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

Abstract

Background: Osteosarcoma (OS) is an aggressive malignant neoplasm that still suffers from poor prognosis in the case of distal metastases or occurrence of multi-drug resistance. It is therefore crucial to find novel therapeutic options able to go beyond these limitations and improve patients' survival. The objective of this study is to exploit the intrinsic properties of mesenchymal stromal cells (MSCs) to migrate and infiltrate the tumor stroma to specifically deliver therapeutic agents directly to cancer cells. In particular, we aimed to test the efficacy of the photoactivation of MSCs loaded with nanoparticles in vitro and in a murine in vivo ectopic osteosarcoma model.

Methods: AlPcS₄@FNPs were produced by adding tetra-sulfonated aluminum phthalocyanine (AlPcS₄) to an aqueous solution of positively charged poly-methyl methacrylate core-shell fluorescent nanoparticles (FNPs). The photodynamic therapy (PDT) effect is achieved by activation of the photosensitizer AlPcS₄ in the near-infrared light with an LED source. Human MSCs were isolated from the bone marrow of five donors to account for inter-patients variability and used in this study after being evaluated for their clonogenicity, multipotency and immunophenotypic profile. MSC lines were then tested for the ability to internalize and retain the nanoparticles, along with their migratory properties in vitro. Photoactivation effect was evaluated both in a monolayer (2D) co-culture of AlPcS₄@FNPs loaded MSCs with human OS cells (SaOS-2) and in tridimensional (3D) multicellular spheroids (AlPcS₄@FNPs loaded MSCs with human OS cells, MG-63). Cell death was assessed by AnnexinV/PI and Live&Dead CalceinAM/EthD staining in 2D, while in the 3D co-culture, the cell killing effect was measured through ATP content, CalceinAM/EthD staining and TEM imaging. We also evaluated the effectiveness of AlPcS₄@FNPs loaded MSCs as delivery systems and the ability of the photodynamic treatment to kill cancer cells in a subcutaneous mouse model of OS by bioluminescence imaging (BLI) and histology.

Results: MSCs internalized AlPcS₄@FNPs without losing or altering their motility and viability in vitro. Photoactivation of AlPcS₄@FNPs loaded MSCs induced high level of OS cells death in the 2D co-culture. Similarly, in the 3D co-culture (MSCs:OS ratios 1:1 or 1:3), a substantial decrease of both MSCs and OS cells viability was observed. Notably, when increasing the MSCs:OS ratio to 1:7, photoactivation still caused more than 40% cells death. When tested in an in vivo ectopic OS model, AlPcS4@FNPs loaded MSCs were able to decrease OS growth by 68% after two cycles of photoactivation.

Conclusions: Our findings demonstrate that MSCs can deliver functional photosensitizer-decorated nanoparticles in vitro and in vivo and inhibit OS tumor growth. MSCs may be an effective platform for the targeted delivery of therapeutic nanodrugs in a clinical scenario, alone or in combination with other osteosarcoma treatment modalities.

Keywords: Aluminium phthalocyanine; Cell-mediated drug delivery system; Mesenchymal stromal cells; Musculoskeletal tumors; Nanoparticles; Osteosarcoma; Photodynamic therapy.

Fig. 1

MSCs internalize AlPcS₄@FNPs nanoparticles without cytotoxic effect. Representative flow cytometry analysis of FNPs uptake at increasing doses (45, 90, 180 μg/mL) 24 h after 1 h-loading in MSCs (a) and cell cytotoxicity analysis (WST-1 assay) of MSCs exposed for 1 h to increasing concentrations of FNPs, AlPcS₄, or AlPcS₄@FNPs, at the end of incubation (day 0) and after 1, 2 and 6 days (b). All data are expressed as mean ± SD (n = 3)

Fig. 2

AlPcS₄@FNPs internalization and retention analysis. Representative flow cytometry analysis of AlPcS₄@FNPs (90 μg/mL) loaded MSCs over time (0, 24, 48, 72 h) (a). Representative images of FNPs internalization in MSCs after 1 h loading (0 h) over the time (up to 72 h) by confocal microscope (merge images of green (FITC of FNP) and blue (Hoechst, nuclei) channels are shown) (scale bar = 200 μm) (b). Representative images and quantification of cells migrated through the porous membrane of a Boyden chamber, in absence (0.2% BSA) or presence (20%FBS) of chemotactic stimuli; MSCs loaded with 90 μg/mL AlPcS₄@FNPs were compared to unloaded MSCs (c). All data are expressed as mean ± SD (n = 3)

Fig. 3

Cell death evaluation after PDT of AlPcS₄@NPs loaded MSCs in co-culture with Saos-2 cells. Graph representing quantification of total cell death (a) and survival rate (b), 24 h after PDT by Annexin V/PI and by Alamar Blue assay respectively, 5 × 10³ MSCs loaded with 90 μg/ml AlPcS₄@NPs were seeded into 24-well plate alone (grey bar) and in co-culture with 5 × 10³ or 15 × 10³ with Saos-2 cells (AlPcS₄@NPs@MSC:Saos-2; black bars) at different ratios (1:1 and 1:3 respectively). Graph representing quantification by flow cytometry of the percentage of live or dead cells for Saos-2 (dark grey bars) and AlPcS₄@NPs loaded MSCs (light gray bars) 24 h upon photoirradiation, 5 × 10³ MSCs loaded with 90 μg/ml AlPcS₄@NPs were seeded into 24-well plate in co-culture with Saos-2 cells at 1:1 and 1:3 ratios (5 × 10³ or 15 × 10³ cells respectively) (c). All data are expressed as mean ± SD (n = 3)

Fig. 4

Cell death evaluation after PDT in a 3D co-culture system. Schematic summary of 3D in-vitro testing (a). Quantification of survival rates observed in multicellular spheroids composed by different ratios of AlPcS₄@NPs loaded MSCs and MG-63 after 10 min irradiation. Data are expressed as mean ± SD (ratio 1:1 n = 5, ratio 1:3 n = 4, ratio 1:7 n = 3) (b). Representative confocal images (scale bar = 100 μm) of Live&Dead staining (green Calcein AM staining of live cells and red EthD-1 staining of dead cells’ nuclei) (c) and representative TEM images (scale bar = 5 μm) (d) of control (−PDT) and irradiated (+PDT) spheroids at 1:1, 1:3 and 1:7 ratios

Fig. 5

In vivo photodynamic therapy of OS tumors. Schematic representation of the in vivo treatments (a). Representative fluorescent luminescent imaging of AlPcS₄ alone or loaded in NPs (AlPcS₄@FNPs) and AlPcS₄@FNPs loaded MSCs (AlPcS₄@FNPs@MSCs) localization after intra-tumor injection (b). Representative BLI images showing the evolution of luciferase-expressing tumor cells treated (c). Quantification of luminescence intensity of regions-of-interest (ROI) (tumor) (the light events recorded in the acquired images expressed in mean ± SD vs time) ** p < 0.001 (AlPcS₄@FNPs alone), * p < 0.01 (AlPcS₄@FNPs loaded MSCs) at day 28 (d). Histological analysis of tumor tissues after treatments: H&E, K_i-67 and TUNEL staining (scale bar = 100 μm, black arrow = necrotic areas) (e). For this study a total of 18 mice were used, mice were divided in 4 group as followed: mice treated with PBS (n = 3), with AlPcS₄ alone (n = 3), with AlPcS₄@FNPs NPs alone (n = 6) and with AlPcS₄@FNPs loaded MSCs (n = 6)