Near-Infrared Light Responsive Folate Targeted ... - ACS Publications

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Near-Infrared Light Responsive Folate Targeted Gold Nanorods for Combined Photothermal-Chemotherapy of Osteosarcoma Anna Li Volsi,† Cinzia Scialabba,† Valeria Vetri,‡,§ Gennara Cavallaro,† Mariano Licciardi,*,†,§ and Gaetano Giammona†,§ †

Laboratory of Biocompatible Polymers, Department of Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF), University of Palermo, Via Archirafi, 32, 90123 Palermo, Italy ‡ Department of Physics and Chemistry, University of Palermo, 90123 Palermo, Italy § Mediterranean Center for Human Health Advanced Biotechnologies (CHAB), Aten Center, University of Palermo, 90129 Palermo, Italy S Supporting Information *

ABSTRACT: Folate-targeted gold nanorods (GNRs) are proposed as selective theranostic agents for osteosarcoma treatment. An amphiphilic polysaccharide based graft-copolymer (INU-LA-PEG-FA) and an amino derivative of the α,βpoly(N-2-hydroxyethyl)-D,L-aspartamide functionalized with folic acid (PHEA-EDA-FA), have been synthesized to act as coating agents for GNRs. The obtained polymer-coated GNRs were characterized in terms of size, shape, zeta potential, chemical composition, and aqueous stability. They protected the anticancer drug nutlin-3 and were able to deliver it efficiently in different physiological media. The ability of the proposed systems to selectively kill tumor cells was tested on U2OS cancer cells expressing high levels of FRs and compared with human bronchial epithelial cells (16HBE) and human dermal fibroblasts (HDFa). The property of the nanosystems of efficiently controlling drug release upon NIR laser irradiation and of acting as an excellent hyperthermia agent as well as Two Photon Luminescence imaging contrast agents was demonstrated. The proposed folate-targeted GNRs have also been tested in terms of chemoterapeutic and thermoablation efficacy on tridimensional (3-D) osteosarcoma models. KEYWORDS: folate-targeted, gold nanorods, photothermal-chemotherapy, osteosarcoma, NIR-laser triggered drug release

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INTRODUCTION

due to early metastases often undetectable using current diagnostic modalities.4 Therefore, the development of novel effective therapeutic and diagnostic strategies are urgently needed to substantially improve disease outcome. Nanotechnology based systems in medicine has been a rapidly growing field in recent years.5 This is mainly because of the possibility to design and produce nanodevices with specific structural and functional features that make them selective toward a determined cancer therapy. In particular with regard to OS therapy, a variety of nanostructures have been exploited in the areas of OS imaging, diagnostics and treatment,6 including liposomal systems7 and (polylactic-co-glycolic acid)-dextran nanoparticles.8 Other groups exploited gold nanoparticles, such as nanospheres and gold nanorods 9 for osteosarcoma treatment, providing remarkable opportunities in the detection and therapy of OS due to their inherently low toxicity10 and due to the strongly enhanced optical properties associated with localized surface

Osteosarcoma (OS) is the most common primary malignant neoplasm of bone. The annual incidence of osteosarcoma is 8− 11 per million in the age group of 15−19 years. Despite its rarity, it has been reported to be the second leading cause of cancer-related deaths in children and young adults.1 Current therapeutic strategies comprise very aggressive multimodal therapy including preoperative (neoadjuvant) chemotherapy followed by surgical removal of all detectable disease, and postoperative (adjuvant) chemotherapy. Currently, the most active chemotherapeutic agents for OS are a combination of high-dose methotrexate, doxorubicin, and cisplatin with the use of IFN-α or the recent immunomodulatory mifamurtide during adjuvant chemotherapy.2 Despite their potent anticancer effect, the chemotherapy treatments of OS are associated with very severe collateral toxic effects due to the lack of selectivity of these conventional anticancer drugs, such as cardiotoxicity, ototoxicity, nephrotoxicity, gonadal disfunction, midollar toxicity, etc. Furthermore, these current aggressive therapeutic protocols frequently fail because of the occurrence of tumor resistance to conventional chemotherapeutics that may result in tumor recurrence3 and/or © 2017 American Chemical Society

Received: March 15, 2017 Accepted: April 6, 2017 Published: April 6, 2017 14453

DOI: 10.1021/acsami.7b03711 ACS Appl. Mater. Interfaces 2017, 9, 14453−14469

Research Article

ACS Applied Materials & Interfaces plasmon resonance (LSPR).11−13 The well-known Surface Plasmon Resonance of GNRs, together with detection ability, confer them the possibility to convert NIR absorbed radiation into heat, and therefore GNRs are characterized by a strong photothermal behavior that can be exploited in tumor hyperthermia treatment. This treatment leads to local temperature values higher than 50 °C, causing the so-called thermal ablation which corresponds to severe cell damage resulting in coagulative necrosis and membrane lysis.14 Besides, hyperthermia treatments additionally provide the possibility to specifically release the loaded drug upon irradiation with an appropriate laser beam,15−17 thus increasing drug delivery efficiency and favoring intracellular drug incorporation thanks to the temperature-induced increase of cell membrane permeability.18 The NIR absorption of GNRs is also exerted for biomedical imaging, probing their NIR photoluminescence using a two-photon laser source.19 This imaging technique facilitates deeper probing, enabling imaging of animal models and human tissues, the results of which are useful for early detection of neoplastic cells and tissues.20 To achieve cancer specificity, targeting ligands have been conjugated to the polymeric coating21,22 of gold nanoparticles. Among targeting ligands, Folic acid is an ideal candidate for ligand-based targeting, as numerous examples of folateconjugated drug carriers have been shown to be transported into cells through receptor mediated endocytosis.23−29 The high-affinity folate receptors (FRs) are highly selective tumor markers overexpressed by over one-third of human cancer cell lines such as osteosarcoma (U2OS) cell lines.30 Folate-targeted GNRs have been successfully proposed in the past for both “in vivo” and “in vitro” highly selective photothermal therapy of tumors and cancer cells with low laser intensities.31,32 In this paper we report the preparation of two novel folate targeted gold nanorods systems functionalized with two versatile cytocompatible polymers, which are inulin and α,βpoly(N-2-hydroxyethyl)-DL-aspartamide (PHEA) derivatives, both bearing folic acid molecules on their backbone. The obtained polymer coated gold nanorods, Au NRs@INU-LAPEG-FA and Au NRs@PHEA-EDA-FA were studied as carrier to efficiently deliver the anticancer drug nutlin-3 to U2OS osteosarcoma cells, which plays a key role in chemo-resistance and tumor recurrence of OS.33 The main finding of the present study is that the proposed systems selectively exert toxic effect toward OS cells and on osteosarcoma three-dimensional (3-D) tumor models expressing high levels of FRs compared with nontumoral cells. Moreover, in this paper we report the ability of the nanosystems to act as an excellent hyperthermia agent as well as TPL imaging contrast agents.

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mamide (DMF) were purchased from Fluka (Switzerland). All reagents were of analytic grade, unless otherwise stated. α,β-Poly(N2-hydroxyethyl)-D,L-aspartamide (PHEA) was prepared and purified according to the previously reported procedure.34 Spectroscopic data (FT−IR and 1H NMR) were in agreement with attributed structure: 1 H NMR (300 MHz, D2O, 25 °C, δ): 2.82 (m, 2H, −CH−CH2− CONH−), 3.36 (t, 2H, −NH−CH2−CH2−OH), 3.66 (t, 2H, −CH2− CH2−OH), 4.72 (m, 1H, −NH−CH−CO−CH2−). Ethylenediamine (EDA) was purchased from Aldrich (Italy). PHEA-EDA copolymer was synthesized as previously published.35 The pure product was obtained with a yield of 97% (w/w) based on starting PHEA. Spectroscopic data (FT−IR, 1H NMR, and SEC) were in agreement with the attributed structure. The degree derivatization in EDA was 25 mol %. The Mw of PHEA-EDA was 26 kDa and polydispersity was 1.57. Inulin-2-aminoethyl-carbamate (INU-EDA) was synthesized as previously reported.36 Apparatus. Molecular weights of INU-LA-PEG-FA, PHEA-EDAFA copolymers and their parent compounds were determined by means of a size exclusion chromatography (SEC) system Agilent 1260 Infinity Multi Detectos Bio-SEC equipped with two Phenogel coloumns from Phenomenex (5 μm particles size, 103 Å and 104 Å of pore size) and Bio Dual Angle LS/DLS and RI Detector. DMF +LiBr 0.1% solution was used as an eluent at 60 °C with a flow rate of 0.8 mL min−1, samples concentration 10 mg/mL. Poly(ethylene oxide) standards (range 10−1.5 kDa) were used to set up calibration curve. 1 H NMR spectra were recorded using a Bruker Avance II 300 spectrometer operating at 300 MHz. High pressure liquid chromatography (HPLC) was carried out using a HPLC 1260 Infinity Agilent apparatus equipped with two C18 Gemini columns (Phenomenex) connected to an UV−vis detector. Transmission electron microscopy (TEM) images were acquired using a 120 keV TEM (JEOL 1010, Japan) equipped with GATAN US1000 CCD camera (2k × 2k). UV−vis spectra were were recorded using an Shimadzu UV-2400 spectrophotometer. Scansion electron microscopy (SEM) and energy dispersive X-ray analysis were carried out using a scanning electron microscope, ESEM Philips XL30. Zeta-potential measurements (mV) were performed at 25 °C using a Malvern Zetasizer NanoZS instrument. Synthesis of Au NRs@CTAB. Gold nanorods were prepared via a seed-mediated synthetic process following the standard CTAB/NaBH4 procedure:37 in a first step, the gold seeds were prepared adding 25 μL of a 0.05 M HAuCl4 solution to 4.7 mL of 0.1 M CTAB solution; 300 μL of a freshly prepared 0.01 M NaBH4 solution was then injected under vigorous stirring. Then, in a second step, freshly prepared gold seeds were used to synthesize gold nanorods as previously described by Scarabelli and co-workers.38 Synthesis of INU-LA-PEG-FA. INU-LA-PEG-FA copolymer was synthesized in two synthetic steps. First, INU-LA was synthesized adding a solution of DCC (153 mg, 0.74 mmol) and DMAP (90 mg, 0.74 mmol) in 2 mL of DMFa to a solution of inulin (200 mg, 1.23 mmol of repetitive units) in the same solvent. The reaction mixture was kept 18 h at 25 °C, then filtered and DMFa was removed by means of a rotavapor. The obtained product, INU-LA, was precipitated and washed three times in diethyl ether, and stored as dried powder. In the second step, BNPC (41.4 mg, 0.136 mmol) was added to 6 mL of INU-LA solution in DMFa (50 mg/mL). The reaction mixture was irradiated for 30 min with microwave at a power of 25W in a CEM Discover Microwave Reactor maintaining the temperature at 60°. Then, the reaction mixture was added dropwise to a PEG-FA solution obtained as previously reported39 (330 mg of PEG-FA (0.136 mmol) in 5 mL of a-DMF). Briefly, folic acid was linked to PEG bis-amine by means of an amidic bond, using EDC and NHSS as coupling agents to activate carboxyl group of FA. The stoichiometric conditions employed were chosen to functionalize just one of the two amino groups of the PEG bis-amine chain: moles of FA/mol of PEG = 0.75; moles of EDC-HCl/mol of FA = 1.5 and moles of NHSS/mol of FA = 1.5. By comparing the integrals of the peak at δ 7.52−6.69 recorded

MATHERIALS AND METHODS

Materials. Inulin from dahlia tubers, bis(4-nitrophenyl)carbonate (BNPC), N-ethyl-N′-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC-HCl), N-hydroxysuccinimide (NHS), Folic acid, Poly(ethylene glycol) diamine 2 kDa, (±)-α-Lipoic acid, Dicyclohexylcarbodiimide (DCC), 4-(Dimethylamino)pyridine (DMAP), Nutlin3, hexadecyltrimethylammonium bromide (CTAB ≥ 96%), 5bromosalicylic acid (5-BrSA) (technical grade, 90%), hydrogentetrachloroaurate trihydrate (HAuCl4·H2O, ≥ 99.9%), silver nitrate (AgNO3, ≥ 99.0%), L-ascorbic acid (≥99%) (AA), dodecanethiol (≥98%), and sodium borohydride (NaBH4, 99%) were purchased from Aldrich. Spectroquant Gold test was purchased from Merck KGaA, (Germany). Milli-Q water (resistivity 18.2 MΩ·cm at 25 °C) was used in all experiments. Sephadex G-25, anhydrous dimethylfor14454


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ACS Applied Materials & Interfaces through 1H NMR spectroscopy, ascribable to the protons of the phenyl ring of FA, to that at δ 3.7 belonging to the protons of PEG, the molar percent of FA covalently linked to PEG (DDFA %) was determined and it was equal to 44 mol %. The reaction mixture of INU-LA and PEG-FA was allowed to react for 4 h at 25 °C. The obtained product was precipitated and washed several times in acetone (5 °C for 5 min, at 11 000 rpm). Then, the product was solubilized in 2 mL of Milli-Q water and purified by GPC using Sephadex G-25 as stationary phase. Yield: 97% w/w based on the starting INU-LA. 1H NMRD2O: 3.60−3.90 (5HINU, −CH2−OH; −CH−CH2−OH; −C−CH2−O−), 3.92−4.30 (2HINU, −C−CH− OH; −CH−OH), δ 1.39 (m, 2HLA, −HO−CO−CH2−CH2−CH2− CH2−cCH2−CH2−CH2−S−S−), 1.59 (2HLA, −HO−CO−CH2− CH 2 −CH 2 −CH 2 −cCH−CH 2 −CH 2 −S−S−), 1.97 (m, 2H LA , −HO−CO−CH2−CH2−CH2−CH2−cCH−CH2−CH2−S−S−), 2.23 (m, 2HLA −HO−CO−CH2−CH2−CH2−CH2−cCH−CH2−CH2− S−S−), 2.54 (m, 2HLA − HO−CO−CH2−CH2−CH2−CH2−cCH− CH2−CH2−S−S−), 3.70 (m, 176HPEG 2000, −NH−CH2−CH2−(O− CH2−CH2)44−), δ 7.52−6.69 4H FA, phenyl group. Synthesis of PHEA-EDA-FA. PHEA-EDA copolymer was synthesized as previously reported.35 To a solution of PHEA-EDA (200 mg ≈ 0.326 mmol of PHEA repeating units) in Milli-Q water (2 mL) a solution of folic acid in Milli-Q water/NaOH 0.1 N 1.8 mL/0.2 mL (43 mg ≈ 0.097 mmol) was added dropwise. After that NHS (16.11 mg, 0.14 mmol) and EDC-HCl (26.8 mg, 0.14 mmol) were added, respectively. Then, the pH of the reaction was fine-tuned to 6.5−6.8 with 0.1 M NaOH, and the reaction was allowed to continue for 24 h at 25 °C under vigorous stirring. The pure product was collected by GPC using Sephadex G-25 and then freeze-dried. Typically a yield of 87−92% was obtained. 1H NMRD2O: δ 2.8 (m, 2H, −CH−CH2−CO−NH), 3.01 (m, 2 H, − NH−CH2−CH2−NH2), 3.38 (m, 2 H, −NH−CH2−CH2−OH), 3.43 (m, 2H, −NH−CH2− CH2−NH2), 3.68 (m, 2H, −NH−CH2−CH2−OH), 4.15 (m, 2 H, −NH−CH2−CH2−O(CO)NH−CH2−CH2−NH2) 4.73 (m, 1 H, −NH−CH(CO)CH2), δ 7.52−6.69 4H FA, phenyl group. Determination of Critical Aggregation Concentration. The critical aggregation concentration (CAC) of INU-LA-PEG-FA and PHEA-EDA-FA was estimated by pyrenyl fluorescence assay, using a Shimadzu RF-5301 PC spectrofluorophotometer. Aliquots of 20 μL of a solution of pyrene (6.0 × 10−5 M in acetone) were placed into vials at 37 °C and subsequently, 2 mL of aqueous solution of copolymers at concentrations ranging from 5 × 10−4 to 10 mg mL−1 were added; the solutions were allowed to equilibrate for 24 h at 37 °C. Pyrene excitation and emission spectra were recorded at 37 °C using an emission wavelength of 373 nm and an excitation wavelength of 333 nm. Preparation of INU-LA-PEG-FA, INU-LA-PEG-FA/Nutlin, PHEA-EDA-FA, and PHEA-EDA-FA/Nutlin coated GNRS. The coated GNRs were obtained by the following procedure: the previously produced AuNRs@CTAB were washed with deionized water twice via centrifugation at 8000 rpm for 15 min, 25 °C to remove the excess of CTAB prior to use. For the preparation of Au NRs@INU-LA-PEG-FA, a solution of INU-LA-PEG-FA copolymer (40 μL, 1 mg mL−1 in Milli-Q water) was added to CTAB capped Au NRs (Au NRs@CTAB), at a ratio INU-EDA-FA molecules/Au nm2 of 10 (assuming that GNRs are perfect cylinders). The colloidal system was incubated for 30 min at 37 °C. The obtained polymer-coated nanorods (Au@INU-LA-PEG-FA) were then purified by centrifugation at 8000 rpm (15 min, 25 °C), dispersing the pellet in Milli-Q water. Au NRs@PHEA-EDA-FA were prepared using a solution of PHEA-EDA-FA copolymer (40 μL, 1 mg mL−1 in Milli-Q water) with PHEA-EDA-FA molecules/Au nm2 ratio of 2 and following the same method reported for INU-LA-PEG-FA. Nutlin loaded Au NRs (Au NRs@INU-LA-PEG-FA/Nutlin and Au NRs@PHEA-EDA-FA/Nutlin) were prepared by adding a Nutlin solution in methanol (35 μL 1 mg mL−1) to a proper amount (50% w/w nutlin/coated GNRs) of dried copolymers. Then the mixtures were dried at room temperature under vacuum, dispersed in Milli-Q water, and added to Au NRs@ CTAB aqueous dispersion (30 μg mL−1) to obtain an R Nutlin/Au0 equal to 50% w/w. After incubation time (3 h, 37 °C) the systems

were centrifuged (15 min, at 8,000 rpm) and washed in Milli-Q/ methanol 1:2 v/v twice to remove the excess of Nutlin. Folate-free GNRs were prepared using the same experimental conditions abovedescribed, except for the use of the correspondents folate-free copolymers, that are INU-LA and PHEA-EDA. Preparation of Alexa Fluor-647 labeled Au NRs@INU-LAPEG-FA and Au NRs@PHEA-EDA-FA GNRs. Alexa Fluor 647 Nhydroxysuccinimide ester (0.5 mg) was dissolved in 200 μL of DMF. 50 μL of this solution were added to 1 mL of colloidal dispersion of AuNRs@INU-LA-PEG-FA or AuNRs@PHEA-EDA-FA GNRs (0.03 mg/mL) in 0.1 M sodium bicarbonate buffer, pH 8.3. The reaction was carried out at room temperature for 3 h. The product was purified by several washes in distilled water. Trasmission Electron Microscopy. Five microliters of GNRs were drop casted onto carbon-coated 400 square mesh copper grids and left to dry in air before examination. All samples were centrifuged twice before blotting on the grid. Observation was performed at an accelerating voltage of 120 kV. Scansion Electron Microscopy and Energy Dispersive X-ray Analysis. Five μL GNRs samples were dusted on a TEM copper grid and allowed to dry before examination. UV−vis Spectroscopy. All polymer coated GNRs and GNRs@ CTAB were placed in a cell and spectral analysis was performed in the 400 to 900 nm range at room temperature, at a concentration of 15 μg mL−1 of Au0. Zeta Potential Measurements. Aqueous dispersion 15 μg mL−1 Au0 of the all GNRs samples were analyzed. The zeta potential (mV) was calculated using the Smoluchowsky relationship and assuming that K × a ≫ 1 (where K and a are the Debye−Hückel parameter and particle radius, respectively). Determination of Drug Payload into Au NRs@INU-LA-PEGFA/Nutlin and Au NRs@PHEA-EDA-FA/Nutlin and Drug Release Studies. The drug loading of Au NRs@INU-LA-PEG-FA/ Nutlin and Au NRs@PHEA-EDA-FA/Nutlin systems was evaluated by HPLC method and UV spectroscopy. In the first method, a known amount of Au NRs@INU-LA-PEGFA/Nutlin or Au NRs@PHEA-EDA-FA/Nutlin was dispersed in 5 mL of acetonitrile to extract the loaded drug. After 3 h extraction under stirring, the dispersion was centrifuged (15 min, at 8000 rpm), the surnatant was filtered on Nylon filter, cut off 0.2 μm, and injected in a C18 Gemini HPLC column. The eluent used for HPLC analysis was 0.02 M KH2PO4/ ACN/MeOH 45:35:20 (v/v) at 1 mL/min. The content of drug loaded (DL) into the systems was calculated by using a calibration curve obtained for serially diluted concentrations of nutlin-3 in the eluent. In the second method, an analogous procedure to that above-described to extract the loaded drug was followed. Then, the surnatant was analyzed by UV spectroscopy measuring the absorbance at 260 nm to determine the amount of nutlin-3 loaded into the polymer coated GNRs systems. A calibration curve was obtained for serially diluted concentrations of nutlin-3 in acetonitrile. The average of drug loading % calculated by means of HPLC and UV spectroscopies and expressed as the amount of loaded nutlin-3 per unit mass of polymer coated GNRs resulted equal to 12.45 ± 1.5% (w/w) with an encapsulation efficiency of 40.95% and of 13.75 ± 0.7% with an encapsulation efficiency of 50.02% for Au NRs@INU-LA-PEG-FA/ Nutlin and Au NRs@PHEA-EDA-FA/Nutlin, respectively. Drug release studies were performed in three different release media: human plasma, phosphate buffer solution pH 7.4 and 5.5, both supplemented with fetal bovine serum (FBS) at 10% v/v. One hundred μL of Au NRs@INU-LA-PEG-FA/Nutlin or Au NRs@ PHEA-EDA-FA/Nutlin or free nutlin in 10 mM phosphate buffer pH 7.4 were added to 1 mL of each release media (plasma, PBS pH 5.5 and pH 7.4 supplemented with FBS 10% v/v. The samples were kept at 37 °C under mild stirring. At scheduled times (from 30 min to 48 h), the samples were centrifuged for 15 min at 8000 rpm, to remove the system. Then, 1.5 mL of acetonitrile were added to a precise volume (500 μL) of the collected supernatants to precipitate plasma and FBS proteins. The mixture was vortexed for 5 min and kept under stirring for 15 min in order to extract nutlin. After centrifugation for 3 min at 3000 rpm, a precise volume of supernatant containing the 14455


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ACS Applied Materials & Interfaces

25,10, 5, and 1 μM. After 72 h, samples were taken away from the wells, organoids were washed with fresh media, and finally 25 μL of fresh media and 5 μL of MTS solution were added. Cells were incubated for 24 h at 37 °C and then the absorbance at 490 nm was measured as above-described. Nutlin solutions at the same concentrations (ranging from 1.87 to 15 μM.) were used as a positive control. Pure cell medium was used as a negative control. Results were expressed as percentage reduction of the control cells. All culture experiments were performed in triplicates. Thermoablation Treatment and Hyperthermia-Triggered Effect on U2OS and HFDa Cells. For thermoablation treatment on 2D cell cultures, U2OS and HDFa cells were seeded in 96-well plate at a density of 2 × 104 cells per well and grown in supplemented McCoy’s medium, as previously described. After overnight attachment, cells were incubated for 4 h with fresh medium containing either AuNRs@INU-LA-PEG-FA, AuNRs@PHEA-EDA-FA or Au NRs@ INU-LA-PEG-FA/Nutlin, AuNRs@PHEA-EDA-FA/Nutlin or free nutlin at concentration of 3.75 μg mL−1 of Au0 corresponding to 0.75 μM of nutlin. After incubation time, the systems were removed, cell washed with DPBS twice, and cells were treated with a 810 nm laser beam, fitted at 3.5 × 10−2 W mm−3 for 10, 30, 60, and 120 s. After the laser exposure, cells were incubated for additional 24 h at 37 °C. Cell viability was evaluated by means of MTS assay as above-described after 24 h of postincubation following the laser treatment. Cells exposed to the same laser, using the same settings, were used as negative control. For thermoablation treatment on 3D U2OS cultures, organoids were formed and cultured for 3 days as described above. On the fourth day, organoids were incubated for 24 h with Au NRs@INULA-PEG-FA, Au NRs@PHEA-EDA-FA or Au NRs@INU-LA-PEGFA/Nutlin, Au nRs@PHEA-EDA-FA/Nutlin, or free nutlin at concentration of 52 μg mL−1 of Au0 corresponding to 10 μM of nutlin. After the incubation time, the systems were removed, organoids were washed with fresh media, and were then treated with with a 810 nm laser beam for 30, 60, 90, and 200 s. After the laser exposure, organoids were incubated for additional 48 h at 37 °C and cell viability was evaluated by means of MTS assay as above-described. Organoids irradiated with the same laser settings were used as negative control. All culture experiments were performed in triplicate. Quantitative Cell Uptake and Enhanced HyperthermiaTriggered Drug Internalization. U2OS and HDFa cell lines were seeded at a density of 2 × 104 cells/well and grown as above-reported. After overnight attachment the medium was replaced with 200 μL of fresh DMEM containing Au NRs@INU-LA-PEG-FA/Nutlin or Au NRs@PHEA-EDA-FA/Nutlin or free Nutlin at the concentration of 15 μg/mL Au0, corresponding to 3 μM of nutlin and cells were incubated for 4 h at 37 °C. For the quantitative uptake, cells were washed twice with DPBS and were lysed in 600 μL lysis buffer (1% Triton X-100 in DPBS) at 37 °C. 575 μL of the collected cell lysate were freeze-dried and then reconstituted with 500 μL of acetonitrile to extract nutlin. After 3 h extraction, the reconstituted cell lysate was centrifuged for 5 min at 5000 rpm, and the surnatant was filtered on 0.45 μm Nylon filter and analyzed by means of HPLC method as above-described. The remaining 25 μL of lysates were transferred to disposable 96 well plates and used to determine the protein content using the bicinchonic acid kit for protein determination (Sigma-Aldrich), according to the protocol of the manufacturer. Free nutlin-3 3 μM was used as positive control for quantitative uptake HPLC analysis. To study the hyperthermia-triggered internalization, an analogous procedure was followed apart from the fact that after adding Au NRs@ INU-LA-PEG-FA/Nutlin or Au NRs@PHEA-EDA-FA/Nutlin or free Nutlin at the same concentrations used (15 μg/mL Au 0 , corresponding to 3 μM of nutlin) to U2OS and HDFa cell cultures, cells were treated with a 810 nm laser, 3.5 × 10−2 W mm−3 for 90 s and then incubated for 4h at 37 °C. Gold Determination into U2OS Organoids. The gold content in U2OS organoids was measured using the Spectroquant gold test kit. U2OS organoids, formed as above-described, were incubated with AuNRs@INU-LA-PEG-FA and AuNRs@PHEA-EDA-FA systems for 24 and 48 h. After that, the organoids were lysed with 5 mL of lysis

organic phase was collected, filtered on a 0.2 cutoff Nylon filter and analyzed by HPLC. The extraction recovery of nutlin was higher than 98% in all the tested matrices. The NIR-laser-induced drug release was assessed exposing a dispersion of Au NRs@INU-LA-PEG-FA/Nutlin or Au NRs@PHEA-EDA-FA/Nutlin in PBS pH 5.5/FBS 10% v/v with a 810 nm laser (3.5 × 10−2 W mm−3) for different times (from 30 to 90 s) and then comparing the release obtained after 1, 2, and 4 h at 37 °C with the untreated control. In Vitro Evaluation of Hyperthermia. Hyperthermia generated by aqueous dispersions of Au NRs@INU-LA-PEG-FA/Nutlin or Au NRs@PHEA-EDA-FA/Nutlin, at different concentrations ranking from 1.6 to 30 μg mL−1 of Au0 upon the exposed to a 810 nm surgical diode laser (GBox 15A/B by GIGA Laser, 2.8 × 10−3 W mm−3), was carried out as already reported.21 Cell Cultures and Reagents. Human osteosarcoma cells (U2OS) were purchased from ATCC. Human bronchial epithelial (16HBE) were purchased from Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna (Italy), and human dermal fribroblast (HDFa) were purchased from Invitrogen. 16HBE and HDFa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% of penicillin/ streptomycin (100 U/mL penicillin and 100 mg/mL streptomycin), 1% glutamine, and 0.5% of amphotericin B, at 37 °C in 5% CO2 humidified atmosphere. U2OS were grown in McCoy’s medium with 10% FBS, 1% penicillin/streptomycin, and 1% glutamine. DMEM, McCoy’s medium, and other constituents were purchased by Euroclone and ATCC respectively. Cell Titer 96 Aqueous One Solution (MTS reagents for cell proliferation assay) was purchased from Thermo Scientific. Bicinchoninic Acid Kit for protein determination, Perfecta3D hanging drop plate and ECM Gel from Engelbreth−Holm−Swarm murine sarcoma were purchased from Sigma-Aldrich. U2OS Organoids Formation. U2OS organoids were formed following the HDP1096 Perfecta3D 96-Well Hanging Drop Plates Protocol as reported in other works.40,41 Briefly, 40 μL of a cell suspension at density of 100 cells/ μL enriched with 2.5% of ECM Gel to improve the compactness of spheroids was pipetted into each well from the top side of the Perfecta 3D hanging drop plate. Cell culture media was exchanged every day by removing 10 μL of media and adding 14 μL of fresh one. Cell Cytotoxicity Assay. The cytotoxicity of the Au NRs systems was assessed by the MTS assay on human osteosarcoma cells (U2OS), on human bronchial epithelial (16HBE), on human dermal fribroblast (HDFa) cell lines and on U2OS organoids using a commercially available kit (Cell Titer 96 Aqueous One Solution Cell Proliferation assay, Promega). For cell cytotoxicity assay on 2D cell cultures, cells were seeded in a 96-multiwell plate at a density of 2 × 104 cells/well. Upon attachment by incubation overnight, 200 μL of fresh culture medium containing Au NRs@INU-LA-PEG-FA or Au NRs@PHEA-EDA-FA at a concentration per well equal to 15, 7.5, 3.75, and 1.87 μg mL−1 of Au0, or Au NRs@INU-LA-PEG-FA/Nutlin or Au NRs@PHEA-EDAFA/Nutlin at different concentrations (15, 7.5, 3.75, and 1.87 μg mL−1 of Au0), corresponding to equivalent concentrations of nutlin-3 equal to 3, 1.5, 0.75, and 0.375 μM, were added. After 24 or 48 h, MTS assay was performed. Nutlin solutions at the same concentrations (ranging from 0.375 to 3 μM.) were used as a positive control. Cell medium was used as a negative control. All culture experiments were performed in triplicate. An analogue cell cytotoxicity assay was conducted incubating the correspondent folate-free systems AuNRs@INU-LA/Nutlin and AuNRs@PHEA-EDA/Nutlin, on the same cell lines, using the same concentrations and incubation times. For cell cytotoxicity assay on 3D U2OS cultures, organoids were formed and cultured for 3 days as described above. The fourth day, 10 μL of media were replaced with fresh culture medium containing Au NRs@INU-LA-PEG-FA or Au NRs@PHEA-EDA-FA at a concentration per well equal to 260, 130, 52, 26, 5, 2 μg mL−1 of Au0, or Au NRs@INU-LA-PEG-FA/Nutlin or Au NRs@PHEA-EDA-FA/Nutlin at different concentrations (260, 130, 52, 26, and 5.2 μg mL−1 of Au0), corresponding to equivalent concentrations of nutlin equal to 50, 14456


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copolymer of the α,β-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) functionalized with primary amine groups and folic acid, named PHEA-EDA-FA. Concerning the synthesis of INULA-PEG-FA, inulin (α-D-glucopyranosyl-[β-D-fructofuranosyl](n-1)-D-fructofuranoside), a natural polysaccharide composed by fructose units (β −1,2), was used as main polymeric chain. Above all, lipoic acid (LA) was bound onto inulin to introduce a hydrophobic chain disulfide terminated such that it can be reduced by exposing thiol groups with high affinity to the GNRs’ surface. In particular, the carboxyl group of LA was activated using DCC/DMAP as a coupling agent and left to react with the hydroxyl groups of the inulin backbone. The amount of LA and carboxylic acid activating agents were used according to the moles of LA/mol repeating units of inulin = 0.5; moles of DCC/mol of LA = 1.2, and moles of DMAP/mol of LA = 1.2. The molar derivatization degree of LA linked to inulin was calculated by means of 1H NMR spectroscopy, making a comparison between the integral of the peaks between δ 2.5−1.4 imputable to the protons of LA, to that of the protons of inulin at δ between 3.6 and 4.3. The molar percent of LA linked to inulin (DDLA%) was equal to 23 mol % referring to inulin repeating units. INU-LA still presents a large amount of hydroxyl groups available for other subsequent functionalization, such as PEGylation. Hence, the following step was the synthesis of PEGylated inulin derivative by employing a poly(ethylene glycol) bis(amine) having an average molecular weight of 2000 Da, previously conjugated with folic acid (PEG-FA). In a second step, the hydroxyl groups of INU-LA copolymer were activated with BNPC by means of microwave reactor and left to react with the available terminal amine group of PEG-FA conjugate to obtain the final product INU-LA-PEG-FA. The stoichiometric conditions employed were as follows: mol BNPC/mol repeating units of inulin = 0.1; mol PEG-FA/mol repeating units of inulin = 0.1. INU-LAPEG-FA copolymer was characterized by 1H NMR spectroscopy, which confirmed the occurred functionalization with PEG-FA chains on the INU-LA backbone and made possible the quantification of the molar derivatization degree (DDPEG‑FA%). The DDPEG‑FA%, indicated as percentage of linked poly(ethylene glycol)-folate chains in comparison with the repeating units of inulin, was determined by comparing the integral of the peak related to protons at δ 3.7 assigned to ethylene protons of PEG with the integral of the peaks related to protons of inulin backbone at δ comprised between 3.6 and 4.20. The DDPEG‑FA% was equal to 4 ± 0.5 mol % referring to the inulin repeating units (Figure S1 of the Supporting Information, SI). With regard to the synthesis of the PHEAEDA-FA copolymer, the activation reaction of hydroxyl groups of PHEA with BNPC followed by the reaction with EDA allowed the derivatization of polymer chains with pendant primary amine groups obtaining the PHEA−EDA copolymer as previously reported,35 with a derivatization degree in EDA equal to 25 mol % referring to PHEA repeating units. PHEAEDA primary amine groups reacted with γ-carboxyl group of folic acid in aqueous medium upon activation with EDC-HCl and NHSS, yielding a partial functionalization of PHEA-EDA primary amine groups with folic acid (Scheme 1). After purification, the obtained PHEA-EDA-FA copolymer was characterized by 1H NMR analysis which confirmed the reaction and the quantitative derivatization of amine groups, by comparing the integrals of the peak at δ 7.52−6.69, ascribable to the protons of the phenyl ring of FA, with the integral of the peaks related to protons at δ 2.85 assignable to −CH−CH2−

buffer (Triton-X) and centrifugated for 5 min at 1000 rpm. The obtained pellet was treated with 1 mL of aqua regia and kept for 2 h at 37° to allow the complete dissolution of gold nanoparticles. Then the content in gold(III) was determined according to the product manual. In Vitro Two Photons Imaging of U2OS Cells with Gold NRs. The cellular uptake of polymer coated GNRs was evaluated by two photon excitation microscopy analysis. Three ×103 cells were added to 8-well chambered coverglass (Nunc Lab-Tek II) and allowed to adhere overnight. AuNRs@INU-LA-PEG-FA and AuNRs@PHEA-EDA-FA were added at a final concentration of 2 μg mL−1 of Au0 and cells were incubated for either 6 or 24 h. After each incubation time, cells were washed using Dulbecco phosphate buffer solution (DPBS), and stained using 40,6-diamidino-2-phenylindole (DAPI) diluted 1/600 in cell media. After 15 min incubation at room temperature, cells were fixed using glycerol/DPBS mixture 80:20 v/v, at room temperature. Both living or glycerol (glycerol/DPBS 80:20 v/v) fixed cells were imaged at 1024 × 1024 pixel resolution using a Leica TCS SP5confocal laser scanning microscope with a 63× oil objectiveNA = 1.4 (Leica Microsystems, Germany). Measurements were acquired at 400 Hz frequency in two channels under at 790 nm (Spectra-Physics Mai-Tai Ti:Sa ultrafast laser. DAPI signal was detected in the spectral range 420−515 nm (blue channel) and the spectral range 605−730 nm was chosen as a good compromise to detect GNRs’ luminescence avoiding cells auto fluorescence and other unsought signals. Confocal Fluorescence Microscopy of U2OS Organoids with Alexafluor 647-Labeled GNRs. The diffusion of polymer coated GNRs into U2OS organoids was evaluated by confocal fluorescence microscopy analysis. U2OS organoids, formed as above-described, were incubated with Alexafluor647-labeled AuNRs@INU-LA-PEG-FA and AuNRs@PHEA-EDA-FA at a final concentration of 260 μg mL−1 of Au0 for 24 h. After incubation time, U2OS organoids were washed using Dulbecco phosphate buffer solution (DPBS), transferred in 8well chambered coverglass (Nunc Lab-Tek II) and imaged using a Leica TCS SP5confocal laser scanning microscope with a 40× oil objectiveNA = 1.4 (Leica Microsystems, Germany). Statistical Analysis. A T Test was applied to compare different sample groups. Data were considered statistically significant with a value of p < 0.05.

■

RESULTS AND DISCUSSION Synthesis and Characterization of INU-LA-PEG-FA and PHEA-EDA-FA Copolymers. Interest in functionalizing gold nanoparticles (GNPs) and in particular gold nanorods (GNRs) for manifold biomedical purposes, such as the delivery of chemotherapeutic in cancer, has risen in recent years. Usually, GNPs have the tendency to aggregate mainly in case of anisotropic particles like GNRs, limiting their application in therapy. For this reason, preparation of a stable colloidal GNR system is a challenge in different application fields. The problem of instability due to aggregation could be overcome by decorating the surface of GNPs with hydrophilic or amphiphilic polymers, containing −SH or amino functional groups, exploiting the binding affinity of GNPs for thiols and amines via the formation of Au−S or Au−N bonds.42 Surface modification with polymers increases circulation lifetime, improves the stability of GNPs, and reduces the cytotoxicity caused by certain stabilizing agents such as cetyl triethylammonium bromide (CTAB) used in the synthetic procedure of GNRs. Besides guaranteeing stability in aqueous media, the polymeric coating can also provide the nanosystems with cancer specificity, by means of conjugation with targeting ligands able to recognize specific receptors overexpressed in malignant cells, thus enabling active tumor targeting. These considerations have encouraged the synthesis of two new folate-conjugate polymers able to act as GNR coating agents. One is an amphiphilic polysaccharide based graft-copolymer, henceforth named INU-LA-PEG-FA, and the second one is a 14457


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ACS Applied Materials & Interfaces Scheme 1. Synthesis of PHEA-EDA-FA

Table 1. Summary of Average Molecular Weight (Mw), Polydispersity, and Molar Derivatization Degree Values of LA, FA, and EDA in the Inulin and PHEA Copolymers and Their Parent Compounds sample

Mw (KDa)

MwMn

inulin INU-LA INU-LA-PEG-FA PHEA PHEA-EDA PHEA-EDA-FA

4.56 10.06 8.96 36.4 26 26.8

1.06 1.54 1.23 1.31 1.57 1.44

DDEDAa (mol %) DDLAa(mol %) DDFAb(mol %) 23 23 25 25

4

5

a

Calculated by 1H NMR spectroscopy. bCalculated by combining 1H NMR spectroscopy and UV spectrophotometry.

respectively available in the side chains of the above-described copolymers. In particular, it is well-known in the literature that the thiol/disulfide redox couple can coexist on the GNP surface being favored by the disulfide formation for high density gold surface coating.42 However, the amino groups of PHEA derivative formed an electrostatic complex between copolymers protonated amino groups and negative charged gold surface for the presence of AuCl4-/AuCl2- ions.43 These polymeric coatings had a variety of tasks. Alternatively, both polymers may act as a colloidal hydrophilic coating stabilizing the dispersion of the nanosystem into aqueous media. Furthermore, they behave as a targeting moiety for the cancer-targeted therapy. Finally, they can provide the loading of drugs by means of physical interactions. The occurrence of polymer coatings on the GNPs surface was studied by qualitative energy dispersion X-ray elemental analysis (Figure S2) that revealed the characteristic peaks of carbon, oxygen, and nitrogen of both the polymeric coatings. The stability of the nanosystems in aqueous media was studied by VIS-NIR spectroscopy. As shown in Figure 1a, absorption

CO−NH− of the polymer backbone (Figure S1). The DDFA% was equal to 4.5 mol %. The molar percent of FA covalently linked into PHEA-EDAFA and INU-LA-PEG-FA (DDFA %) was also determined by means of UV spectroscopy, measuring the absorbance of a weighted amount of copolymers in Milli-Q water pH 8 for NaOH 1N at λ 364 nm, and comparing this value with a calibration curve obtained from standard solutions of folic acid in the same solvent. The DDFA% calculated by means of UV spectroscopy was equal to 6.2%. The average folate derivatization degree (DDFA%) calculated by means of 1H NMR and UV spectroscopies was equal to 5.35 mol %. Weightaverage molecular weights (Mw) and polydispersity of the synthesized copolymers were determined by SEC analyses, and data are reported in Table 1. The molecular weight increment of INU-LA and INU-LA-PEG-FA with respect to the starting inulin confirms the derivatization of the inulin backbone with LA first and, subsequently, with PEG-FA. Preparation and Characterization of INU-LA-PEG-FA and PHEA-EDA-FA Coated GNRs (Au NRs@INU-LA-PEGFA, Au NRs@PHEA-EDA-FA). GNRs were functionalized with INU-LA-PEG-FA and PHEA-EDA-FA copolymers (Au NRs@ INU-LA-PEG-FA, Au NRs@PHEA-EDA-FA) taking advantage of the high affinity of gold for thiols and primary amines

Figure 1. Vis−NIR spectra of AuNRs@CTAB, AuNRs@INU-LAPEG-FA, and Au@PHEA-EDA-FA nanorods (a), showing a sharp longitudinal SPR band fitted at around 770 nm without signs of aggregation in Milli-Q water. TEM image of starting AuNRs@CTAB (b).

spectra of the NRs sample shows the expected shape for GNRs, with a weak band in the visible range centered at about 510 nm and a strong one in the NIR range centered at 770 nm. Within the experimental error AuNRs@INU-LA-PEG-FA and AuNRs@PHEA-EDA-FA are superimposable with the starting AuNRs@CTAB particles, whose TEM is shown in Figure 1b. 14458


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ACS Applied Materials & Interfaces Lipoic acid (LA) tails in the coating polymer INU-LA-PEGFA ended up having a fundamental role in the nanorods stability at pH 7.4 and pH 5.5 at 37 °C. Au@INU-PEG-FA NRS not functionalized with LA tails were prepared using the same procedure (see SI 1.1 for details) and resulted in the loss of the characteristic Vis−NIR absorption properties within 2 h of preparation (see Figure S3) thus pointing out the key role of the thiol groups in stabilizing the gold nanoparticles. A difference between INU-LA-PEG-FA and PHEA-EDA-FA coated GNRs was observed. Particularly, the ratio of INU-LAPEG-FA molecules/nm2 Au0 required to guarantee a good aqueous stability to GNRs is higher (R = 5) than the ratio used to stabilize the particles with PHEA-EDA-FA conjugate (R = 2) (Table S1). This can be explained taking into account the big differences in Mw and structure of the two copolymers. As a matter of fact, INU-LA-PEG-FA has an Mw of 8.9 kDa that is much lower than the Mw of PHEA-EDA-FA of about 30 kDa. Moreover, PHEA-EDA-FA, unlike the amphiphilic characteristics of inulin derivative, is endowed with a highly hydrophilic nature able to create a remarkable hydration shell surrounding GNRs. The SEM microscopy confirmed the absence of aggregation of GNRs in both cases showing well separated particles with an average longitudinal length of about 70 nm, surrounded by organic coatings (Figures 2 and 3).

Figure 3. Vis−NIR spectra of AuNRs@CTAB, AuNRs@INU-LAPEG-FA/Nut, and Au@PHEA-EDA-FA/Nut in Milli-Q water.

Nut nanorods are presumably of different origin which will be investigated in the following. For Au@INU-LA-PEG-FA, the most likely drug loading mechanism is the encapsulation of hydrophobic drug into the hydrophobic domain of the coating consisting of the lipoic acid tails. It is possible to infer that the simultaneous presence in the INU-LA-PEG-FA copolymer of both hydrophobic and hydrophilic portions makes this macromolecule suitable to form polymeric micelles by self-assembling in aqueous dispersion. In order to prove this hypothesis, micelles formation was investigated through fluorescence studies using pyrene as fluorescent probe and it was estimated to be 0.030 mg mL−1 (3.9 × 10−6 M) that is a concentration close to that used to functionalize GNRs (0.035 mg mL−1). Taking the above result into consideration, it is reasonable to assume that hydrophobic gold core as well as the hydrophobic drug nutlin-3 are efficiently encapsulated into polymeric nanoaggregates existing in aqueous dispersion. Concerning Au NRs@PHEA-EDA-FA/Nut the driving forces involved in drug loading procedure are supposed to be most likely physical interactions, for instance hydrogen bonding between the nutlin-3 molecules and the polyamino acidic backbone of PHEA-EDA-FA, as well as hydrophobic interactions between the hydrophobic drug domains and the gold core. Indeed, PHEA-EDA-FA copolymer is not able to aggregate in the conditions used in the coating procedure being the CAC of PHEA-EDA-FA much higher than the concentration of the copolymer used during the coating of GNRs (CAC of PHEA-EDA-FA = 0.95 mg mL−1). The amount of drug loaded in INU-LA-PEG-FA and PHEA-EDA-FA coated systems was evaluated by HPLC analysis, and it was equal to be 12.00% and 13.9% w/w respectively, with an encapsulation efficacy of approximately 40% and 50% for Inulin and PHEA derivatives coated GNRs (Table S1). SEM microscopy (Figure S4) of nutlin-3 loaded GNRs did not evidence significant differences between loaded and unloaded systems shape and morphology. Vis−NIR spectroscopy highlights a tiny red shift of the NIR peak confirming ligand binding on NRs surface, the hallmark shape of these nanorods spectrum is maintained for several days confirming long-term stability of both systems (Figure S5). Zeta potential measurements resulted in values of +9.2 mV for Au NRs@INU-LA-PEG-FA/Nut and +31.3 mV for Au NRs@PHEA-EDA-FA/Nut in contrast with +14.6 mV for Au

Figure 2. SEM micrographs of AuNRs@PHEA-EDA-FA (a) and AuNRs@INU-LA-PEG-FA (b).

The obtained GNRs were able to efficiently load nutlin-3, an anticancer drug currently in phase clinic I. Nutlin-3 is widely employed to treat p53 wild-type tumors such as retinoblastoma and osteosarcoma. It actively inhibits p53-MDM2 interaction, avoiding MDM2-mediated p53 degradation and resulting in p53 accumulation that induce cell apoptosis.33 The driving forces that allowed the loading of nutlin-3 on Au@INU-LA-PEG-FA/Nut and Au NRs@PHEA-EDA-FA/ 14459


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Figure 4. Drug release from AuNRs@INU-LA-PEG-FA/Nut (a) and of AuNRs@PHEA-EDA-FA/Nut (b), obtained from left to right in human plasma, PBS pH 7.4 and PBS pH 5.5; in both media 10% of FBS was added as solubilizing agent.

Figure 5. NIR laser-triggered release of nutlin-3 from AuNRs@PHEA-EDA-FA/Nut (a) and from AuNRs@INU-LA-PEG-FA/Nut (b) in PBS pH 5.5 + FBS 10%, after laser exposure (30−120 s) followed by 1, 2, and 4 h of incubation time at 37 °C. Red line indicates the temperature of the release medium.

tumor environment, 36% of drug release was observed for Au NRs@PHEA-EDA-FA/Nut system versus the 18% at pH 7.4 and the 16% in human plasma after 48 h. On the contrary, INU-LA-PEG-FA coated GNRs did not show marked differences in drug release rate in the three release media, settling between 15 and 20% of drug released after 48 h in all the release media, and a stronger retention of nutlin-3 can be noticed resulting in a slower release rate of the drug. The pH dependent drug release of a PHEA-EDA-FA coated system can be explained with the profile of ionization of primary pendant amino groups of PHEA-EDA-FA. Owing to the high amount of amino groups that can be protonated in the acidic environment, structural organization of the chain may occur, changing the solvent solvent affinity, resulting in larger drug release. Alongside the pH dependent drug release, it could be readily controlled by means of external stimuli such as NIR laser and has therefore been proposed to improve the selectivity of the treatment and reduce unwanted side effects, since accurate and

NRs@CTAB, as a further confirmation of successful modification of the GNR surface (Table S1). The capability of these systems to release the loaded drug was evaluated in three media simulating different human body districts. In particular, PBS solution at pH 7.4, which mimics physiological medium; pH 5.5, mimicking tumor environment; and human plasma to mimic the intravenous administration of the nanosystem. At selected time points, the amount of released nutlin in each medium was quantified. In Figure 4 the cumulative release of nutlin-3, referred to as the total amount of nutlin-3 loaded into the nanosystem, is shown as a function of the incubation time. It is noteworthy that in human plasma, both systems released a small amount of their payload in 1 day (7% for inulin coated system and 12% for PHEA coated GNRs after 24 h), this being in line with data in Figure S2 and guaranteeing good stability in human plasma. This feature is important to limit systemic toxic effects before GNRs are accumulated into tumor mass, where a higher drug amount should be released. At pH 5.5, in conditions that simulate 14460


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Figure 6. Cell viability as a function of nutlin-3 concentration on U2OS (left graphs) and HDFa.

worth noting that after 120” laser exposure and 4 h at 37 °C at pH 5.5, the amount of nutlin-3 released was more than twice as large as that released from the untreated nanosystems for the INU-LA-PEG-GA coated system (from 3.8 to 10%), and four times higher (from 6 to 24%) for PHEA-EDA-FA coated GNRs (Figure 5). These results point out that the highly efficient and localized light-to-heat conversion by plasmonic nanoparticles renders the tested nanosystems extremely useful in the selective treatment of cancer.

selective control of the release rate of encapsulated drugs is possible using light-responsive materials such as GNRs.15 By exploiting the widely known ability of GNRs to transform an NIR light into heat (hyperthermic effect),44 the hyperthermia-triggered drug release was assessed exposing a dispersion of Au NRs@INU-LA-PEG-FA/Nut and AuNRs@ PHEA-EDA-FA/Nut to a NIR laser beam for 30, 60, 90, and 120 s, and incubating the resulting dispersion at 37 °C for 1, 2, and 4 h, so as to measure nutlin-3 release at equilibrium. It is 14461


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Table 2. IC50 Values of AuNRs@INU-LA-PEG-FA/Nut, AuNRs@PHEA-EDA-FA/Nut, AuNRs@INU-LA/Nut, AuNRs@ PHEA-EDA/Nut, and Free Nutlin-3, after 24 and 48 h of Incubation with U2OS and HDFa Cell Lines sample

IC5024h (μM) U2OS

IC5048h (μM) U2OS

IC5024h (μM) HDFa

IC5048h (μM) HDFa

AuNRs@INU-LA-PEG FA/Nut AuNRs@INU-LA/Nut AuNRs@PHEA-EDA-FA/Nut AuNRs@PHEA-EDA/Nut Nutlin

1.3 1.68 1.1 1.68 1.7

0.57 0.82 0.35 0.66 0.46

2.22 1.94 3.42 3.9 0.44

1.27 1.21 1.17 1.1 0.36

In Vitro Biological Evaluations. The in vitro cytotoxic profile of the GNRs based systems, loaded with nutlin-3, and the respective drug-free systems, was evaluated on 2-D and 3-D cell colture models. For 2-D assays, U2OS human osteosarcoma cell line, HDFa dermal fibroblasts and 16 HBE human bronchial epithelial cell lines were used as cell colture models; for 3-D assays, tridimensional tumor models of human osteosarcoma were used (tumor spheroids). Actually, the latter better resemble the histomorphological, functional, and microenvironmental features of in vivo human tumor tissue. For an initial screening of the efficacy of GNRs based systems, MTS viability assay was carried out on the traditional 2-D in vitro coltures of the above-mentioned three cell lines after an incubation time of 24 and 48 h using free nutlin-3 as positive control. Figure 6 shows the cell viability of AuNRs@ INU-LA-PEG-FA/Nut and AuNRs@PHEA-EDA-FA/Nut on U2OS and HDFa cell lines. Empty nanosystems (Figure S6), resulted in cytocompatibility, wherein the cell viability was comparable with the control cells in the whole range of concentrations used and for all cell lines tested, proving also that the highly cytotoxic cationic stabilizing agent CTAB had been successfully replaced by polymeric coating. Differently, Au NRs@INU-LA-PEG-FA/Nut and Au NRs@PHEA-EDA-FA/ Nut exerted a cytotoxic effect on all the cell lines tested, showing a time-dependent and dose-dependent efficacy (Figure 6). A detailed observation reveals interesting differences between cancer and noncancer cell lines. In particular, when nutlin-3 is loaded in Au NRs@INU-LA-PEG-FA/Nut and Au NRs@ PHEA-EDA-FA/Nut systems, it is always more effective on osteosarcoma U2OS cells than on HDFa fibroblasts, while free nutlin-3 being used as positive control could not easily distinguish between cancer and noncancer cell lines. For example, at the intermediate drug concentration of 1.5 μM, already after 24 h incubation time, the cell viability of osteosarcoma cells (U2OS) incubated with Au NRs@INULA-PEG-FA/Nut and Au NRs@PHEA-EDA-FA/Nu is about 38% and 29%, respectively, in contrast with the viability of HDFa cell lines that is, at the same drug concentration, 64% for inulin coated GNRs and 45% for PHEA coated GNRs. By contrast, the cytotoxic effect of free nutlin-3 was higher in noncancer cell lines than on osteosarcoma cells for the same concentration of 1.5 μM used; in fact, U2OS cells showed a cell viability of about 40% whereas that of the HDFa cells was around 25%. The IC5024h and IC5048h values calculated by the dose−response curves show more clearly the notwithstanding higher potency of the folate targeted systems on U2OS cells than on HDFa cells respect to free nutlin-3 (Table 2). Furthermore, in order to prove the effective FA-mediated targeting endocytosis of the proposed systems, a study with FAfree GNRs, in terms of cytotoxicity, was performed. In particular, GNRs were coated with the respective copolymers PHEA-EDA and INU-LA, without folic acid in polymeric

backbone and loaded with nutlin-3 using the same experimental conditions used for FA-conjugated GNRs. As shown in Figure 6, the FA-free GNRs exerted a lower cytotoxic effect and showed a minor drug potency on U2OS cancer cells respect to the FA-targeted systems (as supported by IC50 values reported in Table 2), while showing a similar efficacy on HDFa (Figure 6 and Table 2) and 16 HBE noncancer cell lines (Figure S7 and Table S2) imputable to a lower internalization of the FA-free systems in cancer cells. Overall, these results are consistent with literature findings and showed an evident folate-enhanced cytotoxicity since the conjugation with folate of both coating polymers proved capable of improving the cancer selectivity respect to free nutlin-3. These results can be explained with the ability of the folate targeted system to be preferentially internalized by U2OS cells, which express high levels of FRs, rather than in HDFa noncancer cells, suggesting a folate-mediated endocytosis mechanism which allowed exertion of a selective cytotoxic effect toward cancer cells. A similar selective trend is also seen in another noncancer cell line, that is in human bronchial epithelial cell line (16HBE) (Figure S7 and Table S2). Furthermore, the PHEA-EDA-FA coated GNRs have been proven to be more effective than INU-LA-PEG-FA coated GNRs in killing U2OS cancer cells as showed by IC50 values both at 24 and 48 h. This finding may be imputed to two different reasons: an higher release rate of nutlin-3 from PHEAEDA-FA coated GNRs, both at physiological pH of 7.4 and at acid tumoral and endosomial pH of 5.5, and to an higher internalization of a PHEA coated system into cancer cells. With this aim, two photon luminescence (TPL) imaging was used for the evaluation of cellular uptake of gold NRs by exploiting the property of NRs subjected to multi photon laser excitation to emit a strong fluorescence. Figure 7 shows representative images of living U2OS cancer cells at 6 and 24 h of incubation with AuNRs@INU-LA-PEG-FA (a) and AuNRs@PHEA-EDAFA (b). As can be seen, fluorescence signal of NRs inside cells measured in the red channel significantly grows from 6 to 24 h incubation time for both systems. Interestingly, measurements at 24 h shows the presence of coated NRs in localized region of the cytoplasm with PHEA-EDA-FA-GNRs signal observed to be distributed in the whole cytoplasm (as also evident in Video S1). We also present images obtained when the sample is fixed using glycerol/DPBS 80:20 v/v which allow better visualizing GNRs signal also outside cells. Data in Figure 8 show representative images of uptake U2OS cancer cells fixed with glycerol/DPNS 80:20 v/v after 24 h of incubation time with AuNRs@INU-LA-PEG-FA (a) and AuNRs@PHEA-EDA-FA (b). In panel a) it is possible to detect the presence of INU-LAPEG-FA/Nut coated GNRs outside osteosarcoma cells and see that the most of cytoplasm does not contain NR which appear to be localized in the perinuclear region. In panel (b) in the same conditions it is not possible to distinguish GNRs signal 14462


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concentrations (ranging from 1 to 50 μM) and a longer incubation time were needed to appreciate a biological effect. After 72 h incubation time, the viability of OS microtumors was evaluated by means of MTS assay and some images showing the tumor spheroids before and after treatment with the nanosystems were acquired (Figure 10). Notably, also in 3D colture, AuNRs@PHEA-EDA-FA/Nut has proved to be the most effective system in causing cell death and reducing tumor volume. In particular, at the highest drug concentration (50 μM), cancer spheroid viability was reduced up to 33% when treated with PHEA-EDA-FA/Nut coated GNRs, and up to 47% for INU-LA-PEG-FA/Nut coated system. At the same time, at the highest concentration tested, microtumors surface area (calculated by means of Zeiss microscope software) was reduced of 78% and of 65% for organoids treated with PHEAEDA-FA/Nut and INU-LA-PEG-FA/Nut coated systems, respectively. IC5072h values highlight higher potency of AuNRs@PHEA-EDA-FA/Nut coated system respect to AuNRs@INU-LA-PEG-FA/Nut system on 3D colture and point out a comparable potency of free nutlin-3 and PHEAEDA-FA/Nut coated NRs. Organoids incubated with empty systems, Au NRs@INU-LA-PEG-FA and Au NRs@PHEAEDA-FA and untreated organoids (control) showed a high tumor progression as they increase their volume after 72 h of incubation (Figure S9) The antitumoral activity of GNRs based systems was accompanied by efficient internalization and deep diffusion into tridimensional U2OS organoids as early as 24 h of incubation with GNRs, as confirmed by the fluorescence signal of Alexa-labeled GNRs inside spheroids, measured in the red channel (Figure 11). Obtained images show a gradient of the fluorescence signal from outside to inside the “microtumor” mass for INU-LA-PEG-FA coated GNRs (Figure 11a) while a more uniform localization of the system into the spheroid mass can be observed for PHEA-EDA-FA coated GNRs (Figure 11b). The fluorescence signal inside spheroids is certainly associated with the presence of gold nanorods penetrated into microtumors. Actually, to support this finding, the gold content into U2OS spheroids was determined and the results reported in Figure 12. On the whole, the obtained data showed that the GNRs based systems were efficiently internalized into “microtumor” mass and moreover, an increase of gold amount into the U2OS organoids was detected from 24 to 48 h incubation time for both systems. In Vitro Hyperthermic Effect of Au NRs@INU-LA-PEGFA/Nut. Thermoablation experiments on U2OS cells, and on tridimensional U2OS culture, were performed to demonstrate the high and selective heating efficiency of polymer coated GNRs under NIR laser exposure against the U2OS cells and highlight the advantages of the hyperthermia treatment. U2OS and HDFa cells were incubated for 4 h with Au NRs@INU-LAPEG-FA and Au NRs@PHEA-EDA-FA systems loaded and not loaded with nutlin-3 as well as free nutlin-3 whose concentration provides a low cytotoxic effect after 4 h of incubation (0.75 μM) and subsequently, treated with a 810 nm laser beam with a fixed power for 120 s. Then, the effect of generated hyperthermia was evaluated by MTS assay after 24 h of postincubation following laser treatment. In line with expectations, the nutlin-3 loaded GNRs exerted an higher toxic effect than the empty systems in both cell lines. As a matter of fact, in addition to toxic effects attributable to protein

Figure 7. 1024 × 1024 Representative images of in living U2OS cancer cells at 6 and 24 h of incubation with AuNRs@INU-LA-PEGFA (a) and AuNRs@PHEA-EDA-FA (b). GNRS signal red channel, DAPI fluorescence (nuclear stain) blue channel. Intensity pseudoscale is adapted to better visualize the signal.

Figure 8. 1024 × 1024 Representative images of U2OS cancer cells at 6 and 24 h of incubation with AuNRs@INU-LA-PEG-FA (a) and AuNRs@PHEA-EDA-FA (b). GNRS signal red channel, DAPI fluorescence (nuclear stain) blue channel. Images are acquired after fixation with glycerol/DPNS 80:20 v/v.

outside cells and a different topological distribution of the same signal is evident within the cytoplasm. Although important information has been collected from traditional in vitro studies in which cells are grown on flat surfaces (2D culture), this culture condition does not reflect the essential features of tumor tissues. Thus, in order to better study the biological properties of the proposed systems, the anticancer activity was studied on tridimensional osteosarcoma colture as well, which better mimicks tumor microenivroment, being cancer cells, relevant matrix components, and biochemical and biophysical signals are integrated. In Figure 9 some differences can be noticed in responses from 3D in vitro tumors and conventional monolayer cultures. The “microtumors” are characterized by a certain drug resistance being less sensitive to the toxic effect of nutlin, therefore, higher 14463


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Figure 9. Cytotoxicity assay on tridimensional U2OS organoids incubated for 72 h with AuNRs@INU-LA-PEG-FA/Nut (a) and AuNRs@PHEAEDA-FA/Nut (b). The viability of organoids was determined using the MTS assay. Data represent mean ± standard deviation of triplicate experiments.

Figure 11. Representative fluorescence (left) and brightfield (right) images of living U2OS organoids at 24 h of incubation with AlexaFluor647-labeled AuNRs@INU-LA-PEG-FA (a) and Au NRs@ PHEA-EDA-FA (b). Intensity pseudoscale is adapted in order to better visualize the signal. Magnification 40×.

Figure 10. Brightfield images of tridimensional U2OS organoids incubated with AuNRs@INU-LA-PEG-FA/Nut and AuNRs@PHEAEDA-FA/Nut systems, at a concentration of 50 μM of nutlin-3, before and after 72 h incubation time, showing a significant reduction of microtumors volumes after treatment.

nanoparticles uptake by cancer cells just after 4 h incubation, due to the affinity between folate-functionalized NRs surface and FRs overexpressed in U2OS cells. In addition we have to consider the widely known higher resistance of noncancer cells to damages induced by high temperatures.45 The intracellular uptake of nutlin-3 resulted, as detected by HPLC analysis, is significantly higher in cancer cells for both polymer coated GNRs, thus justifying the selective toxic effect previously found. Worth of noting is that the quantitative uptake studies (Figure 14) confirmed the higher uptake of FA-targeted systems respect to the corresponding ones without FA molecules on GNRs surface, thereby strengthening the folate-mediated endocytosis mechanism which allowed it them exert a selective cytotoxic effect toward cancer cells. The selective drug accumulation observed in U2OS cells, was enhanced by applying an NIR laser for 120 s before incubating

denaturation and physical cell eradication owing to high temperature (see at this regard the formation of a black hole in the well of treated cells, Figure S8), an enhanced drug release and drug sensitization triggered by hyperthermia occurred, provoking additional cell death up to 5% of cell survival for U2OS cells and up to 31% for HDFa after 120 s laser exposure in the case of Au NRs@PHEA-EDAFA/Nut (Figure 13). It is worth noting that both polymer coated systems displayed a lower toxicity to noncancer HDFa cells, whereas it was significantly higher in U2OS cells, above all when we used PHEA-EDA-FA coated GNRs. The selective damage or killing of U2OS cells can be well explained by the enhanced 14464


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for 4 h. As a matter of fact, as shown in Figure 14, nutlin-3 was more efficiently internalized in U2OS cells, especially upon laser treatment and in particular when loaded in PHEA-EDAFA coated GNRs. The higher drug internalization triggered by NIR laser is related principally to two factors: the temperatureinduced increase of cell membrane permeability and the enhancement of drug delivery efficiency, as previously proven (Figure 5), thus inducing an higher and selective toxic effect toward cancer cells. In an attempt to evaluate the therapeutic potentiality of phototermal ablation of solid tumors for an efficient localized cancer treatment, we carried out thermoablation experiments on osteosarcoma tridimensional organoids. Apart from the fact that an higher GNRs concentration was used (52 μg/mL, Nutlin: 10 μM) and that MTS assay was conducted after a longer incubation time following the laser exposure (48 h), an analogous experiment to that carried out for conventional 2D

Figure 12. Gold amount in U2OS organoids after 24 and 48 h incubation with Au NRs@INU-LA-PEG-FA and Au NRs@PHEAEDA-FA.

Figure 13. Cytotoxicity assay, after hyperthermia treatment, on U2OS and HDFa cell lines incubated with drug-free and drug loaded AuNRs@INUA-PEG-FA (a) and AuNRs@PHEA-EDA-FA (b) systems at equivalent concentration of nutlin-3 of 0.75 μM and of Au0 of 3.75 μg mL−1 after 4 h of incubation, followed by laser treatments and 24 h of post incubation. Cells and free nutlin-3 were used as controls. 14465


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Figure 14. Quantitative cell uptake in U2OS (a) and HDFa cells (b) of AuNRs@INU-LA-PEG-FA/Nut, AuNRs@INU-LA/Nut (left graphs), AuNRs@PHEA-EDA-FA/Nut, and AuNRs@PHEA-EDA/Nut (right graphs) compared with free nutlin-3.

Figure 15. Cytotoxicity assay, upon hyperthermia treatment, on tridimensional U2OS organoids incubated with drug-free and drug loaded AuNRs@ INU-A-PEG-FA (a) and AuNRs@PHEA-EDA-FA (b) systems at equivalent concentrations of nutlin-3 of 0.75 μM and of Au0 of 3.75 μg mL−1 after 24 h of incubation, followed by laser treatments and 48 h of postincubation. Cells and free nutlin-3 were used as controls.

colture, was performed. The penetration depth and the toxic effect could be improved by hyperthermia treatment in tumor spheroids. MTS viability assay showed the high toxicity of empty GNRs and GNRs loaded with nutlin-3 on osteosarcoma organoids, demonstrating the greater effectiveness after 200 s

exposure time, corresponding with the almost complete absence of living cells (cell viability close to 1%) (Figure 15). In Figure 16 it can be noticed that most of the organoids treated with GNRs were disgregated and looked “burned” after NIR laser treatment and, notably, the organoids size was almost 14466


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Figure 16. Brightfield images of U2OS organoids incubated with AuNRs@INU-LA-PEG-FA/Nut, AuNRs@PHEA-EDA-FA/Nut and free nutlin-3 at equivalent concentrations of drug of 10 μM and of Au0 of 52 μg mL −1 Au0, acquired before and after 30, 60, and 200 s of NIR laser exposure.

Luminescence (TPL) images. In addition to being efficient chemotherapeutics and selective TPL diagnostics agents, the proposed polymer coated GNRs turned out to be efficient hyperthermia agents, thus producing a selective thermoablation of the tumor mass together with an NIR laser-triggered drug release and sensitization. On the whole, Au NRs@INU-LAPEG-FA/Nut and Au NRs@PHEA-EDA-FA/Nut displayed remarkable anticancer activity both on conventional bidimensional cell culture as well as on tridimensional osteosarcoma tumor models, acting as effective drug delivery systems, hyperthermia agents, and imaging contrast agents. Therefore, these gold NRs have proven to be promising theranostic agents for improving osteosarcoma disease outcome.

completely suppressed by polymer coated GNRs upon 200” laser treatment. This effect is also evident for drug-free GNRs (Figure S10). By contrast, the organoids treated with free nutlin-3 reduce their surface just up to 45% of their starting surface, remarking that the laser treatment enhanced anticancer activity of nutlin-3. As expected, the control groups, showed a continuous proliferation.

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CONCLUSIONS In this work, we developed successfully two different polymer coated GNRs systems targeted with folic acid molecules for achieving cancer specificity toward osteosarcoma cells. Both polymeric coatings, INU-LA-PEG-FA and PHEA-EDA-FA, endowed GNRs with excellent physiochemical stability in aqueous media and allowed a good loading capability of anticancer drug nutlin-3. It is noteworthy that both polymer coated GNRs efficiently protected the drug in human plasma, and enabled a sustained drug release in different media simulating cell cytoplasm and tumor microenvironment. In particular, PHEA-EDA-FA coated GNRs gave rise to a smart pH sensitive release, increasing of the double the drug release % at acidic pH mimicking intratumoral conditions. Both of the studied systems were able to be preferentially accumulated in U2OS cells and to selectively kill malignant cells as proven by quantitative uptake of drug and supported by Two Photon

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b03711. Polymers and nanoparticles characterization; and biological evaluations (PDF) Video showing the presence of coated NRs in a localized region of the cytoplasm with PHEA-EDA-FA-GNRs signals distributed throughout the cytoplasm (ZIP) 14467


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(15) de Aberasturi, D. J.; Serrano-Montes, A. B.; Liz-Marzán, L. M. Modern Applications of Plasmonic Nanoparticles: From Energy to Health. Adv. Opt. Mater. 2015, 3, 602−617. (16) Zhang, W.; Wang, F.; Wang, Y.; Wang, J.; Yu, Y.; Guo, S.; Chen, R.; Zhou, D. pH and near-infrared light dual-stimuli responsive drug delivery using DNA-conjugated gold nanorods for effective treatment of multidrug resistant cancer cells. J. Controlled Release 2016, 232, 9− 19. (17) Wang, F.; Shen, Y.; Zhang, W.; Li, M.; Wang, Y.; Zhou, D.; Guo, S. Efficient, dual-stimuli responsive cytosolic gene delivery using a RGD modified disulfide-linked polyethylenimine functionalized gold nanorod. J. Controlled Release 2014, 196, 37−51. (18) Dinarvand, R.; Khodaverdi, E.; Atyabi, F.; Erfan, M. Thermoresponsive drug delivery using liquid crystal-embedded cellulose nitrate membranes. Drug Delivery 2006, 13, 345−350. (19) Jiang, X. F.; Pan, Y.; Jiang, C.; Zhao, T.; Yuan, P.; Venkatesan, T.; Xu, Q. H. J. Phys. Chem. Lett. 2013, 4, 1634−1638. (20) Tong, L.; Wei, Q.; Wei, A.; Cheng, J. Review Gold Nanorods as Contrast Agents for Biological Imaging: Optical Properties, Surface Conjugation and Photothermal Effects. Photochem. Photobiol. 2009, 85, 21−32. (21) Mauro, N.; Scialabba, C.; Cavallaro, G.; Licciardi, M.; Giammona, G. Biotin-Containing Reduced Graphene Oxide-Based Nanosystem as a Multieffect Anticancer Agent: Combining Hyperthermia with Targeted Chemotherapy. Biomacromolecules 2015, 16, 2766−2775. (22) Licciardi, M.; Li Volsi, A.; Mauro, N.; Scialabba, C.; Cavallaro, G.; Giammona, G. Preparation and Characterization of Inulin Coated Gold Nanoparticles for Selective Delivery of Doxorubicin to Breast Cancer Cells. J. Nanomater. 2016, Article ID 2078315.2016110.1155/ 2016/2078315 (23) Leamon, C. Folate-targeted chemotherapy. Adv. Drug Delivery Rev. 2004, 56, 1127−1141. (24) Paolino, D.; Licciardi, M.; Celia, C.; Giammona, G.; Fresta, M.; Cavallaro, G. Folate-targeted supramolecular vesicular aggregates as a new frontier for effective anticancer treatment in in vivo model. Eur. J. Pharm. Biopharm. 2012, 82, 94−102. (25) Licciardi, M.; Craparo, E. F.; Giammona, G.; Armes, S. P.; Tang, Y.; Lewis, A. L. in vitro Biological Evaluation of Folate-Functionalized Block Copolymer Micelles for Selective Anti-Cancer Drug Delivery. Macromol. Biosci. 2008, 8, 615−626. (26) Cavallaro, G.; Licciardi, M.; Mariano, L.; Salmaso, S.; Caliceti, P.; Gaetano, G. Folate-mediated targeting of polymeric conjugates of gemcitabine. Int. J. Pharm. 2006, 307, 258−269. (27) Yoo, H. S.; Park, T. G. Folate receptor targeted biodegradable polymeric doxorubicin micelles. J. Controlled Release 2004, 96, 273− 283. (28) Zhao, H.; Yung, L. Y. L. Selectivity of folate conjugated polymer micelles against different tumor cells. Int. J. Pharm. 2008, 349, 256− 268. (29) Licciardi, M.; Scialabba, C.; Cavallaro, G.; Sangregorio, C.; Fantechi, E.; Giammona, G. Cell Uptake Enhancement of Folate Targeted Polymer Coated Magnetic Nanoparticles. J. Biomed. Nanotechnol. 2013, 9, 949−964. (30) Zanoni, M.; Piccinini, F.; Arienti, C.; Zamagni, A.; Santi, S.; Polico, R.; Bevilacqua, A.; Tesei, A. The folate receptor is frequently overexpressed in osteosarcoma samples and plays a role in the uptake of the physiologic substrate 5-methyltetrahydrofolate. Coord. Chem. Rev. 2014, 13, 238−244. (31) Huang, P.; Bao, L.; Zhang, C.; Lin, J.; Luo, T.; Yang, D.; He, M.; Li, Z.; Gao, G.; Gao, B.; Fu, S.; Cui, D. Folic acid-conjugated Silicamodified gold nanorods for X-ray/CT imaging-guided dual-mode radiation and photo-thermal therapy. Biomaterials 2011, 32, 9796− 9809. (32) Tong, L.; Zhao, Y.; Huff, T. B.; Hansen, M. N.; Wei, A.; Cheng, J. X. Gold Nanorods Mediate Tumor Cell Death by Compromising Membrane Integrity. Adv. Mater. 2007, 19, 3136−3141.

AUTHOR INFORMATION

Corresponding Author

*Tel.: +39 09123891927. Fax: +39 0916162646. E-mail: [email protected] (M.L.). ORCID

Mariano Licciardi: 0000-0003-4539-9337 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Mediterranean Center for Human Health Advanced Biotechnologies (CHAB), Aten Center, University of Palermo for the use of instrumentations at “Microscopy and Bio-imaging Lab”. The authors thank the Italian Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) (Project code B72I15000020005) and the University of Palermo, Italy (Fund no. 2014-ATE-0178) for funding.

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