Nanoscale structure of poly(ethylene glycol) hybrid block copolymers containing amphiphilic beta-strand peptide sequences

This paper discusses the solid state and melt nanoscale structure of a series of novel poly(ethylene glycol) (PEG) hybrid di- and triblock copolymers, which contain amphiphilic beta-strand peptide sequences. The block copolymers have been prepared via solid-phase synthesis, affording perfectly monodisperse peptide segments with a precisely defined alpha-amino acid sequence. Attenuated total reflection Fourier transform infrared spectroscopy and X-ray scattering experiments indicate that the self-assembly properties of the peptide sequences are retained upon conjugation to PEG and mediate the formation of an ordered superstructure consisting of alternating PEG layers and peptide domains with an highly organized antiparallel beta-sheet structure. The results suggest that combination of biological structural motifs with synthetic polymers may be a versatile strategy for the development of novel self-assembled materials with complex internal structures and the potential to interface with biology.     

Biosynthesis of ethylene glycol in Escherichia coli

Ethylene glycol (EG) is an important platform chemical with steadily expanding global demand. Its commercial production is currently limited to fossil resources; no biosynthesis route has been delineated. Herein, a biosynthesis route for EG production from d-xylose is reported. This route consists of four steps: d-xylose?→?d-xylonate?→?2-dehydro-3-deoxy-d-pentonate?→?glycoaldehyde?→?EG. Respective enzymes, d-xylose dehydrogenase, d-xylonate dehydratase, 2-dehydro-3-deoxy-d-pentonate aldolase, and glycoaldehyde reductase, were assembled. The route was implemented in a metabolically engineered Escherichia coli, in which the d-xylose?→?d-xylulose reaction was prevented by disrupting the d-xylose isomerase gene. The most efficient construct produced 11.7 g?L^?1 of EG from 40.0 g?L^?1 of d-xylose. Glycolate is a carbon-competing by-product during EG production in E. coli; blockage of glycoaldehyde?→?glycolate reaction was also performed by disrupting the gene encoding aldehyde dehydrogenase, but from this approach, EG productivity was not improved but rather led to d-xylonate accumulation. To channel more carbon flux towards EG than the glycolate pathway, further systematic metabolic engineering and fermentation optimization studies are still required to improve EG productivity.     

Solution self-assembly of hybrid block copolymers containing poly(ethylene glycol) and amphiphilic beta-strand peptide sequences

The self-assembly in aqueous solution of hybrid block copolymers consisting of amphiphilic beta-strand peptide sequences flanked by one or two PEG chains was investigated by means of circular dichroism spectroscopy, small-angle X-ray scattering, and transmission electron microscopy. In comparison with the native peptide sequence, it was found that the peptide secondary structure was stabilized against pH variation in the di- and tri-block copolymers with PEG. Small-angle X-ray scattering indicated the presence of fibrillar structures, the dimensions of which are comparable to the estimated width of a beta-strand (with terminal PEG chains in the case of the copolymers). Transmission electron microscopy on selectively stained and dried specimens shows directly the presence of fibrils. It is proposed that these fibrils result from the hierarchical self-assembly of peptide beta-strands into helical tapes, which then stack into fibrils.     

Poly(ethylene glycol) block copolymers

The ubiquitous use of poly(ethylene glycol) in the biomaterials field has also boosted the research activity in the chemical derivatization of this polymer. We focused our interest on the preparation of tailor-made poly(ethylene glycol)-based structures and on the study of structure-activity relationships for its functionalization, as preliminary steps for the preparation of smart functional materials. More specifically, amphiphilic and cationic block copolymers were prepared for prospective use in the preparation of self-assembled carriers, and Michael-type addition of thiols onto acrylates was studied as a model for end-group reaction leading to hydrogel formation.     

Biosynthesis of a repressor/nuclease hybrid protein

The phage T7 endonuclease gene was fused to the 3' end of the lac repressor gene. The hybrid protein exhibits repressor and nuclease functions in a manner dependent on the conformation of the DNA. With supercoiled DNA, nuclease activity is directed to the major cruciform, whereas with linear DNA, the enzyme cleaves preferentially restriction fragments carrying the operator. These properties render the hybrid protein a unique probe of DNA conformation in vitro and in vivo.     

Biosynthesis and accumulation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-polyethylene glycol,a hybrid co-polymer by endophytic Bacillus cereus RCL 02

Bioprocess and Biosystems Engineering - Co-polymerization of microbial polyesters, polyhydroxyalkanoates (PHAs), with synthetic polymers has become an established and promising tool in the recent...     

Monomolecular assembly of siRNA and poly(ethylene glycol)-peptide copolymers

In this work, we design and investigate the complex formation of highly uniform monomolecular siRNA complexes utilizing block copolymers consisting of a cationic peptide moiety covalently bound to a poly(ethylene glycol) (PEG) moiety. The aim of the study was to design a shielded siRNA construct containing a single siRNA molecule to achieve a sterically stabilized complex with enhanced diffusive properties in macromolecular networks. Using a 14 lysine-PEG (K14-PEG) linear diblock copolymer, formation of monomolecular siRNA complexes with a stoichiometric 1:3 grafting density of siRNA to PEG is realized. Alternatively, similar PEGylated monomolecular siRNA particles are achieved through complexation with a graft copolymer consisting of six cationic peptide side chains bound to a PEG backbone. The hydrodynamic radii of the resulting complexes as measured by fluorescence correlation spectroscopy (FCS) were found to be in good agreement with theoretical predictions using polymer brush scaling theory of a PEG decorated rodlike molecule. It is furthermore demonstrated that the PEG coating of the siRNA-PEG complexes can be rendered biodegradable through the use of a pH-sensitive hydrazone or a reducible disulfide bond linker between the K14 and the PEG blocks. To model transport under in vivo conditions, diffusion of these PEGylated siRNA complexes is studied in various charged and uncharged matrix materials. In PEG solutions, the diffusion coefficient of the siRNA complex is observed to decrease with increasing polymer concentration, in agreement with theory of probe diffusion in semidilute solutions. In charged networks, the behavior is considerably more complex. FCS measurements in fibrin gels indicate complete dissociation of the diblock copolymer from the complex, while transport in collagen solutions results in particle aggregation.     

Biosynthesis of polyhydroxyalkanoate copolymers from mixtures of plant oils and 3-hydroxyvalerate precursors

The combination of plant oils and 3-hydroxyvalerate (3HV) precursors were evaluated for the biosynthesis of polyhydroxyalkanoate (PHA) copolymers containing 3HV monomers by Cupriavidus necator H16. Among various mixtures of plant oils and 3HV-precursors, the mixture of palm kernel oil and sodium propionate was suitable for the biosynthesis of high concentration of PHA (6.8gL(-1)) containing 7mol% of 3HV. The 3HV monomer composition can be regulated in the range of 0-23mol% by changing culture parameters such as the initial pH, and the nitrogen source and its concentration. PHA copolymers with high weight-average molecular weights (Mw) ranging from 1,400,000 to 3,100,000Da were successfully produced from mixtures of plant oils and 3HV-precursors. The mixture of plant oils and sodium propionate resulted in PHA copolymers with higher M(w) compared to the mixture of plant oils and sodium valerate. DSC analysis on the PHA containing 3HV monomers showed the presence of two distinct melting temperature (Tm), which indicated that the PHA synthesized might be a blend of P(3HB) and P(3HB-co-3HV). Sodium propionate appears to be the better precursor of 3HV than sodium valerate.     

Diblock copolymers based on dihydroxyacetone and ethylene glycol: synthesis, characterization, and nanoparticle formulation

Polymeric biomaterials have played an integral role in tissue engineering, biomedical devices, and targeted drug delivery. Block copolymers are especially important because their physical and chemical properties can be controlled by adjusting the ratio, size, and type of constituting blocks. Herein, the synthesis and characterization of diblock copolymers composed of poly(ethylene glycol) and a polycarbonate based on the metabolic intermediate, dihydroxyacetone, are reported. The length of the dihydroxyacetone-based block was controlled by adjusting the reactant feed ratios and initiator injection conditions. Intermediates and final products were characterized via (1)H NMR, GPC, DSC, TGA, and diffusion-ordered NMR spectroscopy. The dihydroxyacetone-based hompolymer is insoluble in water and most organic solvents, but is hydrophilic in nature. This, coupled with poly(ethylene glycol)'s solubility characteristics, allows the block copolymer to form nanoparticles in aqueous and organic anti-solvents. Dynamic light scattering and TEM results indicated the formation of spherical nanoparticles.     

Intracellular trafficking of polyamidoamine-poly(ethylene glycol) block copolymers in DNA delivery

The delivery of nucleic acids has the potential to revolutionize medicine by allowing previously untreatable diseases to be clinically addressed. Viral delivery systems have shown immunogenicity and toxicity dangers, but synthetic vectors have lagged in transfection efficiency. Previously, we developed a modular, linear-dendritic block copolymer architecture with high gene transfection efficiency compared to commercial standards. This rationally designed system makes use of a cationic dendritic block to condense the anionic DNA and forms complexes with favorable endosomal escape properties. The linear block provides biocompatibility and protection from serum proteins, and can be functionalized with a targeting ligand. In this work, we quantitate performance of this system with respect to intracellular barriers to gene delivery using both high-throughput and traditional approaches. An image-based, high-throughput assay for endosomal escape is described and applied to the block copolymer system. Nuclear entry is demonstrated to be the most significant barrier to more efficient delivery and will be addressed in future versions of the system.     

Dynamic light scattering study of self-assembly of HPMA hybrid graft copolymers

The time course of self-assembly of a hybrid hydrogel system was investigated using dynamic light scattering (DLS) techniques. The self-assembling system consisted of a hydrophilic synthetic N-(2-hydroxypropyl)methacrylamide (HPMA) polymer backbone and a pair of oppositely charged peptide grafts (CCE and CCK). These two distinct pentaheptad peptides were anticipated to act as physical cross-linkers by the formation of antiparallel coiled-coil heterodimers. Equimolar mixture of HPMA graft copolymers CCE-P and CCK-P solutions (where P is the HPMA copolymer backbone) with total concentration from 1.25 to 10 mg/mL were measured at a scattering angle 90 degrees and room temperature. A critical extension of average relaxation time was observed with increasing concentration and incubation time. To reveal the role of coiled-coil grafts in the self-assembly process, a pair of modified random coil peptides, CCEw and CCKy, was designed. The DLS evaluation of HPMA copolymer conjugates (CCEw-P and CCKy-P) at total concentration of 10 mg/mL demonstrated that no association occurred after 28 h of incubation. Moreover, addition of a competing peptide (CCK) or a denaturant (guanidium chloride, GndHCl) to the self-assembled CCE-P/CCK-P hydrogels resulted in partial disassembly or collapse of the hydrogel clusters. These results correlated to changes in the secondary structure of peptides (grafts) as measured by circular dichroism spectroscopy (CD). These investigations supported the hypothesis that the self-assembly of CCE-P/CCK-P into hybrid hydrogels is mediated by the formation of coiled-coil heterodimers.     

Biosynthesis of poly(3-hydroxybutyrate) copolymers by Azotobacter chroococcum 7B: A precursor feeding strategy

A precursor feeding strategy for effective biopolymer producer strain Azotobacter chroococcum 7B was used to synthesize various poly(3-hydroxybutyrate) (PHB) copolymers. We performed experiments on biosynthesis of PHB copolymers by A. chroococcum 7B using various precursors: sucrose as the primary carbon source, various carboxylic acids and ethylene glycol (EG) derivatives [diethylene glycol (DEG), triethylene glycol (TEG), poly(ethylene glycol) (PEG) 300, PEG 400, PEG 1000] as additional carbon sources. We analyzed strain growth parameters including biomass and polymer yields as well as molecular weight and monomer composition of produced copolymers. We demonstrated that A. chroococcum 7B was able to synthesize copolymers using carboxylic acids with the length less than linear 6C, including poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) (PHB-4MHV) using Y-shaped 6C 3-methylvaleric acid as precursor as well as EG-containing copolymers: PHB–DEG, PHB–TEG, PHB–PEG, and PHB–HV–PEG copolymers using short-chain PEGs (with n?≤?9) as precursors. It was shown that use of the additional carbon sources caused inhibition of cell growth, decrease in polymer yields, fall in polymer molecular weight, decrease in 3-hydroxyvalerate content in produced PHB–HV–PEG copolymer, and change in bacterial cells morphology that were depended on the nature of the precursors (carboxylic acids or EG derivatives) and the timing of its addition to the growth medium.     

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).     

Biosynthesis of polylactic acid and its copolymers using evolved propionate CoA transferase and PHA synthase

For the synthesis of polylactic acid (PLA) and its copolymers by one‐step fermentation process, heterologous pathways involving Clostridium propionicum propionate CoA transferase (Pct_Cp) and Pseudomonas sp. MBEL 6‐19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1_Ps6‐19) were introduced into Escherichia coli for the generation of lactyl‐CoA endogenously and incorporation of lactyl‐CoA into the polymer, respectively. Since the wild‐type PhaC1_Ps6‐19 did not efficiently accept lactyl‐CoA as a substrate, site directed mutagenesis as well as saturation mutagenesis were performed to improve the enzyme. The wild‐type Pct_Cp was not able to efficiently convert lactate to lactyl‐CoA and was found to exert inhibitory effect on cell growth, random mutagenesis by error‐prone PCR was carried out. By employing engineered PhaC1_Ps6‐19 and Pct_Cp, poly(3‐hydroxybutyrate‐co‐lactate), P(3HB‐co‐LA), containing 20–49 mol% lactate could be produced up to 62 wt% from glucose and 3HB. By controlling the 3HB concentration in the medium, PLA homopolymer and P(3HB‐co‐LA) containing lactate as a major monomer unit could be synthesized. Also, P(3HB‐co‐LA) copolymers containing various lactate fractions could be produced from glucose alone by introducing the Cupriavidus necator β‐ketothiolase and acetoacetyl‐CoA reductase genes. Fed‐batch cultures were performed to produce P(3HB‐co‐LA) copolymers having 9–64 mol% of lactate, and their molecular weights, thermal properties, and melt flow properties were determined. Biotechnol. Bioeng. 2010; 105: 150–160. © 2009 Wiley Periodicals, Inc.     

RGD-grafted poly-L-lysine-graft-(polyethylene glycol) copolymers block non-specific protein adsorption while promoting cell adhesion

A novel class of surface-active copolymers is described, designed to protect surfaces from nonspecific protein adsorption while still inducing specific cell attachment and spreading. A graft copolymer was synthesized, containing poly-(L-lysine) (PLL) as the backbone and substrate binding and poly(ethylene glycol) (PEG) as protein adsorption-resistant pendant side chains. A fraction of the grafted PEG was pendantly functionalized by covalent conjugation to the peptide motif RGD to induce cell binding. The graft copolymer spontaneously adsorbs from dilute aqueous solution onto negatively charged surfaces. The performance of RGD-modified PLL-g-PEG copolymers was analyzed in protein adsorption and cell culture assays. These coatings efficiently blocked the adsorption of serum proteins to Nb(2)O(5) and tissue culture polystyrene while specifically supporting attachment and spreading of human dermal fibroblasts. This surface functionalization technology is expected to be valuable in both the biomaterial and biosensor fields, because different signals can easily be combined, and sterilization and application are straightforward and cost-effective.     

Star-shaped poly(ethylene glycol)-block-polyethylenimine copolymers enhance DNA condensation of low molecular weight polyethylenimines

Star-shaped poly(ethylene glycol)-block-polyethylenimine [star-(PEG-b-PEI)] significantly enhance plasmid DNA condensation of low molecular weight (MW) PEIs. The star-block copolymers were prepared via a facile synthesis route using hexamethylene diisocyanate as linker between PEG and PEI blocks. NMR and FT-IR spectroscopy confirmed the structures of intermediately activated PEG and final products. Furthermore, the copolymers were characterized by size exclusion chromatography, static light scattering, and viscosimetry. Their molecular weights (M(w) 19-26 kDa) were similar to high MW PEI (25 kDa). Thermoanalytical investigations (thermogravimetric analysis, differential scanning calorimetry) were also performed and verified successful copolymer synthesis. DNA condensation with the low MW PEIs (800 and 2000 Da) and their 4- and 8-star-block copolymers was studied using atomic force microscopy, dynamic light scattering, zeta-potential measurements, and ethidium bromide (EtBr) exclusion assay. It was found that low MW PEIs formed huge aggregates (500 nm to 2 microm) in which DNA is only loosely condensed. By contrast, the star-block copolymers yielded small (80-110 nm), spherical and compact complexes that were stable against aggregation even at high ionic strength and charge neutrality. Furthermore, as revealed in the EtBr exclusion assay these star-block copolymers exhibited a DNA condensation potential as high as high MW PEI. Since these star-(PEG-block-PEI) copolymers are composed of relatively nontoxic low MW PEI and biocompatible PEG, their potential as gene delivery agents merits further investigations.     

Tuning the properties of elastin mimetic hybrid copolymers via a modular polymerization method

We have synthesized elastin mimetic hybrid polymers (EMHPs) via the step-growth polymerization of azide-functionalized poly(ethylene glycol) (PEG) and alkyne-terminated peptide (AKAAAKA)(2) (AK2) that is abundant in the cross-linking domains of the natural elastin. The modular nature of our synthesis allows facile adjustment of the peptide sequence to modulate the structural and biological properties of EMHPs. Therefore, EMHPs containing cell-binding domains (CBDs) were constructed from α,ω-azido-PEG and two types of alkyne-terminated AK2 peptides with sequences of DGRGX(AKAAAKA)(2)X (AK2-CBD1) and X(AKAAAKA)(2)XGGRGDSPG (AK2-CBD2, X = propargylglycine) via a step-growth, click coupling reaction. The resultant hybrid copolymers contain an estimated five to seven repeats of PEG and AK2 peptides. The secondary structure of EMHPs is sensitive to the specific sequence of the peptidic building blocks, with CBD-containing EMHPs exhibiting a significant enhancement in the α-helical content as compared with the peptide alone. Elastomeric hydrogels formed by covalent cross-linking of the EMHPs had a compressive modulus of 1.06 ± 0.1 MPa. Neonatal human dermal fibroblasts (NHDFs) were able to adhere to the hydrogels within 1 h and to spread and develop F-actin filaments 24 h postseeding. NHDF proliferation was only observed on hydrogels containing RGDSP domains, demonstrating the importance of integrin engagement for cell growth and the potential use of these EMHPs as tissue engineering scaffolds. These cell-instructive, hybrid polymers are promising candidates as elastomeric scaffolds for tissue engineering.     

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.     

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.