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Atopijski dermatitis (AD) je najpogostejša nenalezljiva kronična vnetna bolezen kože. Poleg pravilne/ustrezne in redne nege atopijske kože je zelo pomembna tudi lokalna terapija s protivnetnimi zdravilnimi učinkovinami (ZU). Dostavni sistemi z lamelarno strukturo bi, zaradi svoje podobnosti z medceličnimi lipidi rožene plasti lahko predstavljali ugodno podporno terapijo za bolnike z AD in omogočili podaljšano sproščanje protivnetnih ZU, kot je betametazondipropionat (BDP). V okviru magistrske naloge smo proučevali osem sistemov lamelarnih tekočih kristalov (TK) z različno kvali- in kvantitativno sestavo na osnovi konopljinega ali lanenega olja z vgrajenim BDP, in sicer smo izvedi stabilnostno študijo, sproščanje in vitro ter vrednotenje mikrostrukture. V okviru 10-tedenske pospešene stabilnostne študije smo ugotovili, da je stabilnost vgrajenega BDP odvisna predvsem od deleža vode v posameznih formulacijah. Stabilnost BDP je bila najboljša v formulacijah z 30 % deležem vodne faze, najslabša pa v formulacijah z 80 % deležem vodne faze. Izbira emulgatorja je v manjši meri vplivala na stabilnost BDP, ta je bila nekoliko manjša v vzorcih z Montanov 68 v primerjavi z vzorci s Tween 80. Kinetika razgradnje BDP v lamelarnih TK sledi 1. redu. Z metodo ozkokotnega rentgenskega sipanja smo vrednotili mikrostrukturo vzorcev in potrdili prisotnost lamelarnih mezofaz v vseh vzorcih ter tudi micelarne strukture v sistemih z večjim deležem vode in Montanov 68. Z večanjem deleža vodne faze v vzorcih se je povečevala razdalja med lamelami vzorcev. Mikrostrukturo smo ovrednotili pri različnih temperaturah, pomembnih za dermalno dostavo, tj. 25 °C (sobna temperatura), 32 °C (temperatura površine kože) in 37 °C (telesna temperatura). Temperatura ni imela značilnega vpliva na mikrostrukturo vzorcev, kar pomeni, da se ta ohrani tudi po nanosu formulacije na kožo. Prav tako vgradnja BDP ni vplivala na mikrostrukturo TK. V sklopu sproščanja in vitro, ki smo ga izvedli s pomočjo Franz-ovih difuzijskih celic in Strat-M® membran, smo potrdili podaljšano sproščanje BDP iz vseh osmih formulacij. Na sproščanje BDP je bistveno vplivala mikrostruktura posameznih vzorcev. Iz formulacij z lamelarnimi mezofazami in miceli je bilo sproščanje BDP v 24 h hitrejše in večje ((L/M)Lo80, (L/M)Ko80) kot v ostalih vzorcih. Sproščanje BDP iz formulacij smo primerjali z Beloderm® kremo (vsebnost BDP je enaka kot v TK in znaša 0,64 mg/g formulacije) in potrdili večji obseg sproščanja BDP iz vzorcev TK. Atopic dermatitis (AD) is the most common non-infectious chronic inflammatory skin disease. In addition to suitable and regular care of atopic skin, local therapy with anti-inflammatory drugs is of crucial importance. Due to their similarity to the intercellular lipids of the stratum corneum, delivery systems with lamellar structure could provide a favourable supportive therapy for patients with AD in addition to prolonged release of anti-inflammatory drugs such as betamethasone dipropionate (BDP). Within the scope of this Master's thesis, eight lamellar liquid crystal (LC) systems with different qualitative and quantitative composition based on hemp or flaxseed oil with incorporated BDP was studied, namely a stability study, in vitro release study and microstructure evaluation were performed. As observed within the 10-week accelerated stability study the stability of incorporated BDP depends mainly on the water content in individual formulations. BDP stability was the highest in samples with 30 % aqueous phase and the lowest in formulations with 80% aqueous phase. The choice of emulsifier has a minor effect on the stability of BDP, which was slightly lower in the samples with Montanov 68 compared to the samples with Tween 80. The BDP degradation in lamellar LC systems follows the 1st order kinetics. The microstructure of the samples was evaluated by the small angle X-ray scattering analysis with confirmed lamellar mesophases in all samples in addition to micellar structures in systems with higher water content and Montanov 68. The microstructure was evaluated at different temperatures relevant for dermal delivery, i.e. 25 ° C (room temperature), 32 ° C (skin temperature) and 37 ° C (body temperature). The temperature did not have a significant effect on the microstructure of the samples being preserved following skin application. Also, the incorporation of BDP did not affect the LC systems’ microstructure. Prolonged BDP release from all eight systems tested was observed within in vitro release study using Franz diffusion cells and Strat-M® membranes. The BDP release was significantly influenced by the microstructure of individual samples. From formulations with lamellar mesophases and micelles, the release of BDP was faster and higher ((L / M) Lo80, (L / M) Ko80) after 24h compared to other systems. BDP release from LC systems was compared to Beloderm® cream (with same BDP content, i.e. 0,64 mg/g of formulation) and higher release was observed for the LC systems.

Askorbilpalmitat (AP) je amfifilni derivat vitamina C, ki izkazuje večjo stabilnost in enostavnejše prodiranje v kožo, vendar je njegova stabilnost zaradi hitre oksidacije, nezadovoljiva. Namen magistrskega dela je izboljšati kemijsko stabilnost AP, vgrajenega v lamelarne tekoče kristale za dermalno uporabo. Ovrednotili smo dva različna pristopa za povečanje stabilnosti AP, in sicer dodatek antioksidantov ali povišanje koncentracije AP. V prvem delu smo v okviru preliminarne študije (2 tedna) testirali vpliv dodatka antioksidantov (vitamin C in E, koencim Q10, kvercetin, butilhidroksitoluen, propilgalat), različnih koncentracij AP (5 % in 10 % napram 1 % (m/m)) na stabilnost AP v dveh sistemih tekočih kristalov s konstantnim razmerjem med lipofilno fazo in emulgatorsko zmesjo ob različnem deležu vodne faze (T1 z najmanjšim deležem vode ter T8 z največjim deležem vode). V sisteme z dodanim antioksidantom smo vgradili 1 % (m/m) antioksidanta in 1 % (m/m) AP. Preliminarno stabilnostno študijo smo izvedli v klimatski komori pri 40 °C in 75 % RV, kjer so bili vzorci zaščiteni pred svetlobo, ter vrednotili stabilnost AP s pomočjo tekočinske kromatografije ob času 0 in 2 tedna. Ugotovili smo, da je bil AP najstabilnejši pri obeh višjih koncentracijah in sočasni vgradnji vitamina C v primeru 1 % (m/m) AP. Na podlagi preliminarnih rezultatov smo nadalje zasnovali in izvedli stabilnostno študijo pri višjih koncentracijah AP ali ob dodatku vitamina C v tekočih kristalih z različno kvantitativno sestavo, in sicer v tekočih kristalih z naraščajočim deležem vode (TK1, TK3, TK5 in TK8) in primerjalno v SMES (zmes olja in emulgatorjev brez dodane vode). Vzorce smo shranjevali v klimatski komori pri 40 °C in 75 % RV in določali upad vsebnosti AP s tekočinsko kromatografijo znotraj 8 tednov. Ugotovili smo, da stabilnost AP z nižanjem deleža vode narašča. Najbolj optimalno stabilnost smo po osmih tednih dosegli v TK1 in SMES-u pri višjih koncentracijah AP ali ob dodatku antioksidanta vitamina C. Ker lahko večji delež vgrajenega AP ali dodatek antioksidantov vpliva na mikrostrukturo, smo mikrostrukturo tekočih kristalov, vključenih v stabilnostno študijo, dodatno pregledali pod polarizacijskim mikroskopom ter potrdili njihovo lamelarno strukturo. Zaključimo lahko, da smo z vgradnjo višjih koncentracij AP in dodatkom vitamina C v tekoče kristale z manjšim deležem vode, dosegli uspešno stabilizacijo AP. Ascorbyl palmitate (AP) is an amphiphilic derivative of vitamin C, which shows greater stability and easier penetration into the skin, but its stability is still insufficient due to rapid oxidation. The aim of master’s thesis is to improve the chemical stability of AP incorporated in lamellar liquid crystals for dermal application. Two different approaches to increase the stability of AP were evaluated, the addition of antioxidants or increasing the AP concentration. Firstly, the effect of antioxidants (vitamins C and E, coenzyme Q10, quercetin, butylhydroxytoluene, propyl gallate) and different concentrations of AP (5% and 10% vs. 1% (w/w)) on the stability of AP in two liquid crystal systems was tested within preliminary study (2 weeks). Liquid crystals had a constant ratio between lipophilic phase and emulsifier mixture at different proportions of aqueous phase (T1 with the lowest and T8 with the highest proportion of water). In the systems with added antioxidant, 1% (w/w) antioxidant and 1% (w/w) AP had been incorporated. A preliminary stability study was carried out at 40 °C and 75 % relative humidity, where the samples were protected from light, and the stability of AP was evaluated by liquid chromatography at 0 and 2 weeks. AP was most stable at both higher AP concentrations and with the additional incorporation of vitamin C in the case of 1% (w/w) AP. Based on the preliminary results, a stability study was further designed and carried out at either higher concentrations of AP or with the addition of vitamin C in liquid crystals. The tested liquid crystals differ in their quantitative composition, namely the water content was increasing from TK1, TK3, TK5 to TK8, comparatively the stability was evaluated also in SMES (mixture of oil and emulsifiers with no added water). The samples were stored at 40 °C and 75 % relative humidity and the decrease in concentration of AP was determined by liquid chromatography within 8 weeks. As determined, the stability of AP increased with decreasing water content. The most optimal stability was observed after 8 weeks in TK1 and SMES at higher AP concentrations or with the addition of the antioxidant vitamin C. Because the addition of antioxidants or increasing the concentration of AP influence the microstructure, the microstructure of the liquid crystals included in the stability study was examined under a polarized microscope and the lamellar structure was confirmed. It can be concluded that by incorporating higher concentrations of AP and the addition of vitamin C to the liquid crystals with a lower proportion of water, successful stabilization of AP was achieved.

Lamelarni tekoči kristali zaradi svoje strukture in lastnosti predstavljajo fiziološko sprejemljiv dostavni sistem za dermalno uporabo, v katerega lahko vgradimo lipofilne, hidrofilne ali amfifilne spojine. Askorbilpalmitat je znan antioksidant, ki se pogosto uporablja v farmaciji in kozmetiki. Je zelo občutljiva in nestabilna amfifilna snov, katere razpad povzročijo številni dejavniki, kot so svetloba, kisik, vlaga ali neustrezna sestava formulacije. V prvem delu magistrske naloge smo ovrednotili stabilnost askorbilpalmitata, vgrajenega v osem sistemov tekočih kristalov (TK 1-TK 8), s konstantnim razmerjem lipofilnih in površinsko aktivnih snovi ob naraščajočem deležu vode kot hidrofilne faze. Preden smo vzorce tekočih kristalov z vgrajenim askorbilpalmitatom izpostavili staranju, smo nadgradili že obstoječo HPLC metodo za analizo askorbilpalmitata. HPLC metodo smo ustrezno optimizirali in validirali, in sicer z vidika selektivnosti, ponovljivosti, točnosti, linearnosti in stabilnosti. Nadalje smo v okviru 56-dnevne pospešene stabilnostne študije ugotovili, da stabilnost askorbilpalmitata z večanjem deleža vode v sistemih tekočih kristalov pada. Sistemi tekočih kristalov z manjšim deležem vode so bili stabilnejši (TK 1-TK 5) glede na sisteme z večjim deležem vode (TK 6-TK 8). Po 28 dneh shranjevanja pri 40 °C in 75 % relativni vlažnosti je bil askorbilpalmitat najbolj stabilen, ko je bil vgrajen v TK 1 (20 % (m/m) vode v sestavi), najmanj pa v sistemu TK 8 (55 % (m/m) vode v sestavi). Na podlagi izračunanih konstant reakcijske hitrosti in R2 smo ugotovili, da kinetika kemijskih reakcij razgradnje askorbilpalmitata sledi 1. redu. Iz vrednosti konstant reakcijske hitrosti je bila razvidna padajoča stabilnost od TK 1 (0,023 d-1) proti TK 8 (0,043 d-1), saj se je z večanjem deleža vode konstanta večala. V drugem sklopu naloge smo vrednotili in vitro sproščanje askorbilpalmitata iz izbranih sistemov tekočih kristalov skozi umetno membrano s pomočjo Franz-ovih difuzijskih celic. Predpostavili smo, da bo sproščanje odvisno od notranje strukture in viskoznosti sistemov, kar se spreminja glede na delež vode. Hitrejše sproščanje in v večjem obsegu smo tako potrdili za sisteme TK 1-TK 4, ki imajo manjši delež proste vode v interlamelarnem prostoru in tako manjše razdalje med lamelami, v primerjavi s TK 5-TK 8, kjer se zaradi naraščajočega deleža vode razdalje med lamelami večajo ob sočasni prisotnosti micelov. Zaključili smo, da lahko tekoče kristale uporabljamo kot dostavne sisteme za podaljšano sproščanje in da le-ti predstavljajo obetaven dostavni sistem za dermalno aplikacijo askorbilpalmitata. Lamellar liquid crystals have been due to their unique structure and properties used as dermal delivery system for lipophilic, hydrophilic or amphiphilic compounds. Ascorbyl palmitate is a well-known antioxidant that is widely used in pharmacy and cosmetics. It is considered as a very unstable amphiphilic compound, which degrades under the influence of light, oxygen, temperature, humidity as well as in suboptimal formulations. In the first part of the thesis we evaluated the stability of ascorbyl palmitate incorporated in eight different systems of liquid crystals (TK 1-TK 8), with a constant ratio of lipophilic compounds and surfactants and with increasing water content as hydrophilic compound. Before the stability study the present HPLC method for ascorbyl palmitate determination was upgraded. Namely, the method was optimized and validated by evaluating the selectivity, repeatability, accuracy, linearity and stability. Further, as a part of the 56-day study we showed that the stability of ascorbyl palmitate was decreasing with increased water content in liquid crystals. Stability of ascorbyl palmitate was better in liquid crystals with lower water content (TK 1-TK 5) compared to liquid crystals with higher water content (TK 6-TK 8). After 28 days of storage at 40 °C and 75 % relative humidity TK 1 (20 % (w/w) of water content) was the most stable system and TK 8 (55 % (w/w) of water content) was considered to be the least stable system. Based on calculated reaction rate constants and R2 we determined that kinetics of chemical reactions of ascorbyl palmitate follows 1. order. Values of the reaction rate constants showed a decreasing stability from TK 1 (0,023 d-1) to TK 8 (0,043 d-1) as the constant increased with increasing water content. In the second part of the thesis we evaluated in vitro release of ascorbyl palmitate from eight systems of liquid crystals. The study was performed on an artificial membrane using Franz diffusion cells. We assumed that the release would depend on the internal structure and viscosity of the systems, which varies with the proportion of water. Therefore, a faster release and to a greater extent was confirmed for systems TK 1-TK 4, which contained smaller amounts of free water in the interlamellar spacing and thus shorter distances between lamellaes, compared to systems TK 5-TK 8, where due to increasing water content the distances between lamellaes are increasing in the presence of micelles. We concluded that liquid crystals can be used as delivery system for sustained release and that they show a great potential as dermal delivery system.