br Cellular uptake of nano lipobubbles
3.5. Cellular uptake of nano-lipobubbles
Formulated CHO- and DSPE-NLBs demonstrated a time-dependent cellular uptake, which was more pronounced in the CHO-NLBs. The distinct increase in fluorescence intensity evident in the fluorescence micrographs in the upper panel of Fig. 3 highlights the increase in CHO-NLB uptake by the A2780 V5 Epitope Tag Peptide 30 min post-treatment relative to that depicted 10 min post-treatment. Cellular uptake of the DSPE-NLBs was markedly higher and faster than observed for CHO-NLBs, which was attributed to the smaller size of DSPE-NLBs. Localization of both CHO-
Fig. 3. Qualitative evaluation of cellular uptake of SRB-labeled CHO- and DSPE-NLBs at 10 and 30 min post-treatment.
and DSPE-NLBs highlight the potential for released CPT to be trans-ported to the cell nucleus, which is the target for antineoplastic activity.
3.5.1. Assessment of cytotoxicity by flow cytometry
Scatter plots and histograms depicted in Fig. 4(I) and (II) provide graphical illustrations of the intensity of 7-AAD detected for each sample, which is directly related to the degree of cytotoxicity of the formulation under assessment. Evaluation of untreated cells provided an indication of natural cell death. As expected, the cell viability was > 96% 24 h post treatment and decreased over the next 2 days to 90.65% and 85.47%, respectively. Treatment of cells with PBS and DMSO + PBS did not demonstrate a significant increase in cell death. The high efficacy of CPT against A2780 tumor cells was readily high-lighted, with 71.20% cytotoxicity recorded 24 h post-treatment. How-ever, there was a significant increase in cell viability 48–72 hours post-treatment.
This increase in viability was attributed to the low stability of CPT and antineoplastic activity being restricted to cells in the S-phase of the cell cycle. CPT may have been readily internalized and had exerted high antineoplastic activity following initial treatment. However, rapid in-ternalization may have also caused rapid saturation of cellular inter-nalization mechanisms. CPT that was not initially internalized was converted to the inactive carboxylate form in the surrounding medium and was not able to exert any further antineoplastic effect. Surviving cells were able to recover and proliferate over the remaining analytical period leading to an increase in the ratio of viable cells: dead cells during analysis. On the contrary, SB displayed relatively low but in-creasing cytotoxic potential over the analysis period. SB is hypothesized to induce cell apoptosis; hence the effect is not restricted to a particular
phase of the cell cycle. The cytotoxicity observed with cells treated with SB was 3.91%–8.41% higher than the natural cell death observed with untreated cells over the 72-hour analytical period.
3.5.2. Assessing cellular status through continuous real-time evaluation Several controls were assessed in order to elucidate the potential of their effects on the overall cytotoxicity of the formulated nano-DDS’s. The response of A2780 ovarian cancer cells to test and control for-mulations are presented in Fig. 5A and B. The ionic environment of plain cell culture media remained constant throughout the analytical period and displayed no electrical impedance. Untreated cells were employed as an indication of unhindered cellular growth and pro-liferation within the confines of this study, limited only by the avail-ability of nutrients and space for attachment. The growth pattern for cells following treatment with PBS, the solvent for the formulated NLBs, was analogous to that observed following treatment with the PBS:DMSO solvent combination. The slower growth pattern was at-tributed to the reduced availability of nutrients for cell replication. Cells treated with placebo CHO-NLBs demonstrated a growth pattern ana-logous to that following treatment with PBS, thus confirming the non-toxicity of the materials employed in nano-NLB, as well as the nano-construct itself. However, treatment with placebo DSPE-NLBs resulted in a significantly lower cell index. Greater cellular internalization of the placebo DSPE-NLBs relative to the larger placebo CHO-NLBs is likely to have hindered cellular function to a slight degree, thereby reducing the proliferative capacity of the cells. However, this reduction in cell pro-liferation was not significant enough to have clinical relevance, parti-cularly in such a rapidly proliferating cell line.
The response of A2780 cells to treatment with native CPT has been
Fig. 4. (I) Histograms (A; C; E) and scatter plots (B; D) indicating the intensity of 7-AAD detected as a measure of the cytotoxicity of test controls, CPT and SB formulations. Histograms in the first column provide an indication of the natural cell death that occurred in untreated cells over the analytical period, with an inset of the forward versus side scatter plot for each day, depicting the cell characteristics on each day. (M1 = R2 = cell cytotoxicity). (II) Histograms (A; C; E) and scatter plots (B; D; F) indicating the intensity of 7-AAD detected as a measure of cytotoxicity induced by placebo, uncoated and the CHO- and DSPE-NLB. (M1 = R2 = cell cytotoxicity).