Curcumin Loaded in Niosomal Nanoparticles Improved the Anti-tumor Effects of Free Curcumin on Glioblastoma Stem-like Cells: an In Vitro Study

Abstract

Using a novel curcumin-loaded niosome nanoparticle (CM-NP), the present study was designed to evaluate the effect of curcumin on human glioblastoma stem-like cells (GSCs). CM-NP has a diameter of ~ 60 nm and a zeta potential of ~ - 35 mV with a constant physicochemical stability. The cytotoxic effects of free curcumin (CM) and CM-NP were investigated on GSCs obtained during the removal of a brain tumor. Both CM and CM-NP caused a dose-dependent decrease in cell proliferation and viability of GSCs. The IC50 values of CM and CM-NP on GSCs were 50 and 137 μg/ml after 24 h, respectively. CM-NP exerted significantly higher effects on GSC viability, apoptosis, cell cycle arrest, and the expression of Bax, a pro-apoptotic marker, compared with CM. In addition, the migration of GSCs was significantly impaired following the administration of CM-NP compared with CM. Furthermore, CM-NP significantly increased the values of reactive oxygen species and decreased the mRNA expressions of NF-κB and IL-6 of GSCs compared with CM. Our data also revealed that CM-NP could significantly reduce the invasiveness of GSCs compared with CM, possibly via MCP-1-mediated pathways. In addition, CM-NP exhibited a significantly greater inhibitory effect on colony formation of GSCs compared with CM. These data indicate that CM-NP exhibited stronger anti-tumor effects on GSCs than CM. Although further in vivo investigations are warranted, our results suggest that CM-NP could be an ideal carrier to deliver curcumin for potential therapeutic approaches into glioblastoma.

Keywords: Brain tumor; Cell death; Cellular engineering; Cytokine; Glioma.

Fig. 1

A schematic overview of synthetic procedure of the nano-niosome-loaded curcumin, its delivery path, and its ability of targeting glioblastoma stem-like cells (GSCs). The niosomal carrier (Tween 60-Span 60-cholestrol) enhanced the entrapment efficiency of curcumin and di-acetyl phosphate (DCP) was used for loading curcumin as well as increasing stability and efficiency. Curcumin was encapsulated in the shell of niosome. Anti-tumor activity of the nano-niosome-loaded curcumin was proved by the efficient reduction of the viability, proliferation, migration, and invasion of human GSCs

Fig. 2

Characterization of curcumin-encapsulated noisome nanoparticle (CM-NP). a Size of curcumin (CM, left) and CM-NP (right) was measured by transmission electron microscopy (TEM). TEM images showed particles with a spherical morphology and an average size of 60 nm. b In vitro release profile of CM-NP was assessed in PBS at physiological pH (7.4). The release of CM from the niosome was ~ 14% at 24 h. c Interaction between components of CM-NP was evaluated by fourier transform infrared (FTIR) spectra. FTIR analysis indicated four peaks at 3860 cm⁻¹, 2916 cm⁻¹, 1738 cm⁻¹, and 1104 cm⁻¹, which were corresponding to cholesterol, Tween 60, Span 60, and C–O–C interactions, respectively. d Differential scanning calorimetry (DSC) thermograms of CM-NP were assessed between 5 and 60 °C. DSC analysis pointed to the stability of CM-NP at body temperature

Fig. 3

Cytotoxic effects of curcumin (CM) and curcumin-encapsulated noisome nanoparticle (CM-NP) on glioblastoma stem-like cells (GSCs). a Representative phase contrast micrographs of neurosphere formation of GSCs. b Phase contrast images of GSCs following 24-h treatment with CM (50 and 200 μg/ml) and CM-NP (137 and 411 μg/ml). c Cytotoxicity of CM and CM-NP on GSC proliferation was assessed by the MTT assay. To determine the half maximal inhibitory concentration (IC50) of drugs, the percentage of live GSCs was measured after 24- and 48-h treatment with different concentrations of CM and CM-NP. The data are presented as means ± SD. Single, double, and triple asterisks indicate P < 0.05, P < 0.01, and P < 0.001, respectively

Fig. 4

Analysis of cytotoxicity of curcumin (CM) and curcumin-encapsulated noisome nanoparticle (CM-NP) on glioblastoma stem-like cells (GSCs). The relative number of dead cells was identified by the amount dead-cell protease activity using a fluorogenic peptide substrate crossing into the dead cells. The values are expressed as mean ± SD. Triple asterisks indicate P < 0.001

Fig. 5

Live/dead assay of glioblastoma stem-like cells (GSCs) treated with IC50 concentration of curcumin (CM) and curcumin-encapsulated noisome nanoparticle (CM-NP). a Living cells are labeled green (Calcein AM) and dead cells are labeled red (ethidium homodimer) after treatment with CM (50 μg/ml) and CM-NP (137 μg/ml). b Quantitative analyses of GSC viability in different groups are shown. The values are expressed as mean ±. Single and triple asterisks indicate P < 0.05 and P < 0.001, respectively

Fig. 6

The effects of curcumin (CM) and curcumin-encapsulated noisome nanoparticle (CM-NP) on viability of NIH-3T3 cells. NIH-3T3 cells were incubated with different concentrations of CM and CM-NP for 24 h. The MTT assay was used to determine cell viability of CM- and CM-NP-treated cells. The data are presented as mean ± SD. Double and triple asterisks indicate P < 0.01 and P < 0.001, respectively

Fig. 7

The effects of curcumin (CM) and curcumin-encapsulated noisome nanoparticle (CM-NP) on glioblastoma stem-like cells (GSCs) cell cycle. GSCs treated with different concentrations of CM and CM-NP for 24 h were stained with propidium iodide (PI) and the cell cycle was assessed using flow cytometry. The cell cycle was determined in GSCs treated with CM (50 and 200 μg/ml) and CM-NP (137 and 411 μg/ml) as well as in the control group (non-treated GSCs). Note the strong effect of CM-NP at 411 μg/ml on cell cycle arrest compared with the other groups. The data represent as the mean ± SD. Triple asterisks indicate P < 0.001

Fig. 8

The effects of curcumin (CM) and curcumin-encapsulated noisome nanoparticle (CM-NP) on apoptosis of glioblastoma stem-like cells (GSCs). GSCs were double-stained with Annexin V/ propidium iodide and assessed by flow cytometry to determine apoptosis of GSCs treated with CM (50 and 200 μg/ml) and CM-NP (137 and 411 μg/ml). Untreated cells were indicated as control. Diagrams quarter 1 (Q1) to Q4 indicate necrotic, late apoptotic, early apoptotic, and live cells, respectively. Cells treated with CM-NP exhibited a higher amount of necrotic GSCs

Fig. 9

The effects of curcumin (CM) and curcumin-encapsulated noisome nanoparticle (CM-NP) on the mRNA expression levels of apoptotic markers p53, Bax, and Bcl2 as well as on chemokines NF-κB, and IL-6 of glioblastoma stem-like cells (GSCs). CM-NP significantly enhanced Bax and decreased Bcl2, NF-κB, and IL-6 compared with the control group. CM significantly increased the expression of p53 compared with the CM-NP and control groups, whereas CM-NP did not regulate p53. All data represent as the mean ± SD. Triple asterisks indicate P < 0.001

Fig. 10

The effects of curcumin (CM) and curcumin-encapsulated noisome nanoparticle (CM-NP) on the generation of reactive oxygen species (ROS) in glioblastoma stem-like cells (GSCs). a Photomicrographs of curcumin-, CM-NP-, and tert-Butyl hydroperoxide (TBHP)–mediated ROS production in GSCs were obtained under fluorescence microscopy. b ROS induction in GSCs after 8 h application of CM, CM-NP, and TBHP as well as in the control group was determined by measuring fluorescent intensities in a microplate reader. Note a significant higher generation of ROS by application of CM-NP compared with the CM and control groups. Data are expressed as the mean ± SD. Triple asterisks indicate P < 0.001

Fig. 11

The effects of curcumin (CM) and curcumin-encapsulated noisome nanoparticle (CM-NP) on the migration of glioblastoma stem-like cells (GSCs). a Migration of GSCs was assessed by the wound healing method. After GSCs have reached 90% confluence, a scratch wound across a confluent monolayer of cultured cells was created on the cell surface using a micropipette tip and then CM (6.25 and 12.5 μg/ml) and CM-NP (17.12 and 34.25 μg/ml) were added. The cultures were incubated at 37 °C and images were captured with a microscope at 4, 24, 48, and 72 h of treatments. b The cell migration rate was estimated by the measurement of cell numbers within the wound region. CM-NP at 34.25 μg/ml significantly inhibited the migration of GSCs after 48 h and 72 h of application compared with the CM and control groups. The data are presented as mean ± SD. The asterisk indicates P < 0.05

Fig. 12

The effects of curcumin (CM) and curcumin-encapsulated noisome nanoparticle (CM-NP) on invasiveness of glioblastoma stem-like cells (GSCs). a Gelatinase zymogram and densitometry analysis of MMP-9 (left) and MMP-2 (right) secretion in conditioned media were detected by electrophoresis of soluble protein on a gelatin containing 10% polyacrylamide gel. Areas and relative intensities of gelatin-digested bands by MMP-9 and MMP-2 were quantified by densitometry and expressed as relative MMP-9 and MMP-2 activity compared with that of untreated cells. b The mRNA expression of CXCL3 and MCP-1 in GSCs treated with CM and CM-NP. Histogram represents mean ± SD. Triple asterisks indicate P < 0.001

Fig. 13

The effects of curcumin (CM) and curcumin-encapsulated noisome nanoparticle (CM-NP) on colony formation of glioblastoma stem-like cells (GSCs). The colony formation of GSCs was assessed in soft agar treated with CM (50 μg/ml) and CM-NP (137 μg/ml) for 5 days. CM-NP significantly decreased the size of tumor sphere and inhibited colony formation of GSCs. The data are presented as mean ± SD. The asterisk indicates P < 0.05

Fig. 14

The effects of curcumin (CM) and curcumin-encapsulated noisome nanoparticle (CM-NP) on the expression of Sox2 and nestin in glioblastoma stem-like cells (GSCs). Quantitative analysis of the expression of Sox2 and nestin in GSCs revealed a significant reduction of these two markers after treatment with CM and CM-NP. Cells were stained for markers are shown in green and nuclei were stained with propidium iodide are shown in red. The data are expressed as mean ± SD. The asterisk indicates P < 0.05