Study Of Multi-functional Nanoparticle For Early Diagnosis Of Gastric Cancer By Targeting CD146

It has been reported that CD146 can be a useful marker for the early diagnosis of gastric cancer. In 2012, the research about CD146 was involved into a new field by a paper which was published on PNAS, it was “CD146, an epithelial-mesenchymal transition inducer, is associated with triple-negative breast cancer.” While the newest research pointed out the same opinion that CD146 introduce the EMT in gastric cancer, enhance the metastasis in patients diagnosed with late-stage GC, also it is often accompanied by a higher chance of metastasis. a useful marker to evaluate the stage of GC.The first monoclonal antibody which was mentioned in the paper on PNAS cannot recognize the marker on triple negative breast cancer cell line MB-231, but can only recognize the cell line transfected by CD146 plasmid. This kind of recombinant monoclonal antibody will hard to recognize the marker in natural, more hardly in vivo. Therefore, we plan to immune the mice by antigen which purified from mammary cells, once we get the monoclonal antibody, it will be conjugated with nanoparticle as an in vivo cancer tracer for the early diagnosis and targeting treatment.Results and data:1. To generate anti-human CD146 mAbs, we immunized the mice with CD146 protein from mammary cells.Based on the commercial CD146 antigen, aided by 4 times colonized, enzyme-linked immunosorbent assay(ELISA) screenings and SDS-PAGE, we selected the five hybridoma clones with the highest immunoreactivity towards CD146. Immunofluorescence staining and FACS screening of malignant human melanoma cells(A375), known to overexpress CD146, revealed YY146(the fifth mAb clone) as the mAb with the highest binding affinity. We therefore selected YY146 as the best candidate for in vivo studies.2. Preparation and characterization of 800ZW–SPION@d Si O2–YY146.According to microemulsion method, we introduced the SPION into the dSiO2 nanoparticles preparation system with NP-5 and cyclohexane. The nanoparticle were functionalized first with-NH2 groups by(3-aminopropyl) trimethoxysilane to form SPION@dSiO2–NH2, followed by polyethylene glycolylation(PEGylation) with SCM-PEG5k-maleimide(MAD) to achieve SPION@dSi O2–PEG–Mal, leaving amino(succinimidyl carboxymethyl ester [SCM]) groups on the shell for further antibody conjugations. Afterward, the desired number of 800 ZW, were added to obtain. 800ZW–SPION@d Si O2–PEG-Mal, Lastly, polyethylene glycol(PEG)(Mal-PEG5k-SCM) was used to acquire 800ZW–SPION@d Si O2–YY146. The morphology and the size of pre- and post-labeling SPION@d Si O2 core–shell nanoparticles were characterized using transmission electron microscopy(TEM), FACS, Confocal.3. Determination of binding affinities of 800ZW–SPION@dSiO2–YY146 in vitro.The binding affinities of the immunoreactivity of 800ZW–SPION@d SiO2–YY146 to MKN45(high CD146 expression) were detected by FACS, Confocal and TEM technique. Taken together, these in vitro experiments confirmed that 800ZW–SPION@dSiO2 conjugation did not impact the antigen-binding specificity of YY146.4. In vivo MR/NIRF imaging about tumor targeting。The time points at 4, 16, 24, and 48 hours post 800ZW–SPION@d Si O2–YY146 injection(pi) were chosen for a series of NIRF scanning. T*2-weighted MR images were collected at specific time points on the T system, where 800ZW–SPION@d Si O2–YY146 appears as discontinued, intermittent, and negative contrast ROIs. A significant signal drop in image contrast occurred between the time points 24 and 48 hours followed by a continual decrease thenceforth. The observation of apparent distinction is peaked at 24 hours of 800ZW–SPION@d Si O2–YY146 injection.Prominent NIRF signals from the tumor indicated significant accumulation of 800ZW–SPION@d Si O2–YY146 in the main organs at 48 hours pi, which is consistent with the in vivo findings.. Importantly, accumulation of 800ZW–SPION@d Si O2–YY146 in the tumor happened fast, which can be visible clearly at 4 hours pi and peaked at around 24 hours pi. In comparison, in unconjugated nanoparticles, the MKN45 tumo r uptake of 800ZW–SPION@d Si O2 was found to be significantly lower than that of 800ZW– SPION@dSiO2–YY146 at allConclusion:We have successfully accomplished in vivo noninvasive monitoring of YY146 labeling with dual-modality biofunctionalized d Si O2-coated SPION nanoparticles as MRI/NIRF contrast agents in xenografted GC model in mice. The results show that 800ZW–SPION@d Si O2–YY146 target GC in vivo by binding to a novel tumor marker of CD146. Undoubtedly, such results are important in the clinic to decrease the side effects and treatment costs, specifically with the increasing number of alternative treatments that are only effective in particular groups of patients.