Fabrication of novel magnetic nanoparticles-coated P(styrene-itaconic acid-divinylbenzene) microspheres

In this paper, P(styrene-itaconic acid-divinylbenzene) microspheres (P(St-IA-DVB) microspheres) based on styrene (St), itaconic acid (IA) and divinylbenzene (DVB) were prepared via water-in-oil emulsions method (W/O) in the presence of emulsifiers with the size of 5-10 μm. The magnetic nanoparticles (i.e. Fe₃O₄) coated tightly on the surface of P(St-IA-DVB) microspheres were prepared in water with a continuous stirring. The morphology of blank microspheres and magnetic nanoparticles-coated microspheres was investigated in this work. In vitro drug release behavior was studied using doxorubicin as a model drug, and these magnetic nanoparticles-coated P(St-IA-DVB) (MNPSID) microspheres might have great potential application in magnetically targeted and thermal therapy.     

Reversible immobilization of glucoamylase onto magnetic polystyrene beads with multifunctional groups

A novel and simple process for the surface functionalization of micron-sized monodisperse magnetic polystyrene (PS) microbeads was reported. The polystyrene seed particles were prepared prior to the dispersion polymerization method. Afterwards, series of surface chemical modifications on polystyrene microspheres were conducted, and three end-functional microspheres with carboxyl, imidazolyl and sulphydryl groups were obtained. The functional magnetic polystyrene microspheres were prepared by impregnation and subsequent precipitation of ferric and ferrous ions into the polystyrene particles. Finally, the functional magnetic polystyrene was used for the reversible immobilization of glucoamylase via metal-affinity adsorption. The results indicated that the obtained immobilized glucoamylase presented excellent reusability, applicability, magnetic response and regeneration of supports. The magnetic PS microspheres retained >65% of its initial activity at 65 °C over 6 h; and the lowest residual activity of immobilized glucoamylase prepared by regenerated supports still remained about 50% of the initial activity after the 10th cycles.     

A polystyrene microsphere assay for detecting surface hydrophobicity variations within Candida albicans populations

Adherence of microbial pathogens to host cell surfaces may involve hydrophobic interactions. Here, we describe the development of an assay for detecting cell surface hydrophobicity of populations and individual cells of the opportunistic fungal pathogen Candida albicans. The assay involves mixing polystyrene latex microspheres with cells and subsequent enumeration of cell-attached microspheres. Similar levels of hydrophobicity within a population of yeast cells were obtained with the microsphere assay and with a commonly used aqueous-hydrocarbon biphasic partitioning assay. Various buffers were found to support detection of surface hydrophobicity with the microsphere assay. Complex fungal growth media did not. Serum in test media prevented microsphere attachment. A unique advantage of the assay compared to others is that individual cells can be assessed for surface hydrophobicity. Within a population of C. albicans yeast cells, strongly, moderately and weakly hydrophobic cells were observed. Within some pairs of mother-daughter cells, only one cell was hydrophobic. Germ tbes and hyphae were hydrophobic regardless of the hydrophobic status of the parent cell. These results indicate that the microsphere assay is a useful test evaluating cell surface hydrophobicity of C. albicans.     

Synthesis and characterization of multifunctional poly(glycidyl methacrylate) microspheres and their use in cell separation

Multifunctional poly(glycidyl methacrylate) (PGMA) microspheres containing magnetic, fluorescent, and cancer cell-specific moieties were prepared in four steps: (i) preparation of parent PGMA microspheres by dispersion polymerization and their reaction with ethylenediamine to obtain amino groups, (ii) precipitation of iron ions (Fe²⁺ and Fe³⁺) to form Fe₃O₄ nanoparticles within the microspheres, (iii) consecutive reactions of folic acid with the amino groups on PGMA, and (iv) incorporation of fluorescein isothiocyanate into the microspheres. The microspheres were superparamagnetic, highly monodispersive, intensively fluorescent, and capable of recognizing and binding cancer cells that overexpress folic acid receptors. It was demonstrated that with these microspheres, HeLa cells could be captured from their suspension and easily moved in the direction of the externally applied magnetic field.     

Cell labelling and separation with polyglutaraldehyde microspheres

Non-magnetic and magnetic polyglutaraldehyde microspheres were used in the labelling and separation of mouse and human T and B lymphocytes. The enrichment of the separated T- and B-cell fractions of mouse and human cells was 30 and 40% respectively. After magnetic separation 79% of the mouse B-cell fraction and 23% of the T-cell fraction were surface Ig-positive. The corresponding figures for human T and B cells were 10 and 51–54%, respectively. The labelling of mouse spleen B cells with antibody-conjugated non-magnetic microspheres was between 27–53% depending on the labelling procedures. The best labelling was obtained when mouse spleen cells coated with rabbit anti-mouse Ig were incubated with protein A-coupled microspheres.     

Synthesis and Characterization of Dual-Functionalized Core-Shell Fluorescent Microspheres for Bioconjugation and Cellular Delivery

The efficient transport of micron-sized beads into cells, via a non-endocytosis mediated mechanism, has only recently been described. As such there is considerable scope for optimization and exploitation of this procedure to enable imaging and sensing applications to be realized. Herein, we report the design, synthesis and characterization of fluorescent microsphere-based cellular delivery agents that can also carry biological cargoes. These core-shell polymer microspheres possess two distinct chemical environments; the core is hydrophobic and can be labeled with fluorescent dye, to permit visual tracking of the microsphere during and after cellular delivery, whilst the outer shell renders the external surfaces of the microspheres hydrophilic, thus facilitating both bioconjugation and cellular compatibility. Cross-linked core particles were prepared in a dispersion polymerization reaction employing styrene, divinylbenzene and a thiol-functionalized co-monomer. These core particles were then shelled in a seeded emulsion polymerization reaction, employing styrene, divinylbenzene and methacrylic acid, to generate orthogonally functionalized core-shell microspheres which were internally labeled via the core thiol moieties through reaction with a thiol reactive dye (DY630-maleimide). Following internal labeling, bioconjugation of green fluorescent protein (GFP) to their carboxyl-functionalized surfaces was successfully accomplished using standard coupling protocols. The resultant dual-labeled microspheres were visualized by both of the fully resolvable fluorescence emissions of their cores (DY630) and shells (GFP). In vitro cellular uptake of these microspheres by HeLa cells was demonstrated conventionally by fluorescence-based flow cytometry, whilst MTT assays demonstrated that 92% of HeLa cells remained viable after uptake. Due to their size and surface functionalities, these far-red-labeled microspheres are ideal candidates for in vitro, cellular delivery of proteins.     

Development of a Fluorescent Based Immunosensor for the Serodiagnosis of Canine Leishmaniasis Combining Immunomagnetic Separation and Flow Cytometry

Background

An accurate diagnosis is essential for the control of infectious diseases. In the search for effective and efficient tests, biosensors have increasingly been exploited for the development of new and highly sensitive diagnostic methods. Here, we describe a new fluorescent based immunosensor comprising magnetic polymer microspheres coated with recombinant antigens to improve the detection of specific antibodies generated during an infectious disease. As a challenging model, we used canine leishmaniasis due to the unsatisfactory sensitivity associated with the detection of infection in asymptomatic animals where the levels of pathogen-specific antibodies are scarce.

Methodology

Ni-NTA magnetic microspheres with 1,7 µm and 8,07 µm were coated with the Leishmania recombinant proteins LicTXNPx and rK39, respectively. A mixture of equal proportions of both recombinant protein-coated microspheres was used to recognize and specifically bind anti-rK39 and anti-LicTNXPx antibodies present in serum samples of infected dogs. The microspheres were recovered by magnetic separation and the percentage of fluorescent positive microspheres was quantified by flow cytometry.

Principal Findings

A clinical evaluation carried out with 129 dog serum samples using the antigen combination demonstrated a sensitivity of 98,8% with a specificity of 94,4%. rK39 antigen alone demonstrated a higher sensitivity for symptomatic dogs (96,9%), while LicTXNPx antigen showed a higher sensitivity for asymptomatic (94,4%).

Conclusions

Overall, our results demonstrated the potential of a magnetic microsphere associated flow cytometry methodology as a viable tool for highly sensitive laboratorial serodiagnosis of both clinical and subclinical forms of canine leishmaniasis.     

Novel, fluorescent, magnetic, polysaccharide-based microsphere for orientation, tracing, and anticoagulation: preparation and characterization

A fluorescent, magnetic composite poly(styrene-maleic anhydride) microsphere, suitable for conjugation with polysaccharide, was synthesized using magnetite/europium phthalate particles as seeds by copolymerization of styrene and maleic anhydride. The magnetite/europium phthalate particles were wrapped up by poly(ethylene glycol), which improved the affinity between the seed particles and the monomers. The composite microspheres obtained, with a diameter of 0.15-0.7 microm, contain 586-1013 microg of magnetite/g of microsphere and 0.5-16 mmol surface anhydride groups/g of microsphere. Heparin was conjugated with the reactive surface anhydride groups on the surface of the microspheres by covalent binding to obtain a fluorescent, magnetic, polysaccharide-based microsphere. The microspheres not only retain their bioactivities but also provide magnetic susceptibility and fluorescence. They can be used as a carrier with magnetic orientation and fluorescence tracer for potent drug targeting. The orientation, tracer, and anticoagulation of the fluorescence, magnetic, polysaccharide-based microspheres were studied. The anticoagulant activity of the microspheres and heparin binding capacity reached 54,212.8 U and 607.1 mg/g of dry microspheres. The activity recovery was 50.2%. The anticoagulant activity of the microspheres increases with the increase of the conjugated heparin on the surface of the microspheres and the decrease of the microsphere size. Furthermore, The fluorescent, magnetic, polysaccharide-based microspheres can be easily transported to a given position in a magnetic field and traced via their fluorescence.     

Determination of bacterial cell surface hydrophobicity of single cells in cultures and in wastewater in situ

Bacterial cell surface hydrophobicity is one of the most important factors that influence bacterial adhesion. A new method, microsphere adhesion to cells, for measuring bacterial cell surface hydrophobicity was developed. Microsphere adhesion to cells is based on microscopic enumeration of hydrophobic, fluorescent microspheres attaching to the bacterial surface. Cell surface hydrophobicity estimated by microsphere adhesion to cells correlates well with adhesion of bacteria to hydrocarbons or hydrophobic interaction chromatography for a set of hydrophilic and hydrophobic bacteria (linear correlation coefficients, R², were 0.845 and 0.981 respectively). We also used microsphere adhesion to cells to investigate the in situ properties of individual free-living bacteria directly in activated sludge. Results showed that the majority of the bacteria were hydrophilic, indicating the importance of cell surface hydrophobicity for bacterial adhesion in sludge, and for the overall success of the wastewater treatment process.     

Surface Properties of Toxoplasma gondii Oocysts and Surrogate Microspheres

The physical properties that govern the waterborne transmission of Toxoplasma gondii oocysts from land to sea were evaluated and compared to the properties of carboxylated microspheres, which could serve as surrogates for T. gondii oocysts in transport and water treatment studies. The electrophoretic mobilities of T. gondii oocysts, lightly carboxylated Dragon Green microspheres, and heavily carboxylated Glacial Blue microspheres were determined in ultrapure water, artificial freshwater with and without dissolved organic carbon, artificial estuarine water, and artificial seawater. The surface wettabilities of oocysts and microspheres were determined using a water contact angle approach. Toxoplasma gondii oocysts and microspheres were negatively charged in freshwater solutions, but their charges were neutralized in estuarine water and seawater. Oocysts, Glacial Blue microspheres, and unwashed Dragon Green microspheres had low contact angles, indicating that they were hydrophilic; however, once washed, Dragon Green microspheres became markedly hydrophobic. The hydrophilic nature and negative charge of T. gondii oocysts in freshwater could facilitate widespread contamination of waterways. The loss of charge observed in saline waters may lead to flocculation and subsequent accumulation of T. gondii oocysts in locations where freshwater and marine water mix, indicating a high risk of exposure for humans and wildlife in estuarine habitats with this zoonotic pathogen. While microspheres did not have surface properties identical to those of T. gondii, similar properties shared between each microsphere type and oocysts suggest that their joint application in transport and fate studies could provide a range of transport potentials in which oocysts are likely to behave.     

Steroid transformation using magnetically immobilized Mycobacterium sp.

Mycobacterium sp. (NRRL-B 3683) has been immobilized by adhesion of magnetic materials of submicron size to the bacterial surface. Preparations based on laboratory-prepared magnetic oxide that had been derivatized with hydrophobic octyltrichlorosilane showed the best properties. The magnetically immobilized bacteria were used for side-chain degradation of cholesterol into androsta-1,4-diene-3,17-dione. The magnetic bacteria behaved as free cells in the transformation media and no mass transfer limitations were observed. The magnetic bacteria could be used repeatedly without any cell loss, the cells being retrieved at the end of each transformation cycle by a magnet.     

Synthesis of novel porous magnetic silica microspheres as adsorbents for isolation of genomic DNA

An improved procedure is described for preparation of novel mesoporous microspheres consisting of magnetic nanoparticles homogeneously dispersed in a silica matrix. The method is based on a three-step process, involving (i) formation of hematite/silica composite microspheres by urea-formaldehyde polymerization, (ii) calcination of the composite particles to remove the organic constituents, and (iii) in situ transformation of the iron oxide in the composites by hydrogen reductive reaction. The as-synthesized magnetite/silica composite microspheres were nearly monodisperse, mesoporous, and magnetizable, with as typical values an average diameter of 3.5 microm, a surface area of 250 m(2)/g, a pore size of 6.03 nm, and a saturation magnetization of 9.82 emu/g. These magnetic particles were tested as adsorbents for isolation of genomic DNA from Saccharomyces cerevisiae cells and maize kernels. The results are quite encouraging as the magnetic particle based protocols lead to the extraction of genomic DNA with satisfactory integrity, yield, and purity. Being hydrophilic in nature, the porous magnetic silica microspheres are considered a good alternative to polystyrene-based magnetic particles for use in biomedical applications where nonspecific adsorption of biomolecules is to be minimized.     

A Simple Method for Encapsulating Single Cells in Alginate Microspheres Allows for Direct PCR and Whole Genome Amplification

Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. Rhodobacter sphaeroides cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA) or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to R. sphaeroides. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated cells in common bulk reactions, although further work must be done to improve the amplification coverage of single genomes.     

NMR spectroscopy of recombinant chinese hamster ovary (CHO) cells in a packed bed reactor

Summary Several clones of CHO cells, including recombinant cell lines expressing Hepatitis B surface antigen, were grown in macroporous collagen microspheres. These provided sufficient cell density in a packed bed recirculation system for phosphorus-31 nuclear magnetic resonance spectroscopic estimation of metabolite concentration. Intracellular nucleoside triphosphate as well as nucleoside tri- plus diphosphate levels were higher in the methotrexate-selected clones compared to the dhfr^– cell line.     

Multiunit Floating Drug Delivery System of Rosiglitazone Maleate: Development, Characterization, Statistical Optimization of Drug Release and In Vivo Evaluation

A multiunit floating drug delivery system of rosiglitazone maleate has been developed by encapsulating the drug into Eudragit® RS100 through nonaqueous emulsification/solvent evaporation method. The in vitro performances of microspheres were evaluated by yield (%), particle size analysis, drug entrapment efficiency, in vitro floating behavior, surface topography, drug–polymer compatibility, crystallinity of the drug in the microspheres, and drug release studies. In vitro release was optimized by a {3, 3} simplex lattice mixture design to achieve predetermined target release. The in vivo performance of the optimized formulation was evaluated in streptozotocin-induced diabetic rats. The results showed that floating microspheres could be successfully prepared with good yields (69–75%), high entrapment (78-97%), narrow size distribution, and desired target release with the help of statistical design of experiments from very small number of formulations. In vivo evaluation in albino rats suggested that floating microspheres of rosiglitazone could be a promising approach for better glycemic control.     

In Situ Transplantation of Alginate Bioencapsulated Adipose Tissues Derived Stem Cells (ADSCs) via Hepatic Injection in a Mouse Model

Objective

Adipose tissue derived stem cells (ADSCs) transplantation has recently gained widespread enthusiasm, particularly in the perspective to use them as potential alternative cell sources for hepatocytes in cell based therapy, mainly because of their capability of hepatogenic differentiation in vitro and in vivo. But some challenges remain to be addressed, including whether ADSCs can be provided effectively to the target organ and whether subsequent proliferation of transplanted cells can be achieved. To date, intrasplenic injection is the conventional method to deliver ADSCs into the liver; however, a number of donor cells retained in the spleen has been reported. In this study, our objective is to evaluate a novel route to transplant ADSCs specifically to the liver. We aimed to test the feasibility of in situ transplantation of ADSCs by injecting bioencapsulated ADSCs into the liver in mouse model.

Methods

The ADSCs isolated from human alpha 1 antitrypsin (M-hAAT) transgenic mice were used to allow delivered ADSCs be readily identified in the liver of recipient mice, and alginate was selected as a cell carrier. We first evaluated whether alginate microspheres are implantable into the liver tissue by injection and whether ADSCs could migrate from alginate microspheres (study one). Once proven, we then examined the in vivo fate of ADSCs loaded microspheres in the liver. Specifically, we evaluated whether transplanted, undifferentiated ASDCs could be induced by the local microenvironment toward hepatogenic differentiation and the distribution of surviving ADSCs in major tissue organs (study two).

Results

Our results indicated ADSCs loaded alginate microspheres were implantable into the liver. Both degraded and residual alginate microspheres were observed in the liver up to three weeks. The viable ADSCs were detectable surrounding degraded and residual alginate microspheres in the liver and other major organs such as bone marrow and the lungs. Importantly, transplanted ADSCs underwent hepatogenic differentiation to become cells expressing albumin in the liver. These findings improve our understanding of the interplay between ADSCs (donor cells), alginate (biomaterial), and local microenvironment in a hepatectomized mouse model, and might improve the strategy of in situ transplantation of ADSCs in treating liver diseases.     

Reversible immobilization of glucoamylase onto magnetic chitosan nanocarriers

A simple preparation process for the monodispersed pH-sensitive core-shell magnetic microspheres was carried out consisting of chitosan self-assembled on magnetic iron oxide nanoparticles. Meanwhile, glucoamylase was immobilized as a model enzyme on this carrier of Fe₃O₄/CS microspheres by ionic adsorption. The morphology, inner structure, and high magnetic sensitivity of the resulting magnetic chitosan microspheres were studied, respectively, with a field emission scanning electron microscope (SEM), transmission electron microscope (TEM), FT-IR spectroscopy, thermogravimetric analysis (TGA), and a vibrating sample magnetometer (VSM). Subsequently, the properties of glucoamylase immobilized on the regenerated supports were also investigated by determining storage stability, pH stability, reusability, magnetic response, and regeneration of supports. The results from characterization and determination remarkably indicated that the immobilized glucoamylase obtained presents excellent storage stability, pH stability, reusability, magnetic response, and regeneration of supports. Therefore, this kind of magnetic Fe₃O₄/CS microspheres with perfect monodispersity should be an ideal support for enzyme immobilization.     

The in vitro and in vivo anti-tumor effects of MTX-Fe3O4-PLLA-PEG-PLLA microspheres prepared by suspension-enhanced dispersion by supercritical CO2

The in vitro and in vivo anti-tumor efficacy of methotrexate-loaded Fe3O4-poly-L-lactide-poly(ethylene glycol)-poly-L-lactide magnetic composite microspheres(MTX-Fe3O4-PLLA-PEG-PLLA MCMs,MMCMs),which were produced by co-precipitation(C)and microencapsulation(M)in a supercritical process,was evaluated at various levels:cellular,molecular,and integrated.The results at the cellular level indicate that MMCMs(M)show a better anti-proliferation activity than raw MTX and could induce morphological changes of cells undergoing apoptosis.At the molecular level,MMCMs(M)lead to a significantly higher relative mRNA expression of bax/bcl-2 and caspase-3 than MMCMs(C)at 10μg mL-1(P0.01);and the pro-caspase-3protein expression measured by Western blot analysis also demonstrates that MMCMs(M)can effectively activate pro-caspase-3.At the integrated level,mice bearing a sarcoma-180 tumor are used;in vivo anti-tumor activity tests reveal that MMCMs(M)with magnetic induction display a much higher tumor suppression rate and lower toxicity than raw MTX.Pharmacokinetic studies show that MMCMs(M)with magnetic induction significantly increase the accumulation of MTX in the tumor tissue compared with the other treatments.These results suggest that the MMCMs(M)prepared by the SpEDS process have great potential to play a positive role in the magnetic targeted therapy field.     

A Short Term Quality Control Tool for Biodegradable Microspheres

Accelerated in vitro release testing methodology has been developed as an indicator of product performance to be used as a discriminatory quality control (QC) technique for the release of clinical and commercial batches of biodegradable microspheres. While product performance of biodegradable microspheres can be verified by in vivo and/or in vitro experiments, such evaluation can be particularly challenging because of slow polymer degradation, resulting in extended study times, labor, and expense. Three batches of Leuprolide poly(lactic-co-glycolic acid) (PLGA) microspheres having varying morphology (process variants having different particle size and specific surface area) were manufactured by the solvent extraction/evaporation technique. Tests involving in vitro release, polymer degradation and hydration of the microspheres were performed on the three batches at 55°C. In vitro peptide release at 55°C was analyzed using a previously derived modification of the Weibull function termed the modified Weibull equation (MWE). Experimental observations and data analysis confirm excellent reproducibility studies within and between batches of the microsphere formulations demonstrating the predictability of the accelerated experiments at 55°C. The accelerated test method was also successfully able to distinguish the in vitro product performance between the three batches having varying morphology (process variants), indicating that it is a suitable QC tool to discriminate product or process variants in clinical or commercial batches of microspheres. Additionally, data analysis utilized the MWE to further quantify the differences obtained from the accelerated in vitro product performance test between process variants, thereby enhancing the discriminatory power of the accelerated methodology at 55°C.     

Separation of cells labeled with immunospecific iron dextran microspheres using high gradient magnetic chromatography

Immunospecific magnetic microspheres, consisting of ferromagnetic iron dextran conjugated to Protein A, were used to specifically label red blood cells (RBC) for cell separation studies using high gradient magnetic chromatography ( HGMC ). When 10(7)-10(8) RBC labeled with Protein A-iron dextran microspheres were applied to a column containing 30 mg stainless steel wire placed in a 7.5 kilogauss magnetic field, 96 +/- 2% of the cells were retained in the column. These cells could be eluted by removing the magnetic field and mechanically agitating the column. The retention of labeled cells by HGMC was shown to be dependent on the applied magnetic field and the amount of wire packed into the column. HGMC in conjunction with cell labeling with immunospecific iron dextran microspheres have useful applications for the separation of specific cell types.