赫赛汀靶向载阿霉素/印度墨水多功能分子探针的制备及其对乳腺癌成像诊断及治疗的实验研究

doi:10.3877/cma.j.issn.1672-6448.2020.06.015

目的

制备赫赛汀靶向载阿霉素/印度墨水（DOX-ink-HER-NBs）多功能分子探针，评估其在乳腺癌动物模型成像诊断及治疗中的应用价值。

方法

通过薄膜水化、碳二亚胺法制备DOX-ink-HER-NBs多功能纳米造影剂，检测其基本物理特性。通过激光共聚焦显像及流式细胞术检测靶向纳米分子探针体外靶向乳腺癌细胞能力。选取6周龄雌性BALB/c裸鼠构建乳腺癌移植瘤模型。将18只移植瘤裸鼠随机分为靶向组（DOX-ink-HER-NBs）、非靶向组（DOX-ink-NBs）和对照组（PBS），每组6只，通过尾静脉分别注射200 μl DOX-ink-HER-NBs混悬液、DOX-ink-NBs混悬液、PBS，应用超声诊断仪及光声成像系统观察分子探针在裸鼠体内超声、光声双模态显像情况。通过激光共聚焦对组织冰冻切片的显像，观察分子探针在肿瘤组织内的分布。对18只移植瘤裸鼠尾静脉注射200 μl DOX-ink-HER-NBs、DOX-ink-NBs、PBS，配合超声辐照，每3天注射治疗一次，持续治疗7次，观测分子探针抑制移植瘤生长的效果。

结果

DOX-ink-HER-NBs分子探针粒径约（621.23±42.56）nm，电位约（-25.34±2.56）mV。流式细胞结果显示靶向组的体外寻靶率较非靶向组、对照组高［（71.64±9.32）% vs （16.99±4.84）%、（9.29±3.15）%］，差异均有统计学意义（P均<0.0001）。裸鼠移植瘤体内实验结果表明，与非靶向组和对照组相比，靶向分子探针能够大量聚集在肿瘤组织细胞周围。靶向组移植瘤部位的超声造影信号强度显著强于非靶向组及对照组［（60.33±4.51）dB vs （15.67±4.04）dB、（7.00±2.00）dB］，差异均有统计学意义（P均<0.0001）。与非靶向组及对照组比较，靶向组移植瘤的光声信号显像明显［（0.8567±0.0950）a.u. vs（0.3233±0.0950）a.u.、（0.1033±0.0153）a.u.］，差异均有统计学意义（P=0.0002、<0.0001）。与非靶向组和对照组相比，靶向组肿瘤抑制率最高［（62.93±6.96）% vs（22.73±8.05）%、（18.33±15.88）%］，差异均有统计学意义（P=0.0043、0.0026）。

结论

本研究成功制备了DOX-ink-HER-NBs多功能分子探针。在赫塞汀的介导下纳米分子探针可聚集于靶组织，实现乳腺癌的超声及光声双模式分子成像，同时协同发挥抗肿瘤效应，为乳腺癌的分子诊断和可视化靶向治疗及疗效评估提供了新的思路。

关键词: 分子探针, 乳腺肿瘤, 超声成像, 光声成像, 造影剂

Objective

To prepare a Herceptin targeted doxorubicin/Indian ink conjugated nano-bubbles (DOX-ink-HER-NBs) as a multifunctional nano-probe, and evaluate its application value in the imaging diagnosis and treatment of breast cancer in an animal model.

Methods

DOX-ink-HER-NBs were prepared by film hydration and carbodiimide method, and the basic physical properties of the nano-bubbles were detected. Laser confocal imaging and flow cytometry were used to detect the ability of the nano-probe to target breast cancer cells in vitro. Six-week-old female BALB/c nude mice were randomly divided into three groups (n=6 each): targeted group (DOX-ink-HER-NBs), non-targeted group (DOX-ink-NBs), and control group (PBS). Two hundred microliters of DOX-ink-HER-NBs suspension, DOX-ink-NBS suspension, and PBS were injected through the tail vein, respectively. Ultrasound and photoacoustic imaging system were used to observe the ultrasound and photoacoustic dual-mode imaging of molecular probes in nude mice. The distribution of molecular probes in tumor tissue was observed by laser confocal imaging. Eighteen nude mice with transplanted tumor were injected with DOX-ink-HER-NBs, DOX-ink-NBs, and PBS via the tail vein and underwent ultrasound irradiation, every 3 days for 7 times. The effect of the molecular probe in inhibiting the growth of transplanted tumor was observed.

Results

The particle size of DOX-ink-HER-NBs was about (621.23±42.56) nm, and the potential was about (-25.34±2.56) mV. Flow cytometry showed that the targeted group had a stronger ability to target breast cancer cells than the non-targeted group and the control group [(71.64±9.32)% vs (16.99±4.84)% and (9.29±3.15)%, P<0.0001]. In vivo results showed that compared with the non-targeted group and the control group, the targeted molecular probe could gather around the tumor tissue. The signal intensity of contrast-enhanced ultrasound in the targeted group was significantly higher than that in the non-targeted group and the control group [(60.33±4.51) dB vs (15.67±4.04) dB and (7.00±2.00) dB, P<0.0001]. Compared with the non-targeted group and the control group, the photoacoustic signal imaging of the targeted group was more significant [(0.8567±0.0950) a.u. vs (0.3233±0.0950) a.u. and (0.1033±0.0153) a.u.; P=0.0002 and P<0.0001, respectively]. Compared with the non-targeted group and the control group, the tumor inhibition rate of the targeted group was the highest [(62.93±6.96)% vs (22.73±8.05)% and (18.33±15.88)%; P=0.0043 and 0.0026, respectively].

Conclusion

We have successfully prepared DOX-ink-HER-NBs. Under the guidance of Herceptin, the nano-molecular probes can gather in the target tissue, thus realizing ultrasound and photoacoustic dual-mode molecular imaging of breast cancer. At the same time, the probe synergistically has an anti-tumor effect, which provides a new idea for molecular diagnosis, targeted treatment, and efficacy evaluation of breast cancer.

Key words: Molecular probes, Breast neoplasms, Ultrasonography, Photoacoustic imaging, Contrast agent

图1 赫赛汀靶向载阿霉素/印度墨水分子探针（DOX-ink-HER-NBs）的物理特性。图a为DOX-ink-HER-NBs粒径图；图b为DOX-ink-HER-NBs电位图

图2 激光共聚焦显微镜下不同分子探针在体外乳腺癌细胞中的靶向分布。图a~c分别为靶向分子探针组同一视野下蓝色荧光标记的乳腺癌细胞核分布图、红色荧光标记的靶向分子探针分布图、乳腺癌细胞核与靶向分子探针分布融合图；图d~f分别为非靶向分子探针组同一视野下蓝色荧光标记的乳腺癌细胞核分布图、红色荧光标记的非靶向分子探针分布图、乳腺癌细胞核与非靶向分子探针分布融合图；图g~i分别为对照组（PBS组）同一视野下蓝色荧光标记的乳腺癌细胞核分布图、红色荧光标记的PBS分子探针分布图、乳腺癌细胞核与PBS分子探针分布融合图

图3 激光共聚焦显微镜下多功能分子探针在裸鼠移植瘤内的分布。图a为PBS在裸鼠移植瘤内的分布；图b为非靶向分子探针在裸鼠移植瘤内的分布；图c为靶向分子探针在裸鼠移植瘤内的分布

图4 多功能分子探针在裸鼠移植瘤体内的超声及光声显像。图a、d分别为PBS在裸鼠移植瘤体内的超声、光声显像；图b、e分别为非靶向分子探针在裸鼠移植瘤体内的超声、光声显像；图c、f分别为靶向分子探针在裸鼠移植瘤体内的超声、光声显像

表1 靶向组、非靶向组与对照组的体内超声造影强度值及光声值比较（±s）

组别	只数	超声造影强度值（dB）	光声值（a.u.）
靶向组（DOX-ink-HER-NBs）	6	60.33±4.51	0.8567±0.0950
非靶向组（DOX-ink-NBs）	6	15.67±4.04^a	0.3233±0.0950^a
对照组（PBS）	6	7.00±2.00^b	0.1033±0.0153^b
F值		181.27	73.87
P值		P<0.0001	P=0.0001

表1 靶向组、非靶向组与对照组的体内超声造影强度值及光声值比较（±s）

组别	只数	超声造影强度值（dB）	光声值（a.u.）
靶向组（DOX-ink-HER-NBs）	6	60.33±4.51	0.8567±0.0950
非靶向组（DOX-ink-NBs）	6	15.67±4.04^a	0.3233±0.0950^a
对照组（PBS）	6	7.00±2.00^b	0.1033±0.0153^b
F值		181.27	73.87
P值		P<0.0001	P=0.0001

表2 靶向组、非靶向组与对照组裸鼠在体肿瘤超声辐照治疗效果比较（±s）

组别	只数	治疗第21天的肿瘤体积（mm³）	肿瘤体积生长率（%）	肿瘤抑制率（%）
靶向组（DOX-ink-HER-NBs）	6	716.67±76.37	449.73±58.96	62.93±6.96
非靶向组（DOX-ink-NBs）	6	1516.67±275.38^a	921.13±180.59^a	22.73±8.05^a
对照组（PBS）	6	2000.00±550.00^b	1233.47±386.35^b	18.33±15.88^b
F值		9.84	10.21	14.88
P值		0.0127	0.0084	0.0047

表2 靶向组、非靶向组与对照组裸鼠在体肿瘤超声辐照治疗效果比较（±s）

组别	只数	治疗第21天的肿瘤体积（mm³）	肿瘤体积生长率（%）	肿瘤抑制率（%）
靶向组（DOX-ink-HER-NBs）	6	716.67±76.37	449.73±58.96	62.93±6.96
非靶向组（DOX-ink-NBs）	6	1516.67±275.38^a	921.13±180.59^a	22.73±8.05^a
对照组（PBS）	6	2000.00±550.00^b	1233.47±386.35^b	18.33±15.88^b
F值		9.84	10.21	14.88
P值		0.0127	0.0084	0.0047

图5 裸鼠移植瘤超声辐照治疗第21天的肿瘤体积。图a为PBS组裸鼠治疗后移植瘤体积；图b为非靶向分子探针组裸鼠治疗后移植瘤体积；图c为靶向分子探针组裸鼠治疗后移植瘤体积

图6 3组乳腺癌裸鼠皮下移植瘤肿瘤体积生长曲线

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Preparation of Herceptin targeted doxorubicin/Indian ink conjugated multifunctional molecular probe: application in imaging diagnosis and treatment of breast cancer

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Preparation of Herceptin targeted doxorubicin/Indian ink conjugated multifunctional molecular probe: application in imaging diagnosis and treatment of breast cancer