实体瘤组织的微环境通常以缺氧为特征，缺氧已被认为是抗放射治疗的主要原因。一氧化氮（NO）被认为是放射增敏剂，因为它具有固定辐射诱导的DNA损伤的能力。由于难以直接施用气态NO分子，已经尝试递送外源NO供体。然而，当暴露于含有血红素的蛋白质如血红蛋白（Hb）时，由NO供体产生的NO的体内半衰期极短，限制了其用于放射增敏的应用。为了延长其半衰期，必须立即保护所产生的NO免于Hb的进入。为了解决这个问题，提出了一种空心微球（HM）系统，其包含PLGA壳和含有pH响应性NO供体（DETA-NONOate; NONOate）和表面活性剂分子（癸酸钠; SD）的水性核。 。在酸性肿瘤组织中，环境质子通过HM的PLGA壳渗透并与其包封的NONOate反应，自发产生NO气泡;同时，表面活性剂分子SD在不存在Hb的条件下主动捕获产生的NO气泡以在HM（Micelles @ HM）内形成胶束贮库。由NONOate和SD在本体溶液（Micelles @ BS）中自组装的含NO的胶束贮库和在其中存在Hb的本体溶液中由游离NONOate产生的NO用作对照。在前一种情况下，产生的NO立即在Micelles @ HM内得到保护，而在后两种情况下，由于环境Hb的存在，产生的NO的一部分可能会发生降解。我们的结果表明，Micelles @ HM产生的NO水平显着高于（~80％）Micelles @ BS（~35％）和游离NONOate（~20％）产生的NO水平，这表明Micelles @ HM在缺乏Hb大大提高了NO的稳定性。然后NO气体可以从Micelles @ HM中逐渐释放出来，充当放射增敏剂并增强辐射诱导的细胞死亡。在体外研究中，结果表明NO（60μM）具有放射增敏作用，当联合放射（2Gy）和Micelles @ HM显着改善NO在Hb存在下的稳定性和放射增敏作用。在体内研究中，与治疗后20天的其他组相比，微粒（MP）与放射联合显示出更好的抗肿瘤效果（肿瘤体积下降约20％）。该研究的前瞻性结果为逆转缺氧引起的放射抗性提供了有用的策略。

The microenvironment of solid tumor tissues is generally characterized by hypoxia, which has been regarded as the major cause of resistance to radiotherapy. Nitric oxide (NO) is considered as a radiosensitizer, owing to its capability on fixation of radiation-induced DNA damage. As directly administering gaseous NO molecules is difficult, attempts have been made to deliver exogenous NO donors. Nevertheless, the in vivo half-life of NO that is generated from NO donors is extremely short when exposed to heme-containing proteins such as hemoglobin (Hb), restricting its application for the radiosensitization. To extend its half-life, the generated NO must be immediately protected from the access of Hb. In an attempt to address this concern, a hollow microsphere (HM) system that comprises a PLGA shell and an aqueous core containing a pH-responsive NO donor (DETA-NONOate; NONOate) and a surfactant molecule (sodium decanoate; SD) is proposed. In acidic tumor tissues, environmental protons infiltrate through the PLGA shell of the HMs and react with their encapsulated NONOate to generate NO bubbles spontaneously; meanwhile, the surfactant molecules SD actively trap the generated NO bubbles to form micellar depots within the HMs (Micelles@HM) in a condition that is absence of Hb. The NO-containing micellar depots that are self-assembled by NONOate and SD in bulk solutions (Micelles@BS) and the NO generated from free NONOate in bulk solutions, in which Hb is present, are used as controls. In the former case, the generated NO is immediately protected within the Micelles@HM, while in the latter two cases, part of the generated NO may undergo degradation owing to the presence of environmental Hb. Our results demonstrate that the NO level generated by Micelles@HM was significantly higher (~80%) than those generated by Micelles@BS (~35%) and free NONOate (~20%), suggesting that the Micelles@HM formed in the absence of Hb largely improves the stability of NO. The NO gas could then be gradually released from Micelles@HM, acting as a radiosensitizer and enhancing radiation-induced cell death. In in vitro study, the results demonstrated that NO (60 µM) has radiosensitization effect when combined radiation (2 Gy) and Micelles@HM significantly improved the stability and radiosensitization effect of NO in the presence of Hb. In in vivo study, micropaticles (MPs) combined with radiation showed greatly better anti-tumor effect (~20% decline in tumor volume) compare to other groups 20 days after treatment. The prospective results of this study provide a useful strategy for reversing hypoxia-caused radioresistance.

Contents ii
List of Figures iv
List of Tables v
Chapter 1: Introduction 1
1.1. Radiation therapy and radiosensitizer 1
1.2. The potential of nitric oxide as a radiosensitizer 2
1.3. Limitations of nitric oxide therapy 3
1.4. In Situ self-assembling micellar depots and functional mechanism 3
1.5. Purpose of the research 5
1.6. Experiment design 7
Chapter 2: Materials and methods 8
2.1. Characteristic of microsphere 8
2.1.1. Microsphere marterials 8
2.1.2. The preparation of microsphere 8
2.1.3. Morphology of particles 9
2.1.4. The release of nitric oxide from particles 9
2.1.5. Loading content of particles 9
2.1.6. The stability of particles 9
2.1.7. Nitric oxide release mechanism 9
2.1.8. Anti-degradation activity 10
2.2. Cell experiment 10
2.2.1. Cell culture 10
2.2.2. Cell viability & Cell cytotoxicity 10
2.2.3. Nitric oxide radiosensitization mechanism 10
2.3. In vivo study 10
Chapter 3: Result and discussion 12
3.1. Characteristic of the hollow microsphere 12
3.1.1. Morphology of particles 12
3.1.2. Formulation of particles 12
3.1.3. The release profile of nitric oxide from particles 13
3.1.4. The detection of sodium decanoate 13
3.1.5. Particle stability 14
3.1.6. Nitric oxide release mechanism 15
3.1.7. Anti-degradation activity 19
3.2. In vitro study 20
3.2.1. Cell viability 20
3.2.2. Nitric oxide radiosensitization mechanism 23
3.3. In vivo study 23
Chapter 4: Conclusion 25
References 26

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