Light-Sensitive Ruthenium Complex-Loaded Cross-linked Polymeric Nanoassemblies for the Treatment of Cancer

Abstract

This work focuses on improving the efficacy of photoactivatable Ru complexes for photodynamic therapy by employing cross-linked nanoassemblies (CNAs) as a delivery approach. The effects of complex photoactivation, hydrophobicity, and solution ionic strength and pH on complex loading and release from CNAs were analyzed. The cell cytotoxicity of CNA formulations was similar to free Ru complexes despite reduced or eliminated DNA interactions. The release rate and the amount of each Ru complex released (%) varied inversely with complex hydrophobicity, while the effect of solution ionic strength was dependent on complex hydrophobicity. Premature release of two photoactivatable prodrugs prior to irradiation was believed to account for higher activity in cells studies compared to DNA interaction studies; however, for photostable ¹O₂ generator-loaded CNAs this cannot explain the high cytotoxicity and lack of DNA interactions because release was incomplete after 48 hrs. The cause remains unclear, but among other possibilities, accelerated release in a cell culture environment may be responsible.

Figure 1

Structures of Ru complexes a) [Ru(bpy)₂(dmbpy)]Cl₂ (1), b) [Ru(dmbpy)₂(dip)]Cl₂ (2), and c) [Ru(dip)₃]Cl₂

Figure 2

PEG (5k) (blue lines)-ASP (35 units) (red lines) CNA preparation. 1) PEG-ASP(H⁺) block copolymers were cross-linked (green lines) with diaminooctane (DAO) activated with diisopropylcarbodiimide (DIC)/ N-hydroxysuccinimide (NHS)/dimethylaminopyridine (DMAP); 2) ASP(H⁺) groups of PEG-ASP CNAs converted to ASP(Na⁺); 3) positively charged Ru complexes (red circles) were physically entrapped inside PEG-ASP CNAs through electrostatic interactions with ASP(Na⁺) groups.

Figure 3

a) Empty PEG-ASP CNA diameter determined by DLS, b) empty PEG-ASP CNA molecular weight estimated based on GPC, and c) 1- and 2-loaded CNA stability was determined by DLS (1-loaded PEG-ASP CNAs ( ) and 2-loaded PEG-ASP CNAs ( )).

Figure 4

Photoejection kinetics of a) free 1, b) 1-loaded PEG-ASP CNAs, c) free 2, and d) 2-loaded PEG-ASP CNAs. The blue lines indicate the spectra of initial Ru complex prodrugs, red lines indicate final photoactivated product(s), and the green line in c) indicates a transition between the primary and secondary phases (t = 90 sec).

Figure 5

Percent of a) 1, b) 2, and c) 3 PEG-ASP CNA-entrapped Ru complexes released from 10K MWCO dialysis cassettes in PBS (pH 7.4) at 37 °C (PEG-ASP CNAs protected from light ( ) and light irradiated PEG-ASP CNAs ( )) compared to free Ru complexes released from 10K MWCO dialysis cassettes ( ). Because 3 was a photostable Ru complex, release was only evaluated when protected from light. The MLCT peaks of the entrapped Ru complexes were monitored in order to determine the amount of each Ru complex remaining inside the PEG-ASP CNAs, and this was used to calculate the amount of Ru complexes released as a function of time (N = 3).

Figure 6

The effect of buffer ionic strength on release from PEG-ASP CNAs for a) 1, b) 2, and c) 3 (free complex ( ), dark phosphate buffer ( ), dark PBS ( ), light phosphate buffer ( ), and light PBS ( )); d) shows a magnification of the rapid release of 3 from PEG-ASP CNAs when placed in a solution with low ionic strength (0 mM NaCl) (N = 3). Because 3 was a photostable Ru complex, release was only evaluated when protected from light.

Figure 7

The effect of phosphate buffer pH on complex release from PEG-ASP CNAs through 10K MWCO dialysis cassettes for a) 1, b) 2, and c) 3 (dark pH 6.0 ( ), dark pH 7.4 ( ), light pH 6.0 ( ), and light pH 7.4 ( )) compared to the release of free complexes from 10K MWCO dialysis cassettes ( ) (N = 3). Because 3 was a photostable Ru complex, release was only evaluated when protected from light (N = 3).

Figure 8

Agarose gel electrophoresis of 40 μg/mL pUC19 plasmid (10 mM phosphate buffer, pH 7.4) with light-sensitive Ru complexes. The supercoiled plasmid form migrates at 2000 bp, relaxed circle form is ~4000 bp, and linear form is just below 3000 bp. Dose response profiles: a) free 1 in the dark and b) photoactivated (40 J/cm²; 200 W source (λ> 400 nm)), c) 1-loaded PEG-ASP CNAs in the dark and d) photoactivated, e) free 2 in the dark and f) photoactivated, g) 2-loaded PEG-ASP CNAs in the dark and h) photoactivated, i) free 3 in the dark and j) photoactivated, and k) 3-loaded PEG-ASP CNAs in the dark and l) photoactivated. Lanes 1 and 12, DNA molecular weight standard; lane 2, linear pUC19; lane 3, relaxed circle (Cu(phen)₂ reaction with pUC19); lanes 4–11, 0, 7.5, 15, 30, 60, 120, 240, and 500 μM complexes.

Figure 9

UV/Vis absorbance spectra (hatched lines) and emission spectra (solid lines) in air-equilibrated diH₂O (red) and deoxygenated (Ar purged) diH₂O (black) for a) free 3, and b) 3-loaded PEG-ASP CNAs.