Synthesis Of Peptide-targeted Magnetic-fluorescence Dual-function Molecular Probe And MR Imaging Research

Objective: A new sort of magnetic-fluorescence dual-function carbon dots (C-dots)were synthesized, analysized and their properties, distribution and clearance werestudied. The possibility of Gd doping C-dots was evaluated for potential application invivo. Binding with the CLT1cyclic peptide by covalent bond as molecular probes, it cantarget tumor fibrin–fibronectin actively.Materials and methods: Citric acid was mixed with different concentrationgadolinium chloride, using the hydrothermal method to prepare the Gd-doping C-dots.Then the Gd-Cdots were modification by amido groups, and cyclized by click chemistrychemical to bind CLT1cyclic peptide. It was composed a peptide-targeted probe fortumor fibrin-fibronectin. Inductively coupled plasma atomic emission spectroscopy(ICP) was used to test Gd content. The parameters of transverse and longitudinalrelaxation time and rate were analysized with MRI. The morphological characteristicswas checked with transmission electron microscopy (TEM). The fluorescence propertiesof Gd-Cdots were studied with fluorescence spectrophotometer and UVspectrophotometer. The Gd-doping C-dots exhibited good fluorescence stability underdifferent pH conditions and different ion concentration. Gd-Cdots were applied toimaging in mice, and their biodistribution and clearance were studied. Results:1. Gd-Cdots-CLT1the Synthesis and properties(1) Gd-CDots were numbered according to the content of Gd. The Cdots withoutGd were numbered1#, and2-5#Gd-Cdots were numbered by citric acid withgadolinium chloride ratio:5:1,10:1,20:1,80:1. ICP showed the2-5#sample content ofGd:0.704、0.477、0.243、0.049μ mol/mg. The measured r2were18,19.77,15.85,19.77mM-1S-1, r1:14.51,17.32,14.08,17.32mM-1S-1, and r2/r1:1.24,1.13,1.13,1.04,respectively, for the2-5#samples. Different concentration samples were used for MRimaging in vitro. TEM showed the average size for1#Cdots was about2.9nm,5#Gd-Cdots about5.3nm. The size of Gd-doping C-dots was larger than that of C-dotswithout Gd. The UV-vis spectra showed that there was an apparent absorption for1-5#C-Dots in the wavelengths of200-300nm. The emission light was changed byexcitation light. The functional groups of the sample1#,5#, did not significantly changeduring the synthesis process of nanoparticle and there were a large number of carboxyland hydroxyl groups on their surface. The max excitation wavelength of the sample1#,5#, was320nm and the max emission wavelength for the sample1#-5#were410nm,400nm,400nm,410nm,410nm, respectively. Quantum yields were9.78%,8.11%,6.72%,6.17%and6.17%. Fluorescence lifetime of sample1#and5#were9.7ns and3.6ns, respectively. The fluorescence intensity was stable under different pH conditionsand different ion concentration.(2) Considering magnetic and fluorescent properties, sample5#was chosen for thenext experiment. Then the Gd-Cdots were modification with amino groups and CLT1cyclic peptide by click chemistry. The peptide-targeted probe was specific for tumorfibrin-fibronectin. The max excitation wavelength of Gd-Cdots-CLT1showed ared-shift from320to360nm, and the max emission wavelength from410to443nm. Infrared spectrum reflected the change of its functional groups in the process of reaction.Finally, the Gd content was measured with ICP.2. The distribution of Gd-CDots in normal ratsAfter intravenous injection of the Gd-CDots into Kunming mice, liver, kidney,muscle, heart showed increased T1W signal at10min and30min post-injection ascompared with pre-injection. No signal increase was found in the brain. Quantitativeanalysis of the signal at pre-injection,10min and30min post-injection showed that theSNR of liver were13.58±0.59、20.79±0.09、23.13±0.84, kidney14.03±0.55、17.53±0.14、20.33±0.73, muscle8.30±0.23、9.19±0.15、13.73±0.23, and8.61±0.25、9.11±0.26、9.46±0.11for brain. The SNRs at three time points of the liver, kidney and muscleshowed statistical differences. No significant difference of SNR in the brain wasobserved.Conclusion:1. Hydrothermal method was used for the synthesis of magnetic-fluorescent dual-function nanoparticles. The nanostructure was observed by TEM and showed gooddispersibility. The water-soluble fluorescent nanoparticles were stable. Gd-cdotsexhibited showed high r1.2. Gd-Cdots-CLT1molecular probe was synthesized through the covalent bondingmethod and successfully confirmed by infrared spectra.3. The Gd-Cdots were mainly distributed in liver, kidney, muscle, but not the brainof the normal mice. They were mainly eliminated from the body via the renal clearance.