3.2.7.1 In-Vitro Cytotoxicity (MTT Assay)
To evaluate cytotoxic behaviour of drug delivery system, cell viability assay was performed with MTT assay. Initially blank carriers were evaluated for their biocompatibility and inertness. The same procedure was adopted as used for drug loaded carriers. It was observed that both MSNPs and Lip-MSNPs were non-cytotoxic in nature as shown in figure 24. Here we have used just 4 hr incubation time with MSNPs and Lip-Dox-MSNPs. After evaluation of inertness of blank carriers, the study was extended to drug loaded carriers for longer incubation periods.
Results & Discussion ___________________________________________________________________________
73
Figure 24: Cell viability of blank MSNPs and Lip-MSNPs
The findings of cell viability assay have shown that after 4 hrs of incubation, Dox-MSNPs resulted in more toxicity to the cells as compared to Lip-Dox-MSNPs. But after 24 hrs, 48 hrs and 72 hrs of incubation the results were inverted and Lip-MSNPs have shown increased toxicity compared to Dox-MSNPs carriers as can be seen in Figure 25. The higher toxicity of Dox-Dox-MSNPs after 4 hrs incubation might be due to the relatively faster release of Dox from MSNPs as there was no lipid coating. On the other hand, even after higher cellular uptake of Lip-Dox-MSNPs due to lipid coating, lesser toxicity was observed due to the sustained release effect of lipid layer at the pore opening which hindered the release of drug. After 24 hrs of incubation the cytotoxicity by Lip-Dox-MSNPs was increased as compared to Dox-MSNPs and these effects are due to higher amount of drug uptake by the cells due to biocompatibility of lipid layer. Once the higher amount of carrier was available in the cells it required some time to release higher concentration of Dox as compared to Dox-MSNPs to cause more toxicity. Similar pattern of toxicity was
Chapter 2 - Cell Culture Experiments
______________________________________________________________
74
observed after 48 hrs and 72 hrs of incubation. Our cytotoxic studies have shown toxicity, which was not only concentration but time dependent as well.
Figure 25: In-vitro cytotoxicity evaluation by MTT assay with SKBR-3 breast cancer cells after (A) 4 hrs, (B) 24 hrs, (C) 48 hrs and (D) 72 hrs incubation of Dox-MSNPs, Lip-Dox-MSNPs and Dox. Data is presented as mean ± SD (n=3)
3.2.7.2 Pathway Uptake Analysis
The results of cellular uptake pathway studies are given in figure 26 which indicates that Dox-MSNPs with filipin III has shown no difference in cell viability but with chlorpromazine significant effects were observed. So we can say that Dox-MSNPs were internalized by clathrin mediated endocytosis and not by caveolea/lipid-raft mediated. In comparison, Lip-Dox-MSNPs with chlorpromazine and filipin III have shown higher cell viability, indicating involvement of both clathrin mediated and caveolea/lipid-raft mediated endocytosis. Effects of pure Dox suggest the adaptation of some other
Results & Discussion ___________________________________________________________________________
75
mechanisms instead of both above mentioned pathways. These findings indicate that the internalization of Lip-Dox-MSNPs utilizes not only clathrin mediated but caveolea endocytosis pathway which is advantageous to Dox-MSNPs clathrin mediated endocytosis.
Figure 26: Cellular uptake pathway analysis: where MTT assay of SKBR-3 cells was performed after 30min incubation of two different pathway inhinitors including chlorpromazine (6 µM) and filipin III (3 µM) with Dox-MSNPs, Lip-Dox-MSNPs and Dox.
3.2.7.3 Cellular Uptake Studies
Dox, a fluorescent moiety, interacts with DNA and tends to be internalized into the nucleus. To confirm the localization of Dox in nucleus of the cells, qualitative fluorescence imaging with CLSM was performed.122 Cells were incubated with Dox-MSNPs and Lip-Dox-MSNPs containing 12.5 µg/ml of Dox for 4 hrs and after staining nucleus with DAPI, cells were fixed to glass coverslip and observed under CLSM. The microscopic images are shown in figure 27 where the localization of Dox was observed in the nuclear region along with DAPI. As postulated, stronger red intensity indicates the higher internalization of the Dox by Lip-Dox-MSNPs as compared to Dox-MSNPs. It is obvious that the higher internalization of Lip-Dox-MSNPs was due to more biocompatible protocell nature of lipid layer which enhanced cellular uptake. Although the Dox intensity of Dox-MSNPs is also indicating the delivery of drug to target site but as our
Chapter 2 - Cell Culture Experiments
______________________________________________________________
76
aim was to deliver the drug in higher concentrations, so Lip-Dox-MSPs were able to accomplish it. Apart from localization of Dox in the nuclear region for Lip-Dox-MSNPs, we can observe the higher intensity outside the nucleus as well. This might be due to the availability of the drug which was not completely released due to lipid layer and may require some time to get released and internalized in nucleus. These findings are supportive to our MTT assay results where after 4 hrs of incubation Dox-MSNPs have shown bit higher toxicity as compared to Lip-Dox-MSNPs and this higher toxicity would be due to relative faster release in the absence of lipid layer. Although the lipid layer has increased the cellular uptake but released the drug in a sustained manner which is also evident from 24 hrs, 48 hrs and 72 hrs toxicity results where Lip-Dox-MSNPs have shown significantly higher toxic effects to the cells.
Figure 27: CLSM images after uptake of Dox-MSNPs and Lip-Dox-MSNPs by SKBR-3 cells where blue and red fluorescence in the nuclear region correspond to DAPI and Dox along with merged images. Higher cellular uptake of Lip-Dox-MSNPs can be visualized by higher fluorescence of Dox
DAPI Dox Merged
Do x- MSNP s Lip -Do x- M SNP s
Results & Discussion ___________________________________________________________________________
77