1. Introduction
Cancer is one of the main diseases of this century [
1]. Given the existence of numerous types of tumors, different possible therapies are envisaged, depending on the stage of progress of the pathology [
2,
3,
4].
In the drug delivery field, the need for new more effective therapies has led to scientific progress mainly in two directions: the development of new active molecules and the development of innovative systems for drug administration [
5,
6,
7,
8,
9,
10,
11].
Combining these two approaches, the present study proposes a highly innovative material with synthetic antitumoral gold-based complexes, developing a cutting-edge wearable patch-like device that demonstrates excellent performance against melanoma.
The use of metal-based molecules for cancer treatment is widely accepted, from cisplatin to titanocene dichloride, and they are generally based on Pt, Au, Ag, Rh, etc. [
12]. The complexes selected for this study are based on gold. Gold has been used since ancient times to treat various diseases. The first scientific evidence on the therapeutic efficacy of gold dates back to the 1920s and relates to the compound K[Au(CN)
2], whose antituberculous activity has been clinically tested [
13]. Currently, Auranofin (1-thio-β-D-glucopyranosatotriethylphosphine gold-2,3,4,6-tetraacetate) is used in the treatment of rheumatoid arthritis and has also been tested in experiments carried out to evaluate its antitumor activity, giving important results [
14]. Its target is mainly represented by the mitochondrial enzyme thioredoxin reductase (TrxR) [
15]. Gold complexes with N-heterocyclic carbene (NHC) ligands have the ability to potently inhibit TrxR and decrease tumor cell proliferation [
16,
17]. Furthermore, they interfere with the metabolism of tubulin and/or actin and therefore play an important role in regulating the dynamics of the cytoskeleton [
18,
19,
20,
21,
22]. Previously, two NHC-ligated gold-based molecules, namely AuL20 and AuM1 (
Figure 1) [
19,
22], were identified, which showed remarkable antitumor activity.
In the present study, the applicability of Au-complexes to promote topical drug delivery is investigated. Different percentages of the two complexes were loaded in electrospun polymeric matrices. Membrane’s design and production are tailored to make them easily applied for topical treatments, providing an efficient release directly in the zone needing for the treatment.
In particular, electrospun membranes produced were tested against human malignant melanoma, the MeWo cell line, evaluating their cytotoxicity. MeWo represents one of the most aggressive forms of melanoma tumor; these cells are harvested from a metastatic site in the lymph node tissue. No effective treatment for metastatic melanoma exists, hence currently, an intense effort for new drug evaluation is strongly desirable [
23].
Among the numerous techniques able to produce transdermal drug delivery systems, electrospinning was chosen, mainly for two reasons. On the one hand, for the possibility of producing films composed of small-sized polymer fibers (50–5000 nm), that can well mimic the human tissue and, hence, is also particularly suitable for wound dressing. In this perspective, these patches could be easily applied, for example, before or after surgery, using them for topic chemotherapy and protection of the treated areas [
24]. On the other hand, the electrospinning process is suitable for processing biodegradable and biocompatible materials in several configurations [
25], and is ideal for materials applicable in the biomedical field [
26]. In this study, polycaprolactone (PCL) was chosen, since it allows for obtaining a mat composed of fibers with reduced average dimensions, biocompatibility, biodegradability and good mechanical performance [
27,
28]. PCL-based electrospun membranes were successfully functionalized with different types of filler, from active molecules to nanoparticles [
29,
30,
31,
32,
33], and tested against other aggressive skin cancer [
34,
35].
In the current paper, PCL electrospun membranes, loaded with different percentages of AuM1 and AuL20, are produced, and the process conditions are described. The cytotoxicity of the complexes alone (free) and the functionalized membranes are tested against MeWo cells. By Field Emission Scanning Electron Microscopy (FESEM) and Atomic Force Microscopy (AFM) analyses, the net morphology, the fiber dimensions, and the distribution of the complexes in the nanofibers are studied. Eventually, the obtained release profiles are modeled.
2. Experimental Section
2.1. Materials
Poly(ε-caprolactone) (PCL-CAS N° 24980-41-4; Molecular weight 80,000 Da) was purchased by Perstorp (Warrington, UK). Dimethylformamide (DMF-CAS 68-12-2) was purchased from Sigma Aldrich (Burlington, VT, USA). Acetone was purchased from Aldrich Chemical Corporation (St. Louis, MO, USA). Chloroform was purchased from Carlo Erba (Cornaredo, Italy). Phosphate buffered saline (PBS) (pH 7.3) was purchased by Oxoid (Basingstoke, UK).
Au-complexes were prepared according to the procedure reported by Mariconda et al. [
36]. AuL20 was obtained using styrene-oxide, which by the opening of the epoxy-ring, reacts with imidazole giving the monoalkylated product N-methyl, N-[(2-hydoxy-2-phenyl) ethyl]-imidazolium iodide. The second nitrogen atom is methylated using CH
3I, producing the racemic mixture of the salt. It was reacted with silver oxide (Ag
2O) in an inert nitrogen atmosphere. In these conditions, the silver oxide deprotonates the cationic carbon, giving the corresponding Ag–NHC complex.
The silver complex is reacted with chloro-(dimethylsulfide)-gold(I) [(CH
3)
2SAuCl] in dichloromethane. The reaction was left for 1 h at room temperature, then the mixture was filtered, and the solvent removed in vacuo. The obtained solid AuL20 was characterized by
1H NMR and
13C NMR, mass spectrometry, and elemental analysis. The same procedure was applied in the synthesis of AuM1 using 4,5-dichloroimidazole instead of imidazole. Both complexes were obtained in the form of solid powders. The complete analysis (
1H NMR,
13C NMR, ESI-MS, CHN) of the synthesized complexes is provided in the
Supplementary Materials (Figures S1–S6).
The analysis of the hydrolytic stability of the complexes, carried out at 40 °C in an aqueous solution of DMSO-d6 at 10% on these complexes, by means of
1H NMR spectroscopy, demonstrated that these complexes are particularly stable. In fact, their spectra recorded after 24 h are practically identical to those recorded at zero time (an amount of more than 97% of complexes’ results not hydrolyzed). This ensures that the NHC ligands are ancillary ligands and therefore stably bound to the metal centers [
19].
2.2. Preparation of Solutions for Electrospinning Membrane
PCL pellets were added to Acetone/DMF mixture (3:1 in volume) at 11 wt.%. Different amounts of complexes AuL20 and AuM1 were added in the PCL solution: 0%, 1%, and 3% by weight of active complex obtaining the samples named with the acronyms PCL, 1%AuL20, 3%AuL20, 1%AuM1 and 3%AuM1. The solutions were kept under magnetic stirring at 40 °C for 24 h to obtain homogeneous solutions.
2.3. Electrospinning Procedure
The polymeric solutions were electrospun by the climate-controlled electrospinning equipment (EC-CLI by IME Technologies, Spaarpot 147, 5667 KV, Geldrop, The Netherlands). Each solution was loaded in a syringe of 5 mL and fed to a 0.8 mm diameter needle connected to a power supply. The flow was ejected by the needle in the climate room in the presence of a strong electric field that allows the spinning of the polymer. It was necessary to vary the process conditions among the various membranes depending on the spinnability of the solution; however, the room conditions were set at a temperature of 25 °C and the relative humidity at 35%. The other process parameters are reported in
Table 1 for all the prepared and tested membranes. It is worth noting that samples loaded with a higher percentage of complexes required higher values of the Electrical Potential difference to obtain membranes with a good distribution of the morphological parameters, as discussed in the section “Morphological and structural characterization”.
Table 1 shows only the electrospinning parameters employed for the membranes optimized from the point of view of the electrospinning process and subsequently tested for evaluating the anticancer effectiveness.
2.4. Sample Preparation and Sterilization Protocol
Free metallic complexes were solubilized in dimethyl sulfoxide (DMSO) and diluted in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose (GIBCO, Invitrogen, Walthan, MA, USA) at a final concentration of 1, 5, 10 and 20 μM, for cell treatments.
For adhesion culture, not functionalized and functionalized PCL membranes were cut to obtain a cycle shape of 15 mm of diameter and then they were dipped in 70% ethanol and washed twice in sterile PBS 1X. Samples were dried for 24 h under a laminar flow cabinet.
Silicon rings were cut using a hollow cutter (outer diameter: 14 mm; inner diameter: 11 mm) and sterilized in 70% ethanol. After ethanol evaporation, silicon rings were stuck on PCL membranes using non-corrosive silicon rubber and left to dry overnight. Samples were dipped in 70% ethanol, exposed to UV rays for 5 min on both sides and then used for cell seeding.
2.5. Cell Culture in Adhesion
To test the cytotoxicity of free metallic complexes, MeWo (ATCC®, HTB-65TM; P22) were seeded in 96-well plates at a density of 100.000 cells/mL. Cells were cultured in DMEM high glucose (GIBCO, Invitrogen, Walthan, MA, USA) containing 10% Fetal Bovine Serum (GibcoTM, Walthan, MA, USA), 1% Penicillin/Streptomycin (Corning, Manassas, VA, USA), and 1% GlutagroTM (Corning, Manassas, VA, USA) at 37 °C in a 5% CO2 atmosphere. After 24 h, different concentrations of each complex (1, 5, 10 and 20 µM) were added to their respective wells and incubated for 24 h and 48 h.
To test the cytotoxicity of functionalized PCL membranes, MeWo (ATCC
®, HTB-65
TM; P27) were seeded within silicon rings on PCL membranes, to prevent cells flushing from the membrane area, at a density of 30.000 cells/cm
2. Samples were cultured in DMEM high glucose (GIBCO, Invitrogen, Walthan, MA, USA) supplemented with 10% Fetal Bovine Serum (Gibco
TM, Walthan, MA, USA), 1% Penicillin/Streptomycin (Corning, Manassas, VA, USA) and 1% Glutagro
TM (Corning, Manassas, VA, USA) at 37 °C in a 5% CO
2 atmosphere [
37,
38,
39].
2.6. Cell Viability Assay
For suspension culture, after 24 h and 48 h, 0.5 mg/mL of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was directly added in the culture medium and incubated for 4 h, protecting the plate from the light. Then, the supernatant was removed and 100 µL of 100% DMSO was added to solubilize formazan crystals.
The absorbance was measured at 490 nm using a microplate reader (Infinite F200 PRO, Tecan Group Ltd., Männedorf, Switzerland). Cell viability was calculated as the percentage of the control group, considered as 100%. The percentage viability of cells was calculated according to Equation (1):
2.7. Time-Lapse Live-Cell Imaging System Assay
Specific MeWo culture was performed using a Time-lapse Live-Cell Imaging System formed by a Bold Line Top Stage Incubator for 35 mm Petri dishes (H301-T UNIT BL; Okolab S.r.l., Pozzuoli, Italy), which allows the acquisition of the same images along the culture time in a fixed culture point mapped by the fully-automated stage. The incubator has independent control of gas (CO
2/O
2), humidity and temperature and assures an environment with 37 °C of temperature and 5% of CO
2 atmosphere. The system allows acquisition in brightfield and fluorescence. All images of different points within the culture chamber were reached automatically, using Olympus IX83 time-lapse microscope by motorized stage and CCD monochrome camera (mod. XM10, Olympus, Tokyo, Japan), and with all operations under the control of the X-Excellence advanced live-cell imaging software (rel. 2.0, Olympus Inc., Hamburg, Germany). Cells’ images were captured in brightfield using a 10× objective every 4-h intervals along the 48 h of culture, and further details on cells’ characterization were reported elsewhere [
40]. The related videos reported in additional data were generated with windows movie maker software (Version 2.0, Microsoft) starting from the acquired frames.
2.8. Statistical Analysis
Results from multiple experiments are presented as mean ± standard deviation (SD). Statistical analysis was performed using the two-tailed independent Student’s
t-test for comparisons of two independent groups.
p values less than 0.05 were accepted as significant [
41]. All statistical analysis was conducted using GraphPad Prism software (6.0 for Windows).
2.9. Morphology Analysis
Morphological analysis was performed by using Field emission scanning electron microscopy (FESEM). The samples were coated with a thin gold layer before the FESEM analysis. By the acquired images, geometrical information and the size distribution of the nanofibers were obtained. The analysis of the fiber distribution was performed by using ImageJ, considering at least 150 fibers. The analysis of the pore size was performed by using the software MATLAB (Natick, MA, USA) following the procedure reported in the literature [
31,
42]. The images were collected with a FESEMLEO1550VP microscope (Carl Zeiss SMT AG, Oberkochen, Germany).
For Energy Dispersive X-ray Analysis (EDX) investigation, a FESEM LEO1525 microscope (Oberkochen, Germany) equipped with an EDX detector was used. EDX maps were obtained after sputtering the samples with a thin coating of chromium. This investigation was performed for AuM1-loaded membranes.
Moreover, Atomic Force Microscopy (AFM) was performed to better characterize the morphology of the samples. AFM is a technique able to recreate a topographic map of the sample surface by exploiting the interactions between the tip and the sample surface. By acquiring information on the deflection of the cantilever through a laser, it is possible to obtain morphology accurate data on the surface of the sample. In this case, Bruker NanoScope V multimode AFM (Digital Instruments, Santa Barbara, CA, USA) was used, in tapping mode and ambient atmosphere. The tip used has a nominal spring constant of 20–100 N/m, resonance frequencies of 200–400 kHz and a tip radius of 5–10 nm. This investigation was performed both for blank PCL and AuM1-loaded membranes. The height profiles along the fibers were acquired using NanoScope Analysis 1.40 Software (Bruker Corporation, Billerica, MA, USA), and were elaborated via OriginPro software (OriginLab Corporation, Northampton, MA, USA) to consider the height profile without being affected by the fiber slope in the chosen region. The data elaboration is displayed in
Figure 2.
In this way, the average surface roughness (
Ra) and the root mean square roughness parameter (
Rq) were evaluated for the various membranes, according to (2) and (3):
where
lr is the length of the line,
z is the height and
x is the position.
2.10. Structural Characterization
Structural characterization of electrospun membranes was performed by X-ray Diffraction (XRD) measurements using the diffractometer Bruker D8 Advance diffractometer (Bruker Corporation, Billerica, MA, USA) operating at 35 kV and 40 mA. The analysis was performed in an angle range (2θ) of 10–80°. The spectra presented in the section of the “Results” are shown between 15–35° because the curves are flat out of this range. This investigation was performed both for blank PCL and AuM1-loaded membranes.
Crystallite sizes were determined using Scherrer Equation (4):
where
τ is the mean size of the crystallite,
λ is the wavelength of the X-ray source (0.1542 nm), and
β is the width of the peak at half maximum intensity, whereas θ is the diffraction angle. The data were analyzed with the same methodology reported by Naddeo et al. [
43].
The crystallinity of the sample was obtained according to Sownthari et al. [
44] by deconvoluting the spectra and considering the crystalline and the amorphous areas under the diffractometric curve profile, according to Equation (5):
2.11. Release Profiles
Samples of membranes of 1 cm diameter were placed in PBS and stirred at 300 rpm in an orbital shaker. The release medium was taken at a fixed time and then replaced with fresh PBS. Drug release kinetics were monitored by using a Spectrometer UV-2401 PC (Shimadzu, Kyoto, Japan). The tests were performed using rectangular plates with an exposed area of about 3 cm2 and 1 cm of the light path.
Considering the chemical structure of AuM1 and AuL20, the phenyl group was tracked in the release medium to monitor drug release. However, it is known that benzene absorbs UV radiation causing a π→π* at 180 nm, 203.5 nm and 254 nm [
45,
46]. In this case, the 254 nm peak was monitored for 5 days. By tracking the absorbance of phenyl group of known quantities of AuM1 and AuL20, the calibration curves are well described by the Lambert–Beer Equation (6).
where
A is the absorbance,
ε is the absorptivity of the complex,
c is the concentration of the solution and
l is the light path length.
Table 2 reports the
ε values for the complexes.
The peak monitored for AuL20 is slightly shifted from 254 nm (typical peak of the phenyl group) to 260 nm; whereas the peak value of AuM1 complex is detected around the expected value (252 nm). The spectra are reported in
Figure 3.
Observing the spectra, π→π* transitions around 254 nm and around 200 nm caused by phenyl are evident for both complexes dissolved in the solution. However, for AuL20, a third peak is registered around 240 nm. Since AuM1 shows the presence of two chlorines on the backbone compared to the AuL20 chemical structure, it is reasonable that Cl atoms attract electronic density given its high electronegativity, decreasing and shifting the intensity of the π→π* transition of the electrons involved in the double bond of the two carbonium (see the shoulder at 226 nm visible in AuM1 spectrum).
In AuL20, where there are two hydrogens instead of Cl, the transition is allowed and it is very evident in the spectrum. π→π* double bond transition in literature can be found in very different ranges of the UV-vis spectrum, from 184 nm for ethylene to over 400 nm for molecules such as
β-carotene [
45].
For release profiles fitting, a statistical approach was followed by using a modified Weibull model (7) recently proposed in literature [
31]:
where
m is the substance amount released at a certain time,
m0 is the total substance amount in the sample (evaluated by weighting the sample and knowing the complex percentage in the membrane),
A1,
A2,
b1,
b2 and θ are kinetic constant parameters,
t is the time.
In general, the two parts of the model can describe two contributes, that are usually considered the Case II transport and the Fickian Diffusion, in agreement with other variations of the Weibull Model [
47]. In particular, θ defines which mechanism is more relevant, whereas
A1,
A2,
b1 and
b2 define the kinetic of each mechanism.
Moreover, the first-order kinetic and Ritger–Peppas models were used together to explain the mechanisms that control the release behavior following the approach of Wu et al. [
48]. The models used are reported below in Equations (8) and (9):
where
a and
k are kinetic constants and
n is a descriptive parameter of the diffusive release [
49].
4. Conclusions
In this work, two synthesized complexes were proposed for potential application for contrasting malignant skin melanomas (MeWo cells). The Au-based complexes were incorporated in fibers of Polycaprolactone (PCL) membranes produced by the electrospinning technique. The complexes were also tested, for comparison, in the form of free complexes, directly in contact with the MeWo cell line culture. The tests performed on the functionalized membranes highlighted a very effective antitumoral activity in the first 24–28 h, which was very similar to that found for free complexes.
The peculiar chemical structure of the complexes, combined with the singular structural and morphological organization of the fibers constituting the functionalized electrospun membrane, determines a particularly favorable condition for having a very effective antitumor action in the first few hours of treatment. This favorable condition is due to the almost complete segregation of nanometric complex aggregates on the wall of the electrospun fibers and hence are promptly available for contact with the malignant cells. This occurrence clearly emerges from the Drug Release Curves, very fast in the initial stage, and the AFM analysis by which the evaluation of Ra and Rq parameters allowed for evaluating the presence of aggregates, of a few tens of nanometers, partially protruding from the walls.
Experimental points of the release profiles were perfectly fitted with theoretical curves, which easily allow for interpreting the kinetic phenomena occurring in the release of the complexes in the medium. From the theoretical curves emerge two different behaviors in the release of the complex. In particular, two regions were identified: the initial linear region corresponding to a very fast kinetic, and the subsequent region corresponding to a slowest kinetic. These two regions were fitted with two different models, as the drug release occurs because of different mechanisms. In particular, the first region, the dissolution of the complex aggregates, segregated on the fiber walls, was fitted by using a first-order model, since diffusive models are not suitable for describing the first part of the dissolution of the complex. In the second region, the behavior of the drug release was interpreted by using a Ritger–Peppas model, which confirms a diffusive mechanism for all the analyzed membranes. The overall results of the performed experimentation allow for foreseeing a relevant applicative potentiality. The functional membranes developed could be directly applied in the form of patches on the regions of the skin that needed anticancer treatment. Future research activities will be oriented toward this direction.