Interface Scattering Characteristics Of Micro-nano Bubbles And Their Applications In Fluorescence Enhancement And Photodynamic Therapy

Micro-nano bubbles refer to bubbles with diameters ranging from nanometers to micrometers.Compared with large bubbles with diameter of about 1-10 mm,this kind of bubbles has some special physical and chemical properties.Among them,biomedical microbubbles(MBs)with a diameter of 1-10 μm are usually composed of a gas core encapsulated by a shell with good biocompatibility and biodegradability.Microbubbles are used as ultrasound contrast agents(UCAs)due to the difference in acoustic impedance between the gas and surrounding tissues,which can enhance the contrast of ultrasound images.The diagnostic/therapeutic drugs can also be loaded onto the shell of MBs to realize the combination of multiple diagnostic/therapeutic methods.Nanobubbles(NBs)with size less than 1 μm can enter the tissue through the vascular cavity to achieve targeted ultrasound imaging.Therefore,the size of micro-nano bubbles and the composition of shell may have important effects on their ultrasonic imaging and other component loading.This thesis focuses the research on the gas-liquid interface performance of micronano bubbles,especially the interfacial light scattering characteristics,ultrasonic scattering characteristics,interfacial molecular assembly and its application in biomedicine of micro-nano bubbles.Firstly,based on interface light scattering,imagebased dynamic light scattering(IDLS)and visual particle tracking method are combined to realize the in-situ and real-time size measurement of free micro-nano bubbles during the preparation process and steady state.Furthermore,based on the ultrasonic scattering characteristics of the interface,the acoustic dynamic response of hybrid-shell microbubbles(hybrid MBs)loaded with nanoparticles(NPs)is studied based on the modified Rayleigh Plesset model.And the effects of NPs on the vibration amplitude,harmonic response and scattering cross section of hybrid MBs are confirmed.Finally,the clinical fluorescent dye indocyanine green(ICG)is self-assembled on the gas-liquid interface of free NBs,which has improved the instability of free ICG aqueous solution and reduced the fluorescence quenching caused by concentration aggregation,resulting in the fluorescence enhancement and improved photodynamic therapy(PDT)effect on tumors.The specific research content includes the following parts:(1)A novel size characteristics method is developed to achieve the in-situ and realtime size measurement of free micro-nano bubbles by combining visualization particle tracking with IDLS technology.Based on the visualization method,using dark-field microscopy(DFM)imaging,the in-situ size distribution of NBs is obtained.The IDLS measurement method is used to realize real-time size monitoring during the preparation of NBs.It is found that the average size of micro-nano bubbles compressed 200 times by repeated compression method decreases from 1202.4 nm to 237.7 nm within 1 min,and finally stabilizes to be about 230 nm.In the steady state,the size of NBs in the sample cell is longitudinally distributed due to buoyancy,and the sizes of NBs in the upper,middle and bottom positions are 340.3,347.6 and 228.4 nm,respectively.Compared with the commercial Zetasizer nanoparticle measuring instrument,this method can provide the size information of NBs in the whole sample cell as well as a characterization means for in-situ and real-time size measurement of micro-nano bubble.(2)Based on the existing research on bubble scattering characteristics in ultrasonic field,considering the loading capacity of hybrid MBs on NPs and the influence of NPs density on the shell density of hybrid MBs,the corresponding ultrasonic parameters are selected according to the different shell density,and excellent acoustic performance is obtained.In the experiment,the Rayleigh-Plesset-like equation of hybrid MBs is derived.Under small amplitude ultrasonic excitation,the analytical and numerical solutions to the model are obtained.The fast Fourier transform(FFT)spectrum results show that the shell density can significantly affect the acoustic performance of the hybrid MBs.On the one hand,the impact of the shell density on the harmonic components(subharmonic and ultraharmonic)of the hybrid MBs is greater than the fundamental component.Subharmonics and ultraharmonics are typical characteristics of stable cavitation of microbubbles.The increase of shell density is conducive to enhance the stable cavitation effect of bubbles.On the other hand,the shell density also shows a significant influence on the interface scattering cross section.Therefore,the influence of the shell density should be considered comprehensively,and the ultrasonic excitation frequency close to the resonant frequency should be used to obtain large first and second harmonic scattering cross sections of hybrid MBs.It can be seen that excellent acoustic performance can be obtained by optimizing the shell density at the bubble interface,which provides a new idea for the study of the dynamic response of hybrid MBs in ultrasonic field.(3)Based on the study of gas-liquid interface performance,the near-infrared fluorescent dye ICG,widely used in clinical practice,are assembled on the the interface layer of free sulfur hexafluoride gas NBs in order to overcome the shortcomings of ICG,which is such as poor stability and concentration aggregation leading to fluorescence quenching.Prepared by repeated compression method,the ICG-NBs have an average size about 244.6 nm.At the gas-liquid interface,the hydrophobic end of the ICG molecules in ICG-NBs faces the hydrophobic gas core of the NBs,and the hydrophilic end is combined with water molecules.Experimental results show that ICG-NBs exhibit good monodispersity,excellent fluorescence characteristics and size stability.Compared with the free ICG solution,the fluorescence intensity of ICG-NBs increases by about 25%;after being stored at 4°C for 4 days,the fluorescence intensity of the ICG solution drops to 35.8% of the initial intensity,while the fluorescence intensity of ICG-NBs remains at 82.4% of the initial value.(4)Finally,in order to improve the PDT effect of ICG,free ICG molecules are selfassembled on the gas-liquid interface of free oxygen NBs(NBs-O2)to prepare the ICGNBs-O2 aqueous solution.The results show that the singlet oxygen(1O2)quantum yield of ICG-NBs-O2 is significantly improved,which is 8 times that of the free ICG aqueous solution.At the same time,ICG-NBs-O2 exhibits better aqueous solution stability.After being stored at 4°C for 4 days,the maximum UV absorption peak intensity of ICGNBs-O2 remains at 64% of the initial value,while the free ICG solution drops to 25%of the initial value.The cell counting Kit-8(CCK-8)cell assay is used to study the effect of ICG-NBs-O2 at different concentrations on the survival rate of Cal27 human tongue cancer cells.The results show that under 808 nm laser irradiation,compared with free ICG aqueous solution,the production of ICG-NBs-O2 in cells is increased.The results on the nude mouse tumor model also show that ICG-NBs-O2 display obvious tumor killing effect.Compared with before treatment,the tumor volume in the ICG-NBs-O2 treatment group decreases to 50%,while the tumor volume in the free ICG treatment group increases to 2.4 times that before treatment.Compared with free ICG aqueous solution,ICG-NBs-O2 aqueous solution significantly improves the PDT effect of tumor.