The binding of poly(rA) and poly(rU) to denatured DNA. I. Model studies with homopolymers.

We have compared the properties of the poly(rA).oligo(dT) complex with those of the poly(rU).oligo(dA)n complex. Three main differences were found. First, poly(rA) and oligo(dT)n do not form a complex in concentrations of CsCl exceeding 2 M because the poly(rA) is insoluble in high salt. If the complex is made in low salt, it is destabilized if the CsCl concentration is raised. Complexes between poly(rU) and oligo(dA)n, on the other hand, can be formed in CsCl concentrations up to 6.6 M. Second, complexes between poly(rA) and oligo(dT)n are more rapidly destabilized with decreasing chain length than complexes between poly(rU) and oligo(dA)n. Third, the density of the complex between poly(rA) and poly(dT) in CsCl is slightly lower than that of poly(dT), whereas the density of the complex between poly(rU) and poly(dA) in CsCl is at least 300 g/cm3 higher than that of poly(dA). These results explain why denatured natural DNAs that bind poly(rU) in a CsCl gradient usually do not bind poly(rA).     

Organotin(IV) compounds as intramolecular transesterification catalysts in thermal depolymerization of poly(L-lactic acid) oligomer to form LL-lactide.

Mono-, di-, and tetraalkyl tin(IV) compounds were evaluated for the intramolecular transesterification reaction of the thermal depolymerization of poly(L-lactic acid) oligomer forming lactide by gas chromatography using a beta-cyclodextrin stationary phase capillary column. The most active catalyst was found to be monobutyltin trichloride (BuSnCl3) (8), which contains tin-halogen bonds, and the least effective was the coordinatively saturated monoorganotin derivative, monobutyltin tris(2-ethylhexanoate) (7). Coordination of the carbonyl group in the oligomer to the tin catalysts is an important factor influencing its activity.     

Self-assembled micelles of biodegradable triblock copolymers based on poly(ethyl ethylene phosphate) and poly(-caprolactone) as drug carriers

A series of novel amphiphilic triblock copolymers of poly(ethyl ethylene phosphate) and poly(-caprolactone) (PEEP-PCL-PEEP) with various PEEP and PCL block lengths were synthesized and characterized. These triblock copolymers formed micelles composed of a hydrophobic core of poly(-caprolactone) (PCL) and a hydrophilic shell of poly(ethyl ethylene phosphate) (PEEP) in aqueous solution. The micelle morphology was spherical, determined by transmission electron microscopy. It was found that the size and critical micelle concentration values of the micelles depended on both hydrophobic PCL block length and PEEP hydrophilic block length. The in vitro degradation characteristics of the triblock copolymers were investigated in micellar form, showing that these copolymers were completely biodegradable under enzymatic catalysis of Pseudomonas lipase and phosphodiesterase I. These triblock copolymers were used for paclitaxel (PTX) encapsulation to demonstrate the potential in drug delivery. PTX was successfully loaded into the micelles, and the in vitro release profile was found to be correlative to the polymer composition. These biodegradable triblock copolymer micelles are potential as novel carriers for hydrophobic drug delivery.     

Cross-linked nanoassemblies from poly(ethylene glycol)-poly(aspartate) block copolymers as stable supramolecular templates for particulate drug delivery

Block copolymer cross-linked nanoassemblies (CNAs) were developed as stable supramolecular templates for particulate drug delivery. Poly(ethylene glycol)-poly(aspartate) [PEG-p(Asp)] block copolymers, consisting of PEG (5 or 12 kDa) and Asp (5, 14, 25, 33, and 37 repeating units), were used as scaffolds and grafts in combination to prepare a nanoassembly library of grafted nanoassemblies (GNAs) and CNAs. Four synthesis routes were tested to maximize the number of drug-binding Asp units per nanoassembly. Grafting-onto-scaffold and grafting-from-scaffold methods were used for GNA synthesis. Either partially or completely deprotected PEG-p(Asp) was cross-linked with diamine compounds to prepare CNAs. (1)H NMR and GPC measurements showed that GNAs and CNAs contained the maximum 183 and 253 Asp units, respectively. Initial screening of the nanoassemblies revealed that GNAs would be impractical for further development as drug carriers due to variable grafting efficiency and low product yields. CNAs were obtained in high yields and identified as a promising supramolecular template that can entrap and release ionizable drugs (doxorubicin), enhancing the particle stability of nanoassemblies in the pharmaceutically relevant pH ranges between 4 and 9. Light scattering measurements demonstrated that the particle size of CNAs remained uniform before and after drug entrapment, causing neither aggregation nor dissociation (<5 mg/mL).     

Production of targeted poly(3-hydroxyalkanoates) copolymers by glycogen accumulating organisms using acetate as sole carbon source

One of the main limitations in bacterial polyhydroxyalkanoate (PHA) production with mixed cultures is the fact that primarily polyhydroxybutyrate (PHB) homopolymers are generated from acetate as the main carbon source, which is brittle and quite fragile. The incorporation of different 3-hydroxyalkanoate (HA) components into the polymers requires the addition of additional carbon sources, leading to extra costs and complexity. In this study, the production of poly(3-hydroxybutyrate (3HB)-co-3-hydroxyvalerate (3HV)-co-3-hydroxy-2-methylvalerate (3HMV)), with 7-35C-mol% of 3HV fractions from acetate as the only carbon source was achieved with the use of glycogen accumulating organisms (GAOs). An enriched GAO culture was obtained in a lab-scale reactor operated under alternating anaerobic and aerobic conditions with acetate fed at the beginning of the anaerobic period. The production of PHAs utilizing the enriched GAO culture was investigated under both aerobic and anaerobic conditions. A polymer content of 14-41% of dry cell weight was obtained. The PHA product accumulated by GAOs under anaerobic conditions contained a relatively constant proportion of non-3HB monomers (30+/-5C-mol%), irrespective of the amount of acetate assimilated. In contrast, under aerobic conditions, GAOs only produced 3HB monomers from acetate causing a gradually decreasing 3HV fraction during this aerobic feeding period. The PHAs were characterized by gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). The data demonstrated that the copolymers possessed similar characteristics to those of commercially available poly(3HB-co-3HV) (PHBV) products. The PHAs produced under solely anaerobic conditions possessed lower melting points and crystallinity, higher molecular weights, and narrower molecular-weight distributions, compared to the aerobically produced polymers. This paper hence demonstrates the significant potential of GAOs to produce high quality polymers from a simple and cheap carbon source, contributing considerably to the growing research body on bacterial PHA production by mixed cultures.     

Novel synthesis of functionalized poly(3-hydroxybutanoic acid) and its copolymers.

Novel feasibility of fuctionalized poly(3-hydroxybutanoic acid), PHB, and its copolymers synthesis via ring-opening of beta-butyrolactone (ROP) mediated by activated anionic initiators or enzymes in vitro is presented. Using these new synthetic approaches, PHB with defined chemical structure of the end groups as well as block, graft and random copolymers have been obtained and characterized by IR, NMR, ESI-MS and GPC techniques. The relationship between the structure and properties of the novel polymeric materials prepared is discussed.     

Poly(styrenesulfonate)/poly(allylamine) multilayers: a route to favor endothelial cell growth on expanded poly(tetrafluoroethylene) vascular grafts

Small-diameter synthetic vascular grafts of expanded poly(tetrafluoroethylene) (ePTFE) polymer concern one of the most common alternatives for the replacement of diseased vessels. However, high failure rates arise especially due to the lack of endothelial cells (ECs). EC seeding was developed to build a monolayer on the luminal surface. Because ECs show little or no adhesion on synthetic prostheses, it is necessary to promote their retention. On ePTFE surfaces we successfully deposited polyelectrolyte multilayer films (PMFs) consisting of poly(ethylenimine) (PEI), poly(sodium 4-styrenesulfonate) (PSS), and poly(allylamine hydrochloride) (PAH) to obtain PEI-(PSS-PAH)3 films. EC adhesion and spreading on modified ePTFE were assessed by scanning electron and confocal microscopies. Cell viability was evaluated by Alamar Blue assay. After 7 days of culture, the ePTFE modified with PMF exhibited improvements of EC viability as compared to that of the controls (nonmodified ePTFE) or even ePTFE coated by a PAH monolayer (p < 0.05). Moreover, the spreading of ECs was largely enhanced compared to that of the same controls, resulting in a healthy confluent cell monolayer formation. Positive staining for the von Willebrand factor confirmed the EC phenotype. Promoting EC attachment and function on ePTFE modified with PMFs could become in the future a promising treatment for synthetic small-diameter vascular grafts.     

Novel amphiphilic poly(epsilon-caprolactone)-g-poly(L-lysine) degradable copolymers

As part of the search of novel degradable polymers, amphiphilic and cationic poly(epsilon-caprolactone)-g-poly(l-lysine) (PCL-g-PlL) copolymers have been synthesized following a grafting "onto" or a grafting "from" method both applied to a macropolycarbanionic PCL derivative. The first approach led to PCL-g-PZlL containing 36% of epsilon-caprolactone and 64% of N-epsilon-Z-l-lysine units, by reaction of activated poly(N-epsilon-Z-l-lysine) on the macropolycarbanion derived from PCL. The second route was based on the anionic ring opening polymerization of N-carboxyanhydride of N-epsilon-benzyloxycarbonyl-l-lysine initiated by the macropolycarbanion derived from PCL and led to a similar copolymer containing 45% of of epsilon-caprolactone and 55% of N-epsilon-Z-l-lysine units. After deprotection of the lysine units, PCL-g-PlL copolymers were obtained. These copolymers are water-soluble and form nanometric micelle-like objects with mean diameters between 60 and 500 nm in distilled water depending on the synthesis route.     

Drug release from and hydrolytic degradation of a poly(ethylene glycol) grafted poly(3-hydroxyoctanoate)

Monoacrylate-poly(ethylene glycol)-grafted poly(3-hydroxyoctanoate) (PEGMA-g-PHO) copolymers were synthesized to develop a swelling-controlled release delivery system for ibuprofen as a model drug. The in vitro hydrolytic degradation of and the drug release from a film made of the PEGMA-g-PHO copolymer were carried out in a phosphate buffer saline (pH 7.4) medium. The hydrolytic degradation of the copolymer was strongly dependent on the degree of grafting (DG) of the PEGMA group. The degradation rate of the copolymer films in vitro increased with increasing DG of the PEGMA group on the PHO chain. The copolymer films showed a controlled delivery of ibuprofen to the medium in periods of time that depend on the composition, hydrophilic/hydrophobic characteristics, initial drug loading amount and film thickness of the graft copolymer support. The drug release rate from the grafted copolymer films was faster than the rate of weight loss of the films themselves. In particular, a combination of the low DG of the PEGMA group in the PHO chains with the low ibuprofen solubility in water led to long-term constant release from these matrices in vitro.     

Synthesis and biopharmaceutical characterisation of new poly(hydroxyethylaspartamide) copolymers as drug carriers.

Four new poly(hydroxyethylaspartamide)-based copolymers bearing (a) poly(ethylene glycol) 2000, (b) poly(ethylene glycol) 5000, (c) poly(ethylene glycol) 2000 and hexadecylalkyl, (d) poly(ethylene glycol) 5000 and hexadecylalkyle, as pendant groups were synthesised. The copolymers were obtained by partial aminolysis of polysuccinimide with poly(ethylene glycol) and hexadecylalkyl amino derivatives followed by reaction with ethanolamine. Naked polyhydroxyaspartamide was obtained by polysuccinimide reaction with ethanolamine. The nuclear magnetic resonance, infrared, light scattering and elemental analysis allowed for the extensive physico-chemical characterisation of the carriers. The molecular mass of all the polymers was in the range of 27000-34000 Da, and the polydispersivity was in the range of 1.5-1.7. By intravenous injection to mice bearing a solid tumour, all the polymeric carriers displayed a bi-compartmental pharmacokinetic behaviour. Both the poly(ethylene glycol) and the hexadecylalkyle conjugation prolonged and enhanced the distribution phase of poly(hydroxyethylaspartamide). The poly(ethylene glycol) conjugation was found to promote the carrier elimination by kidney ultrafiltration and to prevent partially the accumulation in the spleen and in the liver. The poly(ethylene glycol)/hexadecylalkyle conjugates localised preferentially in the liver were over 30% of the dose/g of tissue was determined after 144 h from administration. In the tumour all the polymers displayed a relevant accumulation that significantly increased throughout the time to reach high concentrations after 24 h. In particular, the poly(ethylene glycol)/hexadecylalkyle conjugates achieved a concentration of 15-25% of the dose/g of tissue after 24 h from administration that was maintained up to 144 h.     

Physiochemical properties of low and high molecular weight poly(ethylene glycol)-grafted poly(ethylene imine) copolymers and their complexes with oligonucleotides

Inefficient delivery of antisense oligonucleotides (AOs) to target cell nuclei remains as the foremost limitation to their usefulness. Copolymers of cationic poly(ethylene imine) (PEI) and poly(ethylene glycol) (PEG) have been well-studied for delivery of plasmids. However, the properties of PEG-PEI-AO polyplexes have not been comprehensively investigated. Therefore, we synthesized a series of PEG-PEI copolymers and evaluated their physiochemical properties alone and when complexed with AO. The M(w) of PEG was found to be the main determinant of polyplex size, via its influence on particle aggregation. DLS measurements showed that when PEG5000 was grafted to PEI2K and PEI25K, polyplex diameters were extremely small (range 10-90 nm) with minimal aggregation. In contrast, when PEG550 was grafted to PEI2K and PEI25K, polyplexes appeared as much larger aggregates (approximately 250 nm). As expected, the surface charge (zeta potential) was higher for polyplexes containing PEI25K than those containing PEI2K, but decreased with increased levels of PEG grafting. Surprisingly, within the physiological range (pH 7.5-5), the buffering capacity of all copolymers was nearly equivalent to that of unsubstituted PEI2K or PEI25K, and was barely influenced by PEGylation. The stability of polyplexes was evaluated using a heparin polyanion competition assay. Unexpectedly, polyplexes containing PEI2K showed stability equal to or greater than that of PEI25K polyplexes. The level of PEG grafting also had a dramatic effect on polyplex stability. The relationships established between molecular formulations and polyplex size, aggregation, surface charge, and stability should provide a useful guide for future studies aimed at optimizing polymer-mediated AO delivery in cell and animal studies. A summary of the relationships between polyplex structures and recent studies of their transfection capacity is provided.     

Synthesis and characterization of six-arm star poly(delta-valerolactone)-block-methoxy poly(ethylene glycol) copolymers

Novel amphiphilic six-arm star diblock copolymers based on biocompatible and biodegradable poly(delta-valerolactone) (PVL) and methoxy poly(ethylene glycol) (MePEG) were synthesized by a two-step process. First, the hydrophobic star-shaped PVL with hydroxyl terminated functional groups was synthesized using a multifunctional alcohol, dipentaerythritol (DPE), as the initiator and fumaric acid as the catalyst. The amphiphilic six-arm star copolymer of poly(delta-valerolactone)-b-methoxy poly(ethylene glycol), (PVL-b-MePEG)(6), was then synthesized by coupling the hydroxyl terminated six-arm PVL homopolymer with alpha-methoxy-omega-chloroformate-poly(ethylene glycol) (MePEG-COCl). (1)H NMR and GPC analyses confirmed the successful synthesis of star-shaped copolymers with predicted compositions and narrow molecular weight distributions. DSC analysis revealed that the glass transition temperatures of the star PVL homopolymers with M(n) between 5000 and 49 000 are not dependent on their molecular weights, whereas the melting temperatures of both the PVL homopolymers and the amphiphilic (PVL-b-MePEG)(6) copolymers increase with an increase in the PVL molecular weight. Micelles were prepared from the (PVL-b-MePEG)(6) copolymers via the dialysis method and found to have effective mean diameters ranging from 10 to 45 nm, depending on the copolymer composition. In addition, the (PVL-b-MePEG)(6) copolymers having lower PVL content were found to form micelles with a narrow monomodal size distribution, whereas the copolymers having higher PVL content tended to form aggregates with a bimodal size distribution. The noncytotoxicity of the copolymers was also confirmed in CHO-K1 fibroblast cells using a cell viability assay, indicating that the (PVL-b-MePEG)(6) copolymers are suitable for biomedical applications such as drug delivery.     

Biodegradable amphiphilic copolymers based on poly(epsilon-caprolactone)-graft chondroitin sulfate as drug carriers

The goal of this study was to develop a new type of core-shell micelles based on biocompatible and biodegradable amphiphilic copolymers, named PCL-CS, using chondroitin sulfate (CS) as a hydrophilic segment and poly(epsilon-caprolactone) (PCL) as a hydrophobic segment. The copolymers, prepared from the various compositions between CS and PCL, were characterized by Fourier transform infrared spectrometer, proton nuclear magnetic resonance spectrometer, and differential scanning calorimeter. The PCL-CS copolymers could be assembled into micelles using a simple emulsion. With the fluorescent probe technique, the critical micelle concentrations were obtained in the range of 1.26 x 10(-3)-8.86 x 10(-3) mg/mL. The spherical images of micelles were visualized in the presence of polyvinyl alcohol (PVA) with the use of the transmission electron microscope. The particle sizes of micelles were all smaller than 300 nm, neither aggregate nor change in hydrodynamic sizes after 15 days staying in solutions containing salts or PVA by dynamic light scattering. The intracellular uptake of KB cells incubated with PCL-CS micelles was evidenced by confocal laser scanning microscope upon loading fluorescein isothiocyanate labeled bovine serum albumin as a probe.     

Enzymatic degradation of block copolymers prepared from epsilon-caprolactone and poly(ethylene glycol)

Block copolymers were prepared by ring-opening polymerization of epsilon-caprolactone in the presence of monohydroxyl or dihydroxyl poly(ethylene glycol) (PEG), using Zn powder as catalyst. The resulting poly(epsilon-caprolactone) (PCL)-PEG diblock and PCL-PEG-PCL triblock copolymers were characterized by various analytical techniques such as NMR, size-exclusion chromatography, differential scanning calorimetry, and X-ray diffraction. Both copolymers were semicrystalline polymers, the crystalline structure being of the PCL type. Films were prepared by casting dichloromethane solutions of the polymers on a glass plate. Square samples with dimensions of 10 x 10 mm were allowed to degrade in a pH = 7.0 phosphate buffer solution containing Pseudomonas lipase. Data showed that the introduction of PEG blocks did not decrease the degradation rate of poly(epsilon-caprolactone).     

Polyphosphates strongly inhibit the tRNA dependent synthesis of poly(A) catalyzed by poly(A) polymerase from Saccharomyces cerevisiae

Polyphosphates of different chain lengths (P₃, P₄, P₁₅, P₃₅), (1 μM) inhibited 10, 60, 90 and 100%, respectively, the primer (tRNA) dependent synthesis of poly(A) catalyzed poly(A) polymerase from Saccharomyces cerevisiae. The relative inhibition evoked by p₄A and P₄ (1 μM) was 40 and 60%, respectively, whereas 1 μM Ap₄A was not inhibitory. P₄ and P₁₅ were assayed as inhibitors of the enzyme in the presence of (a) saturating tRNA and variable concentrations of ATP and (b) saturating ATP and variable concentrations of tRNA. In (a), P₄ and P₁₅ behaved as competitive inhibitors, with K_i values of 0.5 μM and 0.2 μM, respectively. In addition, P₄ (at 1 μM) and P₁₅ (at 0.3 μM) changed the Hill coefficient (n_H) from 1 (control) to about 1.3 and 1.6, respectively. In (b), the inhibition by P₄ and P₁₅ decreased V and modified only slightly the K_m values of the enzyme towards tRNA.     

Synthesis of temperature-responsive heterobifunctional block copolymers of poly(ethylene glycol) and poly(N-isopropylacrylamide)

Heterobifunctional block copolymers of poly(ethylene glycol) (PEG) and poly(N-isopropylacrylamide) (PNIPAM) were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization of NIPAM using a macromolecular trithiocarbonate PEG-based chain transfer agent. The polymerization showed all the expected features of living radical polymerization and allowed the synthesis of copolymers with different lengths of the PNIPAM block. The synthesized block copolymers contained a carboxylic acid group from L-lysine at the focal point and a trithiocarbonate group at the terminus of the PNIPAM block. The trithiocarbonate functionality was converted into a thiol group and used for conjugation of biotin to the end of the PNIPAM block. The copolymers exhibited temperature-dependent association behavior in aqueous solution with a phase transition of approximately 32 degrees C. The described heterobifunctional block copolymers show promise for surface modifications with the potential for stimulus-controlled surface presentation of ligands attached to the terminus of the PNIPAM block.     

New amphiphilic poly(2-ethyl-2-oxazoline)/ poly(L-lactide) triblock copolymers

A new series of cationic, thermo-sensitive, and biodegradable poly(L-lactide)-poly(2-ethyl-2-oxazoline)-poly(L-lactide) (PLLA-PEOz-PLLA) triblock copolymers were synthesized by ring-opening polymerization. With increasing molecular weight and crystallinity of hydrophobic PLLA blocks, the critical micellization concentrations (CMC) occurred at lower concentration. The PLLA-PEOz-PLLA aqueous solution was transparent at room temperature. Heating the solution resulted in precipitations, which were caused by the combination of dehydration of water around PEOz and the aggregations of PLLA segments. Acid/base titration profiles indicated that PLLA-PEOz-PLLA were protonated at neutral and acidic conditions. Considerable buffering capacity was found over the entire pH range. The specific PLLA-PEOz-PLLA triblock copolymers with thermal- and pH-sensitive properties can be tailored by varying the compositions and can be applied as controlled release carries for biomedical applications.     

新型生物可降解塑料——多聚羟基烷酸研究进展

面对日益严重的白色污染 ,人们迫切需要一种能在自然界较快分解的新型塑料。多聚羟基烷酸是原核生物在不平衡代谢条件下形成的碳源和能源贮藏物质 ,这种贮藏物质如同淀粉、糖原一样 ,当生命活动需要时可以再分解利用。由于多聚羟基烷酸有着与石化塑料相似的理化性质 ,又能在一定条件下被微生物迅速而彻底地降解 ,因此是一种理想的传统石化塑料替代品。1　多聚羟基烷酸的理化及生物学特性1 1　多聚羟基烷酸的分子结构及理化性质多聚羟基烷酸是由羟基脂肪酸单体首尾相联构成的高分图 1　多聚羟基烷酸的分子结构子聚合物。又分为不同种类。如多…     

Degradation of poly(3-hydroxybutyrate) by poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1

The extracellular poly(3-hydroxybutyrate) depolymerase purified from Alcaligenes faecalis T₁ has two disulfide bonds, one of which appears to be necessary for the full enzyme activity. This depolymerase hydrolyzed not only hydrophobic poly(3-hydroxybutyrate) but also water-soluble trimer and larger oligomers of D-(−)-3-hydroxybutyrate, regardless of their solubilities in water. Kinetic analyses with oligomers of various sizes indicated that the substrate cleaving site of the enzyme consisted of four subsites with individual affinities for monomer units of the substrate. Analyses of the hydrolytic products of oligomers, which had labeled D-(−)-3-hydroxybutyrate at the hydroxy terminus, showed that the enzyme cleaved only the second ester linkage from the hydroxy terminus of the trimer and tetramer, and acted as an endo-type hydrolase toward the pentamer and higher oligomers. The enzyme appeared to have a hydrophobic site which interacted with poly(3-hydroxybutyrate) and determined the affinity of the enzyme toward the hydrophobic substrate.