In recent years, there has been a growing search for developing high-efficiency nanomedicines for cancer treatment . Consequently, the scientific community has focused on improving the targeting of nanoparticles (NPs) to tumor cells through surface functionalization with active groups (e.g., folate, monoclonal antibodies) . However, according to the literature, only 0.7% of the injected dose of NPs accumulates in tumors and <0.0014% are internalized by the cells . Once in contact with blood, NPs interact with a series of physiological components (e.g., amino acids, salts, and proteins), which can induce poor colloidal stability or changes in the original chemical and biological identity of these particles, impairing their therapeutic efficiency . Proteins and other biomolecules can be adsorbed on the surface of NPs (protein corona formation), masking their original functionality and hiding their target ability . Protein corona can further lead to the formation of aggregates of NPs and activation of unplanned biological pathways (e.g., the complement system) . The factors mentioned above hamper the biodistribution of nanomedicines and their targeting efficiency, therefore causing adverse effects.

Multiple functionalization strategies to produce colloidally stable NP dispersions with retained targeting capacity have been widely investigated . While a panel of molecules (e.g., polyethylene glycol, zwitterionics, glycans, aptamers) can improve the stability of NPs in biological media by preventing the adsorption of proteins , developing methods that can meet the trade-off between targeting and colloidal stability of NPs in such media has proven to be a major challenge . Our group produced SiO2NPs functionalized with a steric stabilizer (zwitterionic sulfobetaine silane) and biologically active groups (amino, mercapto, or carboxylic functionalities) to interact with cells, viruses, and bacteria . Pan et al. developed mesoporous silica nanoparticles (SiO2NPs) covalently attached to (i) poly(oligo(ethylene glycol) monomethyl ether methacrylate) for improved stabilization and (ii) targeting peptide for targeted delivery aimed at increasing efficiency against cancer. Although valuable, these systems have not been able to provide reduced protein corona formation and targeting ability, or they have not been scrutinized in complex biological environments. In fact, there is a lack of more in-depth studies in complex media to assess the stability of NPs, along with the possible protein corona formation, eventual hemolytic activity, and targeting ability. Parameters such as the length of stabilizing and directing groups and their absolute concentrations and proportions play a key role in obtaining NPs with the desired stability and functionality in bodily fluids .

Here, we proposed developing SiO2NPs colloidally stable in biological media and targetable to tumor cells through selective binding with folate receptors, highly expressed in tumor cell lines. Sulfobetaine zwitterionic (ZW) and folate groups were chosen as kinetic stabilizers and targeting agents, respectively. It is worth highlighting that sulfobetaines were selected specifically for their enhanced hydration properties, effectively preventing protein adsorption on various NPs and contributing to improved colloidal stability . Remarkably, functionalized NPs were stable in a complex medium (cell culture medium and human plasma) and showed greater potential for recognition by tumor cells.

Tetraethyl orthosilicate (TEOS, 98%), (3-aminopropyl)triethoxysilane (APTES, 99%), ammonium hydroxide (NH4OH, 28%), rhodamine B isothiocyanate, 1-ethyl-3-carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and folic acid were purchased from Sigma-Aldrich. (N,N-Dimethylaminopropyl)trimethoxysilane (DMAPTMS), 1,3 propanesultone (99%), ethyl acetate (HPLC), (3-aminopropyl)triethoxysilane (99%) were purchased from Fisher Scientific. Ethanol (absolute) was purchased from Merck. All reagents and chemicals were used as received without further purification. Water used in the described procedures was obtained from a water purification system (Purelab from ELGA, resistivity of 18.2 MΩ·cm–1).

The synthesis of rhodamine-labeled SiO2NPs was similar to the protocol proposed by the group . The dye precursor was synthesized by conjugation of the isothiocyanate group of rhodamine B isothiocyanate to APTES at a molar ratio of 50:1 (dye: APTES) in absolute ethanol under nitrogen. The resulting solution was continuously stirred for 24 h. The conjugate was then hydrolyzed in a basic ethanolic solution with TEOS and catalyzed by ammonia. In summary, the dye precursor (0.1 mL) was added to ethanol (120.0 mL), TEOS (2.0 mL), and ammonia solution (6.1 mL), and the resulting mixture was stirred for 3 h. Afterward, the conjugate and TEOS were added at the same concentrations as described above, and the reaction was stirred overnight. The NPs were centrifuged (10000 rpm, 15 min), followed by a single wash with ethanol and four washes with deionized water. Finally, they were re-dispersed in 120.0 mL of water. After, a silica shell was synthesized around the preformed fluorescent core by the addition of ethanol (60.0 mL), ammonia solution (1.4 mL), and TEOS (1.7 mL). The addition of TEOS was done at a rate of 1.0 mL per minute, using a syringe pump (New Era Pump Systems, NE8000, Farmingdale, NY). Posteriorly, the solution remained under stirring for 24 h. The resulting suspension was purified and re-dispersed in water for storage and subsequent functionalization.

Scanning electron microscopy (SEM) micrographs were obtained in a high-resolution FEI Inspect F50 microscope. A NP suspension (7 μL) was deposited directly onto a copper substrate, dried, and sputter-coated with Au using a Bal-Tec SCD050 Sputter Coater. Secondary electrons were collected after backscattering of the Au-coated samples attained by electron beams with a 5 kV acceleration voltage.

The particle hydrodynamic diameter and zeta potential were evaluated on a Malvern Zetasizer ZS equipment (Malvern Instruments Ltd., UK – detection angle of 173° and laser wavelength of 633 nm). For DLS measurements, NPs were dispersed in MilliQ water (1.0 mg·mL–1). To determine the zeta potential, the NPs were diluted in 10 Mm of phosphate buffer at a concentration of 1.0 mg·mL–1. All measurements were made in triplicates at room temperature.

The 1H NMR spectra were obtained in Bruker spectrometers operating at frequencies of 500 MHz. The NP dispersions (25 mg) were centrifuged and concentrated to 600 μL of D2O with the standard TMSP. The samples were sonicated before measurements. Chemical shifts (δ) were related in part per million (ppm) to the standard TMSP. The signal referring to the solvent used, deuterated water, was omitted from the spectra.

The elemental analysis measurements were performed on a CHN 2400 Elementary Analyzer (Perkin-Elmer). The NP dispersions were dried in an oven at 100 °C and the percentages of carbon, nitrogen, and hydrogen were obtained. It is noteworthy that only the nitrogen percentages were considered for the calculations.

The elemental composition of the NP surface was obtained using a K-Alpha XPS (Thermo Fisher Scientific) which operates with Al Kα X-rays with charge compensation. Spectra were recorded in three distinct areas per sample with 400 μm spatial resolution, using 200 eV pass energy. High-resolution spectra for C 1s, N 1s, Si 2p, and S 2p were recorded with a resolution of 0.1 eV, using a pass energy of 40 eV. All spectra were analyzed using CasaXPS® software.

Dynamic light scattering (DLS) measurements were performed to evaluate the colloidal stability of NPs in complex media. The media used for these measurements were i) Dulbecco’s modified eagle medium (DMEM, Sigma-Aldrich), supplemented with 10.0% fetal bovine serum (FBS, Sigma-Aldrich), with 1.0% of non-essential amino acids (Sigma-Aldrich), and with 1.0% of penicillin and streptomycin (Sigma-Aldrich) and ii) human plasma. The human plasma was in compliance with ethics in research committee (CEP) of UNICAMP/Brazil. Particularly, the plasma was diluted (final percentage of 5.5%) in phosphate-buffered saline (PBS, pH 7.4, 10 mM) for incubation with particles. The plasma concentration was determined based on in vitro experiments found in the literature using animal blood . In such experiments, mouse blood is diluted in PBS to obtain a concentration of 10 mg·mL–1 of hemoglobin. Generally, to achieve this concentration the blood must be diluted to a concentration of 10%. In this case, as the concentration of plasma in the blood is 55% , the plasma must be diluted to 5.5% to carry out the experiments. Analyses were performed in triplicate at 37 °C, with incubation times of 0, 1, 2, 3, and 24 h. The NP concentration used was 0.5 mg·mL–1.

To verify the adsorption of proteins on the functionalized SiO2NPs, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) experiment was performed. For this, the nanomaterials (in concentrations of 2 to 5 mg·mL–1) were incubated in DMEM culture medium (10% FBS) at 37 °C for 10 min, using the thermoblock Thermomixer C (Eppendorf). Posteriorly, the precipitate was washed with PBS (pH 7.4, 10 mM) to remove excess proteins weakly adsorbed on SiO2NPs. The washing procedure was performed by centrifugation (14000 rpm for 10 min at 4 °C) and was repeated four times. Then, Laemmli buffer (1610737, Bio-Rad) containing 50 mM of dithiothreitol (DTT, 1610611, Bio-Rad) was added to the final precipitate, followed by heating at 95 °C for 5 min, and 10 μL of this suspension was applied in a 12% SDS-PAGE gel. The gel was run at a voltage of 200 V for 1 h and then the gel was stained with silver nitrate to visualize the bands. PageRuler™ Prestained Protein Ladder (Thermo Scientific) was used as a standard. The DMEM culture medium used for SiO2NP incubation was also added to the gel for comparison purposes.

To determine the total protein concentration present in the protein corona of each NP, bicinchoninic acid (BCA) assay was used, following the recommendations of the manufacturer (23225, Thermo Scientific). First, the functionalized and non-functionalized SiO2NPs (concentration of 3 mg·mL–1) were incubated in i) DMEM culture medium (10% FBS) and ii) human plasma (5.5% in PBS) at 37 °C for 10 min, using the thermoblock Thermomixer C (Eppendorf). After this period, the precipitate was washed with PBS (pH 7.4, 10 mM) to remove excess proteins weakly adsorbed on the surface of SiO2NPs. The washing procedure was performed by centrifugation (14000 rpm for 10 min at 4 °C) and repeated four times. The SiO2NPs were resuspended in 25 μL of water and transferred to a 96-well plate. Then, 200 µL of working reagent (50:1, reagent A/B) was added. Finally, the plate was incubated at 37 °C for 30 min and quantification was performed using the absorbance value at 562 nm, using the Thermo microplate reader (Multiskan GO model). For quantification, it was necessary to prepare an analytical curve with diluted BSA standards already included in the kit provided by the manufacturer. Analyses were performed in quintuplicate.

To identify the main proteins adsorbed on the SiO2NPs, liquid chromatography coupled with mass spectrometry (LC-MS/MS) was used. The proteins were separated in SDS-PAGE as described limiting the protein run to only enter in the beginning of the resolving gel. Then, the gel was stained with Coomassie brilliant blue, and the unique bands from each sample were cut out. The preparation of samples for analysis by LC-MS/MS was performed according to previously described protocols . Briefly, the protein bands were bleached with a destaining solution and dehydrated in acetonitrile. Reduction and alkylation of disulfide bridges were performed with dithiothreitol and iodoacetamide, respectively, followed by in-gel trypsin digestion.

The peptides were desalted on a C18 Stage Tips column and an aliquot containing 2 µg of each sample was analyzed using an LTQ Orbitrap Velos (Thermo Fisher Scientific) coupled to a nanoflow EASY-nLC (Proxeon Biosystems) liquid chromatography system through a Proxeon nanoelectrospray ion source. Peptides were separated at a flow rate of 300 nL/min in a 2–90% acetonitrile gradient in 0.1% formic acid for a total gradient time of 65 min (35% acetonitrile at 33 min) using a PicoFrit analytical column (20 cm × ID75, 5 μM particle size, New Objective). The source temperature was set to 275 °C and the nanoelectrospray voltage to 2.2 kV. The mass spectrometer operated in data-dependent acquisition mode, where full scan MS1 spectra (m/z 300–1,600) were acquired at resolution r = 60,000 after accumulation of 1 × 106 ions. The 20 most intense peptide ions with charge state ≥2 were sequentially isolated to a target value of 5000 and fragmented by collision-induced dissociation in the linear ion trap using a normalized collision energy of 35%. Dynamic exclusion was enabled with an exclusion size list of 500 peptides, duration of 60 s, and repetition count of 1.

Proteome Data Analysis: Raw data was processed using MaxQuant v1.5.8 22 and protein identification was performed against the UniProt Human Protein Database (97,511 protein sequences, released in 2021) and the Bos taurus Database (6,404 protein sequences, released in 2022) using the Andromeda search engine. Carbamidomethylation of cysteines (+57.02 Da) was set as fixed modifications, and protein N-terminal acetylation (+42.01 Da) and methionine oxidation (+15.99 Da) were set as variable modifications. Trypsin/P was set as the proteolytic enzyme, with a maximum of two missed cleavages allowed. A tolerance of 10 ppm for precursor ions and 1 Da for fragment ions was defined. A maximum of 1% FDR calculated using reverse sequences was set for both protein and peptide identification.

HaCat and KB cells were cultured in a DMEM culture medium and kept in an atmosphere of 5% CO2, and 95% air at 37 °C in a humidified incubator. Subsequently, cells were seeded onto 96 well microplates at a density of 1 × 104 cells/well at a final volume of 200 μL and incubated for 24 h to obtain a subconfluent monolayer. Cells were exposed to SiO2NPs at concentrations of 0.05, 0.10, 0.50, and 1.00 mg·mL–1. After 24 h of incubation, cells were washed three times with PBS 1×, and Alamar Blue (Invitrogen, 10% in DMEM) was added to each well. The samples were incubated for 3 h at 37 °C. The supernatant was then collected from the wells, put in a fresh 96-well plate, and analyzed with a spectrophotometer (EnSpire 2300, Perkin Elmer) setting the excitation wavelength at 560 nm and the emission wavelength at 590 nm. Analyses were performed in triplicate.

Transfusion service human concentrated red blood cell (RBC) (type B+) plastic bags, collected according to their consent form for blood donation, were provided by the Hemocenter from the Faculty of Medicine at the University of Campinas, São Paulo, Brazil. First, the RBCs were washed three times with PBS (pH 7.4, 10 mM) followed by centrifugation at 5000 rpm for 10 min at 4 °C (centrifuge Eppendorf, 5810R model) and supernatant removal. All hemolytic assays were prepared using a stock suspension of 10% RBCs (10 mL, in PBS). Then, a PBS solution was used to dilute functionalized and non-functionalized SiO2NPs at concentrations ranging from 0.5 to 1.0 mg·mL–1. Later, 100 μL of the RBC stock suspension was added to distinct tubes and incubated in the static method for 1 h at room temperature after gentle homogenization. The final volume of the hemolytic assay in all experiments was 1 mL. Then, all tubes were centrifuged (10 min) and 200 μL of supernatant was removed from each tube and transferred to a 96-well plate. The supernatant hemoglobin quantification was done by measuring the absorbance of the solution (540 nm) with a microplate reader (Thermo, Multiskan GO model). Deionized water (900 μL) and RBC stock suspension (100 μL) were used as a positive control, whereas PBS solution (900 μL) and RBC stock suspension (100 μL) were used as a negative control. All hemolytic tests were performed in triplicate. A similar assay was performed but the NPs were incubated in DMEM (10.0% FBS) and not in PBS.

The ability of the particles to cause hemolysis was also evaluated using mouse blood. For these experiments, ten Swiss mice were used, obtained from the Multidisciplinary Center for Biological Investigation of the State University of Campinas (CEMIB/ UNICAMP). All animals obtained from CEMIB received water and food ad libitum (Nuvilab) and were kept in the vivarium of the Department of Cellular and Structural Biology (Area of Anatomy) of the Institute of Biology, under the responsibility of Dr. Valéria Helena Alves Cagnon Quitete, until they reached the experimental age for euthanasia. At 2 months of age, the animals were weighed on a Denver P-214 analytical balance (Denver Instrument Company), anesthetized with 2% xylazine hydrochloride (5 mg·kg–1, intramuscular; König) and 10% ketamine hydrochloride (60 mg·kg–1, intramuscular; Fort Dodge) and had their blood collected by cardiac puncture, followed by euthanasia of the animal. The procedures were approved by the Ethics Committee on Animal Use (protocol 5725-1/2021) and conducted in accordance with the Ethical Principles for Research with Animals, established by the Brazilian College of Animal Experimentation (COBEA). The detailed experimental procedure is described in ASTM standard E2524-08 (Standard test method for analyzing the hemolytic properties of nanoparticles) . Briefly, the total hemoglobin concentration of heparinized blood was measured using the cyanomethemoglobin method (EIAHGBC, Thermo Scientific), based on an analytical curve of hemoglobin concentration at an absorbance wavelength of 540 nm. The blood was then diluted to a hemoglobin concentration of 10 mg·mL–1 with Ca2+/Mg2+-free PBS. Functionalized and non-functionalized SiO2NPs were evaluated at concentrations of 0.5, 1.0, 3.0, and 5.0 mg·mL–1. Aliquots were prepared for a final volume of 1 mL, considering 80 µL of blood and the remainder NP + PBS. The tubes were incubated in an oven at 37 °C for 180 min, with gentle inversion of the sample tubes every 30 min. After incubation, the tubes were centrifuged at 10000 rpm for 10 min (4 °C), and aliquots of the supernatant (200 μL) were carefully removed from the tubes and transferred to a 96-well plate. The quantification of hemoglobin in the supernatant and the preparation of positive and negative controls were performed as described above.

To verify the folate receptor expression, the Western blot technique was used, which consists of transferring proteins from a polyacrylamide gel to an adsorbent membrane . The identification of the target protein is performed through the specific recognition of a specific secondary antibody.

To identify the folate receptor in KB (high receptor expression) and HaCat (low receptor expression) cells, they were initially washed with ice-cold PBS (pH 7.4, 10 mM), followed by the addition of approximately 1 mL of cell lysis buffer (RIPA with protease inhibitor (50×)). Afterward, they were incubated on ice for 30 min and homogenized at predetermined times. Next, the samples were sonicated for 10 s and subsequently centrifuged (12000 rpm, 20 min, 4 °C). The supernatant was recovered, kept on ice, and quantified using the BCA assay. All measurements were performed simultaneously and in triplicate. After quantification of total proteins in the cell lysate, 20 μg of total protein per sample was mixed with Laemmli buffer with DTT (50 mM) and separated by an SDS-PAGE assay. Proteins were transferred to a nitrocellulose membrane (Bio-Rad transfer system) using transfer buffer (2.5 mM TrisHCl, 20 mM glycine, 0.01% SDS and 20% methanol). Transfers were performed for 1 h 30 min (100 V). Next, the membranes were incubated with blocking buffer (Tris saline solution with Tween 20 (TBS-T) + 3% BSA) for 1 h (room temperature). Subsequently, they were washed with TBS-T buffer and incubated with primary antibody anti-folate alpha receptor at a dilution rate of 1:500 for 12 h at 4 °C. The membranes were rewashed with TBS-T buffer and incubated with a secondary antibody (Goat anti-Mouse IgG (H + L) HRP, ThermoFisher) at a ratio of 1:20000 for 2 h (room temperature). After a new series of washes with TBS-T, the peroxidase activity was revealed with a chemiluminescent solution (Clarity™ Western ECL Substrate – 1705060, Bio-Rad) for 5 min. Antibody to β-actin (AC-15, Novus) was used as an endogenous control for all samples.

To assess the internalization of NPs, HaCat and KB cells were cultured in a 96-well plate (1 × 104 cells/well) for 24 h. Subsequently, the SiO2NPs (functionalized and non-functionalized) were dispersed in DMEM (10% FBS) and added to the cells (0.5 mg·mL–1). The incubation was carried out for periods of 3, 6, 12, and 24 h. Afterward, the medium with SiO2NPs was discarded and the cells were washed 3 times with PBS. Finally, the cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 and 2% bovine serum albumin for 40 min. F-actin was stained for 30 min with 10 µg/mL phalloidin-FITC (fluorescein isothiocyanate), and the nuclei were counterstained with 2 µg/mL of 4’,6-diamino-2-phenylindole (DAPI). After that, samples were analyzed on the Operetta High Content Screening System microscope (PerkinElmer Life Sciences).

To obtain quantitative information on the internalization of functionalized and non-functionalized SiO2NPs, the flow cytometry technique was used. HaCat and KB cells were cultured in a 6-well plate (200,000 cells per well) for 24 h. Subsequently, the SiO2NPs (functionalized and non-functionalized) were dispersed in DMEM (10% FBS) and added to the cells (0.5 mg·mL–1). Incubation was carried out for periods of 3 and 24 h. Thereafter, the medium with SiO2NPs was discarded, and the cells were washed 3 times with PBS and removed from culture plates by trypsinization. Next, the cells were centrifuged to remove trypsin and subsequently washed with PBS. Following that, 4% paraformaldehyde was added for 20 min. Finally, the cells were centrifuged again, washed with PBS, and resuspended in PBS.

The Shapiro–Wilk test evaluated the normality of data. The Student t-test was used to make comparisons between two sets of data with a normal distribution (parametric data), while the Mann–Whitney test was used to make comparisons between two non-parametric data sets. One-way and two-way analysis of variance (ANOVA) and Tukey’s test were used to compare three or more parametric data. Differences were considered significant when p < 0.05. All statistical calculations were carried out using the R program version 3.6.2 (R Development Core Team, 2019).

Figure 1: a) Representation of the synthesis steps of NPs with kinetic stabilizer and tumor driver. SiO2NPs: NPs without functionalization; SiO2NPs-ZW: NPs with zwitterionic; SiO2NPs-ZW-NH2: NPs with zwitterionic + APTES and SiO2NPs-ZW-FO: NPs with zwitterionic + APTES + folate. b,c) SEM image and size distribution for SiO2NPs and SiO2NPs-ZW-FO (n ≈ 1000), respectively. Scale bar: 500 nm. d) DLS and zeta potential results for SiO2NPs, SiO2NPs-ZW, SiO2NPs-ZW-NH2, and SiO2NPs-ZW-FO samples. e) Results obtained by the elemental analysis technique. Values in mg of nitrogen present in 1 g of sample for each step of the synthesis. f) High-resolution XPS spectrum (C 1s) for SiO2NPs-ZW-FO. The main peaks for each sample are marked on the spectrum itself. g) Fluorescence intensity for non-functionalized SiO2NPs and ZW-FO-SiO2NPs after incubation with magnetic beads coated with the folate receptor.

Figure 2: (a) Representation of the protein adsorption on the surface of NPs, which can lead to particle aggregation as well as impair their targeting efficiency. Size distribution curves for (b) SiO2NPs, (c) SiO2NPs-ZW, and (d) SiO2NPs-ZW-FO in DMEM (10% FBS). For this analysis, a particle concentration of 0.5 mg·mL–1 was used, and different incubation times were evaluated. The approximate PDI for the NPs incubated in DMEM was 0.15. (e) Quantification of proteins adsorbed on SiO2NPs, SiO2NPs-ZW, and SiO2NPs-ZW-FO per mg of NP after incubation in DMEM medium (10% FBS). Results are displayed as mean ± standard deviation (n = 5). Size distribution curves for (f) SiO2NPs, (g) SiO2NPs-ZW, and (h) SiO2NPs-ZW-FO in human plasma. For this analysis, a particle concentration of 0.5 mg·mL–1 was used, and different incubation times were evaluated. The approximate PDI for the NPs incubated in plasma was 0.18 for SiO2NPs and SiO2NPs-ZW-FO, and 0.38 for SiO2NPs-ZW. (i) Quantification of proteins adsorbed on SiO2NPs, SiO2NPs-ZW, and SiO2NPs-ZW-FO per mg of NP after incubation in DMEM medium (10% FBS). Results are displayed as mean ± standard deviation (n = 5). The distinct symbols at the top of the bars indicate that there is a statistical difference between them. The most abundant proteins identified by LC-MS/MS on the hard corona of non-functionalized and functionalized SiO2NPs in (j) DMEM (10% FBS) and (k) human plasma. (l) Statistical analysis of protein enrichment (negative numbers) and depletion (positive numbers) on SiO2NPs, SiO2NPs-ZW, and SiO2NPs-ZW-FO in relation to DMEM and human plasma. ALB: serum albumin, TL: tissue leakage, C: complement, COA: coagulation, APO: apolipoprotein, AP: acute phase, IGG: immunoglobulin, A1P1: alpha-1-antiproteinase, FGB: fibrinogen beta chain, C1q: complement component 1q.

Figure 3: a) Illustration of intact red blood cells after the hemolysis process. Hemolytic activity of SiO2NPs, SiO2NPs-ZW, and SiO2NPs-ZW-FO diluted in b) DMEM (10% FBS) and c) murine blood. The concentrations of NPs used were 0.5 and 1.0 mg·mL–1 (b) and 0.5, 1.0, 3.0, and 5.0 mg·mL–1 (c). Results are presented as mean ± standard deviation (n = 3 for b and n = 10 for c). The common symbols on the top of the bars indicate that there is no statistical difference between them. For samples marked with an asterisk, no significant hemolysis was observed. Cell viability assay (Alamar Blue) of d) HaCat and e) KB cells after incubation for 24 h with non-functionalized SiO2NPs and with SiO2NPs-ZW-FO at concentrations of 0.05, 0.10, 0.50 and 1.00 mg·mL–1. Cisplatin (Cis) was added as a cell death control. Results are presented as mean ± standard deviation (n = 3). The common symbols on the top of the bars indicate that there is no statistical difference between them.

Figure 4: a) Representation of cellular recognition of SiO2NPs-ZW-FO through the folate receptor. b) Cell targeting assay using non-functionalized SiO2NPs and SiO2NPs-ZW-FO, after 24 h of incubation, obtained by an Operetta microscope. Cells were stained with DAPI (cell nucleus, blue) and phalloidin 488 (actin filaments, green) and SiO2NPs can be visualized in red. Scale bar: 25 μm. Percentage of positive cells for SiO2NPs, SiO2NPs-ZW, and SiO2NPs-ZW-FO, where c) HaCat and d) KB cells.