| Part One Exploration of Ultrasound Contrast Agent ApplicationsObjective:By collecting ultrasound contrast cases,we explore the application of ultrasound contrast agents and provide a basis for the populari-zation of the clinical application of contrast agents and subsequent research.Methods:Patients who attended the Second Hospital of Hebei Medical University from January to December 2020 who underwent Contrast-enhan-ced ultrasound were collected,and basic patient information was recorded.Patients were subgrouped into disease groups according to the examination site in order to observe the value of contrast agent Sono Vue application in different sites.The contrast agent application dose was different according to the patient’s examination site,patient’s weight,etc.Subcontrast observation of the lesion was performed at the beginning of contrast injection,30 seconds,60 seconds and 2 minutes after contrast injection,and the contrast images were retained.The patients’adverse reactions were observed and recorded.Results:A total of 135 patients were collected from January to Decem-ber 2020,the mean age is 56 years.There were 89 males and 46 females.Among them,55 were liver imaging,19 were renal imaging,18 were cholecy-stography,30 were prostate imaging,6 were mammography,and 7 were superficial soft tissue imaging.Among the cases collected in this experiment,31(23.0%)were liver occupations,19(14.1%)were kidney occupations,18(13.3%)were gallbladder lesions,30(22.2%)were prostate occupations,7(5.2%)were superficial soft tissue occupations,6(4.4%)were breast occupa-tions,2(1.5%)were pancreatic occupations,1(0.7%)was bile duct occupa-tions,and 0.7%,and 21 other patients,accounting for 15.6%.Among the examined patients,3 had mild adverse reactions,all of which were mild pain at the injection site;no serious adverse reactions occurred during the exami-nation.The ultrasound contrast agent Sono Vue was better for the observation of abdominal organs such as liver.During the mammography examination,the amount of contrast agent was large,but the ultrasound contrast effect was poor.Conclusions:Although ultrasound contrast agents have been used in a variety of clinical conditions,further research is needed to explore the contrast agents that be used in the breast.Part Two Preparation and screening of ultrasound contrast agentsObjective:To explore the conditions and methods of ultrasound contrast preparation to provide a basis for breast ultrasound contrast preparation and subsequent low intensity ultrasound stimulated breast cancer radiation therapy research.Methods:Based on seven formulations,the lipid suspensions were pre-pared by thin film hydration and stirred dissolution methods,respectively.The vesicle size of the lipid suspensions was measured with a Zetasizer Nano ZS90 analyzer,and the screening of materials,solvents,preparation methods,sonication time and other conditions were carried out to optimize the resulting lipid suspensions as micro-nano-dispersion systems.Subsequently,gas encap-sulation was performed,and the prepared micro-nano vesicles were adminis-tered to BALB/C mice to observe and record the imaging effect of the pre-pared micro-nano vesicles.After performing imaging comparison and quanti-tative analysis,we obtained a formulation with good contrast effect,reprodu-cibility and stability.Results:A total of 34 different combinations of conditions were perfor-med based on 7 of the formulations.The results showed that the system pre-pared by formulation 4 had better reproducibility and stability,as well as a more desirable contrast effect.We used Graph Pad Prism to visualize the para-meter data and obtained the particle size distribution values for the better ima-ging group:Pk1,252.0±175.2nm;Pk2,1861.0±1598.0nm;Pk3,543.1±1540.0nm;Pk1 Area,72.5±23.0%;Pk2 Area 18.7±14.0%;Pk3 Area,5.6±6.9%;PDI,0.53±0.19;Intensity,507.6±118.9 nm;and Z-Ave,148.5±11.5nm.Conclusions:Through prescription screening and process optimization,ideal formulations and operating conditions for the preparation of ultrasound contrast agents applied to the breast were obtained,providing a basis for subsequent exploratory studies of low-intensity ultrasound stimulated breast cancer radiotherapy.The resulting contrast agents are expected to play an active role in ultrasound molecular imaging applications.Part Three The exploration of the structure-echogenicity relationship of ultrasound contrast agents based on neural network modelObjectives:The correlation of each parameter of ultrasound contrast preparation process was explored to provide guidance for subsequent ultra-sound contrast preparation and research.Methods:A neural network-based model was applied to explore the correlation between the structure and echogenicity of ultrasound contrast agents.In this study,seven different formulations were screened 98 times,38of them to obtain lipid suspensions.Measurements were performed by dyna-mic light scattering using a Zetasizer Nano ZS 90 analyzer,and a total of 109measurements were obtained.The size distribution of UCA and its imaging under different production conditions were recorded.We applied Graph Pad Prism for data visualization and applied neural network models to explore the patterns.Results:The ANN-MLP model was applied to analyze the data,and the original data were divided into training and testing groups,which included 73training groups and 36 testing groups.The model has three hidden layers including bias.In the ANN-MLP model,the classification plot shows that the prediction values for the training and testing groups are 76.7%and 66.7%,respectively.The ROC curve shows that the metrics included in different imaging effects can achieve a prediction rate of 88.1～96.5%.The percentage plot shows a gradual convergence of the data.The prediction analysis curves show that the different ultrasound imaging effects gradually approached stable values.The predictive importance contributions of each index obtained from the ANN-MLP model analysis were as follows:Pk1,100%;PDI,98.5%;intensity,89.7%;DSPE-PEG-2000,88.9%;and Pk1 Area,85.8%.Conclusions:We applied the ANN-MLP model combined with Graph-Pad Prism to predict the contrast effects of different contrast agents in mice and verified that our model formulations were successful.These key para-meters can be used to improve the success rate of future contrast agents pre-paration.Part Four Application of low-intensity ultrasound stimulated oxygen microbubbles in breast cancer radiosensitivityObjectives:An exploratory study of the effects of applying low-intensity ultrasound to stimulate oxygen microbubbles for radiation therapy.Methods:In the experiment,4T1-Luc cells were selected for in situ mammary tumor modeling in BALB/C mice.Seven-week-old female BALB/C mice were selected to be inoculated with tumor cells at the fat pad of the right fourth mammary gland.The enrollment criteria were met when the maximum tumor diameter reached 8-10 mm or the tumor volume was≥200mm~3.The enrolled mice were randomly divided into three groups according to the treatment modality:radiation therapy group(XRT;8Gy),low-intensity ultrasound stimulation microbubble destruction oxygenated microbubble group(LF-USMB VFLow)+radiation therapy(XRT;8Gy)and control group.After the preparation of ultrasound contrast lipid suspension according to the method in Part I,the contrast agent inclusion gas treatment was per-formed to produce oxygenated fluorocarbon containing lipid microbubbles for backup.The mice in each treatment group were treated.Before treatment and1-10 days after treatment,2D images,size measurement and blood flow observation were performed on the tumors,and the body weight of the mice were measured and recorded to observe the general condition of the mice;before treatment and at the end of treatment,Contrast-enhanced ultrasound was performed on the tumors,and the maximum length and width of the tumors,the body weight of the mice and the PI and AUC values of the ultra-sonography were recorded.The mice were executed by cervical dislocation method after anesthesia.The tumors of mice were routinely paraffin-embed-ded filmed and observed by light microscopy.Immunohistochemistry was performed,and the tumor vascular marker CD34 responded to tumor micro-vessel density;Ki-67 assay responded to intra-tumor cell proliferation ability.Results:The BALB/C mouse mammary gland carcinoma in situ model was successfully constructed.The experimental results showed that there were differences in the PI values and AUC values of imaging quantitative indexes in the radiation treatment group and radiation treatment with oxygen micro-bubble cavitation treatment group compared with the control group,and the differ-ences were statistically significant.The tumor HE staining,immunohis-tochemical detection,CD34 and Ki-67 expression in the mice of each treatment group before and after treatment:control group>radiotherapy group>radiotherapy with cavitation treatment group.There was no statistical difference in the body weight of mice in each treatment group.The tumor volume changes of mice in each group during the treatment period were diffe-rent in the radiotherapy with cavitation treatment group compared with the control group,and the differences were statistically significant;there were differences in the radiation treatment group compared with the control group,but the differences were not statistically significant.Conclusions:Low-intensity ultrasound stimulation of oxygen micro-bubbles can increase the sensitivity of tumors to radiation therapy.Contrast-enhanced ultrasound quantitative indexes,PI and AUC values,can reflect microvascular density changes before and after tumor treatment.Contrast-enhanced ultrasound can be used as an effective means to assess the effect of radiation therapy,and is expected to provide an earlier evaluation of the response to tumor treatment. |
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