Mesenchymal Stem Cell Membrane-Coated TPCS2a-Loaded Nanoparticles for Breast Cancer Photodynamic Therapy

Abstract

Despite substantial improvements in breast cancer (BC) treatment there is still an urgent need to find alternative treatment options to improve the outcomes for patients with advanced-stage disease. Photodynamic therapy (PDT) is gaining a lot of attention as a BC therapeutic option because of its selectivity and low off-target effects. However, the hydrophobicity of photosensitizers (PSs) impairs their solubility and limits the circulation in the bloodstream, thus representing a major challenge. The use of polymeric nanoparticles (NPs) to encapsulate the PS may represent a valuable strategy to overcome these issues. Herein, we developed a novel biomimetic PDT nanoplatform (NPs) based on a polymeric core of poly(lactic-co-glycolic)acid (PLGA) loaded with the PS meso-tetraphenylchlorin disulfonate (TPCS_2a). TPCS_2a@NPs of 98.89 ± 18.56 nm with an encapsulation efficiency percentage (EE%) of 81.9 ± 7.92% were obtained and coated with mesenchymal stem cells-derived plasma membranes (mMSCs) (mMSC-TPCS_2a@NPs, size of 139.31 ± 12.94 nm). The mMSC coating armed NPs with biomimetic features to impart long circulation times and tumor-homing capabilities. In vitro, biomimetic mMSC-TPCS_2a@NPs showed a decrease in macrophage uptake of 54% to 70%, depending on the conditions applied, as compared to uncoated TPCS_2a@NPs. Both NP formulations efficiently accumulated in MCF7 and MDA-MB-231 BC cells, while the uptake was significantly lower in normal breast epithelial MCF10A cells with respect to tumor cells. Moreover, encapsulation of TPCS_2a in mMSC-TPCS_2a@NPs effectively prevents its aggregation, ensuring efficient singlet oxygen (¹O₂) production after red light irradiation, which resulted in a considerable in vitro anticancer effect in both BC cell monolayers (IC₅₀ < 0.15 µM) and three-dimensional spheroids.

Keywords: biomimetic nanoparticles; breast cancer; photodynamic therapy.

Figure 1

Formulation scheme and characterization of TPCS_2a@NPs and mMSC-TPCS_2a@NPs. (a) Scheme illustrating the formulation procedure of mesenchymal stem cell-coated TPCS_2a@NPs (mMSC-TPCS_2a@NPs). (b) Hydrodynamic diameter, polydispersity index (PDI), and zeta potential (ZP) of TPCS_2a@NPs, mMSC vesicles, and mMSC-TPCS_2a@NPs. (c) Representative silver-stained SDS-PAGE protein pattern of: line 1 = ASC52telo cells lysate, line 2 = isolated mMSC, line 3 = mMSC-TPCS_2a@NPs, M = protein marker. (d) Representative transmission electron microscopy (TEM) images of TPCS_2a@NPs, mMSC vesicles, and mMSC-TPCS_2a@NPs; scale bars: 100 nm.

Figure 2

Physico-chemical characterization of TPCS_2a@NPs and mMSC-TPCS_2a@NPs. Stability of TPCS_2a@NPs and mMSC-TPCS_2a@NPs in (a) H₂O at 4 °C, (b) H₂O at 37 °C, (c) DMEM + 5% FBS at 4 °C and (d) DMEM + 5% FBS at 37 °C. (e) Monitoring of singlet oxygen (¹O₂) generation with an SOSG probe in the presence of free TPCS_2a solution or TPCS_2a@NP and mMSC-TPCS_2a@NP suspensions. Data are expressed as mean percentage ± SD of at least two independent experiments. Statistical analysis (two-way ANOVA, NPs vs. free TPCS_2a): * p < 0.05; ** p < 0.01; *** p < 0.001. (f) Release profiles of free TPCS_2a or TPCS_2a from TPCS_2a@NPs over a period of 17 days. Data are expressed as mean percentage ± SD of three independent experiments.

Figure 3

NP capture by human macrophages. Flow cytometry data showing the mean fluorescence intensity (MFI) of TPCS_2a in human macrophages incubated with 1 μM TPCS_2a@NPs or mMSC-TPCS_2a@NPs and incubated at 37 °C for 2 h. In some cases, NPs were pre-incubated with human serum (HS) for 30 min or 2 h at 37 °C and then placed in contact with cells. Data are expressed as mean percentage ± SD of at least three independent experiments carried out in triplicate. Statistical analysis (one-way ANOVA): **** p < 0.0001.

Figure 4

In vitro uptake and intracellular localization of TPCS_2a. Cell uptake measured by flow cytometry after incubation of MDA-MB-231 (a), MCF7 (b), and MCF10A (c) cells for 24 h with free TPCS_2a, TPCS_2a@NPs, or mMSC-TPCS_2a@NPs. Data are expressed as mean percentage ± SD of at least two independent experiments carried out in triplicate. Statistical analysis (two-way ANOVA, NPs versus free TPCS2a): ** p < 0.01; *** p < 0.001. (d) Confocal microscopy images of MDA-MB-231 cells after incubation with free TPCS_2a, TPCS_2a@NPs, or mMSC-TPCS_2a@NPs showing TPCS_2a (red), LysoTracker Green lysosome probe (green), Hoechst 33342 nuclei staining (blue). Scale bars: 20 μm.

Figure 5

Dark toxicity and phototoxicity of TPCS_2a formulations toward monolayered breast cancer cells. Cytotoxicity profiles of MDA-MB-231 (a,c) and MCF7 (b,d) cells exposed to increasing concentrations of TPCS_2a, TPCS_2a@NPs, or mMSC-TPCS_2a@NPs. Cytotoxicity of TPCS_2a was assessed by MTS assay after incubation of cells in the dark (a,b) as well as after exposure to 10 J/cm² of red light (c,d), and an additional 24 h incubation in a drug-free medium. Data are expressed as mean ± SD of at least three independent experiments carried out in triplicate. Statistical analysis (two-way ANOVA, free TPCS_2a versus TPCS_2a@NPs or mMSC-TPCS_2a@NPs): **** p < 0.0001.

Figure 6

In vitro uptake and cytotoxicity of TPCS_2a formulations in MDA-MB-231 spheroids. (a) Confocal microscopy images of spheroids treated for 24 h with free TPCS_2a, TPCS_2a@NPs, and mMSC-TPCS_2a@NPs at 1 μM TPCS_2a equivalent concentration. The images represent TPCS_2a fluorescence distribution at the equatorial plane of each spheroid and the maximum projection of 20 different focal planes superimposed. Scale bar: 100 μm. (b) Cytotoxicity in spheroids was assessed after 24 h of incubation in the dark or (c) exposure to 10 J/cm² of red light, and an additional 24 h incubation in drug-free medium. Cell viability was measured by CellTiter-Glo 3D Assay. Data are expressed as mean ± SD of at least two independent experiments carried out in quadruplicate. Statistical analysis (two-way ANOVA, free TPCS_2a versus TPCS_2a@NPs and mMSC-TPCS_2a@NPs): **** p < 0.0001. (d) Selected images of spheroids stained with the LIVE/DEAD kit and acquired 24 h after irradiation with 10 J/cm² of red light. Scale bar: 100 μm.