Izdelava in vrednotenje nanovlaken iz polietilenoksida in alginata z vgrajenimi sporami potencialnega probiotika rodu Bacillus za lokalno zdravljenje parodontalne bolezni

Presinger, Ana (author), Kristl, Julijana (mentor), Zupančič, Špela (comentor)

Probiotiki so za ohranjanje človeškega zdravja izredno pomembni, v zadnjem času pa se raziskujejo za zdravljenje nekaterih bolezni, med katere uvrščamo tudi parodontalno bolezen. Pri tem pa velik izziv predstavlja razvoj lokalnega dostavnega sistema, ki bi omogočal vgradnjo velikega števila probiotikov, ohranjal njihovo preživetje med shranjevanjem in na mestu aplikacije nudil učinkovito kolonizacijo. V okviru magistrske naloge smo zato z metodo elektrostatskega sukanja razvili nanovlakna in vanje vgradili spore potencialnega probiotičnega seva Bacillus sp. 25.2.M ter jih podrobno vrednotili. Po začetni optimizaciji formulacijskih, procesnih in okoljskih parametrov smo izdelali nanovlakna iz polietilenoksida (PEO) in kompozitna nanovlakna iz PEO ter alginata v masnem razmerju 60/40 in 20/80 (m/m), v katera smo nato vgradili spore (7,2×105–7,8×106 spor/mg nanovlaken). Njihovo uspešno vgradnjo smo potrdili z vrstičnim elektronskim mikroskopom (SEM) in hkrati ugotovili, da so imela nanovlakna s sporami povprečni premer (164–212 nm) značilno večji od premera enakih nanovlaken brez spor (127–172 nm). Analiza z diferenčno dinamično kalorimetrijo je pokazala, da je elektrostatsko sukanje zmanjšalo kristalnost PEO v vseh vzorcih nanovlaken v primerjavi z izhodnim polimerom. S Fourierjevo transformacijsko infrardečo spektroskopijo smo dokazali nastanek vodikovih vezi med PEO in alginatom v nanovlaknih, na interakcije med polimeri in sporami pa na podlagi spektrov ne moremo sklepati. Spremljali smo preživetje spor med izdelavo nanovlaken in ugotovili, da mehanski stres, aplicirana napetost in osmozne spremembe zmanjšajo preživetje spor za največ 1 logaritemsko enoto. Po 1-mesečnem shranjevanju pri sobni temperaturi, 58 % relativni vlažnosti (% RH) in 40 °C, 75 % RH ni prišlo do statistično pomembnega zmanjšanja števila živih spor. S povečevanjem deleža alginata v nanovlaknih je padala hitrost sproščanja spor v fosfatni pufer, alginat pa je zakasnil sproščanje spor in razraščanje bakterij tudi na trdnem gojišču. Izdelana nanovlakna imajo potencial za lokalno dostavo probiotikov v obzobni žep. So iz biokompatibilnih in bioadhezivnih polimerov, omogočajo vgradnjo velikega števila živih probiotičnih spor ter zagotavljajo njihovo stabilnost tudi med shranjevanjem.

• Language: Slovenian
• Keywords: parodontalna bolezen, nanovlakna, elektrostatsko sukanje, alginat, probiotiki
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-121533
• Publication date in RUL: 14.10.2020

Secondary language

• Language: English
• Title: Formulation and characterisation of polyethylene oxide and alginate nanofibers loaded with potential probiotic spores of the Bacillus genus for local treatment of periodontal disease

Abstract

Probiotics are very important for maintaining human health, whereas in the last years they are extensively studied for the treatment of various diseases, including periodontal disease. However, a challenge is to develop delivery system that would enable the incorporation of large number of probiotics, maintain their survival during storage, and offer effective colonization at the site of application. Therefore, the aim of this master thesis is development and characterisation of nanofibers with spores of the potential probiotic strain Bacillus sp. 25.2.M using the electrospinning method. After the initial optimization of the formulation, process and environmental parameters, we produced nanofibers from polyethylene oxide (PEO) and composite nanofibers from PEO and alginate in weight ratio of 60/40 and 20/80 (m/m) with incorporated spores (7.2×105–7.8×106 spores/mg nanofibers). Their successful incorporation was evaluated using scanning electron microscopy (SEM). The average diameter of spore-loaded nanofibers (164–212 nm) was significantly bigger compared to the diameter of the same nanofibers without spores (127–172 nm). Analysis by differential scanning calorimetry showed that electrospinning reduced the crystallinity of PEO in all nanofibre samples relative to the polymers used. Fourier transform infrared spectroscopy demonstrated the formation of hydrogen bonds between PEO and alginate in nanofibers, whereas no interactions between polymers and spores could be inferred from the spectra. The survival of spores during nanofiber production was reduced for maximum of one logarithmic unit probably due to mechanical stress, applied pressure, and osmotic changes. After one-month storage at room temperature, 58 % relative humidity (RH) and at 40 °C, 75 % RH did not show a statistically significant decrease in the number of viable spores. As the proportion of alginate in nanofibers increased, the rate of spore release into phosphate buffer decreased, and alginate delayed the release of spores and the growth of bacteria even on solid agar plates. The developed nanofibers demonstrate the potential for local delivery of probiotics to the periodontal pocket. They are from bioadhesive and biocompatible polymers, enable the incorporation of a large number of viable probiotic spores, and ensure their stability during storage.

• Keywords: periodontal disease, nanofibers, electrospinning, alginate, probiotics