Microbead Method Research Articles

Colon Targeted Delivery and In Vitro Evaluation of Curcumin for Colon Cancer

Background:: The second most common cause of mortality by cancer is thought to be colorectal cancer, which is one of the most prevalent tumours in the world. Many health advantages have been linked to curcumin, which is the key component of turmeric. The goal of the current study was to create a colon-targeted microbead method coated with Eudragit S100 to improve cur-cumin targeting in the colon by speeding up the rate of its dissolution Methods: The ionotropic gelation process was used to create the formulations. The surface phe-nomena, bead shape, entrapment effectiveness, drug loading, and in vitro drug release were all as-sessed for formulations. Moreover, calcium alginate beads with an improved core were enteric coat-ed with Eudragit S100. The polymer concentration and curing duration significantly affected parti-cle size and entrapment effectiveness, respectively. Results: The particle size of the improved formulation was 705 μm, drug entrapment efficiency was 83.56%, drug loading was 28.64%, and in vitro release was 81.66% after 6 hours in phosphate buff-er at pH 6.8. After 10 hours, enteric coating with Eudragit S100 of optimized calcium alginate mi-crobeads revealed a 64.09 ± 0.16% drug release. The calculated values of the regression coefficients for the Higuchi, first-order, and zero-order models were 0.9494, 0.8913, and 0.9579, respectively. The 50% inhibitory concentration value was 2.676 based on the percentage of cell viability. Conclusion: To effectively treat colorectal cancer, the enteric-coated calcium alginate microbeads can deliver curcumin selectively to the colon when taken orally.

κ-opioid receptor is involved in electroacupuncture analgesia via inhibition of spinal microglial Toll-like receptor 4 in neuropathic pain rats

To observe the effect of electroacupuncture (EA) on the expression of lumbar spinal κ-opioid receptor (KOR) and Toll-like receptor 4(TLR4) in microglia in neuropathic pain rats, so as to explore the role of cross-talk between KOR and TLK4 in EA-induced alleviation of chronic neuropathic pain. Wistar male rats were randomized into control, model, EA and EA plus KOR inhibitor (EA+inhibitor) groups (n=18 in each group). The neuropathic pain model was established in rats by ligature of the right sciatic nerve. EA was applied at bilateral "Zusanli"(ST36) and "Yanglingquan"(GB34) for 30 min, once daily for 5 days. JDTic dihydrochloride (a KOR inhibitor) was administrated by intraperitoneal injection before EA intervention. The difference value of paw withdrawal thermal latency (PWLD) of the bilateral hind-limbs was used as the thermal pain reaction level. At the end of experiments, the rat's lumbar spinal cord (L2-L4) was taken for detecting the expression of CD68 mRNA (a marker of the activated microglia) and Iba-1 (a marker for the activated and resting microglia) immunoactivity, and dynorphin content, and KOR mRNA and TLR4 protein (in immunomagnetic microbead method separated microglia) by using fluorescence quantitative PCR, immunofluorescence, radioimmunoassay and Western blot, separately. Compared with the control group, a strong thermal hyperalgesia was induced, the expression levels of Iba-1 and CD68 mRNA in the spinal cord, TLR4 protein of the spinal microglia were significantly increased(P<0.01) in the model group. The microglia were characterized by somatic hypertrophy and thickened branches in the model group. After EA intervention, the PWLD, the expression of Iba-1, CD68 mRNA and TLR4 protein of the microglia were significantly decreased(P<0.05), while the content of spinal dynorphin and the expression of KOR mRNA of the microglia increased in the EA group relative to the model group(P<0.05). The hypertrophic microglia shrinked slightly in the EA group. After injection of KOR inhibitor, the PWLD and expression levels of Iba-1, CD68 mRNA and TLR4 protein were significantly increased(P<0.05), and the expression of KOR mRNA was significantly decreased(P<0.05) in the EA+inhibitor group in comparison with the EA group. The analgesia effect of EA may partly mediated by spinal microglial KOR and the activation of KOR of microglia may be a target for inhibition of microglial TLR4-induced pro-inflammatory signaling.

Celomic Fluid: Laboratory Workflow for Prenatal Diagnosis of Monogenic Diseases.

Celomic fluid can be considered as an ultra-filtrate of maternal serum, containing a high protein concentration, urea, and many other molecules. It is an important transfer interface and a reservoir of nutrients for the embryo. Celomic fluid contains fetal cells that can be used for prenatal diagnosis of monogenic diseases in an earlier gestational period than villocentesis and amniocentesis. The purpose of this study was to evaluate the characteristics of celomic fluid and to establish a workflow laboratory procedure for very early prenatal diagnosis of monogenic diseases. Three hundred and eighty-five celomatic fluids were collected between the seventh and tenth week of gestation. We sampled 1 mL of celomic fluid in all cases. The embryo-fetal erythroid precursor cells were selected by the anti-CD71 microbead method or by a direct micromanipulator pick-up on the basis of their morphology. We amplified the extracted DNA using a nested polymerase chain reaction. Primers for short tandem repeat amplification were used to perform a quantitative fluorescent polymerase chain reaction evaluation to control maternal contamination. We observed maternal contamination in 95% of celomic fluids with a range between 5 and 100%. No fetal cells were observed in 0.78% of celomic fluids. The number of fetal cells ranged from a few units to several hundred. Isolation of embryo-fetal erythroblasts selected by the micromanipulator made diagnosis feasible in all cases. The selection of fetal cells by a micromanipulator and nested polymerase chain reaction analysis made celomatic fluid suitable for early prenatal diagnosis of monogenic disorders even in the presence of high maternal contamination and few fetal cells. The procedure reported in this study provides the opportunity for the use of celomic fluid sampled by celocentesis as an alternative to chorionic villi sampling and amniocentesis, to allow invasive prenatal diagnosis at a very early stage of pregnancy.

Microscale solute flow probed with rotating microbead trapped in optical vortex

The dynamics of solute flow in the microscopic chamber can be studied with optical tweezers. A method based on the metallic microbeads trapped in the focused optical vortex beam is proposed. This annular beam of a twisted wavefront exerts torque on a reflective object placed inside the dark core of the vortex. The induced rotational movement of the bead is sensitive to local viscosity changes in the surrounding medium, for example, during the ongoing dissolution process. Two experimental configurations are described, both relying on tracing the angular velocity of the bead in time. In one-bead configuration, the dynamics of local solute concentration can be studied. In two-bead case, the direction and speed of solute flow can be probed with a spatial resolution of single micrometers. We approach the elementary problem of sucrose dissolution and diffusion in water. The surprising impression of the reverse solute flow was observed. Further experimental investigation led to the discovery that this phenomenon originates from the sucrose stream-like diffusion in the mid-depth of the measurement chamber. The rotating microbead method applies for various solid and liquid substances and may become a useful technique for microfluidics research.Graphic abstract

Research progress of nano self - cleaning anti-fouling coatings

There are many methods of evaluating the performance of nano self-cleaning anti-fouling coatings, such as carbon blacking method, coating reflection coefficient method, glass microbead method, film method, contact angle and rolling angle method, organic degradation method, and the application of performance evaluation method in self-cleaning antifouling coating. For the more, the types of nano self-cleaning anti-fouling coatings based on aqueous media was described, such as photocatalytic self-cleaning coatings, silicone coatings, organic fluorine coatings, fluorosilicone coatings, fluorocarbon coatings, polysilazane self-cleaning coatings. The research and application of different kinds of nano self-cleaning antifouling coatings are anlysised, and the latest research results are summed.

Integration of acoustic radiation force and optical imaging for blood plasma clot stiffness measurement.

Despite the life-preserving function blood clotting serves in the body, inadequate or excessive blood clot stiffness has been associated with life-threatening diseases such as stroke, hemorrhage, and heart attack. The relationship between blood clot stiffness and vascular diseases underscores the importance of quantifying the magnitude and kinetics of blood’s transformation from a fluid to a viscoelastic solid. To measure blood plasma clot stiffness, we have developed a method that uses ultrasound acoustic radiation force (ARF) to induce micron-scaled displacements (1-500 μm) on microbeads suspended in blood plasma. The displacements were detected by optical microscopy and took place within a micro-liter sized clot region formed within a larger volume (2 mL sample) to minimize container surface effects. Modulation of the ultrasound generated acoustic radiation force allowed stiffness measurements to be made in blood plasma from before its gel point to the stage where it was a fully developed viscoelastic solid. A 0.5 wt % agarose hydrogel was 9.8-fold stiffer than the plasma (platelet-rich) clot at 1 h post-kaolin stimulus. The acoustic radiation force microbead method was sensitive to the presence of platelets and strength of coagulation stimulus. Platelet depletion reduced clot stiffness 6.9 fold relative to platelet rich plasma. The sensitivity of acoustic radiation force based stiffness assessment may allow for studying platelet regulation of both incipient and mature clot mechanical properties.

Glomerular cytokine expression in murine lupus nephritis.

Aberrant expression of T helper cell (Th) cytokines is believed to play a central role in the pathogenesis of systemic lupus erythematosus (SLE). While the glomerulus is one of the major targets of lupus inflammation, little is known about the cytokine expression in glomeruli. The current study aimed to explore the profiles of Th cytokine gene expressions in isolated glomeruli of lupus-prone mice. Glomeruli were purified from lupus-prone MRL/lpr mice using the magnetic microbead method. Expressions of cytokine genes representing the Th subset and FoxP3 were examined using real-time polymerase chain reaction. Serum levels of these cytokines were also measured by enzyme-linked immunosorbent assay. MRL/n mice were used as controls. Histologic glomerular damages were scored semiquantitatively. To examine the role of TNF-α in glomerular damage, we administered etanercept, a TNF-α antagonist, into the subjects. Glomerular gene expressions of TNF-α in lpr mice increased with week postpartum and reached statistically significant levels at 16 weeks compared with those of the glomeruli from control mice. Expressions of IFN-γ, IL-4 and FoxP3 also increased, but the difference was not significant. There was a significant increase in serum levels of TNF-α, IFN-γ, and IL-17 and decrease in those of IL-4. Among the genes examined, TNF-α significantly correlated with glomerular damage score. Administration of etanercept did not affect glomerular cytokine expressions or proteinuria and failed to ameliorate histologic glomerular damages. Our data suggest that Th1 cytokines, especially TNF-α, are dominantly expressed in the glomeruli of lupus-prone mice, but its pathophysiological role remains unclear.

Increased MAPK and NF-κB expression of Langerhans cells is dependent on TLR2 and TLR4, and increased IRF-3 expression is partially dependent on TLR4 following UV exposure

Toll-like receptors (TLRs) and epidermal Langerhans cells (LCs) play a crucial role in innate and adaptive immunity. To date, the pattern of TLR expression has not been fully analyzed. The effects of ultraviolet (UV) light on TLR expression and the downstream signaling cascades of human LC have not been examined. In this study, we purified human epidermal LCs using a density gradient centrifugation method and an immunomagnetic microbead method. We found that cultured purified LCs from human skin express mRNAs encoding TLR2, TLR4, TLR5 and TLR7-9. The expression of TLR2 and TLR4 protein was confirmed by Western blot analysis. The results showed for the first time that UV exposure up-regulated the mRNA and protein expression of TLR2 and TLR4 in human LCs. We also found that UV exposure-induced up-regulated MAPK and NF-κB/p65 expression was dependent on TLR2 and TLR4, and up-regulated IRF-3 expression was partially dependent on TLR4. In conclusion, UV light up-regulates the expression of TLR2, TLR4 and downstream signaling molecules MAPK, NF-κB/p65 and IRF-3 in human LCs. This suggests that UV light has a significant effect on skin immune responses.

Isolation of human eosinophils: microbead method has no impact on IL-5 sustained viability

In a recent issue of Experimental Dermatology (18, 2009, 654), Schefzyk et al. concluded that multi-antibody eosinophil isolation (Miltenyi) should be abandoned, as differential purity was minimal, and eosinophils underwent accelerated apoptosis when compared with those isolated with traditional anti-CD16 microbeads. Our intent was to investigate the universality of these findings. We isolated eosinophils from normal donor granulocyte packs using two methods, and evaluated purity, viability and annexin-V/propidium-iodide staining. Purity was substantially greater when multi-antibody isolation was used for eosinophil isolation from granulocyte packs (98% vs 69%). No differential survival was detected when eosinophils were maintained in culture with or without interleukin-5. Multi-antibody eosinophil isolation represents a substantial advantage over anti-CD-16 microbeads when isolating large numbers of eosinophils from concentrated leucocyte preparations. No differential survival was observed. While appropriate consideration of methods is always crucial, multi-antibody eosinophil isolation should not be abandoned completely.

Rises and Falls in Donor-Specific and Third-Party HLA Antibody Levels After Antibody Incompatible Transplantation

After human leukocyte antigen (HLA) antibody-incompatible transplantation, donor specific and third party HLA antibodies may be found, and their levels fall in a donor-specific manner during the first month. However, these changes have not been previously described in detail. Donor-specific HLA antibody (DSA) and third-party HLA antibody (TPA) levels were measured using the microbead method in 44 presensitized patients who had renal transplantation. DSA+TPA fell in the first 4 days after transplantation, and greater falls in DSA indicated absorption by the graft. This occurred for class I (57.8% fall compared with 20.2% for TPA, P<0.0005), HLA DR (63.0% vs. 24.3%, P<0.0004), and for HLA DP/DQ/DRB3-4 (34% vs. 17.5%, P=0.014). Peak DSA levels occurred at a mean of 13 days posttransplant, and they were higher than pretreatment in 25 (57%) patients and lower in 19 (43%) patients (P=ns). The risk of rejection was associated with peak DSA levels; 15 of 25 (60%) patients with DSA at median fluorescence intensity (MFI) more than 7000U experienced rejection, compared with 4 of 7 (57%) patients with peak DSA MFI 2000 to 7000U, and 2 of 12 (17%) patients with peak DSA MFI less than 2000U (P<0.02). DSA levels subsequently fell in a donor specific manner compared to TPA. DSA levels may change markedly in the first month after antibody incompatible transplantation, and the risk of rejection was associated with higher pretreatment and peak levels.

Lectin binding and effects in culture on human cancer and non-cancer cell lines: Examination of issues of interest in drug design strategies

By using a non-cancer and a cancer cell line originally from the same tissue (colon), coupled with testing lectins for cell binding and for their effects on these cell lines in culture, this study describes a simple multi-parameter approach that has revealed some interesting results that could be useful in drug development strategies. Two human cell lines, CCL-220/Colo320DM (human colon cancer cells, tumorigenic in nude mice) and CRL-1459/CCD-18Co (non-malignant human colon cells) were tested for their ability to bind to agarose microbeads derivatized with two lectins, peanut agglutinin (Arachis hypogaea agglutinin, PNA) and Dolichos biflorus agglutinin (DBA), and the effects of these lectins were assessed in culture using the MTT assay. Both cell lines bound to DBA-derivatized microbeads, and binding was inhibited by N-acetyl-D-galactosamine, but not by L-fucose. Neither cell line bound to PNA-derivatized microbeads. Despite the lack of lectin binding using the rapid microbead method, PNA was mitogenic in culture at some time points and its mitogenic effect displayed a reverse-dose response. This was also seen with effects of DBA on cells in culture. While this is a simple study, the results were statistically highly significant and suggest that: (1) agents may not need to bind strongly to cells to exert biological effects, (2) cell line pairs derived from diseased and non-diseased tissue can provide useful comparative data on potential drug effects and (3) very low concentrations of potential drugs might be initially tested experimentally because reverse-dose responses should be considered.

Characterization of cryopreserved human Langerhans cells

Epidermal Langerhans cells are potent antigen-presenting cells in the epidermis. The establishment of a cryopreservation method for human Langerhans cells would greatly contribute to our ability to successfully conduct various experiments dealing with Langerhans cells. Since Langerhans cells are known to be sensitive to cold injury, there have been no reports concerning the cryopreservation of Langerhans cells. We have investigated the effect of cryopreservation on the function and phenotype of human Langerhans cells. Langerhans cells from human foreskins were isolated with the immunomagnetic microbead method using monoclonal antibodies for CD1a. Langerhans cells were cryopreserved in the presence of dimethylsulfoxide (DMSO) 10% and fetal calf serum 90%. Cryopreserved Langerhans cells were phenotypically assessed by flowcytometry using monoclonal antibodies to HLA-DR and CD1a. The ultrastructures of the Langerhans cells were compared using electron microscopy. An autologous T cell stimulation test was performed to compare the functions of cryopreserved Langerhans cells and fresh Langerhans cells. The viability of the cryopreserved Langerhans cells was able to be maintained at more than 90%. Cryopreserved Langerhans cells expressed high levels of HLA-DR and CD1a antigens and stimulated autologous T cells to an extent almost identical to that obtained from fresh Langerhans cells. These findings indicate that the cryopreservation of human Langerhans cells could lead to a breakthrough in various experiments dealing with human Langerhans cells.

Lack of Effect of Cryopreservation on the Class I and Class II Antigenicities of Skin Allografts

Epidermal Langerhans cells are potent antigen-presenting cells in the epidermis. The establishment of a cryopreservation method for human Langerhans cells would greatly contribute to our ability to successfully conduct various experiments dealing with Langerhans cells. Since Langerhans cells are known to be sensitive to cold injury, there have been no reports concerning the cryopreservation of Langerhans cells. We have investigated the effect of cryopreservation on the function and phenotype of human Langerhans cells. Langerhans cells from human foreskins were isolated with the immunomagnetic microbead method using monoclonal antibodies for CD1a. Langerhans cells were cryopreserved in the presence of dimethylsulfoxide (DMSO) 10% and fetal calf serum 90%. Cryopreserved Langerhans cells were phenotypically assessed by flowcytometry using monoclonal antibodies to HLA-DR and CD1a. The ultrastructures of the Langerhans cells were compared using electron microscopy. An autologous T cell stimulation test was performed to compare the functions of cryopreserved Langerhans cells and fresh Langerhans cells. The viability of the cryopreserved Langerhans cells was able to be maintained at more than 90%. Cryopreserved Langerhans cells expressed high levels of HLA-DR and CD1a antigens and stimulated autologous T cells to an extent almost identical to that obtained from fresh Langerhans cells. These findings indicate that the cryopreservation of human Langerhans cells could lead to a breakthrough in various experiments dealing with human Langerhans cells.

A rapid microbead method for breaking pathogenic mycobacteria: Application in SDS-PAGE and western blot analysis

A rapid method used to obtain cell-free extracts from pathogenic mycobacteria was successfully applied to five slowly growing species. The method, which simply consisted of vortexing of the bacterial suspensions with 130-μm glass microbeads under predefined conditions, resulted in the breaking of bacterial cells as evidenced by electron microscopy and gave about 1 mg/ml of total proteins. The cell-free extracts so obtained were suitable for SDS-PAGE analysis as well as Western blots. In a pilot test system, this method was applied toMycobacterium avium (M. av),M. paratuberculosis (P.tb), and related species isolated from “wood-pigeon” (M. silvaticum) and Crohn's disease (CD). The procedure was used in the analysis of various mycobacterial proteins by SDS-PAGE and Western blotting, and the findings were compatible with recently published data using criteria based on DNA polymorphism and numerical taxonomy.

Evaluation of the silica microbead method for isolation of red beet protoplast plasma membrane sheets

The silica microbead procedure was utilized for the isolation of plasma membrane sheets from protoplasts of a higher plant, the red beet ( Beta vulgaris L.). Membrane yields, as determined by recovery of an exogenous membrane marker were approx. 75%. The plasma membrane fraction contained the enzyme marker, pH 6.5, vanadate-sensitive, K +-stimulated, Mg 2+-ATPase and small amounts of mitochondria, endoplasmic reticulum, and possibly tonoplast. The silica microbead procedure was also used for the isolation of intact vacuoles from microbead-coated protoplasts.