The viability of cells following treatment with each prepara
The viability of Cucurbitacin I following treatment with each preparation was assessed by flow cytometry 24, 48 and 72 h post-treatment. The excess cell culture medium containing detached, no-viable cells was removed and added to sterile test tubes. Cells were rinsed with PBS (pH 7.4; 37 °C) and trypsinized according to standard procedures as outlined. The cell suspensions were added to the respective sterile test tubes containing the previously removed non-viable cells. The total volumes were centrifuged at 800 rpm for 3 min and the supernatant discarded. The cell sediments were resuspended in 100 μL cell culture medium each and transferred to round-bottom polystyrene test tubes. The 7-AAD stain (5 μL) was added to each test sample and incubated at room temperature in darkness for 10 min, in accordance with the manu-facturer’s guidelines. PBS (pH 7.4; 37 °C; 500 μL) was subsequently added to each test sample.
Prior to cell analysis, quality control assessment was undertaken on the flow cytometer (FACS Calibur, BD Biosciences, Tullastrasse, Heidelberg, Germany). Unstained cell counting beads as well as 4 stains, namely FITC, PE, PerCP and APC, were employed to ensure the quality of fluorescence detection by each of the 4 fluorescence detec-tors. The FL3 detector was of particular relevance in this study as the 7-AAD dye employed during analysis is detected by the FL3 detector. Following the successful outcome of the quality control test, the test parameters were determined. Evaluation of the forward scatter and side scatter plots of completely viable and completely non-viable cells en-abled accurate distinction of the voltage threshold and region of in-terest. Test samples were subsequently analyzed at low and medium flow rates with buffer rinses between each sample to avoid results re-flecting the effects of residue from previous samples. No less than 10,000 cells were analyzed in each sample. Cell viability was analyzed employing BD CellQuest™ Pro version 5.1 software (BD Biosciences, Tullastrasse, Heidelberg, Germany).
2.6.6. Real-time evaluation of formulation cytotoxicity
Cell growth and cytotoxicity of the NLB-DDS and relevant controls were determined in real-time as a measure of electrical impedance employing the xCELLigence™ RTCA (Roche Applied Science, Penzberg, Upper Bavaria, Germany and ACEA Biosciences Inc., San Diego, CA, USA). A background scan of the cell culture medium (100 μL/well) was performed prior to each analysis. A cell titration was initially performed in triplicate with cell densities ranging from 1250 to 10,000 cells/well. The growth pattern of cells was observed over a 100-hour period and a density of 10,000 cells/well was deemed suitable for analysis. This further identified the most suitable time period (22–34 hours post-seeding) to initiate drug treatment. All subsequent drug treatment was undertaken 24 h post seeding.
A titration of CPT was subsequently undertaken to establish sensi-tivity of the cells to CPT and to determine an effective concentration of CPT for the achievement of adequate cytotoxic activity. A saturated solution of CPT in DMSO (10 mg/mL) was prepared with subsequent serial dilutions resulting in a concentration range of 2–10 mg/mL. The CPT solutions were diluted 25-fold PBS (pH 7.4; 37 °C) and then 2-fold in cell culture medium due to the toxicity of concentrated DMSO to cells, resulting in final CPT solutions of 0.04- 0.20 mg/mL. Cells were seeded at 10,000 cells/well and treated 24 h later with serial dilutions of CPT and the response pattern of cells to the range of CPT con-centrations was analyzed for 36 h post-treatment. The final concentra-tion of CPT determined to display the most appropriate response by the cells for the purposes of this study (0.20 mg/mL following dilution in cell culture medium), coincided with the concentration employed for analysis by flow cytometry. Hence, all test and control samples were prepared in accordance with the description as above. Colloids and Surfaces B: Biointerfaces 177 (2019) 160–168
Experimentally determined average size, zeta potential and PDI of candidate CHO- and DSPE-NLS and NLB.
Formulation Average Size % Deviation Zeta Potential Average PDI
2.6.7. Hemocompatibility and stability studies
Hemocompatibility and stability analyses were undertaken on the NLB system as described by Shen and co-workers, Elmowafy and co-workers and Sharma and co-workers and are presented in the supple-mentary data [13–15].
3. Results and discussion
3.1. Size and surface charge characterization
Zeta potential analysis detailed an unfavorable decrease in surface charge following conversion of the CHO-NLS to CHO-NLBs which may be attributed to slight destabilization of the lipid membrane during the conversion process (Table 2). However, the zeta potential of formulated DSPE-NLB remained highly favorable, designating a stable formulation that is not inclined to aggregation. The size characteristics of the DSPE-NLBs greatly favors passive targeting of the DDS to tumor tissue by the Enhanced Permeability and Retention (EPR) effect and, hence, may improve the safety and efficacy of CPT delivered by this DDS. Fur-thermore, conversion of the DSPE-NLSs to DSPE-NLBs resulted in a marginal decrease in surface charge, analogous to that observed for CHO-NLBs.