Biomedical Studies and Cellular Imaging via Atomic Force Microscopy

我们继续开发选择性生物物理测量系统(生物原子力显微镜(Bio-AFM)平台、石英晶体微平衡-耗散(QCM-D)以及光学显微镜和光谱分析)，并与NIH内外杰出的科学家合作，将这些技术应用于重要的生物医学研究。我们正致力于多功能、多模式和多平台AFM成像和单分子力谱(SMFS)在细胞和大分子研究中的更广泛和更有洞察力的应用。 在生物医学应用方面，我们继续开展广泛的合作，包括： (1)我们致力于开发更好的临床疫苗，以增强免疫反应并最终根除疟疾。几年来，通过Bio-AFM和相关生物分析，我们与David Narum博士(NIAID，NIH)和其他合作者一起研究了More和疟疾候选疫苗以及病毒样颗粒(VLP)或脂质体载体的大分子结构和纳米机械特性。这些疟疾蛋白抗原和疫苗载体是通过重组蛋白生物技术生产的，经过提纯，并以适合人体试验和扩大生产的方式进行表征。使用Bio-AFM成像和力光谱在单个大分子和组装水平上的生物物理表征有助于在发育阶段定义这些疫苗结构。在这一年里，我们致力于提取和表征恶性疟原虫脂筏和GPI锚定蛋白，以提高对疟疾寄生虫和病原体-宿主相互作用的机制的了解。这项工作是在我们发表的对恶性疟原虫环子孢子蛋白(CSP)可逆构象变化和粘附域掩蔽的观察之后进行的，CSP是疟疾疫苗的主要靶点。 (2)我们扩大了在多功能纳米医学和治疗学方面的合作，与NIBIB分子成像和纳米医学实验室的陈晓元博士以及NIBIB的黄鹏博士和NIBIB的王哲博士等共同研究人员。专注于了解和抗击癌症，我们参与了几项已发表和正在进行的研究，例如使用肿瘤特异性形成的酶指导的超分子组件作为癌症治疗药物。我们正在应用Bio-AFM和QCM-D方法来研究纳米粒子治疗、癌细胞和干细胞以及其他生物医学系统。 (3)我们继续与艾琳·拉弗教授和Rui Sousa教授(Univ.Lafer)等合作者进行了多年的生物原子力显微镜(Bio-AFM)对蛋白质网状蛋白及其组装的研究。Ralph Nossal博士(NICHD)和Dan Sackett博士(NICHD)致力于受体介导的内吞作用和细胞内转运这一关键领域。这种合作已经扩展到微管与几种已批准或正在开发的抗癌药物相互作用的生物AFM研究。与胞吐和内吞有关，我们还与吴令刚博士(NINDS)和同事合作，更好地了解大脑中的突触传递和神经元通讯。 (4)我们与NIDCR细胞与发育生物学实验室的Andrew Doyle博士和Kenneth Yamada博士以及西班牙加泰罗尼亚生物工程研究所的Raimon Sunyer博士合作，通过Bio-AFM力谱研究重组细胞外基质(ECM)样胶原蛋白凝胶的纳米力学和结构特性，以研究细胞黏附、迁移和其他动态行为。我们拥有完善的力谱和定量纳米技术图谱(QNM)方法，以探索更完整的组织模拟软材料和组织特定的细胞外基质。 (5)与柯蒂斯·默斯博士(NIST)及其同事一起，我们推进了对阿尔茨海默病中淀粉样β纤维的生物原子力显微镜和生物物理研究，特别是在生理相关的液体和表面环境中解决组装途径的问题。我们与齐鲁教授(特拉华州立大学)和合作研究人员一起推进了一个项目，关于纳米颗粒对脂膜结构域和膜性质的影响。最后，我们正在将AFM和光学显微镜结合在其他持续和新的合作中，包括生物膜、蛋白质组装、DNA和蛋白质交联、DNA-蛋白质相互作用和细胞周期，这些对疾病机制和生物纳米技术至关重要。

We have continued to develop selective biophysical measurement systems (biological atomic force microscopy (Bio-AFM) platforms, Quartz Crystal Microbalance-Dissipation (QCM-D), and optical microscopy and spectroscopy), and to apply these technologies to important biomedical investigations in collaboration with outstanding NIH intramural and extramural scientists. We are working toward broader and more insightful applications of multifunctional, multimodal, and multiplatform AFM imaging and single molecule force spectroscopy (SMFS) for cellular and macromolecular studies. On biomedical applications, we have continued our broad range of collaborations that include: (1) We have advanced our commitment to developing a better clinical vaccine toward enhanced immunological response and eventual eradication of malaria. Over several years and via Bio-AFM and related bioanalysis, we have investigated the macromolecular structure and nanomechanical properties of more and malaria vaccine candidates and virus-like-particle (VLP) or liposome carriers with Dr. David Narum (NIAID, NIH) and other collaborators. These malaria protein antigens and vaccine carriers are produced via recombinant-protein biotechnology, purified, and characterized in a manner suitable for human trials and scale-up production. Biophysical characterization at single macromolecule and assembly level using Bio-AFM imaging and force spectroscopy are helping to define these vaccine constructs along the developmental phases. In this year, we worked on extracting and characterizing Plasmodium falciparum lipid rafts and GPI-anchored proteins to improve mechanistic understanding of the malaria parasites and pathogen-host interactions. This work follows upon our published observation of a reversible conformation change and adhesion domain masking in the Plasmodium falciparum circumsporozoite protein (CSP), the leading malaria vaccine target. (2) We have expanded our collaborations on multifunctional nanomedicine and theranostics with Dr. Xiaoyuan Chen (laboratory of Molecular Imaging and Nanomedicine, NIBIB) and co-investigators including Dr. Peng Huang (NIBIB) and Dr. Zhe Wang (NIBIB). Focusing on understanding and fighting cancer, we have contributed to several published and ongoing studies such as using tumor-specific formation of enzyme-instructed supramolecular assemblies as cancer theranostics. We are applying Bio-AFM and QCM-D methodology to investigate nanoparticle theranostics, cancer cells and stem cells, and other biomedical systems. (3) We have continued our multi-year Bio-AFM studies of protein clathrin and assemblies with collaborators including Prof. Eileen Lafer and Prof. Rui Sousa (Univ. Texas Health Sciences Center, San Antonio), Dr. Ralph Nossal (NICHD) and Dr. Dan Sackett (NICHD)in the key area of the receptor-mediated endocytosis and intracellular trafficking. This collaboration has expanded toward bio-AFM studies of microtubules interacting with several approved or developing anti-cancer drugs. Related to exocytosis and endocytosis, we are collaborating also with Dr. Ling-gang Wu (NINDS) and coworkers to better understand synaptic transmission and neuronal communications in brain. (4) We have collaborated with Dr. Andrew Doyle and Dr. Kenneth Yamada (Laboratory of Cell and Developmental Biology, NIDCR) and with Dr. Raimon Sunyer (Institute for Bioengineering of Catalonia, Spain) on nanomechanics and structural properties of reconstituted extra cellular matrix (ECM)-like collagen gels via Bio-AFM force spectroscopy to investigate cell adhesion, migration, and other dynamic behaviors. We have refined force spectroscopy and quantitative nanomechnical mapping (QNM) approaches to explore more completely tissue-mimicking soft material and tissue specific extracellular matrices. (5) With Dr. Curtis Meuse(NIST) and coworkers, we have advanced our Bio-AFM and biophysical studies of amyloid-beta fibrils in the Alzheimer's disease, especially to resolve assembly pathways in physiologically relevant fluid and surface environments. We advanced on a project with Prof. Qi Lu (Delaware State University) and co-investigators, on the effect of nanoparticles on lipid domains and membrane properties. Finally, we are combining AFM and optical microscopy in other continuing and new collaborations, including biomembranes, protein assemblies, DNA and protein crosslinking, DNA-protein interactions and cell cycles, that are critically important to disease mechanisms and bionanotechnology.