Pluronic^® P123/F127 mixed micelles delivering sorafenib and its combination with verteporfin in cancer cells

Abstract

Here, we developed Pluronic^® P123/F127 (poloxamer) mixed micelles for the intravenous delivery of the anticancer drug sorafenib (SRB) or its combination with verteporfin (VP), a photosensitizer for photodynamic therapy that should complement well the cytotoxicity profile of the chemotherapeutic. SRB loading inside the core of micelles was governed by the drug:poloxamer weight ratio, while in the case of the SRB–VP combination, a mutual interference between the two drugs occurred and only specific ratios could ensure maximum loading efficiency. Coentrapment of SRB did not alter the photophysical properties of VP, confirming that SRB did not participate in any bimolecular process with the photosensitizer. Fluorescence resonance energy-transfer measurement of micelles in serum protein-containing cell-culture medium demonstrated the excellent stability of the system in physiologically relevant conditions. These results were in line with the results of the release study showing a release rate of both drugs in the presence of proteins slower than in phosphate buffer. SRB release was sustained, while VP remained substantially entrapped in the micelle core. Cytotoxicity studies in MDA-MB231 cells revealed that at 24 hours, SRB-loaded micelles were more active than free SRB only at very low SRB concentrations, while at 24+24 hours a prolonged cytotoxic effect of SRB-loaded micelles was observed, very likely mediated by the block in the S phase of the cell cycle. The combination of SRB with VP under light exposure was less cytotoxic than both the free combination and VP-loaded micelles + SRB-loaded micelles combination. This behavior was clearly explainable in terms of micelle uptake and intracellular localization. Besides the clear advantage of delivering SRB in poloxamer micelles, our results provide a clear example that each photochemotherapeutic combination needs detailed investigations on their particular interaction, and no generalization on enhanced cytotoxic effects should be derived a priori.

Keywords:

• Pluronic^® micelles
• sorafenib
• chemotherapy
• photodynamic therapy
• verteporfin

Supplementary materials

Figure S1 Storage stability of lyophilized Pluronic^® P123/F127 mixed micelle formulations at room temperature.

Note: Results reported as mean values of three independent experiments (n=3) ± standard deviation.

Abbreviations: SRB, sorafenib; VP, verteporfin.

Figure S2 Absorption and emission spectra of VP and NR, respectively.

Notes: Normalized absorption spectrum of verteporfin (VP) and emission spectrum of Nile red (NR; λ_exc =480 nm) coloaded in Pluronic^® P123/F127 mixed micelles dispersed in phosphate-buffered saline at pH 7.4 and 37°C. VP =4 μg·mL⁻¹ and NR =0.8 μg·mL⁻¹. The shaded area represents the overlap of the evaluated spectra.

Figure S3 Release profile of free (A) SRB and (B) VP from Pluronic^® P123/F127 mixed micelles in phosphate-buffered saline (PBS) at pH 7.4 and 37°C.

Notes: The external medium used for dialysis was PBS with polysorbate 80 (5% v:v) at pH 7.4 and 37°C. SRB =100 μg·mL⁻¹ and VP =10 μg·mL⁻¹. Data reported as mean values of three independent experiments (n=3) ± standard deviation.

Abbreviations: SRB, sorafenib; VP, verteporfin.

Figure S4 Release profile of (A) SRB and (B) VP from Pluronic^® P123/F127 mixed micelles dispersed in DMEM enriched with FBS 10%.

Notes: Release profile of free and micelle-loaded (A) SRB and (B) VP from Pluronic^® P123/F127 mixed micelles dispersed in DMEM enriched with FBS 10%. The external medium used for dialysis was phosphate-buffered saline with polysorbate 80 (5% v:v) at pH 7.4 and 37°C. SRB =100 μg·mL⁻¹ and VP =10 μg·mL⁻¹. Data are reported as mean values of three independent experiments (n=3) ± standard deviation.

Abbreviations: SRB, sorafenib; VP, verteporfin; DMEM, Dulbecco’s Modified Eagle’s Medium; FBS, fetal bovine serum.

Figure S5 Dark cytotoxicity in MDA-MB231 cells.

Notes: Cells incubated in the dark with VP alone or to the drug combination (VP + SRB) delivered or not by Pluronic^® P123/F137 mixed micelles for 24 hours (A) or 24+24 hours (B). Cell viability was measured at the end of incubation time with the MTS assay. Data reported as mean values of at least three independent experiments carried out in triplicate ± standard deviation.

Abbreviations: SRB, sorafenib; VP, verteporfin; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium.

Figure S6 Cytotoxicity of MDA-MB231 cells incubated in the dark for 4 hours with VP (free or loaded in micelles) and irradiated with 0.75 J·cm² of red light.

Notes: Some samples were further incubated with SRB (free or loaded in micelles) for 24 hours. Cell viability was measured with the MTS assay at the end of SRB incubation or after 24 hours of cell release in VP-free medium for the samples not incubated with SRB. Data reported as mean values of at least two independent experiments carried out in triplicate ± standard deviation.

Abbreviations: SRB, sorafenib; VP, verteporfin; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium.

Acknowledgments

FQ wishes to thank the Italian Ministry of University and Research (PRIN 2010H834LS) and Italian Association for Cancer Research (IG2014, for 15764) for financial support. SS thanks PON Hippocrates (2013–2015) for financial support. DSP and NH wish to thank the Brazilian agency Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), process number BEX 14036/13-14.

Disclosure

The authors report no conflicts of interest in this work.