trans-[Ru(NO)Cl(cyclam)](PF₆)₂and [Ru(NO)(Hedta)] Incorporated in PLGA Nanoparticles for the Delivery of Nitric Oxide to B16–F10 Cells: Cytotoxicity and Phototoxicity

Abstract

The immobilization and characterization of trans-[Ru(NO)Cl(cyclam)](PF6)2 (cyclam=1,4,8,11-tetraazacyclotetradecane), and [Ru(NO)(Hedta)] (Hedta=ethylenediaminetetraacetic acid) entrapped in poly(d,l-lactic-co-glycolic) acid (PLGA) nanoparticles (NP) using the double emulsification process is described. Scanning electron microscopy and dynamic light scattering revealed that the particles are spherical in shape, have a size distribution between 220 and 840 nm of diameter, and have a tendency to aggregate confirmed by a zeta potential between -3.2 and +3.5 mV. Using this method the loading efficiency was 26% for trans-[Ru(NO)Cl(cyclam)](PF6)2 and 32% for [Ru(NO)(Hedta)]. The release of the complexes from the NPs shows that cyclam-NP and Hedta-NP exhibited a two-phase exponential association release pattern, which was characterized by an initial complex burst during the first 24 h, followed by a slower release phase complex profile, due to a few pores observed in surface of nanoparticles using atomic force microscopy. The in vitro cytotoxic activity of the nitrosyl complexes in solution and incorporated in PLGA nanoparticles on melanoma cancer cells (cell line B16-F10) was investigated. The lower cytotoxicity of trans-[RuCl(cyclam)(NO)]2+ (12.4±2.6%) and [Ru(NO)(Hedta)] (4.0±2.7%) in solution compared to that of trans-[Ru(NO)(NH3)4py]3+ (46.1±6.4%) is consistent with the rate constant release of NO of these complexes (k-NO=6.2×10(-4) s(-1), 2.0×10(-3) s(-1), and 6.0×10(-2) s(-1), respectively); the cytotoxicities are also inhibited in the presence of the NO scavenger carboxy-PTIO. The phototoxicity of these complexes is due to NO release, which lead to 53.8±6.2% of cell death in the presence of trans-[Ru(NO)Cl(cyclam)](PF6)2 and 22.3±5.1% in the presence of [Ru(NO)(Hedta)]. The PLGA nanoparticles loaded with trans-[Ru(NO)Cl(cyclam)](PF6)2 and [Ru(NO)(Hedta)] exerted in vitro a reduced activity against melanoma cells when compared to the activity of complex in solution (nonentrapped in nanoparticles). Blank PLGA nanoparticles did not exhibit cytotoxicity. In the presence of light and of ruthenium nitrosyl complexes or cyclam-NP and Hedta-NP, B16-F10 cells displayed a considerable damage of the surface with rupture of the plasma membrane. This behavior is an indicative of the efficiency of the DDS to deliver the NO from the entrapped complex when photoinduced.

The versatile ruthenium(II/III) tetraazamacrocycle complexes and their nitrosyl derivatives

“…This complex is slightly less phototoxic for B16-F10 melanoma cells than trans-[Ru(NO)(py)(NH 3 ) 4 ](BF 4 ) 3 (1 × 10 −4 M; NO = 0.18) that leads to 63.3% ± 3% cell death [53]. Cell death is inhibited by the NO scavenger c-PTIO in both cases, in the dark and under irradiation [53,54]. Assays with V79 cell show that trans-[Ru(NO)Cl(cyclam)](PF 6 ) 2 is particularly non-toxic; the IC 50 value was not achieved experimentally up to 3.0 mM and the cytotoxicity was inhibited in the presence of c-PTIO [48].…”

“…The in vitro cytotoxic activity of trans-[Ru(NO)Cl(cyclam)](PF 6 ) 2 on B16-F10 melanoma cancer cells [53] is low (IC 50 6.8 mM), and it is non-toxic at ≤10 −4 M. That value is consistent with the low rate constant for NO release from this complex (k NO 6.2 × 10 −4 s −1 ) compared with trans-[Ru(NO)(py)(NH 3 ) 4 ](BF 4 ) 3 (IC 50 1.0 mM, k NO 2.0 × 10 −3 s −1 ) [40]. However, under light irradiation (366 nm, NO = 0.16), trans-[Ru(NO)Cl(cyclam)](PF 6 ) 2 (1 × 10 −4 M) leads to 53.8 ± 6.2% of cell death when incubated with B16-F10 cells [53].…”

“…However, under light irradiation (366 nm, NO = 0.16), trans-[Ru(NO)Cl(cyclam)](PF 6 ) 2 (1 × 10 −4 M) leads to 53.8 ± 6.2% of cell death when incubated with B16-F10 cells [53]. This complex is slightly less phototoxic for B16-F10 melanoma cells than trans-[Ru(NO)(py)(NH 3 ) 4 ](BF 4 ) 3 (1 × 10 −4 M; NO = 0.18) that leads to 63.3% ± 3% cell death [53]. Cell death is inhibited by the NO scavenger c-PTIO in both cases, in the dark and under irradiation [53,54].…”

“…with low toxicity for healthy cells. Both the chemical properties and biological activities of ruthenium nitrosyl complexes have been extensively investigated [40,[48][49][50][51], not only in solution but also incorporated into drug delivery systems such as polymeric nanoparticles [52][53][54]. With that aim, a large number of ruthenium nitrosyl complexes with macrocyclic ligands ([Ru(NO)L(mac)] q+ ) have been reported [40,47,48,50].…”

Evaluation of Antitumor Effects of Folate-Conjugated Methyl-β-cyclodextrin in Melanoma

“…[2][3][4][5] To confer an active targeting-ability, the chemical modification of tumor-specific ligands to a drug carrier is known. Of various tumor-targeting ligands, folic acid (FA) [6][7][8][9][10] has emerged as a remarkable targeting ligand capable of potent interaction with cancer cells expressing the folate receptor-α (FR-α) with high affinity (K d : 10 −9 -10 −10 M).…”