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Biocompatible and Biodegradable Therapeutic Helical Microrobot with MNP Retrieval and Sequential Multi-drug Release

Title
Biocompatible and Biodegradable Therapeutic Helical Microrobot with MNP Retrieval and Sequential Multi-drug Release
Alternative Title
자성나노입자 회수 및 순차적 다중 약물 방출 기능과 생적합성 및 생분해성을 가지는 나선형 마이크로로봇
Author(s)
Hyoryong Lee
DGIST Authors
Hyoryong LeeSukho ParkEunjoo Kim
Advisor
박석호
Co-Advisor(s)
Eunjoo Kim
Issued Date
2023
Awarded Date
2023-08-01
Type
Thesis
Description
마이크로로봇 (microrobot); 전자기장 구동 (electromagnetic manipulation); 자성나노입자 회수 (magnetic nanoparticle retrieval); 다중 약물 방출 (multi-drug release); 통합시스템 (integration system)
Abstract
Various cancer treatment methods have been developed to treat cancer cells, among them, medical drug delivery microrobots with minimal invasiveness and precise targeting are receiving attention. However, existing biomedical microrobots with active magnetic manipulation ability contain magnetic nanoparticles (MNPs), which remain in the body after delivering the therapeutic drug. In such cases, iron ions, which are the component of the MNPs, and hydrogen peroxide react in the body to generate reactive oxygen species, which can affect the growth of normal cells by reacting with glutathione, a nutrient for normal cells. In addition, existing drug delivery microrobots mainly have the limitation of delivering only one type of drug, which can make cancer cells more prone to developing resistance to the drug and reducing the efficacy of cancer treatment. To overcome these limitations, there is a need for precise targeting of microrobots, followed by immediate separation/retrieval of MNPs. There is also a need to improve existing drug delivery methods to overcome limitations such as low drug release from cancer cells and drug resistance.
Therefore, this paper proposes the microrobot that overcomes the limitations of existing drug delivery microrobots and aims to use the helical type microrobot with good mobility in high viscosity fluids such as blood. Firstly, a preliminary study was conducted on a helical type microrobot capable of active drug release, and the feasibility of microrobot manipulation and active drug release was confirmed using the electromagnetic actuation (EMA) system and near-infrared (NIR) integrated system. The performance of liver cancer cell treatment through active drug release was verified using it, and as a result, it was validated that the treatment efficiency was improved. Next, another preliminary study was conducted on a helical type microrobot capable of separating/retrieving MNPs. The microrobot was extremely small and intricately fabricated using two-photon lithography. The microscope, 8-coil, and NIR integrated system was developed, and the feasibility of separating/retrieving magnetic nanoparticles was demonstrated. It was confirmed that the toxicity that could be caused to normal cells was reduced by the retrieval of MNPs. Therefore, the necessity of MNP retrieval were verified.
Finally, based on previous studies, the aim is to propose and verify the method for separating/retrieving MNP with minimal cytotoxicity to normal cells and utilizing active drug release capabilities for the sequential multi-drug release to mitigate drug resistance effects in liver cancer cells and maximize treatment efficacy. In the case of the proposed sequential multi-drug release helical type microrobot, first, using materials that possess biocompatibility and biodegradability, the microrobot is fabricated with high precision and small size using a two-photon lithography method. Second, after targeting of the microrobot, MNPs attached to the surface of the microrobot can be separated from the surface using focused ultrasound (FUS) and can be retrieved through an external magnetic field. Third, using multi-drug, doxorubicin (DOX) and gemcitabine (GEM), active drug release of the first drug, GEM, bound to the surface of the microrobot can be achieved using NIR while the microrobot slowly degrades over time, allowing for the release of the second drug, DOX, which is encapsulated within the microrobot. This sequential release of the multi-drug can enhance the therapeutic effect on liver cancer cells by attacking different stages of the cell growth cycle. About these characteristics, fundamental experiments were conducted to confirm the feasibility of the proposed helical type microrobot for targeting, separation, and retrieval of MNPs, and sequential release of the multi-drug. Through in vitro experiments using normal and liver cancer cells, it was validated that the implemented functions could reduce cell toxicity and improve the efficiency of liver cancer cell treatment. Finally, the MNP separation/retrieval and sequential multi-drug release for liver cancer cell treatment performance of the microrobot were validated through in vitro experiments using the EMA/FUS/NIR integrated system.
Ultimately, the proposed microrobot is expected to be used as one of the methods to improve the efficiency of other cancer cells as well as liver cancer cell treatment by overcoming the limitations of existing microrobots in cancer cell treatment.
| 암세포를 치료하기 위해서 다양한 암세포 치료 방식들이 개발되고 있으며, 그 중에서 최소 침습 수술 및 정밀한 타겟팅이 가능한 의료용 자기구동 마이크로로봇에 대한 연구가 각광받고 있다. 하지만, 기존의 의료용 능동 자기장 구동 마이크로로봇은 약물 방출 속도가 느려 초기 암세포 치료에 있어 효율이 낮아지는 문제가 있었다. 그리고, 자성나노입자를 담지하고 있어 치료 약물 전달 후 체내에 남아있게 된다. 이러한 경우, 체내에서 자성나노입자의 구성 성분인 철 이온과 과산화수소가 반응하여 활성산소를 발생시키게 되고, 이는 정상세포의 영양분인 글루타티온과 반응하여 정상세포의 성장에 영향을 미칠 수 있다는 문제점을 가진다. 또한, 기존의 약물 전달 마이크로로봇의 경우, 주로 한 가지의 약물을 전달하여 암세포가 약물에 대한 내성을 가지기 쉬우며, 이로 인해, 암세포 치료 효율이 떨어질 수 있다는 제한점을 가진다. 이러한 제한점들을 극복하기 위해 마이크로로봇의 정밀한 타겟팅 이후 곧바로 자성나노입자 분리/회수가 가능할 필요성이 있으며, 암세포의 낮은 약물 방출 및 약물 내성으로 인해 발생하는 제한점들을 극복하기 위해 기존의 약물 전달 방식을 개선할 필요성이 있다.
따라서, 본 논문에서는 기존의 약물 전달 마이크로로봇이 가지는 제한점들을 극복하는 마이크로로봇을 제안하고자 하며, 혈액과 같이 점도가 높은 유체내에서 이동 성능이 좋은 나선형 형태의 마이크로로봇을 사용하고자 한다. 먼저, 능동 약물 방출이 가능한 나선형 마이크로로봇에 대한 선행연구를 진행했으며, 전자기장 구동 시스템 및 근적외선이 통합된 시스템을 이용하여 마이크로로봇 구동 및 능동 약물 방출 가능성을 확인했다. 이를 활용하여 능동 약물 방출을 통한 간암세포 치료 성능을 확인했으며, 결과적으로 치료 효율이 향상되는 것을 검증했다. 그 다음, 자성나노입자의 분리/회수가 가능한 나선형 마이크로로봇에 대한 선행연구를 진행했으며, 2광자 리소그래피를 이용하여 크기가 매우 작고 정교하게 제작되었다. 이에 대해 현미경/전자기장 구동/근적외선이 통합된 시스템을 제작하고, 자성나노입자의 분리/회수 가능성을 증명하였으며, 자성나노입자 회수로 인한 정상세포에 일으킬 수 있는 독성이 감소하는 것을 확인했다. 따라서, 자성나노입자 회수 필요성에 대해 검증했다.
최종적으로, 앞선 선행연구들을 바탕으로 자성나노입자들의 분리 및 회수가 가능하여 정상세포의 독성을 최소화하고, 능동 약물 방출 기능을 활용하여 두 가지 약물의 순차적인 방출로 인한 암세포의 약물 내성 효과를 감쇠 시켜 치료 효율을 극대화시키는 방법을 제시 및 검증하고자 한다. 제안된 순차적 다중 약물 방출 나선형 마이크로로봇의 경우, 첫째, 생적합성 및 생분해성을 가지는 재료를 사용하여 2광자 리소그래피 방식으로 크기가 매우 작고 정교하게 제작된다. 둘째, 타겟팅 이후 마이크로로봇의 표면에 부착된 자성나노입자는 집중 초음파를 사용하여 마이크로로봇의 표면으로부터 분리되고, 외부자기장을 통해 회수할 수 있다. 셋째, 두 가지 약물로 독소루비신과 젬시타빈을 사용하며, 순차적인 약물 방출에 대해, 근적외선을 사용하여 마이크로로봇의 표면에 결합되어 있는 첫번째 약물인 젬시타빈의 능동 방출이 가능하고, 시간이 지남에 따라 마이크로로봇이 천천히 분해되면서 담지 되어 있던 두번째 약물인 독소루비신의 순차적인 방출이 가능하다. 이 때, 두 가지의 약물이 간암 세포 성장 주기에서 각각 다른 단계를 공격하여 치료효과를 높일 수 있다. 따라서, 제안된 나선형 마이크로로봇의 타겟팅, 자성나노입자 분리 및 회수, 그리고 두 가지 약물의 순차적인 방출에 대한 기초 실험을 진행하여, 각 기능들이 구현 가능함을 확인했으며, 정상세포 및 간암세포를 이용한 실험들을 통해 자성나노입자 분리 및 두 가지 약물의 순차적인 방출로 세포 독성 감소 및 간암세포 치료 효율을 향상시킬 수 있다는 것을 확인했다. 최종적으로, 전자기장구동/집중초음파/근적외선 통합시스템 및 미세 유체 채널을 이용한 세포 실험을 통해, 제안된 마이크로로봇의 자성나노입자 분리/회수 및 순차적인 약물 방출로 인한 간암세포 치료 성능을 검증하였다.
궁극적으로 제안된 마이크로로봇은 기존 마이크로로봇의 암세포 치료에서의 제한점들을 개선하여 간암세포 뿐만 아니라 다른 암세포 치료 효율을 높이는 방법 중 하나로 사용될 수 있을 것으로 기대한다.
Table Of Contents
Ⅰ. Introduction 1
1.1 Cancer 1
1.1.1 Cell cycle 1
1.1.2 Tumor 1
1.2 Conventional cancer therapy 2
1.2.1 Surgery 2
1.2.2 Radiation therapy 3
1.2.3 Photodynamic therapy 3
1.2.4 Immunotherapy 3
1.2.5 Targeted drug therapy 4
1.2.6 Limitation of conventional cancer therapy 4
1.3 Drug delivery microrobot for cancer therapy 7
1.3.1 Shape of microrobot 7
1.3.2 Fabrication methods of microrobot 8
1.3.3 Materials for microrobot fabrication 9
1.3.4 External stimulus responsive microrobot 11
1.3.5 Electromagnetic actuation system for microrobot manipulation 12
1.3.5.1 Magnetic torque and force generation 12
1.3.5.2 Motion of helical microrobot using EMA system 14
1.4 MNPs using for microrobot manipulation 15
1.4.1 MNP cytotoxicity 15
1.4.2 Microrobot/MNP retrieval after drug delivery 16
1.5 Improvement for conventional cancer therapy 17
1.5.1 State-of-the art of conventional helical-type microrobot 17
1.5.2 Proposal 19
1.6 Dissertation overview 20
IⅠ. Magnetically Actuated Helical Microrobot with Active Drug Release Ability Using NIR 21
2.1 Introduction 21
2.2 Materials and methods 23
2.2.1 Materials 23
2.2.2 Preparation for proposed microrobot fabrication 24
2.2.3 Fabrication of helical type microrobot using PFA microtube 24
2.2.4 Therapeutic drug loading in the temperature responsive helical type microrobot 24
2.2.5 Magnetic property analysis of the microrobot 25
2.2.6 Temperature response fundamental tests of the microrobot 25
2.2.7 System setup for the microrobot manipulation and NIR response 25
2.2.8 Active drug release test of the microrobot 26
2.2.9 In vitro test using Hep3B cells for the therapeutic efficacy of the microrobot 26
2.2.10 Cell viability analysis 27
2.3 Results and discussion 27
2.3.1 Swelling and deswelling properties of the microrobot 27
2.3.2 Microrobot performance tests using EMA/NIR integration system 29
2.3.2.1 Microrobot manipulation 29
2.3.2.2 Swelling and deswelling test in the integration system 30
2.3.3 Active drug release tests of the microrobot 32
2.3.4 In vitro test for the therapeutic efficacy evaluation of the microrobot 32
2.4 Discussion 34
2.5 Conclusion 35
IIⅠ. Magnetically Actuated Drug Delivery Helical Microrobot with MNP Retrieval Ability Using NIR 36
3.1 Introduction 36
3.2 Materials and methods 39
3.2.1 Materials 39
3.2.2 Fabrication of the microstructure 40
3.2.3 Fabrication of the microrobot 41
3.2.4 Fluorescein labeling of amine functionalized MNPs 41
3.2.5 Characterization of the microrobot 42
3.2.6 MNP separation fundamental test of the microrobot 42
3.2.7 Microstructure degradation test 43
3.2.8 HUVECs and Huh-7 cell culture for in vitro tests 43
3.2.9 HUVECs cytotoxicity evaluation of MNPs and microrobots 43
3.2.10 In vitro Therapeutic efficacy evaluation of DOX and DOX loaded microrobots 44
3.2.11 MNP retrieval test of the microrobot using integration system 45
3.3 Results and discussion 45
3.3.1 Fabrication and characterization of the microstructure 45
3.3.2 Fabrication and characterization of the microrobot 47
3.3.3 Electromagnetic manipulation test of the microrobot 49
3.3.4 MNP separation test of the microrobot 50
3.3.4.1 MNP separation fundamental tests of the microrobot 51
3.3.4.2 MNP separation/retrieval test of the microrobot using microscope/NIR integration system 54
3.3.5 In vitro test using HUVECs for MNP and microrobot cytotoxicity evaluation 55
3.3.5.1 Evaluation for MNP Cytotoxicity 55
3.3.5.2 Validation for reduced MNP cytotoxicity through MNP retrieval 57
3.3.6 In vitro test using Huh-7 for released drug performance validation 59
3.4 Discussion 60
3.5 Conclusion 63
IV. Magnetically Actuated Helical Microrobot with MNP Retrieval and Sequential Multi-drug Release Abilities Using NIR and FUS 65
4.1 Introduction 65
4.2 Materials and methods 68
4.2.1 Materials 68
4.2.2 Fabrication of the microrobot 69
4.2.3 Characterization of the microrobot 71
4.2.4 Magnetic manipulation of the microrobot 71
4.2.5 FUS Simulation for FUS system setup 71
4.2.6 MNP separation using FUS system 72
4.2.7 Sequential multi-drug release fundamental test of the microrobot 72
4.2.8 HUVECs and HuCCT1 cell culture for in vitro tests 73
4.2.9 HUVECs cytotoxicity evaluation of MNPs and microrobots 73
4.2.10 In vitro test for therapeutic efficacy evaluation of multi drug and microrobots 74
4.2.11 System setup for the microrobot performance validation 74
4.2.12 In vitro test using the EMA/FUS/NIR integration system 75
4.3 Results and discussion 76
4.3.1 Fabrication and characterization of the microrobot 76
4.3.2 Microrobot manipulation test 80
4.3.3 MNP separation test of the microrobot 81
4.3.3.1 FUS simulation and measurement comparison 81
4.3.3.2 MNP separation analysis of the microrobot 82
4.3.4 In vitro test using HUVECs for MNP and microrobot cytotoxicity evaluation 83
4.3.4.1 Evaluation for MNP cytotoxicity 84
4.3.4.2 Validation for reduced MNP cytotoxicity through MNP retrieval of the microrobot 86
4.3.5 Multi model drug release test of the microrobot 86
4.3.6 In vitro test using HuCCT1 for validation of sequential multi drug release 89
4.3.6.1 Therapeutic efficacy evaluation for using multi drug sequentially 89
4.3.6.2 Therapeutic efficacy evaluation for sequentially released multi drug from microrobots 91
4.3.7 In vitro test for the microrobot performance validation using EMA/FUS/NIR integration system 93
4.3.7.1 EMA/FUS/NIR integration system setup 93
4.3.7.2 In vitro test of the microrobot using EMA/FUS/NIR integration system 96
4.4 Conclusion 98
V. Conclusion and Future Works 99
5.1 Summary and discussion 99
5.2 Future works 100
References 102
요약문 117
URI
http://hdl.handle.net/20.500.11750/46393

http://dgist.dcollection.net/common/orgView/200000687119
DOI
10.22677/THESIS.200000687119
Degree
Doctor
Department
Department of Robotics and Mechatronics Engineering
Publisher
DGIST
Related Researcher
  • 박석호 Park, Sukho 로봇및기계전자공학과
  • Research Interests Biomedical Micro/Nano Robotics; Biomedical Devices and Instruments
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