| Cancer morbidity and mortality have continued to rise and seriously threaten human health in recent years.Developing novel targeting and efficient tumor treatment strategies are essential to prolong patient survival and improve patient quality of life.Photothermal therapy(PTT)is a new anti-tumor therapy emerging in recent years,which uses local light to stimulate materials(photothermal transduction agents)with high photothermal conversion efficiency,and then converts light energy into heat energy to kill tumor cells.PTT has attracted widespread attention due to its advantages such as high efficiency,minimally invasive and not easy to cause drug resistance.However,the uncertainty of the accumulation of photothermal transduction agents in tumor sites and the difficulty of imaging-guided treatment have limited the development of PTT to some extent.Therefore,it's great significance to develop an integrated diagnosis and treatment platform with imaging and targeted PTT treatment for improving the efficiency of PTT and expanding the applications of PTT.Microbubbles(MBs)as contrast agents for ultrasound imaging are widely used in clinical diagnosis.In addition,MBs under ultrasound targeted destruction(UTMD)can achieve targeted delivery of various drugs and can be used as excellent PTT adjuvant.However,the low drug loading and large size of MBs have limited their effective application in PTT antitumor therapy.In this study,the stable pyropheophorbide microbubbles(Pyro-MBs)with high porphyrin loading were synthesized by maxing and shaking the pyropheophorbide-lipid(Pyro-lipid)with inert perfluorohexane gas.Due to the well-organized arrangement of porphyrins in the shell,microbubbles have excellent light-to-heat conversion properties.And ultrasound/near infrared fluorescence(US/NIRF)dual-mode imaging can be achieved because of the presence of porphyrins and inert gases.Besides,Pyro-MBs can be efficiently transformed into high-density porphyrin-loaded nanoparticles(Pyro-NPs)by applying UTMD technology,and Pyro-NPs can be efficiently enriched in tumor tissues with the aid of sonochemical effect.This strategy overcomes the shortcomings of low drug loading and large size of conventional microbubbles for antitumor therapy,and achieves excellent PTT effect under the guidance of US/NIRF dual modality imaging.This article mainly explored the potential of Pyro-MBs for imaging guided PTT antitumor treatment from three aspects:the synthesis and characterization of Pyro-MBs;the imaging performance of Pyro-MBs,and the PTT antitumor effects mediated by Pyro-MBs conversion under ultrasound regulation.The purpose was to provide experimental evidence for enhancing the PTT efficiency and the application of Pyro-MBs in tumor diagnosis and treatment.Research contents and results(1)Synthesis and characterization of Pyro-MBsPyro-lipid was synthesized by acylation reaction,and the synthesized Pyro-lipid was purified and identified by thin-layer chromatography,high-performance liquid chromatography,column chromatography and high-resolution mass spectrometry.And Pyro-lipid was used to prepare Pyro-MBs containing different porphyrin molar ratios by thin film hydration-mechanical shaking method.Dynamic light scattering(DLS)analysis and Zeta potentiometer were used to detect the size distributions,dispersion coefficient and Zeta potential of each proportion of Pyro-MBs.By measuring the size changes of microbubbles,the optimal parameters(ultrasonic intensity and time)for ultrasonic conversion of porphyrin microbubbles to nanoparticles were optimized,and the changes in size distributions,potential,morphology and stability of the particles before and after ultrasound exposure were evaluated.The absorption and fluorescence spectra as well as the fluorescence imaging performance of Pyro-MBs before and after ultrasound treatment were analyzed by using a multifunctional microplate reader and small animal imaging system.An infrared thermal imager was used to evaluate the photothermal conversion performance of each Pyro-MBs with different porphyrin content before and after ultrasound exposure.The main conclusions are as follows:?Pyro-lipid was successfully prepared by porphyrin-phospholipid coupling,and thin layer chromatography showed that Pyro-lipid purified by column chromatography had a single component,which could be used for subsequent experimental research.? By adjusting the molar ratio of Pyro-lipid,Pyro-MBs with different porphyrin ratios were prepared,all of them had uniform particle size,regular morphology and were responsive to ultrasound treatment,their particle size could be changed from 1?2?m to 100?200 nm under the effect of ultrasound(4 W?2 min),and the porphyrin nanoparticles formed by ultrasonic conversion have good stability.? Zeta potential,optical properties and light-to-heat conversion performance of Pyro-MBs with various porphyrin ratios would not change significantly before and after ultrasound exposure.? 5%Pyro-MBs with lower porphyrin content had a small degree of fluorescence quenching and showed excellent fluorescence imaging performance in vitro,but it had lower photothermal conversion efficiency.As the molar ratio of Pyro-lipid in microbubbles increased,the fluorescence signal of Pyro-MBs were weakened while the photothermal conversion performance was significantly enhanced.60%Pyro-MBs had better light-to-heat conversion performance compared to the other ratios of porphyrin microbubbles,and can be used as photothermal transduction agents.(2)Imaging ability of Pyro-MBsUsing ultrasound imaging system to analyze the ultrasound imaging performance of Pyro-MBs in vivo and in vitro.The animal imaging system was used to study the fluorescence imaging characteristics of 5%Pyro-MBs in low porphyrin group and 60%Pyro-MBs in high porphyrin group,and compared the distribution of Pyro-MBs in tumors and major organ tissues with/without ultrasound exposure.The results show that:? Pyro-MBs could significantly enhance the contrast of ultrasound imaging in vivo and in vitro,which can be used for ultrasound imaging in vivo and achieve good diagnosis and localization effects,provides tracking guarantee for the precise regulation of ultrasound-mediated microbubbles conversion in later.?5%Pyro-MBs showed clear fluorescent signal in vivo and displayed little background interference,which can be used for real-time imaging of tumors.60%Pyro-MBs had severe fluorescence quenching in vivo but with good photothermal signal,which can be used for photothermal treatment.? Ultrasound-regulated porphyrin microbubbles conversion into nanoparticles can effectively promote the enrichment and penetration of Pyro-NPs in tumor sites,which provides a basis for subsequent ultrasound-assisted 60%Pyro-MBs to achieve excellent PTT antitumor effects.(3)Ultrasound regulated in situ conversion of Pyro-MBs to Pyro-NPs and PTT antitumor efficacyTaking mouse 4T1 breast cancer as a model,the antitumor effect of PTT mediated by ultrasound regulation of 60%Pyro-MBs conversion to Pyro-NPs was evaluated from both in vitro cell experiments and in vivo animal experiments.The MTT and Calcein-AM/PI double staining method were used to evaluate the cytotoxicity of Pyro-MBs in vitro and killing effect of ultrasound-mediated Pyro-MBs conversion combined with laser treatment on 4T1 cells.Continuous frozen sections and fluorescent stereo microscope were used to analyze the penetration of drugs into the tumor after ultrasound treatment.Thermal infrared camera was used to measure the temperature of the tumor site during PTT in real time.And the growth inhibition of 4T1 breast cancer induced by PTT mediated by ultrasound-regulated Pyro-MBs conversion were discussed in terms of tumor size,tumor weight,survival curve of tumor-bearing mice and tumor tissue pathological sections.A preliminary safety test of the research system was performed by monitoring body weight of tumor-bearing mice and H&E staining of major organ tissues.Studies show that:? 60%Pyro-MBs had almost no toxicity to 4T1 cells,and ultrasound-mediated conversion of Pyro-MBs to nanoparticles combined with light irradiation exhibited a significant killing effect on 4T1 cells.? Ultrasound regulated Pyro-MBs conversion could promote effective enrichment and deep penetration of porphyrin molecules in tumor sites.? Ultrasound-mediated conversion of Pyro-MBs to Pyro-NPs combined with light irradiation caused severe damage to tumor tissues,significantly inhibited tumor growth and prolonged survival time of tumor-bearing mice.? Under the porphyrin dose,ultrasound and laser parameters selected for the research system,different treatments had no significant effect on the growth and main organ tissues of mice,suggesting that the experimental system was relatively safe.In conclusionThis study successfully constructed a Pyro-MBs platform with integrated diagnosis and treatment,achieved US-controlled accumulation of photothermal transduction agents and enhanced the antitumor efficacy of PTT under the guidance of imaging.This strategy overcomes the defects of large size and low drug loading of microbubbles in imaging diagnosis and treatment,and provides data support and theoretical basis for the future application of MBs and PTT in clinical antitumor treatment. |
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