Antitumoral and Antimicrobial Activities of Block Copolymer Micelles Containing Gold Bisdithiolate Complexes

Gold(III) bisdithiolate complexes have been reported as potential antimicrobial and antitumoral agents. The complex [Au(cdc)2]− (cdc=cyanodithioimido carbonate) displayed antimicrobial and outstanding antitumor activity against the ovarian cancer cells A2780 and A2780cisR, which are sensitive and resistant to cisplatin, respectively. However, poor water solubility may hamper its clinical use. Block copolymer micelles (BCMs) may solubilize hydrophobic drugs, improving their bioavailability and circulation time in blood. Aiming to provide water solubility, prolonged availability, and enhanced therapeutic indexes, BCMs loaded with [Au(cdc)2]− were synthesized and characterized. The BCM-[Au(cdc)2] micelles were prepared with a loading efficiency of 64.6% and a loading content of 35.3 mg [Au(cdc)2]−/gBCM. A hydrodynamic diameter of 77.31 ± 27.00 nm and a low polydispersity index of 0.18 indicated that the micelles were homogenous and good candidates for drug delivery. Cytotoxic activity studies against A2780/A2780cisR cells showed that BCM-[Au(cdc)2] maintained relevant cytotoxic activity comparable to the cytotoxicity observed for the same concentration of gold complexes. The Au uptake in A2780 cells, determined by PIXE, was ca. 17% higher for BCMs-[Au(cdc)2] compared to [Au(cdc)2]−. The BCMs-[Au(cdc)2] presented antimicrobial activity against S. aureus Newman and C. glabrata CBS138. These results evidenced the potential of BCM-[Au(cdc)2] for drug delivery and its promising anticancer and antimicrobial activities.


Introduction
One of the main goals of nanomedicine is to develop targeting delivery systems with the ability to reach pathological sites in the body, such as tumors. This strategic approach allows healthy cells to be spared undesired effects, while drug effectiveness and pharmacokinetic profiles can be improved [1]. Nanocarriers, such as polymeric micelles, are appealing drug-delivery systems, particularly for anticancer drugs, due to the reduction in toxicity with the maintenance of therapeutic effects and biocompatibility [2]. These systems can overcome the drawbacks of therapeutic molecules and free drugs, including non-selective activity with respect to targeted tissues, poor water solubility, poor biodistribution and pharmacokinetics (PK), multidrug resistance, dose-limiting toxicity, and fast degradation in vivo [3,4].
gen-or sulphur-donor ligands have the ability to confer stability. On the other hand, their stability can be attained by the careful choice of appropriate inert ligands, such as polydentate ligands with sulphur, oxygen, or nitrogen as electron-donor atoms [21].
The present study reports on the recent advances with TBA[Au(cdc)2] ( Figure 1) as a new chemotherapeutic drug-delivery agent. This complex has shown important therapeutic properties but exhibits poor solubility in water, like many other chemotherapeutic drugs. The association of these complexes with nanosized carriers, such as polymers and micelles, could improve their solubility, pharmacokinetics, and in vivo circulation times, resulting in increased drug accumulation based on the EPR (enhanced permeability and retention) effect [27,31].

Materials and Methods
All chemicals and solvents were of reagent grade and used without further purification, unless otherwise stated. Toluene, obtained from Fisher Chemical (Waltham, MA, USA), was dried by distillation with sodium. Dichloromethane was dried with phosphorus pentoxide. Hydrogen chloride solution 2M in diethyl ether, poly(ethylene glycol) methyl ether (Me-PEG, Mn = 5000), and Ɛ-caprolactone (CL) were all acquired from Sigma-Aldrich. Poly(ethylene glycol) methyl ether (Me-PEG, Mn = 5000) was dried twice by azeotropic distillation in toluene, which was distilled off completely, while Ɛ-caprolactone (CL) was dried using calcium hydride and was distilled prior to use. The gold compound TBA[Au(cdc)2] was prepared as tetrabutyl ammonium (TBA) salt according to a previously described method [32].
The copolymer methoxy-terminated poly(ethylene glycol)-b-poly(ε-caprolactone) (Me-PEG-b-PCL) was synthesized and characterized following reported synthetic methodologies [33,34] using metal-free cationic ring-opening polymerization of ε-caprolactone (ε-CL) via an activated monomer mechanism with HCl-diethyl ether. UV-Vis spectrophotometry was performed on a Cary 60 UV-Vis spectrophotometer from Agilent Technologies with quartz cuvettes (QS high Precision Cell; 10 mm (Hellma ® Analytics, Jena, Germany)), and TBA[Au(cdc)2] was quantified with reference to a calibration curve. However, these complexes have poor solubility in water, which poses some challenges regarding their in vivo administration. The association of these complexes with nanosized carriers, such as micelles, could circumvent the solubility issues and also reduce liganddisplacement reactions with blood proteins [12,[27][28][29][30].
The present study reports on the recent advances with TBA[Au(cdc) 2 ] ( Figure 1) as a new chemotherapeutic drug-delivery agent. This complex has shown important therapeutic properties but exhibits poor solubility in water, like many other chemotherapeutic drugs. The association of these complexes with nanosized carriers, such as polymers and micelles, could improve their solubility, pharmacokinetics, and in vivo circulation times, resulting in increased drug accumulation based on the EPR (enhanced permeability and retention) effect [27,31].

Materials and Methods
All chemicals and solvents were of reagent grade and used without further purification, unless otherwise stated. Toluene, obtained from Fisher Chemical (Waltham, MA, USA), was dried by distillation with sodium. Dichloromethane was dried with phosphorus pentoxide. Hydrogen chloride solution 2M in diethyl ether, poly(ethylene glycol) methyl ether (Me-PEG, Mn = 5000), and E-caprolactone (CL) were all acquired from Sigma-Aldrich. Poly(ethylene glycol) methyl ether (Me-PEG, Mn = 5000) was dried twice by azeotropic distillation in toluene, which was distilled off completely, while E-caprolactone (CL) was dried using calcium hydride and was distilled prior to use. The gold compound TBA[Au(cdc) 2 ] was prepared as tetrabutyl ammonium (TBA) salt according to a previously described method [32].

Preparation and Characterization of BCMs
BCMs loaded with [Au(cdc) 2 ] − were prepared by the thin-film hydration method [33,35]. The polymer (50 mg), Me-PEG-b-PCL, and [Au(cdc) 2 ] − (2-8 mg) were dissolved in CHCl 3 (4 mL) under constant stirring for 4 h at atmospheric pressure and room temperature (RT). Then, the solvent was slowly evaporated overnight under N 2 flux to form the [Au(cdc) 2 ] − / Me-PEG-b-PCL thin film, which was hydrated at 60 • C with H 2 O (or PBS) (1 mL) and stirred (low velocity) for 4 h at RT. After the hydration, the solution was centrifuged for 10 min at 1000× g, and the supernatant was filtered with a SARTORIUS filter (0.20 µm). The last step of this process was the lyophilization of the micelles. Unloaded micelles were prepared using the same procedure, in the absence of the gold complex ( Figure 2).
(B). The eluents were of HPLC grade, and the aqueous solutions were prepared with ultrapure MilliQ water.

Preparation and Characterization of BCMs
BCMs loaded with [Au(cdc)2] − were prepared by the thin-film hydration method [33,35]. The polymer (50 mg), Me-PEG-b-PCL, and [Au(cdc)2] − (2-8 mg) were dissolved in CHCl3 (4 mL) under constant stirring for 4 h at atmospheric pressure and room temperature (RT). Then, the solvent was slowly evaporated overnight under N2 flux to form the [Au(cdc)2] − / Me-PEG-b-PCL thin film, which was hydrated at 60 °C with H2O (or PBS) (1 mL) and stirred (low velocity) for 4 h at RT. After the hydration, the solution was centrifuged for 10 min at 1000× g, and the supernatant was filtered with a SARTORIUS filter (0.20 µ m). The last step of this process was the lyophilization of the micelles. Unloaded micelles were prepared using the same procedure, in the absence of the gold complex ( Figure 2).

Drug Loading Content and Efficiency
The drug loading content (LC) was determined by UV-Vis spectrophotometry (Figure 3) with reference to a standard calibration curve ( Figure 3b). For this, 2-4 mg of BCM-Au(cdc)2 was dissolved in 1.0 mL of acetonitrile, vortexed and centrifuged at 3000× g for 10 min to precipitate the copolymer. The supernatant was then collected and analyzed by UV-Vis spectroscopy. The The calculation of drug loading efficiency (LE) can be determined using Equation (2):

Drug Loading Content and Efficiency
The drug loading content (LC) was determined by UV-Vis spectrophotometry ( Figure 3) with reference to a standard calibration curve ( Figure 3b). For this, 2-4 mg of BCM-Au(cdc) 2 was dissolved in 1.0 mL of acetonitrile, vortexed and centrifuged at 3000× g for 10 min to precipitate the copolymer. The supernatant was then collected and analyzed by UV-Vis spectroscopy.

Sizes and Zeta Potentials of Micelles
The zeta potentials and hydrodynamic diameters (dhs) of the micelles (0.1 g/L) were determined in 0.01 M phosphate buffer (PB) pH 7.4, using a Zetasizer Nano ZS obtained from Malvern with zeta-potential cells. Particle size was measured three times by dynamic light scattering (DLS) at 25 °C with a 173° scattering angle and an optical arrangement known as non-invasive back scatter (NIBS).
At the beginning of this procedure, the micelles were dissolved in 0.01 M phosphate buffer, pH 7.4 (PB) in order to obtain 1.0 g/L solutions that were subsequently sonicated for 20 min before use. Afterwards, the solutions were diluted and filtered using a 0.20 µ m syringe filter.

Release Study
The in vitro release of [Au(cdc)2] − from BCM-Au(cdc)2 was evaluated at pH 7.4 using the dialysis method [36,37]. Briefly, a 3 mg solution of BCM-Au(cdc)2 in 3 mL of 0.01 M phosphate-buffered saline (PBS) pH 7.4 was placed in a regenerated cellulose tubular dialysis membrane (MWCO = 25 kDa), immersed in 200 mL of 0.01 M PBS pH 7.4, and maintained at 37 °C under continuous stirring. At predetermined time points, 500 µ L of the solution inside the dialysis membrane was retrieved and lyophilized, and the membrane was immersed in fresh medium. Afterwards, 500 µ L of acetonitrile was added to the retrieved solutions, and the resultant solutions were vortexed and centrifuged at 3000× g for 10 min to precipitate the copolymer and the PBS salts. The supernatant was collected and analyzed by UV-vis spectrophotometry. The drug-release profile was calculated as the cumulative percentage of released [Au(cdc)2] − over time, and 100% release corresponds to the total amount of [Au(cdc)2] − entrapped in the micelles.

Sizes and Zeta Potentials of Micelles
The zeta potentials and hydrodynamic diameters (d h s) of the micelles (0.1 g/L) were determined in 0.01 M phosphate buffer (PB) pH 7.4, using a Zetasizer Nano ZS obtained from Malvern with zeta-potential cells. Particle size was measured three times by dynamic light scattering (DLS) at 25 • C with a 173 • scattering angle and an optical arrangement known as non-invasive back scatter (NIBS).
At the beginning of this procedure, the micelles were dissolved in 0.01 M phosphate buffer, pH 7.4 (PB) in order to obtain 1.0 g/L solutions that were subsequently sonicated for 20 min before use. Afterwards, the solutions were diluted and filtered using a 0.20 µm syringe filter.

Release Study
The in vitro release of [Au(cdc) 2 ] − from BCM-Au(cdc) 2 was evaluated at pH 7.4 using the dialysis method [36,37]. Briefly, a 3 mg solution of BCM-Au(cdc) 2 in 3 mL of 0.01 M phosphate-buffered saline (PBS) pH 7.4 was placed in a regenerated cellulose tubular dialysis membrane (MWCO = 25 kDa), immersed in 200 mL of 0.01 M PBS pH 7.4, and maintained at 37 • C under continuous stirring. At predetermined time points, 500 µL of the solution inside the dialysis membrane was retrieved and lyophilized, and the membrane was immersed in fresh medium. Afterwards, 500 µL of acetonitrile was added to the retrieved solutions, and the resultant solutions were vortexed and centrifuged at 3000× g for 10 min to precipitate the copolymer and the PBS salts. The supernatant was collected and analyzed by UV-vis spectrophotometry. The drug-release profile was calculated as the cumulative percentage of released [Au(cdc) 2 ] − over time, and 100% release corresponds to the total amount of [Au(cdc) 2 ] − entrapped in the micelles.

Cells and Cell-Culture Media
A2780 (cisplatin-sensitive) and A2780cisR (cisplatin-resistant) ovarian tumor cells were purchased from Sigma-Aldrich. V79 (hamster lung fibroblasts) were purchased from the ATCC (American Type Culture Collection). Cell media and media supplements were purchased from Gibco (Thermo Fisher Scientific). All cell lines were grown in RPMI medium (Gibco) supplemented with 10% FBS (Gibco) and maintained in a humidified incubator (Heraeus, Hanau, Germany) with 5% CO 2 .

Determination of Cytotoxic Activity
The cytotoxic activities of BCMs, BCMs-[Au(cdc) 2 ] − , and [Au(cdc) 2 ] − were evaluated with the matched pair of cisplatin-sensitive/-resistant A2780 cell lines (A2780/A2780cisR) and in normal fibroblasts (V79), using the MTT assay, as previously described [25]. For the assays, cells (1-2 × 10 4 cells/200 µL medium) were seeded in 96-well plates and allowed to adhere for 24 h. Loaded BCMs (drug loading content: 3.56%) were diluted to prepare serial concentrations in the range of 10 ng/mL-2 g/mL-concentrations that correspond to 10 −7 -10 −4 M of [Au(cdc) 2 ] − . Unloaded BCMs were diluted in medium to prepare serial dilutions in the range of 10 ng/mL-2 g/mL. Loaded and unloaded BCMs were added to the cells and incubated for 48 h at 37 • C. At the end of the treatment, the MTT assay was used, following a procedure similar to one previously described [25].

Cellular Uptake Analysis
The concentration of gold in A2780 cell pellets after incubation with [Au(cdc) 2 ] − or BCM-[Au(cdc) 2 ] was determined by particle-induced X-ray emission (PIXE), installed at the Van de Graaff accelerator of the Centro Tecnológico e Nuclear, Instituto Superior Técnico. A2780 cells were incubated with [Au(cdc) 2 ] − and BCM-[Au(cdc) 2 ] at 9.0 µM, and the IC 50 values were determined after 3 h incubation. The cell pellets were obtained by centrifugation after washing of the cells with PBS to remove the medium. The samples were freeze-dried and microwave-assisted acid-digested in aqua regia with yttrium as an internal standard, as previously described [26]. The concentrations of Au in the cell pellets were obtained in µg/g dry weight and converted to ng/10 6 cells.

Antimicrobial Activities of BCMs
The antimicrobial activities of the BCMs and BCMs-Au(cdc) 2 towards S. aureus Newman and the pathogenic fungi Candida glabrata CBS138 were assessed by the determination of minimum inhibitory concentrations (MICs) using microdilution assays, as previously described [25] and according to EUCAST (European Committee on Antimicrobial Susceptibility Testing) recommendations [38,39]. Both strains were isolated from human infections [40,41]. S. aureus Newman was maintained in Lennox Broth (LB) solid medium, composed of 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, and 20 g/L agar. C. glabrata CBS138 was maintained in yeast extract-peptone-dextrose (YPD) solid medium (20 g/L glucose, 20 g/L peptone, 10 g/L yeast extract, and 15 g/L agar).
Briefly Then, for S. aureus, 100 µL aliquots of adequately diluted bacterial suspensions were mixed with the MH broth serially diluted micelle aliquots to obtain 5 × 10 5 CFU/mL. Bacterial suspensions were prepared from cultures grown for 5 h in MH broth at 37 • C and 250 rev·min −1 and adequately diluted with fresh MH broth. After 22 h of incubation at 37 • C, the well contents were resuspended by pipetting, and the optical densities were measured in a SPECTROstar Nano microplate reader (BMG Labtech) at 640 nm.
C. glabrata overnight-grown fungal cultures (carried out in YPD broth at 30 • C and 250 rev. min −1 ) were diluted with fresh RPMI-1640 liquid medium to a final optical density of 0.025, measured at 530 nm (OD 530 ) using a Hitachi U-2000 UV/Vis spectrophotometer. The wells were then inoculated with the addition of 100 µL of fungal suspensions and incubated for 24 h at 35 • C. After incubation, the wells were examined for turbidity (growth) and resuspended, and their optical densities were measured using a SPECTROstar Nano microplate reader (BMG Labtech) at 530 nm.
At least two independent experiments were performed in duplicate for each micelle preparation under study. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of the antimicrobial that inhibited the visible growth of a microorganism after the incubation time. Positive (no micelles) and negative controls (no inoculum) were performed for each experiment. Micelles at the same concentrations used in the assay without inoculum were also tested, as negative controls.

Synthesis and Characterization of Block Copolymer Micelles
The block copolymer micelles (BCMs) prepared in this work were synthesized according to the thin-film hydration method [33,35]. The [Au(cdc) 2 ] − loaded and non-loaded micelles, BCMs-Au(cdc) 2 and BCMs, were formed by self-assembly using Me-PEG-b-PCL (Figure 2), an amphiphilic polymer with polyethylene glycol (PEG) as the hydrophilic chain and polycaprolactone (PCL) as the hydrophobic chain. Due to the low water solubility of [Au(cdc) 2 ] − , the gold complexes are encapsulated inside the micelles when the self-assembly of the polymer occurs in aqueous solution.
To ensure an optimal loading content of the [Au(cdc) 2 ] − gold complexes in the BCMs, various parameters were evaluated. In the first part of the optimization process, the influences of different types of solvents used for the dissolution of the polymer and the gold complex and for the hydration of the thin film were studied (Table 1). For the dissolution of the polymer and [Au(cdc) 2 ] − , CHCl 3 , ACN, or a mixture of DMF with DCM were used, while hydration was evaluated using H 2 O or PBS. The amounts of [Au(cdc) 2 ] − and polymer used were maintained at 2 mg and 25 mg, respectively. In addition, volumes of solvents for the dissolution ranged between 2 and 4 mL, and 1 mL for the solvent was used in the hydration of the thin film. In order to assess the loading contents of the resulting BCMs-[Au(cdc) 2 ] (formulations AS1-AS5), known amounts of the micelles were disassembled using acetonitrile followed by UV-Vis analysis of the solubilized contents ( Figure 3). The results were compared with a standard calibration curve (Figure 3b) previously obtained using solutions of known concentrations of [Au(cdc) 2 ] − , with values for the maximum absorbance of the gold complexes acquired at 303 nm (Figure 3a).
The results indicated a clear influence of the type of solvent used on LC, both for the polymer and gold-complex solubilization, as well as for the thin-film hydration, as can be seen in Table 2. Using ACN as a solubilization agent led to the lowest [Au(cdc) 2 ] − LC results; in fact, in the case of formulation AS2, no gold-complex encapsulation was obtained. In contrast, CHCl 3 led to the highest amount of loaded [Au(cdc) 2 ] − , as evidenced by the results obtained for formulations AS4 and AS5. Regarding the thin-film hydration solvent used, a higher loading was obtained using H 2 O compared to PBS. Water led to the micelle formulation AS4 standing out as the one with the highest [Au(cdc) 2 ] − loading content (LC (mg [Au(cdc)2]−/ g BCM ) = 5.68). The variations in LCs obtained using water or PBS were not as significant as those obtained with the change of the polymer and the gold-complex solubilization solvent. Aiming at the optimization of the [Au(cdc) 2 ] − loading process, we then studied the impact of the drug/copolymer ratio. For this purpose, we used a constant amount of copolymer (50 mg) and amounts of gold complexes in the range of 1-8 mg (Table 3). The results presented in Table 3 show that the drug loading contents and efficiencies were higher for lower quantities of [Au(cdc) 2 ] − , with the highest loading content being found for the micelle formulation AS6 (LC = 35.29 mg [Au(cdc)2] − /g BCM ). The structural integrity of the gold complex [Au(cdc) 2 ] − upon encapsulation in the micelles was investigated by high-performance liquid chromatography (HPLC) analysis. After disassembly of the loaded micelles, the supernatants were analyzed by HPLC and the results were compared to those of the gold complexes before encapsulation (Figure 4). The HPLC chromatograms show that the free gold complexes and the gold complexes collected from the loaded micelles exhibited similar profiles, with single peaks at similar retention times, suggesting that the gold complexes maintained their chemical structures unaltered.
The drug loading content reported for auranofin-loaded nanoparticles was in the range of 1.8-6.2 mg of auranofin per gram of NPs [42] as compared to 35.29 mg of [Au(cdc) 2 ] − per gram of micelles for formulation AS6. These results indicate that [Au(cdc) 2 ] − is encapsulated more efficiently than auranofin using this optimized formulation, which is a remarkable result for these newly developed BCMs.
The hydrodynamic diameters (d h s) and the zeta potentials (Z p s) of the micelles were determined by DLS and LDV, respectively. After sample dilution with PBS (pH = 7.4, 0.01 M), measurements were carried out, and the values obtained for each sample are presented in Table 4. This study was conducted for the micelle formulations AS1, AS4, and AS6.
The morphology of the micelles (BCMs and BCMs-[Au(cdc) 2 ]) was determined by transmission electron microscopy (TEM) ( Figure S2).  The drug loading content reported for auranofin-loaded nanoparticles was in the range of 1.8-6.2 mg of auranofin per gram of NPs [42] as compared to 35.29 mg of [Au(cdc)2] − per gram of micelles for formulation AS6. These results indicate that [Au(cdc)2] − is encapsulated more efficiently than auranofin using this optimized formulation, which is a remarkable result for these newly developed BCMs.
The hydrodynamic diameters (dhs) and the zeta potentials (Zps) of the micelles were determined by DLS and LDV, respectively. After sample dilution with PBS (pH = 7.4, 0.01 M), measurements were carried out, and the values obtained for each sample are presented in Table 4. This study was conducted for the micelle formulations AS1, AS4, and AS6.
The morphology of the micelles (BCMs and BCMs-[Au(cdc)2]) was determined by transmission electron microscopy (TEM) ( Figure S2). For a long circulation half-life, polymeric micelles should have a hydrodynamic diameter in the range of 10-100 nm [4]. The micelles studied herein exhibited hydrodynamic diameters within this range. However, the differences in the LCs for samples A1, A4, and A6 did not translate into significant variations in dh values. This may have been due to the planar molecular geometries of the gold complexes, which, at higher concentrations, may force the complexes into a uniform stacking, instead of a random spatial orientation, favourable at lower concentrations. Nevertheless, the results also showed that the hydrodynamic diameters (dhs) were higher for micelles with higher loading contents (77.31 ± 27.00 nm for AS6). The polydispersity indexes (PdIs) suggest that the samples were homogeneous, with values comparable to those for other micelles synthesized from the copolymer PEG-b-PCL [33,43,44].  For a long circulation half-life, polymeric micelles should have a hydrodynamic diameter in the range of 10-100 nm [4]. The micelles studied herein exhibited hydrodynamic diameters within this range. However, the differences in the LCs for samples A1, A4, and A6 did not translate into significant variations in d h values. This may have been due to the planar molecular geometries of the gold complexes, which, at higher concentrations, may force the complexes into a uniform stacking, instead of a random spatial orientation, favourable at lower concentrations. Nevertheless, the results also showed that the hydrodynamic diameters (d h s) were higher for micelles with higher loading contents (77.31 ± 27.00 nm for AS6). The polydispersity indexes (PdIs) suggest that the samples were homogeneous, with values comparable to those for other micelles synthesized from the copolymer PEG-b-PCL [33,43,44].
Absolute zeta potential (Z p ) values of >30 mV are required for full electrostatic stabilization. Z p values within the range of 5-15 mV are in the region of limited flocculation, while micelles with Z p values lower than 3 mV will exhibit a maximal tendency to flocculate. Hence, particle aggregation is less expected to occur for charged particles (high Z p ) due to electric repulsion. The micelles studied exhibited absolute Z p values > 50 mV, indicative of their suitable stability in aqueous media and low tendency for aggregation.
In vitro release studies were performed at different time points using the AS6 micelles at pH 7.4 and 37 • C, using the dialysis method [36,37]. The results obtained ( Figure 5) showed a steady and controlled release of the gold complexes from the micelles. At 24 h post incubation, about 89% of the [Au(cdc) 2 ] − was already released from the BCM structures.
due to electric repulsion. The micelles studied exhibited absolute Zp values > 50 mV, indicative of their suitable stability in aqueous media and low tendency for aggregation.
In vitro release studies were performed at different time points using the AS6 micelles at pH 7.4 and 37 °C, using the dialysis method [36,37]. The results obtained ( Figure  5) showed a steady and controlled release of the gold complexes from the micelles. At 24 h post incubation, about 89% of the [Au(cdc)2] − was already released from the BCM structures.

Biological Studies
Given the highest LC observed for AS6, as well as the suitable dh and Zp values displayed by this formulation, these micelles were selected for in vitro biological studies. Hence, the results reported below are based on the AS6 BCMs.

Cytotoxic Activity
The gold complex [Au(cdc)2] − was previously shown to exhibit a remarkable antiproliferative activity towards A2780 ovarian cancer cells [25]. Herein, we evaluated the retention of [Au(cdc)2] − activity upon encapsulation in the BCMs. The MTT assay was used to compare the activities of [Au(cdc)2] − and BCM-Au(cdc)2 in the ovarian cancer cells A2780 and A2780cisR and normal V79 fibroblasts (Table 5, Figure S1). Unloaded BCMs were also included as controls in order to confirm whether the cytotoxic activity exhibited by BCM-[Au(cdc)2] was due to the gold complexes and not the unloaded micelles ( Figure 6).

Biological Studies
Given the highest LC observed for AS6, as well as the suitable d h and Z p values displayed by this formulation, these micelles were selected for in vitro biological studies. Hence, the results reported below are based on the AS6 BCMs.

Cytotoxic Activity
The gold complex [Au(cdc) 2 ] − was previously shown to exhibit a remarkable antiproliferative activity towards A2780 ovarian cancer cells [25]. Herein, we evaluated the retention of [Au(cdc) 2 ] − activity upon encapsulation in the BCMs. The MTT assay was used to compare the activities of [Au(cdc) 2 ] − and BCM-Au(cdc) 2 in the ovarian cancer cells A2780 and A2780cisR and normal V79 fibroblasts (Table 5, Figure S1). Unloaded BCMs were also included as controls in order to confirm whether the cytotoxic activity exhibited by BCM-[Au(cdc) 2 ] was due to the gold complexes and not the unloaded micelles ( Figure 6).  The results presented in Table 5 show   The results presented in Table 5 83 vs. 1.69), as well as the selectivity index (SI) values for both compounds in these cells (SI > 2), suggest that these compounds are promising therapeutic agents for ovarian cancer and better candidates when compared with auranofin, the reference drug, which has a lower SI value (<2) [25,45].
Additionally, the BCMs without encapsulated gold complexes ( Figure 6) displayed a ca. 30% loss of cellular viability only at the highest concentration of 2 mg/mL.
Overall, these results show that [Au(cdc) 2 ] − is still able to maintain its cytotoxic activity after micelle encapsulation. Moreover, the results are also in agreement with others found for the reference drug auranofin. In fact, this gold(I) complex encapsulated in a micellar form (a block copolymer system) displayed a similar activity against ovarian cancer cells (OVCAR3) when compared with the complex itself. Using this micellar platform, the instability of auranofin was overcome due to the presence of protein thiols and their unspecific toxicity [30].

Quantification of Cellular Uptake of Free and Encapsulated [Au(cdc) 2 ] − by PIXE
Since it is assumed that the therapeutic effect could depend on the amount of gold complexes within cells, the net uptake of both [Au(cdc) 2 ] − and BCM-[Au(cdc) 2 ] in whole A2780 cells was evaluated using the PIXE technique (Table 6). Table 6. Cellular uptake of Au in whole A2780 cells as determined by PIXE. Cells were previously incubated with the compounds at 9 µM for 3 h. The Au levels in the whole cells are expressed as ng Au/10 6 cells. For these studies, the IC 50 values at shorter incubation times (3 h) were determined as part of a compromise to ensure that the compounds entered the cells and deposited measurable Au levels. The PIXE technique offers advantages in assessing Au contents in cell extracts due to its high sensitivity (in the ppm range) and accuracy in determining elemental concentrations, even with small sample masses. The estimated detection limit for Au < 30 µg/g dry weight was two orders of magnitude below the Au concentration in the sample, and the precision of Au determination was below 5%.

Compound
The results presented in Table 6 were obtained after incubating the cells for 3 h with the compounds at their IC 50 values. The Au uptake by A2780 treated cells was ca. 17% higher for BCM-[Au(cdc) 2 ] compared to [Au(cdc) 2 ] − , which indicates that the rate of uptake of the loaded micelles was faster.

Antimicrobial Activities
The antimicrobial properties of the BCMs and BCMs-[Au(cdc) 2 ] − were assessed based on the determination of the MIC values with respect to the Gram-positive bacteria S. aureus Newman and the yeast C. glabrata CBS138, using the microdilution method (Table 7). The BCMs-[Au(cdc) 2 ] − presented antimicrobial activity against S. aureus Newman and C. glabrata CBS138. As expected, BCMs not loaded with [Au(cdc) 2 ] − had no antimicrobial activity.

Conclusions
Polymeric micelles have emerged as alternatives to deliver therapeutic drugs more selectively, improve bioavailability, enhance drug release, and maintain therapeutic drug plasma levels, with reduced side effects. In addition, BCMs are able to increase drug solubility and stability and control delivery rates. In particular, block copolymer micelles have demonstrated great potential as nanocarriers capable of delivering hydrophobic drugs and controlling their distribution and function. The formulation of such delivery nanocarriers requires reliable characterization and evaluation of their efficacy at a biological level. This paper outlines our recent studies on block copolymer micelles to deliver prospective metal-based complexes with antitumoral and antimicrobial activities.
To sum up, BCMs prepared with Me-PEG-b-PCL chains, loaded and non-loaded with gold bisdithiolate complexes ([Au(cdc) 2 ] − ), were successfully synthesized. The hydrodynamic diameters were below 100 nm, and the zeta potential values were indicative of high stability and a low tendency to form aggregates. The synthesis of the micelles was optimized to achieve a high loading content of [Au(cdc) 2 ] − (35.29 mg[Au(cdc) 2 ] − /gBCM). Additionally, HPLC analysis and UV-vis spectrophotometry data indicated that the gold complexes maintained their stability after encapsulation in the micelles. The loaded micelles displayed significant cytotoxic activity towards the ovarian pair A2780/A2780cisR and equivalent activity for both cell lines. Compared with the reference drug auranofin, the BCMs-[Au(cdc) 2 ] − displayed a better profile in terms of general cytotoxicity, i.e., a higher SI, when cancer cells were compared with normal fibroblasts. The results showed that, after micelle encapsulation, [Au(cdc) 2 ] − was still able to maintain its cytotoxic activity against ovarian cancer cells and its antimicrobial activity against S. aureus Newman and glabrata CBS138.
Overall, the results obtained evidenced the potential of BCM-[Au(cdc) 2 ] as a novel drug-delivery system with promising anticancer and antimicrobial activities that deserve to be further evaluated.