Development of core-shell nanofibers for innovative periodontal disease treatment

Parodontalna bolezen je zelo razširjena bolezen, zahtevna za zdravljenje zaradi številnih dejavnikov, ki vplivajo na njen razvojtrenutno rapoložljivi terapevtski pristopi pa običajno vodijo le v kratkotrajno ozdravitev. Namen doktorske naloge je bil razvoj novih nanodostavnih sistemov za inovativno lokalno zdravljenje parodontalne bolezni, ki temelji na vplivanju na vnetni odziv organizma, na antibiotičnem zdravljenju in vzpostavitvi normalne ustne flore. Signifikanten korak naprej smo naredili k razumevanju vgrajevanja hidrofilnih in lipofilnih učinkovin in njihove kristalizacije med izdelavo različnih eno in dvoslojnih nanovlaken. V dvoslojna nanovlakna iz polikaprolaktona in polimetilmetaakrilata smo uspešno vgradili metronidazol in ciprofloksacin in dokazali 7- ali več dnevno podaljšano sproščanje, kar je pomembno za uspešno zdravljenje parodontalne bolezni. S sistematičnim proučevanjem številnih parametrov smo ugotovili, da na kinetiko sproščanja učinkovine iz nanovlaken vplivajo izbrani polimeri, vsebnost in fizikalno-kemijske lastnosti učinkovine, kvantitativna sestava in premer nanovlaken, razmerje med pretokoma raztopin za sredico in ovojnico dvoslojnih nanovlaken, ter debelina plasti nanovlaken, ki je bila do sedaj pogosto spregledana lastnost. Dodatno smo na podlagi eksperimentalnih podatkov z matematičnimi modeli dokazali mehanizem sproščanja učinkovin iz razvitih hidrofilnih in hidrofobnih nanovlaken, pri čemer sta difuzija in erozija pomembna procesa pri sproščanju iz hidrofilnih nanovlaken in eno- ter dvostopenjska desorpcija pri hidrofobnih. Eden od najobetavnejših razvitih lokalnih dostavnih sistemov so dvoplastna nanovlakna z metronidazolom in ciprofloksacinom. Njihova natančno zasnovana sestava iz biorazgradljivih, bioadhezivnih in biokompatibilnih polimerih je omogočila in vitro podaljšano sproščanje teh dveh protimikrobnih učinkovin več kot 12 dni nad minimalno inhibitorno koncentracijo za Aggregatibacter actinomycetemcomitans, kar smo dokazali z novo razvito pretočno in vitro napravo, ki simulira stanje v obzobnem žepu, idealnem mestu za lokalno zdravljenje parodontalne bolezni. Glede na razpoložljive podatke iz literature smo kot prvi razvili nanovlakna s probiotiki, izoliranimi iz ustne flore zdravih prostovoljcev, za lokalno aplikacijo z namenom preprečevanja napredovanja ali ponovitve parodontalne bolezni. Preživelost probiotikov v obliki spor je bila izjemna, tako tekom priprave nanovlaken kot tudi med 6-mesečnim shranjevanjem pri sobnih pogojih. Za izdelana nanovlakna s probiotiki smo dokazali protimikrobno delovanje proti sevu A. actinomycetemcomitans in že z aplikacijo 10 mg teh nanovlaken z 107 CFU/mg bi predvidoma dosegli terapevtski odmerek v obzobnem žepu. To delo predstavlja razvoj različnih inovativnih formulacij nanovlaken s protivnetno, protimikrobnima učinkovinama in probiotiki za lokalni vnos v obzobne žepe, kar obeta izboljšanje trenutnega zdravljenja parodontalne bolezni.Periodontal disease is a widespread condition that is difficult to treat due to several factors influencing its development. The currently accepted therapeutic approaches usually lead only to short-term recovery. The aim of this doctoral thesis was the development of novel nanodelivery systems for innovative local treatment of periodontal disease, based on the modulation of the host immune response, antimicrobial treatment and restoration of normal oral flora. A significant step forward has been made in the understanding of the incorporation and crystallization of hydrophilic and lipophilic drugs during the production of a number of monolithic and core-shell nanofibers. We successfully incorporated metronidazole and ciprofloxacin into core-shell nanofibers from polycaprolactone and poly(methyl methacrylate), compared them with monolithic nanofibers and confirmed 7+ days of prolonged drug release, which is the duration of suggested treatment for periodontal disease. The important findings for drug release kinetics from nanofibers depend on varying several parameters that relate to the following: the selected polymers, drug loading, physico-chemical drug properties, nanofiber composition and diameter, the flow rate ratio between core and shell solution, and the thickness of the nanofiber mat, a parameter often overlooked previously. Mathematical models were used to determine the mechanism of drug release from the hydrophilic and hydrophobic nanofibers that were developed, pointing out that diffusion and erosion are important processes in drug release from hydrophilic nanofiber mats, whereas, single and two-stage desorption is crucial in drug release from hydrophobic ones. The local delivery system with the highest potential that was developed was the double layer nanofiber mat with metronidazole and ciprofloxacin. Its precisely designed composition based on biodegradable, bioadhesive, and biocompatible polymers enabled in vitro prolonged release of the two antimicrobials over 12 days above the minimal inhibitory concentration for Aggregatibacter actinomycetemcomitans. The release was evaluated in a newly-developed flow-through in vitro apparatus that simulated conditions in a periodontal pocket, the ideal site for local treatment. To prevent progression or recurrence of periodontal disease, an innovative concept of nanofibers with new probiotics, isolated from the oral flora of healthy volunteers, was introduced for the first time to best of our knowledge. The survival of probiotics as spores was remarkable both during nanofiber preparation and over a 6-month storage period at room temperature. The novel nanofibers with probiotics had an antimicrobial activity against A. actinomycetemcomitans and were anticipated to reach therapeutic levels in the periodontal pocket by using only 10 mg of nanofibers with 107 CFU/mg. To conclude, this work represents the development of innovative delivery systems i.e. nanofiber mats loaded with anti-inflammatory drug, antimicrobials and probiotics for local administration into periodontal pockets, which holds much promise for improving current periodontal disease treatment