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Xiaomin Yang - One of the best experts on this subject based on the ideXlab platform.
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cytotoxicity and wound healing properties of pva ws chitosan glycerol hydrogels made by irradiation followed by Freeze Thawing
Radiation Physics and Chemistry, 2010Co-Authors: Xiaomin Yang, Xiliang Chen, Feng Yu, Kang Yang, Shengwei Wu, Jungang LiAbstract:Abstract Hydrogels based on poly(vinyl alcohol), water-soluble chitosan and glycerol made by irradiation followed by Freeze–Thawing were evaluated as wound dressing. MTT assay suggested that the extract of hydrogels was nontoxic towards L929 mouse fibroblasts. Compared to gauze dressing, the hydrogel can accelerate the healing process of full-thickness wounds in a rat model. Wounds treated with hydrogel healed at 11th day postoperatively and histological observation showed that mature epidermal architecture was formed. These indicate that it is a good wound dressing.
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preparation of novel bilayer hydrogels by combination of irradiation and Freeze Thawing and their physical and biological properties
Polymer International, 2009Co-Authors: Xiaomin Yang, Xiliang Chen, Feng Yu, Kang Yang, Shengwei WuAbstract:BACKGROUND: Hydrogels made by irradiation or Freeze-Thawing often exhibit poor mechanical strength; therefore we investigated a novel synthetic method to circumvent this detrimental effect. We report a series of novel bilayer poly(vinyl alcohol) (PVA)/water-soluble chitosan (ws-chitosan)/glycerol hydrogels prepared by a combination of irradiation and Freeze-Thawing. Scanning electron microscopy morphology, swelling behavior, mechanical strength, elongation at break, PVA dissolution behavior and bovine serum albumin (BSA) release profile of the bilayer hydrogels were compared with those of hydrogels made by irradiation and Freeze-Thawing followed by irradiation. The cytotoxicity of the bilayer hydrogels was studied using a tetrazolium salt (MTT) assay. RESULTS: The novel bilayer hydrogels contain one layer made by Freeze-Thawing followed by irradiation and the other layer made by irradiation. The preparation method provides the two layers with good combination force in the wet state. However, the two layers are not combined very well in the Freeze-dried state due to the difference in microstructure. The bilayer hydrogels have large swelling capacity and good mechanical strength, and these properties can be varied by changing Freeze-Thawing cycles, irradiation doses and the relative thickness of the two layers. The PVA and BSA release behaviors show that the bilayer hydrogels have a small amount of dissolved PVA and can prolong the BSA release time. The MTT assay shows that extracts of the bilayer hydrogels are non-toxic towards L929 mouse fibroblasts. CONCLUSION: The novel bilayer hydrogels prepared in this study show good physical properties with no cytotoxicity, indicating that they are suitable for biomedical applications, such as in wound dressings and drug delivery devices. (C) 2009 Society of Chemical Industry
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thermal and rheological properties of poly vinyl alcohol and water soluble chitosan hydrogels prepared by a combination of γ ray irradiation and Freeze Thawing
Journal of Applied Polymer Science, 2008Co-Authors: Xiaomin Yang, Xiliang ChenAbstract:Poly(vinyl alcohol) (PVA)/water-soluble chitosan (ws-chitosan) hydrogels were prepared by a combination of gamma-irradiation and Freeze Thawing. The thermal and rheological properties of these hydrogels were compared with those of hydrogels prepared by pure irradiation and pure Freeze Thawing. Irradiation reduced the crystallinity of PVA, whereas Freeze Thawing increased it. Hydrogels made by Freeze Thawing followed by irradiation had higher degrees of crystallinity and higher melting temperatures than those made by irradiation followed by Freeze Thawing. ws-Chitosan disrupted the ordered association of PVA molecules and decreased the thermal stability of both physical blends and hydrogels. All the hydrogels showed shear-thinning behavior in the frequency range of 0.2-100 rad/s. Hydrogels made by Freeze Thawing dissolved into sol solutions at about 80 degrees C, whereas those made by irradiation showed no temperature dependence up to 100 degrees C. The chemical crosslinking density of the hydrogels made by irradiation followed by Freeze Thawing was much greater than that of hydrogels made by Freeze Thawing followed by irradiation. (c) 2008 Wiley Periodicals, Inc. J Appl Polym Sci 109: 3825-3830,2008.
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investigation of pva ws chitosan hydrogels prepared by combined γ irradiation and Freeze Thawing
Carbohydrate Polymers, 2008Co-Authors: Xiaomin Yang, Xiliang Chen, Feng YuAbstract:γ-Irradiation combined with Freeze-Thawing, i.e. irradiation followed by Freeze-Thawing and Freeze-Thawing followed by irradiation, was applied to prepare poly(vinyl alcohol) (PVA)/water soluble chitosan (ws-chitosan) hydrogels for wound dressing. The properties of these hydrogels were investigated and compared to those prepared by Freeze-Thawing and by irradiation, respectively. Hydrogels made by irradiation followed by Freeze-Thawing show larger swelling capacity and mechanical strength, higher thermal stability, lower water evaporation rate, and are less turbid than those made by pure Freeze-Thawing and Freeze-Thawing followed by irradiation. Hydrogels made by irradiation alone cannot be used as wound dressing due to their poor mechanical strength. SEM results show that the final structure of hydrogels made by combined irradiation and Freeze-Thawing is mainly determined by the first processing step. It is found that the appropriate amount of ws-chitosan can endow hydrogels with large swelling capacity and mechanical strength. The presence of ws-chitosan provides the hydrogels with good antibacterial activity against Escherichia coli (E. coli).
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effects of pva agar contents and irradiation doses on properties of pva ws chitosan glycerol hydrogels made by γ irradiation followed by Freeze Thawing
Radiation Physics and Chemistry, 2008Co-Authors: Xiaomin Yang, Xiliang ChenAbstract:Poly(vinyl alcohol) (PVA)/water soluble chitosan (ws-chitosan)/glycerol hydrogels were prepared by gamma-irradiation and gamma-irradiation followed by Freeze-Thawing, respectively. The effects of irradiation dose and the contents of PVA and agar on the swelling, rheological, and thermal properties of these hydrogels were investigated. The swelling capacity decreases while the mechanical strength increases with increasing PVA or agar content. Increasing the irradiation dose leads to an increase in chemical crosslinking density but a decrease in physical crosslinking density. Hydrogels made by irradiation followed by Freeze-Thawing own smaller swelling capacity but larger mechanical strength than those made by pure irradiation. The storage modulus of the former hydrogels decreases above 50 degrees C and above 70 degrees C it comes to the same value as that prepared by irradiation. The ordered association of PVA is influenced by both chemical and physical crosslinkings and by the presence of ws-chitosan and glycerol. These hydrogels are high sensitive to pH and ionic strength, indicating that they may be useful in stimuli-responsive drug release system. (C) 2008 Elsevier Ltd. All rights reserved.
Xiliang Chen - One of the best experts on this subject based on the ideXlab platform.
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cytotoxicity and wound healing properties of pva ws chitosan glycerol hydrogels made by irradiation followed by Freeze Thawing
Radiation Physics and Chemistry, 2010Co-Authors: Xiaomin Yang, Xiliang Chen, Feng Yu, Kang Yang, Shengwei Wu, Jungang LiAbstract:Abstract Hydrogels based on poly(vinyl alcohol), water-soluble chitosan and glycerol made by irradiation followed by Freeze–Thawing were evaluated as wound dressing. MTT assay suggested that the extract of hydrogels was nontoxic towards L929 mouse fibroblasts. Compared to gauze dressing, the hydrogel can accelerate the healing process of full-thickness wounds in a rat model. Wounds treated with hydrogel healed at 11th day postoperatively and histological observation showed that mature epidermal architecture was formed. These indicate that it is a good wound dressing.
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preparation of novel bilayer hydrogels by combination of irradiation and Freeze Thawing and their physical and biological properties
Polymer International, 2009Co-Authors: Xiaomin Yang, Xiliang Chen, Feng Yu, Kang Yang, Shengwei WuAbstract:BACKGROUND: Hydrogels made by irradiation or Freeze-Thawing often exhibit poor mechanical strength; therefore we investigated a novel synthetic method to circumvent this detrimental effect. We report a series of novel bilayer poly(vinyl alcohol) (PVA)/water-soluble chitosan (ws-chitosan)/glycerol hydrogels prepared by a combination of irradiation and Freeze-Thawing. Scanning electron microscopy morphology, swelling behavior, mechanical strength, elongation at break, PVA dissolution behavior and bovine serum albumin (BSA) release profile of the bilayer hydrogels were compared with those of hydrogels made by irradiation and Freeze-Thawing followed by irradiation. The cytotoxicity of the bilayer hydrogels was studied using a tetrazolium salt (MTT) assay. RESULTS: The novel bilayer hydrogels contain one layer made by Freeze-Thawing followed by irradiation and the other layer made by irradiation. The preparation method provides the two layers with good combination force in the wet state. However, the two layers are not combined very well in the Freeze-dried state due to the difference in microstructure. The bilayer hydrogels have large swelling capacity and good mechanical strength, and these properties can be varied by changing Freeze-Thawing cycles, irradiation doses and the relative thickness of the two layers. The PVA and BSA release behaviors show that the bilayer hydrogels have a small amount of dissolved PVA and can prolong the BSA release time. The MTT assay shows that extracts of the bilayer hydrogels are non-toxic towards L929 mouse fibroblasts. CONCLUSION: The novel bilayer hydrogels prepared in this study show good physical properties with no cytotoxicity, indicating that they are suitable for biomedical applications, such as in wound dressings and drug delivery devices. (C) 2009 Society of Chemical Industry
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thermal and rheological properties of poly vinyl alcohol and water soluble chitosan hydrogels prepared by a combination of γ ray irradiation and Freeze Thawing
Journal of Applied Polymer Science, 2008Co-Authors: Xiaomin Yang, Xiliang ChenAbstract:Poly(vinyl alcohol) (PVA)/water-soluble chitosan (ws-chitosan) hydrogels were prepared by a combination of gamma-irradiation and Freeze Thawing. The thermal and rheological properties of these hydrogels were compared with those of hydrogels prepared by pure irradiation and pure Freeze Thawing. Irradiation reduced the crystallinity of PVA, whereas Freeze Thawing increased it. Hydrogels made by Freeze Thawing followed by irradiation had higher degrees of crystallinity and higher melting temperatures than those made by irradiation followed by Freeze Thawing. ws-Chitosan disrupted the ordered association of PVA molecules and decreased the thermal stability of both physical blends and hydrogels. All the hydrogels showed shear-thinning behavior in the frequency range of 0.2-100 rad/s. Hydrogels made by Freeze Thawing dissolved into sol solutions at about 80 degrees C, whereas those made by irradiation showed no temperature dependence up to 100 degrees C. The chemical crosslinking density of the hydrogels made by irradiation followed by Freeze Thawing was much greater than that of hydrogels made by Freeze Thawing followed by irradiation. (c) 2008 Wiley Periodicals, Inc. J Appl Polym Sci 109: 3825-3830,2008.
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investigation of pva ws chitosan hydrogels prepared by combined γ irradiation and Freeze Thawing
Carbohydrate Polymers, 2008Co-Authors: Xiaomin Yang, Xiliang Chen, Feng YuAbstract:γ-Irradiation combined with Freeze-Thawing, i.e. irradiation followed by Freeze-Thawing and Freeze-Thawing followed by irradiation, was applied to prepare poly(vinyl alcohol) (PVA)/water soluble chitosan (ws-chitosan) hydrogels for wound dressing. The properties of these hydrogels were investigated and compared to those prepared by Freeze-Thawing and by irradiation, respectively. Hydrogels made by irradiation followed by Freeze-Thawing show larger swelling capacity and mechanical strength, higher thermal stability, lower water evaporation rate, and are less turbid than those made by pure Freeze-Thawing and Freeze-Thawing followed by irradiation. Hydrogels made by irradiation alone cannot be used as wound dressing due to their poor mechanical strength. SEM results show that the final structure of hydrogels made by combined irradiation and Freeze-Thawing is mainly determined by the first processing step. It is found that the appropriate amount of ws-chitosan can endow hydrogels with large swelling capacity and mechanical strength. The presence of ws-chitosan provides the hydrogels with good antibacterial activity against Escherichia coli (E. coli).
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effects of pva agar contents and irradiation doses on properties of pva ws chitosan glycerol hydrogels made by γ irradiation followed by Freeze Thawing
Radiation Physics and Chemistry, 2008Co-Authors: Xiaomin Yang, Xiliang ChenAbstract:Poly(vinyl alcohol) (PVA)/water soluble chitosan (ws-chitosan)/glycerol hydrogels were prepared by gamma-irradiation and gamma-irradiation followed by Freeze-Thawing, respectively. The effects of irradiation dose and the contents of PVA and agar on the swelling, rheological, and thermal properties of these hydrogels were investigated. The swelling capacity decreases while the mechanical strength increases with increasing PVA or agar content. Increasing the irradiation dose leads to an increase in chemical crosslinking density but a decrease in physical crosslinking density. Hydrogels made by irradiation followed by Freeze-Thawing own smaller swelling capacity but larger mechanical strength than those made by pure irradiation. The storage modulus of the former hydrogels decreases above 50 degrees C and above 70 degrees C it comes to the same value as that prepared by irradiation. The ordered association of PVA is influenced by both chemical and physical crosslinkings and by the presence of ws-chitosan and glycerol. These hydrogels are high sensitive to pH and ionic strength, indicating that they may be useful in stimuli-responsive drug release system. (C) 2008 Elsevier Ltd. All rights reserved.
John F. Carpenter - One of the best experts on this subject based on the ideXlab platform.
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protein nanoparticles promote microparticle formation in intravenous immunoglobulin solutions during Freeze Thawing and agitation stresses
Journal of Pharmaceutical Sciences, 2018Co-Authors: Neha N Pardeshi, Theodore W. Randolph, Chen Zhou, John F. CarpenterAbstract:Abstract In this study, we investigated the potential roles of nanoparticles ( 1000 nm) in protein formulations under some pharmaceutically relevant stress conditions. Exposure of intravenous immunoglobulin solutions to the interface-associated stresses of Freeze-Thawing or agitation resulted in relatively large increases in microparticle concentrations, which depended directly on the levels of pre-existing nano- and submicron particles. Thus, agglomeration of nanoparticles and submicron particles appears to play a role in microparticle formation under these stresses. In contrast, increases in microparticle concentrations during quiescent incubation at elevated temperatures were independent of the initial nano- and submicron particle concentrations in solution.
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subvisible particle counting provides a sensitive method of detecting and quantifying aggregation of monoclonal antibody caused by Freeze Thawing insights into the roles of particles in the protein aggregation pathway
Journal of Pharmaceutical Sciences, 2011Co-Authors: James G Barnard, Theodore W. Randolph, Satish K Singh, John F. CarpenterAbstract:ABSTRACT The objective of this study was to evaluate microflow imaging (MFI) as a sensitive tool to detect and quantify subvisible particle formation during Freeze-Thawing of an IgG 2 monoclonal antibody (mAb). Solutions of the protein formulated in 20 mM of histidine buffer (pH 5.5) were subjected to three Freeze–thaw cycles and analyzed by MFI and size-exclusion chromatography (SEC). MFI showed increased particle numbers after each Freeze-thaw cycle, whereas aggregates were not detected by SEC. Estimates of the total mass of particles formed revealed that monitoring of particle formation allows for the detection of protein aggregates comprising only hundredths of a percent of the total protein mass. Furthermore, differences in protein aggregation levels due to different formulations or different Freeze-Thawing protocols were resolved, even though protein aggregation could not be detected by SEC. To examine whether SEC and MFI-based estimations of total aggregate mass were in quantitative agreement, mAb was Freeze-thawed in phosphate-buffered saline. This process created sufficient level of insoluble aggregates to be detected by SEC as a reduction in the monomer peak area in the chromatogram. There was good agreement between the loss of monomer detected by SEC and the total mass of subvisible particles detected by MFI
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immunogenicity of aggregates of recombinant human growth hormone in mouse models
Journal of Pharmaceutical Sciences, 2009Co-Authors: Amber Haynes Fradkin, John F. Carpenter, Theodore W. RandolphAbstract:Aggregation of recombinant therapeutic protein products is a concern due to their potential to induce immune responses. We examined the immunogenicity of protein aggregates in commercial formulations of recombinant human growth hormone produced by Freeze-Thawing or agitation, two stresses commonly encountered during manufacturing, shipping and handling of therapeutic protein products. In addition, we subjected each preparation to high-pressure treatment to reduce the size and concentration of aggregates present in the samples. Aggregates existing in a commercial formulation, as well as aggregates induced by Freeze-Thawing and agitation stresses enhanced immunogenicity in one or more mouse models. The use of high-pressure treatment to reduce size and concentrations of aggregates within recombinant human growth hormone formulations reduced their overall immunogenicity in agreement with the “immunon” hypothesis. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3247–3264, 2009
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effects of solution conditions processing parameters and container materials on aggregation of a monoclonal antibody during Freeze Thawing
Journal of Pharmaceutical Sciences, 2008Co-Authors: Lisa A Kueltzo, Theodore W. Randolph, Wei Wang, John F. CarpenterAbstract:ABSTRACT Freeze–Thawing is a potentially damaging stress to which therapeutic proteins can be exposed deliberately during storage of bulk drug substance, and accidentally because of mishandling of commercial product during shipping and/or storage. The primary route of degradation induced by Freeze–Thawing is protein aggregation. We studied the effects of Freeze–Thawing on aggregation of an IgG 2 monoclonal antibody, examining solution conditions (pH, and the presence or absence of 150 mM KCl), protein concentration, cooling and warming rates, and container type and material. In addition, we determined the effect of pH and KCl on protein tertiary structure and thermal stability with second derivative UV spectroscopy. In general, aggregation of the antibody during Freeze–Thawing increased with decreasing pH, which correlated well with T m values. Aggregation was most prevalent at pH 3 and 4, with potential mechanisms involving both the formation of aggregation-prone conformational states as well as adsorption to and denaturation at various interfaces. Although all the parameters examined demonstrated some effect on the formation of soluble aggregates, the effect of container material was especially pronounced. Samples stressed in plastic or glass containers contained low amounts of aggregate. Storage in Teflon or commercial freezing containers, however, led to significantly higher levels of aggregate formation.
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Effect of Tween 20 on Freeze-Thawing- and agitation-induced aggregation of recombinant human factor XIII.
Journal of Pharmaceutical Sciences, 1998Co-Authors: Lotte Krielgaard, Theodore W. Randolph, Latoya S. Jones, Sven Frokjaer, James M. Flink, Mark C. Manning, John F. CarpenterAbstract:Agitation- and Freeze-Thawing-induced aggregation of recombinant human factor XIII (rFXIII) is due to interfacial adsorption and denaturation at the air?liquid and ice?liquid interfaces. The aggregation pathway proceeds through soluble aggregates to formation of insoluble aggregates regardless of the denaturing stimuli. A nonionic surfactant, polyoxyethylene sorbitan monolaurate (Tween 20), greatly reduces the rate of formation of insoluble aggregates as a function of surfactant concentration, thereby stabilizing native rFXIII. Maximum protection occurs at concentrations close to the critical micelle concentration (cmc), independent of initial protein concentration. To study the mechanistic aspects of the surfactant-induced stabilization, a series of spectroscopic studies were conducted. Electron paramagnetic resonance spectroscopy indicates that binding is not occurring between Tween 20 and either the native state or a folding intermediate state of rFXIII. Further, circular dichroism spectroscopy suggests that Tween 20 does not prevent the secondary structural changes induced upon guanidinium hydrochloride-induced unfolding. Taken together, these results imply that Tween 20 protects rFXIII against Freeze-Thawing- and agitation-induced aggregation primarily by competing with stress-induced soluble aggregates for interfaces, inhibiting subsequent transition to insoluble aggregates.
Feng Yu - One of the best experts on this subject based on the ideXlab platform.
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cytotoxicity and wound healing properties of pva ws chitosan glycerol hydrogels made by irradiation followed by Freeze Thawing
Radiation Physics and Chemistry, 2010Co-Authors: Xiaomin Yang, Xiliang Chen, Feng Yu, Kang Yang, Shengwei Wu, Jungang LiAbstract:Abstract Hydrogels based on poly(vinyl alcohol), water-soluble chitosan and glycerol made by irradiation followed by Freeze–Thawing were evaluated as wound dressing. MTT assay suggested that the extract of hydrogels was nontoxic towards L929 mouse fibroblasts. Compared to gauze dressing, the hydrogel can accelerate the healing process of full-thickness wounds in a rat model. Wounds treated with hydrogel healed at 11th day postoperatively and histological observation showed that mature epidermal architecture was formed. These indicate that it is a good wound dressing.
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preparation of novel bilayer hydrogels by combination of irradiation and Freeze Thawing and their physical and biological properties
Polymer International, 2009Co-Authors: Xiaomin Yang, Xiliang Chen, Feng Yu, Kang Yang, Shengwei WuAbstract:BACKGROUND: Hydrogels made by irradiation or Freeze-Thawing often exhibit poor mechanical strength; therefore we investigated a novel synthetic method to circumvent this detrimental effect. We report a series of novel bilayer poly(vinyl alcohol) (PVA)/water-soluble chitosan (ws-chitosan)/glycerol hydrogels prepared by a combination of irradiation and Freeze-Thawing. Scanning electron microscopy morphology, swelling behavior, mechanical strength, elongation at break, PVA dissolution behavior and bovine serum albumin (BSA) release profile of the bilayer hydrogels were compared with those of hydrogels made by irradiation and Freeze-Thawing followed by irradiation. The cytotoxicity of the bilayer hydrogels was studied using a tetrazolium salt (MTT) assay. RESULTS: The novel bilayer hydrogels contain one layer made by Freeze-Thawing followed by irradiation and the other layer made by irradiation. The preparation method provides the two layers with good combination force in the wet state. However, the two layers are not combined very well in the Freeze-dried state due to the difference in microstructure. The bilayer hydrogels have large swelling capacity and good mechanical strength, and these properties can be varied by changing Freeze-Thawing cycles, irradiation doses and the relative thickness of the two layers. The PVA and BSA release behaviors show that the bilayer hydrogels have a small amount of dissolved PVA and can prolong the BSA release time. The MTT assay shows that extracts of the bilayer hydrogels are non-toxic towards L929 mouse fibroblasts. CONCLUSION: The novel bilayer hydrogels prepared in this study show good physical properties with no cytotoxicity, indicating that they are suitable for biomedical applications, such as in wound dressings and drug delivery devices. (C) 2009 Society of Chemical Industry
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investigation of pva ws chitosan hydrogels prepared by combined γ irradiation and Freeze Thawing
Carbohydrate Polymers, 2008Co-Authors: Xiaomin Yang, Xiliang Chen, Feng YuAbstract:γ-Irradiation combined with Freeze-Thawing, i.e. irradiation followed by Freeze-Thawing and Freeze-Thawing followed by irradiation, was applied to prepare poly(vinyl alcohol) (PVA)/water soluble chitosan (ws-chitosan) hydrogels for wound dressing. The properties of these hydrogels were investigated and compared to those prepared by Freeze-Thawing and by irradiation, respectively. Hydrogels made by irradiation followed by Freeze-Thawing show larger swelling capacity and mechanical strength, higher thermal stability, lower water evaporation rate, and are less turbid than those made by pure Freeze-Thawing and Freeze-Thawing followed by irradiation. Hydrogels made by irradiation alone cannot be used as wound dressing due to their poor mechanical strength. SEM results show that the final structure of hydrogels made by combined irradiation and Freeze-Thawing is mainly determined by the first processing step. It is found that the appropriate amount of ws-chitosan can endow hydrogels with large swelling capacity and mechanical strength. The presence of ws-chitosan provides the hydrogels with good antibacterial activity against Escherichia coli (E. coli).
Wim Jiskoot - One of the best experts on this subject based on the ideXlab platform.
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structural properties of monoclonal antibody aggregates induced by Freeze Thawing and thermal stress
European Journal of Pharmaceutical Sciences, 2009Co-Authors: Andrea Hawe, Julia Christina Kasper, Wolfgang Frieß, Wim JiskootAbstract:Abstract Aggregation of monoclonal antibodies can be induced by Freeze–Thawing and elevated temperature, typical stress factors during development, production and storage. Our aim was to characterize structural properties of aggregates formed after Freeze–Thawing and thermal stressing of humanized monoclonal IgG 1 antibody (IgG). Formulations with 1.0 mg/ml IgG in 100 mM phosphate pH 7.2 were subjected to Freeze–Thawing and heating and characterized by spectroscopic techniques (UV-absorption, CD, ATR-FTIR and fluorescence), light obscuration, dynamic light scattering, SDS-PAGE, AF4 with UV and MALLS detection, and HP-SEC with UV and online fluorescent dye detection. Thermal stress led to an increased formation of dimers and soluble oligomers (HP-SEC, AF4). Aggregates smaller than 30 nm were measured (DLS), next to slightly elevated particle levels in the μm range (light obscuration). Aggregates created by heating were in part covalently linked (SDS-PAGE) and made up of conformationally perturbed monomers (CD, ATR-FTIR, extrinsic dye fluorescence). Aggregation after Freeze–Thawing was manifested primarily in particle formation in the μm range. These aggregates were noncovalently linked (SDS-PAGE) and composed of native-like monomers, as obvious from CD, ATR-FTIR and extrinsic dye fluorescence spectroscopy. In conclusion, the complementary methods used in this study revealed that heating and Freeze–Thawing induced aggregates differ significantly in their physico-chemical characteristics.
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Structural properties of monoclonal antibody aggregates induced by Freeze-Thawing and thermal stress
European Journal of Pharmaceutical Sciences, 2009Co-Authors: Andrea Hawe, Julia Christina Kasper, Wolfgang Frieß, Wim JiskootAbstract:Aggregation of monoclonal antibodies can be induced by Freeze-Thawing and elevated temperature, typical stress factors during development, production and storage. Our aim was to characterize structural properties of aggregates formed after Freeze-Thawing and thermal stressing of humanized monoclonal IgG1 antibody (IgG). Formulations with 1.0 mg/ml IgG in 100 mM phosphate pH 7.2 were subjected to Freeze-Thawing and heating and characterized by spectroscopic techniques (UV-absorption, CD, ATR-FTIR and fluorescence), light obscuration, dynamic light scattering, SDS-PAGE, AF4 with UV and MALLS detection, and HP-SEC with UV and online fluorescent dye detection. Thermal stress led to an increased formation of dimers and soluble oligomers (HP-SEC, AF4). Aggregates smaller than 30 nm were measured (DLS), next to slightly elevated particle levels in the μm range (light obscuration). Aggregates created by heating were in part covalently linked (SDS-PAGE) and made up of conformationally perturbed monomers (CD, ATR-FTIR, extrinsic dye fluorescence). Aggregation after Freeze-Thawing was manifested primarily in particle formation in the μm range. These aggregates were noncovalently linked (SDS-PAGE) and composed of native-like monomers, as obvious from CD, ATR-FTIR and extrinsic dye fluorescence spectroscopy. In conclusion, the complementary methods used in this study revealed that heating and Freeze-Thawing induced aggregates differ significantly in their physico-chemical characteristics. © 2009 Elsevier B.V. All rights reserved.
