【作者】 杨莉；

【导师】 顾宁；

【作者基本信息】 东南大学 ， 生物医学工程， 2022， 博士

【摘要】 微纳气泡是指直径从纳米到微米量级的气泡,与直径约为1-10 mm的大气泡结构相比,这种气泡具有一些特殊的物理、化学特性。其中,直径为1–10μm的生物医用微气泡通常由生物相容性良好、可生物降解的膜壳材料封装气体核心组成。微气泡由于气体和周围组织之间存在声阻抗差,可增加超声图像对比度而被用作超声造影剂(ultrasound contrast agent,UCA)。还可将诊断/治疗药物装载到微气泡膜壳上,实现多种诊断/治疗方法的联合。粒径小于1μm的纳米气泡(nanobubble,NB)可透过血管腔进入组织,实现靶向超声显影。由此可见,微纳气泡的粒径、膜壳成分等对其超声显影和其他成分装载具有重要的影响。本论文以微纳气泡的气液界面性能研究为重点,探究了微纳气泡的界面光散射特性、超声散射特性、界面分子组装及其在生物医学中的应用。首先,基于界面光散射,联合动态光散射图像法(image-based dynamic light scattering,IDLS)和可视化粒子追踪方法,实现了对自由微纳气泡在制备过程和稳定状态下的粒径的原位、实时测量。进而,基于界面超声散射特性,通过改进Rayleigh-Plesset模型研究了装载纳米颗粒(nanoparticles,NPs)的复合膜壳微泡的声动力学响应,证实了NPs对复合膜壳微泡振动幅值、谐波响应和散射截面的影响。最后,将可用于临床的荧光染料吲哚菁绿(indocyanine green,ICG)在自由NBs的气液界面上进行自组装,改善了自由ICG水溶液的不稳定和浓度聚集导致的荧光猝灭等缺点,增强其荧光特性,提高了对肿瘤的光动力治疗(photodynamic therapy,PDT)效果。具体研究内容包括以下几个部分:(1)结合可视化粒子追踪和IDLS技术,开发了一种新的粒径测量方法,实现对自由微纳气泡粒径的原位和实时测量。基于可视化方法,利用暗场显微镜(dark-field microscopy,DFM)成像,获得NBs的原位粒径分布。采用IDLS测量方法,实现了对NBs制备过程中的实时粒径监测。研究发现,利用反复压缩法压缩200次制备的微纳气泡,1 min内,其平均粒径由1202.4 nm下降到237.7nm,最终稳定在230 nm左右。稳定状态下,受到浮力作用,样品池中NBs的粒径呈纵向分布,上、中、下层的粒径分别为340.3,347.6和228.4 nm。与商用Zeta Sizer纳米粒子测量仪相比,该测量方法能够提供整个样品池中NBs粒径信息,为微纳气泡粒径的原位、实时测量提供了表征手段。(2)基于已有超声场中气泡散射特性的研究,考虑复合膜壳微泡对NPs装载能力及NPs的密度对复合膜壳微泡膜壳密度的影响,根据膜壳密度的不同,选取相应的超声参数,获得了优良的声学性能。实验中推导了复合膜壳微泡的Rayleigh-Plesset-like方程,在小振幅超声激励下,给出了该模型的解析解和数值解。快速傅立叶变换(fast fourier transform,FFT)频谱结果表明,膜壳密度能显著影响复合膜壳微泡的声学性能。一方面,膜壳密度对复合膜壳微泡谐波分量(次谐波和超谐波)的影响大于基波分量。次谐波和超谐波是微气泡稳态空化的典型表征,膜壳密度的增大有利于增强气泡的稳态空化效应。另一方面,膜壳密度对界面散射截面也表现出显著的影响。因此,应综合考虑膜壳密度的影响,采用接近于共振频率的超声激发以获得较大的复合膜壳微泡一次和二次谐波散射截面。由此可见,通过对气泡界面的膜壳密度优化,可获得优良的声学性能,为复合膜壳微泡在超声场中的动力学响应研究提供新的思路。(3)在气液界面性能研究的基础上,本论文进一步针对临床广泛应用的近红外荧光染料ICG水溶液稳定性差、浓度聚集导致荧光猝灭等缺点,将ICG分子通过反复压缩法组装到包裹六氟化硫气体的自由NBs界面层,制备出平均粒径约为244.6 nm的ICG-NBs。在气液界面上,ICG-NBs中的ICG分子的疏水端朝向NBs的疏水气核,而其亲水端与水分子结合。实验结果表明,ICG-NBs展现出良好的单分散性,优异的荧光特性和粒径稳定性。与游离的ICG溶液相比,ICG-NBs的荧光强度增强了约25%;在4℃放置4天后,ICG溶液的荧光强度下降至初始强度的35.8%,而ICG-NBs的荧光强度仍保持在初始值的82.4%。(4)最后,为提高ICG的PDT疗效,将自由ICG分子在自由纳米氧气泡(NBs-O2)的气液界面上自组装,制备得到ICG-NBs-O2水溶液。结果表明,ICGNBs-O2的单线态氧(singlet oxygen,1O2)量子产率明显提升,为自由ICG水溶液的8倍。同时,ICG-NBs-O2展现出更好的水溶液稳定性。在4℃放置4天后,ICG-NBs-O2的最大紫外吸收峰强度保持在初始值的64%,而自由ICG溶液下降到初始值的25%。在细胞层面,采用CCK-8细胞实验,研究不同浓度下ICGNBs-O2对Cal27人舌癌细胞存活率的影响。结果表明,在808 nm激光照射下,与自由ICG水溶液相比,ICG-NBs-O2在细胞内的1O2的产量增加。在裸鼠肿瘤模型上的结果也表明,ICG-NBs-O2表现出明显的肿瘤杀伤作用,相较于治疗前,ICG-NBs-O2治疗组的肿瘤体积减少至50%,而自由ICG治疗组的肿瘤体积增大至治疗前的2.4倍。与自由ICG水溶液相比,ICG-NBs-O2水溶液对肿瘤的PDT效果明显提升。更多还原

【Abstract】 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.更多还原