Characterization of the soluble nanoparticles formed through Coulombic interaction of bovine serum albumin with anionic graft copolymers at low pH

A static light scattering (SLS) study of bovine serum albumin (BSA) mixtures with two anionic graft copolymers of poly(sodium acrylate-co-sodium 2-acrylamido-2-methyl-1-propanesulphonate)-graft-poly(N,N-dimethylacrylamide), with a high composition in poly(N,N-dimethylacrylamide) (PDMAM) side chains, revealed the formation of oppositely charged complexes, at pH lower than 4.9, the isoelectric point of BSA. The core-corona nanoparticles formed at pH = 3.00 were characterized. Their molecular weight and radius of gyration were determined by SLS, while their hydrodynamic radius was determined by dynamic light scattering. Small angle neutron scattering measurements were used to determine the radius of the insoluble complexes, comprising the core of the particles. The values obtained indicated that their size and aggregation number of the nanoparticles were smaller when the content of the graft copolymers in neutral PDMAM side chains was higher. Such particles should be interesting drug delivery candidates, if the gastrointestinal tract was to be used.     

The immobilization of enzymes and cells of Bacillus stearothermophilus onto poly(maleic anhydride/styrene)-Co-polyethylene and poly(maleic anhydride/vinyl acetate)-Co-polyethylene

The graft copolymer, poly(maleic anhydride/styrene)-co-polyethylene was prepared. The copolymer immobilized bovine serum albumin (BSA), but the amount coupled appeared to be effected by the amount of styrene in the graft copolymer, temperature, and pH of the coupling medium. Competition existed between hydrolysis of the grafted anhydride groups and the protein. A graft copolymer with 66% add-on immobilized 4.5 mg/glucose oxidase/g copolymer, 4.6 mg alkaline phosphates/g copolymer and 0.2 mg cell of Bacillus stearothermophilus/g copolymer. A number of copolymers containing poly(maleic anhydride/vinyl acetate)-co-polyethylene were prepared to cover a range of grafting levels. These immobilized larger quantities of BSA, alkaline phosphatase, and cells of B. stearothermophilus than did the styrene graft copolymer. The copolymer was also hydrolyzed to release the hydroxyl group from the poly(vinyl acetate) component of the grafted chains. Using p-benzoquinone as the "activating agent," the copolymer coupled to BSA and to acid phosphatase. Using p-toluene-sulfonyl chloride, the copolymer was very effective in immobilizing trypsin.     

An acid-labile block copolymer of PDMAEMA and PEG as potential carrier for intelligent gene delivery systems

Intelligent gene delivery systems based on physiologically triggered reversible shielding technology have evinced enormous interest due to their potential in vivo applications. In the present work, an acid-labile block copolymer consisting of poly(ethylene glycol) and poly(2-(dimethylamino)ethyl methacrylate) segments connected through a cyclic ortho ester linkage (PEG- a-PDMAEMA) was synthesized by atom transfer radical polymerization of DMAEMA using a PEG macroinitiator with an acid-cleavable end group. PEG- a-PDMAEMA condensed with plasmid DNA formed polyplex nanoparticles with an acid-triggered reversible PEG shield. The pH-dependent shielding/deshielding effect of PEG chains on the polyplex particles were evaluated by zeta potential and size measurements. At pH 7.4, polyplexes generated from PEG- a-PDMAEMA exhibited smaller particle size, lower surface charge, reduced interaction with erythrocytes, and less cytotoxicity compared to PDMAEMA-derived polyplexes. At pH 5.0, zeta potential of polyplexes formed from PEG- a-PDMAEMA increased, leveled up after 2 h of incubation and gradual aggregation occurred in the presence of bovine serum albumin (BSA). In contrast, the stably shielded polyplexes formed by DNA and an acid-stable block copolymer, PEG- b-PDMAEMA, did not change in size and zeta potential in 6 h. In vitro transfection efficiency of the acid-labile copolymer greatly increased after 6 h incubation at pH 5.0, approaching the same level of PDMAEMA, whereas there was only slight increase in efficiency for the stable copolymer, PEG- b-PDMAEMA.     

Synthesis of well-defined graft copolymers by combination of enzymatic polycondensation and "click" chemistry

Aliphatic polyesters having pendant azide groups were prepared by enzymatic polycondensation in the presence of lipase from Candida antarctica type B (CAL-B). The grafting reaction to the N(3)-functional polyester was carried out quantitatively at room temperature using copper-catalyzed azide-alkyne cycloaddition (CuAAC, "click" reaction) with monoalkyne-functional poly(ethylene oxide) (alkyne-PEO, M(n) = 750 g/mol). Furthermore, both enzymatic polycondensation and "click" reaction were carried out successfully in sequential one-pot reaction. The graft copolymer was surface-active and self-assembled in water. The graft copolymer had a critical aggregation concentration (cac) of 3 × 10(-2) μM in water determined by surface tension measurements. Above cac, the graft copolymer formed single chains and aggregates having a hydrodynamic radius of ～75 nm. Furthermore, the surface activity of the polymers at the air-water interface was studied by Langmuir trough measurements. The Langmuir isotherm of the graft polymer showed a pseudoplateau resulting from desorption of PEO chains into the subphase upon compression.     

Phosphorylcholine-based pH-responsive diblock copolymer micelles as drug delivery vehicles: light scattering, electron microscopy, and fluorescence experiments

The micellization behavior of a diblock copolymer comprising a highly hydrophilic and biocompatible poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) corona-forming block and a pH-sensitive poly(2-(diisopropylamino)ethyl methacrylate) (PDPA) core-forming block (PMPC-b-PDPA) has been studied by static and dynamic light scattering (SDLS), transmission electron microscopy (TEM), and potentiometry. Self-assembly of PMPC-b-PDPA copolymers with two different DPA volume fractions (phi(DPA)) leads to narrowly distributed and structurally distinct spherical micelles, as evidenced by their molecular weight (M(w,mic)), aggregation number (N(agg)), hydrodynamic radius (R(H)), corona width (W), and core radius (R(c)). The excellent potential of these pH-responsive micelles as nanosized drug delivery vehicles was illustrated by the encapsulation of dipyridamole (DIP), a model hydrophobic drug that dissolves in acid solutions and becomes insoluble above pH 5.8, which is comparable to the pK(a) of the PDPA block. The influence of micelle structure (namely M(w,mic), N(agg), R(H), W, and R(c)) on drug loading content, drug loading efficiency, partition coefficient, and release kinetics was investigated and confirmed by fluorescence spectroscopy studies. The maximum dipyridamole loadings within PMPC(30)-b-PDPA(30) (R(H) = 14.0 nm; W = 4.8 nm; R(c) = 9.2 nm) and PMPC(30)-b-PDPA(60) (R(H) = 27.1 nm; W = 11.0 nm; R(c) = 16.1 nm) micelles were 7 and 12% w/w(p), respectively. This preferential solubilization of DIP into micelles formed by copolymer chains having longer core-forming blocks (i.e., possessing larger core volumes) reflects the larger partition coefficient (K(V)) of DIP between the aqueous phase and PMPC(30)-b-PDPA(60) micelles (K(V) = 5.7 x 10(4)) compared to PMPC(30)-b-PDPA(30) micelles (K(V) = 1.1 x 10(4)). This enhanced ability of PMPC(30)-b-PDPA(60) aggregates to entrap/stabilize small hydrophobic molecules also produces slower release kinetics. Rapid release can be triggered by lowering the pH to induce micellar dissociation.     

Aqueous polymer two-phase systems formed by new thermoseparating polymers

A set of new polymers that can be used as phase forming components in aqueous two-phase systems is presented. All polymers studied have thermoseparating properties i.e. form one separate polymer enriched phase and one aqueous solution when heated above the critical temperature. This property makes the polymers attractive alternatives to the polymers used in traditional aqueous two-phase systems such as poly(ethylene glycol) (PEG) and dextran. The thermal phase separation simplifies recycling of the polymers, thus making the aqueous two-phase systems more cost efficient and suitable for use in large scale. Thermoseparating polymers studied have been copolymers of ethylene oxide and propylene oxide (EO-PO), poly (N-isopropylacrylamide) (poly-NIPAM), poly vinyl caprolactam (poly-VCL) and copolymers of N-isopropylacrylamide and vinyl caprolactam with vinyl imidazole (poly(NIPAM-VI) and poly(VCL-VI), respectively). In addition, the copolymer poly(NIPAM-VI) has the property to be uncharged at pH above 7.0 and positively charged at lower pH. This allows the partitioning of protein to be directed by changing the pH in the system instead of the traditional addition of salt to direct the partitioning. Hydrophobically modified EO-PO copolymer (HM-(EO-PO)) with alkyl groups (C₁₄) at both ends forms two-phase system with for example poly(NIPAM-VI). The phase diagram for poly(NIPAM-VI)/HM-(EO-PO) was determined and the model proteins lysozyme and BSA were partitioned in this system. For BSA in poly(NIPAM-VI)/HM-(EO-PO) system a change in pH from 8.0 to 5.4 results in a change of partition coefficient from K=0.8 to K=5.1, i.e. BSA could be transferred from the HM-(EO-PO) phase to the poly(NIPAM-VI) phase. BSA partitioning in poly(NIPAM-VI)/HM-(EO-PO) system allows quantitative BSA recovery, and recoveries of poly(NIPAM-VI) and HM-(EO-PO) were 53% and 92%, respectively, after the thermoseparation step.     

Antibacterial surfaces based on polymer brushes: investigation on the influence of brush properties on antimicrobial peptide immobilization and antimicrobial activity

Primary amine containing copolymer, poly(N,N-dimethylacrylamide-co-N-(3-aminopropyl)methacrylamide hydrochloride) (poly(DMA-co-APMA)), brushes were synthesized on Ti surface by surface-initiated atom transfer radical polymerization (SI-ATRP) in aqueous conditions. A series of poly(DMA-co-APMA) copolymer brushes on titanium (Ti) surface with different molecular weights, thicknesses, compositions, and graft densities were synthesized by changing the SI-ATRP reaction conditions. Cysteine-functionalized cationic antimicrobial peptide Tet213 (KRWWKWWRRC) was conjugated to the copolymers brushes using a maleimide-thiol addition reaction after initial modification of the grafted chains using 3-maleimidopropionic acid N-hydroxysuccinimide ester. The modified surfaces were characterized by X-ray photoelectron spectroscopy (XPS), water contact angle measurements, attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, atomic force microscopy (AFM), and ellipsometry analysis. The conjugation of the Tet213 onto brushes strongly depended on graft density of the brushes at different copolymer brush compositions. The peptide density (peptides/nm(2)) on the surface varied with the initial composition of the copolymer brushes. Higher graft density of the brushes generated high peptide density (pepetide/nm(2)) and lower number of peptides/polymer chain and vice versa. The peptide density and graft density of the chains on surface greatly influenced the antimicrobial activity of peptide grafted polymer brushes against Pseudomonas aeruginosa.     

Immobilization of DNA onto a polymer support and its potentiality as immunoadsorbent

Immobilization of DNA to the surface of poly(ethylene terephthalate) (PET) microfibers with a high specific surface area of 0.83 m(2)/g was carried out to give the fiber surface an affinity for anti-DNA antibody. Following ozone oxidation, the microfibers were subjected to graft polymerization of monomers including acrylic acid, methacryloyloxyethyl phosphate, N,N-dimethylaminoethyl methacrylate, N-vinylformamide, and glycidyl methacrylate. Calf thymus DNA was immobilized to the grafted fiber surface through either covalent binding or polyion complexation with the grafted polymer chains. The highest surface density of DNA immobilized (0.6 mug/cm(2)) was obtained when DNA was immobilized through formation of phosphodiester linkage between the hydroxyl group of DNA and the phosphate group in grafted poly(methacryloyloxyethyl phosphate) using 1,1-carbonyldiimidazole, or through polyion complexation between the anionic DNA and the cationic grafted poly(N,N-dimethylaminoethyl methacrylate) chains. Batch adsorption of anti-DNA antibody to the grafted PET fibers with and without DNA immobilized on their surface was conducted with serum obtained from systemic lupus erythematosus model mice. The DNA-immobilized PET fibers exhibited a higher adsorption capacity and specificity than the others. In addition, the DNA-immobilized fibers effectively adsorbed human anti-DNA antibody.     

Preparation of low molecular weight N-maleated chitosan-graft-PAMAM copolymer for enhanced DNA complexation

Low molecular weight N-maleated chitosan-graft-PAMAM (polyamidoamine) copolymer was prepared through N-maleated chitosan (NMC) by Michael type addition reaction to enhance its solubility in water as well as its cationic character for enhancement of DNA complexation. FTIR, (1)H NMR, XRD and GPC were used to characterize the graft copolymers. The copolymer showed better DNA complexation ability at low N/P ratio than that of chitosan due to increased surface charge density by the incorporation of PAMAM molecule on to chitosan backbone. The copolymer can effectively protect the DNA toward anionic surfactant. In vitro release study showed efficient DNA release occurred at physiological pH (pH 7.4). In vitro cell cytotoxicity test indicated toward less cytotoxicity of NMC-graft-PAMAM copolymers compared to that of 25kDa PEI. Thus, the synthesized NMC-graft-PAMAM copolymers have great potential of finding application in drug and gene delivery.     

Interaction of sugars, polysaccharides and cells with boronate-containing copolymers: from solution to polymer brushes

Interaction of mono- and disaccharides, polysaccharide particles and yeast cells with boronate-containing copolymers (BCC) of N-acryloyl-m-aminophenylboronic acid (NAAPBA) with N,N-dimethylacrylamide (DMAA) or N-isopropylacrylamide (NIPAM) was studied. The binding of saccharides to BCC of NIPAM resulted in a shift of its phase transition temperature (DeltaTP), which provided a quantitative measure for the complex formation. Among the sugars typical of non-reducing ends of glycoproteins the DeltaTP decreased in the order: N-acetylneuraminic acid > xylose approximately galactose > mannose approximately fucose > N-acetylglucosamine. Strong specific adsorption of the BCC on the cross-linked agarose gel Sepharose CL-6B (15-30 mg/ml gel at pH 9.2) was registered. The copolymers adsorption was due to boronate-sugar interactions and decreased with pH. Multivalent interaction of the BCC with the agarose gel has been proven by liquid column chromatography exhibiting a weak reversible adsorption of NAAPBA and almost irreversible adsorption of DMAA-NAAPBA copolymer from 0.1 M sodium phosphate buffer, pH 7.9. The two studied BCCs could be completely desorbed from the gel by 0.1 M fructose in aqueous buffered media with pH from 7.5 to 9.2. In turn, the agarose particles and yeast cells were found to adhere to siliceous supports end-grafted with boronate-BCC of N,N-dimethylacrylamide at pH > or = 7.5, due to the actions. Quantitative detachment of adhered particles or cells could be attained by addition of 20 mM or 100 mM fructose, respectively, in the pH range from 7.5 to 9.2. Affinity adhesion of micron-size carbohydrate particles to boronate-containing polymer brushes fixed on solid supports was considered as a model system suggesting a new approach to isolation and separation of living cells.     

Synthesis of poly (3-hydroxybutyrate-co-3-hydroxyoctanoate) by a Sinorhizobium fredii strain

AIMS: The potential of a Sinorhizobium fredii strain to produce a copolymer from glucose and sodium dodecanoate substrates was investigated. METHODS AND RESULTS: Using an orthogonal design in a flask-shaker culture system, the vital regulation conditions for copolymer synthesis were optimized. These optimal results were applied to further studies in a two-stage fed-batch fermentation with a 10-l fermentor. When the biomass approached 33.5 g l(-1) dry cells at 35 h, 7 mmol l(-1) sodium dodecanoate was added into the broth to trigger the copolymer synthesis. After further culturing for 3 h, the copolymer product could be 17.14 g l(-1). The molecular structure of the copolymer was determined to be a poly (3-hydroxybutyrate-co-3-hydroxyoctanoate) [P (HB-HO)] by nuclear magnetic resonance. The content of HB and HO in P (HB-HO) was 79.2% (w/w) and 20.8% (w/w) respectively. The molecular weight of the P (HB-HO) was measured as 1.85 x 10(5) Da by a viscosity method. CONCLUSION: The results demonstrated that the S. fredii strain used could be a potential candidate for the industrial production of the copolymer. SIGNIFICANCE AND IMPACT OF THE STUDY: Some basic fermentation parameters were acquired through the fed-batch culturing experiments and they should be applicable in developing large-scale fermentation technologies for producing the P (HB-HO) copolymers.     

Promotion of acid-induced membrane fusion by basic peptides. Amino acid and phospholipid specificities

The ability of oligo- and polymers of the basic amino acids L-lysine, L-arginine, L-histidine and L-ornithine to induce lipid intermixing and membrane fusion among vesicles containing various anionic phospholipids has been investigated. Among vesicle consisting of either phosphatidylinositol or mixtures of phosphatidic acid and phosphatidylethanolamine rapid and extensive lipid intermixing, but not complete fusion, was induced at neutral pH by poly-L-ornithine or L-lysine peptides of five or more residues. When phosphatidylcholine was included in the vesicles, the lipid intermixing was severely inhibited. Such lipid intermixing was also much less pronounced among phosphatidylserine vesicles. Poly-L-arginine provoked considerable leakage from the various anionic vesicles and caused significantly less lipid intermixing than L-lysine peptides at neutral pH. When the addition of basic amino acid polymer was followed by acidification to pH 5-6, vesicle fusion was induced. Fusion was more pronounced among vesicles containing phosphatidylserine or phosphatidic acid than among those containing phosphatidylinositol, and occurred also with vesicles whose composition resembles that of cellular membranes (i.e., phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine, 50:30:20, by mol). Liposomes with this composition are resistant to fusion by Ca2+ or by acidification after lectin-mediated contact. The tight interaction among vesicles at neutral pH, resulting in lipid intermixing, does not seem to be necessary for the fusion occurring after acidification, but the basic peptides nevertheless appear to play a more active role in the fusion process than simply bringing the vesicles in contact. However, protonation of the polymer side chains and transformation of the polymer into a polycation does not explain the need for acidification, since the pH-dependence was quite similar for poly(L-histidine)- and poly(L-lysine)-mediated fusion.     

Synthesis and in vitro evaluation of novel star-shaped block copolymers (blocked star vectors) for efficient gene delivery

Novel 4-branched diblock copolymers consisting of cationic chains as an inner domain and nonionic chains as an outer domain were prepared by iniferter-based living radial polymerization and evaluated as a polymeric transfectant. The cationic polymerization of 3-(N,N-dimethylamino)propyl acrylamide (DMAPAAm) using 1,2,4,5-tetrakis( N,N-diethyldithiocarbamylmethyl)benzene as a 4-functional iniferter followed by the nonionic block polymerization of N,N-dimethylacrylamide (DMAAm) afforded 4-branched diblock copolymers with controlled compositions. By changing the solution or irradiation conditions, 4-branched PDMAPAAms with molecular weights of 10,000, 20,000, and 50,000 were synthesized. In addition, by graft polymerization, PDMAPAAm-PDMAAm blocked copolymers with copolymer composition (unit ratio of DMAAm/DMAPAAm) ranging from 0.18 to 1.0 for each cationic polymer were synthesized. All polymers were shown to interact with and condense plasmid DNA to yield polymer/DNA complexes (polyplexes). A transfection study on COS-1 cells showed that the polyplexes from block copolymers with cationic chain length of approximately 50,000 and a nonionic chain length of 30,000, which were approximately 200 nm in diameter and very stable in aqueous media, had the most efficient luciferase activity with minimal cellular cytotoxicity under a charge ratio of 20 (vector/pDNA). The PDMAPAAm-PDMAAm-blocked, star-shaped polymers are an attractive novel class of nonviral gene delivery systems.     

Interaction of poly(L-lysine)-g-poly(ethylene glycol) with supported phospholipid bilayers

Interactions between the graft copolymer poly(L-lysine)-g-poly(ethylene glycol), PLL-g-PEG, and two kinds of surface-supported lipidic systems (supported phospholipid bilayers and supported vesicular layers) were investigated by a combination of microscopic and spectroscopic techniques. It was found that the application of the copolymer to zwitterionic or negatively charged supported bilayers in a buffer of low ionic strength led to their decomposition, with the resulting formation of free copolymer-lipid complexes. The same copolymer had no destructive effect on a supported vesicular layer made up of vesicles of identical composition. A comparison between poly(L-lysine), which did not induce decomposition of supported bilayers, and PLL-g-PEG copolymers with various amounts of PEG side chains per backbone lysine unit, suggested that steric repulsion between the PEG chains that developed upon adsorption of the polymer to the nearly planar surface of a supported phospholipid bilayer (SPB) was one of the factors responsible for the destruction of the SPBs by the copolymer. Other factors included the ionic strength of the buffer used and the quality of the bilayers, pointing toward the important role defects present in the SPBs play in the decomposition process.     

Protein partitioning in aqueous two-phase systems composed of a pH-responsive copolymer and poly(ethylene glycol)

The effect of pH and salt concentration on the partitioning behavior of bovine serum albumin (BSA) and cytochrome c in an aqueous two-phase polymer system containing a novel pH-responsive copolymer that mimics the structure of proteins and poly(ethylene glycol) (PEG) was investigated. The two-phase system has low viscosity. Depending on pH and salt concentration, the cytochrome c was found to preferentially partition into the pH-responsive copolymer-rich (bottom) phase under all conditions of pH and salt concentrations considered in the study. This was caused by the attraction between the positively charged protein and negatively charged copolymer. BSA partitioning showed a more complex behavior and partitioned either to the PEG phase or copolymer phase depending on the pH and ionic strength. Extremely high partitioning levels (partition coefficient of 0.004) and very high separation ratios of the two proteins (up to 48) were recorded in the new systems. This was attributed to strong electrostatic interactions between the proteins and the charged copolymer.     

Hydrolysis of poly(alkylene amidophosphate)s containing amino acid or peptide residues in the side groups. Kinetics and selectivity of hydrolysis

Kinetics of hydrolysis of poly(alkylene amidophosphate)s with amino acids or dipeptides as the side groups was studied by 31P NMR at pH 1.5, 6.5, and 8.5. The direction of hydrolysis and the relative rate coefficients of breaking P-O bonds in the main chain and P-N bonds in the side groups depend strongly on the pH of the medium of hydrolysis. The P-N (amide) bond hydrolyzes much faster than the P-O (ester) bond in acidic and close to neutral conditions (negligible P-O hydrolysis), whereas above pH > or = 8.5 these differences are much smaller. For instance, for 4-Ala the rate coefficients of hydrolysis are equal (in H2O at 37 degrees C and pH 8.5) to 1.9 x 10(-8) s(-1) and 1.0 x 10(-9) s(-1) for the P-N and P-O bonds, respectively, quite different from the values found for the low molecular model 2 (0 and 1.4 x 10(-7) s(-1), respectively).     

Inhibition of cathepsin K with lysosomotropic macromolecular inhibitors

Cathepsin K is the major enzyme responsible for the degradation of the protein matrix of bone and probably for the destruction of articular cartilage in rheumatoid arthritis joints. These processes occur mainly in the resorption lacuna and within the lysosomal compartment. Here, we have designed, synthesized, and evaluated new lysosomotropic (water-soluble) polymer-cathepsin K inhibitor conjugates. In particular, we characterized the relationship between conjugate structures and their activity to inhibit cathepsins K, B, L, and papain. A potent selective cathepsin K inhibitor, 1,5-bis(N-benzyloxycarbonylleucyl)carbohydrazide, was modified to 1-(N-benzyloxycarbonylleucyl)-5-(phenylalanylleucyl)carbohydrazide (I) to facilitate polymer conjugation. It was conjugated to the polymer chain termini of two water-soluble polymers [alpha-methoxy poly(ethylene glycol), abbreviated as mPEG-I; semitelechelic poly[N-(2-hydroxypropyl)methacrylamide], abbreviated as ST-PHPMA-I]. The conjugation of inhibitor I to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer side chains was accomplished via either a Gly-Gly spacer (PHPMA-GG-I) or with no spacer between I and the copolymer backbone (PHPMA-I). Kinetic analysis revealed that free inhibitor I possessed an apparent second-order rate constant against cathepsin K (k(obs)/[I] = 1.3 x 10(6) M(-1) s(-1)) similar to that of unmodified 1,5-bis(Cbz-Leu) carbohydrazide, while I conjugated to the chain termini of mPEG and ST-PHPMA-COOH had slightly lower values (about 5 x 10(5) M(-1) s(-1)). The k(obs)/[I] values for I attached to the side chains of HPMA copolymers (PHPMA-GG-I and PHPMA-I) were about 3 x 10(4) M(-1) s(-1). When tested against cathepsin L, inhibitor I and all its polymer conjugates produced k(obs)/[I] values 1-2 orders of magnitude less than those determined for cathepsin K, while for cathepsin B and papain, the values were 2-4 orders of magnitude lower. The ability of mPEG-I and ST-PHPMA-I to inhibit cathepsin K activity in synovial fibroblasts was also evaluated. Both polymer-bound inhibitors were internalized by endocytosis and were ultimately trafficked to the lysosomal compartment. ST-PHPMA-I was internalized faster than mPEG-I. The inhibitory activity in the synovial fibroblast assay correlated with the rate of internalization.     

In-situ injectable physically and chemically gelling NIPAAm-based copolymer system for embolization

The goal of this work is to make an injectable physically and chemically cross-linking NIPAAm-based copolymer system for endovascular embolization. A copolymer with N-isopropylacrylamide (NIPAAm) and hydroxyethyl methacrylate (HEMA) was synthesized and converted to poly(NIPAAm-co-HEMA-acrylate) functionalized with olefins. When poly(NIPAAm-co-HEMA-acrylate) was mixed with pentaerythritol tetrakis 3-mercaptopropionate (QT) stoichiometrically in a 0.1 N PBS solution of pH 7.4, it formed a temperature-sensitive hydrogel with low swelling through the Michael-type addition reaction and showed improved elastic properties at low frequency compared to physical gelation. This material could be useful for applications requiring water-soluble injection but lower swelling and lower creep properties than available with other soluble in-situ-gelling materials.     

Temperature sensitization of liposomes by use of N-isopropylacrylamide copolymers with varying transition endotherms

Three kinds of copolymers of N-isopropylacrylamide (NIPAM) with the same conformational transition temperature and varying transition endotherms were synthesized with N-acryloylpyrrolidine (APr), N,N-dimethylacrylamide (DMAM), and N-isopropylmethacrylamide (NIPMAM) as the comonomers. Two dodecyl groups were incorporated into the termini of these copolymers as an anchor for the fixation to a liposomal membrane. Egg yolk phosphatidylcholine liposomes having these copolymers were prepared and their temperature-sensitive contents release and association properties were investigated. While these copolymer exhibited a conformational transition at ca. 40 degrees C, DeltaH for the transition increased in the order of poly(APr-co-NIPAM) < poly(DMAM-co-NIPAM) < poly(NIPMAM-co-NIPAM). The liposomes containing poly(NIPMAM-co-NIPAM) showed a drastic release enhancement of entrapped calcein above the transition temperature, whereas the liposomes with poly(DMAM-co-NIPAM) and those with poly(APr-co-NIPAM) exhibited moderate and slight enhancement of calcein release above that temperature, respectively. On the contrary, the liposomes containing poly(APr-co-NIPAM) showed significant aggregation above the transition temperature, but the aggregation was hardly observed for the liposomes having poly(NIPMAM-co-NIPAM), indicating that poly(APr-co-NIPAM) more efficiently made the liposome surface hydrophobic. Thus, we concluded that the copolymer with a large DeltaH is suitable for obtaining functional liposomes with a temperature-sensitive contents release property, whereas the copolymer with a small DeltaH is appropriate for preparing functional liposomes with a temperature-sensitive surface property.     

Solid phase extraction of BSA

Silica with immobilized polyoxyethyle-neisooctylphenol (SiO2-TX) was investigated as an adsorbent for solid phase extraction of bovine serum albumin (BSA). It was shown that efficient BSA extraction (up to 96%) takes place on SiO2-TX from water solution in the form of its ionic associates with cationic (at pH = 8) and anionic (at pH = 1.5) surfactants.