Simultaneous synthesis of temperature-tunable peptide and gold nanoparticle hybrid spheres

Hybrid spheres containing peptides and gold nanoparticles have been simultaneously synthesized in water using AG4 (NPSSLFRYLPSD) peptides that acted as a reducing agent to guide the nucleation and growth of gold nanoparticles and a precursor to form sphere-like structure by self-assembly where the size of hybrid spheres is precisely controlled by adjusting the operating temperature. The self-assembled peptide spheres remain stable even after selective removal of the gold nanoparticles by iodide etching. The amino acids containing the aromatic functional group in the peptide sequence significantly affect the construction of sphere structures. The surface of gold nanoparticles containing hybrid spheres has been functionalized using the thiol group linked to biomolecules. The ability to synthesize nanoparticle and self-assembled peptide structures with controlled size and composition in an environmental benign way will allow us to fabricate a new class of multifunctional organic-inorganic hybrid superstructures for various biomedical and electronic applications.     

Caspase-3 mediated programmed cell death by a gold-stabilised peptide carbene

The synthesis of novel N-heterocyclic carbene complexes derived from a tripeptide ligand (L), containing non-natural amino acid, thiazolylalanine is described here. The peptide ligand was reacted with suitable precursors to generate gold and mercury carbene complexes. The plausible structures of both complexes were predicted by spectroscopic data and DFT calculations. The binding energy data was also analyzed to predict their stability. The gold carbene complex (1A), showed activity against MCF7 breast cancer cell line due to mitochondrial triggered caspase-3 mediated programmed cell death. Its internalization inside cells could be observed due to autofluorescence. This study affords a methodology for successful generation of peptide carbene complexes for their therapeutic potential.     

Mycocrystallization of gold ions by the fungus Cylindrocladium floridanum

The size and morphology determines the thermodynamic, physical and electronic properties of metal nanoparticles. The extracellular synthesis of gold nanoparticles by fungus, Cylindrocladium floridanum, which acts as a source of reducing and stabilizing agent has been described. The synthesized nanoparticles were characterized using techniques such as UV–Vis spectroscopy, X-ray diffraction (XRD), scanning electron microscopy, energy dispersive X-ray analysis (EDAX), and high-resolution transmission electron microscopy (HR-TEM). Based on the evidence of HR-TEM, the synthesized particles were found to be spherical with an average size of 19.05 nm. Powder XRD pattern proved the formation of (111)-oriented face-centered cubic crystals of metallic gold. This microbial approach by fungus for the green synthesis of spherical gold nanoparticles has many advantages such as economic viability, scaling up and environment friendliness.     

FTIR study of secondary structure changes in Epidermal Growth Factor by gold nanoparticle conjugation

Conformation of protein is vital to its function, but may get affected when processing to manufacture products. It is therefore important to understand structural changes during each step of production. In this study, we investigate secondary structure changes in the targeting protein Epidermal Growth Factor (EGF) during synthesis of theranostic bifunctional nanoparticle, devised for Photodynamic therapy of breast cancer. We acquired FTIR spectra of EGF; unconjugated, post treatment with α-lipoic acid, attached to gold nanoparticle, and bound to the bifunctional nanoprobe. We observed decreasing disordered structures and turns, and increasing loops, as the synthesis process progressed. There was an overall increase in β-sheets in final product compared to pure EGF, but this increase was not linear and fluctuated. Previous crystal structure studies on EGF-EGFR complex have shown loops and β-sheets to be important in the binding interaction. Since our study found increase in these structures in the final product, no adverse effect on binding function of EGF was expected. This was confirmed by functional assays. Such studies may help modify synthesis procedures, and thus secondary structures of proteins, enabling increased functionality and optimum results.     

Synthesis of N-terminally linked protein and peptide dimers by native chemical ligation

Dimerization can be utilized to double the molecular weight of proteins and peptides and potentially increase their avidity of binding to target receptors. These dimerization effects may be utilized to increase in vivo half-lives in a manner similar to PEGylation and may also improve biological activity. In this paper, we report a new strategy for the synthesis of N-terminally linked protein and peptide homodimers utilizing native chemical ligation to conjugate a short dithioester linker to the N-terminal cysteines of protein and peptide monomers to form dimers in a single step. This strategy is general and has been applied to the production of dimers from three recombinantly expressed polypeptides, the IgG binding domain Protein G, an HIV entry inhibitor peptide C37H6, and human interleukin-1 receptor antagonist (IL-1ra). The biological activities of the C37H6 and IL-1ra dimers produced by these methods were retained or even slightly increased when compared to their corresponding monomers.     

Modeling of a C‐end rule peptide adsorbed onto gold nanoparticles

The RPAR peptide, a prototype C‐end Rule (CendR) sequence that binds to neuropilin‐1 (NRP‐1), has potential therapeutic uses as internalization trigger in anticancer nanodevices. Recently, the functionalization of gold nanoparticles with CendR peptides has been proved to be a successful strategy to target the NRP‐1 receptor in prostate cancer cells. In this work, we investigate the influence of two gold surface facets, (100) and (111), on the conformational preferences of RPAR using molecular dynamics simulations. Both clustering and conformational analyses revealed that the peptide backbone becomes very rigid upon adsorption onto gold, which is a very fast and favored process, the only flexibility being attributed to the side chains of the two Arg residues. Thus, the different components of RPAR tend to adopt an elongated shape, which is characterized by the pseudo‐extended conformation of both the backbone and the Arg side chains. This conformation is very different from the already known bioactive conformation, indicating that RPAR is drastically affected by the substrate. Interestingly, the preferred conformations of the peptide adsorbed onto gold facets are not stabilized by salt bridges and/or specific intramolecular hydrogen bonds, which represent an important difference with respect to the conformations found in other environments (e.g. the peptide in solution and interacting with NRP‐1 receptor). However, the conformational changes induced by the substrate are not detrimental for the use of gold nanoparticles as appropriate vehicles for the transport and targeted delivery of the RPAR. Thus, once their high affinity for the NRP‐1 receptor induces the targeted delivery of the elongated peptide molecules from the gold nanoparticles, the lack of intramolecular interactions facilitates their evolution towards the bioactive conformation, increasing the therapeutic efficacy of the peptide.     

Varied presentation of the Thomsen-Friedenreich disaccharide tumor-associated carbohydrate antigen on gold nanoparticles

Three-dimensional self-assembled monolayers of gold coated with the Thomsen-Friedenreich antigen (TF(ag)) disaccharide (beta-Galp-(1-->3)-GalpNAc) in a variety of presentations have been prepared and characterized. Anomalies in the size distribution of our originally synthesized TF(ag)-bearing nanoparticles as shown in dynamic light scattering experiments prompted us to explore the effect of antigen density on the uniformity of the particles. Gold nanoparticles containing a range of densities 'diluted' with copies of the PEG-thiol spacer unit showed that lower antigen density affords more uniform particles. We also wanted to study the constitution of the actual antigen by synthesizing nanoparticles not only with the linker-extended disaccharide, but also within the context of the surrounding peptide sequence where it may be presented in vivo. The synthesis of TF(ag)-containing glycopeptide thiols based on a mucin peptide repeating unit were prepared, assembled into gold nanoparticles and their physical properties evaluated. These novel multivalent tools should prove extremely useful in exploring the binding properties and immune response to this important carbohydrate antigen.     

Tat peptide as an efficient molecule to translocate gold nanoparticles into the cell nucleus

The labeling of targeting molecules with nanoparticles has revolutionized the visualization of cellular or tissue components by electron microscopy. A particularly desirable target is the nucleus, because the genetic information is there. To date, utilizing nanoparticles for nuclear targeting has not proved very successful due to the impermeable nature of the plasma and nuclear membranes; thus nanoparticle design and synthesis is a critical factor. We report in this article the synthesis of water-soluble gold nanoparticles functionalized with a Tat protein-derived peptide sequence by a straightforward and economical methodology. The particles were subsequently tested in vitro with a human fibroblast cell line by optical and transmission electron microscopy to determine the biocompatibility of these nanoparticles and whether the functionalization with the translocation peptide allowed particles to transfer across the cell membrane and locate in the nucleus.     

Inhibition of production of macrophage-derived angiogenic activity by the anti-rheumatic agents gold sodium thiomalate and auranofin

We have investigated the effect of gold sodium thiomalate and auranofin, gold compounds employed in the treatment of rheumatoid arthritis, on production of macrophage-derived angiogenic activity. Elicited mouse peritoneal macrophages were cultured in the presence or absence of gold compounds or thiomalic acid, and the macrophages or their conditioned media were then assayed for their angiogenic activity in rat corneas. Control macrophage conditioned medium was potently angiogenic. In contrast, conditioned medium from gold or thiomalic acid treated macrophages was not. Addition of gold compounds or thiomalic acid to control macrophage conditioned medium did not inhibit its angiogenic activity. Drug treatments did not significantly affect macrophage lactate dehydrogenase release, lysozyme release, or protein synthesis. We conclude that gold sodium thiomalate and auranofin potently reduce the detectable angiogenic activity produced by macrophages.     

Gold nanoparticles capped by a GC-containing peptide functionalized with an RGD motif for integrin targeting

Gold nanoparticles were obtained by reduction of a tetrachloroaurate aqueous solution in the presence of a RGD-(GC)(2) peptide as stabilizer. As comparison, the behavior of the (GC)(2) peptide has been studied. The (GC)(2) and RGD-(GC)(2) peptides were prepared ad hoc by Fmoc synthesis. The colloidal systems have been characterized by UV-visible, TGA, ATR-FTIR, mono and bidimensional NMR techniques, confocal and transmission (TEM) microscopy, ζ-potential, and light scattering measurements. The efficient cellular uptake of Au-RGD-(GC)(2) and Au-(GC)(2) stabilized gold nanoparticles into U87 cells (human glioblastoma cells) were investigated by confocal microscopy and compared with the behavior of (GC)(2) capped gold nanoparticles. A quantitative determination of the nanoparticles taken up has been carried out by measuring the pixel brightness of the images, a measure that highlighted the importance of the RGD termination of the peptide. Insight in the cellular uptake mechanism was investigated by TEM microscopy. Various important evidences indicated the selective uptake of RGD-(GC)(2) gold nanoparticles into the nucleus.     

Potential of plant as a biological factory to synthesize gold and silver nanoparticles and their applications

Green synthesis of metallic nanoparticles has become a promising field of research in recent years. Syntheses of gold and silver nanoparticles by various chemical and physical methods as well as the biosynthetic approach mediated by numerous microorganisms have been actively researched. A more scalable and economic route to produce these metallic nanoparticles would be through the plant-mediated synthetic approach. Owing to the biodiversity of plant biomasses, the mechanism by which bioconstituents of plants have contributed to the synthetic process is yet to be fully understood. Nevertheless, the feasibility of controlling the shape and size of nanoparticles by varying the reaction conditions has been demonstrated in many studies. This paper provides an overview of the plant-mediated syntheses of gold and silver nanoparticles, possible compounds and mechanisms that might be responsible for the bioreduction process as well as the potential applications of biosynthesized nanoparticles in different fields. The challenges and limitations of this plant-mediated biosynthetic approach are also discussed.     

Pseudomonas syringae phytotoxins: mode of action, regulation, and biosynthesis by peptide and polyketide synthetases.

Coronatine, syringomycin, syringopeptin, tabtoxin, and phaseolotoxin are the most intensively studied phytotoxins of Pseudomonas syringae, and each contributes significantly to bacterial virulence in plants. Coronatine functions partly as a mimic of methyl jasmonate, a hormone synthesized by plants undergoing biological stress. Syringomycin and syringopeptin form pores in plasma membranes, a process that leads to electrolyte leakage. Tabtoxin and phaseolotoxin are strongly antimicrobial and function by inhibiting glutamine synthetase and ornithine carbamoyltransferase, respectively. Genetic analysis has revealed the mechanisms responsible for toxin biosynthesis. Coronatine biosynthesis requires the cooperation of polyketide and peptide synthetases for the assembly of the coronafacic and coronamic acid moieties, respectively. Tabtoxin is derived from the lysine biosynthetic pathway, whereas syringomycin, syringopeptin, and phaseolotoxin biosynthesis requires peptide synthetases. Activation of phytotoxin synthesis is controlled by diverse environmental factors including plant signal molecules and temperature. Genes involved in the regulation of phytotoxin synthesis have been located within the coronatine and syringomycin gene clusters; however, additional regulatory genes are required for the synthesis of these and other phytotoxins. Global regulatory genes such as gacS modulate phytotoxin production in certain pathovars, indicating the complexity of the regulatory circuits controlling phytotoxin synthesis. The coronatine and syringomycin gene clusters have been intensively characterized and show potential for constructing modified polyketides and peptides. Genetic reprogramming of peptide and polyketide synthetases has been successful, and portions of the coronatine and syringomycin gene clusters could be valuable resources in developing new antimicrobial agents.     

Selective destabilization of acidic phospholipid bilayers performed by the hepatitis B virus fusion peptide

A peptide corresponding to the N-terminal region of the S protein of hepatitis B virus (Met-Glu-Asn-Ile-Thr-Ser-Gly-Phe-Leu-Gly-Pro-Leu-Leu-Val-Leu-Gln) has been previously demonstrated to perform aggregation and destabilization of acidic liposome bilayers and to adopt a highly stable beta-sheet conformation in the presence of phospholipids. The changes in the lipid moiety produced by this peptide have been followed by fluorescence depolarization and electron microscopy. The later was employed to determine the size and shape of the peptide-vesicle complexes, showing the presence of highly aggregated and fused structures only when negatively charged liposomes were employed. 1,6-Diphenyl-1,3,5-hexatriene depolarization measurements showed that the interaction of the peptide with both negatively charged and zwitterionic liposomes was accompanied by a substantial reduction of the transition amplitude without affecting the temperature of the gel-to-liquid crystalline phase transition. These data are indicative of the peptide insertion inside the bilayer of both types of liposomes affecting the acyl chain order, though only the interaction with acidic phospholipids leads to aggregation and fusion. This preferential destabilization of the peptide towards negatively charged phospholipids can be ascribed to the electrostatic interactions between the peptide and the polar head groups, as monitored by 1-(4-(trimethylammoniumphenyl)-6-phenyl-1,3, 5-hexatriene fluorescence depolarization analysis.     

Accumulation of gold nanoparticles in Brassic juncea

Enzymatic digestion is proposed as a method for concentrating gold nanoparticles produced in plants. The mild conditions of digestion are used in order to avoid an increase in the gold particle size, which would occur with a high-temperature process, so that material suitable for catalysis may be produced. Gold nanoparticles of a 5-50-nm diameter, as revealed by transmission electron microscopy (TEM), at concentrations 760 and 1120 ppm Au, were produced within Brassica juncea grown on soil with 22-48 mg Au kg(-1). X-ray absorption near edge spectroscopy (XANES) reveals that the plant contained approximately equal quantities of Au in the metallic (Au0) and oxidized (Au+1) states. Enzymatic digestion dissolved 55-60 wt% of the plant matter. Due to the loss of the soluble gold fraction, no significant increase in the total concentration of gold in the samples was observed. However, it is likely that the concentration of the gold nanoparticles increased by a factor of two. To obtain a gold concentration suitable for catalytic reactions, around 95 wt% of the starting dry biomass would need to be solubilized or removed, which has not yet been achieved.     

Exploitation of marine bacteria for production of gold nanoparticles

Background

Gold nanoparticles (AuNPs) have found wide range of applications in electronics, biomedical engineering, and chemistry owing to their exceptional opto-electrical properties. Biological synthesis of gold nanoparticles by using plant extracts and microbes have received profound interest in recent times owing to their potential to produce nanoparticles with varied shape, size and morphology. Marine microorganisms are unique to tolerate high salt concentration and can evade toxicity of different metal ions. However, these marine microbes are not sufficiently explored for their capability of metal nanoparticle synthesis. Although, marine water is one of the richest sources of gold in the nature, however, there is no significant publication regarding utilization of marine micro-organisms to produce gold nanoparticles. Therefore, there might be a possibility of exploring marine bacteria as nanofactories for AuNP biosynthesis.

Results

In the present study, marine bacteria are exploited towards their capability of gold nanoparticles (AuNPs) production. Stable, monodisperse AuNP formation with around 10?nm dimension occur upon exposure of HAuCl₄ solution to whole cells of a novel strain of Marinobacter pelagius, as characterized by polyphasic taxonomy. Nanoparticles synthesized are characterized by Transmission electron microscopy, Dynamic light scattering and UV-visible spectroscopy.

Conclusion

The potential of marine organisms in biosynthesis of AuNPs are still relatively unexplored. Although, there are few reports of gold nanoparticles production using marine sponges and sea weeds however, there is no report on the production of gold nanoparticles using marine bacteria. The present work highlighted the possibility of using the marine bacterial strain of Marinobacter pelagius to achieve a fast rate of nanoparticles synthesis which may be of high interest for future process development of AuNPs. This is the first report of AuNP synthesis by marine bacteria.     

Cardiac adrenomedullin gene expression and peptide accumulation after acute myocardial infarction in rats

Plasma adrenomedullin (AM) has been shown to increase in the early phase of acute myocardial infarction (MI). However, little information is available regarding cardiac AM synthesis after MI. Accordingly, we examined the time course of ventricular AM production and potential stimulation of AM in the infarcted and noninfarcted regions in MI rats produced by coronary artery ligation. Compared with sham-operated rats, the ventricular AM peptide level 6 h after MI increased 1.5-fold in the infarcted region and 1.7-fold in the noninfarcted region in association with increased left ventricular end-diastolic pressure (EDP). Northern blot analysis also showed marked induction of AM gene expression in the infarcted region (11-fold) and the noninfarcted region (6-fold) 6 h after MI. The AM peptide level in the infarcted region reached its peak (2. 6-fold) 1 wk postinfarction and thereafter decreased to normal. In the noninfarcted region, however, the AM level remained elevated for at least 4 wk. Immunohistochemical studies demonstrated that intense immunostaining for AM was limited to myocytes in both the infarcted and noninfarcted regions. Interestingly, the AM level in the noninfarcted region correlated positively with infarct size (r = 0. 40, P < 0.01) and EDP (r = 0.52, P < 0.001). An oral angiotensin-converting enzyme inhibitor suppressed the overproduction of AM 1 wk postinfarction in association with decreases in EDP and mean arterial pressure. In summary, cardiac AM synthesis was rapidly induced in both the infarcted and noninfarcted regions after MI. The subsequent ventricular AM in the two regions demonstrated different time-concentration curves during 4 wk after MI. AM may be synthesized predominantly by cardiac myocytes, but not by fibroblasts, at least in part, in association with increased ventricular load after MI.     

Mycogenesis of gold nanoparticles using a phytopathogen <Emphasis Type="Italic">Alternaria alternata</Emphasis>

The development of an eco-friendly and reliable process for the synthesis of gold nanomaterials (AuNPs) using microorganisms is gaining importance in the field of nanotechnology. In the present study, AuNPs have been synthesized by bio-reduction of chloroauric acid (HAuCl₄) using the fungal culture filtrate (FCF) of Alternaria alternata. The synthesis of the AuNPs was monitored by UV–visible spectroscopy. The particles thereby obtained were characterized by UV, dynamic light scattering (DLS), X-ray diffraction (XRD), energy dispersive X-ray (EDX) analysis, Fourier transform infrared (FTIR) spectroscopy, atomic force microscopy (AFM) and transmission electron microscopy (TEM). Energy-dispersive X-ray study revealed the presence of gold in the nanoparticles. Fourier transform infrared spectroscopy confirmed the presence of a protein shell outside the nanoparticles which in turn also support their stabilization. Treatment of the fungal culture filtrate with aqueous Au⁺ ions produced AuNPs with an average particle size of 12 ± 5 nm. This proposed mechanistic principal might serve as a set of design rule for the synthesis of nanostructures with desired architecture and can be amenable for the large scale commercial production and technical applications.     

绿色木霉菌合成金纳米颗粒的可能性及影响因素

【背景】金纳米颗粒(AuNPs)凭借其稳定性、抗氧化性能和生物相容性在许多领域有广泛应用。目前关于微生物合成金纳米颗粒的研究较少。【目的】对微生物合成金纳米颗粒的可能性以及影响因素进行探究,有利于揭示具体的合成机制,发现AuNPs的特性以及合成位置与菌丝和影响因素的关系。【方法】以绿色木霉菌(Trichoderma viride)菌株(GIM3.141)为菌种资源,通过目视检测法、紫外可见分光光度计、X射线衍射和透射电镜等手段分析合成AuNPs的特征。探讨细胞内生物合成金纳米颗粒(AuNPs)的可能性,研究生物量、初始金离子浓度、溶液pH等因素对细胞内合成AuNPs的影响。【结果】X射线衍射分析表明AuNPs以金纳米晶体形态存在。透射电镜分析表明AuNPs主要位于细胞壁膜间隙,一小部分附着在细胞壁上。紫外可见分光光度计分析表明,金纳米颗粒粒径随着生物量添加量和溶液pH的升高而变小,随着初始金离子浓度的升高而变大。【结论】非致病性真菌绿色木霉菌可以在细胞内合成AuNPs,其中包括伪球形、三角形、四边形和六边形等多种形状,粒径范围从几纳米到三百纳米,为大规模、低成本、无污染地生物合成纳米颗粒工艺提供了菌种资源。     

Multiple peptide synthesis

The synthesis of large numbers of peptides can be very labor intensive and, if a conventional peptide synthesizer is used, only small numbers of peptides can be produced within a reasonable time. The techniques described below can make large numbers of different peptides simultaneously with varying degrees of mechanization, ranging from the wholly manual methods, to those involving complete mechanization of the whole synthesis process. Most of the multiple synthesis methods are primarily intended for small scale production ranging from microgram amounts up to a few tens of milligrams. All of the systems are economical in use of solvents and reagents, enabling cost-effective synthesis. The techniques described can also be used to prepare peptide libraries, containing several millions of peptide sequences, to enable the rapid screening of all possible permutations of amino acids within short peptides. However, it is considered that multiple synthesis methods are not particularly suited where extreme high purity or very long peptides are required.     

Process design for enzymatic peptide synthesis in near-anhydrous organic media

This work is a case study on a process design for enzymatic peptide synthesis, which is based on and inspired by previously established data about the Alcalase-catalyzed coupling of an amino acid amide and a chemically synthesized activated N-protected amino acid carbamoylmethyl ester in near-anhydrous tetrahydrofuran. The choices with regard to Alcalase formulation, the type of reactor, method of controlling the water content, and whether or not to recycle the enzyme, are discussed. In addition, an estimate is given for the reactor size, volumes of solvent, amount of substrate, enzyme and molecular sieves, needed in order to meet a specific demand for peptides. We believe that this case study gives a good indication of the various choices that have to be made when designing a process for enzymatic peptide synthesis and the implications of these choices.