多肽自组装及其在生物医学中的应用

多肽自组装广泛存在于自然界中。自组装多肽分子成分简单,生物相容性良好,自组装过程受多方面因素的影响,在生物医学材料方面具有巨大的应用前景。介绍了多肽分子自组装技术的概念和多肽自组装过程中的影响因素(氨基酸的组成和序列、温度、pH值、离子强度、多肽浓度、超声波),综述了多肽自组装系统的种类和多肽自组装技术在创伤修复、药物释放载体以及组织工程支架方面的应用。     

Constructing biomaterials using self-assembling peptide building blocks

Molecular self-assembly is ubiquitous in nature and has recently emerged as a new bottom-up approach in constructing biomaterials. Synthetic peptides assemble through specific molecular recognition and form diverse nanostructures. The resulting versatile peptide self-assemblies may be used in a wide range of biological and medical applications. Examples of two self-assembling peptide systems are presented and techniques for self-assembly control are discussed.     

Probing peptide nanotube self-assembly at a liquid-liquid interface with coarse-grained molecular dynamics

Self-assembly at a liquid-liquid interface is a powerful experimental route to novel nanomaterials. We report herein a computational study of peptide nanotube formation at an oil-water interface. We probe interfacial self-assembly and nanotube formation of the cyclic octapeptide, cyclo [(-L-Trp-D-Leu-)4] as an illustrative example. Individual peptide rings are rapidly adsorbed at the liquid-liquid interface where they self-assemble. Monomeric and dimeric peptide rings lie with their molecular planes mostly parallel to the interface. Longer oligomeric nanotubes are increasingly tilted at the interface and grow by an Oswald ripening mechanism to eventually align their tube axis parallel to the interface. The present results on nanotube assembly suggest that computation will be a useful complement to experiment in understanding the nature of self-assembly of nanomaterials at liquid-liquid interfaces.     

A reactive peptidic linker for self-assembling hybrid quantum dot-DNA bioconjugates

Self-assembly of proteins, peptides, DNA, and other biomolecules to semiconductor quantum dots (QD) is an attractive bioconjugation route that can circumvent many of the problems associated with covalent chemistry and subsequent purification. Polyhistidine sequences have been shown to facilitate self-assembly of proteins and peptides to ZnS-overcoated CdSe QDs via complexation to unoccupied coordination metal sites on the nanocrystal surface. We describe the synthesis and characterization of a thiol-reactive hexahistidine peptidic linker that can be chemically attached to thiolated-DNA oligomers and mediate their self-assembly to CdSe-ZnS core-shell QDs. The self-assembly of hexahistidine-appended DNA to QDs is probed with gel electrophoresis and fluorescence resonance energy transfer techniques, and the results confirm high-affinity conjugate formation with control over the average molar ratio of DNA assembled per QD. To demonstrate the potential of this reactive peptide linker strategy, a prototype QD-DNA-dye molecular beacon is self-assembled and tested against both specific and nonspecific target DNAs. This conjugation route is potentially versatile, as altering the reactivity of the peptide linker may allow targeting of different functional groups such as amines and facilitate self-assembly of other nanoparticle-biomolecule structures.     

Formation and electrochemical investigation of ordered cobalt coordinated peptide monolayers on gold substrates

The monolayers composed of cobalt coordinated peptides were prepared on gold substrates by two different approaches. One was the self-assembly method, which was used to prepare a peptide monolayer on the gold substrate via the spontaneous attachment of peptides owing to the interaction between gold and sulfur at the N-terminal of the peptide. The other one was the stepwise polymerization method that was utilized to fabricate the unidirectionally arranged peptide monolayer by the stepwise condensation of amino acids from the initiator fixed on the gold substrate. Leu₂Ala(4-Pyri)Leu₆Ala(4-Pyri)Leu₆ sequence was chosen as the cobalt coordinated peptide. The 4-pyridyl alanines, Ala(4-Pyri)s, were introduced as ligands for cobalt to the leucine-rich sequential peptide. The complexation between cobalt and pyridyl groups of the peptide induced the formation of a stable α-helical bundle, which oriented perpendicularly to the substrate surface. In the case of the monolayer fabricated by the stepwise polymerization method, the direction of the peptide macro-dipole moment aligned unidirectionally, and the cobalt complexes were fixed in the monolayer to form the ordered arrangement. On the other hand, the peptides prepared by the self-assembly method formed the mixture of parallel and antiparallel packing owing to the dipole-dipole interaction. The spatial location of the cobalt complexes in the monolayer prepared by the self-assembly method was distorted, compared with that in the monolayer fabricated by the stepwise polymerization method. The vectorial electron flow through the peptide monolayer was achieved by the regular alignment of the peptide macro-dipole moment and the cobalt complexes in the monolayer fabricated by the stepwise polymerization method.     

Fibrillized peptide microgels for cell encapsulation and 3D cell culture

One of the advantages of materials produced by self-assembly is that in principle they can be formed in any given container to produce materials of predetermined shapes and sizes. Here, we developed a method for triggering peptide self-assembly within the aqueous phase of water-in-oil emulsions to produce spherical microgels composed of fibrillized peptides. Size control over the microgels was achieved by specification of blade type, speed, and additional shear steps in the emulsion process. Microgels constructed in this way could then be embedded within other self-assembled peptide matrices by mixing pre-formed microgels with un-assembled peptides and inducing gelation of the entire composite, offering a route towards multi-peptide materials with micron-scale domains of different peptide formulations. The gels themselves were cytocompatible, as was the microgel fabrication procedure, enabling the encapsulation of NIH 3T3 fibroblasts and C3H10T-1/2 mouse pluripotent stem cells with good viability.     

Presentation and recognition of biotin on nanofibers formed by branched peptide amphiphiles

A branched peptide amphiphile system was designed for enhanced recognition of biotin on nanofibers formed by self-assembly of these molecules. Branching at a lysine residue was used to design peptide amphiphiles that are capable of presenting more than one epitope per molecule. We found that biotinylated branched structures form nanofibers that enhance recognition by the avidin protein receptor relative to similar nanostructures formed by linear peptide analogues. Biotin-avidin binding to the supramolecular nanofibers was characterized by measurement of fluorescence from nanofibers incubated with chromophore-conjugated avidin.     

Enzyme-Instructed Self-Assembly (EISA) and Hydrogelation of Peptides

Self-assembly is a powerful tool for constructing supramolecular materials for many applications, ranging from energy harvesting to biomedicine. Among the methods to prepare supramolecular materials for biomedical applications, enzyme-instructed self-assembly (EISA) has several advantages. Herein, the unique properties and advantages of EISA in preparing biofunctional supramolecular nanomaterials and hydrogels from peptides are highlighted. EISA can trigger molecular self-assembly in situ. Therefore, using overexpression enzymes in disease sites, supramolecular materials can be formed in situ to improve the selectivity and efficacy of the treatment. The precursor may be involved during the EISA process, and it is actually a two-component self-assembly process. The precursor can help to stabilize the assembled nanostructures of hydrophobic peptides formed by EISA. More importantly, the precursor may determine the outcome of molecular self-assembly. Recently, it was also observed that EISA can kinetically control the peptide folding and morphology and cellular uptake behavior of supramolecular nanomaterials. With the combination of other methods to trigger molecular self-assembly, researchers can form supramolecular nanomaterials in a more precise mode and sometimes under spatiotemporal control. EISA is a powerful and unique methodology to prepare supramolecular biofunctional materials that cannot be generated from other common methods.     

Histidine as a key modulator of molecular self-assembly: Peptide-based supramolecular materials inspired by biological systems

Histidine, a versatile proteinogenic amino acid, plays a broad range of roles in all living organisms and behaves as a key mediator of the interactions of biomolecules with inorganic constituents. The self-assembly of histidine-rich peptides and proteins is critical in biology, as the histidine unit is both a multifunctional regulator and an ideal motif for the construction of complex biological structures. In particular, non-covalent interactions between the imidazole ring and other molecular building blocks and metal ions are routinely employed to generate these complexes. Therefore, this strategy can be duplicated in an artificial context to create sophisticated bioactive materials. In this review, we first highlight a clear perspective of the bio-inspired design strategies which can replicate the hierarchical structure of biological systems allowing the engineering of the supramolecular self-assembly of histidine-functionalized peptides. We further summarize advancements in the field of peptide supramolecular structures incorporating histidine residues in the peptide backbone to generate organized functional supramolecular biomaterials with customizable features. We also discuss significant advances and future prospects in supramolecular self-assembly of histidine-functionalized peptides, as well as provide an overview of advanced techniques for the fabrication of histidine-based biomaterials for bio-nanotechnology, optoelectronic engineering, and biomedicine. Overall, artificial supramolecular materials based on histidine functionalized peptides, motivated by the intriguing properties discovered in natural proteins, bear the potential to boost the creation of sustainable bio-inspired materials.     

Exploiting enzymatic (reversed) hydrolysis in directed self-assembly of peptide nanostructures

Enzyme-catalyzed reactions can be exploited to control molecular self-assembly under physiological conditions by converting nonassembling precursors into self-assembly building blocks. Two complementary approaches based on aromatic short-peptide derivatives that form molecular hydrogels are demonstrated. Firstly, it is shown that esterase-directed self assembly via hydrolysis of hydrophobic N-(fluorenyl-9-methoxycarbonyl) (Fmoc)-peptide methyl esters give rise to formation of transparent hydrogels composed of defined peptide nanotubes. The internal and external diameters of these tubes are highly tunable, depending on the amino acid composition and chain length of the building blocks. Secondly, protease-directed self-assembly of Fmoc-peptide esters is achieved via amide-bond formation (reversed hydrolysis) for combinations of Fmoc-threonine and leucine/phenylalanine methyl esters, producing fibrous hydrogels. Upon treatment with an esterase, these systems revert back to solution, thus providing a two-stage solution-gel-solution transition.     

Nanoscale biomimetics: fabrication and optimization of stability of peptide-based thin films

The method of thin film preparation known as layer-by-layer assembly is of growing interest for current and envisioned developments in bionanotechnology. Here, cysteine-containing 32mer peptides have been designed, synthesized, purified, and used to prepare polypeptide films. A range of methods-quartz crystal microbalance, Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and high-performance liquid chromatography-have been used to probe the effect of ionic strength and polymer secondary structure in solution on peptide self-assembly, and on secondary structure formation and disulfide bond cross-linking in the multilayer film. The amount of designed peptide deposited per adsorption step of film fabrication increased with increasing ionic strength, as with conventional polyelectrolytes. Secondary structure content changed from random coil to beta sheet on incorporation of peptides into a film. "Peptide-inherent" cross-linking by disulfide bond formation increased film stability at acidic pH. Conditions for disulfide stabilization have been optimized. The results contribute to exploration of the physical basis of peptide self-assembly broaden the scope of applications of layer-by-layer assembly, particularly where biocompatibility and stability are key design concerns, and provide a basis for mass production of custom polypeptide thin films of high stability, even in harsh environments.     

Dynamic behaviors of lipid-like self-assembling peptide A6D and A6K nanotubes

Nanoscience and nanotechnology require development of nanomaterials that are amiable for molecular design from bottom up. Molecular designer self-assembling peptides are one of such nanomaterials that will become increasingly important for the endeavor. Peptides have not only been used in all aspects of biomedical and pharmaceutical research and medical products, but also have had enormous impact in nascent field of designed biological materials. We here report the dynamic structures of lipid-like designer peptide A6D (AAAAAAD) and A6K (AAAAAAK) that undergo self-assembly into nanotubes in water and salt solution. We not only analyzed their self-assemblies using dynamic light scattering to determine the critical aggregation concentration (CAC), but also use atomic force microscope to observe their nanostructures. We also propose a simple scheme by which these lipid-like peptides self-assemble into dynamic nanostructures. Since the knowledge of CAC is important for uses of these peptides for a variety of applications, these findings may have significant implications in the study of molecular self-assembly and for a wide range of utilities of designer self-assembling peptide materials.     

Cation–π Interaction Trigger Supramolecular Hydrogelation of Peptide Amphiphiles

As an important noncovalent interaction, cation–π interaction plays an essential role in a broad area of biology and chemistry. Despite extensive studies in protein stability and molecular recognition, the utilization of cation–π interaction as a major driving force to construct supramolecular hydrogel remains uncharted. Here, a series of peptide amphiphiles are designed with cation–π interaction pairs that can self-assemble into supramolecular hydrogel under physiological condition. The influence of cation–π interaction is thoroughly investigated on peptide folding propensity, morphology, and rigidity of the resultant hydrogel. Computational and experimental results confirm that cation–π interaction could serve as a major driving force to trigger peptide folding, resultant β-hairpin peptide self-assembled into fibril-rich hydrogel. Furthermore, the designed peptides exhibit high efficacy on cytosolic protein delivery. As the first case of using cation–π interactions to trigger peptide self-assembly and hydrogelation, this work provides a novel strategy to generate supramolecular biomaterials.     

Hierarchical, interface-induced self-assembly of diphenylalanine: formation of peptide nanofibers and microvesicles

To gain insight into the hierarchical self-assembly of peptides and the surface effect on assembly formation, an aromatic peptide of diphenylalanine (FF) was used in this study as the model peptide. We found that the diphenylalanine peptide could self-assemble into a core-branched nanostructure through non-covalent interactions in aqueous solution. The pre-assemblies further assembled into nanofibers and microvesicles on the glass surface and microporous membrane, respectively, showing a significant dependence on surface characteristics. The structural and morphological differences between nanofibers and microvesicles were investigated directly using several spectroscopy and microscopy techniques. Our results revealed a hierarchical and interface-induced assembly behavior of diphenylalanine peptide. The novel strategy based on the surface effect allows one to controllably fabricate various peptide-based nanostructures.     

De novo design of bioactive protein-resembling nanospheres via dendrimer-templated peptide amphiphile assembly

Self-assembling peptide amphiphiles (PAs) have been extensively used in the development of novel biomaterials. Because of their propensity to form cylindrical micelles, their use is limited in applications where small spherical micelles are desired. Here we present a platform method for controlling the self-assembly of biofunctional PAs into spherical 50 nm particles using dendrimers as shape-directing scaffolds. This templating approach results in biocompatible, stable protein-like assemblies displaying peptides with native secondary structure and biofunctionality.     

Nucleation of apatite crystals in vitro by self-assembled dentin matrix protein 1

Bones and teeth are biocomposites that require controlled mineral deposition during their self-assembly to form tissues with unique mechanical properties. Acidic extracellular matrix proteins play a pivotal role during biomineral formation. However, the mechanisms of protein-mediated mineral initiation are far from understood. Here we report that dentin matrix protein 1 (DMP1), an acidic protein, can nucleate the formation of hydroxyapatite in vitro in a multistep process that begins by DMP1 binding calcium ions and initiating mineral deposition. The nucleated amorphous calcium phosphate precipitates ripen and nanocrystals form. Subsequently, these expand and coalesce into microscale crystals elongated in the c-axis direction. Characterization of the functional domains in DMP1 demonstrated that intermolecular assembly of acidic clusters into a beta-sheet template was essential for the observed mineral nucleation. Protein-mediated initiation of nanocrystals, as discussed here, might provide a new methodology for constructing nanoscale composites by self-assembly of polypeptides with tailor-made peptide sequences.     

Generation of protein lattices by fusing proteins with matching rotational symmetry

The self-assembly of supramolecular structures that are ordered on the nanometre scale is a key objective in nanotechnology. DNA and peptide nanotechnologies have produced various two- and three-dimensional structures, but protein molecules have been underexploited in this area of research. Here we show that the genetic fusion of subunits from protein assemblies that have matching rotational symmetry generates species that can self-assemble into well-ordered, pre-determined one- and two-dimensional arrays that are stabilized by extensive intermolecular interactions. This new class of supramolecular structure provides a way to manufacture biomaterials with diverse structural and functional properties.     

Morphology and structural properties of pH-responsive amphiphilic peptides

Protein nanomaterials at the peptide level have shown great potential for medical applications. Peptides change their morphological conformation because of changes in self-assembly properties when they are exposed to changes in solvent composition or pH. Two 15-residue peptide sequences, KhK (KKKFLIVIGSIIKKK) and Alternating Kh (KFLKKIVKIGKKSII), were designed for the purpose of determining the role of peptide sequence on solution morphology and conformation. KhK solutions exhibited a random coil to helical transition when solvent conditions were changed from water to a trifluorethanol/water solution at acidic pH. Alternating Kh solutions, however, demonstrated primarily random coil character under similar solvent and pH conditions as determined by circular dichroism spectroscopy and 2D-1H-1H nuclear magnetic resonance spectroscopy. At basic pH, circular dichroism spectroscopy and nuclear magnetic resonance spectroscopy analysis demonstrated that random coil character increased at basic pH for KhK, whereas Alternating Kh exhibited an increase in beta-sheet character. Further analysis by transmission electron microscopy showed marked differences in the peptide solution morphology. Peptide particle aggregation and fiber formation were significantly affected by solvent composition and pH values for both peptide sequences.     

Self-Assembled Peptide-Based Nanodrugs: Molecular Design,Synthesis, Functionalization,and Targeted Tumor Bioimaging and Biotherapy

Functional nanomaterials as nanodrugs based on the self-assembly of inorganics, polymers, and biomolecules have showed wide applications in biomedicine and tissue engineering. Ascribing to the unique biological, chemical, and physical properties of peptide molecules, peptide is used as an excellent precursor material for the synthesis of functional nanodrugs for highly effective cancer therapy. Herein, recent progress on the design, synthesis, functional regulation, and cancer bioimaging and biotherapy of peptide-based nanodrugs is summarized. For this aim, first molecular design and controllable synthesis of peptide nanodrugs with 0D to 3D structures are presented, and then the functional customization strategies for peptide nanodrugs are presented. Then, the applications of peptide-based nanodrugs in bioimaging, chemotherapy, photothermal therapy (PTT), and photodynamic therapy (PDT) are demonstrated and discussed in detail. Furthermore, peptide-based drugs in preclinical, clinical trials, and approved are briefly described. Finally, the challenges and potential solutions are pointed out on addressing the questions of this promising research topic. This comprehensive review can guide the motif design and functional regulation of peptide nanomaterials for facile synthesis of nanodrugs, and further promote their practical applications for diagnostics and therapy of diseases.     

酶响应水凝胶研究进展

本文简要介绍了环境响应型水凝胶的研究现状,重点分析了酶响应水凝胶,包括谷氨酰胺转氨酶、激酶与磷酸酶、酪氨酸酶、枯草杆菌蛋白酶与嗜热菌蛋白酶、过氧化物酶、酯酶与核酸内切酶等引起水凝胶响应的酶因素,并介绍了酶响应引起自组装的形成、自组装的破坏以及动态自组装等材料响应。此外,分析了酶响应水凝胶研究中存在的问题,并介绍了肽水凝胶的生物学功能。