金纳米棒——从可控制备与修饰到纳米生物学与生物医学应用

金纳米棒因其独特的光学活性(纵向和横向两个等离子体共振吸收峰,可调范围从可见光区到近红外区)、长径比可调,表面易于修饰,生物相容性良好而使得其在纳米生物学和生物医学等领域具有广泛的应用前景。金纳米棒的合成及表面修饰直接决定着其物理化学性质,进而影响其生物相容性及其在生物医学中的应用。本文综述了金纳米棒的可控制备方法(包括模板法、电化学法、光化学法和晶种法)、表面可控修饰方法及其在纳米生物学和生物医学中的应用新进展,重点总结了金纳米棒的表面可控修饰及其在分子探针、生物传感、生物成像、药物载体、基因载体和光热疗法的最新研究进展。最后针对金纳米棒在生物应用过程中的一些瓶颈问题(如:特异性识别能力需要增强和荧光量子产率尚待提高等)提出了将手性分子或智能聚合物引入到金纳米棒表面进行可控修饰,以期增强其特异性识别能力并提高荧光量子产率,为金纳米棒的发展提供了新的思路。     

A Coordinative Dendrimer Achieves Excellent Efficiency in Cytosolic Protein and Peptide Delivery

Cytosolic protein delivery is a prerequisite for the development of protein therapeutics that act on intracellular targets. Proteins are generally membrane‐impermeable and thus need a carrier such as a polymer to facilitate their internalization. However, the efficient binding of proteins with different isoelectric points to polymeric carriers is challenging. In this study, we designed a coordinative dendrimer to solve this problem. The dendrimers modified with dipicolylamine/zinc(II) complex were capable of binding proteins through a combination of ionic and coordination interactions. The best polymer efficiently delivered 30 cargo proteins and peptides into the cytosol, while maintaining their bioactivity after intracellular release. The removal or replacement of zinc ions in the polymer with other transition‐metal ions lead to significantly decreased efficiency in cytosolic protein delivery. This study provides a new strategy to develop robust and efficient polymers for cytosolic protein delivery.     

Facile fabrication of large area of aggregated gold nanorods film for efficient surface-enhanced Raman scattering

An effective and facile method for fabrication of large area of aggregated gold nanorods (AuNRs) film was proposed by self-assembly of AuNRs at a toluene/water interface for the first time. It was found that large area of aggregated AuNRs film could be formed at the interface of toluene and water due to the interfacial tension between the two phases. The obtained large area of aggregated AuNRs film exhibits strong surface-enhanced Raman scattering (SERS) activity with 4-aminothiophenol (4-ATP) and 2-aminothiophenol (2-ATP) as the probe molecules based on the strong electromagnetic coupling effect between the very adjacent AuNRs. Enhancement factors (EF) were used to estimate the SERS activity of the aggregated AuNRs film, which is obtained to be 1.7x10(5) for 7a vibration of 4-ATP. SERS intensity is compared with AuNRs deposited directly on glass, indicating high SERS activity and reproducibility of the aggregated AuNRs film. In addition, SERS activity has also been successfully demonstrated for dye molecule (Rhodamin 6G (R6G)) and biological small molecule (adenine) on the aggregated AuNRs film, showing great potential of the aggregated AuNRs film as a convenient and powerful SERS substrate for biological tags and biological molecular detection.     

Engineering Nucleotidoproteins for Base-Pairing-Assisted Cytosolic Delivery and Genome Editing

Protein therapeutics targeting intracellular machineries hold profound potential for disease treatment, and hence robust cytosolic protein delivery technologies are imperatively demanded. Inspired by the super-negatively charged, nucleotide-enriched structure of nucleic acids, adenylated pro-proteins (A-proteins) with dramatically enhanced negative surface charges have been engineered for the first time via facile green synthesis. Then, thymidine-modified polyethyleneimine is developed, which exhibits strong electrostatic attraction, complementary base pairing, and hydrophobic interaction with A-proteins to form salt-resistant nanocomplexes with robust cytosolic delivery efficiencies. The acidic endolysosomal environment enables traceless restoration of the A-proteins and consequently promotes the intracellular release of the native proteins. This strategy shows high efficiency and universality for a variety of proteins with different molecular weights and isoelectric points in mammalian cells. Moreover, it enables highly efficient delivery of CRISPR-Cas9 ribonucleoproteins targeting fusion oncogene EWSR1-FLI1, leading to pronounced anti-tumor efficacy against Ewing sarcoma. This study provides a potent and versatile platform for cytosolic protein delivery and gene editing, and may benefit the development of protein pharmaceuticals.     

二元混合表面活性剂用于窄吸收金纳米棒的无种子合成

利用十六烷基三甲基溴化铵(CTAB)和油酸钠(NaOL)二元混合表面活性剂体系, 开发了一种高质量金纳米棒(AuNRs)的无种子合成方法. 通过透射电子显微镜(TEM)、 紫外-可见-近红外吸收光谱(UV-Vis-NIR)和热成像仪对金纳米棒的形貌、 光学性质及光热性能进行了表征. 实验结果表明, 当NaOL浓度为8.21~11.5 mmol/L时, 能够获得形貌均匀的AuNRs, 其纵向表面等离子体共振吸收(LSPR)在650~1150 nm范围内可调. 该方法制得的样品具有较窄的LSPR半峰宽, 特别是在制备LSPR在近红外二区(NIR-II, 大于1000 nm)的AuNRs方面具有明显优势. 在1064 nm激光的辐照下, 金纳米棒溶液能够快速升温至67 ℃, 光热转换效率可达31.5%, 同时表现出优秀的光热稳定性, 在近红外二区光声成像和光热治疗方面具有良好的应用价值.     

Magnetically Guided Protein Transduction by Hybrid Nanogel Chaperones with Iron Oxide Nanoparticles

Protein pharmaceuticals show great therapeutic promise, but effective intracellular delivery remains challenging. To address the need for efficient protein transduction systems, we used a magnetic nanogel chaperone (MC): a hybrid of a polysaccharide nanogel, a protein carrier with molecular chaperone‐like properties, and iron oxide nanoparticles, enabling magnetically guided delivery. The MC complexed with model proteins, such as BSA and insulin, and was not cytotoxic. Cargo proteins were delivered to the target HeLa cell cytosol using a magnetic field to promote movement of the protein complex toward the cells. Delivery was confirmed by fluorescence microscopy and flow cytometry. Delivered β‐galactosidase, inactive within the MC complex, became enzymatically active within cells to convert a prodrug. Thus, cargo proteins were released from MC complexes through exchange interactions with cytosolic proteins. The MC is a promising tool for realizing the therapeutic potential of proteins.     

Phosphorothioated DNA Engineered Liposomes as a General Platform for Stimuli-Responsive Cell-Specific Intracellular Delivery and Genome Editing

Intracellular protein delivery is highly desirable for protein drug-based cell therapy. Established technologies suffer from poor cell-specific cytosolic protein delivery, which hampers the targeting therapy of specific cell populations. A fusogenic liposome system enables cytosolic delivery, but its ability of cell-specific and controllable delivery is quite limited. Inspired by the kinetics of viral fusion, we designed a phosphorothioated DNA coatings-modified fusogenic liposome to mimic the function of viral hemagglutinin. The macromolecular fusion machine docks cargo-loaded liposomes at the membrane of target cells, triggers membrane fusion upon pH or UV light stimuli, and facilitates cytosolic protein delivery. Our results showed efficient cell-targeted delivery of proteins of various sizes and charges, indicating the phosphorothioated DNA plug-in unit on liposomes could be a general strategy for spatial-temporally controllable protein delivery both in vitro and in vivo.     

The Early Life of Gold Nanorods: Temporal Separation of Anisotropic and Isotropic Growth Modes

Gold nanorods (AuNRs) are a particularly interesting class of nanomaterials because their dimensions and size-dependent optical properties make them ideally suited for many applications. AuNRs are typically synthesized using seeded growth approaches, in which a small spherical gold nanoparticle seed grows anisotropically into a rod-shaped particle. Using AuNRs themselves as seeds for the growth of other anisotropic shapes has been demonstrated but is relatively little-explored. In this study, we show that AuNRs grown using a common method (silver-assisted seeded growth) cannot be used as seeds in the synthesis of higher aspect ratio AuNRs. Instead, the seed AuNRs grow isotropically, providing a new synthetic approach to precisely tune the absolute dimensions of the final AuNRs. We furthermore show that the dimensions of the AuNRs are determined by the reaction conditions at very early times (<10?min), and that perturbing the growth solution beyond these times has little influence on the final AuNR properties. The observation of these behaviors may be relevant to ongoing investigations of AuNR growth mechanisms.     

Real-time observation of dynamic heterogeneity of gold nanorods on plasma membrane with darkfield microscopy

The plasma membrane possesses a complicated structure, on which the protein clusters are randomly but orderly distributed to maintain the regular morphology and function of cells. Investigating the detailed dynamic behaviors of nanoparticles(NPs) on cytomembrane is of great importance to understand cellular mechanisms and advance the bio-nano technologies for drug delivery, photothermal therapy, immunotherapy, etc. In this work, to study the dynamic heterogeneous interactions between NPs and cell membrane with high resolution, we established a simple method to efficiently track the translational and rotational diffusion of individual gold nanorods(AuNRs) on cell membranes. This method is based on that an anisotropic AuNR appears as a colored spot under a darkfield microscope(DFM) equipped with a color camera. While obtaining its lateral position, the polar angle of the AuNR can be calculated simultaneously from intensity difference between the R and G channels. Careful analysis shows that the lateral motion of single AuNRs do not follow normal Brownian diffusion, which could be attributed to their hop diffusion in the dynamically varying picket-fence structure of the live cell membrane. Furthermore, 4 different rotationtranslation patterns of the AuNR are observed due to spatiotemporal heterogeneity of the cytomembrane. This simple but robust method for simultaneously obtaining the location and orientation of anisotropic plasmonic nanoparticles could be further applied to the analysis of complicated biological and biomedical processes.     

Tailoring Cationic Helical Polypeptides for Efficient Cytosolic Protein Delivery

Protein delivery is of central importance for both diagnostic and therapeutic applications.However,protein delivery faces challenges including poor endosomal escape and thus limited efficiency.Here,we report the facile construction and screening of a small library of cationic helical polypeptides for cytosolic protein delivery.The library is based on a random copolymer poly(γ-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}esteryl-L-glutamate)-randompoly(γ-6-chlorohexyl-L-glutamate)[P(EG3-r-ClC6)Glu],which is then modified with various pyridine derivatives and alkyl thiols.Flow Cytometry,confocal laser scanning microscopy,and viability assay collaboratively identify two leading polymers,showing efficient delivery of enhanced green fluorescent protein(eGFP)and low cytoto-xicity.This finding is further validated by the cytosolic delivery of RNase A and cytochrome C(Cyt C)to HeLa cells in the viability assay.Together,this work demonstrates that high-throughput screening is an effective and viable approach to the selection of cationic helical polypeptides for the cytosolic delivery of functional proteins.     

Platelet‐Facilitated Photothermal Therapy of Head and Neck Squamous Cell Carcinoma

Here, we present a platelet‐facilitated photothermal tumor therapy (PLT‐PTT) strategy, in which PLTs act as carriers for targeted delivery of photothermal agents to tumor tissues and enhance the PTT effect. Gold nanorods (AuNRs) were first loaded into PLTs by electroporation and the resulting AuNR‐loaded PLTs (PLT‐AuNRs) inherited long blood circulation and cancer targeting characteristics from PLTs and good photothermal property from AuNRs. Using a gene‐knockout mouse model, we demonstrate that the administration of PLT‐AuNRs and localizing laser irradiation could effectively inhibit the growth of head and neck squamous cell carcinoma (HNSCC). In addition, we found that the PTT treatment augmented PLT‐AuNRs targeting to the tumor sites and in turn, improved the PTT effects in a feedback manner, demonstrating the unique self‐reinforcing characteristic of PLT‐PTT in cancer therapy.     

Protein Delivery System Containing a Nickel‐Immobilized Polymer for Multimerization of Affinity‐Purified His‐Tagged Proteins Enhances Cytosolic Transfer

Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea‐grafted polyethylenimine (πPEI) with affinity‐purified His‐tagged proteins pre‐organized onto a nickel‐immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His‐tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His‐tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single‐chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions.     

原位液体室透射电镜观察金纳米棒/石墨烯复合物的形成和运动过程

本文利用原位液体室透射电子显微镜实时观察了液态下金纳米棒/石墨烯复合物的动态自组装行为。结果表明,由于电荷吸引力,金纳米棒倾向于通过尖端接近方式靠近石墨烯的边缘。组装结构形成以后,金纳米棒与石墨烯边缘可以发生相对旋转,其中金纳米棒边缘贴合石墨烯边缘的结构更稳定,并且没有显示金纳米棒与石墨烯边缘之间的相对角度随时间的变化。观察到了自组装结构的漂移运动,与较小尺寸的自组装结构相比,较大尺寸的结构显得更难以通过液体流动推动运动,并且其运动更容易因为来自液体室窗口基底的阻力而慢下来。利用液体室透射电镜进一步观察石墨烯折叠结构,观察结果表明折叠结构可随时间在液体中打开和闭合,导致固定在石墨烯层上的金纳米棒表现出与石墨烯之间的明显相对位置变化。总体上,自组装结构非常稳定,并且在液体中没有表现出任何的分离行为。进一步,将金纳米棒/石墨烯复合物用作催化剂,在4-硝基苯酚催化还原实验中显示出比单纯金纳米棒更好的催化性能。投料质量比为1:5的金纳米棒/石墨烯复合物表现出最佳性能,表观速率常数值为0.5570min~(-1),是单纯金纳米棒的8倍。这一显著改善与优化稳定的金纳米棒/石墨烯复合物结构密切相关。原位液体室透射电镜为分析液体中复杂的自组装行为,及未来的高性能复合催化剂材料的开发,提供了一种强有力的表征方法。     

Optimization of Biocompatibility for a Hydrophilic Biological Molecule Encapsulation System

Despite considerable advances in recent years, challenges in delivery and storage of biological drugs persist and may delay or prohibit their clinical application. Though nanoparticle-based approaches for small molecule drug encapsulation are mature, encapsulation of proteins remains problematic due to destabilization of the protein. Reverse micelles composed of decylmonoacyl glycerol (10MAG) and lauryldimethylamino-N-oxide (LDAO) in low-viscosity alkanes have been shown to preserve the structure and stability of a wide range of biological macromolecules. Here, we present a first step on developing this system as a future platform for storage and delivery of biological drugs by replacing the non-biocompatible alkane solvent with solvents currently used in small molecule delivery systems. Using a novel screening approach, we performed a comprehensive evaluation of the 10MAG/LDAO system using two preparation methods across seven biocompatible solvents with analysis of toxicity and encapsulation efficiency for each solvent. By using an inexpensive hydrophilic small molecule to test a wide range of conditions, we identify optimal solvent properties for further development. We validate the predictions from this screen with preliminary protein encapsulation tests. The insight provided lays the foundation for further development of this system toward long-term room-temperature storage of biologics or toward water-in-oil-in-water biologic delivery systems.     

Direct Observation of the Orientation Dynamics of Single Protein‐Coated Nanoparticles at Liquid/Solid Interfaces

We have observed the rotational dynamics of single protein‐coated gold nanorods (AuNRs) on C₁₈‐modified silica surfaces in real time by dual‐channel polarization dark‐field microscopy. Four different rotational states were identified, depending on the apparent strength of interactions between the AuNRs and the surface. The distributions of the states could be regulated by adjusting the salt concentration, and the state transitions were verified by monitoring the entire desorption process of a single AuNR. Our study provides insight into the interfacial orientation and dynamics of nanoparticles and could be useful for in vitro biophysics and the separation of proteins.     

Protein-cation interactions: structural and thermodynamic aspects

Cations are specifically recognized by numerous proteins. Cations may play a structural role, as cofactors stabilizing their binding partners, or a functional role, as cofactors activating their binding partners or being themselves involved in enzymatic reactions. Despite their small size, their charge density and their specific interaction with highly charged residues allow them to induce significant conformational changes on their binding proteins. The protein conformational change induced by cation binding may be as large as to account for the complete folding of a protein (as evidenced in Hepatitis C NS3 protease, or human rhinovirus 2A protease), and they may also trigger oligomerization (as in calcium-binding protein 1). Especially intriguing is the ability of cation-binding proteins of discriminating between very similar cations. In particular, calcium and magnesium are recognized by proteins with markedly different binding affinities and cause significantly different conformational changes and stabilization effects in the binding proteins (as in the fifth ligand binding repeat of the LDL receptor binding domain, calcium-binding protein 1, or parvalbumin). This article summarizes recent findings on the structural and energetic impact of cation binding to different proteins. A general framework can be envisaged in which cations can be considered as a special type of allosteric effectors able to modulate the functional properties of proteins, in particular the ability to interact with biological targets, by altering their conformational equilibrium.     

Seedless synthesis of gold nanorods with tunable plasmonic peaks beyond 1300 nm

Gold nanorods(Au NRs), as relatively common materials used in biomedical areas, have been synthesized by means of many methods. However, the conventional seed-mediated method is limited by complex operations and low yield. Besides, for further applications of Au NRs, well monodispersed Au NRs and tunable longitudinal surface plasmon resonance(LSPR) remain to be improved. Herein, we report a one-pot method for synthesizing Au NRs without seeding agents. In this method, we use phenols as reducing ...     

Protein semi-synthesis in living cells

Incorporation of chemical probes into proteins is a powerful way to elucidate biological processes and to engineer novel function. Here we describe an approach that allows ligation of synthetic molecules to target proteins in an intracellular environment. A cellular protein is genetically tagged with one-half of a split intein. The complementary half is linked in vitro to the synthetic probe, and this fusion is delivered into cells using a transduction peptide. Association of the intein halves in the cytosol triggers protein trans-splicing, resulting in the ligation of the probe to the target protein through a peptide bond. This process is specific and applicable to cytosolic and integral membrane proteins. The technology should allow cellular proteins to be elaborated with a variety of abiotic probes.     

腰接型侧链液晶弹性体研究进展

液晶弹性体是交联型液晶大分子,兼具液晶取向有序性和交联聚合物熵弹性等特性,在传感器、触发器、微流体装置和仿生器件等方面具有很好的应用前景.制备液晶弹性体的微结构,探索其独特的刺激响应性,是目前液晶弹性体研究的重要方向.侧链液晶弹性体的液晶相态类型取决于其液晶基元和主链的连接方式.腰接型侧链液晶弹性体倾向于形成向列型液晶相,具有较快的响应速度和形变程度,是一类独特的液晶弹性体.本文重点介绍腰接型液晶弹性体微结构(如微米柱、微米线等)的制备;利用金纳米粒子的光热转换效应,实现液晶弹性体光响应性的新途径;以及腰接型侧链液晶弹性体仿生微结构的功能性等.同时还对该领域的发展前景进行了展望.     

Self-interaction nanoparticle spectroscopy: a nanoparticle-based protein interaction assay

Solution conditions conducive to protein crystallization are identified mainly in an empirical manner using screening methods. Measurements of a dilute solution thermodynamic parameter, the osmotic second virial coefficient, have been shown to be useful in guiding this search, yet the measurement of this parameter remains difficult. In this work, a nanoparticle-based assay, self-interaction nanoparticle spectroscopy, is presented as an efficient alternative. The method involves adsorbing proteins on the surface of gold nanoparticles and adding the protein/gold conjugates to solutions of interest for crystallization. The optical properties of gold colloid, including macroscopic ones such as color, are sensitive to the interparticle separation distance, and they are demonstrated to correlate with the value of the second virial coefficient for BSA and ovalbumin. Serendipitously, the conditions that correspond to second virial coefficient values within the thermodynamic region ideal for protein crystallization lead to the maximum change in color of the gold suspensions. Given the remarkable efficiency of this method, it holds significant potential to aid in the crystallization of proteins that have not been crystallized previously. Moreover, this method may find utility in the analysis of weak homo- and heterotypic interactions involved in other biological applications, including preventing protein aggregation and formulating therapeutic proteins.