Curing cancer is one of the toughest challenges of modern medicine,1 and chemotherapy occupies the front line of cancer treatment.2-4 Many chemotherapy agents are well-defined, small molecules that typically lead to nonspecific delivery, rapid blood clearance, and low accumulation in tumors, altogether generating severe side effects for cancer patients.5,6 To overcome these limitations, they have been conjugated to tumor-targeting nanocarriers, either covalently or supramolecularly, which in principle enhances drug delivery to the tumor.7-18 However, many nanocarriers show comparatively low drug-loading capacity (typically <20%),19,20 while the resulting tumor accumulation remains disappointingly low: recent studies showed that a median 0.7% of the administered nanoformulated drug dose ends up in solid tumors.20,21 In addition, achieving the reproducible preparation of drug-nanoparticle conjugates is often challenging, which restricts the clinical applications of nanodrugs.
Drug self-delivery systems (DSDS) may solve that issue. They consist of small-molecule drugs that self-assemble into nanostructures without the assistance of dedicated nanocarriers.22 These systems combine the easy preparation of small molecules and the tumor-targeting properties of nanoconjugates, achieving high drug loading efficiency.2 They also require supramolecular forces that hold in the complex biological environment of a living animal. DSDS proposed to date relies on a combination of hydrophobic forces, hydrogen bonding, and/or metal-ligand coordination. The so-called “metallophilic interaction” is another kind of supramolecular interaction occurring specifically between d8 or d10 metal centers. It is well known in optoelectronics and material science23 for its ability to modify the photophysical and photochemical properties of metal compounds; it has also been proposed for biological applications, but only in vitro demonstrations have been made.24 Here, we demonstrate with a new light-activated DSDS that the metallophilic interaction survives blood circulation in living mice, leading to self-assembled nanoparticles that show outstanding tumor-targeting and phototherapeutic properties in a human skin melanoma xenograft.
Synthesis and characterization of a light-sensitive DSDS.
The PdL small molecule (Figure 1a) contains, as in the padeliporfin photosensitizer recently approved for PDT,25 a palladium(II) metal center. In contrast with padeliporfin, however, PdL is a bis-cyclometalated palladium compound characterized by the presence of two Pd-C covalent bonds (see Figure 1b and full characterization in Figure S3-S4 and Table S1). Its X-ray structure (Figure 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 DFT model of the supramolecular dimer converged at a Pd…Pd distance of 3.52 Å (Figure 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 HOMO of the dimer. Meanwhile, TDDFT calculations confirmed the decrease in the HOMO-LUMO gap 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 (Figure 1d).
When dissolved in DMSO, PdL showed a modest absorption in the 434-540 nm region, an absorption maximum at 481 nm, a concentration-independent emission maximum (564 nm) and a low phosphorescence quantum yield (φp = 0.0008) and lifetime (τ = 0.406 ns) characteristic of the monomer (Figure 1e, Table S6). However, 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 (Figure 1f), which are typical for metal-metal-to-ligand charge transfer (MMLCT) excited states induced by Pd…Pd interactions26 and altogether suggest self-assembly. This hypothesis was confirmed by TEM images showing nanorods and nanocubes (Figure 1f, insert). Usually, the formation of Pd…Pd supramolecular bonds is accompanied by a long-wavelength emission peak,26 and indeed, an increase in the H2O content in DMSO:water mixtures (fw = Vwater/Vtotal) led to a gradual replacement of the monomeric emission peak at 564 nm (as observed in pure DMSO, fw = 0.0) 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 (Figure 1g). In THF/H2O solutions, similar self-assembly was observed, though with slower polymerization rates and different morphologies (Figure S6-S7). Overall, PdL appeared as a self-assembling molecule, at least in mixtures of water and organic solvents.
The self-assembly of PdL was then studied in a cell-growing medium called Opti-MEM complete that contained 2.5 vol% fetal calf serum (FCS). At 25 µM, aggregation immediately occurred, as shown by a hydrodynamic diameter of approximately 164 nm determined by dynamic light scattering (DLS, Figure 2a). After 30 min, the maximum hydrodynamic diameter had only slightly shifted to 190 nm, but the number of particles had increased significantly (Figure 2b). The absorption of the solution (Figure 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 (Figure 2d), but these nanodots self-assembled as regular nanofibers that gradually lengthened. Cryo-EM imaging confirmed the formation of nanofibers in such medium, characterized by a well-ordered structure at a repeating distance of ~1.68 nm in the Fourier transform image (Figure 2e). Overall, in cell-growing medium, DLS, EM, and UV-vis spectroscopy demonstrated the time-dependent self-assembly of PdL into nanorods and nanoparticles, which, considering DFT and crystal structure analysis, must involve metallophilic Pd…Pd interactions.
In 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) mechanism.27 Direct detection of the near-infrared emission peak of 1O2 at 1270 nm under blue light irradiation (450 nm) was only possible in CD3OD, hence for the PdL monomer. The corresponding 1O2 generation quantum yield was very low (φΔ = 0.09, Figure 2f and Table S6). In Opti-MEM medium, 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 (ABMDMA) showed no decrease in the absorbance band at 378 nm upon green light irradiation, characteristic of the 1O2 adduct (Figure 2g),28 indicating negligible 1O2 generation (φΔ = 0.04, Figure S8).29 Overall, PdL is a poor PDT type II sensitizer, both as a monomer in methanol and as aggregates in medium. By contrast, type I PDT sensitizers can be characterized by the initial generation of superoxide radicals (O2•−), which can further generate other ROS such as HO• or H2O2.30 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 (Figure 2h, Figure S9).31 These results demonstrated that PdL is capable of photochemically generating superoxide both in the monomeric and aggregated states, which suggested that as light-activated DSDS, it would behave as a PDT type I photosensitizer.
Biological properties in vitro and in vivo. Considering the significant 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 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 (EC50>10 µM) for the three cancer cell lines under normoxic (21% O2) and hypoxic (1% O2) conditions (Table S7). By contrast, upon green light irradiation (520 nm, 13 J/cm2) under normoxia and hypoxia, PdL exhibited high phototoxicity with submicromolar EC50 and high photoindexes (PI = EC50, dark/EC50, light) of 32-72 (Figure 3a, Figure S10 and Table S7), 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 in dark conditions, as determined by Annexin V/propidium iodide double staining experiments (Figure 3b and S11). In the light-irradiated group, no toxicity was observed after 2 h, but after 4 h and 24 h, the number of apoptotic and necrotic cells increased significantly, suggesting that PdL induced cancer cell death 4 h after irradiation via both cell death mechanisms. The cytotoxicity of PdL in 3D multicellular tumor spheroid models (A549 and A375), which better mimic the physical penetration of light and drugs in three dimensions,32 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 the visible collapse of the spheroid cores and dramatic shrinkage of the spheroid diameters (Figure 3c, S12 and Table S7). 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 significantly increased in the green light-irradiated group compared with the dark group (Figure 3c), confirming drug penetration and light-induced cell killing by membrane disruption in 3D environments. Overall, these excellent photocytotoxicity results suggested further in vivo testing in mouse tumor models.
Human skin melanoma is known to be prone to resist PDT type II treatment by a combination of a hypoxic tumor microenvironment33 and melanin-induced quenching of reactive oxygen species (ROS).34 PdL was hence evaluated in vivo using human skin melanoma (A375) tumor xenografts in nude mice. Following intravenous tail injection (100 µL, 420 µM in DMEM (10% FBS), 0.9 mg/kg), the mice showed constant body weight over 20 days (Figure 3d), and the important organs remained healthy, as determined by H&E staining (Figure S13), suggesting low systemic toxicity at this compound dose. In the dark group, PdL showed moderate tumor growth inhibition, but green light irradiation (520 nm, 100 mW/cm2, 10 min, 60 J/cm2) performed 12 h after injection of the self-assembled PdL strongly inhibited tumor growth (Figure 3e). H&E staining of the irradiated tumors at day 5 revealed that the tumor 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 tumor and cell killing via apoptosis (Figure 3f). Overall, these experiments demonstrated not only that PdL showed excellent antitumor efficacy in an A375 melanoma mouse model but also that it showed very low cytotoxicity to healthy organs, highlighting the high potential of PdL DSDS for anticancer PDT application.
Uptake, biodistribution, and tumor targeting. The low systemic dark toxicity and high antitumor PDT efficacy of PdL stimulated us to check drug uptake in vitro and in vivo using 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 (at 2 h) to 172 ng Pd/million cells (at 24 h, Figure 4a). It was also temperature-dependent, with a reduction of 19 ng Pd/million cells at 4 °C 2 h after treatment (5 µM) compared to 44 ng Pd/million cells at 37 °C (Figure 4b). Further coincubation experiments (Figure 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.
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 showed roughly spherical, high-contrast nanoparticles, characterized by an average size of 181±75 nm (Figure S14). Similar to those found in the injected DMEM solution, these nanoparticles were rich in palladium according to EDX analysis (Figure 4c-d), which confirmed that they contained PdL. Altogether, these results suggested that molecules of PdL aggregated into nanoparticles in DMEM and that upon intravenous injection, they remained self-assembled in blood while circulating. Second, 12 h after tail vein injection of PdL, the A375 tumor was sectioned and imaged by EM. These images (Figure 4e, 1 and 0.5 µm scale, indicated by red arrows) showed dark nanosized spots in the cytoplasm of the cancer cells with an average diameter of 260±75 nm, slightly larger than the diameter of nanoparticles in blood. These dark spots were not observed in the untreated control group (Figure S15); thus, we interpret them as palladium-containing nanoparticles. Overall, the presence of nanoparticles both in the blood and in the tumor 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 tumor.
To quantify tumor delivery, the biodistribution of Pd was determined by ICP-MS in A375 mouse xenografts several hours (2, 6, 12, 20, 24 h) after intravenous tail injection of PdL. As shown in Figure 4f, the complex showed low accumulation (below 0.27 µg/g tissue) in the heart, kidney, and lung, while the liver showed significantly higher accumulation (above 1.0 µg/per gram tissue), as expected considering its role in detoxification and metabolism of exogenous substances. Noticeably, the accumulation level of PdL in the liver gradually decreased over time. Meanwhile, the tumor tissue showed an increasing Pd accumulation from 0.17 µg per gram tissue after 2 h to a peak of 0.87 µg per gram tissue at 12 h, which corresponded to an impressive 10.2 %ID/g of the injected drug (Figure 4f and 4g), and finally decreased to 0.17 µg per gram tissue at 24 h. These results highlight both the long circulation time and extraordinary tumor accumulation rate of PdL nanoparticles, which peak in the tumor at 12 h. In conclusion, PdL appears as a particularly well-performing DSDS characterized by an easy synthesis and formulation in biocompatible buffer, a high-drug loading efficiency of the self-assembled nanoparticles, a low systemic toxicity to the tumor-bearing mouse for these nanoparticles, and excellent tumor accumulation and antitumor efficacy upon light irradiation using a drug-to-light interval of 12 h.