Mechanistic probe for siRNA-polyplex delivery towards potent cancer therapeutics

用于 siRNA-多聚复合物递送以实现有效癌症治疗的机制探针

• 批准号: 8144895
• 负责人: Christopher Akinleye Alabi
• 金额: $ 5.13万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-15 至 2012-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8144895
• 关键词: Address; Affect; Alkenes; Attention; Binding; Biological; Biological Assay; Cancer Etiology; Cell Line; Cell-Mediated Cytolysis; Cells; Cellular Membrane; Charge; Chemicals; Chemistry; Clinical; Cytoplasm; DNA; Dependence; Detection; Development; Disease; Due Process; Dyes; Encapsulated; Ensure; FDA approved; Fluorescence; Future; Genes; Goals; Health; Hela Cells; Human; In Vitro; Indocyanine Green; Kinetics; Label; Libraries; Ligation; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Methods; Mind; Modeling; Nature; Physical condensation; Polymers; Process; Property; RNA; RNA Interference; Reaction; Reporter; Reporting; Research; Research Project Grants; Role; Screening procedure; Signal Transduction; Structure; Structure-Activity Relationship; Sulfhydryl Compounds; Surface; Techniques; Testing; Therapeutic; Transfection; Treatment Efficacy; United States National Institutes of Health; Water; analytical tool; base; biodegradable polymer; combat; cytotoxicity; density; design; extracellular; human disease; improved; information gathering; insight; mathematical model; nanoparticle; particle; polycation; prevent; public health relevance; retinal rods; success; therapy development; tool; uptake; vector; zeta potential

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.

描述（由申请人提供）：尽管近年来已经开发了几种用于siRNA递送的阳离子聚合物，但没有一种被FDA批准，并且由于缺乏广泛的结构/功能研究，例如用DNA进行的研究，在递送过程的机理细节中仍然存在空白。虽然siRNA和DNA具有相同的电荷密度，但由于其刚性棒状性质，siRNA在被聚阳离子缩合的能力方面明显不同于DNA。这种差异极大地影响了siRNA纳米颗粒的细胞外和细胞内稳定性，这两个特征对于有效递送至关重要。为此，我建议设计和使用一种新型的传递聚合物，封装siRNA沿着与一种新的解体特异性探针，研究siRNA纳米颗粒的细胞质内解体的动力学。为了促进细胞内纳米颗粒分解的研究，需要将siRNA浓缩成纳米颗粒的递送载体，因为裸siRNA本身不能穿过细胞膜。为了实现这一目标，我设计了一种阳离子聚合物，通过硫醇-烯化学将siRNA浓缩成50-60 nm的颗粒。为了确保这类新型聚合物可生物降解、能够进入细胞并且无毒，我将通过1H NMR研究聚合物降解、通过zeta电位研究纳米颗粒表面电荷以及聚合物和纳米颗粒的细胞毒性程度通过MTT细胞毒性试验研究人宫颈癌（HeLa）细胞系。对于分解动力学研究，待包封在多聚物内的siRNA将用红外荧光吲哚菁绿色（ICG）染料标记。ICG的荧光由于聚集诱导的自猝灭机制而具有浓度敏感性。经标记的siRNA-ICG探针应如下起作用：siRNA分子之间由于其强负电荷而产生的强排斥应防止siRNA-ICG聚集并因此促进荧光。然而，在与阳离子聚合物缩合以形成聚合复合物时，由于强电荷相互作用，几个siRNA-ICG分子在50-60 nm颗粒中的积累应引发聚集诱导的ICG自猝灭，导致其荧光强度急剧降低。因此，ICG的荧光对纳米颗粒分解的依赖性应该提供纳米颗粒稳定性的开/关指示。通过该siRNA-ICG探针沿着数学模型检测细胞内分解将形成定量测定siRNA释放动力学的基础。在使用新设计的聚合物开发和测试该探针后，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效率相关联，以提供有关纳米颗粒拆解的贡献作用的新见解在SiRNA递送中。从这项研究中收集的信息也将用于开发新的结构-功能相关性，这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。 公共卫生相关性：我的研究计划包括通过新设计的探针和聚合物文库进行siRNA递送的机制研究，以改进设计，从而加速发现有效的siRNA治疗方法。这些有效的治疗载体将有助于实现RNAi疗法在通过沉默致癌基因治疗人类疾病（如癌症）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。