In vivo metallophilic self-assembly of a light-activated anticancer drug

Synthesis and characterization of a light-sensitive DSDS

As in the padeliporfin photosensitizer recently approved for clinical photodynamic therapy (PDT)25, the PdL small molecule (Fig. 1a) contains a palladium(ii) metal centre. In contrast with padeliporfin, however, PdL is a bis-cyclometalated palladium compound characterized by the presence of two Pd–C covalent bonds (see Fig. 1b and full characterization in Supplementary Figs. 3 and 4 and Supplementary Table 1). Its X-ray structure (Fig. 1b) shows head-to-tail dimers with a short interplanar distance of 3.4 Å and a short Pd⋯Pd distance of 3.518 Å, characteristic of metallophilic interactions. A density functional theory (DFT) model of the supramolecular dimer converged at a Pd⋯Pd distance of 3.26 Å (Fig. 1c), matching well with that observed in the crystal. The metallophilic interaction derives from the hybridization of both palladium d4z2 orbitals and π orbitals of the aromatic ligand in the highest occupied molecular orbital (HOMO) of the dimer. A dimerization energy of −40.0 kcal mol−1 was found for the dimer [PdL]2, while that of the ligand dimer [H2L]2 (where H2L is (bis(3-(pyridin-2-yl)phenyl)amine)) was −33.7 kcal mol−1. As the ligand H2L can only dimerize via π–π interactions, these calculations suggest that the overlap of the palladium d4z2 orbitals contributed ~16% to the supramolecular interaction between two PdL molecules in the dimer, while π–π stacking contributed ~84%. Time-dependent DFT (TDDFT) calculations at the same degree of theory further confirmed the decrease in the gap between the HOMO and the lowest unoccupied molecular orbital (LUMO) induced by supramolecular dimerization, with a bathochromic shift of the lowest-energy absorption band from 383 nm for the monomer to 502 nm for the dimer (Fig. 1d).

Fig. 1: Synthesis, crystal structure, DFT calculation and photophysical properties of PdL.figure 1

a, Synthesis of H2L and PdL ((i) Pd(dba)2, KOt-Bu, BINAP and toluene at 95 °C under N2 for 72 h with a yield of 67%; (ii) Pd(OAc)2 and CH3COOH at 135 °C for 24 h with a yield of 56%). b, Displacement ellipsoid plot (50% probability level) of PdL and its stacking structure at 110(2) K. c, DFT calculation of HOMOs (bottom) and LUMOs (top) of PdL as a monomer or dimer (calculated Pd⋯Pd distance = 3.26 Å). Occupied orbitals (HOMOs) have red and blue lobes, whereas unoccupied orbitals (LUMOs) have brown and cyan lobes. Element colour code: blue = N, grey = C, brown = Pd and white = H. d, TDDFT-calculated spectra of PdL as a monomer (black line) or dimer (red line). Level of theory: TDDFT/PBE0/TZP/COSMO (in methanol). e, Absorption spectrum (black solid line) and emission spectra of PdL in pure DMSO solution at different concentrations (blue dashed line = 10 µM; black dashed line = 100 µM; red dashed line = 1,000 µM; excitation = 419 nm). f, Time evolution of the absorption spectra of H2O/DMSO solutions (100 µM; fw = Vwater/Vtotal = 0.9) of PdL at 298 K for 30 min (30-s interval; the colours of the spectra change from black (0 min) to red (30 min); the blue line is the absorbance spectrum of PdL (100 µM) in pure DMSO. Inset, TEM images of nanostructures of PdL in H2O/DMSO solution (100 µM; fw = Vwater/Vtotal = 0.9; scale bar, 2 µm). g, Emission spectra of PdL (100 µM) in H2O/DMSO mixtures with different volumetric ratios fw.

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When dissolved in dimethyl sulfoxide (DMSO) in air, PdL showed a low emission quantum yield (φp = 0.0016) with a short lifetime (τ = 0.295 µs) (Fig. 1e and Supplementary Table 6). Under degassed conditions, the emission quantum yield and lifetime increased by 1–2 orders of magnitude (0.07 and 29.6 μs, respectively), demonstrating the triplet character of the excited state of PdL. The corresponding photoluminescence quenching efficiency was ~98%, which suggested that 3PdL* efficiently interacts with molecular oxygen. Transient spectra under aerated and degassed conditions (Supplementary Fig. 5) demonstrated that the initially formed singlet excited state 1PdL* performs fast intersystem crossing to 3PdL* (rate = 34.5 ns−1), which then reacts with O2 via electron transfer to generate a PdL+ cation radical. This result strongly suggested that PdL may engage in PDT type I reactivity to generate superoxide radicals (O2•−) by electron transfer.

In pure DMSO, PdL (100 μM) was mostly monomeric (Supplementary Fig. 6a) and minute variations of the ultraviolet–visible spectrum with temperature tentatively suggested low-affinity dimerization with a dimerization constant of 103–104 M−1 (Supplementary Fig. 6b,c and Supplementary Table 7)26,27. In contrast, in a DMSO/H2O 1/9 mixture (100 µM), a rapid (<1 min) increase in the baseline and the generation of a new absorbance peak at 504 nm were observed (Fig. 1f), which are typical for metal–metal-to-ligand charge transfer excited states induced by Pd⋯Pd interactions28 and altogether suggest efficient and fast self-assembly. This hypothesis was confirmed by transmission electron microscopy (TEM) images showing nanorods and nanocubes (Fig. 1f, insert). Usually, the formation of Pd⋯Pd supramolecular bonds is accompanied by a long-wavelength emission peak28, and indeed an increase in the H2O content in DMSO/H2O mixtures (fw = Vwater/Vtotal is the ratio of the water volume Vwater and the total volume of the solution Vtotal) led to a gradual replacement of the monomeric emission peak at 564 nm (as observed in pure DMSO; fw = 0) by new emission maxima at 593 nm (fw = 0.5) and finally 610–670 nm (fw = 0.9) concomitant with the formation of a precipitate (Fig. 1g). In tetrahydrofuran/H2O solutions, similar self-assembly was observed, albeit with slower polymerization rates and different morphologies (Supplementary Figs. 7 and 8). The two series of self-assembled nanoparticles showed different powder X-ray diffraction patterns that were also different from the pattern calculated from the crystal structure or obtained from a bulk solid sample of PdL (Supplementary Fig. 9). This result indicated that the four types of materials are different polymorphs. Overall, PdL appeared to self-assemble readily in the presence of water, although the resulting self-assembly depended entirely on the detailed composition of the solvent it was dissolved in.

The self-assembly of PdL was then studied in a cell-growing medium called Opti-MEM complete that contained 2.5 vol% foetal calf serum. At 25 µM, aggregation immediately occurred, as shown by a hydrodynamic diameter of approximately 164 nm determined by dynamic light scattering (DLS; Fig. 2a). After 30 min, the maximum hydrodynamic diameter had only slightly shifted to 190 nm, but the number of particles had increased significantly (Fig. 2b). The absorption of the solution (Fig. 2c) showed a gradual baseline increase during the first 2 h, which is characteristic of light scattering by nanoparticles, to remain constant over 24 h. The main nanostructures observed by TEM in the medium were nanodots (Fig. 2d), but these nanodots self-assembled as regular nanofibres that gradually lengthened. Cryogenic electron microscopy (Cryo-EM) imaging confirmed the formation of nanofibres in such medium, characterized by a well-ordered structure at a repeating distance of ~1.68 nm in the Fourier transform image (Fig. 2e). As for the monomer in DMSO, in medium the photoluminescence lifetime of aggregates of PdL was dramatically quenched by O2 from 83.5 to 0.058 μs (Supplementary Fig. 5g,h), suggesting that the aggregates reacted even faster with O2 than molecular PdL (in DMSO). Also, the different shapes of the nanorods in Fig. 2d compared with those in DMSO/H2O (Fig. 1f) or tetrahydrofuran/H2O mixtures (Supplementary Fig. 7d) suggested that nanorod formation may involve proteins in the medium. Hence, we determined the protein content of the nanoaggregates formed in cell medium via protein gel and the commercial Pierce BCA Protein Assay Kit. The results (Supplementary Fig. 10) indicated that the amount of protein involved in the stabilization of the nanoparticles in medium was very low, and that the different shapes observed under such conditions must be due to other factors, such as compound concentration, solvent polarity, pH, ionic force, the presence of salts, viscosity, and so on. Overall, in cell-growing medium, DLS, electron microscopy, ultraviolet–visible spectroscopy and protein determination demonstrated the time-dependent self-assembly of PdL into nanorods and nanoparticles, which, considering DFT and crystal structure analysis, must at least in part be a result of the metallophilic Pd⋯Pd interaction.

Fig. 2: Self-assembly and aggregation nanostructure of PdL in cell medium.figure 2

a, Size distribution of Opti-MEM complete medium and its PdL (25 µM) solution at 0 min (red line) or 30 min (blue line) according to DLS analysis at room temperature. d.nm, particle diameter in nanometer. b, DLS-derived count rate of PdL solution in Opti-MEM complete medium for 0 and 30 min. The data represent means ± s.d. of three replicates. kcps, kilocounts (of photons) per second. c, Observed absorbance spectra of PdL (25 µM) in Opti-MEM complete medium over 24 h (30-s intervals for the first 30 min and 15-min intervals for the remaining 23.5 h). d,e, TEM (d) and cryo-EM images (e) of samples prepared from an Opti-MEM complete medium solution of PdL (25 µM) at room temperature. Insert in e shows the Fourier transform of the atomically resolved area highlighted by a dashed box. nm/c, nanometer per cycle. f, Singlet oxygen emission spectra of [Ru(bpy)3]Cl2 (black) and PdL (red) in CD3OD irradiated with blue light (λex = 450 nm; 50 mW; 0.4 W cm−2). g, Time evolution of the absorption spectrum (left) and of the absorbance at 378 nm (right) of a 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABMDMA) Opti-MEM complete solution (100 µM) in the presence of PdL (25 µM) under green light irradiation (515 nm; 2.0 mW) over 5 min. h, Time evolution of the emission spectrum (left, middle) and of the emission intensity at 615 nm (right) of a DHE solution in DMSO (left) or Opti-MEM complete (middle) in the presence or absence of PdL (25 μM) under green light irradiation (515 nm; 2.0 mW) over 60 s.

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As the next step, the influence of self-assembly on the photochemical properties of PdL was considered. Photodynamic effects may occur either via a type I mechanism (electron transfer) or a type II (energy transfer) mechanism29. Direct detection of the near-infrared emission peak of 1O2 at 1,270 nm under blue light irradiation (450 nm) was only possible in CD3OD and hence for the PdL monomer. The corresponding 1O2 generation quantum yield was very low (φΔ = 0.09; Fig. 2f and Supplementary Table 6). In Opti-MEM medium, and hence for the self-assembled form of PdL (25 µM), indirect 1O2 detection using the chemoselective chemical probe 9,10-anthracenediyl-bis(methylene)dimalonic acid showed no decrease in the absorbance band at 378 nm upon green light irradiation, characteristic of the 1O2 adduct (Fig. 2g)30, indicating negligible 1O2 generation (φΔ = 0.04; Supplementary Fig. 8)31. Overall, PdL is a poor PDT type II sensitizer, both as a monomer in methanol and as aggregates in medium. In contrast, type I PDT sensitizers can be characterized by the initial generation of superoxide radicals (O2•−), which can further generate other reactive oxygen species (ROS), such as HO• or H2O2 (ref. 32). When a DMSO or Opti-MEM solution of PdL (25 µM) was irradiated with green light in the presence of dihydroethidium (DHE)—a chemoselective chemical probe for superoxide—the oxidation product 2-hydroxyethidium was produced efficiently, as shown by its emission at 590–620 nm (Fig. 2h and Supplementary Fig. 9)33. These results clearly demonstrated that PdL is capable of photochemically generating superoxide both in the monomeric and aggregated states, which suggested that as a light-activated DSDS it may behave as a PDT type I photosensitizer, generating its photodynamic effect by electron transfer.

Biological properties in vitro and in vivo

Considering the good absorption of PdL at 520 nm (ε = 915 M−1 cm−1 in DMSO) and its PDT type I properties, its cytotoxicity was evaluated first in vitro using two-dimensional (2D) monolayers of lung carcinoma (A549), epidermoid carcinoma (A431) and skin melanoma (A375) cell lines, both in the dark and under green light irradiation. PdL showed moderate dark cytotoxicity (half-maximal effective concentration (EC50) > 10 µM) for the three cancer cell lines under normoxic (21% O2) and hypoxic (1% O2) conditions (Supplementary Table 8). In contrast, upon green light irradiation (520 nm; 13 J cm−2) under normoxia and hypoxia, PdL exhibited high phototoxicity with submicromolar EC50 and high photoindices (EC50,dark/EC50,light = 32–72; Fig. 3a, Supplementary Fig. 10 and Supplementary Table 8), thus demonstrating outstanding PDT efficacy even at low dioxygen concentrations. Clearly, at the concentrations used (0.5 and 2 µM), PdL showed no or limited cell death under dark conditions, as determined by annexin V/propidium iodide double staining experiments (Fig. 3b and Supplementary Fig. 11). In the light-irradiated group, no toxicity was observed after 2 h, but after 4 h and 24 h, the numbers of apoptotic and necrotic cells had increased, suggesting that PdL induced cancer cell death 4 h after irradiation via both cell death mechanisms. The cytotoxicity of PdL in 3D multicellular tumour spheroid models (A549 and A375), which better mimic the physical penetration of light and drugs in three dimensions34, was nearly 100-fold higher under light irradiation (EC50 = ~0.20 µM) than under dark conditions (EC50 > 25 µM), while light activation was accompanied by visible collapse of the spheroid cores and dramatic shrinkage of the spheroid diameters (Extended Data Fig. 1a, Supplementary Fig. 12 and Supplementary Table 8). A further Hoechst 33342/propidium iodide double staining experiment was carried out to compare the morphology and health status of A375 spheroids after treatment. The red fluorescence of propidium iodide increased in the green light-irradiated group compared with the dark group (Extended Data Fig. 1a for 1 µM), confirming drug penetration and light-induced cell killing by membrane disruption in 3D environments.

Fig. 3: In vitro and in vivo anticancer properties of PdL.figure 3

a, EC50 values of PdL in A375 2D monolayer and 3D spheroid cancer cells incubated either in the dark or upon green light irradiation (13 J cm−2) and under normoxic or hypoxic conditions. The data points represent averages (n = 3) with 95% confidence intervals. Statistical significance was determined by two-way analysis of variance (ANOVA) (*P < 0.05). b, Flow cytometry quantification of healthy, early apoptotic, later apoptotic and necrotic A375 cells after treatment with PdL (2 µM) in the dark or with green light irradiation over a time gradient (2, 4 and 24 h). Cisplatin (7.5 µM; 24 h) was used as a positive control. c, Time evolution of mouse weight up to 20 d post-treatment. d, A375 tumour growth inhibition in different groups of mice treated by tail intravenous injection. The data represent means ± s.d. of three replicates. Statistical significance was determined by two-way ANOVA (**P < 0.05). Light irradiation conditions: 520 nm, 100 mW cm−2, 10 min and 60 J cm−2. Dose: 2.1 µmol kg−1, 420 µM, 100 µl DMEM (10% FBS) and 0.9 mg kg−1.

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To explain the phototoxicity observed in vitro, a few mechanistic studies were undertaken. First, the intracellular ROS (as demonstrated with the non-selective dichlorodihydrofluorescein diacetate (DCFDA) probe), and especially superoxide levels (as demonstrated with the DHE probe), were found to be significantly increased after light irradiation in the presence of PdL (Supplementary Fig. 16), while the glutathione levels decreased (Supplementary Fig. 17). These results confirmed the conclusion of photophysical studies—that electron transfer (PDT type I) is probably the main source of the phototoxicity of PdL aggregates in cells. A cellular fractionation experiment was then realized to see where the palladium was mainly located (Supplementary Fig. 18); clearly, PdL accumulated in the membrane fraction (which includes cell, mitochondria and lysosome membranes) and the nuclei (Supplementary Fig. 18). This result was consistent with the idea that an endocytic uptake mechanism may be working for these aggregates; it also suggested that the DNA photocleavage properties of PdL should be tested. However, DNA agarose gel experiments at different concentrations (20–100 µM), irradiation times (5–60 min) and incubation times (1–24 h) after activation demonstrated that PdL neither interacted with DNA in the dark nor cleaved DNA upon irradiation (Supplementary Fig. 19). Interestingly, after incubating PdL nanoparticles with pUC19 plasmid DNA (2 mg ml−1) at 37 °C in the dark or after green light irradiation, the PdL nanoparticles were found to be stable (Supplementary Fig. 20). Overall, these results suggested that the excellent phototoxicity of PdL may result more from increased ROS levels in membrane-rich organelles, such as the lysozome or mitochondria, than from DNA damage in the nucleus. Considering these promising in vitro results, further in vivo testing in mouse tumour models was undertaken.

Human skin melanoma is known to be prone to resisting PDT type II treatment by the combination of a hypoxic tumour microenvironment35 and melanin-induced quenching of ROS36. Hence, PdL was evaluated in vivo using human skin melanoma (A375) tumour xenografts in nude mice. Following intravenous tail injection (100 µl; 420 µM in Dulbecco’s modified Eagle’s medium (DMEM) and 10% foetal bovine serum (FBS); 0.9 mg kg−1), the mice showed a constant body weight over 20 d (Fig. 3c) and the important organs remained healthy, as determined by haematoxylin and eosin staining (Supplementary Fig. 21), suggesting low systemic toxicity at this compound dose. In the dark group, PdL showed moderate tumour growth inhibition, but green light irradiation (520 nm; 100 mW cm−2; 10 min; 60 J cm−2) performed 12 h after injection of the self-assembled PdL strongly inhibited tumour growth (Fig. 3d). Haematoxylin and eosin staining of the irradiated tumours on day 5 revealed that the tumour tissues were dramatically damaged in the PdL + light group, while the other groups did not show any remarkable effect. TUNEL staining also demonstrated a decrease in cancer cells in the irradiated tumour and cell killing via apoptosis (Extended Data Fig. 1b). Overall, these experiments demonstrated not only that PdL showed excellent antitumour efficacy in an A375 melanoma mouse model but also that it showed very low cytotoxicity to healthy organs, highlighting the high potential of PdL DSDSs for anticancer PDT application.

Uptake, biodistribution and tumour targeting

The low systemic dark toxicity and high antitumour PDT efficacy of PdL stimulated us to check drug uptake in vitro and in vivo using inductively coupled plasma mass spectrometry (ICP-MS). The cellular uptake of PdL (2 µM) was found to be time dependent, with the Pd content in A375 cells increasing from 29 ngPd per million cells at 2 h to 172 ngPd per million cells at 24 h (Fig. 4a). It was also temperature dependent, with a decrease to 23 ngPd per million cells at 4 °C 2 h after treatment (5 µM) compared with 41 ngPd per million cells at 37 °C (Fig. 4b). Further coincubation experiments (Fig. 4b) showed that active internalization occurred via clathrin-mediated endocytosis (pitstop) and micropinocytosis (wortmannin). Altogether, these results highlighted that both energy-independent and energy-dependent cellular uptake took place in vitro, suggesting that PdL may pass through the cell membrane as both isolated molecules and nanoaggregates.

Fig. 4: Cellular uptake in vitro; biodistribution, nanoparticle morphology and Pd content; and tumour accumulation of PdL in vivo in mouse A375 tumour xenografts.figure 4

a, Pd content (ICP-MS) of A375 skin melanoma cell monolayers 2 or 24 h after treatment with PdL (2 µM). The data represent means ± s.d. of three biological replicates. b, Pd content (ICP-MS) of A375 skin melanoma cell monolayers 2 h after treatment with PdL (5 µM) in combination with different uptake inhibitors. The data represent means ± s.d. of three biological replicates. Statistical significance was determined by two-way ANOVA (*P < 0.05). c, Biodistribution of palladium (inductively coupled plasma optical emission spectroscopy) in different organs of mice at different time points after intravenous tail injection of PdL. The data represent means ± s.d. of three replicates. d, Tumour palladium accumulation efficiency in mice at different time points after intravenous tail injection of PdL. The units were determined using the equation %ID g−1 = (Pd content of tumour/Pd content of injection solution) × 100%/mass of measured organs. The data represent means ± s.d. of three replicates. In vivo injection conditions: 2.1 µmol kg−1, 420 µM, 100 µl DMEM (10% FBS) and 0.9 mg kg−1.

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An essential question at this stage was to understand whether the nanoparticles formed by PdL in cell-growing medium in vitro would also form in a living mouse. Thus, the presence and morphology of nanostructures in the bodies of mice injected with PdL were investigated in more detail. First, blood samples taken from the eye socket of mice 5 min after intravenous tail injection of PdL in DMEM solution (100 µl; 0.9 mg kg−1) showed roughly spherical, high-contrast nanoparticles characterized by an average size of 181 ± 75 nm (Supplementary Fig. 22). Similar to those found in the injected DMEM solution, these nanoparticles were rich in palladium according to energy-dispersive X-ray (EDX) element mapping analysis (Extended Data Fig. 2a), which strongly suggested that they contained PdL. As proof of this concept, we also injected the maximum permitted volume (10 µl) of a pure, non-aggregated DMSO solution of PdL (4.2 mM) into a mouse to check whether molecules of PdL would self-assemble as nanoparticles upon introduction in the blood. Blood collection after 5 min, followed by TEM, EDX and scanning electron microscopy analysis (Extended Data Fig. 2b and Supplementary Fig. 23), demonstrated that palladium-rich round nanoparticles with a diameter of ~100 nm had indeed formed, similar to the nanoparticles observed upon intravenous injection of PdL in DMEM. Altogether, these results suggested that molecules of PdL spontaneously aggregated into nanoparticles upon injection in the blood, where they remained self-assembled during circulation. The biological half-time of these PdL nanoparticles in the blood was found to be ~7.9 h (Supplementary Fig. 26), which is rather long and notably much longer than that found for many small-molecule anticancer drugs (typically below 1 h). At 12 h after tail vein injection of PdL, the A375 tumour was sectioned and imaged by electron microscopy. These images (Extended Data Figs. 2c; 1 and 0.5 µm scales; indicated by red arrows) showed dark nanosized spots in the lysozomes of the cancer cells, with an average diameter of 260 ± 75 nm (slightly larger than the diameter of nanoparticles in the blood). These dark spots were not observed in the untreated control group (Supplementary Fig. 24); thus, we interpreted them as palladium-containing nanoparticles. To verify this hypothesis, we performed EDX analysis of these nanoparticles (Supplementary Fig. 25), which confirmed that the cancer cells in the tumour had indeed taken up PdL nanoparticles. Overall, the presence of nanoparticles both in the blood and in the tumour tissue of mice treated with PdL is proof that the Pd⋯Pd interaction causing the self-assembly of the molecule in medium is strong enough to keep the nanostructures in circulating blood, which leads to delivery of the prodrug to the tumour.

To quantify tumour delivery, the biodistribution of Pd was determined by ICP-MS in A375 mouse xenografts several hours (2, 6, 12, 20 and 24 h) after intravenous tail injection of PdL in DMEM. As shown in Fig. 4c, the complex showed low accumulation (below 0.27 µg g−1 tissue) in the heart, kidney and lung, while the liver showed significantly higher accumulation (above 1.0 µg per per gram of tissue), as expected considering its role in the detoxification and metabolism of exogenous substances. Noticeably, the accumulation level of PdL in the liver gradually decreased over time. Meanwhile, the tumour tissue showed an increasing Pd accumulation from 0.17 µg per gram of tissue after 2 h to a peak of 0.87 µg per gram of tissue at 12 h, which corresponded to an impressive 10.2% of the injected dose per gram (% ID g−1) (Fig. 4c,d), and finally decreased to 0.17 µg per gram of tissue at 24 h. These results highlight that the long circulation time in the blood (~8 h) leads to an extraordinary tumour accumulation rate of the PdL nanoparticles, which peaks at 12 h. These data suggest that the enhanced permeability and retention effect, conjugated with the high drug loading of the nanoparticle, explains the high tumour accumulation of the compound, as the PdL nanoparticles do not contain any active tumour-targeting molecules. In conclusion, PdL appears to be a particularly well-performing DSDS characterized by easy synthesis and formulation in biocompatible buffer, high drug-loading efficiency of the self-assembled nanoparticles, low systemic toxicity to the tumour-bearing mouse for these nanoparticles and excellent tumour accumulation and antitumour efficacy upon light irradiation using a drug-to-light interval of 12 h.

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