| Ultrasound medical technology is widely used because of its advantages of safety,speed and low cost.In recent years,micro/nano bubbles as ultrasound contrast agents and drug carrier systems have shown broad application prospects in molecular imaging,drug delivery,mediated gene therapy and thrombolysis.Lipid-encapsulated bubbles have good biocompatibility,and currently approved ultrasound contrast agents,such as Definity,Imagent and Sono Vue,are lipid contrast agents.The lipid-encapsulated bubbles reported in literature are mostly micron-sized and limited to blood vessels;the relatively complicated preparations limit their clinical applications.With the rapid development of medicine and health technology,new demands are constantly being put on micro/nano bubbles with simple and efficient preparation and excellent contrast effects.Based on the preparation of free micro/nano bubbles,a simple and efficient preparation method of lipid ultrasound contrast agent was constructed by using the self-assembly of phospholipids at the bubble interface.Using this method,nanobubbles loading Xenon were prepared and tried for the treatment of ischemic stroke.The research is mainly divided into the following parts: First,a method for preparing free micro/nano bubbles by reciprocating pressure difference method is proposed.The formation,collapse and stability mechanism of the prepared free nanobubbles(NBs)were investigated.Secondly,a new preparation method for micron and nano sized lipid-encapsulated bubbles are constructed by self-assembly of phospholipids at the bubble interface.Thirdly,Xenon was encapsulated in the nanobubbles,and its neuroprotective effects in vitro and in vivo were studied to explore its potential application in the treatment of ischemic stroke.The following points are mainly involved in the dissertation:1.Free micro/nano bubbles were prepared by repeated differential pressure method,and the preparation parameters and stability of nanobubbles were further explored.The results show that microbubbles of about 10 to 50 μm are first formed.Then the microbubbles shrink and the presence of nanobubbles(NBs)is detected in the bulk.The NBs have an average size of 240 ± 9 nm,a PDI of 0.25,and a high negative zeta potential(-40 ± 2 m V).The concentration of NBs was positively correlated with the number of compressions.After repeated compression for 600 times,the concentration of NBs reached about 1.92×1010 /m L.Dynamic light scattering(DLS)and zeta potential indicate that NBs can be stabe for more than 48 hours.Total reflection infrared spectroscopy(ATR-FTIR)showed that the O-H stretching vibration peak of water moved toward the low wave number during the microbubble shrink,forming a stronger hydrogen bond.It is speculated that the stability of NBs is related to the strong hydrogen bonding at the bubble interface.2.Free micro/nano bubbles loading three different gas(Xenon,air and sulfur hexafluoride)was prepared,and the dynamic evolution of NBs was studied using in-situ real-time observation of dark field microscopy(DFM),in vitro ultrasound evaluation and free radical detection.The DFM results show that NBs are formed after the microbubbles shrink,and the microbubbles loading different gases have different shrinkage rates,which are related to the permeability coefficient of gas in water.However,there is no significant difference in the size and zeta potential of different gas NBs.The motion analysis shows passive Brownian motion of NBs.NBs are easily to collapse near the contact line of solid/liquid/gas,during which radiation force are generated that affect the movement of the surrounding bubbles,and hydroxyl radicals are detected by fluorescent probe.These results can be used to differentiate solid particles and droplets.Due to the little effect of inner gas on NBs,it is speculated that the hydrogen bond network of solvent water molecules at the NBs interface is a key factor for NBs stability.3.Based on the gas-liquid interface of free bubbles,temperature-regulated self-assembly of phospholipid molecules was used to prepare encapsulated bubbles.The assembly mechanism of lipids was studied in depth.In vitro and in vivo ultrasound contrast imaging of the bubbles were further investigated.Distearylphospholipidylcholine(DSPC)and poly(ethylene glycol-2000)-grafted distearoylphosphatidylethanolamine(DSPE-PEG2K)were directly dispersed in a buffer solution,heated to 60 °C and mixed with free bubbles,and then cooled to room temperature to obtain lipid-encapsulated bubbles.The bubble concentration was(2.06±0.9)×109 /m L,and can be stable within 5 hours.Further,the curvature of the membrane shell can be adjusted by changing the content of DSPE-PEG2 K in the phospholipid component,thereby adjusting the bubble size.Bubbles with size of 1.68±0.11 μm,704±7 nm and 208±6 nm were successfully prepared.In vitro ultrasound imaging showed good contrast t ability of these bubbles.As the bubble size decreases,the ultrasound imaging time is prolonged.Ultrasound imaging of the mouse liver showed that nano-sized bubbles have a longer imaging duration(up to 8 min)compared to microbubbles,and the contrast imaging area in the liver is enlarged,providing more detailed vascular information.4.Nanobubbles loading Xenon were prepared through lipid self-assembly at free bubble interface,and their neuroprotection during oxygen/glucose deprivation was investigated at cellular and animal levels.The average size of nanobubbles is 205±10 nm,and the total content of Xe is73±2 μL/m L.In vitro experiments show that xenon nanobubbles can restore the survival rate of PC12 cells after glucose and hypoxia treatment to no significant difference from the normal control group.In vivo experiments show that xenon nanobubbles can effectively treat ischemic stroke in mice,reduce the area of cerebral infarction,promote the recovery of nerve function,and continuously improve the recanalization of blood flow on the ischemic side.Immunofluorescence analysis showed that nanobubbles could penetrate blood vessels into nerve cells. |
