Upotreba hidrogelova dobijenih modifikacijom polisaharida fenolnim jedinjenjima za imobilizaciju ćelija i biokatalizatora

Abstract

Hidrogelovi predstavljaju polimerne mreže sa hidrofilnim osobinama, sposobne da apsorbuju velike količine vode, uz povećanje svoje zapremine. Geliranje se može izvesti fizičkim ili hemijskim tipovima umrežavanja polimernih lanaca, koji mogu da vode poreklo od prirodnih i sintetičkih polimera. U oblastima biomedicine, najzastupljeniji su hidrogelovi dobijeni od prirodnih polimera, kao što su polisaharidi i njihovi derivati. Cilj ovog rada je bio modifikovati različite polisaharide (alginat, pektin, karboksimetilceluloza) fenolnim jedinjenjima (tiramin, tirozin, dopamin, L-DOPA), sa ciljem dobijanja polimera sposobnih da grade hidrogelove. Uspešnost modifikacije je okarakterisana UV-Vis, FTIR i 1H NMR spektroskopskim metodama. Ispitana je njihova sposobnost geliranja, nakon čega su oni sa najpogodnijim karakteristikama primenjeni u različitim oblastima. Dopaminski derivati alginata su korišćeni za enkapsulaciju ćelijskih zidova sa enzimom lakazom, dobijenih lizom ćelija Saccharomyces cerevisiae sa prethodno eksprimiranom lakazom na površini ćelije. Dobijeni ćelijski zidovi sa lakazom su pokazali 300 puta veću enzimsku aktivnost u odnosu na nelizirane ćelije. Uspešno je urađena imobilizacija u kalcijum-dopamin-alginatnim kuglicama, koje su pokazale prednost u odnosu na kuglice sa nativnim alginatom. Ovako dobijeni biokatalizatori su korišćeni za dekolorizaciju tekstilnih boja Amido Black 10B, Reactive Black 5, Evans Blue, i Remazol Brilliant Blue. Nakon 10 ciklusa dekolorizacije od po 48 sati, efikasnost dekolorizacije za boje Evans Blue i Amido Black 10 B je iznosila 90 % i 61 %, navedenim redom. Lakaza iz Streptomyces cyaneus je takođe eksprimirana u E. coli, izolovana i prečišćena. Izolovana lakaza je imobilizovana u dopamin-pektinskim mikrokuglicama, metodom enzimske polimerizacije u emulziji. Imobilizovana lakaza je pokazala povećanu termalnu i pH stabilnost u odnosu na slobodan enzim, kao i sposobnost dekolorizacije boja Amido Black 10B, Reactive Black 5 i Evans Blue. U uzastopnim ciklusima dekolorizacije boja je ovaj biokatalizator pokazao najviše potencijala za degradaciju boja Amido Black 10B i Reactive Black 5. Sintetisani dopaminski derivati karboksimetilceluloze i alginata su korišćeni za proizvodnju nanovlakana elektrospining metodom. Dobijena nanovlakna su dodatno umrežavana i korišćena kao podloga za gajenje ćelija fibroblasta (MRC-5). Dopamin-alginatna nanovlakna su se pokazala kao dobar materijal za gajenje ćelija, što je pokazano snimanjem SEM-a i MTT esejem, iz čega bi moglo da se zaključi da su adhezija i proliferacija ćelija moguće na ovim nanovlaknima i da su ona potencijalno dobar materijal za tkivni inženjering.

Hydrogels are polymer networks, which have hydrophilic properties and could absorb large amount of water, increasing their volume. They could form gels either by physical or chemical cross-linking of polymer chains and could be derived from natural and synthetic polymers. The aim of this study was to modificate diverse polysaccharides (alginate, pectin, and carboxymethylcellulose) with phenol compounds (tyramine, tyrosine, dopamine, L-DOPA), in order to obtain polymers which could form hydrogels. The success of modification was confirmed by UV-Vis, FTIR and 1H NMR spectroscopy. Their gelling ability was investigated, afterward polymers with the most suitable characteristics were applied in different areas. Dopamine alginate derivatives were used to encapsulate cell walls with the enzyme laccase, obtained by lysing cells of Saccharomyces cerevisiae, with previously expressed laccase on the cell surface. The obtained cell wall laccase has shown 300 times higher enzyme activity, compared to the whole cells. Immobilization was successfully performed in calcium-dopamine-alginate beads, which showed an advantage compared to beads with native alginate. Thus obtained biocatalyst were used for decolorization of textile dyes Amido Black 10B, Reactive Black 5, Evans Blue, and Remazol Brilliant Blue. After ten cycles of repeated use, decolourization efficiency for dyes Evans Blue and Amido Black 10B was 90 % and 61 %, respectively. Laccase from Streptomyces cyaneus was also expressed in E. coli, isolated and purified. The isolated laccase was immobilized in dopamine-pectin microbeads, using emulsion-based enzymatic crosslinking polymerization. The immobilized laccase showed improved thermal and pH stability in comparison to the free enzyme, and ability of decolorization dyes Amido Black 10B, Reactive Black 5 and Evans Blue. In successive cycles of dyes decolorization, this biocatalyst showed the greatest potential for degradation of Amido Black 10B and Reactive Black 5. The synthesized dopamine derivatives of carboxymethylcellulose and alginate were used for the production of nanofibers by the method of electrospinning. The obtained nanofibers were additionaly crosslinked and used as a scaffold for fibroblast cells (MRC-5) cultivation. Dopamin-alginate nanofibers have been shown as a good material for cell cultivation, as shown by SEM imaging and MTT assay. It could be concluded that cell adhesion and proliferation are possible on these nanofibers and they are potentially good material for tissue engineering.