载IR780靶向纳米粒体外荧光成像及光动力诱导大鼠脉络膜血管内皮细胞凋亡的研究

冉一君; 王梦蕾; 黎梦; 周希瑗; 曹阳; 冉海涛

doi:10.16016/j.1000-5404.201907085

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载IR780靶向纳米粒体外荧光成像及光动力诱导大鼠脉络膜血管内皮细胞凋亡的研究

基础医学 | 更新时间：2026-01-09

- 载IR780靶向纳米粒体外荧光成像及光动力诱导大鼠脉络膜血管内皮细胞凋亡的研究
- In vitro imaging of targeting fluorescent IR780 nanoparticles and their mediated photodynamics in rat choroidal endothelial cells
- 第三军医大学学报 2020年42卷第1期页码：67-74
- 作者机构：
  
  1.400010 重庆，重庆医科大学附属第二医院:眼科
  2.400016 重庆，重庆医科大学超声影像学研究所，超声分子影像重庆市重点实验室
  3.400010 重庆，重庆医科大学附属第二医院:超声科
- 作者简介：
  
  [周希瑗，E-mail:zhouxiyuan2002@aliyun.com ZHOU Xiyuan, E-mail:zhouxiyuan2002@aliyun.com
- 基金信息：
  
  国家自然科学基金重点项目(81630047);国家自然科学基金重点项目(81501487)
- DOI：10.16016/j.1000-5404.201907085
  中图分类号： R944.9;R969.4;R977.6
- 收稿：2019-07-11，
  
  修回：2019-08-26，
  
  纸质出版：2020-01-15
- 稿件说明：
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冉一君, 王梦蕾, 黎梦, 等. 载IR780靶向纳米粒体外荧光成像及光动力诱导大鼠脉络膜血管内皮细胞凋亡的研究[J]. 第三军医大学学报, 2020,42(1):67-74.

Yijun RAN, Menglei WANG, Meng LI, et al. In vitro imaging of targeting fluorescent IR780 nanoparticles and their mediated photodynamics in rat choroidal endothelial cells[J]. Journal of Third Military Medical University, 2020, 42(1): 67-74.
冉一君, 王梦蕾, 黎梦, 等. 载IR780靶向纳米粒体外荧光成像及光动力诱导大鼠脉络膜血管内皮细胞凋亡的研究[J]. 第三军医大学学报, 2020,42(1):67-74. DOI： 10.16016/j.1000-5404.201907085.

Yijun RAN, Menglei WANG, Meng LI, et al. In vitro imaging of targeting fluorescent IR780 nanoparticles and their mediated photodynamics in rat choroidal endothelial cells[J]. Journal of Third Military Medical University, 2020, 42(1): 67-74. DOI： 10.16016/j.1000-5404.201907085.

摘要

目的

制备一种新型载IR780靶向血管内皮生长因子受体2(vascular endothelial growth factor receptor 2，VEGFR2)纳米粒(VEGFR2/IR780/PLGA-NPs)，体外评价其物理性质、荧光成像能力、对大鼠脉络膜血管内皮细胞(rat choroid vascular endothelial cells，RCVECs)的寻靶能力及光动力治疗(photodynamic therapy，PDT)效果。

方法

采用超声乳化法制备包载IR780的非靶向纳米粒(IR780/PLGA-NPs)，运用碳二亚胺法将VEGFR2抗体与纳米粒相连接，最终制备出靶向纳米粒(VEGFR2/IR780/PLGA-NPs)；检测其基本特性；运用CCK-8法检测纳米粒的细胞毒性；定量荧光和生物发光成像系统评价纳米粒的荧光成像能力；激光共聚焦与流式细胞术评价靶向组、非靶向组、抗体封闭组纳米粒体外寻靶能力；单线氧荧光探针(singlet oxygen sensor green reagent，SOSG)评估体外光动力治疗效果；流式细胞仪检测靶向激光组、非靶向激光组、单纯激光组纳米粒介导的光动力诱导RCVECs的凋亡比例。

结果

成功制备出VEGFR2/IR780/PLGA-NPs，不同显微镜下观察呈球形，大小均匀，粒径(273.23±31.55)nm；电位(-8.85±1.60)mV；IR780包封率(87.37±2.44)%。荧光成像结果显示:纳米粒浓度越高，其荧光信号强度越强。CCK-8法检测结果显示纳米粒没有明显的细胞毒性。流式细胞仪检测VEGFR2抗体已成功连接于纳米粒，其连接率为(83.60±4.79)%。激光共聚焦观察发现在靶向组中RCVECs周围的纳米粒明显多于非靶向组和抗体封闭组。流式细胞仪检测发现靶向组纳米粒与细胞连接率为(58.13±1.64)%，明显高于非靶向组(20.64±1.89)%和抗体封闭组(15.12±1.16)%，差异有统计学意义(

0.05)。经过激光辐照后，随纳米粒浓度增加，体外活性氧产量逐渐增加，具备良好的PDT能力。流式细胞仪检测发现靶向激光组诱导RCVECs凋亡的比例为(82.99±0.98)%，明显高于非靶向激光组(30.75±1.81)%和单纯激光组(7.05±0.96)%，差异有统计学意义(

0.05)。证实靶向纳米粒具备更好的PDT效果。

结论

成功制备VEGFR2/IR780/PLGA-NPs，其在体外具有良好的靶向能力，同时可用于荧光成像以及光动力治疗。

Abstract

Objective

To prepare novel IR780-loaded nanoparticles targeting vascular endothelial growth factor receptor 2 (VEGFR2)

and evaluate their physical properties

fluorescence imaging ability

targeting ability to rat choroidal vascular endothelial cells (RCVECs)

and the effects of photodynamic therapy (PDT).

Methods

The non-targeting nanoparticles loaded with IR780 (IR780/PLGA-NPs) were prepared by single emulsification. After anti-VEGFR2 antibodies were coupled with nanoparticle by carbodiimide

then the targeting nanoparticles (VEGFR2/IR780/PLGA-NPs) were prepared finally. CCK-8 assay was carried out to detect the cytotoxicity of nanoparticle; Quantitative fluorescence and bioluminescence imaging system was per

formed to evaluate the fluorescence imaging ability of the nanoparticles

in vitro

. Laser confocal and flow cytometry were employed to evaluate the connectivity and

in vitro

targeting ability of the targeted group

non-targeted group and closed receptor group. The effect of PDT was determined by singlet oxygen sensor green reagent (SOSG); Flow cytometry was applied to detect the percentage of apoptotic RCVECs in the targeted laser group

non-targeted laser group and simple laser irradiated group after nanoparticle-mediated PDT.

Results

The nanoparticles VEGFR2/IR780/PLGA-NPs were successfully prepared

and presented spherical in shape and uniform in size

in a particle size of 273.23±31.55 nm

at a potential of -8.85±1.60 mV

with an encapsulation efficiency of (87.37±2.44)% to IR780. Fluorescence imaging showed that the fluorescence signal intensity was increased with the increment of concentration of the nanoparticles. The result of CCK-8 assay indicated that there was no obvious cytotoxicity of the nanoparticles. Flow cytometry displayed that VEGFR2 antibodies were successfully attached to the nanoparticles

with a ratio of (83.60±4.79)%. Laser confocal microscopy demonstrated that there were more nanoparticles around the RCVECs in the targeted group than the other 2 groups. Flow cytometry also showed that the connectivity of nanoparticles to RCVECs was (58.13±1.64)% in the targeted group

which was significantly higher than that of the non-targeted group [(20.64±1.89)%

0.05

]

and the closed receptor group [(15.12±1.16)%

0.05

]

. After irradiated with 808 nm laser

the production of reactive oxygen species was gradually increased with the increment of nanoparticles

which means the nanoparticles having good PDT ability. Flow cytometry indicated that the apoptotic rate of RCVECs was (82.99±0.98)% in the targeted laser group

obviously higher than those of the non-targeted laser group [(30.75±1.81)%

0.05

]

and the simple laser irradiated group [(7.05±0.96)%

0.05

]

suggesting sound PDT effect of the nanoparticles.

Conclusion

The targeting nanoparticles VEGFR2/IR780/PLGA-NPs are successfully prepared. They have good targeting ability

in vitro

and could be used for fluorescence imaging and PDT.

关键词

Keywords

references

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