超声微泡介导的声动力治疗与基因治疗在癌症诊疗中的应用研究

The combination of photodyanmic therapy and gene therapy can greatly enhance the cancer therapeutic efficiency , so it has become a hot topic. However, there are two big challenge in the medicine field, one is how to solve the penetration depth limitation and enhance the gene delivery efficiency；the other is how to use one carrier to co-deliver both sonosensitizer drug and gene for combining sonodyanmic therapy with gene therapy. Herein, the applicant synthesizes a new type of hybrid lipid by covalent attachment of sonosensitizer of indocyanine green derivative and cationic group of β-hydroxyethylenediamine. Then, a multifunctional perfluorinated microbubbles is fabricated from such functional lipid, followed by adsorption of gene. On one hand, the indocyanine green derivative covalently conjugated in the lipid monolayer of microbubbles act as sonosensitizer for Near infrared fluorescence imaging and sonodynamic therapy without any leaking. On the other hand, apoptin VP3 gene adsorbed on the microbubble surface can be used for controlled gene therapy. Fixed-point monitoring and treatment to tumor can be easily realized by adjusting the ultrasonic parameters. In addition, lipids containing folate targeted groups can be introduced into the microbubble during the preparation. Thus, multifunctional microbubbles can be constructed with integrating different functions including ultrasound stimuli targeting, gene controlled release, multimodal imaging (fluorescent and ultrasound imaging), sonodyanmic therapy and gene therapy, ultimately achieving the purpose of improving the diagnosis and treatment of deep malignant tumors.

光动力治疗与基因治疗联合可以有效改善肿瘤疗效，已成为当前癌症治疗研究的热点。但是，如何解决光的穿透深度问题和提高基因转染效率，以及如何使用同一载体负载声敏剂和基因药物对癌症进行声动力治疗和基因治疗的联合治疗，是医药领域面临的两大挑战。申请人拟通过共价键将声敏剂吲哚菁绿衍生物和阳离子基团羟乙基乙二胺同时引入两亲性脂质分子中，并以此为自组装基元制备能够携载基因药物的全氟丙烷微气泡。一方面，位于微泡壳层中以共价键连接的声敏剂可用于近红外荧光成像和声动力治疗；另一方面，微泡表面吸附的凋亡素基因VP3可实现基因治疗。通过调节不同超声参数即可实现肿瘤的成像监测与定点治疗。在微泡制备过程中，进一步引入含叶酸靶向基团的脂质分子，即可得到超声激发下的集定点靶向、基因药物可控释、双模态诊断（超声和荧光成像结合可深浅兼顾）、声动力治疗与基因治疗于一体的新型功能化微泡造影剂，最终达到改善深部肿瘤诊疗效果的目的。

本研究在功能化微气泡的设计、超声增强药物递送效率、联合基因治疗、光/声动力治疗、免疫治疗等方面开展系统研究，有效地抑制了肿瘤的生长和复发。主要包括如下4个方面：1.超声联合孟加拉红微泡靶向增效肿瘤声动力疗效：以两亲性孟加拉红脂质（ARB）作为壳层材料封装全氟丙烷气体构建新型声敏剂—微泡递送系统，该体系具有高载药量、高稳定性、药物不过早释放等优点，同时又可通过微泡来介导超声能量的传递进一步增强孟加拉红的单线态氧产率，显著提高了声动力治疗的效果；2.将抑制肿瘤细胞增殖的FOXA1基因和阳离子卟啉微泡联合，实现超声增效的肿瘤光/声动力和基因联合治疗：以含两个阳离子氨基的卟啉脂质包载全氟丙烷制备阳离子卟啉微泡（CPMBs）。CpMBs单层中的卟啉基团可作为光/声敏剂，而氨基基团可通过静电作用吸附FOXA1 KD的siRNA，从而抑制雌激素依赖性ER+乳腺癌的增殖。CpMBs/siRNA联合超声可产生声穿孔效应，显著增加卟啉和siRNA在肿瘤的局部积累，对雌激素依赖性ER+乳腺癌显示出良好的治疗效果；3.将改善乏氧基因HIF1α-siRNA和阳离子卟啉微泡联合，改善肿瘤乏氧微环境，增强肿瘤联合疗效：在第二部分所研发的多功能阳离子卟啉脂质（CPGL）的基础上，引入能下调肿瘤组织乏氧的HIF 1αsiRNA基因，构建了siHIF@CpMB微泡体系。在超声靶向微气泡爆破技术的作用下，siHIF@CpMBs可在肿瘤区域原位高效转化为纳米颗粒，通过空化效应促进卟啉和siRNA在肿瘤部位的富集。HIF-1αsiRNA可高效下调肿瘤区域的Hif1α水平，提高光/声动力疗效并有效抑制肿瘤的生长；4.超声联合工程化细胞膜修饰的钛酸钡，实现肿瘤的声动力和免疫协同治疗：以细胞膜稳定表达基质金属肽酶2（MMP2）激活的PD-L1阻断剂装载超小型钛酸钡纳米颗粒（BTO NP）制备BTO@M-αPD-L1纳米粒用于克服肿瘤免疫耐受。该体系表面的细胞膜壳层明显促进了BTO NP的肿瘤蓄积,在肿瘤微环境中MMP2作用下，M-αPD-L1外壳能特异发挥阻断肿瘤细胞上PD-L1过度表达的作用。超声辐照能进一步激发BTO产生ROS杀死肿瘤并触发免疫原性细胞死亡（ICD），协同促进抗肿瘤免疫治疗。