| The development of ultrasound contrast agents(UCAs)has greatly improved the accuracy of ultrasound diagnosis.However,current commercial UCAs are mostly in micron level,which are restricted imaging only in blood pool.Recently,imaging of peripheral lesions is of great expectance because of extavasation of nanoparticles based on enhanced penetration and retention effects.Therefore,the two main focuses involved with nano-size and high-capacity drug loaded are the current development on a new ultrasound diagnostic carrier.Thus,free nanobubbles are employed to prepare lipid encapsulated bubbles in this study based on the concept of self-assemble of lipid molecular on the interface of nanobubbles.We then explore their characters and theranostic applications.This study was opened from the following aspects:Sulfur hexafluoride free nanobubble water was prepared and characterized first,demonstrating the stability of nanobubble water,which paved a way for the subsequent incubation with membrane materials.Secondly,nanobubble water was incubated with different forms of lipids(dry-filmed and freeze dried)to study the probable reaction mechanism and its application.Thirdly,a new generation of IL-6/GP130 inhibitor bazedoxifene(Baze)was used as a model drug to study the combination effects of ultrasound and drug loaded gas-filled liposomes,made by gas nanobubble water,on the cancer cell viability.Results were shown as follows:(1)The inert sulfur hexafluoride gas was selected to prepare the sulfur hexafluoride nanobubble water using a gas-liquid mixed rotary pump.The stability of the nanobubble water was confirmed by means of optical observation,particle size analyzer,atomic force microscope and so on.Results showed nanobubble water size can be measured,the peak of size distribution was at 141 nm.NaCl and Baze could be absorbed at the interface of bubble to increase the particle size,which conferred the larger size,whose peak shifted at 190 and 199 nm,respectively.Dindal phenomenon could reveal that nanobubble maintained in 48 hours in nanobubble water.Thus,it could be concluded the existence and stability of nanobubble water.(2)Nanobubble water was then incubated with film-dried and freeze-dried lipids,respectively,and the obtained liposomes were characterized through microscopy,particle size,potential,and acoustic performances.Three aspects of variation in the system including phospholipid structure,concentration and theoretical calculation were to investigate and verify the incubation mechaaism.When incubation with filmed lipids,it was confirmed that nanobubble water could facility the dispersion of phospholipids,and the dispersion difference between in degassed water and nanobubble water was maximized at 5.7 hours.Combined with the results of transmission electron microscopy,the nano-sized and multi-layer encapsulated gas-filled liposomes were prepared by co-incubation of nanobubble water and phospholipids film.The calculated values of the layers were between 4 and 14.The particle size of liposomes was 194.4± 6.6 nm,PDI was 0.13 ± 0.02 and the zeta potential was-25.2 ± 1.9 mV.The content of gas was fixed,concentrations of lipid were different,which was found the group of 6.0 mg/mL possessed the highest acoustic quality.The nano-bubbles have ability to enhance ultrasound contrast in vitro,and the enhancement was related to gas/lipid ratio.The imaging features are divided into two modes:initial perfusion imaging and later accumulation imaging.When nanobubble water was incubated with freeze-dried lipid,the particle size of liposomes obtained was 302.9 ±15.9 nm,and PDI was 0.5 ± 0.1,both were lower than those obtained by mechanical agitation.In a short time about 5 min,duration after injection,the obtained lipid nanobubbles by nanobubble water have a similar ultrasound capacity with those obtained by mechanical agitation in vitro,no significant difference was observed between them.(3)Combination use of drug loaded gas-filled liposomes and ultrasound was investigated.Firstly,free Baze could inhibit downstream of JIL-6/GP130 protein STAT3,AKT and ERK phosphorylated expression,inhibit cell viabilities.Thus,Baze was evaluated as a IL-6/GP130 inhibitor in treatment of triple negative breast cancer cells.Secondly,Baze was loaded into the gas filled ultrasound sensitive liposomes.The particle size was191.8 ±0.02 nm and PDI was 0.13 ±0.02,without significant change between after drug loaded and before.After screened of the ultrasonic parameters,MDA-MB-231 cells treated with drug loaded gas-filled liposomes were exposed under 42.6 kPa and 60 s ultrasound,the cell viability was(61.46 ±2.61)%,which resulted in an increasing inhibition effect significantly(p<0.05)compared with that in group of non-ultrasound incitement in the same dosage,which was(114.70 ± 11.95%).Therefore,based on the absorption on nanointerface,Gas-filled Ultrasound-sensitive Liposome(GU-Liposome)with the features of nano-size,multilayer,and ultrasound sensitization,was developed in this study.which was father investigated in application in drug delivery combinating of ultrasound.GU-Liposome is expected to be used for the combined delivery of therapeutic gases and drug,multi-drug loaded and controllable release,which have a great promising in clinical ultrasound application. |
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