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.     

Prediction of lower critical solution temperature of N-isopropylacrylamide–acrylic acid copolymer by an artificial neural network model

In this paper, we have investigated the lower critical solution temperature (LCST) of N-isopropylacrylamide–acrylic acid (NIPAAm-AAc) copolymer as a function of chain-transfer agent/initiator mole ratio, acrylic acid content of copolymer, concentration, pH and ionic strength of aqueous copolymer solution. Aqueous solutions with the desired properties were prepared from previously purified polymers, synthesized at 65 °C by solution polymerization using ethanol. The effects of each parameter on the LCST were examined experimentally.In addition, an artificial neural network model that is able to predict the lower cretical solution temperature was develeped. The predictions from this model compare well against both training and test data sets with an average error less than 2.53%.Figure Cross plot of predicted and experimental LCST values for the testing data set.     

Chemical modification of proteinsby “smart” polymers

The modification of duck ovomucoid, a proteinaceous proteinase inhibitor from egg white, by poly-N,N-diethylacrylamide possessing a low critical solution temperature (LCST) has been investigated. The free amino groups of the lysine residues and the N-terminal residue of the ovomucoid molecule were modified; as a result, the inhibitor activity towards trypsin decreased significantly and that towards chymotrypsin decreased slightly. The transformation of ovomucoid antitryptic centers into antichymotryptic centers was observed upon the heating of the solutions of the modified protein above the LCST. The hydrophobization of the lysine residues situated in the reactive centers of the inhibitor was shown to cause this phenomenon. The structure of the binding loop was not distorted and the modified lysine residues could be recognized by chymotrypsin molecules, similarly to the hydrophobic amino acid residues of the antichymotryptic center.     

Chiral chemical absorption property of a cross-linked poly(N-isopropyl acrylamide-co-sodium acrylate) thermoresponsive smart gel

This investigation leads to the chiral chemical absorption property of a thermoresponsive gel material. d ‐(+)‐α‐phenyl ethyl amine was taken as the chiral chemical. The gel material was synthesized by polymerizing 1:1 mole ratio of N‐isopropyl acrylamide and Na‐acrylate along with methylene bisacrylamide (2 wt.% of total monomer) as a cross‐linker using ammonium persulfate as an initiator and N, N, N′, N′‐tetra‐methyl ethylene diamine as an accelerator. The microporous nature of the gel material is observed by scanning electron microscope as well as by surface analysis. It is a pH as well as thermoresponsive gel. The highest gel swelling is observed at around pH 8.2 at room temperature (30 °C). The gel contains carboxylate (―COO^?) group in this slightly alkaline condition. In the ionic state, the mutual repulsion of the ionic groups helps in swelling the gel. The lower critical solution temperature (LCST) of the gel is observed to be about 38 °C, which is higher than that of poly N‐isopropyl acrylamide itself (32 °C). This corroborates with the theory and other reported results that LCST increases with the incorporation of ionic moiety in the cross‐linked copolymer. The chiral chemical absorption of the gel material was monitored by measuring circular dichroism of the chiral compound in the presence and absence of gel using a circular dichroism spectropolarimeter (J‐810 L) (JASCO International Co., Ltd., Tokyo, Japan). About 18% of d ‐(+)‐α‐phenyl ethyl amine is absorbed from its aqueous solution by 0.01 g of dry gel material (particle size: 106–212 µm) at room temperature. The absorption of the chiral compound is reversible with temperature having a sudden jump at LCST (38 °C) of the gel material. Chirality 24:506–511, 2012. © 2012 Wiley Periodicals, Inc.     

Synthesis and secretion of hirudin by Streptomyces lividans

Summary To examine the secretory production of heterologous proteins by Streptomyces lividans, we fused the DNA encoding the signal peptide of the -amylase inhibitor tendamistat, derived from S. tendae with a synthetic gene encoding the thrombin inhibitor hirudin. The analysis of secretion by immunoblots revealed an efficient translocation of hirudin through the membrane, with no detectable immunoreaction among the cellular proteins. The secreted hirudin was stable in the shaking culture for about 6 days. A comparison of the hirudin secreted by S. lividans and recombinant reference hirudin from yeast by immunoblots and thrombin inhibition assays shows that hirudin from Streptomyces has a lower specific activity, which may be due to a different aminoterminal sequence or to inexact processing of the precursor.Offprint requests to: J. Engels     

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.     

The influence of the copolymer composition on the diltiazem hydrochloride release from a series of pH-sensitive poly[(<Emphasis Type="Italic">N</Emphasis>-isopropylacrylamide)-<Emphasis Type="Italic">co</Emphasis>-(methacrylic acid)] hydrogels

A series of poly[(N-isopropylacrylamide)-co-(methacrylic acid)] (P[(N-iPAAm)-co-(MAA)]) hydrogels was investigated to determine the composition that exhibits a better pH-modulated release of diltiazem hydrochloride (DIL.HCl). For this purpose hydrogel slabs were loaded with DIL.HCl by the immersion method, and its release under acidic medium (0.1N HCl, pH 1.2) and in phosphate buffer pH 7.2, using United States Pharmacopeia (USP) 24 Apparatus 1, was investigated. According to the results from the slabs, copolymers with 85% mol N-iPAAm content were selected to prepare tablets with different particle size. The effect of pH and particle size changes on DIL.HCl release from these last hydrogel tablets was investigated by a stepwise pH variation of the dissolution medium. The amount of DIL.HCl released from high N-iPAAm content copolymer slabs under acidic pH medium was not only very low but it was also released at a slow rate. In the 85% N-iPAAm tablets, significant differences between and within release profiles were found as a function of particle size and pH, respectively. A relationship between particle size and release rate has been found. The lower DIL.HCl release at acidic pH from enriched N-iPAAm copolymers is interpreted by a cooperative thermal- and pH-collapse. Although for the whole range of copolymer composition a dependence of the equilibrium of swelling on the pH was found, DIL.HCl release experiments indicated that hydrogels with 85% mol N-iPAAm are the more adequate to be used for modulated drug delivery systems. Additionally, the particle size of the tablet can be used to tailor the release rate.     

Molecular simulation study of the effect of cross-linker on the properties of poly(N-isopropyl acrylamide) hydrogel

Molecular simulation techniques have been utilised to investigate the effect of cross-linker type on the structural and dynamical properties of a temperature-sensitive hydrogel, poly (N-isopropyl acrylamide) (PNIPAM) across its lower critical solution temperature (LCST). PNIPAM exhibits an LCST at ～305 K, above which it collapses and below which it is swollen. Molecular dynamics simulations of PNIPAM hydrogel cross-linked with N, N′-methylene bisacrylamide (BIS) and ethylene glycol dimethacrylate (EGD) cross-linkers were carried out below, at and above its LCST (namely 300, 305 and 310 K, respectively). Structural analysis indicates that the cross-linkers did not affect the temperature of the onset of the LCST, but did affect the degree of swelling and pore size distribution, where the EGD-cross-linked hydrogel exhibited a greater degree of structural change than that of the BIS-cross-linked hydrogel. We believe that this could be attributed to the longer chain length and more flexible nature of the EGD cross-linker compared to the BIS cross-linkers.     

The C-terminal binding domain of hirullin P18. Antithrombin activity and comparison to hirudin peptides

Hirullin P18 is a 61-amino acid hirudin-related protein having potent antithrombin activity. Similar to hirudin, it contains a highly acidic C-terminus, but has a significantly different sequence from any other known hirudin variant. The present study demonstrates that the C-terminal fragment acetyl-hirullin P18(41-62) [corrected] possesses an antithrombin potency similar to that of acetyl-desulfatohirudin(45-65). Additionally, like the hirudin fragment analog, it inhibits fibrin-clot formation by binding to a non-catalytic site on thrombin. Sequential shortening of the hirullin P18 C-terminal fragment demonstrates the critical nature of Phe51, which corresponds to the important Phe56 residue of hirudin. Although the sequences of hirullin P18(54-61) and hirudin(59-65) have substantial differences, the C-terminal functional domain represented by hirullin P18(50-61) appears to be comparable to hirudin(55-65) in terms of its functional role in antithrombin activity.     

嵌合水蛭肽的构建与活性分析

血管成形术或动脉粥样斑块破裂等因素所致血管壁损伤而引起的血栓形成过程中 ,血小板的激活和凝血酶的形成起着关键作用 .因此 ,抗血小板和抗凝是治疗血栓的两个重要方面 .血小板膜糖蛋白GPⅡb Ⅲa受体拮抗剂 ,如含Arg Gly Asp(RGD)序列的多肽 ,在临床上已显示了良好的抗血小板     

Preparation and characterization of novel optically active poly(vinyl alcohol-co-vinyl ester) in nonaqueous medium using <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine as a chiral material

In this investigation, poly(vinyl alcohol) was chemically modified by the introduction of different amounts of N-phthaloyl-l-phenylalanine. The modification was carried out by the reaction of PVA hydroxyl groups with (2S)-3-phenyl-2-phthalimidylpropanoyl chloride using N,N-dimethyl acetamide/lithium chloride as a reaction media. The novel copolymers obtained were characterized by spectroscopic techniques, elemental analysis, X-ray diffraction and thermal methods. Optical rotation and viscosities were also measured. The degree of esterification was determined by ¹H-NMR. The influence of reagent molar ratio on the degree of modification was also evaluated. The vinyl(3-phenyl-2-phthalimidopropanoate) content in the copolymer was attained up to 52%. Thermal stability of the copolymers was checked by thermogravimetric analysis and differential thermogravimetric analysis. All copolymers displayed improved thermal stability compared to the parent polymer.     

Effects of enzymatic deglycosylation on the biological activities of human thrombin and antithrombin

Sequential digestion of human thrombin and antithrombin with neuraminidase, βgalactosidase, β-N-acetylglucosaminidase, and endo-β-N-acetylglucosaminidase D resulted in the successive removal of sialic acid, galactose, N-acetylglucosamine, and mannose and more N-acetylglucosamine residues. The products obtained after each stage of deglycosylation had electrophoretic mobilites that were consistent with the calculated change in mass expected from the cleavage of the sugar moieties. The modified thrombins did not lose fibrinogen-clotting activity, amidolytic activity, nor the ability to form complexes with antithrombin. In addition, asialothrombin and asialoagalactothrombin caused the same extent of platelet release as did control thrombin. The products obtained after removal of sugars from antithrombin retained thrombin-neutralizing activity. In the presence of heparin the inhibition of thrombin as well as factor Xa was enhanced. Thus, the sugar residues of thrombin and antithrombin are not required for the formation of enzyme-inhibitor complexes or for the other activities that were measured.     

Dual stimuli-responsive polypeptide prepared by thiol-ene click reaction of poly(l-cysteine) and N,N-dimethylaminoethyl acrylate

Stimuli-responsive polymers that can undergo conformational changes with external triggers have enabled themselves as smart materials for various utilizations, among which biodegradability is of particular importance to be engineered for biomedical application. In this study, a thermo and pH dual responsive polypeptide (N, N-dimethylaminoethyl acrylate-modified poly(l -cysteine)) (PLC-g-DMAEA) was prepared by the combination of N-carboxyanhydride ring-open polymerization and thiol-ene click chemistry. The biodegradable poly(l -cysteine) (PLC) with pendant thiol groups provided an easily clickable backbone for postmodification, which was demonstrated by reacting with a well-known monomer of N, N-dimethylaminoethyl acrylate (DMAEA) to achieve both temperature and pH responsiveness. The irreversible thermo-response of PLC-g-DMAEA could be attributed to the ordered β-sheets formed upon heating, leading to the trapped side groups with poor water accessibility. Moreover, this copolymer precipitated at pH ranging from 7.5 to 9.7, but protonation of tertiary amine groups (pH < 7.5) and salt forming of masked thiol groups (pH > 9.7) rendered it soluble in water. Our results revealed that a ready available vinyl monomer could be easily clicked onto the biodegradable PLC and its stimuli responsiveness would be reserved. Moreover, the primary and secondary structures of PLC might influence the conformation, thus leading to the unique responsive behavior of the resulted copolymer.     

天然水蛭素冻干粉实验结果的分析

目的研究天然水蛭素冻干粉的生物化学成分及其毒理学的安全性。方法采用《中华人民共和国药典》的抗凝血酶活性测定法、电感耦合等离子体质谱法、《中华人民共和国国家标准》中的食品中水分、微生物、重金属检测、检验法等。结果天然水蛭素具有较高的抗凝血酶活性(809 ATU/g),无致病菌,微生物及重金属不超标,对动物和人体无毒副作用。结论天然水蛭素有潜在的药物开发价值。     

Polymerization of amino acid derivatives by polymer catalysts. II. Polymerization by copolymer catalysts containing a tertiary amine as a component

The polymerizations of D ,-L β-phenylalanine NCA, p–nitro-D ,L -β-phenylalanine NCA, and o,p-dinitro-D ,L -β-phenylalanine NCA were investigated, homopolymers and copolymers of N-vinyl-2-ethylimidazole or 2-Vinylpyridine being used as catalysts. When N-vinylpyrrolidone and N,N-diethylacrylamide, which are capable of forming hydrogen bonds with the NCA's, were used as comonomers with N-vinyl-2-ethylimidazole, the copolymer catalysts were found to bring about a faster polymerization than poly-N-vinyl-2-ethylimidazole. However, when styrene, which has no particular interaction with the NCA's, was used as a comonomer with 2-vinylpyridine, the copolymer catalyst was found to give a slower polymerization than poly-2-vinylpyridine. Electronic spectroscopy showed that the charge-transfer complex between copolymer catalysts and the NCA's plays an important role in the polymerization. The experimental results are discussed in terms of the effectiveness of the copolymer catalysts for forming hydrogen bonds or charge-transfer complexes with the NCA's.     

Self-aggregates of oligoarginine-conjugated poly(amino acid) derivatives as a carrier for intracellular drug delivery

N_ω-2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl (N_ω-Pbf)-protected oligoarginine was directly conjugated to poly(amino acid) derivatives modified with a long alkyl chain. The final concentration of conjugated peptides was easily controlled by the feed ratio of oligoarginine to polymer backbone and a final soluble polymeric system was obtained by the deprotection of N_ω-Pbf groups. The polymeric conjugates formed stable self-aggregates of size range of 8–40 nm in aqueous solution and effectively internalized into HeLa cells by adsorptive endocytosis.Revisions requested 8 April 2005; Revisions received 6 May 2005     

Production of recombinant hirudin in Hansenula polymorpha: variation of gene expression level depends on methanol oxidase and fermentation strategies

Various recombinant Hansenula polymorpha strains were developed and compared for their level of expression of the anticoagulant hirudin. H. polymorpha DL1-57 harboring an autonomously replicating sequence, HARS36, efficiently expressed the gene for recombinant hirudin. The effect of methanol oxidase (MOX) on the expression of the hirudin gene in H. polymorpha DL1-57 was studied, and the fermentation strategies coupled with the MOX activity and an antioxidant, tocopherol, were also examined. Received 4 February 1998/ Accepted in revised form 24 June 1998     

Synthesis of glycuronamides of amino acids,constituents of microbial polysaccharides and their conversion into neoglycoconjugates of copolymer type

Glycopyranosiduronic acids, amidically linked to amino acids (alanine, serine, threonine, and lysine) were prepared.O-tert-Butyl andN-tert-butyloxycarbonyl protected amino acidtert-butyl esters were used in ethyl 2-ethoxy-1,2-dihydroquinoline-1-carboxylate promoted condensation with 2-azidoethyl glycosides of glucuronic and galacturonic acid. Reduction of the azido-function followed byN-acryloylation and removal of blocking groups with trifluoroacetic acid gave the target monomers. These were converted into neoglycoconjugates of copolymer type, potentially useful for immunochemical studies.On leave from the Indian Institute of Chemical Technology, Hyderabad, India.     

Investigation on the microcirculation effect of local application of natural hirudin on porcine random skin flap venous congestion

Medicinal leech therapy is commonly used to treat venous congestion by local application to skin flaps, which are frequently employed in re-construction surgery. In the present study, we investigated the effect of natural hirudin, which was applied locally to treat the venous congestion, in random skin flaps in a Guangxi Bama miniature pig model. The congestive random skin flaps were divided into 3 groups: Group A (control group that was locally applied with saline), Group B (locally applied with 20ATU natural hirudin per each flap), Group C (locally applied with 40ATU natural hirudin per each flap). The microcirculatory changes were observed by measuring myeloperoxidase (MPO), malondialdehyde (MDA) and superoxide dismutase (SOD). The level of MPO, MDA and SOD activities in the tissue in hirudin-treated groups was significantly different compared with control group (P < 0.05), but there was no significant difference between group B and group C. At 12 days, as compared to the control group, flap survival rate in hirudin-treated groups was significantly different (P < 0.05), but there was no significant difference between the groups receiving two different doses of hirudin. Natural hirudin can improve microcirculation by inhibiting oxygen free radical damage and also improve flap survival time.     

Reduction of ferriprotoporphyrin IX to the ferroderivative by copoly(4-vinylpyridine-N-(p-vinylbenzyl)-3-carbamoyl-1,4-dihydropyridine) in dimethyl sulfoxide

The copolymer which has both ligand sites (4-vinylpyridine) and redox sites (N-(p-vinylbenzyl)-3-carbamoyl-1,4-dihydropyridine) was synthesized by the dithionite reduction of the copoly(4-vinylpyridine-N-(p-vinylbenzyl)-3-carbamoylpyridinium chloride) and the reduction of a central ferric-iron of ferriprotoporphyrin IX by the above-described copolymer was studied spectrophotometrically in dimethyl sulfoxide. The rate of the reduction by the copolymer was much faster than by N-benzyl-3-carbamoyl-1,4-dihydropyridine. This acceleration by the copolymer could be explained by the intramolecular reduction of ferriprotoporphyrin IX which was coordinated by the pyridine residue of the copolymer.