The ideal nanoparticle-based drug delivery system assures the safe delivery and selective action of a drug to a target site. Indeed, nanomaterials can add further functionality to the conjugated/loaded drug and, taking advantage of their unique size, are able to play a crucial therapeutic role. This has triggered an increased interest in nanopharmaceuticals [1
] and the development of a wide range of nanoparticle systems, such as liposomes, nanoparticles, micelles, dendrimers, and nanotubes [3
]. However, only a few nanoparticle-based systems have been FDA-approved for cancer therapy to date [6
]. Although nanoscience in drug development is in its early stages, the fusion of engineered nanomaterials and nanopharmaceutical research is paving the way for the development of stimuli-responsive drug delivery systems, especially in cancer treatment. Interestingly, several chemically modified polymers [8
] and cross-linked cyclodextrin-based polymers have been proposed to obtain compounds responsive to the external environment [10
]. In this regard, cyclodextrin-based nanosponges are of particular interest [11
Nanosponges are hyper-cross-linked cyclodextrin polymers generally obtained from α, β, and γ cyclodextrins, containing suitable amounts of linear dextrin cross-linked with a proper cross-linking agent. A cage-like structure is obtained via the cross-linking of cyclodextrins, thus creating nanochannels in the polymer matrix that can be modulated employing different types of cross-linking agents and/or varying the amount used [13
]. It is worth noting that active carbonyl compounds, like carbonyl diimidazole, diphenyl carbonate, and organic dianhydrides such as pyromellitic dianhydride, can be used as cross-linker in the preparation of nanosponges [15
Nanosponges offer several features, such as sustained and controlled release, improvement of aqueous solubility, bioavailability, and stability of the hosted molecules, which could be advantageously exploited for drug delivery [15
]. Indeed, previous research highlighted the capability of nanosponges to encapsulate different active molecules and magnify their activity in either in vitro or in vivo studies [15
]. In particular, several anticancer drugs such as doxorubicin, paclitaxel, and camptothecin have been efficiently incorporated in cyclodextrin-based nanosponges, showing an improved antitumor effect [21
Interestingly, nanosponge-based drug delivery systems can be tuned to form ‘stimuli-responsive’ nanocarriers that modify their structure in response to external changes, such as pH or redox potential [25
]. Therefore, glutathione (GSH)-responsive nanocarriers have been developed for targeted intracellular anticancer drug release [28
], as the GSH tripeptide has a higher intracellular than extracellular concentration [29
]. Several intracellular compartments, such as cytosol, mitochondria, and the cell nucleus, contain higher GSH concentrations than do extracellular fluids and circulation. Moreover, GSH intracellular concentration is further increased in cancer cells and, above all, in chemoresistant cells [30
]. Oxidative stress has long been implicated in cancer development and progression [31
]. An increase in reactive oxygen species (ROS) usually induces a cell adaptive response and the compensatory up-regulation of antioxidant systems to restore redox homeostasis; GSH/GSH disulfide is the major redox combination in mammalian cells [32
]. Moreover, many primary tumors have high levels of overexpression of antioxidant enzymes [33
Trotta et al. have developed a next generation of nanosponges that are bioresponsive to GSH external concentration [35
]. This behavior may be an ideal trigger for rapid nanocarrier destabilization inside cells, leading to efficient intracellular drug release through disulfide-bond cleavage [35
]. Indeed, the disulfide bridge remains stable in extracellular fluids for long periods before being reduced upon internalization in the cytosol, having a higher GSH concentration, thus improving drug bioavailability [36
]. As depleting endogenous antioxidants, like GSH, make cancer cells more chemosensitive, this reduction-sensitive nanosystem is further suited to anticancer therapy owing to its ability to enhance the anticancer activity of such drugs. Previously, the encapsulation of doxorubicin and strigolactone analogues in GSH-responsive nanosponges was in vitro and in vivo evaluated. In vitro release kinetics studies from GSH-NS revealed a GSH concentration-based drug release profile over time. Moreover, the GSH-NSs were able to release the payload as a function of in-cell GSH concentration [37
]. This behavior might favor the selectively controlled release in target cancer cells and enhance the cytotoxic effect. Indeed, both of the compounds loaded in GSH-NS were more effective in inhibiting the cell viability than the corresponding free drugs, particularly in cancer cells presenting a higher GSH content. In addition, a greater reduction of prostate cancer growth was observed for doxorubicin incorporated in GSH-NS compared with the free drug in xenograft mice models [37
The fact that GSH-NS may well represent an efficient stimuli-responsive drug delivery system for anticancer drugs prompted in-depth study into their biological effect per se on cell growth reported herein. As preliminary cellular evaluations of nanocarriers are usually carried out on 2D cell cultures, previously, the effects of cyclodextrin-based nanosponges have been widely tested in 2D cell monolayer cultures. However, 3D cell cultures, such as multicellular spheroids (MCS), have various in vivo tissue characteristics including the production of an extracellular matrix [39
]. This study reports a series of experiments carried out to evaluate the bioeffects of GSH-NS containing two different amounts of disulfide bridges either on 2D cell cultures or on 3D cell cultures of human cancer cells, differing in cancer type and intracellular GSH level, namely, human colorectal, HCT116 and HT-29, and human prostatic, DU145 and PC-3, cancer cell lines.
Understanding the effect that nanoparticles have on cells is crucial to predict their in vivo toxicity and avoid any undesirable nanoparticle activities. Although there are numerous in vitro cytotoxicity assays that can be applied for the general screening of nanoparticles [42
], it is of vital importance that the research covers nanoparticle cytotoxicity itself. In this contest the use of strictly controlled in vitro experimental conditions can ensure that the measured effect is the result of nanoparticle toxicity and not unstable culturing conditions [44
]. Moreover, up to nowadays, there has been no single analysis able to provide sufficient information to correlate the biomaterial chemistry and surface with biological response [45
]. Herein, we investigated the in vitro biological effects of a stimuli-responsive nanosystem, that is, glutathione responsive β-cyclodextrin-based nanosponges (GSH-NS), in various cancer cell lines, characterized by their GSH basal content, as this nanosystem is designed to be a GSH responsive anticancer drug carrier.
GSH plays a key role in cellular defense against oxidative stress [46
] and its increased redox capacity in cancer cells is well-known [34
]. Consequently, GSH has been recognized to be an ideal intracellular trigger for selective drug delivery by responsive nanocarriers, as many compounds exert their therapeutic effects only inside cells. As disulfide chemistry is particularly versatile, a wide range of GSH-responsive nano-vehicles, such as micelles, nanoparticles, and nanogels, have been recently developed [28
]. Among them, glutathione responsive β-cyclodextrin-based nanosponges incorporate high payload and provide controlled drug release over time, with the further advantage of triggered intracellular drug delivery in response to cell GSH content. In addition, GSH-NSs are able to protect degradable drugs from the external environment. It is foreseen that β cyclodextrin-based nanosponges will have a significant positive impact on anticancer therapeutic scenarios [13
]. Taking into account the promising results concerning the efficacy of GSH-NSs as an anticancer drug delivery system [37
], the biological safety of the nanosponge itself is a critical parameter for their future clinical application.
β-cyclodextrin toxicology has been evaluated in in vitro and in vivo studies that have reported it as non-toxic and well tolerated even at very high doses [48
]. Previous in vitro studies showed no signs of cytotoxicity after cell exposure to unloaded nanosponges in the 10–100 μg/mL concentration range used for the delivery of therapeutic drugs [23
]. In addition, in vivo experiments have shown that β-cyclodextrin-based nanosponges prepared with pyromellitic dianhydride as a cross-linking agent have been orally administered to rats without showing any toxic side effects at selected doses in an acute and repeated dose toxicity study [51
]. Previously, GSH-NSs have been investigated as doxorubicin carrier. No acute cardiotoxic effects were observed in mice after the in vivo administration of doxorubicin-loaded GSH-NS [37
]. Recently, the hepatotoxicity of this nanoformulation was investigated either in vitro on human HepG2 cell line or ex vivo on rat precision-cut liver slices (PCLSs), where a good nanosponge safety profile was demonstrated, showing a comparable hepatotoxicity to that of free doxorubicin [52
As no reports have been published on the effects at a cellular level of GSH-NS as such, it was decided to study the effect of GSH-NS per se on HCT116, HT-29, DU145, and PC-3 cancer cell lines with various GSH content in a concentration range that is about fifty times higher than that used in the above mentioned studies to ensure the use of cytotoxic concentrations. HCT116 and DU145 cells showed the highest GSH values in colorectal and prostatic cancer cell lines, respectively; previous research studies have shown that DU145 cells have the highest GSH content [53
]. Non-toxic (IC1
) and cytotoxic (IC50
) GSH-NS concentrations were determined by a 2D cell assay, which measured mitochondrial activity. A decrease in cell growth with significantly different IC1
values was observed when the two nanosponge formulations were compared in all cell lines, except in DU145 cell line, where no statically significant difference was observed.
DU145 cell line was the most sensitive to the GSH-NS D cytotoxic effect among all cell lines tested. Notably, DU145 cells are more resistant to electrophilic toxicity than other cells owing to their high levels of redox-sensitive transcription factor, nuclear factor erythroid 2-related factor-2 (Nrf2), which activates cytoprotective pathways against oxidative injury, such as GSH synthesis [54
]. As this nanosystem has the ability to disrupt itself in the presence of GSH, we can hypothesize that it is the high GSH content in DU145 cells that allows GSH-NS to exert their cytotoxic effect, whatever the disulfide-bond concentration. Further studies are needed to investigate whether agents able to modulate intracellular GSH, such as N
-acetyl cysteine or buthionine sulfoximine [56
] could affect nanosponge intracellular drug release and cytotoxicity.
Our study shows that colorectal cancer cells, in particular HCT116 cells, have the most pronounced GSH-NS B and D cellular uptake. This difference in nanosponge cellular uptake in this cell line may be owing to differing uptake mechanisms, as cell surface thiols have been reported to affect disulfide-conjugated peptide cell entry [57
]. Indeed, disulfide bridge cleavage may start at the cell surface via thiol/disulfide exchange reactions catalyzed by redox proteins such as thioredoxines [58
]. Therefore, the 2D data on IC50
would appear to indicate that prostatic cancer cell lines are more sensitive to GSH-NS cytotoxic effects.
Worthy of note is that cell cycle analyses revealed a significant cell cycle arrest in the G0
phase in all cell lines at 24 h IC50
values. Thus, to further investigate this cell cycle arrest, we analyzed a panel of genes that are involved in cell cycle regulation. Notably, the results show significant mRNA over-expression in the cell cycle progression regulators at G1
, and CDKN2A
, which code for p21 and p16 that inhibit the cyclin-CDK2 and -CDK4 complexes, respectively. Apart from this, the mRNA expression of CDC25A
, and CDK4
was either unaffected or down-regulated in all cell lines. These data demonstrate that GSH-NS inhibition of cell proliferation is essentially owing to G1
cell cycle arrest, in agreement with previous reports by Choi et al. [59
]. Interestingly, only HTC116 cells showed significant ROS production after GSH-NS exposure, which is most likely owing to their high GSH-NS cellular uptake.
Lastly, the investigation of nanosponges effects on MCS growth was carried out. The results were interesting as differences in the 2D study were observed. There were no significant differences between the two GSH-NS formulations in HCT116 and DU145 MCS, whereas there was a significant difference in the 2D HCT-116 culture. Indeed, IC50 values were significantly lower in the 2D cultures than in the 3D cultures, especially after 24 h incubation, where similar values were reached only after 72 h of incubation. For example, IC50 was twofold higher after 24 h in 3D cultures for HCT116 and three-fold higher in DU145 than in their respective monolayers. On the other hand, IC1 concentrations were significantly lower in HCT116 MCS than in HCT116 cell monolayers, whereas IC1 was quite similar both in DU145 spheroids and monolayers.
GSH-NS cytotoxicity might appear to be linked to disulfide-bond content in 2D cell monolayers as the formulation with the higher disulfide-bond content, GSH-NS D, had the lowest cytotoxic effect in all cell lines, except for the DU145 cell line. On the other hand, GSH-NS cytotoxicity was not influenced by the disulfide-bond content in MCS and the most pronounced cell growth decrease was observed in the colorectal cancer cell line, HCT116, after 24 h of exposure to GSH-NS. Tissue-like morphology and phenotypic change may be identified as the major factors in diminishing toxicity on MCS. This means that in vitro 3D cell culture models could act as an intermediate stage and bridge the gap between in vitro 2D and in vivo studies, which would extend current cellular level cytotoxicity to the tissue level and improve the predictive power of in vitro nanoparticle toxicology [60
]. Finally, GSH-NSs showed a limited toxicity, leading to G1
cell cycle arrest, without membrane damage or oxidative stress generation at significantly higher concentrations about fifty times those used for the delivery of anticancer drugs.
4.1. Synthesis of Glutathione Responsive β-cyclodextrin-Based Nanosponges
Glutathione-responsive β-cyclodextrin-based nanosponges (GSH-NSs) were synthetized according to the method developed by Trotta et al. [35
Briefly, GSH-NSs were obtained using a one-step synthetic route by reacting β-cyclodextrin and pyromellitic dianhydride, in the presence of 2-hydroxyethyl disulfide to insert disulfide bridges in the NS nanostructure [35
]. Varying amounts of 2-hydroxyethyl disulfide were used to obtain a series of GSH-NS with different disulfide bridge percentages in the polymer matrix. In particular, 2-hydroxyethyl disulfide/β-cyclodextrin molar ratios of 1:20 and 1:5 were used for the synthesis of GSH-NS with low (GSH-NS B) and high disulfide-bridge content (GSH-NS D), respectively. The reaction was estimated at room temperature under stirring for 24 h. The nanosponges were then purified by Soxhlet extraction with acetone for a few hours. The percentage of sulfur in the two types of GSH-NS was measured by elemental analysis and was 0.62 and 1.90 for GSH-NS B and GSH-NS D, respectively.
4.2. Preparation of Glutathione Responsive β-cyclodextrin-Based Nanosponge Nanosuspension
GSH-NS nanosuspensions were prepared following the preparation protocol previously reported [35
]. A weighed amount of GSH-NSs was suspended in a saline solution (NaCl 0.9%) at a concentration of 10 mg/mL. The suspension was homogenized by a high shear homogenizer (Ultraturrax®
, IKA, Konigswinter, Germany) for 5 min at 24,000 rpm. The sample was then homogenized on a high-pressure homogenizer (EmulsiFlex C5, Avastin, Mannheim, Germany) for 90 min at a back pressure of 500 bar to further reduce the size of the nanosponges. The aqueous nanosponge nanosuspension was subsequently purified by dialysis (membrane cutoff 12,000 Da) to eliminate potential synthesis residues. The nanosuspension was stored at +4 °C and used for all experiments.
4.3. Preparation of Fluorescent Glutathione Responsive β-cyclodextrin-Based Nanosponges
Fluorescent GSH-responsive nanosponges were obtained by adding 6-coumarin (0.1 mg/mL) to the aforementioned aqueous GSH-NS nanosuspensions (previously described) (10 mg/mL) under stirring for 24 h at room temperature in the dark.
4.4. Characterization of Glutathione Responsive β-cyclodextrin-based Nanosponges
The two types of GSH-NS (GSH-NS B and D), either blank or 6-coumarin loaded, were characterized in vitro to measure their physico-chemical parameters. The average diameters, polydispersity indices, and zeta potential values were determined by photon correlation spectroscopy (PCS) and electrophoretic mobility using a 90 Plus Instrument (Brookhaven, NY, USA) at a fixed angle of 90° and a temperature of +25 °C. The analyses were performed on diluted GSH-NS samples (1:30 v/v). For zeta potential determination, the samples were placed in an electrophoretic cell where an electric field of approximately 15 V/cm was applied. Three batches were analyzed for each NS type and each measured value was the average of ten repetitions. Nanosponge morphology was evaluated by transmission electron microscopy (TEM) (Philips CM10 instrument, Eindhoven, Netherlands) after the diluted aqueous nanosponge nanosuspensions were sprayed onto a Form war-coated copper grid and air-dried.
4.5. Cell Culture and Treatment with Glutathione Responsive β-cyclodextrin-Based Nanosponges
Human colorectal cancer cell lines, HCT116 and HT-29 (ICLC, Interlab Cell Line Collection, Genova, Italy), and human prostatic carcinoma cell lines, DU145 and PC-3 (ICLC), were cultured in McCoy’s 5A Medium and RPMI-1640 Medium, respectively. These media were supplemented with 2 mM L-glutamine, 100 UI/mL penicillin, 100 µg/mL streptomycin, and 10% (v/v) heat-inactivated fetal calf serum (Sigma, ST Louis, MO, USA) in a humidified atmosphere of 5% CO2 air at 37 °C. At 85% confluence, cells were harvested with 0.25% trypsin and sub-cultured into 75 cm2 flasks, 6-well plates or 96-well plates according to need. Cells were allowed to attach to the surface for 24 h prior to treatment. GSH-NS B and D were then suspended in a cell culture medium and diluted to the appropriate concentrations. After treatment, the cells were harvested to determine cytotoxicity, cell cycle distribution and ROS production. The cells that were not exposed to GSH-NS were used as control conditions for each experiment.
4.6. Measurement of Basal Intracellular Reduced Glutathione Levels
The total glutathione level (GSSG + GSH) in HT-29, HCT116, DU154, and PC-3 cells were assayed by the Glutathione Assay Kit (Sigma, Milano, Italy), according to manufacturer’s instructions. The protein concentration (μg/mL) was quantified by the Qubit fluorometer (Invitrogen, Milan, Italy) and the Quant-IT Protein Assay Kit (Invitrogen, Milano, Italy). Calibration was performed by the application of a two-point standard curve, according to the manufacturer’s instructions.
Briefly, reduced glutathione (GSH) reacts with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in a recycling assay and produces glutathione disulfide (GSSG) and the 1,3,5-trinitrobenzene (TNB) anion, which can be detected by absorbance. In turn, the enzyme glutathione reductase then reduces GSSG, which release GSH that can react with another DTNB molecule. Therefore, the rate of TNB production is measured rather than a single determination of how much DTNB react with GSH, as it is proportional to the initial amount of GSH [61
]. The plate was read at 412 nm on a microplate reader Asys UV 340 (Biochrom, Cambridge, UK) and the amount of GSH was expressed in nmol/µg protein.
4.7. Cell Proliferation Assay
The effect that GSH-NS B and D had on HCT116, HT-29, DU145, and PC-3 cell growth was evaluated by WST-1 cell proliferation assay (Roche Applied Science, Penzberg, Germany). Briefly, 2.0 × 103 HT-29, 1.5 × 103 HCT116, 5.0 × 102 DU145, and 1.2 × 103 PC-3 cells were seeded in 100 µL of growth medium in replicates (n = 8) in 96-well culture plates; the seeding density of each cell line was chosen according to the best proliferation rate. The medium was removed after 24 h and the cells were incubated with in an experimental medium containing differing GSH-NS B or GSH-NS D concentrations (0.5, 1.0, 2.0, and 3.0 mg/mL). At 24, 48, and 72 h, WST-1 reagent (10 µL) was added and the plates were incubated at 37 °C in 5% CO2 for 1.5 h. Well absorbance was measured at 450 and 620 nm (reference wavelength) on a microplate reader Asys UV 340.
Cell proliferation data were expressed as a percentage of control, that is, untreated cells. At 24, 48, and 72 h, the inhibition concentration 50% (IC50), defined as the dose of compound that inhibited 50% of cell growth, was interpolated from the growth curves, as was the inhibition concentration 1% (IC1), defined as the dose of compound that inhibited 1% of cell growth. Thus, to compare the effects of GSH-NS on the different cell lines, the IC1 and IC50 values obtained were used to carry out the following experiments.
4.8. Nanosponge Cellular Uptake Assays
Coumarin 6-loaded GSH-NS cellular uptake was assessed by cytofluorimetric analysis using a C6 flow cytometer (Accuri Cytometers, Ann Arbor, MI, USA) and imaging analysis using a DMI4000B fluorescence microscopy (Leica, Wetzlar, Germany). For flow cytometry analysis, 5.0 × 104 cells were seeded in a six-well culture plate. Forty-eight hours after seeding, HT-29, HCT116, DU145, and PC-3 cells were treated with the respective not-cytotoxic (IC1) and cytotoxic (IC50) concentrations of either fluorescent GSH-NS B or fluorescent GSH-NS D at 24 h. After a 24 h incubation, the cells were washed three times with phosphate-buffered saline PBS, suspended in 250 µL PBS, and run on the flow cytometer with 488 nm excitation. Intracellular fluorescence was expressed as integrated mean fluorescence intensity (iMFI), which was the product of the frequency of 6-coumarin-loaded GSH-NS positive cells and the mean fluorescence intensity.
Microscopy observation was carried out after glass coverslips were placed in 24-well plates and the cells seeded at a density of 5.0 × 104 cells/coverslip for 48 h of incubation. The coumarin 6-loaded nanosponges were then added at the respective IC50 values for GSH-NS B and D, and incubated for 24 h. The cells were incubated with 1 µg/mL of 4′,6-diamidino-2-phenylindole (DAPI) for nuclear counterstaining 30 min before the programmed stop time. After the cells were washed with PBS, the cells on the coverslip were mounted on a glass slide, observed under a fluorescence microscope, and photographed.
4.9. Lactate Dehydrogenase Leakage Assay
Lactate dehydrogenase (LDH) is an enzyme that is widely present in cytosol and catalyzes the conversion of lactate to pyruvic acid. If plasma membrane integrity is disrupted, the LDH leaks into culture media, increasing its extracellular level, and the amount of LDH release is proportional to the number of damaged cells [62
]. The LDH leakage was evaluated by the LDH-Cytotoxicity Detection Kit (Roche Diagnostic, Indianapolis, USA), according to manufacturer’s instructions. Briefly, 96-well plates were seeded with HT-29, HCT116, DU145, and PC-3 cell lines at a density of 2.0 × 103
, 1.5 × 103
, 5.0 × 102
, and 1.2 × 103
cells/100 µL culture medium, respectively. Twenty-four hours after the seeding, 100 µL of different concentrations (0.5, 1.0, 2.0, and 3.0 mg/mL) of GSH-NS B or GSH-NS D was added to the wells. The plates were then incubated for 24, 48, and 72 h, at 37 °C, in a humidified atmosphere of 5% CO2
air. Cell-free culture media were then collected and incubated with the same volume of reaction mixture for 30 min. LDH activity was measured at 490 nm on a microplate reader Asys UV 340. The background control was obtained by measuring the LDH activity of the assay medium, the untreated control by measuring the LDH activity of untreated cells, and the positive control by measuring the maximum releasable LDH activity after the treatment with the lysis buffer. The LDH leakage percentage was calculated as follows: LDH leakage (%) = (experimental value−untreated control)/(positive control−untreated control) × 100, and is the mean of three independent wells.
4.10. Cell Cycle Analysis
Cell cycle distribution was evaluated 24 h after cell treatment with the respective IC50 of GSH-NS B or GSH-NS D. The occurrence of the so-called sub-G0/G1 peak, which is a distinct cell population characterized by subdiploid DNA fluorescence and might correlate with the internucleosomal DNA fragmentation typical of apoptosis (Pozarowski and Darzynkiewicz, 2004), was also evaluated. Briefly, 1 × 106 HCT116, 1 × 106 HT-29, 1 × 106 DU145, and 1 × 106 PC-3 cells were incubated with 2 µM of the live cell staining Vybrant Dye Cycle Green (Invitrogen) for 30 min at 37 °C. The samples were run on a flow cytometer with 488 nm excitation to measure Vybrant Dye Cycle Green staining and data analysis was performed by FCS Express software version 4 (BD Bioscience, Milano, Italy).
4.11. Real Time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from the HCT116, HT-29, DU145, and PC-3 cells, 24 h after incubation with the respective GSH-NS B or GSH-NS D IC50. Briefly, the cells were collected in RNA Cell Protection Reagent (Qiagen, Milano, Italy) and stored at −80 °C. Total RNA was obtained by the RNeasy Plus Mini Kit (Qiagen Milano, Italy). Total RNA concentration (µg/mL) was determined using the fluorometer Qubit (Invitrogen) and the Quant-IT RNA Assay Kit (Invitrogen). Calibration was carried out by applying a two points standard curve, according to the manufacturer’s instructions. RNA sample integrity was determined by the Total RNA 6000 Nano Kit (Agilent Technologies, Milano, Italy) using the Agilent 2100 Bioanalyzer (Agilent Technologies, Milano, Italy).
Real-time RT-PCR analysis was carried out using 1 µg of total RNA, which was reverse transcribed in a 20 µL cDNA reaction volume using the QuantiTect Reverse Transcription Kit (Qiagen, Milano, Italy). Each 10 µL real-time RT-PCR reaction was obtained using 12.5 ng of cDNA, according to the manufacturer’s instructions. Quantitative RT-PCR was performed by the SsoFast EvaGreen (Bio-Rad, Milan, Italy) and the QuantiTect Primer Assay (Qiagen, Milano, Italy) was used as the gene-specific primer pair for the studied gene panel (Table 4
The transcript of the reference gene 18S ribosomal RNA (RRN18S
) was used to normalize mRNA data and real-time RT-PCR was performed by the MiniOpticon Real Time PCR system (Bio-Rad, Milan, Italy). The PCR protocol conditions were as follows: a HotStarTaq DNA polymerase activation step at +95 °C for 30 s, followed by 40 cycles at +95 °C for 5 s and +55 °C for 10 s. All runs were performed on at least three independent cDNA preparations per sample and all samples were run in duplicate. At least two non-template controls were included in each PCR run. Quantification data analyses were performed by the Bio-Rad CFX Manager software version 1.6 (Bio-Rad, Milan, Italy), according to the manufacturer’s instructions. These analyses were performed in compliance with MIQE guidelines (Minimum Information for Publication of Quantitative Real-time PCR Experiments) [63
4.12. Reactive Oxygen Species Production Assay
The production of intracellular reactive oxygen species (ROS) was measured by flow cytometry using dichlorofluorescein-diacetate (DCFH-DA) (Sigma, Milano, Italy) as the oxidation-sensitive probe. Briefly, after 1, 12, and 24 h cell exposure to the respective GSH-NS B or GSH-NS D IC1 and IC50 at 24 h, HT-29, HCT116, DU145, and PC-3 cells were washed twice with PBS in six-well plates and incubated with 10 µM DCFH at 37 °C in the dark for 30 min. The cells were then washed with PBS, trypsinized, collected in 500 µL of PBS, and analyzed. ROS production was expressed as iMFI ratio, that is, the difference between the iMFI of treated and untreated cells over the iMFI of untreated cells (iMFI is the product of the frequency of ROS-producing cells and the median fluorescence intensity).
4.13. Cell Growth and Nanosponge Cellular Uptake Assays on Three-Dimensional Cell Culture
Cell suspensions (250-cell spheroids) 40 µL were dispensed into the access hole at each cell culture site to form a hanging drop on a Perfecta3D® 96-well Hanging Drop Plate (3D Biomatrix, Ann Arbor, MI, USA). On day 8 of the HCT116 and DU145 spheroid culture, 15 µL of different GSH-NS B or GSH-NS D concentrations (0.5, 2.0, 4.0, and 6.0 mg/mL) was added to each cell hanging drop and MCS growth was analyzed at 24, 48, and 72 h after nanosponge incubation. Noteworthy is the fact that we had to use a different concentration range for 3D cell growth assay (0.5, 2.0, 4.0, and 6.0 mg/mL) to obtain the dose-response data necessary to calculate the IC50 values from the one used in the 2D cell growth assay (0.5, 2.0, 4.0, and 3.0 mg/mL). Phase contrast photographs were taken by the DMI4000B microscope (Leica, Milano, Italy) and the diameter of each MCS was measured by Leica Application Suite Software (Leica) and the volume (V) was calculated using the equation V = 4/3πr3. Coumarin 6-loaded nanosponge uptake by MCS at the respective IC50 at 24 h for GSH-NS B or GSH-NS D was analyzed by fluorescence microscopy using a DMI4000B microscope (Leica).
4.14. Statistical Analysis
The results are expressed as the average value ± standard deviation (St.Dev) of three independent experiments. Median-effect analysis was performed by CalcuSyn software version 2.11 (Biosoft, Cambridge, UK) to calculate the values of the concentration required to cause a 1% inhibition of cell growth (IC1) and for a 50% inhibition of cell growth (IC50) for each nanosponge formulation. Statistical analyses were performed on Prism software version 6 (Graph-Pad, La Jolla, CA, USA) using a Student’s t-test and one-way analysis of variance (ANOVA) to calculate the threshold of significance as appropriate. Statistical significance was set at p < 0.05.