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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.

To further enhance the performance of biodegradable polymer-based medical devices, there is an increasing need to obtain independent control of key properties such as mechanical strength, degradation rate, and bioactivity. In this study, biodegradable copolymers of poly(1,6-bis-p-carboxyphenoxyhexane-co-sebacic anhydride) (CPH:SA) are synthesized, via melt condensation techniques, at three different molar ratios (7 : 3, 5 : 5, and 3 : 7). Tablets of the copolymers are prepared by mold casting at high temperature. Using an in vitro degradation test, copolymer tablets demonstrate a suitable mechanical strength, a slight decrease in pH value, and a slow degradation rate. High cell viability is observed on the surface of the copolymer tablets. The 3-(4,5dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay and live/dead staining demonstrate reduced toxicity and high cell survival. In vitro testing with C2C12 cells reveals good cellular attachment and spreading on the tablet surfaces, with the best properties displayed by the 7 : 3 molar ratio copolymer. Materials composed of CPH:SA have the potential to serve as medical implants. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

This study evaluates the crosslinkage effect of chondroitin sulphate C (CSC) and type II collagen (COL II) on chondrogenesis of mesenchymal stem cells (MSCs) in vitro and in vivo. In the in vitro studies, our results show that the weight ratio CSC:COL II that reaches 1.2:100 (CSC1.2/100–COL II scaffold) can provide an optimal microenvironment for MSC chondrogenesis. When MSCs are cultured in this CSC1.2/100–COL II scaffold, the chondrogenic gene expression of cultured cells is upregulated, while the osteogenic gene expression of these is downregulated. In addition, MSCs cultivated in the CSC1.2/100–COL II scaffold are found to express the highest glycosaminoglycans:DNA ratio as compared to those in scaffolds of other CSC:COL II ratios. Histological and immunohistological evidence also supports the result. In the in vivo study, our results show that MSCs cultivated in the CSC1.2/100–COL II scaffold demonstrate a better repair ability on cartilage lesions than does the COL II scaffold. After 1 month in vivo, the injected MSCs in the CSC1.2/100–COL II scaffold show lacuna structures and stimulate the formation of type II collagen at the defective sites. Six months after transplantation, the generated cells in the CSC1.2/100–COL II group show higher gene expressions of type II collagen and aggrecan but lower gene expression of type I collagen at the defective sites than those in the COL II group. The results strongly suggest that CSC1.2/100–COL II scaffold can serve as a potential candidate for cartilage repair and improve the chondrogenesis of MSCs in general. Copyright © 2012 John Wiley & Sons, Ltd.

Chondroitin sulfate C (CSC) is an important extracellular matrix (ECM) component of native cartilage tissue. Since ECM is considered to play an important role in guiding proper cellular functions, such as proliferation, differentiation, migration, synthesis or degradation of ECM, in specific tissues, we would like to elucidate the effects of CSC on chondrocytes are cultured on type-II collagen (COL II) scaffolds in this study. In particular, we want to investigate if the oligosaccharides of CSC (O-CSC) have much stronger effects on the chondrocytes. In this in vitro study, human articular chondrocytes were cultured on porous scaffolds made of COL II, cross-linked by genipin. Media containing different molecular weights of CSC were used to cultivate the cells. The results were examined mainly from the gene expression profiles of the cultured cells. The expression levels of several genes were examined by real-time PCR. These included genes of COL II, aggrecan, SRY-related high mobility group-box gene 9 (SOX9) and cartilage oligo matrix protein (COMP), alkaine phosphtase (ALP), a disintegrin and metalloproteinases with thrombospondin motifs 4 (ADAMTS-4), a disintegrin and metalloproteinases with thrombospondin motifs 5 (ADAMTS-5), matrix metalloproteinases 3 (MMP-3) and tissue inhibitors of metalloproteinases 3 (TIMP-3). The results suggested that O-CSC is more potent in upregulating genes that promote chondrogenesis and downregulating genes that degrade cartilage ECM. The results suggest low-molecular-weight glycosaminoglycans may have therapeutic values in osteoarthritis treatment and may lead to further understanding of the basic mechanism of the interactions between the chondrocytes and their ECM.

Suitable carrier systems for sustained release of curcumin were studied by using the self-assembled polymeric micelles. Poly(ethylene glycol) methyl ether and poly(aromatic anhydride) were used as the hydrophilic and hydrophobic blocks, respectively, in forming amphiphilic diblock copolymers. Four different types of polymers methoxy poly(ethylene glycol- b-1,3-bis(p-carboxyphenoxy)propane) (mPEG5000CPP, mPEG2000CPP), methoxy poly(ethylene glycol-b-1,6-bis(p-carboxyphenoxy)hexane) (mPEG5000CPH, mPEG2000CPH) were synthesized via melt condensation approach. Micelles were formed at very low polymer concentration with stable hydrophobic cores. The particle sizes of micelles remained stable during degradation period. All four different polymeric micelles showed low cytotoxicity toward human fibroblasts cells and can kill cancer cells in very low concentrations. High loading efficiency and drug content were observed in curcumin-loaded micelles. Curcumin showed mild initial burst (30% of drug loading in the first 24 h) when released from the micelles and its release was sustained for at least 18 days. These micelles, especially those of mPEG5000CPP, show potential to serve as the delivery vehicles for sustained release of curcumin. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011.

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.

A series of biodegradable thermo-sensitive hydrogels were synthesized by ring-opening polymerization of methoxy-poly(ethylene glycol) (mPEG) and various ester monomers, i.e. D,L-lactide, glycolide, β-propiolactone, δ-valerolactone and e-caprolactone. The copolymers were characterized using 1H NMR spectroscopy and gel permeation chromatography. The micelle properties were also measured. The results indicated that the diblock copolymers formed nano-micelles at low concentrations in aqueous phase. The lower critical solution temperatures of the diblock copolymers were above 35 °C at 1 wt%. As the temperature increased above room temperature, the diblock copolymer solutions underwent a sol-to-gel phase transition, which was manifested in viscosity increases, indicative of the formation of a gel. The mPEG–polyester diblock copolymer solutions exhibited sol-gel transition behavior as a function of temperature and polymer concentration. Copyright © 2010 Society of Chemical Industry

Cartilage extracellular matrix (ECM) is composed primarily of type II collagen (COL II) and large, networks of proteoglycans (PGs) that contain glycosaminoglycans such as hyaluronic acid (HA) and chondroitin sulfate (CS). Since cartilage shows little tendency for self-repair, injuries are kept unhealed for years and can eventually lead to further degeneration. During the past decades, many investigations have pursued techniques to stimulate articular cartilage repair or regeneration. The current study assessed the effects of exogenous glycosaminoglycans (GAGs) including CS-A, CS-B, CS-C, heparan sulfate and HA, administration on human chondrocytes in terms of proliferation and matrix synthesis, while the cells were seeded and grown on the genipin-crosslinked collagen type II (COL II) scaffold. DNA content was measured by Hoechst dye intercalation, matrix deposition was evaluated by DMMB dye. Expression of collagen II and aggrecan mRNAs was assessed by RT-PCR, followed by gel electrophoresis. In a 28-day in vitro culture, administration of 5 microg/ml CS-A, 50 microg/ml CS-B, 50 microg/ml CS-C, 5 microg/ml HS, and 500 kDa HA led to significant increase in biosynthesis rate of PGs. Gene expression of aggrecan and collagen II were upregulated by CS-A, CS-C and HA. These results showed considerable relevance of GAGs to the issue of in vitro/ex vivo neo-cartilage synthesis for tissue engineering and regenerative medical applications.

For the limited availability of autologous chondrocytes, a cultured system for expansion in vitro until sufficient cells are obtained must be developed. These cells must maintain their chondrocyte phenotype in vitro as well as in vivo, following implantation to ensure that differentiated chondrocytes synthesize a normal hyaline cartilage matrix and not a fibro-cartilage matrix. This study uses porous three-dimensional (3-D) alginate scaffolds within a perfusion system to culture low-density (5 x 10(5) cells) primary porcine chondrocytes for 1-4 weeks to study their proliferation and differentiation. The results of RT-PCR reveal that most cells could maintain their differentiation state for up to 4 weeks of culturing. Chondrocytes proliferated to 3 x 10(7) cells after 4 weeks in culture. Alginate scaffolds induced the formation of chondrocyte clusters and stimulated the synthesis of matrix, which effects were evaluated using histology and electron microscopy. These findings demonstrate that culturing chondrocytes in alginate scaffolds may effectively prevent the dedifferentiation and improve autologous chondrocyte transplantation.

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.

Various modifications of alginate-poly-L-lysine microcapsules were made, such as the inclusion of polyethylenimine (PEI) or carboxyl methyl cellulose (CMC) in the core and the coating of bis(polyoxyethylene bis-[amine]) (PEGA) onto the microcapsule membrane surface. A characterization of the modified microcapsules in terms of mechanical and mass transfer properties as well as their chemical composition was performed. The PEI treatment greatly enhanced the mechanical stability of the microcapsules, and this treatment did not affect the coating process of poly-L-lysine or PEGA. PEGA was found to be able to coat the microcapsules while the mass transfer property was similar to the poly-L-lysine coated ones.

A fed-batch culture strategy for the production of recombinant Escherichia coli cells anchoring surface-displayed transglucosidase for use as a whole-cell biocatalyst for alpha-arbutin synthesis was developed. Lactose was used as an inducer of the recombinant protein. In fed-batch cultures, dissolved oxygen was used as the feed indicator for glucose, thus accumulation of glucose and acetate that affected the cell growth and recombinant protein production was avoided. Fed-batch fermentation with lactose induction yielded a biomass of 18 g/L, and the cells possessed very high transglucosylation activity. In the synthesis of alpha-arbutin by hydroquinone glucosylation, the whole-cell biocatalysts showed a specific activity of 501 nkat/g cell and produced 21 g/L of arbutin, which corresponded to 76% molar conversion. A sixfold increased productivity of whole cell biocatalysts was obtained in the fed-batch culture with lactose induction, as compared to batch culture induced by IPTG.

The mycelia of Aspergillus niger, cultivated in a medium containing 45 g l(-1) maltose, 66 g l(-1) yeast extract, and 5 g l(-1) K(2)HPO(4) at 30 degrees C and 200 rpm, were used as a biocatalyst in the glucosylation of ascorbic acid. Free mycelia from 3-day-old culture, when used in a 6-h reaction with maltose as the acyl donor, gave 16.07 g l(-1) ascorbic acid glucoside corresponding to a volumetric productivity of 2.68 g l(-1) h(-1) and a conversion of 67%. Mycelia from 3-day-old cultures were entrapped in calcium alginate beads and used as a catalyst in the glucosylation of ascorbic acid. An ascorbic acid-to-maltose molar ratio of 1:9 was found to be optimum, and the conversion reached 75% after 12 h. The concentration of ascorbic acid glucoside produced at this molar ratio was 17.95 g l(-1), and the productivity was 1.5 g l(-1) h(-1). The biocatalyst was repeatedly used in a fixed bed bioreactor for the synthesis of ascorbic acid glucoside and approximately 17 g l(-1) of ascorbic acid glucoside corresponding to a volumetric productivity of 1.42 g l(-1) h(-1) was produced in each use. The conversion was retained at 70% in each use. The entrapped mycelia also exhibited exceptionally high reusability and storage stability. The product was purified to 85% by anion exchange and gel permeation chromatography with a final yield of 75%.

Polyanhydrides have been used in many drug delivery systems because of their biodegradability and biocompatibility. Their degradation pattern of surface erosion made them suitable for stable drug release applications. However, in nanoparticle systems, this degradation pattern may not hold, and the drug release kinetics will be different also. In this study, copolymers of 1,3-bis(p-carboxyphenoxy)propane (CPP) and sebacic acid (SA) were synthesized to investigate the different degradation patterns of disk and nanoparticle forms of polyanhydride, in addition to the study of the method of preparation of nanoparticles from these copolymers. By using oil-in-water emulsion and poly(vinyl alcohol) (PVA) as emulsifier, nanoparticles of the size 200-500 nm were prepared. The size of the particles can be controlled by varying polymer concentration or PVA concentration, but different SA:CPP ratio did not affect the particle size significantly. Degradation was followed by detecting the amount of monomers released to the medium. It was found that CPP and SA were released at approximately the same rate from nanoparticles; while in disk form, SA was released much faster than CPP. It was found that contrary to general trend in disks, higher CPP content, a more hydrophobic component than SA, in the copolymer actually accelerated the degradation of nanoparticles.

In this work, biodiesel synthesis via fatty acids esterification with methanol is conducted by using a heterogeneous catalyst made from cation-exchange resin. The kinetics of esterification is studied at the different levels of catalyst loading (3.65–53.6%, w/w), reaction temperature (333–353 K) and molar ratio of methanol to fatty acids (1:1 to 20:1). The reaction rate and fatty acids conversion increased with increases in catalyst loading, reaction temperature and molar ratio of feeding reactants. A pseudo-homogeneous kinetic model coupling the effect of catalyst loading, reaction temperature and methanol/fatty acids molar ratio used for describing the process gave a correlation coefficient of 0.95 between experimental and predicted data. The proposed model was further used to predict the optimal operating condition for obtaining equilibrium conversion of 0.99. A reaction temperature of 372.15 K, molar ratio of feeding reactants of 14.9:1 and reaction time of 9.5 h was numerically calculated as the optimal operating condition. Under this optimal operating condition, an experimental verification was carried out and a satisfactory match was observed between experimental data and model prediction. © 2007 American Institute of Chemical Engineers AIChE J, 2008

Pluronic F-127 (PF127) is a thermosensitive polymer that has been widely recognized as a potential candidate for various bio-applications. However, in hydrogel form, its rapid disintegration and inhospitality toward cells have significantly limited its usage. As a means to increase the integrity and cell compatibility of a PF127 hydrogel, we propose the introduction of stabilizing secondary structures to the gel network by the addition of secondary structure-forming oligo-alanine and oligo-phenylalanine. Results indicate that increasing the oligo(peptides) attached to PF127 led to a significant decrease in the gelation concentration and temperature. A selected oligo(peptide)-modified PF127 was capable of forming a stable hydrogel network at 5% and suffered only 20% weight loss after 7 days of incubation in media. Scanning electron microscopy (SEM) revealed comparably more interconnected morphology in modified hydrogels which may be attributed to the presence of secondary structures, as verified by circular dichroism (CD) and Fourier-transformed infrared (FT-IR) spectroscopy. Nuclear magnetic resonance (NMR) provided insights into the extensive interactions at the micelle core, which is the key to altered gelation behavior. Furthermore, modified hydrogels maintained structural integrity within culturing media and supported the proliferation of encapsulated chondrocytes. In addition, in vivo residence time was extended to well beyond 2 weeks after oligo(peptide) modification, thereby broadening the application scope of the PF127 hydrogel to encompass long-term drug delivery and cell culturing.

Shih-Heng Tseng,1,2 Min-Yuan Chou,2 I-Ming Chu1 1Department of Chemical Engineering, National Tsing Hua University, 2Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan Abstract: We have developed a theranostic nanoparticle, ie, cet-PEG-dexSPIONs, by conjugation of the anti-epidermal growth factor receptor (EGFR) monoclonal antibody, cetuximab, to dextran-coated superparamagnetic iron oxide nanoparticles (SPIONs) via periodate oxidation. Approximately 31 antibody molecules were conjugated to each nanoparticle. Cet-PEG-dexSPIONs specifically bind to EGFR-expressing tumor cells and enhance image contrast on magnetic resonance imaging. Cet-PEG-dexSPION-treated A431 cells showed significant inhibition of epidermal growth factor-induced EGFR phosphorylation and enhancement of EGFR internalization and degradation. In addition, a significant increase in apoptosis was detected in EGFR-overexpressing cell lines, A431 and 32D/EGFR, after 24hours of incubation at 37°C with cet-PEG-dexSPIONs compared with cetuximab alone. The antibody-dependent cell-mediated cytotoxicity of cetuximab was observed in cet-PEG-dexSPIONs. The results demonstrated that cet-PEG-dexSPIONs retained the therapeutic effect of cetuximab in addition to having the ability to target and image EGFR-expressing tumors. Cet-PEG-dexSPIONs represent a promising targeted magnetic probe for early detection and treatment of EGFR-expressing tumor cells. Keywords: epidermal growth factor receptor, cetuximab, superparamagnetic iron oxide nanoparticle, magnetic resonance imaging, sodium periodate, polyethylene glycol

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.

A novel branched poly(anhydride-co-glycerol) was synthesized by introducing glycerin into a poly(sebacic anhydride-co-ethylene glycol) system via melt condensation without catalyst. The nuclear magnetic resonance spectroscopy (NMR) analysis showed that the reaction between the hydroxyl group on glycerin and the sebacic anhydride pre-polymer was completed without any hydroxyl group remaining. In addition, with more glycerin introduced, intramolecular chain reaction became more remarkable. This resulted in the production of more cyclic chains and lower molecular weight species that hinder the crystal growth of sebacic anhydride chain segments. This in turn decreases melting temperature of sebacic anhydride chain segments in these synthesized materials. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 886–893, 2003

Polyanhydrides are found to be useful in drug delivery systems; however, these aliphatic polyanhydrides are also unstable under humidity and temperature (greater than 0°C) for their low toxicity and surface eroding mode of drug release. In this research, poly(sebacic anhydride) was synthesized for a stability and depolymerization study in high humidity a surrounding temperature of 35°C. According to the results from gel permeation chromatography, IR spectrophotometry, and differential scanning calorimetry investigations, it was found that both self-depolymerization and hydrolysis mechanisms existed during the depolymerization process for poly(sebacic anhydride) and they can be monitored directly. These results were further compared with previous research in a depolymerization study of polyanhydrides in a vacuum and in buffer solution. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 1423–1429, 2003

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.

In this research, hybrid materials based on poly{(butyl methacrylate)-co-[(3-methacryloxypropyl)trimethoxysilane]} were synthesized via a sol-gel process. The physical properties of the materials with various silica contents were investigated. It was found from infrared spectra data that the organic-inorganic interface was bonded not only by covalent bonds but also by a portion of hydrogen bonds. A second glass transition temperature appeared when there was more than 60 mol-% silane in this hybrid system. The thermogravimetric data illustrated that all the hybrid materials decomposed in three stages, whereas the homopolymer of butyl methacrylate decomposed in only one stage. It is deduced that when the silane coontent is 60 mol-% or more, part of organic chain segments was found restricted by the inorganic phase.

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.

Dimethyl sulfoxide (DMSO) (1% v/v) stimulated stable transformed Chinese hamster ovary (CHO) cells to synthesize different recombinant proteins and repress their proliferation rate. The expressions of a fusion protein and β-galactosidase were increased 1.6- and 1.4-fold after adding DMSO. The expression of fusion protein was increased by up to 2.8-fold that of uninduced control by the simultaneous addition of DMSO and pentanoic acid. However, DMSO did not increase the production of the monoclonal antibody (immunoglobulin G) of three hybridoma cell lines (OKM1, OKT4 and HyGPD-YK-1-1), although it could inhibit the growth rates of the hybridoma.

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.

A newly designed apparatus, the J-column, was used to implement the concept of counteracting chromatographic electrophoresis (CACE). The process of separation and focusing was visualized by a model mixture of hemoglobin and ferritin. According to isoelectric focusing analysis, hemoglobin can be purified from the hemolysate mixture by CACE on a J-column.

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.

Countercurrent chromatography was used to separate taxol from cephalomannine, on a preparative scale for the final purification of taxol. A solvent system consisting of hexane:ethyl acetate:methanol: ethanol:water in 5:7:5:1:6.5 was found to be suitable in achieving near base-line resolution of the two components. It was found that the amount of methanol in the solvent system was very critical to a successful separation operation.

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.

In the fermentation of Escherichia coli, a ceramic membrane filter was used in conjunction with a fermenter for cell-recycle operation. The metabolic behaviour, especially the acetic acid production and the specific growth rate, under different concentrations of dissolved O was investigated. An unstructured model was constructed, and it was shown that the observed lower cell density under low dissolved O concentrations was accompanied by large acetic aicd production. However, the concentration of acetic acid was not high enough to cause the observed growth inhibition, and inhibition by other metabolites under low O environment is suspected.

Temperature-sensitive hydrogels are attractive alternatives to porous cell-seeded scaffolds and is minimally invasive through simple injection and in situ gelling. In this study, we compared the performance of two types of temperature-sensitive hydrogels on chondrocytes encapsulation for the use of tissue engineering of cartilage. The two hydrogels are composed of methoxy poly(ethylene glycol)- poly(lactic-co-valerolactone) (mPEG-PVLA), and methoxy poly(ethylene glycol)-poly(lactic- co-glycolide) (mPEG-PLGA). Osmolarity and pH were optimized through the manipulation of polymer concentration and dispersion medium. Chondrocytes proliferation in mPEG-PVLA hydrogels was observed as well as accumulation of GAGs and collagen. On the other hand, chondrocytes encapsulated in mPEG-PLGA hydrogels showed low viability and chondrogenesis. Also, mPEG-PVLA hydrogel, which is more hydrophobic, retained physical integrity after 14 days while mPEG-PLGA hydrogel underwent full degradation due to faster hydrolysis rate and more pronounced acidic self-catalyzed degradation. The mPEG-PVLA hydrogel can be furthered tuned by manipulation of molecular weights to obtain hydrogels with different swelling and degradation characteristics, which may be useful as producing a selection of hydrogels compatible with different cell types. Taken together, these results demonstrate that mPEG-PVLA hydrogels are promising to serve as three-dimensional cell carriers for chondrocytes and potentially applicable in cartilage tissue engineering. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A: 3311-3319, 2013.