Intracellular Delivery of Biologically-Active Fungal Metabolite Gliotoxin Using Magnetic Nanoparticles

materials Article Intracellular Delivery of Biologically-Active Fungal Metabolite Gliotoxin Using Magnetic Nanoparticles Laura Comas 1, Esther Polo 2, M Pilar Domingo 1, Yulán Hernández 3, Maykel Arias 1, Patricia Esteban 1, Luis Martínez-Lostao 4,5,6,7, Julián Pardo 5,7,8,9,10, Jesús Martínez de la Fuente 7,10 and Eva M Gálvez 1,* 1Instituto de Carboquímica (ICB-CSIC), 50018 Zaragoza, Spain; [email protected] (L.C.); [email protected] (M.P.D.); maykelariascabrer[email protected] (M.A.); [email protected] (P.E.) 2Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS), Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain; [email protected] 3Pontificia Universidad Católica del Perú, Departamento de Ciencias - Sección Química, Lima 1761, Perú; [email protected] 4Instituto de Investigaciones Sanitarias de Aragón (IIS), 50009 Zaragoza, Spain; [email protected] 5Departamento de Microbiología, Medicina Preventiva y Salud Pública, Universidad de Zaragoza, 50009 Zaragoza, Spain; [email protected] 6Servicio de Inmunologia, Hospital Clinico Lozano Blesa, 50009 Zaragoza, Spain 7Instituto de Nanociencia de Aragón, Universidad de Zaragoza, 50018 Zaragoza, Spain; [email protected] 8 Centro de Investigación Biomédica de Aragón, Instituto de Investigación Sanitaria Aragón, 50009 Zaragoza, Spain 9Fundacion Agencia Aragonesa para la Investigación y el Desarrollo (ARAID), 50018 Zaragoza, Spain 10 Instituto de Ciencia de Materiales de Aragón, ICMA-CSIC, Universidad de Zaragoza, 50009 Zaragoza, Spain *Correspondence: [email protected] Received: 19 February 2019; Accepted: 27 March 2019; Published: 2 April 2019   Abstract: Gliotoxin (GT), a secondary metabolite produced by Aspergillus molds, has been proposed as a potential anti-tumor agent. Here we have developed a nanoparticle approach to enhance delivery of GT in tumor cells and establish a basis for its potential use as therapeutical drug. GT bound to magnetic nanoparticles (MNPs) retained a high anti-tumor activity, correlating with efficient intracellular delivery, which was increased in the presence of glucose. Our results show that the attachment of GT to MNPs by covalent bonding enhances intracellular GT delivery without affecting its biological activity. This finding represents the first step to use this potent anti-tumor agent in the treatment of cancer. Keywords: magnetic nanoparticles; gliotoxin; therapeutic; drug delivery; cancer cells 1. Introduction Gliotoxin (GT) is a well-known secondary metabolite produced by molds of the Aspergillus, Penicillium, Gliocladium, and Trichoderma families. It belongs to the group of epipolithiodioxopiperazines (ETP) [ 1 ] that are mycotoxins characterized by the presence of a disulfide bridge in their structure, responsible for their biological activity [ 2 ]. Among the different members of this family, GT has been the best characterized and most extensively studied, since its discovery in 1932 [ 3 – 6 ]. It was initially described as an anti-microbial toxin produced by Penicillium spp. [ 7 , 8 ]. Later on, it was shown to be a potent immunosuppressive factor synthesized by Aspergillus fumigatus [ 9 – 13 ], the most ubiquitous species within the Aspergillus genus and most common causative agent of the lethal opportunistic infection Invasive Aspergillosis (IA). GT synthesis is carried out by Materials 2019,12, 1092; doi:10.3390/ma12071092 www.mdpi.com/journal/materials Materials 2019,12, 1092 2 of 17 specific genes, involved in several metabolic pathways, and its production is tightly regulated by mold specific thiol methylases that produce the methyl inactive derivative, bismethylthiogliotoxin (bmGT) [ 14 , 15 ]. In line with its immunosuppressive and toxic effects on the host, GT was characterized as a virulence factor of Aspergillus fumigatus [ 16 ] and its inactive metabolite bmGT has been proposed as a diagnosis biomarker for IA [14,17,18]. As mentioned above, GT is a potent immunosuppressant that affects normal functioning of both the innate and adaptive immune response in vitro and in vivo . It has been shown that GT inhibits phagocytosis, inflammation, neutrophil activation, monocytes, dendritic cell and antigen presentation, and T and B cell activation [ 19 – 22 ]. In addition, GT has been extensively studied as anti-fibrotic, anti-angiogenic, anti-proliferative, and pro-cell death factor [ 20 , 23 ]. Indeed, GT has been proposed as a potent anti-tumor agent that activates both apoptotic and necrotic death pathways in target cells by a mechanism dependent on its redox modulatory activity. The disulfide bridge present in GT is able to interact with thiol groups on proteins initiating the production of reactive oxygen species (ROS) which lead to mitochondrial membrane disruption, DNA damage and cell death [ 24 – 29 ]. Apart from its ability to directly activate cell death, GT has been proposed to inactivate molecular pathways involved in cell proliferation like NFkB [ 30 , 31 ], NOTCH [ 32 ], WNT [ 33 ], or fanesyl transferases. In addition, it has been proposed as a good drug candidate for the treatment of autoimmune and inflammatory diseases like colitis or arthritis [ 34 – 36 ], transplant tolerance [ 37 ], and as an anti-cancer compound. However, GT, as other immunomodulatory and chemotherapeutic agents, is also toxic for healthy tissues and cells and its use for the treatment of human disease has not been further developed. A protocol that would specifically deliver GT to affected cells would reduce exposition of healthy tissue to this toxin and would allow exploiting its biological activity for the treatment of human disease. For that reason, we have used biocompatible MNPs as carriers. MNPs are usually composed of an inorganic core and an organic shell. The inorganic core very often consists of magnetite and maghemite (Fe 3 O 4 , γ -Fe 2 O 3 ), the most common forms. The organic shell, located in the most external part of the MNP, provides the chemical functionality and is responsible for the solubility, stability, charge effect, and interactions with other molecules. These MNPs have obtained significant attention owing to their biocompatibility and relatively low toxicity [ 38 , 39 ]. Furthermore, MNPs can be covalently conjugated to biological molecules and to surface coatings e.g., poly (ethylene glycol) (PEG) or carbohydrates. PEG [ 40 ] confers hydrophilicity, increasing their solubility in water, and also prevents MNP aggregation and non-specific binding with other molecules. Carbohydrate [ 41 ] functionalization modulates cellular uptake for specific recognition or interaction with glucose transporters, which are overexpressed in tumor cells. Glucose is the most common carbohydrate that acts as a selective carrier. Here we have analyzed the potential of using biocompatible MNPs, functionalized with fluorescent dyes and glucose to enhance GT delivery into tumor cells and simultaneously monitor intracellular delivery. We have found it is possible to bind GT to biocompatible MNPs in the presence of specific delivery agents like glucose, modulating its internalization and retaining a good biological activity. Thus, pending of further studies to characterize the in vivo efficacy and safety of these novel nanoconjugates, our findings reveal a novel approach to take advantage of the pharmacological activity of GT, increasing the stock of fungal secondary metabolites for the treatment of human disease. 2. Materials and Methods 2.1. Synthesis of MNPs Synthesis of monodisperse MNPs (nominal diameter of 6–8 nm) was carried out following the seed-mediated growth method reported by [ 42 ]. The resulting MNPs were transferred into water by using an amphiphilic polymer [ 43 ]. Briefly, 250 mg of poly(maleic anhydride-alt-1-octadecene) (PMAO) was added to a flask containing 200 mL of chloroform. After the polymer was completely dissolved under magnetic stirring, 20 mg of nanoparticles were dropped, and the mixture was sonicated Materials 2019,12, 1092 3 of 17 for 15 min. The solvent was removed under vacuum, and then the polymer coated MNPs were resuspended in 20 mL of NaOH 0.05 M. The MNPs were purified by centrifugation at 25,000 rpm for 2 h and characterized by transmission electron microscopy (TEM, under a 200 kV transmission electronmicroscope FEI Tecnai TF20 (FEI Europe, Eindhoven, Netherlands). 2.2. Functionalization of MNPs with Gliotoxin To attach the gliotoxin (GT) or its bismethylated analogue (bmGT) to the MNPs, as shown in Scheme 1, the toxins were first modified with N-(p-maleimidophenyl) isocyanate (PMPI, Thermo Fisher, Waltham, MA, USA) and cysteine (Cys, Sigma Aldrich, St. Louis, MO, USA). For the first step, 1.5 µ M of PMPI and 0.3 µ M of GT/bmGT were mixed in 132 µ L of DMSO and left stirring for 2 h at room temperature. Then, 10 µ L of methanol were added to block the unreacted isocyanate groups (PMPI-GT/bmGT). After that, PMPI-GT/bmGT was incubated with 3 mol of cysteine dissolved in 20 µ L of 50 mM phosphate buffer pH 6.5 for 1 h at room temperature (Cys-PMPI-GT/bmGT). The efficacy of the reaction was monitored by the Ellman’s assay determining the decrease of the concentration of free thiol groups in the sample. Materials 2019, 12, 1092 5 of 16 For TEM analysis L929 cells (2 × 104 cell/well) were plated on 4 well chamber slides with permanox (Nunc-Thermo Fisher Scientific, Waltham, MA USA), washed three times in PBS, fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB) for 2h at 4 °C and finally washed with 0.1 M PB for four times. After, samples were processed for TEM as described in [46]. 2.9. Statistics Statistical analyses were performed using GraphPad software version 7. A one-way ANOVA with Bonferroni post-test was performed comparing the different groups with control cells. In all cases, statistically significant difference was determined as *(p < 0.05), **(p < 0.01) and ***(p < 0.001) respectively. Scheme 1. (a) Chemical structure of gliotoxin and bismethylthiogliotoxin; (b) Schematic representation of magnetic nanoparticles (MNPs) functionalization using EDC/NHS chemistry. 3. Results and Discussion 3.1. Characterization of Magnetic NPs Functionalized with GT and Inactive bmGT MNPs were synthesized in organic media by thermal decomposition and efficiently transferred to aqueous phase by using an amphiphilic polymer (PMAO). To differentially modulate cell MNP uptake and monitoring its internalization, they were further functionalized with either PEG or glucose and a fluorescent marker (TAMRA). This type of MNPs have been used in our group since 2008 and they have been extensively characterized previously as indicated in [41,47–49]. The attachment to the GT or the inactive derivative bmGT was made in a stepwise procedure using the bifunctional linker PMPI and cysteine (Cys-PMPI-GT/bmGT). The effectiveness of this coupling relied on the reaction of the sulfhydryl group of the cys with the maleimide of the PMPI, which could be followed by Ellman’s assay showing an important decrease in the signal due to the smaller amount of free thiols in the sample. We have previously shown that this type of assay is suitable to quantify the free thiols in this type of MNPs and there are not interferences between MNP and Ellman´s assay [50]. Scheme 1. ( a ) Chemical structure of gliotoxin and bismethylthiogliotoxin; ( b ) Schematic representation of magnetic nanoparticles (MNPs) functionalization using EDC/NHS chemistry. In a common functionalization 1 mg of MNPs were mixed with 8 µ M of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Sigma Aldrich) and 10 µ M of either α -metoxyω -aminopolyethyleneglycol (H2N-PEG-OMe 750 Da, Iris Biotech, Marktredwitz, Germany) (MNPs/PEG) or 10 µ M of 4-aminophenylβ -D-glucopyranoside (Sigma Aldrich) (MNPs-Glu) in 450 µ L of 50 mM borate buffer pH 9. After incubating the mixture for 15 min at room temperature, 8 µ M of EDC and 0.03 µ M of 5(6)-TAMRA (tetramethylrhodamine-5-carboxamide) cadaverine (Anaspec, Fremont, CA, USA) were added and left stirring for 2 h followed by a washing step with phosphate buffered saline (PBS) and Tween 20 at 0.1% (w/v). Then, 25 µ M of Tris (Sigma Aldrich) were added to block the remaining carboxylic groups on the MNPs and left stirring overnight at 4 ◦C. Materials 2019,12, 1092 4 of 17 Finally, 0.5 mg of purified MNPs (MNPs-PEG or MNPs-Glu) were mixed with a solution containing 11.7 µ M of EDC and 20.7 µ M of N-hydroxysulfosuccinimide (sulfo-NHS, Sigma Aldrich) in 1 mL of 10 mM MES buffer pH 6 and incubated for 30 min at 37 ◦ C. After that, the excess of EDC and sulfo-NHS was removed by using a PD-10 desalting column (GE Healthcare, Chicago, IL, USA). The activated MNPs were then incubated with the complex previously prepared (Cys-PMPI-GT/bmGT) and 50 mM borate buffer pH 9 up to a final volume of 500 µ L and left stirring for 2 h at room temperature. Lastly, the rest of the activated carboxylic groups were blocked with 20 µL of Trisbuffer 3 M and the mixture was incubated overnight at 4 ◦C (GT/bmGT-MNPs). The final GT/bmGT-MNPs were purified by centrifugation with Amicon ® centrifugal filters (Merck KGaA, Darmstadt, Germany) 100 kDa cut off) and characterized by TEM, Elman’s assay and agarose electrophoresis. 2.3. Characterization of MNPs TEM analysis. 10 µ L of the aqueous solution of MNPs was placed on a copper grid coated with a carbon film. The grid was left to dry in air and the TEM images were obtained by using a Tecnai T20 (FEI, Hillsboro, OR, USA) electron microscope model working at 200 kV. Agarose Electrophoresis. 1% (w/v) agarose gels prepared in 0.5X Tris-Borate-EDTA buffer (TBE). The samples were loaded by mixing with 30% of glycerol and allowed to run for 10–20 min at 100 V. Ellman’s assay. The free thiol groups react with DTNB (5,5 0 -dithio-bis(2-nitrobenzoic) acid) to give a colored product 2-nitro-5-thiobenzoate (TNB) that can be measured spectrophotometrically. In the optimal conditions, 50 µ L of sample solution were mixed with 225 µ L of phosphate buffer 0.1 M pH 8 and 5 µ L of DTNB 4 mg/mL. The mixture was made to react for 15 min and the absorbance at 405 nm was measured. The linear range for the thiol group was 0–1.5 mM (Abs405 = 1.8934 [SH, mM] +0.0186. 2.4. Cell Culture and Reagents L929 (Mouse fibrosarcoma) cell lines were used [ 44 ]. Cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), L-glutamine 2 mM (Gibco) and 1% antibiotics (penicillin 100 U/mL – streptomycin 100 µ g/mL, Sigma-Aldrich). All cell lines were maintained at 37 ◦ C under a humidified atmosphere with 5% CO 2 . Only cells with viability higher than 90% were used in the experiments. GT and bmGT were purchased from Enzo Life Tech., Farmingdale, NY, USA. 2.5. Cell Viability, Cell Proliferation, and Cytotoxicity Assay Cells (10 4 cells/mL) were seeded in 96 well plates (100 µ L/well) to get attached to the bottom. The following day, cells were incubated overnight with different concentrations of GT/bmGT or MNPs functionalized to analyze proliferation and cytotoxicity. Cell viability was evaluated by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously [ 45 ]. All experiments were run in triplicate and repeated at least three times. IC50 (the concentration of compound required to reduce cell survival to 50%) was obtained by linear regression analysis of the dose-response curve plotting concentration (X axes) versus percentage of viability (Y axes) according to the following formula: %V = Abs(cells) −Abs(medium) ×100 (1) Quantification of cell death and apoptosis was carried out using two different parameters, Phosphatidylserine (PS) exposure and membrane permeabilization by using Annexin V (AnnV) and 7-Aminoactinomycin D (7AAD) staining. Cells were analyzed by Flow Cytometry (FACS) employing a FACS Calibur instrument (BD Pharmingen, Franklin Lakes, NJ, United States) and CellQuests software version 6.0 (BD). Briefly, cells were incubated in 100 µ L Annexin binding buffer (ABB) with 1 µ L Materials 2019,12, 1092 5 of 17 Annexin V-FITC (Inmunostep) and 1.5 µ L 7AAD (BD) for 15 min at room temperature. For the analysis cells were washed with PBS and fixed with 1% (v/v). 2.6. Flow Cytometry For quantification of intracellular uptake of TAMRA labelled MNPs, L929 cells (10 4 cells/well) were incubated with MNPs at a final concentration of 10 µ M in a 96 well plate (100 µ L/well). After different incubation times, cells were trypsinized and washed with PBS to remove not internalized MNPs bound to extracellular cell membrane. Afterwards cells were fixed in paraformaldehyde (PFA) 1% for 30 min at 4 ◦ C. All experiments were repeated three times for each group. 2.7. Confocal Microscopy Cells were seeded on 5 mm diameter microscopy cover slides (Electron Microscopy Sciences, Hatfield, PA, USA) placed in 96 well plates and treated under the same conditions as before. At selected times, slides were washed with PBS and cells were fixed with 4% PFA for 20 min at 4 ◦ C. Coverslips were taken out and cells were permeabilized and washed with 0.1% saponin in PBS, PBS and deionized water following the incubation with Lamp1 (rat, BD) (1:500) and cytochrome c antibody (mouse, BD) (1:100) in 0.1% saponin and 10% goat serum for 1 h at room temperature in darkness. Cells were washed again and incubated with the secondary antibody Rat-Alexafluor488 (goat, Invitrogen) (1:500) and Mouse-Alexa633 (goat, Invitrogen) (1:250) for 30 min at room temperature. Then slides were air-dried and mounted on a drop of fluoromount-G (Southern Biotechnology Associates, Inc., Birmingham, AL, USA) containing 20 µ g/mL of nuclear staining Hoechst 33342 (Molecular ProbesTM). Next, the cells were analyzed by confocal microscopy. Fluorescence images were taken on a confocal microscope using 60X oil immersion objective (Olympus FV10-I, Waltham, MA, USA). Data was analyzed using the software FV10i-SW Viewer v3.1 (Olympus) with the same settings for all the samples. 2.8. Transmission Electron Microscopy For TEM analysis L929 cells (2 × 104 cell/well) were plated on 4 well chamber slides with permanox (Nunc-Thermo Fisher Scientific, Waltham, MA USA), washed three times in PBS, fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB) for 2h at 4 ◦ C and finally washed with 0.1 M PB for four times. After, samples were processed for TEM as described in [46]. 2.9. Statistics Statistical analyses were performed using GraphPad software version 7. A one-way ANOVA with Bonferroni post-test was performed comparing the different groups with control cells. In all cases, statistically significant difference was determined as *(p< 0.05), **(p< 0.01) and ***(p< 0.001) respectively. 3. Results and Discussion 3.1. Characterization of Magnetic NPs Functionalized with GT and Inactive bmGT MNPs were synthesized in organic media by thermal decomposition and efficiently transferred to aqueous phase by using an amphiphilic polymer (PMAO). To differentially modulate cell MNP uptake and monitoring its internalization, they were further functionalized with either PEG or glucose and a fluorescent marker (TAMRA). This type of MNPs have been used in our group since 2008 and they have been extensively characterized previously as indicated in [ 41 , 47 – 49 ]. The attachment to the GT or the inactive derivative bmGT was made in a stepwise procedure using the bifunctional linker PMPI and cysteine (Cys-PMPI-GT/bmGT). The effectiveness of this coupling relied on the reaction of the sulfhydryl group of the cys with the maleimide of the PMPI, which could be followed by Ellman’s Materials 2019,12, 1092 6 of 17 assay showing an important decrease in the signal due to the smaller amount of free thiols in the sample. We have previously shown that this type of assay is suitable to quantify the free thiols in this type of MNPs and there are not interferences between MNP and Ellman’s assay [50]. Then this complex was attached to the MNPs through the amide formation between the free amine on cys and the carboxylic groups exposed on the MNPs. To ensure an effective functionalization, the stability of the MNPs was confirmed by TEM (Figure 1a) and the variations of their net charge due to replacing some carboxylic groups by GT/bmGT were analyzed by agarose electrophoresis (Figure 1b). Materials 2019, 12, 1092 6 of 16 Then this complex was attached to the MNPs through the amide formation between the free amine on cys and the carboxylic groups exposed on the MNPs. To ensure an effective functionalization, the stability of the MNPs was confirmed by TEM (Figure 1a) and the variations of their net charge due to replacing some carboxylic groups by GT/bmGT were analyzed by agarose electrophoresis (Figure 1b). Finally, a similar strategy was followed in order to quantify the amount of GT or bmGT attached to the final MNPs. To do so, the functionalization was done in the same way, but cys was attached directly to the MNPs and then coupled with the complex PMPI-GT/bmGT. So, 200 µM of GT/bmGT turned out to be successfully attached to the MNPs as determined by the reduction of free thiol groups on the MNPs by Ellman’s assay (Figure 1c). Figure 1. Characterization of MNPs. (a) TEM micrograph of Gliotoxin (GT)-TAMRA-MNPs; (b) 1% agarose gel electrophoresis of (1) MNPs control, (2) GT-MNPs and (3) bmGT-MNPs run at 100 V for 20 min; and (c) Ellman’s assay for quantifying thiol groups monitored the efficacy of the reaction between MNPs-Cys and the complex PMPI-GT/bmGT (200 µM of the complex were immobilized on the MNPs). 3.2. Magnetic NPs Functionalized with GT Kill L929 Sarcoma Cells To assess the potential of employing GT-functionalized MNPs to kill tumor cells we used the mouse sarcoma model L929, a mouse cell model previously found to be sensitive to soluble GT [17]. First of all, we calculated the IC50 dose of GT by MTT assay in order to have a control value to compare with the activity of GT after MNP functionalization. Cells were incubated over a range of Figure 1. Characterization of MNPs. ( a ) TEM micrograph of Gliotoxin (GT)-TAMRA-MNPs; ( b ) 1% agarose gel electrophoresis of (1) MNPs control, (2) GT-MNPs and (3) bmGT-MNPs run at 100 V for 20 min; and ( c ) Ellman’s assay for quantifying thiol groups monitored the efficacy of the reaction between MNPs-Cys and the complex PMPI-GT/bmGT (200 µ M of the complex were immobilized on the MNPs). Materials 2019,12, 1092 7 of 17 Finally, a similar strategy was followed in order to quantify the amount of GT or bmGT attached to the final MNPs. To do so, the functionalization was done in the same way, but cys was attached directly to the MNPs and then coupled with the complex PMPI-GT/bmGT. So, 200 µ M of GT/bmGT turned out to be successfully attached to the MNPs as determined by the reduction of free thiol groups on the MNPs by Ellman’s assay (Figure 1c). 3.2. Magnetic NPs Functionalized with GT Kill L929 Sarcoma Cells To assess the potential of employing GT-functionalized MNPs to kill tumor cells we used the mouse sarcoma model L929, a mouse cell model previously found to be sensitive to soluble GT [ 17 ]. First of all, we calculated the IC50 dose of GT by MTT assay in order to have a control value to compare with the activity of GT after MNP functionalization. Cells were incubated over a range of concentrations between 0.1 and 1 µ M for 16 h (Figure 2a). We can observe a dose dependent GT toxicity with a calculated IC50 value of 200 nM. In contrast, most cells treated with 1 µ M inactive bmGT as control, remained viable after overnight incubation. Materials 2019, 12, 1092 7 of 16 concentrations between 0.1 and 1 µM for 16 h (Figure 2a). We can observe a dose dependent GT toxicity with a calculated IC50 value of 200 nM. In contrast, most cells treated with 1 µM inactive bmGT as control, remained viable after overnight incubation. Since MTT assay measures cell proliferation, we analyzed if cells incubated with GT died by monitoring phosphatidylserine (PS) translocation and membrane integrity by Annexin-V binding and 7AAD uptake assays, respectively. As shown in Figure 2b, L929 cells incubated with 0.5 µM of GT were positive for AnnV and 7AAD staining confirming that GT actually kill L929 cells. Moreover, the IC50 values calculated by MTT and cell death assays were similar (data not shown). Figure 2. Effect of exposures to gliotoxin on the viability of L929 cell line. (a) L929 cells were exposed to different concentrations, i.e., 0.06, 0.125, 0.25, 0.5, and 1 µM of GT at 37 °C overnight. Cell viability was measured using MTT assay. The IC50 values indicated in the text were determined by extrapolation, which were presented as mean ± SEM of three independent experiments. (b) A representative dot plot is shown. Cell death was analyzed by FACS monitoring AnnV and 7AAD under the same conditions as before with 0.5 µM of GT. Once we confirmed that GT, but not bmGT, killed L929 cells, we analyzed if GT bound to GT-MNPs was still able to kill L929 sarcoma cells. First of all, we confirmed that non-functionalized MNPs were not toxic for L929 cells (data not shown). As shown in Figure 3a, GT bound to MNPs was able to block survival of L929 cells in a dose dependent manner confirming that GT retained anti-tumor activity after binding to MNPs. The results obtained employing the MTT survival assay were confirmed by analyzing Trypan Blue exclusion assay. As shown in Figure 3b, GT-MNPs killed L929 cells in a dose dependent manner. In contrast, bmGT-MNPs had no effect against L929 cells at the highest concentration neither by MTT survival assay nor by Trypan blue exclusion cell death assay. These results confirm that functionalization of MNPs with GT generates a nanoconjugate system with specific anti-tumor activity in L929 sarcoma cells. We have also analyzed the ability of GT-MNPs to kill other cell types like breast carcinoma MCF-7 or cervix carcinoma HeLa. However, Figure 2. Effect of exposures to gliotoxin on the viability of L929 cell line. ( a ) L929 cells were exposed to different concentrations, i.e., 0.06, 0.125, 0.25, 0.5, and 1 µ M of GT at 37 ◦ C overnight. Cell viability was measured using MTT assay. The IC50 values indicated in the text were determined by extrapolation, which were presented as mean ± SEM of three independent experiments. ( b ) A representative dot plot is shown. Cell death was analyzed by FACS monitoring AnnV and 7AAD under the same conditions as before with 0.5 µM of GT. Since MTT assay measures cell proliferation, we analyzed if cells incubated with GT died by monitoring phosphatidylserine (PS) translocation and membrane integrity by Annexin-V binding and 7AAD uptake assays, respectively. As shown in Figure 2b, L929 cells incubated with 0.5 µ M of GT were positive for AnnV and 7AAD staining confirming that GT actually kill L929 cells. Moreover, the IC50 values calculated by MTT and cell death assays were similar (data not shown). Materials 2019,12, 1092 8 of 17 Once we confirmed that GT, but not bmGT, killed L929 cells, we analyzed if GT bound to GT-MNPs was still able to kill L929 sarcoma cells. First of all, we confirmed that non-functionalized MNPs were not toxic for L929 cells (data not shown). As shown in Figure 3a, GT bound to MNPs was able to block survival of L929 cells in a dose dependent manner confirming that GT retained anti-tumor activity after binding to MNPs. The results obtained employing the MTT survival assay were confirmed by analyzing Trypan Blue exclusion assay. As shown in Figure 3b, GT-MNPs killed L929 cells in a dose dependent manner. In contrast, bmGT-MNPs had no effect against L929 cells at the highest concentration neither by MTT survival assay nor by Trypan blue exclusion cell death assay. These results confirm that functionalization of MNPs with GT generates a nanoconjugate system with specific anti-tumor activity in L929 sarcoma cells. We have also analyzed the ability of GT-MNPs to kill other cell types like breast carcinoma MCF-7 or cervix carcinoma HeLa. However, these tumor types are much more resistant to free GT (IC50 values around 1 µ M, data not shown) and, thus, it was required up to 20 µ M of GT-MNPs to see any effect on cell proliferation. This data suggests that although GT retains anti-tumor activity in vitro against sarcoma L929 cells, its potential application to other tumor types might be more limited. Materials 2019, 12, 1092 8 of 16 these tumor types are much more resistant to free GT (IC50 values around 1 µM, data not shown) and, thus, it was required up to 20 µM of GT-MNPs to see any effect on cell proliferation. This data suggests that although GT retains anti-tumor activity in vitro against sarcoma L929 cells, its potential application to other tumor types might be more limited. Figure 3. Biocompatibility of different functionalized nanoparticles with L929 cells. L929 cells were incubated with MNPs functionalized with GT, GT and glucose, and GT and PEG, respectively at different concentrations (1.25, 2.5, 5 and 10 µM) overnight at 37 °C. Subsequently, cells were washed twice with PBS to remove non-internalized MNPs. L929 incubated with the maximum concentration of MNPs functionalized with bmGT, in the presence or absence of glucose or PEG, were used as control. (a) Quantification of cell viability was carried out by MTT as indicated in experimental procedures. The IC50 values were determined by extrapolation, which were presented as mean ± SEM of three independent experiments. Statistical analysis between MNPs functionalized with GT and GT/glucose was performed using paired t test. *, 0.01 < p < 0.05. (b) Analysis of the effect of GT-MNPs, glucose-conjugated GT-MNPs and PEG-conjugated GT-MNPs on the cell viability of L929 cells by Trypan Blue by microscopy. Data are expressed as the mean values ± SEM of three separate experiments. 3.3. Glucose Enhances the Anti-Tumor Activity of Magnetic NPs Functionalized with GT against L929 Sarcoma Cells In order to modulate the selectivity of GT-MNP conjugate against tumor cells we analyzed if glucose, known to enhance the uptake of MNPs in tumor cells [47], would increase the ability of GT-MNPs to kill L929 sarcoma cells. As control, the cytotoxic activity of GT-MNP functionalized with glucose (GT-MNPs-Glu) was compared with PEGylated GT-MNPs (GT-MNPs-PEG). As shown in Figure 3a, inhibition of L929 cell proliferation was significantly higher in GT-MNPs-Glu-treated cells than in those treated with GT-MNPs. A similar result was found when cell death was analyzed by trypan blue exclusion assay (Figure 3b). In contrast, incorporation of PEG into GT-NPs completely abrogated the anti-tumor effect of GT-MNPs. Again, the cytotoxic potential of MNPs-Glu functionalized with bmGT was almost completely reduced, confirming that this effect was due to GT presence. Our results agree with previous findings indicating that PEGylation modifies the size of the particle and prevents intracellular delivery [43]. In contrast, due to changes in the metabolic status, glucose would favor intracellular uptake of MNPs in L929 tumor cells enhancing intracellular delivery of GT and its ability to kill tumor cells. This result agrees with previous reports in which glucose enhanced the uptake of gold NPs in other types of tumor cells [46,51] Figure 3. Biocompatibility of different functionalized nanoparticles with L929 cells. L929 cells were incubated with MNPs functionalized with GT, GT and glucose, and GT and PEG, respectively at different concentrations (1.25, 2.5, 5 and 10 µ M) overnight at 37 ◦ C. Subsequently, cells were washed twice with PBS to remove non-internalized MNPs. L929 incubated with the maximum concentration of MNPs functionalized with bmGT, in the presence or absence of glucose or PEG, were used as control. ( a ) Quantification of cell viability was carried out by MTT as indicated in experimental procedures. The IC50 values were determined by extrapolation, which were presented as mean ± SEM of three independent experiments. Statistical analysis between MNPs functionalized with GT and GT/glucose was performed using paired t test. *, 0.01 < p< 0.05. ( b ) Analysis of the effect of GT-MNPs, glucose-conjugated GT-MNPs and PEG-conjugated GT-MNPs on the cell viability of L929 cells by Trypan Blue by microscopy. Data are expressed as the mean values ± SEM of three separate experiments. Materials 2019,12, 1092 9 of 17 3.3. Glucose Enhances the Anti-Tumor Activity of Magnetic NPs Functionalized with GT against L929 Sarcoma Cells In order to modulate the selectivity of GT-MNP conjugate against tumor cells we analyzed if glucose, known to enhance the uptake of MNPs in tumor cells [ 47 ], would increase the ability of GT-MNPs to kill L929 sarcoma cells. As control, the cytotoxic activity of GT-MNP functionalized with glucose (GT-MNPs-Glu) was compared with PEGylated GT-MNPs (GT-MNPs-PEG). As shown in Figure 3a, inhibition of L929 cell proliferation was significantly higher in GT-MNPs-Glu-treated cells than in those treated with GT-MNPs. A similar result was found when cell death was analyzed by trypan blue exclusion assay (Figure 3b). In contrast, incorporation of PEG into GT-NPs completely abrogated the anti-tumor effect of GT-MNPs. Again, the cytotoxic potential of MNPs-Glu functionalized with bmGT was almost completely reduced, confirming that this effect was due to GT presence. Our results agree with previous findings indicating that PEGylation modifies the size of the particle and prevents intracellular delivery [ 43 ]. In contrast, due to changes in the metabolic status, glucose would favor intracellular uptake of MNPs in L929 tumor cells enhancing intracellular delivery of GT and its ability to kill tumor cells. This result agrees with previous reports in which glucose enhanced the uptake of gold NPs in other types of tumor cells [46,51]. 3.4. Glucose Enhances Cellular Interaction of Magnetic NPs Functionalized with GT in L929 Sarcoma Cells Once we have confirmed that Glucose functionalization enhanced the ability of GT-MNPs conjugates to kill L929 cells, we analyzed if this effect correlated with intracellular uptake of MNPs. To this aim the different MNP conjugates were functionalized with TAMRA and intracellular uptake in L929 cells analyzed by flow cytometry. As shown in Figure 4b, after incubation with the different MNP conjugates most cells were positive for TAMRA fluorescence after 30, 60 or 240 min, indicating interaction of MNPs with cells, irrespectively of the presence of GT, bmGT, and/or glucose. However, when the level of single cell fluorescence intensity (MFI) was analyzed and represented (Figure 4a), it was found that cellular uptake was significantly enhanced by the presence of glucose after 4h. In contrast, the presence of GT and bmGT has no effect on MNP uptake. The same number of cells was recovered at the end of the incubation time indicating that the differences in fluorescence intensity were not due to a lower number of cells. Notably, PEG almost completely abrogated the uptake of MNPs. These results agree with the ability of the different MNP conjugates to kill L929 cells (Figure 3) and confirm that glucose enhances cell uptake of MNPs in L929 cells and the ability of GT-MNPs to kill these types of tumor cells. This result agrees with previous experimental findings [ 43 , 52 ], showing that PEG prevent NP uptake, suggesting that that the lack of GT toxicity using MNPs-PEG might be related to its inability to enter into cells [53]. 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