Biotoxicity And Tumour-targeted Imaging Of Adenosine 5’-monophosphate Functionalized Nanoparticles

Reducing toxicity and enhancing tumour target ability of nanoparticles are important aspects in nanoparticle research field. In this work, adenosine 5’-monophosphate (AMP), as a biocompatible small molecular, was used as a surface ligand to modify nanoparticles, and its roles in reducing the biotoxicity and increasing the tumour-targeted delivery of nanoparticles were assessed systematically.1. AMP reduced the biotoxicity of quantum dotsAMP modified quantum dots (AMP-QDs) were prepared via phase transfer method using AMP as a ligand. Using macrophage J774A.1 as a cell model, AMP-QDs exhibited a prior imaging property to that of mercaptopropanoic acid (MPA) modified QDs (MPA-QDs). Furthermore, AMP-QDs demonstrated lower cytoxicity than MPA-QDs by MTT method. Using BALB/c mice as an animal model, AMP-QDs were injected intravenously and found mainly detained in liver and kidneys at 4 h. After long time (60 d) circulation, they couldn’t be excreted completely from the mice. However, AMP-QDs didn’t evoke robust inflammation responses via histological section and detection of immune related cytokines experiments.2. AMP guided QDs to target to cancer cells in vitroUsing human colon cancer cell CW-2 and breast cancer cell MDA-MB-468 as cancer cell models, AMP-QDs showed significant uptake in CW-2 and MDA-MB-468 cells, indicating its potential as a new tumour-targeted ligand. In order to systematically investigate the tumour-targeted behaviours of AMP-conjugated nanoparticles both in vitro and in vivo, the following experiments were carried out in this wok:(1) Synthesis and characterization of AMP-coupled polymer-based fluorescence nanoparticles (NPs-AMP)1,6-hexanediamine (HDA) was conjugated to the phosphate group site of AMP to get AMPHDA with the amino terminal, and polymer-based fluorescence nanoparticles (NPs) with two emission peaks (MEH-PPV emission peak at 594 nm and a NIR emission peak at 777 nm) were prepared. AMPHDA was then conjugated to NPs to synthesize the AMP-functionalized nanoparticles (NPs-AMP). Conjugation of AMP to NPs slightly increased the average hydrodynamic diameter of nanoparticles from-53 nm (NPs) to-57 nm (NPs-AMP). NPs-AMP preserved high fluorescent brightness comparable to that of NPs.(2) NPs-AMP target to colon cancer model both in vitro and in vivoUsing colon cancer cells CW-2 as the cell model, firstly, NPs-AMP could highly selectively target to CW-2 cells in relative to corresponding intestine epithelial cells (IECs). It is found that NPs-AMP initially bound to the cell membrane and were then internalized into CW-2 cells. Secondly, through RT-PCR and western blot methods, a remarkably 1832-fold higher expression of adenosine A1 receptor (A1R) was found in CW-2 cells compared to IECs. Immunocytochemistry experiments showed that NPs-AMP could colocalize with A1R on CW-2 cells, indicating that the interaction of NPs-AMP with A1R guided nanoparticles to target CW-2. The assumption was further confirmed by inhibition and RNA interference (RNAi) of A1R experiments. Finally, the delivery of NPs-AMP to other colon cancer cells including SW620, Lovo and HCT116 was tested as well, and the corresponding expression levels of the A1R were investigated. Remarkably, it was concluded that NPs-AMP could target to them and the target ability was positively correlated to the A1R expression levels.Afterwards, using nude mice bearing colon tumour xenograft as the animal model, NPs-AMP could efficiently target to CW-2 tumour through in vivo imaging experiment. The ratio of nanoparticles in tumour was correspondingly enhanced from 20.5%(NPs) to 41.6% (NPs-AMP). The subsequent immunohistochemical staining of the colon tumour tissue showed that AMP could further guide the nanoparticles to enter into the tumour cells.(3) NPs-AMP target to breast cancer model both in vitro and in vivoUsing breast cancer cells MDA-MB-468 as the cell model, firstly, NPs-AMP could highly selectively target to MDA-MB-468 cells in relative to corresponding mammary epithelial cells (MECs). It is found that NPs-AMP initially bound to the cell membrane and were then internalized into MDA-MB-468 cells. Secondly, through RT-PCR method, a remarkably 870-fold higher expression of A1R was found in MDA-MB-468 cells compared to MECs. Immunocytochemistry experiments showed that NPs-AMP could colocalize with A1R on MDA-MB-468 cells, indicating that the interaction of NPs-AMP with A1R guided nanoparticles to target MDA-MB-468. The assumption was further confirmed by RNA interference (RNAi) of A1R experiment. Finally, the delivery of NPs-AMP to other breast cancer cells including HS578、T47D and MDA-MB-453 was tested as well, and the corresponding expression levels of the A1R were investigated. Remarkably, it was concluded that NPs-AMP could target to them and the target ability was positively correlated to the A1R expression levels.Afterwards, using nude mice bearing breast tumour xenograft as the animal model, NPs-AMP could efficiently target to MDA-MB-468 tumour through in vivo imaging experiment. The ratio of nanoparticles in tumour was correspondingly enhanced from 15.7%(NPs) to 31.8%(NPs-AMP). The subsequent immunohistochemical staining of the breast tumour tissue showed that AMP could further guide the nanoparticles to enter into the tumour cells.In summary, on one hand, AMP endowed nanoparticles with low immunotoxicity, providing perspectives for designing biocompatible nanoparticles in the future. On the other hand, AMP, as a tumour-targeted ligand, successfully guided nanoparticles to target to colon and breast cancer models both in vitro and in vivo. These data open the possibility that designing tumour-targeted diagnosis and therapy drug and potentially clinical application can be explored.