Enzyme-catalyzed degradation of poly(l-lactide)/poly(?-caprolactone) diblock, triblock and four-armed copolymers

Poly(l-lactide)/poly(?-caprolactone) diblock, triblock and four-armed copolymers with the same monomer feed ratio (50/50) were synthesised by two step ring opening polymerisation of successively added ?-caprolactone and l-lactide, using isopropanol, ethylene glycol, or pentaerythritol as initiator and zinc lactate as co-initiator. The resulting copolymers were characterised by ¹H NMR, DSC, SEC, and FT-IR, which confirmed the blocky characteristic of the copolymers. Solution cast films were allowed to degrade at 37 °C in the presence of proteinase K, and the degradation was monitored by gravimetry, DSC, SEC, ¹H NMR and ESEM. The effects of chain structure, block length and crystallinity on the degradation are discussed. The four-armed block copolymer degrades the most rapidly, while the diblock copolymer exhibited the slowest degradation rate. The difference was related to the crystallinity depending on both the molecular structure and block length. Little compositional or molar mass changes were obtained during degradation, which strongly supports a surface erosion mechanism, in agreement with ESEM observations.     

Blends as a strategy towards tailored hydrolytic degradation of poly(?-caprolactone-co-d,l-lactide)-poly(ethylene glycol)-poly(?-caprolactone-co-d,l-lactide) co-polymers

Binary blends were prepared from poly(?-caprolactone) (PCL), and P(CL-co-d,l-lactic acid)-P(ethylene glycol)-P(CL-co-d,l-lactic acid) co-polymers, where the d,l-LA content in the side chains varied from 0 to 70 mol%. Blend discs were fabricated by melt-molding, and the effect of blend composition on hydrolytic degradation was studied. Variations in medium pH were monitored, and morphological changes were observed using scanning electron microscopy. Blending of these co-polymers was found to constitute a simple means by which intermediate rates of water absorption and mass loss were obtained, compared to those observed in pure co-polymer preparations. In one of the blends, prepared from the two components containing 70 or 0 mol% d,l-LA in the side chains and thereby exhibiting large differences in degradation rate, hydrolysis resulted in the formation of a porous material over time. Furthermore, all blend samples maintained their initial shape throughout the study. Such materials may be interesting for further investigations for applications in cellular therapy and controlled release.     

Synthesis, characterization and degradability of the comb-type poly(4-hydroxyl-ε-caprolactone-co-ε-caprolactone)-g-poly(l-lactide)

The novel comb-type biodegradable graft copolymers based on ε-caprolactone and l-lactide were synthesized. Firstly, 2-oxepane-1,5-dione (OPD) was synthesized by the Baeyer-Villiger oxidation of 1,4-cyclohexanedione, and was subsequently copolymerized with ε-caprolactone (CL) to produce poly(2-oxepane-1,5-dione-co-ε-caprolactone) (POCL) catalyzed by stannous(II) 2-ethylhexanoate in toluene. Then, POCL was converted into poly(4-hydroxyl-ε-caprolactone-co-ε-caprolactone) (PHCL) using sodium borohydride as reductant. Finally, poly(4-hydroxyl-ε-caprolactone-co-ε-caprolactone)-g-poly(l-lactide) (PHCL-g-PLLA) were prepared successfully by bulk ring-opening polymerization of l-lactide using PHCL as a macro-initiator. All the copolymers have been characterized by ¹H and ¹³C NMR, DSC, and GPC. Compared with the random copolymer of poly(CL-co-LA), the elongation is highly increased. And the thermal analysis showed that the crystallization rate of the PCL backbone in the graft copolymers was greatly reduced compared to the PCL homopolymer. The hydrolytic degradation of the copolymer was much faster in a phosphate buffer (pH = 7.4) at 37 °C, which is confirmed by the weight loss and change of intrinsic viscosity.     

Biodegradable poly(dl-lactide)/poly(ε-caprolactone) mixtures: Miscibility at the air/water interface

Mixtures of biodegradable polymers, poly(dl-lactide) and poly(ε-caprolactone) monolayers at the air/water interface have been studied. Surface pressure-area isotherms of poly(dl-lactide), poly(ε-caprolactone) and their mixtures were obtained by monolayer compression at constant temperature. The behavior of the mixed monolayers was analyzed according to the classical addition rule. Good agreement was observed between experimental and ideal behavior except for one composition where a negative deviation was observed. The polymer monolayer miscibility was corroborated by comparison between the surface pressure-area isotherms of the random copolymers (dl-lactide-co-ε-caprolactone) and their mixtures at the same compositions. Brewster angle microscopy (BAM) shows homogeneity in the monolayers in the whole range of compositions. These results also confirm the miscibility of the mixtures.     

Enzymatic degradation of block copolymers obtained by sequential ring opening polymerization of l-lactide and ?-caprolactone

Copolymers of ?-caprolactone and l-lactide with different molar ratios were prepared via sequential ring opening polymerization (ROP) of both monomers. The resulting PCL-PLLA-PCL triblock copolymers were characterized by using NMR, SEC, DSC and XRD. One melting peak corresponding to the PCL block was detected, but the presence of PLLA decreased the crystallinity of PCL. Enzyme-catalyzed biodegradation of solution cast films was investigated at 37 °C in the presence of Pseudomonas lipase. It was observed that the PLLA component retarded the degradation of the block copolymer as compared to the PCL homopolymer. Therefore, the enzymatic degradation rate can be adjusted by varying the composition of the copolymers. ¹H NMR and SEC data showed no significant chemical composition or molecular weight changes during degradation, indicating that the degradation proceeded according to a surface erosion mechanism. ESEM confirmed surface erosion with appearance of a rugged morphology.     

Lipase-catalysed degradation of copolymers prepared from ?-caprolactone and dl-lactide

A series of copolymers were prepared by ring-opening polymerization of ?-caprolactone and dl-lactide, using zinc lactate as catalyst. The resulting PCL/PLA copolymers were characterized by various analytical techniques such as NMR, SEC, DSC and X-ray diffraction. The [CL]/[LA] ratios of the copolymers are very close to those in the feed, indicating a good conversion of monomers. The copolymers with CL contents higher than 50% appear semi-crystalline, the crystalline structure being of the PCL-type. Compression moulded polymer films were allowed to degrade in a pH = 7.6 phosphate buffer containing Pseudomonas lipase. Data show that copolymers with CL contents lower than 25% are not degradable and the degradation rate increases with CL content for CL-rich copolymers. Various soluble degradation products are detected in the degradation medium, including CL₁ to CL₃ and LA₁ to LA₄ homo-oligomers, and CL₂LA₁ co-oligomer. The presence of LA homo-oligomers and CL₂LA₁ co-oligomer suggests that Pseudomonas lipase can not only degrade PCL but also LA short blocks along PCL/PLA copolymer chains. On the other hand, little changes of composition are detected during degradation, in agreement with a surface erosion mechanism as shown by ESEM.     

Effect of partial crosslinking on morphology and properties of the poly(β-hydroxybutyrate)/poly(d,l-lactic acid) blends

Poly(β-hydroxybutyrate) (PHB) is a bio-based and biodegradable aliphatic polyester, however its application is limited by some disadvantages such as high price, brittleness, poor processability and low melt-strength due to serious thermal degradation. Partial crosslinking initiated by dicumyl peroxide (DCP) was applied in this work to improve the performance of poly(β-hydroxybutyrate)/poly(d,l-lactic acid) (PHB/PDLLA) blends. The partial crosslinking of the blends and its effect on the properties, morphology, rheology and thermal behavior of the blends were investigated. The tensile strength and impact toughness of the PHB were increased by incorporation of the PDLLA, which were improved further after the partial crosslinking because of an increased compatibility between the PHB and the PDLLA phases. The rheological study revealed that the storage modulus (G′) and complex viscosity (η*) of the blends were increased after addition of the DCP. On the other hand, the crystallization of PHB in the blends was restricted to a certain extent by the formation of partially crosslinked network while its crystal form was not modified.     

Structure-based study of antiamyloidogenic cyclic d,l-α-peptides

Protein misfolding and aggregation are the hallmarks of many devastating diseases. We have previously shown that cyclic d,l-α-peptide CP-2 reacts and stabilizes less toxic forms of amyloid β (Aβ), and protects the cells from Aβ-induced toxicity. Here, we performed extensive structure-based studies on CP-2 to elucidate the contribution of each residue to the total antiamyloidogenic activity and determine the interactions that are involved between CP-2 and Aβ. We showed that the hydrophobicity of CP-2 analogs correlates with their antiamyloidogenic potency, however, aromatic interactions are even more important for this activity. The antiamyloidogenic activity of CP-2 analogs also correlates with their ability to self-assemble, as shown by the critical micelle concentration measurements. The cell survival studies performed on rat pheochromocytoma PC-12 cells suggest that incorporation of an additional aromatic residue to the CP-2's sequence increases its protective effect against Aβ42-induced toxicity.     

New organotin(IV) complexes with l-Arginine, Nα-t-Boc-l-Arginine and l-Alanyl-l-Arginine: Synthesis, structural investigations and cytotoxic activity

Novel diorganotin(IV) derivatives of l-Arginine (HArg), N_α-(tert-Butoxycarbonyl)-l-Arginine (Boc-Arg-OH) and l-Ala-l-Arg (H₂Ala-Arg), H₂NC(NH)NH(CH₂)₃CH(NHR′)CO₂H, where R′ = H in HArg, R′ = C(O)OC(CH₃)₃ in Boc-Arg-OH, R′ = H₂NCH(CH₃)CO in H₂Ala-Arg and triorganotin(IV) derivatives of Boc-Arg-OH have been synthesized and structurally characterized. The complexes were investigated by FT-IR and ¹¹⁹Sn Mössbauer in the solid state and by ¹H, ¹³C, ¹¹⁹Sn and ¹H-¹H COSY NMR spectroscopy, in solution. The spectroscopic characterization leading to the proposed molecular structures was accomplished on the basis of these experiments. l-Arginine appears to behave as a chelating ligand through carboxylate and -NH₂ groups in Me₂Sn(Arg)₂, while in N_α-t-Boc-l-Arginine complex, the N_α-protected amino group being exempted from coordination, only the carboxylate groups are effectors of bonding to the organometallic moieties. FT-IR spectra give a clear indication that guanidino groups in all the complexes are not involved in coordination, since ν(CN-H) frequency of the terminal guanidino group is fairly constant and unshifted relative to the free ligand. The biological activity of organotin(IV)-complexes was also investigated by use of human HT29 colorectal carcinoma cells. The cytotoxic activity of the compounds was determined by the MTT quantitative colorimetric assay, capable of detecting viable cells in comparison with that exerted by cisplatin. A marked cytotoxic activity for nearly all complexes, is evident being higher than that exerted by cisplatin, while no significant improvement of activity was observed for Me₂Sn(Arg)₂ and Me₂Sn(Ala-Arg), which was confirmed by IC₅₀ values. Then, we assessed whether the cytotoxicity induced by organotin(IV) complexes was associated with the induction of apoptosis. Light microscopy analysis, performed to study the morphological changes induced in HT29 cells, confirmed the results obtained with MTT test. No significant morphological alterations were observed in HT29 cells after treatment with Me₂Sn(Ala-Arg) and Me₂Sn(l-Arg)₂. Cells treated with ⁿBu₂Sn(Boc-Arg)₂, ⁿBu₂Sn(Ala-Arg), ⁿBu₃Sn(Boc-Arg) and Me₃Sn(Boc-Arg), appeared rounded, isolated and detached from culture substrate, indicating the commitment to apoptotic cell death.     

Solvent-induced morphological diversification in poly(l-lactide-b-?-caprolactone) block copolymer thin films

Biodegradable block copolymer of poly(l-lactide-b-?-caprolactone) (P(LA-b-CL)) was dissolved in various solvents with different solubility as well as volatility, and spin-cast on a highly oriented pyrolytic graphite (HOPG) to prepare thin films. The surface morphologies were observed by using atomic force microscopy (AFM) in a dynamic force (tapping) mode. Particle like morphology was found in the thin films prepared form the dichloromethane and acetone. Higher volatility of dichloromethane and acetone resulted in the reflection of the particle like objects in the solution to HOPG substrate. In contrast, the P(LA-b-CL)s in toluene and 1,4-dioxane exhibited different morphologies compared to those in dichloromethane and acetone. Lower volatility of toluene and 1,4-dioxane assisted the epitaxial crystallization of PCL component along the HOPG lattice, that was revealed by enzymatic degradation of PLLA component by proteinase K. Thus, adjusting the solubility and solvent volatility for the film formation provided morphological divergence of the P(LA-b-CL) block copolymer, and this technique would be applicable for the surface patterning of biodegradable polymers.     

Direct access to new β-d-galactofuranoconjugates: application to the synthesis of galactofuranosyl-l-cysteine and l-serine

Galactofuranose post-translational modifications, although quite rare, were detected in some biomolecules produced by parasites. While hexopyranosides were already linked to various peptides and proteins, few hexofuranosides have been artificially conjugated to amino acids. We thus report herein a robust glycosylation methodology to obtain S-alkyl, O-serine and S-cysteine-β-d-galactofuranosides starting from readily available galactofuranose donors. O-Acetyl, thioimidoyl and acetimidoyl donors were compared in terms of yields and selectivity when reacted with mercaptans, l-cysteine and l-serine. Acetimidates turned out to be the best notably for amino acids glycosylation.     

Development of poly(ε-caprolactone-co-l-lactide) and poly(ε-caprolactone-co-δ-valerolactone) as new degradable binder used for antifouling paint

Copolyesters containing ε-caprolactone and l-lactide or ε-caprolactone and δ-valerolactone at different compositions were synthesized by using tetrabutoxytitane Ti(OBu)₄ at high temperature in bulk. A series of copolyesters were prepared by varying the compositions of both comonomers. These copolymers were characterized by using ¹H NMR, ¹³C NMR, gel permeation chromatography (GPC), differential scanning calorimetry (DSC) and MALDI-TOF mass spectrometry. ¹³C NMR analysis gave an insight on their microstructure. Structural parameters of the copolymers were obtained by calculating the triad sequence fractions. Poly(ε-caprolactone-co-l-lactide) has a more alternate structure than poly(ε-caprolactone-co-δ-valerolactone). The potential use of these copolyesters in antifouling coatings was examined because of their solubility in aromatic solvent and their hydration and hydrolytic degradation. Paints based on these new degradable binders had a good antifouling activity in Atlantic Ocean (France).     

Synthesis and characterization of biodegradable pH-sensitive hydrogels based on poly(?-caprolactone), methacrylic acid, and poly(ethylene glycol)

In this work, a novel biodegradable pH-sensitive hydrogel based on poly(?-caprolactone) (PCL), methoxpoly(ethylene glycol) (MPEG) and methacrylic acid (MAA) was prepared by UV-initiated free radical polymerization. The resulting macromonomers and hydrogels were characterized by FTIR and/or ¹H NMR. Swelling behaviour and pH sensitivity of the hydrogels were studied in detail. With increase in pH of aqueous medium from 1.2 to 7.2, swelling ratio of the hydrogels increased accordingly. The hydrolytic degradation behaviour was also investigated. The prepared biodegradable pH-sensitive hydrogel based on PCL, MPEG, and MAA might have great potential application in smart drug delivery system.     

In-depth study on aminolysis of poly(?-caprolactone): Back to the fundamentals

The aminolysis can effectively introduce primary amine (?NH₂) groups onto polyester materials, enabling a variety of subsequent surface biofunctionalization reactions. However, less attention has been paid to the basic knowledge of aminolysis reaction in terms of reaction kinetics and its influences on materials properties. In this study, taking the widely used poly(?-caprolactone) (PCL) as a typical example, the influences of diamines and solvent property on the surface ?NH₂ density are firstly assessed by using X-ray photoelectron spectroscopy (XPS) and colorimetric analysis. Results show that smaller diamine molecules and nonpolar alcohols could accelerate the reaction. The reaction kinetics with 1,6-hexanediamine is further investigated as a function of temperature, reaction time, and diamine concentration. During the initial stage, the reaction shows a 1^st order kinetics with the diamine concentration and has an activation energy of 54.5 kJ/mol. Ionization state of the ?NH₂ groups on the PCL surface is determined, revealing that the pKa of ?NH₃ ⁺ (<5) is much lower than that of the corresponding diamine molecules in solution. After aminolysis, surface hydrophilicity of PCL membrane is significantly enhanced, while surface elastic modulus and average molecular weight are decreased to some extent, and others such as weight, surface morphology and bulk mechanical strength are not apparently changed. The introduced ?NH₂ groups are found to be largely lost at 37 °C, but can be mostly maintained at low temperature.     

A novel amperometric biosensor based on a co-crosslinked l-lysine-α-oxidase/overoxidized polypyrrole bilayer for the highly selective determination of l-lysine

An amperometric biosensor for the determination of l-lysine based on l-lysine-α-oxidase immobilized by co-crosslinking on a platinum electrode previously modified by an overoxidized polypyrrole film is described. The optimization of experimental parameters, such as pH and flow rate, permitted to minimize significantly substrate interferences even using a low specific, commercial enzyme. The relevant biases introduced in the measurement of lysine were just about 1% for l-arginine, l-histidine and l-ornithine, roughly 4% for l-phenylalanine and l-tyrosine. The developed approach allowed linear lysine responses from 0.02 mM up to 2 mM with a sensitivity of 41 nA/(mM × mm²) and a detection limit of 4 μM (S/N = 3). No appreciable loss in lysine sensitivity was observed up to about 40 days. Allowing polypyrrole layer to remove interference from electroactive compounds, the present method revealed suitable to detect l-lysine in a pharmaceutical and cheese sample, showing a good agreement with the expected values.     

A practical synthesis of fully protected l-γ-carboxyglutamic acid

We have developed a new synthetic route for the preparation of Fmoc protected l-γ-carboxyglutamic acid in 60% overall yield (>99% ee) via a six-step synthesis from d-Garner’s aldehyde. An aldol condensation and the selective cleavage of the acetonide protective group are key steps.     

Supramolecular structures from self-assembled poly(γ-benzyl-l-glutamate)-polydimethylsiloxane-poly(γ-benzyl-l-glutamate) triblock copolypeptides in thin films

We describe the self-assembly of A-B-A triblock copolymers in thin films composed of a soft polydimethylsiloxane (PDMS) central block (B) and two polypeptidic (A) blocks, poly(γ-benzyl)-l-glutamate (PBLG). The PBLG segment exhibits depending on the chain length two distinct secondary conformations either a β-sheet or a α-helical conformation. The direct relationship between the surface morphology and the secondary conformation of the polypeptide segment has been evidenced by atomic force microscopy. For chain lengths below 20 U the polypeptide segments adopt preferentially a β-sheet secondary structure and the triblock copolymer self-assembled in fibers. Moreover, the fiber diameters increased with the chain length of the triblock copolymer. For chain lengths above 20, the α-helical structure is stabilized and a lamellar morphology is formed driven by rod-rod interactions in spite of the very asymmetric composition of the triblock copolymer. However, decreasing the film thickness from 25 to 8 nm, i.e., below the L/2 and due to the preferential attraction of the polypeptide block for the hydrophilic substrate employed, instead of a lamellar morphology a rod-like morphology could be found. Thus, the use of hybrid block copolymer containing polypeptides with particular secondary structures offers novel alternatives to control the self-assembly in thin films compared to traditional amorphous block copolymers.     

Hierarchical self-assembly in diblock copolypeptides of poly(γ-benzyl-l-glutamate) with poly poly(l-leucine) and poly(O-benzyl-l-tyrosine)

Block copolypeptides with their inherent nanometer length scale of phase separation, provide means of manipulating the type (α-helices, β-strands) and persistence of peptide secondary structures. Two such examples are employed based on the α-helical poly(γ-benzyl-l-glutamate) (PBLG) polypeptide as one block and poly(l-leucine) (α-helical) or poly(O-benzyl-l-tyrosine) (POBT) (β-strands) as the second block. Although both secondary structures are present in the copolypeptides the effect of nano-scale confinement is to induce folding in the POBT β-sheets and to maintain the defected α-helices of PBLG and PLEU with a limited lateral coherence.     

A novel sensor based on electropolymerization of β-cyclodextrin and l-arginine on carbon paste electrode for determination of fluoroquinolones

An electrochemical sensor for fluoroquinolones (FQs) based on polymerization of β-cyclodextrin (β-CD) and l-arginine (l-arg) modified carbon paste electrode (CPE) (P-β-CD-l-arg/CPE) was built for the first time. Synergistic effect of l-arg and β-CD was used to construct this sensor for quantification of these important antibiotics. Scanning electron microscope (SEM) image shows that polymer of β-CD and l-arg has been successfully modified on electrode. Electrochemical impedance spectroscopy (EIS) and cyclic voltammograms (CV) further indicate that polymer of β-CD and l-arg efficiently decreased the charge transfer resistance value of electrode and improved the electron transfer kinetic between analyte and electrode. Under the optimized conditions, this modified electrode was utilized to determine the concentrations of ciprofloxacin, ofloxacin, norfloxacin and gatifloxacin. The differential pulse voltammogram (DPV) exhibits the oxidation peak currents were linearly proportional to their concentration in the range of 0.05–100 μM for ciprofloxacin, 0.1–100 μM for ofloxacin, 0.1–40 μM for norfloxacin and 0.06–100 μM for gatifloxacin, respectively. This method was also successfully used to detect the concentrations of each drug in pharmaceutical formulations and human serum samples. In addition, this proposed fluoroquinolones sensor exhibited good reproducibility, long-term stability and fast current response.     

l-Galactose as a natural product: isolation from a marine octocoral of the first α-l-galactosyl saponin

Saponine 1 (6′-O-acetyl-3β-pregna-5,20-dienyl-α-l-galactopyranoside), that contains a l-galactose moiety linked to the aglycone through an infrequent α-glycosidic bond, has been isolated from the marine octocoral Muricea c.f. purpurea. This constitutes the first report on the occurrence of l-Gal as a nonpolymeric natural product. A CD procedure for the absolute stereochemical assignment of saponins, based on the CD analysis of its perbenzoylated derivative, is proposed.