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. Author manuscript; available in PMC: 2019 Sep 4.
Published in final edited form as: Inorg Chem. 2018 Aug 22;57(17):10608–10615. doi: 10.1021/acs.inorgchem.8b01225

Photochemotherapeutic Properties of a Linear Tetrapyrrole Palladium(II) Complex displaying an Exceptionally High Phototoxicity Index

Andrea M Potocny †,⊥, Rachel S Riley ‡,⊥, Rachel K O’Sullivan ‡, Emily S Day ‡,§,∥,*, Joel Rosenthal †,*
PMCID: PMC6167929  NIHMSID: NIHMS987999  PMID: 30132325

Abstract

graphic file with name nihms-987999-f0001.jpg

Photodynamic therapy (PDT) represents a minimally invasive and highly localized treatment strategy to ablate tumors with few side effects. In PDT, photosensitizers embedded within tumors are activated by light and undergo intersystem crossing, followed by energy transfer to molecular oxygen, resulting in the production of toxic singlet oxygen (1O2). Previously, we reported a robust, linear tetrapyrrole palladium(II) complex, Pd[DMBil1], characterized by its facile and modular synthesis, broad absorption profile, and efficient 1O2 quantum yield of ΦΔ = 0.8 in organic media. However, the insolubility of this porphyrinoid derivative in aqueous solution prevents its use under biologically relevant conditions. In this work, we report the synthesis of Pd[DMBil1]-PEG750, a water-soluble dimethylbiladiene derivative that is appended with a poly(ethylene) glycol (PEG) functionality. Characterization of this complex shows that this PEGylated biladiene architecture maintains the attractive photophysical properties of the parent complex under biologically relevant conditions. More specifically, the absorption profile of Pd[DMBil1]-PEG750 closely matches that of Pd[DMBil1] and obeys the Beer–Lambert Law, suggesting that the complex does not aggregate under biologically relevant conditions. Additionally, the emission spectrum of Pd[DMBil1]-PEG750 retains the fluorescence and phosphorescence features characteristic of Pd[DMBil1]. Importantly, the PEGylated photosensitizer generates 1O2 with ΦΔ = 0.57, which is well within the range to warrant interrogation as a potential PDT agent for treatment of cancer cells. The Pd[DMBil1]-PEG750 is biologically compatible, as it is taken up by MDA-MB-231 triple negative breast cancer (TNBC) cells and has an effective dose (ED50) of only 0.354 μM when exposed to λex > 500 nm light for 30 min. Impressively, the lethal dose (LD50) of Pd[DMBil1]-PEG750 without light exposure was measured to be 1.87 mM, leading to a remarkably high phototoxicity index of ~5300, which is vastly superior to existing photosensitizers that form the basis for clinical PDT treatments. Finally, through flow cytometry experiments, we show that PDT with Pd[DMBil1]-PEG750 induces primarily apoptotic cell death in MDA-MB-231 cells. Overall these results demonstrate that Pd[DMBil1]-PEG750 is an easily prepared, biologically compatible, and well-tolerated photochemotherapeutic agent that can efficiently drive the photoinduced apoptotic death of TNBC cells.

INTRODUCTION

Photodynamic therapy (PDT) is used clinically as a non-invasive treatment for solid tumors. In PDT, photosensitizing compounds are either intratumorally or intraveneously injected and circulate throughout the body prior to irradiation of the tumor site.1 Photosensitizers present within the illuminated tissue absorb light and transfer energy to ground-state molecular oxygen to produce toxic 1O2 that ultimately induces localized cell death (Scheme 1). PDT is regarded as a promising treatment strategy for certain types of cancers2 and skin conditions,3 because it is less invasive than surgical removal,4 has fewer side effects than radiation or chemotherapy,5–9 and can, in some cases, stimulate an antitumor immune response.10,11

Scheme 1. Photodynamic Therapya.

Scheme 1.

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aPhotosensitizers are intravenously or intratumorally administered. The tumor area is then irradiated, driving the formation of toxic 1O2 to irreversibly damage/kill the surrounding cells.

Several photosensitizers, most of which belong to the porphyrinoid family of macrocyclic tetrapyrroles, have been approved for use in PDT.12–14 However, widespread clinical use of PDT has been hindered, at least in part, because existing 1O2 photosensitizers lack the photophysical and pharmacological attributes required for an optimal phototherapeutic agent. An ideal photosensitizer would be simple to synthesize and purify, demonstrate strong absorption of 600–850 nm light, which can penetrate deeper into tissues, and generate 1O2 with a high quantum yield during irradiation. It is also critical that a photosensitizer for PDT be relatively nontoxic in the absence of light to minimize off-target side effects. Lastly, water solubility is critical to ensure stability during storage or intravenous injection and to prevent aggregation-induced attenuation of the 1O2 quantum yield.

We recently demonstrated that the linear tetrapyrrole metal complex palladium 10, 10-dimethyl-5, 15-bis-(pentafluorophenyl)biladiene (Pd[DMBil1], structure shown in Scheme 2) is capable of absorbing light up to λ ≈ 600 nm and sensitizing 1O2 with a high quantum yield (ΦΔ ≈ 0.8) that is on par with those of photosensitizers currently used in PDT.15 Importantly, Pd[DMBil1] is easily synthesized in four steps from commercially available starting materials,15 and it can be purified and isolated using modular methods. While these traits suggest that the Pd[DMBil1] core is well-suited for interrogation as a PDT agent, this compound’s complete lack of water solubility has precluded such studies.

Scheme 2.

Scheme 2.

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Synthesis of Pd[DMBil1], Pd[DMBil1]-SCH2CO2H, and Pd[DMBil1]-PEG750

The addition of poly(ethylene) glycol (PEG) substituents is commonly employed in the development of therapeutics, since PEGs generally show excellent biocompatibility and lend high water solubility.16–19 In the present study, we modified the Pd[DMBil1] photosensitizer with a PEG functionality to overcome the inherent hydrophobicity of the biladiene architecture. We demonstrate that the addition of this PEG functionality endows the dimethylbiladiene complex with water solubility while having little effect on its photophysical properties, thus generating a biocompatible compound, Pd[DMBil1]-PEG750, that retains the ability to generate 1O2 with a high quantum yield under biologically relevant conditions. Additionally, we demonstrate that, while this biladiene complex is highly nontoxic in the dark, it can serve as an extremely potent chemotherapeutic agent for treatment of triple negative breast cancer (TNBC) cells and drives apoptotic cell death with a remarkable phototoxicity index of ~5300.

RESULTS AND DISCUSSION

Pd[DMBil1]-PEG750 is readily accessible from unfunctionalized Pd[DMBil1], which is readily prepared (Scheme 2) in four steps using methodology previously developed for the synthesis of biladienes15,20 and related tetrapyrroles containing sp3 hybridized meso-carbons.21–23 PEGylation of the parent Pd[DMBil1] architecture was facilitated by the presence of the two pentafluorophenyl substituents at the 5- and 15-positions, which can be derivatized via nucleophilic aromatic substitution of the para-fluorine substituents.24–26 As shown in Scheme 2, treatment of Pd[DMBil1] with mercaptoacetic acid and NEt3 at 90 °C in dimethylformamide (DMF) for 1 h afforded the monomercaptoacetic acid-substituted product (Pd[DMBil]-SCH2CO2H) in better than 75% yield following purification by flash chromatography. PEGylation of Pd[DMBil]-SCH2CO2H was achieved through conversion of the mercaptoacetic acid substituent into an N-(methoxyPEG)mercaptoacetamide using carbodiimide coupling chemistry.27,28 Pd[DMBil]-SCH2CO2H was initially reacted with N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to form an NHS mercaptoacetate intermediate. Further treatment with triethylamine and a methoxy-PEG-amine with an average molecular weight of 750 Da resulted in the formation of Pd[DMBil1]-PEG750, as is shown in Scheme 2. Detailed synthetic procedures and characterization data for all new compounds shown in Figure 2 are detailed in the Supporting Information.

Figure 2.

Figure 2.

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Emission spectra following excitation with 500 nm light of (a) Pd[DMBil1] and (b) Pd[DMBil1]-PEG750 in methanol at 298 K under an atmosphere of air (black) or nitrogen (red).

The visible absorption profile of Pd[DMBil1]-PEG750 has three features centered at 402, 483, and 540 nm in methanol (Figure 1 and Table 1). Aside from showing a slight enhancement of the feature at 483 nm, this absorption profile is nearly identical to that of Pd[DMBil1] in methanol, suggesting that introduction of the thioether and PEG chain at the para position of one biladiene C6F5 substituent has little effect on the electronic structure of the palladium tetrapyrrole core. In a pH 7.4 phosphate-buffered saline (PBS) solution the absorption spectrum of Pd[DMBil1]-PEG750 shows a bathochromic shift relative to its appearance in methanol. Although the full width at half-maximum (fwhm) of the most prominent absorption feature of the PEGylated derivative increases slightly from 63 nm in methanol to 67 nm in PBS, both of these values are smaller than the fwhm of the corresponding absorption feature of Pd[DMBil1] in methanol (73.5 nm). Furthermore, solutions of Pd[DMBil1]-PEG750 behave in accordance with the Beer–Lambert Law when dissolved in either methanol or PBS at concentrations ranging from 4 to 24 μM (Supporting Information Figure S2). Self-aggregation of other tetrapyrroles under aqueous conditions has been associated with broadened absorption features29–31 and deviations from the Beer–Lambert Law,32 so the observation that Pd[DMBil1]-PEG750 does not exhibit such behavior indicates that aggregation for this system should be insignificant. The Pd[DMBil1]-PEG750 complex is also resistant to photodegradation, as the UV–vis absorption spectrum for this construct in PBS (24.0 μM) showed no substantial changes over the course of 2 h of irradiation with 500 or 550 nm light (Figure S3).

Figure 1.

Figure 1.

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Electronic absorption spectra of Pd[DMBil1] in methanol (black line), Pd[DMBil1]-PEG750 in methanol (red line), and Pd[DMBil1]-PEG750 in pH 7.4 PBS (dashed blue line).

Table 1.

Photophysical Properties of Pd[DMBil] Complexes

compound (solvent) λabs, nm (ε × 103 M−1 cm−1) λfl, nm (Φfl) λph, nm (Φph) Φ Δ a
Pd[DMBil1] (methanol) 401 (17.4), 483 (31.9), 540 (7.5) 557 (1.3 × 10−4) 753 (1.3 × 10−4) 0.80
Pd[DMBil1]-PEG750 (methanol) 402 (16.6), 483 (34.6), 540 (7.5) 568 (1.4 × 10−4) 756 (7.8 × 10−5) 0.57
Pd[DMBil1]-PEG750 (PBS) 405 (13.6), 496 (33.2), 551 (8.3) 580 (2.0 × 10−4) 0.23
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a

Measured upon irradiation with λexc = 500 nm.

The emission properties of the Pd[DMBil1] complex in nitrogen-saturated methanol are also largely unaffected by PEGylation. As has been previously observed for Pd-[DMBil1],15 excitation of Pd[DMBil1]-PEG750 with 500 nm light elicits weak fluorescence from 500 to 700 nm as well as phosphorescence from 700 to 850 nm (Figure 2). Table 1 shows that, while the fluorescence emission maximum of Pd[DMBil1]-PEG750 in methanol is red-shifted by 11 nm compared with that of Pd[DMBil1], the fluorescence quantum yield of the PEGylated derivative (Φfl = 1.4 × 10−4) is essentially identical to that of the parent compound (Φfl = 1.3 × 10−4). The phosphorescence emission maximum shows a much smaller red shift from 753 nm for Pd[DMBil1] to 756 nm for Pd[DMBil1]-PEG750, and the phosphorescence quantum yield decreases slightly from Φph = 1.3 × 10−4 for Pd[DMBil1] to Φph = 7.8 × 10−5 for Pd[DMBil1]-PEG750.

The emission characteristics of Pd[DMBil1]-PEG750 in nitrogen-saturated PBS (pH 7.4) were also investigated; however, irradiation with 460 nm light under these conditions only resulted in one emission feature stretching from 500 to 700 nm corresponding to fluorescence (Table 1 and Figure S4). As compared with its fluorescence in methanol, the fluorescence of Pd[DMBil1]-PEG750 in PBS is further red-shifted, with an emission maximum at 580 nm and a quantum yield of Φfl = 2.0 × 10−4 in (Table 1). Given that fluorescence quenching is a well-documented consequence of porphyrinoid self-aggregation,33 the lack of attenuation of Φfl in PBS provides further evidence that Pd[DMBil1]-PEG750 does not tend to aggregate in aqueous environments. Failure to detect phosphorescence from the PEGylated derivative in PBS may be attributed to shortening of the triplet excited-state lifetime via energy transfer to a H2O overtone.

In prior work, we showed that air efficiently quenches the excited triplet state of Pd[DMBil1] in methanol, resulting in quenching of the biladiene’s phosphorescence due to efficient energy transfer to molecular oxygen.15 This phenomenon was also manifest in the ability of Pd[DMBil1] to photosensitize 1O2 generation with an impressive quantum yield of ΦΔ = 0.8, as quantified using diphenylisobenzofuran (DPBF)34 as an 1O2 probe and [Ru(bpy)3][PF6]2 as an actinometer (ΦΔ = 0.81).35 Similarly, the phosphorescence observed for Pd[DMBil1]-PEG750 in methanol under an inert atmosphere was quenched upon introduction of air (Figure 2b) to the sample. Quantification of the ability of the Pd[DMBil1]-PEG750 complex to sensitize the formation of 1O2 produced a quantum yield of ΦΔ = 0.57 following irradiation with 550 nm light. The slight decrease in ΦΔ value for the PEGylated biladiene complex may be attributed to a decrease in the O2 diffusion rate within the immediate vicinity of the photosensitizer due to partial shielding by the PEG moiety.36 Nonetheless, the ability of Pd[DMBil1]-PEG750 to sensitize the formation of 1O2 (ΦΔ = 0.57) is competitive with commercial photosensitizers employed for PDT.13

Although Pd[DMBil1]-PEG750 does not exhibit phosphorescence in PBS solutions (vide supra) this complex is capable of sensitizing the formation of 1O2 under aqueous conditions. Through use of the probe singlet oxygen sensor green (SOSG)37 and methylene blue as a reference photosensitizer (ΦΔ = 0.52),38,39 the ability of Pd[DMBil1]-PEG750 to sensitize singlet oxygen was determined to be ΦΔ ≈ 0.23 upon irradiation with 550 nm light (Table 1). The apparent attenuation of ΦΔ in PBS relative to that in methanol is not surprising, given that detecting 1O2 in aqueous environments is more challenging due to its shorter lifetime40,41 as well as the lower solubility of oxygen in water as compared to organic solvents.42 Importantly, however, the aqueous ΦΔ measured for Pd[DMBil1]-PEG750 is high enough to enable PDT in biological samples (vide infra).

TNBC accounts for ~15–20% of diagnosed breast cancer cases and is associated with earlier relapse, higher mortality rates, and significantly decreased progression-free survival compared to non-TN breast cancers.43 TNBC patients are unsusceptible to available targeted or hormonal therapies, because the cells in these tumors do not express the necessary surface receptors. Therefore, these patients are treated with aggressive chemotherapies and surgeries that have harmful side effects and are often unsuccessful, necessitating the development of new treatment strategies for this disease. Because of its high potency and specificity, PDT has been recognized as a promising therapeutic approach for TNBC.44

To evaluate the inherent toxicity of Pd[DMBil1]-PEG750, TNBC MDA-MB-231 cells were treated with up to 5.0 mM Pd[DMBil1]-PEG750 for 48 h in the dark and then subjected to an Alamar blue cell viability assay. MDA-MB-231 cells were completely viable at Pd[DMBil1]-PEG750 concentrations up to 0.5 mM (Figure 4A). Further, the lethal (LD) dose required for 50% cell death was found to be LD50 = 1.87 mM, an exceptionally high value, which demonstrates that, in the dark, Pd[DMBil1]-PEG750 is biocompatible and well-tolerated by the TNBC cells.

Figure 4.

Figure 4.

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Normalized cell viability after incubation with Pd-[DMBil1]-PEG750 for (a) 48 and (b) 24 h followed by light exposure for 0 (black), 10 (red), 20 (beige), or 30 (blue) min. Cells were incubated overnight post-irradiation and prior to the viability analyses. *p < 0.0001 by two-way ANOVA with post hoc Tukey-Kramer compared to 0 min light exposure for each concentration.

To further demonstrate the safety profile of Pd[DMBil1]-PEG750 and facilitate a comparison with a set of commercially available 1O2 photosensitizers that form the basis for common PDT treatments, cell viability assays were also conducted for hematoporphyrin dihydrochloride (HPDC) and isohematoporphyrin (IHP). Cell viability following treatment with up to 3.0 mM of either HPDC or IHP revealed lethal dose values of LD50 = 1.22 mM and LD50 = 0.64 mM, respectively, which are both lower than the LD50 obtained for Pd[DMBil1]-PEG750 (Figure S5). These results demonstrate that Pd[DMBil1]-PEG750 is less toxic than two commercially available photosensitizers, suggesting that it may be used as a photochemotherapeutic without causing off-target side effects that often limit the efficacy of PDT agents.

In an effort to assess whether Pd[DMBil1]-PEG750 is taken up by TNBC cells, MDA-MB-231 cells were treated with up to 1.5 mM Pd[DMBil1]-PEG750 for 48 h, and the cellular fluorescence was analyzed by fluorescence imaging and flow cytometry. As discussed above, Pd[DMBil1]-PEG750 retains the emission properties of Pd[DMBil1], enabling its detection by fluorescence microscopy and flow cytometry. Fluorescence imaging revealed that Pd[DMBil1]-PEG750 is taken up by cells during the incubation period, as indicated by the red fluorescent signal (Figure 3). Further, flow cytometry analysis confirmed this result, as cells treated with Pd[DMBil1]-PEG750 experienced a twofold increase in median fluorescence intensity (MFI) compared to untreated cells (Figure S6). These results were encouraging given that cellular uptake is important for any compound to be used for PDT,45 and they lead us to probe the efficacy of Pd[DMBil1]-PEG750 as a photochemotherapeutic agent.

Figure 3.

Figure 3.

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Fluorescence images showing Pd[DMBil1]-PEG750 uptake by MDA-MB-231 cells following a 24 h incubation period. Nuclei are stained blue (DAPI), F-actin in the cytoskeleton is stained green (phalloidan), and Pd[DMBil1]-PEG750 appears red. Scale = 25 μm.

To investigate the ability of Pd[DMBil1]-PEG750 to mediate PDT, MDA-MB-231 TNBC cells were treated with the biladiene complex at concentrations ranging from 0 to 10.0 μM for 4 h. At each concentration surveyed, cells were irradiated (λex > 500 nm) for either 0, 10, 20, or 30 min. Viability assays were conducted to assess cell death 16 h after irradiation. Cells incubated with Pd[DMBil1]-PEG750 for 24 h prior to irradiation were highly susceptible to PDT-mediated cell death compared to cells irradiated immediately after adding Pd[DMBil1]-PEG750. More specifically, cells irradiated immediately following the addition of Pd[DMBil1]-PEG750 required concentrations of at least 1 μM and 30 min of light exposure before any loss of cell viability was observed (Figure S7). By contrast, cells incubated with Pd[DMBil1]-PEG750 for 24 h prior to light application were susceptible to PDT with concentrations of photosensitizer as low as 0.25 μM, and 10 μM treatment resulted in complete loss of viability (Figure 4B). This enhancement in photoinduced toxicity in cells incubated with the photosensitizer before light treatment provides further evidence of cellular uptake of Pd[DMBil1]-PEG750. From these results, we also determined that the effective dose (ED) of Pd[DMBil1]-PEG750 required to reduce cell viability by 50% to be ED50 = 0.354 μM for cells incubated with the photosensitizer for 24 h and then subjected to 30 min of light exposure. The ratio of LD50/ED50 delivers the phototoxicity index (PI) of a given photochemotherapeutic agent, which provides an indication of the potency of a photodrug in relation to its inherent dark toxicity. For Pd[DMBil1]-PEG750 the phototoxicity index is determined to be PI ≈ 5300, which is exceptionally high,46 especially when compared to porphyrinoids typically employed for PDT (vide infra).

To compare the phototoxicity index of Pd[DMBil1]-PEG750 with those for the commercially available photosensitizers, we conducted the same photodynamic activity experiments with HPDC and IHP. Treatment of MDA-MB-231 cells with each photosensitizer for 24 h followed by a 30 min period of irradiation (λex > 500 nm) revealed effective doses of ED50 = 48.65 μM for HPDC and ED50 = 327.56 μM for IHP (Figure S8). The LD50 and ED50 values for HPDC and IHP correlate to phototoxicity indicies of PI ≈ 25 for HPDC and PTI ≈ 2 for IHP. By comparison, the phototoxicity index of Pd[DMBil1]-PEG750 is quite impressive, as it is ~200 and 3000 times higher than those of HPDC and IHP, respectively.

With the realization that Pd[DMBil1]-PEG750 is highly potent and effective for PDT via 1O2 sensitization, we sought to evaluate the mechanism by which the biladiene phototriggers cell death. Ideally PDT will induce cellular apoptosis as opposed to necrosis, as the latter can cause the release of intracellular compartments and local inflammation that can stimulate tumor growth.47 By contrast, apoptosis is anti-inflammatory and therefore discourages disease progression.48 To assess the mechanism by which Pd[DMBil1]-PEG750 photoinduces cell death, MDA-MB-231 cells were treated with 4.0, 6.0, or 8.0 μM Pd[DMBil1]-PEG750 for 24 h, irradiated for 30 min (λex > 500 nm), and then incubated for 1 h prior to AnnexinV (fluorescein isothiocyanate (FITC) channel) and PI (PerCP channel) staining. Experiments in which none of the biladiene photosensitizer was added to the cells were also performed as controls. For these experiments, the MDA-MB-231 cells were treated with higher Pd[DMBil1]-PEG750 concentrations than in the viability experiments because we analyzed the cells only 1 h post light treatment (as opposed to overnight). Although these photosensitizer concentrations are higher than required for effective PDT, they are still more than 2 orders of magnitude lower than the LD50 determined for Pd[DMBil1]-PEG750. In these experiments, each mechanism of cell death appears in a distinct quadrant of Figure 5A depending on their level of staining. For example, live cells are negative for both FITC and PI (lower left quadrant), apoptotic cells stain positive for only FITC (lower right quadrant), late apoptotic cells stain positive for both FITC and PI (upper right quadrant), and necrotic cells stain positive for only PI (upper left quadrant).

Figure 5.

Figure 5.

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Apoptosis and necrosis assay after incubation with 0.0, 4.0, 6.0, or 8.0 μM of Pd[DMBil1]-PEG750 for 24 h, followed by light treatment for 30 min and incubation for 1 h. (A) Representative flow cytometry density plot of cells treated with 8.0 μM Pd[DMBil1]-PEG750. (B–D) Flow cytometry analysis of cell populations in early apoptosis, late apoptosis, and necrosis following treatment with the photosensitizer. *p < 0.05 by t-tests compared to no light treatment for each concentration.

The results of the apoptosis and necrosis assay show that PDT with Pd[DMBil1]-PEG750 induces primarily apoptotic cell death and that the percentage of apoptotic cells increases with higher photosensitizer concentrations (Figures 5 and S9). The maximum amount of positively stained cells resulted from treatment with 8.0 μM Pd[DMBil1]-PEG750. The average of three separate assays showed that 20.21% of cells were positive for early apoptosis, 17.08% of cells were positive for late apoptosis, and 8.17% of cells stained positive for necrosis only. Alternatively, cells that did not undergo light treatment experienced only minimal cell death by any mechanism. These results demonstrate that almost half of the total cell populations treated with Pd[DMBil1]-PEG750 that are exposed to light for 30 min experience primarily apoptotic cell death, and ~82% of the cells killed by the phototreatment expire via apoptosis rather than necrosis. Given that apoptosis is much preferred over necrosis for cancer treatment, these results make Pd[DMBil1]-PEG750 even more attractive as a potential photosensitizer for use in PDT.

CONCLUSIONS AND FUTURE DIRECTIONS

The development of new biologically compatible 1O2 sensitizers for use as PDT agents remains a topic of significant interest,49 given the benefits that phototherapeutics can hold over more conventional treatment options for certain cancers. Despite these advantages and the fact that PDT has been approved to treat cancers of the skin, head, neck, digestive system, ovaries, prostate, bladder, lungs, breast, and brain,2 its adoption as a mainstream cancer treatment has been hampered by a general lack of 1O2 photosensitizers that are efficient, biologically tolerated, and pose minimal side effects and toxicity in the dark. In this work we described our efforts to address these shortcomings through development of a water-soluble dimethylbiladiene derivative (Pd[DMBil1]-PEG750) that can sensitize the formation of singlet O2 with high quantum yield under biologically relevant conditions.

Preparation of the Pd[DMBil1]-PEG750 complex was accomplished in two straightforward, high-yielding steps from the parent palladium dimethylbiladiene complex, and photo-physical studies revealed that PEGylation of the tetrapyrrole core has little effect on the electronic properties of the parent Pd[DMBil1] complex. In the absence of oxygen, Pd-[DMBil1]-PEG750 displays both fluorescence and phosphor-escence when dissolved in methanol. Notably, the triplet emission signal is completely quenched upon introduction of air to the sample due to efficient sensitization of 1O2 with a quantum yield of ΦΔ = 0.57, which is competitive with traditional sensitizers employed for PDT, including Photofrin.

Pd[DMBil1]-PEG750 is highly soluble in water as well as in biological media. Measurement of the photophysical properties of Pd[DMBil1]-PEG750 in pH 7.4 PBS solutions showed that the complex does not aggregate and can photosensitize 1O2 under aqueous conditions. The efficacy of Pd[DMBil1]-PEG750 as a photochemotherapeutic agent was assessed using TNBC MDA-MB-231 cells, which readily take up the biladiene construct as evidenced by fluorescence microscopy and flow cytometry. In the dark, the Pd[DMBil1]-PEG750 complex is extremely well-tolerated by the TNBC cells and has an LD50 of 1.87 mM. By contrast, the PEGylated palladium biladiene is an exceptionally potent photochemotherapeutic agent, as the effective dose of this complex required to reduce the TNBC viability by 50% was determined to be ED50 = 0.354 μM after 30 min of irradiation. Consideration of both the ED50 and LD50 metrics delivers the PI of Pd[DMBil1]-PEG750, which is a benchmark of the efficacy of a photochemotherapeutic agent in relation to its inherent dark toxicity. Pd[DMBil1]-PEG750 has a remarkably high phototoxicity index of PI ≈ 5300. This value dwarfs those determined for molecular surrogates of existing commercial PDT agents, as HPDC and IHP were found to have photoxicity indices of just PI ≈ 25 and PI ≈ 2, respectively.

Since comparison of the PI of Pd[DMBil1]-PEG750 to those for common porphyrin-based PDT models suggests that the linear palladium(II) tetrapyrrole can be a vastly superior photochemotherapeutic agent, the mechanism by which Pd[DMBil1]-PEG750 phototriggers TNBC cell death was probed. Cell death assays show that PDT with Pd[DMBil1]-PEG750 induces primarily apoptotic cell death as opposed to necrosis, as ~82% of the TNBC cells that die as a result of PDT do so via apoptosis. This finding serves to further distinguish Pd[DMBil1]-PEG750 as a photochemotherapeutic agent, since apoptosis is much preferred over necrosis for cancer treatments, because the latter can result in local inflammation that stimulates tumor growth.

When viewed as a whole, this study demonstrates that Pd[DMBil1]-PEG750 holds promise as a photochemotherapeutic agent and should warrant further investigation as a PDT photosensitizer for treatment of cancers including TNBC. From a broader perspective, while macrocyclic tetrapyrroles such as porphyrins and phthalocyanines have been well-studied as potential PDT agents, this work demonstrates that properly engineered linear oligopyrrole complexes may represent a privileged class of 1O2 sensitizers for the treatment of human disease. Building off our findings that Pd[DMBil1]-PEG750 is well-tolerated by TNBC cells and is a potent 1O2 sensitizer, future efforts will be focused on pushing the absorption envelope for this and related complexes further into the therapeutic window, either via modulation of the tetrapyrrole core or by using photon upconversion strategies, to better enable the use of such platforms in tissue and animal models.

Supplementary Material

ic8b01225_si_001 (1)

ACKNOWLEDGMENTS

A Strategic Initiatives Grant from the Univ. of Delaware (UD) Research Foundation and the State of Delaware Federal Research and Development Grant Program (16A00471) supported portions of this work. This work was also supported through NSF CAREER Award Nos. CHE1352120 and NIH P20GM104316. NMR and other data were acquired at UD using instrumentation obtained with assistance from the NSF and NIH (NSF-MIR 0421224, NSF-MIR 1048367, NSF-CRIF MU CHE-0840401 and CHE-0541775, NIH P20 RR017716). R.S.R. received support from the Univ. of Delaware Graduate Fellowship and from the American Association of Univ. Women.

Footnotes

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications Web site. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b01225.

Experimental procedures, additional Pd[DMBil1]-PEG750 characterization data, and photodynamic therapy data (PDF)

The authors declare no competing financial interest.

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ic8b01225_si_001 (1)
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