Gadolinium-doped Fluorescence Silica Targeting Prostate Cancer For Bual-mode Imaging

PART Ⅰ PREPARATION AND CHARACTERIZATION OF GADOLINIUM-DOPED FLUORESCENCE SILICA NANOPARTICLESObjective To prepare gadolinium-doped fluorescent silica targeted nanoprobe（Gd@Cy5.5@SiO₂-Ab）by optimizing the reaction conditions.and to detect their basic properties.Methods Using TX-100,cyclohexane and hexanol,TEOS,Cy5.5-APTES and gadopentetic acid solution as microemulsions system and ammonia as catalyst,the fluorescent nanoprobes of different gadolinium content were prepared by inverse microemulsion method.The amino and carboxyl groups were successively introduced onto the surface of the synthesized silica nanoparticles（NPs）;after these steps,a monoclonal antibody（YPSMA-1）of the prostate-specific membrane antigen（PSMA）was conjugated with carboxyl-modified Gd@Cy5.5@SiO₂（Gd@Cy5.5＠SiO₂-COOH）NPs by the carbodiimide method.Gd@Cy5.5＠SiO₂-Ab NPs were thus obtained as MRI/fluorescent specific contrast agents for PCa targeted imaging.Then its surface morphology,particle size,zeta potential and chemical structure etc.were studied.Gadolinium content and gadolinium stability of NPs were detected by inductively coupled plasma atomic emission spectrometry（ICP-AES）.The fluorescence spectrometer was used to analyze the fluorescence signal of the silica NPs,and the immunofluorescence method was used to detect whether the YPSMA-1 antibody was successfully connected to the surface of silica.Results Transmission electron microscopy（TEM）displayed that the prepared NPs exhibited a dispersed spherical morphology with smooth surface and a relatively uniform size distribution.Zeta potential and FTIR spectra exhibited that amino and carboxyl groups have been successfully modified on the surface of silica.Gd@Cy5.5@SiO₂-Ab NPs showed high stability,and gadolinium content（GC）in Gd@Cy5.5@SiO₂-Ab NPs was determined to be 8.21 ± 0.63%by ICP-AES.The longitudinal relaxation rate（r₁）of the Gd@Cy5.5＠SiO₂-Ab NPs was 13.234 mM^-1S¹-by MRI.UV-vis spectrum showed that the fluorescent dye Cy5.5 was successfully doped into the silica NPs.The fluorescence spectrometer proved that the fluorescence peak shape of the NPs had no significant difference from that of the free Cy5.5.YPSMA-1 antibody was successfully linked to the surface of silica by immunofluorescence.Conclusion The best dose can be determined by adding different doses of the reactant.The amino and carboxyl groups were successfully introduced onto the surface of NPs by co-condensation,and then YPSMA-1 was conjugated with carboxyl-modified NPs by the carbodiimide method.In vitro MRI showed that the synthesized NPs had higher relaxation rate（r₁）.The fluorescence spectrometer proved that the fluorescence peak shape of the NPs had no significant difference from that of the free Cy5.5,which was helpful for MRI/fluorescence imaging.PART Ⅱ TARGETING,CYTOTOXICITY AND IN VITRO IMAGING OF GADOLINIUM-DOPED FLUORESCENCE SILICA NPSObjective To observe the cellular targeting and cytotoxicity of nanoprobe in vitro.To study the effect of cell imaging in vitro,and explore its feasibility as an in vivo imaging agent.Methods Cellular targeting of non-targeted and targeted nanoprobe were observed with LNCaP cells,known to be PSMA receptor-positive,and PC3 cells,known to be PSMA receptor-negative.The toxicity of different concentrations of various NPs on the LNCaP and PC3 cells were detected by CCK8 assay.LNCaP and PC3 cells were co-incubated with the targeted nanoprobe,and in vitro MRI/fluorescence imaging were performed to observe the imaging effect of the targeted nanoprobe.Results In vitro cell targeting experiments showed that red fluorescence was observed around LNCaP cells,whereas only individual cell membranes showed a small amount of red fluorescence in PC3 group.A little red fluorescence were also found around the cell membrane in the non-targeted groups.Flow cytometry revealed that the yield of GdgCy5.5@SiO₂-Ab NPs binding LNCaP cells was 99.79%,whereas the yield of Gd@Cy5.5@SiO₂-Ab binding PC3 cells was 2.34%,and in the non-targeted groups the yield of Gd@Cy5.5@SiO₂ binding LNCaP and PC3 cells were 4.58%and 2.74%respectively.CCK8 assay indicated that various NPs had no significant effect on cell viability within the tested concentration and time range.The MRI in vitro showed that the LNCaP and PC3 cells displayed high/low signals on T₁WI after co-incubation with prepared nanoprobe respectively.Fluorescence imaging displayed that the nanoprobe + LNCaP groups became significantly red fluorescence,whereas there were no obvious red fluorescence in the nanoprobe + PC3 groups.Conclusion The surface of the prepared NPs can be modified by the antibody to be targeted,and had good targeting ability to the LNCaP cells with high expression of PSMA receptor.The NPs had no obvious effect on the cell activity in the tested concentration and time range,indicating good safety;and in vitro LNCaP cells co-incubated with nanoprobe showed high signal on MRI imaging,and fluorescence imaging showed significant red fluorescence,suggesting that nanoprobe can lay the foundation for the experiments in vivo.PART Ⅲ IN VIVO IMAGING,BIOSAFETY AND BIOLOGICAL DISTRIBUTION OF NPSObjective To observe the effect of dual-modality imaging of NPs and to investigate the distribution and biosafety of NPs in vivo.Methods Ten nude mice bearing LNCaP and PC3 xenografts respectively were selected to perform MR imaging.The targeted nanoprobe Gd@Cy5.5@SiO₂-Ab and Gd-DTPA were injected slowly via tail vein（Gd concentration 0.01mM/kg body weight）.MR images were collected at different time points（before injection,15 min,1h,3h,6h and 24h after injection）,and their signal intensity were analyzed.The MR images of liver,spleen,kidney and muscle were collected after injecting Gd@Cy5.5@SiO₂-Ab,and their distribution in the body were analyzed.Five nude mice bearing LNCaP and PC3 xenografts respectively were selected to perform fluorescence imaging.The targeted nanoprobes Gd@Cy5.5＠SiO₂-Ab were injected via tail vein about 0.2mL（10mg/mL）.Fluorescence images were collected at different time points,and their signal intensity were analyzed.After tail vein injection 1 d and 3 d,the blood was collected for biochemical analysis,and the nude mice were dissected to obtain the main organs（heart,liver,spleen,lungs,and kidneys）and sent to pathology department for pathological section and HE staining.Results In vivo MR images of LNCaP xenograft model in nude mice showed that the signal intensity of the tumor tissue gradually increased and peaked at 3 h after tail vein injection of the targeted nanoprobe.Whereas MR images of the PC3 xenograft model in nude mice displayed that the signal intensity of the tumor tissue increased slightly at 1h after injection,and the signal enhancement of the LNCaP groups was more obvious than that of the PC3 groups.There were significant statistical differences（P<0.05）.In vivo fluorescence imaging of small animals showed that PC3 groups hardly observed fluorescence at the tumor site,while LNCaP groups distributed red fluorescence at the tumor site.Fluorescence intensity analysis revealed that the fluorescence intensity of LNCaP groups were significantly higher than that of PC3 groups by Living image software system（P<0.05）,indicating the difference was statistically significant.The distribution of nanoprobes in vivo showed that the T,signal of liver,spleen and kidney increased significantly after injection of nanoprobes.The signal to noise ratio of liver,spleen and kidney were higher than that of before injection,indicating that part of the nanoprobes were swallowed by the liver and spleen and excreted via the intestine.Some are excreted by the kidneys.No obvious necrosis and inflammatory reaction were found in the pathological sections of HE staining in the heart,liver,spleen,lung and kidney tissue;and there were no obvious change in biochemical tests（aspartate transaminase（AST）,alanine aminotransferase（ALT）,total bilirubin（T-BIL）,blood urea nitrogen（BUN）and creatinine（CREA））before and after injection（P>0.05）,indicating that the synthesized nanoprobe had good biosafety in vivo.Conclusion Targeting NPs Gd@Cy5.5＠SiO₂-Ab can enhance magnetic resonance imaging and fluorescence imaging in vivo,and can be targeted to aggregate in tumor tissues.The NPs had good biological safety,which provides a new way of thinking for the diagnosis of prostate cancer.