br administration barring the use of a
administration, barring the use of a delivery vehicle. Systemic circula-tion will be minimized due to the passively targeted nature of the NLB and the nano-size of the particles on account of the Enhanced Permeability and Retention (EPR) phenomenon, ultimately favorably altering the biodistribution of antineoplastic drugs.
Tumor targeting will enable the attainment of augmented levels of CPT at the tumor site by favorably altering biodistribution of the drug following intravenous administration, whilst preserving the state of healthy tissue. Hence, anti-tumor efficacy will be substantially in-creased while the side-effects associated with conventional che-motherapy will be dramatically reduced. Furthermore, the existence and concurrent treatment of co-morbidities will pose less of a challenge in the realm of cancer treatment due to site-specific release of che-motherapeutic drugs, which will minimize drug-disease and drug-drug interactions with co-existing morbidities and their treatment. Targeted therapy that displays increased mean residence time (MRT) in tumor tissue, will allow for reduced frequency and duration of therapy, which will provide psychological and physiological benefits for the patient.
2. Material and methods
dioctyl sulfosuccinate sodium salt (≥99% purity; Mw = 444.56), polysorbate 80sorbitan monooleate, silibinin (≥98% purity; Mw = 482.44), sulphur hexafluoride gas (Mw = 146.06), gelatin (˜Mw = 20000–25000), poly-L-lysine (PLL) (˜Mw = 30000–70000), polyethyleneimine (PEI) (50%w/v in H2O; Mw = 750,000), commercial grade Angeli’s Salt (Mw=˜200000), poly(acrylic acid) (PAA) (Mw = 1800), sodium alginate (Mw = 216), lactose (Mw = 360.32), fructose (Mw = 180.16), fluorescein isothiocyanate (FITC) (≥90% purity; Mw = 389.38), fetal bovine serum (FBS), cryoprotective medium (15%v/v DMSO) and sulforhodamine B (SRB) dye were procured from Sigma Chemical Company (St Louis, MO, USA). Chitosan (CHT) (food grade) was obtained from Wellable Group Marine Biological &Chemical Co., Ltd. (Shishi City, Fujian, China). Human epithelial ovarian cancer cell line (A2780), from the European Collection of Cell Cultures, RPMI-1640, fetal bovine serum (FBS), a penicillin-streptomycin antibiotic combination, L-glutamine and Trypsin-EDTA were purchased from Lonza Group Ltd (Basel, Switzerland). Trypan blue (0.4%v/v) was procured from BioRad Laboratories Inc. (Hercules, CA, USA). 7-aminoactinomycin (7-AAD), fluorescein isothiocyanate (FITC), R-phycoerythrin (PE2), peridininchlorophyll proteins (PerCP) and allophycocyanin (APC) stains were purchased from The Scientific Group (Randburg, Gauteng, South Africa). All other chemicals were of analytical grade and used as re-ceived.
2.2. Preparation of the nano-liposomes and nano-lipobubbles
2.2.1. Preparation of the CHO and DSPE nano-liposomes
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), dioctyl sulfo-
succinate sodium salt (DOS) and either cholesterol (CHO) or L-α-dis-tearoylphosphatidylethanolamine-methoxypolyethylene glycol con-jugate (DSPE) (concentrations as per Table 1) were simultaneously dissolved in a chlororform:methanol (9:1; 10 mL) solvent system under continuous stirring at 400 rpm for 5 min. Camptothecin (CPT) (0.05% w/v) was added to the organic solution under continuous agitation. The addition of silibinin (SB) to the nano-liposomes (NLS) preparations in-volved the addition of 30 mg SB dissolved in acetone (5 mL) during the emulsification process. Phosphate buffered saline (PBS) (pH 7.4, 25 °C; 10 mL) was subsequently added to the organic solution under ultra-sonication (amplitude = 80%; 90 s), over an ice-bath, employing a Colloids and Surfaces B: Biointerfaces 177 (2019) 160–168
Composition of the NLS systems.
Vibracell probe ultra-sonicator (Sonics & Materials Inc., Newtown, Connecticut, USA). The emulsion was subsequently subjected to eva-poration under vacuum (60–65 °C) for 2–3 hours, employing a Multi-vapor™ (Buchi Labortechnik AG, Switzerland). PBS (pH 7.4, 25 °C; 10 mL) was thereafter added periodically during the evaporation pro-cess and the formulation was subjected to ultra-sonication as previously outlined for 30 s after each addition. Complete evaporation of the sol-vent resulted in an aqueous NLS suspension. The resultant NLS sus-pension was subjected to three cycles of freezing at - 70 °C and thawing at 37 °C, to convert multilamellar NLS to unilamellar NLS with filtration through a 0.22 μm millipore filter after each freeze-thaw cycle. All ensuing modifications and analyses were conducted in triplicate (n = 3) on these unilamellar NLS.
2.2.2. Polymeric coating by layer-by-layer self-deposition
The prepared NLS suspension was added dropwise to a cationic chitosan solution (0.1%w/v) under constant agitation employing a magnetic stirrer. Coating was allowed for periods of 3–12 h under ambient condition, with zeta potential analysis employing a Zetasizer NanoZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK), un-dertaken in triplicate at 25 °C every 3 h, whilst the sample was main-tained at 37 °C in an orbital shaker bath (20 rpm), to determine suc-cessful polymer coating. The cationic NLS suspension was thereafter subsequently added drop-wise to an anionic polyacrylic acid solution (2%w/v) under constant stirring and adsorption of the polymer was allowed under ambient conditions for periods of 6–18 h, with periodic zeta potential analysis. Lactose (˜0.05%w/v), a lyoprotectant, was added to the polymer coated-NLS suspension and the suspension was frozen at -70 °C for 48 h, followed by lyophilization (Labconco, Kansas City, MO, USA). The lyophilized powder was resuspended in PBS (pH 7.4; 25 °C) to form polymer coated NLS.