Temoporfin-Conjugated PEGylated Poly(N,N-dimethylacrylamide)-Coated Upconversion Colloid for NIR-Induced Photodynamic Therapy of Pancreatic Cancer.

Photodynamic therapy (PDT) has the potential to cure pancreatic cancer with minimal side effects. Visible wavelengths are primarily used to activate hydrophobic photosensitizers, but in clinical practice, these wavelengths do not sufficiently penetrate deeper localized tumor cells. In this work, NaYF4:Yb3+,Er3+,Fe2+ upconversion nanoparticles (UCNPs) were coated with polymer and labeled with meta-tetra(hydroxyphenyl)chlorin (mTHPC; temoporfin) to enable near-infrared light (NIR)-triggered PDT of pancreatic cancer. The coating consisted of alendronate-terminated poly[N,N-dimethylacrylamide-co-2-aminoethylacrylamide]-graft-poly(ethylene glycol) [P(DMA-AEM)-PEG-Ale] to ensure the chemical and colloidal stability of the particles in aqueous physiological fluids, thereby also improving the therapeutic efficacy. The designed particles were well tolerated by the human pancreatic adenocarcinoma cell lines CAPAN-2, PANC-1, and PA-TU-8902. After intratumoral injection of mTHPC-conjugated polymer-coated UCNPs and subsequent exposure to 980 nm NIR light, excellent PDT efficacy was achieved in tumor-bearing mice.

Toxicity of Large and Small Surface-Engineered Upconverting Nanoparticles for In Vitro and In Vivo Bioapplications.

In this study, spherical or hexagonal NaYF4:Yb,Er nanoparticles (UCNPs) with sizes of 25 nm (S-UCNPs) and 120 nm (L-UCNPs) were synthesized by high-temperature coprecipitation and subsequently modified with three kinds of polymers. These included poly(ethylene glycol) (PEG) and poly(N,N-dimethylacrylamide-co-2-aminoethylacrylamide) [P(DMA-AEA)] terminated with an alendronate anchoring group, and poly(methyl vinyl ether-co-maleic acid) (PMVEMA). The internalization of nanoparticles by rat mesenchymal stem cells (rMSCs) and C6 cancer cells (rat glial tumor cell line) was visualized by electron microscopy and the cytotoxicity of the UCNPs and their leaches was measured by the real-time proliferation assay. The comet assay was used to determine the oxidative damage of the UCNPs. An in vivo study on mice determined the elimination route and potential accumulation of UCNPs in the body. The results showed that the L- and S-UCNPs were internalized into cells in the lumen of endosomes. The proliferation assay revealed that the L-UCNPs were less toxic than S-UCNPs. The viability of rMSCs incubated with particles decreased in the order S-UCNP@Ale-(PDMA-AEA) > S-UCNP@Ale-PEG > S-UCNPs > S-UCNP@PMVEMA. Similar results were obtained in C6 cells. The oxidative damage measured by the comet assay showed that neat L-UCNPs caused more oxidative damage to rMSCs than all coated UCNPs while no difference was observed in C6 cells. An in vivo study indicated that L-UCNPs were eliminated from the body via the hepatobiliary route; L-UCNP@Ale-PEG particles were almost eliminated from the liver 96 h after intravenous application. Pilot fluorescence imaging confirmed the limited in vivo detection capabilities of the nanoparticles.

Evaluation of two printing techniques for maxillary removable partial denture frameworks.

STATEMENT OF PROBLEM: Three-dimensional (3D) printing technology has gained popularity in producing removable partial denture (RPD) frameworks, including direct 3D printing of the metal framework and framework printing using castable resin, subsequently cast and processed. However, whether the technology is sufficiently accurate and precise to supersede traditional methods is unclear. PURPOSE: The purpose of this in vitro study was to determine the accuracy and precision of 2 different methods in the fabrication of RPD frameworks, including 3D printing by selective laser melting (SLM) and digital light processing (DLP). MATERIAL AND METHODS: Maxillary casts were digitized to design RPD frameworks. Thereby, 8 frameworks were produced for each group. The SLM group underwent a thermal finishing process after printing. In the DLP group, castable resin was printed but not cast. All frameworks were scanned to generate digital files, which were then compared with the original design using a metrology software program and manual measurements. Statistical analysis was executed using the t-test for independent specimens (α=.05) and by comparing heatmaps of the overlaid meshes. RESULTS: The analysis of the frameworks indicated minor deviations across all specimens. Regarding accuracy, there were no significant differences between the groups (P=.986). The SLM frameworks demonstrated greater precision, with absolute deviation values of 0.13 mm compared with 0.17 mm in the DLP group (P<.001). CONCLUSIONS: The findings underscored a high consistency between the 2 printing techniques, demonstrating a sufficiently advanced production process to yield predictable results. While the accuracy of both techniques was at a comparably high level and did not differ significantly, the SLM technique delivered RPDs with higher precision.

Chemical and Colloidal Stability of Polymer-Coated NaYF4:Yb,Er Nanoparticles in Aqueous Media and Viability of Cells: The Effect of a Protective Coating.

Upconverting nanoparticles (UCNPs) are of particular interest in nanomedicine for in vivo deep-tissue optical cancer bioimaging due to their efficient cellular uptake dependent on polymer coating. In this study, particles, ca. 25 nm in diameter, were prepared by a high-temperature coprecipitation of lanthanide chlorides. To ensure optimal dispersion of UCNPs in aqueous milieu, they were coated with three different polymers containing reactive groups, i.e., poly(ethylene glycol)-alendronate (PEG-Ale), poly(N,N-dimethylacrylamide-co-2-aminoethylacrylamide)-alendronate (PDMA-Ale), and poly(methyl vinyl ether-co-maleic acid) (PMVEMA). All the particles were characterized by TEM, DLS, FTIR, and spectrofluorometer to determine the morphology, hydrodynamic size and ξ-potential, composition, and upconversion luminescence. The degradability/dissolution of UCNPs in water, PBS, DMEM, or artificial lysosomal fluid (ALF) was evaluated using an ion-selective electrochemical method and UV-Vis spectroscopy. The dissolution that was more pronounced in PBS at elevated temperatures was decelerated by polymer coatings. The dissolution in DMEM was relatively small, but much more pronounced in ALF. PMVEMA with multiple anchoring groups provided better protection against particle dissolution in PBS than PEG-Ale and PDMA-Ale polymers containing only one reactive group. However, the cytotoxicity of the particles depended not only on their ability to rapidly degrade, but also on the type of coating. According to MTT, neat UCNPs and UCNP@PMVEMA were toxic for both rat cells (C6) and rat mesenchymal stem cells (rMSCs), which was in contrast to the UCNP@Ale-PDMA particles that were biocompatible. On the other hand, both the cytotoxicity and uptake of the UCNP@Ale-PEG particles by C6 and rMSCs were low, according to MTT assay and ICP-MS, respectively. This was confirmed by a confocal microscopy, where the neat UCNPs were preferentially internalized by both cell types, followed by the UCNP@PMVEMA, UCNP@Ale-PDMA, and UCNP@Ale-PEG particles. This study provides guidance for the selection of a suitable nanoparticle coating with respect to future biomedical applications where specific behaviors (extracellular deposition vs. cell internalization) are expected.

Isolation and identification in human blood serum of the proteins possessing the ability to bind with 48 kDa form of unconventional myosin 1c and their possible diagnostic and prognostic value.

We firstly identified 48 kDa molecular form of the unconventional myosin 1c (p48/Myo1C), and isolated it from blood serum of multiple sclerosis patients. The amount of p48/Myo1C in human blood serum correlated with some autoimmune, hemato-oncological and neurodegenerative diseases and thus may serve as a potential molecular biomarker. The biological functions of this protein in human blood remain unknown. Previously, we used the monodisperse magnetic poly (glycidyl methacrylate)(mag-PGMA-NH2 ) microspheres with immobilized 48/Myo1C and western-blot analysis, which allowed us to identify IgM and IgG immunoglobulins presenting an affinity to this protein. Here, we used mass spectrometry followed by the western blotting in order to identify other blood serum proteins with affinity to 48/Myo1C. The obtained data demonstrate that 48/Myo1C binds to component 3 of the complement and the antithrombin-III proteins. A combination of magnetic microparticle-based affinity chromatography with MALDI-TOF mass spectrometry and an in silico analysis provided an opportunity to identify the partners of interaction of 48/Myo1C with other proteins, in particular those participating in complement and coagulation cascades.

RGDS-Modified Superporous Poly(2-Hydroxyethyl Methacrylate)-Based Scaffolds as 3D In Vitro Leukemia Model.

Superporous poly(2-hydroxyethyl methacrylate-co-2-aminoethyl methacrylate) (P(HEMA-AEMA)) hydrogel scaffolds are designed for in vitro 3D culturing of leukemic B cells. Hydrogel porosity, which influences cell functions and growth, is introduced by adding ammonium oxalate needle-like crystals in the polymerization mixture. To improve cell vitality, cell-adhesive Arg-Gly-Asp-Ser (RGDS) peptide is immobilized on the N-(γ-maleimidobutyryloxy)succinimide-activated P(HEMA-AEMA) hydrogels via reaction of SH with maleimide groups. This modification is especially suitable for the survival of primary chronic lymphocytic leukemia cells (B-CLLs) in 3D cell culture. No other tested stimuli (interleukin-4, CD40 ligand, or shaking) can further improve B-CLL survival or metabolic activity. Both unmodified and RGDS-modified P(HEMA-AEMA) scaffolds serve as a long-term (70 days) 3D culture platforms for HS-5 and M2-10B4 bone marrow stromal cell lines and MEC-1 and HG-3 B-CLL cell lines, although the adherent cells retain their physiological morphologies, preferably on RGDS-modified hydrogels. Moreover, the porosity of hydrogels allows direct cell lysis, followed by efficient DNA isolation from the 3D-cultured cells. P(HEMA-AEMA)-RGDS thus serves as a suitable 3D in vitro leukemia model that enables molecular and metabolic assays and allows imaging of cell morphology, interactions, and migration by confocal microscopy. Such applications can prospectively assist in testing of drugs to treat this frequently recurring or refractory cancer.

The negative effect of magnetic nanoparticles with ascorbic acid on peritoneal macrophages.

Superparamagnetic iron oxide nanoparticles (SPIOn) are widely used as a contrast agent for cell labeling. Macrophages are the first line of defense of organisms in contact with nanoparticles after their administration. In this study we investigated the effect of silica-coated nanoparticles (γ-Fe2O3-SiO2) with or without modification by an ascorbic acid (γ-Fe2O3-SiO2-ASA), which is meant to act as an antioxidative agent on rat peritoneal macrophages. Both types of nanoparticles were phagocytosed by macrophages in large amounts as confirmed by transmission electron microscopy and Prusian blue staining, however they did not substantially affect the viability of exposed cells in monitored intervals. We further explored cytotoxic effects related to oxidative stress, which is frequently documented in cells exposed to nanoparticles. Our analysis of double strand breaks (DSBs) marker γH2AX showed an increased number of DSBs in cells treated with nanoparticles. Nanoparticle exposure further revealed only slight changes in the expression of genes involved in oxidative stress response. Lipid peroxidation, another marker of oxidative stress, was not significantly affirmed after nanoparticle exposure. Our data indicate that the effect of both types of nanoparticles on cell viability, or biomolecules such as DNA or lipids, was similar; however the presence of ascorbic acid, either bound to the nanoparticles or added to the cultivation medium, worsened the negative effect of nanoparticles in various tests performed. The attachment of ascorbic acid on the surface of nanoparticles did not have a protective effect against induced cytotoxicity, as expected.

Versatile Bioconjugation Strategies of PEG-Modified Upconversion Nanoparticles for Bioanalytical Applications.

Lanthanide-doped upconversion nanoparticles (UCNPs) display highly beneficial photophysical features for background-free bioimaging and bioanalysis; however, they are instable in high ionic strength buffers, have no functional groups, and are nonspecifically interacting. Here, we have prepared NIR-excitable UCNPs that are long-term colloidally stable in buffered media and possess functional groups. Heterobifunctional poly(ethylene glycol) (PEG) linkers bearing neridronate and alkyne or maleimide were attached to UCNPs via a ligand exchange. Streptavidin (SA)-conjugates were prepared by click reaction of UCNP@PEG-alkyne and SA-azide. Antihuman serum albumin pAbF antibody was modified with azide groups and conjugated to UCNP@PEG-alkyne via click reaction; alternatively, the antibody, after mild reduction of its disulfide bonds, was conjugated to UCNP@PEG-maleimide. We employed these nanoconjugates as labels for an upconversion-linked immunosorbent assay. SA-based labels achieved the lowest LOD of 0.17 ng/mL for the target albumin, which was superior compared to a fluorescence immunoassay (LOD 0.59 ng/mL) or an enzyme-linked immunoassay (LOD 0.56 ng/mL).

Measurement of Sub-femtomolar Concentrations of Prostate-Specific Antigen through Single-Molecule Counting with an Upconversion-Linked Immunosorbent Assay.

Single-molecule (digital) immunoassays provide the ability to detect much lower protein concentrations than conventional immunoassays. As photon-upconversion nanoparticles (UCNPs) can be detected without optical background interference, they are excellent labels for so-called single-molecule upconversion-linked immunosorbent assays (ULISAs). We have introduced a UCNP label design based on streptavidin-PEG-neridronate and a two-step detection scheme involving a biotinylated antibody that efficiently reduces nonspecific binding on microtiter plates. In a microtiter plate immunoassay, individual sandwich immune complexes of the cancer marker prostate-specific antigen (PSA) are detected and counted by wide-field epiluminescence microscopy (digital readout). The digital detection is 16× more sensitive than the respective analogue readout and thus expands the limit of detection to the sub-femtomolar concentration range (LOD: 23 fg mL-1, 800 aM). The single molecule ULISA shows excellent correlation with an electrochemiluminescence reference method. Although the analogue readout can routinely measure PSA concentrations in human serum samples, very low concentrations have to be monitored after radical prostatectomy. Combining the digital and analogue readout covers a dynamic range of more than 3 orders of magnitude in a single experiment.

Antifouling Microparticles To Scavenge Lipopolysaccharide from Human Blood Plasma.

Currently, one of the most promising treatments of lipopolysaccharides (LPS)-induced sepsis is based on hemofiltration. Nevertheless, proteins rapidly adsorbed on the artificial surface of membranes which leads to activation of coagulation impairing effective scavenging of the endotoxins. To overcome this challenge, we designed polymer-brush-coated microparticles displaying antifouling properties and functionalized them with polymyxin B (PMB) to specifically scavenge LPS the most common endotoxin. Poly[( N-(2-hydroxypropyl) methacrylamide)- co-(carboxybetaine methacrylamide)] brushes were grafted from poly(glycidyl methacrylate) microparticles using photoinduced single-electron transfer living radical polymerization (SET-LRP). Notably, only parts-per-million of copper catalyst were necessary to achieve brushes able to repel adsorption of proteins from blood plasma. The open porosity of the particles, accessible to polymerization, enabled us to immobilize sufficient PMB to selectively scavenge LPS from blood plasma.

Antibacterial Silver-Conjugated Magnetic Nanoparticles: Design, Synthesis and Bactericidal Effect.

PURPOSE: The aim was to design and thoroughly characterize monodisperse Fe3O4@SiO2-Ag nanoparticles with strong antibacterial properties, which makes them a candidate for targeting bacterial infections. METHODS: The monodisperse Fe3O4 nanoparticles were prepared by oleic acid-stabilized thermal decomposition of Fe(III) oleate; the particles were coated with silica shell using a water-in-oil reverse microemulsion, involving hydrolysis and condensation of tetramethyl orthosilicate. Resulting Fe3O4@SiO2 particles were modified by (3-mercaptopropyl)trimethoxysilane to introduce 1.1 mmol SH/g. Finally, the Fe3O4@SiO2-SH nanoparticles were decorated with silver nanoclusters formed by reduction of silver nitrate with NaBH4. The particles were analyzed by FTIR, X-ray photoelectron and atomic absorption spectroscopy, dynamic light scattering and vibrating sample magnetometry. The antibacterial activity of the Fe3O4@SiO2 and Fe3O4@SiO2-Ag nanoparticles was tested against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria cultivated on Luria agar plates or in Luria broth. RESULTS: The superparamagnetic Fe3O4@SiO2-Ag nanoparticles (21 nm in diameter; saturation magnetization 26 A∙m2/kg) were successfully obtained and characterized. Inhibitory and toxic effects against bacteria were documented by incubation of the Fe3O4@SiO2-Ag nanoparticles with Staphylococcus aureus and Escherichia coli. CONCLUSIONS: The combination of magnetic properties together with bactericidal effects is suitable for the disinfection of medical instruments, water purification, food packaging, etc.

Enhanced solid phase extraction of DNA using hydrophilic monodisperse poly(methacrylic acid-co-ethylene dimethacrylate) microparticles.

The efficiency of solid phase extraction (SPE) of DNA on polymer particles is limited by the features of the applied solid support, such as size, hydrophilicity, and functionality and their application in SPE also requires additional steps and compounds to finally obtain sufficient amount of high-quality DNA. The present study describes a preparation of sub-micrometer monodisperse poly(methacrylic acid-co-ethylene dimethacrylate) (PME) particles by precipitation polymerization. The effect of the ethylene dimethacrylate (EDMA) crosslinker concentration on morphology and particle size, which varied from 730 to 900 nm, was investigated. The particles with 5 and 15 wt% EDMA were selected for a study of SPE of plasmid DNA under various adsorption and elution conditions, followed by the enzymatic restriction of isolated DNA to verify a quality the nucleic acid. The particles with 15 wt% EDMA were suitable for the SPE because they retained better colloidal stability during the adsorption without additional induction of DNA conformational change. The quality of isolated DNA was finally verified by enzymatic restriction by restriction endonuclease EcoRI. Moreover, the developed method using PME particles was successfully utilized for DNA isolation from Escherichia coli lysate.

The purification and identification of human blood serum proteins with affinity to the antitumor active RL2 lactaptin using magnetic microparticles.

The cytopoxic effect of RL2 lactaptin (the recombinant analog of proteolytic fragment of human kappa-casein) toward tumor cells in vitro and in vivo presents it as a novel promising antitumor drug. The binding of any drug with serum proteins can affect their activity, distribution, rate of excretion and toxicity in the human body. Here, we studied the ability of RL2 to bind to various blood serum proteins. Using magnetic microparticles bearing by RL2 as an affinity matrix, in combination with mass spectrometry and western blot analysis, we found a number of blood serum proteins possessing affinity for RL2. Among them IgA, IgM and IgG subclasses of immunoglobulins, apolipoprotein A1 and various cortactin isoforms were identified. This data suggests that in the bloodstream RL2 lactaptin takes part in complicate protein-protein interactions, which can affect its activity.

Dynamics of tissue ingrowth in SIKVAV-modified highly superporous PHEMA scaffolds with oriented pores after bridging a spinal cord transection.

While many types of biomaterials have been evaluated in experimental spinal cord injury (SCI) research, little is known about the time-related dynamics of the tissue infiltration of these scaffolds. We analyzed the ingrowth of connective tissue, axons and blood vessels inside the superporous poly (2-hydroxyethyl methacrylate) hydrogel with oriented pores. The hydrogels, either plain or seeded with mesenchymal stem cells (MSCs), were implanted in spinal cord transection at the level of Th8. The animals were sacrificed at days 2, 7, 14, 28, 49 and 6 months after SCI and histologically evaluated. We found that within the first week, the hydrogels were already infiltrated with connective tissue and blood vessels, which remained stable for the next 6 weeks. Axons slowly and gradually infiltrated the hydrogel within the first month, after which the numbers became stable. Six months after SCI we observed rare axons crossing the hydrogel bridge and infiltrating the caudal stump. There was no difference in the tissue infiltration between the plain hydrogels and those seeded with MSCs. We conclude that while connective tissue and blood vessels quickly infiltrate the scaffold within the first week, axons show a rather gradual infiltration over the first month, and this is not facilitated by the presence of MSCs inside the hydrogel pores. Further research which is focused on the permissive micro-environment of the hydrogel scaffold is needed, to promote continuous and long-lasting tissue regeneration across the spinal cord lesion.

Monodisperse magnetic poly(glycidyl methacrylate) microspheres for isolation of autoantibodies with affinity for the 46 kDa form of unconventional Myo1C present in autoimmune patients.

Monodisperse nonmagnetic macroporous poly(glycidyl methacrylate) (PGMA) microspheres were synthesized by multistep swelling polymerization of glycidyl methacrylate, ethylene dimethacrylate and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA). This was followed (a) by ammonolysis to modify the microspheres with amino groups, and (b) by incorporation of iron oxide (γ-Fe2O3) into the pores to render the particles magnetic. The resulting porous and magnetic microspheres were characterized by scanning and transmission electron microscopy (SEM and TEM), atomic absorption and Fourier transform infrared spectroscopy (AAS and FTIR), elemental analysis, vibrating magnetometry, mercury porosimetry and Brunauer-Emmett-Teller adsorption/desorption isotherms. The microspheres are meso- and macroporous, typically 5 µm in diameter, contain 0.9 mM · g-1 of amino groups and 14 wt.% of iron according to elemental analysis and AAS, respectively. The particles were conjugated to p46/Myo1C protein, a potential biomarker of autoimmune diseases, to isolate specific autoantibodies in the blood of patients suffering from multiple sclerosis (MS). The p46/Myo1C loaded microspheres are shown to enable the preconcentration of minute quantities of specific immunoglobulins prior to their quantification via SDS-PAGE. The immunoglobulin M (IgM) with affinity to Myo1C was detected in MS patients. Graphical abstract Monodisperse magnetic poly(glycidyl methacrylate) microspheres were synthesized, conjugated with 46 kDa form of unconventional Myo1C protein (p46/Myo1C) via carbodiimide (DIC) chemistry, and specific autoantibodies isolated from blood of multiple sclerosis (MS) patients; immunoglobulin M (IgM) level increased in MS patients.

Modified Methacrylate Hydrogels Improve Tissue Repair after Spinal Cord Injury.

Methacrylate hydrogels have been extensively used as bridging scaffolds in experimental spinal cord injury (SCI) research. As synthetic materials, they can be modified, which leads to improved bridging of the lesion. Fibronectin, a glycoprotein of the extracellular matrix produced by reactive astrocytes after SCI, is known to promote cell adhesion. We implanted 3 methacrylate hydrogels: a scaffold based on hydroxypropylmethacrylamid (HPMA), 2-hydroxyethylmethacrylate (HEMA) and a HEMA hydrogel with an attached fibronectin (HEMA-Fn) in an experimental model of acute SCI in rats. The animals underwent functional evaluation once a week and the spinal cords were histologically assessed 3 months after hydrogel implantation. We found that both the HPMA and the HEMA-Fn hydrogel scaffolds lead to partial sensory improvement compared to control animals and animals treated with plain HEMA scaffold. The HPMA scaffold showed an increased connective tissue infiltration compared to plain HEMA hydrogels. There was a tendency towards connective tissue infiltration and higher blood vessel ingrowth in the HEMA-Fn scaffold. HPMA hydrogels showed a significantly increased axonal ingrowth compared to HEMA-Fn and plain HEMA; while there were some neurofilaments in the peripheral as well as the central region of the HEMA-Fn scaffold, no neurofilaments were found in plain HEMA hydrogels. In conclusion, HPMA hydrogel as well as the HEMA-Fn scaffold showed better bridging qualities compared to the plain HEMA hydrogel, which resulted in very limited partial sensory improvement.

Functionalized porous silica&maghemite core-shell nanoparticles for applications in medicine: design, synthesis, and immunotoxicity.

AIM: To determine cytotoxicity and effect of silica-coated magnetic nanoparticles (MNPs) on immune response, in particular lymphocyte proliferative activity, phagocytic activity, and leukocyte respiratory burst and in vitro production of interleukin-6 (IL-6) and 8 (IL-8), interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and granulocyte macrophage colony stimulating factor (GM-CSF). METHODS: Maghemite was prepared by coprecipitation of iron salts with ammonia, oxidation with NaOCl and modified by tetramethyl orthosilicate and aminosilanes. Particles were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier-transform infrared (FTIR), and X-ray photoelectron spectroscopy (XPS). Cytotoxicity and lymphocyte proliferative activity were assessed using [3H]-thymidine incorporation into DNA of proliferating human peripheral blood cells. Phagocytic activity and leukocyte respiratory burst were measured by flow cytometry; cytokine levels in cell supernatants were determined by ELISA. RESULTS: γ-Fe2O3&SiO2-NH2 MNPs were 13 nm in size. According to TEM, they were localized in the cell cytoplasm and extracellular space. Neither cytotoxic effect nor significant differences in T-lymphocyte and T-dependent B-cell proliferative response were found at particle concentrations 0.12-75 µg/cm2 after 24, 48, and 72 h incubation. Significantly increased production of IL-6 and 8, and GM-CSF cytokines was observed in the cells treated with 3, 15, and 75 µg of particles/cm2 for 48 h and stimulated with pokeweed mitogen (PHA). No significant changes in TNF-α and IFN-γ production were observed. MNPs did not affect phagocytic activity of monocytes and granulocytes when added to cells for 24 and 48 h. Phagocytic respiratory burst was significantly enhanced in the cultures exposed to 75 µg MNPs/cm2 for 48 h. CONCLUSIONS: The cytotoxicity and in vitro immunotoxicity were found to be minimal in the newly developed porous core-shell γ-Fe2O3&SiO2-NH2 magnetic nanoparticles.

Magnetic beads-based electrochemical immunosensor for monitoring allergenic food proteins.

Screen-printed platinum electrodes as transducer and magnetic beads as solid phase were combined to develop a particle-based electrochemical immunosensor for monitoring the serious food allergen ovalbumin. The standard arrangement of enzyme-linked immunosorbent assay became the basis for designing the immunosensor. A sandwich-type immunocomplex was formed between magnetic particles functionalized with specific anti-ovalbumin immunoglobulin G and captured ovalbumin molecules, and secondary anti-ovalbumin antibodies conjugated with the enzyme horseradish peroxidase were subsequently added as label tag. The electrochemical signal proportional to the enzymatic reaction of horseradish peroxidase during the reduction of hydrogen peroxide with thionine as electron mediator was measured by linear sweep voltammetry. The newly established method of ovalbumin detection exhibits high sensitivity suitable for quantification in the range of 11 to 222nM and a detection limit of 5nM. Magnetic beads-based assay format using external magnets for rapid and simple separation has been proven to be an excellent basis for electrochemical detection and quantification of food allergens in highly complex sample matrices.

Use of specific polysaccharide-immobilized monodisperse poly(glycidyl methacrylate) core-silica shell microspheres for affinity purification of lectins.

Immobilization of polysaccharides (yeast mannan and gum arabic) on the macroporous poly(glycidyl methacrylate) monodisperse microspheres coated with silica (SiO2 )-containing amino groups on the surface was used to prepare affinity sorbents for lectin purification. The efficiency of isolating mannose specific Pisum sativum lectin was demonstrated on sorbent with immobilized yeast mannan and that of galactose specific Glycine hispida lectin on sorbent with immobilized gum arabic. The microspheres with immobilized polysaccharides can be used for selecting an affinity sorbent for purification of other mannose- and galactose-specific lectins. In contrast to yeast mannan, the gum arabic immobilized on the microspheres possesses much narrower specificity and is suitable for purification of only those galactose specific lectins which interact well with l-rhamnose or l-arabinose. The synthesized macroporous particles are capable of immobilizing 50 mg of polysaccharide per 1 g of the matrix, which is 10 times higher than the capacity of epoxy-activated Sepharose 6B. That makes it possible to obtain the same lectin quantity using a column of 10 times smaller volume. Another advantage of novel affinity sorbents comparing corresponding Sepharose gels is the possibility of sorbent drying after use.

Application of an improved magnetic immunosorbent in an Ephesia chip designed for circulating tumor cell capture.

In this study, we describe a particular step in developing a microfluidic device for capture and detection of circulating tumor cells-specifically the preparation of an immunosorbent for implementation into the separation chip. We highlight some of the most important specifics connected with superparamegnetic microspheres for microfluidic purposes. Factors such as nonspecific adsorption on microfluidic channels, interactions with model cell lines, and tendency to aggregation were investigated. Poly(glycidyl methacrylate) microspheres with carboxyl groups were employed for this purpose. To address the aforementioned challenges, the microspheres were coated with hydrazide-PEG-hydrazide, and subsequently anti-epithelial cell adhesion molecule (EpCAM) antibody was immobilized. The prepared anti-EpCAM immunosorbent was pretested using model cell lines with differing EpCAM density (MCF7, SKBR3, A549, and Raji) in a batchwise arrangement. Finally, the entire system was implemented and studied in an Ephesia chip and an evaluation was performed by the MCF7 cell line.