The effect of formulation and electrospinning parameters on viability of Stenotropomonas bacteria in nanofibers

Parodontitis predstavlja velik javnozdravstveni problem, zanj pa je značilna disbioza ustnega biofilma. Eden izmed obetavnejših naprednih načinov ponovne vzpostavitve ravnovesja predstavljajo probiotiki, izolirani iz ustne votline. Probiotike moramo lokalno dostaviti na prizadeto mesto, da s tem omogočimo njihovo kolonizacijo. Nanovlakna predstavljajo obetajoč dostavni sistem za ta namen. Namen te magistrske naloge je bil optimizirati formulacijske in procesne parametre izdelave nanovlaken z vgrajenim potencialno probiotičnim sevom 27.3.S rodu Stenotrophomonas sp. tako, da med postopkom vgradnje čim bolj ohranjamo njegovo živost. Ugotovili smo, da lahko s 6-urnim obsevanjem 4 % (m/V) raztopine polietilenoksida (PEO) z UV svetlobo valovne dolžine 254 nm zmanjšamo možnost kontaminacije polimernih raztopin s kontaminantnimi sevi mikroorganizmov. Uspešno izdelavo PEO nanovlaken z vgrajenimi bakterijami seva 27.3.S smo potrdili z vrstično elektronsko mikroskopijo. Živost bakterij v nanovlaknih se je med njihovo izdelavo zmanjšala za 2,9 log CFU/mg. Osmolarnost polimerne disperzije in temperatura med elektrostatskim sukanjem nista imela vpliva na preživetje bakterij, dokazali pa smo negativen vpliv izpostavitve polimerne disperzije visoki napetosti (upad živosti za 1,3 log CFU/mL) in sušenja (upad živosti za 2,1 log CFU/mL) na preživetje bakterij po elektrostatskem sukanju. Dodatno smo ugotovili, da elektrostatsko sukanje boljše preživijo bakterije, katerih rast smo prekinili v pozni logaritemski fazi v primerjavi s stacionarno fazo. Izdelali smo tudi PEO nanovlakna z dodatkom 55,6 % izbranih stabilizatorjev (saharoze, manitola, glicerola in sorbitola) z namenom zmanjšanja negativnega vpliva dehidracije med elektrostatskim sukanjem na živost bakterij seva 27.3.S. Statistično pomembno izboljšanje preživetja bakterij v nanovlaknih smo dosegli z dodatkom sorbitola in saharoze. Primerjali smo tudi preživetje izbranega seva v polimernih filmih in liofilizatih z nanovlakni takoj po njihovi izdelavi ter ugotovili, da je bilo preživetje bakterij seva 27.3.Z po obeh procesih sušenja boljše kot v primerjavi z elektrostatskim sukanjem. Rezultati magistrske naloge predstavljajo sistematičen pregled vplivov različnih parametrov na preživetje bakterijskega seva 27.3.S med njihovo vgradnjo v PEO nanovlakna z elektrostatskim sukanjem, kar je ključno za načrtovanje naprednih dostavnih sistemov z občutljivimi biološkimi komponentami, kot so probiotiki.Periodontitis represents a major public health problem, and is characterized by dysbiosis of the oral biofilm. Probiotics isolated from the oral cavity represent one of the promising advanced methods to restore the balance. Probiotics must be delivered locally to the affected area to enable their colonization. Nanofibers represent a promising delivery system for this purpose. The aim of this master\u27s thesis was to optimize the formulation and process parameters of the production of nanofibers with incorporated potentially probiotic strain 27.3.S of the genus Stenotrophomonas sp. by keeping its viability as high as possible during the electrospinning process. We found that irradiation of a 4% (m/V) polyethylene oxide (PEO) solution for 6 hours with UV light of a wavelength of 254 nm can reduce the possibility of contamination of polymer solutions with contaminating strains of microorganisms. The successful development of PEO nanofibers with incorporated bacterial strain 27.3.S was confirmed by scanning electron microscopy. The viability of bacteria in the nanofibers decreased by 2.9 log CFU/mg during their preparation. The polymer dispersion osmolarity and temperature during electrospinning did not affect bacterial survival, but we demonstrated a negative effect of exposing the polymer dispersion to high voltage (decrease in viability by 1.3 log CFU/mL) and drying (decrease in viability by 2.1 log CFU/mL) on bacterial survival after electrospinning. In addition, we found that bacteria whose growth was interrupted in the late logarithmic phase compared to the stationary phase survived electrospinning better. PEO nanofibers with an addition of 55.6% of selected stabilizers (sucrose, mannitol, glycerol, and sorbitol) were developed to reduce the negative effects of dehydration during electrospinning on bacteria’s viability. The addition of sorbitol and sucrose resulted in a statistically significant improvement in the survival of bacteria in nanofibers. Moreover, survival of the studied strain in dried polymer films and lyophilizates was compared to survival in nanofibers immediately after their preparation. After both drying processes, bacterial survival was better compared to electrospinning. The results of the master\u27s thesis represent a systematic study of the effects of various parameters on the survival of bacterial strain 27.3.S during their incorporation into PEO nanofibers by electrospinning, which is crucial for the design of advanced delivery systems with sensitive biological components such as probiotics