Dual-Modal Bio-imaging Probe Based On Au Nanoclusters Enriched With Mesoporous Silica Nanoparticles: Construction And Imaging In Vitro And In Vivo

Recently,bio-imaging technology has been widely studied in vivo and in vitro.Fluorescence imaging is a kind of biological imaging technology.It has the advantages of simple operation and intuitive imaging effect.Among many fluorescent imaging media,fluorescent nanoparticles have the advantages of small particle size,high fluorescence stability,and good biocompatibility.Certain research results have been achieved in tracer and drug loading.A single imaging technology is not enough to provide accurate diagnostic information,while dual-modal imaging can make a single imaging technology complementary and combine the advantages of two imaging technologies for scientific research or clinical research.The current dual-modal imaging probes mostly use nanoparticles as carriers and combine two imaging media to obtain the final dual-modal imaging probe through processes such as synthesis,modification,and assembly.However,the complex preparation process often affects the performance and imaging effect of the imaging media.Therefore,it is of great significance to develop a simple dual-modal bio-imaging probe with good imaging function for in vivo and in vitro imaging analysis.Objective:Using the biocompatible mesoporous silicon nanoparticles to enrich the gold nanoclusters that possess fluorescence and CT(computer tomography)imaging functions by a simple method.Thereby,to improve the two imaging signals simultaneously and construct an enhanced fluorescence/CT dual-modal probe for bio-imaging research in vitro and in vivo.Methods:Section 1 Au nanoclusters enriched with mesoporous silica nanoparticles: construction and characterizations The functional protein bovine serum albumin(BSA)was used to prepare the BSA-capped Au nanoclusters(BSA-Au NCs)through a one-step method.BSA-Au NCs were characterized by UV absorption spectroscopy,transmission electron microscopy,particle size potentiometer and three-dimensional fluorescence spectroscopy.Using the cationic surfactant CTAC and TEA as templates,mesoporous silica nanoparticles(MSNs)were synthesized by a two-phase method.Then they were aminated.Through the EDC/NHS system,BSA-Au NCs were enriched on the surface of mesoporous silicon nanoparticles to obtain enriched nanoparticles(MSN@Au NCs).The enriched materials were tested by transmission electron microscopy,Zeta potential,and energy spectrum analysis to characterize the physical and chemical properties and proved the successful synthesis.Section 2 Dual-modal bioimaging probe based on Au nanoclusters enriched with mesoporous silica nanoparticles: imaging in vitroThe concentration of Au nanoclusters and MSN@Au NCs was adjusted by Inductively Coupled Plasma Optical Emission Spectrometer(IPC-OES)so that the concentration of Au in two solutions are equal.Fluorescence and CT intensity were compared under this condition.A fluorescence spectrophotometer was used to compare the changes in fluorescence intensity before and after enrichment,and measure fluorescence stability and fluorescence lifetime.CT intensity of two kinds of nanoparticles was measured by micro-CT.The mouse fibroblast cell line(L929 cells),the human tongue squamous cell carcinoma cell line(CAL-27 cells and SCC-25 cells),and the adenoid cystic carcinoma cell line(ACC-2 cells)were chosen to analyze the cytotoxicity of Au NCs and MSN@Au NCs through CCK-8.The fluorescence of the L929 cells and CAL-27 cells incubated with the two materials was observed under a confocal microscope and a two-photon laser scanning microscope,conducting semi-quantitative analysis.Flow cytometry was used to quantitatively analyze the fluorescence intensity in L929 cells and CAL-27 cells.Section 3 Enhanced Fluorescence/CT dual-modal bio-imaging probe based on Au nanoclusters enriched with mesoporous silica nanoparticles: imaging in vivo The nude mice were used for the studies of the dual-modal bioimaging probe in vivo to compare the dual-modal imaging effects of Au NCs and MSN@Au NCs.First,nude mice were injected through the tail vein with two kinds of imaging media solutions,respectively.After a certain period of time,the experimental nude mice were dissected,and the heart,liver,spleen,lung and kidney were taken for hematoxylin-eosin staining.The pathological conditions of each organ were observed under a microscope to analyze the in vivo toxicity of the two imaging media.Subsequently,nude mice were injected subcutaneously with aqueous solutions of the two imaging media,respectively.Maestro Automated In Vivo Imaging and a High Resolution in-vivo X-ray Microtomography were used to analyze the fluorescence and CT imaging on the part of experimental mice injected with different imaging media,which proved the enhancement effect of MSN@Au NCs on fluorescence and CT dual-modal imaging.Results:Section 1 Au nanoclusters enriched with mesoporous silica nanoparticles: construction and characterizations Au nanoclusters: Au nanoclusters,synthesized by the one-step method,have an obvious absorption peak in the ultraviolet absorption spectrum at an excitation wavelength of about280 nm,which is a typical feature of BSA.The result indicated the successful capped of BSA.Three-dimensional fluorescence spectroscopy characterized the excitation and emission wavelength of Au nanoclusters,which showed that the synthesized gold nanoclusters emitted near-infrared fluorescence and possessed large Stokes shift.Transmission electron microscopy and particle size analyzer showed that the BSA-Au NCs were spherical with a particle size of about 2 nm.Mesoporous silica nanoparticles: White colloidal mesoporous silica nanoparticles with no fluorescence had low solubility in water,good dispersibility.The results of transmission electron microscopy and particle size analyzer showed that it had a spherical,porous structure with a particle size of about 100 nm.They may be agglomerate in water.MSN@Au NCs: After Au nanoclusters were enriched with the mesoporous silicon nanoparticles,the non-luminescent white colloid MSNs became a slightly brownish colloid that emitted red fluorescence.The potential,particle size and element composition of the particles have changed significantly,which proved the successful synthesis of the MSN@Au NCs.Section 2 Dual-modal bioimaging probe based on Au nanoclusters enriched with mesoporous silica nanoparticles: imaging in vitro It can be seen from the photos and ultraviolet–visible spectroscopy that MSN@Au NCs exhibited higher fluorescence intensity under the same Au concentration.The fluorescence lifetime of MSN@Au NCs has no significant change compared with Au nanoclusters,indicating that the enrichment process did not affect the fluorescence performance of the imaging medium.The stability of the two were both better than the fluorescent dye.Micro-CT detection showed that the CT signal of gold nanoclusters was enhanced after enrichment,and the Hounsfield units(HU)value increased linearly with the increase of concentration.CCK-8revealed that Au nanoclusters and MSN@Au NCs were both non-toxic to L929 cells,CAL-27 cells,ACC-2 cells,and SCC-25 cells,and cell viability was no significant decline after incubated at higher concentration for 48 hours.The fluorescence intensity of CAL-27 cells incubated with MSN@Au NCs was significantly stronger than that of Au nanocluster,which was observed by confocal microscope and two-photon microscope.Quantitative analysis of flow cytometry experiments indicated that the average fluorescence intensity of CAL-27 cells cultured with MSN@Au NCs was highest which verified the above results.Section 3 Enhanced Fluorescence/CT dual-modal bio-imaging probe based on Au nanoclusters enriched with mesoporous silica nanoparticles: imaging in vivo The results of Hematoxylin Eosin stain showed that compared with the control groups,the heart,liver,spleen,lung and kidney of nude mice showed no significant difference after injection of Au NCs or MSN@Au NCs solutions,which indicating neither nanomaterial have a toxic effect on nude mice.The results suggested that the bio-imaging probe constructed in this study had good biological safety which can be applied for imaging in vivo.The fluorescence and CT dual-modal imaging effects of nude mice injected with MSN@Au NCs were better than that of Au NCs.The comparation of imaging intensity in vivo indicated that the fluorescence performance and CT signal of Au nanoclusters were improved because of enrichment strategy.The imaging medium exhibited significantly enhanced dual-modal bio-imaging function.We will further modify the performance of the imaging probe and explore the application of MSN@Au NCs in diagnosis and treatment of disease.Conclusion:1.Au nanoclusters and the biocompatible mesoporous silicon nanoparticles were combined through electrostatic adsorption and carboxyl-amine bond.Au NCs were enriched in the pore of mesoporous silicon nanoparticles.The local concentration of Au nanoclusters was increased.2.The fluorescence and CT intensity of MSN@Au NCs were both enhanced utilizing the large Stokes shift of Au nanoclusters.3.In this study,MSN@Au NCs,a novel dual-modal bio-imaging probe,was constructed through a simple method without complicated modification process,which exhibited highly sensitive and biocompatible.The fluorescence intensity of MSN@Au NCs was significantly higher than that of Au nanoclusters in vitro.4.The enrichment strategy optimized the imaging function of the Au nanoclusters.The dual-modal bioimaging probe based on mesoporous silicon nanoparticles had been successfully applied to in vivo,which exhibited higher fluorescence and CT signals in nude mice.The sensitive bio-imaging medium had a powerful potential for applications in the diagnosis and treatment of disease.