The Research Of Duramycin Conjugated Nanoparticle Tracer In Chemotherapy Inducing Apoptosis In Glioma

Objective Apoptosis is a fundamental biologic process of regulated cell death,having a close relationship with treatment of tumor.Apoptosis imaging can noninvasively and dynamically evaluate the effect and prognosis of chemotherapy of malignant tumor in vivo.Phosphatidylethanolamine(PE)is one of the most abundant phospholipid species in mammalian cellular membranes and can be exposed to the outlet of cell membrane at the early stage of apoptosis.Duramycin,a 19 amino acid lantibiotic peptide,can bind PE with high affinity and specificity.We aim to combine^99mTc-Duramycin with dendrimer-entrapped gold nanoparticles,demonstrate its binding efficiency with tumor cell apoptosis in vitro and in vivo and display its value in monitoring treatment efficiency.Methods Poly amidoamine(PAMAM)dendrimers have emerged as an ideal tracer carrier in molecular imaging because of their advantage in high chemical stability,biocompatibility,and high affinity to biomolecules.The surface functional groups of PAMAM dendrimers are abundant and are prone to be conjugated simultaneously with specific ligands,imaging agents and therapeutic materials.In this study,the generation five PAMAM(G5.NH₂)was used as a platform to be sequentially conjugated with polyethylene glycol(PEG),duramycin and DOTA.Then the multifunctional dendrimers were used to entrap gold nanoparticles.¹H nuclear magnetic resonance(NMR)spectrum,zeta potential and dynamic light scattering(DLS)were conducted for characterization of nanoparticles.CCK-8 assay was used to assess the cytotoxicity of the nanoparticles in U87 glioma cells.Flow cytometric analysis was performed to determine the binding of FI-Au DENPs-duramycin and FI–Au DENPs to U87 cells treated with 10 n M paclitaxel for 16 h.Then,the Au DENPs-duramycin and Au DENPs were radiolabeled with ^99mTc.The radiochemicaly purity was assayed by thin-layer chromatography(TLC).The biodistribution of^99mTc Au DENPs-duramycin and ^99mTc Au DENPs were investigated in normal rats using SPECT imaging.The nude mice bearing U87 glioma cells were treated with one dose doxorubicin(20 mg/kg)to induce apoptosis.SPECT imaging by using ^99mTc Au DENPs-duramycin and ^99mTc Au DENPs before treatment and after 2 days of treatment were performed.Imaging results were confirmed by histopathological studies(Hematoxylin and eosin staining,TUNEL staining).Results ¹H NMR spectra confirmed the characteristic peaks of Au DENPs-duramycin and Au DENPs.CCK-8 assay demonstrated that U87 cells displayed a relatively high viability(>90%)after treatment of Au DENPs-duramycin or Au DENPs at the concentration up to 200?M.Flow cytometric analysis suggested that U87 cells treated with paclitaxel displayed significantly stronger fluorescence intensity in FI-Au DENPs-duramycin group than those in FI-Au DENPs group.The yield of^99mTc-Au DENPs-duramycin was 60.4±5.4%(n=3).The yield of ^99mTc-Au DENPs was 64.5±6.8%(n=3)-.The radiochemicaly purity of both two tracers were more than 99%and they were stable in PBS.The in vivo SPECT imaging showed that^99mTc-Au DENPs-duramycin and ^99mTc-Au DENPs mostly accumulated in liver and spleen,while excreted through kidneys and bladder.There was no significant accumulation in thyroid,gastrointestinal tract and soft tissue.Before treatment,both^99mTc-Au DENPs-duramycin and ^99mTc-Au DENPs did not accumulate in tumor.After 2 days of treatment,the tumor showed moderate uptake of ^99mTc-Au DENPs-duramycin while ^99mTc-Au DENPs did not accumulate in tumor.The staining of H&E and TUNEL demonstrated significant increase in apoptotic cells after treatment with doxorubicin.Conclusions Au DENPs-dramycin displays excellent cytocompatibility and can specifically target apoptotic cells.The radiolabeling method of ^99mTc-Au DENPs-duramycin is agile and mild.^99mTc-Au DENPs-duramycin can target apoptotic cells in vitro and in vivo.^99mTc-Au DENPs-duramycin can be applied as a novel apoptotic tracer to monitor the response of chemotherapy.