声致相变纳米探针用于瘢痕疙瘩成纤维细胞级联疗法的实验研究  

Cascade therapy of keloid fibroblasts with acoustic induced phase-change nanoprobes:an experimental study

作　　者：凡正超 夏纪筑 朱炜薇 胥莹 赵香芝 FAN Zhengchao;XIA Jizhu;ZHU Weiwei;XU Ying;ZHAO Xiangzhi(Department of Ultrasound Medicine,Affiliated Hospital of Southwest Medical University,Sichuan 646000,China)

机构地区：[1]西南医科大学附属医院超声医学科,四川省泸州市646000

出　　处：《临床超声医学杂志》2024年第9期705-712,共8页Journal of Clinical Ultrasound in Medicine

基　　金：泸州市指导性科技计划项目(22YYJC0037、2022YJY108);西南医科大学附属医院博士启动资金项目(20123)。

摘　　要：目的制备一种新型脂质载药纳米探针HD@P-NPs,探讨其体外超声成像效果及用于瘢痕疙瘩成纤维细胞(KFs)迁移抑制和级联放大治疗的效果。方法采用超声乳化法制备以全氟乙烷为核心脂质壳层,负载血卟啉单甲醚和阿霉素的脂质载药纳米探针HD@P-NPs,观察其形态、结构并检测粒径和Zeta电位,计算药物包封率和载药率;进一步观察HD@P-NPs在低强度聚焦超声(LIFU)辐照下的二维超声及超声造影成像效果;溶血实验检测HD@P-NPs在不同浓度下的溶血率。取对数生长期的瘢痕疙瘩KFs按照不同实验条件培养,并分为5组:对照组(不予特殊处理)、LIFU辐照组、D@P-NPs组(仅加入阿霉素)、HD@P-NPs组、HD@P-NPs+LIFU辐照组,使用MTT法检测各组细胞存活率;Calcein AM/PI染色观察各组细胞活性;细胞迁移实验检测各组细胞迁移率;活性氧荧光染色观察各组活性氧产生情况,获取其荧光强度。结果成功制备HD@P-NPs,呈球形且大小均一,平均粒径(170.36±6.03)nm,平均Zeta电位(-36.91±3.56)mV;血卟啉单甲醚包封率和载药率分别为67.41%、5.18%,阿霉素包封率和载药率分别为72.80%、11.20%。LIFU辐照后,HD@P-NPs相变产生微气泡,在3 W/cm^(2)、2 min辐照条件下可实现最佳成像效果,后续实验采用此辐照条件。HD@P-NPs在25、50、100、200μg/ml浓度下的溶血率分别为(2.48±0.02)%、(4.87±0.06)%、(5.03±0.03)%、(6.10±0.04)%。对照组、LIFU辐照组、D@P-NPs组、HD@P-NPs组及HD@P-NPs+LIFU辐照组细胞存活率分别为100%、(96.87±0.71)%、(77.94±2.83)%、(77.11±3.53)%、(49.75±1.25)%,HD@P-NPs+LIFU辐照组与对照组细胞存活率比较差异有统计学意义(P<0.05)。HD@P-NPs+LIFU辐照组见明显红色荧光及少量绿色荧光。对照组、LIFU辐照组、D@P-NPs组、HD@PNPs组、HD@P-NPs+LIFU辐照组的细胞迁移率分别为(29.96±3.20)%、(28.62±2.56)%、(18.13±0.89)%、(17.46±0.20)%、(10.04±1.62)%,其中HD@P-NPs+LIFU辐照组细胞迁移率低Objective To prepare a new type of lipid nanoprobes(HD@P-NPs),and to investigate the effects of ultrasound imaging in vitro,migration inhibition as well as amplification therapy on keloid fibroblasts(KFs).Methods HD@PNPs were prepared through ultrasonic emulsification with perfluoroethane as the core lipid shell and loading of hematoporphyrin monomethyl ether(HMME)and doxorubicin(DOX).The morphology,structure,particle size and Zeta potential of HD@P-NPs were measured,and the encapsulation efficiency and loading rate of corresponding drug were calculated.The imaging effects of two-dimensional ultrasound and contrast-enhanced ultrasound(CEUS)under the irradiation of low intensity focused ultrasound(LIFU)were explored.The hemolysis rate of HD@P-NPs at different concentrations were calculated.KFs in the logarithmic growth phase were cultured under different experimental conditions and divided into the following 5 groups:control group(no treatment),LIFU group,D@P-NPs group,HD@P-NPs group and HD@P-NPs+LIFU group.The cell survival rate was detected by MTT assay,the cell activity in each group was observed by Calcein-AM/PI staining method.Cell migration rate in each group was detected by cell migration experiment.Reactive oxygen species fluorescence staining was applied to observe the intracellular reactive oxygen species(ROS)production,and the fluorescence intensity was obtained.Results HD@P-NPs were successfully prepared with uniform dimensions.The average particle size was(170.36±6.03)nm,the average Zeta potential was(-36.91±3.56)mV,respectively.The encapsulation efficiency and loading rate of HMME and DOX were 67.41%,5.18%and 72.80%,11.20%,respectively.Microbubbles were generated by the phase transition of HD@P-NPs after LIFU irradiation.The optimal imaging efficacy was achieved under the irradiation conditions of 3 W/cm^(2) for 2 min and these parameters were subsequently adopted for further experiments.The hemolysis rate of HD@P-NPs at different concentrations(25,50,100,200μg/ml)were(2.48±0.02)%,(4.87±0.06)%,(

关 键 词：纳米探针 瘢痕疙瘩成纤维细胞 血卟啉单甲醚 阿霉素 声动力治疗 

分 类 号：R445.1[医药卫生—影像医学与核医学]