Ultrasound Regulation Of Platelet Bioactivity And Nanoparticles Loading

As circulating sentinels,platelets have played a key role in physiological and pathological processes of hemostasis,wound healing,inflammatory response,thrombosis and organ transplant rejection.In recent years,both the various micro-nano drug delivery systems to target the activated platelets and the micro-nano carriers encapsulated by natural platelets and platelet membranes have shown great potential in the diagnosis and treatment of diseases such as thrombosis and tumors.In this thesis,in order to make use of platelets as the natural drug delivery carriers,we investigated the interaction between natural platelets and ultrasonic energy treatment.Further,platelets loading small molecular components and nanoparticles were performed based on the mechanism of cell membrane permeability enhancement by the regulation of ultrasonic energy.Nanoparticle-loaded platelets retained the biological activity of natural platelets and can be further applied to the dark-field,photoacoustic bioimaging.The main research contents of this thesis are as follows:Firstly,the effects of ultrasonic energy?ultrasound frequency,intensity,and duration of action?on platelet bioactivity were investigated.The response of platelets to ultrasonic energy was studied by the platelet aggregation ability and P-selectin?CD62P?expression on platelet membrane.The results showed that low-frequency ultrasound?45 kHz,80 kHz,100 kHz?had no effect on platelet aggregation.The ultrasonic energy of 1 MHz and 0.25 W/cm² had no influence on platelet aggregation,platelet surface morphology and release of platelet-associated particle.When the ultrasonic intensity was more than 0.25 W/cm²,the aggregation ability,morphology and particle release of platelets were changed.The ultrasonic energy of 0.25 W/cm² and 60 s had no effect on the bioactivity of platelets.When the ultrasonic time was more than 60 s,the aggregation ability,surface morphology and particle release of platelets were changed except the secretion of CD62P by platelet?particles.When the ultrasonic parameters were 1 MHz,0.25 W/cm² and 60 s,the protein content of platelets was also unaffected.Therefore,the parameter of 1 MHz,0.25 W/cm² and 60 s was chosen as the optimized condition for the ultrasound treatment in the following experiments.Secondly,the thesis explored the ultrasonic energy that promote the entry of small molecules such as chloroauric acid and reducing agent?NaBH₄ and sodium citrate?into platelets.The gold nanoparticles?GNPs?were synthesized in situ assisted by intra-platelet reductase and reducing agent under the condition of optimizing ultrasonic energy parameters?1 MHz,0.25 W/cm²,60 s?.The results showed that the permeability of platelet membranes were increased under the ultrasonic energy exposure.GNPs about 5 nm were rapidly synthesized in situ?30 min?and uniformly distributed in the cytoplasm(Au synthesis amount:?12.7±2.4?×10^-3 pg/cell).This kind of GNPs-platelets had the Raman enhancement,dark field imaging and computed tomography?CT?imaging effect.Finally,the thesis further studied the interaction between GNPs and platelets by the regulation of ultrasonic energy.The results showed that GNPs of 36 nm could be rapidly loaded?30 min?into platelets due to the mechanism of endocytosis/open canalicular system?OCS?and sonoporation triggered by an ultrasound energy of 0.25 W/cm²?1MHz,60 s?.Compared to the method of the traditional GNPs co-incubation,the loaded GNPs were increased by 32.1%after the ultrasound energy treatment.And this kind of GNPs-platelets had the effect of photoacoustic and dark field imaging.In addition,the study also found that?-ferumoxytol coated with polyglycoside carboxymethyl ether could not be loaded into the platelets,indicating that different surface modifications of the nanoparticles would affect the loading efficiency.In this thesis,the methods of ultrasound assisted platelets in situ synthesis and the direct loading GNPs were not only maintained the natural activity and biological functions of platelets but also were prompted to load the nanoparticles.The GNPs-platelets biomimetic multifunctional carrier could realize the wide applications of natural high-targeting,multi-modal biological imaging,and had great application potential in the diagnosis and treatment of platelet-related diseases.