Cytosolic Access and Instability of DNA nanoparticles

DNA 纳米颗粒的细胞质进入和不稳定性

• 批准号: 10701061
• 负责人: Divita Mathur
• 金额: $ 24.9万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10701061
• 关键词: Address; Animal Model; Architecture; Award; Behavior; Biological Models; Biological Sciences; Biomedical Engineering; Buffers; CRISPR/Cas technology; Calcium; Cancerous; Cell Culture Techniques; Cell Nucleus; Cells; Confocal Microscopy; Cytosol; DNA; Dictyoptera; Dose; Drug Carriers; Endocytosis; Endosomes; Engineering; Environment; Family suidae; Fibroblasts; Fluorescence Resonance Energy Transfer; Gene Silencing; Genes; Goals; Grant; Hypoxia; Immune; In Vitro; Kinetics; Learning; Ligands; Lysosomes; Mammalian Cell; Maps; Measures; Mentorship; Messenger RNA; Microinjections; Mus; Nanotubes; National Institute of Biomedical Imaging and Bioengineering; Nucleic Acids; Outcome; Physiology; Process; Property; Protein Biosynthesis; Proteins; Regulation; Reporter; Research; Resolution; Role; Science; Series; Site; Small Interfering RNA; Spectrum Analysis; Structure; System; Technical Expertise; Testing; Therapeutic; Therapeutic Agents; Training; Training Programs; Transfection; Transformed Cell Line; Translations; Untranslated RNA; Vesicle; Work; Writing; biomaterial compatibility; cell type; cytotoxic; delivery vehicle; gene therapy; improved; in vitro Model; in vivo Model; innovative technologies; nanoparticle; neoplastic cell; nuclease; nucleic acid-based therapeutics; particle; pharmacologic; physical science; programs; protein expression; receptor mediated endocytosis; recruit; self assembly; targeted treatment; therapeutic RNA; therapeutic nanoparticles; tool; tumor; uptake

PROJECT SUMMARY/ABSTRACT: A number of candidate therapies such as CRISPR-Cas9 and gene silencing require the efficient delivery of functional nucleic acids to the cell cytosol and nucleus. Unfortunately, such therapies currently lack proper delivery mechanisms, precluding their widespread applicability. Self- assembled deoxyribonucleic acid (DNA) nanoparticles have shown potential as minimally cytotoxic therapeutic carriers in cancerous and other in vitro and in vivo models. While evidence suggests that DNA nanoparticles-based drug carriers can be taken up by mammalian cells via endocytosis, it is unknown how these DNA nanoparticles can overcome the fate of endocytosis-triggered degradation to reach the cytosol and, once there, can controllably maintain stability. With the enabling science explaining their behavior and mechanisms of controlling their stability in the cell cytosol it will be possible to make bold advances in engineering therapeutic delivery systems. To that end, the proposed work has two overarching scientific payoffs. Payoff 1, induce endosomal escape and enhanced cytosolic accessibility of DNA nanoparticles by the integration of calcium in their assembly process. Payoff 2, identify the rate of breakdown and mechanisms of stabilization of DNA nanoparticles in different types of cell cytosols. Innovative technologies will be the foci of the PI's training program and will be implemented to achieve the project goals, namely, multi-step Förster resonance energy transfer spectroscopy for high-resolution tracking of DNA nanoparticle inside the cell and in vitro cell microinjections enabling study of these nanoparticles directly in the cytosolic environment. First, a DNA origami based nanotube will be tested for structural stability in calcium-supplemented buffer. Thereafter, the nanotube will be used as a carrier for the delivery of functional RNA molecules to representative fluorescent protein-expressing cells and checked for its cytosolic reachability and efficacy in protein regulation after undergoing endocytosis. Second, small (20 nm) DNA nanoparticles with branched architecture and non-canonical nucleic acids will be embedded with multi-step FRET reporters for measuring structural integrity. These DNA nanoparticles will be microinjected into live cells cytosolic region and their breakage be determined. Last, the cytosolic stability of these DNA nanoparticles will be correlated with different types of mammalian cells with known cytosolic variability (tumor, immune, and other cell types) in order to map the role of structurally diverse DNA nanoparticles in targeting cells with different physiologies. The PI will also receive training in rigorous analysis of in vitro research, lab management, and the prolific grant writing process, which will facilitate their transition to an independent research program. Outcomes of this project will pave the way towards developing more bio-compatible delivery systems, specifically for functional nucleic acid therapeutic agents that are vital in the cell cytosol.

项目摘要/摘要：许多候选疗法，例如CRISPR-CAS9和GENE 沉默需要将功能核酸递送到细胞胞质和核。 目前，此类疗法缺乏适当的交付机制，因此排除了广泛的应用。 组装的脱氧核糖核酸（DNA）纳米颗粒已显示为最小细胞毒性的潜力 癌和其他体外和体内模型中的治疗载体。 基于纳米颗粒的药物载体可以通过哺乳动物细胞通过内吞作用来吸收 这些DNA纳米颗粒如何克服内吞作用触发的降解的命运到达 细胞质和一旦可以控制稳定性。 它们控制其在细胞胞质溶胶中稳定性的行为和机制，可以使大胆 工程治疗系统的进步。 科学收益。 纳米颗粒通过钙在组装过程中的整合。 DNA纳米颗粒稳定的分解和机制，不同类型的细胞胞质中。 创新技术将成为PI培训计划的焦点，并将实施以实现脚趾 项目目标，即高分辨率的多步förster共振能量传递光谱 跟踪细胞内的DNA纳米属和体外注射启用这些 纳米颗粒直接在胞质环境中。 钙补充的缓冲液的结构稳定性。 功能性RNA分子递送到抑制表达荧光蛋白的细胞并检查 因为胞质的蛋白质调节性是胞质的性能和疗效 （20 nm）具有分支结构和非当核酸的DNA纳米颗粒将嵌入 使用多步兵记者进行结构完整性。 将微型注射到活细胞中，并确定其胞质稳定性。 这些DNA纳米颗粒将与不同类型的cymmalian细胞相关 可变性（肿瘤，免疫和其他细胞类型），以绘制结构多样的DNA的作用 具有不同生理的靶向细胞中的纳米颗粒也将在严格的 分析体外研究，实验室管理和Prolitic赠款写作过程，这将有助于 他们过渡到独立研究计划。 开发更多与生物兼容的输送系统，该系统的功能性核酸性治疗剂的特殊性 在细胞细胞质中至关重要。

项目成果

Hybrid Nucleic Acid-Quantum Dot Assemblies as Multiplexed Reporter Platforms for Cell-Free Transcription Translation-Based Biosensors.

• DOI: 10.1021/acssynbio.2c00394
• 发表时间: 2022-11
• 期刊: ACS synthetic biology
• 影响因子: 4.7
• 作者: Divita Mathur;Meghna Thakur;S. A. Díaz;K. Susumu;M. Stewart;E. Oh;Scott A. Walper;Igor L. Medintz

DNA origami presenting the receptor binding domain of SARS-CoV-2 elicit robust protective immune response.

• DOI: 10.1038/s42003-023-04689-2
• 发表时间: 2023-03-23
• 期刊: Communications biology
• 影响因子: 5.9

Structural and optical variation of pseudoisocyanine aggregates nucleated on DNA substrates.

• DOI: 10.1088/2050-6120/acb2b4
• 发表时间: 2023-01-31
• 期刊: Methods and applications in fluorescence
• 影响因子: 3.2
• 作者: Chiriboga M;Green CM;Mathur D;Hastman DA;Melinger JS;Veneziano R;Medintz IL;Díaz SA
• 通讯作者: Díaz SA

Tunable Electronic Structure via DNA-Templated Heteroaggregates of Two Distinct Cyanine Dyes.

• DOI: 10.1021/acs.jpcc.2c04336
• 发表时间: 2022-10-13
• 期刊: JOURNAL OF PHYSICAL CHEMISTRY C
• 影响因子: 3.7
• 作者: Huff, Jonathan S.;Diaz, Sebastian A.;Barclay, Matthew S.;Chowdhury, Azhad U.;Chiriboga, Matthew;Ellis, Gregory A.;Mathur, Divita;Patten, Lance K.;Roy, Simon K.;Sup, Aaron;Biaggne, Austin;Rolczynski, Brian S.;Cunningham, Paul D.;Li, Lan;Lee, Jeunghoon;Davis, Paul H.;Yurke, Bernard;Knowlton, William B.;Medintz, Igor L.;Turner, Daniel B.;Melinger, Joseph S.;Pensack, Ryan D.
• 通讯作者: Pensack, Ryan D.

Uptake and stability of DNA nanostructures in cells: a cross-sectional overview of the current state of the art.

• DOI: 10.1039/d2nr05868e
• 发表时间: 2023-02-09
• 期刊: Nanoscale
• 影响因子: 6.7