Biomedical subjects

Eugene Khor

Publications and source records attributed to Eugene Khor.

11 recordsLinked to original sources

PubMed2005

Transfection efficiency of chitosan vectors: effect of polymer molecular weight and degree of deacetylation.

Chitosans of defined molecular weight (Mw 10-213 kDa) and degree of deacetylation (DD 46-88%) were synthesized, complexed with pEGFP-C2 plasmid into nanoparticles (NP) and evaluated for cellular uptake and transfection efficiency in the A549 cell model. DNA condensation of >90% was achieved at the N/P ratio of 6, independent of the chitosan Mw and DD. However, chitosan vectors of lower Mw or DD were less efficient at retaining the DNA upon dilution, and consequentially, less capable of protecting the condensed DNA from degradation by DNase and serum components. A549 cellular uptake of the NP was also significantly reduced by decreasing the Mw or DD of the chitosan vector. These factors contributed to the low transfection efficiencies for chitosan vectors of low Mw or DD. There was good correlation between transfection efficiency, cellular uptake and zeta potential of the NP, suggesting that cellular uptake mediated by electrostatic interactions with the cell membrane preceded efficient transfection. NP produced with chitosan of Mw 213 kDa and DD of 88% showed the highest zeta potential (+23 mV), cellular uptake (4.1 microg/mg protein) and transfection efficiency (12.1%), while chitosan vector with Mw of 213 kDa and DD of 46% showed the lowest cellular uptake (0.4 microg/mg protein) and transfection efficiency (0.05%). Confocal microscopy images suggested that the chitosan-complexed DNA successfully escaped from the endo-lysosomal compartment for nuclear translocation and expression. Intracellular DNA disassembly appeared to occur at different locations depending on the retentive capacity of the chitosan vector.

Acetylation↗

PubMed2004

Flexible chitin films: structural studies.

Chitin gels were transformed into thin, flexible chitin films with minimal dimensional shrinkage and maximum flexibility and thickness in the range of 25-80 microm by a cold-press process. Solvent residue was removed by heating the films at 50 degrees C for 12 h, followed by rinsing in 95% ethanol. The crystallinity and mechanical properties of the flexible chitin films were found to be a function of the amount of shrinkage from the gel to the final film that was obtained. For 28-microm thick films with 30% shrinkage, transparency of up to 90% was found. X-ray diffractometry (XRD) showed that the number of diffraction peaks appearing at 2theta;=23 degrees and 2theta;=27 degrees became increasingly sharper with shrinkage. Topographical information obtained from scanning electron microscopy (SEM) and atomic force microscopy (AFM) attributed the structural morphology of the films to the formation of sub-microscopic micelles. Scanning transmission electron microscopy (STEM) showed that shrinkage resulted in coarser microstructure, affecting tensile properties, where the ductility and toughness were proportional to the amount of shrinkage. These flexible chitin films have potential as wound dressing materials.

Bandages↗

PubMed2004

Hydroxyapatite-chitin materials as potential tissue engineered bone substitutes.

Hydroxyapatite (HA) in 25%, 50% and 75% w/w fractions was incorporated into chitin solutions and processed into air- and freeze-dried materials. These HA-chitin materials were exposed to cell cultures and implanted into the intramusculature of a rat model. The HA-chitin materials were found to be non-cytotoxic and degraded in vivo. The presence of the HA filler enhanced calcification as well as accelerated degradation of the chitin matrix. The freeze-dried HA-chitin matrixes were selected for further cell seeding experiments because of their porous nature. Mesenchymal stem cells harvested from NZW rabbits were induced into osteoblasts in vitro using dexamethasone. These osteoblasts were cultured for 1 week, statically loaded onto the porous HA-chitin matrixes and implanted into bone defects of the rabbit femur for 2 months. Histology of explants showed bone regeneration with biodegradation of the HA-chitin matrix. Similarly, green fluorescence protein (GFP) transfected MSC-induced osteoblasts were also loaded onto porous HA-chitin matrixes and implanted into the rabbit femur. The results from GFP-transfected MSCs showed that loaded MSCs-induced osteoblasts did not only proliferate but also recruited surrounding tissue to grow in. This study demonstrates the potential of HA-chitin matrixes as a good substrate candidate for tissue engineered bone substitute.

Animals↗

PubMed2004

Uptake and cytotoxicity of chitosan molecules and nanoparticles: effects of molecular weight and degree of deacetylation.

PURPOSE: To evaluate the effects of molecular weight (Mw) and degree of deacetylation (DD) on the cellular uptake and in vitro cytotoxicity of chitosan molecules and nanoparticles. METHODS: Chemical depolymerization and reacetylation produced chitosans of Mw 213,000 to 10,000 and DD 88-46%, respectively. Chitosan was labeled with FITC and transformed into nanoparticles by ionotropic gelation. Uptake of chitosan by confluent A549 cells was quantified by fluorometry, and in vitro cytotoxicity was evaluated by the MTT and neutral red uptakeassays. RESULTS: Nanoparticle uptake was a saturable event for all chitosan samples, with the binding affinity and uptake capacity decreasing with decreasing polymer Mw and DD. Uptake fell by 26% when Mw was decreased from 213,000 to 10,000, and by 41% when DD was lowered from 88% to 46%; the uptake data correlated with the zeta potential of the nanoparticles. Uptake of chitosan molecules did not exhibit saturation kinetics and was less dependent on Mw and DD. Postuptake quenching with trypan blue indicated that the cell-associated chitosan nanoparticles were internalized, but not the cell-associated chitosan molecules. Chitosan molecules and nanoparticles exhibited comparable cytotoxicity, yielding similar IC50 and IC20 values when evaluated against the A549 cells. Cytotoxicity of both chitosan entities was attenuated by decreasing polymer DD but was less affected by a lowering in Mw. CONCLUSIONS: Transforming chitosan into nanoparticles modified the mechanism of cellular uptake but did not change the cytotoxicity of the polymer toward A549 cells. Chitosan DD had a greater influence than Mw on the uptake and cytotoxicity of chitosan nanoparticles because of its effect on the zeta potential of the nanoparticles.

Acetylation↗

PubMed2003

Flexible chitin films as potential wound-dressing materials: wound model studies.

Chitin films possessing increased flexibility, softness, transparency, and conformability have been prepared. These attributes enable the potential application of chitin films as occlusive, semipermeable film wound dressings similar to commercial products such as Opsite trade mark. The chitin films are generally nonabsorbent, exhibiting a total weight gain of only up to 120-160% in physiological fluid. Dry chitin films transpire water vapor at a rate of about 600 g/m(2)/24 h, similar to commercial polyurethane-based film dressings, but rises to 2400 g/m(2)/24 h, when wet, which is higher than the water vapor transmission rate of intact skin. The chitin films are nontoxic to human skin fibroblasts, maintaining 70-80% cell viability. Wound studies using a rat model showed no signs of allergenicity or the high inflammatory response associated with biodegradable biomaterials. The chitin films displayed accelerated wound-healing properties. Based on histological examination, wound sites dressed with the chitin films stabilized and healed faster, and appeared stronger than those dressed with Opsite trade mark and gauze dressings after 7 days of healing.

Animals↗

PubMed2003

Implantable applications of chitin and chitosan.

Chitin, extracted primarily from shellfish sources, is a unique biopolymer based on the N-acetyl-glucosamine monomer. More than 40 years have lapsed since this biopolymer had aroused the interest of the scientific community around the world for its potential biomedical applications. Chitin, together with its variants, especially its deacetylated counterpart chitosan, has been shown to be useful as a wound dressing material, drug delivery vehicle and increasingly a candidate for tissue engineering. The promise for this biomaterial is vast and will continue to increase as the chemistry to extend its capabilities and new biomedical applications are investigated. It is interesting to note that a majority of this work has come from Asia. Japan has been the undisputed leader, but other Asian nations, namely Korea, Singapore, Taiwan and Thailand have also made notable contributions. More recently, China has joined the club to become an increasingly major research source for chitin and chitosan in Asia. This review surveys select works of key groups in Asia developing chitin and chitosan materials for implantable biomedical applications.

Animals↗

PubMed2002

Selection of Gongronella butleri strains for enhanced chitosan yield with UV mutagenesis.

This paper describes the selection of Gongronella butleri strains producing higher chitosan yield using UV mutagenesis. We have devised an enzyme-linked immunosorbent assay for the selection of high chitin deacetylase (CDA) yielding strains. Mutant strains M+1, M+2 and M+7 could produce twice the extractable chitosan yield and double the CDA activity, as compared to the wild type strain.

Amidohydrolases↗

PubMed2002

Chitosan-alginate PEC membrane as a wound dressing: Assessment of incisional wound healing.

Flexible, thin, transparent, novel chitosan-alginate polyelectrolyte complex (PEC) membranes, cast from aqueous suspensions of chitosan-alginate coacervates with CaCl(2), were evaluated as potential wound-dressing materials. MTT and NR assays suggested that the chitosan-alginate PEC membranes and their aqueous extracts were nontoxic towards mouse and human fibroblast cells. Cell growth was also not hindered by co-incubation with the membranes. Compared to conventional gauze dressing, the PEC membranes caused an accelerated healing of incision wounds in a rat model. Wounds closed at 14 days postoperatively, and histological observations showed mature epidermal architecture with keratinized surface of normal thickness and a subsided inflammation in the dermis. This was followed by an excellent remodeling phase with organized thicker collagen bundles and mature fibroblasts at 21 days postoperative. Control wounds continued to show signs of an active inflammatory phase under scab on Day 21. Closure rate and appearance of PEC membrane-treated wounds were comparable with Opsite(R)-treated wounds. On the basis of its biocompatibility and wound-healing efficacy, the chitosan-alginate PEC membrane can be considered for wound-dressing applications.

Alginates↗

PubMed2002

Uptake of FITC-chitosan nanoparticles by A549 cells.

PURPOSE: The objective of this study was to evaluate the extent and mechanism of uptake of fluorescent chitosan nanoparticles by the A549 cells, a human cell line derived from the respiratory epithelium. METHODS: Covalent conjugation with fluorescein-5-isothiocyanate yielded stably labeled chitosan molecules, which were successfully formulated into nanoparticles by ionotropic gelation. Uptake of fluorescein-5-isothiocyanate-chitosan nanoparticles and chitosan molecules by confluent A549 cells was quantified by fluorometry. RESULTS: Cellular uptake of chitosan nanoparticles was concentration and temperature dependent, having Km and Vmax of 3.84 microM and 58.14 microg/mg protein/h, respectively. Uptake of chitosan nanoparticles was up to 1.8-fold higher than that of chitosan molecules alone and was not inhibited by excess unlabeled chitosan molecules. Hyperosmolarity, chlorpromazine and K+ depletion inhibited by 65, 34, and 54%, respectively, the uptake of chitosan nanoparticles at 37 degrees C, but filipin had no influence on the uptake. Confocal imaging confirmed the internalization of the chitosan nanoparticles by the A549 cells at 37 degrees C. CONCLUSIONS: Formulation of chitosan into nanoparticles significantly improved its uptake by the A549 cells. Internalization of chitosan nanoparticles by the cells seems to occur predominantly by adsorptive endocytosis initiated by nonspecific interactions between nanoparticles and cell membranes, and was in part mediated by clathrin-mediated process.

Chitin↗

PubMed2002

Complete cDNA sequence of chitin deacetylase from Gongronella butleri and its phylogenetic analysis revealed clusters corresponding to taxonomic classification of fungi.

A cDNA library containing a chitin deacetylase (CDA) gene from a zygomycete Gongronella butleri was constructed and the complete gene was sequenced. The complete gene contains an open reading frame of 1290 nucleotides which encodes a sequence of 430 amino acid residues. The gene sequence consists of nucleotides encoding a polysaccharide deacetylase domain located in the middle, covering 34% of the entire sequence. This domain shares high sequence similarities with CDAs of fungi and other deacetylases including the nodb-like protein from Sinorhizobium meliloti and Bacillus subtilis, xylanase D from Cellulomonas fimi, and acetylxylan esterase A from Streptomyces lividans. The deduced amino acid sequence shows homology with CDAs from Mucor rouxii (48% identity) and Phycomyces blakesleeanus (30% identity). Phylogenetic analysis reveals that CDAs are clustered in accordance with the taxonomic classification of the fungi.

Journal Article↗

PubMedDate not supplied

Preparation of C-6 substituted chitin derivatives under homogeneous conditions.

Tosylation of chitin under homogeneous conditions was achieved by the reaction of tosyl chloride with chitin in a DMAc/LiCl solvent system. The resultant tosyl-chitin was fully N-acetylated with acetic anhydride in methanol. The fully acetylated tosyl-chitin was subsequently reacted with the sodium salts of ethyl p-hydroxybenzoate, diethyl malonate, and diethyl phosphite in DMAc to give the corresponding chitin derivatives of 6-O-ethyl benzoate-chitin, 6-deoxy-diethyl malonate-chitin, and 6-(deoxydiethyl) phosphite-chitin, respectively. Subsequent hydrolysis of the chitin-ester derivatives with tert-butoxide in dimethyl sulfoxide (DMSO) generated 6-O-carboxyphenyl-chitin and 6-(deoxydicarboxy)methyl-chitin. The structures of the chitin derivatives were assessed by FT-IR, (13)C NMR, and (31)P NMR, while the degree of substitution of the S(N)2 reaction was estimated by elemental analysis. All the chitin derivatives were found to be soluble or swellable in water, DMAc, or DMSO.

Carbohydrate Sequence↗