Mechanistic probe for siRNA-polyplex delivery towards potent cancer therapeutics

描述(申请人提供)：尽管近年来已经开发了几种阳离子聚合物用于siRNA传递，25还没有一种被FDA批准，由于缺乏广泛的结构/功能研究，例如那些用DNA进行的研究，传递过程的机制细节仍然存在空白。虽然siRNA和DNA具有相同的电荷密度，但由于siRNA具有刚性棒状的性质，所以它与DNA的聚阳离子缩合能力明显不同。这种差异极大地影响了siRNA纳米颗粒的细胞外和细胞内的稳定性，这两个特征对于有效的递送至关重要。为此，我提出了一种新型的包裹siRNA的递送聚合物的设计和使用，以及一种新的拆卸特异性探针来研究siRNA纳米颗粒在细胞质内的拆解动力学。为了便于研究细胞内纳米颗粒的分解，需要一种将siRNA浓缩成纳米颗粒的递送载体，因为裸露的siRNA本身不能穿过细胞膜。为了实现这一目标，我通过硫醇-烯化学设计了一种阳离子聚合物，将siRNA凝聚成50-60 nm的颗粒。为了确保这种新型聚合物是可生物降解的，能够进入细胞，并且无毒，我将通过1H核磁共振研究聚合物的降解，通过Zeta电位研究纳米颗粒的表面电荷，并通过四甲基偶氮唑盐细胞毒性试验研究聚合物和纳米颗粒在人宫颈癌(HeLa)细胞系中的细胞毒性程度。在拆解动力学研究中，包裹在多聚体中的siRNA将被红外荧光吲哚青绿(ICG)染料标记。由于聚集诱导的自猝灭机制，ICG的荧光是浓度敏感的。标记的siRNA-ICG探针的功能如下：siRNA分子之间由于其强烈的负电荷而产生的强排斥力应该阻止siRNA-ICG的聚集，从而促进荧光。然而，当与阳离子聚合物缩合形成聚合物时，由于强烈的电荷相互作用，几个siRNA-ICG分子聚集在50-60 nm的颗粒中，可能会引发ICG的聚集诱导自猝灭，导致其荧光强度急剧下降。因此，ICG的荧光对纳米颗粒解体的依赖关系应该提供纳米颗粒稳定性的开/关指示。通过这种siRNA-ICG探针检测细胞内的解离，并建立数学模型，将形成定量确定siRNA释放动力学的基础。在用新设计的聚合物开发和测试这个探针后，我将通过硫醇-烯化学创建新的可降解聚合物模型库，并将从数学模型中获得的拆解动力学参数与转染率关联起来，为纳米粒子拆解在siRNA传递中的贡献提供新的见解。从这项研究中收集的信息还将用于开发新的结构-功能相关性，这些相关性将指导未来聚合物文库的设计，以加速发现用于RNAi癌症治疗的有效siRNA递送载体。 与公共健康相关：我的研究计划涉及通过新设计的探针和聚合物库进行siRNA传递的机制研究，以改进设计，从而加速发现有效的siRNA疗法。这些有效的治疗载体将有助于实现RNAi治疗在通过沉默致癌基因来治疗癌症等人类疾病方面的巨大潜力，从而通过疾病治疗改善人类健康来推进NIH的目标。

DESCRIPTION (provided by applicant): Although several cationic polymers have been developed in recent years for siRNA delivery,25 none have been FDA approved and there still exists a void in the mechanistic details of the delivery process due to the lack of extensive structure/function studies, such as those done with DNA. While both siRNA and DNA have the same charge density, siRNA is distinctly different from DNA in its ability to be condensed by polycations due to its rigid-rod like nature. This difference greatly affects the extracellular and intracellular stability of siRNA- nanoparticles, two features that are critical for efficient delivery. To this end, I propose the design and use of a new type of delivery polymer that encapsulates siRNA along with a new disassembly-specific probe to study the kinetics of siRNA nanoparticle disassembly within the cytoplasm. To facilitate the study of intracellular nanoparticle disassembly, a delivery vector that condenses siRNAs into a nanoparticle is required because naked siRNA on its own cannot cross the cellular membrane. Towards this goal, I have designed a cationic polymer via thiol-ene chemistry that condenses siRNAs into 50-60 nm particles. To ensure that this new class of polymers will be biodegradable, able to get into cells, and non-toxic, I will investigate polymer degradation via 1H NMR, nanoparticle surface charge via zeta potential, and the degree of polymer and nanoparticle cytotoxicity in a human cervical cancer (HeLa) cell line via the MTT cytotoxicity assay. For the disassembly kinetics study, the siRNA to be encapsulated within the polyplex will be labeled with an infra-red fluorescent indocyanine green (ICG) dye. ICG's fluorescence is concentration sensitive due to an aggregation induced self- quenching mechanism. The labeled siRNA-ICG probe should function as follows: strong repulsion between siRNA molecules due to their strong negative charges should prevent siRNA-ICG aggregation and thus promote fluorescence. However, upon condensation with cationic polymers to form polyplexes, the accumulation of several siRNA-ICG molecules in a 50-60 nm particle due to strong charge interactions should initiate aggregation induced self-quenching of ICG leading to a sharp decrease in its fluorescence intensity. Thus, the dependence of ICG's fluorescence on nanoparticle disassembly should provide an on/ff indication of nanoparticle stability. The detection of intracellular disassembly via this siRNA-ICG probe along with a mathematical model will form the basis for quantitatively determining the kinetics of siRNA release. After developing and testing this probe with the newly designed polymer, I will create new model library of degradable polymers via thiol-ene chemistry and correlate the disassembly kinetic parameters obtained from the mathematical model with transfection efficiencies to provide new insights into the contributing role of nanoparticle disassembly in siRNA delivery. Information gathered from this study will also be used to develop new structure-function correlations that will guide the design of future polymer libraries towards accelerating the discovery of potent siRNA delivery vectors for RNAi cancer therapeutics. PUBLIC HEALTH RELEVANCE: My research plan involves a mechanistic study of siRNA delivery via a newly designed probe and polymer library in order to improve the design and thus accelerate the discovery of potent siRNA therapeutics. These potent therapeutic vectors will help realize the great potential of RNAi therapy in the treatment of human diseases such as cancer via silencing of cancer causing genes, thus advancing the goals of the NIH by improving human health through disease treatment.