The Construction And Study Of Drug Loaded Gold Nanoparticles Complex Modificated With Peptide Targeting Mantle Cell Lymphoma Cell Jeko-1

Background: MCL is a subtype of NHL with high degree of malignancy.Most MCL patients were diagnosed at advanced stage because of the few of early symptoms and the poor diagnosis.5-year survival rate of MCL remains low mainly due to the severe side effects of chemotherapy and chemo-resistant relapse.It is of great importance to exploit safer and more effective treatment strategies.Active targeting drug delivery system is the main stream of targeted therapy,and suitable carrier materials and specific targeting molecules are crucial in achieving active targeting.Possessing excellent physicochemical properties,surface modification and biosafety,gold nanoparticles(Au NPs)are widely used in photodynamic therapy,photothermal therapy,imaging and drug delivery.Tumor-targeting peptide is short peptide fragments with low molecular weight and targeted selectivity to targeted tumor cells or tissues and it has promising application value in cancer diagnosis and therapy.This study aimed to construct an active targeting system consisted of Jeko-1(MCL cell line)targeting peptide and Au NPs,and to verify its targeted selectivity and drug delivery.Methods: Firstly,Au NPs were prepared through sodium citrate reduction,then it was modified with ?-mercapto-?-carboxy PEG(HS-PEG-COOH)to prepared carboxylic Au NPs.Finally,to prepare targeted peptide modificated and drug loaded Au NPs,lymphoma specific peptide TUZG12 labeled with FITC(Fluorescein isothiocyanate)and DOX(Doxorubicin)were coupled to the surface of carboxylic Au NPs by esterification reaction catalyzed by 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide(EDC)and N-Hydroxysulfosuccinimide(NHS)(EDC/NHS reaction).The surface modifications of Au NPs were characterized by UV-visible spectrometry,particle size measurement and agarose gel electrophoresis.Targeted peptide modificated and drug loaded Au NPs were introduced to lymphoma cell Jeko-1(targeted cells),Granta-519 and normal human lymphocytes(two negative control cell lines)to identify their targeted selectivity and targeted drug delivery by immunofluorescence assay.The MTT assay was applied to verify the specific cytotoxicity of targeted peptide modificated and drug loaded Au NPs to Jeko-1.Finally,the potential target molecules of peptide TUZG12 were isolated and identified through immunomagnetic beads,SDS-PAGE and mass spectrometry.Results: The particle size of the Au NPs is about 28 nm.After several modifications,the average particle size,UV-visible spectrum and agarose gel electrophoresis behavior of Au NPs changed significantly,indicating that peptides and DOX were most probably coupled to the surface of Au NPs.In immunofluorescence assays,strong green fluorescence was observed in Jeko-1 treated with Au NPs modificated with targeted peptide,while the fluorescence signal was weak in normal human lymphocytes and Granta-519.Highlighted red and green fluorescence were only observed in Jeko-1 treated with targeted peptide modificated and drug loaded Au NPs,while cells treated with other drug loaded Au NPs exhibited diminished fluorescence intensity.The cell viability of Jeko-1 was lower after the treatment of targeted peptide modificated and drug loaded Au NPs than that treated with other drug loaded Au NPs.The captures of magnetic beads coupled with target peptide and untargeted peptide were separated by SDS-PAGE.With the mass spectrometry identification,PYM1(Partner of Y14 and mago)was probably one of the targeted molecules of TUZG12 on Jeko-1.Conclusion: In this study,we have constructed Jeko-1-targeted Au NPs complex consisted of Au NPs,targeted peptide TUZG12 and DOX.This Au NPs complex exhibited high targeted selectivity to Jeko-1 but low to normal human lymphocytes and Granta-519.In addition,targeted peptide modificated and drug loaded Au NPs displayed targeted cytotoxicity to Jeko-1.PYM1 may be one of the potential targeted molecules of peptide TUZG12.