Bioactive Macromolecules Research Articles

Epoxy-derived pHEMA membrane for use bioactive macromolecules immobilization: Covalently bound urease in a continuous model system

Poly(2-hydroxyethylmethacrylate) (pHEMA) membranes were prepared by UV-initiated photopolymerization of HEMA in the presence of an initiator (α-α′-azobisisobutyronitrile, AIBN). The epoxy group, i.e., epichlorohydrin, was incorporated covalently, and the urease was immobilized onto pHEMA membranes by covalent bonding through the epoxy group. The retained activity of the immobilized enzyme was found to be 27%. The Km values were 18 and 34 mM for the free and the immobilized enzymes, respectively, and the Vmax values were found to be 59.7 and 16.2 U mg−1 for the free and the immobilized enzyme. The optimum pHs was 7.2 for both forms, and the optimum temperature for the free and the immobilized enzymes were determined to be 45 and 50°C, respectively. The immobilized urease was characterized in a continuous system and during urea degradation the operational stability rate constant for the immobilized enzyme was found to be 5.83 × 10−5 min−1. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 77: 2000–2008, 2000

Preparation of non-porous microspheres with high entrapment efficiency of proteins by a (water-in-oil)-in-oil emulsion technique

Emulsification–solvent removal methods have been widely used for encapsulating bioactive macromolecules like proteins and polypeptides in biodegradable polymers. We report, a (water-in-oil)-in-oil emulsion technique wherein proteins and polypeptides differing in molecular weight and shape were encapsulated in polymers of current biomedical interest. When an oil was used as the processing medium in combination with a carefully selected mixed solvent system such that a stable (w/o 1/o 2 emulsion is formed and solvents are removed by a combination of extraction and evaporation, the entrapment efficiency was high and the product nonporous. The entrapment efficiency of globular proteins exceeded 90% while that of fibrous proteins was around 70%. Fracture studies revealed that the polymer matrix was dense. The mechanism of entrapment involved solvent-induced precipitation of the protein as the microspheres were being formed. The principle of the method will find use in preparation of non-porous polymer microparticles with reduced burst effect.

Covalent immobilization of biological molecules to maleic anhydride and methyl vinyl ether copolymers?A physico-chemical approach

The covalent grafting of biological molecules to copolymers of maleic anhydride and methyl vinyl ether (MAMVE) has been used in various applications in diagnostics. To tentatively elucidate the phenomena involved in the control of the immobilization of oligodeoxynucleotides and proteins, the physico-chemical properties of MAMVE copolymers were investigated. Because the grafting mixture contains water, to allow dissolution of the biomolecules without loss of biological properties, the anhydride-based copolymer evolves from a neutral to a negatively charged macromolecule due to hydrolysis of the anhydride moities. The properties of both hydrolyzed and nonhydrolyzed polymers were investigated. As demonstrated by light-scattering measurements in batch, the copolymers showed some level of aggregation in DMF, DMSO, and aqueous DMSO. The presence of aggregates was confirmed by size-exclusion chromatography in DMF. However, partial deaggregation occurred for the lowest molecular weight sample, on adding 1% w/v of LiBr. The nonhydrolyzed copolymers exhibited a rigid conformation in a 5% water/DMSO mixture, as well as their hydrolyzed counterpart at a low ionization degree. The rate of the hydrolysis reaction was shown to be dependent on the pH of the reaction medium and on temperature. The activation energy of the hydrolysis reaction was 14 kJ/mol, and the rate constant in the order of 10 -4 s -1 . On the basis of these data, the effect on the grafting reaction of biomolecules of different parameters such as ionic strength and the nature of the solvent, along with some other results were interpreted in terms of interactions between the synthetic and bioactive macromolecules.

Biopolymers as drug carriers and bioactive macromolecules

Biopolymers as drug carriers and bioactive macromolecules

Biopolymers as drug carriers and bioactive macromolecules

Biopolymers as drug carriers and bioactive macromolecules

Enhanced loading and activity retention of bioactive proteins in hydrogel delivery systems

A simple, general and effective technique is developed for increasing the loading of bioactive macromolecules into hydrogels using the principles of aqueous two-phase extraction. Model proteins, ovalbumin and α-amylase, were loaded into hydrated gels by soaking the gels in a buffered solution of protein containing 12 wt% PEG-10 000 and 0.22 M salt (KCl, KBr or KI). The PEG and salts were expected to enhance protein sorption according to aqueous two-phase extraction heuristics. In the absence of the solution additives, the gels absorbed little protein. But protein loading up to 270 mg ovalbumin/g polymer and 67 mg α-amylase/g polymer was obtained when the PEG and salt were added; loading was not significantly dependent upon salt type. Ovalbumin release from hydrated gels was diffusion-controlled. The diffusion coefficient was 1·10 −7 cm 2/s, consistent with protein absorption into the gel rather than adsorption onto the surface. Release kinetics of both proteins from dried, glassy gels matched conventional behavior for release of absorbed drugs from glassy polymers. Finally, α-amylase activity was retained even after drying the loaded gel at 65°C, conditions which denatured the enzyme when not absorbed in the gel.

Novel types of polysaccharidic assemblies

AbstractNew types of polysaccharidic assemblies based on chitosan and uncharged or charged derivatives of scleroglucan are presented. Macroscopic assemblies having a spherical skinlike shape were prepared by controlled mixing of two polyelectrolyte solutions. Hydrogels based exclusively on the above natural derivatized polysaccharides crosslinked via simple chemical processes, avoiding the use of extraneous reticulating agents and organic solvents, are also described. These biocompatible materials may, eventually, be used in drug release formulations or for the incapsulation of bioactive macromolecules. The interesting possibility exists to study the micelle formation of amphiphiles within the spherical skins.

Chiral and Steric Recognition in Optically Active, Isotactic, Alternating .alpha.-Olefin-Carbon Monoxide Copolymers. Effect on Physical Properties and Chemical Reactivity

Chiral and steric recognitions play critical roles in selective binding and resultant high stereo- and enantioselectivity in chemical reactions mediated by bioactive macromolecules such as enzymes and nucleic acids. Herein, we report the presence of both strong chiral and steric recognitions between optically active, highly isotactic, alternating {alpha}-olefin-carbon monoxide copolymers with 1,4-keto groups in the backbone and their effect on both the physical properties and chemical reactivity of the polymers. Thus, an alternating {alpha}-olefin-carbon monoxide copolymer with a given chiral sense for the tertiary carbons in the main chain can distinguish (a) between the two possible chiral senses for the tertiary carbons in the main chain of a second {alpha}-olefin-carbon monoxide copolymer even when the latter has a different pendant alkyl group and (b) between the length of the pendant alkyl group in two different copolymers whose tertiary carbons have the same chiral sense. 7 refs., 3 figs., 2 tabs.

高分子デリバリーを目的とした温度感受性リポソームの開発

It is useful if the bioactive macromolecules, such as cytokines, could be delivered to the desired site. Liposomes have been used successfully as carriers for various agents. Targeting of liposomes to the desirable site has been also achieved to some extent, and the release of the contents at the target site has been attempted by means of thermosensitive liposomes utilizing phase transition of lipid bilayer. Thermosensitiye Iiposomes with internal solution of 2-fold higher osmotic pressure and sized through 200 nm-pore, released encapsulated macromolecules effectively when they were incubated at 40∼42°C, since transient swelling during phase transition of liposomes with hyper-osmotic internal aqueous phase causes permeation of large molecules through lipid bilayer. Long-circulating, serum stable, type of thermosensitive liposomes were also attemped to be prepared for practical usage of liposomes as a tool for macromolecule delivery.

Heparin release from polymer complex

An electro-erodible polyelectrolyte complex was prepared and investigated for a pulsatile drug release system. An insoluble polyelectrolyte complex was formed by combining two water-soluble polymers, poly(allylamine) and heparin. Upon the application of an electric current, a rapid structural change of the complex occurred, dissolving the polymer matrix in proportion to the intensity of an applied electric current. The disruption of ionic bonds in the polymer matrix attached to the cathode and subsequent release of heparin was due to the locally increased pH near the cathode (resulting from hydroxyl ion production). Thus, the release pattern of a model bioactive macromolecule, heparin, followed the applied electric current, primarily due to surface erosion of the polymer matrix.

Anti-T cell immunotoxins: a look at post-endocytotic receptor-mediated routing

Many types of targeted drug delivery systems utilize cell surface receptor binding to achieve targeting specificity. Receptor-mediated endocytosis also provides internalization of the targeted drug. What has not been previously appreciated is the fact that cell surface receptors play an important post-endocytotic role. After endocytosis the receptor ligand interaction determines how the ligand-drug complex will be routed. In many cases this routing uniquely determines the overall efficacy of the drug delivery system. In this paper we will show that a unique anti-T cell immunotoxin, anti-CD3 +-CRM9, constructed with a diphtheria toxin binding site mutant, CRM9, is a highly effective reagent for inducing in vivo T cell ablation. The high degree of efficacy and the wide therapeutic margin of this reagent is a consequence of the obligatory intracellular routing pathway required for diphtheria toxin (DT) intoxication. The DT receptor (DTR) typically fulfills this function in diphtheria toxin intoxication. In the case of the anti-CD3 + toxin conjugate the routing function is performed by CD3. This epitope routes parallel with DTR. This fact accounts for the high toxicity of anti-CD3 conjugates made with diphtheria toxin binding site mutants such as CRM9. CRM9 alone is incapable of entering the optimal routing pathway and hence has very low systemic toxicity. The anti-CD3 antibody doubly complements the CRM9 defect: It facilitates (1) the entrance of CRM9 into the targeted cell endosomes and (2) directs the entrance of CRM9 into the DT intoxication pathway. It is likely that these concepts will have relevance to other cell type-specific immunotoxins and other cell type-specific targeted delivery systems which depend on the membrane translocation of bio-active macromolecules into the cytosol or nuclear compartments.

Coated alginate microspheres: Factors influencing the controlled delivery of macromolecules

AbstractA systematic study of the mild alginate/polycation microencapsulation process, as applied to encapsulation of bioactive macromolecules such as proteins, was conducted. When protein drugs were suspended in sodium alginate solution and sprayed into 1.3% buffered calcium chloride to form cross‐linked microcapsules, large (up to 90%) losses of encapsulation species were encountered, and moderate to strong protein‐alginate interactions caused poor formation of capsules. As a result, a diffusion‐filling technique was adopted in which blank alginate beads, coated twice with small amounts of polycation, were formed prior to drug loading. Protein was then loaded into these capsules by stepwise diffusion from solutions of increasing drug concentration. The drug‐loaded capsules were coated with a final layer of polycation. In all, three polycation coatings were used, two prior to filling and one after filling. The first coating strongly influenced the size, integrity, and loading capacity of the capsules. Low concentrations of polycation resulted in poorly formed capsules with very low retention of the drug in the final capsule, while very high concentrations prevented the drug from entering the capsule at the filling stage. This first coat also affected the duration of drug release from the capsule and the size of the burst effect. The second coat had less effect on the capsule integrity, but it did influence the drug payload and relase profile. The final, sealing‐coat had little effect on drug payload and only limited effect on the release profile up to a critical concentration, above which the release profile was not affected. For all coats, increasing polycation concentration decreased the burst effect, and caused the release profile to be more sustained. Encapsulation of a series of dextrans with increasing molecular weight revealed that the release profile was directly related to the molecular weight of the diffusing species, which was more sustained as molecular weight increased. We have shown that the choice of coating parameters in the diffusion‐filled, alginate/polycation system is critical for successful drug delivery from these capsules. By carefully choosing the coating parameters, both the drug payload and the release profile can be fine‐tuned to meet the desired profile.

Uptake and concentration of bioactive macromolecules by K562 cells via the transferrin cycle utilizing an acid-labile transferrin conjugate

The transferrin cycle was used to attempt the import of bioactive macromolecules into cells with the aid of an acid-labile cross-linking agent. Anti-tetanus F(ab')2 fragments were iodinated and then conjugated to transferrin with a newly developed acid-labile cleavable cross-linking reagent, bismaleimidoethoxy propane, following thiolation of both proteins. Noncleavable conjugates were also prepared. At saturating conjugate concentrations, the uptake rate for both conjugates averaged over the first 2 h is about 6.5 fmol/million cells/min. Incubation of loaded cells in fresh medium for 30 min and analysis of cell pellets and supernatants reveal that 1) of the previously cell-associated label, only intact conjugate (about 50% of the label) is returned to the medium; 2) most of the remaining cell-associated material for the cleavable conjugate is chromatographically coincident with free Fab with some contribution from free F(ab')2 fragments. In contrast, the cell pellets loaded with noncleavable conjugates contained intact transferrin-F(ab'), conjugates. These results are consistent with transferrin receptor-mediated uptake of acid-labile conjugate followed by hydrolysis in acidified endosomes and resulting in concentration of free F(ab')2 and Fab within a prelysosomal intracellular compartment. A protein shuttle such as transferrin may therefore be used with ketal based acid-labile cross-linkers to load foreign molecules into an intracellular compartment. In addition, these data provide independent confirmation of the low pH compartment within the transferrin cycle. This new methodology is applicable to other cases of receptor/ligand trafficking to report low pH compartments independent of morphological analysis. Since transferrin receptors are overexpressed in tumors, antineoplastic agents could be targeted to tumors as transferrin acid-labile conjugates. This import system might be particularly useful in combatting the tumor cell export of antitumor agents occurring in multidrug resistance.

Protein purification by affinity binding to unilamellar vesicles

A novel purification technique is proposed which employs affinity-ligand-modified liposomes to specifically purify bioactive macromolecules from solution. This process is demonstrated with avidin as the model biomolecule and biotin as the affinity ligand. Biotin is covalently bound to the surface of small unilamellar vesicles composed of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylethanolamine (DMPE). The number of accessible binding sites on the liposomes is determined by titration with avidin, and the kinetics of binding are evaluated by monitoring the concentration of free avidin in solution after the addition of biotinylated liposomes. The specificity of the process is determined by following the affinity binding of avidin to biotinylated liposomes in the presence of model impurities (i.e., lysozyme and cytochrome C). Liposome-bound avidin is separated from the impurities by ultrafiltration through a membrane which retains the liposomes.

物質生産の手段としての動物培養細胞 培養細胞によるタンパク質生産の増強

物質生産の手段としての動物培養細胞 培養細胞によるタンパク質生産の増強

物質生産の手段としての動物培養細胞 増殖阻害

物質生産の手段としての動物培養細胞 増殖阻害

物質生産の手段としての動物培養細胞 細胞培養のための細胞接着因子

物質生産の手段としての動物培養細胞 細胞培養のための細胞接着因子

物質生産の手段としての動物培養細胞 培養システム

物質生産の手段としての動物培養細胞 培養システム

物質生産の手段としての動物培養細胞

物質生産の手段としての動物培養細胞

物質生産の手段としての動物培養細胞 プロテアーゼ・インヒビター

物質生産の手段としての動物培養細胞 プロテアーゼ・インヒビター