In vitro biocompatibility study of sub-5 nm silica-coated magnetic iron oxide fluorescent nanoparticles for potential biomedical application

Magnetic iron oxide nanoparticles (IONPs), for their intriguing properties, have attracted a great interest as they can be employed in many different biomedical applications. In this multidisciplinary study, we synthetized and characterized ultrafine 3 nm superparamagnetic water-dispersible nanoparticles. By a facile and inexpensive one-pot approach, nanoparticles were coated with a shell of silica and contemporarily functionalized with fluorescein isothiocyanate (FITC) dye. The obtained sub-5 nm silica-coated magnetic iron oxide fluorescent (sub-5 SIO-Fl) nanoparticles were assayed for cellular uptake, biocompatibility and cytotoxicity in a human colon cancer cellular model. By confocal microscopy analysis we demonstrated that nanoparticles as-synthesized are internalized and do not interfere with the CaCo-2 cell cytoskeletal organization nor with their cellular adhesion. We assessed that they do not exhibit cytotoxicity, providing evidence that they do not affect shape, proliferation, cellular viability, cell cycle distribution and progression. We further demonstrated at molecular level that these nanoparticles do not interfere with the expression of key differentiation markers and do not affect pro-inflammatory cytokines response in Caco-2 cells. Overall, these results showed the in vitro biocompatibility of the sub-5 SIO-Fl nanoparticles promising their safe employ for diagnostic and therapeutic biomedical applications.


X-ray diffraction analysis (XRD)
XRD measurements were performed using a powder diffractometer (Philips X'Pert Pro) operating in the reflection mode with CuKa radiation and equipped with a graphite back monochromator.

Magnetization measurements
The magnetic property of the nanoparticles was analyzed by vibrating sample magnetometry (VSM) and superconducting quantum interference device (SQUID) at a magnetic field of 100 Oe for both field-cooling (FC) and zero-field-cooling (ZFC).

Phase contrast and Fluorescence Microscopy
The CaCo-2 cells (2x10 4 cells/cm 2 ) were seeded on cover glasses in a 24-well plate and allowed to adhere for 24h. Then sub-5 SIO-Fl nanoparticles, treated ultrasonically for 5 min to break up aggregation, were dispersed in cell culture medium and added to the CaCo-2 cells at a concentration of 10 µg/ml, 50µg/ml and 100 µg/ml. 48h after exposure the cells were repeatedly washed with PBS and fixed in paraformaldehyde 4% at 4°C for 10 min, then washed twice in Ca 2+ /Mg 2+ -free PBS and stained for nuclei localization with Hoechst 33342 (trihydrochloride trihydrate). Phase contrast and fluorescence measurements were performed by using an inverted microscope (Olympus IX51, RT Slider SPOT -Diagnostic instruments, Sterling Heights, MI, USA) equipped with a 20X, 40X and 60X objective and with a cooled CCD camera (Spot RT Slider, Diagnostic Instruments).

Fluorescence microscopy analysis of Rhodamine phalloidin-labelled F-actin
The CaCo-2 cells (2x10 4 cells/cm 2 ) were seeded on cover glasses in a 24-well plate and after 24 h of culture incubated with sub-5 SIO-Fl nanoparticles at three different concentrations (10µg/ml, 50µg/ml, and 100 µg/ml). 7 days after exposure the cells were repeatedly washed with PBS and fixed in paraformaldehyde 4% at 4°C for 10 min, washed twice in Ca 2+ /Mg 2+ -free PBS and permeabilized at room temperature for 15 min (0.1% Triton X-100, 1% BSA; Sigma-Aldrich). The cells were also incubated with phalloidin tetramethylrhodamine isothiocyanate conjugated (1:100), an anti-actin toxin (Sigma), in a blocking buffer for 1 h. Cells were washed three times with PBS, counterstained for nuclei localization with Hoechst 33342 (trihydrochloride-trihydrate), and examined. Fluorescence measurements were using an inverted microscope (Olympus IX51, RT Slider SPOT -Diagnostic instruments, Sterling Heights, MI, USA) equipped with a 20X, 40X and 60X objective and with a cooled CCD camera (Spot RT Slider, Diagnostic Instruments).

Real-time quantitative reverse transcriptase polymerase chain reaction analysis (qPCR)
Amount of target was calculated using the 2 -ΔΔCt equation. Δ Ct = (average target Ct -average GAPDH Ct) ΔΔ Ct = (average Δ Ct treated sample -average Δ Ct untreated sample) Before using ΔΔ Ct method for quantification, we performed a validation experiment to demonstrate that efficiency of target genes and reference GAPDH were equal. Real-time PCR was performed with Sybr Green I Mastermix (Applied Biosystems) using an ABI PRISMTM 7000 Sequence Detection System. Each reaction was run in triplicate and contained 0,5 µl of cDNA template along with 250 nM primers in a final reaction volume of 25 µl. The genes investigated were ALP1 and VIL1. The specific primers used are the following: ALP1: 5'-ccaggacatcgccactcag-3'; 5'-tcagtgcggttccacacata-3' VIL1: 5'-gctatctatggtgtgggaag-3'; 5'-cctgtagtctcttggtgttg-3' GAPDH: 5'-catcatctctgccccctct-3'; 5'-caaagttgtcatggatgacct-3' Cycling parameters were 50°C for 2 min., then 95°C for 10 min. in order to activate DNA polymerase, then 40 cycles of 95°C for 15 seconds and finally 60°C for 1 min. The Melting curves were performed using Dissociation Curves software (Applied Biosystems) to ensure that only a single product was amplified. As negative controls, we used tubes where RNA or reverse transcriptase was omitted during the RT reaction.

TEM analysis
The SIO nanoparticles, treated ultrasonically for 5 min to break up aggregation, were dispersed in cell culture medium and added to the CaCo-2 cells at a concentration of 50µg/ml. After 1h exposure the cells were repeatedly washed with PBS and fixed using glutaraldehyde (2.5%) for 1 h. The cells were washed several times with 0.15M cacodylate buffer and post-fixed with 1.5% osmium tetroxide for 1 h. After further washing with cacodylate buffer, the samples were dehydrated through an ethanol gradient from 70% to 100%, embedded and cut into ultrathin sections and examined as previously described.