Synthesis and evaluation of clickable gelatin-oleic nanoparticles for drug delivery
- 주제(키워드) Fattigation-platform nanoparticle based drug delivery
- 발행기관 아주대학교
- 지도교수 이범진
- 발행년도 2018
- 학위수여년월 2018. 2
- 학위명 박사
- 학과 및 전공 일반대학원 약학과
- 실제URI http://www.dcollection.net/handler/ajou/000000026981
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Targeted nanocarriers offer potential promise to improve the efficiency of nanomedicine while reducing systemic undesirable side effects. One of the main requisite for the targeted nano drug delivery system is biocompatibility, high drug loading of hydrophilic and hydrophobic compounds, and promising pharmacokinetics. In the last decade, click chemistry has paved the way for novel synthesis reaction via precise, efficient and faster reaction rate in vivo under mild conditions with limited side reactions and high yield. It provides a novel approach to the researchers to covalently combine the two inert molecules at physiological conditions. Several studies have demonstrated application of biorthogonal click chemistry for diverse purposes which include site-specific tagging of proteins or nucleotides, analysis of metabolic pathways, nanocarriers surface modification, monitoring growth of zebrafish, etc. In this study, metabolic glycoengineering cell surface modification was used to introduce unnatural azide groups on the cell surface which bind to cyclooctyne group of GON-DBCO nanoparticles and form the covalent link on the cell surface between nanoparticles and cell surface. Gelatin and oleic acid are FDA approved biocompatible materials and belongs to ‘generally regarded as safe’ (GRAS) category. Dibenzocyclooctyne (DBCO) was chosen since it is biologically inert with other functional groups in vivo. Doxorubicin (DOX) was used as a model drug. Therefore, we designed a simple, biocompatible, and targeted covalent chemistry-based drug delivery system and physico-chemically characterized by ATR-FTIR spectroscopy, NMR spectroscopy, and TNBS assay, particle size, stability and evaluated its efficacy against two different cell lines (A549 and MCF-7). The clickable nanoparticles ranged in size from ~220 to 240 nm and had positively charged surfaces. The drug (DOX) loading and encapsulation efficiencies of GON-DBCO were 10.35 ± 0.62 and 62.10 ± 3.77, respectively. A sialic acid precursor (Ac4ManNAz) was introduced via metabolic glycoengineering for the click reaction. Cell viability was significantly reduced after 48 and 24 hours for A549 and MCF-7 cell lines. respectively while cellular uptake was increased approimately 23% and 54% for MCF-7 and A549 cell lines as compared with marketed product Caelyx®. In the blood vessels and other microenvironment and extracellular matrix, fluidic shear stress which is caused by the blood flow range from 0.1 dyne/cm2 to 35 dyne/cm2. In the physiological fluid, proteins tend to interact with nanoparticles with different amount and affinity of proteins which controls the in vivo response ultimately. Competition of proteins to bind to the surface of nanoparticles which form the ‘protein corona’ greatly guides the biological identity of the nanoparticles. Nanoparticle-protein interaction in physiological mimicking condition will determine how NPs will behave inside the blood vessels and therefore, overall outcome of the drug delivery system. Therefore in the second chapter of the thesis, we investigated the effect of shear stress (5 dyne/cm2) and protein corona on cell behavior of nanoparticles by in vitro studies. We chose clickable GON-DBCO polymeric nanoparticles as a model delivery system, and docetaxel (DTX) as a model hydrophobic drug. Human serum albumin (HSA) was used to form the protein corona, since it is the most ample protein found in the body. As proved in the first chapter, the clickable GON-DBCO nanoparticles target the cancer cell by means of metabolic glycoengineering and thus serve as an active targeting system. Physicochemical properties of the drug loaded GON-DBCO nanoparticles revealed the spherical shape of the nanoparticles with increasingly negative zeta potential as the concentration of HSA protein increased. Cellular uptake showed approximately 40% and 22% decrease in protein corona treated A549 and MCF-7 cancer cell lines, respectively compared to cells treated in static condition. Similarly, cell-killing efficiency of clickable nanoparticles also significantly increased in both cell lines as compared to static condition.
more목차
Chapter I. Synthesis and evaluation of clickable gelatin-oleic nanoparticles using fattigation-platform for cancer therapy 1
1. Introduction 1
2. Materials and methods 5
2.1. Materials 5
2.2. Methods 6
2.2.1. Synthesis of DBCO-modified gelatin-oleic conjugates (GOC-DBCO) 6
2.2.1.1. Activation of OA 6
2.2.1.2. Preparation of gelatin-oleic conjugates (GOC). 6
2.2.1.3. Synthesis of GOC-DBCO 6
2.2.2. Preparation of DBCO-modified GON (GON-DBCO) 7
2.2.3. Preparation of DOX-loaded GON-DBCO (GON-DBCO-DOX) 7
2.2.4. Characterization of the drug delivery system 8
2.2.4.1. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) analysis 8
2.2.4.2. Proton nuclear magnetic resonance (1H NMR) analysis 8
2.2.4.3. Analysis of the degree of substitution 8
2.2.4.4. Measurement of zeta potential and particle size 9
2.2.4.5. Evaluation of the nanoparticle morphology. 9
2.2.4.6. Evaluation of the nanoparticle stability. 9
2.2.4.7. Measurement of encapsulation efficiency (EE) and drug loading (DL) 10
2.2.4.8. In vitro drug release. 10
2.2.5. Cell culture and maintenance. 11
2.2.5.1. Confirmation of the presence of azide groups by confocal microscopy. 11
2.2.5.2. Confocal imaging of DOX-loaded GON (GON-DOX) and GON-DBCO-DOX... 12
2.2.5.3. Cell viability assay.. 12
2.2.5.4. Flow cytometry studies. 13
2.2.6. Statistics.. 13
3. Results and discussion . 14
3.1. Characterization of the GOC DBCO conjugates. 14
3.2. Characterization of the nanoparticles 21
3.3. DL, EE, and in vitro drug release 26
3.4. Cell viability analysis 29
3.5. Confirmation of the presence of azide groups and intracellular DOX uptake by confocal microscopy . 31
3.6. Flow cytometric analysis of the cellular internalization of nanoparticles. 35
4. Conclusions 38
Chapter II. Exploring the effect of shear stress and protein-corona of clickable nanoparticles behavior in cancer cell lines 39
1. Introduction 39
2. Materials and methods 44
2.1. Materials 44
2.2. Setting up of a dynamic micro-fluidic system (DMS) 44
2.2.1. Design of DMS. 44
2.2.2. Calibration of DMS. 46
2.3. Preparation of DTX-loaded GON-DBCO (GON-DBCO-DTX) 48
2.4. Preparation of coumarin-6-loaded GON-DBCO-DTX 48
2.5. Preparation of HSA-coated GON-DBCO-DTX 48
2.6. Characterization of nanoparticles 49
2.6.1. Determination of the particle size and zeta potential 49
2.6.2. Evaluation of the nanoparticle morphology 49
2.6.3. Determination of the drug encapsulation efficiency (EE) and drug loading (DL) 50
2.6.4. UV spectra of HSA-coated GON-DBCOs 50
2.7. Cell culture and maintenance 51
2.7.1. Biomimetic dynamic microfluidic experiment.. 51
2.8. Characterization of cellular studies 53
2.8.1. Confirmation of the presence of azide groups by confocal microscopy. 53
2.8.2. Confocal imaging of Coumarin-6 loaded GON-DBCO-DTX 53
2.8.3. Flow cytometry evaluation . 55
2.8.4. MTT assay for cell-killing efficiency . 55
2.9. Statistics 56
3. Results and Discussion 57
3.1. Physicochemical properties of NPs. 57
3.2. Interaction between HSA and GON-DBCOs . 62
3.3. Qualitative cellular uptake of the GON-DBCOs. 65
3.4. Quantitative evaluation of cellular uptake. 68
3.5. Cell-killing efficiency.. 73
4. Conclusion. 79
5. References 80
