Assembly of Exosome Membrane and Nanoparticles Using Sonication for Efficient Delivery
효율적인 전달을 위해 초음파 처리를 이용한 엑소좀막 재구성 기반 나노전달체 연구
- 주제(키워드) exosome , nanoparticle
- 발행기관 고려대학교 대학원
- 지도교수 육순홍
- 발행년도 2021
- 학위수여년월 2021. 2
- 학위구분 석사
- 학과 대학원 약학과
- 세부전공 산업약학
- 원문페이지 64 p
- UCI I804:11009-000000235261
- DOI 10.23186/korea.000000235261.11009.0001178
- 본문언어 영어
- 제출원본 000046073990
초록/요약
Docetaxel (DTX) is a taxane-based anticancer agent used for the treatment of lung and breast cancer. However, limited solubility and instability of DTX in an aqueous medium is a remaining issue. To overcome these difficulties, the current study is designed with core/shell NPs. The core is composed of DTX-loaded Solutol nanodroplets (as template NPs) within the vesicles. The shell consists of exosomes extracted from macrophages cells (RAW 264.7). Exosomes are formed during endocytosis in cells. Exosomes have high biocompatibility and low immunogenicity. Because of the unique structure of exosomes, reconstitution (RC) process is possible and forms a bilayer structure with amphiphilic nature. During the RC process, exosomes are split by temporary pressure and then returns to its stable original state. Drug encapsulation process can be added to form RC nanoparticles (NPs) using simple mixing. The morphology and size distribution of the drug-loaded RC exo NPs were observed with transmittance electron microscopy (TEM) and dynamic light scattering (DLS). Western blot was performed to confirm whether the protein was expressed in Reconstitution (RC) exosome. In vitro cytotoxicity, cellular uptake, and release tests were performed to confirm the function of RC exo NPs as nanocarriers for cancer therapy. The toxicity of exosomes extracted from RAW 264.7 was confirmed through the cytotoxicity test. Cellular uptake confirmed the targeting function to parental cells and cancer cells. The release profile of DTX from RC exo NPs were conducted via macroscopic observations and High-performance liquid chromatography (HPLC), and the sustained release pattern was confirmed. Drug-loaded exosomes act as a promising building block for anti-cancer treatments, but need to be improved to target cancer rather than liver with the largest number of macrophage cells (RAW 264.7). In this study, multilayer RC exo NPs were formed using Pluronic F-68 for further improvement of exosome function. Freeze-drying process was employed and glucose, a cryoprotectant, was used to enhance stability. In vivo Biodistribution was observed to confirm the improved targeting functionality of multilayer RC exo NPs. Pluronic F-68 played an important role in improving the targeting functionality of exosomes. Based on the results, we suggested that multilayer RC exo NPs was formed with Pluronic F-68 using RC process which provided a next-generation anticancer treatment.
more초록/요약
세포 독성과 생체적합성 및 표적화를 향상시키기 위해 대식 세포 유래 물질인 엑소좀을 사용하여 나노전달체를 형성하였다. Sonicator 를 이용하여 엑소좀에 압력을 가하여 엑소좀의 이중층 구조를 일시 적으로 쪼갠 뒤, 재구성을 유도하였다. 이 때, 기존의 엑소좀과 재구성한 엑소좀의 엑소좀막 단백질 및 테트 라스파닌을 비교하여 재구성이 변성된 형태가 아님을 검증하였다. Bio-Transmission Electron Microscopy (TEM) 과 Dynamic Light Scattering (DLS) 을 사용하여 엑소좀의 크기와 표면 전하 및 재구성의 가능성 을 확인하였고, cellular uptake를 이용하여 약물 로딩 여부와 엑소좀의 모세포 및 종양 표적화를 확인하였다. 이 때, cellular uptake는 형광 감 지가 요구되어 DTX NPs 대신 ICG/MB/SOL NPs를 이용하여 나노전달 체를 형성하였다. 또한 세포 독성 실험을 통해 엑소좀의 안정성과 제 어된 방출 거동을 확인하였다. 추가적으로 방출거동실험을 통하여 엑 소좀의 Docetaxel 방출 거동을 살펴보았으며 제어된 방출 거동을 갖 는 것을 입증하였다. 나아가, 재구성 엑소좀 나노전달체와 pluronic F-68을 혼합하고 동결건 조를 진행하여 핵/쉘 구조를 갖는 다층 엑소좀 나노전달체를 형성하 였다. Pluronic F-68의 종양 표적화 개선 효과를 입증하기 위해 Biodistribution을 진행하여 종양이 자란 mice로부터 형광세기를 확인 하였다. Biodistribution 또한 형광 감지가 요구되어 ICG/MB/SOL NPs를 이용하여 나노 전달체를 형성하였으며, 장기 적출을 통해 특정 부위 로의 표적화를 확인하였다. 이러한 결과들을 통해 엑소좀 및 Pluronic F-68을 이용한 다층 나노전달체의 표적화 기능을 검증하였으며 항암 효과의 유망한 전략임을 증명하였다
more목차
1. Introduction 1
2. Materials and methods. 9
2.1 Materials. 9
2.2 Methods. 12
2.2.1 Cell culture. 12
2.2.2 Isolation of exosomes 13
2.2.3 Preparation and reconstitution of the NPs . 14
2.2.4 Particle size and morphology of the NPs. 15
2.2.5 Quantification of exosomes. 16
2.2.6 Loading amount and encapsulation efficiency of the NPs. 17
2.2.7 Western blot of the NPs. 18
2.2.8 In vitro cellular uptake behavior of the NPs 19
2.2.9 In vitro cellular cytotoxicity assay of the NPs . 20
2.2.10 In vitro drug release characteristics of the NPs 21
2.2.11 In vivo Biodistribution of the NPs 22
3. Results and discussion 24
3.1 Preparation and characterization of the NPs 24
3.2 Loading amount and encapsulation efficacy of the NPs. 29
3.3 Western blot of the NPs. 31
3.4 In vitro cellular uptake behavior of the NPs. 33
3.5 In vitro cellular cytotoxicity assay of the NPs. 38
3.6 In vitro drug Release characteristics of the NPs. 41
3.7 In vivo biodistribution and tumor-targeting ability of the NPs 43
3.8 Quantification of the NPs in major organs. 46
4. Conclusions . 48
5. References . 49
국문 요약 52
