Distinct Solubility and Cytotoxicity Regimes of Paclitaxel-Loaded Cationic Liposomes at Low and High Drug Content Revealed by Kinetic Phase Behavior and Cancer Cell Viability Studies

Abstract

Lipid-based particles are used worldwide in clinical trials as carriers of hydrophobic paclitaxel (PTXL) for cancer chemotherapy, albeit with little improvement over the standard-of-care. Improving efficacy requires an understanding of intramembrane interactions between PTXL and lipids to enhance PTXL solubilization and suppress PTXL phase separation into crystals. We studied the solubility of PTXL in cationic liposomes (CLs) composed of positively charged 2,3-dioleyloxypropyltrimethylammonium chloride (DOTAP) and neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) as a function of PTXL membrane content and its relation to efficacy. Time-dependent kinetic phase diagrams were generated from observations of PTXL crystal formation by differential-interference-contrast microscopy. Furthermore, a new Synchrotron small-angle x-ray scattering in situ methodology applied to DOTAP/DOPC/PTXL membranes condensed with DNA enabled us to detect the time-dependent depletion of PTXL from membranes by measurements of variations in the membrane interlayer and DNA interaxial spacings. Our results revealed three regimes with distinct time scales for PTXL membrane solubility: hours for > 3 mol% PTXL (low), days for ≈ 3 mol% PTXL (moderate), and ≥ 20 days for < 3 mol% PTXL (long-term). Cell viability experiments on human cancer cell lines using CL PTXL nanoparticles (NPs) in the distinct CL PTXL solubility regimes reveal an unexpected nonmonotonic dependence of efficacy on PTXL content in NPs delivered at short time scales (hours) after liposome hydration, where we see two distinct high-efficacy regimes at low (˂ 2 mol%) and high (9 mol%) drug loading. These newly identified high-efficacy regimes flank the membrane solubility . CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/154948 doi: bioRxiv preprint first posted online Jun. 23, 2017; 2 limit (≈ 3 mol%, where efficacy declines), which has been the focus of most previous physicochemical studies (and clinical trials) of PTXL-loaded CLs. At longer times scales (days), CL PTXL NPs with ≥ 3 mol% PTXL lose efficacy while formulations with 1-2 mol% PTXL maintain high efficacy. Our findings underscore the importance of understanding the relationship of the kinetic phase behavior and physicochemical properties of CL PTXL NPs to efficacy.

PEGylation of Paclitaxel-Loaded Cationic Liposomes Drives Steric Stabilization of Bicelles and Vesicles thereby Enhancing Delivery and Cytotoxicity to Human Cancer Cells

“…This experiment was carried out as previously described 34 . Briefly, cells were plated in clear 96-well plates at a density of 5000 cells/well.…”

“…CL ptx NPs with 0-14 mol% PEG2K-lipid were diluted in cell culture medium to a final PTX concentration of 60 nM (M21) and 50 nM (PC3) and added to cells (CL composition: 30 mol% DOTAP, 3 mol% PTX, 0-14 mol% PEG2K-lipid, and remaining mol% DOPC). Each cell line has a different dose-dependence on PTX, therefore they are treated with different PTX concentrations 34 . The resultant cell viability is reported in Figure 2a.…”

“…Moreover, doxorubicin has functional groups that allow it to be loaded via pH or ion gradient loading methods (which PTX lacks) to form reversibly soluble crystals within the liposomal aqueous pocket 33 . Instead, PTX incorporates within the hydrophobic layer of lipid membranes to variable extents depending on the lipid composition 24,[34][35][36] . PTX is typically soluble in lipid membranes on the order of one day or longer when loaded at or below 3 mol% of the total liposome formulation 24,34 .…”

“…Instead, PTX incorporates within the hydrophobic layer of lipid membranes to variable extents depending on the lipid composition 24,[34][35][36] . PTX is typically soluble in lipid membranes on the order of one day or longer when loaded at or below 3 mol% of the total liposome formulation 24,34 . Once PTX phase separates into stable insoluble crystals, the bioavailability of the drug drops significantly 24,34,[37][38][39] .…”

“…PTX is typically soluble in lipid membranes on the order of one day or longer when loaded at or below 3 mol% of the total liposome formulation 24,34 . Once PTX phase separates into stable insoluble crystals, the bioavailability of the drug drops significantly 24,34,[37][38][39] . Thus, it is critical to evaluate the duration of PTX solubility (an indicator of drug loading and retention) in new lipid formulations to ensure the drug remains soluble on relevant timescales for delivery 34 .…”