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Phenyllactic acid (PLA) is an important organic acid with wide antimicrobial activities against gram-positive and gram-negative bacteria and some fungi. This interesting compound can be synthesized by the microbial fermentation or the bioconversion using phenylpyruvic acid (PPA) as the key substrate and microorganisms as the whole-cell biocatalysts. However, the isolation of high-purity PLA with a high recovery from the crude fermentation or conversion broth is a challenging task. In this work, the separation of PLA from the crude conversion broth prepared by employing Lactobacillus buchneri cells as the whole-cell catalysts was achieved by the chromatography using the poly(hydroxyethyl methacrylate) (pHEMA)-based cryogel with a combination of anion-exchange and hydrophobic benzyl groups. The static adsorption behaviors of PLA under different salt concentrations and the adsorption capacities of PLA on the cryogel were measured experimentally. The chromatographic performance of PLA from the crude conversion broth was compared with that from the clarified broth. The results showed that the pHEMA-based cryogel has a high capacity of PLA, i.e., 14.64 mg mL −1 cryogel, and the adsorption of PLA was influenced by the salt concentration. By using deionized water as running buffer, PLA with a high purity of 97.6% was obtained with one step elution using 0.3 M NaCl as the elution solution with the recovery at the range of 80.2–90.8% from crude feedstock without any pretreatment at various flow velocities. These values were close to those obtained for the clarified broth, i.e., the purity of 98.4% and the recovery of 92.3% under the same chromatography conditions at 1 cm min −1 . The cryogel was then applied to separate PLA from clarified feedstock, high purity (>96.7%) and recovery (>91.4%) of PLA were found with 20 cycles, which verified the selectivity and robustness of prepared pHEMA-VBTAC cryogel. Therefore, the chromatography using pHEMA-based cryogel with the dual functional groups is an effective approach for the isolation of PLA directly from the crude bioconversion broth and thus could be interesting in the separation and production of high-purity PLA in industry. [ABSTRACT FROM AUTHOR]

Supercritical fluid assisted atomization introduced by a hydrodynamic cavitation mixer (SAA-HCM) was used to prepare micrometric particles of a labile protein, i.e. , trypsin from aqueous solution without use of any organic solvents. The trypsin particles precipitated had various morphologies under different process conditions, with particle diameters ranging from 0.2 to 4 μm. FTIR, SDS-PAGE, CD and fluorescence spectra were performed to analyze the structural stability of the protein, and trypsin retained above 70% of the biological activity. Besides, chitosan was selected as the polymer carrier in an effort to prepare trypsin composite microparticles via SAA-HCM process. The influences of chitosan molecular weight, polymer/protein ratio and solution concentration on the particle morphology and size distribution were investigated in detail. Non-coalescing spherical composite microparticles with a narrow particle distribution (0.2–3 μm) could be obtained. The SAA-HCM prepared particles were amorphous as demonstrated by XRD and had a loading efficiency about 90%. The protein release profiles of the composite microparticles were evaluated using both the immersion condition and a Franz diffusion cell. Finally, the distribution of the protein within the particles was characterized through CLSM analysis of FITC-labeled trypsin-loaded chitosan microparticles. The SAA-HCM process is demonstrated to be a protein-friendly and promising technique for production of protein and polymer/protein composite particles formulations from aqueous solutions for drug delivery systems. [ABSTRACT FROM AUTHOR]

Thesolubility of dexamethasone, a synthetic glucocorticoid, insupercritical CO2and together with 3.0 mol % ethanol asa cosolvent was measured at different temperatures (313.2, 318.2,and 323.2) K and pressures ranging from (10.0 to 25.0) MPa by a staticanalytical method. The obtained solubility of dexamethasone was between1.19·10–6and 1.52·10–6in pure supercritical CO2and between 1.97·10–6and 2.98·10–6in supercriticalCO2with a cosolvent. The experimental data in supercriticalCO2in the absence and presence of a cosolvent were correlatedusing four density-based semiempirical models, that is, Chrastil,Kumar and Johnston, Bartle, and Méndez-Santiago and Teja models.The results indicated that the Kumar and Johnston model gave a bettercorrelated result than the other three with an average absolute relativedeviation [AARD (%)] value lower than 2.15 %. [ABSTRACT FROM AUTHOR]

Drug-loaded chitosan films suitable for oral mucosal drug delivery were prepared using supercritical solution impregnation (SSI) technology. Firstly, chitosan films were obtained via casting method, and the film properties including water-uptake, erosion and mucoadhesive were characterized. SSI process was then employed to load the drug of ibuprofen onto the prepared chitosan films, and the effects of impregnation pressure and temperature on morphologies of the ibuprofen-loaded chitosan films and drug loading capacity (DLC) were studied. The SEM and X-ray diffraction patterns suggested that distinct ibuprofen shapes such as microparticles, flake, rod-like and needle-like occurred after impregnation at different pressures, and DLC varied from 7.9% to 130.4% during the SSI process. The ex vivo release profiles showed that ibuprofen-loaded chitosan films could deliver the drug across the rabbit buccal mucosa, and up to 70% of the ibuprofen was released from the matrix in 460 min. SSI process is a promising method to prepare drug-loaded film formulations for oral mucosal drug delivery, which provides the advantages of low solvent residual and sustained- and controlled- release behavior. [ABSTRACT FROM AUTHOR]

Abstract: DsbA (disulfide bond formation protein A) is essential for disulfide bond formation directly affecting the nascent peptides folding to the correct conformation in vivo. In this paper, recombinant DsbA protein was employed to catalyze denatured lysozyme refolding and inhibit the aggregation of folding intermediates in vitro. Statistical methods, i.e., Plackett–Burman design and small central composite design, were adopted to screen out important factors affecting the refolding process and correlating these parameters with the refolding efficiency including both protein recovery and specific activity of refolded lysozyme. Four important parameters: initial lysozyme concentration, urea concentration, KCl concentration and GSSG (glutathione disulfide) concentration were picked out and operating conditions were optimized by introducing the effectiveness coefficient method and transforming the multiple objective programming into an ordinary constrained optimization issue. Finally, 99.7% protein recovery and 25,600U/mg specific activity of lysozyme were achieved when 281.35μg/mL denatured lysozyme refolding was catalyzed by an equivalent molar of DsbA at the optimal settings. The results indicated that recombinant DsbA protein could effectively catalyze the oxidized formation and reduced isomerization of intramolecular disulfide bonds in the refolding of lysozyme in vitro. [Copyright &y& Elsevier]

Abstract: The data of the liquid volume expansion could normally be used as one of the thermodynamic criteria to decide an appropriate process of supercritical fluid based micronization techniques. The liquid volume expansion data were predicted by the vapor–liquid phase equilibrium calculation of two binary systems (CO2/acetone and CO2/ethanol) using Peng–Robinson equation of state. The results indicated that the liquid volume expansion of CO2/acetone mixtures was much larger than that of CO2/ethanol mixtures at the specified temperature and pressure. According to the thermodynamic criterion, acetone was affirmed to be a good solvent for supercritical antisolvent process (SAS) due to the higher liquid volume expansion, while ethanol was suitable for supercritical assisted atomization (SAA). This thermodynamic criterion was applied to the micronization of cholesterol and spherical particles were prepared in various organic solvents by SAA with an enhanced mixer. When the liquid volume expansion was lower than 80%, SAA could be successfully conducted and the good morphology and size distribution of the cholesterol particles were obtained. If the liquid volume expansion was higher than 140%, the solute would precipitate in the mixer before the atomization leading to the blockage of nozzle due to the antisolvent effect. In that case, the cholesterol particles in the mixer maintained the same morphology and crystalline structure as the SAS processed. This criterion is a practical and accessible tool to successfully implement the SAA process avoiding undesirable precipitation in the mixer. [Copyright &y& Elsevier]

Abstract: Supercritical fluid assisted atomization introduced by hydrodynamic cavitation mixer (SAA-HCM) was used to produce lysozyme microparticles with controlled particle size distribution in the range for aerosol drug delivery. The process is based on the atomization effect of carbon dioxide. The solubilization of certain amount of carbon dioxide in the solution plays the key role and the HCM can intensify mass transfer between carbon dioxide and liquid feedstock greatly. Water was used as the solvent to solubilize lysozyme and thus no organic residual was detected. The influences of process parameters on particle formation were investigated including temperature in the precipitator, pressure and temperature in the mixer, concentration of the solution and feed ratio CO2/solution. The particles were characterized with respect to their morphologies and particle size: well defined, spherical and separated particles with diameters ranging between 0.2 and 5μm could be always produced at optimum operating conditions. Bio-activity assay showed that good activity maintenance of higher than 85% for lysozyme was usually achieved. Solid state characterizations were further performed to investigate the changes of lysozyme in the process. Fourier transform infrared spectroscopy indicated that no change in secondary structure had occurred for processed lysozyme. X-ray diffraction analysis showed that the lysozyme particles produced remained similarly amorphous as the raw material. Differential scanning calorimetry and thermogravimetry analysis revealed that there was no significant difference in water association but with the increase of water content after processing. [Copyright &y& Elsevier]

Abstract: 11α-Hydroxylation of 16α,17-epoxyprogesterone (EP) by Rhizopus nigricans is an essential step in the synthesis of many steroidal drugs, while low conversion of the biohydroxylation is a tough problem to be solved urgently in industry. Two ionic liquids (ILs) of [BMIm][PF6] and [BMIm][NTf2] were used in the biotransformation of EP by R. nigricans. The results indicated that the conversion carried out in [BMIm][PF6]-aqueous biphasic system was greatly increased to above 90% at 18g/L feeding concentration. A simplified mechanism was proposed to explain the improvement of the bioconversion in a biphasic ionic liquid aqueous system. Besides, successive three batches of bioconversion were carried out in the biphasic system with a total conversion of 87% at phase ratio 10 and 75% at phase ratio 5, respectively. Since recycling of the [BMIm][PF6] is quite easy, there is a great potential for the application of ILs in fungi biotransformation to implement green production. [Copyright &y& Elsevier]

Abstract: DsbA (disulfide bond formation protein A) located in the periplasm of Escherichia coli is a disulfide isomerase, which is vital to disulfide bonds formation directly affecting the nascent peptides folding to the correct conformation. In this paper, recombinant DsbA was firstly immobilized onto NHS-activated Sepharose Fast Flow gel. Then Sephadex G-100 gel was sequentially packed on the top of recDsbA Sepharose Fast Flow, and a so-called conjoint chromatography column composed of SEC and immobilized recombinant DsbA was constructed. Denatured lysozyme was applied on the conjoint column. The effect of SEC volume, flow rate, loading amount and volume, pre-equilibrium mode and KCl concentration in the buffer on lysozyme refolding were investigated in detail and the stability of DsbA immobilization was evaluated. Finally the reusability of the conjoint refolding column was also tested. When loading 2.4mg denatured lysozyme in 0.5ml solution, the activity recovery reached 92.7% at optimized experimental conditions, and the conjoint column renaturation capacity decreased only 7.7% after six run reuse due to the use of SEC section in the chromatographic refolding process. The conjoint chromatography offers an efficient strategy to refold proteins in vitro with high productivity and column reusability. [Copyright &y& Elsevier]

Abstract: Recycling high speed counter-current chromatography (HSCCC) was successfully applied to resolution of (R, S)-naproxen (NAP) using hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector. The two-phase solvent system composed of n-hexane–ethyl acetate–0.1molL−1 phosphate buffer solution with pH=2.67 (8:2:10, v/v/v) was selected. Influence factors for the chiral separation process were investigated, including concentration of HP-β-CD, equilibrium temperature and pH of aqueous phase. Suitable elution mode was selected for HSCCC enantioseparation of (R, S)-NAP. Under optimum separation conditions, 29mg of (R, S)-NAP was separated using preparative recycling HSCCC with the molar ratio HP-β-CD/NAP racemate 83:1. Technical details for recycling elution mode were discussed as for chiral HSCCC separation. The purities of both (S)-NAP and (R)-NAP were over 99.5% as determined by HPLC. Enantiomeric excess of (S)-NAP and (R)-NAP reached 99.4%. Recovery for NAP enantiomers from HSCCC fractions was 82–89%, yielding 13mg of (S)-NAP and 12mg of (R)-NAP. [Copyright &y& Elsevier]

Abstract: Supercritical fluid assisted atomization introduced by a hydrodynamic cavitation mixer (SAA-HCM) was used to prepare bovine serum albumin (BSA) microparticles. Water was used as the sole solvent. A hydrodynamic cavitation mixer was applied to improve mass transfer and achieve a continuous near-thermodynamic-equilibrium solubilization of SC-CO2 in the liquid solution. Under the different conditions, the prepared BSA microparticles had various morphologies, such as corrugated particles, smooth hollow spherical particles and cup particles, with particle diameters ranging from 0.3 to 5μm. The microparticle formation process was elucidated with the shell formation and central bubble mechanism. Compared to native BSA, BSA microparticles did not show significant change in primary structure, according to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The secondary structure of BSA was characterized by Fourier transform infrared spectroscopy (FT-IR). No new peaks were observed after SAA-HCM processing. In addition, the crystalline structure of the BSA microparticles was demonstrated to be amorphous because of the sudden supersaturation in the precipitation process. The SAA-HCM process is expected to be a promising technique for producing microparticles suitable for pulmonary delivery of therapeutic macromolecules. [ABSTRACT FROM AUTHOR]

Abstract: A novel micronization technique so-called supercritical fluid assisted atomization introduced by hydrodynamic cavitation mixer (SAA-HCM) was used to produce the sodium cellulose sulfate (NaCS) microparticles with well-defined spherical morphologies and controlled particle size distributions in aqueous solution. The process parameters such as mixer temperature and pressure, the mass flow ratio, precipitator temperature, solution concentration and the molecular weight of NaCS were investigated in detail to evaluate their influences on the morphologies and size distributions of precipitates. Spherical NaCS particles with mean diameters ranging from 0.3 to 3.0μm were produced at the optimum operating conditions and narrow particle size distributions were obtained. It is natural to put forward the particle formation mechanism of deflated balloons. Compared with the unprocessed NaCS, there was no significant change on the primary structure and stability of the NaCS processed by SAA-HCM verified by Fourier transform infrared spectroscopy. The X-ray diffraction and thermo-gravimetric analysis were also used to investigate NaCS modifications and a slight change in crystalline state with higher thermal stability was observed when treated by SAA-HCM process. The results indicated that NaCS micronization by SAA-HCM process was expected to be a promising technique for drug delivery system. [Copyright &y& Elsevier]

Abstract: A hydrodynamic cavitation mixer (HCM) was introduced in the supercritical fluid assisted atomization (SAA) process to improve mass transfer. SAA-HCM resulted in a more homogenous primary droplet and, as a consequence, narrower particle size distributions. Levofloxacin hydrochloride (LH) being selected as a model drug, the effects of cavitation generator on morphology, size, and distribution of precipitated particles were evaluated. In addition, the influences of several process parameters, such as the feed ratio between CO2 and liquid solution, solute concentration, etc., were investigated in detail. Spherical amorphous micron-sized particles were obtained at different CO2-solution feed ratio and solute concentrations. In all cases, particles with diameters smaller than 2.1μm were produced. The SAA-HCM is expected to be a promising technique for producing microparticles suitable for pulmonary delivery in drug delivery applications. [Copyright &y& Elsevier]

Abstract: Inorganic salts were introduced into the formation process in the preparation of sodium cellulose sulfate (NaCS)–polydimethyldiallylammonium chloride (PDMDAAC) capsules in an effort to change the NaCS–PDMDAAC capsules properties. Membrane thickness of the capsules increased greatly, especially at higher salt concentrations, while network density of the capsules membrane exhibited the opposite effect. The size of the capsules remained unchanged; however, they were affected by the aforementioned two intrinsic capsules characteristics. The molecular weight cut-off (MWCO) of the capsules was increased in the presence of the salts, while the permeability of the whole capsules decreased. The mechanical strength increased at the lower salt concentrations while a decrease was exhibited at the higher salt concentrations. The ion order necessary for the increase in mechanical strength of the capsules was Mg2+ >Ca2+ >Na+ >K+ for cations and Cl− >SO4 2− for anions, which was inversely proportional to the ion radii. It was found that the properties of NaCS–PDMDAAC membranes could be controlled by merely making adjustments in the concentration of inorganic salts in the process. [Copyright &y& Elsevier]

Abstract: Minichaperone sht GroEL191-345 was covalently coupled to NHS-activated Sepharose Fast Flow gel. Refolding of recombinant human interferon gamma (rhIFN-γ) was carried out on a chromatographic column packed with immobilized minichaperone. The effects of salt concentration, urea concentration gradient, elution flow rate and protein loading on the refolding efficiency were investigated. The results indicated that immobilized sht GroEL191-345 chromatography was an effective protocol for the refolding of rhIFN-γ. When loading 100μl denatured rhIFN-γ (17.8mg/ml), the protein mass recovery and total activity obtained in this optimal process reached 74.25% and 6.74×106 IU/ml, respectively with the immobilized minichaperone column which was reused for 10 times with 25% decrease of renaturation capacity. [Copyright &y& Elsevier]

Graphical abstract Abstract Chemotherapeutics are used extensively in cancer and encapsulating the drug in nanoparticles is an effective method to enhance therapeutic efficacy and reduce side effect. In this work, DOX-loaded chitosan nanoparticles were prepared using supercritical fluid assisted atomization introduced by a hydrodynamic cavitation mixer (SAA-HCM) from aqueous solution. The influences of solution concentration, CO 2 /solution ratio, mixer pressure, chitosan/DOX ratio and chitosan molecular weight on particle morphologies and sizes were investigated in detail. Well defined spherical nanoparticles with average diameter ranging from 120 nm to 250 nm were obtained, and the loading efficiency was up to 90%. FT-IR result showed that the structure of DOX was not changed after the SAA-HCM process. Zeta potential of the nanoparticles was about +50 mV. The in vitro drug release behavior conducted in the media with pH of 4.5, 6.5 and 7.4 respectively showed strongly pH responsive. In vitro cytotoxicity profiles revealed that the activity of DOX was well maintained after loaded into chitosan nanoparticles. The SAA-HCM process was demonstrated to be a promising technique for one-step production of polymer/drug composite nanoparticles suitable for cancer drug delivery from aqueous solution. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Large pores increased in size with extended soaking time from 0.5 to 2 h. • Elongating holding time inceased mean size and decrease the density of large pores. • Pore size change patterns are distinct below and above 7 MPa intermediate pressure. • The pore growth profile in two-step depressurization sc-CO 2 foaming was unfolded. Abstract Bi-modal porous material is considered to have superior physical and tissue-inducing properties. This study reports on the fabrication of bi-modal porous poly(ε-caprolactone) (PCL) scaffolds using two-step depressurization supercritical CO 2 foaming process. In a single process, large pores over 100 μm nucleated in the slow depressurization step, and coalesced during holding stage; small pores below 40 μm were formed in the rapid depressurization step. Investigation on foaming parameters demonstrated that soaking time longer than 0.5 h was necessary to produce bi-modal pores. It was noted that the holding stage had a significant impact on the final pores. In particular, intermediate pressure would affect the plasticizing capabilities of CO 2 to increase (5–7 MPa) or decrease (7–12 MPa) pore size. In all, bi-modal porous PCL foams with around 80% porosity were produced, and this work provided a potential reference to efficient design of bone tissue engineering scaffolds. [ABSTRACT FROM AUTHOR]

Colon-targeted oral drug delivery systems (CDDSs) are desirable for the treatment of ulcerative colitis (UC), which is a disease with high relapse and remission rates associated with immune system inflammation and dysregulation localized within the lining of the large bowel. However, the success of current available approaches used for colon-targeted therapy is limited. Budesonide (BUD) is a corticosteroid drug, and its rectal and oral formulations are used to treat UC, but the inconvenience of rectal administration and the systemic toxicity of oral administration restrict its long-term use. In this study, we designed and prepared colon-targeted solid lipid nanoparticles (SLNs) encapsulating BUD to treat UC by oral administration. A negatively charged surfactant (NaCS-C12) was synthesized to anchor cellulase-responsive layers consisting of polyelectrolyte complexes (PECs) formed by negatively charged NaCS and cationic chitosan onto the SLNs. The release rate and colon-specific release behavior of BUD could be easily modified by regulating the number of coated layers. We found that the two-layer BUD-loaded SLNs (SLN-BUD-2L) with a nanoscale particle size and negative zeta potential showed the designed colon-specific drug release profile in response to localized high cellulase activity. In addition, SLN-BUD-2L exhibited excellent anti-inflammatory activity in a dextran sulfate sodium (DSS)-induced colitis mouse model, suggesting its potential anti-UC applications. [ABSTRACT FROM AUTHOR]

Parathyroid hormone (PTH1–34)-loaded dry powders were fabricated from aqueous solution for pulmonary administration using supercritical fluid assisted atomization introduced by a hydrodynamic cavitation mixer (SAA-HCM). Herein, chitosan oligosaccharide (CSO) was selected as a carrier in an effort to enhance transmucosal absorption of the drug. Well-defined, separated and spherical PTH(1–34)/CSO composite microparticles were obtained, and the particles size could be well controlled with narrow distribution. Aerodynamic performance was determined using next generation impactor (NGI), and the mass median aerodynamic diameter (MMAD) ranged strictly 1–5 μm range with fine particle fraction (FPF) up to 63.51%. The structural integrity of coprecipitated PTH(1–34) was validated by HPLC, FT-IR and circular dichroism, and a high loading efficiency up to 92.8% was obtained. TGA analyses revealed its thermal stability was preserved and XRD patterns showed amorphous structure of particles. The SAA-HCM process is proposed as a green technique for preparation of inhalable protein/polymer composite dry powders without use of any organic solvents. [ABSTRACT FROM AUTHOR]

Drug-loaded hydrogels with wound environment stimuli-responsive property can significantly promote wound healing and are widely applied in dressings. Two natural polymers, i.e. sodium alginate and sodium hyaluronate, were modified by 3-aminophenylboric acid and then cross-linked with polyvinyl alcohol to prepare ROS responsive hydrogels. Doxycycline hydrochloride was loaded onto hydrogels using supercritical solvent impregnation (SSI) technology, and the effects of immersion time, pressure and temperature on drug loading capacity (DLC) were investigated. DLC firstly increased and then decreased with the elevation of supercritical CO 2 pressure, and maximum value was up to 11 mg/g at 15 MPa and 50 °C. In the in vitro drug release study, doxycycline hydrochloride was controllably released from hydrogels for 10 h via the sensitivity of boronate ester bonds to ROS. This work expands the application of SSI in wound dressing, which provides a new approach for the design of composite hydrogel formulations with uniform drug distribution. [Display omitted] • ROS-responsive hydrogels were fabricated via crosslinking Alg-PBA, HA-PBA and PVA. • Doxy was loaded onto hydrogels using supercritical solution impregnation. • The dynamic mechanical properties of drug-loaded hydrogels can be adjusted. • Dug-loaded hydrogels exhibited excellent in vitro antibacterial properties. [ABSTRACT FROM AUTHOR]

Reteplase is the third generation of thrombolytic medicine and has many advantages over commercial t-PA. However, over-expressing recombinant reteplase in E. coli always accumulates as inclusion bodies due to nine pairs of disulfide bonds formation that is the main obstacle for correct folding. In this paper, in order to enhance soluble expression of recombinant reteplase in E. coli , DsbA/DsbC foldases were used to introduce disulfide bonds into the reduced polypeptide chain and catalyze their isomerization to the native disulfide linkage during the folding process. Firstly multiple E. coli protein expression systems, i.e. DsbA, DsbC and DsbA/DsbC co-expression were constructed. Subsequently, IPTG and l -arabinose were added to induce expression of foldases and reteplase accordingly, and experimental parameters such as culture temperature and inducer concentration were optimized. As a result, the co-expression system markedly enhanced soluble expression of recombinant reteplase, and up to 60% of reteplase achieved soluble expression especially for the DsbC co-expression system. The fibrin plate method for active reteplase quantification showed that ∼70 mg soluble reteplase per liter fermentation broth was obtained with 2.35 × 10 5 IU/mg thrombolytic activity. Finally, fluorescence spectra indicated that the structural conformation of soluble reteplase was identical to its native state. The soluble expression of recombinant reteplase in E. coli was accomplished by co-expression with DsbA/DsbC, which contributes to further research in clinical application and folding mechanism, and provides guidance for production of other proteins with disulfide bonds. [ABSTRACT FROM AUTHOR]

Abstract: Supercritical fluid assisted atomization introduced by a hydrodynamic cavitation mixer (SAA–HCM) was used to micronize insulin from aqueous solution without use of any organic solvents. Insulin microparticles produced under different operating conditions including solution type, solution concentration and precipitator temperature presented distinct morphologies such as highly folded, partly deflated, corrugated or smooth hollow spherical shape. Solution concentration had a striking influence on particle size, and insulin microparticles produced from acidic solution had mean diameters increasing from 1.4μm to 2.7μm when protein concentration increased from 3g/L to 50g/L. HPLC chromatograms showed no degradation of insulin after SAA–HCM processing and FTIR, CD and fluorescence data further confirmed the structural stability. TGA analysis revealed that insulin microparticles remained moderate moisture content compared with raw material. In vivo study showed that insulin processed by SAA–HCM from acidic solution retained identical bioactivity. SAA–HCM is demonstrated to be a very promising process for insulin inhaled formulation development. [Copyright &y& Elsevier]

Abstract: Recombinant human interferon gamma (rhIFN-γ) is a protein with great potential for clinical therapy, but rhIFN-γ expressed in Escherichia coli is usually in the form of insoluble inclusion bodies which should be refolded in vitro. A novel type of hairy particles (PNIPAM-grafted-PS) consisted of submicron polystyrene cores and brushes of thermo-sensitive poly(N-isopropylacrylamide) grafted onto the cores was prepared and then applied to assist the refolding of rhIFN-γin vitro. Two kinds of PNIPAM-grafted-PS particles with different thickness of brush layer (55nm and 110nm) were synthesized, which were spherical shape with good dispersion properties and the LCST was about 33°C. The effect of thickness of brush layer, particle concentration and temperature on the refolding process was investigated, it was shown that particles with larger thickness of brush layer were more effective and the final rhIFN-γ activity could be up to more than 21 times of that in dilution refolding when initial rhIFN-γ concentration was 50μg/mL. The optimal refolding condition was the concentration ratio of particle to rhIFN-γ 1:1 and refolding temperature of 15°C. All results above demonstrated that PNIPAM-grafted-PS particles could assist rhIFN-γ refolding which presented an alternative way to facilitate recombinant protein refolding in vitro. [Copyright &y& Elsevier]

Recombinant human interferon-gamma (rhIFN-γ) is a protein of great potential for clinical therapy due to its multiple biological activities. However, overexpressing rhIFN-γ in Escherichia coli was found to accumulate as cytoplasmic inclusion bodies. In this work, a system for soluble and active expression of rhIFN-γ was constructed by coexpressing chaperonin GroEL/GroES in E. coli. The rhIFN-γ gene was fused to a pET-28a expression vector, and rhIFN-γ was partially expressed as the soluble form following coexpression with a second vector producing chaperonin GroEL/GroES. The fermentation of recombinant E. coli harboring rhIFN-γ and GroEL/GroES plasmids was investigated, and the optimized conditions were as follows: culture temperature of 25°C, incubation time of 8 h, isopropyl-β- d-thio-galactoside concentration of 0.2 mM, and l-arabinose concentration of 0.5 g/L. As a result, the expression level of rhIFN-γ was improved accordingly by 2.2-fold than the control, while a significantly positive correlation was also found between the ratio of supernatant to precipitate of rhIFN-γ and the amount of chaperonin. Circular dichroism spectra, fluorescence spectra, size exclusion chromatography, and chemical cross-linking method were applied to characterize rhIFN-γ, indicating that the three-dimensional structure of rhIFN-γ was identical to that of the native rhIFN-γ. The enzyme-linked immunosorbent assay for active rhIFN-γ quantification showed that coexpression yielded 72.91 mg rhIFN-γ per liter fermentation broth. Finally, protein-protein interactions between rhIFN-γ and chaperonin were analyzed using the yeast two-hybrid system, which provided the direct evidence that chaperonin GroEL/GroES interacted with rhIFN-γ to increase the soluble expression and presented the potential in producing efficiently recombinant proteins. [ABSTRACT FROM AUTHOR]

Abstract: High speed counter-current chromatography (HSCCC) was successfully applied to resolution of phenylsuccinic acid (PSA) with hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector (CS). The two-phase solvent system composed of n-hexane–methyl tert-butyl ether–0.1molL−1 phosphate buffer solution with pH=2.51 (0.5:1.5:2, v/v/v) was selected. Influence factors involved in the chiral separation were investigated, including the concentration of chiral selector, pH value of the aqueous phase, the separation temperature, and the thermodynamic parameters of inclusion complex were calculated. The complex formation constants were determined using analytical instrument. Two HSCCC elution modes were studied and peak resolution equation was discussed. Under optimum separation conditions, 712mg of PSA racemate was separated using preparative apparatus. The purities of both of the fractions including (+)-PSA and (−)-PSA from the preparative CCC separation were over 98.5% determined by HPLC and enantiomeric excess of (+)-PSA and (−)-PSA reached 97.6% and 98.6%, respectively. Recovery for the target compounds from the CCC fractions reached 80–82% yielding 285mg of (+)-PSA and 292mg of (−)-PSA. [Copyright &y& Elsevier]

Abstract: This work concentrates on a chiral separation technology named biphasic recognition applied to resolution of α-cyclohexylmandelic acid enantiomers by high-speed counter-current chromatography (HSCCC). The biphasic chiral recognition HSCCC was performed by adding lipophilic (−)-2-ethylhexyl tartrate in the organic stationary phase and hydrophilic hydroxypropyl-β-cyclodextrin in the aqueous mobile phase, which preferentially recognized the (−)-enantiomer and (+)-enantiomer, respectively. The two-phase solvent system composed of n-hexane-methyl tert-butyl ether–water (9:1:10, v/v/v) with the above chiral selectors was selected according to the partition coefficient and separation factor of the target enantiomers. Important parameters involved in the chiral separation were investigated, namely the types of the chiral selectors (CS); the concentration of each chiral selector; pH of the mobile phase and the separation temperature. The mechanism involved in this biphasic recognition chiral separation by HSCCC was discussed. Langmuirian isotherm was employed to estimate the loading limits for a given value of chiral selectors. Under optimum separation conditions, 3.5–22.0mg of α-cyclohexylmandelic acid racemate were separated using the analytical apparatus and 440mg of racemate was separated using the preparative one. The purities of both of the fractions including (+)-enantiomer and (−)-enantiomer from the preparative CCC separation were over 99.5% determined by HPLC and enantiomeric excess reached 100% for the (±)-enantiomers. Recovery for the target compounds from the CCC fractions reached 85–88% yielding 186mg of (+)-enantiomer and 190mg of (−)-enantiomer. The overall experimental results show that the HSCCC separation of enantiomer based on biphasic recognition, in which only if the CSs involved will show affinity for opposite enantiomers of the analyte, is much more efficient than the traditional monophasic recognition chiral separation, since it utilizes the cooperation of both of lipophilic and hydrophilic chiral selectors. [Copyright &y& Elsevier]

Abstract: This work concentrates on pH-zone-refining counter-current chromatography of two isomeric dicaffeoylquinic acids, 3,5-dicaffeoylquinic acid and 3,4-dicaffeoylquinic acid along with 3-caffeoylquinic acid, from crude extracts of Flos Lonicerae. The elution sequence of the isomeric dicaffeoylquinic acids, the mixing zone and mechanism of separation are discussed. The separation of 2.136g of the crude sample from Flos Lonicerae yielded two isomeric compounds: 0.289g 3,5-dicaffeoylquinic acid and 0.106g 3,4-dicaffeoylquinic acid plus 0.690g 3-caffeoylquinic acid at a high purity of over 92.9%, 94.2% and 97.5%, respectively. [Copyright &y& Elsevier]

Abstract: A novel capsule system composed of sodium cellulose sulfate (NaCS), carboxymethyl cellulose (CMC) and poly[dimethyl(diallyl)ammonium chloride] (PDMDAAC) was prepared for improving the properties of NaCS/PDMDAAC capsules. The process parameters, such as CMC concentration (0, 2, 4, 6 and 8g/L), NaCS concentration (20, 25, 30, 35 and 40g/L), PDMDAAC concentration (20, 30, 40, 50, 60, 70 and 80g/L), reaction time and temperature were investigated to understand their effects on the diameter, membrane thickness and mechanical strength of capsules. The optimum operation conditions for preparing NaCS–CMC/PDMDAAC capsules were determined as 6–8g/L CMC, 35–40g/L NaCS, 60g/L PDMDAAC and polymerization for 30–40min. Diffusion of substances with low molecular weight into capsules was investigated, and diffusion coefficients were calculated according to the developed model. The yeast of Candida krusei was chosen as representative cell to evaluate the effects of different cell loading on capsule mechanical strength. Meanwhile the encapsulated osmophilic C. krusei cells were cultured in 250mL shaking flasks for 72h to determine the cell leaking properties in short and long term. [Copyright &y& Elsevier]

Abstract: Macroporous capsules composed by sodium cellulose sulphate (NaCS) and poly(dimethyldiallylammonium chloride) (PDMDAAC) were prepared by the addition of pore forming agent starch in the encapsulation solution. The starch was firstly immobilized in the membrane of the capsules and then degraded by amylase. The molecular weight cut-off (MWCO) of the macroporous capsules was about 70kDa for globular proteins, which was about five times larger than that of capsules prepared by standard method. Correspondingly, the mass transfer behavior of the capsules improved significantly, especially for the substances with relatively higher molecular weight. Moreover, the characteristics of the macroporous capsules, such as mechanical strength, size and the thickness of the membrane remained nearly the same as those of the standard capsules. When the macroporous capsules were applied in the immobilization of yeast, the cell density inside these capsules increased 20% than that inside the standard capsules. [Copyright &y& Elsevier]

Abstract: (1→3)-β-d-Glucans have received increasing attentions with respect to their biological functions. Previously, a method combining cellular induced autolysis with subsequent oxidation by sodium hypochlorite to prepare (1→3)-β-d-glucan from Saccharomyces cerevisiae was developed. However, the poor aqueous solubility of the resultant polysaccharide was a huge hindrance to its application especially in pharmaceuticals. Herein, a novel glucan sulphation process, also a heterogeneous reaction system, composed of sulfuric acid and n-propanol with a molar ratio of 1.4, is proposed to improve polysaccharide polarity and its aqueous solubility. After the heterogeneous sulphation at -6°C for 90min, (1→3)-β-d-glucan sulphate yield was about 37.4% (w/w), the degree of sulphation 0.36, and its empirical formula was (C6H10O5)25·9SO3·7H2O according to elemental analysis. In addition, sulphation made the polysaccharide morphologically loosened according to observation under scanning electron microscopy. The presence of characteristic absorptions at 1246 and 813cm-1 in the FTIR spectrum, as well as the 1H NMR spectrum demonstrated that the material was a sulphate ester of glucan. [Copyright &y& Elsevier]

E. coli and S. cerevisiae were encapsulated within alginate/cellulose sulphate–CaCl2/poly(methylene-co-guanidine) (SA/CS–CaCl2/PMCG) capsules. Although PMCG inhibited strongly the growth of E. coli and S. cerevisiae in free cultivation, PMCG coated capsules had no negative effects on the growth of free E. coli and S. cerevisiae or on the consumption of glucose in the cultivation of S. cerevisiae. If commercial PMCG agent was pre-treated by the method specified in this paper, E. coli and S cerevisiae immobilised in the SA/CS–CaCl2/PMCG capsules grew almost as normally as in free cultivation. Thus, PMCG was shown to be a suitable membrane material for microorganism encapsulation after it was purified under appropriate conditions. [Copyright &y& Elsevier]

Natural polymers like polysaccharides, polypeptides and their derivatives are broadly applied in drug delivery due to excellent biocompatibility and biodegradability. In this study, the dissolving tablets, formed with carboxymethylcellulose/poly- l -lysine/tripolyphosphate (CMC/PLL/TPP) complex, were prepared using metformin hydrochloride (MetHCl) as model drug. Confocal laser scanning microscopy observation manifested that FITC-labeled PLL interacted with CMC and formed a uniform interior microstructure. Scanning electron microscope images showed the drug-loaded tablets had well-formed shapes with smooth surfaces. MetHCl embedded interior the microstructures of the tablets and represented in a crystal form. Thermo-gravimetric analysis and differential scanning calorimetry indicated that the drug-loaded tablets had stable thermal properties with less moisture content (3.52%). Fourier transform infrared spectrometer confirmed that the CMC/PLL/TPP complex was fabricated via the electrostatic interactions between -NH 3 +, -COO− and -[P 2 O 5 4-]− groups. The drug-loaded tablets had a high drug loading efficiency of 85.76% and drug encapsulation efficiency of 81.47%, and a shorter wetting time of 2.16 min in SSF (pH 6.8) and lower swelling ratio of 233.34%. The drug loaded in the samples could be released completely within 10 min in simulated saliva fluid (SSF pH 6.8), indicating a rapid drug release and dissolving profile in the environment, which could be developed for dissolving tablets. • Novel carboxymethylcellulose/poly- l -lysine/tripolyphosphate tablets were prepared. • FITC-labeled PLL interacted with CMC and formed a uniform interior microstructure. • The drug-loaded tablets had a higher drug loading efficiency of 85.76%. • The drug released completely within 10 min in simulated saliva fluid (SSF pH 6.8). • CMC/PLL/TPP complex could be used to develop dissolving tablets as drug vehicles. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is one of the most serious neurodegenerative diseases with no effective treatment options available. The formation of insoluble amyloid fibrils of the hyperphosphorylated tau protein is intimately associated with AD, hence the tau protein has been a key target for AD drug development. In this work, hematoxylin was discovered as a dual functional compound, that is, acting in the inhibition of repeat domain of tau (tau-RD) protein fibrillogenesis and remodeling of pre-formed tau-RD fibrils in vitro. Meanwhile, hematoxylin was able to reduce the accumulation of tau-RD aggregates in Saccharomyces cerevisiae. Experimental and computational studies indicated that hematoxylin directly interacts with tau-RD protein through hydrophobic forces, hydrogen bonds, π -cation interactions, and π - π stackings. In addition, cellular viability assays showed that hematoxylin greatly reduced cytotoxicity induced by tau-RD aggregates. In summary, hematoxylin might be a promising candidate for further development as a potential therapeutic drug for AD patients. [ABSTRACT FROM AUTHOR]

Poly(ε-caprolactone) (PCL) is recently developed for bone tissue engineering applications due to its lasting degradation rate which matches the natural growth rate of a bone. In this work, batch foaming of PCL using supercritical CO 2 was employed, and the effects of processing variables, namely soaking time, foaming temperature and pressure, depressurization rate on the pore morphology and structure were studied in detail. Results demonstrated that morphologies and structures of pores were significantly distinct astride foaming temperature of 40 °C, which were characterized by melt-state foaming and solid-state foaming respectively. The dramatic reduction of melting temperature ( T m ) caused by the presence of supercritical CO 2 was found to be the key for this specific phenomenon. Above 40 °C, the enhanced melting of PCL caused by the reduction of T m led to wider pore size distribution, i.e. melt-state foaming, and the domino effect of variables on pore size was amplified; whilst below 40 °C the foams showed dense and small pore morphology, i.e. solid-state foaming. By tuning processing variables, scaffolds with diameter of 12.5 ± 0.1 to 344 ± 54 μm and porosity of 60.8 ± 3.2% to 83.4 ± 1.3% were fabricated. Furthermore, the interconnectivity of neat PCL scaffolds were mostly around 70%, which showed potential application in bone tissue engineering. [ABSTRACT FROM AUTHOR]

Supercritical fluid assisted atomization introduced by a hydrodynamic cavitation mixer (SAA-HCM) was proposed as a green technique to fabricate insulin-loaded dry powders for inhalation administration. N -trimethyl chitosan (TMC), a polymeric mucoadhesive absorption enhancer, was synthesized and successfully micronized from aqueous solution using SAA-HCM. The prepared well-defined spherical TMC microparticles with preserved structure and thermal stability were potential carriers for delivery of proteins. Then, insulin-loaded TMC microparticles with high loading efficiency were coprecipitated from aqueous solutions using SAA-HCM without use of any organic solvents. The polymer/protein ratio revealed to be a factor influencing the particle morphology, and non-coalescing composite microparticles in amorphous state mainly ranging from 1 μm to 5 μm could be obtained in this work. Aerodynamic properties were assessed by next generation impactor (NGI) and the mass median aerodynamic diameter (MMAD) lied inside the inhalable range of 1–5 μm, while fine particle fraction (FPF) reached above 60%. The structural integrity of encapsulated insulin was confirmed by HPLC, circular dichroism and fluorescence spectroscopy. In vivo study demonstrated that TMC could enhance the absorption and bioavailability of the pulmonarily administered insulin formulation for SD rats. These results suggest that TMC microparticles could be efficiently prepared as a promising vehicle for drug delivery, and SAA-HCM is a promising technique to prepare inhalable polymer/protein composite dry powders. [ABSTRACT FROM AUTHOR]

The preparation and characterization of mixed-mode adsorbents for a typical separation purpose are of great importance in bioseparation areas. In this work, we prepared a new monolithic cryogel with a combination of ion-exchange and hydrophobic functions by employing benzyl-quaternary amine groups. The fundamental cryogel properties, protein equilibrium adsorption isotherm and chromatographic adsorption in the cryogel were measured experimentally. The results showed that, by using bovine serum album as the model protein, the dual functional cryogel has protein binding capability even in salt solution and the buffer with pH close or below the protein isoelectric point due to both the electrostatic and hydrophobic interactions. A capillary-based adsorption model was developed, which provided satisfied insights of the microstructure, axial dispersion, mass transfer as well as protein adsorption characteristics within the cryogel bed. The chromatographic isolation of bioactive proteins from rabbit blood serum was carried out by the cryogel. Immunoglobulin G antibody with a purity of 98.2% and albumin with a purity of 96.8% were obtained, indicating that the cryogel could be an interesting and promising adsorbent in bioseparation areas. [ABSTRACT FROM AUTHOR]

Abstract: Supercritical fluid assisted atomization introduced by hydrodynamic cavitation mixer (SAA-HCM) was used to prepare chitosan microparticles with the well-defined spherical morphology and the controlled particle size distribution as a promising carrier for drug delivery system. Chitosan with different molecular weights (3kDa, 50kDa, 300kDa) was successfully micronized, in which water was used as the solvent for 3kDa chitosan, while 1.0% (v/v) acetic acid aqueous solution was used for 50kDa and 300kDa chitosan, respectively. The process parameters including operating pressure and temperature in the mixer, mass flow ratio of CO2/solution, precipitator temperature and solution concentration were explored to evaluate their influences on the morphologies and size distributions of precipitated particles. Results showed that particle size tailoring (ranging between 0.2 and 5μm) could be achieved through modulation of the process parameters. After processing by SAA-HCM, Fourier transform infrared spectroscopy did not indicate significant change in the main structure of chitosan microparticles. Compared with raw materials, a decrease in crystallinity and thermal stability was observed for the SAA-HCM processed chitosan microparticles, as demonstrated by X-ray diffraction, thermogravimetric analysis and differential scanning calorimetry. [Copyright &y& Elsevier]

Highlights: [•] Separated and spherical chitosan oligomers microparticles were produced from water. [•] Particle size could be tailored and distribution was narrow in the range of 0.5–3μm. [•] FTIR, TGA and DSC results confirmed chitosan oligomers molecular structure stability. [•] Mass median aerodynamic particle size ranged inside 1–2μm suitable for inhalation. [ABSTRACT FROM AUTHOR]

A novel super-macroporous monolithic composite cryogel was prepared by embedding macroporous cellulose beads into poly(hydroxyethyl methacrylate) cryogel. The cellulose beads were fabricated by using a microchannel liquid-flow focusing and cryopolymerization method, while the composite cryogel was prepared by cryogenic radical polymerization of the hydroxyethyl methacrylate monomer with poly(ethylene glycol) diacrylate as cross-linker together with the cellulose beads. After graft polymerization with (vinylbenzyl)trimethylammonium chloride, the composite cryogel was applied to separate immunoglobulin- G and albumin from human serum. Immunoglobulin- G with a mean purity of 83.2% and albumin with a purity of 98% were obtained, indicating the composite cryogel as a promising chromatographic medium in bioseparation for the isolation of important bioactive proteins like immunoglobulins and albumins. [ABSTRACT FROM AUTHOR]

Abstract: Counter-current chromatography (CCC) was applied for preparative enantioseparation of three β-blocker drugs, including propranolol, pindolol and alprenolol. The two-phase solvent system was composed of chloroform–0.05molL−1 acetate buffer containing 0.10molL−1 boric acid (1:1, v/v), in which 0.10molL−1 di-n-hexyl l-tartrate was added in the organic phase as chiral selector. Influence factors in the enantioseparation of propranolol were investigated. The chromatographic retention mechanism based on borate coordination complex was proposed. 116mg of racemic propranolol was completely enantioseparated using conventional high speed CCC in a single run, yielding 48mg of (+)-propranolol with HPLC purity of 98.9% and 47mg of (−)-propranolol with HPLC purity of 96.3%. Recovery for propranolol enantiomers from CCC fractions was in the range of 75–82%. pH-zone-refining CCC was also successfully applied in enantioseparation of propanolol and it was found that 356mg of racemic propranolol could be completely enantioseparated. 145mg of (+)-enantiomer with HPLC purity of 95.6% and 148mg of (−)-enantiomer with HPLC purity of 98.2% were recovered from pH-zone-refining mode. Separation mechanism about chiral separation by pH-zone-refining CCC was discussed. [Copyright &y& Elsevier]

Abstract: Polymeric cryogels are sponge-like materials with supermacroporous structure, allowing them to be of interest as new chromatographic supports, cell scaffolds and drug carriers in biological and biomedical areas. The matrices of cryogels are always prepared in the form of monoliths by cryo-polymerization under frozen conditions. However, there are limited investigations on the production of cryogels in the form of adsorbent beads suitable for bioseparation. In this work, we provide a new approach by combining the microchannel liquid-flow focusing with cryo-polymerization for the preparation of polyacrylamide-based supermacroporous cryogel beads with a narrow particle size distribution. The present method was achieved by introducing the aqueous phase solution containing monomer, cross-linker and redox initiators, and the water-immiscible organic oil phase containing surfactant simultaneously into a microchannel with a cross-shaped junction, where the aqueous drops with uniform sizes were generated by the liquid shearing and the segmentation due to the steady flow focusing of the immiscible phase streams. These liquid drops were in situ suspended into the freezing bulk oil phase for cryo-polymerization and the cryogel matrix beads were obtained by thawing after the achievement of polymerization. By grafting the polymer chains containing sulfo binding groups onto these matrix beads, the cation-exchange cryogel beads for protein separation were produced. The results showed that at the aqueous phase velocities from 0.5 to 2.0cm/s and the total velocities of the water-immiscible phase from 2.0 to 6.0cm/s, the obtained cryogel beads by the present method have narrow size distributions with most of the bead diameters in the range from 800 to 1500μm with supermacropores in sizes of about 3–50μm. These beads also have high porosities with the averaged maximum porosity of 96.9% and the mean effective porosity of 86.2%, which are close to those of the polyacrylamide-based cryogel monoliths. The packed bed using the cryogel beads with mean diameter of 1248μm, as an example, has reasonable and acceptable liquid dispersion, but high water permeability (4.29×10−10 m2) and high bed voidage (90.2%) owing to the supermacropores within the beads, enhanced the rapid binding and separation of protein from the feedstock even at high flow velocities. The purity of the obtained lysozyme from chicken egg white by one-step chromatography using the packed bed was in the range of about 78–92% at the flow velocities of 0.5–15cm/min, indicating that the present cryogel beads could be an effective chromatographic adsorbent for primary bioseparation. [Copyright &y& Elsevier]