Use of magnetic poly(glycidyl methacrylate) monosize beads for the purification of lysozyme in batch system

The hydrophobic affinity ligand L-tryptophan immobilized magnetic poly(glycidyl methacrylate) [m-poly(GMA)] beads in monosize form (1.6 microm in diameter) were used for the affinity purification of lysozyme from chicken egg white. The m-poly(GMA) beads were prepared by dispersion polymerization in the presence of Fe3O4 nano-powder. The epoxy groups of the m-poly(GMA) beads were converted into amino groups with 1,6 diaminohexane (i.e., spacer arm). l-tryptophan was then covalently immobilized on spacer arm attached m-poly(GMA) beads. Elemental analysis of immobilised L-tryptophan for nitrogen was estimated as 42.5 micromol/g polymer. Adsorption studies were performed under different conditions in a batch system (i.e., medium pH, protein concentration and temperature). Maximum lysozyme adsorption amount of m-poly(GMA) and m-poly(GMA)-L-tryptophan beads were 1.78 and 259.6 mg/g, respectively. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium adsorption capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. It was observed that after 10 adsorption-elution cycle, m-poly(GMA)-L-tryptophan beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. It was found to be successful in achieving purification of lysozyme in a high yield of 76% with a purification fold of 71 in a single step. The specific activity of the eluted lysozyme (62,580 U/mg) was higher than that obtained with a commercially available pure lysozyme (Sigma (60,000 U/mg).     

Magnetic poly(glycidyl methacrylate) microspheres for protein capture

Immunoprecipitation of protein p53 from cell lysate on magnetic PGMA microspheres.

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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.     

Spray-dried poly(D,L-lactide) microspheres containing carboplatin for veterinary use: In vitro and in vivo studies

The aim of this study was the development of a veterinary dosage form constituted by injectable biodegradable microspheres designed for the subcutaneous release of carboplatin, a chemotherapeutic drug. Poly(D,L-lactide) (PDLLA) microspheres were prepared by an emulsification/spray-drying method, using the drug-to-polymer weight ratios 1∶9 and 1∶5; blank microspheres (1% w/v) were prepared as a comparison. Microparticles were characterized in terms of morphology, encapsulation efficiency, and in vitro drug release behavior. In vivo tests were conducted on rats by subcutaneous injection of microsphere aqueous suspensions. Levels of carboplatin were evaluated both in the skin and in serum. The microparticles obtained had a spherical shape; particle size ranged from 5 to 7 μm, dependent on drug loading. Microspheres were able to control the in vitro release of the drug: approximately 90% to 100% of the carboplatin was released over 30 days. In vivo results showed that the microspheres were able to release high drug amounts locally, and sustained serum levels of drug were also achieved. Based on these results, carboplatin-loaded PDLLA microspheres may be useful for local delivery of the antineoplastic drug to the tumor, avoiding tumor recurrence in small animals, and may decrease the formation of distant metastases. Published: September 20, 2005     

Dye-incorporated poly(EGDMA-HEMA) microspheres as specific sorbents for aluminum removal

Aluminum [Al(III)] adsorption onto dye-incorporated poly(ethylene glycol dimethacrylate-hydroxyethyl methacrylate) [poly(EGDMA-HEMA)] microspheres was investigated. Poly(EGDMA-HEMA) microspheres, in the size range of 150–200 μm, were produced by a modified suspension polymerization of EGDMA and HEMA. The reactive dyes (i.e., Congo Red, Cibacron Blue F3GA and Alkali Blue 6B) were covalently incorporated to the microspheres. The maximum dye load was 14.5 μmol Congo Red/g, 16.5 μmol Cibacron Blue F3GA/g and 23.7 μmol Alkali Blue 6B/g polymer. The maximum Al(III) adsorption on the dye microspheres from aqueous solutions containing different amounts of Al(III) ions were 27.9 mg/g, 17.3 mg/g and 12.2 mg/g polymer for the Congo Red, Cibacron Blue F3GA and Alkali Blue 6B, respectively. The maximum Al(III) adsorption was observed at pH 7.0 in all cases. Non-specific Al(III) adsorption was about 0.84 mg/g polymer under the same conditions. High desorption ratios (95%) were achieved in all cases by using 0.1 M HNO₃. It was possible to reuse these dye-incorporated poly(EGDMA-HEMA) microspheres without significant losses in the Al(III) adsorption capacities.     

The use of titanium (IV) oxide for the immobilisation of carbohydrate-directed enzymes.

The use of particles of porous titanium (IV) oxide as a suitable matrix for enzyme immobilisation has been investigated with dextranase. Treatment of the particles with enzyme in the presence and absence of ammonium ions showed that the presence of ammonia induced a greater coupling of protein, whereas a greater retention of enzyme specific activity was achieved in the absence of ammonia. Properties of the immobilised enzyme include a pH-dependence and reversibility of the coupling between enzyme and matrix. The immobilised dextranase was most stable at pH 5.0. Automated analytical techniques for measuring the activity of dextranase and other polysaccharidases in soluble and insoluble forms are also reported.     

Hydrazide-functionalized poly(2-hydroxyethyl methacrylate) microspheres for immobilization of horseradish peroxidase

Nonporous cross-linked poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (poly(HEMA-co-EDMA)) microspheres were prepared by dispersion polymerization of HEMA and EDMA. The polymerization was performed in toluene/2-methylpropan-1-ol in the presence of cellulose acetate butyrate as a steric stabilizer and dibenzoyl peroxide initiator. The particle size may be increased by decreasing the toluene/2-methylpropan-1-ol ratio and by increasing polymerization temperature. Adipohydrazide was attached to the microspheres activated with 2,4,6-trichloro-1,3,5-triazine. After periodate oxidation of its carbohydrate moieties, horseradish peroxidase was coupled to the hydrazide-functionalized poly(HEMA-co-EDMA) microparticles up to 7.3 microgram of enzyme/g of carrier without a significant loss of its activity. Immobilized peroxidase was found to be stable, retaining more than 97% of its initial activity when stored for 23 days after the preparation.     

Temperature-dependent reversible transition of poly(dCdG) · poly(DcdG) in ethanolic and methanolic solutions

Using CD we investigated the transitions of poly(dCdG) · poly(dGdC) from B-to-Z form and from Z-to-Z′ form. We have found experimental conditions that allow the cooperative transition to occur as a function of temperature in ethanolic solutions. The transition is reversible and can be repeated as often as desired. There is no evidence of strand separation during the cooperative transition as monitored by absorbance. For purposes of calculation, we have assumed a two-state model for the B-to-Z transition, although the data indicate that such a model is too simplistic. The calculations allow the estimation of the change in enthalpy per mole of cooperative unit for the transition as a function of ethanol concentration. The values range from ±140 to ±200 kcal/mol for ethanol concentrations between 10 and 20%. Investigations of the noncooperative Z-to-Z′ transition show that it is a reversible two-state transition. The different forms of poly(dCdG) · poly(dGdC) give no scattering contributions to the CD as shown by fluorescent-detected CD or fluorscat techniques. This indicates that the CD spectra are true spectra, and contain no contributions from differential scattering of the polynucleotide. This is particularly significant in the case of the Z′ form, since it exists at high ethanol concentrations (80%) where condensation of polynucleotides can provide large contributions to the CD spectra. Analogous investigations using methanol show that the two transitions also occur, but the final Z′ form in methanol is qualitatively different from the ethanol form.     

Holmium-loaded poly(L-lactic acid) microspheres: in vitro degradation study

The clinical application of holmium-loaded poly(L-lactic acid) (PLLA) microspheres for the radionuclide treatment of liver malignancies requires in depth understanding of the degradation characteristics of the microspheres. To this end, an in-vitro degradation study was conducted. PLLA-microspheres with and without HoAcAc loading, and before and after neutron or gamma irradiation, were incubated in a phosphate buffer at 37 degrees C for 12 months. In contrast with the other microsphere formulations, only the neutron-irradiated Ho-PLLA-MS disintegrated. At the end of the experiment (52 weeks) highly crystalline fragments, as evidenced from Differential Scanning Calorimetry, were present. Infrared spectroscopy showed that these fragments consisted of holmium lactate. In conclusion, this study demonstrates that the degradation of neutron-irradiated Ho-PLLA-MS was substantially accelerated by the HoAcAc incorporation and subsequent neutron irradiation. The degradation of these microspheres in aqueous solution resulted in the formation of insoluble holmium lactate microcrystals without release of Ho3+.     

Production of poly(l-lactide)-degrading enzyme by Amycolatopsis orientalis for biological recycling of poly(l-lactide)

Efficient production of poly(l-lactide)(PLA)-degrading enzyme was achieved by addition of 0.1% (w/v) silk fibroin powder into a liquid culture medium of an actinomycete, Amycolatopsis orientalis, without other complex nitrogen sources, such as yeast extract and peptone. Scaled-up production of the enzyme in a 5-l jar fermenter showed the possibility of producing this enzyme on an industrial scale at low production cost. The extracellular PLA-degrading enzyme showed potent degrading activity, which is effective for biological recycling of PLA, i.e., 2,000 mg/l of PLA powder was completely degraded within 8 h at 40°C using 20 mg/l purified enzyme. An optically active l-lactic acid with 600 mg/l was obtained as degradation product of PLA without undesirable racemization.     

Highly branched poly(N-isopropylacrylamide) for use in protein purification

Poly(N-isopropylacrylamide)s with imidazole endgroups were used to separate a histidine-tagged protein fragment directly from a crude cell lysate. The polymers display a lower critical solution temperature that can be tuned to occur at a range of subambient temperatures. UV-visible spectra indicated differences in the binding in aqueous media of Cu(II) and Ni(II) to the imidazole endgroups. These changes in the UV-visible spectra were reflected in the solution/aggregation behavior of the polymers as studied by dynamic light scattering. The addition of Cu(II) disaggregated the polymers, and the polymer coil swelled. On the other hand, when Ni(II) was added the polymers remained aggregated in aqueous media. The polymers were used to purify residues 230-534 of the histidine-tagged breast cancer susceptibility protein his6-BRCA1. Cu(II) was found to be better suited to the formation of useful polymer-metal ion-protein complexes that display cloud points, since Ni(II)/polymer mixtures generated very little purified protein. The polymers were synthesized using a previously reported variation of the reversible addition-fragmentation chain termination (RAFT) methodology, using the chain transfer agent 3H-imidazole-4-carbodithioic acid 4-vinyl benzyl ester with N-isopropylacrylamide (NIPAM).     

Novel biodegradable and pH-sensitive poly(ester amide) microspheres for oral insulin delivery

Biodegradable and pH-sensitive PEAs based on dual amino acids are designed, synthesized, and characterized. Insulin can be loaded into the PEA microspheres by a solid-in-oil-in-oil technique with high encapsulation efficiency. The feasibility of PEA microspheres as oral insulin delivery carriers is evaluated in vitro and in vivo. The hydrophobic leucine groups on PEA seem to play an important role in the pH-dependent release mechanism and cytotoxicity of PEA microspheres. Oral administration of insulin-loaded PEA microspheres to streptozotocin-induced diabetic rats at 60 IU kg(-1) is able to reduce fasting plasma glucose levels to 49.4%. These results indicate that PEA microspheres are potential new vehicles for insulin oral delivery.     

Antibody purification with protein A attached supermacroporous poly(hydroxyethyl methacrylate) cryogel

Immunoglobulin G (IgG) purification from human plasma with protein A attached supermacroporous poly(hydroxyethyl methacrylate) [PHEMA] cryogel has been studied. PHEMA cryogel was prepared by bulk polymerization which proceeds in aqueous solution of monomer frozen inside a plastic syringe (cryo-polymerization). After thawing, the PHEMA cryogel contains a continuous matrix having interconnected pores of 10–200 μm size. Protein was covalently attached onto the PHEMA cryogel via cyanogen bromide (CNBr) activation. The maximum IgG adsorption on the PHEMA/protein A cryogel was found to be 83.2 mg/g at pH 7.4 from aqueous solutions. The non-specific IgG adsorption onto the PHEMA cryogel was about 0.38 mg/g. The macropore size of the cryogel makes it possible to process blood cells without blocking the column. Higher adsorption capacity was observed from human plasma (up to 88.1 mg/g). Adsorbed IgG was eluted using 0.1 M glycine–HCl buffer (pH 3.5) with a purity of 85%. PHEMA–protein A cryogel was used for repetitive adsorption/desorption of IgG without noticeable loss in IgG adsorption capacity after 10 cycles. PHEMA–protein A cryogel showed several advantages such as simpler preparation procedure, good selectivity for IgG purification from human plasma and good stability throughout repeated adsorption–desorption cycles.     

Synthesis and properties of star-shaped polylactide attached to poly(amidoamine) dendrimer

Star-shaped polylactide was synthesized by bulk polymerization of lactide with poly(amidoamine) (PAMAM) dendrimer as initiator, which was marked as PAMAM-g-PLA for simplicity. The nonlinear architecture of PAMAM-g-PLA was confirmed by gel permeation chromatograph, nuclear magnetic resonance, and differential scanning calorimetry analysis. Unlike the linear polylactide (PLA) with similar molecular weight, PAMAM-g-PLA had a higher hydrophilicity and a faster degradation rate because of shortened polymer chains and increased polar terminal endgroups due to its branch structure. The highly branched structure significantly accelerated the release of water-soluble bovine serum albumin from PAMAM-g-PLA microspheres, whereas the linear PLA with similar molecular weight exhibited an initial time lag release. This star polymer may have potential applications for hydrophilic drug delivery in tissue engineering, including growth factor and antibodies to induce tissue regeneration, by adjusting the chain lengths of PLA branches.     

Preparation and characterization of temperature-sensitive poly(N-isopropylacrylamide)-b-poly(D,L-lactide) microspheres for protein delivery

Temperature-sensitive diblock copolymers, poly(N-isopropylacrylamide)-b-poly(D,L-lactide) (PNIPAAm-b-PLA) with different PNIPAAm contents were synthesized and utilized to fabricate microspheres containing bovine serum albumin (BSA, as a model protein) by a water-in-oil-in-water double emulsion solvent evaporation process. XPS analysis showed that PNIPAAm was a dominant component of the microspheres surface. BSA was well entrapped within the microspheres, and more than 90% encapsulation efficiency was achieved. The in vitro degradation behavior of microspheres was investigated using SEM, NMR, FTIR, and GPC. It was found that the microspheres were erodible, and polymer degradation occurred in the PLA block. Degradation of PLA was completed after 5 months incubation in PBS (pH 7.4) at 37 degrees C. A PVA concentration of 0.2% (w/v) in the internal aqueous phase yielded the microspheres with an interconnected porous structure, resulting in fast matrix erosion and sustained BSA release. However, 0.05% PVA produced the microspheres with a multivesicular internal structure wrapped with a dense skin layer, resulting in lower erosion rate and a biphasic release pattern of BSA that was characterized with an initial burst followed by a nonrelease phase. The microspheres made from PNIPAAm-b-PLA with a higher portion of PNIPAAm provided faster BSA release. In addition, BSA release from the microspheres responded to the external temperature changes. BSA release was slower at 37 degrees C (above the LCST) than at a temperature below the LCST. The microspheres fabricated with PNIPAAm-b-PLA having a 1:5 molar ratio of PNIPAAm to PLA and 0.2% (w/v) PVA in the internal aqueous phase provided a sustained release of BSA over 3 weeks in PBS (pH 7.4) at 37 degrees C.     

Red cell enzyme polymorphisms in Friuli Venezia Giulia (northeast Italy)

Seven erythrocyte enzyme polymorphisms (ACP1, ADA, ESD, GLO1, PGD, PGM1 and PGM2) were investigated in a sample of 673 unrelated adult individuals from Friuli Venezia Giulia (or Friuli) and Istria. The gene frequencies found in the four provincial samples of Friuli and Istria fall within the range previously reported for Italy, showing a genetic homogeneity among the considered samples. However, comparisons with data from ex-Yugoslavian samples--using the chi 2 test--showed rather marked differences, probably due to a real different genetic structure of the compared samples. A significant association was found assuming a linear relation between the ADA*2 allele frequencies and longitude (r = +0.5503) and between the PGD*C frequencies and latitude (r = -0.6483), suggesting the existence of a clinal trend for these allele frequencies in Italy. These results seem to disagree with foregoing conclusions stated by other authors, probably because these studies were carried out in an area either rather narrow from the geographical point of view or affected by small size migration movements.     

Immobilization of enzyme onto poly(ethylene-vinyl alcohol) membrane

Invertase was ionically bound to the poly(ethylene-vinyl alcohol) membrane surface modified with two aminoacetals with different molecular length, 2-dimethyl-aminoacetoaldehyde dimethylacetal (AAA) and 3-(N, N-dimethylamino-n-propanediamine) propionaldehyde dimethylacetal (APA). Immobilization conditions were determined with respect to enzyme concentration in solution, pH value, ionic strength in immobilization solution, and immobilization time. Various properties of immobilized invertase were evaluated, and thermal stability was found especially to be improved by immobilization. The apparent Michaelis constant, K(m), was smaller for invertase bound by APA with longer molecular lengths than for invertase bound by AAA. We attempted to bind glucoamylase of Rhizopus delemar origin in the same way. The amount and activity of immobilized glucoamylase were much less than of invertase.     

Storage stabilization of enzyme activity by poly(ethyleneimine)

The effect of mixing penicillin acylase with poly(ethyleneimine) is discussed. The properties of the polymer-enzyme system were evaluated for a wide range of enzyme concentrations (0.3–45.5 mg/cm³) and poly(ethyleneimine) concentrations (0.0001–10% wt). It was shown that addition of poly(ethyleneimine) to crude enzyme preparation caused precipitation of ballast protein and stabilization of the enzyme fraction remaining in the supernatant. The soluble fraction had stable activity for 21 days storage at 37 °C while the native enzyme lost about 80% of its initial activity. Additionally, it was ascertained that the polymer very slightly affected the properties of penicillin acylase in the PEI-enzyme preparations. Finally, possible ways of using the polymer-enzyme preparations in a membrane reactor are suggested.This work was supported by Government Committee of Science: Grant KBN # 3 0321 91 1     

The use of titanium(IV) oxide coated with diazotised 1,3-diaminobenzene for the immobilisation of carbohydrate-directed enzymes

The utility of porous titanium(IV) oxide particles as a matrix for the immobilisation of enzymes on column packings has been extended. On coating the particles with diazotised 1,3-diaminobenzene, their capacity for binding dextranase was increased two-fold. The stability of the enzyme-matrix bridge was enhanced by the covalent bond so formed. Excess diazonium groups were reacted with 2-naphthol. Investigations of the effects of change of dextran concentration, pH, temperature, and flow rate upon a continuously operated column of the immobilised dextranase permitted assessment of the kinetic aspects of the enzyme via Lineweaver-Burk plots. The change of reaction rate with temperature showed, according to Arrhenius plots, an abrupt change at 28°. Possible sources of the kinetic characteristics of the immobilised dextranase are discussed.