Highly Branched Polymers Based on Poly(amino acid)s for Biomedical Application.

Polymers consisting of amino acid building blocks continue to receive consideration for biomedical applications. Since poly(amino acid)s are built from natural amino acids, the same building blocks proteins are made of, they are biocompatible, biodegradable and their degradation products are metabolizable. Some amino acids display a unique asymmetrical AB2 structure, which facilitates their ability to form branched structures. This review compares the three forms of highly branched polymeric structures: structurally highly organized dendrimers, dendrigrafts and the less organized, but readily synthesizable hyperbranched polymers. Their syntheses are reviewed and compared, methods of synthesis modulations are considered and variations on their traditional syntheses are shown. The potential use of highly branched polymers in the realm of biomedical applications is discussed, specifically their applications as delivery vehicles for genes and drugs and their use as antiviral compounds. Of the twenty essential amino acids, L-lysine, L-glutamic acid, and L-aspartic acid are asymmetrical AB2 molecules, but the bulk of the research into highly branched poly(amino acid)s has focused on the polycationic poly(L-lysine) with a lesser extent on poly(L-glutamic acid). Hence, the majority of potential applications lies in delivery systems for nucleic acids and this review examines and compares how these three types of highly branched polymers function as non-viral gene delivery vectors. When considering drug delivery systems, the small size of these highly branched polymers is advantageous for the delivery of inhalable drug. Even though highly branched polymers, in particular dendrimers, have been studied for more than 40 years for the delivery of genes and drugs, they have not translated in large scale into the clinic except for promising antiviral applications that have been commercialized.

Modification of polyhydroxyalkanoates: Evaluation of the effectiveness of novel copper(II) catalysts in click chemistry.

Copper(I) catalyzed azide-alkyne cycloadditions, click reactions, are an established synthetic tool to derivatize polymers. Only a few catalytic systems have been explored towards the derivatization of functionalized poly(3­hydroxyalkanoate)s, PHAs, using click reactions. Here, the performances of three Cu(II)-catalysts supported by tetradentate polypyridyl ligands, [Cu(L1)ClO4]ClO4, [Cu(L2)ClO4]ClO4 and [Cu(L3)ClO4]ClO4, were examined in click reactions on functionalized PHAs carrying either terminal azido or alkyne groups in the side chain and the results were compared to the traditional CuSO4·5H2O/Na ascorbate and the organo-soluble Cu(I) bromotris(triphenylphosphine)copper(I), CuBr(PPh3)3 catalysts. It was determined that the effectiveness of the catalytic systems depended on the molecular architecture of the polymer and the nature of the small molecule reactants to be clicked onto the PHA. Click reactions on PHAs with terminal azido groups were catalyzed with Cu(II)-catalysts, but not with CuBr(PPh3)3. For alkyne-containing polymers CuBr(PPH3)3 effected 65% conversion in contrast to Cu(II) catalysts that were ineffective. While no strong trend was found, differences in the effectiveness were related to dissimilarities in the accessibility of the alkyne moiety for the reactive Cu(I) species. Propargyl benzoate was most effectively clicked onto a azido PHA (100% conversion) when catalyzed by CuSO4·5H2O/Na ascorbate, however the click reaction with a similar reactant, propargyl acetate, was more effectively catalyzed by a Cu(II)-catalyst supported by a tetradentate polypyridyl ligand (44% conversion).

Impact of Cationic Charge Density and PEGylated Poly(Amino Acid) Tercopolymer Architecture on Their Use as Gene Delivery Vehicles. Part 2: DNA Protection, Stability, Cytotoxicity, and Transfection Efficiency.

The impact of the molecular architecture on the transfection efficiency of PEGylated poly(amino acid) block copolymers was investigated for PEG-b-p(l-Lys)x -b-p(l-Leu)y , PEG-b-p(l-Leu)x -b-p(l-Lys)y , and PEG-b-p((l-Leu)x -co-(l-Lys)y ). The block lengths of p(l-Lys) and p(l-Leu) were varied between 10, 20, and 40; and 10 and 20, respectively, to study the influence of the ionic/hydrophobic balance. The results show that ABC triblock copolymers form smaller and more stable polyplexes with plasmid DNA than AB diblock copolymers-as verified by long-term aggregation and ethidium bromide exclusion studies-protect the DNA more effectively against nucleases, and provide better transfection efficiencies, as indicated by total protein as well as luciferase expression. More detailed studies revealed that triblock copolymers with p(l-Leu) forming the C-block were most efficient in DNA complexation with a 2.3 times higher transfection rate. Furthermore, increasing the cationic character by increasing the p(l-Lys) chain length led to up to 25% higher transfection but at the same time induced some cytotoxicity. Diblock copolymers, where the amino acid-building blocks exist as a random copolymer, bind more loosely with DNA leading to less compact and less stable aggregates with lower transfection efficiencies.

Impact of Cationic Charge Density and PEGylated Poly(amino acid) Tercopolymer Architecture on Their Use as Gene Delivery Vehicles. Part 1: Synthesis, Self-Assembly, and DNA Complexation.

The interaction of PEGylated poly(amino acid)s with their biological targets depends on their chemical nature and spatial arrangement of their building blocks. The synthesis, self-assembly, and DNA complexation of ABC terblock copolymers consisting of poly(ethylene glycol), (PEG), poly(l-lysine), and poly(l-leucine), are reported. Block copolymers are produced by a metal-free, living ring-opening polymerization of respective amino acid N-carboxyanhydrides using amino-terminated PEG as macroinitiator: (PEG-b-p(l-Lys)x -b-p(l-Leu)y , PEG-b-p(l-Leu)x -b-p(l-Lys)y , and PEG-b-p((l-Lys)x -co-p(l-Leu)y ). Sizes of self-assembled nanoparticles depend on the formation method. The nanoprecipitation method proves useful for copolymers with the poly(l-lysine) block protected as trifluoroacetate, effective diameters range between 92 and 132 nm, while direct dissolution in distilled water is suitable for the deprotected copolymers, yielding effective diameters between 52 and 173 nm. Critical micelle concentration (CMC) analyses corroborate particle size analyses and show a distinct impact of the molecular architecture; the lowest CMC (8 µg mL-1 ) is observed when the poly(l-leucine) segment forms the C-block and the hydrophilic, disassembly driving poly(l-lysine) segment is short. DNA complexation, evaluated by gel motility and RiboGreen analyses, depends strongly on the molecular architecture. A more efficient DNA complexation is observed when poly(l-lysine) and poly(l-leucine) form individual blocks as opposed to them forming a copolymer.

Synthesis and Application of Aurophilic Poly(Cysteine) and Poly(Cysteine)-Containing Copolymers.

The redox capacity, as well as the aurophilicity of the terminal thiol side groups, in poly(Cysteine) lend a unique characteristic to this poly(amino acid) or polypeptide. There are two major application fields for this polymer: (i) biomedical applications in drug delivery and surface modification of biomedical devices and (ii) as coating for electrodes to enhance their electrochemical sensitivity. The intended application determines the synthetic route for p(Cysteine). Polymers to be used in biomedical applications are typically polymerized from the cysteine N-carboxyanhydride by a ring-opening polymerization, where the thiol group needs to be protected during the polymerization. Advances in this methodology have led to conditions under which the polymerization progresses as living polymerization, which allows for a strict control of the molecular architecture, molecular weight and polydispersity and the formation of block copolymers, which eventually could display polyphilic properties. Poly(Cysteine) used as electrode coating is typically polymerized onto the electrode by cyclic voltammetry, which actually produces a continuous, pinhole-free film on the electrode via the formation of covalent bonds between the amino group of Cysteine and the carbon of the electrode. This resulting coating is chemically very different from the well-defined poly(Cysteine) obtained by ring-opening polymerizations. Based on the structure of cysteine a significant degree of cross-linking within the coating deposited by cyclic voltammetry can be assumed. This manuscript provides a detailed discussion of the ring-opening polymerization of cysteine, a brief consideration of the role of glutathione, a key cysteine-containing tripeptide, and examples for the utilization of poly(Cysteine) and poly(Cysteine)-containing copolymers, in both, the biomedical as well as electrochemical realm.

Chemical modification of functionalized polyhydroxyalkanoates via "Click" chemistry: A proof of concept.

A novel approach to the post-biosynthetic chemical modification of bromo and alkyne functionalized poly(3-hydroxyalkanoates), (PHAs), via copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain promoted azide alkyne cycloaddition (SPAAC) is reported. Optimum conditions for the biosynthesis of the PHA copolymers, poly(3-hydroxynonanoate-co-3-hydroxy-11-bromoundecanoate) (PHNUBr) and poly(3-hydroxynonanoate-co-3-hydroxy-10-undecynoate) (PHNUD), using Pseudomonas oleovorans as cell factories were 20h of fermentation time and a total carbon substrate concentration of 40mM. Percent incorporation of brominated repeat units and alkyne repeat units were 38.5% and 50% respectively, as determined by 1H NMR. PHNUBr was converted into an azido-terminated precursor for the polymer analogous "click" reactions via SN2 nucleophilic substitution reaction using sodium azide with a yield of 96.7% and analyzed by FTIR and 1H NMR. CuAAC reactions were used to attach propargyl benzoate and methyl-2-azidoacetate to the PHAs with terminal azido and alkyne functional side groups respectively, with yields of 78.2% and 65.4% respectively. FTIR analysis of the products showed the disappearance of the azide peak at 2093.5cm-1 and the alkynyl CCH stretch at 3292cm-1. 1H NMR analysis confirmed the formation of the expected triazole linkage; showing the expected proton chemical shift corresponding to the triazole proton at 7.73 and 7.47ppm respectively. A strain promoted azide-alkyne reaction was used to attach (1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN-OH) to the azido-terminated PHA with an average yield of 94.5%. Products were analyzed by FTIR and 1H NMR.

Cooperative effects of fatty acids and n-butanol on lipid membrane phase behavior.

Biodiesel-derived crude glycerol can be fermented to produce n-butanol, which is a platform chemical for biorefining and a biofuel. One limitation to crude glycerol fermentation is the presence of long-chain fatty acids (FAs) that can partition into cellular membranes, leading to membrane fluidization and interdigitation, which can inhibit cellular function. In this work, we have examined the phase behavior of dipalmitoylphosphatidylcholine (DPPC, C16:0) membranes and the membrane partitioning of n-butanol as a function of FA degree of unsaturation (steric, oleic, and linoleic acids) using differential scanning calorimetry (DSC) and monolayer surface pressure studies. All three FAs at 15mol% (85mol% DPPC) prevented interdigitation by n-butanol based on the DSC results. n-Butanol partitioning and membrane expansion was greatest for DPPC/oleic acid membranes, where monounsaturated oleic acid (OA, C18:1) was miscible in gel and fluid phase DPPC. Saturated steric acid (SA, C18:0), which ordered the membranes and yielded a SA-rich phase during melting, led to a modest increase in n-butanol partitioning compared to DPPC alone. Di-unsaturated linoleic acid (LA, C18:2), which disordered the membranes and phase separated, had little affect on n-butanol partitioning into the DPPC-rich phases. The effects of OA and LA are attributed to the additional interfacial area provided by these FAs due to acyl tail 'kinks' at the carbon double bonds. These results show that exogenous FAs can partition into membranes, impacting n-butanol partitioning and acting cooperatively with n-butanol to alter membrane structure.

Homeoviscous response of Clostridium pasteurianum to butanol toxicity during glycerol fermentation.

Clostridium pasteurianum ATCC 6013 achieves high n-butanol production when glycerol is used as the sole carbon source. In this study, the homeoviscous membrane response of C. pasteurianum ATCC 6013 has been examined through n-butanol challenge experiments. Homeoviscous response is a critical aspect of n-butanol tolerance and has not been examined in detail for C. pasteurianum. Lipid membrane compositions were examined for glycerol fermentations with n-butanol production, and during cell growth in the absence of n-butanol production, using gas chromatography-mass spectrometry (GC-MS) and proton nuclear magnetic resonance ((1)H-NMR). Membrane stabilization due to homeoviscous response was further examined by surface pressure-area (π-A) analysis of membrane extract monolayers. C. pasteurianum was found to exert a homeoviscous response that was comprised of an increase lipid tail length and a decrease in the percentage of unsaturated fatty acids with increasing n-butanol challenge. This led to a more rigid or stable membrane that counteracted n-butanol fluidization. This is the first report on the changes in the membrane lipid composition during n-butanol production by C. pasteurianum ATCC 6013, which is a versatile microorganism that has the potential to be engineered as an industrial n-butanol producer using crude glycerol.

n-Butanol partitioning into phase-separated heterogeneous lipid monolayers.

Cellular adaptation to elevated alcohol concentration involves altering membrane lipid composition to counteract fluidization. However, few studies have examined the biophysical response of biologically relevant heterogeneous membranes. Lipid phase behavior, molecular packing, and elasticity have been examined by surface pressure-area (π-A) analysis in mixed monolayers composed of saturated dipalmitoylphosphatidylcholine (DPPC) and unsaturated dioleoylphosphatidylcholine (DOPC) as a function of DOPC and n-butanol concentration. n-Butanol partitioning into DPPC monolayers led to lipid expansion and increased elasticity. Greater lipid expansion occurred with increasing DOPC concentration, and a maximum was observed at equimolar DPPC:DOPC consistent with n-butanol partitioning between coexisting liquid expanded (LE, DOPC) phases and liquid condensed (LC, DPPC) domains. This led to distinct changes in the size and morphology of LC domains. In DOPC-rich monolayers the effect of n-butanol adsorption on π-A behavior was less pronounced due to DOPC tail kinking. These results point to the importance of lipid composition and phase coexistence on n-butanol partitioning and monolayer restructuring.

Role of ionic strength on n-butanol partitioning into anionic dipalmitoyl phosphatidylcholine/phosphatidylglycerol vesicles.

Bacteria adjust their membrane lipid composition to counteract the fluidizing effects of alcohol and to adapt to elevated alcohol concentrations during fermentation. Bacterial membranes are rich in anionic phosphatidylglycerols (PGs), but little is known regarding alcohol partitioning into anionic membranes, particularly for n-butanol. This work examines the effects of lipid charge on n-butanol partitioning into anionic membrane vesicles composed of dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylglycerol (DPPG) in the absence and presence of salt (phosphate-buffered saline, PBS; 0.152 and 1.52 M). Above 0.135 M n-butanol, the membranes were interdigitated irrespective of DPPG or salt concentration, consistent with previous results for neutral membranes, such as DPPC. Increasing salt concentration led to greater n-butanol partitioning in DPPC membranes and caused aggregation/fusion. However, aggregation/fusion was prevented with increasing DPPG concentration (i.e., increasing membrane charge) and small vesicles were observed. The results suggest that n-butanol partitioning, and subsequent changes in membrane and vesicle structure, was driven by a balance between the "salting-out" of n-butanol, interlipid electrostatic interactions, and interfacial cation binding and hydration. This is the first study to the best of our knowledge to examine the effects of n-butanol partitioning on model cell membranes composed of negatively charged lipids in the presence of salts.

n-Butanol partitioning and phase behavior in DPPC/DOPC membranes.

Membrane phase behavior and fluidization have been examined in heterogeneous membranes composed of dipalmitoylphosphatidylcholine (DPPC, a saturated lipid) and dioleoylphosphatidylcholine (DOPC, an unsaturated lipid) at n-butanol concentrations below and above the interdigitation threshold of DPPC. Our results show that the presence of DOPC did not influence the interdigitation concentration of n-butanol on DPPC (0.1-0.13 M) despite the fact that DOPC increased n-butanol partitioning into the membranes. When DPPC was the continuous phase, up to equimolar DPPC:DOPC, n-butanol partitioning into gel or interdigitated DPPC was only slightly affected by the presence of DOPC. In this case a "cooperative effect" of DOPC + n-butanol eliminated the DPPC pretransition phase and yielded an untilted gel-like phase. When DOPC was the continuous phase, more n-butanol was needed to cause DPPC interdigitation (0.2 M), which was attributed to n-butanol residing at the interface between DOPC and DPPC domains. To our knowledge, this is the first study to examine the effects of n-butanol partitioning on membranes composed of saturated and unsaturated lipids that exhibit coexisting phase states.

Impact of impurities in biodiesel-derived crude glycerol on the fermentation by Clostridium pasteurianum ATCC 6013.

During the production of biodiesel, crude glycerol is produced as a byproduct at 10% (w/w). Clostridium pasteurianum has the inherent potential to grow on glycerol and produce 1,3-propanediol and butanol as the major products. Growth and product yields on crude glycerol were reported to be slower and lower, respectively, in comparison to the results obtained from pure glycerol. In this study, we analyzed the effect of each impurity present in the biodiesel-derived crude glycerol on the growth and metabolism of glycerol by C. pasteurianum. The crude glycerol contains methanol, salts (in the form of potassium chloride or sulfate), and fatty acids that were not transesterified. Salt and methanol were found to have no negative effects on the growth and metabolism of the bacteria on glycerol. The fatty acid with a higher degree of unsaturation, linoleic acid, was found to have strong inhibitory effect on the utilization of glycerol by the bacteria. The fatty acid with lower or no degrees of unsaturation such as stearic and oleic acid were found to be less detrimental to substrate utilization. The removal of fatty acids from crude glycerol by acid precipitation resulted in a fermentation behavior that is comparable to the one on pure glycerol. These results show that the fatty acids in the crude glycerol have a negative effect by directly affecting the utilization of glycerol as the carbon source, and hence their removal from crude glycerol is an essential step towards the utilization of crude glycerol.

Synthesis and self-assembly of well-defined poly(amino acid) end-capped poly(ethylene glycol) and poly(2-methyl-2-oxazoline).

Poly(ethylene glycol) (PEG) and poly(2-methyl-2-oxazoline) (PMOx) are water-soluble, biocompatible polymers with stealth hemolytic activities. Poly(amino acid) (PAA) end-capped PEG and PMOx were prepared using amino-terminated derivatives of PEG and PMOx as macroinitiators for the ring-opening polymerization of γ-benzyl protected l-glutamate N-carboxyanhydride and S-benzyloxycarbonyl protected l-cysteine N-carboxyanhydride, respectively, in the presence of urea, at room temperature. The molecular weight of the PAA moiety was kept between M(n) = 2200 and 3000 g mol(-1). PMOx was polymerized by cationic ring-opening polymerization resulting in molecular weights of M(n) = 5000 and 10,000 g mol(-1), and PEG was a commercial product with M(n) = 5000 g mol(-1). Here, we investigate the self-assembly of the resulting amphiphilic block copolymers in water and the effect of the chemical structure of the block copolymers on the solution properties of self-assembled nanostructures. The PEG-block-poly(amino acid), PEG-b-PAA, and PMOx-block-poly(amino acid), PMOx-b-PAA, block copolymers have a narrow and monomodal molecular weight distribution (PDI < 1.3). Their self-assembly in water was studied by dynamic light scattering and fluorescence spectroscopy. In aqueous solution, the block copolymers associate into particles with hydrodynamic radii (R(H)) ranging in size from R(H) 70 to 130 nm, depending on the block copolymer architecture and the polymer molecular weight. Larger R(H) and critical association concentration values were obtained for copolymers containing poly(S-benzyloxycarbonyl-l-cysteine) compared to their poly(γ-benzyl-L-glutamate) analogue. FTIR investigations revealed that the poly(γ-benzyl-L-glutamate) block adopts a helical conformation, while the poly(S-benzyloxycarbonyl-L-cysteine) block exists as ß-sheet.

Perspectives to produce positively or negatively charged polyhydroxyalkanoic acids.

An overview is provided on the possibilities of producing positively and negatively charged poly(ß-hydroxyalkanoates), PHAs. A large variety of bacterial polyesters with functionalized terminal side chains can be produced in microbial fermentation processes by a direct polymerization of respective carbon sources, that is, carbon sources that carry functional groups in their ω-position. However, charged PHAs are not accessible by a direct approach and must be synthesized via polymer-analogous reactions of functionalized bacterial polyesters. PHA polyanions are produced by converting the terminal functional groups into carboxylate groups, while PHA polycations are produced by introducing terminal amino groups. PHAs with terminal vinyl groups emerged as most suitable PHA precursors, as they can be produced in relatively high yields and the double bonds are sufficiently reactive. The oxidation of vinyl groups yields PHA polyanions. The conversion of terminal vinyl groups into epoxides with a subsequent ring-opening reaction with an amine yields PHA polycations. Other functionalized PHA that potentially lend themselves to polymer-analogous reactions are reviewed.

Evaluation of a cationic poly(ß-hydroxyalkanoate) as a plasmid DNA delivery system.

Poly(ß-hydroxyalkanoates) (PHAs) are biodegradable polymers produced by a wide range of bacteria. The structures of these polymers may be tuned by controlling the available carbon source composition, but the range of functional groups accessible in this manner is limited to those that the organism is able to metabolize. Much effort has been made to chemically modify the side chains of these polymers to achieve new materials with new applications. We have previously reported the synthesis of the first cationic PHA, poly(ß-hydroxyoctanoate)-co-(ß-hydroxy-11-(bis(2-hydroxyethyl)-amino)-10-hydroxyundecanoate) (PHON). Here, we report the use of this polymer as a plasmid DNA delivery system. PHON was found to bind and condense the DNA into positively charged particles smaller than 200 nm. In this manner, PHON was shown to protect plasmid DNA from nuclease degradation for up to 30 min. In addition, treatment of mammalian cells in vitro with PHON/DNA complexes resulted in luciferase expression as the result of the delivery of the encoded gene.

Synthesis and characterization of a cationic poly(beta-hydroxyalkanoate).

Poly(beta-hydroxyalkanoates) (PHAs) are biodegradable polyesters produced by a wide range of bacteria. The structures of these polymers may be tuned by controlling the carbon source composition in the feed stock, but the range of functional groups accessible in this manner is limited to those that the organism is able to metabolize. Much effort has been made to chemically modify the side chains of these polymers to achieve new materials. Here, we report the synthesis of the first cationic PHA, poly(beta-hydroxy-octanoate)- co-(beta-hydroxy-11-(bis(2-hydroxyethyl)-amino)-10-hydroxyundecanoate) (PHON). Pseudomonas putida Gpo1 was used to produce poly(beta-hydroxy-octanoate)- co-(beta-hydroxy-10-undecenoate) (PHOU), whose vinyl-terminated side chains were first converted to terminal epoxides and then modified with diethanolamine. The modification of PHOU was examined using (1)H, COSY, and HSQC NMR and GPC and resulted in a loss of molecular weight due to aminolysis and also in the introduction of side chains terminated with tertiary amine groups, which are protonated at physiological pH. The polycationic PHA is soluble in polar solvents such as DMSO, DMF, and water. The new biodegradable cationic polymers are envisioned as nucleic acid delivery systems.

Cloning, expression, purification, and characterization of a designer protein with repetitive sequences.

An artificial protein containing alternating hydrophilic-hydrophobic blocks of amino acids was designed in order to mimic the structure of synthetic multiblock copolymers. The hydrophobic block consisted of the six amino acids Ala Ile Leu Leu Ile Ile (AILLII) and the hydrophilic block of the eight amino acids Thr Ser Glu Asp Asp Asn Asn Gln (TSEDDNNQ). The coding DNA sequence of the cluster was inserted into an commercial pET 30a(+) vector using a two step strategy. The expression of the artificial protein in Escherichia coli was optimized using a temperature shift strategy. Only at cultivation temperature of 24 degrees C after induction expression was observed, whereas at 30 and 37 degrees C no target protein could be detected. Cells obtained from a 15L bioreactor cultivation of E. coli were disintegrated by mechanical methods. Interestingly, glass bead milling and high pressure homogenization resulted in a different solubility of the target protein. The further purification was carried out by affinity chromatography using the soluble homogenized protein. Extreme conditions (6M urea, 0.5M NaCl) were applied in order to prevent aggregation to insoluble particles. The designer protein showed an extremely high tendency to form dimers or trimers caused by intermolecular interactions which were even not broken under the conditions of SDS-polyacrylamide gel electrophoresis, rendering the behavior during purification different from proteins usually found in nature. The protein preparation was not completely pure according to SDS-PAGE stained by Coomassie blue or silver. In MALDI-TOF-MS, nano-ESI qTOF-MS of the entire protein preparation and nano-ESI-MS after digestion by trypsin and chymotrypsin impurities were not detectable.

Biocompatibility of materials implanted into the subretinal space of Yucatan pigs.

PURPOSE: To assess the biocompatibility of materials for possible use in subretinal prostheses. METHODS: Strips (0.5 x 5 mm; 10-microm thick) of either plain poly(imide) or poly(imide) coated with amorphous aluminum oxide (AAO), amorphous carbon (AC), parylene, poly(vinyl pyrrolidone) (PVP), or poly(ethylene glycol) (PEG) were each implanted into the subretinal space of four Yucatan miniature pigs. Two types of control surgery without implantation were performed in four other animals. Electroretinograms (ERGs) were performed before and after surgery. All animals were euthanatized 3 months after surgery, and histologic slides of the retina were assessed for 15 criteria. Paired, two-tailed Student's t-tests were used for statistical analyses. RESULTS: Across all animals, the mean amplitude of the ERG b-wave did not differ from baseline after 3 months. In implanted animals, the histologic analyses revealed that (1) all the implanted materials produced abnormalities that were significantly greater than in the control subjects; (2) overall, PEG, parylene, and PVP produced less histologic disruption than the other three materials; (3) parylene and PEG did not differ significantly from the control in disturbing retinal anatomy; (4) only PI and AAO produced RPE alterations that were significantly greater than in control subjects; and (5) AAO and PI produced a significantly greater degree of peri-implant cellular responses than did the other materials. CONCLUSIONS: All implants produced some alteration of the retina, but there were clear differences among the materials in the degree to which their presence disturbed the normal anatomy of the retina or RPE or incited tissue reactions around the implant.

Contact angle, WAXS, and SAXS analysis of poly(beta-hydroxybutyrate) and poly(ethylene glycol) block copolymers obtained via Azotobacter vinelandii UWD.

This study investigated and correlated physical properties and cell interactions of copolymers obtained by a poly(ethylene glycol) (PEG)-modulated fermentation of Azotobacter vinelandii UWD. PEGs with molecular weights of 400 and 3400 Da and di(ethylene glycol) (DEG) were used to modulate the bacterial synthesis of poly(beta-hydroxybutyrate) (PHB). The PHB crystallinity was determined by wide-angle X-ray scattering (WAXS). Small-angle X-ray scattering (SAXS) showed that lamellar distances decreased between the PHB and the PHB modulated with PEG or DEG. Furthermore, the contact angle of water on the PHB/PEG polymer surfaces decreased when compared to that of PHB. The significant decrease of the contact angle and corresponding increase in surface tension, as well as significant decrease in cell adhesion, suggest the presence of hydrophilic PEG and DEG within the hydrophobic surface.

Biosynthesis of natural-synthetic hybrid copolymers: polyhydroxyoctanoate-diethylene glycol.

A new natural-synthetic hybrid biomaterial has been isolated from the growth of Pseudomonas oleovorans in the presence of diethylene glycol (DEG). DEG was consumed by P. oleovorans with 20 mM sodium octanoate in modified E* medium, but its presence in the fermentation medium retarded cell growth and viability, influencing production and composition of polyhydroxyalkanoates with medium chain length substituents (mclPHAs) and consequently attenuating PHA yield. DEG affected the composition of the mclPHA with an increase in the C8 component: polyhydroxyoctanoate (PHO). Gas chromatography-mass spectrometry (GC-MS) was used to quantitatively monitor DEG in the system and reveal its cellular adsorption and penetration. Intracellularly, the DEG significantly reduced the molar mass of the mclPHA; PHO with a bimodal distribution of high and low molecular weight fractions was observed. 1H NMR, 2-D COSY, and heteronuclear single quantum coherence spectra confirmed that the high molecular weight fraction consisted of PHO chains terminated by DEG. Thus, the synthesis of this natural-synthetic hybrid copolymer, PHO-DEG, opens the way for microbial synthesis of a wide variety of PHA-DEG copolymers with a range of bioactive properties.