Preparation Of Shear-stress Sensitive Liposomes And Evaluation Of Thrombosis Diagnosis

Thrombosis is a common chronic disease,which is closely related to various cardiovascular disease,have been the most common cause of death in the world.Traditional vascular contrast agent includes iodinated contrast material,gadolinium contrast material and so on,but it may lead to allergic reations and cause kidney damage with significant dose of vascular contrast agent,which limited the application and also affect the diagnosis of thrombosis.If we enhance the targeting ability of vascular contrast agent,we could reduce the dose while highlight the diagnosis effect of thrombosis.Liposomes are common and well-established drug delivery systems and are formed spontaneously when amphiphatic phospholipids contact water and align into mechanically stable bilayers.Through some synthenic modifications,different chemical structures can lead to liposomes with outstanding nature,such as p H-sensitive liposomes,temperature-sensitive liposomes,enzymessensitive liposomes,ultrasound-sensitive liposomes,photochemistry-sensitive liposomes,redox-sensitive liposomes and so on.With the development of medical,molecular biology technology and materialogy,it was found that platelets are elliptical and have shear-stress sensitivity,which change they shape then are locally activated by high shear stress.It has been concerned that the shear-stress sensitive drug delivery system made by proper materials can effectively enhance the drug release specificity.In this research,calcein or Di I as a model drug,a kind of shear-stress sensitive drug delivery system,i.e.shear-stress sensitive liposomes,were designed and prepared by synthetized fully functional phospholipids,i.e.Pad-PC-Pad.The delivery system with shear-stress sensitivity made by Pad-PC-Pad released contents to emit fluorescence,which provided the valuable reference for the design of function carriers for the diagnosis of thrombosis.In the first chapter of this research,Pad-PC-Pad were synthetized fully,and the intermediate products and target products were characterized by TLC,1H NMR and MS.Firstly,Boc group and ethanolamine were covalently linked,characterized by TLC results,the Boc group protected ethanolamine was synthesized successfully.Secondly,the Boc group protected ethanolamine,1,3-dichloropropanol and POCl3 were covalently linked,characterized by TLC and 1H NMR spectra,the compound 3 was synthesized successfully.Helium atoms of the compound 3 substituted by azide group,hydrogenation products and palmitoyl chloride were covalently linked,characterized by TLC,1H NMR and MS spectra,the compound 6 was synthesized successfully.Then the target products prepared from the compound 6 through deprotection and methylation,characterized by 1H NMR and MS spectra,Pad-PC-Pad was synthesized successfully.In the second chapter of this research,SSSL and TL were prepared by filming-rehydration method,treated PC-98 T and Pad-PC-Pad as film material,meanwhile we filtered the hydration temperature,sonication time and power while evaluate physicochemical property and stability of TL and SSSL.The screening results of SSSL preparation showed that hydration at 60℃,probe ultrasonic(50W,work for 2s,pause for 2s)for 5 times was optimized preparation condition,the particle size of SSSL was uniform and stable under that preparation condition.TL and SSSL showed particle size of(105.5±3.8)nm and(106.9±1.2)nm respectively,the zeta potential were(-12.5 ± 1.5)m V and(+14.6 ± 2.1)mV.The TEM morphology of TL and SSSL were spheroidal and elliptical respectively,and distributed uniformly.Placed at room temperature for a week,the particle size of TL and SSSL have no change and obvious aggregation,there was no significant difference between the two groups.With the extension of resting time,the release rate of TL and SSSL both had a small increase but no more than 5%,which indicated that TL and SSSL both were stable.In the third chapter of this research,the sensitivity of TL and SSSL were evaluated.Firstly,the TL and SSSL were prepared by filming-rehydration method,which loaded calcein with aggregation-caused quenching(ACQ)characteristics to evaluate shear-stress sensitivity of liposomes at different temperature under different ultrasonic power.The results indicated the release rate of TL and SSSL both relate to temperature,high release rate corresponded to higher temperature.But at the same condition,the release rate of SSSL was much higher than TL.When at 37℃,the release rate of TL was about 20% but SSSL reach up to 65%.The shear-stress sensitivity also related to the content of Pad-PC-Pad,shear-stress sensitivity was higher with higher content of Pad-PC-Pad.The release behavior of liposomes in obstructive region was investigated using in model system with local constriction,the results showed that the release of TL increase slightly while the release of SSSL increased remarkably in the obstructive region,which indicated that SSSL is shear-stress sensitive.In the fourth chapter of this research,HUVEC cell used as the cell model,we studied the cytotoxicity and cellular uptake of TL and SSSL before and after high shear stress via fluorescent probe.Cytotoxicity test showed that TL and SSSL had no obvious effect on cell survival rates,which indicated TL and SSSL is biological safety.The results of cellular uptake showed the intracellular fluorescence intensity of A-SSSL was higher than other groups,which indicated cellular uptake ability about SSSL after high shear stress is much stronger than other three groups.In the fifth chapter of this research,FeCl3 method leaded to mesenteric artery obstruction locally,the male KM mice were used as animal model to evaluate the capacity of diagnosis of thrombosis of TL and SSSL.After thrombosis models established successfully,the fluorescent intensity was observed under microscope.The results showed SSSL after high shear stress was higher than TL,which indicated that SSSL can be applied to diagnosis of thrombosis.Above all,we synthesized functional phospholipids Pad-PC-Pad,the liposomes made by Pad-PC-Pad were shear-stress sensitive and could be applied to diagnosis of thrombosis effectively.