br Conversion of formulated nano liposomes
2.2.3. Conversion of formulated nano-liposomes to nano-lipobubbles
NLS was converted to NLB as described by du Toit et al. .
Briefly, NLS suspensions (10 mL) as prepared earlier were filtered and injected into 20 mL vials. Sulfur hexafluoride (SF6) gas was thereafter introduced into the headspace of the vials, which were subsequently sealed. Ultra-sonication of the vials was then undertaken in a bath type sonicator (Sonics & Materials Inc., Newtown, Connecticut, USA) for 3 min causing the SF6 gas to penetrate the lipid membrane of the NLS, forming a gaseous core, thereby creating NLBs . Furthermore, for morphological analysis, lyophilized powders of formulated NLS were reconstituted with PBS (pH 7.4; 25 °C) in the presence of FITC dye and subsequently converted to NLBs. The NLB suspension was allowed to dry onto a cover-slip covered with a slide for 1 h, followed by imaging employing an inverted immunofluorescence microscope (Olympus IX71, Olympus, Tokyo, Japan) after 100 mS exposure.
2.3. Morphological characterization of formulated nano-systems
The shape of formulated NLS and NLB was confirmed by Transmission Electron Microscopy (TEM). Briefly, formvar-coated copper grids were coated with the nano-liposomal suspension, em-ploying a micro-pipette and allowed to dry for approximately one hour. The grids were then inserted into the loading chamber of a Transmission Electron Microscope (TEM-100S, Jeol Ltd., Japan).
Photomicrographs were obtained at different magnifications to illus-trate the structure of the individual NLS.
2.4. Camptothecin and silibinin incorporation efficiency
The formulated NLS and NLB suspensions were double filtered through 0.22 μm filters to remove free drug. The filtrate was subse-quently sonicated (Amplitude = 80%, 5 min) and, thereafter, dissolved in DMSO (1:1) to prevent precipitation of CPT. Drug incorporation ef-ficiency was elucidated, in triplicate, by UV photospectroscopy (366 nm; ε = 51.03). The addition of SB to NLB preparations initially involved the addi-tion of 15–200 mg SB dissolved in acetone (5 mL) during the emulsifi-cation process. Quantification of SB incorporated into the NLB followed the principles of CPT quantification as outlined above, due to the poor aqueous solubility of SB. Following double 96187-53-0 through 0.22 μm filters to remove unincorporated SB, NLB suspensions were ultra-soni-cated to disrupt the structural integrity of the NLBs. DMSO was added to the suspensions, creating a 1:1 dilution, to solubilize the SB. SB in-corporation efficiency was determined in triplicate by UV photo-spectroscopy, against a constructed standard reference curve of SB in DMSO:PBS (pH 7.4; 37 °C) (1:1).
2.5. In vitro release analysis
Following removal of free drug from the NLS suspension, 5 mL samples were enclosed in treated dialysis tubing (Mw cut-off = 12,000 Da) and suspended in PBS (pH 7.4; 37 °C; 500 mL). The receptacle was maintained at 37 °C in an orbital shaker bath rotating at 20 rpm. At pre-determined intervals, 5 mL aliquots were removed from the external PBS phase and added to DMSO (5 mL), creating a 1:1 ratio, to prevent precipitation of the drug. Fresh buffer (5 mL) was replaced to the external phase to maintain sink conditions. Vortexed samples were analyzed by UV spectroscopy. This procedure was repeated on the formulated NLB system.
2.6.1. Cell culturing and expansion of the A2780 ovarian cancer cell line A2780 ovarian cancer cells were cultured in RPMI-1640 medium supplemented with FBS (10%v/v), L-glutamine (0.2%v/v) and a peni-cillin-streptomycin antibiotic combination (0.2%v/v). Cells were in-cubated at 37 °C in a humidified incubator with 95%O2 and 5%CO2 (Afrox, Johannesburg, Gauteng, South Africa) and monitored daily through microscopic observation. Cells were sub-cultured at 80–90% confluence, under sterile conditions with aseptic technique being ob-served throughout the culturing and analyses of the cells. Briefly, cell culture medium was removed from the culture flask with the cells thoroughly rinsed with phosphate buffered saline (PBS) (pH 7.4; 37 °C; 3–5 mL) to remove remnants of serum. Trypsin-EDTA (˜3 mL) was thereafter added to the cell culture flask with the cells incubated for 3 min in the humidified incubator to facilitate detachment of the cells. Fresh cell culture medium (˜3 mL) was then introduced into the cell culture flask to re-suspend the cells with the entire volume transferred to a sterile test tube. The suspension was subsequently centrifuged at 800 rpm for 3 min, the supernatant discarded, and the pellet of cells re-suspended in fresh cell culture medium. The cell suspension was then subsequently split into 3 cell culture flasks and allowed to attach and proliferate in the humidified incubator. Following adequate expansion of the cell line, cells were suspended in a combination of cell culture medium and 15%v/v DMSO cryoprotectant medium (1:1) and frozen at -70 °C until further analysis. This was undertaken as per the provided