Mesenchymal stromal cells mediated delivery of photoactive nanoparticles inhibits osteosarcoma growth in vitro and in a murine in vivo ectopic model

Dulbecco’s Modified Eagle’s Medium-high glucose (DMEM-HG, glucose 4500 mg/L), McCoy’s medium, and Ficoll®-Paque PREMIUM 1.073 reagents were purchased by Sigma Aldrich (Saint Luis, Missouri, USA). α-Minimum Essential Medium Eagle (α-MEM) was purchased by Lonza (Verviers, Belgium). Fetal Bovine Serum (FBS), GlutaMAX, penicillin/streptomycin solution, Dulbecco’s phosphate buffered solution without calcium and magnesium (D-PBS), Puromycin, Alexa Fluor® 488 Annexin V/PI Dead Cell Apoptosis Kit, LIVE/DEAD® Viability/Cytotoxicity Kit (Calcein-AM and Ethidium homodimer-1), Alamar Blue, and WST-1 assay reagents were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). CellTiter-GLO® was purchased from Promega (Milano, Italy). Tetra-sulfonated aluminum phthalocyanine (AlPcS₄) was purchased from LivChem Logistics GmbH (Frankfurt, Germany).

Poly-methyl methacrylate (PMMA) core-shell fluorescent nanoparticles (FNPs) were obtained by an emulsion-co-polymerization reaction as previously described [26, 41]. Briefly, 2-(dimethyloctyl)-ammonium ethyl-methacrylate bromide (0.52 g, 1.5 mmol) in water (50 mL) was placed into a 250 mL three-necked reactor equipped with a mechanical stirrer, a condenser, a thermometer and a nitrogen inlet. The mixture was heated to 80 °C under stirring (300 rpm), and 2-aminoethyl methacrylate hydrochloride (AEMA, 0.25 g, 1.48 mmol) was added to the solution. Afterward, a mixture of allyl 2-(3-allyloxy-6-oxo-6H-xanthen-9-yl) benzoate [44] (0.003 g, 0.007 mmol) and methyl methacrylate (0.93 mL, 9.35 mmol) was added to the previously obtained solution. After 10 min, 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. Whenever needed, and to avoid interference with fluorescent staining, non-fluorescent PMMA nanoparticles (NPs) were prepared by the same procedure without the addition of the fluorescent comonomer, i.e. allyl 2-(3-allyloxy-6-oxo-6H-xanthen-9-yl) benzoate AlPcS₄@FNPs or AlPcS₄@NPs stock solution was prepared by adding 50 μl of AlPcS₄ (1 mg/mL in milliQ water) to 50 μl of FNPs or NPs (10 mg/mL) and milliQ water to a final 1 mL volume. The stock solution was diluted in complete cell culture medium to the desired concentration. Where not explicitly stated, the indicated concentrations refer to the amount of FNPs/NPs per volume unit, resulting in an equivalent AlPcS₄ concentration of 1/10 (e.g. 90 μg/ml AlPcS₄@FNPs are equivalent to 9 μg/mL AlPcS₄).

Human osteosarcoma cell lines, MG-63 (CRL-1427), Saos-2 (HTB-85) and U-2 OS (HTB-96) were purchased from ATCC (Manassas, Virginia, USA). Briefly, cells were cultured respectively in DMEM-HG (MG-63) or McCoy’s medium (Saos-2, U-2 OS) containing 10% of FBS, 1% GlutaMAX and 50 U/ml penicillin/streptomycin at 37 °C in a humidified atmosphere with 5% CO₂.

Saos-2-Luc/GFP cell line was generated by transduction with Lentivirus particles containing the CMV promoter for the expression of humanized firefly luciferase (hLUC) and SV40 promoter for the expression of GFP protein according to manufacturer’s protocol (GeneCopoeia). Three days after infection, cells with high levels of GFP expression were selected by Cell Sorter NIR Aria II (BD Bioscience) and expanded for a week in culture medium supplied with Puromycin to generate a stable cell line.

MSCs were obtained from bone marrow samples of five patients undergoing surgery at Rizzoli Orthopedic Institute (Bologna, Italy). Isolation and culture expansion of human MSCs was performed as previously described in Pierini et al. [45] with minor modifications. Briefly, mononucleated cells were isolated from bone marrow through gradient separation with Ficoll®-Paque PREMIUM 1.073, then placed in 150 cm² culture flasks in complete growth medium (αMEM+ 20% FBS) at a density of 4 × 10⁵ cells/cm² and incubated at 37 °C in 5% CO₂ atmosphere. Medium was changed every 3–4 days; after the first passage sub-culturing was performed at 2 × 10³ cell/cm² every time the cells reached a 70–80% confluence. Complete characterization in terms of fibroblast-colony forming unit (CFU-F) efficiency, immunophenotypic profile, proliferation rate, and trilineage-differentiation potential of each MSC line was performed. Since ex-vivo expanded MSCs are a heterogeneous population and are known to be highly sensitive to the protocols used to isolate and expand the cells in culture [46, 47], detailed protocols are provided as Supplementary Methods (Additional file 1), and all MSCs’ characterization and in-process data are provided for review in Table 1S (Additional file 2), as suggested by Reger and Prockop [48]. In order to account for unpredictable variations on test results due to the well-known donor-to-donor variability of MSCs [49], at least 3 different MSCs lines were tested in independent experiments. When stated, all the 5 MSCs lines were tested, in order to strengthen the reproducibility of the results. Only cells from the third to the sixth passage were used in all experiments.

MSCs were seeded in 96 well plates and incubated for 1 h with AlPcS₄@FNPs, FNPs or AlPcS₄ at increasing doses (45, 90, 180 μg/mL for AlPcS₄@FNPs or 4.5, 9, 18 μg/mL for AlPcS₄). Cells were then washed twice with D-PBS and new complete medium was added to each well. WST-1 assay was performed 1, 2 and 6 days after loading following the manufacturer’s instructions. The optical density of each well was measured by a microplate reader (Synergy HT, BioTek Winooski, VT, USA) set at 450 nm with the correction wavelength set at 690 nm.

MSCs were seeded at 10⁴ cells/cm² in complete medium and allowed to adhere to the plates overnight before loading. MSCs were exposed to AlPcS₄@FNPs or AlPcS₄@NPs diluted in complete medium for 1 h, then washed two times with D-PBS. AlPcS4@FNPs or AlPcS4@NPs loaded MSCs were allowed to recover for a time varying from 2 h to overnight (o/n) in complete medium, before being detached from culture flasks, for subsequent experiments. Before each experiment, AlPcS4@FNPs loaded MSCs were checked through automated cell counter Countess II® FL (Thermo Scientific, Waltham, Massachusetts, USA) in order to verify the loading efficiency.

The percentage of loaded MSCs were determined by BD FACScanto II cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) and by the automated cell counter Countess II® FL (Thermo Scientific, Waltham, Massachusetts, USA), taking advantage of FITC fluorescence of FNPs. For confocal microscopy (Nikon, Amsterdam, Netherlands) analysis, MSCs were seeded onto glass coverslips, loaded with FNPs and at the indicated time points fixed for 10 min in 10% Neutral Buffered Formalin at room temperature, thoroughly washed with D-PBS, stained with Hoechst and imaged.

Cell migration was assessed by the Boyden chamber technique. Cell culture inserts for 24-well plate with 8 μm pore diameter were used (Millipore, Darmstadt, Germany). After a recovery period of 2 h in complete medium, AlPcS₄@FNPs loaded MSCs, along with unloaded MSCs used as control, were exposed to o/n starvation, switching the complete medium to αMEM+ 0.2%FBS. Then 10⁴ cells were placed in the upper chamber in 200 μL of αMEM+ 0.2%BSA. Six hundred microliters of αMEM supplemented with 20% FBS (chemo-attractant) or 0.2% BSA (neutral) was added into the lower chamber. After overnight incubation, cells on the upper face of the membrane were removed with a cotton swab while the ones on the bottom surface were fixed in 100% methanol and stained with Hema-stain kit (Fischer Scientific, Hampton, New Hampshire, USA). Cells migrated through the microporous membrane were counted in 10 randomly chosen fields under an inverted Nikon Eclipse TE2000-U microscope (Nikon, Amsterdam, The Netherlands).

MSCs were loaded with 90 μg/ml AlPcS₄@FNPs and left o/n to recover in complete medium. AlPcS₄@FNPs loaded MSCs were then trypsinized, counted with Countess II® FL and 5 × 10³ cells were seeded into 24-well plate mixed with 5 × 10³ or 15 × 10³ Saos-2 cells, i.e 1:1 or 1:3 ratio respectively. Photoirradiation was delivered after overnight cell adhesion.

MSCs were loaded with 90 μg/ml AlPcS₄@FNPs and left for a recovery period of 4 h in complete medium. AlPcS₄@FNPs loaded MSCs were then trypsinized, counted and mixed with MG-63 in different ratios (1:1, 1:3 and 1:7) to a final concentration of 10⁵ mixed cells/mL in DMEM-HG + 10%FBS. One hundred microliters aliquots of the suspension were dispensed in an ultra-low attachment U-bottom 96-well plate (Corning Costar, Amsterdam, The Nederlands) and allowed to aggregate for 4 days to form regularly shaped spheroids.

In in vitro experiments, AlPcS₄@NPs loaded MSCs were photoactivated using a LED light source (λmax = 668 ± 3 nm) at room temperature, with the light-emitting unit placed directly under the tissue culture plates (radiant power: 140 mW). Monolayer cultures (2D) received photoactivation for 5 min, while spheroids (3D) for 10 min. Viability assays were performed, in all experiments, 24 h after PDT treatment.

In in vivo model, the tumor bearing area was irradiated for 20 min using the same LED source but with the addition of a focusing device (i.e. a cylinder of 0.6 cm diameter and 2 cm length, with a light-reflecting internal surface). The end of the focusing device was placed in close proximity to the mouse skin (Radiant power: 130 mW). Treatment was repeated twice, once a week.

In 2D co-culture, cell death was evaluated by Alexa Fluor® 488 Annexin V/Propidium Iodide Dead Cell Apoptosis Kit according to the manufacturer’s protocol and analyzed with BD FACScanto II cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). Cell survival rate was determined by Alamar blue assay following the manufacturer’s instructions. The fluorescence of each well was measured by a microplate reader (Synergy HT, BioTek Winooski, VT, USA) with excitation/emission wavelengths of 530/590 nm. The fluorescence intensity from the samples was corrected using a cell-free control as blank.

For 3D co-culture system, cell death was evaluated through the ATP content–based assay CellTiter-Glo® 3D following the manufacturer’s protocol. Additionally, a LIVE/DEAD® staining was performed. Spheroids were incubated with 2.5 μM Calcein-AM in DMEM Phenol Red-free for 2 h, then Ethidium homodimer-1 (EthD-1) was added to a 5 μM final concentration for 10 min. Z-stacks images, for a total depth of 100-120 μm, were acquired with an A1R confocal laser scanner (Nikon, Amsterdam, The Netherlands) using Nikon Plan Apo VC 20x/0.75 NA DIC N2 objective lens and 3D rendering was performed with NIS elements software using the Alpha-blending algorithm.

Spheroids were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate pH 7.6 buffer for 1 h at room temperature. After post-fixation with 1% OsO₄ in cacodylate buffer for 1 h, cells were dehydrated in an ethanol series and embedded in Epon resin. Semithin sections of 0.8 μm were cut using an ultramicrotome and stained with toluidine blue. Ultrathin sections (70 nm) were contrasted with uranyl acetate and lead citrate and observed with a Jeol Jem-1011 transmission electron microscope (Jeol Jem, USA).

Eighteen female Athymic-nude mice, aged 6–8 weeks, were subcutaneously injected into the left flank with a mixture of Saos-2/Luc cells (1 × 10⁶) and MSCs (1 × 10⁶) in 50 μL of PBS/Matrigel. When tumors reached 100–150 mm³, approximately 2 weeks post-injection, the mice were divided into four groups: two control groups (group I and II respectively PBS and AlPcS₄), group III AlPcS₄@FNPs alone and group IV AlPcS₄@FNPs loaded into MSCs. Fifty microliters of PBS, AlPcS₄ (9 μg/mL), AlPcS₄@FNPs (90 μg/mL) and AlPcS₄@FNPs loaded-MSCs (1 × 10⁶) were intra-tumorally injected. The next day, the mice were exposed for 20 min to PDT. Intra-tumor injection and PDT treatment were performed weekly for 2 weeks. All animals were euthanized 1 week after the last treatment. After intra-tumor administration of test substances, the whole animal fluorescent imaging (excitation/emission wavelengths: 640/680 nm) was performed using the IVIS Lumina II (PerkinElmer, Waltham, MA) to observe AlPcS₄@FNP nanoparticles biodistribution. The same instrumentation was used to monitor tumor growth through bioluminescence imaging (BLI). D-luciferin (GolBio, St Louis, MO) dissolved in PBS (1.5 mg luciferin/100 μL PBS) was injected intraperitoneally at a dose of 150 mg D-luciferin/kg. The BLI imaging was performed prior NPs/NPs loaded MSCs injections and after PDT treatment. Regions of interest (ROIs) were drawn within the tumor to measure average radiance (expressed as photons/s/cm²/sr) using Living Image® 4.2 software (Caliper Life Sciences, Hopkinton, MA).

Tumors were collected, fixed in 4% paraformaldehyde solution, and embedded in paraffin. Samples were sectioned at a thickness of 4 μm and hematoxylin and eosin (H&E) staining was performed for a general inspection of the pathologic specimens. To evaluate the extent of tumor apoptosis and validate the BLI results, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed with a commercial kit (Roche, Mannheim, Germany). K_i-67 staining for cell proliferation was also performed. Images of tumor tissue were taken by a NIKON Upright BF&Fluorescent light microscope.

Fluorescent core-shell PMMA nanoparticles, namely FNPs, were characterized in terms of size, zeta potential, and morphology. In particular, FNPs were obtained with an average hydrodynamic diameter of 75 ± 0.92 nm (five measurements, PDI = 0.16 ± 0.01; Table 2S, Additional file 3), and a zeta-potential of 54 ± 2 mV (five measurements; Table 3S, Additional file 3). The number of ammonium group available for AlPcS₄ loading was determined by potentiometric titration of the bromide ions obtained after complete ion exchange and was found to be 571 μmol per gram of nanospheres. The morphological analysis, performed through scanning electron microscopy (SEM), confirmed the spherical shape of FNPs (Figure 1S, Additional file 3).

In order to determine the AlPcS₄@FNPs concentration to guarantee a 90% uptake into MSCs without altering their viability, we optimized the NPs loading parameters. Several MSC lines were incubated for 1 h with different concentrations of AlPcS₄@FNPs (45, 90 and 180 μg/mL) and 24 h after the loading, the FITC fluorescence intensity was quantified by flow cytometry. Unloaded cells were used as control. As shown in the representative histograms of Fig. 1a, 98 to 100% of the MSCs internalized the NPs at all tested concentrations. In addition, for all concentrations, 1 h loading was sufficient for AlPcS₄@FNPs internalization, therefore this incubation time was used in all the experiments.

To evaluate the potential cytotoxicity of AlPcS₄@FNPs on MSCs without light irradiation, i.e. dark toxicity, cells were incubated with 45, 90 and 180 μg/mL of AlPcS₄@FNPs for 1 h, as well as with each of the NP components alone (FNPs and AlPcS₄). WST-1 assay was performed at 1, 2 and 6 days after loading. As shown in Fig. 1b, the viability of MSCs was not affected by the internalization of AlPcS₄@FNPs or the single components. In fact, WST-1 values in MSCs control cells were comparable to those of MSCs exposed to AlPcS₄@FNPs, FNPs or AlPcS₄ at different concentrations and at all tested times (Fig. 1b).

The loading efficiency was further investigated using an automated cell counter to measure the percentage of AlPcS₄@FNPs positive cell and the average fluorescence intensity of 5 different MSCs lines, incubated with 45, 90 and 180 μg/mL of AlPcS₄@FNPs. Results shown in Table 1 demonstrate that, regardless of the MSC lines used, the 90 μg/mL concentration ensures the highest and most uniform internalization rate, therefore this concentration was selected for all subsequent experiments.

The retention of AlPcS₄@FNPs (90 μg/mL) in MSCs over time (1, 2 and 3 days) was determined by flow cytometry (Fig. 2a) and by fluorescence microscopy (Fig. 2b). Both assays demonstrated that the fluorescence intensity remains constant for 3 days. In particular, the internalization was close to 100% at all tested time points, and the FNPs localization was intracellular (Fig. 2b).

Since one of the major aims of this study was to exploit MSCs as delivery vehicles of AlPcS₄@FNPs, the migration of NPs loaded MSCs was investigated using the Boyden chamber assay. Results in Fig. 2c showed that nanoparticles internalization does not affect MSCs migration profile in both neutral and chemoattractant conditions (0.2% BSA or 20% FBS respectively), suggesting that AlPcS₄@FNPs do not modify the migratory potential of MSCs.