Effects of Ipriflavone-Loaded Mesoporous Nanospheres on the Differentiation of Endothelial Progenitor Cells and Their Modulation by Macrophages

Abstract

Angiogenic biomaterials are designed to promote vascularization and tissue regeneration. Nanoparticles of bioactive materials loaded with drugs represent an interesting strategy to stimulate osteogenesis and angiogenesis and to inhibit bone resorption. In this work, porcine endothelial progenitor cells (EPCs), essential for blood vessel formation, were isolated and characterized to evaluate the in vitro effects of unloaded (NanoMBGs) and ipriflavone-loaded nanospheres (NanoMBG-IPs), which were designed to prevent osteoporosis. The expression of vascular endothelial growth factor receptor 2 (VEGFR2) was studied in EPCs under different culture conditions: (a) treatment with NanoMBGs or NanoMBG-IPs, (b) culture with media from basal, M1, and M2 macrophages previously treated with NanoMBGs or NanoMBG-IPs, (c) coculture with macrophages in the presence of NanoMBGs or NanoMBG-IPs, and (d) coculture with M2d angiogenic macrophages. The endocytic mechanisms for nanosphere incorporation by EPCs were identified using six different endocytosis inhibitors. The results evidence the great potential of these nanomaterials to enhance VEGFR2 expression and angiogenesis, after intracellular incorporation by EPCs through clathrin-dependent endocytosis, phagocytosis, and caveolae-mediated uptake. The treatment of EPCs with basal, M1, and M2 macrophage culture media and EPC/macrophage coculture studies also confirmed the angiogenic effect of these nanospheres on EPCs, even in the presence of phagocytic cells.

Keywords: endocytosis; endothelial progenitor cells; ipriflavone; macrophages; mesoporous nanospheres; vascular endothelial growth factor receptor 2.

Scheme 1

Cocultures of endothelial progenitor cells (EPCs) and RAW 264.7 macrophages in the presence of 50 μg/mL of NanoMBG nanospheres with or without ipriflavone.

Figure 1

Representative SEM micrograph obtained from Nano-MBGs (a). Representative TEM images of Nano-MBGs before loading with ipriflavone (b) and after loading with iproflavone (Nano-MBG-IPs) (c).

Figure 2

Phenotypic characterization of EPCs in culture. The expression of (A) CD31, CD34, and eNOS and (B) VEGFR2 and vWF, was evaluated in EPCs as endothelial phenotype markers by immunofluorescence labeling and flow cytometry after 23 and 30 days of differentiation. In each sample, 10,000 cells were analyzed. Statistical significance: *** p < 0.005.

Figure 3

(A) Incorporation of FITC-NanoMBGs by EPCs evaluated by flow cytometry. Fluorescence intensity of EPCs with intracellular FITC-NanoMBGs after treatment with 50 µg/mL for different times (30, 60, and 90 min). Statistical significance: *** p < 0.005. (B) Effects of specific endocytosis inhibitors on FITC-NanoMBG incorporation by EPCs. EPCs were treated for 2 h with each inhibitor before treatment with 50 µg/mL FITC-NanoMBGs for 30 min. FITC-NanoMBG incorporation in each case was quantified by flow cytometry. Control cells without inhibitors were carried out in parallel. C = control cells, W = cells treated with wortmannin, G = cells treated with genistein, CB = cells treated with cytochalasin B, CD = cells treated with cytochalasin D, PAO = cells treated with phenylarsine oxide, Chl = cells treated with chlorpromazine. Statistical significance: * p < 0.05, *** p < 0.005.

Figure 4

Incorporation of FITC-NanoMBGs by EPCs observed by confocal microscopy. Images of EPCs after 24 h of incubation with 50 μg/mL of FITC-NanoMBGs. Nuclei were stained with DAPI (blue), F-actin filaments were stained with rhodamine-phalloidin (red), and FITC-NanoMBGs are observed in green.

Figure 5

Effects of unloaded (NanoMBGs) and ipriflavone-loaded nanospheres (NanoMBG-IPs) on VEGFR2 expression in EPCs and modulating role of M1 and M2 macrophages. Percentages of VEGFR2⁺ EPCs were analyzed by flow cytometry after 10 days of treatment under the following conditions: (A) direct treatment with 50 µg/mL of either NanoMBGs or NanoMBG-IPs; (B) treatment with culture media of basal macrophages previously cultured for 24 h in the absence or in the presence of 50 μg/mL of NanoMBGs or NanoMBG-IPs; (C) treatment with culture media of E. coli LPS-stimulated M1 macrophages previously cultured for 24 h in the absence or in the presence of 50 μg/mL of NanoMBGs or NanoMBG-IPs; (D) treatment with culture media of IL-10-stimulated M2 macrophages previously cultured for 24 h in the absence or in the presence of 50 μg/mL of NanoMBGs or NanoMBG-IPs. Statistical significance: *** p < 0.005 (comparisons with control EPCs directly treated with either NanoMBGs or NanoMBG-IPs), ### p < 0.005 (comparison with EPCs in the same culture conditions without macrophage conditioned medium).

Figure 6

Effects of unloaded (NanoMBGs) and ipriflavone-loaded nanospheres (NanoMBG-IPs) on VEGFR2 expression in EPCs cocultured with RAW 264.7 macrophages. Percentages of VEGFR2⁺ EPCs after 7 days under the following conditions: (A) EPCs in monoculture; (B) EPCs cocultured with RAW 264.7 macrophages in the absence of nanospheres and without stimuli; (C) EPCs cocultured with M2d angiogenic macrophages obtained from RAW 264.7 macrophages treated with 1 μM NECA and 100 ng/mL of E. coli LPS; (D) EPCs cocultured with RAW 264.7 macrophages in the presence of 50 μg/mL of NanoMBGs; (E) EPCs cocultured with RAW 264.7 macrophages in the presence of 50 μg/mL of NanoMBG-IPs. Statistical significance: ### p < 0.005, ## p < 0.01 (comparisons with EPC RAW).

Figure 7

Effects of unloaded (NanoMBGs) and ipriflavone-loaded nanospheres (NanoMBG-IPs) on CD206 expression in RAW 264.7 macrophages cocultured with EPCs. Percentages of CD206⁺ macrophages after 7 days under the following conditions: (A) RAW 264.7 macrophages in monoculture; (B) RAW 264.7 macrophages cocultured with EPCs in the absence of nanospheres and without stimuli; (C) M2d angiogenic macrophages obtained from RAW 264.7 macrophages treated with 1 μM NECA and 100 ng/mL of E. coli LPS and cocultured with EPCs; (D) RAW 264.7 macrophages cocultured with EPCs in the presence of 50 μg/mL of NanoMBGs; (E) RAW 264.7 macrophages cocultured with EPCs in the presence of 50 μg/mL of NanoMBG-IPs. Statistical significance: ** p < 0.01 (comparisons with control RAW cells in monoculture).