Surface-anchored microbial enzyme-responsive solid lipid nanoparticles enabling colonic budesonide release for ulcerative colitis treatment.

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.

Poly(hydroxyethyl methacrylate)-based composite cryogel with embedded macroporous cellulose beads for the separation of human serum immunoglobulin and albumin.

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.

Hematoxylin modulates tau-RD protein fibrillization and ameliorates Alzheimer's disease-like symptoms in a yeast model.

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.

Understanding the Molecular Mechanisms of Polyphenol Inhibition of Amyloid ß Aggregation.

Alzheimer's disease (AD) is highly associated with self-aggregation of amyloid ß (Aß) proteins into fibrils. Inhibition of Aß aggregation by polyphenols is one of the major therapeutic strategies for AD. Among them, four polyphenols (brazilin, resveratrol, hematoxylin, and rosmarinic acid) have been reported to be effective at inhibiting Aß aggregation, but the inhibition mechanisms are still unclear. In this work, these four polyphenols were selected to explore their interactions with the Aß17-42 pentamer by molecular dynamics simulation. All four polyphenols can bind to the pentamer tightly but prefer different binding sites. Conversion of the ß-sheet to the random coil, fewer interchain hydrogen bonds, and weaker salt bridges were observed after binding. Interestingly, different Aß17-42 pentamer destabilizing mechanisms for resveratrol and hematoxylin were found. Resveratrol inserts into the hydrophobic core of the pentamer by forming hydrogen bonds with Asp23 and Lys28, while hematoxylin prefers to bind beside chain A of the pentamer, which leads to ß-sheet offset and dissociation of the ß1 sheet of chain E. This work reveals the interactions between the Aß17-42 pentamer and four polyphenols and discusses the relationship between inhibitor structures and their inhibition mechanisms, which also provides useful guidance for screening effective Aß aggregation inhibitors and drug design against AD.

Coexpression of chaperonin GroEL/GroES markedly enhanced soluble and functional expression of recombinant human interferon-gamma in Escherichia coli.

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-ß-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 crosslinking 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.

Development of metformin hydrochloride loaded dissolving tablets with novel carboxymethylcellulose/poly-l-lysine/TPP complex.

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.

Preparation of pH-responsive DOX-loaded chitosan nanoparticles using supercritical assisted atomization with an enhanced mixer.

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.

Preparative separation of isomeric caffeoylquinic acids from Flos Lonicerae by pH-zone-refining counter-current chromatography.

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.

Preparation, characterization and refolding in vitro of a recombinant human cyclophilin A mutant: effect of a single Pro/Ser substitution on cyclophilin A structure and properties.

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.

Preparation of micrometric powders of parathyroid hormone (PTH1-34)-loaded chitosan oligosaccharide by supercritical fluid assisted atomization.

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.

Chromatographic separation of phenyllactic acid from crude broth using cryogels with dual functional groups.

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.

Chromatographic refolding of recombinant human interferon gamma by an immobilized sht GroEL191-345 column.

Minichaperone sht GroEL191-345 was covalently coupled to NHS-activated Sepharose Fast Flow gel. Refolding of recombinant human interferon gamma (rhIFN-gamma) 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-gamma. When loading 100 microl denatured rhIFN-gamma (17.8 mg/ml), the protein mass recovery and total activity obtained in this optimal process reached 74.25% and 6.74 x 10(6)IU/ml, respectively with the immobilized minichaperone column which was reused for 10 times with 25% decrease of renaturation capacity.

Formulation of insulin-loaded N-trimethyl chitosan microparticles with improved efficacy for inhalation by supercritical fluid assisted atomization.

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 1µm to 5µm 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 1-5µm, 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.

Production of minichaperone (sht GroEL191-345) and its function in the refolding of recombinant human interferon gamma.

The recombinant minichaperone sht GroEL191-345 was cultivated in a 3.7 L stirred bioreactor with the high yield of 216.2 mg/L broth. In the refolding of recombinant human interferon gamma (rhuIFN-gamma) inclusion bodies, more than 2-3 fold enhancement in protein mass recovery and total activity were observed in the presence of free or immobilized minichaperone to the refolding buffer.

Preparation and characterization of NaCS-CMC/PDMDAAC capsules.

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 8 g/L), NaCS concentration (20, 25, 30, 35 and 40 g/L), PDMDAAC concentration (20, 30, 40, 50, 60, 70 and 80 g/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 6-8 g/L CMC, 35-40 g/L NaCS, 60 g/L PDMDAAC and polymerization for 30-40 min. 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 250 mL shaking flasks for 72 h to determine the cell leaking properties in short and long term.

Supercritical fluid assisted production of micrometric powders of the labile trypsin and chitosan/trypsin composite microparticles.

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.

Chromatographic adsorption of serum albumin and antibody proteins in cryogels with benzyl-quaternary amine ligands.

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.

Novel isoelectric precipitation of proteins in a pressurized carbon dioxide-water-ethanol system.

A novel isoelectric precipitation of proteins in a pressurized carbon dioxide-water-ethanol system was developed where carbon dioxide was used as a volatile acid. The pH-pressure curves of the system with the absence and presence of proteins were investigated. By introducing the pressurized carbon dioxide to a solution containing protein, the pH value in the solution was decreased to the isoelectric region of the model protein BSA. Addition of ethanol could lower the buffer capacity of the protein, which made the precipitation concentration of protein go beyond the limits in a system without ethanol and well exploited the application field of the technique. In addition, ethanol in solution played the role of aiding precipitation in the process. Another model protein, hen egg white lysozyme, was also studied but could not be precipitated in the above system. All of these phenomena prove that isoelectric precipitation is the key point in the pressurized carbon dioxide-water-ethanol system.

On-column refolding of recombinant human interferon-gamma with an immobilized chaperone fragment.

The chaperone mini-GroEL is a soluble recombinant fragment containing the 191-345 amino acid sequence of GroEL with a 6xHis tag. The refolding protocol assisted with mini-GroEL was studied for the activity recovery of rhIFN-gamma inclusion bodies. In a suspended system, mini-GroEL showed significant enhancement of the activity recovery of rhIFN-gamma, applyed with a 1-5:1 stoichiometry of mini-GroEL to rhIFN-gamma at 25 degrees C. Moreover, 1 M urea in the renaturation buffer had a synergistic effect on suppressing the aggregation and improving the activity recovery. Finally, a novel chromatographic column, containing 1 cm height of Sephadex G 200 at the top of column and packed with immobilized mini-GroEL to promote refolding, was devised. The total activity recovered per milligram of denatured rhIFN-gamma was up to 3.93 x 10(6) IU with the immobilized mini-GroEL column, which was reused four times without evident loss of renaturation ability. A convenient technique with the integrated process of chaperon preparation and rhIFN-gamma folding in vitro was developed.

Lysozyme refolding at high concentration by dilution and size-exclusion chromatography.

This study of renaturation by dilution and size exclusion chromatography (SEC) addition of urea to improve yield as well as the initial and final protein concentrations showed that although urea decreased the rate of lysozyme refolding, it could suppress protein aggregation to sustain the pathway of correct refolding at high protein concentration; and that there existed an optimum urea concentration in renaturation buffer. Under the above conditions, lysozyme was successfully refolded from initial concentration of up to 40 mg/mL by dilution and 100 mg/mL by SEC, with the yield of the former being more than 40% and that of the latter being 34.8%. Especially, under the condition of 30 min interval time, i.e. tau > 2(t(R2) - t(R1)), the efficiency was increased by 25% and the renaturation buffer could be recycled for SEC refolding in continuous operation of downstream process.