More than half of the hospital-associated infections worldwide are related to the adhesion of bacteria cells to biomedical devices and implants. To prevent these infections, it is crucial to modify biomaterial surfaces to develop the antibacterial property. In this study, chitosan (CS) and chondroitin sulfate (ChS) were chosen as antibacterial coating materials on polylactic acid (PLA) surfaces. Plasma-treated PLA surfaces were coated with CS either direct coating method or the carbodiimide coupling method. As a next step for the combined saccharide coating, CS grafted samples were immersed in ChS solution, which resulted in the polyelectrolyte complex (PEC) formation. Also in this experiment, to test the drug loading and releasing efficiency of the thin film coatings, CS grafted samples were immersed into lomefloxacin-containing ChS solution. The successful modifications were confirmed by elemental composition analysis (XPS), surface topography images (SEM), and hydrophilicity change (contact angle measurements). The carbodiimide coupling resulted in higher CS grafting on the PLA surface. The coatings with the PEC formation between CS-ChS showed improved activity against the bacteria strains than the separate coatings. Moreover, these interactions increased the lomefloxacin amount adhered to the film coatings and extended the drug release profile. Finally, the zone of inhibition test confirmed that the CS-ChS coating showed a contact killing mechanism while drug-loaded films have a dual killing mechanism, which includes contact, and release killing.
The study presents a novel vancomycin-releasing collagen wound dressing derived from Cyprinus carpio collagen type I cross-linked with carbodiimide which retarded the degradation rate and increased the stability of the sponge. Following lyophilization, the dressings were subjected to gamma sterilization. The structure was evaluated via scanning electron microscopy images, micro-computed tomography, and infrared spectrometry. The structural stability and vancomycin release properties were evaluated in phosphate buffered saline. Microbiological testing and a rat model of a wound infected with methicillin-resistant Staphylococcus aureus (MRSA) were then employed to test the efficacy of the treatment of the infected wound. Following an initial mass loss due to the release of vancomycin, the sponges remained stable. After 7 days of exposure in phosphate buffered saline (37°C), 60% of the material remained with a preserved collagen secondary structure together with a high degree of open porosity (over 80%). The analysis of the release of vancomycin revealed homogeneous distribution of the antibiotic both across and between the sponges. The release of vancomycin was retarded as proved by in vitro testing and further confirmed by the animal model from which measurable concentrations were observed in blood samples 24 hours after the subcutaneous implantation of the sponge, which was more than observed following intraperitoneal administration. The sponge was also highly effective in terms of reducing the number of colony-forming units in biopsies extracted from the infected wounds 4 days following the inoculation of the wounds with the MRSA solution. The presented sponges have ideal properties to serve as wound dressing for prevention of surgical site infection or treatment of already infected wounds.
- MeSH
- Anti-Bacterial Agents pharmacokinetics MeSH
- Wound Healing drug effects MeSH
- Carps MeSH
- Carbodiimides pharmacokinetics MeSH
- Collagen pharmacokinetics MeSH
- Rats MeSH
- Methicillin-Resistant Staphylococcus aureus drug effects MeSH
- Bandages MeSH
- Vancomycin pharmacokinetics MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Extracellular matrix (ECM) hydrogels, produced by tissue decellularization are natural injectable materials suitable for neural tissue repair. However, the rapid biodegradation of these materials may disrupt neural tissue reconstruction in vivo. The aim of this study was to improve the stability of the previously described ECM hydrogel derived from human umbilical cord using genipin and N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), crosslinking at concentration of 0.5-10 mM. The hydrogels, crosslinked by genipin (ECM/G) or EDC (ECM/D), were evaluated in vitro in terms of their mechanical properties, degradation stability and biocompatibility. ECM/G, unlike ECM/D, crosslinked hydrogels revealed improved rheological properties when compared to uncrosslinked ECM. Both ECM/G and ECM/D slowed down the gelation time and increased the resistance against in vitro enzymatic degradation, while genipin crosslinking was more effective than EDC. Crosslinkers concentration of 1 mM enhanced the in vitro bio-stability of both ECM/G and ECM/D without affecting mesenchymal stem cell proliferation, axonal sprouting or neural stem cell growth and differentiation. Moreover, when injected into cortical photochemical lesion, genipin allowed in situ gelation and improved the retention of ECM for up to 2 weeks without any adverse tissue response or enhanced inflammatory reaction. In summary, we demonstrated that genipin, rather than EDC, improved the bio-stability of injectable ECM hydrogel in biocompatible concentration, and that ECM/G has potential as a scaffold for neural tissue application.
- MeSH
- Extracellular Matrix chemistry MeSH
- Hydrogels chemistry MeSH
- Iridoids * MeSH
- Carbodiimides administration & dosage MeSH
- Humans MeSH
- Mesenchymal Stem Cells cytology MeSH
- Cell Proliferation physiology MeSH
- Umbilical Cord cytology MeSH
- Nerve Regeneration physiology MeSH
- Tissue Engineering MeSH
- Tissue Scaffolds chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Infections of the musculoskeletal system present a serious problem with regard to the field of orthopedic and trauma medicine. The aim of the experiment described in this study was to develop a resorbable nanostructured composite layer with the controlled elution of antibiotics. The layer is composed of collagen, hydroxyapatite nanoparticles, and vancomycin hydrochloride (10 wt%). The stability of the collagen was enhanced by means of cross-linking. Four cross-linking agents were studied, namely an ethanol solution, a phosphate buffer solution of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide, genipin, and nordihydroguaiaretic acid. High performance liquid chromatography was used so as to characterize the in vitro release rates of the vancomycin and its crystalline degradation antibiotically inactive products over a 21-day period. The maximum concentration of the released active form of vancomycin (approximately 265 mg/L) exceeded the minimum inhibitory concentration up to an order of 17 times without triggering the burst releasing effect. At the end of the experiment, the minimum inhibitory concentration was exceeded by up to 6 times (approximately 100 mg/L). It was determined that the modification of collagen with hydroxyapatite nanoparticles does not negatively influence the sustainable release of vancomycin. The balance of vancomycin and its degradation products was observed after 14 days of incubation.
- MeSH
- Durapatite MeSH
- Carbodiimides chemistry MeSH
- Collagen chemistry MeSH
- Drug Delivery Systems methods MeSH
- Delayed-Action Preparations chemistry MeSH
- Methylamines chemistry MeSH
- Nanoparticles chemistry MeSH
- Nanostructures chemistry MeSH
- Drug Carriers chemistry MeSH
- Vancomycin chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
In this study a strategy to immobilize phospholipids onto a polymer-based stationary phase is described. Methacrylate-based monoliths in capillary format (150×0.1mm) were modified by soybean phosphatidylcholine through 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide coupling to obtain stationary phases suitable to mimic cell surface membranes. The covalent coupling reaction involves the phosphate group in phospholipids; therefore, the described methodology is suitable for all types of phospholipids. Immobilization of soy bean phosphatidylcholine on the monolith was confirmed by attenuated total reflectance Fourier transform infrared spectroscopy and gas chromatography-mass spectrometry of the fatty alcohol profile, generated upon reductive cleavage of the fatty acyl side chains of the phospholipid on the monolith surface with lithium aluminium hydride. The prepared stationary phases were evaluated through studies on the retention of low-molar mass model analytes including neutral, acidic, and basic compounds. Liquid chromatographic studies confirmed predominant hydrophobic interactions between the analytes and the synthesized stationary phase; however, electrostatic interactions contributed to the retention as well. The synthesized columns showed high stability even with fully aqueous mobile phases such as Dulbecco's phosphate-buffered saline solution.
- MeSH
- Biomimetics MeSH
- Chemistry Techniques, Analytical instrumentation methods MeSH
- Chromatography, Liquid instrumentation MeSH
- Ethyldimethylaminopropyl Carbodiimide chemistry MeSH
- Phosphatidylcholines chemistry MeSH
- Phospholipids chemistry MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Methacrylates chemistry MeSH
- Polymers chemistry MeSH
- Water chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
OBJECTIVES: The cytochrome P450 (P450) and cytochrome b5 are membrane hemoproteins composing together with flavoprotein NADPH:P450 reductase a mixed function oxidase (MFO) system. The knowledge of the interaction between P450 and its redox partners within a MFO system is fundamental to understand P450 reaction mechanism, an electron transport from its redox partner and also detoxification of xenobiotics and/or metabolism of endogenous substrates with all positive or negative aspects for organisms. METHODS: The chemical cross-linking by soluble carbodiimide (EDC) in combination with the liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) has been employed to characterize the contact surface regions involved in the transient interaction between two catalytic domains of P450 2B4 and cytochrome b5. RESULTS: The cross-linking reaction was accomplished in an equimolar catalytic complex of P450 2B4:cytochrome b5 and the covalent hetero-dimers detected on SDS-PAGE electrophoresis were analyzed (after in gel trypsin digestion) using LC-HRMS to identify cross-linked amino-acid residues. The computed in silico models of P450 2B4:cytochrome b5 complex using amino-acids participating in cross-links (Asp134, Lys139, Glu424 and Glu439 located on a proximal surface of P450 2B4) suggest interpretation that two different types of cytochrome b5 orientations are present in the studied interaction within a MFO system: the first allowing potential cytochrome b5 electron donation to P450, the second one inducing cytochrome b5 modulation of P450 structural changes. CONCLUSIONS: The results demonstrated the capability of the used experimental approach to map the interaction between P450 and cytochrome b5 suggesting the formation of multi-meric structures within a MFO system as interpretation of the two observed mutual orientations.
- MeSH
- Aryl Hydrocarbon Hydroxylases chemistry metabolism MeSH
- Models, Chemical MeSH
- Chromatography, Liquid methods MeSH
- Cytochromes b5 chemistry metabolism MeSH
- Dimerization MeSH
- Electrons MeSH
- Mass Spectrometry methods MeSH
- Protein Interaction Domains and Motifs MeSH
- Microsomes, Liver enzymology MeSH
- Carbodiimides chemistry MeSH
- Rabbits MeSH
- Oxidation-Reduction MeSH
- Cross-Linking Reagents chemistry MeSH
- Protein Structure, Tertiary MeSH
- Structure-Activity Relationship MeSH
- Animals MeSH
- Check Tag
- Rabbits MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Hydrogen/deuterium (H/D) exchange or chemical cross-linking by soluble carbodiimide (EDC) was employed in combination with high-resolution mass spectrometry (MS) to extend our knowledge about contact surface regions involved in the well-characterized model of interaction between two molecules of human 14-3-3ζ regulatory protein. The H/D exchange experiment provided low resolution mapping of interaction in the homodimeric 14-3-3ζ complex. A lower level of deuteration, suggesting structural protection, of two sequential segments has been demonstrated for dimeric 14-3-3ζ wild type relative to the monomeric mutant 14-3-3ζ S58D. The N-terminal sequence (the first 27 residues) from one subunit interacts with region αC'and αD'-helices (residues 45-98) of the other molecule across the dimer interface. To identify interacting amino acid residues within the studied complex, a chemical cross-linking reaction was carried out to produce the covalent homodimer, which was detected by SDS-PAGE. The MS analysis (following tryptic in-gel digestion) employing both high resolution and tandem mass spectrometry revealed cross-linked amino acid residues. Two alternative salt bridges between Glu81 and either Lys9 or the N-terminal amino group have been found to participate in transient interactions of the 14-3-3ζ isotype homodimerization. The data obtained, which have never previously been reported, were used to modify the published 14-3-3 crystal structure using molecular modeling. Based on our findings, utilization of this combination of experimental approaches, which preserve protein native structures, is suitable for mapping the contact between two proteins and also allows for the description of transient interactions or of regions with flexible structure in the studied protein complexes.
- MeSH
- Mass Spectrometry methods MeSH
- Carbodiimides chemistry MeSH
- Protein Conformation MeSH
- Humans MeSH
- Protein Interaction Mapping MeSH
- Molecular Sequence Data MeSH
- Protein Multimerization MeSH
- Mutation MeSH
- 14-3-3 Proteins chemistry genetics isolation & purification metabolism MeSH
- Cross-Linking Reagents chemistry MeSH
- Recombinant Proteins chemistry genetics isolation & purification metabolism MeSH
- Amino Acid Sequence MeSH
- Molecular Dynamics Simulation MeSH
- Deuterium Exchange Measurement methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- Avidin diagnostic use MeSH
- Biosensing Techniques methods instrumentation utilization MeSH
- Biotin diagnostic use MeSH
- Cyanogen Bromide diagnostic use MeSH
- Epichlorohydrin diagnostic use MeSH
- Glutaral diagnostic use MeSH
- Carbodiimides diagnostic use MeSH
- Clinical Laboratory Techniques trends utilization MeSH
- Periodic Acid diagnostic use MeSH
- Macromolecular Substances isolation & purification MeSH
- Cysteamine diagnostic use MeSH
- Nanoparticles diagnostic use MeSH
- Polysaccharides diagnostic use MeSH
- Streptavidin diagnostic use MeSH
- Succinimides diagnostic use MeSH
Modifications of poly(2-hydroxyethyl methacrylate) (PHEMA) with cholesterol and laminin have been developed to design scaffolds that promote cell-surface interaction. Cholesterol-modified superporous PHEMA scaffolds have been prepared by the bulk radical copolymerization of 2-hydroxyethyl methacrylate (HEMA), cholesterol methacrylate (CHLMA) and the cross-linking agent ethylene dimethacrylate (EDMA) in the presence of ammonium oxalate crystals to introduce interconnected superpores in the matrix. With the aim of immobilizing laminin (LN), carboxyl groups were also introduced to the scaffold by the copolymerization of the above monomers with 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA). Subsequently, the MCMEMA moiety in the resulting hydrogel was hydrolyzed to [2-(methacryloyloxy)ethoxy]acetic acid (MOEAA), and laminin was immobilized via carbodiimide and N-hydroxysulfosuccinimide chemistry. The attachment, viability and morphology of mesenchymal stem cells (MSCs) were evaluated on both nonporous and superporous laminin-modified as well as laminin-unmodified PHEMA and poly(2-hydroxyethyl methacrylate-co-cholesterol methacrylate) P(HEMA-CHLMA) hydrogels. Neat PHEMA and laminin-modified PHEMA (LN-PHEMA) scaffolds facilitated MSC attachment, but did not support cell spreading and proliferation; the viability of the attached cells decreased with time of cultivation. In contrast, MSCs spread and proliferated on P(HEMA-CHLMA) and LN-P(HEMA-CHLMA) hydrogels.
- MeSH
- Cholesterol metabolism MeSH
- Hydrogels chemistry MeSH
- Carbodiimides chemistry MeSH
- Microscopy, Confocal MeSH
- Rats MeSH
- Methacrylates pharmacology chemistry MeSH
- Mesenchymal Stem Cells cytology drug effects MeSH
- Microscopy, Electron, Scanning MeSH
- Cell Count MeSH
- Porosity drug effects MeSH
- Solutions MeSH
- Spectrophotometry, Ultraviolet MeSH
- Tissue Engineering MeSH
- Tissue Scaffolds chemistry MeSH
- Cell Survival drug effects MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
A spontaneous mutant of Methanothermobacter thermautotrophicus resistant toward the ATP-synthase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) was isolated. DCCD normally inhibits methanogenic electron-transport-driven ATP synthesis, however, the DCCD-resistant strain exhibited methanogenesis in the presence of 300 micromol/L DCCD. Total ATP synthesis was shown to be higher in the mutant strain, both in the presence and absence of DCCD. These results suggested a modification in the ATP-synthesizing system of the mutant strain. Using Blue Native PAGE combined with MALDI TOF/TOF mass spectrometry, increased concentrations of both the A(1) and A(o) subcomplexes of the A(1)A(o)-type synthase were identified in the mutant strain. However, no alterations were found in the structural genes (atp) for the A(1)A(o) ATP synthase. The results imply that DCCD resistance is a consequence of increased A(1)A(o) ATP synthase expression, and suggest that genes involved in regulating synthase expression are responsible for DCCD resistance.
- MeSH
- Adenosine Triphosphate metabolism MeSH
- Archaeal Proteins biosynthesis MeSH
- Dicyclohexylcarbodiimide toxicity MeSH
- Electrophoresis, Polyacrylamide Gel MeSH
- Gene Expression MeSH
- Financing, Organized MeSH
- Enzyme Inhibitors toxicity MeSH
- Drug Resistance MeSH
- Methane metabolism MeSH
- Methanobacteriaceae MeSH
- Mutation MeSH
- Oxidation-Reduction MeSH
- Proton-Translocating ATPases biosynthesis MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
- Up-Regulation MeSH
