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: 7996798
• 负责人: Christopher Akinleye Alabi
• 金额: $ 4.76万
• 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/7996798
• 关键词: Affect; Biological Assay; Cancer Etiology; Cell Line; Cells; Cellular Membrane; Charge; Chemistry; Cytoplasm; DNA; Dependence; Detection; Disease; Due Process; Dyes; Encapsulated; Ensure; FDA approved; Fluorescence; Future; Genes; Goals; Health; Hela Cells; Human; Indocyanine Green; Kinetics; Label; Libraries; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Modeling; Nature; Physical condensation; Polymers; RNA Interference; RNAi vector; Research; Role; Small Interfering RNA; Structure; Sulfhydryl Compounds; Surface; Testing; Therapeutic; Transfection; United States National Institutes of Health; base; biodegradable polymer; cytotoxicity; density; design; extracellular; human disease; improved; information gathering; insight; mathematical model; nanoparticle; particle; polycation; prevent; public health relevance; retinal rods; 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阳离子聚合物，用于siRNA的递送，但由于缺乏广泛的结构/功能研究，例如使用DNA完成的，但仍存在FDA批准，并且在交付过程的机理细节中仍然存在空隙。尽管siRNA和DNA都具有相同的电荷密度，但siRNA与DNA在其刚性类似于的刚性杆上凝结的能力明显不同。这种差异极大地影响了sirna-Nanoparpicles的细胞外和细胞内稳定性，这对于有效递送至关重要。为此，我提出了一种新型的递送聚合物的设计和使用，该聚合物将siRNA封装在一起，以及新的拆卸特异性探针，以研究细胞质内脱离siRNA纳米颗粒的动力学。为了促进细胞内纳米颗粒拆卸的研究，需要将siRNA凝结到纳米颗粒中的递送载体，因为裸siRNA本身无法越过细胞膜。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，该化学将siRNA凝结成50-60 nm颗粒。为了确保这种新的聚合物将是可生物降解的，能够进入细胞，并且无毒，我将通过1H NMR来调查聚合物通过ZETA电位通过ZETA电位的纳米颗粒表面电荷，以及通过人类颈椎癌（Hela）细胞系中的聚合物和纳米粒子细胞毒性的程度。在拆卸动力学研究中，将封装在流载体内的siRNA将用红外荧光氨基氨基氨基绿（ICG）染料标记。由于聚集引起的自淬灭机制，ICG的荧光对浓度敏感。标记的siRNA-ICG探针应如下：siRNA分子之间强烈的抑制作用，因为它们的强负电荷应防止siRNA-ICG聚集，从而促进荧光。然而，在与阳离子聚合物凝结以形成多流胎后，由于强电荷相互作用而导致几个siRNA-ICG分子在50-60 nm粒子中的积累，应启动聚集诱导ICG的自我淬火，从而导致其荧光强度急剧下降。因此，ICG荧光对纳米颗粒拆卸的依赖性应提供纳米颗粒稳定性的ON/FF指示。通过此siRNA-ICG探针以及数学模型检测细胞内拆卸将构成定量确定siRNA释放动力学的基础。在与新设计的聚合物开发和测试此探测器之后，我将通过硫醇化学创建新的可降解聚合物的模型库，并将从数学模型获得的拆卸动力学参数与转染效率相关联，以提供新的见解，以提供新的见解，以促进NanApartiles sibsemplys incirna sirna sirna sirna sirna sirna sirna sirna sirna incememplys的作用。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库的设计，以加速发现RNAi Cancer Therapeatics的有效siRNA递送向量。 公共卫生相关性：我的研究计划涉及通过新设计的探针和聚合物库对siRNA传递的机械研究，以改善设计，从而加速发现有效的siRNA疗法。这些有效的治疗媒介将有助于实现RNAi治疗在癌症等癌症等癌症等癌症治疗中的巨大潜力，从而通过疾病治疗改善人类健康，从而促进NIH的目标。