2 and [Ru(NO)(Hedta)] Incorporated in PLGA ... - ACS Publications

Article pubs.acs.org/molecularpharmaceutics

trans-[Ru(NO)Cl(cyclam)](PF6)2 and [Ru(NO)(Hedta)] Incorporated in PLGA Nanoparticles for the Delivery of Nitric Oxide to B16−F10 Cells: Cytotoxicity and Phototoxicity Anderson J. Gomes,† Enilza M. Espreafico,‡ and Elia Tfouni*,§ †

Faculdade de Ceilândia, Universidade de Brasília, Brasília, DF, Brazil Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil § Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil ‡

S Supporting Information *

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. KEYWORDS: ruthenium nitrosyl, nitric oxide, nanoparticles, PLGA, cytotoxicity, phototoxicity, B16−F10, cyclam, Hedta, nitric oxide donors

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(cyclam)]2+. They can be tailored to have the desired properties by adequate choice of ligands and can also be incorporated in matrices as drug delivery systems (DDS).12−14,23,24 In aqueous solution, trans-[Ru(NO)(NH3)4L]3+ undergoes one electron reduction with NO release (eqs 1 and 2) and with potentials between ∼-0.3 to ∼+0.1 V vs NHE,12−14,23,24 which are accessible to several biological reducing agents, such as glutathione, for example. A second reduction process, followed

INTRODUCTION Nitric oxide (NO) plays key roles in several physiological processes and pathologies;1 hence several NO donors and scavengers have been developed.2−22 Among the several NO donors, ruthenium tetraamine and tetraazamacrocyclic nitrosyl compounds,12−14,23,24 such as trans-[Ru(NO)(NH3)4(py)]3+ (py = pyridine) and trans-[Ru(NO)Cl(cyclam)]2+ (cyclam = 1,4,8,11-tetraazacyclotetradecane), were shown to be effective NO-donors with biological activity. They are water-soluble and stable as solids or in solution in the dark even at physiological pH, and they release NO after reduction and/or by irradiation with light, with only the solvent product besides NO or NO and HNO, and chloride in the case of trans-[Ru(NO)Cl© 2013 American Chemical Society

Received: Revised: Accepted: Published: 3544

September 29, 2012 July 3, 2013 July 18, 2013 July 18, 2013 dx.doi.org/10.1021/mp3005534 | Mol. Pharmaceutics 2013, 10, 3544−3554

Molecular Pharmaceutics

Article

light irradiation and whose product is in the hydroxo form at this pH, but unlike what occurs upon reduction, there is no release of NO12−14,24 (eq 8).

by HNO release, can occur for these nitrosyl complexes at potentials more negative than −1.0 V vs NHE,13 which is below those of most biological reducing agents. The rates of NO release are dependent on the trans effect and trans influence promoted by the trans ligand L, and the rate constants, k2 are in the range of 0.02−5.1 s−1 at 25 °C. n+

trans‐[Ru(NO)(NH3)4 L]

hν

trans‐[RuCl(NO)cyclam]2 + → trans‐ [RuCl(OH)cyclam]2 + + NO

k1

+ e → trans‐

(n − 1) +

[Ru(NH3)4 (NO)L]

The quantum yields of NO release are dependent on the other ligands and on the solution pH, and, as seen, in addition to NO, there is only one other photoproduct, a ruthenium complex, and chloride, in the case of the cyclam complex, but for biological applications, chloride is a not a problem because it is normally present in the human body. The photochemical release of NO is especially important as far as biological applications are concerned, as happens with photodynamic therapy (PDT), because it introduces a further control by focusing the light beam on the target, light intensity, and time of irradiation.14,22,34 The energy of irradiation for NO photolabilization in the above nitrosyl complexes (300−350 nm) is not suitable for PDT applications, which normally require irradiation in the therapeutic window (600−1100 nm).35 Nonetheless, there are some cases were topical irradiation is needed and suffices36 as in the case of skin melanoma B16−F10 cells, and in these cases, these ruthenium complexes would be suitable. The ruthenium nitrosyls, such as trans-[Ru(NO)(NH3) 4(py)]3+ (py = pyridine) and trans-[Ru(NO)Cl(cyclam)]2+, were shown to be active, either photochemically or by reduction, in biological assays, such as evoking potential in mouse hippocampus, reactions with mitochondria, vasodilation effect, toxicity, biodistribution and bioavailability, antiparasitic effects, cytotoxicity, and phototoxicity against tumor cells in solution or incorporated in materials.12,14,23 It should be noted that ruthenium-based complexes are metallo drugs that received considerable attention for medical applications because their kinetic behavior is similar to platinum with respect to cellular division processes,37−40 but their toxicity is lower than that of the commercially available cisplatin,41 probably due to their ability to mimic iron and therefore bind to many biomolecules, such as human serum albumin and the iron transport protein transferrin,14,42 whose side effects prompted investigations on improvement on platinum compounds and alternatives.43−47 Nanoparticles of biodegradable polymers have attracted great interest in the recent years for clinical administration of anticancer drugs for the treatment of human glioma, breast cancer, ovarian carcinoma, prostate cancer, brain cancer, and others.40−43 The use of nanostructured materials as drug delivery systems impacts in medicine due to beneficial sizedependent physical and chemical properties14,23,48 and targeted selectivity. The advantages of nanoparticle formulation as drug delivery systems (DDS) include the preservation of the drug activity until reaching the target, controlled drug release, sustained drug action on the lesion, reduced systemic side effects, facilitated extravasation into the tumor, and high capability to cross various physiological barriers as well as controlled and targeted delivery of the drug.49 There are cases where the ruthenium nitrosyl actions are in solution, such occurs with trans-[Ru(NO)Cl(cyclam)]2+ that reduces the blood pressure of hypertensive male Wistar rats.50 However, in other situations, incorporation in DDS is desirable for targeting and to avoid premature reduction that leads release of NO,23 because the ubiquitous nitric oxide can be deleterious or

(1) k2

trans‐[Ru(NO)(NH3)4 L](n − 1) + + H 2O → trans‐ [Ru(NH3)4 (H 2O)L](n − 1) + + NO

(2) 2+

In the case of trans-[Ru(NO)Cl(cyclam)] (cyclam = 1,4,8,11tetraazacyclotetradecane), the first reduction (at −0.078 vs NHE) (eq 3) is followed by a fast chloro release (k2 = 1.5 s−1) (eq 4). The chloride loss from trans-[RuCl(cyclam)(NO)]+ is suppressed by 0.1 M chloride, and trans-[Ru(H2O)(cyclam)(NO)]+ releases NO (eq 5), with specific rate constants, k3, of 6.2 × 10−4 s−1 at 25 °C, and 2.2 × 10−3 s−1 at 35 °C, which are independent of chloride concentration up to 0.25 M,25 lower than that of trans-[Ru(NO)(NH3)4(py)]3+ (k−NO = 0.06 s−1 at 25 °C).13 k1

trans‐[RuCl(NO)(cyclam)]2 + + e → trans‐ [RuCl(NO)(cyclam)]+

(3) k2

trans‐[RuCl(NO)(cyclam)]+ + H 2O → trans‐ [Ru(NO)(H 2O)(cyclam)]2 + + Cl−

(4) k3

trans‐[Ru(NO)(H 2O)(cyclam)]2 + + H 2O → trans‐ [Ru(H 2O)2 (cyclam)]2 + + NO

(5)

In its turn, [Ru(Hedta)(NO)] (Hedta = ethylenediaminetetraacetic acid) (Hedta-NO) exhibits a slow release of NO to the medium (k−NO = 2.0 × 10−3 s−1),26 but the product of the reaction,27 [Ru(Hedta)H2O], reacts quickly with NO to form [Ru (NO)(Hedta)], k+NO = 2.24 × 107 M−1 s−1 at, pH 7.4, showing that the aquo ion is an efficient and effective NO scavenger,26−31 potentially useful for the therapeutic treatment of septic shock, tissue injury, inflammatory bowel disease, and rheumatoid arthritis. Considering that the NO effects depend on its concentration and bioavailability, and accordingly classified as direct or indirect,32 fast or slow release of NO is important, depending on the target. The delivery of NO from the ruthenium nitrosyl complexes can also be activated by light irradiation, yielding NO and either trans-[Ru(H2O)(NH3)4L]3+ (at pH ≤ 2) or trans-[Ru(OH)(NH3)4L]2+ (at pH ≥ 6) as the only photoproducts (eqs 6 and 7)33 hν

trans‐[Ru(NO)(NH3)4 L]n + → trans‐ [Ru(H 2O)(NH3)4 L]n + + NO

(6)

hv

trans‐[Ru(NO)(NH3)4 L]n + → trans‐ [Ru(OH)(NH3)4 L](n − 1+) + NO

(8)

(7) 2+

In the case of trans-[Ru(NO)Cl(cyclam)] , NO release also occurs with a quantum yield of 0.16 at pH 7.4 under 355 nm 3545

dx.doi.org/10.1021/mp3005534 | Mol. Pharmaceutics 2013, 10, 3544−3554


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ruthenium nitrosyl complexes (trans-[Ru(NO)Cl(cyclam)](PF6)2 or [Ru(NO)(Hedta)]) were prepared by the double emulsification method.51,53 The ruthenium nitrosyl complex (6.3−10.0 mg) was solubilized in 5.0 mL of 2.0% PVA aqueous solution and emulsified in organic phase (acetone−methylene chloride (5.0:5.0 mL) containing PLGA (500 mg) using a high speed homogenizer (Ultra-Turrax T18-S18N-19G, IKA) for 60 s, at 14 000 rpm. The primary water/oil emulsion was immediately transferred to 20 mL of a 3.0% (w:v) aqueous solution of PVA and homogenized under ice cooling, with stirring at 14 000 rpm for 2 min, using the Ultra-Turrax homogenizer. Afterward, solvent evaporation was carried out by gentle magnetic stirring at room temperature, usually for 3−5 h. Nanoparticles were recovered by centrifugation for 10 min, at 6000 rpm and 4 °C, and washed (three times) with distilled water. After centrifugation, the NPs were stored at 4 °C. The encapsulation process was carried out under aseptic conditions. Nanoparticles without the complex were also prepared by the same procedure in order to evaluate the damage promoted by the nanoparticle itself. Determination of Encapsulation Efficiency. The encapsulation efficiency was calculated using eq 9, where the initial amount of drug was 6.3 mg for [Ru(NO)(Hedta)] and 10.0 mg for (trans-[Ru(NO)Cl(cyclam)](PF6)2. A total of 5.0 mg of ruthenium nitrosyl complex loaded nanoparticles were dissolved in 1.0 mL of methylene chloride, and the complex was extracted from the NP with 3.0 mL of phosphate buffer saline (PBS) medium (pH 7.4). A nitrogen stream was introduced to evaporate the methylene chloride. The PBS solution containing the extracted ruthenium nitrosyl was determined spectrophotometrically (trans-[Ru(NO)Cl(cyclam)](PF6)2, ε262nm = 2.5 × 103 M−1 cm−1; [Ru(NO)(Hedta)], ε370nm =1.8 × 102 M−1 cm−1).31,32 The amount of Ru nitrosyl in the NP was determined by the difference between the initial Ru added and that extracted.

beneficial depending on its concentration which demands for targeted and selective NO donors.14,23 The approach of using ruthenium nitrosyl complexes may also improve the in vivo residence time of ruthenium nitrosyl complexes and avoid undesired premature release of NO and side reactions, such as nucleophilic attack on the coordinated nitrosyl, and especially premature reduction that leads to NO release.23 The trans-[Ru(NO)(NH3)4(py)]3+ complex releases NO upon reduction with a rate constant of 6 × 10−2 s−1 and a quantum yield of 0.18 with 330 nm light irradiation at pH 6.4, and it was entrapped in poly(D,L-lactic-co-glycolic) acid (PLGA) delivery system51 and tested against B16−F10 cells. Upon light irradiation, a considerable damage of cell surface by rupture of the cell membrane was observed and assigned to NO release. This behavior is an indicative of the efficiency of the DDS to deliver the NO from the entrapped complex when photoinduced. Thus, it would be interesting to compare this complex in solution and entrapped in PLGA paper with trans[Ru(NO)Cl(cyclam)](PF6)2 (cyclam-NO) and [Ru(NO)(Hedta)] also in solution and entrapped in PLGA against B16−F10 cells. Compared to trans-[Ru(NO)(NH3)4(py)]3+, trans-[Ru(NO)Cl(cyclam)]2+ has a lower rate of release of NO upon reduction and similar quantum yield, and [Ru(NO)(Hedta)] has a lower quantum yield and also a lower rate of NO release upon reduction and whose product is a good NO scavenger. Therefore, in this paper, we describe the immobilization and characterization of trans-[Ru(NO)Cl(cyclam)](PF6)2 (cyclam = 1,4,8,11-tetraazacyclotetradecane) (cyclam-NO), and [Ru(NO)(Hedta)] (Hedta = ethylenediaminetetraacetic acid) (Hedta-NO) entrapped in poly(D,Llactic-co-glycolic) acid (PLGA) nanoparticles, by the double emulsification process,51 and in vitro cytotoxicity evaluation under light irradiation of the complexes in solution and of the loaded PLGA NPs against B16−F10 skin melanoma cells.

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MATERIALS AND METHODS Chemicals. Ruthenium trichloride (RuCl3·nH2O) and 1,4,8,11-tetraazacyclotetradecane (cyclam) (Strem); poly(D,Llactic-co-glycolic acid) (PLGA 50:50), 17 kDa MW), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and ethylenediaminetetraacetic acid disodium salt (Na2Hedta) (Sigma); dimethylsulfoxide-d6 solution (Acros); polyvinyl alcohol (PVA) (13−23 kDa MW, 87−89% hydrolyzed) (Aldrich); and analytical grade dichloromethane (Merck) were used as supplied. Ham’s F-10, RPMI-1640, fetal bovine serum (FBS), L-glutamine, penicillin, and streptomycin were obtained from GIBCO-BRL (Gaithersburg, MD). All other chemicals were of analytical grade and were used without further purification. Syntheses of Ruthenium Complexes. trans-[RuCl(NO)(cyclam)](PF6)2 and [Ru(NO)(Hedta)] were prepared, respectively, by a modification of a previously reported method25 and as described elsewhere52 (see Supporting Information). Spectroscopy. UV−vis spectra were recorded on a Hewlett-Packard 8452A diode array spectrophotometer in H2O using quartz cells. 1H NMR spectra were obtained in 5 mm NMR tubes on Bruker DRX-400 Unity 400 MHz FT spectrometers in D2O (unless otherwise noted). Infrared (IR) absorption spectra were obtained in KBr pellets on a Bomem MB-102 IR spectrophotometer. Preparation of the Nanoparticles by the Double Emulsion Technique. The nanoparticles loaded or not with

encapsulation efficiency(%) amount of drug in microparticle = × 100 initial amount of drug

(9)

Particle Size and Surface Charge (Zeta Potential). Particle size and size distribution were determined by photon correlation spectroscopy (PCS) using the quasi-elastic light scattering technique, in a Zetasizer 3000 equipment (Malvern Instrument). The zeta potential of the NPs in PBS buffer, (1.0 × 10−3 M, pH 7.4) was determined by using ZetaPlus in the zeta potential analysis mode.51 Nanoparticle Morphology: SEM Analysis. Scanning electron microscopy (SEM) was used to evaluate the shape and size of PLGA nanoparticles using an EVO 50 (Zeiss) scanning electron microscope operating at 20 kV in the traditional mode (SE1 detector). Atomic Force Microscope (AFM) Imaging. The shape and surface morphology of the nanoparticles were determined by using atomic force microscope (AFM). The dried nanoparticle samples were suspended in distilled water and were sonicated before measurement to obtain a homogeneous suspension. A drop of the nanoparticle suspension was mounted on the metal slabs, air-dried, and scanned by the atomic force microscope multimode scanning probe microscope (Shimadzu, SPM-9600) maintained in a constanttemperature and vibration-free environment. 3546



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Cell Morphology. B16−F10 cells were grown on glass pieces and placed in Petri dishes. Preparation for SEM was carried out as follows. The cells were fixed in 2% glutaraldehyde for 1 h at room temperature (RT) and washed with 0.1 M phosphate buffer (pH 7.4). Following washing, the cells were postfixed in 0.10% OsO4 for 30 min and washed with 0.1 M phosphate buffer (pH 7.4) at 4 °C. Dehydration was carried out in ethanol 70−100% and acetone P.A. for 15 min each. A Blazers SCD-050 coater sputter was used to coat the specimens with 5 nm of gold. The cells were examined and photographed in a Zeiss EVO50. The microscope was operated at 5 kV. A similar procedure was used with cells−nanoparticles after irradiation. Unstimulated cells were used as controls. Data Report. The results are expressed as the means and standard error (SE). Differences among the experimental groups were analyzed by ANOVA, and when the differences were significant we applied the Student−Newman−Keuls multiple comparison test; P < 0.05 was considered significant. Statistical analysis was performed using Prism 5 software for Windows (Graph-Pad Software, San Diego, CA).

Energy Dispersive Spectroscopic (EDS) Analysis. Bulk compositional analysis of the Ru was performed using an EVO 50 (Zeiss) SEM equipped with an X-ray detector (IXRF Systems “Iridium” EDS system) for energy dispersive spectroscopy analysis. An accelerating voltage of 10 kV was used in all of the EDS experiments.51 In Vitro Drug Release. Drug-loaded nanoparticles (100 mg) were added to 10 mL of 1.0 × 10−3 M, pH 7.4 phosphate buffer inside a dialysis bag (Sigma, cutoff: 12 000 Da), which was placed in a vessel containing 50 mL phosphate buffer at constant temperature (37 °C), protected from light, and stirred at 200 rpm. At time intervals, 3.0 mL samples were withdrawn from the solution outside the bag in order to follow the change in ruthenium nitrosyl complex concentration by spectrophotometry. The removed volume from the vessel was replaced with phosphate buffer. The cumulative amount of complex release was calculated by using eq 10: ⎛D ⎞ cumulative amount release(%) = ⎜ t ⎟ × 100 ⎝ D∞ ⎠

■

(10)

RESULTS AND DISCUSSION Preparation of the Nanoparticles and Determination of Drug Content. A large number of investigations have shown that both tissue and cell distribution of anticancer drugs can be controlled by entrapment in submicrometer colloidal systems such as liposomes and nanoparticles.54−58 The rationale behind this approach is to increase antitumor efficacy, while reducing systemic side effects.58,59 In the present study, the nanoparticles were prepared by a modified double emulsion process, which resulted in reduced loading (26% ± 2.5% for trans-[Ru(NO)Cl(cyclam)](PF6)2 and 32% ± 2.3% of [Ru(NO)(Hedta)]) of nanoparticles with these water-soluble nitrosyl complexes. Similar results were observed by others authors using hydrophilic compounds.60−64 It is generally accepted that lipophilic drug substances load with higher efficiencies into polymeric NP, such as those composed of PLGA. Water-soluble counterparts are more likely to remain in the aqueous continuous phase, giving high amounts of free drug in supernatant fractions.65 trans-[Ru(NO)Cl(cyclam)](PF6)2 and [Ru(NO)(Hedta)] are no exception, but nevertheless, results obtained confirm its incorporation into polymeric nanoparticles. Particle Size and Surface Charge (Zeta Potential). Particles size plays a key factor in the long circulation and biodistribution of nanoparticles. Results of sizing, zeta potential, and polydispersity analyses are shown in Table 1. The

where Dt is the amount of complex released at time t and D∞ is the total amount of complex released at infinite time, which is the actual loading of drug determined in the loading efficiency experiment. Cell Culture. The highly metastatic mouse melanoma cell line B16−F10 were routinely cultured as monolayers at 37 °C in HAM F10 (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 2.0 × 10−3 M L-glutamine, penicillin (100 U/mL), and streptomycin (100 mg/mL), in an atmosphere of 95% air and 5% CO2. Toxicity Assays. B16−F10 cells were obtained from exponentially growing cultures (90−95% confluence) and seeded at 5000 cells/well in a 96-well plate. The toxicity assay were performed in the presence of NO donor solution (1.0 × 10−7 to 1.0 × 10−3 M) and also in the presence of complex-loaded nanoparticle at 1.0 × 10−4 g/mL at 37 °C for 2 h with Hank’s buffer in the dark (without light irradiation). Following incubation, cells were washed to remove excess NO donor solution and unbound nanoparticles and were placed in HAM-F10 medium. After 24 h incubation, the medium was removed, and the wells were washed using PBS media before adding 50 μL of 1.0 mg/mL yellow tetrazolium dye [3-(4,5dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide, MTT]. The phototoxicity studies were performed, using the same conditions as those used in the dark toxicity assay. After incubation, the cells were irradiated at 366 nm using an ultraviolet light (Black-Ray lamp model UVL-56), placed 5 cm away from the sample. From this distance, the lamp delivered an intensity of 6.4 × 1015 einstein s−1. Immediately after light exposure, the cells were incubated in HAM-F10 for 48 h, at 37 °C. Cell viability was determined by an MTT assay. Control cells were incubated for 2 h in the medium in the absence of phenol red and irradiated under the same conditions. Samples of these cell suspensions were assayed in triplicate. We also investigated the action of the nitric oxide donors in solution and of the nanoparticles against B16−F10 cells, using the NO scavenger carboxy-PTIO [2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide] (C-PTIO). An amount of 1.0 × 104 cells were plated in 96-well with the presence of NO donors/nanoparticles and 1 × 10−4 M carboxy-PTIO. After treatment, the cell viability was determined by MTT assay. The same procedures were done in the phototoxicity assay.

Table 1. Particle Size and Surface Charge of Particles at pH 7.4 complex containing NP

size distribution/ nm

polydispersity

zeta potential/ mV

empty-NP cyclam-NP Hedta-NP

220−660 270−780 220−840

0.26 0.53 0.38

−3.2 ± 1.2 +3.5 ± 1.7 +2.3 ± 1.0

entrapment process into the nanoparticles does not promote a significant difference in the size between empty-NP and complex-loaded NP. Cyclam-NP and Hedta-NP had, respectively, a mean diameter of 270−780 nm and 220−840 nm, and a polydispersity index of 0.38 and 0.53. In our assay the NP produced have a high index of polydispersity, which is an 3547



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inherent problem of the method of preparation used,66 where the size distributions of particles are generally reproducible but nonuniform. The empty-NP were slightly smaller, with a size diameter between 220 and 660 nm, and were negatively charged (−3.2 ± 1.2 mV). In this work a significant reduction of the size of nanoparticles were achieved when compared to previous work (diameters varying from 1519 to 1747 nm)51 by the addition of acetone to the medium which reduced the viscosity of organic phase. The dispersion of size observed in this preparation is characteristic of this method.67 Nanoparticles smaller than 10 nm can be rapidly cleared by the kidneys or through extravasation, while larger nanoparticles (>200 nm) may have a higher tendency to be cleared by cells of the mononuclear phagocyte system (MPS also referred to as reticuloendothelial system, RES).68 The NP colloidal stability was analyzed by measuring the NP zeta potential. The zeta potential of cyclam-NP and Hedta-NP displayed low positive zeta-potential in PBS, while the emptyNP had a negative value (Table 1). In theory, more pronounced zeta potential values, either positive or negative, tend to stabilize particle suspension, since the electrostatic repulsion between particles with the same electric charge prevents aggregation of the spheres.69 The values shown in Table 1 are considered to be associated with a nonstable colloid (∼30 mV).70 The low variation in zeta potential can be explained by residual PVA that is still present on the surface of the particles even after three washings.60,71−73 On the other hand, the entrapment of the NO donor conduced to a positive zeta potential value; this positive zeta potential indicates that these NO donors may be located not only inside the NP but also on the external surface of the PLGA nanoparticle. Another problem inherent in size can arise due to the phenomenon of aggregation. Aggregation, which includes formation of dimers and higher-order aggregates, can dramatically affect the subcutaneous bioavailability and pharmacokinetics of a therapeutic drug.74 The zeta potential near zero obtained in our trial indicates the tendency of aggregation. As the concentration of the microspheres in the suspension increases, so does the likelihood of collisions and of the hydrophobic interactions which cause aggregation. Moreover this value of zeta may be due to the use of phosphate buffer. Ions from buffer salts in solution will bind to either positively or negatively charged microspheres, decreasing their surface charge.75 Similarly, divalent cations (with negatively charged microspheres) or divalent anions (with positively charged microspheres) can cause bridging between microspheres and hence aggregation.76 Nanoparticle Morphology: SEM Analysis. Figure 1 shows a representative SEM image of cyclam-NP used in this study. As can be seen, under SEM inspection, the nanoparticles had generally a spherical shape with a smooth surface with formation of aggregates. Similar results were observed also for Hedta-NP and empty nanoparticles. The aggregation process is favored by the low zeta potential, near to zero. Similar results were observed for trans-[Ru(NO)(NH3)4(py)]3+ using this method.51 Atomic Force Microscope (AFM) Imaging. The analysis of the surfaces of the nanoparticles was performed using atomic force microscopy. The image of a sample of cyclam-NP is shown in Figure 2. This study indicated that the surface was relatively smooth in the scale of observation and with spherical shape. The absence of pores in the surfaces of the nanoparticles indicated probably that the NO donors were stably associated

Figure 1. Scanning electron microscopy (SEM) of external morphology of nanoparticles loaded with trans-[Ru(NO)Cl(cyclam)](PF6)2 without light irradiation. The scale bar corresponds to 3.0 μM. Magnification 5000×.

Figure 2. Three-dimensional atomic force microscopy (AFM) images of surface of cylam-NP.

with the surface. Similar results were observed for Hedta-NP and empty-NP. Energy Dispersive Spectroscopic (EDS) Analysis. The EDS technique was used to confirm that the nitrosyl complexes are associated to the nanoparticles produced. The EDS spectra of cyclam-NP and Hedta-NP (Figure 1A and B, Supporting Information) showed the energy dispersive value of each element present in the NPs. The typical Ru energy value (2.55 keV)77 was found for the ruthenium nitrosyl complexes loaded in the PLGA nanoparticle, showing the entrapment of the complex. The EDS analyses show low percentages of ruthenium ( 0.05) was reached in a 1.0 × 10−3 M trans-[Ru(NO)Cl(cyclam)](PF6)2 solution in B16−F10 cells. A negligible decrease in cell viability was observed when B16−F10 cells were incubated with [Ru(NO)(Hedta)] in the dark (Figure 6 in the Supporting Information). The IC50 values against B16−F10 cells are 1.0, 6.8, and >10 mmol L−1 for trans-[Ru(NO)(NH3)4(py)]3+, trans-[Ru(NO)Cl(cyclam)]2+, and [Ru(NO)(Hedta)], respectively (Table 2), reflecting the rates of NO release from the complexes and the scavenging property of the [Ru(NO)(Hedta)] product.

32% for HEDTA); and (3) elevated ratio between polymer (500 mg) and complex (∼10 mg). The appearance of Na, Al, Si, S, K, Ti, and Zn peaks in the spectra is due to the SEM glass grid supporting the sample. This analysis showed that the complexes were entrapped into these nanoparticles. In Vitro Drug Release. The cumulative percentages of drug release from NP loaded with the ruthenium complexes are shown in Figure 3. The release kinetics of the drug from the

Table 2. IC50 Values of Complexes for B16−F10 Cell Lines

Figure 3. Release profiles of (○) trans-[Ru(NO)Cl(cyclam)](PF6)2 and (□) [Ru(NO)(Hedta)] loaded PLGA nanoparticles during 8 days using a 1.0 × 10−3 M pH 7.4 phosphate buffer.

nanoparticles 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 (Figure 3). A plateau is reached at 48 h when the concentration of NO released is ∼7.7 × 10−4 M for cyclam-NO and ∼7.5 × 10−4 M for Hedta-NO. After eight days these concentrations are ∼8.6 × 10−4 M and ∼9.1 × 10−4 M for cyclam-NO and Hedta-NO, respectively. It should be noted that the NO release was inferred indirectly. This behavior can be attributed to the burst effect in the initial stage of the release, where the initial drug release phase is primarily controlled by the NO-donors diffusion, which takes place due to the dissolution of NO donors molecules associated with the surface (i.e., molecules that are not entrapped but are adsorbed on the surface of the NP). The burst release stage is followed by a slow release of NO for up to 7 days, caused by hydrolytic degradation of the PLGA matrix. The release of NO donors from the PLGA nanoparticle was found to be slow, due to the low porosity of the NP (Figure 3). In this step, the complete release of NO donor complexes from the surface of the NP was observed. However, the NO donor complexes that were completely entrapped in the PLGA matrix could not be released until the polymer matrix started to lose its integrity, which is reported to be ∼15 days.78 A similar release profile was observed for the trans-[Ru(NO)(NH3)4(py)]3+ complex,51 so this behavior must be associated with the composition of the polymer matrix. Other authors observed comparable release behavior with regard to PLGA-NP.79−81 Toxicity Assay. Low dark toxicity is one of the important criteria for assessing the usefulness of compounds, since the major side effects in clinical therapy result from the dark toxicity of photosensitizers toward normal tissues. For in vitro cytotoxicity, all complexes and components that were used for the nanoparticles preparation were tested by the MTT assay, which was used to evaluate the cytotoxicity against B16−F10

complex

IC50

ref

trans-[Ru(NO)(NH3)4(py)](BF4)3 trans-[Ru(NO)Cl(cyclam)](PF6)2 [Ru(NO)(Hedta)] cisplatin

1.0 mmol L−1 6.8 mmol L−1 >10 mmol L−1 0.0074 mmol L−1

14, 38 14 14 14

Carboxy-PTIO was reported to react rapidly and irreversibly with NO in vitro82 with a rate constant of 1.0 × 104 M−1 s−1 and was often used as a NO scavenger in vivo in various tissues.70,83,84 In this case, the addition of 1.0 × 10−4 M carboxy-PTIO in the tests with trans-[Ru(NO)Cl(cyclam)]2+, [Ru(NO)(Hedta)], and trans-[Ru(NO)(NH3)4(py)](BF4)3· H2O, resulted in the absence of cell death without irradiation (Figure 6 in the Supporting Information). The empty-NP did not decrease the viability of cells. Also, the PLGA nanoparticles loaded with ruthenium nitrosyl complexes were nontoxic in the dark (Figure 7, Supporting Information), showing the level of cell viability of more than 95% even at the maximal incubation times used. According to the data obtained (Figure 7, Supporting Information), even after prolonged contact with the cells (2 h), the loaded PLGA nanoparticles were statistically nontoxic, as expected, because the complexes are entrapped in the particles and out of the reach of reducing agents. Similar behavior observed for PLGANP nanoparticles against human breast cancer MCF-7, prostate cancer LNCaP, and melanoma cancer B16−F10 cells was reported recently, in agreement with our results.51,85,86 The same profile was observed for PLGA-NP in the presence of carboxy-PTIO (Figure 7, Supporting Information). Phototoxicity Assay. In this assay, the NO donors (trans[Ru(NO)Cl(cyclam)](PF6)2 and [Ru(NO)(Hedta)]) in solution or the NO donors-loaded PLGA nanoparticles were incubated with B16−F10 cell lines for a definite period of time (2 h), and then cells were thoroughly washed with PBS to remove complexes non associated to the cell prior to photoirradiation. Then, photoirradiation (λ = 366 nm) was performed during 10 min, and cell viability was determined after the specified time period (48 h). Irradiation of B16−F10 cells treated with the NO donor in solution (nontoxic concentration 1.0 × 10−4 M), in the absence and presence of 1 × 10−4 M carboxy-PTIO, and with light irradiation (Figure 4A), resulted in a higher cell damage (53.8 3549



Article

Figure 4. Phototoxicity assay determined by incubation of B16−F10 cells (A) with 1.0 × 10−4 M cyclam-NO and 1.0 × 10−4 M Hedta-NO in solution, in the presence and absence of C-PTIO with light irradiation. (B) Empty-NP, 1.0 × 10−4 g/mL cyclam-NP, and 1.0 × 10−4 g/mL HedtaNP, in the presence and absence of C-PTIO with light irradiation. Control with/without C-PTIO stands for B16−F10 cells. Cells with empty nanoparticles with/without C-PTIO were used as nanoparticle control. Survival rate of the different batches of particles on cancer cells, MTT test (mean of eight wells per experimental set), is shown; *P < 0.05 in relation to both controls.

Figure 5. Scanning electron microscopy. (A) Control; (B) surface morphology of B16−F10 cells incubated with cyclam-NP (1000× magnification) without irradiation. (C) After light irradiation of B16−F10 cells incubated with trans-[Ru(NO)Cl(cyclam)](PF6)2 (15 000× magnification).

± 6.2%, n = 8 of cell death in the presence of trans[Ru(NO)Cl(cyclam)](PF6)2 and 22.3 ± 5.1%, n = 8 [Ru(NO)(Hedta)]) in relation to control (*P < 0.05). In the presence of C-PTIO no cell death was observed. In Figure 4B the phototoxicity of NO donors encapsulated in the absence and absence of 1.0 × 10−4 M C-PTIO is displayed. The results in terms of cell death showed that cyclam-NP produced 13.6 ± 3.6% of cell death and Hedta-NP 6.3 ± 4.2% upon light irradiation in relation to control (*P < 0.05). In the presence of C-PTIO no cell death was observed. Our results shows that upon light irradiation cell death occurs without carboxy-PTIO, but in its presence no cell death occurs with trans-[Ru(NO)Cl(cyclam)]2+ and [Ru(NO)(Hedta)] in solution, which means that the released NO is fully captured by carboxy-PTIO. These results confirm that the toxicity observed in B16−F10 cells is due to the release of nitric oxide from the NO donors. The addition of 1.0 × 10−4 M carboxy-PTIO in the tests with py-NP, cyclam-NP, and HedtaNP (Figure 4B) resulted in the absence of cell death before irradiation, as expected, and no cell death was observed upon

irradiation, which means that, as in solution, the photochemically released NO is rapidly scavenged by carboxy-PTIO. Our results presented here show that free trans-[Ru(NO)Cl(cyclam)](PF6)2 in solution, with a quantum yield of NO release, ϕNO, of 0.16,87 is slightly less phototoxic for melanoma cells than trans-[Ru(NO)(NH3)4(py)](BF4)3 (ϕNO = 0.18)33 (63.3% cell death). 51 Irradiation of trans-[Ru(NO)(NH3)4(py)]3+ and trans-[Ru(NO)Cl(cyclam)]2+ with light of 355 nm results in the respective aquoRu(III) complex and NO as the only products. On its side, [Ru(NO)(Hedta)] showed a lower cell death of 22.3 ± 5.1%, compatible with the lower quantum yield of release of NO from [Ru(NO)(Hedta)] (0.0002 ± 0.0001 (λir = 365 nm),88 and it was suggested that at pH 5.0, besides [Ru(H2O)(Hedta)] and NO, it may also have other products photochemically.89 Upon irradiation [Ru(NO)(Hedta)] releases NO forming the good NO scavenger27,29−31 [Ru(H2O)(Hedta)], which, as in the dark assay, reacts with NO forming back [Ru(NO)(Hedta)], with a high formation rate constant kf = 2.24 × 107 M−1 s−1 at pH 7.4, reducing the availability of NO in solution which induces cell death. A similar decrease in cell death is seen for trans-[Ru(NO)Cl3550



Article

(cyclam)](PF6)2 and trans-[Ru(NO)(NH3)4(py)](BF4)3 in the presence of the NO scavenger carboxi-PTIO (see below). The results indicated that the used PLGA preparations of NO donors provided a significant reduction of the phototoxicity in vitro, when compared with that of the complex in solution. When encapsulated, cyclam-NP produced 13.6 ± 3.6% of cell death and Hedta-NO 6.3 ± 4.2% upon light irradiation. The toxicity of both compounds was reduced in 74 and 72%, respectively, which is comparable to trans-[Ru(NO)(NH3)4(py)]3+.51 The smaller phototoxicity could be related to the nitrosyl complex being internalized into the polymeric matrix. This behavior makes the complexes less available to photostimulus once PLGA absorbs radiation in the same region. The fact that these complexes were localized inside the matrix makes them preserved to the action of reductases avoiding reduction and consequent undesirable premature NO release. In this context, ruthenium nitrosyl complex loaded PLGA nanoparticles could result in materials that may be used in association with light to provide controlled NO release at specific target sites, resulting in a useful tool to tumor cell death. Cell Morphology. Figure 5A (control) shows B16−F10 cells in the dark and in the absence of cyclam-NP and HedtaNP and shows that the particles are localized adhered in the cell surface. No significant morphological alteration was observed in the absence of light, indicating that this drug delivery system is adequate for use with NO donors. Figure 5B shows a micrograph of B16−F10 cells incubated with NPs for 2 h without irradiation; after this incubation time, it is observed a large quantity of nanoparticles adhered to the cell surface. The PLGA nanoparticles loaded with NO donors did not show any significant surface alteration that could induce cell death. This result was confirmed by the MTT assay showing no toxicity below than 1 × 10−4 M. Significant morphological changes were observed 48 h after exposure of tumor cells to NO donors and light irradiation (Figure 5C). The results indicate that the effect of the light irradiation on B16−F10 cells in the presence of cyclam-NO and Hedta-NO in solution or cyclam-NP and Hedta-NP results in NO release that leads to irreversible photodamage to the tumor cell with damage by rupture of the cell membrane, with loss of cytoplasmic material and cell death.

result of the entrapment of the complexes in the particles, nondiffusing out of the particle into the solution, and protecting them from environmental reductases that could promote an early release of NO. However, in the presence of light these drug delivery systems loaded with cyclam-NO exerted in vitro cytotoxic activity against cancer cells. Under light irradiation these embedded complexes are able to release sufficient NO that can diffuse out of the matrix, reach the adjacent cell membranes, and kill the tumor cell, but in the presence of carboxy-PTIO, cell death does not occur. The results show that the cytotoxicities observed with the NO donors in solution and embedded in the PLGA NPs are related to the release of NO. Control of the NO release at the target was achieved by using the PLGA NPs as DDS and by use of light irradiation. Further control can be achieved by focusing the light beam on the target, light intensity, and time of irradiation. Thus, the loaded NPs combined with photochemical delivery can serve as a model for controlled NO release at specific targets such as the skin tumor cells.

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ASSOCIATED CONTENT

* Supporting Information S

Syntheses of ruthenium complexes, energy-dispersive spectra, UV−vis spectra, morphological assay after irradiation, and toxicity assay details with figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

́ Facul*E-mail: [email protected]. Departamento de Quimica, dade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo Address: Av. Bandeirantes, 3900, Ribeirão Preto, SP Brazil 14040-901. Phone numbers: +55-1636023478; +55-16-36023877. Fax: +55-16-36024838. Author Contributions

All authors contributed equally. Notes

The authors declare no competing financial interest.

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CONCLUSIONS In this work, we reported a water/oil/water process to obtain ruthenium nitrosyl complexes effective loading into PLGA nanoparticles. These nanoparticles were spherical, polydisperse, with a mean diameter between 220 and 840 nm. Our results show that the NO donors trans-[RuCl(NO)(cyclam)]2+ and [Ru(NO)(Hedta)] can be entrapped with preservation of its molecular structure and properties. On the basis of these results, as well as the relatively good encapsulation efficiencies to hydrophilic drugs, the currently described nanoparticles might represent a promising formulation as anticancer agents under irradiation. The lower cytotoxicity of trans-[RuCl(cyclam)(NO)]2+ and [Ru(NO)(Hedta)] in solution compared to that of trans-[Ru(NO)(NH3)4py]3+ is consistent with the rate constant release of NO of these complexes, and with the fact that [Ru(Hedta)(H2O)], which is product of [Ru(NO)(Hedta)], is an excellent NO scavenger; the cytotoxicities are also inhibited in presence of the NO scavenger carboxy-PTIO. We showed that the PLGA nanoparticles loaded with ruthenium nitrosyl complex are nontoxic in the dark, compared to the complexes in solution. This is a

ACKNOWLEDGMENTS The authors thank the Brazilian agencies FAPESP, FAPDF, CNPq, and CAPES for financial support.

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ABBREVIATIONS Cyclam, 1,4,8,11-tetraazacyclotetradecane; Hedta, ethylenediaminetetraacetic acid; cyclam-NP, trans-[Ru(NO)Cl(cyclam)](PF6)2 entrapped in PLGA nanoparticles; HedtaNP, [Ru(NO)(Hedta)] entrapped in PLGA nanoparticles; PLGA, poly(D,L-lactic-co-glycolic) acid; NP, nanoparticles; SEM, scanning electron microscopy; DLS, dynamic light scattering; B16−F10, melanoma cancer cells; DDS, drug delivery system; NO, nitric oxide; k−NO = constant release of NO; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PVA, polyvinyl alcohol; PBS, phosphate buffer saline; PCS, photon correlation spectroscopy; AFM, atomic force microscopy; EDS, energy dispersive spectroscopic; carboxy-PTIO, [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide]; ϕNO, quantum yield of NO release; kf, formation rate constant 3551



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2 and [Ru(NO)(Hedta)] Incorporated in PLGA ... - ACS Publications

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