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In this study, Saccharomyces cerevisiae was engineered to degrade and utilize xylan, one of the major polysaccharide chains present in hemicellulose. Different hemicellulases from Trichoderma reesei, namely: endoxylanase, β-xylosidase, acetylxylan esterase, α- d -glucuronidase and α- l -arabinofuranosidase, were heterologously secreted by S. cerevisiae. A mixture experimental design was adapted to statistically describe the synergistic interactions between the hemicellulases and to determine the optimum formulations for the hydrolysis of xylan substrates. The hydrolytic activities of the hemicellulase mixtures were then improved by displaying the hemicellulases on the yeast surface as whole-cell biocatalysts. The engineered yeast strains displaying hemicellulases were further engineered to express xylose-utilization genes xylose isomerase (XI) and xylulokinase (XK) which enable its utilization of xylose as a sole carbon source. The resulting consortium was then able to grow and produce ethanol from different xylan substrates.

The aim of this study was to utilize a new and highly effective bioreactor system, i.e., simultaneous saccharification and fermentation (SSCF), for bioethanol production by the cocultivation of Trichoderma reesei, Aspergillus niger, and Zymomonas mobilis by using a direct conversion process of pretreated-wood dust medium. Wood dust has been effectively used to obtain reducing sugars (glucose, xylose, and other byproducts) through the use of either a supercritical fluid extraction (SFE) or steam explosion (SE) pretreatment step. In addition, experimental results showed that polyurethane as a porous carrier could enhance total saccharification enzyme activity at an inoculum proportion of 1/1 of T. reesei, and A. niger and at a total inoculum concentration of 6.5 × 106 spores/ml. Furthermore, the concentration of alginate beads (3%) and immobilized proportion of Z. mobilis to alginate beads (1:4) were also examined. In accordance with previous reports, bioethanol production was carried out in a SSCF bioreactor by the cocultivation of T. reesei and A. niger in the polyurethane carrier and Z. mobilis immobilized in alginate beads using pretreated-wood dust medium. Experimental results revealed that, after 24 h of cultivation, the yield of bioethanol produced using pretreated-wood dust medium (1%) were 0.069 g/g and 0.049 g/g, for SFE and SE, respectively. Meanwhile, the sugar conversion rate reached 20.72% and 24.39% for SFE and SE, respectively. Thus, the results of this study show that pretreated-wood dust medium has significant potential for use in bioethanol production.

Poly (1,6-bis- (p-carboxyphenoxy hexane)-co- (sebacic anhydride)) (PANH), is a polyanhydride copolymer which has good biocompatibility, and degrades to non-toxic products with a predictable rate of degradation. Nano-sized hydroxyapatite (HAP) is a well-known biomaterial which has been applied in bone regeneration due to its osteoconductivity. However, nano-sized HAP has poor colloidal stability which leads to agglomeration when incorporated into polymeric composites. In this work we describe the surface grafting of poly(ɛ-caprolactone) to HAP (PCL-gHAP) to improve the interfacial adhesion and dispersion of HAP particles in a PANH matrix to form a composite material. The use of scanning electron microscopy-backscattered electron (SEM-BSE) detector and the combination of focused ion beam (FIB)/ transmission electron microscopy (TEM) provided a powerful approach for observing the dispersion of HAP particles in the polymer matrix. We show that surface modification of HAP with PCL improved the homogeneity of the dispersion of HAP particles in the composites and affected the composite morphology during hydrolytic degradation. Composites with high HAP content displayed high compressive strength and a fast rate of degradation. The PCL-gHAP/PANH composites showed superior maintenance of mechanical properties compared with HAP/PANH composites during degradation. A preliminary in vivo study on rat calvaria repair, demonstrated the superior performance of PCL-gHAP/PANH composites. The results suggest that the newly developed PCL-gHAP/PANH composite materials have great potential of serving as a new substrate for bone tissue engineering.

5-hydroxymethylfurfural (HMF) is a versatile biomass-derived chemical for synthesizing useful compounds. In this work an attempt is made to produce HMF by developing a low-cost process for doing that using cellulosic waste and deep eutectic solvents (DESs). The most effective DES was choline chloride/citric acid (ChCl/citric acid, 2/1), which provided a greater glucose conversion percentage (around 100 mol%), HMF yield (8 mol%), and HMF selectivity (8 %) than other DESs with 0.1 g of pure glucose. To increase the HMF yield further, the species of catalyst, reaction time and reaction temperature were varied. The highest glucose conversion percentage (91.76 mol%), HMF yield (15.02 g/g) and HMF selectivity (8%) were obtained with a reaction time of five minutes, a reaction temperature of 130 ℃, sulfuric acid as the catalyst, and a ChCl/citric acid ratio of the DES of 2/1. These optimal conditions were used with the enzymatic hydrolysate of pretreated wood dusts as a substrate. The experimental results thus obtained revealed a glucose conversion percentage, HMF yield and HMF selectivity of 34.86 mol%, 16.46 g/g and 67.43 %, respectively, under these conditions. These results reveal that using enzymatic hydrolysate from pretreated wood in the production of HMF can yield approximately as much (16.46 g/g) as the use of pure glucose, indicating that this low-cost process may be commercially feasible.

Tissue engineering can provide alternatives to current methods for tracheal reconstruction. Here we describe an approach for ectopic engineering of vascularized trachea based on the implantation of co-cultured scaffolds surrounded by a muscle flap. Poly(L-lactic-co-glycolic acid) (PLGA) or poly(ε-caprolactone) (PCL) scaffolds were seeded with chondrocytes, bone marrow stem cells and co-cultured both cells respectively (8 groups), wrapped in a pedicled muscle flap, placed as an ectopic culture on the abdominal wall of rabbits (n = 24), and harvested after two and four weeks. Analysis of the biochemical and mechanical properties demonstrated that the PCL scaffold with co-culture cells seeding displayed the optimal chondrogenesis with adequate rigidity to maintain the cylindrical shape and luminal patency. Histological analysis confirmed that cartilage formed in the co-culture groups contained a more homogeneous and higher extracellular matrix content. The luminal surfaces appeared to support adequate epithelialization due to the formation of vascularized capsular tissue. A prefabricated neo-trachea was transferred to the defect as a tracheal replacement and yielded satisfactory results. These encouraging results indicate that our co-culture approach may enable the development of a clinically applicable neo-trachea.

In this study, a biodegradable in situ gel-forming controlled drug delivery system based on a thermosensitive methoxy polyethylene glycol-co-poly (lactic acid-co-aromatic anhydride) (mPEG-PLCPPA) hydrogel was studied. The hydrogels were formed by micelle aggregation with rising temperature. The hydrogels underwent a temperature-dependent sol–gel–sol transition, which was a flowing sol at ambient temperature and a non-flowing gel at the physiological body temperature. The residual weight and pH value changes after degradation and the viscosity properties of the hydrogel were investigated. The in vitro release behavior of vancomycin from the mPEG-PLCPPA hydrogels at different concentrations was also investigated. The results showed that the mPEG-PLCPPA amphiphilic copolymer could self-assemble to form micelles at low concentrations, and that the particle sizes gradually increased with increasing temperature. The hydrogel maintained a stable degradation rate and provided a moderate pH microenvironment after degradation for 30 days. Vancomycin sustained a stable release profile from the hydrogel over a 10-day period. Furthermore, good biocompatibility was proven by MTT assay and live and dead test. Therefore, the mPEG-PLCPPA hydrogel shows promise as an injectable local antibiotic delivery system.

Bioceramic or inorganic nanoparticles made of SiO2, TiO2, SrO, and hydroxyapatite (HAP) have been reported to improve cell adhesion onto polymers. However, direct mixing of these nanoparticles with polymers often leads to their aggregation within the polymer matrix and subsequent deterioration of the material’s mechanical strength. A novel method for modifying the surfaces of the nanoparticles by grafting e-caprolactone using a ring-opening condensation reaction was developed to improve the interconnection of the nanoparticles within the polymer matrix. The mechanical studies showed that adding grafted nanoparticles into the polycaprolactone (PCL) matrix improved the initial mechanical strength. MTT assay and a live/dead stain showed higher cell viability in the tablets with grafted SiO2, TiO2, and HAP nanoparticles, except the SrO-containing tablets. The cell adhesion and alkaline phosphatase activity assay confirmed that the composite tablets with PCL-grafted HAP nanoparticles had better osteoconductivity. HE stains showed that composite tablets with PCL-grafted SiO2, TiO2, and HAP nanoparticles produce less immune response than the pure PCL. We thus conclude that a PCL matrix incorporating PCL-grafted HAP nanoparticles has enhanced mechanical strength, improved osteoconductivity, and a slower degradation rate than pure nanoparticles.

Human embryonic stem cells (hESCs), due to their self-renewal capacity and pluripotency, are an important source of cells for regenerative medicine. The immediate obstacles that need to be addressed are the poor cell survival rate of hESCs and their cell quality after cryopreservation. In this study, we used the Cell Alive System (CAS) which combines a programmed freezer with an oscillating magnetic field to reduce cryo-injury during the freezing process. The hESC clumps suspended in freezing medium were divided into three groups: (i) cells frozen by a conventional freezing container, Mr. Frosty and kept in a −80 °C freezer (MF); (ii) cells frozen to −32 °C by CAS, and then transferred to a −80 °C freezer (CAS); (iii) cells frozen to −32 °C by CAS, and then transferred to a pre-cooled Mr. Frosty and kept in a −80 °C freezer (CAS-MF) for overnight. All cryovials were placed in liquid nitrogen for one week, and hESCs were then thawed and cultured on feeder for 7 days. The results of alkaline phosphatase (AP) staining showed that the attachment efficiency of the cells cryopreserved by CAS and CAS-MF was significantly higher (29.0% and 44.0%) than in the MF method (7.0%). Furthermore, we confirmed the cells cryopreserved using CAS-MF could be subcultured while expressing pluripotent markers, differentiate into three germ layers, and maintain a normal karyotype. These results demonstrate that the use of CAS-MF offers an efficient method of hESC banking.

The purpose of this study was to design a thermosensitive composite gel to be used as a bone graft substitute. This gel can provide a more suitable microenvironment by using the amphiphilic triblock copolymer (mPEG 550 PLGA 1405 ) consisting of methoxy poly(ethylene glycol) (mPEG), poly(lactic- co -glycolic acid) (PLGA). An aqueous dispersion of mPEG 550 PLGA 1405 mixed with different ratios of HAP/β-TCP (composite gel) underwent a sol–gel–sol transition as the temperature was increased from 4 to 70 °C. The particle size and critical micellization concentration (CMC) were increased by adding ceramics. During the in vitro degradation process, composite gels demonstrated a slight decrease in pH value, a slower degradation rate, less toxicity, and a higher cell survival rate. The biocompatibility of the composite gels was validated by hemolysis test. In vivo animal studies demonstrated both radiographic and gross bone union when the ratio of HAP/β-TCP was 7:3. Based on the results, we have developed novel thermosensitive composite gels as bone substitutes.

This paper reports a gel-free multi-well microfluidic chip for cell cultures. Polydimethylsiloxane (PDMS) material was used because of its hydrophobic and gas-permeable features. Surface tension and fluidic channel resistance simplified the procedure of cell injection into the system. The shear stress and the mass-transfer perfusion medium reached a balance through the multi-row square-pillar microstructure and the driven pressure. The cell seeding was completed in 10-mm scaled culture wells in less than 6 s using the microstructure composed of PDMS. The adjustable initial cell density and length of the culture area facilitate its use in desired biological experiments. To verify whether the operation caused cell damage or not, the biological index of supernatants with glutamic oxaloacetic transaminase (GOT) and blood urea nitrogen (BUN) were monitored for 4 days. The 1–4-mm-long multi-rows square-pillar microstructure can sustain HA22T cells for 5 days and still maintain cell viability for up to 90% of seeded cells.

Background/Purpose Durable mechanical strength and biocompatibility are the two major requirements for osteogenic scaffolds. Polyanhydrides are a class of biodegradable polymers characterized by anhydride bonds that connect repeating units of the polymer backbone chain. Hydroxyapatite (HAP) is the main component of human bone and is a good osteoinductive factor that promotes bone mineralization. This work validates the combination of polyanhydrides and HAP for biomedical application. Methods Polyanhydride copolymers were fabricated from sebacic acid (SA) and 1,6-bis(p-carboxyphenoxy)hexane (CPH). HAP was surface-modified by polycaprolactone (PCL), and testing tablets were made using different ratios of copolymers and surface-grafted HAP (g-HAP). Degradation tests were performed to evaluate mechanical strength, pH, and weight loss. Biocompatibility was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and live/dead stain test. Cell affinity was measured using scanning electronic microscopy (SEM). Results The favorable surface erosion property of polyanhydrides prevented marked changes in the mechanical properties over time. In addition, the degradation byproducts of the copolymer did not cause a serious decline in pH and were less harmful to the cells. g-HAP increased cell affinity for the polymer surface. Conclusion The research team synthesized polyanhydride/g-HAP composites with high mechanical strength, slow degradation, and excellent biocompatibility. The result showed that a CPH/SA ratio of 7:3 in combination with 10 wt% g-HAP was optimal as bone substitute.

The composite of methoxy polyethylene glycol (mPEG) and poly(lactic-co-glycolic acid) (PLGA) thermosensitive hydrogel mixed with various portions of hydroxyapatite (HAP) or β-tricalcium phosphate (β-TCP) were used as bone graft substitutes. The physical properties of a series of composite gels, including the critical micelle concentration (CMC), particle sizes, zeta potential, rheological behavior, morphology of composite gels, and sol–gel transition, were characterized in vitro . These composite gels could form a gel at body temperature and could be controlled easily at room temperature, but showed only a small decline in pH, to between 6.33 and 6.66, whereas mPEG–PLGA gel without ceramic exhibited a more significant decrease in pH over a period of 5 days. The dissolution of ceramics results in an increase in the concentration of calcium and phosphate, which can buffer the degradation of mPEG–PLGA. Higher cell viability was observed in the composite gels with more bioceramics, as shown in the MTT assay and the live and dead stain. Mixing mPEG–PLGA with HAP or β-TCP may hold greater promise than mPEG–PLGA alone for repairing bone defects.

During the fermentation process, dissolved oxygen values and carbon-to-nitrogen ratios are critical factors influencing DHA productivity. This study employed an intermittent oxygen feeding method to maintain a 50% dissolved oxygen level and produced a dissolved oxygen fluctuation environment to facilitate both Aurantiochytrium limacinum SR21 growth and lipid accumulation. Study results indicated that at a 1.25 C:N ratio and medium composition of 100 g L −1 glycerol, 40 g L −1 yeast extract, and 40 g L −1 peptone, A. limacinum SR21 achieved biomass at 61.76 g L −1 , lipid content at 65.2%, DHA concentration at 20.3 g L −1 , and DHA productivity at 122.62 mg L −1 h −1 , this result were better than most similar researches. Dissolved oxygen fluctuation environment also altered the fatty acid composition of A. limacinum SR21. In the late period of the fermentation process, C16:0 fatty acid ratios decreased significantly to below 5%, and C22:6 fatty acid ratios increased to 70%.

mPEG–PLLA (poly l -lactic acid) is synthesized by ring-opening polymerization of lactide and conjugation with mPEG. Sebacic acid is modified with acetic anhydride and condensed with mPEG to form mPEG–PSA (poly sebacic anhydride). The micelles formed by mPEG–PLLA are characterized by slow degradation and low drug encapsulation efficiency; on the contrary, mPEG–PSA micelles are characterized by rapid degradation but high encapsulation efficiency. They can merge into spherical micelles ( Φ = 140 nm) by self-assembly in water. The mixed micelles can successfully encapsulate a typical hydrophobic drug (curcumin), and significantly improve its solubility. Experimental results show that the mixed micelles have the features of high encapsulation efficiency and slow degradation.

This paper presents a microfluidic chip platform with electrochemical carbon nanotube electrodes for preclinical evaluation of antibiotics nanocapsules. Currently, there has been an increasing interest in the development of nanocapsules for drug delivery applications for localized treatments of diseases. So far, the methods to detect antibiotics are liquid chromatography (LC), high performance liquid chromatography (HPLC), mass spectroscopy (MS). These conventional instruments are bulky, expensive, not ease of access, and talented operator required. In order to help the development of nanocapsules and understand drug release profile before planning the clinical experiments, it is important to set up a biosensing platform which could monitor and evaluate the real-time drug release profile of nanocapsules with high sensitivity and long-term measurement ability. In this work, a microfluidic chip platform with electrochemical carbon nanotube electrodes has been developed and characterized for rapid detection of antibiotics teicoplanin nanocapsules. Multi-walled carbon nanotubes are used to modify the gold electrode surfaces to enhance the performance of the electrochemical biosensors. Experimental results show that the limit of detection of the developed platform using carbon nanotubes electrodes is 0.1 μg/ml with a linear range from 1 μg/ml to 10 μg/ml. The sensitivity of the developed system is 0.023 mA ml/μg at 37°C. The drug release profile of teicoplanin nanocapsules in PBS shows that the antibiotics nanocapsules significantly increased the release of drug on the 4th day, measuring 0.4858 μg/(ml hr). The release of drug from the antibiotics nanocapsules reached 34.98 μg/ml on the 7th day. The results showed a similar trend compared with the measurement result using the HPLC instrument. Compared with the traditional HPLC measurements, the electrochemical sensing platform we developed measures results with increased flexibility in controlling experimental factors for long-term preclinical measurement of nanocapsules in real time and at low cost.

Ex vivo generation of megakaryocytes from hematopoietic stem cells (HSCs) is crucial to HSC research and has important clinical potential for thrombocytopenia patients to rapid platelet reconstruction. In this study, factorial design and steepest ascent method were used to screen and optimize the effective cytokines (10.2 ng/ml TPO, 4.3 ng/ml IL-3, 15.0 ng/ml SCF, 5.6 ng/ml IL-6, 2.8 ng/ml FL, 2.8 ng/ml IL-9, and 2.8 ng/ml GM-CSF) in megakaryocyte induction medium that facilitate ex vivo megakaryopoiesis from CD34(+) cells. After induction, the maximum fold expansion for accumulated megakaryocytes was almost 5000-fold, and the induced megakaryocytes were characterized by analysis of gene expression, polyploidy and platelet activation ability. Furthermore, the combination of megakaryocyte induction medium and HSC expansion medium can induce and expand a large amount of functional megakaryocytes efficiently, and might be a promising source of megakaryocytes and platelets for cell therapy in the future.

Amphiphilic block copolymers, methoxy poly(ethylene glycol)-b-poly(valerolactone) (mPEG-b-PVL), were synthesized via ring opening polymerization of δ-valerolactone in the presence of methoxy poly(ethylene glycol) (mPEG). The copolymers form micelle-like nanoparticles by their amphiphilic characteristics and their structures were examined by Nuclear Magnetic Resonance (NMR). The sizes of nanoparticles ranged from 60 to 120 nm as measured by dynamic light scattering detection, and were larger with higher molecular weight of the copolymers. The Critical Micelle Concentration (CMC) of these nanoparticles in water decreased with increasing molecular weight of hydrophobic segment. Stability analysis showed that the micellar solutions maintain their sizes at 37 °C for six weeks without aggregation or dissociation. The lyophilization method was better than the evaporation method when camptothecin (CPT) was incorporated to the micelles. The former method yielded higher CPT loading efficiency and lower aggregation. The loading efficiency of CPT could be more than 96% and a steady release rate of CPT was kept for twenty six days. Moreover, the mPEG-b-PVL polymeric micelles offered good protection of CPT lactone form at 37 °C for sixteen days. The copolymers showed no cytotoxicity towards L929 mouse muscular cells when incubated for one day. Taken together, the mPEG-b-PVL copolymer has potential to be used for the delivery of CPT or other similar drugs.

Poly(sebacic anhydride-co-ethylene glycol) was synthesized by using sebacic anhydride prepolymer and poly(ethylene glycol) for encapsulation of p-nitroaniline and brilliant blue G as modeling drugs to investigate the behavior of hydrophobic and hydrophilic drug release, respectively. Since p-nitroaniline is likely located in the sebacic anhydride-rich phase and brilliant blue G in the PEG-rich phase, respectively, their incorporation would affect the phase behavior of the host polymer. Different pore structure of eroded polymer matrix and drug release behavior were identified for hydrophobic and hydrophilic compounds. With a certain amount of PEG in the copolymer matrix, low drug release rate was accomplished for hydrophobic drug incorporation.

S-acetylthio-2-methylpropionic acid (S-AMPA) is an important chiral intermediary for numerous hypertension drugs such as captopril. S-AMPA can be produced by hydrolyzing the corresponding racemic methyl MAMP (S,R-methyl-β-acetylthioisobutyrate) by lipases or esterases that have the appropriate stereo specificity. Psudomonas fluorescens IFO 12055 possessing a highly specific lipase was used to process this reaction in the form of immobilized cells. Reaction kinetic and immobilization methods were also studied. Strong product inhibition was observed, that is, at 3% S-AMPA (namely 183 mM), activity was reduced by 50%. Spontaneous hydrolysis of the ester and thioester bonds was also observed, and was independent of the cells. Thus, reaction selectivity and yield must be optimized through adjusting the substrate concentration and total biocatalyst activity. Conventional calcium alginate (3% w/w) encapsulation was modified by adding 3% w/w polyethyleneimine (PEI) and cross-linked by a biologically derived agent, genipin (5.6 mM). This method was found to be satisfactory to produce stable and functioning biocatalyst and can maintain high reactivity for repeated 25 batches with e.e. values above 90%.

In this study, in vitro degradation behavior in buffer solutions of poly(sebacic anhydride- co -ethylene glycol), synthesized with sebacic acid and poly(ethylene glycol) (PEG) via melt-condensation process, was investigated. These copolymers degraded in a surface layer erosion mode within 300 μm from the edge of polymeric matrix that differs from that of neat poly(sebacic anhydride) (PSA), which exhibits simple surface erosion behavior. On the early stage of degradation, synthesized copolymers lost more weight than neat PSA, due to the loss of hydrophilic PEG segments located near surface layer of the polymeric matrices. However, because of the enhanced crystallinity of sebacic anhydride chain segments by the introduction of soft PEG chain segments, degradation rates of copolymers were slower than neat PSA at later stages after 96 h. In addition, degradation rate of copolymeric matrix was determined by the degradation of sebacic anhydride chain segments, which protect PEG chain segments located in the inner part of copolymeric matrices.

In this research, hybrid materials constructed by poly(vinyl pyrrolidone) (PVP) and inorganic tetraethyloxysilane (TEOS) or methyltriethyloxyslane (MTEOS) were synthesized via sol–gel process. From visual and scanning electron microscopic (SEM) observation, inorganic silicate was not separated from the organic phase in PVP/MTEOS hybrid system and in PVP/TEOS hybrid system with 20 wt.% or less TEOS content. With 40 wt.% TEOS content, microphase separation occurred; furthermore, phase separation and phase inversion can be observed with TEOS content increased to 60 wt.% or more in PVP/TEOS hybrid system. These results were confirmed by the variation of glass transition behavior. From infrared spectra analysis, hydrogen bonds were formed in both PVP/MTEOS and PVP/TEOS hybrid systems. However, higher hydrogen bonding interaction in PVP/TEOS did not induce higher compatibility as compared to PVP/MTEOS. This was due to the greater non-polar interaction between methyl group of MTEOS and polymer backbone in PVP/MTEOS hybrid system. D 2004 Elsevier B.V. All rights reserved.

The development of ex vivo culture systems that facilitate the expansion of hematopoietic stem and progenitor cells is crucial to stem cell research and clinical application. In this study, a serum-free, stroma-free and cytokine-containing culture system for CD34+ and colony-forming cell (CFC) expansion was systematically developed and optimized using the two-level factorial design and steepest ascent methods. The experimental results show that the optimal compositions of the serum substitutes and the cytokine cocktail were BIT2 (1.5 g/l BSA, 4.39 μg/ml insulin, 60 μg/ml transferrin, and 25.94 μM 2-ME), and CC-S6 (8.46 ng/ml TPO, 4.09 ng/ml IL-3, 15 ng/ml SCF, 6.73 ng/ml FL, 0.78 ng/ml IL-6, 3.17 ng/ml G-CSF, and 1.30 ng/ml GM-CSF) in the Iscove’s modified Dulbecco’s medium, respectively. After one-week culture, the increases in the total number of white blood cells (WBC), CD34+cells and CFC were 64-, 27- and 22-fold, respectively. Its expansion ability of CD34+ cells and CFC was comparable to that of X-vivo 20™, Stemline™, and Stemspan™ commercial media. These systematic methodologies are helpful in improving the ex vivo expansion system for hematopoietic stem cell and progenitor cells.

Distearoylphosphatidylcholine (DSPC) spread at the air/water interface is used as a model membrane and to study the lateral interaction between DSPC and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000). DSPE-PEG2000 was found to be miscible with DSPC by our measurements of surface pressure–area per molecule (Π–A) isotherms at different temperatures. At different temperatures the nonideality and miscibility of mixed monolayer were determined by the analysis of excess area as a function of compositions, and the temperature effects on these deviations from ideality were evaluated. Furthermore, the interfacial thermodynamic characteristics of this mixed system including the change of entropies, the change of latent heats, and excess and mixing free energies during the compression process were calculated from the isotherms as a function of temperature in order to understand factors that affect the stability of mixed monolayer. It was found that increasing temperature and incorporation of DSPE-PEG2000 both may make the mixed monolayer more compressible.

A novel biomaterial: poly(sebacic anhydride-co-ethylene glycol) was synthesized by introducing poly(ethylene glycol) (PEG) into a polyanhydride system. This copolymer was synthesized using sebacic acid and PEG via melt-condensation polymerization. The crystalline behavior of these synthesized products was studied, and compared to that of polymer blends of poly(sebacic anhydride) (PSA) and PEG. The crystallinity of PSA chain segments can be significantly enhanced by increasing chain mobility via the introduction of PEG. The crystallinity of the PSA component in copolymers was substantially greater than that of blends. However, the crystalline growth of the PEG segments was totally hindered by the presence of PSA chain segments, such that no crystal for PEG component was found in these copolymers. Besides, a dynamic mechanical analysis of these materials was also performed to provide additional information concerning visco-elastic behavior for other biomedical applications, where it was found that the viscous behavior in copolymers was more significant than in neat PSA and PEG.

Liposomal irinotecan was prepared by pH gradient loading. The parameters that govern this process, including drug loading time, incubation temperature, buffer composition for hydration, and deltapH, were studied. The uptake of irinotecan into liposomal systems in response to the magnitude of the pH gradient was examined. The drug uptake was maximum when the magnitude of deltapH approached 3.7. The effect of the formulation of the liposomes on the stability of the drug delivery system was also studied. Liposomes composed of lipids with a high phase transition temperature, L-alpha-distearoyl-phosphatidyl-choline and hydrogenated soy phosphatidylcholine, were more stable than those composed of lipids with lower phase transition temperatures. Incorporating distearoyl phosphatidylethanolamine-poly (ethylene glycol)2000 into liposomes helped to reduce the size of the liposomes. In addition, the retention of a drug within liposomes was found to be slightly enhanced by including dimyristoylphosphatidylglycerol or dextran sulfate in the liposome formulation.

Measurements of the film pressure–area ( ∏ – A ) isotherms for paclitaxel/ l -α-distearoyl phosphatidylcholine (DSPC) mixed monolayers were conducted at 24.5 and 37 °C. Thermodynamic properties of paclitaxel and DSPC in a monolayer at the air/water interface were studied from data obtained. The ∏ – A isotherms showed that paclitaxel and DSPC were miscible at 24.5 °C and at higher temperature, desorption of paclitaxel from the interface into subphase was observed. Incorporation of paclitaxel had a profound influence on the shape of isotherms and the compressibility of a mixed monolayer. These observations were directly dependent on the concentration of paclitaxel in the monolayer. Comparison with the data of compressibility for mixed monolayers suggests that a monolayer containing 5% paclitaxel possessed the strongest attractive interaction, and also the most stable state one according to the estimation of the free energy and the excess free energy of mixing. Additionally, the relevance of these observations to the behavior of paclitaxel-lipid liposomes was discussed.

Hybrid materials based on poly( n -butyl methacrylate-co-(3-(methacryloxypropyl)) trimethoxysilane) were synthesized via a sol–gel process. From a phase behavior study of these hybrid materials using a differential scanning calorimeter (DSC) and solid-state nuclear magnetic resonance (NMR) spectroscopy, it was found that the hybrid system was miscible for organic and inorganic phases with low silane content, but exhibited heterogeneously when silane content was increased to 40 mol% or more. Furthermore, the hybrid system became more complicated when 50 mol% or more silane introduced. It was also found that an inorganic Si–O–Si network developed, which resulted in side chain segments into two different molecular environments and with different relaxation behaviors. The development of an inorganic matrix of a Si–O–Si structure and the corresponding polymer chain environment were also discussed in detail.

Poly(acrylic acid) is a water soluble polymer, which has a high glass transition temperature of ca. 102°C. However, when silica (20% of TEOS) was incorporated via sol–gel process, it was found that glass transition temperature of the hybrid material decreased drastically. From the analysis of infrared spectra, this phenomenon can be attributed to the decreasing of hydrogen bonding between carboxyl and carbonyl groups of the poly(acrylic acid). Furthermore, with the TEOS content of 40–60%, silica matrix was formed and resulted in the occurrence of phase separation. With TEOS content further increased to 80%, molecular chains of PAA were aggregated together to form poly(acrylic acid) matrix. Hence, the chain segments of PAA were no more confined by the silica matrix and only single Tg was observed during DSC thermo-scan.

In this research, hybrid materials based on poly(methyl methacrylate) and silica were synthesized via sol–gel process. Their glass transition behavior was investigated by differential scanning calorimeter. In DSC measurement, 180°C heat-treated hybrid materials exhibited higher miscibility as compared to untreated materials. Relationship between miscibility variation of hybrid materials and heat treatment was discussed with the data from thermogravimetric analysis and infrared spectra. It was found that degradation behavior and silica structure was affected by heat treatment. At the same time, content of hydrogen bonds were elevated at the organic–inorganic interface, which may enhance the phase miscibility.

A serum free medium for recombinant CHO NTHU 108 cell growth and fusion protein (CD20 linked to a human IgG-Fc γ4 fragment) synthesis were systematically developed using factorial designs combined with the steepest ascent method. Experimental results indicate that the optimal composition of serum replacement for specific fusion protein production was 1% SITE (selenium, insulin, transferrin, ethanolamine), 0.3 g/L yeast extract, and 0.09% linoleic acid-BSA. Cell growth and fusion protein production of the adapted CHO NTHU 108 cultured in Iscove’s modified Dulbecco’s medium supplemented with these serum substitutes were comparable to those in the Ex-Cell 301 commercial serum-free medium. These serum substitutes can also promote CHO cell growth and fusion protein production in nine kinds of commercial media. The low protein content of the developed medium facilitates downstream processing and product purification.

When aurintricarboxylic acid (ATA) was added at a concentration of 30 mg/l in DME/F12 medium to Chinese hamster ovary (CHO) NTHU-108 cells in static six-well plates, some of the cells exhibited adherent growth while others grew in suspension. Beyond the critical concentration of ATA, CHO cells grew in single-cell suspension. In the same serum-free medium, insulin at a level of 0.5 mg/l was found to be the most important protein ingredient promoting cell growth. We used the respective kinase inhibitors to investigate their influence on the cell proliferation induces by ATA and insulin. It is interesting that the inhibition by seven kinase inhibitors of ATA-induced proliferation is similar to that of insulin-induced proliferation. It is possible that ATA mimics insulin and influences the mitogen activated signal transduction to induce the proliferation of CHO cells. Although the actual mechanism of the proliferation of CHO cells by ATA is unclear, ATA supported the long-term proliferation of CHO cells under serum-free conditions and thus could be used as a good substitute for insulin in the formulation of protein-free media.

Nine carboxylic acids were tested to evaluate their effects on recombinant fusion protein production and cell growth of Chinese Hamster Ovary (CHO) cells. Pentanoic acid was demonstrated to have the highest enhancement effect on the protein biosynthesis of CHO cells among the acids tested. Pentanoic acid also had less growth suppression effects compared with butyrate. The optimal induction time and concentration of pentanoic acid for a 120-h batch culture were 72 h and 1 mM, respectively. Apoptosis (programmed cell death) was observed in the serum-free batch culture of CHO cells using a cell death detection ELISA kit. The addition of butyrate accelerated the rate of apoptosis of CHO cells whereas the addition of pentanoate did not. These results confirmed that pentanoic acid was a better stimulant for protein biosynthesis in animal cell culture than butyrate.

Soybean oil, white mineral oil and tricaprylin were employed with various emulsifiers to create o/w (oil-in-water) emulsion with particle size ranging from 70 to 200 nm for potential drug delivery applications. Suitable formulation of this o/w emulsion was sought for, with aims of good shelf stability (in particle size and drug retention), and high drug loading capacity. Nonionic surfactants, including Cremophore EL, Pluronic F68 and Tween 80, were compared when used in combination with egg phosphatidylcholine (EPC) as the emulsifiers in this study. The effect of types of emulsifiers and oils used on the particle size and shelf stability was investigated. The results revealed a synergistic effect of surfactants EPC and Tween 80 on emulsion stability, with an optimum ratio of ca. 1:1 wt/wt. Emulsion made of the following formulation was found to posses small size around 80–120 nm and good storage stability: tircaprylin/Tween 80/EPC (1 g/0.3 g/0.4 g) in 40 ml of 2.25% glycerol aqueous solution.

Paclitaxel is an anticancer agent with low aqueous solubility. More extensive clinical use of this drug is somewhat delayed due to lack of appropriate delivery vehicles. An attempt was made to adopt an o/w emulsion as the drug carrier which incorporated paclitaxel in the triacylglycerol stabilized by a mixed-emulsifier system. A suitable formulation was found in this study: 0.75 mg/ml paclitaxel, 10% (w/v) oil blend, 4% (w/v) EPC, 3% (w/v) Tween 80 in 2.25% (w/v) glycerol solution. The formulated emulsion has very good stability when stored at 4 degrees C, and the paclitaxel containment efficiency can be maintained above 95% and the mean emulsion diameter around 150 nm for at least 3 months. Paclitaxel-emulsion displayed cytotoxicity against HeLa cells with IC50 at 30 nM. The average life span of ascitic-tumor-bearing mice was prolonged significantly by the treatment of paclitaxel-emulsion (P0.05). The formulated emulsion is a promising carrier for paclitaxel and other lipophilic drugs.

Production of a lipopeptide surfactant, surfactin, by Bacillus subtilis ATCC 21332 was highly enhanced when iron concentration in the medium was raised from 4 μ m to the few m m level. This iron enrichment of the medium also resulted in increased biomass concentration. With 1.7 m m iron sulfate added to the media of different initial iron concentrations or at different stages of growth, surfactin concentration can be raised to levels of several hundred mg l −1 in general. Acidification of the broth was usually observed following the iron addition, and surfactin in the broth disappeared rapidly due to precipitate as soon as the pH level dropped below 5.0; however, if this acidification phenomenon was delayed, avoided (for reasons still unknown), or reversed (by alkaline addition), surfactin production was highly enhanced to as high as 3,500 mg l −1 which was almost ten times over the previously reported level for this strain and higher than most reported values for genetically improved strains.

Two positively charged derivatives of poly(ethylene glycol) (PEG) were coated onto alginate-poly( l -lysine)-alginate (A-P-A) microcapsules by allowing them to interact them with the negatively charged alginate on the capsule surface. The polymers are methoxypolyoxyethylene amine (PEGA1) and polyoxyethylene bis(amine) (PEGA2), which contain charged amine groups at one or both ends, respectively, with PEG as the backbone. The coating of the microcapsules with PEG-amine resulted in a much smoother capsule surface than A-P-A microcapsule surfaces as examined under a scanning electron microscope. The diffusivity of bovine serum albumin into the microcapsules remained the same after PEGA1 coating. But the diffusivity decreased to less than one-fifth that in A-P-A microcapsules coated with PEGA2. The biocompatibility of the microcapsules also improved as investigated by an in vivo study. Microcapsules were implanted in the peritoneal cavity of BALB c mice and retrieved 120 d after implantation. The fibrotic action against A-P-A microcapsules was severe and the capsules retrieved by peritoneal lavage aggregated into clusters. In contrast, the surface-modified capsules were free-flowing and free of cell overgrowth. Secretion of insulin from rat islets within A-P-A-PEGA microcapsules responded well to changes in glucose concentration in a static glucose test. Intraperitoneal transplantation of the microencapsulated islets into streptozotocin-induced diabetic mice could maintain normal blood glucose levels in test animals for up to 200 d without immunosuppression.

Encapsulation of taxol (paclitaxel) by liposomes provides an environment that enhances the solubility of taxol and avoids side effects caused by Cremophor EL, the emulsifier currently used. In this study, a suitable formula and production method for the liposomal delivery system were investigated. A combination of bath-type sonication and passage through ultrafiltration membranes was found to be the best way to produce liposomes particles of uniform size in the range of 100–200 nm. Phosphatidylcholine and phosphatidylglycerol in a 7 : 3 lipid molar ratio together with 40%, cholesterol, 25% α-tocopherol and 3% taxol (in mol/mol lipids), further resulted in liposomes with homogeneous and stable particle sizes. This taxol delivery method was found to have superior therapeutic effects in tests conducted on mice with transplanted liver tumors in comparison with taxol delivered in ethanol/Cremophor EL formula.

Microfiltration (MF) of a fermentation broth containing Escherichia coli is reported in this article. We used a ceramic membrane filter (zirconia on sintered carbon) having a nominal pore size of 0.2 μm. Our results indicate that the filtration resistance was mainly caused by the cake formed on the membrane surface. Both transmembrane pressure (TMP) and fluid sweeping velocity influenced this cake resistance. Resistances due to membrane itself and due to internal pore blockage by E. coli were less important and insensitive to both TMP and fluid sweeping velocity. Preliminary results also showed that the cell density could be significantly increased when we connected such a ceramic filter on-line with our fermentation system. In particular it was found that the gas bubbles entrained in the broth side of the filter could increase the filtration flux by as much as 80%.

Magnetic beads coated with monoclonal antibodies (MAb) were used to specifically remove cells carrying the common acute lymphoblastic leukemia antigen (cALLA). The target-cell elimination effectiveness (TEE) was found to be kinetically controled, and can be quantitatively described by a model where antigen-carrying cells and MAb-coated magnetic beads are described as colloidal particles. This model can correctly explain the temperature effect on the immunomagnetic cell adhesion where models based on antigen-antibody interaction failed.

The production of alkaline protease from Bacillus subtilis ATCC 14416 was studied. Inorganic nitrogen compounds in the medium were found to show inhibitory effects on the enzyme formation. The pH variations of the culture broth gave indications of the start and the end of the enzyme production period and thus provided a useful indicator for process control. The rapid deactivation of protease after the end of the production period was studied, and ways to stabilize the enzyme were discussed.

Objective A complete process for mass generation of megakaryocytes from hematopoietic stem cells under serum-free conditions has great clinical potential for rapid platelet reconstruction in thrombocytopenia patients. We have previously reported on the generation of an optimized serum-free medium (serum-free hematopoietic stem cell medium) for ex vivo expansion of CD34 + cells. Here, we further generated large amounts of functional megakaryocytes from serum-free expanded CD34 + cells under a complete and optimal serum-free condition for complying with clinical regulations. Materials and Methods Serum substitutes and cytokines were screened and optimized for their concentration for megakaryocyte generation by systemically methods. Serum-free induced megakaryocytes were characterized by surface antigens, gene expression, ex vivo megakaryocyte activation ability, and ability of megakaryocyte and platelet recovery in nonobese diabetic/severe combined immunodeficient mice. Results The optimal serum-free megakaryocyte induction medium was Iscove's modified Dulbecco's medium containing serum substitutes (i.e., human serum albumin, human insulin, and human transferrin) and a cytokine cocktail (i.e., thrombopoietin, stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, interleukin-9, and granulocyte-macrophage colony-stimulating factor). After induction, induced megakaryocytes expressed CD41a and CD61 surface antigens, nuclear factor erythroid-derived 2 and GATA-1 transcription factors and megakaryocyte activation ability. Importantly, transplantation of induced megakaryocytes could accelerate megakaryocyte and platelet recovery in irradiated nonobese diabetic/severe combined immunodeficient mice. Conclusion In conclusion, we have developed a serum-free megakaryocyte induction medium, and the combination of serum-free megakaryocyte and serum-free hematopoietic stem cell media can generate a large amount of functional megakaryocytes efficiently. Our method represents a promising source of megakaryocytes and platelets for future cell therapy.