Background The cellular plasma membrane represents a natural barrier to many exogenous molecules including magnetic resonance （MR） contrast agent. Cell penetrating peptide （CPP） is used to internalize proteins, pe...Background The cellular plasma membrane represents a natural barrier to many exogenous molecules including magnetic resonance （MR） contrast agent. Cell penetrating peptide （CPP） is used to internalize proteins, peptides, and radionuclide. This study was undertaken to assess the value of a new intracellular MR contrast medium, CPP labeled diethylenetriamine pentaacetic acid gadolinium （Gd-DTPA） in molecular imaging in vitro. Methods Fluorescein-5-isothiocyanate （FITC） and Gd-DTPA respectively labeled with CPP （FITC-CPP, Gd-DTPA-CPP） were synthesized by the solid-phase method. Human hepatic cancer cell line-HepG2 was respectively stained by FITC-CPP and FITC to observe the uptake and intracellular distribution. HepG2 was respectively incubated with 100 nmol/ml Gd-DTPA-CPP for 0, 10, 30, 60 minutes, and imaged by MR for studying the relationship between the incubation time and T：W1 signal. The cytotoxicity to NIH3T3 fibroblasts cells was measured by 3-（4,5-dimethylthiazol-2-yl）- 2,5-diphenyltetrazolium bromide reduction assay （MTr）. Results The molecular weights of CPP labeled imaging agents, which were determined by MALDI mass spectrometry （FITC-CPP MW=2163.34, Gd-DTPA-CPP MW=2285.99）, were similar to the calculated molecular weights. Confocal microscopy suggested HepG2 translocated FITC-CPP in cytoplasm and nucleus independent with the incubation temperature. MR images showed HepG2 uptaken Gd-DTPA-CPP had a higher T1 weighted imaging （T1W1） signal, and that the T1W1 signal intensity was increasing in a time-dependent manner （r=0.972, P=0.001）, while the signal intensity between the cells incubated by Gd-DTPA for 60 minutes and the controlled cells was not significantly different （P=0.225）. By MTT, all concentrations from 50 nmol/ml to 200 nmol/ml had no significant （F=0.006, P=1.000） effect on cell viability of mouse NIH3T3 fibroblasts, compared with the control group. Conclusions The newly constructed CPP based on polyarginine can translocate cells by carrying FITC and MR contrast agent Gd-DTPA, and the intracellular concentrations are readily detectable by MR imaging, suggesting a new way for MR molecular imaging.展开更多
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 param...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 （111） （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.展开更多
基金This work was supported by a grant from Doctoral Special Research Foundation of Ministry of Education(No.20060698049)Doctoral Foundation of Xi'an Jiao Tong University(No.dfxjtu2005-09)
文摘Background The cellular plasma membrane represents a natural barrier to many exogenous molecules including magnetic resonance （MR） contrast agent. Cell penetrating peptide （CPP） is used to internalize proteins, peptides, and radionuclide. This study was undertaken to assess the value of a new intracellular MR contrast medium, CPP labeled diethylenetriamine pentaacetic acid gadolinium （Gd-DTPA） in molecular imaging in vitro. Methods Fluorescein-5-isothiocyanate （FITC） and Gd-DTPA respectively labeled with CPP （FITC-CPP, Gd-DTPA-CPP） were synthesized by the solid-phase method. Human hepatic cancer cell line-HepG2 was respectively stained by FITC-CPP and FITC to observe the uptake and intracellular distribution. HepG2 was respectively incubated with 100 nmol/ml Gd-DTPA-CPP for 0, 10, 30, 60 minutes, and imaged by MR for studying the relationship between the incubation time and T：W1 signal. The cytotoxicity to NIH3T3 fibroblasts cells was measured by 3-（4,5-dimethylthiazol-2-yl）- 2,5-diphenyltetrazolium bromide reduction assay （MTr）. Results The molecular weights of CPP labeled imaging agents, which were determined by MALDI mass spectrometry （FITC-CPP MW=2163.34, Gd-DTPA-CPP MW=2285.99）, were similar to the calculated molecular weights. Confocal microscopy suggested HepG2 translocated FITC-CPP in cytoplasm and nucleus independent with the incubation temperature. MR images showed HepG2 uptaken Gd-DTPA-CPP had a higher T1 weighted imaging （T1W1） signal, and that the T1W1 signal intensity was increasing in a time-dependent manner （r=0.972, P=0.001）, while the signal intensity between the cells incubated by Gd-DTPA for 60 minutes and the controlled cells was not significantly different （P=0.225）. By MTT, all concentrations from 50 nmol/ml to 200 nmol/ml had no significant （F=0.006, P=1.000） effect on cell viability of mouse NIH3T3 fibroblasts, compared with the control group. Conclusions The newly constructed CPP based on polyarginine can translocate cells by carrying FITC and MR contrast agent Gd-DTPA, and the intracellular concentrations are readily detectable by MR imaging, suggesting a new way for MR molecular imaging.
基金This work was supported by National Natural Science Foundation of China （No. 30770619, 30900364） and Beijing Municipal Commission of Education Research Foundation （No. KM200910025017）.
文摘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 （111） （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.
