Effect of peptides' binding on the antimicrobial activity and biocompatibility of protein-based substrates Maja Kaisersberger Vincek
, 2017, doctoral dissertation
Abstract: This work reveals the effect of coupling approach (chemical by using carbodiimide chemistry and grafting-to vs. grafting-from synthesis routes, and enzymatic by using transglutaminase) of a hydrophilic ε-poly-L-lysine (εPL) and an amphiphilic oligo-acyl-lysyl (OAK) derivative (K-7α12-OH) to wool fibers and gelatine (GEL) macromolecules, respectively, and substrates antibacterial activity against Gram-negative E. coli and Gram-positive S. aureus bacteria after 1–24 h of exposure, as well as their cytotoxicity. Different spectroscopic (ultraviolet-visible, infrared, fluorescence and electron paramagnetic resonance) and separation techniques (size-exclusion chromatography and capillary zone electrophoresis) as well as zeta potential and potentiometric titration analysis, were performed to confirm the covalent coupling of εPL/OAK, and to determine the amount and orientation of its immobilisation.
The highest and kinetically the fastest level of bacterial reduction was achieved with wool/GEL functionalised with εPL/OAK by chemical grafting-to approach. This effect correlated with both the highest grafting yield and conformationally the highly-flexible (brush-like) orientation linkage of εPL/OAK, implicating on the highest amount of accessible amino groups interacting with bacterial membrane. However, OAK`s amphipathic structure, the cationic charge and the hydrophobic moieties, resulted to relatively high reduction of S. aureus for grafting-from and the enzymatic coupling approaches using OAK-functionalised GEL.
The εPL/OAK-functionalised GEL did not induce toxicity in human osteoblast cells, even at ~25-fold higher concentration than bacterial minimum inhibitory (MIC) concentration of εPL/OAK, supporting their potential usage in biomedical applications.
It was also shown that non-ionic surfactant adsorbs strongly onto the wool surface during the process of washing, thereby blocking the functional sites of immobilized εPL and decreases its antibacterial efficiency.
Keywords: wool, gelatine, antimicrobial peptides, ε-poly-L-lysine, oligo-acyl-lysyl, grafting chemistry, grafting approach, peptide orientation, antibacterial activity, cytotoxicity
Published: 17.08.2017; Views: 966; Downloads: 92
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Native cellulose nanofibrills induce immune tolerance in vitro by acting on dendritic cellsSergej Tomić
, Vanja Kokol
, Dušan Mihajlović
, Aleksandar Mirčić
, Miodrag Čolić
, 2016, original scientific article
Abstract: Cellulose nanofibrills (CNFs) are attractive biocompatible, natural nanomaterials for wide biomedical applications. However, the immunological mechanisms of CNFs have been poorly investigated. Considering that dendritic cells (DCs) are the key immune regulatory cells in response to nanomaterials, our aim was to investigate the immunological mechanisms of CNFs in a model of DC-mediated immune response. We found that non-toxic concentrations of CNFs impaired the differentiation, and subsequent maturation of human monocyte-derived (mo)-DCs. In a co-culture with CD4+T cells, CNF-treated mo-DCs possessed a weaker allostimulatory and T helper (Th)1 and Th17 polarizing capacity, but a stronger capacity to induce Th2 cells and CD4+CD25hiFoxP3hi regulatory T cells. This correlated with an increased immunoglobulin-like transcript-4 and indolamine dioxygenase-1 expression by CNF-treated mo-DCs, following the partial internalization of CNFs and the accumulation of CD209 and actin bundles at the place of contacts with CNFs. Cumulatively, we showed that CNFs are able to induce an active immune tolerance by inducing tolerogenic DCs, which could be beneficial for the application of CNFs in wound healing and chronic inflammation therapies.
Keywords: nanocellulose, cytotoxicity, T-cell proliferation, cytokine production, in vitro
Published: 23.06.2017; Views: 655; Downloads: 300
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Antimicrobial medical textiles based on chitosan nanoparticles for gynaecological treatmentTijana Ristić
, 2014, doctoral dissertation
Abstract: The aim of this dissertation was to develop a novel medical tampon for alternative gynaecological treatment using chitosan nanoparticles as an antimicrobial agent or as a drug delivery system. For this purpose viscose tampon band was used and functionalized with chitosan and trimethyl chitosan nanoparticles. A comprehensive of interactions between chitosan and cellulose as well as characterization of prepared materials were done. At the beginning, chitosan (CS) and trimethyl chitosan (TMC) solutions, as well as nanoparticles synthesised by ionic gelation were studied. Their characterization was focused on determining the charge and antimicrobial properties against common pathogenic microorganism. The influence of cationic charge on the inhibition of microbial growth was confirmed. Since CS and TMC solutions and nanoparticles dispersions exhibited antibacterial activity against Lactobacillus, a detailed investigation in chitosan’s antimicrobial mode of action was performed using a novel diffusion nuclear magnetic resonance (D-NMR). D-NMR allowed the monitoring of intra- and extracellular water exchange from the cells indicating the membrane alteration and leakage of intracellular constituencies. Further, in order to study the adsorption phenomena and molecular interactions between CS/TMC (solution or nanoparticles) and cellulose material, model cellulose surfaces were used, and adsorption was studied by quartz crystal microbalance with dissipation. CS and TMC were favourably deposited onto cellulose model surface at higher ionic strength, higher pH values, i.e. factors causing lower solubility, where the presence of electrostatic interactions was negligible and non-electrostatic interactions were dominant. The knowledge gained from the model surfaces was extremely helpful in characterization of real systems, i.e. functionalized cellulose fibres and for understanding the obtained results. Immobilization of CS and TMC (in the form of solution and/or nanoparticles) onto cellulose viscose fibres was confirmed with several analytical methods. The attachment of chitosan onto fibres was reversible, as endorsed with desorption studies mimicking the conditions of vaginal usage. Evaluation of antimicrobial properties was performed using two different techniques, both revealing a high inhibition of the tested microorganism. In addition, Lactobacillus susceptibility testing has shown that chitosan-coated fibres do not have any negative influence on the resident microbiota. Assessment of in-vitro cytotoxicity demonstrated that samples do not cause a cytotoxic effect in direct contact. Additionally, model drug was incorporated into chitosan nanoparticles and subsequently attached onto fibres in order to create modern, vaginal drug delivery systems. Antimicrobial medical textiles investigated in the scope of this dissertation show the potential for their exploitation in gynaecological field as preventive or curative treatment without triggering any adverse effects for the user.
Keywords: chitosan, N, N, N-trimethyl chitosan, nanoparticles, antimicrobial activity, cellulose model films, regenerated cellulose fibres, diffusion nuclear magnetic resonance, quartz crystal microbalance, vaginal infections, drug delivery systems, cytotoxicity
Published: 13.02.2014; Views: 2503; Downloads: 111
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