Functionalized Silver-based Nanoparticles For Computed Tomography Imaging And Radiosensitization Treatment Of Glioma

Background:Glioma is the most common malignant tumor in central nervous system,with increasing morbidity and mortality.Despite great progress in the diagnosis and therapy of gliomas in recent years,the overall survival of glioma patients is short,and long-term survival patients are even rarer.Thus,innovative technologies and methods are urgently needed to improve the diagnosis and treatment effect of patients with glioma.The rapid development of nanotechnology in the medical field is expected to provide accurate diagnosis and efficient treatment for glioma.Due to their unique physical,chemical and biological properties,silver nanoparticles(AgNPs)possess advantages in the detection and treatment of certain diseases,thus becoming a research hotspot in recent years.Unique X-ray absorption properties make AgNPs have huge application space and great development potential in computed tomography(CT)imaging and radiosensitization of tumors.Previous studies of our group confirmed AgNPs as an excellent radiosensitizer,which could significantly enhance the radiosensitivity of glioma cells.However,their abilities to target tumor cells and efficiently enrich in the tumor area still need to be improved.In addition,in CT imaging,specific targeted modification of AgNPs or preparation with other nanomaterials into silver-based nanocomposites could exert better imaging effects.Therefore,this study performed some researches on the application of functionalized silver-based nanoparticles in CT imaging and radiosensitization of glioma.The main aspects are shown below.Part Ⅰ Enhancement of radiosensitization by silver nanoparticles functionalized with polyethylene glycol and aptamer AS1411 for glioma irradiation therapyObjective: The research concentrated on constructing AgNPs functionalized with polyethylene glycol(PEG)and aptamer AS1411(ASNPs),and then assessing their glioma-targeting property and radiosensitizing effect both in vitro and in vivo.Methods: The ASNPs were synthesized and subsequently characterized by transmission electron microscopy,ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy.Then the tumor targeting property of ASNPs was evaluated by dark-field imaging,confocal microscopy and in vivo imaging.Both colony formation assay and glioma-bearing mouse model were employed to study the radiosensitizing effect of ASNPs.Results: The characterization results revealed a spherical shape of AgNPs with an average diameter of 18 nm and the successful construction of ASNPs.ASNPs were confirmed to specifically target C6 glioma cells,but not normal human microvascular endothelial cells.Moreover,ASNPs could not only internalize into tumor cells,but also penetrate into the core of tumor spheroids.In vitro experiments showed that ASNPs exhibited a better radiosensitizing effect than AgNPs and PEGylated AgNPs(PNPs),inducing a higher rate of apoptotic cell death.In vivo imaging demonstrated that Cy5-ASNPs preferentially accumulated at the tumor site,and the ratio of fluorescence intensity of Cy5-ASNPs to that of Cy5-PNPs reached the maximum at 6 h post-systemic administration.Furthermore,the combination of ASNPs with irradiation significantly prolonged the median survival time of C6 glioma-bearing mice.Conclusion: ASNPs could be an effective nano-radiosensitizer for glioma targeting treatment,which might provide an important basis for the potential application of ASNPs as an effective nano-radiosensitizer for the targeted treatment of glioma and other tumors.Part Ⅱ Application of aptamer AS1411-and verapamil-functionalized silver nanoparticles in radiosensitization of gliomaObjective: The research was focusing on constructing bovine serum albumin(BSA)coated AgNPs functionalized with AS1411 and verapamil(VRP)(AgNPs@BSA-AS-VRP),and then evaluating their enrichment in glioma cells and radiosensitization effect.Methods: AgNPs@BSA-AS-VRP were synthesized and characterized.Dark-field imaging and inductively coupled plasma mass spectrometry were applied to investigate the accumulation of AgNPs@BSA-AS and AgNPs@BSA-AS-VRP mixed in different ratios in U251 glioma cells.Rhodamine 123 accumulation assay was carried out to assess the influence of 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP on the P-glycoprotein(P-gp)activity.Colony formation assay and in vivo anti-tumor tests were conducted to evaluate the radiosensitizing effect of 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP.Thioredoxin Reductase(Trx R)Assay Kit was used to detect the Trx R activity in cells treated with different functionally modified AgNPs.Results: Characterization results revealed that AgNPs@BSA-AS-VRP were successfully constructed.When AgNPs@BSA-AS and AgNPs@BSA-AS-VRP were mixed in a ratio of 19:1,nanoparticles were efficiently enriched in cells via increasing endocytosis and inhibiting efflux.In vitro and in vivo experiments results showed that the radiosensitization efficacy of 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP was much stronger than that of AgNPs@BSA and AgNPs@BSA-AS.It was also found that 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP significantly inhibited intracellular Trx R activity.Conclusion: These results indicated that 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP could effectively accumulate in glioma cells by increasing endocytosis and inhibiting efflux,thereby enhancing the radiosensitivity of cells.The mechanism underlying radiosensitization of AgNPs may be involved with the inhibition of intracellular Trx R activity.These findings might provide a novel approach and strategy for designing highly effective nano-radiosensitizers.Part Ⅲ Application of silver-gold core-shell nanoparticles in computed tomography imaging of gliomaObjective: The research aimed to synthesize polyethylene glycolylated silver-gold core-shell nanoparticles(Ag@Au NPs),and evaluate their computed tomography(CT)contrast properties in in vivo and in vitro experiments.Methods: A range of Ag@Au NPs with varying molar ratios of Ag and Au were prepared and characterized by transmission electron microscopy,ultraviolet-visible spectroscopy,dynamic light scattering and inductively coupled plasma mass spectrometry.Then,micro CT was used to evaluate the CT contrast properties of Ag@Au NPs with different molar fractions of Ag and Au.The Ag@Au NPs with the better performance were selected for targeted modification with GMT8 and their CT imaging effect at the cellular level was detected.In addition,a subcutaneous xenograft tumor model of glioma in nude mice was constructed,and CT images of tumor-bearing mice intratumorally injected with Ag@Au NPs were observed.Results: Characterization results showed that synthesized Ag@Au NPs were spherical,and the diameters of more than 90 % of the nanoparticles were between 12~16 nm.All Ag@Au NPs prepared with different molar ratios of Ag(75 %,50 % and 25 %)exhibited better CT contrast properties than iohexol,a clinical commonly used CT contrast agent.Then,Ag@Au NPs with a silver molar ratio of 50 % were selected for further research,because of their excellent CT contrast performance,morphology and dispersion.After treatment with Ag@Au NPs and GMT8-Ag@Au NPs,the attenuation coefficient of U87 cells was increased in a concentration-dependent manner.Moreover,at the same mass concentration,GMT8-Ag@Au NPs displayed a stronger contrast enhancement effect than Ag@Au NPs.The results of in vivo experiments showed that the contrast in the tumor area injected with Ag@Au NPs was significantly enhanced in CT images.Conclusion: Our results suggested that Ag@Au NPs could be an excellent CT contrast agent,and the targeted modification of GMT8 can further improve the CT imaging effect.Our study might provide an important theoretical basis for the development of efficient contrast agents in tumor CT imaging.