I am writing to submit our manuscript entitled，＂Self－Assembled Nanodrugs for Reversing Heat Tolerance of Photothermal Tumor Therapy in Vitro＂，for consideration for publication in RSC Advances． 我写信是为了提交我们的手稿，题为“用于逆转体外光热肿瘤治疗的耐热性”的自组装纳米药物，以供考虑在 RSC Advances 上发表。
In this paper，We synthesized a crocin dye（CRI）with excellent near infrared（NIR）absorption， which can be used to study the photothermal therapy of tumors in vitro．Atovaquinone（ATO），an inhibitors of oxidative phosphorylation，could self－assembled into nanodrugs CRI／ATO NPs with CRI through pi-pi\pi-\pi stacking，showing excellent near－infrared absorption and good biocompatibility． CRI／ATO NPs had an ideal size and could aggregate to tumor tissues through EPR effect．It had been proved that CRI／ATO NPs could release CRI and ATO in tumor acid environment，inhibit the electron transfer in mitochondrial respiratory chain and down－regulating the expression level of ATP and HSP70 in tumor cells．Finally，reversing the heat resistance of tumor was realized 本文合成了一种具有优异近红外（NIR）吸收性能的藏红花素染料（CRI），可用于体外肿瘤的光热治疗研究。 pi-pi\pi-\pi CRI/ATO NPs 具有理想的大小，可以通过 EPR 聚集到肿瘤组织 effect.It 已被证明 CRI/ATO NPs 在肿瘤酸性环境中能释放 CRI 和 ATO，抑制线粒体呼吸链中的电子转移，下调肿瘤细胞中 ATP 和 HSP70 的表达水平，最终实现逆转肿瘤的耐热性
This manuscript had not been previously published and is not currently under consideration by any other journal．All authors approved the manuscript and this submission．This research was fully sponsored by Project to Improve Central Medical Service And Support Capacity． 此手稿以前未发表过，目前未被任何其他期刊考虑。所有作者都批准了该手稿和此提交。本研究由提高中央医疗服务和支持能力项目全额赞助。
1 Department of General Surgery，The First Affiliated Hospital of Anhui Medical University，Hefei，China． 2 Anhui Public Health Clinical Center，Hefei，China． 1 安徽医科大学第一附属医院普外科，合肥。2 安徽省公共卫生临床中心，合肥
3 The Second Hospital of Anhui Medical university，Hefei，China． 3 安徽医科大学附属第二医院，合肥，中国.
＊Corresponding authors at：Department of General Surgery，The First Affiliated Hospital of Anhui Medical University North District， 100 Huaihai Avenue，Hefei 230012，Anhui，China *通讯作者：安徽省合肥市淮海大道100号安徽医科大学第一附属医院北区普外科 230012
E－mail address： 1662386945@qq.com\mathbf{1 6 6 2 3 8 6 9 4 5 @ q q . c o m} 电子邮件地址： 1662386945@qq.com\mathbf{1 6 6 2 3 8 6 9 4 5 @ q q . c o m}
ABSTRACT The heat tolerance of tumor is the main factor affecting the effect of tumor photothermal therapy（PTT）．In this study，we synthesized a croconaine dyes（CRI）with excellent near infrared（NIR）absorption，which could be used for photothermal therapy of tumors．Then oxidative phosphorylation inhibitor atovaquinone（ATO）and CRI were self－assembled into nano－ drug CRI／ATO NPs through pi-pi\pi-\pi stacking，so as to reverse the photothermal tolerance of tumors and improve the photothermal therapy effect．Specifically，CRI／ATO nanoparticles（NPs）showed excellent NIR absorption and good biocompatibility．In addition，CRI／ATO NPs had ideal sizes which were beneficial to penetrate into tumor tissues．Studies have confirmed that CRI／ATO NPs could inhibit the electron transfer in mitochondrial respiratory chain to downregulate the expression levels of ATP and HSP70 in tumor cells，and reverse tumor heat tolerance．The synergistic effect of CRI and ATO showed enhanced PTT effect and CRI／ATO NPs could provide a new theoretical basis for the clinical application of PTT． 摘要 肿瘤的耐热性是影响肿瘤光热疗法（PTT）效果的主要因素，本研究合成了一种具有优异近红外（NIR）吸收性能的三合碱染料（CRI），可用于肿瘤的光热疗法，然后通过 pi-pi\pi-\pi 氧化磷酸化抑制剂阿托伐醌（ATO）和 CRI 自组装成纳米药物 CRI/ATO NPs 研究证实，CRI/ATO biocompatibility.In NPs 可抑制线粒体呼吸链中的电子转移，下调肿瘤细胞中 ATP 和 HSP70 的表达水平，逆转肿瘤细胞中 ATP 和 HSP70 的表达水平 CRI 和 ATO 的协同作用显示 PTT 效应增强，CRI/ATO NPs 可为 PTT 的临床应用提供新的理论基础。
In recent years，nanodrugs have been widely studied for tumor treatment because of their unique physical and chemical properties and liable functional modification ^([1-3]){ }^{[1-3]} ．Drug delivery mediated by enhanced permeability and retention（EPR）effect was currently considered as an effective way to 近年来，纳米药物因其独特的理化性质和易受的功能修饰 ^([1-3]){ }^{[1-3]} 而被广泛研究用于肿瘤治疗，目前被认为是一种有效的方法，由增强渗透性和保留 （EPR） 效应介导的药物递送是
摘要 肿瘤的热耐受性是影响肿瘤光热治疗（PTT）效果的主要因素。在本研究中，我们合成了一种具有优异近红外（NIR）吸收性能的克罗康因染料（CRI），可用于肿瘤的光热治疗。然后通过 À À 堆叠，将氧化磷酸化抑制剂阿托喹酮（ATO）和 CRI 自组装成纳米药物 CRI／ATO NPs，以逆转肿瘤的热耐受性并提高光热治疗效果。具体来说， CRI／ATO 纳米颗粒（NPs ）具有优异的 NIR 吸收性能和良好的生物相容性。此外，CRI／ ATO NPs 具有理想的大小，有利于渗透到肿瘤组织中。研究表明，CRI／ATO NPs 可以抑制线粒体呼吸链中的电子传递，下调肿瘤细胞中 ATP 和 HSP70 的表达水平，逆转肿瘤热耐受性。CRI 和 ATO 的协同作用增强了 PTT 效果，CRI／ATO NPs 为 PTT 的临床应用提供了新的理论依据。
关键词：克罗康因染料；纳米药物；合成；热耐受性；光热治疗；
1 引言
近年来，由于纳米药物独特的物理化学性质和易于功能化，纳米药物在肿瘤治疗方面得到了广泛的研究。目前，通过增强渗透和滞留（EPR）效应介导的药物递送被认为是提高治疗效果的有效方法。
introduce nanodrugs into tumors，and nanodrugs accumulated preferentially in tumor tissues to reduce side effects and produce better therapeutic effects ^([4-6]){ }^{[4-6]} ．Many nanomedicine，such as iron oxide nanoparticles ^([7]){ }^{[7]} and gold nanorods ^([8]){ }^{[8]} ，most of them were difficult to be used in clinical trials， mainly because of their complex structure and potential toxicity to normal tissues ^([9]){ }^{[9]} ．Photothermal therapy（PTT）was a promising tumor treatment method，which mainly used the heat generated by photothermal agent（PTA）under laser irradiation to ablate tumor tissue ^([10-12]){ }^{[10-12]} ．In the process of photothermal therapy of tumor，the increase of temperature made cells produce heat shock proteins （HSPs）rapidly，which would lead to the tolerance to thermal stimulation with the results of the decreasing of photothermal therapy effect．The synthesis of heat shock protein required ATP energy ^([13]){ }^{[13]} ，so some studies tried to reduce the synthesis of HSPs by cutting ATP production in tumor cells through some ways ^([14]){ }^{[14]} ．Recently，croconaine dyes stood out among the near－infrared dyes with excellent photothermal performance because of their small molecular weight，simple synthesis， strong near－infrared absorption ^([15,16]){ }^{[15,16]} ，high PTC efficiency and good stability ^([17]){ }^{[17]} ，but there were few reports on the treatment of cancer with croconaine dyes in vitro．In this paper，self－assembled nanoparticles（designated as CRI／ATO NPs）based on oxidative phosphorylation inhibitor atovaquinone（ATO）and croconaine dye（CRI）were new nanodrugs developed to reverse the photothermal tolerance of tumors．By optimizing the ratio of ATO and CRI，ATO and CRI could self－assembled into uniform nanodrug CRI／ATO NPs through pi-pi\pi-\pi stacking which had excellent near infrared（NIR）absorption capacity and good long－term stability．CRI／ATO NPs could penetrate into tumor tissues through EPR effect and release ATO and CRI．ATO inhibited oxidative phosphorylation to cut ATP synthesis，so as to reduce the expression level of HSP70 protein and reverse the heat resistance of tumor cells．CRI／ATO NPs enhanced the photothermal efficacy of CRI （Scheme 1），providing new research ideas and methods for photothermal treatment of tumors． 将纳米药物引入肿瘤，纳米药物优先积累在肿瘤组织中，以减少副作用，产生更好的治疗效果 ^([4-6]){ }^{[4-6]} ，许多纳米药物，如氧化铁纳米颗粒 ^([7]){ }^{[7]} 和金纳米棒 ^([8]){ }^{[8]} ，大多难以用于临床试验，主要是因为它们结构复杂，对正常组织 ^([9]){ }^{[9]} 有潜在毒性 .光热疗法（PTT）是一种很有前途的肿瘤治疗方法，它主要利用光热剂（PTA）在激光照射下产生的热量消融肿瘤组织 ^([10-12]){ }^{[10-12]} 。在肿瘤光热疗法过程中，温度的升高使细胞迅速产生热激蛋白（HSPs），这会导致对热刺激的耐受性，结果光热疗法效果减弱。 ^([13]){ }^{[13]} 因此，一些研究试图通过一些方法通过某种方式 ^([14]){ }^{[14]} 切断肿瘤细胞中 ATP 的产生来减少 HSPs 的合成，近年来，色合碱染料因其分子量小、合成简单、近红外吸收 ^([15,16]){ }^{[15,16]} 能力强、PTC 效率高、稳定性 ^([17]){ }^{[17]} 好等优点，在近红外染料中脱颖而出，具有优异的光热性能 ，但关于 vitro.In 克罗康因染料治疗癌症的报道较少，基于氧化磷酸化抑制剂阿托伐醌（ATO）和克罗康因染料（CRI）的自组装纳米颗粒（命名为 CRI/ATO NPs）是针对优化 ATO 和 CRI 比例的 tumors.By 光热耐受性而开发的新型纳米药物，ATO 和 CRI 可以通过 pi-pi\pi-\pi 堆叠自组装成均匀的纳米药物 CRI/ATO NPs，具有优异的近红外（NIR）吸收性能 CRI/ATO NPs 可通过 EPR 效应渗透到肿瘤组织，释放 ATO 和 CRI.ATO 抑制氧化磷酸化，切断 ATP 合成，从而降低 HSP70 蛋白的表达水平，逆转肿瘤细胞的耐热性。
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