Izdelava in vrednotenje nanovlaken iz polietilenoksida in hitosana z vgrajenimi sporami probiotika rodu Bacillus

Kumar, Jan (author), Kristl, Julijana (mentor), Zupančič, Špela (comentor)

Človeška mikrobiota ima pomembno vlogo pri ohranjanju človeškega zdravja. Disbioza, s čimer poimenujemo spremembe v sestavi mikrobiote in sočasno porušitev homeostaze med človekom in mikroorganizmi, pa lahko vodi v nastanek bolezni, ki bi jih lahko potencialno zdravili s probiotiki. Za razliko od peroralne uporabe, lokalni vnos probiotikov omejuje pomanjkanje dostavnih sistemov, ki bi ohranili njihovo preživetje, se dovolj dolgo zadržali na tarčnem mestu in nudili učinkovito kolonizacijo. V okviru magistrske naloge smo zato z metodo elektrostatskega sukanja izdelali nanovlakna ter vanje vgradili spore modelnega potencialnega probiotičnega seva Bacillus sp. 25.2.M. Z optimizacijo formulacijskih, procesnih in okolijskih parametrov smo pripravili nanovlakna iz polietilenoksida (PEO) in kompozitna nanovlakna iz PEO in hitosana v razmerjih 60/40 ter 20/80 (m/m), v katera smo uspešno vgradili spore (2,7×105–7,3×105 spor/mg nanovlaken). Tekom priprave nanovlaken smo opazili upad preživetja spor, ki je bil izrazitejši v primeru nanovlaken s hitosanom, kar je posledica nizkega pH raztopine in ostrejših pogojev elektrostaskega sukanja. Z vrstičnim elektronskim mikroskopom smo potrdili vgradnjo spor v nanovlakna in določili signifikantno razliko v povprečnem premeru nanovlaken s sporami (167–342 nm) in enakih vlaken brez spor (110–177 nm). Z diferenčno dinamično kalorimetrijo smo potrdili manjšo kristaliničnost PEO v vseh pripravljenih nanovlaknih v primerjavi z izhodnim polimerom. S Fourierjevo transformacijsko infrardečo spektroskopijo smo potrdili tvorbo vodikovih vezi med hitosanom in PEO v nanovlaknih, medtem ko tvorba interakcij med sporami in polimeri ni bila enoznačno razvidna. S povečanjem deleža hitosana v nanovlaknih sta se zmanjševala hitrost in obseg sproščanja spor v fosfatni pufer, podaljševal pa se je čas, potreben za kalitev spor in izraščanje kolonij iz nanovlaken na trdnih gojiščih. Po 1 mesecu shranjevanja nanovlaken pri sobni temperaturi in 58 % relativni vlagi ter 40 °C in 75 % relativni vlagi se vsebnost živih spor v nanovlaknih ni zmanjšala za več kot 0,32 log CFU/mg. Razvita nanovlakna predstavljajo obetaven pristop za lokalno zdravljenje z disbiozo povezanih bolezni, saj so biorazgradljiva, omogočajo vgradnjo velikega števila potencialnih probiotikov in ohranjajo njihovo viabilnost med shranjevanjem.

• Language: Slovenian
• Keywords: disbioza, probiotiki, nanovlakna, elektrostatsko sukanje, polietilenoksid, hitosan
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-117067
• Publication date in RUL: 23.06.2020

Secondary language

• Language: English
• Title: Formulation and characterisation of polyethylene oxide and chitosan nanofibers loaded with probiotic spores of the Bacillus genus

Abstract

Human microbiota plays an important role in maintaining human health. Dysbiosis, namely changes in composition of microbiota and accompanying breakage of homeostasis between human and microorganisms, may lead to development of several diseases. The latter could be potentially treated with probiotics. Unlike well-established per os application, local application of probiotics is limited due to the lack of delivery systems that could ensure preservation of probiotic viability, sufficient retention at the targeted areas and effective colonisation. Thus, in the scope of master thesis, we produced electrospun nanofibers with incorporated spores of model potential probiotic strain Bacillus sp. 25.2.M. By optimizing formulation, process and environment parameters, polyethyleneoxide (PEO) nanofibers and composite nanofibers from PEO and chitosan in ratio 60/40 or 20/80 (w/w), were developed with subsequent incorporation of spores (2.7×105–7.3×105 spores/mg of nanofibers). During the production of nanofibers, a decrease in spore viability was observed, being the highest for nanofibers with chitosan due to lower pH value of the dispersions and harsher parameters of electrospinning. Using scanning electron microscopy, successful incorporation of spores into nanofibers was confirmed and significant difference in the average diameter observed between spore-loaded nanofibers (167–342 nm) and the same nanofibers without spores (110–177 nm). Results of differential scanning calorimetry showed lower crystallinity of PEO within all nanofibers. Using Fourier transform infrared spectroscopy, formation of hydrogen bonds between chitosan and PEO in nanofibers was confirmed, whereas interactions between spores and polymers could not be observed. Increasing the content of chitosan in nanofibers decreased the rate and extent of spore release from nanofibers in phosphate buffer and increased the time required for spore germination and outgrowth of bacteria from nanofibers in the solid agar. After 1-month storage at room temperature, 58% relative humidity and 40 °C, 75% relative humidity the content of viable spores in nanofibers did not decrease more than 0.32 log CFU/mg. Biodegradability, high loading capacity for potential probiotics and preservation of their viability during storage, demonstrate the developed nanofibers as an innovative approach for potential local treatment of diseases, associated with dysbiosis.

• Keywords: dysbiosis, probiotics, nanofibers, electrospinning, polyethylene oxide, chitosan