Study On PH-Responsive In Situ Gas-Generating Targeted Ultrasound Contraet Agents For Ultrasound Imaging

Contrast-enhanced ultrasound（US）is a widely used imaging modality for tumor diagnosis and treatment.Currently used ultrasound contrast agents（UCAs）have limitations such as single function,poor stability and lack of targeting,which limit their application.Targeted UCAs can accumulate and enhance the ultrasound signal in tumor tissue,in which case,it can expand the dimension of tumor ultrasound imaging at the cellular level.Hence,based on the characteristics of the tumor microenvironment and the chemical properties of the materials,we prepared pH-responsive functionalized calcium carbonate nanocomposite particles（ICG@CaCO₃-PEG/FA NPs）,with load of indocyanine green（ICG,a near-infrared photosensitizer）.The NPs were then modified with polyethylene glycol（PEG）and folic acid（FA）to fabricate the contrast ultrasound system with long-circulating property and targeting ability.The main research work includes:1.Based on the characteristics of the tumor microenvironment and the physicochemical properties of CaCO₃,we designed a pH-responsive targeted UCAs.First,functionalized CaCO₃-PEG/FA NPs modified with FA and PEG were prepared;Rod-shaped CaCO₃ NPs were prepared by secondary growth in Na₂CO₃ solution using polyacrylic acid（PAA）as a template to control the reaction of Ca²⁺and CO₃^2-;Coating polyvinylamine（PVAm）by electrostatic interaction and glutaraldehyde cross-linking to introduce the amino reactive sites onto the surface of CaCO₃ NPs;To prepare CaCO₃-PEG/FA NPs,PEG and FA was attached onto the surface of NPs by ring‐opening and amidation reaction.2.The ultrasound imaging and in situ gas generation of CaCO₃-PEG/FA NPs was investigated by simulating the tumor acid microenvironment in vitro.pH-dependent gas generation experiment showed that the decomposition gas generation of CaCO₃-PEG/FA NPs has obvious acid responsiveness.Based on the characteristics of hyperechoic,CO₂microbubbles can be used as ultrasound contrast agent for tumor tissue.The agar model was designed to simulate tumor tissue,and the experimental results showed that NPs has acid-responsive ultrasound imaging.Cellular targeting analysis showed that FA-modified CaCO₃-PEG/FA NPs have tumor targeting.3.The light responsiveness of ICG@CaCO₃-PEG/FA NPs loaded with ICG was investigated.The experimental results showed that ICG@CaCO₃-PEG/FA NPs could efficiently generate reactive oxygen species（ROS）and have good photothermal ability under 808 nm NIR;The results showed that FA-modified ICG@CaCO₃-PEG/FA NPs were specifically recognized and taken up by MCF-7 cells via FR receptor-mediated endocytosis,which could generate ROS in MCF-7 cells and cytotoxicity through light stimulation;The contrast-enhanced ultrasound of the ICG@CaCO₃-PEG/FA NPs can guide the phototherapy of the tumor and may realize the integration of tumor diagnosis and treatment.