Human Hepatocarcinoma Cell Targeting by Glypican-3 Ligand

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Human Hepatocarcinoma Cell Targeting by Glypican-3 Ligand Peptide Functionalized Silica Nanoparticles: Implications for Ultrasound Molecular Imaging Marco Di Paola, Alessandra Quarta, Francesco Conversano, Enzo Antonio Sbenaglia, Simona Bettini, Ludovico Valli, Giuseppe Gigli, and Sergio Casciaro Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b00327 • Publication Date (Web): 18 Apr 2017 Downloaded from http://pubs.acs.org on April 20, 2017

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Human

Hepatocarcinoma

Cell

Targeting

by

Glypican-3 Ligand Peptide Functionalized Silica Nanoparticles:

Implications

for

Ultrasound

Molecular Imaging Marco Di Paola,*,

†

Alessandra Quarta,‡ Francesco Conversano,† Enzo Antonio Sbenaglia,†

Simona Bettini,¶ Ludovico Valli,§ Giuseppe Gigli‡ and Sergio Casciaro† †Institute of Clinical Physiology, National Research Council, c/o Campus Ecotekne, via Monteroni, 73100 Lecce, Italy.

ABSTRACT

Silica nanoparticles (SiNPs) are widely studied nanomaterials for their potential employment in advanced biomedical applications, like selective molecular imaging and targeted drug delivery. SiNPs are generally low cost and high biocompatible, can be easily functionalized with a wide variety of functional ligands and have been demonstrated to be effective in enhancing ultrasound contrast at clinical diagnostic frequencies. Therefore, SiNPs might be used as contrast agents in echographic imaging. In this work, we have developed a SiNPs-based system for the in vitro

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molecular imaging of hepatocellular carcinoma cells that express high levels of Glypican-3 protein (GPC-3) on their surface. At this regard, a novel GPC-3 targeting peptide was designed and conjugated to fluorescent silica nanoparticles. The physicochemical properties, acoustic behavior and biocompatibility profile of the functionalized SiNPs were characterized; then, binding and uptake of both naked and functionalized SiNPs were analyzed by laser scanning confocal microscopy and transmission electron microscopy in GPC-3 positive HepG2 cells, a human hepatocarcinoma cell line. The results obtained showed that GPC-3-functionalized fluorescent SiNPs significantly enhanced the ultrasound contrast and were effectively bound and taken up by HepG2 cells without affecting their viability.

INTRODUCTION Recent advances in the design and synthesis of innovative smart nanomaterials boosted the development of new diagnostic and therapeutic approaches, such as selectively targeted molecular imaging and controlled drug release.1-5 Among them, silica nanoparticles (SiNPs) seem to be particularly promising thanks to their low cost, low toxicity,6,7 ease of synthesis protocols as well as of functionalization.8 Indeed, different techniques such as magnetic resonance imaging (MRI), optical fluorescent imaging, positron emission tomography, X-ray computed tomography, and ultrasound imaging have benefited of the advances in the development of imaging agents based on silica nanoparticles and silica-derived hybrid nanostructures, in fields spanning from cancer diagnosis to detection of labelled stem cells in vivo.9-12 Interestingly, as demonstrated by our group, owing to their acoustic properties, SiNPs from 160 to 660 nm diameters behaved, at least in agarose gel phantoms, as effective ultrasound


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contrast agents at routine diagnostic frequencies.13 More recently, we found that the ultrasound contrast enhancement power of SiNPs was optimal for nanoparticles of around 240 nm diameter, at concentrations fully biocompatible, as assessed by in vitro cytotoxicity tests.14 This is an aspect of crucial importance for their potential safe employment in non-ionizing echographic molecular imaging. Furthermore, dual-mode imaging, combining both magnetic resonance and ultrasound imaging has been obtained by coating silica nanoparticles with an outer shell of smaller superparamagnetic nanoparticles.15,16 Moreover, recent reports have demonstrated that silica-based fluorescent nanoprobes functionalized with specific ligands were effective in early apoptosis detection17 and in intracellular microRNAs imaging.18,19 Although several pathological conditions have been identified as potential targets of these nanomaterials, their most important applications are those related to cancer diagnosis and therapy at cellular and molecular level through functionalization-dependent specific targeting.20-26 Hepatocellular carcinoma (HCC) accounts for around 75% of all liver cancer types and is the fifth among all the most common cancer cases.27,28 Hepatocarcinoma cells are known to express on their surface high levels of Glypican-3 protein (GPC-3), a member of the glypican family, which, therefore, is considered a marker of hepatocellular carcinoma29,30 and an ideal candidate for specific targeting.31,32 Indeed, recent literature data have shown that streptavidin-conjugated, fluorescent superparamagnetic iron oxide nanoparticles were successfully targeted to liver cancer cells preincubated with a biotinylated anti GPC-3 monoclonal antibody allowing dual-mode optical and MR imaging.33 Furthermore, Lee et al. have reported that a synthetic fluorescent peptide of seven aminoacidic residues binds to GPC-3 protein with very high affinity, allowing fluorescence imaging of liver cancer cells.34 On these bases, we have studied the targeting effectiveness of hepatocellular carcinoma cells by novel fluorescein isothiocyanate (FITC)-loaded silica nanoparticles


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functionalized with the above mentioned GPC-3 ligand peptide, in order to assess their potential suitability as contrast agents for ultrasound molecular imaging. Physicochemical properties of SiNPs were characterized by transmission electron microscopy (TEM), Z-potential measurements and fluorescence spectra analysis. Their acoustic behavior was also investigated. Binding and uptake of both naked and functionalized SiNPs were evaluated on HepG2 cells, a GPC-3 positive human hepatocarcinoma cell line, by laser-scanning confocal microscopy and transmission electron microscopy. MTT cell viability assay was also carried out to assess doseand time-dependent cytotoxicity of SiNPs. EXPERIMENTAL SECTION Chemicals.

Tetraethylorthosilicate

Aminopropyl)triethoxysilane)

(TEOS),

(APTES),

Mercaptopropyl)trimethoxysilane

(MPTS)

ammonium

Fluorescein and

hydroxide

isothiocyanate

(NH4OH),

(3-

(FITC),

(3-

3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl

tetrazolium bromide (MTT), BCA assay and protease were purchased from Sigma-Aldrich (Milan, Italy). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), L-glutamine, penicillin-streptomycin, trypsin, thiazolyl blue tetrazolium bromide (>97.5% TLC, and dimethyl sulfoxide (DMSO) were purchased from Euroclone (Milan, Italy). GPC-3 ligand peptide (N-Maleoyl-β-alanine-Tyr-Phe-Leu-Thr-ThrArg-Gln, purity grade >95%) was synthesized and functionalized with a maleimide moiety by Espikem s.r.l. (Sesto Fiorentino, Italy; www.espikem.com). HPLC and ESI-MS analysis of GPC3 ligand peptide is shown in Fig.S1 (Supporting Information). Phycoerythrin-conjugated, mouse monoclonal anti-human GPC-3 antibody was purchased from R&D Systems (Milan, Italy). All the solvents used were of analytical grade.


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Synthesis and physicochemical characterization of SiNPs. First, FITC molecules (20 mg) were derivatized with 3-aminopropyltriethoxysilane (APTES, 100 μL) in anhydrous ethanol (EtOH, 10 mL) under dark conditions for 24 h. The solution was then dried under vacuum.35,36 Thereafter, silica NPs were synthesized through the Stöber method.37 In a typical synthesis, 250 μL of TEOS, the functionalized FITC and 5 mL of NH4OH were added to 10 mL of EtOH (95% v/v): the mixture was stirred for 12 h. The SiNPs were washed five times by centrifugation at 2000 rpm in a mixture of water/acetone (1:1). Under these conditions SiNPs with an average diameter of 220 nm and a standard deviation below 10% (as statistically determined by means of ImageJ program analysis) were obtained. The functionalization of the obtained nanoparticles with the GPC-3 ligand peptide was carried out by using the maleimide-thiol coupling chemistry.38 For this purpose, prior to conjugation to the maleimide-derivatized peptide, the surface of the fluorescent silica nanoparticles was modified with SH-groups by direct adding of (3-Mercaptopropyl)trimethoxysilane, 5% v/v in methanol. The mixture was stirred for 2 h and then washed 2 times with methanol and 3 times with ultrapure water. Soon after, surfacemodified fluorescent silica nanoparticles were dispersed in MES buffer (40 mM MES, 10 mM EDTA, pH 6.0) and conjugated to the maleimide-derivatized peptide (molar ratio 1/500): the reaction mixture was kept under stirring overnight at 4 °C. The peptide-conjugated fluorescent SiNPs were washed 5 times by centrifugation and the collected particles were then dispersed in PBS for cellular studies. Characterization of naked and GPC-3 ligand peptide-functionalized SiNPs was carried out by transmission electron microscopy (TEM), Dynamic Light Scattering (DLS) and Z-potential measurements, Fourier transform infrared (FTIR) and fluorescence spectroscopy. In particular, TEM and DLS analysis provided measurements of particle shape, size and hydrodynamic


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diameter, whereas particle surface charge evaluation, as determined by Z-potential measurements, and FTIR spectroscopy confirmed the successful functionalization of the nanoparticle surface. Fluorescence spectra were collected to confirm the loading of SiNPs with the fluorochrome. Samples for both TEM and Z-potential measurements were prepared and analyzed as reported previously.39 Samples for TEM analysis were prepared by dropping a PBS solution (0.1 mg/mL) of nanoparticles on carbon-coated copper grids (Formvar/Carbon 300 Mesh Cu). After the evaporation of the solvent, samples were imaged on a Hitachi HD 2000 STEM operated at an accelerating voltage of 80 kV. Z-potential measurements and DLS analysis were carried out on 0.1 mg/mL SiNPs suspension in PBS, at pH 7.4, 25°C by using a Zetasizer Nano ZS90 (Malvern, PA) equipped with a 4.0 mW He–Ne laser operating at 633 nm and an Avalanche photodiode detector. In the case of DLS analysis, the nanoparticles were filtered (0.8 µm) prior to the measure, and three distinct readouts were collected per each sample. Infrared measurements were recorded by use of a PerkinElmer Spectrum One Fourier Transform spectrophotometer with an ATR plate. Aqueous suspensions of SiNP, GPC-3 ligand peptide and of GPC-3 SiNP were deposited directly on the ATR plate and each spectrum was acquired by averaging 64 scans. Fluorescence spectra were acquired with a Jasco FP 6200 spectrofluorimeter, by exciting at 494 nm a PBS solution of SiNPs (0.1 mg/mL) in a 1 cm light path cuvette. In all the experiments, SiNPs were sonicated in a bath sonicator (1 h, 25°c, 100% US power) immediately before their use. The stability of functionalized SiNPs was assayed by measuring the spontaneous release of GPC3 ligand peptide. Briefly, both naked and functionalized nanoparticles were suspended in PBS at 0.5 mg/mL and incubated at different time points (24-96 h), at 37°C. After each incubation time, the samples were spun down on Microcon centrifugal filters (MWCO 10.000 Da) to separate


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nanoparticles from released peptide, and the collected filtrate was qualitatively analyzed by UV spectrophotometry. Quantitative analysis on the collected filtrates was carried out by BCA protein assay, using pure peptide solution as standard to build a calibration curve. The colorimetric detection was carried out, as specified by the assay provider, at 562 nm. As internal positive control, nanoparticle suspension was incubated overnight at 37 °C in the presence of protease cocktail (protease type XIV from Streptomyces griseus, 5 mg mL-1 in PBS) to induce the complete release of targeting peptide. Thereafter, the nanoparticle suspension was processed as already specified on centrifugal filters to separate nanoparticles and proteases from the released peptide. Ultrasound Imaging. The ultrasound signal enhancement of both naked and functionalized SiNPs was assessed in agarose gel phantoms by using a conventional echographic device essentially as described by Chiriacò et al.14, with some minor modifications. Phantoms for ultrasound detection were prepared in Eppendorf tubes (2 mL volume, 1 cm diameter) as follows: firstly, each tube was filled with 0.5 mL of pure agarose solution (0.4% w/v), sonicated for 30 sec to avoid the formation of air bubbles and left to jellify overnight at 2-8°C. Afterwards, 1 mL of SiNPs-containing agarose solution, at two different concentrations of SiNPs (0.25 and 0.5 mg/mL), was layered on the top and subjected to the same sonication and jellification steps. Eppendorf tubes containing 1.5 mL of pure agarose gel were used as control. The experimental set-up for both ultrasound signal acquisition and analysis is described in details in Fig. S2 (Supporting Information). Cell Culture. All cell lines used in this study were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine and 5 mg/mL penicillin–streptomycin, at 37 °C in a 95% humidified,


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5% CO2 atmosphere. For all experiments, cells were harvested by trypsinization and resuspended in fresh medium before plating. Biocompatibility Test. For MTT viability assay, cells were plated into 96-well plates at a density of 2 104 cells/mL in 100 μL of culture medium and allowed to attach for 12 h. After the incubation, the medium was removed from each well and replaced with 100 μL of SiNP suspension prepared in complete medium, at concentration ranging from 0.05 to 0.50 mg/mL. Cells were then incubated for 1 to 72 h in the presence of SiNPs before the MTT viability assay was performed. For each incubation time untreated cells served as negative internal control. At least three independent experiments were carried out for each tested cell line and three replicate wells were employed for each tested SiNP concentration. At the end of each incubation time, the cells were washed two times with PBS and 100 μL of fresh culture medium containing 0.5 mg/mL of MTT were added to each well. After a further 2 h incubation at 37 °C, the MTT, reduced by the mitochondrial reductases of viable cells, formed a dark insoluble formazan precipitate. The precipitate was dissolved in 100 μL DMSO and the solution was centrifuged at 12.500 rpm for 1 min to remove pelleted debris and nanoparticles; thus, the resulting supernatant was assayed spectrophotometrically, by measuring the absorbance changes at 570 nm with an Epoch™ Multi-Volume Spectrophotometer System (BioTek, Winooski, VT, USA). Cell viability was expressed as percentage compared to control (untreated cells). Binding and uptake of SiNPs as a function of surface functionalization. Binding and uptake of fluorescent SiNPs were analyzed by confocal as well as electron microscopy. For both experiments HepG2 cells were plated at a density of 1  105/ml into 6 wells Nunclon Sphera plates (Thermo Scientific), which do not allow cell adhesion, and incubated 12 h to restore the


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membrane protein supply cleaved by trypsin treatment. Cells were then collected by pipetting, washed three times with PBS, resuspended in fresh medium containing 0.1 mg/ml FITC-loaded SiNPs and further incubated in the dark for 1, 6 and 24 h. At the end of each incubation time cells were washed three times and resuspended in 0.5 ml of PBS. Confocal analysis was performed with a Leica confocal scanning system (TCS-SP5, Leica, Mannheim, Germany). The samples were transferred onto a glass microscope slide and imaged under 488 nm laser excitation, with an acquisition window at 530 ± 15 nm. Fluorescent signal emitted by FITC was quantified with the Leica Application Suite AF 2.6.3 software (Leica, Mannheim, Germany), by measuring the fluorescence intensity of the pixels within a defined region of interest (ROI) enclosing a single cell. The fluorescence intensity of each cell was normalized with respect to the background signal of the scanned area. Samples for TEM analysis were prepared exactly as described above, and processed as reported in Quarta et al.39 Briefly, at the end of treatment with SiNPs cells were washed with PBS and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer at 4°C for 1 h. Fixed samples were washed three times with the same buffer and resuspended in 1% osmium tetroxide in cacodylate buffer. After 1h incubation, cells were washed again and dehydrated with 25%, 50%, 75% and 100% acetone. Two steps of infiltration in a mixture of resin/acetone (1/1 and 2/1 ratios) followed, and finally the specimens were embedded in 100% Epoxy resin at 60 °C for 48 h. 70 nm ultra-thin sections were cut with a diamond knife on a LKB-V ultra-tome, stained with lead citrate and examined under the electron microscope at 100 kV. Analysis of GPC-3 expression. Cell surface GPC-3 expression was studied on four different cell lines, namely HepG2, MDA-MB-231 (breast cancer), MG-63 (osteosarcoma) and SKOV-3 (ovarian carcinoma) cells, by confocal microscopy analysis using a phycoerythrin-conjugated


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mouse anti-human GPC-3 monoclonal antibody. Cells (1  105/ml) were grown for 24h on a glass coverslip placed at the bottom of each well of a 6 well plate. Then, the medium was removed, cells were washed three times with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. After further three washing, cells were incubated with anti GPC-3 antibody (ten times dilution in PBS) for 1 h in the dark at 2-8°C. Finally, cells were washed with PBS and imaged under 543 nm (PE-conjugated antibody) laser excitation, with acquisition windows at 600±25 nm. Binding and uptake of peptide-conjugated SiNPs as a function of cell surface GPC-3 expression. Binding and uptake of SiNPs as a function of GPC-3 expression was assessed by confocal microscopy in GPC-3 positive HepG2 cells and MG-63 (GPC-3 negative) cells. Cells were grown on a glass coverslip as described in the previous paragraph. After 24 h incubation, the medium was removed and replaced by fresh medium containing 0.1 mg/mL functionalized SiNPs. Cells were then further incubated for 1, 6 and 24 h. At the end of each incubation time, cells were fixed with paraformaldehyde as described previously, washed and imaged under 488 nm laser excitation, with acquisition windows at 530±15 nm. RESULTS AND DISCUSSION Preparation and physicochemical characterization of SiNPs. Fluorescent tracking of nanostructures devised for cellular studies is highly desired to allow quick and easy follow up of their intracellular localization and long-term fate. To this aim, monodisperse FITC fluorescent silica nanoparticles were prepared: FITC was first derivatized with APTES35 and then incorporated into silica NP during the synthesis process, based on Stöber method.37 Thereafter, the surface of the fluorescent SiNPs was functionalized with MPTS; the outward SH-groups of


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the nanoparticle coating enabled direct conjugation to the maleimide-activated GPC-3 targeting peptide. A schematic representation of nanoparticles synthesis and functionalization is shown in Fig.1. The peptide-coated fluorescent SiNPs were then carefully characterized prior to be tested in cellular models. The shape, size and surface charge of the SiNPs were determined by means of TEM, DLS and Z-potential measurements, whereas their fluorescence emission was assessed spectrofluorimetrically. Surface modification of the nanoparticles was further investigated by FTIR spectroscopy. TEM images (Fig. 2 A and B) of SiNPs clearly show their spherical shape, and underline a significant difference in the surface of naked particles as compared to functionalized SiNPs. Indeed, while naked SiNPs exhibit a quite smooth surface (A), those coated with the GPC-3 ligand peptide display a rather rough contour (B). Statistical analysis applied to the obtained TEM images provided an accurate measurement of the nanoparticles diameter, which resulted to be 220±10 nm for naked SiNPs, and 250±25 nm for GPC-3 SiNPs (Table 1). Successful functionalization of the SiNPs was confirmed by DLS and zeta-potential measurements. Indeed, as shown in Table 1, the overall hydrodynamic diameter of the nanoparticles resulted broadened, shifting from average 350 nm in the case of naked SiNPs to 450 nm for GPC3-targeted nanoparticles (see also Fig. S3 A). This significant broadening of the nanoparticle size as compared to that measured by TEM is not surprising, since the peptide coating is a dynamic volume and the DLS measure estimates the contribution of the surface coating and of the hydration layer to the overall particle diameter. This significant broadening of the nanoparticle size as compared to that measured by TEM may be attributed to the thickness of the peptide layer around the nanoparticles that is highly hydrated in solution, and likely to interparticles interaction due to the sticky nature of the peptide coating. Moreover, the surface charge of the naked nanoparticles was highly negative (about -44 mV, at pH 7.0), owing to the presence


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of hydroxylic groups on particle surface. On the other hand, surface functionalization with the peptide gave rise to nanospheres with more positive surface charge (-16 mV), likely due to shielding of the hydroxylic groups and to the net positive charge of the native peptide, which was found to be around +10 mV (Table 1). It is worth to remind that the size and the surface charge of a nanostructure are crucial aspects that determine their interactions at cellular level, and most importantly, in vivo. Sizes below 250 nm and neutral or slightly positive surface charge are generally considered optimal for effective cellular internalization without affecting system integrity or causing adverse effects.39,40 Fluorescence spectra analysis of both naked and functionalized FITC-loaded SiNPs showed the appearance of typical 518 nm peak that was substantially identical for both types of nanoparticles. This suggested that surface modification with the peptide did not affect their fluorescence emission (Fig. S3 B) which, in turn, was found to be stable over 24 h incubation at room temperature. Notably, fluorescence measurements were recorded in PBS, thus supporting the use of these particles as fluorescent tools in physiological conditions. Furthermore, the samples preserved their colloidal stability and fluorescence for more than one year, upon storage at 4°C in the dark. FTIR spectroscopy analysis was performed to further confirm the functionalization of naked SiNP with the GPC-3 ligand peptide. Panel A of Figure 3 illustrates the IR spectra of naked SiNP, GPC-3 peptide and GPC-3 SiNP, black, red and blue lines respectively, in the 3700-550 cm-1 frequency range and the principal peaks are highlighted by the arrows. The blue spectrum is characterized by signals typical of both SiO2 and protein chain, with some modifications. In fact, the signal centered at 1080 cm-1 in the SiNP spectrum can be attributed to Si-O-Si asymmetrical stretching modes, while the peaks at about 950 cm-1 and 800 cm-1 to silanol Si-O and Si-O-Si


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symmetrical stretching mode, respectively.41 In the GPC-3 SiNP spectrum the Si-O-Si stretching is centered at about 1093 cm-1, with a blue shift of 13 cm-1. Such a behavior could be imputable to GPC-3 binding to the silica surface, as already reported in the literature for similar systems.42,43 Furthermore, the C-O stretching (1200-1130 cm-1 range) and the C-H bending (836 and 800 cm-1) modes of the protein backbone44 almost disappear in the adduct spectrum, suggesting the direct involvement of these groups in the binding process. Moreover, the bending modes of NH moiety of the secondary amine, 721 and 695 cm-1 peaks of the peptide spectrum, become a single signal centered at 691 cm-1 in the adduct. Panel B shows a magnification of the 1800-1250 cm-1 range. Amide peaks are visible only in the spectrum of the peptide and of the adduct. The whole protein backbone IR peaks (1270-1480 cm-1) result changed when bonded to the SiNP. The C=O stretching mode centered at 1709 cm-1 in the spectrum of GPC-344 results shifted to 1760 cm-1 in the GPC-3 SiNP spectrum, confirming that carboxylic groups were strongly involved in the binding. Interestingly, the linkage of ligand peptide to nanoparticle surface, as determined by peptide release assay, was found to be stable over 96 h incubation at 37°C. In fact, as shown in the upper panels of Fig. S4 (Supporting Information), UV spectra of samples from both naked and functionalized nanoparticles at each time point were fairly comparable. Indeed, the full release of GPC-3 peptide was obtained only after protease treatment of functionalized nanoparticle suspension, as revealed by the appearance of a clearly detectable absorption peak at 280 nm (left upper panel). Moreover, quantitative analysis of released peptide (lower histogram) allowed to estimate an average peptide/nanoparticle ratio of 160 g/mg, respectively. The acoustic behavior of functionalized SiNPs was investigated by performing ultrasound imaging experiments. As shown in Figure S5 (Supporting Information), the nanoparticle-


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containing phantoms (B and C) produced an evident echographic contrast enhancement respect to the pure agarose gel (A), substantially confirming the results obtained in an our previous study.14 Frame by frame analysis of backscatter amplitude (D) confirmed these observations and provided quantitative estimation of image enhancement, which showed a three-fold increase respect to the control. Ultrasound measurements carried out on agarose gel phantoms containing naked SiNPs gave rise to almost identical results. Biocompatibility of SiNPs. In vitro biocompatibility of both naked and functionalized SiNPs was evaluated by means of MTT reduction assay on HepG2 cells. The dose- and time-dependent effects on cell viability was determined by performing the tests at different concentrations (from 0.05 to 0.5 mg/mL) and time intervals (1, 6, 24, 48 and 72 h). As shown in Fig. 4 A, HepG2 cell viability was substantially unaffected by treatment of naked SiNPs up to 48 h, at any concentrations tested. A slight decrease of viability, although not statistically significant, was observed only at 72 h incubation, at higher concentration (0.5 mg/mL). On the other hand, when HepG2 cells were treated with functionalized SiNPs (Fig.4 B) their viability was always comparable to the control, at any concentrations and incubation times. Thus, on the basis of the ISO 10993-5 international guide, which settles a reduction in cell viability of 30% as cytotoxic threshold, we can state that both type of nanoparticles tested in this study are highly biocompatible in HepG2 cells. In a recent paper, Kim et al. have shown that silica nanoparticle toxicity depends on both nanoparticle diameter and cells type tested.45 In particular, the authors found that cell toxicity was in inverse correlation with the particle diameter, and that was significantly pronounced in mouse embryonic fibroblast (NIH-3T3) and human alveolar carcinoma (A549) cells. Whereas, and in good agreement with our results, the effect of SiNPs on HepG2 cell viability resulted to be only negligible, likely due to the innate ability of these cells


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to resist external stresses. Therefore, we have also assayed the biocompatibility of our SiNPs in HeLa cells, a human cervical cancer cell line, differing from HepG2 cells for their embryological origin. The results obtained, illustrated in Fig. 5, showed that naked SiNPs caused a progressive dose- and time-dependent drop in HeLa cells viability (up to 40%), which became statistically significant at 48-72 h incubation with 0.5 mg/mL SiNPs (Fig. 5 A). Interestingly, no statistically significant effects on HeLa cell viability were observed when functionalized SiNPs were tested (Fig. 5 B), this confirming the general concept that functionalization may increase the biocompatibility of nanomaterials. Binding and uptake of SiNPs as a function of surface functionalization. The capability of functionalized SiNPs to selectively target human hepatocellular carcinoma was tested by studying their binding and uptake in GPC-3 positive HepG2 cells. The experiment was performed by keeping cells in suspension to reduce nonspecific internalization due to gravity force-driven nanoparticle accumulation on the cell membrane. Cells were incubated in the presence of 0.1 mg/mL fluorescent SiNPs at three different time intervals (1, 6, and 24 h) and imaged by laser scanning confocal microscopy. The results of these experiments showed that a significant fluorescence of cells incubated with functionalized SiNPs was already detectable after 1 h incubation (Fig. 6, upper images). At 6 and 24 h incubation cell fluorescence increased only slightly, and a progressive appearance of bright intracellular spots was also observed (arrows). However, when naked SiNPs were tested, cell fluorescence was largely lower (Fig. 6, lower images), becoming significant and comparable to that observed with functionalized SiNPs only at the longest incubation time. Higher magnification of confocal images (Fig. 7) showed that the bright spots observed in Fig. 6 consisted of large clusters of GPC-3 functionalized SiNPs taken up by cells as endosomal bodies (Fig. 7, left panels), which were almost undetectable in cells


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incubated with naked SiNPs (Fig. 7, right panels). As a general remark, it could be noted that in Figure 6 the fluorescent spots look distributed not uniformly in the imaged samples. This behavior may be explained taking into consideration the experimental conditions employed: indeed, cells being in suspension (not adherent) were distributed on different focal planes that do not allow the simultaneous focusing of all fluorescent spots. In addition, only large ( 1 μm) endocytic vesicles, derived by the fusion of smaller nanoparticle-containing vesicles, can be detected at low magnification (Fig. 6). Consistently, the higher magnification Figure 7 shows the presence of small fluorescent spots in almost all the cells at the same focal plane. Furthermore, it is well known that membrane receptors and antigens, such as GPC-3 protein,46 are not uniformly expressed within the same cell population (see next paragraph and Figure S7), thus explaining the observed phenomenon. Quantitative analysis (performed statistically on 100 cells from two distinct experimental setup) confirmed the qualitative outcome (histograms of Fig. 6). Indeed, while naked nanoparticles showed a clear time-dependent uptake, as expected in the case of nonspecific endocytosis,47 GPC-3 targeting nanoparticles interacted faster with the cell membrane and were internalized at higher extent even at 1 h, likely due to enhanced uptake mediated by GPC3-binding. Parallel experiments in which binding and uptake were investigated by TEM analysis substantially confirmed the results obtained and showed that the treatment of cells with both naked and functionalized SiNPs did not induce appreciable ultrastructural alterations (Figure S6). Binding and uptake of peptide-conjugated SiNPs as a function of cell surface GPC-3 expression. To demonstrate the capability of peptide-conjugated SiNPs to be internalized prominently through interaction with GPC-3 protein expressed on the cell membrane, comparative binding studies were performed with GPC-3 positive and negative cell lines. To this


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aim, preliminary analysis of GPC-3 expression was performed on four different cell lines, namely, HepG2, MDA-MB-231 (breast cancer), MG-63 (osteosarcoma) and SKOV-3 (ovarian carcinoma) cells, using a phycoerythrin-conjugated mouse anti-human GPC-3 monoclonal antibody. As reported in Figure S7, HepG2 cells showed the highest expression level of GPC-3, while MDA-MB-231 cells were slightly positive and MG-63 and SKOV-3 cells resulted completely negative to GPC-3 expression. Thus, the binding experiment was performed incubating either HepG2 or MG63 cells with GPC-3 SiNPs for 1, 6 and 24 h. Figure 8 shows that at shorter incubation times (1 and 6 h) the GPC-3 mediated binding and endocytosis accelerates the internalization of the nanoparticles that are localized, as expected, into endocytic vesicles in the perinuclear region of HepG2 cells. On the other hand, confocal images of MG63 cells denote the scarce presence of intracellular fluorescent spots up to 6 h, while being the fluorescence after 24 h incubation almost comparable to that of HepG2 cells due to nonspecific uptake. These findings confirm that the functionalization with the GPC-3 ligand peptide effectively enhances SiNPs targeting to HepG2 cells on one hand and, on the other, speeds up their uptake considerably. SUMMARY AND CONCLUSIONS The selective targeting of diseased cells or tissues by functionalized nanomaterials is one the most critical aspects for their employment in biomedical applications such as molecular imaging and selective drug delivery. Due to their particular physicochemical characteristics and ease of functionalization, silica nanoparticles are one of the most promising classes of nanomaterials with potential use in cancer diagnosis and therapy. Furthermore, their acoustic behavior allows ultrasound detectability at routinely applied frequencies. In this work, the specific targeting of


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hepatocellular carcinoma by fluorescent SiNPs functionalized with GPC-3 ligand peptide was studied. Binding and uptake experiments carried out on GPC-3 positive HepG2 cells by confocal microscopy, as well as transmission electron microscopy, have shown that, at concentrations useful for ultrasound detection, GPC-3-targeted silica nanoparticles were effectively bound and taken up by HepG2 cells, without affecting their viability. Furthermore, comparative experiments carried out on GPC-3 negative cells showed that this targeting system was also highly selective. Therefore, owing to their high biocompatibility and targeting effectiveness SiNPs seem to be promising contrast agents for ultrasound molecular imaging. It is worth to note that compared to other non-ionizing imaging techniques such as optical imaging and MRI, ultrasound detection is a low cost and widespread clinically available technique that would greatly benefit from the development of new safe contrast tools. At this particular regard, work is in progress aimed at investigating the ultrasound detectability of functionalized SiNPs in cultured cells, as well as in in vivo model organisms. ASSOCIATED CONTENT Supporting Information. HPLC and ESI-MS analysis of GPC-3 ligand peptide; Experimental setup for ultrasound detection of SiNPs; DLS measurements and fluorescence spectra of naked and GPC-3 functionalized SiNPs; Qualitative and quantitative estimation of peptide release; Ultrasound imaging of SiNPs; TEM analysis of HepG2 cells treated with SiNPs; Confocal analysis of cell surface GPC-3 protein expression. This material is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; Tel: +39 0832 422319; Fax: +39 0832 422340. Present Addresses ‡Institute of Nanotechnology, CNR-NANOTEC, c/o Campus Ecotekne, via Monteroni, 73100 Lecce, Italy ¶Department of Engineering for Innovation, University of Salento, c/o Campus Ecotekne, via Monteroni, 73100 Lecce, Italy §Department of Biological and Environmental Sciences and Technologies, University of Salento, c/o Campus Ecotekne, via Monteroni, 73100 Lecce, Italy ACKNOWLEDGMENTS This work was partially funded by the grant N° DM18604 – Bando Laboratori – DD MIUR 14.5.2005 n.602/Ric/2005 of the Italian Ministry of Instruction and Research, by FESR PO Apulia Region 2007-2013-Action 1.2.4 (grant number 3Q5AX31), by the Progetto Bandiera NANO¬MAX ENCODER and by the Italian project MAAT “Nanotecnologie Molecolari per la Salute dell’Uomo e l’Ambiente” (PON R&C 2007-2013, Contract number 02_00563_3316357). The skillful technical assistance of Dr. Antonio Greco in ultrasound imaging experiments is also acknowledged. REFERENCES (1) Liu, Y.; Miyoshi, H.; Nakamura, M. Nanomedicine for drug delivery and imaging: A promising avenue for cancer therapy and diagnosis using targeted functional nanoparticles. International Journal of Cancer 2007, 120 (12), 2527.


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(17) Wu, Y.; Zhou, H.; Wei, W.; Hua, X.; Wang, L.; Zhou, Z.; Liu, S. Signal Amplification Cytosensor for Evaluation of Drug-Induced Cancer Cell Apoptosis. Analytical Chemistry 2012, 84 (4), 1894. (18) Li, H.; Mu, Y.; Lu, J.; Wei, W.; Wan, Y.; Liu, S. Target-Cell-Specific Fluorescence Silica Nanoprobes for Imaging and Theranostics of Cancer Cells. Analytical Chemistry 2014, 86 (7), 3602. (19) Li, H.; Mu, Y.; Qian, S.; Lu, J.; Wan, Y.; Fu, G.; Liu, S. Synthesis of fluorescent dye-doped silica nanoparticles for target-cell-specific delivery and intracellular MicroRNA imaging. Analyst 2015, 140 (2), 567. (20) Wickline, S. A.; Neubauer, A. M.; Winter, P. M.; Caruthers, S. D.; Lanza, G. M. Molecular imaging and therapy of atherosclerosis with targeted nanoparticles. Journal of magnetic resonance imaging : JMRI 2007, 25 (4), 667. (21) Puvanakrishnan, P.; Park, J.; Chatterjee, D.; Krishnan, S.; Tunnell, J. W. In vivo tumor targeting of gold nanoparticles: effect of particle type and dosing strategy. International journal of nanomedicine 2012, 7, 1251. (22) Shin, S. J.; Beech, J. R.; Kelly, K. A. Targeted nanoparticles in imaging: paving the way for personalized medicine in the battle against cancer. Integrative biology : quantitative biosciences from nano to macro 2013, 5 (1), 29. (23) Benachour H, S. A., Bastogne T, Frochot C, Vanderesse R, Jasniewski J, Miladi I, Billotey C, Tillement O, Lux F, Barberi-Heyob M. Multifunctional Peptide-Conjugated Hybrid Silica Nanoparticles for Photodynamic Therapy and MRI. Theranostics 2012, 2 (9), 889. (24) Tang, L.; Cheng, J. J. Nonporous silica nanoparticles for nanomedicine application. Nano Today 2013, 8 (3), 290. (25) De Oliveira, L. F.; Bouchmella, K.; Gonçalves, K. D. A.; Bettini, J.; Kobarg, J.; Cardoso, M. B. Functionalized Silica Nanoparticles As an Alternative Platform for Targeted Drug-Delivery of Water Insoluble Drugs. Langmuir 2016, 32 (13), 3217. (26) Yin, D.; Wang, S.; He, Y.; Liu, J.; Zhou, M.; Ouyang, J.; Liu, B.; Chen, H.-Y.; Liu, Z. Surface-enhanced Raman scattering imaging of cancer cells and tissues via sialic acidimprinted nanotags. Chemical Communications 2015, 51 (100), 17696. (27) Raza, A.; Sood, G. K. Hepatocellular carcinoma review: Current treatment, and evidence-based medicine. World Journal of Gastroenterology : WJG 2014, 20 (15), 4115. (28) Waller, L. P.; Deshpande, V.; Pyrsopoulos, N. Hepatocellular carcinoma: A comprehensive review. World Journal of Hepatology 2015, 7 (26), 2648. (29) Filmus, J.; Capurro, M. Glypican-3: a marker and a therapeutic target in hepatocellular carcinoma. The FEBS journal 2013, 280 (10), 2471. (30) Mu, H.; Lin, K.-X.; Zhao, H.; Xing, S.; Li, C.; Liu, F.; Lu, H.-Z.; Zhang, Z.; Sun, Y.-L.; Yan, X.-Y.; Cai, J.-Q.; Zhao, X.-H. Identification of biomarkers for hepatocellular carcinoma by semiquantitative immunocytochemistry. World Journal of Gastroenterology : WJG 2014, 20 (19), 5826. (31) Ho, M.; Kim, H. Glypican-3: a new target for cancer immunotherapy. European journal of cancer (Oxford, England : 1990) 2011, 47 (3), 333. (32) Haruyama, Y.; Kataoka, H. Glypican-3 is a prognostic factor and an immunotherapeutic target in hepatocellular carcinoma. World Journal of Gastroenterology 2016, 22 (1), 275.


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(33) Park, J. O.; Stephen, Z.; Sun, C.; Veiseh, O.; Kievit, F. M.; Fang, C.; Leung, M.; Mok, H.; Zhang, M. Glypican-3 targeting of liver cancer cells using multifunctional nanoparticles. Molecular imaging 2011, 10 (1), 69. (34) Lee, Y. L.; Ahn, B. C.; Lee, Y.; Lee, S. W.; Cho, J. Y.; Lee, J. Targeting of hepatocellular carcinoma with glypican-3-targeting peptide ligand. Journal of peptide science : an official publication of the European Peptide Society 2011, 17 (11), 763. (35) Verhaegh, N. A. M.; Blaaderen, A. v. Dispersions of Rhodamine-Labeled Silica Spheres: Synthesis, Characterization, and Fluorescence Confocal Scanning Laser Microscopy. Langmuir 1994, 10 (5), 1427. (36) Wang, S.; Yin, D.; Wang, W.; Shen, X.; Zhu, J.-J.; Chen, H.-Y.; Liu, Z. Targeting and Imaging of Cancer Cells via Monosaccharide-Imprinted Fluorescent Nanoparticles. Scientific Reports 2016, 6, 22757. (37) Stöber, W.; Fink, A.; Bohn, E. Controlled growth of monodisperse silica spheres in the micron size range. Journal of Colloid and Interface Science 1968, 26 (1), 62. (38) Ghosh, S. S.; Kao, P. M.; McCue, A. W.; Chappelle, H. L. Use of maleimide-thiol coupling chemistry for efficient syntheses of oligonucleotide-enzyme conjugate hybridization probes. Bioconjugate Chemistry 1990, 1 (1), 71. (39) Quarta, A.; Curcio, A.; Kakwere, H.; Pellegrino, T. Polymer coated inorganic nanoparticles: tailoring the nanocrystal surface for designing nanoprobes with biological implications. Nanoscale 2012, 4 (11), 3319. (40) He, C.; Hu, Y.; Yin, L.; Tang, C.; Yin, C. Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials 2010, 31 (13), 3657. (41) Singh, S.; Barick, K. C.; Bahadur, D. Surface engineered magnetic nanoparticles for removal of toxic metal ions and bacterial pathogens. Journal of Hazardous Materials 2011, 192 (3), 1539. (42) Bettini, S.; Santino, A.; Valli, L.; Giancane, G. A smart method for the fast and low-cost removal of biogenic amines from beverages by means of iron oxide nanoparticles. RSC Advances 2015, 5 (23), 18167. (43) Lu, H.-T. Synthesis and characterization of amino-functionalized silica nanoparticles. Colloid Journal 2013, 75 (3), 311. (44) Smith, B. C. Infrared Spectral Interpretation. A systematic approach; CRC Press: Boca Raton, 1998. (45) Kim, I. Y.; Joachim, E.; Choi, H.; Kim, K. Toxicity of silica nanoparticles depends on size, dose, and cell type. Nanomedicine : nanotechnology, biology, and medicine 2015, 11 (6), 1407. (46) Capurro, M.; Wanless, I. R.; Sherman, M.; Deboer, G.; Shi, W.; Miyoshi, E.; Filmus, J. Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma. Gastroenterology 2003, 125 (1), 89. (47) Zhu, J.; Liao, L.; Zhu, L.; Zhang, P.; Guo, K.; Kong, J.; Ji, C.; Liu, B. Sizedependent cellular uptake efficiency, mechanism, and cytotoxicity of silica nanoparticles toward HeLa cells. Talanta 2013, 107, 408.


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For Table of Contents only


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Figure 1. Schematic representation of (A) FITC-doped silica nanoparticle synthesis and (B) their functionalization with the GPC-3 ligand peptide.


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Figure 2. TEM images of (A) naked and (B) GPC-3 ligand peptide functionalized SiNPs. Upper images show a high magnification of the framed region in lower pictures. Scale bars = 200 nm.


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Figure 3. FTIR spectra of naked SiNP (black spectrum), GPC-3 ligand peptide (red spectrum) and GPC-3 SiNP (blue spectrum) in the 3600-550 cm-1 (A) and 1800-1000 cm-1 (B) frequency range. In panel B, the GPC-3 SiNP and SiNP curves were multiplied by 25 to make the comparison easier. Arrows highlight the most significant signals of the corresponding curves.


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Figure 4. Viability of HepG2 cells evaluated by the MTT assay after 1, 6, 24, 48 or 72 h exposure to increasing doses (from 0.05 to 0.5 mg/mL) of naked SiNPs (A) or functionalized SiNPs (B). Data, expressed as percentage of control, are mean ± SD of three independent experiments, each in triplicate. Error bars represent SDs. Statistical analysis was performed according to the t test.


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Figure 5. Viability of HeLa cells evaluated by the MTT assay after 1, 6, 24, 48 or 72 h exposure to increasing doses (from 0.05 to 0.5 mg/mL) of naked SiNPs (A) or functionalized SiNPs (B). Data are expressed as percentage of control, and are mean ± SD of three independent experiments, each in triplicate. Statistical analysis was performed according to the t test. *: p 0.05 vs control.


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Figure 6. Confocal microscopy images of HepG2 cells treated with fluorescent SiNPs for 1, 6 and 24 h. Upper pictures: cells treated with GPC-3 ligand peptide functionalized SiNPs. The arrows highlight intracellular clusters of SiNPs. Lower pictures: cells treated with naked SiNPs. Typical images from two different experiments are reported. Scale bars = 75 μm. The correspondent histograms show the quantitative analysis of cell fluorescence. Values of fluorescence intensities for each incubation time are mean ± SD (n = 100). Statistical analysis was performed according to the t test. *: p 0.05 vs 1 h and 6 h.


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Figure 7. Confocal analysis of binding and uptake of fluorescent SiNPs in HepG2 cells: effect of functionalization. Binding and uptake of GPC-3 ligand peptide functionalized SiNPs after 1 , 6 and 24 h incubation are shown in left panels. The correspondent effects of naked SiNPs are shown in right panels. Typical images from two different experiments are reported. For all images, confocal fluorescence micrographs (dark) and their overlay on the correspondent optical images are shown. Scale bars = 7.5 μm.


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Figure 8. Confocal analysis of binding and uptake of GPC-3 ligand peptide functionalized SiNPs: effect of cell surface GPC-3 protein expression. Left panels show the images of GPC-3 positive HepG2 cells treated with functionalized SiNPs for 1, 6 and 24 h. The images of GPC-3 negative MG-63 cells are shown in right panels. For all images, confocal fluorescence micrographs (dark) and their overlay on the correspondent optical images are shown.. Typical images from two different experiments are reported. Scale bars = 25 μm.


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Table 1. Average diameter of SiNPs, determined by TEM and DLS, and Zeta Potential values of nanoparticles and GPC-3 ligand peptide (PBS suspension). TEM diameter (nm)

DLS diameter (nm)

Zeta Potential

Naked SiNPs

220

350

-44±2.4

GPC-3 SiNPs

250

450

-16±1.7

GPC-3 ligand peptide

-

-

9.6±2.5

sample

(mV)


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