The Self-Assembly of a Cyclometalated Palladium Photosensitizer into Protein-Stabilized Nanorods Triggers Drug Uptake In Vitro and In Vivo

Abstract

Enhanced passive diffusion is usually considered to be the primary cause of the enhanced cellular uptake of cyclometalated drugs because cyclometalation lowers the charge of a metal complex and increases its lipophilicity. However, in this work, monocationic cyclometalated palladium complexes [1]OAc (N^N^C^N) and [2]OAc (N^N^N^C) were found to self-assemble, in aqueous solutions, into soluble supramolecular nanorods, while their tetrapyridyl bicationic analogue [3](OAc)₂ (N^N^N^N) dissolved as isolated molecules. These nanorods formed via metallophilic Pd···Pd interaction and π-π stacking and were stabilized in the cell medium by serum proteins, in the absence of which the nanorods precipitated. In cell cultures, these protein-stabilized self-assembled nanorods were responsible for the improved cellular uptake of the cyclometalated compounds, which took place via endocytosis (i.e., an active uptake pathway). In addition to triggering self-assembly, cyclometalation in [1]OAc also led to dramatically enhanced photodynamic properties under blue light irradiation. These combined penetration and photodynamic properties were observed in multicellular tumor spheroids and in a mice tumor xenograft, demonstrating that protein-stabilized nanoaggregation of cyclometalated drugs such as [1]OAc also allows efficient cellular uptake in 3D tumor models. Overall, serum proteins appear to be a major element in drug design because they strongly influence the size and bioavailability of supramolecular drug aggregates and hence their efficacy in vitro and in vivo.

Scheme 1. Structures of the Metal Complexes

Figure 1

Molecular view of the cationic complexes (a) and their stacking (b) in the crystal structures of [1]PF₆, [2]PF₆, and [3](BF₄)₂. Displacement ellipsoids are shown at the 50% probability level. Pd···Pd distances are indicated in angstroms. Counterions and disorder have been omitted for clarity.

Figure 2

(a) Absorption (solid line, left axis) and normalized emission spectra (dashed line, right axis) of [1]OAc–[3](OAc)₂ in water (50 μM, 350 nm excitation). (b) Singlet oxygen emission for solutions of [1]⁺–[3]²⁺ in CD₃OD (450 nm excitation, A₄₅₀ = 0.1).

Figure 3

(a) Dynamic light scattering derived count rate of [1]⁺–[3]²⁺ at 5 or 50 μM in pure water, PBS, and Opti-MEM medium with or without FCS (2.5% v/v). Size distribution of the DLS analysis of solutions of [1]⁺–[3]²⁺ (50 μM) in Opti-MEM medium with (b) or without (c) FCS. The X axis is the hydrodynamic diameter (in nm); the Y axis is intensity (%).

Figure 4

Time evolution of the absorption spectra of the H₂O/MeCN solution (100 μM, 9:1, v/v) of complexes [1]PF₆ (a), [2]PF₆ (b), and [3](PF₆)₂ (c) at 298 K for 30 min. (d) Time evolution of the absorption at 428 nm (black stars, [1]PF₆), 332 nm (red dots, [2]PF₆), 360 nm (green triangles, [3](PF₆)₂) of these solutions. The absorption spectra were measured every 30 s. (e) TEM images of [1]PF₆ (a), [2]PF₆ (b), and [3](PF₆)₂ (c) after aggregation in the H₂O/MeCN solution (100 μM, 9:1, v/v) for 30 min (scale bar 5 μm, inset 1 μm).

Figure 5

Cryo-TEM images of complexes [1]OAc and [3](OAc)₂ (50 μM) in Opti-MEM medium with or without FCS or in pure FCS solution.

Figure 6

Singlet oxygen generation of aggregates of [1]OAc in Opti-MEM complete medium. (a) The absorbance change of ABMDMA (100 μM) in Opti-MEM complete in the presence of [1]OAc (50 μM) in the dark (top) or upon blue light irradiation (bottom). (b) Evolution of the absorbance at 378 nm vs irradiation time of ABMDMA (100 μM) in Opti-MEM complete medium in the absence or presence of [1]OAc (50 μM) or [Ru(bpy)₃]Cl₂ (50 μM) in the dark or under blue light irradiation. Irradiation conditions: 298 K, 450 nm, 5.23 mW cm^–2, and 1 min.

Figure 7

(a) Pd contents (expressed in μg Pd/million cells) of A549 cells after treatment with NaN₃ or dynasore for 1 h and compounds [1]⁺–[3]²⁺ (5 μM) for 3 h. (b) Distribution (expressed in ng Pd/million cells) of palladium compounds [1]⁺–[3]²⁺ in the cytosol (black), membranes (red), nucleus (blue), and cytoskeleton (green) of A549 cells after treatment at 1 μM for 24 h.

Figure 8

(a) Mean fluorescence intensity of cells treated first with [1]OAc–[3](OAc)₂ (5 μM, 24 h) and then with DCFDA (20 μM, 30 min) and analyzed by flow cytometry. (b) Flow cytometry quantification of alive (Annexin −, PI −), early apoptotic (Annexin +, PI −), later apoptotic (Annexin +, PI +), and necrotic (Annexin −, PI +) A549 cells after treatment with [1]⁺–[3]²⁺ (15 μM) or cisplatin (15 μM) in the dark or after irradiation for 5 min with blue light (455 nm, 5.66 mW cm^–2, 1.7 J cm^–2).

Figure 9

Dose–response curves for A549 3D tumor spheroids incubated with complex [1]⁺ irradiated for 10 min with blue light (in blue) or kept in the dark (in black). EC_50,dark = 13 μM (95% confidence intervals +7.7 μM, −6.0 μM), EC_50,light = 2.1 μM (95% confidence intervals +0.7 μM, – 0.7 μM), PI = 6.2.

Figure 10

(a) Relative 4T1 breast tumor volumes of Balb/c mice and (b) visual tumor sizes at day 10 of Balb/c mice treated with vehicle control, [1]⁺, or [2]⁺ at day 0 and left in the dark or irradiated with blue light. Mice were treated on days 0 and 2 and irradiated with blue light (450 nm, 50 mW cm², 20 min, 60 J cm^–2) 1 h after injection. Statistical significance was set to p < 0.01 (**) and 0.001 (***).