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Development of Spheroid-on-a-Chip for Evaluating the Apoptosis of Lung Cancer Cells Using Sericin Nanoparticles with SHMT1 siRNA : 세리신 나노입자 유전자 전달체의 SHMT1 siRNA를 통한 폐암 세포의 세포사멸 유도 효과를 확인하기 위한 스페로이드 배양 플랫폼 개발

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dc.contributor.advisor정종훈-
dc.contributor.author임재운-
dc.date.accessioned2024-05-31T18:01:04Z-
dc.date.available2024-05-31T18:01:04Z-
dc.date.issued2023-
dc.identifier.other000000176253-
dc.identifier.urihttps://hdl.handle.net/10371/204009-
dc.identifier.urihttps://dcollection.snu.ac.kr/common/orgView/000000176253ko_KR
dc.description학위논문(박사) -- 서울대학교대학원 : 농업생명과학대학 바이오시스템·소재학부(바이오시스템공학), 2023. 2. 정종훈.-
dc.description.abstractLung cancer adenocarcinoma is one of the most aggressive type of cancer with low survival rate and treatment strategies are often marred by poor prognosis. Depending on the disease progression, the conventional therapies such as radiotherapy, chemotherapy and immunotherapy might not be able to completely cure the disease which necessitates the intervention of gene editing using gene therapy. However, delivery vectors used in gene therapy have their own limitations such as oncogenicity and toxicity in case of viral vectors and higher toxicity and low biocompatibility incase cationic polymers non-viral vectors. Thus, biodegradable non-viral vectors with low toxicity and high delivery efficiency could be a pragmatic approach for gene therapy. Silk derived natural polymer sericin can be used for vector fabricating as nanoparticle, which have many functional groups like other protein, and also can be used as wound dressing and supplement for serum-free media indicating its biocompatibility.
In therapeutic research, animal models have more reliable and better performance compared to in vitro assays on monolayer cells. The promising results obtained during invitro experiments often do not translate into the in vivo results using animal models. However, it is not easy to develop models even asides from ethical issues or high costs, and survival of animals can hinder the experimental process. 3D culture of cancer cells, which is also called spheroids could be solution to this when combined with microfluidic based biochip. The spheroids inside microfluidic device can mimic more precise microenvironment around the cells similar to in vivo tumors. Therefore, it is more relevant to use on-chip spheroids instead of monolayer culture for chemotherapy during in vitro experiments. But the operation of microfluidic device can be challenging, which requires special equipment design such as pumps and operational skilled enough to obtain desired results.
In this research, we developed microfluidic biochips that can culture cancer spheroid and evaluated the efficiency of sericin based nanoparticles to deliver therapeutic agents. Nanoparticles were successfully fabricated and showed better transfection of reporter genes compared to positive control, Polyethylenimine (PEI). Cancer spheroids cultured inside the biochip showed prominent cell viability and transfection using sericin/siRNA-based nanoparticles on the spheroid inside biochip showed apoptotic events. The experimental finding could be promising in the field of 3D culture-based cancer gene therapy research.
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dc.description.abstract폐선암은 가장 생존률이 낮은 암 중 하나로, 현재 치료법이 효과적이지 않아 예후 역시 나쁜 편이다. 진행 정도에 따라 기본 요법인 방사선 요법, 화학 요법, 그리고 면역 요법은 이를 완전히 치료할 수 없고, 따라서 유전자 요법을 이용한 유전자 편집 기술을 이용할 필요가 있다. 그러나 유전자 전달체 중 바이러스성 전달체는 각각 발암성과 세포 독성이 있고, 다중양이온(Polycation) 고분자 비바이러스성 전달체의 경우에도 독성이 있는 등의 한계가 있다. 따라서 독성이 낮고 전달 효율이 높은 생분해성 비바이러스성 벡터를 제작할 필요가 있다. 실크 기반 단백질인 세리신은 다른 단백질처럼 풍부한 작용기를 가지고 있으며, 창상피복재나 무혈청 배지의 보충제로 들어갈 정도로 생체적합성 역시 뛰어나다.
치료법 연구에서, 동물 모델은 단일 층으로 배양된 세포에 대한 시험관 내 (In vitro) 시험법에 비해 더 신뢰성이 있고 나은 결과를 얻을 수 있다. 시험관 내 시험법에서 확실해 보이던 결과도 동물 모델을 이용한 생체 내에서의 결과와 상이해지는 경우가 빈번하다. 그러나 동물 모델의 개발은 윤리적 문제나 비용 문제, 그리고 동물의 생존성 문제 때문에 어려울 수 있다. 스피로이드(Spheroid)라고 불리는, 3D 배양된 암세포의 경우 미세유체 기술 기반 바이오칩에 배양될 경우 해법이 될 수 있다. 미세유체 장치 내부의 스피로이드는 생체 내의 종양과 유사하게 보다 정확한 미세환경을 모사할 수 있다. 따라서 이는 시험관 내 시험법을 통해 화학요법을 시행하는 것보다 더욱 신뢰성이 높다. 그러나 미세유체 장치의 운용은 어려울 수 있는데, 이는 펌프나 튜브, 그리고 원하는 결과를 내기 위해서 충분한 숙달을 필요로 하기 때문이다.
이 연구에서는 종양 스피로이드를 배양할 수 있는 미세유체 바이오칩을 개발하고 약물 전달용 세리신 기반 나노입자의 효율성을 확인하였다. 나노입자는 성공적으로 제작되었으며, 양성 대조군인 폴리에틸렌이민 (Polyethyleneimine, PEI)에 비해 리포터 유전자의 발현이 보다 뛰어났다. 바이오칩에서 배양된 종양 스피로이드는 세포 활성이 뛰어났으며, 바이오칩 내부의 스피로이드에 siRNA와 결합한 세리신 기반 나노입자를 투여했을 경우 세포 사멸의 징후를 보였다. 이 연구는 3D 배양 기반 암 유전자치료 연구에 이후 기여할 수 있을 것이다.
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dc.description.tableofcontentsChapter 1. Introduction 1
1.1. General Introduction 1
1.2. Objectives 3
Chapter 2. Literature Review 6
2.1. Lung Cancer and Current Anticancer Therapies 6
2.2. Gene Therapy for Cancer Treatment 8
2.3. Gene delivery vectors 11
2.4. 3D cell culture method for cancer research 12
2.5. Nanoparticles as gene delivery vector 15
2.6. Drug delivery systems (DDS) 17
2.7. Passive pump systems on microfludics 18

Chapter 3. Induction of Apoptosis of Cancer Cells Using the Cisplatin-based Electrospray (CDES) System 26
3.1. Summary 26
3.2. Introduction 26
3.3. Material and Methods 28
3.4. Results 31
3.5. Discussion 32
3.6. Conclusion 33

Chapter 4. Fabrication of 3D-Printing Assisted Spheroid-on-a-chip Platform 42
4.1. Summary 42
4.2. Introduction 42
4.3. Material and Methods 45
4.4. Results 50
4.5. Discussion 52
4.6. Conclusion 54

Chapter 5. Design and Fabrication of Sericin Nanoparticle for Gene Therapy 62
5.1. Summary 62
5.2. Introduction 62
5.3. Material and Methods 65
5.4. Results 71
5.5. Discussion. 74
5.6. Conclusion 76
Chapter 6. Conclusion 87
Abstract (Korean) 89
Bibliography 91

Tables
Table 2.1. Brief history of microfluidic systems and organ-on-a-chip system. 24
Table 2.2. Brief history of spheroid culture. 25
Table 4.1. Primer sequence used in qPCR. 61
Table 5.1. Primer sequence used in qPCR. 86

Figures

Figure 1.1. Overall schematic presentation of the study. 5
Figure 2.1. Spheroid-on-a-chip for culture of A549 and MRC-5 device design. 20
Figure 2.2. The schematic of the microchannel with passive pump system. 21
Figure 2.3. Schematic and image of passive pumping system 22
Figure 2.4. Schematic of gene therapy method discovery by years. 23
Figure 3.1. Schematic diagram of the cisplatin delivery-based electrospray (CDES) system.
35
Figure 3.2. Comparison of cell viability of 6 kV CDES and cisplatin. 36
Figure 3.3. The results of fluorescence-activated cell sorting (FACS) for Annexin V staining after 6 kV CDES (Control, 6 kV ES, Cisplatin, and 6 kV CDES). 37
Figure 3.4. Proposed CDES mechanism. 38
Figure 3.5. Components of CDES system. 39
Figure 3.6. Cell viability after electrospray (ES) at various voltage. 40
Figure 3.7. Comparison of cell viability at various voltage CDES. 41
Figure 4.1. Schematic of the spheroid-on-a-chip platform and its application. 55
Figure 4.2. Cell culture conditioning on the biochip. 56
Figure 4.3. Day 3 and Day 5 ICC results of CD133 expression. 57
Figure 4.4. Day 3 and Day 5 qPCR results of stemness marker expression compared with in vitro results. 58
Figure 4.5. Drug resistance assay on biochips compared with in vitro results. 59
Figure 4.6. Comparison of cell viability in vitro and on the biochip before and after 6 kV doxorubicin ES. 60
Figure 5.1. Schematic of sericin nanoparticle (SNP) and polyethylenimine conjugated sericin (PEIS) fabrication and its application. 78
Figure 5.2. Characteristics of particle properties. 79
Figure 5.3. Cell viability, NMR, FT-IR and EMSA images of the nanoparticles. 80
Figure 5.4. GFP transfection images of nanoparticles. 81
Figure 5.5. qPCR results of the A549 cells treated with RNAi by nanoparticle. 82
Figure 5.6. Cell viability and TUNEL assay images of the nanoparticles. 83
Figure 5.7. Comparison of cell viability in vitro and on the biochip before and after siRNA transfection by nanoparticles. 84
Figure 5.8. Comparison of cell viability in vitro and on a biochip before and after siRNA transfection by nanoparticles with electrospray. 85
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dc.format.extentvi, 111-
dc.language.isoeng-
dc.publisher서울대학교 대학원-
dc.subjectBiochip-
dc.subjectCancer-
dc.subjectSericin-
dc.subjectGene delivery-
dc.subjectNanoparticle-
dc.subjectSpheroid-
dc.subject.ddc660.6-
dc.titleDevelopment of Spheroid-on-a-Chip for Evaluating the Apoptosis of Lung Cancer Cells Using Sericin Nanoparticles with SHMT1 siRNA-
dc.title.alternative세리신 나노입자 유전자 전달체의 SHMT1 siRNA를 통한 폐암 세포의 세포사멸 유도 효과를 확인하기 위한 스페로이드 배양 플랫폼 개발-
dc.typeThesis-
dc.typeDissertation-
dc.contributor.AlternativeAuthor세리신 나노입자 유전자 전달체의 SHMT1 siRNA를 통한 폐암 세포의 세포사멸 유도 효과를 확인하기 위한 스페로이드 배양 플랫폼 개발-
dc.contributor.department농업생명과학대학 바이오시스템·소재학부(바이오시스템공학)-
dc.description.degree박사-
dc.date.awarded2023-02-
dc.contributor.major바이오시스템공학-
dc.identifier.uciI804:11032-000000176253-
dc.identifier.holdings000000000049▲000000000056▲000000176253▲-
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