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Effect of Methotrexate-Loaded Formulations on Real-Time ROS and Cell Viability Using Microfluidic Chip System

초록/요약

This study sought to examine the impact of methotrexate (MTX)-loaded nanoparticles (MTX-AONs) and pharmaceutical excipients on the survival of breast cancer cells (MCF-7) and human non-tumorigenic breast epithelial cells (MCF-10A) in real-time ROS sensing. The desolvation technique was used to form self- assembled albumin-oleic acid nanoparticles (AONs), giving particle size, 184±2 nm, PDI, 0.23±0.01, and zeta potential, -37.07±0.9. When MTX was loaded into AONs with a (93%) encapsulation efficiency and (9.3%) loading content, the particle size increased to 306.8±2 nm, but PDI (0.15±0.01) and zeta potential -30.3±0.6 had been almost unchanged. MTX solubility was measured alone or combined with 1% (w/v) pharmaceutical excipients (PEs) examples involve sodium dodecyl sulfate (SLS), D- α-tocopheryl polyethylene glycol 1000 succinate (D-α-TPGS), 2-hydroxypropyl-β- cyclodextrin (HP-β-CD), along with sodium oleate (SO) in pH 7.4 PBS. Sodium oleate gives us the highest solubility with 5.89±0.12 mg/mL, which is nearly ten times higher than the free MTX, which was 0.58±0.04 mg/mL, because it’s a fatty acid salt that enhances solubility via hydrophobic interactions and micelle formation. Under dynamic conditions, the cellular viability of MTX-AON in MCF-7 cell lines increased as compared to static conditions. Furthermore, the cellular viability of MTX-AONs was greatly decreased. When SLS and D-α-TPGS were combined with MTX-AONs, the cellular viability was the lowest in both static and dynamic environments in MCF-7 cells, while maintaining cellular viability in MCF-10A cells lines. As the drug concentration increased, the cell viability decreased accordingly while increasing real-time ROS production, giving a good correlation. The microfluidic chip system can be utilized to investigate the cellular viability along with real-time ROS recognizing of diverse dosage forms for the prediction of clinical efficacy.

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목차

1. Introduction 1
2. Materials and Methods 4
2.1. Materials 4
2.2. Preparation of AONs, MTX-AONs, MTX-AON-PE, and MTX-PE-AONs. 5
2.2.1. Synthesis of HSA-OA conjugates (AOCs) 5
2.2.2. Conversion of AOCs to AONs Via a Desolvation Technique 6
2.2.3. Encapsulation of MTX into AONs (MTX-AONs) 6
2.2.4. Preparations of MTX-AON-PE 7
2.2.5. Encapsulation of MTX-PE into AONs (MTX-PE-AONs) 7
2.2.6. Encapsulation efficiency (EE) and drug loading content (LC) 8
2.3. Physicochemical characterizations of AOCs, AONs, MTX-AONs, MTX-AON-PE, and MTX-PE-AONs 9
2.3.1. Fourier Transform-Infrared (FT-IR) Spectrometer 9
2.3.2. Matrix-Assisted Laser Desorption Ionization - Time of Flight Mass Spectrometry (MALDI-TOF MS) 10
2.3.3. Dynamic light scattering (DLS) measurement 11
2.3.4. Field emission scanning electron microscopy (FE-SEM) along with field emission transmission electron microscopy (FE-TEM) analysis 11
2.4. Phase Solubility Studies 12
2.5. Cell culture and maintenance 13
2.6. Establishment and calibration of microfluidic monolayer chip system 13
2.6.1. Calibration of monolayer chip system 13
2.6.2. Cell culturing on microfluidic chip system 19
2.6.3. Cell viability assay 23
2.6.4. Real-time ROS generation detection by sensor 24
2.6.5. Cellular images using confocal laser scanning microscopy 25
2.6.6. Statistical Analysis of IC50 and LD50 26
3. Results and discussions 27
3.1. Characterizations of AOCs, AONs, MTX-AONs, MTX-AON-PE, and MTX-PE-AONs. 27
3.1.1. Evaluation of AOC 27
3.1.2. Physicochemical characteristics of AONs, and each formulation 31
3.2. Phase Solubility Studies 37
3.3. Efficacy of Cells Based on Drug Formulations and Shear Stress 40
3.4. Real-Time ROS Sensing of Methotrexate Drug. 49
3.5. Association between Real-Time ROS and Cellular Viability. 52
4. Conclusions 54
5. References 55

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