생체모사시스템에서의 나노입자의 분포 및 세포 내 약물 전달
Biodistribution and cellular behavior of nanoparticles in a biomimetic microfluidic system
- 주제(키워드) Biomimetic microfluidic system , Fluidic shear stress , Cell dynamic environment , Cellular drug delivery
- 발행기관 아주대학교
- 지도교수 이범진
- 발행년도 2016
- 학위수여년월 2016. 2
- 학위명 박사
- 학과 및 전공 일반대학원 약학
- 실제URI http://www.dcollection.net/handler/ajou/000000021915
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Different nanoparticle properties, such as size, shape, material and surface coating, as well as the cell type, interaction with other cells and the cellular environment, influence nanoparticle uptake and the cellular behavior. Additionally, cells are affected by fluid shear stress generated by interstitial flows in the cellular microenvironment. Therefore, it requires an understanding of the interactions between nanoparticles and cells within a physiological cell environment for the cellular uptake of drug and drug delivery systems. In this study, effects of shear stress on the intracellular uptake of nanoparticles were investigated using a calibrated biomimetic microfluidic system (BMS) that mimics the dynamic environment of cells. Positively or negatively charged polystyrene nanoparticles (PSNs) were chosen as a model. PSNs were delivered to normal cell lines (HEK 293T and MS1) and cancer cell lines (Panc-1, A549, and HT29) under static and biomimetic dynamic conditions (0.5 dyne/cm2) using the BMS. Endocytosis experiment was done in the presence of endocytosis inhibitors specific for one of the endocytosis pathways. Furthermore, to evaluate how the shear stress in dynamic environment affects efficacy of drugs to cells using biomimetic microfluidic system (BMS), we prepared gelatin-oleic nanoparticles (GON) from gelatin-oleic conjugate (GOC) by desolvation method with glutaraldehyde used as a crosslinker. Coumarin-6 and paclitaxel were used as a fluorescence marker and model drug, respectively, to load into GONs using incubation method. Properties of GON, coumarin-6 loaded GON (C-GON), paclitaxel loaded GON (PTX-GON) were measured morphology, size, and zeta potential. The results showed that spherical and uniformed GONs are about 150nm and below -20 mV. To evaluate cellular uptake of nanoparticles under static and dynamic conditions, the fluorescence intensity and fluorescence images of nanoparticles were measured by flow cytometer and confocal laser scanning microscopy (CLSM), respectively. Additionally, we evaluated the drug efficacy by MTT assay. When delivered cationic PSNs to cells, the cellular uptake increased as the exposure time and PSN concentration increased under both static and dynamic conditions. Nevertheless, under dynamic conditions, the cellular uptake of cationic PSN was highly increased in all different type of cell lines compared to static conditions. In comparing between cell lines, normal cell line was more influenced by fluidic shear stress than cancer cell lines, even cancer cell lines shows higher mean fluorescence intensity in both static and dynamic conditions than normal cell lines. Form this results, it is suggested that biomimetic dynamic condition stimuli specific endocytosis and prompted cellular uptake of cationic PSN. In endocytosis study, dynasore (inhibits dynamin-GTPase) inhibited cellular uptake of cationic PSN only in dynamic condition, and thus, we could suggest that fluidic shear stress prompted dynamin dependent endocytosis pathway. Contrarily, the anionic PSNs showed no significant difference of cellular uptake in presence of shear stress. Cellular uptake of negatively charged GONs were also revealed a slightly decreased mean fluorescence intensity but there was no significant difference. Additionally, negatively charged paclitaxel loaded GONs had no significant difference in cell killing effect in dynamic condition than static condition. From those results, cellular uptake and efficiency of anionic nanoparticles are not significantly affected from the fluidic shear stress. It suggests that cationic nanoparticles are more influenced by fluidic shear stress than anionic nanoparticles. The results of the present study suggest the interaction of nanoparticles and cells considering in presence of fluidic shear stress. First of all, in terms of physical force, the cells interact more with cationic nanoparticles than anionic nanoparticles (Verma and Stellacci, 2010) because of negatively charged groups on surface of cell membrane. Then, shear stress could be stimulate the cellular uptake of cationic nanoparticles. Next, fluidic shear stress contribute interactions between nanoparticles and cells and may affect cellular uptake of nanoparticles during drug delivery. Various physicochemical and biochemical properties, including pH, temperature, oxygen tension, soluble factors, gradients and shear stress within the cellular microenvironment influence interactions between nanoparticles and cells (Young and Beebe, 2010), and complex those factors may be affected by nanoparticles properties such as their size, charge and concentration (Doorley and Payne, 2010). Among them, fluidic shear stress, which is one of critical factors to show the difference of in vitro and in vivo cellular distribution of drug and nanoparticles, may change concentration gradients of nanoparticles and stimulate diffusion of nanoparticles to cells. Thus, investigation of nanoparticles behaviors with cells under biomimetic microfluidic conditions, where shear stress and physicochemical factors may change the cellular uptake of nanoparticles, is important. Additionally, fluidic shear stress affect to cell signaling. Nanoparticles and drugs are transported into the cells not only by passive transport, but also via active transport. The active transport process requires energy in the form of adenosine triphosphate (ATP), or it may require receptor-mediated endocytosis. Shear stress could induce release of ATP from the cell (Bodin and Burnstock, 2001) and activate specific cell signaling pathways (Fleischer and Payne, 2012). This evidence may support our results of endocytosis study that fluidic shear stress prompted dynamin dependent endocytosis pathway with dynasore inhibitor. In comparison in cell signaling of different charged nanoparticles, cationic and anionic nanoparticles induced different cell surface receptors, even though the final charge of both cationic and anionic PSNs with adsorbed albumin proteins were negative (Fleischer and Payne, 2014). Because cellular uptake of cationic nanoparticles was accelerated under dynamic biomimetic conditions, cationic nanoparticles may be more affect cellular behavior. Thus, shear stress should be considered in terms of cellular uptake, and it is influenced by not only the cell–drug contact time but also cell signaling i.e. different receptors can be activated (Chachisvilis et al., 2006; Nguyen et al., 2001). Therefore, fluidic shear stress should be considered to investigate the cellular distribution of various nanoparticles and drug delivery systems. A better understanding of shear stress and delivery system should be crucial in drug and cellular interaction under biomimetic environments, leading to drug action. Future approaches utilizing biomimetic dynamic system may better predict cellular drug delivery and drug efficacy in such in vivo dynamic environment.
more목차
ChapterⅠ. Investigation of shear stress on the cellular distribution of polystyrene nanoparticles in a biomimetic microfluidic system
Abstract 2
1. Introduction 3
2. Materials and methods 7
2.1. Materials 7
2.2. Cell culture 7
2.3. Establishment of a biomimetic microfluidic system (BMS) 9
2.3.1. BMS Design 9
2.3.2. Calibration of BMS as a function of shear stress 11
2.4. PSN delivery 14
2.5. Evaluation of intracellular PSN uptake 14
2.5.1. Flow cytometer 15
2.5.2. Confocal Laser Scanning Microscopy 15
2.6. Statistical Analysis 16
3. Results and discussion 17
3.1. Identification of fluorescence quenching effect 17
3.2. Comparison of static and dynamic conditions for intracellular uptake 19
3.2.1. PSN concentration 19
3.2.2. Cell types 21
3.2.3. PSN charges 24
3.2.4. Level of shear stress 27
4. Conclusions 29
ChapterⅡ. Cellular drug delivery of polystyrene nanoparticles to different type of cancer cell lines under biomimetic microfluidic system 32
Abstract 33
1. Introduction 34
2. Materials and methods 37
2.1. Materials 37
2.2. Cell culture 37
2.3. Biomimetic microfluidic experiment 38
2.4. Inhibition of endocytosis 39
2.5. Determination of PSN cellular uptake 39
2.5.1. Flow cytometer 39
2.5.2. Confocal Laser Scanning Microscopy 40
2.6. Statistical Analysis 41
3. Results and discussion 42
3.1. Cellular uptake of cationic PSN in three types of cancer cells 42
3.2. Endocytosis study of cationic PSN 48
4. Conclusions 52
Chapter Ⅲ. Effects fluidic shear stress on drug delivery of gelatin oleic acid nanoparticles to cancer cell lines under biomimetic microfluidic system 53
Abstract 54
1. Introduction 56
2. Materials and methods 59
2.1. Materials 59
2.2 Preparation of GON, C-GON and PTX-GON 59
2.2.1. Synthesis of GOC 59
2.2.2. Preparation of GON using a desolvation method 60
2.2.3. Preparation of C-GON and PTX-GON 61
2.3 Characterization of GON, C-GON, and PTX-GON 61
2.3.1. Dynamic light scattering (DLS) 61
2.3.2. Morphology: Transmission electron microscopy (TEM) and Scanning electron microscopy(SEM) 62
2.3.3. Drug loading content (DL) and encapsulation efficiency (EE) 62
2.4. Cell culture 63
2.5. Biomimetic microfluidic experiment 63
2.6. Determination of cellular uptake 64
2.6.1. Flow cytometer 64
2.6.2. Confocal Laser Scanning Microscopy 65
2.7. Cell viability assay 65
2.8. Statistical Analysis 66
3. Results and discussion 68
3.1. Physicochemical properties GONs 68
3.2. Physicochemical properties C-GONs and PTX-GONs 73
3.3. Cellular uptake of coumarin-6 loaded GONs 76
3.4. Cell-killing efficiency of paclitaxel loaded GONs 79
4. Conclusions 83
References 84
국문초록 97
