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Synthesis of segmented poly(ether urethane)s and poly(ether urethane urea)s incorporating various side-chain or backbone functionalities.

A series of linear and branched functionalized high-molecular-weight segmented poly (ether urethane)s and poly(ether urethane urea)s were prepared by chain-extending isocyanate pre-polymers based on poly(tetramethylene oxide) with molecular weight 1000 and 4,4'-diphenylmethane diisocyanate. Different functional groups were incorporated within the polymer backbone or on side-chains by using several chain extenders during the synthesis: glycerol, 2,2-bis (hydroxymethyl) propionic acid, 1H, 1H,2H,3H,3H-perfluoroundecane-1,2-diol, 1H,1H,8H,8H-dodecanefluoro-1,8-octanediol, 1,3-diamino-2-hydroxypropane and 3,5-diaminobenzoic acid, using the method of gradual approach to stoichiometry. In some cases, pendant functional groups were used as reactive sites for the further attachment of side groups. Polymers were characterized using 1H-NMR and FT-IR spectroscopies and GPC in conjunction with chemical structural confirmation by a model compound comparison study of 4,4'-diphenylmethane diisocyanate or trifluoro-p-tolyl isocyanate reacted with 1,3-diamino-2-hydroxypropane and 1,4-butanediol.

Chromatography, Gel↗

Poly (ethylenimine)-grafted-poly [(aspartic acid)-co-lysine], a potential non-viral vector for DNA delivery.

A potential non-viral gene-transfer vector, poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine] (PSL), has been developed by thermal polycondensation of aspartic acid and lysine under reduced pressure. Low-molecular-mass branch poly(ethylenimine) (PEI600) was conjugated to the backbone. The chemical structure of the resulting co-polymer was identified by 1H-NMR, FT-IR, TGA and X-ray diffraction. The results of the MTT assay showed that at concentration up to 4000 nmol/l of the vector cell viability was over 80% and showed low toxicity. Electrophoretic retardation and ethidum bromide assay showed that at N/P ratios 12-15 (w/w) the DNA could be condensed and neutralized. Using the zeta potential assay we discovered that it had a high positive charge on its surface of the particle (over 30 mV). The particle sizes of the co-polymer/DNA complexes were 150-170 nm, as measured by DLS and AFM. Compared with PEI600, co-polymer/DNA complexes showed a significant enhancement of transfection activity in the absence and presence of serum in NT2 and COS7 cell lines. This means that the PEI600-PSL co-polymer is a promising candidate for gene delivery.

Animals↗

Modification of poly(ether urethane)elastomers by incorporation of poly(isobutylene)glycol. Relation between polymer properties and thrombogenicity.

Non-polar hydrophobic poly(isobutylene)glycol (PIBG) was substituted for poly(tetramethylene ether)glycol (PTMEG) in poly(ether urethanes) based on 4,4'-methylenebis-(phenylisocyanate) (MDI) and 1,4-butanediol (BD) as chain extender. Two series of polyurethanes differing in their soft segment length, polymer composition, and hard segment content were studied by dynamic mechanical analysis (DMA) and static, as well as dynamic, contact angle measurements. The thrombogenicity of these polymers was characterized by studying the adhesion and activation of platelets using ELISA for GMP 140 and fluorescence microscopy. It was found by DMA that in PIBG-containing polyurethanes (PUE) exist soft domains containing hard segments, strictly separated hard segment domains, and hard segments partially mixed with soft segments. Contact angle measurements revealed that 25% PIBG or even less, are sufficient for a remarkable enrichment of these non-polar soft segments on the polymer surface. The platelet adhesion/activation on these materials was demonstrated to increase with the rise in hard segment content, as well as with an enhancement of the PIBG content. However, comparison of PIBG-containing PUE with medical applied polypropylene and pellethane expressed that PUE with PIBG content equal or less 25% have excellent haemocompatibility.

Biocompatible Materials↗

Synthesis of size-tunable gold nanoparticles by poly(vinylphenol) and electrostatic multilayer deposition of the gold-poly(vinylphenol) nanocomposites.

Water-dispersible gold nanoparticles (GNPs) were synthesized by the polymer in situ redox technique at room temperature using poly(4-vinylphenol) (PVPh) as a simultaneous template/stabilizer and reducing agent. GNPs were characterized by its surface plasmon absorption peak at 536 nm. The average particle size of the gold-poly(4-vinylphenol) (Au-PVPh) nanocomposites decreases with increase in the ratio of PVPh to gold salt. A uniform multilayer of Au-PVPh nanocomposites was fabricated on glass surface by electrostatic layer-by-layer assembly using poly(diallyldimethylammonium chloride) (PDDAC) as the oppositely charged polycation. UV-vis spectra of the consecutive multilayer showed that the absorbance at 549 nm corresponding to GNPs increases linearly with the number of Au-PVPh nanocomposite layers, indicating regular bilayer growth.

Cations↗

Photorefractivity of poly-N-vinylindole-based materials as compared with that of poly-N-vinylcarbazole-based blends.

The asymmetric two-beam coupling technique has been employed to measure the photorefractivity of thin films of polymer blends containing 2,5-dimethyl-4-(p-nitrophenylazo)anisole as the nonlinear optical component. Poly-(1-vinylindole) and poly-(2,3-dimethyl-1-vinylindole) were the photoconductive polymer counterparts. The values of the photorefractivity are reported. It appears that they are comparable with those of similar blends based on the well-known poly-(9-vinylcarbazole) (PVK), here considered as a reference standard. Careful differential scanning calorimetry analyses have been accomplished on the different blends taken into account to rationalize the significantly longer shelf lifetime of the indolyl-based films with respect to the PVK-based blends.

Journal Article↗

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.

Adsorption↗

Simplex lattice design for the optimization of the microencapsulation of a water soluble drug using poly(lactic acid) and poly(lactide co-glycolide) copolymer.

An emulsion solvent evaporation technique was applied to prepare microspheres of nicotinic acid as a water soluble vitamin. Poly(lactic acid) of two molecular weights, 2000 and 100,000 and poly(lactide-co-glycolide) (50:50) of molecular weight 18,000 were used. A simplex lattice design with the aid of a computer program was applied to study the effect of the previously mentioned polymers on different characteristics of the prepared microcapsules. The studied characteristics included the yield, the percentage drug loading and the particle size of microcapsules. Microcapsules were examined by light microscope for particle size determination and by electron scanning microscope for surface morphology characterization. The release pattern of the drug from the microcapsules was evaluated on the basis of the burst effect, the rate of release and the extent of release after 24 h. A model equation was developed to be the best representation of the relationship between the above polymers and the measured characteristics. The goodness of fit of the developed equations was checked, both statistically and experimentally. The release of the drug from microcapsules mainly followed the Higuchi diffusion model. Polylactic acid of a molecular weight 100,000 had the most pronounced delaying effect on the release rate of the drug. However, polylactic acid of a molecular weight 2,000 showed the highest improvement effect on the yield and the drug loading characteristics of the microcapsules. Poly(lactide-co-glycolide) copolymer exhibited the lowest burst effect and the most steady drug release pattern.

Drug Compounding↗

In vitro controlled release kinetics of local anaesthetics from poly(D,L-lactide) and poly(lactide-co-glycolide) microspheres.

Poly(D,L)lactide and polylactide-co-glycolide drug-loaded microspheres were prepared with lipopholic (bupivacaine and etidocaine) and hydrophilic (mepivacine and lidocaine) local anaesthetics. Formulations of drug-loaded microspheres were characterized by the drug content, the in-vitro release kinetics and by the physical state of the drug within the microspheres. Release rates of the local anaesthetics from the microspheres were different and could not be accounted for by the intrinsic dissolution rates of the drugs. The encapsulation efficiency was highly dependent on the lipophilicity of the drugs, reaching the maximum for the lipophilic drugs and the poly(lactide-co-glycolide) polymers. The influence of the molecular weight of the poly(lactide-co-glycolide) polymers on the release rate and on the release mechanism depended on the drug studied and its physical state within the polymeric matrices. Diffusion-controlled release was evidenced in various formulations as a result of the linearity of the release as a function of the square root of time.

Anesthetics, Local↗

[Effect of moulding and extruding conditions on mechanical properties of poly(D,L-lactide) and MDI chain-extending poly(D,L-lactide)/hydroxyapatite composite].

In this paper, poly(D,L-lactide) (PDLLA), MDI chain-extending poly(D,L-lactide) (PDLLA/MDI) and MDI chain-extending poly(D,L-lactide)/hydroxyapatite composite (PDLLA/HA/MDI) were prepared respectively and the effects of moulding and extruding conditions on their mechanical properties were also investigated. At the optimal conditions, bending strength of PDLLA and PDLLA/MDI is 35.1 MPa and 51.3 MPa, respectively, and their bending modulus is 2413.6 MPa and 1830.9 MPa, respectively. Bending strength of PDLLA/HA and PDLLA/HA/MDI is 31.2 MPa and 55.4 MPa, respectively, and their bending modulus is 1735.0 MPa and 2068.5 MPa, respectively. These results have shown that the mechanical properties of PDLLA/MDI and PDLLA/HA/MDI have enhanced significantly by MDI chain-extending.

Biocompatible Materials↗

[Synthesis and characterizations of poly (ethylene glycol)-block-poly (glutamate)].

Amine-terminated poly (ethylene glycol) (PEG) was prepared by two steps. Firstly, potassium naphthalene was added to a solution of methoxypolyethylene glycol 5,000 in benzene until the solution maintains green in half of an hour, then excess tosylchloride was introduced; secondly, the conversion of the tosylate into an amine was carried out by Gabriel synthesis. The block copolymer poly (ethylene glycol)-co-poly (gamma-benzyl L-glutamate ) could be obtained by ring-opening polymerization of gamma-benzyl-L-glutamate N-carboxy anhydride with amine-terminated PEG as macroinitiator. And the benzyl group could be removed by sodium hydroxide. The product structure was characterized by IR, 1HNMR, GPC. The cisplatin-loaded micelle was observed by transmission electron microscope (TEM). And the block copolymer is expected to be useful as functional materials including carrier systems in drug controlled delivery applications.

Drug Carriers↗

Autoantibodies reacting with poly(ADP-ribose) and with a zinc-finger functional domain of poly(ADP-ribose) polymerase involved in the recognition of damaged DNA.

Poly(ADP-Ribose) polymerase (PARP) is a chromatin-associated enzyme that specifically binds to DNA strand breaks in a zinc-dependent manner. We describe here the presence of IgG antibodies reacting with recombinant human PARP in the serum of patients with systemic lupus erythematosus (SLE) and primary and secondary Sjögren's syndrome (pSS and sSS). The reactivity of patients' sera was further studied in ELISA with a synthetic peptide of 44 residues corresponding to the second zinc finger (F2) present in the DNA-binding domain of PARP and which was shown to effectively bind 65Zn. Thirty-five percent of SLE sera (n = 97), 42% of pSS sera (n = 67), and 56% of sSS sera (n = 16) were found to contain raised levels of IgG antibodies reacting with peptide F2 which corresponds to the domain in PARP that is directly involved in the specific recognition of single and double strand breaks in DNA. Antibodies reacting with the whole enzyme and/or peptide F2 occurred independently from antibodies reacting with poly(ADP-ribose) which is rapidly synthesized in vivo by PARP from NAD and then degraded in response to DNA strand breaks.

Amino Acid Sequence↗

Changes in body temperature and vasopressin content of brain neurons, in pregnant and non-pregnant guinea pigs, during fevers produced by Poly I:Poly C.

The synthetic polyribonucleotide pyrogen Poly I:Poly C (800 micrograms.kg-1) was injected intramuscularly on alternate days into pregnant and non-pregnant female guinea pigs. Pregnant animals, close to term, had smaller fevers in response to the pyrogen than did non-pregnant animals. Repeated injections of the pyrogen caused sequentially smaller fevers for the first 3-4 injections, particularly in non-pregnant animals, and this appeared to be like the tolerance usually developed to repeated injections of endotoxin. Continued pyrogen injections then caused, in non-pregnant animals, fevers of increasing magnitude until the original fever levels were reached, whereas in pregnant guinea pigs the fever responses remained reduced until parturition. The development of tolerance was associated with an increase in immunoreactivity for arginine vasopressin (AVP) in some neurons in the medial part of the paraventricular nucleus, and in terminals in the lateral septum and amygdala similar to changes found in these areas at term of pregnancy. These observations raise the possibility that AVP in these regions may have a role in the development of tolerance to pyrogens, and further quantitative studies of the AVP content of, and release from, nerve terminals projecting to the limbic system seem warranted.

Animals↗

A misaligned double-stranded RNA, poly(I).poly(C12,U), induces accumulation of 2',5'-oligoadenylates in mouse tissues.

2',5'-Oligoadenylate synthetase was induced 3-2000-fold in spleen, liver, kidney and brain of NIH Swiss mice injected intravenously with 2-200 micrograms of the misaligned dsRNA, poly(I).poly(C12,U). Levels of 2',5'-oligoadenylates extracted from these tissues were also elevated, although the amount of 2',5'-oligoadenylates extracted did not correlate directly with the amount of enzyme present. These results suggest that double-stranded portions of the misaligned polymer survived intracellularly and activated the 2',5'-oligoadenylate synthetase, and that the level of dsRNA may contribute to the control of 2',5'-oligoadenylate metabolism.

2',5'-Oligoadenylate Synthetase↗

Detection of mutations using microarrays of poly(C)10-poly(T)10 modified DNA probes immobilized on agarose films.

Allele-specific hybridization to a DNA microarray can be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation to an agarose film grafted onto unmodified glass. Microarrays of TC-tagged probes immobilized on the agarose film can be used to diagnose mutations in the human beta-globin gene, which encodes the beta-chains in hemoglobin. Although the probes differed widely regarding melting point temperature ( approximately 20 degrees C), a single stringency wash still gave sufficiently high discrimination signals between perfect match and mismatch probes to allow robust mutation detection. In all, 270 genotypings were performed on patient materials, and no genotype was incorrectly classified. Quality control experiments conducted using a target DNA specific for the TC tag of the immobilized probes showed that the spotting and hybridization procedure had a variance of 20%, indicating that signal differences as low as twofold could be detected between perfect match and mismatch. Together, our results show that the use of microarrays of TC-tagged probes that have been immobilized on agarose films grafted onto glass is a robust and inexpensive genotyping method.

Alleles↗

Dual role for poly(ADP-ribose)polymerase-1 and -2 and poly(ADP-ribose)glycohydrolase as DNA-repair and pro-apoptotic factors in rat germinal cells exposed to nitric oxide donors.

We describe the involvement of poly(ADP-ribose)polymerase 1 and 2 (PARP-1 and -2) and poly(ADP-ribose)glycohydrolase (PARG) in the response of rat germinal cells to the action of the NO donors, 3-morpholino-sydnonimine (SIN-1) and spermine nonoate (SNO). Primary spermatocytes and round spermatids showed a differential sensitivity to DNA damage induced by acute exposure to SIN-1 and SNO. Spermatocytes were able to repair DNA damage caused by the release of NO from SNO but neither spermatocytes nor spermatids could recover from the release of NO and O2*- from SIN-1. Addition of the PARPs inhibitor, 3-aminobenzamide, and the PARG inhibitor, gallotannin (GT), to germ cell cultures impaired DNA repair significantly. Consistent with the DNA repair seen in primary spermatocytes, both SIN-1 and SNO induced PARPs activation in these cells. In the case of SIN-1, there was an immediate but transient response while SNO induced a delayed but more sustained increase in PARPs activity. Chronic exposure of spermatocytes to SIN-1 and SNO, however, committed the cells to apoptosis, which coincided with proteolysis of PARP-1. The data indicate a dual role for PARPs and PARG in germinal cells as key proteins in processes that sense and repair DNA damage as well as in the commitment to apoptosis following prolonged oxidative stress.

Animals↗

The binding of DNA intercalating and non-intercalating compounds to A-form and protonated form of poly(rC).poly(rG): spectroscopic and viscometric study.

Polymorphic RNA conformations may serve as potential targets for structure specific antiviral agents. As an initial step in the development of such drugs, the interaction of a wide variety of compounds which are characterized to bind to DNA through classical or partial intercalation or by mechanism of groove binding, with the A-form and the protonated form of poly(rC).poly(rG), been evaluated by multifaceted spectroscopic and viscometric techniques. Results of this study suggest that (i) ethidium intercalates to the A-form of RNA, but does not intercalate to the protonated form, (ii) methylene blue intercalates to the protonated form of the RNA but does not intercalate to the A-form, (iii) actinomycin D does not bind to either conformations of the RNA, and (iv) berberine binds to the protonated form by partial intercalation process, while its binding to the A-form is very weak. The DNA groove binder distamycin A has much higher affinity to the protonated form of the RNA compared to the A-form and binds to both structures by non-intercalative mechanism. We conclude that the binding affinity characteristics of these DNA binding molecules to the RNA conformations are vastly different and may serve as data for the development of RNA based antiviral drugs.

Berberine↗

Purification from normal human plasma and biochemical characterization of a ribonuclease specific for poly(C) and poly(U).

A new specific ribonuclease from normal human plasma has been purified to homogeneity, following a five-step purification protocol that included DEAE-Sepharose, CM-Sepharose, and Heparin-Sepharose chromatographies. The purified enzyme was found to be glycosylated and appeared as a single 25-kDa band on a SDS polyacrylamide gel. This RNase is poly(C) preferential, degrading poly(U) at a lower rate. Activity of this RNase toward cleavage of native substrates such as ribosomal RNA was also detected. The human plasma ribonuclease is a thermolabile molecule, exhibiting maximum activity at pH 6.5. Comparison between other known plasma RNases and the human plasma ribonuclease described here indicated a variety of differences in their biochemical and catalytic properties.

Animals↗