CN117224673A - Titanium-based nano material for enhancing acoustic power treatment of bladder cancer - Google Patents
Titanium-based nano material for enhancing acoustic power treatment of bladder cancer Download PDFInfo
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Abstract
Description
技术领域Technical field
本发明涉及生物医学材料技术领域，具体涉及一种可用于增强膀胱癌声动力治疗的钛基纳米材料。The invention relates to the technical field of biomedical materials, and in particular to a titanium-based nanomaterial that can be used to enhance sonodynamic therapy for bladder cancer.
背景技术Background technique
膀胱癌是我国泌尿系统常见肿瘤，全球十大癌症之一，发病率持续上升，复发率高，生活治疗差等这些问题依旧困扰着膀胱癌的治疗。随着中国老年化人口的增加，膀胱癌的患病率可能持续上升，膀胱癌的新型治疗方式亟待探索。根据2020年欧洲外科学会(EAU)指南，膀胱癌的治疗手段主要包括手术治疗、化疗药物灌注、靶向药物治疗等。尽管这些传统治疗方式在临床上取得不错的治疗效果，但手术等有创治疗大大增加患者心理压力和治疗成本，而化疗则因周期尿流稀释、偏离靶点和全身副作用明显等问题严重削弱药物的抗肿瘤效果。治疗膀胱癌后患者生活治疗差、治疗成本高、巨大的患者心理压力依旧存在改善的空间。因此，寻找一种膀胱癌治疗的新策略，提高膀胱灌注药物的稳定性和渗透能力，达到无创治疗的目的，对防止膀胱癌的转移和复发具有重要的临床价值和科学意义。Bladder cancer is a common tumor of the urinary system in my country and one of the top ten cancers in the world. The incidence rate continues to rise, the recurrence rate is high, and life treatment is poor. These problems still plague the treatment of bladder cancer. As China's aging population increases, the prevalence of bladder cancer may continue to rise, and new treatments for bladder cancer need to be explored urgently. According to the 2020 European Association of Surgery (EAU) guidelines, the treatment of bladder cancer mainly includes surgical treatment, chemotherapy drug infusion, targeted drug therapy, etc. Although these traditional treatments have achieved good clinical results, invasive treatments such as surgery greatly increase patients' psychological pressure and treatment costs, while chemotherapy seriously weakens the drug due to problems such as periodic urinary dilution, deviation from the target, and obvious systemic side effects. anti-tumor effect. After treatment for bladder cancer, there is still room for improvement in terms of poor living conditions, high treatment costs, and huge psychological pressure on patients. Therefore, finding a new strategy for bladder cancer treatment, improving the stability and penetration ability of intravesical drugs, and achieving the purpose of non-invasive treatment has important clinical value and scientific significance in preventing the metastasis and recurrence of bladder cancer.
近年来，随着纳米技术的发展，相应基于纳米技术的治疗手段逐渐引起了研究人员的关注和重视。为了提高膀胱癌的精确治疗效果，降低膀胱癌的转移和复发的风险，各种新型的治疗方法应用于膀胱癌的治疗中。其中光动力疗法(Photodynamic therapy，PDT)是用特定的激光照肿瘤部位，使选择性聚集在肿瘤组织的光敏剂活化，引起光化学反应杀死肿瘤的方法。该方法能够实现精确有效的治疗，副作用小。虽然在治疗非肌层浸润性膀胱癌中具有一定的效果，但是受到激光穿透能力的限制，即使通过尿道将光纤插入膀胱腔内，能够提高一定的治疗效果，但是仍会给病人带来痛苦，且对发生转移的膀胱癌治疗效果不显著。此外，卟啉类光敏剂的持续毒性也极大限制了PDT在膀胱癌中的应用。In recent years, with the development of nanotechnology, corresponding treatments based on nanotechnology have gradually attracted the attention and attention of researchers. In order to improve the precise treatment effect of bladder cancer and reduce the risk of metastasis and recurrence of bladder cancer, various new treatment methods are used in the treatment of bladder cancer. Among them, photodynamic therapy (PDT) is a method that uses a specific laser to illuminate the tumor site to activate the photosensitizer that selectively accumulates in the tumor tissue, causing a photochemical reaction to kill the tumor. This method enables precise and effective treatment with few side effects. Although it has a certain effect in the treatment of non-muscle invasive bladder cancer, it is limited by the penetration ability of the laser. Even if the optical fiber is inserted into the bladder cavity through the urethra, it can improve the treatment effect to a certain extent, but it will still cause pain to the patient. , and the therapeutic effect on metastatic bladder cancer is not significant. In addition, the persistent toxicity of porphyrin photosensitizers also greatly limits the application of PDT in bladder cancer.
为了提高组织穿透性和降低光敏剂的毒性，声动力疗法(Sonodynamic therapy，SDT)被开发用于膀胱癌治疗，这是一种在无创条件下利用超声波(US)激发聚集于肿瘤细胞的声敏剂，使其发生超声化学反应产生单线态氧(1O2)，造成肿瘤细胞死亡的一种治疗方法。与传统的膀胱灌注放化疗和光动力治疗相比，SDT被认为是一种高效的非侵入性癌症治疗方式之一，超声辐射可以穿透数十厘米厚的软组织，从而不需要内窥镜或其他膀胱内干预手段。鉴于声动力治疗定位精确、穿透性良好、无创治疗、依从性良好、副作用较少；同时膀胱癌独特的解剖位置在超声穿透深度范围中，应用声动力疗法治疗膀胱癌具有巨大潜能，有望成为膀胱癌治疗的新策略。但是，现有的声敏剂仅限于有机染料(如卟啉)或TiO2衍生的纳米材料，它们往往稳定性差或产生1O2的效率低。因此，发展生物相容性好，声敏效率高，稳定性好，作用原理清楚的纳米声敏剂是目前声动力治疗的研究热点。In order to improve tissue penetration and reduce the toxicity of photosensitizers, sonodynamic therapy (SDT) has been developed for bladder cancer treatment. It is a method that uses ultrasound (US) to excite sound waves accumulated in tumor cells under non-invasive conditions. A treatment method that uses a sensitizer to cause an ultrasonic chemical reaction to produce singlet oxygen ( 1 O 2 ), causing tumor cell death. Compared with traditional intravesical chemoradiotherapy and photodynamic therapy, SDT is considered to be one of the most efficient non-invasive cancer treatments. Ultrasound radiation can penetrate tens of centimeters of soft tissue, eliminating the need for endoscopes or other treatments. Intravesical interventions. In view of the fact that sonodynamic therapy has precise positioning, good penetration, non-invasive treatment, good compliance, and few side effects; at the same time, the unique anatomical location of bladder cancer is within the range of ultrasound penetration depth, so the application of sonodynamic therapy to treat bladder cancer has great potential and is promising. Becoming a new strategy for bladder cancer treatment. However, existing sonosensitizers are limited to organic dyes (such as porphyrins) or TiO2- derived nanomaterials, which often have poor stability or low efficiency in producing 1O2 . Therefore, the development of nano-sound sensitizers with good biocompatibility, high sonosensitivity efficiency, good stability, and clear principles of action is a current research hotspot in sonodynamic therapy.
发明内容Contents of the invention
本发明的目的在于克服现有技术的不足，提供一种可用于增强膀胱癌声动力治疗的钛基纳米材料。The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a titanium-based nanomaterial that can be used to enhance sonodynamic therapy of bladder cancer.
为实现上述目的，本发明采取的技术方案为：一种钛基纳米材料，所述钛基纳米材料包括二氧化钛和Ru。本发明通过中空纳米球TiO2装载钌配合物Ru后显著降低TiO2-Ru-PEG的带隙，从4.73eV降至3.76eV活跃了电子之间的传递，从而增强电子空穴对的分离，提高ROS生成率，在超声照射下与细胞内的氧气结合后爆发性产生1O2、O2.-损伤线粒体引起肿瘤细胞凋亡，从而达到增强声动力治疗膀胱癌的效果。In order to achieve the above object, the technical solution adopted by the present invention is: a titanium-based nanomaterial, and the titanium-based nanomaterial includes titanium dioxide and Ru. The present invention significantly reduces the band gap of TiO 2 -Ru-PEG by loading the hollow nanosphere TiO 2 with the ruthenium complex Ru, from 4.73eV to 3.76eV, which activates the transfer between electrons, thereby enhancing the separation of electron-hole pairs. Increase the ROS generation rate, and explosively produce 1 O 2 and O 2 after combining with intracellular oxygen under ultrasound irradiation. - Damage mitochondria and cause tumor cell apoptosis, thus achieving the effect of enhancing sonodynamic treatment of bladder cancer.
作为本发明所述钛基纳米材料的优选实施方式，所述二氧化钛为中空纳米球二氧化钛。As a preferred embodiment of the titanium-based nanomaterial of the present invention, the titanium dioxide is hollow nanosphere titanium dioxide.
本发明还提供所述钛基纳米材料的制备方法，包括以下步骤：The invention also provides a preparation method of the titanium-based nanomaterial, which includes the following steps:
(1)分别将二氧化钛和Ru溶于DMSO，得二氧化钛溶液和Ru溶液；(1) Dissolve titanium dioxide and Ru in DMSO respectively to obtain titanium dioxide solution and Ru solution;
(2)将Ru溶液滴加到二氧化钛溶液溶液中，反应后离心得TiO2-Ru沉淀；(2) Add the Ru solution dropwise into the titanium dioxide solution, and centrifuge after the reaction to obtain TiO2-Ru precipitation;
(3)取DSPE-PEG-2000溶于超纯水得DSPE-PEG-2000溶液；采用DSPE-PEG-2000溶液重悬TiO2-Ru沉淀并反应，离心得沉淀，即为所述钛基纳米材料。(3) Dissolve DSPE-PEG-2000 in ultrapure water to obtain a DSPE-PEG-2000 solution; use the DSPE-PEG-2000 solution to resuspend and react the TiO2-Ru precipitate, and centrifuge to obtain the precipitate, which is the titanium-based nanomaterial .
作为本发明所述钛基纳米材料的制备方法的优选实施方式，所述步骤(2)中的反应为避光搅拌反应12hAs a preferred embodiment of the preparation method of titanium-based nanomaterials of the present invention, the reaction in step (2) is a light-protected stirring reaction for 12 hours.
作为本发明所述钛基纳米材料的制备方法的优选实施方式，所述步骤(3)中的反应于300-1000r的转速下搅拌反应3-8h。As a preferred embodiment of the preparation method of titanium-based nanomaterials of the present invention, the reaction in step (3) is carried out with stirring at a rotation speed of 300-1000r for 3-8 hours.
本发明还提供所述的钛基纳米材料在制备声敏剂中的应用。The invention also provides the application of the titanium-based nanomaterial in preparing sonosensitizer.
本发明还提供一种声敏剂，所述声敏剂包括所述的钛基纳米材料。The present invention also provides a sound sensitizer, which includes the titanium-based nanomaterial.
本发明还提供所述的钛基纳米材料在肿瘤声动力治疗中的应用。The invention also provides the application of the titanium-based nanomaterial in sonodynamic therapy of tumors.
本发明还提供所述的钛基纳米材料在制备抗肿瘤药物中的应用。The invention also provides the application of the titanium-based nanomaterial in preparing anti-tumor drugs.
作为本发明所述应用的优选实施方式，所述肿瘤包括膀胱癌。As a preferred embodiment of the application of the present invention, the tumor includes bladder cancer.
本发明的有益效果：本发明首次用钌配合物Ru与中空纳米球TiO2开发一种安全无毒、低成本、制备简单的钛基纳米材料TiO2-Ru-PEG。本发明的钛基纳米材料TiO2-Ru-PEG能够显著提高ROS的生成率，为改善声动力治疗提供新策略；本发明的钛基纳米材料TiO2-Ru-PEG可被膀胱癌细胞有效摄取，超声照射下与细胞内的氧气结合后爆发性产生1O2、O2.-损伤线粒体引起肿瘤细胞凋亡，从而达到增强声动力治疗膀胱癌的效果；本发明的钛基纳米材料TiO2-Ru-PEG制备方法简单，产率高，可重复性好，具备大规模生产的潜力。Beneficial effects of the present invention: For the first time, the present invention uses ruthenium complex Ru and hollow nanospheres TiO 2 to develop a safe, non-toxic, low-cost, and simple-to-prepare titanium-based nanomaterial TiO 2 -Ru-PEG. The titanium-based nanomaterial TiO 2 -Ru-PEG of the present invention can significantly increase the generation rate of ROS and provide a new strategy for improving sonodynamic therapy; the titanium-based nanomaterial TiO 2 -Ru-PEG of the present invention can be effectively taken up by bladder cancer cells , combines with intracellular oxygen under ultrasonic irradiation and explosively produces 1 O 2 and O 2. - damages mitochondria and causes tumor cell apoptosis, thereby achieving the effect of enhancing sonodynamic treatment of bladder cancer; the titanium-based nanomaterial TiO 2 of the present invention -The preparation method of Ru-PEG is simple, has high yield and good reproducibility, and has the potential for large-scale production.
附图说明Description of drawings
图1为TiO2-Ru-PEG的制备过程及其用于增强膀胱癌声动力治疗的原理示意图。Figure 1 is a schematic diagram of the preparation process of TiO 2 -Ru-PEG and its principle for enhancing sonodynamic therapy of bladder cancer.
图2为TiO2-Ru-PEG的TEM、SEM、zeta电位、傅里叶红外光谱、mapping元素分析、UV-VIS-NIR、BET介孔分析、XRD、XPS表征结果。Figure 2 shows the TEM, SEM, zeta potential, Fourier transform infrared spectrum, mapping element analysis, UV-VIS-NIR, BET mesopore analysis, XRD, and XPS characterization results of TiO 2 -Ru-PEG.
图3：A-C分别为Ru3P1、Ru3d5、Ti2P3价态的高分辨率XPS图谱；D为Ru、TiO2、TiO2-Ru的单性态氧；E为Ru、TiO2、TiO2-Ru的带隙；F为Ru、TiO2、TiO2-Ru的价带；G为TiO2-Ru-PEG的带隙和电子转移示意图。Figure 3: AC is the high-resolution XPS spectrum of the valence states of Ru3P1, Ru3d5, and Ti2P3 respectively; D is the singlet oxygen of Ru, TiO 2 , and TiO 2 -Ru; E is the band of Ru, TiO 2 , and TiO 2 -Ru Gap; F is the valence band of Ru, TiO 2 and TiO 2 -Ru; G is the band gap and electron transfer diagram of TiO 2 -Ru-PEG.
图4：A-C为流式细胞仪检测TiO2-Ru-PEG在MB49细胞和SV-HUC-1细胞中的吸收情况；D-I为TiO2-Ru-PEG对不同细胞的活力的影响。Figure 4: AC is a flow cytometer to detect the absorption of TiO 2 -Ru-PEG in MB49 cells and SV-HUC-1 cells; DI is the effect of TiO 2 -Ru-PEG on the viability of different cells.
图5为TiO2-Ru-PEG的细胞毒性结果。Figure 5 shows the cytotoxicity results of TiO 2 -Ru-PEG.
图6：A为细胞(Calcein-AM/PI)染色结果图；B为细胞克隆染色图；C为显微镜拍摄划痕情况图；D为半定量分析克隆结果；E为半定量分析克隆结果。Figure 6: A is the result of cell (Calcein-AM/PI) staining; B is the staining of cell clones; C is the picture of scratches taken by a microscope; D is the result of semi-quantitative analysis of clones; E is the result of semi-quantitative analysis of clones.
图7：A为MB49荷瘤小鼠的声动力治疗过程示意图；B-D为小动物成像仪检测TiO2-Ru-PEG在体内的近红外荧光分布情况；E为各组小鼠肿瘤实体照片；F-G为各组小鼠肿瘤体积变化；H-I为各组小鼠肿瘤肿瘤抑制率。Figure 7: A is a schematic diagram of the sonodynamic treatment process of MB49 tumor-bearing mice; BD is a small animal imager detecting the near-infrared fluorescence distribution of TiO 2 -Ru-PEG in the body; E is a photo of the tumor entities of mice in each group; FG is the change in tumor volume of mice in each group; HI is the tumor inhibition rate of mice in each group.
图8：A为各组肿瘤切片H&E染色结果；B为各分组的裸鼠心脏、肝脏、脾脏、肺脏及肾脏的组织形态；C为各组小鼠肝肾功能指标。Figure 8: A is the H&E staining results of tumor sections in each group; B is the tissue morphology of the heart, liver, spleen, lungs and kidneys of nude mice in each group; C is the liver and kidney function indicators of mice in each group.
具体实施方式Detailed ways
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。The above contents of the present invention will be further described in detail below through specific implementation methods in the form of examples. However, this should not be understood to mean that the scope of the above subject matter of the present invention is limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention.
实施例1钛基纳米材料TiO2-Ru-PEG的制备Example 1 Preparation of titanium-based nanomaterial TiO 2 -Ru-PEG
本实施例提供一种钛基纳米材料TiO2-Ru-PEG的制备方法，如图1所示，包括以下步骤：This embodiment provides a preparation method of titanium-based nanomaterial TiO 2 -Ru-PEG, as shown in Figure 1, including the following steps:
(1)中空纳米球形TiO2的制备：常温下将0.86ml硅酸四乙酯(TEOS)、0.46ml氨水、23ml乙醇和4.3ml超纯水置于烧瓶中低速400r搅拌4h后，用10000r离心10min并用5ml乙醇重悬，得重悬溶液A；将0.2g羟丙基纤维素(HPC)加入90ml乙醇和0.48ml超纯水的混合溶液中低速400r搅拌3h，得混合溶液A；将4ml钛酸四丁酯溶于18ml乙醇溶液中，得混合溶液B；将重悬溶液A缓慢滴加到混合溶液A中低速600r搅拌0.5h，然后将混合溶液B在低速600r搅拌下续滴(0.5ml/min)滴入上述混合溶液；滴加结束后置于硅油中加热到85℃回流100min后立刻离心(4000r 10min)并用20ml超纯水重悬，得重悬溶液B；向重悬溶液B中续滴加入4ml浓度为2.5M的NaOH并低速300r搅拌6h后立刻离心(8000r 10min)取沉淀，将沉淀置于65℃干燥室内12h称量沉淀质量并按照150mg/ml计算所需的超纯水重悬；根据重悬所需的超纯水体积V1，按照V2＝V1/12000后计算所需的浓盐酸体积V2，并加入重悬溶液中低速搅拌0.5h后离心(8000r 10min)取沉淀，用40ml离子水反复清洗沉淀3次，用10ml超纯水重悬，得中空纳米球形TiO2。(1) Preparation of hollow nanospherical TiO 2 : Place 0.86ml tetraethyl silicate (TEOS), 0.46ml ammonia, 23ml ethanol and 4.3ml ultrapure water in a flask at room temperature, stir at low speed 400r for 4 hours, and then centrifuge at 10000r 10 min and resuspend with 5 ml ethanol to obtain resuspension solution A; add 0.2g hydroxypropyl cellulose (HPC) to a mixed solution of 90 ml ethanol and 0.48 ml ultrapure water and stir at low speed 400r for 3 hours to obtain mixed solution A; add 4 ml titanium Dissolve tetrabutyl acid in 18 ml of ethanol solution to obtain mixed solution B; slowly add the resuspended solution A dropwise into the mixed solution A and stir at a low speed of 600r for 0.5h, then continue to drop the mixed solution B (0.5ml) at a low speed of 600r. /min) dropwise into the above mixed solution; after the dropwise addition, place it in silicone oil and heat to 85°C, reflux for 100min, then immediately centrifuge (4000r for 10min) and resuspend with 20ml of ultrapure water to obtain resuspension solution B; add to resuspension solution B Add 4 ml of NaOH with a concentration of 2.5M drop by drop and stir at low speed 300r for 6h. Immediately centrifuge (8000r for 10min) to collect the precipitate. Place the precipitate in a drying room at 65°C for 12h. Weigh the precipitate mass and calculate the required ultrapure water according to 150mg/ml. Resuspend; According to the ultrapure water volume V 1 required for resuspension, calculate the required concentrated hydrochloric acid volume V 2 according to V 2 = V 1 /12000, and add it to the resuspension solution, stir at low speed for 0.5h, and then centrifuge (8000r 10min ) Take the precipitate, wash it three times with 40 ml of ionized water, and resuspend it with 10 ml of ultrapure water to obtain hollow nanosphere TiO 2 .
(2)Ru的制备：将dppz(0.100g,0.355mmol)和cis-Ru(phen)2Cl2(0.188g,0.355mmol)加入乙醇和水(3:1)的组分溶剂中，得到[Ru(phen)2dppz](PF6)2；将[Ru(phen)2dppz](PF6)2在氮气气氛下加热回流8小时，使产物为橙色固体；然后用PF6-交换Cl-，用硅胶柱(CH3CN:甲苯＝1:1)纯化原料，减压去除挥发物，其余固体用CH3CN和乙醚重结晶得到红色晶体，即Ru。(2) Preparation of Ru: Add dppz (0.100g, 0.355mmol) and cis-Ru(phen) 2 Cl 2 (0.188g, 0.355mmol) into the component solvent of ethanol and water (3:1) to obtain [ Ru(phen) 2 dppz](PF6) 2 ; [Ru(phen) 2 dppz](PF6) 2 was heated and refluxed for 8 hours under a nitrogen atmosphere to make the product an orange solid; then PF6 - was used to exchange Cl - and silica gel was used The raw material was purified by column (CH3CN:toluene=1:1), volatiles were removed under reduced pressure, and the remaining solid was recrystallized with CH3CN and diethyl ether to obtain red crystals, namely Ru.
(3)TiO2-Ru-PEG的制备：(3) Preparation of TiO 2 -Ru-PEG:
1)将20mg中空纳米球TiO2溶于1ml DMSO溶液，得TiO2溶液；将9mg Ru溶于1ml DMSO溶液，得Ru溶液；将Ru溶液缓慢续滴加TiO2溶液中并避光搅拌12h，离心(8000r 10min)后采用超纯水清洗一次得TiO2-Ru沉淀；1) Dissolve 20 mg of hollow nanosphere TiO 2 in 1 ml of DMSO solution to obtain a TiO 2 solution; dissolve 9 mg of Ru in 1 ml of DMSO solution to obtain a Ru solution; slowly add the Ru solution dropwise into the TiO 2 solution and stir in the dark for 12 hours. Centrifuge (8000r for 10min) and wash once with ultrapure water to obtain TiO 2 -Ru precipitate;
2)称取20mg DSPE-PEG-2000溶于10ml超纯水得溶液，采用该溶液重悬TiO2-Ru沉淀并低速(500r)搅拌5h后离心，采用超纯水清洗沉淀一次后用10ml超纯水重悬，得所述TiO2-Ru-PEG。2) Weigh the solution obtained by dissolving 20 mg DSPE-PEG-2000 in 10 ml ultrapure water. Use this solution to resuspend the TiO 2 -Ru precipitate and stir it at low speed (500r) for 5 hours and then centrifuge. Use ultrapure water to wash the precipitate once and then use 10 ml ultrapure water to Resuspend in pure water to obtain the TiO 2 -Ru-PEG.
TiO2-Ru-PEG的表征：利用透射电镜(TEM)、扫描电镜(SEM)、紫外可见光分光光度计(UV-VIS-NIR)、傅里叶红外光谱仪、mapping元素分析、介孔分析、X射线衍射(XRD)、射线光电子能谱(XPS)、电子自旋共振(ESR)、ZETA电位评估TiO2-Ru-PEG的稳定性以及增强声动力的机制。结果如图2所示。由图2A-B可知，TiO2-Ru-PEG为表面凹凸不平，直径约250nm的中空球状形貌，壳层厚度约为40-50nm，最外表明修饰的一层PEG厚度约为2-5nm；图2C为Ru-PEG、TiO2-PEG、TiO2-Ru-PEG的zeta电位；图2D为Ru、TiO2、TiO2-Ru的傅里叶红外光谱；图2E-F为利用元素分析检测TiO2-Ru-PEG以及酶标仪检测TiO2-Ru-PEG在波长410nm的吸收情况；上述结果均表明中空纳米球TiO2可成功装载钌配合物Ru，证明新型声敏剂TiO2-Ru-PEG的成功合成。通过计算在410nm波长Ru-PEG和TiO2-Ru-PEG吸收强度，算得TiO2-Ru-PEG装载Ru的装载率约为40％，这为中空纳米球TiO2与金属配合物Ru的结合提供了基础。从元素分析结果显示，装载的金属钌配合物Ru主要集中在中空纳米球的壳层中。由图2G、H可知，通过介孔分析检测TiO2、TiO2-Ru，TiO2装载Ru后TiO2-Ru仍然为介孔材料，其比表面积由29.3368m2/g增大至271.0138m2/g，而孔径则由13.4774nm减小至4.5933nm，表明金属钌配合物可富集在中空纳米球的壳层孔隙中，这与元素分析的结果一致，装载Ru配合物后TiO2-Ru比表面积显著增大，增大与氧气的接触面增加ROS的生成效率，从而增强声动力治疗。通过紫外光可见光分度计测量其在不同波长的吸收强度(图2I)，XRD(图2J)以及XPS总谱(图2K)分析Ru、TiO2、TiO2-Ru的结构，结果表明TiO2-Ru合成后具有稳定的结构且其价态主要为Ru3P、Ru3d、Ti2P，这些结果也间接证明TiO2成功装载Ru合成了中空纳米球TiO2-Ru，验证了中空纳米球TiO2-Ru-PEG可成功低成本、稳定的制备。Characterization of TiO 2 -Ru-PEG: using transmission electron microscope (TEM), scanning electron microscope (SEM), ultraviolet-visible spectrophotometer (UV-VIS-NIR), Fourier transform infrared spectrometer, mapping element analysis, mesopore analysis, X X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), electron spin resonance (ESR), and zeta potential were used to evaluate the stability of TiO 2 -Ru-PEG and the mechanism of enhanced sonodynamic force. The results are shown in Figure 2. As can be seen from Figure 2A-B, TiO 2 -Ru-PEG has a hollow spherical shape with an uneven surface and a diameter of about 250nm. The thickness of the shell layer is about 40-50nm. The thickness of the outer layer of PEG modified is about 2-5nm. ; Figure 2C is the zeta potential of Ru-PEG, TiO 2 -PEG, and TiO 2 -Ru-PEG; Figure 2D is the Fourier transform infrared spectrum of Ru, TiO 2 , and TiO 2 -Ru; Figure 2E-F is the use of elemental analysis Detect TiO 2 -Ru-PEG and use a microplate reader to detect the absorption of TiO 2 -Ru-PEG at a wavelength of 410nm; the above results indicate that hollow nanospheres TiO 2 can be successfully loaded with ruthenium complex Ru, proving that the new sonosensitizer TiO 2 - Successful synthesis of Ru-PEG. By calculating the absorption intensity of Ru-PEG and TiO 2 -Ru-PEG at a wavelength of 410 nm, it is calculated that the loading rate of TiO 2 -Ru-PEG loaded with Ru is about 40%, which provides a good basis for the combination of hollow nanospheres TiO 2 and metal complex Ru foundation. The elemental analysis results show that the loaded metal ruthenium complex Ru is mainly concentrated in the shell of the hollow nanospheres. As can be seen from Figure 2G and H, TiO 2 and TiO 2 -Ru were detected through mesoporous analysis. After TiO 2 is loaded with Ru, TiO 2 -Ru is still a mesoporous material, and its specific surface area increases from 29.3368m 2 /g to 271.0138m 2 /g, while the pore diameter decreased from 13.4774nm to 4.5933nm, indicating that the metal ruthenium complex can be enriched in the shell pores of the hollow nanospheres, which is consistent with the results of elemental analysis. After loading the Ru complex, TiO 2 -Ru The specific surface area is significantly increased, increasing the contact surface with oxygen and increasing the generation efficiency of ROS, thereby enhancing sonodynamic therapy. The structure of Ru, TiO 2 and TiO 2 -Ru was analyzed by measuring the absorption intensity at different wavelengths with a UV-visible light protractor (Figure 2I), XRD (Figure 2J) and XPS spectrum (Figure 2K). The results showed that TiO 2 -Ru has a stable structure after synthesis and its valence states are mainly Ru3P, Ru3d, and Ti2P. These results also indirectly prove that TiO 2 was successfully loaded with Ru to synthesize hollow nanospheres TiO 2 -Ru, verifying that hollow nanospheres TiO 2 -Ru- PEG can be successfully prepared in a low-cost and stable manner.
为了探究TiO2-Ru-PEG增强声动力治疗的机理，通过比较Ru3P1、Ru3d5、Ti2P3价态的高分辨率XPS图谱，结果由图3A-C所示：由于Ti和Ru之间的相互作用，Ru3d5、Ti2P3的特征峰均发生了轻微的位移，距离分别为0.38eV、0.35eV，表明Ru通过Ti-Ru配位的方式与中空纳米球TiO2结合，这与介孔分析以及mapping元素分析的结果相一致，即Ru与TiO2中以Ti-Ru配位方式结合在TiO2中空纳米球的壳层孔隙中导致TiO2-Ru比表面积增大而孔径则相应变小。TiO2-Ru的Ru3P1价态为495.18eV，而Ru的Ru3P1价态为484.28eV，表明装载后的钌配合物Ru在TiO2-Ru体系中发生显著的结合能迁移处于高氧化态，这种高氧化态的TiO2-Ru与氧气接触后可高效的产生单性态氧，在超声的照射下可显著增强声动力治疗。得益于化学键形式的结合赋予中空纳米球TiO2-Ru-PEG稳定的结构，至少一个月没有颜色变化，这种稳定的结构对于生物应用是至关重要的。通过ESR以及紫外可见光吸收光谱、XPS测量了Ru、TiO2、TiO2-Ru的单性态氧、带隙以及价带，由图3D-F可知，TiO2-Ru-PEG产生单性态氧的效率更高。Ru、TiO2、TiO2-Ru带隙分别为3.99eV、4.73eV、3.76eV。Ru、TiO2、TiO2-Ru导带分别为2.85eV、2.69eV、2.26eV。显然，TiO2装载金属钌配合物Ru后显著降低了TiO2-Ru的带隙。通过计算Ru、TiO2、TiO2-Ru的价带和导带，结合XPS高分辨中Ru3d5、Ti2P3价态结合能的变化，得出TiO2-Ru-PEG的带隙和电子转移示意图，如图3G所示，在超声照射下，TiO2-Ru-PEG在超声照射后TiO2和Ru的电子传递增强电子空穴对的分离效率，从而显著增强单线态氧等活性氧的生成，增强了声动力疗效。In order to explore the mechanism of TiO2-Ru-PEG enhanced sonodynamic therapy, the high-resolution XPS spectra of the valence states of Ru3P1, Ru3d5, and Ti2P3 were compared. The results are shown in Figure 3A-C: Due to the interaction between Ti and Ru, Ru3d5 The characteristic peaks of Ti2P3 and Ti2P3 are slightly shifted, with distances of 0.38eV and 0.35eV respectively, indicating that Ru is combined with the hollow nanosphere TiO2 through Ti-Ru coordination. This is consistent with the results of mesopore analysis and mapping element analysis. Consistent with this, that is, Ru is combined with TiO 2 in the Ti-Ru coordination mode in the shell pores of TiO 2 hollow nanospheres, resulting in an increase in the TiO 2 -Ru specific surface area and a corresponding decrease in pore size. The Ru3P1 valence state of TiO 2 -Ru is 495.18eV, while the Ru3P1 valence state of Ru is 484.28eV, indicating that the loaded ruthenium complex Ru undergoes significant binding energy migration in the TiO 2 -Ru system and is in a high oxidation state. This Highly oxidized TiO 2 -Ru can efficiently generate singlet oxygen after contact with oxygen, which can significantly enhance sonodynamic therapy under ultrasound irradiation. Thanks to the combination of chemical bonds that give the hollow nanospheres TiO 2 -Ru-PEG a stable structure, there is no color change for at least a month. This stable structure is crucial for biological applications. The singlet oxygen, band gap and valence band of Ru, TiO 2 and TiO 2 -Ru were measured through ESR, UV-visible absorption spectroscopy and XPS. As shown in Figure 3D-F, TiO 2 -Ru-PEG produces singlet oxygen. higher efficiency. The band gaps of Ru, TiO 2 and TiO 2 -Ru are 3.99eV, 4.73eV and 3.76eV respectively. The conduction bands of Ru, TiO 2 and TiO 2 -Ru are 2.85eV, 2.69eV and 2.26eV respectively. Obviously, loading TiO 2 with the metal ruthenium complex Ru significantly reduces the band gap of TiO 2 -Ru. By calculating the valence band and conduction band of Ru, TiO 2 and TiO 2 -Ru, combined with the changes in the valence state binding energy of Ru3d5 and Ti2P3 in XPS high resolution, the band gap and electron transfer schematic diagram of TiO 2 -Ru-PEG is obtained, as shown As shown in Figure 3G, under ultrasonic irradiation, the electron transfer of TiO 2 and Ru in TiO 2 -Ru-PEG after ultrasonic irradiation enhances the separation efficiency of electron-hole pairs, thereby significantly enhancing the generation of reactive oxygen species such as singlet oxygen and enhancing Sonodynamic efficacy.
实施例2Example 2
本实施例对实施例1制备得到的钛基纳米材料TiO2-Ru-PEG的安全性及声动力疗效进行体外验证，具体实验方法如下：This example conducts in vitro verification of the safety and sonodynamic efficacy of the titanium-based nanomaterial TiO 2 -Ru-PEG prepared in Example 1. The specific experimental methods are as follows:
(1)细胞培养：本实验所使用的细胞为正常膀胱上皮细胞SV-HUC-1、鼠源膀胱癌细胞MB49。所有细胞于pH7.4，37℃，5％CO2的含有10％胎牛血清的DMEM培养基中培养，当达到80％-90％细胞密度时用胰酶消化细胞后轻轻吹打细胞形成用于后续细胞实验的细胞悬浮液。(1) Cell culture: The cells used in this experiment were normal bladder epithelial cells SV-HUC-1 and mouse bladder cancer cell MB49. All cells were cultured in DMEM medium containing 10% fetal calf serum at pH 7.4, 37°C, 5% CO2 . When reaching 80%-90% cell density, digest the cells with trypsin and gently pipet to form cells. cell suspension for subsequent cell experiments.
(2)TiO2-Ru-PEG的细胞摄取过程：(2) Cellular uptake process of TiO2-Ru-PEG:
1)合成香豆素-6标记的TiO2-Ru-PEG：将终浓度为2ug/mL的香豆素-6加入到TiO2-Ru-PEG溶液中，避光搅拌8h，8000r离心10min，收集沉淀，用超纯水清洗三次，最后用10ml超纯水重悬，即得香豆素-6标记的TiO2-Ru-PEG；1) Synthesis of coumarin-6 labeled TiO 2 -Ru-PEG: Add coumarin-6 with a final concentration of 2ug/mL into the TiO 2 -Ru-PEG solution, stir in the dark for 8 hours, and centrifuge at 8000r for 10 minutes. Collect the precipitate, wash it three times with ultrapure water, and finally resuspend it in 10 ml of ultrapure water to obtain coumarin-6 labeled TiO 2 -Ru-PEG;
2)将MB49细胞以5x104cell/ml的密度接种在12孔板培养板中(1ml/孔)，贴壁后用贴壁后用14μg/mL(以Ru定量)香豆素-6标记的TiO2-Ru-PEG(绿色)分别处理不同的时间；用溶酶体探针Lyso-tracker(红色)、细胞核探针DPAI(蓝色)分别处理细胞1h和15min，用荧光显微镜观察TiO2-Ru-PEG进入细胞的过程。2) Seed MB49 cells in a 12-well culture plate at a density of 5x10 4 cells/ml (1ml/well). After adhesion, use 14 μg/mL (quantitated by Ru) coumarin-6 labeled TiO 2 -Ru-PEG (green) was treated for different times respectively; cells were treated with lysosome probe Lyso-tracker (red) and nuclear probe DPAI (blue) for 1h and 15min respectively, and TiO 2 - was observed with a fluorescence microscope. The process of Ru-PEG entering cells.
3)流式细胞仪检测TiO2-Ru-PEG的选择性吸收：MB49细胞和SV-HUC-1细胞以2×104cells/mL的密度接种在6cm的培养皿(6mL/皿)，贴壁后用香豆素-6标记的TiO2-Ru-PEG(以Ru定量，14μg/mL)分别处理不同的时间(0、2、4、8、12h)；弃上清用PBS清洗三次，胰酶消化收集细胞，继续用PBS清洗一次，用200目的尼龙网过滤细胞悬液，流式细胞仪检测TiO2-Ru-PEG在MB49细胞和SV-HUC-1细胞中的吸收情况。3) Flow cytometry to detect the selective absorption of TiO2-Ru-PEG: MB49 cells and SV-HUC-1 cells were seeded in a 6cm culture dish (6mL/dish) at a density of 2×10 4 cells/mL and adhered to the wall. Afterwards, the coumarin-6 labeled TiO 2 -Ru-PEG (quantitated by Ru, 14 μg/mL) was treated for different times (0, 2, 4, 8, 12 h); the supernatant was discarded and washed three times with PBS. Collect the cells after enzymatic digestion, continue to wash them once with PBS, filter the cell suspension with a 200-mesh nylon mesh, and detect the absorption of TiO 2 -Ru-PEG in MB49 cells and SV-HUC-1 cells with a flow cytometer.
结果如图4所示。由图4A可知，MB49对TiO2-Ru-PEG的摄取具有时间依赖性，TiO2-Ru-PEG(香豆素-6标记)孵育细胞1h后，溶酶体中显示出微弱的绿色荧光信号，随着药物孵育时间的延长，溶酶体出的绿色荧光信号逐渐增强，孵育8h后TiO2-Ru-PEG的绿色信号扩展到整个胞浆环境，表明TiO2-Ru-PEG对MB49细胞具有良好的渗透能力，容易被膀胱癌细胞MB49摄取，这种内吞作用具有时间依赖性。由图4B-C可知，MB49细胞和SV-HUC-1细胞均在摄取TiO2-Ru-PEG 8h后达到基本饱和状态，但相同时间下MB49细胞摄取TiO2-Ru-PEG的能力较于SV-HUC-1细胞更强，表明TiO2-Ru-PEG具有良好的生物相容性。The results are shown in Figure 4. As can be seen from Figure 4A, the uptake of TiO 2 -Ru-PEG by MB49 is time-dependent. After cells were incubated with TiO 2 -Ru-PEG (labeled with coumarin-6) for 1 hour, a weak green fluorescence signal was displayed in lysosomes. , with the prolongation of drug incubation time, the green fluorescence signal from lysosomes gradually increased, and after 8 hours of incubation, the green signal of TiO 2 -Ru-PEG expanded to the entire cytoplasmic environment, indicating that TiO 2 -Ru-PEG has an effect on MB49 cells. It has good penetration ability and is easily taken up by bladder cancer cell MB49. This endocytosis is time-dependent. As can be seen from Figure 4B-C, both MB49 cells and SV-HUC-1 cells reached a basically saturated state after 8 hours of uptake of TiO 2 -Ru-PEG, but the ability of MB49 cells to uptake TiO 2 -Ru-PEG was lower than that of SV at the same time. -HUC-1 cells are stronger, indicating that TiO 2 -Ru-PEG has good biocompatibility.
(3)TiO2-Ru-PEG对不同细胞的活力的影响(3) Effect of TiO 2 -Ru-PEG on the viability of different cells
在96孔板中每孔分别接种100ul细胞浓度为4x104cell/ml的SV-HUC-1、MB49的细胞悬浮液体，并在5％CO2湿润的环境中于37℃温育24小时。将SV-HUC-1、MB49分为control组、US组、Ru-PEG组、TiO2-PEG组、TiO2-Ru-PEG组。待细胞贴壁，以Ru浓度为定量分别向各组加入不同浓度(2、4、7、14、28)ug的Ru-PEG、TiO2-PEG、TiO2-Ru-PEG与细胞共同孵育8h后，让超声组的的细胞接受强度为1MHz，1.5w/cm2，占空比40％超声照射1min，并在37℃下孵育48h，孵育结束后，向每孔加入MTT(5mg/mL,20μL/孔)在37℃环境下孵育4～5小时后，吸弃上清，向每孔中继续加入DMSO(150μL/孔)于恒温摇床上震荡10min以充分溶解水不溶性的蓝紫色甲臜结晶，最后在多功能酶标仪上于570nm处读取吸光值。结果如图4D所示。为了确认TiO2-Ru-PEG进行声动力治疗的超声准确时间，设置不同超声时间照射MB49细胞，比较有无TiO2-Ru-PEG孵育的情况下细胞存活率变化，结果显示超声强度为1MHz，1.5w/cm2，占空比40％，照射时间为1min时，单独US组细胞存活率为95.2％，而TiO2-Ru-PEG+US组细胞存活率为52.9％，可见超声时间为1min时超声对细胞的损伤较低可忽略，而声敏剂的存在显著增强了对MB49细胞的毒性，抑制MB49细胞活性。如图4E所示，单独加入TiO2-Ru-PEG与细胞孵育48h，MB49细胞活力分别为94.5％、93％、89.2％、80.5％、70.7％，在超声照射下MB49细胞活力分别80.1％、71.5％、60.7％、52.4％、32.1％，显然TiO2-Ru-PEG浓度越高，MB49细胞活力越低，在超声的照射后，MB49细胞活力进一步降低，显示TiO2-Ru-PEG具有出色的声动力治疗效应。如图4F-G所示，相同浓度下Ru-PEG、TiO2-PEG、TiO2-Ru-PEG对MB49细胞活力影响最明显是TiO2-Ru-PEG，特别的是在超声照射后MB49细胞的活力US+TiO2-Ru-PEG组更低，由此可见TiO2-Ru-PEG较于Ru-PEG、TiO2-PEG可显著增强声动力效应。如图4H-I所示，不同浓度TiO2-Ru-PEG与SV-HUC-1单独孵育48h后细胞活力分别为98.7％、95.3％、92.9％、91.6％、89.9％；而在超声照射后孵育48h，MB49细胞活力分别为96.9％、94.4％、92.8％、89.9％、86.7％，证明TiO2-Ru-PEG在浓度为28ug/ml以下对细胞SV-HUC-1细胞活力几乎没有影响，TiO2-Ru-PEG具有良好的生物相容性。Inoculate 100ul of SV-HUC-1 and MB49 cell suspension in each well of a 96-well plate with a cell concentration of 4x10 4 cells/ml, and incubate at 37°C for 24 hours in a humidified environment of 5% CO2 . SV-HUC-1 and MB49 were divided into control group, US group, Ru-PEG group, TiO 2 -PEG group, and TiO 2 -Ru-PEG group. After the cells have adhered, Ru-PEG, TiO 2 -PEG, and TiO 2 -Ru-PEG of different concentrations (2, 4, 7, 14, 28) ug were added to each group based on the Ru concentration and incubated with the cells for 8 hours. Finally, the cells in the ultrasound group were exposed to ultrasonic irradiation with an intensity of 1MHz, 1.5w/cm 2 and a duty cycle of 40% for 1 min, and incubated at 37°C for 48h. After the incubation, MTT (5mg/mL, After incubating for 4 to 5 hours at 37°C, discard the supernatant, add DMSO (150 μL/well) to each well, and shake on a constant temperature shaker for 10 minutes to fully dissolve the water-insoluble blue-violet formazan crystals. , and finally read the absorbance value at 570nm on a multifunctional microplate reader. The results are shown in Figure 4D. In order to confirm the accurate ultrasound time of TiO 2 -Ru-PEG for sonodynamic therapy, different ultrasound times were set to irradiate MB49 cells, and the changes in cell survival rate were compared with and without TiO 2 -Ru-PEG incubation. The results showed that the ultrasound intensity was 1MHz. 1.5w/cm 2 , duty cycle 40%, and irradiation time of 1 min, the cell survival rate of the US group alone was 95.2%, while the cell survival rate of the TiO 2 -Ru-PEG+US group was 52.9%. It can be seen that the ultrasound time is 1 min. The damage to cells caused by ultrasound was low and negligible, but the presence of sonosensitizer significantly enhanced the toxicity to MB49 cells and inhibited the activity of MB49 cells. As shown in Figure 4E, when TiO 2 -Ru-PEG was added alone and incubated with cells for 48 h, the viability of MB49 cells were 94.5%, 93%, 89.2%, 80.5%, and 70.7%, respectively. Under ultrasound irradiation, the viability of MB49 cells was 80.1%, 71.5%, 60.7%, 52.4%, 32.1%. Obviously, the higher the concentration of TiO2-Ru-PEG, the lower the viability of MB49 cells. After irradiation with ultrasound, the viability of MB49 cells further decreased, showing that TiO2 -Ru-PEG has excellent Sonodynamic therapy effects. As shown in Figure 4F-G, at the same concentration, Ru-PEG, TiO 2 -PEG, and TiO 2 -Ru-PEG have the most obvious effect on the viability of MB49 cells, especially TiO 2 -Ru-PEG, especially after ultrasound irradiation. The activity of US+TiO 2 -Ru-PEG group is lower, which shows that TiO 2 -Ru-PEG can significantly enhance the sonodynamic effect compared with Ru-PEG and TiO 2 -PEG. As shown in Figure 4H-I, the cell viability of different concentrations of TiO 2 -Ru-PEG and SV-HUC-1 after incubation alone for 48 h were 98.7%, 95.3%, 92.9%, 91.6%, and 89.9% respectively; and after ultrasound irradiation After incubation for 48 hours, the viability of MB49 cells were 96.9%, 94.4%, 92.8%, 89.9%, and 86.7% respectively, proving that TiO 2 -Ru-PEG has almost no effect on the viability of SV-HUC-1 cells at a concentration below 28ug/ml. TiO 2 -Ru-PEG has good biocompatibility.
(4)TiO2-Ru-PEG的细胞毒性(4) Cytotoxicity of TiO 2 -Ru-PEG
1)细胞内ROS含量检测1) Detection of intracellular ROS content
将MB49细胞以5x104cell/ml的密度接种在12孔板培养板中(1ml/孔)，待细胞贴壁后分成8组，加入9.4ug/ml Ru-PEG、TiO2-PEG、TiO2-Ru-PEG孵育细胞5h，让超声组的细胞接受强度为1MHz，1.5w/cm2，占空比40％的超声照射1min，细胞继续培养0.5h后去除上清，用PBS清洗3次，加入0.5ml用无血清培养基1：1000稀释DCFH-DA(碧云天S0033M)探针、DHE(二氢乙啶)、DPBF(单线态氧检测探针)避光染色30min，利用荧光显微镜对各组进行拍照记录。用DCFH-DA检测各处理组的ROS生成。MB49 cells were seeded in a 12-well culture plate at a density of 5x10 4 cells/ml (1ml/well). After the cells adhered, they were divided into 8 groups. Add 9.4ug/ml Ru-PEG, TiO 2 -PEG, and TiO 2 - Ru-PEG incubated the cells for 5 hours, and allowed the cells in the ultrasound group to receive ultrasonic irradiation with an intensity of 1 MHz, 1.5 w/cm 2 and a duty cycle of 40% for 1 minute. The cells continued to be cultured for 0.5 hours, then the supernatant was removed and washed three times with PBS. Add 0.5 ml of DCFH-DA (Beyotime S0033M) probe, DHE (dihydroethidine), and DPBF (singlet oxygen detection probe) diluted 1:1000 in serum-free culture medium for 30 minutes in the dark, and use a fluorescence microscope to detect each The group takes photos and records. DCFH-DA was used to detect ROS production in each treatment group.
结果如图5A所示，各个分组MB49细胞内的绿色荧光变化，未经超声照射的Ru-PEG、TiO2-PEG、TiO2-Ru-PEG与细胞单独孵育，各组细胞内显示出轻微的绿色荧光。经超声照射后相同浓度下Ru-PEG+US、TiO2-PEG+US、TiO2-Ru-PEG+US组细胞内荧光强度对比未经超声照射时明显增强，特别的是TiO2-Ru-PEG+US组细胞内荧光强度对比其他组更显著，同时对各组的荧光进行半定量分析(图5E)可见TiO2-Ru-PEG+US组荧光最强，说明TiO2-Ru-PEG在MB49细胞摄取后在超声照射可产生大量的ROS。用DPBF检测各处理组的单性态氧1O2生成情况。结果如图5B所示，各个分组MB49细胞内的绿色荧光变化，未经超声照射的Ru-PEG、TiO2-PEG、TiO2-Ru-PEG与细胞单独孵育，各组细胞内显示出强烈的绿色荧光。经超声照射后相同浓度下Ru-PEG+US、TiO2-PEG+US、TiO2-Ru-PEG+US组细胞内荧光强度对比未经超声照射时明显减弱，特别的是TiO2-Ru-PEG+US组细胞内荧光强度对比其他组最弱，同时对各组的荧光进行半定量分析(图5F)可见TiO2-Ru-PEG+US组绿色荧光最弱，说明TiO2-Ru-PEG在MB49细胞摄取后在超声照射可产生大量的单性态氧1O2。用DHE检测各处理组的超氧阴离子O2 .-生成情况，结果如图5C所示，各个分组MB49细胞内的红色荧光变化，未经超声照射的Ru-PEG、TiO2-PEG、TiO2-Ru-PEG与细胞单独孵育，各组细胞内显示出轻微的红色荧光。经超声照射后相同浓度下Ru-PEG+US、TiO2-PEG+US、TiO2-Ru-PEG+US组细胞内荧光强度对比未经超声照射时明显增强，特别的是TiO2-Ru-PEG+US组细胞内荧光强度对比其他组最强，同时对各组的荧光进行半定量分析(图5G)可见TiO2-Ru-PEG+US组红色荧光最强，说明TiO2-Ru-PEG在MB49被细胞摄取后在超声照射可产生大量的超氧阴离子O2 .-。The results are shown in Figure 5A. The green fluorescence changes in MB49 cells in each group. Ru-PEG, TiO 2 -PEG, and TiO 2 -Ru-PEG without ultrasonic irradiation were incubated with cells alone. The cells in each group showed slight changes. Green fluorescence. After ultrasonic irradiation, at the same concentration, the intracellular fluorescence intensity of the Ru-PEG+US, TiO 2 -PEG+US, and TiO 2 -Ru-PEG+US groups was significantly enhanced compared to that without ultrasonic irradiation, especially for TiO 2 -Ru- The intracellular fluorescence intensity of the PEG+US group was more significant than that of other groups. At the same time, semi-quantitative analysis of the fluorescence of each group (Figure 5E) showed that the TiO 2 -Ru-PEG+US group had the strongest fluorescence, indicating that TiO 2 -Ru-PEG MB49 cells can produce a large amount of ROS after ultrasound irradiation after uptake. DPBF was used to detect the production of singlet oxygen 1 O 2 in each treatment group. The results are shown in Figure 5B. The green fluorescence changes in MB49 cells in each group. Ru-PEG, TiO 2 -PEG, and TiO 2 -Ru-PEG without ultrasonic irradiation were incubated with cells alone. The cells in each group showed strong Green fluorescence. After ultrasonic irradiation, the intracellular fluorescence intensity of the Ru-PEG+US, TiO 2 -PEG+US, and TiO 2 -Ru-PEG+US groups at the same concentration was significantly weaker than that without ultrasonic irradiation, especially for TiO 2 -Ru- The intracellular fluorescence intensity of the PEG+US group was the weakest compared to other groups. At the same time, a semi-quantitative analysis of the fluorescence of each group (Figure 5F) showed that the green fluorescence of the TiO 2 -Ru-PEG+US group was the weakest, indicating that TiO 2 -Ru-PEG After uptake by MB49 cells, a large amount of singlet oxygen 1 O 2 can be produced under ultrasound irradiation. DHE was used to detect the production of superoxide anion O 2 .- in each treatment group. The results are shown in Figure 5C. The red fluorescence changes in MB49 cells in each group, Ru-PEG, TiO 2 -PEG, and TiO 2 without ultrasonic irradiation -Ru-PEG was incubated with cells alone, and cells in each group showed slight red fluorescence. After ultrasonic irradiation, at the same concentration, the intracellular fluorescence intensity of the Ru-PEG+US, TiO 2 -PEG+US, and TiO 2 -Ru-PEG+US groups was significantly enhanced compared to that without ultrasonic irradiation, especially for TiO 2 -Ru- The intracellular fluorescence intensity of the PEG+US group was the strongest compared to other groups. At the same time, semi-quantitative analysis of the fluorescence of each group was performed (Figure 5G). It can be seen that the red fluorescence of the TiO 2 -Ru-PEG+US group was the strongest, indicating that TiO 2 -Ru-PEG After MB49 is taken up by cells, a large amount of superoxide anion O 2 .- can be produced under ultrasound irradiation.
2)线粒体膜电位及形貌变化2) Mitochondrial membrane potential and morphology changes
将MB49细胞以5x104cell/ml的密度接种在12孔板培养板中(1ml/孔)，待贴壁细胞后分成8组，加入9.4ug/ml Ru-PEG、TiO2-PEG、TiO2-Ru-PEG孵育细胞5h，让超声组的细胞接受强度为1MHz，1.5w/cm2，占空比40％的超声照射1min，置于37℃培养箱中继续培养1h，根据试剂盒说明说(碧云天C2006)加入0.5ml JC-1染色工作液在37℃培养箱中避光孵育20分钟，孵育结束后吸除上清，用1ml冰浴的JC-1染色缓冲液洗涤细胞2次，洗涤结束后加入1ml细胞培养基置于荧光显微镜下观察。孵育结束后用荧光显微镜下观察各处理组的细胞染色效果。线粒体膜电位的降低(Δψm)与细胞早期凋亡有关。JC-1标记线粒体中的J-聚集体或单体分别呈现红色和绿色荧光，通过JC-1从红色荧光到绿色荧光的转变可检测细胞(Δψm)的下降情况。Seed MB49 cells in a 12-well culture plate at a density of 5x10 4 cells/ml (1ml/well). After the cells have adhered, they are divided into 8 groups. Add 9.4ug/ml Ru-PEG, TiO 2 -PEG, and TiO 2 -Ru-PEG incubate the cells for 5 hours, let the cells in the ultrasound group receive ultrasonic irradiation with an intensity of 1MHz, 1.5w/cm 2 and a duty cycle of 40% for 1 minute, then place them in a 37°C incubator and continue to culture for 1 hour, according to the instructions of the kit. (Beyotime C2006) Add 0.5 ml JC-1 staining working solution and incubate in a 37°C incubator in the dark for 20 minutes. After the incubation, remove the supernatant and wash the cells twice with 1 ml of JC-1 staining buffer in an ice bath. After washing, add 1 ml of cell culture medium and observe under a fluorescence microscope. After the incubation, the cell staining effects of each treatment group were observed under a fluorescence microscope. The decrease in mitochondrial membrane potential (Δψm) is related to early cell apoptosis. J-aggregates or monomers in JC-1 labeled mitochondria exhibit red and green fluorescence respectively, and the decrease in cell (Δψm) can be detected through the transition of JC-1 from red fluorescence to green fluorescence.
结果如图5D所示，未经超声照射时各组和正常细胞发出强橙红色荧光，仅TiO2-Ru-PEG组发出较微弱的绿光，其表明各组药物对细胞(Δψm)的下降有限。经超声照射后，Ru-PEG+US、TiO2-PEG+US、TiO2-Ru-PEG+US组细胞内红光减弱，绿光增强，其中TiO2-Ru-PEG+US组变化最显著，表明TiO2-Ru-PEG在超声的照射下能显著引起线粒体的去极化从而促进细胞早期凋亡，验证了TiO2-Ru-PEG增强声动力治疗的效果。The results are shown in Figure 5D. Without ultrasonic irradiation, each group and normal cells emitted strong orange-red fluorescence, and only the TiO 2 -Ru-PEG group emitted weaker green light, which indicated the decrease in the effects of drugs on cells (Δψm) in each group. limited. After ultrasonic irradiation, the red light in the cells of the Ru-PEG+US, TiO 2 -PEG+US, and TiO 2 -Ru-PEG+US groups was weakened and the green light was enhanced. Among them, the TiO 2 -Ru-PEG+US group had the most significant change. , indicating that TiO 2 -Ru-PEG can significantly cause mitochondrial depolarization and promote early cell apoptosis under ultrasound irradiation, verifying the effect of TiO 2 -Ru-PEG in enhancing sonodynamic therapy.
(5)TiO2-Ru-PEG抗肿瘤效应(5)TiO 2 -Ru-PEG anti-tumor effect
1)活死细胞(Calcein-AM/PI)染色1) Live and dead cells (Calcein-AM/PI) staining
将MB49细胞以5x104cell/ml的密度接种在12孔板培养板(1ml/孔)，待贴壁细胞后分成8组，加入9.4ug/ml Ru-PEG、TiO2-PEG、TiO2-Ru-PEG孵育细胞5h，让超声组的细胞接受强度为1MHz，1.5w/cm2，占空比40％的超声照射1min，细胞继续培养5h。根据试剂盒说明书(碧云天C2015M)配制Calcein-AM/PI检测工作液，各组细胞分别加入0.5ml Calcein-AM/PI检测工作液，37℃避光孵育30min，孵育结束后用荧光显微镜下观察各处理组的细胞染色效果。MB49 cells were seeded in a 12-well culture plate (1ml/well) at a density of 5x10 4 cells/ml. After the cells adhered, they were divided into 8 groups. Add 9.4ug/ml Ru-PEG, TiO 2 -PEG, and TiO 2 - The cells were incubated with Ru-PEG for 5 hours, and the cells in the ultrasound group were exposed to ultrasound with an intensity of 1MHz, 1.5w/cm 2 and a duty cycle of 40% for 1 minute. The cells were continued to be cultured for 5 hours. Prepare Calcein-AM/PI detection working solution according to the instructions of the kit (Beyotime C2015M). Add 0.5ml Calcein-AM/PI detection working solution to each group of cells, incubate at 37°C in the dark for 30 minutes, and observe under a fluorescence microscope after the incubation. Cell staining effects of each treatment group.
结果如图6A所示，未经超声照射的各组细胞密度80-90％，细胞轮廓形态好数量多，呈绿色荧光，表明多数细胞是活的，其中只有TiO2-Ru-PEG组显示微弱的红色荧光，表明TiO2-Ru-PEG单独处理细胞可导致少量细胞死亡。经超声照射后，TiO2-Ru-PEG+US组细胞密度显著性下降约为40％，绿光明显减弱，红光明显增强，说明在超声照射下TiO2-Ru-PEG可显著引起细胞凋亡，说明TiO2-Ru-PEG具有增强声动力治疗的效果。The results are shown in Figure 6A. The cell density of each group without ultrasonic irradiation was 80-90%. The cells had good outlines and large numbers. They showed green fluorescence, indicating that most cells were alive. Among them, only the TiO 2 -Ru-PEG group showed weak The red fluorescence indicates that treating cells with TiO 2 -Ru-PEG alone can cause a small amount of cell death. After ultrasound irradiation, the cell density of the TiO 2 -Ru-PEG+US group significantly decreased by about 40%, the green light was significantly weakened, and the red light was significantly enhanced, indicating that TiO 2 -Ru-PEG can significantly cause cell apoptosis under ultrasound irradiation. death, indicating that TiO 2 -Ru-PEG has the effect of enhancing sonodynamic therapy.
2)克隆形成2) Clone formation
将密度为2000cells的MB49细胞接种在6孔板中(2mL/孔)，待完全贴壁后，分别加入9.4μg/mL(以Ru定量)的Ru-PEG、TiO2-PEG、TiO2-Ru-PEG继续孵育5h，用强度为1MHz，1.5w/cm2，占空比40％超声照射1min后37℃培养，每天观察克隆形成情况，14天后抽掉培养基用预冷的PBS55清洗三次，用4.0％多聚甲醛固定细胞15min，用0.5％的甲基紫避光染色15min，等自然风干后，拍照并计数，通过(克隆数/接种细胞数)*100％计算克隆形成率。MB49 cells with a density of 2000 cells were seeded in a 6-well plate (2 mL/well). After complete attachment, 9.4 μg/mL (quantitated by Ru) of Ru-PEG, TiO2-PEG, and TiO2-Ru-PEG were added. Continue to incubate for 5 hours, use ultrasonic irradiation with an intensity of 1MHz, 1.5w/cm 2 and a duty cycle of 40% for 1 minute and then culture at 37°C. Observe the colony formation every day. After 14 days, remove the culture medium and wash it three times with pre-cooled PBS55, and wash it with 4.0 Cells were fixed with % paraformaldehyde for 15 minutes, stained with 0.5% methyl violet for 15 minutes to protect from light, and allowed to air dry naturally before being photographed and counted. The colony formation rate was calculated by (number of clones/number of seeded cells)*100%.
结果如图6B所示，未经超声照射的各组细胞增殖形成的紫色面积明显扩大，而经超声照射后各组细胞增殖形成的紫色面积均有下降，其中TiO2-Ru-PEG+US组最为明显，通过半定量分析克隆结果如图6D所示，TiO2-Ru-PEG在超声照射下可显著抑制了MB49细胞的增殖。The results are shown in Figure 6B. The purple area formed by cell proliferation in each group without ultrasonic irradiation significantly expanded, while the purple area formed by cell proliferation in each group decreased after ultrasonic irradiation. Among them, the TiO 2 -Ru-PEG+US group Most obviously, as shown in Figure 6D through semi-quantitative analysis of the cloning results, TiO 2 -Ru-PEG can significantly inhibit the proliferation of MB49 cells under ultrasound irradiation.
3)划痕实验3) Scratch test
将密度为5x104cells的MB49细胞，70μL接种在置于与六孔板中的划痕插件孔内(Ibidi GmbH)，孵育24h细胞贴壁后将划痕插件去除并向每个孔内加入2ml完全培养基，分别加入9.4μg/mL(以Ru定量)的Ru-PEG、TiO2-PEG、TiO2-Ru-PEG并在显微镜下拍照记录0h划痕情况，在培养箱继续孵育5h后用强度为1MHz，1.5w/cm2，占空比40％超声照射1min后37℃培养。每隔2h用显微镜拍摄划痕情况并记录。70 μL of MB49 cells with a density of 5x10 4 cells were seeded into the scratch insert wells (Ibidi GmbH) placed in a six-well plate. After incubation for 24 hours, the scratch insert was removed and 2 ml was added to each well. Complete culture medium, add 9.4 μg/mL (quantitated by Ru) Ru-PEG, TiO 2 -PEG, and TiO 2 -Ru-PEG respectively and take photos under a microscope to record the scratches at 0h. Continue incubating in the incubator for 5 hours before using The intensity was 1MHz, 1.5w/cm 2 , duty cycle 40%, ultrasonic irradiation for 1 minute and then cultured at 37°C. Use a microscope to photograph the scratches every 2 hours and record them.
结果如图6C所示，孵育细胞12h后，未经超声照射的各组细胞对比0h的划痕，细胞已发生显著的迁移，对照组已出现细胞接触，而经超声照射后各组细胞划痕较于没有超声处组别的划痕距离相对明显，其中TiO2-Ru-PEG+US组最为明显，通过半定量分析克隆结果如图6E所示，TiO2-Ru-PEG在超声照射下可显著抑制了MB49细胞的增殖。The results are shown in Figure 6C. After incubating the cells for 12 hours, the cells in each group without ultrasonic irradiation were compared with the scratches at 0 h. The cells had significantly migrated, and cell contact had occurred in the control group. However, the cells in each group were scratched after ultrasonic irradiation. Compared with the scratch distance of the group without ultrasound, the scratch distance is relatively obvious, among which the TiO 2 -Ru-PEG+US group is the most obvious. The cloning results through semi-quantitative analysis are shown in Figure 6E. TiO 2 -Ru-PEG can be Significantly inhibited the proliferation of MB49 cells.
实施例3Example 3
本实施例对实施例1制备得到的钛基纳米材料TiO2-Ru-PEG的安全性及声动力疗效进行体内验证，具体实验方法如下：This example conducts in vivo verification of the safety and sonodynamic efficacy of the titanium-based nanomaterial TiO 2 -Ru-PEG prepared in Example 1. The specific experimental methods are as follows:
(1)MB49荷瘤小鼠模型构建(1) Construction of MB49 tumor-bearing mouse model
雌性BALB/c小鼠(4～6周龄，体重18～20g，购自广东省医学实验动物中心，SPF级别)，饲养在广州吉妮欧生物科技技术有限公司(动物合格证编号：SYXK(粤)2022-0298)，以下所有实验均符合动物保护、动物福利和伦理原则，符合国家实验动物福利伦理的相关规定。小鼠检疫7天后，150μl密度为7.5×107cells/ml的MB49细胞悬液(PBS和基质胶体积为1:1)皮下植入小鼠左侧腋下(血流充足、皮肤松弛)，隔天观察小鼠状态及成瘤情况，约7天后肿瘤大小达6～8mm可随机分组并进行相关实验。Female BALB/c mice (4 to 6 weeks old, weighing 18 to 20 g, purchased from Guangdong Medical Experimental Animal Center, SPF level) were raised in Guangzhou Genio Biotechnology Co., Ltd. (animal certificate number: SYXK ( Guangdong) 2022-0298), all the following experiments comply with the principles of animal protection, animal welfare and ethics, and comply with the relevant national regulations on experimental animal welfare ethics. After the mice were quarantined for 7 days, 150 μl of MB49 cell suspension with a density of 7.5×10 7 cells/ml (the volume of PBS and Matrigel was 1:1) was subcutaneously implanted into the left armpit of the mice (with sufficient blood flow and loose skin). Observe the status and tumor formation of the mice the next day. After about 7 days, when the tumor size reaches 6 to 8 mm, the mice can be randomly divided into groups and related experiments can be conducted.
(2)TiO2-Ru-PEG安全性体内验证(2) In vivo verification of safety of TiO2-Ru-PEG
吲哚菁绿(ICG)是目前唯一被美国食品药物管理局(FDA)批准用于临床的近红外成像试剂，是一种具有近红外特征吸收峰的三碳花菁染料，最大发射波长在795～845nm之间。采用100ul ICG标记的1.5μg/ml TiO2-Ru-PEG溶液对两只BALB/C荷瘤小鼠进行瘤内注射，分别在0、10min、30min、2h、4h、6h、8h、12h、24h、30小时使用小动物成像仪检测TiO2-Ru-PEG在体内的近红外荧光分布情况，在4h将其中一个裸鼠解剖取出重要脏器以及肿瘤。Indocyanine green (ICG) is currently the only near-infrared imaging reagent approved for clinical use by the U.S. Food and Drug Administration (FDA). It is a three-carbon cyanine dye with a characteristic near-infrared absorption peak and a maximum emission wavelength of 795 ~845nm. Two BALB/C tumor-bearing mice were injected intratumorally with 100ul ICG-labeled 1.5μg/ml TiO 2 -Ru-PEG solution at 0, 10min, 30min, 2h, 4h, 6h, 8h, 12h, and 24h respectively. , use a small animal imager to detect the near-infrared fluorescence distribution of TiO 2 -Ru-PEG in the body at 30 hours, and dissect one of the nude mice at 4 hours to remove important organs and tumors.
结果如图7B-D所示，在0小时未注射纳米药物时，裸鼠体内无荧光分布；瘤内注射后30min内荧光最强，在4h后相对减弱并保持相对稳定的荧光强度，24h后裸鼠体内荧光开始显著减弱，表明TiO2-Ru-PEG可在瘤内稳定扩散且较长时间在瘤内停留，并可安全的代谢出体外，具有较好的生物相容性。The results are shown in Figure 7B-D. When no nanomedicine was injected at 0 hours, there was no fluorescence distribution in the nude mice; the fluorescence was strongest within 30 minutes after intratumoral injection, then weakened relatively after 4 hours and maintained a relatively stable fluorescence intensity after 24 hours. The fluorescence in nude mice began to weaken significantly, indicating that TiO 2 -Ru-PEG can diffuse stably in the tumor and stay in the tumor for a long time, and can be safely metabolized out of the body and has good biocompatibility.
(3)TiO2-Ru-PEG体内声动力疗效(3) In vivo sonodynamic efficacy of TiO 2 -Ru-PEG
将40只体重约为20g的MB49荷瘤小鼠分成8组，每组5只，其中：第一组：瘤内注射生理盐水作为对照组(Control)；第二组：瘤内注射Ru-PEG；第三组：瘤内注射TiO2-PEG；第四组：瘤内注射TiO2-Ru-PEG；第五组：瘤内脏注射生理盐水后超声照射组(US)；第六组：瘤内注射Ru-PEG后8h超声照射(Ru-PEG+US)；第七组：瘤内注射TiO2-PEG后8h超声照射(TiO2-PEG+US)；第八组：瘤内注射TiO2-Ru-PEG后8h超声照射(TiO2-Ru-PEG+US)；如图7A所示，整个MB49荷瘤小鼠的声动力治疗过程，注射浓度以Ru计算，每只小鼠(20g)注射10mg/kg，瘤内注射1小时后用超声强度为1MHz，1.5w/cm2，10min，占空比40％照射肿瘤。隔2天称量小鼠体重，量取瘤大小并计算瘤体积：V＝ab2/2，a为瘤长度，b为瘤宽度。治疗14天后，眼球取血，离心后收集血清保存于-80℃进行血液生化指标检测；收集重要脏器(心肝脾肺肾)和肿瘤，PBS清洗后，取一部分用4％多聚甲醛固定24h以后，进行H&E染色，另外切肿瘤空白片进行免疫组化和TUNEL染色分析，进而验证TiO2-Ru-PEG用于体内增强声动力治疗的作用。结果如图7、图8所示。Forty MB49 tumor-bearing mice weighing approximately 20 g were divided into 8 groups, with 5 mice in each group, among which: the first group: intratumoral injection of normal saline as the control group (Control); the second group: intratumoral injection of Ru-PEG ; The third group: intratumoral injection of TiO 2 -PEG; the fourth group: intratumoral injection of TiO 2 -Ru-PEG; the fifth group: intratumoral injection of normal saline followed by ultrasound irradiation group (US); the sixth group: intratumoral injection Ultrasound irradiation 8 hours after injection of Ru-PEG (Ru-PEG+US); Group 7: Ultrasound irradiation 8 hours after intratumoral injection of TiO 2 -PEG (TiO 2 -PEG+US); Group 8: Intratumoral injection of TiO 2 - Ultrasonic irradiation (TiO 2 -Ru-PEG+US) 8 hours after Ru-PEG; as shown in Figure 7A, the entire sonodynamic treatment process of MB49 tumor-bearing mice, the injection concentration was calculated as Ru, and each mouse (20g) was injected 10mg/kg, 1 hour after intratumoral injection, the tumor was irradiated with ultrasound intensity of 1MHz, 1.5w/cm 2 , 10min, and duty cycle 40%. Weigh the mouse body weight every 2 days, measure the tumor size and calculate the tumor volume: V=ab 2 /2, a is the tumor length, and b is the tumor width. After 14 days of treatment, blood was taken from the eyeballs, and the serum was collected after centrifugation and stored at -80°C for blood biochemical index testing; important organs (heart, liver, spleen, lungs and kidneys) and tumors were collected, washed with PBS, and a portion was fixed with 4% paraformaldehyde for 24 hours. Afterwards, H&E staining was performed, and blank tumor sections were cut for immunohistochemistry and TUNEL staining analysis to verify the role of TiO 2 -Ru-PEG in enhancing sonodynamic therapy in vivo. The results are shown in Figures 7 and 8.
如图7E所示从上到下分别是Control、Ru-PEG、TiO2-PEG、TiO2-Ru-PEG、US、Ru-PEG+US、TiO2-PEG+US、TiO2-Ru-PEG+US组；从肿瘤实体照片可知，未经超声照射的Control、Ru-PEG、TiO2-PEG、TiO2-Ru-PEG组的肿瘤体积明显增大，而在超声照射下的US、Ru-PEG+US、TiO2-PEG+US、TiO2-Ru-PEG+US组，除了US组肿瘤体积与对照组几乎无差别，Ru-PEG+US、TiO2-PEG+US、TiO2-Ru-PEG+US组的肿瘤体积均有所减小，其中TiO2-Ru-PEG+US组的肿瘤体积最小，表明TiO2-Ru-PEG在超声照射后显著增强声动力治疗，明显抑制肿瘤的增殖。As shown in Figure 7E, from top to bottom they are Control, Ru-PEG, TiO 2 -PEG, TiO 2 -Ru-PEG, US, Ru-PEG+US, TiO 2 -PEG+US, and TiO 2 -Ru-PEG. +US group; it can be seen from the tumor solid photos that the tumor volume of the Control, Ru-PEG, TiO 2 -PEG, and TiO 2 -Ru-PEG groups without ultrasound irradiation increased significantly, while the US, Ru- PEG+US, TiO 2 -PEG+US, TiO 2 -Ru-PEG+US groups, except that the tumor volume of the US group was almost no different from the control group, Ru-PEG+US, TiO 2 -PEG+US, TiO 2 -Ru The tumor volume in the -PEG+US group decreased, and the tumor volume in the TiO 2 -Ru-PEG+US group was the smallest, indicating that TiO 2 -Ru-PEG significantly enhanced sonodynamic therapy and significantly inhibited tumor growth after ultrasound irradiation. proliferation.
由图7F可知Control、Ru-PEG、TiO2-PEG、TiO2-Ru-PEG、US组肿瘤体积随着时间的变化不断增大，而Ru-PEG+US、TiO2-PEG+US组相对抑制了肿瘤体积的增大，但效果不明显。而TiO2-Ru-PEG+US组则明显抑制了肿瘤体积的增大，可见TiO2-Ru-PEG在超声照射下具有优异的声动力疗效，可显著抑制肿瘤的生长。It can be seen from Figure 7F that the tumor volume of the Control, Ru-PEG, TiO 2 -PEG, TiO 2 -Ru-PEG, and US groups continued to increase over time, while the Ru-PEG+US, TiO 2 -PEG+US group was relatively The increase in tumor volume was inhibited, but the effect was not obvious. The TiO 2 -Ru-PEG+US group significantly inhibited the increase in tumor volume. It can be seen that TiO 2 -Ru-PEG has excellent sonodynamic efficacy under ultrasound irradiation and can significantly inhibit tumor growth.
由图7G可知，经过14天的不同治疗后Control、Ru-PEG、TiO2-PEG、TiO2-Ru-PEG、US组肿瘤重量相较大，而Ru-PEG+US、TiO2-PEG+US、TiO2-Ru-PEG+US组肿瘤重量则相对较小，其中TiO2-Ru-PEG+US组肿瘤重量下降最明显，表明TiO2-Ru-PEG在超声照射具有显著的声动力疗效，显著抑制肿瘤的生长。As can be seen from Figure 7G, after 14 days of different treatments, the tumor weights in the Control, Ru-PEG, TiO 2 -PEG, TiO 2 -Ru-PEG, and US groups were relatively larger, while those in the Ru-PEG+US, TiO 2 -PEG+ The tumor weights in the US and TiO 2 -Ru-PEG+US groups were relatively small, and the tumor weight in the TiO 2 -Ru-PEG+US group decreased most significantly, indicating that TiO 2 -Ru-PEG has significant sonodynamic efficacy in ultrasound irradiation. , significantly inhibiting tumor growth.
由图7H-I可知，Control、Ru-PEG、TiO2-PEG、TiO2-Ru-PEG、US、Ru-PEG+US、TiO2-PEG+US、TiO2-Ru-PEG+US组的肿瘤抑制率分别为0％、18％、22.7％、22.8％、11％、28.6％、29.4％、66.7％，表明TiO2-Ru-PEG在超声照射后显著抑制肿瘤的生长，TiO2-Ru-PEG具有增强膀胱癌声动力治疗疗效。It can be seen from Figure 7H-I that the Control, Ru-PEG, TiO 2 -PEG, TiO 2 -Ru-PEG, US, Ru-PEG+US, TiO 2 -PEG+US, and TiO 2 -Ru-PEG+US groups The tumor inhibition rates were 0%, 18%, 22.7%, 22.8%, 11%, 28.6%, 29.4%, and 66.7% respectively, indicating that TiO 2 -Ru-PEG significantly inhibited tumor growth after ultrasound irradiation. TiO 2 -Ru -PEG can enhance the efficacy of sonodynamic therapy for bladder cancer.
如图8A所示，为了更好评价TiO2-Ru-PEG增强声动力治疗的作用，对各组肿瘤切片H&E染色结果进行仔细观察，Control、Ru-PEG、TiO2-PEG、TiO2-Ru-PEG、US组可见大量核仁明显的肿瘤细胞，肿瘤组织非常致密。而Ru-PEG+US、TiO2-PEG+US、TiO2-Ru-PEG+US组肿瘤细胞数目减少、排列疏松、边界模糊、细胞核固缩，同时存在坏死现象，TiO2-Ru-PEG+US组最为明显，进一步佐证了TiO2-Ru-PEG具有更好的声动力治疗效果。Tunel(细胞凋亡标志物)是检测细胞凋亡的重要指标，如图所示Control、Ru-PEG、TiO2-PEG、TiO2-Ru-PEG、US组均没有出现明显的细胞凋亡情况，而联合超声照射后Ru-PEG+US、TiO2-PEG+US、TiO2-Ru-PEG+US组可见细胞凋亡现象，而TiO2-Ru-PEG+US组则明显出现细胞凋亡现象，表明TiO2-Ru-PEG在超声照射可引起细胞凋亡，TiO2-Ru-PEG具有增强声动力治疗效果。Ki67(细胞增殖标志物)是组织标本活检分级的重要指标，如图所示，Control、Ru-PEG、TiO2-PEG、TiO2-Ru-PEG、US组均高表达Ki67表明肿瘤恶性程度较大，且处于活跃的增殖状态，而当超声照射后，Ru-PEG+US、TiO2-PEG+US、TiO2-Ru-PEG+US组Ki67表达水平有所下降，表明肿瘤增殖有所抑制，其中TiO2-Ru-PEG+US组Ki67表达水平下降最明显，表明TiO2-Ru-PEG在超声照射后显著抑制肿瘤的生长。As shown in Figure 8A, in order to better evaluate the effect of TiO 2 -Ru-PEG in enhancing sonodynamic therapy, the H&E staining results of tumor sections in each group were carefully observed. Control, Ru-PEG, TiO 2 -PEG, TiO 2 -Ru A large number of tumor cells with obvious nucleoli were seen in the -PEG and US groups, and the tumor tissue was very dense. However, in the Ru-PEG+US, TiO 2 -PEG+US, and TiO 2 -Ru-PEG+US groups, the number of tumor cells decreased, the arrangement was loose, the boundaries were blurred, the nuclei were pyknotic, and necrosis was present. The TiO 2 -Ru-PEG+ The most obvious effect in the US group further proves that TiO 2 -Ru-PEG has a better sonodynamic therapeutic effect. Tunel (apoptosis marker) is an important indicator for detecting cell apoptosis. As shown in the figure, there was no obvious cell apoptosis in the Control, Ru-PEG, TiO 2 -PEG, TiO 2 -Ru-PEG, and US groups. , and after combined ultrasound irradiation, cell apoptosis was seen in the Ru-PEG+US, TiO 2 -PEG+US, and TiO 2 -Ru-PEG+US groups, while cell apoptosis was obvious in the TiO 2 -Ru-PEG+US group. phenomenon, indicating that TiO 2 -Ru-PEG can cause cell apoptosis under ultrasound irradiation, and TiO 2 -Ru-PEG has the effect of enhancing sonodynamic therapy. Ki67 (cell proliferation marker) is an important indicator of tissue specimen biopsy grading. As shown in the figure, high expression of Ki67 in the Control, Ru-PEG, TiO 2 -PEG, TiO 2 -Ru-PEG, and US groups indicates that the tumor is more malignant. large, and in an active proliferation state. After ultrasound irradiation, the expression level of Ki67 decreased in the Ru-PEG+US, TiO 2 -PEG+US, and TiO 2 -Ru-PEG+US groups, indicating that tumor proliferation was inhibited. , among which the expression level of Ki67 decreased most significantly in the TiO 2 -Ru-PEG+US group, indicating that TiO 2 -Ru-PEG significantly inhibited tumor growth after ultrasound irradiation.
如图8B所示，各分组的裸鼠心脏、肝脏、脾脏、肺脏及肾脏的组织形态正常，结构清晰，未见异常组织；高倍镜下可看出细胞分界清楚，排列规整，未见炎性改变，表明TiO2-Ru-PEG具有良好的生物相容性。而通过收集各分组裸鼠血液并检测了ALB、AST、ALB肝功能重要指标以及UREA、CREA、UA重要肾功能指标，如图8C所示，Ru-PEG、TiO2-PEG、TiO2-Ru-PEG、US、Ru-PEG+US、TiO2-PEG+US、TiO2-Ru-PEG+US组与Control组的肝肾功能指标并无明显差异，进一步佐证了TiO2-Ru-PEG具有良好的生物相容性。As shown in Figure 8B, the tissue morphology of the hearts, livers, spleens, lungs, and kidneys of nude mice in each group was normal, the structure was clear, and no abnormal tissue was found; under high-power microscope, it can be seen that the cells are clearly demarcated and arranged regularly, and no inflammation is found. changes, indicating that TiO 2 -Ru-PEG has good biocompatibility. By collecting the blood of nude mice from each group and detecting the important liver function indicators of ALB, AST, and ALB, as well as the important renal function indicators of UREA, CREA, and UA, as shown in Figure 8C, Ru-PEG, TiO 2 -PEG, and TiO 2 -Ru -PEG, US, Ru-PEG+US, TiO 2 -PEG+US, TiO 2 -Ru-PEG+US group and Control group had no significant difference in liver and kidney function indicators, which further proved that TiO 2 -Ru-PEG has Good biocompatibility.
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art will understand that Modifications or equivalent substitutions may be made to the technical solution of the present invention without departing from the essence and scope of the technical solution of the present invention.
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