A Method To Detect Circulating Tumor Cells By Nanostructured Substratesgrafted With Aptamers

Circulating tumor cells(CTCs)play an important role in tumor metastasis and recurrence.Early detection and diagnosis could be offer timely and effective treatment for tumor patients,and also reduce the fatality rate,improve the prognosis.As a kind of real-time detection of "liquid biopsy",CTCs carryimportant and valuablemessage for diagnosis of tumor diseases and individualized treatment.However,CTCs are rare cells in the peripheral blood andthe methods to detecting CTCs require high sensitivity and specificity.Currently CTCs detection methods havemany defects such as low detection rate,poor sensitivity and lack of specificity.In view of significant clinical significance of CTCs,to find better detection method is very urgent.Recently,nanostructured substrates thatmimic the natural extracellular matrix or basement membranehave emerged as very promising tools for CTCsdetection and isolation.We developed a newrecipe using C3F8 and C4F8 as etchant gas and successfullyprepared homogeneous nanostructures with silicon boron glass.Bi-specific aptamersand a biomimetic combination of antibodies have beenused for targeted drug delivery and isolation of CTCs,respectively.However,it is unknown whether multispecificaptamers are able to improveisolation efficiencyof cancer cells yet.LongDNA molecules containing multiple aptamer units(anti-EpCAM aptamer unit(AEA)or anti-prostate specificmembrane antigen(PSMA)aptamer unit(APA))are preparedby rolling circle amplification(RCA)and graftedonto nanostructured glass substrates.weselected three kinds of human prostate cancer cell LNCaP(PSMA+/EpCAM +),PC3(PSMA-/EpCAM),Ramos(EpCAM-/PSMA-)to test the isolation efficiency.Objective:Nanostructures glass substratesareobtained from one step dry etching,Bi-specific aptamers targetingepithelial cell adhesion molecule and prostate specific membrane antigen fixed insubstrates arealso tested the isolation efficiency for tumor cells.Methods:For preparing nanostructured glass substrates,we used C3F8 and C4F8 as etchant gas to etching silicon oxide and then characterizedby atomic force microscopy.APA and AEA were produced by RCA and tested by gel electrophoresis.Four kinds of certain quantity tumor cells(PC3?MCF7?HepG2?SW620)were mixed into medium and were used to test thecapture rate of nanostructured glass substrates.APA and AEA aptamer graftedontonanostructured glass substrates by chemical reaction.Three kinds of prostate cancer cells were incubated withAEA,APA and contrast of DNA which labeled with Cy3 and fluorescence imaging was focused on aptamers.The fluorescence images were taken using appropriate filtersfollowed by analysis of fluorescence intensity withImageJ.Cells capture assay was divided into four groups:AEA + cells,APA + cells,AEA/APA + cell group,and DNA + cells,three kinds of prostate cancer cells were incubatedwith nanostructured substrates and thenumber of captured cellswas countedthroughDAPI stained nuclei of cells by MD high-content imaging system using 10× objectives.The formation ofheterodimers between AEA and APAwhether or not was detected by electrophoresis.Nanostructured glass substrates fixed with AEA to capture the peripheral blood of healthy human mixed with three kinds of tumor cells which expressed highlevel of EpCam(human hepatocellular carcinoma HepG2 cells and human breast cancer MCF7 cells and colon cancer cells SW620),the positive cells(CTCs)weredetected and then counted by high intension screening system microscope.The criteria of CTCs is staining with immunofluorescence CK18+/CD45-/DAPI+,the appearance of the cells are round,elliptic,or oblong withcomplete edge.Results:We successful created nanostructures substrates and imaged by JPK atomic force microscope(AFM)with diameters ranging from 126.8nm to 412.9nm,andthe nanostructures with 374 nm produced in the condition with 5 mins and 100 Vwas the best for capture cells theoretically.Theadherence rate of the four kinds of tumor cells which captured by nanostructured glass substrates were more than 93%.Electrophoresis assay showed we successfully amplified the APA and AEA aptamers by rolling circle amplification(RCA)within 30min.The results of APA and AEA aptamers combined with three kinds of tumor cell showed the highest fluorescence intensity were from LNCaP cells,and the fluorescence intensity of AEA was higher than that in APA.The fluorescent signal from PC3 cells was detected only in AEA group.No obvious fluorescence signal of Ramos cells group and DNA groupswhich played as control groups were detected.Cells capture assayshowed that cell capture rate of the LNCaP cells captuered by nanostructured substratesgrafted with AEA and APAaptamers were 85%±5.3%? 68%±7.5%.The capture rate of PC3 cells captuered by nanostructured substrates fixed with AEA aptamers is 76.2%±8.5%,but the capture rate of APA group was only 11.2%±0.4%.The capture rate of Ramos cells incubated with nanostructured substratesgrafted with AEA or APA were lower than 2%.However,nanostructured substratesgrafted with Bi-aptamers(AEA/APA)to LNCaP and PC3 cells were 8.7%±2.3%and 4.5%±1.8%.We used different mixing ratio of AEA/APA(1:3,1:1 and 3:1)to capture the PC3 cells and the results showed that the capture rates were 0.5%,6.7%and 42.1%,respectively.Electrophoresis assay showed that there was a weaken stripe appeared in the position about 120-nt.Mimic peripheral blood of tumor patients showed that detection rate of MCF7,HepG2 and SW620 were 84%15.78%?73.6%±5.41%.70%±6.28%respectively,the average detection rate of the three cells was 75.87%.ConclusionReliable and homogeneousnanostructuredsurface on borosilicate glass substrateswith large area were prepared rapidly and successfully by using dry etching methodwithetchant gas.The diameter of nanostructurescould becontrolledbetween 126.8nm and 412.9nm by changingthe reaction conditions.The average diameter of nanostructures wasapproximately 374 nm with condition of 100V and 5mins,which was more close to the nanostructures of natural extracellular matrix.Specific single-aptamergrafted on nanostructured substratescan improve the capture rate of tumor cells.However,bi-specific aptamers co-functionalized nanostructured substratesfail to capture tumor cells probably due to the formation of hetero-dimers.Mimic peripheral blood of tumor patients assay established that nanostructured substrates combining with specific single-aptamer have a better efficiency ofdetecting CTCs.The potential shortcoming may impair the application of multi-specific aptamers for detection and isolation of molecules unless the hetero-dimers between selected aptamers can be strictly excluded.Nanostructured substrateswhich prepared by RIE were high efficiency,reliable and large area(approximately 80cm2).Because of rapid producing,coating and avioding CTCs isolation,CTCs captured by the substracts can be detected derictly and The loss of CTCs were reduced.Nanostructured substrates can not only detect the number of CTCs,also can analyze the expression of tumor molecules inCTCs,and providemore accurate information for clinical diagnosis,monitoring therapeutic effect andprognosis of tumor.Radiation therapy is one of the important treatments for unresectable primary hepatic carcinoma(PHC).However,radiation-induced liver disease and radiation resist have severe impacts on liver cancer radiotherapy effect.The radiosensitization method is significant for improving the effect of liver cancer radiotherapy.Asialoglycoprotein receptor(Asialoglycoprotein Receptor,ASGPR)is mainly expressed in mammalian liver cell surface,is commonly used in the target-mediated endocytosis.Gold nanoparticles(GNPs)have been proved to have radiotherapy sensitization effect in many experiments in vivo and in vitro.We designed new compound--GAL-PEG-GNPs which were carried with galactose(GAL,ASPGR specificity ligand)and modified by polyethylene glycol(PEG).We confirmed that the GAL-PEG-GNPs have remarkable good radiotherapy sensitization effect with ASGPR positive HepG2 cells in vitro experiment and the sensitization mechanism is discussed.On the basis of ASGPR positive nude mouse model of hepatocellular carcinoma was established in our research,we explore the targeting,toxicity,tissue distribution,radiotherapy sensitization mechanism of GAL-PEG-GNPs and evaluate its radiotherapy sensitization effect in vivo.The results will build an experimental basis for liver targeted delivery system as well as provide a theoretical basis for HCC radiosensitization.ObejeciveBase on the establishment of ASGPR positive nude mouse model load with live cancer,to research the pharmacokinetic,organ distribution and radiotherapy sensitization of GAL-PEG-GNPs in vivo drug,which provides theoretical basis for liver cancer radiotherapy sensitization.MethodsBalb/C nude micemodel loadedwith subcutaneous transplantation liver tumor was established by using ASGPR positive liver cancer cells-HepG2 cell.0.2 mL 2 mg/mL of GAL-PEG-GNPs and GNPs were injected into nude mice model by tail veinrespectively,drug metabolism of the two nanomaterial in different time points were tested by ICP-MS.To separated the heart,liver,spleen,lung,kidney,tumor tissue and muscle of nude mice model after they were executed,the tissue distribution of Au at different time points was detected by ICP-MS.Micewere fixed in a special box after the establishment of subcutaneous transplantation tumor,we used 6 MeV X-ray to irradiate the tumor(2.5Gy/time,ltimes every 3 days,total dose was 20 Gy).The radiotherapy sensitization of nanomaterials was tested by the weight of mice,the tumor volume and the weight of theeventually tumor.ResultsTumor in the right front alar of Nude mice can be measured after the injection of HepG2 cells,tumor volume was measured after 5 days of injection,and all the tumor volume reached 60 mm3 to 340 mm3.Half-life of the two kinds of nanomaterials in the metabolism of nude mice blood was t1/2 GNPs = 1.007 h and t1/2GAL-PEG-GNPs=3.406 hrespectively,Tissue distribution tests revealed that GNPs and GAL-PEG-GNPs mostly were intook by liver and spleen after they were injected into nude mice in vivo,the Au of GAL-PEG-GNPs group in tumor tissue was significantly higher than GNPs groups,difference were statistically significant in the three time points.Radiotherapy sensitization assayshowed that the speed increase oftumor volume in radiation group is bigger than the two nanomaterial groups which combined with radiation,Tumor volumesof GAL-PEG-GNPs combined radiotherapy group weresignificantly smaller than radiation group and GNPs combined radiotherapy groupat the end of the Radiation therapy(p<0.05).The weight of three groups of nude mice are growing steadilyafter the starting of the radiotherapy,and the weight of GAL-PEG-GNPs combined radiotherapy group grows best and mostheaviest,but there no significant difference in the three groups(p>0.05).At the end of treatment after 22 days,tumor volume inhibition rate of GAL-PEG-GNPs combined radiotherapy group was 82.22%,GNPs combined radiotherapy group was 47.24%,and it was statistically significant between three groups(p<0.05).ConclusionWe confirmed that GAL-PEG-GNPs waslow toxicity and targeted to ASGPR positivenude mouse model loaded with liver cancer,GAL-PEG-GNPs was more effectively to control the tumor inhibition volume of mice than GNPs,the experimental results provides theoretical basis for the liver cancer radiotherapy sensitization and the applications of liver targeting drug.