纸质出版日期:2019-6,
网络出版日期:2019-3-28,
收稿日期:2019-1-3,
修回日期:2019-2-12
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为了解决花菁类(Cypate)近红外染料作为光热试剂的一些固有缺点,例如疏水性、毒性、不稳定性等,以牛血清白蛋白和Cypate染料作为原料,在水热条件下制备出尺寸较小的稳定杂化纳米粒. 纳米结构显著改善了Cypate染料的水溶性,也提升了染料在水相溶液中的稳定性. 与小分子染料相比,染料杂化蛋白纳米粒具有良好的生物相容性和光稳定性. 此外,蛋白纳米粒具有优于小分子染料的光热转换能力,并展现了良好的光热细胞杀伤能力.
To make up for the inherent drawbacks (e.g. hydrophobicity, toxicity, and instability) of near-infrared Cypate dyes, as photothermal agents, stable hybrid nanoparticles (CBNPs) with small size were prepared from bovine serum albumin (BSA) and Cypate dyes under hydrothermal conditions. Free Cypate exhibited strong fluorescence whereas fluorescence quenching in CBNPs caused a sharp decrease in emission peak intensity, so the dye molecules were successfully assembled into protein nanoparticles. As-prepared CBNPs were characterized with dynamic light scattering (DLS) and transmission electron microscopy (TEM). The hydrodynamic diameter of CBNPs was around 25 – 40 nm with a low polydispersity index of 0.2, slightly higher than the measured values from TEM observation. BSA encapsulation could significantly improve the water solubility of Cypate dye and the stability of dye aqueous solutions. Besides, these nanoparticles showed good colloidal stability and biocompatibility. Photothermal experiments suggested that the protein nanoparticles had good photothermal performance and were able to generate enough heat under near-infrared laser irradiation. The photothermal conversion efficiency (η) of CBNPs under 808 nm laser irradiation reached up to 50%, which made them outperform the small molecules of neat Cypate dyes in terms of their photothermal conversion capabilities. Confocal laser scanning microscopy further revealed that the protein nanoparticles could be efficiently internalized by cancer cells in a time-dependent manner, which is very important for their therapeutic functions. Photothermal treatment toward human cervical carcinoma (HeLa) cells and human liver hepatocellular carcinoma (HepG2) cells under laser irradiation was extamined by MTT method, in which the protein nanoparticles showed an effective inhibition effect on tumor cell proliferation at the cellular level. Live/dead cell staining experiments conducted on HeLa cells also showed the same results intuitively. The dye-protein hybrid nanoparticles developed in this study as a novel nano-agent possess promising prospects in the field of tumor photothermal therapy.
Dye-protein hybrid nanoparticles with small size were successfully prepared under hydrothermal conditions. The experimental procedure was greatly simplified and the hybrid nanoparticles showed good biocompatibility and stability. Such dye-protein hybrid nanoparticles hold great potential in the application field of photothermal therapy.
光热治疗(PTT)是近几年被广泛研究的一种新兴肿瘤治疗策略. 病灶区域蓄积的光热试剂在特定波长的激光照射下,可以将吸收的光能转换为热能,从而产生局部高温,引起病灶组织细胞凋亡或坏死,而且可以有效降低对正常组织细胞的副作用[
花菁类(Cypate)染料是被广泛研究的一种近红外染料[
白蛋白,作为含量最高的血浆蛋白,在调节渗透压和运输内源物质上扮演着重要角色[
我们在先前的研究工作中发现,白蛋白与具有负电性的染料通过简单的水热法可以形成性能优良的蛋白纳米粒[
Fig 1 Scheme of the preparation of CBNPs and their application in photothermal therapy
BSA和噻唑蓝(MTT)以及青霉素-链霉素-两性霉素B混合液购自源叶生物. 所用的Cypate染料参照文献中的方法进行合成[
将BSA溶于水中,配成浓度为1 mmol/L的溶液. 将Cypate溶于乙醇中,配成浓度为1 mmol的溶液. 取5.6 mL水,置于反应釜中,分别加入0.8 mL的BSA和1.6 mL的Cypate,混合均匀后,将反应釜置于烘箱中,程序升温到100 °C,加热5 min后,取出反应釜,室温下降至可处理温度,取出釜中溶液置于透析袋中(截留分子量3500),用去离子水透析约24 h,每3 h换1次透析液. 之后使用0.45 μm的滤头过滤.
纳米粒的粒径和粒径分布均由英国马尔文电位粒度仪(Zetasizer Nano ZS90)通过测定DLS得到. 测量温度选择标准室温25 °C,散射角固定在90°. TEM图由日本的JEOL JEM-1011透射电镜在加速电压100 kV时测量得到,用于观察纳米粒的形貌和尺寸分布情况. 为了制备用于测量透射电镜的样品,把10 μL的纳米粒样品滴加在碳膜覆盖的铜网上. 在空气中室温晾干. 紫外-可见光吸收光谱在Shimadzu UV-2450PC紫外-可见光扫描光谱仪上测得. 荧光发射光谱在LS-55荧光光谱仪上测得. 狭缝宽度根据需要进行调节,扫描速度是500 nm/min. 傅里叶变换红外光谱(FTIR)的测试仪器为德国Bruker Vertex 70真空傅里叶变换红外光谱仪. 样品制备的方法是将样品和溴化钾充分混合后压片. XRD广角X射线衍射仪(D8 ADVANCE 德国BRUKER公司)Cu靶波长为0.154 nm. 细胞培养箱的型号是ESCO Cell culture CO2 Incubator,Singapore. 酶标仪的型号是BioTeK ELX808TM (USA),测定96孔细胞板在490 nm处的吸光度值. 细胞的共聚焦图片使用蔡司共聚焦激光显微镜(ZEISS LSM 700)测得.
为了评价CBNPs的光热能力,取0.3 mL CBNPs和游离的染料Cypate (5 μg/mL)使用激光(808 nm, 1 W/cm2) 照射5 min. 每隔30 s使用温度探测器测一次溶液温度. 为了计算纳米粒的光热转换效率(η),激光照射样品2 min,当溶液温度形成平台后,撤掉激光,记录溶液回复到室温过程中的温度变化,与时间做成降温曲线. 然后参照文献的方法[
1.5.1 细胞培养
人宫颈癌细胞系(HeLa)和人肝癌细胞系(HepG2)购买自中国科学院上海生物化学与细胞生物学研究所. 细胞使用DMEM培养基进行培养,其中添加有链霉素和青霉素以及10%的胎牛血清. 培养箱条件设定为37 °C和5%的CO2.
1.5.2 纳米粒细胞摄取测定
纳米粒的细胞摄取使用共聚焦荧光显微镜检测. 收集对数生长期的细胞,按照每孔2.5 × 105个细胞的浓度加入到六孔板的孔中,加入细胞前,在每个孔中要先加入一个无菌处理的盖玻片. 然后在培养箱中继续培养24 h. 让细胞充分贴在盖玻片上. 然后将纳米粒溶液使用培养基稀释到所需的浓度,吸去原有的培养基,然后用纳米粒溶液对细胞进行孵育,按照设定的浓度和时间进行处理,依旧在培养箱中进行培养. 对于将要进行共聚焦的样品组,细胞使用磷酸盐缓冲液(PBS)洗涤3次后,使用4%的多聚甲醛对细胞进行固定10 min. 然后用PBS洗涤细胞3次. 用DAPI对细胞核进行染色,染5 min即可. 然后仍旧用PBS洗涤细胞3次. 取干净的载玻片,在片子上滴加10 μL甘油和水的等体积混合液,然后将孔板中的盖玻片取出来,注意细胞所在层面,这个层面要面对载玻片,然后放置于液体上,使用滤纸压一下,吸掉多余的液体,使用封片剂封片并做好标记. 然后将片子放在共聚焦荧光显微镜下进行观察和拍照.
1.5.3 纳米粒的生物相容性以及光毒性检测
将处于对数生长期的细胞消化收集,离心洗涤,细胞计数,然后以每孔8000个细胞种在96孔板中,进行培养,当细胞重新贴壁并达到要求的汇合度后,先把CBNPs按照设定的浓度梯度配置好,先将原有的培养基吸走,然后将配置好的药物按顺序加好,并做好标记. 在培养箱中继续培养24 h或者48 h,然后吸去孔中的溶液,PBS洗一次,在每个孔中加入150 μL的新鲜培养基,然后每个孔中加入20 μL的MTT (5 mg/mL),37 °C下孵育4 h. 取出孔板,吸去孔中溶液,每个孔中加入150 μL的DMSO,在酶标仪上用中等振板速度振板5 min,然后在490 nm处检测吸收值. 不加入药品的组作为对照组. 之后对细胞存活率进行计算. 细胞存活率 = 样品组的吸收值/对照组的吸收值 × 100%.
对于光热细胞毒性,肿瘤细胞与不同浓度的纳米粒孵育6 h后,吸出溶液,用PBS洗涤3次,更换为新鲜培养基,然后进行5 min激光照射 (808 nm, 1 W/cm2). 继续培养24 h后,使用MTT法评价细胞存活率.
1.5.4 活死细胞染色
为了进一步证明纳米粒的生物相容性,将孵育过纳米粒的细胞分别使用活死细胞检测试剂盒按照说明书的操作流程进行染色. 没有使用纳米粒孵育的细胞作为对照组. 然后使用荧光显微镜观察结果并拍摄图片. 红色荧光代表死细胞,绿色荧光代表活细胞.
在前期的工作中[
Fig 2 (a) DLS intensity-weighted diameter of CBNPs (Inset: TEM image of CBNPs); (b) Absorbance spectra, (c) PL spectra, and (d) FTIR spectra of Cy, BSA and CBNPs
接下来使用粉末X射线衍射和傅里叶转换红外光谱进一步研究染料和蛋白的组装. 如
为了证明纳米粒的光热转换能力,我们监测了CBNPs水溶液在激光(808 nm, 1 W/cm2)照射下的变化并以游离小分子Cypate作为对照组. 如
Fig 3 Photothermal conversion behavior of CBNPs in water and Cypate in DMF under irradiation (808 nm, 1 W/cm2) for 5 min with DMF and water as the control
Fig 4 IR thermal images of the CBNPs aqueous dispersions with various power densities (upper row, with CBNPs concentration fixed at 5 μg/mL)or concentrations (lower row, with power density fixed at 1 A) under 808 nm laser irradiation for 5 min
良好的稳定性对于纳米粒维持它们的固有功能是非常重要的. 首先,我们利用DLS评价了CBNPs在水相溶液和含有20% FBS的溶液中的胶体稳定性. 如
Fig 5 Stability of CBNPs in (a) water or (b) 20% FBS; UV-Vis spectra of (c) free Cypate and (d) CBNPs determined right after the dilution process or 1 day of storage (black: fresh solution; red: 1 day of storage in darkness; blue: 1 day of storage in light) (The online version is colorful.)
细胞摄取对于纳米材料执行其功能是非常必要的. 使用共聚焦显微镜对CBNPs在HeLa和HepG2细胞上的细胞摄取行为进行了研究. 细胞在37 °C用CBNPs孵育不同时间后,使用Hoechst33258对细胞核进行染色处理. 如
Fig 6 Confocal microscopy images of the HeLa cells incubated with CBNPs for different time (The blue and red colors represent the fluorescence of Hoechst 33258 and CBNPs images, respectively.) (The online version is colorful.)
良好的生物相容性是光疗纳米药物必备的先决条件之一,CBNPs蛋白纳米粒的生物相容性以及在激光照射下的光毒性测试都采用MTT法. 选择了HeLa和HepG2两种细胞系进行测定. 首先,我们对BSA的细胞毒性进行了研究. 如
Fig 7 The cell viability of (a) HeLa or (b) HepG2 cells incubated with BSA at different concentrations; The cell viability of (c) HeLa or (d) HepG2 cells incubated with CBNPs without and with irradiation by 808 nm laser (1 W/cm2, 5 min)
为了更直观地显示CBNPs在肿瘤细胞光热消除上的效果,肿瘤细胞分别使用PBS和CBNPs处理后,用激光照射5 min. 使用活死细胞染色试剂盒对光照后的细胞进行处理,钙黄绿素染活细胞,PI染死细胞.
Fig 8 Fluorescence microscopy images of HeLa cells co-cultured with calcein AM (green, dye of live cells) and propidium iodide (red, dye of dead cells), in the absence (upper row) or presence (lower row) of CBNPs, and subjected to irradiation (right column) or not (left column) (The online version is colorful.)
本文设计并制备得到一种平均尺寸较小的包封有花菁类染料的蛋白纳米粒,纳米粒具有较好的胶体稳定性和生物相容性,使用BSA包封不仅提升了Cypate染料的水溶性也提升了染料在水相溶液中的稳定性. 光热实验结果表明,蛋白纳米粒具有良好的光热性能,能够在近红外光照射下快速产生足够的热量,展现了良好的光热转换能力. 此外,蛋白纳米粒能够有效地被细胞摄取. 重要的是,在细胞水平上,蛋白纳米粒在激光照射下能够很好地抑制肿瘤细胞的增殖活性. 总之,这种新型的染料-蛋白杂化纳米粒作为肿瘤光热治疗的纳米试剂在肿瘤治疗领域具有广泛的应用前景.
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