Mesenchymal stem cells as delivery vehicle of porphyrin loaded nanoparticles: Effective photoinduced in vitro killing of osteosarcoma

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S. Duchi a,⁎, G. Sotgiu b,⁎, E. Lucarelli a, M. Ballestri b, B. Dozza a, c, S. Santi d, e, A. Guerrini b, P. Dambruoso b, S. Giannini a, c, D. Donati a, c, G. Varchi b

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Mesenchymal stem cells as delivery vehicle of porphyrin loaded nanoparticles: Effective photoinduced in vitro killing of osteosarcoma

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Osteoarticolar Regeneration Laboratory, Rizzoli Orthopaedic Institute (IOR), Via di Barbiano 1/10, 40136, Bologna, Italy National Research Council (CNR), Institute for the Organic Synthesis and Photoreactivity (ISOF), Via Gobetti 101, 40129, Bologna, Italy Department of Human Anatomy and Physiopathology of the Locomotor Apparatus, University of Bologna, via Irnerio 48, 40126, Bologna, Italy d Institute of Molecular Genetics (CNR-IOR), Via di Barbiano 1/10, 40136, Bologna, Italy e Laboratory of Muscoskeletal Cell Biology, Via di Barbiano 1/10, 40136, Bologna, Italy b

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Article history: Received 21 November 2012 Accepted 14 March 2013 Available online xxxx

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Mesenchymal stem cells (MSC) have the unique ability to home and engraft in tumor stroma. These features render them potentially a very useful tool as targeted delivery vehicles which can deliver therapeutic drugs to the tumor stroma. In the present study, we investigate whether fluorescent core-shell PMMA nanoparticles (FNPs) post-loaded with a photosensitizer, namely meso-tetrakis (4-sulfonatophenyl) porphyrin (TPPS) and uploaded by MSC could trigger osteosarcoma (OS) cell death in vitro upon specific photoactivation. In co-culture studies we demonstrate using laser confocal microscopy and time lapse imaging, that only after laser irradiation MSC loaded with photosensitizer-coated fluorescent NPs (TPPS@FNPs) undergo cell death and release reactive oxygen species (ROS) which are sufficient to trigger cell death of all OS cells in the culture. These results encourage further studies aimed at proving the efficacy of this novel tri-component system for PDT applications. © 2013 Published by Elsevier B.V.

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Keywords: Nanoparticles (NPs) Porphyrins (TPPS) Photodynamic therapy (PDT) Osteosarcoma (OS) Mesenchymal stem cells (MSC)

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1. Introduction

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Osteosarcoma (OS) is the most common primary tumor of human bone [1]. Current treatments consist of multiple modalities, traditionally including amputation or limb sparing surgery as well as chemotherapy. Despite great strides in the diagnosis and treatment of OS to date, substantial improvement in overall survival rate has been elusive and has remained constant for over two decades [2–4]. Various chemotherapeutics have been investigated for the treatment of OS in the adjuvant setting. Efficacy has been observed with platinum analogues, e.g. cisplatin and carboplatin, the anthracycline antibiotic doxorubicin (DOX), and combinations of these drugs [2,5]. However, patients receiving these agents frequently experience severe side effects, such as hypersensitivities, extravasation injuries, arrhythmias, gastrointestinal toxicity, myelosuppression, and cumulative cardiotoxicity. Such a scenario is worsened by lack of OS-associated/ specific markers, which has hampered the development of targeted therapeutics. Although alternatives to conventional chemotherapeutic

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Abbreviations: NPs, Nanoparticles; OS, Osteosarcoma; MSC, mesenchymal stem cells; PDT, photodynamic therapy; TPPS, meso-tetrakis(4-sulfonatophenyl) porphyrin; FNPs, Fluorescent nanoparticles; TPPS@FNPs, Fluorescent nanoparticles loaded with TPPS. ⁎ Corresponding authors. E-mail address: [email protected] (G. Sotgiu).

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treatments have been proposed and studied [6–8], there is still a large need to identify new, safer and more effective therapeutic approaches for OS. Despite progress in basic research which has given a better understanding of tumor biology and has led to the design of new generations of targeted drugs, recent large clinical trials for cancer have only been able to detect small differences in treatment outcomes [9]. These facts indicate that to promote further progress it is necessary to emphasize on other therapeutic approaches. To this regard, photodynamic therapy (PDT) has the potential to meet many currently unmet medical needs. Although still emerging, PDT is already a successful and clinically approved therapeutic modality used for the management of neoplastic and other malignant diseases [10]. PDT is a minimally invasive procedure in which target cells are destroyed by reactive oxygen species (ROS) and especially by singlet oxygen, generated by excitation of nontoxic photosensitizer with light at an appropriate wavelength. There are very few reports describing the use of PDT to treat structural lesions within bone [11,12]. However, a promising pilot study in spontaneous canine OS has recently demonstrated that PDT induces tumor necrosis in treated animals [13]. In order for PDT to be both effective and safe, it is crucial that the photosensitizers are delivered in therapeutic concentrations preferentially to targeted cells, while being scarcely absorbed by non-target tissues, thus minimizing undesirable side effects [14,15]. To this regard, nanotechnology has been

0168-3659/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jconrel.2013.03.012

Please cite this article as: S. Duchi, et al., Mesenchymal stem cells as delivery vehicle of porphyrin loaded nanoparticles: Effective photoinduced in vitro killing ..., J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.03.012

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2-(Dimethylamino)ethyl methacrylate (DMAEMA), 1-bromooctane, 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AIBA), methyl methacrylate (MMA, 99.0%) (distilled before use), TPPS, 2′,7′dichlorodihydrofluorescein diacetate (H2DCFDA), human osteosarcoma cell line U2OS-RFP-TUBA1B (we will refer to these cells as U2OSTubRFP cells), mouse anti-βTubulin [T4026], anti-mouse IgG Cy3 [C2181], and TRITC-Phalloidin [P1951] were purchased from Sigma-Aldrich. α-Modified minimum Essential Medium (α-MEM), and fetal bovine serum (FBS) were purchased from Lonza (Verviers, Belgium). McCoy's 5A, GlutaMAX™ 1%, TripLe™ Select, Hoechst 33342 [H1399], and LIVE/DEAD viability kit (L3224) were purchased from Invitrogen-Life Technologies (Paisley, UK). Mouse anti-Lamp1/ CD107a [H4A3] was purchased from BD Pharmingen™ (Frankiln Lakes, NJ, USA). U2OS (HTB-96) cells were purchased from ATCC (Teddington, UK). Fluoromount-G was purchased from Southern Biotech (Birmingham, AL, USA). WST-1 was purchased from Roche (Mannheim, Germany).

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2.2. Fluo nanoparticles (FNPs) synthesis and characterization

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2.2.1. Synthesis FNPs were obtained using the following procedure. Briefly, an aqueous solution (50 ml) of 2-(dimethyloctyl)-ammonium ethylmethacrylate bromide (0.52 g, 1.5 mmol) was introduced, at room temperature, into a 250 ml three-neck reactor equipped with a condenser, a mechanical stirrer, a thermometer and nitrogen inlet. The mixture was purged with nitrogen, at a stirring rate of 300 rpm, and heated to 80 °C. Subsequently, 2-aminoethyl methacrylate hydrochloride (AEMA, 0.25 g, 1.48 mmol) was added to the previously obtained solution until complete solubilization was obtained. The fluorescent co-monomer allyl 2-(3-allyloxy-6-oxo-6H-xanthen9-yl) benzoate [36] (0.003 g, 0.007 mmol) was then added to methylmethacrylate (0.93 ml, 9.35 mmol) and the obtained mixture was added to the water solution [37,38]. After additional 10 min equilibration time, 15 mg (0.05 mmol) of 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AIBA), dissolved in 0.5 ml of mQ water was added to the mixture, which was then allowed to react for 4 h. The reaction product was purified by dialysis (against water) to remove residual monomer and stabilizer.

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2.2.2. Characterization The FNPs UV spectrum and the TPPS release amount were determined by spectrophotometric measurement with a Lamda 20 Perkin Elmer spectrophotometer (Waltham, MA, USA). Supernatants were filtered with an Amicon Ultra 0.5 ml 100 K Millipore Filter (Billerica, MA, USA) by using a EBA 12 HETTIC centrifuge equipped with a F-205 FALC tubes rotator. The hydrodynamic diameter of the nanospheres was determined by photon correlation spectroscopy (PCS) at 25 °C using a Zetasizer 3000 HS system (Malvern, UK) equipped with a 10 mV He\Ne laser. Data was analyzed based on the viscosity and refractive index of pure water at 25 °C. The instrument was calibrated with standard polystyrene latex particles of 200 nm in diameter. To achieve a constant ionic background, the sample was diluted in 10 mM NaCl to a concentration of 20 mg/ml. As far as the electrophoretic mobility is concerned, ζ-potential was measured at 25 °C by means of the same Zetasizer 3000 HS system. The instrument calibration was checked using standard polystyrene latexes,

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this system to induce controlled cell death when stimulated with laser light. In order to roughly mimic in vivo conditions, we carried out the co-culture experiment with a 1:5 (MSC vs OS cells) ratio, demonstrating the high effectiveness of our system even in this environment.

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shown to play a crucial role. Nanomedicine has recently drawn strong interest from the scientific community because of its potential to radically change cancer therapies [16–18]. Within the last decade many studies have described nanoparticles (NPs) as effective drugs carriers which allow to considerably decrease drug dosages and therefore toxicity effects associated with free chemotherapeutic agents administration [19]. However, their ability to deliver chemotherapeutic agents to subcellular targets and to control their behavior within the cell requires further studies. In addition, quantitative descriptions on the kinetics, amount, mechanisms, and trajectories of NPs uptake and trafficking are lacking. These barriers continue to hamper the clinical utility and benefit of nanoparticle-based technologies [20]. From this starting point, cells and in particular non-hematopoietic mesenchymal stem cells (MSC) could represent an ideal vehicle for targeted drug delivery, since they can be loaded with therapeutic agents, while maintaining their ability to migrate to sites of disease [21,22]. MSC are primary cells that can be expanded ex-vivo to reach a clinically relevant number. They are found in different tissues, such as bone marrow, fat and muscle [23,24], and these cell types are intimately involved in tissue regeneration and repair [25,26]. The therapeutic potential of MSC is linked to a broad spectrum of biological activities such as anti-inflammatory, immunomodulative and tissue reparative activities combined with low immunogeniticy. In addition MSC express genes encoding a large variety of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms [27]. Besides these activities, MSC have the unique ability to home to sites of inflammation/injury and to tumors stroma, which makes them useful for the targeted delivery of therapeutics to these sites [28,29]. Furthermore, in the last decade researchers have taken advantage of MSC tropism to inflamed tissues to target and kill cancer cells [30–32]. Considering these preliminary remarks, we hypothesized that combining photodynamic therapy, nanoparticles, and MSC would represent a very promising tool for targeted drug delivery. In fact, a suitably designed photosensitizer@NPs system loaded into MSC would at the same time overcome the still unascertained issues associated with NPs administration alone and be safe for cells activity, being active only once MSC reach the target site and when they are exposed to an appropriate stimulus. Moreover, we considered that a system with these characteristics could potentially overcome many of the limits associated with current therapeutic approaches, both in terms of inefficient cells accumulation into the tumor stroma and of ineffective/ uncontrolled drug release. In the present study we describe a novel tri-component biomaterial system, composed of core-shell PMMA nanoparticles (FNPs) postloaded with a photosensitizer, namely meso-tetrakis (4-sulfonatophenyl) porphyrin (TPPS) and incorporated into MSC for targeted osteosarcoma photo-dynamic treatment. Our NPs are fluorescently labeled through incorporation of a fluorescein derivative in the inner hydrophobic core, while the external shell is decorated with primary and quaternary ammonium salts which are able to electrostatically bind anionic porphyrins [33]. The aim of our study is therefore to demonstrate that MSC can be loaded with TPPS@FNPs and that, upon light irradiation, an energy transfer occurs from the TPPS@FNPs–MSC system to the free oxygen, resulting in the generation of ROS that cause cell death. In principle this system can overcome all the issues still associated with selective NPs biodistribution and uptake by exploiting the tumor homing ability of MSC and their low immunogenicity [34]. Moreover, the high photosensitizer payload on the NPs ensures the transport of a massive pro-drug quantity to the tumor site. The internalization and cytotoxicity of TPPS@FNPs was determined by FACS analysis and Methylene blue/WST-1 assays respectively, whereas the amount of released ROS was established by monitoring H2DCFDA activation [35]. Then, through laser confocal microscopy and time lapse imaging of TPPS@ FNPs–MSC co-cultured with OS cells in vitro, we tested the ability of

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Please cite this article as: S. Duchi, et al., Mesenchymal stem cells as delivery vehicle of porphyrin loaded nanoparticles: Effective photoinduced in vitro killing ..., J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.03.012

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2.3. TPPS loading and release experiments

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2.3.1. Loading calibration curve Briefly, TPPS was combined with FNPs using an electrostatic interaction: a certain amount of porphyrin (typically 50 μg/ml to 600 μg/ml in mQ H2O), was added to an aqueous solution of positively charged FNPs (0.5 mg/ml) and stirred at room temperature in a Vortex apparatus for 20 s. Each sample was then centrifuged (4 min, 4722RCF) with a 100 kDa filter. The filtrate was spectrophotometrically analyzed at the appropriate wavelength, thus a calibration curve was generated for further determination of the loading values.

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2.3.2. Release procedure TPPS release from FNPs was studied to ensure that ROS species during the experiments are generated from TPPS@FNPs and not from free TPPS eventually released from FNPs. To this aim release experiments were performed as a function of saline concentration and time at 37 °C and analyzed by spectrophotometric analysis (absorbance and fluorescence). For the release experiments we needed to use a 100 KDa centrifugal filter tube which is permeable by TPPS but not by TPPS@FNPs and FNPs. The release procedure, which for more clarity has been depicted in the integrated figure below, was performed as follows. 1) (A): 0.1 mL of TPPS@FNPs in mQ water, corresponding to 25 mg of FNPs loaded with 25 μg di TPPS, were placed in a 100 kDa centrifugal filter tube and suspended with 0.4 mL of releasing medium (PBS, pH 7.4, 150 mM or cell culture medium); 2) (B): the whole assembly was shaken at 37 °C in a rotative stirring flat plate for predetermined time intervals; 3) (C) the solution was centrifuged-filtered for 4 min at 4722 RCF and the resulting bottom portion of medium (0.4 mL), possibly containing TPPS, was spectrophotometrically analyzed (absorbance and florescence); 4) (D) 0.4 mL of fresh medium was added to the 100 kDa centrifugal filter tube to keep volume constant.

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2.5. Isolation, culture and immunophenotypic characterization of human 274 mesenchymal stem cells (MSC) 275

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To ensure a quantitative binding of porphyrin onto FNPs (see calibration curve, Fig. S1B), 50 μl of TPPS (1 mg/mL in mQ water) were added to 37 μl of FNPs mother solution (13.5 mg/mL). mQ water was then added in order to reach a total volume of 1 mL. 90 μl of this TPPS@FNPs mother solution (0.5mgFNPs/mL; 100 μgTPPS/mgFNPs) were then withdrawn and added to the plate which contains 1 mL of culture medium. In this way the FNPs concentration used for cells treatment is 45 μgFNPs/mL, while for all the other experiments the TPPS loading on the NPs is 100 μgTPPS/mgFNPs.

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Bone marrow (BM) samples were obtained from patients undergoing surgery at Rizzoli Orthopaedic Institute after obtaining informed consent according to a protocol approved by the Ethics Committee. Isolation and culture expansion of human MSC was performed as previously described [39], through gradient separation and plastic adherence. Briefly, cells were transferred to 150-cm 2 culture flasks with α-MEM supplemented with 20% lot-selected FBS and GlutaMAX™ 1%, incubated in a humidified atmosphere at 37 °C with 5% CO2 and medium changed every 3–4 days. When the cells reached approximately 70–80% confluence, they were detached using mild trypsinization for 5 min at 37 °C and counted. Then, 1/3 of them were reseeded into a new 150-cm2 flask. MSC were recognized by their ability to proliferate in culture with adherent, spindle-shape morphology. Further characterization of MSC was performed for phenotypical characterization via cytofluorimetric analysis of cell surface markers at passage 2. MSC resulted positive for CD44, CD73, CD90, CD105, CD146 and negative for CD34 and CD45. The analysis was performed using a FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). The multilineage potential was evaluated by differentiation assays [40].

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2.6. Human osteosarcoma cell line

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Human osteosarcoma cells (U2OSTubRFP and U2OS cells) were cultured according to the Sigma-Aldrich instructions. Briefly, the culture medium was McCoy's 5A and was supplemented with 10% FBS and 1% GlutaMAX™. The cells were maintained at 37 °C, 95% humidity, and 5% CO2 and split 1:5 every 3 days.

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2.7. FNPs and TPPS@FNPs uptake

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MSC were plated at a density of 2.5 × 10 4 cells in 35 mm Glass Bottom Dishes (MatTek Corporation, Ashland, MA, USA) in 2 ml of complete growth medium (α-MEM supplemented with 20% lot-selected FBS and GlutaMAX™ 1%), and incubated at 37 °C in 5% CO2 atmosphere. Cells were allowed to adhere to the plates for 24 h before the addition of NPs. Cells were then incubated for 1 h with i) FNPs alone (45 μg/mL) and ii) TPPS@FNPs 100 μg/mg (unless otherwise stated, concentration for TPPS@FNPs is referred as μg of TPPS for mg of particles, whereas the total particles concentration is stated at 45 μg/mL), washed several times with PBS 1 ×, and prepared for immunostaining and time lapse imaging assays.

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2.8. Immunofluorescence analysis and confocal laser scanning microscopy (CLSM)

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supplied by Malvern Instruments with known ζ-potential. The loading per gram of nanospheres of primary and quaternary ammonium groups was determined by potentiometric titration of the chloride and bromide ions, respectively obtained after complete ion exchange. The ionic exchange was accomplished by dispersing 67.5 mg of the nanosphere sample in 25 ml of 1 M KNO3 at room temperature for 6 h. The mixture was then adjusted to pH 2 with dilute H2SO4 and bromide and chloride ions were titrated with a 0.0201 M solution of AgNO3. The samples for the atomic force microscopy (AFM) measurement were prepared by spin-coating deposition of 0.67 mg/ml of NPs solution on atomic flat silicon substrate at 1500 rpm. These experimental conditions allow for the prevention of the aggregation of nanospheres and allow for a very uniform deposition over 1 cm 2 of isolated nanospheres, which can be clearly distinguished from the substrate. AFM images are performed in air and recorded using a Multimode IIIA (Bruker, Billerica, MA, USA) scanning probe. Imaging was done in tapping mode using silicon RTESPA probe (Bruker, frequency f0 = 270 KHz and nominal tip radius ≤ 15 nm).

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Considering that TPPS is completely water soluble (≤ 6 mg/mL), and that the quantity present on our NPs is 0.1 mg/mL, we can assume

For immunostaining analysis, 2.5 × 10 4 FNPs or TPPS@FNPs loaded 315 MSC were seeded onto glass coverslips and fixed for 10 min in 4% 316 formaldehyde at RT or for 7 min in ice cooled 100% Methanol for 317

Please cite this article as: S. Duchi, et al., Mesenchymal stem cells as delivery vehicle of porphyrin loaded nanoparticles: Effective photoinduced in vitro killing ..., J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.03.012

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2.11.1. WST-1 assay In vitro cytotoxicity of FNPs and TPPS@FNPs on MSC was evaluated using a cell metabolic assay by the reagent water soluble tetrazolium salt-1 (WST-1) following the manufacturer's instructions. Briefly, cells were seeded in 96-well plates at a density of 2000 cells/well in 100 μl a-MEM containing 20% FBS. After overnight attachment, cells were treated with different concentrations of NPs for 1 h and then immersed in 90 μl culture medium supplemented with 10 μl WST-1 (day 0). For proliferation assay cells were allowed to grow in complete medium for 1, 2, and 6 days followed by incubation with WST-1. After 4 h of WST-1-incubation at 37 °C and 5% CO2, the optical density of each well was measured by a microplate reader (Synergy HT, BioTek Winooski, VT, USA) set at 450 nm with a correction wavelength set at 690 nm. The absorbance of the samples was measured against a background control as blank.

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2.9. Flow cytometry

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To determine the cellular uptake of the nanoparticles, we analyzed the fluorescent signal from the FNPs using a BD FACSCanto II (BD Bioscience, Frankiln Lakes, NJ, USA). MSC loaded with 45 μg/ml FNPs at the indicated time points were washed three times with PBS 1× and harvested by trypsinization. After centrifugation, the loaded cells were washed twice with PBS 1×, counted with NucleoCounter® (Chemotec, Lillerod, Denmark) and 1 × 106 MSC suspended in fresh PBS 1×, and directly analyzed.

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2.10. Time lapse in vivo imaging and cellular irradiation (photoactivation)

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2.12. ROS analysis

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Fluorescence cell imaging for the detection of ROS was performed using the non-fluorescent molecule 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), which permeates live cells and is de-acetylated by nonspecific intracellular esterases. In the presence of ROS the reduced fluorescein compound is oxidized and emits green fluorescence. Briefly, 10 mM H2DCFDA stock solution made in dimethyl sulfoxide (DMSO) was diluted in culture medium without serum or other additive to yield a 10 μM working solution. After 1 h of exposure to TPPS@ FNPs (100 μg/mg), MSC were washed twice with PBS 1× and then incubated in 1 ml working solution of H2DCFDA at 37 °C for 45 min. Photoactivation and video recording was performed as stated above.

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MSC were plated at a density of 2.5 × 10 4 cells per 35 mm Glass Bottom Dishes (MatTek Corporation) and loaded with FNPs or TPPS@FNPs as described above. Photoactivation and subsequent video recording was performed using a NikonTi Eclipse microscope equipped with temperature and CO2 controllers (Okolab, Ottaviano, Napoli, Italy), a DS-QiMc-U2 12 bit camera and an AR1 confocal laser. Total time duration of movies, time frame interval and objectives used are indicated in the text. Photoactivation of FNPs or TPPS@FNPs loaded MSC has been performed by exposing cells to 10% of the maximum power of 405 nm laser (0.02 J/s) for the indicated time points. Photoactivation of FNPs, TPPS and TPPS@FNPs loaded MSC in co-culture with U2OS cells to quantify the cell death amount after photostimulation, has been performed by exposing cells to 0.16 J/s 405 nm laser for the indicated time points in the selected areas. All the images and videos shown in this paper are representative of at least three independent experiments carried out under the same conditions.

2.11.2. Methylene blue assay In vitro cytotoxicity and cell proliferation were also measured by methylene blue assay [41]. Briefly, MSC were seeded and treated with different concentrations of FNPs as described in Section 2.11.1. Cells were fixed by adding 10% formol saline to each well for 30 min. Cells were then stained with filtered 1% (w/v) methylene blue in 0.01 M borate buffer (pH 8.5). After 30 min, excess dye was removed and the remaining dye was rinsed three times in 0.01 M borate buffer (pH 8.5). At this point, the cells were examined microscopically. The dye was then eluted by adding 1:1 (v/v) absolute ethanol and 0.1 M HCl to each well. The plates were gently shaken and the absorbance at 650 nm (A650) was measured using a microplate spectophotometer (Synergy HT, BioTek Instruments Inc., Winooski, VT USA). The photometer was blanked on control wells containing elution solvent alone. Linear relationship between absorbance and cell number was verified.

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2.11. In vitro cytotoxicity and proliferation assays Various assays which take advantage of different strategies to measure cell proliferation are available. The obvious limitation of metabolic assays (such as WST-1) is that cellular metabolic activity changes throughout cells lifecycle and under different cell culture conditions. Methylene Blue is a basic dye that is positively charged at pH 8.5. It binds electrostatically to negatively charged groups within cells, predominately phosphate moieties of nucleic acids and some charged groups in proteins. An advantage of methylene blue assay is a direct correlation with cell proliferation.

MSC were plated at a density of 2.5 × 10 4 cells in a 6 well plate and treated with FNPs or TPPS@FNPs as described above. After 1 h incubation, in order to eliminate any residual NPs containing medium, cells were washed three times with PBS 1× and harvested by trypsinization. After centrifugation, the labeled MSC were washed twice with PBS 1×, counted with NucleoCounter®, and 2.5 × 104 cells were reseeded in 35 mm Glass Bottom Dishes (MatTek Corporation) in complete medium. In the same plates, U2OSTubRFP or U2OS cells were seeded in a 1:1 or 1:5 ratio, let adhere overnight and then imaged as described above for photoactivation and time lapse imaging.

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Therefore, we used both the above mentioned assays to have a complete view of cell response to treatment with NPs. The assays were performed in a quadruplicate for WST-1 and in a quintuplicate for Methylene Blue on MSC originated from 3 independent healthy donors to evaluate cytotoxicity right after MSC loading (day 0) and after 1, 2 and 6 days.

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anti-βTubulin staining. After three washes in PBS 1 ×, cells were permeabilized for 10 min in PBT (PBS 1 × + 0.1% Triton × 100), then incubated in blocking solution (PBS 1 × + 5% BSA) for 30 min. Primary antibodies diluted in blocking solution were added and incubated for 1 h at RT or overnight at 4 °C for anti-βTubulin. After three washes in PBS 1×, secondary antibody was added diluted in PBS 1 × and incubated for 45 min at RT. Primary antibodies used were: mouse antiLamp1/CD107a [1:1000]; mouse anti-βTubulin [1:50]. Secondary antibody used was anti-mouse IgG-Cy3 [1:100]. Cortical actin was stained using TRITC-Phalloidin [1:500] and added directly during secondary antibody incubation. Nuclei were stained by incubating cells with Hoechst 33342 [1:2000] for 10 min at RT after secondary antibody step. Coverslips were mounted after washes in PBS 1× in Fluoromount-G solution, and analyzed with a Nikon TE2000-U inverted epifluorescence microscope or a NikonTiE microscope equipped with a fully automated A1 confocal laser which incorporates the resonant scanner with a resonance frequency of 7.8 kHz which allows high-speed imaging (A1R, Nikon, Amsterdam, Netherlands), and equipped with DS-QiMc-U2 12 bit camera. Digital images were processed using ImageJ software for the analysis and Photoshop software for visualization purposes without biased manipulations.

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3.2. TPPS release evaluation under different experimental conditions

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The LIVE⁄DEAD® Viability⁄Cytotoxicity Kit quickly discriminates live cells from dead ones by simultaneously staining with greenfluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 (EthD1) to indicate loss of plasma membrane integrity. For our post-photoactivation analysis, cells were incubated with the staining solutions according to the manufacturer's protocol. The apoptotic index was calculated as the percentage of ethidium-positive apoptotic nuclei divided by the total number of nuclei visualized through counterstaining with Hoechst and obtained from counts of randomly chosen microscopic fields in the irradiated areas.

In order for TPPS@FNPs to be a valuable system given our purposes, the TPPS must be tightly associated with NPs over time as well as under different experimental conditions. By roughly mimicking physiological environment, TPPS@FNPs were subsequently treated, at 37 °C, with PBS (150 mM) r with cells growth culture medium. The volume of release medium used was enough to dissolve at least four times the quantity of drug present in nanoparticles (100 μg × mg of particles). Collected data showed that under the reported conditions no release of TPPS from the nanospheres was detectable analysis (Figs. S1C–D and S3). Thus, we are confident that TPPS, even if electrostatically loaded onto the particles, is tightly bounded to the cationic shell and release from the spheres is not likely to occur during in vitro experiments.

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Fluorescent core-shell PMMA nanoparticles, namely FNPs (Fig. 1A), were obtained by emulsion polymerization reaction and were characterized by an average AFM diameter of 14 ± 5 nm (Fig. 1B, C) and by an average hydrodynamic diameter of 39 nm (five measurements; Table S1). The difference in the NPs radii observed with PCS and AFM techniques could be ascribed to the different environments in which the measurements are performed, e.g. water and air, respectively. The Stokes diameter measured with PCS corresponds to the size of the solvated nanoparticle, which is expanded due to the presence of the external ammonium groups while AFM directly measures the dry radius [42,43]. TPPS@FNPs average hydrodynamic diameter was also measured just before incubation with cells. As expected, dynamic light scattering data (Table S2) showed an increase in the particles hydrodynamic radius. The ammonium group loading, available for the interaction with TPPS, was determined by potentiometric titration of the bromide and chloride ions obtained after complete ion exchange and was found to be 670 μmol of quaternary ammonium bromides and 387 μmol of primary ammonium chlorides per gram of nanosphere (Fig. S1A). Finally, the ζ-potential of NPs (28.5 mV), which was measured after dialysis purification, confirmed that the cationic co-monomer employed in the polymerization reaction is covalently bound to the nanoparticle surface (Table S3). ζ-potential measurements of the TPPS@FNPs (Table S4) confirm that particles retain a positive external charge even after TPPS loading. This data is quantitatively confirmed from the titration of the FNPs sample once loaded with the sensitizer (Fig. S2).

Fluorescent FNPs uptake by MSC cells was determined by exposing MSC to increasing dosage of FNPs (Fig. 2) and TPPS@FNPs (Fig. S5) (30 μg/mL–100 μg/mL). Internalization was observed using a fluorescent microscope. Nanoparticles were clearly visible in MSC exposed to a concentration of FNPs higher than 20 μg/mL. In particular, at 45 μg/mL, the nanoparticles were observed in 100% of the analyzed MSC (green in Figs. 2A and S5A). In order to determine FNPs intracellular localization, right after FNPs incubation cells were fixed, stained with anti-Lamp1 late endocytic marker and fluorescence images taken every 30 min by time lapse confocal imaging. Endocytosis analysis is relevant in order to assess NPs optimal cellular uptake, as the rate and mechanism of uptake is cell-type dependent and varies between nanoparticles of different size, charge, and other surface properties. Importantly, the polyvalent surface of nanoparticles may induce cross-linking of cellular receptors, start signaling processes, cause structural alterations at the cell surface, and interfere with normal cell function (for a review see [44]). As shown in Figs. 2A and S5A, FNPs fluorescent signal was nicely distributed intracellularly (green in Fig. 2A b, g, m and S5A b, g, m). Our results demonstrate that MSC do uptake FNPs within 30 min (Figs. 2A a-e and S5A a-e), with the majority of FNPs being localized to the plasma membrane (white lines in Figs. 2A b and S5A b), intracellular space (asterisks in Figs. 2A b and S5A b) and in Lamp1 positive endocytic compartments (asterisks in Fig. 2A d, higher magnification in e and Fig. S5A e). Within 60 min, FNPs do evade late endocytic compartimentalization with few FNPs localized in Lamp1 positive organelles (asterisks in Fig. 2A j and higher magnification in k). FNPs are still intracellularly distributed and not degraded (asterisks in Figs. 2A g and S5A g), and only a minor percentage of FNPs could be identified

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Fig. 1. (A) Schematic representation of FNPs. (B) Distribution (%) of nanoparticle radius. (C) AFM image of nanospheres deposited on atomic flat silicon Z-range = 14 ± 5 nm.

Please cite this article as: S. Duchi, et al., Mesenchymal stem cells as delivery vehicle of porphyrin loaded nanoparticles: Effective photoinduced in vitro killing ..., J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.03.012

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images show that MSC are mildly affected by FNPs internalization, but overall cells maintain a cell morphology comparable to that of untreated cells, namely that tubulin fibers are regularly shaped along cell axis (Fig. S8B) and actin bundles are only partially altered (Fig. S8D). Moreover, cell motility in FNPs loaded MSC is not altered. Live cell time lapse observations clearly show that the motility of loaded MSC is not altered by the presence of intracellular FNPs when compared to unloaded cells (compare Video 1A, unloaded MSC, with Video 1B, FNPs loaded MSC). Viewed together, these results suggest that exposure of MSC to 45 μg/mL FNPs or TPPS@FNPs for 60 min did not alter the major features associated with MSC, such as cell motility and migration potential nor does induce any sign of morphology alteration. Our results strongly support the hypothesis that FNPs have no cytotoxic effect on MSC in culture and do not alter their migratory capacity, at least at the concentration tested. Migratory capacity is a crucial requirement for MSC to home and deliver the therapeutic agent to the tumor site.

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To evaluate the citotoxicity of FNPs and their effect on cell proliferation, WST-1 and Methylene Blue assays were carried out in MSC culture. MSC were exposed to increasing concentration of FNPs or TPPS@ FNPs for 1 h in the dark in order to avoid TPPS activation. In this way, the maximum dose which does not interfere with cell proliferation is discovered. Equivalent effects are observed from both assays (Fig. 3). Overall, our results show no significant decrease in cell proliferation during a time period up to 6 days from the initial loading in MSC exposed to FNPs and TPPS@FNPs at 45 μg/mL (100 μgTPPS/mgFNPs) compared to the cell proliferation of unloaded cells (0 in the graphs) (Fig. 3A–B). The differences in cytotoxicity observed over time while using the higher dose (90 μg/mL, concentration referred to the NPs) are associated with the different principles of the assays (see Section 2.11). The toxic effect observed in the WST-1 assay indicates a significant degree of toxicity associated with FNPs only at the concentration

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on the cell surface. After 90 min, FNPs tend to form aggregates (asterisk in Figs. 2A m and S5A p), to accumulate on the plasma membrane (white lines in Fig. 2A m), and to elude endolysosomal compartimentalization (Fig. 2A o and higher magnification in p). Therefore, in light of these observations, we establish the 60 min time point as the length of time to reach a sufficient intracellular FNPs incorporation to be used even for the incorporation of TPPS@ FNPs. At this time length and dosage, NPs are not accumulated on the cell surface which avoids the risk of intercellular crosslinking. This time length and dosage is also ideal due to the fact that both FNPs and TPPS@FNPs are not stuck in the lysosomal compartment (Lamp1), which avoids the risk of degradation or recycling back to the plasma membrane which would result in secretion into the extracellular space throughout exocytosis. After 72 h for instance, nanoparticles are still intracellularly retained in the cytoplasm and are not trapped in the lysosomal compartments (data not shown). As a result, these nanoparticles are still active and ready to be activated by laser light. Moreover, FNPs incorporation seems to modify neither cell morphology nor nuclear shape (compare Fig. 2A a, f, l). In order to confirm the labeling efficiency of MSC and to obtain a quantitative measurement of FNPs and TPPS@FNPs (data not shown) uptake, we used flow cytometry with a FACS scan analyzer. MSC were incubated with 45 μg/mL FNPs for 30 and 60 min. After 30 min around 72% of cells in the labeled human MSC population displayed the green-fluorescence signal, whereas after 60 min fluorescence signal was expressed by 85% of labeled cells (Fig. 2B). This data confirms the results obtained with the previously shown immunostaining analyses and quantifies the NPs incorporation in MSC over time. Our data clearly show that MSC efficiently internalize FNPs, which indicates that these NPs can be taken up physiologically by the cells and be intracellularly retained. To further detect subtle changes that could likely occur in cellular morphology due to possible toxicological effect of FNPs, we also exposed both the control as well as the FNPs loaded MSC to antiβTubulin (Fig. S8A, B) and PhalloidinTRITC (Fig. S8C, D) staining to respectively mark and observe microtubule and actin networks. The

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Fig. 2. Intracellular distribution of FNPs in loaded MSC. A. Immunostaining analysis on MSC loaded with 45 μg/mL FNPs and imaged with confocal laser microscope with a 60× PlanApo VC Oil DIC N2 objective. (a–e) Within 30 min FNPs accumulate on the plasma membrane (white lines in b) and within late endocytic compartments (asterisks in b and e). (f–k) Within 60 min, FNPs are more concentrated in cells cytoplasm (asterisk in g) and are less concentrated in Lamp1 positive organelles (asterisk in k). (l–p) Within 90 min, FNPs tend to concentrate onto the plasma membrane (white lines in m) and to strongly aggregate in the cell cytoplasm (asterisk in m). Cells have been fixed and stained, right after FNPs loading, at the indicated time points, with anti-Lamp1 monoclonal antibody (red in all panels) to detect possible colocalization with FNPs (green in all panels). Hoechst staining (blue in A, F, L) was used to detect cell nuclei and is shown merged with bright field images. Scale bar is 50 μm in all panels. B. MSC labelling efficiency assay was performed with FACS analysis on MSC control (mock) and MSC loaded for 30 and 60 min with 45 μg/ml FNPs. The percentage of loaded cells was obtained by selecting Fluorescein positive cells and by subtracting from the fluorescence intensity of loaded NPs (green curve) the autofluorescence intensity coming from not loaded MSC (black curve). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: S. Duchi, et al., Mesenchymal stem cells as delivery vehicle of porphyrin loaded nanoparticles: Effective photoinduced in vitro killing ..., J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.03.012

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Fig. 3. Cytotoxicity and cell proliferation evaluation of FNP and TPPS@FNP loaded MSC. 2 × 103 seeded MSC were loaded for 1 hr with the indicated concentrations of NPs. Absorbance lectures were performed right after NP loading (day 0) and after 1, 2 and 6 days. (A) WST-1 assay. (B) Methylene blue assay. Data is expressed as the mean of four wells ± SEM. A time point comparison between NP-loaded cells and cells without NPs was carried out and evaluated (*P b 0.05; ***P b 0.001).

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In order to assess the functionality of TPPS photoactivation, confocal time lapse imaging of MSC-TPPS@FNPs and MSC-FNPs control activated by laser light was performed. MSC were subjected to illumination with a 405 nm laser (10% of the maximum power, 0.02 J/s) for 2 s causing specific activation of TPPS as revealed by emission of a signal at around 550–600 nm wavelength (Fig. 4D–E and Video 2B) and according to the TPPS emission spectrum (See Fig. S4 for details). Laser light activation of TPPS@FNPs loaded MSC resulted in rapid cell damage of MSC (Fig. 4F and Video 2B) as evidenced by the formation of bubble-like structures arising from the simulated cells in a short time frame from the initial photoactivation (white brackets in

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of 90 μg/mL after 0, 1, and 2 days (Fig. 3A, left panel) and with TPPS@ FNPs only after 6 days (Fig. 3A, right panel). Cellular metabolism is found to be altered only by FNPs immediately after loading, while cell recovery was seen after 6 days. When FNPs are coated with TPPS, cellular metabolism is not altered immediately after loading (0, 1 and 2 days), and is affected only after a longer period of time (6 days). As a result, we speculate that the binding of TPPS to FNPs masks some of positive charges on the outer NPs shell, thus preserving cell metabolism. It is likely that the experimental conditions (light during the observation time) could produce a slight phototoxicity effect and induce the cell metabolism impairment observed after 6 days upon treatment with TPPS@FNPs. As a consequence of these observations, our proceeding analyses were carried out with the higher dose for both FNPs and TPPS@FNPs having been discarded. We conclude that uptake of FNPs or TPPS@FNPs at 45 μg/mL does not influence the proliferation behavior of the MSC. These results strongly support the hypothesis that NPs loaded either with or without the sensitizer, have no cytotoxic effect on MSC in culture, at least under our experimental condition.

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Fig. 4F). These events are peculiar of the necrotic process that occurs in a characteristic sequence: coalescence of membrane bubbles into a single larger one that detaches from cells, which remain on the substrate [45]. FNPs loaded MSC and photoactivated as for TPPS@FNPs, were used as a control. In the same time frame shown for TPPS@FNPs loaded MSC, laser light stimulation did not cause any cell damage (Fig. 4A–C and Video 2A). TPPS@FNPs activation is specific and precise since we direct laser light toward a single or few nanoparticles (red square area in Fig. 4) and ROS intracellular release and spreading is able to rapidly kill the stimulated cell. Our results confirm that TPPS@FNPs are biocompatible and useful tool for MSC photodynamic therapy. Cells are able to properly move and proliferate until laser stimulation occurs. As a consequence of TPPS light activation and the release of toxic free oxygen species, MSC die. The generation of intracellular ROS was monitored using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) probe [35]. In order to perform this analysis, we used non-fluorescent NPs to avoid crosstalk with the green-fluorescence signals from FNPs and the ROS probe. We loaded MSC for 1 h with TPPS@NPs, wash them and incubated with 10 μM H2DCFDA probe for 45 min. Photoactivation with time lapse confocal microscope was subsequently carried out as in the previous experiments. In Fig. 5 and Video 3 A, B 1 min after photoactivation, performed in the small white box in Fig. 5A, we observed the wave of ROS generation as a wave of green fluorescent signal emitted from the oxidized probe (Fig. 5B) and the subsequent appearance of membrane bubbles from the stimulated cell (Fig. 5A and white brackets in Fig. 5C). 5 min after the first photoactivation, ROS probe fluorescent signal appeared in the second cell (Fig. 5D) and its subsequent cell death at 7 min time frame (with brackets in Fig. 5E) was observed. These data confirm the highly efficient generation of ROS post-photostimulation and their diffusion (red arrow in Fig. 5A and B), which is compatible with the conventional

Please cite this article as: S. Duchi, et al., Mesenchymal stem cells as delivery vehicle of porphyrin loaded nanoparticles: Effective photoinduced in vitro killing ..., J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.03.012

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Fig. 4. Photoactivation of MSC-TPPS@FNPs and MSC-FNPs was carried out through time lapse confocal microscope equipped with temperature and CO2 controllers (still images from Video 2). Few nanoparticles (red area in all panels) are subjected to light stimulation for 24 s with 405 nm laser source and then imaged with a Plan Apo 60× Oil DIC N2 objective right before stimulation (A and D, 0 min) and right after (B and E, 0.20 min) and followed until 12 min (C and F) time frame. (A–C) FNPs stimulated with laser light do not cause any photoactivation nor was bubbling of stimulated cells observed, consistent with the fact that there was no cytotoxic effect caused by laser light stimulation. (D–F) TPPS@FNPs are strongly activated (red signal in E that merging with green FNPs becomes yellow signal) under stimulation and subsequent rapid formation of bubble-like structures is observed (white brackets in F). Scale bar is 60 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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3.6. Functionality of TPPS@FNPs over time

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In order to assess the efficacy of our tri-combined strategy over time, we verified the retention rate of nanoparticles into MSC by optical imaging observation and cytofluorimetric analysis. 24 h after the initial incorporation with either FNPs or TPPS@FNPs, almost 100% of MSC are still labeled, whereas after 72 h (Fig. S6A,B), we observe a reduction of around 50%. This decrease is probably ascribed to cell doubling event of MSC during the 72 h. In order to exclude possible leaking of TPPS in the extracellular space, a photophysical study was conducted on the conditioned medium (CM) obtained 72 h after MSC were loaded with 45 μg/mL TPPS@FNPs. We performed the above experiment at 72 h since we considered that this should have been the appropriate time-length for MSC to migrate to the tumor site [46]. The electronic absorption spectra of CM sample and of the corresponding reference porphyrin (10 −6 M in water) show split soret peaks, which are characteristic of porphyrin aggregates (Fig. S7A). The photoluminescence measurements were carried out with the

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life-time and diffusion length of ROS [45]. This data also suggests that ROS species migrate within the medium and kill cells. Our experiment clearly shows that upon activation of the first MSC, the second MSC “lights up” after approximately 3.5 min. This behavior matches with ROS species migration from cell 1 to cell 2. In fact, light refraction or diffusion phenomena, which in principle could independently activate cell 2, should occur at the same time of cell 1 irradiation and not after 3.5 min.

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same samples, and upon excitation at 410 nm (high-energy soret peak of porphyrin), both samples show emission peaks characteristic of freebase porphyrins (Fig. S7B). This data clearly indicates that the emission intensity of the CM sample is 40 times lower than that of the reference porphyrin sample, under the same experimental conditions. Thus, the concentration of free porphyrin in the CM sample should be approximately 1 × 10 −6 M/40. Despite the fact that the absorbance of the CM sample at 410 nm is approximately 4 times higher than that of the reference porphyrin, we can safely assume that the porphyrin in the CM absorbs only 1/4 of the incident light. Moreover, the same CM was used to reload new MSC. Upon photo-stimulation of these MSC at 405 nm (0.02 J/s for 24 s), no significant cell death was observed even after several minutes, thus confirming that the concentration of TPPS in the extracellular space is not sufficient to produce the critical concentration of ROS. Overall, our data clearly indicates that photoinduced stimulation of the CM in the presence of new MSC does not produce evident cell death, thus confirming that ROS species responsible for cells death are those produced upon photoactivation of TPPS@FNPs internalized into MSC.

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3.7. TPPS@FNPs loaded MSC induce death of OS cells upon photoactivation

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FNPs or TPPS@FNPs loaded MSC cells were co-cultured with U2OSTubRFP tumor cell line in order to test the efficacy of PDT in a tumor environment, by seeding MSC and OS cell in a 1:5 ratio (for protocol details see Materials and methods section and Suppl. Fig. 9, for experiment Fig. 6A–D and Video 4). Surprisingly, specific and direct

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Please cite this article as: S. Duchi, et al., Mesenchymal stem cells as delivery vehicle of porphyrin loaded nanoparticles: Effective photoinduced in vitro killing ..., J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.03.012

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4. Discussion

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The successful management of osteosarcoma (OS) would be greatly aided by novel agents and/or therapeutic techniques with the ability to reduce side effects associated with conventional chemotherapy as well as to be effective in drug resistant conditions [47,48]. Among unconventional therapeutic approaches, photodynamic therapy (PDT) has emerged has a potential effective treatment for solid tumors [10], due to its low mutagenic potential and few adverse effects [49,50]. However, some main issues need to be addressed in order for PDT to be considered a consolidated therapeutic procedure for OS and solid tumors treatment. In particular, developing a way to enhance efficacy at tumor site is one of the major challenges to be solved [51]. To this regard, nanotechnology has been shown to play a crucial function. The use of NPs for molecular transport is advantageous in that they are able to provide an inert environment which protects the drug from being recognized by and cleared from the human body before having the chance to reach the target site [52,53]. Although the in vitro and in vivo data from NPs-based PDT agents seem to promise a very high potential for clinical application, none of these agents have been used in the clinic to date [51]. The major reliable delivery procedure for sensitizers-loaded nanocarriers remain the passive way, which exploits the leaky-vasculature of the tumor tissues as well as their lack of effective lymphatic drainage (Enhanced Permeability and Retention (EPR) effect). However, this passive mechanism impairs the treatment of micro-metastases, which are known for being non-vascularized, and as such are not subjected to EPR effect [54]. Moreover, few examples related to NPs-mediated PDT treatment of OS are currently known [55,56], and none of them

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amount of cell death of MSC loaded with FNPs and TPPS alone. As expected, MSC loaded with non-coated FNPs do not respond to photoactivation (Fig. 6A). In addition, TPPS molecules in loaded MSC could not be detected at the same concentration and time length incorporation of coated FNPs (45 μg/ml for 60 min). To perform this quantification, we photoactivated different areas in a 1:5 ratio (MSC:U2OS) background by using 405 laser power at 0.16 J/s for 1.5 min. After 24 h, we performed the Live and Dead Assay. The apoptotic index was calculated as the percentage of etidium-positive apoptotic nuclei divided by the total number of nuclei visualized by counterstaining with Hoechst obtained from counts of randomly chosen microscopic fields in the corresponding irradiated areas (Fig. 6E, F). We also verified the efficiency of PDT by photoactivating MSC after 7 days from loading treatment (Fig. 7A–D and Video 5A, B). As previously demonstrated, MSC are still loaded and TPPS@FNPs (red area in Fig. 7A) can be efficiently photostimulated, generating ROS and killing surrounding tumor cells (Fig. 7A, B and Video 5A). Remarkably, previously stimulated nanoparticles, can be photoactivated a second time, even if trapped in the dead MSC attached to the bottom of the plate (Fig. 7C, D and Video 5B). The second photoactivation is sufficient to completely eliminate tumor cells in the microscope observation field next to the first one. To perform this stimulation, we slightly moved the microscope stage in order to visualize the OS cells which were not eliminated by the first photoactivation. In addition, after 7 days in culture, U2OSTubRFP cells proliferate at higher rate compared to MSC, but even few photostimulated TPPS@FNPs are able to induce massive tumor cells death. Overall our results strongly demonstrate the in vitro efficacy of the PDT strategy in which MSC are used as a delivery vehicle. The 1:5 ratio of MSC:U2OS shows that even a low dosage of MSC compared to tumor cells is sufficient to induce tumor cell death. Moreover, the efficacy of the second photostimulation on the same TPPS@FNPs demonstrates the opportunity to induce several and repeated cycles of photostimulation, able to eliminate both vehicle cells (MSC) and tumor cells at the same time.

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Fig. 5. Intracellular ROS generation and intercellular ROS diffusion were monitored through confocal time lapse imaging of activated TPPS@NPs loaded MSC. Bright field (A, C, E) and 488 channel (B, D, F) still images from Video 3 are shown. (A, B) MSC bearing TPPS over the non-fluorescent equivalent FNPs were imaged 1 min after photostimulation (small white box in A). (C, D) 3.5 min after photostimulation, the fluorescent signal visible in panel B developed from the ROS probe in the stimulated cell, diffuses to the adjacent cell (D). (C) At 3.5 min post-photoactivation bubbling of the first stimulated MSC becomes evident (white brackets). (E, F) After 7 min, also the second cell begins to release membrane bubbles (white bracket in C) and fluorescent signal from ROS probe become more intense. Videos and still images were taken with confocal microscope equipped with temperature and CO2 controllers and a Plan Apo 60× Oil DIC N2 objective. Scale bar is 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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photoactivation of few TPPS@FNPs in the loaded MSC (red box in Fig. 6C, D) was sufficient to release an amount of ROS able to kill the surrounding tumor cells in a short time frame (Fig. 6C, D and Video 4B). As expected, in non-coated FNPs loaded MSC (Fig. 6A, B and Video 4A) photoactivation does not induce the release of any ROS and therefore does not induce any cell death. As shown in Video 4B, after photostimulation U2OSTubRFP cells rapidly become rounded, detach from plastic surface and lose their RFP signal due to cytoskeleton injury and concomitantly cell death. We also performed a quantitative evaluation of cell death through Live and Dead assay by quantifying dead cells present after photoactivation in TPPS@FNPs loaded MSC. As a control we evaluated the

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administration of photosensitizers. In fact, we reasoned that the use of such a system could allow escaping the well documented shortcomings associated with incorporation of traditional chemotherapeutics, such as the intrinsic toxicity of the drugs to the host cells, the scarce drug payload eventually reaching the target site and the inherent inhability to effectively control the drug release mechanism [32,60]. Accordingly, the use of NPs coated with a photosensitizer, allows the release of the drug (OS) in a controlled manner, since NPs are generated

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resulted particularly efficient also due to a lack of OS-associated/ specific markers. Therefore, we selected non-hematopoietic mesenchymal stem cells (MSC) for the specific delivery of NPs coated with porphyrins to the tumor site [34,57–59]. The tumor homing ability of MSC improves the specific biodistribution of TPPS@FNPs directly within the tumor stroma, while diminishing their dispersion in non-target tissues. In principle, this system has the ability to limit the occurrence of undesirable side effects associated to the systemic

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Fig. 6. (A–D) Co-culture assay of TPPS@FNPs and FNPs loaded MSC with U2OSTubRFP cells upon photostimulation. MSC and U2OSTubRFP were seeded in a 1:5 ratio (for protocol details see Materials and methods section) and videos were recorded using a confocal microscope equipped with temperature and CO2 controllers and a SFluor 40× Oil DIC HN2 objective. Still images from video 6 are shown. (A, B) Stimulated FNPs loaded MSC (red area, 24 s with 405 nm laser source at 0.02 J/s) does not result in the induction of any photoactivation and consequently any cell death. (C, D) Stimulated TPPS@FNPs loaded MSC (red area) after 5 min induce massive tumor cell death, as appreciable from detachment of OS cell from the plastic surface and loss of red signal from TubulinRFP. Scale bar is 50 μm. (E) Apoptosis evaluation through Live and Dead assay. Calcein-AM (green signal), ethidium (red signal) and Hoechst (blue signal) stainings were performed after photostimulation of TPPS@FNPs loaded MSC (red area, 405 nm laser power at 0.16 J/s for 1.5 min) and 10 randomly chosen microscopic fields with a 10× objective has been counted to quantify cell death. (F) Graphical view of apoptosis measurement. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7. (A–D) Co-culture assay of TPPS@FNPs loaded MSC with U2OSTubRFP cells upon photostimulation performed 7 days after MSC loading. MSC and U2OSTubRFP were seeded in a 1:5 ratio (for protocol details see Materials and methods section) and videos were recorded using a confocal microscope equipped with temperature and CO2 controllers and a SFluor 40× Oil DIC HN2 objective. Still images from video 7 are shown. (A, B) Stimulated TPPS@FNPs loaded MSC (red box) were still able to induce massive tumor cells death 7 days after the initial loading, even though U2OS cells proliferated at a higher rate compared to MSC. (C, D) A second photostimulation in the same first stimulated MSC loaded cell (red area), is able to kill tumor cells in the microscope observation field next to the first one. Scale bar is 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: S. Duchi, et al., Mesenchymal stem cells as delivery vehicle of porphyrin loaded nanoparticles: Effective photoinduced in vitro killing ..., J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.03.012

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In the current study we demonstrated that TPPS@FNPs are efficiently taken up by MSC at a concentration of 45 μg/ml without evident sign of toxicity. Laser confocal microscopy and time lapse imaging of MSC loaded with TPPS@FNPs co-cultured with OS cells in vitro, show that this novel tri-component system is able to induce controlled and massive cells death in a short time frame when stimulated with laser light. Collectively these encouraging preliminary results indicate that our bio-system could represent an efficient targeted delivery strategy in killing human OS cells. Our results propone a novel yet therapeutic option for the treatment of bone sarcomas and other tumors, such as breast cancer and gliomas, all of which currently require different therapeutic approaches to overcome recurring and drug treatment failures.

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available when they are generated from the surface, rather than they have to diffuse from the NPs [71]. TPPS@FNPs release experiments clearly indicate that, at least under physiological conditions (150 mM PBS and culture medium), the electrostatic bond between TPPS and the ammonium salts on the FNPs external shell is strong enough to avoid any TPPS leakage. Moreover, argentometric titration of TPPS@FNPs confirmed their positive ζ-potential (See supplementary material, Table S5), which can promote cellular uptake through electrostatic interactions with the cell membrane. Initial internalization studies allowed us to assess the most suitable concentration of FNPs and TPPS@FNPs which need to be incorporated into MSC, in order to preserve cells viability, motility and in turn tumor-homing capability. In our study we show that 40 nm FNPs at a concentration of 45 μg/mL can enter MSC as a nanoparticular material, which afterwards agglomerate inside the perinuclear region associated with the endo-lysosomal cell compartment, without affecting cells properties. Due to particles size and their intracellular agglomeration phenomena, cell nucleus internalization is unlikely to occur. These results are in accordance with those of previous reports [72–74], showing that internalization of nanoparticles of these sizes are uptaken by MSC through normal endocytosis. Through confocal and photophysical analysis of the culture medium obtained from loaded MSC, we can assume that TPPS@FNPs are not escaping MSC, while only traces amount of TPPS can be detected after a time period of 72 h (see supporting material Figs. S6 and S7). This length of time should be sufficient for MSC to migrate to the tumor site [34,46]. In order to determine the suitability of our system, we co-cultured in vitro MSC-TPPS@FNPs with OS cells in a 1:5 ratio, with the aim of miming a pathological condition in which tumor cells are more abundant than the MSC which reach the tumor site. Upon irradiation with a low power potency 405 nm laser (10% of the maximum power, 0.02 J/s), we observed a dramatic and fast cell death. Even given such a low dosage of loaded MSC, we were able to induce an adequate ROS which in turn lead to efficient tumor cells elimination. Our data thus demonstrate the excellent ability of MSC to function as a carrier of photo-killing agents in vitro. Overall our study demonstrates that the incorporation of appropriate doses of TPPS@FNPs is nontoxic for the guest cell, until an external stimulus (light) is applied. TPPS@FNPs are internalized through endocytosis mechanism without evident signs of motility alteration. We demonstrate that upon the first light irradiation MSCs undergo cell death, while the TPPS@FNPs continue to be available at the diseased site for successive irradiations. This behavior renders the MSC-TPPS@FNPs a very promising system for the use of MSC-immolative technique, a technique which is able to ensure the safe clearance of stem cells from the body, while still maintaining its therapeutic activity [75]. Future investigations are required to understand more deeply the potential and the efficiency of our combined PDT strategy in a tumor environment.

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only after the occurance of external light activation, and ultimately only at the desired body region. Our study focused on the preparation, uptake and photo-toxicity action of a novel tri-component biomaterial system, composed of fluorescent core-shell poly-methyl methacrylate (PMMA) nanoparticles (FNPs) post-loaded with a photosensitizer (PS), namely mesotetrakis(4-sulfonatophenyl) porphyrin (TPPS), which were incorporated into mesenchymal stem cells (MSC) for targeted OS treatment. Very recently we have reported a thorough study on the photophysical and photochemical properties of a TPPS–PMMA NPs system similar to that selected for the present work [61]. Despite being aware that TPPS might not be the ideal sensitizer for in vivo application, due to its low excitation wavelength (405 nm) which results in low tissue penetration, we hypothesized that TPPS@FNPs could represent an ideal point of reference for our in vitro proof-of-concept. However, one way around the 405 nm wavelength limitation is to use hollow needles or properly engineered devices which are able to get light into deeper tissues [62]. In fact, with particular regard to OS treatment, Burch and coworkers demonstrated that optical fibers connected to a laser source could be placed directly into the osseous tumor without compromising the native tissue [13]. The assembly of our tri-component system is quite straightforward since: i) FNPs synthesis is easily achieved from commercially available and inexpensive starting materials, ii) the process of loading the porphyrin onto the particles shell is fast and readily obtained by simply mixing the two components, iii) 100% incorporation into MSCs occurs within 1 h and iv) MSC scale up is feasible. In a previously published study [30] 0.5 × 10 6 interferon-beta expressing MSC were able to suppress tumor growth in nude mice. Moreover, Huang and coworkers [34] demonstrated that 1 × 10 6 MSC loaded with mesoporous silica nanoparticles are sufficient in concentration to reach the tumor site and allow cells to be visualized by optical imaging techniques. These MSC numbers are easily achievable. The advantages of using such an organized system are multifold. Firstly, as compared with the tumortargeted nanocarriers, which simply involve the ligand–receptor interaction, more factors are implicated in the homing of MSC to the tumor sites, and therefore, a higher tumor target efficiency of MSC is expected [22,28]. Besides, the use of polymeric non-degradable carriers offers the advantage to always protect the sensitizer from the surrounding environment [61] and in this way is more likely able to perform multiple photoactivations without the need of additional pro-drug administrations. PMMA nanoparticles are biocompatible, non-biodegradable materials [63–65]. So far, we do not have a proved understanding of their excretion pathway; however, recently reported results using similar nanoparticles on animal models suggest that up to 80% or 100% of the particles are excreted in feces when intraperitoneally or orally administrated respectively [66]. Moreover, when considering an OS affected patient with terminal tumors and pulmonary metastasis, the issue of non-biodegradable NPs may indeed be considered trivial compared to the potential improvement of patients life-span and the potential of intense pain relief obtained through the use of our strategy. The FNPs design was performed while taking into account that their size, shape, and surface charge could have played an important role in cellular uptake [67,68]. In fact, studies have shown that MSC uptake of negatively charged NPs is substantially lower compared to the uptake of the cationic ones [69]. Moreover, it has been demonstrated that for NPs with similar ζ-potential, the uptake was significantly enhanced when their surface was decorated with additional primary amino groups with respect to surface-plain NPs [70]. For our purpose, we intentionally chose to externally bind the PS instead of incorporating it into the hydrophobic core. The high number of external cationic charges allows a much higher loading percentage of porphyrin without loss of singlet oxygen production [61]. In addition, the ROS produced during the photodynamic reaction are promptly

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The authors are grateful to Dr. Panagiota Dimopoulou, for editorial assistance, Dr. Andrea Liscio for atomic force microscopy measurements, Dr. Luca Cattini for FACS analysis measurements, Sabrina Paoletti for graphical abstract, and Dr. Mohanraj John for support on photophysical measurments. Research was supported in part by Progetto FIRB-Accordi di programma 2010 COD. RBAP10447, Italian Ministry of Health (Project IOR-2006-422755, and CNR Project PM.P03.011.002 (MISO).

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Please cite this article as: S. Duchi, et al., Mesenchymal stem cells as delivery vehicle of porphyrin loaded nanoparticles: Effective photoinduced in vitro killing ..., J. Control. Release (2013), http://dx.doi.org/10.1016/j.jconrel.2013.03.012

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