Synthesis of IrO<sub>2</sub>@SF Nanoparticle and Its Antitumor Applications

HE Jie; YANG Ruihao; YU Yunlong; ZHONG Li; XUE Peng; KANG Yuejun

doi:10.13718/j.cnki.xdzk.2024.06.004

2024 Volume 46 Issue 6

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HE Jie, YANG Ruihao, YU Yunlong, et al. Synthesis of IrO2@SF Nanoparticle and Its Antitumor Applications[J]. Journal of Southwest University Natural Science Edition, 2024, 46(6): 40-50. doi: 10.13718/j.cnki.xdzk.2024.06.004

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HE Jie, YANG Ruihao, YU Yunlong, et al. Synthesis of IrO₂@SF Nanoparticle and Its Antitumor Applications[J]. Journal of Southwest University Natural Science Edition, 2024, 46(6): 40-50. doi: 10.13718/j.cnki.xdzk.2024.06.004

Synthesis of IrO₂@SF Nanoparticle and Its Antitumor Applications

1.
Yibin Academy of Southwest University, Yibin Sichuan 644005, China
2.
School of Materials and Energy, Southwest University, Chongqing 400715, China
3.
Burns Research Institute, The First Affiliated Hospital of the Army Medical University, Chongqing 400038, China
4.
School of Bioengineering, Chongqing University, Chongqing 400044, China

More Information

Corresponding author: XUE Peng ;
Received Date: 25/03/2024
Available Online: 20/06/2024
MSC: R737.9

Abstract

Hypoxic tumor microenvironment usually promotes tumor proliferation and leads to the resistance of tumor cells to reactive oxygen species (ROS) therapy. In this paper, silk fibroin (SF) was selected as the template, and a layer of iridium oxide (IrO₂) was mineralized on the surface of SF by biomineralization. A nanocomplex of IrO₂@SF nanoparticles (IOS) was obtained. The loading of natural SF endowed the IOS with multiple antitumor activities. The results show that the IOS have high photothermal conversion efficiency, catalase and peroxidase activities, and promoted sonodynamic effect. IOS can reverse the hypoxic state of tumor microenvironment by decomposing excess hydrogen peroxide. Cell experiments (in vitro) show that IOS achieves two antitumor therapeutic modes, including photothermal therapy incorporated with nanocatalytic therapy, and sonodynamic therapy incorporated with nanocatalytic therapy. Both treatment modalities show excellent antitumor effects. Meanwhile, due to the loading of native SF, IOS also has high biocompatibility. This study provides an effective and reliable treatment regimen for antitumor therapy.
- tumor microenvironment,
- silk fibroin protein,
- iridium oxide,
- antitumor therapy

References

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肿瘤微环境因其具有一些独特的理化性质而受到研究人员的广泛关注，例如乏氧、酸性微环境，高表达H₂O₂和高表达肿瘤细胞分泌物等. 其中，肿瘤微环境乏氧被认为是能直接影响肿瘤进展和肿瘤治疗的一大难题. 研究表明，肿瘤微环境乏氧能够促进肿瘤细胞生长增殖和转移，并且活性氧(ROS)介导的氧化应激疗法也受肿瘤乏氧所抑制，因此乏氧的解除对肿瘤治疗至关重要^[1-4]. 例如，缓解肿瘤乏氧能够增强化疗疗效，氧气的供应还能促进光动力和声动力等需氧反应的活性等.

近年来，已经开发了多种基于纳米材料的抗肿瘤治疗方法，比如光热疗法^[5-9]、声动力疗法^[10-11]、化学动力疗法^[12-14]和纳米酶催化疗法^[15-17]等. 但是，单一的疗法往往难以起到彻底根除肿瘤的目的. 因此，多模式协同疗法因其具有使用尽可能少的纳米药物就能起到多重抗肿瘤疗效的特点而受到广大科研人员的青睐. 目前已经开发了各种具有多种功能协同抗肿瘤的纳米药物^[18-22]. 然而，这些抗肿瘤纳米药物在兼顾了多种治疗模式的同时，其对机体的潜在毒性不可忽略. 因此，在设计开发这类多模式协同治疗的纳米粒子时，科研人员还需兼顾其安全性与应用转化价值.

基于此，本研究设计了一种结构简单的IrO₂@SF纳米粒子(IOS)，其具有多重抗肿瘤效应，同时其稳定的结构和较为安全的原材料还决定了其较高的生物安全性. 相较于已报道过的IrO₂基纳米粒子具有的光热效应^[23]，本研究合成的IOS还具有较高的类过氧化氢酶活性、过氧化物酶活性和声动力活性. 同时，从天然蚕茧提取的丝素蛋白(SF)还赋予了IOS较高的生物相容性. 与传统基于牛血清蛋白(BSA)为模板进行矿化制备无机纳米颗粒相比，SF具有成本低、安全性高、具有免疫活性等性能优势. 经过一系列的研究表明，本研究合成的具有多模式协同治疗效应的IOS能够逆转肿瘤微环境的乏氧状况且具有较高的抗肿瘤活性. 同时IOS还具有良好的生物安全性，使其具有极高的临床转化潜力.

1. 实验部分

1.1. 实验仪器与试剂

仪器：场发射扫描电子显微镜(JSM-7800F)，紫外可见分光光度计(UV-1800)，傅里叶变换红外光谱仪(FT-IR Nicolet 6700)，X射线粉末衍射仪(Rigaku Smartlab)，X射线光电子能谱仪(Thermo Kalpha)，共聚焦激光显微镜(Zeiss LSM 800)，倒置荧光显微镜(IX73).

试剂：二水合氯化钙(CaCl₂·2H₂O，AR)、碳酸钠(NaCO₃，AR)和氯化铱(IrCl₃，AR)购自上海阿拉丁公司. 氢氧化钠(NaOH，AR)购自美国Sigma-Aldrich公司. MTT细胞活力测定试剂盒、双醋酸荧光素(FDA，＞97%)、碘化丙啶(PI，＞94%)和Hoechst 33342(HPLC)由美国赛默飞世尔科技公司提供. 所有试剂都可直接用于实验.

1.2. IOS的合成

1.2.1. 丝素蛋白溶液的提取

第一步对蚕丝进行脱胶，先将家蚕茧在质量分数为0.5%的Na₂CO₃溶液中煮沸30 min(100 ℃)，随后得到絮状白色丝素蛋白，将絮状丝素蛋白用去离子水洗涤并烘干. 然后将得到的1 g丝素蛋白加入到125 mL的Ajisawa溶液中(CaCl₂·2H₂O∶CH₃CH₂OH∶H₂O，摩尔比为1∶2∶8)，再加热2 h(90 ℃). 最后将溶液透析3 d，以去除杂质，最后得到纯净的丝素蛋白溶液.

1.2.2. IOS的合成

首先在8 mL丝素蛋白溶液(15 mg/mL)中加入2 mL IrCl₃水溶液(50 mmol/L)，室温搅拌30 min. 然后用2 M NaOH将溶液pH值调到12，再加热搅拌12 h(80 ℃). 最后用去离子水透析24 h即得到IOS.

1.2.3. IOS的产ROS检测

通过亚甲基蓝(MB)、邻苯二胺(OPD)和3，3′，5，5′-四甲基联苯胺(TMB) 3种比色试剂对IOS的过氧化物酶活性和声动力效果进行探究. 分别将不同药物与MB、TMB和OPD在室温下共同孵育15 min，其中分组为：① H₂O₂，② IOS，③ IOS+H₂O₂，④ IOS+H₂O₂+L和⑤ IOS+H₂O₂+U. IOS和H₂O₂的浓度分别为400 μg/mL和10 mmol/L；激光波长、功率和时间分别为：1 064 nm，1.5 W/cm²，5 min；超声功率和时间分别为：1 MHz，1 W/cm²，50%空占比，5 min. 反应结束后使用紫外可见分光光度计分别测量在652 nm(TMB)，665 nm(MB)和492 nm(OPD)的光吸收变化情况.

1.2.4. IOS的细胞摄取实验

小鼠乳腺癌细胞4T1对IOS的内吞作用，通过共价偶联的方式将Cy5.5与IOS进行连接. 将EDC/NHS活化的Cy5.5(1 mg/mL)与IOS(200 μg/mL)在室温下搅拌6 h，然后通过离心(1×10⁴ rpm，5 min)去除未反应的Cy5.5，再用去离子水多次洗涤得到Cy5.5标记的IOS. 随后用共聚焦激光显微镜观察IOS的细胞摄取情况. 简单地说，将4T1细胞在12孔板(每孔7 × 10⁴个细胞)中培养12 h(37℃)，然后在不同时间点(第0，0.5，1，2，4 h)加入Cy5.5标记的IOS(200 μg/mL). 用Hoest 33342染色20 min后，在共聚焦激光显微镜下进行观察拍摄.

1.2.5. IOS的细胞相容性

以小鼠成纤维细胞(L929)和人脐静脉内皮细胞(HUVEC)为实验对象，通过3-(4，5-二甲基噻唑-2-基)-2，5-二苯基溴化四唑(MTT)测定IOS对正常体细胞的细胞毒性. 简单地说，将96孔板上的细胞(每孔1×10⁴个细胞)分别用不同剂量的IOS(0~800 μg/mL)处理24 h，PBS冲洗后，用MTT(100 μL，0.5 mg/mL)孵育4 h，吸取掉培养基后用二甲基亚砜(DMSO)(100 μL)溶解蓝紫色结晶甲臜，在摇床上摇晃15 min后使用酶标仪测其在490 nm下的吸收光强度.

1.2.6. IOS体外细胞毒性实验

通过活/死差异荧光染色研究IOS的抗肿瘤活性. 将4T1细胞置于12孔板(每孔5×10⁴个细胞)中，在37 ℃下培养12 h. 然后使用不同分组的药物与细胞进行共孵育培养4 h. 药物分组为：① H₂O₂，② IOS，③ IOS+H₂O₂，④ IOS+H₂O₂+L和⑤ IOS+H₂O₂+U. IOS和H₂O₂的浓度分别为400 μg/mL和10 mmol/L；激光波长、功率和时间分别为：1 064 nm，1.5 W/cm²，5 min；超声功率和时间分别为：1 MHz，1 W/cm²，50%空占比，5 min. 在第4 h分别使用激光光源和超声声源对第4组和第5组细胞进行处理，之后所有细胞继续培养2 h. 随后细胞经钙黄素-AM/碘化丙啶(PI)共染色15 min后，在荧光显微镜下观察和拍摄.

3. 结论

本研究成功制备了IrO₂@SF纳米粒子，并在此基础上探究了其各项抗肿瘤活性. 实验结果表明IOS具有高效的光热转化效率，实际计算其光热效率达到了35.64%. 此外，IOS还具有较强的类过氧化氢酶活性、类过氧化物酶活性以及声动力效果. 通过体外的光热或声动力结合IOS的酶催化效应进行抗肿瘤实验，都取得了优异的抗肿瘤效果. 基于IOS的多模式协同治疗有望在体内研究中达到彻底消除肿瘤的目的. 总而言之，IOS是一种能够有效杀伤肿瘤细胞且生物相容性良好的抗肿瘤纳米材料，具有广阔的临床转化前景和商业化价值.

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Synthesis of IrO₂@SF Nanoparticle and Its Antitumor Applications