Synthesis of polyhydroxyalkanoate in the peroxisome of Pichia pastoris.

Polyhydroxyalkanoates (PHAs) are polyesters naturally produced by bacteria that have properties of biodegradable plastics and elastomers. A PHA synthase from Pseudomonas aeruginosa modified at the carboxy-end for peroxisomal targeting was transformed in Pichia pastoris. The PHA synthase was expressed under the control of the promoter of the P. pastoris acyl-CoA oxidase gene. Synthesis of up to 1% medium-chain-length PHA per g dry weight was dependent on both the expression of the PHA synthase and the presence of oleic acid in the medium. PHA accumulated as inclusions within the peroxisomes. P. pastoris could be used as a model system to study how peroxisomal metabolism needs to be modified to increase PHA production in other eukaryotes, such as plants.

Ocular drug delivery targeting the retina and retinal pigment epithelium using polylactide nanoparticles.

PURPOSE: To study the kinetics of polylactide (PLA) nanoparticle (NP) localization within the intraocular tissues and to evaluate their potential to release encapsulated material. METHODS: A single intravitreous injection (5 micro L) of an NP suspension (2.2 mg/mL) encapsulating either Rh-6G (Rh) or Nile red (Nr) was performed. Animals were killed at various times, and the NPs localization within the intraocular tissues was studied by environmental scanning electron microscopy (ESEM), confocal microscopy, light microscopy histology, fluorescence microscopy, and immunohistochemistry. Eyes injected with blank NPs, free Rh, or PBS solution were used as the control. RESULTS: ESEM showed the flow of the NPs from the site of injection into the vitreous cavity and their rapid settling on the internal limiting membrane. Histology demonstrated the anatomic integrity of the injected eyes and showed no toxic effects. A mild inflammatory cell infiltrate was observed in the ciliary body 6 hours after the injection and in the posterior vitreous and retina at 18 to 24 hours. The intensity of inflammation decreased markedly by 48 hours. Confocal and fluorescence microscopy and immunohistochemistry showed that a transretinal movement of the NPs was gradually taking place with a later localization in the RPE cells. Rh encapsulated within the injected NPs diffused and stained the retina and RPE cells. PLA NPs were still present within the RPE cells 4 months after a single intravitreous injection. CONCLUSIONS: Intravitreous injection of PLA NPs appears to result in transretinal movement, with a preferential localization in the RPE cells. Encapsulated Rh diffuses from the NPs and stains the neuroretina and the RPE cells. The findings support the idea that specific targeting of these tissues is feasible. Furthermore, the presence of the NPs within the RPE cells 4 months after a single injection shows that a steady and continuous delivery of drugs can be achieved.

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Plants naturally produce the lipid-derived polyester cutin, which is found in the plant cuticle that is deposited at the outermost extracellular matrix of the epidermis covering nearly all aboveground tissues. Being at the interface between the cell and the external environment, cutin and the cuticle play important roles in the protection of plants from several stresses. A number of enzymes involved in the synthesis of cutin monomers have recently been identified, including several P450s and one acyl-CoA synthetase, thus representing the first steps toward the understanding of polyester formation and, potentially, polyester engineering to improve the tolerance of plants to stresses, such as drought, and for industrial applications. However, numerous processes underlying cutin synthesis, such as a controlled polymerization, still remain elusive. Suberin is a second polyester found in the extracellular matrix, most often synthesized in root tissues and during secondary growth. Similar to cutin, the function of suberin is to seal off the respective tissue to inhibit water loss and contribute to resistance to pathogen attack. Being the main constituent of cork, suberin is a plant polyester that has already been industrially exploited. Genetic engineering may be worth exploring in order to change the polyester properties for either different applications or to increase cork production in other species. Polyhydroxyalkanoates (PHAs) are attractive polyesters of 3-hydroxyacids because of their properties as bioplastics and elastomers. Although PHAs are naturally found in a wide variety of bacteria, biotechnology has aimed at producing these polymers in plants as a source of cheap and renewable biodegradable plastics. Synthesis of PHA containing various monomers has been demonstrated in the cytosol, plastids, and peroxisomes of plants. Several biochemical pathways have been modified in order to achieve this, including the isoprenoid pathway, the fatty acid biosynthetic pathway, and the fatty acid β-oxidation pathway. PHA synthesis has been demonstrated in a number of plants, including monocots and dicots, and up to 40% PHA per gram dry weight has been demonstrated in Arabidopsis thaliana. Despite some successes, production of PHA in crop plants remains a challenging project. PHA synthesis at high level in vegetative tissues, such as leaves, is associated with chlorosis and reduced growth. The challenge for the future is to succeed in synthesis of PHA copolymers with a narrow range of monomer compositions, at levels that do not compromise plant productivity. This goal will undoubtedly require a deeper understanding of plant biochemical pathways and how carbon fluxes through these pathways can be manipulated, areas where plant "omics" can bring very valuable contributions.

Polymers of 3-hydroxyacids in plants

Polyhydroxyalkanoates (PHA) are polyesters of bacterial origin that have properties of biodegradable plastics and elastomers. Synthesis of PHA in crop plants would allow the large-scale production and use of these biodegradable and renewable polymers as substitutes for petroleum-derived plastics. Synthesis of a diversity of PHAs in plants, such as Arabidopsis thaliana, rapeseed, corn and cotton, has been demonstrated through the genetic engineering of metabolic pathways in the cytoplasm, plastid and peroxisome. PHA can also be used as a novel tool to study various aspects of plant metabolism, such as the regulation of carbon flux to the fatty acid biosynthetic and degradation pathways.

Ocular biocompatibility of novel Cyclosporin A formulations based on methoxy poly(ethylene glycol)-hexylsubstituted poly(lactide) micelle carriers.

Topical ocular drug delivery has always been a challenge for pharmaceutical technology scientists. In the last two decades, many nano-systems have been studied to find ways to overcome the typical problems of topical ocular therapy, such as difficult corneal penetration and poor drug availability. In this study, methoxy poly(ethylene glycol)-hexylsubstituted poly(lactides) (MPEG-hexPLA) micelle formulations, which are promising nanocarriers for poorly water soluble drugs, were investigated for the delivery of Cyclosporin A (CsA) to the eye. As a new possible pharmaceutical excipient, the ocular compatibility of MPEG-hexPLA micelle formulations was evaluated. An in vitro biocompatibility assessment on human corneal epithelial cells was carried out using different tests. Cytotoxicity was studied by using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), and clonogenic tests and revealed that the CsA formulations and copolymer solutions were not toxic. After incubation with MPEG-hexPLA micelle formulations, the activation of caspase-dependent and -independent apoptosis as well as autophagy was evaluated using immunohistochemistry by analyzing the localization of four antibodies: (1) anti-caspase 3; (2) anti-apoptotic inducing factor (AIF); (3) anti-IL-Dnase II and (4) anti-microtubule-associated protein 1 light chain 3 (LC3). No apoptosis was induced when the cells were treated with the micelle solutions that were either unloaded or loaded with CsA. The ocular tolerance was assessed in vivo on rabbit eyes by Confocal Laser Scanning Ophthalmoscopy (CLSO), and very good tolerability was seen. The observed corneal surface was comparable to a control surface that was treated with a 0.9% NaCl solution. In conclusion, these results demonstrate that MPEG-hexPLA micelles are promising drug carriers for ocular diseases involving the activation of cytokines, such as dry eye syndrome and autoimmune uveitis, or for the prevention of corneal graft rejection.

Analysis of the alternative pathways for the beta-oxidation of unsaturated fatty acids using transgenic plants synthesizing polyhydroxyalkanoates in peroxisomes.

Degradation of fatty acids having cis-double bonds on even-numbered carbons requires the presence of auxiliary enzymes in addition to the enzymes of the core beta-oxidation cycle. Two alternative pathways have been described to degrade these fatty acids. One pathway involves the participation of the enzymes 2, 4-dienoyl-coenzyme A (CoA) reductase and Delta(3)-Delta(2)-enoyl-CoA isomerase, whereas the second involves the epimerization of R-3-hydroxyacyl-CoA via a 3-hydroxyacyl-CoA epimerase or the action of two stereo-specific enoyl-CoA hydratases. Although degradation of these fatty acids in bacteria and mammalian peroxisomes was shown to involve mainly the reductase-isomerase pathway, previous analysis of the relative activity of the enoyl-CoA hydratase II (also called R-3-hydroxyacyl-CoA hydro-lyase) and 2,4-dienoyl-CoA reductase in plants indicated that degradation occurred mainly through the epimerase pathway. We have examined the implication of both pathways in transgenic Arabidopsis expressing the polyhydroxyalkanoate synthase from Pseudomonas aeruginosa in peroxisomes and producing polyhydroxyalkanoate from the 3-hydroxyacyl-CoA intermediates of the beta-oxidation cycle. Analysis of the polyhydroxyalkanoate synthesized in plants grown in media containing cis-10-heptadecenoic or cis-10-pentadecenoic acids revealed a significant contribution of both the reductase-isomerase and epimerase pathways to the degradation of these fatty acids.

Schwann cell strip for peripheral nerve repair.

Many strategies have been investigated to provide an ideal substitute to treat a nerve gap injury. Initially, silicone conduits were used and more recently conduits fabricated from natural materials such as poly-3-hydroxybutyrate (PHB) showed good results but still have their limitations. Surgically, a new concept optimising harvested autologous nerve graft has been introduced as the single fascicle method. It has been shown that a single fascicle repair of nerve grafting is successful. We investigated a new approach using a PHB strip seeded with Schwann cells to mimic a small nerve fascicle. Schwann cells were attached to the PHB strip using diluted fibrin glue and used to bridge a 10-mm sciatic nerve gap in rats. Comparison was made with a group using conventional PHB conduit tubes filled with Schwann cells and fibrin glue. After 2 weeks, the nerve samples were harvested and investigated for axonal and Schwann cell markers. PGP9.5 immunohistochemistry showed a superior nerve regeneration distance in the PHB strip group versus the PHB tube group (> 10 mm, crossed versus 3.17+/- 0.32 mm respectively, P<0.05) as well as superior Schwann cell intrusion (S100 staining) from proximal (> 10 mm, crossed versus 3.40+/- 0.36 mm, P<0.01) and distal (> 10 mm, crossed versus 2.91+/- 0.31 mm, P<0.001) ends. These findings suggest a significant advantage of a strip in rapidly connecting a nerve gap lesion and imply that single fascicle nerve grafting is advantageous for nerve repair in rats.

Fibrinmatrix erhöht Adhärenz von regenerativen peripheren Nervenzellen [Fibrin matrix enhances adherence of peripheral nerve regenerative cells]

Optimal seeding of a nerve conduit with cells is a core problem in tissue engineering of constructing an artificial nerve substitute to gap lesions in the peripheral nerve system. An ideal nerve gap substitute would have to present an equally distributed number of cells that can activate the regrowing axons. This work shows a new in vitro technique of two-step seeding of cells inside a conduit and on layered mats that allows a valuable targeting of the cells and a proven survival in the environment of poly-3-hydroxybutyrate (PHB) conduits. The technique uses two components of diluted fibrin glue Tisseel. Initially, the chosen area on the mat was coated with thrombin followed from the seeding of a fibrinogen-cell compound. Using Sprague Dawley rat cells, we could demonstrate with immunohistochemistry (S100, DAPI) techniques that undifferentiated (uMSC) and Schwann cells (SC) mimicking differentiated mesenchymal stem cells (dMSC) as well as SC can be suspended and targeted significantly better in dissolvable diluted fibrin glue than in growth medium. Analysis showed significantly better values for adherence (p < 0.001) and drop off (p < 0.05) from seeded cells. Using this two-step application allows the seeding of the cells to be more precise and simplifies the handling of cell transplantation.

Polyhydroxyalknoate synthesis in plants as a tool for biotechnology and basic studies of lipid metabolism.

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyacids naturally synthesized in bacteria as a carbon reserve. PHAs have properties of biodegradable thermoplastics and elastomers and their synthesis in crop plants is seen as an attractive system for the sustained production of large amounts of polymers at low cost. A variety of PHAs having different physical properties have now been synthesized in a number of transgenic plants, including Arabidopsis thaliana, rape and corn. This has been accomplished through the creation of novel metabolic pathways either in the cytoplasm, plastid or peroxisome of plant cells. Beyond its impact in biotechnology, PHA production in plants can also be used to study some fundamental aspects of plant metabolism. Synthesis of PHA can be used both as an indicator and a modulator of the carbon flux to pathways competing for common substrates, such as acetyl-coenzyme A in fatty acid biosynthesis or 3-hydroxyacyl-coenzyme A in fatty acid degradation. Synthesis of PHAs in plant peroxisome has been used to demonstrate changes in the flux of fatty acids to the beta-oxidation cycle in transgenic plants and mutants affected in lipid biosynthesis, as well as to study the pathway of degradation of unusual fatty acids.

Synthesis of polyhydroxyalkanoate in the peroxisome of Saccharomyces cerevisiae by using intermediates of fatty acid beta-oxidation.

Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads. Saccharomyces cerevisiae was transformed with the Pseudomonas aeruginosa PHAC1 synthase modified for peroxisome targeting by the addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. The PHAC1 gene was put under the control of the promoter of the catalase A gene. PHA synthase expression and PHA accumulation were found in recombinant S. cerevisiae growing in media containing fatty acids. PHA containing even-chain monomers from 6 to 14 carbons was found in recombinant yeast grown on oleic acid, while odd-chain monomers from 5 to 15 carbons were found in PHA from yeast grown on heptadecenoic acid. The maximum amount of PHA accumulated was 0.45% of the dry weight. Transmission electron microscopy of recombinant yeast grown on oleic acid revealed the presence of numerous PHA inclusions found within membrane-bound organelles. Together, these data show that S. cerevisiae expressing a peroxisomal PHA synthase produces PHA in the peroxisome using the 3-hydroxyacyl coenzyme A intermediates of the beta-oxidation of fatty acids present in the media. S. cerevisiae can thus be used as a powerful model system to learn how fatty acid metabolism can be modified in order to synthesize high amounts of PHA in eukaryotes, including plants.

Modification of the monomer composition of polyhydroxyalkanoate synthesized in Saccharomyces cerevisiae expressing variants of the beta-oxidation-associated multifunctional enzyme.

Expression by Saccharomyces cerevisiae of a polyhydroxyalkanoate (PHA) synthase modified at the carboxy end by the addition of a peroxisome targeting signal derived from the last 34 amino acids of the Brassica napus isocitrate lyase (ICL) and containing the terminal tripeptide Ser-Arg-Met resulted in the synthesis of PHA. The ability of the terminal peptide Ser-Arg-Met and of the 34-amino-acid peptide from the B. napus ICL to target foreign proteins to the peroxisome of S. cerevisiae was demonstrated with green fluorescent protein fusions. PHA synthesis was found to be dependent on the presence of both the enzymes generating the beta-oxidation intermediate 3-hydroxyacyl-coenzyme A (3-hydroxyacyl-[CoA]) and the peroxin-encoding PEX5 gene, demonstrating the requirement for a functional peroxisome and a beta-oxidation cycle for PHA synthesis. Using a variant of the S. cerevisiae beta-oxidation multifunctional enzyme with a mutation inactivating the B domain of the R-3-hydroxyacyl-CoA dehydrogenase, it was possible to modify the PHA monomer composition through an increase in the proportion of the short-chain monomers of five and six carbons.

Unsaturated Polyester Resins from Recycled Polyethyleneterephthalate

Tämän diplomityön tarkoituksena oli tutkia miten kuluttajien kierrättämästä polyeteenitereftalaatista ( PET ) voi valmistaa tyydyttymättömiä polyesterihartseja. Työssä valmistettiin yleiskäyttöön soveltuva laminointihartsi sekä 'gel coat' -hartsi jota käytetään esim. veneiden pintamaalina. Yleishartsin depolymerointiin käytettiin propyleeniglykolia ja 'gel coat' -hartsin valmistamiseen neopentyyliglykolia. Polykondensaatiovaiheessa reaktioon lisättiin maleiinihappoa ja lopuksi hartsit liuotettiin styreeniin. Kirjallisuusosassa esitetään eri menetelmiä PET:n depolymeroimiseksi. Lisäksi esitetään eri vaihtoehtoja glykolien, happojen, katalyyttien ja vinyylimonomeerien valitsemiseksi tyydyttymättömien polyesterihartsien valmistuksessa. Analyysimenetelmiä nestemäisten ja kovetettujen hartsien tutkimiseen ja vertailuun käydään läpi kuten myös erilaisia sovelluksia polyesterihartsien käyttämiseksi. Kokeellinen osa todisti että PET-pullojäte voidaan prosessoida hartsiksiilman uusia investointeja prosessilaitteistoon. PET:n glykolyysi kesti viidestäseitsemään tuntia ja polykondensaatiovaihe kahdesta ja puolesta viiteen tuntiin. Hartsien molekyylipainot ja mekaanisten testien tulokset olivat vertailukelpoisia kaupallisten hartsien antamien tulosten kanssa. Glykolyysivaiheen momomeeri- ja oligomeeripitoisuudet mitattiin geelipermeaatiokromatografialla, jotta nähtiin miten pitkälle depolymerisaatio oli edennyt. Tätä tietoa voidaan hyödyntää uusien hartsireseptin suunnittelussa. Polymeeriketjussa jäljellä olevien C=C kaksoissidosten määrä ja niiden isomeraatioaste maleaattimuodosta fumaraattimuotoon mitattiin 1H-NMR -menetelmällä. Tislevesien koostumus määritettiin kaasukromatografialla, ja tulokset kertoivat katalyytin sisältämän kloorin reagoineen glykolien kanssa, johtaen suureen glykolikulutukseen ja muihin ei-toivottuihin sivureaktioihin. Hartsien sietokykyä auringon valolle mitattiin niiden UV-absorption avulla. Kummastakin hartsista valmistettiin 'gel coat' -maalit jotkalaitettiin sääkoneeseen, joka simuloi auringonpaistetta ja vesisadetta vuorotellen. Näistä 'gel coateista' mitattiin niiden kellastumista. Kummastakin hartsista tehdyt valut asetettiin myös sääkoneeseen ja IR-spektreistä ennen jajälkeen koetta nähtiin että C=O ja C-O esterisidoksia oli hajonnut.

New fibrin conduit for peripheral nerve repair.

An ideal substitute to treat a nerve gap has not been found. Initially, silicone conduits were employed. Later, conduits were fabricated from collagen or polyesters carbonates. More recently, it has been shown that a bioresorbable material, poly-3-hydroxybutyrate (PHB), can enhance nerve repair. The present investigation shows the use of fibrin as a conduit to guide nerve regeneration and bridge nerve defects. In this study we prepared and investigated a novel nerve conduit made from fibrin glue. Using a rodent sciatic nerve injury model (10-mm gap), we compared the extent of nerve regeneration through the new fibrin conduits versus established PHB conduits. After 2 and 4 weeks, conduits containing proximal and distal stumps were harvested. We evaluated the initial axon and Schwann cell stimulation using immunohistochemistry. The conduits presented full tissue integration and were completely intact. Axons crossed the gap after 1 month. Immunohistochemistry using the axonal marker PGP 9.5 showed a superior nerve regeneration distance in the fibrin conduit compared with PHB (4.1 mm versus 1.9 mm). Schwann cell intrusion (S100 staining) was similarly enhanced in the fibrin conduits, both from the proximal (4.2 mm versus 2.1 mm) and distal ends (3.2 mm versus 1.7 mm). These findings suggest an advantage of the new fibrin conduit for the important initial phase of peripheral nerve regeneration. The use of fibrin glue as a conduit is a step toward a usable graft to bridge peripheral nerve lesions. This might be clinically interesting, given the widespread acceptance of fibrin glue among the surgical community.

PEN as substrate for new solar cell technologies

The possible use of polyethylene naphthalate as substrate for low-temperature deposited solar cells has been studied in this paper. The transparency of this polymer makes it a candidate to be used in both substrate and superstrate configurations. ZnO:Al has been deposited at room temperature on top of PEN. The resulting structure PEN/ZnO:Al presented good optical and electrical properties. PEN has been successfully textured (nanometer and micrometer random roughness) using hot-embossing lithography. Reflector structures have been built depositing Ag and ZnO:Al on top of the stamped polymer. The deposition of these layers did not affect the final roughness of the whole. The reflector structure has been morphologically and optically analysed to verify its suitability to be used in solar cells.

Poly(butylene terephthalate-co-5-tert-butyl isophthalate) Copolyesters: Synthesis, Characterization and Properties

A series of poly(butylene terephthalate) copolyesters containing 5-tert-butyl isophthalate units up to 50%-mole, as well as the homopolyester entirely made of these units, were prepared by polycondensation from the melt. The microstructure of the copolymers was determined by NMR to be at random for the whole range of compositions. The effect exerted by the 5-tert-butyl isophthalate units on thermal, tensile and gas transport properties was evaluated. Both Tm and crystallinity as well as the mechanical moduli were found to decrease steadily with copolymerization whereas Tg increased and the polyesters became more brittle. Permeability and solubility sligthly increased also with the content in substituted units whereas the diffusion coefficient remained practically constant. For the homopolyester poly(5-tert-butyl isophthalate), all these properties were found to deviate significantly from the general trend displayed by copolyesters suggesting that a different chain mode of packing in the amorphous phase is likely adopted in this case.