Phase behaviour of l-lactic acid based polymers of low molecular weight in supercritical carbon dioxide at high pressures

The results of investigations of phase behaviour in the systems l-lactic acid based polymers + carbon dioxide at high pressures are presented. The measurements have been performed in wide temperature and composition ranges. Two samples of the polymer differing in molecular weight (M_n: 1080 and 3990 g/mol) have been investigated. Both samples of the polymer were characterized with the gel permeation chromatography and NMR spectroscopy. The influence of the structure of the polymer on the solubility in supercritical carbon dioxide has been discussed. The results obtained suggest that the solubility of low molecular weight l-lactic acid based polymers in supercritical carbon dioxide is not controlled by its size, but to a large extent by the character of its terminal groups. The phase behaviour in the system l-lactic acid + carbon dioxide has been also investigated and the results were compared with those for the systems composed of l-lactic acid based polymers and carbon dioxide.     

Supercritical fluids extraction and anti-solvent purification of carotenoids from microalgae and associated bioactivity

This study investigated co-solvent modified supercritical carbon dioxide extraction of lipids and carotenoids from the microalgal species of Nannochloropsis oculata. Supercritical carbon dioxide (SCCO₂) anti-solvent precipitation of carotenoids from the extracts following purification of Zeaxanthin was also examined. Continuous modification by ethanol of supercritical carbon dioxide extractions showed that the addition ratio was important for extraction efficiency of lipids and carotenoids. SCCO₂ extraction at 350 bar, 323 K and 16.7 wt% of ethanol addition yielded 239.7 mg of triglycerides and 7.61 mg of carotenoids per gram extract with a total yield of 15.5%. SCCO₂ anti-solvent experiments showed that the content of Zeaxanthin in the precipitate was greater than that in the fraction of normal phase column chromatography. The purest Zeaxanthin (93.8%) was then successfully isolated from the purified fraction by using a reverse-phase HPLC column chromatography. Rat macrophages treated by ultra-sonicated water extracts of the SCCO₂ defatted algae showed a positive phagocytotic activity.     

Titanium complexes based on aminodiol ligands for the ring opening polymerization of l- and d,l-lactide

A series of new titanium isopropoxide complexes (1-4-Ti(OⁱPr)₂ based on enantiopure (1-H₂), racemic (2-H₂), meso (3-H₂) and diastereomeric (4-H₂) aminodiol ligands have been prepared and tested as initiators for the ring opening polymerization (ROP) of l/rac-lactide in solution and in bulk conditions. All complexes were shown to have significant activity in solution at 70 °C and higher activity in bulk at 130 °C with a good control over the molar mass distribution and molecular weights. The complex derived from the racemic-aminodiol ligand gave partially heterotactic polylactide in ROP of rac-lactide and afforded atactic polylactide in the bulk, whereas all other complexes yielded atactic polylactides both in solution and in bulk. Ligand variation (chirality) in the complexes has little effect on either the activity or selectivity of the initiators. The polymerization kinetics using (1-Ti(OⁱPr)₂) as an initiator indicated a first order reaction with respect to the monomer concentration.     

Lipase-catalyzed synthesis of poly-l-lactide using supercritical carbon dioxide

The present work shows that the enzyme mediated ring-opening polymerization of l-lactide at 65 °C can be achieved using supercritical carbon dioxide. It is reported a biphasic media system where the supercritical phase coexists with a liquid organic phase, which is mainly composed of melted monomer, wherein the growing poly-l-lactide chains are soluble. The immobilized lipase B from Candida antartica was used as the biocatalyst. The results indicated that semi-crystalline polymers with a molecular weight (M_w) up to 12,900 g mol⁻¹ can be attained and that the monomer conversion is related to the biocatalyst concentration and its initial water activity (a_wi). Experiments carried out with denatured enzyme gave no monomer conversion which confirms that the enzymatic mechanism is only involved in our system.     

Acylated and hydroxylated polyamides derived from l-tartaric acid

A series of polyamides 6,4 were prepared from 1,6-hexanediamine and active esters of 2,3-di-O-acylated l-tartaric acid by polycondensation in solution. Both O-alkoyl and O-benzoyl esters were used as hydroxyl protecting groups. The resulting acylated polytartaramides were found to be semicrystalline polymers with T_m between 100 and 200 °C and T_g slightly above 100 °C. Controlled hydrolysis of the ester side group led to the preparation of poly(hexamethylene l-tartaramide)s with different content in free hydroxyl groups. These polyamides continue being crystalline but their properties largely differ from those displayed by their parent acylated polymers.     

Crystallization behavior of poly(l-lactic acid)

The crystallization behavior of poly(l-lactic acid) was studied in the range of 80-160 °C. The peak crystallization time (τ_p) was defined and obtained from the crystallization isotherm measured with a differential scanning calorimeter (DSC). Isothermal crystallization temperature (T_c) dependence of log(τ_p) discretely changed at 113 °C (= T_b). The linear growth rate of spherulite, G, was measured with a polarizing microscope. The T_c dependence of G and the size of the spherulite also discretely changed at T_b. Crystal structures for samples isothermally crystallized at temperatures which were higher and lower than T_b were orthorhombic (α-form) and trigonal (β-form), respectively. The discrete change of the crystallization behavior was explained by the formation of different crystal.     

Direct synthesis of poly(l-lactic acid) in supercritical carbon dioxide with dicyclohexyldimethylcarbodiimide and 4-dimethylaminopyridine

The direct synthesis of poly(l-lactic acid) (PLLA) from an l-lactic acid oligomer has been performed in supercritical carbon dioxide (scCO₂) using an esterification promoting agent, dicyclohexyldimethylcarbodiimide (DCC), and 4-dimethylaminopyridine (DMAP) as a catalyst. PLLA within M_n of 13,500 g/mol was synthesised in 90% yield at 3500 psi and 80 °C after 24 h. The molecular weight distribution of the products was narrower than PLLA prepared with melt-solid phase polymerisation under conventional conditions. Both DCC and DMAP showed high solubility in scCO₂ (DCC: 7.6 wt% (1.63×10⁻² mol/mol CO₂) at 80 °C, 3385 psi, DMAP: 4.5 wt% (1.62×10⁻²mol/mol CO₂) at 80 °C, 3386 psi) and supercritical fluid extraction was found to be effective at removing excess DMAP and DCC after the polymerisation was complete. We show that DCC and DMAP are effective esterification promoting reagents with further applications for condensation polymerisations in scCO₂.     

pH affected migrational transport of ferrocene derivative of l-cysteine in aqueous solutions: Voltammetric studies

The mass transport of biologically-active l-cysteine derivatized with the ferrocenyl group (FcCH₂Cys) was investigated voltammetrically at a microelectrode in aqueous solutions under the conditions of varying content of supporting electrolyte and at different pH values. By varying the pH conditions one could obtain samples containing differently ionized forms of the l-cysteine derivative, from a monovalent cation via a neutral form (zwitterion) to a monovalent anion. Due to the acid-base equilibrium the obtained solutions were, in fact, mixtures composed predominantly of either anionic or cationic, or neutral species of FcCH₂Cys, respectively. Under the conditions of low ionic support the mass transport of these forms is differently affected by the migrational contribution. The results obtained experimentally were in good agreement with the theoretical predictions. In the calculations it was assumed that the FcCH₂Cys forms, coexisting in the solution, contribute independently to the steady-state transport-limited current. It was also assumed that the diffusion coefficients of the FcCH₂Cys forms were equal. This was validated by the voltammetric measurements at the supporting electrolyte excess (purely diffusional conditions). The diffusion coefficients of different forms of FcCH₂Cys are very similar and the average diffusion coefficient is (5.35 ± 0.05) × 10⁻¹⁰ m²/s.The studies clearly show that the variation in the conditions of pH or/and concentration of electrolyte can change the transport rate of l-cysteine even by several tens percent.     

Preparation in supercritical CO2 of porous poly(methyl methacrylate)-poly(l-lactic acid) (PMMA-PLA) scaffolds incorporating ibuprofen

Partially biodegradable porous scaffolds incorporating bioactive molecules prepared by clean techniques posses an enormous interest in tissue engineering applications. Poly(methyl methacrylate)-poly(l-lactic acid) (PMMA-PLA) blends were submitted to CO₂ supercritical conditions (P = 160-260 bar, T = 60 °C) after certain time and then rapidly depressurized to obtain porous structures that have been related with the supercritical parameters and to the polymer blend composition. In some cases ibuprofen was also incorporated to the formulations previously to the CO₂ treatment and studied the appropriate conditions for avoiding its extraction in SCCO₂. Scaffolds purity, thermal transitions, swelling and degradation behaviour, and the ibuprofen release were also studied to determine the appropriate scaffolds with a desired porosity for cell seeding. Cell culture was performed on the selected porous scaffolds using human fibroblast examined by scanning electron microscopy (SEM).     

Crystallization, structure and properties of plasticized poly(l-lactide)

Poly(l-lactide) (PLA) was plasticized with poly(ethylene glycol)s having M_w of 400 and 600 g/mol. In addition to poly(ethyne glycol)s with hydroxyl end groups, monomethyl ethers of poly(ethylene glycol) having M_w of 550 and 750 g/mol, with chains terminated with hydroxyl groups and methyl groups, were used. The effect of different end groups on the plasticization of both amorphous and semicrystalline PLA was studied. The crystallization, structure, thermal and tensile properties of PLA and PLA with 5 and 10 wt% of plasticizers were explored. No marked effect induced by different end groups of plasticizers was found. All the plasticizers used decreased T_g and increased the ability of PLA to cold crystallization. While an amorphous plasticized PLA could be deformed to about 550%, a semicrystalline PLA with the same total plasticizer content exhibited nonuniform plasticization of the amorphous phase and less ability to the plastic deformation. Nevertheless, a 20% elongation at break was achieved for a semicrystalline PLA with 10 wt% of the plasticizer. The plastic deformation of both neat and plasticized PLA was associated with crazing.     

Spherulite growth of l-lactide copolymers: Effects of tacticity and comonomers

The spherulite growth behavior and mechanism of l-lactide copolymers, poly(l-lactide-co-d-lactide) [P(LLA-DLA)], poly(l-lactide-co-glycolide) [P(LLA-GA)], and poly(l-lactide-co-ε-caprolactone) [P(LLA-CL)] have been studied using polarization optical microscopy in comparison with poly(l-lactide) (PLLA) having different molecular weights to elucidate the effects of incorporated comonomer units. The incorporation of comonomer units reduced the radius growth rate of spherulites (G) and increased the induction period of spherulite formation (t_i), irrespective of the kind of comonomer unit. Such effects became remarkable with the content of comonomers. At a crystallization temperature (T_c) of 130 °C, the disturbance effects of comonomers on the spherulite growth decreased in the following order: d-lactide>glycolide>ε-caprolactone, when compared at the same comonomer unit or reciprocal of averaged l-lactyl unit sequence length (l_l). The t_i estimation indicated that the glycolide units have the lowest disturbance effects on the formation of spherulite (crystallite) nuclei. The PLLA having the number-average molecular weight (M_n) exceeding 3.1×10⁴ g mol⁻¹ showed the transition from regime II to regime III at T_c=120 °C, whereas PLLA with the lowest M_n of 9.2×10³ g mol⁻¹ crystallized solely in regime III kinetics and the copolymers excluding P(LLA-DLA) with 3% of d-lactide units crystallized solely according to regime II kinetics. The nucleation and front constant for regime II and III [K_g(II), K_g(III), G₀(II), and G₀(III), respectively] estimated with each (not with a fixed for high-molecular-weight PLLA) decreased with increasing the amount of defects per unit mass of the polymer for crystallization, i.e. with increasing the comonomer content and the density of terminal group through decreasing the molecular weight.     

Electrochemical detection of l-cysteine using a boron-doped carbon nanotube-modified electrode

A boron-doped carbon nanotube (BCNT)-modified glassy carbon (GC) electrode was constructed for the detection of l-cysteine (L-CySH). The electrochemical behavior of BCNTs in response to l-cysteine oxidation was investigated. The response current of L-CySH oxidation at the BCNT/GC electrode was obviously higher than that at the bare GC electrode or the CNT/GC electrode. This finding may be ascribed to the excellent electrochemical properties of the BCNT/GC electrode. Moreover, on the basis of this finding, a determination of L-CySH at the BCNT/GC electrode was carried out. The effects of pH, scan rate and interferents on the response of L-CySH oxidation were investigated. Under the optimum experimental conditions, the detection response for L-CySH on the BCNT/GC electrode was fast (within 7 s). It was found to be linear from 7.8 × 10⁻⁷ to 2 × 10⁻⁴ M (r = 0.998), with a high sensitivity of 25.3 ± 1.2 nA mM⁻¹ and a low detection limit of 0.26 ± 0.01 μM. The BCNT/GC electrode exhibited high stability and good resistance against interference by other oxidizable amino acids (tryptophan and tyrosine).     

Controlled crystal nucleation in the melt-crystallization of poly(l-lactide) and poly(l-lactide)/poly(d-lactide) stereocomplex

Among the various inorganic nucleators examined, Talc and an aluminum complex of a phosphoric ester combined with hydrotalcite (NA) were found to be effective for the melt-crystallization of poly(l-lactide) (PLLA) and PLLA/poly(d-lactide) (PDLA) stereo mixture, respectively. NA (1.0 phr (per one hundred resin)) can exclusively nucleate the stereocomplex crystals, while Talc cannot suppress the homo crystallization of PLLA and PDLA in the stereo mixture. Double use of Talc and NA (in 1.0 phr each) is highly effective for enhancing the crystallization temperature of the stereo complex without forming the homo crystals. The stereocomplex crystals nucleated by NA show a significantly lower melting temperature (207 °C) than the single crystal of the stereocomplex (230 °C) in spite of recording a large heat of crystallization ΔH_c (54 J/g). Photomicrographic study suggests that the spherulites with a symmetric morphology are formed in the stereo mixture added with NA while the spherulites do not grow in size in the mixture added with Talc. The exclusive growth of the stereocomplex crystals by the melt-crystallization process will open a processing window for the PLLA/PDLA.     

Kinetics and mechanism of ?-caprolactone and l,l-lactide polymerization coinitiated with zinc octoate or aluminum acetylacetonate: The next proofs for the general alkoxide mechanism and synthetic applications

Following our previous papers on mechanism of cyclic esters' polymerization coinitiated by tin(II) octoate [tin(II) bis-(2-ethylhexanoate), (Sn(Oct)₂)] in the presence of either the low molar mass coinitiator (an alcohol, hydroxy acid, or H₂O) or a macromolecule fitted with a hydroxy end group (ROH), the present work deals with ?-caprolactone (CL) and l,l-lactide (LA) polymerizations coinitiated with zinc octoate (Zn(Oct)₂) or aluminum acetylacetonate (Al(Acac)₃). A series of kinetic measurements revealed that similarly as in the Sn(Oct)₂ coinitiated process, these polymerizations proceed by simple monomer insertion into the …Mt-OR bond, reversibly formed in the reaction -Mt-L + ROH ? …-Mt-OR + LH (where Mt = Sn, Zn or Al; L = Oct or Acac), taking place throughout the whole polymerization process. MtL_n itself does not play an active role in the polymerization. Applicability of the commercially available Zn(Oct)₂ or Al(Acac)₃ for the aliphatic polyester (10³ ≤ M_n ≤ 4 × 10⁵) synthesis is also discussed.     

Formation of retinyl palmitate-loaded poly(l-lactide) nanoparticles using rapid expansion of supercritical solutions into liquid solvents (RESOLV)

Poly(l-lactide) (PLLA) nanoparticles loaded with retinyl palmitate (RP) were successfully prepared by rapid expansion of a supercritical carbon dioxide (CO₂) solution into an aqueous receiving solution containing a stabilizing agent (RESOLV). Three stabilizing agents, Pluronic F127, Pluronic F68, and sodium dodecyl sulfate (SDS) have been employed and the Pluronic F127 was found to be more effective for stabilizing PLLA/RP nanoparticles than Pluronic F68 and SDS, as RESOLV into a 0.1 wt% Pluronic F127 solution produced a stable nanosuspension consisting mainly of well-dispersed, individual nanoparticles. The effect of rapid expansion processing conditions (i.e., degree of saturation (S), pre-expansion temperature (T_pre), and concentrations of PLLA and RP (C_PLLA, C_RP)) on the particle size, form, and RP loading was systematically investigated. It was found that spherical PLLA/RP nanoparticles with an average size range of ∼40-110 nm and RP loadings of 0.9-6.2 wt% were consistently produced by RESOLV. The size of PLLA/RP nanoparticles increased from ∼30-80 to ∼30-160 nm as the solution degree of saturation changed from S < 1 to S > 1, independent of T_pre, C_PLLA, and C_RP. The entrapment capacity of RP in PLLA nanoparticles was predominantly determined by T_pre and C_RP. Increasing the T_pre from 70 to 100 °C and the C_RP from 0.05 to 0.15 wt% increased the encapsulated RP content at least twofold. Our results show that the technique with benign supercritical CO₂ should be generally applicable to nanoparticle fabrications of other important active ingredients, especially in liquid form, in polymeric nanoparticles.     

Solubility and diffusivity of CO2 in poly(l-lactide)-hydroxyapatite and poly(d,l-lactide-co-glycolide)-hydroxyapatite composite biomaterials

Solubility and diffusivity of supercritical CO₂ in poly(l-lactide)-hydroxyapatite (PLLA-HA) and poly(d,l-lactide-co-glycolide)-hydroxyapatite (PLGA-HA) composite materials were measured using a magnetic suspension balance at a temperature of 313 K and a pressures range of 10-30 MPa. The effect of the HA concentration on the solubility and diffusivity was investigated by varying filler content in the range of 0-50 wt%. For the PLLA-HA composites the solubility decreases with the increase of filler concentration. Diffusivity of the gas in the substrate is also lower as the HA content increases. In the case of PLGA-HA composites, small filler content favors the solubility and diffusivity of CO₂ due to incomplete wetting of the solid particles by the polymer. As the amount of HA increases solubility decreases. The results suggest that dense CO₂ could be used as a ‘green’ processing agent for composite biomaterials when organic solvents or high temperatures should be avoided.     

Melting behavior of poly(l-lactic acid): Effects of crystallization temperature and time

Effects of crystallization temperature and time on the melting behavior of poly(l-lactic acid) were studied with differential scanning calorimetry (DSC). The isothermal crystallization was performed at various temperatures (T_cs), and DSC melting curves for the isothermally crystallized samples were obtained at 10 K min⁻¹. When T_c was lower than T_d (∼135 °C), the double melting peaks appeared. The melting behavior, especially T_c dependence of the melting temperature (T_m), discretely changed at T_b (=113 °C), in accordance with the discrete change of the crystallization behavior at T_b, which was previously reported. When T_c was higher than T_d, a single melting peak appeared. In addition, T_c dependence of dT_m/dT_c discretely changed at T_d. That is, the melting behavior, especially T_c dependence of T_m and dT_m/dT_c, are different in three temperature regions of T_c divided by T_b and T_d: Regions I (T_c ≤ T_b), II (T_b ≤ T_c ≤ T_d), and III (T_d ≤ T_c). The effect of crystallization time on the melting behavior, melting temperature and heat of fusion in each temperature region of T_c is also discussed.     

Production of l-asparaginase from Escherichia coli ATCC 11303: Optimization by response surface methodology

This paper discusses the studies carried out for the optimal production of enzyme l-asparaginase (l-asparagine amidohydrolase, EC 3.5.1.1) from Escherichia coli (ATCC 11303). It was found that inoculum age of 18 h and inoculum size of 10% were the most favorable operating conditions for enzyme production. Lactose, yeast extract and KH₂PO₄ were found to be the best carbon, nitrogen and ion sources, respectively. Statistical method was used to survey how various medium conditions affect the enzyme production. By response surface methodology, the values of lactose, tryptone, yeast extract, KH₂PO₄ and l-asparagine concentration were investigated to obtain the maximum enzyme activity. The highest enzyme activity, 1.03 U mL⁻¹ enzyme, was determined under the following conditions: 1.08% lactose, 1.79% tryptone, 1.6% yeast extract, 2% KH₂PO₄ and 0.19% l-asparagine. Response surface methodology proved to be a powerful tool in optimizing the medium and by this method, more than 10-fold (from 0.1 to 1.03 U mL⁻¹) enhancement in l-asparaginase activity was achieved as compared to that obtained in the basal medium (Luria-Bertani media, inoculum age of 24 h and inoculum size of 10%).     

Broadband dielectric spectroscopic characterization of the hydrolytic degradation of carboxylic acid-terminated poly(d,l-lactide) materials

Broadband dielectric spectroscopy was used to examine carboxylic acid-terminated poly(d,l-lactide) samples that were hydrolytically degraded in 7.4 pH phosphate buffer solutions at 37 °C. The dielectric spectral signatures of degraded samples were considerably more distinct than those of undegraded samples and a T_g-related relaxation associated with long range chain segmental mobility was seen. For both degraded and undegraded samples, a relaxation peak just beneath a DSC-based T_g was observed, which shifts to higher frequency with increasing temperature. Thus, this feature is assigned as the glass transition as viewed from the dielectric relaxation perspective. Linear segments on log-log plots of loss permittivity vs. frequency, in the low frequency regime, are attributed to d.c. conductivity. An upward shift in relaxation peak maximum, f_max, observed especially after 145 d of immersion in buffer, implies a decrease in the time scale of long range segmental motions with increased degradation time.Permittivity data for degraded and undegraded materials were fitted to the Havriliak-Negami equation with subtraction of the d.c. conductivity contribution to uncover pure relaxation peaks. Parameters extracted from these fits were used to construct Vogel-Fulcher-Tammann-Hesse (VFTH) curves and distribution of relaxation time, G(τ), curves for all samples. It was seen that the relaxation times for the α-transition in both degraded and undegraded samples showed VFTH temperature behavior. G(τ) curves showed a general broadening and shift to lower τ with degradation, which can be explained in terms of a broadening of molecular weight within degraded samples and faster chain motions.     

Towards the conception of an amperometric sensor of l-tyrosine based on Hemin/PAMAM/MWCNT modified glassy carbon electrode

A novel amperometric sensor was fabricated based on the immobilization of hemin onto the poly (amidoamine)/multi-walled carbon nanotube (PAMAM/MWCNT) nanocomposite film modified glassy carbon electrode (GCE). Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and ultraviolet visible (UV-vis) adsorption spectroscopy were used to investigate the possible state and electrochemical activity of the immobilized hemin. In the Hemin/PAMAM/MWCNT nanocomposite film, MWCNT layer possessed excellent inherent conductivity to enhance the electron transfer rate, while the layer of PAMAM greatly enlarged the surface average concentration of hemin (Γ) on the modified electrode. Therefore, the nanocomposite film showed enhanced electrocatalytical activity towards the oxidation of l-tyrosine. The kinetic parameters of the modified electrode were investigated. In pH 7.0 phosphate buffer solution (PBS), the sensor exhibits a wide linear range from 0.1 μM to 28.8 μM l-tyrosine with a detection limit of 0.01 μM and a high sensitivity of 0.31 μA μM⁻¹ cm⁻². In addition, the response time of the l-tyrosine sensor is less than 5 s. The excellent performance of the sensor is largely attributed to the electro-generated high reactive oxoiron (IV) porphyrin (O = Fe^IV-P) which effectively catalyzed the oxidation of l-tyrosine. A mechanism was herein proposed for the catalytic oxidation of l-tyrosine by oxoiron (IV) porphyrin complexes.