pHIFU介导载药声敏脂质体增强药物靶向释放应用机制实验研究

Drug loaded microbubbles targeted therapy mediated by high intensity focused ultrasound (HIFU) normally achieved by using thermal and cavitation effects, which had shortcomings such as non-temperature control and likely to cause damage to the surrounding normal tissues，etc. Our study designed a novel drug delivery system, called ultrasound sensitive liposomes. Through adjusting the ratio of DPPC and HSPC, the phase-transition temperature of liposomes matched the thermal transmission mediated by pHIFU. By controlling the working state of pHIFU system, beginning with interaction between ultrasonic and drug loaded ultrasound sensitive liposomes, according to the theories and experimental data of acoustics, material science and biomedicine, establishing the physical model based on ultrasonic resonance therapy, and revealing the action mechanism and law of ultrasonic by means of experiments, so that the drugs can be delivered to targeted spatiality and interacted with liposomes which resulted in the non-linear resonance effect. Thus，the goals that delivering chemotherapeutic drugs quantitatively, spatially, safely and effectively are accomplished.The project achieved accurate control of ultrasound sensitive liposomes'vibrational state in living tissues by regulating the intensity of ultrasonic. By comparing the thermal, cavitation and resonance effects initiatd by different ultrasonic intensities, and analyzing the rusults of non-linear resonance effect on enhancing the local concentration of chemotherapeutic drugs in tumor sites, effective killing of tumor tissues, reducing the damage to normal cells and systemic adverse reactions. Further study on related mechanisms of targeting therapy mediated by ultrasound.

由于高强度聚焦超声（HIFU）介导的载药微泡靶向治疗一般是利用超声波的热效应和空化效应，具有温度不易控制及易对周围正常组织造成损伤等缺点，本研究将完善具有专属性热效应和振动效应的脉冲式高强度聚焦超声（pHIFU）设备的软硬件，并构建一种新型声敏脂质体给药系统，通过调整DPPC与HSPC的配比，使其相变温度与pHIFU介导的热传递相匹配，通过控制pHIFU系统的工作状态，建立基于超声共振治疗的物理模型,使其能在药物靶向投送区域与脂质体作用并产生非线性共振效应，由此达到化疗药物能定点定量和安全有效投送的目的，并通过比较不同超声波强度所引发的热效应、空化效应和共振效应，分析应用可控的非线性共振效应在提高化疗药物在肿瘤部位的局部浓度，有效杀伤肿瘤组织和减少对正常细胞的伤害的效果，并通过实验揭示超声波介导靶向治疗的相关机制。

pHIFU(脉冲式聚焦超声）具有聚焦性高、组织穿透力强、指向性好、可体外聚焦等优点，是一项具有临床转化潜能的可显著提高肿瘤细胞对药物反应性的无创技术。本项目首先进行自主研发球面自聚焦超声治疗仪超声波作用效应研究，研究超声波与生物组织作用时所产生的热效应和非线性振动效应，获得不同频率、强度、分布和作用周期的超声波与生物组织作用时所产生的温度变化规律，进行超声波生物效应研究和超声治疗剂量学研究，通过研制各类测试模块和建立计量测试系统，完成对超声波声场、强度的测量以及作用效果的观察和测试。构建一种新型声敏脂质体给药系统，通过调整DPPC与HSPC的配比，使其相变温度与pHIFU介导的热传递相匹配。我们的pHIFU探头频率为1.18MHz,能量设置为3w/cm2(0.21MPa)，占空比20%，辐照时间3分钟，这样避免过高的超声能量和过强的微泡爆破力对细胞产生机械性损伤和热效应损伤，同时在一个治疗周期后，受pHIFU 辐照的细胞存活率为空白组的80%左右，研究结果表明pHIFU 可促进耐紫杉醇卵巢癌细胞的死亡及改变细胞的生长状态、降低耐紫杉醇卵巢癌细胞自噬激活，减弱自噬流。通过 Western Blot、共聚焦荧光显微镜、透射电镜等技术检测受 pHIFU 辐照和未受辐照的耐紫杉醇卵巢癌细胞自噬相关蛋白 LC3-II、Beclin1、Atg5的表达、自噬小泡的数量和形态，阐明 pHIFU 对激活耐药卵巢癌细胞自噬活性，比较与传统单药紫杉醇作用后细胞的存活率，pHIFU 引起的自噬活性受抑制后，耐紫杉醇卵巢癌细胞的对紫杉醇的敏感性增强，从另一个角度表明pHIFU 通过抑制自噬，促进耐药卵巢癌细胞的死亡；通过自噬相关基因 PCR 芯片，比较受 pHIFU 辐照和空白组的耐紫杉醇卵巢癌细胞自噬相关基因 mRNA 的表达变化，发现涉及自噬和凋亡相关基因有明细差异。进一步通过 qRT-PCR 验证，确定了相关通路及关键调控分子，实验证明pHIFU能在药物靶向投送区域与脂质体作用并产生非线性共振效应，由此达到化疗药物能定点定量和安全有效投送的目的，达到在提高化疗药物在肿瘤部位的局部浓度，有效杀伤肿瘤组织、逆转肿瘤耐药和减少对正常细胞的伤害的效果，并揭示超声波其治疗的相关机制。