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：一些候选疗法，如CRISPR-Cas9和GINE 沉默需要有效地将功能核酸输送到细胞、胞浆和细胞核。不幸的是， 这种疗法目前缺乏适当的传递机制，使其无法广泛应用。自我- 组装的脱氧核糖核酸(DNA)纳米颗粒显示出最小的细胞毒性潜力 肿瘤和其他体外和体内模型中的治疗性载体。虽然有证据表明DNA 纳米粒药物载体可通过内吞作用被哺乳动物细胞摄取，目前尚不清楚 这些DNA纳米颗粒如何克服内吞作用引发的降解的命运 细胞液，一旦到达，就可以控制地保持稳定。用使能科学来解释 它们的行为和控制它们在细胞胞浆中稳定性的机制将有可能大胆地 工程化治疗给药系统的研究进展。为此，拟议的工作有两个主要方面 科学回报。收益1，诱导内体逃逸和提高DNA的胞浆可及性 纳米颗粒通过在其组装过程中整合钙来实现。收益2，确定 DNA纳米颗粒在不同类型细胞胞浆中的分解及其稳定机制。 创新技术将是PI培训方案的重点，并将实施以实现 项目目标，即用于高分辨率的多步Förster共振能量转移光谱 追踪细胞内的DNA纳米颗粒和体外细胞微注射使这些研究成为可能 纳米粒子直接存在于胞浆环境中。首先，将测试一种基于DNA折纸的纳米管 补钙缓冲液的结构稳定性。此后，纳米管将被用作 将功能性RNA分子运送到具有代表性的荧光蛋白表达细胞并检查 因为它在细胞内的可达性和在接受内吞作用后对蛋白质调节的有效性。第二，小规模 具有分支结构和非规范核酸的(20 Nm)DNA纳米粒子将被嵌入 用多步FRET记者测量结构的完整性。这些DNA纳米粒子将是 显微注射到活细胞胞浆区，并测定其破坏情况。最后，胞浆稳定性。 这些DNA纳米粒子将与不同类型的哺乳动物细胞相关联，这些细胞具有已知的胞浆 可变性(肿瘤、免疫和其他细胞类型)，以确定结构不同的DNA的作用 纳米颗粒靶向不同生理状态的细胞。PI还将接受严格的培训 分析体外研究、实验室管理和多产的拨款申请过程，这将有助于 他们向独立研究项目的过渡。该项目的成果将为 开发更多生物相容的递送系统，特别是功能核酸治疗剂 在细胞胞浆中是至关重要的。

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.

Determining interchromophore effects for energy transport in molecular networks using machine-learning algorithms.

• DOI: 10.1039/d2cp04960k
• 发表时间: 2023-02-01
• 期刊: PHYSICAL CHEMISTRY CHEMICAL PHYSICS
• 影响因子: 3.3
• 作者: Rolczynski, Brian S. S.;Diaz, Sebastian A.;Kim, Young C. C.;Mathur, Divita;Klein, William P. P.;Medintz, Igor L. L.;Melinger, Joseph S. S.
• 通讯作者: Melinger, Joseph S. S.