A Nonanuclear Pd-Based Coordination Cage Featuring Ni-Chelated Porphyrin Ligand for Cancer Therapy via Combination of Chemodynamic and Photodynamic Modalities

1. Introduction

Reactive oxygen species (ROS), particularly hydroxyl radical (•OH), singlet oxygen (¹O₂), and hydrogen peroxide (H₂O₂), have emerged as promising agents for cancer therapy due to their ability to induce oxidative stress and disrupt cellular homeostasis, offering distinct advantages in tumor selectivity and the potential to overcome conventional drug resistance [1,2]. Among various ROS-generating strategies, transition metal-catalyzed Fenton or Fenton-like reactions have garnered significant attention due to their ability to convert H₂O₂ into cytotoxic •OH [3,4,5]. Nickel complexes have demonstrated remarkable catalytic activity in this context [6,7,8]. The ability of Ni(II) to undergo reversible redox cycling with Ni(III) enables efficient decomposition of H₂O₂ to generate •OH radicals, a mechanism that has been extensively exploited in environmental remediation for the degradation of tough organic pollutants [7].

In the context of ROS-based cancer therapies, Ni(II)-induced •OH generation also offers an advantage as it can be produced by consumption of endogenously overexpressed H₂O₂ (typically 10–50 μM) in the tumor microenvironment (TME) [9,10]. This TME-dependent ROS production circumvents the O₂ limitation and penetration depth limitations associated with conventional O₂-dependent therapies such as type II photodynamic therapy (PDT) [11,12,13]. For example, NiCl₂ has long been documented to induce lipid peroxidation and DNA damage via Fenton-like process [14]. Liu et al recently reported that nickel-based single-atom-metal-clusters (NSAMCs) exhibit peroxidase-like activity, catalyzing H₂O₂ in the TME to generate •OH, which synergizes with glutathione (GSH) depletion to boost ferroptosis [15].

Complementary to Ni-induced •OH production, photoinduced ROS generation, and ¹O₂ in particular, have become a cornerstone of PDT, an approach that relies on photosensitizers activated by specific light wavelengths [16,17]. Porphyrins and their derivatives are among the most widely used photosensitizers due to their strong absorption in the visible-near-infrared region and high ¹O₂ quantum yields [18,19]. However, their application is hindered by inherent limitations, including self-aggregation resulting from π–π stacking, poor water solubility, and low tumor targeting efficiency [20]. To address these challenges, researchers have integrated porphyrins into metal-organic frameworks or discrete coordination cages via coordination with metal ions [21,22,23,24]. These hybrid structures not only inhibit porphyrin stacking to preserve photosensitizer activity but also enhance ROS generation efficacy by optimizing light absorption, improving cellular uptake, and enabling controlled release of ROS in the TME [25,26,27].

Herein, we report a coordination cage of Pd₆-TMPP(Ni) constructed from Ni-chelated porphyrin metalloligand TMPP(Ni) and Pd²⁺ (H₂TMPP = meso-tetrakis (6-methylpyridin-3-yl) porphyrin). Pd₆-TMPP(Ni) features a hexagonal prismatic structure wherein the three TMPP(Ni) metalloligand occupy alternative lateral faces of the prism, juxtaposing three pairs of PdCl₂ units via binding to the trans positions of the Pd²⁺ ion (Scheme 1 and Figure 1a). The TMPP(Ni) moieties in Pd₆-TMPP(Ni) serve as both a catalyst for •OH production via Fenton-like reaction and a photosensitizer for light-induced ¹O₂ generation (Scheme 1b). Our in vitro cytotoxicity assays against multiple cancer cell lines NCI-H82 (lung cancer), A549 (lung cancer), KYSE-510 (esophageal cancer), and Te-1 (esophageal cancer) reveal potent dark toxicity of Pd₆-TMPP(Ni) with half maximal inhibitory concentration (IC₅₀) in the range of 1.867–2.107 μmol L⁻¹, which can be further lowered to 0.835–1.528 μmol L⁻¹ with the presence of 650 nm light irradiation. The cytotoxicity of Pd₆-TMPP(Ni) outperforms that of free ligand H₂TMPP and the Pd congener of Pd₆-TMPP(Pd).

2. Results and Discussion

2.1. Synthesis and Structure of Pd₆-TMPP(Ni)

The coordination cage of Pd₆-TMPP(Ni) was directly obtained in 16.4% yield as red needle-like crystals from the solvothermal reaction of TMPP(Ni) and PdCl₂ in CH₂Cl₂/MeOH in the presence of HAc as the modulator. Pd₆-TMPP(Ni) is soluble in CH₂Cl₂, DMF, and DMSO, slightly soluble in MeOH and MeCN, but insoluble in H₂O.

Single-crystal X-ray diffraction analysis revealed that Pd₆-TMPP(Ni) crystallizes in the monoclinic P2₁/c space group (Table 1). The asymmetric unit of Pd₆-TMPP(Ni) contains a full molecule (C₁ point group, Figure 1a) instead of the expected D_3h or C_2v symmetry as that found for Pd₆-TMPP(Au) [28].

In Pd₆-TMPP(Ni), three pairs of PdCl₂ units were juxtaposed by three TMPP(Ni) metalloligand to give a hexagonal prismatic structure. The side lengths of the two Pd-based triangles from the base planes, i.e., Pd1∙∙∙Pd2/Pd2∙∙∙Pd3/Pd1∙∙∙Pd3 being 12.20/11.99/13.52 Å, and Pd4∙∙∙Pd5/Pd5∙∙∙Pd6/Pd4∙∙∙Pd6 being 12.12/11.80/13.61 Å (Table S1), are similar but both deviate from an equilateral triangle or an isosceles triangle. In addition, the triangle formed by three Ni ions, viz., Ni1∙∙∙Ni2/Ni2∙∙∙Ni3/Ni1∙∙∙Ni3, is 10.30/10.66/12.46 Å (Table S1).

It is notable that the small ionic radius for Ni²⁺ (0.63 Å) also caused the saddle-shaped ruffling of the three porphyrin backbones (the twelve Ni−N bond distances are in the range of 1.904(12)−1.983(12) Å, Table S1) [29], featuring six pyrrole rings pointing inwards to the centers of the two base planes to create a hydrophobic pocket that may be suitable for storing small molecules [30]. Platon void calculation also suggested that the guest-accessible void of Pd₆-TMPP(Ni) was sufficiently large, occupying 43.6% (8369.2 ų of 19210.0 ų) of the total cell volume [31].

2.2. Spectroscopic and Spectrometric Characterization of Pd₆-TMPP(Ni)

The ¹H nuclear magnetic resonance (NMR) spectroscopy indicated that the characteristic peak of pyrrole protons at −2.81 ppm in H₂TMPP disappeared after the formation of Pd₆-TMPP(Ni) (Figure S1), which directly confirmed that Ni²⁺ had been successfully incorporated into the center of the porphyrin center [18,28]. The diffusion ordered spectroscopy (DOSY) for Pd₆-TMPP(Ni) shows a narrow diffusion bands with diffusion coefficients of 3.83 × 10⁻¹⁰ m² s⁻¹ (Figure 1e), suggesting the formation of single assembles.

In the ultraviolet-visible (UV-Vis) absorption spectrum (Figure 1b), the Soret band of Pd₆-TMPP(Ni) blue-shifted to 408 nm as compared to those of H₂TMPP (420 nm) and Pd₆-TMPP(Pd) (417 nm) [32]. Notably, the four Q bands of H₂TMPP in the range of 500−700 nm coalesced into two peaks (525/559 nm) in Pd₆-TMPP(Ni) (Figure 1b, inset) due to the metalation that elevated the symmetry of the porphyrin ring from the original D_2h to D_4h [29,33].

The X-ray photoelectron spectroscopy (XPS) shows that in the Pd 3d region, peaks at binding energies of 337.51 eV and 343.77 eV were assigned to Pd 3d_5/2 and Pd 3d_3/2 (Figure 1c), which were in line with the characteristic peaks of Pd²⁺ [34]. Meanwhile, in the Ni 2p region, peaks at binding energies of 855.34 eV and 872.46 eV corresponded to the spin-orbit split peaks of Ni 2p_3/2 and Ni 2p_1/2 (Figure 1d), consistent with those reported in the literature [35]. The above data collectively confirmed that both Pd and Ni existed in the +2 oxidation state in Pd₆-TMPP(Ni).

In the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Figure 1f and Figure S2), the peaks at a mass-to-charge ratio (m/z) of 3258.421 and 3280.443 were observed for Pd₆-TMPP(Ni), consistent with those calculated for [M+H]⁺ (3258.706 m/z) and [M+Na]⁺ (3280.695 m/z).

The EDS analysis results (Figure S3) showed that the atomic ratio of Pd to Ni in Pd₆-TMPP(Ni) was approximately 4.1 : 1.9 (equal to 2.2 : 1.0), which was close to the theoretical ratio in its molecular formula (2.0 : 1.0).

In the FT-IR spectra (Figure S4), the characteristic peak attributed to the in-plane vibration of N-H at the porphyrin center (964 cm⁻¹) in H₂TMPP disappeared in Pd₆-TMPP(Ni), while new peaks appeared at 1002 cm⁻¹ and 1012 cm⁻¹, which directly confirmed that Ni²⁺ had replaced the protons at the porphyrin center [36,37]. In addition, the stretching vibration peak of the C=N bond in the porphyrin ring was also red-shifted from 1593 cm⁻¹ in H₂TMPP to 1610 cm⁻¹ in Pd₆-TMPP(Ni) due to the coordination of pyridine nitrogen atoms with Pd²⁺ [38,39].

2.3. Synthesis and Characterizations of Pd₆-TMPP(Ni) Nanoparticles

Water-soluble nanoparticles were prepared by encapsulating H₂TMPP, Pd₆-TMPP(Pd), and Pd₆-TMPP(Ni) with Pluronic F127, an FDA-approved triblock polymer for therapeutic use (Scheme 1a) [40,41], in a mixture of DMSO and H₂O, followed by dialysis. The transmission electron microscopy (TEM) showed that the obtained nanoparticles H₂TMPP-F127, Pd₆-TMPP(Pd)-F127, and Pd₆-TMPP(Ni)-F127 all exhibited regular spherical morphology and good dispersibility in water (Figure 2), with their diameters being 190 ± 15 nm, 140 ± 15 nm, and 230 ± 15 nm, respectively. The hydrodynamic diameters as revealed by dynamic light scattering (DLS) were marginally larger due to the hydration of these nanoparticles, featuring 231.9 nm for H₂TMPP-F127, 184.6 nm for Pd₆-TMPP(Pd)-F127, and 311.7 nm for Pd₆-TMPP(Ni)-F127 (Figure S5).

The zeta potentials of these nanoparticles were 9.6 mV for H₂TMPP-F127, 14.9 mV for Pd₆-TMPP(Pd)-F127, and 27.4 mV for Pd₆-TMPP(Ni)-F127, respectively (Figure S6). The highest zeta potential value of Pd₆-TMPP(Ni)-F127, as compared to H₂TMPP-F127 and Pd₆-TMPP(Pd)-F127, indicates its best colloidal stability, which is critical during the drug delivery stage [42,43].

As shown in Figure S7a, the Soret bands of H₂TMPP-F127, Pd₆-TMPP(Pd)-F127, and Pd₆-TMPP(Ni)-F127 remained constant when exposed to 650 nm laser for 10 min, suggesting that these particles are photostable. In addition, these particles were stable in H₂O, PBS (0.1×), and RPMI 1640 (0.1×) for 0, 8, 12, and 24 h, indicated by only insignificant changes in particle size as revealed by DLS (Figure S8).

2.4. Detection of H₂TMPP-F127, Pd₆-TMPP(Pd)-F127, and Pd₆-TMPP(Ni)-F127 Generation of ROS in Solution

The generation of •OH can be chemically assayed by 3, 3′, 5, 5′-tetramethylbenzidine (TMB) [22,44,45,46]. As shown in Figure S9, when H₂TMPP-F127, Pd₆-TMPP(Pd)-F127, and Pd₆-TMPP(Ni)-F127 respectively mixed with a TMB solution containing 100 μM H₂O₂, H₂TMPP-F127 produced no •OH within 3 h, while the amount of •OH generated by Pd₆-TMPP(Pd)-F127 and Pd₆-TMPP(Ni)-F127 increased with the rise of concentration. At the same TMPP concentration, Pd₆-TMPP(Ni)-F127 produced significantly more •OH, which may be attributed to the fact that Ni(II) can activate the Fenton-like reaction to generate cytotoxic •OH [9].

As shown in the Figure 3a, the electron paramagnetic resonance (EPR) spectroscopy of both Pd₆-TMPP(Pd)-F127 and Pd₆-TMPP(Ni)-F127 exhibited four peaks with a intensity ratio of 1 : 2 : 2 : 1, which confirmed the presence of •OH induced both types of metal ions wherein Ni(II) outperforms Pd(II) [47,48,49]. The above experiments showed that both Pd₆-TMPP(Ni)-F127 and Pd₆-TMPP(Pd)-F127 could induce the chemodynamic therapeutic effect by generating •OH, whereas Pd₆-TMPP(Ni)-F127 was superior to Pd₆-TMPP(Pd)-F127 in this aspect.

We further used DPBF as a probe to detect and compare the ability of H₂TMPP-F127, Pd₆-TMPP(Pd)-F127, and Pd₆-TMPP(Ni)-F127 to generate ¹O₂ in aqueous solutions [50,51]. As shown in Figures S7b−7f, the absorbance of DPBF decreased after 1 min of irradiation, with the decrease rate being similar among these particles.

2.5. Cell Cytotoxicity and in Vitro ROS Generation Assay

The cytotoxicity of H₂TMPP-F127, Pd₆-TMPP(Pd)-F127, and Pd₆-TMPP(Ni)-F127 against A549 (Figure 4a and Figure 4b), KYSE-510 (Figure 4c and Figure 4d), NCI-H82 (Figure 4e and Figure 4f) and Te-1 (Figure 4g and Figure 4h) cells under light and light-free conditions was evaluated and compared. For all these cell lines, H₂TMPP-F127 shows negligible cytotoxicity under both light and light-free conditions. In contrast, both Pd₆-TMPP(Pd)-F127 and Pd₆-TMPP(Ni)-F127 show concentration-dependent cytotoxicity against all these cell lines, with the exception that A549 is insensitive to Pd₆-TMPP(Pd)-F127 treatment within the tested concentration range. Pd₆-TMPP(Ni)-F127 also outperforms Pd₆-TMPP(Pd)-F127 for all these cell lines in terms of both dark and light toxicities, with Pd₆-TMPP(Ni)-F127 nearly killing all the cells with its TMPP concentrations above 3.33 μmol L⁻¹ in the dark (equivalent to 1.11 μmol L⁻¹ of Pd₆-TMPP(Ni)). It is also feasible that the light irradiation further augments the toxicity of both Pd₆-TMPP(Pd)-F127 and Pd₆-TMPP(Ni)-F127, albeit for Pd₆-TMPP(Ni)-F127, the light contribution to the toxicity against esophageal cancer cell lines KYSE-510 and Te-1 is insignificant. The low PDT contribution of porphyrin-chelated Ni(II) than Pd(II) can be explained by the fact that Ni²⁺ can quench the excited state of TMPP ligand via fast non-radiative decay due to the presence of metal-to-ligand charge transfer (MLCT) and d-d excited states that lie below or close to the π-π* states of TMPP [52,53].

The half maximal inhibitory concentration (IC₅₀) for Pd₆-TMPP(Ni)-F127 against cell lines NCI-H82 (dark: 2.015 μmol L⁻¹; light: 1.100 μmol L⁻¹), A549 (dark: 2.024 μmol L⁻¹; light: 0.835 μmol L⁻¹), KYSE-510 (dark: 2.107 μmol L⁻¹; light: 1.528 μmol L⁻¹), and Te-1 (dark: 1.867 μmol L⁻¹; light: 1.194 μmol L⁻¹) are comparable to those reported in the literature (Table 2).

2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) was used as a fluorescent probe to estimate the ROS-generating capacity of the nanomaterials in Te-1 cells [54]. As shown in Figure 3b and Figure 3c, in the absence of laser irradiation, both Pd₆-TMPP(Pd)-F127 and Pd₆-TMPP(Ni)-F127 were able to generate a certain level of ROS. The presence of Ni in Pd₆-TMPP(Ni)-F127 further enhanced this activity. After laser irradiation, the amount of ROS generated by Pd₆-TMPP(Pd)-F127 and Pd₆-TMPP(Ni)-F127 increased significantly, while H₂TMPP-F127 produced almost no ROS.

2.6. Cellular Uptake of Pd₆-TMPP(Pd)-F127 and Pd₆-TMPP(Ni)-F127

The cellular uptake of Pd₆-TMPP(Pd)-F127 and Pd₆-TMPP(Ni)-F127 was evaluated by determining the Pd²⁺ via inductively coupled plasma mass spectrometry (ICP-MS). As shown in Table 3, both particles can be internalized by KYSE-510 cells in a time-dependent manner. It is also notable that the uptake efficiency of Pd₆-TMPP(Ni)-F127 was higher than that of Pd₆-TMPP(Pd)-F127, which probably also contributed to the higher cytotoxicity of Pd₆-TMPP(Ni)-F127 observed in the cytotoxicity assays.

3. Materials and Methods

3.1. General

Ligand H₂TMPP [28,66] and Pd₆-TMPP(Pd)-F127 [32] were synthesized as described in our previous reports. PdCl₂ (99%, TCI (Shanghai) Development Co., Ltd., Shanghai, China), Ni(NO₃)₂·6H₂O (≥ 98%, Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China), Pluronic F-127 (MW: 2000, Shanghai Macklin Biochemical Co., Ltd, Shanghai, China), 3,3’,5,5’-tetramethylbenzidine (TMB, AR, Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China), 1,3-diphenylisobenzofuran (DPBF, AR, Shanghai Maokang Biotechnology Co., Ltd., Shanghai, China), 5,5-dimethyl-1-pyrroline N-oxide (DMPO, 97%, Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China), and hydrogen peroxide (30%, ≥ 95%, Yonghua Chemical Co., Ltd., Shanghai, China) were available from the corresponding suppliers without further purification. MeOH, HAc, MeCN, Et₂O, DMSO, and N, N-dimethylformamide (DMF), all in AR grade, were procured from respective sources and used as received.

NCI-H82, A549, KYSE-510, and Te-1 cell lines were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Phosphate buffer solution (PBS), cell culturing medium RPMI 1640 (10% FBS + 1% P/S), RPMI 1640 (1% P/S), Ham’s F-12 (10% FBS + 1% P/S), Ham’s F-12 (1% P/S), and 0.25% trypsin solution (containing EDTA, dissolved in PBS) were purchased from Shanghai Basal Media Technologies Co., Ltd (Shanghai, China). The cell counting kit-8 (CCK-8) was available from APEXBIO. The reactive oxygen species detection kit and DCFH-DA were purchased from Shanghai Beyotime Biotechnology Co., Ltd (Shanghai, China).

¹H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III HD 400 MHz superconducting NMR spectrometer (Bruker AXS GmbH, Germany). Fourier-transform infrared (FT-IR) spectra were measured using a Bruker Vertex 70 FTIR-spectrometer with a Hyperion 2000 IR-Microscope (Bruker AXS GmbH, Germany), employing the attenuated total reflection (ATR) technique. Ultraviolet-visible (UV-Vis) spectra were acquired using a Varian Cary-50 UV-Vis spectrophotometer (Varian, Inc., Palo Alto, CA, USA). X-ray photoelectron spectroscopy (XPS) was performed on an Thermo Scientific EXCALAB 250 XI X-ray photoelectron spectrometer (Thermo Scientific, Waltham, MA, USA). Energy-dispersive X-ray spectroscopy (EDS) was conducted using a Zeiss EVO 18 scanning electron microscope (ZEISS Group, Oberkochen, Germany). Transmission electron microscopy (TEM) images were obtained using a Hitachi HT7700 transmission electron microscope (Hitachi, Japan), and samples were prepared by dropping aqueous solutions onto copper grids. Dynamic light scattering (DLS) and zeta potential measurements were performed using a Horiba LA-95052 laser particle size analyzer (Horiba, Kyoto, Japan). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was carried out on a Bruker UltrafleXtreme MALDI-TOF/TOF (Bruker AXS GmbH, Germany). Electron paramagnetic resonance (EPR) spectroscopy was carried out on a JEOL JES-X320 electron spin resonance spectrometer (JEOL Ltd., Akishima, Japan). Inductively coupled plasma-mass spectrometry (ICP-MS) was performed with an iCAP PRO instrument (Thermo Scientific, Waltham, MA, USA). Photodynamic therapy experiments were conducted using a PR-CPC2-635NM cellular phototoxicity irradiator at 650 nm (PURI Materials, Guangdong, China). Cytotoxicity assays were performed on a Tecan M1000PRO microplate reader (Tecan, Switzerland) by measuring absorbance at 450 nm. Fluorescence imaging of reactive oxygen species was conducted using an MF52 inverted biological microscope (Guangzhou Micro-shot Technology Co., Ltd., Guangdong, China).

3.2. Synthesis of TMPP(Ni)

Ni(NO₃)₂·6H₂O (2.0 mg, 6.88 μmol), H₂TMPP (2.0 mg, 2.96 μmol) were introduced to a mixture of DMF/EtOH (v : v = 0.75 : 0.75 mL), and 2-pyridinemethanol (30 μL) was sequentially introduced as a modulator. The resulting mixture was transferred into a Pyrex glass tube, sealed, and transferred to a programmable oven. The temperature of the oven was gradually increased from r.t. to 120°C in 4 hours. This is followed by a stable heating at 120°C for 36 hours before cooling back to r.t. within 12 hours to yield purple plate crystals of TMPP(Ni). The tubes (50 parallel reactions) were carefully opened, and the resulting product was washed thoroughly with anhydrous ether and dried in an oven. Yield: 43.2 mg from 50 parallel reactions (39.8% based on Ni). FT-IR (ATR, cm⁻¹): 3050 (m), 3011 (w), 2922 (w), 2852 (w), 2162 (w), 1667 (w), 1595 (m), 1557 (w), 1487 (w), 1366 (m), 1349 (m), 1295 (m), 1249 (w), 1204 (w), 1132 (m), 1081 (w), 1036 (w), 1020 (w), 1000 (s), 884 (w), 863 (w), 798 (s), 732 (w), 716 (s), 649 (w), 612 (w). ¹H NMR (400 MHz, CDCl₃, ppm): δ 9.07 (d, J = 4.0 Hz, 4H), 8.69 (s, 8H), 8.13 (q, J = 8.0 Hz, 4H), 7.47 (d, J = 8.0 Hz, 4H), 2.81 (s, 12H).

3.3. Synthesis of Pd₆-TMPP(Ni)

TMPP(Ni) (1.5 mg, 2.10 μmol) and PdCl₂ (1.0 mg, 5.63 μmol) were dissolved in a mixed solvent of CH₂Cl₂/MeOH (v : v = 0.5 : 1.5 mL). Acetic acid (HAc, 20 μL) was subsequently introduced as a modulator. The resulting solution was transferred into a Pyrex glass tube, sonicated for 30 min, and sealed. The tube was then transferred to a programmable oven, with its temperature gradually increased from r.t. to 120°C in 4 hours. This is followed by a stable heating at 120°C for 48 hours, before cooling back to r.t. within 24 hours to yield red needle-like crystals of Pd₆-TMPP(Ni). The tubes (70 parallel reactions) were carefully opened, and the resulting crystals were filtered off and washed with diethyl ether 2−3 times to afford Pd₆-TMPP(Ni). Yield: 25.6 mg from 70 parallel reactions (16.4% based on TMPP(Ni)). FT-IR (ATR, cm⁻¹): 3323 (w), 3010 (w), 2923 (w), 1593 (m), 1548 (m), 1490 (s), 1471 (m), 1438 (w), 1384 (m), 1379 (m), 1338 (m), 1294 (m), 1240 (m), 1178 (m), 1151 (m), 1130 (m), 1085 (w), 1031 (m), 985 (m), 964 (s), 885 (m), 850 (m), 798 (s), 723 (s), 643 (s), 634 (m). UV-Vis (DMSO): λ_max (log ε) 417 nm (4.81), 525 nm (3.65), 559 nm (3.11). MALDI TOF-MS: m/z calcd for [M+H]⁺: 3258.706, found 3258.421; calcd for [M+Na]⁺: 3280.725, found 3280.443. ¹H NMR (400 MHz, CDCl₃, ppm): δ 9.36 (s, 12H), 8.72 (s, 12H), 8.54 (s, 12H), 8.26 (d, J = 8.0 Hz, 12H), 7.59 (d, J = 8.0 Hz, 12H), 3.76 (s, 36H).

3.4. Single-crystal X-ray Crystallography

Diffraction data for Pd₆-TMPP(Ni) were acquired on a Bruker APEX II CCD X-ray diffractometer using Ga-Kα (λ = 1.34139 Å) irradiation. Refinement and reduction of the collected data were achieved using the program SAINT, and absorption corrections were performed using a multi-scan method [67]. The crystal structure of Pd₆-TMPP(Ni) was solved by direct methods and refined on F² by full-matrix least-squares techniques with SHELXTL-2016 [68].

During refinement, some spatially delocalized electron density was observed within the crystal lattice, but it proved difficult to obtain satisfactory results. The solvent contribution was then modeled using SQUEEZE in the Platon program suite [69].

Crystallographic data for Pd₆-TMPP(Ni) have been deposited in the Cambridge Crystallographic Data Center (CCDC) as supplementary publication numbers 2525223. These data can be obtained free of charge either from the CCDC via www.ccdc.cam.ac.uk/data_request/cif or from the Supporting Information. A summary of the key crystallographic data for Pd₆-TMPP(Ni) is listed in Table 1.

3.5. The Formation of H₂TMPP-F127, Pd₆-TMPP(Pd)-F127, and Pd₆-TMPP(Ni)-F127 Nanoparticles

The formation of H₂TMPP-F127 follows our previously published procedures. [41] Specifically, Pluronic F-127 (10.6 mg, 5.30 μmol) was introduced to a 1 mL DMSO containing H₂TMPP (5.00 mg, 7.41 μmol). Upon dissolution, the mixture was added dropwise into deionized water (10 mL) under tip sonication. The formed mixture was then dialyzed using a membrane with a molecular weight cutoff of 2000 Da for 24 h to obtain H₂TMPP-F127 aqueous solution for subsequent use.

The preparation procedures for Pd₆-TMPP(Pd)-F127 and Pd₆-TMPP(Ni)-F127 were similar to that of H₂TMPP-F127, except that Pd₆-TMPP(Pd) (3.5 mg, 1.03 μmol) and Pd₆-TMPP(Ni) (3.5 mg, 1.07 μmol) were used as the starting material.

3.6. Photostability Assay

H₂TMPP-F127, Pd₆-TMPP(Pd)-F127, and Pd₆-TMPP(Ni)-F127 aqueous solutions with the same porphyrin concentration (7 μg mL⁻¹) were prepared, and then irradiated with 650 nm laser (25 mW cm⁻²). The absorbance intensity at 416 nm was monitored over a period of 10 min, with data recorded every 1 min.

3.7. Singlet Oxygen (¹O₂) Detection

The same concentration of DPBF (33 μg mL⁻¹) was added to aqueous solutions of H₂TMPP-F127, Pd₆-TMPP(Pd)-F127, and Pd₆-TMPP(Ni)-F127 with an equivalent concentration of TMPP (7 μg mL⁻¹) to give uniform mixtures. The solutions were then irradiated with a 650 nm laser at an intensity of 25 mW cm^–2. After irradiation for every 1 min, the characteristic absorption intensity of DPBF at 416 nm was immediately measured.

3.8. Hydroxyl Radical (•OH) Detection

Chemical method: Different concentrations of H₂TMPP-F127, Pd₆-TMPP(Pd)-F127, and Pd₆-TMPP(Ni)-F127 were added to TMB solution containing 100 μM H₂O₂. After reacting for 3 hours at r.t., the reaction solutions were centrifuged to remove the influence of nanoparticles on absorbance. The UV-Vis curve of the supernatant at the wavelength of 652 nm was determined.

Spectroscopic method: Pd₆-TMPP(Pd)-F127 and Pd₆-TMPP(Ni)-F127 with the equivalent concentration of TMPP (7 μg mL⁻¹) were co-incubated with 100 μM H₂O₂, and after 3 hours, the spin trap 0.1 mL DMPO (97%) was added and stirred for 15 min. The resulting solution was then sealed in capillaries for EPR measurement.

3.9. In Vitro Cytotoxicity Evaluation by CCK-8 Assay

The NCI-H82 cell line (suspension-grown) was cultured in RPMI 1640 medium supplemented with 10% FBS, 1% P/S, or serum-free medium (RPMI 1640 + 1% P/S). Specifically, the suspension-grown cells were centrifuged, and the supernatant was discarded. The cells were re-suspended in serum-supplemented RPMI 1640 medium at a concentration of 1 × 10⁵ cells per milliliter. These cells were cultured at 37°C under a 5% CO₂ atmosphere for the CCK-8 assays.

NCI-H82 cells were seeded at a density of 3.5 × 10⁴ cells per well in 100 µL of serum-free medium containing various concentrations of H₂TMPP-F127, Pd₆-TMPP(Pd)-F127, and Pd₆-TMPP(Ni)-F127. All experiments were conducted with five replicates (n = 5), using untreated cells as the 100% cell viability control and cell-free medium (RPMI 1640 + 10 µL PBS + 1% P/S + CCK-8) as the blank.

For the PDT group, the cells were irradiated with a laser (650 nm, 25 mW cm⁻²) for 5 min after 24 hours of incubation, followed by an additional incubation time of 48 hours. By contrast, the non-PDT group was incubated continuously for 72 hours. After the incubation period, 10 µL FBS and 10 µL CCK-8 were added to the wells, and the plates were further incubated for 3.5 hours before analysis at 450 nm using a microplate reader. The relative cell viability (V%) was calculated using equation (1) as described below:

$$\mathrm{V}\mathrm{\%}={\displaystyle \frac{{\left[A\right]}_{experimental}-{\left[A\right]}_{blank}}{{\left[A\right]}_{control}-{\left[A\right]}_{blank}}}\times 100\mathrm{\%}\left(\mathrm{E}\mathrm{q}\mathrm{u}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}1\right)$$

in which V% is the percentage of cell viability, [A]_experimental is the absorbance of the wells culturing the treated cells, [A]_blank is the absorbance of the blank, and [A]_control is the absorbance of the wells culturing untreated cells.

The A549 cell line (adherent cells) was cultured in Ham’s F-12 medium supplemented with 10% FBS and 1% P/S, or serum-free medium (Ham’s F-12 + 1% P/S). Specifically, the adherent cells grew as a monolayer and were detached at confluence using trypsin (0.5% w/v in PBS). After trypsinization, the cells were incubated for 3 min, centrifuged, and the supernatant was discarded. A 3 mL portion of serum-supplemented culture medium was added to neutralize any residual trypsin. The cells were re-suspended in serum-supplemented Ham’s F-12 medium at a concentration of 1 × 10⁵ cells mL^–1 and cultured under standard conditions (37°C, 5% CO₂) for the CCK-8 studies.

A549 cells were seeded at a density of 1 × 10⁴ cells per well in 100 µL of culture medium and incubated for 24 hours at 37°C and 5% CO₂ to allow cell attachment. The culture medium was then replaced with serum-free medium containing various concentrations of the H₂TMPP-F127, Pd₆-TMPP(Pd)-F127, and Pd₆-TMPP(Ni)-F127. All experiments were performed with five replicates (n = 5), using untreated cells as the 100% cell viability control and cell-free medium (Ham’s F-12 + 10% FBS + 1% P/S + CCK-8) as the blank.

For the PDT group, the cells were irradiated with a laser (650 nm, 25 mW cm^–2) for 5 minutes after 24 hours of incubation, followed by an additional incubation of 48 hours. For the non-PDT group, cells were directly incubated for 72 hours. After the incubation period, 100 µL of culture medium and 10 µL of CCK-8 were added to each well, and the plates were incubated for an additional 1 hour before being analyzed at 450 nm using a microplate reader. The relative cell viability (V%) was calculated using Equation 1 above.

The cytotoxicity assessment for the KYSE-510 (adherent cells) and Te-1 (adherent cells) cell lines follow a similar protocol to that of the A549 cells, except that the culture media are different: KYSE-510 cells were cultured in a 1 : 1 (v : v) mixture of RPMI 1640 and Ham’s F-12 media, while Te-1 cells were cultured in RPMI 1640 medium. The exact compositions were: RPMI 1640 : Ham’s F-12 = 1 : 1 + 10% FBS + 1% P/S and RPMI 1640 : Ham’s F-12 = 1 : 1 + 1% P/S for KYSE-510; RPMI 1640 + 10% FBS + 1% P/S and RPMI 1640 + 1% P/S for Te-1.

3.10. Detection of Intracellular Reactive Oxygen Species

Te-1 cells were seeded in a 6-well plate at a density of 8 × 10⁵ cells per well in 2 mL culture medium (RPMI 1640 : Ham’s F-12 = 1 : 1 + 10% FBS + 1% P/S). The cells were incubated at 37°C under 5% CO₂ for 24 hours to allow proper attachment. After incubation, the culture medium was removed, and the wells were replenished with 1.5 mL serum-free culturing medium (RPMI 1640 : Ham’s F-12 = 1 : 1 + 1% P/S) containing H₂TMPP-F127, Pd₆-TMPP(Pd)-F127, and Pd₆-TMPP(Ni)-F127 at a concentration of 10 μmol L⁻¹.

For the PDT group, one plate of cells was irradiated with a 650 nm laser at a light intensity of 25 mW cm⁻² for 5 min, while cells in the non-PDT group were not subjected to laser irradiation. After irradiation, all cells were further incubated for 24 h, followed by the addition of 2 mL of fresh medium to each well. In the negative control wells, 1.0 μL of PBS was added, while 1.0 μL of Rosup-containing medium was added to the positive control wells. After 30 min of incubation, 1 μL of DCFH-DA was added to each well, and the culture was continued for another 20 min. The cells were then rinsed three times with serum-free medium to remove uninternalized reagents. Finally, the fluorescent performance of the cells was observed and recorded under an inverted fluorescence microscope.

3.11. Cellular Uptake

KYSE-510 cells were prepared in a 1 : 1 mixture of Ham’s F-12 and RPMI 1640 medium, and seeded into cell culture dishes (9 parallel dishes) with a volume of 10 mL medium each containing a density of 1 × 10⁷ cells per dish. After the cells adhered to the dish and proliferated to 95% confluency, the medium was replaced with serum-free medium. Subsequently, Pd₆-TMPP(Pd)-F127 and Pd₆-TMPP(Ni)-F127 were added directly to the medium at a porphyrin concentration of 10 μmol L⁻¹. After drug addition, the cells were incubated for 2 h, 4 h, and 6 h, with three replicate groups (n = 3) for each time point.

After incubation, the cells were washed three times with PBS, harvested by trypsin digestion, transferred to 15 mL centrifuge tubes, and centrifuged at 900 rpm for 3 min. The collected cells were rinsed twice with PBS, centrifuged, and reserved for subsequent use. The cells were lysed with 1 mL of nitric acid, then diluted to 10 mL with deionized water and filtered. The intracellular contents of Pd were analyzed by ICP-MS.

4. Conclusion

In this work, we synthesized a nonanuclear Pd-based coordination cage of Pd₆-TMPP(Ni) that elicits its anticancer effect via dual ROS generation strategies, viz., catalytic •OH via the Ni²⁺ center and the photoinduced ¹O₂ generation via the TMPP ligand. These results validate that Pd₆-TMPP(Ni) is a promising candidate for hypoxia-tolerant cancer therapy. Our future work is to equip Pd₆-TMPP(Ni) with targeting functions via conjugation with tumor-specific ligands for more effective cancer therapies.