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Title:Razvoj bioaktivnih membran na osnovi bakterijske nanoceluloze za uporabo pri opeklinah : doktorska disertacija
Authors:ID Jančič, Urška (Author)
ID Gorgieva, Selestina (Mentor) More about this mentor... New window
ID Trček, Janja (Comentor)
Files:.pdf DOK_Jancic_Urska_2025.pdf (14,79 MB)
MD5: 68B66AE20A4EBF42280898804A0F2DB6
 
Language:Slovenian
Work type:Doctoral dissertation
Typology:2.08 - Doctoral Dissertation
Organization:FS - Faculty of Mechanical Engineering
Abstract:V doktorski disertaciji smo se osredotočili na razvoj novih bioaktivnih membran na osnovi bakterijske nanoceluloze (BnC), in situ, modificirane s karboksimetil celulozo (CMC), in ex situ, modificirane s proteolitskim encimom bromelainom (Br) in/ali protimikrobnim peptidom nizinom (N) za nekirurško odstranitev mrtvega kožnega tkiva (mrtvine) pri opeklinah ter hitrejše, učinkovitejše in netoksično celjenje opeklin. Membrane BnC smo producirali s pred kratkim opisano novo vrsto ocetnokislinske bakterije Komagataeibacter melomenusus AV436T, saj je bila ta izmed testiranih ocetnokislinskih bakterij najproduktivnejša. Za učinkovito odstranitev ostankov bakterij in komponent gojišča smo optimizirali alkalno obdelavo membran (1 h 0,5 M NaOH), s CMC-Br konjugati pa smo določili optimalno masno razmerje zamreževalca EDC/NHS (2/1) za najvišjo ohranitev specifične proteolitske aktivnosti bromelaina. In situ modifikacijo membran BnC smo izvedli z dodajanjem CMC v gojišče RAE za namnoževanje bakterij, kar je vodilo v nastanek membran BnC-CMC s karboksilnimi skupinami, gostejšo razporeditvijo fibrilov in izboljšanim rehidracijskim razmerjem v primerjavi z membrano BnC. Bromelain smo ex situ imobilizirali na (modificirane) membrane BnC z adsorpcijo in kovalentno vezavo z uporabo zamreževalca EDC/NHS, pri čemer smo za identifikacijo optimalne kombinacije procesnih parametrov (koncentracije CMC, bromelaina in zamreževalca EDC/NHS) z maksimalno specifično proteolitsko aktivnostjo bromelaina uporabili metodologijo odzivnih površin (RSM). S koimobilizacijo bromelaina in nizina smo proučevali sinergistični učinek obeh komponent s ciljem razširitve protimikrobnega delovanja in pridobitve proteolitskega delovanja. Optimalne bioaktivne membrane in njihove reference smo okarakterizirali s fizikalno-kemijskimi analiznimi metodami, kar je vključevalo ATR-FTIR in mikroanalizo FTIR, SEM, EDS, XPS, XRD, stične kote, kapaciteto zadrževanja vode, rehidracijsko razmerje, vsebnost vode, zeta potencial, TGA, mehanske lastnosti in specifično proteolitsko aktivnost. Biološko aktivnost membran smo analizirali s testiranjem protimikrobne aktivnosti in z različnimi in vitro (celična viabilnost in morfologija celic NHDF in HaCaT, celjenje ran) in ex vivo metodami (testiranje bioadhezije na modelu piščančje kože, testiranja toksičnosti na ekspantatih človeške kože – histološke študije, aktivnost LDH, sproščanje IL-8). Optimalni imobilizacijski pogoji (9 mg/mL CMC in 10 mg/mL bromelaina) so omogočili hiperaktivacijo encima s specifično proteolitsko aktivnostjo 2,3 U/mg in 39,1 % učinkovitostjo imobilizacije. Imobilizacija bromelaina in/ali nizina na membrano BnC-CMC je povečala povprečen premer nanovlaken, rehidracijsko razmerje in vsebnost vode ter izboljšala protimikrobno delovanje in adhezijske lastnosti. Membrani BnC-CMC in BnC-CMC-N sta bili in vitro citokompatibilni za celice NHDF in HaCaT ter sta povzročili hitrejšo proliferacijo celic HaCaT in/ali migracijo celic NHDF kot komercialni produkt Vivamel. Ex vivo analiza na ekspantatih človeške kože je za membrano BnC-CMC pokazala citokompatibilnost brez akutne toksičnosti in iritacije kože, membrana BnC-CMC-N je bila prav tako citokompatibilna brez akutne toksičnosti, vendar z zmernim dražilnim učinkom. Membrana BnC-CMC-Br je na ekspantatih človeške kože izkazovala aktivnost, ki se je odražala v razgradnji medceličnih stikov v epidermisu, vendar ni povzročila inducirajoče akutne toksičnosti in iritacije kože. Pripravljene membrane BnC-CMC, BnC-CMC-Br in BnC-CMC-N izkazujejo potencial za uporabo v različnih fazah zdravljenja opeklin.
Keywords:bakterijska nanoceluloza, bioaktivnost, bromelain, nizin, protimikrobna aktivnost, proteolitska aktivnost, membrana, opekline
Place of publishing:Maribor
Place of performance:Maribor
Publisher:[U. Jančič]
Year of publishing:2024
Number of pages:XXII, 180 str.
PID:20.500.12556/DKUM-89696 New window
UDC:[577:576.314]:620.3(043.3)
COBISS.SI-ID:223225859 New window
Publication date in DKUM:14.01.2025
Views:0
Downloads:17
Metadata:XML DC-XML DC-RDF
Categories:KTFMB - FS
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Licences

License:CC BY-NC-ND 4.0, Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
Link:http://creativecommons.org/licenses/by-nc-nd/4.0/
Description:The most restrictive Creative Commons license. This only allows people to download and share the work for no commercial gain and for no other purposes.
Licensing start date:06.08.2024

Secondary language

Language:English
Title:Development of bacterial nanocellulose-based bioactive membranes for application in burns
Abstract:In this doctoral dissertation, we focused on developing new bioactive membranes based on bacterial nanocellulose (BnC), in situ modified with carboxymethyl cellulose (CMC) and ex situ modified with the proteolytic enzyme bromelain (Br) and/or the antimicrobial peptide nisin (N) for use in burns, specifically for non-surgical removal of necrotic skin tissue (eschar) and faster, more effective and non-toxic burn healing. BnC membranes were produced using the recently described novel acetic acid bacterium species Komagataeibacter melomenusus AV436T, as it was the most productive among the tested acetic acid bacteria. The optimal alkaline treatment of the membranes was determined (1 h 0.5 M NaOH) to remove bacterial residues and media components effectively. Furthermore, CMC-Br conjugates were prepared to determine the optimal mass ratio of the crosslinker EDC/NHS (2/1) for the highest retention of specific proteolytic activity of bromelain. The in situ modification of BnC membranes was performed by adding CMC to the RAE bacterial culture medium, resulting in BnC-CMC membranes with carboxyl groups, denser fibril distribution and an improved rehydration ratio compared to the BnC membrane. Bromelain was ex situ immobilized on (modified) BnC membranes by adsorption and covalent binding using EDC/NHS crosslinker, and response surface methodology (RSM) was used to identify the optimal combination of process parameters (the concentration of CMC, bromelain and EDC/NHS crosslinker) with maximum specific proteolytic activity of bromelain. The co-immobilization of bromelain and nisin was studied to explore the synergistic effect of both components, expand the antimicrobial activity and gain proteolytic activity. The optimal bioactive membranes and their references were characterized according to physico-chemical analysis methods, which included ATR-FTIR and micro FTIR analysis, SEM, EDS, XPS, XRD, contact angles, water retention capacity, rehydration ratio, water content, zeta potential, TGA, mechanical properties and specific proteolytic activity. The biological activity of the membranes was analyzed using antimicrobial activity and various in vitro (the cell viability and morphology of NHDF and HaCaT cells, wound healing) and ex vivo methods (bioadhesion testing on a chicken skin model, testing on a human skin explants model – histological analysis, LDH activity, IL-8 release). The optimal immobilization conditions (9 mg/mL CMC and 10 mg/mL bromelain) led to hyperactivation of the enzyme with a specific proteolytic activity of 2.3 U/mg and immobilization efficiency of 39.1%. The immobilization of bromelain and/or nisin on the BnC-CMC membrane increased the average nanofiber diameter, rehydration ratio and water content, and improved the antimicrobial activity and adhesion properties. The BnC-CMC and BnC-CMC-N membranes were in vitro cytocompatible for NHDF and HaCaT cells and induced faster HaCaT cell proliferation and/or NHDF cell migration than the commercial Vivamel product. Ex vivo analysis on human skin explants showed a cytocompatible BnC-CMC membrane without acute toxicity and skin irritation, while the BnC-CMC-N membrane was also cytocompatible without acute toxicity but with a moderate irritant effect. The BnC-CMC-Br membrane showed activity on human skin explants, which manifests in the digestion of intercellular junctions in the epidermis but did not induce acute toxicity and skin irritation. The prepared membranes BnC-CMC, BnC-CMC-Br and BnC-CMC-N show potential for use in different stages of burn treatment.
Keywords:bacterial nanocellulose, bromelain, nisin, bioactivity, antimicrobial activity, proteolytic activity, membrane, burns


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