Preparation Of Dendrimer-entrapped Gold Nanoparticles As A Probe For Detection And Evaluation Of T Cell Activity

Tumor immunotherapy has various advantages such as fewer side effects,less recurrence and better therapeutic effect on patients with advanced tumors than traditional treatment methods,greatly improving the survival rate and life quality of cancer patients.The principle of adoptive cell transfer therapy(ACT),which is a tumor immunotherapy hotspot in recent years,is to modify and activate autologous or allogeneic T cells in vitro by artificial intervention,and then to transfer the modified T cells back into patients to activate the human immune system,so as to kill the tumor cells.With the rapid development of tumor immunotherapy,researchers are paying more and more attention to T lymphocytes.Therefore,a novel nanomaterial that can monitor and trace T lymphocytes is a key to promote development of T cell-based tumor immunotherapy.A suitable carrier is essential to construct nanomaterials.Poly(amidoamine)(PAMAM)dendrimers have a highly branched structure with abundant modifiable moieties on the surface and a cavity for loading various metal nanoparticles,enabling them an excellent carrier to fabricate diagnostic and therapeutic agents.As a molecular imaging technique,computed tomography(CT)is widely used.With the development of nanoscience and nanotechnology,a variety of nanoparticles(NPs),such as PAMAM-based gold nanoparticles,can be used as CT angiography for CT imaging.These NPs usually have high X-ray attenuation coefficient,longer in vivo blood circulation time,and excellent biocompatibility.Calcium ion probes are commonly used for cytosolic calcium signal monitoring due to their high affinity and sensitivity to calcium ions.Some researchers perform fluorescence imaging by labeling calcium ion probes on NPs.In this study,based on the previous work of our research group and related literatures,the fifth generation(G5)PAMAM dendrimer is used as a platform to entrap gold nanoparticles and covalently conjugate calcium ion probes to fabricate nanomaterials.Then the synthesized nanomaterials were incubated with T lymphocytes to explore the potential to detect and track T cell activity in tumor immunotherapy.In chapter 2,G5 PAMAM dendrimer was first modified with hydroxy polyethylene glycol(PEG),and then used as a platform to entrap gold NPs in the interior by in situ reduction,followed by acetylation of the excess amino groups on the dendrimer surface.Subsequently,the calcium ion probe was covalently attached to the dendrimers through the reaction of the hydroxyl group of the PEG with the carboxyl group of the calcium ion probe.Various techniques were utilized to characterize the physicochemical properties of the NPs.The results showed that the dendrimerentrapped gold NP calcium ion probe was successfully synthesized,and had excellent water solubility and stability,as well as high X-ray attenuation coefficient.In Chapter 3,a series of evaluations were carried out on T cells that uptook dendrimer-entrapped gold nanoparticle calcium ion probe.Biocompatibility experiment showed that the synthesized nanomaterials had low cytotoxicity and flow cytometry result indicated that the probe was sensitive to the fluctuation of cytoplasmic calcium signal of T cells,resulting in a potential to effectively monitor the changes of calcium signal.In vivo and in vitro imaging results demonstrated that the nanomaterial can simultaneously perform CT/fluorescence imaging on T cells for cell monitoring.In summary,we successfully synthesized a dendrimer-entrapped gold NP calcium ion probe.Through CT and fluorescence imaging of T cells which were incubated with nanoprobes and subcutaneously injected back into mice,T cell tracking and monitoring in tumor immunotherapy can be achieved,providing a new idea for evaluating the efficacy of immunotherapy.