利用发夹穿透肽标记的顺磁颗粒对 SKOV-3 细胞进行成像。

SKOV-3 cell imaging by paramagnetic particles labeled with hairpin cell-penetrating peptides.

机构信息

Imaging Center, the First Affiliated Hospital of Medical School, Xi'an Jiao Tong University, Xi'an 710004, Shannxi, China.

出版信息

Chin Med J (Engl). 2011 Jan;124(1):111-7.

PMID:21362318

Abstract

BACKGROUND

The hairpin cell-penetrating peptides (hCPPs) demonstrate an interesting characteristic of conditioned activation by molecules. We hypothesized that hCPPs have the potential to selectively deliver a paramagnetic gadolinium probe into the matrix metalloproteinase 2 (MMP-2) positive human ovary adenocarcinoma cell lines, SKOV-3.

METHODS

hCPPs were synthesized and labeled with 1,4,7,10-tetraazacyclododecane-N,N',N'',N''' tetraacetic acid gadolinium (III) (Gd-DOTA) and fluorescein isothiocyanate (FITC) by f-moc strategy using a standard solid phase peptide synthesis protocol. MMP-2 expression and activity were demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) and zymography. Internalization and location of hCPPs in SKOV-3 cells were observed by fluorescein imaging and flow cytometery. Selective delivery of Gd-DOTA in SKOV-3 cells was observed by magnetic resonance imaging (MRI) and transmission electron microscopy (TEM).

RESULTS

The uptake of hCPPs by SKOV-3 cells depended on the activity of MMP-2. T1WI signals of SKOV-3 cells treated with Gd-DOTA-hCPPs suggested the uptake of Gd-DOTA-hCPPs increased in a time- (r = 0.990, P < 0.01) and concentration-dependent manner (r = 0.964, P < 0.001), but was inhibited by a MMP-2 inhibitor. Electron-dense particles observed in the cytoplasm and nucleus by transmission electron microscopy proved the intracellular penetration of gadolinium.

CONCLUSIONS

hCPPs can be used as an effective vector for an MRI molecular probe to assess the activity of MMP-2.

摘要

背景

发夹状细胞穿透肽（hCPPs）表现出一种有趣的分子条件激活特性。我们假设 hCPPs 有可能选择性地将顺磁钆探针递送至基质金属蛋白酶 2（MMP-2）阳性的人卵巢腺癌 SKOV-3 细胞系中。

方法

hCPPs 通过 f-moc 策略使用标准固相肽合成方案进行合成，并通过 1,4,7,10-四氮杂环十二烷-N,N',N'',N'''四乙酸钆（III）（Gd-DOTA）和异硫氰酸荧光素（FITC）标记。通过逆转录聚合酶链反应（RT-PCR）和酶谱法证明 MMP-2 的表达和活性。通过荧光成像和流式细胞术观察 hCPPs 在 SKOV-3 细胞中的内化和定位。通过磁共振成像（MRI）和透射电子显微镜（TEM）观察 Gd-DOTA 在 SKOV-3 细胞中的选择性递药。

结果

hCPPs 被 SKOV-3 细胞摄取取决于 MMP-2 的活性。用 Gd-DOTA-hCPPs 处理的 SKOV-3 细胞的 T1WI 信号表明，Gd-DOTA-hCPPs 的摄取呈时间依赖性（r = 0.990，P < 0.01）和浓度依赖性（r = 0.964，P < 0.001）增加，但被 MMP-2 抑制剂抑制。透射电子显微镜下观察到的细胞质和细胞核中的电子致密颗粒证明了镧系元素的细胞内渗透。