| Circulating tumor cells(CTCs)are those that are shed from solid tumors and thenmigrate to distant organs through the circulatory system.Screening for these CTCs in peripheral blood is of proven importance for earlycancer diagnosis,treatment monitoring,metastasis diagnosis,and prognostic evaluation.Unfortunately,the occurrence rate of CTCs is at an estimated level of one cell among billionsof surrounding background cells.It is very challengingto efficiently enrich CTCs due to their extremely low abundance and serious interference from the background cells.Researchers have developed several strategies to selectivelyenrich CTCs from peripheral blood.Among these strategies,the immuno-magnetic approach is oneof the most frequently used.For example,the Cell Search? system,the only FDA-approved technique for CTC detection,enriches CTCs by using 120-200 nm ferrofluid linked with anti-EpCAM antibodies,but this system still yields background of 1000-3000 leukocytes per sample,which makes subsequent CTC analysis problematic.In detail,this thesis mainly consisted of two parts:Construction and characterization of biomimetic immuno-magnetosomes(IMSs).First,the Cho analogue azide-choline(azide-cho)was biosynthetically bonded to phospholipids and incorporated into the J774 A.1 cells.The azide-modified membranes were harvested by osmotic shock,sonication and centrifugation.Secondly,the positive charge of the MNCs facilitatedsubsequent coating with extracted leukocyte membrane fragments(magnetosomes)through electrostatic interaction,which was demonstrated by reversed zeta potential.The magnetosomes exhibited good magnetic controllability.Thirdly,we conjugated the EpCAM antibody to magnetosomes using a copper free click chemistry reaction.Above all,we had constructed biomimetic IMSs successfully.The application of IMSs in CTCs enrichment.Under the optimized condition,we selected EpCAM+ cells and J774 A.1 cells to evaluate the specificity of CTCs and nonspecific interaction with leukocytes.All results showing that IMSs had good stability and specificity.The well-preserved activity of the anchored antibody and the fluidity of the coated membraneendowed the IMSs with satisfactory binding avidity to tumor cells.The homology of the biomimetic magnetosome significantly inhibits nonspecific binding to leukocytes.As a result,~90% of the rare tumor cells can be captured from whole blood in 15 min with undetectable leukocyte background,thus demonstrating great promise for the high-performance enrichment of CTCs.Meanwhile,we were glad to find that our IMSs werevery biocompatible because almost all of the captured cells were still viable.The IMS-captured CTCs can be used for subsequent molecular analysis directly. |
