| Backgrounds and objectives:Ultrasound contrast agents(UCA)in deed is suspension of microbubbles.It brings marked improvement of ultrasonic diagnosis on the sensitivity and specificity. Nowadays,research on UCA is one of the hotspot in the ultrasonic medicine with rapid development.Great application potential of UCA has showed to us in the targeted imaging and therapy,along with the progress of other molecular imaging.Gene transfer technology,as a key procedure,hindered the development of gene therapy owing to its safety,efficiency and targeting problems.Viruses are attractive delivery vectors because of their ability to efficiently transfer genes with sustained expression.However,this very same attribute also serves as one of the greatest drawbacks due to the immunogenicity and potential for insertional mutagenesis. Non-virus delivery vectors are safe and convenient,but the low transfer efficiency can not meet the requirements of therapy.Liposomes and microbubbles are often used as non-virus delivery vector.It was proved that sonification of liposomes and microbubbles can raise the gene transfer efficiency.However,the efficiency and methods should be optimized further.Second generation UCA contain high-molecular weight gas with less solubility and diffusivity,which improves microbubble persistence to surviving for several minutes within the bloodstream,and micron size allows trans-pulmonary passage and peripheral vein injection.The tissue blood perfusion information can be captured by detecting the ultrasonic signal of microbubbles.Commercially available UCA,i.e. SonoVue?,were utilized in clinic generally.Third generation UCA are characterized by the conjugation of specific ligand to the surface of microbubbles,loading drugs or gene in microbubbles to realize the targeted imaging and therapy.The ideal microbubble may accomplish targeted imaging and therapy simultaneously with great specificity and accuracy.In order to acquire good enhancement effect the microbubble diameter most likely is micron degree,yet hard to penetrate the capillary wall and carry the load to tumor parenchyma.The conflict between penetration ability and enhancement effect can be solved by the combination of micron-bubble and nano-liposome small enough to penetrate the capillary,the former served as provider of targeted image and the latter carrier of drugs or gene.Ultrasound-targeted microbubble destruction will release the nano-liposome to lesions as they reach and bind to the target through blood circulation.In this research,we try to build the schema of combination between commercially available UCA SonoVue?and self-made liposomes,and determine the efficiency;establish the foundation for further research of liposome-loaded microbubbles as targeted contrast agents and delivery vehicle in vivo,even in clinic.Material and methods:1.The common properties of SonoVue?microbubbles were observed. Microbubbles was labeled by carbocyanines dye DiI,and then observed them under Fluorescence Microscope.The effect of DiI labelling was determined by Flow Cytometer.2.The insertion of DSPE-PEG(2000)Amine into the lipid shell of SonoVue? microbubbles was determined.The effect of DSPE-PEG-FITC labelling was determined by Flow Cytometer to evaluate the insertion efficiency of the amphipathic molecule DSPE-PEG(2000)Amine into lipid monomers shell.So the post-formation modification methods for lipid microbubbles were established,which provided a linker for microbubbles to other bioactive molecules.The target effect of microbubbles after post-formation modification was evaluated by determining the binding of biotinylated-microbubble to avidin-coated polystyrene cell culture dishes.3.The combination of SonoVue?microbubbles and liposomes:Aminated fluorescent liposomes were preparation by rotary evaporation-sonification.Laser Particle Size Analyzer determined the liposome size.Liposomes and microbubbles were combined by two-step glutaraldehyde crosslinking.After floation,we got the complex of SonoVue?conjugated to liposomes.4.The impact of liposome concentration on the construction efficiency of the complex of SonoVue?conjugated to liposomes by Flow Cytometer.150μL DSPE-PEG(2000)Amine solution(100μM)was added into 200μL SonoVue suspension per tube,and labeled by DSPE-PEG-FITC,let SonoVue?microbubbles become aminated fluorescent microbubbles.0μL～300μL glutaral-liposome(DiI labeling)solution were added into each tube respectively.Flow Cytometer determined the FITC and DiI double-fluorescence positive rate.And then we drew and analyzed the curve.5.The impact of DSPE-PEG(2000)Amine concentration on the construction efficiency of the complex of SonoVue?conjugated to liposomes by Multifunctional ELIASA.0～250μL DSPE-PEG(2000)Amine solution(100μM)were added into 200μL SonoVue suspension per tube,and then 200μL glutaral-liposome(DiI labeling) solution were added,thus we got the complex of SonoVue?conjugated to liposomes with difference concentration of DSPE-PEG(2000)Amine.Multifunctional ELIASA determined the DiI fluorescence intensity.According the fluorescence intensity and standard curve,we calculated the carry efficiency of liposomes by SonoVue? microbubbles.6.Determining the time-intensity of microbubbles under the mode of CPS to evaluate the influence of microbubbles amination and the conjugation of liposomes to microbubbles.7.The effects of the complex of SonoVue?conjugated to liposomes contacted with MCF-7 and SMMC-7721 cell lines were observation.After sonification, fluorescent signal within the cells were determined,and compared with the group that cells were cultured with liposomes only or microbubbles only. Results:1.The common configuration of SonoVue?microbubbles showed fine.DiI labeling succeeded with(92.31±3.62)%of fluorescent positive microbubbles,as the DiI concentration was 5μM.2.20μL/mL was the optimal concentration of DSPE-PEG-FITC(100μM),with (94.7±6.4)%positive rate of fluorescence on microbubbles showed as Flow Cytometer.More biotinylated-microbubbles stayed on the avidin-coated polystyrene cell culture dishes than non-biotinylated microbubbles after cleansing.3.The fluorescent liposomes were prepared by rotary evaporation-sonification successfully with mean peak diameter as 272.6nm.The combination between liposomes and microbubbles was realized by glutaraldehyde crosslinking.4.The Flow Cytometer showed that 200μL liposomes solution per 200μL microbubbles suspension was the optimal concentration,with the peak positive rate as (87.80±5.91)%.5.Multifunctional ELIASA showed that 150μL DSPE-PEG(2000)Amine (100μM)solution per 200μL microbubbles suspension was the optimal concentration, with the peak carry efficiency as(83.41±2.21)%.6.The change of the time-intensity of microbubbles under the mode of CPS showed that the difference between original microbubbles and aminated microbubbles was not significant statistically(P＞0.05),the conjugation of liposomes to microbubbles fasten the decrease of sound intensity,the difference was significant statistically(P＜0.05).7.Fluorescent signal within the cells were more intensive and strong in the group of MCF-7 and SMMC-7721 cell lines contacted with the complex of SonoVue? conjugated to liposomes after sonification,compared with the group that cells were cultured with liposomes only or microbubbles only. Conclusions:We could realize the amination of commercially available UCA SonoVue?by the insertion of DSPE-PEG(2000)Amine molecule.Liposomes,as cartier of drugs or gene,can get amino easily by selection of molecular materials.Glutaral crosslinking the liposomes and microbubbles is feasible.Moreover,we could choose other active groups to modify the microbubbles and liposomes,construct more convenient methods with higher efficiency.Usually we need varied microbubbles with different targeted molecules or drugs, as means of imaging or delivery vehicle,to meet different diseases requirements.In this research,we showed that through post-formation modification of microbubbles with different targeted molecules or drugs,we only need one kind of microbubbles enough to accomplish the task.It simplifies the procedure remarkably and fulfil the application purpose. |
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