Delineation of physical methods and carriers for dermal drug delivery enhancement

Koža kot največji in lahko dostopen človeški organ predstavlja za vnos zdravilnih učinkovin privlačno alternativo drugim načinom vnosa molekul v telo. Ker pa je poglavitna funkcija kože zaščita in obramba telesa pred vplivom zunanjega makro in mikro okolja, je pasivni prehod molekul skozi kožo praktično nemogoč. Za zagotovitev uspešnega vnosa molekul skozi kožo in/ali v celice kože je potrebno skrbno izbrati metodo ali kombinacijo metod pospeševanja dermalnega in transdermalnega vnosa in jo/jih optimizirati za določeno ciljno aplikacijo. Glavni namen in cilj raziskovalnega dela je bil razvoj učinkovite kombinacije metod za pospešitev vnosa molekul skozi in v kožo ter primerjava le-teh. Osredotočila sem se na tri različne fizikalne pristope pospešitve vnosa: i) Elektroporacija: povečanje prehodnosti zunanje rožene plasti kože (stratum corneum), in/ali membran celic v nižjeležečih plasteh kože ob izpostavitvi kožnega tkiva električnim pulzom, zaradi lokalnih sprememb v mikrostrukturi lipidov celičnih membran. Te spremembe so odvisne od amplitude, trajanja in števila pulzov in so lahko velikosti reda nekaj nano do mikrometrov, pa vse tja do večjih lokalnih transportnih področij (LTR – angl.: local transport regions) velikosti nekaj 100 mikrometrov. Pri električno nabitih molekulah lahko z električnimi pulzi dodamo tudi potisno silota pojav imenujemo elektroforezaii) Laserska mikroablacija: izpostavitev kože laserskemu žarku z namenom ustvarjanja mikro-kanalčkov v roženi plastiiii) Ultrazvok: uporaba nizkofrekvenčnega ultrazvoka (20-40 kHz) z namenom ustvarjanja prehodnih poti v roženi plasti kože zaradi kavitacije (sonoporacija), termalnih učinkov in mehanskega potiska (sonoforeza). Optimiziranim protokolom fizikalnih metod pospešitve vnosa smo dodali primerjavo dermalnega vnosa molekul, vgrajenih v nano-dostavne sisteme (liposome in etosome) in tako združili uporabo kombinacij različnih fizikalnih pristopov in dostavnih sistemov. V prvem delu doktorske naloge je delo potekalo in vitro na dermatomirani koži prašičjih ušes v dvoprostornem sistemu t.i. Franz-ovih difuzijskih celic, kjer smo preizkusili uspešnost pospešitve vnosa modelne molekule kalceina z elektroporacijo. Osredotočili smo se na primerjavo različnih kombinacij pravokotnih električnih pulzov, in sicer kratkih visokonapetostnih (angl. high voltage – HV) in dolgih nizkonapetostnih (angl. low voltage – LV) pulzov. Ugotovili smo, da več dolgih LV pulzov bistveno poveča kasnejši pasivni transport kalceina skozi kožo, medtem ko je učinkovitost pospešitve kalceina pri uporabi kratkih HV pulzov zanemarljivo majhna. Dokazali smo tudi, da kratki HV pulzi, ki jim sledijo daljši pulzi, transport molekule preko kože v primerjavi s samo dolgimi LV pulzi, bistveno zmanjšajo. To dokazujemo z rezultati numeričnega modela, ki temelji na razvoju lokalnih transportnih regij (LTR). V nadaljni študiji smo z elektroporatorjem »Green skin pore«, s katerim smo v kožo vnašali barvilo patent blue in le-tega kasneje v histoloških preparatih dokazali z mikroskopijo, da lahko z elektroporacijo zagotovimo enakomeren vnos in porazdelitev molekul predvsem v zgornjih plasteh kože. Metoda elektroporacije je tako primerna za dermalni vnos molekul in za aplikacije pri katerih moramo zagotoviti vnos molekul ne samo v kožno tkivo temveč v notranjost celic. V nadaljevanju smo na enakem eksperimentalnem modelu z lasersko mikroablacijo zgornjih plasti kože s pomočjo laserja Er:YAG primerjali različne dolžine laserskih pulzov pri konstantni ali variabilni energiji in njihovo uspešnost pospešitve transdermalnega vnosa treh različno velikih FITC dekstranov (4 kDa, 10 kDa in 20 kDa). Ugotovili smo, da energija dovedenih pulzov večinoma narekuje velikost/globino lasersko povzročenih mikrokanalčkov, medtem ko dolžina laserskih pulzov narekuje predvsem obseg termično spremenjenega tkiva. Spremembe parametrov laserskih pulzov imajo različne vplive na molekule različnih fizikalno-kemijskih lastnosti, zato je potrebna optimizacija protokolov laserskih pulzov za vsako molekulo in medicinsko aplikacijo posebej. V nadaljni študiji smo prešli na ex vivo model celotne debeline prašičje kože, s čemer smo se bolj približali in vivo pogojem. Razvili in optimizirali smo elektroporacijske (dovajanje enosmernih električnih pulzov) in sonoporacijske (dovajanje nizkofrekvenčnega ultrazvoka) protokole in raziskali uspešnost kombinacij metod za pospeševanje transdermalnega vnosa molekul. Rezultati so pokazali statistično signifikantno povečanje vnosa kalceina v kožo že z uporabo 6x100 kratkih visokonapetostnih električnih pulzov (amplitude 200 V in trajanja 100 μs), ali po 5 minutah izpostavitve nizkofrekvenčnemu ultrazvoku. Kasneje smo z željo, da bi dosegli sinergistični efekt metodi uporabili v kombinaciji, tako da je ena neposredno sledila drugi. Rezultati so nas presenetili, saj vnos molekule pri kombinaciji obeh metod skupaj ni bil večji od vnosa molekule pri vsaki metodi posebej. Mehanizem pospešitve vnosa pri obeh metodah temelji na ustvarjanju novih transportnih poti preko rožene plasti, pri čemer je pri kombinaciji obeh (ne glede na vrstni red) druga metoda neuspešna pri tvorbi dodatnih transportnih poti v koži. Zaradi že obstoječih transportnih poti ustvarjenih s prvo metodo je namreč koža spremenjena do te mere da so začetni pogoji ob uporabi druge metode drugačni in neugodni za nastanek novih transportnih poti. V zadnjem delu doktorske naloge smo poleg razvitih protokolov preverjali uspešnost pospešitve transdermalnega vnosa molekul kalceina, vgrajenih v nano-dostavne sisteme. Liposomi so se izkazali kot uspešni pospeševalci vnosa kalceina v epidermis kože, kjer služijo kot nekakšen rezervoar učinkovine. Po drugi strani so se etosomi izkazali kot izredno uspešni dermalni in transdermalni pospeševalci vnosa, ki lahko dostavijo molekule tudi v nižje predele kože. Z raziskovalnim delom smo s kombinacijo teoretičnega in eksperimentalnega pristopa dodatno osvetlili mehanizme delovanja vseh treh fizikalnih metod pospešitve dermalnega in transdermalnega vnosa posebej in nekaterih njihovih kombinacij. Znanje, pridobljeno na omenjenem področju predstavlja pomemben prispevek k znanosti in bo pripomoglo k nadaljnemu razvoju novih načinov vnosa molekul v telo preko kože.The skin is the largest and most easily accessible organ of the human body and transdermal drug delivery represents an attractive alternative over other drug administration routes. Since one of the most important functions of the skin is the protection of the body against the influence of the external macro and micro environment, passive molecular transport through the skin is practically impossible. Quite a few enhancement techniques have been developed so far to overcome the stratum corneum barrier and to facilitate transdermal drug delivery and they include various passive (penetration enhancers, liposomes) and active approaches (electroporation, iontophoresis, microneedles). However, to ensure successful transport of molecules through the skin or into the skin cells it is necessary to carefully choose the method or their combination and to optimize it for a specific target application. The main purpose of my research work was gaining new insights into the mechanisms and the underlying physics of some of the transdermal enhancement methods, as well as to compare them and use them in combination where appropriate. Our focus was on three different physical enhancement methods: i) Electroporation: increases the permeability of the outermost layer of the skin (stratum corneum), and/or cell membranes in lower skin layers when exposed to electric pulses, due to local changes in the microstructure of the cell membrane lipids. These changes depend on the magnitude, duration and number of pulses and may be the size of a few nano to micrometers, also up to a few 100 micrometers (local transport regions – LTR). For electrically charged molecules, electric pulses also provide driving force to push molecules into the skin (electrophoresis)ii) Laser microablation: the exposure of the skin to a fractional laser beam in order to create micro-channels in the stratum corneumiii) Ultrasound: the use of low-frequency ultrasound (20-40 kHz) in order to increase skin permeability by creating aqueous pathways in the skin by cavitation (sonoporation), skin heating and mechanical effects (sonophoresis). Apart from the physical enhancement methods, we added a passive transdermal enhancement method: the encapsulation of molecules in nano-delivery systems (liposomes and ethosomes), and used combinations of different physical approaches with nano-delivery systems. The first part of the experimental work of my doctoral thesis was carried out in vitro on dermatomed pigs\u27 ear skin, in a two-compartment Franz diffusion cell system, where electroporation was used to enhance transdermal transport of flourecent molecule calcein. We focused on comparison of different combinations of square wave electric pulses using short high voltage pulses (HV) and longer low voltage pulses (LV). Our results show, that the calcein transport through the skin significantly increased using more long LV pulses, while the efficiency of short HV pulses compared to the passive diffusion only was negligibly small. We also showed that the combination of HV pulses, followed by LV pulses do not work in synergy as we hypothesized. Surprisingly, when LV pulses are preceded by HV pulses, calcein delivery was lowered compared to LV pulses alone. We explained the mechanism with a numerical model that is based on the development of local transport regions (LTR). In a further study we were using Green skin pore electroporator for pulse delivery (together with electrodes, both developed exclusively for use on skin) and Patent Blue dye later observed by cryo-histology to assess the distribution of dye in the skin. We have shown that a uniform distribution of the dye can be observed primarily in the upper layers of the skin, when using protocols suitable for possible clinical use. We conclude that electroporation is suitable for the dermal molecular delivery and also for the applications that need to ensure the introduction of molecules not only in the skin tissue but also inside the cells. Further, using the same experimental system, Er:YAG laser with fractional output beam profile was used as enhancement method for transdermal drug delivery of three model molecules of different sizes: FITC-dextrans with average molecular weights of 4 kDa, 10 kDa and 20 kDa. The Er:YAG laser is used for controlled removal of the thin dead outer layer of the skin, the stratum corneum, taking advantage of the ablative effects of laser light on tissue while causing minimal thermal damage, thus sparing viable underlying layers (epidermis, dermis). Further, laser beam of fractional lasers is split into microbeams, so even larger portion of viable skin tissue is spared. First, we used protocols among which pulse duration was varied while keeping pulse energy constant (shorter pulses of equal energy mean higher peak power). Following that, we kept the duration of the pulses constant while varying their energy. We showed that the energy of the delivered pulses is the most important parameter for the size/depth of the microchannels, while differing pulse duration/power dictates the extent of thermally-altered tissue and with that the partitioning of the permeant into tissue. Also, laser pulse parameters have different impacts on the molecules of different sizes and physico-chemical properties, therefore each molecule and each application requires optimization of the laser protocols. Further, we introduced a different experimental system using ex vivo full thickness pigs\u27 ear skin, in order to move one step closer to the potential in vivo conditions. We developed and optimized electroporation (application of square wave electric pulses) and sonoporation (application of low-frequency ultrasound) protocols and investigated the effectiveness of a combination of methods to further enhance transdermal molecular transport. The results showed a statistically significant increase in calcein transport into the skin using already 6x100 short high voltage electrical pulses (amplitude of 200 V and duration 100 μs), or 5 minutes of low-frequency ultrasound exposure. Later, to achieve a synergistic effect, the combination of the methods was delivered to skin so that one method is directly followed by the other one. Surprisinglly, the results showed no evident improvement over a single method. The mechanism of action of both methods is the creation of aqueous pathways in the stratum corneum leading to increased skin permeability. However, when used in combination (regardless of the order of methods), the second method was unsuccessful in adding many new aqueous pathways in the stratum corneum, as it acted preferentially near the sites of the existing ones. In the last part of the doctoral thesis, we used previously developed protocols to verify the transdermal enhancement efficay of calcein encapsulated in two different nano-delivery systems. Liposomes showed to be a successfull dermal enhanceres of the calcein, where they can serve as a sort of reservoir of the molecules. On the other hand, the ethosomes proved to be extremely successful as dermal and transdermal molecular enhancers and can deliver the molecules also into the lower skin layers. Our research work contains the combination of theoretical and experimental approaches to shed light on the mechanisms of three physical dermal and transdermal drug delivery enhacement methods used separately and some in combination, as well as a passive approach: different encapsulation techniques. The knowledge gained in this field represents new knowledge that will further contribute to development of new methods for dermal and transdermal drug delivery enhancement tehniques