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アイテム
エラスターゼの効率的な近赤外検出とバイオイメージングを目指した新規スクアレイン-ペプチド複合体の研究
https://doi.org/10.18997/0002001059
https://doi.org/10.18997/000200105928cd956c-f5b3-4aa9-b01e-40afd6a6c840
| 名前 / ファイル | ライセンス | アクション |
|---|---|---|
sei_k_500.pdf (5.8 MB)
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| アイテムタイプ | 学位論文 = Thesis or Dissertation(1) | |||||||
|---|---|---|---|---|---|---|---|---|
| 公開日 | 2024-11-21 | |||||||
| 資源タイプ | ||||||||
| 資源タイプ識別子 | http://purl.org/coar/resource_type/c_db06 | |||||||
| 資源タイプ | doctoral thesis | |||||||
| タイトル | ||||||||
| タイトル | Investigation of Novel Squaraine-Peptide Conjugates Aiming Towards the Efficient NIR Detection and Bioimaging Of Elastase | |||||||
| 言語 | en | |||||||
| タイトル | ||||||||
| タイトル | エラスターゼの効率的な近赤外検出とバイオイメージングを目指した新規スクアレイン-ペプチド複合体の研究 | |||||||
| 言語 | ja | |||||||
| 言語 | ||||||||
| 言語 | jpn | |||||||
| 著者 |
Mavileti Chilamakuru, Sai Kiran
× Mavileti Chilamakuru, Sai Kiran
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| 抄録 | ||||||||
| 内容記述タイプ | Abstract | |||||||
| 内容記述 | In an era characterized by rapid lifestyle changes and environmental influences, the prevalence of diseases is on the rise. Chronic conditions such as diabetes, cardiovascular disorders, autoimmune/inflammatory diseases, and certain types of cancer have become increasingly prevalent, posing significant challenges to healthcare systems worldwide. Thus, the timely diagnosis and effective monitoring of diseases have emerged as the most crucial aspect of promoting public health and improving patient outcomes. In this context the existence and knowledge of Biomarkers are essential. In simple terms, biomarker refers to the presence or absence of a particular biomolecule in a specific disease. The identification and analysis of biomarkers have revolutionized the fields of disease diagnostics, disease monitoring, drug development, and personalized medicine. The growing prevalence of chronic diseases, along with the demand for more reliable, accurate, cost-efficient, and improved diagnostic solutions has propelled the market for the detection of disease biomarkers. Among various sensing methods, the optical method of sensing offers high selectivity and sensitivity, real-time monitoring, and more importantly activatable sensing. Since light is the main transducing element, optical imaging can be performed simultaneously, creating a visual representation providing crucial information on the local microenvironment. Among the various optical imaging techniques, the fluorescence mode of detection is non-invasive, easy, and cost-effective. The growing demand for reliable near-infrared (NIR) probes that maintain fluorescence in living systems and are easily compatible with biomolecules is fueled by the increasing use of NIR imaging in clinical diagnostics, owing to the enhanced spatial resolution and deeper tissue penetration in the NIR region. Five benzo[e]indole-based unsymmetrical squaraine dyes with -COOH functionalization and one without were synthesized and evaluated for their biocompatibility through BSA interactions, interactions with molecules of biological origin, in-vitro and in-vivo studies. All the dyes showed complete fluorescence quenching in aqueous solutions due to aggregation-induced quenching (AIQ). Dyes with -COOH functionalization showed strong fluorescence upon interaction with BSA, compared to the one without -COOH. Among these dyes, SQ-212 and SQ-215 showed distinct cell penetration behaviors, biocompatibility, non-toxic, and stable signal in-vitro and in-vivo, making them suitable for biomedical applications. Neutrophil Elastase (NE) and Neutrophil Extracellular Traps (NETs) are critical in immune responses. Neutrophils secrete NETs as an extracellular matrix to capture and neutralize pathogens like bacteria, fungi, and viruses. NETs are characterized by condensed DNA, Histones, NE, Cathepsin G, Myeloperoxidase, etc. Excessive or prolonged NETs formation can contribute to Chronic inflammatory diseases and cancer. Recently, NETs have also been accounted for lung damage in COVID-19 patients. Hence, it is important to detect and monitor NETs. Neutrophil elastase (NE), is a serine protease expressed in NETs. Hence, the protease activity of NE is exploited as a fundamental mechanism for targeting NETs. Understanding NE and NETs interactions offers insights into disease development, leading to diagnostic and therapeutic advances. Foster Resonance Energy Transfer (FRET)-based squaraine-peptide conjugates Homo-APA and Hetero-APA containing the Ala-Pro-Ala as the NE recognition sequence were designed and synthesized. The probes were self-quenching (fluorescent-off) state through AIQ and FRET mechanisms. Upon incubating with NE, a tremendous increase in fluorescence intensity was seen, with Hetero-SQ-APA being better than Homo-APA. In-vitro and in-vivo studies in NETs-induced mice models showed good activity with NE and were able to detect NETs specifically. Intraperitoneal injection of Hetero-APA showed the detection of NETs and selective accumulation in the gout-induced mice model. The probes showed stable, strong, and intense ex-vivo signals while being non-toxic. However, no significant activity when tested in pathological samples. To further refine the NETs targeting probe, a non-FRET-based SQ-215-NETP incorporating the NE recognition sequence GEAIPMSIPPEVK that binds covalently to NE was designed and synthesized. When tested in-vitro with purified NE, and in-vivo in gout-induced mice SQ-215-NETP showed enhanced activity than Hetero-APA. SQ-215-NETP demonstrated high-resolution bioimaging capabilities across various ex-vivo samples, including human eye-derived NETs and human aortal thrombi aiding in detailed visualization of NET structures and NE activity. SQ-215-NETP was also able to detect NETs in flow cytometry setup as well. Thus this thesis presents the first small molecule-based probe for the detection and imaging of NETs in a wide range of samples having a great market potential in the diagnosis and monitoring of NET-induced diseases. |
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| 目次 | ||||||||
| 内容記述タイプ | TableOfContents | |||||||
| 内容記述 | 1 Introduction| 2 Experimental| 3 Synthesis, BSA Interactions, In-vitro and In-vivo Studies of Newly Designed Squaraine Dyes| 4 Investigation of FRET-Based Squaraine-Peptide Conjugates for the Specific Detection of Neutrophil Elastase| 5 Synthesis, Characterization, and Evaluation of non-FRET-based Squaraine-Peptide Conjugate Probe| 6 General Conclusion and Future Outlook | |||||||
| 備考 | ||||||||
| 内容記述タイプ | Other | |||||||
| 内容記述 | 九州工業大学博士学位論文 学位記番号:生工博甲第500号 学位授与年月日:令和6年9月25日 | |||||||
| 学位授与番号 | ||||||||
| 学位授与番号 | 甲第500号 | |||||||
| 学位名 | ||||||||
| 学位名 | 博士（学術） | |||||||
| 学位授与年月日 | ||||||||
| 学位授与年月日 | 2024-09-25 | |||||||
| 学位授与機関 | ||||||||
| 学位授与機関識別子Scheme | kakenhi | |||||||
| 学位授与機関識別子 | 17104 | |||||||
| 学位授与機関名 | 九州工業大学 | |||||||
| 言語 | ja | |||||||
| 学位授与年度 | ||||||||
| 内容記述タイプ | Other | |||||||
| 内容記述 | 令和6年度 | |||||||
| 出版タイプ | ||||||||
| 出版タイプ | VoR | |||||||
| 出版タイプResource | http://purl.org/coar/version/c_970fb48d4fbd8a85 | |||||||
| アクセス権 | ||||||||
| アクセス権 | open access | |||||||
| アクセス権URI | http://purl.org/coar/access_right/c_abf2 | |||||||
| ID登録 | ||||||||
| ID登録 | 10.18997/0002001059 | |||||||
| ID登録タイプ | JaLC | |||||||
