Protimikrobne medicinske tekstilije na osnovi hitozanskih nanodelcev za ginekološko zdravljenje

The aim of this dissertation was to develop a novel medical tampon for alternative gynaecological treatment using chitosan nanoparticles as an antimicrobial agent or as a drug delivery system. For this purpose viscose tampon band was used and functionalized with chitosan and trimethyl chitosan nanoparticles. A comprehensive of interactions between chitosan and cellulose as well as characterization of prepared materials were done. At the beginning, chitosan (CS) and trimethyl chitosan (TMC) solutions, as well as nanoparticles synthesised by ionic gelation were studied. Their characterization was focused on determining the charge and antimicrobial properties against common pathogenic microorganism. The influence of cationic charge on the inhibition of microbial growth was confirmed. Since CS and TMC solutions and nanoparticles dispersions exhibited antibacterial activity against Lactobacillus, a detailed investigation in chitosan’s antimicrobial mode of action was performed using a novel diffusion nuclear magnetic resonance (D-NMR). D-NMR allowed the monitoring of intra- and extracellular water exchange from the cells indicating the membrane alteration and leakage of intracellular constituencies. Further, in order to study the adsorption phenomena and molecular interactions between CS/TMC (solution or nanoparticles) and cellulose material, model cellulose surfaces were used, and adsorption was studied by quartz crystal microbalance with dissipation. CS and TMC were favourably deposited onto cellulose model surface at higher ionic strength, higher pH values, i.e. factors causing lower solubility, where the presence of electrostatic interactions was negligible and non-electrostatic interactions were dominant. The knowledge gained from the model surfaces was extremely helpful in characterization of real systems, i.e. functionalized cellulose fibres and for understanding the obtained results. Immobilization of CS and TMC (in the form of solution and/or nanoparticles) onto cellulose viscose fibres was confirmed with several analytical methods. The attachment of chitosan onto fibres was reversible, as endorsed with desorption studies mimicking the conditions of vaginal usage. Evaluation of antimicrobial properties was performed using two different techniques, both revealing a high inhibition of the tested microorganism. In addition, Lactobacillus susceptibility testing has shown that chitosan-coated fibres do not have any negative influence on the resident microbiota. Assessment of in-vitro cytotoxicity demonstrated that samples do not cause a cytotoxic effect in direct contact. Additionally, model drug was incorporated into chitosan nanoparticles and subsequently attached onto fibres in order to create modern, vaginal drug delivery systems. Antimicrobial medical textiles investigated in the scope of this dissertation show the potential for their exploitation in gynaecological field as preventive or curative treatment without triggering any adverse effects for the user.Cilj predstavljene doktorske disertacije je razvoj medicinskega tampona za alternativno ginekološko zdravljenje z uporabo hitozanskih nanodelcev, ki bodo delovali kot protimikroben agent ali kot dostavni sistemi za zdravilne učinkovine. V ta namen smo uporabili viskozni tamponski trak, ki smo ga funkcionalizirali s hitozanskimi in trimetil hitozanskimi nanodelci. Sledila je poglobljena karakterizacija pripravljenih materialov. Na začetku smo analizirali raztopine hitozana (CS) in trimetil hitozana (TMC), kakor tudi nanodelce sintetizirane s postopkom ionskega geliranja. Njihova karakterizacija je bila usmerjena predvsem v določitev naboja in protimikrobne aktivnosti na najpogostejše patogene mikroorganizme. Vpliv prisotnosti pozitivnega naboja na inhibicijo rasti mikrobov je bila potrjena. Ker so CS in TMC raztopine ter disperzije nanodelcev pokazale protimikrobno delovanje na laktobacile, je bila izvedena podrobna študija mehanizma protimikrobnega delovanja hitozana z uporabo nove, difuzijske nuklearne magnetne resonance (D-NMR). S tem smo lahko spremljali intra- in ekstracelularno izmenjavo vode v celicah, ki kaže na poškodbo celične membrane in izhajanje celularnih komponent. Nadalje smo z namenom, da bi raziskali pojav adsorpcije in molekulskih interakcij med CS/TMC in celulozo, uporabili celulozne modelne filme, na katerih smo z uporabo kremenove mikrotehnice spremljali adsorpcijo. CS in TMC sta se na celulozne filme prednostno odlagala pri višjih ionskih jakostih in višjih pH vrednostih, t.j. karakteristikah, ki vodijo v manjšajo topnost polimera, kjer je prisotnost elektrostatskih interakcij zanemarljiva in prevladujejo ne-elektrostatske interakcije. Ugotovitve pridobljene na modelnih površinah so bile izjemno koristne za prenos na realne sisteme, kakor tudi pri karakterizaciji funkcionaliziranih vlaken in dobljenih rezultatov. Imobilizacija CS in TMC (v obliki raztopine in/ali nanodelcev) na celulozna vlakna je bila potrjena s številnimi analiznimi metodami. Vezava hitozana na vlakna se je izkazala za reverzibilno, kar smo ugotovili s spremljanjem desorpcije v simuliranih pogojih vaginalne uporabe. Evaluacija protimikrobnih lastnosti je bila izvedena z dvema mikrobiološkima tehnikama, ki sta obe pokazali učinkovito inhibicijo testiranih mikroorganizmov. Testiranje občutljivosti laktobacilov na vlakna obdelana s hitozanom ni pokazalo negativnega učinka na normalno vaginalno mikrobioto. Obdelani materiali prav tako niso pokazali citotoksičnega efekta pri testiranju citotoksičnosti v direktnem kontaktu. Hitozanske nanodelce, v katere smo pripeli modelno zdravilo, smo naknadno nanesli na celulozna vlakna z namenom kreiranja modernih, vaginalnih dostavnih sistemov. Protimikrobne medicinske tekstilije raziskane v sklopu te disertacije kažejo potencialno uporabo na ginekološkem področju, kot preventiva ali kurativa, brez neželenih stranskih učinkov za uporabnika