Preparation And In Vitro Assay Of Microbubbles Targeting Solid Carcinoma By KDR With Biotin-avidin System

Purposes:The diagnosis of Tumor in early stage is the issue urgently need to be settled in imageology.As the molecular mechanism of tumor angiogenesis to be understood gradually,the progressive development of vascular functional molecular markers which based on the heterogeneity of tumor blood vessels,and applied to the diagnosis and treatment of tumor through molecular imaging.KDR as the most important vascular endothelial growth factor(VEGF) main receptor,highly expressed in endothelial cells in tumor angiogenesis,plays a key role in the early stages of tumor angiogenesis.We used KDR which expressed specifically on tumor vascular endothelial cells as the specific target,through the bridging of biotinavidin and signal multi-amplification,conjugded the small peptide K237 which has a high degree affinity of KDR to liposome microbubbles.To build a new kind of targeted microbubbles and identify the physical and chemical properties and biological activity,observe the specific imaging effect of microbubbles targeted to tumor angiogenesis.With a view to establishing a new type of ultrasound molecular imaging technology for early diagnosis of tumor.The biotin - avidin bridge system in construction of the targeted microbubble may overcome the shortcomings of the existing contrast agent targeting poor sensitivity and unstability,have significance to the diagnosis of other diseases. 1.Preparation of targeted microbubbles(MB_K237)1.1 biotinylated liposome microbubbles(MB_B) Preparation1.1.1 DPPC,PEG,DSPE-PEG2000-Biotin and other lipid materials at a certain percentage of the amount dissolved in distilled water,at the same time give perfluoropropane(C3F8) gas,acoustic wave oscillation to form a milky white liquid, 4℃refrigerator hierarchical standing,abandoned follow fluid,injected with an equal volume of pure water to purify microbubble repeatedly to remove free liposomes.1.1.2 Microbubble solution at 1:10000 dilution ratio,Coulter particle size analyzer (Mutisizerâ…¡,USA) determinate the particle size,microsphere concentration,particle size distribution.1.2 Preparation of biotin - avidin bridge-mediated targeted liposomes microbubble with small peptide(MB_K237)1.2.1 Corporation Hangzhou synthetic fluorescent-labeled peptide of 12 biotinylated peptides(FITC-Biotin-K237),the molecular weight of 2523.6 D,amino acid sequences for fitc-C6-His-Thr-Met-Tyr-Tyr-His-His-Tyr - Gln-His-His-Leu-Lys (Biotin) ^[10].K237 is a peptide,Lei Hetian etal people isolated from phage display peptide library.Screening methods are:use KDR as the target,the use of affinity selection of phage peptide library technology,through ongoing reduce the concentration of KDR,improved the strength of washing fluid and competitive elution,to obtain strong binding of phage clones.From the two screening results, randomly selected 40 phage clones was amplified,using ELISA method to identify their binding activity,selected with the highest affinity phage clones sequenced, according to sequencing results of the chemical preparation of small peptide elements.1.2.2 Preparation of targeted microbubbles(MB_K237)â‘ Avidin-Biotin-Microbubbles(Av-MB_B) Preparation and detection: MB_B purified then shaked into milky white suspension,0.3ml of each suspension (about 10⁸ microbubbles) as a sample,5 groups and 3 samples each group.respectively add 0μg,2μg,6μg,10μg,30μg fluorescent-labeled avidin (FITC-avidin) into each sample,after 30min incubation.Purified twice,flow cytometry detect the rate of avidin of microbubbles,evaluate the optimum dose.MB(0.3ml) and Av- MB_B(0.3ml) were deal with adding an equivalent FITC-Avidin for 30min incubation,full washing,the fluorescence microscope to observe two fluorescence effection of the microbubble.â‘¡.Targeted Microbubble Preparation and Detection: MB_B in the suspension,10⁸/ml microbubbles adding 6μg no fluorescent avidin, incubated 30min,purifiy twice,shaken to the liquid,one sample is 0.3ml,7 groups and 3 samples in each group,add respectively 0μg,30μg,40μg,50μg,60μg, 70μg,l00μg of FITC-Biotin-K237 into each sample,flow cytometry to detect the rate of a small peptide bubble brought,to evaluate the optimal dose ratio.Kurt observed MB_k237 of physical properties.Light microscopy and fluorescence microscopy in general the appearance of targeted microbubbles.2.Targted Microbubbles(MB_k237) in vitro assay2.1 Cell culture and Immunohistochemistry2.1.1 LOVO colorectal cancer cells,human umbilical vein endothelial cells(HUVE) and human colon cancer LS174T cells from logarithmic phase,0.25%trypsin digestion and inoculated in 6-well plates(3×105 / hole),plus 10%newborn calf 1640 serum-wide liquid-pui,5%CO2 incubator incubated until the cells in 80%of saturation.2.1.2 PBS washed 3 times,4%paraformaldehyde fixed,KDR conventional immunohistochemical staining.2.2 Targeted in vitro experiments:2.2.1 In vitro binding assay:â‘ Three cells(KDR expression strong positive LOVO cells,positive HUVE cells, and KDR expression negative LS174T cells) were incubated with MB_k237 for 30min, after washing observed under a light microscope and fluorescence microscope, analysing the effect of the combination of the three cell lines with targeted microbubbles.â‘¡Three types of microbubbles(targeted microbubbles MB_k237,simply K237 modified liposome microbubblesK237-MB_B,control microbubble MB) incubated with KDR expression strong positive LOVO cells for 30min,after washing observed under light microscope and fluorescent microscope,analysing the effect of the combination of the three microbubbles targeted with LOVO cells.2.2.2 Blocking experiment: 10μg,50μg of K237 to deal with LOVO cells for 1h,incubated these treated LOVO cells with targeted microbubbles for 30min,then observed after washing,ananlysing the effect of MB_K237combined with teated LOVO cells.2.2.3 Mav = 6μg,Mk237 =(50μg,70μg,90ug,100μg) preparation targeted microbubble in four doses programs,LOVO cells were incubated with the four MB_k237 for 30min,then observed after washing,annalysing the MB_k237 in different doses the effect of targeted combination.2.2.4 Four speed(5ml/h,20ml/h,50ml/h,99ml/h) controlled by pump flushing microinjection,washing rosette structure formatted by LOVO cells and MB_k237 at these four speed,then observed the changes in rosette structure under light microscope.3.Results Standard3.1 Immunohistochemicstry:Immunohistochemicstry was performed according to the protocol of the kit.The positive cell was determined by the appearances of light brown granules in cytoplasm or cell membrane.Semi-quantitative integral method used to determination[9],the number of positive cells≤5%,0 score;6%-25%,1 score;26%-50%,2 score;51%-75%,3 score;＞75%,4 score.The score of stain:non-specific staining for 0 score,a yellow staining intensity for 1 score,light brown for 2 score,brown for 3 score.To multipliy the two scores were denifinited as flow:0-1,negative(－),2 - 4,positive(+),more than 5 score,strongly positive(++).3.2 Rosette formation rate:Random field of vision from 10 the total number of cells,each cell-binding five or more microbubbles as a positive rosette formation,rosette formation rate calculation.4.Statistical analysisSPSS 13.0 package used for statistical analysis.The measurement data of the whole experiment was present with percentage.The Games Howell test was used to examine the normality of the value' distribution.A value of P＜0.05 was considered statistically significant.5.Results5.1 Preparation of targeted microbubbles MB_k2375.1.1 Biotinylated liposome contrast agents(MB_B):MB_B appearance was white, about the concentration of microbubbles(2-6.3)×10⁹ / mL,the average diameter (2.0 - 8.3)μm.Diameter of＜5μm microbubble was not less than 94.6%.200×optical microscope observation shows that microbubbles were well- distributed,no aggregation phenomenon,the appearance of a single spherical microbubble was well. MBb has a certain stability,4℃refrigerator can be stored for one month or more, and the form and concentration of microbubbles remain basically unchanged.5.1.2 Avidin - biotin - microbubble(Av-MB_B):â‘ Under fluoroscope:two microbubbles adding equivalent of FITC-Avdin were washed fully,MB shell without fluorescence,FITC-Avdin-MB_B shell with a bright green fluorescent.â‘¡Screening adaptive dose:The bingding rate of FITC-avidin in 5 groups of microbubbles has correlation with avidin dosage,the binding rate gradually increase with the avidin dosage increased until they reach saturation.5 groups microbubble binding rate:χ²=12.767,v=4,P=0.012＜0.05,there were statistically significant in 5 groups.Microbubble combination of avidin as a dose dependent manner,with the accession to avidin dose increase,combined with the rate gradually increased. Games-Howell multiple comparison results,0μg,2μg group were significantly different(P value of 0.000),6μg,10μg,30μg the' combination of the rate between the each two groups were no statistically significant(P=0.781,0.882,0.793). Demomtrate that Av- MB_B systems,for about 10⁷ MB_B,when the M_Av＜6μg when, with the avidin dose increase,combined with a gradual increase in rates,and rising faster binding rate curve;when the M_Av≥6μg,combined with a view to curve into the platform,increasing the dose of avidin,binding rate curve was no longer a significant upward trend.5.1.3 Targeted microbubbles(MB_K237) Preparation:â‘ targeting the physical properties of microbubbles:MB_k237 milky appearance was targeted,microbubbles concentration of about(3-8.4)×10⁸/mL,the average diameter(2.6～8.0)μm.Diameter of＜5μm microbubble of not less than 90.3%.200 optical microscope observation shows that microbubbles diameter distribution,no aggregation phenomenon,the appearance of a single spherical microbubbles. Fluorescence microscope,targeted microbubble shell inspired bright green fluorescence.â‘¡The bingding rate of FITC-Biotin-K237 in 7 groups of microbubbles has correlation with FITC-Biotin-K237 dosage,the binding rate gradually increase with the FITC-Biotin-K237 dosage increased until they reach saturation.MB_K237 binding rate of peptide results:M_AV=6μg,M_k237=(0μg,10μg,20μg,30μg,40μg,50μg,70μg, 100μg)/10⁸ microbubbles under the ratio of the dose in each group microbubble Connect the small peptides have significantly different rates(χ²=18.113,v=6,P =0.006＜0.05),it concluded that the 7 groups were significantly different;further multiple comparisons,among group 6,7,8 the multiple Comparison the differences were not statistically(P＜0.05),that when the M_k237≥50μg when connecting curve into the plateau.KDR adding high affinity of small peptide M_K237=50μg,can make 83.4600%±1.58218%percent of the microbuubbles'surface be marked fluorescent signals. 5.2 Identification of the results of in vitro5.2.1 Cell CultureLOVO cell growth strong,good shape,showed a typical fusiform,both ends of thin, uniform and transparent cytoplasm,with little change in time.HUVECs can be seen after vaccination 2h adherent,24h after the completely adherent,the cells were short spindle or polygonal,with close,like pebbles or "cobblestone"-like for a typical endothelial cell morphology.24h completely LS174T cell-adherent,spindle or polygonal cells,showing uneven distribution of clusters is the typical characteristic of the cells5.2.2 Immunohistochemical stainingLOVO cells was KDR strongly positive expression,positive positioning in the membrane material;HUVECs were positive expression,the positive material located in the cell membrane and cytoplasm;LS174T cells were KDR negative expression.5.2.3 Targeted contrast agent in vitro to identify:5.2.3.1 In vitro binding assay:â‘ Under a light microscope:around the LOVO cells we can see the targeted microbubble around the cells and formed the rosettes formation;under fluorescence microscopy the microbubbles issued bright green fluorescent,rosette formation rate up to 90.52%;around human umbilical vein endothelial cells(HUVE) the positive rosette formation rate of up to 53.46%;a small number micro-bubble adhesion around LS174T cells in the control group can be seen,rosette formation rate is 5.57%.â‘¡targeted microbubbles targeted LOVO cells with the combination of positive rosette test,rosette formation rate of 87.62%;small peptide - microbubble (K237-Mbs) were incubated with celis can be seen a little microbubble adhesion, rosette formation rate of 1.43%,relative microbubble targeting was significantly reduced;the surface of non-modified gaps biotinylated microbubbles were incubated with LOVO cells,rosette formation assay was negative.5.2.3.2 Blocking experiments,10μg closed group of target cells can be seen around a small number of targeted microbubble adhesion,50μg closed group of target cells around the basic non-targeted microbubble adhesion,rosette formation rate for the negative.5.2.3.3.Four doses of the program were targeted microbubble preparation,the effect of targeting among the for groups had no significant statistical difference(P＞0.05).5.2.3.4.Erosion rate of water flow in the next four LOVO cells and targeted microbubbles combined with the formation of rosettes formation structure gradually reduced.ConclusionTargeted microbubbles ultrasound contrast agent that carried peptide K237 be of high-affinity with KDR were constructed successfully in this study.The targeted microbubbles can made specific binding with KDR positive expression cells(LOVO cells,HUVEC_s) and had a certain stability,while no-specific binding with the KDR negative expression LS174T cells.There was hope for the realization of specific molecular imaging of tumor angiogenesis(such as:breast cancer) by targeting KDR, and laid the foundation for the next step experiments of targeted ultrasound molecular imaging in breast cancer transplanted in nude mice model...