Design Of ^99mTc-Labeled Low Generation Dendrimer-entrapped Gold Nanoparticles For Targeted SPECT/CT Imaging Of Gliomas

THE FIRST PART DESIGN AND PREPARATION OF ^99mTc-LABELED LOW GENERATION DENDRIMER-ENTRAPPED GOLD NANOPARTICLESObjective:^99mTc-labeled multifunctional low generation Au DENPs were designed and synthesized for targeted SPECT/CT imaging of CXCR4-overexpressing tumors.Methods:Firstly,in order to synthesize FC131-PEG-COOH,COOH-PEG-COOH was used to modify FC131.Then FC131-PEG-COOH was obtained.Secondly,in order to synthesize the G2-DTPA,cDTPAA was added to the G2.NH₂.Then FC131-PEG-COOH was added to the above G2.NH₂ solution to acquire the product of the G2-DTPA-PEG-FC131 dendrimers.Finally,these prepared dendrimers were subsequently used as a template to synthesize the{（Au⁰）₆-G2-DTPA-（PEG-FC131）}DENPs with a dendrimers/Au salt molar ratio of 1:6 through NaBH₄ reduction chemistry.In addition,{（Au⁰）₆-G2-DTPA-mPEG}DENPs without FC131 conjugation were also synthesized for comparison.Results:Prior to ^99mTc radiolabeling,the modification degrees of DTPA and FC131-PEG onto the G2 PAMAM dendrimer surface were characterized via ¹H NMR spectroscopy.Through NMR integration,the average number of FC131 peptides attached onto each PEG molecule was 0.7,and the number of DTPA and FC131-PEG conjugated onto each G2 dendrimer were estimated to be 7.1 and 3.2,respectively.In regard to the non-targeted dendrimers modified with mPEG-COOH,there were 7.9mPEG moieties modified onto each G2 dendrimer.The hydrodynamic diameters of the{（Au⁰）₆-G2-DTPA-（PEG-FC131）}and{（Au⁰）₆-G2-DTPA-mPEG}DENPs were analyzed by DLS,and the data revealed that the formed targeted and non-targeted Au DENPs had a hydrodynamic size of 193.9±7.76 and 153.5±9.06 nm with a low polydispersity index,respectively.The Au core size and morphology of the targeted and non-targeted Au PENPs were also measured by TEM.Both of the Au DENPs possessed a spherical shape with a narrow size distribution,and the mean diameters of the targeted and non-targeted Au PENPs were 1.8±0.36 and 2.1±0.45 nm,respectively.Moreover,strong characteristic peaks at 540 nm were observed in the UV-vis spectrum of the synthesized Au DENPs,demonstrating the successful entrapment of Au NPs within the dendrimers.Furthermore,the radiochemical yield of{（Au⁰）₆-G2-^99mTc-DTPA-（PEG-FC131）}and{（Au⁰）₆-G2-^99mTc-DTPA-mPEG}DENPs was 72.6±4.1 and 71.3±3.4%,respectively,and the ITLC data indicated that both of the ^99mTc-labeled Au DENPs could be efficiently purified by the PD-10 columns with high radiochemical purities,which are above 99%.In addition,after 12 h exposure to PBS at room temperature,the radiochemical purities of both NPs were still above 90%,exhibiting high radiochemical purity and good radiochemical stability.Conclusion:In this study,^99mTc-labeled dendrimer gold nanoparticles modified by FC131 were successfully prepared.Prior to ^99mTc radiolabeling,FC131-modified Au DENPs have a uniform size distribution and acceptable biocompatibility in the given concentration range.FC131 modified Au DENPs can be easily radiolabeled with ^99mTc with good radiochemical stability and high radiochemical purity.Therefore,this probe can be used for SPECT/CT imaging in vivo and in vitro.THE SECOND PARTTHE TARGETING SPECIFICITY FOR CANCER CELLS AND CYTOCOMPATIBILITY OF {（Au⁰）₆-G2-DTPA-（PEG-FC131）} DENPs IN VITROObjective: The targeting specificity for cancer cells and cytocompatibility of {（Au⁰）₆-G2-DTPA-（PEG-FC131）} DENPs.Methods: The cytocompatibility of {（Au⁰）₆-G2-DTPA-（PEG-FC131）} DENPs was analyzed by CCK-8 and confocal microscope.The glioma targeting in vitro of {（Au⁰）₆-G2-99 m Tc-DTPA-（PEG-FC131）} DENPs was evaluated by ICP-OES and SPECT/CT imaging.Results: The cytotoxicity of the {（Au⁰）₆-G2-DTPA-（PEG-FC131）} and {（Au⁰）₆-G2-DTPA-m PEG} DENPs was determined by CCK-8 assay.As shown in results,the C6 cells treated with the targeted and non-targeted Au DENPs had a viability above 95% at a concentration up to 200 μM and was not significantly different when compared with those treated with PBS（P > 0.05）,confirming their desired cytocompatibility.Then in order to further validate the cytocompatibility of the prepared Au DENPs,the cell morphology was also evaluated.The C6 cells treated with the targeted and non-targeted Au DENPs at different Au concentrations maintained a normal cytoskeleton and nucleus without cytoskeletal damage and cellular membrane disruption,similar to the morphology of cells treated with PBS,further demonstrating the satisfying cytocompatibility.To investigate the targeting specificity of NPs modified with FC131 to CXCR4-expressing cells,the Au uptake of {（Au⁰）₆-G2-^99mTc-DTPA-（PEG-FC131）} and {（Au⁰）₆-G2-^99mTc-DTPA-m PEG} DENPs in C6 cells was quantitatively investigated using ICP-OES and SPECT/CT.Notably,it was observed that C6 cells treated with the targeted Au DENPs displayed a higher uptake than those treated with the non-targeted Au DENPs in the given Au concentration range of 0 to 16 μM,and the difference in the cellular uptake between the two was more significant with increasing Au concentrations.The FC131-modified Au DENPs for CT imaging of CXCR4-overexpressing cancer cells in vitro was evaluated.With increasing Au concentrations,the higher CT value of C6 cells treated with the targeted Au DENPs could be found in the quantitative CT value measurements,compared to those treated with the non-targeted Au DENPs at the same Au concentration.The CT contrast enhancement of C6 cells treated with targeted Au DENPs was 2.2 times higher than that of cells treated with the non-targeted Au DENPs at the Au concentration 20 μM.After 99 m Tc radiolabeling,quantitative analysis at the 99 mTc concentration of 400 Ci/m L indicated that the SPECT signal intensity of C6 cells treated with the {（Au⁰）₆-G2-^99mTc-DTPA-（PEG-FC131）} DENPs was 7.3 times higher than that of cells treated with the {（Au⁰）₆-G2-^99mTc-DTPA-m PEG} DENPs.This further demonstrated that the formed {（Au⁰）₆-G2-^99mTc-DTPA-（PEG-FC131）} DENPs enabled the targeted SPECT/CT imaging of CXCR4-expressing cancer cells in vitro.Conclusion: {（Au⁰）₆-G2-DTPA-（PEG-FC131）} DENPs has excellent cytocompatibility.Compared with {（Au⁰）₆-G2-^99mTc-DTPA-m PEG} DENPs,{（Au⁰）₆-G2-99 m Tc-DTPA-（PEG-FC131）} DENPs has good targeting specificity for CXCR4-expressing cancer cells.THE THIRD PART THE TARGETING SPECIFICITY FOR CANCERS AND CYTOCOMPATIBILITY OF {（Au⁰）₆-G2-DTPA-（PEG-FC131）} DENPs IN VIVOObjectives: The study is worked to investigate the targeting specificity for gliomas and cytocompatibility of {（Au⁰）₆-G2-DTPA-（PEG-FC131）} DENPs in vivo.Methods: The glioma tumor mouse xenograft model was established.The cytocompatibility of {（Au⁰）₆-G2-DTPA-（PEG-FC131）} DENPs was analyzed by H&E staining.The glioma targeting in vivo of {（Au⁰）₆-G2-^99mTc-DTPA-（PEG-FC131）} DENPs was evaluated by SPECT/CT imaging.Results: H&E staining was used to investigate the potential histological toxicity of the formed {（Au⁰）₆-G2-DTPA-（PEG-FC131）} and {（Au⁰）₆-G2-DTPA-m PEG}DENPs after in vivo SPECT/CT imaging.The results demonstrated that there was no obvious tissue damage,and no necrotic areas or abnormalities were detected in the major organs（heart,liver,spleen,lungs,and kidneys）of the mice at 2 weeks after injection,revealing that the developed nanoprobes displayed excellent organ compatibility.Notably,the mice injected with the {（Au⁰）₆-G2-^99mTc-DTPA-（PEG-FC131）} DENPs had a much higher SPECT signal intensity of tumors than those injected with the non-targeted ^99mTc-labeled Au DENPs.Quantitative SPECT intensity measurements revealed that the tumor SPECT signal intensity of mice injected with the ^99mTc-labeled targeted Au DENPs was 3.3 times higher than those injected with the ^99mTc-labeled non-targeted Au DENPs at 8 h post-injection.Furthermore,the majority of the ^99mTc-labeled Au DENPs accumulated in the liver and spleen,with relatively low levels of radioactivity in the other organs,including the heart,lung,tumor,kidney,intestines,stomach and soft tissue.Likewise,a similar tendency was observed in the CT imaging.As shown in results,thanks to the specific targeting of the FC131 peptide,the tumor CT value increased steadily and reached its peak at 8 h in the mice treated with the targeted Au DENPs,while no significant changes were identified in the CT value of the tumors from mice treated with the non-targeted Au DENPs during the investigated period.Quantitative analysis of the CT images revealed that the tumor CT value in the mice treated with the targeted Au DENPs was 2.1 times higher than that of those treated with the non-targeted Au DENPs at 8 h post-injection.Overall,the combined SPECT results and CT data suggest that the prepared {（Au⁰）₆-G2-^99mTc-DTPA-（PEG-FC131）} DENPs are able to be used as a nanoprobe for SPECT/CT imaging of CXCR4 expressing tumors.H&E staining was used to investigate the potential histological toxicity of the formed {（Au⁰）₆-G2-^99mTc-DTPA-（PEG-FC131）} and {（Au⁰）₆-G2-^99mTc-DTPA-m PEG} DENPs after in vivo SPECT/CT imaging.The results demonstrated that there was no obvious tissue damage,and no necrotic areas or abnormalities were detected in the major organs（heart,liver,spleen,lungs,and kidneys）of the mice at 2 weeks after injection,revealing that the developed nanoprobes displayed excellent organ compatibility.Conclusions: {（Au⁰）₆-G2-DTPA-（PEG-FC131）} DENPs has excellent organ compatibility.Compared with {（Au⁰）₆-G2-^99mTc-DTPA-m PEG}DENPs {（Au⁰）₆-G2-^99mTc-DTPA-（PEG-FC131）}DENPs has good targeting specificity for CXCR4-expressing tumors in vivo.