Effect of nano formulation and shear stress on the cellular behavior and real- time ROS sensing in a liver-bilayer microfluidic system
- 주제(키워드) Bilayer liver-on-chip , HepG2 cell , real-time ROS sensing , nanoformulation , shear stress , doxorubicin , cell viability , IC50 value , predictive LD50
- 주제(DDC) 615.1
- 발행기관 아주대학교 일반대학원
- 지도교수 Beom-Jin Lee
- 발행년도 2024
- 학위수여년월 2024. 2
- 학위명 석사
- 학과 및 전공 일반대학원 약학과
- 실제URI http://www.dcollection.net/handler/ajou/000000033504
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
This study investigated the effects of drug formulation and shear stress on cellular uptake, viability, and real-time reactive oxygen species (ROS) sensing using a microfluidic bilayer liver-on-chip (BLC) system. To improve the biomimetics of the liver chip model and form vascular structures, we designed a bilayer model in which human umbilical vein endothelial cells and epithelial hepatocarcinoma cells (HepG2) were co-cultured through a porous membrane. Human serum albumin (HSA)-oleic acid nanoparticles (AONs) were synthesized using the desolvation method, and doxorubicin hydrochloride (DOX-HCl) was efficiently loaded into the nanoparticles (NPs) via an incubation process involving electrostatic interactions. Because of continuous blood flow in the human body, differences in cellular uptake and ROS generation of NPs occur between dynamic and static environments, affecting delivery efficiency. Therefore, cellular internalization and cell viability based on the drug formulation were evaluated under both static and dynamic conditions. Under dynamic conditions, we observed a notable enhancement in the cellular internalization of DOX-AONs compared to that under static conditions, leading to improved efficiency in killing cancerous cells. Simultaneously, real-time ROS production was detected using a sensor, and it was confirmed that with an increase in the drug concentration, cell viability decreased, and ROS production increased. It was concluded that there was a correlation between ROS production and cell survival rate. Additionally, to verify in vivo correlation of the liver chip data, IC50 values were used to predict LD50 values according to the regression formula in ICCVAM (log LD50 (mg/kg) = 0.7092 log IC50 (μg/mL) + 0.4005). This value was similar to LD50 of DOX in a mouse model, indicating that the model can be used as an alternative to animal testing. Keywords: bilayer liver-on-chip, HepG2 cell, real-time ROS sensing, nanoformulation, shear stress, doxorubicin, cell viability, IC50 value, predictive
more목차
1. Introduction 1
2. Materials and Methods 4
2.1. Materials 4
2.2. Synthesis of OA-HSA conjugates (AOCs) 4
2.3. Preparations of AONs and DOX-AONs 5
2.3.1. Self-assembled AONs using desolvation method 5
2.3.2. Encapsulation of DOX into AONs (DOX-AONs) 6
2.3.3. Encapsulation efficiency (EE) and drug loading content (LC) 7
2.4. Physicochemical characterizations of AOCs, AONs, and DOX-AONs 7
2.4.1. Fourier transform-infrared (FT-IR) spectroscopy 7
2.4.2. Matrix-Assisted Laser Desorption Ionization - Time of Flight Mass Spectrometry (MALDI-TOF MS) 8
2.4.3. Dynamic light scattering (DLS) measurement 8
2.4.4. Field emission scanning electron microscopy (FE-SEM) and field emission transmission electron microscopy (FE-TEM) analysis 9
2.5. Cell culture and maintenance 9
2.6. Establishment and calibration of microfluidic BLC system 10
2.6.1. Calibration of BLC system 10
2.6.2. Establishment of microfluidic BLC system 14
2.6.3. Cell culturing on microfluidic BLC 18
2.6.4. Cell viability assay 19
2.6.5. Real-time ROS generation detection by sensor 19
2.6.6. Cellular images using confocal laser scanning microscopy 20
2.6.7. Estimation of IC50 and LD50 21
3. Results and discussions 22
3.1. Characterization of AOCs 22
3.2. Physicochemical characteristics of blank AONs and DOX-AONs 26
3.3. Comparison of the effectiveness of DOX-AONs under dynamic and static conditions 31
3.4. Effect of shear stress on cellular uptake 40
4. Conclusions 42
5. References 43
국문초록 47
