Valérie Langlois

Poly(β-malic acid): obtaining high molecular weights by improvement of the synthesis route

Abstract Poly(β-malic acid), PMLA, was first synthesized with the aim of being used as a macromolecular prodrug. However, this biodegradable polymer is now the parent compound of a large family functional polymers, copolymers and polystereoisomers. The requirement for high molecular weight polymers for a series of temporary therapeutic or specific applications needs to be conducted to examine the different steps of the synthesis route starting from either racemic or chiral aspartic acid. Drastic purifications of the intermediate products and of the β-substituted-β-lactone used as monomer has allowed the synthesis of polymers with high molecular weights, in a reproducible manner. In the case of racemic poly(β-malic acid benzyl ester), a precursor of PMLA, it is now possible to prepare polymers with a MSEC superior to 150 000 (polystyrene standards). The specific catalytic hydrogenolysis of the lateral benzyl protecting groups can be carried out and leads to the corresponding PMLA with long molecular chains, which are necessary for certain applications in vivo. The results have been extended to different racemic and optically active derivatives of this poly(3-hydroxy aci) ester type. Consequently, reproducible characteristics of the corresponding polymeric materials can be obtained.

Bacterial poly-3-hydroxyalkenoates with epoxy groups in the side chains

Pseudomonas oleovorans was grown on 10-undecenoic acid and on a mixture of sodium octanoate and 10-undecenoic acid. With either or both of these substrates, cells produced polyesters containing unsaturated side chains in the repeating units. In the case of 10-undecenoic acid as the sole nutrient, the microorganism produced a polymer containing only repeating units with unsaturated side chains. A second bacterium, Rhodospirillum rubrum was grown on 4-pentanoic acid alone and it produced polyesters with shorter pendant groups in the repeating units, including both saturated and unsaturated side chains. Epoxidation of these different bacterial polyesters with m-chloroperbenzoic acid, as 4 chemical reagent, yielded to quantitative conversions of the unsaturated groups into epoxy groups as determined by 1H- and 13C-NMR, no side reaction was observed on the macromolecular chain by molecular weights measurements. More important, it has been possible to produce new functional bacterial polyesters containing terminal epoxy groups in the side chains, in variable proportions up to 37% by growing P. oleovorans on a 10-epoxyundecanoic acid and sodium octanoate culture feed mixture.

Biocomposite scaffolds based on electrospun poly(3-hydroxybutyrate) nanofibers and electrosprayed hydroxyapatite nanoparticles for bone tissue engineering applications.

The electrospinning technique combined with the electrospraying process provides a straightforward and versatile approach for the fabrication of novel nanofibrous biocomposite scaffolds with structural, mechanical, and biological properties potentially suitable for bone tissue regeneration. In this comparative investigation, three types of poly(3-hydroxybutyrate) (PHB)-based scaffolds were engineered: (i) PHB mats by electrospinning of a PHB solution, (ii) mats of PHB/hydroxyapatite nanoparticle (nHA) blends by electrospinning of a mixed solution containing PHB and nHAs, and (iii) mats constituted of PHB nanofibers and nHAs by simultaneous electrospinning of a PHB solution and electrospraying of a nHA dispersion. Scaffolds based on PHB/nHA blends displayed improved mechanical properties compared to those of neat PHB mats, due to the incorporation of nHAs within the fibers. The electrospinning/electrospraying approach afforded biocomposite scaffolds with lower mechanical properties, due to their higher porosity, but they displayed slightly better biological properties. In the latter case, the bioceramic, i.e. nHAs, largely covered the fiber surface, thus allowing for a direct exposure to cells. The 21 day-monitoring through the use of MTS assays and SEM analyses demonstrated that human mesenchymal stromal cells (hMSCs) remained viable on PHB/nHA biocomposite scaffolds and proliferated continuously until reaching confluence.

Polyhydroxyalkanoate (PHA) biosynthesis in Thermus thermophilus: Purification and biochemical properties of PHA synthase

The biosynthesis of polyhydroxyalkanoates (PHAs) was studied, for the first time, in the thermophilic bacterium Thermus thermophilus. Using sodium gluconate (1.5% w/v) or sodium octanoate (10 mM) as sole carbon sources, PHAs were accumulated to approximately 35 or 40% of the cellular dry weight, respectively. Gas chromatographic analysis of PHA isolated from gluconate-grown cells showed that the polyester (Mw: 480,000 g.mol−1) was mainly composed of 3-hydroxydecanoate (3HD) with a molar fraction of 64%. In addition, 3-hydroxyoctanoate (3HO), 3-hydroxyvalerate (3HV) and 3-hydroxybutyrate (3HB) occurred as constituents. In contrast, the polyester (Mw: 391,000 g mol−1) from octanoate-grown cells was composed of 24.5 mol% 3HB, 5.4 mol% 3HO, 12.3 mol% 3-hydroxynonanoate (3HN), 14.6 mol% 3HD, 35.4 mol% 3-hydroxyundecanoate (3HUD) and 7.8 mol% 3-hydroxydodecanoate (3HDD). Activities of PHA synthase, a β-ketothiolase and an NADPH-dependent reductase were detected in the soluble cytosolic fraction obtained from gluconate-grown cells of T. thermophilus. The soluble PHA synthase was purified 4271-fold with 8.5% recovery from gluconate-grown cells, presenting a Km of 0.25 mM for 3HB-CoA. The optimal temperature of PHA synthase activity was about 70°C and acts optimally at pH near 7.3. PHA synthase activity was inhibited 50% with 25 μM CoA and lost all of its activity when it was treated with alkaline phosphatase. PHA synthase, in contrary to other reported PHA synthases did not exhibit a lag phase on its kinetics, when low concentration of the enzyme was used. Incubation of PHA synthase with 1 mM N-ethyl-maleimide inhibits the enzyme 56%, indicating that cysteine might be involved in the catalytic site of the enzyme. Acetyl phosphate (10 mM) activated both the native and the dephosphorylated enzyme. A major protein (55 kDa) was detected by SDS-PAGE. When a partially purified preparation was analyzed on native PAGE the major band exhibiting PHA synthase activity was eluted from the gel and analyzed further on SDS-PAGE, presenting the first purification of a PHA synthase from a thermophilic microorganism.

A first overview of textile fibers, including microplastics, in indoor and outdoor environments.

Studies about microplastics in various environments highlighted the ubiquity of anthropogenic fibers. As a follow-up of a recent study that emphasized the presence of man-made fibers in atmospheric fallout, this study is the first one to investigate fibers in indoor and outdoor air. Three different indoor sites were considered: two private apartments and one office. In parallel, the outdoor air was sampled in one site. The deposition rate of the fibers and their concentration in settled dust collected from vacuum cleaner bags were also estimated. Overall, indoor concentrations ranged between 1.0 and 60.0 fibers/m3. Outdoor concentrations are significantly lower as they range between 0.3 and 1.5 fibers/m3. The deposition rate of the fibers in indoor environments is between 1586 and 11,130 fibers/day/m2 leading to an accumulation of fibers in settled dust (190-670 fibers/mg). Regarding fiber type, 67% of the analyzed fibers in indoor environments are made of natural material, primarily cellulosic, while the remaining 33% fibers contain petrochemicals with polypropylene being predominant. Such fibers are observed in marine and continental studies dealing with microplastics. The observed fibers are supposedly too large to be inhaled but the exposure may occur through dust ingestion, particularly for young children.

Preparation of a novel artificial bacterial polyester modified with pendant hydroxyl groups.

The Poly(hydroxyalkanoate) (PHA) chemical modifications represent an alternative route to introduce functional groups, which cannot be introduced by bioconversion. PHAs containing unsaturated chains were readily converted into polyesters containing a terminal hydroxyl group on the side chains. With the use of the borane-tetrahydrofuran complex, the pendant side chain alkenes were quantitatively transformed into hydroxyl functions. The conversion proceeded to completion without a significant decrease in molecular weight. The introduction of hydroxyl groups in the products was confirmed from Fourier transform infrared and 1H NMR analysis. The presence of repeating units containing pendant hydroxyl groups in the proportion 25 mol % caused an increase in hydrophilicity of these new PHAs because they were soluble in polar solvents such as ethanol. Besides, these reactive PHAs can be used to bind bio-active molecules or to prepare novel graft copolymers with desired properties.

Bioactive functionalized polymer of malic acid for bone repair and muscle regeneration.

A bioactive poly(β-hydroxyalkanoate) derived from malic acid was prepared and tested on bone repair and muscle regeneration. This functionalized and hydrolyzable polymer was obtained after several steps, the first one being the anionic copolymerization of three malolactonic acid esters. Chemical modifications were carried out on the terpolymer to turn benzyl-protecting groups into carboxyl groups and allyl groups into sulfonate groups. The resulting polymer bore carboxylate, sulfonate, and sec-butyl pendent groups in 65/25/10 molar proportions and were aimed at interacting with heparan binding growth factors. This polymer did not present any toxic effect in cell viability of HepG2 cells, over a large range of concentrations (0.01-0.25 mg l-1). Its ability to improve wound healing was tested in vivo and positive results are reported. Furthermore, the bioactivity of this polymer was evaluated using the regeneration model of Extensor digitorum longus (EDL) rat muscle. The study displayed a significant increase in the muscle regeneration and maturation.

Poly(β -malic acid) derivatives with unsaturated lateral groups: epoxidation as model reaction of the double bonds reactivity

Poly(β-malic acid) derivatives bearing a lateral allyl or 3-methyl-3-butenyl ester group have been prepared by anionic ring opening polymerization or copolymerization of 4-allyloxycarbonyl-2-oxetanone or 4-[3-methyl-3-butenyloxycarbonyl]-2-oxetanone and 4-benzyloxycarbonyl-2-oxetanone as comonomer. These new chiral β-substituted-β-lactones with unsaturated lateral groups have been synthesized from aspartic acid as precursor and by using allylic alcohol or 3-methyl-3-buten-1-ol for opening bromosuccinic acid anhydride, an intermediate compound in the monomer synthesis route. The proportion of unsaturated lateral groups in the polymer was strictly controlled by the proportion of the corresponding lactone in the initial monomers mixture and can vary up to 100%. Functionalized polyesters have been prepared and characterized, and to test the reactivity of the present double bonds, epoxidation has been carried out by using m-chloroperbenzoic acid and dimethyldioxirane as chemical reagents. The activation of the double bond was depending on its chemical environment and on the polymer composition, but quantitative epoxidation has been achieved.

4-Carboxy-2-oxetanone as a new chiral precursor in the preparation of functionalized racemic or optically active poly(malic acid) derivatives

SummaryRacemic and optically active 4-carboxy-2-oxetanones have been prepared, starting from racemic, (R)-4-benzyloxycarbonyl-2-oxetanone, by catalytic hydrogenolysis of the lateral benzyl protecting group. This new β-substituted-β-lactone (malolactonic acid), which was considered as totally unstable due to the presence of a carboxyl group, has been isolated, characterized and prepared in large quantities. The liberated carboxylic acid function can be used for coupling reactions with different types of molecules in the goal to tailor make functionalized multimeric macromolecules (reactive polymers, supported catalysts, liquid crystals polymers, macromolecular prodrugs). This possibility has been examplified by using 2, 4, 5-trichlorophenol as activating agent and chloramphenicol as bioactive molecule, which have been bound to malolactonic acid and then copolymerized by anionic ring opening polymerization in the presence of 4-benzyloxycarbonyl-2-oxetanone. It has been shown that this new route conducts to activated derivatives of poly (malic acid) and polymeric drug carriers patterns.

Facile Synthesis of Multicompartment Micelles Based on Biocompatible Poly(3-hydroxyalkanoate)

In this paper, a straightforward method to produce poly(3-hydroxyalkanoate)-based multicompartment micelles (MCMs) is presented. Thiol-ene addition is used to graft sequentially perfluorooctyl chains and poly(ethylene glycol) oligomers onto poly(3-hydroxyoctanoate-co-hydroxyundecenoate) oligomers backbone. Well-defined copolymers are obtained as shown by ¹H NMR and size-exclusion chromatography. After nanoprecipitation in water, novel PHA-based MCMs are evidenced by cryo-transmission electron microscopy. Moreover, the cytocompatibility of MCMs is demonstrated in vitro via cell viability assay.