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1 ISSN Том/Volume LIV 2007 Книжка/Number 1-2 СПИСАНИЕ НА БЪЛГАРСКОТО НАУЧНО ДРУЖЕСТВО ПО ФАРМАЦИЯ Главен редактор: Ст. Николов Секретар: Ал. Златков Редакционна колегия: Зл. Димитрова, Св. Богданова, И. Иванов, Г. Китанов, И. Йонкова, Н. Данчев, Г. Петрова, Д. Обрешкова, Ст. Титева, И. Костадинова, Ф. Клерфьой, Е. Х. Хансен, М. Шефер, Р. Грьонинг, Л. Пистели, М. Унзета JOURNAL OF THE BULGARIAN PHARMACEUTICAL SCIENTIFIC SOCIETY Editor in Chief: St. Nikolov Assistant Editor: Al. Zlatkov Editorial Board: Zl. Dimitrova, Sv. Bogdanova, I. Ivanov, G. Kitanov, I. Jonkova, N. Danchev, G. Petrova, D. Obreshkova, St. Titeva, I. Kostadinova, F. Clerfeuille, E. H. Hansen, M. Schaefer, R. Gröning, L. Pistelli, M. Unzeta Адрес на редакцията Address of Editorial Board Фармацевтичен факултет Faculty of Pharmacy ул. "Дунав" 2, София , Dunav str., Sofia 1000 Факс (02) Fax (02) Гл. редакпор: (02) Editor in Chief: (+359 2) snikolov@mbox.pharmfac.acad.bg snikolov@mbox.pharmfac.acad.bg

2 СЪДЪРЖАНИЕ Оригинални статии Alaa M. Hayallah. Дизайн и синтез на нови 1,8-дисубституирани пурин-2,6-диони и 3,6-дисубституирани тиазоло[2,3- f]пурин-2,4-диони като потенциални антиноцицептивни и противовъзпалителни средства...3 К. Йончева, Й. Вандервоорт и А. Лудвиг. Влияние на карбопол върху мукоадхезивните свойства на наночастици за очно приложение, приготвени на базата на съполимер на полимлечна-полигликолова киселина...14 Д. Дренска, М. Варадинова и Н. Бояджиева. Сравняване на антидепресивната активност на антоциани и миансерин при експериментален модел на оксидативен стрес у плъхове...20 Т. Шумкова-Тучева и Н. Бояджиева. Хистоморфологични изследвания върху развитието на хипофизен тумор при третиране с естрогени и с алкохол...25 К. Тодорова, В. Петкова, Зл. Димитрова, С. Захариева и Н. Доганов. Фармакоикономически анализ на профилактичната стратегия за планиране на бременност при жени с инсулинозависим захарен диабет...29 Е. Кръстева, И. Костадинова и Н. Матева. Самолечение с анксиолитици и хипнотици сред студенти и влияние на нервното напрежение...35 От редакционната колегия Инструкции към авторите...39 CONTENTS Original Articles Alaa M. Hayallah. Design and synthesis of new 1,8-disubstituted purine-2,6-diones and 3,6-disubstituted thiazolo[2,3- f]purine-2,4-diones as potential antinociceptive and anti-inflammatory agents...3 K. Yoncheva, J. Vandervoort and A. Ludwig. The influence of carbopol on the mucoadhesive properties of poly(dl-lactide-coglycolide) nanoparticles for ocular purpose...14 D. Drenska, M. Varadinova and N. Boyadjieva. Comparison of antidepressant activity of anthocyanins and mianserin in experimental model of oxidative stress in rats...20 T. Shumkova-Tucheva and N. Boyadjieva. Histomorphological studies on the development of pituitary tumor after treatment with estrogens and alcohol...25 K. Todorova, V. Petkova, Zl. Dimitrova, S. Zaharieva and N. Doganov. Pharmacoeconomic analysis of the prophylactic strategy for pregnacy planning in women with insulin-dependent diabetes mellitus...29 E. Krasteva, I. Kostadinova and N. Mateva. Self-administration of anxiolytics and hypnotics among students and influence of the stress...35 From the Editorial Board Instructions to authors...42 ФАРМАЦИЯ 1-2/2007 ISSN УДК 615 Организационен секретар и стилов редактор Св. Цветанова Корекция Д. Танчева и Св. Цветанова Терминологичен и семантичен контрол д-р Б. Станчева Подписана за печат на г. Печатни коли 5.5, формат 60 x 90/8 Централна медицинска библиотека 1431 София, ул. Св. Г. Софийски 1, тел Fax: nmi@medun.acad.bg; svetlamu@mail.bg

3 14 ФАРМАЦИЯ, том LIV, кн. 1-2/2007 THE INFLUENCE OF CARBOPOL ON THE MUCOADHESIVE PROPERTIES OF POLY(DL-LACTIDE-CO-GLYCOLIDE) NANOPARTICLES FOR OCULAR PURPOSE K. Yoncheva 1,2, J. Vandervoort 2 and A. Ludwig 2 1 Department of Pharmaceutical Technology, Faculty of Pharmacy, Medical University Sofia, Bulgaria 2 Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium Summary. Poly(lactide-co-glycolide) nanoparticles containing pilocarpine hydrochloride as a model drug were prepared by a double emulsification method using polyvinylalcohol (PVA), Carbopol or their mixture either as stabilizers or as coating agents. The larger size and more negative zeta-potential of the nanoparticles formulated with Carbopol suggested the formation of a coating layer on their surface. This process was not well pronounced in the case of PVA/Carbopol prepared nanoparticles due to intermolecular interaction between both agents. The mucoadhesive properties of Carbopol-modified nanoparticles were considered comparing their surface charge before and after incubation into mucin dispersion. The reduction of the negative value denoted that mucin and Carbopol-coated nanoparticles interacted, which could lead to a longer precorneal residence time. Key words: mucoadhesive nanoparticles, mucin, Carbopol, surface charge, coating ВЛИЯНИЕ НА КАРБОПОЛ ВЪРХУ МУКОАДХЕЗИВНИТЕ СВОЙСТВА НА НАНОЧАСТИЦИ ЗА ОЧНО ПРИЛОЖЕНИЕ, ПРИГОТВЕНИ НА БАЗАТА НА СЪПОЛИМЕР НА ПОЛИМЛЕЧНА-ПОЛИГЛИКОЛОВА КИСЕЛИНА К. Йончева 1,2, Й. Вандервоорт 2 и А. Лудвиг 2 1 Катедра по технология на лекарствените средства с биофармация, Фармацевтичен факултет, Медицински университет София 2 Катедра по фармацевтични науки, Антверпенски университет, Антверпен, Белгия Резюме. Наночастици на базата на съполимера полимлечна-полигликолова киселина бяха приготвени по метода на емулгиране/изпаряване на разтворителя. Поливиниловият алкохол, карбополът, както и тяхната смес бяха използвани като стабилизатори и обвиващи агенти. По-големият размер и отрицателният заряд на наночастиците, модифицирани с карбопол, предполагат наличие на повърхностен обвиващ слой около тези частици. Това явление се наблюдава в по-малка степен при наночастиците, обвити с ПВА/карбопол, поради взаимодействие между двата агента. Мукоадхезивните свойства на наночастиците, обвити с карбопол, са предположени, като са сравнени промените в повърхностния им заряд преди и след инкубирането им в муцинова дисперсия. Намаляването на отрицателната стойност на заряда им илюстрира тяхното взаимодействие с муцина, което би могло да доведе до удължаване на прекорнеалното време на приложените наночастици. Ключови думи: мукоадхезивни наночастици, муцин, карбопол, повърхностен заряд, обвиване Introduction Topical administration of drug delivery systems is the most popular way for the treatment of ocular diseases. The main disadvantage of this application is the rapid drug elimination from the precorneal area due to drainage through the naso-lacrimal duct and dilution by tear turnover. These processes result in a very low percentage of the drug administered (less than 5%) which could penetrate through cornea and reaches intraocular tissues. An effective approach to improve the ocular availability of topically applied drugs is to insert them in liposomes [8] or in polymeric systems like nanoparticles [4]. Nanoparticles based on synthetic or natural polymer carriers as poly(lactic-co-glycolic acid), polyalkylcyanoacrylates or gelatin and albumin have been widely studied

4 The influence of carbopol on the mucoadhesive ФАРМАЦИЯ, том LIV, кн. 1-2/ for ocular application [2, 15]. The nanoparticles demonstrated an enhanced accumulation in the conjunctival cul-de-sac and better drug bioavailability compared to conventional dosage forms like solutions and ointments. Although they possess more desirable characteristics, the problem of the fast elimination out of the absorption site still occurred [7]. Various properties of nanoparticles as their size, charge and hydration may influence both the residence time and the penetration of drugs through the cornea. The formation of a coating layer around particles is a promising approach to prolong their residence precorneal time. The use of mucoadhesive polymers as coating agents has been developed since they may interact with the corneal mucus layer [14]. Calvo et al. [1] prepared indomethacin loaded poly(ε-caprolactone) nanocapsules coated either with chitosan or with poly-l-lysine. An improved interaction of the nanocapsules with the corneal epithelium was attempted using the coated particles. In another research, acyclovir loaded PEG-coated polycyanoacrylate nanospheres showed a significant increase of drug levels compared to the application of free drug or a physical mixtures of compounds [3]. The aim of the present study was to prepare poly(lactide-co-glycolide) mucoadhesive nanoparticles for topical ocular application. Carbopol was chosen as a mucoadhesive compound and its mixture with PVA was also studied. Carbopol can prolong the residence time because of interaction with the eye mucosal surface, while PVA may improve the contact between particles and cornea mainly because of its wetting properties. To combine the microencapsulation procedure with the coating process, Carbopol as well as its mixture with PVA were included into the water phases either during the emulsification or evaporation step. For comparison, particles formulated only with PVA in both water phases were prepared. The mucoadhesive properties of the nanoparticles were examined by measurements of surface charge after incubation of nanoparticles into mucin dispersion in simulated lacrimal fluid. Materials and methods Materials. Pilocarpine hydrochloride was obtained from Federa (Brussels, Belgium). Poly(DL-lactideco-glycolide) (PLGA, Resomer RG 503, lactic:glycolic ratio 52:48, MW ) was purchased from Boehringer Ingelheim (Ingelheim, Germany). Since ocular mucin is not commercially available, porcine gastric mucin (type II: crude) was used (Sigma Chemical Co., St. Louis, USA). Carbopol 980 NF was obtained from BF Goodrich (Cleveland, USA). Methanol and acetonitrile (HPLC grade) were from Acros Organics (New Jersey, USA) and methylene chloride from Aldrich (Gillingham, UK). Preparation of nanoparticles. Nanoparticles were prepared by combination of a double emulsification and homogenization procedure [12]. Firstly, an aqueous solution of pilocarpine hydrochloride (2.5% w/v) was dispersed in 10 ml of a methylene chloride phase, containing 1.0 g PLGA-copolymer. The emulsification was carried out by sonication for 1 min at 80 W (Branson Sonifier B-12, Danbury, USA). The resulting emulsion was poured into 50 ml of an aqueous phase containing PVA (1% w/v), Carbopol (0.012% w/v) or PVA/Carbopol mixture. The multiple emulsions obtained were then subjected to a high pressure (500 bar) using a microfluidizer (M-110L, Microfluidics, Newton, USA) and were treated three cycles. The homogenized emulsions were placed in water phases (w 3 - phase) containing PVA (0.3%), Carbopol (0.004%) or their mixture. The procedure was carried out at room temperature under stirring at 700 rpm (IKA Eurostar digi-visc, IKA Labortechnik, Staufen, Germany). The resulting suspensions were cooled down at 18 C and then freeze dried. Determination of drug loading. Freeze dried nanoparticles (20 mg), accurately weighed, were dispersed in 10 ml of distilled water by sonication during 10 min. The samples were centrifuged at 3000 rpm for 3 h and the drug content in the supernatant was determined by an HPLC method. The HPLC system was a Gilson 321 pump (Villiersle-Bel, France). The mobile phase consisted of a water/methanol mixture (97:3, v/v) and potassium dihydrogen phosphate (5%, w/v). Determinations were performed using a column µbondapak C Å 10 µm (Waters) at a flow rate of 2 ml/min and sensitivity 0.005%, respectively. Pilocarpine hydrochloride was detected at 216 nm and its concentration was calculated according to the calibration curve prepared under the same conditions. Particle size measurements. The size of nanoparticles was determined by photon correlation spectroscopy with a Zetasizer 3000 (Malvern Instruments, Malvern, UK). The freeze dried samples were diluted 25 times with distilled water before measurements. Each sample was determined four times and average values were calculated.

5 16 ФАРМАЦИЯ, том LIV, кн. 1-2/2007 K. Yoncheva, J. Vandervoort and A. Ludwig Determination of the surface charge of nanoparticles. The surface charge of the nanoparticles was examined by laser doppler anemometry using a Zetasizer 3000 apparatus (Malvern Instruments, Malvern, UK). Before measurement, the freezedried nanoparticles were dispersed in distilled water, simulated lacrimal fluid (SLF), and 0.1% mucin dispersion in SLF. In vitro drug release studies. The in vitro release studies were carried out using diffusion cells. The acceptor and donor compartments of the cells were separated by a dialysis membrane (Mw cut off D, Medicell, UK). The membranes were washed with distilled water for 30 min before the experiments. The nanoparticles (20 mg) were placed as an aqueous suspension in the donor compartments of the cells. The acceptor compartments were filled with 18 ml distilled water and stirred magnetically at 200 rpm. At suitable time intervals, aliquots of 0.8 ml were withdrawn from the acceptor compartments and replaced by the same volumes of fresh distilled water. The concentrations of samples were determined by the above described HPLC method. Results and discussion In the present study, poly(dl-lactide-co-glycolide) nanoparticles with PVA as a stabilizer were obtained by high-pressure emulsification method (Fig. 1). Similar particles were prepared using mixed water phase of PVA and Carbopol as well as with the phase containing only Carbopol. The addition of Carbopol aimed to improve the mucoadhesive properties of the nanoparticles. Drug loading of nanoparticles. The drug loading extent could be influenced depending on the properties of the coating agent, which also functioned as a stabilizer of the w/o/w emulsion. In our studies, a small difference was obtained between drug loading levels of the particles (Table 1). Lower drug loadings were determined for the PLGA-nanoparticles prepared with PVA and PVA/Carbopol mixed phase and only a little higher loading was observed in the case of the Carbopol nanoparticles. These results were expected because the concentrations of both agents were chosen as to give an equal viscosity to the water phases (1.45 mpa.s). Therefore, the viscosity did not influence drug diffusion out from polymeric droplets to the water phase (w 2 -phase) during emulsification. The higher loading of Carbopol particles could be explained considering the properties of the model drug. Since pilocarpine hydrochloride is a positively charged drug molecule, it may interact with negatively charged polymers [10]. Our investigations showed that Carbopol nanoparticles possessed more negative potential than the other series (Table 2). Hence, charge interaction could contribute to the attraction and entrapment of pilocarpine in the case of nanoparticles prepared with Carbopol. Table 1. Drug loading and encapsulation efficiency (EE) of the PLGA-nanoparticles formulated with PVA, PVA/carbopol or carbopol in the water phases during preparation Water phase (during preparation) Drug loading (%) EE (%) PVA 0.95 ± PVA/Carbopol 0.99 ± Carbopol 1.41 ± Pilocarpine.HCl aqueous solution w 1-phase homogenization freeze-drying sonication in CH 2Cl 2 w 1/o emulsion sonication in w 2-phase w 1/o/w 2 emulsion Fig. 1. Preparation of nanoparticles by double emulsification method. W 2 and w 3 phases contained PVA, carbopol or their mixture either as stabilizers or as coating agents

6 The influence of carbopol on the mucoadhesive ФАРМАЦИЯ, том LIV, кн. 1-2/ Size and surface charge of nanoparticles. Nanoparticle size and zeta-potential measurements were performed to get insights about possible surface coating especially for nanoparticles formulated with Carbopol or PVA/Carbopol mixture. The results from particle size and zeta-potential measurements in two different media are given in Table 2. It was found that the size of PVA and PVA/Carbopol formulated nanoparticles was similar. The data suggested that the presence of Carbopol in the mixture with PVA did not influence the size of particles at all. On the other hand, the size of Carbopol nanoparticles differed by approximately 100 nm in diameter compared to the other series. Hence, the larger size of these particles could be due to the presence of Carbopol at their surface. Similar results were reported in a study where amino-functionalized-peg was applied as a coating agent for polymethyl vinyl ether-co-maleic anhydride nanoparticles [13]. The pegylated nanoparticles were larger than non-coated and their surface charge reached value of 11.5 mv compared to 58.8 mv for the non-coated. Here, the surface charge of the nanoparticles was examined in a simulated lacrimal fluid (SLF) and in water. SLF was chosen as a medium because it possesses a similar composition of ions as the physiological lacrimal fluid. The results revealed that the values of zeta-potential for all series of particles were approximately the same in SLF (Table 2). However, the measurements in water showed different characteristics. This phenomenon was due to the high salt concentration of the SLF-medium which reduced and even equalised the surface charge of the nanoparticles. The process was probably due to the compression of the electric double layer as it was reported by Hoffmann et al. [6]. The surface charge of PVA and PVA/Carbopol nanoparticles in water was more negative in water than in SLF but almost identical for both samples. Only a small difference of approximately 3.0 mv was observed between them. In the same time, nanoparticles formulated with Carbopol alone possessed much more negative zeta-potential in water than the other series. Hence, the addition of PVA to the Carbopol in the same phase strongly changed the properties of particles and their characteristics became identical to those of nanoparticles formulated only with PVA. The explanation could be a possible intermolecular interaction between PVA and Carbopol which blocked the Carbopol functional groups. This assumption is in agreement with the results of nuclear magnetic resonance spectroscopy of PVA/Carbopol film formulations [9]. The authors described the formation of hydrogen bonds between the hydroxyl or carbonyl groups of PVA and the carboxyl groups or carboxylate ions of the polyacrylic acid. The same interaction could be considered between PVA and Carbopol into the water phases during nanoparticle preparation. The value of zeta-potential of Carbopol nanoparticles was 63.5 mv compared to 26.3 mv of nanoparticles formulated with the mixed PVA/Carbopol phase. The reduction of zeta-potential values was a result from the blockage of the negatively charged functional groups of Carbopol. In vitro drug release. In vitro drug release profiles of the three series of particles are given in Fig. 2. The model drug was released from PVA and PVA/Carbopol nanoparticles in a biphasic manner initial burst effect and further slower release, which was previously observed [11]. However, some differences could be made between both series. The drug was released slower from PVA/Carbopol nanoparticles compared to PVA nanoparticles during the first eight hours. After this phase, the pilocarpine was released from both samples in a similar way. As it was mentioned, both kinds of nanoparticles were characterized by identical size and drug loading extent. Consequently, the size and drug loading levels were not responsible for the different release behaviour. The presence of mixed PVA/Carbopol layer around particles could be the main factor influencing the release process. Probably, Carbopol chains swelled and hindered the drug diffusion out of the particles in the beginning. After eight hours when the coating layer was desorbed or dissolved, the drug diffusion was enabled. The same dependence could be considered in the case of nanoparticles formulated with Carbopol. The drug release rate was the slowest till the 8 th h, but almost the same pilocarpine concentration was released at the 24 th h as by the other series. Two reasons could be mentioned to explain the slower drug release rate from Carbopol formulated nanoparticles. The smaller PVA and PVA/Carbopol nanoparticles proposed more surface area favouring a faster release of the drug compared to larger Carbopol nanoparticles. On the other hand, the release rate was lower from nanoparticles prepared with Carbopol because of the surface layer formed by Carbopol chains around these particles. According to the different nanoparticle size observed (Table 2), the thickness of Carbopol layer was probably higher than the thickness of the mixed PVA/Carbopol layer resulting in a lower release rate.

7 18 ФАРМАЦИЯ, том LIV, кн. 1-2/2007 K. Yoncheva, J. Vandervoort and A. Ludwig Table 2. Size and surface charge of the PLGA-nanoparticles formulated with PVA, PVA/Carbopol or Carbopol Water phase (during preparation) Mean diameter (nm) Zeta-potential in water (mv) Zeta-potential in SLF (mv) PVA ± ± ± 2.7 PVA/Carbopol ± ± ± 2.8 Carbopol ± ± ± 6.7 cumulative released (%) time (hours) z-potential (mv) PVA PVA/ Carbopol Carbopol mucin/slf PVA PVA/Carbopol Carbopol nanoparticles in SLF nanoparticles/mucin in SLF Fig. 2. In vitro drug release profiles of pilocarpine hydrochloride from the PLGA-nanoparticles depending on their surface characteristics. Each point represents the mean value of triplicate runs In vitro interaction with mucin. One of the easiest ways to examine mucoadhesive potential of nanoparticles is an in vitro determination of their interaction with a mucin. In the present study, the changes in the surface charge of the nanoparticles after their placing in mucin dispersion were measured. It was assumed that if any changes occur they could be due to an interaction between particles and mucin chains. Simulated lacrimal fluid (SLF) was chosen as a medium for the dispersion because its ion composition may influence the interaction process [5]. The zeta-potential values obtained after one-hour incubation were compared with the values for nanoparticles placed in a mucin-free SLF medium. The results showed that all types of nanoparticles denoted lowering of surface charge in the presence of mucin (Fig. 3). Reduction of the negative value for Carbopol-NP was more pronounced, taking in account the lower charge of these particles. An electrostatic interaction could be excluded because of the negative charge either of mucin chains or coated nanoparticles. Most probably, another phenomenon like a physical entanglement might occur. Conclusion Poly(lactide-co-glycolide) nanoparticles containing pilocarpine hydrochloride as a model drug were prepared by double emulsification method using PVA, Carbopol or their mixture either as stabi- Fig. 3. Zeta-potential values of the nanoparticles in SLF and nanoparticle/mucin dispersion in SLF after one hour incubation, mean values (n=3). The zeta-potential value of mucin in SLF-medium was used as a reference lizers or as coating agents. In the case of PVA/Carbopol as well as Carbopol formulated nanoparticles, a coating layer on the particle surface was probably formed. The mucoadhesive properties of the nanoparticles were considered comparing the surface charge of nanoparticles before and after their incubation into mucin dispersion. Acknowledgments. This research was supported by the Fund for Scientific Research Flanders (F. W. O. Vlaanderen). Dr. K. Yoncheva wishes to thank the Academic Authority of the University of Antwerp (Belgium) for providing a postdoctoral fellowship. References 1. C a l v o, P., J. L. Vila-Jato et M. J. Alonso. Evaluation of cationic polymer-coated nanocapsules as ocular drug carriers. Int. J. Pharm., 153, 1997, D e s h p a n d e, A. A., J. Heller et R. Gurny. Biodegradable polymers for ocular drug delivery. Crit. Rev. Ther. Drug Carrier Syst., 15, 1998, F r e s t a, M. et al. Ocular tolerability and in vivo bioavailability of poly(ethylene glycol)(peg)-coated polyethyl-2- cyanoacrylate nanosphere-encapsulated acyclovir. J. Pharm. Sci., 90, 2001, G u r n y, R. et al. Design and evaluation of controlled release system for the eye. J. Contr. Rel., 6, 1987, H a g e r s t r o m, H., M. Paulsson et K. Edsman. Evaluation of mucoadhesion for two polyelectrolyte gels in

8 The influence of carbopol on the mucoadhesive ФАРМАЦИЯ, том LIV, кн. 1-2/ simulated physiological conditions using a rheological method. Eur. J. Pharm. Sci., 9, 2000, H o f f m a n n, F. et al. Preparation, characterization and citotoxicity of methylmethacrylate copolymer nanoparticles with a permanent positive surface charge. Int. J. Pharm., 157, 1997, L i, V. H. et al. Ocular drug delivery of progesterone using nanoparticles. J. Microencapsul., 3, 1986, S a s a k i, H. et al. Delivery of drugs to the eye by topical application. Progress Ret. Eye Res., 15, 1996, S o l o m o n i d o u, D. et al. Effect of carbomer concentration and degree of neutralization on the mucoadhesive properties of polymer films. J. Biomater. Sci. Polymer Edn., 12, 2001, V a n d e r v o o r t, J., K. Yoncheva et A. Ludwig. Influence of homogenization procedure on the physicochemical properties of PLGA nanoparticles. Chem. Pharm. Bull., 52, 2004, Y o n c h e v a, K. et N. Lambov. Development of biodegradable poly(?-methylmalate) microspheres. Pharmazie, 55, 2000, Y o n c h e v a, K., J. Vandervoort et A. Ludwig. Influence of process parameters of high-pressure emulsification method on the properties of pilocarpine-loaded nanoparticles. J. Microencapsul., 20, 2003, Y o n c h e v a, K. et al. Bioadhesive properties of pegylated nanoparticles. Exp. Opinion Drug Deliv., 2, 2005, Z i m m e r, A. K. et al. Evaluation of pilocarpine-loaded albumin particles as controlled drug delivery systems for the eye. II. Co-administration with bioadhesive and viscous polymers. J. Contr. Rel., 33, 1995, Z i m m e r, A. and J. Kreuter. Microspheres and nanoparticles used in ocular delivery systems. Adv. Drug Deliv. Rev., 16, 1995, Постъпила 15 декември 2006 г. Address for correspondence: Dr. K. Yoncheva Department of Pharmaceutical Technology Faculty of Pharmacy 2 Dunav Str Sofia Bulgaria Адрес за кореспонденция: Д-р К. Йончева Катедра по технология на лекарствените средства с биофармация Фармацевтичен факултет ул. Дунав София