Polymeric Systems with Antithrombin Activity for Thermally Activated Targeting

Copolymers of N,N-diethylacrylamide and N-acryloylphthalimide with a lower critical solution temperature (LCST) were synthesized by radical copolymerization. Polymeric systems with antithrombin activity and an LCST were prepared via a reaction of amino groups of hirudin with phthalimide groups of the copolymers. On increasing hirudin content, the LCST of the polymeric systems increased. The antithrombin activity of polymeric systems obtained by hirudin immobilization on copolymer carriers was inversely related to the content of the copolymer, amounting to 6% of the activity of native hirudin.     

Polymeric systems with antithrombin activity for thermally activated targeting

Copolymers of N,N-diethylacrylamide and N-acryloylphthalimide with lower critical solution temperature (LCST) were synthesized by radical copolymerization. Polymeric systems with antithrombin activity and LCST were prepared via a reaction of amino groups of hirudin with phthalimide groups of the copolymers. On increasing hirudin content, LCST of the polymeric systems increased. The antithrombin activity of polymeric systems obtained by hirudin immobilization on copolymer carriers was inversely related to the content of the copolymer, amounting to 6% of the activity of native hirudin.     

"Schizophrenic" Hemocompatible Copolymers via Switchable Thermoresponsive Transition of Nonionic/Zwitterionic Block Self-Assembly in Human Blood

"Schizophrenic" diblock copolymers containing nonionic and zwitterionic blocks were prepared with well-controlled molecular weights via atom-transfer radical polymerization (ATRP). In this work, we report a systematic study of how morphological changes of poly(N-isopropylacrylamide)-block-poly(sulfobetaine methacrylate) (PNIPAAm-b-PSBMA) copolymers affect hemocompatibility in human blood solution. The "schizophrenic" behavior of PNIPAAm-b-PSBMA was observed by (1)H NMR, dynamic light scattering (DLS), and turbidity measurement with double morphological transition, exhibiting both lower critical solution temperature (LCST) and upper critical solution temperature (UCST) in aqueous solution. Below the UCST of PSBMA block, micelles were obtained with a core of insoluble PSBMA association and a shell of soluble PNIPAAm, whereas the opposite micelle structure was observed above the LCST of PNIPAAm block. In between the UCST and LCST, unimers with both soluble blocks were detected. Hydrodynamic size of prepared polymers and copolymers is determined to illustrate the correlations between intermolecular nonionic/zwitterionic associations and blood compatibility of PNIPAAm, PNIPAAm-b-PSBMA, and PSBMA suspension in human blood. Human fibrinogen adsorption onto the PNIPAAm-b-PSBMA copolymers from single-protein solutions was measured by DLS to determine the nonfouling stability of copolymer suspension. The new nonfouling nature of PNIPAAm-b-PSBMA copolymers was demonstrated to show extremely high anticoagulant activity and antihemolytic activity in human blood over a wide range of explored temperatures from 4 to 40 °C. The temperature-independent blood compatibility of nonionic/zwitterionic block copolymer along with their schizophrenic phase behavior in aqueous solution suggests their potential in blood-contacting applications.     

Preparation and properties of immobilized pectinase onto the amphiphilic PS-b-PAA diblock copolymers

Well-defined amphiphilic block copolymers poly(styrene-b-acrylic acid) (PS-b-PAA) with controlled block length were synthesized using atom transfer radical polymerization (ATRP). Pectinase enzyme was immobilized on the well-defined amphiphilic block copolymers PS-b-PAA. The carboxyl groups on the amphiphilic PS-b-PAA diblock copolymers present a very simple, mild, and time-saving process for enzyme immobilization. Various characteristics of immobilized pectinase such as the pH and temperature stability, thermal stability, and storage stability were valuated. Among them the pH optimum and temperature optimum of free and immobilized pectinase were found to be pH 6.0 and 65 degrees C.     

Temperature-controlled properties of DNA complexes with poly(ethylenimine)-graft-poly(N-isopropylacrylamide)

End-functionalized poly(N-isopropylacrylamide) (PNIPA) was synthesized by living free radical polymerization and conventional free radical polymerization and was used to prepare graft copolymers with poly(ethylenimine) (PEI). The copolymers exhibited lower critical solution temperature (LCST) behavior between 30 and 32 degrees C and formed complexes with plasmid DNA. The LCST of the copolymers in the DNA complexes increased slightly to approximately 34-35 degrees C. Cytotoxicity of the copolymers was evaluated by measuring lactate dehydrogenase (LDH) release from cells. The copolymers exhibited temperature-dependent toxicity, with higher levels of LDH release observed at temperatures above the LCST. Cellular uptake and transfection activity of the DNA complexes with the PEI-g-PNIPA copolymers were lower than those of the control PEI/DNA complexes at temperature below the LCST but increased to the PEI/DNA levels at temperatures above the LCST.     

Immobilization of Escherichia coli cells with penicillin-amidohydrolase activity on solid polymeric carriers

Whole cells of Escherichia coli containing the enzyme penicillinamidohydrolase EC 3.5.1.11 were immobilized on the surface of modified macroporous copolymers of glycidylmethacrylate with ethylenedimethacrylate and of copolymers of methacrylaldehyde (MA) with divinylbenzene (DVB) by means of glutaraldehyde. These polymeric carriers were modified before cell binding by using ammonia or polyamines, especially ethylenediamine and hexamethylenediamine (HMDA). The highest specific activity and the largest yield in cell immobilization were achieved with the macroporous copolymer of MA and DVB modified with HMDA. The material thus obtained was used in repeated conversions of benzylpenicillin to 6-aminopenicillanic acid in a stirred batch reactor.     

Stabilization of enzyme immobilized in temperature-sensitive hydrogels

-Galactosidase was immobilized in a crosslinked poly(N-isopropylacrylamide-co-acrylamide) hydrogel which exhibits an LCST(lower critical solution temperature) behavior. The hydrogel collapses above the LCST, and expands below the LCST. The temperature-dependent phase transition was around 37 °C. The stability of immobilized enzyme was investigated at different temperatures which allow different degrees of collapse in the hydrogel matrix. It was hypothesized that the immobilzed enzyme is more stable in the collapsed matrix due to the physical restraint imposed on the enzyme entrapped.     

Immobilization of Arthrobacter simplex in a thermally reversible hydrogel: effect of temperature cycling on steroid conversion

Arthrobacter simplex cells, which convert the steroid hydrocortisone to prednisolone, have been entrapped in a thermally reversible hydrogel. Such hydrogels exhibit a lower critical solution temperature (LCST) wherein the gel shrinks and deswells when it is warmed through its LCST, and then reversibly expands and reswells when it is cooled below the LCST. The immobilized cell-hydrogel system has been thermally cycled between two temperatures, each below the LCST. The upper temperature was selected to be just below the LCST, where the gel deswells but does not collapse, as it does at the LCST. The thermal cycling acts like a "hydraulic pump" which enhances mass transfer of the substrate (hydrocortisone) in and the product (prednisolone) out of the gel, thereby increasing steroid conversion dramatically relative to isothermal operation at either the upper or lower temperature. The increased conversion can also be due in part to reduced product inhibition. Mass transfer resistance and product inhibition are among the most serious problems in immobilized biocatalyst technology and thermal cycling of LCST hydrogels is both a novel and useful approach to minimizing these problems.     

Synthesis and characterization of triblock copolymers of methoxy poly(ethylene glycol) and poly(propylene fumarate)

Amphiphilic block copolymers were synthesized by transesterification of hydrophilic methoxy poly(ethylene glycol) (mPEG) and hydrophobic poly(propylene fumarate) (PPF) and characterized. Four block copolymers were synthesized with a 2:1 mPEG:PPF molar ratio and mPEGs of molecular weights 570, 800, 1960, and 5190 and PPF of molecular weight 1570 as determined by NMR. The copolymers synthesized with mPEG of molecular weights 570 and 800 had 1.9 and 1.8 mPEG blocks per copolymer, respectively, as measured by NMR, representing an ABA-type block copolymer. The number of mPEG blocks of the copolymer decreased with increasing mPEG block length to as low as 1.5 mPEG blocks for copolymer synthesized with mPEG of molecular weight 5190. At a concentration range of 5-25 wt % in phosphate-buffered saline, copolymers synthesized with mPEG molecular weights of 570 and 800 possessed lower critical solution temperatures (LCST) between 40 and 45 degrees C and between 55 and 60 degrees C, respectively. Aqueous solutions of copolymer synthesized with mPEG 570 and 800 also experienced thermoreversible gelation. The sol-gel transition temperature was dependent on the sodium chloride concentration as well as the mPEG block length. The copolymer synthesized from mPEG 570 had a transition temperature between 40 and 20 degrees C with salt concentrations between 1 and 10 wt %, while the sol-gel transition temperatures of the copolymer synthesized from mPEG molecular weight 800 were higher in the range 75-30 degrees C with salt concentrations between 1 and 15 wt %. These novel thermoreversible copolymers are the first biodegradable copolymers with unsaturated double bonds along their macromolecular chain that can undergo both physical and chemical gelation and hold great promise for drug delivery and tissue engineering applications.     

Regulation of growth and adhesion of cultured cells by insulin conjugated with thermoresponsive polymers

We developed a new biomaterial for use in cell culture. The biomaterial enabled protein-free cell culture and the recovery of viable cells by lowering the temperature without the aid of supplements. Insulin was immobilized and a thermoresponsive polymer was grafted onto a substrate. We investigated the effect of insulin coupling on the lower critical solution temperature (LCST) of the thermoresponsive polymer, poly(N-isopropylacrylamide-co-acrylic acid), using polymers that were ungrafted, or coupled with insulin. The insulin conjugates were precipitated from an aqueous solution at high temperatures, but they were soluble at low temperatures. The LCST was not significantly affected by the insulin coupling. The thermoresponsive polymer was grafted to glow-discharged polystyrene film and covalently conjugated with insulin. The surface wettability of the conjugate film was high at low temperatures and low at high temperatures. The amounts of immobilized insulin required to stimulate cell growth were 1-10% of the amount of free insulin required to produce the same effect. The maximal mitogenic effect of immobilized insulin was greater than that of free insulin. About half of the viable cells was detached from the film only by lowering the temperature. The recovered cells proliferated normally on new culture dishes. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 339-344, 1997.     

Controlling the aggregation of conjugates of streptavidin with smart block copolymers prepared via the RAFT copolymerization technique

Block copolymers containing stimuli-responsive segments provide important new opportunities for controlling the activity and aggregation properties of protein-polymer conjugates. We have prepared a RAFT block copolymer of a biotin-terminated poly(N-isopropylacrylamide) (PNIPAAm)-b-poly(acrylic acid) (PAA). The number-average molecular weight (M(n)) of the (PNIPAAm)-b-(PAA) copolymer was determined to be 17.4 kDa (M(w)/M(n) = 1.09). The PNIPAAm block had an M(n) of 9.5 kDa and the poly(acrylic acid) (PAA) block had an M(n) of 7.9 kDa. We conjugated this block copolymer to streptavidin (SA) via the terminal biotin on the PNIPAAm block. We found that the usual aggregation and phase separation of PNIPAAm-SA conjugates that follow the thermally induced collapse and dehydration of PNIPAAm (the lower critical solution temperature (LCST) of PNIPAAm is 32 degrees C in water) is prevented through the shielding action of the PAA block. In addition, we show that the cloud point and aggregation properties (as measured by loss in light transmission) of the [(PNIPAAm)-b-(PAA)]-SA conjugate also depended on pH. At pH 7.0 and at temperatures above the LCST, the block copolymer alone was found to form particles of ca. 60 nm in diameter, while the bioconjugate exhibited very little aggregation. At pH 5.5 and 20 degrees C, the copolymer alone was found to form large aggregates (ca. 218 nm), presumably driven by hydrogen bonding between the -COOH groups of PAA with other -COOH groups and also with the -CONH- groups of PNIPAAm. In comparison, the conjugate formed much smaller particles (ca. 27 nm) at these conditions. At pH 4.0, however, large particles were formed from the conjugate both above and below the LCST (ca. 700 and 540 nm, respectively). These results demonstrate that the aggregation properties of the block copolymer-SA conjugate are very different from those of the free block copolymer, and that the outer-oriented hydrophilic block of PAA shields the intermolecular aggregation of the block copolymer-SA bioconjugate at pH values where the -COOH groups of PAA are significantly ionized.     

Thermo-sensitive amphiphilic block copolymer poly (styrene-<Emphasis Type="Italic">b</Emphasis>-N-isopropylacrylamide) with switchable catalytic activity immobilizing pectinase

Pectinase was immobilized onto thermo-sensitive amphiphilic block copolymers poly(styrene-b-Nisopropylacrylamide) PS-b-poly(N-isopropylacrylamide) (PNIPAM) by covalent attachment. Biochemical studies have found that the stability of the PS-b-PNIPAM support is not impeded by the bound proteins despite that up to 242.5 mg of enzyme is immobilized per gram of carrier particles. The immobilized enzyme retained nearly 65% of its initial activity over 30 days, and the optimum temperature and pH also increased to the range of 60 ∼ 70°C and 4.0 ∼ 6.0, respectively. The immobilized enzyme also exhibited great operational stability, and more than 60% residual activity was observed in the immobilized enzyme after 10 batch reactions. Moreover, the lower critical solution temperature of the PS-b-PNIPAM support could be switched on or off by a small change in solution temperature. Thus, the immobilized pectinase could be recovered and showed durable activity during the recycle process.     

Immobilization of adenosine deaminase onto agarose and casein

In the present study adenosine deaminase (ADA) was immobilized onto two different polymeric materials, agarose and casein. The factors affecting the amount of enzyme attachment onto the polymeric supports such as incubation time were investigated. The maximum amount of enzyme immobilized onto different polymeric supports occurred at incubation pH value 7.5 and ADA concentration 42 units/g and the incubation time needed for the maximum amount of enzyme attachment to the polymeric supports was found to be 8 h. Some phsicochemical properties of the free and immobilized ADA such as operational stability, optimum temperature and thermal stability, pH optimum and stability, storage stability, and the effect of gamma-radiation were studied. The operational stability of the free and immobilized enzyme showed that the enzyme immobilized by a cross-linking technique using gultaric dialdehyde showed poor durability and the relative activity decreased sharply due to the leakage after repeated washing, while the enzymes immobilized by covalent bonds to the carriers showed a slight decrease in most cases in the relative activity (around 20%) after being used 10 times. Storage for 4-6 months, showed that the free enzyme lost its activity, while the immobilized enzyme showed the opposite behavior. Subjecting the immobilized enzyme to a dose of gamma radiation of 0.5-10 Mrad showed complete loss in the activity of the free enzyme at a dose of 5 Mrad, while the immobilized enzymes showed relatively high resistance to gamma radiation up to a dose of 5 Mrad.     

Surface-active and stimuli-responsive polymer--Si(100) hybrids from surface-initiated atom transfer radical polymerization for control of cell adhesion

A simple two-step method was developed for the covalent immobilization of atom-transfer radical polymerization (ATRP) initiators on the hydrogen-terminated Si(100) (Si-H) surface. Well-defined functional polymer-Si hybrids, consisting of covalently tethered brushes of poly(ethylene glycol) monomethacrylate (PEGMA) polymer, N-isopropylacrylamide (NIPAAm) polymer, and NIPAAm-PEGMA copolymers and block copolymers on Si-H surfaces, were prepared via surface-initiated ATRP. Kinetics study revealed that the chain growth from the silicon surface was consistent with a "controlled" process. Surface cultures of the cell line 3T3-Swiss albino on the hybrids were evaluated. The PEGMA graft-polymerized silicon [Si-g-P(PEGMA)] surface is very effective in preventing cell attachment and growth. At 37 degrees C [above the lower critical solution temperature (LCST, approximately 32 degrees C) of NIPAAm], the seeded cells adhered, spread, and proliferated on the NIPAAm graft polymerized silicon [Si-g-P(NIPAAm)] surface. Below the LCST, the cells detached from the Si-g-P(NIPAAm) surface spontaneously. Incorporation of PEGMA units into the NIPAAm chains of the Si-g-P(NIPAAm) surface via copolymerization resulted in more rapid cell detachment during the temperature transition. The "active" chain ends on the Si-g-P(PEGMA) and Si-g-P(NIPAAm) hybrids were also used as the macroinitiators for the synthesis of diblock copolymer brushes. Thus, not only are the hybrids potentially useful as stimuli-responsive adhesion modifiers for cells in silicon-based biomedical microdevices but also the active chain ends on the hybrid surfaces offer opportunities for further surface functionalization and molecular design.     

Engineering soft nanostructured functional materials via orthogonal chemistry

Nanostructured amphiphilic block copolymers, graft copolymers, polymeric thermally responsive molecular brushes and polymer stars are only few examples of macromolecular architectures accessible either via controlled/living radical polymerization (CLRP) techniques or the combination of CLRP mechanisms with efficient post-polymerization routes including click chemistry. Precise control over the composition, molecular weight and functionalities is a prerequisite for soft polymeric materials to self-organize into ordered morphologies. This contribution describes novel orthogonal chemical routes for the synthesis of macromolecular architectures and self-assembly of functional soft polymeric materials. Emerging potential applications of well-defined block and graft copolymers are outlined as well.     

De novo synthesis and cellular uptake of organic nanocapsules with tunable surface chemistry

Water-soluble organic nanocapsules were prepared from bottlebrush copolymers with triblock terpolymer side chains composed of a degradable inner block (polylactide), a cross-linkable middle block (poly(4-butenylstyrene)), and a functional outer block (poly(styrene-co-maleic anhydride)). Bottlebrush copolymers are macromolecules with a long linear backbone and shorter polymeric side chains densely grafted onto the backbone. Hollow cylindrical nanoparticles were prepared by peripheral cross-linking of the bottlebrush copolymers and subsequent selective removal of the core. Reactive anhydride groups of the outer functional layer allowed for the preparation of nanocapsules with tunable surface characteristics. Cellular uptake of negatively charged organic nanocapsules showed strong surface chemistry dependence. The presence of hydrophobic groups on the nanocapsule surface was necessary for their nonspecific association with the cell membrane and subsequent internalization by endocytosis. The length of surface grafted oligoethylene glycol chains also had a dramatic influence on the intracellular accumulation of nanocapsules. Macropinocytosis was shown to be the predominant pathway for the cellular uptake of organic nanocapsules.     

Galactosylated N-2-hydroxypropyl methacrylamide-b-N-3-guanidinopropyl methacrylamide block copolymers as hepatocyte-targeting gene carriers

As alternatives of viral and cationic lipid gene carriers, cationic polymer-based vectors may provide flexible chemistry for the attachment of targeting moieties. In this report, galactosylated N-2-hydroxypropyl methacrylamide-b-N-3-guanidinopropyl methacrylamide block copolymers (galactosylated HPMA-b-GPMA block copolymers, or abbreviated as GHG) were prepared in order to develop hepatocyte targeting gene transfection carriers. The block copolymers were synthesized by aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization of N-2-hydroxypropyl methacrylamide (HPMA) and N-3-aminopropyl methacrylamide (APMA), followed by galactosylation and guanidinylation. The molecular weight of GHG copolymers determined by static light scattering method was in the range from 48?600 to 76?240 g/mol. In addition, the galactose content in the GPMA block in the copolymers was determined to be 6.5-8.0 mol % according to the sulfuric acid method. The GHG copolymers complexed completely with plasmid DNA (pDNA) to show positive zeta-potential values with diameter 100-250 nm from charge ratio of 4, which demonstrated the excellent DNA condensing ability of guanidino groups. Furthermore, the MTT assay data of GHG/pDNA complexes on HepG2 cells and HeLa cells indicated that GHG copolymers had significantly lower cytotoxicity than PEI. In addition, the copolymers with GPMA component from 30.23% showed higher transfection efficiency than PEI at charge ratio of 12 in HepG2 cells. The result revealed that the conjugation of galactose groups in the copolymers brought asialoglycoprotein-receptor (ASGP-R) mediated transfection. The employing of HPMA component decreased the aggregation of protein in transfection presence of serum. The GHG copolymers combined the advantages of galactose moieties, guanidino groups, and HPMA component might show potential in safe hepatocyte targeting gene therapy.     

Preparation and characterization of UV-curable polymeric support for covalent immobilization of xylanase enzyme

The hydroxyl group of poly(ethylene glycol) monoacrylate (PEGMA) was activated by 1,1′-carbonyldiimidazole (CDI) and then a xylanase enzyme was immobilized to amine active PEGMA. UV-curable polymeric support formulation was prepared by mixing the xylanase bonded PEGMA, aliphatic polyester, 2-hydroxyethyl methacrylate (HEMA), poly(ethylene glycol) diacrylate (PEGDA) and photoinitiator. After UV irradiation, the enzymatic activity of the polymeric matrix was evaluated and compared with the corresponding free enzyme. By immobilization, the temperature resistance of the enzyme was improved and showed maximum activity at 60 °C. pH dependent activities of the free and immobilized enzymes were also investigated, and it was found that the pH of maximum activity for the free enzyme was 6.0, while for the optimal pH of the immobilized enzyme was 6.5. The immobilized enzyme retained 75% of its activity after 33 runs. The morphology of the polymeric support was characterized by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) coupled with SEM was used to explore the chemical composition. The results have confirmed the evidence of enzyme in the structure of the polymeric material.     

Modulate the phase transition temperature of hydrogels with both thermosensitivity and biodegradability

The synthesis and characterization of thermoresponsive hydrogels on the basis of N-isoproplyarylamide (NIPAAm) and acrylamide (AAm) copolymers crosslinked with a novel biodegradable crosslinker (PEG-co-PLA) were carried out in this study. Swelling measurement results demonstrated that four gels of PNAM5, PNAM10, PNAM12 and PNAM15 are thermoresponsive. The equilibrium swelling ratio and degradation of the hydrogels strongly depend on hydrogels composition. The morphology of the hydrogels was observed by scanning electron microscopy (SEM), and their thermal property was characterized by differential scanning calorimetry (DSC). The results show that the proportion of AAm in the copolymer has notable effect on the low critical solution temperature (LCST) of the hydrogel. When the molar ratio of AAm to NIPAAm was increased from 1:10 to 3:10 the LCST of the copolymer increased from 39.7 to 64.2 °C. The compression modulus of PNAM15 is of the highest among other hydrogels, because PNAM15 hydrogel has a more compact structure.     

金属螯合载体定向固定化木瓜蛋白酶的研究

以磁性金属螯合琼脂糖微球为载体,利用金属螯合配体（IDACu²⁺）与蛋白质表面供电子氨基酸相互作用的原理,定向固定了木瓜蛋白酶。固定化最适条件为Cu²⁺1.5×10^-2mol/g载体、固定化时间4h、固定化pH7.0、给酶量30mg/g载体。固定化酶的最适反应温度70℃、最适反应pH8.0,固定化酶的热稳定性明显高于溶液酶,固定化酶活力回收为68.4％,且有较好的操作稳定性,载体重复使用5次后固定化酶酶活为首次固定化酶79.71%。