Induction of apoptosis and inhibition of breast cancer cell growth and multicellular tumor spheroids by paclitaxel combined curcumin-loaded PLGA-TPGS-based nanoparticles
- Curcumin-loaded PLA-TPGS-based Nanoparticles. - Here, we presented a novel combination of paclitaxel and curcumin-loaded PLA-TPGS (PTX-Cur/PLA-TPGS) nanoparticles prepared by a modified solvent extraction/evaporation technique. - Furthermore, PTX-Cur/PLA-TPGS nanoparticles exhibited a powerful ability in preventing MCF7 spheroids growth. - Based on the autofluorescence of curcumin, the absorption of PTX-Cur/PLA-TPGS nanoparticles into MCF7 spheroids could be followed and calculated.. - Keywords: Paclitaxel, curcumin, PLA-TPGS nanoparticles, breast cancer, apoptosis, multicellular tumor spheroid.. - One of the solutions is to encapsulate paclitaxel or curcumin in polymeric nanoparticle [10].. - It was found that poly(lactide)-vitamin E TPGS (PLA-TPGS) copolymer can greatly increase the drug encapsulation efficiency and PLA-TPGS-based nanoparticle formulation avoids using toxic adjuvant Cremophor EL. - In addition, PLA-TPGS micelles co-loaded Curcumin and paclitaxel showed a dual-function nano system for theranostics [12]. - This study concentrated on exploring the ability of Curcumin as a label compound for cellular uptaking and penetrating in the tumor spheroids [12].. - In this study, (PTX combined Cur)-loaded PLA-TPGS nanoparticle is the main subject.. - The cytotoxicity and the ability to induce the apoptosis process of cancer cells of the complex were evaluated. - Our results showed that Paclitaxel and Curcumin encapsulated in PLA-TPGS nanoparticle effectively inhibited breast cancer cells in both 2D and 3D culture and increased the nanoparticle-induced apoptosis.. - Cells were grown in a humidified chamber in the presence of 5% CO 2 , at 37 °C. - Cells were incubated with DNA staining solution for 30 minutes at room temperature and in the dark. - After 48 hours of incubation in a humidified chamber in the presence of 5% CO 2 , at 37 °C, spheroids were transferred from the cover into each well of the agarose-coated plate and further cultivated in fresh growth medium.. - MCF7 spheroids grown on agarose-coated 96-well plate were cultured in the presence of the combination at the concentration of 0.09 µM paclitaxel and 0.43 µM curcumin. - One-way ANOVA was used to compare the IC50 values of the compounds. - Cytotoxicity of PTX-Cur/PLA-TPGS We have checked the cytotoxicity of compounds: PTX, Cur, PTX/PLA-TPGS, Cur/PLA-TPGS, PTX-Cur (normal mixture of paclitaxel and curcumin with the corresponding concentrations) and PTX-Cur/PLA-TPGS on two breast cancer cell lines - KPL4 and MCF7 cells. - and, more importantly, is there an increase in synergism when the compounds are co-coated with PLA-TPGS. - The MTS assay, the very efficient method for screening anti-proliferating compounds, was used to determine the cell viability in the presence of four compounds with different concentration range from 8.3 nM to 0.625 µM of paclitaxel and 0.05 to 2.9 µM of curcumin. - The concentrations of paclitaxel and curcumin were similar in Cur, PTX-Cur and PTX-Cur/PLA-TPGS. - However, at the lower doses, there is a big change in the cell viability between the 4 compounds. - The obtained results revealed that PTX alone induced much less cytotoxicity in comparison with the combination of paclitaxel and curcumin in PTX-Cur and PTX-Cur/PLA-TPGS. - For example, in KPL4 cells, PTX at 0.3 µM induced about 23.5% cytotoxicity while the combination of 0.15 µM paclitaxel and 0.72 µM curcumin induced 35% cytotoxicity in PTX-Cur and 47% in PTX-Cur/PLA-TPGS (Figure 1). - Moreover, PTX-Cur/PLA-TPGS nanoparticles had better effect in inhibition of breast cancer cell growth than PTX/PLA-TPGA and Cur/PLA-TPGS alone (Table 1). - The IC50 value of PTX-Cur/PLA-TPGS nanoparticles. - The enhanced effect of the combination between paclitaxel and curcumin on cell viability was proved before by Smitha et al . - Dose-response curves of PTX, Cur, PTX-Cur and PTX-Cur/PLA-TPGS for KPL4 cell growth. - Our results showed that this combination in nanoparticulate formulation with PLA-TPGS coating increased even much more the cytotoxicity of the wo compounds. - Even in water, PTX-Cur/PLA-TPGS showed significant advantages in inhibiting cancer cell proliferation compared to PTX or PTX-Cur in DMSO solution (p<0.05).. - PTX/PLA-TPGS Cur/PLA-TPGS . - PTX-Cur PTX-Cur/. - PLA-TPGS . - PTX-CUR/PLA-TPGS Induced Apoptosis in Breast Cancer Cells. - Checking the ability of PTX-Cur/PLA-TPGS in increasing PTX or Cur-induced apoptosis is one of the major aims of our work. - In the control without treatment, most of the cellular nuclei were round and similar in size whereas in PTX or PTX and Cur combination - treated cells, chromatin condensation and DNA fragmentation were observed (Figure 2A). - We used a flow cytometry method to determine the percentage of apoptotic cells following administration of either PTX, PTX-Cur, Cur or PTX-Cur/PLA-TPGS at the same concentration (0.36 µM for paclitaxel and 1.68 µM for curcumin) for 48 hours. - Cells were harvested only when we observed the DNA fragmentation and cells still stayed in the complete membrane. - The percentage of apoptotic nuclear is highest in PTX-Cur/PLA-TPGS (37. - nearly 3 times more than in PTX and PTX-Cur (13% and 13.5%, relatively). - Cells in the presence of paclitaxel can double their DNA but cannot divide during the M phase. - After two rounds of mitotic slippage, most of the cells will go to apoptosis [23].. - Cells treated with PTX-Cur/PLA-TPGS spent less time in the G2/M phase or diploid G1 (51%) but more in apoptosis. - That means PTX-Cur/PLA-TPGS with the equivalent concentration not only increased the apoptosis but also induced this process to happen earlier than PTX and PTX-Cur.. - PTX+Cur/PLA-TPGS Inhibited MCF7 Spheroid Growth. - That is the reason we carried out the experiment on MCF7 spheroid to check the anti-spheroid growth activity of PTX-Cur/PLA-TPGS and to compare it to commercial PTX. - observed that all of the treated spheroids decreased in size compared to the control and that PTX-Cur/PLA-TPGS had greater effect on inhibiting MCF7 spheroid growth than PTX alone. - Some cells of the outer layer - the highest proliferated part in spheroid structure - became more loosely bound together. - There was no increase in size or volume of spheroids treated with any dose of PTX-Cur/PLA-TPGS even from day 1 of treatment, whereas the controls were frequently larger (Figure 3A).. - PTX-Cur/PLA-TPGS induced apoptosis in KPL4 cells. - (A) Effect of PTX, Cur, PTX-Cur and PTX-Cur/PLA-TPGS at the same concentration (0.36 µM for paclitaxel and 1.68 µM for curcumin) for 48 hours on the cell cycle of KPL4 cells. - and size of nuclei in the cells treated with PTX, Cur, PTX-Cur and PTX-Cur/PLA-TPGS.. - Moreover, from day 3 of the treatment, the size of spheroids at all treated doses became smaller than the first day of treatment.. - Especially at the highest dose of 2.3 µM, there was a sharp decrease in the volumes of spheroids. - For example, at the lowest dose of 37 nM paclitaxel, the MCF7 spheroid growth delay was about one day when treated with PTX, but 4 days when treated with PTX-Cur/PLA-TPGS in comparison to the control. - indicated that PTX-Cur/PLA-TPGS not only inhibited the growth of the outer layer but also affected the second layer of spheroids - the layer consists of quiescent cells, and made the spheroid smaller. - Figure 3B showed the dose-response curves of MCF7 spheroid volume under the effects of PTX and PTX-Cur/PLA-TPGS at day 7 of treatment with IC50 of 1.108 µM ± 0.5 and 40.013 µM ± 5.3, respectively.. - Curcumin in the Complex Plays the Second Function as the Label of Nanoparticles. - Based on the autofluorescence of Curcumin, we checked the presence of PTX-Cur/PLA-TPGS when incubated with cells at concentration of 0.09 µM paclitaxel and 0.43 µM curcumin in 2 h, 4 h and 24 h. - As shown in Figure 4, the level of PTX-Cur/PLA-TPGS increased in. - The nanoparticles concentrated in the cytoplasm of KPL4 cells around the nucleus.. - Using confocal laser scanning microscope, we found a significant difference in the distribution of nanoparticles inside the MCF7 spheroids with time.. - (A) The morphology of MCF7 spheroids changed under the treatment with PT and PTX-Cur/PLA-TPGS at the concentration of 2.3 µM from day 2 to day 6 of treatment.. - (B) Dose-response curve of MCF7 spheroid volume under the effects of PTX and PTX-Cur/PLA-TPGS after day 7 of treatment.. - After 2 h, very little of the drugs stayed in cells of the outer layer of spheroids. - It means that after 24 hours, PTX-Cur/PLA-TPGS successfully absorbed. - These results also fit with our analysis of the ability of PTX-Cur/PLA-TPGS in attacking the second layer cells of the MCF7 spheroids.. - These results showed the potential of PTX-Cur/PLA-TPGS nanoparticles for applying in cancer theranostics. - The absorption of PTX-Cur/PLA-TPGS into MCF7 spheroids can be visualized by the autofluorescence of Curumin in the nanoparticle complex. - The upper number on the left of each image represents the depth of the layer from the top to the bottom of the spheroid (i.e. - the number is 0.0 µm means that it is the surface of the spheroid).. - (B) Fluorescence could not be detected in the control and PTX spheroids but presented in the PTX-Cur/PLA-TPGS one. - and PTX-Cur/PLA-TPGS in comparison with the control.. - We have successfully investigated and evaluated the bioactivity of (PTX combined Cur)- loaded PLA-TPGS nanoparticles. - Moreover, PTX-Cur/PLA-TPGS showed very high ability in inhibiting MCF7-spheroids growth or even preventing the regrowth of the spheroids following the time of treatment.. - 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