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HCE-T cell line is the most extensively characterized human-derived cell line of corneal epithelium. 3D HCE-T models are commonly cultured on 12-well insert plates and are widely used for permeability and biocompatibility testing [1, 2]. The aim of this research is transfer of a 3D HCE-T model from 12 to 96-well insert plate, in order to develop a high-throughput screening (HTS) model of human corneal epithelium. The cells were stained using 4′, 6-diamidino-2-phenylindole (DAPI, Invitrogen, USA). Fluoroshield™ mounting medium with DAPI was purchased from Sigma-Aldrich (Germany) and 3-[4, 5-dimethylthiazol-2-yl]-2, 5- diphenyl tetrazolium bromide (MTT) was purchased from Carbosynth (UK). For nanoemulsion (NE) preparation the following substances were used: ibuprofen (Hubei Biocause Pharmaceutical, China), Miglyol® 812 (Kemig, Croatia), lecithin S45 (Lipoid, Germany), LMW chitosan (Sigma-Aldrich), Kolliphor® EL (BASF, Germany), glycerol (T.T.T., Croatia) and purified water (SG, Germany). HCE-T cells (RIKEN Cell Bank, Japan) were cultivated in DMEM/F12 medium (Sigma-Aldrich) supplemented with FBS (5%, Capricorn, Germany), insulin (5 μg/ml, Sigma-Aldrich), dimethyl sulfoxide (0.5%, Applichem, Germany), epidermal growth factor (10 ng/ml, Sigma-Aldrich) and antibiotic-antimycotic solution (Sigma-Aldrich) at 37 °C in a humidified atmosphere with 5% CO2. The cells were seeded (1×104 cells per well) on polycarbonate membranes of a 96-well insert plate (PSHT004S5, Merck, Germany) pre-coated with rat tail type I collagen (Sigma-Aldrich) and human fibronectin (Sigma- Aldrich). The cells were cultivated submerged in the medium (75 µL apical and 250 µL basolateral volume) during 7 days and were subsequently exposed to the air-liquid interface (ALI) for 3 days. The cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) at different time points after seeding and the cell nuclei were stained with DAPI. The membranes with cells were mounted on slides using Fluoroshield™ mounting medium with DAPI and the cover slips were sealed with nail polish. The cell nuclei were imaged using confocal fluorescence microscope (ImageXpress® Micro Confocal, Molecular Devices, USA) at 60× magnification. Ophthalmic nanoemulsions (NEs) were produced using microfluidizer (Microfluidics LM20, USA) at 1000 bar and 5 cycles and their composition is shown in table 1. The NEs were diluted 10× in HBSS buffer (pH 6) and the HTS model was exposed to the diluted formulations 30 min at 37°C. The viability of the model was determined using MTT assay and the results are compared with the results obtained previously on a 12-well plate HCE-T model. The HCE-T model was successfully cultivated on a 96- well insert plate. After a 7-day cultivation period in submerged conditions a confluent monolayer was formed and 3 days after ALI exposure multiple layers are observed (Figure 1a). However, HTS HCE-T model was characterized with fewer layers (3-5) than the 12-well plate model [1]. Furthermore, the uppermost layer of the HTS model was not confluent after 3 days of ALI exposure. The viability of the HTS HCE-T model after treatment with ophthalmic NEs was high (about 80%). However, it was lower than the viability of a 12-well plate model treated with the same formulations, under the same conditions (Figure 1b) [2]. A HTS 3D HCE-T model of human corneal epithelium can be cultivated using 96-well insert plates. However, slower proliferation rate and greater sensitivity to the ophthalmic NE formulation is observed in comparison to the standard 12-well plate HCE-T model. In conclusion, further optimisation of cultivation conditions is necessary to obtain higher similarity with the standard 12-well plate HCE-T model. References: 1. M. Juretić et al., HCE-T cell-based permeability model: A well-maintained or a highly variable barrier phenotype?, Eur. J. Pharm. Sci., 2017. vol. 104, pp. 23–30 2. B. Jurišić Dukovski et al., Functional ibuprofen-loaded cationic nanoemulsion: Development and optimization for dry eye disease treatment, Int. J. Pharm., 2020. vol. 576, 118979

Background Donepezil hydrochloride (DH) is commonly used for the treatment of symptoms of Alzheimer’s disease as an oral dosage form (1). Nasal DH administration provides the potential for direct and efficient DH delivery to the central nervous system, while reducing its systemic side effects (2). Purpose The aim of this study is to investigate the potential of spray dried DH loaded chitosan/mannitol microspheres for nasal DH delivery by relevant biopharmaceutical in vitro and ex vivo characterisation. Methods DH loaded chitosan/mannitol microspheres were characterised in terms of DH in vitro release profile, ex vivo mucoadhesiveness using porcine nasal mucosa, in vitro biocompatibility with Calu- 3 cells and DH permeability across Calu-3 cell monolayer. DH loaded chitosan microspheres, pure DH powder and/or pure mannitol were used as controls where appropriate. Results Both, chitosan and chitosan/mannitol microspheres provided prolonged DH release in comparison to pure DH powder. DH loaded chitosan/mannitol microspheres showed prominent mucoadhesive properties. Calu-3 cells exposed to microsphere suspensions prepared at chitosan concentration 10– 30 μg/mL retained viability above 90% in relation to negative control. The Papp values of DH from chitosan/mannitol and chitosan microspheres showed 1.16 and 1.30-fold higher DH permeation in relation to DH solution, respectively. Conclusion Formulating DH as chitosan/mannitol microspheres resulted in prolonged drug release, increased mucoadhesiveness and enhanced drug permeation across Calu-3 monolayer. Results on in vitro biocompatibility confirmed the formulation potential for safe and efficient DH nasal delivery.

Cationic nanoemulsions (NEs) are a new type of artificial tears and a technological platform for poorly soluble drugs that functionally mimic a healthy tear film (TF) [1]. However, their fate after application on the ocular surface is not fully understood and the mechanism of their interaction with the TF is only based on theory or in silico modeling [1, 2]. The aim of this research is getting a deeper insight into cationic NE interaction with the TF components. Chitosan- coated NE (CNE) was prepared using microfluidizer LM20 (Microfluidics, USA) under the pressure of 1000 bar and 5 cycles. CNE was composed of Miglyol 812 (2.5 %, w/w, Kemig, Croatia), ibuprofen (0.2 %, w/w, Hubei Biocause Pharmaceutical, China), lecithin S45 (0.05 %, w/w, Lipoid, Germany), LMW chitosan (0.05 %, w/w, Sigma-Aldrich, Germany), Kolliphor EL (0.25 %, w/w, BASF, Germany), glycerol (2.5 %, w/w, T.T.T., Croatia) and purified water (up to 100 %, w/w). Cationic NE commercially available as artificial tears in EU (caNE) was also used. Artificial tear solution composed of salts only (ATS) or salts and lipids (ATS + lipids) naturally present in the TF was prepared as previously described [3]. The NEs were mixed with ATS or ATS + lipids in 25:7 (V/V) ratio and incubated during 40 min at 34 °C and 50 rpm. Each minute 16 % (V/V) of the mixture was removed and replaced with fresh ATS or ATS + lipids, to simulate TF turnover in vivo [4]. At predetermined time points NE droplet size, PDI and zeta- potential were measured using Zetasizer Ultra (Malvern, UK). NEs were characterized using Zetasizer Ultra and the results are shown in Table 1. Both, CNE and caNE had similar droplet size and PDI and a positive zeta-potential. After mixing with ATS or ATS + lipids both NEs showed changes in their physico-chemical properties, as shown in Figure 1. The zeta-potential of CNE and caNE dropped significantly, which could be caused by high concentration of salts in both ATSes. While caNE did not show bigger changes in droplet size or PDI, CNE showed sudden increase in droplet size after 40 min of incubation and a gradual PDI increase in ATS + lipids. This could be caused by destabilization of the NE droplets due to zeta- potential decrease or merging with lipids present in the ATS. The results presented in this research are a first step toward development of a biopharmaceutical method for testing of NE stability on the eye surface. Further research using more complex ATS (including TF mucins and proteins) is necessary to upgrade the current method. References: 1. Daull, P. et al. Cationic Emulsion-Based Artificial Tears as a Mimic of Functional Healthy Tear Film for Restoration of Ocular Surface Homeostasis in Dry Eye Disease. J. Ocul. Pharmacol. Ther. 36, 355–365 (2020). 2. Gan, L. et al. Recent advances in topical ophthalmic drug delivery with lipid-based nanocarriers. Drug Discov. Today 18, 290–297 (2013). 3. Lorentz, H. et al. Contact lens physical properties and lipid deposition in a novel characterized artificial tear solution. Mol. Vis. 17, 3392–3405 (2011). 4. Pepić, I., Lovrić, J., Cetina-Čižmek, B., Reichl, S. & Filipović-Grčić, J. Toward the practical implementation of eye-related bioavailability prediction models. Drug Discov. Today 19, 31–44 (2014).

To improve ocular bioavailability of eye drops, viscosity-increasing polymers with improved functional properties have been used (1,2). The aim of this study was formulation and evaluation of viscous eye drops using vehicles containing hydroxypropyl guar gum (HPG) (0.25% w/v), sodium hyaluronate (SH) (0.4% w/v), and their combination (HPG-SH) as carriers for olopatadine (0.1% w/v). Physicochemical properties (appearance, clarity, pH, osmolality, viscosity) of the prepared formulations were tested. The drug permeability was estimated in vitro using HCE-T cell-based models and the parallel artificial membrane permeability assay (PAMPA). MTT cytotoxicity assay was performed at the end of the permeability study. Physicochemical stability of the formulated eye drops was tested during 12 months. An accelerated stability study was performed using the heating/cooling cycles test. The clarity, pH and osmolality of all formulations were in acceptable range for ophthalmic preparations. Formulation HPG-SH showed significantly higher viscosity (73.1 mPa·s) than formulations with single polymer (7.4 mPa·s for HPG i.e 3.7 mPa·s for SH) pointing to synergistic effect of polymers. The addition of polymers led to a decrease in transcorneal permeability and lower permeability coefficients with prolonged residence of drug at the ocular surface. The results of MTT assay demonstrated that the tested formulations were biocompatible and well tolerated. Formulated eye drops showed satisfactory physicochemical stability under all storage conditions. Olopatadine was successfully formulated in biocompatible viscous ophthalmic solutions. The combination of viscosity enhancer HPG with mucoadhesive polymer SH has a potential to improve precorneal residence time and therapeutic effect of olopatadine. Polimeri poboljšanih funkcionalnih karakteristika koji povećavaju viskozitet koriste se u kapima za oči kako bi se povećala okularna raspoloživost (1,2). Cilj ove studije bio je formulacija i ispitivanje viskoznih kapi za oči korišćenjem vehikuluma koji sadrže hidroksipropil guar gumu (HPG) (0,25% v/v), natrijum hijaluronat (SH) (0,4% v/v) i njihovu kombinaciju (HPG-SH) kao nosača za olopatadin (0,1% v/v). Ispitivane su fizičkohemijske osobine (izgled, bistrina, pH, osmolalnost, viskozitet) pripremljenih formulacija. Permeabilnost leka je procenjena in vitro korišćenjem modela zasnovanih na HCE-T ćelijama i testa permeabilnosti na paralelnim veštačkim membranama (PAMPA). MTT citotoksični test sproveden je na kraju studije permeabilnosti. Fizičkohemijska stabilnost formulisanih kapi za oči ispitivana je tokom 12 meseci. Sprovedena je ubrzana studija stabilnosti korišćenjem cikličnog testa grejanja/hlađenja. Bistrina, pH i osmolalnost svih formulacija bili su u prihvatljivom opsegu za oftalmološke preparate. Formulacija HPG-SH je pokazala značajno viši viskozitet (73,1 mPa·s) od formulacija sa pojedinačnim polimerom (7,4 mPa·s za HPG, tj. 3,7 mPa·s za SH) ukazujući na sinergistički efekat polimera. Dodatak polimera je doveo do smanjenja transkornealne permeabilnosti i nižih koeficijenata permeabilnosti uz produženo zadržavanje leka na površini oka. Rezultati MTT testa su pokazali da su testirane formulacije biokompatibilne i dobro podnošljive. Formulisane kapi za oči imale su zadovoljavajuću fizičkohemijsku stabilnost pri svim uslovima čuvanja. Uspešno su formulisani biokompatibilni viskozni rastvori za oftalmološku primenu sa olopatadinom. Kombinacija HPG kao sredstva za povećanje viskoziteta sa mukoadhezivnim polimerom SH ima potencijal da poboljša prekornealno vreme zadržavanja i terapijski efekat olopatadina. VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beograd

Oftalmičke nanoemulzije (NE) predstavljaju inovativni pristup liječenju bolesti suhog oka. Složen su farmaceutski oblik koji čine kapljice ulja nanometarske veličine raspršene u vodenoj fazi i stabilizirane površinski aktivnim tvarima (PAT). S obzirom na složenost NE te složenost i dinamiku površine oka, dostupnost biorelevantnih in vitro alata, konstruiranih na temelju ključnih fizioloških i anatomskih aspekata površine oka, osobito je važna za uspješan razvoj oftalmičkih NE. Cilj ovog istraživanja bio je razviti biorelevantnu in vitro metodu za predviđanje stabilnosti NE nakon primjene, a koja uključuje najvažnije fiziološke aspekte površine oka, tj. treptanje i dinamiku izmjene suznog filma. U tu svrhu ispitano je pet različitih NE, od kojih jedna komercijalno dostupna (Cationorm®). Pripravljena je umjetna suzna tekućina (engl. artificial tear solution, ATS), koja sadrži soli, glukozu, ureu, lipide i proteine, koji su fiziološki prisutni u suznom filmu. NE su u inkubatoru (pri 34 °C i 50 okretaja u minuti) razrijeđene ATS-om u volumnom omjeru 25 : 7 (omjer volumena kapi za oko i volumena suznog filma) te je kroz 40 minuta svake minute uklonjeno 16 % volumena smjese NE i ATS-a i nadopunjeno svježim ATS-om, kako bi se simulirala in vivo izmjena suzne tekućine. U predodređenim vremenskim točkama izmjerena je veličina kapljica ulja i indeks polidisperznosti metodom fotonske korelacijske spektroskopije. Nadalje, za odabrane NE korišten je i reološki test koji simulira utjecaj treptanja na formulaciju. NE su prije podvrgavanja visokom gradijentu brzine smicanja razrijeđene ATS-om u omjeru 25 : 7 (v/v). Ciklus simuliranog treptanja, s pauzom između dvaju treptaja, ponovljen je 10 puta pri temperaturi površine oka (34 °C). Rezultati ispitivanja jasno pokazuju razliku u stabilnosti ispitanih NE na osnovu promjene prosječne veličine kapljica uljne faze i indeksa polidisperznosti. Predložena metoda predstavlja vrijedan doprinos razvoju biorelevantnih in vitro alata za pouzdan te vremenski i financijski isplativ razvoj inovativnih i generičkih oftalmičkih proizvoda.

INTRODUCTION Strategies to improve nasal drug delivery include development of formulations which reduce mucociliary clearance [1] and careful selection of nasal delivery device [2]. In situ gelling nanosuspensions present combined approach to increase mucoadhesion and prolong drug retention at nasal mucosa [1]. The aim of this study is to develop an in situ gelling fluticasone propionate nanosuspension and assess its nasal deposition after spraying with jet-flow nebulizer. MATERIALS AND METHODS Fluticasone propionate nanosuspensions prepared by wet media milling, were mixed with pectin and sodium hyaluronate solutions at different ratios. Formulations were characterised in terms of particle size, zeta- potential, surface tension, rheological properties, in vitro biocompatibility using Calu-3 cells, mucoadhesiveness with porcine mucosa and nasal deposition pattern using a jet- flow nebulizer and a 3D printed nasal cast. RESULTS In situ gelling fluticasone propionate nanosuspensions were characterised by particle diameter between 140.3 to 166.8 nm, zeta-potential between –89.5 and –83.2 mV and surface tension of about 37 mN m-1 at 25 °C. Rheological studies confirmed the gelation of formulations when mixed with simulated nasal fluid. The formed gel showed appropriate strength and stability profile. All formulations were biocompatible (cell viability > 90%) and mucoadhesive. Spraying the formulations with jet-flow nebulizer resulted in targeted deposition within the 3D printed nasal cast. CONCLUSION Biocompatible in situ gelling fluticasone propionate nanosuspensions have been successfully prepared. Their rheological properties imply instant gelling in contact with nasal fluid, which, combined with mucoadhesive properties, indicate the potential for extended residence time at nasal mucosa. Nasal deposition profiles proved the suitability of jet-flow nebulizer for nasal delivery of in situ gelling nanosuspensions. Acknowledgement This work has been supported in part by Croatian Science Foundation under the project UIP-2017-05-4592 and European Social Fund under the Croatian Science Foundation project DOK- 2020-01-2473. REFERENCES 1. Alshweiat, A, et al. Int J Pharm (2020) 579:119-166 2. Xi, J, et al. Pharm Res (2016) 33:1527- 1541

Introduction: Nasal route of administration offers a variety of therapeutic opportunities. Recently, it has been proposed as a route of choice for direct nose-to-brain glucocorticoid delivery to control neuroinflammation in patients with severe Covid-19 (1). Nasal powders have shown encouraging results in nose-to-brain drug delivery (2). This work presents the development of nasal powder delivery platform for dexamethasone sodium phosphate (DSP), employing Quality by Design (QbD) principles and including consideration of nasal deposition in the early phase of formulation development. Methods: QbD approach was employed in optimization of spray-dried DSP-loaded pectin and hypromellose microspheres. The optimized microspheres were blended with lactose monohydrate or mannitol at particle size of 45-63 µm as an inert carrier. Powder blends were characterized in terms of homogeneity, flow properties, spray cone angle, in vitro biocompatibility using Calu-3 cells and mucoadhesion using porcine nasal mucosa. Nasal deposition studies were performed using a 3D printed nasal cast connected to respiratory pump. Results: Swellable DSP-loaded polymeric microspheres (Dv50=2.88±0.01 mm) ensuring diffusion-controlled drug release completed in 90 minutes were developed based on QbD approach. Optimised microspheres/inert carrier powder blends were shown to be homogenous and suitable for administration by spraying. SEM micrographs of powder blends revealed microspheres adhered to carrier particles, fitting into the surface within the capsule upon device actuation (1.7-6.3% vs. 14.8%) and narrower spray cone angle (19.6-22.0° vs. 26.5°), which, coupled with appropriate paricle size range, resulted in improved nasal deposition profile. Microspheres were proven to be mucoadhesive showing 12-fold higher work of adhesion in relation to pure drug powder. Blending with inert carriers did not impair their mucoadhesive properties. Powder blends were shown to be biocompatible, inducing no decrease in cell viability. Conclusion/Impact: QbD approach enabled rational desing of spray-dried DSP-loaded polymeric microspheres. Optimised microspheres/inert carrier powder blends showed favourable biopharmaceutcal and sprayability properties. Nasal deposition studies revealed the potential of strategy employed to deliver drug to the targeted regions of nasal cavity.

Svrha rada Razvoj topikalnih oftalmičkih lijekova veliki je izazov zbog slabe topljivosti oftalmičkih djelatnih tvari te anatomskih i fizioloških barijera (suzenje, treptanje, nazolakrimalna drenaža) prednjeg segmenta oka. Kationske U/V nanoemulzije jedan su od inovativnih formulacijskih pristupa za oblikovanje slabo topljivih djelatnih tvari u farmaceutski oblik kapi za oko. Pozitivni naboj na površini uljnih kapljica s uklopljenom djelatnom tvari omogućuje njihovo produljeno zadržavanje zahvaljujući elektrostatskim interakcijama s negativno nabijenom površinom oka. Pri razvoju kationskih nanoemulzija poseban naglasak je na odabiru vrste i koncentracije nositelja pozitivnog naboja kako bi se osigurao dovoljan pozitivni naboj za uspješnu interakciju kapljica s površinom oka, a da se pri tome ne javlja toksični učinak. U sklopu ovog rada izrađene su nanoemulzije sa stearilaminom kao nositeljem pozitivnog naboja. Svrha rada bila je fizičko-kemijska karakterizacija te ispitivanje biokompatibilnosti nanoemulzija na 3D in vitro modelu humanog epitela rožnice. Materijali i metode Kationske nanoemulzije izrađene su visokoenergetskom metodom mikrofluidizacije (M- 110EH-30, Microfluidics, SAD). U njihovoj pripremi korišten je stearilamin (Sigma- Aldrich, Njemačka), Miglyol 812 (Kemig, Hrvatska) i Cremophor® EL (BASF, Njemačka). Izrađenim nanoemulzijama izmjerena je veličina kapljica, indeks polidisperznosti (PDI) i zeta potencijal (Zetasizer Nano Series, Malvern Instruments, Velika Britanija) te pH. Kako bi se ispitala stabilnost nanoemulzija navedeni parametri izmjereni su i nakon 30 dana skladištenja pri 4°C. Za uzgoj 3D in vitro modela epitela humane rožnice korištena je HCE- T stanična linija (RIKEN Cell Bank, Japan) uzgajana na Transwell® pločama (Corning, SAD). Izlaganje modela nanoemulzijama provedeno je tijekom 30 minuta, a vijabilnost modela određena je 24 sata nakon izlaganja kolorimetrijskim MTT testom. Rezultati Kationske nanoemulzije s veličinom kapljica od 81, 2 do 95, 6 nm (PDI 0, 110-0, 251) uspješno su izrađene pomoću mikrofluidizatora. Zeta potencijal nanoemulzija je u rasponu od 3, 1 do 25, 5 mV, ovisno o udjelu stearilamina u formulaciji. Izmjereni pH nanoemulzija je između 6, 8 i 8, 8 te također ovisi o udjelu stearilamina, što je u skladu s literaturnim podacima. Korištenjem 3D in vitro modela epitela humane rožnice dokazana je biokompatibilnost izrađenih nanoemulzija, pri čemu vijabilnost modela nakon tretiranja iznosi oko 90%. Izmjerene vrijednosti veličine kapljica, PDI, zeta potencijala i pH nakon 30 dana skladištenja pri 4 °C ne odstupaju značajno od vrijednosti izmjerenih neposredno nakon izrade, što ukazuje na stabilnost formulacija. Zaključak Rezultati ovog istraživanja ukazuju na potencijal primjene kationskih nanoemulzija sa stearilaminom kao tehnološke platforme za uklapanje teško topljivih oftalmičkih djelatnih tvari i njihovo produljeno zadržavanje na površini oka.

The main objective of this Winter School is to provide basic knowledge of the concepts and conditions needed to fulfil the requirements for the commercialization of scientific research. The Winter School organization comprises a three-day course for PhD students, postdoctoral fellows and early career academics from all disciplines involved in the management and development of healthcare solutions. The Winter School aims to provide a supportive and stimulating international setting where participants can present their business ideas based on their research, receive feedback, and establish valuable business and academic networks for future career development.

Cilj projekta Pharma5.0 je razviti visokokvalitetan suvremeni studij farmacije na engleskom jeziku, ojačati jezične kompetencije osoblja, izraditi didaktički materijal te promovirati studij. Uvođenjem predloženog studijskog programa postavlja se temelj dugotrajnoj internacionalizaciji.

Purpose Cationic nanoemulsions (NEs) are one of the formulation approaches to improve solubility of lipophilic ophthalmic drugs, eye-related bioavailability and drug residence at the ocular surface. The positive charge on the NE droplet surface enables interaction with negatively charged mucins at the ocular surface, which is the proposed mechanism of mucoadhesion 1. The aim of this study was preparation of optimized chitosan coated NE appropriate for dry eye disease treatment, employing lecithin (a natural lipid mixture of phospholipids commonly found in tears2) as a surfactant with overall negative charge that enables droplet interaction with positively charged chitosan chains. Materials and Methods For preparation of NEs the following substances were used: Miglyol 812 (Kemig, Croatia), lecithin S 45 (Lipoid, Germany), Kolliphor EL (BASF, Germany), chitosan (75 % DD ; LMw and MMw ; Sigma-Aldrich, Germany) and double- distilled water. NEs were produced using microfluidizer (Model M-110EH-30, Microfluidics, USA) and characterized in terms of droplet size, PDI and zeta potential (Zetasizer 3000 HS, Malvern Instruments, UK). In the first method, NEs without chitosan were produced first and were further coated with chitosan by its direct addition to the formulation and subsequent short magnetic stirring. In the second method, chitosan was added to the formulation prior processing on microfluidizer. Results NE without chitosan was successfully prepared (droplet size 181.1±2.9 nm ; PDI 0.092±0.026 ; zeta-potential -15.9±0.4 mV). When chitosan (0.05 – 0.5 % (w/w)) was added to the anionic NE the zeta-potential turned positive (29.2±.2 – 44.0±2.0) while the droplet size and PDI increased. The increase was more pronounced when MMw chitosan was used. Therefore, LMw chitosan was used for further studies. When NEs with 0.05 and 0.3 % (w/w) LMw chitosan were produced with the second method the obtained droplet sizes and PDI values were even smaller (183.9±1.5 nm, 0.116±0.008 ; 203.1±8.4 nm, 0.339±0.0.052). Conclusions Chitosan coated NEs were successfully produced using microfluidizer. Smaller droplets and PDIs were obtained when LMw chitosan was used. Chitosan Mw did not seem to have an influence on NE zeta-potential. The moment of chitosan addition was shown to have a major influence on NE droplet size and PDI. References 1. Gan, L. et al. Recent advances in topical ophthalmic drug delivery with lipid- based nanocarriers. Drug Discov. Today 18, 290– 297 (2013). 2. Willcox, M. D. P. et al. TFOS DEWS II Tear Film Report. Ocular Surface (2017). doi:10.1016/j.jtos.2017.03.006

Olive pomace has a composition very similar to olive oil. Thus, it may exert biological effects comparable to those of olive oil (antioxidative, antinflammatory, etc.). However, in complex matrices, such as olive pomace extract (OPE), bioavailability is largely dictated by the release of active ingredients from matrix during the digestion process. Therefore, we simulated in vitro digestion of OPE encapsulated with several cyclodextrins and predicted intestinal permeability of hydroxytyrosol and tyrosol (main olive phenolic compounds).

Bolest suhog oka je multifaktorijalna bolest površine oka čije su glavne značajke nestabilnost i hiperosmolarnost suznog filma, što uzrokuje upalu i oštećenje površine oka [1]. Kationske U/V nanoemulzije važan su formulacijski pristup za unaprjeđenje liječenja suhog oka jer mogu uklopljenom lijeku produljiti vrijeme zadržavanja na površini oka te osigurati uklanjanje simptoma uz smanjenu učestalost doziranja [2]. Ibuprofen je izabran kao modelni nesteroidni protuupalni lijek koji djeluje na upalnu komponentu bolesti. Cilj istraživanja bio je razvoj funkcionalne kationske nanoemulzije za liječenje bolesti suhog oka specifično dizajnirane s obzirom na fizičko-kemijska svojstva koja određuju biokompatibilnost, bioadhezivnost, oslobađanje lijeka i stabilnost. Kationske nanoemulzije s ibuprofenom izrađene su koristeći mikrofluidizator. Kao nositelj pozitivnog naboja korišten je kitozan niske molekulske mase. Od ostalih pomoćnih tvari korišteni su Miglyol 812, lecitin S45 i Cremophor® EL. Lecitin S45 izabran je kao surfaktant jer, osim što su njegove sastavnice prirodno prisutne u suznom filmu, sadrži i negativno nabijene slobodne masne kiseline te omogućuje uklapanje kitozana u nanoemulzije. Cremophor® EL izabran je kao oftalmički prihvatljiv sekundarni, neionogeni surfaktant jer dodatno stabilizira nanoemulziju. Određena je veličina kapljica, indeks polidisperznosti (PDI) i zeta-potencijal te osmolarnost, pH, viskoznost i površinska napetost nanoemulzija. Ispitana je i uspješnost uklapanja lijeka, in vitro oslobađanje te stabilnost. Mukoadhezivnost je ispitana reološkom metodom, a biokompatibilnost koristeći in vitro stanični model humanog epitela rožnice [3] te ex vivo model rožnice svinje. Veličina kapljica kitozanskih nanoemulzija iznosila je 175, 1 – 208, 4 nm (PDI 0, 127 – 0, 356), a zeta-potencijal 24, 6 – 37, 0 mV. Utvrđena je biokompatibilnost i mukoadhezivnost kitozanskih nanoemulzija. [1] J.P. Craig, K.K. Nichols, E.K. Akpek, B. Caffery, H.S. Dua, C.K. Joo, Z.Liu, J.D. Nelson, J.J. Nichols, K. Tsubota, F. Stapleton. TFOS DEWS II Definition and Classification Report. Ocul Surf 15 (2017) 276-83. [2] L. Gan, J. Wang, M. Jiang, H. Bartlett, D. Ouyang, F. Eperjesi, J. Liu, Y. Gan. Recent advances in topical ophthalmic drug delivery with lipid-based nanocarriers. Drug Discov Today 18 (2013) 290-7. [3] M. Juretić, B. Jurišić Dukovski, I. Krtalić, S. Reichl, B. Cetina-Čižmek, J. Filipović-Grčić, J. Lovrić, I. Pepić. HCE-T cell-based permeability model: A well- maintained or a highly variable barrier phenotype? Eur J Pharm Sci 104 (2017) 23-30.