Immune cell profiling of silica and carbon coated silica nanosheets

C T Silica nanosheets (SiO ⁠2 NS) are considered to be a promising material in clinical practice for diagnosis and ther- apy applications. However, an appropriate surface functionalization is essential to guarantee high biocompatibility and molecule loading ability. Although SiO ⁠2 NS are chemically stable, its effects on immune systems are still being explored. In this work, we successfully synthesized a novel 2D multilayer SiO ⁠2 NS and SiO ⁠2 NS coated with carbon (C/SiO ⁠2 NS), and evaluated their impact on human Peripheral Blood Mononuclear Cells (PBMCs) and some immune cell subpopulations. We demonstrated that the immune response is strongly dependent on the surface functionalities of the SiO ⁠2 NS. Ex vivo experiments showed an increase in biocompatibility of C/SiO ⁠2 NS compared to SiO ⁠2 NS, resulting in a lowering of hemoglobin release together with a reduction in cellular toxicity and cellular activation. However, none of them are directly involved in the activation of the acute inflammation process with a consequent release of pro-inflammatory cytokines. The obtained results provide an important di-rection towards the biomedical applications of silica nanosheets, rendering them an attractive material for the development of future on


Introduction
With the rapid development of nanotechnology, nanomaterials provide important source of tools for multidisciplinary applications [1].The finite small size and high surface to volume ratio of nanomaterials together with the enhanced electronic, optical, magnetic, and catalytic properties, making them very attractive for application in a broader range of disciplines [2]. In medicine, the use of nanomaterials can provide a powerful tool with high carrier loading capacity, in vivo stability which elevates the potential for early detection and diagnostics and treatment of diseases [3,4]. Nanoparticles can provide a new way of cancer treatment and drug delivery [5][6][7][8]. In particular, silica nanoparticles have attracted attention in the past decades because of their extraordinary physical and chemical properties such as the feasibility of surface functionalization, size-dependent multicolor light emission, stability against photo bleaching and intriguingly, biocom patibility [9]. Mesoporous silica nanoparticles emerge as a biodegradable drug carrier to improve efficacy and reduce side-effects due to their unique structure that allows high drug loading and surface modifications [10,11]. These intrinsic features, added to the low cost production and ability to be functionalized with a wide range of molecules and polymers, make them good tools for biomolecule detection and separation. Although several studies were focused on cell biocompatibility of these materials [12][13][14][15][16][17][18], a crucial step is represented by the evaluation of the impact of nanomaterials on the immune system, independent of their specific application. In fact, after parenteral administration (e.g. intravenous, intramuscular, subcutaneous, etc.), nanomaterials interact directly with peripheral immune cells, either in the blood or in the peripheral tissues [19]. This interaction is widely influenced by different factors, including the chemical property of nanomaterials which can modify the compatibility with the immune system, stimulating and/or suppressing the immune responses [19,20]. It has been demonstrated that an alteration of the surface chemistry can signifi cantly reduce immunotoxicity and change the immune impact of nanoparticles, making them useful platforms for different applications in biomedicine [21]. Therefore, material construction represents a powerful means to produce more innovative materials able to fulfil various functions in bio related systems [22].
In this work, we designed and synthesized novel 2D multilayer SiO 2 nanosheets (SiO 2 NS) and SiO 2 nanosheets coated with carbon (C/SiO 2 NS) with high surface area and uniform pore size, exploring and clarifying the interactions with immune cells. The morphology and porosity of these nanosheets could be controlled through the use of different concentrations of surfactant or changing the reaction time during synthesis, or through post synthesis heat treatments. Variations of the  structure was studied by investigations using scanning electron microscope (SEM), Transmission Electron microscope (TEM), X-Ray diffraction (XRD), Energy Dispersive X-Ray spectroscopy (EDX), X-ray photoelectron spectroscopy (XPS), Brunauer-Emmett-Teller (BET) surface area measurements and pore structure determination. The main purpose of our study was to produce and characterize highly stable and re-dispersible silica nanosheets following its coating with carbon molecules. We aimed at understanding the role of carbon in the interaction between these materials and the immune system, studying its effects on the biocompatibility. This study paves the way for the use of silica nanosheets in nanomedicine for the prospective development as future medical biosensors or diagnostic/therapeutic tools that will be in contact with blood and immune cells.

Synthesis of SiO 2 nanostructures
The synthesis of the SiO 2 nanosheets was preformed using Microwave Assisted Precipitation Method. The method is based on the reduction of SiBr 4 using NaOH as reducing agent in a microwave reactor. The synthesis parameters such as power, microwave radiation exposure time, and pH of the solution were optimized. The SiO 2 nanosheets were obtained via homogeneous wet-chemical precipitation in the presence of a dispersant CTAB acting as a directional agent and a surfactant, to help the dispersion and prevent aggregation. In a typical synthesis, 4 g of analytical grade SiBr 4 (0.115 M), 2 g NaOH (0.5 M) and 2.37 g (0.065 M) of CTAB were mixed and dissolved in 100 ml of Ethanol: distilled water (H 2 O) in the ratio of (1:1) by volume, at room temperature, and stirred vigorously for 10 min. The completion of precipitation reaction is evidenced by the appearance of a white precipitate in the reaction medium. The mixture was subsequently heated in a microwave chemical reactor (MCR-3, China) where the microwave power was adjusted to 1/3 of full 800 W power rating. The solution remained exposed to microwave for approximately 10-15 min and was removed upon reaching boiling. The resulting solution was left to cool down naturally to room temperature. Finally, the precipitate was filtered and collected after cleaning thoroughly several times with deionized water and ethanol resulting in the silica nanosheets. The resultant precipitate was calcined at 600°C under atmospheric conditions for 2 h to remove the CTAB from the precipitated sheets. For carbon functionalization, graphite was mixed with the precipitates obtained by the method described above in a high temperature furnace (Lab Box Furnace, Michigan, USA). The high temperature (1500°C) permits the reaction between Carbon and silica to form carbon coated nanostructures. The process starts by mixing of graphite and precipitated silica to form a mixture of C/SiO 2 with a molar ratio of 3.6:1 and placed in 20 ml of water. Water was removed from this mixture by heating at 120°C for 24 h. The mixture was then placed in Alumina crucible, and heated to experimental temperature (approximately 1500°C) in Argon atmos phere for 15 min. Finally, the mixture was cooled down to room temperature prior to storage in a desiccator until further use.

Materials characterization
Scanning electron microscopy (SEM) was carried out in a (NOVA NANOSEM 450, FEI company, USA) microscope operated at 12 mA and 5 kV. The samples were coated with a gold film prior to the observation. Transmission electron microscopy (TEM) measurements was carried out using a (Tecnai TF20, FEI company, USA) transmission electron microscope operated at an acceleration voltage of 60 kV. XRD analysis were carried out using (X'Pert PRO X-ray diffractometer, USA). The N 2 adsorption/desorption isotherms at 77 K was measured using a Nanometric instrument (Micrometrics ASAP 2420, USA). All samples were outgassed at 100°C for 6 h in vacuum prior to nitrogen adsorption/ desorption measurements.

Cell culture
Peripheral blood mononuclear cells (PBMCs) were obtained from ethylene diamine tetra acetic acid (EDTA)-venous (Sigma-Aldrich, USA) blood samples from informed healthy donors (aged 25-50 years). Cell separation and experiments were performed immediately after blood drawing. PBMCs were isolated from whole blood using Ficoll-Paque PLUS (GE Healthcare, USA) density gradient centrifugation. Purified PBMCs were cultured in RPMI 1640 medium (Sigma-Aldrich, USA) supplemented with 1% Penicillin/Streptomycin (Sigma-Aldrich, USA) and 10% heat-inactivated fetal bovine serum (Invitrogen, Thermo Fisher Scientific, USA). Both the uncoated and coated silica particles were homogeneously dispersed at the concentration of 1 mg/ml in sterile phosphate-buffered saline (PBS) 1 × . Initially, the compounds were sonicated for 45 min in a Branson 3200 water bath sonicator (Kimco Distributing, USA) and vortexed for a few seconds. An additional sonication was performed for 10 min prior to each experiment. The experiments were performed on blood samples from at least three different donors to average variations of the immune system. All the experiments were performed at least in triplicate.

Viability and apoptosis assay
Viability assays were performed using Annexin V/ 7-amino actinomycin D (7AAD) labeling (Thermo Fisher Scientific, USA). Annexin V is a 35-36 kDa Ca 2+ -dependent phospholipid-binding protein with high affinity for the phospholipid phosphatidylserine expressed on apoptotic cells membrane. The second component is the DNA-binding dye molecule 7AAD, which can only enter cells when their membranes present Soubaihi et al. Colloids and Surfaces B: Biointerfaces xxx (2018) xxx-xxx characteristics of necrosis and late apoptosis. Annexin V was conjugated with Alexa Flour 488 and apoptotic cells were detected in the green channel; 7AAD binds necrotic or late apoptotic cells were detected in the red channel. The cells were treated as previously reported [51] with the silica nanosheets. The samples were then incubated for 6 h and 24 h using, for each compound and each time point for two different concentrations: 5 and 50 µg/ml. PBMCs were stained with Annexin V/7AAD staining and major immune cell populations were detected with fluorescently labelled monoclonal antibodies purchased from BD Biosciences (USA): CD14+ for monocytes, CD20+ for B cells, CD3+ for T cells and CD56+ for NK cells. The cells were incubated for 20 min in the dark and suspended in Annexin V 1X buffer. Ethanol 70% treated samples were used as positive controls. Analysis were performed by flow cytometry (FACS Canto II BD, Biosciences, USA) and 20,000 to 50,000 events were collected.

Biocompatibility assays
Fresh human heparinized whole blood was obtained from informed healthy volunteer donors. Red Blood Cells (RBCs) were purified from serum by centrifugation at 200 g for 5 min. RBCs were washed five times in sterile PBS 1X, then diluted in 1:10 ratio using the same solution. In order to determine the hemolytic activity of the silica particles, RBCs were treated with two different concentrations for each compound (5, 50 µg/ml) and vortexed for 6 and 24 h, respectively in a thermocycler at RT. Ultrapure water was used to induce hemolysis for positive control. The release of hemoglobin was analyzed measuring the absorbance in the supernatant at 570-690 nm with a microplate reader (Thermo Scientific, Applied Biosystems, Invitrogen) [52].

TNFα secretion assay
PBMCs were cultured (1 × 10 6 cells/well) at increasing doses of particles (5-50 µg/ml) or left untreated. Concanavalin A (ConA; 10 μg/ml) and bacterial endotoxin lipopolysaccharides (LPS; 2 μg/ml) were used as positive controls (Sigma-Aldrich, USA). Cell culture supernatants were collected to analyze tumor necrosis factor-alpha (TNFα) secretion by ELISA kit following the manufacturer's protocol (Boster Biological Technology Co. Ltd, USA). Briefly, a monoclonal antibody specific for TNFα was already pre-coated into the 96-well plates. Standards and test samples were then added to the wells diluted in 1:10 ratio, a biotinylated detection polyclonal antibody from goat specific for TNFα was subsequently added and then washed with PBS or TBS buffer. Horseradish peroxidase (HRP) substrate with 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma-Aldrich, USA) was used to visualize HRP enzymatic reaction since TMB can act as a hydrogen donor. TMB was catalyzed by HRP to produce a blue colored product that changed into yellow color after adding acid stop solution. The density of yellow color is proportional to the human TNFα amount of sample captured in plate. The optical density (O.D.) was recorded at 450 nm using a microplate reader (Thermo Scientific, Applied Biosystems, Invitrogen).

Morphology and microstructure of the synthesized silica
Powder X-ray diffraction (XRD) patterns of as-synthesized precipitated powders are shown in Fig. 1 which upon indexing to JCPDS Card #850335 for SiO 2 , shows that the material formed to be silica. A broad peak with the equivalent Bragg angle at 2θ = 23°was observed in both the coated or uncoated samples, suggesting that the synthesized materials are amorphous. The X-ray diffraction pattern of samples shows a shoulder at 2θ = 5°which correspond to 2θ = 4.8°of amorphous and mesoporous silica structures with empty channels, as reported by Martinez et al [53]. Xu et.al reported that the SiO 2 crystal layers are distributed in each nanosheets or in the lamellar structure of the nanosheets [54]. The diffraction patterns of carbonized nanosheets (C/ SiO 2 NS) exhibited a strong peak at 2θ = 26.5°corresponding to plane (002) of graphitic carbon and two weak peaks at 2θ = 44.3°and 54.5°, respectively, corresponding to graphitic carbon planes (101), and (004), as indexed with JCPDS Card #751621.
Scanning Electron Micrographs (SEM) and Energy Dispersive X-ray (EDX) spectra of the synthesized mesoporous SiO 2 and carbonized SiO 2 are shown Fig. 2. Both samples resemble sheet morphology with several micrometers size ranges. The silicon sheets appear to be very thin though the exact thickness cannot be estimated from the images. The morphology of SiO 2 shows nanosheets structures stacked together (Fig.  2a), resembling flat, irregular, and multilayered sheets of nanometer thicknesses. Closer inspection of Fig. 2b reveals that the C/SiO 2 NS's are made up of individual and irregularly shaped platelets or thin nanosheets. It can be observed that despite the carbon functionalization, the carbonated samples retain the morphology of the parent SiO 2 NS. Also, the carbon deposition interrupts the layered structure of SiO 2 NS and exfoliate them suggesting that the carbon diffuses between the SiO 2 NS layers.
Chemical composition of the synthesized nanosheets were quantified by Energy Dispersive X-ray (EDX) (Fig. 2). EDX spectrum of mesoporous silica SiO 2 NS reveals that the SiO 2 NS's ( Fig. 2a) are composed of silicon and oxygen (beside the carbon peak from the tape used to hold samples on the holder), in the atomic (O:Si) ratio of: 2.2:1, which is close to the stoichiometric ratio due to the crystallization of the precipitated SiO 2 at high temperature, removal of the water, and the condensation of Si−OH groups [55]. On the other hand, the atomic (O: Si) ratio in C/ SiO 2 NS sample (Fig. 2b) is approximately 3:1, while most of the sample consist of carbon. Also, the EDX spectrum did not show the presence of chloride or sodium ions in either samples, indicating that these ions were effectively removed from the precipitated SiO 2 samples. To determine in final details of microstructure of the mesoporous SiO 2 NS and C/SiO 2 NS samples, High Resolution Transmission Electron Microscopy (HRTEM) was used. The micrographs of typical SiO 2 NS samples (Fig. 3a, b) shows multilayered nanosheet structures with aspect ratio of~300 x 500 nm and a thickness of 1-2 nm with well defined small pores. The C/SiO 2 NS (Fig. 3c, d) samples in retrospect, are more like exfoliated nanosheets with aspect ratio~100 × 200 nm and a thickness of 1-2 nm and large non-uniform pores. None of the samples studied showed any ordered structural arrangement confirming the poor crystalline nature which is in good agreement with the results obtained with XRD.
The microstructures of the SiO 2 and C/SiO 2 NS were further characterized by N 2 adsorption-desorption analysis. The adsorption/desorption isotherms with the corresponding Brunauer-Emmett-Teller (BET) surface area analysis and the Barrett-Joyner-Halenda (BJH) pore size and volume are summarized in Fig. 4. The isotherms obtained for SiO 2 NS sample is similar to MCM-41 structure that consists of a regular arrangement of cylindrical mesopores [56]. This structure is characterized by a sharp pore distribution, a large surface and pore volume with diameters between 2-6.5 nm. The mesopores are vertically aligned cylindrical with typical type IV isotherm with a steep increase due to capillary condensations, according to the IUPAC classification (Fig. 4a) [57,58]. The curve also showed a pronounced knee with adsorption at ca. 0.2 and 0.3 P/P 0 indicating the formation of monolayer and uniform cylindrical mesopores respectively [59]. Also, the adsorption and desorption branches show an uptake of N 2 due to capillary condensation in the relative pressure (P/P 0 ) range of 0.2-0.3, demonstrating that the mesopores are uniform and open without any pore-blocking effects occurring during desorption [60]. The absence of a hysteresis loop in this interval between 0.3-0.9 P/P 0 (relative pressure) and the narrow pore size distribution, with an average pore mesopore diameter centered at of 2.56 nm indicates the consistency of the observations made with high resolution TEM (HRTEM) (Fig. 4b) [57,61]. However, the isotherms obtained for C/SiO 2 NS samples show a typical type III isotherm in adsorption-desorption isotherms (according to the IUPAC classification) characteristic of mesoporous solids, which is characterized by the hysteresis loop which is normally obtained for microporous materials [62][63][64][65][66]. Also, the absence of knee in C/SiO 2 NS curve is indicative of a week adsorbate-adsorbent interaction which prevent monolayer formation [67] Textural properties of the synthesized samples, BET surface area, BJH pore diameter, total pore volume can be summarized as follow: for the SiO 2 nanosheets, 971.4 m 2 /g, 2.56 nm, and 0.727 nm, respectively.
These results is in a good agreement with the typical textural properties of mesoporous silica nanosheets such as MCM-41 and SBA-15 nanosheets reported by many researchers [58,68]. The BET surface area, BJH pore diameter, and BJH total pore volume of C/SiO 2 NS samples were found to be 69.7178 m 2 /g, 6.1137 nm, and 0.061774 cm³/g, respectively. The lower surface area in the C/SiO 2 NS is attributed to arise from the carbon functionalization and blocking of the small pores similar results reported for SBA-15 [69]. The surface area and the total pore volume of C/SiO 2 NS sample were smaller than those of the SiO 2 NS. The decrease in BET surface area and porosity and the increase in pore diameter may be attributed to arise from the adherence of the carbon to the inner walls of the pores and the agglomeration of carbon on the surface of the SiO 2 NS results in the pore size distribution to be shifted approximately from 2.6 to 40 nm as shown in the inset of Fig. 4b. The agglomeration of carbon coated nanosheets lead to blockage of some micro-and mesopores [70].

Surface characteristics of the synthesized silica nanosheets
The surface composition of the samples was further confirmed by X-ray photoelectron spectra (XPS). The spectra attributable to C1 s, Si 2p and O1 s are shown in Fig. 5a and b respectively, confirming the elemental composition while providing the oxidation states of SiO 2 and C/ SiO 2 nanosheets. Fig. 5a shows the survey scan spectrum and high resolution XPS spectra of Si 2p and O 1 s of SiO 2 NS where only Si and O core levels are observed. Both the peaks are symmetrical after deconvolution, centered at approximately 103.00 eV and 532.00 eV, similar to what has been reported in the literature for binding energy values of Si 2p and O 1 s for SiO 2 [71]. No other obvious XPS peaks are observed here. The (O:Si) ratio is calculated to be (3.85:1), which is higher than the ideal proportion of Si and O elements for SiO 2 observed in SEM-EDX due to some atomic oxygen adsorption on the surface during storage of the samples prior to the XPS measurements [72]. However, the C 1 s peak at 284.79 eV was observed in C/SiO 2 NS as shown in Fig. 5b. High resolution XPS spectra of Si 2p and O 1 s for C/SiO 2 NS show a symmetrical peak after deconvolution, centered at approximately 103.00 eV and 533.00 eV with a major C1 s Peak at 288 eV. The atomic percentages of Si (13.65%), O (41.27%) and C (45.08%) and the calculated (O:Si) ratio is 3.3:1, which is similar to what was observed for the as prepared SiO 2 NS. The spectrum of Si 2p consisting of two peaks can be attributed to the C-Si bonding, located at the binding energy of about~103.053 eV and SiO 2 located at the binding energy of about~103.998 [48,49]. The spectrum of C1 s revealed that the amorphous carbon from C/SiO 2 NS sample represent the two possible bonds of carbon C O (~287.6 eV) with atomic percentage of 74.49% and C-Si (~283.3) with atomic percentage of 24.51%, respectively [73]. As can be observed, carbon exist in two chemical environments i.e. carbon bonded to Si and another peak at 287.6 is most probably due to carbon bound to oxygen species (around 75.46%). The shift of the Si 2p and O1 s peaks occur towards the greater binding energies indicating a chemical interaction of the introduced carbon atoms with atoms of Si and O in the synthesized samples [74].

Cell viability studies
Physico-chemical features of nanomaterials play a crucial role in defining their impact on the biological systems [75]. Nevertheless, the microenvironment with which they interact can affect the functional activity, including kinetics, signaling, transport, accumulation, and toxicity [76,77]. Successful commercialization of nanomedicines ultimately depend on demonstrating their superiority over existing approaches and on demonstrating their biosafety [78,79]. Indeed, a detailed understanding of the biological interactions of nanomaterials, not least the interactions with cellular components, is important both from the efficacy and safety points of view [80]. Several reports show that physical and chemical properties of nanomaterials can also regulate the interaction with immune cells, influencing their response [81].
Following the necessity to understand the immunological fate of different nanomaterials, we investigated in depth, the interaction of the two silica nanosheets, SiO 2 NS and C/SiO 2 NS, on peripheral blood mononuclear cells (PBMCs) obtained from informed healthy donors. The influence of the nanosheets was first analyzed on the viability of PBMCs (evaluating necrotic and apoptotic cells), followed by investigation of the functional effects, analyzing their whole blood biocompatibility and the expression level of one of the most critical cytokines (TNF-α). PBMCs were chosen as experimental model owing to their accepted clinical relevance compared to other cell lines or specific populations. Thus the ex vivo approach allows to perform assays and measurements directly on human cells, using experimental conditions resembling in-vivo conditions.
After exposure to SiO 2 NS and C/ SiO 2 NS samples, Annexin V staining shows no significant differences in the percentage of apoptotic cells (Fig. 6a). However, a significant increase of cellular apoptosis (p value < 0.05) was detected in B, T and NK cells after 6 and 24 h of incubation, when 50 µg/ml of SiO 2 NS samples were used (Fig. 6b). The total toxicity of PBMCs was not statistically significant for either type of samples, as observed following the analysis of each population (monocytes and B, T, NK cells) using different markers (CD14, CD20, CD3 and CD56, respectively). After 6 h of incubation with 50 µg/ml of SiO 2 NS indicate a statistically significant increase in percentage of necrotic cells in monocytes. Also at 24 h following the treatment with SiO 2 (5-50 µ/ml), the cells were positive for 7AAD. On the other hand, C/SiO 2 NS showed an enhancement in biocompatibility, compared to the control samples (attributed to the presence of the carbon functionalization) [15,40]. These results suggest that SiO 2 NS and C/ SiO 2 NS showed a favorable toxicity profile and did not affect the viability of primary immune cells [82]. 7-AAD viability staining was also used to detect cells with compromised membranes (late apoptotic and necrotic cells) as a separate test. Both nanomaterials did not show significant cell toxicity (Fig. 7a). An exception was found in monocytes with a reduction in cellular viability after the treatment with SiO 2 NS (5-50 µg /ml) after both 6 h and 24 h exposure (Fig. 7b).

Whole blood biocompatibility
Interestingly, the above data confirm what has been argued till now, namely that nanomaterial structure and composition may be engineered to minimize effects on biochemical and cellular components [83]. In fact, our results emphasize the important role of carbon functionalization in enhancing the compatibility with the living system and in reducing the immunological reactions.
To further confirm this hypothesis, we then investigated the SiO 2 NS and C/SiO 2 NS biocompatibility on whole blood cells. Hemolysis tests were performed on human red blood cells (RBCs). The samples 9 were treated using two different concentrations for each of the materials synthesized in this work (5-50 µg/ ml) and vortexed for 6 and 24 h.
As expected, carbon coated silica nanosheets presented a higher level of biocompatibility compared to silica nanosheets. Following these findings, we can reconfirm the assumption that carbon is a relevant component to enhance the function of biomaterials significantly. In fact, hemolysis values (reported in absorbance 570-690 nm) displayed no release of hemoglobin in supernatant following treatment with C/SiO 2 NS at either time points used in this study. However, a statistically significant hemolytic activity (p value < 0.01 and p value < 0,001 respectively) was found in cells treated with high concentrations of SiO 2 NS (50ug/ml) after either 6 h or 24 h (Fig. 8).

TNFα secretion
The immune-compatibility of silica nanosheets was also investigated by measuring TNFα expression levels, which is a potent cytokine normally secreted by monocytes/macrophages and directly involved in the activation of the acute inflammation process [84]. Interestingly, SiO 2 NS and C/SiO 2 NS did not stimulate the release of TNFα (Fig. 9), although carbon functionalization provides an increase in the biocompatibility of silica nanosheets compared to those which were not coated; results showed that both the silica samples did not enhance the innate immune response and TNFα production.

Conclusions
Despite recent progress in the field of nanomedicine, basic understanding of the interaction of nanomaterials with the immune system remains insufficient. This work reports the wet chemical synthesis and characterization of novel 2D multilayer silica nanosheets (SiO 2 NS) and SiO 2 NS coated with carbon (C/SiO 2 NS) of high surface area, narrow pore size distribution and uniform pore diameters. The materials synthesized are potential vehicle for drug delivery and treatment as demonstrated by a wide-ranging approach aimed at analyzing the immunotoxicological impact of on human Peripheral Blood Mononuclear Cells (PBMCs). We further demonstrate in this work that carbon atoms, used to functionalize SiO 2 NS, improved the biosafety of the silica nanosheets, resulting in a reduction of cell toxicity. Thus functionalization of SiO 2 NS with biocompatible molecules increases their stability, minimizing the interactions with other biomolecules and reducing the risk of immunological responses. In summary, we synthesized SiO 2 NS and C/SiO 2 NS and carefully evaluated biological activities which could be correlated to microstructure of the synthesized materials and assessed their potential toxicity by a pilot ex-vivo study on primary immune cells. It was demonstrated that silica nanosheets show potential to be applied in drug delivery that needs to be further verified by clinical practice. The absence of toxicity and activation stimuli, of C/SiO 2 NS, provides interesting proofs regarding the overall immune-compatibility of these structures as potential drug carriers.