负载白藜芦醇自组装多肽水凝胶的抗菌性能研究

[目的]制备一种负载白藜芦醇的自组装多肽水凝胶并探讨其抗菌性能。[方法]通过自组装制备多肽(FmocFFGGRGD)水凝胶和载有白藜芦醇的多肽水凝胶(Pep/RES);通过扫描电子显微镜和透射电子显微镜观察水凝胶的形貌和内部结构;通过流变仪检测水凝胶的流变性质;通过高效液相色谱检测Pep/RES的释放速率;通过细胞毒性试验研究该水凝胶的生物相容性;通过抑菌圈实验和活死细菌染色研究Pep/RES对大肠杆菌和金黄色葡萄球菌的抗菌性能。[结果]多肽溶液可在30 min内自组装形成稳定的水凝胶,水凝胶内部的三维结构密度随多肽浓度的增加而增加,2.0wt%浓度的多肽水凝胶稳定效果最好。白藜芦醇从Pep/RES水凝胶中缓慢释放7 d释放量达到50%,Pep/RES浸泡液对NIH/3T3细胞表现出良好的生物相容性。Pep/RES水凝胶中负载的白藜芦醇浓度为512μg/m L时,对金黄色葡萄球菌的抑菌圈直径即可达到5.41±0.18 mm,但即使白藜芦醇浓度达到1 024μg/m L,对大肠杆菌的抑菌圈直径仅为4.27±0.22 nm。[结论]Pep/RES结构稳定,安全无毒,能缓释白藜芦醇,并对金黄...     

促细胞黏附生长的自组装肽水凝胶

随着细胞与组织工程的迅猛发展,能够促进细胞黏附、生长和分化的生物材料基质支架的研究日益重要。具有生物相容性且含水量超过99％的自组装肽水凝胶因其很好地符合理想的生物材料基质支架标准而备受重视。这类自我互补的两亲寡肽含50％的带电残基,并且以交替的离子亲水性和不带电的氨基酸残基周期性重复为特征;在其寡肽的氨基末端可用直接固相合成法修饰几个短序列生物活性模体进行功能化,用以促进不同细胞的黏附生长和靶向定位。现对自组装肽水凝胶的结构特征、自组装机制、对细胞黏附生长的影响以及未来自组装肽生物材料设计的目标进行综述．     

功能化自组装肽水凝胶在细胞培养中应用

自组装肽水凝胶是一类能够在特定的条件下利用疏水作用、静电作用、氢键、范德华力等非共价作用力来形成内部高度有序结构的多肽分子凝胶。自组装多肽凝胶具有易于设计合成、低免疫原性、低炎症反应、良好组织相容性、生物利用度高等特点,可为各种细胞提供近似于天然的细胞外基质,促进细胞增殖、分化、黏附等细胞生物学行为。因此,自组装肽水凝胶是细胞培养理想支架,具有广阔应用前景。本文主要对功能化自组装肽凝胶在干细胞、成骨细胞、内皮细胞、肿瘤细胞培养中的应用进行综述,期望为功能性自组装肽水凝胶在细胞培养中应用提供理论依据。     

超分子多肽自组装在生物医学中的应用

近年来,自组装多肽纳米技术因其可形成规则有序的结构、具有多样的功能而备受关注.研究发现自组装多肽能在特定的条件下形成具有确定结构的聚集体,这种聚集体具备生物相容性好、稳定性高等优点,表现出不同于单体多肽分子的特性和优势,因此其在药物传递、组织工程、抗菌等领域具有良好的应用前景.文中介绍了 自组装多肽形成的分子机理、类型...     

基于自组装交联的水凝胶在皮肤损伤中的应用

自组装水凝胶具有高吸水性、高保水性、良好的生物相容性、生物降解性和三维立体结构等物理优势,同时具备止血、抗菌、抗炎、抗氧化等功能优势。因此自组装水凝胶作为一种新型伤口敷料,在皮肤损伤的创面愈合和调节再生中具有广阔的应用前景。本文通过分析讨论自组装水凝胶的交联机制,阐述自组装水凝胶的功能,明确其作为伤口敷料在皮肤损伤中的优势,总结自组装水凝胶在皮肤损伤应用中的发展趋势,展望自组装水凝胶的未来方向,有助于更全面地了解自组装水凝胶,为自组装水凝胶的多技术联合应用提供新思路。     

功能核酸DNA水凝胶的制备与组装

功能核酸DNA水凝胶是一种以DNA为构建单元通过化学反应或物理缠结自组装而成的新型柔性材料,其构建单元中包含1种或多种能够形成功能核酸的特定序列。功能核酸是通过碱基修饰和DNA分子之间的相互作用力组合的一类特定核酸结构,包括核酸适配体、DNA核酶、G-四联体(G-quadruplex,G4)和i-motif结构等。传统上,高浓度的长DNA链是制备DNA水凝胶的必要条件,而核酸扩增方法的引入为DNA水凝胶的组装方式提供了新的可能。因此,对常用于制备DNA水凝胶的多种功能核酸以及核酸的提取、合成和扩增手段进行了详细的介绍。在此基础上,综述了通过化学或物理交联方式组装功能核酸DNA水凝胶的制备方法。最后,提出了DNA纳米材料的组装所面临的挑战和潜在的发展方向,以期为开发高效组装的功能核酸DNA水凝胶提供参考。     

生物分子自组装

分子自组装是一种普遍存在于生命体系中的现象,是生命科学最本质的内容之一。开展分子自组装的研究具有重要意义,有助于人们从分子水平上认识自然界中生命形成和演变的过程,并为人们提供新的思路,开展生物医学基础研究、新材料合成及分子器件研制等。该文介绍了自组装的基本含义,对分子自组装技术在生物材料、生物分子器件研究方面的进展作了综述。     

酶响应型肽水凝胶及应用研究进展

酶响应型肽水凝胶可用于缓控释药物的释放,并具有抗菌、抗肿瘤等作用,是目前材料领域新兴的的研究热点之一.本文总结了近年来国内外开发的酶响应型肽水凝胶材料,重点介绍了包括谷氨酰胺转氨酶、激酶、磷酸酶、赖氨酸氧化酶(血浆氨氧化酶)、蛋白酶、酯酶、β内酰胺酶、基质金属蛋白酶等酶响应型肽水凝胶,以及在酶的催化作用下,水凝胶的形成、破坏或动态转换,同时总结了它们的响应机理.此外,介绍了酶响应型肽水凝胶的应用.酶响应型肽水凝胶具有广阔的发展前景,是未来智能响应材料的发展方向之一.     

小鼠前成骨细胞MC3T3-E1在自组装多肽水凝胶支架中的成骨分化

目的研究MC3T3-E1细胞在自组装多肽水凝胶支架上的生长和成骨分化.方法在多肽水凝胶支架RADA16上接种MC3T3-E1细胞,荧光染色观察细胞形态和存活情况;组织化学染色检测MC3T3-E1细胞碱性磷酸酶活性以及细胞外钙质沉积;RT-PCR分析成骨特异性基因的表达.结果 MC3T3-E1细胞在水凝胶支架RADA16上粘附铺展良好,呈纺锤样形态.诱导培养后支架上的细胞有较高水平的碱性磷酸酶表达和矿化基质沉积.此外,骨分化特异性基因骨桥蛋白和骨涎蛋白也有表达,且表达量随培养时间的延长而增多.结论 在自组装水凝胶内MC3T3-E1细胞可向成骨方向分化,并能在凝胶内产生矿化的细胞外基质.     

融合自组装双亲短肽对重组过氧化氢酶酶学性质的影响

【目的】利用融合自组装双亲短肽策略对源自枯草芽孢杆菌(Bacillus subtilis)的过氧化氢酶Kat A进行改性,以强化重组过氧化氢酶在工业中的应用适应性。【方法】将自组装双亲短肽S1vw通过连接肽PT-linker融合在Kat A的N端,构建重组质粒p HT254-S1vw-PT-kat A,将其与携带天然酶基因的p HT254-kat A分别转入枯草芽孢杆菌WB800N中进行分泌表达,之后将分离纯化得到的纯酶进行酶学性质研究。【结果】成功构建出工程菌并将胞外粗酶液通过乙醇沉淀、DEAE阴离子交换层析、疏水层析和凝胶过滤层析4步纯化,最终获得电泳纯的重组酶蛋白。酶学性质研究结果显示,融合酶S1vw-PT-Kat A和天然酶Kat A的最适反应温度均为30°C,最适反应p H值均为11.0。然而,融合酶在p H 12.0下孵育30 min的相对酶活为77.3%,是相同处理条件下天然酶相对酶活的14.9倍,在65°C和70°C下孵育30 min的相对酶活分别为19.8%和17.5%,是相同处理条件下天然酶相对酶活的1.8倍和1.7倍。此外,融合酶在4°C储存14 d后相对酶活为8...     

Heterochiral tetrapeptide self-assembly into hydrogel biomaterials for hydrolase mimicry

Self-assembling short peptides have attracted great interest as enzyme mimics, especially if the catalytic activity resides solely in the supramolecular structure so that it can be switched on/off as needed by controlling assembly/disassembly. Among the various enzyme classes, hydrolases find wide application in biomaterials, and their mimetics often contain His residues, in addition to either divalent cations or other amino acids to mimic the catalytic site. This work reports two self-assembling tetrapeptides based on the Ser-His motif for catalysis and the Phe-Phe motif to drive amyloid structure formation. Both peptides form thermoreversible hydrogels in phosphate buffer at neutral pH that display a mild esterase-like activity, as demonstrated on the hydrolysis of 4-nitrophenyl acetate as a model substrate, although presence of Ser did not enhance catalytic activity. The systems are characterised by circular dichroism, transmission electron microscopy, oscillatory rheology and Thioflavin T fluorescence as an amyloid stain, to provide further insights that may assist the future design of improved supramolecular catalysts.     

Layer-by-layer self-assembly of supramolecular and biomolecular films

In this paper, we give a short account on recent studies of layer-by-layer self-assembly of supramolecular and biomolecular films. Such films are built up from layers of macro-ions with opposing charge. A simple film can be obtained by alternating the adsorption of two components: a flexible, synthetic polycation chains and a supramolecular or biomolecular moiety. We focus on three examples, in which the second component consists either of a supramolecular metal-organic complex (MOC), a nucleic acid, or a biological membrane patch (purple membrane). While the flexible polvcation chains (as well as eventual annealing layers) ensure a uniform build-up of the chain, the second macromolecular component may be used to functionalize the films. The combination of layer-by-layer self-assembly and biotechnologically relevant macromolecules may lead to new devices or biomaterial applications. To this end, precise studies of the deposition process and the film structure are needed. Here, we focus on interface sensitive scattering techniques for the structural analysis.     

Microtubules: dissipative structures formed by self-assembly

Microtubules are hollow fibres that form the track upon which chromosomes or proteins, such as kinesins, are transported in the cell. They are formed by the self-assembly of the protein tubulin both in vitro and in vivo. In the cell, their appearance in time and space is strictly controlled by the presence of nucleation centres. Microtubules are very dynamic structures, a property that is obtained by coupling the self-assembly process to the hydrolysis of the nucleotide, guanosine 5′-triphosphate, (GTP). After assembly, GTP is hydrolysed and guanosine 5′-diphosphate, (GDP)-microtubule structure is formed which, although intrinsically very unstable, is stabilised by a small remaining tubulin-GTP-cap at both ends. As such, the ends of microtubules can be considered as gates for entry into the polymeric state. These gates can be blocked by sub-stoichiometric amounts of drugs such as colchicine.

As biological devices, microtubules differ considerably from man-made devices: they are dynamic dissipative structures, made by spontaneous self-assembly. It has been suggested that microtubules could play a role in the conduction and dynamic storage of information. This implies the existence of different conformational states of tubulin.     

Enhanced stability of heterologous proteins by supramolecular self-assembly

Recently, we reported on the dual function of human ferritin heavy chain (hFTN-H) used for the fusion expression and solubility enhancement of various heterologous proteins: (1) high-affinity interaction with HSP70 chaperone DnaK and (2) formation of self-assembled supramolecules with limited and constant sizes. Especially the latter, the self-assembly function of hFTN-H is highly useful in avoiding the undesirable formation of insoluble macroaggregates of heterologous proteins in bacterial cytoplasm. In this study, using enhanced green fluorescent protein (eGFP) and several deletion mutants of Mycoplasma arginine deiminase (ADI_132–410) as reporter proteins, we confirmed through TEM image analysis that the recombinant fusion proteins (hFTN-H::eGFP and hFTN-H::ADI_132–410) formed intracellular spherical particles with nanoscale diameter (≈10 nm), i.e., noncovalently cross-linked supramolecules. Surprisingly, the supramolecular eGFP and ADI showed much enhanced stability in bioactivity. That is, the activity level was much more stably maintained for the prolonged period of time even at high temperature, at high concentration of Gdn–HCl, and in wide range of pH. The stability enhancement by supramolecular self-assembly may make it possible to utilize the protein supramolecules as novel means for drug delivery, enzymatic material conversion (biotransformation), protein chip/sensor, etc. where the maintenance of protein/enzyme stability is strictly required. Jin-Seung Park and Ji-Young Ahn contributed equally to this work.     

A saccharide-based supramolecular hydrogel for cell culture

It is well known that the saccharides forming the intricate sugar coat that surrounds the cells play important biological roles in intercellular communication and cell differentiation. Therefore, it is worthwhile developing saccharide-based hydrogels for cell culture study. In this study, three novel saccharide-based compounds were designed and synthesized. It was found that one of them could form hydrogels efficiently, while the other two precipitated from water. The stability of the resulting hydrogel was tested, and the supramolecular nanofiber with fiber diameters in the range of 80–300 nm was characterized as the structural element by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Fluorescence microscopy revealed that extensive hydrogen bonds between sugar rings assisted the formation of efficient π–π stacking between aromatic naphthalene groups, thus resulting in the formation of a stable hydrogel in aqueous solution. When the gel was applied for mouse embryonic fibroblast (NIH 3T3), human hepatocellular carcinoma (HepG2), AD293 and HeLa cells culture in two dimensional environments, all of them showed a very good adhesion and good proliferation rate on the top of the hydrogel. These results indicates that the biocompatible hydrogel reported here has a potential to be developed into useful materials for in vitro cell culture, drug delivery, and tissue engineering.     

登革病毒衣壳蛋白在大肠杆菌中的表达及其自组装活性的研究

采用高保真RT-PCR自登革2型病毒43株基因组RNA中扩增全长C基因及缺失羧基端Cv片段,分别构建可表达C及Cv的重组质粒pLEX—C和pLEX—Cv,转化E．coliGI724后用色氨酸诱导表达。经SDS—PAGE分析,表达的C及Cv蛋白相对分子质量分别约为12000和10000,分别约占菌体蛋白总量的19％和13％。Western印迹检测表明重组表达的C蛋白均可被特异识别登革病毒衣壳蛋白的单克隆抗体特异识别。表达的蛋白经过硫酸铵沉淀和蔗糖密度梯度离心后,通过琼脂糖凝胶电泳和负染电镜均未能检测到衣壳样颗粒的存在,说明登革病毒衣壳蛋白可能不具体外自组装活性。     

Modelling and simulation of DNA-mediated self-assembly for superlattice design

ABSTRACT

Functionalisation of colloidal particles with DNA provides a powerful and flexible path towards self-assembly of ordered materials. Given the nearly limitless possibilities for constructing DNA-functionalised particles, and the wide range of conditions under which they can be assembled, it is crucial to gain an understanding of the principles governing self-assembly of these particles and how their properties affect the structures produced. A number of computational models for DNA-functionalised systems have successfully described their properties, and molecular simulation techniques have provided a unique insight into the factors underlying their assembly. Here, we discuss a variety of efforts using simulations to solve an important design problem in DNA-mediated assembly: how the properties of individual DNA-functionalised particles affect their interactions with each other, and ultimately how these interactions determine what structures can be produced.