Content Of Microparticles Research Articles

Circulating serum-derived microparticles provide novel proteomic biomarkers of spontaneous preterm birth.

The purpose of this study was to determine whether the proteomic biosignature of circulating microparticles in maternal serum obtained in the second trimester could identify pregnancies that result in spontaneous preterm birth (SPTB). Microparticles were isolated from blinded biorepository-sourced serum samples from 48 pregnant women at 15 to 17 weeks of gestation. Microparticle proteins were extracted and analyzed using label-free liquid chromatography/mass spectrometry. Peptide features were analyzed to assess the association of specific protein patterns with subjects delivering at term (≥ 37 weeks gestation; n = 24) and those experiencing SPTB (≤ 34 weeks gestation; n = 24). We found 99 proteins that had statistically significant differences in signal intensity between term and SPTB women in both first (n = 26) and second (n = 22) singleton gestation pregnancy cohorts. Additional evaluation identified 18 biomarkers that met criteria for further priority evaluation (12 preterm, 6 term). Pathway analysis showed that differentiating SPTB biomarker proteins were predominantly associated with inflammation and cell injury, while differentiating term biomarkers were associated with cell growth and hematological parameters. This study shows for the first time that the proteomic content of serum microparticles isolated in the second trimester can identify with a high degree of accuracy pregnancies that result in SPTB.

Stimuli-responsive hybrid composites based on CaCO3 microparticles and smart polyelectrolytes for controllable drug delivery

CaCO3/aliphatic poly(urethane-amine) (PUA)/sodium polystyrene sulfonate (PSS) hybrid composites with dual-responsive controlled drug delivery property had been prepared via electrostatic interaction. Aliphatic PUA and PSS were employed as smart polyelectrolytes. PSS was also served as crystal growth additive to control the morphology of CaCO3 microparticles. The electrostatic interaction between PSS and aliphatic PUA under weak-acid condition improved the bonding force between PSS-doped CaCO3 microparticles and polyelectrolytes. pH-/thermo-responsive drug delivery property and the high drug loading capacity (around 90 %) could be achieved from the prepared hybrid composites. More importantly, the relative content of CaCO3 microparticles and polyelectrolytes had a significant effect on the morphology and controllable release properties of the hybrid composites. The smart drug release mechanism of the hybrid composites was also analyzed by fitting the cumulative release data to Retger–Peppas equation.

Mechanisms and behavioral regularities of the vacuum-arc microparticles near and on a potential electrode immersed in plasma

It is experimentally revealed that the pulse-periodic bias potential provides a multifold decrease in the surface content of microparticles (MPs). It is ascertained that a decrease in the MP concentration at the target can be explained by several physical mechanisms. From experiments with a fine-structure grid, it is found that less than 10% of MPs negatively charged in the plasma can be reflected in the electric field of the charge-separation layer near the sample. A substantial decrease in the MP density occurs after direct interaction between a MP and the sample under the action of a negative HF short-pulse bias potential. Almost half the MP surface density is caused by ion sputtering. A twelvefold reduction in the MP surface density is attained when the target is irradiated for 2 min.

The properties of microparticles from RAW 264.7 macrophage cells undergoing in vitro activation or apoptosis.

Microparticles (MPs) are small, membrane-bound vesicles that arise from dead and dying cells, and display pro-inflammatory and pro-thrombotic activity. As shown previously, the RAW 264.7 murine macrophage cell line can release MPs following stimulation with LPS or polyinosinic:polycytidylic acid [poly (I:C)], ligands of TLR4 and TLR3 respectively. To determine the relationship of these MPs to those released during apoptosis, the nucleic acid content of MPs from cultures stimulated with LPS or poly (I:C) was compared with the nucleic acid content of MPs from untreated cells or cells induced to undergo apoptosis by treatment with etoposide or staurosporine (STS). As results of these studies showed, MPs from activated, apoptotic and untreated cells had features in common, as demonstrated by binding of the nucleic acid dyes SYTO 13 and propidium iodide; molecular mass of DNA; and binding of monoclonal anti-DNA and anti-nucleosome Abs. While MPs from the different culture conditions all contained ribosomal RNA, ribosomal RNA from MPs from STS-treated cells showed cleavage and degradation. Taken together, these studies indicate that the nucleic acid content of MPs from activated and apoptotic cells have important similarities, suggesting that events during TLR activation may lead to apoptosis and subsequent MP release.

Preliminary Studies on Solid Lipid Microparticles of Loratadine for the Treatment of Allergic Reactions via the Nasal Route

Purpose: To formulate solid lipid microparticles (SLM) of loratadine (LRT) for the treatment of allergic reactions via the nasal route.Methods: Microparticles were prepared by emulsion congealing technique. The drug content of microparticles was analysed. Drug/excipient compatibility and crystallinity characteristics of microparticles were investigated by Fourier Transform Infrared Spectroscopy (FT-IR) and differential scanning calorimetry (DSC). Particle size distribution was determined by laser diffraction (LD). Drug release from microparticles was compared to that from conventional vehicles (O/W emulsion, gel andoleageneous cream) using Franz-type diffusion cells.Results: Drug content of microparticles was > 87.96 %. FT-IR and DSC analysis indicated that the drug and excipients were compatible for at least 6 months at room temperature after production. Microparticle size was between 86 ± 5.63 ìm and 184 ± 13.21 ìm while mean droplet size of O/Wemulsion was 76 ± 3.45 ìm. Release profiles of LRT from microparticles were significantly different from those of O/W emulsion, gel and oleageneous cream (p < 0.05). In the case of conventional vehicles,increase in the hydrophilicity of the vehicles led to increase in drug release rate. Drug release fitted generally to zero order kinetics as well as Korsmeyer-Peppas model for one of the SLM formulations, indicating non-Fickian drug release (super case II transport).Conclusion: SLM provided LRT release for a longer period than the conventional vehicles. However, in vivo studies are required to ascertain the effectiveness of the formulations.Keywords: Loratadine, Solid lipid microparticles, Allergy, Controlled drug delivery, Transnasal delivery

Spray-Dried Chitosan Microparticles for Cellular Delivery of an Antigenic Protein: Physico-chemical Properties and Cellular Uptake by Dendritic Cells and Macrophages

Spray-dried chitosan microparticles for cellular delivery of antigen to dendritic cells (DC) and macrophages (Mϕ) were investigated. Chitosan microparticles were prepared by spray drying. For comparison, poly(lactic-co-glycolic acid) (PLGA) and poly(α-butyl cyanoacrylate) (BCA) micro-/nanoparticles were generated. Bovine serum albumin (BSA) was used as a model antigen. The particles were characterized in terms of size, morphology, surface charge, surface composition, protein content, entrapment efficiency, in vitro release, and protein integrity. Additionally, they were subject to cell viability and cellular uptake study with DC and Mϕ. Size of chitosan, PLGA, and BCA micro-/nanoparticles ranged between 3.11-7.18, 0.94-6.26, and 0.30-6.34μm, respectively. Particle morphology and in vitro protein release varied, depending on polymer type, particle composition and preparation process parameters. Chitosan microparticles were cationic, while PLGA microparticles were neutral. BCA micro-/nanoparticles were either anionic or cationic, according to polymerization pH. Protein content and entrapment efficiency of chitosan and PLGA microparticles were relatively consistent. Only integrity and conformational structure of protein encapsulated in chitosan microparticles were completely retained. Chitosan and PLGA microparticles were non-toxic to DC and Mϕ, but the former were internalized more efficiently. Spray-dried chitosan microparticles delivered the antigen efficiently to DC and Mϕ.

Increased Shedding of Microvesicles from Intimal Smooth Muscle Cells in Athero-Prone Areas of the Human Aorta: Implications for Understanding of the Predisease Stage

Objective: This study evaluated whether a change in the content of matrix microvesicles might occur at the preatherosclerotic stage. Methods: Applying quantitative electron microscopic and immunohistochemical analyses, two areas of grossly normal segments of the thoracic aorta were compared: atherosclerosis-prone (AP) areas, situated at the dorsal aspect of the aorta along the rows of intercostal branch origins, and atherosclerosis-resistant (AR) areas, situated at the corresponding sites of the ventral aspect of the aorta. Results: The electron microscopic analysis showed that there were 1.4 times more microvesicles in AP areas than AR areas (p = 0.019). It was found that matrix microvesicles originated as a result of blebbing and shedding of surface membranes of smooth muscle cells. A quantitative analysis of the expression of ADP-ribosylation factor 6 (ARF6), which is known to be involved in membrane trafficking and microvesicle formation, showed that ARF6 expression was 1.3 times higher in AP areas than that in AR areas (p = 0.006). There was a positive correlation between the content of matrix microparticles and the expression of ARF6 by intimal smooth muscle cells (r = 0.61; p < 0.0001). Conclusion: The present study supports the concept that alterations of the arterial intima occur at the predisease stage.

Multivariate design for the evaluation of lipid and surfactant composition effect for optimisation of lipid nanoparticles

Physicochemical properties of lipid nanoparticles (LN), such as size, size distribution and surface charge, have a major influence both, on in vitro stability and delivery of the incorporated drug in vivo. With the purpose of understanding how these properties are influenced by variations of LN composition (e.g. lipid and surfactant type and concentration) 22 factorial designs with centre point were applied for several types of lipids and surfactants in the present study. Tested factors and levels were the type and concentration of lipid (cetyl palmitate, Dynasan 114 and Witepsol E85) at the concentrations of 5%, 10% and 15%, in combination with type and concentration of surfactant (polysorbate 20, 40, 60 and 80 and poloxamer 188 and 407) at concentrations of 0.8%, 1.2% and 2.0%. Responses measured within the design space were the mean size and polydispersity index (photon correlation spectroscopy), content of microparticles (optical single particle sizing), macroscopic appearance, pH and zeta potential on the day of production, 1 and 2years after production. Multivariate evaluation and modelling were performed starting with a principal component analysis (PCA) and followed by partial least square regression analysis (PLS) to assess both qualitative and quantitative influence of the investigated factors in the LN. Our study showed that both, lipid and surfactant concentration and the type of surfactant are crucial parameters for the particle size of the LN prepared by high pressure homogenisation (HPH). For LN stability during 2years both, lipid and surfactant types and concentrations were identified as the most relevant parameters. Among the surfactants most suitable for producing LN with small sizes were the polysorbates and the lipid yielding best storage stability was cetyl palmitate. Furthermore, the models allowed the prediction of the mean size of LN that could be achieved with a certain lipid/surfactant combination and concentration. The obtained results are considered useful for future design of stable LN formulations without the need of extensive empirical testing of formulation parameters within the given HPH technology.

Subcellular Analysis of the Platelet Proteome

Blood platelets are key players mainly involved in physiological thrombus formation as well as pathological events, such as atherosclerosis, coronary artery disease, or myocardial infarction. Consequently, numerous studies have focused on gaining insight into the platelet proteome, with biomolecular mass spectrometry providing an efficient technology to cope with this task as it nowadays enables both protein identification and differential quantification of cellular material. To assemble a comprehensive proteomic dataset, platelet-centric studies have to be performed not only on the global scale, but also on the subcellular level, which implies subfractionation techniques and targeted approaches prior to detailed proteomic analyses. In this review we present an overview of advances in platelet subcellular proteomics focussing on the platelet membrane proteome and the protein content of both platelet granules and platelet-derived microparticles. Since posttranslational modifications are furthermore known to decisively participate in platelet function e.g. upon activation, analytical methods for the platelet phospho- as well as the glycoproteome will also be discussed. Keywords: Glycosylation, granule, mass spectrometry, membrane, microparticle, phosphorylation, platelet, proteome, hemostasis, megakaryocytes, atherosclerosis, coronary artery disease, myocardial infarction

Use of SYTO 13, a fluorescent dye binding nucleic acids, for the detection of microparticles in in vitro systems

Microparticles (MPs) are small membrane-bound vesicles that are released from activated or dying cells by a blebbing process. These particles contain nuclear and cytoplasmic components and represent unique biomarkers for disease. The small size of particles, however, limits detection using flow cytometry with either light scatter or staining for surface markers. Because MPs contain DNA and RNA, we have explored the use of SYTO 13, a member of the class of SYTO dyes, for particle detection. SYTO 13 is cell permeable and has a high fluorescent yield when bound to DNA or RNA. In this study, we compared detection of MPs using either light scatter or SYTO 13 staining, testing the hypothesis that, with fluorescence detection with SYTO 13, problems of "noise" with light scatter are reduced and the range of MP sizes detected is increased. In these experiments, particles were obtained from lymphoid cell lines treated in vitro to undergo apoptosis. As these results showed, STYO 13 allowed the detection of 1.5-2.9 times as many particles as did light scatter. The increased sensitivity was observed with three different cell lines and was independent of inducing stimulus. Treatment of fixed and permeabilized MPs with DNase and RNase showed that SYTO 13 binding resulted from interaction with both DNA and RNA. Together, these findings indicate that the nucleic acid content of MPs provides the basis for their detection in in vitro systems and suggests the utility of fluorescent dyes like SYTO 13 for more sensitive quantitative assays.

Effects of Multi-modal Filler Size Distributions on Thermal Conductivity of Electrically Conductive Adhesives Containing Ag Micro and Nanoparticles

Electrically conductive adhesives composed of a multi-functional epoxy-based matrix containing micro- and nano-fillers with bimodal and trimodal size distributions were prepared in order to investigate their electrical and thermal conductivities. When Ag flakes were used as the filler, anisotropy was clearly observed in the thermal conductivities due to the alignment of the flakes along the in-plane direction. A bimodal adhesive containing Ag flakes and spherical micro-particles exhibited a maximal value for thermal conductivity in the vertical direction when the content of the micro-particles was 50–60 wt% of the total filler loading, although its conductivity in the in-plane direction decreased monotonically with increasing content of micro-particles. With trimodal adhesives containing Ag flakes, micro- and nanoparticles, the Ag nanoparticles could be sintered during the curing process. Adequate dispersion and sufficient sintering of the nanoparticles were found to be essential in order to improve the electrical and thermal conductivities of these adhesives.

Endothelial microparticles in diseases

Microparticles are submicron vesicles shed from plasma membranes in response to cell activation, injury, and/or apoptosis. The measurement of the phospholipid content (mainly phosphatidylserine; PSer) of microparticles and the detection of proteins specific for the cells from which they are derived has allowed their quantification and characterization. Microparticles of various cellular origin (platelets, leukocytes, endothelial cells) are found in the plasma of healthy subjects, and their amount increases under pathological conditions. Endothelial microparticles (EMP) not only constitute an emerging marker of endothelial dysfunction, but are also considered to play a major biological role in inflammation, vascular injury, angiogenesis, and thrombosis. Although the mechanisms leading to their in vivo formation remain obscure, the release of EMP from cultured cells can be caused in vitro by a number of cytokines and apoptotic stimuli. Recent studies indicate that EMP are able to decrease nitric-oxide-dependent vasodilation, increase arterial stiffness, promote inflammation, and initiate thrombosis at their PSer-rich membrane, which highly co-expresses tissue factor. EMP are known to be elevated in acute coronary syndromes, in severe hypertension with end organ damage, and in thrombotic thrombocytopenic purpura, all conditions associated with endothelial injury and pro-thrombotic state. The release of EMP has also been associated with endothelial dysfunction of patients with multiple sclerosis and lupus anticoagulant. More recent studies have focused on the role of low shear stress leading to endothelial cell apoptosis and subsequent EMP release in end-stage renal disease. Improved knowledge of EMP composition, their biological effects, and the mechanisms leading to their clearance will probably open new therapeutic approaches in the treatment of atherothrombosis.

Reverse self-assemblies based on amphiphilic polyphosphazenes for encapsulation ofwater-soluble molecules

A novel series of amphiphilic polyphosphazenes (PNIPAm/AA-PPP) containing hydrophilicoligo-(N-isopropylacrylamide) (oligo-NIPAm) and various hydrophobic aliphatic amines asco-substitutes was synthesized via a two-step substitution reaction. The extraction andsolubilization of water-soluble substances such as fluorescein sodium and trypan blue froman aqueous phase into the chloroform phase were supposed to result from theformation of polyphosphazene reverse self-assemblies in the organic phase. Afield emission scanning electronic microscope was adopted to characterize themorphology of reverse assemblies in chloroform. Additionally, a significant improvementof encapsulation and release profiles of water-soluble substances was found forpoly(lactic-co-glycolic acid) (PLGA) microparticles in the presence of amphiphilic copolymers, which wasassociated with the chemical structure of copolymers as well as the content of copolymer inthe microparticles.

Grinding Properties of Abandoned Concrete

The grinding properties of abandoned concrete, which consists primarily of hardened cement, limestone aggregate and river sand, are studied. Theoretical models of grinding are used to explain the experimental observation. The results show that 1) The principle disintegration mechanism of hardened cement and river sand is volumetric grinding, although at later stages grinding of cement becomes difficult because of its flaked structure; 2) The limestone grinding process can be divided into two steps. First, volumetric grinding, with an obvious component of surface grinding, followed by primarily surface grinding as the micro-particle content increases; 3) Initially, the principle mechanism of grinding limestone and river sand is volumetric grinding, albeit less efficient grinding than if these components were ground separately, and; 4) After 10 to 20 min of grinding the grinding bottleneck phenomenon appears and after 20 min of grinding the content of micro-particles is large and surface grinding is the main mechanism while the particle size of the mixture is smaller than that of separately ground river sand and cement but bigger than that of separately ground limestone.

The phospholipid composition and cholesterol content of platelet-derived microparticles: a comparison with platelet membrane fractions

The processes that govern the distribution of molecules between platelets and the microparticles (MP) they release are unknown. Certain proteins are sorted selectively into MP, but lipid sorting has not been studied. To compare the phospholipid composition and cholesterol content of platelet-derived MP obtained with various stimuli with that of isolated platelet membrane fractions. Washed platelets from venous blood of healthy individuals (n = 6) were stimulated with collagen, thrombin, collagen plus thrombin, or A23187. Platelet activation, MP release and antigen exposure were assessed by flow cytometry. MPs were isolated by differential centrifugation. Platelet plasma-, granule- and intracellular membranes were isolated from platelet concentrates (n = 3; 10 donors each) by pressure homogenization and Percoll density gradient fractionation. The phospholipid composition and cholesterol content of MPs and membrane fractions were analyzed by high performance thin layer chromatography. The phospholipid composition of MPs was intermediate compared with that of platelet plasma- and granule membranes, and differed significantly from that of intracellular membranes. There were small but significant differences in phospholipid composition between the MPs produced by the various agonists, which paralleled differences in P-selectin exposure in case of the physiological agonists collagen, thrombin, or collagen plus thrombin. The cholesterol content of MPs tended to be higher than that of the three-platelet membrane fractions. Regarding its phospholipid content, the MP membrane is a composite of the platelet plasma- and granule membranes, showing subtle differences depending on the platelet agonist. The higher cholesterol content of MPs suggests their enrichment in lipid rafts.

Buparvaquone mucoadhesive nanosuspension: preparation, optimisation and long-term stability

The poorly soluble drug buparvaquone is used in experimental clinics against the gastrointestinal persisting parasite Cryptosporidium parvum. It was produced as nanosuspension by high pressure homogenisation. Main advantages of nanosuspensions (amongst others) are their increase of saturation solubility and dissolution velocity, improving the bioavailability of drugs. The buparvaquone nanosuspension had a bulk population of about 600 nm (analysed by photon correlation spectroscopy (PCS)). The additional analysis performed with laser diffraction showed that only a very small content of microparticles occurred, which is, for the special features of nanosuspensions, negligible because they were still below 3 μm. Another feature of nanosuspensions is the adhesion properties to surfaces, e.g. mucosa. To further increase the adhesion time of the buparvaquone nanosuspension to C. parvum, the nanosuspension was formulated with hydrogels made from mucoadhesive polymers, e.g. different types of Carbopol® and chitosan. Only a small increase of the particle size of the bulk population occurred directly after the incorporation of buparvaquone nanosuspension into the hydrogels. The nanosuspension/hydrogel systems were physically long-term stable over a period of 6 months as indicated by the unchanged particle sizes.

Nanosuspensions of poorly soluble drugs — reproducibility of small scale production

The major problem of many newly developed pharmaceutical drugs is their poor solubility in water and simultaneously in organic media. To solve these problems formulation as nanosuspensions is an attractive alternative. During the drug development process screening for an optimal formulation by homogenisation is essential. Time and cost effective production in an initial phase of R&D can be conducted on lab scale by using the Micron Lab 40 in its discontinuous version. In this report reproducibility of small scale production parameters (particle size, size distribution, content of microparticles) was exemplary studied for the drug RMKP22.

Protein-loaded poly( dl-lactide-co-glycolide) microparticles for oral administration: formulation, structural and release characteristics

FITC-labelled bovine serum albumin has been entrapped in sub-5 micron particles of poly( dl-lactide-co-glycolide copolymer) (PLG) using a water-in-oil-in-water (w/o/w) emulsification-solvent evaporation technique. The concentration of PVA stabiliser in the external continuous phase was found to affect not only the particle size, size distribution and protein content but also the release characteristics and internal structure of the microparticles. The importance of primary emulsification was underlined by the finding that the protein content of microparticles with mean size 1 μm could be increased from about 1% w/w to around 12% w/w by increasing the amount of protein added to the primary emulsion and the homogenisation time in this stage. Under conditions of low stabiliser concentration, multi-nucleate particles formed by polymer precipitation and envelopment of the droplets of the primary w/o emulsion. In this case surface protein loading was of the order of 30% w/w. Under conditions of high PVA stabiliser concentration, disruption of the primary emulsion occurred, resulting in sub-micron particles which were characterised by a high surface protein loading of the order of 70% w/w. A mechanism for protein microencapsulation is presented which is heavily influenced by the shear stresses induced during the process of secondary emulsification. This can explain certain aspects of the relationship between microparticle size and size distribution, protein content and release and the structural characteristics of microparticles produced using the w/o/w emulsification/solvent evaporation technique.

The Greenland ice-sheet margin — A mine of ice for paleo-environmental studies

Stable isotope analysis of surface-ice samples from three locations on the margin of the Greenland ice sheet have provided δ 18O records dating back into the Wisconsinan. The samples were collected in profiles transverse to the ice margin on a location about 40 km northeast of Jakobshavn, West Greenland, and at two locations in Warming Land, North Greenland. The Holocene/Wisconsinan transition, as detected by a large aburpt δ-shift, was found between 470 and more than 850 m from the ice edge. The magnitude of the δ-shift was about 8% and 12% in the West and North Greenland records, respectively. The West Greenland record, which is the more detailed, documents that some δ-variations in the Wisconsinan are apparently preserved in the ice margin isotopic records. By linking locations in the accumulation and ablation zones with the same observed δ-value, the surface ice of the ablation zone can be dated back to at least the Wisconsinan/Holocene transition, if the velocity of the ice motion is measured or estimated by ice flow modelling. This means that dated records of ice constituents (gas-composition, chemistry, content of microparticles, micro-meteorites, etc.) can be established by mining the easily accessible surface ice of the ablation zone.