The Construction Of Functionalized Nylon Guide Wire, Biocompatibility Evaluation And Experimental Study On Capturing Circulating Tumor Cells In Blood Vessels

Objectives:Tumor cells that are shed from the primary site for a variety of reasons are so-called circulating tumor cells?CTCs?.It is documented that CTCs detection have prognostic value in many types of cancer.Meanwhile,CTCs could provide large amounts of real-time biological information of the tumor and possess the potential to guide cancer therapy.Because of the low frequency in the peripheral blood,the detection of CTCs remains to be a technical challenge.Traditional strategies to separate CTCs are based on blood samples of several milliliters.Herein,we planed to eliminate the limits caused by sample volume by means of practicing a new conception for CTCs seperation which introduces a piece of functionalized vein remaining Nylon wire to capture CTCs.We designed to graft CBMA on Nylon surface to promote the biocompability of the material and to make it possible for immobilizing antibodies.In the present study,we research the synthesis and identificatioin of CBMA,the CBMA grafting,the biocompability assessment of CBMA grafted Nylon surface and the simulation of in vivo CTCs capture step by step to verify the idea.Methods:CBMA was synthesised by the reaction of DMAEMA and 3-chloropropionic acid.FTIR and 1H NMR were applied to verify the structure and purity of product.By introducing KH550 as coupling agent,CBMA was grafted onto the Nylong surface according to our design.The grafting was documented by ART-FTIR and XPS.The WCA determination,BSA adsorption determination,platelets adsorption determination and PRT determination were conducted to estimate the blood compatibility of CBMA grafted Nylon surface.The cytotoxicity test of CBMA grafted Nylon to HUVECs was done to reveal the biological safty of the material.After the immobilization of anti-EpCAM antibody on the CBMA grafted Nylon,tumor cell adhension tests were carried out in 24 well cell cultrue cluster and a flowing system made by a peristaltic pump seperately.Finally,the simulation of in vivo CTCs capture using the functionalized Nylon wire was achieved on nude mouse orthotopic tumor model of human gastric cancer cell line SGC7901.Results:The product of the reaction of DMAEMA and 3-chloropropionic acid was tested by FTIR and 8 peaks at 3435 cm^-1?2979 cm^-1?1650 cm^-1?1592 cm^-1?1395 cm^-1?1320 cm^-1?1168 cm^-1?966 cm^-1 belong to characteristic groups of CBMA could be found.In the 1H NMR spectrum,each H atom of CBMA was exactly showed:6.06?s,1H,=CH?,5.68?s,1H,=CH?,4.55?t,2H,OCH₂?,3.70?t,2H,CH₂N?,3.59?t,2H,NCH₂?,3.10?s,6H,NCH₃?,2.64?t,2H,CH₂COO?,1.84?s,3H,=CCH₃?.The ATR-FTIR spectrum of CBMA grafted Nylon showed an additional peak at 1724cm-1 and enhancement of the peak at 1027 cm-1 compaired with pristine Nylon.Typical changes could also be found in the XPS spectrum of CBMA gaafted Nylon.After CBMA grafting,the water contact angle of Nylon surface decreased from 75.05±0.77°to 50.07±1.17°.The amount of adsorbed BSA decreased from 94.32±17.85?g/cm²?1 mg/mL?,66.66±10.56?g/cm²?0.5 mg/mL?,and 25.98±6.54?g/cm²?0.1 mg/mL?to 48.76±7.79?g/cm²?p<0.01?,21.92±7.80?g/cm²?p<0.01?,and 8.14±2.25?g/cm²?p<0.01?,respectively.The platelets adhension test showed the density of adhesive platelets on Nylong surface also decreased significantly after CBMA grafting??1.33±0.29?×10⁴ cells/mm² for pristine Nylon,?1.42±0.20?×10⁴ cells/mm² for?-APS-grafted Nylon,and?4.24±1.0?×10³cells/mm² for CBMA-grafted Nylon?.The PRT of pristine Nylon,KH550 grafted Nylon and CBMA grafted Nylon were 834.17±31.56 s,827.50±35.46 s and1000.33±58.69 s respectively.The prolonged PRT was statistically significant.No obvious toxicity effects were found in the toxicity test of HUVECs.Under the participation of EDC/NHS,anti-EpCAM antibodies were immobilized on the CBMA grafted Nylon surface.The immunohistochemistry results showed human gastric cancer cell line SGC7901 was EpCAM possitive while the MKN45 cell was negative.Tumor cell adhension test showed a significant increase of adhesive SGC7901 cells on anti-EpCAM immobilized Nylon surface?from 20.0±5.5 cells/version to 740.4±42.1 cells/version?.In the flowing system,the adhesive SGC7901cells were 22.3±7.6 cells/version for 10min,73.3±19.6 cells/version for 30min,66.0±8.5 cells/version for 60min,152.7±36.1 cells/version for 90min,172.7±20.6 cells/version for 120min and 163.3±26.7 cells/version for 150min.In the in vivo CTCs capture simulation,3 CTCs were found on one of the six functionalized Nylon wires that had been remained for 30min in the postcava of the nude mice with orthotopic gastric tumor.Conclusion:CBMA can be accurately synthesised by the reaction of DMAEMA and3-chloropropionic acid,and the purity was acceptable.By use of KH550 as coulping agent,CBMA was grafted onto the Nylon surface successfully and conveniently.The hydrophilia,the ability of anti nonspecific protein adsorption and the anti thrombin property of Nylon surface are enhanced after CBMA grafting.No toxicity effects to HUVECs exist accroding to our result focusing on the CBMA grafted Nylon.The anti-EpCAM antibodies immobilized Nylon surface possess the ability to adhere EpCAM possitive cells such as SGC7901 in both static and flowing systme.The in vivo CTCs capture simulation confirmed the feasibility of our design.However,further improvement of the device and much deeper studies are eagerly needed.