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 incorporation and 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 CLPTXL nanoparticles (NPs) in the distinct CLPTXL solubility regimes reveal an unexpected dependence of efficacy on PTXL content in NPs. Remarkably, formulations with lower PTXL content and thus higher stability show higher efficacy than those formulated at the membrane solubility limit of ≈ 3 mol% PTXL (which has been the focus of most previous physicochemical studies and clinical trials of PTXL-loaded CLs). Furthermore, an additional high-efficacy regime is seen on occasion for liposome compositions with PTXL ≥ 9 mol% applied to cells at short time scales (hours) after formation. At longer time scales (days), CLPTXL 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 CLPTXL NPs to efficacy.

Dynamically Monitoring Cell Viability in a Dual‐Color Mode: Construction of an Aggregation/Monomer‐Based Probe Capable of Reversible Mitochondria‐Nucleus Migration

“…Monitoring cell viability is a highly valuable task essential for the fundamental researches in biology, pathology, and medicine . For instance, detecting the cell viability is necessary to evaluate the efficacy of drugs and the cytotoxicity of biological reagents, including antibiotics, drugs, nanoparticles, and fluorophores . Meanwhile, changing of cell viability such as apoptosis and necrosis process under physiological conditions is indispensable to maintain the homeostasis of cell population, and to defend organisms in immune reactions .…”

Organic Nanocarriers for Delivery and Targeting of Therapeutic Agents for Cancer Treatment

“…EndoTAG‐1 (ET) is a cationic liposome, which prepared by using 2,3‐dioleyloxypropyltrimethylammonium chloride (DOTAP), DOPC, and PTX with a 50:47:3 molar ratio. ET entered Phase III clinical trial (NCT03002103) in Taiwan to determine the efficacy and safety of ET in metastatic triple negative breast cancer and study is in progress . Wu et al.…”

Enhanced Loading of Paclitaxel in Cationic Liposomes by Replacement of Oleoyl with Linoleoyl Lipid Tails with Benefits in Cancer Therapeutics fromIn VitroStudies

“…47 Instead, hydrophobic drugs such as PTX are solubilized by and incorporated into the nonpolar (hydrocarbon chain) bilayer membrane of lipid-based carriers (red spheres in Figure 1C). 16,37,48–50…”

“…Once PTX has phase-separated into stable, water-insoluble crystals, the drug loses efficacy. 37,48,84–86 Thus, it is crucial that PTX remains soluble in the membrane on timescales relevant for delivery. Relatively few studies have investigated the PTX solubility limit in different types of membranes, and not many common themes have emerged.…”

“…Such vectors require less lipid to deliver a given amount of PTX, reducing costs and side effects. Carriers with high solubility have also shown increased efficacy, 48 allowing administration of lower total doses of PTX. We obtained promising initial results of increased PTX membrane solubility using CLs prepared from DOTAP (oleoyl (C18:1) tails; see Figure 2) and DLinPC (linoleoyl (C18:2) tails; see Figure 2) (at a molar ratio of DOTAP/DLinPC/PTX=30/70–x/x).…”