Polymeric nanoparticles enhance the sonodynamic activity of meso-tetrakis (4-sulfonatophenyl) porphyrin in an in vitro neuroblastoma model

Abstract

Purpose: Sonodynamic therapy is a developing noninvasive modality for cancer treatment, based on the selective activation of a sonosensitizer agent by acoustic cavitation. The activated sonosensitizer agent might generate reactive oxygen species leading to cancer cell death. We investigated the potential poly-methyl methacrylate core-shell nanoparticles (NPs) loaded with meso-tetrakis (4-sulfonatophenyl) porphyrin (TPPS) have to function as an innovative sonosensitizing system, ie, TPPS-NPs.

Methods: Shockwaves (SWs) generated by a piezoelectric device were used to induce acoustic cavitation. The cytotoxic effect of the sonodynamic treatment with TPPS-NPs and SWs was investigated on the human neuroblastoma cell line, SH-SY5Y. Cells were exposed for 12 hours to TPPS-NPs (100 μg/mL) and then to SWs (0.43 mJ/mm(2) for 500 impulses, 4 impulses/second). Treatment with SWs, TPPS, and NPs alone or in combination was carried out as control.

Results: There was a statistically significant decrease in SH-SY5Y cell proliferation after the sonodynamic treatment with TPPS-NPs and SWs. Indeed, there was a significant increase in necrotic (16.91% ± 3.89%) and apoptotic (27.45% ± 3.03%) cells at 48 hours. Moreover, a 15-fold increase in reactive oxygen species production for cells exposed to TPPS-NPs and SWs was observed at 1 hour compared with untreated cells. A statistically significant enhanced mRNA (messenger ribonucleic acid) expression of NRF2 (P<0.001) and a significant downregulation of TIGAR (P<0.05) and MAP3K5 (P<0.05) genes was observed in cells exposed to TPPS-NPs and SWs at 24 hours, along with a statistically significant release of cytochrome c (P<0.01) at 48 hours. Lastly, the sonosensitizing system was also investigated in an in vitro three-dimensional model, and the sonodynamic treatment significantly decreased the neuroblastoma spheroid growth.

Conclusion: The sonosensitizing properties of TPPS were significantly enhanced once loaded onto NPs, thus enhancing the sonodynamic treatment's efficacy in an in vitro neuroblastoma model.

Keywords: cancer; poly-methyl methacrylate nanoparticles; shockwaves; sonodynamic therapy; ultrasound.

Figure 1

TPPS release experiments. Note: TPPS-NPs in MilliQ® water (Merck Millipore, Billerica, Ma, USA) are placed in a 100 kDa centrifugal filter tube and suspended with releasing medium (A); the whole mixture was shaken at 37°C (B); the solution was centrifuged and the resulting bottom portion analyzed by spectrophoto metric analysis (C); an equal volume of fresh medium was added to the tube to maintain a constant volume (D). Abbreviation: TPPS, meso-tetrakis (4-sulfonatophenyl) porphyrin.

Figure 2

(A) Representation of polymethyl methacrylate core-shell nanoparticles; (B) atomic force microscopy image of nanospheres deposited on atomic flat silicon Z-range =72 ± 17 nm; (C) distribution (%) of nanoparticle radius.

Figure 3

Effect of sonodynamic treatment on SH-SY5Y proliferation as a function of sonosensitizer dose and formulations. Cells were incubated for 12 hours with increasing concentration of TPPS or TPPS-NPs (25, 50, and 100 μg/ml) and then exposed to SWs at 0.43 mJ/mm² for 500 impulses (4/second). Notes: Cell proliferation was evaluated after 48 hours by WST-1 assay (Roche Applied Science, Penzberg, Germany). Statistically significant difference versus sonodynamic treatment with TPPS: **P<0.01; ***P<0.001. Abbreviations: TPPS, meso-tetrakis (4-sulfonatophenyl) porphyrin; NPs, nanoparticles; SW, shockwave.

Figure 4

Effect of different treatment conditions on SH-SY5Y proliferation as a function of time. Sonodynamic treatment was performed with SWs at 0.43 mJ/mm² for 500 impulses (4/second) and photodynamic therapy with an LB at 15 mW, for 5 minutes. The control conditions are reported on the upper panels (A and B), ie, treatments without the sensitizer, and the lower panels (C and D) report treatment on cells incubated for 12 hours with the sensitizer, ie, TPPS (100 μg/ml). Notes: Cell proliferation was evaluated by WST-1 assay (Roche Applied Science, Penzberg, Germany). Statistically significant difference versus untreated cells: **P<0.01; ***P<0.001. Abbreviations: NPs, nanoparticles; SW, shockwave; LB, light beam; TPPS, meso-tetrakis (4-sulfonatophenyl) porphyrin; SDT, sonodynamic treatment; PDT, photodynamic treatment; Abs, absorbance; h, hours.

Figure 5

Effect of sonodynamic treatment on SH-SY5Y cell death. Representative images of (A) viable cells stained with acetoxymethyl ester of calcein, (B) nonviable cells with propidium iodide, and (C) overlay image, after 48 hours from sonodynamic treatment with TPPS-NPs. Cells were incubated for 12 hours with TPPS-NPs (100 μg/ml) and then exposed to SWs at 0.43 mJ/mm² for 500 impulses (4/second). Scale bars: 100 μm (63× magnification). (D) Representative flow cytometry density plots of SH-SY5Y at 48 hours from different treatment conditions: cells were exposed to SWs (0.43 mJ/mm² for 500 impulses, 4/second) alone or after cell incubation for 12 hours with TPPS (100 μg/ml) or TPPS-NPs (100 μg/ml). The cells were stained with Annexin V-APC (FL-4) and Sytox® Green (FL-1). Each density plot represents viable cells in the lower left quadrant (negative to APC-Annexin V and Sytox Green), apoptotic cells in the lower right quadrant (positive to APC-Annexin V and negative to Sytox Green), and necrotic cells in the upper right quadrant (positive to Annexin V and Sytox Green). Notes: Sytox® Green (Life Technologies, Milano, Italy). Annexin V-APC (Life Technologies). Abbreviations: TPPS, meso-tetrakis (4-sulfonatophenyl) porphyrin; NPs, nanoparticles; SW, shockwave; APC, allophycocyanin.

Figure 6

SH-SY5Y ROS production after different treatment conditions as a function of time. The cells were exposed to SWs at 0.43 mJ/mm² for 500 impulses (4/second) alone or after cell incubation for 12 hours with TPPS (100 μg/ml), unloaded NPs (1 mg/ml), or TPPS-NPs (100 μg/ml). Notes: ROS levels were determined according to the dichlorofluorescin-diacetate assay by flow cytometry and expressed as an iMFI ratio. Statistically significant difference versus untreated cells: *P<0.05; **P<0.01; ***P<0.001. Abbreviations: ROS, reactive oxygen species; iMFI, integrated median fluorescence intensity; SW, shockwave; TPPS, meso-tetrakis (4-sulfonatophenyl) porphyrin; NPs, nanoparticles; min, minutes.

Figure 7

(A) mRNA expression analysis in SH-SY5Y cells at 24 hours from sonodynamic treatment. Cells were incubated for 12 hours with TPPS, NPs, or TPPS-NPs at the same concentration (100 μg/ml) and then exposed to SWs at 0.43 mJ/mm² for 500 impulses (4/second). RRN18S (ribosomal RNA 18S) was used as a reference gene to normalize the data. The sonodynamic therapy-induced alterations in mRNA levels were compared with those of the untreated cells stated as 1 and are shown by the dotted line. (B) Cytochrome c release at 48 hours post sonodynamic treatment with TPPS-NPs. The cells were incubated for 12 hours with TPPS-NPs (100 μg/ml) and then exposed to SWs at 0.43 mJ/mm² for 500 impulses (4/second). Notes: Statistically significant difference versus untreated cells: *P<0.05; **P<0.01; ***P<0.001. Abbreviations: TPPS, meso-tetrakis (4-sulfonatophenyl) porphyrin; SW, shockwave; NPs, nanoparticles; mRNA, messenger RNA; RNA, ribonucleic acid.

Figure 8

The average volume of SH-SY5Y spheroids after different treatment conditions. The spheroids were exposed to SW alone or after incubation of spheroids for 12 hours with TPPS or TPPS-NPs. Statistically significant difference versus untreated spheroids: **P<0.01, ***P<0.001. Abbreviations: TPPS, meso-tetrakis (4-sulfonatophenyl) porphyrin; NPs, nanoparticles; SW, shockwave; h, hours.