| Background&ObjectiveAs the most commonly used method in medical imaging diagnosis,ultrasound diagnosis technology is a technology that uses the intensity difference of reflected and scattered signals produced by ultrasound at the human tissue interface to display the normal and abnormal information of the structure and function of the intermal tissues and organs of organisms,so as to achieve the purpose of disease diagnosis.Contrast-enhanced ultrasonography(CEUS)uses substances that are significantly different from the echo characteristics of human soft tissues or acoustic impedance to inject into the body cavity,pipeline or blood vessel to enhance the display of viscera or lesions,as well as the information of blood perfusion,so as to improve the accuracy,accuracy and sensitivity of diagnosis.Conventional ultrasound contrast agents are usually gas-encapsulated microbubbles,with a diameter of 1-8 um,injected into blood vessels or lumens,which produce liquid-gas interface with great difference in acoustic impedance characteristics and significantly enhance the backscattering intensity.In addition,their elastic shell can produce abundant second harmonic signals.Selective reception of second harmonic by contrast harmonic imaging technology can significantly improve the signal-to-noise ratio of signals.It improves the quality of contrast enhanced image.Compared with CT,MRI and other detection techniques,ultrasound microbubbles have many advantages in perfusion detection and imaging of human microvessels and tissues,such as better imaging effect,less real-time high frame-frequency artifacts,no radiation damage,relatively cheap,wide application and easy bedside examination.It provides a new idea for searching for an effective,safe and highly targeted cancer treatment method,and provides a basis for the study of targeted,localized and controlled release technology based on ultrasound microbubbles.According to the different preparation materials and methods,the development of ultrasound microbubble contrast agents has experienced the first generation of free microbubble ultrasound contrast agents,the second generation of air-encapsulated ultrasound contrast agents,and the third generation microbubble contrast agents represented by SonoVue(SonoVue)of Bracco Company,which contain low-solubility and low-dispersibility gases,such as sulfur hexafluoride,which improve the stability of microbubbles in the blood.Ultrasound can be used for tissue perfusion angiography.Protein,phospholipids and polysaccharides are commonly used as membrane materials for the preparation of microbubble contrast agents.The microbubbles prepared by these materials generally have some problems,such as poor stability in vivo and short half-life in plasma.In recent years,studies have shown that microbubbles made of macromolecule materials have better stability in vivo,good biocompatibility and biodegradability.It is a potential film-forming material and has become a hot spot in the research of ultrasound contrast agents.The purpose of this study is to improve the flexibility of the shell by lipid modification of polymer materials,and finally to obtain a new ultrasound contrast agent with good ultrasound response and stable circulation in vivo.Methods&Materials1.Part ? Preparation and optimization of porous lipid/PLGA microbubblesLipid/PLGA microbubbles were prepared by improved emulsified solvent evaporation method and vacuum freeze-drying technology.The physicochemical and acoustic properties of the microbubbles were tested,and the preparation process and parameters were optimized.The surface structure of Lipid/PLGA microbubbles was observed,the particle size potential and Young's modulus of the shell membrane were measured,and solutions with different concentration gradients(0.025,0.05,0.1,0.2 mg/mL)were prepared.The intensity of the second harmonic signal produced by Lipid/PLGA microbubbles was measured,and the contrast-enhanced effect and duration in vitro were recorded.Lipid/PLGA microbubbles were injected into tail vein,and then the development of different organs in mice was observed.In contrast mode,the Lipid/PLGA microbubbles were acted on by a certain intensity of ultrasound pulse,and then the changes of image and signal intensity in and out of the microbubbles before and after blasting were compared.2.Part ? Application of porous Lipid/PLGA microbubbles as drug deliveryLipid/PLGA microbubbles(Dox-Lipid/PLGA MBs)loaded with doxorubicin(Dox)were prepared by the above-mentioned optimized methods and parameters,and their physicochemical and acoustic properties were tested.The particle size,potential,drug loading and encapsulation efficiency were measured.The contrast-enhanced ultrasound effects of drug-loaded and drug-free microbubbles at the same concentration were compared in vitro.The release of Dox in vitro was measured by changing pH(3.0,5.0,7.4)and sound pressure(0,0.39,0.52,0.65 MPa).The cultured 4T1 tumor cells were mixed with PBS,Lipid/PLGA MBs and Dox-Lipid/PLGA MBs,respectively.The survival rates of the cells in each group were detected by CCK8.Fifty BALB/C mice models of subcutaneous breast cancer were established by cell transplantation.They were randomLy divided into control group,Lipid/PLGA+US group,Free Dox group,Dox-Lipid/PLGA group and Dox-Lipid/PLGA+US group,with 10 mice in each group.In the drug-loaded microbubble ultrasound group,the tumors were irradiated by ultrasound(frequency 1 MHz,sound pressure 0.52 MPa,duty cycle 25%)while the drug-loaded microbubbles were slowly injected into the tail vein;in the blank control group,0.1 mL saline was injected into the tail vein;and in the blank microbubbles ultrasound group,the tumors were irradiated by ultrasound under the same conditions as those in the drug-loaded microbubbles group when the drug-loaded microbubbles were slowly injected into the tail vein.In the simple drug group,0.1 mL Dox solution was injected into the tail vein(5mg/mL);in the drug-loaded microbubbles group,0.1 mL drug-loaded microbubbles were injected into the tail vein.All mice were irradiated once every two days for 3 minutes,three times.During the treatment,the weight changes and the growth of the tumors were observed,and the survival analysis was carried out.At the end of the first treatment,the weight of mice was weighed by electronic scale(2 days/time)for 9 times;the diameter of tumors was measured by vernier caliper(2 days/time),and the volume of tumors was calculated for 9 times;after the death of mice,the tumors were stained with H&E,the apoptosis was detected by TUNEL,and the proliferation inhibition of tumors was evaluated by Ki-67.Results1.Part I Preparation and optimization of porous lipid/PLGA microbubblesLipid/PLGA microbubbles dissolved in PBS buffer showed milky white suspension.The morphology of the microbubbles was regular,uniform and well dispersed.Scanning electron microscopy(SEM)showed that the surface of microbubbles was smooth before freeze-drying and porous after freeze-drying.Transmission electron microscopy(TEM)showed the internal cavity.The average particle size of Lipid/PLGA microbubbles was 736.8-1± 118.3 nm,the dispersion index was 0.19,the Zeta potential was(-5.32 ± 3.56)mV,and atomic force microscopy(AFM)showed that the shell film of LiPid/PLGA microbubbles was softer than that of ordinary PLGA microbubbles.At the same concentration,the second harmonic signal intensity produced by Lipid/PLGA microbubbles is close to that of lipid microbubbles,which is much better than that of ordinary PLGA microbubbles.In vitro ultrasound imaging experiments showed that Lipid/PLGA microbubbles could produce strong contrast-enhanced ultrasound signals,which varied with the concentration of Lipid/PLGA microbubbles,and had stable performance.The continuous development time could reach 36 hours.Lipid/PLGA microbubbles can be developed stably in vivo.The perfusion imaging signals of liver,kidney and spleen are strong,and disappear after 30 minutes.The results of in-vitro and in-vitro ultrasonic blasting experiments show that the signal intensity of Lipid/PLGA microbubbles after blasting is significantly lower than that before blasting,and the development signal almost disappears.2.Part ? Application of porous Lipid/PLGA microbubbles as drug deliveryLipid/PLGA microbubbles were dissolved in PBS buffer and showed a red emulsion.Light microscopy and scanning electron microscopy(SEM)showed that the morphology of the microbubbles was uniform and the pores were slightly smooth Laser confocal microscopy(LSCM)showed that the microbubbles were bright red with good dispersion.The average particle size of Dox-Lipid/PLGA microbubbles was 623 ± 105 nm,the dispersion index was 0.15,and the Zeta potential was(-5.82±2.72)mV.Dox-Lipid/PLGA and Lipid/PLGA microbubbles have no statistical difference in the contrast experiment of in vitro imaging.The entrapment efficiency and drug loading were 72 ± 1.5 and 8±0.23%respectively by high performance liquid chromatography.The cumulative drug release rates were 5±3.2%14± 1.3%,21±2.2%and 26±1.8%respectively after 125 hours of ultrasound irradiation at 0,0.39,0.52 and 0.65 MPa.When pH = 3 or 5(sound pressure is 0.52 MPa),the cumulative drug release rate is 47± 2.5%and 35± 1.3%after 125 hours.When 4T1 breast cancer cells were mixed with drug-loaded microbubbles,obvious red fluorescence was observed in the cells treated with ultrasound(0.52 MPa,1 min),while no red fluorescence was observed in the control group.Lipid/PLGA microbubbles showed no significant cytotoxicity to tumor cells after 1 minute exposure to sound pressure of 0,0.39,0.52 and 0.65 MPa.The cell survival rates were 98 ± 2.70%,96 ±6.3%,91 ±2.9%and 89 ± 7.5%respectively.In contrast,Dox-Lipid/PLGA microbubbles irradiated with the same ultrasound could significantly reduce the cell survival rates,which were 88 ± 6.7%,50 ±1.9%,31 ±4.3%and 28± 3.1%respectively.The prepared ox-Lipid/PLGA microbubbles can develop stably in vivo,and can improve the concentration and retention time of microbubbles in tumor area through EPR effect of tumor tissue.Compared with the control group,the inhibition effect of the blank microbubble ultrasound group was not obvious,and the free drug group or the Dox-Lipid/PLGA group had inhibitory effect on the tumors.The drug-loaded microbubble ultrasound group had the strongest inhibitory effect on tumor growth,reaching 86%inhibition rate(calculated by tumor volume).The median survival time of mice increased to 46 days,compared with 36 days in the control group.These results indicate that Dox-Lipid/PLGA microbubbles combined with ultrasound can effectively promote drug release and have a good therapeutic effect on tumors.During the animal experiment,no significant changes in body weight were observed in all groups.Histochemical analysis showed that nuclear contraction,fragmentation and necrosis were found in the tumor cells of the drug-loaded microbubble ultrasound group,while no malignant necrosis was found in the control group and the microbubble ultrasound group.Similar results were obtained by TUNEL assay,which showed that there were a large number of apoptotic tumor cells in the drug-loaded microbubble ultrasound group.Ki-67 immunohistochemical staining further showed that the level of Ki-67 positive cells in the drug-loaded microbubble ultrasound group was the lowest.Compared with the control mice,no obvious pathological abnormalities were found in the main organs(including heart,liver,spleen,lung and kidney)after intravenous injection of Dox-Lipid/PLGA microbubbles.Conclusion1.Part I Preparation and optimization of porous lipid/PLGA microbubblesLipid/PLGA microbubbles were successfully prepared with regular morphology,uniform size and good dispersion.The freeze-dried Lipid/PLGA microbubbles have porous surface and cavity-like structure.The intensity of harmonic signal generated by the microbubbles is similar to that of lipid microbubbles,but much better than that of ordinary PLGA microbubbles.The lipid-modified Lipid/PLGA microcellular shell membrane is more flexible and softer than ordinary PLGA microcellular membrane.Lipid/PLGA microbubbles have good acoustic properties,can enhance contrast imaging in vivo and in vitro,and have good circulatory stability.They can be cracked by a certain energy of ultrasound.They are a multifunctional ultrasound contrast agent with good application prospects.2.Part ? Application of porous Lipid/PLGA microbubbles as drug deliveryDox-loaded Dox-Lipid/PLGA microbubbles were successfully prepared.The microbubbles have high encapsulation efficiency,uniform particle size,good dispersion and good ultrasonic response.Dox-Lipid/PLGA microbubbles can develop stably in vivo.Through EPR effect of tumor tissue,the concentration and retention time of microbubbles in tumor area can be increased.Ultrasound irradiation can rupture microbubbles,promote drug release in microbubbles,and increase the release concentration of chemotherapeutic drugs in target area.Dox-Lipid/PLGA microbubbles combined with UTMD effectively inhibited the proliferation of tumor cells,promoted cell apoptosis and prolonged the survival cycle of tumor-bearing mice. |
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