br Fig Ultraviolet visible calibration curve
Fig. 2. Ultraviolet-visible calibration curve of Doxorubicin (DOX) and Sildenafil Citrate (SC).
cytotoxicity in comparison to [SC + DOX] and [SC + DOX co-loaded NLC] respectively (Fig. 3c–e). These differences are statistically sig-nificant (p < 0.05).
Cellular uptake studies by flow cytometry and fluorescence micro-scopy were performed to evaluate the ability of NLC and NLC-RGD in enhancing accumulation of DOX into the A549 lung cancer cells. As shown in Fig. 4a, SC causes more accumulation of DOX into cell during 3 h incubation, suggesting that SC may decrease efflux of drug by in-hibiting drug extrusion pumps. Results also showed that the cellular uptake of [DOX + SC]-coloaded NLC-RGD was significantly higher than those of [DOX + SC]-coloaded NLC in cancerous CAY 10566 (Fig. 3a), sug-gesting that NPs were absorbed into the tumor cells more efficiently using RGD mediated endocytosis. This results confirmed by fluores-cence microscopy imaging which shows that cells treated with NLC-RGD display more strength red fluorescence in their cytoplasm, in comparison with cells treated with NLC and free DOX (Fig. 4b).
In order to compare the strength of apoptotic effect of DOX, DOX + SC and [DOX + SC]-coloaded NPs, flow cytometry analysis of Annexin V/PI double staining was performed. In normal viable cells phosphatidyl serine (PS) is located on the cytoplasmic surface of the cell membrane. Upon induction of apoptosis, rapid alterations in the
organization of phospholipids occurs leading to exposure of PS on the cell surface. In vitro detection of externalized PS can be achieved through interaction with the anticoagulant annexin V. In the presence of calcium, rapid high affinity binding of annexin V to PS occurs. Propidium iodide is used to distinguish between viable, early apoptotic, and necrotic or late apoptotic cells. Propidium iodide will be excluded from viable (FITC negative) and early apoptotic (FITC positive) cells. Results of flow cytometry are displayed in Fig. 5a. Viable cells are shown in bottom left quadrant, early stage apoptotic cells bind pri-marily to Annexin V are shown bottom right quadrant, the late apop-totic cells binds to both of Annexin V and PI, are shown in top left quadrant and necrotic cells are shown in top left quadrant. As seen in Fig. 5a. more than 91% of cells in control group are viable. Treating cells with DOX, DOX + SC, [DOX + SC]-coloaded NLC and [DOX + SC]-coloaded NLC-RGD induced apoptosis in 29.87%, 34.69%, 38.37% and 44.32% of cells respectively. These results confirmed higher programmed cell death by combination therapy with DOX and SC compared to pure DOX. Furthermore, obtained result concludes that co-delivery with NLC-RGD is highly competent compared to NLC. In other word, co-treatment of cells with DOX and SC intensify apoptotic effects of DOX, suggesting Inhibition of ABC transporters. Co-delivery of DOX and SC by NLC-RGD may enhances drugs uptake through in-tegrin mediated endocytosis which lead to more apoptotic effects of DOX in compared with free DOX and SC.
Fig. 3. Cell viability of non-small cell lung cancer (A549) after 48 h incubation with (a) SC and SC loaded nanoparticles, (b) Different concentration of doxorubicin,
(c) 1 μM DOX + different concentration of SC, and (d) combination of 1.26 μM doxorubicin and 15 μM SC in free form and in the form of loaded NLC and NLC-RGD. e) phase-contrast image of A549 cells when treated with DOX and SC in free form and in the form of loaded NPs. The figure illustrates that [DOX + SC]-coloaded NLC-RGD are more anti-proliferative when compared with free form of DOX + SC (p < 0.05). Data is presented as mean ± standard deviation (n = 3), * Showing Significant Differences. (DOX, Doxorubicin; SC, Sildenafil Citrate; NLC-RGD, Arginyl-glycyl-aspartic acid containing nanostructured lipid carriers; NPs, Nanoparticles).
Fig. 4. Doxorubicin uptake into cells. DOX fluorescence intensity graphs (a) and fluores-cence microscope images (b) when cells were treated with DOX and SC in the free from and in the form of loaded NLC and NLC-RGD. (DOX, Doxorubicin; SC, Sildenafil Citrate; NLC-RGD, Arginyl-glycyl-aspartic acid containing nanostructured lipid carriers).
Fig. 5. The apoptosis induction flow cytometry graphs (a) and fluorescent images of DAPI stained cancerous cells following a 48 h treatment of cells with DOX and SC in the free from and in the form of loaded into NLC and NLC-RGD. (DOX, Doxorubicin; SC, Sildenafil Citrate; NLC-RGD, Arginyl-glycyl-aspartic acid containing nanostructured lipid carriers).
In order to compare the influence of DOX, DOX + SC and [DOX + SC]-coloaded NPs on morphologic feature of the nucleus, as a simple factor for the determination of healthy and apoptotic cells, DAPI staining was performed. Based on the obtained results, the frequency of nucleus condensation and chromatin fragmentation in DOX + SC treated cells was more than free DOX, suggesting synergistic effect of SC and DOX in inducing apoptosis (Fig. 5b). Furthermore, the cells treated with [DOX + SC]-coloaded NLC-RGD showed nucleus condensation or chromatin degradation more noticeably than the cells grown in the presence of [DOX + SC]-coloaded NLC which suggest more absorbance of [DOX + SC]-coloaded NLC-RGD in compared with [DOX + SC]-coloaded NLC (Fig. 5b).