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Androst-4-ene-3,17-dione (AD) is an important intermediate of steroid drugs, and the microbial side-chain cleavage of sterols to produce AD by Mycobacterium sp. has attracted much attention. Unfort...

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, CO2/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.

A Lactobacillus buchneri GBS3 strain isolated from the traditional Chinese pickles was used for the production of 3-phenyllactic acid (PLA), an important compound with antimicrobial activities against a wide species of gram-positive and gram-negative bacteria and some fungi. The growth performance of this strain in the de Man, Rogosa and Sharpe (MRS) medium, the production of metabolites of valuable organic acids, and the biosynthesis of PLA using this strain as the whole-cell biocatalyst and phenylpyruvic acid (PPA) as the precursor, were investigated experimentally. The uniform design method with overlay sampling was developed for the optimization of the biotransformation conditions. The results showed that although it produced naturally lactic acid with the maximum concentration of 1.84 g·L− 1 and PLA with the concentration of 0.015 g·L− 1 after 66 to 72 h cultivation in MRS broth by fermentation, the present strain displayed an effective utilization ability by transforming PPA to PLA. By the uniform design method with overlay sampling for the design and optimization of transformation conditions, a maximum yield of 10.93 g·L− 1 PLA with the mole conversion ratio of 83.07% from PPA to PLA was achieved under the optimized condition, i.e., 20 g·L− 1 glucose, 270 g·L− 1 cells, 13 g·L− 1 PPA, pH 8.0 and the reaction time of 15 h, indicating that Lactobacillus buchneri GBS3 was an interesting strain for the biosynthesis of PLA via the microbial transformation. The prediction of PLA yield under different conditions was achieved successfully based on the limited information of only a small number of experiments by the uniform design with overlay sampling. Therefore, the present methodology is effective and helpful for the optimization of the biosynthesis processes of PLA.

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(e-caprolactone) (PCL) scaffolds using two-step depressurization supercritical CO2 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 CO2 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.

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 -NH3+, -COO− and -[P2O54-]− 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.

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.

Due to a worldwide focus on sustainable materials for human health and economy services, more and more natural renewable biomass are regarded as promising materials that could replace synthetic polymers and reduce global dependence on petroleum resources. Cellulose is known as the most abundant renewable polymer in nature, varieties of cellulose-based products have been developed and have gained growing interest in recent years. In this review, a kind of water-soluble cellulose derivative, i.e., sodium cellulose sulfate (NaCS) is introduced. Details about NaCS’s physicochemical properties like solubility, biocompatibility, biodegradability, degree of substitution, etc. are systematically elaborated. And promising applications of NaCS used as biomaterials for microcarriers’ designing, such as microcell- carriers, micro-drug-carriers, etc., are presented.

Scaffolds with multimodal pore structure are essential to cells differentiation and proliferation in bone tissue engineering. Bi-/multi-modal porous PLGA/hydroxyapatite composite scaffolds were prepared by supercritical CO2 foaming in which hydroxyapatite acted as heterogeneous nucleation agent. Bimodal porous scaffolds were prepared under certain conditions, i.e. hydroxyapatite addition of 5%, depressurization rate of 0.3 MPa·min− 1, soaking temperature of 55 °C, and pressure of 9 MPa. And scaffolds presented specific structure of small pores (122 μm ± 66 μm) in the cellular walls of large pores (552 μm ± 127 μm). Furthermore, multimodal porous PLGA scaffolds with micro-pores (37 μm ± 11 μm) were obtained at low soaking pressure of 7.5 MPa. The interconnected porosity of scaffolds ranged from (52.53 ± 2.69)% to (83.08 ± 2.42)% by adjusting depressurization rate, while compression modulus satisfied the requirement of bone tissue engineering. Solvent-free CO2 foaming method is promising to fabricate bi-/multi-modal porous scaffolds in one step, and bioactive particles for osteogenesis could serve as nucleation agents.

Nano-in-microparticles used for inhalation lung cancer therapy combine the advantages of microparticles with aerodynamic diameters of 1∼5 μm for deep lung deposition and nanoparticles for long residence time in lung, high mucus penetrating and cancer cells uptake efficiency. In this work, nano-in-microparticles composed of chitosan nanoparticles and mannitol were prepared using a modified supercritical CO2 assisted atomization (SAA-HCM) technique. Influences of process parameters of CO2/liquid mass flow ratio, total mass concentration and nanoparticles/mannitol ratio on size and morphologies of nano-in-microparticles were investigated in detail. Well-defined spherical nano-in-microparticles with theoretical mass median aerodynamic diameter of 1∼2 μm were obtained under optimized conditions. Fraction of about 40 % of nanoparticles was redispersed in deionized water at nanoparticles/mannitol ratio of 10:90. Meanwhile, quick disintegration of nano-in-microparticles was observed under conditions simulating high humidity in lung. In summary, dissociation of nano-in-microparticles and redispersibility of nanoparticles were determined by the structure of nano-in-microparticles according to particles formation mechanism. This work will expand the applicability of SAA-HCM to the preparation of nano-in-microparticles from nanosuspension and provide more references for designing of particles with elaborate structures.

Poly(e-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.

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.

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.

The microchannel liquid-flow focusing and cryo-polymerization is an efficient method for the preparation of cryogel beads with a narrow diameter distribution. In order to prepare cryogel beads with expected diameters, it is necessary to get insights in the liquid–liquid immiscible flow characteristics of the flow-focusing fluid and the monomer solution in microchannels. In this work, the slug flow behaviors of two immiscible liquids regarding the preparation of poly(2-hydroxyethylmethacrylate) (pHEMA) cryogel beads in a rectangular cross-junction microchannel were investigated experimentally by the high-speed imaging method. Correlations of the immiscible liquid–liquid slug flow parameters like the aqueous slug velocity and length, the aqueous slug nose and tail lengths, the water-immiscible slug length as well as the aqueous droplet size were obtained. The pHEMA cryogel beads were prepared under certain flow conditions and the bead sizes were measured by laser particle size analyzer. The obtained correlations were then employed to estimate the bead sizes and compared with those obtained experimentally. The results showed that the present correlations gave reasonable estimations of the mean bead diameters at various conditions and thus, could be useful and helpful in the preparation of cryogel beads with expected size distributions in rectangular microchannels.

Microbial side-chain cleavage of natural sterols to 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) by Mycobacteria has received much attention in pharmaceutical industry, while low yield of the reaction owing to the strong hydrophobicity of sterols is a tough problem to be solved urgently. Eight kinds of vegetable oils, i.e., sunflower, peanut, corn, olive, linseed, walnut, grape seed, and rice oil, were used to construct oil/aqueous biphasic systems in the biotransformation of phytosterols by Mycobacterium sp. MB 3683 cells. The results indicated that vegetable oils are suitable for phytosterol biotransformation. Specially, the yield of AD carried out in sunflower biphasic system (phase ratio of 1:9, oil to aqueous) was greatly increased to 84.8 % with 10 g/L feeding concentration after 120-h transformation at 30 °C and 200 rpm. Distribution coefficients of AD in different oil/aqueous systems were also determined. Because vegetable oils are of low cost and because of their eco-friendly characters, there is a great potential for the application of oil/aqueous two-phase systems in bacteria whole cell biocatalysis.

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 (3 kDa, 50 kDa, 300 kDa) was successfully micronized, in which water was used as the solvent for 3 kDa chitosan, while 1.0% (v/v) acetic acid aqueous solution was used for 50 kDa and 300 kDa 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 5 μm) 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.

BACKGROUND Mycelia pellet has been exploited as a biological carrier for whole-cell immobilization due to its unique structural characteristics. An innovative two-species whole-cell immobilization system was developed initially, which was achieved simply by inoculating the marine-derived fungus Pestalotiopsis sp. J63 spores into culture medium containing another fungus Penicillium janthinellum P1 pre-grown mycelia pellets for 2 days. RESULTS The resulting co-immobilization system exhibited stronger biocatalytic activity, broader spectrum of substrates and remained more stable in media when subjected to hostile environment than free mycelia. In order to examine the potential function and prospects of this co-immobilization system, the treatment of paper mill effluent was applied as a practical example. Numerous insoluble fine fibers in the wastewater were successfully and rapidly biodegraded and removed using this novel co-immobilization system. CONCLUSION Overall, the present study offers a simple and reproducible way to architect a two-species whole-cell immobilization system, which is highly promising for biochemical and industrial processes. © 2013 Society of Chemical Industry

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.

Equilibrium sorption amount, desorption diffusion coefficients and sorption diffusion coefficients of CO 2 in poly( l -lactic acid) (PLLA) films at elevated pressures were determined by the gravimetric method, in which the Fick's diffusion model was applied to analyze both the desorption and sorption processes. The equilibrium sorption amount of CO 2 in PLLA increased with lowering temperature and elevating pressure at the temperature range from 40 to 60 °C and pressure from 10 4 to 2×10 4 kPa. Desorption diffusion coefficients were greatly influenced by the equilibrium sorption amount, and they were in the same order of magnitude as the sorption diffusion coefficients. The scan electron microscope (SEM) photos demonstrated that there was no foaming phenomenon of the PLLA film during desorption and sorption processes. The XRD spectra implied that the crystalline degree of PLLA film decreased after CO 2 processing. It was concluded that PLLA polymer could be well swollen and plasticized by supercritical CO 2 .

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 1.4 μm to 2.7 μm when protein concentration increased from 3 g/L to 50 g/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.

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-γ in vitro . Two kinds of PNIPAM-grafted-PS particles with different thickness of brush layer (55 nm and 110 nm) 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 50 μg/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 .

Disulfide bond formation protein A (DsbA) is one of the important helper proteins for folding in protein synthesis in vivo . In this study, purification of recombinant DsbA was investigated by examining four important factors with Box-Behnken design method, a statistic-based design of experiments. The optimal operation conditions were obtained by adopting the effectiveness coefficient method on the multi-objective problem, which takes the protein recovery, purification efficiency and throughput of ion-exchange chromatography into account. After the optimization, protein recovery of 96.8% and purity higher than 95% DsbA was achieved, and the productivity was (377.9±1.7) mg soluble DsbA per liter broth. The purified protein was identified by peptide mass fingerprinting matching the record of gi|2624856, a mutant of DsbA. The DsbA was preliminarily applied to the refolding of denatured lysozyme in vitro .

Specific pore structure is a vital essential for scaffolds applied in tissue engineering. In this article, poly(lactide-co-glycolide) (PLGA) scaffolds with a bimodal pore structure including macropores and micropores to facilitate nutrient transfer and cell adhesion were fabricated by combining supercritical CO2 (scCO2) foaming with particle leaching technique. Three kinds of NaCl particles with different scales (i.e., 100–250

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-0.05 mol L(-1) acetate buffer containing 0.10 mol L(-1) boric acid (1:1, v/v), in which 0.10 mol L(-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. 116 mg of racemic propranolol was completely enantioseparated using conventional high speed CCC in a single run, yielding 48 mg of (+)-propranolol with HPLC purity of 98.9% and 47 mg 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 356 mg of racemic propranolol could be completely enantioseparated. 145 mg of (+)-enantiomer with HPLC purity of 95.6% and 148 mg 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.

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 25,600 U/mg specific activity of lysozyme were achieved when 281.35 μg/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 .

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 (CO 2 /acetone and CO 2 /ethanol) using Peng–Robinson equation of state. The results indicated that the liquid volume expansion of CO 2 /acetone mixtures was much larger than that of CO 2 /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.

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 5 μm 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.

Supercritical solution impregnation (SSI) was applied to prepare drug-loaded biodegradable films where poly(l-lactic acid) (PLLA) was used as the matrix and roxithromycin as the model drug. The eff...

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.

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 2.4 mg denatured lysozyme in 0.5 ml 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.

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 18 g/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.

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–0.1 mol L−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, 29 mg 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 13 mg of (S)-NAP and 12 mg of (R)-NAP.

A novel hydrogel of P(NIPAM-co-SA) copolymer was synthesized by inverse suspension polymerization by adding sodium acrylate (SA) to improve the phase transition properties of poly(N-isopropylacrylamide) (PNIPAM). The morphologies, size distribution and thermosensitive characteristics of gel particles were studied and the maximal swelling ratio and LCST (Lower Critical Solution Temperature) of gel particles increased obviously with the addition of SA comonomer. When the protein concentration was 250 μg/mL, the optimized refolding conditions of denatured lysozyme with P(NIPAM-co-SA) hydrogel were that operating at the temperature of 35°C and a urea concentration of 2M, in which the mass ratio of P(NIPAM-co-SA) hydrogel with 4% SA copolymerized to lysozyme was 10 : 1. Under the optimized conditions, the activity recovery of lysozyme increased to 76.5% assisted by P(NIPAM-co-SA) gel particles compared with 55.6% by simple dilution. When refolding finished, the gel particles could be removed and recovered easily and the activity recovery of lysozyme was still as high as 61.5% after reused for 5 batches. With the addition of different amounts of SA comonomer, the hydrophobicity of the copolymer could be varied. Then the copolymerized hydrogel inhibits protein molecules aggregation more effectively through the moderate hydrophobic interactions between copolymers and protein molecules in the course of lysozyme refolding compared with the presence of PNIPAM polymer. All results above demonstrate that the P(NIPAM-co-SA) is a cost effective additive with tunable hydrophobicity for application in the refolding of recombinant proteins expressed as inclusion bodies in vitro. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

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 5 μm. 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.

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 3.0 μm 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.

Ginkgo flavonoids were extracted from Ginkgo biloba leaves using supercritical carbon dioxide extraction technology (SFE) with ethanol as the entrainer. The effects of important process parameters on SFE were examined, and the crude extracts were further refined. At optimized experimental conditions, i.e., 20 MPa of extraction pressure, a 40 °C extraction temperature, a 10 g/min CO2 flow rate, and a 6 mL/min ethanol flow rate, the extraction yield could reach 0.36%. Supercritical fluid assisted atomization introduced by a hydrodynamic cavitation mixer (SAA-HCM) was used to obtain microparticles of Ginkgo flavonoids. The influences of the solute concentration as well as the mass flow ratio of the CO2/liquid solution on particle size (PS) and particle size distribution (PSD) were investigated. Well-defined, spherical, and separated particles with a controlled particle size ranging between 0.2 and 3.0 μm were successfully obtained in this work. The micronization of Ginkgo flavonoids can improve the drug abso...

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, 3.5–22.0 mg of α-cyclohexylmandelic acid racemate were separated using the analytical apparatus and 440 mg 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 186 mg of (+)-enantiomer and 190 mg 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.

The renaturation of lysozyme assisted by a novel type of hairy particles was investigated. The particles used consisted of polystyrene (PS) cores onto which long chains of thermosensitive poly(N-isopropylacrylamide) (PNIPAM) were grafted as shell. Here, two kinds of particles were synthesized as sub-micron PS cores and 89 nm brush thickness (Particle A) and 122 nm brush thickness (Particle B), respectively. Thermosensitive characteristics of the above prepared hairy uniform PS particles with PNIPAM brushes were studied and the results indicated that the lower critical solution temperature (LCST) of PNIPAM brushes grafted on the surface of uniform PS cores was between 33 and 34°C. It was proved that the particles were quite efficient in assisting lysozyme renaturation at high initial protein concentration. When the protein concentration was increased to 500 μg/mL, the refolding yield of the recovery of lysozyme activity could achieve 58.0% (Particle A) and 71.5% (Particle B), compared with only 34.8% by simple dilution refolding. Furthermore, the kinetics of lysozyme refolding in the absence and the presence of the colloidal particles were studied accordingly. The results indicated that the time required for the refolding with colloidal particles was a little bit delayed than that by the simple dilution method owing to the hydrophobic interactions between lysozyme and the PNIPAM brushes. The mechanism of the enhancement for the hairy uniform PS particles with PNIPAM brushes assisted refolding was further discussed. All results above demonstrated that the spherical PNIPAM brushes presented an alternative way to assist protein renaturation in vitro. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci 114:1270-1277,2009.

BACKGROUND: A process combining biocatalytic reaction and cell activity regeneration was designed for the asymmetric reduction of 3-oxo ester. By immobilizing resting baker's yeast (Saccharomyces cerevisiae) in calcium alginate beads, the high yield and long catalyst life were achieved in the aqueous phase in this process with methyl acetoacetate (MAA) as the model substrate. RESULTS: Two combined fixed-bed reactors were able to work steadily for at least 16 days. The activity of immobilized baker's yeast could be retained by re-culture with culture medium regularly. The re-culture time for bead reactivation was optimized to be 30 h. High yield (about 80%) and high enantiomeric excess (>95%) were maintained after 12 batches of asymmetric reduction. The immobilized beads retained their original shapes even after a long reaction time in the fixed-bed reactor, while the beads broke after reaction of five batches in a flask. CONCLUSION: The combined process of biocatalysis and cell activity regeneration was successfully achieved in the asymmetric reduction and decreased the breakage of beads as well as increased the efficiency of catalyst. Copyright © 2008 Society of Chemical Industry

BACKGROUND:Arthrobacter simplex cells immobilised in sodium cellulose sulfate/poly-dimethyl-diallyl-ammonium chloride microcapsules were used for the microbial dehydrogenation of 11α-hydroxy-16α,17-epoxyprogesterone to 11α-hydroxy-16α,17α-epoxypregn-1,4-diene-3,20-dione in an aqueous/organic solvent two-liquid-phase system, which is a key reaction in the production of glucocorticoid pharmaceuticals. The aim of the study was to establish a suitable aqueous/organic solvent two-liquid-phase system for performing semi-continuous production in an airlift loop reactor by encapsulated A. simplex cells with the addition of suitable surfactants to achieve a higher yield of the product. RESULTS:n-Hexane was selected as the most suitable organic solvent. In optimised Tween-80 emulsion feed mode the conversion in the airlift loop reactor was as high as 97.54% when the time of reaction was 2 h, and the reaction time was greatly shortened. In semi-continuous production the cultivation with immobilised cells was carried out for five batches in total. The conversion in each batch was above 95% and the enzymatic activity still remained quite high after five batches of biotransformation. CONCLUSION: The results showed that performing the conversion by this method shortened the reaction time and increased the productivity, thus demonstrating the great potential of the method for the dehydrogenation of 11α-hydroxy-16α,17-epoxyprogesterone. Copyright © 2008 Society of Chemical Industry

A recombinant cyclophilin A (CypA) mutant, which carries a serine instead of proline at sequence 16, was prepared for structural and functional assessment for human CypA. Soluble expression of the recombinant CypA mutant in E. coli was obtained under 30 degrees C, 180 rpm culture condition after being induced by IPTG. Ion exchange chromatography was used to purify the CypA mutant in a single step, and a high activity recovery of target protein with a high purity was achieved. Peptide fragments produced by trypsin proteolysis were applied to MALDI-TOF-MS, and searching results from the NCBI protein databank confirmed the protein attribution as well as the mutation sequence. Peptidyl-prolyl cis-trans isomerase activity was assayed for the CypA mutant using tetrapeptide substrate Suc-Ala-Ala-Pro-Phe-p-nitroanilide, and the calculated kcat/Km value was 1.5 x 106 M-1 s-1 at 10 degrees C, which was 10-fold lower than the previously reported constant for wild-type CypA. An Eyring plot was also carried out. Inhibition by cyclosporine A demonstrated that the IC50 value was 26.5 nM. Meanwhile the expected enhancement of intrinsic tryptophan fluorescence was quenched by the mutation. The effect of CypA mutant on accelerating protein refolding in vitro was investigated in ribonuclease A refolding process, and it was found that 10% slow phase could be catalyzed by CypA. The protein was subject to urea and GdmCl denaturation, where both activity and fluorescence served as structural probes. Activity recovery indicated this CypA mutant was extremely sensitive to GdmCl and the susceptibility to urea was increased. Low pH could also destabilize CypA. Furthermore the refolding of this CypA mutant itself was studied. Although the activity yield was nearly unchanged, the former proposed folding/assembly pathway might be altered. Fluorescence chart also demonstrated that the folding time was extended, and fast-folding and slow-folding analysis indicated the slow-folding rate constant presented a concentration dependence property denoting the autocatalysis of the foldase.

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 2.1 μm were produced. The SAA-HCM is expected to be a promising technique for producing microparticles suitable for pulmonary delivery in drug delivery applications.

Microorganisms are inactivated remarkably by carbon dioxide at the elevated pressure. The process can be divided into two phases based on the different rate of inactivation: the rate of inactivation is slower at the initial stage and increased sharply at the later stage. The dissolution of CO 2 into the aqueous solution and then diffusion into cells controls the duration and inactivation rate at the early stage. An increase of pressure and temperature enhances the antimicrobial effect of CO 2 under pressure. Inactivation is also affected by agitation and release of pressure. Ethanol is an effective and novel aid to intensify inactivation of microorganisms in CO 2 . E. coli cells after CO 2 treatment are subjected to injury on membranes so as to lose salt tolerance and induce a leakage of intracellular materials. The changes of cell structure and inactivation of several enzymes are observed after treatment. But the most important inactivation mechanism may be the lowering of pH due to the penetration of CO 2 , which leads to the inhibition of certain metabolic and regulating processes.