Synthesis Of Gold Nanohybrids And Their Applications To In Vitro/in Vivo Targeted Imaging And Enhancing Microwave Ablation Thermo-sensitizing Of Pancreatic Ductal Adenocarcinoma

Nanotechnology,which is potentially used in biomedicine,has peaked interests in biomedical field.At present,common nanomaterials include liposomes,graphene,carbon nanotubes,magnetic nanoparticles,metallic nanoparticles and so on.Among them,gold nanoparticles show high surface activity,good biocompatibility,excellent optical and electrical properties.In addition,gold nanoparticles could be easily synthesized in various shapes ranging from 1nm to 100nm,such as spherical,rod-like,cage-like,and so on.The optical and electrical properties of gold nanoparticles depend on their shape and size.All of these particular properties made gold nanoparticles become the most potential material for various biomedical applications,including targeted imaging and cancer therapy.Pancreatic ductal adenocarcinoma(PDA),also known as pancreatic cancer,is one of the lethal tumors all around the world.And the symptoms and signs of PDA in early stage are not specific.The majority of patients once diagnosed with PDA are not candidates for surgery because PDA is in advanced stage.The lack of suitable therapies of PDA also contribute to the poor prognosis,the five-year survival rate of PDA patients is nearly 5%.In the thesis,two kinds of gold nanohybrids have been synthesized via chemical solution routs.The applications of these nanohybrids in diagnosis and therapy of PDA have been addressed.The long-term plan is to combine different gold nanohybrids as a integration nanostructure with multiple functional constituents.Part 1ObjectiveThis research aims to synthesize the new nanohybrid,Gd-Au NCs modified with GPC-1 antibody(Gd-Au-NC-GPCAb-1).Then we observe its characterization and biocompatibility.Finally,we evaluate its feasibility and possibility as FI and MRI contrast agent to diagnose PDA,based on its in vitro and in vivo targeted imaging to PDA cells and nude mice PDA xenografts.Methods1.Synthesis of Gd-Au-NC-GPCAb-1We chose a biomineralization method to finish the synthesis of Gd-Au NCs,then modified GPC-1 antibody on Gd-Au NCs’surface activated by EDC.Finally we got Gd-Au-NC-GPCAb-1.Morphological characteristics of Gd-Au-NC-GPCAb-1 nanohybrid were observed by transmission electron microscope(TEM).Stability,relaxation rate and other properties of this nanohybrid were measured one by one.2.Cell toxicity of Gd-Au-NC-GPCAb-1The cytotoxicity of the various concentrations ranging from 0 to 150mg/L Gd-Au-NC-GPCAb-1 and Gd-Au NCs nanohybrids solution against human pancreatic cancer cell line COLO-357 and human embryonic kidney cell line 293T at different time were determined by measuring the inhibition of the cell growth with a Cell Counting Kit-8(CCK-8)assay,then absorbance at 450nm was measured by ELSA Plate Reader;We chose some nude mice treated by intravenous injecting Gd-Au-NC-GPCAb-1 solution and saline solution separately.One day later,major organs of mice were collected to hematoxylin and eosin(H&E)staining to observe their pathological changes;We also collected the blood samples of mice for blood chemistry analysis.3.In vitro and in vivo targeting test of Gd-Au-NC-GPCAb-1At first,we built the nude mice xenografts model.Female nude mice(4 to 5 weeks old)were chosen,a 0.2 mL of the cell suspension(5×10~6/mL)COLO-357 cells were subcutaneously injected into the axillary of each mouse,these mice were raised under SPF conditions in the Laboratory Animal Center.The growth of tumors was observed.When the tumor volumes reached about 60mm~3,we chose suitable mice with tumors and cut the xenografts tissue for IHC staining,integrated optic density(IOD)was measured by software Image-pro plus 6.0.In order to test the targeting ability of the Gd-Au-NC-GPCAb-1 to COLO-357 cells in vitro,we mixed the water with Gd-Au-NC-GPCAb-1 and Gd-Au NCs separately,these two kind of mixture were added to the COLO-357 cells and 293T cells in the logarithmic phase.The COLO-357 cells were incubated with nanohybrids for 24 hours,293T cells were incubated with nanohybrids for 24 hours as control.Then all the cells were visualized through fluorescence microscope to determine the different effects of Gd-Au-NCs and Gd-Au-NC-GPCAb-1 on COLO-357 and 293T cells.After COLO-357 and 293T cells were digested by Trypsin,they were incubated with Gd-Au NCs and Gd-Au-NC-GPCAb-1separately for 1 h at 37℃.Finally the samples were measured by flow cytometry to observe the targeting ability of nanocomposites to cells quantitatively.We chose some other nude mice to build mice xenografts model in the same way.Suitable mice with xenografts were divided into 3 groups,some of them were scanned in an IVIS Lumina II for FI,others were scanned in a Siemens 3.0 T magnetic resonance imaging machine for MRI.3 groups of mice were received intravenous injection 0.2mL of saline,Gd-Au NCs and Gd-Au-NC-GPCAb-1 via tail veins,respectively.FI images were obtained before and 10min,30min,40min after injection,while MRI images were obtained before and 10min,30min,60min,120min after injection.Then we compared the changes of fluorescence intensity and T2WI signal intensity of tumor regions separately.Results1.Characterization of Gd-Au-NC-GPCAb-1ThenanohybridsGd-AuNCsmodifiedwithanti-GPC-1antibodies(Gd-Au-NC-GPCAb-1)were successfully prepared.They were spherical and regular in shape.The size of nanohybrids was uniform,and most of the hydrodynamic diameters were ranging between 13.5 nm and 24.4 nm.XPS detected that antibodies were conjugated on the nanohybrids.Gd-Au-NC-GPCAb-1 had high stability of diffusion.The T2relaxation rate r2 of Gd-Au-NC-GPCAb-1 nanohybrid was 24.434 mM/s and its T1relaxation rate r1 was 17.722mM/s.The feasible ratio of r2/r1 stood for potential T1positive contrast.2.Cytotoxicity of Gd-Au-NC-GPCAb-1Results of CCK-8 assays indicated there was no significant cytotoxicity of Gd-Au NCs against both COLO-357 and 293T.But Gd-Au-NC-GPCAb-1 was cytotoxicity to COLO-357 depending on concentration;H&E result showed no significant differences in histology between the experimental group of Gd-Au-NC-GPCAb-1 and the control group of saline;Results of serum biochemistry analysis also showed that there was no significant difference between saline solution group and Gd-Au-NC-GPCAb-1 group.Therefore,the targeting nanohybrid was nontoxic to mice,and manifested potential for clinical application.3.In vitro and in vivo targeting test of Gd-Au-NC-GPCAb-1The IHC staining results showed GPC-1 were higher expressed on tumor tissues than nearby normal tissues.Results of IOD analysis showed that the expression level of GPC-1in COLO-357 tumor tissues was significantly higher than that of normal tissues.The red fluorescence intensity was stronger on the surface of COLO-357 cells than that of 293T,after incubation with Gd-Au-NC-GPCAb-1.The other two groups,COLO-357 cells with non-antibody-linked Gd-Au NCs and 293T cells with Gd-Au-NC-GPCAb-1 nanohybrids showed only a little red fluorescent signals.Flow cytometric analysis showed that the combination rate of Gd-Au-NC-GPCAb-1nanohybrids with COLO-357 cells was the highest in all groups.The fluorescent intensities in tumor sites of Gd-Au-NCs groups gradually increased during the first 10 minutes after injection and decreased slightly during the following 30minutes.While the fluorescence intensity in tumor sites of Gd-Au-NC-GPCAb-1 groups increased with time,reached a peak at 30 minutes after injection,and then subsequently decreased during the next 10 minutes;From the T2WI images of nude mice xenografts,we found that significant signal increased in Gd-Au-NC-GPCAb-1 groups after intravenous injection,and reached peak at 30min after injection,while not in Gd-Au NCs and saline groups.The changes of fluorescent intensities and T2WI signals by Gd-Au-NC-GPCAb-1solution were much more significant than that by saline and Gd-Au NCs solution.ConclusionThe Gd-Au NCs nanohybrids were successfully prepared and conjugated with anti-GPC-1 antibody.Results showed stable Gd-Au-NC-GPCAb-1 with regular morphology,excellent biocompatibility.The continued experiments results showed that Gd-Au-NC-GPCAb-1 successfully recognized the GPC-1-expressing cells in vitro.In addition,Gd-Au-NC-GPCAb-1 also exhibited good MRI property and targeting potential to human pancreatic cancer xenografts of nude mice in vivo.Therefore,Gd-Au-NC-GPCAb-1 nanohybrids could effectively target PDA cells in vitro and in vivo.In conclusion,Gd-Au-NC-GPCAb-1 nanohybrids represented a potential FI and MRI contrast agent for early diagnosis of PDA.Part 2ObjectiveWe try to synthesize the simple nanohybrid GO/GNR as reported previously.Then we evaluate its possibility in enhancing the thermo-sensitizing effect of MWA.Methods1.Synthesis of GO/GNR nanohybridsGO/GNR nanohybrids were synthesized with a gold-seed-mediated in situ growth method at room temperature.GO was modified with PSS,we called it GO-PSS,the growth process began with gold seeds adhering on the GO-PSS and continued with the growth of the gold seeds into GNR on the GO-PSS.Then,GO/GNRs were modified with PSS,we got GO/GNR-PSS.2.Characterization and cytotoxicity of GO/GNRMorphological and structural characteristics of GO/GNR nanohybrids were observed using a TEM,a scanning electron microscope(SEM),X-ray photoelectron spectroscopy(XPS),a Veeco NanoScope Multimode IIIa and so on.The cytotoxicity of the various concentrations ranging from 0 to 200mg/L GO/GNR nanohybrids against human embryonic kidney cell line 293T was determined by measuring the inhibition of the cell growth with a CCK-8 assay,then absorbance at 450nm was measured by ELSA Plate Reader;We chose some nude mice treated by intravenous injecting GO/GNR solution and PBS solution separately.24 hours later,major organs of mice were collected to H&E staining to observe their pathological changes.3.The GO/GNRs used in MWA of PDA xenograftWe chose some other nude mice to build mice xenografts model in the same way.When the tumor volumes reached about 100mm~3,we chose suitable mice with xenografts.These mice were divided into 3 groups,3 groups were received intra-tumoral injected with0.2mL GO/GNRs solution before MWA at 10W for 2min,intra-tumoral injected with0.2mL saline solution before MWA at 10W for 2min and simply MWA at 10W for 2min separately.Then measured the max diameter of ablation areas at the position of coronal and axial with magnetic resonance imaging.Results1.Synthesis of GO/GNRsThe GO/GNR nanohybrids were successfully prepared.2.Characterization and cytotoxicity of GO/GNRGO/GNRs were smooth and regular in shape,GNRs were rod-like and incorporated compactly into the surface of GO-PSS.The average size of the GNRs was approximately31 nm×8 nm on a few layers of GO.XPS detected that GNRs conjugated with GO-PSS was based on covalent bonds not throughπ–πstacking interactions.Results of CCK-8 assays showed GO/GNR was a bit low toxicity to 293T cells at a concentration of 200μg/mL(based on Au).H&E staining exhibited the GO/GNRs did not appear to be toxic to normal organs,there was no obvious inflammation,cell necrosis,or apoptosis.3.The GO/GNRs in enhancing the thermo-sensitizing effect of MWAFrom the MRI images of nude mice xenografts,we could clearly find that ablation zones in intra-tumoral injected with GO/GNR solution and MWA group were significantly bigger than other groups(p<0.05).And there is no significant difference between ablation areas of intra-tumoral injected with saline solution and MWA group and that of simply MWA group(p>0.05).ConclusionThe GO/GNR nanohybrids were synthesized with regular morphology and good biocompatibility as former experiments of our team.The MWA experiments on nude mice xenografts showed that intra-tumoral injection of GO/GNRs before MWA could be significance to thermos-sensitizing enhanced of MWA,with bigger ablation zones.GO/GNR represents a promising enhancing thermos-sensitizing agent for MWA of tumors.