Preparation Of Targeted Lipid Nano Ultrasound Contrast Agents With HSV-TK Suicide Gene And Its Target-finding Experiments In Vitro

Objective:This study aims to prepare a lipid nano-ultrasound contrast agents that targets human epidermal growth factor receptor 2(HER2)and carries the herpes simplex virus thymidine kinase(HSV-TK)gene and has a nuclear localization effect.The physical and chemical properties of the ultrasound contrast agent were characterized,and its ability to target HER2-positive gastric cancer cells(NCI-N87 cells)in vitro and its ultrasound imaging effect in vitro were explored.Methods: According to the requirements of Tiangen high-purity granules small medium-volume kit to extract the prosurgery HSV-TK,using the pre-task group to build the arginoid-based SS-PAAs(polyaminoamines containing dithione bonds)class of cation gene vectors in the nihminoid base AGM-CBA,a polymer product of butamine monosomy(AGM)and N,N,N’-biacrylamide(CBA),prepares the HSV-TK/AGM-CBA gene carrier complex and determines its particle size.Using thin-film hydration-mechanical oscillation,di palmyl phosphatidylcholine(DPPC),biotinized diosteryl phosphatidyl ethanolamine(biotinized-DSPE-PEG2000),cholesterol(CH)is dissolved in aqueous ethanol in accordance with a certain proportion,evaporated into fat by rotating evaporator The membrane,which dissolves and dissociates the lipid membrane with a hydrated solution of the gene carrier complex,obtains the biotinized lipid body,and then adds liquid fluorocarbon perfluorohexane to it one by one,oscillating through an ultrasonic oscillator to obtain ultrasound contrast agents(Ultrasound contrast agents,UCAs).Using the bridge-affine-biotin method,the biotinized HER2 antibody is connected with the biotin liposome with the help of affinity,i.e.,the target nano-lipid microboules UCAs with HER2 as the target are obtained.The appearance form,particle size,and likeness of UCAs are indicated by scanning electron microscope and Malvern laser particle size meter.The effect of nuclear positioning and its in-body targeting ability of HER2-positive stomach cancer cells were observed by laser confocus microscope.Use a flow cytometer to quantitatively detect the connection between HER2 antibodies and lipid nanosound imaging agents.Finally,the ultrasound effect of target lipid nano-imaging agent was observed by Doppler color ultrasound instrument.Results: Malvern laser particle size meter measured the average particle size of the gene carrier complex(96.73±1.27)nm,the prepared nanolipid UCAs size is the same,the average particle size is(550.93±10.19)nm,Zeta potential is(-9.75±0.74)m V.The UCAs are observed under a scanning electron microscope as a relatively well-rounded icing.Flow cytometer tests showed that HER2 antibodies were successfully connected to lipid nano UCAs,and the laser concentration microscope showed that HSV-TK gene-targeted lipid nano UCAs carrying fluorescent dye markers were able to gather on the nuclei of cells,showing excellent nuclear positioning effects and targeting her2-positive stomach cancer cells.In-body ultrasound imaging showed better ultrasonic contrast enhancement,and nano UCAs showed fine and uniform dot-like dense high echo.Conclusions:In this experiment,the nano-ultrasound contrast agent with HER2 targeted HSV-TK gene and nuclear localization effect was successfully prepared,and the particle size was uniform,the shape was good.In vitro it can be specific to HER2-positive gastric cancer(NCI-N87)cells,and the ultrasound imager in vitro ultrasound development effect is ideal,this study provides a preliminary basis for the subsequent cell killing experiments and the study of tumor targeted contrast-enhanced ultrasound in animals.