Hydroxyapatite-binding albumin nanoclusters for enhancing the targeting ability to the bone tumor microenvironment and antitumor effects

학위논문(박사) -- 서울대학교대학원 : 약학대학 약학과, 2022.2. 김대덕.Malignant bone tumors feature a highly mineralized extracellular matrix, causing microenvironment-induced resistance to drug accumulation. Herein, alendronate-decorated human serum albumin (HSA-AD) nanoclusters (NCs) were developed for targeting hydroxyapatites in the bone tumor microenvironment and enhancing the tumor suppressive effects. Doxorubicin (DOX) was loaded onto the surface of HSA-AD via the ball-milling technology, a novel drug-loading method in which the secondary structures of HSA can be preserved at more than 90%, based on circular dichroism analysis. The targeting ability of NCs was confirmed in a novel in vitro bone cancer model, wherein hydroxyapatite and collagen, the major components of the bone matrix representing the highly mineralized bone tumor microenvironment, were co-cultured with HOS/MNNG cells, a human osteosarcoma cell line. The binding affinity of HSA-AD/DOX to hydroxyapatite was evaluated based on DOX binding efficiency; HSA-AD/DOX showed a 5.04-fold higher affinity than HSA/DOX. The enhanced distribution of HSA-AD/DOX to bone tumors was verified in a newly developed mouse model bearing HOS/MNNG tumors with hydroxyapatite beads. HSA-AD/DOX led to a 52.0% increase in tumor accumulation compared to the unmodified NCs (HSA/DOX). This was mainly due to the hydroxyapatite-binding affinity of the AD moiety, which is supported by histological analysis performed on the dissected tumors. Taken together, the targeting ability of HSA-AD/DOX is effectively translated into improved therapeutic efficacy in bone tumor-xenografted mice, suggesting that the developed NCs are a promising delivery system for bone tumor treatment.골 암은 뼈와 유사한 세포 외 기질을 특징으로 한 종양 미세 환경을 특징으로 하며, 이는 항암치료 효과를 떨어뜨리는 주요 원인으로 평가된다. 본 연구의 목표는 골 암의 미세환경의 주요 성분 중 하나인 하이드록시아파타이트에 대한 표적성을 높여 암으로의 약물 분포를 증가시키고 항암 활성이 증가된 알부민 기반 나노 전달체를 개발하는 것이다. 이를 위해 알렌드로네이트를 알부민 표면에 수식하였으며, 새롭게 개발한 Ball-milling technology (BMT)방식을 도입하여 독소루비신을 표면에 흡착시킴과 동시에 나노클러스터를 형성하도록 디자인하였다. 제조된 나노 클러스터는 알부민의 2차 구조를 90 % 이상 유지하였으며, 신생혈관의 알부민 수용체에 대한 친화력이 유지되는 것을 in vitro 세포 실험 수준에서 확인하였다. 암세포 및 하이드록시아파타이트에 대한 표적성은 골 암의 미세 환경을 모방하는 세포 실험계에서 평가되었으며, 증가된 암세포 흡수율과 하이드록시아파타이트 결합능이 확인되었다. 나노클러스터의 하이드록시아파타이트에 대한 증가된 표적성은 유세포 분석기 및 자외 및 가시선 분광분석법을 통해 HSA-AD/DOX 의 경우 HSA/DOX에 비해 5.04배 높은 친화력을 보이는 것을 확인하였다. 이를 기반으로 골 암 조직으로의 분포를 in vivo 상에서 검증하기 위해 암세포와 하이드록시아파타이트가 혼합되어 주입된 이종이식 마우스 모델을 구축하여 평가하였다. HSA-AD/DOX는 HSA/DOX에 비해 52% 가량 증가된 암 조직 분포를 보였으며, 암 조직의 조직학적 분석을 통해 하이드록시아파타이트에 대한 높은 친화력이 원인임을 증명하였다. 본 모델에서의 항암활성 평가를 진행하였을 때, 독소루비신 수용액 및 HSA/DOX에 비해 더욱 적은 독성으로 암세포의 성장을 유의미하게 감소시킨 것으로 평가되었다. 항체를 이용한 마이크로어레이 기법을 도입하여 제형 처리 후 암세포 내 세포사멸 관련 단백질의 발현패턴을 관찰 및 GSEA 분석하였으며, HSA-AD/DOX에서 통계적으로 유의미한 관련성을 확인하였다. 조직 독성은 주요 장기의 조직학적 평가 및 전혈 분석을 통해 평가하였으며, DOX 수용액 그룹에 비해 유의미한 심장 및 전신 독성 감소를 확인하였다. 본 연구의 결과들을 종합하여 평가할 때, 새롭게 개발된 알렌드로네이트가 수식된 알부민 기반 나노입자는 골 암 치료에 효과적이고 안전성이 증가된 것을 확인하였다.1. Introduction 1 1.1. Bone tumor 1 1.2. Nano delivery platforms to enhance the bone tumor accumulation 3 1.3. Bisphosphonate as a targeting moiety for hydroxyapatites in bone tumor microenvironment 4 1.4. Limitations of previous nano delivery platforms for bone tumor treatment 4 1.5. Albumin-based drug delivery system 5 2. Materials and Method 7 2.1. Materials 7 2.2. Synthesis of HSA-AD 7 2.3. Fabrication of DOX-loaded albumin nanoclusters (NCs) 8 2.4. Characterization 10 2.5. In vitro drug release test 11 2.6. Cell culture conditions 12 2.7. Confocal Laser Scanning Microscope (CLSM) analysis 13 2.8. Flow cytometry analysis 14 2.9. Transwell study 14 2.10. In vitro antitumor efficacy and cytotoxicity 15 2.11. Affinity to hydroxyapatite 15 2.12. Animal models 16 2.13. Near-infrared fluorescence imaging 17 2.14. In vivo antitumor efficacy 19 2.15. Antibody microarray analysis 19 2.16. Statistical analysis 20 3. Results and Discussion 22 3.1. Synthesis of HSA-AD 22 3.2. Ball-milling technology (BMT) for fabricating the NCs 22 3.3. Physicochemical properties 24 3.4. Microscopic and atomic analyses 24 3.5. Investigation of secondary structures 25 3.6. Stability test 26 3.7. In vitro drug release test 26 3.8. In vitro cellular uptake and hydroxyapatite-binding ability test using bone tumor microenvironment-mimicking cell culture model 27 3.9. In vitro cellular uptake test using HUVECs 29 3.10. Transwell study 30 3.11. In vitro antitumor efficacy and cytotoxicity tests 31 3.12. Near-infrared fluorescence (NIRF) imaging in a bone tumor-mimicking xenografted mouse model 31 3.13. Histological analysis of the dissected tumors 32 3.14. Near-infrared fluorescence (NIRF) imaging in an orthotopic mouse model 33 3.15. In vivo antitumor efficacy 34 3.16. Histological analyses of tumors and major organs 35 3.17. Complete blood counting (CBC) analysis 36 3.18. Antibody microarray analysis of NCs-treated tumors 36 4. Conclusion 38 5. References 39 6. Tables 50 7. Figures 56 국문초록 91박

Critical Viewing of Television Debate Programs and its Implications on Media Education

The present study explored ways of critical viewing of the television debate program on current public affairs by examining the three broadcast debate programs-Late Night Debate(Sim-ya-to-ron), 100 Minute Debate(100-boon-to-ron), and Offense/Defense Debate(To-ron-gong-bang) - aired respectively by KBS, MBC, and SBS. The critical viewing of the television rebate program; was exclusively~in respect of media education which was understood in the present study as the practice to enhance the audience's ability to decode, evaluate, and analyze the media contents and their production processes, as well as to cultivate media users with civic and democratic skills. Three specific research questions were asked: firstly, what were the basic program formation or structure of each broadcast debate program? Secondly, what were the characteristics in topics or subjects of each debate program? Thirdly, how did the debate panels consist of in the three debate programs? To answer these questions, a totaI of 61 program units from Late Night Debate, 100 Minute Debate, and Offense/Defense Debate aired from June to October :ro1 were secured from the web sites of KBS, MBC, and SBS. The basic program structures of each debate program were found analogous to each other with minor variations. Each program was started with facilitators' opening comments introducing the specific debate topics and appealing to the audience's interests. Then, with or without showing a brief edited material, most proportion of the whole program time of 80-120 minutes was allocated for debates and argumentations across each panel comprising 4 to 7 people for whom facilitators presented main controversial issues. In the midst of the debate, facilitators asked several spectators in their studios and introduced a coup e of viewers who wanted to show their opinions on the phone. Topics covered in the debate programs were diverse current public affairs and issues from the social impact of the film Friend (Chin-goo) to the North-South issue. Specifically, the topics were identified into six categories such as issues of domestic politics, international politics, economics, society, culture, and education. Table 1 showed dominant proportion of social, economic, and political (domestic and international) issues. Presumably, debate program viewers could become knowledgeable on the issues which the debate programs dealt with and conceive those issues as salient national agendas. For the analysis of the composition of debate panels, five categories of groups were produced according to panels' social and occupational characteristics: groups from the administration, political parties, social organizations (interest groups, civil organizations, etc.), professors, and experts. The finding showed that approximately 60% of the whole panels of the three debate programs were professors and experts (see Table 2), implicating that the three debate programs comoosed panel members with similar occupational backgrounds and professors and experts were easy to persuade to participate in panels. The findings beared several implications in terms of critical viewing, one of media education objectives, of broadcast debate programs. First, related to program structures, the finding that the time allocation and participation format for spectators and viewers were very much limited compared to those for panel members made it doubtful whether each debate program, as the broadcasting companies emphasized, functioned as a public sphere or a sphere for debate or communication in order to have their viewers vigorously participate in broadcast debates. Accordingly, judging from the analysis of panel composition, spectators and viewers were not regarded as a important constituent of the panel who were much engaged in topics. To have some of the general public be involved as panel members in debates potentially indicates to make viewers of debate programs more conscious of and get engaged in their public life and public issues as empowered citizens or democratic viewers