肿瘤是全世界主要的公共卫生问题之一，严重威胁人类健康。目前常见的肿瘤治疗方法包括手术治疗、放疗和化疗等。乏氧是肿瘤微环境的典型特征，可促进肿瘤细胞的转移，增强肿瘤的耐受性和耐药性，严重影响肿瘤的治疗效果。光热疗法（Photothermal therapy，PTT）是一种新兴的肿瘤治疗方法，它利用光热剂在近红外光辐射下进行局部热疗，从而杀死肿瘤细胞。虽然目前已有少量研究证实了缓解肿瘤乏氧能够增强光热疗效，但大都是在离体组织层面进行的验证，随着肿瘤的演变生长，难以反映肿瘤乏氧环境的实时变化。因此，对于缓解肿瘤乏氧增强光热疗效和动态实时监测效果的研究是十分必要的。鉴于此，本课题以非小细胞肺癌为研究模型，通过一种近红外二区（NIR II）有机染料IRFE-PEG-DOTA（IPD）作为光热剂进行自组装，包载褪黑素，螯合Mn²⁺，然后修饰靶向肿瘤的核酸适配体，最终成功构建了一种能够缓解肿瘤乏氧以增强光热疗效并实现动态监测的新型多功能纳米探针MLT@IPD-Apt-Mn²⁺（IMAM NPs）。本研究主要内容如下：

    （1）MLT@IPD-Apt-Mn²⁺的构建与表征：通过IPD自组装，包载褪黑素，螯合Mn²⁺并配位核酸适配体构建了多功能纳米探针IMAM NPs。通过紫外吸收光谱和荧光光谱验证了IMAM NPs的成功合成。透射电镜图结果表明IMAM NPs的形态为球形，粒径大小为150 nm。通过稳定性实验研究验证了IMAM NPs具有良好的稳定性，在不同溶剂中7天内粒径无明显变化。光热实验结果表明IMAM NPs具有良好的光热稳定性和光热性能。通过体外释药实验研究验证IMAM NPs具有缓释药物的能力。类芬顿反应结果表明IMAM NPs能够与H₂O₂进行反应产生活性氧，具有化学动力学治疗特性。通过体外成像实验研究验证了IMAM NPs具有良好的MRI和NIR II成像性能。

    （2）MLT@IPD-Apt-Mn²⁺的体外生物学研究：细胞毒性实验结果表明IMAM NPs具有良好的生物安全性。通过细胞摄取和亲和实验研究验证了IMAM NPs在细胞水平具有良好的递送能力，可以更好的靶向肿瘤细胞。细胞在常氧、乏氧条件下培养实验研究验证了IMAM NPs具有缓解肿瘤细胞乏氧的能力。化疗/光热联合治疗实验结果表明IMAM NPs具有较强的细胞杀伤能力。蛋白印迹分析实验研究从分子水平上验证了IMAM NPs能够抑制HIF-1α的表达，具有缓解肿瘤细胞乏氧的能力。

   （3）MLT@IPD-Apt-Mn²⁺的在体生物学研究：体内毒性实验结果表明IMAM NPs具有良好的生物安全性，可以安全地用于在体水平。通过体内MRI/NIR II双模态成像实验研究验证了IMAM NPs具有良好的体内成像性能，能够监测肿瘤生长，在24 h时，探针积累在小鼠肿瘤部位到达最高峰，且信号强度能够维持到48 h。通过体内乏氧成像监测实验验证了IMAM NPs能够缓解小鼠肿瘤部位的乏氧情况。通过体内光热效果实验研究验证了IMAM NPs具有良好的体内光热性能，肿瘤部位温度达到43 ℃左右。通过抗肿瘤实验验证了在IMAM NPs缓解肿瘤部位乏氧时，加以光热治疗，能够对肿瘤产生更好的杀伤效果，从而杀死肿瘤。

Cancer is one of the major public health problems in the world, which seriously threatens human health. Currently, common cancer treatment methods include traditional surgery, radiotherapy and chemotherapy. However, due to the tumor hypoxic microenvironment, the tolerance and drug resistance of the tumor will be improved, seriously affecting the therapeutic effect of the tumor. Photothermal therapy (PTT) is an effective tumor therapy. It uses photothermal agents to perform local heat therapy under near infrared radiation and then kill tumor cells. Therefore, it is of great significance to explore whether alleviating tumor hypoxia can enhance photothermal efficacy. Although a few studies have discussed the relationship between the two, most of them are verified at the tissue level in vitro. With the evolution and growth of tumors, it is difficult to reflect the real-time change of tumor oxygen deficiency. Therefore, it is very necessary to study the effect of alleviating tumor hypoxia and enhancing photothermal efficacy and dynamic real-time monitoring. In view of this, in this study, non-small cell lung cancer was used as the research model, and a near infrared region II (NIR II) organic dye IRFE-PEG-DOTA (IPD) was used as the photothermal agent for self-assembly, embedding melatonin, then chelating Mn²⁺, and finally modifying the nucleic acid aptamer targeting tumor. Finally, a novel multifunctional nanoprobe MLT@IPD-Apt-Mn²⁺ (IMAM NPs) was successfully constructed, which can relieve tumor hypoxia to enhance photothermal efficacy and achieve dynamic monitoring. The main contents of this study are as follows:

 

(1) A bimodal diagnostic and therapeutic nanoprobe IMAM NPs was successfully constructed by IRFE-PEG-DOTA self-assembly containing melatonin, coordinating Mn²⁺ and aptamers. The IMAM NPs were characterized was being spherical with a particle size of 200 nm. Stability experiments show that IMAM NPs had a good stability and Mn²⁺ chelating stability. The photothermal experiments show that the IMAM NPs had good photothermal stability and photothermal performance at temperatures up to 60 ℃. In vitro drug release studies show that IMAM NPs had the ability to controlled release drugs. Fenton-like reaction shows that IMAM NPs could react with H₂O₂ to produce hydroxyl radical. In vitro imaging studies show that IMAM NPs had good MRI and NIR II imaging performance.

 

(2) Cell culture studies: IMAM NPs were investigated in cell culture studies. Cytotoxicity tests show that IMAM NPs had good biosafety and biocompatibility. Cell uptake and affinity studies show that IMAM NPs can be delivered well in cell culture studies and be better to target tumor cells. Hypoxia and normoxia cell culture studies demonstrate that the ability of IMAM NPs to relieve hypoxia in tumor cells. The combination of chemo/photothermal show that IMAM NPs had strong cell killing ability and could effectively kill tumor cells and induce cell apotosis, which could be used for subsequent in vivo anti-tumor experiments. Western blot analysis show that IMAM NPs inhibited HIF-1α expression, and had a certain ability to relieve oxygen deficiency in tumor cells.

 

(3) Animal studies: In vivo toxicity tests show that IMAM NPs had good biosafety and could be safely used in vivo. In vivo MRI/NIR II dual-mode imaging show that IMAM NPs had good in vivo imaging performance and could monitor tumor growth. At 24 h, the probe accumulation reached its peak at the tumors in mice, and the signal intensity could maintain until 48 h. Imaging monitoring of hypoxia in vivo show that IMAM NPs could alleviate hypoxia of tumors in mice. In vivo photothermal effects of IMAM NPs injected into mice shows that temperature of tumors reached about 43 ℃ after laser irradiation with good photothermal properties. Anti-tumor studies indicate that IMAM NPs, while alleviating the hypoxia of tumors, had a better killing effect on the tumors with laser irradiation, and thus achieved the effect of killing the tumors.

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