2,† free RhB presented a higher quantum yield (0.34 vs. 0.18 and 0.19) and a longer fluorescence lifetime (2.0 vs. 1.0 and 1.1 ns) than rhodaplatins, indicating electron transfer from the excited rhodamine moiety to the Pt center.18 After photoactivation, the absorption and fluorescence intensity of the completely photoactivated products were very close to those from the same amount of free RhB (Fig. S36 and S37†); no fragment of RhB was detected in the photoreduction products (Fig. S22, S23 and S25†), indicating the RhB ligand remained intact during the photoactivation process. Therefore, electron transfer from reducing agents to the Pt center through the excited RhB ligand may occur. For this reason, we measured the interaction between free RhB and sodium ascorbate. In the presence of sodium ascorbate, the absorption of RhB rapidly changed (Fig. S38†), indicating the formation of the reduced counterpart.19 The formation of ascorbate radicals in the mixture of rhodaplatin 2 and sodium ascorbate upon irradiation was confirmed by electron paramagnetic resonance (EPR) spectroscopy (Fig. S39†), ascertaining that ascorbate is the electron donor for the photoreduction of rhodaplatins. Based on these observations, we propose a possible photoreduction mechanism of rhodaplatins in the presence of reducing agents (e.g., sodium ascorbate). As shown in Scheme 1, rhodaplatins were designed to be activated in physiological environments, in which rhodamine and its derivatives are present in the cationic form (I).20 Upon visible light irradiation, the rhodaplatin is first transformed to its excited state (II), then an electron from ascorbate is transferred to the RhB ligand to form the Pt(IV) intermediate containing the RhB radical (III).13a Since RhB and the Pt(IV) center are connected by a covalent bond, which can facilitate the transfer of the extra electron to the Pt(IV) center to yield the Pt(III) intermediate (IV) along with the release of a hydroxyl group. After that, the Pt(III) intermediate is promoted to its excited state by irradiation, and steps (II) to (IV) are repeated to generate the Pt(II) drug and free RhB ligand.
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. |
As rhodaplatins presented significantly greater reduction efficiency than the “RhB plus Pt(IV)” platform, we subsequently analyzed how this boosted photoconversion efficiency would affect their biological activities. We first measured the cellular accumulation of rhodaplatins in A2780cisR platinum-resistant ovarian cancer cells. Most rhodaplatins accumulated in the cells within 6 h (Fig. S40†); thus 6 h was chosen as the treatment time for the following cell-based assays. More importantly, after incubation for 6 h, more than 90% of rhodaplatins have remained stable in the culture medium or cancer cells; whereas after irradiation for 10 min with white light, more than 93% of rhodaplatins were reduced (Fig. S41†), indicating that rhodaplatins are sufficiently stable in the physiological environment but can be rapidly photoactivated in cells. The high stability of 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 significant change in cell viability was observed after irradiation (Fig. S42†). RhB presented poor photocytotoxicity with an IC50 value of 2.46 × 10−4 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, significantly 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, indicating the effective intracellular activation of rhodaplatins. Compared with Pt(II) drugs, rhodaplatins also displayed significantly 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 significantly 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.
Cell line | IC50 [μM] | PIc | FId | IC50 [μM] | PIc | FId | |||||
---|---|---|---|---|---|---|---|---|---|---|---|
Carboplatin | RhB | RhB + 2cb | Rhodaplatin 1 | Oxaliplatin | RhB + 3cb | Rhodaplatin 2 | |||||
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 μM 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. | |||||||||||
A2780 | 301 ± 28 | 258 ± 19 | 242 ± 19 | 220 ± 17 | 5.0 | 7.3 | 68 ± 6 | 263 ± 18 | 108 ± 9 | 4.4 | 2.6 |
322 ± 33 | 222 ± 14 | 204 ± 19 | 44 ± 5 | 64 ± 6 | 235 ± 9 | 25 ± 2 | |||||
A2780cisR (RF)a | >400 | 265 ± 27 | 254 ± 21 | 250 ± 18 | 6.1 | >9.8 | 187 ± 19 | 284 ± 17 | 136 ± 13 | 6.7 | 9.8 |
>400 (—) | 246 ± 18 (1.1) | 234 ± 19 (1.1) | 41 ± 5 (0.9) | 199 ± 21 (3.1) | 242 ± 19 (1.0) | 20 ± 7 (0.8) | |||||
MCF-7 | >400 | 301 ± 30 | 325 ± 22 | 245 ± 17 | 3.2 | >5.2 | 113 ± 13 | 311 ± 22 | 133 ± 11 | 3.1 | 2.4 |
>400 | 255 ± 15 | 279 ± 18 | 77 ± 9 | 103 ± 12 | 264 ± 25 | 43 ± 3 | |||||
A549 | >400 | 288 ± 23 | 291 ± 16 | 251 ± 15 | 4.4 | >7.0 | 95 ± 12 | 277 ± 20 | 104 ± 15 | 3.7 | 3.1 |
>400 | 247 ± 17 | 219 ± 18 | 57 ± 5 | 87 ± 6 | 211 ± 15 | 29 ± 5 | |||||
A549cisR (RF)a | >400 | 305 ± 29 | 284 ± 19 | 289 ± 17 | 4.7 | >6.5 | 212 ± 9 | 274 ± 17 | 142 ± 10 | 4.0 | 5.0 |
>400 (—) | 265 ± 19 (1.1) | 228 ± 19 (1.0) | 61 ± 6 (1.1) | 218 ± 10 (2.5) | 232 ± 16 (1.1) | 33 ± 5 (1.1) | |||||
HCT116 | >400 | 246 ± 24 | 257 ± 20 | 248 ± 18 | 5.3 | >10.7 | 58 ± 4 | 239 ± 26 | 112 ± 6 | 6.3 | 3.4 |
>400 | 184 ± 16 | 201 ± 17 | 47 ± 5 | 60 ± 4 | 163 ± 16 | 18 ± 1 | |||||
MRC-5 | >400 | 262 ± 32 | 277 ± 21 | >300 | — | — | 82 ± 7 | 242 ± 19 | 116 ± 7 | — | — |
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 considerable fluorescence in an aqueous solution (Fig. S37†), which enabled us to monitor its subcellular distribution. Since rhodaplatin 2 is presented as a lipophilic cation, which may easily cross the phospholipid bilayers and accumulate in the mitochondria or endoplasmic reticulum (ER),21 we treated the cells with rhodaplatin 2 and co-stained the cells with fluorescent 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†), indicating 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 μg 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 μg mtDNA) than rhodaplatin 2 (2.7 ng Pt per μg 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 targeted Pt drugs with controllable activation properties. Subsequently, the loss of mitochondrial membrane potential, a major event after intense mtDNA damage,24 was also detected in the cells treated with photoactivated rhodaplatin 2 but not oxaliplatin (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 condensation (Fig. S55†).26 At the same time, the photoactivated 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 rhodaplatin 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 confirmed that rhodaplatin 2 could induce mtDNA damage and activate the nDNA-damage-independent intrinsic apoptosis to overcome drug resistance.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0sc06839j |
This journal is © The Royal Society of Chemistry 2021 |