Impact of methotrexate-loaded fattigated albumin nanoparticles and pharmaceutical excipients on real-time reactive oxygen species and cell viability in a microfluidic chip system

Abstract

This study examined the impact of methotrexate (MTX)-loaded albumin-oleic acid nanoparticles (MTX-AONs) and pharmaceutical excipients (PEs) on cell viability and real-time reactive oxygen species (ROS) sensing in breast cancer cells (MCF-7) and human non-tumorigenic breast epithelial cells (MCF-10A). Water-soluble PEs, such as sodium dodecyl sulfate (SLS), D-α-tocopheryl polyethylene glycol 1000 succinate (D-α-TPGS), 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), and sodium oleate (SO), were also screened for their ability to improve MTX solubility and influence cellular responses. The self-assembled albumin-oleic acid nanoparticles (AONs) prepared by desolvation, exhibited a mean particle size of 184 ± 2 nm, polydispersity index (PDI) of 0.23 ± 0.01, and zeta potential of -37.07 ± 0.9. MTX loading yielded an encapsulation efficiency of 93 % and loading of 9.3 %, increasing particle size greatly to 306.8 ± 2 nm with minimal changes in PDI (0.15 ± 0.01) and zeta potential (-30.3 ± 0.6). The PEs (1 %, w/w) influenced aqueous MTX solubility (mg/mL), giving in the order: SO (5.89 ± 0.12), SLS (1.52 ± 0.11), HP-β-CD (0.97 ± 0.03) and D-α-TPGS (0.64 ± 0.03) compared with free MTX (0.58 ± 0.04 mg/mL). Under dynamic shear stress, MTX-AONs exhibited enhanced anticancer activity compared with static conditions. Furthermore, MCF-7 cell viability was decreased in a dose-dependent manner, while MCF-10A cells were spared, suggesting better cellular uptake than that of free MTX. Co-treatment with PEs decreases MCF-7 cell viability; however, their effectiveness is not affected by MTX solubility. For example, SLS and D-α-TPGS combined with MTX-AONs showed the strongest effects on the cellular viability of MCF-7 cells in both static and dynamic environments, while maintaining MCF-10A cells. As the drug concentration increased, cell viability decreased accordingly, whereas real-time ROS production increased, indicating a good correlation. Interestingly, the expected lethal dose (LD₅₀) calculated from the half-maximal inhibitory concentration (IC₅₀) correlated with the reported LD₅₀ of MTX. The microfluidic chip system can be utilized to screen the impact of formulation design and PEs in cancer therapy by simultaneously measuring cellular viability and real-time ROS levels to predict clinical relevance.

Keywords: Albumin-oleic acid nanoparticles; Cellular viability; Expected lethal dose; Methotrexate; Microfluidic chip system; Pharmaceutical excipient; Real-time ROS sensor.