open access

https://doi.org/10.14943/doctoral.k15136

https://hdl.handle.net/2115/90556

細胞イメージングと一重項酸素センシングに向けた蛍光分子とナノバイオコンジュゲートの開発

英語

2022-09-26

Hokkaido University

xii, 103p.

Recently, theranostics, a combination of diagnosis and therapy, has become a key modality in cancer management. Certain intrinsic limitations in conventional cancer diagnosis/therapy strategies lead to the development of nanomaterial-based therapeutics. Different nanoparticles have been developed into theranostics by combining multimodal contrast/imaging agents, drugs, and targeting moieties for cancer diagnosis, monitoring, therapy, and treatment follow-up. Theranostics for fluorescence imaging or fluorescence molecular tomography (FMT) using organic dyes and semiconductor nanocrystals with ligands/antibodies against cancer markers receive much attention in basic research and clinical applications. Also, nanomaterials combining FMT with chemotherapy, hyperthermia, or phototherapy enter the clinical stage. This thesis focuses on nanobioconjugates combining fluorescence probes, photosensitizers, fluorogenic sensors, and cancer-targeting biomolecules. I use fluorescence probes such as semiconductor quantum dots (QDs) and nucleus staining Syto dyes to detect or image cancer cells. Also, QDs and porphyrins generate singlet oxygen (1O2), an essential reactive oxygen species (ROS) in photodynamic therapy (PDT), which is detected using a high sensitivity electron donor-acceptor (D-A) fluorogenic molecule. The cancer-targeting biomolecules include anticancer antibodies and a peptide. Rationally designed nanobioconjugates using the above components help me enrich and efficiently detect cancer cells in blood samples, and produce, detect, store and release 1O2 in a solution or cells. This thesis has five chapters, including general conclusions and perspectives. Chapter 1 of the thesis provides a general introduction to fundamental aspects of cancer management. Next, I discuss the significance of circulating tumor cells (CTCs)-based liquid biopsy and the current detection technologies for CTC isolation and enrichment. The importance of nanomaterials-based immunocapturing and optical detection based on the fundamental properties of nanomaterials are also discussed. Next, I discuss the role of 1O2 in cancer therapeutics due to its cytotoxic effect on various biological substrates. I also discuss a few biological and chemical processes involved in the generation of 1O2 followed by its detection using fluorescent molecular probes. Chapter 2 provides the experimental procedures and techniques in this study. I discuss the procedure for the functionalization of silica, attachment of antibodies on functionalized silica microparticles, QD labeling with cancer-specific antibodies, and the attachment of QD-antibody conjugates on the functionalized silica particles. I also discuss the synthesis of a 1O2 sensor molecule, preparation of silica-1O2 sensor nanoassemblies, and the conjugation of cell-penetrating peptides on the nanoassembly. Next, I discuss the procedure for cell culture and cell labeling. I also discuss time-resolved fluorescence spectroscopy used in the characterization of CTCs. Finally, I discuss UV-vis absorption spectroscopy, steady-state and time-resolved fluorescence spectroscopy, single-particle microspectroscopy, laser scanning confocal microscopy, nuclear magnetic resonance spectroscopy, and scanning electron microscopy used in this thesis. In chapter 3, I discuss a multimodal fluorescence microspectroscopic and mesenchymal-antigen specific detection, collection, and characterization of cancer cells. I use self-segregating immunosilica microparticles to capture the pre-labeled cells and the cells are identified from modalities such as multicolor images, multimodal fluorescence spectra, and fluorescence decay profile of nucleus staining dyes or QDs. The large size of silica microparticles prevents their endocytosis and help avoid an external force for cell separation, and the CD44 antigen-selective cell capturing help in an error-free cancer cell detection. The CD44-targeted cell collection combined with the above modalities shows a 9-fold detection accuracy for CTCs among blood cells. In chapter 4, I synthesize a 1O2 sensor composed of an aminomethyl anthracene and a coumarin moieties to increase the efficiency of intracellular 1O2 generation, detection, and release. I construct a nanoassembly of a sensitizer and the sensor and investigate the ability of the assembly to generate, store, sense, and release 1O2 at the ensemble, single-particle, and cell levels. In all cases, the sensor shows an enormous fluorescence enhancement due to the reaction of 1O2 generated by the photosensitizer. The mechanisms behind 1O2 sensing and releasing are explained in detail in this chapter. The intracellular uptake ability of the nanoassembly and 1O2 generation are studied after conjugating an RGD peptide to the assembly. The single-particle and cell imaging reveal continuous 1O2 release and efficient cell death. In addition, the fluorescence from the photosensitizer and the sensor help colocalized cell imaging. Thus, this work highlights the utilization of programed nanocarriers for multimodal cancer therapeutic strategies. Chapter 5 is the general summary of the thesis and future prospects of nanobioconjugates and 1O2 sensing-releasing probes for cancer therapy. Also, I explain the toxicity aspects and the significance to analyze the pharmacokinetics of nanobioconjugates.

2022-09-26

甲第15136号

博士(環境科学)

(主査) 教授 BIJU VASUDEVAN PILLAI, 教授 小西 克明, 教授 野呂 真一郎

環境科学院（環境物質科学専攻）

博士論文

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