Razvoj terapevtskih nanovlaken in vrednotenje njihove imunogenosti in vitro

Zidar, Anže (avtor), Kristl, Julijana (mentor), Jeras, Matjaž (komentor)

V disertaciji smo se osredotočili na razvoj nanovlaken in vrednotenje njihovega vpliva na humane imunske celice in vitro. Konvencionalne metode za in vitro testiranje nanovlaken na celicah, še posebej imunskih, izkazujejo slabo ponovljivost. Za premostitev te ovire smo razvili in optimizirali nov celični model in vitro, ki omogoča boljšo ponovljivost od dosedanjih, saj zagotavlja standardizirano testno površino, izključuje robove preskušanih membran iz nanovlaken ter omogoča neprekinjen stik med celicami in nanovlakni. Fizikalno kemijske lastnosti nanovlaken izdelanih iz polikaprolaktona (PCL) ter kombinacij alginata s polietilenoksidom (ALG/PEO2MDa), hitosana s polietilenoksidom (CS/PEO900kDa) in zeina s polivinilpirolidonom (zein/PVP) smo preučevali na mononuklearnih imunskih celicah iz periferne krvi (PBMC). Ugotovili smo, da ima na PBMC največji vpliv debelina membrane nanovlaken, sledita pa ji velikost por v membrani ter premer nanovlaken. Na dozorevanje dendritičnih celic so najbolj vplivale membrane nanovlaken iz PCL in CS/PEO900kDa, tem so sledile tiste, izdelane iz kombinacije zein/PVP, za najbolj nevtralna pa so se v tem smislu izkazala nanovlakna ALG/PEO2MDa. V nadaljevanju smo v membrane, izdelane iz izbranih nanovlaken ALG/PEO2MDa, vgradili liposome s simvastatinom kot protivnetno učinkovino in antioksidantom butiliranim hidroksianizolom, s čimer smo izboljšali topnost in stabilnost simvastatina. Ta nanodostavni sistem je zagotovil podaljšano sproščanje učinkovine v terapevtskem območju, dober varnostni profil in učinkovitost pri zaviranju prekomernega vnetja in vitro. Poleg tega smo razvili tudi različne membrane izdelane tako iz nanovlaken PEO kot iz kombinacije ALG/PEO, v katere smo že tekom elektrostatskega sukanja vgradili spore probiotičnih sevov Bacillus 25.2.M in 27.3.Z. Pri tem smo dokazali, da imajo bakterijske spore enak vpliv na PBMC kot proste bakterije ter da se njihova imunomodulatorna funkcija ne spremeni kljub izpostavljenosti elektrostatskemu sukanju. Naše raziskave prispevajo k razumevanju interakcij različnih vrst nanovlaken z imunskimi celicami in ponujajo nov, zanesljiv model in vitro preverjanja učinkovitosti in varnosti nanovlaken za biomedicinsko uporabo.

• Jezik: Slovenski jezik
• Ključne besede: Nanovlakna, imunske celice, in vitro model, mononuklearne celice periferne krvi (PBMC), elektrostatsko sukanje, fizikalno-kemijske lastnosti, simvastatin, bakterijske spore, imunomodulacija, dendritične celice
• Vrsta gradiva: Doktorsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-166799
• Datum objave v RUL: 25.01.2025

ZIDAR, Anže, 2025, Razvoj terapevtskih nanovlaken in vrednotenje njihove imunogenosti in vitro [na spletu]. Doktorska disertacija. [Dostopano 7 april 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=166799

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Development of therapeutic nanofibers and in vitro evaluation of their immunogenicity

Izvleček

In the dissertation, we focused on the development of nanofibers and the evaluation of their impact on immune cells in vitro. Conventional in vitro methods for nanofiber cells assays, especially with immune cells, showed poor reproducibility. To overcome this obstacle, we developed and optimized a new in vitro cell model that provides better reproducibility than conventional ones by ensuring a standardized test surface, excluding the edges of the tested nanofiber membranes, and allowing continuous contact between the cells and the nanofibers. The physicochemical properties of nanofibers made from polycaprolactone (PCL), alginate with polyethylene oxide (ALG/PEO2MDa), chitosan with polyethylene oxide (CS/PEO900kDa), and zein with polyvinylpyrrolidone (zein/PVP), were studied on peripheral blood mononuclear cells (PBMC). We found that the thickness of the nanofiber membrane had the greatest impact on PBMC, followed by interfibrillar pore size and nanofiber diameter. The maturation of monocyte derived dendritic cells was most affected by nanofiber membranes made of PCL and CS, followed by zein, and ALG nanofibers proved to be the most neutral in this regard. Furthermore, we incorporated liposomes containing simvastatin into ALG nanofibers, which improved the solubility and stability of simvastatin. This nano delivery system provided an extended-release profile of simvastatin within the therapeutic range, a good safety profile, and effectiveness in inhibiting excessive inflammation in vitro. Additionally, we developed nanofiber mats from ALG and/or polyethylene oxide, into which we incorporated spores of Bacillus strains 25.2.M and 27.3.Z during electrospinning. We demonstrated that bacterial spores had the same impact on PBMCs as free bacteria and that their bacterial immunomodulatory function did not change during electrospinning. Our research offers a new, reliable in vitro model for testing nanofiber efficacy and safety, contributes new insights into the interactions between nanofibers and immune cells and paves the way for the preparation of safe and effective nanofiber formulations.

• Ključne besede: Nanofibers, immune cells, in vitro model, peripheral blood mononuclear cells (PBMC), electrospinning, physicochemical properties, simvastatin, bacterial spores, immunomodulation, dendritic cells