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Effects of mesoporous SiO2-CaO nanospheres on the murine peritoneal macrophages -- Candida albicans interface

The use of nanoparticles for intracellular drug delivery could reduce the toxicity and side effects of the drug but, the uptake of these nanocarriers could induce adverse effects on cells and tissues after their incorporation. Macrophages play a central role in host defense and are responsible for in vivo nanoparticle trafficking. Assessment of their defense capacity against pathogenic micro-organisms after nanoparticle uptake, is necessary to prevent infections associated with nanoparticle therapies. In this study, the effects of hollow mesoporous SiO2-CaO nanospheres labeled with fluorescein isothiocyanate (FITC-NanoMBGs) on the function of peritoneal macrophages was assessed by measuring their ability to phagocytize Candida albicans expressing a red fluorescent protein. Two macrophage-fungus ratios (MOI 1 and MOI 5) were used and two experimental strategies were carried out: a) pretreatment of macrophages with FITC-NanoMBGs and subsequent fungal infection; b) competition assays after simultaneous addition of fungus and nanospheres. Macrophage pro-inflammatory phenotype markers (CD80 expression and interleukin 6 secretion) were also evaluated. Significant decreases of CD80+ macrophage percentage and interleukin 6 secretion were observed after 30 min, indicating that the simultaneous incorporation of NanoMBG and fungus favors the macrophage noninflammatory phenotype. The present study evidences that the uptake of these nanospheres in all the studied conditions does not alter the macrophage function. Moreover, intracellular FITCNanoMBGs induce a transitory increase of the fungal phagocytosis by macrophages at MOI 1 and after a short time of interaction. In the competition assays, as the intracellular fungus quantity increased, the intracellular FITC-NanoMBG content decreased in a MOI- and timedependent manner...


Introduction
Nanoparticles are currently designed for intracellular drug delivery in order to reduce the toxicity and side effects of the loaded drug to the patient. Silica-based mesoporous nanoparticles present high loading capacity within their pores and the possibility of modifying their surface to target the damaged tissues [1]. However, depending on the characteristics of these nanocarriers, their interaction with tissues and cells could induce adverse effects [2]. Thus, recent studies evidence that micro-and nanoparticles can produce immunosuppression that initially limits early inflammation but contributes later to fibrosis, cancer, and infection [3].
Macrophages are among the first cell types which take up nanoparticles [4][5][6][7][8] and they are primarily responsible for their trafficking in vivo [9,10]. These phagocytic cells play a central role in host defense, removal of dead cells and pathogens, inflammatory response regulation and modulation of adaptive immunity [11]. Macrophages have functional plasticity that allows them to respond to their local environment, changing their phenotype between two extremes defined as pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes, characterized by different cell surface markers and cytokines [12]. Thus, the evaluation of the in vitro response of macrophages after exposure to nanomaterials is an important issue due to the key role of these phenotypes in both the host response and the inflammation/healing processes [13].
Phagocytosis by macrophages is a critical process for the uptake and degradation of infectious agents and senescent cells, and it participates in tissue remodeling, immune response, and inflammation [14]. A defective phagocytic ability of macrophages is considered as a critical determinant in the progression of different diseases because this fact is related to a suppressive adaptive immune response [15,16].
On the other hand, the evaluation of the possible macrophage capability impairment against pathogens after nanoparticle uptake is necessary to prevent infections that could occur during treatments based on the administration of these nanocarriers. In this sense, different strategies have been developed to minimize risk of infections related with the use of biomedical materials and their complications [17].
Candida albicans is a fungal pathogen that easily colonizes host tissues and implant devices forming mature biofilms directly involved in pathogenesis and drug resistance [18]. On the other hand, the pathogenicity of this opportunistic human fungal pathogen depends on the competence of host immune response that if diminishes, C. albicans behaves like a true pathogen. Both innate and adaptive components are involved in the immune response to microbial pathogens [19]. Thus, although the innate immune response plays a key role in the initial containment of the fungus, both cellular and humoral responses are needed to finally finish the infection [20][21][22][23][24]. The importance of phagocytes in the immune response against the fungus and the resolution of the infection has been accurately demonstrated [25].
Phagocytosis is a very important step by which macrophages destroy pathogens and a huge amount of scientific data defines this process as a very complex and essential phase for the immune response [26,27]. In this context, it is relevant to note that many pathogens have evolved strategies to avoid or subvert phagocytosis at various stages of this process [28,29].
Similarly, C. albicans has developed different mechanisms and one of these well-known strategies is the morphogenetic switch inside macrophages [30,31]. Other important fungal evasion strategies have been described [32,33], indicating the necessity to improve antifungal treatments against this pathogen.
In the present study, we have analyzed by flow cytometry and confocal microscopy the phagocytic capacity of murine peritoneal macrophages after treatment with mesoporous SiO2-CaO nanospheres labeled with fluorescein isothiocyanate (FITC-NanoMBGs) and subsequent infection with the fungus Candida albicans expressing a red fluorescent protein (RFP-Candida albicans) at two macrophage/fungus ratios (multiplicity of infection, MOI): MOI 1 and MOI 5. We have also carried out competition assays, after simultaneous addition of fungus and nanoparticles to peritoneal macrophages, in order to study the modulation of intracellular FITC-NanoMBG content by intracellular RFP-Candida albicans yeasts or vice versa. In these assays, we have also analyzed the effects of FITC-NanoMBGs and Candida albicans on macrophage polarization towards the pro-inflammatory M1 phenotype by measuring the CD80 expression (as specific M1 marker) and the secretion of interleukin-6 (IL-6, as proinflammatory cytokine).
It is important to highlight that the biomedical treatments with nanoparticles require the study of the effects of nanomaterials on the macrophage / microbial pathogen interface to know if the intracellular uptake of these nanoparticles could alter the immune response. On the other hand, the present study will allow us to obtain information for the possible future biomedical employment of these mesoporous SiO2-CaO nanospheres in an infection scenario. We have recently shown the utility of this nanomaterial for intracellular drug delivery [34,35]. These previous works have opened the way for future potential profits of this nanomaterial for antifungal delivery by the analysis of the uptake by macrophages of both nanospheres and C.
albicans. The present work will contribute for the better understanding of the functional state of macrophages after treatment with these potential nanocarriers.

Synthesis and characterization of FITC-NanoMBGs
The synthesis of hollow mesoporous SiO2-CaO nanospheres (NanoMBGs) was carried out by the method described by Li et al. [36], using a double template strategy to obtain particles with a hollow core surrounded by a shell containing a radial porous structure (NanoMBGs). For the subsequent internalization studies, NanoMBGs were labelled with FITC thus obtaining FTIC-NanoMBGs particles (see supporting information for a complete description of the synthesis and labelling methods).
NanoMBGs were characterized by scanning electron microscopy (SEM) using a JEOL F-6335 microscope (JEOL Ltd., Tokyo, Japan), operating at 20 kV and equipped with an energy dispersive X-ray spectrometer (EDS). Previously, the samples were mounted on stubs and gold coated in vacuum using a sputter coater (Balzers SCD 004, Wiesbaden-Nordenstadt, Germany). Transmission electron microscopy (TEM) was carried out using a JEOL-1400 microscope, operating at 300 kV (Cs 0.6mm, resolution 1.7 Å). Images were recorded using a CCD camera (model Keen view, SIS analyses size 1024 X 1024, pixel size 23.5mm x 23.5mm) at 60000X magnification using a low-dose condition. Nitrogen adsorption/desorption isotherms were obtained with an ASAP 2020 porosimeter. NanoMBGs were previously degassed under vacuum for 15 h, at 150 ºC. The surface area was determined using the Brunauer-Emmett-Teller (BET) method. The pore size distribution between 0.5 and 40 nm was determined from the adsorption branch of the isotherm by means of the Barret-Joyner-Halenda (BJH) method. The surface area was calculated by the BET method and the pore size distribution was determined by the BJH method using the adsorption branch of the isotherm. Particle size was determined from SEM micrographs using the intercept technique.

Isolation and culture of murine peritoneal macrophages
Primary macrophages were obtained from the peritoneum of untreated mice as described elsewhere [37]. Briefly, naive non treated CL57/BL mice were killed, and the skin was removed from the abdominal area. Mice were then injected intraperitoneally with 4-5 mL of phosphate buffered saline (PBS) using an 18 gauge needle. Without extracting the needle, the abdomen was gently massaged and then as much fluid from the peritoneum as possible was slowly withdrawn with the syringe. After removing, the peritoneal cells were gently washed with PBS before use. All procedures were approved by Institutional Animal Care and Use Committees.

Candida albicans strains
The C. albicans strains used in this study were the clinical isolate SC5314 and the CAF2-dTOM2 derived from SC5314 expressing a red fluorescent protein (RFP) called RFP-Candida albicans [38], kindly provided by Dr. D. Prieto and Dr. J. Pla. This red fluorescent strain was synthesized by codon optimization of the DsRed-derived RFP dTomato gene [39] using the tetracycline-dependent integrative plasmid pNIM1R. Expression in C. albicans was also detectable in SD plates as pink-reddish colored colonies after two days of growth [40].

Treatment of murine peritoneal macrophages with FITC-NanoMBGs for 24 h and subsequent infection with RFP-Candida albicans for different times and multiplicity of infection
Prior to Candida infection, murine peritoneal macrophages were seeded a) onto 24 well culture plates (for confocal microscopy studies), containing sterile 12-mm-diameter round glass coverslips, at a density of 5 × 10 5 cells per well, and b) onto 6 well culture plates (for flow cytometry studies), at a density of 1 × 10 6 cells per well. Then, macrophages were treated with a FITC-NanoMBGs suspension (50g/mL), which involves approximately 5.3ꞏ10 9 FITC-NanoMBGs/mL, for 24 h at 37 ºC under a 5% CO2 atmosphere. After treatment, some wells were washed to retire the nanomaterial, meanwhile in others the nanomaterial was not removed, in order to compare the effects produced by the previous uptake of nanospheres and by the continuous presence of them. Controls with macrophages without FITC-NanoMBG treatment were carried out in parallel. On the other hand, RFP-C. albicans was grown overnight at 30 ºC on solid YED medium (to maintain cells in the yeast form). Yeast cells, harvested and washed twice with phosphate buffered saline (PBS), were counted and diluted to the desired density in complete culture media.
In all the conditions described previously, macrophages were infected for different times (30,45 and 90 min) with C. albicans using two macrophage/fungus ratios (multiplicity of infection, MOI): MOI 1 and MOI 5. All the experiments were performed at 37 °C and 5% CO2. We employed controls with macrophages without C. albicans infection or without nanoesphere treatment in parallel.

Infection of murine peritoneal macrophages with RFP-Candida albicans and simultaneous treatment with FITC-NanoMBGs: competition assay
Similarly to the previous experimental conditions, murine peritoneal macrophages were seeded either onto 24 well culture plates (for confocal microscopy studies) and onto 6 well culture plates (for flow cytometry studies). Then, macrophages were simultaneously treated with both the fungus RFP-Candida albicans (MOI 1 and MOI 5) and the nanomaterial (FITC-NanoMBGs, 50μg/mL) for different times (30,45 and 90 min) at 37 ºC under a 5% CO2 atmosphere. We designed this experiment, considered as a competition assay, to evaluate the possible modulation of both macrophage fungal phagocytosis and FITC-NanoMBG uptake by the simultaneous presence of the nanomaterial and the yeasts, respectively. We employed controls with macrophages without C. albicans infection or without nanoesphere treatment in parallel.

Uptake of FITC-NanoMBGs and phagocytosis of RFP-Candida albicans by murine peritoneal macrophages evaluated by flow cytometry and confocal microscopy
To evaluate FITC-NanoMBG uptake and RFP-Candida albicans phagocytosis by murine peritoneal macrophages by flow cytometry, cells were washed three times with ice-cold phosphate buffered saline (PBS) and harvested using cell scrapers. Then, cell suspensions were centrifuged at 300xg for 5 min and the pellets were suspended in 300 l of PBS with 2% FBS. In the table, the percentage of phagocytosis corresponds to the % of macrophages that presented the fungus inside and therefore exhibited red fluorescence above the horizontal line (UL% + UR%). The percentage of FITC-NanoMBG uptake corresponds to the % of macrophages that presented nanospheres inside and therefore exhibited green fluorescence to the right of the vertical line (UR% + LR%). X Mean and Y Mean correspond to the green and red fluorescence values respectively, in arbitrary units, emitted by each cell population (each quadrant).
For confocal microscopy studies, cells were fixed with 3.7% paraformaldehyde in PBS and observed using a Leica SP2 Confocal Laser Scanning Microscope. The fluorescence of RFPexpressing C. albicans was excited at 488 nm and measured at 584/663 nm. DAPI fluorescence was excited at 405 nm and measured at 409/468 nm. FITC fluorescence of FITC-NanoMBGs was excited at 488 nm and measured at 505/547 nm.

M1 phenotype
In order to assess the effects of FITC-NanoMBGs and Candida albicans on macrophage polarization towards M1 phenotype, the CD80 expression as specific marker of proinflammatory M1 macrophages [8,41]  The amount of IL-6 in the culture medium was quantified by ELISA (Gen-Probe, Diaclone) with pre-coated strip plates, biotinylated secondary antibodies and streptavidin-avidin conjugated to horseradish peroxidase for colorimetric reaction, according to the manufacturer's instructions. Recombinant cytokine was used as standard. The absorbance at 450 nm of the samples and standards was measured using an ELISA Plate Reader. The sensitivity of this assay was 10 pg/mL and its inter-assay variation coefficient was < 10%.

Statistics
Data are expressed as means ± standard deviations of a representative of three experiments carried out in triplicate. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 19 software. Statistical comparisons were made by analysis of variance (ANOVA). Scheffé test was used for post hoc evaluations of differences among groups. In all of the statistical evaluations, p ˂ 0.05 was considered as statistically significant.  The inset shows a TEM image evidencing the hollo core-radial shell porous structure of the spheres. Nitrogen adsorption analysis confirm the highly porous structure of NanoMBG spheres, showing high values of surface area (more than 540 m 2 ꞏg -1 ) and pore volume (see Table 1).

Influence of previous 24 h FITC-NanoMBG treatment on RFP-Candida albicans phagocytosis by murine peritoneal macrophages
In order to know if the uptake or/and the presence of FITC-NanoMBGs in the culture medium alter the function of murine peritoneal macrophages, we analyze RFP-Candida albicans phagocytosis by these cells after 24 h of treatment with this nanomaterial by performing two experimental approaches. Thus, after FITC-NanoMBG treatment, some wells were washed to eliminate the nanomaterial and fresh medium was added, meanwhile other wells were maintained with nanospheres in the medium. After these two treatments, a fungal phagocytosis assay using a RFP-Candida albicans strain was settled down.     Figures 2A and   2B. The results obtained after FITC-NanoMBG treatment allow us to affirm that these nanospheres do not exhibit toxicity on the macrophage function in the tested conditions because their phagocytosis capability was time-and MOI-dependent after treatment with these nanospheres, in agreement with previous studies [6]. Interestingly, this macrophage function increases after FITC-NanoMBG treatment at the shortest time of fungal interaction and MOI 1, thus suggesting an initial stimulation induced by these nanospheres on the activity of peritoneal macrophages that decreases along the time, probably due to the nanomaterial uptake ( Figure   2A). Moreover, in these experiments with and without the presence of nanospheres, we did not detect significant differences in fungal phagocytosis, thus indicating that macrophages discern between the nanomaterial and the fungus.

RFP-Candida albicans phagocytosis and FITC-NanoMBG uptake by macrophages in a competition assay
We performed a competition assay in order to evaluate the possible modulation of both RFP-Candida albicans phagocytosis and FITC-NanoMBG uptake by macrophages due to the simultaneous presence of the nanomaterial and the yeasts, respectively. In this kind of assay, we cultured murine peritoneal macrophages with FITC-NanoMBGs (50μg/mL) and fungal cells (at the desired MOI) simultaneously during different times (30 min, 45 min and 90 min) and we detected intracellular RFP-Candida albicans and FITC-NanoMBGs by flow cytometry and confocal microscopy as described above. We carried out controls with macrophages without C. albicans infection or without nanomaterial treatment in parallel.
As it is shown in Figure 3A, after 30 min of the competition assay and at MOI 1, we detected a significant increase of the phagocytic capability of macrophages (45%) induced by the presence of nanospheres comparing with the of control macrophages without nanomaterial (25%).
However, after 45 min and 90 min of interaction at MOI 1, fungal phagocytosis by macrophages diminished in the presence of nanospheres from 60% and 70% phagocytosis by control macrophages to 50% and 61% phagocytosis, respectively ( Figures 3B and 3C). When employing a higher macrophage/fungus ratio (MOI 5), the presence of nanospheres also induced a significant fungal phagocytosis increase at the shortest time of interaction (30 min, Figure 3A). Thus, phagocytosis by control macrophages at MOI 5 was around 63% (white bar) and in the competition assay there was a phagocytosis stimulation to 72% (barred bar). decreased significantly along the time (52%, 49% and 36%). Thus, at the longest time of interaction of 90 min, we can observe a significant increase (p < 0.005) of the +Sph/+Cand macrophages, revealing that the macrophage capability of phagocytosis is not altered by the uptake of these nanospheres.
With respect to the results obtained at MOI 5 ( Figure 4B), when a higher dose of C. albicans is employed but using the same quantity of the nanomaterial, we detected higher percentage of the +Sph/+Cand population (comparing to MOI 1) that is maintained along the time (70%, 72% and 69%) and lower values of the +Sph/-Cand population (27%, 24% and 22%).
To conclude, the results represented in Figure 4 [47]. After the fifth day of culture, a decrease in the fluorescence intensity was observed and was attributed to the high mitotic index of HUVEC cells, that dilutes the nanoparticles per cell and leads to reduced fluorescence intensity. In our study, the maximum time of the assay was 90 min. This time is short enough so that the photoluminescence of the nanospheres is not affected. As these authors indicates, another notable factor for loss of fluorescence can be attributed to the efficient exocytosis of these nanomaterials by HUVEC cells [47].

Effects of FITC-NanoMBGs and Candida albicans on macrophage polarization towards M1 phenotype
Given the importance of the balance between the M1-mediated inflammation and M2-mediated regeneration after the treatment with any nanomaterial, we have evaluated the macrophage polarization process after nanospheres treatment. Thus, to assess the effects of FITC-NanoMBGs and Candida albicans on macrophage polarization towards M1 phenotype, we detect by flow cytometry the CD80 expression, as specific marker of pro-inflammatory M1 macrophages. In addition, we also quantify by ELISA the interleukin-6 (IL-6) levels in the culture medium. Two approaches were carried out: a) after 24 hours treatment of macrophages with NanoMBGs, cells were washed to eliminate the nanomaterial and then incubated 30 min with Candida albicans at the desired density (MOI 1 or MOI 5); b) simultaneous treatment of macrophages with NanoMBGs and C. albicans (MOI 1 or MOI 5) during 30 min. Control cells in the absence of nanomaterial or/and fungus were also analyzed.

Table 2. Effects of FITC-NanoMBGs and Candida albicans on macrophage polarization towards M1 phenotype
As it can be observed in Table 2 macrophages have a lower phagocytic capability [48]. These results are also in agreement with previous studies with Raw 264.7 macrophages treated with these nanospheres, which induced also a decrease of the M1 phenotype [34]. On the other hand, we have observed a similar macrophage behavior after treatment with graphene oxide nanoparticles [8]. In previous studies, we have evaluated the effect of different GO nanosheets functionalized with poly(ethylene glycol)-amine and labelled with fluorescein isothiocyanate (FITC-PEG-GO) on macrophage and lymphocyte immune response. We observed that these GO nanosheets produced significant dose-dependent decreases of cell proliferation and IL-6 levels, revealing weak inflammatory properties of this nanomaterial, in the same way that we observed with nanospheres in the present work [4,8]. On the other hand, we have studied the polarization of murine peritoneal macrophages towards M1 and M2 phenotypes in the presence of these GO evidencing that FITC-PEG-GO uptake did not induce the macrophage polarization towards the M1 proinflammatory phenotype, promoting the control of the M1/M2 balance with a slight shift towards M2 reparative phenotype. Similar results have also been observed with the nanospheres in the present study. The uptake of NanoMBGs by macrophages produces a decrease of CD80 + macrophage percentage and less IL-6 secretion, thus suggesting a non-inflammatory macrophage phenotype related to the simultaneous incorporation of NanoMBGs and the phagocytosis of the fungus [6,7]. Regarding the evaluation of the macrophage capacity to phagocytize the fungus Candida albicans, the effects of GO uptake on this macrophage activity has been evaluated in previous studies of our group. FITC-PEG-GO nanosheets were efficiently taken up by peritoneal macrophages inducing a significant increase of C. albicans phagocytosis by both pro-inflammatory macrophages (M1) and reparative macrophages (M2). On the other hand, after FITC-PEG-GO treatment and C. albicans infection, the percentages of GO + macrophages diminished when Candida uptake increased, thus suggesting the exocytosis of this nanomaterial. Similar results to those obtained in the present study indicates that the exocytosis of the nanomaterial is a dynamic mechanism favoring fungal phagocytosis [6,7].
Macrophages release different cytokines related with the modulation macrophage functions and the expression of cell surface markers [49]. In this sense, we have investigated the effects of the treatment with these nanospheres and C. albicans on the secretion of interleukin 6 (IL-6) by peritoneal macrophages, by measuring this pro-inflammatory cytokine in the culture medium.
We have selected the simultaneous treatment with NanoMBGs and C. albicans (MOI 1 and 5) for 30 min, as the most representative condition related to the observed CD80 decrease. Figure 6. Effects of NanoMBGs (50μg/mL) or/and Candida albicans at MOI 1 and MOI 5 on IL-6 production by peritoneal macrophages after 45 min of treatment. Statistical significance: ***p<0.005 (comparison between each condition and control cells without nanomaterial and without fungus). ϕϕϕ p<0.005 (comparison between cells treated with nanomaterial + fungus and cells treated only with nanomaterial).
As it can be observed in Figure 6, a significant decrease of IL-6 was detected after treatment with NanoMBGs when compared with control macrophages (p<0.005). The infection of macrophages with the fungus also induces a significant decrease of the IL-6 levels (p<0.005).
When comparing macrophages only treated with NanoMBGs and macrophages simultaneously treated with nanospheres and Candida, a similar and significant decrease (p<0.005) is detected.
Again, the higher number of yeasts (MOI 5) in the presence of NanoMBGs produces the most pronounced IL-6 decrease, in agreement with the observed reduction of macrophage CD80 + population ( Table 2). Previous studies with graphene oxide nanosheets evidenced a significant dose-dependent decreases of cell proliferation and IL-6 secretion by primary splenocytes and Saos-2 osteoblasts, revealing weak inflammatory properties of this other nanomaterial [4,50].
The decrease of both CD80 expression and IL-6 secretion by macrophages due to the presence of NanoMBGs and C. albicans, evidences the absence of a macrophage pro-inflammatory phenotype thus suggesting the potential utility of these nanospheres for drug delivery without harmful side effects.

Conclusions
The present work highlights the importance of evaluating the effects of macrophage infection on their functional capability after the uptake of nanoparticles and vice versa. This study demonstrates that the exocytosis of NanoMBGs in the presence of C. albicans is a dynamic mechanism which favors the fungal phagocytosis, in agreement with previous studies with other nanoparticles [5,6]. In summary, the NanoMBG uptake by macrophages does not alters their phagocytosis capability and phenotypic plasticity. The following conclusions reflect the importance of studying the nanomaterial / macrophage / pathogen interface to design efficient therapies based on these nanocarriers potentially useful for drug delivery: 1. After assessing if the internalization of NanoMBGs decreases the phagocytosis capability of macrophages, we have demonstrated that the previous uptake of these nanospheres does not alter the macrophage function.
2. An efficient and maintained in time incorporation of the nanomaterial by macrophages was observed even in the presence of high doses of the fungus (MOI 5), thus ensuring the intracellular location of these nanospheres.
3. The significant decrease of CD80 expression and IL-6 secretion evidences a noninflammatory macrophage phenotype related to the simultaneous incorporation of NanoMBGs and the fungus. 5. Once we had settled down a phagocytosis competition assay (between nanospheres and fungus), necessary to validate macrophage functional capability, our results have confirmed that macrophages clearly distinguish between the inert material and the live yeast, being a dynamic process and favoring the microbial phagocytosis.
The present study evidences that phagocytosis of macrophages can be closely connected to their functional state and, it has opened the way for future potential profits of these nanospheres for antifungal drug delivery.