Characterisation of the Poly-(Vinylpyrrolidone)-Poly-(Vinylacetate-Co-Crotonic Acid) (PVP:PVAc-CA) Interpolymer Complex Matrix Microparticles Encapsulating a Bifidobacterium lactis Bb12 Probiotic Strain

The method of producing poly-(vinylpyrrolidone)-poly-(vinylacetate-co-crotonic acid) (PVP:PVAc-CA) interpolymer complex matrix microparticles in supercritical carbon dioxide (scCO₂), encapsulating bacteria, has recently been developed. This study was aimed at probing the external and internal structure of these microparticles, which can be used in food. The encapsulation efficiency and distribution of encapsulated Bifidobacterium lactis Bb12 within these microparticles were also investigated. Scanning electron microscopy (SEM) revealed irregular, mostly small, smooth microparticles with no visible bacterial cells on the surface. However, some of the microparticles appeared to have porous surfaces. The results of a Microtrac S3500 particle size analyzer showed that the PVP:PVAc-CA interpolymer complex matrix microparticles encapsulating B. lactis Bb12 had an average particle size of 166.1 μm (<350 μm designated standard size for microparticles). The D ₁₀, D ₅₀ and D ₉₀ values for these microparticles were 48.16, 166.06 and 382.55 μm, respectively. Both SEM and confocal laser scanning microscopy showed a high density of bacterial cells within the microparticles. An average encapsulation efficiency of 96% was achieved. Consequently, the microparticles have the potential to be evenly distributed in foods, deliver adequate amounts of probiotics and produce minimal adverse effects on the texture and mouth feel of the foods into which they are incorporated.     

Fabricating PLGA microparticles with high loads of the small molecule antioxidant N‐acetylcysteine that rescue oligodendrocyte progenitor cells from oxidative stress

Reactive oxygen species (ROS), encompassing all oxygen radical or non‐radical oxidizing agents, play key roles in disease progression. Controlled delivery of antioxidants is therapeutically relevant in such oxidant‐stressed environments. Encapsulating small hydrophilic molecules into hydrophobic polymer microparticles via traditional emulsion methods has long been a challenge due to rapid mass transport of small molecules out of particle pores. We have developed a simple alteration to the existing water‐in‐oil‐in‐water (W/O/W) drug encapsulation method that dramatically improves loading efficiency: doping external water phases with drug to mitigate drug diffusion out of the particle during fabrication. PLGA microparticles with diameters ranging from 0.6 to 0.9 micrometers were fabricated, encapsulating high loads of 0.6–0.9 µm diameter PLGA microparticles were fabricated, encapsulating high loads of the antioxidant N‐acetylcysteine (NAC), and released active, ROS‐scavenging NAC for up to 5 weeks. Encapsulation efficiencies, normalized to the theoretical load of traditional encapsulation without doping, ranged from 96% to 400%, indicating that NAC‐loaded external water phases not only prevented drug loss due to diffusion, but also doped the particles with additional drug. Antioxidant‐doped particles positively affected the metabolism of oligodendrocyte progenitor cells (OPCs) under H₂O₂‐mediated oxidative stress when administered both before (protection) or after (rescue) injury. Antioxidant doped particles improved outcomes of OPCs experiencing multiple doses of H₂O₂ by increasing the intracellular glutathione content and preserving cellular viability relative to the injury control. Furthermore, antioxidant‐doped particles preserve cell number, number of process extensions, cytoskeletal morphology, and nuclear size of H₂O₂‐stressed OPCs relative to the injury control. These NAC‐doped particles have the potential to provide temporally‐controlled antioxidant therapy in neurodegenerative disorders such as multiple sclerosis (MS) that are characterized by continuous oxidative stress.     

Physicochemical Characterization and Water Vapor Sorption of Organic Solution Advanced Spray-Dried Inhalable Trehalose Microparticles and Nanoparticles for Targeted Dry Powder Pulmonary Inhalation Delivery

Novel advanced spray-dried inhalable trehalose microparticulate/nanoparticulate powders with low water content were successfully produced by organic solution advanced spray drying from dilute solution under various spray-drying conditions. Laser diffraction was used to determine the volumetric particle size and size distribution. Particle morphology and surface morphology was imaged and examined by scanning electron microscopy. Hot-stage microscopy was used to visualize the presence/absence of birefringency before and following particle engineering design pharmaceutical processing, as well as phase transition behavior upon heating. Water content in the solid state was quantified by Karl Fisher (KF) coulometric titration. Solid-state phase transitions and degree of molecular order were examined by differential scanning calorimetry (DSC) and powder X-ray diffraction, respectively. Scanning electron microscopy showed a correlation between particle morphology, surface morphology, and spray drying pump rate. All advanced spray-dried microparticulate/nanoparticulate trehalose powders were in the respirable size range and exhibited a unimodal distribution. All spray-dried powders had very low water content, as quantified by KF. The absence of crystallinity in spray-dried particles was reflected in the powder X-ray diffractograms and confirmed by thermal analysis. DSC thermal analysis indicated that the novel advanced spray-dried inhalable trehalose microparticles and nanoparticles exhibited a clear glass transition (T _g). This is consistent with the formation of the amorphous glassy state. Spray-dried amorphous glassy trehalose inhalable microparticles and nanoparticles exhibited vapor-induced (lyotropic) phase transitions with varying levels of relative humidity as measured by gravimetric vapor sorption at 25°C and 37°C.     

Preparation of budesonide and budesonide-PLA microparticles using supercritical fluid precipitation technology

The objective of this study was to prepare and characterize microparticles of budesonide alone and budesonide and polylactic acid (PLA) using supercritical fluid (SCF) technology. A precipitation with a compressed antisolvent (PCA) technique employing supercritical CO₂ and a nozzle with 100-μm internal diameter was used to prepare microparticles of budesonide and budesonide-PLA. The effect of various operating variables (temperature and pressure of CO₂ and flow rates of drug-polymer solution and/or CO₂) and formulation variables (0.25%, 0.5%, and 1% budesonide in methylene chloride) on the morphology and size distribution of the microparticles was determined using scanning electron microscopy. In addition, budesonide-PLA particles were characterized for their surface charge and drug-polymer interactions using a zeta meter and differential scanning calorimetry (DSC), respectively. Furthermore, in vitro budesonide release from budesonide-PLA microparticles was determined at 37°C. Using the PCA process, budesonide and budesonide-PLA microparticles with mean diameters of 1 to 2 μm were prepared. An increase in budesonide concentration (0.25%–1% wt/vol) resulted in budesonide microparticles that were fairly spherical and less aggiomerated. In addition, the size of the microparticles increased with an increase in the drug-polymer solution flow rate (1.4–4.7 mL/min) or with a decrease in the CO₂ flow rate (50–10 mL/min). Budesonide-PLA microparticles had a drug loading of 7.94%, equivalent to ∼80% encapsulation efficiency. Budesonide-PLA microparticles had a zeta potential of— 37±4 mV, and DSC studies indicated that SCF processing of budesonide-PLA microparticles resulted in the loss of budesonide crystallinity. Finally, in vitro drug release studies at 37°C indicated 50% budesonide release from the budesonide-PLA microparticles at the end of 28 days. Thus, the PCA process was successful in producing budesonide and budesonide-PLA microparticles. In addition, budesonide-PLA microparticles sustained budesonide release for 4 weeks.     

Drying Using Supercritical Fluid Technology as a Potential Method for Preparation of Chitosan Aerogel Microparticles

Supercritical fluid technology offers several advantages in preparation of microparticles. These include uniformity in particle size, morphology, and drug distribution without degradation of the product. One of the recent advantages is preparation of porous aerogel carrier with proper aerodynamic properties. In this study, we aimed to prepare chitosan aerogel microparticles using supercritical fluid (SCF) technology and compare that with microparticles produced by freeze drying (FD). Loading the prepared carriers with a model drug (salbutamol) was also performed. Comparisons of the particle properties and physicochemical characterizations were undertaken by evaluating particle size, density, specific surface area, and porosity. In vitro drug release studies were also investigated. The effect of many variables, such as molecular weight of chitosan oligomers, concentrations of chitosan, and concentrations of tripolyphosphate on the release, were also investigated. Chitosan aerogels were efficiently produced by SCF technology with an average particle size of 10 μm with a tapped density values around 0.12 g/mL, specific surface area (73–103) m²/g, and porosity (0.20–0.29) cc/g. Whereas, microparticles produced by FD method were characterized as cryogels with larger particle size (64 microns) with clear cracking at the surface. Sustained release profile was achieved for all prepared microparticles of salbutamol produced by the aforementioned methods as compared with pure drug. The results also demonstrates that chitosan molecular weight, polymer concentration, and tripolyphosphate concentration affected the release profile of salbutamol from the prepared microparticles. In conclusion, SCF technology was able to produce chitosan aerogel microparticles loaded with salbutamol that could be suitable for pulmonary drug delivery system.KEY WORDS: aerodynamic, aerogels, chitosan, salbutamol, supercritical fluid technology     

The Expression of HMGB1 on Microparticles Released during Cell Activation and Cell Death In Vitro and In Vivo

High mobility group box protein 1 (HMGB1) is a nonhistone nuclear protein that is a prototypic alarmin that can stimulate innate immunity and drive the pathogenesis of a wide range of inflammatory diseases. While HMGB1 can be released from both activated and dying cells, its biochemical and immunological properties differ depending on the release mechanism, resulting from redox changes and posttranslational modifications including acetylation. In addition to release of HMGB1, cell death is associated with the release of microparticles. Microparticles are small membrane-bound vesicles that contain cytoplasmic, nuclear and membrane components. Like HMGB1, microparticles display immunological activity and levels are elevated in diseases characterized by inflammation and vasculopathy. While studies have addressed the immunological effects of HMGB1 and microparticles independently, HMGB1, like other nuclear molecules, is a component of microparticles. Evidence for the physical association of HMGB1 comes from Western blot analysis of microparticles derived from RAW 264.7 macrophage cells stimulated by lipopolysaccharide (LPS) or induced to undergo apoptosis by treatment with etoposide or staurosporine in vitro. Analysis of microparticles in the blood of healthy volunteers receiving LPS shows the presence of HMGB1 as assessed by flow cytometry. Together, these findings indicate that HMGB1 can be a component of microparticles and may contribute to their activities. Furthermore, particle HMGB1 may represent a useful biomarker for in vivo events that may not be reflected by measurement of the total amount of HMGB1 in the blood.     

T lymphocytes export proteasomes by way of microparticles: a possible mechanism for generation of extracellular proteasomes

The 20S proteasome is almost exclusively localized within cells. High levels of extracellular proteasomes are also found circulating in the blood plasma of patients suffering from a variety of inflammatory, autoimmune and neoplastic diseases. However, the origin of these proteasomes remained enigmatic. Since the proteome of microparticles, small membrane enclosed vesicles released from cells, was shown to contain proteasomal subunits, we studied whether intact proteasomes are actively released into the extracellular space. Using human primary T lymphocytes stimulated with CaCl₂ and the calcium ionophore A23187 to induce membrane blebbing we demonstrate that microparticles contain proteolytically active 20S proteasomes as well as the proteasome activator PA28 and subunits of the 19S proteasome regulator. Furthermore, our experiments reveal that incubation of in vitro generated T lymphocyte‐microparticles with sphingomyelinase results in the hydrolysis of the microparticle membranes and subsequent release of proteasomes from the vesicles. Thus, we here show for the first time that functional proteasomes can be exported from activated immune cells by way of microparticles, the dissolution of which may finally lead to the generation of extracellular proteasomes.     

Zeta potential mediated reaction monitoring on nano and microparticles

Nano and microparticles are widely used across the life science interface, with applications ranging from chemical probes of biological function to fluorescent particles for flow cytometry and cellular tracking. Increasingly, particles are modified with a variety of chemistries to boost their functionality and broaden their biological applicability. However, although particle modification has become standard laboratory practice, the ability to determine the extent and efficiency of chemical modification is often very limited and empirical in nature. Herein, we report the use of zeta potential analysis as a simple and rapid "direct-on-particle" approach allowing levels of bead modification and derivatization to be evaluated. As a proof-of-concept, aminomethyl-functionalized nano and microparticles were derivatized to display a variety of surface functionalities and their zeta potentials measured, allowing verification of the applicability of the approach for particle analysis. We demonstrate that zeta potential measurement is a convenient approach which allows multistep reaction sequences to be followed, and show that this method can be used to verify and validate successful particle modification.     

Continuous supercritical emulsions extraction: a new technology for biopolymer microparticles production

Supercritical emulsion extraction (SEE) was recently proposed for the production of biopolymer microparticles starting from oil‐in‐water emulsions. This technology can improve the product quality because of the fast and selective extraction of the dispersed oily phase by using supercritical carbon dioxide (SC‐CO₂). However, until now, SEE was proposed in batch configuration, sharing with the traditional processes an intrinsically discontinuous operation and problems of batches reproducibility and process yield. In this study, by using a countercurrent packed column, the SEE process was proposed in a continuous operating mode (SEE‐CM) for the production of poly‐lactic‐co‐glycolic acid (PLGA) microparticles. The new process design takes advantage of the large contact area between the SC‐CO₂ and emulsion allowing the production of PLGA microparticles with controlled and narrow size distributions in only few minutes. SEE‐CM operating parameters such as pressure, temperature, and flow rate ratios were analyzed and the process efficiency in terms of recovered material and its size distribution compared with SEE (batch mode operation) and conventional evaporation technology. PLGA microparticles showed a mean particle size between 1–3 µm (depending on the droplet sizes) with a SD that was always smaller than that associated with particles produced by discontinuous processes. Single and double emulsions were successfully treated and the microparticles physico‐chemical properties showed no morphological and structural differences between the SEE‐CM‐produced microparticles and the ones obtained by conventional evaporation technology. Biotechnol. Bioeng. 2011; 108:676–686. © 2010 Wiley Periodicals, Inc.     

Detection and Quantification of Microparticles from Different Cellular Lineages Using Flow Cytometry. Evaluation of the Impact of Secreted Phospholipase A2 on Microparticle Assessment

Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A₂ (sPLA₂), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA₂ can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA₂ enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes.     

Magnetic fluorescent microparticles as markers for particle transfer in extracorporeal blood purification

The microspheres-based detoxification system (MDS) is a combined membrane-adsorption system for extracorporeal blood purification in which adsorbent microparticles are recirculated in an extracorporeal filtrate circuit. Because the plasma filter represents the only barrier between the adsorbents and the patient's blood, there is the potential risk of particle entrance into the patient in case of a membrane rupture. To guarantee first fault safety of the system required for clinical application, magnetic fluorescent microparticles are added as markers to the adsorbent circuit. Detection of these particles in the venous blood line results in immediate shutdown of the pumps. Magnetic beads were functionalized with cresyl violet and tested with an in vitro setup of the particle detector to assess the detection limit in different matrices (water versus blood) as well as the influence of flow rate and particle size on the signal. In addition, biocompatibility and influence of sterilization on the performance of the particles were assessed. Functionalization of the magnetic particles with cresyl violet yielded fluorescent particles that were stable at 4 degrees C for at least 12 months. No leakage of dye was detectable, and the particles were neither cytotoxic nor mutagenic. The particles could be steam sterilized without significant loss in fluorescence intensity. With an in vitro setup of the particle detector, 0.1 mg and 5 mg of particles were reproducibly detectable in water and blood, respectively.     

Microparticle‐mediated gene delivery for the enhanced expression of a 19‐kDa fragment of merozoite surface protein 1 of Plasmodium falciparum

The 19 kDa carboxyl‐terminal fragment of merozoite surface protein 1 (MSP1₁₉) is a major component of the invasion‐inhibitory response in individual immunity to malaria. A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic‐co‐glycolic acid) (PLGA) microparticles of malaria DNA vaccines encoding MSP1₁₉ is presented here. After condensing the plasmid DNA (pDNA) molecules with a cationic polymer polyethylenimine (PEI), a 40 kHz ultrasonic atomization frequency was used to formulate PLGA microparticles at a flow rate of 18 mL h^?1. High levels of gene expression and moderate cytotoxicity in COS‐7 cells were achieved with the condensed pDNA at a nitrogen to phosphate (N/P) ratio of 20, thus demonstrating enhanced cellular uptake and expression of the transgene. The ability of the microparticles to convey pDNA was examined by characterizing the formulated microparticles. The microparticles displayed Z‐average hydrodynamic diameters of 1.50–2.10 μm and zeta potentials of 17.8–23.2 mV. The encapsulation efficiencies were between 78 and 83%, and 76 and 85% of the embedded malaria pDNA molecules were released under physiological conditions in vitro. These results indicate that PLGA‐mediated microparticles can be employed as potential gene delivery systems to antigen‐presenting cells in the prevention of malaria. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Proteomic analysis of serum opsonins impacting biodistribution and cellular association of porous silicon microparticles

Mass transport of drug delivery vehicles is guided by particle properties, such as size, shape, composition, and surface chemistry, as well as biomolecules and serum proteins that adsorb to the particle surface. In an attempt to identify serum proteins influencing cellular associations and biodistribution of intravascularly injected particles, we used two-dimensional gel electrophoresis and mass spectrometry to identify proteins eluted from the surface of cationic and anionic silicon microparticles. Cationic microparticles displayed a 25-fold greater abundance of Ig light variable chain, fibrinogen, and complement component 1 compared to their anionic counterparts. Anionic microparticles were found to accumulate in equal abundance in murine liver and spleen, whereas cationic microparticles showed preferential accumulation in the spleen. Immunohistochemistry supported macrophage uptake of both anionic and cationic microparticles in the liver, as well as evidence of association of cationic microparticles with hepatic endothelial cells. Furthermore, scanning electron micrographs supported cellular competition for cationic microparticles by endothelial cells and macrophages. Despite high macrophage content in the lungs and tumor, microparticle uptake by these cells was minimal, supporting differences in the repertoire of surface receptors expressed by tissue-specific macrophages. In summary, particle surface chemistry drives selective binding of serum components impacting cellular interactions and biodistribution.     

Constitutive expression of murine c-FLIPR causes autoimmunity in aged mice

Death receptor-mediated apoptosis is a key mechanism for the control of immune responses and dysregulation of this pathway may lead to autoimmunity. Cellular FLICE-inhibitory proteins (c-FLIPs) are known as inhibitors of death receptor-mediated apoptosis. The only short murine c-FLIP splice variant is c-FLIP_Raji (c-FLIP_R). To investigate the functional role of c-FLIP_R in the immune system, we used the vavFLIP_R mouse model constitutively expressing murine c-FLIP_R in all hematopoietic compartments. Lymphocytes from these mice are protected against CD95-mediated apoptosis and activation-induced cell death. Young vavFLIP_R mice display normal lymphocyte compartments, but the lymphocyte populations alter with age. We identified reduced levels of T cells and slightly higher levels of B cells in 1-year-old vavFLIP_R mice compared with wild-type (WT) littermates. Moreover, both B and T cells from aged vavFLIP_R animals show activated phenotypes. Sera from 1-year-old WT and transgenic animals were analysed for anti-nuclear antibodies. Notably, elevated titres of these autoantibodies were detected in vavFLIP_R sera. Furthermore, tissue damage in kidneys and lungs from aged vavFLIP_R animals was observed, indicating that vavFLIP_R mice develop a systemic lupus erythematosus-like phenotype with age. Taken together, these data suggest that c-FLIP_R is an important modulator of apoptosis and enforced expression leads to autoimmunity.     

The role of membrane lipids in the induction of macrophage apoptosis by microparticles

Microparticles are membrane-derived vesicles that are released from cells during activation or cell death. These particles can serve as mediators of intercellular cross-talk and induce a variety of cellular responses. Previous studies have shown that macrophages undergo apoptosis after phagocytosing microparticles. Here, we have addressed the hypothesis that microparticles trigger this process via lipid pathways. In these experiments, microparticles induced apoptosis in primary macrophage cells or cell lines (RAW 264.7 or U937) with up to a 5-fold increase. Preincubation of macrophages with phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)BP) reduced the microparticle-induced apoptosis in a dose-dependent manner. PtdIns(3,5)BP is a specific inhibitor of the acid sphingomyelinase and thus can block the generation of pro-apoptotic ceramides. Similarly, the pre-incubation of macrophages with PtdIns(3,5)BP prevented microparticle-induced upregulation of caspase 8, which is a major target molecule of ceramide action in the apoptosis pathway. PtdIns(3,5)BP, however, had no effect on the spontaneous rate of apoptosis. To evaluate further signaling pathways induced by microparticles, the extracellular signal regulated kinase (ERK-) 1 was investigated. This kinase plays a role in activating phospholipases A2 which cleaves membrane phospholipids into arachidonic acid; microparticles have been suggested to be a preferred substrate for phospholipases A2. As shown in our experiments, microparticles strongly increased the amount of phosphorylated ERK1/2 in RAW 264.7 macrophages in a time-dependent manner, peaking 15 min after co-incubation. Addition of PD98059, a specific inhibitor of ERK1, prevented the increase in apoptosis of RAW 264.7 macrophages. Together, these data suggest that microparticles perturb lipid homeostasis of macrophages and thereby induce apoptosis. These results emphasize the importance of biolipids in the cellular cross-talk of immune cells. Based on the fact that in clinical situations with excessive cell death such as malignancies, autoimmune diseases and following chemotherapies high levels of circulating microparticles might modulate phagocytosing cells, a suppression of the immune response might occur due to loss of macrophages.     

Casein Microparticles from Blend Films Forming Casein/α-Tocopherol Emulsion Droplets in Solution

Microparticles continue to receive widespread attention from food professionals because of their potential use as carriers of bioactive substances. This study demonstrates a novel method to prepare casein microparticles, which co-assemble with α-tocopherol (αT) into emulsion droplets. When the particles were extracted from the pectin matrix via enzymatic degradation, they remained stable in a buffer solution for at least 3 weeks. Optical microscopy showed that the size distribution of the microparticles is between 5 μm and 50 μm, which is in accordance with previous observations in blend films. High-performance liquid chromatography revealed that the amounts of α_S1- and κ-casein in the microparticles are significantly higher than those in native casein micelles. Confocal Raman microscopy showed that in the presence of α-tocopherol, the microparticles assemble into emulsion droplets, with phenol in the core and casein in the shell. Herein, we demonstrate a new method to form casein-based emulsion droplets for potential use as carriers of bioactive substances.     

Investigation of processing parameters of spray freezing into liquid to prepare polyethylene glycol polymeric particles for drug delivery

The objective of this study was to investigate the influence of processing parameters on the morphology, porosity, and crystallinity of polymeric polyethylene glycol (PEG) microparticles by spray freezing into liquid (SFL), a new particle engineering technology. Processing parameters investigated were the viscosity and flow rate of the polymer solution, nozzle diameter, spray time, pressure, temperature, and flow rate of the cryogenic liquid. By varying the processing parameters and feed composition, atomization and heat transfer mechanisms were modified resulting in particles of different size distribution, shape, morphology, density, porosity, and crystallinity. Median particle diameter (M50) varied from 25 μm to 600 μm. Particle shape was spherical or elongated with highly irregular surfaces. Granule density was between 0.5 and 1.5 g/mL. In addition to producing particles of pure polymer, drug particles were encapsulted in polymeric microparticles. The encapsulation efficiency of albuterol sulfate was 96.0% with a drug loading of 2.4%, indicating that SFL is useful for producing polymeric microparticles for drug delivery applications. It was determined that the physicochemical characteristics of model polymeric microparticles composed of PEG could be modified for use as a drug delivery carrier.     

Interaction of P-selectin and PSGL-1 generates microparticles that correct hemostasis in a mouse model of hemophilia A

High plasma levels of soluble P-selectin are associated with thrombotic disorders and may predict future cardiovascular events. Mice with high levels of soluble P-selectin have more microparticles in their plasma than do normal mice. Here we show that chimeras of P-selectin and immunoglobulin (P-sel-Ig) induced formation of procoagulant microparticles in human blood through P-selectin glycoprotein ligand-1 (PSGL-1; encoded by the Psgl1 gene, officially known as Selpl). In addition, Psgl1-/- mice produced fewer microparticles after P-sel-Ig infusion and did not spontaneously increase their microparticle count in old age as do wild-type mice. Injected microparticles specifically bound to thrombi and thus could be involved in thrombin generation at sites of injury. Infusion of P-sel-Ig into hemophilia A mice produced a 20-fold increase over control immunoglobulin in microparticles containing tissue factor. This significantly improved the kinetics of fibrin formation in the hemophilia A mice and normalized their tail-bleeding time. P-sel-Ig treatment could become a new approach to sustained control of bleeding in hemophilia.     

Circulating mesenchymal stem cells microparticles in patients with cerebrovascular disease

Preclinical and clinical studies have shown that the application of CD105(+) mesenchymal stem cells (MSCs) is feasible and may lead to recovery after stroke. In addition, circulating microparticles are reportedly functional in various disease conditions. We tested the levels of circulating CD105(+) microparticles in patients with acute ischemic stroke. The expression of CD105 (a surface marker of MSCs) and CXCR4 (a CXC chemokine receptor for MSC homing) on circulating microparticles was evaluated by flow cytometry of samples from 111 patients and 50 healthy subjects. The percentage of apoptotic CD105 microparticles was determined based on annexin V (AV) expression. The relationship between serum levels of CD105(+)/AV(-) microparticles, stromal cells derived factor-1α (SDF-1α), and the extensiveness of cerebral infarcts was also evaluated. CD105(+)/AV(-) microparticles were higher in stroke patients than control subjects. Correlation analysis showed that the levels of CD105(+)/AV(-) microparticles increased as the baseline stroke severity increased. Multivariate testing showed that the initial severity of stroke was independently associated with circulating CD105(+)/AV(-) microparticles (OR, 1.103 for 1 point increase in the NIHSS score on admission; 95% CI, 1.032-1.178) after adjusting for other variables. The levels of CD105(+)/CXCR4(+)/AV(-) microparticles were also increased in patients with severe disability (r = 0.192, p = 0.046 for NIHSS score on admission), but were decreased with time after stroke onset (r = -0.204, p = 0.036). Risk factor profiles were not associated with the levels of circulating microparticles or SDF-1α. In conclusion, our data showed that stroke triggers the mobilization of MSC-derived microparticles, especially in patients with extensive ischemic stroke.     

Optically induced flow cytometry for continuous microparticle counting and sorting

This paper reports a new microfluidic device capable of performing optically induced flow cytometry (OIFC). This enables it to continuously count and to sort microparticles based on optically induced dielectrophoretic (ODEP) forces. Gravity was used to drive the particles instead of using syringe pumps. The particles were then focused inside a sample channel by the ODEP forces and then passed through a detection region. A pair of optical fibers were embedded into fiber channels to count the number of particles and analyze the particle size in real time. Using 20.9 and 9.7 microm polystyrene microparticles, the average light intensity were about 63.67 and 8.80 units, with a coefficient-of-variation (CV) of 7.46 and 25.57%, respectively. This demonstrated that these two particle sizes could be successfully distinguished. After detecting the number and size of the microparticles, an optically induced dynamic switch (ODS) was used to sort microparticles to downstream fluidic outlets. The ODS used ODEP forces generated by different illumination intensities or optical line widths. The ODS was composed of two virtual electrodes which controlled particle movement in two dimensions. The ODS can successfully sort microparticles with different sizes continuously. The development of the OIFC device is a major advancement in the design of microparticle counting and sorting devices. Applications in future biomedical applications for cell counting and manipulation are envisioned.