Biomedical potential of silver nanoparticles synthesized from calli cells of Citrullus colocynthis (L.) Schrad

Background

Transmission electron microscopy (TEM) remains an important technique to investigate the size, shape and surface characteristics of particles at the nanometer scale. Resulting micrographs are two dimensional projections of objects and their interpretation can be difficult. Recently, electron tomography (ET) is increasingly used to reveal the morphology of nanomaterials (NM) in 3D. In this study, we examined the feasibility to visualize and measure silica and gold NM in suspension using conventional bright field electron tomography.

Results

The general morphology of gold and silica NM was visualized in 3D by conventional TEM in bright field mode. In orthoslices of the examined NM the surface features of a NM could be seen and measured without interference of higher or lower lying structures inherent to conventional TEM. Segmentation by isosurface rendering allowed visualizing the 3D information of an electron tomographic reconstruction in greater detail than digital slicing. From the 3D reconstructions, the surface area and the volume of the examined NM could be estimated directly and the volume-specific surface area (VSSA) was calculated. The mean VSSA of all examined NM was significantly larger than the threshold of 60 m²/cm³. The high correlation between the measured values of area and volume gold nanoparticles with a known spherical morphology and the areas and volumes calculated from the equivalent circle diameter (ECD) of projected nanoparticles (NP) indicates that the values measured from electron tomographic reconstructions are valid for these gold particles.

Conclusion

The characterization and definition of the examined gold and silica NM can benefit from application of conventional bright field electron tomography: the NM can be visualized in 3D, while surface features and the VSSA can be measured.     

The effects of dipalmitoyl phosphatidyl choline on the precipitation of native fibrils and segment-long-spacing aggregates from collagen solution

The effect of dipalmitoyl phosphatidyl choline (DPPC), the major phospholipid component of pulmonary surfactant, on the precipitation of collagen in the form of native fibrils and segment-long-spacing (SLS) aggregates was studied in vitro. The effects of DPPC on both phases of collagen fibrillogenesis were analyzed spectrophotometrically, and alterations in the morphology of precipitated fibrils and SLS aggregates were ascertained by transmission electron microscopy (TEM). Low concentrations of DPPC inhibited the growth phase of fibrillogenesis, while higher concentrations were required to inhibit nucleation. Both the meshwork density and mean width of precipitated fibrils were altered by DPPC, as was the size of SLS aggregates. Segment-long-spacing aggregates prepared from pepsin-treated collagen were inhibited to a greater degree than SLS aggregates prepared from untreated collagen, indicating that the pepsin-susceptible residues of the telopeptide extensions of tropocollagen molecules stabilize SLS aggregates against the effects of DPPC. Based on these results and the inhibition of the growth phase at lower concentrations than those which inhibited the nucleation phase of fibrillogenesis, it was concluded that the primary mechanism of DPPC inhibition is electrostatic interference between the positively charged phospholipid molecules and the net positive charge of collagen. It is proposed that pathological conditions involving the pulmonary epithelium may allow interaction between surfactant and collagen, which could further weaken the interstitial connective tissue.     

Inclusion of proteins into polyelectrolyte microparticles by alternative adsorption of polyelectrolytes on protein aggregates

A new method of protein immobilization into polyelectrolyte microparticles by alternative adsorption of the oppositely charged polyelectrolytes on the aggregates obtained by salting out of protein is proposed. The model protein -chymotrypsin (ChT) was included in the polyelectrolyte microparticles obtained by various number of polyelectrolyte adsorption steps (from 1 to 11). The main parameters of ChT inclusion into microparticles were calculated. Scanning electron and optical microscopy were used for characterization of morphology and determination of particle size which was from 1 to 10 m in most cases. It was shown that the size and shape of protein-containing particles and protein aggregates used as a matrix were similar. Change in ChT enzymatic activity during entrapment into polyelectrolyte particles and activity of released protein were studied. The effect of pH on release of incorporated proteins was investigated; it was shown that change in pH and the number of polyelectrolyte adsorption steps allows protein release to be manipulated.     

Detection of prion protein particles in blood plasma of scrapie infected sheep

Prion diseases are transmissible neurodegenerative diseases affecting humans and animals. The agent of the disease is the prion consisting mainly, if not solely, of a misfolded and aggregated isoform of the host-encoded prion protein (PrP). Transmission of prions can occur naturally but also accidentally, e.g. by blood transfusion, which has raised serious concerns about blood product safety and emphasized the need for a reliable diagnostic test. In this report we present a method based on surface-FIDA (fluorescence intensity distribution analysis), that exploits the high state of molecular aggregation of PrP as an unequivocal diagnostic marker of the disease, and show that it can detect infection in blood. To prepare PrP aggregates from blood plasma we introduced a detergent and lipase treatment to separate PrP from blood lipophilic components. Prion protein aggregates were subsequently precipitated by phosphotungstic acid, immobilized on a glass surface by covalently bound capture antibodies, and finally labeled with fluorescent antibody probes. Individual PrP aggregates were visualized by laser scanning microscopy where signal intensity was proportional to aggregate size. After signal processing to remove the background from low fluorescence particles, fluorescence intensities of all remaining PrP particles were summed. We detected PrP aggregates in plasma samples from six out of ten scrapie-positive sheep with no false positives from uninfected sheep. Applying simultaneous intensity and size discrimination, ten out of ten samples from scrapie sheep could be differentiated from uninfected sheep. The implications for ante mortem diagnosis of prion diseases are discussed.     

Relationship of turbidity to the stages of platelet aggregation

The process of platelet aggregation as detected by turbidity changes in the platelet aggregometer was studied relative to light scattering by large particles. For latex beads a plot of light scattering intensity/unit mass versus particle size gave increased light scattering intensity for small particle sizes but decreased scattering at large particle size. This behavior is predicted by Rayleigh-Gans theory. These results were related to the platelet aggregometer, an optical instrument used to measure the association of small particles (monomeric platelets) to large particles (platelet aggregates). Formalin-fixed platelets do not show changes in light transmission due to energy-requiring processes, such as shape change, so that turbidity changes in the presence of aggregating agents could be attributed to a change in platelet aggregation state. Small platelet aggregates showed increased turbidity compared to a similar mass of monomeric platelets. In fact, very large platelet aggregates that were visible to the unaided eye were needed to produce a decrease in light scattering intensity. Thus, turbidity can either increase or decrease with platelet aggregation depending on the size of the aggregates. Studies of platelet aggregation that show no initial increase in turbidity must be characterized by dominance of large platelet aggregates and monomeric platelets.     

Multichain aggregates in dilute solutions of associating polyelectrolyte keeping a constant size at the increase in the chain length of individual macromolecules

Multichain aggregates together with individual macromolecules were detected by light scattering in dilute aqueous solutions of chitosan and of its hydrophobic derivatives bearing 4 mol % of n-dodecyl side groups. It was demonstrated that the size of aggregates and their aggregation numbers increase at the introduction of hydrophobic side groups into polymer chains. The key result concerns the effect of the chain length of individual macromolecules on the aggregation behavior. It was shown that for both unmodified and hydrophobically modified (HM) chitosan, the size of aggregates is independent of the length of single chains, which may result from the electrostatic nature of the stabilization of aggregates. At the same time, the number of macromolecules in one aggregate increases significantly with decreasing length of single chains to provide a sufficient number of associating groups to stabilize the aggregate. The analysis of the light scattering data together with TEM results suggests that the aggregates of chitosan and HM chitosan represent spherical hydrogel particles with denser core and looser shell covered with dangling chains.     

Quantitative characterization of virus-like particles by asymmetrical flow field flow fractionation, electrospray differential mobility analysis, and transmission electron microscopy

Here we characterize virus-like particles (VLPs) by three very distinct, orthogonal, and quantitative techniques: electrospray differential mobility analysis (ES-DMA), asymmetric flow field-flow fractionation with multi-angle light scattering detection (AFFFF-MALS) and transmission electron microscopy (TEM). VLPs are biomolecular particles assembled from viral proteins with applications ranging from synthetic vaccines to vectors for delivery of gene and drug therapies. VLPs may have polydispersed, multimodal size distributions, where the size distribution can be altered by subtle changes in the production process. These three techniques detect subtle size differences in VLPs derived from the non-enveloped murine polyomavirus (MPV) following: (i) functionalization of the surface of VLPs with an influenza viral peptide fragment; (ii) packaging of foreign protein internally within the VLPs; and (iii) packaging of genomic DNA internally within the VLPs. These results demonstrate that ES-DMA and AFFFF-MALS are able to quantitatively determine VLP size distributions with greater rapidity and statistical significance than TEM, providing useful technologies for product development and process analytics.     

Amorphous mineral phases in magnetotactic multicellular aggregates

Magnetotactic multicellular aggregates consist of several bacteria that produce iron sulfide magnetosomes through a complex and poorly understood process. We observed new amorphous mineral particles within the cytoplasm of magnetotactic multicellular aggregates. Elemental mapping and electron energy loss spectroscopy detected iron and oxygen, but not sulfur, in these particles. These amorphous particles were about the same size as mature magnetosomes, around 50-70 nm in diameter. No membranes were observed surrounding the amorphous minerals. Partially crystalline inclusions composed of a crystalline core and an amorphous region around them similar in texture to the amorphous particles were also present. The shape of these amorphous regions followed the shape of the crystalline cores they enveloped. These regions also contained oxygen and iron. The crystalline phase, as previously reported, contained sulfur and iron. The presence of independent amorphous particles has not been reported before in magnetotactic multicellular aggregates.     

Developmental features of calcium oxalate crystal sand deposition inBeta vulgaris L. Leaves

Summary Sugar beet (Beta vulgaris L.) leaf has a layer of cells extended laterally between the palisade parenchyma and spongy mesophyll that develop numerous small crystals (crystal sand) within their vacuoles. Solubility studies and histochemical staining indicate the crystals are calcium oxalate. The crystals are deposited within the vacuoles early during leaf development, and at maturity the cells are roughly spherical in shape and 2 to 3 times larger than other mesophyll cells. Crystal deposition is preceeded by formation of membrane vesicles within the vacuole. The membranes are synthesizedde novo in the vacuole and have a typical trilaminate structure as viewed with the TEM. The membranes are formed within paracrystalline aggregates of tubular particles (6–8nm outer diameter) as membrane sheets, but are later organized into chambers or vesicles. Calcium oxalate is then precipitated within the membrane chambers. The tubular particles involved in membrane synthesis are usually present in the vacuoles of mature crystal cells, but in very small amounts.     

Electron microscopy of nuclear matrices prepared in situ under oxidizing conditions

Nuclear matrices (NM) were prepared from mouse 3T3 fibroblasts attached to growth support film in the continuous presence of the disulfide cross-linking reagent, sodium tetrathionate. Intact cells and samples at each stage of NM preparation were fixed, embedded in Epon-Araldite, sectioned and stained conventionally with uranyl-lead. Nuclear size and shape changed little during extraction, but nuclei showed a gradual reduction in internal fibrogranular elements up to 1M NaCl, after which larger spaces were visible in the nucleoplasm. In contrast to similar samples prepared under reducing conditions in other studies, final NMs contained a highly ramified internal network of fibers and granular aggregates.     

Influence of Particle Size and Concentration on Rheological Behaviour of Reconstituted Apple Purees

This work investigates the impact of structural parameters on the rheological behaviour of apple purees. Reconstructed apple purees from 0 g/100 g up to 2.32 g/100 g of insoluble solids content and varying in particle size were prepared. Three different particle size distributions were obtained by mechanical treatment only, to modify both size and morphology of the particles without modifying the intrinsic rigidity of the cell walls. Rheological measurements showed that the insoluble solids content have a first order effect on the rheological behaviour of the suspensions: three concentrations domains were observed in both dynamic and flow measurements. A model is proposed for each domain. The existence of a weak network between particles is clearly shown over a critical concentration of insoluble solids (cell walls) depending on particle size distribution (semi-diluted domain). In a concentrated domain, particles are on close packing conditions and their apparent volume begin to shrink. Particle size and shape also play an important role on the rheological behaviour of reconstructed apple puree. Due to their irregular shape, cell clusters clog the medium at lower concentration compared to individual cells.     

Inert phosphorescent nanospheres as markers for optical assays.

A simple encapsulation technique is presented to produce highly phosphorescent, inert nanospheres that are suitable luminescent markers. It is based on the coprecipitation of phosphorescent ruthenium(II)-tris(polypyridyl) complexes and polyacrylonitrile (PAN) derivatives from a solution in N,N-dimethylformamide. The beads precipitate in the form of very small aggregates of spherical shape and a typical particle diameter of less than 50 nm. This process allows the encapsulation of phosphorescent and fluorescent dyes in an individual nanosphere provided that they are sufficiently lipophilic. Quenching by oxygen is negligible due to the use of PAN. The nanospheres were characterized with respect to their spectral properties (quantum yields of the luminophores, brightness, luminescence decay time), stability in aqueous buffered suspensions, and in terms of size, shape, and surface charge of the particles, as well as storage stability, quenching by oxygen, and dye leaching.     

LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review¹ and recent examples^2-4). Leaves are the primary photosynthetic organ, and their size and shape vary developmentally and environmentally within a plant. For these reasons studies on leaf morphology require measurement of multiple parameters from numerous leaves, which is best done by semi-automated phenomics tools^5,6. Canopy shade is an important environmental cue that affects plant architecture and life history; the suite of responses is collectively called the shade avoidance syndrome (SAS)⁷. Among SAS responses, shade induced leaf petiole elongation and changes in blade area are particularly useful as indices⁸. To date, leaf shape programs (e.g. SHAPE⁹, LAMINA¹⁰, LeafAnalyzer¹¹, LEAFPROCESSOR¹²) can measure leaf outlines and categorize leaf shapes, but can not output petiole length. Lack of large-scale measurement systems of leaf petioles has inhibited phenomics approaches to SAS research. In this paper, we describe a newly developed ImageJ plugin, called LeafJ, which can rapidly measure petiole length and leaf blade parameters of the model plant Arabidopsis thaliana. For the occasional leaf that required manual correction of the petiole/leaf blade boundary we used a touch-screen tablet. Further, leaf cell shape and leaf cell numbers are important determinants of leaf size¹³. Separate from LeafJ we also present a protocol for using a touch-screen tablet for measuring cell shape, area, and size. Our leaf trait measurement system is not limited to shade-avoidance research and will accelerate leaf phenotyping of many mutants and screening plants by leaf phenotyping.     

Q-band EPR investigations of neuromelanin in control and Parkinson's disease patients

New insights into the understanding of the changes induced in the iron domain of neuromelanin (NM) upon development of Parkinson's disease (PD) have been gained by electron paramagnetic spectroscopy (EPR). The results of this study are compared with a previously reported variable temperature analysis of X-band EPR spectra of a NM specimen obtained from control brain tissues. The availability of high sensitivity instruments operating in the Q-band (34.4 GHz) allows us to deal with the low amounts of NM available from PD brains. The organization of iron in NM is in the form of polynuclear superparamagnetic/antiferromagnetic aggregates, but the lack of one or more signals in the EPR spectra of NM from PD suggests that the development of the pathology causes NM to decrease its ability to bind iron. Furthermore, the detection of the Mn(II) signal in the Q-band spectra is exploited as an additional internal probe to assess minor structural differences in iron domains of PD and control NM specimens.     

Preparation and characterization of chitosan-based nanoparticles

The present investigation describes the synthesis and characterization of novel biodegradable nanoparticles based on chitosan for biomedical applications. Natural di- and tricarboxylic acids were used for intramolecular cross-linking of the chitosan linear chains. The condensation reaction of carboxylic groups and pendant amino groups of chitosan was performed by using water-soluble carbodiimide. This method allows the formation of polycations, polyanions, and polyampholyte nanoparticles. The prepared nanosystems were stable in aqueous media at low pH, neutral, and mild alkaline conditions. The structure of products was determined by NMR spectroscopy, and the particle size was identified by laser light scattering (DLS) and transmission electron microscopy (TEM) measurements. It was found that particle size depends on the pH, but at a given pH, it was independent of the ratio of cross-linking and the cross-linking agent. Particle size measured by TEM varied in the range 60-280 nm. In the swollen state, the average size of the particles measured by DLS was in the range 270-370 nm depending on the pH. The biodegradable cross-linked chitosan nanoparticles, as solutions or dispersions in aqueous media, might be useful for various biomedical applications.     

Measuring Floc Structural Characteristics

A review is presented of a range of techniques for the structural characterisation of flocs. Flocs may be considered as highly porous aggregates composed of smaller primary particles. The irregular size and shape of flocs makes them difficult to measure and quantify. A range of different equivalent diameters are often used to define the floc size and allow comparison with other floc systems. The application of a range of floc sizing methods has been described. Microscopy is time consuming, requiring large sample size and considerable preparation but gives good information on floc shape and form. Light scattering and transmitted light techniques have been used to good effect to measure floc size on-line whilst individual particle sensors have limited applicability to measuring floc size. Fractal dimension can be measured using one of three major techniques: light scattering, settling and two dimensional (2D) image analysis. Light scattering is ideally suited for small, open flocs of low refractive index whilst settling may be applied to most floc systems of low porosity. 2D image analysis requires flocs to have good contrast between the solid in the floc and the background.     

Effects of mixing during acid addition on fractionally precipitated protein

Isoelectric fractionation of the two major proteins of soy is characterized. Fractions are acid precipitated and cen-trifugally collected at pH 6.0 (glycinin) and pH 4.8 (beta-conglycinin). Two extremes in the speed of acid addition (rapid, with no mixing, and slow, via acid dialysis, with complete mixing) are compared to determine their effects on the properties of the precipitate. Total protein yield, fraction composition, and aggregate microstructure do not depend significantly on the method of acid addition. Particle size distribution and hindered settling behavior do differ and are explained using a model of aggregate strength. The rapid acid addition produces larger primary particles, because of higher supersaturation, and yields larger aggregates, because of higher interparticle potential and stronger aggregates. Further aggregation in low-shear hindered settling is faster for the slowly precipitated aggregate because few of these bonding sites could survive the high-shear precipitator, whereas more can contribute to aggregation during hindered settling.     

Non-native aggregation of recombinant human granulocyte-colony stimulating factor under simulated process stress conditions

Effective inhibition of protein aggregation is a major goal in biopharmaceutical production processes optimized for product quality. To examine the characteristics of process-stress-dependent aggregation of human granulocyte colony-stimulating factor (G-CSF), we applied controlled stirring and bubble aeration to a recombinant non-glycosylated preparation of the protein produced in Escherichia coli. We characterized the resulting denaturation in a time-resolved manner using probes for G-CSF conformation and size in both solution and the precipitate. G-CSF was precipitated rapidly from solutions that were aerated or stirred; only small amounts of soluble aggregates were found. Exposed hydrophobic surfaces were a characteristic of both soluble and insoluble G-CSF aggregates. Using confocal laser scanning microscopy, the aggregates presented mainly a circular shape. Their size varied according to incubation time and stress applied. The native intramolecular disulfide bonds in the insoluble G-CSF aggregates were largely disrupted as shown by mass spectrometry. New disulfide bonds formed during aggregation. All involved Cys(18) , which is the only free cysteine in G-CSF; one of them had an intermolecular Cys(18(A)) -Cys(18(B)) crosslink. Stabilization strategies can involve external addition of thiols and extensive reduction of surface exposition during processing.     

The Hofmeister effect on amyloid formation using yeast prion protein

A variety of proteins are capable of converting from their soluble forms into highly ordered fibrous cross‐β aggregates (amyloids). This conversion is associated with certain pathological conditions in mammals, such as Alzheimer disease, and provides a basis for the infectious or hereditary protein isoforms (prions), causing neurodegenerative disorders in mammals and controlling heritable phenotypes in yeast. The N‐proximal region of the yeast prion protein Sup35 (Sup35NM) is frequently used as a model system for amyloid conversion studies in vitro. Traditionally, amyloids are recognized by their ability to bind Congo Red dye specific to β‐sheet rich structures. However, methods for quantifying amyloid fibril formation thus far were based on measurements linking Congo Red absorbance to concentration of insulin fibrils and may not be directly applicable to other amyloid‐forming proteins. Here, we present a corrected formula for measuring amyloid formation of Sup35NM by Congo Red assay. By utilizing this corrected procedure, we explore the effect of different sodium salts on the lag time and maximum rate of amyloid formation by Sup35NM. We find that increased kosmotropicity promotes amyloid polymerization in accordance with the Hofmeister series. In contrast, chaotropes inhibit polymerization, with the strength of inhibition correlating with the B‐viscosity coefficient of the Jones‐Dole equation, an increasingly accepted measure for the quantification of the Hofmeister series.     

New methods allowing the detection of protein aggregates: A case study on trastuzumab

Aggregation compromises the safety and efficacy of therapeutic proteins. According to the manufacturer, the therapeutic immunoglobulin trastuzumab (Herceptin®) should be diluted in 0.9% sodium chloride before administration. Dilution in 5% dextrose solutions is prohibited. The reason for the interdiction is not mentioned in the Food and Drug Administration (FDA) documentation, but the European Medicines Agency (EMEA) Summary of Product Characteristics states that dilution of trastuzumab in dextrose solutions results in protein aggregation. In this paper, asymmetrical flow field-flow fractionation (FFF), fluorescence spectroscopy, fluorescence microscopy and transmission electron microscopy (TEM) have been used to characterize trastuzumab samples diluted in 0.9% sodium chloride, a stable infusion solution, as well as in 5% dextrose (a solution prone to aggregation). When trastuzumab samples were injected in the FFF channel using a standard separation method, no difference could be seen between trastuzumab diluted in sodium chloride and trastuzumab diluted in dextrose. However, during FFF measurements made with appropriate protocols, aggregates were detected in 5% dextrose. The parameters enabling the detection of reversible trastuzumab aggregates are described. Aggregates could also be documented by fluorescence microscopy and TEM. Fluorescence spectroscopy data were indicative of conformational changes consistent with increased aggregation and adsorption to surfaces. The analytical methods presented in this study were able to detect and characterize trastuzumab aggregates.Key words: immunoglobulin, aggregation, stability, protein, trastuzumab, herceptin®, methodology