| Lymphoma is a heterogeneous group of malignancies of the lymphoid tissue,affecting both children and adults with a high mortality rate.90% lymphomas are non-Hodgkin lymphomas(NHL),which are very difficult to be diagnosed and treated efficiently.Therefore,there is in dire need of accurate and appropriate approaches for early detection and treatment of this disease.Herein,based on nanophotonics and antibody therapy for cancer,we report a kind of stable and reliable Rituxan-Ag nanoconjugation,which integrates the ability of sensitive surface enhanced Raman scattering(SERS)detection with targeted ablation for CD20-positive lymphoma cells in vitro,and has higher potency compared with Rituxan(anti-CD20 monoclonal antibody)at the same concentration.The main research contents of this dissertation are presented as following:(1)Design and synthesis of the nanoconjugation.The Rituxan-Ag Nanoconjugation was synthesized by coating the Raman reporter p-Methylthiobenzoic acid(p-MBA)and Rituxan on the surface of silver nanoparticles respectively via covalent coupling.(2)Characterization and evaluation of the nanoconjugation.The nanoconjugation were characterized by absorption spectrum,TEM,Bradford assay,XPS,DLS,Zeta potential analysis and SERS measurements to confirm the success of synthesis and to analyse its size,shape,drug loading capacity,oxidation,stability and SERS spectrum,respectively.Besides,it has been confirmed that the nanoconjugation retains the same antibody property as intact drug via Rituxan anti-CD20 ELISA assay and antibody-dependent cell-mediated cytotoxicity analysis.(3)SERS detection of lymphoma cells in vitro.The nanoconjugates were used to specifically detect single live CD20-positive lymphoma cell by SERS based on the excellent plasmons property of the silver nanoparticles and the connection with Raman reporter.The spatial-resolved SERS mapping and photoluminescence imaging of the same cell were also performed to further confirm that the enhanced Raman signal was indeed derived from the nanoconjugate-associated cell.(4)Simultaneous depletion of lymphoma cells.It has been discovered that treatment of lymphoma cells with nanoconjugates resulted in a higher rate of cell ablation and apoptosis compared to treatment with free Rituxan by MTS assay and apoptosis analysis using imaging-flow cytometry.(5)Analysis of the mechanism of the nanoconjugation’s enhanced potency.Internalization of the nanoconjugates is evaluated via imaging flow cytometry analysis and laser scanning confocal microscopic analysis of fluorescently labeled nanoconjugates.And it has been found that the nanoconjugation prolonged the drug action time on the cell surface,and caused enhanced capping of CD20 molecules on the cell membrane,which may contribute to the enhanced cell death.Targeting monoclonal antibody has been widely concerned,since it can react with the specific cancer site,following the death of cancer cells without influencing the nearby normal cells.However,clinical researches indicate that,after being treated with this kind of drugs for a long time,most of the remained cancer cells will have resistance to the drugs,which is a big challenge for targeting cancer therapy using monoclonal antibody.Our data confirmed that the spatial structure design of the nanodrugs not only enhances the drug efficiency and fights resistance,but can also detect tumour cells simultaneously,which provides a new insight into cancer therapy in the future. |
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