br previously expounded The presence of
previously expounded. The presence of native CPT may have led to saturation of cellular internalization of CPT, leading to rapid killing of cells in the S-phase of the cell cycle. However, the short duration of the cytotoxic stress presented by CPT had a marginal effect on cells in other phases of the cell cycle, resulting in expeditious recovery of the cells and subsequent proliferation. The cytotoxic activity of SB was notably less intense and of a shorter duration to that observed for CPT. SB does not work on a particular phase of the AUY 922 and thus will have an indiscriminate cytotoxic effect on all cells. The initial decrease in cell index was attributed to this indirect effect. The solubility constraints of SB and the high dilution required due to the DMSO solvent resulted in a
very low final concentration of SB for cell treatment and low cytotoxic activity. However, the analysis established a distinct sensitivity of A2780 cells to SB. Recovery and proliferation of cells was of a fairly swift nature with marginal differences in cell index relative to that of cells treated with PBS:DMSO from approximately 18 h post-treatment.
Treatment of cells with the CHO- and DSPE-NLBs proved sub-stantially favorable. The slower release pattern of both CPT and SB following polymer coating translated into augmented and sustained cytotoxic activity of both formulations. The achievement of superior cytotoxicity with DSPE-NLBs was associated with enhanced cellular internalization due to the smaller size of this formulation. The
Fig. 5. The growth and response of A2780 cells to various control preparations establishing the cytotoxic potential of each preparation (in all cases n = 3 and SD < 0.02).
enhanced cytotoxicity may also be attributed to a higher concentration of SB within the formulations relative to that of native SB. The cell index curve of the DSPE-NLBs treated cells displayed a steeper gradient over the initial 16 h post-treatment, with a marginal decrease in cyto-toxicity thereafter. The cell index profile of cells treated with CHO-NLBs highlighted a fairly constant decrease in cell index, indicative of sus-tained cytotoxic activity. The slower release of CPT and SB from the CHO- and DSPE-NLB formulations prevented saturation of cell uptake of the compounds at an early stage resulting in constant exposure to the antineoplastic compounds. Moreover, prolonged release of CPT in particular allowed for more cells to be exposed to CPT whilst in the S-phase of the cell cycle, where CPT acts.
3.5.3. Hemocompatibility and stability analyses
Hemocompatibility and stability studies were conducted on the NLB
formulations (presented in supplementary data). The NLBs were found to exhibit concentration-dependent hemolysis. CHO-NLBs displayed an increase in hemolysis from 4.63% to 6.87% with an increase in con-centration from 10 mg/mL to 20 mg/mL (Fig. S1). A similar increase in concentration of DSPE-NLBs resulted in a concomitant increase in he-molysis from 5.36% to 7.03%. Stability studies (Figs. S2 and S3) in-dicated favorable formulation stability both after reconstitution as well as after long-term (3 months) storage. Interestingly, the storage stability of CHO-NLBs with respect to SB incorporation was slightly less favor-able than observed with DSPE-NLBs.
The undertaking of both in vitro and ex vivo analyses was essential in the systematic characterization of formulated CHO- and DSPE-NLBs, and the potential of these nano-DDS to enhance the cytotoxic activity of CPT and SB. The cell cytotoxicity evaluation of control formulations highlighted the inactivity of materials employed in the formulation of the NLBs, the structure of the nano-system in the absence of anti-neoplastic compounds and the solvents employed in the assays. Cells displayed a marginally slower growth rate when treated with control formulations due to reduced availability of nutrients to meet the high demand of rapidly proliferating cells. Additionally, the formulated polymer-coated CHO- and DSPE-NLBs remained below the benchmark size initially outlined, displayed satisfactory incorporation of both CPT and SB and successfully enhanced the drug release characteristics of CPT and SB to facilitate more effective passive targeting and drug ef-ficacy.
The author reports no conflicts of interest in this work.
This work was supported by the National Research Foundation (NRF) of South Africa.
Appendix A. Supplementary data
 L.C. du Toit, T. Govender, V. Pillay, Y.E. Choonara, T. Kodama, Investigating the effect of polymeric approaches on circulation time and physical properties of